WO2017206688A1 - Substituted pyrrolidine compound, pharmaceutical composition, and applications thereof - Google Patents

Substituted pyrrolidine compound, pharmaceutical composition, and applications thereof Download PDF

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Publication number
WO2017206688A1
WO2017206688A1 PCT/CN2017/083882 CN2017083882W WO2017206688A1 WO 2017206688 A1 WO2017206688 A1 WO 2017206688A1 CN 2017083882 W CN2017083882 W CN 2017083882W WO 2017206688 A1 WO2017206688 A1 WO 2017206688A1
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Prior art keywords
compound
hepatitis
virus
hydrogen
hcv
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PCT/CN2017/083882
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French (fr)
Chinese (zh)
Inventor
王义汉
赵九洋
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深圳市塔吉瑞生物医药有限公司
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Priority to CN201780004349.4A priority Critical patent/CN108368042A/en
Publication of WO2017206688A1 publication Critical patent/WO2017206688A1/en

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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • 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
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals

Definitions

  • the invention belongs to the technical field of medicine, and in particular relates to a hepatitis C virus inhibitor, a pharmaceutical composition and application thereof.
  • HCV Hepatitis C Virus
  • the encapsulated HCV virion contains a positive-stranded RNA genome that encodes all known virus-specific proteins in a single uninterrupted open reading frame.
  • the open reading frame comprises approximately 9500 nucleotides and encodes a single large polyprotein of approximately 3000 amino acids.
  • Polyproteins include core proteins, envelope proteins E1 and E2, membrane-bound protein P7, and non-structural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B.
  • the therapeutic drugs for HCV were interferon and ribavirin. These two drugs, by modulating the immune system or opening various antiviral genes in the cell, create an environment that is not conducive to viral replication, actually targeting host cells. These drugs have many side effects because they act on every cell in the body. New direct-acting antiviral drugs can directly target viruses, which has great advantages.
  • protease inhibitors, NS5A inhibitors and NS5B polymerase inhibitors are the three major classes of anti-HCV drugs under investigation. There were two HCV drugs approved by the US FDA in 2014, namely Jelly's Harvoni and Abbott's Viekira Pak, all of which are compound preparations.
  • Aibowei's Viekira Pak is mainly composed of ombitasvir, paritaprevir and dasabuvir, and can be used for the treatment of patients with chronic genotype 1 (GT1) hepatitis C virus infection, and also for HCV/HIV-1 co-infection (hepatitis C virus combined with humans).
  • GT1 chronic genotype 1
  • HCV/HIV-1 co-infection hepatitis C virus combined with humans.
  • Immunodeficiency virus type 1 patients and liver transplant patients.
  • Ombitasvir is an indispensable NS5A polymerase inhibitor in this combination.
  • sofosbuvir is currently the first medicine in the world that can completely cure hepatitis C in the short term. It takes the oral route directly to the lesion, and the method has simple side effects and is highly sought after by patients. Sofibuvir is produced by Gilead, USA, and is marketed in the United States in 2013. It has been clinically proven to be effective in treating hepatitis C, type 1, 2, 3, and 4, including liver transplantation, liver cancer, and HCV/HIV-1 co-infection. Clinical Trials. This breakthrough is for patients with hepatitis C worldwide Come the gospel. The US Gilead company has extended the authorization of the new hepatitis C drug Sofibuvir to Egypt last year.
  • Egypt is the country with the highest incidence of hepatitis C in the world. Subsequently agreed to produce sofibru generic drugs in India. Up to now, a total of eight Indian pharmaceutical companies have obtained Gilead's authorization to sell Sophie Buwei to 91 developing countries around the world (China is excluded). Why Sophie Buwei is currently in a blank space in China. On the one hand, there is a large demand, and on the other hand, it is drug-free. In the face of such awkward dilemma, some large hospitals are trying to solve this problem through video remote overseas medical treatment. Some patients who can't wait, have to consider going abroad to see a doctor.
  • the present invention discloses a substituted pyrrolidine compound and a pharmaceutical composition thereof as a hepatitis C virus inhibitor which has better hepatitis C virus protein NS5A inhibitory activity and/or has better pharmacodynamics/ Pharmacokinetic properties.
  • a hepatitis C virus inhibitor such as a pyrrolidine compound represented by the formula (1), or a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotope variant, a hydrate or Solvent compound,
  • R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 , R 58 , R 59 , R 60 , R 61 , R 62 and R 63 are each independently hydrogen and hydrazine. , halogen or trifluoromethyl;
  • the shape and volume of the ruthenium in the drug molecule are substantially the same as those of the hydrogen. If the hydrogen in the drug molecule is selectively replaced with hydrazine, the deuterated drug generally retains the original biological activity and selectivity. At the same time, the inventors have confirmed through experiments that the binding of carbon-germanium bonds is more stable than the combination of carbon-hydrogen bonds, which can directly affect the absorption, distribution, metabolism and excretion of some drugs, thereby improving the efficacy, safety and tolerability of the drugs.
  • the strontium isotope content of the cerium in the deuterated position is at least greater than the natural strontium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, and even more preferably greater than 95. %, more preferably greater than 99%.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently hydrazine or hydrogen.
  • R 10 , R 11 , R 12 and R 13 are each independently hydrazine or hydrogen.
  • R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 and R 21 are each independently hydrazine or hydrogen.
  • R 22 , R 23 , R 24 , R 25 , R 26 and R 27 are each independently hydrazine or hydrogen.
  • R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 and R 41 are each independently hydrazine or hydrogen.
  • R 42 , R 43 , R 44 , R 45 , R 46 , R 47 , R 48 , R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 and R 57 are each independently hydrazine or hydrogen.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are ⁇ .
  • R 11 , R 12 and R 13 are deuterium.
  • R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 and R 21 are ⁇ .
  • R 22 , R 23 , R 24 , R 25 , R 26 , and R 27 are ⁇ .
  • R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 is ⁇ .
  • R 42 , R 43 , R 44 , R 45 , R 46 , R 47 , R 48 , R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 is ⁇ .
  • the compound is selected from the group consisting of the following compounds or a pharmaceutically acceptable salt thereof:
  • the compound does not include a non-deuterated compound.
  • the compound does not include a compound in which R 58 , R 59 , R 60 , R 61 , R 62 , and R 63 are all ruthenium, and the other R is not ruthenium.
  • the present invention also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the hepatitis C virus inhibitor as described above, or a crystalline form, a pharmaceutically acceptable salt, a hydrate or a solvent thereof
  • a pharmaceutical composition of a compound, stereoisomer, prodrug or isotopic variation comprising a pharmaceutically acceptable carrier and the hepatitis C virus inhibitor as described above, or a crystalline form, a pharmaceutically acceptable salt, a hydrate or a solvent thereof.
  • the pharmaceutically acceptable carrier includes a glidant, a sweetener, a diluent, a preservative, a dye/colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersant At least one of a disintegrant, a suspending agent, a stabilizer, an isotonic agent, a solvent or an emulsifier.
  • the pharmaceutical composition is a tablet, a pill, a capsule, a powder, a granule, an ointment, an emulsion, a suspension, a solution, a suppository, an injection, an inhalant, a gel, a microsphere or Aerosol.
  • Typical routes of administration of the pharmaceutical compositions of the invention include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal , intramuscular, subcutaneous, intravenous administration. Oral administration or injection administration is preferred.
  • the pharmaceutical composition of the present invention can be produced by a method known in the art, such as a conventional mixing method, a dissolution method, a granulation method, a sugar-coating method, a pulverization method, an emulsification method, a freeze-drying method, and the like.
  • it further comprises other active compounds which are immunomodulatory or antiviral pharmaceutical compounds.
  • the immunomodulator is an interferon drug compound.
  • the antiviral drug compound is ribavirin, amantadine, other inhibitors of NS5A, helicase in the HCV life cycle, protease, polymerase, metalloproteinase or internal ribosome entry.
  • the present invention provides a method of inhibiting HCV replicon activity, inhibiting HCV viral replication and/or HCV viral production, treating HCV infection, and/or reducing the likelihood or severity of HCV infection, using a compound of the invention in combination with a Or a plurality of therapeutic agents and pharmaceutical compositions comprising a compound of the invention and one or more therapeutic agents selected from the group consisting of HCV antiviral agents, immunomodulators, and anti-infective agents.
  • Such therapeutic agents act against HCV, including, but not limited to, ribavirin, levovirin, verapamil, thymosin alpha-1, R7025 (enhanced interferon, Roche), interferon-beta, interference Combination of p- ⁇ , pegylated interferon- ⁇ (peginterferon- ⁇ ), interferon- ⁇ and ribavirin, peginterferon- ⁇ and ribavirin , a combination of interferon- ⁇ and levovirin, and a combination of peginterferon- ⁇ and levovirin.
  • Ribavirin, levovirin, and verapamil can exert their anti-HCV effects by modulating the intracellular pool of guanine nucleotides via inhibition of the intracellular enzyme inosine monophosphate dehydrogenase (IMPDH).
  • IMPDH is the rate-limiting enzyme on the biosynthetic pathway in the nucleotide biosynthesis of the guanine.
  • Ribavirin is susceptible to intracellular phosphorylation and the monophosphate derivative is an inhibitor of IMPDH.
  • inhibition of IMPDH represents another for the development of HCV replication inhibitors.
  • the compounds of the invention may also be administered in combination with inhibitors of IMPDH, as disclosed in WO 97/41211, WO 01/00622 and WO 00/25780.
  • the compounds of the invention may also be administered in combination with an agent that is an HCV NS3 serine protease inhibitor.
  • HCV NS3 serine protease is the major viral enzyme and has been described as an excellent target for HCV replication inhibition.
  • Exemplary substrate-based and non-substrate-based inhibitors of HCV NS3 protease inhibitors are disclosed in the following: International Patent Application Publication No.
  • WO 98/22496 WO 98/46630, WO 99/07733, WO 99/07734, WO 99/38888, WO 99/50230, WO 99/64442, WO 00/09543, WO 00/59929, WO 02/48116, WO 02/48172, WO 2008/057208 and WO 2008/057209, British Patent No. GB 2 337 262 and U.S. Patent Nos. 6,323,180, 7,470,664 and 7,012,066.
  • the compounds of the invention may also be used in combination with nucleosides having anti-HCV properties for the treatment of HCV infection, such as disclosed in the following patents: International Patent Application Publication No. WO 02/51425, WO 01/79246, WO 02/32920, WO 02 /48165 and WO 2005/003147; WO 01/68663, WO 99/43691, WO 02/18404, and WO 2006/021341, and US Patent Application Publication No. US 2005/0038240, US 2002/0019363, US 2003/0236216, US 2004 /0006007, US 2004/0063658 and US 2004/0110717; US Patent Nos. 7,105,499, 7,125,855, 7,202,224; and International Patent Application Publication No. WO 02/100415, WO 03/026589, WO 03/026675, WO 03/093290, WO 04/ 011478, WO 04/013300 and WO 04/028481.
  • the compounds of the invention may also be administered in combination with an agent that is an inhibitor of HCV NS5B polymerase.
  • HCV NS5B polymerase inhibitors that can be used as a combination therapy include, but are not limited to, those disclosed in the following: International Patent Application Publication No. WO 02/057287, WO 02/057425, WO 03/068244, WO 2004/000858, WO 04 / 003 138 and WO 2004/007512; U.S. Patent Nos. 6,777,392, 7,105,499, 7,125,855, 7,202,224, and U.S. Patent Application Publication Nos. 2004/0067901 and US 2004/0110717.
  • the present invention also provides a method of preparing a pharmaceutical composition comprising the steps of: administering a pharmaceutically acceptable carrier to a hepatitis C virus inhibitor as described above, or a crystalline form thereof, a pharmaceutically acceptable salt, or a hydrate thereof Or the solvate is mixed to form a pharmaceutical composition.
  • the invention also discloses the use of a hepatitis C virus inhibitor as described above, characterized in that it is used for the preparation of a medicament for the treatment of hepatitis C virus infection.
  • the HCV includes a plurality of genotypes thereof and a plurality of gene subtypes, such as 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a, 6a.
  • halogen means F, Cl, Br, and I unless otherwise specified. More preferably, the halogen atom is selected from the group consisting of F, Cl and Br.
  • deuterated means that one or more hydrogens in the compound or group are replaced by deuterium; deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted.
  • deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted.
  • deuterated is used interchangeably with “one or more deuterated”.
  • non-deuterated compound means a compound containing a proportion of germanium atoms not higher than the natural helium isotope content (0.015%).
  • the invention also includes isotopically labeled compounds (also referred to as "isotopic variants"), equivalent to the original compounds disclosed herein.
  • isotopes which may be listed as compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine isotopes such as 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, respectively. , 31 P, 32 P, 35 S, 18 F and 36 Cl.
