WO2014131371A1 - Composé azétidinone utilisé dans la prévention et/ou le traitement de l'hépatite c, et composition pharmaceutique de ce composé - Google Patents

Composé azétidinone utilisé dans la prévention et/ou le traitement de l'hépatite c, et composition pharmaceutique de ce composé Download PDF

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WO2014131371A1
WO2014131371A1 PCT/CN2014/072736 CN2014072736W WO2014131371A1 WO 2014131371 A1 WO2014131371 A1 WO 2014131371A1 CN 2014072736 W CN2014072736 W CN 2014072736W WO 2014131371 A1 WO2014131371 A1 WO 2014131371A1
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group
fluorophenyl
compound
azetidinone
ene
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PCT/CN2014/072736
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English (en)
Chinese (zh)
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白骅
赵旭阳
刘晓宇
徐肖杰
郑晓鹤
刘礼飞
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浙江海正药业股份有限公司
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Priority to CN201480002991.5A priority Critical patent/CN104780918B/zh
Publication of WO2014131371A1 publication Critical patent/WO2014131371A1/fr
Priority to HK15111078.5A priority patent/HK1210057A1/xx

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/397—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having four-membered rings, e.g. azetidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
    • C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D205/06—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D205/08—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with one oxygen atom directly attached in position 2, e.g. beta-lactams

Definitions

  • the present invention relates to the field of medicine, and in particular to an azetidinone compound and a pharmaceutical composition thereof for use in the prevention and/or treatment of hepatitis C, and a method of using the same for preventing and/or treating a hepatitis C virus infection.
  • HCV refers to the hepatitis C virus, which belongs to the Flaviviridae family and is a single-stranded positive-strand RNA virus that is one of the leading causes of liver disease. Although acutely infected patients appear asymptomatic, approximately 80% of patients fail to clear the virus, which translates into chronic hepatitis with other liver diseases, including hepatic steatosis, insulin resistance, liver fibrosis, cirrhosis and hepatocellular carcinoma. (Alter & Seeff (2000) semin. Liver Dis. 20: 17-35).
  • HCV infection has become a serious public health problem, there is currently no effective vaccine to prevent HCV infection.
  • HCV-infected host cells are first conjugated to a series of receptors on the cell surface and then receptor-mediated endocytosis.
  • receptors include the four-molecule cross-linked cell membrane protein CD81 (Pileri, et al. (1998) Science 282: 938-41), the B-type I scavenger receptor (SR-B I) (Scavenger, et, al. ( 2002) EMBO 121:5017-25), tight junction protein claudin-1 (Evants, et, al. (2007) Nature 446:801-5) and tight junction protein claudin-6 (Liu, et, al. (2009) J. Virol. 83: 2011-4; Ploss, et al.
  • HCV virions are not only enriched in cholesterol (Aizaki, et al. (2008) J. Virol. 82:5715-24), but the loss of cholesterol reduces the virus infects cells (Aizaki, et Al. (2008) supra; Kapadia, et al. (2007) J. Virol. 81:374-83).
  • HCV virus invasive inhibitors there are no approved HCV virus invasive inhibitors in the clinic, but existing preparations can inhibit HCV The replication of the virus in the body.
  • HCV is currently being treated with a combination of interferon and ribavirin, but the combination has limited side effects and marginal effects that limit its range of application (Firpi & Nelson (2007) Arch. Med. Res. 38:678 -690; Foster & Mathurin (2008) Antivir. Ther. 13:3-8).
  • a series of HCV viral inhibitors that inhibit viral replication are described in US 2008/0161324. However, these compounds do not prevent the infestation and spread of HCV virus. Therefore, it is imperative to find new and promising antiviral drugs from other aspects of the viral life cycle, such as inhibition of viral invasion.
  • Patent document WO2011017907A1 discloses an azetidinone compound having the structure of the following formula (I), a preparation method thereof and use thereof, and the data show that the compound of the formula (I) is a plasma-lowering cholesterol agent for lowering plasma cholesterol content.
  • the invention provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of a hepatitis virus infection, in particular a hepatitis C virus: Formula (I)
  • R 1 is independently selected from the group consisting of 1-3 or less: hydrogen, halogen, trifluoromethyl, cyano, C r C 6 alkyl, C 2 -C 6 alkenyl, (3 ⁇ 4-( 6 cycloalkyl, Hydroxy, C r C 6 alkoxy, benzyloxy and -OCOR 7 ;
  • R 2 is independently selected from the group consisting of 1-3 or less: hydrogen, halogen, trifluoromethyl, cyano, C r C 6 alkyl, C 2 -C 6 alkenyl, C 3 -C 6 cycloalkyl, Hydroxy, (, - alkoxy, C 6 -C 1 () aryloxy, C 6 - aryl methoxy and -OCOR 7 ;
  • R 3 is independently selected from the group consisting of 1-3 or less: hydrogen, halogen, trifluoromethyl, cyano, dC 6 alkyl, c 2 -c 6 alkenyl, c 3 - c 6 cycloalkyl, c r c 6 methoxy and benzyloxy;
  • R 4 is hydrogen, C r C 6 alkyl, C 2 -C 6 alkenyl, and C 3 -C 6 cycloalkyl;
  • R 5 is hydrogen, CrC 6 ⁇ group, C 2 -C 6 alkenyl group and C 3 -C 6 cycloalkyl group;
  • R 6 is hydrogen or -COR 7 ;
  • ⁇ is ⁇ .
  • n 0, 1, 2 or 3;
  • n 1, 2 or 3;
  • the carbon-carbon double bond is in the Z configuration or the E configuration.
  • R 2 is independently selected from the group consisting of 1-3 or less: hydrogen, halogen, trifluoromethyl, cyano, C 6 alkyl, C 2 -C 6 yl, C r C 6 cycloalkyl, hydroxy, CC 6 alkoxy, benzyloxy and -OCOR 7 ;
  • R 7 is d-Cio alkyl, phenyl or substituted phenyl, said substituent being selected from : ! 3 ⁇ 4, trifluoromethyl, cyano, hydroxy, C r C 6 alkyl, C 2 -C 6 alkenyl, C 3 -C 6 cycloalkyl and C Cg aerated base
  • compounds of Formula (I) compound wherein R 1 is independently preferably from 1 to 3 [3 ⁇ 4 atom, and more preferably from 1 to 3 fluorine or chlorine atom, most preferably fluorine atom.
