WO2015126808A2

WO2015126808A2 - Hbv assay

Info

Publication number: WO2015126808A2
Application number: PCT/US2015/016110
Authority: WO
Inventors: John J. Baldwin
Original assignee: Baldwin John J
Priority date: 2014-02-18
Filing date: 2015-02-17
Publication date: 2015-08-27
Also published as: WO2015126808A3

Abstract

This invention relates to the discovery that members of the pyrrolotriazinyl class of IGF-1R kinase inhibitors also are inhibitors of the NCTP transporter and block taurocholic acid entry into hepatocytes. An IGF-1R kinase inhibitor such as described in U.S.P. 7,534,792, especially BMS-754807, blocks the NTCP transporter and is therefore useful in preventing Hepatitis B disease, cirrhosis, and hepatic cellular cancer. This preferred compound is (S)-1-(4-(5-cyclopropyl-1H-pyrazol-3-ylamino)pyrrolo[1,2-f][1,2,4]triazin-2-yl)-N-(6-fluoropyridin-3-yl)-2-methylpyrrolidine-2-carboxamide.

Description

HBV ASSAY

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and takes priority to U.S. Application 61/966,188 filed February 18, 2014, the contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Hepatitis B (HB) is a major worldwide health problem. The causative, infective agent is the Hepatitis B virus (HBV). The virus is composed of a viral core surrounded by a surface coat. This core contains circular double stranded DNA and the enzyme DNA polymerase. The envelope is composed of three proteins named L, M, and S. The virus replicates within the nucleus of an infected hepatocyte yielding core particles, with the surface coat being added in the cytoplasm. The coat proteins are generated in excess and can be detected in the serum of patients by immunologic methods as the Hepatitis B surface antigen.

The viral disease is spread by multiple vectors, including food, waterborne exposure, person-to-person contact, drug needles, and mother-to- child transmission. It has been estimated that about 10% of acute cases of HBV infection result in chronic hepatitis. The disease has infected 2 billion people. Approximately 350 million people are chronic carriers who may progress to cirrhosis and hepatic cellular carcinoma.

The current treatment for Hepatitis B infection is interferon alpha and

nucleos(t)ide polymerase inhibitors such as entecavir and adefovir, and the non- nucleos(t)ide polymerase inhibitors (NNTI) such as Lamivudine. Prolonged treatment periods, side effects, and resistance seen with current therapy suggest that new and superior agents targeting different steps in the virus life cycle are needed to treat this infective disease. (See: Taylor J, WJG 28, 19, 6730 (2013), Merck Index 17th Ed., p 377, and Hu Y, Ann. Reports of Med.Chem., 48, 265, (2013).)

One possible approach is through an effect on viral entry. It has been shown that the pre-Sl domain of the coat L protein is important for infectivity. Based on the structure of this recognition unit, a peptide, Myrelodex, was designed. Pretreatment with this peptide has been shown to prevent viral entry in vitro. It appears that this recognition unit of the viral coat protein may interact with a specific element on the hepatocyte as the initiation step for viral entry. It has been suggested that cyclosporine, an

immunosuppressant, may influence HBV inactivity through multiple mechanisms, one of which may be through blockade of the taurocholate co-transporting peptide NTCP. This Na⁺-dependent bile acid transporter is critical in the movement of bile acids from the portal blood into the liver.

Transporters are membrane proteins that are key determinants of drug entry and distribution. The function of the enterohepatic cycle depends on NTCP for the movement of bile salts from the blood into the liver. The NTCP protein is composed of 335 amino acids arranged into nine trans-membrane domains. This transporter is classified as a member of the SLC super family (Transporter Consortium, Nature Rev., 9,215, (2010)).

SUMMARY OF THE INVENTION

It has now been discovered that compounds of the present invention have the ability to inhibit the inflow of taurocholic acid into hepatocytes through inhibition of the NTCP transporter and are therefore useful in blocking the entry of HBV into liver cells, thereby preventing infection by the virus and resulting hepatitis B disease. DETAILED DESCRIPTION

One embodiment of the invention is a method of selecting compounds in the molecular weight range of about 400 to 650 daltons that are placed in class 3 or 4 of the Biopharmaceutics classification system (BCS) and the Biopharmaceutics Drug

Disposition classification system (BDDES) based on solubility and permeability.

