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WO2012093174A1 - Dérivés de 1-(azo-hétérocyclique à 6 chaînons)-2,5-dihydro-1h-pyrrol-2-one à titre d'anti-virus de l'hépatite c, composition pharmaceutique les contenant et leur utilisation thérapeutique - Google Patents

Dérivés de 1-(azo-hétérocyclique à 6 chaînons)-2,5-dihydro-1h-pyrrol-2-one à titre d'anti-virus de l'hépatite c, composition pharmaceutique les contenant et leur utilisation thérapeutique Download PDF

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Publication number
WO2012093174A1
WO2012093174A1 PCT/EP2012/050195 EP2012050195W WO2012093174A1 WO 2012093174 A1 WO2012093174 A1 WO 2012093174A1 EP 2012050195 W EP2012050195 W EP 2012050195W WO 2012093174 A1 WO2012093174 A1 WO 2012093174A1
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alkyl group
atom
phenyl
mmol
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Inventor
Paola Ciapetti
Nathalie Trouche
Subhash VENKATA PITHANI
Philippe Guedat
Amaya Berecibar
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Vivalis SA
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Vivalis SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

Definitions

  • the present invention concerns antiviral compounds, in particular anti hepatitis C compounds.
  • Viral proteins constitute a group of biologically active proteins with high pharmacological value. Drugs to deal with viral infections are a field of medicine that has been traditionally weak. However since the 1980s, the full genetic sequences of viruses began to be available to researchers, and they began to learn how viruses worked in detail, and to envision what kind of molecules were needed to j am their machinery. The general idea behind modern antiviral drug design is to identify viral proteins, or parts of proteins, that can be disabled. The targets should also be common across many strains of a virus, or even among different species of virus in the same family, so a single drug will have broad effectiveness. Dozens of "antiviral" treatments are now available, and a lot are currently under development. Most of the antivirals now available are designed to help deal with HIV, herpes virus, hepatitis B and C viruses and influenza viruses.
  • Viral life cycles vary in their precise details depending on the species of virus, but they all share a general pattern:
  • One of the major antivirals development approach is to interfere with the ability of a virus to get into a target cell.
  • the virus has to take a sequence of actions to do this, beginning with binding to a specific receptor molecule on the surface of the host cell and ending with the virus "un-coating" inside the cell and releasing its payload.
  • Viruses that have a lipid envelope must also fuse their envelope with the target cell, or with a vesicle that transports them into the cell, before they can uncoated. All these steps involve the binding of viral proteins with one or more binding partners. Indeed, a number of "entry-inhibiting" or "entry-blocking" drugs are being developed to fight HIV.
  • Amantine and rimantadine are two entry-blockers that have been developed to combat influenza virus. Amantine and rimantadine are thought to interfere with influenza A virus M2 protein, an ion channel protein, and to inhibit virus uncoating. However, amantine and rimantadine does not work on influenza B viruses and the two drugs have been associated with gastro-intestinal and central nervous system adverse effects. Pleconaril, another entry-blocker, works against rhinoviruses, which cause most colds, by blocking a pocket on the surface of the virus that controls the un-coating process. This pocket is similar in most strains of rhinoviruses, and the drug also seems to work against "entero-virus", which can cause diarrhea, meningitis, conjunctivitis, and encephalitis.
  • a second approach is to target the processes that synthesize virus components after a virus invades a cell.
  • Nucleotide or nucleoside analogues are antivirals that will interfere and block the enzymes that synthesize the RNA or DNA once the analogue is incorporated.
  • the first successful antiviral, "acyclovir” is a nucleoside analogue, and is effective against herpes virus infections.
  • Another nucleoside analogue named “zidovudine” or “AZT” has been approved for treating HIV.
  • Another class of antivirals that has been proven effective is the viral proteases inhibitors. Viral proteases act through binding to a target protein. However, protease inhibitors may have odd side- effects, for example causing fat to build up in unusual places. Then there is a need for improved protease inhibitors.
  • the final stage in the life cycle of a virus is the release of completed viruses from the host cell, and of course this step has also been targeted by antiviral drug developers.
  • Two drugs named "zanamivir” and “oseltamivir” that have been recently introduced to treat influenza prevent the release of viral particles by blocking a molecule named “neuraminidase” that is found on the surface of flu viruses, and also seems to be constant across a wide range of flu strains. Those two drugs block the active site of the influenza viral enzyme neuraminidase.
  • oseltamivir has been associated with adverse effects such as nausea and vomiting.
  • Zanamivir showed adverse respiratory events in persons with chronic pulmonary disease. Therefore there is a great need to extend the activity, the specificity and the efficacy of current antivirals, but also to extend the range of antivirals to other families of pathogens.
  • Hepatitis C is a global health problem with 170 million carriers' worldwide, 3 to 4 million new cases each year and a worldwide mortality estimated to 500,000 persons a year. 30% of liver grafts are currently prescribed to patients infected with HCV (Hepatitis C Virus).
  • HCV is spread primarily by direct contact with human blood. Transmission through blood transfusions that are not screened for HCV infection, through the re-use of inadequately sterilized needles and syringes or other medical equipment or through needle-sharing among drug users, i s well documented. Sexual and perinatal transmission may also occur, although less frequently.
  • the incubation period of HCV infection before the onset of clinical symptoms ranges from 15 to 150 days. About 80 % of infected patients progress to develop chronic infection which can also be asymptomatic. Cirrhosis develops in about 10% to 20% of persons with chronic infection and liver cancer develops in 1% to 5% of persons with chronic infection over a period of 20 to 30 years.
  • Hepatitis C virus is an enveloped virus from the Flaviviridae family and is the only member of hepacivirus genus. HCV comprises 6 genotypes, more than 45 subtypes and quasi-species patient-specific. Its positive single strand linear RNA has about 9,600 nucleotides. RNA genome is flanked by two untranslated regions (UTR) that play a maj or role in translation and replication of the viral genome. Upon interaction and fusion of viral and cellular membranes, RNA genome is released into the cytoplasm of a newly infected cell and serves as template for RNA replication.
  • UTR untranslated regions
  • Viral genome replication is a two step process: the positive RNA strand is used as a matrix for the synthesis of a negative polarity RNA which in turn serves as matrix for the synthesis of positive RNA strands that will be incorporated in new virions.
  • Translation of HCV genome depends on an internal ribosome entry site and produces a large polyprotein which is proteolytically cleaved by cellular and viral proteases to produce 10 viral proteins.
  • the amino terminal one third of the polyprotein encodes the structural proteins: core protein glycoproteins El + E2. After the structural region, comes a small integral protein, P7, which seems to function as an ion chemical.
  • the remainder of the genome encodes the non structural proteins NS2, N3, NS4A, NS4B, NS5A & NS5B which coordinate the intracellular processes of the virus life cycle (Lindenbach et al., 2005).
  • Replication complex is associated with membranes of the endoplasmic reticulum.
