Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
  • C07D209/52—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring condensed with a ring other than six-membered
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
  • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
  • C07D491/04—Ortho-condensed systems

Definitions

• the present invention relates to novel hepatitis C virus (“HCV”) protease inhibitors, pharmaceutical compositions containing one or more such inhibitors, methods of preparing such inhibitors and methods of using such inhibitors to treat hepatitis C and related disorders.
• HCV hepatitis C virus
• This invention additionally discloses novel compounds as inhibitors of the HCV NS3/NS4a serine protease.
• NANBH is to be distinguished from other types of viral- induced liver disease, such as hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV), as well as from other forms of liver disease such as alcoholism and primary biliar cirrhosis.
• HCV hepatitis A virus
• HBV hepatitis B virus
• HDV delta hepatitis virus
• CMV cytomegalovirus
• EBV Epstein-Barr virus
• This approximately 3000 amino acid polyprotein contains, from the amino terminus to the carboxy terminus, a nucleocapsid protein (C), envelope proteins (E1 and E2) and several non-structural proteins (NS1 , 2, 3, 4a, 5a and 5b).
• NS3 is an approximately 68 kda protein, encoded by approximately 1893 nucleotides of the HCV genome, and has two distinct domains: (a) a serine protease domain consisting of approximately 200 of the N-terminal amino acids; and (b) an RNA-dependent ATPase domain at the C-terminus of the protein.
• the NS3 protease is considered a member of the chymotrypsin family because of similarities in protein sequence, overall three- dimensional structure and mechanism of catalysis.
• Other chymotrypsin-like enzymes are elastase, factor Xa, thrombin, trypsin, plasmin, urokinase, tPA and PSA.
• the HCV NS3 serine protease is responsible for proteolysis of the polypeptide (polyprotein) at the NS3/NS4a, NS4a/NS4b, NS4b/NS5a and NS5a/NS5b junctions and is thus responsible for generating four viral proteins during viral replication.
• HCV hepatitis C virus
• NS3/NS4a junction contains a threonine at P1 and a serine at P1 ⁇
• the Cys ⁇ Thr substitution at NS3/NS4a is postulated to account for the requirement of cis rather than trans processing at this junction. See, e.g., Pizzi et al. (1994) Proc. Natl. Acad. Sci (USA) 91:888-892, Failla et al.
• NS3/NS4a cleavage site is also more tolerant of mutagenesis than the other sites. See, e.g., Kollykhalov et al. (1994) J. Virol. 68:7525-7533. It has also been found that acidic residues in the region upstream of the cleavage site are required for efficient cleavage. See, e.g., Komoda et a (1994) J. Virol. 68:7351-7357. Inhibitors of HCV protease that have been reported include antioxidants (see, International Patent Application Publication No.
• HCV has been implicated in cirrhosis of the liver and in induction of hepatocellular carcinoma.
• the prognosis for patients suffering from HCV infection is currently poor.
• HCV infection is more difficult to treat than other forms of hepatitis due to the lack of immunity or remission associated with HCV infection.
• Current data indicates a less than 50% survival rate at four years post cirrhosis diagnosis.
• Patients diagnosed with localized resectable hepatocellular carcinoma have a five-year survival rate of 10-30%, whereas those with localized unresectable hepatocellular carcinoma have a five-year survival rate of less than 1%.
• WO 00/59929 US 6,608,027, Assignee: Boehringer
• the present invention provides a novel class of inhibitors of the HCV protease, pharmaceutical compositions containing one or more of the compounds, methods of preparing pharmaceutical formulations comprising one or more such compounds, and methods of treatment or prevention of HCV or amelioration of one or more of the symptoms of hepatitis C using one or more such compounds or one or more such formulations. Also provided are methods of modulating the interaction of an HCV polypeptide with HCV protease. Among the compounds provided herein, compounds that inhibit HCV NS3/NS4a serine protease activity are preferred.
• the present invention discloses compounds, or enantiomers, stereoisomers, rotamers, tautomers, diastereomers or racemates of said compounds, or a pharmaceutically acceptable salt, solvate or ester of said compounds, said compounds having the general structure shown in structural Formula 1 :
• R 1 is H, OR 8 , NR 9 R 10 , or CHR 9 R 10 , wherein R 8 , R 9 and R 10 can be the same or different, each being independently selected from the group consisting of H, alkyl-, alkenyl-, alkynyl-, aryl-, heteroalkyl-, heteroaryl-, cycloalkyl-, heterocyclyl-, arylalkyl-, and heteroarylalkyl;
• a and M can be the same or different, each being independently selected from R, OR, NHR, NRR', SR, SO 2 R, and halo; or A and M are connected to each other such that the moiety:
• R, R', R 2 , and R 3 can be the same or different, each being independently selected from the group consisting of H, alkyl-, alkenyl-, alkynyl-, cycloalkyl-, heteroalkyl-, heterocyclyl-, aryl-, heteroaryl-, (cycloalkyl)alkyl-, (heterocyclyl)alkyl-, aryl-alkyl-, and heteroaryl-alkyl-; or alternately R and R' in NRR' are connected to each other such that NRR' forms a
• u is a number 0-1 ;
• X is selected from O, NR 15 , NC(O)R 16 , S, S(O) and S(O 2 );
• G is NH or O; and
• R 15 , R 16 , R 17 , R 18 , R 19 , Ti, T 2 , and T 3 can be the same or different, each being independently selected from the group consisting of H, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, or alternately, R 17 and R 18 are connected to each other to form a three to eight-membered cycloalkyl or heterocyclyl; wherein each of said alkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl can be unsubstituted or optionally independently substituted with
• Formula I forms a six -membered cycloalkyl (cyclohexyl), Formula I can be depicted as:
• M-L-E-A taken together are connected to form a three, four, seven or eight- membered cycloalkyl, a four to eight-membered heterocyclyl, a six to ten-membered aryl, or a five to ten-membered heteroaryl.
• HCV hepatitis C virus
• inventive compounds can inhibit such protease. They can also modulate the processing of hepatitis C virus (HCV) polypeptide.
• R 1 is NR 9 R 10
• R 9 is H
• R 10 is H
• R 14 wherein R 14 is H, alkyl, aryl, heteroalkyl, heteroaryl, cycloalkyl, alkyl-aryl, alkyl-heteroaryl, aryl- alkyl, alkenyl, alkynyl or heteroaryl-alkyl.
