HK1181390A

HK1181390A - Viral polymerase inhibitors

Info

Publication number: HK1181390A
Application number: HK13108827.7A
Authority: HK
Inventors: 尤拉.S.桑特里佐斯; 凯瑟琳.沙博特; 皮埃尔.博利厄; 克里斯琴.布罗丘; 马丁.波伊里尔; 蒂莫西.A.斯坦默斯; 邦克汉姆.撒沃尼克汉姆; 琼.兰库尔特
Original assignee: 贝林格尔.英格海姆国际有限公司
Priority date: 2004-02-20
Filing date: 2007-07-12
Publication date: 2013-11-08

Description

Viral polymerase inhibitors

The application is a divisional application of Chinese invention application (title of the invention: viral polymerase inhibitor; application date: 2005-2-18, application number: 200580005539.5).

Technical Field

The present invention relates to inhibitors of RNA-dependent RNA polymerases, particularly those viral polymerases of the Flaviviridae (Flaviviridae) family, more particularly to HCV polymerase.

Background

An estimated 30,000 new cases of Hepatitis C Virus (HCV) infection occur annually in the United states (Kolykhalov, A.A.; Mihalik, K.; Feinstone, S.M.; Rice, C.M.;2000; J.Virol.74: 2046-. HCV is not readily cleared by the host's immune defenses; 85% of the population infected with HCV became chronically infected. Many of these persistent infections lead to chronic liver disease, including cirrhosis and hepatocellular carcinoma (Hoofnagle, J.H.;1997; Hepatology 26: 15S-20S). About 17000 million people worldwide are estimated to be HCV carriers, and end-stage liver disease associated with HCV is now the leading cause of liver transplantation. Hepatitis c has approximately 8,000 to 10,000 deaths per year in the united states alone. Without effective intervention, it is expected that this amount will triple now within the next 10 to 20 years. No vaccine can prevent HCV infection.

Treatment of chronically infected patients with interferon or interferon in combination with ribavirin is currently the only approved therapy. Recently, pegylated interferon (pegylated interferon alpha-2 a (Pegasys)^TMRoche) and PEGylated Interferon alpha-2 b (PEG-Intro)^TMSchering)) are approved in some countries for the treatment of chronic hepatitis c infection, both alone and in combination with ribavirin. But these treatments are reported to achieve a sustained response in less than 60% of cases.

HCV belongs to the Flaviviridae family, the hepatitis C virus (genus Hepacivirus), which includes three small, enveloped, positive-strand RNA viruses (Rice, C.M.;1996; "Flaviviridae: the viruses and the replication"; pp.931-960in Fields Virology; Fields, B.N.; Knipe, D.M.; Howley, P.M. (eds.); Lippincott-Raven Publishers, Philadelphia Pa.). The 9.6kb genome of HCV consists of a long Open Reading Frame (ORF) flanked by 5 'and 3' untranslated regions (NTRs). The HCV 5' NTR is 341 nucleotides in length and functions as an internal ribosome entry site for cap-independent translation initiation (Lemon, S.h.; Honda, M.;1997; Semin. Virol.8: 274-. The HCV polyprotein is cleaved into at least 10 molecules in a co-and post-translational mannerPolypeptide alone (Reed, K.E.; Rice, C.M.;1999; Curr. Top. Microbiol. Immunol.242: 55-84). The structural protein is derived from the signal peptidase of the N-terminal part of the polyprotein. Two viral proteases mediate downstream cleavage to produce a non-structural (NS) protein that functions as a component of the HCV RNA replicase. The NS2-3 protease spans the C-terminal half of NS2 and the N-terminal third of NS3 and catalyzes cis cleavage at the NS2/3 site. The same portion of NS3 also encodes the catalytic region of NS3-4A serine protease, which cleaves at 4 downstream sites. The C-terminal two-thirds of NS3 is highly conserved among HCV isolates (isolates), with RNA-binding, RNA-stimulated NTPase, and RNA melting activities. Although NS4B and NS5A phosphoproteins may also be components of replicase, their specific roles are unknown. This C-terminal polyprotein cleavage product, NS5B, is the elongation subunit of the HCV replicase with RNA-dependent RNA polymerase (RdRp) activity (Behrens, S.E.; Tomei, L.; DeFrance sco, R.;1996; EMBO J.15:12-22; and Lohmann, V.;herian, U.S., Bartenschlager, R.1997, J.Virol.71: 8416-8428). Mutations that disrupt NS5B activity have recently been shown to eliminate RNA infectivity in chimpanzee models (Kolykhalov, a.a.; Mihalik, k.; Feinstone, s.m.; Rice, c.m.;2000; j.virol.74: 2046-.

The development of new, specific anti-HCV therapies is a high priority, and virus-specific functions necessary for replication are the most attractive targets for drug development. The fact that no RNA-dependent RNA polymerase is present in mammals and that this enzyme appears to be essential for viral replication would suggest that this NS5B polymerase is an ideal target for anti-HCV therapy. WO 01/47883, WO 02/04425, WO 03/000254, WO03/0007945, WO 03/010140, WO 03/026587, WO 03/101993, WO 04/005286, WO 2004/064925, WO 2004/065367 and WO 2004/087714 suggest that NS5B inhibitors may be useful in the treatment of HCV.

Indole inhibitors of the NS5B polymerase of HCV are disclosed in WO 03/010141. However, the inhibitors of the present invention differ from the inhibitors disclosed in WO03/010141 in that they show unexpectedly good activity in cell-based HCV RNA replication experiments.

Summary of The Invention

The present invention provides a novel series of compounds with good to excellent inhibitory activity against HCV polymerase and/or unexpectedly good activity in cell-based HCV RNA replication experiments.

A further object of the invention will be derived from the following description and examples for a person skilled in the art.

In a first aspect of the invention, there is provided a compound represented by formula I, or an enantiomer, diastereomer or tautomer thereof, including salts or acids thereof.

Wherein:

a or B is N and the other B or A is C, wherein- - -between two C-atoms represents a double bond and- - -between the C-atom and the N-atom represents a single bond;

R¹is H or (C)_1-6) An alkyl group;

R²selected from halogen, cyano, (C)_1-6) Alkyl, (C)_2-6) Alkenyl, (C)_2-6) Alkynyl, (C)_3-7) Cycloalkyl, aryl and Het; the aryl and Het are optionally substituted by R²¹Substitution;

wherein R is²¹Is a mono-, di-or tri-substituent, each of which is selected from-OH, -CN, -N (R)^N2)R^N1Halogen, (C)_1-6) Alkyl, (C)_1-6) Alkoxy group, (C)_1-6) Alkylthio, Het and-CO-N (R)^N2)R^N1；

Wherein (C) is_1-6) Alkyl, (C)_1-6) Alkoxy radicalAnd (C)_1-6) Each alkylthio group is optionally substituted with one, two or three halogen atoms;

R³is (C)_5-6) Cycloalkyl optionally substituted with one to four halogen atoms;

R⁴and R⁷Each independently selected from H, (C)_1-6) Alkyl, (C)_1-6) Alkoxy group, (C)_1-6) Alkylthio, -NH₂、-NH(C_1-6) Alkyl, -N ((C)_1-6) Alkyl radical)₂And halogen;

R⁵and R⁶One is selected from COOH, -CO-N (R)^N2)R^N1Aryl, Het and (C)_2-6) Alkenyl, wherein aryl, Het, (C)_2-6) Alkenyl and R^N1Or any at R^N2And R^N1In which the heterocyclic radical is each optionally substituted by R⁵⁰Substitution;

wherein R is⁵⁰Is a mono, di or tri substituent each independently selected from (C)_1-6) Alkyl, -COOH, -OH, oxo, -N (R)^N2)R^N1,-CO-N(R^N2)R^N1And halogen, wherein (C)_1-6) Alkyl is optionally substituted by aryl or-N (R)^N2)R^N1Substitution;

and R is⁵And R⁶Is selected from H, (C)_1-6) Alkyl, (C)_1-6) Alkoxy group, (C)_1-6) Alkylthio and N (R)^N2)R^N1；

R⁸Is (C)_1-6) Alkyl, (C)_3-7) Cycloalkyl or (C)_3-7) Cycloalkyl- (C)_1-6) Alkyl-;

wherein the alkyl, cycloalkyl and cycloalkyl-alkyl are each optionally mono-, di-or three independently selected from halogen, (C)_1-6) Alkoxy and (C)_1-6) Substituted by a substituent of alkylthio;

R⁹and R¹⁰Each independently selected from (C)_1-6) An alkyl group; or R⁹And R¹⁰Are linked and form together with the carbon atom to which they are linked (C)_3-7) Cycloalkyl group, (C)_3-7) Cycloalkenyl or a 4-, 5-or 6-membered heterocyclic ring having 1 to 3 heteroatoms each independently selected from O, N and S;

wherein the cycloalkyl, cycloalkenyl or heterocyclyl is optionally substituted at each occurrence by (C)_1-4) Alkyl substituted;

R^N1selected from H, (C)_1-6) Alkyl, (C)_3-7) Cycloalkyl group, (C)_3-7) Cycloalkyl- (C)_1-6) Alkyl-, -CO- (C)_1-6) Alkyl, -CO-O- (C)_1-6) Alkyl and Het;

wherein (C) is_1-6) Alkyl, (C)_3-7) Cycloalkyl group, (C)_3-7) Cycloalkyl- (C)_1-6) Alkyl, -CO- (C)_1-6) Alkyl and-CO-O- (C)_1-6) Each alkyl and cycloalkyl moiety in the alkyl group is optionally substituted with one, two or three substituents each independently selected from halogen, (C)_1-6) Alkoxy and (C)_1-6) Substituted by a substituent of alkylthio; and is

R^N2Is H or (C)_1-6) Alkyl, or

R^N2And R^N1May be linked and taken together with the nitrogen atom to which they are attached form a 4-,5-, 6-or 7-membered saturated, unsaturated or aromatic N-containing heterocyclyl or an 8-, 9-, 10-or 11-membered N-containing saturated, unsaturated or aromatic heterobicyclic ring, each optionally further having from 1 to 3 heteroatoms each independently selected from O, N and S;

wherein is represented by R^N2And R^N1The heterocyclic or heterobicyclic ring formed being optionally substituted by one, two or three each independently

Is selected from halogen, (C)_1-6) Alkyl, (C)_1-6) Alkoxy and (C)_1-6) Alkylthio group substituent; wherein Het is defined as a 4-,5-, 6-or 7-membered heterocycle having 1 to 4 heteroatoms, each selected from O, N and S, which heterocycle may be saturated, unsaturated or aromatic, or an 8-, 9-, 10-or 11-membered heterobicyclic ring having, where possible, positionsThere are 1 to 5 heteroatoms each selected from O, N and S, and the heterocyclic ring may be saturated, unsaturated or aromatic.

Included within the scope of the present invention are compounds of formula (I) as described hereinbefore having at least one of a "detectable label", "affinity tag" and "photoactive group" attached thereto.

The compounds according to the invention generally show inhibitory activity against HCV polymerase. Particular compounds according to the present invention can utilize the RNA-dependent RNA polymerase of HCV to inhibit RNA synthesis, particularly the HCV-encoded NS5B enzyme. Furthermore, the compounds according to the invention show unexpectedly good activity in cell-based HCV RNA replication experiments. Another benefit of the compounds provided by the present invention is their low to very low or even no significant activity towards other polymerases.

In a second aspect of the present invention there is provided the use of a compound of formula I, or a pharmaceutically acceptable salt or ester thereof, or a composition thereof, according to the compounds of the present invention, as an HCV polymerase inhibitor, preferably as an inhibitor of the RNA-dependent RNA polymerase activity of the enzyme NS5B encoded by HCV.

In a third aspect of the invention, there is provided the use of a compound of formula I, or a pharmaceutically acceptable salt or ester thereof, or a composition thereof, according to the compounds of the invention, as an inhibitor of HCV replication.

In a fourth aspect of the invention, there is provided the use of a compound of formula I, or a pharmaceutically acceptable salt or ester thereof, or a composition thereof, according to a compound of the invention, for the treatment or prophylaxis of HCV infection in a mammal.

In a fifth aspect of the invention, there is provided a method of inhibiting the RNA-dependent RNA polymerase activity of the enzyme NS5B encoded by HCV, comprising exposing the enzyme NS5B to an effective amount of a compound of formula I under conditions in which the RNA-dependent RNA polymerase activity of the enzyme NS5B is inhibited.

In a sixth aspect of the invention, there is provided a method of inhibiting HCV replication comprising exposing a cell infected with HCV to an effective amount of a compound of formula I under conditions in which HCV replication is inhibited.

In a seventh aspect of the invention, there is provided a method of treating or preventing HCV infection in a mammal, comprising administering to the mammal an effective amount of a compound of formula I according to the invention, or a pharmaceutically acceptable salt or ester thereof, or a composition thereof.

In an eighth aspect of the invention, there is provided a method of treating or preventing HCV infection in a mammal, comprising administering to the mammal an effective amount of a compound of formula I, or a pharmaceutically acceptable salt or ester thereof, or a combination thereof, in combination with at least one antiviral agent.

In a ninth aspect of the invention, there is provided a pharmaceutical composition for the treatment or prophylaxis of HCV infection, comprising an effective amount of a compound of formula I according to the invention, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier.

According to a particular embodiment, the pharmaceutical composition of the invention further comprises a therapeutically effective amount of one or more antiviral drugs. Examples of antiviral drugs include, but are not limited to, ribavirin and amantadine (amantadine).

According to another specific embodiment, the pharmaceutical composition of the invention further comprises at least one other anti-HCV agent as antiviral agent.

According to another embodiment, the pharmaceutical composition of the invention comprises an additional immunomodulator as other anti-HCV agent. Examples of other immunomodulators include, but are not limited to, alpha-, beta-, delta-, gamma-, tau, and omega-interferons and pegylated forms thereof.

According to another embodiment, the pharmaceutical composition of the invention further comprises at least one other inhibitor of HCV polymerase as an additional anti-HCV agent.

According to another embodiment, the pharmaceutical composition of the invention further comprises at least one inhibitor of HCV ns3 protease as an additional anti-HCV agent.

According to another embodiment, the pharmaceutical composition of the invention further comprises at least one inhibitor of another target in the HCV life cycle as an additional anti-HCV agent. Examples of such inhibitors of other targets include, but are not limited to, drugs that inhibit a target selected from HCV helicase, HCV NS2/3 protease, and HCV IRES, as well as drugs that interfere with the function of other viral targets, including, but not limited to, the NS5A protein.

In a tenth aspect of the present invention there is provided the use of a compound of formula I according to the present invention, or a pharmaceutically acceptable salt or ester thereof, or a composition thereof, for the manufacture of a medicament for the treatment and/or prophylaxis of a flavivirus infection, preferably an HCV infection.

In an eleventh aspect the present invention is the subject of an article of manufacture comprising a composition of NS5B polymerase effective for treating or preventing HCV infection or inhibiting HCV, and a packaging material comprising a label indicating that the composition can be used for treating hepatitis c virus, wherein the label states that the composition comprises a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof according to the present invention.

Detailed Description

The following definitions will be used unless otherwise stated:

as used herein, the term "(C)_1-n) Alkyl ", wherein n is an integer, alone or in combination with other groups, refers to an acyclic straight or branched alkyl group containing from 1 to n carbon atoms, respectively. Examples of such alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (i-propyl), n-butyl, 1-methylpropyl, 2-methylpropyl, 1-dimethylethyl (t-butyl), n-pentyl, and the like. In the following, the term Me represents methyl.

If alkyl is substituted by halogen, it is preferably mono-, di-or trisubstituted by fluorine or monosubstituted by chlorine or bromine.

As used herein, the term "(C)_2-n) Alkenyl ", wherein n is an integer, aloneOr in combination with other groups, means unsaturated, acyclic, linear or branched groups containing from 2 to n carbon atoms, wherein at least two carbon atoms are bonded to each other by a double bond. Examples of such alkenyl groups include, but are not limited to, ethenyl (ethenyl), 1-propenyl, 2-propenyl, 1-butenyl, and the like. The term includes (C)_2-n) Cis and trans isomers of alkenyl groups and mixtures thereof. (C)_2-n) Alkenyl groups may be substituted on any of their carbon atoms, which would otherwise carry a hydrogen atom.

As used herein, the term "(C)_2-n) Alkynyl "wherein n is an integer, alone or in combination with other groups, refers to an acyclic, straight chain, or branched group containing 2 to n carbon atoms, wherein at least two carbon atoms are bonded through a triple bond. Examples of such alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and 1-butynyl. (C)_2-n) An alkynyl group may be substituted on any of its carbon atoms otherwise it would carry a hydrogen atom.

As used herein, the term "(C)_3-n) Cycloalkyl ", wherein n is an integer, alone or in combination with other groups, refers to cycloalkyl groups containing from 3 to n carbon atoms. Examples of such cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

As used herein, the term "(C)_5-n) Cycloalkenyl ", wherein n is an integer, alone or in combination with other groups, refers to an unsaturated cyclic group containing from 5 to n carbon atoms. Examples include, but are not limited to, cyclopentenyl and cyclohexenyl.

As used herein, the term "(C)_3-m) Cycloalkyl- (C)_1-n) Alkyl- ", wherein n and m are integers, alone or in combination with other groups, means that a cycloalkyl group containing three to m carbon atoms is covalently bonded to a branched or straight chain alkyl group having 1 to n carbon atoms. (C)_3-7) Cycloalkyl- (C)_1-6) Examples of alkyl-include, but are not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 1-cyclopropylethyl, 2-cyclopropylethyl, 1-cyclobutanMethylethyl, 2-cyclobutylethyl, 1-cyclopentylethyl, 2-cyclopentylethyl, 1-cyclohexylethyl, 2-cyclohexylethyl and the like.

As used herein, The term "Protective group" is defined as a Protective group that can be used during synthetic transformations, examples of which are listed in Greene, "Protective Groups in Organic Chemistry", John Wiley & Sons, New York (1981) and "The Peptides: Analysis, Synthesis, Biology", Vol.3, Academic Press, New York (1981).

The carboxyl group is usually protected as an ester, which can be cleaved to give the carboxylic acid. Protecting groups that may be used include (but are not limited to): 1) alkyl esters such as methyl, ethyl, trimethylsilylethyl and tert-butyl, 2) aralkyl esters such as benzyl and substituted benzyl, or 3) esters cleavable with a weak base treatment or a weak reductive process, such as trichloroethyl and phenacyl.

The term "aryl", alone or in combination with other groups, as used herein, refers to a 6-or 10-membered aryl group, i.e., an aryl group containing six or ten carbon atoms. Examples include, but are not limited to, phenyl, 1-naphthyl, or 2-naphthyl.

As used herein, the term "Het" is defined as a 4-,5-, 6-, or 7-membered heterocyclic group having 1 to 4 heteroatoms each independently selected from O, N, and S, and may be saturated, unsaturated, or aromatic, or an 8-, 9-, 10-, or 11-membered heterobicyclic ring having 1 to 5 heteroatoms each independently selected from O, N and S at possible positions, which ring may be saturated, unsaturated, or aromatic unless otherwise specified.

As used herein, the term "heteroatom" refers to O, S or N.

As used herein, the term "heterocycle", alone or in combination with other groups, refers to a monovalent radical derivable by removal of a hydrogen atom from a five-, six-or seven-membered saturated or unsaturated (including aromatic) heterocyclic ring containing one to four heteroatoms selected from nitrogen, oxygen and sulfur. Examples of such heterocycles include (but are not limited to)Not limited to acridine (azetidine), pyrrolidine, tetrahydrofuran, thiazolidine, pyrrole, thiophene (thiophene), hydantoin (hydantoin), diaza(diazepine), 1H-imidazole, isoOxazole, thiazole, tetrazole, piperidine, piperazine, homopiperidine (homopiperidine), homopiperazine (homopiperazine), 1, 4-bis (piperazine)An alkane, 4-morpholine, 4-thiomorpholine, pyridine-N-oxide or pyrimidine, or the following heterocycles:

as used herein, the term "9-or 10-membered heterobicyclic" or "heterobicyclic", alone or in combination with other groups, refers to a heterocyclic group as defined above fused to one or more other rings (heterobicyclic or any other ring). Examples of such heterobicyclics include, but are not limited to, indole, benzimidazole, thiazolo [4,5-b ] -pyridine, quinoline (quinoline), or coumarin, or as shown below:

as used herein, the term "halo" or "halogen" refers to a halogen atom and includes fluorine, chlorine, bromine, and iodine.

As used herein, the term "OH" refers to a hydroxyl group. It is well known to those skilled in the art that hydroxyl groups may be substituted with equivalent functional groups. Examples of such functional valencies included within the scope of the present invention include, but are not limited to, ethers, thiols, thioethers and primary, secondary or tertiary amines.

As used herein, the term "SH" refers to a sulfhydryl group. It is within the scope of the present invention that any occurrence of an "SH" or "SR" group may be substituted by any other suitable oxidation state, e.g., SOR, SO₂R or SO₃R。

As used herein, the term "(C)_1-n) Alkoxy "means also bonded to (C)_1-n) Oxygen atom of alkyl group. (C)_1-6) Examples of alkoxy groups include, but are not limited to, methoxy (CH)₃O-), ethoxy (CH)₃CH₂O-), n-propoxy (CH)₃CH₂CH₂O-), 1-methylethoxy (isopropoxy; (CH)₃)₂CHO-), 1-dimethylethoxy (tert-butoxy; (CH)₃)₃CO-) and the like. When (C)_1-n) When the alkoxy radical is substituted, it is understood that in (C) thereof_1-n) The alkyl moiety is substituted.

As used herein, the term "(C)_1-n) Alkylthio "means also bonded to (C)_1-n) Sulfur atom of an alkyl group. (C)_1-6) Examples of alkylthio groups include, but are not limited to, methylthio (CH)₃S-), ethylthio (CH)₃CH₂S-), n-propylthio (CH)₃CH₂CH₂S-), 1-methylethylthio (isopropylthio; (CH)₃)₂CHS-), 1-dimethylethylthio (tert-butylthio (CH)₃)₃CS-), and the like. When (C)_1-n) When the alkylthio group is substituted, it is understood to be in (C) thereof_1-n) Alkyl moieties are substituted.

The term "oxo" as used herein refers to an oxygen atom (= O) attached to a carbon atom through a double bond as a substituent.

The term "substituted" is used in connection with a group having more than one moiety, e.g., (C)_3-7) Cycloalkyl- (C)_1-6) Alkyl-, such substitution applies to both moieties, i.e., one or both of the alkyl and cycloalkyl moieties can be substituted with defined substituents.

As used herein, the term "COOH" refers to a carboxylic acid group. It is well known to those skilled in the art that carboxylic acid groups can be replaced by functional group equivalents. Examples of such functional equivalents encompassed by the present invention include, but are not limited to, esters, amides, imides, boronic acids, phosphonic acids, sulfonic acids, tetrazoles, triazoles, N-acylsulfonyl diamides (RCONHSO)₂NR²) And N-acylsulfonamides (RCONHSO)₂R)。

As used herein, the term "functional group equivalent" refers to an element or group or substituted derivative thereof that may be replaced by an element or group having similar electronic, hybrid, or bonding properties.

The following symbols- - - - - - -, andmay be used interchangeably in the subformulae to represent the bond, or in the case of a spiro group, the atom bonded to the rest of the molecule as defined above.

As used herein, the term "detectable label" refers to any group that can be attached to a polymerase or to a compound of the invention, such attachment being such that when the compound is contacted with the polymerase target, the label allows the compound to be directly or indirectly recognized, such that the compound can be detected, measured and quantified. Examples of such "labels" include, but are not limited to, fluorescent labels, chemiluminescent labels, colorimetric labels, enzyme labels, radioisotopes, and affinity labels, such as biotin. The label is attached to the compound or polymerase using well known methods.

As used herein, the term "affinity tag" refers to a ligand (that can be attached to a polymerase or a compound of the invention) whose strong affinity for a receptor can be used to extract the entity to which the ligand is attached from solution. Examples of such ligands include, but are not limited to, biotin or a derivative thereof, a histamine polypeptide, polyarginine, an amylose sugar moiety, or a defined epitope that can be recognized by a particular antibody. The affinity tag is attached to the compound or polymerase using well known methods.

As used herein, the term "photoreactive group" refers to a group that is activated by light to convert from an inert group to a reactive species, e.g., a free radical. This group can be used, for example, as a photoaffinity label. Examples of such groups include, but are not limited to, benzophenones, azides, and the like.

