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  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

• c- AbI tyrosine kinase inhibitor STI571 (Gleevec or Imanitib, Novartis Pharma) for the treatment of Chronic Myeloid Leukemias (CML) has promoted development of protein kinase inhibitors (PKIs) as targeted therapy for other types of leukemia as well as for solid tumors.
• PKIs protein kinase inhibitors
• CML is a rare, malignant, myeloproliferative disorder that is characterized by the acquisition of the reciprocal (9:22) (q34;ql 1) chromosomal translocation (Philadelphia chromosome) in hematopoietic stem cells and constitutive activation of protein tyrosine kinase Bcr-Abl (Heisterkamp N, Stam K, Groffen J, de Klein A and Grosveld G 1985 Structural organization of the bcr gene and its role in the Ph 1 translocation. Nature 315:758-61; Barila D and Superti-Furga G 1998.
• FLT-3 FLT-3 (FMS-like tyrosine kinase 3) is a receptor tyrosine kinase that catalyzes the phosphorylation of hydroxy groups on tyrosine residues of proteins.
• Receptor tyrosine kinases comprise a large family of transmembrane receptors with diverse biological activity. At present, at least nineteen (19) distinct subfamilies of receptor tyrosine kinases have been identified.
• FLT-3 is a member of the platelet derived growth factor receptor (PDGFR) tyrosine kinase subfamily, and shares the structural features of KIT, FMS, and PDGFR. Structurally, FLT-3 has five immunoglobulin-like domains in the extracellular region, a single transmembrane sequence, and, intracellularly, a short juxtamembrane portion followed by an intracellular kinase domain (Levis M, Allebach J,, Tse K-F, Zheng R, Baldwin BR, Douglas Smith B, Jones-Bolin S, Ruggeri B, Dionne C and Small D (2002).
• PDGFR platelet derived growth factor receptor
• a FLT-3 -targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. Blood 99(11): 3885-3891). Its activation signals are transduced through phosphorylation of itself and cytoplasmic proteins in biochemical signaling pathways that promote uncontrolled cell growth and inhibit apoptosis. On binding to FLT-3 ligand, the wild type receptor dimerizes, activating its tyrosine kinase domain with resultant phosphorylation.
• RAS-GAP, PLCD, PI3- kinase, STAT5, and ERK1/2 are important signaling proteins linked to activation of FLT-3.
• EGF receptor tyrosine kinases include EGF receptor tyrosine kinases, insulin receptor tyrosine kinases, and FGF receptor tyrosine kinases.
• EGF receptor kinase for example is overexpressed in many cancers such as lung and breast cancers.
• agents such as Astrazeneca's Iressa, Genentech's Herceptin (for HER2-positive metastatic breast cancer), and OSI-Genentech's Tarceva (for nonsmall cell lung cancer) specifically target the EGFR receptor pathway.
• a number of other small molecule receptor tyrosine kinase inhibitors that are in clinical development include: SU5416, SU6668 and SUl 1248 (Sugen-
• benzoxazole, benzothiazole, and benzimidazole derivative have also been reported useful for the treatment of cancer and other diseases as well but have not been shown to be suitable targeted inhibitors of specific kinases, which is also the case for many other herocyclic compounds described in many patents.
• the present invention relates to a patentable class of kinase inhibitors with certain substituted benzoxazole derivatives that inhibit Flt-3 with high efficacy, and specifically kill leukemic cells that carry Flt-3 mutation in vitro and in vivo.
• these compounds inhibited effectively c-Kit (including Gleevec-resistant mutants), and/or RET, and/or PDGFRD (platelet derived growth factor receptor beta) in in vitro tests and can therefore be expected to be useful for the treatment of cancers in which these kinases play a role for the proliferation of these cancers.
• c-Kit including Gleevec-resistant mutants
• RET RET
• PDGFRD platelet derived growth factor receptor beta
• the compounds described here are structurally, and, because of their selective anticancer activity, different from compounds reported in PCT WO 02/072543 A2. Having only targeted anti-cancer activity and not broad anti-cancer activity is a major advantage since such compounds can be expected to have minimum side effects and toxicity. Described in particular are compounds that inhibit FLT-3 in vitro and which in vivo are inhibitors of the growth and progression of human AML tumors that carry Flt-3 mutation. Furthermore, some of these heterocyclic compounds also appear to be potent inhibitors of other kinases, including c-Abl, PDGFR, and c-Kit and/or RET known to be involved in CML, gastrointestinal stromal tumors, and/or thyroid cancer.
• kinases that were observed to be inhibited with high affinity in vitro were AURKA, FGFR3, JAK2, PDGFRQRET, and VEGFR.
• AURKA AURKA
• FGFR3, JAK2, PDGFRQRET PDGFRQRET
• VEGFR VEGFR
• the general structures and specific examples shown in this application cover molecules that belong to a new class of kinase inhibitors that can serve as "targeted anticancer" agents and function by defined mechanisms, inhibiting specific kinases, and thus markedly differ from drugs that are currently available for the treatment of myeloid leukemia and other cancers. Therefore, the heterocyclic compounds described herein are useful in the treatment of diseases of uncontrolled proliferation including AML, CML, gastrointestinal stromal cancers, and thyroid cancer, as well as other cancers and other diseases including inflammation and atherosclerosis.
• the compounds analyzed here were selective kinase inhibitors with good oral bio-availability, these compounds can thus be expected to have limited side-effects and be suitable for the treatment of many cancers, alone or in combination with other cancer therapies.
• This invention thus also relates to the usage of such compounds for the treatment of solid tumor cancers and other diseases.
• Pharmaceutical compositions comprising these compounds, methods of treating diseases, and methods of preparing them are also described.
• the present invention is directed to certain patentable substituted benzoxazole derivatives that exhibit protein kinase (PK) inhibition activity or modulating ability and are therefore useful in treating disorders related to abnormal PK activity.
