WO2010118367A2 - Antiviral pyrimidines - Google Patents

Antiviral pyrimidines Download PDF

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Publication number
WO2010118367A2
WO2010118367A2 PCT/US2010/030598 US2010030598W WO2010118367A2 WO 2010118367 A2 WO2010118367 A2 WO 2010118367A2 US 2010030598 W US2010030598 W US 2010030598W WO 2010118367 A2 WO2010118367 A2 WO 2010118367A2
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Prior art keywords
compound
hcv
aryl
amine
composition
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PCT/US2010/030598
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French (fr)
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WO2010118367A3 (en
Inventor
Amy Qi Han
Eric Wang
Carla Gauss
Walter Xie
Glen Coburn
Jean-Marc Demuys
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Progenics Pharmaceuticals, Inc.
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Publication of WO2010118367A3 publication Critical patent/WO2010118367A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/32—One oxygen, sulfur or nitrogen atom
    • C07D239/42—One nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/47—One nitrogen atom and one oxygen or sulfur atom, e.g. cytosine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/48—Two nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • C07D239/545—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/553—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms with halogen atoms or nitro radicals directly attached to ring carbon atoms, e.g. fluorouracil
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the present disclosure generally pertains to derivatives of nitrogen-containing heterocyclic compounds, pyrimidines, pyrazolopyrimidines and imidazoiopyrimidines, their stereoisomers, polymorphs, salts, solvates, metabolites, synthetic methods for their preparation, pharmaceutical compositions of the same, and methods for their therapeutic utilization.
  • Such compounds may be useful in general embodiment for the treatment of viral diseases of the flavivirus family, and in one embodiment, for therapy for acute and chronic infections by hepatotrophic virions of the Hepatitis C class (NANB, Non-A, Non-B virus).
  • NANB Hepatitis C class
  • All references cited in this specification, including copending PCT application PCT/US2008/013964, and their references, are incorporated by reference herein where appropriate for teachings of additional alternative details, features, and/or technical background.
  • HCV Hepatitis C virus
  • Flaviviridae and genus Hepacivirus is responsible for chronically infecting approximately 200 million persons worldwide, roughly 3% of the current population of 6.6 billion (1 ). Infection predominantly occurs via the percutaneous exchange of infected blood. The initial infection fails to clear in most instances, and chronic hepatitis, resulting in decompensated liver disease or hepatocellular carcinoma occurs in many cases. Other morbidities associated with chronic HCV infection are mixed cryoglobulinemia, overt B-cell non-Hodgkin's lymphoma, and idiopathic pulmonary fibrosis (2).
  • HCV is structurally related to hepatitis G (HGV-C), GBV-A and GBV-B viruses that infect Tamarin monkeys, West-Nile virus, dengue fever, and yellow fever viruses (3).
  • HCV shows considerable intra-genomic diversity, existing in at least 6 major genotypes, with at least 50 subtypes having been identified.
  • the most effective, proven therapeutic regimen for HCV infection is a combination therapy incorporating alpha-interferon (IFN- ⁇ ) or pegylated IFN- ⁇ and ribavirin, 1-( ⁇ -D-Ribofuranosyi)-1 H-1 ,2,4-triazole-3-carboxamide.
  • This regimen is substantially more efficacious against infections of HCV genotypes 2 and 3, compared to genotype 1 , as measured by sustained viral response.
  • Genotype 1 comprised of subtypes 1a and 1 b, is the major infective agent in the United States, constituting roughly 80% of reported cases (4),
  • IFN- ⁇ probably functions as a genera! inhibitor of viral replication as weli as favorably modulating the host's antiviral immune response (4).
  • HCV is an enveloped, positive sense RNA virus possessing a ⁇ 9.6 kb genome with a single open reading frame.
  • the virus is approximateiy spherical in shape with a diameter of about 60 nm. in the intact virus the genome resides in an icosahedral core.
  • the genome is translated into a single ⁇ 3,000 amino acid polyprotein directed from an internal ribosome entry site (!RES) located within the 5' non-translated region.
  • !RES internal ribosome entry site located within the 5' non-translated region.
  • the structural proteins are released from the poiyprotein by cellular peptidases, whereas non-structural proteins are cleaved by viraily encoded proteases.
  • the envelope protein E2 possesses a binding site for CD81 , a tetraspannin receptor expressed on the cell surface of hepatocytes that acts as a receptor or co-receptor of the HCV viral particle (6).
  • CD81 is necessary but not sufficient for HCV entry. The expression of CD81 alone cannot explain the ce ⁇ ular tropism exhibited by HCV, because this receptor is ubiquitously expressed by a large number of tissue types (6). VanCampemoile et a/.
  • Fusion of the viral capsule with the lipid membrane of a potential host cell is important for viral entry into the cell and is thought to occur by a iow-pH endocytotic process mediated by CD81 (7).
  • HCV research has been hampered by the lack of suitable infectivity models, but recent advances have demonstrated that unmodified HCV envelope proteins can pseudotype retroviral particles and thereby mediate cell entry. Details of HCV tropism and cell entry can now be studied, because such HCV pseudovirus particles (HCVpp) seem to accurately replicate early stages of the viral life cycle (6-10). It has been demonstrated that human immunodeficiency virus (HIV) readily forms pseudotypes, bearing native HCV E1 and E2 glycoproteins, that are infectious for human hepatoma cell lines in vitro (7).
  • HCV human immunodeficiency virus
  • HCVpp accurately reproduce the essential biology of HCV entry into cells susceptible to infection by HCV, (See, e.g., reference 7) and serve as an authentic source of native, fusogenic forms of HCV envelope glycoproteins.
  • HCVpp also provide a means by which to assess HCV entry into cells and to screen small molecule compounds for inhibitory activity. The findings obtained using HCVpp have been substantiated using authentic HCV (12-15).
  • HCVpp entry into liver cells requires co-expression of both the E1 and E2 HCV envelope glycoproteins; neither individual protein is sufficient for entry. Similar to authentic HCV and related viruses, HCVpp fusion does not occur at the cell surface but rather requires endocytosis of virus into mildly acidic endosomes, where fusion is triggered by exposure to low pH (7,16). HCVpp have been shown to be specifically inhibited by monoclonal antibodies directed against £2, as well as by HCV patient sera (7-8,17-18). Studies with HCVpp have identified the presence of naturally-occurring, broad and cross-genotype neutralizing antibodies in sera from HCV-infected individuals (16-18).
  • HCVpp infect CD81 -positive primary hepatocytes and liver cell lines, and monoclonal antibodies directed against CD81 inhibit HCVpp infection (6-8,19-20).
  • CD81 -negative human hepatoma cells are resistant to HCVpp entry, but such ceils become permissive when modified to express CD81.
  • non-hepatic cells are resistant to infection regardless of CD81 expression.
  • CD81 expression is necessary but not sufficient for HCVpp to enter target cells. It has been demonstrated that CD81 functions as a post-attachment co-receptor for HCV as shown by the potent inhibitory activity of CD81 monoclonal antibodies added to HCVpp that was pre-bound to target cells (6). In addition, certain mutations in E2 abolish binding to CD81 but not to target cells (5, 21 ).
  • L 2 , L 4 , and L ⁇ are independently none, H, O, S, NRR', (CH 2 V 5 , CN, CRR', SO 2 , CO, CONHR or CONR'R, NHCONR R, halide, cycloalkyl, heterocycle, aryl, alkyne, alkene;
  • R 2 , R 4 , and R 6 are independently none, R, OR, amine, alkoxy, (CH 2 )o- 3 CF 3 , (CH 2 ) 0 - 3 W, alky!, aryl, cycloalkyl, heterocycle, fused alkyiaryl or heteroaikylaryl, substituted with 0-2 W;
  • W is H, halide, OR, CF 3 , NO 2 , CN, amine, aniline, ester, amide, sulfonamide, sulfone, amino acid, ether, urea acid, heterocycfe, alkyl, aryl, arylalkyl, alkyiaryl; and
  • R or R' is independently H, alkyl, aryl.
  • R 1 H, (u ⁇ )substit ⁇ ted alkyl, (un)substituted aryl, (un)substituted heterocycle;
  • R 3 H, (un)substit ⁇ ted alkyl, (un)substituted aryl, (un)substituted heterocycte;
  • R 4 , and R 6 are independently none, R, OR 1 amine, (CH 2 ) n CF 3 , CF 3 , CH 2 CF 3 , ⁇ CH 2 ) n W, alkyl, aryl, phenyl, cycfoaiky ⁇ ,
  • W is H, halide, OR, CF 3 , NO 2 , CN, SO 2 NRR', SO 2 R, amine, aniline, ester, amide, sulfarn ⁇ yl, amino acid, ether, urea, acid, heterocycle, heteroaromatic, alky), aryl, arylaikyt, afkylaryl, sulfone, sulfonamide substituted with alkyi, aryl, heterocycle, amino, aniline; and R or R 1 is independently H 1 alky!, aryl.
  • Ri H, (un)substituted alky!, (un)substituted aryl, (un)substituted heterocycle;
  • R 3 H, (un)substituted alkyl, (un)substituted aryl, (un)substituted heterocycle;
  • L 5 and L 7 are independently H, O, S, NRR', (CH 2 ),,, CN, CRR', SO 2 , CO, CONR'R,
  • R 5 , and R 7 are independently none, R, OR, amine, ⁇ CH 2 ) n CF 3 , (CH z ) n W, alky!, aryl, phenyl, cycloalkyl, piperidinyl, heterocycle, fused aryl, alkylaryl, or heteroaikylaryl, all of which are optionally substituted with 0-5 W, wherein n is chosen independently to be 0-5; W is H, halide, OR, CF 3 , NO 2 , CN, SO 2 NRR', SO 2 R, sulfamoyl, sulfone, amino acid, ether, urea, heterocycie, heteroaromatic, alkyl, aryi, arylalkyi, alkyiary!, suifone, sulf
  • R 1 H, (un)substituted alkyl, (un)substituted aryl, (un)substituted heterocycie;
  • R 2 H, (un)substituted alkyl, (un)substituted aryi, (un)substituted heterocycie;
  • L 4 and L 6 are independently H, O, S, NRR', (CH 2 J n , CN, CRR', SO 2 , CO, CONR'R,
  • R 4 , and R 6 are independently none, R, OR, amine, (CH 2 J n CF 3 , (CH 2 J n W, alkyl, aryl, phenyl, cycloalkyl, piperidinyl, heterocycie, fused aryl, alkylaryl, or heteroalkylaryl, all of which are optionally substituted with 0-5 W, wherein n is chosen independently to be 0-5;
  • W is H 1 haiide, OR, CF 3 , NO 2 , CN, SO 2 NRR', SO 2 R, amine, aniline, ester, amide, sulfamoyl, amino acid, ether, urea, heterocycie, heteroaromatic, alkyl, aryl, arylalkyi, alkylaryl, sulfone, sulfonamide substituted with alkyl, aryl, heterocycie, amino, aniline; and
  • R or R' is independently H, alkyl, aryl.
  • R 1 H, (un)substituted alkyl, (un)substituted ary!, (un)substituted heterocycie;
  • R 2 H, (un)substituted alkyl, (un)substituted aryl, (unjsubstituted heterocycie;
  • L 5 and L 7 are independently H, O, S, NRR', ⁇ CH 2 ) ni CN, CRR 1 , SO 2 , CO, CONR'R,
  • R 5 , and R 7 are independently none, R, OR, amino, amine, (CH 2 J n CF 3 , (CHa) n W, alkyl, aryl, phenyl, cycioalkyl, piperidinyl, heterocycle, fused aryl, alkylaryl, or heteroalkylaryl, all of which are optionally substituted with 0-5 W, wherein n is chosen independently to be 0-5;
  • W is H, halide, OR, CF 3 , NO 2 , CN, SO 2 NRR', SO 2 R, amino, amine, ani ⁇ ne, ester, amide, sulfonamide, sulfamoyl, sulf ⁇ ne, amino acid, ether, urea, acid, heterocycle, heteroaromatic, alkyl, aryl, arylalkyl, alkylaryl, suifone, sulfonamide substituted with alkyl, aryl, heterocycle, amino, aniline; and R or R' are independently H, alkyl, aryl.
  • composition comprising at least one of a compound, stereoisomer, hydrate, polymorph, or a salt thereof, of a compound of the invention, as exemplified in Tables 1-35, below.
  • compositions of a compound of the invention comprising a pharmaceutically acceptable carrier, the composition further comprising a solid formulation, a semisolid formulation, a solution formulation, an aqueous formulation, an immediate release formulation, a sustained release formulation, an enteric coating formulation or a lyophilized formulation, such formulation optionally being a packaged unit dosage.
  • a pharmaceutical dosage unit composition comprising a pharmaceutical carrier and a therapeutically effective amount of a compound of the invention suitable for treating viral infections of the family Flaviviridae, and, in one aspect, infections by HCV.
  • a further embodiment of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention and a pharmaceutically acceptable carrier or excipient.
  • the composition of the invention may further comprise at least one additional antiviral active ingredient such as an interferon, an anti-HCV monoclonal antibody, an anti-HCV polyclonal antibody, a HCV RNA polymerase inhibitor, a HCV protease inhibitor, an IRES inhibitor, a helicase inhibitor, an antisense compound, an anti-viral small molecule, or a ribozyme.
  • additional antiviral active ingredient such as an interferon, an anti-HCV monoclonal antibody, an anti-HCV polyclonal antibody, a HCV RNA polymerase inhibitor, a HCV protease inhibitor, an IRES inhibitor, a helicase inhibitor, an antisense compound, an anti-viral small molecule, or a ribozyme.
  • antiviral active ingredients may be provided in
  • an antiviral active ingredient may be selected from one or more of the following in any combination: ribavirin, interferon- ⁇ , interferon- ⁇ -2 ⁇ , or a combination thereof.
  • ribavirin interferon- ⁇ , interferon- ⁇ -2 ⁇ , or a combination thereof.
  • Different doses of a compound of the invention may be needed depending on the status of the viral infection, such that different dosages may be needed prior to potential infection, or for early, post infection. Other, different dosages may be needed for obtaining a sustained viral response in the case of a long-term, chronic infection.
  • a compound of the invention may be co-adrninistered with one or more chemotherapeutic drugs or therapeutic or antiviral agents, in one example, with antiviral agents such as ribavirin and/or interferon-alpha (IFN- ⁇ ), and in other examples, with microbial anti- infective agents or with anti-cancer agents.
  • chemotherapeutic drugs or therapeutic or antiviral agents in one example, with antiviral agents such as ribavirin and/or interferon-alpha (IFN- ⁇ ), and in other examples, with microbial anti- infective agents or with anti-cancer agents.
  • IFN- ⁇ interferon-alpha
  • compounds and/or compositions of the invention are administered via an entry portal of the subject body, for example, via oral, enteral, parenteral, subcutaneous, topical and/or intravenous routes,
  • a compound of the invention when a viral infection is being treated, or prophylaxis is desired, a compound of the invention may be administered by any route of administration, including, intravenously, parenteraily, subcutaneously, intramuscularly, or orally.
  • a compound of the invention may be formulated to provide an immediate release dosage form that predominantly releases compound in the stomach.
  • a compound of the invention may be coated to provide an enteric dosage form designed to preferentially release in the intestine, with little or no release in the stomach, [0028] Also provided in one embodiment of the invention is provided a method for treating or preventing infection by a virus of the family Flavivi ⁇ dae, comprising administering to a patient in need thereof a compound and/or composition in an amount effective to treat or prevent the infection.
  • an effective amount of a compound of the invention and/or composition of the invention inhibits or blocks entry of HCV into a susceptible cell.
  • the HCV is of genotype 1 , and more preferably of genotype 1a, genotype 1b, or combinations thereof.
  • the susceptible cell is in a patient, and the compound and/or composition is administered to the patient.
  • [0030] in an embodiment of the invention is a method comprising contacting an HCV-infected cell with a compound and/or composition of the invention, in an amount effective to inhibit or block exit of newly formed HCV particles from an HCV-infected cell.
  • a method of inhibiting HCV infection of a cell susceptible to HCV infection comprising contacting the cell with the compound and/or composition of the invention, in an amount effective to inhibit HCV infection of the cell
  • a method of preventing or diminishing HCV infection in a subject comprising administering to the subject a compound and/or composition of the invention in an amount effective to prevent or diminish the HCV infection.
  • the compound and/or composition of the invention may be administered to the subject before, after, or during exposure of the subject to HCV.
  • an embodiment of the invention is a method of reducing exposure of a subject to HCV infection outside or on the external body surface of the subject, comprising contacting the outside or external body surface of the subject with a compound and/or composition of the invention, in an amount effective to inactivate or inhibit the virus so as to reduce exposure of the subject to HCV infection.
  • In one embodiment of the invention is a method of reducing the occurrence of HCV infection in a population of individuals, comprising administering to the population of individuals in need thereof a compound of the invention in an amount effective to reduce the occurrence of HCV infection in the population.
  • In one embodiment of the invention is a method of treating or preventing a liver disease in a subject, which comprises administering to the subject compound and/or composition of the invention in an amount effective to inhibit infection of the subject's HCV susceptible cells, thereby treating or preventing the liver disease in the subject.
  • a method of treating or preventing an HCV associated disorder in a subject which comprises administering to the subject a compound and/or composition of the invention in an amount effective to inhibit infection of the subject's HCV susceptible cells, thereby treating or preventing an HCV associated disorder in the subject
  • methods for prophylactically preventing or diminishing HCV infections in a warm-blooded animal which comprises administering before, after or during the exposure of said animal to the infective viral agent, a prophylactically effective amount of a compound of the invention.
  • kits for reducing the occurrence of HCV infection in a population of individuals comprising administering to the population of individuals in need thereof a compound and/or composition of the invention in an amount effective to reduce the occurrence of HCV infection in the population.
  • kits for reducing exposure of a subject to HCV infection outside or on the external body surface of the subject comprising contacting the outside or external body surface of the subject with a compound of the invention in an amount effective to inactivate or inhibit the virus so as to reduce exposure of the subject to HCV infection.
  • a liver disease in a subject comprises administering to the subject a compound and/or composition of the invention, in an amount effective to inhibit infection of the subject's HCV susceptible cells, thereby treating or preventing the liver disease in the subject.
  • a method for inhibiting infection of a susceptible cell wherein the cell is contacted with a compound or composition of the invention, in an amount effective to inhibit HCV infection of the cell; further, wherein the cell is in a patient and a compound of the invention is administered to the patient.
  • a method of reducing or diminishing the severity of HCV infection in a subject infected or exposed to HCV comprising administering a compound of the invention to the patient in effective amount.
  • a method of inactivating, inhibiting, decontaminating, or rendering inactive or weakly infective objects, spaces, surfaces, or substances that have been contaminated with HCV which comprises contacting the objects, spaces, surfaces, or substances with a compound of the invention, in an amount effective to inactivate, inhibit, decontaminate, or render inactive or weakiy infective the HCV.
  • compounds of the invention inhibit HCV entry by blocking the interaction of HCV E2 envelope glycoprotein with the scavenger receptor B1 (SR- B1 ) integral membrane, cell surface receptor.
  • [0045] in an embodiment of the invention is a method of inhibiting HCV entry via the SR-B1 receptor into a ceil susceptible to HCV infection, comprising contacting the ceil with a compound of Tables 1-35 S infra.
  • Compounds of the invention may also be used solely or in combination with other antiviral, inactivating or decontaminating agents or drugs to render inactive or weakly infective surfaces or substances that have been contaminated with Flaviviridae such as HCV or other viruses.
  • prodrugs may enhance a number of desirable pharmaceutical qualities (e.g., solubility, bioavailability, manufacturing, efc.).
  • Prodrugs of the compounds of the invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Metabolites of the disclosed compounds may possess pharmacological activity.
  • the compounds are also useful as research or diagnostic reagents, as radioisomers or otherwise, whereby such compounds can be used, for example, to establish competitive binding constants for other compounds, or, for example, as quantitative reagents to assess viral titer.
  • Fig. 1 depicts a schematic representation of various HlV- 1 based pseudoviral particles utilized in assays to evaluate the compounds of the invention.
  • Test and control pseudoparticles include HCVpp, VSVpp (Vesicular Stomatitis Virus pp), MLVpp (Murine Leukemia Virus pp), HiV-i pp, and human patient specific HCVpp, The pseudoviruses are capable of only a single round of infection in target cells. Of the pseudoparticles, HCVpp only recapitulate the process of HCV entry.
  • Fig. 2 illustrates the inhibition of viral pseudoparticle entry into target cells by pyrimidine compounds of the invention and a control (antibody JS-81) as a function of concentration of the inhibitors.
  • Fig. 3 shows the inhibition of viral pseudoparticle entry into target cells by pyrazolopyrimidine and imidazolopyrimidines compounds of the invention and a control (antibody JS-81 ) as a function of concentration of the inhibitors.
  • Fig. 4 shows that a representative pyrazolopyrimidine compound of Table 35 potently neutralizes binding of sE2-6xHis to surface expressed SR-BI on Huh7.5.1 cells.
  • Fig. 5 shows that a compound of the invention, Compound B, maintains its ability to potently neutralize binding of sE2-6xHis to surface expressed SR-BI on Huh7.5.1 cells following pre-treatment and a washout.
  • the present invention provides in one embodiment the selective inhibition or blockade of HCV entry into hepatocytes and other permissive cell types.
  • heterocyclic compounds of the pyrimidine type surprisingly were discovered to be highly active in preventing the entry of HCV genotype 1 pseudoviruses into potential eukaryotic hosts, activity that may include other virions of the Flaviviridae family. This property of the inventive compounds, as described herein, is advantageous because current therapies are poorly efficacious against HCV genotype 1 , the predominant genotype in the United States of America.
  • the blocking mechanism of the compounds may involve direct interaction of HCV with the ectopic domain of the CD81 receptor, or alternatively a direct binding of the compounds with the viral structural proteins E1/E2 such that virion docking is impeded.
  • the compounds may also potentially block the assembly and/or facile release of viral particles from infected cells via intracellular binding to newly formed E1/E2 structural proteins. In this therapeutic modality, the compounds would function as virion assembly or exit inhibitors.
  • the invention further provides the stereoisomers of the compounds disclosed herein, as well as to prodrugs, polymorphs, solvates, ail salts thereof, particularly pharmaceutically acceptable salts, synthetic methods for the preparation of compounds of the invention, pharmaceutical compositions of the same, and methods for therapeutic and/or prophylactic utilization, preparation, and pharmaceutical compositions.
  • the invention also provides methods for utilizing these compounds in anti-viral treatment, therapy, or prophylaxis, either as monotherapy or in combination with other antiviral or chemotherapeutic and/or prophylactic agents. Further, the invention provides for any human and/or animal subject or patient that may be treated with compounds according to the invention.
  • therapeutic and “therapy” are used to describe the administration of medicaments to a patient to correct, treat, ameliorate, or eradicate a condition or infection that has already initiated.
  • prophylactics I Iy and “prophylaxis” describe protective medications or preventive treatments that are administered to a subject and/or applied to an object before contact with HCV, for example, to prevent or diminish the intensity of a subsequent infection of the patient by the virus, or to prevent or diminish contamination of the object by the virus.
  • acyl denotes a radical provided by the residue after removal of hydroxy) from an organic acid.
  • acylamino embraces an amine radical substituted with an acyi group.
  • aryloxy denotes a radical provided by the residue after removal of hydrido from a hydroxy-substituted aryl moiety (e.g., phenol).
  • alkanoyl groups include acetyl (ethanoyi), n-propanoyl, n-butanoyl, 2- methylpropanoyi, n-pentanoyl, 2-methylbutanoyi, 3-methylbutanoyi, 2,2- dimethylpropanoyl, heptanoyl, decanoyl, and palmitoyl.
  • alkenyl includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyfs described below, but that contain at least one double bond and must contain at least two carbon atoms.
  • alkenyl includes straight- chain alkenyl groups (e.g., ethylenyi, propeny!, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycioalkenyi (alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cyclo heptenyl, cyclooctenyl), afkyl or alkenyl substituted cycioalkenyi groups, and cycloaikyl or cycioalkenyi substitute
  • lower aikylene herein refers to those alkyiene groups having from about 1 to about 6 carbon atoms.
  • alkenyj includes both "unsubstituted alkenyls” and “substituted alkenyis”, the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, aikyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyioxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyi, arninocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, aSkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, diaikylamino, arylamino, diarylamino, and alkylaryfamino), acyiamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthi
  • alkenylene in general, refers to an alkyiene group containing at least one carbon- carbon double bond.
  • Preferred alkenylene groups have from 2 to about 4 carbons.
  • alkoxy and alkoxyalkyl embrace linear or branched oxy-containing radicals each having alkyl portions of one to about ten carbon atoms, such as methoxy radical.
  • alkoxyalkyl also embraces alkyl radicals having two or more alkoxy radicals attached to the alkyl radical, that is, to form monoalkoxyaikyl and dialkoxyalkyl radicals.
  • alkoxy or alkoxyalkyl radicals may be further substituted with one or more halo atoms, such as fluoro chloro or bromo to provide "haloalkoxy” or "haloalkoxyalkyl” radicals.
  • halo atoms such as fluoro chloro or bromo to provide "haloalkoxy" or "haloalkoxyalkyl” radicals.
  • alkoxy radicals include methoxy butoxy and trifluoromethoxy.
  • Alkyl in general, refers to an aliphatic hydrocarbon group which may be straight, branched or cyclic having from 1 to about 10 carbon atoms in the chain, and all combinations and subcombinations of ranges therein, e.g., a cycioalkyl, branched cycloalkyialky!, a branched alkylcycloalky having 4-10 carbon atoms.
  • alkyl includes both "unsubstituted alkyls" and “substituted alkyls,” the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the backbone.
  • “Lower alkyl” refers to an alkyl group having 1 to about 6 carbon atoms.
  • Alkyl groups include, but are not limited to, methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyi, t-butyl, n-pentyl, cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl, cyciohexyl, cyclooctyl, adamantyi, 3-methyipentyl, 2-dimethylbutyl, and 2,3- d ⁇ methyibutyi, cyclopropylmethyl and cyclobutyimethyl,
  • Alkyl substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxy
  • araikyl embraces aryl-substituted alkyl radicals such as benzyl, diphenylmethyl, triphenylmethyi, phenethyl, phenylpropyl, and diphenethyl.
  • benzyl and phenylmethyl are interchangeabie.
  • n-alkyl means a straight chain (i.e. unbranched) unsubstituted alkyl group.
  • Branched refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.
  • alkynyi includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond and two carbon atoms.
  • alkynyl includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyi, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloaikyl or cycSoalkenyl substituted alkynyl groups.
  • amido when used by itself or with other terms such as “amidoalkyi", “N- monoalkylamido”, “N-monoarylamido”, “N,N-dtalkylarnido", “N-aikyl-N-arylamido", “N-alkyl-N- hydroxyamido” and “N-alkyl-N-hydroxyamidoalkyl”, embraces a carbonyl radical substituted with an amino radical.
  • N-alkyiamido and “N,N-dialkyiamido M denote amido groups which have been substituted with one alkyl radical and with two aikyt radicals, respectively.
  • N-monoarylamido and “N-a!kyI-N-aryiamido” denote amido radicals substituted, respectively, with one aryl radical, and one alkyl and one aryl radical.
  • N-alkyl-N-hydroxyamido embraces amido radicals substituted with a hydroxyl radical and with an alkyl radical.
  • N-alkyl-N-hydroxyamidoalkyl embraces aikyl radicals substituted with an N-alkyi-N- bydroxyamido radical.
  • amidoalkyl embraces alkyl radicals substituted with amido radicals.
  • amine or "amino” have their common, ordinary meaning.
  • the amines useful in the invention have the general formula:
  • R 3 wherein R 1 , R 2 , and R 3 are identical or a combination of different hydrido, straight or branched chain alkyl groups, alkenyl groups, alkylene groups, alkenylene groups, cycloalkyl groups, cycloalkyl-substituted alkyl groups, cycloafkenyl groups, alkoxy groups, alkoxy-aikyl groups, acyl groups, aryl groups, aryl-substituted alkyl groups, and heterocyclic groups, such as morpholine. If none of R r3 are hydrido, the compound is a tertiary amine.
  • Exemplary tertiary amines useful according to the invention also are cycloalkyl tertiary amines (e.g., N-methylmorphoiine, N- methylpyrrolidine, N-methylpiperidine), pyridine and Proton Sponge® (N 1 N, N', N'-tetramethyl- 1 ,8- naphthalene).
  • aminoalkyl embraces alkyl radicals substituted with amine radicals.
  • alkylaminoalkyi embraces aminoalkyl radicals having the nitrogen atom substituted with an alkyl radical.
  • anti-HCV compound refers to any compound showing the effect of inactivating the virus or diminishing its' infectivity or replication in any way.
  • anti-HCV activity for example, is a compound that interferes with the entry of HCV into an animal cell; such a compound is an "entry inhibitor”. If such a compound interferes with the exit of vira! replicons from the cell, after infection by the virus, the compound is an "exit inhibitor”.
  • a third possibility is a compound that enhances the effectiveness of the subject's immune system in attacking and neutralizing the virus.
  • Yet another possibility for example, is a compound that interferes with the viral life cycle once the virus has gained cellular entry.
  • ⁇ CV-metalloprotease a viral enzyme that is thought to cleave the vira! polypeptide at its NS2-NS3 junction.
  • NS5b HCV encoded RNA dependent RNA polymerase
  • the viral HCV-polymerase, NS5b is essential for viral replication.
  • an "HCV-serine protease” inhibitor Such a compound interferes with the virally encoded serine protease known as NS3-4A that is essential for viral polypeptide cleavage.
  • HCV-he ⁇ case an "HCV-he ⁇ case” inhibitor which prevents the unwinding of the virat genome by interfering with the enzyme HCV-helicase encoded by the virus.
  • Anti-HCV monoclonal antibodies are antibodies that are reactive toward HCV.
  • the antibodies are identical, having been produced by cells that are all genetically identical clones of a single parent cell.
  • Anti-HCV polyclonal antibodies are antibodies that are reactive against HCV. Such antibodies are derived from different cell lines, and are a mixture of immunoglobulin molecules secreted against the virus, each reconizing a specific antigenic site or epitope on the virus.
  • anti-infective agent refers to a compound, composition, substance, reagent, drug, and the like, which acts therapeutically or prophylactically against infectious virat ⁇ e.g. HCV), bacterial, protozoal, or other agents by inhibiting their growth, replication, and survival.
  • Anti-infective agents may comprise preparations that contain natural or synthetic antibiotic agents.
  • anti-cancer agent or “cancer chemotherapeutic agent” refers to a compound, composition, substance, reagent, drug, and the like, which acts therapeutically or prophylactically by inhibiting the growth, replication, spread, and survival of cancer cells.
  • Anticancer agents may comprise preparations that contain natural or synthetic materials that act therapeutically singly or in combination to achieve their effect,
  • antisense molecule refers to a nucleic acid molecule (DNA, RNA, or a chemical analogue) that will complementarily bind to viral RNA, thus preventing the translation of viral proteins, thereby interfering with the viral life cycle. More generally, an antisense molecule binds to or pairs with messenger RNA (mRNA), e.g., an mRNA transcript, to block the expression of a gene, thus effectively turning off that gene and inhibiting its function.
  • mRNA messenger RNA
  • the interfering molecule typically an oligonucleotide, is termed “antisense” because its base sequence is complementary to the RNA, i.e., the "sense” sequence. (Thus, for instance, a sense segment of RNA “ 5'-AAGGUC-3' " would be blocked by the complementary antisense oligonucleotide " 3'-UUCCAG-S' ").
  • aryl alone or in combination, means a carbocyciic aromatic system containing one, two or three rings wherein such rings may be attached together in a pendent manner or may be fused.
  • aryl embraces aromatic radicals such as phenyl, naphthyl, tetrahydronapthyl, indane and biphenyl.
  • Aryl-substituted aikyl in general, refers to an linear alkyl group, preferably a lower alky! group, substituted at a carbon with an optionally substituted aryl group, preferably an optionally substituted phenyl ring.
  • exemplary aryi-substitutedl aikyt groups include, for example, phenylmethyl, phenylethyl and 3- ⁇ 4-methylphenyl)propyl,
  • associated liver disorder refers to any liver dysfunction or malady associated with infection by a virus of the Flaviviridae family, in one example HCV.
  • cycloalkyl embraces radicals having three to ten carbon atoms, such as cyclopropyi cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl,
  • carbocycle is intended to mean any stable 3- to 7-membered monocyclic or bicyclic or 7- to 13-membered bicydic or tricyclic, any of which may be saturated, partially unsaturated, or aromatic.
  • carbocycles include, but are not limited to, cyclopropyi, cyclobutyl, cyclopentyl, cydohexyl, cycloheptyl, adamantyl, cyclooctyl,
  • Carbocycle are cyclopropyi, cyclobutyl, cyclopentyi, and cyclohexyl.
  • Cydoalkyl-substituted alkyl in general, refers to a linear alky! group, preferably a lower alkyl group, substituted at a terminal carbon with a cycioalkyl group, preferably a C 3 -C 8 cycioalkyl group.
  • Typical cydoalkyl-substituted alkyl groups include cyclohexylmethyl, cyclohexylethyl, cyclopentylethyl, cyclopentylpropyl, cyclopropylmethyl and the like.
  • Cycloalkenyl in general, refers to an olefinicaily unsaturated cycioalkyl group having from about 4 to about 10 carbons, and all combinations and subcombinations of ranges therein.
  • the cycloaikenyl group is a C 5 -C 8 cycloalkenyl group, i.e., a cycloalkenyl group having from about 5 to about 8 carbons.
  • halo means halogens such as fluorine, chlorine, bromine or iodine atoms.
  • haloalkyl embraces radicals wherein any one or more of the alkyl carbon atoms is substituted with halo as defined above. Specifically embraced are monohaloalkyl, dihaloalkyl and polyhaloalkyl radicals.
  • a monohaloalkyl radical for one example, may have either a bromo, chloro or a fluoro atom within the radical.
  • Dihalo radicals may have two or more of the same halo atoms or a combination of different halo radicals and polyhaloalkyl radicals may hav ⁇ more than two of the same halo atoms or a combination of different halo radicals.
  • HCV associated disorder is a illness or disorder that is caused or happens as a result of HCV infection.
  • heterocycle or “heterocyclic ring” is intended to mean a stable 5- to 7- membered monocyclic or bicyclic or 7- to 14-membered bicyclic heterocyclic ring which is saturated, partially unsaturated, or unsaturated (aromatic), and which consists of carbon atoms and 1 , 2, 3 or 4 heteroatoms independently selected from the group consisting of
  • hydroxyalkyi embraces linear or branched alkyl radicals having one to about ten carbon atoms any one of which may be substituted with one or more hydroxy! radicals.
  • hydro denotes a single hydrogen atom (H)
  • This hydrido radical may be attached, for example, to an oxygen atom to form a hydroxyl radical or two hydrido radicals may be attached to a carbon atom to form a methylene (-CH 2 - ⁇ radical.
  • interferon denotes a natural protein produced by the cells of the immune response of most vertebrates when challenged by foreign agents such as viruses, bacteria, parasites and tumor cells.
  • interferons e.g., interferon-alpha, interferon-beta, interferon-garnma
  • cytokines cytokines
  • interferons are biological defense modifiers, which inhibit viral replication within cells of the body and thereby assist immune response, e.g., the eradication of virus and viral infection
  • interferons are antiviral and anti-oncogenic, assist macrophage and natural killer lymphocyte activation, and enhance major histocompatibility complex glycoprotein classes I and II, and thereby the presentation of foreign (microbial) peptides to T ceils, which have immune effector function to combat infection.
  • liver disease refers to any pathology, dysfunction, illness, inflammation, cancer or malady of the liver.
  • Non-limiting examp ⁇ es are amebic liver abscess, autoimmune hepatitis, biliary atresia, cirrhosis, dessiminated coccidioido-mycosis, delta agent (hepatitis D), drug-induced cholestasis, hemochromatosis, hepatitis A, hepatitis B, hepatitis C, hepatocellular carcinoma, liver disease due to alcohol, primary biliary cirrhosis, pyogenic liver abscess, Reye's syndrome, sclerosing cholangitis, and Wilson's disease.
  • N-alkylamino and “N,N-dialkyIamino” denote amino groups which have been substituted with one alkyl radical and with two alkyl radicals, respectively.
  • Organic solvent has its common ordinary meaning to those of skill in the art.
  • organic solvents useful in the invention include, but are not limited to, tetrahydrofuran, acetone, hexane, ether, chloroform, acetic acid, acetonitrtle, chloroform, cyclohexane, methanol, and toluene.
  • Anhydrous organic solvents are included.
  • patient refers to animals, including mammals, e.g., rodents (mice, rats) dogs, rabbits, sheep, goats, and non-human primates.
  • rodents mice, rats
  • rabbits sheep, goats
  • non-human primates e.g., monkey, rats
  • patient refers preferably to humans.
  • prodrug refers to compounds specifically designed to maximize the amount of active species that reaches the desired site of reaction that are of themselves typically inactive or minimally active for the activity desired, but through biotransformation or chemical reaction are converted into biologically active molecules.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salts refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • the pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • s ⁇ ch conventional nontoxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, giycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfontc, methanesulf ⁇ nic, ethane disulfonic, oxalic, ethylenediaminetetraacet ⁇ c , and the like.
  • physiologically acceptable salts are prepared by methods known in the art, e.g., by dissolving the free amine bases with an excess of the acid in aqueous alcohol, or neutralizing a free carboxylic acid with an alkali metal base such as a hydroxide, or with an amine.
  • Certain acidic or basic compounds of the present invention may exist as zwitterions. All forms of the compounds, including free acid, free base and zwitterions, are contemplated to be within the scope of the present invention. It is well known in the art that compounds containing both amino and carboxyl groups often exist in equilibrium with their zwitterionic forms.
  • ribozyme derived from a contraction of ribonucleic acid enzyme, refers to a RNA molecule that catalyzes a chemical reaction, typically either the hydrolysis of one of its' own phosphodiester bonds, or the hydrolysis of bonds in other RNAs. Ribozymes are naturally occuring or synthetic.
  • Non-limiting examples of naturally occuring ribozymes are Peptidyl transferase 23S rRNA, Rnase P, G1R1 branching ribozyme, Hairpin ribozyme, Hammerhead ribozyme, HDV ribozyme, Mammalian CPEB3 ribozyme, VS ribozyme, glmS ribozyme and CoTC ribozyme.
  • a "susceptible cell” is a cell which is subject to infection by a virus, in one example, HCV.
  • virion refers to a matured viral particle, either existing outside a cell, or nascent within a cell prior to release.
  • the subjects or patients to which the compounds of the present invention may be administered are vertebrates, in particular mammals.
  • the mammal is a human, nonhuman primate, dog, cat, sheep, goat, horse, cow, pig or rodent, In one embodiment, the mammal is a human.
  • compositions of the invention when used alone or in cocktails, are administered in therapeutically effective amounts.
  • a effective amount will be determined by the parameters discussed below; but typically is that amount which establishes a level of the drug(s) effective for treating a subject, such as a human subject, having one of the conditions described herein.
  • An effective amount means that amount alone, as a single dose, or multiple doses, necessary to delay or prevent the onset of, lessen the severity of, inhibit completely, lessen or reduce the progression of, eradicate or halt altogether the onset or progression of the condition being treated or a symptom associated therewith.
  • an effective therapeutic amount for example, is that amount which relieves a symptom of infection, which induces a decrease in viral load, which increases the time before relapse, or which decreases circulating viral RNA
  • an effective amount for example, would be an amount that prevents active infection, lowers the frequency of active infection, slows the time before an active infection occurs, or diminishes the intensity of the infection.
  • a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular combination of drugs selected, the severity of the condition being treated, or prevented, the condition of the patient, and the dosage required for therapy and/or efficacy.
  • the methods of this invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
  • Such modes of administration include, without limitation, oral, rectal, topical, transdermal, subungual, intravenous infusion, pulmonary, intra-arterial, intra-adipose tissue, intra-lymphatic, intramuscular, intracavity, intraperitoneal (IP), intrathecal, subcutaneous (SC), aerosol, aural (e.g., via eardrops), intranasal, inhalation, intra-articular, needleless injection, subcutaneous or intradermal (e.g., transdermal) delivery.
  • a patient- controlled device or an implantable drug delivery device may be employed.
  • the administration may be by the patient, using an injection device for SC self-administration.
  • Oral, rectal, or topical administration may be important for long-term treatment.
  • Preferred rectal modes of delivery include administration as a suppository or enema wash.
  • the pharmaceutical preparations may conveniently be provided in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. In such form, the entire unit is intended to be administered to the patient as a separate dose. All methods include the step of bringing the compounds of the invention into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compounds of the invention into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. [00101] When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically acceptable compositions.
  • Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, lubricants, and optionally other therapeutic substances and/or ingredients.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically acceptable saits include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulfonic, tartaric, citric, methanesulfonic, formic, succinic, naphthalene-2-sulfonic, pamoic, 3-hydroxy-2-naphthalenecarboxylic, and benzene sulfonic.
  • acids hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulfonic, tartaric, citric, methanesulfonic, formic, succinic, naphthalene-2-sulfonic, pamoic, 3-hydroxy-2-naphthalenecarboxylic, and benzene sulfonic.
  • the salts When used in pharmaceutical preparations, the salts preferably are pharmaceutically- acceptable for use in humans.
  • a bromide salt is an example of one such salt in the case that the parent compound is basic.
  • a sodium salt is an example of one such salt in the case that the parent compound is acidic.
  • radioisomers of the same are encompassed.
  • Such isomers obtained by replacing one or more component atoms of the compound by a radioactive atom, are of a variety well known to those or ordinary skill in the art.
  • such radioisomers can be used therapeutically to deliver localized radiation to a tissue, in one embodiment, a tissue infected with HCV; or in another example, a radioisomer may be used as a tracer to measure metabolic pathways in an animal, or to measure competitive binding in a laboratory sample of tissue.
  • Non-radioactively labeled compounds, produced by replacing one or more of the component atoms with an atomic isotope thereof, are also encompassed.
  • Hydrates are formed when water binds to the crystal structure of a compound in a fixed stoichiometric ratio, although generally this ratio will change depending on the surrounding humidity with which the hydrate is in equilibrium. Hydration is a more specific form of solvation.
  • Solvates are crystalline solid adducts containing either stoichiometric or nonstoichiometric amounts of a solvent incorporated within the crystal structure. If the incorporated solvent is water, the solvates are also commonly known as hydrates. Hydrates and solvates are welt known to those or ordinary skill in the art.
  • Polymorphism is characterized as the ability of a compound or drug substance to exist as two or more crystalline phases that have different arrangements and/or conformations of the molecules in the crystal lattice.
  • Amorphous solids consist of disordered arrangements of molecules and do not possess a distinguishable crystal lattice.
  • Polymorphism refers to the occurrence of different crystalline forms of the same drug substance. Polymorphs are well know to those of ordinary skill in the art.
  • Polymorphs or solvates of a solid can have different chemical and physical properties such as melting point, chemical reactivity, apparent solubility, dissolution rate, optical and electrical properties, vapor pressure, and density, for example. These properties can have a direct impact on the processing of drug substances and the quality or performance of drug products. Chemical and physical stability, dissolution, and bioavailability are some of these qualities.
  • a metastable solid form may change crystalline structure or solvate or desolvate in response to changes in environmental conditions, processing, or over time. New polymorphs can develop spontaneously over time.
  • Infection by Hepatitis C virus predominantly occurs via the percutaneous exchange of infected blood from an outside source, such as a contaminated syringe needle.
  • a compound of the disclosure is capable of reducing exposure of a patient to HCV infection.
  • a compound of the invention produces inactivation or inhibition, or diminishment of infectivity of the virus outside or on the body of a subject by bringing the virus in contact with a compound of the invention in effective amount.
  • An effective amount would be the amount of a compound of the invention that diminishes the infectivity of a virally contaminated outside source, upon the contact of the source with the subject.
  • Compounds of the invention may also be used solely or in combination with other antiviral, inactivating or decontaminating agents or drugs to render inactive or weakly infective spaces, sources, surfaces or substances that have been contaminated with Ftavivi ⁇ dae such as HCV or other viruses.
  • Ftavivi ⁇ dae such as HCV or other viruses.
  • allograft or xenograft tissues, blood, surgical instrument surfaces, syringes, garments, and transfusion apparatuses that pose a viral infective risk to others may be rendered virally inactive or weakly infective by use of the compounds.
  • airborne virally-contarninated blood particles pose an infective risk.
  • a compound of the invention may be dispersed as an aerosol in the contaminated space in an effective amount to inactivate or diminish the infectivity of the airborne virus by contact with the virus.
  • the pharmaceutical preparations of the present invention may include, or be diluted into, a pharmaceutica ⁇ y-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a human or other mammal such as non- human primate, for example, a dog, cat, horse, cow, sheep, pig, or goat.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the carriers are capable of being commingled with the preparations of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy or stability.
  • Carrier formulations suitable for oral administration, for suppositories, and for parenteral administration, etc. can be found in Remington: The Science and Practice , of Pharmacy, 20th Edition. (Aifanso R. Gennaro): Uppincott Williams & Wilkins, Baltimore, MD, 2000.
  • Aqueous formulations may include one or more chelating agents, buffering agents, antioxidants and, optionally, isotonicity agents, preferably pH adjusted, for example, to between 3.0 and 3.5.
  • Chelating agents include, for example and without limitation, ethylenediaminetetraacetic acid (EDTA) and derivatives thereof, citric acid and derivatives thereof, niacinamide and derivatives thereof, sodium desoxycholate and derivatives thereof, and L-glutamic acid, N, N ⁇ diacetic acid and derivatives thereof.
  • EDTA ethylenediaminetetraacetic acid
  • citric acid and derivatives thereof citric acid and derivatives thereof
  • niacinamide and derivatives thereof sodium desoxycholate and derivatives thereof
  • L-glutamic acid N, N ⁇ diacetic acid and derivatives thereof.
  • Buffering agents include, without limitation, those selected from the group consisting of citric acid, sodium citrate, sodium acetate, acetic acid, sodium phosphate and phosphoric acid, sodium ascorbate, tartaric acid, maleic acid, glycine, sodium lactate, lactic acid, ascorbic acid, imidazole, sodium bicarbonate and carbonic acid, sodium succinate and succinic acid, histidine, and sodium benzoate and benzoic acid, and combinations thereof.
  • Antioxidants include, without limitation, those selected from the group consisting of an ascorbic acid derivative, butylated hydroxy anisole, butylated hydroxy toluene, alkyl gallate, sodium meta-bisulfite, sodium bisulfite, sodium dithionite, sodium thioglycollate acid, sodium formaldehyde sulfoxylate, tocopherol and derivatives thereof, monothiogiycerol, sodium sulfite, and combinations thereof.
  • an ascorbic acid derivative butylated hydroxy anisole, butylated hydroxy toluene, alkyl gallate, sodium meta-bisulfite, sodium bisulfite, sodium dithionite, sodium thioglycollate acid, sodium formaldehyde sulfoxylate, tocopherol and derivatives thereof, monothiogiycerol, sodium sulfite, and combinations thereof.
  • Isotonicity agents include, without limitation, those selected from the group consisting of sodium chloride, mannitol, lactose, dextrose, glycerol, and sorbitol and combinations thereof.
  • Preservatives that can be used with the present compositions include, without limitation, benzyl alcohol, parabens, thimerosal, chlorobutanol and preferably benzalkonium chloride and combinations thereof.
  • the preservative is present in a composition in a concentration of up to about 2% by weight. The exact concentration of the preservative, however, will vary depending upon the intended use and can be easily ascertained by one skilled in the art.
  • the compounds of the invention can be prepared in lyophilized compositions, preferably in the presence of one or more cryoprotecting agents such as trehalose, mannitol, lactose, sucrose, polyethylene glycol, and polyvinyl pyrrolidines, Cryoprotecting agents which result in a reconstitution pH of 6.0 or less are preferred.
  • the invention therefore provides a lyophilized preparation of compounds and/or compositions of the invention.
  • the preparation can contain a cryoprotecting agent, such as mannitol or lactose, which is preferably neutral or acidic in water.
  • Oral, parenteral and suppository formulations of agents are well known and commercially available.
  • the therapeutic compounds and/or compositions of the invention can be added to such well known formulations, which can be mixed together in solution or semisolid solution in such formulations, can be provided in a suspension within such formulations, or can be contained in particles within such formulations.
  • a product containing one or more therapeutic compounds of the invention and, optionally, one or more other active agents can be configured as an oral dosage.
  • the oral dosage may be a liquid, a semisolid or a solid.
  • the oral dosage may be configured to release the therapeutic compound of the invention before, after, or simultaneously with the other agent.
  • the oral dosage may be configured to have the therapeutic compound of the invention and the other agents release completely in the stomach, release partially in the stomach and partially in the intestine, in the intestine, in the colon, partially in the stomach, or wholly in the colon.
  • the oral dosage also may be configured whereby the release of the therapeutic compound of the invention is confined to the stomach or intestine while the release of the other active agent is not so confined or is confined differently from the therapeutic compound of the invention.
  • the therapeutic compound of the invention may comprise an enterically coated core or pellets contained within a pill or capsule that releases the other agent first and releases the therapeutic compound of the invention only after the therapeutic compound of the invention passes through the stomach and into the intestine
  • a therapeutic compound of the invention also can be in a sustained release material, whereby the therapeutic compound of the invention is released throughout the gastrointestinal tract and the other agent is released on the same or a different schedule.
  • the same objective for a therapeutic compound of the invention can be achieved with an immediate release of the therapeutic compound of the invention, combined with an enteric coated therapeutic compound of the invention. In this instance, the therapeutic compound could be released immediately in the stomach, throughout the gastrointestinal tract, or only in the intestine.
  • a therapeutic compound of the invention could be coated on the surface of the controlled release formulation in any pharmaceutically acceptable carrier suitable for such coatings and for permitting the release of the therapeutic agent of the invention, such as in a temperature sensitive pharmaceutically acceptable carrier used for controlled release routinely.
  • any pharmaceutically acceptable carrier suitable for such coatings and for permitting the release of the therapeutic agent of the invention such as in a temperature sensitive pharmaceutically acceptable carrier used for controlled release routinely.
  • Other coatings which dissolve when placed in the body are well known to those of ordinary skii! in the art.
  • a therapeutic compound of the invention also may be mixed throughout a controlled release formulation, whereby it is released before, after or simultaneously with another agent.
  • the therapeutic compound of the invention may be free, that is, solubilized within the material of the formulation.
  • the therapeutic compound of the invention also may be in the form of vesicles, such as wax coated micropellets dispersed throughout the material of the formulation.
  • the coated pellets can be fashioned to immediately release the therapeutic compound of the invention based on temperature, pH, or the like.
  • the pellets also can be configured so as to delay the release of the therapeutic compound of the invention, allowing the other agent a period of time to act before the therapeutic compound of the invention exerts its effects.
  • the therapeutic compound of the invention pellets also can be configured to release the therapeutic compound of the invention in virtually any sustained release pattern, including patterns exhibiting first order release kinetics or sigmoidal order release kinetics using materials of the prior art and well known to those of ordinary skill in the art.
  • a therapeutic compound of the invention also can be contained within a core within the controlled release formulation.
  • the core may have any one or any combination of the properties described above in connection with the pellets.
  • the therapeutic agent of the invention may be, for example, in a core coated with a material, dispersed throughout a material, coated onto a material or adsorbed into or throughout a material. It should be understood that the pellets or core may be of virtually any type. They may be drug coated with a release material, drug interspersed throughout material, drug adsorbed into a material, and so on. The material may be erodible or nonerodible.
  • a therapeutic compound of the invention may be provided in particles.
  • Particles as used herein means nano- or microparticles (or in some instances larger) which can consist in whole or in part of a compound of the invention or other agents as described herein.
  • the particles may contain the therapeutic compounds in a core surrounded by a coating, including, but not limited to, an enteric coating. Such compounds also may be dispersed throughout the particles. These compounds also may be adsorbed into the particles.
  • the particles may be of any order release kinetics, including zero order release, first order release, second order release, delayed release, sustained release, immediate release, and any combination thereof, etc.
  • the particle may include, in addition to the therapeutic compound, any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable materia! or combinations thereof.
  • the particles may be microcapsules which contain the antiviral compound in a solution or in a semisolid state.
  • the particles may be of virtually any shape.
  • Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering the therapeutic compounds of the invention.
  • Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired.
  • Btoadhesive polymers of particular interest include bioerodible hydrogels described by H. S.
  • the therapeutic compounds of the invention may be contained in controlled release systems.
  • controlled release is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controiled. This refers to immediate as well as nonimmediate release formulations, with nonimmediate release formulations including but not limited to sustained release and delayed release formulations.
  • sustained release ⁇ also referred to as "extended release” is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period.
  • delayed release is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug therefrom. "Delayed release” may or may not involve gradual release of drug over an extended period of time, and thus may or may not be “sustained release.” These formulations may be for any mode of administration.
  • Delivery systems specific for the gastrointestinal tract are roughly divided into three types: the first is a delayed release system designed to release a drug in response to, for example, a change in pH; the second is a timed-release system designed to release a drug after a predetermined time; and the third is a microflora enzyme system making use of the abundant enterobacteria in the lower part of the gastrointestinal tract (e.g., in a colonic site-directed release formulation).
  • An example of a delayed release system is one that uses, for example, an acrylic or cellulosic coating material and dissolves on pH change. Because of ease of preparation, many reports on such "enteric coatings" have been made, In general, an enteric coating is one which passes through the stomach without releasing substantial amounts of drug in the stomach (i.e., less than 10% release, 5% release and even 1 % release in the stomach) and sufficiently disintegrating in the intestinal tract (by contact with approximately neutral or alkaline intestine juices) to allow the transport (active or passive) of the active agent through the walls of the intestinal tract. [0125] Various in vitro tests for determining whether or not a coating is classified as an enteric coating have been published in the pharmacopoeia of various countries.
  • a coating which remains intact for at least 2 hours, in contact with artificial gastric juices such as HCI of pH 1 at 36 to 38 0 C and thereafter disintegrates within 30 minutes in artificial intestinal juices such as a KH 2 PO 4 buffered solution of pH 6,8 is one example.
  • artificial gastric juices such as HCI of pH 1 at 36 to 38 0 C and thereafter disintegrates within 30 minutes in artificial intestinal juices such as a KH 2 PO 4 buffered solution of pH 6,8
  • EUDRAGIT® material commercially available and reported on by Behringer, Manchester University, Saaie Co., and the like.
  • Enteric coatings are discussed further, below.
  • the enteric coating is typically, although not necessarily, a polymeric material.
  • Preferred enteric coating materials comprise bioerodible, gradually hydrolyzable and/or gradually water-soluble polymers.
  • the "coating weight,” or relative amount of coating material per capsule, generally dictates the time interval between ingestion and drug release.
  • any coating should be applied to a sufficient thickness such that the entire coating does not dissolve in the gastrointestinal fluids at pH below about 5, but does dissolve at pH about 5 and above. It is expected that any anionic polymer exhibiting a pH-dependent solubility profile can be used as an enteric coating in the practice of the present invention.
  • the selection of the specific enteric coating material will depend on the following properties: resistance to dissolution and disintegration in the stomach; impermeability to gastric fluids and drug/carrier/enzyme while in the stomach; ability to dissolve or disintegrate rapidly at the target intestine site; physical and chemical stability during storage; non-toxicity; ease of application as a coating (substrate friendly); and economical practicality.
  • Suitable enteric coating materials include, but are not limited to: cellulosic polymers such as cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethy! cellulose phthalate, hydroxypropyhmethyl cellulose succinate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ammonium methylacrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate ⁇ e.g., those copolymers sold under the trade name EUDRAGIT®); vinyl polymers and copolymers such as polyvinyl acetate, polyvinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymers; and shellac (purified lac).
  • cellulosic polymers such as cellulose acetate phthalate, cellulose acetate trimellitate,
  • enteric coating material for use herein are those acrylic acid polymers and copolymers available under the trade name EUDRAGIT® from Rohm Pharma (Germany).
  • EUDRAGIT® series E, L, S, RL, RS and NE copolymers are available as solubilized in organic solvent, as an aqueous dispersion, or as a dry powder.
  • the EUDRAGIT® series RL, NE, and RS copolymers are insoluble in the gastrointestinal tract but are permeable and are used primarily for extended release.
  • the EUDRAGIT® series E copolymers dissolve in the stomach.
  • the EUDRAGIT® series L, L-30D and S copolymers are insoluble in stomach and dissolve in the intestine, and are thus most preferred herein.
  • a particular methacrylic copolymer is EUDRAGIT® L, particularly L-30D and EUDRAGIT® L 100-55.
  • EUDRAGIT® L-30D the ratio of free carboxyl groups to ester groups is approximatefy 1 :1.
  • the copolymer is known to be insoluble in gastrointestinal fluids having pH below 5.5, generally 1.5-5.5, i.e., the pH generally present in the fluid of the upper gastrointestinal tract, but readily soluble or partially soluble at pH above 5.5, i.e., the pH generally present in the fluid of lower gastrointestinal tract.
  • EUDRAGiT® S Another particular methacrylic acid polymer is EUDRAGiT® S, which differs from EUDRAGIT® L-30D in that the ratio of free carboxyl groups to ester groups is approximately 1 :2.
  • EUDRAGIT® S is insoluble at pH below 5.5, but unlike EUDRAGIT® L-30D, is poorly soluble in gastrointestinal fluids having a pH in the range of 5.5 to 7.0, such as in the small intestine.
  • This copolymer is soluble at pH 7.0 and above, i.e., the pH generally found in the colon.
  • EUDRAGIT® S can be used alone as a coating to provide drug delivery in the large intestine.
  • EUDRAGIT® S being poorly soluble in intestinal fluids beiow pH 7, can be used in combination with EUDRAGIT® L-3QD, soluble in intestinal fluids above pH 5.5, in order to provide a delayed release composition which can be formulated to deliver the active agent to various segments of the intestinal tract.
  • EUDRAGIT L-30D used, the more proximal release and delivery begins, and the more EUDRAGIT® S used, the more distal release and delivery begins.
  • both EUDRAGIT® L-30D and EUDRAGIT® S can be replaced with other pharmaceutically acceptable polymers having similar pH solubility characteristics.
  • the preferred enteric coating is ACRYL-EZE TM (methacrylic acid co-polymer type C; Coiorcon, West Point, PA).
  • the enteric coating provides for controlled release of the active agent, such that drug release can be accomplished at some generally predictable location.
  • the enteric coating also prevents exposure of the therapeutic and/or agent and carrier to the epithelial and mucosal tissue of the buccal cavity, pharynx, esophagus, and stomach, and to the enzymes associated with these tissues.
  • the enteric coating therefore helps to protect the active agent, carrier and a patient's internal tissue from any adverse event prior to drug release at the desired site of delivery.
  • the coated material of the present invention allows optimization of drug absorption, active agent protection, and safety.
  • the coating can, and usually does, contain a plasticizer to prevent the formation of pores and cracks that would permit the penetration of the gastric fluids.
  • Suitable plasticizers include, but are not limited to, triethyl citrate (Citroflex® 2), triacetin (glyceryl triacetate), acetyl triethyl citrate (Citroflec® A2), CarbowaxTM 400 (polyethylene glycol 400), diethyl phthalate, tributyl citrate, acetylated monoglycerides, glycerol, fatty acid esters, propylene glycol, and dibutyl phthalate.
  • Triethyl citrate Citroflex® 2
  • triacetin glyceryl triacetate
  • acetyl triethyl citrate Citroflec® A2
  • CarbowaxTM 400 polyethylene glycol 400
  • diethyl phthalate diethyl phthalate
  • tributyl citrate acetylated monoglycerides
  • glycerol glycerol
  • a coating comprised of an anionic carboxylic acrylic polymer will usually contain approximately 10% to 25% by weight of a plasticizer, particularly dibutyl phthalate, polyethylene glycol, triethyl citrate and triacetin.
  • the coating can also contain other coating excipients such as detackifiers, antifoaming agents, lubricants (e.g., magnesium stearate), and stabilizers (e.g., hydroxypropyiceliulose, acids and bases) to soiubilize or disperse the coating material, and to improve coating performance and the coated product.
  • the coating can be applied to particles of the therapeutic and/or agent(s), tablets of the therapeutic and/or agent(s), capsules containing the therapeutic agent(s)and the like, using conventional coating methods and equipment.
  • an enteric coating can be applied to a capsule using a coating pan, an airless spray technique, fluidized bed coating equipment, or the like.
  • Detailed information concerning materials, equipment and processes for preparing coated dosage forms may be found in Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al, (New York; Marcel Dekker, Inc., 1989), and in Ansei et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th Ed. (Media, PA: Williams & Wiikins, 1995).
  • drug dosage forms comprise an enterically coated, osmotically activated device housing a formulation of the invention.
  • the drug-containing formulation is encapsulated in a semipermeable membrane or barrier containing a small orifice.
  • the semipermeable membrane allows passage of water, but not drug, in either direction. Therefore, when the device is exposed to aqueous fluids, water will flow into the device due to the osmotic pressure differential between the interior and exterior of the device.
  • the drug-containing formulation in the interior will be "pumped” out through the orifice.
  • the rate of drug release will be equivalent to the inflow rate of water times the drug concentration.
  • the rate of water influx and drug efflux can be controlled by the composition and size of the orifice of the device.
  • Suitable materials for the semipermeable membrane include, but are not limited to, polyvinyl alcohol, polyvinyl chloride, semipermeable polyethylene glycols, semipermeable poiyurethanes, semipermeable poiyamides, semipermeable sulfonated polystyrenes and polystyrene derivatives; semipermeable poly(sodium styrenesulfonate), semipermeable poly(vinylbenzyltrimethylammonium chloride), and cellulosic polymers such as cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose propionate, cellulose acetate propionate, cellulose acetate butyrate, cellulose trivalerate, cellulose trilmate, cellulose tripalmitate, cellulose trioctanoate, cellulose tripropionate, cellulose disuccinate, cellulose dipalm ⁇ tate, cellulose dicylate, cellulose acetate succinate, cellulose propionate succinate
  • drug dosage forms are provided that comprise a sustained release coated device housing a formulation of the invention.
  • the drug- containing formulation is encapsulated in a sustained release membrane or film.
  • the membrane may be semipermeable, as described above.
  • a semipermeable membrane allows for the passage of water inside the coated device to dissolve the drug.
  • the dissolved drug solution diffuses out through the semipermeable membrane.
  • the rate of drug release depends upon the thickness of the coated film and the release of drug can begin in any part of the GI tract. Suitable membrane materials for such a membrane include ethylcellulose.
  • drug dosage forms are provided that comprise a sustained release device housing a formulation of the invention.
  • the drug-containing formulation is uniformly mixed with a sustained release polymer.
  • sustained release polymers are high molecular weight water-soluble polymers, which when in contact with water, swell and create channels for water to diffuse inside and dissolve the drug. As the polymers swell and dissolve in water, more of drug is exposed to water for dissolution.
  • sustained release matrix Such a system is generally referred to as sustained release matrix.
  • Suitable materials for such a device include hydropropyl m ethylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose and methyl cellulose.
  • drug dosage forms comprise an enteric coated device housing a sustained release formulation of the invention.
  • the drug containing product described above is coated with an enteric polymer.
  • Such a device would not release any drug in the stomach and when the device reaches the intestine, the enteric polymer is first dissolved and only then would the drug release begin. The drug release would take place in a sustained release fashion.
  • osmotically activated devices can be manufactured using conventional materials, methods and equipment.
  • osmotically activated devices may be made by first encapsulating, in a pharmaceutically acceptable soft capsule, a liquid or semi-solid formulation of the compounds of the invention as described previously, This interior capsule is then coated with a semipermeable membrane composition (comprising, for example, cellulose acetate and polyethylene glycol 4000 in a suitable solvent such as a methylene chloride-methanol admixture), for example using an air suspension machine, until a sufficiently thick laminate is formed, e.g., around 0.05 mm. The semipermeable laminated capsule is then dried using conventional techniques.
  • a semipermeable membrane composition comprising, for example, cellulose acetate and polyethylene glycol 4000 in a suitable solvent such as a methylene chloride-methanol admixture
  • an orifice having a desired diameter e.g., about 0.99 mm
  • a desired diameter e.g., about 0.99 mm
  • the osmotically activated device may then be enterically coated as previously described.
  • the interior capsule is optional; that is, the semipermeable membrane may be formed directly around the carrier-drug composition.
  • preferred carriers for use in the drug-containing formulation of the osmotically activated device are solutions, suspensions, liquids, immiscible liquids, emulsions, sols, colloids, and oils.
  • Particularly preferred carriers include, but are not limited to, those used for enterically coated capsules containing liquid or semisolid drug formulations.
  • Cellulose coatings include those of cellulose acetate phthalate and trimeiiitate; methacrylic acid copolymers, e.g. copolymers derived from methylacrylic acid and esters thereof, containing at least 40% methylacrylic acid; and especially hydroxypropyl methylceliulose phthalate.
  • Methylacrylates include those of molecuiar weight above 100,000 daltons based on, e.g. methylacrylate and methyl or ethyl methyiacrylate in a ratio of about 1 :1.
  • Typical products include Endragit L, e.g. L 100-55, marketed by Rohm GmbH, Darmstadt, Germany.
  • Typical cellulose acetate phthalates have an acetyl content of 17-26% and a phthalate content of from 30-40% with a viscosity of ca. 45-90 cP.
  • Typical cellulose acetate trimeiiitates have an acetyl content of 17-26%, a trimellityi content from 25-35% with a viscosity of ca. 15-20 cS.
  • An example of a cellulose acetate trimeiiitate is the marketed product CAT (Eastman Kodak Company, USA).
  • Hydroxypropyl methylceliulose phthalates typically have a molecuiar weight of from 20,000 to 130,000 daltons, a hydroxypropyl content of from 5 to 10%, a methoxy content of from 18 to 24% and a phthalyl content from 21 to 35%,
  • An example of a cellulose acetate phthalate is the marketed product CAP (Eastman Kodak, Rochester N.Y., USA).
  • hydroxypropyl methylceliulose phthalates are the marketed products having a hydroxypropyl content of from 6-10%, a methoxy content of from 20-24%, a phthalyl content of from 21-27%, a molecular weight of about 84,000 daltons, sold under the trademark HP50 and available from Shin-Etsu Chemical Co. Ltd., Tokyo, Japan, and having a hydroxypropyl content, a methoxyl content, and a phthalyl content of 5-9%, 18-22% and 27-35%, respectively, and a molecular weight of 78,000 daltons, known under the trademark HP55 and available from the same supplier.
  • a timed release system is represented by Time Erosion System (TES) by Fujisawa Pharmaceutical Co., Ltd. and Pulsincap by R. P. Scherer. According to these systems, the site of drug release is decided by the time of transit of a preparation in the gastrointestinal tract. Since the transit of a preparation in the gastrointestinal tract is largely influenced by the gastric emptying time, some time release systems are also enterically coated. [0139] Systems making use of the enterobacteria can be classified into those utilizing degradation of azoaromatic polymers by an azo reductase produced from enterobacteria as reported by the group of Ohio University (M. Saffran, e ⁇ a/., Science, Vol.
  • the therapeutic compounds may be provided in capsules, coated or not.
  • the capsule material may be either hard or soft, and as will be appreciated by those skilled in the art, typically comprises a tasteless, easily administered and water soluble compound such as gelatin, starch or a ceilulosic material.
  • the capsules are preferably sealed, such as with gelatin bands or the like. See, for example, Remington: The Science and Practice of Pharmacy, Nineteenth Edition (Easton, Pa.: Mack Publishing Co., 1995), which describes materials and methods for preparing encapsulated pharmaceuticals.
  • a product containing one or more therapeutic compounds of the invention can be configured as a suppository.
  • the therapeutic compound of the invention can be placed anywhere within or on the suppository to favorably affect the relative release of the therapeutic compound.
  • the nature of the release can be zero order, first order, or sigmoidal, as desired.
  • Suppositories are solid dosage forms of medicine intended for administration via the rectum. Suppositories are compounded so as to melt, soften, or dissolve in the body cavity (around 98.6 0 F) thereby releasing the medication contained therein.
  • Suppository bases should be stable, no n irritating, chemically inert, and physiologically inert.
  • suppositories contain oily or fatty base materials, such as cocoa butter, coconut oil, palm kernel oil, and palm oil, which often melt or deform at room temperature necessitating cool storage or other storage limitations.
  • U.S. Patent No. 4,837,214 to Tanaka et al. describes a suppository base comprised of 80 to 99 percent by weight of a lauric-type fat having a hydroxyl value of 20 or smaller and containing glycerides of fatty acids having 8 to 18 carbon atoms combined with 1 to 20 percent by weight diglycerides of fatty acids (which erucic acid is an example of). The shelf life of these type of suppositories is limited due to degradation.
  • suppository bases contain alcohols, surfactants, and the like which raise the melting temperature but also can lead to poor absorption of the medicine and side effects due to irritation of the local mucous membranes (see for example, U.S. Patent No. 6,099,853 to Hartelendy ef a/., U.S. Patent No. 4,999,342 to Ahmad ef at., and U.S. Patent No. 4,765,978 to Abidi ef a/.).
  • the base used in the pharmaceutical suppository composition of this invention includes, in general, oils and fats comprising triglycerides as main components such as cacao butter, palm fat, palm kernel oil, coconut oil, fractionated coconut oil, lard and WITEPSOL®, waxes such as lanolin and reduced lanolin; hydrocarbons such as VASELINE®, squalene, squalane and liquid paraffin; long to medium chain fatty acids such as caprylic acid, lauric acid, stearic acid and oleic acid; higher alcohols such as lauryl alcohol, cetanol and stearyl alcohol; fatty acid esters such as butyl stearate and dtlauryl malonate; medium to long chain carboxylic acid esters of glycerin such as triolein and tristearin; glycerin-substituted carboxylic acid esters such as glycerin acetoacetate; and polyethylene glycols and its derivatives such as macrogols
  • the pharmaceutical composition of this invention may be prepared by uniformly mixing predetermined amounts of the active ingredient, the absorption aid and optionally the base, etc, in a stirrer or a grinding mill, if required at an elevated temperature.
  • the resulting composition may be formed into a suppository in unit dosage form by, for example, casting the mixture in a mold, or by forming it into a gelatin capsule using a capsule filling machine.
  • the compositions according to the present invention also can be administered as a nasal spray, nasal drop, solution, suspension, gel, ointment, cream or powder.
  • the administration of a composition can also include using a nasal tampon or a nasal sponge containing a composition of the present invention.
  • the nasal delivery systems that can be used with the present invention can take various forms including aqueous preparations, non-aqueous preparations and combinations thereof.
  • Aqueous preparations include, for example, aqueous gels, aqueous suspensions, aqueous liposomal dispersions, aqueous emulsions, aqueous microemulsions and combinations thereof.
  • Non-aqueous preparations include, for example, non-aqueous gels, nonaqueous suspensions, non-aqueous liposomal dispersions, non-aqueous emulsions, nonaqueous microemulsions and combinations thereof.
  • the various forms of the nasal delivery systems can include a buffer to maintain pH, a pharmaceutically acceptable thickening agent and a humectant.
  • the pH of the buffer can be selected to optimize the absorption of the therapeutic agent(s)across the nasal mucosa.
  • suitable forms of buffering agents can be selected such that when the formulation is delivered into the nasal cavity of a mammal, selected pH ranges are achieved therein upon contact with, e.g., a nasal mucosa.
  • the pH of the compositions may be maintained from about 2.0 to about 6.0. It is desirable that the pH of the compositions is one which does not cause significant irritation to the nasal mucosa of a recipient upon administration.
  • the viscosity of the compositions of the present invention can be maintained at a desired level using a pharmaceutically acceptable thickening agent.
  • Thickening agents that can be used in accordance with the present invention include methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.
  • concentration of the thickening agent will depend upon the agent selected and the viscosity desired. Such agents can also be used in a powder formulation discussed above.
  • compositions of the present invention can also include a humectant to reduce or prevent drying of the mucus membrane and to prevent irritation thereof.
  • Suitable humectants that can be used in the present invention include sorbitol, mineral oil, vegetable oil and glycerol; soothing agents; membrane conditioners; sweeteners; and combinations thereof.
  • the concentration of the humectant in the present compositions will vary depending upon the agent selected.
  • One or more therapeutic and/or agents may be incorporated into the nasal delivery system or any other delivery system described herein.
  • composition formulated for topical administration may be iiquid or semi-solid
  • a gel, lotion, emulsion, cream, ointment, spray or aerosol may be provided in combination with a "finite" carrier, for example, a non-spreading material that retains its form, including, for example, a patch, bioadhesive, dressing or bandage. It may be aqueous or non-aqueous; it may be formulated as a solution, emulsion, dispersion, a suspension or any other mixture.
  • compositions provided herein may be applied topically or locally to various areas in the body of a patient.
  • topical application is intended to refer to application to the tissue of an accessible body surface, such as, for example, the skin (the outer integument or covering) and the mucosa (the mucous-producing, secreting and/or containing surfaces).
  • mucosal surfaces include the mucosal surfaces of the eyes, mouth (such as the lips, tongue, gums, cheeks, sublingual and roof of the mouth), larynx, esophagus, bronchial, nasal passages, vagina and rectum/anus; in some embodiments, preferably the mouth, larynx, esophagus, vagina and rectum/anus; in other embodiments, preferably the eyes, larynx, esophagus, bronchial, nasal passages, and vagina and rectum/anus.
  • local application refers to application to a discrete internal area of the body, such as, for example, a joint, soft tissue area (such as muscle, tendon, ligaments, intraocular or other fleshy internal areas), or other interna! area of the body.
  • a discrete internal area of the body such as, for example, a joint, soft tissue area (such as muscle, tendon, ligaments, intraocular or other fleshy internal areas), or other interna! area of the body.
  • soft tissue area such as muscle, tendon, ligaments, intraocular or other fleshy internal areas
  • local application refers to applications to discrete areas of the body.
  • desirable efficacy may involve, for example, penetration of therapeutic agent(s) of the invention into the skin and/or tissue to substantially reach systemic circulation or a peripheral or central locus.
  • the compositions may also contain a glycol, that is, a compound containing two or more hydroxy groups.
  • a glycol which may be particularly useful for use in the compositions is propylene glycol.
  • the glycol may be included in the compositions in a concentration of from greater than 0 to about 5 wt. %, based on the total weight of the composition.
  • the compositions are preferably formulated as a solution or a suspension in an aqueous-based medium, such as isotonically buffered saline or are combined with a biocompatible support or bioadhesive intended for internal administration.
  • Lotions which, for example, may be in the form of a suspension, dispersion or emulsion, contain an effective concentration of one or more of the compounds. The effective concentration is preferably to deliver an effective amount.
  • the compound of the present invention may find use at a concentration of between about 0.1-50% [by weight] or more of one or more of the compounds provided herein.
  • the lotions may contain, for example, [by weight] from 1% to 50% of an emollient and the balance water, a suitable buffer, and other agents as described above.
  • emollients known to those of skill in the art as suitable for application to human skin may be used. These include, but are not limited to, the following: (a) Hydrocarbon oils and waxes, including mineral oil, petrolatum, paraffin, ceresin, ozokerite, microcrystailine wax, polyethylene, and perhydrosqualene.
  • Silicone oils including dimethylpoiysiloxanes, methylphenylpolysiloxanes, water-soluble and alcohol-soluble silicone- glycol copolymers
  • Triglyceride fats and oils including those derived from vegetable, animal and marine sources. Examples include, but are not limited to, castor oil, safflower oil, cotton seed oil, corn oil, olive oil, cod liver oil, almond oil, avocado oil, palm oil, sesame oil, and soybean oil.
  • Acetoglyceride esters such as acetylated monoglycerides.
  • Ethoxylated glycerides such as ethoxylated glyceryl monostearate.
  • esters of fatty acids having 10 to 20 carbon atoms Methyl, isopropyl and butyl esters of fatty acids are useful herein. Examples include, but are not limited to, hexyl laurate, isohexyl laurate, isohexyl paimitate, isopropyl paimitate, isopropyl myristate, decyl oleate, isodecyl oleate, hexadecyl stearate, decyl stearate, isopropyi isostearate, diisopropyl adipate, diisohexyl adipate, dihexyldecyl adipate, diisopropy!
  • Alkenyl esters of fatty acids having 10 to 20 carbon atoms examples thereof include, but are not limited to, oleyl myristate, oleyl stearate, and oleyl oleate.
  • Suitable examples include, but are not limited to, peiargonic, lauric, myristic, palmitic, stearic, isostearic, hydroxystearic, oleic, linoleic, ricinoleic, arachidonic, behenic, and erucic acids, (t) Fatty alcohols having 10 to 22 carbon atoms, such as, but not limited to, lauryl, myristyi, cetyl, hexadecyl, stearyl, isostearyi, hydroxystearyf, oleyl, ricinoleyl, behenyl, erucyl, and 2-octyl dodecyl alcohols.
  • Fatty alcohol ethers including, but not limited to ethoxylated fatty alcohols of 10 to 20 carbon atoms, such as, but are not limited to, the lauryl, cetyl, stearyl, isostearyi, oleyl, and cholesterol alcohols having attached thereto from 1 to 50 ethylene oxide groups or 1 to 50 propylene oxide groups or mixtures thereof, (k) Ether-esters, such as fatty acid esters of ethoxylated fatty alcohols.
  • Lanolin and derivatives including, but not limited to, lanolin, lanolin oil, lanolin wax, lanolin alcohols, lanolin fatty acids, isopropyl lanolate, ethoxylated lanolin, ethoxylated lanolin alcohols, ethoxylated cholesterol, propoxylated lanolin alcohols, acetylated lanolin, acetylated lanolin alcohols, lanolin alcohols linoleate, lanolin alcohols ricinoleate, acetate of lanolin alcohols ricinoleate, acetate of ethoxylated alcohols-esters, hydrogenolysis of lanolin, ethoxylated hydrogenated lanolin, ethoxylated sorbitol lanolin, and liquid and semisolid lanolin absorption bases, (m) poiyhydric alcohols and polyether derivatives, including, but not limited to, propylene glycol, dipropylene glycol, polyethylene glycol
  • polyoxyethylene polyoxypropylene glycols poiyoxypropylene polyoxyethylene glycols, glycerol, ethoxylated glycerol, propoxylated glycerol, sorbitol, ethoxylated sorbitol, hydroxypropyl sorbitol, polyethylene glycol [M.W. 200-6000], methoxy polyethylene glycols 350, 550, 750, 2000, 5000, poly(ethylene oxide) homopolymers [M.W.
  • polyalkylene glycols and derivatives polyalkylene glycols and derivatives, hexylene glycol (2-methyi-2,4-pentanediol), 1 ,3-butylene glycol, 1 ,2,6,-hexanetrtol, ethohexadiol USP (2-ethyl-1 ,3-hexanediol), C 15 -C 18 vicinal glycol and poiyoxypropyte ⁇ e derivatives of trim ethylol propane, (n) poiyhydric alcohol esters, including, but not limited to, ethylene glycol mono- and di-fatty acid esters, diethylene glycol mono- and di- fatty acid esters, polyethylene glycol [M.W.
  • the lotions further preferably contain [by weight] from 1% to 10%, more preferably from 2% to 5%, of an emulsifier.
  • the emulsifiers can be nonionic, anionic or cationic. Examples of satisfactory nonionic emulsifiers include, but are not limited to, fatty alcohols having 10 to 20 carbon atoms, fatty alcohols having 10 to 20 carbon atoms condensed with 2 to 20 moles of ethylene oxide or propylene oxide, alkyl phenols with 6 to 12 carbon atoms in the alkyl chain condensed with 2 to 20 moles of ethylene oxide, mono- and di-fatty acid esters of ethylene oxide, mono- and di-fatty acid esters of ethylene glycol where the fatty acid moiety contains from 10 to 20 carbon atoms, diethylene glycol, polyethylene glycols of molecular weight 200 to 6000, propylene glycols of molecular weight 200 to 3000, glycerol, sorbitol, sorbitan,
  • Suitable anionic emulsifiers include, but are not limited to, the fatty acid soaps, e.g., sodium, potassium and triethanolamine soaps, where the fatty acid moiety contains from 10 to 20 carbon atoms.
  • Other suitable anionic emulsifiers include, but are not limited to, the alkali metal, ammonium or substituted ammonium alkyl sulfates, alkyl arylsulfonates, and alkyl ethoxy ether sulfonates having 10 to 30 carbon atoms in the alkyl moiety.
  • the alkyl ethoxy ether sulfonates contain from 1 to 50 ethylene oxide units.
  • cationic emulsifiers are quaternary ammonium, morpholinium and pyridinium compounds. Certain of the emollients described in preceding paragraphs also have emulsifying properties. When a lotion is formulated containing such an emollient, an additional emulsifier is not needed, though it can be included in the composition.
  • the balance of the lotion is water or a C 2 or C 3 alcohol, or a mixture of water and the alcohol.
  • the lotions are formulated by simply admixing all of the components together.
  • the compound is dissolved, suspended or otherwise uniformly dispersed in the mixture,
  • a thickening agent at a level from 1 % to 10% by weight of the composition.
  • suitable thickening agents include, but are not limited to: cross-linked carboxypolymethylene polymers, ethyl cellulose, polyethylene glycols, gum tragacanth, gum kharaya, xanthan gums and bentonite, hydroxyethyl cellulose, and hydroxypropyl cellulose.
  • Creams can be formulated to contain a concentration effective to deliver an effective amount of therapeutic agent(s) of the invention to the treated tissue, typically at between about 0.1 %, preferably at greater than 1% up to and greater than 50%, preferably between about 3% and 50%, more preferably between about 5% and 15% therapeutic agent(s) of the invention.