Another embodiment of the invention is a strategy for the identification of anti-

HBV drugs through targeting the interaction of the virus with the taurocholic acid portal, the goal being to identify and develop orally available drugs through an in vitro assay that measures the inhibition of taurocholic acid transport into the hepatocyte and therefore prevent HBV entry Such an assay must express the physiologically relevant transporter NTCP and provide for a measurement of taurocholic acid uptake alone and in the presence of an inhibitor. A further embodiment is to provide a method to evaluate the transporter's function in hepatocytes under specified culture conditions such as that described in Liu X, Am. J. Physiol. 277, G12-G21 (1999). This assay, the uptake assay, uses fresh or cryopreserved human, rat, chimpanzee, or tree schrew (Tupaia belangeri) hepatocytes and allows for a measurement of taurocholic acid uptake in the presence of an experimental compound. This provide the information needed for the calculation of an IC50 for the compound of interest.

An additional embodiment is to provide random screening or selective structural analysis followed by testing using the uptake inhibition assay with suspension or plated hepatocytes as a convenient method to search for compounds that inhibit the function of the NCTP transporter. In this case the structural analysis of potential inhibitors provides a select group of drug-like compounds for testing versus taurocholic acid transport in both the suspension and plated hepatocyte uptake assays.

This invention relates to the discovery of novel NTCP inhibitors.

This invention relates to the discovery of NTCP inhibitors within the insulin- like growth factor type 1 receptor (IGF-1R) antagonist class.

This invention relates to the discovery that members of the pyrrolotriazinyl class of IGF-IR kinase inhibitors also are inhibitors of the NTCP transporter and block taurocholic acid entry into hepatocytes.

Another embodiment of the invention is that an IGF-IR kinase inhibitor such as defined as formula I in U.S.P. 7,534,792, especially BMS-754807, blocks the NTCP transporter and is therefore useful in preventing Hepatitis B disease, cirrhosis, and hepatic cellular cancer. This preferred compound is (S)-l-(4-(5-cyclopropyl-lH-pyrazol-3- ylamino)pyrrolo [ 1 ,2-f] [ 1 ,2,4]triazin-2-yl)-N-(6-fluoropyridin-3 -yl)-2-methylpyrrolidine- 2-carboxamide.

Another embodiment of the invention is that the use of said IGF-IR inhibitors (especially BMS-754807) in the treatment of HBV infection.

Another embodiment of the invention is that the combination of said IGF-IR inhibitors with other antiviral agents, especially those that operate by a different mechanism and work synergistically with the viral entry blockers, especially interferon alpha, HBV ccc DNA transcription modulators, ccc DNA biosynthesis inhibitors, natural inhibitors such as magnolol and swertia mileensis extract, and HBV capsid assembly inhibitors.

The compounds of formula I are as follows:

wherein

Q¹ is aryl, substituted aryl, heteroaryl, or substituted heteroaryl; X is CO, C=S, C=NR⁹, or CH₂;

1 2 3

R , R , and R are independently hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, hydroxy, alkoxy, substituted alkoxy, halogen, haloalkyl, haloalkoxy, alkanoyl, substituted alkanoyl, amino, substituted amino, aminoalkyl, substituted aminoalkyl, alkylamino, substituted alkylamino, amide, substituted amide, carbamate, ureido, cyano, sulfonamido, substituted sulfonamido, alkylsulfone, nitro, thio, thioalkyl, alkylthio, disubstituted amino, alkylsulfonyl, alkylsulfinyl, carboxy, alkoxycarbonyl, alkylcarbonyloxy, carbamoyl, substituted carbamoyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, or alkylcarbonyl;

4

R is hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, halogen, haloalkyl, haloalkoxy, oxo, aryloxy, arylalkyl, arylalkyloxy, alkanoyl, substituted alkanoyl, alkanoyloxy, amino, substituted amino, aminoalkyl, substituted aminoalkyl, alkylamino, substituted alkylamino, hydroxyalkyl, disubstituted amino, amide, substituted amide, carbamate, substituted carbamate, ureido, cyano, sulfonamide, substituted sulfonamide, alkylsulfone, heterocycloalkyl, substituted heterocycloalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, cycloalkylalkyl, cycloalkylalkoxy, nitro, thio, thioalkyl, alkylthio, alkylsulfonyl, alkylsulfinyl, carboxy, alkoxycarbonyl,

alkylcarbonyloxy, carbamoyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heteroaryloxy, arylheteroaryl, arylalkoxycarbonyl, heteroarylalkyl, heteroarylalkoxy, aryloxyalkyl, aryloxyaryl, heterocycle, substituted heterocycle, alkylcarbonyl, substituted heteroalkyl,

heteroalkenyl, substituted heteroalkenyl, heteroalkynyl, substituted heteroalkynyl, arylamino, arylalkylamino, alkanoylamino, aroylamino, arylalkanoylamino,

arylthio, arylalkylthio, arylsulfonyl, arylalkylsulfonyl, alkylsulfonyl, arylcarbonylamino, or alkylaminocarbonyl;

R⁵ is hydrogen, halogen, cyano, alkyl, or substituted alkyl;