• R 14 is selected from the group consisting of:
• R 2 is selected from the group consisting of the following moieties:
• R 31 is OH or O-alkyl; and R 32 is H, C(O)CH 3 , C(O)OtBu or C(O)N(H)tBu.
• R 3 is selected from the group consisting of the following moieties:
• G is NH
• Y is selected from the following moieties
• Y-2 is selected from the group consisting of:
• Y ,30 is selected from where u is a number 0-1 ; and R >1'9 a ;is selected from H, alkyl, phenyl or benzyl.
• T-i and T 2 can be the same or different, each being independently selected from the group consisting of:
• T ⁇ N " ⁇ " taken together represents ⁇ 2
• T 3 is selected from:
• R 1 is NHR 14 , where R 14 is selected from the group consisting of:
• R is selected from the group consisting of the following moieties:
• R is selected from the group consisting of the following moieties:
• Y ,30 is selected from the group consisting of:
• Y 30 is selected from the group consisting of
• Y ⁇ U is selected from the group consisting of:
• Y . 32 is selected from the group consisting of:
• Y 12 is selected from the group consisting of H, CO 2 H, C ⁇ 2 Me, OMe, F, Cl, Br, NH 2 , N(H)S(O 2 )CH 3) N(H)C(O)CH 3 , NO 2) NMe 2 , S(O 2 )NH 2 , CF 3> Me, OH, OCF 3 , and C(O)NH 2 ;
• Y 33 is selected from the group consisting of:
• H ' ⁇ . or . Me' and T 3 is selected from: and the moiety:
• Yet another embodiment of the invention discloses compounds shown in Table 1 and later in Table 2. Also shown in the Table 2 are the biological activities of several inventive compounds (as Ki* values).
• alkyl means an aliphatic hydrocarbon group which may be straight or branched and comprising about 1 to about 20 carbon atoms in the chain. Preferred alkyl groups contain about 1 to about 12 carbon atoms in the chain. More preferred alkyl groups contain about 1 to about 6 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, are attached to a linear alkyl chain.
• “Lower alkyl” means a group having about 1 to about 6 carbon atoms in the chain which may be straight or branched.
• substituted alkyl means that the alkyl group may be substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkyl, aryl, cycloalkyl, cyano, hydroxy, alkoxy, alkylthio, amino, -NH(aikyl), -NH(cy oalkyl), -N(alkyl) 2 , -N(alkyl) 2> carboxy and -C(O)O- alkyl.
• alkyl groups include methyl, ethyl, n- propyl, isopropyl and t-butyl.
• alkenyl means an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched and comprising about 2 to about 15 carbon atoms in the chain. Preferred alkenyl groups have about 2 to about 12 carbon atoms in the chain; and more preferably about 2 to about 6 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, are attached to a linear alkenyl chain.
• “Lower alkenyl” means about 2 to about 6 carbon atoms in the chain which may be straight or branched.
• substituted alkenyl means that the alkenyl group may be substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkyl. aryl, cycloalkyl, cyano, alkoxy and -S(alkyl).
• suitable alkenyl groups include ethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n- pentenyl, octenyl and decenyl.
• Alkynyl means an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond and which may be straight or branched and comprising about 2 to about 15 carbon atoms in the chain.
• Preferred alkynyl groups have about 2 to about 12 carbon atoms in the chain; and more preferably about 2 to about 4 carbon atoms in the chain.
• Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, are attached to a linear alkynyl chain.
• “Lower alkynyl” means about 2 to about 6 carbon atoms in the chain which may be straight or branched.
• Non-limiting examples of suitable alkynyl groups include ethynyl, propynyl, 2-butynyl and 3- methyl butynyl.
• substituted alkynyl means that the alkynyl group may be substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of alkyl, aryl and cycloalkyl.
• Aryl means an aromatic monocyclic or multicyclic ring system comprising about 6 to about 14 carbon atoms, preferably about 6 to about 10 carbon atoms. The aryl group can be optionally substituted with one or more "ring system substituents" which may. be the same or different, and are as defined herein.
• Suitable aryl groups include phenyl and naphthyl.
• Heteroaryl means an aromatic monocyclic or multicyclic ring system comprising about 5 to about 14 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the ring atoms is an element other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination. Preferred heteroaryls contain about 5 to about 6 ring atoms.
• the "heteroaryl” can be optionally substituted by one or more "ring system substituents" which may be the same or different, and are as defined herein.
• the prefix aza, oxa or thia before the heteroaryl root name means that at least a nitrogen, oxygen or sulfur atom respectively, is present as a ring atom.
• a nitrogen atom of a heteroaryl can be optionally oxidized to the corresponding N-oxide.
• Non- limiting examples of suitable heteroaryls include pyridyl, pyrazinyl, furanyl, thienyl, pyrimidinyl, pyridone (including N-substituted pyridones), isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazolyl, 1 ,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, oxindolyl, imidazo[1 ,2-a]pyridinyl, imidazo[2,1-b]thiazolyl, benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinolinyl, imidazolyl, thienopyri
• heteroaryl also refers to partially saturated heteroaryl moieties such as, for example, tetrahydroisoquinolyl, tetrahydroquinolyl and the like.
• “Aralkyl” or “arylalkyl” means an aryl-alkyl- group in which the aryl and alkyl are as previously described. Preferred aralkyls comprise a lower alkyl group. Non-limiting examples of suitable aralkyl groups include benzyl, 2- phenethyl and naphthalenylmethyl. The bond to the parent moiety is through the alkyl.
• Alkylaryl means an alkyl-aryl- group in which the alkyl and aryl are as previously described.
• Preferred alkylaryls comprise a lower alkyl group.
• Non- limiting example of a suitable alkylaryl group is tolyl.
• the bond to the parent moiety is through the aryl.
• Cycloalkyl means a non-aromatic mono- or multicyclic ring system comprising about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms.
• Preferred cycloalkyl rings contain about 5 to about 7 ring atoms.
• the cycloalkyl can be optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined above.
• Non-limiting examples of suitable monocyclic cycloalkyls include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.