  • the creams also contain from 5% to 50%, preferably from 10% to 25%, of an emollient and the remainder is water or other suitable non-toxic carrier, such as an isotonic buffer.
  • the emollients, as described above for the lotions can also be used in the cream compositions.
  • the cream may also contain a suitable emulsifier, as described above.
  • the emulsifier is included in the composition at a level from 3% to 50%, preferably from 5% to 20%.
  • These compositions that are formulated as solutions or suspensions may be applied to the skin, or, may be formulated as an aerosol or foam and applied to the skin as a spray-on.
  • the aerosol compositions typically contain [by weight] from 25% to 80%, preferably from 30% to 50%, of a suitable propellant.
  • propellants are the chlorinated, fluorinated and chlorofluorinated lower molecular weight hydrocarbons. Nitrous oxide, carbon dioxide, butane, and propane are also used as propellant gases. These propellants are used as understood in the art in a quantity and under a pressure suitable to expel the contents of the container.
  • solutions and suspensions may also be topically applied to the eyes and mucosa.
  • Solutions particularly those intended for ophthalmic use, may be formulated as 0.01%-10% isotonic solutions, pH about 5-7, with appropriate salts, and preferably containing one or more of the compounds herein at a concentration of about 0.1%, preferably greater than 1%, up to 50% or more.
  • Suitable ophthalmic solutions are known [see, e.g., U.S. Pat. No, 5,116,868, which describes typical compositions of ophthalmic irrigation solutions and soiutions for topical application].
  • Such solutions which have a pH adjusted to about 7,4, contain, for example, 90-100 mM sodium chloride, 4-6 mM dibasic potassium phosphate, 4-6 mM dibasic sodium phosphate, 8-12 mM sodium citrate, 0.5-1.5 mM magnesium chloride, 1.5-2.5 mM calcium chloride, 15-25 mM sodium acetate, 10-20 mM D,L-sodium ⁇ -hydroxybutyrate and 5- 5.5 mM glucose.
  • Gel compositions can be formulated by simply admixing a suitable thickening agent to the previously described solution or suspension compositions.
  • suitable thickening agents have been previously described with respect to the lotions.
  • the gelled compositions contain an effective amount of therapeutic agent(s) of the invention, typically at a concentration of between about 0.1-50% by weight or more of one or more of the compounds provided herein.; from 5% to 75%, preferably from 10% to 50%, of an organic solvent as previously described; from 0.5% to 20%, preferably from 1% to 10% of the thickening agent; the balance being water or other aqueous or non-aqueous carrier, such as, for example, an organic liquid, or a mixture of carriers.
  • a compound or composition of the invention may be administered to a subject at least once per day, daily, every other day, every 6 to 8 days, weekly, bi-weekly, monthly, or bi-monthly.
  • the formulations can be designed and provided to create steady state plasma levels.
  • Steady state plasma concentrations can be measured using HPLC techniques, as are known to those of skill in the art.
  • Steady state is achieved when the rate of drug availability is equal to the rate of drug elimination from the circulation, in typical therapeutic and/or settings, the therapeutic agent(s) of the invention will be administered to patients either on a periodic dosing regimen or with a constant infusion regimen.
  • the concentration of drug in the plasma will tend to rise immediately after the onset of administration and will tend to fail over time as the drug is eliminated from the circulation by means of distribution into cells and tissues, by metabolism, or by excretion.
  • Steady state will be obtained when the mean drug concentration remains constant over time.
  • the pattern of the drug concentration cycle is repeated identically in each interval between doses with the mean concentration remaining constant.
  • the mean drug concentration will remain constant with very little oscillation.
  • the achievement of steady state is determined by means of measuring the concentration of drug in plasma over at least one cycle of dosing such that one can verify that the cycle is being repeated identically from dose to dose.
  • maintenance of steady state can be verified by determining drug concentrations at the consecutive troughs of a cycle, just prior to administration of another dose.
  • steady state can be verified by any two consecutive measurements of drug concentration.
  • Example 1 Preparation of Example 1: ⁇ 4-(4-(2-(Phenylamino)-6-2,2 I 2-trifluoroethoxy)pyrimidin-4- ylam ⁇ no)piperidin-1-lsulfonyl)phenyl)(pyrrolidin-1-yl)methanone
  • Example 2 4-(4-(4-fluorophenethyl)piperazin-1-y1)-6-(2,2,2-trifluoro ethoxy)-N-(3-(trifluorornethyl)phenyl)pyrimidin-2-amine [0179]
  • Example 3 4- ⁇ 4-(4-fluorobenzyl)p ⁇ perazin-1-yl)-6-(2,2,2-tr ⁇ fluoro ethoxy)-N-
  • Furfuryl amine (143 mg, 1.5 mmol) was added to a solution of 2 (400 mg, 1.5 mmol) and DIPEA (0.26 m!_, 1.5 mmot) in THF (10 ml_) at room temperature. The mixture was stirred for 24 hours at room temperature (TLC control), diluted with water and extracted with dichloromethane. The combined organic phases were concentrated at reduced pressure.
  • Furfuryl amine (143 mg, 1.5 mmol) was added to a solution of 3 (400 mg, 1.5 mmol) and DIPEA (0.26 mL, 1.5 mmol) in THF (10 mL) at room temperature. The mixture was stirred for 24 hours at room temperature (TLC control), diluted with water and extracted with dichloromethane. The combined organic phases were concentrated at reduced pressure.
  • the first approach started from benzenecarboxamidine or aminecarboxamtdine by introducing aryl or amine at C6-position in the first step.
  • the carboxamidine cyclized with diethylmalonate and further underwent chlorinated-oxidation to form dichioroaldehyde 4.
  • cyclization of 4 with various hydrazines formed the pyrazole- pyrimidine core and also introduced an N1 substituent.
  • the mono-chlorine in 6 was replaced with various primary and secondary amines or boronic acids to generate libraries with C4 variation in libraries A, C, E, S, F, K, N, H, AB, wherein:
  • R1 alkyl and aryl (benzyl, Me, Et, Pr, Phenethyl, Piperidine ethyl);
  • R4 anilines (m-, p-arnidoanilines, m-sutfamidoanilines, m-suifoneanilines etc.), primary amines (1 -substituted piperidin-4-amines), secondary amines (substituted piperazines), substituted aryi;
  • R1 alkyl and aryl (benzyl, Me, Pr, o-C!-Phenyl, p-F-Phenyl);
  • R4 anilines (p-Me-aniline, m-Me-aniline, p-F-aniline, m-F-ani)ine, m- s ⁇ lfonyiamidoanilines), primary amines (1 -substituted piperidi ⁇ -4-amines, o-Me- benzylamine);
  • R6 anilines (3,4-diF-aniline, m-F-aniline, etc), primary amines (1 -substituted piperidin-4- amines), secondary amines (substituted piperazines, pyrrolidine), Ar, ethoxy-.
  • R4 OH, anilines, p-F-benzylamine
  • R6 m-, p-sulfonyiamidophenyls, m-amidophenyls.
  • R5 m-sulfonylamidophenyts
  • reaction was monitored by TLC.
  • the reaction mixture was cooled to RT, the precipitate was filtered, and the residue was concentrated under reduced pressure.
  • the key intermediate (8) was obtained as a white precipitate, it was crystallized from ether and dried giving 8 with quantitative yield (0.430 g).
  • LCMS analysis of the reaction mixture demonstrated the presence of starting material only in the case of primary amine, and about 70% of target compound in cases of secondary amine and substituted piperazine.
  • the target compounds were purified by column chromatography with methylene chloride as eluent giving white precipitates in yields of 10-55 %.
  • reaction mixture was added 1.38 g (4,51 mmol) of TEA and 3.76 mmol of amine. Reaction mixture was stirred at 80 0 C overnight. LCMS demonstrated total conversation of starting material. The reaction mixture was evaporated and purified by column chromatography.
  • Compound 7 Compound 6 (200 mg, 0.63 mmol), boronic acid (150 mg, 0.72 mmol), and triphenylphosphine (25 mg, 0.10 mmol) were charged into the flask containing 4 mL of dioxane and 1.5 mL of 2 M aqueous Na 2 CO 3 solution. After purging the mixture with Ar for 20 min, [Pd(PPh 3 ) 4 ] catalyst (36 mg, 0,03 mmol, 5 mo! %) was added. The reaction mixture was heated at 100 0 C for 4 h under an Ar atmosphere. After cooling of the reaction mixture to RT the solvent was removed under reduced pressure to produce yellow oil. The residue was washed with water (20 mL) and extracted with CHCI 3 (20 mL). The organic layer was isolated and purified by coiumn chromatography.
  • reaction mixture was poured into water acidified with HCI and the organic phase was extracted with chloroform twice.
  • the target compound was purified by column chromatography with methylene chloride-ethanol (100:1 ) as an eluent affording oily products in 5-30 % yield (3-15 mg).
  • the 4- and 6-positions were further derivatized by O- and N-nucleophiles.
  • the chlorine at 6-position was attacked by sodium ethoxide, furfurylamine and 4-methoxyaniline to provide intermediates 4a, 5a, 6a correspondingly.
  • the last chlorine was replaced by amines to give the desired compounds.
  • PCI5 (1.2 g) was stirred at refiuxing for 2 hours and then cooied to room temperature. POCI3 was removed at reduced pressure. Crushed ice was added to the residue and the obtained mixture was stirred at room temperature. The formed solid was collected by filtration, washed with water and dried in high vacuum to give compound 3. Yield 160 mg, 47%.

Abstract

Disclosed herein are novel compounds comprising substituted pyrimidines, pyrazolopyrimtdines, and imidazolopyrimidines, the syntheses thereof, and compositions thereof, including pharmaceutical compositions, comprising the novel pyrimidines, pyrazolopyrimtdines, imidazolpyrimidines and related compounds. Such compounds function to inhibit entry of viruses of the Flaviviridae family, including Hepatitis C virus (HCV), into cells that are susceptible to virus infection. These compounds are useful for the treatment, therapy and/or prophylaxis of viral diseases and infection, including HCV infection.

Description

ANTIVIRAL PYRtMIDINES
Technical Field of Disclosure
[0001] The present disclosure generally pertains to derivatives of nitrogen-containing heterocyclic compounds, pyrimidines, pyrazolopyrimidines and imidazoiopyrimidines, their stereoisomers, polymorphs, salts, solvates, metabolites, synthetic methods for their preparation, pharmaceutical compositions of the same, and methods for their therapeutic utilization. Such compounds may be useful in general embodiment for the treatment of viral diseases of the flavivirus family, and in one embodiment, for therapy for acute and chronic infections by hepatotrophic virions of the Hepatitis C class (NANB, Non-A, Non-B virus). [0002] All references cited in this specification, including copending PCT application PCT/US2008/013964, and their references, are incorporated by reference herein where appropriate for teachings of additional alternative details, features, and/or technical background.
Introduction
[0003] Hepatitis C virus (HCV), a virus of the family Flaviviridae and genus Hepacivirus, is responsible for chronically infecting approximately 200 million persons worldwide, roughly 3% of the current population of 6.6 billion (1 ). Infection predominantly occurs via the percutaneous exchange of infected blood. The initial infection fails to clear in most instances, and chronic hepatitis, resulting in decompensated liver disease or hepatocellular carcinoma occurs in many cases. Other morbidities associated with chronic HCV infection are mixed cryoglobulinemia, overt B-cell non-Hodgkin's lymphoma, and idiopathic pulmonary fibrosis (2). [0004] HCV is structurally related to hepatitis G (HGV-C), GBV-A and GBV-B viruses that infect Tamarin monkeys, West-Nile virus, dengue fever, and yellow fever viruses (3). HCV shows considerable intra-genomic diversity, existing in at least 6 major genotypes, with at least 50 subtypes having been identified.
[0005] The US Center for Disease Control estimates that 1.8% of United States inhabitants show seropositivity for HCV antibodies. Roughly 3 out of 4 of these seropositive individuals are also virerrtic, presenting acute or chronic active infections. HCV infection accounts for roughly 30,000 new, acute infections and 8000 to 10,000 deaths yearly in the United States. [0006] The continued failure to develop a highly efficacious treatment for chronic HCV infection is well known, as are the difficulties and experimental uncertainties in developing efficacious medicaments (4). The most effective, proven therapeutic regimen for HCV infection is a combination therapy incorporating alpha-interferon (IFN-α) or pegylated IFN-α and ribavirin, 1-(β-D-Ribofuranosyi)-1 H-1 ,2,4-triazole-3-carboxamide. This regimen is substantially more efficacious against infections of HCV genotypes 2 and 3, compared to genotype 1 , as measured by sustained viral response. Genotype 1 , comprised of subtypes 1a and 1 b, is the major infective agent in the United States, constituting roughly 80% of reported cases (4), The detailed mechanism of ribavirin interaction with the viral life-cycle is not well defined, but IFN-α probably functions as a genera! inhibitor of viral replication as weli as favorably modulating the host's antiviral immune response (4).
[0007] HCV is an enveloped, positive sense RNA virus possessing a ~9.6 kb genome with a single open reading frame. The virus is approximateiy spherical in shape with a diameter of about 60 nm. in the intact virus the genome resides in an icosahedral core. The genome is translated into a single ~3,000 amino acid polyprotein directed from an internal ribosome entry site (!RES) located within the 5' non-translated region. The structural proteins are released from the poiyprotein by cellular peptidases, whereas non-structural proteins are cleaved by viraily encoded proteases.
[0008] Two of the structural subunits, the envelope glycoproteins, E1 and E2, form heterodϊmers and mediate the process of viral attachment, fusion and entry (5,6). The envelope protein E2 possesses a binding site for CD81 , a tetraspannin receptor expressed on the cell surface of hepatocytes that acts as a receptor or co-receptor of the HCV viral particle (6). [0009] CD81 is necessary but not sufficient for HCV entry. The expression of CD81 alone cannot explain the ceϋular tropism exhibited by HCV, because this receptor is ubiquitously expressed by a large number of tissue types (6). VanCampemoile et a/. (5) proposed that cellular permissivity relates to sympathetic mutations in a helical segment of the second extracellular loop of CD81 , known as helix D (5). Therein, residues ((182), N(184), and F(186) have been shown to be particularly important for CD81 binding to HCV-E2 (11 ), and as such, are targets for competitive inhibition and pharmaceutical intervention.
[0010] Fusion of the viral capsule with the lipid membrane of a potential host cell is important for viral entry into the cell and is thought to occur by a iow-pH endocytotic process mediated by CD81 (7).
[0011] HCV research has been hampered by the lack of suitable infectivity models, but recent advances have demonstrated that unmodified HCV envelope proteins can pseudotype retroviral particles and thereby mediate cell entry. Details of HCV tropism and cell entry can now be studied, because such HCV pseudovirus particles (HCVpp) seem to accurately replicate early stages of the viral life cycle (6-10). It has been demonstrated that human immunodeficiency virus (HIV) readily forms pseudotypes, bearing native HCV E1 and E2 glycoproteins, that are infectious for human hepatoma cell lines in vitro (7).
[00121 As indicated above, such HCVpp accurately reproduce the essential biology of HCV entry into cells susceptible to infection by HCV, (See, e.g., reference 7) and serve as an authentic source of native, fusogenic forms of HCV envelope glycoproteins. HCVpp also provide a means by which to assess HCV entry into cells and to screen small molecule compounds for inhibitory activity. The findings obtained using HCVpp have been substantiated using authentic HCV (12-15).
[0013] HCVpp entry into liver cells requires co-expression of both the E1 and E2 HCV envelope glycoproteins; neither individual protein is sufficient for entry. Similar to authentic HCV and related viruses, HCVpp fusion does not occur at the cell surface but rather requires endocytosis of virus into mildly acidic endosomes, where fusion is triggered by exposure to low pH (7,16). HCVpp have been shown to be specifically inhibited by monoclonal antibodies directed against £2, as well as by HCV patient sera (7-8,17-18). Studies with HCVpp have identified the presence of naturally-occurring, broad and cross-genotype neutralizing antibodies in sera from HCV-infected individuals (16-18).
[0014] HCVpp infect CD81 -positive primary hepatocytes and liver cell lines, and monoclonal antibodies directed against CD81 inhibit HCVpp infection (6-8,19-20). CD81 -negative human hepatoma cells are resistant to HCVpp entry, but such ceils become permissive when modified to express CD81. In contrast, non-hepatic cells are resistant to infection regardless of CD81 expression. Thus, CD81 expression is necessary but not sufficient for HCVpp to enter target cells. It has been demonstrated that CD81 functions as a post-attachment co-receptor for HCV as shown by the potent inhibitory activity of CD81 monoclonal antibodies added to HCVpp that was pre-bound to target cells (6). In addition, certain mutations in E2 abolish binding to CD81 but not to target cells (5, 21 ). SUMMARY
[0015] In one embodiment of the invention are compounds of formula (I), salts, including pharmaceutically acceptable salts, polymorphs, hydrates, stereoisomers, metabolites and prodrugs thereof:
Figure imgf000004_0001
wherein:
L2, L4, and Lθ are independently none, H, O, S, NRR', (CH2V5, CN, CRR', SO2, CO, CONHR or CONR'R, NHCONR R, halide, cycloalkyl, heterocycle, aryl, alkyne, alkene;
R2, R4, and R6 are independently none, R, OR, amine, alkoxy, (CH2)o-3CF3, (CH2)0-3W, alky!, aryl, cycloalkyl, heterocycle, fused alkyiaryl or heteroaikylaryl, substituted with 0-2 W; W is H, halide, OR, CF3, NO2, CN, amine, aniline, ester, amide, sulfonamide, sulfone, amino acid, ether, urea acid, heterocycfe, alkyl, aryl, arylalkyl, alkyiaryl; and R or R' is independently H, alkyl, aryl.
[0016] In another embodiment of the invention are compounds of formula (Ii), salts, including pharmaceuticafly acceptable salts, polymorphs, hydrates, stereoisomers, and prodrugs thereof
Figure imgf000005_0001
(H) wherein:
R1 = H, (uπ)substitυted alkyl, (un)substituted aryl, (un)substituted heterocycle; R3 = H, (un)substitυted alkyl, (un)substituted aryl, (un)substituted heterocycte; L4 and L6 are independently H, O, OR1 S, NRR', (CH2Jn, CN, CRR', SO2, CO, CONR1R, NHCONR'R, halide, cycloalkyl, heterocycle, aryl, alkyne, alkene, wherein n = 0-5 ; R4, and R6 are independently none, R, OR1 amine, (CH2)nCF3, CF3, CH2CF3, {CH2)nW, alkyl, aryl, phenyl, cycfoaikyϊ, piperidinyl, heterocycle, fused aryl, alkylaryl, or heteroaikylaryl, al! of which are optionally substituted with 0-5 W, wherein n is chosen independently to be 0-5;
W is H, halide, OR, CF3, NO2, CN, SO2NRR', SO2R, amine, aniline, ester, amide, sulfarnαyl, amino acid, ether, urea, acid, heterocycle, heteroaromatic, alky), aryl, arylaikyt, afkylaryl, sulfone, sulfonamide substituted with alkyi, aryl, heterocycle, amino, aniline; and R or R1 is independently H1 alky!, aryl.
[0017] In a further embodiment of the invention compounds of formula (Ha), salts, including pharmaceutically acceptable salts, polymorphs, hydrates, stereoisomers, and prodrugs thereof
Figure imgf000005_0002
wherein,
Ri = H, (un)substituted alky!, (un)substituted aryl, (un)substituted heterocycle; R3 = H, (un)substituted alkyl, (un)substituted aryl, (un)substituted heterocycle; L5 and L7 are independently H, O, S, NRR', (CH2),,, CN, CRR', SO2, CO, CONR'R,
NHCONR'R, halide, cycloalkyl, heterocycle, aryl, alkyne, alkene, wherein n = 0-5; R5, and R7 are independently none, R, OR, amine, {CH2)nCF3, (CHz)nW, alky!, aryl, phenyl, cycloalkyl, piperidinyl, heterocycle, fused aryl, alkylaryl, or heteroaikylaryl, all of which are optionally substituted with 0-5 W, wherein n is chosen independently to be 0-5; W is H, halide, OR, CF3, NO2, CN, SO2NRR', SO2R, sulfamoyl, sulfone, amino acid, ether, urea, heterocycie, heteroaromatic, alkyl, aryi, arylalkyi, alkyiary!, suifone, sulfonamide substituted with alkyl, aryl, heterocycie, amino, aniline; and R or R' is independently H, alkyl, aryl.
[0018] In an additional embodiment of the invention are compounds of formula (III), salts, including pharmaceutically acceptable salts, polymorphs, hydrates, stereoisomers, and prodrugs thereof
Figure imgf000006_0001
(III) wherein,
R1 = H, (un)substituted alkyl, (un)substituted aryl, (un)substituted heterocycie;
R2 = H, (un)substituted alkyl, (un)substituted aryi, (un)substituted heterocycie;
L4 and L6 are independently H, O, S, NRR', (CH2Jn, CN, CRR', SO2, CO, CONR'R,
NHCONR'R, halide, cycloalkyl, heterocycie, aryl, alkyne, alkene, wherein n = 0-5,;
R4, and R6 are independently none, R, OR, amine, (CH2JnCF3, (CH2JnW, alkyl, aryl, phenyl, cycloalkyl, piperidinyl, heterocycie, fused aryl, alkylaryl, or heteroalkylaryl, all of which are optionally substituted with 0-5 W, wherein n is chosen independently to be 0-5;
W is H1 haiide, OR, CF3, NO2, CN, SO2NRR', SO2R, amine, aniline, ester, amide, sulfamoyl, amino acid, ether, urea, heterocycie, heteroaromatic, alkyl, aryl, arylalkyi, alkylaryl, sulfone, sulfonamide substituted with alkyl, aryl, heterocycie, amino, aniline; and
R or R' is independently H, alkyl, aryl.
[0019] In another embodiment of the invention are compounds of formula (Ilia), salts, pharmaceutically acceptable salts, polymorphs, hydrates, stereoisomers, and prodrugs thereof
Figure imgf000006_0002
wherein,
R1 = H, (un)substituted alkyl, (un)substituted ary!, (un)substituted heterocycie;
R2 = H, (un)substituted alkyl, (un)substituted aryl, (unjsubstituted heterocycie; L5 and L7 are independently H, O, S, NRR', <CH2)ni CN, CRR1, SO2, CO, CONR'R,
NHCONR'R, haltde, cycloalkyi, heterocycle, aryi, alkyne, alkene, wherein n = 0-5; R5, and R7 are independently none, R, OR, amino, amine, (CH2JnCF3, (CHa)nW, alkyl, aryl, phenyl, cycioalkyl, piperidinyl, heterocycle, fused aryl, alkylaryl, or heteroalkylaryl, all of which are optionally substituted with 0-5 W, wherein n is chosen independently to be 0-5;
W is H, halide, OR, CF3, NO2, CN, SO2NRR', SO2R, amino, amine, aniϋne, ester, amide, sulfonamide, sulfamoyl, sulføne, amino acid, ether, urea, acid, heterocycle, heteroaromatic, alkyl, aryl, arylalkyl, alkylaryl, suifone, sulfonamide substituted with alkyl, aryl, heterocycle, amino, aniline; and R or R' are independently H, alkyl, aryl.
[0020] Also, as an embodiment of the invention, is a compound, salt thereof, pharmaceutically acceptable salt, polymorph, hydrate, stereoisomer, metabolite or prodrug thereof, which is selected from Tables 1-35, below,
[0021] Further embodied in the invention is a composition comprising at least one of a compound, stereoisomer, hydrate, polymorph, or a salt thereof, of a compound of the invention, as exemplified in Tables 1-35, below.
[0022] Also embodied in the invention is a composition of a compound of the invention comprising a pharmaceutically acceptable carrier, the composition further comprising a solid formulation, a semisolid formulation, a solution formulation, an aqueous formulation, an immediate release formulation, a sustained release formulation, an enteric coating formulation or a lyophilized formulation, such formulation optionally being a packaged unit dosage. [0023] In accordance with embodiments of the present invention there is provided a pharmaceutical dosage unit composition comprising a pharmaceutical carrier and a therapeutically effective amount of a compound of the invention suitable for treating viral infections of the family Flaviviridae, and, in one aspect, infections by HCV. [0024] A further embodiment of the invention is a pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention and a pharmaceutically acceptable carrier or excipient. The composition of the invention may further comprise at least one additional antiviral active ingredient such as an interferon, an anti-HCV monoclonal antibody, an anti-HCV polyclonal antibody, a HCV RNA polymerase inhibitor, a HCV protease inhibitor, an IRES inhibitor, a helicase inhibitor, an antisense compound, an anti-viral small molecule, or a ribozyme. Such antiviral active ingredients may be provided in any combination. Additionally, an antiviral active ingredient may be selected from one or more of the following in any combination: ribavirin, interferon-α, interferon-α-2β, or a combination thereof. [0025] Different doses of a compound of the invention may be needed depending on the status of the viral infection, such that different dosages may be needed prior to potential infection, or for early, post infection. Other, different dosages may be needed for obtaining a sustained viral response in the case of a long-term, chronic infection. To obtain therapeutic or prophylactic effects, a compound of the invention may be co-adrninistered with one or more chemotherapeutic drugs or therapeutic or antiviral agents, in one example, with antiviral agents such as ribavirin and/or interferon-alpha (IFN-α), and in other examples, with microbial anti- infective agents or with anti-cancer agents.
[0026] In embodiments of the invention, compounds and/or compositions of the invention are administered via an entry portal of the subject body, for example, via oral, enteral, parenteral, subcutaneous, topical and/or intravenous routes,
[0027] In an embodiment of the invention, when a viral infection is being treated, or prophylaxis is desired, a compound of the invention may be administered by any route of administration, including, intravenously, parenteraily, subcutaneously, intramuscularly, or orally. When oral administration is utilized, a compound of the invention may be formulated to provide an immediate release dosage form that predominantly releases compound in the stomach. Alternatively, a compound of the invention may be coated to provide an enteric dosage form designed to preferentially release in the intestine, with little or no release in the stomach, [0028] Also provided in one embodiment of the invention is provided a method for treating or preventing infection by a virus of the family Flaviviήdae, comprising administering to a patient in need thereof a compound and/or composition in an amount effective to treat or prevent the infection.
[0029] In an embodiment of the invention is a method wherein an effective amount of a compound of the invention and/or composition of the invention inhibits or blocks entry of HCV into a susceptible cell. Preferably the HCV is of genotype 1 , and more preferably of genotype 1a, genotype 1b, or combinations thereof. Preferably, the susceptible cell is in a patient, and the compound and/or composition is administered to the patient.
[0030] in an embodiment of the invention is a method comprising contacting an HCV-infected cell with a compound and/or composition of the invention, in an amount effective to inhibit or block exit of newly formed HCV particles from an HCV-infected cell. [0031] In an embodiment of the invention is a method of inhibiting HCV infection of a cell susceptible to HCV infection, comprising contacting the cell with the compound and/or composition of the invention, in an amount effective to inhibit HCV infection of the cell, [0032] In an embodiment of the invention is a method of preventing or diminishing HCV infection in a subject, comprising administering to the subject a compound and/or composition of the invention in an amount effective to prevent or diminish the HCV infection. The compound and/or composition of the invention may be administered to the subject before, after, or during exposure of the subject to HCV.
[0033] In an embodiment of the invention is a method of reducing exposure of a subject to HCV infection outside or on the external body surface of the subject, comprising contacting the outside or external body surface of the subject with a compound and/or composition of the invention, in an amount effective to inactivate or inhibit the virus so as to reduce exposure of the subject to HCV infection.
[0034] In one embodiment of the invention is a method of reducing the occurrence of HCV infection in a population of individuals, comprising administering to the population of individuals in need thereof a compound of the invention in an amount effective to reduce the occurrence of HCV infection in the population.
[0035] In one embodiment of the invention is a method of treating or preventing a liver disease in a subject, which comprises administering to the subject compound and/or composition of the invention in an amount effective to inhibit infection of the subject's HCV susceptible cells, thereby treating or preventing the liver disease in the subject. [0036] In one embodiment of the invention is a method of treating or preventing an HCV associated disorder in a subject, which comprises administering to the subject a compound and/or composition of the invention in an amount effective to inhibit infection of the subject's HCV susceptible cells, thereby treating or preventing an HCV associated disorder in the subject [0037] In accordance with a further embodiment of the present invention there are provided methods for prophylactically preventing or diminishing HCV infections in a warm-blooded animal, which comprises administering before, after or during the exposure of said animal to the infective viral agent, a prophylactically effective amount of a compound of the invention. [0038] In one embodiment of the invention are methods of reducing the occurrence of HCV infection in a population of individuals, comprising administering to the population of individuals in need thereof a compound and/or composition of the invention in an amount effective to reduce the occurrence of HCV infection in the population.
[0039] In one embodiment of the invention are disclosed methods of reducing exposure of a subject to HCV infection outside or on the external body surface of the subject, comprising contacting the outside or external body surface of the subject with a compound of the invention in an amount effective to inactivate or inhibit the virus so as to reduce exposure of the subject to HCV infection.
[0040] In one embodiment of the invention is further disclosed methods of treating or preventing a liver disease in a subject, which comprises administering to the subject a compound and/or composition of the invention, in an amount effective to inhibit infection of the subject's HCV susceptible cells, thereby treating or preventing the liver disease in the subject. [0041] In one embodiment is disclosed a method for inhibiting infection of a susceptible cell, wherein the cell is contacted with a compound or composition of the invention, in an amount effective to inhibit HCV infection of the cell; further, wherein the cell is in a patient and a compound of the invention is administered to the patient.
[0042] Disclosed in one embodiment is a method of reducing or diminishing the severity of HCV infection in a subject infected or exposed to HCV comprising administering a compound of the invention to the patient in effective amount. [0043] In one embodiment of the invention is disclosed a method of inactivating, inhibiting, decontaminating, or rendering inactive or weakly infective objects, spaces, surfaces, or substances that have been contaminated with HCV, which comprises contacting the objects, spaces, surfaces, or substances with a compound of the invention, in an amount effective to inactivate, inhibit, decontaminate, or render inactive or weakiy infective the HCV. [0044] In an embodiment of the invention, compounds of the invention inhibit HCV entry by blocking the interaction of HCV E2 envelope glycoprotein with the scavenger receptor B1 (SR- B1 ) integral membrane, cell surface receptor.
[0045] In an embodiment of the invention, is a method of inhibiting HCV entry via the SR-B1 receptor into a ceil susceptible to HCV infection, comprising contacting the ceil with a compound of Tables 1-35S infra.
[0046] Compounds of the invention may also be used solely or in combination with other antiviral, inactivating or decontaminating agents or drugs to render inactive or weakly infective surfaces or substances that have been contaminated with Flaviviridae such as HCV or other viruses. For instance, allograft or xenograft tissues, blood, surgical instrument surfaces, syringes, garments, and transfusion apparatuses that pose an viral infective risk to others may be rendered virally inactive or more weakly infective by use of the compounds, [0047] Also included as useful for the conditions discussed herein are the prodrugs, metabolites, pharmaceutically acceptable salts, radioisomers, stereoisomers, hydrates, solvates, and acid hydrates of the compounds of the invention. For example, prodrugs may enhance a number of desirable pharmaceutical qualities (e.g., solubility, bioavailability, manufacturing, efc.). Prodrugs of the compounds of the invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Metabolites of the disclosed compounds may possess pharmacological activity. The compounds are also useful as research or diagnostic reagents, as radioisomers or otherwise, whereby such compounds can be used, for example, to establish competitive binding constants for other compounds, or, for example, as quantitative reagents to assess viral titer.
[0048] In one embodiment of the invention are provided methods of synthesis for compounds of the invention, including salts, pharmaceutically acceptable salts, radioisomers, stereoisomers, hydrates, solvates, and acid hydrates of the compounds of the invention. BRIEF DESCRIPTION OF FIGURES
[0049] Fig. 1 depicts a schematic representation of various HlV- 1 based pseudoviral particles utilized in assays to evaluate the compounds of the invention. Test and control pseudoparticles include HCVpp, VSVpp (Vesicular Stomatitis Virus pp), MLVpp (Murine Leukemia Virus pp), HiV-i pp, and human patient specific HCVpp, The pseudoviruses are capable of only a single round of infection in target cells. Of the pseudoparticles, HCVpp only recapitulate the process of HCV entry. [0050] Fig. 2 illustrates the inhibition of viral pseudoparticle entry into target cells by pyrimidine compounds of the invention and a control (antibody JS-81) as a function of concentration of the inhibitors.
[0051] Fig. 3 shows the inhibition of viral pseudoparticle entry into target cells by pyrazolopyrimidine and imidazolopyrimidines compounds of the invention and a control (antibody JS-81 ) as a function of concentration of the inhibitors.
[0052] Fig. 4 shows that a representative pyrazolopyrimidine compound of Table 35 potently neutralizes binding of sE2-6xHis to surface expressed SR-BI on Huh7.5.1 cells. [0053] Fig. 5 shows that a compound of the invention, Compound B, maintains its ability to potently neutralize binding of sE2-6xHis to surface expressed SR-BI on Huh7.5.1 cells following pre-treatment and a washout. DETAILED DESCRIPTION
[0054] The present invention provides in one embodiment the selective inhibition or blockade of HCV entry into hepatocytes and other permissive cell types. According to the invention, heterocyclic compounds of the pyrimidine type surprisingly were discovered to be highly active in preventing the entry of HCV genotype 1 pseudoviruses into potential eukaryotic hosts, activity that may include other virions of the Flaviviridae family. This property of the inventive compounds, as described herein, is advantageous because current therapies are poorly efficacious against HCV genotype 1 , the predominant genotype in the United States of America. [0055] Without wishing to be bound by the mechanism of receptor blockade, the blocking mechanism of the compounds may involve direct interaction of HCV with the ectopic domain of the CD81 receptor, or alternatively a direct binding of the compounds with the viral structural proteins E1/E2 such that virion docking is impeded. In a different modality, prior to the release of newly-formed viral repiicons, the compounds may also potentially block the assembly and/or facile release of viral particles from infected cells via intracellular binding to newly formed E1/E2 structural proteins. In this therapeutic modality, the compounds would function as virion assembly or exit inhibitors.
[0056] The invention further provides the stereoisomers of the compounds disclosed herein, as well as to prodrugs, polymorphs, solvates, ail salts thereof, particularly pharmaceutically acceptable salts, synthetic methods for the preparation of compounds of the invention, pharmaceutical compositions of the same, and methods for therapeutic and/or prophylactic utilization, preparation, and pharmaceutical compositions. The invention also provides methods for utilizing these compounds in anti-viral treatment, therapy, or prophylaxis, either as monotherapy or in combination with other antiviral or chemotherapeutic and/or prophylactic agents. Further, the invention provides for any human and/or animal subject or patient that may be treated with compounds according to the invention.
[0057] In this application it will be understood that the terms "therapeutic" and "therapy" are used to describe the administration of medicaments to a patient to correct, treat, ameliorate, or eradicate a condition or infection that has already initiated. The terms "prophylactics I Iy" and "prophylaxis" describe protective medications or preventive treatments that are administered to a subject and/or applied to an object before contact with HCV, for example, to prevent or diminish the intensity of a subsequent infection of the patient by the virus, or to prevent or diminish contamination of the object by the virus.
[0058] The term "acyl", whether used alone, or within a term such as "acylamino", denotes a radical provided by the residue after removal of hydroxy) from an organic acid. The term "acylamino" embraces an amine radical substituted with an acyi group. An example of an "acyiamino" radical is acetylamine (CH3 C(=O)~-NH--). The term "aryloxy" denotes a radical provided by the residue after removal of hydrido from a hydroxy-substituted aryl moiety (e.g., phenol).
[0059] As used herein, "alkanoyl" refers to a-C (=O)-alkyl group, wherein alkyl is as defined below. Exemplary alkanoyl groups include acetyl (ethanoyi), n-propanoyl, n-butanoyl, 2- methylpropanoyi, n-pentanoyl, 2-methylbutanoyi, 3-methylbutanoyi, 2,2- dimethylpropanoyl, heptanoyl, decanoyl, and palmitoyl.
[0060] The term "alkenyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyfs described below, but that contain at least one double bond and must contain at least two carbon atoms. For example, the term "alkenyl" includes straight- chain alkenyl groups (e.g., ethylenyi, propeny!, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycioalkenyi (alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cyclo heptenyl, cyclooctenyl), afkyl or alkenyl substituted cycioalkenyi groups, and cycloaikyl or cycioalkenyi substituted alkenyl groups. The term lower aikylene" herein refers to those alkyiene groups having from about 1 to about 6 carbon atoms. The term "alkenyj" includes both "unsubstituted alkenyls" and "substituted alkenyis", the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, aikyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyioxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyi, arninocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, aSkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, diaikylamino, arylamino, diarylamino, and alkylaryfamino), acyiamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyi, cyano, azido, heterocyclyl, aikylaryl, or an aromatic or heteroaromatic moiety. [0061] "Alkenylene", in general, refers to an alkyiene group containing at least one carbon- carbon double bond. Exemplary atkenylene groups include, for example, ethenylene (-CH=CH-) and propenylene (-CH=CHCH2-). Preferred alkenylene groups have from 2 to about 4 carbons. [0062] The terms "alkoxy" and "alkoxyalkyl" embrace linear or branched oxy-containing radicals each having alkyl portions of one to about ten carbon atoms, such as methoxy radical. The term "alkoxyalkyl" also embraces alkyl radicals having two or more alkoxy radicals attached to the alkyl radical, that is, to form monoalkoxyaikyl and dialkoxyalkyl radicals. The "aikoxy" or "alkoxyalkyl" radicals may be further substituted with one or more halo atoms, such as fluoro chloro or bromo to provide "haloalkoxy" or "haloalkoxyalkyl" radicals. Examples of "alkoxy" radicals include methoxy butoxy and trifluoromethoxy.
[0063] "Alkyl" in general, refers to an aliphatic hydrocarbon group which may be straight, branched or cyclic having from 1 to about 10 carbon atoms in the chain, and all combinations and subcombinations of ranges therein, e.g., a cycioalkyl, branched cycloalkyialky!, a branched alkylcycloalky having 4-10 carbon atoms. The term "alkyl" includes both "unsubstituted alkyls" and "substituted alkyls," the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the backbone. "Lower alkyl" refers to an alkyl group having 1 to about 6 carbon atoms. Alkyl groups include, but are not limited to, methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyi, t-butyl, n-pentyl, cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl, cyciohexyl, cyclooctyl, adamantyi, 3-methyipentyl, 2-dimethylbutyl, and 2,3- dϊmethyibutyi, cyclopropylmethyl and cyclobutyimethyl, Alkyl substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, aikylthiocarbonyl, alkoxyi, phosphate, phosphonato, phosphinato, cyano, amino (including alkyi amino, dialkylamϊno, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonyiamino, aryScarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, aryithio, thiocarboxylate, sulfates, alkyisulfinyl, sulfonato, sulfamoyl, sulfonamide nttro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. The term "araikyl" embraces aryl-substituted alkyl radicals such as benzyl, diphenylmethyl, triphenylmethyi, phenethyl, phenylpropyl, and diphenethyl. The terms benzyl and phenylmethyl are interchangeabie. The term "n-alkyl" means a straight chain (i.e. unbranched) unsubstituted alkyl group. "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.
[0064] The term "alkynyi" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond and two carbon atoms. For example, the term "alkynyl" includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyi, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloaikyl or cycSoalkenyl substituted alkynyl groups. J0065] The term "amido" when used by itself or with other terms such as "amidoalkyi", "N- monoalkylamido", "N-monoarylamido", "N,N-dtalkylarnido", "N-aikyl-N-arylamido", "N-alkyl-N- hydroxyamido" and "N-alkyl-N-hydroxyamidoalkyl", embraces a carbonyl radical substituted with an amino radical. The terms "N-alkyiamido" and "N,N-dialkyiamidoM denote amido groups which have been substituted with one alkyl radical and with two aikyt radicals, respectively. The terms "N-monoarylamido" and "N-a!kyI-N-aryiamido" denote amido radicals substituted, respectively, with one aryl radical, and one alkyl and one aryl radical. The term "N-alkyl-N-hydroxyamido" embraces amido radicals substituted with a hydroxyl radical and with an alkyl radical. The term "N-alkyl-N-hydroxyamidoalkyl" embraces aikyl radicals substituted with an N-alkyi-N- bydroxyamido radical. The term "amidoalkyl" embraces alkyl radicals substituted with amido radicals.
[0066] The terms "amine" or "amino" have their common, ordinary meaning. In general, the amines useful in the invention have the general formula:
N
R3 wherein R1, R2, and R3 are identical or a combination of different hydrido, straight or branched chain alkyl groups, alkenyl groups, alkylene groups, alkenylene groups, cycloalkyl groups, cycloalkyl-substituted alkyl groups, cycloafkenyl groups, alkoxy groups, alkoxy-aikyl groups, acyl groups, aryl groups, aryl-substituted alkyl groups, and heterocyclic groups, such as morpholine. If none of Rr3 are hydrido, the compound is a tertiary amine. Exemplary tertiary amines useful according to the invention are those where R1-3 is an alkyl group of the formula (CnH2n+!, n=1-4), or arafkyl group of the formula (C6H5 (CH2Jn- [n=1-2]. Exemplary tertiary amines useful according to the invention also are cycloalkyl tertiary amines (e.g., N-methylmorphoiine, N- methylpyrrolidine, N-methylpiperidine), pyridine and Proton Sponge® (N1N, N', N'-tetramethyl- 1 ,8- naphthalene).
[0067] The term "aminoalkyl" embraces alkyl radicals substituted with amine radicals. The term "alkylaminoalkyi" embraces aminoalkyl radicals having the nitrogen atom substituted with an alkyl radical. The term "amidino" denotes an -C(=NH)— NH2 radical. The term "cyanoamidino" denotes an ~C(=N--C N)-NH2 radical.
[0068] The term "anti-HCV compound" refers to any compound showing the effect of inactivating the virus or diminishing its' infectivity or replication in any way. One possibility for anti-HCV activity, for example, is a compound that interferes with the entry of HCV into an animal cell; such a compound is an "entry inhibitor". If such a compound interferes with the exit of vira! replicons from the cell, after infection by the virus, the compound is an "exit inhibitor". A third possibility is a compound that enhances the effectiveness of the subject's immune system in attacking and neutralizing the virus. Yet another possibility, for example, is a compound that interferes with the viral life cycle once the virus has gained cellular entry. An example of such a compound is a ΗCV-metalloprotease" inhibitor which inhibits the virus1 metalloprotease, a viral enzyme that is thought to cleave the vira! polypeptide at its NS2-NS3 junction. Another example is an ΗCV-polymerase" inhibitor which inhibits the HCV encoded RNA dependent RNA polymerase (known as NS5b) that the virus needs in order to replicate its genome. The viral HCV-polymerase, NS5b, is essential for viral replication. Yet another example is an "HCV-serine protease" inhibitor. Such a compound interferes with the virally encoded serine protease known as NS3-4A that is essential for viral polypeptide cleavage. And yet another such compound is an "HCV-heϋcase" inhibitor which prevents the unwinding of the virat genome by interfering with the enzyme HCV-helicase encoded by the virus.
[0069] "Anti-HCV monoclonal antibodies"are antibodies that are reactive toward HCV. The antibodies are identical, having been produced by cells that are all genetically identical clones of a single parent cell. " Anti-HCV polyclonal antibodies" are antibodies that are reactive against HCV. Such antibodies are derived from different cell lines, and are a mixture of immunoglobulin molecules secreted against the virus, each reconizing a specific antigenic site or epitope on the virus.
[0070] The term "anti-infective agent" refers to a compound, composition, substance, reagent, drug, and the like, which acts therapeutically or prophylactically against infectious virat {e.g. HCV), bacterial, protozoal, or other agents by inhibiting their growth, replication, and survival. Anti-infective agents may comprise preparations that contain natural or synthetic antibiotic agents.
[0071] The term "anti-cancer agent" or "cancer chemotherapeutic agent" refers to a compound, composition, substance, reagent, drug, and the like, which acts therapeutically or prophylactically by inhibiting the growth, replication, spread, and survival of cancer cells. Anticancer agents may comprise preparations that contain natural or synthetic materials that act therapeutically singly or in combination to achieve their effect,
[0072J The term "antisense molecule" refers to a nucleic acid molecule (DNA, RNA, or a chemical analogue) that will complementarily bind to viral RNA, thus preventing the translation of viral proteins, thereby interfering with the viral life cycle. More generally, an antisense molecule binds to or pairs with messenger RNA (mRNA), e.g., an mRNA transcript, to block the expression of a gene, thus effectively turning off that gene and inhibiting its function. The interfering molecule, typically an oligonucleotide, is termed "antisense" because its base sequence is complementary to the RNA, i.e., the "sense" sequence. (Thus, for instance, a sense segment of RNA " 5'-AAGGUC-3' " would be blocked by the complementary antisense oligonucleotide " 3'-UUCCAG-S' ").
[0073] The term "aryl", alone or in combination, means a carbocyciic aromatic system containing one, two or three rings wherein such rings may be attached together in a pendent manner or may be fused. The term "aryl" embraces aromatic radicals such as phenyl, naphthyl, tetrahydronapthyl, indane and biphenyl.
[0074] "Aryl-substituted aikyl", in general, refers to an linear alkyl group, preferably a lower alky! group, substituted at a carbon with an optionally substituted aryl group, preferably an optionally substituted phenyl ring. Exemplary aryi-substitutedl aikyt groups include, for example, phenylmethyl, phenylethyl and 3-{4-methylphenyl)propyl,
[0075] As used herein, the term "associated liver disorder" refers to any liver dysfunction or malady associated with infection by a virus of the Flaviviridae family, in one example HCV.
[0076] The term "cycloalkyl" embraces radicals having three to ten carbon atoms, such as cyclopropyi cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl,
[0077] The term "carbocycle" is intended to mean any stable 3- to 7-membered monocyclic or bicyclic or 7- to 13-membered bicydic or tricyclic, any of which may be saturated, partially unsaturated, or aromatic. Examples of such carbocycles include, but are not limited to, cyclopropyi, cyclobutyl, cyclopentyl, cydohexyl, cycloheptyl, adamantyl, cyclooctyl,
[3.3.0]bicyclooctane, [4.3.0]bicyclononane» [4.4.0]bicyclodecane (decalin), [2.2.2]bicyclooctane, fluorenyl, phenyl, naphthyl, indanyl, adamantyl, or tetrahydronaphthyl (tetraiin). Preferred
"carbocycle" are cyclopropyi, cyclobutyl, cyclopentyi, and cyclohexyl.
[0078] "Cydoalkyl-substituted alkyl", in general, refers to a linear alky! group, preferably a lower alkyl group, substituted at a terminal carbon with a cycioalkyl group, preferably a C3 -C8 cycioalkyl group. Typical cydoalkyl-substituted alkyl groups include cyclohexylmethyl, cyclohexylethyl, cyclopentylethyl, cyclopentylpropyl, cyclopropylmethyl and the like.
[0079] "Cycloalkenyl", in general, refers to an olefinicaily unsaturated cycioalkyl group having from about 4 to about 10 carbons, and all combinations and subcombinations of ranges therein.
In some embodiments, the cycloaikenyl group is a C5 -C8 cycloalkenyl group, i.e., a cycloalkenyl group having from about 5 to about 8 carbons.
[0080] The term "halo" means halogens such as fluorine, chlorine, bromine or iodine atoms.
The term "haloalkyl" embraces radicals wherein any one or more of the alkyl carbon atoms is substituted with halo as defined above. Specifically embraced are monohaloalkyl, dihaloalkyl and polyhaloalkyl radicals. A monohaloalkyl radical, for one example, may have either a bromo, chloro or a fluoro atom within the radical. Dihalo radicals may have two or more of the same halo atoms or a combination of different halo radicals and polyhaloalkyl radicals may hav© more than two of the same halo atoms or a combination of different halo radicals.
[0081 J The term "HCV associated disorder" is a illness or disorder that is caused or happens as a result of HCV infection.
[0082] As used herein, the term "heterocycle" or "heterocyclic ring" is intended to mean a stable 5- to 7- membered monocyclic or bicyclic or 7- to 14-membered bicyclic heterocyclic ring which is saturated, partially unsaturated, or unsaturated (aromatic), and which consists of carbon atoms and 1 , 2, 3 or 4 heteroatoms independently selected from the group consisting of
N, O and S and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. Examples of saturated heterocyclic radicals include pyrrolidyl and morpholinyl. [0083] The term "hydroxyalkyi" embraces linear or branched alkyl radicals having one to about ten carbon atoms any one of which may be substituted with one or more hydroxy! radicals.
[0084] The term "hydrido" denotes a single hydrogen atom (H), This hydrido radical may be attached, for example, to an oxygen atom to form a hydroxyl radical or two hydrido radicals may be attached to a carbon atom to form a methylene (-CH2 -} radical.
[0085] The term "interferon" denotes a natural protein produced by the cells of the immune response of most vertebrates when challenged by foreign agents such as viruses, bacteria, parasites and tumor cells. There are different types of interferons (e.g., interferon-alpha, interferon-beta, interferon-garnma), which belong to the class of glycoproteins known as cytokines, interferons are biological defense modifiers, which inhibit viral replication within cells of the body and thereby assist immune response, e.g., the eradication of virus and viral infection, interferons are antiviral and anti-oncogenic, assist macrophage and natural killer lymphocyte activation, and enhance major histocompatibility complex glycoprotein classes I and II, and thereby the presentation of foreign (microbial) peptides to T ceils, which have immune effector function to combat infection.
[0086] The term "liver disease" refers to any pathology, dysfunction, illness, inflammation, cancer or malady of the liver. Non-limiting exampϊes are amebic liver abscess, autoimmune hepatitis, biliary atresia, cirrhosis, dessiminated coccidioido-mycosis, delta agent (hepatitis D), drug-induced cholestasis, hemochromatosis, hepatitis A, hepatitis B, hepatitis C, hepatocellular carcinoma, liver disease due to alcohol, primary biliary cirrhosis, pyogenic liver abscess, Reye's syndrome, sclerosing cholangitis, and Wilson's disease.
[0087] The terms "N-alkylamino" and "N,N-dialkyIamino" denote amino groups which have been substituted with one alkyl radical and with two alkyl radicals, respectively.
[0088] Organic solvent" has its common ordinary meaning to those of skill in the art.
Exemplary organic solvents useful in the invention include, but are not limited to, tetrahydrofuran, acetone, hexane, ether, chloroform, acetic acid, acetonitrtle, chloroform, cyclohexane, methanol, and toluene. Anhydrous organic solvents are included.
[0089] As used herein, "patient" or "subject" refers to animals, including mammals, e.g., rodents (mice, rats) dogs, rabbits, sheep, goats, and non-human primates. The term "patient" refers preferably to humans.
[0090] As used herein, "prodrug" refers to compounds specifically designed to maximize the amount of active species that reaches the desired site of reaction that are of themselves typically inactive or minimally active for the activity desired, but through biotransformation or chemical reaction are converted into biologically active molecules.
[0091] As used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio. As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, sυch conventional nontoxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, giycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfontc, methanesulfσnic, ethane disulfonic, oxalic, ethylenediaminetetraacetϊc , and the like. These physiologically acceptable salts are prepared by methods known in the art, e.g., by dissolving the free amine bases with an excess of the acid in aqueous alcohol, or neutralizing a free carboxylic acid with an alkali metal base such as a hydroxide, or with an amine. Certain acidic or basic compounds of the present invention may exist as zwitterions. All forms of the compounds, including free acid, free base and zwitterions, are contemplated to be within the scope of the present invention. It is well known in the art that compounds containing both amino and carboxyl groups often exist in equilibrium with their zwitterionic forms. Thus, any of the compounds described herein throughout that contain, for example, both amino and carboxyl groups, also include reference to their corresponding zwitterions. [0092] The term "ribozyme", derived from a contraction of ribonucleic acid enzyme, refers to a RNA molecule that catalyzes a chemical reaction, typically either the hydrolysis of one of its' own phosphodiester bonds, or the hydrolysis of bonds in other RNAs. Ribozymes are naturally occuring or synthetic. Non-limiting examples of naturally occuring ribozymes are Peptidyl transferase 23S rRNA, Rnase P, G1R1 branching ribozyme, Hairpin ribozyme, Hammerhead ribozyme, HDV ribozyme, Mammalian CPEB3 ribozyme, VS ribozyme, glmS ribozyme and CoTC ribozyme.
[0093] As used herein, the term "stereoisomers" refers to compounds that have identical chemical constitution, but differ as regards the arrangement of the atoms or groups in space. [0094] As used herein, a "susceptible cell" is a cell which is subject to infection by a virus, in one example, HCV.
[0095] As used herein, the term "virion" refers to a matured viral particle, either existing outside a cell, or nascent within a cell prior to release.
[0096] The subjects or patients to which the compounds of the present invention may be administered are vertebrates, in particular mammals. In embodiments the mammal is a human, nonhuman primate, dog, cat, sheep, goat, horse, cow, pig or rodent, In one embodiment, the mammal is a human.
[0097] The pharmaceutical preparations or compositions of the invention, when used alone or in cocktails, are administered in therapeutically effective amounts. A effective amount will be determined by the parameters discussed below; but typically is that amount which establishes a level of the drug(s) effective for treating a subject, such as a human subject, having one of the conditions described herein. An effective amount means that amount alone, as a single dose, or multiple doses, necessary to delay or prevent the onset of, lessen the severity of, inhibit completely, lessen or reduce the progression of, eradicate or halt altogether the onset or progression of the condition being treated or a symptom associated therewith. In the case of an active viral infection, an effective therapeutic amount, for example, is that amount which relieves a symptom of infection, which induces a decrease in viral load, which increases the time before relapse, or which decreases circulating viral RNA, In the case of prophylactic usage, either before transmission or soon after transmission, an effective amount, for example, would be an amount that prevents active infection, lowers the frequency of active infection, slows the time before an active infection occurs, or diminishes the intensity of the infection. [0098] Patients amenable to the therapy of the present invention also include but are not limited to patients suffering from other dysfunctions
[0099] A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular combination of drugs selected, the severity of the condition being treated, or prevented, the condition of the patient, and the dosage required for therapy and/or efficacy. The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include, without limitation, oral, rectal, topical, transdermal, subungual, intravenous infusion, pulmonary, intra-arterial, intra-adipose tissue, intra-lymphatic, intramuscular, intracavity, intraperitoneal (IP), intrathecal, subcutaneous (SC), aerosol, aural (e.g., via eardrops), intranasal, inhalation, intra-articular, needleless injection, subcutaneous or intradermal (e.g., transdermal) delivery. For continuous infusion, a patient- controlled device or an implantable drug delivery device may be employed. The administration may be by the patient, using an injection device for SC self-administration. Oral, rectal, or topical administration may be important for long-term treatment. Preferred rectal modes of delivery include administration as a suppository or enema wash.
[00100] The pharmaceutical preparations may conveniently be provided in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. In such form, the entire unit is intended to be administered to the patient as a separate dose. All methods include the step of bringing the compounds of the invention into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compounds of the invention into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. [00101] When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically acceptable compositions. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, lubricants, and optionally other therapeutic substances and/or ingredients. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable saits include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulfonic, tartaric, citric, methanesulfonic, formic, succinic, naphthalene-2-sulfonic, pamoic, 3-hydroxy-2-naphthalenecarboxylic, and benzene sulfonic. [00102] It should be understood that when referring to compounds of the invention, saits of the same are encompassed. Such salts are of a variety well known to those or ordinary skill in the art. When used in pharmaceutical preparations, the salts preferably are pharmaceutically- acceptable for use in humans. A bromide salt is an example of one such salt in the case that the parent compound is basic. A sodium salt is an example of one such salt in the case that the parent compound is acidic.
[00103] it should be understood that when referring to compounds of the invention, radioisomers of the same are encompassed. Such isomers, obtained by replacing one or more component atoms of the compound by a radioactive atom, are of a variety well known to those or ordinary skill in the art. In the present case, such radioisomers can be used therapeutically to deliver localized radiation to a tissue, in one embodiment, a tissue infected with HCV; or in another example, a radioisomer may be used as a tracer to measure metabolic pathways in an animal, or to measure competitive binding in a laboratory sample of tissue. Non-radioactively labeled compounds, produced by replacing one or more of the component atoms with an atomic isotope thereof, are also encompassed.
[0100] It should also be understood that when referring to compounds of the invention, hydrates, solvates, and polymorphs of the same are encompassed. Hydrates are formed when water binds to the crystal structure of a compound in a fixed stoichiometric ratio, although generally this ratio will change depending on the surrounding humidity with which the hydrate is in equilibrium. Hydration is a more specific form of solvation. Solvates are crystalline solid adducts containing either stoichiometric or nonstoichiometric amounts of a solvent incorporated within the crystal structure. If the incorporated solvent is water, the solvates are also commonly known as hydrates. Hydrates and solvates are welt known to those or ordinary skill in the art. [0101] Polymorphism is characterized as the ability of a compound or drug substance to exist as two or more crystalline phases that have different arrangements and/or conformations of the molecules in the crystal lattice. Amorphous solids consist of disordered arrangements of molecules and do not possess a distinguishable crystal lattice. Polymorphism refers to the occurrence of different crystalline forms of the same drug substance. Polymorphs are well know to those of ordinary skill in the art.
[0102] Polymorphs or solvates of a solid can have different chemical and physical properties such as melting point, chemical reactivity, apparent solubility, dissolution rate, optical and electrical properties, vapor pressure, and density, for example. These properties can have a direct impact on the processing of drug substances and the quality or performance of drug products. Chemical and physical stability, dissolution, and bioavailability are some of these qualities. A metastable solid form may change crystalline structure or solvate or desolvate in response to changes in environmental conditions, processing, or over time. New polymorphs can develop spontaneously over time.
[0103] Infection by Hepatitis C virus predominantly occurs via the percutaneous exchange of infected blood from an outside source, such as a contaminated syringe needle. After being applied in an effective amount to such a source, a compound of the disclosure is capable of reducing exposure of a patient to HCV infection. In this embodiment, a compound of the invention produces inactivation or inhibition, or diminishment of infectivity of the virus outside or on the body of a subject by bringing the virus in contact with a compound of the invention in effective amount. An effective amount would be the amount of a compound of the invention that diminishes the infectivity of a virally contaminated outside source, upon the contact of the source with the subject.
[0104] Compounds of the invention may also be used solely or in combination with other antiviral, inactivating or decontaminating agents or drugs to render inactive or weakly infective spaces, sources, surfaces or substances that have been contaminated with Ftaviviήdae such as HCV or other viruses. For instance, allograft or xenograft tissues, blood, surgical instrument surfaces, syringes, garments, and transfusion apparatuses that pose a viral infective risk to others may be rendered virally inactive or weakly infective by use of the compounds. In another example, airborne virally-contarninated blood particles pose an infective risk. In the presence of such a risk, a compound of the invention may be dispersed as an aerosol in the contaminated space in an effective amount to inactivate or diminish the infectivity of the airborne virus by contact with the virus.
[0105] The pharmaceutical preparations of the present invention may include, or be diluted into, a pharmaceuticaϋy-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a human or other mammal such as non- human primate, for example, a dog, cat, horse, cow, sheep, pig, or goat. [0106] The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The carriers are capable of being commingled with the preparations of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy or stability. Carrier formulations suitable for oral administration, for suppositories, and for parenteral administration, etc., can be found in Remington: The Science and Practice, of Pharmacy, 20th Edition. (Aifanso R. Gennaro): Uppincott Williams & Wilkins, Baltimore, MD, 2000.
[0107] Aqueous formulations may include one or more chelating agents, buffering agents, antioxidants and, optionally, isotonicity agents, preferably pH adjusted, for example, to between 3.0 and 3.5.
[0108] Chelating agents include, for example and without limitation, ethylenediaminetetraacetic acid (EDTA) and derivatives thereof, citric acid and derivatives thereof, niacinamide and derivatives thereof, sodium desoxycholate and derivatives thereof, and L-glutamic acid, N, N~diacetic acid and derivatives thereof.
[0109] Buffering agents include, without limitation, those selected from the group consisting of citric acid, sodium citrate, sodium acetate, acetic acid, sodium phosphate and phosphoric acid, sodium ascorbate, tartaric acid, maleic acid, glycine, sodium lactate, lactic acid, ascorbic acid, imidazole, sodium bicarbonate and carbonic acid, sodium succinate and succinic acid, histidine, and sodium benzoate and benzoic acid, and combinations thereof. [0110] Antioxidants include, without limitation, those selected from the group consisting of an ascorbic acid derivative, butylated hydroxy anisole, butylated hydroxy toluene, alkyl gallate, sodium meta-bisulfite, sodium bisulfite, sodium dithionite, sodium thioglycollate acid, sodium formaldehyde sulfoxylate, tocopherol and derivatives thereof, monothiogiycerol, sodium sulfite, and combinations thereof.
[0111] Isotonicity agents include, without limitation, those selected from the group consisting of sodium chloride, mannitol, lactose, dextrose, glycerol, and sorbitol and combinations thereof. [0112J Preservatives that can be used with the present compositions include, without limitation, benzyl alcohol, parabens, thimerosal, chlorobutanol and preferably benzalkonium chloride and combinations thereof. Typically, the preservative is present in a composition in a concentration of up to about 2% by weight. The exact concentration of the preservative, however, will vary depending upon the intended use and can be easily ascertained by one skilled in the art.
[0113] The compounds of the invention can be prepared in lyophilized compositions, preferably in the presence of one or more cryoprotecting agents such as trehalose, mannitol, lactose, sucrose, polyethylene glycol, and polyvinyl pyrrolidines, Cryoprotecting agents which result in a reconstitution pH of 6.0 or less are preferred. The invention therefore provides a lyophilized preparation of compounds and/or compositions of the invention. The preparation can contain a cryoprotecting agent, such as mannitol or lactose, which is preferably neutral or acidic in water. [0114] Oral, parenteral and suppository formulations of agents are well known and commercially available. The therapeutic compounds and/or compositions of the invention can be added to such well known formulations, which can be mixed together in solution or semisolid solution in such formulations, can be provided in a suspension within such formulations, or can be contained in particles within such formulations.
{0115] A product containing one or more therapeutic compounds of the invention and, optionally, one or more other active agents can be configured as an oral dosage. The oral dosage may be a liquid, a semisolid or a solid. The oral dosage may be configured to release the therapeutic compound of the invention before, after, or simultaneously with the other agent. The oral dosage may be configured to have the therapeutic compound of the invention and the other agents release completely in the stomach, release partially in the stomach and partially in the intestine, in the intestine, in the colon, partially in the stomach, or wholly in the colon. The oral dosage also may be configured whereby the release of the therapeutic compound of the invention is confined to the stomach or intestine while the release of the other active agent is not so confined or is confined differently from the therapeutic compound of the invention. For example, the therapeutic compound of the invention may comprise an enterically coated core or pellets contained within a pill or capsule that releases the other agent first and releases the therapeutic compound of the invention only after the therapeutic compound of the invention passes through the stomach and into the intestine, A therapeutic compound of the invention also can be in a sustained release material, whereby the therapeutic compound of the invention is released throughout the gastrointestinal tract and the other agent is released on the same or a different schedule. The same objective for a therapeutic compound of the invention can be achieved with an immediate release of the therapeutic compound of the invention, combined with an enteric coated therapeutic compound of the invention. In this instance, the therapeutic compound could be released immediately in the stomach, throughout the gastrointestinal tract, or only in the intestine.
[0116] The materials useful for achieving these different release profiles are well known to those of ordinary skill in the art. Immediate release is obtainable by conventional tablets with binders which dissolve in the stomach. Coatings which dissolve at the pH of the stomach or which dissolve at elevated temperatures will achieve the same purpose. Release only in the intestine is achieved using conventional enteric coatings such as pH sensitive coatings which dissolve in the pH environment of the intestine (but not the stomach) or coatings which dissolve over time. Release throughout the gastrointestinal tract is achieved by using sustained-release materials and/or combinations of the immediate release systems and sustained and/or delayed intentional release systems (e.g., pellets which dissolve at different pHs). [0117] In the event that it is desirable to release the therapeutic compound of the invention first, a therapeutic compound of the invention could be coated on the surface of the controlled release formulation in any pharmaceutically acceptable carrier suitable for such coatings and for permitting the release of the therapeutic agent of the invention, such as in a temperature sensitive pharmaceutically acceptable carrier used for controlled release routinely. Other coatings which dissolve when placed in the body are well known to those of ordinary skii! in the art.
[0118] A therapeutic compound of the invention also may be mixed throughout a controlled release formulation, whereby it is released before, after or simultaneously with another agent. The therapeutic compound of the invention may be free, that is, solubilized within the material of the formulation. The therapeutic compound of the invention also may be in the form of vesicles, such as wax coated micropellets dispersed throughout the material of the formulation. The coated pellets can be fashioned to immediately release the therapeutic compound of the invention based on temperature, pH, or the like. The pellets also can be configured so as to delay the release of the therapeutic compound of the invention, allowing the other agent a period of time to act before the therapeutic compound of the invention exerts its effects. The therapeutic compound of the invention pellets also can be configured to release the therapeutic compound of the invention in virtually any sustained release pattern, including patterns exhibiting first order release kinetics or sigmoidal order release kinetics using materials of the prior art and well known to those of ordinary skill in the art.
[0119] A therapeutic compound of the invention also can be contained within a core within the controlled release formulation. The core may have any one or any combination of the properties described above in connection with the pellets. The therapeutic agent of the invention may be, for example, in a core coated with a material, dispersed throughout a material, coated onto a material or adsorbed into or throughout a material. It should be understood that the pellets or core may be of virtually any type. They may be drug coated with a release material, drug interspersed throughout material, drug adsorbed into a material, and so on. The material may be erodible or nonerodible.
{012OJ A therapeutic compound of the invention may be provided in particles. Particles as used herein means nano- or microparticles (or in some instances larger) which can consist in whole or in part of a compound of the invention or other agents as described herein. The particles may contain the therapeutic compounds in a core surrounded by a coating, including, but not limited to, an enteric coating. Such compounds also may be dispersed throughout the particles. These compounds also may be adsorbed into the particles. The particles may be of any order release kinetics, including zero order release, first order release, second order release, delayed release, sustained release, immediate release, and any combination thereof, etc. The particle may include, in addition to the therapeutic compound, any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable materia! or combinations thereof. The particles may be microcapsules which contain the antiviral compound in a solution or in a semisolid state. The particles may be of virtually any shape. [0121] Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering the therapeutic compounds of the invention. Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Btoadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, (1993) 26:581-587, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, poiyacrylic acid, alginate, chitosan, poly{methyf methacrylates), poly{ethy! methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexyimethacrylate), poty(isodecyi methacrylate), poly(iaury( methacryiate), poly(phenyi methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyi acrylate).
[0122] The therapeutic compounds of the invention may be contained in controlled release systems. The term "controlled release" is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controiled. This refers to immediate as well as nonimmediate release formulations, with nonimmediate release formulations including but not limited to sustained release and delayed release formulations. The term "sustained release" {also referred to as "extended release") is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period. The term "delayed release" is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug therefrom. "Delayed release" may or may not involve gradual release of drug over an extended period of time, and thus may or may not be "sustained release." These formulations may be for any mode of administration. [0123] Delivery systems specific for the gastrointestinal tract are roughly divided into three types: the first is a delayed release system designed to release a drug in response to, for example, a change in pH; the second is a timed-release system designed to release a drug after a predetermined time; and the third is a microflora enzyme system making use of the abundant enterobacteria in the lower part of the gastrointestinal tract (e.g., in a colonic site-directed release formulation).
[0124] An example of a delayed release system is one that uses, for example, an acrylic or cellulosic coating material and dissolves on pH change. Because of ease of preparation, many reports on such "enteric coatings" have been made, In general, an enteric coating is one which passes through the stomach without releasing substantial amounts of drug in the stomach (i.e., less than 10% release, 5% release and even 1 % release in the stomach) and sufficiently disintegrating in the intestinal tract (by contact with approximately neutral or alkaline intestine juices) to allow the transport (active or passive) of the active agent through the walls of the intestinal tract. [0125] Various in vitro tests for determining whether or not a coating is classified as an enteric coating have been published in the pharmacopoeia of various countries. A coating which remains intact for at least 2 hours, in contact with artificial gastric juices such as HCI of pH 1 at 36 to 38 0C and thereafter disintegrates within 30 minutes in artificial intestinal juices such as a KH2PO4 buffered solution of pH 6,8 is one example. One such well known system is EUDRAGIT® material, commercially available and reported on by Behringer, Manchester University, Saaie Co., and the like. Enteric coatings are discussed further, below. [0126] The enteric coating is typically, although not necessarily, a polymeric material. Preferred enteric coating materials comprise bioerodible, gradually hydrolyzable and/or gradually water-soluble polymers. The "coating weight," or relative amount of coating material per capsule, generally dictates the time interval between ingestion and drug release. Any coating should be applied to a sufficient thickness such that the entire coating does not dissolve in the gastrointestinal fluids at pH below about 5, but does dissolve at pH about 5 and above. It is expected that any anionic polymer exhibiting a pH-dependent solubility profile can be used as an enteric coating in the practice of the present invention. The selection of the specific enteric coating material will depend on the following properties: resistance to dissolution and disintegration in the stomach; impermeability to gastric fluids and drug/carrier/enzyme while in the stomach; ability to dissolve or disintegrate rapidly at the target intestine site; physical and chemical stability during storage; non-toxicity; ease of application as a coating (substrate friendly); and economical practicality.
[0127] Suitable enteric coating materials include, but are not limited to: cellulosic polymers such as cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethy! cellulose phthalate, hydroxypropyhmethyl cellulose succinate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ammonium methylacrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate {e.g., those copolymers sold under the trade name EUDRAGIT®); vinyl polymers and copolymers such as polyvinyl acetate, polyvinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymers; and shellac (purified lac). Combinations of different coating materials may also be used. Well known enteric coating material for use herein are those acrylic acid polymers and copolymers available under the trade name EUDRAGIT® from Rohm Pharma (Germany). The EUDRAGIT® series E, L, S, RL, RS and NE copolymers are available as solubilized in organic solvent, as an aqueous dispersion, or as a dry powder. The EUDRAGIT® series RL, NE, and RS copolymers are insoluble in the gastrointestinal tract but are permeable and are used primarily for extended release. The EUDRAGIT® series E copolymers dissolve in the stomach. The EUDRAGIT® series L, L-30D and S copolymers are insoluble in stomach and dissolve in the intestine, and are thus most preferred herein. [0128] A particular methacrylic copolymer is EUDRAGIT® L, particularly L-30D and EUDRAGIT® L 100-55. In EUDRAGIT® L-30D, the ratio of free carboxyl groups to ester groups is approximatefy 1 :1. Further, the copolymer is known to be insoluble in gastrointestinal fluids having pH below 5.5, generally 1.5-5.5, i.e., the pH generally present in the fluid of the upper gastrointestinal tract, but readily soluble or partially soluble at pH above 5.5, i.e., the pH generally present in the fluid of lower gastrointestinal tract. Another particular methacrylic acid polymer is EUDRAGiT® S, which differs from EUDRAGIT® L-30D in that the ratio of free carboxyl groups to ester groups is approximately 1 :2. EUDRAGIT® S is insoluble at pH below 5.5, but unlike EUDRAGIT® L-30D, is poorly soluble in gastrointestinal fluids having a pH in the range of 5.5 to 7.0, such as in the small intestine. This copolymer is soluble at pH 7.0 and above, i.e., the pH generally found in the colon. EUDRAGIT® S can be used alone as a coating to provide drug delivery in the large intestine. Alternatively, EUDRAGIT® S, being poorly soluble in intestinal fluids beiow pH 7, can be used in combination with EUDRAGIT® L-3QD, soluble in intestinal fluids above pH 5.5, in order to provide a delayed release composition which can be formulated to deliver the active agent to various segments of the intestinal tract. The more EUDRAGIT L-30D used, the more proximal release and delivery begins, and the more EUDRAGIT® S used, the more distal release and delivery begins. It will be appreciated by those skilled in the art that both EUDRAGIT® L-30D and EUDRAGIT® S can be replaced with other pharmaceutically acceptable polymers having similar pH solubility characteristics. In certain embodiments of the invention, the preferred enteric coating is ACRYL-EZE ™ (methacrylic acid co-polymer type C; Coiorcon, West Point, PA).
[0129] The enteric coating provides for controlled release of the active agent, such that drug release can be accomplished at some generally predictable location. The enteric coating also prevents exposure of the therapeutic and/or agent and carrier to the epithelial and mucosal tissue of the buccal cavity, pharynx, esophagus, and stomach, and to the enzymes associated with these tissues. The enteric coating therefore helps to protect the active agent, carrier and a patient's internal tissue from any adverse event prior to drug release at the desired site of delivery. Furthermore, the coated material of the present invention allows optimization of drug absorption, active agent protection, and safety. Multiple enteric coatings targeted to release the active agent at various regions in the gastrointestinal tract would enable even more effective and sustained improved delivery throughout the gastrointestinal tract, [0130] The coating can, and usually does, contain a plasticizer to prevent the formation of pores and cracks that would permit the penetration of the gastric fluids. Suitable plasticizers include, but are not limited to, triethyl citrate (Citroflex® 2), triacetin (glyceryl triacetate), acetyl triethyl citrate (Citroflec® A2), Carbowax™ 400 (polyethylene glycol 400), diethyl phthalate, tributyl citrate, acetylated monoglycerides, glycerol, fatty acid esters, propylene glycol, and dibutyl phthalate. In particular, a coating comprised of an anionic carboxylic acrylic polymer will usually contain approximately 10% to 25% by weight of a plasticizer, particularly dibutyl phthalate, polyethylene glycol, triethyl citrate and triacetin. The coating can also contain other coating excipients such as detackifiers, antifoaming agents, lubricants (e.g., magnesium stearate), and stabilizers (e.g., hydroxypropyiceliulose, acids and bases) to soiubilize or disperse the coating material, and to improve coating performance and the coated product. [0131] The coating can be applied to particles of the therapeutic and/or agent(s), tablets of the therapeutic and/or agent(s), capsules containing the therapeutic agent(s)and the like, using conventional coating methods and equipment. For example, an enteric coating can be applied to a capsule using a coating pan, an airless spray technique, fluidized bed coating equipment, or the like. Detailed information concerning materials, equipment and processes for preparing coated dosage forms may be found in Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al, (New York; Marcel Dekker, Inc., 1989), and in Ansei et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th Ed. (Media, PA: Williams & Wiikins, 1995). The coating thickness, as noted above, must be sufficient to ensure that the oral dosage form remains intact until the desired site of topical delivery in the lower intestinal tract is reached. [0132] In another embodiment, drug dosage forms are provided that comprise an enterically coated, osmotically activated device housing a formulation of the invention. In this embodiment, the drug-containing formulation is encapsulated in a semipermeable membrane or barrier containing a small orifice. As known in the art with respect to so-called "osmotic pump" drug delivery devices, the semipermeable membrane allows passage of water, but not drug, in either direction. Therefore, when the device is exposed to aqueous fluids, water will flow into the device due to the osmotic pressure differential between the interior and exterior of the device. As water flows into the device, the drug-containing formulation in the interior will be "pumped" out through the orifice. The rate of drug release will be equivalent to the inflow rate of water times the drug concentration. The rate of water influx and drug efflux can be controlled by the composition and size of the orifice of the device. Suitable materials for the semipermeable membrane include, but are not limited to, polyvinyl alcohol, polyvinyl chloride, semipermeable polyethylene glycols, semipermeable poiyurethanes, semipermeable poiyamides, semipermeable sulfonated polystyrenes and polystyrene derivatives; semipermeable poly(sodium styrenesulfonate), semipermeable poly(vinylbenzyltrimethylammonium chloride), and cellulosic polymers such as cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose propionate, cellulose acetate propionate, cellulose acetate butyrate, cellulose trivalerate, cellulose trilmate, cellulose tripalmitate, cellulose trioctanoate, cellulose tripropionate, cellulose disuccinate, cellulose dipalmϊtate, cellulose dicylate, cellulose acetate succinate, cellulose propionate succinate, cellulose acetate octanoate, cellulose valerate palmttate, cellulose acetate heptanate, cellulose acetaldehyde dimethyl acetal, cellulose acetate ethylcarbamate, cellulose acetate methylcarbamate, cellulose dimethylaminoacetate and ethylceliulose.
[0133] In another embodiment, drug dosage forms are provided that comprise a sustained release coated device housing a formulation of the invention. In this embodiment, the drug- containing formulation is encapsulated in a sustained release membrane or film. The membrane may be semipermeable, as described above. A semipermeable membrane allows for the passage of water inside the coated device to dissolve the drug. The dissolved drug solution diffuses out through the semipermeable membrane. The rate of drug release depends upon the thickness of the coated film and the release of drug can begin in any part of the GI tract. Suitable membrane materials for such a membrane include ethylcellulose. [0134] In another embodiment, drug dosage forms are provided that comprise a sustained release device housing a formulation of the invention. In this embodiment, the drug-containing formulation is uniformly mixed with a sustained release polymer. These sustained release polymers are high molecular weight water-soluble polymers, which when in contact with water, swell and create channels for water to diffuse inside and dissolve the drug. As the polymers swell and dissolve in water, more of drug is exposed to water for dissolution. Such a system is generally referred to as sustained release matrix. Suitable materials for such a device include hydropropyl m ethylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose and methyl cellulose.
[0135] In another embodiment, drug dosage forms are provided that comprise an enteric coated device housing a sustained release formulation of the invention. In this embodiment, the drug containing product described above is coated with an enteric polymer. Such a device would not release any drug in the stomach and when the device reaches the intestine, the enteric polymer is first dissolved and only then would the drug release begin. The drug release would take place in a sustained release fashion.
[0136J Enterically coated, osmotically activated devices can be manufactured using conventional materials, methods and equipment. For example, osmotically activated devices may be made by first encapsulating, in a pharmaceutically acceptable soft capsule, a liquid or semi-solid formulation of the compounds of the invention as described previously, This interior capsule is then coated with a semipermeable membrane composition (comprising, for example, cellulose acetate and polyethylene glycol 4000 in a suitable solvent such as a methylene chloride-methanol admixture), for example using an air suspension machine, until a sufficiently thick laminate is formed, e.g., around 0.05 mm. The semipermeable laminated capsule is then dried using conventional techniques. Then, an orifice having a desired diameter (e.g., about 0.99 mm) is provided through the semipermeable laminated capsule wall, using, for example, mechanical drilling, laser drilling, mechanical rupturing, or erosion of an erodible element such as a gelatin plug. The osmotically activated device may then be enterically coated as previously described. For osmotically activated devices containing a solid carrier rather than a liquid or semi-solid carrier, the interior capsule is optional; that is, the semipermeable membrane may be formed directly around the carrier-drug composition. However, preferred carriers for use in the drug-containing formulation of the osmotically activated device are solutions, suspensions, liquids, immiscible liquids, emulsions, sols, colloids, and oils. Particularly preferred carriers include, but are not limited to, those used for enterically coated capsules containing liquid or semisolid drug formulations.
[0137] Cellulose coatings include those of cellulose acetate phthalate and trimeiiitate; methacrylic acid copolymers, e.g. copolymers derived from methylacrylic acid and esters thereof, containing at least 40% methylacrylic acid; and especially hydroxypropyl methylceliulose phthalate. Methylacrylates include those of molecuiar weight above 100,000 daltons based on, e.g. methylacrylate and methyl or ethyl methyiacrylate in a ratio of about 1 :1. Typical products include Endragit L, e.g. L 100-55, marketed by Rohm GmbH, Darmstadt, Germany. Typical cellulose acetate phthalates have an acetyl content of 17-26% and a phthalate content of from 30-40% with a viscosity of ca. 45-90 cP. Typical cellulose acetate trimeiiitates have an acetyl content of 17-26%, a trimellityi content from 25-35% with a viscosity of ca. 15-20 cS. An example of a cellulose acetate trimeiiitate is the marketed product CAT (Eastman Kodak Company, USA). Hydroxypropyl methylceliulose phthalates typically have a molecuiar weight of from 20,000 to 130,000 daltons, a hydroxypropyl content of from 5 to 10%, a methoxy content of from 18 to 24% and a phthalyl content from 21 to 35%, An example of a cellulose acetate phthalate is the marketed product CAP (Eastman Kodak, Rochester N.Y., USA). Examples of hydroxypropyl methylceliulose phthalates are the marketed products having a hydroxypropyl content of from 6-10%, a methoxy content of from 20-24%, a phthalyl content of from 21-27%, a molecular weight of about 84,000 daltons, sold under the trademark HP50 and available from Shin-Etsu Chemical Co. Ltd., Tokyo, Japan, and having a hydroxypropyl content, a methoxyl content, and a phthalyl content of 5-9%, 18-22% and 27-35%, respectively, and a molecular weight of 78,000 daltons, known under the trademark HP55 and available from the same supplier.
[0138] A timed release system is represented by Time Erosion System (TES) by Fujisawa Pharmaceutical Co., Ltd. and Pulsincap by R. P. Scherer. According to these systems, the site of drug release is decided by the time of transit of a preparation in the gastrointestinal tract. Since the transit of a preparation in the gastrointestinal tract is largely influenced by the gastric emptying time, some time release systems are also enterically coated. [0139] Systems making use of the enterobacteria can be classified into those utilizing degradation of azoaromatic polymers by an azo reductase produced from enterobacteria as reported by the group of Ohio University (M. Saffran, e^ a/., Science, Vol. 233: 1081 (1986)) and the group of Utah University (J. Kopecek, et al., Pharmaceutical Research, 9(12), 1540-1545 (1992)); and those utilizing degradation of polysaccharides by beta-gaiactosidase of enterobacteria as reported by the group of Hebrew University (unexamined published Japanese patent application No. 5-50863 based on a PCT application) and the group of Freiberg University (K. H. Bauer et ai, Pharmaceutical Research, 10(10), S218 (1993)). In addition, the system using chitosan degradable by chitosanase by Teikoku Seiyaku K. K. (unexamined published Japanese patent application No, 4-217924 and unexamsned published Japanese patent application No. 4-225922) is also included.