  • isotopically-labeled compounds of the present invention such as the radioisotopes of 3 H and 14 C, are also among them, useful in tissue distribution experiments of drugs and substrates. ⁇ , ie 3 H and carbon-14, ie 14 C, are easier to prepare and detect and are preferred in isotopes. In addition, heavier isotopic substitutions such as guanidine, or 2 H, are preferred in certain therapies due to their good metabolic stability, such as increased half-life or reduced dosage in vivo, and therefore may be preferred in certain circumstances. Isotopically labeled compounds can be prepared in a conventional manner by substituting a readily available isotopically labeled reagent with a non-isotopic reagent using the protocol of the examples.
  • Pharmaceutically acceptable salts include inorganic and organic salts.
  • a preferred class of salts are the salts of the compounds of the invention with acids.
  • Suitable acids for forming salts include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid; formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, Organic acids such as fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid; Amino acids such as amino acid, phenylalanine, aspartic acid, and glutamic acid.
  • salts of the compounds of the invention with bases such as alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example magnesium or calcium salts), ammonium salts (for example lower alkanolammonium).
  • bases such as alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example magnesium or calcium salts), ammonium salts (for example lower alkanolammonium).
  • Salts and other pharmaceutically acceptable amine salts such as methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tert-butyl a base amine salt, an ethylenediamine salt, a hydroxyethylamine salt, a dihydroxyethylamine salt, a trishydroxyethylamine salt, and a morpholine, piperazine, and lysine, respectively.
  • Amine salt such as methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tert-butyl a base amine salt, an ethylenediamine salt, a hydroxyethylamine salt, a dihydroxyethylamine salt, a trishydroxyethylamine salt, and a morpholine, piperazine, and lysine, respectively.
  • Amine salt such as
  • polymorph refers to a different arrangement of chemical drug molecules, generally expressed as the presence of a pharmaceutical material in a solid state.
  • a drug may exist in a plurality of crystalline forms, and different crystal forms of the same drug may have different dissolution and absorption in the body, thereby affecting the dissolution and release of the formulation.
  • solvate refers to a complex of a compound of the invention that is coordinated to a solvent molecule to form a specific ratio.
  • Hydrophilate means a complex formed by the coordination of a compound of the invention with water.
  • prodrug refers to a compound that is converted in vivo to an active form having its medical effect by, for example, hydrolysis in blood.
  • a prodrug is any covalently bonded carrier which, when administered to a patient, releases the compound of the invention in vivo.
  • Prodrugs are typically prepared by modifying functional groups that cleave the prodrug in vivo to yield the parent compound.
  • Prodrugs include, for example, a compound of the invention wherein a hydroxy, amino or thiol group is bonded to any group which, when administered to a patient, can be cleaved to form a hydroxy, amino or thiol group.
  • prodrugs include, but are not limited to, covalent derivatives of the compounds of the invention formed by the hydroxyl, amino or thiol functional groups thereof with acetic acid, formic acid or benzoic acid.
  • acetic acid formic acid or benzoic acid.
  • an ester such as a methyl ester, an ethyl ester or the like can be used.
  • the ester itself may be active and/or may hydrolyze under conditions in humans.
  • Suitable pharmaceutically acceptable in vivo hydrolysable esters include those which readily decompose in the human body to release the parent acid or its salt.
  • the compounds of the invention may include one or more asymmetric centers, and thus may exist in a variety of "stereoisomer" forms, for example, enantiomeric and/or diastereomeric forms.
  • the compounds of the invention may be in the form of individual enantiomers, diastereomers or geometric isomers (e.g., cis and trans isomers), or may be in the form of a mixture of stereoisomers, A racemic mixture and a mixture rich in one or more stereoisomers are included.
  • the isomers can be separated from the mixture by methods known to those skilled in the art, including: chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of a chiral salt; or preferred isomers can be passed Prepared by asymmetric synthesis.
  • HPLC high pressure liquid chromatography
  • the beneficial effects of the present invention compared to the prior art are: First, the compound of the present invention has excellent inhibitory effect on the hepatitis C virus protein NS5A. Second, by deuteration this technique changes the metabolism of the compound in the organism, giving the compound better pharmacokinetic parameter characteristics. In this case, the dosage can be changed and a long-acting preparation can be formed to improve the applicability. Third, replacing the hydrogen atom in the compound with hydrazine increases the drug concentration of the compound in the animal due to its strontium isotope effect, thereby improving the drug efficacy. Fourth, replacing the hydrogen atom in the compound with hydrazine can inhibit certain metabolites and improve the safety of the compound.
  • the preparation method of the structural compound of the formula (1) of the present invention is more specifically described below, but these specific methods are not The invention constitutes any limitation.
  • the compounds of the present invention may also be conveniently prepared by combining various synthetic methods described in the specification or known in the art, and such combinations are readily made by those skilled in the art to which the present invention pertains.
  • N-methoxycarbonyl-L-proline (1.75 g, 10 mmol) and N-hydroxysuccinimide (1.21 g, 10.5 mmol) were added to the reaction flask, dissolved in 22 mL of ethyl acetate, and cooled to 0- At 5 ° C, diisopropylcarbodiimide (1.26 g, 10 mmol) was added, and the reaction was stirred for 1 hour. After the TLC reaction was completed, the mixture was allowed to warm to room temperature, and the by-product was removed by filtration and concentrated to give the crude compound 7 which was directly purified without purification. Go to the next step.
  • Step 9 Dimethyl(2S,2'S)-1,1'-((2S,2'S)-2,2'-(4,4'-((2S,5S)-1-(4-tert-butyl) -2,6-d2-phenyl)pyrrolidine-2,5-diyl)bis(4,1-phenyl))bis(azanediyl)bis(oxomethyl)bis(pyrrolidine-2 Synthesis of 1,1-diyl)bis(3-methyl-1-oxobutane-2,1-diyl)dicarboxylate (Compound O-1).
  • the inventors used the HCV Replicon System as an evaluation model. Since its first report in Science in 1999, the HCV replication system has become one of the most important tools for studying HCV RNA replication, pathogenicity and viral persistence, for example, the use of replicons has successfully demonstrated the 5' required for HCV RNA replication. - NCR minimum region, and the HCV replication subsystem has been successfully used as an evaluation model for antiviral drugs. The inventors of the present invention verified according to the methods described in Science, 1999, 285 (5424), 110-3, and J. Virol, 2003, 77(5), 3007-19.
  • the inhibitory activities of the recombinant hepatitis C virus genotype 1a and 1b replicons were detected by stable transfection of replicon cells with HCV-1a and HCV-1b. This experiment will use the NS5A inhibitor Ombitasvir as a positive control compound.
  • Step 1 The compound was diluted 1:3 in 8 series points, double-replicated, and added to a 96-well plate.
  • the DMSO was set to no compound control.
  • the final concentration of DMSO in the cell culture was 0.5%.
  • Step 2 HCV-1a and 1b cells were separately suspended in a culture medium containing 10% FBS, and seeded into a 96-well plate containing the compound at a density of 8,000 cells per well. The cells were cultured for 3 days at 5% CO 2 at 37 °C.
  • Step 3 The cytotoxicity of the compound against GT1b replicon was determined using CellTiter-Fluor (Promega).
  • Step 4 Detection of luciferase assay by Bright-Glo (Promega) for anti-hepatitis C virus activity.
  • Step Five using GraphPad Prism data analysis software, the curve fitting and EC 50 values were calculated and the 50 value CC.
  • Example 1 The compound O-1 of Example 1 and the control Ombitasvir were analyzed according to the above procedure to calculate the EC 50 value and the CC 50 value, and the results are shown in Table 1.
  • This infection model is referred to as A novel luciferase and GFP dual reporter virus for rapid and convenient evaluation of HCV replication.
  • Example 1 The compound O-1 of Example 1 and the control Ombitasvir were analyzed according to the above procedure to calculate EC 50 value, and the results are shown in Table 1.
  • the experimental results show that the compound of the present invention can inhibit multiple genotypes of HCV and exert superior anti-HCV effects by inhibiting the mechanism of HCV NS5A protein.
  • Microsomal experiments human liver microsomes: 0.5 mg/mL, Xenotech; rat liver microsomes: 0.5 mg/mL, Xenotech; coenzyme (NADPH/NADH): 1 mM, Sigma Life Science; magnesium chloride: 5 mM, 100 mM phosphate buffer Agent (pH 7.4).
  • phosphate buffer 100 mM, pH 7.4.
  • the pH was adjusted to 7.4, diluted 5 times with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100 mM) containing 100 mM potassium phosphate, 3.3 mM magnesium chloride, and a pH of 7.4.
  • NADPH regeneration system containing 6.5 mM NADP, 16.5 mM G-6-P, 3 U/mL G-6-P D, 3.3 mM magnesium chloride, placed on wet ice before use.
  • Formulation stop solution acetonitrile solution containing 50 ng/mL propranolol hydrochloride and 200 ng/mL tolbutamide (internal standard). Take 25057.5 ⁇ L of phosphate buffer (pH 7.4) into a 50 mL centrifuge tube, add 812.5 ⁇ L of human liver microsomes, and mix to obtain a liver microsome dilution with a protein concentration of 0.625 mg/mL. 25057.5 ⁇ L of phosphate buffer (pH 7.4) was taken into a 50 mL centrifuge tube, and 812.5 ⁇ L of SD rat liver microsomes were added and mixed to obtain a liver microsome dilution having a protein concentration of 0.625 mg/mL.
  • the corresponding compound had a reaction concentration of 1 ⁇ M and a protein concentration of 0.5 mg/mL.
  • 100 ⁇ L of the reaction solution was taken at 10, 30, and 90 min, respectively, and added to the stopper, and the reaction was terminated by vortexing for 3 min.
  • the plate was centrifuged at 5000 x g for 10 min at 4 °C.
  • 100 ⁇ L of the supernatant was taken into a 96-well plate to which 100 ⁇ L of distilled water was previously added, mixed, and sample analysis was performed by LC-MS/MS.
  • OBJECTIVE To investigate the pharmacokinetic behavior of the compounds of the present invention after administration of Ombitasvir and Compound O-1 to rats.
  • SD rat grade SPF grade
  • Weight range 180 ⁇ 220g (actual weight range is 187 ⁇ 197g)
  • the experimental results show that the present inventors have found that the compound of the present invention has superior activity to Ombitasvir and has excellent pharmacokinetic properties as compared with Ombitasvir, and thus is more suitable as a compound for inhibiting the hepatitis C virus protein NS5A, and is suitable for use. Preparation of a medicament for treating hepatitis C virus infection.

Abstract

The present invention provides a substituted pyrrolidine compound, a pharmaceutical composition, and applications thereof. The pyrrolidine compound is a compound represented by formula (1), or a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotope variant, a hydrate or a solvent compound thereof. The compound in the present invention can be used as a hepatitis C virus inhibitor, has better hepatitis C virus protein NS5A inhibitory activity and better pharmacokinetics/pharmacokinetics performance, and can be used in the preparation of drugs for treating hepatitis C virus infection.

Description

一种取代的吡咯烷化合物及药物组合物及其应用Substituted pyrrolidine compound and pharmaceutical composition and application thereof 技术领域Technical field
本发明属于医药技术领域，尤其涉及一种丙型肝炎病毒抑制剂、药物组合物及其应用。The invention belongs to the technical field of medicine, and in particular relates to a hepatitis C virus inhibitor, a pharmaceutical composition and application thereof.
背景技术Background technique
HCV(Hepatitis C Virus，丙型肝炎病毒)是一种RNA病毒，其属于黄病毒科(Flaviviridae family)中的丙型肝炎病毒属(Hepacivirus genus)。包裹HCV病毒粒子包含正股RNA基因组，其在单个不间断的开放读码框中编码全部已知的病毒—特异的蛋白质。开放读码框包括大约9500个核苷酸并且编码单个约3000个氨基酸的巨大多蛋白。多蛋白包括芯蛋白，包裹蛋白E1和E2，膜结合蛋白P7，和非结构性蛋白NS2、NS3、NS4A、NS4B、NS5A和NS5B。HCV (Hepatitis C Virus) is an RNA virus belonging to the genus Hepacivirus genus in the Flaviviridae family. The encapsulated HCV virion contains a positive-stranded RNA genome that encodes all known virus-specific proteins in a single uninterrupted open reading frame. The open reading frame comprises approximately 9500 nucleotides and encodes a single large polyprotein of approximately 3000 amino acids. Polyproteins include core proteins, envelope proteins E1 and E2, membrane-bound protein P7, and non-structural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B.
近年来，丙肝的发病人数呈逐年上升趋势，2006年－2010年分别为70681例、92378例、108446例、131849例和163174例。In recent years, the number of hepatitis C patients has increased year by year. From 2006 to 2010, there were 70,681 cases, 92,378 cases, 108,446 cases, 131,849 cases and 163,174 cases.