• Non-limiting examples of suitable multicyclic cycloalkyls include 1-decalinyl, norbomyl, adamantyl and the like, as well as partially saturated species such as, for example, indanyl, tetrahydronaphthyl and the like.
• "Halogen” or “halo” means fluorine, chlorine, bromine, or iodine.
• Ring system substituent means a substituent attached to an aromatic or non-aromatic ring system which, for example, replaces an available hydrogen on the ring system. Ring system substituents may be the same or different, each being independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, alkylaryl, heteroaralkyl, heteroarylalkenyl, heteroarylalkynyl, alkylheteroaryl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, acyl, aroyl, halo, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylthio, arylthio, hetero
• Ring system substituent may also mean a single moiety which simultaneously replaces two available hydrogens on two adjacent carbon atoms (one H on each carbon) on a ring system.
• Examples of such moiety are methylene dioxy, ethylenedioxy, -C(CH 3 ) 2 - and the like which form moieties such as, for example:
• Heterocyclyl means a non-aromatic saturated monocyclic or multicyclic ring system comprising about 3 to about 10 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the atoms in the ring system is an element other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination. There are no adjacent oxygen and/or sulfur atoms present in the ring system.
• Preferred heterocyclyls contain about 5 to about 6 ring atoms.
• the prefix aza, oxa or thia before the heterocyclyl root name means that at least a nitrogen, oxygen or sulfur atom respectively is present as a ring atom.
• Any -NH in a heterocyclyl ring may exist protected such as, for example, as an -N(Boc), -N(CBz), -N(Tos) group and the like; such protections are also considered part of this invention.
• the heterocyclyl can be optionally substituted by one or more "ring system substituents" which may be the same or different, and are as defined herein.
• the nitrogen or sulfur atom of the heterocyclyl can be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide.
• Non-limiting examples of suitable monocyclic heterocyclyl rings include piperidyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1 ,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, lactam, lactone, and the like. It should be noted that in hetero-atom containing ring systems of this invention, there are no hydroxyl groups on carbon atoms adjacent to a N, O or S, as well as there are no N or S groups on carbon adjacent to another heteroatom. Thus, for example, in the ring:
• Alkynylalkyl means an alkynyl-alkyl- group in which the alkynyl and alkyl are as previously described. Preferred alkynylalkyls contain a lower alkynyl and a lower alkyl group. The bond to the parentrnoiety is through the alkyl. Non-limiting examples of suitable alkynylalkyl groups include propargylmethyl.
• Heteroaralkyl means a heteroaryl-alkyl- group. in which the heteroaryl and alkyl are as previously described. Preferred heteroaralkyls contain a lower alkyl group.
• Non-limiting examples of suitable aralkyl groups include pyridyl methyl, and quinolin-3-ylmethyl.
• the bond to the parent moiety is through the alkyl.
• "Hydroxyalkyl” means a HO-alkyl- group in which alkyl is as previously defined. Preferred hydroxyalkyls contain lower alkyl.
• suitable hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl.
• “Acyl” means an H-C(O)-, alkyl-C(O)- or cydoalkyl-C(O)-, group in which the various groups are as previously described.
• the bond to the parent moiety is through the carbonyl.
• acyls contain a lower alkyl.
• suitable acyl groups include formyl, acetyl and propanoyl.
• Aroyl means an aryl-C(O)- group in which the aryl group is as previously described. The bond to the parent moiety is through the carbonyl.
• suitable groups include benzoyl and 1- naphthoyl.
• Alkoxy means an alkyl-O- group in which the alkyl group is as previously described.
• suitable alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy and n-butoxy. The bond to the parent moiety is through the ether oxygen.
• Aryloxy means an aryl-O- group in which the aryl group is as previously described.
• suitable aryloxy groups include phenoxy and naphthoxy.
• the bond to the parent moiety is through the ether oxygen.
• Aralkyloxy means an aralkyl-O- group in which the aralkyl group is as previously described.
• suitable aralkyloxy groups include benzyloxy and 1- or 2-naphthalenemethoxy.
• the bond to the parent moiety is through the ether oxygen.
• Alkylthio means an alkyl-S- group in which the alkyl group is as previously described.
• Non-limiting examples of suitable alkylthio groups include methylthio and ethylthio.
• the bond to the parent moiety is through the sulfur.
• Arylthio means an aryl-S- group in which the aryl group is as previously described.
• suitable arylthio groups include phenylthio and naphthylthio.
• the bond to the parent moiety is through the sulfur.
• Aralkylthio means an aralkyl-S- group in which the aralkyl group is as previously described.
• Non-limiting example of a suitable aralkylthio group is benzylthio.
• the bond to the parent moiety is through the sulfur.
• Alkoxycarbonyl means an alkyl-O-CO- group. Non-limiting examples of suitable alkoxycarbonyl groups include methoxycarbonyl and ethoxycarbonyl. The bond to the parent moiety is through the carbonyl.
• Aryloxycarbonyl means an aryl-O-C(O)- group. Non-limiting examples of suitable aryloxycarbonyl groups include phenoxycarbonyl and naphthoxycarbonyl. The bond to the parent moiety is through the carbonyl.
• Aralkoxycarbonyl means an aralkyl-O-C(O)- group. Non-limiting example of a suitable aralkoxycarbonyl group is benzyloxycarbonyl.
• alkylsulfonyl means an alkyl-S(O 2 )- group. Preferred groups are those in which the alkyl group is lower alkyl.
• the bond to the parent moiety is through the sulfonyl.
• Arylsulfonyl means an aryl-S(O 2 )- group. The bond to the parent moiety is through the sulfonyl.
• substituted means that one or more hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded, and that the substitution results in a stable compound.
• the term “optionally substituted” means optional substitution with the specified groups, radicals or moieties.
• isolated or “in isolated form” for a compound refers to the physical state of said compound after being isolated from a synthetic process or natural source or combination thereof.
• purified or “in purified form” for a compound refers to the physical state of said compound after being obtained from a purification process or processes described herein or well known to the skilled artisan, in sufficient purity to be characterizable by standard analytical techniques described herein or well known to the skilled artisan. It should also be noted that any heteroatom with unsatisfied valences in the text, schemes, examples and Tables herein is assumed to have the hydrogen atom(s) to satisfy the valences.