  • R 2 in the compound of formula (I) is independently preferably from 1-3 hydroxy, ⁇ -( 6 alkoxy or -OCOR 7 wherein R 7 has the definitions as described above; more preferably independently It is selected from 1-3 hydroxyl groups, methoxy, phenoxy or -OCOR 7 .
  • R 3 is independently selected from 1-3! 3 atomic atoms, more preferably from 1 to 3 fluorine or chlorine atoms, most preferably fluorine.
  • R 4 in the compound of formula (I) is preferably hydrogen or -C 6 alkyl, more preferably hydrogen or methyl.
  • R 5 in the compound of formula (I) is preferably hydrogen or C r C 6 sulphonyl, more preferably hydrogen or methyl.
  • R 6 in the compound of formula (I) is preferably hydrogen.
  • R 6 in the compound of formula (I) is preferably -COR 7 ; wherein R 7 is as defined above, more preferably d-o alkyl, most preferably methyl.
  • n is preferably 1.
  • the compound of formula (I) is preferably selected from:
  • Halogen means fluorine, chlorine, bromine and iodine.
  • Alkyl as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group. The most preferred option is C r C ⁇ , unless otherwise indicated. Examples of straight or branched d-alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, t-butyl, hexyl and the like.
  • alkenyl as a group or part of a group refers to a fatty group containing at least one carbon-carbon siloxane, which may be straight or branched. The most preferred is the c 2 -c 6 alkenyl group. The group may contain multiple double bonds in its backbone and its geometry may each be E or Z. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, allyl, propenyl, and the like.
  • Cycloalkyl means a saturated or partially saturated monocyclic, fused or spiro carbon ring. A ring consisting of 3-6 carbon atoms is preferred. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
  • Alkoxy means a group of (alkyl-0-). Among them, alkyl groups are defined in the text.
  • the alkoxy group of c r c 6 is preferred. Examples thereof include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy and the like.
  • the present invention includes the compounds represented by the formula (I) and various possible isomeric forms thereof. These include: non-imaged isomers, mirror image isomers, and geometric isomers of the "Z" or "F" configuration.
  • pharmaceutically acceptable salt means a salt of the above compounds which retains its original biological activity and is suitable for medical use.
  • the pharmaceutically acceptable salt of the compound represented by the formula (I) is a salt formed with a metal.
  • the metal forming a pharmaceutically acceptable salt with the compound represented by the formula (I) includes lithium, sodium, potassium, magnesium, calcium, aluminum, zinc, and the like.
  • a pharmaceutical composition comprising a compound of the above formula (I), which comprises a prophylactically and/or therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, And optionally, a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises one or more other active ingredients capable of inhibiting hepatitis virus infection, such as type I interferon, ribavirin, ribavirin, nucleotide analog R1479, a nucleotide inhibitor R1626, a diphenyl heterocyclic compound, etc., wherein the type I interferon is IFN- ⁇ or lFN- ⁇ »
  • the invention provides the use of a compound of the above formula (I), or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prophylaxis and/or treatment of hepatitis viruses, in particular hepatitis C virus infection.
  • the present invention provides the use of a compound of the above formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for reducing or preventing the entry of a hepatitis virus, particularly a hepatitis C virus, into a cell.
  • the present invention provides a method for preventing and/or treating a hepatitis virus, particularly a hepatitis C virus infection, the method comprising administering an effective dose to a subject in need of treatment as described above
  • a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same is administered to a subject in need of treatment as described above.
  • the present invention provides a method for reducing or preventing entry of a hepatitis virus, particularly a hepatitis C virus, into a cell, the method comprising administering a cell or an effective amount of the compound of the above formula (I) or a pharmaceutically thereof thereof Acceptable salts, or pharmaceutical compositions containing them, are contacted.
  • the present invention describes a method of preventing liver virus infection.
  • the i-hai method prevents or reduces the invasion of the liver virus by administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • the subject is a liver transplanter.
  • the present invention is also applicable as a synergistic pharmaceutical composition for treating hepatic virus infection.
  • the composition comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof, and at least one other agent which inhibits hepatic viral infection.
  • Other drugs for inhibiting hepatic virus infection described therein are selected from the group consisting of type I interferon, ribavirin, ribavirin, nucleotide analog R1479, nucleotide inhibitor R1626, diphenyl heterocyclic compound and the like.
  • the other agent that inhibits hepatitis virus infection is type I interferon, and the type I interferon is IFN-[alpha] or IFN-P.
  • the present invention is also applicable to a combination of a certain effective amount of another drug or a pharmaceutical composition, synergistically acting on cells infected with a hepatic virus, and effectively removing hepatic viruses in the cells.
  • a specific embodiment of the present invention provides a method for preventing hepatitis virus infection, which is mainly by administering to a "subject" in need of prevention a certain effective amount of a compound of the formula (I) of the present invention or a pharmaceutically acceptable compound thereof. Salts, or pharmaceutical compositions containing them, thereby reducing or preventing liver virus infection of cells.
  • a “subject” herein may refer to any animal (e.g., including a mammal such as a human).
  • hepatitis virus infection mainly refers to adsorption and internalization of hepatitis virus, has been confirmed by viral replication and continuous infection of the virus.
  • prevention in the present invention means preventing or delaying the prevention of clinical symptoms associated with HCV. Treatment.
  • the compound (1-1 Z) of the present invention exhibits a synergistic effect with an interferon
  • the synergistic composition of the present invention refers to a compound of the formula (I) or a pharmaceutically acceptable salt thereof.
  • a composition that combines at least one inhibitor that inhibits hepatic viral infection e.g., inhibits expression of a viral gene, replicates, or inhibits assembly and release of viral particles.
  • the total therapeutic effect of the present invention in combination with other pharmaceutical compositions is greater than the sum of the therapeutic effects when they are used alone.
  • in combination with at least one other inhibitor that inhibits hepatic viral infection refers to a viral replication inhibitor.