2011年以前，HCV的治疗药物为干扰素和利巴韦林。这两种药物，通过调节免疫***或开启细胞内的各种抗病毒基因，建立了一个不利于病毒复制的环境，实际上是针对宿主细胞。由于药物作用于体内的每一个细胞，因此这些药物有许多副作用。新的直接作用抗病毒药物可直接靶向病毒，这一点有很大的优势。在直接作用的抗病毒药物中，蛋白酶抑制剂，NS5A抑制剂和NS5B聚合酶抑制剂是正在研究的抗HCV三大类药物。2014年获美国FDA批准的HCV药物有两个，分别是吉利德的Harvoni和艾伯维的Viekira Pak，均为复方制剂。Prior to 2011, the therapeutic drugs for HCV were interferon and ribavirin. These two drugs, by modulating the immune system or opening various antiviral genes in the cell, create an environment that is not conducive to viral replication, actually targeting host cells. These drugs have many side effects because they act on every cell in the body. New direct-acting antiviral drugs can directly target viruses, which has great advantages. Among the direct acting antiviral drugs, protease inhibitors, NS5A inhibitors and NS5B polymerase inhibitors are the three major classes of anti-HCV drugs under investigation. There were two HCV drugs approved by the US FDA in 2014, namely Jelly's Harvoni and Abbott's Viekira Pak, all of which are compound preparations.
其中，艾伯维的Viekira Pak主要由ombitasvir，paritaprevir和dasabuvir组成，可以用于治疗慢性基因1型(GT1)丙型肝炎病毒感染患者，也可用于HCV/HIV-1合并感染(丙肝病毒合并人类免疫缺陷病毒1型)患者和肝移植患者。Ombitasvir是该复方制剂中不可缺少的NS5A聚合酶抑制剂。Among them, Aibowei's Viekira Pak is mainly composed of ombitasvir, paritaprevir and dasabuvir, and can be used for the treatment of patients with chronic genotype 1 (GT1) hepatitis C virus infection, and also for HCV/HIV-1 co-infection (hepatitis C virus combined with humans). Immunodeficiency virus type 1) patients and liver transplant patients. Ombitasvir is an indispensable NS5A polymerase inhibitor in this combination.
此外，索非布韦是目前世界上第一个在短期内可以彻底治愈丙肝的良药。它以口服途径直达病灶，方法简单副作用很小，深受患者的追捧。索非布韦由美国吉利德公司生产，2013年在美国上市，经临床试验证实可有效治疗基因1、2、3、4型丙肝，包括对肝移植、肝癌以及HCV/HIV-1合并感染的临床试验。这一突破为全世界的丙肝患者带 来了福音。美国吉利德公司去年已经将丙肝新药索非布韦的授权扩大至埃及，埃及是全球丙肝发病率最高的国家。随后同意印度生产索菲布韦仿制药。截至目前，共有8家印度制药公司拿到了吉利德的授权，可以将索菲布韦销往全球91个发展中国家(中国被排除在外)。索非布韦目前在我国处于空白，一边是大量的需求，一边是无药上市，面对如此尴尬的窘境，一些大医院正在尝试通过视频远程海外医疗解决这一难题。一些等不及的患者，不得不考虑出国看病。In addition, sofosbuvir is currently the first medicine in the world that can completely cure hepatitis C in the short term. It takes the oral route directly to the lesion, and the method has simple side effects and is highly sought after by patients. Sofibuvir is produced by Gilead, USA, and is marketed in the United States in 2013. It has been clinically proven to be effective in treating hepatitis C, type 1, 2, 3, and 4, including liver transplantation, liver cancer, and HCV/HIV-1 co-infection. Clinical Trials. This breakthrough is for patients with hepatitis C worldwide Come the gospel. The US Gilead company has extended the authorization of the new hepatitis C drug Sofibuvir to Egypt last year. Egypt is the country with the highest incidence of hepatitis C in the world. Subsequently agreed to produce sofibru generic drugs in India. Up to now, a total of eight Indian pharmaceutical companies have obtained Gilead's authorization to sell Sophie Buwei to 91 developing countries around the world (China is excluded). Sophie Buwei is currently in a blank space in China. On the one hand, there is a large demand, and on the other hand, it is drug-free. In the face of such awkward dilemma, some large hospitals are trying to solve this problem through video remote overseas medical treatment. Some patients who can't wait, have to consider going abroad to see a doctor.
因此，本领域仍需要开发对丙型肝炎病毒蛋白NS5A有抑制活性或更好药效学性能的化合物。Therefore, there is still a need in the art to develop compounds having inhibitory activity or better pharmacodynamic properties against the hepatitis C virus protein NS5A.
发明内容Summary of the invention
针对以上技术问题，本发明公开了一种取代的吡咯烷化合物及其药物组合物作为丙型肝炎病毒抑制剂，其具有更好的丙肝病毒蛋白NS5A抑制活性和/或具有更好药效学/药代动力学性能。In view of the above technical problems, the present invention discloses a substituted pyrrolidine compound and a pharmaceutical composition thereof as a hepatitis C virus inhibitor which has better hepatitis C virus protein NS5A inhibitory activity and/or has better pharmacodynamics/ Pharmacokinetic properties.
对此，本发明采用的技术方案为：In this regard, the technical solution adopted by the present invention is:
一种丙型肝炎病毒抑制剂，如式(1)所示的吡咯烷化合物化合物，或其多晶型、药学上可接受的盐、前药、立体异构体、同位素变体、水合物或溶剂化合物，A hepatitis C virus inhibitor, such as a pyrrolidine compound represented by the formula (1), or a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotope variant, a hydrate or Solvent compound,
Figure PCTCN2017083882-appb-000001
Figure PCTCN2017083882-appb-000001
其中，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49、R50、R51、R52、R53、R54、R55、R56、R57、R58、R59、R60、R61、R62、 R63各自独立地为氢、氘、卤素或三氟甲基；Wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 And R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , R 42 , R 43 , R 44 , R 45 , R 46 , R 47 , R 48 , R 49. R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 , R 58 , R 59 , R 60 , R 61 , R 62 and R 63 are each independently hydrogen and hydrazine. , halogen or trifluoromethyl;
附加条件是R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49、R50、R51、R52、R53、R54、R55、R56、R57、R58、R59、R60、R61、R62和R63中至少一个是氘代的或氘。Additional conditions are R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 And R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , R 42 , R 43 , R 44 , R 45 , R 46 , R 47 , R 48 , At least one of R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 , R 58 , R 59 , R 60 , R 61 , R 62 and R 63 is a generation Or 氘.
采用此技术方案，氘在药物分子中的形状和体积与氢基本上相同，如果药物分子中氢被选择性替换为氘，氘代药物一般还会保留原来的生物活性和选择性。同时发明人经过实验证实，碳氘键的结合比碳氢键的结合更稳定，可直接影响一些药物的吸收、分布、代谢和***等属性，从而提高药物的疗效、安全性和耐受性。With this technical solution, the shape and volume of the ruthenium in the drug molecule are substantially the same as those of the hydrogen. If the hydrogen in the drug molecule is selectively replaced with hydrazine, the deuterated drug generally retains the original biological activity and selectivity. At the same time, the inventors have confirmed through experiments that the binding of carbon-germanium bonds is more stable than the combination of carbon-hydrogen bonds, which can directly affect the absorption, distribution, metabolism and excretion of some drugs, thereby improving the efficacy, safety and tolerability of the drugs.
优选的，氘在氘代位置的氘同位素含量至少是大于天然氘同位素含量(0.015％)，较佳地大于30％，更佳地大于50％，更佳地大于75％，更佳地大于95％，更佳地大于99％。Preferably, the strontium isotope content of the cerium in the deuterated position is at least greater than the natural strontium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, and even more preferably greater than 95. %, more preferably greater than 99%.
具体地说，在本发明中R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49、R50、R51、R52、R53、R54、R55、R56、R57、R58、R59、R60、R61、R62和R63中，各氘代位置中氘同位素含量至少是5％，较佳地大于10％，更佳地大于15％，更佳地大于20％，更佳地大于25％，更佳地大于30％，更佳地大于35％，更佳地大于40％，更佳地大于45％，更佳地大于50％，更佳地大于55％，更佳地大于60％，更佳地大于65％，更佳地大于70％，更佳地大于75％，更佳地大于80％，更佳地大于85％，更佳地大于90％，更佳地大于95％，更佳地大于99％。Specifically, in the present invention, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 and R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 And R 32 , R 33 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , R 42 , R 43 , R 44 , R 45 , R 46 , R 47 , R 48 , R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 , R 58 , R 59 , R 60 , R 61 , R 62 and R 63 The strontium isotope content in each metamorphic position is at least 5%, preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, more preferably greater than 25%, and even more preferably greater than 30%, more Preferably more than 35%, more preferably more than 40%, more preferably more than 45%, more preferably more than 50%, more preferably more than 55%, more preferably more than 60%, more preferably more than 65%, more preferably More than 70%, more preferably more than 75%, more preferably more than 80%, more preferably more than 85%, more preferably more than 90%, more preferably more than 95%, More preferably greater than 99%.
优选的，式(I)中化合物的R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49、R50、R51、R52、R53、R54、R55、R56、R57、R58、R59、R60、R61、R62和R63，至少其中一个R含氘，更佳地两个R含氘，更佳地三个R 含氘，更佳地四个R含氘，更佳地五个R含氘，更佳地六个R含氘，更佳地七个R含氘，更佳地八个R含氘，更佳地九个R含氘，更佳地十个R含氘，更佳地十一个R含氘，更佳地十二个R含氘，更佳地十三个R含氘，更佳地十四个R含氘，更佳地十五个R含氘，更佳地十六个R含氘，更佳地十七个R含氘，更佳地十八个R含氘，更佳地十九个R含氘，更佳地二十个R含氘，更佳地二十一个R含氘，更佳地二十二个R含氘，更佳地二十三个R含氘，更佳地二十四个R含氘，更佳地二十五个R含氘，更佳地二十六个R含氘，更佳地二十七个R含氘，更佳地二十八个R含氘，更佳地二十九个R含氘，更佳地三十个R含氘，更佳地三十一个R含氘，更佳地三十二个R含氘，更佳地三十三个R含氘，更佳地三十四个R含氘，更佳地三十五个R含氘，更佳地三十六个R含氘，更佳地三十七个R含氘，更佳地三十八个R含氘，更佳地三十九个R含氘，更佳地四十个R含氘，更佳地四十一个R含氘，更佳地四十二个R含氘，更佳地四十三个R含氘，更佳地四十四个R含氘，更佳地四十五个R含氘，更佳地四十六个R含氘，更佳地四十七个R含氘，更佳地四十八个R含氘，更佳地四十九个R含氘，更佳地五十个R含氘，更佳地五十一个R含氘，更佳地五十二个R含氘，更佳地五十三个R含氘，更佳地五十四个R含氘，更佳地五十五个R含氘，更佳地五十六个R含氘，更佳地五十七个R含氘，更佳地五十八个R含氘，更佳地五十九个R含氘，更佳地六十个R含氘，更佳地六十一个R含氘，更佳地六十二个R含氘，更佳地六十三个R含氘。Preferably, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R of the compound of formula (I) 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , R 42 , R 43 , R 44 , R 45 , R 46 , R 47 , R 48 , R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 , R 58 , R 59 , R 60 , R 61 , R 62 and R 63 , at least one of R contains ruthenium, more preferably two R contains ruthenium, more preferably three R contains ruthenium, more preferably four R contains ruthenium, more preferably five R contains ruthenium, more preferably six R contains 氘, more preferably seven R contains 氘, more preferably eight R contains 氘, more preferably nine R contains 氘, more preferably ten R contains 氘, more preferably eleven 氘, More preferably, twelve R 氘, more preferably thirteen R 氘, more preferably fourteen R 氘, more preferably fifteen R 氘, more preferably sixteen R 氘, More preferably, seventeen R-containing sputum, more preferably eighteen R contains 氘, more preferably 19 R contains 氘, more preferably 20 R contains 氘, more preferably twenty-one R contains 氘, more preferably twenty-two R contains 氘, more preferably Twenty-three R contains 氘, more preferably twenty-four R contains 氘, more preferably twenty-five R contains 氘, more preferably twenty-six R contains 氘, more preferably twenty-seven R contains氘, preferably twenty-eight R 氘, more preferably twenty-nine R 氘, more preferably thirty R 氘, more preferably thirty-one R 氘, better thirty The two Rs contain ruthenium, more preferably thirty-three R 氘, more preferably thirty-four R 氘, more preferably thirty-five R 氘, more preferably thirty-six R 氘, More preferably thirty-seven R contains 氘, more preferably thirty-eight R contains 氘, more preferably thirty-nine R contains 氘, more preferably forty R contains 氘, more preferably forty-one R contains 氘, more preferably forty-two R 氘, more preferably forty-three R 氘, more preferably forty-four R 氘, more preferably forty-five R 氘, better Forty-six R 氘, more preferably forty-seven R 氘, more preferably forty-eight R 氘, more preferably forty-nine R 氘, more preferably fifty R 氘, Better Eleven R contains 氘, more preferably fifty-two R contains 氘, more preferably fifty-three R contains 氘, more preferably fifty-four R contains 氘, more preferably fifty-five R contains 氘More preferably, fifty-six R contains 氘, more preferably fifty-seven R contains 氘, more preferably fifty-eight R contains 氘, more preferably fifty-nine R contains 氘, more preferably sixty R contains 氘, more preferably sixty-one R contains 氘, more preferably sixty-two R contains 氘, more preferably sixty-three R contains 氘.