[0140] The therapeutic compounds may be provided in capsules, coated or not. The capsule material may be either hard or soft, and as will be appreciated by those skilled in the art, typically comprises a tasteless, easily administered and water soluble compound such as gelatin, starch or a ceilulosic material. The capsules are preferably sealed, such as with gelatin bands or the like. See, for example, Remington: The Science and Practice of Pharmacy, Nineteenth Edition (Easton, Pa.: Mack Publishing Co., 1995), which describes materials and methods for preparing encapsulated pharmaceuticals.
[0141] A product containing one or more therapeutic compounds of the invention can be configured as a suppository. The therapeutic compound of the invention can be placed anywhere within or on the suppository to favorably affect the relative release of the therapeutic compound. The nature of the release can be zero order, first order, or sigmoidal, as desired. [0142] Suppositories are solid dosage forms of medicine intended for administration via the rectum. Suppositories are compounded so as to melt, soften, or dissolve in the body cavity (around 98.6 0F) thereby releasing the medication contained therein. Suppository bases should be stable, no n irritating, chemically inert, and physiologically inert. Many commercially available suppositories contain oily or fatty base materials, such as cocoa butter, coconut oil, palm kernel oil, and palm oil, which often melt or deform at room temperature necessitating cool storage or other storage limitations. U.S. Patent No. 4,837,214 to Tanaka et al. describes a suppository base comprised of 80 to 99 percent by weight of a lauric-type fat having a hydroxyl value of 20 or smaller and containing glycerides of fatty acids having 8 to 18 carbon atoms combined with 1 to 20 percent by weight diglycerides of fatty acids (which erucic acid is an example of). The shelf life of these type of suppositories is limited due to degradation. Other suppository bases contain alcohols, surfactants, and the like which raise the melting temperature but also can lead to poor absorption of the medicine and side effects due to irritation of the local mucous membranes (see for example, U.S. Patent No. 6,099,853 to Hartelendy ef a/., U.S. Patent No. 4,999,342 to Ahmad ef at., and U.S. Patent No. 4,765,978 to Abidi ef a/.). [0143] The base used in the pharmaceutical suppository composition of this invention includes, in general, oils and fats comprising triglycerides as main components such as cacao butter, palm fat, palm kernel oil, coconut oil, fractionated coconut oil, lard and WITEPSOL®, waxes such as lanolin and reduced lanolin; hydrocarbons such as VASELINE®, squalene, squalane and liquid paraffin; long to medium chain fatty acids such as caprylic acid, lauric acid, stearic acid and oleic acid; higher alcohols such as lauryl alcohol, cetanol and stearyl alcohol; fatty acid esters such as butyl stearate and dtlauryl malonate; medium to long chain carboxylic acid esters of glycerin such as triolein and tristearin; glycerin-substituted carboxylic acid esters such as glycerin acetoacetate; and polyethylene glycols and its derivatives such as macrogols and cetomacrogol. They may be used either singly or in combination of two or more. If desired, the composition of this invention may further include a surface-active agent, a coloring agent, etc, which are ordinarily used in suppositories.
[0144] The pharmaceutical composition of this invention may be prepared by uniformly mixing predetermined amounts of the active ingredient, the absorption aid and optionally the base, etc, in a stirrer or a grinding mill, if required at an elevated temperature. The resulting composition, may be formed into a suppository in unit dosage form by, for example, casting the mixture in a mold, or by forming it into a gelatin capsule using a capsule filling machine. [0145] The compositions according to the present invention also can be administered as a nasal spray, nasal drop, solution, suspension, gel, ointment, cream or powder. The administration of a composition can also include using a nasal tampon or a nasal sponge containing a composition of the present invention.
[0146] The nasal delivery systems that can be used with the present invention can take various forms including aqueous preparations, non-aqueous preparations and combinations thereof. Aqueous preparations include, for example, aqueous gels, aqueous suspensions, aqueous liposomal dispersions, aqueous emulsions, aqueous microemulsions and combinations thereof. Non-aqueous preparations include, for example, non-aqueous gels, nonaqueous suspensions, non-aqueous liposomal dispersions, non-aqueous emulsions, nonaqueous microemulsions and combinations thereof. The various forms of the nasal delivery systems can include a buffer to maintain pH, a pharmaceutically acceptable thickening agent and a humectant. The pH of the buffer can be selected to optimize the absorption of the therapeutic agent(s)across the nasal mucosa.
[0147] With respect to the non-aqueous nasal formulations, suitable forms of buffering agents can be selected such that when the formulation is delivered into the nasal cavity of a mammal, selected pH ranges are achieved therein upon contact with, e.g., a nasal mucosa. In the present invention, the pH of the compositions may be maintained from about 2.0 to about 6.0. It is desirable that the pH of the compositions is one which does not cause significant irritation to the nasal mucosa of a recipient upon administration.
[0148] The viscosity of the compositions of the present invention can be maintained at a desired level using a pharmaceutically acceptable thickening agent. Thickening agents that can be used in accordance with the present invention include methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof. The concentration of the thickening agent will depend upon the agent selected and the viscosity desired. Such agents can also be used in a powder formulation discussed above.
[0149] The compositions of the present invention can also include a humectant to reduce or prevent drying of the mucus membrane and to prevent irritation thereof. Suitable humectants that can be used in the present invention include sorbitol, mineral oil, vegetable oil and glycerol; soothing agents; membrane conditioners; sweeteners; and combinations thereof. The concentration of the humectant in the present compositions will vary depending upon the agent selected.
[0150] One or more therapeutic and/or agents may be incorporated into the nasal delivery system or any other delivery system described herein.
[0151] A composition formulated for topical administration may be iiquid or semi-solid
(including, for example, a gel, lotion, emulsion, cream, ointment, spray or aerosol) or may be provided in combination with a "finite" carrier, for example, a non-spreading material that retains its form, including, for example, a patch, bioadhesive, dressing or bandage. It may be aqueous or non-aqueous; it may be formulated as a solution, emulsion, dispersion, a suspension or any other mixture.
[0152] Important modes of administration include topical application to the skin, eyes or mucosa. Thus, typical vehicles are those suitable for pharmaceutical or cosmetic application to body surfaces. The compositions provided herein may be applied topically or locally to various areas in the body of a patient. As noted above, topical application is intended to refer to application to the tissue of an accessible body surface, such as, for example, the skin (the outer integument or covering) and the mucosa (the mucous-producing, secreting and/or containing surfaces). Exemplary mucosal surfaces include the mucosal surfaces of the eyes, mouth (such as the lips, tongue, gums, cheeks, sublingual and roof of the mouth), larynx, esophagus, bronchial, nasal passages, vagina and rectum/anus; in some embodiments, preferably the mouth, larynx, esophagus, vagina and rectum/anus; in other embodiments, preferably the eyes, larynx, esophagus, bronchial, nasal passages, and vagina and rectum/anus. As noted above, local application herein refers to application to a discrete internal area of the body, such as, for example, a joint, soft tissue area (such as muscle, tendon, ligaments, intraocular or other fleshy internal areas), or other interna! area of the body. Thus, as used herein, local application refers to applications to discrete areas of the body.
[0153] With respect to topical and/or local administration of the present compositions, desirable efficacy may involve, for example, penetration of therapeutic agent(s) of the invention into the skin and/or tissue to substantially reach systemic circulation or a peripheral or central locus.
[0154] Also in certain embodiments, including embodiments that involve aqueous vehicles, the compositions may also contain a glycol, that is, a compound containing two or more hydroxy groups. A glycol which may be particularly useful for use in the compositions is propylene glycol. The glycol may be included in the compositions in a concentration of from greater than 0 to about 5 wt. %, based on the total weight of the composition.
[0155] For local internal administration, such as intra-articular administration, the compositions are preferably formulated as a solution or a suspension in an aqueous-based medium, such as isotonically buffered saline or are combined with a biocompatible support or bioadhesive intended for internal administration. [0156] Lotions, which, for example, may be in the form of a suspension, dispersion or emulsion, contain an effective concentration of one or more of the compounds. The effective concentration is preferably to deliver an effective amount. For example, the compound of the present invention may find use at a concentration of between about 0.1-50% [by weight] or more of one or more of the compounds provided herein. The lotions may contain, for example, [by weight] from 1% to 50% of an emollient and the balance water, a suitable buffer, and other agents as described above. Any emollients known to those of skill in the art as suitable for application to human skin may be used. These include, but are not limited to, the following: (a) Hydrocarbon oils and waxes, including mineral oil, petrolatum, paraffin, ceresin, ozokerite, microcrystailine wax, polyethylene, and perhydrosqualene. b) Silicone oils, including dimethylpoiysiloxanes, methylphenylpolysiloxanes, water-soluble and alcohol-soluble silicone- glycol copolymers, (c) Triglyceride fats and oils, including those derived from vegetable, animal and marine sources. Examples include, but are not limited to, castor oil, safflower oil, cotton seed oil, corn oil, olive oil, cod liver oil, almond oil, avocado oil, palm oil, sesame oil, and soybean oil. (d) Acetoglyceride esters, such as acetylated monoglycerides. (e) Ethoxylated glycerides, such as ethoxylated glyceryl monostearate. (f) Alky! esters of fatty acids having 10 to 20 carbon atoms. Methyl, isopropyl and butyl esters of fatty acids are useful herein. Examples include, but are not limited to, hexyl laurate, isohexyl laurate, isohexyl paimitate, isopropyl paimitate, isopropyl myristate, decyl oleate, isodecyl oleate, hexadecyl stearate, decyl stearate, isopropyi isostearate, diisopropyl adipate, diisohexyl adipate, dihexyldecyl adipate, diisopropy! sebacate, lauryl lactate, myristyi lactate, and cetyl lactate, (g) Alkenyl esters of fatty acids having 10 to 20 carbon atoms. Examples thereof include, but are not limited to, oleyl myristate, oleyl stearate, and oleyl oleate. (h) Fatty acids having 9 to 22 carbon atoms. Suitable examples include, but are not limited to, peiargonic, lauric, myristic, palmitic, stearic, isostearic, hydroxystearic, oleic, linoleic, ricinoleic, arachidonic, behenic, and erucic acids, (t) Fatty alcohols having 10 to 22 carbon atoms, such as, but not limited to, lauryl, myristyi, cetyl, hexadecyl, stearyl, isostearyi, hydroxystearyf, oleyl, ricinoleyl, behenyl, erucyl, and 2-octyl dodecyl alcohols. G) Fatty alcohol ethers, including, but not limited to ethoxylated fatty alcohols of 10 to 20 carbon atoms, such as, but are not limited to, the lauryl, cetyl, stearyl, isostearyi, oleyl, and cholesterol alcohols having attached thereto from 1 to 50 ethylene oxide groups or 1 to 50 propylene oxide groups or mixtures thereof, (k) Ether-esters, such as fatty acid esters of ethoxylated fatty alcohols. (I) Lanolin and derivatives, including, but not limited to, lanolin, lanolin oil, lanolin wax, lanolin alcohols, lanolin fatty acids, isopropyl lanolate, ethoxylated lanolin, ethoxylated lanolin alcohols, ethoxylated cholesterol, propoxylated lanolin alcohols, acetylated lanolin, acetylated lanolin alcohols, lanolin alcohols linoleate, lanolin alcohols ricinoleate, acetate of lanolin alcohols ricinoleate, acetate of ethoxylated alcohols-esters, hydrogenolysis of lanolin, ethoxylated hydrogenated lanolin, ethoxylated sorbitol lanolin, and liquid and semisolid lanolin absorption bases, (m) poiyhydric alcohols and polyether derivatives, including, but not limited to, propylene glycol, dipropylene glycol, polypropylene glycol [M.W. 2000-4000], polyoxyethylene polyoxypropylene glycols, poiyoxypropylene polyoxyethylene glycols, glycerol, ethoxylated glycerol, propoxylated glycerol, sorbitol, ethoxylated sorbitol, hydroxypropyl sorbitol, polyethylene glycol [M.W. 200-6000], methoxy polyethylene glycols 350, 550, 750, 2000, 5000, poly(ethylene oxide) homopolymers [M.W. 100,000-5,000,000], polyalkylene glycols and derivatives, hexylene glycol (2-methyi-2,4-pentanediol), 1 ,3-butylene glycol, 1 ,2,6,-hexanetrtol, ethohexadiol USP (2-ethyl-1 ,3-hexanediol), C15 -C18 vicinal glycol and poiyoxypropyteπe derivatives of trim ethylol propane, (n) poiyhydric alcohol esters, including, but not limited to, ethylene glycol mono- and di-fatty acid esters, diethylene glycol mono- and di- fatty acid esters, polyethylene glycol [M.W. 200-6000], mono- and di-fatty esters, propylene glycol mono- and di-fatty acid esters, polypropylene glycol 2000 monooleate, polypropylene glycol 2000 monostearate, ethoxylated propylene glycol monostearate, glyceryl mono- and di- fatty acid esters, polyglycerol poly-fatty acid esters, ethoxylated glyceryl monostearate, 1 ,3- butylene glycol monostearate, 1 ,3-butylene glycol distearate, polyoxyethylene polyol fatty acid ester, sorbitan fatty acid esters, and polyoxyethylene sorbitan fatty acid esters, (o) Wax esters, including, but not limited to, beeswax, spermaceti, myristyl myristate, and stearyl stearate and beeswax derivatives, including, but not limited to, polyoxyethylene sorbitol beeswax, which are reaction products of beeswax with ethoxylated sorbitol of varying ethylene oxide content that form a mixture of ether-esters, (p) Vegetable waxes, including, but not limited to, camauba and candelilla waxes, (q) phospholipids, such as lecithin and derivatives, (r) Sterols, including, but not limited to, cholesterol and cholesterol fatty acid esters, (s) Amides, such as fatty acid amides, ethoxylated fatty acid amides, and solid fatty acid alkanolamides. [0157] The lotions further preferably contain [by weight] from 1% to 10%, more preferably from 2% to 5%, of an emulsifier. The emulsifiers can be nonionic, anionic or cationic. Examples of satisfactory nonionic emulsifiers include, but are not limited to, fatty alcohols having 10 to 20 carbon atoms, fatty alcohols having 10 to 20 carbon atoms condensed with 2 to 20 moles of ethylene oxide or propylene oxide, alkyl phenols with 6 to 12 carbon atoms in the alkyl chain condensed with 2 to 20 moles of ethylene oxide, mono- and di-fatty acid esters of ethylene oxide, mono- and di-fatty acid esters of ethylene glycol where the fatty acid moiety contains from 10 to 20 carbon atoms, diethylene glycol, polyethylene glycols of molecular weight 200 to 6000, propylene glycols of molecular weight 200 to 3000, glycerol, sorbitol, sorbitan, polyoxyethylene sorbitol, polyoxyethylene sorbitan and hydrophilic wax esters. Suitable anionic emulsifiers include, but are not limited to, the fatty acid soaps, e.g., sodium, potassium and triethanolamine soaps, where the fatty acid moiety contains from 10 to 20 carbon atoms. Other suitable anionic emulsifiers include, but are not limited to, the alkali metal, ammonium or substituted ammonium alkyl sulfates, alkyl arylsulfonates, and alkyl ethoxy ether sulfonates having 10 to 30 carbon atoms in the alkyl moiety. The alkyl ethoxy ether sulfonates contain from 1 to 50 ethylene oxide units. Among satisfactory cationic emulsifiers are quaternary ammonium, morpholinium and pyridinium compounds. Certain of the emollients described in preceding paragraphs also have emulsifying properties. When a lotion is formulated containing such an emollient, an additional emulsifier is not needed, though it can be included in the composition.
[0158] The balance of the lotion is water or a C2 or C3 alcohol, or a mixture of water and the alcohol. The lotions are formulated by simply admixing all of the components together. Preferably the compound, is dissolved, suspended or otherwise uniformly dispersed in the mixture,
[0159] Other conventional components of such lotions may be included. One such additive is a thickening agent at a level from 1 % to 10% by weight of the composition. Examples of suitable thickening agents include, but are not limited to: cross-linked carboxypolymethylene polymers, ethyl cellulose, polyethylene glycols, gum tragacanth, gum kharaya, xanthan gums and bentonite, hydroxyethyl cellulose, and hydroxypropyl cellulose.
[0160] Creams can be formulated to contain a concentration effective to deliver an effective amount of therapeutic agent(s) of the invention to the treated tissue, typically at between about 0.1 %, preferably at greater than 1% up to and greater than 50%, preferably between about 3% and 50%, more preferably between about 5% and 15% therapeutic agent(s) of the invention. The creams also contain from 5% to 50%, preferably from 10% to 25%, of an emollient and the remainder is water or other suitable non-toxic carrier, such as an isotonic buffer. The emollients, as described above for the lotions, can also be used in the cream compositions. The cream may also contain a suitable emulsifier, as described above. The emulsifier is included in the composition at a level from 3% to 50%, preferably from 5% to 20%. [0161] These compositions that are formulated as solutions or suspensions may be applied to the skin, or, may be formulated as an aerosol or foam and applied to the skin as a spray-on. The aerosol compositions typically contain [by weight] from 25% to 80%, preferably from 30% to 50%, of a suitable propellant. Examples of such propellants are the chlorinated, fluorinated and chlorofluorinated lower molecular weight hydrocarbons. Nitrous oxide, carbon dioxide, butane, and propane are also used as propellant gases. These propellants are used as understood in the art in a quantity and under a pressure suitable to expel the contents of the container. [0162] Suitably prepared solutions and suspensions may also be topically applied to the eyes and mucosa. Solutions, particularly those intended for ophthalmic use, may be formulated as 0.01%-10% isotonic solutions, pH about 5-7, with appropriate salts, and preferably containing one or more of the compounds herein at a concentration of about 0.1%, preferably greater than 1%, up to 50% or more. Suitable ophthalmic solutions are known [see, e.g., U.S. Pat. No, 5,116,868, which describes typical compositions of ophthalmic irrigation solutions and soiutions for topical application]. Such solutions, which have a pH adjusted to about 7,4, contain, for example, 90-100 mM sodium chloride, 4-6 mM dibasic potassium phosphate, 4-6 mM dibasic sodium phosphate, 8-12 mM sodium citrate, 0.5-1.5 mM magnesium chloride, 1.5-2.5 mM calcium chloride, 15-25 mM sodium acetate, 10-20 mM D,L-sodium β-hydroxybutyrate and 5- 5.5 mM glucose.
[0163] Gel compositions can be formulated by simply admixing a suitable thickening agent to the previously described solution or suspension compositions. Examples of suitable thickening agents have been previously described with respect to the lotions.
[0164] The gelled compositions contain an effective amount of therapeutic agent(s) of the invention, typically at a concentration of between about 0.1-50% by weight or more of one or more of the compounds provided herein.; from 5% to 75%, preferably from 10% to 50%, of an organic solvent as previously described; from 0.5% to 20%, preferably from 1% to 10% of the thickening agent; the balance being water or other aqueous or non-aqueous carrier, such as, for example, an organic liquid, or a mixture of carriers.
[0165J The dosing regimens, as well as the timing for administering the compounds and/or compositions of the present invention are able to be determined by the skilled practitioner in the art, Illustratively and without limitation, a compound or composition of the invention may be administered to a subject at least once per day, daily, every other day, every 6 to 8 days, weekly, bi-weekly, monthly, or bi-monthly.
[0166] The formulations can be designed and provided to create steady state plasma levels. Steady state plasma concentrations can be measured using HPLC techniques, as are known to those of skill in the art. Steady state is achieved when the rate of drug availability is equal to the rate of drug elimination from the circulation, in typical therapeutic and/or settings, the therapeutic agent(s) of the invention will be administered to patients either on a periodic dosing regimen or with a constant infusion regimen. The concentration of drug in the plasma will tend to rise immediately after the onset of administration and will tend to fail over time as the drug is eliminated from the circulation by means of distribution into cells and tissues, by metabolism, or by excretion. Steady state will be obtained when the mean drug concentration remains constant over time. In the case of intermittent dosing, the pattern of the drug concentration cycle is repeated identically in each interval between doses with the mean concentration remaining constant. In the case of constant infusion, the mean drug concentration will remain constant with very little oscillation. The achievement of steady state is determined by means of measuring the concentration of drug in plasma over at least one cycle of dosing such that one can verify that the cycle is being repeated identically from dose to dose. Typically, in an intermittent dosing regimen, maintenance of steady state can be verified by determining drug concentrations at the consecutive troughs of a cycle, just prior to administration of another dose. In a constant infusion regimen where oscillation in the concentration is low, steady state can be verified by any two consecutive measurements of drug concentration. [0167] This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Aiso, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing", "involving", and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
EXPERIMENTAL SECTION
EXAMPLES OF SYNTHESES OF COMPOUNDS OF THE INVENTION [0168] The following general and specific examples illustrate the syntheses of compounds of the present invention, and should not be construed to limit the scope of the claims which follow. Variations of the synthetic procedures to produce the same or similar compounds in a somewhat different manner should be evident to one skilled in the art. Also, the procedures and methods may be suitably adapted to produce the compounds of this invention, including those not specifically disclosed.
[0169] Common abbreviations used to identify the chemical compounds and chemical techniques used herein include: diameter (d), diisopropylethylamine (DIPEA), dichloromethane (DCM), trifluoroacetic acid (TFA), ethyl acetate (EA), petroleum ether (PE), thin-layer chromatography (TLC), high performance liquid chromatography (HPLC), photodiode array (PDA), evaporative light-scattering detector (ELSD), tetrahydrofuran (THF), hexa-deuterio dimethylsulfide (DMSOd6), proton (H), Hertz (Hz), liquid chromatography-mass spectrometry (LCMS)1 high performance liquid chromatography (HPLC), retention time(tR), mass spectrum (MS), (MW) microwave, molecular weight, total ion current (TIC), atmospheric pressure chemical ionization (APCI), coupling constant, Hz (J), mass of parent ion + 1 proton ([M+H+]), charge to mass ratio (m/z), melting point (Mp), mega-Hertz (MHZ).
A. PREPARATION OF PYRIMIDINE DERIVATIVES AS HCV ENTRY INHIBITORS Preparation of Examples 1-5
Figure imgf000038_0001
Preparation of 4,6-Dirnethoxy-N-phenylpyrimidin-2«amine (2)
[0170] To a solution of NaH (60% in oil, 2.1Og, 29 mrnol) in 20 ml_ of anhydrous THF was added aniline (3.0Og, 29 mmol) and the mixture was refluxed for 0.5 hour. After cooling to room temperature, 1 (5,0Og1 29mmol) in 10 ml_ of anhydrous THF was added to the above solution and the resulting reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then poured into 5OmL of water and extracted with EtOAc {50 ml_ x 3). The combined organic phase was washed with brine and dried over MgSO4 Removal of solvent in vacuo gave 6.Og of crude product which was purified by chromatography (PE/EA =50/1 to 10/1 ).
3.50 g of 2 (52%) was obtained as a white solid. LCMS: rn/z = 232.2 (M+ ■§• 1 ).
Preparation of 2-(Phenylarnino)pyrimϊdine-4,6-dϊoI (3)
[0171] To a solution of 2 (3.50 g, 15.4 mmol) and NaI (12.00 g, 80.5 mmol) in 80 mL of anhydrous CH3CN was added MesSiCI (16.1 mL, 126.0 mmol) slowly and the resulted reaction mixture was refluxed for 3 hours. The reaction solution was concentrated under reduced pressure to give a residue, which was poured into 50 mL of water and extracted with EtOAc
(100 mL x 3). The combined organic phase was washed with brine and dried over MgSO4.
Removal of solvent in vacuo gave crude product which was purified by chromatography
(PE/EA=5Q/1) to afford 2.60 g of 3 (85%) as a white solid. LCMS: m/z = 204.1 (M* + 1 ).
Preparation of 4, 6-Dichloro-N-phenyIpyrinidin-2-amϊne (4)
[0172] A mixture of 3 (2.50 g, 12.5 mmol) in 30 mL of POCI3 was refluxed for 3 hours. After cooling, the mixture was poured into ice-water slowly and extracted with EtOAc (150 mL x 3).
The combined organic phase was washed with brine, and dried over MgSO4. Removal of solvent in vacuo gave 6.0 g of crude product, which was purified by chromatography to afford
2.50 g of 4 (83%) as a white solid. LCMS: m/z = 240.0 (M+ + 1 ).
Preparation of tert-Butyl 4-{6-chloro-2-(phenylamino)pyrinidin-4-ylamino) ptperidine-1- carboxylate (6)
[0173] A mixture of 4 (0.523 g, 2.2 mmol), DIPEA (0.8 mL) and N-boc-4- aminopiperidine 5
(0.658 g, 3.3 mmol) in 3.0 mL of butan-1-ol was microwaved for 40 minutes at 12O0C. After cooling to room temperature, the reaction solution was concentrated under reduced pressure to give a residue which was poured into water (50 mL) and extracted with EtOAc (100 mL x 3).
The combined organic phase was washed with brine and dried over MgSO4. Removal of solvent in vacuo gave crude product which was purified by chromatography to afford 0.70 g of 6 (79%) as a white solid. LCMS: m/z = 404.2 (M+ + 1 ).
Preparation of tert-Butyl 4-{2-phenylamino)-6-(2,2,2-trifluoroethoxy)pyrimidm-4- y]amino)ρiperidine-4~ylamino)piperidine-1 -carboxylate (7)
[0174] To a solution of 6 (1.40 g, 3.5 mmol) in 40 mL of anhydrous THF was added
CF3CH2OH (1.06 g, 10.5 mmol) and 4.1 mL of 2.5N KOH solution. The mixture was refluxed for
3 hours. The reaction mixture was concentrated to afford a residue, which was diluted with 100 mL of EtOAc, The resulting solution was washed with brine, dried over MgSO4 and concentrated under reduced pressure to give crude product which was purified by coiumn chromatography (PE/EA=5/1 ). 0.40 g of 7 (24.4%) was obtained as a white solid. LCMS: m/z =
468.2 (M+ + 1 ).
Preparation of N-phenyI-Nt-(piperidin-4-yl}-6-<2,2,2-trifluoroethoxy)pyrimidine-2J4-diamine
(8)
[0175] To a solution of 7 (0.40 g, 0.86 mmoi) in 10mL of CH2Ci2 was added 10 mL of TFA.
The resulting reaction mixture was stirred at room temperature overnight. The reaction mixture was then poured into water (100 mL), basified with KOH to pH 13 and extracted with CH2CI2 (50 mL x 3). The combined organic layers were washed with brine and dried over MgSO4. Removal of solvent produced 8 (0.285 g, 90%) which was used without purification in next step. LCMS: m/z = 368.2 (M* + 1 ).
Preparation of 4-(4-(2-Phenylamino)-6-(2,2,2-trifluoroethoxy)pyrimidin-4-ylamino) piperidin-1-ylsulfonyl)benzoic acid (10)
[0176] To a solution of 8 (0.174 g, 0.45 mmoi) in 20 mL of dried CH2Cl2 was added DIPEA
(0.117g, 0.90 mmoi) and 9 (0.110 g, 0.50 mmoi) slowly. The resulting reaction mixture was stirred at room temperature for 0.5 hour. The mixture was then washed with 2N HCI, NaHCO3
(sat.), brine and dried over MgSO4. Removal of solvent in vacuo afforded 10 (0.218 g, 88%), which was used in next step without purification. LCMS: m/z = 552.2 (M+ + 1 ).
Preparation of 4-(4-(2-Phenylamino)-6-(2,2,2-trofluoroethoxy)pyrϊmidiπ-4-ylamino) piperidin-1-yisulfonyl)benzoyl chloride (11)
[0177] To a solution of 10 (0.218 g, 0.40 mmoi) in 5.0 mL of anhydrous CH2CI2 was added oxalyl chloride (0.083 g, 0.64 mmoi) slowly at O0C. The mixture was stirred for 2 hours (00C to room temperature). The reaction solution was then employed in vacuo to remove the solvent and the excess oxalyl chloride, 0.215 g of 11 was obtained, which was used in the next step without purification.
Preparation of Example 1: {4-(4-(2-(Phenylamino)-6-2,2I2-trifluoroethoxy)pyrimidin-4- ylamϊno)piperidin-1-lsulfonyl)phenyl)(pyrrolidin-1-yl)methanone
[0178] The compound 11 (0.215 g) in 5.0 mL of anhydrous CH2CI2 was slowly added to a solution of pyrrolidine (0,213 g, 3.0 mmoi) in 5.0 mL of anhydrous CH2CI2 at O0C and the reaction mixture was stirred at room temperature for 0.5 hour. Removal of solvent in vacuo gave the crude product which was purified by preparative HPLC (prepHPLC). 0.100 g of final compound (40%) was obtained as a white solid: 1HNMR (CDCI3): 6 9.85 (s, 1 H), 8.64-8.58 (m,
4H), 8.46-8.42 (m, 2H), 7.96-7.93 (m, 3H), 7.70-7.66 (m, 1 H), 6.15-6.13 (m, 1 H), 5.80-5.61 (m,
2H), 4.44-4.31 (m, 5H), 4.20-4.15 (m, 2H), 3.32-3.25 (m, 2H), 2.77-2.62 (m, 6H), 2.33-2.30 (m,
2H); LCMS: m/z = 605.3 (M+ + 1).
Preparation of Example 2: 4-(4-(4-fluorophenethyl)piperazin-1-y1)-6-(2,2,2-trifluoro ethoxy)-N-(3-(trifluorornethyl)phenyl)pyrimidin-2-amine [0179] 1HNMR (CDCI3): δ 8.12 (s, 1 H), 7.37-7.32 (m, 2H), 7.23-7.22 (m, 1 H), 7.12-7.10 (m,
2H), 6.99-6.93 (m, 3H), 5.52 (s, 1 H), 4.86-4.63 (m, 2H), 4.32-4.22 (m, 2H), 3.71-3.51 (m, 4H),
3.16-3.13 (m, 2H), 3.04-3.01 (m, 2H), 2.82-2.70 (m, 2H); LCMS: m/z = 544.0 (M+ + 1 ).
Preparation of Example 3: 4-{4-(4-fluorobenzyl)pϊperazin-1-yl)-6-(2,2,2-trϊfluoro ethoxy)-N-
(3-(trifluoromethyl)phenyl)pyrimidin-2-amine
1HNMR (CDCI3): δ 8.26 (s, 1 H), 7,39-7.38 (m, 2H), 7.32-7.27 (m, 2H), 7.25-7,24 (m, 1 H), 7.04-
7.01 (m, 2H), 6,86 (s, 1 H), 5.54 (s, 1 H), 4.72-4.69 (m, 2H), 3.73-3.41 (m, 6H), 2,58-2.39 (m.
4H). LCMS: m/z = 530.0 (M+ + 1).
Preparation of Example 4: N4-{pyrϊdin-2-ylmethyl)-6-(2,2,2-trifluoroethoxy}-N2-(3-
(trifluoromethyI)phenyl)pyrimidine-2,4-diamine
[0180] NMR (MeOD): δ 8.52-8.51 (m, 1 H), 8.18-8.16 (m, 1 H), 7,82-7.79 (m, 1 H), 7.67 (s,
1 H), 7.46-7.43 (m, 1 H), 7.37-7.26 (m, 2H), 7.18-7.16 (m, 1H), 5.53 (s, 1 H), 4.87-4.81 (m, 2H),
4.72 (S, 2H). LCMS: m/z = 444.1 (M+ + 1 ).
Preparation of Example 5: N4-(1-(4-fluorophenylsulfonyl)piperidin-4-yi)-6-(2,2,2- trifluoroethoxy)-N2-(3-(tπfluoromethyl)phenyl)pyrimidine-2,4-diamine
[0181] NMR (CDCI3): δ 8.27 (s, 1 H), 7.81-7.79 (m, 2H), 7.38-7.36 (m, 1 H), 7.27-7.22 (m,
5H), 6.88-6.87 (m, 1H), 6.34 (m, 1 H), 4.71-4.63 (m, 2H), 4.61-4.57 (m, 1 H), 3.79-3.76 (m, 2H),
2.49-2.44 (m, 2H), 2.18-2.06 (m, 2H), 1.66-1.58 (m, 2H). LCMS: m/z = 594.2 (M+ + 1 ).
Preparation of Examples 6-9
min
Figure imgf000041_0001
Preparation of {4,6-Dichloro-pyrimidϊn-2-yl)-furaπ-2-ylmethyl-amtne {3}
[0182] A solution of furfuryl amine (543 mg, 5.6 mmol) in THF (5 mL) was added slowly dropwise to a solution of compound 2,4,6-trichloro-pyrimidine (1.024 g, 5.6 mmol) and DIPEA
(1.1 mL, 6.2 mmol) in THF (20 mL) at -200C, The resulting mixture was stirred at -2O0C for 2 hours and at room temperature for 16 hours, diluted with water, and extracted with dichioromethane. The combined organic phases were concentrated at reduced pressure. The target isomer is less polar on TLC (ethyl acetate/hexane=1/1 ). Purification by column chromatography (silica gel, ethyl acetate/hexane) gave 3 (497 mg, 36%).
Preparation of 6:6-Chloro-N-furan-2-ylmethyI-N -{4-methoxy-phenyl)-pyrimidine-2,4- diamlne (4)
[0183] A mixture of 3 (100 mg, 0.4 mmol), p-methoxyaniline (50 mg, 0.4 mmol), DIPEA (0.4 mL, 2.3 mol) was stirred for 24 hours at 15O0C, cooled to room temperature and diluted with water. The formed solid was collected by filtration, washed with water and diethyl ether, recrystallized from ether to give 4 (75 mg, 57%). 1 H-NMR (400MHz, DMSO-D6) δ 3.72 (3H, s),
4.42 (2H, d, J=7.5 Hz), 5.93 (1 H, s), 6.19 (1 H1 broad, ZJE forms), 6.37 (1 H1 broad), 6.87 (2H, d,
J=8.5 Hz), 7.42-7.52 (3H, broad), 7.53 (1 H, s), 9.12 (1 H, broad). LCMS tR 1.78 (min). MS
(APCI), m/z 330.95 [M+H]+. Mp 165-1670C.
P reparat ion of 6-Ethoxy-N-f u ran-2-y lmethy I- N -(4-methoxy-pheny l)-py rim id ine-2 ,4-di amine
(5)
[0184] Sodium (115 mg, 5 mmol) was dissolved in ethanol (3 mL). Then compound 4 (1 mmol) was added to the solution. The mixture was irradiated with MW at 1350C for 40 seconds, cooϊed to room temperature, diluted with water and extracted with dichioromethane. The combined organic phases were concentrated at reduced pressure. Purification by column chromatography (silica gel, dichloromethane/hexane) gave the product (26%). 1 H-NMR
(400MHz, DMSO-D6) δ 1.25 (3H, t, J=7.5 Hz), 3.70 (3H, s), 4.20 (2H, q, J=7.5 Hz), 4.43 (2H, broad, ZJE forms), 5.28 (1 H, s), 6.19 (1H, broad, ZJE forms), 6.35 (1 H, broad, Z/E forms), 6.84
(2H, d, J=8.5 Hz)1 6.94 (1 H, t, J=7.5 Hz)1 7.43 (2H1 d, J=8.5 Hz), 7.53 (1 H1 s), 8.61 (1 H, broad).
LCMS tR 1.56 (min). MS (APCI), m/z 341.01 [M+H]+. Mp 98-1 OOoC.
Preparation of 2-Ethoxy-N-furan-2-ylmethyI-N'-(4-methoxy-phenyl)-pyrimidine-4,6-diamine
(8)
[0185] Sodium (46 mg, 2.0 mmol) was dissolved in anhydrous ethanol (1 mL) at room temperature. The obtained solution was added dropwise to a solution of 6 (122 mg, 0,37 mmol) in anhydrous ethanol (3 mL). The resulting mixture was stirred at room temperature for 20 minutes, and then at refluxing for 2 hours. After completion of the reaction (TLC control) the solvent was removed at reduced pressure. The reaction mixture was washed with water (10 mL) and extracted with chloroform (3x5 mL). The combined organic phases were concentrated at reduced pressure. Purification by column chromatography on silica gel gave the final compound (60 mg, 47%). 1 H-NMR (400MHz, DMSO-D6) δ 1.25 (3H, t, J=7.5 Hz), 3.70 (3H, s), 4.20 (2H, q, J=7.5 Hz), 4.38 (2H, broad, Z/E forms), 5.42 (1H, s), 6.20 (1H, broad, Z/E forms), 6,37 (1H1 broad, Z/E forms), 6.84 (2H1 d, J=8.5 Hz)5 7.07 (1 H, broad), 7.35 (2H, d, J=8.5 Hz), 7.53 (1 H, s),
8.55 {1 H, broad). LCMS tR 1.50 (min). MS (APCI), m/z 340.85 [M+H]+. Mp 112-1140C.
Preparation of N-Furan-2-ylmethyI-N')N"-bIs-{4-methoxy-phenyi}-pyrimidine-2,4,6- triaminθ (9)
[0186] The procedure is the same as used to prepare 5.
[0187] (98 mg, 32%). 1 H-NMR (400MHz, DMSO-D6) δ 3.70 (6H, s), 4.43 (2H, d, J=7.5 Hz),
5.33 (1H, s), 6.18 (1 H, broad, Z/E forms), 6.34 (1 H, broad), 6.55 (1 H, broad, Z/E forms), 6.81
(4H, d, J=8.5 Hz), 7.42 (4H, d, J=8.5 Hz), 7.52 (1 H, s), 8.32 (2H, broad). LCMS tR 1.63 (min).
MS (APCI), m/z 418.08 [M+H]+. Mp 145-1470C.
Preparation of Example 10-11
Figure imgf000043_0001
Preparation of {2,6-Dichloro-pyrimidin-4-yϊH4-methoxy-phenyl)-amine (2)
[0188] A mixture of 2,4,6-trichloro-pyrimidine (4.080 g, 22 mmol), p-methoxyaniline (2.750 g,
22 mmol}, DIPEA (3.76 mL, 22 mmol) was stirred for 1.5 hour at room temperature, and diluted with water. The formed solid was collected by filtration and washed with diethyl ether and hexane to give 2 (4.681 g, 78%).
Preparation of (4,6-Dichloro-pyrimidin-2-yi)-{4-methoxy-phenyi)-amine (3)
[0189] A mixture of 2,4,6-trichloro-pyπmidine (535 mg, 2.9 mmol), p-methoxyaniline (360 mg,
2.9 mmol), DIPEA (0.5 mL, 2.9 mmol) and THF (10 mL) was stirred for 8 hours at 5O0C, diluted with water and extracted with dtchloromethane. The combined organic phases were concentrated at reduced pressure. A major part of isomer 2 was separated by recrystallization from ethyl acetate. Mother liquids were concentrated at reduced pressure. Purification by column chromatography (silica gel ethyl acetate/hexane) gave 3 (86 mg, 11%).
Preparation of 6-Chloro-N-furan-2-ylmethyl-N -(4-methoxy-phenyl)-pyrimidine-2,4-diamine
(4)
[0190] Furfuryl amine (143 mg, 1.5 mmol) was added to a solution of 2 (400 mg, 1.5 mmol) and DIPEA (0.26 m!_, 1.5 mmot) in THF (10 ml_) at room temperature. The mixture was stirred for 24 hours at room temperature (TLC control), diluted with water and extracted with dichloromethane. The combined organic phases were concentrated at reduced pressure.
Purification by column chromatography (silica gel, dichloromethane) gave 4 (410 mg, 84%),
2-ChIoro-N-fυran-2-ylmethyl-N'-{4-methoxy-phenyl)-pyrimidine-4,6-diamine (5)
[0191] A mixture of 2 (2.000 g, 7.4 mmol), furfuryl amine (719 mg, 7.4 mmo!) and DIPEA (1.3 mL) was refiuxed for 16 hours, cooled to room temperature, diluted with mixture of methanol/water. The formed solid was collected by filtration. The mother liquid was extracted with dichloromethane. The organic phase was concentrated at reduced pressure. Separation from the isomer 4 and fine purification by column chromatography (silica gel, ethyl acetate/hexane) gave 5 (168 mg, 7%).
Preparation of 6-Ethoxy-N-furan~2-ylmethyI-N'-{4-methoxy-phenyl)-pyrimϊdine-2,4- diamine (6)
[0192] Sodium (115 mg, 5 mmol) was dissolved in ethanol (3 mL). Thereafter, compound 5
(330 mg, 1 mmol) was added to the solution. The mixture was irradiated with MW for 15 min at
12O0C and at 1350C for 30 seconds, cooled to room temperature, diluted with water and extracted with dichloromethane. The combined organic phases were concentrated at reduced pressure. Purification by column chromatography (silica gel, dichloromethane/hexane) gave 6
(110 mg, 32%).1H-NMR (400MHz, DMSO-D6) δ 1.28 (3H, t, J=7.5 Hz), 3.70 (3H, s), 4.22 (2H1 q,
J=7.5 Hz), 4.44 (2H, d, J=7.5 Hz), 5.30 (1 H, s), 6.26 (1 H, dd, J=3.6 1.8 Hz), 6.38 (1 H, d, J=3.6
Hz), 6.80 (2H1 d, J=8.5 Hz), 7.18 (1 H1 broad), 7.55 (1 H, d, J=1.8 Hz), 7.60 (2H, d, J=8.5 Hz),
8.60 (1H, s). LCMS tR 1.56 (min). MS (APCI), m/z 341.05 [M+Hf. Mp 112-1140C.
Preparation of 6-Chloro-N-furan-2-ylmethyl-N'-(4-methoxy-phenyl)-pyrimidine-2,4-diamine
(7)
[0193] Furfuryl amine (143 mg, 1.5 mmol) was added to a solution of 3 (400 mg, 1.5 mmol) and DIPEA (0.26 mL, 1.5 mmol) in THF (10 mL) at room temperature. The mixture was stirred for 24 hours at room temperature (TLC control), diluted with water and extracted with dichloromethane. The combined organic phases were concentrated at reduced pressure.
Purification by column chromatography (silica gel, dichloromethane) gave 7 (410 mg, 84%).
P re paration of 6-Ethoxy-N-f u ran-2-y lmethy I- N ' -(4-m ethoxy-phen yl )-py rim i d i ne-2,4- diamine (8)
[0194] Sodium (115 mg, 5 mmol) was dissolved in ethanol (3 mL). Then compound 7 (330 mg, 1 mmol) was added to the solution. The mixture was irradiated with MW for 15 min at 12O0C and at 1350C for 30 seconds, cooled to room temperature, diluted with water and extracted with dlchtoromethane. The combined organic phases were concentrated at reduced pressure. Purification by column chromatography (silica gel, dichloromethane/hexane) gave compound 8. Yield 110 mg, 32%. 1H-NMR (400MHz, DMSO-D6) δ 1.28 (3H1 1, J=7.5 Hz), 3.70 (3H, s), 4.22 (2H1 q, J=7.5 Hz), 4.44 (2H, d, J=7.5 Hz), 5.30 (1 H, s), 6.26 (1 H1 dd, J=3.6 1.8 Hz), 6.38 (1 H, d, J=3.6 Hz), 6.80 (2H, d, J=8.5 Hz), 7.18 (1 H1 broad), 7.55 (1 H, d, J=1.8 Hz), 7.60 (2H, d, J=8.5 Hz), 8.60 (1 H, s).LCMS tR 1.56 (miπ). MS (APCi), m/z 341.05 [M+H]*. Preparation of Examples 12-16.
Figure imgf000045_0001
Preparation of 6-Trifluoromethyl«pyrimidine-2,4-diol (4)
[0195] Sodium (7.18 g, 312 mmol) was dissolved in methanol (150 ml_) and 2-ethyi-isourea (2) (36,8 g, 149 mmol) was added to the resulting solution at room temperature. The obtained mixture was stirred at room temperature for 30 minutes. Then 4,4,4-Trifiuoro-3-oxo-butyric acid ethyl ester (1) (50 g, 272 mmol) was added. The reaction mixture was stirred at refluxiπg for 4 hours, cooled to room temperature and concentrated at reduced pressure. The residue was dissolved in water and acidified to pH 4. The formed solid was collected by filtration, washed with water and hexane and dried at 500C. To the resulting solid 20% aqueous solution of HC! (200 ml_) was added and the obtained suspension was stirred at refluxing for 3 hours and cooled to room temperature. Precipitate was collected by filtration, washed with water and dried at 5O0C to produce compound 4. Yield 23.3 g, 48%. Preparation of 2,4-Dichloro-6-trifluoromethyl-pyrimidine (5)
[0196] A mixture of compound 4 (23.0 g, 128 mmol), SOCI2 (100 ml.) and DMF (12 mi_) was stirred at refluxing for 4 hours, cooled to O0C. Thereafter, hexane and crushed ice were added to the resulting mixture, followed by careful addition of NaHCO3 until CO2 had stopped evolving. The organic layer was separated, dried over sodium sulfate and carefully concentrated at reduced pressure with bath temperature below 350C, giving compound 5 used in the next stage without additional purification. Yield 20.1 g, 72%.
Preparation of (4-Chloro-6-trϊfluoromethyi-pyrimidin-2-yt)-furan-2-ytmethyl-amine (6) [0197] To a solution of compound 5 (1.0 g, 4.6 mmol) and 4-methoxyaniline (624 mg, 5.1 mmoi) in acetonitrile (10 mL) NaHCOS was added and the resulting mixture was stirred at room temperature for 1 hour. The solvent was removed at reduced pressure. Water was added to the residue and the mixture was extracted with chloroform. The combined organic phases were dried over sodium sulfate and concentrated. The residue was washed with hexane and dried to give compound 6, used in the next step without additional purification. Yield 1.19 g, 85%. 1 H- NMR (400MHz, DMSO-D6) δ H: 3.77 (3H, s), 6.99 (2H, d, J=8.5 Hz) 7.01 (1 H, s), 7.50 (2H, broad), 10.35 (1H, broad). LCMS tR (min): 1.90. MS (APCI), m/z 304.19, 306.17 [M+H]+. HPLC tR (min): 14.76. Mp 152-1540C.
P reparation of N-Furan-2-ylm ethyl-N '-{4-methoxy-phenyl )-6-trif luoromethy t-pyr i m i d i ne- 2,4-diamine (7)
[0198] A mixture of compound 6 (500 mg, 1.8 mmol), 4-methoxyani!ine (488 mg, 4.0 mmol) and acetic acid (3 mL) was stirred at refluxing for 4 hours and cooled to room temperature. Acetic acid was removed at reduced pressure. The residue was diluted with water, extracted with chloroform. The combined organic phases were dried over sodium sulfate and concentrated. Purification by column chromatography (silica gel, chloroform /acetone) gave compound 7. Yield 585 mg, 89%. 1 H-NMR (400MHz, DMSO-D6) δ 3.73 (3H, s), 4.58 (2H, broad doublet, J=7.5 Hz), 6.30 (1 H1 broad), 6.32 (1 H, s), 6.40 (1H, broad), 6.84 (2H, d, J=8.5 Hz), 7.59 (1 H, s), 7.62 (2H, d, J=8.5 Hz), 8.07 (1 H, broad), 9.25 (1 H, s). LCMS tR (min): 1.96. MS (APCI), m/z 365.09 [M+H]+. HPLC tR (min): 14.98. Mp 147-1490C.
Preparation of N-Furan-2"ylmethyl-N'-(3-morpholin-4-yi-phenyi)-6-trifluoromethyl- pyrimidine-2,4-diamine (3) [0199] Yield 80 mg, 39%, 1 H-NMR (400MHz, DMSO-D6) δ 3.04 (4H, m), 370 (4H, m), 4.62 (2H, broad), 6.33 (1 H1 broad}, 6.36 (1 H, s), 6.40 (1 H1 broad), 6.54 (1H, d, J=8.5 Hz), 7.09 (1H1 1, J=8.5 Hz), 7.12 (1 H, broad), 7.54 (1 H, broad), 7.60 (1 H, s), 8.11 (1 H, broad), 9.29 (1 H, s). LCMS tR (rnin): 1.91. MS (APCI), m/z 420.16 [M+H]+. HPLC tR (min): 13.81. Mp 124-126°C. Preparation of Furan-2-ylmethyf-[4-<4-methoxy-phenyl)-6-trifluoromethyl-pyrimidin -2-yf]- amine (9)
[0200] A mixture of compound 6 (390 mg, 1.4 mmol), 4-methoxy boronic acid (92) (256 mg, 1.7 mmol), Pd(PPh3)4 (81 mg, 0.07 mmol), K2CO3 (787 mg, 5.62 mmol), dioxane (5 mL) and water (0.1 mL) was stirred at 900C for 3 hours, cooled to room temperature and concentrated. Purification of the residue by column chromatography (silica gel, ethyl acetate/hexane) and prepTLC (ethyl acetate/hexane) gave compound 9-Yieid 70 mg,14 %. 1 H-NMR (400MHz, DMSO-D6) 5 3.84 (3H, s), 4.72 (2H, broad), 6.38 (1 H, broad), 6.41 (1 H, broad), 6.82 (1H, s),
7.04 (2H, d, J=8.5 Hz), 7.59 (1 H, s), 8.30 (2H1 d, J=8.5 Hz), 8.36 (1 H, broad). LCMS tR (min): 2.09. MS (APCI), m/z 350.05 [M+HJ+. HPLC tR (min): 16.38. Mp 115-1170C.
P re paration of (4-C h loro-6-trtfl u oromethy I-py r i m id in-2-yl )-(3-morph ol i n-4-y Im ethyl - phenyt)-amine (10)
[0201] By the same method to prepare 6, 10 was obtained in 40% (437 mg).
Preparation of N-Furan-2-ylmethyl-N*-(3-morpholin-4-ylmethyl-phenyI)-6-trifluoro methyl- pyrimidine-2,4-dϊamine (11)
{0202| Yield 75 mg, 28%. 1H-NMR (400MHz, DMSO-D6) δ 2.38 (4H, m), 3.42 (2H, broad),
3.57 (4H, m), 4,52 (2H, broad), 6.22 (1 H, broad), 6.35 (1 H, broad), 6.39 (1 H, s), 6.97 (1 H1 d,
J=8.5 Hz), 7.25 (1 H, t, J=8.5 Hz), 7.53 (1 H, s), 7.55 (1 H, broad), 7.74 (2H1 broad, ZJE forms),
9.63 (1 H, broad). LCMS tR (min): 1.55. MS (APCI), m/z 434.14 [M+Hf. HPLC tR (min): 10.65.
Preparation of [4-(4-Methoxy-phenyl)-6-trifluoromethyl-pyrimidin-2-yl]-(3-morpholin-4- ylmethyi-phenyl)-amine (12)
[0203] Compound 12 was prepared according to procedure for 9. Yield 100 mg, 42%. 1 H-
NMR (400MHz, DMSO-D6) 6 2.41 (4H, m), 3.51 (2H1 S)1 3.60 (4H, m), 3.86 (3H1 s), 7.00 (1 H, s),
7.05 (1 H, broad), 7.07 (2H, d, J=8.5 Hz), 7.38 (1 H, t, J=8.5 Hz), 7.65 (1 H, broad), 7.82 (1H, s), 8.42 (2H, d, J=8.5 Hz), 10.05 (1 H, s). LCMS tR (min): 1.66. MS (APCl), m/z 445.15 [M+H]+. HPLC tR (min): 12.13. Mp 151 -1530C.
Preparation of 5-(4-Chloro-6-trifluoromethyl-pyrimidin-2-ylamino)-1 ,3-dihydro- benzoimidazol-2-one (13)
[0204] To a solution of compound 5 (300 mg, 1.382 mmol) and 5-amino-1 ,3-dihydro- benzoimidazol-2-one (227 mg, 1.521 mmol) in DMF (3 mL) NaHCO3 (232 mg, 2.765 mmol) was added. The mixture was stirred at room temperature for 3 hours, diluted with water and filtered. The formed solid was washed with water and dried, giving compound 13. Yield 400 mg, 88%. Preparation of 5-{4-[(5-Methyi-furan-2-ylmethyl)-amino]-6-trifluoromethyl-pyrimidin-2- ylamino}-1,3-dihydro-benzoimidazot-2-one (14) (0205] A solution of compound 13 (331 mg, 1.0 mmol) and 5-methylfurfuryiamine (247 mg, 2.2 mmol) in a mixture of EtOH (7 mL) and DMSO (0.5 ml_) was stirred at refiuxing for 16 hours, cooled to room temperature, diluted with water and filtered. The formed precipitate was extracted with a hot mixture of ethanol and acetone (1 :1 ). The solvent was evaporated and crystallized from ethanol, giving compound 14, Yield 155 mg, 38%.1 H-NMR (400MHz, DMSO- D6) δ 2.20 (3H, s), 4.40 (2H1 broad Z/E forms), 5.91 (1H, d, J=3.6 Hz), 6.05 (1 H, d, J=3.6 Hz), 6,30 (1 H, s), 6.85 (1 H, d, J=8.5 Hz), 7.20 (1 H, broad), 7.31 (1 H, broad), 7.50 (1 H, broad), 9.45 (1 H, broad), 10.40 (1 H1 s), 10.50 (1 H, broad). MW 404.35. LCMS tR (min): 1.71. MS (APCI), m/z 404.94 [M+H]+. HPLC tR (min): 11.56. MP >250°C decomp.
Preparation of Examples 18-20.
Figure imgf000048_0001
(4,6-Dichloro-pyrimfdϊn-2-yl)-(3-trifluoromethyt-phenyl)-amine (3) [0206J To a solution of cyanuric chloride (1) (10 g, 54.5 mmol) and m-trifluoromethyf aniline (2) (8.78 g, 54.5 mmol) in acetonitrile (100 mL) NaHCO3 (9.1 g, 109 mmol) was added. The mixture was stirred at refiuxing for 3 hours. The solvent was evaporated. Then water and chloroform were added to the mixture, the organic layer was separated and the water layer was extracted with chloroform. The combined organic phases were dried over Na2SO4 and concentrated at reduced pressure. Purification by column chromatography (silica gel, 20% ethyl acetate/hexane) gave compound 3. Yield 1.98 mg, 12%.
[4-Chloro-6-(2,2,2-trifluoro-ethoxy)-pyrimidin-2-yll-(3-trifluoromethyl-phenyl)-amine (5) [0207] To a solution of compound 3 (500 mg, 1.623 mmol) in 2,2,2-trifluoroethanol (5.0 mL) K2CO3 (224 mg, 1.623 mmol) was added. The reaction mixture was stirred at refiuxing for 6 hours, cooled to room temperature and concentrated. The residue was triturated with water and cold hexane and dried to give compound 5 as white crystals. Yield 420 mg, 70%. MW 371.61. LCMS tR (min): 2,17. MS (APCI+), m/z 372.37 [M+Hf.
Preparation of N-{4-{4-[6-(2,2,2-Trifluoro-ethoxy)-2-(3-trifluoromethyi-phenylamtno) - pyrimidIn-4-ylamiπo]-ρiρeridine-1-sulfonyl}-phenyI)-acetamide (8) [0208] A mixture of compound 5 (100 mg, 0.27 mmol), compound 6 hydrochloride (125 mg, 0.37 mmol), NaHCO3 (50 mg, 0.60 mmoi) in DMSO (0.5 mL) was heated at 1300C for 2 hours, cooled to room temperature, diluted with water and filtered. The crude product was purified by column chromatography on silica gel (dichloromethane/methano!, 30/1) and recrystallized from methanol to give compound 8 as white powder. Yield 85 mg, 50 %.1 H-NMR (400MHz, DMSO- De) δ 1.51 (2H, m), 1.92 (2H, m), 2.19 (3H, s), 2.41 (2H, m), 3.51 (2H, m), 3.71 (1 H, broad peak, 2VE forms), 4.92 (2H, q, J=7.5 Hz), 5.41 (1H, s), 7.19 (1 H5 d, J=8.5 Hz), 7.38 (1 H1 1, J=8.5 Hz), 7.69 (2H, d, J=8.5 Hz), 7.71 (1 H, broad peak, Z/E forms), 7.82 (2H, d, J=8.5 Hz), 8.21 (1 H, broad peak, Z/E forms), 9.25 (1 H, broad peak, Z/E forms), 10.25 (1 H, broad peak, Z/E forms). MW 632.59. LCMS tR (min): 2.03. MS (APCI+), m/z 633.15 [M+H]+. HPLC t* (min): 16.66. Mp 225-227°C.
Preparation of N-(3-{4-[6-(2,2,2-Trifluoro-ethoxy)-2-{3-trifiuoromethy!-phenyIamino) - pyrimidin-4-yIamino]-plperidlne-1-sulfonyl}-phenyt)-acetamfde (9) [0209] A mixture of compound 5 (250 mg, 0.67 mmol), compound 7 hydrochloride (270 mg, 0.81 mmol), NaHCO3 (200 mg, 2.38 mmol) in DMSO (1 mL) was stirred at 1400C for 1 hour, cooied to room temperature, diluted with water and filtered. The crude product was purified by column chromatography on silica gel (dichloromethane/methanoi, 30/1 ) and recrystallized from DMSO/water to give compound 9 as a cream-colored powder. Yield 250 mg, 59 %.1H-NMR (400MHz, DMSO-D6) δ 1.51 (2H, m), 1.92 (2H, m), 2.11 (3H, m), 2.51 (2H, m), 3.61 (2H, m), 3.79 (1 H, broad peak, Z/E forms), 5.41 (1 H, s), 5.49 (2H, q, J=7.5 Hz), 7.11 (1 H, broad peak, Z/E forms), 7.15 (1 H, d, J=8.5 Hz), 7.41 (2H, broad peak, Z/E forms), 7.58 (1 H, t, J=8.5 Hz), 7.72 (1 H, broad peak, Z/E forms), 7.88 (1H, d, J=8.5 Hz), 8.09 (1 H, s), 8.28 (1 H, broad peak, Z/E forms), 9.29 (1 H, broad peak, Z/E forms), 10.21 (1H, broad peak, Z/E forms). MW 632.59. LCMS tR (min): 2.04. MS (APCi+), m/z 633.05 [M+Hf . HPLC tR (min):16.80. Mp 195-1970C. Preparation of N-(1-BenzenesulfonyI-ptperidin-4-yl)-β-(2,2,2-trifluoro-ethoxy)-N'-(3- trifluoromethyl-phenyI)-pyrimidme-2,4-dϊamine {11)
[021 OJ A mixture of compound 5 (240 mg, 0.645 mmol), 1-benzenesulfonyi-piperidin-4- ylamine hydrochloride (10) (360 mg, 1.3 mmol), NaHCO3 (218 mg, 2.6 mmol) and DMSO (1.0 mL) was stirred at 850C for 1.1 hour (TLC control), cooled to room temperature and diluted with water. The obtained mixture was extracted with chloroform and concentrated at reduced pressure. Purification by column chromatography on silica gel (ethyl acetate/hexane) gave compound 11 as white crystals. Yield 123 mg, 33%.1H-NMR (400MHz, DMSO-D6) δ 1.60 (2H, m), 1.93 (2H, m), 2.43 (2H, m), 3.58 (2H, m), 3.75 (1 H, broad peak, Z/E forms), 4.90 (2H, q, J=7.5 Hz), 5.42 (1 H, broad peak, Z/E forms), 7.07 (1 H, broad peak, Z/E forms), 7.16 (1 H, d, J=8.5 Hz), 7.38 (1 H, t, J=8.5 Hz), 7.66 (2H, d, J=8.5 Hz), 7.73 (4H, m), 8.28 (1 H, broad peak, Z/E forms), 9.28 (1 H, broad peak, Z/E forms). MW 575.53. LCMS tR (min): 2.23. MS (APCI+), m/2 576.15, 564.12 [M+H]+. HPLC fe (min): 19.03. MP 196-1970C. Preparation of Examples 21-23
O
Figure imgf000050_0001
[ΦChloro-e-t∑^^-triftuoro-ethoxyJ-pyrimicJm^-yll-fS-tπfluoromethyl-phenyO-amine te) [0211] To a soiutton of compound 3 (0.5 g, 1.623 mmol) in 2,2,2-trifluoroethanoI (5) (5.0 mL) K2CO3 (0.224 g, 1.623 mmol) was added. The reaction mixture was stirred at refluxing for 6 hours, cooled to room temperature and concentrated. The residue was triturated with water and cofd hexane to give compound 6. Yield 0.42 g, 70%.
Preparation of N-fS-Methyl-furan-a-yfmethylJ-e-ta^^-trifluoro-etrioxyJ-N'-fa- trifluoromethyl~phenyl)-pyrimidine-2.4~diamine (8)
[0212] A mixture of compound 6 (50 mg, 0.134 mmol), 5-methylfurfuryiamine (7) (50 mg, 0.45 mmol), K2CO3 (23 mg, 0.269 mmol) and DMSO (1.0 mL) was stirred at 900C for 1 hour (TLC control), cooled to room temperature and diluted with water (250 mL). The obtained mixture was extracted with chloroform. The combined organic layers were washed with water and 2M NaHSO4, dried over Na2SO4 and concentrated at reduced pressure. Purification by column chromatography (silica gel, 50% acetone/chloroform) gave compound 8. Yield 25 mg, 42%,1 H- NMR (400MHz1 DMSO-D6) δ 2.21 (3H, s), 4.45 (2H, broad, Z/E forms), 4.95 (2H, q, J=7.5 Hz), 5.49 (1 H, s), 5.95 (1 H, broad, Z/E forms), 6.13 (1 H, broad, Z/E forms), 7.21 (1H, d, J=8.5 Hz), 7.44 (1 H, t, J=8.5 Hz), 7.50 (1 H, broad, Z/E forms), 7.83 (1 H, broad d, J=8,5 Hz), 8.32 (1 H, broad), 9,42 (1 H, broad), MW 446,36. LCMS tR (min); 2.27. MS (APCI), m/z 446.97 [M+HJ+. HPLC tR (min): 17.48. MP 117-1190C.
Preparation of N-( 5-Methyi-isoxazol-3-yimethyi)-6-(2,2,2-trifluoro-ethoxy)-N'-(3- trϊfluoromethyi-phenyl)-pyrimidine-2,4-diamine (10)
[0213] A mixture of compound 6 (0.150 mg, 0.404 mmoi), C-(5-methyl-isoxazoI-3-yl)- methylamine (9) (136 mg, 1.215 mmol), NaHCOS (68 mg, 0.810 mmol) and DMSO (3.0 mL) was stirred at 9O0C for 1 hour (TLC control), cooled to room temperature and diluted with water. The reaction mixture was extracted with chloroform. The combined organic layers were washed with water and 2M NaHSO4 aqueous solution, dried over Na2SO4 and concentrated at reduced pressure. Purification by column chromatography (silica get, 50% acetone/chloroform) gave compound 10. Yield 90 mg, 50%.1 H-NMR (400MHz, DMSO-D6) δ 2.35 (3H, s), 4.52 (2H, broad), 4.92 (2H, q, J=7.5 Hz), 5.51 (1 H1 s), 6.11 (1 H, s), 7.20 (1 H, d, J=8.5 Hz), 7.42 (1 H, t, J=8.5 Hz)1 7.60 (1 H1 broad), 7.82 (1 H, d, J=8.5 Hz), 8.27 (1 H, broad), 9.45 (1 H, broad). MW 447.34. LCMS tR (min): 2.09. MS (APCI), m/z 448.08 [M+H]+. HPLC IR (min): 16.46. MP 155- 1570C.
Preparation of N-[1-(4-Dϊmethylamino-benzenesulfonyl)-piperidin-4-yl]-6-(2I2(2-trifluoro- ethoxy}-N'-(3-trifIuoromethyt-pheπyl)-pyrimicline-2,4-cliamϊne (12)
[0214] A mixture of compound 6 (250 mg. 0.67 mmol), 1-(4-dimethyiamiπo-benzenesulfonyl)- piperidin-4-ylamϊπe (11) (285 mg, 0.80 mmoi) and K2CO3 (220 mg, 1 ,60 mmoi) in DMSO (5 mL) was stirred at 1200C for 12 hours, cooled to room temperature and diluted with water. The formed solid was collected by filtration and washed with hexane. The crude product was purified by column chromatography (dichforomethane/acetone, 50/1 ) and recrystallized from dichloromethane to give compound 12 as a white powder. Yield 105 mg, 25%. 1H-NMR (400MHz, OMSO-O6) δ: 1.51 (2H1 m), 1.95 (2H, m), 2.41 (2H, m), 3.11 (6H, s), 3.52 (2H, m), 3.75 (1 H, broad peak), 4.91 (2H, q, J=7.5 Hz), 6.81 (2H, d, J=8.5 Hz), 7.11 (2H, broad peak), 7.21 (1H, d, J=8.5 Hz), 7.35 (1 H, t, J=8.5 Hz), 7.51 (2H, d, J=8.5 Hz), 7.71 (1 H, broad peak), 8.32 (1 H, broad peak), 9.31 (1H, broad peak). MW 618.60. LCMS fe (min): 2.23. MS (APCI+), m/z 619.03, 620.14 [M+H]+. HPLC fe (min): 18.59. MP 252-2540C. Preparation of Examples 24-25
Figure imgf000051_0001
6-Propyl-2-thioxo-2,3-dihydro-1H-pyrim«din-4-one (2)
[0215] 3-Oxohexanoic acid methyl ester (1) (2.95 ml_, 20.81 mmol) and K2CO3 (2.819 g, 20,39 mmol) were added to a solution of thiourea (1.044 g, 13.73 mmol) in water (1.8 ml_) at 700C. The obtained mixture was stirred at 1050C for 3 hours, cooled to room temperature and diluted with water (7,5 mi_). Thereafter, concentrated aqueous HCI solution (6.75 ml_) was added carefully. The resulting precipitate was collected by filtration, washed with water and dried, giving compound 2 as a white-yellow solid. Yield 1.815 g, 78%. MW 170.23. LCMS tR (min): 1.17. MS (APCI+), m/z 171.12 [M-t-hTf. 2-{Methylthio)-6-propylpyrimidin-4(3H)-one (3)
[0216] Compound 2 (1.815 g, 10.66 mmol) was dissolved in 1.0 N aqueous NaOH solution (10.8 mL, 10.8 mmol). Then methyl iodide (0.675 mL, 10.80 mL) was added to the obtained solution at room temperature. The reaction mixture was stirred at room temperature for 2 hours, then at 400C for 2 hours and left overnight at room temperature. The resulting precipitate was collected by filtration, washed with water and dried, giving the first portion of compound 3. The mother liquid was treated with acetic acid and the resulting precipitate was washed with water and dried, giving the second portion of compound 3. The two portions were combined. Yield 1.610 g, 82%. MVV 184.26. LCMS tR (min): 1.44. MS (APCI+), m/z 185.10 [M+Hf . 6-Propyl-2-(3-trifiuoromethyl-phenyiamino)-3H-pyrimidin-4-one (4) [0217] A mixture of compound 3 (1.604 g, 8.71 mmol) and m~CF3-aniline (36) (2.50 mL, 20 mmol) was stirred at 22O0C for 5 hours in Ar atmosphere and cooled to room temperature. The resulting solid was dissolved in 1.0 N aqueous NaOH solution and extracted with dichloromethane. The aqueous phase was acidified with acetic acid and the resulting precipitate was collected by filtration, washed with water and hexane and dried, giving compound 4 as a white solid. Yield 2.356 g, 91 %. MW 297.28. LCMS tR (min): 1.85. MS (APCI+), m/z 298.15
(4-Chloro-6-propyl-pyrimidin-2-yl)-{3-trifluoromethyt-phenyl)-amine (5) [0218] A mixture of compound 4 (1.486 g, 5.0 mmol) and POCI3 (1.865 mL, 20.0 mmol) was stirred at 1500C for 6 hours, cooled to room temperature, poured onto an ice-water mixture and extracted with ethyl acetate. The organic phase was washed with water, brine, dried over sodium sulfate, concentrated and dried, giving compound 5 as a white soiid. Yield 1.515 g, 96%. MW 315.73. LCMS fe (min): 2.30. MS (APCI+), m/z 316.19, 318.19 [M+H]+.
Preparation of N-(4-Fluoro-phenyl)-6-propyi-N'-{3-trifluoromethyl-phenyl)-pyrimidine-2,4- diamine (6)
[0219] A mixture of compound 5 (316 mg, 1.0 mmol) and p-fluoro-aniline (275 mg, 2.5 mmol) was stirred at 17O0C for 10 minutes. The formed solid was cooled to room temperature, triturated with water and extracted with ethyl acetate. The organic phase was washed with water, brine, dried over sodium sulfate and concentrated. The residue was purified by column chromatography (silica gel, dichioromethane), giving compound 6. Yield 102 mg, 26%. 1H-NMR
{400MHz, DMSO-D6) δ 0.95 (3H, t, J=7.5 Hz), 1.72 {2H, m), 2.45 (2H1 m) 6.12 (1 H, s), 7.10 (2H1 d/d, 8.5/8.0 Hz), 7.18 (1 H, d, J=8.5 Hz), 7.43 (1 H, t, J=8.5 Hz), 7.66 (2H, broad m), 7.93 (1 H, d,
J=8.5 Hz), 8.28 (1 H, s), 9.25 (1 H, broad peak, ZJB forms), 9.38 (1 H, broad peak, ZJB forms).
MW 390.38. LCMS tR (min): 1.84. MS (APCt+), m/z 391.23 [M+H]+. HPLC tR (min): 13.16. MP
81-830C.
Preparation of N-(4-FIuoro-benzyl)-6-propyI-N'-(3-trifluoromethyl-phenyl)-pyrimidine-2,4- diamine (7)
[0220] A mixture of compound 5 (316 mg, 1.0 mmol) and p-fluoro-benzyl amine (400 mg,
3.15 mmol) was stirred at 17O0C for 30 minutes. The obtained mixture was cooled to room temperature, poured onto water and extracted with ethyl acetate. The organic phase was washed with water, brine, dried over sodium sulfate and concentrated. The residue was purified by column chromatography (silica gel, dichioromethane), giving compound 7 as an oil. Yield 310 mg, 77%. 1H-NMR (400MHz, DMSO-D6) δ 0.92 (3H1 1, J=7.5Hz), 1.68 (2H, m), 2.41 (2H1 1,
J=7.5 Hz), 4.57 (2H, broad d, J=7.5 Hz, ZJE forms), 5.93 (1 H, s), 7.13 (3H, broad m), 7.36 (3H, broad m, ZJE forms), 7.57 (1 H, broad peak, ZJE forms), 7.86 (1 H, broad d, J=8.5 Hz, ZJE forms),
8.42 (1 H, s), 9.25 (1 H, broad peak, Z/E forms). MW 404.41. LCMS tR (min): 1.84. MS (APCI+), m/z 405.20 [M+Hf. HPLC tR (min): 13.16.
Table 1. Examples 1-25 of Pyrimidine Compounds
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
B. PREPARATION OF PYRAZOLOPYRIMIDiNES DERIVATIVES AS HCV ENTRY INHIBITORS General Approaches
[0221] Exemplary chemical intermediates and compounds of pyrazolopyrimidines of the present invention were prepared according to the following methods. Three major approaches were developed to synthesize 1 ,4,6-trtsubstituted pyrazolopyrimidine derivatives (I):
Figure imgf000056_0002
(I)
[0222] The first approach (Scheme 1 , below) started from benzenecarboxamidine or aminecarboxamtdine by introducing aryl or amine at C6-position in the first step. The carboxamidine cyclized with diethylmalonate and further underwent chlorinated-oxidation to form dichioroaldehyde 4. Then cyclization of 4 with various hydrazines formed the pyrazole- pyrimidine core and also introduced an N1 substituent. Finally, the mono-chlorine in 6 was replaced with various primary and secondary amines or boronic acids to generate libraries with C4 variation in libraries A, C, E, S, F, K, N, H, AB, wherein:
R1 = alkyl and aryl (benzyl, Me, Et, Pr, Phenethyl, Piperidine ethyl);
R4 = anilines (m-, p-arnidoanilines, m-sutfamidoanilines, m-suifoneanilines etc.), primary amines (1 -substituted piperidin-4-amines), secondary amines (substituted piperazines), substituted aryi;
R6 = pyrrolidine, morphoiine, substituted phenyl. Scheme 1. R6-R1-R4 Approach
Figure imgf000056_0003
[0223] The second approach (Scheme 2, below) started from intermediate 3. Cyclization of 3 with hydrazine formed aminoamide 5, which was further cyciized with diphosgene and chlorinated with POCI3-PCI5 to form intermediate 7. In this common intermediate, C4-chloro appeared to be more reactive, which can generate libraries with C4 variation. Further substitution at the C6-position generated various libraries with C6 variation. This approach provided seven C6 libraries (B, J, R, T, W, R1 , Z), four C4 libraries (L, D, M, X), and one N1 library; wherein:
R1 = alkyl and aryl (benzyl, Me, Pr, o-C!-Phenyl, p-F-Phenyl);
R4 = anilines (p-Me-aniline, m-Me-aniline, p-F-aniline, m-F-ani)ine, m- sυlfonyiamidoanilines), primary amines (1 -substituted piperidiπ-4-amines, o-Me- benzylamine);
R6 = anilines (3,4-diF-aniline, m-F-aniline, etc), primary amines (1 -substituted piperidin-4- amines), secondary amines (substituted piperazines, pyrrolidine), Ar, ethoxy-.
Scheme 2, R1-R4-R6 Approach
Figure imgf000057_0001
[0224] The third approach (Scheme 3, below) started from the intermediate 5 in Scheme 2. Cyclization of aminoamide 1 with substituted aldehyde 2 formed pyrazole-pyrimidine core with N1- and C6-substitutions. Compound 3 can be further chlorinated with POCI3-PCI5 to form intermediate 4, which was further replaced with aniline, amine, or boronic acid to generate five libraries with C6 variations, and C6, C4 variation in libraries I, Q, Y, Zb, AA; wherein:
R1 = Pr;
R4 = OH, anilines, p-F-benzylamine;
R6 = m-, p-sulfonyiamidophenyls, m-amidophenyls.
Scheme 3. R1-R6-R4 Approach
Figure imgf000057_0002
[0225] Another approach leading to structure 5 is depicted in Scheme 4, below. Starting from commercially available aminoamide 1, cyclization of 1 with aldehyde 2 or ester 2a formed intermediate 3. Intermediate 3 was chlorinated with POCl3-PCI5 and the chlorine was further substituted with any aniline or boronic acid to generate three libraries of O, P, V; wherein:
R1 = Me;
R5 = m-sulfonylamidophenyts;
R7 = OH.
Scheme 4. R1-R3-R4-R6
Figure imgf000058_0001
I R6-R1-R4 Approach Library A
HN' NH,
Figure imgf000058_0002
OMF POCt3 9O =C 12 h
Figure imgf000058_0003
Preparation of 1-pyrrolidinecarboximidamide hydrobromide (3)
[0226] Ethyl isothiourea (40.0 g, 0.216 mol) was dissolved in water (200 m!_) and pyrrolidine (18.4 g, 0.258 mol) was added at once. The reaction mixture was refluxed overnight. The solvent was removed in vacuo and compound 3 was obtained in quantitative yield (40 g) as a white precipitate.
Preparation of 2-(1-pyrrolidinyl)-4,δ-pyrimidinediol (4)
[0227] 20.0 g (0.870 mol) of sodium were dissolved carefully in 1.2 L of dry ethanol. The solution was cooled to 40-45 0C and 37.8 g (0.236 mol) of dtethylmalonate was added dropwise. At this time, 40.0 g (0.206 mol) of 1-pyrrolidinecarboximidamide hydrobromide (3) was added in portions to the reaction mixture. The reaction mixture was refluxed overnight, and the white solid was filtered, dissolved in water and acidified with acetic acid to pH 5-6, The resulting formed white precipitate was filtered, washed with ether and iyophilized to provide a pale pink precipitate (37 g, 99 %).
Preparation of 2.6-dichloro-4-(1 -pyrrolidine) benzaldehyde (5)
[0228] 10 ml of dry DMF was cooled to 0-5 0C, then 25 mL of POCI3 were added dropwise. This mixture was allowed to react at 20 0C for 1 h and then 5.78 g of 2-(1-pyrrolidinyI)-4,6- pyrimidinediol (4) was added in portions for one hour. After 30 min, the reaction mixture was heated on a steam bath overnight. The excess phosphoryl chloride was removed under reduced pressure, the reaction mixture was poured into ice, and the yellow precipitate with yield 63 % (5.0 g) was collected and lyophilized.
Preparation of 4-chloro-1-aIkyl-6-(1-pyrrolidtnyi)-1H-pyrazolo-[3,4-d]-pyrimidine {6) [0229] 0.0022 mol of hydrazine were suspended in 30 mL of dry dioxane; then 0.5 g (0.0020 mol) of 2.6-dichloro-4-{1-pyrrolidini!) benzaldehyde (5) was added at once. The reaction mixture was stirred for 60 h at room temperature, then an orange precipitate was filtered and the residue was concentrated in vacuo. The residue was purified by column chromatography with methylene chloride as an eluent, A white precipitate (0.25 g) was obtained in 40% yield. Preparation of 1-alkyl-4-{anilino)~6«{1-pyrrolidinyl)-1H-pyrazolo-[3,4d] pyrimidϊne (7) [0230] 0.100 g (0.00032 moi) of 1-methyl-4-chloro-6-{1-pyrrolidinyl)-1 H-pyrazolo-[3,4d] pyrimidine (6) was dissolved in 5 mL of dry dioxane and 0.00064 mol of aniline was added at once. The reaction mixture was stirred overnight at 100 0C. LCMS analysis of the reaction mixture demonstrated the total conversion of the starting material to the target compound. The reaction mixture was poured into water; the precipitate was filtered, carefully washed with ether, and crystallized from hexanes. A white precipitate was obtained with yield 52 % (23.2 g). HNMR in DMSO-d6: 51.88 m (4H), 2.38 s (3H)5 3.52 bs (4H), 3.86 m (2H), 4.24 m (2H), 7.18 d (2H), 7.72 d (2H), 7.86 s (1 H), 9.24 s (1 H).
Figure imgf000059_0001
Library C
Figure imgf000060_0001
Preparation of 4-chloro-1-methyI-6-{1-pyrroHdinyl)-1H-pyrazoIo-[3,4-d]φyrimid!ne (6) [0231] 2.325 g {0,016 moϊ) of methyihydrazine sulphate was suspended in 250 ml_ of dry dioxane and 1.630 g {0.0032 mol) of TEA was added; then 4,0 g (0.016 mol) of 2.6-dichloro-4- {1-pyrrolidinil) benzaldehyde {5) was added at once. The reaction mixture was stirred for 60 h at RT, and the solvent was removed in vacuo. The residue was purified by column chromatography with methylene chloride as an eluent. The yield was 35-40 % {1.5 g). Preparation of 1-methyl-4»(substituted phertyiamino)-6-(1-pyrrolidinyi)-1H-pyrazolo-[3,4d] pyrϊmϊdine (7)
[0232] 0.05 g {0.00021 mol) of 1-methyl-4-chloro-6-(1-pyrrolidinyl)-1H-pyrazolo-[3,4d] pyrimidine (6) was dissolved in 5 mL of dry dioxane and 0.00042 mol of corresponding aniline was added at once. The reaction mixture was stirred overnight at 130 0C. LCMS analysis of the reaction mixture demonstrated the total conversion of the starting material to target compound. The reaction mixture was poured into water; the precipitate was filtered, carefully washed with ether, and crystallized from hexanes to provide white precipitates in yields of 10-45 %.
Figure imgf000060_0002
Table 3. Examples 2-18 of Pyrazolopyrimidines . Library C
Figure imgf000060_0003
Figure imgf000061_0001
Figure imgf000062_0001
Analytical Data for Library C.
2. methyI-N-(4-methyIphenyl)-6-pyrrolidin-1-yl-1H-pyrazoio[3,4-dJpyrimidin-4-amine
HNMR in DMSO-d6: δ 1.95 m (4H); 2.30 S (3H); 3.55 m (4H); 3.75 s (3H); 7.15 d (2H); 7.73 d (2H); 7.88 s (1 H); 9.15 s (1 H),
3. N-<4-chlorophenyf)-1-methyl-6-pyrrolidin-1-yl-1H-pyrazolo[3,4~d]pyrimidin-4-amine HNMR in DMSO-d6: δ 1.95 m (4H); 3.50 m (4H); 3.75 s (3H); 7.36 d (2H); 7.92 m (3H); 9.40 s (1 H).
4. N-(4-fluorophenyl)-1-methyl-6-pyrrolidin-1-yi-1H-pyrazolo[3,4-d]pyrimidin-4-amine HNMR in DMSO-d6; δ 1.95 m (4H); 3.55 m (4H); 3.75 s (3H); 7.14 t (2H); 7.88 m (3H); 9.25 s (1H).
5. rnethyl-6-pyrroiidiπ-1-y(-N-[4-{trifluoromethy!)phenyi]-1H-pyrazoto[3,4-d]ρyrim(din-4- amine HNMR in DMSO-d6: δ 2.00 m (4H); 3.60 m (4H); 3.70 s (3H); 7.68 d (2H); 8.04 s (1H); 8.15 d (2H); 9.65 s (1 H).