• PK protein kinase
• One aspect of this invention concerns compounds of Formula (1):
• A is -CR 2 I R 22 -, -NR 23 -, -O-, or -S- ;
• B is -OR 24 , -SR 25 , Or-NR 28 R 29 ;
• D and E are together or independently -CR 30 -, or -N-;
• L is -CH 2 -
• n 0 to 1 ;
• n 1 to 2;
• Ri, R 2 , R3,R ⁇ , Ri2, R2i,R22, R23, R24, R2 5 , R28, R29, and R 30 are independently or together hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogen, cyano, nitro, hydroxyl, acyl, substituted acyl, acyloxy, amino, mono- substituted amino, di-substituted amino, alkylsulfonamide, aryl sulfonamide, alkylurea, arylurea, alkylcarbamate, arylcarbamate, heteroaryl, alkoxy, substituted alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy, alkylcarboxamide, and substituted alkylcarboxamide, dialkylcarboxamide, or substituted
• Another aspect of the invention provides methods of synthesizing such compounds.
• this invention relates to the use of the compounds described herein for inhibiting uncontrolled cellular proliferation, such as various forms of cancer and leukemias, as well as for the treatment inflammatory diseases or atherosclerosis.
• This invention also relates to methods for inhibiting uncontrolled cellular proliferation, e.g., a cancer or leukemia, by administering to a mammal, preferably a human, diagnosed as needing such an inhibitor.
• the invention also provides for methods of treatment for diseases of uncontrolled cellular proliferation, such as cancer and leukemias, comprising administering an effective amount of a compound of the invention to a mammal diagnosed as having such a disease, as well as of methods of treating an inflammatory disease comprising administering to a mammal diagnosed as having an inflammatory disease an effective amount of a compound of the invention.
• this invention relates to pharmaceutical compositions comprising a compound described herein in admixture with one or more pharmaceutically acceptable carriers, excipients, etc.
• kits which comprise, for example, a unit dosage of a pharmaceutical composition according to the invention packaged in a suitable container (e.g., a glass vial), which may optionally be packaged in a box or the like for shipment and storage prior to use.
• a suitable container e.g., a glass vial
• such kits typically further include a package insert providing details on the particular compound, its administration, and use.
• FIGURE 1 shows the kinase inhibition profile of compound 1.
• FIGURE 2 shows killing of MV4;11 human AML cell line by compounds 2, 3, 4, and 5.
• FIGURE 3 shows selectivity of cell killing.
• Compound 1 specifically kills AML (MV4;11) but not prostate (PC-3) and pancreatic (Bx-PC-3) cells.
• FIGURE 4 shows the kinase inhibition profiles of compounds 2, 5, and 8.
• FIGURE 5 shows oral bioavailability and pharmacokinetics profile of compound 2 in rats.
• FIGURE 6 shows prevention of AML tumor progression in the athymic nude mouse model by compound 2.
• FIGURE 7 shows inhibition of large established AML tumors in the nude mouse model by compound 2.
• FIGURE 8 shows selectivity of cell killing by compound 5.
• FIGURE 9 shows oral bioavailability and pharmacokinetics profile of compound 5 in rats.
• FIGURE 10 shows efficacy studies in nude mouse model by compound 5.
• FIGURE 11 shows comparison of compound 2 and compound 5 in reducing tumor growth in nude mouse model.
• FIGURE 12 shows inhibition of large established tumors in nude mouse model by compound 5.
• FIGURE 13 shows a representative scheme of the synthetic pathway for the compounds disclosed herein in Formula 1, wherein m is 0 and n is 1.
• the present invention provides compounds that are useful, for example, to prevent, alleviate, or otherwise treat cancer, and in particular AML and gastro-intestinal cancer — including Gleevec resistant mutants- and certain thyroid cancer, in humans and other mammals.
• compounds of the invention have demonstrated oral bioavailability as exhibited by their high blood levels after oral dosing, either alone or in the presence of an excipient. Oral bioavailability allows oral dosing for use in chronic diseases, with the advantage of self- administration and decreased cost over other means of administration. Definitions
• alkyl denotes a radical containing 1 to 12 carbons, straight or branched chain group such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, t-butyl, amyl, t-amyl, n- pentyl, and the like.
• alkenyl denotes a straight or branched chain hydrocarbon radical containing 1 to 12 carbons and a carbon-carbon double bond, such as vinyl, allyl, 2- butenyl, 3-butenyl, 2- ⁇ entenyl, 3-pentenyl, 4-pentenyl, 2-hexenyI, 3-hexenyI, 4-hexenyl, 5- hexanyl, 2-heptenyL 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, and the like.
• alkenyl includes dienes and trienes of straight and branched chains.
• alkynyl denotes a straight or branched chain hydrocarbon radical containing 1 to 12 carbons and a carbon-carbon triple bond such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3- butynyl, 1-penty ⁇ yl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, A- hexynyl, 5-hexynyl, and the like.
• alkynyl includes di- and tri-ynes.
• substituted alkyl denotes a radical containing 1 to 12 carbons of the above definitions that are substituted with one or more groups, but preferably one, two or three groups, selected from hydroxyl, holagen, cycloalkyl, amino, mono- substituted amino, di-substituted amino, acyloxy, nitro, cyano, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfmyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, or haloalkoxy. When more than one group is present then they may be the same or different.
• substituted alkenyl denotes a radical containing 1 to 12 carbons of the above definitions that are substituted with one or more groups, but preferably one, two or three groups, selected from halogen, hydroxyl, cycloalkyl, amino, mono-substituted amino, di-substituted amino, acyloxy, nitro, cyano, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfmyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, or haloalkoxy.
• substituted alkynyl denotes a radical containing 1 to 8 carbons of the above definitions that are substituted with one or more groups, but preferably one or two groups, selected from halogen, hydroxyl, cycloalkyl, amino, mono-substituted amino, di-substituted amino, acyloxy, nitro, cyano, carboxy, carboalkoxy, alkylcarboxamide, sub- stituted alkylcarboxamide, dialkylcarboxamide, substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, or haloalkoxy.