作为本发明的进一步改进，R1、R2、R3、R4、R5、R6、R7、R8和R9各自独立地为氘或氢。As a further improvement of the present invention, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently hydrazine or hydrogen.
作为本发明的进一步改进，R10、R11、R12和R13各自独立地为氘或氢。As a further improvement of the present invention, R 10 , R 11 , R 12 and R 13 are each independently hydrazine or hydrogen.
作为本发明的进一步改进，R14、R15、R16、R17、R18、R19、R20和R21各自独立地为氘或氢。As a further improvement of the present invention, R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 and R 21 are each independently hydrazine or hydrogen.
作为本发明的进一步改进，R22、R23、R24、R25、R26和R27各自独立地为氘或氢。As a further improvement of the present invention, R 22 , R 23 , R 24 , R 25 , R 26 and R 27 are each independently hydrazine or hydrogen.
作为本发明的进一步改进，R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40和R41各自独立地为氘或氢。As a further improvement of the present invention, R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 and R 41 are each independently hydrazine or hydrogen.
作为本发明的进一步改进，R42、R43、R44、R45、R46、R47、R48、R49、R50、R51、R52、R53、R54、R55、R56和R57各自独立地为氘或氢。As a further improvement of the present invention, R 42 , R 43 , R 44 , R 45 , R 46 , R 47 , R 48 , R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 and R 57 are each independently hydrazine or hydrogen.
在另一优选例中，R1、R2、R3、R4、R5、R6、R7、R8、R9是氘。 In another preferred embodiment, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are 氘.
在另一优选例中，R11、R12、R13是氘。In another preferred embodiment, R 11 , R 12 and R 13 are deuterium.
在另一优选例中，R14、R15、R16、R17、R18、R19、R20、R21是氘。In another preferred embodiment, R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 and R 21 are 氘.
在另一优选例中，R22、R23、R24、R25、R26、R27是氘。In another preferred embodiment, R 22 , R 23 , R 24 , R 25 , R 26 , and R 27 are 氘.
在另一优选例中，R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41是氘。In another preferred embodiment, R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 is 氘.
在另一优选例中，R42、R43、R44、R45、R46、R47、R48、R49、R50、R51、R52、R53、R54、R55、R56、R57是氘。In another preferred embodiment, R 42 , R 43 , R 44 , R 45 , R 46 , R 47 , R 48 , R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 is 氘.
作为本发明的进一步改进，所述化合物选自下述化合物或其药学上可接受的盐：As a further improvement of the present invention, the compound is selected from the group consisting of the following compounds or a pharmaceutically acceptable salt thereof:
Figure PCTCN2017083882-appb-000002
Figure PCTCN2017083882-appb-000002
Figure PCTCN2017083882-appb-000003
Figure PCTCN2017083882-appb-000003
在另一优选例中，所述化合物不包括非氘代化合物。In another preferred embodiment, the compound does not include a non-deuterated compound.
在另一优选例中，所述化合物不包括R58、R59、R60、R61、R62、R63全是氘，而且其它R不是氘的化合物。In another preferred embodiment, the compound does not include a compound in which R 58 , R 59 , R 60 , R 61 , R 62 , and R 63 are all ruthenium, and the other R is not ruthenium.
本发明还公开了一种药物组合物，其含有药学上可接受的载体和如上所述的所述的丙型肝炎病毒抑制剂，或其晶型、药学上可接受的盐、水合物或溶剂合物、立体异构体、前药或同位素变体的药物组合物。 The present invention also discloses a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the hepatitis C virus inhibitor as described above, or a crystalline form, a pharmaceutically acceptable salt, a hydrate or a solvent thereof A pharmaceutical composition of a compound, stereoisomer, prodrug or isotopic variation.
作为本发明的进一步改进，所述药学上可接受的载体包括助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味增强剂、表面活性剂、润湿剂、分散剂、崩解剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂中的至少一种。As a further improvement of the present invention, the pharmaceutically acceptable carrier includes a glidant, a sweetener, a diluent, a preservative, a dye/colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersant At least one of a disintegrant, a suspending agent, a stabilizer, an isotonic agent, a solvent or an emulsifier.
作为本发明的进一步改进，所述药物组合物为片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、溶液剂、栓剂、注射剂、吸入剂、凝胶剂、微球或气溶胶。As a further improvement of the present invention, the pharmaceutical composition is a tablet, a pill, a capsule, a powder, a granule, an ointment, an emulsion, a suspension, a solution, a suppository, an injection, an inhalant, a gel, a microsphere or Aerosol.
给予本发明药物组合物的典型途径包括但不限于口服、直肠、透黏膜、经肠给药，或者局部、经皮、吸入、肠胃外、舌下、***内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药。优选口服给药或注射给药。Typical routes of administration of the pharmaceutical compositions of the invention include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal , intramuscular, subcutaneous, intravenous administration. Oral administration or injection administration is preferred.
本发明的药物组合物可以采用本领域周知的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等。The pharmaceutical composition of the present invention can be produced by a method known in the art, such as a conventional mixing method, a dissolution method, a granulation method, a sugar-coating method, a pulverization method, an emulsification method, a freeze-drying method, and the like.
作为本发明的进一步改进，其还包含其他活性化合物，所述活性化合物为免疫调节剂或抗病毒药物化合物。As a further improvement of the invention, it further comprises other active compounds which are immunomodulatory or antiviral pharmaceutical compounds.
作为本发明的进一步改进，所述免疫调节剂为干扰素类药物化合物。As a further improvement of the present invention, the immunomodulator is an interferon drug compound.
作为本发明的进一步改进，所述抗病毒药物化合物为利巴韦林、金刚烷胺、NS5A的其他抑制剂、HCV生命周期中的解旋酶、蛋白酶、聚合酶、金属蛋白酶或内部核糖体进入位点靶标的抑制剂，其中，所述NS5A的其他抑制剂为雷迪帕韦或达卡他伟中至少一种。As a further improvement of the present invention, the antiviral drug compound is ribavirin, amantadine, other inhibitors of NS5A, helicase in the HCV life cycle, protease, polymerase, metalloproteinase or internal ribosome entry. An inhibitor of a site target, wherein the other inhibitor of NS5A is at least one of radipavir or daclitaxel.
本发明提供了一种抑制HCV复制子活性、抑制HCV病毒复制和/或HCV病毒产生、治疗HCV感染和/或减少HCV感染的可能性或症状严重性的方法，其应用本发明化合物联合一种或多种治疗剂以及包含本发明化合物和选自HCV抗病毒剂、免疫调节剂和抗感染剂的一种或多种治疗剂的药物组合物。此类治疗剂针对HCV起作用，包括但不限于，利巴韦林、左旋韦林、韦拉米啶、胸腺素α-1、R7025(增强的干扰素，Roche)、干扰素-β、干扰素-α、聚乙二醇化干扰素-α(聚乙二醇干扰素-α)、干扰素-α和利巴韦林的联合、聚乙二醇干扰素-α和利巴韦林的联合、干扰素-α和左旋韦林的联合、以及聚乙二醇干扰素-α和左旋韦林的联合。The present invention provides a method of inhibiting HCV replicon activity, inhibiting HCV viral replication and/or HCV viral production, treating HCV infection, and/or reducing the likelihood or severity of HCV infection, using a compound of the invention in combination with a Or a plurality of therapeutic agents and pharmaceutical compositions comprising a compound of the invention and one or more therapeutic agents selected from the group consisting of HCV antiviral agents, immunomodulators, and anti-infective agents. Such therapeutic agents act against HCV, including, but not limited to, ribavirin, levovirin, verapamil, thymosin alpha-1, R7025 (enhanced interferon, Roche), interferon-beta, interference Combination of p-α, pegylated interferon-α (peginterferon-α), interferon-α and ribavirin, peginterferon-α and ribavirin , a combination of interferon-α and levovirin, and a combination of peginterferon-α and levovirin.
利巴韦林、左旋韦林和韦拉米啶可经由细胞内酶肌苷一磷酸脱氢酶(IMPDH)的抑制通过调节鸟嘌呤核苷酸的胞内库施加它们的抗-HCV作用。IMPDH是从头鸟嘌呤核苷酸生物合成中的生物合成途径上的限速酶。利巴韦林容易在胞内磷酸化，并且单磷酸衍生物是IMPDH的抑制剂。因而，IMPDH的抑制代表用于发HCV复制抑制剂的另 一有用的目标。因此，本发明化合物可也与IMPDH的抑制剂联合施用，(如WO 97/41211、WO 01/00622和WO 00/25780中所公开的)。Ribavirin, levovirin, and verapamil can exert their anti-HCV effects by modulating the intracellular pool of guanine nucleotides via inhibition of the intracellular enzyme inosine monophosphate dehydrogenase (IMPDH). IMPDH is the rate-limiting enzyme on the biosynthetic pathway in the nucleotide biosynthesis of the guanine. Ribavirin is susceptible to intracellular phosphorylation and the monophosphate derivative is an inhibitor of IMPDH. Thus, inhibition of IMPDH represents another for the development of HCV replication inhibitors. A useful goal. Thus, the compounds of the invention may also be administered in combination with inhibitors of IMPDH, as disclosed in WO 97/41211, WO 01/00622 and WO 00/25780.
对于HCV感染的治疗，本发明化合物可也与为HCV NS3丝氨酸蛋白酶抑制剂的药剂联合施用。HCV NS3丝氨酸蛋白酶是主要的病毒酶并且已被描述为对于HCV复制抑制的优异目标。HCV NS3蛋白酶抑制剂的示范性的基于底物和非基于底物的抑制剂在下列中公开：国际专利申请公开WO 98/22496、WO 98/46630、WO 99/07733、WO 99/07734、WO 99/38888、WO 99/50230、WO 99/64442、WO 00/09543、WO 00/59929、WO 02/48116、WO 02/48172、WO 2008/057208和WO 2008/057209，英国专利号GB 2 337 262及美国专利号6,323,180、7,470,664和7,012,066。For treatment of HCV infection, the compounds of the invention may also be administered in combination with an agent that is an HCV NS3 serine protease inhibitor. The HCV NS3 serine protease is the major viral enzyme and has been described as an excellent target for HCV replication inhibition. Exemplary substrate-based and non-substrate-based inhibitors of HCV NS3 protease inhibitors are disclosed in the following: International Patent Application Publication No. WO 98/22496, WO 98/46630, WO 99/07733, WO 99/07734, WO 99/38888, WO 99/50230, WO 99/64442, WO 00/09543, WO 00/59929, WO 02/48116, WO 02/48172, WO 2008/057208 and WO 2008/057209, British Patent No. GB 2 337 262 and U.S. Patent Nos. 6,323,180, 7,470,664 and 7,012,066.
本发明化合物可也与具有抗-HCV性质的核苷联合用于HCV感染的治疗，诸如下列专利所公开的：国际专利申请公开WO 02/51425、WO 01/79246、WO 02/32920、WO 02/48165和WO 2005/003147；WO 01/68663、WO 99/43691、WO 02/18404和WO 2006/021341，及美国专利申请公开US 2005/0038240、US 2002/0019363、US 2003/0236216、US 2004/0006007、US 2004/0063658和US 2004/0110717；美国专利号7,105,499、7,125,855、7,202,224；和国际专利申请公开WO 02/100415、WO 03/026589、WO 03/026675、WO 03/093290、WO 04/011478、WO 04/013300和WO 04/028481。The compounds of the invention may also be used in combination with nucleosides having anti-HCV properties for the treatment of HCV infection, such as disclosed in the following patents: International Patent Application Publication No. WO 02/51425, WO 01/79246, WO 02/32920, WO 02 /48165 and WO 2005/003147; WO 01/68663, WO 99/43691, WO 02/18404, and WO 2006/021341, and US Patent Application Publication No. US 2005/0038240, US 2002/0019363, US 2003/0236216, US 2004 /0006007, US 2004/0063658 and US 2004/0110717; US Patent Nos. 7,105,499, 7,125,855, 7,202,224; and International Patent Application Publication No. WO 02/100415, WO 03/026589, WO 03/026675, WO 03/093290, WO 04/ 011478, WO 04/013300 and WO 04/028481.