  • Drugs that inhibit hepatic virus replication may be published inhibitors that target various aspects of viral replication.
  • inhibitors of HCV viral replication are inhibited by inhibitors that reduce the efficiency of each replication of the virus, or by inhibiting factors required for viral replication, including but not limited to viral genome replication, viral RNA transcription, and proteolysis. process.
  • inhibitors of hepatic virus infection include, but are not limited to, iminothiazolones (US) , Patent No.
  • Type I Interferon Zeuzem, et al, (1996), Hepatology, 23(2): 366-71), ribavirin (Gish (2006), J Antimicrob Chemother, 5(l): 8 -13), nucleotide analogue R1479 (klumpp, et al, (2006) J Biol Chem, 281(7): 3793-3797), Telaprevir (Weisberg & Jacobson (2009 Clin Liver Dis, 13(3):441- 52; Serrazin, et al. (2007) Gastroenterology 132(5): 1767-77), Boceprevir (Mederacke, et al. (2009) Curr Opin. Investig.
  • Drugs 10(2): 181-9 Nucleotide inhibition Agent R1626 (Toniutoo, et al. (2008) IDrugs ll (10): 738-49), ITMN-191 (R7227; Seiwert, et al. (2008) Antimicrob. Agents Chemother. 52(12): 4432-41) , an alternative diphenyl heterocyclic compound (Huang, et al. (2008) Antimicrob. Angents Chemother. 52(4): 1419-29), beta-D-2-Deoxy-2-fluoro-2-C -methylcytidiine (PSI-6130; Asif, et al. (2007) Antimicrob. Angents Chemother.
  • statins Ikeda, et al. (2006) Hepatology 44(1): 117-25
  • bisindolylmaleimides and indolocarbazoles Murakami, et al. (2009) Antiviral Res 83:112-117.
  • the synergistic composition comprises at least one type I interferon (eg, IFN-[alpha] or IFN-[beta]), a combination of ribavirin or type I interferon and ribavirin .
  • the synergistic composition in a specific embodiment of the present invention comprises: 1) a compound of the formula (I) or a pharmaceutically acceptable salt thereof; 2) an interferon or ribavirin; 3) a hepatitis C virus infection inhibitor.
  • Type I interferons described herein are a family of interferon proteins that inhibit viral replication, cell proliferation, and modulate immune responses.
  • alpha interferons including but not limited to, Roferon A interferon (Hoffinan-La Roche, Nutley NJ), interferon a-2 (Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, CT), rDNA-interferon (Sumitomo, Japan).
  • Roferon A interferon Hoffinan-La Roche, Nutley NJ
  • interferon a-2 Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, CT
  • rDNA-interferon Suditomo, Japan
  • alpha interferon 2b is approved by most countries in the world and is widely used to treat HCV.
  • An interferon 2b has been described in U.S. Patent No. 4,530,901.
  • the desired compound of the formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, preferably a synergistic composition is formulated for administration to a subject.
  • a pharmaceutically acceptable carrier can be optionally used as needed. These carriers are widely known, such as physiological saline, cellulose, lactose, caramel, mannitol, sorbitol and phosphoric acid.
  • Selective additives include lubricants, binders such as silicic acid, silica, talc, stearic acid, magnesium stearate, stearic acid, PEG; disintegrants such as starch, carboxymethyl starch, Cross-linked polyvinylpyrrolidone, agar, alginic acid and its salts; pigments; flavoring agents and fuming agents. Pigments or natural colors are added to tablets or icings, such as to distinguish between different doses of the active ingredient or active ingredient.
  • the compound of formula (I) or a pharmaceutically acceptable salt thereof and the other at least one agent for inhibiting hepatitis C virus infection may be formulated with one or more carriers in an appropriate ratio.
  • the active ingredient of the drug accounts for 1-95% of the total weight of the therapeutic administration.
  • the dosage form of the drug should be suitable for different administration modes, such as oral administration, injection (intravenous injection, intramuscular injection), rectal administration, administration of human appendages, transdermal administration, intranasal, vaginal administration, inhalation administration, skin localization. Administration (patch), eye drops. Therefore, the pharmaceutical dosage forms include tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels, pastes, ointments, creams, transdermal patches, suppositories , injections, infusions, inhalants and aerosols.
  • Formulations can be prepared using conventional processes (e.g., emington: The Science and Practice of Pharmacy, 20 th edition, 2000, ed AR Gennaro, Lippincot Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds J. Swarbrick and JC.. Boylan, 1988-1999, Marcel Dekker, New York) 0
  • the compound of the formula (I) or a pharmaceutically acceptable salt thereof and the other at least one drug for inhibiting hepatitis C virus infection may be prepared unilaterally or continuously administered in a plurality of times (minutes, hours or one day). Compound dosage form.
  • the dosage and combination regimen will depend on factors such as the treatment regimen, type of hepatitis virus, viral infectivity, treatment with hepatitis or prevention of viral infection, and age, weight, and health of the patient.
  • the synergistic composition of the invention can simultaneously target the invasion of the virus and the expression and replication of the viral gene. And the assembly and release of viral particles, so the synergistic composition can effectively remove hepatitis virus from cells and treat liver virus infection. Therefore, indications for this patent include the removal of hepatitis C virus in cells.
  • the use of a certain effective amount of the synergistic composition of the present invention acts on virus-infected cells to effectively remove hepatitis C virus from the cells. It is expected that the viral load in liver cells can be reduced by 20%, 30%, 50%, 70%, 80%, 90%, 95%, 99% by a reasonable treatment regimen.
  • hepatocytes are used for testing, and hepatitis C virus can be effectively eliminated from its host cells. Therefore, the present invention is applicable to hepatitis C virus infection.
  • Another embodiment of the present invention is useful for treating hepatitis C virus infection by administering an effective amount of a synergistic composition to a patient infected with hepatitis C virus, which can significantly prevent or reduce the rate of viral replication and transmission, and reduce the viral load (such as the virus mentioned herein, Including liver virus A, B, C, D, E), at least to alleviate a symptom of liver infection in patients.