  • Viral proteins involved in this complex are the NTPase/helicase/serine protease NS3-4A, NS4B which is involved in the formation of the replication web, NS5A whose function still remains to be elucidated and the RNA-dependent RNA polymerase (RdRp) NS5B.
  • No vaccine is currently available to prevent hepatitis C.
  • the standard treatment consists in a combination between interferon, a cytokine with immuno-modulatory and antiviral activity (Moussalli et al., 1998) and ribavirin, a synthetic guanosine nucleoside analogue (Hugle et al, 2003).
  • HCV genotype la/lb the predominant one in USA, Japan and Europe
  • the sustained viral response Loss of serum HCV RNA following 24 weeks of antiviral therapy
  • is at best 42-46% (Walker et al., 2002; Gordon et al., 2005; Lake-Bakaar et al., 2003).
  • NS5B RNA polymerase is a 66 kD oligomeric, tail-anchored protein (Ivashkina et al., 2002; Schmidt-Mend e et al . , 200 1 ) .
  • RNAs Unlike many cellular and other viral polymerase, interactions between the fingers and thumb subdomains result in a completely encircled catalytic site that ensures synthesis of positive- and negative- strand HCV RNAs (Lesburg et al., 1999).
  • a unique feature is the presence of a B-harpin in the thumb subdomain that protrudes toward the active site and may thus restrict binding of the template/primer at the active site.
  • NS5B catalyzes de novo, primer- independent initiation of RNA synthesis followed by elongation, termination of polymerization and release of nascent strand.
  • l-(6-membered azo- heterocyclic)-2,5-dihydro-lH-pyrrol-2-one derivatives could be used for such a purpose. Similar derivatives are described in WO 03/030897 as compounds useful in the treatment of conditions mediated by CCR2, MCP-1 or the interaction thereof, such as alcoholic hepatitis. However, no mention is made in this document to the use of these derivatives to treat hepatitis C, which has a viral origin contrary to alcoholic hepatitis.
  • the present invention concerns thus a compound of the following formula (I) or a salt, solvate, tautomer, isotope, the enantiomer, diastereoisomer or racemic mixture thereof:
  • Z represents a nitrogen atom or a -CH group, advantageously a nitrogen atom
  • Rl represents -OH or a - RSO 2 R group in which R represents a Ci-C 6 alkyl group or a hydrogen atom.
  • R represents a-CH 3 group.
  • Rl represents -OH;
  • R2 represents a-CF 3 group or a -OCF 3 group, advantageously a -OCF 3 group;
  • R4 represents an isopropyl group or a cyclopropyl group, advantageously an isopropyl group;
  • R3 represents a 6-membered heteroaryl group containing as the only heteroatom(s), at least two nitrogen atoms,
  • heteroaryl group being optionally substituted, advantageously at the para position, by
  • Ci-C 6 alkyl group optionally substituted by one or more halogen atom, in particular a fluorine atom, by a -0-(Ci-C 6 )alkyl group, by a -0-(C 3 -C 6 ) cycloalkyl group, by -N 3 , by a 5-, 6- or 7-membered heterocyclic group containing one, two or three heteroatoms selected in the group consisting of nitrogen, sulfur and oxygen atom or by a - R'R" group in which R' and R" represent independently of each other a hydrogen atom, a Ci-C 6 alkyl group, a C 3 -C 6 cycloalkyl group, a -S0 2 R5 group, or a -COR6 group in which R5 and R6 represents a Ci-C 6 alkyl group, a C 3 -C 6 cycloalkyl group, a - H-(Ci- C 6 )alkyl group in
  • - a -0-(Ci-C 6 )alkyl group in which the alkyl group is optionally substituted by one or more halogen atom, in particular a fluorine atom or by a -0-(Ci-C 6 )alkyl group, in particular -OCH 3 ;
  • alkyl group is optionally substituted by one or more halogen atom, in particular a fluorine atom;
  • RIO represent a phenyl group or 5- or 6- membered heteroaryl group containing one, two or three heteroatoms selected in the group consisting of nitrogen, sulfur and oxygen atom, in which the phenyl or heteroaryl groups are optionally substituted by one or more halogen atom, in particular a fluorine atom or by a -0-(Ci-C 6 )alkyl group, in particular -OCH 3 ;
  • heteroaryl group of R3 is substituted, advantageously at the para position by
  • Ci-C 6 alkyl group in particular a -CH 3 group, a -CH 2 CH 3 group or a -CH(CH 3 ) 2 group, optionally substituted by a -0-(Ci-Ce)alkyl group, by a -0-(C 3 -C 6) cycloalkyl group, by -N 3 , by a 5-, 6- or 7-membered heterocyclic group containing one, two or three heteroatoms selected in the group consisting of nitrogen, sulfur and oxygen atom, in particular a 6-membered heterocyclic group containing one or two heteroatoms selected in the group consisting of nitrogen and oxygen, or by a - R'R" group in which R' and R" represent independently of each other a hydrogen atom, a Ci-C 6 alkyl group, a C3-C6 cycloalkyl group, a -S0 2 R5 group or a -COR6 group in which R5 and R6 represents a Ci-
  • - a -0-(Ci-C 6 )alkyl group in particular a -OCH(CH 3 ) 2 group and a -0-CH 2 -CH 3 group, in which the alkyl group is optionally substituted by a -0-(Ci-C 6 )alkyl group, in particular a -OCH 3 group;
  • heteroaryl group of R3 is substituted by
  • Ci-C 6 alkyl group in particular a -CH 3 group, a -CH 2 CH 3 group or a -CH(CH 3 ) 2 group, optionally substituted by -N 3 , by a 6-membered heterocyclic group containing one or two heteroatoms selected in the group consisting of nitrogen and oxygen, in particular a morpholinyl group, or by a - H 2 group;
  • - a -0-(Ci-C 6 )alkyl group in particular a -OCH(CH 3 ) 2 group and a -0-CH 2 -CH 3 group, in which the alkyl group is optionally substituted by a -0-(Ci-C 6 )alkyl group, in particular a -OCH 3 group;
  • the heteroaryl group of R3 is substituted by a -CH 3 group, a -CH 2 CH 3 group, a -CH(CH 3 ) 2 group, a -OCH(CH 3 ) 2 group, a SCH 3 group, a -S-phenyl group, a - 0-(CH 2 ) 2 -0-CH 3 group, an ethynyl group, a -CH 2 -morpholinyl group, a -CH 2 -N3 group or a -CH 2 - H 2 group, more advantageously the heteroaryl group of R3 is substituted by a -CH 3 group.
  • the compound according to the present invention is a compound of the following formula Ibis or a salt, solvate, tautomer, isotope, enantiomer, diastereoisomer or racemic mixture thereof; and in particular or a salt, solvate, tautomer, or isotope thereof:
  • Rl, R2, R3, R4 and Z are as defined above.
