CN108368123B - Substituted imidazolyl compound and pharmaceutical composition thereof - Google Patents

Abstract

A substituted imidazolyl compound and a pharmaceutical composition thereof are disclosed, wherein the compound is shown as a formula (I), or a crystal form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof. The compound has better hepatitis C virus protein NS5A inhibition activity, better pharmacodynamics/pharmacokinetics performance, good applicability and high safety, and can be used for preparing medicines for treating hepatitis C virus infection.

Description

Substituted imidazolyl compound and pharmaceutical composition thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a substituted imidazolyl compound, a pharmaceutical composition thereof, and application thereof in preparation of a medicine for treating hepatitis C.

Background

HCV (Hepatitis C Virus) is an RNA Virus belonging to the genus Hepatitis C Virus (Hepacivirus genus) in the family Flaviviridae (Flaviviridae family). The encapsulated HCV virions comprise a positive strand 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 about 3000 amino acids. The polyprotein includes the core protein, the envelope proteins E1 and E2, the membrane bound protein P7, and the nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS 5B.

HCV infection is associated with progressive liver disease symptoms, including cirrhosis and hepatocellular carcinoma. Harvoni, a fixed dose of the combination of the novel antiviral NS5A inhibitor Ledipasvir (GS5885) and the NS5B blocker Sofosbuvir doublet, is a heavy pound hepatitis C treatment approved by Gilidde 2013 at 12 months. Harvoni was the first approved oral anti-hepatitis C regimen for treatment of genotype 1 hepatitis C infection that did not require combination with interferon or ribavirin. Harvoni can be used either as a single agent or in combination with other oral formulations such as ribavirin.

Harvoni produced by Producer of Gelidder, USA is expensive, and the price of the drug alone is not affordable for ordinary families, so that the development of compounds having inhibitory activity or better pharmacodynamic properties on the hepatitis C virus protein NS5A is still needed in China.

Disclosure of Invention

In view of the above technical problems, the present invention discloses a hepatitis c virus inhibitor, a pharmaceutical composition and use thereof, which has better hepatitis c virus protein NS5A inhibitory activity and/or better pharmacodynamic/pharmacokinetic properties.

In contrast, the technical scheme adopted by the invention is as follows:

a hepatitis C virus inhibitor is a compound shown as a formula (I) or a crystal form, a pharmaceutically acceptable salt, a hydrate or a solvent compound thereof,

wherein R is¹、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、R³²Each independently is hydrogen, deuterium, halogen;

X¹、X²、X³、X⁴、X⁵、X⁶each independently is one or more deuterated methyl, ethyl;

with the proviso that 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、X¹、X²、X³、X⁴、X⁵And X⁶At least one of which is deuterated or contains deuterium.

Preferably, R of the compound of formula (I)¹、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、X¹、X²、X³、X⁴、X⁵And X⁶At least one of R/X, preferably two of R/X, more preferably three of R/X, more preferably four of R/X, more preferably five of R/X, more preferably six of R/X, more preferably seven of R/X, more preferably eight of R/X, more preferably nine of R/X, more preferably ten of R/X, more preferably eleven of R/XMore preferably twelve R/X deuterium containing, more preferably thirteen R/X deuterium containing, more preferably fourteen R/X deuterium containing, more preferably fifteen R/X deuterium containing, more preferably sixteen R/X deuterium containing, more preferably seventeen R/X deuterium containing, more preferably eighteen R/X deuterium containing, more preferably nineteen R/X deuterium containing, more preferably twenty one R/X deuterium containing, more preferably twenty two R/X deuterium containing, more preferably twenty three R/X deuterium containing, more preferably twenty four R/X deuterium containing, more preferably twenty five R/X deuterium containing, more preferably twenty six R/X deuterium containing, more preferably twenty seven R/X deuterium containing, more preferably twenty eight R/X deuterium containing, more preferably twenty nine R/X deuterium containing, more preferably thirty R/X contain deuterium, more preferably thirty one R/X contain deuterium, more preferably thirty two R/X contain deuterium, more preferably thirty three R/X contain deuterium, more preferably thirty four R/X contain deuterium, more preferably thirty five R/X contain deuterium, more preferably thirty six R/X contain deuterium, more preferably thirty seven R/X contain deuterium, more preferably thirty eight R/X contain deuterium.

Preferably, the deuterium isotope content of deuterium at the deuterated position is at least greater than the natural deuterium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, more preferably greater than 99%.