The term "salt thereof" refers to any acid and/or base addition salt of a compound according to the invention; preferably a pharmaceutically acceptable salt thereof.

The term "pharmaceutically acceptable salt" refers to salts of the compounds of formula (I) which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or lower animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable benefit/risk ratio, and generally water-soluble or oil-soluble or dispersible and effective for such intended use. The term includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. Examples of suitable salts can be found in, for example, s.m. berge et al, j.pharm.sci.,1977,66found in pp.1-19.

The term "pharmaceutically acceptable acid addition salts" means that these salts retain the properties of biological effectiveness and free base and are not biologically or otherwise undesirable, as they are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid and the like, as well as with organic acids such as acetic acid, trifluoroacetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, butyric acid, camphoric acid, camphorsulfonic acid (camphosulfonic acid), cinnamic acid, citric acid, diglucosic acid, ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphonic acid, hemisulfuric acid (hemisulfic acid), hexanoic acid, formic acid, fumaric acid, 2-hydroxyethanesulfonic acid (isethionic acid), lactic acid, hydroxymaleic acid, malic acid, malonic acid, mandelic acid, mesitylenesulfonic acid (mesitylenesulfonic acid), methanesulfonic acid, naphthalenesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, sulfuric acid, trifluoroacetic acid, succinic, Oxalic acid, pamoic acid (pamoic acid), pectinic acid, phenylacetic acid, 3-phenylpropionic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid (sulfanilic acid), tartaric acid, p-toluenesulfonic acid, undecanoic acid, and the like.

The term "pharmaceutically acceptable base addition salts" means that these salts retain the properties of biological effectiveness and free acid and are not biologically or otherwise undesirable and are formed with inorganic bases such as ammonia or metal cations such as sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, magnesium, aluminum, and the like or ammonium hydroxides, carbonates or bicarbonates. Particularly preferred are ammonium, potassium, sodium, calcium and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, quaternary ammonium compounds, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N-dimethylaniline, N-methylpiperidine, N-methylammonium compounds, and mixtures thereof, N-methylmorpholine, dicyclohexylamine, benzhydrylamine, N-benzhydrylbenzethylamine, 1-benzhydrylamine (1-ephenamine), N' -benzhydrylethylenediamine, polyamine resins, and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

The term "ester thereof" refers to any ester of a compound in which any carboxyl functionality of the molecule is replaced by an alkoxycarboxyl functionality, including, but not limited to, pharmaceutically acceptable esters thereof.

The term "pharmaceutically acceptable ester" as used herein, alone or in combination with another substituent, refers to an ester of a compound of formula (I) in which any carboxyl functionality (but preferably the carboxyl terminus) is replaced by an alkoxycarbonyl functionality in the molecule:

the R portion of the ester is selected from alkyl (e.g., methyl, ethyl, n-propyl, t-butyl, n-butyl); alkoxyalkyl (e.g., methoxymethyl); alkoxyacyl (e.g., acetoxymethyl); aralkyl (e.g., benzyl); aryloxyalkyl (e.g., phenoxymethyl); aryl (e.g. phenyl), optionally substituted by halogen, (C)_1-4) Alkyl or (C)_1-4) Alkoxy substitution. Other suitable esters may be found in Design of produgs, Bundgaard, h.ed.elsevier (1985). These pharmaceutically acceptable esters are generally hydrolyzed in vivo and converted to the acid form of the compound of formula (I) when injected into a mammal. With respect to the esters described above, any alkyl moiety present advantageously contains from 1 to 16 carbon atoms, especially from 1 to 6 carbon atoms, unless otherwise specified. Any aryl moiety present in the ester advantageously contains a phenyl group. Specifically, the ester may be (C)_1-16) Alkyl esters, unsubstituted benzyl esters or substituted by at least one halogen, (C)_1-6) Alkyl, (C)_1-6) Alkoxy, nitro or trifluoromethyl substituted benzyl esters.

The term "antiviral drug" as used herein refers to a drug (compound or biologic) that is effective in inhibiting the formation and/or replication of a virus in a mammal. Such agents include those that interfere with the host or interfere with the viral mechanisms required for virus formation and/or replication in the mammalian body. Antiviral drugs include, but are not limited to, ribavirin, amantadine, VX-497(merimepodib, Vertex Pharmaceuticals), VX-498(Vertex Pharmaceuticals), Lyocell (Levovirin), Viramidine (Viramidine), ceprine (Ceplene) (histamine dihydrochloride), XTL-001, and XTL-002 (XTLBiopharmaceuticals).

The term "other anti-HCV drugs" as used herein refers to those drugs that are effective in alleviating or preventing the symptoms of hepatitis c related diseases. The drug may be selected from: immunomodulatory drugs, inhibitors of HCV NS3 protease, other inhibitors of HCV polymerase, or inhibitors of other targets in the HCV life cycle.

The term "immunomodulator" as used herein refers to such drugs (compounds or biologicals) which are effective in potentiating or enhancing the immune system response in a mammal. These immunomodulators include, but are not limited to, class I interferons (e.g., alpha-, beta-, delta-, and omega interferons, tau-interferons, consensus interferons (consensus interferons), and asialo-interferons), class II interferons (e.g., gamma-interferons), and pegylated forms thereof (pegylated forms).

The term "inhibitor of HCV NS3 protease" as used herein refers to a drug (compound or biologic) that is effective in inhibiting the function of HCV NS3 protease in a mammal. These hcv ns3 protease inhibitors include, but are not limited to, the compounds described below: WO 99/07733, WO 99/07734, WO 00/09558, WO 00/09543, WO 00/59929, WO 02/060926, US2002/0177725, WO 03/053349, WO 03/062265, WO 03/064416, WO 03/064455, WO 03/064456, WO 03/099316, WO 03/099274, WO 2004/032827, WO2004/037855, WO 2004/043339, WO 2004/072243, WO 2004/093798, WO2004/094452, WO 2004/101602, WO 2004/101605, WO 2004/103996, the Boehringer Ingelheim clinical candidates identified as BILN 1 and the Vertex clinical candidate identified as VX 206950.

The term "other inhibitor of HCV polymerase" as used herein refers to a drug (compound or biologic) effective to inhibit HCV polymerase function in a mammal, wherein the drug has a structure different from a compound according to the present invention and preferably binds to a site of HCV polymerase different from the target site of a compound according to the present invention. Other inhibitors of HCV polymerase include non-nucleosides such as those described below: WO 2004/087714(IRBM), WO 04/005286(Gilead), WO 04/002977(Pharmacia), WO 04/002944(Pharmacia), WO04/002940(Pharmacia), WO 03/101993(Neogenesis), WO 03/099824(Wyeth), WO 03/099275(Wyeth), WO 03/099801(GSK)), WO 03/099801(GSK), WO 03/099801/03/099801 (Pfizer), WO 03/099801 (Viropharma), WO 03/099801 (B & CBiopharmarm), WO 03/099801 (Fujisawa), WO 03/099801 (Pfizer), WO 03/099801/062211 (Merck), WO 03/099801(GSK), EP 03/099801 (Shire), WO 03/099801/03/099801 (Rigel), WO 03/099801(GSK), WO 03/099801/03/099801 (GSK), WO 03/099801 (BMS), WO 03/099801/03/099801 a) and WO 03/099801 (Dong) a, WO 03/000254(Japan Tobacco), WO 02/100846A1 (fire), WO 02/100851A2 (fire), WO 02/098424A1(GSK), WO02/079187(Dong Wha), WO 03/02/20497(Shionogi), WO 02/06246(Merck), WO 01/47883(Japan Tobacco), WO 01/85172A1(GSK), WO 01/85720(GSK), WO 01/77091(Tularik), WO 00/18231(Viropharma), WO00/13708(Viropharma), WO 01/10573(Viropharma), WO 00/06529(Merck), EP 1256628A2(Agouron), WO 02/04425(Boehringer Ingelheim), WO03/007945(Boehringer Ingelheim), WO 03/010140(Boehringer Ingelheim), WO03/010141(Boehringer Ingelheim), WO 2004/064925(Boehringer Ingelheim), and WO 2004/065367(Boehringer Ingelheim). Other inhibitors of HCV polymerase also include nucleoside analogs, such as those described below: WO 04/007512(Merck/Isis), WO04/003000(Idenix), WO 04/002999(Idenix), WO 04/0002422(Idenix), WO04/003138(Merck), WO 03/105770(Merck), WO 03/105770(Merck), WO03/093290(Genelabs), WO 03/087298(Biocryst), WO 03/062256(Ribapharm), WO 03/062255(Ribapharm), WO 03/061385(Ribapharm), WO03/026675(Idenix), WO 03/026589(Idenix), WO 03/020222(Merck), WO03/000713(Glaxo), WO 02/100415(Hoffmann-La Roche), WO02/1094289(Hoffmann-La Roche), WO 02/051425 (Mitsishi), WO02/18404 (Hoffmann-Roche), WO 02/069903 (Biocris), WO 02/057287/Isceus 25 (Incenix), WO 365925/Irpex) WO 02/057425(Merck/Isis), WO 01/90121(Idenix), WO 01/60315 (fire) and WO 01/32153 (fire).

The term "inhibitor of another target in the HCV life cycle" as used herein refers to a drug (compound or biologic) that is effective in inhibiting the formation and/or replication of HCV in a mammal, rather than utilizing an RNA-dependent RNA polymerase that inhibits HCV, including drugs that interfere with the host or with the viral mechanisms required for virus formation and/or replication in a mammal. Inhibitors of additional targets in the HCV life cycle include, but are not limited to, drugs that inhibit a target selected from HCV helicase, HCV NS2/3 protease, and HCV IRES, as well as drugs that interfere with the function of other viral targets, including, but not limited to, the NS5A protein.

The term "HIV inhibitor" as used herein refers to a drug (compound or biologic) that is effective in inhibiting HCV formation and/or replication in a mammal. The agents include those that interfere with the host or interfere with the viral mechanisms required for the formation and/or replication of the HIV virus in the mammal. HIV inhibitors include, but are not limited to, nucleoside inhibitors, non-nucleoside inhibitors, protease inhibitors, fusion inhibitors, and integrase inhibitors.

The term "HAV inhibitor" as used herein refers to an agent (compound or biologic) that is effective to inhibit HAV formation and/or replication in a mammal. The agent includes agents that interfere with the host or interfere with the viral mechanisms required for HAV formation and/or replication in the mammalian body. Such HAV inhibitors include, but are not limited to, hepatitis A vaccines, e.g.(Glaxo SmithKline)、(Merck) and(Aventis Pasteur)。

the term "HBV inhibitor" as used herein refers to a drug (compound or biologic) that is effective in inhibiting HBV formation and/or replication in a mammal. The medicament comprises interfering with the host or interfering with HBV formation and/orDrugs that replicate the desired viral mechanisms. The HBV inhibitor comprises a medicament or an HBV vaccine for inhibiting HBV viral DNA polymerase. Specific examples of HBV inhibitors include, but are not limited to, lamivudine (II)) Adefovir Dipivoxil (Adefoviir Dipivoxil), Entecavir (Entecavir), FTC (F:))、DAPD(DXG)、L-FMAU() AM365 (Amrad), Ldt (Telbuvudine (Telbivudine)), Melovine-LdC (monoval-LdC) (Friedel-crafts), ACH-126,443(L-Fd4C) (Achillion), MCC478 (Eli Lilly), Racivir (RCV), fluorine (Fluoro) -L and D nucleosides, Robostata flavone (Robustaflavone), ICN 2001-3(ICN), Bam (Bam)205 (Novelos), XTL-001(XTL), iminosugar (Imino-Sugars) (Nonyl-DNJ) (Synergy), HepBzyme; and immunomodulator products such as: interferon alpha 2b, HE2000(Hollis-Eden), epitope polypeptide (Theradigm) (epimum), EHT899(Enzo Biochem), Thymosin (Thymosin) alpha-1 (r) (interferon alpha 2b, HE2000(Hollis-Eden), Thymosin peptide (Thymosin) alpha-1 (r)) HBV DNA vaccine (PowderJect), HBV DNA vaccine (Jefferon Center), HBV antigen (OraGen),(Bayer)), (Bayer (R))),(Nabi) and anti-hepatitis b (Cangene); and HBV vaccine products, such as the following: hepatitis B vaccine (Engerix B), hepatitis B vaccine (Recombivax HB) of Recombivax,Kinhavir hepatitis B vaccine (GenHevac B), hepatitis Care vaccine (Hepacare), Sheng-hepatitis B vaccine (Bio-Hep B), Loles vaccine (TwinRix), Kangweix (Comvax), and Haisekey (Hexavac).

The term "class I interferon" as used herein refers to an interferon selected from all interferons that bind to type I receptors. These include both naturally and synthetically produced class I interferons. Examples of class I interferons include, but are not limited to, alpha-, beta-, delta-, omega-interferons, tau-interferons, consensus interferons, asialo-interferons and pegylated forms thereof.

The term "class II interferon" as used herein refers to an interferon selected from all of the group of type II receptor-binding interferons. These include both naturally and synthetically produced class II interferons. Examples of class II interferons include, but are not limited to, gamma interferon and pegylated forms thereof.

As discussed above, combination therapies are also included wherein a compound of formula (I), or a pharmaceutically acceptable salt or ester thereof, is co-administered with at least one other agent selected from the group consisting of: antiviral drugs, immunomodulators, inhibitors of HCV NS3 protease, additional inhibitors of HCV polymerase, inhibitors of other targets in the HCV life cycle, HIV inhibitors, HAV inhibitors, and HBV inhibitors. Examples of such drugs are provided in the definitions section above. Particularly preferred examples of these drugs are listed below:

■ antiviral drugs: ribavirin or amantadine;

■ immunomodulator: class I interferons, class II interferons or pegylated forms thereof (pegylated forms);

■ HCV NS3 protease inhibitors;

■ other inhibitors of HCV polymerase: a nucleoside or non-nucleoside inhibitor;

■ an inhibitor of another target in the HCV life cycle which inhibits a target selected from the group consisting of: NS3 helicase, HCV NS2/3 protease, and an Internal Ribosome Entry Site (IRES), or is a drug that interferes with the function of the NS5A protein;

■ HIV inhibitor: a nucleoside inhibitor, a non-nucleoside inhibitor, a protease inhibitor, a fusion inhibitor or an integrase inhibitor; or

■ HBV inhibitor: a drug for inhibiting HBV virus DNA polymerase or a drug for HBV vaccine.

These other drugs may be combined with the compounds of the present invention to create a single pharmaceutical dosage form. Alternatively, these other agents may be administered to the patient separately as part of a plurality of dosage forms, for example using a kit. These additional agents may be administered prior to, simultaneously with, or after administration of the compound of formula (I) or a pharmaceutically acceptable salt or ester thereof.

As used herein, the term "treatment" refers to the administration of a compound or composition according to the present invention to reduce or eliminate the symptoms of a hepatitis C disease and/or to reduce the viral load in a patient.

As used herein, the term "prevention" refers to administration of a compound or composition according to the present invention after exposure of an individual to the virus but before symptoms of the disease appear and/or before a detectable level of the virus in the blood is reached.

PREFERRED EMBODIMENTS FOR CARRYING OUT THE INVENTION

Unless otherwise stated, including (but not limited to) R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R²¹、R⁵⁰、R^N1、R^N2All groups and substituents of A, B and Het have the definitions as defined hereinbefore or hereinafter. In the following, preferred embodiments, groups and substituents according to the present invention are described.

Core(s)：

The present invention comprises compounds of formula Ia:

alternatively, the invention comprises a compound of formula Ib:

R ¹：

according to a preferred embodiment R of the invention¹Selected from H, methyl and ethyl.

More preferably, R¹Is methyl.

R ²：

Preferably, R²Selected from halogen, cyano, (C)_1-4) Alkyl, (C)_2-4) Alkenyl, (C)_2-4) Alkynyl, (C)_3-6) Cycloalkyl, phenyl and Het selected from the following formulae:

wherein the phenyl and Het are unsubstituted or substituted by R²¹Is substituted in which R²¹As defined herein.

More preferably, R²Selected from the group consisting of Br, Cl, cyano, methyl, ethyl, propyl, 1-methylethyl, ethenyl, 1-methylethenyl, ethynyl, cyclopropyl, phenyl, and Het selected from the group consisting of:

R ²¹：

Preferably, R²¹Is a1, 2 or 3 substituent each independently selected from:

-1 to 3 substituents, each independently selected from halogen; and

-1 to 2 substituents each independently selected from:

a) hydroxy, (C)_1-4) Alkyl or (C)_1-4) An alkoxy group; wherein the alkyl and alkoxy groups are each optionally substituted with one, two or three halogen atoms;

b)-NR^N2R^N1wherein

R^N1Selected from H, (C)_1-3) Alkyl, -CO- (C)_1-3) Alkyl, -CO-O- (C)_1-3) Alkyl and Het;

wherein each of (C)_1-3) Alkyl, -CO- (C)_1-3) Alkyl and-CO-O- (C)_1-3) The alkyl portion of the alkyl group is optionally substituted with one, two or three groups selected from halogen and (C)_1-6) Substituted by a substituent of alkoxy; and wherein Het is a 5-or 6-membered monocyclic saturated, unsaturated or aromatic heterocyclic group having 1 to 2 heteroatoms each independently selected from N, O and S; and is

R^N2Is H or (C)_1-3) An alkyl group;

c)-CONR^N2R^N1wherein R is^N2And R^N1Each independently selected from H and (C)_1-3) An alkyl group; and

d) het, wherein Het is a 5-or 6-membered monocyclic heterocyclyl having 1,2 or 3 heteroatoms each independently selected from N, O and S.

More preferably, R²¹Is 1,2 or 3 substituents each independently selected from:

-1 to 2 substituents, each independently selected from fluorine, chlorine and bromine; and

-1 to 2 substituents each independently selected from:

a) hydroxy, methyl, ethyl, propyl, 1-methylethyl, methoxy, ethoxy, propoxy, or 1-methylethoxy; wherein the methyl, ethyl, propyl, 1-methylethyl, methoxy, ethoxy, propoxy, and 1-methylethoxy groups are optionally substituted with one, two, or three halogen atoms;

b)-N(CH₃)₂or-NHR^N1Wherein

R^N1Selected from H, methyl, ethyl, propyl, 1-methylethyl, -CO-CH₃2-pyridyl, 3-pyridyl and 4-pyridyl;

wherein said methyl, ethyl, propyl and 1-methylethyl are each optionally substituted by one, two or three members selected from halogen and (C)_1-3) Substituted by a substituent of alkoxy;

c)-CONH₂(ii) a And

d) 3-pyridyl, 4-pyridyl, 5-pyrimidyl, 2-furyl, 1-pyrrolyl and 1-morpholino.

Thus preferably, R²Selected from Br, Cl, cyano, methyl, ethyl, propyl, 1-methylethyl, cyclopropyl, vinyl, 1-methylvinyl, ethynyl, and,

More preferably, R²Selected from cyclopropyl, vinyl, 1-methylvinyl,

More preferably, R²Selected from:

most preferably, R²Selected from:

R ³：

preferably, R³Is cyclopentyl or cyclohexyl, each optionally substituted with one or two fluorine atoms.

More preferably, R³Is cyclopentyl or cyclohexyl.

R ⁴ And R ⁷：

Preferably R⁴Is H or halogen and R⁷Is H.

More preferably, R⁴Is H or Cl and R⁷Is H.

Most preferably, R⁴And R⁷Are all H.

R ⁵ And R ⁶：

Preferably, R⁵And R⁶One of which is selected from:

a) by COOH or CONHR^N1Substituted (C)_2-4) Alkenyl, wherein R^N1Is selected from H and (C)_1-3) Alkyl, said alkenyl optionally being further substituted by one or two substituents independently chosen from (C)_1-3) Alkyl and halogen;

b) phenyl or Het, each optionally substituted with one or two substituents each independently selected from:

i. -OH, oxo, COOH;

optionally substituted by phenyl or-N (R)^N2)R^N1Substituted (C)_1-3) Alkyl radical, wherein R^N1And R^N2Each independently selected from H and (C)_1-3) Alkyl, or R^N1And R^N2Together with the nitrogen atom to which they are attached form a 5-or 6-membered monocyclic, saturated, unsaturated or aromatic N-containing heterocycle, which heterocycle optionally further has one or two heteroatoms each independently selected from N, O and S; and

iii.-N(R^N2)R^N1(ii) a Wherein R is^N1Selected from H, (C)_1-3) Alkyl and-CO (C)_1-3) Alkyl and R^N2Is H or (C)_1-3) An alkyl group;

wherein Het is a 5-or 6-membered monocyclic, saturated, unsaturated or aromatic heterocycle having 1 to 3 heteroatoms each independently selected from O, N and S; and

c)COOH；

and R is⁵And R⁶Is selected from H, NHR^N1、(C_1-3) Alkyl and (C)_1-3) Alkoxy radical, wherein R^N1Selected from H and-CO-O- (C)_1-6) An alkyl group.

More preferably, R⁵And R⁶One of which is selected from:

a) by COOH or-CONH₂Substituted (C)_2-4) Alkenyl, optionally further substituted by one or two groups selected from (C)_1-3) Alkyl and halogen; and

i. -OH, oxo, COOH;

ii.(C_1-3) Alkyl, optionally substituted by phenyl, -N (CH)₃)₂OrSubstitution; and

iii.-NH₂,-N(CH₃)₂and-NHCOCH₃；

Wherein Het is selected from the following formulae:

c)COOH；

and R is⁵And R⁶Is selected from H, methyl, methoxy, ethoxy, -NH₂and-NHCO-O-CH (CH)₃)₂。

More preferably, R⁵And R⁶One of which is selected from:

a) -CH = CH-COOH or-CH = CH-CONH₂Each optionally substituted with one or two substituents selected from methyl, ethyl and fluoro; and

b) optionally by NH₂Substituted phenyl, or

Het, optionally substituted with one or two substituents each independently selected from:

i. -OH, oxo, COOH;

methyl or ethyl, each optionally substituted by phenyl, -N (CH)₃)₂OrSubstitution; and

iii.-NH₂、-N(CH₃)₂and-NHCOCH₃；

Wherein Het is selected from the following formulae:

c)COOH；

Still more preferably, R⁵And R⁶One is selected from-COOH,

Most preferably, R⁵And R⁶One isOrAnd R is⁵And R⁶And the other is H.

Or most preferably, R⁵And R⁶One of which is selected from:

and R is⁵And R⁶And the other is H.

R ⁸：

Preferably, R⁸Is selected from (C)_1-5) Alkyl, (C)_4-6) Cycloalkyl and (C)_3-4) Cycloalkyl- (C)_1-3) Alkyl group of which (C)_1-5) Alkyl is optionally substituted by (C)_1-3) Alkoxy groups or substituted with one to three fluorine atoms.

More preferably, R⁸Selected from the group consisting of methyl, ethyl, propyl, 1-methylethyl, 2-methylpropyl, 3-methylbutyl, cyclobutyl, cyclopropylmethyl, 2-fluoroethyl, 2,2, 2-trifluoroethyl and 2-methoxyethylAnd (3) an ethyl group.

Most preferably R⁸Is methyl.

R ⁹ And R ¹⁰：

Preferably, R⁹And R¹⁰Each independently selected from (C)_1-3) Alkyl, or R⁹And R¹⁰Are linked and form together with the carbon atom to which they are linked (C)_3-6) Cycloalkyl group, (C)_5-6) Cycloalkenyl or a 5-or 6-membered monocyclic heterocycle having 1 to 2 heteroatoms each independently selected from O and N; wherein the cycloalkyl, cycloalkenyl or heterocycle is each optionally substituted by (C)_1-4) Alkyl substitution.