  • the compound according to the present invention or a salt, solvate, tautomer, isotope, enantiomer, diastereoisomer or racemic mixture thereof
  • R3 represents a group of the following formula II: ( ⁇ ) in which
  • X represents a nitrogen atom and Y represents a -C-R8 group or
  • X represents a -C-R9 group and Y represents a nitrogen atom or
  • X represents a -C-R9 group and Y represents a -C-R8 group
  • X represents a -C-R9 group and Y represents a -C-R8 group, in another preferred embodiment X or Y represents a nitrogen atom;
  • R7, R8 and R9 represent independently of each other
  • a Ci-C 6 alkyl group optionally substituted by one or more halogen atom, in particular a fluorine atom, by a -0-(Ci-Ce)alkyl group, by a -0-(C 3 -C 6 ) cycloalkyl group, by -N 3 , by a 5-, 6- or 7-membered heterocyclic group containing one, two or three heteroatoms selected in the group consisting of nitrogen, sulfur and oxygen atom or by a - R'R" group in which R' and R' ' represent independently of each other a hydrogen atom, a Ci- C 6 alkyl group, a C3-C6 cycloalkyl group, a -S0 2 R5 group or a -COR6 group in which R5 and R6 represents a Ci-C 6 alkyl group, a C3-C6 cycloalkyl group, a - H-(Ci- C
  • alkyl group is optionally substituted by one or more halogen atom, in particular a fluorine atom or by a -0-(Ci-C 6 )alkyl group, in particular a -OCH 3 group;
  • alkyl group is optionally substituted by one or more halogen atom, in particular a fluorine atom;
  • RIO represent a phenyl group or 5- or 6- membered heteroaryl group containing one, two or three heteroatoms selected in the group consisting of nitrogen, sulfur and oxygen atom, in which the phenyl or heteroaryl groups are optionally substituted by one or more halogen atom, in particular a fluorine atom or by a -0-(Ci-C 6 )alkyl group, in particular -OCH 3 ; and
  • R8 and/or R9 represent a hydrogen atom.
  • R7, R8 and R9 represent independently of each other
  • Ci-C 6 alkyl group in particular a -CH 3 group, a -CH 2 CH 3 group or a -CH(CH 3 ) 2 group, optionally substituted by a -0-(Ci-C 6 )alkyl group, by a -0-(C 3 -C 6 ) cycloalkyl group, by -N 3 , by a 5-, 6- or 7-membered heterocyclic group containing one, two or three heteroatoms selected in the group consisting of nitrogen, sulfur and oxygen atom, in particular a 6-membered heterocyclic group containing one or two heteroatoms selected in the group consisting of nitrogen and oxygen, or by a - R'R" group in which R' and R" represent independently of each other a hydrogen atom, a Ci-C 6 alkyl group, a C3-C6 cycloalkyl group, a -S0 2 R5 group, or a -COR6 group in which R5 and R6 represents
  • - a -0-(Ci-C 6 )alkyl group in particular a -OCH(CH 3 ) 2 group and a -0-CH 2 -CH 3 group, in which the alkyl group is optionally substituted by a -0-(Ci-C 6 )alkyl group, in particular a -OCH 3 group;
  • R7, R8 and R9 represent independently of each other
  • Ci-C 6 alkyl group in particular a -CH 3 group, a -CH 2 CH 3 group or a -CH(CH 3 ) 2 group, optionally substituted by -N 3 , by a 6-membered heterocyclic group containing one or two heteroatoms selected in the group consisting of nitrogen and oxygen, in particular a morpholinyl group, or by a - H 2 group;
  • - a -0-(Ci-C 6 )alkyl group in particular a -OCH(CH 3 ) 2 group and a -0-CH 2 -CH 3 group, in which the alkyl group is optionally substituted by a -0-(Ci-C 6 )alkyl group, in particular a -OCH 3 group;
  • R7, R8 and R9 represent independently of each other a hydrogen atom, a - CH 3 group, a -CH 2 CH 3 group, a -CH(CH 3 ) 2 group, a -OCH(CH 3 ) 2 group, a -SCH 3 group, a -S-phenyl group, a -0-(CH 2 ) 2 -0-CH 3 group, an ethynyl group, a -CH 2 - morpholinyl group, a -CH 2 -N 3 group or a -CH 2 - H 2 group.
  • R8 and/or R9 represent a hydrogen atom.
  • R7 represents a -CH 3 group, a -CH 2 CH 3 group, a -CH(CH 3 ) 2 group, a -OCH(CH 3 ) 2 group, a -SCH 3 group, a -S-phenyl group, a -0-(CH 2 ) 2 -0-CH 3 group, an ethynyl group, a -CH 2 -morpholinyl group, a -CH 2 -N3 group or a -CH 2 - H 2 group. Still more advantageously R7 represents a -CH 3 group.
  • R4 represents an isopropryl group
  • Rl represents OH
  • R2 represents OCF 3
  • Z represents a nitrogen atom
  • R3 represents a group of formula II
  • R7 represents a -Ci-C 6 alkyl group, in particular a -CH 3 group.
  • the compound according to the present invention a salt, solvate, tautomer, isotope, enantiomer, diastereoisomer or racemic mixture thereof, is chosen from the group consisting of the compounds of the following formula 1-25, and notably of the following formula 1-19:
  • Advantageous compounds are compounds 6, 7, 9, 11, 12, 15, 16, 17, 20, 21, 24 and 25 which are particularly active, compounds 11, 17, 21 and 25 showing the highest activities.
  • the compounds according to the present invention can be prepared by methods well known in the art. In particular they can be prepared by the general procedure as described below in the experimental part.
  • the present invention concerns thus also a process to prepare a compound of formula (I) as described above comprising the following successive steps:
  • R4 is as defined previously and Alk represents a Ci-C 6 alkyl group, such as an ethyl group,
  • This step of condensation can be carried out in the presence of an acid such as acetic acid.
  • EtOH can be used as solvent.
  • the reaction medium is refluxed.
  • ketoester (A), the aldehyde (B) and the amine (C) are either commercially available, or prepared according to methods well known to the person skilled in the art.
  • the conversion of the OH group into a RSO 2 R group can be carried out in two steps.
  • the OH group can be converted into a halogen atom, such as a chlorine atom, by methods well known to the person skilled in the art, notably by use of oxalyl chloride.
  • a step of nucleophilic substitution allows than replacing the chlorine atom with a RSO 2 R group, notably by reaction of the chlorinated derivative with HRSO 2 R in the presence of a base such as NaH.
  • step b further steps of substitution of the R3 group can be carried out before or after step b), if present, by methods well known to the person skilled in the art.
  • the Rl group notably when representing an OH group, can be protected beforehand and will be deprotected after the substitution step. Examples of such protection / deprotection are presented in the experimental part below.
  • Suitable protecting groups are well known for the skilled person in the art.
  • a general review of protecting groups in organic chemistry is provided by Wuts, P.G.M. and Greene T.W. in Protecting groups in Organic Synthesis, 4th Ed. Wiley-Interscience, and by Kocienski P.J. in Protecting Groups, 3rd Ed. Georg Thieme Verlag. These references provide sections on protecting groups for OH, amino, and SH groups.