  • a synergistic composition such as the virus mentioned herein, Including liver virus A, B, C, D, E
  • the viral load such as the virus mentioned herein, Including liver virus A, B, C, D, E
  • the replication of the virus can be slowed down and reduced by at least 20%, 30%, 50%, 70%, 80%, 90%, by a reasonable treatment regimen (as mentioned above for the inhibition of viral replication or inhibition of infection in the cell). 95%, 99% viral load.
  • Treatment beneficiaries include patients diagnosed with hepatitis C virus (as evidenced by hepatitis C virus markers).
  • hepatitis C virus detection markers have been reported in the literature and can be tested by professionals.
  • hepatitis C virus infection can be confirmed by detecting hepatitis C virus nucleic acid or protein in the liver or blood.
  • a compound of the formula ( ⁇ ) of the present invention as described herein for the prevention and/or treatment of hepatitis virus infection, wherein "effective dose” refers to a dose of a drug that reduces viral load or eliminates a virus, thereby reducing, alleviating or Eliminate chronic infections that can lead to cancer or cirrhosis (symptoms of hepatomegaly, splenomegaly, jaundice, muscle weakness, hepatic ascites, ankle edema).
  • the effective therapeutic dose can be determined by reference to the relevant dosages and methods reported in the literature.
  • appropriate animal models are selected for testing to determine effective doses, such as in vivo testing to detect the inhibitory effect of drugs on HCV.
  • the advantages and specific applications of a large number of such model tests have been reported in the literature.
  • clinical studies can be dose-designed based on the safe dose and dose-effect relationship of the animal.
  • Such clinical trials can be designed according to the Spilker ((2000) Guid to Clinical Trials. Lippincott Williams & Wilkins: Philadelphia) clinical trial protocol.
  • the dosage of the therapeutic regimen of the present invention can be based on the results of the clinical phase I experimental data of formula (I), and other interferon or hepatitis C virus inhibitors can be used as a reference for the pharmacological and toxicological protocols of clinical use.
  • the amount of medication varies depending on the individual patient, and you should follow the doctor's advice.
  • the compounds of the invention can be used in mechanistic studies to verify whether other pharmaceutical compositions or single agents have the same therapeutic effect as a compound or composition of the invention for the treatment of viral infections (detected by literature reporting methods).
  • the drug candidate can be administered alone or in combination (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof), followed by cells (e.g., Huh7, Huh2, Huh8, Sk-Hep-1, Huh7 lunet> HepG2 , WRL-68, FCA-1, LX-1, LX-2, Hunh7-derived and other stem cell lines) level detection.
  • the virus invades the cells, replicates, and then examines the amount of virus in the cells. If the test compound or composition has the effect of inhibiting viral invasion (as detected by colony formation assays), replicating or reducing the number of viruses,
  • the compounds or compositions are useful as effective agents for the treatment of viral infections.
  • the invention can be used as a tool for studying and elucidating the biological signaling pathway of viral diseases. Such studies can further develop drugs that treat, prevent, or reduce the combination of viral infections or single agents.
  • the present invention can be used to identify signal pathways or infection signaling networks of virus-infected cells, such as hepatocytes. These methods include the detection and analysis of the cell components of the administration group of the present invention and the negative control group, the positive control group or the combination of other single drugs and the combination of the compound drugs, and the cell or virus activity (such as enzyme activity and nutrient absorption). And proliferation) for detection.
  • Cell component analysis includes gene transcription and protein expression.
  • Commonly used methods include standard biochemical techniques, radiolabeling of the present invention, and compound-protein binding assays, such as two-dimensional gel electrophoresis or gene expression profiling.
  • the compound can be tested in vivo (eg, gene knockout or transgenic mice) to further validate the effectiveness of the compound as a tooling study, as well as to develop new drugs for the treatment of prion diseases, and to study new treatment options. Effectiveness.
  • test methods include, but are not limited to, the methods described in the examples:
  • FIG. 1 Compound (1-1 Z) prevents HCV invasion of Huh7 cells: Huh7 cells infected with HCV according to Example 2 were pretreated with different concentrations of compound (1-1 Z) for 6 hours, and intracellular virus was infected 8 h and 24 h after infection. content. It was shown that the compound (1-1 Z) can effectively inhibit the internalization of HCV in cells at a concentration of 10 uM to 40 uM/L.
  • the monolayer human hepatoma cell line HuH7 was pretreated with different concentrations of compound (I-1 Z), and was infected with the HCV virus derived from JFH-1 at a concentration of 0.1 FFU/CELL. The cells were infected with the virus.
  • HCV RNA was determined by real-time quantitative PCR (RT-qPCR), and the content was expressed by HCV RNA copy number/ug cell total RA, and GAPDH was used as an internal reference.
  • RT-qPCR real-time quantitative PCR
  • the results of the three trials are expressed in mean sem, and the negative control (detection of HCV RA content in uninfected cells) is approximately 0.5-l.OxlO 2 copy ⁇ t/ug total cell RA.
  • the compound (1-1 Z) was able to significantly reduce the content of HCV RNA in total cellular RNA (PO.05, one-way variance versus outcome analysis, and t-test).
  • FIG. 2 Compound (1-1 Z), Compound (1-12 Z), Compound ( ⁇ -6) prevent HCV invasion into Huh7 cells: Huh7 cells infected with HCV according to Example 2, 20 uM of compound (1-1 Z) , the compound (1-12 ⁇ ), the compound (1-6 Z) was pretreated for 6 hours and the virus content of the virus was infected for 24 hours. It is shown that the compound (1-1 Z), the compound (1-12 Z), and the compound (1-6 Z) can effectively inhibit the internalization of HCV in cells.
  • the monolayer human hepatoma cell line Huh7 was added with 20 uM/L compound (I-1 Z), compound (1-12 Z), and compound (1-6 Z) for 6 hours, and the concentration was 0.1FFU/CELL.
  • HCV virus infection JFH-1, a HCV full-length gene sequence isolated from a hepatitis C patient in Japan
  • JFH-1 a HCV full-length gene sequence isolated from a hepatitis C patient in Japan
  • JFH-1 a HCV full-length gene sequence isolated from a hepatitis C patient in Japan
  • the content of HCV RNA was determined by real-time quantitative PCR (RT-qPCR), and the content was expressed by HCV RNA copy ug cell total RNA, and GAPDH was used as an internal reference.