More preferably, the groupSelected from:

more preferably, the groupSelected from:

most preferably, the groupIs composed of

Compounds of formula I are included within the scope of the present invention:

wherein:

a or B is N and the other of B or A is C, wherein- - -between two C-atoms represents a double bond and- - -between the C-atom and the N-atom represents a single bond;

R¹is H or (C)_1-6) An alkyl group;

R²is halogen, aryl or Het; the aryl and Het are optionally substituted by R²¹Substitution;

wherein R is²¹Is a mono-, di-or tri-substituent each independently selected from-OH, -CN,

-N(R^N2)R^N1halogen, (C)_1-6) Alkyl, (C)_1-6) Alkoxy group, (C)_1-6) Alkylthio, Het and-CO-N (R)^N2)R^N1；

Wherein the alkyl, alkoxy and alkylthio groups are each optionally substituted with one, two or three halogen atoms;

R⁵and R⁶One is selected from COOH, -CO-N (R)^N2)R^N1Het and (C)_2-6) Alkenyl, wherein Het, (C)_2-6) Alkenyl and R^N1Or at R^N2And R^N1Any heterocyclic ring formed betweenEach is optionally substituted by R⁵⁰Substitution;

wherein R is⁵⁰Is a mono, di or tri substituent each independently selected from (C)_1-6) An alkyl group, -COOH,

-N(R^N2)R^N1,-CO-N(R^N2)R^N1and halogen;

R⁹and R¹⁰Each independently selected from (C)_1-6) An alkyl group; or R⁹And R¹⁰Are linked and form together with the carbon atom to which they are linked (C)_3-7) Cycloalkyl group, (C)_3-7) Cycloalkenyl or a 4-, 5-or 6-membered heterocyclic ring having 1 to 3 heteroatoms, each independently selected from O, N and S;

wherein the cycloalkyl, cycloalkenyl or heterocyclyl are each optionally substituted by (C)_1-4) Alkyl substitution;

wherein all of said alkyl and cycloalkyl groups are optionally substituted by one, two or three each independently selected from halo

Element, (C)_1-6) Alkoxy and (C)_1-6) Substitution of alkylthio groupsSubstituted by radicals; and is

R^N2Is H or (C)_1-6) Alkyl, or

R^N2And R^N1May be linked and taken together with the nitrogen atom to which they are attached form a 4-,5-, 6-, or 7-membered saturated or unsaturated N-containing heterocycle, or an 8-, 9-, 10-, or 11-membered N-containing heterobicyclic ring, each having from 1 to 3 heteroatoms each independently selected from O, N and S;

wherein the group R^N2And R^N1The heterocyclic or heterobicyclic ring formed being optionally substituted by one, two or three each

Independently selected from halogen, (C)_1-6) Alkyl, (C)_1-6) Alkoxy and (C)_1-6) Substituted by a substituent of alkylthio; wherein Het is defined as a 4-,5-, 6-or 7-membered heterocycle having 1 to 4 heteroatoms each independently selected from O, N and S, which may be saturated, unsaturated or aromatic, or an 8-, 9-, 10-or 11-membered heterobicyclic ring having 1 to 5 heteroatoms each independently selected from O, N and S-reducing at possible positions, which may be saturated, unsaturated or aromatic;

or a salt thereof.

Also included within the scope of the present invention are compounds of formula (I), in particular formula Ia or Ib, wherein:

R¹selected from H, methyl and ethyl;

R²selected from halogen, cyano, (C)_1-4) Alkyl, (C)_2-4) Alkenyl, (C)_2-4) Alkynyl, (C)_3-6) Cycloalkyl, phenyl and Het selected from the following formulae:

wherein the phenyl and Het are unsubstituted or substituted by R²¹Is substituted in which R²¹Is 1,2 or 3 substituents each independently selected from:

-1,2 or 3 substituents, each independently selected from halogen; and

-1 or 2 substituents, each independently selected from:

b)-NR^N2R^N1wherein

wherein each of (C)_1-3) Alkyl, -CO- (C)_1-3) Alkyl and-CO-O- (C)_1-3) The alkyl portion of the alkyl group is optionally substituted with one, two or three groups selected from halogen and (C)_1-6) Substituted by a substituent of alkoxy; and wherein Het is a 5-or 6-membered monocyclic saturated, unsaturated or aromatic heterocycle having 1 or 2 heteroatoms each independently selected from N, O and S; and is

R^N2Is H or (C)_1-3) An alkyl group;

d) het, wherein Het is a 5-or 6-membered monocyclic heterocycle having 1,2 or 3 heteroatoms each independently selected from N, O and S;

R³is cyclopentyl or cyclohexyl, each optionally substituted with one to four fluorine atoms;

R⁴is H or halogen and R⁷Is H;

R⁵and R⁶One of which is selected from:

a) by COOH or CONHR^N1Substituted (C)_2-4) Alkenyl, wherein R^N1Is selected from H and (C)_1-3) Alkyl, said alkenyl being optionally further substituted by one or two substituents each independently selected from (C)_1-3) Alkyl and halogen substituents;

i. -OH, oxo, COOH;

optionally substituted by phenyl or-N (R)^N2)R^N1Substituted (C)_1-3) Alkyl radical, wherein R^N1And R^N2Each independently selected from H and (C)_1-3) Alkyl, or R^N1And R^N2(ii) are linked and taken together with the nitrogen atom to which they are attached form a 5-or 6-membered monocyclic, saturated, unsaturated or aromatic N-containing heterocycle optionally further bearing one or two heteroatoms each independently selected from N, O and S; and

wherein Het is a 5-or 6-membered monocyclic saturated, unsaturated or aromatic heterocycle having 1 to 3 heteroatoms each independently selected from O, N and S; and

c)COOH；

and R is⁵And R⁶Is selected from H, NHR^N1、(C_1-3) Alkyl and (C)_1-3) An alkoxy group,

wherein R is^N1Selected from H and-CO-O- (C)_1-6) An alkyl group;

R⁸is selected from (C)_1-5) Alkyl, (C)_4-6) Cycloalkyl and (C)_3-4) Cycloalkyl- (C)_1-3) Alkyl radical, wherein(C_1-5) Alkyl is optionally substituted by (C)_1-3) Alkoxy or one to three fluorine atoms; and is

R⁹And R¹⁰Each independently selected from (C)_1-3) Alkyl, or R⁹And R¹⁰Are linked and form together with the carbon atom to which they are linked (C)_3-6) Cycloalkyl group, (C)_5-6) Cycloalkenyl or a 5 or 6 membered heterocyclic ring having 1 to 2 heteroatoms selected from O and N; wherein the cycloalkyl, cycloalkenyl or heterocycle is optionally substituted by (C)_1-4) Alkyl substitution.

More preferably still, the first and second liquid crystal compositions are,

R¹selected from H, methyl and ethyl;

R²selected from Br, Cl, cyano, methyl, ethyl, propyl, 1-methylethyl, ethenyl, 1-methylethenyl, ethynyl, cyclopropyl, phenyl and Het selected from the following:

-1 to 2 substituents each independently selected from:

b)-N(CH₃)₂or-NHR^N1Wherein

wherein the methyl, ethyl, propyl and 1-methylethyl are each optionally substituted by one, two or three members selected from halogen and (C)_1-3) Alkoxy reducing substituent;

c)-CONH₂(ii) a And

d) 3-pyridyl, 4-pyridyl, 5-pyrimidinyl, 2-furyl, 1-pyrrolyl and 1-morpholino;

R³is cyclopentyl or cyclohexyl, each optionally substituted by one or two fluorine atoms;

R⁴is H or halogen and R⁷Is H;

R⁵and R⁶One of which is selected from:

i. -OH, oxo, COOH;

ii(C_1-3) Alkyl, optionally substituted by phenyl, -N (CH)₃)₂OrSubstitution; and

iii.-NH₂,-N(CH₃)₂and-NHCOCH₃；

Wherein Het is selected from the following formulae:

c)COOH；

and R is⁵And R⁶Is selected from H, methyl, methoxy, ethoxy, -NH₂and-NHCO-OCH (CH)₃)₂；

R⁸Selected from the group consisting of methyl, ethyl, propyl, 1-methylethyl, 2-methylpropyl, 3-methylbutyl, cyclobutyl, cyclopropylmethyl, 2-fluoroethyl, 2,2, 2-trifluoroethyl and 2-methoxyethyl; and is

The groupSelected from:

more preferably

R¹Selected from H, methyl and ethyl;

R²selected from the group consisting of Br, Cl, cyano, methyl, ethyl, propyl, 1-methylethyl, ethenyl, 1-methylethenyl, ethynyl, cyclopropyl, phenyl, and Het selected from the group consisting of:

-1 to 2 substituents each independently selected from:

b)-N(CH₃)₂or-NHR^N1Wherein

wherein the methyl, ethyl, propyl and 1-methylethyl are each optionally substituted by one, two or three members selected from halogen and (C)_1-3) Substituted by a substituent of alkoxy;

c)-CONH₂(ii) a And

d) 3-pyridyl, 4-pyridyl, 5-pyrimidinyl, 2-furyl, 1-pyrrolyl and 1-morpholino;

R³is cyclopentyl or cyclohexyl, optionally substituted by one or two fluorine atoms;

R⁴is H or Cl and R⁷Is H;

R⁵and R⁶One of which is selected from:

b) optionally by NH₂Substituted phenyl, or

Het optionally substituted with one or two substituents each independently selected from:

i. -OH, oxo, COOH;

iii.-NH₂、-N(CH₃)₂and-NHCOCH₃；

Wherein Het is selected from the following formulae:

c)COOH；

Radical (I)Selected from:

most preferably, the first and second substrates are,

R¹is methyl;

R²selected from:

R³is cyclopentyl or cyclohexyl;

R⁴and R⁷Are all H;

R⁵and R⁶One isOrAnd R is⁵And R⁶Is H;

R⁸is methyl; and is

Radical (I)Is composed of

Or most preferably,

R¹is methyl;

R²selected from:

R³is cyclopentyl or cyclohexyl;

R⁴and R⁷Are all H;

R⁵and R⁶One is

And R is⁵And R⁶Is H;

R⁸is methyl; and a groupIs composed of

Each of the individual compounds of formula I present in tables 1 to 4 is also included within the scope of the present invention.

Polymerase activity

The ability of the compound of formula (I) to inhibit RNA synthesis using the RNA-dependent RNA polymerase of HCV can be confirmed by any method capable of measuring the HCV RNA-dependent RNA polymerase activity. Suitable experiments are described in the examples.

Specialization of RNA-dependent RNA polymerase Activity

To demonstrate that the compounds of the present invention act by specific inhibition of HCV polymerase, the compounds can be tested for inhibitory activity in assays that measure the activity of RNA-dependent RNA polymerase rather than HCV polymerase, or in DNA-dependent RNA polymerase assays.

Cell-based HCV RNA replication Activity

The ability of the compounds of the present invention to inhibit HCV RNA replication in a cell can be demonstrated by the inhibitory activity of the compounds in cell-based HCV RNA replication experiments. Suitable experiments are described in the examples.

When a compound of formula (I) or one of its therapeutically acceptable salts is used as an antiviral agent, it may be administered orally, topically or systemically to a mammal, including but not limited to a human, bovine, porcine, canine, feline, rabbit or mouse, in a medium (vehicle) that includes one or more pharmaceutically acceptable carriers, in proportions determined by the solubility and chemical properties of the compound, the chosen route of administration and standard biological practice.

For oral administration, the compound or therapeutically acceptable salt thereof may be formulated in unit dosage form, such as capsules or tablets, each containing a predetermined amount of the active ingredient in a pharmaceutically acceptable carrier, ranging from about 1 to about 500 mg.

For topical administration, the compounds may be formulated in a pharmaceutically acceptable medium containing from about 0.1% to about 5%, preferably from about 0.5% to about 5%, of the active agent. Such formulations may be in the form of solutions, creams or lotions.

For systemic administration, the compound of formula (I) may be administered in combination with a pharmaceutically acceptable vehicle or carrier via intravenous, subcutaneous or intramuscular injection. For injectable administration, it is preferred that the compounds are used in solution in a sterile aqueous medium which may also contain other solutes such as buffers or preservatives, and sufficient pharmaceutically acceptable salts or glucose to render the solution isotonic.

Suitable media or carriers for use in The above mentioned formulations are described in The Pharmaceutical literature, for example in "Remington's The Science and Practice of Pharmacy", 19 th edition, Mack publishing Company, Easton, Penn, 1995 or in "Pharmaceutical Dosage Forming Drugs Delivery Systems", 6 th edition, H.C.Ansel et al, eds., Williams & Wilkins, Baltimore, Maryland, 1995.

The dosage of the compounds will vary with the form of administration and the particular active agent selected. Again, it will vary with the particular host undergoing treatment. In general, treatment is initiated with small increases until the optimum effect is reached in this case. In general, the most desirable concentration level at which the compound of formula I is administered is that which will generally provide antiviral efficacy without causing any injurious or deleterious side effects.

For oral administration, the compound or therapeutically acceptable salt may be administered in the range of about 0.01 to about 200mg per kg of body weight per day, with a preferred range of about 0.05 to about 100mg per kg.

For systemic administration, the compound of formula (I) may be administered in a dose of from about 0.01mg to about 100mg per kg of body weight per day, although the foregoing variations will occur. Dosage levels in the range of about 0.05mg to about 50mg per kilogram of body weight per day are the most desirable dosage levels to achieve effective results.

When the compositions of the present invention comprise a compound of formula I in combination with one or more other therapeutic or prophylactic agents, the compound and the other agent should generally be present in a dosage of between about 10% to 100%, and more preferably between about 10% and 80%, administered in a monotherapy regimen.

When these compounds, or their pharmaceutically acceptable salts, are formulated with a pharmaceutically acceptable carrier, the resulting compositions can be administered in vivo to a mammal, such as a human, to inhibit HCV polymerase or to treat or prevent HCV viral infection. The treatment can also be achieved using the compounds of the present invention in combination with the following drugs, including (but not limited to): immunomodulators, including (but not limited to) alpha-, beta-, delta-, gamma-, tau-and omega-interferons or pegylated forms thereof; other antiviral drugs, such as ribavirin, amantadine; other inhibitors of HCV NS5B polymerase; inhibitors of other targets in the HCV life cycle, including (but not limited to) drugs that inhibit targets including (but not limited to) HCV helicase, HCV NS2/3 protease, HCV NS3 protease, and HCV IRES, as well as drugs that interfere with the function of other viral targets including (but not limited to) the NS5A protein; or a combination thereof. Other drugs may be combined with the compounds of the present invention to create a single dosage form. Or these other drugs may be administered separately to the mammal as part of a variety of dosage forms.

Methods and Synthesis

The synthesis of the compounds according to the invention is preferably accomplished by the following general procedure described in scheme 1.

Reaction scheme 1

A compound of formula I, wherein R¹，R²，R³，R⁴，R⁵，R⁶，R⁷，R⁸，R⁹And R¹⁰As defined hereinbefore, are preferably prepared by coupling carboxylic acids of formula II with amines of formula III as illustrated above in scheme 1, using carboxy-activating reagents well known to those skilled in the art. These agents include, but are not limited to, TBTU, HATU, BOP, BrOP, EDAC, DCC, isobutyl chloroformate, and the like. Alternatively, the carboxylic acid of formula II may be converted to the corresponding acid chloride using standard reagents and then coupled with the amine derivative of formula III. At R⁵Or R⁶In the case of ester-protected carboxylic acid groups, saponification reactions are carried out (using protocols well known to those skilled in the art) to obtain the final inhibitor product of the free carboxylic acid.

The intermediate carboxylic acids of formula II can be prepared using the methods described in WO03/010141 or using the methods described in the examples below. The intermediate amines of formula III can be prepared according to the methods outlined in scheme 2 and scheme 3 below.

Reaction formula 2

The amine intermediates of formula III in scheme 1 can be prepared from the corresponding diamine precursors of formula IV by coupling with the appropriate α, α -disubstituted amino acid chloride hydrochloride. The preparation of the chloride hydrochloride of the appropriate α, α -disubstituted amino acid from the corresponding α, α -disubstituted amino acid can be carried out as described in WO03/007945 or WO03/010141, or as described in e.s.uffelman et al (org.lett.1999,1,1157) or variations thereof. The amide intermediate formed in this coupling reaction is then cyclized using heating with acetic acid to provide the amine intermediate of formula III.

Reaction formula 3

Alternatively, the amine intermediates of formula III in scheme 1 can be prepared from the corresponding diamine precursors of formula IV by coupling with the appropriate Boc-protected α, α -disubstituted amino acid as illustrated in scheme 3, using coupling reagents well known to those skilled in the art, such as TBTU, HATU, BOP, BrOP, EDAC, DCC, isobutyl chloroformate, and the like. Suitable Boc-protected α, α -disubstituted amino acids can be prepared from free α, α -disubstituted amino acids using standard conditions well known to those skilled in the art, e.g., and Boc₂O (di-t-butyl dicarbonate) is reacted in the presence of a tertiary amine such as triethylamine and the like. The amide intermediate formed in the coupling reaction is then cyclized by heating with acetic acid. Deprotection of the Boc group to provide the amine intermediate of formula III in scheme 1 can be performed using standard reagents well known to those skilled in the art. Such reagents include, but are not limited to, trifluoroacetic acid, HCl in bisSolutions in alkanes, and the like.

The diamine precursors of general formula IV in equations 2 and 3 can preferably be prepared using the methods described in the examples, including any variations of these methods, and/or using other synthetic procedures well known to those skilled in the art.

An amine intermediate of formula III in scheme 1 wherein R⁵And R⁶One is-CH = C (R)^50a) -COOR, wherein R^50aSelected from H, (C)_1-6) Alkyl and halogen and where R is, for example, methyl or ethyl, can be prepared from the corresponding amine intermediates of the general formula III or suitably protected derivatives thereof, where R is⁵And R⁶One is-COOR, wherein R is, for example, methyl or ethyl, prepared by the method of the following reaction formula 4. Reaction scheme 4 specifically illustrates the preparation of an amine intermediate of formula III wherein R⁵is-CH = C (R)^50a) -COOR, as understood by those skilled in the art when R is⁶When it is-COOR, R will be obtained by the method of the formula⁶is-CH = C (R)^50a) -COOR products. Also, those skilled in the art will appreciate that when diamine precursors of the general formula IV in the above schemes 2 and 3, or suitably protected derivatives thereof, or intermediates useful in their preparation, are used, wherein R is⁵And R⁶One is-COOR, into a diamine precursor of formula IV, or a suitably protected derivative thereof or an intermediate useful in the preparation thereof, wherein R is⁵And R⁶One is-CH = C (R)^50a) -COOR, wherein R^50aAnd R is as defined above, the process of equation 4 or a variation thereof may also be used.

Reaction formula 4

The appropriately protected amine intermediate of formula IIIa in scheme 4 above may be converted to an alcohol intermediate of formula IIIb by treatment with a suitable reducing agent such as DIBAL-H and the like. Suitable Protecting Groups (PG) include, but are not limited to, urethane protecting groups such as Boc (t-butyloxycarbonyl) and the like. Preparation of the protected amine intermediate of formula IIIa from the amine intermediate of formula III in scheme 1 above can be prepared by standard methods well known to those skilled in the art. The alcohol intermediate IIIb can be converted to the aldehyde intermediate IIIc using standard oxidizing agents well known to those skilled in the art, such as 1,1, 1-tris (acetyloxy-1, 1-dihydro-1, 2-benzodioxol) -3- (1H) -one (also known as Dess-Martin Periodinane) and the like.

The aldehyde intermediate IIIc can be converted to the amine intermediate of formula IIId using standard Horner-Emmons procedures or related procedures well known to those skilled in the art, such as the Wittig method and the like, followed by deprotection of the PG group using well known standard methods. In the case where the PG group is Boc, such methods include, but are not limited to, treatment with an acid, such as trifluoroacetic acid, dissolved in bisHCl of an alkane, and the like.

The amine intermediate of formula III in reaction scheme 1, wherein R⁵And R⁶One of them is-C (R)⁵⁰) = CH-COOR, wherein R⁵⁰Is (C)_1-6) Alkyl groups, wherein R is, for example, methyl or ethyl, can be prepared from intermediate IIIc in the above reaction scheme 4 using the method of the following reaction scheme 5. Although equation 5 specifically illustrates amine intermediates of formula III wherein R⁵is-C (R)⁵⁰) Preparation of = CH-COOR, as R will be understood by those skilled in the art⁶In the case of-CHO, the method illustrated by this equation or a variation thereof will result in a reaction wherein R is⁶is-C (R)⁵⁰) Products of = CH-COOR. Also, those skilled in the art will appreciate that the process of scheme 5 or a variation thereof may also be used when a diamine precursor of formula IV in scheme 2 or 3 above, or a suitably protected derivative thereof or an intermediate useful in the preparation thereof, wherein R is⁵And R⁶One is-CHO, before conversion to a diamine of the formula IVA compound of formula (I), or a suitably protected derivative thereof, or an intermediate useful in the preparation thereof, wherein R is⁵And R⁶One of them is-C (R)⁵⁰) Case of = CH-COOR, where R⁵⁰And R is as defined hereinbefore.

Reaction formula 5

The aldehyde intermediate IIIc (from equation 4) can be alkylated with a suitable nucleophilic alkylating agent well known to those skilled in the art, such as an alkyl lithium and the like, and then the secondary alcohol is oxidized to a ketone with an oxidizing agent well known to those skilled in the art, such as 1,1, 1-tris (acetyloxy-1, 1-dihydro-1, 2-benzodioxol-3- (1H) -one (also known as Dess-Martin periodinane) and the like, to achieve the conversion to a ketone of general formula IIIf. E.g. trifluoroacetic acid, in bisHCl in alkanes, etc.

Alternatively, the amine intermediate of formula III in scheme 1, wherein R⁵And R⁶One is-CH = C (R)^50a) -COOR, wherein R^50aSelected from H, (C)_1-6) Alkyl and halogen and wherein R is (C)_1-6) Alkyl, from the corresponding amine intermediate of formula III or a suitably protected derivative thereof, wherein R⁵And R⁶One is X, where X is a leaving group such as a halogen atom, sulfonate ester, etc., prepared using typical conditions for the Heck reaction described in equation 6 and further described in the examples below. Although equation 6 specifically illustrates amine intermediates of formula III wherein R⁵is-C (R)⁵⁰) Preparation of = CH-COOR, as R will be understood by those skilled in the art⁶When X, the process illustrated by this equation or a variation thereof will result in R wherein⁶is-C (R)⁵⁰) Products of = CH-COOR. At the same time, it will be appreciated by those skilled in the art that the process of scheme 6 or a variation thereof may also be used when a diamine precursor of formula IV in scheme 2 or 3 above, or a suitably protected derivative thereof or an intermediate useful in the preparation thereof, wherein R is⁵And R⁶One is X, into a diamine precursor of the general formula IV, or a suitably protected derivative thereof, or an intermediate useful in the preparation thereof, wherein R is⁵And R⁶One of them is-C (R)⁵⁰) Case of = CH-COOR, where R⁵⁰And R is as defined hereinbefore.

Reaction formula 6

Examples

The invention is illustrated in further detail in the following non-limiting examples. As is well known to those skilled in the art, the reaction is carried out in nitrogen or argon when it is desired to protect the reaction components from exposure to air or moisture. Temperatures are expressed in degrees celsius. Flash chromatography was performed on silica gel. The percent or ratio of the solution represents a volume to volume relationship unless otherwise indicated. The mass spectrometry results were recorded using an electrospray mass spectrometer. Analytical HPLC was performed under standard conditions using Combioscreen ODS-AQ C₁₈Reverse phase column, YMC, 50X 4.6mm i.d., 5. mu.M, 120A at 220nM, with a linear gradient described in the table below (solvent A0.1% TFA in H)₂O is in; solvent B is 0.1% TFA in CH₃CN) elution:

time (min)	Flow rate (mL/min)	Solvent A (%)	Solvent B (%)
				0	3.0	95	5
0.5	3.0	95	5
				6.0	3.0	50	50
10.5	3.5	0	100

The following abbreviations or symbols may be used in the foregoing and in the following text:

AcOH: acetic acid;

Ac₂o: acetic anhydride;

BOC or BOC: tert-butyloxycarbonyl;

BOP: benzotriazol-1-yloxy-tris (dimethylamino)A hexafluorophosphate salt;

and (3) BroP: bromo tri (dimethylamino)A hexafluorophosphate salt;

bu: a butyl group;

CPS: counting per second;

DAST: (diethylamino) sulfur trifluoride;

dba: dibenzylidene acetone;

DCC: 1, 3-dicyclohexylcarbodiimide;

DCM: dichloromethane;

DCMA: dicyclohexylmethylamine;

DIBAL-H: di-isobutyl aluminum hydride;

DMEM: modified Earle medium;

DMF: n, N-dimethylformamide;

DMSO, DMSO: dimethyl sulfoxide;

EC₅₀: 50% effective concentration;

DEAC: see EDC;

EDC: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride;

ES^-: electrospray (negative ionization);

ES⁺: electrospray (positive ionization);

et: an ethyl group;

Et₂o: diethyl ether;

EtOAc: ethyl acetate;

EtOH: ethanol;

FBS: fetal bovine serum;

fmoc: 9-fluorenylmethoxycarbonyl;

HATU: o- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylureaA hexafluorophosphate salt;

HBTU: O-benzotriazol-1-yl-N, N, N ', N' -tetramethylureaA hexafluorophosphate salt;

HOAT: 1-hydroxy-7-azabenzotriazole;

HOBt: 1-hydroxybenzotriazole;

HPLC: high performance liquid chromatography;

ⁱpr or i-Pr: isopropyl group;

me: a methyl group;

MeCN: acetonitrile;

MeOH: methanol;

ms (es): an electrospray mass spectrometer;

NMR: a nuclear magnetic resonance spectrometer;

PBS: phosphate buffer;

ph: a phenyl group;

PG: a protecting group;

PVDF: polyvinylidene fluoride;

RT: room temperature (about 25 ℃);

TBME: tert-butyl methyl ether;

TBTU: 2- (1H-benzotriazol-1-yl) -N, N, N ', N' -tetramethylureaA tetrafluoroborate salt;

tBu: a tertiary butyl group;

tf: a trifluoromethylsulfonyl group;

TfO: trifluoromethanesulfonate;

TFA: trifluoroacetic acid;

THF: tetrahydrofuran;

TLC: thin layer chromatography;

TMS: a trimethylsilyl group;

troc: trichloroethoxycarbonyl.