R⁶ is independently hydrogen, alkyl, substituted alkyl, alkylidene, substituted alkylidene, hydroxy, alkoxy, halogen, haloalkyl, haloalkoxy, oxo, aryloxy, arylalkyl, arylalkyloxy, alkanoyl, substituted alkanoyl, alkanoyloxy, amino, aminoalkyl, substituted aminoalkyl, alkylamino, substituted alkylamino, hydroxyalkyl, disubstituted amino, amide, substituted amide, carbamate, substituted carbamate, ureido, cyano, sulfonamide, substituted sulfonamide, alkylsulfone, heterocycloalkyl, substituted heterocycloalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,

cycloalkylalkyl, cycloalkylalkoxy, nitro, thio, thioalkyl, alkylthio, alkylsulfonyl, alkylsul?nyl, carboxy, alkoxycarbonyl, alkylcarbonyloxy, carbamoyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heteroaryloxy, arylheteroaryl, arylalkoxycarbonyl, heteroarylalkyl, heteroarylalkoxy, aryloxyalkyl, aryloxyaryl, heterocycle, substituted heterocycle, alkylcarbonyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroalkynyl, substituted heteroalkynyl, arylamino, arylalkylamino, alkanoylamino, aroylamino, arylalkanoylamino, arylthio, arylalkylthio, arylsulfonyl, arylalkylsulfonyl, alkylsulfonyl, arylcarbonylamino, or alkylaminocarbonyl; n is 0, 1,2, 3, 4, 5, or 6; or when n is 2 and R are geminal substituents, they may together form an optionally substituted 3-6 membered saturated or unsaturated carbocyclic or heterocyclic ring; or when n is 2 and R⁶ are 1,2-cis substituents, they may together form an optionally substituted 3-6 membered fused saturated carbocyclic or heterocyclic ring; or when n is 2 and R⁶ are 1 ,3-cis substituents they may together form an optionally substituted 1-4 membered alkyl or heteroalkyl bridge; or when there are two R⁶'s on the same carbon, they may together form a carbonyl (C=0) or alkylidene group (C=CHR );

7 8

R and R are independently hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycloalkyl, substituted heterocycloalkyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroalkynyl, or substituted heteroalkynyl, or

7 8

R and R may be taken together to form an optionally substituted monocyclic 4-8 membered saturated or unsaturated carbocyclic or heterocyclic ring, or

an optionally substituted bicyclic 7-12 membered saturated or unsaturated carbocyclic or heterocyclic ring;

R is hydrogen or lower alkyl;

or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

In an another embodiment of the invention, the compounds of formula I are those wherein, independently,

R¹, R², R³, and R⁴ is each H;

Q¹ is aryl or heteroaryl;

R⁵, R⁶, R⁷, R⁸, and R⁹ is each independently H or lower alkyl; and

X is CH₂, C=0, or C=NR⁹;

or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

The compounds of the invention possess activity to prevent infection when a person is exposed to HBV or to treat such infections at early or later stages of the disease. Thus, another embodiment of the invention is a method of preventing HBV infection in a mammal which comprises administering to a mammal in need thereof, a therapeutically effective amount of one or more compounds of formula I. Terms used herein have the following meanings.

"Alkyl" refers to straight or branched chain unsubstituted hydrocarbon groups of 1 to 20 carbon atoms, preferably 1 to 7 carbon atoms.

"Lower alkyl" refers to unsubstituted alkyl groups of 1 to 4 carbon atoms.

"Substituted alkyl" refers to an alkyl group substituted by one to four substituents, such as, halo, hydroxy, alkoxy, oxo, alkanoyl, aryloxy, alkanoyloxy, amino, alkylamino, arylamino, arylalkylamino, disubstituted amino in which the 2 amino substituents are selected from alkyl, aryl or arylalkyl, alkanoylamino, aroylamino, aralkanoylamino, substituted alkanoylamino, substituted arylamino, substituted aralkanoylamino, thiol, alkylthio, arylthio, arylalkylthio, alkylthiono, arylthiono, arylalkylthiono, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, sulfonamido, substituted sulfonamido, nitro, cyano, carboxy, carbamyl, substituted carbamyl (e.g., CONHalkyl, CONHaryl, CONHarylalkyl, or cases where there are two substituents on the nitrogen are selected from alkyl, aryl or arylalkyl), alkoxycarbonyl, aryl, substituted aryl, guanidino, heterocyclyl, (e.g., indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, homopiperazinyl, and the like), and substituted heterocyclyl. Where a substituent is further substituted it may be with alkyl, alkoxy, aryl or arylalkyl.

"Halogen" or "halo" refers to fluorine, chlorine, bromine, and iodine.