• cycloalkyl denotes a cyclic alkyl moieties containing 3 to 8 carbons, wherein alkyl is defined above, to include such groups as cyclopropyl, cyclobutyl, cyclopentyl, cyclopenyl, cyclohexyl, cycloheptyl, and the like.
• substituted cycloalkyl denotes a cycloalkyl as defined above that is further substituted with one or more groups, selected from halogen, alkyl, hydroxyl, alkoxy, substituted alkoxy, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, substituted dialkylcarboxamide, amino, mono-substituted amino, or di-substituted amino.
• groups selected from halogen, alkyl, hydroxyl, alkoxy, substituted alkoxy, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, substituted dialkylcarboxamide, amino, mono-substituted amino, or di-substituted amino.
• combination therapy refers to a therapeutic regimen that involves the provision of at least two distinct therapies to achieve an indicated therapeutic effect.
• a combination therapy may involve the administration of two or more chemically distinct agents, for example, a compound according to the invention and another chemotherapeutic agent.
• a combination therapy may involve the administration of one or more agents according to the invention, alone or in conjunction with another agent as well as the delivery of another therapy, e.g., surgery, radiation, etc.
• another therapy e.g., surgery, radiation, etc.
• the active ingredients may be administered as part of the same composition or as different compositions.
• compositions comprising the different active ingredients may be administered at the same or different times, by the same or different routes, using the same of different dosing regimens, all as the particular context requires and as determined by the attending physician.
• drug(s) may be delivered before, during, and/or after the period the subject is in therapy.
• “monotherapy” refers to a treatment regimen based on the delivery of one therapeutically effective compound, whether administered as a single dose or several doses over time.
• cycloalkenyl denotes a radical containing 3 to 8 carbons, such as cyclopropenyl, 1-cyclobutenyl, 2-cyclobutenyl, 1-cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl, 1- cyclohexenyl, 2-cyclohexenyl, 3-cyclohexenyl, and the like.
• substituted cycloalkenyl denotes a cycloalkenyl as defined above further substituted with one or more groups selected from halogen, alkyl, hydroxyl, alkoxy, substituted alkoxy, haloalkoxy, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, substituted. dialkylcarboxamide, amino, mono-substituted amino, or di-substituted amino.
• the cycloalkenyl is substituted with more than one group, they may be the same or different.
• alkoxy denotes a radical alkyl, defined above, attached directly to an oxygen such as, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, t-butoxy, iso-butoxy, and the like.
• substituted alkoxy denotes a radical alkoxy of the above definition that is substituted with one or more groups, but preferably one or two groups, selected from, hydroxyl, cycloalkyl, amino, mono-substituted amino, di-substituted amino, acyloxy, nitro, cyano, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, or haloalkoxy.
• substituted amino denotes an amino substituted with one group selected from alkyl, substituted alkyl, or arylalkyl wherein the terms have the same definitions found throughout.
• di-substituted amino denotes an amino substituted with two radicals that may be same or different selected from aryl, substituted aryl, alkyl, substituted alkyl, or arylalkyl, wherein the terms have the same definitions found throughout. Some examples include dimethylamino, methylethylamino, diethylamino, and the like.
• haloalkyl denotes a radical alkyl, defined above, substituted with one or more halogens, preferably fluorine, such as a trifiuoromethyl, pentafluoroethyl, and the like.
• haloalkoxy denotes a haloalkyl as defined above, that is directly attached to an oxygen to form trifluoromethoxy, pentafluoroethoxy, and the like.
• acyl denotes a radical containing 1 to 8 carbons such as formyl, acetyl, propionyl, butanoyl, iso- Ie butanoyl, pentanoyl. hexanoyl, heptanoyl, benzoyl, and the like.
• acyloxy denotes a radical containing 1 to 8 carbons of an acyl group defined above directly attached to an oxygen such as acetyloxy, propionyloxy, butanoyloxy, iso-butanoyloxy, benzoyloxy, and the like.
• aryl denotes an aromatic ring radical containing 6 to 10 carbons that include phenyl and naphthyl.
• substituted aryl denotes an aromatic radical as defined above that is substituted with one or more selected from hydroxyl, cycloalkyl, aryl, substituted aryl, heteroaryl, heterocyclic ring, substituted heterocyclic ring, amino, 1 mono-substituted amino, di- substituted amino, acyloxy, nitro, cyano, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, substituted I dialkylcarboxamide, alkylsulfonyl, alkylsulfmyl, alkylthio, I alkoxy, substituted alkoxy, or haloalkoxy, wherein the terms are defined herein.
• halo or “halogen” refers to a fluoro, chloro, bromo or iodo group.
• thioalkyl denotes a sulfide radical containing 1 to 8 carbons, linear or branched. Examples include methyl sulfide, ethyl sulfide, isopropyl sulfide, and the like.
• thiohaloalkyl denotes a thioalkyl radical substituted with one or more halogens. Examples include trifluoromethylthio, 1,1-difluoroethylthio, 2,2,2- trifluoroethylthio, and the like.
• the te ⁇ n "carboalkoxy” refers to an alkyl ester of a carboxylic acid, wherein alkyl has the same definition as found above.
• alkylcarboxamide denotes a single alkyl group attached to the amine of an amide, wherein alkyl has the same definition as found above. Examples include N-methylcarboxamide, N-ethylcarboxamide, N-isopropylcarboxamide, and the like.
• substituted alkylcarboxamide denotes a single "substituted alkyl” group, as defined above, attached to the amine of an amide.
• dialkylcarboxamide denotes two alkyl or arylalkyl groups that are the same or different attached to the amine of an amide, wherein alkyl has the same definition as found above.
• Examples of a dialkylcarboxamide include N,N-dimethylcarboxamide, N-methyl-N - ethyl carboxamide, and the like.