• protecting groups When a functional group in a compound is termed "protected", this means that the group is in modified form to preclude undesired side reactions at the protected site when the compound is subjected to a reaction. Suitable protecting groups will be recognized by those with ordinary skill in the art as well as by reference to standard textbooks such as, for example, T. W. Greene et al, Protective Groups in organic Synthesis (1991 ), Wiley, New York. When any variable (e.g., aryl, heterocycle, R 2 , etc.) occurs more than one time in any constituent or in Formula 1 , its definition on each occurrence is independent of its definition at every other occurrence.
• any variable e.g., aryl, heterocycle, R 2 , etc.
• composition is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
• Prodrugs and solvates of the compounds of the invention are also contemplated. herein.
• prodrug denotes a compound that is a drug precursor which, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to yield a compound of Formula 1 or a salt and/or solvate thereof.
• Solvate means a physical association of a compound of this invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate” encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like.
• “Hydrate” is a solvate wherein the solvent molecule is H2O.
• Effective amount or “therapeutically effective amount” is meant to describe an amount of compound or a composition of the present invention effective in inhibiting the CDK(s) and thus producing the desired therapeutic, ameliorative, inhibitory or preventative effect.
• the compounds of Formula 1 can form salts which are also within the scope of this invention. Reference to a compound of Formula 1 herein is understood to include reference to salts thereof, unless otherwise indicated.
• the term “salt(s)" denotes acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases.
• salts when a compound of Formula 1 contains both a basic moiety, such as, but not limited to a pyridine or imidazole, and an acidic moiety, such as, but not limited to a carboxylic acid, zwitterions ("inner salts") may be formed and are included within the term "salt(s)" as used herein.
• Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful. Salts of the compounds of the Formula 1 may be formed, for example, by reacting a compound of Formula 1 with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
• Exemplary acid addition salts include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates, naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartarates, thiocyanates, toluenesulfonates (also known as tosylates,) and the like.
• Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as dicyclohexylamines, t-butyl amines, and salts with amino acids such as arginine, lysine and the like.
• Basic nitrogen-containing groups may be quartemized with agents such as lower alkyl halides (e.g. methyl, ethyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g.
• esters of the present compounds include the following groups: (1 ) carboxylic acid esters obtained by esterification of the hydroxy groups, in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, acetyl, n-propyl, t-butyl, or n-butyl), alkoxyalkyl (for example, methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl optionally substituted with, for example, halogen, C ⁇ -4 alkyl, or C- ⁇ - alkoxy or amino); (2) sulfonate esters, such as alkyl- or aralkylsulfonyl (for example, methanesulfonyl); (3) amino acid esters (for example, L-valyl or L-isoleucyl);
• the phosphate esters may be further esterified by, for example, a C 1 - 20 alcohol or reactive derivative thereof, or by a 2,3-di (C 6 - 24 )acyl glycerol.
• Compounds of Formula 1 , and salts, solvates, esters and prodrugs thereof, may exist in their tautomeric form (for example, as an amide or imino ether). All such tautomeric forms are contemplated herein as part of the present invention.
• All stereoisomers for example, geometric isomers, optical isomers and the like
• of the present compounds including those of the salts, solvates, esters and prodrugs of the compounds as well as the salts and solvates of the prodrugs
• those which may exist due to asymmetric carbons on various substituents including enantiomeric forms (which may exist even in the absence of asymmetric carbons), rotameric forms, atropisomers, and diastereomeric forms, are contemplated within the scope of this invention, as are positional isomers (such as, for example, 4-pyridyl and 3-pyridyl).
• Individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers, or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers.
• the chiral centers of the present invention can have the S or R configuration as defined by the IUPAC 1974 Recommendations.
• the use of the terms "salt”, “solvate” "prodrug” and the like, is intended to equally apply to the salt, solvate and prodrug of enantiomers, stereoisomers, rotamers, tautomers, positional isomers, racemates or prodrugs of the inventive compounds.
• polymorphic forms of the compounds of Formula I, and of the salts, solvates, esters and prodrugs of the compounds of Formula I, are intended to be included in the present invention. It is to be understood that the utility of the compounds of Formula 1 for the therapeutic applications discussed herein is applicable to each compound by itself or to the combination or combinations of one or more compounds of Formula 1 as illustrated, for example, in the next immediate paragraph. The same understanding also applies to pharmaceutical composition(s) comprising such compound or compounds and method(s) of treatment involving such compound or compounds.
• the compounds according to the invention can have pharmacological properties; in particular, the compounds of Formula 1 can be inhibitors of HCV protease, each compound by itself or one or more compounds of Formula 1 can be combined with one or more compounds selected from within Formula 1.
• the compound(s) can be useful for treating diseases such as, for example, HCV, HIV, (AIDS, Acquired Immune Deficiency Syndrome), and related disorders, as well as for modulating the activity of hepatitis C virus (HCV) protease, preventing HCV, or ameliorating one or more symptoms of hepatitis C.
• HCV hepatitis C virus
• the compounds of Formula 1 may be used for the manufacture of a medicament to treat disorders associated with the HCV protease, for example, the method comprising bringing into intimate contact a compound of Formula 1 and a pharmaceutically acceptable carrier.
• this invention provides pharmaceutical compositions comprising the inventive compound or compounds as an active ingredient.
• the pharmaceutical compositions generally additionally comprise at least one pharmaceutically acceptable carrier diluent, excipient or carrier (collectively referred to herein as carrier materials). Because of their HCV inhibitory activity, such pharmaceutical compositions possess utility in treating hepatitis C and related disorders.
• the present invention discloses methods for preparing pharmaceutical compositions comprising the inventive compounds as an active ingredient.
• the active ingredients will typically be administered in admixture with suitable carrier materials suitably selected with respect to the intended form of administration, i.e.
• the active drug component may be combined with any oral non-toxic pharmaceutically acceptable inert carrier, such as lactose, starch, sucrose, cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, talc, mannitol, ethyl alcohol (liquid forms) and the like.
• suitable binders, lubricants, disintegrating agents and coloring agents may also be incorporated in the mixture.
• Powders and tablets may be comprised of from about 5 to about 95 percent inventive composition.
• Suitable binders include starch, gelatin, natural sugars, corn sweeteners, natural and synthetic gums such as acacia, sodium alginate, carboxymethylcellulose, polyethylene glycol and waxes.