  • an OH protecting group is defined to be the O-bonded moiety resulting from the protection of the OH group through the formation of a suitable protected OH group.
  • protected OH groups include ethers, silyl ethers, esters, sulfonates, sulfenates and sulfinates, carbonates, and carbamates.
  • the compound thus obtained can be separated from the reaction medium by methods well known to the person skilled in the art, such as by extraction, evaporation of the solvent or by precipitation or crystallisation (followed by filtration).
  • the compound can be also purified if necessary by methods well known to the person skilled in the art, such as by recrystallisation, by distillation, by chromatography on a column of silica gel or by high performance liquid chromatography (HPLC).
  • methods well known to the person skilled in the art such as by recrystallisation, by distillation, by chromatography on a column of silica gel or by high performance liquid chromatography (HPLC).
  • the present invention also concerns a pharmaceutical composition
  • a pharmaceutical composition comprising a compound according to the present invention or a salt, solvate, tautomer, isotope, enantiomer, diastereoisomer or racemic mixture thereof and pharmaceutically acceptable excipients, such as diluents or carriers.
  • the pharmaceutical composition according to the present invention contains a further therapeutic agent, preferably at least one antiviral agent, which is preferably selected in the group consisting of ribavirin, interferon, inhibitors of HCV helicase, inhibitors of HCV protease, inhibitors of HCV NS4A, inhibitors of HCV NS5B, inhibitors of HCV NS5A, anti-HIV agent and a mixture thereof.
  • a further therapeutic agent preferably at least one antiviral agent, which is preferably selected in the group consisting of ribavirin, interferon, inhibitors of HCV helicase, inhibitors of HCV protease, inhibitors of HCV NS4A, inhibitors of HCV NS5B, inhibitors of HCV NS5A, anti-HIV agent and a mixture thereof.
  • the present invention concerns also a pharmaceutical composition according to the present invention, or a compound according to the present invention, a salt, solvate, tautomer, isotope, enantiomer, diastereoisomer or racemic mixture thereof for use as a drug, in particular for use as an antiviral drug.
  • Said compound or composition being, advantageously intended for use in the treatment of hepatitis C, in particular as having an hepatitis C virus inhibitory activity, for example as a hepatitis C NS5B polymerase inhibitor.
  • the present invention also concerns a compound according to the present invention, a salt, solvate, tautomer, isotope, enantiomer, diastereoisomer or racemic mixture thereof and a further therapeutic agent, preferably at least another antiviral agent, which is preferably selected in the group consisting of ribavirin, interferon, inhibitors of HCV helicase, inhibitors of HCV protease, inhibitors of HCV NS4A, inhibitors of HCV NS5B, inhibitors of HCV NS5A, inhibitors of HCV polymerase, anti-HIV agent and a mixture thereof, for use as a combined preparation for simultaneous, separate or sequential use in hepatitis C therapy, preferably, in patients having the HIV disease.
  • a further therapeutic agent preferably at least another antiviral agent, which is preferably selected in the group consisting of ribavirin, interferon, inhibitors of HCV helicase, inhibitors of HCV protease, inhibitors of HCV NS
  • the compound according to the present invention is used as a bi- or tri-therapy in order to treat hepatitis C with another anti-hepatitis C antiviral agent (ribavirin, interferon, inhibitors of HCV helicase, inhibitors of HCV protease, inhibitors of HCV NS4A, inhibitors of HCV NS5B, inhibitors of HCV NS5A, inhibitors of HCV polymerase or mixture thereof).
  • the compound according to the present invention is used as a bi or tri-therapy with one or several anti-HIV antiviral agent in order to treat hepatitis C in a patient having HIV disease.
  • the compound according to the present invention is used as a tri-therapy with another anti-hepatitis C antiviral agent and an anti-HIV antiviral agent in order to treat hepatitis C in a patient having HIV disease.
  • the present invention also concerns the compounds of the following formula IV or a salt, solvate, tautomer, isotope, enantiomer, diastereoisomer or racemic mixture thereof
  • antiviral agent any of several drugs used to treat or prevent viral infections.
  • the drugs act by interfering with a virus's ability to enter a host cell and replicate itself with the host cell's DNA. Some drugs block the virus's attachment or entry into the cell; others inhibit replication or prevent the virus from shedding the protein coat that surrounds the viral DNA.
  • Antiviral agents or drugs are now available for a wide variety of viral diseases.
  • Ribavirin available since the mid-1980s, is used to treat respiratory syncytial virus (RSV), a cause of severe childhood respiratory infections. It is thought to inhibit messenger RNA. Amantadine and rimantadine, which are effective against strains of influenza A, act by interfering with viral uncoating.
  • the compounds of the present invention may also be present in the form of pharmaceutically acceptable salts.
  • the salts of the compounds of this invention refer to non-toxic "pharmaceutically acceptable salts".
  • Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic/anionic or basic/cationic salts.
  • Pharmaceutically acceptable salts of the acidic or basic compounds of the invention can of course be made by conventional procedures, such as by reacting the free base or acid with at least a stochiometric amount of the desired salt-forming acid or base.
  • Pharmaceutically acceptable salts of the acidic compounds of the invention include salts with inorganic cations such as sodium, potassium, calcium, magnesium, zinc, and ammonium, and salts with organic bases.
  • Suitable organic bases include N- methyl-D-glucamine, arginine, benzathine, diolamine, olamine, procaine and tromethamine.
  • Pharmaceutically acceptable salts of the basic compounds of the invention include salts derived from organic or inorganic acids.
  • Suitable anions include acetate, adipate, besylate, bromide, camsylate, chloride, citrate, edisylate, estolate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hyclate, hydrobromide, hydrochloride, iodide, isethionate, lactate, lactobionate, maleate, mesylate, methylbromide, methylsulphate, napsylate, nitrate, oleate, pamoate, phosphate, polygalacturonate, stearate, succinate, sulphate, sulphosalicylate, tannate, tartrate, terephthalate, tosylate and triethiodide. Hydrochloride salts are particularly preferred.
  • the compounds of the invention can be administered by oral or parenteral routes, intestinal, ocular, vaginal, rectal nasal (intranasal), pulmonary or other mucosal, transdermal and topical administration, and inhalation, advantageously by oral route.
  • Primary routes for parenteral administration include intravenous, intramuscular, and subcutaneous administration.
  • Secondary routes of administration include intraperitoneal, intra-arterial, intra-articular, intracardiac, intracisternal, intradermal, intralesional, intraocular, intrapleural, intrathecal, intrauterine, and intraventricular administration.
  • the compounds of the invention will generally be provided in the form of tablets or capsules or as an aqueous solution or suspension.
  • Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives.
  • suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate and lactose.
  • Corn starch and alginic acid are suitable disintegrating agents.
  • Binding agents may include starch and gelatine.