"Aryl" refers to monocyclic or bicyclic aromatic hydrocarbon groups having 6 to

12 carbon atoms in the ring, such as phenyl, naphthyl, biphenyl, and diphenyl, each of which may be substituted.

"Aaryloxy", "arylamino", "arylalkylamino", "arylthio", "arylalkanoylamino", "arylsulfonyl", "arylalkoxy", "arylsulfmyl", "arylheteroaryl", "arylalkylthio",

"arylcarbonyl", "arylalkenyl", and "arylalkylsulfonyl" refer to an aryl or substituted aryl bonded to oxygen; amino; alkylamino; thio; alkanoylamino; sulfonyl; alkoxy; sulfmyl; heteroaryl or substituted heteroaryl; alkylthio; carbonyl; alkenyl; or alkylsulfonyl, respectively.

"Arylsulfonylaminocarbonyl" refers to an arylsulfonyl bonded to an

aminocarbonyl. "Aryloxyalkyl", "aryloxycarbonyl", or "aryloxyaryl" refer to an aryloxy bonded to an alkyl or substituted alkyl; a carbonyl; or an aryl or substituted aryl, respectively.

"Arylalkyl" refers to an alkyl or substituted alkyl in which at least one of the hydrogen atoms bonded to at least one of the carbon atoms is replaced with an aryl or substituted aryl. Typical arylalkyls include, but are not limited to benzyl, 2-phenylethan- 1-yl, 2-phenylethen-l-yl, naphthylmethyl, 2-naphthylethan-l-yl, 2-naphthylethen-l-yl, naphthobenzyl, and 2-naphthophenylethan-l-yl.

"Arylalkyloxy" refers to an arylalkyl bonded through an oxygen linkage (-0- arylalkyl).

"Substituted aryl" refers to an aryl group substituted by, for example, one to four substituents such as alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, arylalkyl, halo, trifluoromethoxy,

trifluoromethyl, hydroxy, alkoxy, alkanoyl, alkanoyloxy, aryloxy, arylalkyloxy, amino, alkylamino, arylamino, arylalkylamino, dialkylamino, alkanoylamino, thiol, alkylthio, ureido, nitro, cyano, carboxy, carboxyalkyl, carbamyl, alkoxycarbonyl, alkylthiono, arylthiono, arylsulfonylamine, sulfonic acid, alkylsulfonyl, sulfonamido, aryloxy, and the like. The substituent may be further substituted by hydroxy, halo, alkyl, alkoxy, alkenyl, alkynyl, aryl, or arylalkyl.

"Heteroaryl" refers to an optionally substituted aromatic group which is a 4 to 7 membered monocyclic, a 7 to 11 membered bicyclic, or a 10 to 15 membered tricyclic ring system, which has at least one heteroatom and at least one carbon atom-containing ring, for example, pyridine, tetrazole, indazole.

"Alkenyl" refers to straight or branched chain hydrocarbon groups of 2 to 20 carbon atoms, preferably 2 to 15 carbon atoms, and most preferably 2 to 8 carbon atoms, having one to four double bonds.

"Substituted alkenyl" refers to an alkenyl group substituted by one to two substituents, such as, halo, hydroxy, alkoxy, alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino, alkanoylamino, thiol, alkylthio, alkylthiono, alkylsulfonyl, sulfonamido, nitro, cyano, carboxy, carbamyl, substituted carbamyl, guanidino, indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl, and the like. "Alkynyl" refers to straight or branched chain hydrocarbon groups of 2 to 20 carbon atoms, preferably 2 to 15 carbon atoms, and most preferably 2 to 8 carbon atoms, having one to four triple bonds.

"Substituted alkynyl" refers to an alkynyl group substituted by halo, hydroxy, alkoxy, alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino, alkanoylamino, thiol, alkylthio, alkylthiono, alkylsulfonyl, sulfonamido, nitro, cyano, carboxy, carbamyl, substituted carbamyl, guanidino and heterocyclyl, e.g. imidazolyl, furyl, thienyl, thiaZolyl, pyrrolidyl, pyridyl, pyrimidyl and the like.

"Alkylidene" refers to an alkylene group consisting of at least two carbon atoms and at least one carbon-carbon double bond. Substituents on this group include those in the definition of "substituted alkyl".

"Cycloalkyl" refers to an optionally substituted, saturated cyclic hydrocarbon ring systems, preferably containing 1 to 3 rings and 3 to 7 carbons per ring which may be further fused with an unsaturated C3-C7 carbocylic ring. Exemplary groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, and adamantyl. Exemplary substituents include one or more alkyl groups or one or more groups described herein as alkyl substituents.