6. N-(3-ch loro-4-f luorophe ny i}-1 -methyl-6-py rrol id i n- 1 -y!-1 H-py razol o{3,4-d] py rim i d i n-4- amine HNMR in DMSO-d6: 6 1.95 m (4H); 3,50 m (4H); 3.75 s (3H); 7.36 d (2H); 7.92 m (3H); 9.40 s (1 H). 7. methyl-6-pyrrolidin-1-yl-N-t3-(triftuoromethyf)phenyl3-1H-pyra2olo[3,4-d3pyrimidin-4- amine HNMR in DMSO-d6: δ 1.95 m (4H); 3.50 m (4H); 3.75 S (3H); 7,32 d (1 H); 7.56 t (1 H); 8.00 m (2H); 8.62 s (1 H); 9.60 s (1 H).
8. N-(3,5-dimethoxyphenyl)-1-methyl-6-pyrrolidtn-1-yl-1H-pyrazoio[3,4-d]pyrimJdin-4- amine HNMR in DMSO-d6: 5 1.95 m (4H); 3.60 m (4H); 3.75 s (9H); 6.15 s (1 H); 7.20 s (2H); 8.00 s (1 H); 9.20 s (1 H).
9. N-{2-fluorophenyl)-1-methyl-6-pyrroIidin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4-amine HNMR in DMSO-cl6: δ 1.95 m (4H); 2.30 s (6H); 3.50 m (4H); 3.75 s (3H); 7.20 m (3H); 7.76 s (1 H); 7.94 m (1H); 9.00 s (1H).
10. N-(3-fluorophenyi)-1-methyl-6-pyrroiidin-1-yl-1H-pyrazoϊo[3,4-d]pyrimidin-4-amine HNMR in DMSO-d6: δ 1.95 m (4H); 3.60 m (4H); 3.75 s (3H); 6.80 t (1 H); 7.34 m (1 H); 7.58 d (1 H); 8.00 m (2H); 9.45 s (1 H).
11. N-(3,5-dimethyiphenyl)-1-methyl-6-pyrrolidin-1-yl-1H-pyra2θto[3,4-d3pyπmidin-4-amine HNMR in DMSO-d6: δ 1.95 m (4H); 2.30 s (6H); 3.50 m (4H); 3.75 s (3H); 6.70 s (1H); 7.55 s (1 H); 7.92 s (1H); 9.05 s (1H).
12. N-{4-isopropylphenyl)-1-methyI-6-pyrrolidin-1-yI~1H-pyrazoIot3,4"d]pyrimidtn-4-amine HNMR in DMSO-d6: δ 1.20 m (6H); 1.95 m (4H); 2.30 s (6H); 3.60 m (4H); 3.75 s (3H); 7.20 rn (2H); 7.64-8.00 m (3H); 9,15 s (1 H).
13. methyl-6-pyrroiidin-1 -yf-N-[4-(trif luoromethoxy)phenyl]-1 H-pyrazolo[3,4-d]pyrimidϊn-4- amine HNMR in DMSO-d6: δ 1.95 m (4H); 2.30 s (6H); 3.60 m (4H); 3.75 s (3H); 7.30 d (2H); 8.00 m (3H); 9.45 s (1H).
14. N-fSsS-difluorophenylϊ-i-methyi-θ-pyrroIidin-i-yl-IH-pyrazoloIS^-dlpyrimidin^amine HNMR in DMSO-d6: δ 1.95 m (4H); 2.30 s (6H); 3.60 m (4H); 3.75 s (3H); 6.70 1 (1 H); 7.70 d (2H); 8.00 s (1 H); 9.54 s (1H).
15. N-(3-isopropylphenyl)-1-methyl-β-pyrrolidϊn-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4-amine HNMR in DMSO-d6: δ 1.20 m (6H); 1.95 m (4H); 3.60 m (4H); 3,75 s (3H); 6.92 m (1 H); 7.24 m (1 H); 7.64 d (1 H); 7.76-8.04 m (2H); 9.20 s (1H).
16. methyl-N-[4-(methyisulfonyl)phenyl]-6-pyrrolidin-1-y1-1 H-pyrazoto[3,4-d3pyrimtdin-4- amine HNMR in DMSO-d6: δ 1.95 m (4H); 2.30 s (6H);3.48 s (3H), 3.60 m (4H); 3.75 s (3H); 7.30 d (2H); 8.00 m (3H); 9.45 s (1 H).
17. N-(4-fIuoro-3-methylpheny1)-1-methyi-6-pyrrolidϊn-1-yl-1H-pyrazolo[3,4-dlpyrimidin-4- amine HNMR in DMSO-d6: δ 1.95 m (4H); 2.25 s (1 H); 3.60 m (4H); 3.75 s (3H); 7.08 t (1 H); 7.65 m (1 H); 7.82 d (1 H); 7.90 m (2H); 9.20 s (1H).
18. N-{2-fluoro-5-methylphenyf)-1-methyl-6-pyrroiϊdin-1-yI-1 H-pyrazolo[3,4-d]pyrimidin-4- amϊne HNMR in DMSO~d6: δ 1.95 m (4H); 2.30 s (1H); 3.50 m (4H); 3.75 s (3H); 6.94-7.18 m (2H); 7.70-7.86 m (2H); 8.95 s (1 H). Library E
Figure imgf000064_0001
Preparation of methyl-3-{[1-methyl-6-[1-pyrroIidinyl]-1 H-pyrazolo-[3J4-d3-pyrtmidin»4-yi] aminojbenzoate (7)
[0233] 0-300 g (0.0013 mol) of 4-chJoro~1-methyl-6-{1-pyrrolidinyl)-1 H-pyrazolo--[3,4-d]- pyrimidine (6) was suspended in 5 mL of dry dioxane, followed by 0.382 g (0.0026 mol) of methyl-3-aminobenzoate and 20 mg of CuI. The reaction mixture was stirred for 2 h at 120 0C in a microwave oven (800 watts). LCMS analysis of the reaction mixture demonstrated total conversion of starting material. The precipitate was filtered, carefully washed with ether and iyophilized. A white precipitate was obtained with yield 98 % (0,450 g).
Preparation of 3-{[1-methyi-6-[1-pyrrolidinyl]-1H-pyrazolo-[3,4-d]-pyrimidin-4-yl]amino} benzoic acid (3)
[0234] 0.450 g (0.0013 mol) of (7) was suspended in 10mL of ethanol and 10mL of water, and 0.107 g (0.0019 mol) of potassium hydroxide was added at once. The reaction mixture was stirred for 15 min at RT at which time it was heated to 70 0C. At this temperature total consumption of starting material was observed (reaction was monitored by TLC). The reaction mixture was cooled to RT, the precipitate was filtered, and the residue was concentrated under reduced pressure. Key intermediate (8) was obtained as a white precipitate. It was crystallized from ether and dried, giving 8 with quantitative yield (0.430 g).
Preparation of 3-{[1-methyl-6-[1-pyrrolidinyI]-1H-pyrazolo-[3,4-d]-pyrimidin-4-yl] amino}- benzamides (9)
[0235] 0.100 g (0.00029 mol) of (8) was dissolved in 5 mL of dry dioxane, then 0.00036 mol of amine and 0.040 g (0.00038 mol) of TEA were added at once. The reaction mixture was stirred for about 3-5 min at RT, then 0.072 g (0.00047 mol) of POCI3 was added. The reaction mixture was stirred overnight at 50-60 0C. LCMS analysis of the reaction mixture demonstrated the presence of starting material only in the case of primary amine, and about 70% of target compound in cases of secondary amine and substituted piperazine. The target compounds were purified by column chromatography with methylene chloride as eluent giving white precipitates in yields of 10-55 %. b
Figure imgf000065_0001
Table 4. Examples 19-34 of Pyrazolopyrimidines. Library E.
Figure imgf000065_0002
Figure imgf000066_0001
Analytical Data for Library E,
19. methyl-N^S-^-propylpiperazin-i-ylJcarbonyllpheny^-β-pyrrolidin-i-yi-IH- pyrazolo[3,4-d]pyrimϊdin-4-amine HNMR in DMSO-Cl6 δ (ppm): 0.90 m (3H); 1.45 m (2H); 1.95 m (4H); 2.30 m (6H); 3.55 m (8H); 3.75 s (3H); 7.00 d (1H); 7.40 t (1 H); 7.80-8.10 m (3H); 9.35 s (1 H).
20. methyl- N-{3-[(4-pyridin-2-ylpiperazin-i-yl)carbonyl]phenyl}-6-pyrrolidin-1-yl-1H- pyrazolo[3,4-d]pyrimidin-Φamme LCMS: M÷1 = 494.5; HNMR in DMSOd6 δ (ppm): 1.90 m (4H); 3.60 m (11 H); 3.75 m (3H); 6.60 s (1 H); 6.92 d (1 H); 7,05 d (1 H); 7.401 (1 H); 7.60 1 (1 H); 7.95 m (2H); 8.10 m (2H); 9.45 s (1 H).
21. ethyl 1-{3-[(4-methyl-6-pyrrolidin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]amino] benzoy!}-piperidine-3-carboxyIate HNMR in DMSCRl6 δ (ppm): 1.15 t (3H); 1.20 s (1 H); 1.70 m (2H); 1.95 m (6H); 3.20 m (4H); 3.55 m (4H); 3.75 m (4H); 4.05 m (3H); 7.00 d (1 H); 7.35 t (1 H); 7.95 m (3H); 9.45 s (1 H).
22. {3-<[l-methyl-6-(pyrrolidin-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]amino}phenyl)[4-(4- nitrσphenyi)piperazin-1-yl]methanone HNMR in DMSOd6 δ (ppm): 0..90 m (4H); 3.55 m (8H); 3.80 m (7H); 6.90-7.16 m (3H); 7.451 (1 H); 7.78-8.16 m (5H); 9.50 s (1H).
23. ethyl 4-{3-[(1-methyl-6-pyrrolidm-1-yl-1 H-pyrazoloβAdtøyrimidirM-yOamino] benzoyl}-piperazine-1-carboxyJate HNMR in DMSOd6 δ (ppm): 1.20 m (4H); 1.95 m (4H); 3.35-3,70 m (13H); 3.80 s (3H); 4.10 m (2H); 7.04 d (1H); 7.40 t (1H); 7.90-8.06 m (3H); 9.40 s (1H).
24. N-{3-t(4-ethylpϊperazin-1-yl)carbonyi]phenyI}-1-methyl-6-pyrrolidin-1-yl-1H- pyrazoIo[3,4-d]pyrimidin-4-amine HNMR in DMSOd6 δ (ppm): 1.00 m (3H); 1.95 m (4H); 2.45 m (6H); 3.55 m (8H); 3.75 m (3H); 7.00 m (1 H); 7.30 m (1H); 7.80-8.10 m (3H); 9.45 s (1 H).
25. N-tS-fH-fS-chtorophenyOpiperazin-i-yηcarbony^pheπyO-i-methyl-β-pyrrolidin-i-yl-IH- pyrazoio[3,4-d]pyπmidin-4-amϊne HNMR ϊn DMSOd6 δ (ppm): 1.90 m (4H); 3.20 m (4H); 3.50-3.70 m (8H); 3.75 s (3H); 6.74-6.98 m (4H); 7.16-7.38 m (2H); 7.88-8.02 m (2H); 8.10 s (1H); 9.45 s (1 H).
26. N-{3-[(4-benzylpiperidin-1-yi)carbonyl]phenyl}-1-methyl-6-pyrrolidin-1-yl-1H- pyrazolotS^-dJpyrimidm^-amine HNMR in DMSOd6 6 (ppm): 0.85 1 (1H); 1.15 m (2H); 1.30 s (1H); 1.65 d (2H); 1.90 m (5H); 2.60 d (2H); 3.55 m (4H); 3.75 s (3H); 4.05 d (2H); 6.95 d (1 H); 7.10-7.50 m (6H); 7.85-8.10 m (3H); 9.35 s (1 H).
27. N-(3,5-difluorobenzyl)-3-[(1-methyl-6-pyrroIϊdin-1-yl-1H-pyrazolo[3,4-d]pyrimidϊn-4- yl)amϊno]benzamϊde HNMR in DMSOd6 δ (ppm): 1.95 m (4H); 3.52 m (4H); 3.80 s (3H); 4.50 d (2H); 7.00 m (3H); 7.35-7.60 m (2H); 8.00 m (2H); 8.50 s (1 H); 8.75 m (1 H); 9.50 s (1 H). 28. N^S-^-cyclohexylpiperazϊn-i-ylJcarbonylJpheny^-i-methyl-β-pyrroIidin-i-yi-IH- pyrazolo[3,4-d]pyrimidin-4-amine HNMR in DMSO-Ci6 δ (ppm): 1.20 m (6H); 1.50-1.90 m (5H); 1.95 m (4H); 2.30 m (1 H); 3.40-3.70 m (9H); 3.80 s (3H); 7.00 d (1 H); 7.40 t (1 H); 7.80-8.10 m (3H); 9.45 s (1 H).
29. methyi-6-pyrrolidtn-1-yl-N-[3-(pyrrolidin-1-yIcarbonyl)phenyl]-1H-pyrazolo[3,4- dJpyrimidtn-4-amine HNMR in DMSOcI6 δ (ppm): 1.80-2.00 m (8H); 3.40 m (2H); 3.45 m (2h); 3.55 m (4 H); 3.75 s (3H); 7.14 d (1 H); 7.40 1 (1 H); 8.03 m (2H); 8.12 s (1 H); 9.70 s (1 H).
30. 3-[(1-methyl-6-pyrroiϊdin-1-yl-1H-pyrazoIot3,4-d]pyrimidin-4-yl)amino]-N-(4-prperidin-1- yibenzyl)benzamide HNMR in DMSO-Ci6 δ (ppm): 1.60 m (2H); 1.90 m (7H); 3.30 m (4H); 3.60 m (4H); 3.75 s (3H); 4.50 d (2H); 7.40 m (5H); 7.56 d (1 H); 8.02 m (2H); 8.46 S (1 H); 8.66 m (1H); 9.65 s (1 H)
31. methyl-N-{3-[(4-phenylpϊperazin-1-yt)carbonyIJphenyl}-β-pyrroIidin-1-yl-1H- pyrazolo[3,4-d3pyrimidin~4-amine HNMR in DMSO-d6 δ (ppm): 1.85 m (4H); 3.20 1 (2H); 3.50-3.90 m (11 H); 6.80 t (1 H); 6.94 d (2H); 7.08 d (1 H); 7.24 1 (2H); 7.42 1 (1 H); 7.90-8.04 m (2H); 8.12 s (1 H).
32. N-(3-{t4-{4-fluorophenyI)piperazin-1-yl]carboπyl}phenyl)-1-methyl-6-pyrrotidin-1-yl-1H- pyrazolo[3,4-d]pyrimidin-4-amϊne HNMR in DMSO-d6 δ (ppm): 1.90 m (4H); 3.20 m (4H); 3.50-3.90 m (11 H); 7.00 rn (5H); 7.45 1 (1H); 7.85-8.10 m (3H); 9.45 S (1 H).
33. methyl-N-ta-^^-methylphenylJpiperazin-i-yqcarbony^phenyO-e-pyrrolidin-i-yl-IH- pyrazolo[3,4-d]pyrimidin-4-amine HNMR in DMSO-d6 δ (ppm): 1.95 m (4H); 2.35 s (3H); 2.80 m (7H); 3.20 m (4H); 3.50-3.90 m (11 H); 6.86-7.26 m (5H); 7.4O t (1 H); 7.84-8.16 m (3H); 9.45 s (1 H).
34. methyl-N-(3-{[4-(3-methylphenyl)piperazin-1 -yl]carbonyi}phenyl)-6-pyrrotidin-1 -yl-1 H- pyrazolo[3,4-d]pyrimidiπ-4-amine HNMR in DMSO-d6 δ (ppm): 1.95 m (4H); 2.35 s (3H); 3.20 m (4H); 3.50-3.90 m (11 H); 6.55-6.85 m (5H); 7.10 m (2H); 7.90 t (1 H); 7.85-8.05 m (2H); 8.10 s (1 H); 9.45 s (1 H).
Library S
Figure imgf000068_0001
[0236] Preparation of 2-(3-methyiphenyi)-4,6-pyriniidinediol (4a) 13.8 g (0.600 mol) of sodium was dissolved carefully in 650 mi_ of dry ethanol. The solution was cooled to 40-450C and 26.4 g (0.165 mol) of diethylmalonaie was added dropwise. Next, 25.4 g (0.150 mol) of 3- methyiphenyicarboximidamide hydrochloride was added in portions. The reaction mixture was reftuxed overnight and cooled to rt The white solid was filtered, dissolved in water, and the resulting solution was acidified with acetic acid to pH 5-6. The formed white precipitate was filtered, washed with hexane and iyophilized to provide the desired compound (17.9 g, 60 % of yield). 1HNMR in DMSOd6 δ (ppm): 2.30 s (3H); 5.35 S (1 H); 7.45 m (2H); 8.00 m (2H). [0237J Preparation of 2.6-dichloro-4-(3-methylphenyl) benzaldehyde (5a) 15 mL of dry OMF was cooled to 0-5 0C, and 35 mL of POCI3 were added dropwise. This mixture was allowed to react at 20 0C for 1 h and then 7.00 g of 2-(3-methylphenyl)-4,6-pyrimidinedio! (4a) was added in portions. After 30 mtn, the reaction was heated on a steam bath for 6 h. The excess phosphoryl chloride was removed in vacuo and the residue was treated with ice water. The crystalline pale yellow product was collected on a filter and lyophiiized. Yield was 65 % (6.0 g). 1HNMR in DMSOd6 δ (ppm): 2.30 s (3H); 5.35 s (1 H); 7.45 m (2H); 8.00 m (2H). [0238] Preparation of 1-methyl-4-chloro-6-(3-methylphenil)-1H-pyrazolo- [3,4d]pyrimidiπe (6a) 0.647 g (0.0044 mol) of methyihydrazine sulphate was suspended in 60 mL of dry dioxane and 0.907 g (0.0090 mol) of TEA was added; then 1.000 g (0.0037 mol) of 2.6-dichloro-4-(3-methylphenil)benzaIdehyde (5a) was added at once. The reaction mixture was stirred overnight at RT, then the solvent was removed in vacuo. The residue was purified by column chromatography with methylene chloride as eiuent. A white precipitate of 6a was obtained in a yield of 40 % (0.390 g). NMR 1H, DMSOd6 δ, ppm: 2.40 s (3H); 4.10 s (3H); 7.35- 7.50 m (2H); 8.25 m (2H); 8.40 s (1H).
[0239] Preparation of 1-methyl-4-(3-fluorofenylammo)-6-(3-methyIfenyll)-1H-pyrazolo- [3,4d] pyrϊmidine (7a) 0.06 g (0.00025 mo!) of 1-methyl-4-chloro-6-(3-methylfenyl)-1 H- pyrazolo-[3,4d] pyrimidin (6a) was dissolved in 5 mL of dry dioxane and 0.0 (0.00050 mol) of 3- fluorophenyl aniline was added at once. The reaction mixture was stirred overnight at 130 0C. LCMS analysis of the reaction mixture demonstrated the total conversion of the starting material to the target compound. The reaction mixture was poured into water, and a white precipitate was collected by filtration, carefully washed with ether and crystallized from hexane. Yield was 55 % (26.7 g).
b
Figure imgf000069_0001
Table 5. Examples 35-39 of Pyrazolopyrimidines. Library S
Figure imgf000069_0002
Figure imgf000070_0001
Analytical Data for Library S
35. methyl-6-(3-methyiphenyl)-N-[3-(trifluoromethyl)phenyl]-1H-pyrazolo[3,4-d]pyrimidin-
4-amine HNMR in DMSOd6 δ (ppm): 2.40 s (3H); 4.00 s (3H); 7.26-7.50 s (3H); 7.64 d (1 H); 7.80 s (1 H); 8.30 m (3H); 8.76 s (1 H); 9,55 s (1 H).
36. N-(3-fluorophenyl)-1-methyl-6-(3"methylphenyl}-1H-pyrazoio[3,4-dlpyrimidin-4-amtne HNMR in DMSOd6 δ (ppm): 2.40 S (3H); 4.05 s (3H); 6.90 s (1 H); 725-752 m (3H); 7.65 d (1 H); 8.00 d (1 H); 8.30 m (3H); 9.95 s (1 H).
37. N-(3,5-dimethylpheπyl}-1-methyl-6-{3-methylphenyi}-1H-pyrazolo[3,4-d]pyrimidin-4- amine HNMR in DMSOd6 δ (ppm): 2.40 m (6H); 4.00 s (3H); 6.80 s (1 H); 7.15-7.85 m (4H); 8.00-8.50 m (3H); 9.65 s (1H).
38. N-{4-fluoro-3-methy[phenyI)-1-methyl-6-(3-methyiphenyl)-1H-pyrazolo[3,4-d]pyrimidin- 4-amϊne HNMR in DMSOd6 δ (ppm): 2.35 s (3H); 2.40 s (3H); 4.00 s (3H); 7.15 s (1 H); 7.26-7.44 m (2H); 7.92 m (1 H); 8.12 s (1 H); 8.20-8.30 m (2H); 9.70 s (1 H).
39.N-(2-fluoro-5-methylphenyl)-1-methyl-6-(3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidiπ- 4-amine HNMR in DMSOd6 δ (ppm): 2.40 s (6H); 4.00 s (3H); 7.06-7.40 s (4H); 7.76 d (1 H); 7.96 s (1 H); 8.12-8.26 m (2H); 9.55 s (1 H).
Library F
Figure imgf000071_0001
[0240] Preparation of 3a. Boohydrazine (1a) (50 g, 0.385 mmoi) was dissolved in acetone (500 mL) and the reaction mixture was stirred at 50 0C overnight. Then, the solvent was removed under reduced pressure, and the residue was re-suspended in Et2O and filtered. A white precipitate was obtained with quantity yield (64 g).
[0241] Preparation of 4a. (3a) (15 g, 0.087 mol) was dissolved in toϊuene (150 mL); KOH powder 14.6 g (0.267 mol), 2.105 g (0.0087 mol) BTEA-HOSO3 (10 mol %) and R-ethyl bromide 19.3 g (0.104 rnol) were added. The reaction mixture was stirred at 80 0C overnight. The solids were removed by filtration, and the filtrate was washed twice with the same volume of cold water. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Intermediate 4a contained about 20% elimination by-product, but was used in the next step without further purification.
[0242] Preparation of 5a. (4a) (5.8 g, 0.021 moi) was dissolved in THF (100 mL) and a 2 M aqueous solution of HCI (40 mL) was added. The reaction mixture was refluxed overnight. Thereafter, the solvent was removed under reduced pressure, and the product was dried by concentration from benzene. LCMS analysis of the reaction mixture demonstrated about 80 % of (5a). The precipitate was crystallized from ether to give the target compound 5a (3.0 g, 68 % of yield) as a hydrochloride salt (pale gray precipitate).
[0243] Preparation of (6) 0.523 g (0,0025 mol) of phenethyl hydrazine dihydrochloride was suspended in 60 mL of dry dioxane and 0.505 g (0,0050 mol) of TEA was added; then 0.5 g (0.0021 mol) of 2.6-dichloro-4-(1-pirrolidinyl) benzaldehyde (5) was added at once. The reaction mixture was stirred overnight at RT, at which time the solvent was removed in vacuo. The residue was purified by column chromatography with methylene chloride as eluent to provide a pale yellow precipitate (0.270 g) in a yield averaged 35-40 %.
[0244] Preparation of (7) 0.164 g (0.00050 mol) of 1 -phenethyl-4-chloro-6-(3-methyIphenyl)- 1 H-pyrazolo-[3,4d]pyrimidine (6) was dissolved in 5 mL of dry dioxane, and 0.054 g (0.00050 mol) of 3-methylaniline was added at once. The reaction mixture was stirred overnight at 70 0C. The reaction mixture was poured into water, then extracted with chloroform, concentrated under reduced pressure and purified by column chromatography with methylene chloride as an eluent to provide a white precipitate with yield 32 % (6.3 g).
Figure imgf000072_0001
Figure imgf000072_0002
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Analytical Data for Library F
40. N-{3-methylphenyl)-1-(2-phenylethyl)-6-pyrrolidin-1-yl-1H-pyrazolo[3,4-dIpyrimidtn-4- amine HNMR in DMSOd6 δ (ppm): 1.95 m (4H); 2.35 S (3H); 3.15 m (2H); 3.60 m (4H); 4.40 m (2H); 6.88 d (1 H); 7.10-7.32 m (6H); 7.64 d (1 H); 7.74 s (1 H); 7.95 s (1 H); 9.25 s (1H). 41. N-(4-{[1-(2-phenylethyl)-6-pyrrolidin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4- yi]amino}phenyl)-acetamide HNMR in DMSOd6 δ (ppm): 1.90 m (4H); 3.20 1 (2H); 3.55 m
(4H); 4.40 1 (2H); 6.96 d (1 H); 7.10-7.30 m (5H); 7,55 d (2H); 7.75 d (2H); 7.90 s (1 H); 9.25 s
(1 H); 9.55 s (1 H). 42. N-(4-bromophenyl)-1-(2-phenylethyl)-6-pyrroiidin-1-yI-1H-pyra2olo[3J4-d]pyrimidin-4- amine HNMR in DMSOd6 δ (ppm): 1.95 m (4H); 3.15 t (2H); 3.55 m (4H); 4.50 t (2H); 7.10-
7.30 m (5H); 7.50 d (2H); 7.85 d (2H); 8.10 s (1H); 9.90 S (1 H). 43. 4-<[1-{2-phenylelhyI)-β-pyrro!idin-1-yϊ-1H-pyrazolot3,4-d]pyrimidin-4-yl]amino}benzene- sulfonamide HNMR in DMSOd6 δ (ppm): 1.95 m (4H); 3.20 t (2H); 3.60 m (4H); 4.40 t (2H);
7.10-7.30 m (5H); 7.80 d (2H); 8.10 d (2H); 9.70 s (1 H). 44. N-{3-bromophenyl)-1-(2-phenylethyl)-6-pyrrolidJn-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4- amine HNMR in DMSOd6 δ (ppm): 1.95 m (4H); 3.151 (2H); 3.60 m (4H); 4.50 1 (2H); 7.10-
7.40 m (8H); 7.80 d (2H); 8.10 s (1 H); 8.40 s (1 H); 9.80 s (1 H). 45. 1-{2-phenylethyl)-6-pyrrolidin-1-yl-N-{3-(trifluoromethoxy)phenyt]-1H-pyrazolo[3J4- d]pyrimidϊn-4-amine HNMR in DMSO-de δ (ppm): 1.95 m (4H); 3.20 t (2H); 3.55 m (4H);
4.45 t (2H); 6.96 d (1 H); 7.12-7.28 m (5H); 7.45 1 (1 H); 7.80 d (1H); 8.08 s (1 H); 8.18 s (1 H);
9.60 s (1H). 46. 1-(2-phenylethyl)-6-pyrrolidin-1-yl-N-t4-{trifluoromethyl)phenyl]-1H-pyrazoio[3,4- d]pyrimrdin-4-amine HNMR in DMSOd6 δ (ppm): 1.95 m (4H); 3.20 1 (2H); 3.55 m (4H);
4.45 t (2H); 7.10-7.30 m (7H); 7.66 d (2H); 8.02 s (1 H); 8.14 d (2H); 9.65 S (1 H), 47. 1-(2-phenylethyl)-6-pyrrolidtn-1-yl-N-[4-(trϊfluoromethoxy)phenyll-1H-pyra2θIo[3I4- d]pyrimidin-4-amine HNMR in DMSOd6 δ (ppm): 1 ,95 m (4H); 3.20 t (2H); 3.55 m (4H);
4.55 1 (2H); 7.10-7.35 m (7H); 8.00 d (3H); 9.50 s (1H). 48. 1-(2-phenylethyl)-6-pyrroiidin-1-yi-N-[3-(1H-pyrrol-1-yi)pheny)]-1H-pyrazolo[3!4- d]pyrimidin-4-amine HNMR in DMSOd6 δ (ppm): 1.95 m (4H); 3.20 t (2H); 3.55 m (4H);
4.45 1 (2H); 6.30 m (2H); 7.10-7.30 m (8H); 7.40 t (1 H); 7.70 d (1 H); 8.10 s (1 H); 8.30 s (1 H);
9.65 s (1 H). 49. N"{4-morpho!in»4-ylphenyl}-1 -(2-phenyl-ethy!)-6-pyrrolidin-1-yl-1 H-pyrazolo[3,4- d]pyrimidin-4-amine HNMR in DMSO-de δ (ppm): 1.95 m (4H); 3.10-3.30 m (4H); 3.60 m
(3H); 3.85 m (3H); 4.50 1 (2H); 7.00-7.30 m (5H); 7.80 d (2H); 10.1 s (1 H). 50. 1 "(2-piperidin-1 -ylethyi}-6-pyrrolidϊn-1 -yl-N-[3-(trifluoromethoxy)pheny I]-IH- pyrazolo[3,4-d]pyrimidin-4-amine HNMR in DMSOd6 δ (ppm): 1.57 (s, 2H); 1.79 (d, J=5.9,
4H); 1.99 (m, 4H); 3.30 (m, 2H); 3.58 (m, 6H); 4.61 (t, J=6.3, 2H); 6.99 (m, 1 H); 7.45 (t, J=8.2,
1 H); 7.80 (m, 1 H); 8.19 (s, 2H); 9.83 (s, 1H). 51. N-benzyI-1-{2-phenyiethyl)-6-pyrrolidin-1-yl-1H-pyrazoIoJ3t4-d]pyrimidin-4-amine HNMR in DMSOd6 δ (ppm): 1.90 m (4H); 3.15 t (2H); 3.50 m (4H); 4.35 t (2H); 4.70 d (2H); 7.10-
7.44 m (10H); 7.75-7.95 m (2H). 52. N-tS-methyibenzylϊ-i-tZ-phenylethylJ-e-pyrroIidin-i-yl-IH-pyrazoloEa^-dlpyrimidin-^ amine HNMR in DMSO-(J6 S (ppm): 1.90 m (4H); 2.30 s (1 H); 3.15 t (2H); 3.50 rn (4H); 4,35 1 (2H); 4.65 d (2H); 7.00-7.30 m (9H); 7.80 m (2H).
53. N-{4-methy!benzyI)-1-{2-phenylethyl)-6-pyrrolidin-1-yI-1H-pyrazo!o[3,4-d3pyrimidin-4- amine HNMR in DMSO-Cf6 δ (ppm): 1.90 m (4H); 2.3 s (3H); 3.151 (2H); 3.55 m (4H); 4.30 t (2H); 4.60 d (2H); 7.05-7.30 m (9H); 7.75-7.95 m (2H).
54. 1-(2-pheny)ethyl)-6-pyrrolidin-1-yl-N-[2-(trifluoromethyl)benzyl]-1H-pyrazo)o[3,4- d]pyrimidin-4-amine HNMR in DMSO-d6 δ (ppm): 1.85 m (4H); 3.15 t (2H); 3.45 m (4H);
4.40 t (2H); 4.90 d (2H); 7.10-7.30 m (5H); 7.45 1 (1H); 7.55-7.75 m (3H); 7.85-8.00 m (2H). 55. 1-{2-phenylethyl)-6-pyrrolidin-1-yl-N-t3-(trifluoromethyl)benzyI]-1H-pyrazolo[3,4- d]pyrimidin-4-amine HNMR in DMSO-d6 δ (ppm): 1.90 m (4H); 3.15 t (2H); 3.50 m (4H);
4.40 t (2H); 4.75 d (2H); 7.10-7.30 m (5H); 7.54 m (2H); 7.65-7.90 m (3H); 8.04 m (1 H). 56. 1-(2-phenylethyl)-6-pyrrolidin-1-yl-N-[4-{trifluoromethyl)benzyI]-1H-pyrazolo{3,4- d]pyrimidin-4-amine HNMR in DMSO-d6 δ (ppm): 1.85 m (4H); 3.20 t (2H); 3.50 m (4H);
4.35 1 (2H); 4.75 d (2H); 7.05-7.30 m (5H); 7.55-7.75 m (4H); 7.85 s (1 H); 8.05 m (1 H). 57. 1-{2-phenytethyl)-6-pyrroiidin-1-yl-N-(quinolin-5-yImethyl)-1H-pyrazolot3,4-d]pyrimidin-
4-amϊne HNMR in DMSO-d6 δ (ppm): 1.89 (m, J1=3.7, J2=3.4, 4H); 3.15 (t, J=7.2, 2H); 3.52
(m, J1=3.7, J2=3.4, 4H); 4.36 (t, J=7.2, 2H); 5.15 (d, J=5.5, 2H); 7.10-7.40 (m, 5H); 7.52 (m,
1 H); 7.70 (m, 2H); 7.80 (s, 1 H); 7.95 (m, 2H); 8.70 (d, J=8.9, 1 H); 8.90 (d, J=4.4, 1 H). 58. N-(isoquinoIin"5-ylmethyl)-1-(2-phenylethyl)-6-pyrroltdin-1-yl-1H-pyrazolo[3,4- d]pyrimidϊπ-4-amine HNMR in DMSO-de 6 (ppm): 1.85 m (4H); 3.15 t (2H); 3.50 m (4H);
4.35 1 (2H); 5.15 d (2H); 7.10-7.30 m (5H); 7.64 1 (1 H); 7.82 1 (2H); 7.88-8.16 m (3H); 8.52 d
(1 H). 59, 1-{2-phenylethyl)-6-pyrrolidin-1-yl-N-{quinolin-8-yImethyl)-1H-pyrazolo[3,4-d3pyrimidin-
4-amine HNMR in DMSO-d6 δ (ppm): 1.85 m (4H); 3.20 t (2H); 3.45 m (4H); 4.35 1 (2H);
5.40 d (2H); 7.10-7.30 m (5H); 7.55 m (2H); 7.70-7.90 m (4H); 8.30 d (2H); 9.00 d (1H).
60. N-{4-methoxybenzyl)-1-(2-phenylethyi)-6-pyrrotidin-1-yl-1H-pyrazo[o[3,4-d]pyrimidin-4- amine HNMR in DMSO-d6 δ (ppm): 1.90 m (4H); 3.15 1 (2H); 3.55 m (4H); 3.75 s (3H); 4.40 1 (2H); 4.65 d (2H); 6.80 d (2H); 7.10-7.40 m (6H); 7.70-7.85 m (2H).
61. N-(3-methoxybenzyl)-1-(2-phenyIethyl)-6-pyrrolidin-1-yI-1H-pyrazolo[3,4-d]pyrimidin-4- amine HNMR in DMSO-de δ (ppm): 1.90 m (4H); 3.15 t (2H); 3.55 m (4H); 3.75 s (3H); 4.35 1 (2H); 4.65 d (2H); 6.80 d (1H); 7.00 s (1 H); 7.10-7.28 m (6H); 7.80 m (2H).
62. N-(2!5-dimethoxybenzyl)-l-(2-phenylethyi)-6»pyrrolidin-1-yl-1H-pyrazoIo[3I4- d3pyrimidin-4-amine HNMR in DMSO-d6 δ (ppm): 1.85 m (4H); 3.15 m (2H); 3.55 m (4H); 3.60-3.95 d (6H); 4.35 m (2H); 4.65 d (2H); 6.75-7.05 m (3H); 7.10-7.40 m (5H); 7.60-8.00 m (2H). 63. i^-phenylethyiJ-N-tpyridin-S-ylmethylJ-e-pyrrolidin-i-yl-IH-pyrazolotSjΦdlpyrimϊdin^- amine HNMR in DMSO-d6 δ (ppm): 1.90 m (4H); 3.15 t (2H); 3.50 m (4H); 4.35 1 (2H); 4.65 d (2H); 7.10-7.40 m (6H); 7.80 m (3H); 7.95 t (1 H); 8.45 d (1 H); 8.60 s (1H).
64. 1-{2-phenylethyl)-N-(pyridin-4-ylmethyl)-6-pyrrolidin-1-yl-1H-pyrazolo[3J4-d]pyrimidin-4- amine HNMR in DMSOd6 δ (ppm): 1.85 m (4H); 3.15 t (2H); 3.45 m (4H); 4.35 1 (2H); 4.70 d (2H); 7.10-7.35 m (7H); 7.80 s (1H); 7.95 t (1 H); 8.50 d (1 H).
65. 4-(1,3-dihydro-2H-isoindoI-2-yi)-1-(2-phenylethyl)-6-pyrroiιdin-1-yl-1H-pyra2θlo[3,4- d]pyrimidine HNMR in DMSO-Cl6 δ (ppm): 1.94 (m, 4H); 3.18 (t, J=7.1 , 2H); 3.59 (m, 4H); 4.41 (t, J=7.6, 2H); 5.04 (s, 4H); 7.12-7.30 (m, 5H); 7.34 <m, 2H); 7.45 (m, 2H); 7.95 (s, 1 H).
66. N-(3-fluorobenzyl)-1-(2-phenylethyl)-6-pyrrolrdtn-1-yl-1H-pyrazolo{3,4-d]pyrimidin-4- amine HNMR in DMSO-de δ (ppm): 1.85 m (4H); 3.15 t (2H); 3.50 m (4H); 4.35 1 (2H); 4.7O d (2H); 7.00 t (1 H); 7.10-7.30 m (7H); 7.40 t (1 H); 7.80 s (1 H); 7.95 t (1 H).
67. N-{2-fluorobenzyl)-1-{2-phenyiethyl)-6-pyrrolidin-1-yl-1H-pyrazolo[3,4-d3pyrtmidin-4- amine HNMR in DMSCKl6 δ (ppm): 1.85 m (4H); 3.15 t (2H); 3.50 m (4H); 4.35 t (2H); 4.70 d (2H); 7.05-7.35 m (7H); 7.45 t (1 H); 7.75-7.90 m (2H).
68. N-(4-fluorobenzyl)-1-(2-phenylethyl}-6-pyrro!ϊdin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4- amine HNMR in DMSOd6 δ (ppm): 1.90 m (4H); 3.15 t (2H); 3.50 m (4H); 4.35 1 (2H); 4.65 d (2H); 7.00-7.30 m (6H); 7.45 1 (2H); 7.75-7.95 m (2H).
69. 4-chioro-1-(2-phenylethyl)-6-pyrrolidin-1-yi-1H-pyrazolo[3>4-d]pyrimidine HNMR 1H, DMSOd6 δ, ppm: 1.95 m (4H); 3.15 t (2H); 3.55 m (4H); 4.45 t (2H); 7.10-7.26 m (5H); 7.90 s (1 H).
70. 4-chloro-1-(2-piperidin-1-ylethyi)-6-pyrrotidin-1-yl-1H-pyrazolo[3,4-d]pyrimidine HNMR in DMSOd6 δ (ppm): 1.42 (m, 6H); 2.51 (m, 4H); 2,76 (t, J=6.6; 2HO; 2.95 (s, 4H); 3.58 (t, J=6.7, 4H); 4.31 (t, J=6.7, 2H); 7.90 (s, 1 H).
71. 4-chloro-6-pyrrolidin-1-yl-1H-pyrazolo(3,4-d]pyrimidine HNMR in DMSOd6 δ (ppm): 1.96 (m, 4H); 3.56 (m, 4H); 7.89 (s, 1 H).
72. propyl-6-pyrrolidin-1-yI-N-[2-(trifluoromethyl)benzyl]-1H-pyrazolo[3,4-d]pyrimidin-4- amine HNMR in DMSO-d6 δ (ppm): 0.85 t (3H), HNMR 1H, DMSOd6 δ, ppm: 1.80 m (2H), 3.05 t (4H), 3.45 1 (4H), 4.1O t (2H), 4.90 d (2H), 7.60 m (4H), 7.90 m (2H)
73. N-{4-fIuorobenzyl)-1-propy)-6-pyrrolfdin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4-amine HNMR in DMSOd6 δ (ppm): 0.60 t (3H); 1.75-2.00 m (6H); 3.50 m (4H); 4.05 t (2H); 4.70 d (2H); 7.10 1 (2H); 7.45 1 (2H); 7.75-7.95 m (2H).
74. N-{2-methylbenzyl)-1-propyl-6-pyrrolidrn-1-yl-1H-pyrazoIo[3,4-d]pyrimidin-4-aniine HNMR in DMSOd6 δ (ppm): 0.84 t (3H, CH3), HNMR 1H, DMSOd6 δ, ppm: 1.82 q (2H1 CH2), 1.88 m (4H, 2CH2), 2.36 s (3H, CH3), 3.52 m (4H, 2CH2), 4.06 1 (2H, CH2), 4.68 d (2H, CH2), 7.14 t (3H, Ar), 7.34 t (1 H, Ar), 7.66 s (1 H, Ar), 7.82 s (1 H, Ar). 75. N-phenyl-1-propyl-6-pyrrolidm-1-yI-1H-pyrazoio[3,4-d]pyrimidin-4-amine 90 oC: HNMR in DMSO-d6 δ (ppm): 0.82 1 (3H, CH3), 1.80 m (2H, CH2), 1.92 t (4H, 2CH2), 3.56 m (4H, 2CH2), 4.1O t (2H, CH2), 7.02 t (1 H1 Ar), 7.34 1 {2H, Ar), 7.92 d (2H, Ar), 8.02 s <1H, Ar), 9.58 s (1 H, Ar),
Library K
Figure imgf000079_0001
[0245] Preparation of 1. 50 g (0,658 mol) of thiourea was suspended in dry ethanol (100 mL), and then 78 g (0.724 mol) of ethyibromide was added at once. The reaction mixture was stirred at 500C until the starting material was totally dissolved. Then the reaction mixture was stirred at this temperature for 1 h more and the solvent was removed under reduced pressure and compound 1 was obtained in quantitative yield.
[0246] Preparation of 3. Ethyl isothiourea 1 (40.0 g, 0.216 mol) was dissolved in water (200 mL) and morpholine 2 (22.3 g, 0.258 mol) was added at once. The reaction mixture was refluxed overnight, then the reaction mixture was cooled, the solvent was removed in vacuo and compound 3 was crystallized from ether and isolated as hydrobromide in quantitative yield. [0247J Preparation of 4, 20.0 g (0.870 mol) of sodium was dissolved carefully in 1.2 L of dry ethanol, and this solution was cooled to 40-45 0C and 41.0 g (0.256 mol) of diethylmalonate was added dropwise. Next, 40.0 g (0.206 mol) 1-morpholine-carboximidamide hydro bromide 3 was added in portions to the reaction mixture. The reaction mixture was refluxed overnight, and the resulting white solid was filtered and dissolved in water. The solution was acidified with acetic acid to pH 5-6. The white precipitate was filtered, washed with ether, and lyophilized. [0248] Preparation of 5. 10 mL of dry DMF was cooled to 0-5 0C, and 25 mL of POCI3 were added dropwise. This mixture was allowed to react at 20 0C for 1 h at which time 5 g of 2-{1- pyrrolidtnyl)-4,6-pyrimidinediol 4 was added in portions. After 30 min, the reaction was heated on a steam bath overnight. The excess phosphoryl chloride was removed in vacuo and the residue was treated with ice water. The crystalline product was collected by filtration and lyophilized. [0249] Preparation of 6. 0.365 g (0.0018 mol) of phenethyl hydrazine dϊhydrαchioride was suspended in 60 mL of dry dioxane and 0.295 g (0.0029 mol) of TEA was added; then 0.5 g (0.0015 mol) of 2.6-dichioro-4-(1-pirrolidinil) benzaldehyde 5 was added at once. The reaction mixture was stirred overnight at RT, at which time the solvent was removed in vacuo. Residue was purified by column chromatography with methylene chloride as eluent. The yield averaged 45-60%.
[0250] Preparation of 7, 0.100 g (0.00029 mol) of compound 6 was suspended in 5 mL of water, then (0.00058 mol) of aniline and 0.1 mL of concentrated HCi were added. The reaction mixture was refluxed for 48 h. LCMS analysis of the reaction mixtures demonstrated the 30-40 % conversion of starting material. An additional amount of aniline, HCI and 3 mL of dry dioxane were added in order to increase the solubility and the reaction mixtures were allowed to react for 12 h, more at 80 0C. At this time LCMS analysis of the reaction mixtures demonstrated total conversion of the starting material into target compound. The reaction mixture was poured into water and extracted with chloroform. The solvent was removed in vacuo and the residue was crystallized from ether.
Figure imgf000080_0001
Table 7. Examples 76-83 of Pyrazoiopyrimidines. Library K
Figure imgf000080_0002
Figure imgf000081_0001
Analytical Data for Library K
76. 6-morpholiπ-4-yl-N-phenyl-1-{2-phenylethyl)-1H-pyrazoio[3,4-d]pyrimidin-4-amϊne
HNMR 1H, DMSOd6 δ, ppm: 3.20 t (2H); 3.60-3.70 m (8H); 4.40 1 (2H); 7.05 1 (1 H); 7.10- 7.30 m (5H); 7.35 t (2H); 7.70 d (2H); 7,90 s (1H); 9.25 s (1 H).
77. N-(2-methylbenzyi)-6-morpholin-4-yl-1 -(2-phenylethyl)-1 H-pyrazolo[3,4-d]pyrimidin-4- amine HNMR 1H, DMSOd6 6, ppm: 2.30 s (3H); 3.15 t (2H); 3.60-3.80 m (8H); 4.35 1 (2H); 4.70 d (2H); 7.05-7.40 m (5H); 7,75-7.90 m (2H).
78. N-{3,5-dimethylphenyl)~6-morphoIi n-4-yl-1 -(2-phenylethyl)-1 H-pyrazolo[3,4- d]pyrimidin-4-amineb HNMR 1H, DMSO-d6 δ, ppm: 2.30 s (6H); 3.15 1 (4H); 3.60-3.80 m (8H); 4,40 t (2H); 6.70 s (1 H); 7.10-7.30 m (5H); 7.40 s (2H); 7.95 s (2H); 9.25 s (1 H).
79. N-(4-methylphenyl)-6-morphoiin-4-yi-1-(2-phenylethyl)-1H-pyra2Olo[3,4-d]pyrimidin-4- amine HNMR 1H, DMSOd6 δ, ppm: 2.30 s (3H); 3.15 t (2H); 3.60-3.80 m (8H); 4.40 t (2H); 7.10-7.30 m (7H); 7.60 d (2H); 7.90 s (1 H); 9.30 s (1 H).
80. N-(3-methylphenyl)-6-morpholin-4-yl-1-<2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4- amine HNMR 1H, DMSOd6 δ, ppm: 2.30 s (3H); 3.151 (2H); 3.60-3.80 m (8H); 4.40 1 (2H); 6.90 d (1 H); 7.10-7.30 m (6H); 7.50 d (1H); 7.60 s (1 H); 7.95 s (1 H); 9.30 s (1 H). 81. N-{3-methoxyphenyl)-6-morpholin-4-yl-1-(2-phenylethyl)-1H-pyrazoio[3,4-d]pyrimidin-
4-amine HNMR 1H, DMSO-Cl6 δ, ppm: 3.20 m (2H); 3.60-3.80 m (8H); 4.45 1 (2H); 6.70 d (1 H); 7,10-7.30 m (6H); 7.45 s (1 H); 8.00 s (1H); 9.45 s (1 H).
82. N~(4-methoxyphenyϊ)-6-morpholin-4-yl-1-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidin- 4-amine HNMR 1H, DMSOd6 δ, ppm: 3.15 t (2H); 3.60-3.80 m (8H); 3.85 s (3H); 4.45 1 (2H); 6.90 d (1H); 7.10-7.30 m (5H); 7.60 d (2H); 7.80 s (1 H); 9.30 s (1 H).
83. N-(3-ethyIphenyl)-6-morphoiin-4-yl-1 -(2-phenylethyl)-1 H-pyrazolo[3,4-d]pyπmidin-4- amine HNMR 1H, DMSOd6 δ, ppm: 1.25 1 (3H); 2.65 m (2H); 3.20 t (2H); 3.60-3.80 m (8H); 4.451 (2H); 6.95 d (1H); 7.10-7.30 m (6H); 7.50 d (2H); 7.65 S (1 H); 7.95 s (1H); 9.30 s (1 H).
Library N (derived from Library F)
Figure imgf000082_0001
Alternative Approach Il
Figure imgf000082_0002
Synthesis of Building Blocks
Figure imgf000082_0003
boc boc
1a 2a 3a 4a
Figure imgf000083_0001
|0251| Preparation of propyl-3-{[1-methyl-6-[1-pyrrolidinyl]-1H-pyrazolo-[3,4-d]- pyrimϊdm-4-yl] amino}-benzoate {7) 0.0013 mol of 4-chloro-1-propyl-6-(1-pyrroiidinyi)-1 H- pyrazoIo-[3,4-d]-pyrimidine (6) was suspended in 5 mL of dry dioxane, followed by 0.382 g (0.0026 mol) of methyl-3-aminobenzoaie and 20 mg of CuL The reaction mixture was stirred for 2 h at 120 0C in a microwave oven (800 watts). LCMS analysis of the reaction mixture demonstrated total conversion of starting materiat. The precipitate was filtered, carefully washed with ether and fyophϋized. A white precipitate was obtained with yield 98 % (0.450 g). [0252] Preparation of 3-{[1- propyi-6-[1-pyrroiidinyl]-1 H-pyrazolo~[3,4-d]-pyrimidin-4- yljaminø} benzoic acid (8) 0,0013 mol of (7) was suspended in 10 mL of ethanol and 10 mL of water, and 0.107 g (0.0019 mol) of potassium hydroxide was added at once. The reaction mixture was stirred for 15 min at RT at which time it was heated to 70 0C. At this temperature total consumption of starting material was observed (reaction was monitored by TLC). The reaction mixture was cooled to RT, the precipitate was filtered, and the residue was concentrated under reduced pressure. The key intermediate (8) was obtained as a white precipitate, it was crystallized from ether and dried giving 8 with quantitative yield (0.430 g). [0253J Preparation of 3-{[1-propyI-6-[1-pyrrolidinyi]-1H-pyrazofo-[3,4-d]-pyrimidin-4- yl]amino}-benzamides (9) 0.00029 mol of (8) was dissolved in 5 mL of dry dioxane, then 0.00036 mol of amine and 0.040 g (0,00038 mol) of TEA were added at once. The reaction mixture was stirred for about 3-5 min at RT, then 0.072 g (0.00047 mol) of POCI3 was added. The reaction mixture was stirred overnight at 50-600C. LCMS analysis of the reaction mixture demonstrated the presence of starting material only in the case of primary amine, and about 70% of target compound in cases of secondary amine and substituted piperazine. The target compounds were purified by column chromatography with methylene chloride as eluent giving white precipitates in yields of 10-55 %.
[0254J Preparation of 3a. 2.0 g (0.01 mol) of 1a was dissolved in 100 ml of dry dioxane, then 1.01 g (0.01 mol) of TEA was added. Reaction mixture stirred at RT for about 15 min, after this 2.12 g (0.012 mol) of 2a was added at once. Reaction mixture was stirred overnight at 8OO. Reaction was poured into aqueous solution of sodium carbonate; precipitate was filtered off and lyophilized. Yield was 84 % (2.840 g). [0255] Preparation of 4a 1 g (0.0029 mol) of compound 2 was suspended in 50 ml of dioxane with HCI. Reaction mixture stirred overnight at RT. LCMS of reaction mixture demonstrated total conversion of starting material; precipitate was filtered, washed with ether and lyophilized. Yield was 80 % (0.710 g). Compound 3 was obtained as hydrochloride. [0256] Preparation of 3b. 2.0 g (0.011 moi) of 1 b was dissolved in 100 ml of dry THF, then 1.81 g (0.013 mol) of 2b and 1.958 g (0.013 mol) of DBU were added at once. Reaction mixture was stirred overnight at 100 C. Reaction was poured into aqueous solution of sodium carbonate, precipitate was filtered off and ϊyophiiized. Yield was 76 % (2.630 g). [0257] Preparation of 3c. 2.0 g (0.011 mol) of 1c was dissolved in 100 ml of dry dioxane, and to it was added 1.111 g (0.011 mol) of TEA. After 2.30 g (0.013 mol) of 2c was added at once, the reaction mixture stirred at RT for about 15 min and then stirred overnight at 80° C. The mixture was poured into aqueous solution of sodium carbonate, the precipitate was filtered off and lyophilized to provide the desired compound (3.25 g, 91% of yield).
[0258] Preparation of 4b,c 1 g (0.0031 mol, 0.0034) of compound 2 was suspended in 50 ml of dioxane with HCi. Reaction mixture stirred overnight at RT. LCMS of reaction mixtures demonstrated total conversion of starting material; precipitates were filtered, washed with ether and lyophilized. Yields were 79, 69 % (0.700 g, 0.637 g). Compounds 4 were obtained as hydrochlorides.
[0259] Preparation of Compound 11 : Compound 10 (0.01 mol) was treated with CDI (0.01 mol) in 100 ml of DMF for 2 hours, then an amine was added. After 7 hours of stirring, the solvent was removed and the desired compound was obtained.
[0260] Preparation of Compound 12. To a suspension of 6 (0.001 mol) in 5 ml of water were added (0,001 mol) of aniline and 0.1 ml of concentrated HCI. After the mixture was refluxed for 48 h, it was cooled; and the formed precipitate was filtered off, washed with water and iyophilized to provide the desired products.
Figure imgf000084_0001
Table 8, Examples 84-104 of Pyrazoiopyrimidines. Library N.
Figure imgf000084_0002
Figure imgf000085_0001
N,N-diethyl-2-[4-(1- propyl-6-pyrrolidin-1 -yl-
C22H36N8O / 1H-pyrazolo[3,4-
95 429,5 428.6 d]pyrimidin-4- yl)piperazin-1- yljacetamide
N-[1-
(phenylsulfonyi)piperidin-
C23H31 N7O2 4-yl]-1-propyl-6-
96 470.6 S / 469.6 pyrrolidin-1-yl-1 H- pyrazolo[3,4-d]pyrimidin-
4-amine
1-propyl-6-pyrrolidSn-1 -yl-
N-{1-{[4-(pyrrolidin-1-
C28H38N8O3 ylcarboπy!)pheny!3sulfony
97 567.7 S / 566.7 i}piperidin-4-yi)-1 H- pyrazolo[3 ,4-d]py rim id in-
4-amine
4-(4-benzoylpiperazin-1 -
C23H29N7O /
98 yl)-1 -propyl-6-pyrrolidin-
420.5 419.5 1-yl-1 H-pyrazolo[3,4- d]pyrimidine
4-[4-(phenylsulfonyl)-
C22H29N7O2 piperazin-1 -yl]-1 -propyi-
99 456.5
S / 455.6 6-pyrro[idiπ-1-y!-1 H- pyrazolo[3,4-d]pyrimidine
N-{4-[(4-phenylpiperazin- 1-yl)carbonyl]pheny!}-1 -
C29H34N8O /
100 511.6 propyl-6-pyrroIidin-1 -yl- 510.6 1 H-pyrazolo[3,4- d]pyrimidin-4-amtne
1-methyI-N-{3-[(4- phenylpiperazin-1-
C27H30N8O / yl)carbonyl]phenyl}-6-
101 483.5 482.6 pyrroltdin-1 -y!-1 H- pyrazolo[3,4-d]pyrimidin-
4-amine
N-{3-[(4-m ethy Ip i pe razi n- x v^v 1 -yl)carbonyl]phenyl}-1 -
C24H32N8O /
102 449.5 propyl-6~pyrroiidin-1 -yl- 448.6 1 H-pyrazolo[3,4- d]pyrimidin-4-amine
1 -propyi-6-pyrrolidtn-1 -yl- N-[3-(pyrroifdin-1-
C23H29N7O / 03 420.5 ylcarbonyl)phenyl]-1 H- 419.5 pyrazoio[3,4-d]pyrimidin- 4-amtne
Figure imgf000087_0001
Analytical Data for Library N
84. N-{4-bromophenyi)-1-propyl-6-pyrrolidin-1-yI-1H-pyrazolo[3,4-d]pyrimiclin-4-amine
HNMR 1H, DMSOd6 δ, ppm: 0.85 (t, J= 7.4 Hz, 3H), 1.83 (m, 2H), 1.94 (m, 4H), 3.56 (m, 4H), 4.11 (t, J= 7.0 Hz, 2H), 7.50 (d, J= 8.9, 2H), 7.89 (d, J= 8.6 Hz, 2H), 8.00 (s, 1H), 9.53 (s, 1 H).
85. N-(3-bromophenyl)-1-propyi-6-pyrrolidin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4-amine HNMR 1H, DMSOd6 8, ppm: 0.85 (t, J= 7.4 Hz, 3H), 1.83 (m, 2H)1 1.96 (m, 4H), 3.58 (m, 4H), 4.12 (t, J= 6.8 Hz, 2H), 7.18 (d, J= 7.8 Hz, 1 H), 7.29 (t, J= 8.1 Hz, 1 H), 7.78 (d, J= 8.0 Hz, 1 H), 8.02 (s, 1 H), 8.46 (s, 1 H), 9.58 (s, 1 H),
86. propyl-6-pyrrolidin-1-yl-N-[4-{trifluoromethoxy)phenyl3-1H-pyrazolo[3,4-d]pyrimidin-4- amine HNMR 1H, DMSOd6 6, ppm: 0.88 t (3H), 1.90 m (6H), 3.60 t (4H), 4.16 1 (2H), 7.28 d (2H), 7.98 m (3H), 9.44 m (1 H).
87. N-(4-morpholin-4-ylphenyI)-1-propyl-6-pyrrolidin-1-yl-1H-pyrazofo[3,4-d]pyrϊmidin-4- amine HNMR 1H, DMSOd6 δ, ppm: 0.84 (t, J= 7.3 Hz, 3H), 1.82 (m, 2H)1 1.92 (m, 4H)1 3.55 (m, 4H), 3.74 (m, 4H)1 4.09 (t, J= 6.7 Hz, 2H), 6.93 (d, J= 8.5 Hz, 2H), 7.72 (d, J= 8.2 Hz, 2H), 7.84 (s, 1 H), 9.19 (S1 1 H).
88. N-benzyl-1-propyl-6-pyrrolidin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4-amine HNMR 1H, DMSO-dβ δ, pprn: 0.86 (t, J= 7.4, 3H), 1.82 (m, 2H), 1.88 (m, 4H), 3.52 (m, 4H), 4.07 (t, J= 6.9 Hz, 2H), 4.68 (d, J= 5.9 Hz, 2H), 7.22 (t, J= 7.1 Hz1 1 H), 7.31 (t, J= 7.2 Hz, 2H), 7.39 (d, J= 7.3 Hz1 2H), 7.81 (m, 2H).
89. propyl~6-pyrroIidin-1-yl-N-[3-{trifluoromethyl)benzyf]-1H-pyrazolo[3,4-d]pyrimidin-4- amine HNMR 1H, DMSOd6 δ, ppm: 0.83 (t, J= 7.3 Hz, 3H), 1.80 (m, 2H), 1.87 (m, 4H), 3.47 (m, 4H), 4.05 (t, J= 6.9 Hz, 2H), 4.72 (d, J= 6.0 Hz, 2H)1 7.56 (m, 2H), 7.68 (m, 1H), 7.74 (s, 1H), 7.80 (s, 1 H), 8.17 (m, 1 H).
90. N-(4-methyibenzyl)-1-propyl-6-pyrrolidin-1-yi-1H-pyrazolo[3,4-d]pyrimidin-4-amine HNMR 1H, DMSO-d6 δ, ppm: 0.86 (t, J= 7.2 Hz, 3H), 1.80 <m, 2H), 1.87 (m, 4H), 2.27 (s, 3H), 3.50 (m, 4H), 4.04 (t, J= 6.5 Hz, 2H), 4.62 (d, J= 5.2 Hz, 2H), 7.12 (d, J= 7.0 Hz, 2H), 7.26 (d, J= 7.0 Hz, 2H), 7.79 (s, 1 H), 7.97 (m, 1 H).
91. N-(3-methylbenzyl)-l-propyl-6-pyrrotidin-1-yl-1H-pyrazolo{3,4-d]pyrimidin-4-amine HNMR 1H, DMSOde δ, ppm: 0.86 (t, J= 7.4 Hz, 3H), 1.82 (m, 2H), 1.88 (m, 4H), 2,29 (s, 3H), 3.52 (m, 4H), 4.07 (t, J= 6.9 Hz, 2H), 4.65 (d, J= 5.8 Hz, 2H), 7.04 (m, 1 H), 7.19 (m, 3H), 7.80 (s, 2H). 92. N^isoquinolin-S-ylmethylJ-i-propyl-e-pyrrolidin-i-yl-IH-pyrazolop^-dlpyrimidϊn-Φ amine HNMR 1H, DMSOd6 δ, ppm: 0.83 (t, J= 7.3 Hz1 3H)1 1.80 (m, 2H), 1.87 (m, 4H), 3.49 (m, 4H), 4.05 (t, J= 6.8 Hz, 2H), 5.13 (d, J= 5.6 Hz, 2H), 7.64 (t, J= 7.6 Hz, 1 H), 7.81 (m, 2H), 8.03 (d, J= 8.0 Hz, 1 H), 8.10 (m, 2H), 8.53 (d, J= 5.9 Hz, 1 H), 9.31 (s, 1 H).
93. N-{4-methoxybenzyl}-1-propyl-6-pyrroIidin-1-yI-1H-pyrazolot3,4-d]pyrimidin-4-amine HNMR 1H, DMSOd6 δ, ppm: 0.83 (t, J= 7.1 Hz, 3H), 1.79 (m, 2H), 1.88 (m, 4H), 3.50 (m, 4H), 3.73 (s, 3H), 4.05 (t, J= 6.8 Hz, 2H), 4.59 (d, J= 5.2 Hz, 2H), 6.87 (d, J= 7.5 Hz, 2H), 7.30 (d, J= 7.5 Hz, 2H), 7.79 (s, 1 H), 7.95 (m, 1 H).
94. N-{3-methoxybenzy!)-1-propyI-6-pyrroIidin-1-yI-1H-pyrazolo[3,4-d]pyrimidin-4-amine HNMR 1H, DMSOd6 δ, ppm: 0.83 (t, J= 7.4 Hz, 3H)1 1.79 (m, 2H), 1.87 <m, 4H), 3.50 (m, 4H), 3.73 (S1 3H), 4.06 <t, J= 6.9 Hz, 2H), 4.64 (d, J= 5.9 Hz, 2H), 6.80 (m, 1 H)1 6.96 (m, 2H), 7.22 (t, J= 8.1 Hz, 1 H), 7.81 (S, 1 H), 8.02 (m, 1 H).
95. N,N-diethyϊ-2-[4~(1-propyI-6-pyrrotidin-1-yl-1H-pyrazolo{3>4-d]pyrimidϊn-4-yl)piperazin- 1-yl]acetamide HNMR 1H, DMSOd6 δ, ppm: 0.83 (t, J=7.4, 3H); 1.00-1.15 (m, 6H); 1.80 <q, J1=7, J2=7.2, 2H); 1.89 (m, 4H); 2.58 (m, 4H); 3.19 (s, 2H); 3.20-3.45 (m, 4H); 3.50 {m, 4H); 3.83 (m, 4H); 4.10 (t, J=6.9, 2H); 7.90 (s, 1 H).
96. N-t1-(phenylsulfonyi)piperidin-4-yi]-1-propyl-6-pyrrolidin-1-yl-1H-pyrazolot3,4- d]pyrimidin-4-amine HNMR 1H, DMSOd6 δ, ppm: 0.85 t (3H); 1.65 m (2H); 1.75-2.00 m (6H); 2.05 d (2H); 2.65 t (2H); 3.50 m (4H); 3.65 d (2H); 4.00 t (3H); 7.15 d (1 H); 7.60-7.84 m (6H).
97. i-propyl-β-pyrrolidin-i-yi-N-ti-l^fpyrroIidϊn-i-ylcarbonyOphenyllsulfoiiyilpiperidin^- yl)-1H-pyrazoloE3,4-d]pyrimidin-4-amine HNMR 1H, DMSOd6 δ, ppm: 0.85 t (3H); 1.65 m (2H); 1.75-2.00 m (6H); 2.10 d (2H); 2.75 t (2H); 3.35-3.60 m (8H); 3.70 d (2H); 4.05 1 (3H); 7.15 d (1 H); 7.65-7.90 m (5H).
98. 4-{4-benzoylpiperazin-1-yI)-1-propyl-6-pyrrolidin-1-yl-1H-pyrazolo[3f4-d]pyrimidme HNMR 1H, DMSOd6 δ, ppm: 0.85 1 (3H); 1.80-2.00 m (6H); 3.45-3.70 m (8H); 3.95 m (4H); 4.15 1 (2H); 7.45 m (5H); 7.90 m (1 H).
99. 4-[4-{phenylsuIfonyl)-piperazin-1-yl]-1-propyl-6-pyrroIidtn-1-yl-1H-pyrazolo[3,4- d]pyrimidϊne 0.851 (3H); 1.75-2.00 m (6H); 3.20 1 (4H); 3.50 m (4H); 3.85-4.15 m (6H); 7.55-7.95 m (6H).
100. N^-K^-phenylptperazin-i-yOcarbonylJpheny^-i-propyl-e-pyrroIidin-i-yl-IH- pyrazolo[3,4-dlpyrimidin-4-amine HNMR 1H, DMSO-dg δ, ppm: 0.91 (t, J=7.3 Hz1 3H); 1.90 (m, 6H); 3.24(m, 4H); 3.65 (m, 8H); 4.05 (t, J=6.9Hz, 2H); 6.80(bt, 1 H); 6.96 (d, J=8.1 Hz. 2H); 7.20 (bt, 2H); 7.46 (d, J=8.1 Hz, 2H); 8.00 (m, 3H); 9.45 (bs, 1 H).
101. methyl-N-{3-t(4-phenylpiperazin-1-yl)carbonyl]phenyl}-6-pyrrolidin-1-yl-1H- pyrazolo[3,4-d]pyrimϊdin-4-amϊne HNMR 1H, DMSOd6 δ, ppm: 1.85 m (4H); 3.20 1 (2H); 3.50-3,90 m {11 H); 6.80 t (1 H); 6.94 c! (2H); 7.08 d (1 H); 7.24 1 (2H); 7.42 t (1 H); 7.90-8.04 m (2H); 8.12 s (1 H).
102. N-{3-[{4-methylpiperazin-1-yi)carbonyl]phenyI}-1-propyl-6-pyrrolitJin-1-yl-1H- pyrazoIo[3,4-d]pyrimidiπ-4-amine HNMR 1H, DMSO-d6 δ, ppm: 0.88 (t, J=7.4, 3H); 1.75- 2.10 (m, 6H); 2.20-2.50 (m, 7H); 3.45-3.75 (m, 8H); 4.13 (t, J=6.9, 2H); 7.02 (d, J=7.7, 1 H); 7.40 (t, J=8.0, 1 H); 7.92 (d, J=7.8, 1 H); 7.80 (s, 1 H); 8.05 (s, 1 H); 9.40 (s, 1 H).
103. propyl-6-pyrrolidin-1-yl-N-[3-{pyrrolidin-1-ylcarbonyl)phenyl]-1 H-pyrazolo[3,4- d]pyrimidin-4-amine HNMR 1H5 DMSOd6 δ, ppm: 0.88 (t, J=7.4, 3H); 1.80-2.05 (m, 10H);
2.95 (s, 2H); 3.40-3.65 (m, 9H); 4.13 (t, J=6.9, 2H); 7.14 (d, J=7.5, 1 H); 7.40 (t, J=7.8, 1 H);
7.96 (d, J=8.7S 2H); 8.09 (s, 1 H); 9.37 (s, 1 H).
104. N-{3-[(4-phenyIpiperazin-1-yl)carbonyl]phenyl}-1-propyi-6-pyrrolidin-1-yI-1H- pyrazolo[3,4-d]pyrimidin-4-amine HNMR 1H, DMSOd6 δ, ppm: 0.88 (t, J=7.6, 3H); 1.80- 2.00 (m, 6H); 3.20 (m, 4H); 3.62 (m, 8H); 4.13 (t, J=6.7, 2H); 6.82 (t, J=7.2, 1 H); 6.96 (d, J=8.3, 2H); 7.08 (d, J=6.7, 1 H); 7.23 (t, J=7.7, 2H); 7.42 (t, J=7.9, 1 H); 9.41 (s, 1H) .
Library H
Figure imgf000089_0001
[0261] Preparation of 3a. 5.0 g (0.040 mol) of methyl phenyl mercaptan 2 was suspended in 100 mL of dry chloroform, and then 24.8 g (0.101 mol) of 3-chloro perbenzoic acid was added carefully in portions. The reaction mixture was stirred for 2 h at RT, and then refluxed for 36 h. The reaction progress was monitored by TLC. After total conversion of starting material to target compound, the reaction mixture was carefuliy treated with an equal volume (100 mL) of aqueous sodium carbonate (0.101 mol). The organic layer was extracted and the solvent was removed in vacuo. The product was crystallized from ether. Compound 3 was prepared in 2 g quantity,
[0262] Preparation of 4a, 2.0 g (0.013 mol) of methyl sulfone 3 was dissolved in 6 mL of H2SO4, then reaction mixture was heated to 100 0C. Potassium nitrite, 4.940 g (0.019 mol), was carefully added in portions to the heated solution while the temperature was maintained at 100 0C. The reaction mixture was stirred for 1.5 h at 100 0C and then was stirred overnight at RT. At this time, the reaction mixture was poured into 50 mL of ice water, stirred for an hour, and the resulting precipitate was filtered and iyophilized. The observed crude yield was 80% (2.1 g). [0263] Preparation of 5a. 2.0 g (0.0098 mol) of 4 was dissolved in 100 mL of methanol, and an aqueous suspension of Pd/C (10 %) was added under Ar atmosphere. The reaction mixture was additionally purged under Ar for 20 min. At this time, the reaction mixture was hydrogenated until the reduction was compiete. The reaction was stirred for 2 h. The precipitate was carefully filtered and the residue was collected under reduced pressure. Compound 5 was crystallized from ether and dried on air. The crude yield was 89 % (1.5 g). [0264] Preparation of 2b 3 g (4.51 rrtmoi) of 3-nitro-1-benzenesulfonyl chloride was dissolved in 30 ml of dioxane. Then to reaction mixture was added 1.38 g (4,51 mmol) of TEA and 3.76 mmol of amine. Reaction mixture was stirred at 80 0C overnight. LCMS demonstrated total conversation of starting material. The reaction mixture was evaporated and purified by column chromatography.
[0265] Preparation of 3b Compounds 2a was reduced by H2/Pd/C, then the solution was filtered off and evaporated.
[0266] Preparation of 7 1.5 mmol of aniline was dissolved in 5 ml of dioxane. Then to reaction mixture was added 0.2 ml of HCI, and then after 15 minutes of stirring 1.0 mmol of compound was added. Reaction mixture was stirred at 80 0C overnight. LCMS demonstrated total conversation of starting material.
Figure imgf000090_0001
Table 9. Examples 105-109 of Pyrazolopyrimϊdines. Library H.
Figure imgf000090_0002
Figure imgf000091_0001
Analytical Data for Library H
108. methy)-N-{3-[(4-propylpϊperaztn-1-yl)sυlfonyl]phenyl}-6-pyrrolidin-1-yl~1H- pyrazoio[3»4-d]pyrimidin-4~aminen HNMR 1H, DMSOd6 δ, ppm: 0.77 t <3H, CH3), 1 ,35 q (2H, CH2), 1.94 t (4H, 2CH2), 2.20 m (2H1 CH2), 2.40 m (4H, 2CH2), 2.90 m (AH, 2CH2), 3.60 m (4H, 2CH2), 3.76 S <3H, CH3), 7.35 d (1 H, Ar)1 7.62 1 (1 H, Ar), 8.06 s (1 H, Ar), 8.10 d {1 H, Ar), 8.72 s (1 H, Ar)1 9.96 s <1 H, NH).
109. N-|3-(methyIsulfonyl)phenyi]-1-propyl-6-pyrrolidϊn-1-yl-1H-pyrazolo[3,4-d]pyrimidin- 4-amine HNMR 1H1 DMSOd6 δ, ppm: 0.90 m (3H); 1.70-2.00 m (6H); 3.20 s (3H); 3.65-3.80 m (4H); 4.20 1 (2H); 7.50-7.70 m (2H); 8,15 1 (2H); 8.80 s (1H); 10.0 s (1 H).
110. 4-chloro-1-propyl-6-pyrrolidϊn-1-yl-1H-pyrazolo[3,4-d]pyrimidine HNMR 1H, DMSO- d6 δ, ppm: 1.9O t (2H); 1.75-2.00 m (6H); 3.60 m (4H); 4.201 (2H); 7.95 s (1 H).
111. N"[3-{morpholin-4-ylsulfonyl}phenyt]-1-propyi-6-pyrroIidin-1-yI-1 H-pyrazoio[3,4- d]pyrimidϊrt-4-aminβ HNMR 1H1 DMSOd6 δ, ppm: 0.92 (t, J=7.4, 3H); 1.94 (m, 6H); 3.67 (m, 8H); 4.18 (t, J=6.9, 4H); 4.28 (s, 3H); 7.37 (d, J=7.7, 1 H); 7.56 (t, J=8.0, 1 H); 8.05 (s, 1 H); 8.15 (m, 1 H); 8.62 (s, 1 H); 9.73 (s, 1 H).
112. N-(3,5-difluorobenzyl)-3-[(1-propyl-6-pyrroiidin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4- yl)amir»o]-benzenesulfonamide HNMR 1H, DMSOd6 δ, ppm: 0.88 (t, J=7.4, 3H); 1.90 (m, 6H); 3.61 (t, J=6.7, 4H); 4.13 (m, 4H); 6.93 (m, 3H); 7.49(m, 2H); 7.89 <m, 1 H); 8.03 (m, 2H); 8.66 (s, 1H); 9.60 (s, 1 H). Library AB
Figure imgf000092_0002
[0267] Compound 7: Compound 6 (200 mg, 0.63 mmol), boronic acid (150 mg, 0.72 mmol), and triphenylphosphine (25 mg, 0.10 mmol) were charged into the flask containing 4 mL of dioxane and 1.5 mL of 2 M aqueous Na2CO3 solution. After purging the mixture with Ar for 20 min, [Pd(PPh3)4] catalyst (36 mg, 0,03 mmol, 5 mo! %) was added. The reaction mixture was heated at 100 0C for 4 h under an Ar atmosphere. After cooling of the reaction mixture to RT the solvent was removed under reduced pressure to produce yellow oil. The residue was washed with water (20 mL) and extracted with CHCI3 (20 mL). The organic layer was isolated and purified by coiumn chromatography.
Figure imgf000092_0001
Figure imgf000092_0003
Analytical Data for Library AB
110. 4-phenyl-1-propyI-6-pyrrolidin-1-yl-1H-pyrazolo[3,4-d]pyrimidine HNMR 1H, OMSO- d6 δ, ppm: 0.91 (t, J=7.3 Hz, 3H); 1.88-2.02 (m, 6H); 3.68 (m, 4H); 4.24 (t, J=6,9Hz, 2H);
7.58 (m, 3H); 8.20 (m, 3H). II. R1-R4-R6 APPROACH
Library 8
[0268] Compound 2 To a stirred compound 1 (400 ml) 25 % aqueous ammonia was added portionwise. The mixture was allowed to react overnight. The white precipitate obtained was filtered off and washed with water. Yield 72 % (23.2 g).
[0269] Compound 4 was prepared according to the following procedure: Equimolar mixture of 2 (0.3 mmol) and 3 (0.3 mmoi) was stirred at 60 0C overnight. Then the yeifow reaction mixture was diluted with cooled methanol (200 ml). The yellow precipitate formed was filtered and washed with ether giving crude compound 4 in 53 % yield (15 g).
[0270J Preparation of ethyl S-amino-i-benzyl-IH-pyrazole^carboxylate 6.
Benzylhydrazine 5 (0.5 mol) was mixed with ethyl(ethoxymethylene)cyanoacetate (0.5 mol) 4 in ethanoi (600 mL) and refluxed for 12 hours. Then, ethanoi was removed under reduced pressure, the light yellow precipitate was washed with ether and recrystallized from ethanoi. 65 g, 82 %.1H NMR (DMSO, ppm) δ: 1.34 m (3H), 4.22 m (2H), 5.22 s (2H), 6.42 s (2H), 7.18 m
(2H), 7.24 m (3H), 7.48 s (1 H).
[0271 J Preparation of 1 -benzyl-1 H-pyrazolo-β^-dføyrimidϊne^β-diol 7. 5-amino~1 - benzyl-4H-pyra-zol-4-carboxamide 6 (1g, 4.6 mmol) was dissolved in 1 , 4- dioxane (15 mL) and diphosgene (10 mmol) was added. Reaction mixture was heated to reflux and stirred for 3 hours. LCMS analysis after this time demonstrated that the major product of the reaction was
1 -benzyl-1 H-pyrazolo[3,4-d]pyrimidine~4,6(5H, 7H)-dione 4. The solvent was removed under reduced pressure and the precipitate was washed with ether. A white precipitate was obtained in 79% of yield (2.5 g). LCMS: M+1 =243, 1H NMR (DMSO, ppm) δ: 5.02 bs (2H), 7.04 bs (5H),
7.80 s (1 H), 10.8 s (1 H), 12.2 (1H).
[0272] Preparation of 1-benzyl-4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine 8. 1-benzyl-
1 H-pyrazolo-[3,4-d]pyrimidine-4,6-diol 7 (0.5 g, 2 mmol) was dissolved in POCI3 and 4 mmol of
PCI5 were added. The reaction mixture was stirred at 80 0C overnight, followed by dilution with toluene, and the solvents were removed under reduced pressure to give a brown oil (0.6 g, 81
%).
£0273] Preparation of N-(1-benzyl-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N-(4- methylphenyl)-amϊne 9. 1-benzyI-4,6-dichloro-1 H-pyrazolo[3,4-d]pyrimidine 8 (2 mmol) was dissolved in 1 ,4-dioxane (40 mL). 4-methylaniline (2 mmol) and triethylamine (2 mmol) were added to the reaction mixture. A dark brown solution (0.3 g, 81 %) was obtained.
[0274] Preparation of N-{1-benzyl-6-amϊno-1H-pyrazolo[3,4-d]pyrimϊdin-4-yl)-N-(4- methy!-phenyl)-amines 10. 9 (0.1 g, 0.28 mmol) was dissolved in 1 ,4-dioxane (5 mL).
Corresponding amine (0.3 mmol) and triethylamine (0.3 mmol) were added and the reaction mixture was stirred overnight at 80 0C. Then, the reaction mixture was filtered and the solvent was removed under reduced pressure to provide white precipitates in yields of 32-60 %.
Figure imgf000094_0001
Figure imgf000094_0002
Analytical Data for Library B
111. benzyl-N-(4-methylpheπyl)-6-pyrrofidin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
1H NMR (DMSO, ppm) δ: 1.95 m (4H); 2.30 s (3H); 3.60 m (4H); 5.30 s (2H); 7.13-7.27 m (7H); 7.72 d (2H); 7.92 s (1 H); 9.40 s (1 H).
112. benzyl-N-{4-methyiphenyJ)-6-morpholin-4-yl-1 H-pyrazolo[3,4-d]pyrimidin-4-amine 1H NMR (DMSO, ppm) δ: 2.12 s (3H), 2.96 m (2H), 3.82 m (8H), 5.38 s (2H), 7,22 m (7H), 7.58 m (2H), 7.86 s (1 H)
113. benzyl-N-(4-methylphertyl)-6-piperidin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4-amine 1H NMR (DMSO, ppm) δ: 1.62 m (6H), 2.18 s (3H), 3.86 m (4H), 5.42 s (2H), 7.22 m (7H), 7.60 m (2H), 7.86 s (1 H). Library J
Figure imgf000095_0001
[0275] Preparation of S-amino-i-methyi-IH-pyrazole-^carboxamide (3) 20 g {0.144 mol) of 2-cyano-3-(dimethylamino)-2-propenamicle 1a was dissolved in dry ethanol and 43.6 g (0.432 moi) of TEA was added at once. The solution was stirred for 5-10 min and then 31.5 g {0.216 mol) of methylhydrazine sulfate was added. The reaction mixture was refluxed overnight, and then an aliquot was isolated and analyzed. The reflux was ongoing until total conversion of the starting material. Then the reaction mixture was cooled, the solvent was removed in vacuo and the residue was carefully extracted with plenty ethyl acetate. The desired intermediate 3 is of extremely low solubility in any organic solvent so the extraction took two days. Then the organic layer was concentrated, the yellow precipitate was carefully heated with ether and filtered to afford crude compound 3 in yield 40 % (8.10 g). 1H NMR, DMSOd6 δ, ppm: 3.50 s {3H); 6.05 s (2H); 6.45-7.25 d (2H); 7.55 s (1H).
[0276J Preparation of 1-methyl-1 H-pyrazoIo[3,4-d]pyrimidϊne~4,6(5H,7H)-dione 4. Diphosgene (50 mmol) was dissolved in 1 ,4-dioxane (150 mL) and 3g (21 mmol) of 3 was added; the reaction mixture was heated to 80 0C and stirred at this temperature for 12 hours, then cooled and concentrated under reduced pressure. The white precipitate was filtered and washed with ether. The yield was 2.1 g, 62 %. 1H NMR DMSO-d6 δ, ppm: 3.80 s (3H), 7.76 s (1 H)1 10.80 s (1H), 12.0 s (1 H).
[0277] Preparation of 4,6-dichloro-1-methyI-1 H-pyrazolo[3,4-d]pyrimidine 5. 1 -methyl- 1 H-pyrazolo-[3,4-d]pyrimidine-4,6(5H,7H)-dione 4 (2.1g, 13 mrnoϊ) was dissolved in POCI3 and PCI5 (26 mmol) was added to the solution. The reaction mixture was stirred at 1000C for 12 hours. Then the solvent was removed under reduced pressure and the yellow oily product was dried by concentration from benzene. The yield was 1.8 g, 74 %, 1H NMR (CDCI3, ppm) δ: 4.12 s (3H), 8.20 s (1H).