  • the lubricating agent if present, will generally be magnesium stearate, stearic acid or talc.
  • the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
  • Capsules for oral use include hard gelatine capsules in which the active ingredient is mixed with a solid diluent and soft gelatine capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.
  • the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity.
  • Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride.
  • Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin.
  • Suitable preservatives for aqueous suspensions include ethyl and n-propyl p- hy droxyb enzoate .
  • compositions of the present invention may, in particular, comprise more than one agent (multiple) of the present invention, e.g., two or more agents.
  • the invention also provides a pharmaceutical preparation or system, comprising (a) a first agent, which is an agent of the invention; and (b) a second pharmaceutical agent. Said multiple agents of the invention or said first and second agents are formulated either in admixture or as separate compositions, e.g. for simultaneous though separate, or for sequential administration (see below).
  • compositions of the present invention can be delivered directly or in pharmaceutical compositions containing excipients (see above), as is well known in the art.
  • the present methods of treatment involve administration of a therapeutically effective amount of an agent of the present invention to a subject.
  • therapeutically effective amount refers to an amount of an agent according to the present invention needed to treat or ameliorate the targeted disease condition, or to exhibit a detectable therapeutic effect or a prolongation of survival in a patient.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, for example, in non-human primates, mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration.
  • Effective doses of the compounds of the present invention may be ascertained by conventional methods.
  • the specific dosage level required for any particular patient will depend on a number of factors, including severity of the condition being treated, the route of administration, the general health of the patient (i.e. age, weight and diet) in particular if he is a HIV patient, the gender of the patient, the time and frequency of administration, and tolerance/response to therapy.
  • the daily dose (whether administered as a single dose or as divided doses) will be in the range 0.001 to 5000 mg per day, more usually from 1 to 2500 mg per day, and most usually from 10 to 1500 mg per day.
  • dosages can be administered per unit body weight and in this instance a typical dose will be between 0.01 ⁇ g/kg and 50 mg/kg, especially between 10 ⁇ g/kg and 10 mg/kg, between 100 ⁇ g/kg and 2 mg/kg.
  • An advantage of the compounds of the present invention is that they permit administration to be limited to one, two, three or four times weekly or monthly.
  • compositions may, if desired, be presented in a pack or dispenser device containing one or more unit dosage forms containing the active ingredient.
  • a pack or device may, for example, comprise metal or plastic foil, such as a blister pack, or glass and rubber stoppers such as in vials.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • Compositions comprising an agent of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition.
  • composition means “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.
  • the compounds according to this invention may accordingly exist as enantiomers. Where the compounds possess two or more chiral centres, they may additionally exist as diastereomers. Where the processes for the preparation of the compounds according to the invention give rise to mixture of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The compounds may be prepared in racemic form or individual enantiomers may be prepared by standard techniques known to those skilled in the art, for example, by enantiospecific synthesis or resolution, formation of diastereomeric pairs by salt formation with an optically active acid, followed by fractional crystallization and regeneration of the free base.
  • the compounds may also be resolved by formation of diastereomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary.
  • the compounds may be resolved using a chiral HPLC column (T. E. Beesley and R. P. W. Scott, Chiral Chromatography, John Wiley and Sons, Chichester-New York, (1998)39). It is to be understood that all such isomers and mixtures thereof in all proportion are encompassed within the scope of the present invention.
  • substituted for example a phenyl group comprising a substituent on the aryl ring, unless specified otherwise, the term "substituted" contemplates all possible isomeric forms.
  • substituted phenyl includes all of the following ortho-, meta- and para- permutations:
  • tautomer » refers to isomers of the compounds according to the present invention that readily interconvert by a chemical reaction called tautomerization. Commonly this reaction results in the formal migration of a hydrogen atom or proton, accompanied by a switch of a single bond and adjacent double bond. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers will be reached. The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. The concept of tautomers that are interconvertible by tautomerizations is called tautomerism.
  • Common tautomeric pairs are: ketone - enol; amide - imidic acid; lactam - lactim, an amide - imidic acid tautomerism in heterocyclic rings; enamine - imine; enamine - enamine. In particular it can include ring-chain tautomerism which occurs when the movement of the proton is accompanied by a change from an open structure to a ring.
  • isotope » refers to two molecules which differ only in the isotopic nature of their atoms i.e. their atom have a different atomic mass (mass number).
  • Isotopes of an atom have nuclei with the same number of protons (the same atomic number) but different numbers of neutrons. Therefore, isotopes have different mass numbers, which give the total number of nucleons, the number of protons plus neutrons.
  • an isotope of a compound can comprise one deuterium atom in place of a hydrogen atom.
  • halogen is used herein to refer to any of fluorine, chlorine, bromine and iodine. Most usually, however, halogen substituents in the compounds of the invention are chlorine, bromine and fluorine substituents, in particular fluorine substituents.
  • alkyl refers to a straight or branched saturated monovalent hydrocarbon radical, having the number of carbon atoms as indicated.
  • Ci-C 6 -alkyl includes Ci, C 2 , C 3 , C 4 , C 5 and C 6 alkyl groups.
  • suitable alkyl groups include methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, pentyl and hexyl.
  • alkyl groups are: Ci-C 6 -alkyl, Ci-C 5 -alkyl, Ci-C 4 -alkyl, Ci-C 3 -alkyl and Ci-C 2 -alkyl, in particular Ci-C 3 -alkyl.
  • alkynyl refers to a straight or branched monovalent hydrocarbon radical, having the number of carbon atoms as indicated and at least a triple bond.
  • C 2 -C 6 -alkynyl includes C 2 , C 3 , C 4 , C 5 and C 6 alkynyl groups.
  • suitable alkynyl groups include ethynyl, propynyl, iso-propynyl, butynyl, iso-butynyl, tert-butynyl, pentynyl and hexynyl.
  • alkynyl groups are: C 2 -C 6 - alkynyl, C 2 -C 5 -alkynyl, C 2 -C 4 -alkynyl and C 2 -C 3 -alkynyl in particular C 2 -C 3 -alkynyl.
  • cycloalkyl refers to a cyclic saturated hydrocarbon radical, having the number of carbon atoms as indicated.
  • C 3 -C 6 - cycloalkyl includes C 3 , C 4 , C 5 and C 6 cycloalkyl groups.
  • suitable cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclobutylethyl and cyclopentylmethyl, in particular cyclopropyl.
  • ranges of alkyl groups are: C 3 -C 6 -cycloalkyl, C 3 -C 5 -cycloalkyl and C 3 -C 4 -cycloalkyl.
  • heteroaryl refers to monovalent unsaturated aromatic heterocyclic radicals having one or two ring.
  • the term "6-membered heteroaryl” encompasses heteroaryl moieties that are aromatic monocyclic ring systems containing six members of which at least one member is a N, O or S atom and which optionally depending of the case can contain one, two or three additional N, O or S atoms, advantageously N atoms.