• lubricants there may be mentioned for use in these dosage forms, boric acid, sodium benzoate, sodium acetate, sodium chloride, and the like.
• Disintegrants include starch, methylcellulose, guar gum and the like. Sweetening and flavoring agents and preservatives may also be included where appropriate.
• compositions of the present invention may be formulated in sustained release form to provide the rate controlled release of any one or more of the components or active ingredients to optimize the therapeutic effects, i.e. HCV inhibitory activity and the like.
• Suitable dosage forms for sustained release include layered tablets containing layers of varying disintegration rates or controlled release polymeric matrices impregnated with the active components and shaped in tablet form or capsules containing such impregnated or encapsulated porous polymeric matrices.
• Liquid form preparations include solutions, suspensions and emulsions. As.an example may be mentioned water or water-propylene glycol solutions for parenteral injections or addition of sweeteners and pacifiers for oral solutions, suspensions and emulsions.
• Liquid form preparations may also include solutions for intranasal administration.
• Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier such as inert compressed gas, e.g. nitrogen.
• a pharmaceutically acceptable carrier such as inert compressed gas, e.g. nitrogen.
• a low melting wax such as a mixture of fatty acid glycerides such as cocoa butter is first melted, and the active ingredient is dispersed homogeneously therein by stirring or similar mixing. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby solidify.
• solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration.
• Such liquid forms include solutions, suspensions and emulsions.
• the compounds of the invention may also be deliverable transdermally.
• the transdermal compositions may take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.
• the compounds of the invention may also be administered orally, intravenously, intranasally or subcutaneously.
• the pompounds of the invention may also comprise preparations which are in a unit dosage form. In such form, the preparation is subdivided into suitably sized unit doses containing appropriate quantities of the active components, e.g., an effective amount to achieve the desired purpose.
• the quantity of the inventive active composition in a unit dose of preparation may be generally varied or adjusted from about 1.0 milligram to about 1 ,000 milligrams, preferably from about 1.0 to about 950 milligrams, more preferably from about 1.0 to about 500 milligrams, and typically from about 1 to about 250 milligrams, according to the particular application.
• the actual dosage employed may be varied depending upon the patient's age, sex, weight and severity of the condition being treated. Such techniques are well known to those skilled in the art.
• the human oral dosage form containing the active ingredients can be administered 1 or 2 times per day. The amount and frequency of the administration will be regulated according to the judgment of the attending clinician.
• a generally recommended daily dosage regimen for oral administration may range from about 1.0 milligram to about 1 ,000 milligrams per day, in single or divided doses.
• Capsule - refers to a special container or enclosure made of methyl cellulose, polyvinyl alcohols, or denatured gelatins or starch for holding or containing compositions comprising the active ingredients.
• Hard shell capsules are typically made of blends of relatively high gel strength bone and pork skin gelatins. The capsule itself may contain small amounts of dyes, opaquing agents, plasticizers and preservatives.
• Tablet- refers to a compressed or molded solid dosage form containing the active ingredients with suitable diluents.
• the tablet can be prepared by compression of mixtures or granulations obtained by wet granulation, dry granulation or by compaction.
• Oral gel- refers to the active ingredients dispersed or solubilized in a hydrophillic semi-solid matrix.
• Powder for constitution refers to powder blends containing the active ingredients and suitable diluents which can be suspended in water or juices.
• Diluent - refers to substances that usually make up the major portion of the composition or dosage form. Suitable diluents include sugars such as lactose, sucrose, mannitol and sorbitol; starches derived from wheat, corn, rice and potato; and celluloses such as microcrystalline cellulose.
• the amount of diluent in the composition can range from about 10 to about 90% by weight of the total composition, preferably from about 25 to about 75%, more preferably from about 30 to about 60% by weight, even more preferably from about 12 to about 60%.
• Disintegrant - refers to materials added to the composition to help it
• Suitable • -disintegrants include starches; "cold water soluble" modified starches such as : sodium carboxymethyl starch; natural and synthetic gums such as locust bean, karaya, guar, tragacanth and agar; cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose; microcrystalline celluloses and cross-linked microcrystalline celluloses such as sodium croscarmellose; alginates such as alginic acid and sodium alginate; clays such as bentonites; and effervescent mixtures.
• the amount of disintegrant in the composition can range from about 2 to about 15% by weight of the composition, more preferably from about 4 to about 10% by weight.
• Binder - refers to substances that bind or "glue” powders together and make them cohesive by forming granules, thus serving as the "adhesive" in the formulation. Binders add cohesive strength already available in the diluent or bulking agent. Suitable binders include sugars such as sucrose; starches derived from wheat, corn rice and potato; natural gums such as acacia, gelatin and tragacanth; derivatives of seaweed such as alginic acid, sodium alginate and ammonium calcium alginate; cellulosic materials such as methylcellulose and sodium carboxymethylcellulose and hydroxypropylmethylcellulose; polyvinylpyrrolidone; and inorganics such as magnesium aluminum silicate.
• sugars such as sucrose
• starches derived from wheat, corn rice and potato natural gums such as acacia, gelatin and tragacanth
• derivatives of seaweed such as alginic acid, sodium alginate and ammonium calcium alginate
• the amount of binder in the composition can range from about 2 to about 20% by weight of the composition, more preferably from about 3 to about 10% by weight, even more preferably from about 3 to about 6% by weight.
• Lubricant - refers to a substance added to the dosage form to enable the tablet, granules, etc. after it has been compressed, to release from the mold or die by reducing friction or wear.
• Suitable lubricants include metallic stearates such as magnesium stearate, calcium stearate or potassium stearate; stearic acid; high melting point waxes; and water soluble lubricants such as sodium chloride, sodium benzoate, sodium acetate, sodium oleate, polyethylene glycols and d'l-leucine.
• Lubricants are usually added at the very last step before compression, since they must be present on the surfaces of the granules and in between them and the parts of the tablet press.
• the amount of lubricant in the composition can range from about 0.2 to about 5% by weight of the composition, preferably from about 0.5 to about 2%, more preferably from about 0.3 to about 1.5% by weight.
• ⁇ lident - material that prevents caking and improve the flow characteristics of granulations, so that flow is smooth and uniform.