  • RT-qPCR real-time quantitative PCR
  • GAPDH GAPDH was used as an internal reference.
  • the results of three independent experiments are indicated by means sem, and the negative control (detection of HCV NA content in: infected cells) is approximately 0.5-l.OxlO 2 copies of 3 ⁇ 4/ug total cellular RNA.
  • the compound (1-1 ⁇ ), the compound (1-12 ⁇ ), and the compound (1-6 ⁇ ) with a concentration of 20 uM/L were able to Significantly reduced the amount of HCV RNA in total cellular RNA (PO.05, one-way variance versus outcome analysis, and t-test).
  • FIG. 3 Compound (1-1 Z) and interferon- ⁇ synergistically treat chronic HCVcc infection: According to Example 2 chronic HCVcc infection experiment, Huh7 cells were infected with HCV and the intracellular virus content was changed by drug at different times. It is shown that the compound (1-1 Z) has a synergistic effect with interferon- ⁇ , which can reduce the RNA content of human hepatoma cell line ⁇ 7 chronically infected with HCV. After 60 days of continuous administration, the intracellular HCV RNA content is close to that of uninfected HCV cells. HCV 11 RNA background value. Huh7 was co-cultured with JFH-1 strain containing HCV infectious virus particles at a virus concentration of 0.1 FFU/CELL.
  • Incubation was continued for 10 days after infection to bring the HCV R A content to a stable state.
  • the test was divided into four groups: model group, compound (1-1 ⁇ ) (20 ⁇ ) group, interferon- ot (100 U/ml) group, compound (1-1 ⁇ ) (20 ⁇ ) and interferon- ⁇ ( 100 U/ml) combination group.
  • Cell passage was continued during the administration period.
  • HCV RNA was quantitatively detected by real-time quantitative PCR.
  • GAPDH was used as an internal reference, and the content was expressed by HCV RA copy 3 ⁇ 4/ug total RNA. A duplicate hole, the experimental results are expressed in the mean sem.
  • HCV background values for uninfected HCV cells.
  • three cells were collected in equal amounts, and HCV RNA was quantitatively detected by real-time quantitative PCR.
  • GAPDH was used as an internal reference, and the content was expressed by total RNA of HCV RNA copy Mig cells.
  • the experimental results are expressed in mean ⁇ sem. The experimental results on the graph represent two independent experiments.
  • FIG. 5 Compound (I-1 Z) prevents HCV RNA content in the fourth week of HCV infection in tree shrews: According to the tree shrub of Example 4, the risk of HCV infection was prevented, and 15 trees were set up, 5 in each group, respectively Drug group, I-lZ lm/kg group, I-lZ 5m/kg group. Before the HCV infection, the unadministered group was orally administered with normal saline, and the administration group was administered once a day (the administration method was oral administration), and the administration was continued for 2 weeks. After two weeks, the tree was infected, and the infection was stopped. medicine. HCV RNA was measured by blood sampling from the second week to the sixth week after infection.
  • the results of the experiment showed that the amount of HCV RNA in the I-lZ lm/kg group and 5 trees in the I-lZ lm/kg group was compared with the unadministered animals. Below the detection limit, only one of the tree shrews in the I- 1Z 5m/kg group detected HCV RNA at a very low concentration.
  • FIG. 6 Compound (1-1 Z) Prevents HCV-infected Tree Shrews The frequency of HCV infection positive detection from the second week to the sixth week: According to the case 4, the prevention of HCV infection experiment, 15 trees, 5 in each group, respectively For the unadministered group, the I-lZ lm/kg group and the I-lZ 5m7 kg group. Before the HCV infection, the unadministered group was orally administered with normal saline, and the administration group was administered once a day (the administration method was oral administration), and the administration was continued for 2 weeks. After two weeks, the tree was infected, and the infection was stopped. medicine. HCV RNA was measured by blood sampling from the second week to the sixth week after infection.
  • the blood HCV RNA positive test from the second week to the sixth week showed that the I-lZ lm/kg group and the I-lZ 5m/kg group
  • the frequency of positive infection detection was significantly lower than that of the non-administered group.
  • the positive infection of the untreated group was detected 12 times
  • the positive infection of the I- 1Z lm/kg group was detected 3 times
  • the positive infection of the I-1Z 5m/kg group Check out 2 times.
  • Figure 7 shows the infection of HCV and the administration of compound (1-1 Z) inhibits HCV infection of tree shrews: Simultaneous administration of tree shrews according to Example 4: 10 experimental tree shrews, 5 per group, in HCV infection Simultaneous administration, the non-administered group was orally administered with normal saline per day, and the administration group was administered once a day (administered by oral administration), I-1Z 3 mg/kg, for 2 weeks, for two weeks after administration. Stop administration. On the day before HCV infection, blood was drawn weekly for 4 weeks after infection. At week 4, 60% of the tree's HCV RNA content was below the detection limit compared to the unadministered animals. detailed description
  • the compound of the formula (I) is produced in accordance with the production method described in WO2011017907A1.
  • Huh7 cells Huh7 cells (CCTCC); hepatitis C replicon JFH-1, sg2a, sglb (a gift from Zhejiang University Hospital); JFH-1 linearized by Xbal, transcribed in vitro with TRANSCRIPT T7 ( OC3 ⁇ 4 US,;) kit
  • the cells were transfected by a modified electroporation method (rieger, et al. (2001) J. VIRO; 75(10): 4614-24).
  • Reagents Recombinant human interferon 2a (ROCH, US) and beta factor (MERK, GM) were resuspended in DMEM containing 10% fetal bovine serum to a concentration of 50 ⁇ / ⁇ 1. Transfer all to a test tube, -80. C save. Compound (1-1 Z) was diluted to 20 mM with DMSO, 4. C save.
  • o cells were transfected with HCVcc with a multiplicity of infection of 1 or 0.1 FFU for 12 hours of infection.
  • Three sets of experimental design 1) cells were treated with compounds (1-1 ⁇ ) at concentrations of 40, 20, 10, 5 ⁇ for 6 hours (pre-infection) before infection, and a negative control was set; 2) virus infection in cells At the same time, immediately add the compound (I-1 Z) with the concentration of 40, 20, 10, 5 ⁇ , and co-culture for 12 hours (simultaneous infection and simultaneous administration); 3) The cells were added to the concentration of 40, 20 immediately after infection with HCVcc virus. 10, 5 ⁇ of the compound (1-1 Z) was treated for 60 hours (post-infection administration).