• dialkylcarboxamide denotes two alkyl groups attached to the amine of an amide, where one or both groups are a "substituted alkyl", as defined above. It is understood that these groups may be the same or different. Examples include N 5 N- dibenzylcarboxamide, N-benzyl-N-methylcarboxamide, and the like.
• alkylamide denotes an acyl radical attached to an amine or monoalkylamine, wherein the term acyl has the same definition as found above.
• alkylamide include acetamido, propionamido, and the like.
• arylalkyl defines an alkylene, such as -CH2- for example, which is substituted with an aryl group that may be substituted or unsubstituted as defined above.
• alkylene such as -CH2- for example
• arylalkyl examples include benzyl, phenethylene, and the like.
• a residue of a chemical species refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species.
• an ethylene glycol residue in a polyester refers to one or more -OCH 2 CH 2 O- repeat units in the polyester, regardless of whether ethylene glycol is used to prepare the polyester.
• a 2,4-thiazolidinedione residue in a chemical compound refers to one or more -2,4-thiazolidinedione moieties of the compound, regardless of whether the residue was obtained by reacting 2,4-thiazolidinedione to obtain the compound.
• a "patentable" composition, process, machine, or article of manufacture according to the invention means that the subject matter satisfies all statutory requirements for patentability at the time the analysis is performed. For example, with regard to novelty, non-obviousness, or the like, if later investigation reveals that one or more claims encompass one or more embodiments that would negate novelty, non-obviousness, etc., the claim(s), being limited by definition to “patentable” embodiments, specifically exclude the unpatentable embodiment(s). Also, the claims appended hereto are to be interpreted both to provide the broadest reasonable scope, as well as to preserve their validity.
• a “subject” or “patient” refers to an animal in need of treatment that can be effected by molecules of the invention.
• Animals that can be treated in accordance with the invention include vertebrates, with mammals such as bovine, canine, equine, feline, ovine, porcine, and primate (including humans and non-humans primates) animals being particularly preferred examples.
• A is -CR 21 R 22 -, -NR 23 -, -O-, or -S- ;
• D and E are together or independently -CR 30 -, or -N-;
• L is -CH 2 -
• Ar is formula (C) or (d)
• n is 1 to 2;
• Ri, R 2 , R3, R11, Ri2, R 2 I; R22, R23 > R24, R ⁇ s, R28, R29, and R30 are independently or together hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogen, cyano, nitro, hydroxyl, acyl, substituted acyl, acyloxy, amino, mono- substituted amino, di-substituted amino, alkylsulfonamide, arylsulfonamide, alkylurea, arylurea, alkylcarbamate, arylcarbamate, heteroaryl, alkoxy, substituted alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, or substituted dialkyl
• the present invention also includes other forms of the compounds of the invention, including prodrug forms.
• a “prodrug” is a compound that contains one or more functional groups that can be removed or modified in vivo to result in a molecule that can exhibit therapeutic utility in vivo.
• a “polymorph” refers to a compound that has an identical chemical composition (i.e., it is of the same compound species) as compared to another compound but that differs in crystal structure.
• the compound is a benzilidine compound having the structural Formula:
• the compound when W is 2,4-thiazolidinedione and " " is absent, the compound is a benzyl compound having the structural Formula:
• R] i and R ⁇ are independently or together hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogen, cyano, nitro, hydroxyl, acyl, substituted acyl, acyloxy, amino, mono-substituted amino, di-substituted amino, alkylsulfonamide, arylsulfonamide, alkylurea, arylurea, alkylcarbamate, arylcarbamate, heteroaryl, alkoxy, substituted alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, or substituted dialkylcarboxamide
• A is -CR 2 iR 22 -, -NR 23 -, -O-, or -S- ;
• B is -OR 24 , -SR25, -NR 28 R29, D and E are together or independently -CR 30 -, or -N-.
• Preferred heterobicyclic residues may be selected from
• compounds of formula (1) disclosed herein may exist in the form of tautomers (1) and (2), which are within the scope of the invention.
• 2, 4-thiazolidinedione-containing compounds disclosed herein may exist in the form of tautomers (XI) ((XII), and (XIII), which are within the scope of the invention.
• both Z and E configurations of the 2, 4-thiazolidinedione-containing compounds disclosed herein are within the scope of the invention both Z and E configurations formula may have the following structures, respectively:
• the compounds disclosed herein may also include salts of the compounds, such as salts with cations.
• Cations with which the compounds of the invention may form pharmaceutically acceptable salts include alkali metals, such as sodium or potassium; alkaline earth metals, such as calcium; and trivalent metals, such as aluminum.
• alkali metals such as sodium or potassium
• alkaline earth metals such as calcium
• trivalent metals such as aluminum.
• the only constraint with respect to the selection of the cation is that it should not unacceptably increase the toxicity. Due to the tautomerism described above for the compounds, mono-, di-, or tri-salts may be possible depending on the corresponding alkali metal.
• one or more compounds disclosed herein may include salts formed by reaction of a nitrogen contained within the compound, such as an amine, aniline, substituted aniline, pyridyl and the like, with an acid, such as HCI, carboxylic acid and the like. Therefore, all possible salt forms in relationship to the tautomers and a salt formed from the reaction between a nitrogen and acid are within the scope of the invention.
• the present invention provides, but is not limited to, the specific compounds set forth in the Examples as well as those set forth below, and a pharmaceutically acceptable salt thereof:
• FIG. 13 A representative scheme of the synthetic pathway in the production of the compounds disclosed herein is shown in FIG. 13.
• Coupling reaction such as that described for the formation of Biaryl (XXIV) may be conducted using boronic esters, such as where R15 together with the boron form a pinacol borate ester (formation of pinacol esters: Ishiyama T., et al.,J. Org. Chem.