Example 1

3- (3, 3-Difluorocyclopentyl) -1-methyl-1H-indole-6-carboxylic acid methyl ester

Step 1:

indole-6-carboxylic acid 1-1(5.0g,31.0mmol) was dissolved in MeOH (100mL) and a catalytic amount of H was added₂SO₄(1.0mL) and the reaction mixture was stirred at reflux for 16 h. A small amount of solid K₂CO₃Is added to neutralize excess H₂SO₄And stirring was continued at RT for 1 h. The reaction mixture was concentrated in vacuo to remove MeOH and taken up with NaHCO₃Saturated aqueous solution (. about.50 mL) was diluted and extracted with EtOAc (. about.200 mL). The organic layer was washed with brine (100mL) over anhydrous MgSO₄Medium drying and concentratingAnd (5) condensing to dry. The resulting residue was purified by flash column chromatography using 30% EtOAc in hexanes as eluent to give pure methyl ester 1-2(4.78g,88% yield).

Step 2:

the methyl ester 1-2(3.31g,18.9mmol) obtained in step 1 was dissolved in MeCN (50mL) and a catalytic amount of Yb (OTf) was added₃(586mg,0.95 mmol). 2-Cyclopenten-1-one (7.76mL,94.5mmol) was added and the reaction mixture was stirred at reflux for 16 h. MeCN solvent was removed under vacuum, the residue was redissolved in EtOAc (. about.200 mL), and saturated NaHCO was used₃Aqueous solution (. about.100 mL), H₂O (50mL) and brine (50 mL). The organic layer was washed with anhydrous MgSO₄Dried and concentrated to dryness in vacuo. After purification of the residue by flash column chromatography, the desired cyclopentanone adduct 1-3 was isolated using 40% EtOAc in hexanes as a solution gradient to give a beige powder (3.4g,70% yield).

And step 3:

NaH (60% dispersion in oil, 770mg,19.2mmol) was slowly added to a solution of cyclopentanone adduct intermediates 1-3(3.81g,14.8mmol) obtained in step 2, in anhydrous DMF (150mL) at 0 deg.C. The reaction mixture was stirred at 0 ℃ for 5 minutes, then MeI (1.2mL,19.2mmol) was added dropwise and stirring continued at 0 ℃ for 3 hours. The mixture was warmed to RT and quenched by addition of NH₄The reaction was terminated with saturated aqueous Cl (200 mL). The mixture was extracted with EtOAc (2X 500mL) and taken with NH₄Saturated aqueous Cl solution (2X 200mL) H₂The organic layer was washed with O (200mL) and brine (200 mL). The combined organic layers were over anhydrous MgSO₄Dry, evaporate to dryness and purify the residue by flash column chromatography (using 30% EtOAc in hexanes as eluent) to isolate the N-methylindole intermediates 1-4 as a beige solid (3.1g,77% yield).

And 4, step 4:

the N-methylindole intermediates 1-4 from step 3 (1.4g,5.16mmol) and DAST (2.7mL, 20.6mmol) were dissolved in CH in a sealed tube₂Cl₂(50mL) and stirred at reflux for 3 days. The mixture was slowly poured into NaHCO₃Saturated aqueous solution (-50 mL) and when CO is present₂When no longer generating, using CH₂Cl₂The mixture was extracted (2X 100 mL). The combined organic layers were washed with brine (50mL) over anhydrous MgSO₄Dried and concentrated to dryness. The residue was purified by flash column chromatography (using from 10% to 20% EtOAc in solvent gradient hexanes) to isolate methyl 3- (3, 3-difluorocyclopentyl) -1-methyl-1H-indole-6-carboxylate 1-5(750mg,50 yield).

Conversion of methyl 3- (3, 3-difluorocyclopentyl) -1-methyl-1H-indole-6-carboxylate 1-5 to the carboxylic acid intermediate of formula IIa, wherein R is R, using the method described in WO03/010141²As hereinbefore defined. These intermediates can be converted to compounds of formula I using reaction scheme 1 above and the methods described in WO 03/010141.

Example 2

(E) -3- (3-amino-4-methylaminophenyl) acrylic acid methyl ester

Step 1:

a mixture of 4-chloro-3-nitrocinnamic acid 2-1(500mg,2.2mmol) and a solution of methylamine in THF (2M,8mL,16mmol) was heated in a sealed tube at 80 ℃ for 20 h. The mixture was then cooled to room temperature and concentrated to give 2-2 as an orange solid, which was used without further purification in the following step.

It will be readily apparent to those skilled in the art that other diamine intermediates of formula IV in schemes 2 and 3 above, wherein R is⁸Instead of methyl, a suitable R may be utilized⁸-NH₂Instead of methylamine (CH) in the above step 1₃NH₂) And then the preparation.

Step 2:

the crude 4-methylamino-3-nitrocinnamic acid intermediate 2-2(488mg,2.2mmol) from step 1 was dissolved in methanol (20mL) and diazomethane in ether was added until HPLC analysis showed complete conversion of the acid to the methyl ester. The solution was concentrated to dryness to give 540mg of methyl ester 2-3 as an orange solid, which was used in step 3 without further purification.

And step 3:

crude methyl ester 2-3(540mg,. about.2.2 mmol) from step 2 and SnCl₂The dihydrate (2.25g,10mmol) was dissolved in ethanol (20mL) and the mixture was stirred at 80 ℃ for 4 hours. After that period, the mixture was cooled to room temperature and slowly added to NaHCO₃In a saturated aqueous solution. The reaction mixture was extracted with ethyl acetate (100mL), and the organic layer was extracted with anhydrous MgSO₄Dried and the solvent removed under reduced pressure. The residue was purified by flash column chromatography using a gradient of hexane in ethyl acetate (from 50% to 30%) to give methyl (E) -3- (3-amino-4-methylaminophenyl) acrylate 2-4 as a yellow solid (245 mg).

Example 3

(E) -3- [2- (1-Aminocyclobutyl) -1-methyl-1H-benzimidazol-5-yl ] acrylic acid methyl ester

Methyl (E) -3- (3-amino-4-methylaminophenyl) acrylate 2-4(40mg,0.194mmol) obtained in example 2 was suspended in CH₂Cl₂(3mL) and 1-aminocyclobutanecarboxylic acid chloride hydrochloride (31mg,0.18mmol) prepared according to the applicable method described in E.S. Uffelman et al (org.Lett.1999,1,1157). The reaction mixture was stirred at room temperature for 2 hours, then concentrated to give a white solid. The solid was then dissolved in acetic acid (5mL) and heated to 60 ℃ for 20 hours. The crude product of the reaction is taken up in NaHCO₃Diluting with saturated water solutionReleasing and using CH₂Cl₂(2X 50mL) and brine, and the organic layer was extracted with anhydrous MgSO₄Drying and removing the solvent under reduced pressure to give (E) -3- [2- (1-aminocyclobutyl) -1-methyl-1H-benzimidazol-5-yl]Light brown foam of methyl acrylate 3-1 (53 mg).

Example 4

(E) -3- (2- {1- [ (3-cyclopentyl-1-methyl-2-pyridin-2-yl-1H-indole-6-carbonyl) amino ] cyclobutyl } -1-methyl-1H-benzimidazol-5-yl) acrylic acid

3-cyclopentyl-1-methyl-2-pyridin-2-yl-1H-indole-6-carboxylic acid 4-1(31.1mg,0.97mmol), prepared by the method described in WO03/010141, (E) -3- [2- (1-aminocyclobutyl) -1-methyl-1H-benzimidazol-5-yl prepared from the ethyl ester analog of compound 2-4 using the analogous method described in example 3]Methyl acrylate 4-2(27.7mg,0.97mmol), HATU (47.9mg,0.126mmol) and Et₃A solution of N (58. mu.L, 0.42mmol) in DMSO (2mL) was stirred at RT for 3 h. After this period, NaOH (280 μ L,2.5N) was added and the reaction mixture was stirred at RT for 16 h. The reaction mixture is neutralized by adding a few drops of acetic acid and in reverse phase C₁₈Semi-preparative HPLC column (using a solvent gradient from 5% to 100% MeCN in water (all solvents containing 0.1% trifluoroacetic acid)) to isolate the final inhibitor (E) -3- (2- {1- [ (3-cyclopentyl-1-methyl-2-pyridin-2-yl-1H-indole-6-carbonyl) amino]Cyclobutyl } -1-methyl-1H-benzimidazol-5-yl) acrylic acid 4-3 (compound 4001, Table 4) as a white anhydrous solid with > 95% homogeneity (homogeneity) (45mg, 78% yield).

¹H NMR(400MHz,DMSO):δ1.48-1.58(m,2H),1.75-1.85(m,6H),1.85-1.95(m,1H),2.05-2.15(m,1H),2.69-2.76(m,2H),2.98-3.10(m,3H),3.63(s,3H),3.82(s,3H),6.59(d,J=16Hz,1H),7.42(dd,J=0.8&5.7Hz,1H),7.51(d,J=7.7Hz,1H),7.53(d,J=8.5Hz,1H),7.65(d,J=8.5Hz,1H),7.71(d,J=8.4Hz,1H),7.76(d,J=16Hz,1H),7.82(d,J=8.4Hz,1H),7.92(ddd,J=1.6 & 7.8Hz,1H),8.01(s,1H),8.04(s,1H),8.73(d,J=4.1Hz,1H),9.45(s,1H)。

Example 5

3-cyclopentyl-1-methyl-2-pyrazin-2-yl-1H-indole-6-carboxylic acid {1- [5- ((E) -2-carbamoylvinyl) -1-methyl-1H-benzimidazol-2-yl ] cyclobutyl } amide

Step 1:

coupling of 3-cyclopentyl-1-methyl-2-pyrazin-2-yl-1H-indole-6-carboxylic acid 5- (prepared using the procedure described in WO 03/010141) and (E) -3- [2- (1-aminocyclobutyl) -1-methyl-1H-benzimidazol-5-yl ] acrylic acid ethyl ester 4-2 followed by saponification of the ethyl ester yielded (E) -3- (2- {1- [ (3-cyclopentyl-1-methyl-2-pyrazin-2-yl-1H-indol-6-carbonyl) amino ] cyclobutyl } -1-methyl-1H-benzimidazol-5-yl) acrylic acid 5- (prepared using the procedure described in example 4) 5-methyl-1H-benzimidazol-5-yl) acrylate -2 (compound 4003, table 4).

¹H NMR(400MHz,DMSO-d₆):δ1.50-1.58(m,2H),1.78-1.20(m,7H),2.05-2.15(m,1H),2.65-2.75(m,2H),2.97-3.10(m,3H),3.66(s,3H),3.18(s,3H),6.57(d,J=16.0Hz,1H),7.55(dd,J=1.0&8.4Hz,1H),7.68(2d,J=8.4Hz,2H),7.75(d,J=16.0Hz,1H),7.78(d,J=11.0Hz,1H),8.00(s,1H),8.07(s,1H),8.68(d,J=2.3Hz,1H),8.78(d,J=1.2Hz,1H),8.82(dd,J=0.8&2.2,1H),9.44(brs,1H)。

Step 2:

reacting (E) -3- (2- {1- [ (3-cyclopentyl-1-methyl-2-pyrazin-2-yl-1H-indole-6-carbonyl) amino]Cyclobutyl } -1-methyl-1H-benzimidazol-5-yl) acrylic acid 5-2 (compound 4003, table 4; 60mg,0.087mmol), TBTU (68mg,0.18mmol), ammonium bicarbonate (20mg,0.26mmol) and Et₃N (36. mu.L, 0.26mmol) in DMSO (23mL)The solution was stirred at RT for 3 h. The reaction mixture is neutralized by adding a few drops of acetic acid and in reverse phase C₁₈Semi-preparative HPLC column (using a solvent gradient from 5% to 100% MeCN in water (all solvents containing 0.1% trifluoroacetic acid)) to isolate the inhibitor 3-cyclopentyl-1-methyl-2-pyrazin-2-yl-1H-indole-6-carboxylic acid {1- [5- ((E) -2-carbamoylvinyl) -1-methyl-1H-benzimidazol-2-yl-]Cyclobutyl } amide 5-3 (compound 1005, table 1) as a pale yellow anhydrous solid with > 95% homogeneity (17mg, 34% yield).

¹H NMR(400MHz,DMSO-d₆):δ1.65-1.75(m,2H),1.92-2.15(m,8H),2.73-2.82(m,2H),3.04-3.10(m,2H),3.15-3.25(m,1H),3.79(s,3H),3.81(s,3H),6.65(d,J=15.8Hz,1H),7.06(brs,1H),7.53(brs,3H),7.61(d,J=15.7Hz,1H),7.68(dd,J=1.0&8.4Hz,1H),7.80(d,J=8.4Hz,1H),7.90(s,1H),8.20(s,1H),8.80(d,J=2.5Hz,1H),8.91(d,J=1.2Hz,1H),8.95(dd,J=2.1 & 3.7,1H),9.23(s,1H)。

Example 6

4-amino-2-methyl-5- (methylamino) benzoic acid methyl ester

Step 1:

2-methyl-5-nitrobenzoic acid 6-1(10.0g,55.2mmol) in MeOH (200mL) and H₂SO₄(1.0mL) the solution was heated to reflux while stirring for-3 days. Evaporate solvent under vacuum and redissolve the residue in EtOAc (. about.200 mL) with cold H₂O (50mL), Cold NaHCO₃A saturated aqueous solution (-50 mL) and cold brine (-50 mL). Then using anhydrous MgSO₄The organic layer was dried and concentrated to dryness to give methyl ester 6-2 as a white solid which was used in step 2 without purification.

Step 2:

to obtain in step 1To a solution of crude methyl ester 6-2 (. about.55.2 mmol) in MeOH (200mL) was added Pearlman's catalyst (20% palladium hydroxide on carbon, 1.0g) and the mixture was taken up in H₂The mixture was stirred at RT for 20 h. The mixture was filtered through Celite and concentrated to dryness. The residue was redissolved in THF (200mL) and Ac was added₂O (6.2mL,66mmol) and the solution stirred at RT for 3 h. The reaction mixture was concentrated to dryness under vacuum and the residue was redissolved in a minimum volume of t-Bu methyl ether (-150 mL). The ether suspension was stirred at RT for 1 hour and hexane (100mL) was added to precipitate the desired acetylated intermediate as a white solid. The solid was washed with hexane and dried to give acetylated compound 6-3 with high purity (10.1g, 88% yield).

And step 3:

the acetylated ester 6-3 from step 2 (8.42g,40.6mmol) and potassium nitrate (5.0g,50mmol) in AcOH: H₂SO₄(1:1 ratio, 200mL) solution was stirred at RT for 2 hours and at 40 ℃ for another two hours. The crude reaction mixture was then poured slowly onto ice (. about.1L) and mixed for 20 minutes. The precipitate formed was filtered and washed several times with water to give a mixture of predominantly two products, the desired 4-nitro isomer 6-4 and the undesired 6-nitro isomer 6-5(1:2 ratio), separated in flash column chromatography using 30% EtOAc in hexanes as eluent. Pure 4-nitro isomer 6-4 was isolated as a yellow solid (2.05g, 20% yield).

And 4, step 4:

the 4-nitro intermediate 6-4 from step 3 (2.05g,8.13mmol) was dissolved in THF (50mL) and upon cooling the solution to 0 deg.C, MeI (2.51mL,40.6mmol) and t-BuONa (4.46g,46.4mmol) were added slowly. The reaction mixture was stirred at RT for 15 hours and H was added₂O (. about.50 mL) and washing the aqueous mixture with tert-butyl methyl ether (. about.20 mL). The aqueous layer was acidified to pH 3 with 1N HCl and then extracted with EtOAc (. about.100 mL). The organic layer was washed with brine (. about.50 mL), anhydrous MgSO₄Drying and concentrating to dryness to give a colloidal foam of N-methylated compound 6-6, which may beDirectly used in step 5 without purification.

It will be appreciated by those skilled in the art that other diamine intermediates of formula IV in the above schemes 2 and 3, wherein R is⁸Instead of methyl, an appropriate R may be utilized in step 4 above⁸-X instead of methyl iodide (CH)₃I) Prepared wherein X is a leaving group such as Cl, Br, I, mesylate (mesylate), tosylate (tosylate), triflate (triflate), and the like.

And 5:

a solution of the methylated derivative from step 4, 6-6 (. about.8 mmol) in MeOH (10mL) and HCl (8N,15mL) was stirred at 70 ℃ for 20 h. The solvent was evaporated under vacuum and the residue was taken up in NaHCO₃The saturated aqueous solution (20mL) and EtOAc (50mL) were partitioned. The organic layer was washed with brine, anhydrous MgSO₄Drying and concentration gave methyl ester 6-7 as an orange solid (1.54g), which was used in step 6 without purification.

Step 6:

a solution of crude methyl ester 6-7 from step 5 (1.54g,6.7mmol) in MeOH (30mL) under catalytic hydrogenation conditions using Pd/C (10%,150mg) in H₂The treatment was carried out under atmospheric pressure at RT for 2 hours. The reaction mixture was filtered through Celite and concentrated to give methyl 4-amino-2-methyl-5- (methylamino) benzoate 6-8 as a purple solid (1.33g), which was sufficiently purified (confirmed by NMR) to be used without further purification.

Example 7

2- (1-tert-Butoxycarbonylaminocyclobutyl) -3, 6-dimethyl-3H-benzimidazole-5-carboxylmethyl ester

1- ((1, 1-Dimethylethoxycarbonyl) amino) cyclobutanecarboxylic acid (1.40g,6.5mmol) was dissolved in CH₂Cl₂(45mL) to neutralize TBTU in Et₃The acid was pre-activated by reaction in the presence of N for 30 minutes. Methyl 4-amino-2-methyl-5- (methylamino) benzoate 6-8 from example 6 (1.33g,6.85mmol) in CH₂Cl₂(10mL) solution was added slowly over 30 minutes and the reaction mixture was stirred continuously for 20 hours. The reaction mixture was concentrated to dryness and the residue was redissolved in AcOH (10.0mL) and stirred at 70 ℃ for 2 hours to achieve cyclization of the benzimidazole ring. The reaction mixture was concentrated to dryness and the residue was dissolved in EtOAc (. about.250 mL) with NaHCO₃Saturated aqueous solution (2X 100mL) and brine (100 mL). The organic layer was washed with anhydrous MgSO₄Dried and evaporated to dryness. The residue was purified by flash column chromatography (from 40% to 50% EtOAc in hexanes using a solvent gradient) to give pure methyl 2- (1-tert-butoxycarbonylaminocyclobutyl) -3, 6-dimethyl-3H-benzimidazole-5-carboxylate 7-1 as a beige solid (1.41g, 55% yield) and the unreacted diamine-based starting material was recovered.

Methyl 2- (1-tert-butoxycarbonylaminocyclobutyl) -3, 6-dimethyl-3H-benzimidazole-5-carboxylate 7-1 was converted to the amine intermediate of formula III in equation 1 using standard reagents well known to those skilled in the art. The reagent includes, but is not limited to, trifluoroacetic acid, HCl in bisObtain solution in the alkane, etc. The corresponding amine intermediate of formula III in scheme 1 can be further made into the inhibitor of formula I in scheme 1 using the procedure of example 4.

Example 8

(E) -3- [2- (1-amino-cyclobutyl) -3, 6-dimethyl-3H-benzimidazol-5-yl ] -2-methyl-acrylic acid ethyl ester

Step 1:

the 2- (1-tert-butoxy) obtained in example 7 was reactedCarbonylaminocyclobutyl) -3, 6-dimethyl-3H-benzimidazole-5-carboxylic acid methyl ester 7-1(1.41g,3.8mmol) was dissolved in THF (40mL) and the solution was cooled to 0 ℃. DIBAL-H (18mL,1M in THF, 18mmol) solution was added slowly and the reaction mixture was stirred at 0 ℃ for 1 hour, followed by 50 ℃ for 4 hours. The reaction mixture was cooled to RT. A solution of potassium sodium tartrate (1M,50mL) was added very slowly and stirring was continued slowly at RT for 1 hour. The solution was concentrated in vacuo to remove most of the THF and extracted with EtOAc (. about.200 mL). With NaHCO₃The organic layer was washed with saturated aqueous solution (50mL) and brine (50mL), dried over anhydrous MgSO₄Dried and concentrated to dryness. The residue was purified by flash column chromatography with a solvent gradient from 50% EtOAc in hexanes to pure EtOAc and then to 3% MeOH in EtOAc to give pure alcohol 8-1 as a yellow solid (1.09g, 84% yield).

Step 2:

the alcohols 8-1 from step 1(1.09g,3.16mmol) and Dess-Martin periodinane (1.70g,4.0mmol) were combined in CH₂Cl₂The solution (40mL) was stirred at RT for 2 h. The solvent was evaporated under vacuum and the residue was purified by flash column chromatography using EtOAc: hexane (1:1 ratio) to give the pure aldehyde 8-2(605mg, 56% yield).

And step 3:

a solution of triethyl-2-phosphonopropionate (0.228mL,1.06mmol) in THF (5.4mL) was cooled to 0 deg.C and NaH (42.5mg, 60% in oil, 1.06mmol) was added. The mixture was stirred at 0 ℃ for 30 minutes, then a solution of aldehyde 8-2 from step 2(300mg in 3mL of THF,0.874mmol) was added slowly and stirring continued at RT for 20 hours. The mixture was diluted with EtOAc (. about.100 ml) and NaHCO₃Washed with saturated aqueous solution (2X 30mL) and brine (30 mL). The organic layer was washed with anhydrous MgSO₄Dried and concentrated to a brown residue, which was then purified by flash column chromatography using a solvent gradient from 40% to 60% EtOAc in hexanes to give N-Boc-protected ester 8-3 as a yellow foam (85mg, 23% yield).

And 4, step 4:

hydrolysis of the BOC protecting group may be achieved by addition of a solvent in bisAlkane (2mL) in 4N HCl and stir the solution at RT for 1 hour, done quantitatively. After evaporation of the solvent under vacuum, the pure (E) -3- [2- (1-aminocyclobutyl) -3, 6-dimethyl-3H-benzimidazol-5-yl radical is isolated]-ethyl 2-methacrylate 8-4 as a yellow solid (79 mg).

It will be appreciated by those skilled in the art that the triethyl-2-phosphonopropionate used in step 3 of the process may be replaced by an appropriately substituted derivative to prepare an analogue of formula IIIe in the above reaction scheme, wherein R is⁵⁰As hereinbefore defined. Alternatively, methyl esters can be prepared in a similar manner using appropriate reagents. Compounds 8-4 of general formula IIIe and analogs thereof can be prepared as inhibitors of general formula I in scheme 1 using the procedure of example 4.

Example 9

3-fluoro-4-nitrobenzaldehyde

Step 1:

in a two-necked flask (equipped with an internal thermometer) were added glacial AcOH (252mL), acetic anhydride (252.0mL) and 2-fluoro-4-methyl-1-nitrobenzene 9-1(25.0g,161mmol) at-10 ℃. Concentrated sulfuric acid (40mL) was added dropwise to the cold solution over a period of 5 minutes, followed by very slow addition of chromium (VI) oxide (45g,450 mmol); the rate of addition must be very slow (-1.5 hours) to maintain the temperature below 10 ℃. Adding CrO₃At the time, the clear colorless solution turned amber and after the end of the addition turned dark brown. After the addition was complete, the reaction mixture was stirred for an additional 45 minutes (HPLC analysis showed reaction to be complete-70%). A tar-like partial suspension was poured into ice (1.6L) and washed with H₂Diluting the obtained mud-like substance with O,up to a total of 3L, at which point the product will start to precipitate. After filtration, with cold H₂O wash off the beige solid to give a white solid. The solid was then suspended in cold 2% NaHCO₃(250mL), filtered and chilled H₂O rinse to give diacetate 9-2(22g, containing some unreacted starting material) as a white solid, which was used in step 2.