  • Indicator detection 1) HCV RNA content analysis, total RNA was cleaved three times after infection with lx nucleic acid cleavage purification reagent (Invitrogen), and RNA was extracted for RT-qPCR analysis. 2) Detection of HCV viral envelope protein E2, the medium was removed, and fixed with 4% paraformaldehyde (w/v) (sigma) for 72 hours to immunohistochemically analyze the HCV virus E2 envelope protein.
  • HCVcc infection is real-risk.
  • Cell growth group Huh7 cells were seeded in T75 (Coming) cell culture In the bottle, the inoculation amount was 6 , and the cells were infected with a multiplicity of HCVcc (JFH -1 ) at 0.01 FFU/cell, and the culture of 12 angel HCV RNA was continued to reach a steady state level. On the 12th day, the cells were seeded in a ratio of 1:4 in 4 T25 (Coming) cell culture flasks. After 24 hours of culture, 4 bottles of cells were completely treated with DMEM complete medium and DMEM containing IFN-a (100 U/ml).
  • DMEM complete medium containing compound (1-1 ⁇ ) (20 ⁇ )
  • DMEM complete medium containing both IFN-a (100 U/ml) and compound (I-1 ⁇ ) (20 ⁇ ).
  • the medium was changed every two days in the experiment. Before the cells were full, they were digested with trypsin, 1:3 passage, and the cells were kept viable. At each passage, cells were collected, centrifuged at 1200 rpm for 5 minutes, total RNA was extracted with nucleic acid extraction reagent (Invitrogen), and after reverse transcription, RT-qPCR was performed.
  • Cell non-growth group Huh7 cells, inoculated in 48-well BIOCOAT culture plate (BD), the cell seeding density was ⁇ 4 /well, cultured for 20 days in a 1% DMS0 environment, and the cells were infected with 0.01 FFU/cell. Infection with JFH-1 HCVcc resulted in a steady level of HCV RNA after 14 days. On the 14th day after infection, in the same cell growth group, the cells were completely treated with DMEM complete medium, DMEM complete medium containing IFN-a (100 U/ml), and DMEM containing compound (1-1 ⁇ ) (20 ⁇ ;).
  • the medium was cultured in DMEM complete medium containing both IFN-a (100 U/ml) and compound (1-1 ⁇ ) (20 ⁇ M). The medium was changed every two days. After incubation to the set time, total RNA was extracted from the three parallel wells using RNA nucleic acid extraction reagent (Invitrogen), and after reverse transcription, RT-qPCR detection was performed.
  • DMEM complete medium containing both IFN-a (100 U/ml) and compound (1-1 ⁇ ) (20 ⁇ M.
  • the medium was changed every two days. After incubation to the set time, total RNA was extracted from the three parallel wells using RNA nucleic acid extraction reagent (Invitrogen), and after reverse transcription, RT-qPCR detection was performed.
  • RNA extraction and RT-qPCR detection Total cellular RNA Total RNA was extracted with a nucleic acid extraction reagent (Invitrogen) as indicated in the instructions. Each microgram of purified RNA was subjected to cDNA synthesis using the K1622 reverse transcription kit (Thermo), followed by SYBR green quantitative RT-qPCR using a CFX CONNECT (BIO-rad) quantitative PCR machine. The PCR cycle included 10 min denaturation at 95 ° C followed by 40 amplification cycles (95 ° C, 15 s) and annealing / extension (60 ° C, lmin).
  • the determination of the transcription levels of HCV JFH-1 and GAPDH was based on a standard curve prepared after the respective series of plasmids of the JFH-1 HCV cDNA or the human GAPDH gene were diluted.
  • the PCR primers used to detect GAPDH and HCV are: human GAPDH, 5'-GAA GGT GAA GGT CGGAGT C-3, (forward primer) (Seq. ID. No. 1) and 5,-GAA GAT GGT GAT GGG ATT TC-3, (reverse primer) (Seq. ID. No. 2); JFH-1 HCV, 5'-TCT GCG GAA CCG GTG AGT A-3' (forward primer) (Seq. ID. No. 3) And 5'-TCA GGC AGT ACC ACA AGG C-3, (reverse primer) (Seq. ID. No. 4).
  • the Huh7 cell supernatant was diluted 10-fold in DMEM complete medium, and ⁇ was used to dip cells inoculated in 96-well plates (BD Biosciences) at a cell density of 4 ⁇ 10 3 /well, 3 replicate wells per concentration. Incubate for 24 h at 37 ° C, then add 150 ⁇ l of DMEM complete medium containing 0.4% methylcellulose (w/v) (Fhika BioChemika, Switzerland) with a final concentration of methylcellulose of 0.25%.
  • lx phosphate buffer PBS pH 7.2
  • the cells were supplemented with 0.5% (v/v) TRITON X-100 (Fisher), 3% (w/v) bovine serum albumin (BSA) (Sigma). ) and 10% (v/v) fetal bovine serum blocked for 1 hour.
  • lx human anti-HCV E2 monoclonal antibody C1 containing 0.5% (v/v) TRITON X-100, 3% (w/v) BSA and 1:500 was incubated at room temperature.
  • TRITON X-100 lysate TRITON X-100, 50 mM TRis-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA
  • 50 ⁇ g of protein was taken for SDS-PAGE electrophoresis and then transferred to a nylon nitrocellulose membrane (O-RAD electrophoresis apparatus, US).
  • mice were then blocked with 5% skim milk powder for 2 hours, then incubated with a 1:1000 dilution of mouse anti-HCV NS3 monoclonal antibody (9-G2Clone, ViroGen, Watertown, MA) and washed 3 times with PBS containing 0.05% Tween 20. Incubate with horseradish peroxidase-conjugated goat anti-mouse antibody (Pierce, Rockford, Illinois) and wash again. Protein antibody complexes were detected using SUPERSIGNAL chemiluminescent substrate (Pierce).