• Some embodiments of the invention provide a process for the preparation of a compound of the Formula (XXII):
• A is -CR2 1 R22-, -NR 23 -, -O-, or -S- ;
• D and E are together or independently -CR 3 0-, or -N-;
• Ar is formula (c) or (d)
• Ri, R 2 , R3, R21, R23, R2 4J R25, R 2 8, R 2 9, and R 30 are independently or together hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogen, cyano, nitro, hydroxyl, acyl, substituted acyl, acyloxy, amino, mono-substituted amino, di-substituted amino, alkylsulfonamide, arylsulfonamide, alkylurea, arylurea, alkylcarbamate, arylcarbamate, heteroaryl, alkoxy, substituted alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, or substituted dialkylcarboxamide
• R 1 and R 2 are hydrogen.
• the above product (XXVIII) can be alkylated with alkyl halide in the presence of NaH.
• Formula I which comprises coupling two aromatic rings to give a biaryl wherein one of the aryl rings contains a carbonyl moiety, preferably an aldehyde.
• the resulting biaryl product may be subsequently condensed with an active methylene compound, such as 2,4-thiazolidinedione to give a benzylidene compound of Formula (1) where" " is present.
• the benzylidene compound may be reduced to give a benzyl compound of Formula (1) where "- " is absent.
• Coupling of two aryl rings may be conducted using an aryl boronic acid or esters with an aryl halide (such as, iodo, bromo, or chloro), triflate or diazonium tetrafluoroborate; as described respectively in Suzuki, Pure & Applied Chem., 66:213-222 (1994), Miyaura and Suzuki, Chem. Rev. 95:2457-2483 (1995), Watanabe, Miyaura and Suzuki, Syn- lett. 207-210 (1992), Littke and Fu.Angew. Chem. Int.
• an aryl halide such as, iodo, bromo, or chloro
• triflate or diazonium tetrafluoroborate as described respectively in Suzuki, Pure & Applied Chem., 66:213-222 (1994), Miyaura and Suzuki, Chem. Rev. 95:2457-2483 (1995), Watanabe, Miyaura and Suzuki,
• (X) is either a triflate, a halide (such as, iodo, bromo, or chloro), or diazonium tet- rafluoroborate or hydrogen and Rl 5 is either alkyl or hydrogen.
• a halide such as, iodo, bromo, or chloro
• Rl 5 is either alkyl or hydrogen.
• the coupling groups may be reversed.
• the boronic ester may be prepared from an aryl halide by conversion into the corresponding aryl lithium, followed by treatment with a trialkyl borate.
• the boronic ester is hydrolyzed to the boronic acid.
• the coupling reaction may also be conducted between an arylzinc halide and an aryl halide or triflate. Alternately, the coupling reaction may also be executed using an aryl trialkyltin derivative and an aryl halide or triflate.
• These coupling methods are reviewed by Stanforth, Tetrahedron 65 54:263-303 (1998) and incorporated herein by reference. In general, the utilization of a specific coupling procedure is selected with respect to available precursors, chemoselectivity, regioselectivity and steric considerations.
• biaryl carbonyl containing derivatives e.g., FIG. 13, compound (XXI) with a suitable active methylene compound, such as, 2,4-thiazolidinedione
• a suitable active methylene compound such as, 2,4-thiazolidinedione
• the biaryl carbonyl product from the coupling reaction may be condensed with an active methylene compound to give a benzylidene compound of Formula (I) (i.e., " " is present) as described by Tietze and
• the hydroxyl groups of such intermediates are often eliminated (as water) during the condensation reaction, to form the desired benzylidene compound, Nevertheless, the conditions of the reaction may be modified for the isolation or further use of hydroxyl containing intermediates, and such embodiments are within the scope of the invention.
• the reaction shown above depicts the formation of the condensation intermediate for the reaction between compound (XXI) and an active methylene compound.
• Effective catalysts for the condensation may be selected from ammonia, primary, secondary, and tertiary amines, either as the free base or the amine salt with an organic acid, such as acetic acid. Examples of catalysts include pyrrolidine, piperidine, pyridine, diethylamine, and the acetate salts, thereof.
• Inorganic catalysts may also be used for the condensation.
• Inorganic, catalysts include, but are not limited to, titanium tetrachloride and a tertiary base, such as pyridine; and magnesium oxide or zinc oxide in an inert solvent system.
• This type of condensation can be strongly solvent- dependent and it is understood that routine experimentation may be necessary to identify the optimal solvent with a particular catalyst, preferable solvents include ethanol, tetrahydrofuran, dioxane, or toluene; or mixtures thereof.
• the active methylene compound of the present invention is 2,4- thiazolidinedione.
• the resulting benzylidene e.g., FIG. 13, compound (XXII) may be reduced, if desired, to a compound of Formula (I) wherein " — "is absent (e.g., FIG. 13, compound (XXIII).
• the compounds of the present invention have been found to be potent compounds in a number of biological assays, both in vitro and in vivo, that correlate to, or are representative of, human diseases.
• the compounds that inhibit FLT-3 kinase are cytotoxic to leukemic cells.
• Compound 1 has a specific kinase inhibition profile when tested against approximately 180 different kinases including mutated kinases as for instance in the case of FLT3 and KIT. Only six of all the kinases were inhibited with inhibition constants (or binding constants Kd ) in the sub micro molar range (see Figure 1), as measured using the Ambit kinase screening test ( M.A.
• Results shown in Figure 4 demonstrate that when tested against approximately 180 kinases, using the Ambit test, compound 2 inhibits only 3 different kinases and some of their mutated forms at or near micro molar concentrations.
• a single oral dose of 10 mg/Kg of compound 2 was given to three rats. Blood samples were removed at various time points for analysis of drug levels.
• the single oral dose of compound 2 produced plasma drug concentration of more than 2 DM within 2 hours and reached a maximal concentration (C max ) of 3 DM within 10 hours and an overall peak that extended over 24 hours ( Figure 5).
• C max maximal concentration
• Figure 5 The maintenance of high plasma drug levels for several hours and good bioavailability suggest that compound 2 could be administered orally once a day.