  • the term "5-membered heteroaryl” encompasses heteroaryl moieties that are aromatic monocyclic ring systems containing five members of which at least one member is a N, O or S atom and which optionally depending of the case can contain one, two or three additional N, O or S atoms, advantageously N atoms.
  • heteroaryl encompasses heteroaryl moieties that are aromatic monocyclic ring systems containing seven members of which at least one member is a N, O or S atom and which optionally depending of the case can contain one, two or three additional N, O or S atoms, advantageously N atoms.
  • suitable heteroaryl groups include furanyl, pyridyl, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyrazinyl, oxazinyl, tetrazol, oxadiazol pyridazinyl and triazol.
  • heterocyclic refers to a saturated or partially unsaturated ring having five members of which at least one member is a N, O or S atom and which optionally contains one additional O atom or one or two N atoms; a saturated or partially unsaturated ring having six members of which one, two or three members are an N, O or S atom and which optionally contains one additional O atom or one or two additional N atoms; a saturated or partially unsaturated ring having seven members of which one, two or three members are an N, O or S atom and which optionally contains one additional O atom or one or two additional N atoms.
  • heterocycles comprising peroxide groups are excluded from the definition of heterocyclic.
  • suitable heterocyclic groups include pyrrolinyl, pyrrolidinyl, dioxolanyl, tetrahydrofuranyl, morpholinyl, imidazolinyl, imidazolidinyl, pyrazolidinyl, piped dinyl, dihydropyranyl, tetrahydropyranyl and piperazinyl.
  • morpholinyl imidazolinyl, imidazolidinyl, pyrazolidinyl, piped dinyl, dihydropyranyl, tetrahydropyranyl and piperazinyl.
  • it is morpholinyl.
  • the reactants and commercials compounds were purchased from Acros Organics, Sigma-Aldrich, Alfa Aesar, Interchim, Atlantic Chemicals, Apollo Scientific, Enamine and Maybridge.
  • a NaOEt solution was prepared from sodium (1.93 g; 84.0 mmol; 1.36 eq) in ethanol (215 mL). At room temperature, l-[4-(propan-2-yl)phenyl]ethan-l-one (10.3 mL; 61.6 mmol; 1 eq) was added and the resulting solution was stirred for 30 minutes after which diethyl oxalate (10.87 g; 80.0 mmol; 1.3 eq) was added. The reaction mixture was refluxed for 16 hours then concentrated to dryness.
  • a suspension of l-(6-chloropyridazin-3-yl)-3-hydroxy-4-(4-isopropylbenzoyl)-5-(4- trifluoromethoxy)phenyl)-lH-pyrrol-2(5H)-one of example 1 (517 mg; 1 mmol; 1 eq) in isopropanol (3.8 mL; 48 mmol; 48 eq) was micro-waved at 70°C for 5 minutes. Dimethylformamide (5.2 mL) was then added and the mixture was micro-waved at 100°C for additional 15 minutes.
  • Example 2 Preparation of 3-hydroxy-l-(6-isopropoxypyridazin-3-yl)-4-(4-isopropyl benzoyl)-5-(4-(trifluoromethoxy)phenyl)-lH-pyrrol-2(5H)-one.
  • Example 3 Preparation of 3-hydroxy-4-(4-isopropylbenzoyl)-l-(6- henylth ⁇ o)pyridaz ⁇ n-3-yl)-5-(4-(trifluoromethoxy)phenyl)-lH-pyrrol-2(5H)-one.
  • Example 5 Preparation of l-(6-ethynylpyridazin-3-yl)-3-hydroxy-4- ⁇ [4-(propan-2- yl)phenyl]carbonyl ⁇ -5-[4-(trifluoromethoxy)phenyl]-2,5-dihydro-lH-pyrrol-2-one a) 3-hydroxy-4- ⁇
  • a LDA solution was prepared from n-BuLi 2.5M in hexane (22 mL; 56.80 mmol; 1.1 eq) and diisopropylamine (8 mL; 56.80 mmol; 1.1 eq) in THF (40 mL) at 0°C.
  • a solution of 2-chloro-6-(trifluoromethoxy)pyridine (10.2 g; 51.64 mmol; 1 eq) in THF (40 mL) was added dropwise over 30 minutes.
  • the aqueous phase was extracted with dichloromethane (2 x 25 mL) and the combined organic layers were dried over sodium sulphate, filtered and concentrated to dryness.
  • the crude product was purified on silica gel using petroleum ether/ethyl acetate (7/3 to 6/4) as an eluent to afford N-methoxy-N- methyl-6-(trifluoromethoxy)nicotinamide in 90% yield as a colorless oil.
  • Example 8 Preparation of 3-hydroxy-l-(6-isobutylpyridazin-3-yl)-4-(4-isopropyl benzoyl)-5-(4-(trifluoromethoxy)phenyl)-lH-pyrrol-2(5H)-one. a) 6-isobutylpyridazin-3 -amine (1-12)
  • the resulting residue was diluted with ethyl acetate (50 mL) and a saturated solution of sodium hydrogenocarbonate (50 mL). Resulting salts were filtered and washed with ethyl acetate (150 mL). The 2 layers were separated and the aqueous layer was extracted the combined organic layers were dried over sodium sulphate, filtered and concentrated under reduced pressure.
  • the crude product was purified by silica gel chromatography using dichloromethane/methanol (95/5 ⁇ 90/10) as an eluent.
  • Example 9 Preparation of l-(6-ethylpyridazin-3-yl)-3-hydroxy-4-(4-isopropyl benzoyl)-5-(4-(trifluoromethoxy)phenyl)-lH-pyrrol-2(5H)-one. a) 6-ethylpyridazin-3 -amine (1-13)
  • reaction mixture was filtered and the solid was washed with diethylether to give the title compound l-(6-methylpyridazin-3-yl)-3- hydroxy-4-(4-isopropylbenzoyl)-5-(44rifluoromethoxy)phenyl)-lH-pyrrol-2(5H)-one in 47% yield as a white solid.
  • the crude product was purified by silica gel chromatography using petroleum ether/ethyl acetate (85/15 to 8/2) as an eluent.
  • the title compound 3-(tert-butoxy)-l-[6-(chloromethyl)pyridazin-3-yl]-4- ⁇ [4-(propan- 2-yl)phenyl]carbonyl ⁇ -5-[4-(trifluoromethoxy)phenyl]-2,5-dihydro-lH-pyrrol-2-one was obtained in 72% yield as a white solid.
  • Example 11 Preparation of 3-hydroxy-l-[6-(2 ⁇ 4 ⁇ -triaz-2-yn-l- ylmethyl)pyridazin-3- yl]-4- ⁇ [4-(propan-2- yl)phenyl]carbonyl ⁇ -5-[4-(trifluoromethoxy)phenyl]- 2, 5-dihydro- lH- rrol-2-one
  • the reaction mixture was filtered and the solid wa s wa s h e d wi th d i ethy l eth er to g i v e th e ti tl e c om p ou n d , l-[(2,4- dimethoxyphenyl)methyl] -3 -hydroxy-4- ⁇ [4-(propan-2-yl)phenyl] carbonyl ⁇ -5 - [4- (trifluoromethoxy)phenyl]-2,5-dihydro-lH-pyrrol-2-one in 77% yield as a white solid.