• Suitable glidents include silicon dioxide and talc.
• the amount of glident in the . composition can range from about 0.1 % to about 5% by weight of the total composition, preferably from about 0.5 to about 2% by weight.
• Coloring agents - excipients that provide coloration to the composition or the dosage form.
• excipients can include food grade dyes and food grade dyes adsorbed onto a suitable adsorbent such as clay or aluminum oxide.
• the amount of the coloring agent can vary from about 0.1 to about 5% by weight of the composition, preferably from about 0.1 to about 1 %.
• Bioavailability - refers to the rate and extent to which the active drug ingredient or therapeutic moiety is absorbed into the systemic circulation from an administered dosage form as compared to a standard or control.
• Conventional methods for preparing tablets are known. Such methods include dry methods such as direct compression and compression of granulation produced by compaction, or wet methods or other special procedures.
• inventive compounds or pharmaceutical compositions disclosed above for treatment of diseases such as, for example, hepatitis C and the like.
• the method comprises administering a therapeutically effective amount of the inventive compound or pharmaceutical composition to a patient having such a disease or diseases and in need of such a treatment.
• the compounds of the invention may be used for the treatment of HCV in humans in monotherapy mode or in a combination therapy (e.g., dual combination, triple combination etc.) mode such as, for example, in combination with antiviral and/or immunomodulatory agents.
• antiviral and/or immunomodulatory agents examples include Ribavirin (from Schering-Plough Corporation, Madison, New Jersey) and LevovirinTM (from ICN Pharmaceuticals, Costa Mesa, California), VP 50406TM (from Viropharma, Incorporated, Exton, Pennsylvania), ISIS 14803TM (from ISIS Pharmaceuticals, Carlsbad, California), HeptazymeTM (from Ribozyme Pharmaceuticals, Boulder, Colorado), VX 497TM (from Vertex Pharmaceuticals, Cambridge, Massachusetts), ThymosinTM (from SciClone Pharmaceuticals, San Mateo, California), MaxamineTM (Maxim
• PEG- interferon alpha conjugates are interferon alpha molecules covalently attached to a PEG molecule.
• Illustrative PEG-interferon alpha conjugates include interferon alpha-2a (RoferonTM, from Hoffman La-Roche, Nutley, New Jersey) in the form of pegylated interferon alpha-2a (e.g., as sold under the trade name PegasysTM), interferon alpha-2b (IntronTM, from Schering-Plough Corporation) in the form of pegylated interferon alpha-2b (e.g., as sold under the trade name PEG-IntronTM), interferon alpha-2c (Berofor AlphaTM, from Boehringer Ingelheim, Ingelheim, Germany) or consensus interferon as defined by determination of a consensus sequence of naturally occurring interferon alphas (InfergenTM, from Amgen, Thousand Oaks, California).
• Interferon alpha-2a RosferonTM, from Hoffman La-Roche, Nutley, New Jersey
• PegasysTM interferon alpha-2a
• IntronTM from Schering-Plough
• the invention includes tautomers, rotamers, diastereomers, enantiomers and other stereoisomers of the inventive compounds also.
• inventive compounds may exist in suitable isomeric forms. Such variations are contemplated to be within the scope of the invention.
• Another embodiment of the invention discloses a method of making the compounds disclosed herein.
• the compounds may be prepared by several techniques known in the art. Illustrative procedures are outlined in the following reaction schemes. The illustrations should not be construed to limit the scope of the invention which is defined in the appended claims.
• ADDP 1 ,1 '-(Azodicarbobyl)dipiperidine
• HOBt N-Hydroxybenzotriazole
• PyBrOP Bromo-tris-pyrrolidinophosphonium hexafluorophosphate
• HATU O-(7-azabenzotriazol-1-yl)-1 ,1.,3,3-tetramethyluronium hexafluorophosphate
• BOP Benzotriazol-1 -yl-oxy-tris(dimethylamino)hexafluorophosphate
• PCC Pyridiniumchlorochromate
• KHMDS Potassium Hexamethyldisilazide or Potassium bis(trimethylsilylamide)
• NaHMDS Sodium Hexamethyldisilazide or Sodium bis(trimethylsilylamide)
• LiHMDS Lithium Hexamethyldisilazide or Lithium bis(trimethylsilylamide) 10%
• Pd/C 10% Palladium on carbon (by weight).
• TG Thioglycerol
• Method D in yet another variation, the hydrochloride salt 1.13 was converted to the 4-nitrophenyl carbamate 1.15 by reaction with 4-nitrophenyl chloroformate. Subsequent treatment with an amine (or amine hydrochloride salt) of choice provided the target compound 1.14.
• Method E in yet another variation, the dipeptide hydrochloride salt 1.03 was converted to the 4-nitrophenyl carbamate as described above. Treatment with an amine (or amine hydrochloride salt) of choice provided the urea derivative 1.05. Hydrolysis and further elaboration as described in Methods A B provided the target compounds 1.14.
• Step 9 10.09 10.10 A solution of the acid 10.09 (from above) in CH 2 CI 2 (50 mL) and DMF (25 mL) was treated with NH 4 CI (2.94 g, 55.5 mmol), EDCI (3.15 g, 16.5 mmol), HOOBt (2.69 g, 16.5 mmol), and NMM (4.4 g, 44 mmol). The reaction mixture was stirred at room temperature for 3 d. The solvents were removed under vacuo and the residue was diluted with aq. HCl (250 mL) and extracted with CH 2 Cl 2 . The combined organic layers were washed with aq.
• 10.10 can also be obtained directly by the reaction of 10.06 (4.5 g, 17.7 mmol) with aq. H 2 O 2 (10 mL), LiOH»H 2 O (820 mg, 20.8 mmol) at 0 °C in 50 mL of CH 3 OH for 0.5 h.). Step 10.