• a subcutaneous tumor xenograft model was used to assess the effects of compound 2 in vivo.
• Athymic nude mice injected with cells expressing constitutively activated Flt-3 is a proven model of leukemia (O'Farrell A-M, Abrams TJ, Yuen HA, Ngail TJ, Louie SG, Yee KWH, Wong LM, Hong W, Lee LB, Town A, Smolich BD, Manning WC, Murray LJ, Heinrich MC and Cherrington JM (2003) SUl 1248 is a novel Flt-3 tyrosine kinase inhibitor with potent activity in vitro and in vivo. Blood 101 : 3597-3605).
• mice typically sicken and die, with large spleens full of leukemic cells, within a few weeks following injection.
• MV4;11 a human leukemia cell lines that expresses a Flt-3 -internal tandem repeat mutation were harvested during exponential growth and were resuspended in matrigel (BD Biosciences, Bedford, MA).
• Athymic nude mice were injected with 5 x 10 6 MV4; 11 cells near the hind flank on day 0. The therapeutic effects of daily oral administration of Compound 2 were evaluated in two different experiments.
• mice were injected subcutaneously with MV4:11 cells. Two weeks after tumor initiation, and when tumors were established in the animals, one group of mice was treated with 30 mg/kg of compound 2 administered orally once a day for 8 and 14 days, a second group was treated with placebo. Tumor volumes were measured twice/week using a vernier caliper for the duration of treatment and volumes were calculated as ellipsoid volumes (Tomayko MM 5 Reynolds CP (1989) Determination of subcutaneous tumor size in athymic nude mice. Cancer Chemother Pharmacol 24(3): 148-54) determined from the size of the subcutaneous tumors grown as xenografts. Bars represent five animals/group.
• mice were injected subcutaneously with about 5,000,000 MV4: 1 1 cells. After 3 weeks, when the tumors had reached a size of approximately 100 mm3, mice were divided into 2 groups (6 animals/group). One group was treated with 50 mg/kg of compound 2 administered orally once a day for two weeks, the other group was given placebo. Tumors were then grouped as non-detectable/regressed, static or progressed based on the tumor sizes. Tumors in placebo treated mice progressed to a size of 1000 mm3 within two weeks, whereas 66% of the mice that were treated with compound 2 (50 mg/kg) showed an effect on the tumor size. Of those mice that responded, 50% showed tumors that were static and in the remaining 50% tumors were completely eliminated (Figure 7). Compound 5 selectively killed MV4;11 leukemic cells but not prostate and pancreatic cells.
• Another aspect of the invention relates to the use of the compounds described herein.
• These compounds may be either used singularly or plurally, and pharmaceutical compositions thereof for the treatment of mammalian diseases, particularly those that occur in humans.
• Compounds described herein and compositions that contain them may be administered by various methods including, for example, orally, enterally, parentally, topically, nasally, vaginally, ophthalinically, sublingually, or by inhalation for the treatment of leukemic and other cancers including AML, CML, and solid tumors.
• Routes of administration and dosages known in the art may be found in comprehensive: medicinal chemistry, volume S, Hanscb, C. Pergamon Press, 1990; incorporated herein by reference.
• the compositions may also be used as regulator in diseases of uncontrolled proliferation.
• a representative but non-limiting list of cancers is lymphoma, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, head and neck cancer, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, myeloma, neuroblastoma glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, colon cancer, cervical carcinoma, breast cancer, and epithelial cancer.
• the compounds described herein may be administered as pure chemicals, it is preferable to present the active ingredient as a pharmaceutical composition.
• another aspect of the invention concerns the use of a pharmaceutical composition
• a pharmaceutical composition comprising one or more compounds and/or a pharmaceutically acceptable salt thereof, together without more pharmaceutically acceptable carriers thereof and, optionally, other therapeutic and/or prophylactic ingredients.
• the carrier(s) must be 'acceptable' in the sense of being compatible with the other ingredients of the composition and not overly deleterious to the recipient thereof.
• compositions include those suitable for oral, enteral, parental (including intramuscular, subcutaneous and intravenous), topical, transmucosally, nasal, vaginal, ophthalinical, sublingually, bucally, or by inhalation administration.
• the compositions may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with liquid carriers, solid matrices, semi- solid carrier, finely divided solid carrier or combination thereof, and then, if necessary, shaping the product into the desired delivery system.
• compositions suitable for oral administration may be presented in a discrete unit dosage form, such as hard or soft gelatin capsules, cachets, or tablets each containing a predetermined amount of the active ingredient; as a powder or as granules; or as a solution, suspension, or emulsion.
• the active ingredient may also be presented as a bolus, electuary, or paste.
• Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents.
• the tablets may be coated according to methods well known in the art, e.g., with enteric coatings.
• Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solution, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
• Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or one or more preservative.
• the compounds may also be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small bolus infusion containers or in multi-does containers with an added preservative.
• the composition may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
• the com- pounds may be formulated as ointments, creams or lotion, or as the active ingredient of a transdermal patch.
• Suitable transdermal delivery systems are disclosed, for example, in Fisher et al. (U.S. Pat. No. 4,788,603, incorporated herein by reference) or Bawas et al. (U.S. Pat. Nos. 4,931,279, 4,668,504 and 4,713,224; all incorporated herein by reference).
• Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition or suitable thickening and/or gelling agents.
• Lotion may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
• the active ingredient may also be delivered via iontophoresis, e.g., as disclosed in U.S. Pat. Nos. 4,140,122, 4383,529, or 4,051,842; incorporated herein by reference.
• compositions suitable for topical administration in the mouth include unit dosage forms such as lozenges comprising active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; mucoadherent gels, and mouthwashes comprising the active ingredient in a suitable liquid carrier.
• unit dosage forms such as lozenges comprising active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; mucoadherent gels, and mouthwashes comprising the active ingredient in a suitable liquid carrier.