  • Cell proliferation and cytotoxic bioluminescence detection Cell proliferation and cytotoxic bioluminescence detection.
  • Cell proliferation ATP assay kit (Peomega, US), which is a method for detecting intracellular ATP levels as a measure of cell viability and proliferation. The assay was performed using the cdltiter-glo@luminesent cell viability assay kit according to the instructions.
  • Experimental step drive Add lOOul of ATP detection reagent to the lOOul culture negative control group and drug treatment group for 15 minutes to perform fluorescence detection.
  • an ELISA kit (Shanghai Suobaolai Biotechnology Co., Ltd.) for detecting human alanine transferase (ALT) released from damaged cells was used, according to the method of operation of the kit. The results showed that the compound (1-1 ⁇ ) treated Huh7 cells for 12 h had no cytotoxic effect in the concentration range of 1- 40 ⁇ M.
  • Example 2 Compound ( ⁇ -1 ⁇ ), Compound ⁇ -12 ⁇ ), Compound (I-6 Z) prevents HCV virus from invading Cell compound of formula (I) is azetidinone, which is a high resistance Blood lipids, cholesterol-lowering drugs, the experimental results show that it can inhibit the absorption of cholesterol in the body, in the golden hamster, monkeys have the effect of lowering blood plasma low-density lipoprotein (LDL) and total cholesterol (WO2011017907A1), the same in humans It has the effect of lowering plasma low density lipoprotein (LDL) and total cholesterol.
  • LDL blood plasma low-density lipoprotein
  • WO2011017907A1 total cholesterol
  • HCV virions are enriched in intracellular cholesterol (Aizaki, et al. (2008) J. Virol. 82: 5715-24), and it is speculated that the compound of formula (I) has an effect of inhibiting the invasion of HCV virus into cells.
  • the activity of the compound (1-1 Z) to inhibit the entry of HCV into cells can be translated by detecting the number of HCV lesions in the cells.
  • Huh7 cells were inoculated with a multiplicity of infection (MOI) of 1.0 or 0.14 FFU/ceil derived from cell culture-derived HCV (HCVcc), pre-infection, simultaneous infection, and three groups after infection, in increasing concentrations. (0, 5, 10, 20 and 40 ⁇ M) Compound (1-1 Z) was treated.
  • the drug was treated 6 h before infection and then the compound was removed for 6 h or the compound was infected for 6 h.
  • the compound (1-1 ⁇ ) significantly reduced the number of HCV lesions and existed. Dose effect.
  • the intracellular virus content was detected 24 hours after infection, inhibiting 30%, 89.9% and 97, respectively.
  • % of HCV lesions were formed (Fig. 1), and when cells were incubated with HCVcc and 10, 20, 40 ⁇ M compound (1-1 ⁇ ), inhibition rates of 92%, 95%, and 99%, respectively, were observed.
  • infected infection Post-administration
  • the compound (1-1 Z) was added to the cells, the degree of inhibition was markedly lowered.
  • the cells were pretreated with 20 ⁇ compound (I-1 Z), compound (1-12 Z) and compound (1-6 Z) 6 h before infection. After infecting the virus for 24 hours, the intracellular virus content was detected, and the compound inhibited 88.3%, respectively. 73.2% and 23.5% of HCV lesions were formed (Fig. 2).
  • HCV RNA levels at 24, 48, and 72 h after infection were quantified, and HCV infection inhibition showed a dose effect and infection time effect.
  • Compound (1-1 Z) protects cells from viral infection for at least 48 h after treatment with 20 ⁇ M compound (1-1 ⁇ ) 6 h before cell virus infection.
  • the simultaneous infection group also effectively inhibited HCV infection, consistent with the experimental results of preventing virus invading cells.
  • the addition of the compound (1-1 Z) after infection did not effectively protect the cells from viral infection, and a slight decrease in HCV RNA levels was detected only 72 hours after infection.
  • the compound (1-1 Z) inhibited the entry of another flavivirus dengue virus (DNV, Wuhan Institute of Virology, Chinese Academy of Sciences) into cells. Unlike HCV, cells were treated with 10, 20, 40 ⁇ M compound (1-1 Z) and no significant reduction in DNV plaque formation was observed. Therefore, the above results indicate that the compound (1-1 ⁇ ) is an effective and specific inhibitor of the early stage of HCVcc infection.
  • DNV flavivirus dengue virus
  • Compound (1-1 Z) did not inhibit the HCV R A replication assay. Other antihyperlipidemic and cholesterol-lowering drugs are known to act on the cholesterol biosynthesis pathway rather than inhibit free cholesterol uptake. Compound (1-1 Z) inhibits HCV replication assay directly by treatment with dose-increasing compound (1-1 Z).
  • the addition of the compound (1-1 Z) alone did not reduce the RNA level of the HCV homeostasis in the cells, but the intracellular HCV RNA level after the 30th day of the IFN-a (100 U/ml) group alone was added. 2 orders of magnitude lower.
  • IFN-a and 20 ⁇ compound (I-1 ⁇ ) were used in combination (Fig. 3), they showed strong synergistic effects.
  • the combination of IFN-a and 20 ⁇ M compound (I-l Z) effectively reduced HCV RNA to background values ( ⁇ 4 magnitude reduction), indicating that co-administration cured chronically infected cells.
  • HCV RNA levels were continuously monitored for 20 days.
  • the level of HCV RNA in the cells added to the 100 U/ml IFN-a group immediately rebounded to the level of the negative control group (Fig. 4), and was administered in combination with 10 OU/ml IFN-a and compound (1-1 ⁇ ) (20 ⁇ ).
  • the group of cells had no rebound in HCV RNA (Fig. 4), and the HCV protein immunohistochemical staining test confirmed this.
  • RNA level is more than an order of magnitude.
  • IFN-p alone (20 U/mi) or in combination (20 or 40 ⁇ M compound (I-l ⁇ ) and 20 U/ml IFN- ⁇ ).
  • Drugs were treated with HCV-infected cells for 8 weeks, and 3 replicate wells of intracellular RNA were collected weekly, and HCV RNA was detected by RT-qPCR.
  • the results of the experiment showed that the compound (I-l Z) alone did not reduce HCV infection (i.e., intracellular HCV RNA level in a steady state).