Step 2:

in a screw-cap sample vial, diacetate 9-2 from step 1(1.0 g,3.7mmol) was dissolved in glacial acetic acid (10.0mL) followed by addition of H₂O (1.0mL) and concentrated HCl (1.0 mL). The resulting partially soluble mixture was heated at 115 ℃ for 45 minutes. Most of the solvent was removed under vacuum to give a gummy residue, remaining acid and H₂O and CH₂Cl₂Hexane was azeotroped twice to give the desired semi-pure 3-fluoro-4-nitrobenzaldehyde 9-3 as a yellow solid (600 mg). This compound was purified by flash column chromatography (using 20% EtOAc in hexanes as eluent) to remove a small amount of unreacted 2-fluoro-4-methyl-1-nitrobenzene 9-1 (-35% overall yield).

Example 10

(E) -3- (4-amino-3- (methylamino) phenyl) acrylic acid ethyl ester

Step 1:

NaH (60% dispersion in oil, 314mg,7.84mmol) was added to a solution of triethylphosphonoethyl ester (1.37mL,6.90mmol) in THF (13mL) at 0 deg.C and the mixture was stirred for 30 min. After this period 3-fluoro-4-nitrobenzaldehyde 9-3 from example 9 (1.06g,6.27mmol) was added and stirring continued at room temperature for 16 h. By addition of H₂O (20mL) stopped the reaction and the product was extracted into EtOAc (2X 100 mL). The combined organic layers were washed with brine, anhydrous MgSO₄Dried and concentrated to give cinnamate 10-1 as a pale orange solid which can be used in step 2 without purification.

It will be appreciated by those skilled in the art that the preparation of analogues bearing various substituents on the cinnamic acid double bond may be carried out using appropriate substituent derivatives in place of triethylphosphonoethyl ester as used in the present process or using appropriate ketones in place of the aldehyde 9-3. In addition, methyl cinnamate can also be prepared in a similar manner using appropriate reagents.

Step 2:

cinnamate 10-1 (. about.6.27 mmol) from step 1 and methylamine (2M in THF, 6.3mL,12.5mmol) were dissolved in DMSO (6mL) and the reaction mixture was stirred at room temperature for 2 hours. After that period of reaction, the mixture was diluted with EtOAc (100mL) and washed with H₂The organic layer was washed with O (3X 30mL) and brine (50mL), dried over anhydrous MgSO₄Drying and concentration gave the crude methylamino intermediate 10-2 as an orange solid. This product was used in step 3 without purification.

It will be appreciated by those skilled in the art that other diamine intermediates of formula IV in the above schemes 2 and 3, wherein R is⁸Instead of methyl, an appropriate R may be used⁸-NH₂Instead of methylamine (CH) in step 2₃NH₂) And then the process is carried out.

And step 3:

the 3-methylamino-4-nitrocinnamate 10-2(2-2, 150mg) from step 2 and SnCl₂The dihydrate (950mg,4.2mmol) was dissolved in ethanol (10mL) and the reaction mixture was stirred at 80 ℃ for 20 hours. The mixture was cooled to room temperature and concentrated to dryness. The residue was dissolved in ethyl acetate (100mL) and slowly added to NaHCO₃Saturated aqueous solution and stirred for 30 minutes. The organic layer was then extracted with ice cold brine and anhydrous MgSO₄Dried and the solvent removed under reduced pressure. Purifying the residue by flash column chromatography (using a gradient from 70% to 60% hexane in ethyl acetate) to obtain (E) -3- (4-amino-3- (methylamino) phenyl) acrylic acid ethyl esterYellow solid of ester 10-3 (100 mg).

(E) Ethyl (E) -3- (4-amino-3- (methylamino) phenyl) acrylate can be converted to the amine derivative of formula III in equation 1 according to the method of example 3 or 7 and the inhibitor of formula 1 in equation 1 can be prepared again using the method of example 4.

Example 11

5-amino-2-methyl-4-methylaminobenzoic acid methyl ester

Step 1:

2-methyl-4-nitrobenzonitrile 11-1(2.53g,15.6mmol) was dissolved in aqueous NaOH (10% m31.0mL) and H₂O₂The solution in aqueous solution (10%,16mL) was stirred at reflux for 2.5 hours. After the water circulation in the condenser was stopped for 5-10 minutes (to remove dissolved ammonia) and the water flow was resumed and continued to reflux for another 1.5 hours. The reaction mixture was cooled to RT and HCl (concentrated) was added dropwise until pH-3, at which time carboxylic acid 11-2 precipitated as an orange solid (3.60 g). The carboxylic acid can be used in step 2 without purification.

Step 2:

acid 11-2 from step 1 (3.60g,15.6mmol) in MeOH (30mL) and HCl (4N HCl in bis)2.0mL) was heated to reflux for 48 hours. The solvent was evaporated to dryness in vacuo and the resulting residue was redissolved in EtOAc (200 mL). The solution is treated with NaHCO₃The mixture was washed with saturated aqueous solution (100mL) and brine (100mL) over MgSO₄Drying and evaporation to dryness gave ester intermediate 11-3 as a yellow solid (2.38 g). This material can be used in step 3 without purification.

And step 3:

mixing KNO₃(760mg,7.5mmol) was added very slowly to the H of the ester 11-3(1.27g,6.5mmol) from step 2 precooled to 0 deg.C₂SO₄(concentrated, 13.0mL) in solution. After stirring for a few minutes, the ice bath was removed and the reaction mixture was stirred at RT for 20 hours. The reaction mixture was then slowly poured onto ice (-50 mL) and stirred until the ice dissolved and the desired dinitro product 11-4 was precipitated and filtered (-1.55 g of a pale yellow and slightly moist solid). This compound can be used as such in step 4.

And 4, step 4:

to a solution of the dinitro intermediate 11-4 from step 3 (1.55g,6.45mmol) in THF (15.0mL) at 0 deg.C was added a solution of methylamine (2M in THF, 15.2mL,32.3mmol), the ice bath was removed and the reaction mixture was stirred at RT for 1.5 h. The solution was concentrated to remove some THF before dilution with EtOAc (. about.100 mL). By means of H₂The organic layer was washed with O (. about.50 mL) and brine (. about.50 mL), anhydrous MgSO₄Drying and concentration gave methylamino intermediate 11-5 as an orange solid (1.26 g). This compound was used in step 5 without further purification.

Those skilled in the art will appreciate that other diamine intermediates of formula IV in schemes 2 and 3 above, wherein R is⁸Instead of methyl, a suitable R may be utilized⁸-NH₂Instead of methylamine (CH) in the above step 4₃NH₂) And then the preparation.

And 5:

to methylamino derivative 11-5 from step 4 (1.25g,5.58mmol) in EtOH-H₂Adding K to O (110mL, 1:1 ratio) solution₂CO₃(4.62g,33.5mmol) and Na₂S₂O₄And the mixture was stirred at RT for 3 hours. Adding more H₂O (. about.30 mL) and the mixture was concentrated in vacuo to remove most of the EtOH. The reaction mixture was then diluted with EtOAc (-200 mL) and the organic layer was separated and extracted with brine. The organic layer was washed with anhydrous MgSO₄Dried and concentrated in vacuo to give 5-amino-2-methyl-4Methyl- (methylamino) benzoate 11-6(927mg, 86% yield) as a brown solid.

Compounds 11-6 may be converted to the corresponding amine intermediates of formula III in scheme 1, wherein R is⁶Is CH₃And R is⁵is-COOCH₃. These amine intermediates can be reconverted to amine intermediates of formula III in scheme 1, wherein R is⁶Is CH₃And R is⁵is-CH = C (R)⁵⁰) -COOR. All amine intermediates of formula III in equation 1 can be further made into inhibitors of formula I in equation 1 using the procedure of example 4.

Example 12

(E) -3- (5-amino-2-ethoxy-4- (methylamino) phenyl) acrylic acid methyl ester

Step 1:

2-ethoxy-4-nitrobenzoic acid 12-1(1.56g;7.38mmol) was dissolved in methanol (15mL) and the resulting solution was stirred at 0 ℃. The methyl azide solution in ether was added slowly until the solution continued to be yellow and stirred for an additional 20 minutes. Evaporation of the solvent gave methyl ester 12-2 as a pale yellow solid (1.66g, quantitative), which was used in step 2 without further purification.

Step 2:

the ester 12-2 from step 1 (1.60g;7.10mmol) was dissolved in anhydrous toluene and the solution was cooled to-78 ℃ under nitrogen. A solution of diisobutylaluminum hydride in tetrahydrofuran (1M;8mL;8mmol) was added and the reaction mixture was allowed to warm to ambient temperature. After 1 hour and after another 1.5 hours, two additional portions of DIBAL-H were added in this manner (7 and 10 mL). 0.5 h after the last addition, the reaction was cooled to 0 ℃ and 1N HCl (25mL) was added slowly and the mixture was stirred vigorously for 0.5 h. The organic solvent is then evaporated and treated with ethyl acetateThe aqueous residue was extracted with ester (2X 50mL) and washed with water (50mL) and brine (50 mL). The combined extracts were then extracted with MgSO₄Drying and evaporation gave the alcohol 12-3 as a pale yellow fibrous solid (1.40 g; quantitative), which was used as such in step 3.

And step 3:

a cloudy solution of 1,1, 1-tris (acetyloxy-1, 1-dihydro-1, 2-benzodioxol-3- (1H) -one (Dess-Martin periodinane) (2.32g;5.47mmol) in dichloromethane (40mL +5mL wash) was added to a stirred solution of the alcohol 12-3 from step 2 (0.98g;4.97mmol) in DCM (40mL) and the reaction stirred under nitrogen at ambient temperature after 4 hours, saturated NaHCO was added₃/10%Na₂S₂O₃(1:1,160mL) and the mixture was vigorously stirred until the phase layer was clear (about 0.5 h). The organic phase was separated and the aqueous phase was extracted with dichloromethane (50mL) and saturated NaHCO₃(2X 150mL) was washed. The combined organic phases were then washed with MgSO₄Drying and evaporation gave aldehyde 12-4 as a pale yellow solid (960mg;99%), which was used as such in step 4.

And 4, step 4:

sodium hydride (95% dry powder; 158mg;6.25mmol) was suspended in a solution of anhydrous THF (10mL) and trimethylphosphonoacetate (0.945mL;5.84mmol) was added at 0 deg.C under nitrogen to give a non-stirrable white solid material. A solution of aldehyde 12-4 from step 3 (950mg;4.87mmol) in THF (7mL +3mL wash) was then added dropwise to give a yellow and white solid which slowly dissolved. After addition was complete, the reaction was warmed to ambient temperature. After 15 h, the cloudy reaction mixture was evaporated to a pale yellow solid, which was extracted with ethyl acetate (2X 50mL) and saturated NaHCO₃(3X 75mL) was washed. The combined extracts were extracted with MgSO₄Drying and evaporation gave cinnamate 12-5 as a pale yellow solid (1.212g;99%) which was used in step 5 without further purification.

It will be appreciated by those skilled in the art that the trimethylphosphonoacetate used in the process can be replaced by appropriately substituted derivatives to prepare analogs with various substituents at the cinnamate double bond.

And 5:

4-Nitro-2-ethoxycinnamate 12-5 from step 4 (303mg,1.206mmol) was dissolved in concentrated sulfuric acid (3mL) and the solution was cooled to 0 ℃. Potassium nitrate (128mg,1.27mmol) was added and the mixture was stirred at room temperature for 3.5 hours. After completion the reaction mixture was poured onto ice and the precipitated solid was collected by filtration. The crude product 12-6 was washed with water, dried under vacuum and used in step 6(390mg) without purification.

Step 6:

the dinitro derivative 12-6 from step 5 (390mg) was dissolved in THF (3mL) and methylamine in THF (2M in THF, 3.02mL) was added. After stirring for 30 minutes, the volatiles were removed under reduced pressure and an orange solid 12-7 was used as such in step 7.

It will be appreciated by those skilled in the art that other diamine intermediates of the general formula IV in the above schemes 2 and 3, wherein R is⁸Instead of methyl, a suitable R may be utilized⁸-NH₂Instead of methylamine (CH) in the above step 6₃NH₂) And then the preparation.

And 7:

the nitroaromatic 12-7 from step 6 was suspended in a mixture of EtOH (12mL) and water (12mL) and K was added₂CO₃(1.00g,6 equiv.) followed by the addition of sodium bisulfite (1.26g,6 equiv.). The mixture was stirred at room temperature for 4 hours and the EtOH was removed under reduced pressure. The residue was extracted with EtOAc and the organic phase was washed with brine and dried (MgSO)₄). The solvent was removed and the residue was purified by flash chromatography (50 to 75% EtOAc in hexane) to give methyl (E) -3- (5-amino-2-ethoxy-4- (methylamino) phenyl) acrylate 12-8(162 mg).

Methyl (E) -3- (5-amino-2-ethoxy-4- (methylamino) phenyl) acrylate 12-8 can be converted to the amine intermediate of formula III in example 1 using the method described in example 3 or 7 and the inhibitor of formula 1 in equation 1 can be reproduced using the method of example 4.

Those skilled in the art will also be aware of such inhibitors of the general formula I in scheme 1, wherein R⁶is-OCH₃And R is⁵is-CH = C (R)⁵⁰) -COOR can be prepared using the procedure of example 12, starting with the same precursor as compound 12-1, except that in this case the ethoxy group is replaced by methoxy.

Example 13

4-amino-2-methoxy-5- (methylamino) benzoic acid methyl ester

Step 1:

methyl 2-methoxy-5-nitrobenzoate 13-1(6.21g,29.4mmol) was suspended in MeOH (100mL) and 20% Pd (OH) added₂C (500 mg). The mixture was stirred under hydrogen (1atm) for 18 hours. The catalyst was removed by filtration and the solvent was evaporated under reduced pressure to give a residue of compound 13-2(5.256g), which was used as such in step 2.

Step 2:

aniline 13-2 from step 1 (5.23g) was dissolved in THF (50mL) and acetic anhydride (2.984g) was added. The mixture was stirred at room temperature overnight. The white suspension was concentrated under reduced pressure to a white slurry, t-butyl methyl ether (TBME,20mL) was added and hexane (100mL) was added slowly while stirring. The suspension was then stirred for a further 2 hours and the solid was collected by filtration. Product 13-3 was washed with hexane and dried in air (6.372 g).

And step 3:

90% nitric acid (9mL) was diluted with water (9mL) and cooled to 0 ℃. The anilide compound 13-3(5.905g) from step 2 was added in one portion and the mixture was stirred in an ice-water bath for 30 minutes. Then mixing the reactionThe mixture was added dropwise to ice water (700mL) and the precipitated yellow solid was collected by filtration, washed with water and dried in air.¹H NMR showed the orange solid (5.907g) to consist of a 2:1 mixture. The aqueous filtrate from above was extracted with EtOAc to give an additional 1g of material, and combined with that obtained for the first time, followed by flash chromatography on silica gel using 0.1% EtOAc in CHCl₃The solution was purified as eluent. Orange solid 13-4(4.11g) (one isomer) was obtained.

And 4, step 4:

the nitroanilide 13-4 from step 3 (3.580g) was dissolved in THF (50mL) and the solution was cooled in ice. Methyl iodide (4.155mL,66.7mmol,5 equivalents) and sodium tert-butoxide (6.414g,66.7mmol,5 equivalents) were added in two portions at 3.5 hour intervals. Stirring was continued for another 20 hours at room temperature after the second addition. THF was evaporated under reduced pressure and water (100mL) was added. The dark red solution was washed with TBME (100 mL). The aqueous phase was acidified with concentrated HCl and extracted with EtOAc (2X 100 mL). The combined organic extracts were dried and concentrated to give compound 13-5 as a dark red powder (3.78g), which was used directly in step 5.

It will be appreciated by those skilled in the art that other diamine intermediates of formula IV in the above schemes 2 and 3, wherein R is⁸Instead of methyl, an appropriate R may be utilized in step 4 above⁸-X instead of methyl iodide (CH)₃I) And wherein X is a leaving group, such as Cl, Br, I, mesylate (mesylate), tosylate (tosylate), triflate (triflate), and the like.

And 5:

the free carboxylic acid 13-5 from step 4 (3.75g) was suspended in 8M HCl (100mL) and the mixture was stirred at 100 ℃ for 8 hours. Cooled to room temperature, volatiles were evaporated in vacuo and the residue was co-evaporated three times with MeOH.

Step 6:

the residue from step 5 was resuspended in MeOH (100mL) and cooled in ice water. Thionyl chloride (5.10mL,5 equivalents) was added dropwise and the suspension was stirred at 65 ℃ for 4 hours. The volatiles were removed under reduced pressure and the residue 13-6 and MeOH (100mL) were then co-evaporated twice with toluene (2X 100 mL).

And 7:

the residue from step 6, 13-6, was then dissolved in MeOH (200mL) and 20% Pd (OH) added₂/C (500mg), and the mixture was stirred under 1atm hydrogen overnight. The catalyst was then removed by filtration and the solution was evaporated to dryness. The residue was dissolved in EtOAc and taken up with NaHCO₃The solution was washed with aqueous solution and dried (MgSO₄). The solvent was removed to give a solid, suspended in TBME (50mL) and heated to 60 ℃ for 30 min. An equal volume of hexane was then slowly added to the hot solution and the precipitated methyl 4-amino-2-methoxy-5- (methylamino) benzoate 13-7 was collected by filtration, washed with TBME-hexane and dried (2.00 g).

Methyl 4-amino-2-methoxy-5- (methylamino) benzoate 13-7 can be converted to the corresponding amine intermediate of general formula III in scheme 1 according to the methods of examples 3 or 7. These amine intermediates can be reconverted to amine intermediates of formula III in scheme 1, wherein R is⁵is-OCH₃And R is⁶is-CH = C (R)⁵⁰) -COOR. All of these amine intermediates of formula III in equation 1 can be further made into inhibitors of formula I in equation 1 using the procedure of example 4.

It will be appreciated by those skilled in the art that the method of example 13 can be used with compound 12-2 from example 12, or an analog thereof, wherein the ethoxy group is replaced with a methoxy group to produce a diamine precursor of formula IV in scheme 2 or 3, wherein R is⁶Is OCH₃Or OEt. The diamine precursor can also be converted to an amine intermediate of formula III in scheme 1 according to the methods of examples 3 or 7 and reformed into an inhibitor of formula I in scheme 1 using the method of example 4.

Example 14

N²-methyl-4-(1H-[1,2,3]Triazol-4-yl) benzene-1, 2-diamines

Step 1:

3-fluoro-4-nitrobenzaldehyde 9-3 from example 9 (2.0g,11.8mmol) was dissolved in THF (30mL) and an excess of methylamine (2M in THF, 21mL,42mmol) was added. The reaction mixture was stirred at RT until complete conversion was confirmed by HPLC (-2-3 hours). The cloudy solution was then evaporated to an orange solid, extracted with ethyl acetate (2X 50mL) and washed with 1N HCl (shaken until the deep red bristlegrass color (burgundy colour) dissipated; 100mL), water (100mL) and brine (60 mL). The combined extracts were extracted with anhydrous MgSO₄Drying and evaporation gave methylamino intermediate 14-1 as an orange powder, which was used in step 2 without purification.

It will be appreciated by those skilled in the art that other diamine intermediates of formula IV, wherein R is in the above schemes 2 and 3, are provided⁸Instead of methyl, an appropriate R may be used⁸-NH₂Instead of methylamine (CH) in the above step 1₃NH₂) And then the preparation.

Step 2:

a solution of n-BuLi (2.5M in THF, 14.4mL,36.0mmol) in dry THF (60mL) was added slowly to a solution of TMS-azidomethane (10% in hexanes, 18mL,36.0mmol) at-78 ℃. The mixture was stirred at-78 ℃ for 30 minutes, after which the methylamino intermediate 14-1 from step 1(2.16g,12.0mmol, dissolved in 2mL THF) was added slowly. The reaction mixture was stirred at-78 ℃ for 1 hour, then warmed to RT and stirred for a further 3 hours, after which H was added₂O stops the reaction. The crude mixture was washed with NaHCO₃Partition between saturated aqueous solution (30mL) and EtOAc (60mL), extract the aqueous layer with EtOAc (2X 60mL) and wash the combined organic layers with brine, anhydrous MgSO₄Dried and concentrated to dryness. The residue is quickly utilizedPurification by flash column chromatography (using 20% EtOAc in hexanes as eluent) afforded the desired alkyne 14-2 as a light brown solid (445mg, 21% yield).

And step 3:

alkyne 14-2 from step 2(260mg,1.48mmol) was dissolved in anhydrous DMSO (6.0mL) and TMS-azide (0.392mL,2.96mmol) was added in thick-walled pressure tube. The reaction was heated to 140 ℃ for 2h, then cooled and extracted with EtOAc (50mL) and washed with brine (2X 50 mL). The organic layer was washed with anhydrous MgSO₄Drying and evaporation gave the crude triazole 14-3 as a yellow-brown solid which was used in step 4 without further purification.

And 4, step 4:

crude triazole intermediate 14-3 (. about.1.10 mmol) from step 3 was dissolved in EtOH (10mL) and H₂Some precipitation of the starting material was obtained in O (6mL) and K was added₂CO₃(0.91g,6.58mmol) and sodium bisulfite (1.15g,6.58mmol) were added and the reaction mixture was stirred at RT for 2 hours. The reaction mixture was then extracted with EtOAc (50mL) using H₂The organic layer was washed with O (50mL) and brine (30mL), and anhydrous MgSO₄Drying and evaporating to obtain a brown gum containing N²-methyl-4- (1H- [1,2, 3)]Triazol-4-yl) benzene-1, 2-diamine 14-4 (among other secondary products).

Coarse N²-methyl-4- (1H- [1,2, 3)]Triazol-4-yl) benzene-1, 2-diamine was converted without purification to the corresponding amine intermediate of general formula III in scheme 1 according to the methods of examples 3 or 7 and was then further processed to the inhibitor of general formula I in scheme 1 using the method of example 4.

Example 15

N²-methyl-4- (4-methylpiperazin-1-yl) benzene-1, 2-diamine

Step 1:

to a solution of 4-chloro-2-fluoro-1-nitrobenzene 15-1(1.18g,6.72mmol) in DMSO (7mL) was added a solution of methylamine (2M in THF, 13.6mL,26.9mmol) and the reaction mixture was stirred at RT for 24 h. The solution was diluted with EtOAc (. about.300 mL) and washed with H₂The organic layer was washed with O (3X 50mL) and brine (50mL), dried over anhydrous MgSO₄Dried and concentrated in vacuo to give methylamino derivative 15-2 as a yellow solid (1.19 g). The crude material was used in step 2 without further purification.

Those skilled in the art will appreciate that other diamine intermediates of formula IV in schemes 2 and 3 above, wherein R is⁸Instead of methyl, an appropriate R may be used⁸-NH₂Instead of methylamine (CH) in the above step 1₃NH₂) And then the preparation.

Step 2:

the mixture of methylamino derivative 15-2 from step 1 (105mg,0.56mmol) and N-methylpiperazine (0.5mL) was heated to 90 ℃ and stirred for 3 hours, followed by another 15 hours of stirring at RT. The reaction mixture was diluted with EtOAc (. about.50 mL) and washed with H₂The organic layer was washed with O (3X 10mL) and brine (20mL), dried over anhydrous MgSO₄Drying and concentration in vacuo afforded piperazine derivative 15-3 as a yellow solid (140mg), which was used in step 3 without further purification.

It will be appreciated by those skilled in the art that piperazine derivatives bearing other substituents may be used in place of N-methylpiperazine in step 2 above to prepare intermediates useful in forming other compounds of formula (I).

And step 3:

to a solution of piperazine derivative 15-3 from step 2 (140mg) in EtOH (6mL) was added Pd/C (10%,25mg) and dissolved in H₂The mixture was stirred at RT under air for 15 h. The mixture was filtered and the solution was evaporated to give the desired product N in fairly pure form²A violet oil of-methyl-4- (4-methylpiperazin-1-yl) benzene-1, 2-diamine 15-4 (133 mg).