对于HCV感染的治疗，本发明化合物可也与为HCV NS5B聚合酶抑制剂的药剂联合施用。可用作联合治疗的此类HCV NS5B聚合酶抑制剂包括但不限于下列中公开的那些：国际专利申请公开WO 02/057287、WO 02/057425、WO 03/068244、WO 2004/000858、WO 04/003138和WO 2004/007512；美国专利号6,777,392、7,105,499、7,125,855、7,202,224及美国专利申请公开US 2004/0067901和US 2004/0110717。For treatment of HCV infection, the compounds of the invention may also be administered in combination with an agent that is an inhibitor of HCV NS5B polymerase. Such HCV NS5B polymerase inhibitors that can be used as a combination therapy include, but are not limited to, those disclosed in the following: International Patent Application Publication No. WO 02/057287, WO 02/057425, WO 03/068244, WO 2004/000858, WO 04 / 003 138 and WO 2004/007512; U.S. Patent Nos. 6,777,392, 7,105,499, 7,125,855, 7,202,224, and U.S. Patent Application Publication Nos. 2004/0067901 and US 2004/0110717.
本发明还提供了一种制备药物组合物的方法，包括步骤：将药学上可接受的载体与如上所述的丙型肝炎病毒抑制剂，或其晶型、药学上可接受的盐、水合物或溶剂合物进行混合，形成药物组合物。The present invention also provides a method of preparing a pharmaceutical composition comprising the steps of: administering a pharmaceutically acceptable carrier to a hepatitis C virus inhibitor as described above, or a crystalline form thereof, a pharmaceutically acceptable salt, or a hydrate thereof Or the solvate is mixed to form a pharmaceutical composition.
本发明还公开了一种如上所述的丙型肝炎病毒抑制剂的用途，其特征在于：用于制备治疗丙型肝炎病毒感染的药物中的用途。The invention also discloses the use of a hepatitis C virus inhibitor as described above, characterized in that it is used for the preparation of a medicament for the treatment of hepatitis C virus infection.
所述的HCV包括其多种基因型以及多种基因亚型，例如1a、1b、2a、2b、3a、3b、4a、5a、6a。The HCV includes a plurality of genotypes thereof and a plurality of gene subtypes, such as 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a, 6a.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体 描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above various technical features of the present invention and the following (as in the embodiments) are specific The various technical features described can be combined with each other to form a new or preferred technical solution. Due to space limitations, we will not repeat them here.
本文中，如无特别说明，“卤素”指F、Cl、Br、和I。更佳地，卤原子选自F、Cl和Br。Herein, "halogen" means F, Cl, Br, and I unless otherwise specified. More preferably, the halogen atom is selected from the group consisting of F, Cl and Br.
本文中，如无特别说明，“氘代”指化合物或基团中的一个或多个氢被氘所取代；氘代可以是一取代、二取代、多取代或全取代。术语“一个或多个氘代的”与“一次或多次氘代”可互换使用。As used herein, unless otherwise specified, "deuterated" means that one or more hydrogens in the compound or group are replaced by deuterium; deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted. The terms "one or more deuterated" are used interchangeably with "one or more deuterated".
本文中，如无特别说明，“非氘代的化合物”是指含氘原子比例不高于天然氘同位素含量(0.015％)的化合物。As used herein, unless otherwise specified, "non-deuterated compound" means a compound containing a proportion of germanium atoms not higher than the natural helium isotope content (0.015%).
本发明还包括同位素标记的化合物(也称为“同位素变体”)，等同于原始化合物在此公开。可以列为本发明的化合物同位素的例子包括氢，碳，氮，氧，磷，硫，氟和氯同位素，分别如2H，3H，13C，14C，15N，17O，18O，31P，32P，35S，18F以及36Cl。其中含有上述同位素或其他同位素原子的本发明的式(I)的化合物或其多晶型、药学上可接受的盐、前药、立体异构体、同位素变体、水合物或溶剂合物都在本发明的范围之内。本发明中某些同位素标记化合物，例如3H和14C的放射性同位素也在其中，在药物和底物的组织分布实验中是有用的。氚，即3H和碳-14，即14C，它们的制备和检测比较容易，是同位素中的首选。此外，较重同位素取代如氘，即2H，由于其很好的代谢稳定性在某些疗法中有优势，例如在体内增加半衰期或减少用量，因此，在某些情况下可以优先考虑。同位素标记的化合物可以用一般的方法，通过用易得的同位素标记试剂替换为非同位素的试剂，用示例中的方案可以制备。The invention also includes isotopically labeled compounds (also referred to as "isotopic variants"), equivalent to the original compounds disclosed herein. Examples of isotopes which may be listed as compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine isotopes such as 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, respectively. , 31 P, 32 P, 35 S, 18 F and 36 Cl. A compound of the formula (I) of the present invention containing the above isotope or other isotopic atom, or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvate thereof It is within the scope of the invention. Certain isotopically-labeled compounds of the present invention, such as the radioisotopes of 3 H and 14 C, are also among them, useful in tissue distribution experiments of drugs and substrates.氚, ie 3 H and carbon-14, ie 14 C, are easier to prepare and detect and are preferred in isotopes. In addition, heavier isotopic substitutions such as guanidine, or 2 H, are preferred in certain therapies due to their good metabolic stability, such as increased half-life or reduced dosage in vivo, and therefore may be preferred in certain circumstances. Isotopically labeled compounds can be prepared in a conventional manner by substituting a readily available isotopically labeled reagent with a non-isotopic reagent using the protocol of the examples.
药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸；甲酸、乙酸、三氟乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、苯甲酸、甲磺酸、乙磺酸、对甲苯磺酸、苯磺酸、萘磺酸等有机酸；以及脯氨酸、苯丙氨酸、天冬氨酸、谷氨酸等氨基酸。另一类优选的盐是本发明化合物与碱形成的盐，例如碱金属盐(例如钠盐或钾盐)、碱土金属盐(例如镁盐或钙盐)、铵盐(如低级的烷醇铵盐以及其它药学上可接受的胺盐)，例如甲胺盐、乙胺盐、丙胺盐、二甲基胺盐、三甲基胺盐、二乙基胺盐、三乙基胺盐、叔丁基胺盐、乙二胺盐、羟乙胺盐、二羟乙胺盐、三羟乙胺盐，以及分别由吗啉、哌嗪、赖氨酸形成 的胺盐。Pharmaceutically acceptable salts include inorganic and organic salts. A preferred class of salts are the salts of the compounds of the invention with acids. Suitable acids for forming salts include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid; formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, Organic acids such as fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid; Amino acids such as amino acid, phenylalanine, aspartic acid, and glutamic acid. Another preferred class of salts are the salts of the compounds of the invention with bases, such as alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example magnesium or calcium salts), ammonium salts (for example lower alkanolammonium). Salts and other pharmaceutically acceptable amine salts), such as methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tert-butyl a base amine salt, an ethylenediamine salt, a hydroxyethylamine salt, a dihydroxyethylamine salt, a trishydroxyethylamine salt, and a morpholine, piperazine, and lysine, respectively. Amine salt.
术语“多晶型”是指化学药物分子的不同排列方式，一般表现为药物原料在固体状态下的存在形式。一种药物可以多种晶型物质状态存在，同一种药物的不同晶型，在体内的溶解和吸收可能不同，从而会对制剂的溶出和释放产生影响。The term "polymorph" refers to a different arrangement of chemical drug molecules, generally expressed as the presence of a pharmaceutical material in a solid state. A drug may exist in a plurality of crystalline forms, and different crystal forms of the same drug may have different dissolution and absorption in the body, thereby affecting the dissolution and release of the formulation.
术语“溶剂合物”指本发明化合物与溶剂分子配位形成特定比例的配合物。“水合物”是指本发明化合物与水进行配位形成的配合物。The term "solvate" refers to a complex of a compound of the invention that is coordinated to a solvent molecule to form a specific ratio. "Hydrate" means a complex formed by the coordination of a compound of the invention with water.
术语“前药”是指在体内通过例如在血液中水解转变成其具有医学效应的活性形式的化合物。前药为任何共价键合的载体，当将这种前药给予患者时，其在体内释放本发明化合物。通常通过修饰官能团来制备前药，该修饰使得前药在体内裂解产生母体化合物。前药包括，例如，其中羟基、氨基或巯基与任意基团键合的本发明化合物，当将其给予患者时，可以裂解形成羟基、氨基或巯基。因此，前药的代表性实例包括但不限于，本发明化合物通过其中的羟基、氨基或巯基官能团与乙酸、甲酸或苯甲酸形成的共价衍生物。另外，在羧酸(-COOH)的情况下，可以使用酯，例如甲酯、乙酯等。酯本身可以是有活性的和/或可以在人体体内条件下水解。合适的药学上可接受的体内可水解的酯包括容易在人体中分解而释放母体酸或其盐的那些。The term "prodrug" refers to a compound that is converted in vivo to an active form having its medical effect by, for example, hydrolysis in blood. A prodrug is any covalently bonded carrier which, when administered to a patient, releases the compound of the invention in vivo. Prodrugs are typically prepared by modifying functional groups that cleave the prodrug in vivo to yield the parent compound. Prodrugs include, for example, a compound of the invention wherein a hydroxy, amino or thiol group is bonded to any group which, when administered to a patient, can be cleaved to form a hydroxy, amino or thiol group. Thus, representative examples of prodrugs include, but are not limited to, covalent derivatives of the compounds of the invention formed by the hydroxyl, amino or thiol functional groups thereof with acetic acid, formic acid or benzoic acid. Further, in the case of a carboxylic acid (-COOH), an ester such as a methyl ester, an ethyl ester or the like can be used. The ester itself may be active and/or may hydrolyze under conditions in humans. Suitable pharmaceutically acceptable in vivo hydrolysable esters include those which readily decompose in the human body to release the parent acid or its salt.
本发明化合物可包括一个或多个不对称中心，且因此可以存在多种“立体异构体”形式，例如，对映异构体和/或非对映异构体形式。例如，本发明化合物可为单独的对映异构体、非对映异构体或几何异构体(例如顺式和反式异构体)，或者可为立体异构体的混合物的形式，包括外消旋混合物和富含一种或多种立体异构体的混合物。异构体可通过本领域技术人员已知的方法从混合物中分离，所述方法包括：手性高压液相色谱法(HPLC)以及手性盐的形成和结晶；或者优选的异构体可通过不对称合成来制备。The compounds of the invention may include one or more asymmetric centers, and thus may exist in a variety of "stereoisomer" forms, for example, enantiomeric and/or diastereomeric forms. For example, the compounds of the invention may be in the form of individual enantiomers, diastereomers or geometric isomers (e.g., cis and trans isomers), or may be in the form of a mixture of stereoisomers, A racemic mixture and a mixture rich in one or more stereoisomers are included. The isomers can be separated from the mixture by methods known to those skilled in the art, including: chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of a chiral salt; or preferred isomers can be passed Prepared by asymmetric synthesis.
与现有技术相比，本发明的有益效果为：第一，本发明的化合物对丙型肝炎病毒蛋白NS5A具有优异的抑制性。第二，通过氘化这一技术改变化合物在生物体中的代谢，使化合物具有更好的药代动力学参数特性。在这种情况下，可以改变剂量并形成长效制剂，改善适用性。第三，用氘取代化合物中的氢原子，由于其氘同位素效应，提高化合物在动物体内的药物浓度，提高了药物疗效。第四，用氘取代化合物中的氢原子，可以抑制某些代谢产物，提高了化合物的安全性。The beneficial effects of the present invention compared to the prior art are: First, the compound of the present invention has excellent inhibitory effect on the hepatitis C virus protein NS5A. Second, by deuteration this technique changes the metabolism of the compound in the organism, giving the compound better pharmacokinetic parameter characteristics. In this case, the dosage can be changed and a long-acting preparation can be formed to improve the applicability. Third, replacing the hydrogen atom in the compound with hydrazine increases the drug concentration of the compound in the animal due to its strontium isotope effect, thereby improving the drug efficacy. Fourth, replacing the hydrogen atom in the compound with hydrazine can inhibit certain metabolites and improve the safety of the compound.
具体实施方式detailed description
下面更具体地描述本发明式(1)结构化合物的制备方法，但这些具体方法不对本 发明构成任何限制。本发明化合物还可以任选将在本说明书中描述的或本领域已知的各种合成方法组合起来而方便地制得，这样的组合可由本发明所属领域的技术人员容易地进行。The preparation method of the structural compound of the formula (1) of the present invention is more specifically described below, but these specific methods are not The invention constitutes any limitation. The compounds of the present invention may also be conveniently prepared by combining various synthetic methods described in the specification or known in the art, and such combinations are readily made by those skilled in the art to which the present invention pertains.