[0278] Preparation of 1-methyl-1H-pyrazolo[3,4-d]pyrimidine-4-(4-methylaniline)-6- (5H,7H)-chϊoride (6) 0.414 g (0.0020 mol) of 1-methyl-1 H-pyrazoIo[3,4-d]pyrimidine~ 4,6(5H,7H)-dichloride 5 was suspended in 20 ml of dioxane and 0.216 g (0.0020 mol) of 4- methylanilinβ and 0.204 g (0.0020 mol) of TEA were added at once. The reaction mixture was stirred overnight at 50 0C. Then the solid was filtered, the residue was concentrated and crystallized from ether to give a white precipitate in yield of 80 % (0.440 g). [0279J Preparation of 1-methyl-1H-pyrazolo(3,4-d]pyrimidϊne-4-(4-methylaniUπe)-6- (5H,7H)-aπy aniline (7) 0.160 g (0.00058 mol) of 1-methyl-1 H-pyrazolo[3,4-d]pyrSmidine-4-(4- methylaniline)-6-(5H,7H)-chforide 6 was suspended in 0.5 m! of an aniline. The reaction mixture was stirred overnight at 150 0C. LCMS analysis of the reaction mixture demonstrated total conversion of the starting material. At this time the reaction mixture was poured into water acidified with HCI and the organic phase was extracted with chloroform twice. The target compound was purified by column chromatography with methylene chloride-ethanol (100:1 ) as an eluent affording oily products in 5-30 % yield (3-15 mg).
Figure imgf000096_0001
Table 12. Examples 114-120 of Pyrazolopyrimidines. Library J
Figure imgf000096_0002
1-methyi-N4-(4-
C2GH17F3N6O methy!phenyl)-N6-[4-
118 415 (trifluoromethoxy)ρheπyl]-
/ 414.4 1 H-pyrazolo[3,4- d]pyrimidine-4,6-diamine
N 6-{3-isopropy!phenyl)-1 -
C22H24N6 / methyl-N4-(4-
119 373 methylphenyl)-1 H- 372 5 pyrazolo[3,4-d]pyrimidine-
4,6-diamιne
N6-(2-fiuoro-5-
C20H19FN6 / rnethylphenyl)~1 -methyl-
120 363 N4-(4-meihyiphenyl)-1 H- 362
-..A-. pyrazolo[3,4-d]pyrimidine- 4,6-diamine
Analytical Data for Library J
114. methy!-N4-{4-methylphenyl}-N6-[3-(trifluoromethyl)phenyi]-1 H-pyrazo)o[3,4- d]pyrimidine-4,6-diamiπe 1HNMR (DMSO, ppm) δ: 1 ,80 m (4H); 2.35 s (3H); 3.25 m (4H); 4.05 s (3H); 7.25 d (2H); 7.68-7.96 m (4H); 8.10 s (1 H); 8.64-8.88 m (2H); 9.10 s (1H); 9.85 s (1 H).
115. N6-{2-fluorophenyI)-1-methyl-N4-{4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-4J6- dϊamin 1HNMR (DMSO, ppm) δ: 1.15 s (6H); 2.30 s (3H); 2.85 m (1 H); 3.85 s (3H); 6.72 1 (1 H); 6.90 d (1 H); 7.55 d (1H); 7.68 d (3H); 8.12 s (1H); 9.55 s (1 H); 10.40 s (1 H).
116. N6-(3-fluorophenyl)-1-methyl-N4-(4-methylphenyi)-1H-pyrazolo[3,4-d]pyrimidine-4,6- diamine diamine 1HNMR (DMSO, ppm) δ: 2.30 s (3H); 3.85 s (3H); 6.72 t (1 H); 7.14-7.30 m (3H); 7.42 d (1 H); 7.70 d (2H); 8.10 s (1 H); 9.55 s (1 H); 10.00 s (1 H).
117. N6-(4-fluorophenyl)-1-methyl-N4-(4-methylphenyl)-1H-pyrazolo[3,4-dlpyrϊmidine-4,6- diamine diamine 1HNMR (DMSO, ppm) δ: 2.30 s (3H); 3.85 s (3H); 7,04 t (2H); 7.18 d (2H); 7.58-8.00 m (5H); 9.60 s (1 H).
118. methyl-N4-(4-methylphenyi)-N6-[4-(trifluoromethoxy)phenyl]-1 H-pyrazoto[3,4- d]pyrimidϊne-4,6-diamme diamine 1HNMR (DMSO1 ppm) δ: 2.30 s (3H); 3.85 s (3H); 7.18 d (2H); 7.28 d (2H); 7.70 d (2H); 7.90 d (2H); 8.12 s (1 H); 9.65 s (1 H); 10.20 s (1H).
119. N6-(3-isopropylphenyl)-1-methyl-N4-(4-methylphenyl)-1 H-pyrazoio[3,4- d]pyrimidine-4,6-diamine diamine 1HNMR (DMSO, ppm) δ: 2.25 m (6H); 3.85 s (3H); 6.95 t (1 H); 7.02-7.20 m (3H); 7.54-7.76 m (3H); 8.15 s (1H); 9.05 s (1 H); 10.45 s (1H).
120. N6-{2-fluoro-5-methylphenyl)-1-methyl-N4-(4-methylphenyl)-1H-pyrazolot3,4- d]pyrimidine~4,6-diamine diamine 1HNMR (DMSO, ppm) δ: 1.95 m (4H); 3.55 m (4H); 3.75 s (3H); 7.141 (2H); 7.88 m (3H); 9.25 s (1 H) Library L
Figure imgf000098_0001
[0280] Preparation of 6. 4,6-Dichloro-1 -methyi-1 H-pyrazolo[3,4-d]pyrimidine 5 (0.9 g, 10 mmo!) was dissolved in 1 ,4-dioxane (10 mL). tert-Butyl 4-aminotetrahydro-1(2H)- pyridinecarboxyiate (10 mmol) and triethylamine (10 mmol) were added to the reaction mixture and stirred at 50 0C for 12 hours. Thereafter, the solvent was removed under reduced pressure and the precipitate was filtered off and washed with ether afforded 6 as an orange solid (1 g,
61 %).
[0281] Preparation of 7. Compound 6 (1 g) was dissolved in 1 ,4-dioxane saturated with gaseous HC! (5 mL). The reaction mixture was stirred at RT overnight and concentrated in vacuo. The precipitate formed was filtered and washed with hexane afforded 7 as a yellow solid
(0.7 g, 94%).
[0282] Compound 8 A solution of 10 mmol of 7 in 10 mL of dioxane was treated with sulfonyl chloride (10 mmol) and triethylamine (22 mmol). This mixture was stirred at reflux for 3 hours, then cooled and concentrated. The residue was treated with water in order to form a precipitate.
The former was filtered off, carefully washed with water, dried and purified by column chromatography afforded 8 as a yellow soiid. The crude yield was 0.3-0.35 g, 74-84%
[0283] Compound 9 Compound 8 (0.01 mol) was treated with 3, 4-difluoroanitine (10 ml) at reflux for 48 hours. Thereafter the reaction mixture was cooled and diluted with ether. The precipitate formed was filtered and purified by column chromatography afforded 8 as a yellow solid. The yield was 0.15-0.3 g, 30-60%.
Table 13. Examples 121-128 of Pyrazolopyrimidines. Library L.
Figure imgf000098_0002
Figure imgf000099_0001
Analytical Data for Library L 121. N6-{3,4-difluorophenyl)-1-methyl-N4-[1-(phenyI-sulfonyt)piperidiπ-4-yt]-1H- pyrazolo[3,4-d]pyrimidine-4,6-diamine1HNMR(DMSO,ppm)δ: 1.65m(2H);2.10d(2H); 2.60 m (2H); 3.75 m (5H); 4.10 m (1 H); 7.15 m (1H); 7.45 d (2H); 7.65-7.80 m (6H); 7.85 s (1 H); 8.02 m (1 H); 8.95 S (1 H)
122. N^a.Φdifluoro-phenylJ-N^I-t(4-fluorophenyOsulfonyπpiperidin^-yl^i-methyl-IH- pyrazolo[3,4-d]pyrimidine-4,6-diamine 1HNMR (DMSO1 ppm) δ: 1.65 m (2H); 2,1O d (2H); 2.60 m (2H); 3.75 m (5H); 4.10 m (1H); 7.15 m (1 H); 7.45 m (3H); 7.65 d (1 H); 7.85 m (3H);
8.02 m (1 H); 8.95 s (1H).
123. N6-{3,4-dif luoro-phenyl) -1-methyl-N4-{1-[(3-nitrophenyl)-sulfonyI]piperidin-4-yl}-1H- pyrazoIofS^-dlpyrinnidine^β-diamine 1HNMR (DMSO, pprn) δ: 1.65 m (2H); 2.10 d (2H); 2.60 m (2H); 3.75 m (5H); 4.10 m (1 H); 7.15 m (1H); 7.4O d (1 H); 7.65 d (1 H); 7.90 s (1 H);
8.03 m (2H); 8.22 d (1 H); 8.45 s (1 H); 8.55 d(1 H); 8.95 s (1 H)
124. N4-{1 -[3-chlorophenyl)sulfonyl]piperidin-4-yl}-N6-(SΛ-cϋfluorophenylJ-i-methyl-IH- pyrazolo[3,4-d]pyrimJdine-4,6-diamtne 1HNMR (DMSO, ppm) δ: 1.65 m (2H); 2.10 d (2H); 2.60 m (2H); 3.75 m (5H); 4.10 m (1H); 7.15 m (1 H); 7.40 d (1 H); 7.75 m (4H); 7.90 s (1 H); 8.03 m (1H); 8.95 s (1 H)
125. N6-{3,4-dif luoro-phenyl)-N4-{1 -[(3-fluorophenyI)sulfonyl]piperidin-4-yl}-1 -methyl-1 H- pyrazolo[3,4-dJpyπmidϊne-4,6-diamine 1HNMR (DMSO, ppm) δ: 1.65 m (2H); 2.10 d (2H); 2.60 m (2H); 3.75 m (5H); 4.10 m (1 H); 7.15 m (1H); 7.42 d (1 H); 7.65 m (4H); 7.90 s (1 H); 8.03 m (1 H); 8.95 s (1 H)
126. N6-(3,4-dffluoro-phenyl)-1-methyl-N4-{1-{[3-{trifluoromethyl)- phenylϊsulfoπyl}piperidm-4-yl)-1 H-pyrazolo{3,4-d]pyrimidine-4,6-diamine 1HNMR (DMSO, ppm) δ: 1.65 m (2H); 2.1O d (2H); 2.60 m (2H); 3.75 m (5H); 4.10 m (1 H); 7.15 m (1 H); 7.40 d (1 H); 7.65 d (1 H); 7.90-8.15 m (6H); 8.95 s (1 H).
127. N6-(3,4-difiuoro-phenyi)-1-methyl-N*-{1-[(3-methylphenyl)-sulfonyl]ptperidin-4-yl}- 1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine 1HNMR (DMSO, ppm) δ: 1.65 m (2H); 2.1O d (2H); 2.60 m (2H); 3.75 m (5H); 4.10 m (1 H); 7.15 m (1 H); 7.40 d (1 H); 7.50-7.70 m (4H); 7.90 s (1 H); 8.02 m (1H); 8.95 s (1 H).
128. 3-{[4-({6-[(3,4-difluorophenyl)aminoI-1-methyl-1H-pyrazoIo[3,4-d]pyrimidin-4- yl}amϊno)piperidin-1-yl]sulfonyl}benzonitrile 1HNMR (DMSO, ppm) δ: 1.65 m (2H); 2.10 d (2H); 2.60 m (2H); 3.75 m (5H); 4.10 m (1H); 7.15 m (1 H); 7.40 d (1 H); 7.65 d (1 H); 7.90-8.15 m (6H); 8.95 s (1 H).
Library R
Figure imgf000100_0001
[0284] Preparation of N-[1-methyi-6-(3-methyIphenyi)-1H-pyra2Olot3,4-d]pyrimidin-4-yl]- N-{4-methyl~phenyl)amine (7) 0.150 g (0.00055 mo!) of 1-methyl-1 H-pyrazolo[3,4-d]pyrimidine- 4-(4-methylaniline)-6-(5H,7H)-chioride (6) was suspended in 4 ml of dioxane and 0.5 mi of 10 % aqueous Na2CO3 was added. Next, 0.00066 mol of an appropriate boronic acid and JPd Cl2 (PPh3)2] catalyst (20 mg, 0.032 mmoi, 5 mol %) were added successively under argon atmosphere. The reaction mixture was stirred overnight at 80 0C. LCMS analysis of the reaction mixture demonstrated 65% of the target compound and 35% of starting material, After additional overnight stirring at 1000C the conversion amounted to 90%. At this time the solvent was removed in vacuo, the residual crude was purified by column chromatography with methylene chloride as eluent. The yields averaged 5-45 mg, (10-50 %).
Figure imgf000101_0001
Table 14, Examples 129-147. Library R.
Figure imgf000101_0002
Figure imgf000102_0001
Figure imgf000103_0001
Analytical Data for Library R
129. 6-{4-chloro-3-fiuorophenyI)-1-methyl-N-(4-methylphenyl)-1 H-pyrazolo[3,4- d]pyrimidin-4.amine 1HNMR (DMSO, ppm) δ: 2.35 s (3H); 4.00 s (3H); 7.26 d (2H); 7.62- 7.74 m (3H); 8.08 s (1 H); 8.26 d (2H); 9.80 s (1 H).
130. 6-(3-f!uorophenyl)-1 -methyϊ-N-(4-methyipheny l)-1 H-pyrazolot3,4-d]pyrimidin-4- amine 1HNMR (DMSO1 ppm) 5: 1.95 m (4H); 2.30 s (3H); 3.55 m (4H); 3.75 s (3H); 7.15 d (2H); 7.73 d (2H); 7.88 s (1 H); 9.15 s (1 H).
131. 6-(3,5-difIuorophenyl)-1-methyl-N-(4-methylphenyl)-1H-pyrazo(o[3,4-d]pyrimidin-4- amine 1HNMR (DMSO, ppm) 6: 2.35 s (3H); 4.00 s (3H); 7,20-7.36 m (3H); 7.68 d (2H); 7.94-8.12 m (3H); 9.70 s (1 H).
132. methyl-N-(4-methylphenyl)-6-(3-(trifϊuorornethyl)phenyf]-1 H-pyrazolo[3,4- d]pyrimidin-4-amine 1HNMR (DMSO, ppm) δ: 2.35 s (3H); 4.05 s (3H); 7.24 d (2H); 7.66- 7.88 m (4H); 8.10 s (1H); 8.66-8.78 m (2H); 9.85 s (1 H).
133. 6-<3,4-difluorophenyl)-1 -methyl»N-{4~methy!phenyi)»1 H-pyrazolo[3,4»d3pyrimidin-4- amine 1HNMR (DMSO1 ppm) δ: 2.35 s (3H); 4.00 s (3H); 7.26 d (2H); 7.48 m (2H); 7.68 d
(1 H); 8.08 s (1 H); 8.22-8.30 m (2H); 9.80 s (1 H).
134. 6-(4-fluorophenyI)-1-methyl-N-(4-methyiphenyl)-1 H-pyrazolo[3,4-d]pyrimidin-4- aminE 1HNMR (DMSO1 ppm) δ: 2.35 s (3H); 3.98 s (3H); 7.20-7.36 m (4H); 7.70 d (2H); 8.10 s (1 H); 8.48 1 (2H); 9.70 s (1 H). 135. 3-{1"methyl~4-[(4-methylphenyl)amino]-1H-pyrazolo[3,4-d]pyrimidin-6- yl}benzonitrile 1HNMR (DMSO, ppm) δ: 2.35 s (3H); 4.05 s (3H); 7.25 d (2H); 7.70 m (3H); 7.90 d (1 H); 8.10 s (1 H); 8.65 m (2H); 9.85 s (1 H).
136. 6-(3-fluoro-4-methylphenyl)-1-methyl-N-{4-methylphenyI)-1H-pyrazolo[3,4- d]pyrimidin-4-amine 1HNMR (DMSO, ppm) δ; 2.30 d (6H); 4.05 s (3H); 7.24 d (2H); 7.38 1 (1 H); 7.72 d (2H); 8.00-8.25 m (3H); 9.75 s (1 H).
137. 6-(4-isopropylphenyI)-1-methyl-N-(4-methyiphenyI)-1H-pyrazolo[3,4-d]pyrimidin-4- amine 1HNMR (DMSO1 ppm) δ: 1.25 m (6H); 2.35 s (3H); 3.05 m (1 H); 4.00 s (3H); 7.24 d (2H); 7.48 d (2H); 7.76 d (2H); 8.10 s (1H); 8.38 d (2H); 9.70 s (1 H).
138. 6-(3-isopropyfphenyl)-1-methyi-N-(4-methylphenyl)-1H-pyrazoIo[3,4-d]pyrimidϊn-4- amine 1HNMR (DMSO, ppm) δ: 1.35 m (6H); 2.35 s (3H); 3.05 m (1H); 4.00 s (3H); 7.20- 7.48 m (4H); 7.78 d (2H); 8.06-8.42 m (3H); 9.70 s (1 H).
139. N-methyl-4-{1-methyl-4-[(4-methylphenyt}amino|-1 H-pyrazolo[3,4-d]pyrimidin-6-yl>- benzenesulfonamide 1HNMR (DMSO, ppm) δ: 2.35 s (3H); 4.05 s (3H); 7.10-7.34 m (3H); 7.66-8.00 m (4H); 8.10 s (1 H); 8.60 d (2H); 9.85 s (1 H).
140. N-methyl-3-{1-methyl-4-[(4-methylphenyl)amino]-1H-pyrazoio[3,4-d]pyrimidin-6- yl}bertzenesuifonamide 1HNMR (DMSO, ppm) δ: 2.35 s (3H); 2.45 s (3H); 4.05 s (3H); 7.15-7.35 m (3H); 7.45-7.95 m (4H); 8.10 s (1 H); 8.65 d (1 H); 8.95 s (1 H); 9.80 s (1 H).
141. methyl-N-{4-methylphenyI)-6-[4"(pyrrolIdin-1-yisulfonyi)phenyl]-1H-pyrazolo[3,4- d]pyrimidin-4-amϊne 1HNMR (DMSO, ppm) δ: 1.70 m (4H); 2.30 s (3H); 3.30 m (4H); 4.05 s (3H); 7,25 d (2H); 7.75 d (2H); 7.95 d (2H); 8.15 s (1 H); 8.65 d (2H); 9.90 s (1 H).
142. methyl-N-(4-methylphenyl)-6-[3-(pyrroiidin-1-ylsuIfonyl)pheny(]-1H-pyrazoIot3,4- d]pyrimidin-4-amine 1HNMR (DMSO, ppm) δ: 1.80 m (4H); 2.35 s (3H); 3.25 m (4H); 4.05 s (3H); 7.25 d (2H); 7.68-7.96 m (4H); 8.10 s (1 H); 8.64-8.88 m (2H); 9.10 s (1 H); 9.85 s (1 H).
143. N-(3-{1 -methyl-4-[(4-methylphenyl)amino]-1 H-pyrazoio[3,4-d3pyrimidin-6-yl}phenyl)- methanesulfonamide 1HNMR (DMSO1 ppm) δ:
144. 1-methyl-N-(4-methyIphenyl)-6-(4-phenoxypheny[)-1H-pyrazo(o[3,4"d]pyrimid(n-4- amine 1H NMR (DMSO, ppm) δ: 2.35 s (3H); 4.00 s (3H); 7.05-7.30 m (8H); 7.35-7.50 m (2H); 7.75 d (2H); 8.15 s (1 H); 8.45 d (2H); 9.60 s (1 H).
145. methyI-6-(3-methyIphenyI)-N-{4-methyIphenyi)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 1HNMR (DMSO, ppm) δ: 2.40 s (6H); 4.00 s (3H); 7.20-7.46 m (4H); 7.78 d (2H); 8.10 s (1 H); 8.20-8.32 m (2H); 9.75 s (1 H).
146. 6-{3-ch!oro-4-fluorophenyl)-1-methyl-N-(4-methylphenyl)-1 H-pyrazo!o[3,4- d]pyrϊmidϊn-4-amine 1HNMR (DMSO, ppm) δ: 2.35 s (3H); 4.00 s (3H); 7.24 d (2H); 7.48 1 (1 H); 7.70 d (2H); 8.08 s (1 H); 8.40 m (1 H); 8.50 d (1 H); 9.80 s (1H).
147. 6-(2-<[sec-butyl(methyl)-amino]methyI}phenyl)-1-methyl-N-{4-methyl-phenyl)-1H- pyrazoIo[3f4-d]pyrimidϊn-4-amine 1HNMR (DMSO, ppm) δ: 0.75-2.00 m (10H); 2.30 s (3H); 3.15 s (2H ); 4.00 s (3H); 4.70 m (1 H); 7.24 d (2H); 7.48-7.90 m (5H); 8.04-8.18 s (2H); 9.80- 10.40 m (2H).
Library R1
Figure imgf000105_0001
[0285] Preparation of S-amϊno-i-propyl-IH-pyrazole-^-carboxamide (3) 20 g (0.144 moi) of 2-cyano-3-(dimethylamino)~2-propenamide 1a was dissolved in dry ethanol and 43.6 g (0.432 mol) of TEA was added at once. The solution was stirred for 5-10 min and then 0.216 mol of p ropy I hydrazine was added. The reaction mixture was refluxed overnight, thereafter an aliquot was isolated and analyzed. The reflux was ongoing until total conversion of the starting material. Then the reaction mixture was cooled, the solvent was removed in vacuo and the residue was carefully extracted with plenty ethyl acetate. The desired intermediate 3 is of extremely low solubility in any organic solvent so the extraction took two days. Then the organic layer was concentrated, the yellow precipitate was carefully heated with ether and filtered to afford crude compound 3 in yield 40 % (9.10 g).
[0286] Preparation of 1-propyi-1 H-pyrazolo[3,4-d]pyrimrdirte-4,6(5H,7H)-dione 4. Diphosgene (50 mmol) was dissolved in 1 ,4-dioxane (150 ml) and 21 mmol of 3 was added; the reaction mixture was heated to 80 0C and stirred at this temperature for 12 hours, then cooled and concentrated under reduced pressure. The white precipitate was filtered and washed with ether. The yield was 2.1 g, 62 %.
[0287] Preparation of 4,6-dichloro-1- propyl-1H-pyrazolo[3,4-d]pyrimidine 5. 1-propyl- 1 H-pyrazolo-[3,4-d]pyrimidine-4,6(5H,7H)-dione 4 (13 mmol) was dissolved in POCI3 and PCIS (26 mmol) was added to the solution. The reaction mixture was stirred at 100 0C for 12 hours. Then the solvent was removed under reduced pressure and the yellow oily product was dried by concentration from benzene. The yield was 2.1 g, 54 %.
[0288] Preparation of 1 -propyl-1 H-pyrazoio[3,4-d]pyrimidine-4-{4-methyiani[ine)-6- (5H,7H)-chioride (6) 0.414 g (0.0020 mol) of 1- propyi-1 H-pyrazolo[3,4-d]pyrimidine- 4,6(5H,7H)-dtchioride 5 was suspended in 20 ml of dioxane and 0.216 g (0.0020 mol) of 4- methylaniϋne and 0.204 g (0.0020 mol) of TEA were added at once. The reaction mixture was stirred overnight at 50 0C. Then the solid was filtered, the residue was concentrated and crystallized from ether to provide a white precipitate in yield of 80 % (0.440 g). [02893 Preparation of N-[1-propyl-6-aryl-1H-pyrazolo[3,4-dJpyrimidin-4-yi]-N-(4- methylphenyl- amine (7) 0.00055 mol of 1-propyl-1 H-pyrazoIo[3,4-d]pyrimid)ne-4-(4- methyianiline)-6-(5H,7H)-chloride (6) was suspended in 4 ml of dioxane and 0.5 ml of 10 % aqueous Na2CO3 was added. Next, 0.00066 mol of an appropriate boronic acid and [Pd Cl2 (PPrtøfe] catalyst (20 mg, 0.032 mmof, 5 mol %) were added under argon atmosphere. The reaction mixture was stirred overnight at 80 0C. LCMS analysis of the reaction mixture demonstrated 65% of the target compound and 35% of starting material. After additional overnight stirring at 100 0C the conversion amounted to 90%. The solvent was removed in vacuo, the residual crude compound (7) was purified by column chromatography with methylene chloride as eluent. The yields averaged 5-45 mg, (10-50 %).
Figure imgf000106_0001
Table 15. Examples 148-149 of Pyrazolopyrimidines. Library R1.
Figure imgf000106_0002
Analytical Data for Library RI
148. 6-(4-fluorophenyl)-N-(4-methylphenyi)-1-propyl-1H-pyrazolo{3,4-d]pyrimidin-4- amine 1H NMR (DMSO, ppm) δ: 0.91 (t, J=7.3 Hz, 3H); 1.96 (m, 2H); 2.35(s, 3H); 4.39 (t, J=6.9Hz, 2H); 7.27(m, 4H); 7.75 (d, J=8.1 Hz, 2H); 8.12 (s, 1 H); 8.47 (m, 2H); 9.72 (bs, 1 H).
149. 6-(3-fluorophenyi)-N-(4-methylphenyi)-1-propyl-1H-pyrazolo[3,4-d]pyrimidin-4- amine 1H NMR (DMSO, ppm) δ: 0.91 (t, J=7.3 Hz, 3H); 1.96 (m, 2H); 2.35(S1 3H); 4.39 (t, J=6.9Hz, 2H); 7.25(m, 3H); 7.52 (m,1 H); 7.75 (d, J=8.1 Hz, 2H); 8.12 (m, 2H); 8.27 (d, J=7.7 Hz, 1 H); 9.72 (bs, I H). Library T
Figure imgf000107_0001
[0290] Compound 4 3c (15 g) was dissolved in 1 ,4-dioχane (300 ml) and diphosgene (21 mi) was added. Reaction mixture was heated at reflux for 15 hours. The solvent was removed under reduced pressure and precipitate obtained was filtered off and washed with dioxane afforded 4 as white solid. Yield 12 g (69%).
[0291] Compound 5 To a stirred solution of 4 (12 g) in POCI3 (100 ml.) PCI5 (25.4 g) was added. The reaction mixture was heated for 10 hours at reflux, then POCI3 was removed under reduced pressure and brown oil was used for the next step. Yield 9 g (63%).
[0292] Compound 6 To a stirred solution of o-methylbenzylamine (8.23g) and triethyiamine
(10 ml) in dioxane (200 mt) a solution of 5 (9 g) in 100 ml of dioxane was added. After 5 hours of stirring at 500C, the solvent was removed and the residue was dissolved in dichloromethane and water. The organic iayer was dried over sodium sulphate, and concentrated in vacuo to afford 6 as brown oil (15g, 76 %).
[0293] Compound 7 0.5 g of 6 and 0.5 g of amine was heated at 1900C for 30 min, poured onto acetonitrile, filtered, the solvent was removed and residue solid was used for the next step without further purification. Yield 0.4 g (52 %).
[0294] Compound 8 0.4 g of 7 was dissolved in 10 ml of HCI and stirred at RT for 7 hours, then concentrated in vacuo. The residue was neutralized with sodium carbonate, and extracted with dichloromethane. The organic layer was purified by column chromatography to afford S as brown oil (0.19 g, 52 %).
[0295] Compound 9 To a stirred solution of 8 (0.001 mol) and triethyiamine (0,5 ml) in 10 ml of dioxane a solution of sulfochloride (0.001 mo!) was added. After 5 hours of stirring, the solvent was removed and residue was treated with water, extracted with dichloromethane, and purified by column chromatography.
Figure imgf000108_0001
Table 16. Example 150 of Pyrazolopyrimidines. Library T
Figure imgf000108_0003
Analytical Data for Library T
150. N4-{2-methylbenzyI)-N6-{1-[{3-methylphenyl)sulfonyl]piperidϊn-4-yi}-1-propyl-1H- pyrazolot3,4-d]pyrimidine-4,6-diamϊne 1HNMR (DMSO, ppm) δ: 0.83 (t, J=7.3 Hz, 3H); 1.25-1.40 (m, 3H); 1.62 (bd, 2H); 1.80 (m, 2H); 2.32 (s, 3H); 2.40 (s, 3H); 3.00 (m, 2H); 3.30 (m, 1 H); 4.05<t, J=6.9Hz, 2H); 4.40 (m, 2H); 4.62 (d, J=5.6 Hz, 2H); 7.12 (m, 3H); 7.30 (m, 2H); 7.46 (m, 2H); 7.60- 7.80 (m, 4H). Library W
Figure imgf000108_0002
[0296] Compound 3 Acid 2 {16 mmol) was dissolved in dioxane and treated with CDi (16 mmoi). The reaction mixture was stirred for 1 hour at an ambient temperature, then treated with a solution of amine 1 (17 mmol). This mixture was stirred at 70 0C for 8 hours, then cooled and concentrated under reduced pressure. The residue was washed with 10 % aqueous NaHCOs, then washed with water and dried. [0297] Compound 4 A suspension of 3 was stirred for 8 hours at RT in HCl solution of dioxane, then the mixture was concentrated under reduced pressure. Target compound was obtained based on LCMS.
[0298] Compound 7 To a solution of 5 (9 mmol) in dioxane was added NEt3 (9 mmoi) and aniline 6 (9 mmol). The reaction mixture was stirred for 12 hours at 800C and then used without further purification,
[0299] Compound 8 To a solution of 7 (1 mmol) in dioxane was added NEt3 (1 mmoi) and 4
(1 mmol). The reaction mixture was stirred for 12 hours at reflux.
Figure imgf000109_0001
Table 17. Example 151 of Pyrazolopyrimidines. Library W
Figure imgf000109_0003
Analytical Data for Library W
151. 6-{4-benzoylpϊperazin-1-yl)-N-(3-fluorophenyi)-1 -propyl-1 H-pyrazolo[3,4- d]pyrimidtn-4-amine 1HNMR (DMSO1 ppm) δ: 0.88 (t, J= 7.4 Hz, 3H), 1.86 (m, 2H)1 3.62 (m,
4H), 3.87 (m, 4H), 4.15 (t, J = 7.0 Hz, 2H), 6.84 (m, 1 H)1 7.37 (m, 1 H), 7.46 (m, 5H), 7.54 (d, J=
8.2 Hz, 1 H), 7.69 (d, J= 11.8 Hz, 1 H), 8.01 (S, 1 H).
Library Z
Figure imgf000109_0002
5 [0300] Compound 3 To a solution of 1 (9 mmo!) in dioxane was added NEt3 (9 mmol) and aniline 2 (9 mmol). The reaction mixture was stirred for 12 hours at 80 0C and then used in further step.
[0301] Compound 5 To a solution of 3 (1 mmot) in n-butano[ was added (CH3COO)2Cu (1 mmol) and 4 (2 mmol). The reaction mixture was stirred under microwave irradiation for 2 hours at 120 0C.
[0302] Compound 6 A suspension of 5 was stirred for 8 h. at RT in HC! solution of dioxane, then concentrated under reduced pressure. Target compound was checked by LCMS.
[0303] Compound 7 A solution of 16 mmol of 6 in 50 ml of dioxane was treated with sulphochloride (16 mmol) and triethylamine (16 mmol). This mixture was stirred at reflux for 8 hours, then cooled and evaporated.
Table 18. Example 152 of Py razolopyrimidines. Library Z.
Figure imgf000110_0002
Analytical Data for Library Z
152. N4-(4-fluorophenyl)-N6-[1-(phenylsuifonyl)pϊperidin-4-yl]-1-propyl-1H-pyrazolo[3,4- d]pyrϊmidine-4,6-diamine 1H NMR (DMSO, ppm) : 0.84 (t, J= 7.3 Hz, 3H), 1.60 (m, 2H)1 1.81 (m, 2H), 1.99 (m, 2H), 2.60 (m, 2H), 3.64 (m, 2H)1 3.76 (m, 1 H), 4.06 (t, J = 6.8 Hz, 2H), 6.42 (d, J= 6.7 Hz, 1 H), 7.05 (t, J= 8.8 Hz, 2H), 7.72 (m, 7H)5 7.87 (s, 1H), 9.27 (s, 1 H). Library D
Figure imgf000110_0001
[0304] Compound 2a Boc-hydrazine 1a (50 g, 0.385 mmo!) was dissolved in acetone (500 mL) and the reaction mixture was stirred at 50 0C overnight. The solvent was removed under reduced pressure and the residue was re-suspended in Et2O and filtered. White crystals were obtained with yield 60.2 g (91 %). 1H NMR (DMSO, ppm) δ: 1.48 s (9H), 1.82 S (3H), 1.86 S (3H), 9.18 s (1 H).
[0305] Compound 3a 2a (63.5 g, 0.35 mol) was dissolved in toluene (500 mL); KOH powder (1 mol), BTEA-HOSO3 (10 mol %) and 1-bromopropane (0.4 mol) were added. The reaction mixture was stirred at 80 0C overnight. Ail solids were removed by filtration and the filtrate was washed twice with an equal volume of cold water. Then, the organic layer was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to provide light brown oil with yield 63.5 g (84 %). 1H NMR (DMSO, ppm) δ:0.82 m (3H), 1.4 s (9H), 1.52 m (2H), 1.8 s (3H), 2.0 s (3H), 3.38 m (2H).
[0306] Compound 4 3 (63.5 g, 0.296 mol ) was dissolved in THF (1 I) and 2 M aqueous HCI (500 mL) was added. The reaction mixture was refluxed overnight. At this time, the solvent was removed under reduced pressure and the product was dried by concentration from benzene to provide target compound as a hydrochloride (a gray precipitate) with yield 25 g (61 %). 1H NMR (DMSO, ppm) δ: 0.88 m (3H), 1.54 m (2H), 2.82 m (2H), 6.7 bs (3H). [0307] Compound 5 To a solution of 3 (144 mmol) in ethanol was added propylhydrazine (144 mmol) and sodium acetate (360 mmol). The reaction mixture was stirred for 12 hours at reflux. The ethanol was removed under reduced pressure; the precipitate was washed with ether and recrystallized from ethanol light to provide yellow precipitate with yield 16 g (66 %). [0308] Compound 6 5 (5 g, 30 mmol) was dissolved in 1 , 4- dioxane (30 mL) and diphosgene (11.87 g) was added. The reaction mixture was heated to reflux and stirred for 5 hours. Solvent was removed under reduced pressure and precipitate washed by ether to provide a brown precipitate with yield 3.5 g (60 %).
[0309] Compound 7 To a solution of 6 (2.8 g, 14 mmol) in POCI3 was added PCI5 (5.83 g, 28 mmol). The reaction mixture was stirred for 10 hours at reflux, then POCI3 was removed under reduced pressure, and the precipitate was washed with ether and used in the next step without further purification. LCMS analysis of the reaction mixture demonstrated presence of target compounds in quantity >70%. A dark brown oil was obtained in yield of 72 % (3 g). [03101 Compound 8 was To a solution of 7 (1 rnmoi) in dioxane was added NEt3 (1 mmol) and aniϋne (1 mmol). The reaction mixture was stirred for 12 hours at reflux and then used in the next step. LCMS analysis of the reaction mixture demonstrated presence of target compounds in quantity >60%. A dark brown solution was obtained with approximate yield of target compound 0.25 g (67 %),
[0311] Compound 9 To a solution of 8 (1 mmol) in dioxane was added NEt3 (1 mmol) and pyrrolidine (1 mmol). The reaction mixture was stirred for 12 hours at reflux. Then dioxane was removed under reduced pressure and precipitate was purified by silica gei chromatography using CH2CI2 as an eluent A white precipitate was obtained with yield 0.25 g (62%).
Figure imgf000112_0001
Table 19. Examples 153-164 of Pyrazolopyrimϊciines. Library D.
Figure imgf000112_0002
Figure imgf000113_0001
Analytical Data for Library D
153. 4-[(1-propyl-6-pyrrolidin-1-yl-1H-pyrazolo{3,4-d]pyrimidin-4-yi)amino]-N-pyrimidin-
2-yl-benzenesulfonamide 1HNMR (DMSO, ppm) δ. 0.86 t (3H, CH3), 1.86 q (2H1 CH2), 1.96 m (4H, 2CH2), 3.58 t <4H, 2CH2), 4.14 1 (2H1 CH2), 7.02 1 (1 H, Ar), 8.02 m (6H, Ar), 8.48 d (2H5 Ar), 9.7O d (1H1 Ar).
154. N-(2-methyfphenyl)-4-[(l-propyl-6-pyrrolidin-1-yl-1H-pyra2θlo[3,4-d3pyrimϊdin-4- yl)amino]benzenesulfonamide 1HNMR (DMSO, ppm) δ: 0.86 1 (3H, CH3), 1.86 q (2H, CH2), 1.96 rn (4H, 2CH2), 2.08 s (3H, CH3), 3.58 1 (4H, 2CH2), 4.14 t (2H1 CH2), 7.06 m (4H, Ar), 7.66 d (2H, Ar), 8.04 d (3H, Ar), 9.04 d (1 H1 Ar)1 9.70 s (1H, Ar)
155. N-(4-methy!phenyl)-4-[(1-propyi-6-pyrrolidin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4- yl)amino]benzenesulfonamide 1HNMR (DMSO, ppm) δ: 0.86 t (3H, CH3), 1.86 q (2H, CH2), 1.96 m (4H, 2CH2), 2.20 S (3H, CH3), 3.58 1 <4H, 2CH2), 4.14 t (2H, CH2), 7.02 t (4H, Ar), 7.70 d (2H, Ar), 8.04 d (3H, Ar), 9.64 d (2H1 Ar).
156. 4-{(1-propyl-6-pyrrolidϊn-1-yI-1 H-pyrazolo[3,4-d]pyrimidin-4- yl)amino]benzenesulfonamide 1HNMR (DMSO, ppm) δ: 0.86 t (3H1 CH3), 1.86 q (2H, CH2), 1.96 m (4H, 2CH2), 3.58 t (4H, 2CH2), 4.14 1 (2H, CH2), 6.94 s (2H, Ar), 7.82 d (2H, Ar), 8.06 m (3H, Ar), 9.64 d (2H, Ar). 157. N-[4-{methylsulfonyl)phenyl]-1-propyi-6-pyrrolidin-1-yl-1H-pyrazoIo{3,4-d]pyrimidin-
4-amine 1HNMR (DMSO, ppm) δ: 0.86 t (3H, CH3), 1.86 q (2H, CH2), 1.96 m (4H, 2CH2), 3.14 s (3H, CH3), 3.62 m (4H, 2CH2), 4.14 1 (2H, CH2), 7.88 d (2H, Ar), 8.06 s (1H, Ar), 8.18 d (2H, Ar), 9.74 s (1 H1 Ar).
158. N-(4-fIuorophenyl}-4-[(1-propyl-6-pyrrolidin-i-yl-1H-pyrazolo[3,4-d]pyrimidin-4- yl)amino]benzenesuifonamide 1HNMR (DMSO, ppm) δ: 0.86 1 (3H1 CH3), 1.86 q (2H, CH2), 1.96 m (4H, 2CH2), 3.58 1 (4H, 2CH2), 4.34 1 (2H, CH2), 7.02 1 (2H, Ar), 7.12 1 (2H, Ar), 7.68 d (2H, Ar), 8.04 t (3H, Ar), 9.66 s (2H, Ar)
159. N-{2-fluorophenyi)-4-[(1-propyl-6-pyrrolidin-1-yl-1H-pyrazolo[3,4-d]pyrimidm-4- yl)amino]benzenesuifonamide 1HNMR (DMSO, ppm) δ: 0.86 1 (3H, CH3), 1.86 q (2H, CH2), 1 ,96 m (4H, 2CH2), 2.96 1 (4H, 2CH2), 4.14 1 (2H, CH2), 7.12 t (3H, Ar), 7.28 1 (1 H, Ar), 7.70 d (2H, Ar), 8.04 t (3H, Ar), 9.58 d (2H, Ar)
160. N-{3-methoxyphenyl)-4-[(l-propyl-6-pyrroIidin-1-yI-1H-pyrazolo[3,4-d]pyrϊmidin-4- yl)amino]benzeπesuifonamϊde 1HNMR (DMSO1 ppm) δ: 0.86 1 (3H, CH3), 1.86 q (2H, CH2), 1.96 m (4H, 2CH2), 3.58 1 (4H, 2CH2), 3.68 s (3H, CH3), 4.34 1 (2H, CH2), 6.58 d (1 H, Ar)1 6.72 s (2H, Ar), 7.12 t (1H1 Ar), 7.74 d (2H, Ar), 8.041 (3H, Ar), 9.64 s (1 H, Ar), 9.72 s (1 H1 Ar).
161. 6-chloro-N-<3-fluorophenyl)-1-propyt-1H-pyrazolo[3,4-d]pyrimidin-4-amine 1HNMR (DMSO, ppm) δ: 0.90 t (3H), 1.90 m (2H), 4.26 t (2H), 6.98 1 (1 H), 7.48 m (2H), 7.74 d (1 H), 8.18 s (1H), 10.28 s (1 H)
162. 6-chloro-N-{4-fluoropheπyl)-1-propyl-1H-pyrazoiof3,4-dJpyrimidin-4-amine 1HNMR (DMSO, ppm) δ; 0.90 t (3H), 1.90 m (2H), 4.26 t (2H), 7.22 1 (2H), 7.72 1 (2H), 8.00 s (1 H), 10.18 s (1 H)
163. 6-chloro-N-phenyI-i-propyI-i H-pyrazoiotS^-dlpyrirnidin-^-amine 1HNMR (DMSO, ppm) δ: 0.80 t (3H); 1.85 m (2H); 4.25 t (2H); 7.20 t (1H); 7.45 1 (2H); 7.70 d (2H); 8.05 s (1 H); 10.20 S (1H).
164. {4-[(6-chloro1-propyM H-pyrazolo[3,4-d]ρyrimidin-4-yl)amino]phenyl}acetonitrile 1HNMR (DMSO, ppm) δ: 0.88 t (3H), 1.88 q (2H), 3.96 s (2H), 4.26 t (2H), 7.42 d (2H), 7.78 d (2H), 8.10 s (1 H).
Library M
Figure imgf000115_0001
[0312] Compound 2 A solution of 10 mmol of 1 in 10 ml of dioxane was treated with 4- carboxyphenyisulphonyl chloride (10 mmol) and triethy famine (22 mmoi). This reaction mixture was stirred at reflux for 3 hours, then cooled and concentrated. The residue was treated with water, and acidified with HCt. The formed precipitate was filtered off, carefully washed with water and dried, affording 2 as a white solid (2 g, 52 %).
[0313] Compound 3 Compound 2 (0.01 mol) was dissolved in DMF (75 mL) and CDI (0.01 moi) was added to the solution. The reaction mixture was heated at reflux for 2 hours; then amine (0,01 mol) was added to the mixture. After 5 hours of heating the reaction mixture was poured onto crushed ice and extracted with dichioromethane. The organic layer was separated and concentrated. The residue was purified by column chromatography, affording 3 as a white soiid (2.9 g, 66 %).
[0314] Compound 4 Compound 3 (0.01 mol) was treated with 10 % aqueous HCl at 600C for 7 hours; then the mixture was concentrated and the residue was air dried affording 4 as a white solid (2.9 g, 88 %).
[0315] Compound 5 Compound 4 (0,01 mol) was treated with corresponding dichloride (0.01 moi) and triethyiamine (0.02 mol) in dioxane for 5 hours. At this time, the solvent was removed and the residue brown OiI(LCMS H+1= 533.1 , 90 % purity) was used in the next step without further purification. The yield of crude 5 was 3.9 g, 71 %.
[0316] Compound 6 Compound 5 (0.001 mol) was treated with aniline (0.05 moi) for 60 hours at 120 0C, then the excess of aniiine was removed by distillation and the residue was purified by column chromatography and then by HPLC, affording 6 as pale yellow solid (0.19 g,
35 %).
Figure imgf000116_0001
Table 20. Example 165 of Pyrazoiopyπmϊdines. Library M
Figure imgf000116_0003
Analytical Data for Library IVI
165. N6-(3-fluorophenyl)-1-propyl-N4-{l-{[4-(pyrrolidin-1- yicarbonyiΪphenylJsϋifonyQpiperidϊn-^ylJ-IH-pyrazoloJS.Φdlpyrimidϊne^^-diamϊne
1HNMR (DMSO, ppm) δ: 0.851 (3H); 1.65 m (2H); 1.85 m (6H); 2.10 d (2H); 2.60 1 (2H); 3.10 m (4H); 3.74 d (2H); 4.10 t (3H); 6.62 t (1 H); 7.16 m (1 H); 7.44 d (1 H); 7.64 d (1 H); 7.74 d (2H); 7.85 d (3H); 7.94 s (1 H); 8,90 s (1H).
Library G
hr
Figure imgf000116_0002
[0317] Compound 5 To a solution of 3 (14 mmol) in ethanol was added chlorophenylhydrazine (15 mmol) and sodium acetate (21 mmol). The reaction mixture was stirred for 12 hours at reflux. The ethanol was removed under reduced pressure; the precipitate was washed with ether and recrystallized from ethanol. A light yellow precipitate was obtained with yield 2.9 g (87 %), [0318] Compound 6 5 (2.9 g, 12 mmoi) was fused with 5.05 g. of urea at 200 0C until the fused mass solidified. The sofid was cooled to RT and was dissolved in sodium hydroxide; the solution was treated with charcoal and filtered. The filtrate was acidified with glacial acetic acid and the white precipitate collected. The solid was washed with water and dried. A white precipitate was obtained with yield 2.5 g (79%).
[0319] Compound 7 To a solution of 6 (0.8 g, 3 mmol) in POCt3 was added PCI5 (1.25 g, 6 mmol). The reaction mixture was stirred for 10 hours at reflux, and the POCI3 was removed under reduced pressure, the precipitate was washed with ether and used in the next step without further purification. LCMS analysis of the reaction mixture demonstrated presence of target compounds in quantity >70%. A dark brown oil was obtained with approximate yield of target compound 0.7 g (77 %).
[0320] Compound 8 was synthesized according to the foiiowing procedure: To a solution of 7 (1.3 mmol) in dioxane was added NEt3 (1.3 mmol) and corresponding aniline (1.3 mmol). The reaction mixture was stirred for 12 hours at reflux and immediately subjected to the next step. LCMS analysis of the reaction mixture demonstrated presence of target compounds in quantity > 80%. A dark brown solution was obtained with approximately 81 % yield (0.3 g). [0321] Compound 9 To a solution of 8 (1 mmoi) in dioxane was added NEt3 (1 mmol) and pyrrolidine (1 mmol). The reaction mixture was stirred for 12 hours at reflux. The dioxane was removed under reduced pressure, and the precipitate was purified by silica gel chromatography using CH2CI2 as an eluent to provide a white precipitate with yield 0.25 g (62 %).
Figure imgf000117_0001
Table 21. Example 166 of Pyrazolopyrimidines. Library G.
Figure imgf000117_0002
Analytical Data for Library G
166. 1-(2-chlorophenyl)-N-(3-methy(phenyl)"6-pyrroitdin-1-yf-1H-pyrazolo[3,4- d]pyrimϊdin-4-amme 1H NMR (DMSO, ppm) δ: 1.90 t (4H, 2CH2), 2.34 s (3H, CH3), 3.50 m (4H, 2CH2), 6.88 d (1 H, Ar), 7.24 t (1 H, Ar), 7.50 m (3H, Ar), 7.66 t (2H, Ar), 7.80 s (1 H5 Ar), 8.20 s (1 H1 Ar), 9.34 s (1 H, Ar). Library X
Figure imgf000118_0001
Amine
Dioxane
Figure imgf000118_0003
Figure imgf000118_0002
[0322] Compound 2 Compound 1 {50.45 g) was treated with ethoxymethylenecyanoacetic ether (50 m!) in EtOH (500 ml) at reflux for 36 hours, the solvent was removed and residue was treated with water. The formed precipitate was filtered off, washed with acetonitrile and water and dried to afford 2 (45 g, 61 %) as white solid.
[0323] Compound 3 To a stirred solution of 2 ( 3.8 g) in 170 ml of THF at 00C 0.8 g of sodium hydride was added portionwise. After 1 hour of stirring, diphosgene (2 ml) was added dropwise to the mixture. After 2 days of stirring at RT, the reaction mixture was concentrated and treated with ammonia saturated dioxantc solution. After an additional 4 hours of stirring, the solvent was removed, and residue was filtered off and washed with acetonitrile to afford 3 (2.8 g, 63%) as white solid.
[0324] Compound 4 Compound 3 (2.8 g) was treated with sodium ethyiate (1.3 g) in EtOH
(30m!) at reflux for 36 hours, diluted with water, acidified with acetic acid, precipitate obtained was filtered off and dried to afford 4 (1.7 g,72%) as white solid.
[0325] Compound 5 To a solution of 4 (1.7 g) in POCI3 PCI5 (2.87 g) was added. The reaction mixture was stirred for 10 hours at reflux. Thereafter, POCI3 was removed under reduced pressure and the residue brown oil (1.3 g, 66%) was used for the next step.
[0326] Compound 6 To a solution of 5 (1 mmol) in dioxane was added NEt3 (1 mmol) and amine (1 mmol). The reaction mixture was stirred for 12 hours at reflux, the solvent was removed and residue was used for further step.
[0327] Compound 7 0.001 mol of 6 was treated with 0.001 moi of sodium ethyiate in 20 m! of ethanoi for 37 hours at reflux, the solvent was removed and residue was purified by column chromatography.
Figure imgf000119_0001
Figure imgf000119_0003
Analytical Data for Library X
167. 6-ethoxy-N-(4-fluorobenzyl)-1-{4-f!uorophenyI}-1H-pyrazolo{3,4-d]pyrimidin-4- amine 1H NMR {DMSO, ppm) δ: 1.34 (t, J=7.0 Hz, 3H); 4,40 (m, J=7.0Hz, 2H); 4.73 (d, J=5.9Hz, 2H); 7.13 (t, J=8.8Hz, J=9.1 , 2H); 7.32 (t, J=8.8Hz, J=9.1 , 2H); 7.82-7.92 (m, 3H); 8.17 (s, 1H); 8.67 (m,1H); 8.82 {bt, 1 H); 9.96(bs,1 H). Library G1 Preparation of Entries 168-169
Figure imgf000119_0002
Preparation of 1-(4-Methoxy-phenyl)»1H"pyrazolo[3,4-d]pyrimidin-4-ol (2)
[0328] A mixture of 5-amino-1 -(4-methoxy-phenyl)-1 H-pyrazole-4-carboxylic acid amide (1 )
(2.0 g, 8.6 mrrtol) and formamide (40 mL) was stirred at 150-170oC for 3 hours, cooled down to room temperature, diiuted with water. The formed solid was collected by filtration and recrystaJlized form ethanol to give compound 2. Yield 1.8 g, 86%.
Preparation of 4-Chioro-1-(4-methoxy-phenyi)-1H-pyrazoløt3I4-d]pyrimidme (3)
[0329] A mixture of compound 2 (1.5 g, 6.2 mmo!) and POCI3 (15 mL) was stirred at refiuxing for 4 hours, cooled down to room temperature, concentrated at reduced pressure and poured onto ice. The formed solid was collected by filtration and recrystatiized from ethanol to give 3.
Yield 1.5 g, 93%. 1 H-NMR (400MHz, DMSO-D6) δH: 3.82 (3H, s), 7.15 (2H, d, J=8.5 Hz), 7.96
(2H, d, J=8.5 Hz), 8.67 (1 H, s), 8.92 (1 H, s). LCMS tR (min): 1.79. MS (APCI), m/z 261.03,
263.02 [M+H]+. HPLC tR (min): 14.09. Mp 135-137oC Preparation of Furaπ-2-ylmethyI-[1-{4-methoxy-phenyI)-1 H-pyrazolo[3,4-d]pyrimidin-4-yI]- amine {4a-Entry 168)
[0330] A mixture of compound 3 (400 mg, 1.5 mmol), NEt3 (430 ml_, 3.0 mmol), furfurylamine (2) (300 mg, 3.0 mmoi) and acetornitrϊle (20 mL) was stirred for 3 hours at 1000C, cooied to room temperature, diluted with water. The formed solid was collected by filtration and recrystallized form ethanoi to give 4a. Yield 240 mg, 49%. 1 H-NMR (400MHz, DMSO-D6) δH: 3.80 (3H, s), 4.78 (2H, d, J=7.5 Hz), 6.35 (1 H1 1, J=3.6/1.8 Hz), 6.41 (1 H, d, J=3.6 Hz), 7.09 (2H, d, J=8.5 Hz), 7.58 (1 H, d, J-1.8 Hz), 8.01 (2H1 d, J=8.5 Hz), 8.37 (2H, m), 8.75 (1 H, t, J=7.5 Hz). LCMS tR (min): 1 ,66. MS (APCi), m/z 322.03 [M+HJ+. HPLC tR (min): 11.19. Mp 160-162oC. Preparation of [1-(4-Methoxy-phenyl)-1 H-pyrazoIo[3,4-d]pyrtmidin-4-yI]-thiophen-2- ylmethyϊ-amine (4b-Eπtry 169)
[0331] Yield 295 mg, 57%. 1H-NMR (400MHz, DMSO-D6) δH: 3.79 (3H, s), 4.93 (2H, d, J=7.5 Hz), 6.98 (1H, d/d, J=5.4/4.0 Hz)1 7.08 (2H1 d, J=8.5 Hz), 7.10 (1 H, d, J=3,5 Hz), 7.35 (1 H, d, J=4.0 Hz), 8.01 (2H, d, J=8.5 Hz), 8.33 (1 H, s), 8.38 (1 H, s), 8.89 (1H, t, J=7.5 Hz, broad). LCMS tR (min): 1.72. MS (APCi), m/z 337.97 [M+H]+. HPLC tR (min): 12.20. Mp 180- 1820C
Preparation of Entries 170-172
Figure imgf000120_0001
[0332] The 4- and 6-positions were further derivatized by O- and N-nucleophiles. First, the chlorine at 6-position was attacked by sodium ethoxide, furfurylamine and 4-methoxyaniline to provide intermediates 4a, 5a, 6a correspondingly. Then, the last chlorine was replaced by amines to give the desired compounds.
1 -(4-Methoxy-phenyI)-1 H-pyrazolo[3,4-dJpyrimidine-4,6-diol (2)
[0333] A mixture of 5-amino-1 -(4-methoxy-phenyl)-1 H-pyrazole-4-carboxyiic acid amide (1 )
(3.0 g, 13 mmol) and urea (6.2 g, 100 mmol) was stirred at 25O0C for 3 hours, cooled to room temperature and washed with 1 N aqueous NaOH solution. The formed solid was filtered off.
The mother liquid was acidified with acetic acid. The formed solid was collected by filtration, washed with water and dried in high vacuum to give compound 2. Yield 3.0 g, 90%,
4,6-Dichloro-1-{4-methoxy-pheπyϊ)-1H-pyrazolo[3)4-d]pyrimidϊne (3)
[0334] A mixture of compound 2 (300 mg, 1.1 mmol), freshly distilled POCI3 (20 ml_) and
PCI5 (1.2 g) was stirred at refiuxing for 2 hours and then cooied to room temperature. POCI3 was removed at reduced pressure. Crushed ice was added to the residue and the obtained mixture was stirred at room temperature. The formed solid was collected by filtration, washed with water and dried in high vacuum to give compound 3. Yield 160 mg, 47%.
6-C h loro-4-eth oxy-1 -(4-methoxy-ph eny l)-1 H-py razol o[3 ,4-cf] py rim id i ne (4a)
[0335] Sodium (14 mg, 0.6 mmol) was dissolved in ethanol (5 ml_) at room temperature. To the obtained solution compound 3 (120 mg, 0.4 mmol) was added at room temperature. The resulting mixture was stirred at RT for 3 hours. The solvent was removed at reduced pressure.
The residue was washed with ethano I/water, ether and dried giving compound 4a. Yield 38 mg,
30%. 1 H-NMR (400MHz, CDCI3) 6H: 1.52 (3H, t, J=7.5 Hz)1 3.89 (3H, s), 4.70 (2H, q, J=7.5
Hz), 7.06 (2H, d, J=8.5 Hz), 8.01 (2H, d, J=8.5 Hz), 8.16 (1 H, s), LCMS tR (min): 2.04. MS
(APCI), m/z 304.98, 306.95 [M+H]+. HPLC tR (min): 16.28. Mp 148-150oC. t4-Ethoxy-1-(4-methoxy-phenyl)-1H-pyrazolo[3,4-d]pyrirnrdin-6-yf]-furan-2-ylmethyl-amϊne
(4b-Entry 170)
[0336] A mixture of compound 4a (40 mg, 0.13 mmol), furfurylamine (2) (38 mg, 0.39 mmol),
NEtS (0.055 mL0.39 mmol) and acetonitrile was stirred at refiuxing for 32 hours, cooled to RT, concentrated and extracted with chloroform. The combined organic solutions were concentrated and the residue was purified by prepTLC (50% chloroform/ethanol) giving compound 4b. Yieid
13 mg, 27%. 1 H-NMR (400MHz1 CDCI3) δH: 1.45 (3H, t, J=7.5 Hz)1 3.88 (3H, s), 4.55 (2H1 q,
J=7.5 Hz)1 4.70 (2H, d, J=7.5 Hz), 5.42 (1 H, broad), 6.28 (1 H, broad), 6.34 (1 H, broad), 7.01
(2H, d, J=8.5 Hz), 7.38 (1 H, s), 7.94 (1 H, s), 8.10 (2H, d, J=8.5 Hz). LCMS tR (min): 2.06, MS
(APCI), m/z 366.03 [M+HJ+. HPLC tR (min): 16.09.
[6-Chloro-1-(4-methoxy-phenyl)-1H-pyra2oIo[3,4-d]pyrimidin-4-yl]-furan-2-ylmethyt-amine
(5a)
[0337] A mixture of compound 3 (100 mg, 0.33 mmol), furfurylamine (2) (33 mg, 0.33 mmol),
NEt3 (0.048 mL, 0.33 mmol) and acetonitrϋe (2 mL) was stirred at room temperature for 24 hours. The solvent was removed at reduced pressure. The residue was diluted with water. The formed solid was collected by filtration, washed with ethanoi/water (1/1 ) and dried in high vacuum to give compound 70. Yield 55 mg, 46%.
N4-Furan-2-ylmethyl-1-(4-methoxy-phenyl)-N6,N6-dimethyl-1H-pyrazolo[3,4-d3pyrimϊdine-
4,6-diamine (5b-Entry 172)
[0338] A mixture of compound 5a (20 mg, 0.056 mmoi) and 40% aqueous NMe2 solution (2.5 mL) was stirred at 1000C for 8 hours, cooled to room temperature and concentrated at reduced pressure. The residue was purified by prepTLC (20% ethy! acetate/hexane) to give compound 5b. Yield 5 mg, 24%. 1 H-NMR (400MHz1 DMSO-D6) δH: 3 12 (6H, s), 3 78 (3H, s), 4.65 (2H, d, J=7.5 Hz), 6.30 (1 H, broad), 6.40 (1 H, broad), 7.03 (2H, d, J=8.5 Hz), 7.55 (1 H, s), 8.05 (1 H, s), 8.10 (2H, d, J=8.5 Hz), 8.30 (1 H, broad). LCMS tR (min): 2.10. MS (APCi), m/z 365.14 [M+H]+. HPLC tR (min): 13.98. Mp 116-1180C.
[6-Chloro-1-(4-methoxy-phenyl)-1H-pyrazolo[3,4-d]pyrimidtn-4-yl]-(4-methoxy-phenyl)- amine (6a)
[0339] A mixture of compound 3 (110 mg, 0.37 mmol), acetonitrite (2 mL), p-methoxyaniline (61 ) (46 mg, 0.37 mmol) and NEtS (0,052 mL, 0.37 mmoi) was stirred at room temperature for 24 hours. The solvent was removed at reduced pressure. The residue was diluted with water. The formed solid was collected by filtration, washed with ethanol/water (1/1) and dried in high vacuum to give compound 6a. Yield 53 mg, 38%.
1,N4-Bis-(4-methoxy-phenyl)-Nβ,Ne-dimethyl-1H-pyrazolθE3,4-d]pyrimidine-4,6-dϊamme (6b-Entry 172)
[0340] A mixture of compound 6a (130 mg, 0.34 mmol), HNMe2 hydrochloride (83 mg, 1 mmol), DIPEA (220 mg, 1.7 mmol) and ethanol (20 mL) was stirred at room temperature for 30 minutes. Then the reaction mixture was irradiated in MW at 19O0C for 1 hour, cooled to room temperature and diluted with water. The formed solid was collected by filtration and purified by prepTLC (5% methanoi/chloroform) to give compound 6b. Yield 8 mg, 6 %. 1 H-NMR (400MHz, CDC13) δH: 3.29 (6H, broad), 3.88 (6H1 s), 6.76 (1 H, broad), 6.78-7.13 (1 H, broad, Z/E forms), 6.99 (4H, d, J=8,5 Hz), 7.43 (2H, broad), 8.15 (2H, broad). LCMS tR (min): 2.04. MS (APCi), m/z 391.03 [M+H]+. HPLC tR (min): 14.67. Table 23. Examples 168-172 of Pyrazotopyrimidines. Library G1
Figure imgf000122_0001
Figure imgf000123_0002
Library G3 {Preparation of Entries 173-186)
Figure imgf000123_0001
5-Amino-1-benzyl-1 H-pyrazo!e-4-carbonϊtriie (2a)
[0341] To a solution of t-BuOK (11 g, 50 mmol) in anhydrous methanol (100 mL) benzyl- hydrazine (4.88 g, 25 mmol) was added. The obtained suspension was stirred at room temperature for 15 minutes. Then 2-ethoxymethylene-malononitrile (1 ) (3.05 g, 25 mmol) was added. The resulting mixture was stirred at refluxing for 3 hours and cooled down to RT, The formed solid was collected by filtration and washed with water, giving compound 2a. Yield 3.0 g,
61 %.
S-Amino-i-benzyl-IH-pyrazoie^-carboxyiic acid amide (3a)
[0342] To a solution of potassium hydroxide (10.0 g, 18 mmol) in water (100 mL) hydrogen peroxide (25 mL) and the solution of compound 2a (3 g, 15.2 mmol) in dioxane were added. The resulting mixture was stirred at room temperature for 8 hours. The formed solid was collected by filtration, washed with water and dried, giving compound 3a. Yield 2.74 g, 84%, i-Benzyl-IH-pyrazolofS^-dlpyrimidine^.e-diol (4a)
[0343] Compound 3a (2.74 g, 13 mmol) was fused with urea (10.0 g, 17 mmol) at 2000C for 3 hours. The cooled solid mass was dissolved in 1 N aqueous solution of sodium hydroxide (100 ml_) and acidified with acetic acid. The precipitate formed was collected by filtration, washed with water and dried at 50°C for 10 hours, furnishing compound 4a. Yield 4 g, -100%.
1-Benzyl-4,6-dichioro-1H-pyrazoio[3,4-dJpyrimidine (5a)
[0344] A mixture of compound 4a (2 g, 5.8 mmol), POCI3 (6 mL) and PCI5 (8.0 g) was stirred at refluxing for 14 hours, cooled down to room temperature, poured onto crushed ice and extracted with dichloromethane. The combined organic phases were washed with water, dried over sodium sulfate and concentrated. The residue was purified by column chromatography
(silica gel, DCM/hexane) to give compound 5a. Yield 620 mg, 39%.
(1 -Benzyl-6-ch!oro-1 H-pyrazolo[3,4-d]pyrimidin-4-yl)-(3-chloro-4-fluoro-phenyl)-amine (6a)
[0345] To a solution of compound 5a (150 mg, 0.54 mmol) in acetonitrile (5 mL) 3-chloro-4- fluoro-phenylamine (78 mg, 0.54 mmol) and NEt3 (61 mg, 0.6 mmol) were added. The reaction mixture was stirred for 24 hours at room temperature and diluted with water. The precipitate formed was collected by filtration and washed with water and hexane. Purification by column chromatography (silica gel, 2% acetone/DCM) gave compound 6a, Yield 75 mg, 36%.
(1-Benzyl-6-pyrrolidin-1-yf-1H-pyrazolo[3,4-d]pyrimidϊn-4-yI)-{3-chloro-4-fluoro-phenyl)- amine (7a-Entry 173)
[0346] A mixture of compound 6a (75 mg, 0.19 rnrnoi) and pyrrolidine (0.3 mL) was stirred at refluxing for 3 hours, cooled down to room temperature and concentrated. Purification by recrystallization from acetonitrile gave compound 7a. Yield 60 mg, 75%. 1H-NMR (400MHz,
DMSO-D6) δH: 1 ,94 (4H, broad peak), 3.68 (4H, broad peak), 5.32 (2H, s), 7.27 (5H, m), 7.37
(1 H, d/d, J=8.5/8.0 Hz), 7.72 (1 H, broad peak), 8.02 (1 H, s), 8.40 (1H, broad peak), 9.72 (1 H, broad peak). MW 422.9. LCMS tR (min): 2.35. MS (APCi), m/z 423.19; 425.19 [M+Hf . HPLC tR
(min): 17.43. Mp 218-2200C.
5-Amino-1-(3-chloro-phenyl)-1 H-pyrazole-4-carbonitrϊle (2b)
[0347] 2-Ethoxymethylene-malononttrile 1 (3.44 g, 28.17 mmol) was added slowly to hot ethanolic solution of (3-chloro-phenyl)-hydrazine (5.044 g, 28.17 mmol) and NEt3 (3.96 mL,
28.17 mmol) in ethanol (20 mL). The mixture was stirred at RT for 3 hours and cooled. The formed precipitate was filtered off, washed with small amount of ethanol and ether, dried in drying oven, to give the pure compound (2b). Yield 6.22 g, 99%.
5-Amino~1-(3~ch!orophenyl)-1H-pyrazole-4-carboxylic acid amide (3b)
[0348] The compound 46b (6.02 g, 27.53 mmol) was added gradually to the iced sulfuric acid
(24 mL) within 3 hours, and the reaction temperature was allowed to warm to room temperature.
The reaction mixture was stirred at room temperature for 2 hours. Then, the resulting solution was poured into crushed ice, cooled and neutralized with concentrated aqueous ammonia keeping the reaction temperature below 3O0C. Then, the formed precipitate was filtered off, washed with water, and dried in drying oven at 650C to give the pure compound 3b. Yield 4.204 g, 64%.
1-(3-Chforo-phenyi)-1H-pyrazolo[3,4-d]pyrimϊdine-4,6-dio! (4b) [0349] Yield 239 mg, 72%.
4,6-Dichloro-1 -{3-chloro-phenyl)-1 H-pyrazolo[3,4-d]py rimidine (5b)
[0350] Yield 216 mg, 90%.
[B-Chloro-i-tS-dichlorophenylJ-IH-pyrazoloIS^-dJpyrimidrn^-ylJ-m-tolyl-amine (6b)
[0351] The mixture of the compound 5b (200 mg, 0,67 mmol), m-tolylamine (105 mg, 0.73 mmol) and K2CO3 (202 mg, 1.46 mmol) in acetonitriie (5 mL) was stirred at room temperature for 5 hours. Then, the reaction mixture was diluted with water, and the product was extracted with ethyl acetate. Extract was washed with water, brine, dried over Na2SO4. Purification by chromatography on silica gel (DCM) gave the pure compound 6b as white solid. Yield 216 mg,
59%.
[i-IS-Chloro-pheπylJ-β-pyrrolidin-i-yl-IH-pyrazoiotS^-dlpyrtmidin^-ylJ-m-tolyl-amine tZb-
Entry 174)
[0352] A mixture of the compound 6b (150 mg, 0.41 mmol) and pyrrolidine (1 mL, 11 ,98 mmol) was stirred at 800C for 40 minutes. When the reaction was over according to TLC, the reaction mixture was cooled, poured into water. The precipitated product was filtered off, washed with water. Re-crystallization from ethanol gave the pure compound 7b. Yield 62 mg,
37%. 1H-NMR (400MHz, DMSO-D6) δH: 1.95 (4H, m), 2.32 (3H, s), 3.60 (4H, broad), 6.87 (1 H, d, J=8.5 Hz), 7.22 (1 H, t, J=8.5 Hz), 7.26 (1 H, d, J=8.5 Hz), 7.50 (1 H1 1, J=8.5 Hz), 7.68 (1 H, d,
J=8.5 Hz), 7.80 (1 H, s), 8.30 (2H, broad peak), 8.50 (1H, S)1 9.62 (1 H, broad peak). MW 404.90.
LCMS tR (min): 2.52. MS (APCI), m/z 405.26; 407.25 [M+H]\ HPLC fe (min): 20.27. Mp 243-
2440C.
5-Amino-1-(4-chloro-phenyt)-1H-pyrazole-4-carbonitrile (2c)
[0353] 2-Ethoxymethylene-malononitriie 1 (3.44 g, 28.17 mmol) was added slowly to hot ethanolic solution of (4-chloro-pheny!)-hydrazine (5.044 g, 28.17 mot) and triethanolarnine (3.96 mL, 2.851 g, 28.17 mmol) in ethanol (20 mL). The mixture was stirred at RT for 3 hours and cooled. The formed precipitate was filtered off, washed with a smal! amount of ethanol and ether, and dried in a drying oven to give the pure compound 2c. Yield 6.15 g, 99%.
5-Amino-1-(4-chIoro-phenyl)-1H-pyrazole-4-carboxylic acid amide (3c)
[0354] The compound 2c (6.107 g, 27.93 mmol) was added gradually to the iced sulfuric acid
(24 mL) within 3 hours and then the reaction temperature was allowed to warm to room temperature. The reaction mixture was stirred at room temperature for 2 hours and left overnight. Thereafter, the resulting solution was poured into crushed ice, cooled and neutralized with concentrated aqueous ammonia, keeping the reaction temperature below 300C, and left overnight. Then, the formed precipitate was filtered off, washed with water, and dried in drying oven at 650C to give the pure compound 3c. Yield 4.664g, 71 %.
1-{4-Chloro-phenyi)-1 H-pyrazolo[3,4-d3pyrimidiπe-4,6-diol (4c)
[0355] Yield 298 mg, 89%.
4,6-DichIoro-1-(4-chIoro-phenyl)-1H-pyrazoIo[3,4-d]pyrimidine (5c) [0356J Yield 221 mg, 74%.
[θ-Chioro-i-tΦdichioro-phenyO-IH-pyrazolotS^-dJpyrimϊdin-Φyll-m-tolyl-amine (6c) [0357J The mixture of the compound 5c (200 mg, 0.67 mmol), m-tolylamine (105 mg, 0.73 mmol) and potassium carbonate (202 mg, 1.46 mmol) in acetonitrile {5 ml_) was stirred at room temperature for 5 hours. Thereafter, the reaction mixture was diluted with water, and the product was extracted with ethyl acetate. Extract was washed with water, brine, dried over Na2SO4. Purification by chromatography on silica gel (DCM) gave the pure compound 6c as white solid. Yield 221 mg, 89%.
[i-t-Φ-Chloro-phenylJ-θ-pyrrolidin-i-yl-IH-pyrazolofSjΦdlpyrimidin-ΦylJ-ni-tolyl-amine (7c- Entry 175)
[0358] A mixture of the compound 6c (146 mg, 0.39 mmoi) and pyrrolidine (1 mL, 11.98 mmol) was stirred at 8O0C for 40 minutes. When the reaction was over according to TLC, the reaction mixture was cooled and poured into water. The precipitated product was filtered off, and washed with water. Purification by recrystallization with ethanol gave the pure compound 7c as white solid. Yield 58 mg, 36%. 1H-NMR (400MHz, DMSO-D6) δH: 1.95 {4H, m), 2.32 <3H, s), 3.60 {4H, m), 6.87 (1 H, d, J=8.5 Hz), 7.22 (1 H, t, J=8.5 Hz), 7,52 (2H, d, J=8.5 Hz), 7.68 {1 H, d, J=8.5 Hz), 7.80 (1 H, s), 8.27 (1 H, s), 8.34 (2H, d, J=8.5 Hz), 9.60 (1 H, broad). MW 404.90. LCMS tR (min): 2.51. MS (APCi), m/z 405.25; 407.25 [M+Hf. HPLC tR (min): 19.76. Mp 246- 2470C.
5-Amino-1-(2,5-dtchIoro-phenyl)-1H-pyrazole-4-carbonitrile {2d) [0359] 2-Ethoxymethylene-malononϊtriie 1 (1 mg, 8.5 mmol) was added slowly to hot ethanolic solution of (2,5-dichloro-phenyl)-hydrazine (1. 5 g, 8.5 mmoi) and NEt3 (860 mg, 8.5 mmol) in ethanol (10 mL). The mixture was stirred at RT for 3 hours and cooled. The formed precipitate was filtered off, washed with small amount of ethanol and ether, and dried in a drying oven to give the pure compound 2d. Yield 2.0 g, 93%.
5-Amiπo-1-(2,5-dichloro-phenyl)-1H-pyrazote-4-carboxylic acid amide (3d) [0360] The compound 2d (2 g, 7.9 mmoi) was added gradually to the iced sulfuric acid (24 mL) within 3 hours, and then the reaction temperature was allowed to warm to room temperature. The reaction mixture was stirred at room temperature for 2 hours and left overnight. Thereafter, the resulting solution was poured into crushed ice, cooled and neutralized with concentrated aqueous ammonia keeping the reaction temperature below 3O0C, and left overnight. Then the formed precipitate was filtered off, washed with water, and dried in a drying oven at 650C to give the pure compound 3d. Yield 1.2 g, 59%. 1-(2,5-Dichloro-phenyl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-dioi (4d) [0361] The compound 3d (1.2 g, 4.4 mmol) was added in small portions to the melted urea (6.5 g, 0.108 mol), and heated for 1 hour at 21O0C till the reaction mixture became soϊid. Then, an additional amount of urea was added (3 g, 50 mmol) and the reaction mixture was heated for 1.5 hour at 2100C until it became solid again. Then, the reaction mixture was cooled, dissolved in 10% aqueous solution of NaOH, warming at 500C. Unresolved solid was filtered off. The mother liquid was cooled, and neutralized with concentrated acetic acid. The formed precipitate was filtered off, washed with water, with refluxing methanol, ether, and dried at 5O0C. As a result, the product {4.5 g) was obtained as a mixture with unknown admixtures, and used in the next step without additional purification.
4,6-Dichloro-1-(2,5-dichioro-phenyl)-1H-pyrazolo[3,4-d]pyrimidine (5d) [0362] Yield 650 mg, 43%.
[6-Chloro-1-(2,5-dichloro-phenyl)-1H-pyrazolo[3,4-d]pyπmidin-4-yi3-m-toiyl-amine (6d) [0363] To a solution of m-tolylamine hydrochloride (145 mg, 1 mmol) and K2CO3 (290 mg, 2 mmol) in ethanoi (3 mL) and in acetonitrile (1 mL) the compound 5d (334 mg, 1 mmoi) was added. The reaction mixture was stirred at 70-800C for 2 h, cooled to room temperature, and diluted with water. The formed precipitate was filtered off, washed with water, dried, and purified by column chromatography on silica gel (DCM/hexane, 1/1 ) that gave the compound 5Od. Yield 374 mg, 92%.
[1-(2,5-Dichloro-phenyl)-6-pyrrolidin-1-yl-1H-pyrazolo{3,4-d]pyrimidin-4-yl]-m-tolyI-amine (7d-Entry 176)
[0364] Yield 230 mg, 93%. 1H-NMR (400MHz, DMSO-D6) δH: 1.90 (4H, m), 2.32 (3H1 s), 3.49 (4H, broad m), 6.88 (1 H, d, J=8.5 Hz), 7.24 (1 H, t, J=8.5 Hz), 7.58 (1 H, d, J=8.5 Hz), 7.69 (3H, superposition of one s and two d), 7.81 (1 H, s), 8.27 (1 H, s), 9.60 (1 H, broad). MW 439,35, LCMS .R (min): 1.36. MS (APCI), m/z 439.18; 441.18 [M+Hf . HPLC tR (min): 17.78. MP 103- 1040C.
5-Amino-1-(2-chloro-phenyl)-1H-pyrazole-4-carbonitrile (2e)
[0365] To a solution of t-BuOK (4.6 g, 40 mmol) in anhydrous EtOH (30 mL) (2-chloro- phenyl)-hydrazine (7,33 g, 40 mmol) was added. The obtained suspension was stirred at room temperature for 15 minutes. Then 2-ethoxymethylene-mafononitrile (3.05 g, 25 mmol) was added. The resulting mixture was stirred at refluxing for 2 hours and cooled down to room temperature. The formed solid was collected by filtration and washed with water, ethanoi and ether to give compound 2e. Yield 4.3 g, 49%.
5-Amino-1-{2-chIoro-phenyI)-1H-pyrazole-4-carboxylic acid amide (3e) [0366] To pre-cooled in ice-bath, concentrated H2SO4 (20 mL), compound 2e (4.2 g, 19.0 mmol) was added portionwise keeping internal temperature of the reaction mixture 8-100C. The mixture was stirred at room temperature for 1.5 hours, poured onto crushed ice, neutralized with concentrated aqueous ammonia solution, keeping temperature below 500C, and cooled to room temperature. The formed solid was collected by filtration and dried giving compound 3e, used in the next stage without additional purification. Yield 3.554 g, 91 %. 1-{2-Chloro-phenyl)-1 H-pyrazolo[3,4-d]pyrimidine-4,6-diol (4e)
[0367] Compound 3e (4 g, 14.0 mmol) was fused with urea (25 g, 416 mmol) at 2000C for 2 hours. The cooled solid mass was dissolved in 1 N aqueous solution of sodium hydroxide (100 mL) and acidified with acetic acid. The precipitate formed was collected by filtration, washed with water and dried at 900C for 6 hours, furnishing compound 4e as a mixture with undetectable byproducts (15 g). The material was used in the next stage without additional purification,
4,6-Dichioro-1-{2-chloro-phenyl)-1H-pyrazolot3,4-d]pyrtmidine (5e)
[0368] A mixture of compound 4e (15 g), POCI3 (15 mL) and PCI5 (25 g) was stirred at refluxing for 4 hours, cooled to room temperature, poured onto crushed ice and extracted with dichloromethane. The combined organic phases were washed with water, dried over sodium sulfate and concentrated. The residue was purified by column chromatography (silica gel,
DCIWhexane) to give compound 5ea. Yield 3.989 g, 95%.
[e-Chloro-i^-chloro-phenyO-IH-pyrazoiofS^dJpyrimidin-ΦylJ-m-tolyl-amiπe (6e)
[0369] To a solution of compound 22a (1.018 g, 3.4 mmoi) in acetonttrile (3 mL) a solution of
3-methylani!ine (500 mg, 3.5 mmol) and K2CO3 (968 mg, 7.0 mmol) in ethanol (8 mL) was added. The reaction mixture was stirred for 2 hours at 8O0C and diluted with water. The precipitate formed was collected by filtration, washed with water and dried giving compound 6e.
Yield 1.175 g, 93%.
[1~{2-Chloro-phenyl)-6-pyrroltdin-1-yi-1H-pyrazoϊo[3,4-d]pyrlmidin-4-yl3-m-tolyI-amine (7e-
Entry 177)
[0370] A mixture of compound 6e (370 mg, 1.0 mmol) and pyrrolidine (8) (500 mg, 7.0 mmol) was stirred at refluxing for 1 hour, cooled down to room temperature and concentrated.
Purification by column chromatography (silica gel, DCM) gave compound 7e.
[0371] Yield 125 mg, 31%. 1H-NMR (400MHz, DMSO-D6) δH: 1.89 (4H, m), 2.33 (3H, s), 3.48
(4H, broad m), 6.87 (1 H, d, J=8.5 Hz), 7.23 (1 H, t, J=8.5 Hz), 7.50 (1 H, broad), 7.55 (2H, broad), 7.67 (1 H, broad), 7.71 (1 H, d, J=8.5 Hz), 7.82 (1 H, s), 8.26 (1 H, s), 9.60 (1 H, broad).
MW 404.91 , LCMS tR (min): 2.21. MS (APCI), m/z 405.21 , 407.23 [M+Hf. HPLC t1, (min): 15.78.
Figure imgf000128_0001
5-Amino-1-(3-fluoro-phenyl)-1H-pyrazole-4-carbonitri!e {2f)
[0372] To a solution of t-BuOK (3.45 g, 30.7 mmol) in anhydrous EtOH (20 mL) 3-fluoro- phenyi-hydrazine (5 g, 30.7 mmol) was added. The obtained suspension was stirred at room temperature for 15 minutes. Then 2-ethoxymethytene-malononitriie (3.75 g, 30.7 mmol) was added. The resulting mixture was stirred at refluxing for 3 hours and cooled down to room temperature. The formed solid was collected by filtration, washed with water and dried giving compound 2f. Yield 3.78 g, 61 %.
5"Amrno-1-(3-fluoro-phenyI)-1H-pyrazole-4-carboxylic acid amide (3f)
[0373] To a 15 ml of concentrated H2SO4 cooled in ice-bath compound 2f (3.75 g, 18.5 mmol) was added portionwise keeping internal temperature of the reaction mixture 15-2O0C. The mixture was stirred at room temperature for 1 hour, poured onto crushed ice, neutralized with concentrated aqueous ammonia solution keeping temperature below 500C and cooled to room temperature. The formed soiid was collected by filtration and dried, giving compound 3f. Yield
3.6 g, 88%.