Will N²-methyl-4- (4-methylpiperazin-1-yl) benzene-1, 2-diamine 15-4 was converted without further purification according to the method of example 3 or 7 to the corresponding amine intermediate of general formula III in reaction scheme 1 and reused as inhibitor 1 of general formula I in reaction scheme 1 using the method of example 4.

Example 16

4-imidazol-1-yl-N²-methylbenzene-1, 2-diamine

Preparation of 4-imidazol-1-yl-N using the procedure of example 15²-methylbenzene-1, 2-diamine 16-1 except that imidazole was used in place of N-methylpiperazine in step 2. 4-imidazol-1-yl-N²-methylbenzene-1, 2-diamine 16-1 can be converted to the corresponding amine intermediate of formula III in scheme 1 according to the method of example 3 or 7 and reused as an inhibitor of formula I in scheme 1 using the method of example 4.

Example 17

4- (2-aminothiazol-4-yl) -N¹-methylbenzene-1, 2-diamine

Step 1:

a mixture of 4-chloro-3-nitroacetophenone 17-1(3.00g,15.0mmol) and methylamine (15.0mL,2M in THF,30.0mmol) was placed in a sealed pressure tube and stirred at 80 ℃ for 6 h and at RT for 20 h. The reaction mixture was concentrated to dryness and the residue was purified by flash column chromatography (using 20-30% hexanes in EtOAc) to isolate the desired pure product 17-2 as an orange solid (980mg, 34% yield).

Those skilled in the art will appreciate that the above reaction scheme2 and 3 of formula IV wherein R⁸Instead of methyl, an appropriate R may be used⁸-NH₂Instead of methylamine (CH) in the above step 1₃NH₂) And then the preparation.

Step 2:

to the 4-methylamino-3-nitroacetophenone intermediate 17-2 from step 1 (700mg,3.6mmol) in bisTo a solution of alkyl ether (10mL, 1:1 ratio) Br was added slowly₂(0.20mL,3.96mmol) and the reaction mixture was stirred at RT for 20 h. The reaction mixture was concentrated to dryness and the residue was redissolved in EtOAc (200 mL). With NaHCO₃The solution was washed with saturated aqueous solution (2X 100mL) and brine (100mL), and anhydrous MgSO₄Drying and concentration to dryness gave crude bromoketone intermediate 17-3(1.0g), which was used in step 3 without purification.

And step 3:

a solution of bromoketone intermediate 17-3(1.0g) from step 2 and thiourea (548mg,7.2mmol) in i-PrOH (30mL) was stirred at 70 ℃ for 1 hour. The mixture was cooled to RT and the precipitate formed was filtered, washed with diethyl ether and dried to give the desired aminothiazole intermediate 17-4 as an orange solid (-1.0 g). This compound was used in step 4 without purification.

And 4, step 4:

nitro intermediate 17-4(500mg,. about.2 mmol) from step 3 and SnCl₂A solution of the dihydrate (2.25g,10mmol) in EtOH (15mL) was stirred at 80 ℃ for 16 h. The mixture was poured slowly into NaHCO₃Neutralized and stirred vigorously for 30 minutes. By means of CH₂Cl₂(2X 200mL) the mixture was extracted and the combined organic layers were taken over anhydrous Na₂SO₄Dried and concentrated to dryness. The residue was purified by flash column chromatography (using a solvent gradient from 30% hexane in EtOAc to 100% EtOAc, then to 3% MeOH in EtOAc) to recover unreacted starting material and pure diamine product 4- (2-ammonia)Thiazol-4-yl) -N¹-methylbenzene-1, 2-diamine 17-5(167mg, 38% yield).

Reacting 4- (2-aminothiazole-4-yl) -N¹-methylbenzene-1, 2-diamine 17-5 is converted to the corresponding amine intermediate of formula III in scheme 1 according to the method of example 3 or 7 and then used to make the inhibitor of formula I in scheme 1 using the method of example 4.

The free amino group of the aminothiazole substituent of the inhibitor of general formula I in scheme 1 or a suitable intermediate in its preparation may be alkylated using methods known to those skilled in the art or acetylated using methods known to those skilled in the art, for example treatment with acetic anhydride, acetyl chloride, and the like. Or replacing thiourea with the applicable N-substituted thiourea in step 3 above will give an intermediate wherein the free amino group is substituted.

Example 18

4-amino-2- (9H-fluoren-9-ylmethoxycarbonylamino) -5- (methylamino) benzoic acid methyl ester

Step 1:

to a solution of m-chlorobenzoic acid 18-1(12.5g,79.8mmol) in sulfuric acid (100mL) at 40 deg.C was slowly added potassium nitrate (about half of the total; 22.0g,218mmol) in portions while stirring (temperature maintained below 70 deg.C). The solution was then slowly heated to 105 ℃ and the remaining KNO was removed₃Add slowly (maintain temperature below 110 ℃), finally heat the solution to 130 ℃ for 15 minutes, cool to RT and pour onto ice (-500 mL). The yellow solid formed was filtered, washed with water (50mL), and air dried for 2 hours to give 13.25g (67%) of a mixture of the desired product 18-2 and unknown by-products mixed at 2: 1. This mixture was used in step 2.

Step 2:

the crude dinitrocarboxylic acid 18-2 from step 1 (. about.13 g) was dissolved in methanol (100mL) and sulfuric acid (13.0mL) was added very slowly, since the reaction was very exothermic. The reaction mixture was stirred at reflux for 18 hours. The solution was poured onto ice (. about.500 mL) and the product was extracted with EtOAc (2X 100 mL). The organic layer was washed with 5% NaHCO₃(3X 100mL) of an aqueous solution, and washing with anhydrous MgSO₄Drying and evaporation gave the desired dinitromethyl ester intermediate 18-3(9.54g, 69% yield).

And step 3:

to a solution of the dinitroaryl chloride 18-3(9.5g,36.5mmol) described above in DMF (20mL) was added methylamine (2M in THF,39.2mL,74.7mmol) with stirring at 0 ℃. After a few minutes a crystalline solid formed and the suspension was warmed to RT and stirring continued for 2 hours. Reaction mixture is reacted with H₂Partition between O (200mL) and EtOAc (100mL) and use 5% NaHCO₃The organic layer was washed with aqueous solution (100mL) and brine (3X 100mL), dried over anhydrous MgSO₄Drying and evaporation of the solvent to dryness gave the desired product 18-4 as a yellow-orange solid (7.09g, 76% yield).

Those skilled in the art will appreciate that other diamine intermediates of formula IV in schemes 2 and 3 above, wherein R is⁸Instead of methyl, an appropriate R may be used⁸-NH₂Instead of methylamine (CH) in the above step 3₃NH₂) And then the preparation.

And 4, step 4:

EtOH/H on the dinitroaniline intermediate 18-4 obtained above₂O (100mL,1:1 ratio) suspension was added with K under vigorous stirring₂CO₃(10.3g,74.5mmol) followed by the addition of sodium bisulfite (13.0g,74.5mmol) in portions. The yellow suspension turned to a blood red and then black, became more homogeneous (slightly exothermic), then two-phase, and a white precipitate formed. After stirring for 30 min at RT, the EtOH was partially evaporated and washed with H₂The residue was diluted with O (100 mL). The reaction mixture was extracted with EtOAc (2X 75mL) and the combined organic layers were dried over anhydrous MgSO₄Drying and evaporating to give a black amorphous formSolid 18-5(1.26g,55%), which was used as such in step 5.

And 5:

to a stirred ice-cooled solution of triphenylamine 18-5(400mg,2.05mmol) obtained above in acetonitrile (5mL) was added triethylamine (0.57mL) under nitrogen, followed by dropwise addition of TrocCl (0.282mL,2.05 mmol). The dark purple solution was stirred and warmed to RT over 2 hours. The solvent was evaporated and the residue was added to EtOAc (30mL) with 5% NaHCO₃Aqueous solution (2X 20mL) and brine (20mL) and washed with anhydrous MgSO₄The solvent was dried and evaporated to dryness. The residue was purified by flash chromatography (elution with TLC grade silica gel and a solvent gradient from 30% to 60% EtOAc in hexanes) to give the desired product 18-6 as a beige amorphous solid (459mg, 60% yield).

Step 6:

troc protected Aniline derivative 18-6(100mg,0.27mmol) in CH as above₂Cl₂(1mL) to a stirred solution was added pyridine (0.032mL,0.4mmol) followed by Fmoc-Cl (80mg,0.31 mmol). The reaction mixture was stirred at RT for 2 hours. The mixture was diluted with EtOAc (30mL) and NaHCO with 5% aqueous solution₃(2X 10mL) the suspension was washed with anhydrous MgSO₄Dried and evaporated to dryness. Purification of the residue by flash chromatography (elution with TLC grade silica gel and solvent gradient from 20% to 30% EtOAc in hexanes) afforded two samples of the desired Fmoc-protected product 18-7; 47mg of very pure product and 100mg of slightly less pure product.

And 7:

doubly protected (Troc-and Fmoc-protected) triphenylamine (trianniile) derivatives 18-7(100mg, ~0.17mmol) were dissolved in THF (1mL) and acetic acid (0.25mL), followed by addition of freshly activated zinc (20.0mg,0.31 mmol). The reaction mixture was stirred vigorously under nitrogen at RT for 2 hours. The progress of the reaction was monitored by HPLC and only-30% conversion was observed after 2 hours, so additional zinc (15mg) was added and stirring continued at 60 ℃ for 4 hours. The reaction mixture was diluted with EtOAc (30mL), filtered through Celite and the filtrate was cooled in an ice bath and dissolved in 5% waterLiquid NaHCO₃(20mL) washing; care must be taken to prevent excessive pressure build-up. The organic layer was washed with brine, anhydrous MgSO₄Drying and evaporation of the solvent gave methyl 4-amino-2- (9H-fluoren-9-ylmethoxycarbonylamino) -5- (methylamino) benzoate, a single protected intermediate, 18-8 as a white crystalline solid (68mg, 96% yield).

Compounds 18-8 were converted to the corresponding Fmoc-protected amine intermediates of general formula III in scheme 1 according to the methods of examples 3 or 7, and then the Fmoc-protected inhibitors of general formula Ic above were prepared using the method of example 4. These Fmoc-protected inhibitors of formula Ic, or the Fmoc-protected amine intermediates appropriate for use in these syntheses, can also be converted to Fmoc-protected inhibitors of formula Id above using the procedures of steps 1,2 and 3 of example 8. In both cases, the Fmoc protecting group can be removed by treatment with piperidine, well known to those skilled in the art, and saponification of the ester group can occur under basic conditions (the following protocol well known to those skilled in the art) to yield inhibitors such as compound 1032 (table 1) and 3060 (table 3). The free amino groups of these inhibitors are then combined with reagents known to those skilled in the art, such as isopropyl chloroformate and the like, to form inhibitors such as compound 1033 (Table 1).

Example 19

2- (5-bromopyrimidin-2-yl) -3-cyclopentyl-1-methyl-1H-indole-6-carboxylic acid

Step 1:

bromoindole 19-1 (prepared as described in example 12 of WO 03/010141) (3.0g, 8.9mmol,1 equiv.) is dissolved in anhydrous DME (20mL) and tris- (2-furyl) phosphine (260mg, 1.1mmol,0.12 equiv.), triethylamine (3.0mL,21.5mmol,2.4 equiv.) and Pd (OAc)₂(65mg, 0.28mmol,0.03 equiv.). The mixture is cleaned 10 with bubbling Ar therethroughFor a while, and pinacolborane (4,4,5, 5-tetramethyl-1, 3, 2-di-methyl-)Borane (diobolane); 3.0mL,20mmol,2.2 equiv.) was added using a syringe. The resulting dark brown mixture was stirred at 68 ℃ under argon for 16 hours. The reaction mixture was then cooled to RT and 5-bromo-2-iodopyrimidine (3.0g, 10.5mmol,1.18 equiv.) was added as a solid, followed by careful slow addition of K₃PO₄(10.5g, 47.1mmol,5.4 equiv.) of a cold suspension in water (7 mL). Or, K₃PO₄May be added in advance of the addition of 5-bromo-2-iodopyrimidine. The dark brown mixture was then heated to 80 ℃ under argon for 24 hours. The reaction mixture was cooled to RT and poured into 10% aqueous NaCl (100 mL). The brown suspension was extracted with EtOAc (150mL), and the extracts were washed with water (2X 50mL) and brine (100mL), dried and concentrated to 50 mL. Cooling in the refrigerator for 2 hours gave a beige precipitate, which was collected by filtration, washed with a small amount of EtOAc and dried. The filtrate was concentrated in vacuo and the residue was slurried in acetone (20mL), heated to boiling and cooled in a refrigerator overnight. The solids were filtered and the combined solids were purified by chromatography using CHCl₃As solvent, the desired indole ester 19-2 was obtained as a beige solid with 77% yield.

Step 2:

ester 19-2(300mg, 0.72mmol) was suspended in DMSO (10mL) and the suspension was gently heated to dissolve the solid. The slightly cloudy yellow solution was cooled and 2.5N NaOH (2.0mL,5.0mmol,8.6 equiv.) was added with stirring and stirring continued at RT for 4 h. The mixture was poured slowly into 0.5N HCl (200 mL). The yellow precipitate was collected by filtration and washed with water and dried to give compound 19-3(273mg, 94% yield, 100% homogeneity).

Example 20

3-cyclopentyl-1, 2-dimethyl-6-indolecarboxylic acid

2-Bromoindole derivative 19-1(1.009g,3.00mmol, prepared as described in example 12 in WO 03/010141) was dissolved in anhydrous THF (25mL) under argon and the solution was cooled to-78 ℃. n-BuLi (2.0M in hexanes, 1.60mL,3.20mmol) was added dropwise and the mixture stirred for 15 min. MeI (0.37mL,2.00mmol) was added and stirring continued for an additional 30 minutes. The reaction mixture was then warmed to RT and the volatiles removed under reduced pressure. The residue was dissolved in TBME (100mL) and the solution was washed with brine (2X 25 mL). The extract was dried (MgSO)₄) Concentrated under reduced pressure and the residue purified by flash chromatography using 0-15% EtOAc in hexanes as eluent. Desired 2-methylindole derivative 20-1 was obtained as a waxy solid (0.658g,80% yield): MS-ES m/z272.1 (MH)⁺). Saponification of methyl ester 20-1 in the usual way (NaOH/DMSO) gives the corresponding carboxylic acid 20-2 in 96% yield: MS-ES m/z 258.1 (MH)⁺)。

Example 21

3-cyclopentyl-2-vinyl-1-dimethyl-6-indolecarboxylic acid

2-Bromoindole 19-1 (prepared as described in example 12 of WO 03/010141) (5.000g,14.87mmol) was dissolved in anhydrous bisTo an alkane (50mL) was added ethylene tributyltin (4.82mL,16.50 mmol). Pumping the solution into N₂The bubbles were degassed for 15 minutes. Bis (triphenylphosphine) palladium (II) chloride (0.350g,0.50mmol) was added and the mixture was heated to 100 ℃ under nitrogen overnight. Additional catalyst (0.350g,0.50mmol) was added and heating continued for a further 48 hours, at which pointPoint TLC analysis showed the reaction was almost complete. The reaction mixture was cooled to RT and filtered through a small pad of silica gel, washed with THF. The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography using 5-15% EtOAc in hexanes as eluent. The desired 2-vinyl indole ester 21-1 was obtained as a brown solid (2.92g,69% yield): MS-ES m/z 284.1 (MH)⁺). Saponification of methyl ester 21-1 by the general procedure (NaOH/DMSO) gives the corresponding carboxylic acid 21-2 in 93% yield: MS-ESm/z 270.1 (MH)⁺)。

Example 22

3-cyclopentyl-2-ethyl-1-methyl-6-indolecarboxylic acid

2-Vinylindole ester 21-1 (example 21) (0.250g,0.88mmol) was dissolved in MeOH (15mL) and the solution was taken up in 10% Pd (OH)₂Hydrogenation with/C (50mg) (1atm H)₂Gas) for 18 h. The catalyst was then removed by filtration and the filtrate evaporated under reduced pressure to give the crude ester 22-1. The residue was dissolved in DMSO and saponified with NaOH in the general method to give the desired 2-ethylindole derivative 22-2 as a white solid (0.211g,88% yield): MS-ES m/z272.1 (MH)⁺)。

Example 23

3-cyclopentyl-2- (2-propenyl) -1-methyl-6-indolecarboxylic acid

2-Stannylalkylindole 23-1(1.280g,2.34 mmol; prepared using the method described in WO 03/010141), triphenylphosphine (0.065g,0.25mmol), CuI (0.045g,0.24mmol), LiCl (0.200g,4.72mmol) and 2-bromopropene (0.444mL,5.00mmol) were dissolved in DMF (6mL) and the suspension was suspended usingArgon gas was bubbled through for 20 minutes to degas. Adding Pd₂(dba)₃(0.035g,0.034mmol) and degassing for a further 10 minutes, the reaction mixture is heated to 100 ℃ overnight. The suspension was then diluted with TBME (100mL) and washed with brine (2X 25 mL). The extract was dried (MgSO)₄) And concentrated under reduced pressure to give a residue which was purified by flash chromatography using 5-10% EtOAc in hexanes as eluent. The desired 2- (2-propenyl) indole 23-2 was obtained as a beige solid (0.57g,81% yield): MS-ES m/z 298.1 (MH)⁺). Saponification of methyl ester 23-2 in the general procedure (NaOH/DMSO) gave the corresponding carboxylic acid 23-3 in 96% yield: MS-ES m/z 284.1 (MH)⁺)。

Example 24

3-cyclopentyl-2-isopropyl-1-methyl-6-indolecarboxylic acid

Following a similar procedure as described for the 2-ethyl analogue in example 22, 2-isopropylindole derivative 24-2 was obtained as a white solid (88% yield): MS-ES m/z 286.1 (MH)⁺)。

Example 25

3-cyclopentyl-2-cyclopropyl-1-methyl-6-indolecarboxylic acid

Cyclopropyl bromide (0.471g,3.90mmol) was dissolved in anhydrous THF (20mL) and the solution was cooled to-78 ℃ under argon pressure. nBuLi (0.1M in hexanes, 3.60mL,3.60mmol) was added and the mixture was stirred for 15 minutes. Then ZnBr is added₂(0.878g,3.90mmol) in THF (15mL), the mixture was warmed up to RT and the reaction was stirred for 15 min. 2-bromoindole 19-1 (as in WO 03/010141)Prepared as described in example 12) (1.009g,3.00mmol) in THF (15mL) was added followed by tetrakis (triphenylphosphine) palladium (0) (0.289g,0.25 mmol). The mixture was stirred at reflux for 24 hours, at which point the starting material was still present, but the reaction was stopped by the addition of AcOH (2 mL). The volatiles were removed under reduced pressure and the residue was extracted with TBME (100 mL). With NaHCO₃The extract was washed with saturated aqueous solution and dried (MgSO₄). Evaporation under reduced pressure gave a residue which could be purified by flash chromatography using 0-15% EtOAc in hexanes as eluent to give the desired 2-cyclopropylindole ester 25-1 as a pale green solid (0.540g,60% yield): MS-ES m/z 298.1 (MH)⁺). The methyl ester 25-1 can be saponified by the general method (NaOH/DMSO) to give the corresponding carboxylic acid 25-2 in 80% yield: MS-ES m/z 284.1 (MH)⁺)。

Example 26

3-cyclopentyl-1-methyl-2- (1-pyrazolyl) -6-indolecarboxylic acid

2-bromoindole 19-1 (prepared as described in example 12 in WO 03/010141) (1.00g,2.97mmol) and pyrazole (2.00g,20.4mmol,9.9 equiv.) were added to a sealed tube and the mixture was heated to 160 ℃ for 72 h. The reaction mixture was then cooled to RT and loaded onto a flash chromatography column. The product was eluted using 40-100% EtOAc in hexane as eluent. The recovered material (1.60g), containing the pyrazole, was dissolved in a mixture of THF/MeOH/water and basic with 1N NaOH. The organic layer was then evaporated under reduced pressure and the residue treated with concentrated HCl to precipitate the desired 2-pyrazolyl indolecarboxylic acid 26-1(0.400g, 43% yield).

Analogs containing other N-linked heterocyclic substituents at C-2 of the indole ring can be prepared using analogous methods, starting with nitrogen-based heterocycles such as imidazoles and triazoles.

Example 27

(E) -3- [2- (1-Aminocyclobutyl) -3-methyl-3H-benzimidazol-5-yl ] acrylic acid methyl ester

Step 1:

a solution of 2, 4-dichloronitrobenzene (27-1) (61g,0.32mmol), triethylamine (68mL, 0.48mol) and 2.0M methylamine in THF (500mL, 1.0mol) was combined under argon in a 3-L round bottom flask equipped with a Graham condenser. The solution was then stirred with heating at 40 ℃ and had a white solid (Et)₃NH⁺Cl^-) Formation begins. After heating for 6 hours, TLC (in 20% ethyl acetate in hexanes) showed the reaction to be about-60% complete. An additional two equivalents of methylamine in THF (330mL) were added and the mixture was heated with additional stirring at 40 ℃ for 16 hours. TLC showed all starting material was consumed. The reaction mixture was allowed to cool to room temperature and the white solid was removed by filtration and washed with THF. The filtrate was concentrated under reduced pressure and redissolved in 800mL of dichloromethane, washed with water and brine and washed with Na₂SO₄And (5) drying. The solvent was removed in vacuo to give compound 27-2 as an orange solid (59.5g, quantitative) which was pure enough to be used in the next step.

Step 2:

compound 27-2(2.88g,15mmol), Pd were added under argon in a dry tube₂(dba)₃(414mg,0.45mmol)、P(t-Bu)₃(0.1M in IIA solution of alkane, 18mL,1.8mmol) and N, N-dicyclohexylmethylamine (3.6mL,16.5 mmol). N-butyl acrylate (2.4mL,16.5mmol) was degassed with argon for 35 min and then added to the mixture. The tube was then sealed and the mixture was heated with stirring at 110 ℃ over the weekend. The reaction was cooled to ambient temperature and usedDilute with ethyl acetate (200 mL). The mixture was filtered through a pad of silica gel to remove the solid residue and washed with ethyl acetate (700 mL). The filtrate was concentrated in vacuo and co-evaporated with hexane three times. The red solid was then stirred with hexane (40mL) at 60 ℃. And the mixture was cooled to 0 ℃ for 15 minutes and the red solid was collected by filtration and washed with hexane, then dried again under high vacuum (3.4g,81% yield). The product 27-3 was about 90% pure by NMR, and other products were obtained from the filtrate using flash column purification.

And step 3:

compound 27-3 was converted to compound 27-4 using the procedure of step 5 of example 11.

And 4, step 4:

compound 27-4 was converted to compound 27-5 using the procedure of example 3.

Example 28

(E) -3- [2- (1-Aminocyclobutyl) -7-chloro-3-methyl-3H-benzimidazol-5-yl ] acrylic acid methyl ester

Step 1:

4-amino-3-nitrobenzoic acid 28-1(15.00g,82mmol) was dissolved in AcOH (200mL) and sulfonyl chloride (6.62mL,82mmol) was added. The mixture was stirred at RT for 2 hours, after which additional sulfuryl chloride (1.5mL) was added to complete the reaction. After stirring at RT for a further 1h, the reaction mixture was poured onto ice and the precipitate was collected by filtration. The product 28-2 was washed with water, air dried and used directly in the next step.

Step 2:

crude 28-2 was dissolved in MeOH (300mL) and concentrated H was added₂SO₄(1 mL). The mixture was refluxed for 2 days, after which point the conversion was about-75% complete. Under reduced pressureThe volatiles were removed and partitioned between EtOAc and water. By using Na₂CO₃The slow addition of the saturated aqueous solution alkalinizes the mixture and allows the organic phase to separate. The extract was washed with brine and dried (Na)₂SO₄) And concentrated to give 28-3 as a beige solid (12.32g), which was used directly in the next step.

And step 3:

nitroaniline 28-3(11.32g,49mmol), sodium bisulfite (35.54g,204mmol) and NaHCO₃(17.15g,204mmol) was dissolved in 3:2 EtOH/water (600 mL). The orange mixture was stirred at RT for 20 h. The EtOH was then removed under reduced pressure and the product was extracted with EtOAc. The extract was washed with water and brine, dried (Na)₂SO₄) And evaporated to give compound 28-4 as a brown solid (4.60g, 46% yield), which was used in the next step without purification.