• Butoxycarbonylmethylene)triphenylphosphorane (26.75g; Aldrich). The resulting dark reaction was stirred overnight, diluted with EtOAc), washed with aq. sodium sulfite. sat. aq. NaHCO3, water, brine and dried. The volatiles were removed under reduced pressure and the residue was purified by silica gel column chromatography using 1% EtOAc in hexanes as eluent to give the desired compound, 11.03 (3.92g). Some impure fractions were also obtained but set aside at this time. Step 2
• Step 3 13.04 13.05 To a stirred solution of the amino acid 13.04 (2.0 g, 13.6 mmol) in dioxane (10 mL) and H2O (5mL) at 0°C, was added 1 N NaOH solution (4.3 mL, 14.0 mmol). The resulting solution was stirred for 10 minutes, followed by addition of di-_-butyldicarbonate (0.110 g, 14.0 mmol) and stirred at 0°C for 15 minutes. The solution was then warmed to room temperature, stirred for 45 minutes and kept at refrigerator overnight and concentrated to dryness to give a crude material.
• the amino ester 20.01 was prepared following the method of R. Zhang and J. S. Madalengoitia (J. Org. Chem. 1999, 64, 330), with the exception that the Boc group was cleaved by the reaction of the Boc-protected amino acid with methanolic HCl.
• Step 2 20.06 20.07 A solution of methyl ester 20.06 (4.0 g, 10.46 mmol) was dissolved in 4M HCl in dioxane and stirred at rt. for 3 h. The reaction mixture was concentrated in vacuo to obtain the amine hydrochloride salt, 20.07 which was used without purification.
• the carbamate 22.05 (8.81 g; 28.4mmol) was dissolved in acetonitrile (45ml) and the solution was cooled to -40°C under an atmosphere of nitrogen. Pyridine (6.9ml; 85.3mmol) followed by nitrosium tetrafluoroborate (6.63g; 56.8mmol) were added and the resulting reaction mixture maintained below 0°C until TLC indicated that no starting material remained (approx. 2.25h.). Pyrrolidine (20ml; 240mmol) was added and the cooling bath was withdrawn and stirring was continued at room temperature for 1 h. and then the volatiles were removed under reduced pressure.
• the .residue was quickly passed through a pad of silica gel to provide a yellow oil.
• the yellow oil was dissolved in anhydrous benzene (220ml) and palladium acetate (0.317g; 1.41 mmol) was added before heating the resulting mixture to reflux, under an atmosphere of nitrogen for a period of 1.5h. After cooling, the volatiles were removed under reduced pressure and the dark residue was purified by silica gel column chromatography using EtOAc- hexane (1 :4) to provide the I) the trans- pyrrolidinone 22.06 (1.94g) followed by ii) the cis-pyrrolidinone 22.07 (1.97g).
• the alcohol 22.09 (2.00g; 5.67mmol) was dissolved in acetone (116ml) and cooled in an ice bath for 10min. This solution was then added to a cooled Jones reagent (14.2ml; approx 2mmol/ml) and the resulting mixture was stirred at 5°C for 0.5h and the cooling bath was removed. The reaction was stirred for a further 2h. at room temp., before adding to sodium sulfate (28.54g), celite (15g) in EtOAc (100ml). Isopropanol (15ml) was added after 1 min and then stirred for a further 10min. and filtered. The filtrate was concentrated under reduced pressure, providing a brown oil which was dissolved in EtOAc. This solution was washed with water, 3% aq. citric acid, brine, dried and concentrated to provide the desired carboxylic acid 22.01 (1.64g) as a white solid.
• Step 2 50.03 50.04 The methyl ester 50.03 from above was dissolved in methylene chloride (100 mL) and cooled to -78°C, under nitrogen atmosphere. DIBAL (1.0 M solution in methylene chloride, 200 mL) was added dropwise over 2 h period. The reaction mixture was warmed to room temperature over 16 h. The reaction mixture was cooled to 0°C and MeOH (5-8 mL) was added dropwise. A solution of aqueous 10% sodium potassium tartarate (200 mL) was slowly added with stirring. Diluted with methylene chloride (100 mL) and separated the organic layer (along with some white precipitate).
• DIBAL 1.0 M solution in methylene chloride, 200 mL
• Step 3 50.04 50.05
• the alcohol 50.04 from above was dissolved in methylene chloride (400 mL) and cooled to 0°C under nitrogen atmosphere.
• PCC (22.2 g) was added in portions and the reaction mixture was slowly warmed to room temperature over 16 h.
• the reaction mixture was diluted with diethyl ether (500 mL) and filtered through a pad of celite. The filtrate was concentrated and the residue was taken in diethyl ether (500 mL). This was passed through a pad of silica gel and the filtrate was concentrated to provide the aldehyde 50.05 which was carried forward without further purification.
• Step 1
• Step3 Et 3 N (3 equiv., 5.2 mmol, 0.72 mL) is added to a mixture of crude 100-15 (1.06g, 1.73 mmol theor.) and EDCI (4equiv., 6.92 mmol, 1.33g) in EtOAc (12 mL) at RT. After addition, DMSO (4.5 mL) was slowly charged. This was followed by addition of methanesulfonic acid (3.6equiv, 6.22 mmol, 0.4 mL) with temperature between 20 and 30°C. The reaction was agitated for 1 h. After 1 h, TLC shows reaction completed.
• HCV inhibitors 110. 113 and 133 described in Table 1 were prepared using intermediate of formula 100-5 according to the general procedures described before.
• Amine 106-1 was prepared according the procedure outlined in preparative example 100 by replacing in stepl the commercially N-t-BOC-L- Cyclohexylglycinol 100-2 with the corresponding commercially available N-t- BOC-L-tert-Butylglycinol.
• Amine 106-2 was prepared according the procedure outlined in preparative example 100, part II by replacing in step 2 amine of formula 100-1 by amine of formula 106-1 described before. Part III: Preparation of compound of formula 106
• Compound of formula 106 was prepared following the procedure outlined in part IV of preparative example 100 by replacing amine 100-5 with amine salt 106-2.
• HCV inhibitors 115, 121 , 135, 147 and 148 described in Table 1 were prepared using intermediate of formula 106-1 according to the general procedures described before.
• Step 1
• Compound of formula 112 was prepared according preparative example 106 by replacing in part III amine of formula 106-2 by amine of formula 104.3.
• HCV inhibitors 103, 107, 109, 111 , 114, 117, 119, 123, 128, 137 ? 140 and 145 described in Table 2 were prepared using intermediate of formula 104-3 according to the general procedures described before.