1-(3-Fluoro-phenyl)-1H-pyrazolot3,4-d]pyrimidine-4,6-dio! {4f)
[0374] Compound 3f (3.6 g, 16.3 mrnol) was fused with urea (20.0 g , 33.33 mmol) at 2000C for 3 hours. The cooled solid mass was dissolved in 1 N aqueous solution of sodium hydroxide
(50 m L) and acidified with formic acid. The precipitate formed was collected by filtration, washed with water, dried and purified by recrystallization from methanol, furnishing compound 4f. Yieid
3.04 g, 76%.
4,6-Dichloro-1 -{3-fluoro-phenyl)-1 H-pyrazolo[3,4-d]pyrimidine (5f) [0375] A mixture of compound 4f (2.3 g, 9.35 mmol), POCI3 (9 mL) and PCI5 (30 g) was stirred at refiuxing for 20 hours, cooled down to room temperature, poured onto crushed ice and extracted with dichloromethane. The combined organic phases were washed with water, dried over sodium sulfate and concentrated. The residue was purified by column chromatography (silica gel, DCM/hexane) to give compound 5f. Yield 1.7 g, 64%. [6-Chloro-1-(3-fluoro-phenyI)-1H-pyrazolo[3,4-d]pyrimidin-4-ylI-m-tolyl-amine (6f) [0376] To a solution of compound 5f (500 mg, 1.77 mmol) in acetonitrile (5 mL) 3- methylaniline (254 mg, 1.77mmol) and NEt3 (393 mg, 3.89 mmol) were added. The reaction mixture was stirred for 8 hours at room temperature and diluted with water. The precipitate formed was collected by filtration, washed with water and acetonitrile and dried giving compound 6f. Yield 530 mg, 85%.
[1-(3-Fluoro-phenyl)-6-pyrrolidin-1-yl-iH-pyrazoio[3,4-d]pyrimidin-4-yl]-m-toiyl-amine {7f- Entry 178)
[0377] A mixture of compound 6f (250 mg, 0.65 mmol), pyrrolidine (1 mL) and acetonitrile (2 mL) was stirred at refiuxing for 3 hours, cooled down to room temperature and concentrated. Purification by recrystailizatton from acetonitrile gave compound 7f. Yield 235 mg, 93%.1H- NMR (400MHz, DMSO-D6) δH: 1.97 (4H, m), 2.31 (3H, s), 3.60 (4H, m), 6.87 (1 H, d, J=8.5 Hz),
7.05 (1 H, t, J=8.5 Hz), 7.23 (1 H, dd, J=8.5/8.0 Hz), 7.51 (1 H, superposition of two d, J=8.5/8.0 Hz), 7.69 (1 H, d, J=8.5 Hz), 7.80 (1 H, s), 8.17 (1 H, d, J=8.0 Hz), 8.23 (1 H, d, J=8.5 Hz), 8.29 (1 H, s), 9.62 (1H1 broad). MW 388.45. LCMS fe (min); 2.42. MS (APCl), m/z 389.25 [M+Hf. HPLC tR (min): 19.27. MP 254-2550C.
5-Amino-1-benzyl-1 H-pyrazole-4-carbonitrile (2g)
[0378] To a solution of t-BuOK (5.6 g, 50 mmol) in anhydrous methanol (100 mL) benzyl- hydrazine (4.88 g, 25 mmol) was added. The obtained suspension was stirred at room temperature for 15 minutes. Then 2-ethoxymethylene-malononitri!e (3.05 g, 25 mmol) was added. The resulting mixture was stirred at refiuxing for 3 hours and cooled down to room temperature. The formed solid was collected by filtration and washed with water giving compound 2g. Yield 3.0 g, 61 %. S-Amino-i-benzyi-IH-pyrazole-Φcarboxylic acid amide (3g) [0379] To a solution of potassium hydroxide (10.0 g, 18 mrnol) in water (100 ml_) hydrogen peroxide (25 ml_) and the solution of compound 2g (3 g, 15.2 mmot) in dioxane were added. The resulting mixture was stirred at room temperature for 8 hours. The formed soiid was coiiected by filtration, washed with water and dried, giving compound 3g. Yield 2.74 g, 84%. 1-Benzyl-1H-pyrazolo[3,4-d]pyrimidine-4,6-diol (4g)
[0380J Compound 3g (2.74 g, 13 mmol) was fused with urea (10.0 g, 17 mmo!) at 2000C for 3 hours. The cooled solid mass was dissolved in 1 N aqueous solution of sodium hydroxide (100 mL) and acidified with acetic acid. The precipitate formed was coiiected by filtration, washed with water and dried at 50°C for 10 hours furnishing compound 4g. Yield 4 g, -100%.
1 -Benzyl-4,6-dichioro-1 H-py razolo[3,4-d]pyrimidine (5g)
(0381] A mixture of compound 4g (2 g, 5.8 mmol), POCi3 (6 mL) and PCI5 (8.0 g) was stirred at refluxing for 14 hours, cooled down to room temperature, poured onto crushed ice and extracted with dichioromethane. The combined organic phases were washed with water, dried over sodium sulfate and concentrated. The residue was purified by column chromatography
(silica get, DCM/hexane) to give compound 5g. Yield 620 mg, 39%.
(1 -Benzyl-6-chloro-i H-pyrazolo{3,4-d]pyrimidin-4-yl)-m-tolyI-amine (6g)
[0382] To a solution of compound 5g (200 mg, 0.72 mmoi) in acetonitrile (3 mL) 3- methyianitine (103 mg, 0.72 mmol) and NEt3 (0.2 mL) were added. The reaction mixture was stirred for 20 hours at room temperature and diluted with water. The precipitate formed was collected by filtration and washed with water and hexane. Purification by coiumn chromatography (silica gel, ethyl acetate/hexane, 1/4) gave compound 6g. Yieid 190 mg, 76%.
(1-tert-Beπzyl-6-pyrrolldin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4-yi)-m-tolyl-amine (7g-Entry
179)
[0383] A mixture of compound δg (180 mg, 0.23 mmoi) and pyrrolidine (8) (2 mL) was stirred at refluxing for 2 hours, cooled down to room temperature and concentrated. Purification by recrystallization from acetonitrile gave compound 7g Yieid 95 mg, 48%. 1H-NMR (400MHz,
DMSO-D6) δH: 1.92 (4H, m), 2.30 (3H, s), 3.58 (4H, m), 5.32 (2H, broad), 6.85 (1H, d, J=8.5 Hz),
7.26 (6H, m), 7.68 (1 H, d, J=8.5 Hz), 7.81 (1 H, s), 8.19 (1 H, s), 9.45 (1 H, broad). MW 384.48.
LCMS tfi (min): 2.19. MS (APCI), m/z 385.25 [M+Hf. HPLC tR (min): 14.93. MP 173-1750C.
5-Amino-1-tert-butyl-1 H-pyrazole-4-carbonitrile (2h)
[0384] To a solution of t-BuOK (5.6 g, 50 mmol) in anhydrous methanol (100 mL) tert-butyl- hydrazine (2.2 g, 25 mmoi) was added. The obtained suspension was stirred at room temperature for 15 minutes. Then 2-ethoxymethylene-malononitrsle (3.05 g, 25 mmol) was added. The resulting mixture was stirred at refluxing for 3 hours and cooled down to room temperature. The formed solid was collected by filtration, washed with water and dried giving compound 2h. Yield 4.8 g, 75%. 5-Ammo-1-tert-butyl-1H-pyrazo!e-4-carboxylic acid amide (3h)
[0385] To a solution of potassium hydroxide (10.0 g, 18 mmol) in water (100 ml_) hydrogen peroxide (25 ml) and the solution of compound 2h (2,5 g, 15,2 mmol) in dioxane were added.
The resulting mixture was stirred at room temperature for 8 hours. The formed solid was collected by filtration, washed with water and dried, giving compound 3h. Yield 1.5 g, 90%.
1-tert-Butyl-1 H-pyrazolo{3,4~d]pyrimidine-4,6-dio1 (4h)
[0386] Compound 3h (2.36 g, 13 mmol) was fused with urea (10.0 g, 17 mmoi) at 2000C for 3 hours. The cooled solid mass was dissolved in 1 N aqueous solution of sodium hydroxide (100 ml) and acidified with acetic acid. The precipitate formed was collected by filtration, washed with water and dried at 5O0C for 10 hours furnishing compound 4h. Yield 550 mg, 48%.
Method A l-tert-Butyl-4,6-dichtoro-1H-pyrazoIo[3,4-d]pyrimidine (5h)
[0387] A mixture of compound 4h (1.2 g, 5.8 mmol), POCI3 (6 mL) and PCI5 (8.0 g) was stirred at refluxing for 14 hours, cooled down to room temperature, poured onto crushed ice and extracted with dichloromethane. The combined organic phases were washed with water, dried over sodium sulfate and concentrated. The residue was purified by column chromatography
(silica get, DCM/hexane) to give compound 5h. Yield 70 mg, 11%.
Method 8
1-tert-Butyl-4,6-dichloro-1H-pyrazoIo[3,4-dJpyrimidine (5h)
[0388] To a solution of compound 4h (500 mg, 3.05 mmol) in acetonitriie (4 mL) diphosgene
(900 mg, 4.6 mmol) was added. The reaction mixture was stirred at 1000C for 16 hours in argon atmosphere, cooled down to room temperature and diluted with water and extracted with chloroform. The combined organic phases were dried over Na2SO4 and concentrated.
Purification by column chromatography (silica gel, DCM/hexane, 1/1 ) gave compound 5h. Yield
90 mg, 12%.
{1-tert-Butyl-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yI)-m-toIyl-amine (6h)
[0389] To a solution of compound 5h (176 mg, 0.72 mmol) in acetonitriie (3 mL) 3- methylaniline (23) (103 mg, 0.72 mmol) and Et3N (0.2 mL) were added. The reaction mixture was stirred for 20 hours at room temperature and diluted with water. The precipitate formed was collected by filtration and washed with water and hexane. Purification by column chromatography (silica gel, ethyl acetate/hexane, 1/4) gave compound 6h. Yield 100 mg, 50%.
{1 -tert~Butyl-6-pyrrolidin-1 -yl-1 H-pyrazolo[3,4-d]pyrimidtn-4-y l)-m-tolyl-amine (7h-Entry
180}
[0390] A mixture of compound 6h (72 mg, 0.23 mmol) and pyrrolidine (2 mL) was stirred at refluxing for 2 hours, cooled to room temperature and concentrated at reduced pressure.
Purification by recrystallization from acetonitriie gave compound 7h. Yield 50 mg, 45%. 1H-NMR
(400MHz, DMSO-D6) δH: 1.70 (9H, s), 1.95 (4H, m), 2.30 (3H, s), 3.57 (4H1 m), 6.83 (1 H, d,
J=8.5 Hz), 7.20 (1 H, t, J=8.5 Hz), 7.69 (1 H, d, J=8.5 Hz), 7.80 (1 H, s), 7.95 (1H, s), 9.35 (1 H, s). MW 350.47. LCMS tR (mtn): 2.31. MS (APCl), m/z 351.10 [M+Hf. HPLC tR (min): 16.25. MP
220-2220C.
5-Amino-1-ethyl-1 H-pyrazole-4-carbonitriie (4i)
[0391] To a solution of t-BuOK (5.6 g, 50 mmol) in anhydrous methanol (100 mL) ethyl- hydrazine (1.5 g, 25 mmol) was added. The obtained suspension was stirred at room temperature for 15 minutes. Then 2-ethoxymethylene-malononitrile (3.05 g, 25 mmol) was added. The resulting mixture was stirred at refluxing for 3 hours and cooled down to room temperature. The formed solid was collected by filtration and washed with water. The residue was purified by column chromatography (silica gel, methanol/DCM, 1/20) and recrystallization from ethanol to give compound 4i. Yield 2.54 g, 57%.
5-Amino-1-ethyI-1H-pyrazole-4-carboxy!ic acid amide (5i)
[0392] To a solution of compound 4i (150 mg, 1.10 mmol) in acetonttrile (4 mL) was added diphosgene (325 mg, 1.65 mmol). The reaction mixture was stirred at 1000C for 16 hours in argon atmosphere, cooled down to room temperature and diluted with water and extracted with chloroform. The combined organic phases were dried over Na2SO4 and concentrated.
Purification by column chromatography (silica ge!, DCM/hexane, 1/1 ) gave compound 5i. Yield
150 mg, 16%.
{1-Ethyl-6-chloro-1H-pyrazoiot3,4-d]pyrimidin-4-yl)-m-tolyl-amine (6i) [0393J To a solution of compound 5i (156 mg, 0.72 mmol) in acetonitrile (3 mL) 3- methylaniline (103 mg, 0.72 mmol) and NEt3 (0.2 mL) were added. The reaction mixture was stirred for 20 hours at room temperature and diluted with water. The precipitate formed was collected by filtration and washed with water and hexane. Purification by column chromatography (silica gel, ethyl acetate/hexane, 1/4) gave compound 6i. Yield 183 mg, 95%. (1-Etyi-6-pyrrolidϊn-1-yl-1H-pyrazoio|;3,4-d]pyrimidin-4-yl)-m-tolyl-amine (7i-Entry 181) [0394] A solution of compound 6r (180 mg, 0.62 mmol) and pyrrolidine (3 mL) was stirred at refluxing for 2 hours, cooied to room temperature and concentrated at reduced pressure. Purification by recrystallization from acetonitrile gave compound 7L Yield 180 mg, 90%. 1H-NMR (400MHz, DMSO-D6) δH: 1.35 (3H, t, J=7.5 Hz), 1.92 (4H, m), 2.31 (3H, s), 3.58 (4H, m), 4.19 (2H, broad q, J=7.5 Hz), 6.84 (1 H, d, J=8.5 Hz), 7.21 (1 H, t, J=8.5 Hz), 7.68 (1 H, d, J=8.5 Hz), 7.82 (1 H, s), 7.98 (1 H, s), 9.40 (1 H, broad). MW 322.42. LCMS fe (min): 1.88. MS (APCI), m/z 323.14 [M+H]+. HPLC tR (mtn): 11.77. MP 188-1890C. 5-Amino-1-phenethyl-1H-pyrazote-4-carbonitιile (2j) [0395] To a solution of t-BuOK (1.9 g, 1.7 mmol) in anhydrous MeOH (7 mL) phenethylhydrazine (3.0 g, 1.7 mmol) was added. The obtained suspension was stirred for 20 minutes. Then 2-ethoxymethyiene-malononitrile (2.11 g, 1.7 mmol) was added. The resulting mixture was stirred at refluxing for 2.5 hours and cooled down to room temperature. The formed solid was collected by filtration and washed with water, cold ethanol and ether giving compound 2j. Yield 2.2 g, 61 %.
S-Amino-i-phenethyl-IH-pyrazoIe-^carboxyfic acid amide (3j) [0396] To pre-cooled in ice-bath concentrated H2SO4 (10 mL) compound 2j (2.2 g, 10.0 mmol) was added portionwise keeping internal temperature of the reaction mixture 15-200C. The mixture was stirred at room temperature for 2 hours, poured onto crushed ice, neutralized with concentrated aqueous ammonia solution keeping temperature below 5O0C and cooled to room temperature. The formed solid was collected by filtration and dried giving compound 3j used in the next stage without additional purification. Yield 2.06 g, 82%. 1-Phenethyt-1 H-pyrazoIo[3,4-d]pyrimidine-4,6-diol (4j)
[0397] Compound 3j (2.0 g, 8.7 mmol) was fused with urea (4.0 g , 67 mmol) at 2000C for 3 hours. The cooled solid mass was dissolved in 1 N aqueous solution of sodium hydroxide (100 mL) and acidified with acetic acid. The precipitate formed was collected by filtration, washed with water and dried at 500C for 72 hours, furnishing compound 4j. Yield 1.9 g, 85%. 4,6-Dich)oro-l-phenethyMH-pyrazoIø[3,4-d]pyrimidine (5j)
[0398] A mixture of compound 4j (1.9 g, 7.42 mmol), POCI3 (8 mL) and PCi5 (15.0 g) was stirred at refluxing for 20 hours, cooled to room temperature, poured onto crushed ice and extracted with dichloromethane. The combined organic phases were washed with water, dried over sodium sulfate and concentrated. The residue was purified by column chromatography (silica gel, DCM/hexane) to give compound 5j. Yield 800 mg, 37%,
Figure imgf000133_0001
[0399] To a solution of compound 5j (400 mg, 1.36 mmol) in acetonitrile (7.0 mL) 3- methylaniline (196 mg, 1.4 mmol) and K2CO3 (250 mg, 1.8 mmol) were added. The reaction mixture was stirred for 3 hours at 6O0C and for 10 hours at room temperature, diluted with water and extracted with DCM. The combined organic phases were dried over K2CO3. Purification by column chromatography (silica gel, DCM), prepTLC (ethy! acetate/hexane, 1/2) gave compound 6j. Yield 85 mg, 17%.
(1-Phenethyi-6-pyrrolidin-1-yl-1H-pyrazolo[3,4-d]pyrimidϊn-4-yl)-m-toiyi-amine (7j-Entry 182)
[0400] A solution of compound 6j (85 mg, 0.23 mmol), pyrrolidine (2 mL) in dioxane (2 mL) was stirred at refluxing for 2 hours. The reaction mixture was concentrated. Purification by column chromatography (silica gel, DCM/acetone, 10/1 ) and recrystallization from acetonitrile gave compound 7j Yield 54 mg, 58%.1H-NMR (400MHz, DMSO-D6) δH: 1.93 (4H, m), 2.31 (3H, S), 3.15 (2H, t, J=7.5 Hz), 3.57 (4H, m), 4.39 (2H, t, J=7.5 Hz), 6.84 (1 H, d, J=8.5 Hz), 7,20 (6H, m, J=8.5 Hz), 7.67 (1 H, d, J=8.5 Hz), 7.81 (1 H, s), 7.98 (1 H, s), 9.40 (1 H, broad). MW 398.52. LCMS tR (min): 2.14. MS (APCI), m/z 399.30 [M+H]+. HPLC fe (min): 13.86. MP 188-1900C. 5-Amino-1-butyi-1H-pyrazole-4-carbonitriIe (2k) [0401] To a solution t-BuOK (1.9 g, 1 ,7 mmoi) in anhydrous MeOH (7 mL) butyihydrazine (3.0 g, 1.7 mmoi) was added. The obtained suspension was stirred for 20 minutes. Then 2- ethoxymethylene-malononitrile (2.11 g, 1.7 mmol) was added. The resulting mixture was stirred at refluxing for 2.5 hours and cooled down to room temperature. The formed solid was collected by filtration and washed with water, cold ethanol and ether giving compound 2k. Yield 2.9 g,
70%.
S-Amino-i-butyl-IH-pyrazole-^carboxylic acid amide (3k)
[0402] To a solution of KOH (9.5 g) in water (100 mL) H2O2 (12.5 mL), 2k (2.7 g, 16.0 mmol) were added at room temperature. The obtained mixture was stirred at room temperature for 6 hours. The formed solid was collected by filtration, washed with water and hexane and dried giving compound 3k.Yield 2.0 g, 67%,
1-Butyl~1 H-pyrazoIo[3,4-d]pyrimidine-4,6-diol (4k)
[0403] Compound 3k (2.0 g, 11.0 mmol) was fused with urea (9.0 g , 150 mmol) at 2000C for
3 hours. The cooled solid mass was dissolved in 1 N aqueous solution of sodium hydroxide (100 mL) and acidified with acetic acid. The precipitate formed was collected by filtration, washed with water and dried at 500C for 72 hours, furnishing compound 4k. Yield 2.3 g, 99%.
1-Butyl-4,6-dichtoro-1H-pyrazolo[3,4-d]pyrimidine (5k)
[0404] A mixture of compound 4k (1.9 g, 7.42 mmol), POCl3 (8 mL) and PCi5 (15,0 g), DMF
(0.1 mL) was refluxed for 20 hours, cooled to room temperature, poured onto crushed ice and extracted with DCM. The combined organic phases were washed with water, dried over sodium sulfate and concentrated. The residue was purified by column chromatography DCM to give compound 5k. Yield 1.45 g, 54%.
(1 -Butyl-6-chloro-1 H-pyrazolo[3,4-d]pyrimidin-4-yl)-m-tolyi-amine (6k)
[0405] To a solution of compound 5k (200 mg, 0.82 mmol) in acetonitrile (3.0 mL) 3- methylaniline (118 mg, 0.82 mmol) and NEt3 (0.25 mL) were added. The reaction mixture was stirred at room temperature for 48 hours and diluted with water. The formed solid was collected by filtration and dried, giving compound 6k. Yield 220 mg, 85%.
(1-Butyl-6-pyrrolidin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-m-tolyl-amine (7k-Entry 183)
[0406] A mixture of compound 6k(210 mg, 0.67 mmol) and pyrrolidine (2 mL) was stirred at refluxing for 3 hours, cooled down to room temperature and concentrated. Purification by recrystallization from acetonithfe gave compound 7k. Yield 170 mg, 73%.1H-NMR (400MHz,
DMSO-D6) δH: 0.90 (3H, t, J=7.5 Hz), 1.24 (2H, m), 1.79 (2H, t, J=7.5 Hz), 1.95 (4H, m), 2.30
(3H, s), 3.58 (4H, m), 4.14 (2H, t, J=7.5 Hz), 6.84 (1 H, d, J=8.5 Hz), 7.21 (1 H, t, J=8,5 Hz), 7.68
(1 H, d, J=8.5 Hz), 7.82 (1 H, s), 7.98 (1 H, s), 9.40 (1 H, broad). MW 350.47. LCMS tR (min): 2.13.
MS (APCi), m/z 351.25 [M+Hf. HPLC tR (min): 13.61. MP 178-1790C.
1-Phenyl-1H-pyrazolo[3,4-d]pyrimidine-4,6-diol {4I)
[0407] Compound 3t (3.0 g, 14.8 mmoi) was fused with urea (6.0 g, 100 mmol) at 2000C for 3 hours. The cooled solid mass was dissolved in 1 N aqueous solution of sodium hydroxide (100 mi_) and acidified with acetic acid. The precipitate formed was collected by filtration, washed with water and dried at 500C for 72 hours, furnishing compound 41. Yield 1.1 g, 33%. 4,6-Dichloro-1-phenyl-1 H-pyrazolo[3,4-dJpyrimidine (5I)
[0408] A mixture of compound 4! (1 ,1 g, 4.82 mmol), POCI3 (15 mL) and PCl5 (5.0 g) was stirred at refluxing for 3 hours, cooled to room temperature, poured onto crushed ice and extracted with DCM. The combined organic phases were washed with water, dried over sodium sulfate and concentrated. The residue was purified by column chromatography (silica gel, DCM) to give compound 5t. Yield 710 mg, 55%.
Figure imgf000135_0001
[0409] To a solution of compound 51 (200 mg, 0.75 mmol} in acetonitrile (1 ,0 mL), 3- methylaniline (108 mg, 0.75 mmol), K2CO3 (150 mg, 1.08 mmol) and ethanol (3 mL) were added. The reaction mixture was stirred at 700C for 5 hours and diluted with water. The formed soϋd was collected by filtration. Purification by column chromatography (silica gel, DCM) gave compound 6i. Yield 163 mg, 65%.
(l-PhenyI-6-pyrroIldin-1-yl-1H-pyrazolo[3,4-dJpyrSmϊdin-4-yO-m-toiyl-amine (7I-Entry 184) [0410] A mixture of compound 61 (150 mg, 0.45 mmol) and pyrrolidine (2 mL) was stirred at refluxing for 3 hours and concentrated. Purification by recrystaliizatton from acetonitrile/water (1/1) gave compound 7I. Yield 73 mg, 47%.1 H-NMR (400MHz, DMSO-D6) δH; 1.98 (4H, broad), 2.35 (3H, s), 3.63 (4H, broad), 6.88 (1 H, d, J=8.5 Hz), 7.24 (2H1 superposition of two t, J=8.5 Hz), 7.50 (2H, t, J=8.5 Hz), 7.71 (1 H, d, J=8.5 Hz)1 7.82 (1H, broad), 8.28 (1 H, s), 8.32 (2H, d, J=8.5 Hz), 9.58 (1 H1 broad). MW 370.46. LCMS tR (min): 2.34. MS (APCI), m/z 371.27 [M+H]\ HPLC tR (mtn): 17.54. MP 221-2220C.
(6-Chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yi)-(3-chloro-4-piperidin-1-yl-phenyf)- amine (6m)
[0411] To a solution of 4,6-DichIoro-1-methyl-1 H-pyrazolo[3,4-d]pyrimtdine (203 mg, 1.0 mmoi) in acetonitrile (20.0 mL) 3-chloro-4-piperidin-1-yl-phenylamine (35) (252 mg, 1.5 mmol) and K2CO3 (150 mg, 1.08 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour, diluted with water and extracted with DCM. The combined organic phases were dried over Na2SO4 and concentrated at reduced pressure. Purification by column chromatography (silica gel, acetone/DCM) and recrystallization from acetone/water (1/1 ) gave compound 6m. Yield 200 mg, 53%.
(S-Chloro^-piperidin-i-yl-phenyO-ti-methyl-β-pyrrolidin-i-yl-IH-pyrazoloIS^-dJpyrimidϊn- 4-yi)-amine {7m-Entry 185)
[0412] A mixture of compound 6m (200 mg, 0.54 mmoi) and pyrrolidine (0.40 mL) was stirred at 5O0C for 10 hours in argon atmosphere, cooled to room temperature and concentrated. Purification by column chromatography (silica gel, DCM/acetone, 1/1 ) and recrystaliization from DCM/hexane gave compound 7m. Yield 190 mg, 85%.1H-NMR (400MHz, DMSO-D6) δH: 1.51 (2H, m), 1.65 (4H, m), 1.93 (4H, m), 2.88 (4H, m), 3.58 (4H, m), 3.73 (3H, s), 7.11 (1 H, d, J=8.5 Hz), 7.70 (1 H, d, J=8.5 Hz), 7.95 (1 H, s), 8.21 (1 H, s), 9.51 (1 H, broad). MW 411.94. LCMS tR (min): 2.13. MS (APCI), m/z 412.11 , 414.12 [M÷Hf. HPLC fo (min): 12.67. MP 116-1170C. (6-Chtoro-1-methyi-1 H-pyrazolo[3,4-dlpyrimidIn-4-yI)-(1 H-indazol-6-yl)-amine (6n) [0413] To a solution of 4,6-Dϊchloro-1-methyi-1 H-pyrazo!o[3,4-d]pyrimidine (203 mg, 1.0 mmol) in acetonitrile (20 mL) 1 H-indazol~6-ylamine (150 mg, 1.2 mmol) and K2CO3 (150 mg, 1.08 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour, diluted with water and extracted with DCM. The combined organic phases were dried over Na2SO4 and concentrated. Purification by column chromatography (silica gel, acetone/DCM) and recrystallization from acetone/water (1/1 ) gave compound 6n. Yield 200 mg, 67%. {1H-lndazol-6-yl)-(1-methyf-6-pyrroiidin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4-yt)-amine (7n- Entry 186)
[0414] A mixture of compound 6n (200 mg, 0.67 mmo!) and pyrrolidine (0.40 mL) was stirred at 5O0C for 10 hours in argon atmosphere, cooled down to room temperature and concentrated. Purification by column chromatography (silica gel, DCM/acetone-ethano!/DCM) and recrystallization from propane-2-ol/hexane gave compound 7n. Yield 150 mg, 67%.1H-NMR (400MHz5 DMSO-D6) δH: 1.96 (4H, m), 3.62 (4H1 m), 3.76 (3H, s), 7.39 (1 H, d, J=8.5 Hz), 7.65 (1 H, d, J=8.5 Hz), 7.93 (1 H, s), 8.03 (1 H, s), 8.51 (1 H, s), 9.60 (1 Hr broad), 12.86 (1 H, broad). MW 334.39. LCMS fe (min): 1.48. MS (APCI), m/z 335.08 [M+H]*. HPLC tR (min): 8.77. MP 331- 3350C. Table 24. Examples 173-186 of Py razolopyrϊmi dines. Library G3.
Figure imgf000136_0001
Figure imgf000137_0001
III. RI -R6-R4 APPROACH
Library 1
Figure imgf000138_0001
[0415] Compound 3 A mixture of amtnoamide 1 (5 g, 0.0298 mol) and the aldehyde 2 (8.94 g, 0,06 moi) in the presence of methanesuifonic acid (5 drops) was refluxed in xylene (50 ml) for 48 h. The reaction mixture was chilled and the precipitate was filtered, carefully washed with ether and dried. The pale yellow precipitate was obtained with 85% (7.2 g) yield. [0416J Compound 4 The pyrazopyrimidine 3 (3 g, 0.01 moi) was added to excess SO3HCI (10 eq.) portionwise on careful cooling of the reaction mixture {0 0C). When the addition was complete, the reaction mixture was stirred at RT for 24 h. Then it was diluted with ice water, the organic layer was extracted with EtOAc and concentrated under reduced pressure. The residue was obtained as a yellow precipitate with 90 % (3.8 g) yield,
[0417J Compound 5 0.00054 mol of 4 was dissolved in 6 mL of dry chloroform, then 0.054 g (0.00054 mol) of TEA were added. The reaction mixture was stirred at RT for 3 min, after which time 0.00054 mol of amine was added at once. The reaction mixture was stirred overnight at 80 0C, then cooled and poured into aqueous sodium carbonate solution. The organic layer was extracted, concentrated under reduced pressure and the solid was crystallized from ether. The target compound was carefully washed with hot hexane and air dried. The final compounds were obtained with 40-70 % yields.
Figure imgf000138_0002
Table 25. Examples 187-194 of Pyrazolopyrimidines. Library I.
Entry Structure MF / MW LCMS IUPAC
3-(4-hydroxy-1-propyl-
1 H-pyrazolo[3,4-
C21 H21 N5O3S
: 187 424.4 d]pyrimidin-6-y!)-N- / 423.5 methyl-N-phenyl- benzenesulfonamide
Figure imgf000139_0001
Analytical Data for Library I
187. 3-(4-hydroxy-1-propyl-1H-pyra2θio[3,4-d]pyrϊm!dϊn-6-yl)-N-methy[-N-phenyi- benzenesulfonamide 1H NMR {DMSO, ppm) δ: 0.89 (t, J=7.4; 3H); 1.91 (sx, J1=7.4, J2=6.8, 2H); 3.25 (s, 3H); 4.30 (t, J=6.8, 2H); 7.20 (d, J=7.0, 2H); 7.33 (m, 3H); 7.70 (d, J=4.9, 2H); 8.03 (s, 1 H); 8.39 {s, 1 H); 8.45 (m, 1 H); 12.18 (s, 1 H).
188. N-{4-bromophenyl)-3-(4-hydroxy-1-propyl-1 H-pyrazolo[3,4-d]pyrimidin-6-yl)-N- methyi-benzenesulfonamide 1H NMR (DMSO, ppm) δ: 0.90 (t, J=7.5, 3H); 1.92 (sx, J1=7.5, J2=6,3, 2H); 3.23 (s, 3H); 4.30 (t, J=6.3, 2H); 7.15 (d, J=8.6, 2H); 7.53 (d, J=8.6, 2H); 7,75 (d, J=4.8, 2H); 8.03 (s, 1 H); 8.40 (s, 1 H); 8.47 (t, J=4.8, 1 H); 12.28 (s, 1 H). 189. 4-ethoxy-3-(4-hydroxy-i-propyl-1 H-pyrazolo[3,4-d]pyrimidm-6-yl)-N-rnethyI-N- phenyl-benzenesulfonamide 1H NMR (DMSO, ppm) δ: 0.9 t (3H); 1.45 t (3H); 1.9 m (2H); 3.2 s (3H); 4.3 m (4H); 7.25 m (6H); 7.7 q (1 H); 7.96 m (2H); 11.4 s (1H).
190. N-(4-faromophenyl)-4-ethoxy-3-{4-hydroxy-1-propyi-1 H-pyrazolo[3,4-d]pyrimidin-6~ y!)-N-methyl-benzenesulfonamide 1H NMR (DMSO, ppm) δ: 0.9(3H,t); 1.45 1 (3H); 1.94 m (2H); 3.15 s (3H); 4,3 m (4H); 7,14 d (2H); 7.32 d (1 H); 7.48 d (2H); 7.72 q (1H); 7.94 s (1 H); 7.98 d (1H); 11.5 s (1 H).
191. 4-ethoxy-3-(4-hydroxy-1-propyl-1H-pyrazolo[3,4-d]pyrimidin-6-yi)-N-(4- methoxyphenyl)-N-methylbenzenesulfonamide 1H NMR (DMSO1 pprn) δ: 0.9 1 (3H); 1 ,46 t (3H); 1.92 m (2H); 3.16 s (3H); 3.76 s (3H); 4.30 m (4H); 6.86 d (2H); 7.05 d (2H); 7.32 d (1 H); 7.70 d (1 H); 7.96 d (2H); 11.50 s (1H).
192. 6-{3-t(4-methylpiperazin-l-yl)su(fonyI]phenyl}-1-propyI-1 H-pyrazoIo[3,4- d]pyrϊmidin-4-ol 1H NMR (DMSO, ppm) δ: 0.90 (t, J=7.4, 3H); 1.93 (sx, J1 =7.4, J2=6.9, 2H); 2.18 (s, 3H); 2.38 (t, J=4,7, 4H); 3.07 (t, J=4.7, 4H); 4.33 (t, J=6.9, 2H); 7.81 (t, J=7.8, 1 H); 7.95 (d, J=7.8, 1 H); 8.04 (s, 1 H); 8.47 (m, 2H),
193. 6-{2-ethoxy-5-[(4-methylpϊperazin-1-yi}sulfonyl]phenyI}-1-propyl-1H-pyrazolo[3,4- d]pyrimidin-4-ol 1H NMR (DMSO, ppm) δ: 0.80 m (3H); 1.30 m (3H); 1.90 m (2H); 2,15 m (4H); 2.50 m (3H); 3.00 s (4H); 4.30 s (4H); 7.35 d (1 H); 7.80 d (1 H); 8.00 m (2H); 11 ,50 s (1 H).
194. N-[4-(dimethylamino)phenyll-4-ethoxy-3-(4-hydroxy-l-propy)-1H-pyrazoloE3,4- d]pyrimidin-6-yl)-benzenesulfonamide 1H NMR (DMSO, ppm) δ: 0.80 1 (3H); 1.30 1 (3H); 1.80 m (2H); 2.80 s (6H); 4.50 m (4H); 6.60 d (2H); 6.95 d (2H); 7.30 d (1H); 7.75 d (1 H); 8.00 s (1 H); 8.20 s (1 H), 9.30 s (1 H), 11 ,6 s (1H).
Library Q
Figure imgf000140_0001
[0418] Preparation of 7. Ester 6a (8 mmol) was carefully added to the suspension of 5 (4 mmol) in sodium ethoxide (prepared from 0.18 g of Na and 10 mL of dry C2H5OH on cooling), The reaction mixture was refluxed on an oil bath with stirring overnight. The resulting solution was concentrated under reduced pressure to dryness and the residue was dissolved in 20 mL of water and neutralized with cone. HCI. Then the precipitate was filtered and the product 7 was recrystailized from methanol to obtain a white precipitate in quantity of 0.7 g, 31 %. [04191 Preparation of 8. 7 (200 mg, 0.54 mmol) was dissolved in POCl3 (5 mL) and 0.54 mmol of PCl5 was added. The reaction mixture was stirred at reflux overnight ant then diluted with 20 mL of toluene. Then the solvents were removed under reduced pressure and the residue was used in the next step without purification. Crude yield was 76 %, 170 mg. [0420] Preparation of 9. 8 (100 mg, 0,25 mmol) was dissolved in 1 ,4-dioxane (3 mL) and 0.5 mmol of an appropriate amine was added. The reaction mixture was stirred at 8O0C overnight. LCMS analysis demonstrated the presence of 80 % target compound in the reaction mixture. The reaction mixture was poured into water and extracted with chloroform. The solvent was removed in vacuo and the residue was crystallized from ether. Then the precipitate was filtered and the product 9 was obtained as a white precipitate in quantity of 18 mg, 29 %.
Figure imgf000141_0001
pp01
Table 26. Examples 195-196 of Pyrazolopyrimidines. Library Q.
Figure imgf000141_0002
Analytical Data for Library Q
195. 3-{4-anilino-1 -propyl-1 H-pyrazolo[3,4-d]pyrϊmidin-6-yl)-N-(4-methoxyphenyl)-N- methylbenzenesulfonamide 1H NMR (DIvISO, ppm) δ: 0.88 m (3H), 1.98 m (2H), 3.06 s (3H), 3.70 s (3H), 4.42 m (2H), 6.88 d (2H), 7.02 d (2H), 7.18 m (1H), 7.48 m (2H), 7.70 m (2H), 7.80 d (2H), 8.20 s (1 H)1 8.75 s (1 H), 10.0 s (1 H) 196. 3-(4-hydroxy-1-propyi-1 H-pyrazoIo[3,4<l]pyrimidm-6-yl)-N-(4-methoxypheriyl)-N- methylbenzenesulfonamide 1H NMR (DMSO, ppm) δ: 0.98 s (3H), 1.88 m 2H), 3.06 s (3H), 3.80 s (3H), 4.12 m (2H), 6.98 d (2H), 7.02 d (2H), 7,60 s (2H), 8.0 s (1 H), 8.48 m (2H), 12 bs (1 H).
Library Y
Figure imgf000142_0001
[0421] Preparation of 1a To a stirred solution of 1(3g) and triethylamiπe(1.5 ml) in 70 mi of dioxane, a solution of 3.64 g of corresponding chloroanhydride in 50 ml of dioxane was added dropwise at 5°C. After 5 hours of stirring the solvent was removed and the residue was treated with water. The precipitate obtained was filtered off and dried to produce 1a as white solid. Yield 4 g (70%).
[0422] Preparation of 2 Compound 1a from previous stage (4 g) was treated with 50 ml of 1 N NaOH water solution at reflux for 9 hours, cooled to RT, acidified with acetic acid, filtered off and dried to produce 2 as white solid. Yield 2.7 g (71 %).
[0423] Preparation of 3 2.7 g of 2 were hydrogenated (2 atm.) in ethanol (80 ml) over Pd/C catalyst at RT for 54 hours and filtered. The solvent was removed to produce 3 as yellow solid. Yield 1.4 g (58 %).
[0424] Preparation of 4. A diazonium salt was prepared by adding a solution of 1.0 g of sodium nitrite in 2 ml of water to a suspension of 1.650 g (0.0058 mo!) of compound 3 in 3 ml of hydrochloric acid and 11 ml of acetic acid cooled at 0-50C. After stirring about 20 min., the diazonium salt suspension was poured in one portion into a mixture of 50 mi of acetic acid with SO2 and cupric chloride dihydrate and cooled to 10 0C. The mixture was stirred for 30 min at 10- 15°C and then for 1 h at RT. Thereafter, this reaction mixture was heated to 500C and allowed to stir for 2 h. The suspension was poured into ice water (500 ml) and the precipitate was filtered off and dried. [0425] Preparation of 5 To a stirred solution of 4 (0.001 mot) and triethylamine (0.5 ml) in 10 ml of dioxane, a solution of amine (0.001 mol) was added . After 5 hours of stirring the solvent was removed and the residue was treated with water. The precipitate obtained was filtered off. [0426] Preparation of 6 To a solution of 5 (2.8 g, 14 mmol) in POCI3 was added PCI5 (5.83 g, 28 mmol). The reaction mixture was stirred for 10 hours at reflux, then POCI3 was removed under reduced pressure; the precipitate washed with ether and used in a further step. [0427] Preparation of 7 To a stirred solution of 6 (0.001 mol) and triethylamine (0.5 ml) in 10 ml of dioxane, a solution of amine (0,001 mol) was added. After 5 hours of stirring the solvent was removed and the residue was treated with water, extracted with dichioromethane and purified by column chromatography.
Figure imgf000143_0001
Table 27. Examples 197-198 of Pyrazolopyrimidines. Library Y.
Figure imgf000143_0002
Analytical Data for Library Y
197. propyI-6-[4-(pyrrolidin-1-ylsuϊfonyI)phenyiJ-1H-pyrazolo{3,4-d]pyrimidin-4-oI 1H
NMR (DMSO, ppm) δ: 0.92 (t, J=7.3 Hz, 3H); 1.73 (m, 4H); 1.95 (m, 2H); 3.24 (m, 4H); 4.35 (t, J=6,9Hz, 2H); 7.95 (d, J=8.7Hz, 2H); 8.05 (s, 1 H); 8.40 (d, J=8.7Hz, 2H).
198. N-{4-fluorobenzyl)-1-propyl-6-[4-(pyrrolidin-1-ylsuifonyl)phenyl]-1 H-pyrazoio[3,4- d]pyrimidin-4-amine 1H NMR (DMSO, ppm) δ: 0.92 (t, J=7.3 Hz, 3H); 1.73 (m, 4H); 1.95 (m, 2H); 3.24 (m, 4H); 4.35 (t, J=6.9Hz, 2H); 4.85 (d, J=5.9 Hz, 2H); 7.12 (t, J=8.8Hz, 2H); 7.50 (m, 2H); 7.80 (d, J=8.4Hz, 2H); 8.15 (s, 1 H); 8.65 (m,3H).
Library Zb
Figure imgf000144_0001
[0428] Preparation of 9 To a stirred solution of 8(0.001 mol) and triethyiamine (0.5 ml) in 10 m! of dioxane, a solution of amine (0.001 mol) was added. After 5 hours of stirring the solvent was removed and the residue was washed with water. The formed precipitate was filtered off and dried.
[0429] Preparation of 10 To a solution of 9 (2.8 g, 14 mmoi) in POCl3 , PCI5 (5.83 g, 28 mrnol) was added. The reaction mixture was stirred for 10 hours at reflux, then POCI3 was removed under reduced pressure, the precipitate was washed in ether and used in a further step.
[0430] Preparation of 11 To a stirred solution of 10 (0.001 moi) and triethyiamine (0.5 mi) in 10 mi of dioxane, a solution of amine (0.001 mol) was added. After 5 hours of stirring the solvent was removed and the residue was treated with water, extracted with dichloromethane and purified by column chromatography.
Figure imgf000144_0002
Figure imgf000144_0003
Analytical Data for Library Zb 199. N-fΦfluorophenylJ-i-propyt-β-tS-tpyrroiidin-i-yisuifonyiJphenylj-IH-pyrazofotS^- d]pyrimidin-4-amine 1H NMR (DMSO1 ppm) δ: 0.92 (t, J=7,3 Hz, 3H); 1.73 (m, 4H); 1.97 (m, 2H); 3,24 (m, 4H); 4.42(t, J=6.9Hz, 2H); 7.23 (t, J=8.8Hz, 2H); 7.76 (t, J=7.7Hz, J=7.5 Hz, 1 H); 7.82-7.92 (m, 3H); 8.17 (s, 1 H); 8.67 (m,1 H); 8.82 (bt, 1 H); 9.96{bs,1 H).
200. N-(4-fluorobenzyl)"1-propyl-6-[3-{pyrrolidin-1-ylsuifonyl)phenyl]-1H-pyrazolo[3,4- d]pyrimidin-4-amtne 1H NMR (DMSO, ppm) δ: 0.92 (t, J=7.3 Hz5 3H); 1.73 (m, 4H); 1.95 (m, 2H); 3.24 (m, 4H); 4.35 (t, J=6.9Hz, 2H); 4.85 (d, J=5.9 Hz, 2H); 7.12 (t, J=8.8Hz, 2H); 7.50 (m, 2H); 7.72 (t, J=7.8Hz, 1H); 8.05 (s, 1 H); 8.58-8.70 (m,2H); 8.82 (bt, 1H).
Library AA
Figure imgf000145_0001
[0431] Preparation of 5: Compound 4 (0.01 mol) was treated with urotropine (0.01 mo!) in
100 ml of chloroform for 5 hours, the solvent was removed and the residue was treated with HCI at reflux for 24 hours.
[0432J Preparation of 2: A mixture of 1 (0.01 moi), aldehyde (0.01 mol), methanesulfonic acid
(5 drops), and xylenes (50 ml) was refluxed for 48 h. The reaction mixture was chilled and the precipitate was filtered, washed by ether and dried.
[0433] Preparation of 3: A mixture of 2 (0.01 mol), and sulfuric acid (15 ml) was refluxed for
48 h. The reaction mixture was poured onto ice, the precipitate obtained was filtered off and dried.
[0434] Preparation of 6 Compound 3 (0.01 mol) was treated with CDI (0.01 moi) in 100 ml of
DMF for 2 hours, then amine was added. After 7 hours of stirring the solvent was removed.
R
N
O
O υ
N '
PP-R6-25 Table 29. Example 201 of Pyrazolopyrimidines. Library AA.
Figure imgf000146_0002
Analytical Data for Library AA
201. 3-{4-hydroxy-1-propyl-1 H-pyrazolo[3,4-d]-pyrimidin-6-yl)-N-[4-(morpho(in-4-yl- carbortyl)phenyl]«benzamide 1H NMR (DMSO, ppm) 5 0.92 (t, J=7,3 Hz, 3H); 1.95 (m, 2H); 3.56 (m, 8H); 4.35 (t, J=6.9Hz, 2H); 7.46 (d, J=7.4Hz, 2H); 7.70 (bt, 1 H); 7.85 (d, J=7.4Hz, 2H); 8.01 (s, 1 H); 8.15 (d, J= 7.4Hz,1 H); 8.37 (d, J=7.4Hz, 1 H); 8.70 (s, 1 H); 10.20 (bs, 1 H).
IV. R1-R3-R4-R6 APPROACH
Library O
Figure imgf000146_0001
[0435] Compound 2 A mixture of aminoamidβ 1 (5g 0.0275 mole) and the aldehyde (5.2g 0.055 mole) in the presence of methanesulfonic acid (5 drops) was refluxed in xylene (50ml) for 48 h. The reaction mixture was chilled and the precipitate was filtered, carefully washed with ether and dried. A pale brown precipitate was obtained with 80 % (5.9 g) yield. [0436] Compound 3 The pyrazopyrimidtne 2 (3g, 0.0111 mole) was added to the excess of SO3HCI (10 eq.) portionwise on careful cooling of the reaction mixture (0C). When the addition was complete, the reaction mixture was stirred at 2000C for 48 h. Then it was diluted with ice water, the organic layer was extracted with EtOAc and concentrated. The yellow precipitate of compound 2 was obtained with 90 % (3.6 g) yield.
[0437] Compound 4 0.15 g (0.00041 mol) of 3 was dissolved in 6 mL of dry dioxane; then 0.041 g (0.00041 mol) of TEA was added. The reaction mixture was stirred at RT for 3 min., then 0.00049 mol of an appropriate amine was added at once. The reaction mixture was stirred overnight at 50 0C, The reaction mixture was poured into aqueous sodium carbonate solution and extracted with methylene chloride. The organic layer was concentrated under reduced pressure and crystallized from ether. The final compounds were obtained with 55-65 % yields. OH
R2 O
O
Table 30. Examples 202-215 of Pyrazoiopyrimidines. Library O.
Figure imgf000147_0001
Figure imgf000148_0001
Analytical Data for Library O
202 N-[4-(dimethylamino)-phenyl]-3-(7-hydroxy-1 -methyl-3-propyM H-pyrazolo[4,3- d]pyrimidϊn-5-yl)benzenesulfonamϊde 1H NMR (DMSO, ppm) δ 1.00 t (3H); 1.80 m (2H);
2.75 m (8H); 4.15 s (3H); 6.55 d (2H); 6.90 d (2H); 7.45 t (1 H); 7.60 d (1 H); 8.50 d (1 H); 8.70 s (1 H).
203. methyl-5-{3-[(4-methyipipera2in-1-yl)sulfonyI]phenyl}-3-propyl-1H-pyrazolo[4,3- d]pyπmidin-7-oi 1H NMR (DMSO, ppm) δ 1.0O t (3H); 1.85 m (2H); 2.20 s (3H); 2.70-3.20 m (9H); 4.20 s (3H); 7.70-8.0 m (2H); 8.45 t (2H).
204. methyI-5-[3-(morpholin-4-ylsulfonyi)phenyl]-3-propyl-1H-pyrazolo[4,3-d]pyrimidin- 7-oI 1H NMR (DMSO, ppm) δ 0.90 t (3H); 1.84 m (2H); 2.82 t (2H); 3.05 t (4H); 3.65 t (4H); 4.15 s (3H); 7.71 t (1 H); 7 8O d (1 H); 8.48 d (1 H); 8.52 s (1 H).
205. 3-(7-hydroxy-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N-(4-morpholin-4- yfphenyi)-benzenesulfonamide 1H NMR (DMSO, ppm) δ 0.90 t (3H); 1.80 m (2H); 2.86 1 (2H); 3.00 m (4H); 3.60 m (4H); 4.15 s (3H); 6.75 d (2H); 6.95 d (2H); 7.64 t (1 H); 7.80 d (1H); 8.20 d (1 H); 8.40 s (1 H); 9.55 s (1H), 12,20 s (1H).
206. N-[3-K[3-(7-hydroxy-1-methyl-3-propyl-1H-pyrazolo[4,3-dJpyrimidin-5- y!)phenyt]sulfonyl}-amino)phenyl]acetamide 1H NMR (DMSO, ppm) δ 0.90 1 (3H); 1.82 m (2H); 2.00 s (3H); 2.82 t (2H); 4.18 s (3H); 6.80 d (1 H); 7.15 t (1 H); 7.21 d (1 H); 7.50 S (1 H); 7.65 1 (1 H); 7.90 d (1 H); 8.25 d (1 H); 8.55 s (1 H); 9.58 s (1 H).
207. N-[4-(cyanomethy))-phenyl]-3-(7-hydroxy-1-methyl-3-propyf-1H-pyra2θIoE4,3- d]pyrimidin-5-yI)benzenesulfonamide 1H NMR (DMSO, ppm) δ 1.00 (t, J=7.7, 3H); 1.83 (SX, J1=7.7, J2=7.4, 2H); 2.85 (t, J=7.4, 2H); 3,85 (s, 2H); 4.17 (s, 3H); 7.20 (m, 4H); 7.67 (t, J=7.8, 1 H); 7.89 (d, J=7.8, 1 H); 8.26 (d, J=7.8, 1 H); 8.54 (s, 1 H); 10.12 (s, 1 H); 12.25 (s, 1 H).
208. 3-(7-hydroxy-1-methyI-3-propyl-1H-pyrazolo[4,3~dJpyrimtdin-5-yl)-N-(4- phenoxyphenyl)benzenesulfonamide 1H NMR (DMSO, ppm) δ 1.00 (t, J=7.3, 3H); 1.83 (sx, J=7.3, 2H); 2.83 (t, J=7.3, 2H); 4.19 (s, 3H); 6.86 (m, 4H); 7.05 (t, J=7.4, 1 H), 7.14 (d, J=9.1 , 2H); 7.27 (t, J=7.4, 2H); 7.63 (t, J=7.8, 1 H); 7.86 (d, J=7.8, 1H); 8.30 (d, J=7.8, 1 H); 8.53 (s, 1 H); 9.78 (s, 1 H); 12.20 (s, 1 H).
209. methyf-3-propyl"5"I3-(pyrrolidin-1-ylsulfoπyl)phenyl]-1H-pyrazolo[4,3-d]pyriιnidin-7- 0I 1H NMR (DMSO, ppm) δ 0.9O t (3H); 1.70 m (4H); 1.82 m (2H); 2.79 t (2H); 3,24 m (4H); 4.18 s (3H); 7.59 1 (1 H); 7.70 d (1 H); 8.58 d (1 H); 8.70 s (1 H).
210. 3-(7-hydroxy-l-methyl-3-propyl-1H-pyrazoio[4,3-d]pyrimtdin"5-yl)-N,N- dimethylbenzenesuffonamide 1H NMR (DMSO, ppm) δ 0.98 1 (3H); 1.82 m (2H); 2.72 s (6H); 2.84 t (2H); 4.18 s (3H); 7.74 1 (1 H); 7.85 d (1 H); 8.42 d (1 H); 8.50 s (1 H).
211. 5-t3-(azepan-1-ylsulfonyl)phenyl]-1-methyi-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7- ot 1H NMR (DMSO, ppm) δ 0.96 t (3H); 1 ,52 m (4H); 1.66 m (4H); 1.82 m (2H); 2.801 (2H); 3.34 t (4H); 4.16 s (3H); 7.60 1 (1 H); 7.72 d (1 H); 8.46 d (1 H); 8.62 s (1 H).
212. N-(4-bromophenyI)-3-(7-hydroxy-1-methyt-3-propyl-1H-pyrazolo[4,3-dΪpyrimid(n-5- y[)-N-methylbenzenesuIfonamide 1H NMR (DMSO, ppm) δ 0.99 (t, J=7.4, 3H); 1.82 (sx, J1=7,4, J2=7.2, 2H); 2.84 (t, J=7.2, 2H); 3,23 (s, 3H); 4.17 (s, 3H); 7.15 (d, J=8.6. 2H); 7.52 (d, J=8.6, 2H); 7.70 (m, 2H); 8.37 (m, 2H); 12.34 (s, 1 H).
213. 3-(7-hydroxy-1-methy!-3-propyi-1H-pyrazoIot4,3-d3pyrimfdin-5-yl)-N-(4- methoxyphenyf}-N-methyibenzenesulfon-amide 1H NMR (DMSO, ppm) δ 0.98 (t, J=7.4, 3H); 1.80 (sx, J=7.4, 2H); 2.82 (t, J=7.4, 2H); 3.19 (s, 3H); 3.75 (S, 3H); 4.17 (s, 3H); 6.88 {d, J=8.8, 2H); 7.06 (d, J=8.8, 2H); 7.68 (m, 2H); 8.36 (m, 2H); 12.38 (s, 1 H).
214. 3-(7-hydroxy-1-methyl-3-propyl-1H-pyrazo(o[4,3-d]pyrimidiπ-5-yi)-N-methyl-N- phenyl-benzenesulfonamide 1H NMR (DMSO, ppm) δ 0.98 (t, J=7.4, 3H); 1.80 (sx, J=7.4, 2H); 2.83 (t, J=7.4, 2H); 3.24 (s, 3H); 4.16 (s, 3H); 7.19 (d, J=7.4, 2H); 7.32 (m, 3H); 7.68 (m, 2H); 8.37 (m, 2H); 12.36 (s, 1H). 215. N-(3-chlorophenyi)-3-(7-hydroxy-1-methyl"3-propyl-1 H-pyrazolo[4,3-d]pyrimidin-5- yl)benzenesulfonamide 1H NMR (DMSO, ppm) δ 0.99 (t. J=7.4, 3H); 1.82 (sx, J=7.4, 2H); 2.84 (t, J=7.4, 2H); 4.16 (s, 3H); 7.08 (d, J=8.0, 1 H); 7.12 (d, J=8.0, 1 H); 7.20 (s, 1 H); 7.26 (t, J=8.Q, 1 H); 7.70 (t, J=7.9, 1 H); 7.91 (d, J=7,9, 1 H); 8.29 (d, J=7.9, 1 H); 8.53 (s, 1 H); 10.30 (s, 1 H); 12.28 (s, 1 H). Library P
Figure imgf000150_0001
[0438] Preparation of 2. 10.0 g (0.060 mol) of 3-nitrobenzoic acid was suspended in 40 mL of SOCI2. The reaction mixture was refluxecl for 2 h, then the solvent was removed in vacuo, the residue was washed twice with benzene and dried by concentration from benzene. Thereafter, this crude material was dissolved in 50 mL of dry dioxane and the solution was carefully added to 250 mL of dry methanol. The reaction mixture was refluxed overnight, then cooled and concentrated in vacuo. The crude compound 2 was crystallized from ether and lyophilized. The obtained crude yellow oil was used in the following reaction without purification. The yield was 10.1 g. (94 %).
[0439] Preparation of 3. 0.506 g (0.022 mol) of sodium was carefully dissolved in 100 mL of dry methanol, followed by adding 4.0 g (0.022 mol) of compound 1 and 7.970 g (0.044 mol) of compound 2. The reaction mixture was refluxed overnight. LCMS analysts of the reaction mixture demonstrated presence of 20 % target compound and 70 % non-cyclic product. Then 0.253 g (0.011 mol) of sodium in 50 mL of dry methanol was added to the reaction mixture and the reaction mixture was refluxed overnight. At this time LCMS analysis of the reaction mixture demonstrated 45 % of target compound. 1 more eq. of sodium methylate was added. The reaction mixture was refluxed for 2 days and total conversion of starting material into target compound was reached according to LCMS analysis. The reaction mixture was poured into water and acidified to pH 5 with HCI. The precipitate formed was filtered and lyophilized to produce 3 as a white solid. The yield averaged 43 % (2.950 g).
[0440] Preparation of 4. 2.1 g (0.0067 mo!) of 3 was dissolved in 100 mL of methanol, and an aqueous suspension of Pd/C (10%) was added under Ar atmosphere. The reaction mixture was additionally purged under Ar for 20 min. At this time, the reaction mixture was hydrogenated until the reduction was complete. The reaction was stirred overnight at 40 0C. Then the precipitate was carefully filtered and the residue was collected under reduced pressure. The iatter was crystallized from ether and air dried to produce 4 as a white solid. The crude yield was 87 % (1.650 g).
[0441] Preparation of 5. A diazonium salt was prepared by adding of a solution of 1.0 g of sodium nitrite in 2 mL of water to a suspension of 1.650 g (0.0058 mol) of compound 4 in 3 mL of hydrochloric acid and 11 mL of acetic acid on cooiing to 0-5 0C. After stirring for 20 min., the diazonium salt suspension was poured in one portion into 50 mL of acetic acid saturated with SO2 in the presence of copper chloride dihydrate on cooling to 10 0C. The mixture was stirred for 30 min. at 10-15 0C and for 1 h at RT. Next, the reaction mixture was heated to 50 0C and stirred for 2 h. The suspension was poured into ice water (500 mL). The precipitate was extracted with methylene chloride and concentrated under reduced pressure to produce 5 as a white solid. The crude yield was 75 % (1.60 g).
[0442] Compound 1b Methyl pi perazine (10 g) and potassium carbonate (20 g) were dissolved in 70 mL of DMSO on stirring and a solution of 14 g of 1a in 30 mL of DMSO was added dropwise. After 5 hours of stirring the reaction mixture was treated with water. The precipitate obtained was filtered and washed with water to produce 1b as a yellow solid. The yield was 64 % (14.2O g).
[0443] Compound 2b 5 g of 1b were dissolved in 200 mL of methanol, and an aqueous suspension of Pd/C (10%) was added under Ar atmosphere. The reaction mixture was additionally purged under Ar for 20 min. At this time, the reaction mixture was hydrogenated for 54 hours. The precipitate was carefully filtered and the residue was collected under reduced pressure affording 2b as a yellow solid. The yield was 74 % (3.2 g).
[04443 Compound 3c To a stirred solution of 2b (0.001 mol) and triethylamine (0.5 mL) in 10 mL of dioxane a solution of sulfochforide 5 (0.001 mol) was added dropwise at 5 0C. After 5 hours of stirring the solvent was removed and the residue was carefully treated with water. The precipitate obtained was filtered and treated with 50 mL of 1 N aqueous NaOH. The reaction mixture was stirred at reflux for 9 hours; then cooled to RT and acidified with acetic acid. The precipitate obtained was filtered and air dried to produce 3c as a pale yellow soiid. The yield was 34 % (0.19 g).
Figure imgf000152_0001
Table 31. Example 216 of Pyrazolopyri mi dines. Library P.
Figure imgf000152_0003
Analytical Data for Library P
216. 3-(7-h yd roxy-1 -met hyl -3-propy 1-1 H- py razol o[4,3-d]-py ri m idin-5-y l)-N-[4-(4- methylpiperazin-1-yi)phenyl]benzene-sulfonamtde 1H NMR (DMSO, ppm) δ 0.98 1 (3H); 1.82 m (2H); 2.20 s (3H); 2,40 1 (4H); 2.80 1 (2H); 3.1O t (4H); 4.18 s (3H); 6.70 d (2H); 7.00 d (2H); 7.65 1 (1 H); 7.82 d (1 H); 8.25 d (1H); 8.50 s (1H); 10.75 bs (1H).
Library V
Figure imgf000152_0002
[0445] Compound 2 To a stirred solution of 1 (50 g) SOCI2 (50 ml) was added dropwise at
RT for 1 hour. After 30 hours of stirring at 700C1 the reaction mixture was redistilled in vacuo and produced 2 as brown oil. Yield 10 g.
[0446] Compound 4 To a stirred solution of 3 (0.1 mol) and triethylamine (5 ml) in dichloromethane (100 ml), 2 (0.1 mot) was added dropwise at RT for 1 hour. After 10 hours of stirring at 7O0C, the reaction mixture was treated with water, the organic layer was concentrated in vacuo and 4 was produced as yellow solid.
[0447] Compound 5 0.240 g (0.0060 moi) of sodium hydride was suspended in 15 ml of dry
THF, then a solution of 0.900 g (0.0020 mol) of compound 4 in 30 ml of dry THF was added dropwise. The reaction mixture was stirred overniαht at 50-600C. LCMS of the reaction mixture demonstrated about 85-90% of target compound. Thereafter, 1 eq. of ethano! was added, the reaction mixture was poured into water and extracted with chloroform. The residue was concentrated under reduced pressure and crystallized from ether.
[0448] Compound 6 1.5 g of 5 was hydrogenated in methanol (20 mi) over Pd/C catalyst for
54 hours, filtered, and the solvent was removed. The residue was purified by column chromatography.
[0449] Compound 7 To a stirred solution of 6 (0.001 mol) and triethylamine (0.5 ml) in 10 ml of dioxane, a solution of suifochloride (0.001 mol) was added. After 5 hours of stirring the solvent was removed and the residue was treated with water. The precipitate obtained was filtered off.
Table 32. Example 217 of Pyrazolopyrimidines. Library V.
Figure imgf000153_0001
Analytical Data for Library V
217. 3-(7-hydroxy-1-methyi-3-propyl-1 H-pyrazoIo[4,3-d]pyrimidin-5-yi)-N-[4-<2- oxopiperidin-1-yl)-phenyl]benzene-sulfonamide 1HNMR (DMSO, ppm) δ 0.98 1 (3H); 1.82 m (6H); 2.35 m (2H); 2.85 t (2H); 3.53 m (2H); 4.18 S (3H); 7.13 s (4H); 7.65 t (1 H); 7.92 d (1 H); 8.25 d (1 H); 8.55 S (1 H); 10.07 bs (1 H); 12.25 bs (1 H).
Library V2
Figure imgf000153_0002
Preparation of Compounds 218-223
S-fT-Chloro-i-methyl-S-propyt-IH-pyrazolo^.S-dJpyrtmidin-S-ylJ-N-t'i-dimethylamino- phenyl)-4-ethoxy-benzenesulfonamide (2)
[0450] A mixture of compound 1 (200 mg, 0.392 mmol) and freshly distilled POCI3 (3.5 mL) was stirred at refluxing for 1 hour, then cooled to room temperature, stirred for 1 hour at room temperature and concentrated at reduced pressure. The obtained residue was dissolved in dichioromethane (3 mL) and cooled to O0C. To the resulting mixture NEt3 (400 mg, 3.95 mmol) was carefully added at 00C1 warmed up to room temperature and concentrated at reduced pressure. Purification by column chromatography on silica gel (ethyl acetate) gave compound 2. Yield 109 mg, 53 %. 1H-NMR (400MHz, DMSO-D6) δH: 0.95 (3H, t, J=7.5 Hz), 1.27 (3H, t, J=7.5 Hz), 1.82 (2H1 q, J=7.5 Hz), 2.80 (6H, s), 2.95 (2H, t, J=7.5 Hz), 4.15 (2H, q, J=7.5 Hz), 4.30 (3H, s), 6.63 (2H, broad peak), 6.91 (2H, d, J=8.5 Hz), 7.27 (1 H, d, J=8.5 Hz), 7.70 (1 H1 d, J=8.5 Hz)1 8,02 (1 H, s), 9.63 (1H, broad peak). MW 529.07. LCMS tR (min): 1.89. MS (APCl+), m/z 529.07, 531.07 [M+H]+. HPLC fe (min): 12.58, MP 132-1330C.
N^-Dimethylamino-phenylH-ethoxy-S-II-methyl-a-propyl-T-^^^-trifluoro-ethoxyJ-IH- pyrazolo[4,3-d]pyrimidϊn-5-yl]-benzenesulfonamide (3-Entry 217)
[0451] A mixture of compound 1 (200 mg, 0.392 mmol) and freshly distilled POCI3 (3.5 mL) was stirred at refluxing for 1 hour, then cooled to room temperature, stirred for 1 hour at room temperature and concentrated at reduced pressure. The obtained residue was dissolved in 2,2,2-trifiuoroethanol (2 mL). To the resulting mixture DIPEA (0.4 mL, 2.29 mmol) was carefully added dropwise. The obtained mixture was stirred at room temperature for 1.5 hour and at refluxing for 1.5 hour, cooled to room temperature and concentrated. Purification by prepHPLC (acetonttrile/water) gave compound 2. Yield 200 mg, 86 % (for two steps). 1H-NMR (400MHz, DMSO-D6) δH: 0.95 (3H, t, J=7.5 Hz), 1.28 (3H, t, J=7.5 Hz), 1.83 (2H, m), 2.80 (6H, s), 2.92 (2H, t, J=7.5 Hz), 4.12 (2H, q, J=7.5 Hz), 4.19 (3H, s), 5.30 (2H, q, J=7.5 Hz), 6.56 (2H, d, J=8.5 Hz), 6.92 (2H, d, J=8.5 Hz), 7.23 (1 H, d, J=8.5 Hz), 7.70 (1 H, d, J=8.5 Hz), 8.07 (1 H, d, J=8.5 Hz), 9,52 (1H, broad peak). MW 592.65. LCMS tR (min): 2.02. MS (APCI+), m/z 593.11 [M+Hf. HPLC U (min):14.34. MP 82-830C.
3-(7-Dimethylamino-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N-(4- dimethylamino-phenyl)-4-ethoxy-benzenesulfonamide(4a-Entry 218) [0452] A mixture of compound 3 (200 mg, 0.392 mmol) and freshly distilled POCI3 (3.5 mL) was stirred at refluxing for 1 hour, then cooled down to room temperature, stirred for 1 hour at room temperature and concentrated at reduced pressure. The obtained residue was dissolved in dichioromethane (2 mL). To the resulting mixture dimethylamine hydrochloride (326 mg, 4,0 mmol) and DIPEA (1 mL) were carefully added dropwise at 00C. The obtained mixture was stirred at 9O0C for 2 hours in sealed tube, cooled down to room temperature and concentrated. Purification by prepHPLC (acetonitrile/water) gave compound 4a. Yield 120 mg, 57 % (for two steps). 1H-NMR {400MHz, DMSO-D6) δH: 0.95 (3H, t, J=7.5 Hz), 1.26 (3H1 1, J=7.5 Hz), 1.79 (2H, m), 2.78 (6H, s). 2.83 (2H, t, J=7.5 Hz), 3.10 {6H, s), 4.10 (5H, superposition of q and s), 6.56 (2H, d, J=8.5 Hz), 6.91 (2H, d, J=8.5 Hz), 7.18 <1 H, d, J=8.5 Hz), 7.65 (1 H, d, J=8.5 Hz), 7.99 (1 H, s), 9.50 (1 H, broad). MW 537.69. LCMS fe (min):1.52. MS (APCi+), m/z 538.16 [M+Hf . HPLC tR (min):9.42. MP 89-900C.
N-(4-Dimethylamino-pherϊyl)-4-ethoxy-3-[7-(4-fluoro-benzylamino)-1-methyl-3-propyl-1H- pyrazo1o[4,3-d]pyrimidin-5-yl]-benzenesulfonamide {4b-Entry 219)
[0453] A mixture of compound 1 (156 mg, 0.305 mmol) and freshly distilled POCI3 (3 ml.) was stirred at room temperature for 5 hours and concentrated at reduced pressure. The obtained residue was dissolved in acetonitrile (2 ml_). To the resulting mixture a soiution of DIPEA (0.079 mg, 0.610 mmol) was carefully added dropwise at 100C. To the obtained mixture, p- fluorobenzylamine (1 mL, 8.709 mmol) was added and the mixture was stirred at room temperature for 2 hours and at refiuxing for 5 hours, cooled down to room temperature, diluted with water and extracted with dichloromethane. The combined organic phases were dried over sodium sulfate and concentrated. Purification by prepHPLC (acetonitrile/water) and prepTLC (hexane/ethyl acetate, 1/1 ) gave compound 4b (60 mg). MW 617.75. LCMS tR (min):1.72. MS (APCi+), m/z 618.17 [M+ H]+. HPLC tR (min): 10.95. The compound had 90% purity. N -{4-D i methy I am ino-phe ny l)-4-ethoxy-3-( 1 -methy l-3-propy i-7-py rrolid i n-1 -y I -1 H- pyrazolo[4,3-d]pyrimidin-5-yl)-benzenesuffonamide(4c»Entry 220) [0454] A mixture of compound 1 (200 mg, 0.392 mmoi) and freshly distilled POCI3 (3.5 mL) was stirred at refiuxing for 1 hour, then cooled down to room temperature, stirred for 1 hour at room temperature and concentrated at reduced pressure. The obtained residue was dissolved in dichloromethane (3 mL). To the resulting mixture pyrrolidine (4 mL) and DIPEA (1 mL) were carefully added dropwise at O0C. The obtained mixture was stirred at 900C for 1 hour, cooted down to room temperature and concentrated. The residue was washed with cold hexane and diluted with dichloromethane. The resulting solution was washed with water, dried over sodium sulfate and concentrated at reduced pressure. Purification by column chromatography on silica gel (ethyl acetate) and preparatory HPLC (acetonitrile/water) gave compound 4c. Yield 163 mg, 74 % (for two steps).1H-NMR (400MHz, DMSO-D6) δH: 0.92 (3H, t, J=7.5 Hz), 1.21 (3H, t, J=7.5 Hz), 1.81 (2H, m), 1.95 (4H, m), 2.89 (6H, s), 2.91 (2H, t, J=7.5 Hz), 3.71 (4H, m), 4.05 (2H, q, J=7.5 Hz), 4.19 (3H, s), 6.51 (2H, d, J=8.5 Hz), 6.91 (2H, d, J=8.5 Hz), 7.15 (1H, d, J=8.5 Hz)1 7.61 (1 H, d, J=8.5 Hz), 8.01 (1 H, s), 9.49 (1 H, broad peak). MW 563.73. LCMS tR (min): 1.59. MS (APCI+), m/z 564.22 [M+H]+. HPLC tR (min): 10.14. MP 96-970C. N-(4-DimethyIamino-phenyl)-4-ethoxy-3-(1-methyl-7-morpholin-4-yl-3-propyt-1H- pyrazoio[4,3-d]pyπmidin-5-yl)-benzenesulfonamϊde (4d-Entry 221) [0455] Compound 4d was prepared according to the procedure for compound 4c. [0456] Yield 191 mg, 84 % (for two steps). 1H-NMR (400MHz, DMSO-D6) 6H: 1.01 (3H, t, J=7.5 Hz), 1.29 (3H, t, J=7.5 Hz), 1.81 (2H, m), 2.75 (6H, s), 2.82 (2H, t, J=7.5 Hz), 3.49 (4H, m), 3.81 (4H, m), 4,11 (5H, superposition of q (2H) (J=7.5 Hz) and s (3H)), 6.51 (2H, d, J=8.5 Hz), 6.91 (2H, df J=8.5 Hz), 7.19 (1H, d, J=8.5 Hz), 7.69 (1 H, d, J=8.5 Hz)1 8.01 (1 H, s), 9.51 (1 H, broad peak). MW 579.73. LCMS tR (min): 1.64. MS (APCI+), m/z 580.20 [M+Hf. HPLC tR (min): 9.93. Mp 99-100°C.
N-{4-Dimethylamino-phenyl)-4-ethoxy-3-{1-methyl-7-phenylamino-3-propyl-1H- pyrazolo[4,3-d]pyrimidin-5-yl)-benzenesulfonamide (4e-Entry 222) [0457] Compound 4e was prepared according to the procedure for compound 4c. [0458] Yield 170 mg, 74 % (for two steps).1H-NMR (400MHz, DMSO-D6) δH: 0.98 (3H, t, J=7.5 Hz)1 1.21 (3H1 t, J=7.5 Hz)1 1.81 (2H, m), 2.71 (6H1 s), 2.89 (2H, t, J=7.5 Hz), 4.11 (2H, q, J=7.5 Hz), 6.52 (2H, d, J=8.5 Hz), 6.86 (2H, d, J=8.5 Hz), 7.06 (1 H, t, J=8.5 Hz), 7.19 (1 H, d, J=8.5 Hz), 7.41 (2H, t, J=8.5 Hz), 7.61 (1 H, d, J=8.5 Hz), 7.85 (2H, d, J=8.5 Hz), 7.91 (1 H, s), 8.72 (1 H, broad peak), 9.45 (1 H, broad peak). MW 585.73. LCMS fe (min): 1.76. MS (APCI+), m/z 586.19 [M+Hf. HPLC tR (min): 10.60. MP 108-1100C.
Preparation of Entries 224-225
Figure imgf000156_0001
1-Methyl-3-propyl-1,4-dihydro-pyrazoio[4,3-d]pyrimidine-5,7-dione (2)
[0459] A mixture of compound 1 (1.2 g, 6.59 mmol) and urea (3.95 g, 65.9 mmol) was fused at 2000C for 1 hour, cooled, dissolved in 30 ml of 5% NaOH and neutralized with acetic acid.
The formed solid was filtered and washed with water, dried at 55°C for 20 hours to give compound 2 as a white powder. Yield 1.40 g, 99%. MW 208.22. LCMS tR (min): 1.30. MS
(APCI+), m/z 209.22 [M+Hj+.
5,7-Dichloro-1 -methyl-3-propyI-1 H-pyrazolo[4,3-d]pyrimidine (3)
[0460] A mixture of compound 2 (1.40 g, 6.59 mmol), POCl3 (5 mL) and PCI5 (1.0 g) was reftuxed for 15 hours, cooled to room temperature, poured into crushed ice and extracted with ether (100 mL). The organic layer was separated, washed with aqueous NaHCO3 (30 mL) and water (30 mL), dried over Na2SO4 and concentrated. Purification by column chromatography on silica gel (ethyl acetate/hexane, 1/10) gave compound 3 as yellow oil (purity according LCMS approx. 80%, used in the next stage without additional purification). Yield 850 mg. MW 245.11.
LCMS tR (min): 1.89. MS (APCI+), m/z 245.19 [M+H]+. (5-Chloro-1-methyl-3-propyI-1H-pyrazoIo[4,3-d]pyrimidin-7-yl)-(4-fluoro-phenyl)-amine (4) [0461] A mixture of compound 3 (250 mg, 1.02 mmol) and 4-fluoro-phenylamine (125 mg, 1.35 mmol) in acetonitrile (5 mL) was stirred at room temperature for 10 hours and at 500C for 8 hours, diluted with water and extracted with dichioromethane {50 mL). The organic layer was separated, dried over K2CO3 and concentrated. The residue oil was triturated with ether and dried to give compound 4 as a yellow powder. Yield 122 mg, 37 %. MW 319.77. LCMS tR (min): 1.86. MS (APCiH-), m/z 320.12 [M+H]+.
(4-Fluoro-phenyf)-(1-methyl-3-propyl-5-pyrrolidin-1-yi-1H-pyrazolo[4,3-dJpyrimidin-7-yl)- amine (5-Entry 223)
[0462J A solution of compound 4 (120 mg, 0.38 mmol) in pyrrolidine (2.5 mL) was refluxed for 4 hours and concentrated. The residue was dissolved in dichioromethane (50 mL) and washed with aqueous solution of NH4CI (2x10 mL). The organic layer was separated, dried over Na2SO4 and concentrated. Purification by column chromatography on silica gel
(dichloromethane/acetone, 10/1 ) and recrystallization from hexane gave compound 5 as a white powder. Yield 60 mg, 44 %. 1H-NMR (400MHz, DMSO-D6) δH: 0.90 (3H, t, J=7.5 Hz), 1 ,73 (2H, m), 1 ,88 (4H, broad peak, Z/E forms), 2.70 (2H, t, J=7.5 Hz), 3.42 (4H, broad peak, Z/E forms), 4.15 (3H, s), 7.18 (2H, d/d, J=8.5/8.0 Hz), 7.80 (2H, broad m, Z/E forms), 8.42 (1 H, broad peak). MW 354.43. LCMS fe (min): 1.57. MS (APCl+), m/z 355.28 [M+H]+, HPLC k (min): 11.68. Mp. 111-112°C.
5-Chloro-1-methyI-3-propyl-7-pyrroiϊdin-1-yl-1H-pyrazolo[4,3-d]pyrimidine (6) [0463] A mixture of compound 3 (250 mg, 1.02 mmol), DIPEA (145 mg, 1.13 mmol) and pyrrolidine (80 mg, 1.13 mmol) in acetonitrile (5 mL) was stirred at room temperature for 1 hour and diluted with water. The formed solid was collected by filtration and washed with water and hexane giving compound 6 as a cream-colored solid. Yield 215 mg, 75 %. MW 279.77. LCMS tR (min): 1.74. MS (APCI+), m/z 280.24, 282.23 [M+H]+.
(4-FIuoro-phenyl)-(1-methyl-3-propyl-7-pyrrolidin-1-yI-1H-pyrazolo[4,3-d]pyrimidin-5-yI)- amine (7-Entry 224)
[0464] A mixture of compound 6 (200 mg, 0.71 mmol), DMSO (0.3 mL) and 4-fluoro- phenylamine (102 mg, 0.92 mmol) was heated at 1500C for 4 hours, cooled down and diluted with water. The formed solid was collected by filtration. The crude product was purified by column chromatography on silica gel (dichloromethane/acetone, 30/1 ) and recrystallized from hexane/ether giving compound 7 as white crystals. Yield 60 mg, 24%. 1H-NMR (400MHz, DMSO-D6) δH: 0.92 (3H, t, J=7.5 Hz), 1 ,75 (2H, m), 1 ,95 (4H, broad peak, Z/E forms), 2.72 (2H, t, J=7.5 Hz), 3.75 (4H, broad peak, Z/E forms), 4.05 (3H, s), 7.03 (2H, d/d, J=8.5/8.0 Hz), 7.82 (2H, broad m, Z/E forms), 8.77 (1 H, broad peak). MW 357.43. LCMS fe (min): 1.60. MS (APCI+), m/z 358.25 [M-^H]+, HPLC tR (min): 11.80. Mp. 135-137X. Table 33. Examples 218-225 of Pyrazolopyrimidines. Library V2.
Figure imgf000158_0001
C. PREPARATION OF EXEMPLARY IMlDAZOLOPYRIMiDINE DERIVATIVES AS HCV ENTRY INHIBITORS
Figure imgf000159_0001
(2-Chloro-7H-purin-6-yl)-furan~2-ylmethyi-amine {2)
[0465] A mixture of 2,6-dichloropuriπe (1 ) (200 mg, 1.0 mmol) and furfurylamine (2) (100 mg,
1.0 mmoi), NEtS (0.15 mL, 1 mmol) and acetonitrile (20 ml_) was stirred at room temperature for
3 hours, concentrated at reduced pressure and diluted with water. The formed precipitate was filtered, washed with 50% ethanol and diethyl ether and dried giving compound 2. Yield 164 mg, 62%.
Nβ-Furan-2-ylmethyl-N2-(4-methoxy-phenyl)-7H-purine-2,6-diamine hydrochloride (3-Entry
225)
[0466] A mixture of compound 2 (80 mg, 0.3 mmol) and 4-methoxy-phenylamine (110 mg,
0.9 mmoi) was heated at 2000C for 5 minutes, cooled to room temperature and washed with ethanol. The precipitate was collected by filtration, washed with ethanol and dried, giving compound 3 as a hydrochloride. Yield 60 mg, 56%. 1 H-NMR (400MHz, DMSO-D6) δH: 3.40
(2H, broad), 3,75 (3H, s), 4.75 (2H, broad), 6.32 (1 H, broad), 6.40 (1 H, broad), 6.87 (2H, d,
J=8.5 Hz), 7.58 (1 H, s), 7.60 (2H, d, J=8.5 Hz), 8.12 (1 H1 broad), 8.32 (1 H, broad), 9.01 (1 H, broad). LCMS tR (min): 1.42. MS (APCI), m/z 337.05 [M+H]+. HPLC tR (min): 9.45. Mp 276oC.
(2-Chloro-7H-purin-6-yl)-{4-methoxy-phenyl)-amϊne (4)
[0467] A mixture of 2,6-dichloropurine 1 (520 mg, 2.80 mmol) and 4-methoxy-phenylamine (399 mg, 3.24 mmol) in pentanoi (12 mL) was refiuxed for 2 hours, cooled to room temperature and diluted with water. The precipitate was filtered, washed with water, ethanol and diethyl ether giving compound 4. Yield 640 mg, 83%. N2-Furan-2-ylmethyl~N6-(4-methoxy-phenyl)-7H-purine-2,6-diamϊne (5-Eπtry 226) [0468] A mixture of compound 3 (100 mg, 0.36 mmol) and furfυrylamine (2 mL) was refluxed for 1 hour, concentrated under vacuum and diluted with water. The formed precipitate was fiitered and washed with diethyl ether (2 ml). Purification by coiumn chromatography (chloroform/methanol, 20:1 ) and preparative TLC (chioroform/methanol/ammonia, 10:1 :0.5) gave compound δ.Yield 55 mg, 23%. 1 H-NMR (400MHz, DMSO-D6) δH: 3.75 (3H, s), 4.50 (2H, d, J=7.5 Hz), 6.20 (1 H, broad), 6.35 (1 H, broad), 6.78 (1 H, broad), 6.88 (2H, d, J=8.5 Hz), 7.51 (1 H, s), 7.82 (1H, broad), 7 82 (2H, d, J=7 5 Hz), 9.15 (1 H, broad), 12.32 (1 H, broad). LCMS tR (min): 1.4. MS (APCI), m/z 337 12 [M+H]+. HPLC tR (min): .43. Mp 190-1930C.