实施例1 制备二甲基(2S,2’S)-1,1’-((2S,2’S)-2,2’-(4,4’-((2S,5S)-1-(4-叔丁基-2,6-d2-Example 1 Preparation of dimethyl(2S,2'S)-1,1'-((2S,2'S)-2,2'-(4,4'-((2S,5S)-1-(4-tert-butyl) Base-2,6-d2- 苯基)吡咯烷-2,5-二基)二(4,1-苯基))二(氮烷二基)二(氧代甲基)二(吡咯烷-2,1-二基)二(3-Phenyl)pyrrolidine-2,5-diyl)bis(4,1-phenyl))bis(azanediyl)bis(oxomethyl)bis(pyrrolidine-2,1-diyl)di (3- 甲基-1-氧代丁烷-2,1-二基)二羧酸酯(化合物O-1)Methyl-1-oxobutane-2,1-diyl)dicarboxylate (Compound O-1)
Figure PCTCN2017083882-appb-000004
Figure PCTCN2017083882-appb-000004
具体合成步骤如下：The specific synthesis steps are as follows:
Figure PCTCN2017083882-appb-000005
Figure PCTCN2017083882-appb-000005
Figure PCTCN2017083882-appb-000006
Figure PCTCN2017083882-appb-000006
步骤1.1,4-二(4-硝基苯基)丁烷-1,4-二酮(化合物3)的合成。Step 1. Synthesis of 1,4-bis(4-nitrophenyl)butane-1,4-dione (Compound 3).
向反应瓶中加入无水氯化锌(5.46g，40mmol)、二乙胺(3.2mL，30mmol)、叔丁醇(2.8mL，30mmol)和30mL甲苯，氮气保护下搅拌反应1.5小时。加入2-溴-1-(4-硝基苯基)乙酮(4.88g，20mmol)和4-硝基苯乙酮(4.96g，30mmol)，室温下搅拌反应16小时，薄层色谱(TLC)检测反应完毕后，加入少量稀硫酸淬灭反应，再加入过量水，抽滤，滤饼依次用甲苯、水和甲醇洗涤，真空干燥得黄色固体4.0g，收率：61％。Anhydrous zinc chloride (5.46 g, 40 mmol), diethylamine (3.2 mL, 30 mmol), tert-butanol (2.8 mL, 30 mmol) and 30 mL of toluene were added to the reaction mixture, and the mixture was stirred under a nitrogen atmosphere for 1.5 hours. 2-Bromo-1-(4-nitrophenyl)ethanone (4.88 g, 20 mmol) and 4-nitroacetophenone (4.96 g, 30 mmol) were added, and the reaction was stirred at room temperature for 16 hours, thin layer chromatography (TLC) After the completion of the reaction, a small amount of dilute sulfuric acid was added to quench the reaction, and excess water was added thereto, and the mixture was filtered with suction. The filter cake was washed with toluene, water and methanol, and dried in vacuo to give a white solid (4.0 g, yield: 61%).
步骤2.(1R,4R)-1,4-二(4-硝基苯基)丁烷-1,4-二醇(化合物5)的合成。Step 2. Synthesis of (1R,4R)-1,4-bis(4-nitrophenyl)butane-1,4-diol (Compound 5).
向反应瓶中加入S-α,α-二苯基脯氨醇(271mg，1.07mmol)，加入8mL无水四氢呋喃(THF)溶解，滴加硼酸三甲酯，加毕，室温反应1小时。降温至16-19℃，滴加二乙基苯基硼烷复合物(2.15g，13.2mmol)，搅拌反应15-20分钟。在另一个三口瓶中加入化合物3(2.09g，6.38mmol)，加入8mL无水THF得到黄色浑浊液体，降温至11℃，缓慢滴加上述硼烷溶液(控制内温不超过16℃)，加毕，升至室温反应2.5小时。TLC检测反应完毕后，降温至0-5℃，滴加1.67g甲醇，待气体停止析出后，升至室温搅拌使固体全部溶解，加入乙酸乙酯(30mL)和1.0M稀盐酸(12mL)，有机相依次用1M稀盐酸、水和饱和食盐水洗涤，浓缩，硅胶柱层析纯化得到黄色固体1.65g，收率：77.8％。S-α,α-diphenyldecanol (271 mg, 1.07 mmol) was added to the reaction flask, and dissolved in 8 mL of anhydrous tetrahydrofuran (THF), and trimethyl borate was added dropwise thereto, and the mixture was reacted at room temperature for 1 hour. The temperature was lowered to 16 to 19 ° C, and diethylphenylborane complex (2.15 g, 13.2 mmol) was added dropwise, and the reaction was stirred for 15-20 minutes. Add compound 3 (2.09g, 6.38mmol) to another three-necked flask, add 8mL anhydrous THF to obtain yellow turbid liquid, cool down to 11 ° C, slowly add the above borane solution (control internal temperature does not exceed 16 ° C), add After completion, the reaction was allowed to rise to room temperature for 2.5 hours. After the TLC detection reaction was completed, the temperature was lowered to 0-5 ° C, and 1.67 g of methanol was added dropwise. After the gas stopped to precipitate, the mixture was stirred at room temperature to dissolve all the solids, and ethyl acetate (30 mL) and 1.0 M diluted hydrochloric acid (12 mL) were added. The organic phase was washed successively with 1M EtOAc EtOAc EtOAc.
步骤3.(1R,4R)-1,4-二(4-硝基苯基)丁烷-1,4-二磺酸酯(化合物6)的合成。Step 3. Synthesis of (1R,4R)-1,4-bis(4-nitrophenyl)butane-1,4-disulfonate (Compound 6).
在反应瓶中加入化合物5(200mg，0.6mmol)，加入10mL二氯甲烷溶解，降温至0℃，加入三乙胺(182.1mg，1.8mmol)，氮气保护下滴加甲基磺酰氯(171.8mg，1.5mmol)，加毕搅拌反应1.5小时，TLC检测反应完毕后，加入饱和氯化铵淬灭反应，有机相用水和饱和食盐水洗涤，无水硫酸钠干燥，过滤浓缩和得产物，无需纯化直接投 入到下一步。Compound 5 (200 mg, 0.6 mmol) was added to the reaction flask, dissolved in 10 mL of dichloromethane, cooled to 0 ° C, triethylamine (182.1 mg, 1.8 mmol) was added, and methylsulfonyl chloride (171.8 mg) was added dropwise under nitrogen. , 1.5 mmol), and the reaction was stirred for 1.5 hours. After the reaction was completed by TLC, the reaction was quenched with saturated aqueous ammonium chloride. Direct investment Go to the next step.
步骤4.4-叔丁基-2,6-d2-苯胺(化合物8)的合成。Step 4. 4-Synthesis of tert-butyl-2,6-d2-aniline (Compound 8).
向微波反应管中加入4-叔丁基苯胺(597mg，4mmol)，20mL重水和浓盐酸(0.33mL，4mmol)，置于微波反应器中，180℃反应0.5小时，降至室温后加入饱和碳酸钠溶液调PH至弱碱性，加入乙酸乙酯萃取3-4次，合并有机相，饱和食盐水洗涤，浓缩，硅胶柱层析纯化得产物590mg，收率：97.5％。To the microwave reaction tube was added 4-tert-butylaniline (597 mg, 4 mmol), 20 mL of heavy water and concentrated hydrochloric acid (0.33 mL, 4 mmol), placed in a microwave reactor, reacted at 180 ° C for 0.5 hour, and then added to saturated carbonic acid at room temperature. The sodium solution was adjusted to a weakly basic pH, and extracted with ethyl acetate for 3-4 times. The organic phase was combined, washed with brine, concentrated, and purified by silica gel column chromatography to yield 590 mg, yield: 97.5%.
步骤5.(2S,5S)-N-(4-叔丁基-2,6-d2-苯基)-2,5-二(4-硝基苯基)吡咯烷(化合物9)的合成。Step 5. Synthesis of (2S,5S)-N-(4-tert-butyl-2,6-d2-phenyl)-2,5-bis(4-nitrophenyl)pyrrolidine (Compound 9).
向反应瓶中加入化合物6(293mg，0.6mmol)和化合物8(590mg，3.91mmol)，加入10mL无水THF溶解，加热至60℃搅拌反应过夜，TLC检测反应完毕后浓缩除去溶剂，硅胶柱层析纯化得产物124mg，收率：46.2％。Compound 6 (293 mg, 0.6 mmol) and Compound 8 (590 mg, 3.91 mmol) were added to the reaction flask, and dissolved in 10 mL of anhydrous THF. The mixture was heated to 60 ° C, and the reaction was stirred overnight. After the reaction was completed by TLC, the solvent was removed and concentrated. The purified product was isolated in 124 mg, yield: 46.2%.
步骤6.(2S,5S)-N-(4-叔丁基-2,6-d2-苯基)-2,5-二(4-氨基苯基)吡咯烷(化合物10)的合成。Step 6. Synthesis of (2S,5S)-N-(4-tert-butyl-2,6-d2-phenyl)-2,5-bis(4-aminophenyl)pyrrolidine (Compound 10).
向反应瓶中加入化合物9(170mg，0.38mmol)，用10mL四氢呋喃溶解，加入催化量兰尼镍，氮气鼓泡10分钟除氧，充氢气球，室温下搅拌反应2-4小时，TLC检测反应完毕后，过滤除去催化剂，滤液浓缩，硅胶柱层析纯化得产物116mg，收率：79.2％。Compound 9 (170 mg, 0.38 mmol) was added to the reaction flask, dissolved in 10 mL of tetrahydrofuran, a catalytic amount of Raney nickel was added, nitrogen was bubbled for 10 minutes to remove oxygen, and a hydrogen balloon was charged. The reaction was stirred at room temperature for 2-4 hours, and the reaction was confirmed by TLC. After completion, the catalyst was removed by filtration, the filtrate was concentrated, and purified by silica gel column chromatography to yield product 116 mg, yield: 79.2%.
步骤7.(S)-2,5-二氧代吡咯烷-1-基-2-(甲氧羰基氨基)-3-甲基丁酸酯(化合物12)的合成。Step 7. Synthesis of (S)-2,5-dioxopyrrolidin-1-yl-2-(methoxycarbonylamino)-3-methylbutyrate (Compound 12).
向反应瓶中加入N-甲氧羰基-L-结氨酸(1.75g，10mmol)和N-羟基丁二酰亚胺(1.21g，10.5mmol)，加入22mL乙酸乙酯溶解，降温至0-5℃，加入二异丙基碳二亚胺(1.26g，10mmol)，搅拌反应1小时，TLC检测反应完毕后，升至室温，过滤除去副产物，浓缩得化合物7粗品，不需纯化直接投入到下一步反应。N-methoxycarbonyl-L-proline (1.75 g, 10 mmol) and N-hydroxysuccinimide (1.21 g, 10.5 mmol) were added to the reaction flask, dissolved in 22 mL of ethyl acetate, and cooled to 0- At 5 ° C, diisopropylcarbodiimide (1.26 g, 10 mmol) was added, and the reaction was stirred for 1 hour. After the TLC reaction was completed, the mixture was allowed to warm to room temperature, and the by-product was removed by filtration and concentrated to give the crude compound 7 which was directly purified without purification. Go to the next step.
步骤8.(S)-1-((S)-2-(甲氧羰基氨基)-3-甲基丁酰)吡咯烷-2-羧酸(化合物12)的合成。Step 8. Synthesis of (S)-1-((S)-2-(methoxycarbonylamino)-3-methylbutyryl)pyrrolidine-2-carboxylic acid (Compound 12).
向反应瓶中加入L-脯氨酸(1.21g，10.5mmol)和N,N-二异丙基乙胺(DIPEA，2.58g，20mmol)，加入5mL乙腈和5mL水溶解，缓慢滴加化合物11(2.72g，10mmol)的5mL乙腈溶液，室温下搅拌反应过夜。TLC检测反应完毕后，浓缩除去溶剂，加入稀盐酸调PH至弱酸性，用乙酸乙酯萃取3-4遍，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，过滤，滤液浓缩，加入正庚烷，浓缩，再加入甲基叔丁基醚打浆，析出白色固体，过滤，真空干燥得产物1.69g，收率：62％。 L-valine (1.21 g, 10.5 mmol) and N,N-diisopropylethylamine (DIPEA, 2.58 g, 20 mmol) were added to the reaction flask, dissolved in 5 mL of acetonitrile and 5 mL of water, and the compound 11 was slowly added dropwise. (2.72 g, 10 mmol) in 5 mL of acetonitrile solution was stirred at room temperature overnight. After the reaction was completed by TLC, the solvent was concentrated to remove the solvent, and the mixture was diluted with hydrochloric acid to adjust to a weak acidity, and extracted with ethyl acetate for 3-4 times. The organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. n-Heptane, concentrated, and then added with methyl tert-butyl ether. The white solid was precipitated, filtered, and dried in vacuo to yield 1.69 g, yield: 62%.
步骤9.二甲基(2S,2’S)-1,1’-((2S,2’S)-2,2’-(4,4’-((2S,5S)-1-(4-叔丁基-2,6-d2-苯基)吡咯烷-2,5-二基)二(4,1-苯基))二(氮烷二基)二(氧代甲基)二(吡咯烷-2,1-二基)二(3-甲基-1-氧代丁烷-2,1-二基)二羧酸酯(化合物O-1)的合成。Step 9. Dimethyl(2S,2'S)-1,1'-((2S,2'S)-2,2'-(4,4'-((2S,5S)-1-(4-tert-butyl) -2,6-d2-phenyl)pyrrolidine-2,5-diyl)bis(4,1-phenyl))bis(azanediyl)bis(oxomethyl)bis(pyrrolidine-2 Synthesis of 1,1-diyl)bis(3-methyl-1-oxobutane-2,1-diyl)dicarboxylate (Compound O-1).