"Heterocycle", "heterocyclic", and "heterocyclyl" refer to an optionally substituted, fully saturated or unsaturated, aromatic or non-aromatic cyclic group, which is a 4 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 15 membered tricyclic ring system which has at least one heteroatom in at least one carbon atom- containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, and sulfur, where the nitrogen and sulfur heteroatoms may also optionally be oxidized and the nitrogen heteroatoms may also optionally be quatemized. The heterocyclic group may be attached at any

heteroatom or carbon atom.

Exemplary monocyclic heterocyclic groups include pyrrolidinyl, pyrrolyl, indolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydro furyl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2- oxopiperazinyl, 2-oxopiperidinyl, homopiperazinyl, 2-oxohomopiperazinyl, 2- oxopyrrolidinyl, 2-oxazepinyl, azepinyl, 4-piperidonyl, pyridyl, N-oxo-pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinylsulfone, 1,3-dioxolane, tetrahydro-1,1- dioxothienyl, dioxanyl, isothiazolidinyl, thietanyl, thiiranyl, triazinyl, and triazolyl, and the like.

Exemplary bicyclic heterocyclic groups include 2,3-dihydro-2-oxo-lH-indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinuclidinyl, quinolinyl, quinolinyl-N- oxide, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,l-b]pyridinyl] furo[2,3-b]pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl), benzisothiazolyl, benzisoxazolyl, benzodiazinyl, benzofurazanyl, benzothiopyranyl, benzotriazolyl, benzopyrazolyl, 1,3-benzodioxolyl, dihydrobenzofuryl,

dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, dihydrobenzopyranyl, indolinyl, indazolyl, isochromanyl, isoindolinyl, naphthyridinyl, phthalazinyl, piperonyl, purinyl, pyridopyridyl, pyrrolotriazinyl, quinazolinyl, tetrahydroquinolinyl, thienofuryl, thienopyridyl, thienothienyl, and the like.

Exemplary substituents include one or more alkyl or arylalkyl groups as described supra or one or more groups described supra as alkyl substituents and smaller heterocyclyls, such as, epoxides and aziridines.

"Carbocyclic ring" or "carbocyclyl" refers to stable, saturated, partially saturated or unsaturated, mono or bicyclic hydrocarbon rings that contain 3-12 atoms. This includes a monocyclic ring containing 5 or 6 atoms or a bicyclic ring containing 9 or 10 atoms. Suitable values include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, dihydroindenyl, and tetrahydronaphthyl.

"Optionally substituted" as it refers to "carbocyclic ring" or "carbocyclyl" indicates that the carbocyclic ring may be substituted at one or more substitutable ring positions by one or more groups independently selected from alkyl (preferably lower alkyl), alkoxy (preferably lower alkoxy), nitro, monoalkylamino (preferably lower alkylamino), dialkylamino (preferably a di[lower]alkylamino), cyano, halo, haloalkyl (preferably trifluoromethyl), alkanoyl, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, alkyl amido (preferably lower alkyl amido), alkoxyalkyl

(preferably a lower alkoxy [lower] alkyl), alkoxycarbonyl (preferably lower

alkoxycarbonyl), alkylcarbonyloxy (preferably lower alkylcarbonyloxy) and aryl (preferably phenyl), said aryl being optionally substituted by halo, lower alkyl and lower alkoxy groups.

"Heteroatoms" includes oxygen, sulfur, and nitrogen.

k k k

"Alkylsulfone" refers to -R S (=0)₂R , wherein R is alkyl or substituted alkyl. "Oxo" refers to the divalent radical =0.

"Carbamate" refers to -OC(=0)NH₂.

"Amide" refers to -C(=0)NH₂.

"Sulfonamide" refers to -S0₂NH₂.

"Substituted amide", "substituted sulfonamide", or "substituted carbamate" refer to an amide, sulfonamide, or carbamate, respectively, having at least one hydrogen replaced with a group chosen from alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, and substituted cycloalkyl.

A substituted amide refers to -C(=0)NR"'R" wherein R" and R" are

independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, and substituted cycloalkyl, provided at least one of R'" or R" is a substituted moiety.

Substituted sulfonamide refers to the group -S0₂NR°R^p wherein R°and R^pare independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, and substituted cycloalkyl, provided at least one of R° or R^p is a substituted moiety.

Substituted carbamate refers to -OC(=0)NR^qR^r wherein R^q and R^r are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, and substituted cycloalkyl, provided at least one of R^q or R^r is a substituted moiety.

"Ureido" refers to the group -NHC(=0)NH₂.

"Cyano" refers to the group -CN. "Cycloalkylalkyl" or "cycloalkylalkoxy" refer to a cycloalkyl or substituted cycloalkyl bonded to an alkyl or substituted alkyl; or an alkoxy, respectively.

"Nitro" refers to the group -N(0)₂.

"Thio" refers to the group -SH.