Table 34. Examples 1-2 of Imidazolopyrimidines.
Figure imgf000160_0001
Figure imgf000160_0002
Figure imgf000161_0001
Figure imgf000161_0002
Figure imgf000162_0001
Figure imgf000162_0002
Figure imgf000163_0001
Figure imgf000163_0002
Figure imgf000164_0001
Figure imgf000165_0001
Figure imgf000166_0001
Figure imgf000167_0001
Figure imgf000167_0002
Figure imgf000168_0002
Synthesis Scheme for Entries 4, 6, and 7 of Table 35
Figure imgf000168_0001
4-aminθ"6<hloro-2-(1-pyrrolidinyl)-5-pyrimidinecarbaldehyde (5a).
[0469] A mixture (0.0040 mol) of compound 5 was dissolved in 10 mL of ethanol. NH3 in dioxane was then added slowly, dropwise, while the reaction mixture was started and the process was monitored by TLC. The reaction mixture was heated to 50-60° C and was stirred overnight at this temperature. Solvent was removed in vacuo, a yellow precipitate was filtered off and the residue was evaporated to dryness. The yellow precipitate was then crystallized from ether. Pale yellow precipitate was obtained with yield 69% (0.630 g), HNMR (400MHz,
DMSO-D6) δH: 1.89 (m, 4H); 3.46 (m, 4H); 7.98 (s, 1 H); 8.42 (s, 1 H); 9.92 (s, 1 H).
4-chloro-1-propyl-6-(1-pyrrolidinyl)-1W-pyrazolo(3,4-cfjpyrimidine (7).
[0470] 0.250 g (0.0034 mol) of propyl hydrazine was suspended in 10 mL of dry dioxane and 0.630 g (0.0028 mol) of 2.-chloro-4-(1~pirrolidiniI)-6-amino benzaldehyde (5) was added; then 0.283 g (0,0028 mol) of TEA was added at once. Reaction mixture was stirred overnight at room temperature. White precipitate was filtered off, solvent was removed in vacuo. Target compound was crystallized from hexanes and dried. Yellow precipitate was obtained with yield
65% (0.450 g). HNMR (400MHz, DMSO-D6) δH: 0.86 (t, J= 7.5, 3H); 1.77-1.93 (m, 6H); 3.52
(m, 4H); 4.06 (t, J= 6.8, 2H); 6.69 (s, 2H); 7.78 (s, 1 H).
Λ/-phenyl-Λf-[1-propyl-6-(1-pyrroliclinyI)-1H-pyrazoto[3,4-€/]pyrimidin-4-yl]urea (8).
[0471] 0.150 g (0.00061 mol) of 7 was dissolved in 5 mL of dry dioxane and 0.073 g (0.00061 mol) of phenylisocianate was added. Reaction mixture was stirred at 50-60° C for 48 h. Pale yellow precipitate was filtered off and dried. Target compound was crystallized from hexanes.
Pale yellow precipitate was obtained with yield 65% (68.6 mg), HNMR (400MHz1 DMSO-D6) δH: 0.86 (t, J=JA1 3H); 1.85 (rn, 2H); 2.01 (m, 4H); 3.64 (m, 4H); 4.14 (t, J= 6.9, 4H); 7.08 (t,
J=7.1 , 1 H); 7.35 (t, J=8.1 , 2H); 7.54 (d, J=8.1 , 2H); 8.24 (s, 1 H); 10.04 (s, 1 H); 11.37 (s, 1 H).
£0472] /^-[i-propyt-e-ti-pyrrolidmyO-IH-pyrazolota^-dJpyrimidϊn^-yllbenzamϊde tθ) ets.
[0473] 0.15O g (0.00061 mol) of 7 was dissolved in 5 mL of dry dioxane and 0.00073 mol of benzoyl chloride and 0.112 g (0.00073 mol) of DBU were added. Reaction mixture was stirred at 70° C for 48 h. Reaction mixture was poured into water and extracted with chloroform. Target compound was purified by column chromatography with methylene chloride as eluant. White precipitate was obtained with yield 30% (1.3 mg). HNMR (400MHz, DMSO-06) δH: 0.87 (t,
J=8.3, 3H); 1.75-2.00 (m, 6H); 2.62 (m, 4H); 3.58 (m, 4H); 4.18 (t, J= 6.7, 2H); 7.50-7.70 (m,
3H); 8.01 (d, J=Jl, 2H); 8.10 (s, 1 H); 10.38 (s, 1 H).
Entry 4. 4-chloro-N-[1-propyl-6-(pyrrolidϊn-1-yI)-1 H-pyrazo!o[3,4-d]pyrimidϊn-4« yljbenzamide.
[0474] HNMR (400MHz, DMSO-D6) δH: 0.82 (m, 4H); 1.74-1.97 (m, 6H); 3.54 (m, 4H); 4.15
(m, 2H); 7.58 (m, 2H); 8.03 (m, 3H); 10.97 (s, 1 H).
Entry 6. 4-chIoro-N-[1-propyl-6-(pyrrolidin-1-yl)-lH-pyrazolo[3,4-d]pyrimidin-4- yl]benzamide [0475] HNMR (400MHz, DMSO-D6) δH: 0.88 (t, J=7.3, 3H); 1.88 (m, 6H); 3.41 (m, 4H); 4.17
(t, J=6.9, 2H); 7.40-7.60 (m, 4H); 8,16 (s, 1 H); 10.86 (s, 1 H).
(0476] Entry 7. 2-phenyi-N-[1-propyl-6-(pyrrolidin-1-yl)-1H-pyrazo)o[3,4-d]pyrimidin-4- yijacetamide
[0477] HNMR (400MHz, DMSO-D6) δH; 0.86 (t, J=7.9, 3H); 1.85 (m, 2H); 1.95 (m, 4H); 3.58
(m, 4H); 3.95 (s, 2H); 4.14 (t, J=6.8, 2H); 7.21-7.39 (m, 5H); 8.07 (s, 1 H); 10.39 <s, 1H).
Synthesis Scheme for Entries 5, 8, 9, and 19 of Table 35
Figure imgf000170_0001
Preparation of 3a.
[0478J Boc-hydrazine (1a) (50 g, 0.385 mmol) was dissolved in acetone (500 mL) and the reaction mixture was stirred at 50 °C overnight. Then, the solvent was removed under reduced pressure, and the residue was resuspended in Et2O and filtered. A white precipitate was obtained with quantity yield (64 g). HNMR, DMSO-d6 δ, ppm: 1.45 (s, 9H); 1.79 (s, 3H); 1.88 (s, 3H); 9.11 (s, 1 H). Preparation of 4a.
(3a) (0.087 moi) was dissolved in toiuene (150 mL); KOH powder 14.6 g (0.267 mol), 2.105 g (0.0087 mol) BTEA-HOSO3 (10 mol %) and phenylethyl bromide 19.3 g (0.104 mol) were added. The reaction mixture was stirred at 80°C overnight. The solids were removed by fiitration, and the filtrate was washed twice with the same volume of cold water. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Intermediate 4a contained about 20% elimination byproduct, but was used in the next step without further purification. Orange oil was obtained with crude yield of 70-80 %. R1 =Ph: HNMR, DMSO-Cl6 δ, ppm: 1.33 (s, 9H); 1.66 (s, 3H); 1.97 (s, 3H); 2.30 (t, J=7.1 , 2H); 2.76 {t, J=7.1 , 2H); 7,23 (m, 5H). Preparation of 5a.
[0479J (4a) (5.8 g, 0.021 mo!) was dissoived in THF (100 mL) and a 2 M aqueous solution of HCI (40 mL) was added. The reaction mixture was refluxed overnight. At this time, the solvent was removed under reduced pressure, and the product was dried by concentration from benzene. LCMS analysis of the reaction mixture demonstrated about 80% of (5a). The precipitate was crystallized from ether to give the target compound 5a (3.0 g, 68% of yield) as a hydrochloride salt (pale gray precipitate). RI=Ph: HNMR, DMSO-d6 δ, ppm: 2.87 (t, J=7.3, 2H); 3.13 (t, J=7.3. 2H); 5.87 (s, 2H); 7.26 (m, 5H). Preparation of 6
[0480] 0.523 g (0.0025 mol) of phenethyi hydrazine dihydrochloride was suspended in 60 mL of dry dioxane and 0.505 g (0.0050 mol) of TEA was added; then 0.5 g (0.0021 moi) of 2.6- dschloro-4-(1-pirrolidiny!) benzaldehyde (5) was added at once. The reaction mixture was stirred overnight at room temperature, at which time the solvent was removed in vacuo. The residue was purified by column chromatography with methylene chloride as eSuant to provide a pale yellow precipitate (0.270 g) in a yield that averaged 35-40%. RI=Ph: HNMR, DMSOd6 δ, ppm: 1.95 (m, 4H); 3.12 (t, J=7.3, 2H); 3.51 (m, 4H); 4.41 (t, J=7.6, 2H); 7.18 (m, 5H); 7,84 (s, 1 H). Preparation of 7a1
[0481] 0.150 g (0.00046 mol) of 1-phenethyl-4-chforo-6-(pyrrolϊdinyl)-1 H-pyrazoio- [3,4d]pyrimidin 6 was suspended in 5 ml of dry dioxane, then (0.00092 mol) of aniline and 0.1 ml of concentrated HC! were added. The reaction mixture was refluxed for 48 h. LCMS demonstrated total conversion of starting material to target compound. Reaction mixtures were poured into water and extracted with chloroform. Targets were purified by column chromatography with methylene chloride as eiuant. White precipitates were obtained with yields 41 %, 59% (14.1 , 35.6 mg). Preparation of 7a2
[0482] 0.150 g (0.00046 moi) of 1-phenethyl-4-chloro-6-(pyrrolϊdinyl)-1 H-pyrazok> [3,4d]pyrimidin 6 was dissolved in 5 ml of dry dioxane, then (0.00092 mol) of benzyl amine 20 and 0.070 g (0.00046 mol) of DBU were added at once. Reaction mixture was stirred at 8O0C for 48 h. LCMS demonstrated total conversion of starting material to target compound. Reaction mixture was poured into water and extracted with chloroform. Target compound was purified by column chromatography with methylene chloride as eiuant. White precipitate was obtained with yields 44% (15.3 mg),
[0483] Entry 5. N-(3-chlorophenyl)-1-(2-phenylethyl)-6-(pyrrolidin-1-yl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine HNMR (400MHz, DMSO-D6) δH: 1.97 (m, 4H); 3.18 (t, J= 7.4, 2H); 3.59 (m, 4H); 4.42 (t, J= 7.1 , 2H); 7.04 (d, J= 7.7, 1 H); 7.13-7.38 (m, 5H); 7.74 (d, J= 8.3, 1 H); 7.99 (s, 1 H); 8.23 (m, 1 H); 9.44 (s, 1 H). Entry 8. 4-ftuoro-N-(4-{[1-{2-phenylethy!)-6-{pyrrolidin-1-yl)-1H-pyrazoIo[3J4-cl3pyrimϊdtn- 4-yl]amino}phenyl)benzamicie
[0484] HNMR (400MHz, DMSO-D6) δH: 1.95 (m, 4H); 3.18 (t, J= 7.4, 2H); 3.58 (m, 4H); 4.41 (t, J= 7.5, 2H); 7.13-7.36 (m, 5H); 7.71 (d, J= 9.0, 2H); 7.84 (d, J= 7.4, 2H); 7.92 {s, 1 H); 8.05 (m, 2H); 9.26 (s, 1 H); 9.91 (s, 1 H).
Entry 9. 4-fluoro-N-(4-{E1-<2-phenylethyi)-6-pyrro-lidin-1-yi-1H-pyra2o-fo[3,4-dJpyrimidin- 4-yl]-amiπo}phenyl)benzenesutfonamide
HNMR (400MHz, DMSO-D6) δH: 1.94 (m, 4H); 3.16 (t, 2H); 3.53 (m, 4H); 4.40 (t, J= 7.6, 2H); 7.07 (d, J= 8.1 , 2H); 7.13-7.38 (m, 6H); 7.73 (d, J= 8.8, 2H); 7.80 (m, 2H); 7.88 (s, 1 H); 9.25 (s, 1 H), 9.74 (s, 1 H).
Entry 19. N-(4-{[1-(2-pheny!ethyi)-6-pyrrol-ldin-1-yi-1H-pyrazo-Io[3,4-d]-pyrimidin-4- yl]amino}phenyl)methanesulfonamide
[0485] HNMR (400MHz, DMSO-D6) 5H: 1.95 (m, 4H); 3.17 (t, 2H); 3.57 (m, 4H); 4.40 (t, 2H); 7.21 (m, 6H); 7.85 (d, J= 8.6, 2H); 7.93 (s, 1 H); 9.18 (s, 1 H); 9.27 (s, 1 H). Synthesis Scheme for Entries 10, 11, 15, 40, 42, 53. and 41 of Table 35
Figure imgf000172_0001
Figure imgf000172_0002
Et3N, dioxane, 500C f
Figure imgf000172_0003
Preparation of 3,
[0486] A mixture of aminoamide 2.1 (5 g, 0.0298 mol) and the aldehyde 3.2 (8.94 g, 0.06 mol) in the presence of methanesulfonic acid (5 drops) was refiuxed in xylene (50 ml) for 48 h. The reaction mixture was chilled and the precipitate was filtered, carefully washed with ether and dried. The pale yellow precipitate was obtained with 85 % (7.2 g) yield. HNMR (400MHz, 90DC, DMSO-D6) δH: 0.89 (t, J=7.4, 3H); 1.90 (sx, J1=7.4, J2=7.0, 2H); 4.28 (t, J=7.0, 2H); 7.54 (m, 3H); 8.08 (m, 2H); 8.20 (s, 1 H); 11.60 (s, 1 H). Preparation of 4.
[0487] The pyrazopyrimidine 2.2 (3g, 0.01 mol) was added to plenty of SO3HCI (10 eq.) portionwise on careful cooling of the reaction mixture (O0C). When the addition was complete, the reaction mixture was stirred at RT for 24 h. The mixture was then diluted with ice water; the organic layer was extracted with EtOAc and concentrated under reduced pressure. The residue was obtained as a yellow precipitate with 90 % (3.8 g) yield. HNMR (400MHz, 900C, DMSO- D6) 5H: 0.92 (t, J=7.4, 3H); 1.90 (sx, J1=7.4, J2=7.0, 2H); 4.28 (t, J=7.0, 2H); 7.89 (t, J-8.0,
1 H); 8.26 (s, 1 H); 8.30 (d, J=8.0, 1 H); 8.77 (d, J=8.0, 1 H); 9.18 (s, 1 H); 12.87 (S1 1 H),
Preparation of 5. 3-(4-hydroxy-1-propy!-1H-pyrazoIo[3,4-djpyrϊmidin-6-yl)-Λ/1-R-1- benzenesulfonamide:
[0488] 0.00054 mol of 2.3 was dissolved in 6 mL of dry chloroform, then 0.054 g (0.00054 moi) of TEA were added. The reaction mixture was stirred at RT for 3 min, after this 0,00054 mol of amine was added at once. The reaction mixture was stirred overnight at 800C, then cooled and poured into aqueous sodium carbonate solution. The organic layer was extracted, concentrated under reduced pressure and the solid was crystallized from ether. The target compound was carefully washed with hot hexane and air dried. The final compounds were obtained with 40-70 % yields.
Preparation of 6. 3-{4-chloro-1-propyI-1W-pyrazoio[3,4-<flpyrimidtn~6-y1)"tø1~R-1- benzenesu If on am i de :
[0489] 1eq.(0.3g) of the sulfonamide was suspended in 10 ml of POCI3 and 2eq. of the PCI5 was added. The reaction mixture was refluxed overnight. Excess POCI3 was removed in vacuo. The obtained product was used for the next stage without additional purification.
Preparation of 7. 3-(4~anilino-1-propyl-1W-pyrazoio[3,4-cf]pyrimidin-6-yl)-Λ/1-R-1- benzenesulfonamide:
[0490] Compound 6 was dissolved in dioxane and an excess of aniline was added. The reaction mixture was stirred overnight at 500C. Then it was filtered off and filtrate was evaporated under reduced pressure. The final compounds were purified by HPLC and were obtained with 40-54 % yields.
Entry 10. N-phenyt-1-propyl-6-[3"(pyrrolidin-1-ylsυIfonyl)phenyl]-1H-pyrazolo[3,4- d]pyrimidin-4~amine
[0491] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.93 (t, J=7.4, 3H), 1.73 (m, 4H)5 1.98 (sx,
J1=7.4, J2=6.8, 2H), 3,26 (m, 4H), 4.42 (t, J=6.8, 2H), 7.19 (t, J=7.0, 1 H), 7.44 (t, J=8.5, 2H),
7.79 (t, J=7,8, 1 H), 7.90 (m, 3H), 8.20 (s, 1 H), 8.70 (d, J=7.8, 1 H), 8.85 (s, 1 H), 9.92 (s, 1 H).
Entry 11. N-{4-methylphenyl)-1-propyl-6-[3-(pyrrolidin-1-ylsulfony!)phenyl]-1H- pyrazolo[3,4-d]pyrimidin-4-amine
[0492] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.86 (t, J=7.4, 3H), 1.66 (s, 4H), 1.90 (sx,
J1=7.4, J2=6.8, 2H), 2.34 (s, 3H), 3.20 (s, 4H), 4.38 (t, J=6.8, 2H), 7.24 (d, J=7.9, 2H), 7.77 (m,
3H), 7.93 (m, 1 H), 8.24 (s, 1 H), 8.71 (d, J=7.8, 1 H), 8.81 (s, 1 H), 10.14, (s, 1 H).
Entry 15. 4-(2-chIorophenyl)-1-propyi-6-(pyrroiϊdin~1-yi}-1H-pyrazolo[3,4-d]pyrirnfdine
[0493] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.87 (t, J= 7.4 Hz, 3H), 1.79-1.89 (m, 2H),
1.97 (t, J= 6.7 Hz, 4H), 3.96 (s, 2H), 4.19 (t, J= 6.9 Hz5 2H), 7.35 (d, J= 8.5 Hz5 2H), 7.90 (d, J=
8.5 Hz, 2H), 8.02 (s, 1H)1 9.90 (s, 1H).
Entry 40. N-[4-(morpholin-4-yl)phenyl]-3-[4-(phenyIamino)-1-propyl-1H-pyrazoIo[3,4- d] py ri m id i n-6-y IJbenzen es u If on am i de [0494] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.92 (t, J=7.4, 3H); 1 97 (sx, J1=7.4, J2=6,8,
2H); 2.96 <m, 4H); 3.66 (t, J=4.β, 4H); 4.40 (t, J=6.8, 2H); 6.75 (d, J=9.0, 2H); 7.01 (d, J=9.0,
2H); 7.19 (t, J=7.5, 1 H); 7.45 (t, J=8.0, 2H); 7.66 (t, J=7.8, 1 H); 7.84 (m, 3H); 8.18 (s, 1 H); 8.60
(d, J=7.2, 1 H); 8.82 (s, 1 H); 9.56 (s, 1 H0; 9.88 fs, 1 H).
Entry 42. 3-{4-[(2-methylbenzyl)amino]-l-propyI-1 H-pyrazolo[3,4-d]pyrimidin-6-yi}-N- phenylbenzenesulfonamide
[0495] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.90 (t, J=7.4, 3H); 1.94 (sx, J1=7.4, J2=6 8,
2H); 2.40 (s, 3H); 4.34 (t, J=6.8, 2H); 4.87 (d, J=5.2, 2H); 6.99 <m, 1 H); 7.18 (m, 6H); 7.41 (m,
1H); 7.63 (t. J=7.8, 1 H); 7.85 (d, J=8.0, 1 H); 8.15 (s, 1 H); 8.36 (S, 1 H); 8.62 (d, J=8.0, 1 H); 8.85
(s, 1 H); 9.99 (s, 1 H).
Entry 53. Ethyl 1-({3-[4-(phenylamino)-1-propyl-1H-pyrazoϊo[3,4-d]pyrtmidin-6- yl]phenyl}sulfonyl)piperidine-4-carboxylate
[0496] HNMR (400MHz, DMSO-D6) δH: 0.91 (3H, t, J=7.4 Hz), 1.12 (3H, t, J=7 0 Hz); 1.62
(2H, m); 1.93 (4H1 m); 2.40 (1 H, m); 2.67 (2H, t, J=11.0 Hz); 3.57 (2H, m); 4.03 (2H, q, J=7.0
Hz); 4.41 (2H1 1, J=6.8 Hz); 7.19 (1 H, t, J=7.3 Hz); 7.44 (2H, t, J=7.6 Hz); 7.77 (1 H, t, J=7.7 Hz);
7.87 (3H, t, J=7.7 Hz); 8 22 (1 H, s); 8.70 (1 H, d, J=7.8 Hz); 8 79 (1 H, s); 9.98 (1 H, s). LCMS tR
(min) 3.06. MS (APCI), m/z 549.60 [M+H]+.
Entry 41. N-methyl-N-phenyl~3-[4-(phenyiamino)-1-propyl-1H-pyrazolo[3,4-d]pyrimidin-6- yljbenzenesulfonamide
[0497] HNMR (400MHz, 90 0C1 DMSO-D6) δH: 0.91 (t, J=7.4, 3H); 1.96 (sx, J1=7.4, J2=6.8,
2H), 3.24 (s, 3H); 4.38 (t, J=6.8, 2H); 7.19 (m, 3H); 7.30 (m, 3H); 7.41 (t, J=7.8, 2H); 7.70 (m,
2H); 7.84 (d, J=7.8, 2H); 8.19 (s, 1 H); 8.69 (m, 2H); 9.89 (s, 1 H).
Synthesis Scheme for Entry 12 of Table 35
/
Figure imgf000174_0001
4 5 6
Preparation of 2.
[0498] 5.Og (0.030 mol) of 1 was suspended in 100 ml of dry xylene and then 6.4g (0.060 moi) of benzaldehyde was added at once. 5 drops of toluene sulfuric aod were then added and the reaction mixture was stirred at reflux overnight. LCMS demonstrated about 60% of target compound; an additional amount of benzaldehyde (1 eq.) was added, and the reaction mixture was refluxed for 48h. The reaction mixture was cooled down and a paie yellow precipitate was filtered off and dried. A pale yellow precipitate was obtained with yield 42% (3.2 g). HNMR (400MHz, DMSO-D6) δH: 0.85 (t, J=7.5 Hz, 3H), 1.87 (m, 2H), 4.30 (t, J= 7.1 Hz, 2H), 7.52-7.63 (m, 3H); 8.08 (s, 1 H); 8.15 (m, 2H); 12.33 (s, 1 H), Preparation of 3.
[0499] 3.2 g (0.013 mol) of compound 2 was suspended in 50 ml of POCi3 and then 5.2 g (0.026 mof) of PCI5 was added at once. The reaction mixture was refluxed overnight. Solvent was then removed in vacuo. A paie yellow precipitate was obtained with yield 95 % (3.0 g). HNMR (400MHz, DMSO-D6) δH: 0.85 (t, >8.0 Hz, 3H), 1.91 (m, 2H), 4.44 (t, J= 7.1 Hz, 2H), 7.53 (m, 3H); 8.37-8.44 (m, 3H). Preparation of 4.
[0500] 3.0 g (mol) of 3 was suspended in 5O0C dioxane and 3.5 g (mol) of and 10 mg of CuI were added at once. Reaction mixture was stirred for 48 h at 800C. Solvent was removed in vacuo. Precipitate was filtered off, residue was purified by column chromatography with methylene chloride as eluant. Paie yellow precipitate was obtained with yield 40% (1.680 g). HNMR (400MHz, DMSO-D6) δH: 0.87 (t, J=7.5 Hz, 3H), 1.92 (m, 2H), 3.93 (s, 3H); 4.40 (m, 2H), 7.51-7.61 (m, 4H); 7.71 (d, J=8.2 Hz, 1 H); 8.14 (d, J=8.2 Hz, 1 H); 8.41 (s, 1 H); 8.50-8.54 (m, 2H); 9.01 (s, 1 H); 10.42 (s, 1 H). Preparation of 5.
[0501] 1.680 g (0.0043 mol) of compound 4 was suspended in 20 ml of water and 10 ml of methanol, 0.700 g (0.0108 mol) of KOH was added at once. The reaction mixture was heated to 8O0C and was stirred at this temperature until all starting material disappeared (reaction monitored by TLC). Then the reaction mixture was acidified and the white precipitate was filtered off and dried. White precipitate was obtained with yield 75 % (1.2 g). HNMR (400MHz, DMSO-D6) δH: 0.89 (t, 3H), 1.93 (m, 2H), 4.40 (m, 2H), 7.40-7.79 {m, 5H); 8.04-8.66 (m, 4H); 8.95 (s, 1 H); 10.33 (s, 1 H). Preparation of 6.
[0502] 0.300 g (0.00080 mol) of 5 was dissolved in 5 ml of dry dioxane. 0.156 g (0.00096 mot) of CDI was then added. The reaction mixture was stirred at 800C overnight. Then 0.114 g (0.00160 mol) of amine was added at once. The reaction mixture was stirred overnight at 800C and then was poured into a 5% mass, solution of Na2CO3 and extracted with methylene chloride. Target was purified by column chromatography with methylene chloride as eluant. White precipitate was obtained with yield 42 % (17.4 mg). HNMR (400MHz, DMSO-D6) δH: 0.93 (t, J=7.4, 3H); 1.87 (m, 4H); 1.97 (m, 2H); 3.50 (m, 4H); 4.41 (t, J=6.8, 2H); 7.26 (d, J=7.8, 1 H); 7.50 (m, 4H); 7.95 <d, J=8.9, 1 H); 8.18 (s, 1 H); 8.25 (s, 1 H); 8.44 (m, 2H); 9.91 (s, 1 H). Entry 12. {3-[(6-phenyl-1-propyI-1H-pyrazolα[3,4-d]pyrimidin-4- yl)amino]phenyl}(pyrrolidϊn-1-yl)methanone
[0503] HNMR (400MHz, DMSO-D6) δH: 0.93 (t, J=7.4, 3H); 1.87 (m, 4H); 1.97 (m, 2H); 3.50
(m, 4H); 4.41 (t, J=6,8, 2H); 7.26 (d, J=7.8, 1H); 7,50 (m, 4H); 7.95 (d, J=8.9, 1 H); 8.18 (s, 1 H);
8.25 (s, 1 H); 8.44 (m. 2H); 9.91 (s, 1 H).
Figure imgf000176_0001
1a 2a 3a 72 h
4a 5a
\ s N' *HBr
*H8r + \
N J
N reffux 12 h 3 o
Ra refiux
1
Rb 2 * 12 h N'
/ \
\ y
Cl R1
N" Ra 1^Rb
N N POCI3
_ I N^ >
N N N' DMF N'
/ "N 900C
N Λ Cl' A O ^ ^O
\ I 12 h
7 6 O 4
Preparation of 3a.
[0504] Boohydrazine (1a) (50 g, 0.385 mmol) was dissolved in acetone (500 ml_) and the reaction mixture was stirred at 5O0C overnight. The solvent was then removed under reduced pressure, and the restdue was resuspended in Et2O and filtered. A white precipitate was obtained with quantity yield (64 g). HNMR 1H, DMSO-d6 δ, ppm: 1.45 (s, 9H); 1 79 (s, 3H); 1 88 (s, 3H); 9.11 (s, 1 H). Preparation of 4a.
[0505] (3a) (0.087 mol) was dissolved in toluene (150 mL). KOH powder 14.6 g (0.267 mo!), 2.105 g (0.0087 moi) BTEA-HOSO3 (10 mo! %) and n-propyl bromide (0.104 mol) was added. The reaction mixture was stirred at 80αC overnight. The solids were removed by filtration, and the filtrate was washed twice with the same volume of cold water. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Intermediate 4a contained about 20% elimination byproduct, but was used in the next step without further purification. Orange oil was obtained with crude yield of 70-80 %. RI =Me: LCMS, M-H : 146.3 Preparation of 5a. [0506] (4a) (5.8 g, 0.021 mol) was dissolved in THF (100 ml_) and a 2 M aqueous solution of HCI (40 mL) was added. The reaction mixture was refluxed overnight. The solvent was then removed under reduced pressure, and the product was dried by concentration from benzene. LCMS analysis of the reaction mixture demonstrated about 80% of (5a). The precipitate was crystallized from ether to give the target compound 5a (3.0 g, 68 % of yield) as a hydrochloride salt (pale gray precipitate). RI =Me: LCMS, M+1 : 146.3. Preparation of 6
[0507] 0.523 g (0.0025 mol) of phenethyl hydrazine dihydrochloride was suspended in 60 mL of dry dioxane. 0.505 g (0.0050 mol) of TEA was added, then 0.5 g (0.0021 mol) of 2,6- dichloro-4-(1-pirrolidinyl) benzaldehyde (5) was added at once. The reaction mixture was stirred overnight at RT, at which time the solvent was removed in vacuo. The residue was purified by column chromatography with methylene chloride as eluant to provide a pale yellow precipitate (0.270 g) in a yield that averaged 35-40 %. RI =Me: HNMR 1H, DMSOd6 δ, ppm: 0.80 (t, J=7.3 Hz, 3H), 1.82 (q, 2H), 1.94 (m, 4H)1 3.52 (m, 4H), 4.16 (t, J= 6.9 Hz1 2H), 7.98 (s, 1H). R1 =pipertdine: HNMR 1H, DMSOd6 δ, ppm: 1.42 (m, 6H); 2.51 (m, 4H); 2.76 (t, J=6.6; 2HO; 2.95 (s, 4H); 3.58 (t, J=6.7, 4H); 4,31 (t, J=6.7, 2H); 7.90 (s, 1 H). LCMS: M+1 = 335.8. Preparation of 7
[0508] 0.135 g (0.00046 mol) of 1-propyl-4-chloro-6-(pyrrolidinyl)-1 H-pyrazolo-[3,4d]pyrimidin 6 was dissolved in 5 ml of dry dioxane, then (0.00092 mol) of benzyl amine 20 and 0.070 g (0.00046 mol) of DBU were added at once. The reaction mixture was stirred at 8O0C for 48 h. LCMS demonstrated total conversion of starting material to target compound. The reaction mixture was poured into water and extracted with chloroform. Target compound was purified by column chromatography with methylene chloride as eluant. White precipitates were obtained with yields 40-50 %. Preparation of 7.1
[0509] 0.135 g (0,00046 mol) of 1- propyl-4-chloro-6-(pyrrolidinyl)-1 H-pyrazolo- [3,4d]pyrimidin 6 was suspended in 5 ml of dry dioxane, then (0.00092 mol) of aniline and 0.1 ml of concentrated HCI were added. The reaction mixture was refluxed for 48 h. LCMS demonstrated total conversion of starting material to target compound. Reaction mixtures were poured into water and extracted with chloroform. Targets were purified by column chromatography with methylene chloride as eluant. White precipitates were obtained with yields 41 %, 59% (14.1 , 35.6 mg). Preparation of 7.2.
[0510] 1.0 g (0.0038 mol) of 6 was dissolved in 30 ml of dry dioxane; 0.0075 mol of piperazine was added at once. The reaction mixture was stirred overnight at 700C and then was poured into water and extracted with methylene chloride, concentrated under reduced pressure. The target compound was purified by column chromatography with methylene chloride as eluant. Yield was 44 % - 67 %. Entry 13. 4-fluoro-N-(4-{[1-propyI-6-(pyrrolidin-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-4- yl]amino}phenyl)benzamide
[0511] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.88 (t, J= 7.4 Hz, 3H), 1.80-1.92 (m, 2H),
1.97 (t, J= 6.6 Hz, 4H), 3.62 (t, J= 6.4, 6.6 Hz, 4H), 4,17 (t, J= 6.9 Hz, 2H), 7.31 (t, J= 8.9 Hz,
2H), 7.71-7.85 (m, 4H), 7.92 (s, 1H), 8.02-8.11 (m, 2H), 9.65 (s, 1 H), 9.98 (s, 1 H).
Entry 15. 4-(2-chlorophenyI)-1-propyl-6-(pyrrolidin-1-yl)-1H-pyrazofo[3,4-d]pyrimidine
[0512] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.87 (t, J= 7.4 Hz, 3H), 1.79-1.89 (m, 2H),
1.97 (t, J= 6.7 Hz, 4H), 3.96 (s, 2H)1 4.19 (t, J= 6.9 Hz1 2H), 7.35 (d, J= 8.5 Hz, 2H), 7.90 (d, J=
8.5 Hz, 2H), 8.02 (s, 1H), 9.90 (s, 1 H).
[0513] Entry 17. 4-[4-(2-methylphenyi)piperazin-1-yl]-1-propyl-6-{ρyrroiidin-1-yi)-1H- pyrazolo{3,4-d] pyrimidi ne
[0514] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.85 (t, J=7.4, 3H), 1.82 (sx, J1=7.4, J2=6.8,
2H), 1.91 (m, 4H), 2.24 <s, 3H), 3.55 (m, 4H), 4.07 (t, J=6.8, 2H), 4.60 (d, J=5.7, 2H), 5.96 (s,
1 H), 6.16 (m, 1 H), 7.72 (s, 1H), 7.82 (s, 1 H).
Entry 48. 4-{4-[4-(methylsulfonyl)phenyl]piperazin-1-yt}-1-propyl-6-(pyrrolidin-1-yl)-1 H- pyrazolo[3,4~d]pyrimidine
[0515] HNMR (400MHz, DMSO-D6) δH: 0.87 (t, J=7.5, 3H); 1.79-1.96 (m, 6H); 3.05 (s, 3H);
3.53-3.63 (m, 8H); 4.02-4.14 (m, 6H); 7.05 (d, J=9.3, 2H); 7.72 (d, J=9.3, 2H); 7.90 (s, 1 H).
Entry 34. 1-propyl-4-[4-(pyridin-2-ylrrtethyl)piperazin-1-yl]-6-(pyrroHdin-1-yI)-1H- pyrazolo[3,4-d]pyrimidine
[0516] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.86 (t, J=7.4 Hz, 3H); 1.82 (m, 2H); 1.90 (t,
J=6.7 Hz, 4H); 2.61 (m, 4H); 3.52 (t, J=6.7 Hz5 4H); 3.69 (s, 2H); 3.86 (m, 4H); 4.10 (t, J=6.9 Hz,
2H); 7.24 (m, 1 H); 7.47 (d, J=7.8 Hz, 1 H); 7.75 (m, 1H); 7.85 (s, 1 H); 8.50 (d, J=5.1 Hz5 1 H).
Entry 20. 1-[4-{3-{[1-propyi-6-(pyrroiidrn-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-4- yl]amino}benzoyl)piperazin-1-yi]ethanone
[0517] HNMR (400MHz, DMSO-D6) δH: 0.87 (t, J=7.3 Hz, 3H); 1.84 (m, 2H); 1.91 (m, 4H);
3.14 (m, 4H); 3.55 (m, 4H); 3.84 (s, 3H); 3.99 (m, 4H); 4.11 (t, J=6.9 Hz, 2H); 6.94 <m, 4H); 7.92
(s, 1 H).
Entry 26. N-{4-[(1-propyI-6-pyrrolidin-1-yl-1H-pyrazoloϊ3,4-d]pyrϊmidin~4- yl)amiπo]phenyl}methanesulfonamide
[0518] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.87 (t, J= 7.4 Hz, 3H)1 1.80-1.90 (m, 2H),
1.95 (t, J= 5.9, 6.2 Hz, 4H), 2.98 (s, 3H), 3.58 (t, J= 6.3 Hz, 4H), 4.12 (t, J= 6.7, 6.9 Hz, 2H), 7.22 (d, J= 8.7 Hz, 2H), 7.85 <d, J= 8.6 Hz, 2H)5 7.94 (s, 1 H), 9.25 (m, 2H).
Entry 27. N-{2-methyl-1H-benzimidazo!-6-yl)-1-propy]-6-(pyrroiidiπ-1-yi)-1H-pyrazofo[3,4- d]pyrimidin-4-amine
[0519] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.87 (t, J= 7.4 Hz, 3H), 1.79-1.89 (m, 2H),
1.96 (t, J= 6.7 Hz, 4H), 3.60 (t, J= 6.7 Hz, 4H), 4.11 (t, J= 6.9 Hz, 2H), 7.43 (m, 2H), 7.81 (s, 1 H), 8.13 (S, 1 H), 9.17 (s, 1 H). Entry 29. 2-{4-[1-propyl-6-(pyrro!idin-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]piperazin-1- yl}benzonitrile
[0520] HNMR (400MHz, DMSO-D6) δH: 0.88 (t, J=7.4 Hz, 3H); 1.85 (m, 2H); 1.93 (m, 4H);
3.38 (m, 4H); 3.56 (t, J=6.7Hz, 4H); 4.05 (m, 4H); 4.12 (t, J=6,9 Hz, 2H); 7,11 (m, 1H); 7.21 (d,
J=8.2 Hz, 1 H); 7.60 (m, 1 H); 7.68 (dd, 7.6, 1.4 Hz, 1 H); 7.94 (s, 1 H).
Entry 32. N-(4-<t1-propyl-6-(pyrrolidin-1-yl)-1H-pyra2olo[3,4-d|pyrimidin-4- yl]amino}phenyl)benzamϊde
[0521] HNMR (400MHz, 90 0C1 DMSO-D6) δH: 0.87 (t, J= 7.3, 7.4 Hz, 3H), 1.79-1.90 (m,
2H), 1.91-2.01 (m, 4H), 3.59-3.69 (m, 4H), 4.19 (t, J= 6.9, 7.0 Hz, 2H), 7.53 (m, 3H), 7.79 (s,
4H), 7.90 (S, 1 H), 7.98 (d, J= 7.3 Hz, 2H), 9.80 (s, 1 H), 9.97 (s, 1 H).
Entry 33. 1-propyl-4-[4^pyrimidin~2-y))piperazin-1-yt]-6-{pyrroiidin-1-yl)-1H-pyrazolo[3ir4- d]pyrimidtne
[0522] HNMR (400MHz1 90 0C, DMSO-D6) δH: 0.87 (t, J=7.4 Hz, 3H); 1.84 (m, 2H); 1.92 (t,
J=6.8 Hz, 4H); 3.55 (t, J=6.7 Hz, 4H); 3.96 (m, 8H); 4.11 (t, J=6.9 Hz, 2H); 6.64 (t, J=4.8 Hz,
1 H); 7.92 (s, 1 H); 8.38 (d, J=4.8 Hz,
Synthesis Scheme for Entries 14, 28, 30. and 31 of Table 35
Figure imgf000179_0001
Preparation of 5
[0523] Compound 4 (200 mg, 0,63 tnmol), boronic acid (150 mg, 0.72 mmol) and triphenylphosphine (25 mg, 0.10 mmol) were charged into the flask containing 4 mL of dioxane and 1.5 mL of 2 M aqueous Na2CO3, After purging the mixture with Ar for 20 min, [Pd(PPh3)J catalyst (36 mg, 0.03 mmoi, 5 mol %) was added. The reaction mixture was heated at 1000C for 4 h under Ar atmosphere. After cooling the reaction mixture to RT, the solvent was removed under reduced pressure to produce a yellow oil. The residue was washed with water (20 mL) and extracted with CHCi3 (20 mL). The organic layer was isolated and purified by column chromatography.
Entry 14. {3-[1-propyl-6-(pyrro!tdin-1-yl)-1H-pyrazo)o[3,4-d]pyrimidin-4- yl]phenyi}(pyrroltdin-1-y1)methanone
[0524] HNMR (400MHz, DMSO-D6) δH: 0.90 (t, J=7.3 Hz, 3H); 1.89 (t, J=6.3 Hz, J=6.9 Hz,
4H); 1.97 (m, 2H); 2.02 (t, J=6.3 Hz, J=6.9 Hz1 4H); 3.47-3.53 (m, 4H); 3.65- 3.74 (m, 4H); 4.25
(t, J=6.9 Hz, 2H); 7,60- 7.72 (m, 2H); 8.19 (s, 1H); 8,24- 8.30 (m, 2H). Entry 28. {4-[1-propyl-6-(pyrrolidiπ-1-yI)-1H-pyrazoio[3,4-d]pyrϊmidin-4- yl]phenyl}{pyrrolidin-1-yl)methanone
[0525] HNMR (400MHz, DMSO-D6) δH: 0.90 (t, J=7.3 Hz, 3H); 1.85-2.05 (m, 10H); 3.49 (m, 4H); 3.72 (m, 4H); 4.25 (t, J=6,9 Hz, 2H); 7,69 (d, J=7.3Hz, 2H); 8.26 (t, J=8.0Hz, J=7.3 Hz, 3H). Entry 30. N-{3-[1-propyl-6-{pyrrolidin-1-yt)-1H-pyrazoIo[3,4-d]pyrimidin-4- y I] phe ny l}acetam ide
[05261 HNMR (400MHz1 DMSO-D6) δH: 0.90 (t, J=7.3 Hz, 3H); 1.85-1.95 (m, 2H); 2.01 (m, 4H); 2.11 (s, 3H); 3.69 (m, 4H); 4,25 (t, J=6.9 Hz, 2H); 7.48 (t, J=8.QHz, J=7.8 Hz, 1 H); 7.75 (d, J=8.0Hz, 1 H); 7.90 (d, J=7.8 Hz, 1 H); 8.21 (s, 1 H); 8.49 (m, 1 H); 9.84 (s,1 H). Entry 31. N-[3-(1-propyl-6-pyrrolidϊn-1-yl-1 H-pyrazolo[3,4-d]pyrimidin-4- yi)phenyfjmethanesulfonamide
[0527] HNMR (400MHz, 90 0C, DMSOD6) δH: 0.90 (t, J=7.3 Hz, 3H); 1.85-1.95 (m, 2H); 2.01 (m, 4H); 3.06 (s, 3H); 3.69 (m, 4H); 4.25 (t, J=6.9 Hz, 2H); 7.42- 7.48 (m, 1 H); 7.50- 7.58 <m, 1H); 7.97 (dt, J=7.4 Hz, J=1.5 Hz, 1 H); 8.15 (t, J= 1.5 Hz1 1 H); 8.20 (s, 1 H); 9.63 (s,1 H). Synthesis Scheme for Entry 18 of Table 35
Figure imgf000180_0001
4-chloro-1-propyl-6-{1-pyrrolidiny))-1H-pyrazolo[3,4-cf]pyrimidine (6). [0528] 5.42 g (0.073 mol) of propyl hydrazine was suspended in 300 mL of dry dioxane and 15 g (0.061 mol) of 2.6-dichioro-4-(1-pirrolϊdinil) benzaldehyde (5) was added. 6.2 g (0.061 mol) of TEA was then added at once. The reaction mixture was stirred overnight at room temperature, then solvent was removed in vacuo. The target compound was purified by coiumn chromatography with methylene chloride as eluant. Pale yellow precipitate was obtained with yield 60% (9.8 g). HNMR (400MHz, DMSO-D6) δH: 1.90 1 (2H); 1.75-2.00 m (6H); 3.60 m (4H); 4.2O t (2H); 7.95 s (1 H).
Methyl 3-[1-propyl-6-(1-pyrrolidinyl)-1 H-pyra2olo[3,4-cf]pyrimidfn-4-yl3aminobenzoate (7) [0529] 1.0 g (0.0038 mol) of 4-chioro-1 -methyl-6-(1 -pyrroiidinyl)-1 H-pyrazolo-[3,4-d]- pyrimidine 6 was suspended in 10 mL of dry dioxane, 1.1 g (0.0075 mol) of methyl-3- aminobenzoate and 10 mg of CuI were added; then the reaction mixture was stirred overnight at 7O0C. Precipitate was filtered off and dried. Beige precipitate was obtained with yield 95%
(1.370 g). HNMR (400MHz, DMSO-D6) δH: 0.83 (t, J=7.5, 3H); 1.80 (m, 2H); 1.97 (m, 4H); 3.64
(m, 4H); 3.85 (s, 3H); 4.26 (m, 2H); 7.50 (t, J=7.8, 1H); 7.64 (d, J=6.9, 1 H); 8.07 (d, J=9.3, 1 H);
8.30 (ss 1 H); 8.90 (s, 1 H).
3-[1-propyl-6-(1-ρyrrolϊdinyl)-1 W-pyrazolo[3,4-cf]pyrirnidin-4"yI]aminobenzoic acid (8).
[0530] 1.370 g (0.0036 mol) of compound 7 was suspended in 20 ml_ of water and 0.403 g
(0.0072 mol) of KOH was added at once. The reaction mixture was heated to 800C and was stirred at this temperature untii all starting material disappeared (reaction monitored by TLC).
Reaction mixture was cooled down, reaction mixture was acidified to pH 5-6 and white precipitate was filtered off and dried. White precipitate was otained with yield 93% (1.230 g).
HNMR (400MHz, DMSO-D6) 6H: 0.82 (t, J=7.5, 3H); 1.80 (m, 2H); 1 ,92 (m, 4H); 3.57 (m, 4H);
4.09 (d, J=6.9, 2H); 7.21 (t, J=7.8, 1 H); 7.52 (d, J=6.9, 1 H); 8.32 (d, J=9.3, 1 H); 8.38 (s, 1 H);
8.45 (s, 1 H); 10.20 (s, 1 H).
(4-substϊtuted piperazino)(3-[1-propyl-6-{1-pyrrolidinyl)-1W-pyrazolo[3,4-d]pyrimidin-4- yl]aminophenyE)methanone (9).
[0531] 0.250 g (0.00068 mol) of 8 was dissolved in 5 mL of dry dioxane, then 0.00082 mol of amine and 0.103 g (0.00102 mol) of TEA were added at once. The reaction mixture was stirred for about 3-5 min at room temperature, and then 0.251 g (0.00164 mol) of POCI3 was added.
Reaction mixture was stirred at 800C for 48 h. The reaction mixtures were poured into water and extracted with chloroform. Target compounds were purified by column chromatography with methylene chloride as eiuant.
Entry 18. N-{3-[(4-acetylpiperazin-1-yl)carbonyl3phenyl}-1-propyl-6-pyrrolidin-1-yl-1H- pyrazolo[3,4»d]pyrimidin-4-amine
[0532] HNMR(400MHz, DMSO-D6) δH: 0.88 (t, J=7.2, 3H); 1.86 (m, 2H); 1.95 (m, 4H); 2.03
(s, 3H); 3.45-3.62 (m, 12H); 4.13 (t, J=7.1 , 2H); 7.06 (d, J=8.1 , 1 H); 7.42 (t, J=7.8, 1 H); 7.96 (m,
2H); 8.05 (m, 1H); 9.42 (s, 1 H).
Synthesis Scheme for Entries 36 and 44 of Table 35
Figure imgf000181_0001
BB synthesis
'
Figure imgf000181_0002
N
PdCl2[PPh3]2 dioxaπe f ^
(69%) 1 ^ # N'
1M sol 0f Na2C03
80 C
POC!3 \ LιAIH4
\ THF Py
(92%) It / (59%> \ 60°C
o 60 C
<^~ P O
'"V
Figure imgf000182_0001
1-[(4-bromophenyl)sulfonyl]pyrrolidine 2.
[0533J 6.7 g (0.093 moi) of pyrrolidin was dissolved in 200 ml_ of dioxane and 7.9 g (0.078 mol) of TEA was added at once. The reaction mixture was stirred for 20 min and 20.0 g (0.078 mol) of 4-bromobenzyisulfochloride was added at once. The reaction mixture was stirred overnight at 800C and then was poured into water. A pale brown precipitate was formed. The precipitate was filtered off and crystallized from ether. A pale brown precipitate was obtained with yield 95% (18.7 g). HNMR (400 MHz, DMSO-D6) SH: 1.65 (m, 4H); 3.14 <m, 4H); 7 73 (d, J=8.4, 2H); 7.83 (d, J=8.4, 2H); LCMS tR (min) 1.50. MS (APCI), m/z 291.7 [M+H]. 4-(1-pyrroiidinylsulfonyl)phenylboronic acid 3.
[0534] 14.5 g (0.059 mol) of 2 was dissolved in 600 mL of dry THF. The reaction mixture was cooled down to -70°C and then 28 mL of 2.5 M solution of butyl lithium in hexanes was added in portions under argon atmosphere. The reaction mixture was slowly heated to -300C and was stirred at this temperature for about 1h. Then 26.7 g (0.118 mol) of triϊsopropyl borate was dissolved in 100 mL of dry THF in another flask and both reaction mixtures were again cooled down to -700C. The solution of ϋthium derivative was then gently added in portions to the solution of triisopropyl borate under argon atmosphere while temperature was maintained at - 650C. The reaction mixture was then slowly heated to room temperature and stirred overnight under argon atmosphere. Thereafter the reaction mixture was cooled down to O0C and tert- butanot was added. The solvent was removed in vacuo and residue was dissolved in acidic water and extracted with ether. Precipitate was crystallized from hexanes. White precipitate - crude material - was obtained with iow yield 10 % (about 1.0 g). LCMS tR (min) 1.50. MS
(APCi), m/z 256.5 [M+ H]+.
{4'-(pyrrolidin-1-ylsulfonyI)-1,1'-biphenyl-4-yl]amid 5.
[0535J 2,0 g (0.0081 mol) of 2 was dissolved in 50 mL of dioxane and 5 mL of 1 M aqueous solution of Na2CO3 was added at once. Then 1.6 g (0.0097 moi) of 4 and 0.02 g of Pd Ci2EPPh3J2 as catalyst were added under argon atmosphere. The reaction mixture was stirred overnight at
800C. Then white precipitate was filtered off, washed with water and chloroform. A white precipitate was obtained with yield 69 % (1.85 g). HNMR (400MHz, DMSO-D6) δH: 1.66 (m,
4H); 3.18 (m, 4H); 7.44 (bs, 1 H); 7.79-8.11 (m, 9H); LCMS tR (min) 1.50, MS (APCt), m/z 331.5
[M+H]+.
^'-(pyrroϋdln-i-ylsuifonylJ-i^'-biphenyM-yllnitrite δ.
[0536] 1.66 g (0.0050 moi) of 5 was dissolved in 30 m L of dry Py and then 1.20 g (0.0075 mol) of POCI3 was added dropwise to a stirred solution of starting material. The reaction mixture was stirred overnight at RT and was then poured into water. A brown precipitate was filtered off and dried. A pale brown precipitate was obtained with yield 92% (1.430 g). HNMR (400MHz,
DMSO-D6) δH: 1.67 (m, 4H); 3.18 (m, 4H); 7.88-8.02 (m, 8H); LCMS tR (min) 1.50. MS (APCI), m/z 313.5 [M+H]+.
[4'-(pyrrolidin-1-yIsulfonyi)-1,1'-bipheπyI-4-yi]methyfamine 7.
[0537] 1.430 g (0.0046 mol) of 3a was dissolved in 50 mL of dry THF and then Ni/Re was added under argon atmosphere. The reaction mixture was heated to 50-600C, H2 was purged into the flask with stirring until there was total conversion of starting material to target compound. The precipitate was filtered off and the residue was concentrated under reduced pressure. Target was crystallized from ether. A white precipitate was obtained with yield 59%
(0.860 g). HNMR (400MHz, DMSO-D6) δH: 1.73 (m, 4H); 3.23 (m, 4H); 4.13 (s, 2H); 7.60 (d,
J=7.9, 2H); 7.80 (d, J=7.9, 2H); 7.90 (m, 4H); 8.27 (bs, 2H); LCMS tR (min) 1.50. MS (APCI), m/z 317.1 [M+H]+.
^'-(pyrrolidin-i-ylsuIfonylJ-i.i'-biphenyM-ylJamine 9.
[0538] 2.0 g (0.0081 mol) of 8 was dissolved in 50 mL of dioxane, and 5 mL of 1 M aqueous solution of Na2CO3 was added at once. Then 2.1 g (0.0097 mol) of 4 and 0.02 g of Pd
CI2[PPh3]2 as catalyst were added under argon atmosphere. The reaction mixture was stirred overnight at 80°C. The reaction mixture was then poured into water and extracted with chloroform, The target was purified by column chromatography with chloroform as eluant. A white precipitate was obtained with yield 61 % (1.47 g). HNMR (400MHz, DMSO-D6) δH: 1.64
(m, 4H); 3.14 (m, 4H); 5.43 (bs, 2H); 6.67 (d, J=8.6, 2H); 7.47 (d, J=8.6, 2H); 7.76 (m, 4H);
LCMS tR (min) 1.50. MS (APCI), m/z 303.5 [M+Hf .
Entry 36, 1-propyl-6-{pyrrolidin-1-yl)-N-[4'-(pyrroitdin-1-yisulfonyl)bipheny)-4-yl]-1H- pyrazoto[3,4-d]pyrimidin-4-amine [0539] HNMR(400MHz, DMSO-D6) 6H- 0.86 (t, J=7 6, 3H); 1.74 (m, 4H); 1.83-2.00 (m, 6H);
3 23 (m, 4H); 3.63 (m, 4H); 4.15 (t, J=7.2, 2H); 7.76 (d, J=9.0, 2H); 7.85 (d, J=9.0, 2H); 7.91 (d,
J=9.0); 8.03-8.09 (m, 3H); 9.50 (s, 1H).
Entry 44. 1-propyl-6-{pyrrolidin-1-yl)-N-{[4'-(pyrrolidin-1-yIsulfonyl)biphenyl-4-yi]methyl}-
1H-pyrazolo[3,4-d]pyrimidin-4-amine
[05403 HNMR(400MHz, DMSO-D6) δH: 0.86 (t, J=7.4, 3H); 1 ,73-1.91 (m, 10H); 3.22 (m, 4H);
3.55 (m, 4H); 4.12 (t, J=6.7, 2H); 4.76 (m, 2H); 7.52 <d, J=7.8, 2H); 7.70 (t, J=7.8, 2H); 7.86 (m,
5H); 8.24 {s, 1 H).
Synthesis Scheme for Entry 37 of Table 35
Figure imgf000184_0001
8a a d 8b
1-propyl-1 H-pyrazolop^-dJpyrimidine^jβ-diol 2.
[0541 J 8.6 g (0.051 mol) of 1 was dissolved in dry dioxane and then 20.2 g (0.102 mol) of diphosgene was added. The reaction mixture was refluxed for 3 days and then was cooled. A yellow precipitate was formed. The precipitate was filtered off and washed with ether. A yeϋow precipitate was obtained with yield 42% (4.2g). HNMR (400MHz, DMSO-06) δH: 0 82 (t, J=7 1 ,
3H); 1.72 <m, 2H); 4.08 (t, J=6.8, 2H); 7.77 (s, 1 H); LCMS tR (min) 1.50. MS (APCI), m/z 195.2
[M+H]+.
4,6-dichloro-1-propyl-1H-pyrazolo[3,4-d]pyrimidine 3.
[0542] 4 2 g (0.022 mo!) of 6 was suspended in 70 rrtL of POCI3 and then 9.0 g (0.044 mol) of
PCS5 was added. The reaction mixture was stirred at reflux overnight and then was cooled and the solvent was removed in vacuo. A dark yellow oil was obtained with yield 86% (4.4 g).
LCMS tR (min) 1.50. MS (APCI), m/z 232.1 [M+H]+.
N-(6-chtoro-1-propyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N-(4-fluorophenyl)amine 4.
[0543] 1.3 g (0.0056 mol) of compound 7 was dissolved in 10 mL of dry dioxane and then
0.0056 mol of aniline and 0.6 g (0.0056 mol) of TEA were added. The reaction mixture was stirred overnight at 800C and then was poured into water. A pale yellow precipitate was filtered off and dried. A pale yellow precipitate was obtained with yield 59 %.
1-[(4-fluorophenyI)sulfonyiJpyrrolidine 7
[0544J A solution of the sulfochioride 1 (5 g 0.026 mole), amine (2.2g 0.031 mole) and Et3N
(3.1g 0.031 mole) in chloroform was stirred overnight at 600C. The mixture was diluted with water and the organic layer was separated and dried with Na2SO4 and then evaporated. Yield:
4.2g (70%). 1 H-NMR (400MHz, DMSO-D6) 5H: 1.68 <p, J1 =3.1 , J2=6.8, 4H); 3.15 (t, J=6.8,
4H); 7.40 (m, 2H); 7.85 (m, 2H). te/f-butyl ΛM-[4^1-pyrrolidϊnyIsulfonyl)phenyI]-4-piperidylcarbamate 8a, 1-4-[4-(1- pyrrolidinylsulfonyl)phenyl]piperazino-1-ethanoπe 8b
[0545] Compound 7 (0.5g 0.0022mole) and BOC-protected- (or acetyl) amine (0.0033mole) was dissolved in N-methyl-pirrolidone and the reaction mixture was stirred at 1800C for 2 days.
Then the reaction mixture was diluted with water and the precipitate was filtered off. Compound
8a: LCMS: MS (APCI), m/z 410.6 [M+H]; Compound 8b: LCMS: MS (APCi), m/z 338.4
[M+H].
1-[4-(1-pyrrolidinylsulfonyl)phenyi]-4-piperidinamine 9a.
[0546] BOC-protected compound 8a 1g (0.00246mole) was stirred in dioxane with HCI at room temperature overnight. Then the solvent was evaporated, dissolved in an aqueous solution of Na2CO3 and then extracted with CHCi3. The organic layer was evaporated and a white solid was washed by ether. 0.4g (52 %). 1 H-NMR (400MHz, DMSO-D6) δH: 1.47 (m,
2H); 1.67 (m, 4H); 1.92 (m, 2H); 3.01 (m, 9H); 3.83 (d, J=13.3, 2H); 7.01 (d, J=9.1 , 2H); 7.56 (d,
J=9.1 , 2H).
1-[4-(1-pyrrolidinylsuifonyl)phenyl]piperazine 9b
[0547] NaOH 0.6 g (0.015 mole) was dissolved in the mixture EtOH :H2O (1 :1 ). To this mixture compound 8b 1g (0.00297 mole) was added and the reaction mixture was stirred at
100βC overnight. The reaction mixture was diluted with chloroform and the organic layer was separated, dried and evaporated under reduced pressure. 0.7 g (80 %). 1 H-NMR (400MHz,
DMSO-D6) 6H: 1.67 (m, 4H); 2.85 (t, J=5.0, 4H); 3.11 (m, 4H); 3.24 (m, 4H); 6.99 (d, J=8.8,
2H); 7.57 (d, J=8.8, 2H).
Entry 37. N-(4-fluorophenyi)-1-propyi-6-{4-[4-(pyrrolϊdin-1-yisulfonyl)phenyi]piperazin-1- y I}- 1 H -py razol o[3 ,4-d] py rim id i n-4-am i ne
[0548] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.89 (t, J=7.4, 3H); 1.69 (s, 4H); 1.88 (sx,
J1=7.4, J2=6.8, 2H); 3.13 (s, 4H); 3.48 (s, 4H); 3.94 (s, 4H); 4.15 (t, J=6.8, 2H); 7.07 (d, J=8.6,
2H); 7.18 (t, J=8.6, 2H); 7.62 (d, J=8.6, 2H); 7.76 (m, 2H); 7.91 (s, 1 H); 9.43 (s, 1 H). Synthesis Scheme for Entries 38 and 39 of Table 35
Cl R4
Figure imgf000186_0001
Preparation of 2
[0549] 0.0017mole of amine (9a or 9b) was suspended in 5 mL of dry dioxane. Et3N (0.11 g,
0.00113 mole) and 0.3 g (0.00113 mole) of the 4-chioro-1 -methyl-6-(1 -pyrrolidinyl)-1 H-pyrazo!o-
[3,4-d]-pyrimidine 1 were added, and the reaction mixture was stirred overnight at 500C. LCMS demonstrated total conversion of starting material. The mixture was diluted with water, extracted with CHCI3, and the organic layer was separated and dried with Na2SO4 and then evaporated. The precipitate was filtered off and dried. The final compound was purified by column chromatography.
Entry 38. 1-propyl-6-pyrrolidin-1-yl~4-{4-t4-(pyrrolldin-1-ylsuIfony!)phenyl]piperaz!n-1-yl}-
1 H-pyrazolo[3,4-d]pyrimidine
[0550] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.87 (t, J=7.4, 3H); 1.70 (m, 4H); 1.84 (sx,
J1=7.4, J2=6.8, 2H); 1.93 (m, 4H); 3.14 (t, J=6.8, 4H); 3.57 (m. 8H); 4.05 (t, J=5.4, 4H); 4.12 <t,
J=6.8, 2H); 7.04 (d, J=9.1 , 2H); 7.62 (d, J=9.1 , 2H); 7.90 (s, 1 H).
Entry 39. 1-propyt-6-pyrrolidin-1-yl-N-{1-I4-(pyrrolIdin-1-ylsu!fonyl)phenyl]piperidin-4-yI}-
1H"pyrazolo[3I4-d]pyπmidin-4-amine
[0551] HNMR (400MHz, 90 0C1 DMSO-D6) δH: 0.86 (t, J=7.4, 3H); 1.70 <m, 6H); 1.86 (m,
6H); 2.10 (m, 2H); 3.14 (m, 6H); 3.54 (s, 4H); 3.91 (m, 2H); 4.07 (t, J=6.8, 2H); 4.27 (s, 1 H);
7.06 (d, J=8.6, 2H); 7.20 (d, J=7.0, 1H); 7.60 (d, J=8.6, 2H); 7.81 (s, 1 H).
Synthesis Scheme for Entries 43, 46, 49, and 57 of Table 35
Cl ' N <
Figure imgf000186_0002
Figure imgf000186_0003
[0552] Preparation of 4. 6-chloro-N-phenyl-1-propyl-1H-pyrazolo[3,4-d]pyrimidin-4- amine
[0553] To a stirred solution of 3 (4.6 g, 0.02 moi) and triethylamine (2.5 ml) in 70 ml of dioxane a solution of amine (0.02 mol) was added. After 5 hours of stirring the solvent was removed and residue was treated with water. Precipitate obtained was filtered off and washed with acetonitrile afforded crude 6 as brown solid. Yield 3.2 g (56 %). LCMS tR (min) 2.06. MS
(APCi), m/z 288.10 [M+H].
Preparation of 5. Methyl 3-(4-anilino-1-propyl-1 H-pyrazolo[3,4-d]pyrtmidin-6-yl)benzoate. [0554] 4 (1O g, 0.033 moi), boronic acid (7 g, 0.035 moi), and triphenylphosphine (1.25 g,) were charged into the flask containing 120 mL of dioxane and 45 mL of 2 M Na2CO3 solution.
After purging the mixture with Ar for 20 min, [PdO(PPh3)4] catalyst (1.1 g) was added. The reaction mixture was heated at 100°C for 4 h under an Ar atmosphere. After cooling the reaction mixture to RT the solvent was removed under reduced pressure to produce a yeliow oil.
The residue was washed with water {200 mL) and extracted with CHCI3 (200 mL). The organic layer was isolated and purified by column chromatography and afforded 7 as a yellow solid.
Yield 9 g (68 %).
[0555] 1H-NMR (400 MHz, DMSO-D6) 6H: 0.91 (3H, t, J=7.3Hz), 1.97 (2Hr q, J=7.3Hz,
J=6.9Hz), 3.94 (3H, s), 4.41 (2H1 1, J=6.9 Hz), 7.19 (1 H, t, J=7,4 Hz), 7.46 (2H, t, J=7.8Hz),
7.67 (1 H, t, J=7.8Hz), 7.91 (2H, d, J=7.8Hz), 8.08 (1 H, d, J=7.4Hz), 8.20(1 H, s), 8,68 (1 H, d,
J=7.8Hz), 9.06 (1 H, s), 9.96 (1H, s). LCMS tp (min) 2.77. MS (APCI), m/z 388.50 [M+H].
Preparation of 6. 3-(4-anilino-1-propyl-1H-pyra2oIo[3,4-d]pyrimidin-6-yl)benzoic acid.
[0556] 5 (4.5 g, 0,011 moi) was suspended in water (100 ml) and KOH (0.8 g., 0.013 moi) was added. The reaction mixture was heated for 3 hours, cooled to r.t. and acidified with HCI.
The precipitate obtained was filtered off and dried and afforded 8 as an orange solid. Yield 3 g
(69 %).
J0557J ^ H-NMR (400 MHz, DMSO-D6) δH: 0.91 (3H, t, J=7.3Hz), 1.97 (2H, q, J=7.3Hz,
J=6.9Hz), 4.41 (2H1 1, J=6.9 Hz), 7.18 (1 H1 1, J=7.4 Hz), 7.46 (2H, t, J=7.8Hz), 7.64 (1 H, t,
J=7.8Hz), 7.91 (2H, d, J=7.8Hz), 8.06 (1 H, d, J=7.4Hz), 8.18 (1 H, S)1 8.64 (1 H, d, J=7.8Hz),
9.07 (1 H, s), 9.91 (1 H, s).LCMS \R (min) 2.41. MS (APCI)1 m/z 374.50 [M+H].
Preparation of 7. 3-(4-aniiino-1-propy[-1H-pyrazolo[3.4-d]pyrimidin-6-yl)-N-(4-pyrrolidin-1- yfphenyl)-benzamide,
[0558] 6 (0,3 g, 0.8 mmol) was dissolved in dichloromethane (10 ml) and oxalyl chloride {0.12 g, 0.09 moi)) was added. The reaction mixture was stirred for 3 hours, concentrated, dissolved in 10 ml of dioxane and 0,3 ml of triethylamine, and 0.8 mmol of corresponding aniline were added. After 4 hours of heating the reaction mixture was concentrated in vacuo and purified by column chromatography.
Entry 43. N-{4-cyanophenyl)-3-{4-[(2-methylbenzyI)amino]-1-ρropyI-1H-pyrazolo[3,4- d]pyrimidin-6-yl}benzamide
[0559] HNMR (400MHz1 DMSO-D6) δH: 0.89 <t, J=7.3 Hz, 3H); 1.00 (t, J=7.1 Hz, 3H); 1.96
(q, J=7,3, 7.0 Hz1 2H); 2.40 (s, 3H); 4.09 (m, 2H); 4.48 (t, J=6.9 Hz, 2H); 5.44 (s, 2H); 7.18 (m,
4H); 7.82 (m, 5H); 8.01 (d, J=8.4 Hz, 2H); 8.13 (d, J=7.6 Hz, 1 H); 8.59 (d, J=7.8 Hz, 1H); 8.91
(s, 1 H).
Entry 46. 3-{4-[{2-methylbenzyl)amino]-1-propyl-1 H-pyrazoIo[3f4-d]pyrirnϊdin-6-y)}-N-[4-
(pyrrolidin-1~yl)phenyl]benzamide
[0560] HNMR (400MHz, DMSO-D6) δH: 1.98 (m, 6H); 2.43 (s, 3H); 2.97 (m, 3H); 3.25 (m,
4H); 4.37 (t, J=6.8 Hz, 2H); 4.91 (d, J=5.4 Hz, 2H); 6.57 (d, J= 8.5 Hz1 2H); 7.17 (m, 3H); 7.43 (m, 1 H); 7.57 (m, 3H); 7.99 (d, J=7.7 Hz, 1 H); 8.15 (s, 1H); 8.33 (s, 1 H); 8.56 (d, J=8.0 Hz1 1 H);
8.93 (s, 1 H); 9.57 {s, 1 H).
Entry 49. 3-[4-(phenylamlno)-1 -propyl-1 H-pyrazoio[3,4-dlpyrimidin-6-yl3-N-[4-(pyrrolidin-1 - yl)phenyl]benzamϊde
[0561] HNMR (400MHz, DMSO-D6) δH: 1.90-2.05 (6H. m). 3.25 (4H. t. J=6,2 Hz). 4.41 (2H. t. J=6.9 Hz). 6.58 (2H. d. J=8.5 Hz). 7.16 (1 H. t. J=7.3Hz). 7.44 (2H. t. J=7.4Hz). 7.52-7.67 (3H. m). 7.92 (2H. d. J=8.2Hz). 8.04 (1 H. d. J=7.3Hz). 8.18 (1H. s). 8.57 (1 H. d. J=7.3Hz). 8.95 (1 H.
S). 9.80 (1 H. s). 9.88 (1 H. s).
Entry 57. methyl 3-[4-{phenylamino)-1-propyI-1H-pyrazolo[3,4-d]pyrimtdin-6-yI]benzoate
[0562] HNMR (400MHz1 DMSO-D6) δH: 0.91 (3H. t. J=7.3Hz). 1.97 (2H. q. J=7.3Hz.
J=6.9Hz), 3.94 (3H. s). 4.41 (2H. t. J=6.9 Hz). 7.19 (1 H. t. J=7.4 Hz). 7.46 (2H. t. J=7.8Hz).
7.67 (1 H. t. J=7.8Hz). 7.91 (2H. d. J=7.8Hz). 8.08 (1H. d. J=7.4Hz). 8.20(1H. s). 8.68 (1 H. d.
J=7.8Hz). 9.06 (1 H, s). 9.96 (1 H. s).
Synthesis Scheme for Entry 50 of Table 35
Figure imgf000188_0001
[0563] HNMR (400MHz, DMSO-D6) δH: 1.95 (m, 4H); 3.17 (t, 2H); 3.57 (m, 4H); 4.40 (t, 2H); 7.21 (m, 6H); 7.85 (d, J= 8.6, 2H); 7.93 (s, 1 H); 9.18 (s, 1 H); 9.27 (s, 1 H). 5-amino-1 -propyl-1 H-pyrazoie-4-carboxamide 1.
[0564] 21.O g (0.150 mol) of 8 was dissolved in 200 mL of dry ethanol and 15.2 g (0.205 mol) of propyl hydrazine was added at once. The reaction mixture was refluxed overnight. Then the reaction mixture was cooled down and solvent was removed in vacuo. The yellow precipitate was washed with ethyl acetate and dried. A yellow precipitate was obtained with yield 40% (8.6g). HNMR (400MHz, DMSO-D6) δH: 0.81 (t, J=8.0, 3H); 1.64 (m, 2H); 3.78 (t, J=7.3, 2H); 6.14 (s, 2H); 6.67 (s, 1 H); 7.15 (s, 1 H); 7.61 (s, 1 H); LCMS tR (min) 1.50. MS (APCl), m/z 169.1 [M+H]+. 1-proρyl-1 H-pyrazo!o{3,4-d]pyrimSdine-4,6-dioI 2,
[0565] 8.6 g {0.051 mol) of 1 was dissolved in dry dioxane and then 20.2 g {0.102 mol) of diphosgene was added. The reaction mixture was refluxed for 3 days and then was cooled. A yeiiow precipitate was formed. The precipitate was filtered off and washed with ether. A yeliow precipitate was obtained with yield 42% (4.2g). HNMR (400MHz, DMSO-D6) δH: 0.82 (t, J=7.1 ,
3H); 1.72 (m, 2H); 4.08 (t, J=6.8, 2H); 7.77 (s, 1 H); LCMS tR {min) 1.50. MS (APCI), m/z 195.2
[M+ H]+.
4,6-dichloro-1-propyl-1 H-pyrazolo[3,4-dJpyrimidine 3.
[0566] 4.2 g (0.022 moi) of 2 was suspended in 70 mL of POCI3 and then 9.0 g (0.044 mol) of
PCI5 was added. The reaction mixture was stirred at reflux overnight and then was cooled. S olvent was removed in vacuo. A dark yellow oil was obtained with yield 86% (4.4 g). LCMS tR
(min) 1.50. MS (APCI), m/z 232.1 [M+H|+.
N-(6-chloro-1-propyl-1H-pyrazoloI3,4-d]pyrimidin-4-yl)-N-{4-fIuorophenyl)amine 4.
[0567] 1.3 g (0.0056 moi) of 2 was dissolved in 10 mL of dry dioxane and then 0.7 g (0.0056 mot) of 4-fluoro aniline and 0.6 g (0.0056 mol) of TEA were added. The reaction mixture was stirred overnight at 80°C and then was poured into water. A pale yellow precipitate was filtered off and dried. A paie yeiiow precipitate was obtained with yield 59% (1.0 g). HNMR (400MHz,
DMSO-D6) 5H: 0.83 (t, J=7.1 , 3H); 1.83 (m, 2H); 4.23 (t, J=7.1 , 2H); 7.27 (t, J=9.1 , 2H); 7.73
(m, 2H); 8.22 (s, 1 H); 10.45 (s, 1 H); LCMS tR (min) 1 ,50. MS (APCI), m/z 306.1 [M+H]+.
N-[6-(benzylsulfanyt)-1-propyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-N-(4-fluorophenyI)-amine
5.
[0568] 0.1 g (0.0033 mol) of Na was gently dissolved in 50 mL of dry methanol and 0.405 g
(0.0033 moi) of benzyl mercaptane was added at once. The reaction mixture was stirred at RT for 30 min and then 1.O g (0.0033 mol) of 4 was added. The reaction mixture was refluxed overnight and then was poured into water and extracted with chloroform. The target was purified by column chromatography with methylene chloride as eluant. A paie yellow oil was obtained with yield 38% (0.5 g). HNMR (400MHz, DMSO-D6) δH: 0.82 (t, J=7.5, 3H); 1.82 (m,
2H); 4.25 <t, J=6.8, 2H); 4.38 (s, 2H); 7.18-7.30 (m, 5H); 7.41 (d, J=7.9, 2H); 7.72 (m, 2H); 8.10
(s, 1 H); 10.12 (s, 1 H).
N-[6-{benzylsuIfonyI)-1-propyl-1H-pyrazolo[3,4-d]pyrimϊdin-4-yl]-N-(4-fluorophenyt)-amine
6.
[0569] 1.3 g (0.0033 mol) of 5 was dissolved in 50 mL of dry chloroform and 4.0 g (0.016 mol) of 3-chloroperbenzoic acid was added in portions. The reaction mixture was stirred overnight at
RT, and then the precipitate was filtered off and the residue was poured into a 5% solution of
Na2CO3, extracted and concentrated under reduced pressure, Target was purified by column chromatography with methylene chloride as eiuant.
N-{6-amino or aniline-1-propyf-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-N-(4-fIuoroρhenyl)amine
7. [0570] 0,160 g (0.00051 mol) of 4 was treated with 0.00051 mol of amine and the reaction mixture was melted for about 15 minutes. The reaction mixture was then cooled and the target was purified by HPLC.
N-t^fluorophenyO-i-propyl-β-ΪΦtpyrroliclin-i-yicarbonylΪphenyil-IH-pyrazoiotS.Φdlpyri- midin-4-amine 7a,
[0571] Compound 4 (192 mg, 0.63 mmol), boronic acid pinacole ester (216 mg, 0.72 mmol), and tripheπyiphosphine (25 mg, 0.10 mmol) were charged into the flask containing 4 mL of dioxane and 1.5 mL of 2 M Na2CO3 solution. After purging the mixture with Ar for 20 min,
[Pd(PPh3)4] catalyst (36 mg, 0.03 mmol, 5 mol %) was added. The reaction mixture was heated at 100°C for 4 h under an Ar atmosphere. After the reaction mixture was cooled to RT, the solvent was removed under reduced pressure. The residue was washed with water (20 mL) and extracted with CHCI3 (20 mL). The organic layer was isolated and purified by column chromatography.
Entry 50. N~4~(4-fluorophenyl)-1-propyi-N~6~- [4"-(pyrrolidin-1-yIsulfonyt)biphenyl-4-yl]-
1 H-pyrazo!o[3,4-d] py rϊmid i ne-4,6-d i am i ne,
[0572] HNMR (400MHz, DMSO-D6) δH: 0.92 (3H, t, J=7.3 Hz), 1.73 (4H, m, broad), 1.93
(2H, m, broad), 3.22 (4H, m, broad), 7.20 (2H, t, J=9.2 Hz), 7.68 (2H, d, J=8.2 Hz)1 7.80-8.03
(9H, m, broad), 9.14 (1 H, s, broad), 9.59 (1 H, s, broad).
Synthesis Scheme for Entries 51, 54, 55, 61, and 63 of Table 35
c- J
S -HBr ™F(50*-!
dioxane
Figure imgf000190_0001
1-pyrrolidinecarboximϊdamide hydrobromide 3.
[0573] Ethyl isothiourea (310.0 g, 1.58 mol) was dissolved in water (1 L) and pyrrolidine
(112.0 g, 0.378 mol) was added at once. The reaction mixture was refluxed overnight and evaporated. The mass of white precipitate is 306 g (94%). 1 H NMR (DMSO-dθ): δ 1.89 (m,
4H); 3.31 (m, 4H); 7.18 (s, 4H).
2-(1-pyrroiidinyl)-4,6"pyrimidinedio! 4. [0574] 34.78 g (1.512 moi) of sodium was dissolved gently in 1.6 L of dry ethanol; the solution was then cooled down to 40-450C and 66.5 g (0.415 mo)) of diethylmaionate was added dropwise. Thereafter, 73.4 g (0.378 mol) 1-pyrrolidinecarboximidamtne hydro bromide (3) was added in portions to the reaction mixture. The reaction mixture was reffuxed overnight, and then the white solid was filtered, dissolved in water and acetic acid was added to pH 5-6. White precipitate was filtered and washed with ether and lyophilized. The mass of white precipitate is
45 g (65%). 1 H NMR (DMSOdδ): δ 1.90 (m, 4H); 3.41 (m, 4H); 4.57 (s, 1 H); 10.47 (s, 1 H).
2.6-dichloro-4-{1-pyrrolidinyl) benzaldehyde 5.
[0575] 79 ml of dry DMF was cooled to 0-50C. 191 ml of POCI3 was then added dropwise.
The mixture was allowed to react at 200C for 1 h and then 45.0 g of 2-(1-pyrrolidinyl)-4,δ- pyrimtdinediol (4) was added in portions. After 30 min the reaction was heated on a stream bath overnight. Thereafter the excess of phosphoryl chloride was removed under reduced pressure, the reaction mixture was poured into ice and the yellow precipitate was collected and lyophilized. The mass of yellow precipitate is 29 g (53 %). 1 H NMR (DMSO-d6): δ 2.00 (m, 4H);
3.60 (m, 4H); 10.10 (s, 1 H).
4-amino-6-chIoro-2-(1-pyrrolidinyl)-5-pyrimidine-carbaldehyde 6.
[0576] 2,6-dichloro-4-(1-pyrro-lidinyl)benzaldehyde (5, 6g, 0.024 mol) was dissolved in dioxane (100 ml_). NH3 was passed through this solution until full conversation of 2,6-dichloro-
4-(1-pyrrolidinyl)benzaIdehyde. Solvent was evaporated. (5.3 g, 97%) 1 H NMR (DMSO-d6): δ
1.47 (m, 2H); 1.65 (m, 4H); 2.79 (m, 5H); 3.01 (t, J=5.9 Hz, 2H); 4.42 (t, J=6.3 Hz, 2H); 10.7 (s,
1 H).
1-propyI-6-(1-ρyrrolidinyl}-1H-pyrazolot3,4-d]pyrimϊdin-4-amine 7.
[0577] Propyihydrazine (1.63 g, 0.022 mol) was suspended in 300 mL of dry dioxane; 5 g
(0.020 mol) of 4-amino-6-chloro-2-(1-pyrrolidinyl)-5-pyrimidine-carbaldehyde (6, 4.8 g, 0.021 mol) 5 was then added at once. The reaction mixture was stirred for 60 min. at RT, then Et3N
(2.02 g, 0.02 mol) was added and the reaction mixture was stirred overnight at 60 0C. Solvent was removed under reduced pressure. The reaction mixture was purified by column chromatography eluting with chloroform.
Final compound 8.
[0578] 0.150 g (0.00061 mol) of 7 was dissolved in 5 ml of dry dioxane and (0.00073 mol) of chloro anhydrides and 0.112 g (0.00073 mol) of DBU were added. The reaction mixture was stirred at 700C for 48 h. The reaction mixture was poured into water and extracted with chloroform. Target compound was purified by column chromatography with methylene chloride as eluant. A white precipitate was obtained with yield 40-60 %.
Entry 51. 2-methyl-N-[1-propyl-6-(pyrroIidin-1- yl)-1H-pyrazolo[3,4-d]pyrimidin-4- yl]faenzamide [0579] HNMR (400MHz, DMSO-D6) δH: 0.87 (3H, t, J=7.4 Hz); 1.89 (6H, m); 2.42 {3H, s);
3.46 (4H, bs); 4.16 (2H, t, J=6.8 Hz); 7.29 (2H, m); 7.39 (1H, tn); 7.49 (1 H1 d, J=7.3 Hz); 8.13
(1 H, s); 10.59 (1H, bs).
[0580] Entry 54. 2-fluoro-N-[1-propyi-6-(pyrroIidin-1-yi)-1H-pyrazoIo[3,4-cl]pyrimidin-4- yl]benzamide
[0581] HNMR (400MHz, 90 0C, DMSO-D6) δH: δH: 0.87 (3H, t, J=7.4 Hz), 1.87 (6H, m), 3.47
(4H, t, J=6.7 Hz), 4.17 (2H, t, J=6.8Hz), 7.31 (2H, m), 7.60 (1 H, m), 7.74 (1 H, m), 7.94 (2H1 m),
8.16 (1H, s), 10.62 (1 H, s).