向反应瓶中加入化合物10(165mg，0.426mmol)、化合物13(299.5mg，1.1mmol)和2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU，418.3mg，1.1mmol)，加入8mL无水DMF溶解，加入三乙胺(111.3mg，1.1mmol)，室温下搅拌反应2小时，TLC检测反应完毕后，加入过量水析出白色固体，过滤，滤饼干燥后经硅胶柱层析纯化得目标产物221.4mg，收率：58％。LC-MS(APCI):m/z＝475(M+1)+。1H NMR(400MHz,DMSO-d6)δ9.98(s,2H),7.50(d,J＝8.5Hz,4H),7.32(d,J＝8.4Hz,2H),7.13(d,J＝8.5Hz,4H),6.94(s,2H),5.14(d,J＝6.3Hz,2H),4.43(dd,J＝7.8,4.9Hz,2H),4.02(dt,J＝10.5,3.4Hz,2H),3.81(dd,J＝15.6,6.5Hz,2H),3.62(dd,J＝15.6,6.9Hz,2H),3.52(s,6H),2.45(s,2H),2.19–2.06(m,2H),1.89(ddd,J＝19.5,13.8,6.4Hz,6H),1.62(d,J＝5.3Hz,2H),1.11(s,9H),0.91(dd,J＝21.2,6.7Hz,12H)。Compound 10 (165 mg, 0.426 mmol), compound 13 (299.5 mg, 1.1 mmol) and 2-(7-benzotriazole)-N,N,N',N'-tetramethyl were added to the reaction flask. Urea hexafluorophosphate (HATU, 418.3 mg, 1.1 mmol), dissolved in 8 mL of anhydrous DMF, added triethylamine (111.3 mg, 1.1 mmol), and stirred at room temperature for 2 hours. After TLC detection, add excess water. The white solid was precipitated, filtered, and the filter cake was dried and purified by silica gel column chromatography to give the desired product 221.4 mg, yield: 58%. LC-MS (APCI): m / z = 475 (M + 1) +. 1 H NMR (400MHz, DMSO- d6) δ9.98 (s, 2H), 7.50 (d, J = 8.5Hz, 4H), 7.32 (d, J = 8.4Hz, 2H), 7.13 (d, J = 8.5 Hz, 4H), 6.94 (s, 2H), 5.14 (d, J = 6.3 Hz, 2H), 4.43 (dd, J = 7.8, 4.9 Hz, 2H), 4.02 (dt, J = 10.5, 3.4 Hz, 2H) ), 3.81 (dd, J = 15.6, 6.5 Hz, 2H), 3.62 (dd, J = 15.6, 6.9 Hz, 2H), 3.52 (s, 6H), 2.45 (s, 2H), 2.19 - 2.06 (m, 2H), 1.89 (ddd, J = 19.5, 13.8, 6.4 Hz, 6H), 1.62 (d, J = 5.3 Hz, 2H), 1.11 (s, 9H), 0.91 (dd, J = 21.2, 6.7 Hz, 12H) ).
实施例2Example 2
对以上实施例的化合物进行生物活性评价。The compounds of the above examples were evaluated for biological activity.
为了验证本文所述的化合物对HCV的作用，发明人采用HCV复制子***(HCV Replicon System)作为评价模型。自Science1999年首次报道以来，HCV复制子***已经成为研究HCV RNA复制、致病性和病毒持续性的最重要的工具之一，例如已经利用复制子成功地证明了HCV RNA复制所必须的5'-NCR最小区域，并且HCV复制子***已经成功地被用作抗病毒药物的评价模型。本发明的发明人按照Science，1999，285(5424)，110-3，以及J.Virol，2003，77(5)，3007-19所描述的方法进行验证。To verify the effect of the compounds described herein on HCV, the inventors used the HCV Replicon System as an evaluation model. Since its first report in Science in 1999, the HCV replication system has become one of the most important tools for studying HCV RNA replication, pathogenicity and viral persistence, for example, the use of replicons has successfully demonstrated the 5' required for HCV RNA replication. - NCR minimum region, and the HCV replication subsystem has been successfully used as an evaluation model for antiviral drugs. The inventors of the present invention verified according to the methods described in Science, 1999, 285 (5424), 110-3, and J. Virol, 2003, 77(5), 3007-19.
(1)检测化合物抗HCV 1a和1b基因型复制子活性(1) Detection of compound anti-HCV 1a and 1b genotype replicon activity
应用HCV-1a和HCV-1b稳定转染复制子细胞检测化合物丙型肝炎病毒基因型1a和1b复制子的抑制活性。本实验将以NS5A抑制剂Ombitasvir作为阳性对照化合物。The inhibitory activities of the recombinant hepatitis C virus genotype 1a and 1b replicons were detected by stable transfection of replicon cells with HCV-1a and HCV-1b. This experiment will use the NS5A inhibitor Ombitasvir as a positive control compound.
步骤一：对化合物进行1:3系列稀释8个浓度点，双复孔，加入96孔板中。设置DMSO为无加化合物对照。细胞培养液中的DMSO最终浓度为0.5％。Step 1: The compound was diluted 1:3 in 8 series points, double-replicated, and added to a 96-well plate. The DMSO was set to no compound control. The final concentration of DMSO in the cell culture was 0.5%.
步骤二：将HCV-1a和1b细胞分别悬浮在含10％FBS的培养液中，以每孔8,000个细胞的密度种到含有化合物的96孔板中。细胞在5％CO2、37℃条件下培养3天。Step 2: HCV-1a and 1b cells were separately suspended in a culture medium containing 10% FBS, and seeded into a 96-well plate containing the compound at a density of 8,000 cells per well. The cells were cultured for 3 days at 5% CO 2 at 37 °C.
步骤三：用CellTiter-Fluor(Promega)测定化合物对GT1b复制子细胞毒性。 Step 3: The cytotoxicity of the compound against GT1b replicon was determined using CellTiter-Fluor (Promega).
步骤四：用Bright-Glo(Promega)检测荧光素酶测定化合物抗丙型肝炎病毒活性。Step 4: Detection of luciferase assay by Bright-Glo (Promega) for anti-hepatitis C virus activity.
步骤五：采用GraphPad Prism软件分析数据，拟合曲线并计算EC50值和CC50值。Step Five: using GraphPad Prism data analysis software, the curve fitting and EC 50 values were calculated and the 50 value CC.
对实施例1的化合物O-1和对照样Ombitasvir按照如上步骤进行分析计算EC50值和CC50值，结果如表1所示。The compound O-1 of Example 1 and the control Ombitasvir were analyzed according to the above procedure to calculate the EC 50 value and the CC 50 value, and the results are shown in Table 1.
(2)测定化合物抗感染性病型肝炎病毒(GT2a)活性(2) Determination of compound anti-infectious disease hepatitis virus (GT2a) activity
该感染模型参考已报道文献(A novel luciferase and GFP dual reporter virus for rapid and convenient evaluation of HCV replication.Virus Res 2011.155:406)。用HCVcc(MOI＝0.2)感染Huh-7.5.1细胞后，化合物处理3天(8个浓度，3倍稀释,双复孔)。抗病毒活性将以荧光素酶法测定。同时测定化合物对Huh7.5.1细胞活性。本实验将以NS5A抑制剂BMS-790052作为阳性对照化合物。采用GraphPad软件计算EC50。This infection model is referred to as A novel luciferase and GFP dual reporter virus for rapid and convenient evaluation of HCV replication. Virus Res 2011. 155: 406. After Huh-7.5.1 cells were infected with HCVcc (MOI = 0.2), the compounds were treated for 3 days (8 concentrations, 3 fold dilution, double duplicate wells). Antiviral activity will be determined by the luciferase method. The activity of the compound on Huh7.5.1 cells was also determined. This experiment will use the NS5A inhibitor BMS-790052 as a positive control compound. Using GraphPad software to calculate EC 50.
对实施例1的化合物O-1和对照样Ombitasvir按照如上步骤进行分析计算EC50值值，结果如表1所示。The compound O-1 of Example 1 and the control Ombitasvir were analyzed according to the above procedure to calculate EC 50 value, and the results are shown in Table 1.
表1 实施例0-1与对照品Ombitasvir的抗HCV基因型复制子活性对比表Table 1 Comparison of anti-HCV genotype replicon activity between Example 0-1 and the control Obmitasvir
编号Numbering GT1a EC50(nM)GT1a EC 50 (nM) GT1b EC50(nM)GT1b EC 50 (nM) GT2a EC50(nM)GT2a EC 50 (nM) CC50(nM)CC 50 (nM)
OmbitasvirOmbitasvir 0.01140.0114 0.00120.0012 0.00090.0009 >1>1
O-1O-1 0.01240.0124 0.00150.0015 0.00250.0025 >1>1
实验结果表明，本发明的化合物可抑制HCV的多个基因型，并通过抑制HCVNS5A蛋白的机制，发挥了优越的抗丙肝病毒作用。The experimental results show that the compound of the present invention can inhibit multiple genotypes of HCV and exert superior anti-HCV effects by inhibiting the mechanism of HCV NS5A protein.
(3)代谢稳定性评价(3) Metabolic stability evaluation
微粒体实验：人肝微粒体：0.5mg/mL，Xenotech；大鼠肝微粒体：0.5mg/mL，Xenotech；辅酶(NADPH/NADH)：1mM，Sigma Life Science；氯化镁：5mM，100mM磷酸盐缓冲剂(pH为7.4)。Microsomal experiments: human liver microsomes: 0.5 mg/mL, Xenotech; rat liver microsomes: 0.5 mg/mL, Xenotech; coenzyme (NADPH/NADH): 1 mM, Sigma Life Science; magnesium chloride: 5 mM, 100 mM phosphate buffer Agent (pH 7.4).
储备液的配制：精密称取一定量的化合物实施例化合物的粉末，并用DMSO分别溶解至5mM。Preparation of the stock solution: A powder of the compound of the compound example was accurately weighed and dissolved in DMSO to 5 mM.
磷酸盐缓冲液(100mM，pH7.4)的配制：取预先配好的0.5M磷酸二氢钾150mL和700mL的0.5M磷酸氢二钾溶液混合，再用0.5M磷酸氢二钾溶液调节混合液pH值至7.4，使用前用超纯水稀释5倍，加入氯化镁，得到磷酸盐缓冲液(100mM)，其中含100mM磷酸钾，3.3mM氯化镁，pH为7.4。Preparation of phosphate buffer (100 mM, pH 7.4): Mix 150 mL of pre-formed 0.5 M potassium dihydrogen phosphate and 700 mL of 0.5 M potassium dihydrogen phosphate solution, and adjust the mixture with 0.5 M potassium dihydrogen phosphate solution. The pH was adjusted to 7.4, diluted 5 times with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100 mM) containing 100 mM potassium phosphate, 3.3 mM magnesium chloride, and a pH of 7.4.
配制NADPH再生***溶液(含有6.5mM NADP，16.5mM G-6-P，3U/mL G-6-P D， 3.3mM氯化镁)，使用前置于湿冰上。Prepare a solution of NADPH regeneration system (containing 6.5 mM NADP, 16.5 mM G-6-P, 3 U/mL G-6-P D, 3.3 mM magnesium chloride), placed on wet ice before use.
配制终止液：含有50ng/mL盐酸***和200ng/mL甲苯磺丁脲(内标)的乙腈溶液。取25057.5μL磷酸盐缓冲液(pH7.4)至50mL离心管中，分别加入812.5μL人肝微粒体，混匀，得到蛋白浓度为0.625mg/mL的肝微粒体稀释液。取25057.5μL磷酸盐缓冲液(pH7.4)至50mL离心管中，分别加入812.5μL SD大鼠肝微粒体，混匀，得到蛋白浓度为0.625mg/mL的肝微粒体稀释液。Formulation stop solution: acetonitrile solution containing 50 ng/mL propranolol hydrochloride and 200 ng/mL tolbutamide (internal standard). Take 25057.5 μL of phosphate buffer (pH 7.4) into a 50 mL centrifuge tube, add 812.5 μL of human liver microsomes, and mix to obtain a liver microsome dilution with a protein concentration of 0.625 mg/mL. 25057.5 μL of phosphate buffer (pH 7.4) was taken into a 50 mL centrifuge tube, and 812.5 μL of SD rat liver microsomes were added and mixed to obtain a liver microsome dilution having a protein concentration of 0.625 mg/mL.