"Alkylthio" refers to the group -SR^S where R^s is an alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

"Thioalkyl" refers to the group -R^lS where R^l is an alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

"Alkylsulfonyl" refers to the group

where R^u is an alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

"Alkylsulfinyl" refers to the group -S(=0)R^v where R^v is an alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

"Carboxy" refers to the group -C(=0)OH.

"Carboxyalkoxy" or "alkoxycarbonylalkoxy" refer to a carboxy, or an alkoxycarbonyl, respectively, bonded to an alkoxy.

"Alkoxycarbonyl" refers to the group -C(=0)OR^w where R^w is an alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl.

"Arylalkoxycarbonyl" refers to an aryl or substituted aryl bonded to an alkoxycarbonyl.

"Alkylcarbonyloxy" or "arylcarbonyloxy" refer to the group -OC(=0)R^x, where R is an alkyl or substituted alkyl, or an aryl or substituted aryl, respectively.

"Carbamoyl" refers to the groups -OC(=0)NH₂, -OC(=0)NHR^x, and/or -C(=0)NR^yR^z, wherein R^y and R^z are independently selected from alkyl and substituted alkyl.

The group -NR⁶(C=0)R⁹ refers to a group where R⁶ is selected from hydrogen, lower alkyl and substituted lower alkyl, and R⁹ is selected from hydrogen, alkyl, substituted alkyl, alkoxy, aminoalkyl, substituted aminoalkyl, alkylamino, substituted alkylamino, aryl and substituted aryl. "Carbonyl" refers to a C(=0).

"Alkylcarbonyl", "aminocarbonyl", "alkylaminocarbonyl",

"aminoalkylcarbonyl", or "arylaminocarbonyl" refer to alkyl, substituted alkyl, amino; alkylamino, substituted alkylamino, aminoalkyl, substituted aminoalkyl, or arylamino, respectively, bonded to a carbonyl.

"Aminocarbonylaryl" or "aminocarbonylalkyl" refer to an aminocarbonyl bonded to an aryl or substituted aryl; or an alkyl or substituted alkyl, respectively.

"Sulfonyl" refers to the group S(=0)₂.

"Sulfmyl" refers to the group S(=0).

"Carboxy alkyl" refers to alkyl or substituted alkyl bonded to a carboxy.

The compounds of formula I may form salts which are also within the scope of this invention. Pharmaceutically acceptable (i.e. non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g., in isolating or purifying the compounds of the invention. The compounds of formula I may form salts with alkali metals such as sodium, potassium and lithium, with alkaline earth metals such as calcium and magnesium, with organic bases such as dicyclohexylamine, tributylamine, pyridine and amino acids such as arginine, lysine and the like. Such salts can be formed by methods known to those skilled in the art.

The compounds for formula I may form salts with a variety of organic and inorganic acids. Such salts include those formed with hydrogen chloride, hydrogen bromide, methane sulfonic acid, sulfuric acid, acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, benzenesulfonic acid, toluene sulfonic acid and various others (e.g., nitrates, phosphates, borates, tartrates, citrates, succinates, benzoates, ascorbates, salicylates, and the like). Such salts can be formed by methods known to those skilled in the art. In addition, zwitterions ("inner salts") may be formed.

All stereoisomers of the compounds of the invention are contemplated, either in admixture or in pure or substantially pure form. The definition of compounds according to the invention embraces all possible stereoisomers and their mixtures. It very particularly embraces the racemic forms and the isolated optical isomers having the specified activity. The racemic forms can be resolved by physical methods, such as, fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. The individual optical isomers can be obtained from the racemates from the conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization.

Compounds of formula I may also be administered in prodrug forms. Thus, the invention is intended to cover prodrugs of the compounds of formula 1 , methods of administering the same, and compositions containing the same. "Prodrugs" are intended to include any covalently bonded carriers that release an active parent drug in vivo when such prodrug is administered to a mammalian subject. Prodrugs of the invention are 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. Prodrugs include compounds of the invention wherein a hydroxy, amino, or sulfhydryl group is bonded to any group that, when the prodrug of the invention is administered to a mammalian subject, it cleaves to form a free hydroxyl, free amino, or free sulfhydryl group, respectively. Examples of prodrugs include acetate, formate, and benzoate derivatives of alcohol and amine functional groups in the compounds of the invention. Various forms of prodrugs are well known in the art. For example, see Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in

Enzymology, Vol. 112, pp. 309-396, edited by K. Widder, et al. (Academic Press, 1985).

Solvates (e.g., hydrates) of the compounds of formula I are also within the scope of the invention. Methods of solvation are known in the art.

The pharmaceutical compositions of the invention containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with a water soluble carrier such as polyethyleneglycol or an oil medium, such as peanut oil, liquid parafm, or olive oil.