• the above-described compositions may be adapted to provide sustained release of the active ingredient employed, e.g., by combination thereof with certain hydrophilic polymer matrices, e.g., comprising natural gels, synthetic
• the agent is part of composition, which includes at least a carrier and an amount of such compound sufficient to achieve the desired effect for which it is to be administered in at least a subset of the population of subjects to whom it is administered (i.e., an "effective amount" of the particular compound or other active agent, as the case may be).
• an "effective amount” of the particular compound or other active agent i.e., an "effective amount” of the particular compound or other active agent, as the case may be.
• a “therapeutically effective amount” refers to an amount of an agent sufficient to effect treatment when administered to a subject in need of such treatment.
• a “therapeutically effective amount” is one that produces an objective response in evaluable patients. Determination of therapeutically effective dosages of a composition comprising a compound according to the invention may be readily made by those of ordinary skill in the art.
• the amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
• a model organism such as an athymic nude mouse
• extrapolations are not simply based on the weights of the two organisms, but rather incorporate differences in metabolism, differences in pharmacological delivery, and administrative routes.
• a suitable dose will, in alternative embodiments, typically be in the range of from about 0.5 to about 100 mg/kg/day, from about 1 to about 75 mg/kg of body weight per day, from about 3 to about 50 mg per kilogram body weight of the recipient per day, or in the range of 6 to 90 mg/kg/day.
• the compound is conveniently administered in unit dosage form, for example, in alternative embodiments, containing 0.5 to 5000 mg, 5 to 750 mg, most conveniently, or 10 to 500 mg of active ingredient per unit dosage form.
• the active ingredient may be administered to achieve peak plasma concentrations of the active compound of from about 0.5 to about 75 DM, about: 1 to 50 DM, or about 2 to about 30 DM. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 0.5-500 mg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the active ingredients.
• the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
• the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
• treatment or “treating” means any treatment of a disease or disorder, including preventing or protecting against the disease or disorder (that is, causing the clinical symptoms not to develop); inhibiting the disease or disorder (i.e., arresting or suppressing the development of clinical symptoms; and/or relieving the disease or disorder (i.e., causing the regression of clinical symptoms).
• Example 1 Representative compounds and their synthesis
• the intermediate 6-(2-Ethylamino-7-isopropoxy-benzooxazol-5-yl)-pyridine-3- carbaldehyde was prepared as follows: The mixture of 6-(2-Amino-7-isopropoxy-benzoxazol-5-yl)-pyridine-3-carbaldehyde (0.860 g, 2.893 mmol) and K 2 CO 3 (0.960 g, 6.942 mmol) in DMF 20 ml) was stirred at room temperature for 1 1/2 hours, lodoethane (278 Dl, 3.472 mmol) was added. The reaction was stirred at room temperature for 3 days.
• aqueous phase was brought to pH ⁇ 1 with saturated NaHCO 3 and extracted from ethyl acetate (15 ml).
• Organic phases combined and washed with H 2 O (50 ml), brine (50 ml), dried over anhydrous MgSO 4 , filtered and evaporated.
• the residue was chromatographed on silica gel (eluent: ethyl acetate/ hexane, 4: 1) to give 1.118g of 5-(5-Diethoxymethyl-pyridin-2-yl)-7-isopropoxy-benzoxazol-2- ylamine (81% yield).
• the intermediate 2-amino-4- (5-[l,3] dioxolan-2-yl-pyridin-2-yl)-6-isopropoxy-phenol was prepared as follows: To a mixture of 4-(5-[l,3] dioxolan-2-yl-pyridin-2-yl)-2-isopropoxy-6-nitro-phenol (1.596g,
• the completed reaction mixture cooled to 23 0 C and brought to pH ⁇ 7 with a solution of 10% K 2 CO3 then diluted with ethyl acetate (30 ml), and H 2 O (20 ml).
• the aqueous phase was separated and extracted with ethyl acetate (3 x 15 ml).
• Organic phases combined and washed with H 2 O (30 ml), brine (30 ml), dried over anhydrous MgS ⁇ 4 , filtered and evaporated.
• the intermediate 6-(4-Hydroxy-3-isopropoxy-phenyl)-pyridine-3-carbaldehyde was prepared as follows: To a mixture of 6-[4-(tert-Butyl-dimethyl-silanyloxy)-3-isopropoxy-phenyl]-pyridine-3- carbaldehyde (3.Og, 8.07 mmol), dissolved into anhydrous THF (60ml) was added Tetrabutylammonium fluoride (1.0M in THF, 9.69 ml, 9.69 mmol) drop-wise. The resulting mixture was then stirred at 23 0 C under argon for 1.5 hrs.
• the intermediate (4-Bromo-2-isopropoxy-phenoxy)-tert-butyl-dimethyl-silane was prepared as follows: Tert-butyl-dimethyl silylchloride (21.4 g, 0.142 mol), 4-N-dimethylamininopyridine (0.461 g, 0.004 mol) and triethylamine (19.8 ml, 0.142 mol) were added to a solution of 4-Bromo-2- isopropoxy-phenol (23.5 g, 0.102 mol) in 160 ml DMF. The resulting mixture was stirred for 17 hrs at room temperature.
• reaction mixture was diluted with ethyl acetate (200 ml) and washed with water (150 ml), brine (150 ml) and dried over anhydrous magnesium sulfate. After filtering off magnesium sulfate, the filtrate was evaporated. The residue was chromatographed on silica gel (eluent: hexane) to give 32.3 g of (4-Bromo-2-isopropoxy-phenoxy)-tert-butyl- dimethyl-silane.