Entry 55. 4-chloro-2-fiuoro-N-[1-propyI-6-(pyrroiiclin-1 -yl)-1 H-pyrazoIo[3,4-d]pyrimidin-4- yljbenzamide
[05821 HNMR (400MHz1 90 0C, DMSO-D6) δH: 0.87 (3H, t, J=7.3 Hz)1 1.87 (6H, m), 3.45
(4H, t, J=6.7 Hz)1 4.17 (2H, t, J=6.7Hz), 7.44 (2H1 m), 7,75 (1H, t, J=8.0 Hz), 8.13 (1 H, s), 10.74
(1 H, S).
Entry 61. 4-chloro-2-methoxy-N-[1-propyl-6-(pyrrolidin-1-yI)-1H-pyrazolo[3,4-d]pyrimidin-
4-y)]benzamide
[0583] HNMR (400MHz, DMSO-D6) δH: 0.89 (3H, t, J=7.4 Hz); 1.92 (6H, m); 3.54 (4H, t,
J=6.6 Hz); 4.01 (3H, s); 4.18 (2H, t, J=6.8 Hz); 7.19 (1H, dd, J1=8.3 Hz, J2=1.5 Hz); 7.31 (1 H, d,
J=1.5 Hz); 7.89 (1 H, d, J=8.3 Hz); 8.23 (1 H, s); 8.46 (1 H, bs), 10.43 (1H, bs).
Entry 63. 2-chIoro-4-fluoro-N-[1-propyl-6-(pyrroIidϊn-1-yi)-1H-pyrazolo[3,4-d]pyrimidin-4- yl]benzamide
[0584] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.87 (3H, t, J=7.3 Hz), 1.87 (6H, m), 3.42
(4H, t, J=6.5 Hz), 4.16 (2H, t, J=6.8Hz), 7.28 (1 H, m), 7.45 (1 H, dd, J=8.9, 2.5 Hz), 7.66 (1 H, dd,
J=8.6, 6.1 Hz), 8.14 (1 H, s), 10.95 (1 H, s).
Synthesis Scheme for Entry 52 of Table 35
Figure imgf000192_0001
Entry 52. 4-chloro-N-[1-propyI-6-(pyrroIidin-1-yI)-1H-pyrazoIo[3,4-d]pyrimidin-4- yl]benzenesuifonamtde.
[0585] 0.15O g (0.00060 mol) of Kl 7 was dissolved in 5 mL of dry pyridine and 0.00067 mol of 4-substituted benzylsuifo chlorides were added in each flask. Reaction mixtures were stirred at 6O0C for 48h and then were poured into water and extracted with chloroform. Targets were purified by column chromatography with methylene chioride as eluent. This compound was purified by column chromatography with methylene chioride as eluant and was obtained with yield 48% (11.7 mg). HNMR (DMSO-d6): 0.84 (3H, t, J=7.8 Hz), 1.80 (2H, m, broad), 1.94 (4H, m, broad), 3.49 <4H, m, broad), 4.08 (2H, t, J=6.6 Hz), 7.61 (2H, d, J=8.5 Hz), 7.95 {2H, d, J=8.5 Hz), 8.05 (1 H, s).
Synthesis Scheme for Entries 56 and 58 of Table 35
O POCI3 Cf
O -^N Aldehyde, xylenes PCiS reflux , / N , N ^ - N / \
N s N N & N N O O
) 1 2 3
F //
F // - aniline, Et3N / N H2, Pd/C ^ / N dioxarse / N EtOH
/ N
< &
/ N
O / N
"- 4
5 aniline Et3N dioxane
Figure imgf000193_0001
6-{4-nitrophenyl}-1-propyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one 2. [0586] 5.0 g (0.030 mol) of 1 was suspended in 100 mL of dry xylene and then 3.5 g (0.036 mol) of aldehyde was added. After that 5 drops of toluene methanesulfonic acid were added and the reaction mixture was stirred at reflux overnight. The reaction mixture was cooled and pale yellow precipitate was filtered off and dried. A pale yellow precipitate was obtained with 79 % (7,0 g) yield. 1H-NMR (400 MHz, DMSO-D6) δH: 0.85 (3H, t, J=7.3Hz), 1.88 (2H, q, J=7 3Hz, J=6.9Hz), 4.32 (2H, t, J=6.9 Hz), 8.12 (1 H, s), 8.38 (4H, s), 12 65 (1 H, s). LCMS tR (min) 1 52. MS (APCI)1 m/z 300.20 [M+H].
4-chloro-6-(4-nitrophenyl)-1-propyI-1H-pyrazolo{3,4-d]pyrimidine 3, [0587] To a solution of 2 (1.4 g, 0.0047 mol) in POCI3 was added PCI5 (1.00 g, 0.0047 mmol). The reaction mixture was stirred for 10 hours at reflux, then POCI3 was removed under reduced pressure, precipitate was washed with ether and used in further step. Yield 1.5 g (100 %). 1H-NMR (400 MHz, DMSO-D6) δH: 0.92 (3H, t, J=7.3Hz), 2.00 (2H, q, J=7.3Hz, J=6.9Hz), 4.54 (2H, t, J=6.9 Hz), 8.36 (2H, t, J=9.0 Hz), 8.42 (1 H, s), 8.68 (2H, t, J=9.0 Hz). LCMS tR (min) 2.99. MS (APCI), m/z 318.40 [M+H].
N-(4-fluorophenyl)-6-{4-nJtrophenyl)-1-propyI-1H-pyrazolo[3,4-d]pyrimidin-4-amine 4. [0588] To a stirred solution of 8 (1 5 g, 0.0047 mol) and triethylamine (5 ml) in 70 ml of dioxane a solution of amine (0.00047 mol) was added. After 5 hours of stirring the solvent was removed and residue was treated with water, extracted with dichiorornethane, concentrated in vacuo and dried to afford 9 as yellow solid. Yieid 0.5 g (27 %). 1H-NMR (400 MHz, DMSO-D6) δH: 0.93 (3H, t, J=7.3Hz), 1.95 (2H, q, J=7.3Hz, J=6.9Hz), 4.41 <2H, t, J=6,9 Hz), 7.27 (2H, t,
J=8.8 Hz), 7.80-7.90 (2H, m), 8.19 (1 H, S)1 8.34 <2H, d, J=8.8Hz), 8.61 (2H1 d, J=8.8Hz), 10.00
(1 H1S). LCMS tR (min) 2.98. MS (APCI)1 m/z 393.30 [M+H].
Entry 56. 6-{4-aminophenyi)-N-(4-fluorophenyi) -1-propyl-1H-pyrazolo[3,4-d]pyrimidin-4- amϊne (5).
[0589] 1.5 g (0.0036 mol) of 2 was dissolved in 50 mL of ethanol, and an Pd/C (10%) was added under Ar atmosphere. The reaction mixture was additionally purged under Ar for 20 min.
At this time, the reaction mixture was hydrogenated until the reduction was completed. Then the precipitate was carefuily filtered and the residue was concentrated under reduced pressure to afford 3 as a white solid. Yield 1.0 g (65 %). 1H-NMR (400 MHz, DMSO-D6) δH: 0.90 (3H, t,
J=7.3Hz), 1.92 (2H, q, J=7.3Hz, J=6.9Hz), 4.34 (2H1 1, J=6,9 Hz)1 5.00-5.40 (2H1 broad), 6.65
(2H, d, J=8.8 Hz)1 7.23 (2H, t, J=8,5Hz), 7.80-7.95 (2H, m), 8.15 (3H, t, J=8.5Hz, J=8.8Hz), 9.65
(1 H,s). LCMS tR (min) 1.98. MS (APCI)1 m/z 363.50 (M+H].
4-flu orθ"N-(4-{4-[(4-f I uorop he ny l)am jno] -1 -propy 1-1 H-py razol o[3 ,4-d] py rim id i n-6-y I}- phenyljbenzenesulfonamide 6.
[05903 To a stirred solution of 10 (1 g, 0.0028mo!) and triethylamine (5 ml) in 70 ml of dioxane, suifochloride (0.0028 mol) was added. After 5 hours of stirring, the solvent was removed and the residue was treated with water, extracted with dichloromethane, concentrated in vacuo and dried to afford 9 as a yellow solid. Yield 0.5 g(34%); LCMS fe (min) 2.19. MS
(APCI), m/z 521.70 [M+H].
[0591] Entry 58. 4-{dimethyIamino)-N-(4-{4-[(4-fluorophenyl)amino]-1-propyl-1H- pyrazolo-[3.4-d]pyri-midin-6~yi>phenyl)benzenesulfonamide (7)
[0592] To a stirred solution of 6 (0.001 mol) and triethylamine (0.5 ml) in 10 ml of N-rnethyl- pyrrolidone a solution of amine (0.001 mol) was added. After 5 hours of stirring, the solvent was removed and the residue was treated with water, extracted with dichloromethane and purified by column chromatography. HNMR (400MHz, 90 0C1 DMSO-D6) 5H: 0.90 (3H. t. J=7.3Hz). 1.95
(2H. q. J=7.3Hz. J=6.9Hz). 4.36 (2H. t. J=6.9 Hz). 6.73 (2H. d. J=9.2 Hz). 7.25 (4H. t. J=7.8Hz.
J=7.3Hz). 7.60 (2H. d. J=8.5Hz. J=8.8Hz). 7.80-7.90 (m. 2H). 8.13 (1 H. s). 8.26 (2H. d. J=8.8
Hz). 9.74 (1 H.s). 9.84 (1 H.s). MS (APCI)1 m/z 546.6 [M+H].
Synthesis for Entries 59 and 60 of Table 35
Figure imgf000194_0001
Figure imgf000195_0001
C!
Figure imgf000195_0002
Cl
2-chloro-Λ/1 -(2-hydroxy-5-nitrophenyi)acetamide 5, 2-chloro-W1 -(2-hydroxy-4-nitro- phenyl)acetamide 10
[0593] Compound 3 1Og (0.065mo)e), Et3N 7.9g (0.078 mole) and compound 4 8.8g (0.078 mole) were mixed together in dioxane. The reaction mixture was stirred at 600C for 12 h. TLC control. The mixture was then cooled and diluted with water. The precipitate was collected and dried. 1 H-NMR (400MHz, DMSO-D6) δH: 4.38 (s, 2H), 7.03 (d, J=8 8, 1 H), 7.89 (dd, J1=2.4,
J2=8.8, 1 H); 8.96 (d, J=2.4, 1 H); 9.65 (S, 1H); 11.63 (s, 1 H).
6-nϊtro-2H-1,4-benzoxazin-3(4W)-one 6, 7-nitro-2tf-1,4-benzoxazin-3(4W)-one 11
Compound 5
[0594] 1 g (0.0043 mole) was dissolved in CH3CN, K2CO3 1.8 g (0.0129 mole) was added and the reaction mixture was refiuxed overnight. The reaction mixture was then cooled and diluted with water and the obtained precipitate was filtered off and dried to provide final product 0.66g
(80%). 1 H-NMR (400MHz, DMSO-D6) δH: 4. 70 (S, 2H); 7.08 (d, J=9.1 , 1 H); 7,74 (s, 1 H); 7.80
(d, J=9.0, 1H), 11.03 (s, 1H).
Compound 11 0.7 g (84 %).
[0595] 1 H-NMR (400MHz, DMSO-D6) δH: 4.75 (s, 2H); 7.13 {d, J=8.8, 1 H); 7.73 (d, J=2.7,
1 H); 7 82 (dd, J1 =2.7, J2=8.8, 1 H).
6-amino-2W-1,4-benzoxazin-3(4H)-one 7, 7-amino-2W-1,4-benzoxazin~3(4W)-one 12
[0596] 3 g (0.0155 mol) of 6(11) was dissolved in 80 m L of THF, and an Pd/C (10%) was added under Ar atmosphere. The reaction mixture was additionally purged under Ar for 20 min.
After this the reaction mixture was hydrogenated until the reduction was completed (TLC control). Then the precipitate was carefuϋy filtered and the residue was concentrated under reduced pressure to afford 7 as a yellow solid.
Compound 7. Yield 2.2 g (88 %).
[0597] 1 H-NMR (400MHz, DMSO-D6) δH: 4 36 (s, 2H); 4.98 (bs, 2H); 6.12 (dd, J1=2.4,
J2=8.2, 1 H); 6.18 (d, J=2.4, 1 H); 6.62 (d, J=8.2, 1 H), 10.44 (s, 1 H).
Compound 12, Yield 1.8g (72%).
[0598] 1 H-NMR (400MHz, DMSOD6) δH: 4.40 (s, 2H); 4.84 (bs, 2H); 6.17 (m, 2H); 6,57 (d,
J=8.2, 1 H), 10.24 (s, 1H). N-R-W-[1-propyI-6-{1-pyrroIidinyl)-1W-pyrazolo[3»4-t/]pyrimidin-4-yl]amine 2
[0599] 0,0017 mole of amine (bb2) was suspended in 5 ml_ of dioxane. HCI (5drops) and
0.3g 0.00113 mole of the 4-chloro-1-methyl-6-(1-pyrrolidinyl)-1H-pyrazolo-[3,4-d]-pyrimidine were added and the reaction mixture was stirred overnight at 1000C. LCMS demonstrated total conversion of starting material. Then the mixture was diluted with water and extracted with
CHCI3, the organic layer was separated and dried with Na2SO4 and then evaporated. The precipitate was filtered off and dried. The final compound was purified by column chromatography.
Entry 59. 3-(4-anilino-1 -propyM H-pyrazolo[3,4-d]pyrimidin-6-yl)-N-methyl-N- phenylbenzenesulfonamide
[0600] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.87 (3H, t, J=7.4 Hz); 1.82 (2H, sx, J1 =7.4
Hz, J2=6.8 Hz); 1.98 (4H1 m); 3.65 (4H; t, J=6.6 Hz); 4.23 (2H, t, J=6.8 Hz); 4.57 (2H, s); 6.96
(1H, d, J=8.5 Hz); 7.37 (1 H, d, J=8.5 Hz); 7.54 (1 H, s); 7.96 (1 H, s); 10.37 (1 H1 bs); 10.49 (1 H1 s). MS (APCI), m/z 394.4 [M+H].
Entry 60. 3-{4-[(2-methylbenzyl)amino]-1-propyl-1H-pyrazolo[3,4HJ]pyrimidin-6-yi}-N- phenylbenzenesulfonamide
[0601] HNMR (400MHz, 90 0C, DMSO-D6) δH: 0.88 (3H, t, J=7.4 Hz); 1.86 (2H, sx, J1 =7.4
Hz1 J2=6.8 Hz); 1.94 (4H, m); 3.59 (4H; t, J=6.5 Hz); 4.11 (2H, t, J=6.8 Hz); 4.52 (2H, s); 6.91
(1 H, d, J=8.5 Hz); 7.36 (1H, dd, J1 =8.5 Hz, J2=2.5 Hz); 7.51 (1 H, d, J=2.5 Hz); 7.93 (1 H, s);
8.72 (1 H, bs); 9.30 (1 H, bs); 10.50 (1 H, s). MS (APCI), m/z 394.4 [M+H].
Synthesis Schemes for Entries 62 and 64 of Table 35
Figure imgf000196_0001
F- / } aniline, Et3N "~-^ N H2, PύlC dioxane / εtoH o . N N > N
N N- N
O I s
4
5 aniline, Et3N dioxane
Figure imgf000196_0002
6-(4-nitrophenyi)-1 -propyl-1 ,5-dihydro-4H-pyrazolo[3,4-d]pyrimtdin-4-one 2.
[0602] 5.0 g (0.030 mol) of 1 was suspended in 100 ml of dry xylene, and then 3.5 g (0.036 mot) of aldehyde was added. Thereafter, 5 drops of toiuene methanesulfonic acid were added and the reaction mixture was stirred at reflux overnight. The reaction mixture was cooled and a pale yeϊlow precipitate was filtered off and dried. A paie yellow precipitate was obtained with 79 % (7.0 g) yield. 1H-NMR (400 MHz, DMSO-D6) δH: 0.85 (3H1 t, J=7.3Hz), 1.88 (2H, q, J=7.3Hz, J=6.9Hz), 4.32 (2H, t, J=6.9 Hz), 8.12 (1 H, s), 8.38 (4H, s), 12.65 (1 H, s). LCMS fe (min) 1.52. MS (APCI)1 m/z 300,20 [M+H].
4-chtoro-6-(4"nitrophenyl)-1-propyl-1H-pyrazoio[3,4-dJpyrϊmidine 3. [0603] To a solution of 2 (1 A g, 0.0047 mol) in POCΪ3 was added PCI5 (1.00 g, 0.0047 mmol). The reaction mixture was stirred for 10 hours at reflux, then POCi3 removed under reduced pressure, precipitate was washed with ether and used in further step. Yield 1.5 g (100 %). 1H-NMR (400 MHz, DMSO-D6) δH: 0.92 (3H, t, J=7.3Hz), 2.00 (2H1 q, J=7.3Hz, J=6.9Hz), 4.54 (2H, t, J=6.9 Hz), 8.36 (2H1 1, J=9.0 Hz), 8.42 (1 H, s), 8.68 (2H1 1, J=9,0 Hz). LCMS tR (min) 2.99. MS (APCi)1 m/z 318.40 [M+H].
N-{4-fluorophenyl)-6-(4-nitropheny!)-1-propyf-1H-pyrazoIo[3)4-d]pyrimidin-4-amine 4. [0604] To a stirred solution of 8 (1.5 g, 0.0047 mol) and triethylamine (5 mi) in 70 ml of dioxane a solution of amine (0.00047 mo!) was added. After 5 hours of stirring the solvent was removed and residue was treated with water, extracted with dichloromethane, concentrated in vacuo and dried afforded 9 as yellow solid. Yield 0.5 g (27 %). 1H-NMR (400 MHz, DMSO-D6) δH: 0.93 (3H, t, J=7.3Hz), 1.95 (2H, q, J=7.3Hz, J=6.9Hz), 4.41 (2H1 1, J=6.9 Hz), 7.27 (2H1 1, J=8.8 Hz), 7.80-7.90 (2H1 m), 8.19 (1 H, s), 8.34 (2H, d, J=8.8Hz), 8.61 (2H1 d, J=8.8Hz)t 10.00 (1 H1S). LCMS tR (min) 2.98. MS (APCI), m/z 393.30 [M+H].
6-(4-amϊnophenyl)»N-{4~fluoropheny() -1-propyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5. [0605] 1.5 g (0.0036 mol) of 2 was dissolved in 50 mL of ethanoi, and Pd/C (10%) was added under Ar atmosphere. The reaction mixture was additionally purged under Ar for 20 min. At this time, the reaction mixture was hydrogenated until the reduction was completed. Then the precipitate was carefully filtered and the residue was concentrated under reduced pressure to afford 3 as a white solid. Yield 1.0 g (65 %). 1H-NMR (400 MHz, DMSO-D6) δH: 0.90 (3H, t, J=7.3Hz), 1.92 (2H, q, J=7.3Hz, J=6.9Hz), 4.34 (2H, t, J=6.9 Hz), 5.00-5.40 (2H, broad), 6.65 (2H1 d, J=8.8 Hz), 7.23 (2H1 1, J=8.5Hz), 7.80-7.95 (2H, m), 8.15 (3H, t, J=8.5Hz, J=8.8Hz), 9.65 (1 H1S). LCMS .R (min) 1.98. MS (APCI)1 m/z 363.50 [M+H].
4-fluoro-N-(4-{4-[(4-fluorophenyi)amino]-1-propyI-1H-pyrazo!o[3,4-d]pyrimidm-6-yl}- phenyt)benzenesulfonamide 6.
[0606] To a stirred solution of 10 (1 g, 0.0028 mol) and triethytamine (5 ml) in 70 ml of dioxane a solution of sulfochloride (0.0028 mol) was added. After 5 hours of stirring the solvent was removed and the residue was treated with water, extracted with dichloromethane, concentrated in vacuo and dried to afford 9 as a yellow solid. Yield 0.5 g(34%); LCMS IR (min) 2.19. MS (APCI), m/z 521.70 [M+Hj.
4-(dimethyIamino)-N-(4-{4-[(4-fluorophenyl)amino]-1-propyI-1H-pyrazolo-[3.4-d]pyri- midin-6-yl}phenyl)benzenesulfonamide 7 [0607] To a stirred solution of 6 (0.001 mol) and triethyiamine {0.5 ml) in 10 ml of N-methyi- pyrrolidone a solution of amine (0.001 mol) was added. After 5 hours of stirring the solvent was removed and the residue was treated with water, extracted with dichloromethane and purified by column chromatography.
Entry 62. 4-{dimethylamino)-N-{3-{4-[(4-fluorophenyl)amino]-1-propyl-1 H-pyrazolo[3,4- d] py ri m id i n-6-y l}pheny I }benzenes uff onam ide
[0608] NHMR (400MHz, 90 0C1 DMSO-D6) δH; 0.91 (3H, t, J=7.4 Hz)1 1.95 (2H, m), 2.92
(6H, s), 4.38 (2H, t, J=6.7Hz), 6.66 (2H, d, J=8.7Hz), 7.26 (4H1 m), 7.59 (2H, d, J=8.2 Hz), 7,94
(2H, m), 8.05 (1 H1 d, J=8.8 Hz)1 8.20 (1H, s), 8.31 (1 H1 S)1 9.78 (1H, s), 9.86 (1 H, s). m/z 546.6
[M+H].
Entry 64, N-(4-fIuorophenyl)-6~{4-nitrophenyl)-1-propyl-1 H-pyrazoIo[3,4-d]pyrimidin-4- amine
[0609] NHMR (400MHz, 90 0C1 DMSO-D6) δH: 0.93 (3H. t. J=7.3Hz). m/z 393.4 [M+H]. 1.95
(2H. q. J=7.3Hz. J=6.9Hz). 4,41 (2H. t. J=6.9 Hz). 7.27 (2H. t. J=8.8 Hz). 7.80-7.90 (2H. m).
8.19 {1 H. s). 8.34 (2H. d. J=8.8Hz). 8.61 (2H. d. J=8.8Hz). 10.00 (1H.s).
Synthesis Scheme for Entry 65 of Table 35
Figure imgf000198_0001
6-chloro-1-propyl-/V-[4'-(pyrrolidin1-ylsulfonyl)-1,1'-biphenyl-4-y!]-1H-pyrazolo[3)4-d]- pyrimϊdin-4-amine 4.
[0610] 0.3 g (0.0013 mol) of compound 3 was dissolved in 10 ml of dry dioxane and then 0.4 g (0.0013 mol) of compound 2 and 0.13 g (0.0013 mol) of TEA were added. The reaction mixture was stirred overnight at 800C and then was poured into water and extracted with chloroform. Target was purified by column chromatography with methylene chloride as eluant.
A white precipitate was obtained with yield 31 % (0.2 g).
Entry 65. N~4~-{4'-[dihydroxy(pyrrolidin-1-yl)-lambda~4~-suifanyl]-1,1"-biphenyi-4-yl}-
N~6~-(4-fluoropheny!}-1-propyi-1H-pyrazolo(3,4-d]pyrimidine-4,6-diamine (5).
[0611] 0.2 g (0.00040 mol) of 4 was treated with 0.067 g (0.00060 mol) of 4-fluoro aniline and the reaction mixture was melted and then cooled. Target was purified by HPLC and then by column chromatography. 1H-NMR (400MHz1 DMSO-D6) δH: 0.92 (3H1 1, J=7.3 Hz)1 1.75 (4H, m, broad), 1.90 (2H, m, broad), 3.24 (4H, m, broad), 4.20 (2H1 1, J=6.6), 7.08 (2H, t, J=9.1 Hz),
7.74 (2H1 d, J=8.8 Hz), 7.86 (6H, m, broad), 8.00 (2H, d, J=8.8), 8.08 (1H, s), 8.93 (1 H, s, broad), 9.62 (1 H, s, broad). LCMS tR (min) 1.50. MS (APCI), m/z 572.7 [M+H]*. Synthesis Scheme for Entry 66 of Table 35
N
Figure imgf000199_0001
Ethyl irni doth iocarbam ate 2.
[0612] 50.Og (0.657 mol) of compound 1a was dissolved in 400 mL of ethanol and 71.6 g (0.657 mol) of ethyl bromide was added at once. The reaction mixture was stirred at reflux overnight and then was cooled. Solvent was removed in vacuo. A white precipitate was obtained with yield 99% (12O g). HNMR (400MHz, DMSO-D6) δH: 1.19 (t, J=7.1 , 3H); 3.16 (m, 2H); 8.94 (s, broad, 2H); 9.07 (s, broad, 2H); LCMS tR (min) 1.50, MS (APCI)1 m/z 186.2 [M+H]+.
W-(4-flυorophenyl)guanidine 3.
[0613] 120 g (0.671 mol) of compound 2 was dissolved in 400 mL of water and 75.0 g (0.671 mol) of 4-fluoroanilin was added at once. The reaction mixture was refluxed for 3 days and then was cooled. Solvent was removed in vacuo. Target compound was dissolved in 400 mL of water again and washed with chloroform; layers were separated and water was again removed in vacuo. Target was obtained as yellow oil with yield 51% (81 g - imp 20%). LCMS tR (min) 1.50. MS (APCI), m/z 154.2 [M+H]+. 2-[(4-fluorophenyl)amino]pyrimicline-4,6-clioI 4.
[0614] 40.0 g (1.752 mol) of sodium was gently dissolved in 1500 mL of dry ethanol. The reaction mixture was then cooled to 40-450C and 84.0 g (0.526 mol) of diethyl malonate was added in portions. Then 81.0 g (0.438 mol) of crude compound 3 was added at once to the reaction mixture. The reaction mixture was refluxed overnight and then was cooled. The precipitate was filtered off; the residue was concentrated under reduced pressure, dissolved in water and acidified with acetic acid to pH 5-6. Precipitate was filtered off, washed with water and chloroform and dried. A white precipitate was obtained with yield 30% (20.1 g). HNMR (400MHz, DMSOD6) δH: 3.30 (m, 2H); 4,83 (s, 1 H); 7.05 (t, J-9.1 , 2H); 7.63 (m, 2H), LCMS tR (min) 1.50. MS (APCi), m/z 222.2 [M+H]+. 4,6-dichloro-2-(4-fIuoroanilino)-5-pyrimiclϊnecarbaIdehyde 5.
[0615] 10 ml of dry DMF was cooled to Q-5°C,and than 25 m L of POC13 was added dropwise. This mixture was allowed to react at 20°C for 1 h, and then 5.0 g (0.0226 moi) of crude 4 was added in portions. After 30 min the reaction was heated in a steam bath for 48h.
The reaction mixture was cooled and poured into iced water. A brown precipitate was filtered off and dried. A brown precipitate was obtained with yield 61 % (4.0 g), 1 H-NMR (400MHz,
0MSO-D6) δH: 7.15 (2H, t, J=8.7Hz); 7.67 (2H1 m); 10.15 (1 H, s), 11.09 (1 H, s).
A/.^.chloro-i-propyl-IH-pyrazoioϊS^-t/lpyrimidin-θ-ylJ-W^ΦfluorophenylJamine e.
[0616] To a solution of 5 (4g; 0.014mol) in dioxane was added 1-propylhydrazine (0.07g; 5 mol). The reaction mixture was stirred for 24 h at 800C with 1.2 equiv. of NEt3 (1 Jg; 0.0168 mol) and then filtered. A supernatant solution was concentrated under reduced pressure and compound 14 was purified by coiumn chromatography. A white precipitate was obtained with yield 2.8 g (65 %). 1 H-NMR (400MHz, DMSO-D6) δH: 0.89 (3H, t, J=7.4Hz); 1.92 (2H, sx,
J1=7.4Hz, J2=6.8Hz); 4.27 (2H, t, J=6.8Hz); 7.08 (2H, t, J=8.8Hz); 7.84 (2H, m); 7.99 (1 H, s),
10.17 (1 H, s).
Entry 66. N~6~-(4-fluorophenyl)-1-propyl-N~4~-{[4f-{pyrrolidin-1-yIsulfonyl)-1,1*-biphenyl-
4-yi]methyl}-1H-pyrazolo{3,4-d]pyrimidine-4,6-diamine
[06173 NMR (400MHz, 90 0C1 DMSO-D6) δH: 0.89 (3H, t, J=7.6 Hz), 1.72 (4H1 m, broad),
1.86 (2H, m, broad), 3.21 (4H, m, broad), 4.15 (2H, t, J=6.6), 4.81 (2H1 d, J=6.1 ), 7.01 (2H, t,
J=8.6 Hz), 7.53 (2H, d, J=8.1 Hz), 7.84 (10H, m, broad), 8.27 (1 H, m, broad), 8.77 (1 H, s, broad), m/z 586.7 [M+H]+.
Synthesis Scheme for Entries 67 and 71 of Table 35
Figure imgf000200_0001
1 ~[(4-f Iuorophenyl)su1fonyl]pyrrolidine 2 [0618] A solution of the sulfochioride 1 (5 g 0.026 mote), amine (2.2g 0.031 moie) and Et3N
(3.1 g 0.031 mole) in chloroform was stirred overnight at 600C. The mixture was diluted with water and the organic layer was separated, dried with Na2SO4 and then evaporated. Yield: 4.2 g (70 %). 1 H-NMR (400 MHz, DMSO-D6) δH: 1.68 (p, J1 =3.1 , J2=6.8, 4H); 3.15 (t, J=6.8, 4H);
7.40 (m, 2H); 7.85 (m, 2H).
1-4-[4-{1-pyrrolidinyIsuIfonyl)phenyl]piperazino-1-ethanone 3a, fert-butyl W-1-[4-{1- pyrroIidinylsulfonyl)phenyl]-4-piperidylcarbamate 3b
[0619] Compound 2 (0.5 g 0.0022 mole) and BOC-protected- (or acetyl) amine (0.0033 mole) was dissolved in N-methyl-pirrolidone; the reaction mixture was stirred at 1800C for 2 days.
Then the reaction mixture was diluted with water and the precipitate was filtered off. 3a: LCMS:
MS (APCI), m/z 410.6 [M+HJ+ . 3b: LCMS: MS (APCl), m/z 338.4 [M+H]+.
1-[4-(1-pyrrolidinyIsulfonyl)phenyl]-4-piperϊdinamine 4a
[0620] BOC-protected compound 3a 1g (0.00246 mole) was stirred in dioxane with HCf at room temperature overnight. Then the solvent was evaporated, dissolved in an aqueous solution of Na2CO3, and then extracted with CHCI3. The organic layer was evaporated and a white solid was washed with ether. 0.4g (52 %). 1 H-NMR (400MHz, DMSO-D6) δH: 1.47 (m,
2H); 1.67 (m, 4H); 1.92 (m, 2H); 3.01 (m, 9H); 3.83 (d, J=13.3, 2H); 7.01 (d, J=9.1 , 2H); 7.56 (d,
J=9.1 , 2H).
1-[4-(1-pyrrolidϊnylsulfonyl)phenyl]piperazine 4b
[0621] NaOH 0.6 g (0.015 mole) was dissolved in the mixture EtOH:H2O (1 :1 ). To this mixture compound 3b 1g (0.00297 mole) was added and the reaction mixture was stirred at
1000C overnight. The reaction mixture was diluted with clorofomn and the organic layer was separated, dried and evaporated under reduced pressure. 0.7 g (80 %). 1 H-NMR (400MHz,
DMSO-D6) δH: 1.67 (m, 4H); 2.85 (t, J=5.0, 4H); 3.11 (m, 4H); 3.24 (m, 4H); 6.99 (d, J=8.8,
2H); 7.57 (d, J=8.8, 2H).
Entry 71. W-(6-chloro-1 -propyl-1 tf-pyrazolo[3,4-d]pyrimidin-4-yl)-ΛM -[4-(1 - pyrrolidinyisulfonyi)-phenyl]-4-piperidyIamine (6)
[0622] Compound 3 (0.3 g, 0.0013 mole) was dissolved in 1 , 4- dioxane (10 mL); amine (0.29 g, 0.0016 mole) was added; and then Et3N (0.16 g, 0.0016 mole) was added. The reaction mixture was heated to 6O0C and stirred at this temperature overnight. The Reaction mixture was then diluted with water and a yellow precipitate was filtered off, washed by ether and air dried. The target compound was purified by column chromatography. Yield: 0.3 g (45 %). 1 H-
NMR (400 MHz, DMSO-D6) δH: 0.86 (3H, t, J=7.4 Hz); 1.70 (6H, m); 1.86 (2H, sx, J1=7.4Hz,
J2=6.8Hz); 2.08 (2H, d, J=13.2Hz); 3.14 (6H, m); 3.94 (2H, d, J=13.2Hz); 4.20 (2H, t, J=6.8Hz);
4.30 (1 H, bs); 7.08 (2H, d, J=8.7Hz); 7.60 (2H, d, J=SJHz); 8.12 (1 H, s); 8.22 (1 H, d., J=7.2Hz).
Preparation of 7
[06231 Compound 6 (0.1 g, 0.0002 moie) was mixed with 4-fluoroaniline (0.02 g, 0.0002 mole) and this mixture was melted. The reaction mixture was cooled and treated with ether. The white precipitate was filtered off and air dried. Target compound was pure enough without additional purification.
Entry 67. W-[6-(4-fIuoroanilino)-1-propyI-1H-pyra2olot3,4-cf|pyrimidin-4-yl]-W-1-[4-{1- pyrroiidinyl-sulfonyi)phenyi]-4-piperidylamine
Yield; 0.045g, 38%. 1 H-NMR (400MHz, DMSO-D6) δH: 0,89 (3H, t, J=7,4 Hz); 1.71 (6H, m); 1.87 <2H, sx, J1=7.4Hz, J2=6.8Hz); 2.13 {2H, d, J=9.5Hz); 3.00-3.34 (6H, m); 3.96 (2H, d, J=12.7Hz); 4.14 (2H, t, J=6.8Hz); 4.35 (1 H1 bs); 7.07 (5H, m); 7.61 (2H, d, J=8.8Hz); 7.80 (3H, m); 7.98 (1 H, s); 8,99 (1 H, bs). m/z 57.7 [M+H]+. Synthesis Scheme for Entries 68, 69, 70, and 74 of Table 35
Figure imgf000202_0001
E ntry 68. 4-[4-{6-ch I oro-1 -propy 1-1 W-py razol o[3 ,4-cfJ py ri m id in-4-y I Jtetrahyd ro-1 - pyrazinyl]phenyl (1-pyrrolidϊnyl) sulfone (8)
[0624] Compound 3 (0.8 g, 0.00346 mole) was dissolved in 1 , 4- dioxane (30 mi_); amine
(1.33 g, 0.005 mole) was added; and then Et3N (0.5 g, 0.005 mole) was added. The reaction mixture was heated to 6O0C and stirred at this temperature overnight. The reaction mixture was diluted with water and a beige precipitate was filtered off, washed with ether and air dried. The target compound was purified by column chromatography. Yield: 1.2g (70 %). 1 H-NMR
(400MHz1 DMSO-D6) δH: 0.87 (3H, t, J=7.4 Hz); 1.69 (4H, m); 1.87 (2H, sx, J1=7.4Hz,
J2=6.8Hz); 3.14 (4H, t, J=6,6Hz); 3.64 (4H, t, J=5.4Hz); 4.12 (4H, t, J=5.4Hz); 4.24 (2H, t,
J=6.8Hz); 7.03 (2H, d, J=8.7Hz); 7.64 (2H, d, J=8.7Hz); 8.25 (1H, s).
Entry 69. W-(4-fluorophenyl)-/V-(1-propyl-4-4-[4-(1-pyrrolidϊnyl-sulfonyl)phenyl]piperazino-
1 H-pyra-zolo[3,4-crJpyrimidin-6-yl)amine (9)
[0625] 0.1 g (0.0002 mol) of the compound 8 was treated with 0.02 g (0.0002 mol) of the A- fluoroaniϋne, and the reaction mixture was melted. The reaction mixture was then cooled.
Target was purified by column chromatography and was obtained as a white precipitate. Yield:
0.06 g (54 %). 1 H-NMR (400MHz, DMSO-D6) δH: 0.90 (3H, t, J=7.4 Hz); 1.70 (4H, m); 1.87
(2H, sx, J1=7.4Hz, J2=6.8Hz); 3.14 (4H5 1, J=6.4Hz); 3.62 (4H, t, J=5.2Hz); 4.10 (4H1 1,
J=5.2Hz); 4.19 (2H1 1, J=6.8Hz); 7.07 (4H, m); 7.64 (2H, d, J=8.6Hz); 7.80 (2H, m); 8.01 (1H, s);
8.76 (1 H, s).
Entry 70. W-(3-chloro-4-fluorophenyl)-W-(1-propyl-4-4-[4-(1-pyrrolidinylsuIfonyl) phenyl]- pipera-zino-1 W-pyrazolo[3,4-d]pyrimidin-6-yl}amine (10)
F [0626] 0.2 g (0.00041 mole) of the compound 8 was treated with 0.071 g (0.00049 mole) of the 3-chioro-4-fluoroaniiine, and the reaction mixture was melted and then cooied. Target was purified by column chromatography and was obtained as a white precipitate. Yield 32 mg. 1H- NMR (400MHz, DMSO-D6) δH: 0.92 (3H, t, J=7.4 Hz); 1.70 (4H, bs); 1.90 (2H1 sx, J1=7.4 Hz, J2=6.8 Hz); 3.14 (4H1 bs); 3.63 (4H; t, J=5.0 Hz); 4.11 (4H; t, J=5.0 Hz); 4.20 (2H, t, J=6.8 Hz); 7.05 (2H1 d, J=7,8 Hz); 7.25 (1 H, t, J=8.9 Hz); 7.65 (3H, m); 8.03 (1 H, s); 8.20 (1 H, m). LCMS tR (min) 2.86. MS (APCI), m/z 599.40 [M+Hf.
Entry 74. N-{3-chloro-4-fluorophenyl)-4-{4-(4-(methylsulfonyi) phenyl]piperazϊn-1-yl}-1- propyl-1H-pyrazolo[3,4-d]pyrimidin-6-amine
[0627] 1H-NMR (400MHz, DMSO-D6) δH: 0.91 (3H, t, J=7,3 Hz), 1.89 (2H, m, broad), 3.07 (3H, s), 4.12 (4H, m, broad), 4.20 (2H, d, J=6.6), 7.07 (2H, d, J=9.5), 7.26 (1 H, t, J=9.6), 7.69 (3H, m, broad), 8.05 (1 H, s), 8.22 (1 H, d, J=8.9), 9.07 (1 H, s, broad).
Synthesis Scheme for Entry 72 of Table 35
O ^N O Cl N
PCI5
POCI3 ^s, dsoxane
N ^\ N
N N toiuene -"">■ reflux 80°C reflux
/ 2 -^" Sr F "Br
1 boromc actd /""""^
/ ^
Na2CO3 \
Figure imgf000203_0001
6-{4-brom ophenyl)-1-propyl-1 ,5-dIh ydro-4H~pyrazol o[3 ,4-efl py rim id i ne-4-one 2. [0628] 10 g (0.059 mol) of 1 and 22.0 g (0.119 moi) of 4-bromobenzaIdehyde were suspended in 100 mL of dry toluene. Then 8 drops of methanesulfonic acid were added and the reaction mixture was refluxed for 3 days. The reaction mixture was then cooled and a pale yeiiow precipitate was formed. The precipitate was filtered off and the residue was concentrated under reduced pressure. Target was crystallized from ether and washed with hot i-propanol, A white precipitate was obtained with very low yield (about 1.0 g). 1H-NMR (400MHz, DMSO-D6) δH: 0.85 (3H, t. J=7.5Hz). 1.87 (2H. m). 4.30 (2H. t. J=6.4Hz). 7.75 (2H. d. J=8.4Hz). 8.09 (3H. m). 12.36 (1 H, s).
6-(4-bromophenyI)-4-chloro-1-propyl-1H-pyra2oIo[3,4-c(|pyrirr!idine 3. [0629] 2.0 g {0.020 mol) of 2 was dissolved in 30 mL of POCI3 and then 2.5 g (0.0030 mol) of PCI5 was added at once. The reaction mixture was refluxed overnight and then was cooled. Excess POCI3 was removed under reduced pressure. The reaction mixture was poured into a 5% solution of Na2CO3 and extracted with chloroform. Solvent was removed in vacuo. Target was crystallized from ether. A pale yellow precipitate was obtained with yield 98% (2.140 g). 1H-NMR (400MHz, DMSO-D6) δH: 0.85 (3H. t. J=7.0Hz). 1.90 (2H. m), 4.41 (2H. t. J=7.5Hz). 7.66 (2H. d. J=8.6Hz). 8.25 (2H. d. J=8.6Hz). 8.35 (1 H. s).
6-(4-bromophenyl)-N-(2»methylbenzyl)-1«propyl-1 H-pyrazolo[3,4-dJpyrim!dine- 4-amine 4. [0630] 1.O g (0,0028 mol) of compound 3 was dissolved in 50 mL of dry dioxane and then 0.34 g (0,0028mol) of o-methylbenzylamine and 0.5 ml of TEA were added. The reaction mixture was stirred overnight at 80° C and then was poured into water. A pale yellow precipitate was filtered off and dried. A pale yeliow precipitate was obtained with yield 1g (80%). 1H-NMR (400 MHz. DMSO-D6) δH: 0.83 (3H. t. J=7.3Hz). 1.87 (2H. q. J=7.3Hz. J=6.9Hz). 2.38 (3H. s), 4.32 (2H. t. J=6.9 Hz). 4.85 (2H. d. J=5.4 Hz). 7.10-7,30 (4H. m). 7.35- 7.40 (1 H. broad), 7.65 (2H. d. J=8.6Hz). 8.17 {1 H. s), 8.35 (2H. d. J=8.6Hz). 8.60-8.70 (1 H. broad). LCMS IR (min) 2.66. MS (APCI). m/z 436.50 [M+H],
N-(2-methylbenzyI)-1-propyl-6-[4f-{pyrrolidin-l-ylsulfonyl)-1,1'-biphenyt-4-yl]-1H-pyrazo- lo[3,4-d]pyπmidin-4-amine 5.
[0631] 4 (500 mg, 0.0015mol), boronic acid ester (285 mg, 0.0015mol), and triphenySphosphsne {50 mg) were charged into the flask containing 40 mL of dioxane and 15 mL of 2 M Na2CO3 solution. After the mixture was purged with Ar for 20 min, [PdO(PPh3J4] catalyst (72 mg) was added. The reaction mixture was heated at 1000C for 4 h under an Ar atmosphere. After the reaction mixture was cooled to RT, the solvent was removed and the residue was suspended in 50 ml of water and treated with 300 mg of KOH. The reaction mixture was stirred at 700C for 5 hours, cooled to RT and extracted with 20 mL of dichloromethane. The aqueous layer was separated and acidified with HCI. The obtained precipitate was filtered off, dried and furnished 5 as a white solid. Yield 0.3g (47%). Entry 72. N-(2-methy1benzyl)-1-propyl-6-[4"-(pyrroJidin-1-y[sulfonyl)biphenyl-4-yl]-1 H- pyrazoϊo[3,4-d]pyrϊmidin-4-amine
[0632] NMR (400MHz, 90 0C, DMSO-D6) 5H: 0.91 (3H. t. J=7.3Hz). 1.75 (4H. m). 1.97 (2H. q. J=7.3Hz. J=6.9Hz). 2.42 (3H. s). 3.25 (4H. m). 4.37 (2H. t. J=6.9 Hz). 4.90 (2H. d. J=5.4 Hz). 7.15-7.25 (3H. m). 7.40-7.45 (1 H. m). 7.84 {2H. d. J=8.6Hz). 7.91 (2H. d. J=8,4Hz). 7.98 (2H. d. J=8.4Hz). 8.15 (1 H. s). 8.28-8.38 (1 H. broad). 8.55 (2H. d. J=8.4Hz). MATERIALS AND METHODS FOR BIOLOGICAL PROFILING OF COMPOUNDS OF THE
INVENTION Production of HCVpp
[0633] Functional HCV pseudoparticles (HCVpp) were produced in 293T cells via co- transfection of optimized HCV E1/E2 expression constructs and a non-replicating HIV-1 -based reporter vector as described. (See, e.g., J, Dumonceaux ef a/., 2003; E. Cormier et al., 2004; T. Dragic et a/., 1996; U.S. Patent Application No. 20050266400 to J. Dumonceaux ef a/., the contents of which are hereby incorporated by reference in their entirety). Plasmids for transfection included NLIuc+env- vector (R.I. Conner et a/., 1995) and a construct encoding HCV-ΔC-E1/E2 (HCV isolate H77, genotype 1a), lacking putative splice acceptor sites (Cormier, E.G. et a/., 2004). The HIV-1 backbone NLIuc+env- contains a frameshift mutation in the HiV-1 envelope glycoprotein gene (env), which has the potential to revert. In order to generate vectors with enhanced safety properties, a 299bp deletion in env was introduced by excision of the Nhe1/BsaB1 fragment. This construct, designated NLIuc+Δ299, encodes a packageable HIV core particle that is decorated with the E1/E2 envelope glycoproteins from HCV [06341 The finding of a cryptic intron excision site within HCV E1 aided in the development of an HTS amenable HCVpp assay for screening small molecule compounds. This cryptic splice site is not utilized during natural infection by HCV because RNA splicing occurs in the nucleus and HCV replicates exclusively in the cytoplasm. However, in plasrnid-based expression systems such as those used to generate HCVpp, RNA splicing results in the expression of aberrant, non-fusogenic forms of E1 along with native E1. When the putative splice acceptors were removed from E1/E2 by conservative mutagenesis, plasmid expression generated single E1 and E2 protein species that formed noncovalent heterodimers on the cell surface. HCVpp produced using splice-modified E1 was observed to mediate 5-10-fold higher levels of entry into cells (Dumonceaux, J., 2003; U.S. Patent Application No. 20050266400 to Dumonceaux et a/., published on Dec. 1 , 2005). Accordingly, the described HCVpp involved E1/E2 from the HCV genotype 1a isolate H77, modified by conservative mutagenesis to eliminate cryptic splice sites in E1 , which resulted in a more uniform expression of E1 (Dumonceaux, J. et ai, 2003; U.S. Patent Application No. 20050266400 to Dumonceaux et ai., published on Dec. 1 , 2005). [0635] For HCVpp production, 293T cells were co-transfected with NLIuc+Δ299 env" reporter vector and E1/E2 expression vector (E1/E2 pcDNA 3.1 ) in a 1 :2 or 1 ;3 ratio using Lipofectamine 2000 in serum-free OPTlMEM medium (Gibco BRL; ϊnvitrogen) as described. (See, E.G. Cormier et at, 2004). Typically, 5 x 106 293T cells were cotransfected with 4 μg of the NLIuc+Δ299(env") reporter vector (R.I. Conner er a/,, 1995), and 8 μg of the E1/E2 expression vector in a 10 cm2 dish (BD Falcon, Bedford, MA), (Id.), which is described in detail by Dumonceaux, J. ef at., 2003, U.S. Application No. 20050266400 to Dumonceaux et ai., and E. Cormier et a/., 2004. Four hours post-transfection, medium (Gibco BRL; Invitrogen) supplemented with 10% FBS was added to the transfected cells. Cell culture supernatants containing HCVpp were collected at 48 hours post-transfection and centrifuged at 1000 rpm for 5 minutes to clarify and pellet cell debris. Viral HCVpp containing supernatants were sterile filtered and stored at -800C. HCVpp containing supernatants were quantified for HIV-1 p24 protein content by ELISA, for protein content by BCA assay (Pierce, Rockford, IL) or for E2 content by Western blot assay. In the latter assay, purified HCVpp were heat-denatured at 1000C for 5 minutes and subjected to SDS-polyacrylamide gel electrophoresis using known quantities of purified recombinant soluble E2 (rsE2, Austral Biologicals, San Ramon, CA) as a standard. Proteins were transferred to nitrocellulose membranes, blocked, and then probed with anti-E2 monoclonal antibody (MAb) (MAb 303F76) followed by detection with alkaline- phosphatase-conjugated goat anti-mouse IgG.
Cloning infectious HCV E1/E2 envelope glycoproteins from patient sera for the production of HCVpp of different genotypes
[0636] To examine the breadth of antiviral activity of potent and selective hits, multiple infectious E1/E2 variants were cioned and amplified from from sera of individuals infected with different HCV 1a or 1 b genotypes. Utilizing a modified high-throughput, miniaturized version of an E1//E2 cloning strategy, HCV E1/E2 gene sequences representing amino acids 170 to 746 were amplified by nested RT-PCR using genotype specific primers as described. (tavsllette, D. et a)., 2005, Hepatology, 41:265-274). Purified viral RNA isolated from 150 μL of HCV+ patient serum using the QIAamp Viral RNA mini Kit (QiAGEN) was subjected to reverse transcription using the Superscript™ III First-Strand Synthesis System (Invitrogen). The resulting cDNA served as a template for a nested PCR amplification of the E1/E2 envelope with genotype- specific primer pairs. Briefly, a first round of amplification was performed with genotype-specific outer primers followed by a second round of amplification with genotype-specific inner primers. The forward inner primer contained a 5' CACC sequence to allow directional cloning of nucleic acid encoding HCV E1/E2 into the pcDNA3.1 TOPO vector (Invitrogen). Both rounds of amplification were performed with the high fidelity Platinum Pfx DNA polymerase (Invitrogen). The 1.7 kb PCR product was gel purified using the QIAquick Gel extraction kit (Qiagen) and lϊgated into the pcDNA3.1-TOPO expression vector (Invitrogen). Multiple piasmid DNA clones encoding unique E1/E2 quasispecies were isolated from each of the patient sera and verified by DNA sequencing. Co-transfectton of 293T cells and production of HCVpp were adapted to a 96-well format from previously described methods. Cleared viral supernatant (200 μL) was incubated with Hep3B target cells (2,000 cells/well) and luciferase activity was measured 72 hrs post-infection using BriteGlo reagent (Promega).
[0637] HCVpp-based assays were developed representing 15 authentic HCV viral envelopes from different patients infected with genotype 1. Multiple quasispecies were identified within each serum. A relatively low percentage of the isolated E1/E2 expression constructs were fusogenic when expressed on HCVpp. Quasispecies were verified to be unique by sequencing and typically differed from one another at 5-10 positions within the 130-amino~acid HVR1 region of E2. Transfection conditions for each envelope were optimized and individual HCVpp assays were validated with JS-81. The genotype spectrum pane! was used to evaluate the potency and breadth of the antiviral activity of compounds of the invention.
High Throughput Screening Assay (HTS) for Inhibitors of HCVpp Entry [0638] Compounds were tested for their ability to inhibit HCVpp entry into the human hepatoma cell line Hep3B. HCVpp were generated as described above. HCVpp-containing supematants were stored at -8O0C and then thawed at 25°C for thirty minutes prior to use in the inhibition of infection assay. Equal volumes (20 μl) of HCVpp and Hep3B cells (ATCC), (2 x 103 cells/well), were plated in solid white 384-we!i plates (Perkin Elmer) in DMEM/2%FCS. Test compounds at 5μM concentration in 0.5%DMSO (final) or control samples (5 μl) (DMSO alone or JS-81 ) were added to the HCVpp and Hep38 cells. After incubating the plates at 37°C for 3 days, medium was removed from the weils and equal volumes of PBS and Bright-Glo (Promega, Madison Wl) (25 μl) were added. Luciferase activity (Relative Light Units, R.L.U.) was measured by a luminescence plate reader (Victor2, Perkin Elmer). Percent neutralization of entry was calculated from the R.L.U. values using the following formula: 100- [(R.L.U. (compound)-R,L,U. (minimum signal: cells no virus)) /RLU (maximum signal: cells + virus)- R.L.U. (minimum signai: cells no virus)) x 100]. The formulas used for data analysis were: Maximum signal = average RLU values from weils with HCVpp and celts (vehicle alone) Minimum signai = average RLU values from wells with cells only (no HCVpp or sample) % inhibition of entry = 100- [(R.L.U. (compound)-R.L.U.(minimum signal: cells no virus)) / RLU(maximum signal: cells + virus)-R.L.U. (minimum signal: cells no virus)) x 100]. [0639] During HTS, test compounds were evaluated in parallel for inhibition of Sentiviral particles pseudotyped with an envelope from an irrelevant virus, vesicular stomatitis virus (VSV- G) in order to eliminate from further consideration samples with non-specific activity. The resultant VSV pseudoparticles, VSVpp, used in screening potential HCV small molecule inhibitor compounds for specificity are well suited for this purposes because (a) VSV/G is unrelated to HCV E1/E2, (b) VSVpp possess a broad cellular tropism and efficiently infect Hep3B cells, and (c) VSVpp stocks can be produced at high titer and cryopreserved for later use in screening.
[0640] Primary HTS is a single point measurement that calculates % inhibition only at a 5 micromolar drug concentration. For dose response and !C50 determination, compounds were subjected to 10 serial 0.5log10 dilutions in DMSO. The diluted compounds were added to target cells as described which were then infected with HCVpp. After 72 hours viral entry as measured by luciferase activity was determined using a 4-parameter curve fitting program within a customized template in Activity Base (IDBS) (see Fig. 2 and Fig. 3 as examples). [0641] When tested in the HCVpp assay, compounds of the invention generally exhibited virus infection inhibitory activity against HCV of genotype 1 with a median IC50 value in the range of about 0.001 uM to greater than about 10 μM.
Assessment of Anti-Hepatitis C Virus Activity in an HCV Cell Culture (HCVcc) Assay
[0642] Prior to the development of the HCVcc assay, patient sera were utilized to infect primary hepatocytes or hepatoma cell lines, typically resulting in low level and poorly reproducible viral replication. Substantial efforts were made to develop systems that reliably and robustly produced infectious HCV in vitro. Such systems using an HCV clone (JFH1 , genotype 2) derived from a Japanese patient with fulminant hepatitis were reported several years ago. {Kato, T, et al., 2003, Gastroenterology, 125:1808-1817; Lindenbach, B. D. et a!., 2005, Science, 309:623-626; Wakita, T. et at., 2005, Nat Med, 11 :791-796; Zhong, J. et al., 2005, Proc Natl Acad Sci USA, 102:9294-9299). The subgenomic (replicon) clone of this genotype 2 isolate replicates efficiently in cell culture in the absence of adaptive mutations typically associated with HCV replicon sequences. (Krieger, N. et al., 2001 , J. Virol., 75:4614- 24). Full-length clones, containing either the complete JFH1 consensus sequence or JFH1 nonstructural proteins in association with the core-thro ugh-NS2 regions of another genotype 2 clone, J6, demonstrated robust replication in Huh-7-derived celt lines. Ceils transfected with the cloned viral genome secreted HCV particles that were infectious in vitro and in chimpanzees, HCVcc could be inhibited with tFN-α and by small-molecule inhibitors of the HCV serine protease NS3. HCVcc could be propagated in vitro, particularly on Huh-7 sublines that had been transfected with and then cured of HCV replicons.
[0643] HCVcc enabled the study of entry by authentic HCV in vitro, and the findings have been remarkably convergent with those obtained using HCVpp. HCVcc entry is pH dependent and is restricted to CD81-positive liver cells. CD81 -negative HeρG2 cells become permissive to HCVcc infection when modified to express CD81. HCVcc infection is inhibited by MAbs to CD81 and to recombinant forms of the large extracellular loop of CD81. Likewise, MAbs directed against SR-BI or tagged claudin-1 also inhibit HCVcc entry. HCVcc infection is inhibited by sera from HCV-infected individuals but not by normal human sera. Infection is inhibited by MAbs directed against the E1 and E2 envelope glycoproteins. The findings corroborate those obtained using HCVpp and support the view that HCVpp accurately recapitulate the essential biology of HCV entry.
[0644] In addition to studying the HCV biology and assessing the antiviral activity of HCV drugs, the HCVcc system has also been used successfully in drug resistance studies. The JFH1 HCVcc system was used to develop drug resistance against the protease inhibitor BILN- 2061 (Cheng, G. et al., 2008, Efficient In Vitro Selection of Drug-Resistant Mutants Using the HCV Infection System. 15th International Symposium on Hepatitis C Virus and Related Viruses Oct 5-9, 2008 San Antonio, TX). The in vitro resistance profile for 8ILN-2061 correlated with viral resistance patterns obtained in the replicon with BiLN-2061 and in the clinic with other protease inhibitors. The recent availability of chimeric full-length constructs containing the nonstructural proteins of JFH-1 and the structural proteins of genotype 1 clones such as H77C, J4 or Con1 have provided model, chimeric HCVcc systems in which to determine antiviral activity, mechanism of action and determinants of drug resistance for inhibitors of HCV entry using genotype 1 HCV strains.
[0645] To examine the anti-HCV activity of the small molecule compounds according to the present invention, an HCV cell culture (HCVcc) assay (or model) is performed using an HCV susceptible ceil line, such as Huh7.5, infected with a genetically engineered HCV, e.g., HCV of genotype 2a or genotype 1a/2a, The HCVcc system affords the opportunity to study the ability of compounds of the invention to inhibit HCV of different genotypes from infecting target cells, e.g., in a manner that is more akin to in vivo virus infection conditions.
Inhibition of HCV Entry into Susceptible Cells.
[0646] Compounds of the invention were tested for their ability to inhibit infection of susceptible cells by various and representative genotype 1 envelopes of HCV in a pseudovirus infectivity assay empioying HCVpp as described. The anti-CD81 JS-81 monoclonal antibody (BD Biosciences, San Jose, CA) was used as a reference standard and was tested in parallel with the compounds for inhibition of HCVpp (virus) entry into cells. Compounds were diluted in DMSO and were added to Hep3B celts immediately prior to the addition of HCVpp derived from different genotype isolates, e.g., genotype 1a (HCV strain H77) or genotype 1 b. Plates were incubated for 72 hours prior to measurement of luciferase activity.
[0647] HCVpp containing supernatants were stored at -8O0C and then thawed at 25°C for thirty minutes prior to use in the virus infection inhibition assay. Equal volumes (20 μl) of HCVpp and the cells, (2 x 103 cells/well), were plated in solid white 384-weli plates (Perkin Elmer) in DMEM/2%FCS. JS-81 was added to the HCVpp and HEP3B cells. After incubating the plates at 37°C for 3 days, medium was removed from the wells and equal volumes of PBS and Bright-Glo (Promega, Madison Wl) (25 μl each) were added. Luciferase activity (Relative Light Units, R.L.U.) was measured by a luminescence plate reader (Victor2, Perkin Elmer). Percent inhibition of virus entry was calculated from the R.L.U. values using the following formula: 100- [(R.L.U. (compound)-R.L.U. (minimum signal: celis no virus)) / RLU (maximum signal: cells + virus)-R.L.U. (minimum signal: cells no virus)) x 100].
[0648] The compounds of the invention, in particular, the compounds presented in Table 35, inhibited entry of HCV of genotypes 1a and 1b into susceptible cells as determined by pseudoparticle- and cell culture-based assays that measure inhibition of HCV infection by the tested compounds, as described below. For example, in pseudoparticle-based assays to assess inhibition of HCV entry, neutralization values (IC50) for compounds of the invention against HCV of genotype 1a ranged from 6 nM to 1 μM, or from 6 nM to 525 nM, or from 25 nM to 355 nM, or from 28 nM to 67 nM, or from 89 nM to 195 nM, while neutralization values (IC50) for compounds of the invention against HCV of genotype 1 b ranged from 40 nM to 1 μM, or from 56 nM to 625 nM, or from 56 nM to 320 nM, or from 40 nM to 110 nM. [0649] In cell culture-based assays to assess inhibition of HCV entry, neutralization values (IC50) for compounds of the invention, and in particular, the compounds presented in Table 35, against HCV of genotype 1a ranged, for example, from 27 nM to 1 μM, or from 27 nM to 891 nM, or from 27 nM to 317 nM, or from 27 nM to 106 nM, or from 67 nM to 891 nM, or from 67 nM to 106 nM, while neutralization values (IC50) for compounds of the invention against HCV of genotype 1b ranged, for example, from 5.6 nM to 840 nM, or from 78 nM to 840 nM, or from 83 nM to 528 nM, or from 84 nM to 498 nM.. [0650] In another aspect, compounds of the invention have been found to inhibit HCV entry by blocking the interaction of HCV E2 envelope glycoprotein with the scavenger receptor B1 (SR-B1 ) integral membrane, ceil surface receptor. SR-8I is a 509 amino acid glycoprotein comprised of a large extracellular loop anchored to the plasma membrane at both the N- and C- termini by two transmembrane domains, it is a multϊ-ligand receptor that is highly expressed in liver and steroidogenic tissues (22) and is involved in bidirectional transport of cholesterol.. In addition, SR-BI has been shown to be an essential cofactor required for entry of hepatitis C virus (HCV) into hepatocytes (23-27); thus, SR-B1 may play a role in mediating HCV infection. [0651] To determine whether HCV entry inhibitors of the invention, particularly one or more compounds of Table 35, inhibited binding of HCV to a susceptible cell through cell surface SR- Bl, a FACS-based neutralization of binding assay was employed (27). For this assay, a recombinant, C-terminal, histidine-tagged soluble form of the HCV envelope glycoprotein E2, sE2-6xHlS, (Immune Technologies, ) was used. sE2-6xHIS encompasses amino acid residues 383-663 from the HCV genotype 1b strain (NCBI: Accession Number: AY460204). Similar to other genotype 1 b strains, sE2 derived from AY460204 specifically binds to cells that express SR-BI, but not to cells that lack SR-BI, including MOLT-4 and iMR-90. [Q652J Briefly, Huh7.5.1 cells were harvested, washed and diluted in binding buffer (1X phosphate buffered saline, pH 7.4, containing 0.1% sodium azide and 1% bovine serum albumin). Huh7.5.1 cells (200,000 cells) were mixed with binding buffer containing sE2-6xHis at a final concentration of 10μg/mL in the presence or absence of DMSO or small-molecule SR-BI inhibitors. After 30 minutes of incubation at room temperature, unbound sE2-6xHis was removed by washing with 200 μL assay buffer followed by centrifugation at 500xg for 2 minutes to collect the cells. Huh7,5.1 cells were counterstained with an anti-Histidine antibody conjugated to a phycoerythrin fluorescent label (Abeam) (1 :220). After a 30 minute incubation at room temperature, unbound antibody was removed by washing with 200 μL of assay buffer. The bound fraction of Huh7.5.1 cells was determined by quantifying the positively stained cells by FACS using the FL-2 channel on a FACS-Calibur (Becton-Dickson). Data were analyzed using FlowJo software.
[0653] To evaluate the ability of a representative compound of Table 35 to inhibit sE2-6xHis binding to SR-Bi, the compound was subjected to ten serial 0,5log dilutions in DMSO. Various concentrations of the compound were measured for their ability to neutralize binding of sE2- 6xHϊs to cell surface expressed SR-BI. As shown in Fig. 4, the compound was able to block binding of sE2-6xHis to SR-BI expressed on the surface of Huh7.5.1 cells with a calculated EC50= 126nM. This compound having a MW of 350-650, not only blocked the binding of sE2 to SR-BI, but also exhibited antiviral activity against HCV genotype 1a (IC50 of 89 nM) and 1 b {iC5o of 40 nM) in in vitro HCVpp assays, as well as significant antiviral activity against HCV genotype 1 a (EC50 of 88 nM) and 1 b (EC5O of 84 nM) in cell culture-based in vitro neutralization assays. [0654] To confirm that the compound bound to surface-expressed SR-BI on Huh7.5.1 cells rather than to sE2-6xHis, a washout study was performed. Huh7.5,1 cells were pre-treated with 5 μM of a negative control compound, Compound A, which is inactive in both the HCVpp or HCVcc antiviral assays, or the above-described compound of Table 35 (Compound B in Fig. 5) for 30 minutes at room temperature and then subjected to a washout with assay buffer to remove unbound compound. The results showed that Compound A did not block binding of sE2-6XHis to SR-B) expressed on Huh7.5,1 cells, white a high concentration of Compound B potently blocked binding of sE2 to surface expressed SR-BI on Huh7.5.1 cells (No Wash). Pre- treatment of Huh7.5.1 cells with Compound B followed by a washout did not significantly affect the inhibitory activity of Compound B in the assay (compare No Wash with 2X Wash, i.e., the rightmost and the leftmost bar graphs, respectively, for Compound B in Fig. 5). These results suggest that Compound B targets SR-BI on the surface of Huh7.51 cells and blocks a key interaction with the HCV E2 glycoprotein.
Time of Addition Assay to Evaluate HCV Entry into Cells
[0655] A tsme-of-addition assay may be performed to evaluate inhibition of virus entry into cells and to assess the inhibitory effect of test compound addition as a function of time following exposure of susceptible target cells to HCVpp. Without wishing to be bound by theory, the process of HCV entry can be subdivided into at least four distinct stages: 1 ) attachment to liver cells; 2) E2 binding to a co-receptor (e.g. CD81 and/or SR-Bl); 3) rearrangement of tight junctions and co-localization of CD81/HCV complex with claudin-1 ; and 4) low pH induced E1 (or E2) mediated membrane fusion. The mechanism of action of an entry inhibitor can be inferred, in part, by establishing the time period that it is able to block entry of HCVpp into target cells. Time of addition studies may be performed to show that a compound of the invention potently inhibits a particular step of HCV entry. The effect of anti-CD81 antibody JS-81 is used as a positive control, while a non-active compound serves as a negative control.
Cytotoxicity Assay for Testing HCV Inhibitor Compounds
[0656] Profiling antiviral compounds in cytotoxicity assays is a critical step to determine that the antiviral activity observed in the primary assay is not due to overt cytotoxicity. To profile the HCV inhibitor compounds of the invention, a panel of cytotoxicity assays was developed based on nine cell lines (Hep3B, Huh-7, Huh7.5.1 , Ramos, Daudi, Jurkat, 293T, HeLa, and U87), as well as primary hepatocytes grown in culture. All cytotoxicity assays were benchmarked against the pan-kinase inhibitor staurosporine. The cytotoxic concentration required to reduce cell viability by 50% (CC50) for staurosporine was determined concurrently in all cytotoxicity assays and served as a reference compound for assay validation and data acceptance. [0657] The compounds of the invention were profiled in cytotoxicity assays against Hep3B cells. These assays were carried out for 72 hours under conditions that were identical to those used in the HCVpp primary assay. Briefly, cells were plated in 384-well plates in the presence or absence of serially diluted compounds. After 72 hours, cell proliferation was determined
I using the CellTiter-Glo assay (Promega). In this assay, luminescence is a direct read-out of the number of viable cells in the micropiate. Dose-response curves were generated and the CC50 was determined from the curve for each cell line. A cytotoxicity-based therapeutic index (T!=CC5O/IC5o) was determined for ail compounds. Compounds with Tl >10 were determined to have a high likelihood of being specific antivirals.
[0658] Fig. 2 (pyrimidines) and Fig. 3 (pyrazolopyrimidines and imidazolopyrimidines) illustrate entry inhibition curves for exemplary compounds of the invention as a function of their concentrations. From such inhibition curves IC50 values are determined, as described above.
BiaCore Based Binding Assays
[0659] In order to determine whether HCV inhibitor compounds of the invention bind to the E2 envelope of HCV, the binding of entry inhibitors to a truncated soluble form of the E2 glycoprotein (sE2) is examined by BIACore. Soluble E2 is immobilized to BIACore chips according to the manufacturer's instructions. A binding assay using the E2 bound chip is established and validated with PA-25, a mouse MAb generated against sE2 {positive control) and two negative controls: PA-1 , a MAb that recognizes HIV-1 gp120 and an isotype control. Surface plasmon resonance is monitored during the flow of antibody controls or small-molecule inhibitors over immobilized sE2. PA-25 is expected to demonstrate strong binding to sE2 in the assay, while representative small-molecule entry inhibitors may or may not demonstrate binding to E2 in the BIACore assay. The negative control, PA-1 and the isotype control, are not expected to exhibit specific binding to sE2 in the study.
STATEMENT REGARDING PREFERRED EMBODIMENTS
(0660] While the invention has been described with respect to preferred embodiments, those skilled in the art will readily appreciate that various changes and/or modifications can be made to the invention without departing from the spirit or scope of the invention as defined by the appended claims.
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Claims

CLAIMSWHAT IS CLAIMED IS:
1. One or more compounds of formula (I), salts, including pharmaceutically acceptable salts, polymorphs, hydrates, stereoisomers, metabolites and prodrugs thereof:
Figure imgf000214_0001
(i)
wherein:
L2, L4, and L6 are independently none, H, O, S, NRR', (CH2)O-5, CN, CRR', SO2, CO, CONR, NHCONR or CONR1R, NHCONR1R, halide, cycloalkyl, heterocycle, aryl, alkyne, alkene;
R2, R4, and R6 are independently none, H, R, OR, amino, amine, alkoxy, (CH2)o-3CF3, (CH2)(XjW, alkyl, aryl, cycloalkyl, heterocycle, fused aSkylaryl or heteroalkylaryl, substituted with 0-2 W;
W is H1 halide, OR, CF3, NO2, CN, amino, amine, aniline, ester, amide, sulfonamide, sulfone, amino acid, ether, urea acid, heterocycle, alkyl, aryl, arylalkyl, alkylaryl; and
R or R' is independently none, H, alkyl, aryl.
2. One or more compounds of formula (II), their salts, including pharmaceutically acceptable salts, polymorphs, hydrates, stereoisomers, metabolites, and prodrugs thereof
Figure imgf000214_0002
(ii)
wherein:
R1 is H, aikyl, aryl, heterocycie, each of which may be optionally substituted; R3 is H1 alkyl, aryl, heterocycle, each of which may be optionally substituted;
L4 and L6 are independently none, H, O, OR, S, NRR!, NHRm, (CH2)n, CN, CRR1, SO2, CO, CONR'R, NHCONR'R, halide, cycloalky!, heterocycle, aryi, aikyne, alkene, wherein n = 0-5, and m is independently 1-2;
R4, and R6 are independently none, H, R, OR, amino, amine, (CH2)nCF3, CF3, CH2CF3, (CH2JnW, alkyl, aryl, phenyl, cycloalkyl, piperidinyl, heterocycle, fused aryl, alkylaryl, or heteroalkylaryl, ail of which are optionally substituted with 0-5 W, wherein n is chosen independently to be 0-5;
W is H1 halide, OR, CF3, NO2, CN, SO2NRR', SO2R, amino, amine, aniline, ester, amide, sulfonamide, sulfamoyl, sulfone, amino acid, ether, urea, acid, heterocycle, heteroaromatic, alkyl, aryl, arylalkyl, alkylaryl, sulfone, sulfonamide substituted with alkyl, aryl, heterocycle, amino, aniline; and
R or R' are independently none, H, alkyl, aryl, amide.
3. One or more compounds of formula (Ha), salts, including pharmaceutically acceptable salts, polymorphs, hydrates, stereoisomers, metabolites, and prodrugs thereof
Figure imgf000215_0001
wherein,
R1 is H, alkyl, aryl, heterocycle, each of which may be optionally substituted;
R3 is H, alkyl, aryl, heterocycle, each of which may be optionally substituted;
L5 and L7 are independently none, H, O, S, NRR1, NHRm, (CH2Jn, CN, CRR1, SO2, CO, CONR'R, NHCONR'R, halide, cycloalkyl, heterocycle, aryl, aikyne, alkene, wherein n = 0-5, and m is independently 1-2;
R5, and R7 are independently none, H, R, OR, amino, amine, (CH2)nCF3, CF3, CH2CF3, (CH2)nW, aikyl, aryl, phenyl, cycloalkyl, piperidinyl, heterocycle, fused aryl, alkylaryl, or heteroalkylaryl, all of which are optionally substituted with 0-5 W, wherein n is chosen independently to be 0-5;
W is H, halide, OR, CF3, NO2, CN, SO2NRR', SO2R, amino, amine, aniline, ester, amide, sulfonamide, sulfamoyl, suifone, amino acid, ether, urea, acid, heterocycle, heteroaromatic, alkyl, aryl, arylalkyl, alkyiaryl, sulfone, sulfonamide substituted with alkyl, aryl, heterocycte, amino, aniline; and
R or R' are independently none, H, alkyl, aryl, amide.
4. One or more compounds of formula (III), salts, including pharmaceutically acceptable salts, polymorphs, hydrates, stereoisomers, metabolites and prodrugs thereof
Figure imgf000216_0001
(Hi)
wherein,
R1 is H, aikyl, aryl, heterocycle, each of which may be optionally substituted;
R2 is H, alkyl, aryl, heterocycle, each of which may be optionally substituted;
L4 and L6 are independently none, H, O, S, NRR', NHRm, (CH2)n, CN, CRR!, SO2, CO, CONR1R, NHCONR'R, halide, cycloalkyl, heterocycle, aryl, alkyne, alkene, wherein n = 0-5, and m is independently 1-2;
R4, and R6 are independently none, H, R, OR, amino, amine, (CH2)nCF3, CF3, CH2CF3, (CH2JnW, alkyl, aryl, phenyl, cyctoalkyl, piperidinyl, heterocycle, fused aryl, alkylaryi, or heteroalkylaryi, all of which are optionally substituted with 0-5 W, wherein n is chosen independently to be 0-5;
W is H, halide, OR, CF3, NO2, CN, SO2NRR', SO2R, amino, amine, aniline, ester, amide, sulfonamide, sulfamoyl, sulfone, amino acid, ether, urea, acid, heterocycle, heteroaromatic, alkyl, aryl, arylalkyl, alkylaryi, sulfone, sulfonamide substituted with alkyl, aryl, heterocycle, amino, aniline; and
R or R' are independently none H, aikyl, aryl, amide.
5. One or more compounds of formula (Ilia), salts, pharmaceutically acceptable salts, polymorphs, hydrates, stereoisomers, metabolites and prodrugs thereof
Figure imgf000216_0002
wherein,
R1 is H, alky!, aryl, heterσcycle, each of which may be optionally substituted;
R2 is H, aikyl, aryl, heterocycle, each of which may be optionally substituted;
L5 and L7 are independently none, H, O, S1 NRR', NHRm, (CH2)n, CN, CRR', SO2, CO, CONR'R, NHCONR'R, halide, cycloalkyl, heterocycle, aryl, alkyne, alkene, wherein n = 0-5, and m is independently 1-2;
R5, and R7 are independently none, H, R, OR, amino, amine, (CH2)nCF3, CF3, CH2CF3, (CH2JnW, alkyl, aryl, phenyl, cycloalkyl, piperidinyl, heterocycle, fused aryl, aikylaryl, or heteroalkylaryl, all of which are optionally substituted with 0-5 W, wherein n is chosen independently to be 0-5;
W is H, halide, OR, CF3, NO2, CN, SO2NRR', SO2R, amino, amine, aniline, ester, amide, sulfonamide, sulfamoyl, sulfone, amino acid, ether, urea, acid, heterocycle, heteroaromatic, alkyl, aryl, arylalkyl, aikylaryl, sulfone, sulfonamide substituted with alkyl, aryl, heterocycle, amino, aniline; and
R or R' are independently none, H, alkyl, aryl, amide.
6. A compound, salts thereof, pharmaceutically acceptable salts, polymorphs, hydrates, stereoisomers, metabolites or prodrugs thereof, which is selected from Tables 1-35.
7. A composition comprising at least one of a compound, stereoisomer, hydrate, polymorph, metabolites, prodrugs or a salt thereof, selected from Tables 1-35.
8. A composition comprising the compound of claim 1.
9. A composition comprising the compound of claim 2.
10. A composition comprising the compound of claim 3.
11. A composition comprising the compound of claim 4.
12. A composition comprising the compound of claim 5.
13. A composition comprising the compound of claim 6.
14. The composition of any of claims 7-13 and a pharmaceutically acceptable carrier.
15. The composition of claim 14, further comprising a solid formulation, a suspension, a semisolid formulation, a solution formulation, an aqueous formulation, an immediate release formulation, a sustained release formulation, an enteric coating formulation or a lyophilized formulation.
16. The composition of claim 15, wherein the formulation is a packaged unit dosage.
17. The composition of claim 16, wherein the packaged unit dosage is a solution, solid, powder, aerosol, liquid or gel.
18. The composition of claim 14, further comprising at least one additional antiviral agent.
19. The composition of claim 18, wherein the at least one additional antiviral agent is selected from anti-Hepatitis C virus compounds, Hepatitis C virus metalloprotease inhibitors, Hepatitis C virus polymerase inhibitors, Hepatitis C virus serine protease inhibitors, Hepatitis C virus helicase inhibitors, or combinations thereof.
20. The composition of claim 18 or 19, wherein the at least one additional antiviral agent is selected from interferon, pegyiated interferon, interferon-aipha, pegylated interferon- alpha, ribavirin, or a combination thereof.
21. The composition of claim 14, further comprising at least one pharmaceutical agent that is not an antiviral agent.
22. The composition of claim 21 , wherein the at least one pharmaceutical agent is an anti-infective agent or an anti-cancer agent.
23. A method for treating or preventing infection by a virus of the family Fiaviviridae, comprising administering to a patient in need thereof the composition of claim 14 in an amount effective to treat or prevent the infection.
24. The method of claim 23, wherein the virus is Hepatitis C Virus (HCV).
25. The method of claim 23 or claim 24, wherein the effective amount of the composition inhibits or blocks (i) entry of HCV into a susceptible cell or (ii) exit of HCV virions from an infected cell,
26. The method of claim 24, wherein HCV is of genotype 1 or genotype 2.
27. The method of claim 26, wherein HCV is of genotype 1a, genotype 1 b, or a combination thereof.
28. The method of claim 23, wherein the composition is administered by a route of administration selected from one or more of oral, enteral, parenteral, topical, subcutaneous, or intravenous routes of administration,
29. A method of inhibiting HCV infection of a cell susceptible to HCV infection, comprising contacting the cell with the compound of any of claims 1-6, in an amount effective to inhibit HCV infection of the ceil.
30. A method of inhibiting HCV infection of a cell susceptible to HCV infection, comprising contacting the cell with the composition of any of claims 7-21 , in an amount effective to inhibit HCV infection of the cell.
31. The method of claim 29 or claim 30, wherein the susceptible cell is in a subject and the effective amount of the compound or composition is administered to the subject.
32. A method of preventing or reducing HCV infection in a subject, comprising administering to the subject a compound of any of claims 1-6 in an amount effective to prevent or reduce the HCV infection.
33. The method of claim 32, wherein the compound is administered to the subject before, after, or during exposure of the subject to HCV,
34. A method of reducing exposure of a subject to HCV infection outside or on the external body surface of the subject, comprising contacting the outside or external body surface of the subject with a compound of any of claims 1-6, in an amount effective to inactivate or inhibit the virus so as to reduce exposure of the subject to HCV infection.
35. A method of inactivating, inhibiting, decontaminating, or rendering inactive or weakly infective objects, surfaces, or substances that have been contaminated with HCV, which comprises contacting the objects, surfaces, or substances with a compound of any one of ciaims 1-6, in an amount effective to inactivate, inhibit, decontaminate, or render inactive or weakly infective the HCV.
36. A method of reducing the occurrence of HCV infection in a population of individuals, comprising administering to the population of individuals in need thereof a compound of any one of claims 1-6 in an amount effective to reduce the occurrence of HCV infection in the population.
37. A pharmaceutical composition comprising a therapeutically effective amount of a compound of any of claims 1-6 and a pharmaceutically acceptable carrier or excipient.
38. The pharmaceutical composition of claim 37, in combination with an additional antiviral active ingredient selected from interferons, anti-HCV monoclonal antibodies, anti-HCV polyclonal antibodies, HCV RNA polymerase inhibitors, HCV protease inhibitors, IRES inhibitors, helicase inhibitors, antisense compounds, anti-viral small molecules, ribozymes, or a combination thereof.
39. The pharmaceutical composition of claim 38, wherein the at least one antiviral active ingredient is selected from ribavirin, interferon, pegylated interferon, interferon-α, interferon-α-2β, or a combination thereof.
40. A method of treating or preventing a liver disease in a subject, which comprises administering to the subject a compound of any of claims 1-6, in an amount effective to inhibit infection of the subject's HCV susceptible ceils, thereby treating or preventing the liver disease in the subject.
41. A method of treating or preventing a liver disease in a subject, which comprises administering to the subject a composition of any of claims 7-21 , in an amount effective to inhibit infection of the subject's HCV susceptible cells, thereby treating or preventing the liver disease in the subject.
42. A method of treating or preventing an HCV associated disorder in a subject, which comprises administering to the subject a compound of any of claims 1-6, in an amount effective to inhibit infection of the subject's HCV susceptible cells, thereby treating or preventing the HCV associated disorder.
43. A method of treating or preventing an HCV associated disorder in a subject, which comprises administering to the subject a composition of any of claims 7-21 , in an amount effective to inhibit infection of the subject's HCV susceptible cells, thereby treating or preventing the HCV associated disorder.
44. A method of inhibiting HCV entry via an SR-B1 receptor into an SR-B1- expressing cell susceptible to HCV infection, comprising contacting the cell with a compound of any of claims 1-6 or a composition of any of claims 7-21.
45. A method of inhibiting HCV entry via an SR-B1 receptor into an SR-B1- expressing ceil susceptible to HCV infection, comprising contacting the cell with a compound of Tables 1-35 or a composition comprising the compound.
46. The method of claim 45, comprising contacting the cell with a compound of Table 35,
47. The method of any of ciaims 44-46, wherein the susceptible cell is in a subject and the compound or composition is administered to the subject in an amount effective to inhibit HCV entry into the SR-B1 -expressing cell.
48. The method of any of ciaims 44-47, further comprising providing an antiviral active ingredient selected from interferons, anti-HCV monoclonal antibodies, anti-HCV polyclonal antibodies, HCV RNA polymerase inhibitors, HCV protease inhibitors, IRES inhibitors, helicase inhibitors, antisense compounds, anti-viral small molecules , ribozymes, or a combination thereof.
49. The pharmaceutical composition of claim 48, wherein the antiviral active ingredient is ribavirin, interferon, pegyiated interferon, interferon-α, interferoπ-α-2β, or a combination thereof.
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