The pharmaceutical compositions may be in the form of sterile injectable aqueous solutions. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.

The sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion where the active ingredient is dissolved in the oily phase. For example, the active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil solution then introduced into a water and glycerol mixture and processed to form a microemulation.

The injectable solutions or microemulsions may be introduced into a patient's blood- stream by local bolus injection.

Alternatively, it may be advantageous to administer the solution or microemulsion in such a way as to maintain a constant circulating concentration of the instant compound. In order to maintain such a constant concentration, a continuous intravenous delivery device may be utilized.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension for intramuscular and subcutaneous administration. This suspension may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents such as mentioned supra.

When a compound according to the invention is administered into a human subject, the daily dosage will normally be determined by the prescribing physician with the doage generally varying according to the age, weight, sex and response of the individual patient, as well as the severity of the patient's symptoms.

If formulated as a fixed dose, such combination products employ the compounds of the invention within the dosage range described herein and the other pharmaceutically active agent or treatment within its approved dosage range. Compounds of formula I may also be administered sequentially.

The compounds of formula 1 may be administered in a dosage range of about 0.05 to 200 mg/kg/ day, preferably less than 100 mg/kg/day, in a single dose or in 2 to 4 divided doses. All documents cited herein are incorporated by reference in their entirety.

The examples are intended to be illustrative and not limiting.

EXAMPLE 1

Using the uptake inhibition assay with suspension or plated hepatocytes, compound BMS 754807 yields the following data:

The uptake inhibition assay is performed according to the method of Liu et al. using fresh rat hepatocytes isolated from the liver of the Sprague-Doley strain. It is found that BMS 754807 inhibits the entry of taurocholic acid into the hepatocytes, dosing from 100 to 0.1 μΜ with a calculated IC₅₀ of 51.1 and 1.9 μΜ, using suspension and plated hepatocytes, respectively. With the same method using human plated hepatocytes, a calculated IC₅₀ of 12.1 μΜ is obtained.

In addition, BMS 754807 is taken up by rat hepatocytes (active contribution to total is 33.01 ± 18.70% ) using the suspension assay as described in Takao Watanabe et al, DMD 38, 215-222, 2010.

The aqueous solubility (-150 μΜ in the pH range from 2-7.4), lipophilicity (Log D 7.4 =2.35 ± 0.13), and permeability (Caco-2 P_app (A-to-B, pH 7.4) = 2.32 ± 0.27 10^"6cm/s nm/s) of BMS 254807 is determined by standard methods and found to fit the Class 3-Class 4 definition. EXAMPLE 2

The Na⁺-dependent taurocholate co-transporting polypeptide (NTCP) is the major transporter that takes up bile acids from the portal blood, which represents the first step in hepatocellular bile acid handling. Stably transfected HEK-293 cells and mock control cells are used to measure the effects of BMS-754807 in the inhibition of NTCP-mediated bile acid uptake.

STEP 1 : Stable Transfection of Human NTCP in HEK-293 Cells

HEK-293 cells are grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum in a humidified incubator at 37°C and 5% C0₂. The stably transfected human epithelial kidney cell lines are established by using the Flp-In™ expression system (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol.

STEP 2: Bile Acid Transport in hNTCP-Expressing HEK-293 Cells

For uptake measurements, the stably transfected HEK-293 cells from Stepl and mock control cells are seeded on Biocoat poly-D-lysine-coated 96-well plates at a density

4

of 2.0 x 10 cells per well and cultured overnight to reach 90% confluence. The cells are then washed twice with 100 μΕ Hank's Balanced Salt Solution (HBSS, 137 mM NaCl, 5.36 mM KCl, 0.20 mM MgS0₄, 0.34 mM Na₂HP0₄, 0.44 mM KH₂P0₄, 4.17 mM NaHC0₃, 1.26 mM CaCl₂, and 5.6 mM glucose) and incubated with deuterated sodium taurocholate (d5-STC) in the presence or absence of inhibitor (BMS-754807 or cyclosporine) at 37°C for 1 min, during which the rate of taurocholate uptake is linear. After incubation, the cells are washed 3 times with cold HBSS and then lyzed with the addition of chilled acetonitrile containing internal standard. Concentrations of d5-STC are determined by LC-MS/MS analysis. The %> inhibition of d5-STC uptake is used to calculate the IC₅₀ of BMS-754807 using the Hill's equation in SigmaPlot. The IC₅₀ of BMS-754807 is 16.6 ± 1.4 μΜ, n=3. Using the same method, the IC₅₀ of Cyclosporin A is 3.8 ± 0.5 μΜ, η = 3

Claims

WHAT IS CLAIMED IS

1) A method of selecting anti-HBV drugs in the molecular weight range of about 400 to 650 daltons that are placed in class 3 or 4 of the Biopharmaceutics classification system (BCS) and the Biopharmaceutics Drug Disposition classification system

(BDDES) based on solubility and permeability, which comprises measuring the inhibition of taurocholic acid transporter in the hepatocyte thereby preventing the entrance of the virus HBV through the taurocholic acid portal.