样品的孵育：用含70％乙腈的水溶液将相应化合物的储备液分别稀释至0.25mM，作为工作液，备用。分别取398μL的人肝微粒体或者大鼠肝微粒体稀释液加入96孔孵育板中(N＝2)，分别加入2μL 0.25mM的的工作液中，混匀。Incubation of the sample: The stock solution of the corresponding compound was diluted to 0.25 mM with an aqueous solution containing 70% acetonitrile as a working solution, and was used. 398 μL of human liver microsomes or rat liver microsome dilutions were added to 96-well incubation plates (N=2), and 2 μL of 0.25 mM working solution was added and mixed.
代谢稳定性的测定：在96孔深孔板的每孔中加入300μL预冷的终止液，并置于冰上，作为终止板。将96孔孵育板和NADPH再生***置于37℃水浴箱中，100转/分钟震荡，预孵5min。从孵育板每孔取出80μL孵育液加入终止板，混匀，补充20μL NADPH再生***溶液，作为0min样品。再向孵育板每孔加入80μL的NADPH再生***溶液，启动反应，开始计时。相应化合物的反应浓度为1μM，蛋白浓度为0.5mg/mL。分别于反应10、30、90min时，各取100μL反应液，加入终止板中，涡旋3min终止反应。将终止板于5000×g，4℃条件下离心10min。取100μL上清液至预先加入100μL蒸馏水的96孔板中，混匀，采用LC-MS/MS进行样品分析。Determination of metabolic stability: 300 μL of pre-cooled stop solution was added to each well of a 96-well deep well plate and placed on ice as a stop plate. The 96-well incubation plate and the NADPH regeneration system were placed in a 37 ° C water bath, shaken at 100 rpm, and pre-incubated for 5 min. 80 μL of the incubation solution was taken from each well of the incubation plate, added to the stopper plate, and mixed, and 20 μL of the NADPH regeneration system solution was added as a sample of 0 min. Then, 80 μL of the NADPH regeneration system solution was added to each well of the incubation plate to start the reaction and start timing. The corresponding compound had a reaction concentration of 1 μM and a protein concentration of 0.5 mg/mL. 100 μL of the reaction solution was taken at 10, 30, and 90 min, respectively, and added to the stopper, and the reaction was terminated by vortexing for 3 min. The plate was centrifuged at 5000 x g for 10 min at 4 °C. 100 μL of the supernatant was taken into a 96-well plate to which 100 μL of distilled water was previously added, mixed, and sample analysis was performed by LC-MS/MS.
数据分析：通过LC-MS/MS***检测相应化合物及内标的峰面积，计算化合物与内标峰面积比值。通过化合物剩余量的百分率的自然对数与时间作图测得斜率，并根据以下公式计算t1/2和CLint，其中V/M即等于1/蛋白浓度。Data analysis: The peak area of the corresponding compound and the internal standard was detected by LC-MS/MS system, and the ratio of the peak area of the compound to the internal standard was calculated. The slope is measured by the natural logarithm of the percentage of the remaining amount of the compound versus time, and t 1/2 and CL int are calculated according to the following formula, where V/M is equal to 1/protein concentration.
Figure PCTCN2017083882-appb-000007
Figure PCTCN2017083882-appb-000007
实验结果表明，本发明化合物在人肝微粒体与大鼠肝微粒体实验中都表现出优异的代谢稳定性。The experimental results show that the compounds of the present invention exhibit excellent metabolic stability in both human liver microsomes and rat liver microsomes.
(4)大鼠药代动力学实验(4) Rat pharmacokinetic experiments
实验目的：研究大鼠给予Ombitasvir、化合物O-1后，考察本发明化合物的药代动力学行为。OBJECTIVE: To investigate the pharmacokinetic behavior of the compounds of the present invention after administration of Ombitasvir and Compound O-1 to rats.
实验动物：Experimental animals:
种类及品系：SD大鼠等级：SPF级 Type and strain: SD rat grade: SPF grade
性别及数量：雄性，6只Gender and quantity: male, 6
体重范围：180～220g(实际体重范围为187～197g)Weight range: 180 ~ 220g (actual weight range is 187 ~ 197g)
来源：上海西普尔必凯实验动物有限公司Source: Shanghai Xipuer Bikai Experimental Animal Co., Ltd.
实验及动物合格证号：SCXK(沪)2013-0016。Laboratory and animal certificate number: SCXK (Shanghai) 2013-0016.
实验过程：experiment procedure:
在血样采集之前，预先在EDTA-K2抗凝管中加入20L的2M氟化钠溶液(酯酶抑制剂)，于80度烘箱内烘干后，置于4度冰箱存放。Before the blood sample was collected, 20 L of 2M sodium fluoride solution (esterase inhibitor) was previously added to the EDTA-K2 anticoagulation tube, dried in an 80 degree oven, and stored in a 4 degree refrigerator.
大鼠，雄性，体重187～197g，随机分为2组，于实验前一天下午开始禁食过夜但可自由饮水，给药后4h给食物。A组给予Ombitasvir 3mg/kg，B组给予式T-1化合物3mg/kg，分别于给药后15min、30min、1、2、3、5、8、10h从大鼠眼眶静脉取血100-200L左右，置于经EDTA-K2抗凝的0.5mL的Eppendorf管中，立即混匀，抗凝后，尽快将试管轻轻颠倒混匀5-6次后,血取好后放置在冰盒中，30min内把血样本在4000rpm，10min，4℃条件下离心分离血浆，收集全部血浆后立即于-20℃保存。所有时间点样品采集后测定每个时间点的血浆中的血药浓度。Rats, males, weighing 187-197 g, were randomly divided into 2 groups. They were fasted overnight in the afternoon before the experiment but were free to drink water. Food was given 4 h after administration. Group A was given 3 mg/kg of Obmitasvir, and group B was given 3 mg/kg of compound of formula T-1. Blood was taken from the orbital vein of rats 100-200 L at 15 min, 30 min, 1, 2, 3, 5, 8 and 10 h after administration. Left and right, placed in an EDTA-K2 anticoagulated 0.5mL Eppendorf tube, mix immediately, after anticoagulation, gently invert the tube as soon as possible 5-6 times, the blood is taken and placed in the ice box, Blood samples were centrifuged at 4000 rpm, 10 min, and 4 ° C for 30 min, and all plasma was collected and stored at -20 ° C immediately. Plasma concentrations in plasma at each time point were determined after sample collection at all time points.
根据上述所得的给药后平均血药浓度-时间数据，采用Winnonin软件，按非房室统计矩理论求算雄性SD大鼠分别i.g给予Ombitasvir(3mg/kg)、实施例化合物(3mg/kg)后的药代动力学相关参数。According to the average blood drug concentration-time data obtained after the above, the male SD rats were treated with Winnonin software according to the non-compartmental statistical moment theory to give Ombitasvir (3 mg/kg) and the compound of the example (3 mg/kg). Post-pharmacokinetic related parameters.
实验结果表明，与Ombitasvir相比，本发明人发现本发明化合物具有比Ombitasvir更优的活性，并且具有优异的药代动力学性质，因此更适合作为抑制丙型肝炎病毒蛋白NS5A的化合物，进而适合制备治疗丙型肝炎病毒感染的药物。The experimental results show that the present inventors have found that the compound of the present invention has superior activity to Ombitasvir and has excellent pharmacokinetic properties as compared with Ombitasvir, and thus is more suitable as a compound for inhibiting the hepatitis C virus protein NS5A, and is suitable for use. Preparation of a medicament for treating hepatitis C virus infection.
应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围，实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则份数和百分比为重量份和重量百分比。It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention, and the experimental methods in which the specific conditions are not indicated in the examples, usually in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer. Parts and percentages are parts by weight and percentage by weight unless otherwise stated.
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。 The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims (10)

  1. 一种丙型肝炎病毒抑制剂，其特征在于：如式(1)所示的吡咯烷化合物化合物，或其多晶型、药学上可接受的盐、前药、立体异构体、同位素变体、水合物或溶剂化合物，A hepatitis C virus inhibitor characterized by a pyrrolidine compound represented by the formula (1), or a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotope variant thereof , hydrate or solvent compound,
    Figure PCTCN2017083882-appb-100001
    Figure PCTCN2017083882-appb-100001
    其中，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49、R50、R51、R52、R53、R54、R55、R56、R57、R58、R59、R60、R61、R62、R63各自独立地为氢、氘、卤素或三氟甲基；Wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 And R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , R 42 , R 43 , R 44 , R 45 , R 46 , R 47 , R 48 , R 49. R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 , R 58 , R 59 , R 60 , R 61 , R 62 and R 63 are each independently hydrogen and hydrazine. , halogen or trifluoromethyl;
    附加条件是R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49、R50、R51、R52、R53、R54、R55、R56、R57、R58、R59、R60、R61、R62和R63中至少一个是氘代的或氘。Additional conditions are R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 And R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , R 42 , R 43 , R 44 , R 45 , R 46 , R 47 , R 48 , At least one of R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 , R 58 , R 59 , R 60 , R 61 , R 62 and R 63 is a generation Or 氘.
  2. 根据权利要求1所述的丙型肝炎病毒抑制剂，其特征在于：R1、R2、R3、R4、R5、R6、R7、R8和R9各自独立地为氘或氢。 The hepatitis C virus inhibitor according to claim 1, wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently anthracene or hydrogen.
  3. 根据权利要求1所述的丙型肝炎病毒抑制剂，其特征在于：R10、R11、R12和R13各自独立地为氘或氢。The hepatitis C virus inhibitor according to claim 1, wherein each of R 10 , R 11 , R 12 and R 13 is independently hydrazine or hydrogen.
  4. 根据权利要求1所述的丙型肝炎病毒抑制剂，其特征在于：R14、R15、R16、R17、R18、R19、R20和R21各自独立地为氘或氢。The hepatitis C virus inhibitor according to claim 1, wherein R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 and R 21 are each independently hydrazine or hydrogen.
  5. 根据权利要求1所述的丙型肝炎病毒抑制剂，其特征在于：R22、R23、R24、R25、R26和R27各自独立地为氘或氢。The hepatitis C virus inhibitor according to claim 1, wherein R 22 , R 23 , R 24 , R 25 , R 26 and R 27 are each independently hydrazine or hydrogen.
  6. 根据权利要求1所述的丙型肝炎病毒抑制剂，其特征在于：R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40和R41各自独立地为氘或氢。The hepatitis C virus inhibitor according to claim 1, wherein R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 33 , R 34 , R 35 , R 36 , R 37 And R 38 , R 39 , R 40 and R 41 are each independently hydrazine or hydrogen.
  7. 根据权利要求1所述的丙型肝炎病毒抑制剂，其特征在于：R42、R43、R44、R45、R46、R47、R48、R49、R50、R51、R52、R53、R54、R55、R56和R57各自独立地为氘或氢。The hepatitis C virus inhibitor according to claim 1, wherein R 42 , R 43 , R 44 , R 45 , R 46 , R 47 , R 48 , R 49 , R 50 , R 51 , R 52 R 53 , R 54 , R 55 , R 56 and R 57 are each independently hydrazine or hydrogen.
  8. 根据权利要求1所述的丙型肝炎病毒抑制剂，其特征在于：所述化合物选自下述化合物或其药学上可接受的盐：The hepatitis C virus inhibitor according to claim 1, wherein the compound is selected from the group consisting of the following compounds or a pharmaceutically acceptable salt thereof:
    Figure PCTCN2017083882-appb-100002
    Figure PCTCN2017083882-appb-100002
    Figure PCTCN2017083882-appb-100003
    Figure PCTCN2017083882-appb-100003
    Figure PCTCN2017083882-appb-100004
    Figure PCTCN2017083882-appb-100004
  9. 一种药物组合物，其特征在于：其含有药学上可接受的载体和如权利要求1～8任意一项所述的丙型肝炎病毒抑制剂，或其晶型、药学上可接受的盐、水合物或溶剂合物、立体异构体、前药或同位素变体的药物组合物。A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a hepatitis C virus inhibitor according to any one of claims 1 to 8, or a crystalline form thereof, a pharmaceutically acceptable salt, A pharmaceutical composition of a hydrate or solvate, stereoisomer, prodrug or isotopic variation.
  10. 一种如权利要求1～8任意一项所述的丙型肝炎病毒抑制剂的用途，其特征在于：用于制备治疗丙型肝炎病毒感染的药物中的用途。 Use of a hepatitis C virus inhibitor according to any one of claims 1 to 8, which is for use in the preparation of a medicament for treating hepatitis C virus infection.
PCT/CN2017/083882 2016-05-30 2017-05-11 Substituted pyrrolidine compound, pharmaceutical composition, and applications thereof WO2017206688A1 (en)

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CN104030959A (en) * 2014-06-24 2014-09-10 苏州东南药业股份有限公司 Deuterated hepatitis c virus inhibitor

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* Cited by examiner, † Cited by third party
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CN104030959A (en) * 2014-06-24 2014-09-10 苏州东南药业股份有限公司 Deuterated hepatitis c virus inhibitor

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