  • a NaOMe solution was prepared from sodium (1.3 g; 58.1 mmol; 3.3 eq) in methanol (10 mL).
  • methanol 10 mL
  • Triethylamine (61 mL; 0.438 mol; 1.5 eq.) was added to the crude compound ethyl (2Z)-3-amino-2-( ⁇ [(tert-butoxy)carbonyl] amino ⁇ imino)propanoate (0.292 mol; 1 eq.) (1-36) solubilized in DCM (2 L).
  • the reaction mixture was cooled to 0°C, then, acetyl chloride (20.74 mL; 0.292 mol; 1 eq.) was added dropwise.
  • reaction mixture was refluxed for 64 hours.
  • the resulting reaction mixture was taken up in ethyl acetate (30 ml) and a saturated solution of sodium chloride (20 ml).
  • the aqueous layer was extracted with ethyl acetate (2 x 30 ml).
  • the combined organic layers were washed with a saturated solution of sodium chloride (3 x 20 ml), dried over sodium sulphate, filtered and concentrated under reduced pressure.
  • Examples 17 and 18 Preparation of 3-hydroxy-4-(4-tsopropylbenzoyl)-l-(6- methylpyridazin-3-yl)-5(R)-(6-(trifluoromethoxy)pyridtn-3-yl)-lH-pyrrol-2(5H)-one (Example 17) and 3-hydroxy-4-(4-tsopropylbenzoyl)-l-(6-methylpyridazin-3-yl)-5(S)- (6-(trifluoromethoxy)pyridin-3-yl)-lH-pyrrol-2(5H)-one (Example 18)
  • the two enantiomers of Example 6 were separated by preparative HPLC using the following conditions:
  • the two enantiomers were then taken up in ethyl acetate (30 mL), washed with an aqueous solution of HC1 IN (10 mL), dried over sodium sulphate and concentrated to dryness.
  • the two enantiomers were analyzed by chiral HPLC using the following conditions:
  • Example 20 Preparation of l-[6-(aminomethyl)pyridazin-3-ylJ-3-hydroxy-4- ⁇ [4- ropan-2-yl)phenyl]carbonyl ⁇ -5-[4-(trifluoromethoxy)phenyl]-2,5-dihydro-lH- pyrrol-2-one trifluoroacetic acid
  • the aqueous layer was extracted with dichloromethane (3 x 30 ml). The combined organic layers were washed with a saturated solution of sodium chloride (1 x 50 ml), dried over sodium sulphate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel chromatography using dichloromethane/methanol (9/1 to 8/2) as an eluent. Few drops of TFA were added to combined fractions.
  • Examples 21 and 22 Preparation of l-[6-(aminomethyl)pyridazin-3-ylJ-3-hydroxy-4- ⁇ [4- (propan-2-yl)phenyl]carbonyl ⁇ -5-(R)-[4- (trifluoromethoxy)phenyl]-2, 5-dihydro- lH-pyrrol-2- one trifluoroacetic acid (example21) and l-[6-(aminomethyl)pyridazin- 3-yl]-3-hydroxy-4- ⁇ [4 ⁇ ropan-2-yl)phenyl]carbonyl ⁇ -5-(S)-[4-(trifluoromethoxy) phenylJ-2,5-dihydro-lH-pyrrol-2- one trifluoroacetic acid (example 22).
  • the reaction mixture was stirred at room temperature for 1 hour then diluted by the addition of dichloromethane (50 ml) and water (15 ml). The aqueous layer was extracted with dichloromethane (2 x 30 ml). The combined organic layers were dried over sodium sulphate, filtered and concentrated under reduced pressure.
  • Example 23 Preparation of N- ⁇ [6-(3-hydroxy-2-oxo-4- ⁇ [4-(propan-2-yl)phenyl] carbonylJ-5-[4-(trifluoromethoxy)phenylJ-2,5-dihydro-lH-pyrrol-l-yl)pyridazin-3-ylJ methyljmethanesulfonamide a) N-((6-r3-(fert-butoxy)-2-oxo-4-( r4-(propan-2-vnphenyllcarbonyl
  • the reaction mixture was stirred at room temperature for 1 hour then concentrated to dryness.
  • the resulting residue was taken up in ethyl acetate (30 ml) and a saturated solution of hydrogenocarbonate (30 ml).
  • the aqueous layer was extracted with ethyl acetate (2 x 30 ml).
  • the combined organic layers were washed with a saturated solution of sodium chloride (1 x 20 ml), dried over sodium sulphate, filtered and concentrated under reduced pressure.
  • Trifluoroacetic acid (2 ml) was added to a solution of N-( ⁇ 6-[3-(tert-butoxy)-2-oxo-4- ⁇ [4-(propan-2-yl)phenyl] carbonyl ⁇ -5 - [4-(2,2,2-trifluoroethyl)phenyl] -2, 5-dihydro- 1 H- pyrrol- l-yl]pyridazin-3-yl ⁇ methyl)methanesulfonamide (1-29) (50 mg; 0.08 mmol; 1 eq) in dichloromethane (2 ml). The reaction mixture was stirred at room temperature for 1 hour then concentrated to dryness .
  • Example 24 Preparation of N- ⁇ [6-(3-hydroxy-2-oxo-4- ⁇ [4-(propan-2-yl)phenyl] carbonylJ-5-[4-(trifluoromethoxy)phenylJ-2,5-dihydro-lH ⁇ yrrol-l-yl)pyridazin-3-ylJ methyl ⁇ acetamide a) N-((6-r3-(fert-butoxy)-2-oxo-4-( r4-(propan-2-vnphenyl1carbonyl
  • the reaction mixture was stirred at room temperature for 4 hours then diluted by the addition of dichloromethane (15 ml) and water (15 ml). The aqueous layer was extracted with dichloromethane (2 x 30 ml). The combined organic layers were dried over sodium sulphate, filtered and concentrated under reduced pressure.
  • Trifluoroacetic acid (1 ml) was added to a solution of N-( ⁇ 6-[3-(tert-butoxy)-2-oxo-4- ⁇ [4-(propan-2-yl)phenyl]carbonyl ⁇ -5-[4-(trifluoromethoxy)phenyl]-2,5-dihydro-lH- pyrrol-l-yl]pyridazin-3-yl ⁇ methyl)acetamide (1-30) (54 mg; 0.088 mmol ; 1 eq) in dichloromethane (1 ml). The reaction mixture was stirred at room temperature for 1 hour, then, concentrated to dryness .