• the intermediate 4-Bromo-2-isopropoxy-phenol was prepared as follows: To a suspension of pyridinium tribromide (1 16g, 0.362 mol) in 250 ml dichloromethane, was added 2-isopropoxyphenol (50 g, 0.329 mol) in 150 ml dichloromethane. The mixture was stirred at room temperature for 6 hrs. The reaction mixture was quenched with aqueous HCl (IN, 200ml). After separation, the organic phase was washed with saturated sodium thiosulfate (150 ml), and brine (150 ml), and dried over anhydrous magnesium sulfate. After filtering off magnesium sulfate, the filtrate was evaporated to give 71g 4-Bromo-2-isopropoxy-phenol (94% yield).
• Example 2 Kinase Inhibition Profile Assay As a primary screen, compounds were tested at 10 uM against a panel of 180 kinases. This collection of kinases included the most common FLT-3 and cKit mutations. Kinase assays and binding constant measurements were done as described in Fabian et al (A small molecule-kinase interaction map for clinical kinase inhibitors. Nature Biotechnology 23:329-36 (2005). Briefly, the human kinases are expressed as T7 bacteriphage fusion protein and a small set of immobilized probe ligand is combined with the free test compounds.
• Example 3 Cellular proliferation inhibition assays The biological response of FLT-3 inhibition was tested in a cell proliferation assay with the human AML cell line MV4;1 1. This cell line carries the most frequent human FLT-3 mutation. The selectivity of cell killing was tested by comparing the effect of compounds on the MV4;11 cells with their effects on proliferation of the prostate cancer cell line (PC-3) and pancreatic cancer cell line (Bx-PC3) cells. Cell proliferation was measured using 3-4,5-dimethylthiazol- 2,5-diphenyltetrazoilum (MTT) assay. Briefly, cells were aliquoted into 96 well plates.
• MTT 3-4,5-dimethylthiazol- 2,5-diphenyltetrazoilum
• MV4;11 cells were grown in RPMI medium containing 4500 mg/L glucose; 4 mM L-glutamine; 10 U/ml Pen-G; 10 mcg/ml and 20% fetal bovine serum (FBS).
• PC-3 and Bx-PC3 cells were grown in RPMI medium 1640 containing 2 mM L-glutamine; 10 U/ml Pen-G; 10 mcg/ml Streptomycin and 10% FBS.
• Cells were seeded at 300-500 cells/well in 96-well tissue culture plates and maintained at 6% CO 2 and 37 0 C. Cells were treated with kinase inhibitors or vehicle for three days. The percentage of surviving cells was then measured colorimetrically.
• the assay is based on the cleavage of the yellow tetrazolium salt MTT to purple formazan crystals by dehydrogenase activity in active mitochondria. Therefore, this conversion only occurs in living cells with intact/functional mitochondria.
• the formazan crystals formed are solubilized and the resulting colored solution is quantified using a scanning multiwell spectrophotometer at 595 nm. Briefly, 10 Dl of 5 mg/ml MTT dye are added to each well and incubated for 4 hours and the reaction was stopped by adding 100 G I/well of solubilization solution, consisting of 10% Sodium Dodecyl Sulfate (SDS) and 10 mM HCl].
• SDS Sodium Dodecyl Sulfate
• Compound 1-5 strongly inhibited cellular proliferation and killed MV4;11 cells in a dose dependent manner and compound 1 did not inhibit the proliferation of PC-3 and Bx-PC-3 cancer cell lines ( Figure 2 and 3).
• a single dose of compound 5 when given at 12 mg/Kg produced plasma drug concentrations of more than 10 DM within 1 hour and reached a Cmax of 14 DM within 3 hours and an overall peak that extended over 10 hours. Overall, compound 5 showed approximately 3 to 4 fold increased bioavailability over compound 2 (Figure 9).
• Example 5 Oral administration of compound 2 and compound 5 in the treatment of AML tumors in an animal model
• a subcutaneous tumor xenograft model was used to assess the effects of compounds 2 and 5 in vivo.
• MV4;11 cells a human leukemia cell lines that expresses the most frequent FLT- 3 -ITD mutation
• Matrigel BD Biosciences, Bedford, MA.
• Athymic nude mice were injected with 5 million MV4;11 cells near the hind flank on day 0.
• the therapeutic effects of daily oral administration of compounds 2 and 5 were evaluated in prevention of AML tumors and the treatment of large pre- existing tumors in the athymic mouse.
• nude mice were injected subcutaneously with 5 million MV4:11 cells.
• Two weeks after tumor injection one group of mice was treated with 30 mg/kg of compound 2 administered orally once a day for 8 and 14 days (Figure 6).
• Tumor volumes were measured twice/week using vernier caliper for the duration of treatment, and volumes were calculated as ellipsoid volumes (Tomayko MM and Reynolds CP 1989 Determination of subcutaneous tumor size in athymic nude mice. Cancer Chemother Pharmacol 124: 148-54). Bars represent 5 animals/group.
• Compound 2 dramatically inhibited the growth of FLT3- ITD mutation xenografts at 30 mg/Kg.
• mice were injected subcutaneously with 5 million MV4:11 cells. After the tumors had reached a size of approximately 100 mm3, mice were divided into 2 groups (6 animals/group). One group was treated with 50 mg/kg of compound 2, the other group was treated with placebo, administered orally once a day for two weeks. Tumors were then grouped as non-detectable/regressed, static or progressed based on the tumor sizes in placebo treated group ( Figure 7). Tumors in placebo treated mice progressed to a size of 1000 mm3 within two weeks, whereas, 66% of the mice that were treated with MC-2002 (50 mg/kg) showed an effect on the tumor size. Of those mice that responded, 50% showed tumors that were static and in the remaining 50% tumors were completely eliminated (data not shown).
• Compound 5 was administered orally twice a day at 15 mg/Kg to nude mice injected with MV4; 11 cells. Eighteen days after tumor initiation animals were sorted based on tumor sizes into two groups (7-9 animals per group) and treated with vehicle or 15 mg/kg of compound 5. Tumor sizes were measured at the start of treatment and after 5 and 10 days. Animals treated with 15 mg/Kg of compound 5 showed a significant inhibition of the growth of AML xenografts ( Figure 10).

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