2) A method of measuring taurocholic acid uptake inhibition using hepatocytes either in suspension or in plated cultures.

3) A method of discovering compounds within the insulin- like growth factor type 1 receptor (IGF-1R) antagonist class which are inhibitors of the NCTP transporter and which block taurocholic acid entry into hepatocytes.

4) A method of preventing HBV infection in a mammal which comprises administering to a mammal in need thereof a therapeutically effective amount of a one or more compounds of formula I, either alone or in combination with an antiviral agent, especially those that operate by a different mechanism and work synergistically with the viral entry blockers, especially interferon alpha, HBV ccc DNA transcription modulators, ccc DNA biosynthesis inhibitors, natural inhibitors such as magnolol and swertia mileensis extract, and HBV capsid assembly inhibitors,

wherein formula I is:

wherein

Q¹ is aryl, substituted aryl, heteroaryl, or substituted heteroaryl; X is C=0, C=S, C=NR , or CH₂;

1 2 3

4

R⁵ is hydrogen, halogen, cyano, alkyl, or substituted alkyl;

cycloalkylalkyl, cycloalkylalkoxy, nitro, thio, thioalkyl, alkylthio, alkylsulfonyl, alkylsul?nyl, carboxy, alkoxycarbonyl, alkylcarbonyloxy, carbamoyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heteroaryloxy, arylheteroaryl, arylalkoxycarbonyl, heteroarylalkyl, heteroarylalkoxy, aryloxyalkyl, aryloxyaryl, heterocycle, substituted heterocycle, alkylcarbonyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroalkynyl, substituted heteroalkynyl, arylamino, arylalkylamino, alkanoylamino, aroylamino, arylalkanoylamino, arylthio, arylalkylthio, arylsulfonyl, arylalkylsulfonyl, alkylsulfonyl, arylcarbonylamino, or alkylaminocarbonyl;

n is 0, 1,2, 3, 4, 5, or 6; or when n is 2 and R⁶ are geminal substituents, they may together form an optionally substituted 3-6 membered saturated or unsaturated carbocyclic or heterocyclic ring; or when n is 2 and R⁶ are 1,2-cis substituents, they may together form an optionally substituted 3-6 membered fused saturated carbocyclic or heterocyclic ring; or when n is 2 and R are 1 ,3-cis substituents they may together form an optionally substituted 1-4 membered alkyl or heteroalkyl bridge; or when there are two R⁶'s on the same carbon, they may together form a carbonyl (C=0) or alkylidene group (C=CHR );

7 8

R is hydrogen or lower alkyl;

or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

5) A method of claim 4 wherein at least one of the compounds is interferon alpha, an HBV ccc DNA transcription modulator, a ccc DNA biosynthesis inhibitor, a natural inhibitor such as magnolol and swertia mileensis extract, or an HBV capsid assembly inhibitor.

6) A method of claim 4 wherein one of the compounds is (S)-l-(4-(5-cyclopropyl- lH-pyrazol-3-ylamino)pyrrolo[l,2-f][l,2,4]triazin-2-yl)-N-(6-fluoropyridin-3-yl)-2- methylpyrrolidine-2-carboxamide.

7) A method of claim 4 wherein a compound of formula I is such that

independently,

1 2 3 4

R , R , R , and R is each H;

Q¹ is aryl or heteroaryl;

R⁵, R⁶, R⁷, R⁸, and R⁹ is each independently H or lower alkyl; and

X is CH₂, C=0, or C=NR⁹;

or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. 8) A method of preventing HBV infection in a mammal which comprises administering to a mammal in need thereof a therapeutically effecti ve amount of one or more compounds within the insulin- like growth factor type 1 receptor (IGF-1R) antagonist class which are inhibitors of the NCTP transporter and which block taurocholic acid entry into hepatocyte and the hepatocyte B virus.

9) A method of claim 8 for in preventing Hepatitis B disease, cirrhosis, and hepatic cellular cancer.

10) A method of claim 8 wherein the mammal is man.

11) A method of claim 8 is the combination of said IGF-1R inhibitors with other antiviral agents, especially those that operate by a different mechanism and work synergistically with the viral entry blockers, especially interferon alpha, HBV ccc DNA transcription modulators, ccc DNA biosynthesis inhibitors, natural inhibitors such as magnolol and swertia mileensis extract, and HBV capsid assembly inhibitors.