• amines of formula 101-1 , 116-1 , 129-1 and 132-1 were prepared in the same manner than amine of formula 104- of preparative example 104 by replacing in step 1 , t-BuLi by other reagents like (but not limited to) clopropyl-magnesiumchloride, isopropyl-magnesiumchloride, MeLi, ethyl- magnesium chloride to prepare respectively intermediates of formula 101-1 , 116-1 , 129-1 and 132-1.
• HCV inhibitors 101 , 105, 116, 118, 120, 122, 124, 125, 129, 130, 131 , 132, 134, 136, 143, 144 and 146 described in Table 2 were prepared using intermediate of formula 101-1 , 116-1 , 129-1 and 132-1 according to the general procedures described before.
• the Ki* range shown is: Range A: Ki* ⁇ 75 nM; Range B: Ki*> 75 nM.
• the present invention relates to novel HCV protease inhibitors. This utility can be manifested in their ability to inhibit the HCV NS2/NS4a serine protease. A general procedure for such demonstration is illustrated by the following in vitro assay.
• Spectrophotometric assay for the HCV serine protease can be performed on the inventive compounds by following the procedure described by R. Zhang et al, Analytical Biochemistry, 270 (1999) 268-275, the disclosure of which is incorporated herein by reference.
• the assay based on the proteolysis of chromogenic ester substrates is suitable for the continuous monitoring of HCV NS3 protease activity.
• Reagents for peptide synthesis were from Aldrich Chemicals, Novabiochem (San Diego, California), Applied Biosystems (Foster City, California) and Perseptive Biosystems (Framingham, Massachusetts). Peptides are synthesized manually or on an automated ABI model 431 A synthesizer (from Applied Biosystems).
• UV/VIS Spectrometer model LAMBDA 12 was from Perkin Elmer (Norwalk, Connecticut) and 96-well UV plates were obtained from Corning (Corning, New York).
• the prewarming block can be from USA Scientific (Ocala, Florida) and the 96-well plate vortexer is from Labline Instruments (Melrose Park, Illinois).
• Spectramax Plus microtiter plate reader with monochrometer is obtained from Molecular Devices (Sunnyvale, California).
• Enzyme Preparation Recombinant heterodimeric HCV NS3/NS4A protease (strain 1a) is prepared by using the procedures published previously (D. L. Sali et al, Biochemistry, 37 (1998) 3392-3401 ). Protein concentrations are determined by the Biorad dye method using recombinant HCV protease standards previously quantified by amino acid analysis.
• the enzyme storage buffer 50 mM sodium phosphate pH 8.0, 300 mM NaCl, 10% glycerol, 0.05% lauryl maltoside and 10 mM DTT
• the assay buffer 25 mM MOPS pH 6.5, 300 mM NaCl, 10% glycerol, 0.05% lauryl maltoside, 5 ⁇ M EDTA and 5 ⁇ M DTT
• the assay buffer 25 mM MOPS pH 6.5, 300 mM NaCl, 10% glycerol, 0.05% lauryl maltoside, 5 ⁇ M EDTA and 5 ⁇ M DTT
• the N-acetylated and fully protected peptide fragments are cleaved from the resin either by 10% acetic acid (HOAc) and 10% trifluoroethanol (TFE) in dichloromethane (DCM) for 30 min, or by 2% trifluoroacetic acid (TFA) in DCM for 10 min.
• the combined filtrate and DCM wash is evaporated azeotropically (or repeatedly extracted by aqueous Na2CO3 solution) to remove the acid used in cleavage.
• the DCM phase is dried over Na2SO4 and evaporated.
• the ester substrates are assembled using standard acid-alcohol coupling procedures (K. Holmber et al, Ada Chem. Scand., B33 (1979) 410- 412).
• Peptide fragments are dissolved in anhydrous pyridine (30-60 mg/ml) to which 10 molar equivalents of chromophore and a catalytic amount (0.1 eq.) of para-toluenesulfonic acid (pTSA) were added.
• pTSA para-toluenesulfonic acid
• Dicyclohexylcarbodiimide (DCC, 3 eq.) is added to initiate the coupling reactions.
• Product formation is monitored by HPLC and can be found to be complete following 12-72 hour reaction at room temperature. Pyridine solvent is evaporated under vacuum and further removed by azeotropic evaporation with toluene.
• the peptide ester is deprotected with 95% TFA in DCM for two hours and extracted three times with anhydrous ethyl ether to remove excess chromophore.
• the deprotected substrate is purified by reversed phase HPLC on a C3 or C8 column with a 30% to 60% acetonitrile gradient (using six column volumes). The overall yield following HPLC purification can be approximately 20-30%.
• the molecular mass can be confirmed by electrospray ionization mass spectroscopy.
• the substrates are stored in dry powder form under desiccation.
• Spectra of Substrates and Products Spectra of substrates and the corresponding chromophore products are obtained in the pH 6.5 assay buffer.
• Extinction coefficients are determined at the optimal off-peak wavelength in 1- cm cuvettes (340 nm for 3-Np and HMC, 370 nm for PAP and 400 nm for 4- Np) using multiple dilutions.
• the optimal off-peak wavelength is defined as that wavelength yielding the maximum fractional difference in absorbance between substrate and product (product OD - substrate OD)/substrate OD).
• Protease Assay HCV protease assays are performed at 30°C using a 200 ⁇ l reaction mix in a 96-well microtiter plate.
• Assay buffer conditions 25 mM MOPS pH 6.5, 300 mM NaCl, 10% glycerol, 0.05% lauryl maltoside, 5 ⁇ M EDTA and 5 ⁇ M DTT are optimized for the NS3/NS4A heterodimer (D. L. Sali et al, ibid.)).
• 150 ⁇ l mixtures of buffer, substrate and inhibitor are placed in wells (final concentration of DMSO ⁇ 4 % v/v) and allowed to preincubate at 30 °C for approximately 3 minutes.
• the plates are monitored over the length of the assay (60 minutes) for change in absorbance at the appropriate wavelength (340 nm for 3-Np and HMC, 370 nm for PAP, and 400 nm for 4-Np) using a Spectromax Plus microtiter plate reader equipped with a monochrometer (acceptable results can be obtained with plate readers that utilize cutoff filters).
• Proteolytic cleavage of the ester linkage between the Nva and the chromophore is monitored at the appropriate wavelength against a no enzyme blank as a control for non-enzymatic hydrolysis.

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