And 4, step 4:

diamine 28-4(1.00g,5.0mmol), N-Boc-1-aminocyclobutanecarboxylic acid (1.07g,5.0mmol), HATU (2.20g,5.8mmol) and Et₃N (2.10mL,15.0mmol) was dissolved in DMF (30mL) and the mixture was stirred at RT for 2 days. The reaction mixture was poured onto ice and the precipitated solid was collected by filtration. The material was washed with water, dissolved in EtOAc and extracted with brine. The solution was then dried (Na)₂SO₄) And concentrated under reduced pressure. The residue was dissolved in AcOH and heated to 80 ℃ for 3 hours. HPLC analysis showed complete conversion to the desired benzimidazole derivative. AcOH was removed under reduced pressure, the residue was extracted with EtOAc and with NaHCO₃The solution was washed with water and brine, dried (MgSO)₄) Thereafter, the solvent was removed to give compound 28-5 as an orange solid (563mg), which was used directly in the next step.

And 5:

benzimidazole 28-5(1.63g,4.29mmol) and K₂CO₃(2.96g,21.45mmol) was suspended in DMF (10mL) and iodomethane (0.27mL,4.30mmol) was added. The mixture was stirred at RT for 3 hours. Then will beThe reaction mixture was poured onto ice and the precipitated solid was collected by filtration. The material was washed with water, dissolved in EtOAc and the solution was washed twice with 5% aqueous citric acid and brine, dried (MgSO)₄) Volatiles were then removed under reduced pressure to give compound 28-6 as a brown solid (1.44g), which was used directly in the next step.

Step 6:

methyl ester 28-6(1.22g,3.10mmol) was dissolved in THF (30mL) and LiBH was added in small portions at RT₄(0.243g,11.14 mmol). The mixture was then stirred at 40 ℃ for 16 hours. Since the conversion is not yet complete, additional LiBH is added₄(0.100g,4.6mmol) and the mixture was stirred at 70 ℃ for a further 3 hours. The reaction mixture was cooled to RT and the residue was diluted with EtOAc. Water was carefully added and the organic phase was allowed to separate. The extract was washed with water and brine and dried (MgSO₄). Crude alcohol 28-7(961mg) and other batches of product were combined and purified using flash chromatography.

And 7:

purified alcohol 28-7 from above (0.450g,1.02mmol) was dissolved in DCM (20mL) and Dess-Martin periodinane (0.551g,1.30mmol) was added. The mixture was stirred at RT for 2 hours. (ethyloxymethylene) triphenylphosphorane (0.550g,1.58mmol) was then added, and the mixture was refluxed for 20 hours. The volatiles were then removed under reduced pressure and the residue was dissolved in 1:1 TFA-DCM to remove the Boc protecting group. After stirring at RT for 1 hour, the volatiles were removed under reduced pressure and the residue was partitioned between EtOAc and 1N HCl. The aqueous phase containing the product was separated and washed with 2M Na₂CO₃Neutralized and extracted 2X with EtOAc. The extract was dried (Na)₂SO₄) And concentrated to give compound 28-8 as a white foam (212mg) which was purified by flash chromatography using 80-100% EtOAc in hexanes as eluent. A white solid (66mg) of the desired benzimidazole fragment (fragment).

Example 29

5-[2-(1-Aminocyclobutyl) -3-methyl-3H-benzimidazol-5-yl]-3H-[1,3,4]Diazol-2-ones

Step 1:

10N HCl (2mL) was added to a solution of 3-fluoro-4-nitro-benzoic acid (29-1) (10g,54.0mmol) in 300mL MeOH and the solution refluxed for 15 hours. The mixture was then concentrated, the residue diluted with EtOAc, and the organic phase was washed with 2X water and NaHO₃Washing with saturated aqueous solution, and drying (MgSO)₄) Filtration and evaporation gave 10.45g (97% yield) of compound 29-2 as a white solid. This compound can be used in the next reaction.

Step 2:

methylamine (2N solution in THF, 80mL) was added dropwise to a solution of compound 29-2(10g,50.2mmol) in 100mL THF at 0 ℃. The mixture was stirred at 0 ℃ for a further 20 minutes, then at room temperature for a further 15 hours. The volatiles were then evaporated and the residue was diluted with EtOAc and washed with 2X water, NaHO₃The organic phase was washed with saturated aqueous solution and dried (MgSO)₄) Filtration and evaporation gave 10.21g (96% yield) 29-3 as an orange solid. This compound can be used in the next reaction.

And step 3:

palladium (1g on 10% carbon) was added to a solution of compound 29-3(10g,47.6mmol) in 400mL of a 1/1 mixture of THF-anhydrous EtOH. The mixture was stirred under hydrogen for 16 hours, then the solution was filtered to remove the catalyst and concentrated to give 8.5g (99% yield) of compound 29-4 as an off-white solid. This compound can be used in the next reaction.

And 4, step 4:

compound 29-4 was converted to compound 29-5 using the procedure of example 7.

And 5:

a mixture of compound 29-5(730g,2.03mmol) and hydrazine monohydrate (500. mu.L, 10.3mmol) in 5mL ethanol was heated at 85 ℃ for 72 hours in a vial with a screw cap. The solution was then concentrated with CH₂Cl₂Dilute and wash the organic layer with water. The organic layer was dried (Na)₂SO₄) Filtration and evaporation gave 642mg (88%) of compound 29-6 as an off-white solid. Which can be used in the next step.

Step 6:

triethylamine (190 μ L,1.36mmol) was added to a solution of compound 29-6(350mg,0.97mmol) and 1, 1' -carbonyldiimidazole (190mg,1.17mmol) in THF (5 mL). The mixture was stirred at room temperature for 15 hours. The volatiles were removed and the residue was diluted with EtOAc, washed with water, brine and the organic layer was dried (Na)₂SO₄) Filtration and evaporation gave 318mg (85% yield) of compound 29-7 as a waxy white solid, which was used in the next step.

And 7:

TFA (3mL) was added dropwise to a suspension of compound 29-7(150mg,0.39mmol) in dichloromethane (10mL) and the resulting solution was stirred for 1 hour. The volatiles were evaporated to give 150mg (quantitative yield) of the trifluoroacetate salt of the desired compound 29-8 as a beige solid.

Example 30

5- [2- (1-Aminocyclobutyl) -3-methyl-3H-benzimidazol-5-yl]-3-methyl-3H-1, 3,4-Diazol-2-ones

Potassium carbonate (32mg,0.32mmol) was added to a solution of compound 29.7(80mg,0.21mmol) in DMF (1 mL). The suspension was stirred at room temperature for 15 minutes. Methyl iodide (12.5 μ L,0.2mmol) was then added and the mixture stirred at room temperature for 3 hours. The mixture was diluted with EtOAc, washed with water (3 ×), brine, and the organic phase was dried (MgSO)₄) Filtered and evaporated to yield 67mg (81% yield) of an off-white solid. Treatment with TFA as described in example 29, step 7, afforded 57mg (quantitative yield) of the trifluoroacetate salt of desired compound 30-1 as an off-white solid.

Compound 30-1 can be coupled to the indole intermediate of formula II using the methods of examples 4 and 34, step 1, to afford the compound of formula I.

Example 31

5- [2- (1-aminocyclobutyl) -3-methyl-3H-benzimidazol-5-yl ] -2-methyl-2H-pyrazol-3-ol

Step 1:

NaOH (10N, 11mL, 110mmol) was added to a solution of compound 29-5(5.0g,13.9mmol) in 3:2:1THF, MeOH, and water (180mL) and the solution was stirred at room temperature overnight. The mixture was then concentrated, the pH adjusted to 4 using 1N HCl and the mixture extracted with EtOAc. The organic layer was washed with brine and dried (MgSO)₄) Filtration and evaporation gave compound 31-1(3.94g,82% yield) as a white solid. This compound can be used in the next reaction.

Step 2:

1, 1' -carbonyldiimidazole (702mg,4.33mmol) was added to a solution of compound 31-1(1g,2.90mmol) in THF (24 mL). The solution was stirred for 15 hours and then added dropwise to the malonate anion (Et (R) at 0 ℃₃N (0.81mL,5.80mmol) and MgCl₂(690mg,7.25mmol) was added to malonic acid monoethyl ester potassium salt (1g,5.96mmol) in acetonitrile (10mL)To the solution, followed by stirring at room temperature for 2.5 hours). The resulting mixture was then slowly warmed to room temperature and stirred for a total of about 48 hours. The mixture was concentrated and toluene was added. The mixture was cooled to 10-15 ℃ and slowly hydrolyzed by adding 1M HCl until the pH reached 3-4. The phase layers were then separated and the organic layer was diluted with EtOAc, washed with water, dried and evaporated to give a yellow oil. The product was purified by flash chromatography (eluent: hexane: AcOEt 4:6) to give 885mg (74% yield) of compound 31-2 as a white solid.

And step 3:

methylhydrazine (29. mu.L, 0.55mmol) was added to a solution of compound 31-2(100mg,0.24mmol) in EtOH (2.5 mL). The mixture was stirred at 80 ℃ for 15 hours. The mixture was then concentrated and water was added, followed by adjustment of the pH to 6-7 with the addition of 1N HCl. The aqueous layer was extracted 3 times with EtOAc and the organic layer was dried (MgSO₄) And concentrated to give 94mg (98% yield) of a pale yellow solid. Treatment with TFA in dichloromethane afforded 93mg (quantitative yield) of the trifluoroacetate salt of compound 31-3 by the method described in step 7 of example 29.

Compound 31-3 can be coupled to the indole intermediate of formula II using the methods of example 4 and 34, step 1, to give the compound of formula I.

Example 32

5- [2- (1-Aminocyclobutyl) -3-methyl-3H-benzimidazol-5-yl ] -3H-1,3, 4-thiadiazol-2-one

Step 1:

TBTU (380mg,1.18mmol) and triethylamine (380. mu.L, 2.73mmol) were added to a solution of compound 31-1(350mg,1.01mmol) and ethyl carbazate (120mg,1.15mmol) in DMF (5 mL). The mixture was stirred at rt for 15 h, then diluted with EtOAc.The resulting organic suspension was washed with 2X water and 1X NaHCO₃And (5) washing with saturated aqueous solution. THF was then added to the organic layer to give a solution, which was dried (MgSO)₄) Filtering and concentrating. The residue was triturated with EtOAc to give 290mg (66%) of Compound 32-1 as an off-white solid. This compound can be used in the next reaction.

Step 2:

lawesson's reagent (70mg,0.17mmol) was added to compound 32-1(150mg,0.35mmol) in bis at 100 deg.CAlkane (10 mL). The resulting mixture was stirred at 100 ℃ for 8 hours and then at 140 ℃ for 4 hours. The mixture was then cooled to 100 ℃ and an additional portion of Lawesson's reagent (70mg,0.17mmol) was added. The solution was then heated at 100 ℃ for 15 hours. The mixture was concentrated to dryness and the solid residue was triturated with EtOAc and filtered. Treatment of the resulting beige solid (100mg) with TFA as described in step 7 of example 29 afforded 93mg of compound 32-2 as the trifluoroacetate salt.

Compound 32-2 can be coupled to the indole intermediate of formula II using the methods of example 4 and 34, step 1, to give the compound of formula I.

Example 33

[1- (1-methyl-6-pyrimidin-2-yl-1H-benzimidazol-2-yl) cyclobutyl ] carbamic acid tert-butyl ester

Step 1:

commercially available 1, 3-dibromobenzene 33-1(4.1mL,33.9mmol) was dissolved in concentrated sulfuric acid (35mL) and cooled in an ice bath. Potassium nitrate (3.4g,33.9mmol) was added slowly (in small portions) so that the internal reaction temperature could be maintained below 10 ℃. The reaction mixture was stirred for a further 30 minutes, after which it was poured into 1L of ice. The yellow precipitate (33-2) formed was filtered and washed with water. Dried under reduced pressure and used directly in the following step.

Step 2:

a mixture of compound 33-2(6.3g,22.4mmol) and methylamine hydrochloride (3.0g,44.8mmol) in DMF (50mL) was cooled to 0 ℃. Triethylamine (9.4mL,67mmol) was added and the mixture was stirred at RT for 3.5 h and then heated to 70 ℃ overnight. The mixture was poured into water and the resulting precipitate was filtered. The filtrate was extracted with EtOAc (3X) and the extracts were washed with water (3X) and saturated NaCl and dried (MgSO)₄) Filtration and concentration gave a mixture of compounds 33-3 and 33-4 as an orange solid (4.8g) which was used in the next step.

And step 3:

nitro Compound 33-3 with Na was allowed to proceed using the method described in step 5 of example 11₂S₂O₄/K₂CO₃And (3) reduction reaction of (2). Compound 33-5(1.5g, ~20%, 3 steps yield) was isolated from the reaction mixture by column chromatography using a solvent gradient EtOAc in hexanes from 17% to 25%.

And 4, step 4:

diphenylamine 33-5 was converted to compound 33-6 using the procedure described in example 7.

And 5:

argon gas was bubbled into compound 33-6(300mg,0.79mmol), lithium chloride (67mg,1.6mmol), PPh₃(31mg,0.12mmol) and 2-tributylstannyl pyrimidine (365mg,0.99mmol) in DMF (6.0mL) for 15 min. Pd (PPh)₃)₄(91mg,0.079mmol) and CuI (15mg,0.079mmol) were added and the mixture was heated at 100 ℃ for 24 hours. The mixture was diluted with EtOAc and the organic phase was washed with water and brine, then dried (MgSO)₄) And concentrated to give a yellow oil which was purified by flash chromatography (hexanes: EtOAc3:7 to 2:8) to give compound 33-7 as a yellow solid (100mg, 24%).

Compounds 33-7 may be deprotected using standard conditions described in step 7 of example 29 and the resulting amine coupled to the indole intermediate of formula II using the methods of examples 4 and 34 step 1 to give the compound of formula I.

It will be appreciated by those skilled in the art that the preparation of analogous intermediates with similar heterocyclic or aromatic groups can be carried out using this method or variations thereof. Alternatively, the coupling reaction of step 5 can be carried out using well-known conditions in the Suzuki reaction (A.Suzuki, Pure appl. chem. (1994)66,213; N.Miyaura and A.Suzuki, chem.Rev. (1995)95，2457)。

Example 34

(E) -3- [2- (1- { [2- (5-bromopyrimidin-2-yl) -3-cyclopentyl-1-methyl-1H-indole-6-carbonyl ] -amino } -cyclobutyl) -3-methyl-3H-benzimidazol-5-yl ] -acrylic acid

Compound 19-3 (example 19) and compound 34-1 (prepared from compound 10-2 using the method of example 3) were coupled using the method of example 4 to give compound 34-2 (compound 3085, Table 3) as a dark yellow solid (9.3%).

¹H NMR(400MHz,DMSO-d₆),δ1.63(bs,2H),1.80-1.95(m,6H),1.95-2.10(m,2H),2.70(ddd,J=9.3&10.6Hz,2H),2.99(m,2H),3.65-3.75(m,1H),3.76(s,3H),3.85(s,3H),6.54(d,J=15.6Hz,1H),7.52(d,J=8.4Hz,1H),7.59(d,J=8.4Hz,1H),7.64(d,J=8.2Hz,1H),7.70(d,J=15.9Hz,1H),7.74(d,J=8.4Hz,1H),7.86(s,1H),8.12(s,1H),9.18(s,2H),9.20(s,1H),12.25(s,1H)。

Example 35

3-cyclopentyl-1-methyl-2-pyridin-2-yl-1H-indole-6-carboxylic acid {1- [ 1-methyl-6- (5-oxo-4, 5-dihydro-1, 3,4-Oxadiazol-2-yl) -1H-benzimidazol-2-yl]-cyclobutyl } -amide

Step 1:

TBTU (350mg,1.09mmol) and triethylamine (380mL, 2.73mmol) were added to a solution of compound 35-1 (compound 1025, Table 1) (487mg,0.89mmol) and tert-butyl carbazate (130mg,0.98mmol) in DMF (8 mL). And the mixture was stirred at room temperature for 2 hours, then diluted with EtOAc. The resulting organic suspension was washed with 2X water and 1X NaHCO₃And (5) washing with saturated aqueous solution. THF was then added to the organic layer and the resulting solution was dried (MgSO₄) Filtered and concentrated. The residue was triturated with EtOAc to give 421mg (72%) of compound 35-2 as an off-white solid. This compound can be used in the next reaction.

Step 2:

TFA (3mL) was added dropwise to a solution of compound 35-2(200mg,0.3mmol) in dichloromethane (3mL) and the resulting solution was stirred for 2 h. Evaporation of the volatiles gave 170mg (quantitative yield) of the trifluoroacetate salt of compound 35-3, which was used directly without further purification.

And step 3:

1, 1' -carbonyldiimidazole (25mg,0.15mmol) was added in one portion to a solution of compound 35-3(100mg,0.13mmol) and triethylamine (80. mu.L, 0.57mmol) in 2mL THF and the resulting solution was stirred at room temperature for 4 hours. The mixture was then concentrated under reduced pressure, diluted with 4mL of DMSO and directly at reverse phase C₁₈Semi-preparative HPLC column (from 5% H using solvent gradient)₂O to 100% MeCN in MeCN) to isolate compound 35-4 (compound 1128, table 1) as a yellow amorphous solid with>95% homogeneity (29mg,39% yield).

¹H NMR(400MHz,DMSO)：δ1.54-1.68(m,2H),1.79-1.93(m,6H),1.94-2.05(m,1H),2.09-2.21(m,1H),2.75-2.85(m,2H),3.05-3.25(m,3H),3.69(s,3H),3.90(s,3H),7.49(m,1H),7.57-7.72(m,3H),7.82-7.92(m,2H),7.94-8.02(m,1H),8.06-8.15(m,2H),8.78(d,J=3.9Hz,1H),9.45(s,1H),12.62(s,1H)。

Example 36

3-cyclopentyl-1-methyl-2-pyridin-2-yl-1H-indole-6-carboxylic acid {1- [6- (5-amino-1, 3, 4-)Oxadiazol-2-yl) -1-methyl-1H-benzimidazol-2-yl]Cyclobutyl amides

C- (di-imidazol-1-yl) -methylidene amine (25mg,0.16mmol) was added in one portion to a solution of compound 35-3 (example 35) (80mg,0.14mmol) in THF (4 mL). The resulting solution was heated to 70 ℃ for 16 hours, after which a white precipitate was observed. The reaction was then concentrated under reduced pressure and dissolved in 4mL DMSO and directly in reverse phase C₁₈Semi-preparative HPLC column (from 5% H using solvent gradient)₂O to 100% MeCN in MeCN) to isolate compound 36-1 (compound 1129, table 1) as a yellow amorphous solid with>95% homogeneity (19mg,23% yield).

¹H NMR(400MHz,DMSO)：δ1.54-1.67(m,2H),1.79-1.94(m,6H),1.95-2.06(m,1H),2.11-2.23(m,1H),2.74-2.84(m,2H),3.19-3.05(m,3H),3.69(s,3H),3.91(s,3H),7.49(dd,J=1.8&5.7Hz,1H),7.59-7.71(m,3H),7.86-7.92(m,2H),7.96-8.01(m,1H),8.06-8.10(m,1H),8.10(s,1H),8.78(d,J=4.3Hz,1H),9.51(s,1H)。

Example 37

3-cyclopentyl-1-methyl-2-pyridin-2-yl-1H-indole-6-carboxylic acid [1- (1-methyl-6-1, 3,4-oxadiazol-2-yl-1H-benzimidazol-2-yl) cyclobutyl]Amides of carboxylic acids

Di(s) of Compound 35-3 (example 27) (50mg,0.09mmol) and triethyl orthoformate (1mL, 6mmol)The suspension of alkane (3mL) was heated at reflux for 18 h. The resulting almost clear solution was evaporated to dryness and the residue was dissolved in DMSO (1mL) and in reverse phase C₁₈Semi-preparative HPLC column (from 5% H using solvent gradient)₂O to 100% MeCN in MeCN) to isolate compound 37-1 (compound 1130, table 1) as a yellow amorphous solid with>95% homogeneity (27mg,53% yield).

¹H NMR(400MHz,DMSO)：δ1.55-1.68(m,2H),1.79-1.93(m,6H),1.95-2.04(m,1H),2.12-2.20(m,1H),2.82-2.74(m,2H),3.15-3.05(m,3H),3.69(s,3H),3.92(s,3H),7.49(dd,J=2.2&5.3Hz,1H),7.56-7.73(m,3H),7.93-8.05(m,3H),8.11(s,1H),8.33(s,1H),8.79(d,J=4.3Hz,1H),9.37(s,1H),9.44(s,1H)。

Example 38

3-cyclopentyl-2- (5-fluoro-pyridin-2-yl) -1-methyl-1H-indole-6-carboxylic acid {1- [ 1-methyl-6- (5-oxo-4, 5-dihydro-1, 3,4-Oxadiazol-2-yl) -1H-benzimidazol-2-yl]-cyclobutyl } -amide

TBTU (45mg,0.14mmol) and trisEthylamine (49mL, 0.35mmol) was added to a solution of compound 38-1 (prepared using the method described in WO 03/010141) (45mg,0.13mmol) and compound 29-8 (example 29) (45mg,0.11mmol) in DMF. The solution was stirred for 15 hours and directly in reverse phase C₁₈Semi-preparative HPLC column (from 5% H using solvent gradient)₂O to 100% MeCN in MeCN) to isolate compound 38-2 (compound 1143, table 1) as a yellow amorphous solid with>95% homogeneity (23mg, 34% yield).

¹H NMR(400MHz,DMSO):δ1.54-1.68(m,2H),1.79-1.93(m,6H),1.93-2.04(m,1H),2.07-2.20(m,1H),2.72-2.82(m,2H),3.00-3.15(m,3H),3.67(s,3H),3.89(s,3H),7.57-7.72(m,3H),7.79-7.95(m,3H),8.10(s,2H),8.80(d,J=2.9Hz,1H),9.45(s,1H),12.63(s,1H)。

Example 39

Inhibition of NS5B RNA-dependent RNA polymerase activity

The compounds of the present invention were tested for activity as inhibitors of hepatitis c virus RNA-dependent polymerase (NS5B) as described in WO 03/010141.

Example 40

Specificity of NS5B RNA-dependent RNA polymerase inhibition

The compounds of the invention were tested for activity against a poliovirus RNA-dependent RNA polymerase inhibitor in the format described for HCV polymerase except that the HCV NS5B polymerase was replaced with a poliovirus polymerase as described in WO 03/010141. The compounds may also be characterized as inhibiting calf thymus DNA-dependent RNA polymerase II (McKercher et al, 2004Nucleic Acids Res.32: 422-431) in the previously described experimental format.

EXAMPLE 41

Cell-based luciferase reporter HCV RNA replication experiments

Cell culture:

huh-7 cells with a stable subgenomic HCV replicon encoding a modified luciferase reporter (expressed as luciferase-FMDV 2A-neomycin phosphotransferase fusion gene) were established as described previously (Lohman et al, 1999, Science 285:110-113; Vrojik et al, 2003J. Virol Methods110:201-209) except that replicon cells were selected using 0.25mg/mL G418. The amount of luciferase expressed by the selected cells is directly related to the level of HCV replication. These cells, designated MP-1 cells, were maintained in Dulbecco's Modified Early Medium (DMEM) (standard Medium) supplemented with 10% FBS and 0.25mg/mL neomycin. These cells were passaged by trypsinization and frozen in 90% FBS/10% DMSO. During the experiment, DMEM medium supplemented with 10% FBS and containing 0.5% DMSO and lacking neomycin was used (experimental medium). On the day of the experiment, MP-1 cells were trypsinized and diluted to 100000 cells/mL in the experimental medium. 100 μ L was distributed to a black 96-well Viewplate^TM(Packard) in each well. The plates were then placed at 37 ℃ with 5% CO₂The culture was carried out for two hours.

Reagents and materials:

preparation of test compounds:

test compounds in 100% DMSO were first diluted in experimental medium to a final DMSO concentration of 0.5%. The solution was sonicated for 15 minutes and filtered with a 0.22 μ M Millipore filter pack onto column 3 of a polypropylene Deep Well Titer Plate (Polypropylene Deep-Well Titer Plate) and the appropriate volume was transferred to the assay medium to give the starting concentration (2X) for the assay. In columns 2 and 4 to 12, 200. mu.L of experimental medium (containing 0.5% DMSO) was added. A series of dilutions (1/2) was prepared using 200 μ Ι _ transferred from column 3 to column 4, then from column 4 to column 5, and series through to column 11. Columns 2 and 12 are no inhibition controls.