  • Example 25 Preparation of l-[6-(2-ethoxyethyl)pyridazin-3-yl]-3-hydroxy-4- ⁇ [4 ⁇ ropan-2-yl)phenyl]carbonyl ⁇ -5-[4-(trifluoromethoxy)phenyl]-2,5-dihydro-lH- pyrrol-2- one
  • Huh-7 cell line Human Hepatoma Huh-7 cell line was maintained in DMEM/HAMF-12 supplemented with 10% SVF, 4 mM glutamine, 0.5M Na pyruvate, 1% penistreptomycine.
  • HCV replicon containing Huh-7 cell lines Huh-9. 13 and Luc Neo ET (Reblikon) were maintained in DMEM supplemented with 10% SVF, 2 mM glutamine, and 1 X NEAA, 100 U / ml penicillin, and 100 ⁇ g / ml streptomycine.
  • Replicon cells were maintained in medium supplemented with 1 mg/ml G418 for replicon Huh-9.13 and 0.5 mg/ml for Luc Neo and replicon unless indicated otherwise.
  • Huh-7 and HCV replicon cell lines were maintained at 37°C and 5% C0 2 in a humidified atmosphere. Cells were dissociated at sub confluence with trypsin EDTA IX.
  • cDNA encoding HCV NS5B genotype lb was cloned in frame with Gal4-DNA Binding Domain. The protein was expressed with a 21 amino acid C-terminal deletion to remove transmembrane domain. Expression of NS5BA21/Gal4DBD fusion protein was under control of SV40 early promoter. 3D-Sensor peptide was cloned in frame with VP 16 activation domain. Expression of 3D-Sensor / VP 16 AD fusion protein was under control of CMV promoter. Expression of the firefly luciferase reporter gene was inducible by the [Target protein / conformation sensitive peptide / VP16AD] complex. 3D-SCREEN assay:
  • 3D-SCREEN assay is a reporter gene assay designed to identify chemical entities that modify the 3D-structure of target proteins and hence inhibit their biological activity (WO 2006/046134). It is a single-target, cell based assay. Briefly, expression of a reporter gene depends on the interaction of a short peptide, thereafter named 3D-Sensor, and native conformation of the target protein. Whenever the conformation of the target protein is modified, interaction between 3D-sensor and target protein is disrupted and reporter gene is not expressed anymore. Conformation modifiers are identified by loss of expression of reporter gene.
  • NS5B 3D-Screen platform was generated in Huh-7 cell lines by transient transfection of three expression vectors encoding respectively
  • Huh-7 cells were dissociated the day before transfection and seeded in T 175 flasks at a density of 10 7 cells in 30 ml culture medium. Equimolar ratios of vectors were transfected in cell according to optimized j etPEI transfection protocol (PolyPlus Transfection, Illkirch, France) and 10 ⁇ g total DNA / 10 6 cells. Transfection was performed for 2 hours at 37°C and 5% C0 2 in a humidified atmosphere. After two hours cells were dissociated and seeded in 96 wells plates at a density of 25,000 cells per well and 90 ⁇ culture medium. 10 ⁇ of compounds to be tested were added 2 hours after seeding. Final concentration of DMSO was 1%.
  • Cells were incubated in the presence of compounds for 24 hours after which expression of firefly luciferase reporter gene was quantified. Briefly, culture medium was removed and cells were lysed by addition of 100 ⁇ of lysis buffer containing 125 mM Tris Phosphate ph 7.8, 10 mM EDTA, 5 mM DTT, 50 % glycerol and 5 % Triton. Plates were vortexed 10 min at 1300 rpm. Cell lysat was transferred in OpaqueWhite Assay 96 well Flat Bottom plates. ⁇ ⁇ of luciferin solution IX were added to each well.
  • Luciferin solution contained 40 mM Tris Phosphate ph 7.8, 0.2 mM EDTA, 67 mM DTT, 2.14 mM MgC12, 5.4 mM MgS04, 4.7 x 10 "4 M luciferin, 5.3 x 10 "4 M ATP and 2.7 x 10 "4 M Acetyl co enzyme A.
  • Replicon Luc Neo ET is a bicistronic expression constructs (Lohmann et al, 1999, Science 285, 110-113).
  • the structural genes of the HCV genome were replaced by heterologous sequences; the gene encoding the neomycin phosphotransferase (NPT) and the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV).
  • the bicistronic construct is therefore composed of the following elements: HCV-IRES nucleotides 1-389, the NPT gene, the EMCV-IRES directing translation of downstream HCV sequences from NS2 or NS3 up to the authentic 3 ' end of the genome.
  • HCV Polyprotein harbours the cell culture adaptive mutations E1202G, T1280I, K1846T. G418-resistance is only possible with cells containing high amounts of replicon.
  • Cells were dissociated the day before addition of compounds and seeded in 96 well- plates at a final concentration of 77 777.77 cells. ml "1 . well "2 in 90 ⁇ final volume of culture medium per well and were maintained at 37°C and 5% C0 2 in a humidified atmosphere for 24 hours. 10 ⁇ of compounds to be tested were added 24 hours after seeding. Final concentration of DMSO was 1%. Cells were incubated in the presence of compounds for 72 hours after which expression of firefly luciferase reporter gene was quantified.
  • culture medium was removed and cells were lysed by addition of 100 ⁇ of lysis buffer containing 125 mM Tris Phosphate ph 7.8, 10 mM EDTA, 5 mM DTT, 50 % glycerol and 5 % Triton. Plates were vortexed 10 min at 1300 rpm. Cell lysat was transferred in OpaqueWhite Assay 96 well Flat Bottom plates. ⁇ ⁇ of luciferin solution IX were added to each well.
  • Luciferin solution contained 40 mM Tris Phosphate ph 7.8, 0.2 mM EDTA, 67 mM DTT, 2.14 mM MgC12, 5.4 mM MgS04, 4.7 x 10 "4 M luciferin, 5.3 x 10 "4 M ATP and 2.7 x 10 "4 M Acetyl co enzyme A.
  • hepatitis C virus RNA-dependent RNA polymerase membrane insertion sequence is a transmembrane segment. J Virol. 2002 Dec;76(24): 13088-93.

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Abstract

Cette invention concerne des composés de 1-(azo-hétérocyclique à 6 chaînons)-2,5-dihydro-1H-pyrrol-2-one répondant à la formule (I) suivante ou un sel, solvate, tautomère, isotope, énantiomère, diastéréomère ou mélange racémique de ceux-ci : ; une composition pharmaceutique les contenant et leur utilisation thérapeutique à titre d'inhibiteurs du virus de l'hépatite C.
PCT/EP2012/050195 2011-01-07 2012-01-06 Dérivés de 1-(azo-hétérocyclique à 6 chaînons)-2,5-dihydro-1h-pyrrol-2-one à titre d'anti-virus de l'hépatite c, composition pharmaceutique les contenant et leur utilisation thérapeutique Ceased WO2012093174A1 (fr)

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WO2020179859A1 (fr) 2019-03-06 2020-09-10 第一三共株式会社 Dérivé de pyrrolopyrazole

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