  • the IFN- ⁇ alone administration group significantly reduced intracellular HCV R A levels (about 100-fold) from week 3 to week 8, demonstrating that IFN- ⁇ has pharmacological activity for inhibiting chronic HCV infection.
  • Hepatitis C virus infection and related drug research to prevent or treat infection with hepatitis C virus infection are limited and hindered by the lack of a small animal model of hepatitis C virus infection.
  • Tree ⁇ Xie, et al. (1998) Virology 244:513-520, Zhao, et al. (2002) J. Clin. Invest. 109:221-232
  • ⁇ Monkey/Tamarin Fdnstone, et al. (1982) J. Infect. Dis.l44: 588-598
  • this method transplants human primary hepatocytes into lethal defects with endogenous hepatocytes In the liver of immunodeficient mice. As the mouse endogenous hepatocytes die, the transplanted human primary hepatocytes can refill the mouse liver, causing the mice to form a chimeric liver, allowing HCV infection.
  • mice with triple mutants including Fag-/- / RAG2-/- /I12rg-/- mice
  • mice were Proven to accept hepatic heterogeneous implantation (Azuma, et al (2007) Nat. Immunol Biotechnol.: 25:903-910; Schultz, et al. (2007) Nat. Rev, Imm nol. 7: 118 - 130)
  • SCID immunodeficiency
  • uPA urokinase-type plasminogen activator
  • mice were given the following two modes of administration prior to infection with hepatitis C virus: 1) mice were orally administered 10 mg/kg/day of compound (1-1 Z) one day prior to infection, 2) mice were The compound (1-1 Z) was orally administered at 1 mg/kg/day, 3 mg/kg/day for 7 days before infection. Mice were injected with any HCV genotype HCV-positive human serum, and serum HCV RNA levels (daily and/or weekly) were quantified to assess the effect of the drug in reducing HCV R A levels.
  • mice were infected with HCV-positive human serum (any HCV genotype). Regular monitoring of serum HCV RA confirms successful infection and establishes a stable model of chronic infection in vivo.
  • Chronic hepatitis C virus infection in mice were administered the following parallel processes (1) Type 1 interferons (e.g., subcutaneous injection PEGASYS 30 ⁇ ⁇ /] ⁇ 3 ⁇ 4, twice a week), (2) administration of compound ( 1-1 ⁇ ) (single use), dose 10 mg kg / day (3) type 1 interference (for example, PEGASYS (3 ( ⁇ g / kg, twice a week) and compound (1-1 Z) ( 10mg/kg/day) joint Medication.
  • Type 1 interferons e.g., subcutaneous injection PEGASYS 30 ⁇ ⁇ /] ⁇ 3 ⁇ 4, twice a week
  • type 1 interference for example, PEGASYS (3 ( ⁇ g / kg, twice a week) and compound (1-1 Z) ( 10mg/kg/day) joint Medication.
  • Serum HCV RNA was quantified weekly during treatment to assess the efficacy of each group's dosing regimen for reduced steady-state HCV RNA levels.
  • the results of the experiment showed that the compound (1-1 Z) alone or in combination with IFN can reduce the level of HCV in vivo, suggesting that the compound (1-1 Z) can effectively treat patients infected with HCV.
  • a combination of type I interferon and compound (1-1 Z) will synergistically enhance clearance of HCV infection as compared to type I interferon or compound (1-1 Z) alone.
  • the present invention is directed to the compound (I-IZ) on the well-developed hepatitis C susceptible model strain, Tupaia belangeri (Li et al. 201i). A pharmacodynamic study was conducted.
  • Preventive experiment 15 experimental trees, 5 in each group, were unadministered group, lm/kg group, I- 1Z 5m/kg group.
  • the unadministered group was orally administered with normal saline, and the administration group was administered once a day (administered by oral administration) for 2 weeks, and the tree was infected two weeks later.
  • Blood was taken from the second week to the sixth week after infection to determine HCV R A. The results showed that compared with the unadministered animals, at the 4th week, the content of HCV RNA in the I-1Z lm/kg group and the 5 tree shrews was lower than the detection limit, and only one in the I-1Z 5m/kg group.
  • Simultaneous administration test 10 experimental trees, 5 in each group, were administered simultaneously at the time of HCV infection, the normal administration group was orally administered daily, and the administration group was administered once a day (administration was oral administration) , I-1Z 3mg/kg, continuous administration for 2 weeks.
  • administration was oral administration
  • I-1Z 3mg/kg
  • blood was drawn weekly for 4 weeks after infection.
  • 60% of the tree's HCV RNA content was below the detection limit compared to the unadministered animals, indicating that the compound (I- 1Z) can effectively prevent or treat HCV-infected infections (Fig. 7), and the results suggest that compound (I-1Z) can also effectively inhibit HCV infection when administered simultaneously.

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Abstract

La présente invention concerne un composé azétidinone tel que représenté par la formule (I) qui peut être utilisé dans la prévention et/ou le traitement de l'hépatite C, une composition pharmaceutique de ce composé et un procédé d'utilisation de celui-ci dans la prévention et/ou le traitement de l'infection par le virus de l'hépatite C.
PCT/CN2014/072736 2013-03-01 2014-02-28 Composé azétidinone utilisé dans la prévention et/ou le traitement de l'hépatite c, et composition pharmaceutique de ce composé WO2014131371A1 (fr)

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WO2015188727A1 (fr) * 2014-06-09 2015-12-17 浙江海正药业股份有限公司 Procédé de préparation d'un composé d'azétidinone et intermédiaire d'un composé d'azétidinone

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WO2015188727A1 (fr) * 2014-06-09 2015-12-17 浙江海正药业股份有限公司 Procédé de préparation d'un composé d'azétidinone et intermédiaire d'un composé d'azétidinone
US9926268B2 (en) 2014-06-09 2018-03-27 Zhejiang Hisun Pharmaceutical Co., Ltd. Method for preparing azetidinone compound and intermediate of azetidinone compound
US10364219B2 (en) 2014-06-09 2019-07-30 Zhejiang Hisun Pharmaceutical Co., Ltd. Method for preparing azetidinone compound and intermediate of azetidinone compound

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