Test compounds were added to the cells:

transfer of 100. mu.L volume from each well in compound dilution plates to Cell plates (Cell plates) (using two columns as "no inhibition control", ten [10 ]]The column was used for dose response). Cell culture plates were incubated at 37 ℃ and 5% CO₂The culture was carried out for 72 hours.

Luciferase assay:

following a 72 hour incubation period, the medium was removed from the 96-well assay plate and a volume of 100 μ L of 1 XGlo lysis buffer (Promega) pre-warmed to room temperature was added to each well. The plates were incubated at room temperature for 10 minutes with occasional shaking. And sticking the black adhesive tape to the bottom of the plate. Add 100. mu.L of Bright-Glo luciferase substrate (Promega) pre-warmed to room temperature to each plate, followed by gentle mixing. The fluorescence was measured using a Data Mode Luminescence (CPS) in a Packard Topcount instrument with a1 minute count delay and a2 second count time.

Fluorescence measurements (CPS) in each well of the plate measure the amount of HCV RNA replicated in the presence of various concentrations of inhibitors. Percent inhibition was calculated using the following equation:

inhibition% =100- [ CPS (inhibitor)/CPS (control) × 100]

Nonlinear curves consistent with Hill mode were used for inhibition-concentration data, with 50% Effective Concentration (EC)₅₀) May be calculated using SAS Software (Statistical Software; SAS Institute, inc.

Compound watch

All compounds listed in tables 1 to 4 below were found to have unexpectedly good activity in the cell-based HCV RNA replication experiment described in example 41.

Retention time (t) of each compound_R) Can be measured using standard analytical HPLC conditions described in the examples. As is well known to those skilled in the art, retention time values are sensitive to specific measurement conditions. Thus, even if the same conditions of solvent, flow rate, linear gradient, etc. are used, the residence time values may change at the time of measurement, due to e.g. the use of different HPLC instruments. Even if the same instrument is used for measurement, the value may change at the time of measurement, for example, because different respective HPLC columns are used, or when the same instrument and the same respective column are used for measurement, the value may change because, for example, different contingencies occur between the respective measurements.

TABLE 1

Wherein R is²、R³、R⁵、R⁶、R⁹And R¹⁰As shown in the table.

TABLE 2

Wherein R is²、R³、R⁵、R⁶、R⁹And R¹⁰As shown in the table.

TABLE 3

Wherein R is²、R⁴、R⁵、R⁸、R⁹And R¹⁰As shown in the table.

TABLE 4

Wherein R is¹、R²、R³、R⁵、R⁶、R⁹And R¹⁰As shown in the table.

Claims

1. A compound of formula (I), or an enantiomer, diastereomer or tautomer thereof, including salts or esters thereof:

wherein:

a or B is N and the other B or A is C, wherein- - -between two C-atoms represents a double bond and- - -between a C-atom and an N-atom represents a single bond;

R¹is H or (C)_1-6) An alkyl group;

wherein R is²¹Is a mono, di or tri substituent, each independently selected from-OH, -CN, -N (R)^N2)R^N1、

Halogen, (C)_1-6) Alkyl, (C)_1-6) Alkoxy group, (C)_1-6) Alkylthio, Het and-CO-N (R)^N2)R^N1；

Wherein (C) is_1-6) Alkyl, (C)_1-6) Alkoxy and (C)_1-6) Each alkylthio group is optionally substituted with one, two or three halogen atoms;

R⁵and R⁶One is selected from COOH, -CO-N (R)^N2)R^N1Aryl, Het and (C)_2-6) Alkenyl, wherein aryl, Het, (C)_2-6) Alkenyl and R^N1Or any at R^N2And R^N1Each of the heterocyclic rings formed therebetween is optionally substituted by R⁵⁰Substitution;

wherein R is⁵⁰Is one, two or three substituents, each independently selected from (C)_1-6) Alkyl, -COOH, -OH, oxo, -N (R)^N2)R^N1,-CO-N(R^N2)R^N1And halogen, wherein (C)_1-6) Alkyl being optionally substituted by aryl or-N (R)^N2)R^N1Substitution;

and R is⁵And R⁶Is selected from H, (C)_1-6) Alkyl, (C)_1-6) Alkoxy group, (C)_1-6) Alkylthio andN(R^N2)R^N1；

R⁹and R¹⁰Each independently selected from (C)_1-6) An alkyl group; or R⁹And R¹⁰Are linked and form together with the carbon atom to which they are linked (C)_3-7) Cycloalkyl group, (C)_5-7) Cycloalkenyl or a 4-, 5-or 6-membered heterocyclic ring having 1 to 3 heteroatoms each independently selected from O, N and S;

wherein the cycloalkyl, cycloalkenyl or heterocyclyl is optionally substituted in each case by (C)_1-4) Alkyl substituted;

wherein (C) is_1-6) Alkyl, (C)_3-7) Cycloalkyl group, (C)_3-7) Cycloalkyl- (C)_1-6) Alkyl, -CO- (C)_1-6) Alkane (I) and its preparation method

A radical and-CO-O- (C)_1-6) Each alkyl and cycloalkyl moiety in the alkyl group is optionally substituted by one, two or three substituents each independently selected from halogen, (C)_1-6) Alkoxy and (C)_1-6) Substituted by a substituent of alkylthio; and is

R^N2Is H or (C)_1-6) Alkyl, or

R^N2And R^N1May be linked, together with the nitrogen atom to which they are attached, to form a 4-,5-, 6-or 7-membered saturated, unsaturated or aromatic N-containing heterocycle or an 8-, 9-, 10-or 11-membered saturated, unsaturated or aromatic N-containing heterobicycle, each optionally also having 1 to 3 heteroatoms each independently selected from O, N and S;

wherein is represented by R^N2And R^N1The heterocyclic or heterobicyclic ring formed being optionally substitutedOne, two or three are independently selected from halogen and (C)_1-6) Alkyl, (C)_1-6) Alkoxy and (C)_1-6) Alkylthio group substituent;

wherein Het is defined as a 4-,5-, 6-or 7-membered heterocyclic ring having 1 to 4 heteroatoms each independently selected from O, N and S, which heterocyclic ring may be saturated, unsaturated or aromatic, or an 8-, 9-, 10-or 11-membered heterobicyclic ring having 1 to 5 heteroatoms each independently selected from O, N and S, which heterobicyclic ring may be saturated, unsaturated or aromatic, at possible positions.

2. A compound of formula (Ia) according to claim 1:

wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰And R²¹As defined in claim 1.

3. A compound of formula (Ib) according to claim 1:

4. A compound according to one or more of the preceding claims, wherein R¹Selected from H, methyl and ethyl.

5.A compound according to one or more of the preceding claims, wherein R²Selected from halogen, cyano, (C)_1-4) Alkyl, (C)_2-4) Alkenyl, (C)_2-4) Alkynyl, (C)_3-6) Cycloalkyl, phenyl and Het, Het being selected from the following groups:

wherein the phenyl and Het are unsubstituted or substituted by R²¹Is substituted in which R²¹As defined in claim 1.

6. A compound of claim 5, wherein R²Selected from the group consisting of Br, Cl, cyano, methyl, ethyl, propyl, 1-methylethyl, ethenyl, 1-methylethenyl, ethynyl, cyclopropyl, phenyl, and Het selected from the group consisting of:

wherein the phenyl and Het are unsubstituted or substituted by R²¹And (4) substitution.

7. A compound according to one or more of the preceding claims, wherein R²¹Is 1,2 or 3 substituents each independently selected from:

-1 to 3 substituents, each independently selected from halogen; and

-1 to 2 substituents, each independently selected from:

a) hydroxy, (C)_1-4) Alkyl or (C)_1-4) An alkoxy group; wherein the alkyl and alkoxy radicals are each optionally taken by one, two or three halogen atomsGeneration;

b)-NR^N2R^N1wherein

wherein each of (C)_1-3) Alkyl, -CO- (C)_1-3) Alkyl and-CO-O- (C)_1-3) The alkyl moiety of the alkyl group is optionally substituted with a substituent selected from the group consisting of halogen and (C)_1-6) Substituted with one, two or three substituents of alkoxy; and is

Wherein Het is a 5-or 6-membered monocyclic saturated, unsaturated or aromatic heterocycle having 1 or 2 heteroatoms each independently selected from N, O and S; and is

R^N2Is H or (C)_1-3) An alkyl group;

d) het, wherein Het is a 5-or 6-membered monocyclic heterocycle having 1,2 or 3 heteroatoms each independently selected from N, O and S.

8. A compound of claim 7, wherein R²¹Is 1,2 or 3 substituents each independently selected from:

-1 to 2 substituents, each independently selected from:

a) hydroxy, methyl, ethyl, propyl, 1-methylethyl, methoxy, ethoxy, propoxy, or 1-methylethoxy; wherein the methyl, ethyl, propyl, 1-methylethyl, methoxy, ethoxy, propoxy, and 1-methylethoxy groups are each optionally substituted with one, two, or three halogen atoms;

b)-N(CH₃)₂or-NHR^N1Wherein

wherein the methyl, ethyl, propyl and 1-methyl groupsEthyl is optionally mono-, di-or tri-substituted by one or more groups selected from halogen and (C)_1-3) Substituted by a substituent of alkoxy;

c)-CONH₂(ii) a And

d) 3-pyridyl, 4-pyridyl, 5-pyrimidyl, 2-furyl, 1-pyrrolyl and 1-morpholino.

9. A compound according to one or more of the preceding claims, wherein R³Is cyclopentyl or cyclohexyl optionally substituted by one or two fluorine atoms.

10. A compound according to one or more of the preceding claims, wherein R⁴Is H or halogen and R⁷Is H.

11. The compound of claim 10, wherein R⁴Is H or Cl and R⁷Is H.

12. A compound according to one or more of the preceding claims, wherein R⁵And R⁶One of which is selected from:

a) by COOH or CONHR^N1Substituted (C)_2-4) Alkenyl, wherein R^N1Is selected from H and (C)_1-3) Alkyl, said alkenyl being optionally further substituted by one or two substituents each independently selected from (C)_1-3) Alkyl and halogen;

i. -OH, oxo, COOH;

optionally substituted by phenyl or-N (R)^N2)R^N1Substituted (C)_1-3) Alkyl radical, wherein R^N1And R^N2Each independently selected from H and (C)_1-3) Alkyl, or R^N1And R^N2To and together with the nitrogen atom to which they are attachedA 5-or 6-membered monocyclic, saturated, unsaturated or aromatic N-containing heterocycle optionally further having one or two heteroatoms each independently selected from N, O and S; and

c)COOH；

13. The compound of claim 12, wherein R⁵And R⁶One of them is selected from:

a) by COOH or-CONH₂Substituted (C)_2-4) Alkenyl, which is optionally further substituted by one or two groups selected from (C)_1-3) Alkyl and halogen; and

b) phenyl or Het, each optionally further substituted with one or two substituents each independently selected from:

i. -OH, oxo, COOH;

optionally phenyl, -N (CH)₃)₂OrSubstituted (C)_1-3) An alkyl group; and

iii.-NH₂、-N(CH₃)₂and-NHCOCH₃；

Wherein Het is selected from the following formulae:

c)COOH；

14. The compound of claim 13, wherein R⁵And R⁶One of them is selected from:

b) optionally by NH₂Substituted phenyl, or

i. -OH, oxo, COOH;

iii.-NH₂、-N(CH₃)₂and-NHCOCH₃；

Wherein Het is selected from the following formulae:

c)COOH；

15. A compound according to one or more of the preceding claims, wherein R⁸Is selected from (C)_1-5) Alkyl, (C)_4-6) Cycloalkyl and (C)_3-4) Cycloalkyl- (C)_1-3) Alkyl group of which (C)_1-5) Alkyl is optionally substituted by (C)_1-3) Alkoxy or one to three fluorine atoms.

16. The compound of claim 15, wherein R⁸Selected from the group consisting of methyl, ethyl, propyl, 1-methylethyl, 2-methylpropyl, 3-methylbutyl, cyclobutyl, cyclopropylmethyl, 2-fluoroethyl, 2,2, 2-trifluoroethyl and 2-methoxyethyl.

17. A compound according to one or more of the preceding claims, wherein R⁹And R¹⁰Each independently selected from (C)_1-3) Alkyl, or R⁹And R¹⁰Are linked and form together with the carbon atom to which they are linked (C)_3-6) Cycloalkyl group, (C)_5-6) Cycloalkenyl or a 5-or 6-membered monocyclic heterocycle having 1 to 2 heteroatoms each independently selected from O and N; wherein the cycloalkyl, cycloalkenyl or heterocycle is each optionally substituted by (C)_1-4) Alkyl substitution.

18. The compound of claim 17, wherein the groupSelected from:

19. a compound of formula (I) according to claim 1, wherein

R¹is H or (C)_1-6) An alkyl group;

R²is halogen, aryl or Het; the aromatic hydrocarbonAnd Het is optionally substituted by R²¹Substitution;

wherein R is²¹Is a mono, di or tri substituent, each independently selected from-OH, -CN, -N (R)^N2)R^N1Halogen, (C)_1-6) Alkyl, (C)_1-6) Alkoxy group, (C)_1-6) Alkylthio, Het and-CO-N (R)^N2)R^N1；

R⁵and R⁶One is selected from COOH, -CO-N (R)^N2)R^N1Het and (C)_2-6) Alkenyl, wherein Het, (C)_2-6) Alkenyl and R^N1Or any at R^N2And R^N1Each of the heterocyclic rings formed therebetween is optionally substituted by R⁵⁰Substitution;

wherein R is⁵⁰Is one, two or three substituents, each independently selected from (C)_1-6) Alkyl, -COOH, -N (R)^N2)R^N1、-CO-N(R^N2)R^N1And halogen;

R⁹and R¹⁰Each independentlyIs selected from (C)_1-6) An alkyl group; or R⁹And R¹⁰Are linked and form together with the carbon atom to which they are linked (C)_3-7) Cycloalkyl group, (C)_5-7) Cycloalkenyl or a 4-, 5-or 6-membered heterocyclic ring having 1 to 3 heteroatoms each independently selected from O, N and S;

wherein the cycloalkyl, cycloalkenyl or heterocycle is each optionally substituted by (C)_1-4) Alkyl substituted;

wherein all alkyl and cycloalkyl groups are optionally substituted by one, two or three groups each independently selected from halogen, (C)_1-6) Alkoxy and (C)_1-6) Alkylthio group substituent; and is

R^N2Is H or (C)_1-6) Alkyl, or

R^N2And R^N1May be linked and taken together with the nitrogen atom to which they are attached form a 4-,5-, 6-or 7-membered saturated or unsaturated N-containing heterocycle, or an 8-, 9-, 10-or 11-membered N-containing heterobicyclic ring, each having from 1 to 3 heteroatoms independently selected from O, N and S;

wherein is represented by R^N2And R^N1The heterocyclic or heterobicyclic ring formed is optionally substituted by one, two or three substituents each independently selected from halogen, (C)_1-6) Alkyl, (C)_1-6) Alkoxy and (C)_1-6) Alkylthio group substituent; wherein Het is defined as a 4-,5-, 6-or 7-membered heterocycle having from 1 to 4 heteroatoms each independently selected from O, N and S, which heterocycle may be saturated, unsaturated or aromatic, or an 8-, 9-, 10-or 11-membered heterobicyclic ring having from 1 to 5 heteroatoms each independently selected from O, N and S, which heterobicyclic ring may be saturated, unsaturated or aromatic, where possible.

20. A compound of formula (I) according to claim 1, wherein

R¹Selected from H, methyl and ethyl;

R²selected from halogenElement, cyano group, (C)_1-4) Alkyl, (C)_2-4) Alkenyl, (C)_2-4) Alkynyl, (C)_3-6) Cycloalkyl, phenyl and Het selected from the group consisting of:

-1,2 or 3 substituents, each independently selected from halogen; and

-1 or 2 substituents, each independently selected from:

b)-NR^N2R^N1wherein

wherein each of (C)_1-3) Alkyl, -CO- (C)_1-3) Alkyl and-CO-O- (C)_1-3) The alkyl portion of the alkyl group is optionally substituted with one, two or three members selected from the group consisting of halogen and (C)_1-6) Substituted by a substituent of alkoxy; and wherein Het is a 5-or 6-membered monocyclic saturated, unsaturated or aromatic heterocycle having 1 to 2 heteroatoms each independently selected from N, O and S; and is

R^N2Is H or (C)_1-3) An alkyl group;

c)-CONR^N2R^N1wherein R is^N2And R^N1Each independently selected from H and (C)_1-3) Alkyl radicals, and

d) het, wherein Het is a 5-or 6-membered monocyclic heterocycle having 1,2 or 3 heteroatoms independently selected from N, O and S;

R⁴is H orHalogen and R⁷Is H;

R⁵and R⁶One of which is selected from:

i. -OH, oxo, COOH;

optionally substituted by phenyl or-N (R)^N2)R^N1Substituted (C)_1-3) Alkyl radical, wherein R^N1And R^N2Each independently selected from H and (C)_1-3) Alkyl, or R^N1And R^N2(ii) are linked, taken together with the nitrogen atom to which they are attached, to form a 5-or 6-membered monocyclic saturated, unsaturated or aromatic N-containing heterocycle, which heterocycle optionally further has one or two heteroatoms each independently selected from N, O and S; and

wherein Het is a 5-or 6-membered monocyclic saturated, unsaturated or aromatic heterocycle having 1 to 3 heteroatoms independently selected from N, O and S; and

c)COOH；

wherein R is^N1Selected from H and-CO-O- (C)_1-6) An alkyl group;

R⁸is selected from (C)_1-5) Alkyl, (C)_4-6) Cycloalkyl and (C)_3-4) Cycloalkyl- (C)_1-3) Alkyl group of which (C)_1-5) Alkyl is optionally substituted by (C)_1-3) Alkoxy or one to three fluorine atoms; and is

R⁹And R¹⁰Each independently selected from (C)_1-3) Alkyl, or R⁹And R¹⁰Are linked and form together with the carbon atom to which they are linked (C)_3-6) Cycloalkyl group, (C)_5-6) Cycloalkenyl or a 5-or 6-membered heterocyclic ring having 1 to 2 heteroatoms selected from O and N; wherein the cycloalkyl, cycloalkenyl or heterocycle is optionally substituted by (C)_1-4) Alkyl substitution.

21. A compound of formula (I) according to claim 1, wherein

R¹Selected from H, methyl and ethyl;

wherein the phenyl and Het are unsubstituted or substituted by R²¹Is substituted in which R²¹Is 1,2 or 3 substituents, each independently selected from:

-1 to 2 substituents, each independently selected from:

b)-N(CH₃)₂or-NHR^N1Wherein

wherein the methyl, ethyl, propyl and 1-methylethyl are each optionally substituted by one, two or three members selected from halogen and (C)_1-3) Substituent substitution of alkoxy;

c)-CONH₂(ii) a And

d) 3-pyridyl, 4-pyridyl, 5-pyrimidinyl, 2-furyl, 1-pyrrolyl and 1-morpholino;

R⁴is H or halogen and R⁷Is H;

R⁵and R⁶One of which is selected from:

a) by COOH or-CONH₂Substituted (C)_2-4) Alkenyl, and optionally further substituted by one or two groups selected from (C)_1-3) Alkyl and halogen; and

i. -OH, oxo, COOH;

ii.(C_1-3) Alkyl, optionally substituted by phenyl, -N (R)^N3)₂) OrSubstitution; and

iii.-NH₂,-N(CH₃)₂and-NHCOCH₃；

Wherein Het is selected from the following formulae:

c)COOH；

R⁸Selected from the group consisting of methyl, ethyl, propyl, 1-methylethyl, 2-methylpropyl, 3-methylbutyl, cyclobutyl, cyclopropylmethyl, 2-fluoroethyl, 2,2, 2-trifluoroethyl and 2-methoxyAn ethyl group; and is

Radical (I)Selected from:

22. a compound of formula (I) according to claim 1, wherein

R¹Selected from H, methyl and ethyl;

-1 to 2 substituents, each independently selected from:

b)-N(CH₃)₂or-NHR^N1Wherein

c)-CONH₂(ii) a And

d) 3-pyridyl, 4-pyridyl, 5-pyrimidinyl, 2-furyl, 1-pyrrolyl and 1-morpholino;

R⁴is H or Cl and R⁷Is H;

R⁵and R⁶One of which is selected from:

b) optionally by NH₂Substituted phenyl or

i. -OH, oxo, COOH;

iii.-NH₂、-N(CH₃)₂and-NHCOCH₃；

Wherein Het is selected from the following formulae:

R⁸Selected from methyl and ethylPropyl, 1-methylethyl, 2-methylpropyl, 3-methylbutyl, cyclobutyl, cyclopropylmethyl, 2-fluoroethyl, 2,2, 2-trifluoroethyl and 2-methoxyethyl; and is

The group is selected from:

23. a pharmaceutical composition for the treatment or prevention of HCV infection comprising an effective amount of a compound according to one or more of claims 1 to 22, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier.

24. The composition of claim 23, further comprising a therapeutically effective amount of one or more antiviral drugs.

25. The composition of claim 24, wherein the antiviral drug is selected from the group consisting of: ribavirin and amantadine.

26. The composition of claim 24, wherein the antiviral drug is an additional anti-HCV drug.

27. The pharmaceutical composition of claim 26, wherein the other anti-HCV agent is an immunomodulator selected from α -, β -, δ -, γ -, τ -and ω -interferons and pegylated forms thereof.

28. The composition of claim 26, wherein the additional anti-HCV drug is an additional HCV polymerase inhibitor.

29. The composition of claim 26, wherein the additional anti-HCV drug is an additional HCV ns3 protease inhibitor.

30. The composition of claim 26, wherein the other anti-HCV drug is an inhibitor of an additional target in the HCV life cycle.

31. The composition of claim 30, wherein the inhibitor of an additional target in the HCV life cycle is selected from a drug that inhibits a target selected from HCV helicase, HCV NS2/3 protease, and HCV IRES, and a drug that interferes with the function of MS5A protein.

32. Use of a compound according to one or more of claims 1 to 22, or a pharmaceutically acceptable salt or ester thereof, or a composition thereof, as an HCV polymerase inhibitor.

33. Use of a compound according to one or more of claims 1 to 22, or a pharmaceutically acceptable salt or ester thereof, or a composition thereof, as an inhibitor of the RNA-dependent RNA polymerase activity of the enzyme NS5B encoded by HCV.

34. Use of a compound according to one or more of claims 1 to 22, or a pharmaceutically acceptable salt or ester thereof, or a composition thereof, as an inhibitor of HCV replication.

35. Use of a compound according to one or more of claims 1-22, or a pharmaceutically acceptable salt or ester thereof, or a composition thereof, for treating or preventing HCV infection in a mammal.

36. Use of a compound according to one or more of claims 1-22, or a pharmaceutically acceptable salt or ester thereof, or a composition thereof, in combination with an additional antiviral agent, for treating or preventing an HCV infection in a mammal.

37. A method of inhibiting the RNA-dependent RNA polymerase activity of the enzyme NS5B encoded by HCV, comprising exposing the enzyme NS5B to an effective amount of a compound according to one or more of claims 1 to 22, under conditions in which the RNA-dependent RNA polymerase activity of the enzyme NS5B is inhibited.

38. A method of inhibiting HCV replication comprising exposing a cell infected with HCV to a compound according to one or more of claims 1-22 under conditions in which HCV replication is inhibited.

39. A method of treating or preventing HCV infection in a mammal, comprising administering to the mammal an effective amount of a compound according to one or more of claims 1 to 22, or a pharmaceutically acceptable salt or ester thereof, or a composition thereof.

40. A method of treating or preventing HCV infection in a mammal, comprising administering to the mammal an effective amount of a compound according to one or more of claims 1 to 22, or a pharmaceutically acceptable salt or ester thereof, or a composition thereof, and an additional antiviral agent.

41. Use of a compound according to one or more of claims 1 to 22, or a pharmaceutically acceptable salt or ester thereof, or a composition thereof, for the manufacture of a medicament for the treatment and/or prevention of a flavivirus infection.

42. Use of a compound according to one or more of claims 1 to 22, or a pharmaceutically acceptable salt or ester thereof, or a composition thereof, for the manufacture of a medicament for the treatment and/or prophylaxis of HCV infections.

43. An article of manufacture, comprising:

compositions and methods for effective treatment of HCV infection or inhibition of NS5B polymerase

Comprising a packaging material indicating that the composition is useful for treating hepatitis c virus infection,

wherein the composition comprises a compound according to one or more of claims 1-22, or a pharmaceutically acceptable salt or ester thereof.