More specifically, in the present invention 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、X¹、X²、X³、X⁴、X⁵And X⁶The deuterium isotope content in each deuterated position is at least 5%, preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, more preferably greater than 25%, more preferably greater than 30%, more preferably greater than 35%, more preferably greater than 40%, more preferably greater than 20%At 45%, more preferably greater than 50%, more preferably greater than 55%, more preferably greater than 60%, more preferably greater than 65%, more preferably greater than 70%, more preferably greater than 75%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, more preferably greater than 99%.

As a further improvement of the invention, R¹、R²、R³、R⁴And R⁵Each independently is deuterium or hydrogen.

As a further improvement of the invention, R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³And R¹⁴Each independently is deuterium or hydrogen.

As a further improvement of the invention, R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³And R²⁴Each independently is deuterium or hydrogen.

As a further improvement of the invention, R²⁵、R²⁶、R²⁷、R²⁸、R²⁹、R³⁰Each independently is deuterium or hydrogen.

As a further improvement of the invention, R³¹、R³²Each independently is deuterium or hydrogen.

As a further development of the invention, X¹Is a once or multiply deuterated methyl group.

As a further development of the invention, X⁴And X⁵Each independently is one or more deuterated methyl groups.

More preferably, R³¹、R³²Is deuterium, and X⁴And X⁵Is a three-deuterated methyl group.

More preferably, X¹Is a tri-deuterated methyl group

As a further improvement of the present invention, the compound is selected from the following compounds or pharmaceutically acceptable salts thereof:

the invention also discloses a pharmaceutical composition which contains a pharmaceutically acceptable carrier and the hepatitis C virus inhibitor, or a crystal form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof.

As a further improvement of the invention, the pharmaceutically acceptable carrier comprises at least one of a glidant, a sweetener, a diluent, a preservative, a dye, a colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersing agent, a disintegrant, a suspending agent, a stabilizer, an isotonic agent, a solvent, or an emulsifier.

As a further improvement of the present invention, the pharmaceutical composition is a tablet, pill, capsule, powder, granule, paste, emulsion, suspension, solution, suppository, injection, inhalant, gel, microsphere or aerosol. The pharmaceutical compositions of the present invention may be formulated as solid, semi-solid, liquid or gaseous formulations.

Typical routes of administration of the pharmaceutical compositions of the present 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 compositions of the present invention may be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

As a further development of the invention, it also comprises an active compound which is an immunomodulator or antiviral drug compound.

As a further improvement of the invention, the immunomodulator is an interferon drug compound.

As a further improvement of the present invention, the antiviral drug compound is ribavirin, amantadine, another inhibitor of NS5A, helicase, protease, polymerase, metalloprotease in the HCV life cycle, or an inhibitor of an internal ribosome entry site target, wherein the other inhibitor of NS5A is ledipasvir or daclatavia.

The invention discloses application of the hepatitis C virus inhibitor, and application of the hepatitis C virus inhibitor in preparing a medicament for treating hepatitis C virus infection.

Preferably, the hepatitis c virus HCV includes a plurality of genotypes and a plurality of gene subtypes, preferably 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a, 6 a. "

Herein, "halogen" means F, Cl, Br, and I, unless otherwise specified. More preferably, the halogen atom is selected from F, Cl and Br.

Herein, "deuterated", unless otherwise specified, means that one or more hydrogens of a compound or group are replaced with deuterium; deuterium can be mono-, di-, poly-, or fully substituted. The terms "deuterated one or more" and "deuterated one or more" are used interchangeably.

Herein, unless otherwise specified, "non-deuterated compound" means a compound containing deuterium at an atomic ratio of deuterium not higher than the natural deuterium isotope content (0.015%).

The invention also includes isotopically-labeled compounds, equivalent to those disclosed herein as the original compound. Examples of isotopes that can be listed as compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, respectively²H，³H，¹³C，¹⁴C，¹⁵N，¹⁷O，¹⁸O，³¹P，³²P，³⁵S，¹⁸F and³⁶and (4) Cl. The compounds of the present invention, or enantiomers, diastereomers, isomers, or pharmaceutically acceptable salts or solvates thereof, wherein isotopes or other isotopic atoms containing such compounds are within the scope of the present invention. Certain isotopically-labelled compounds of the invention, e.g.³H and¹⁴placing of CRadioisotopes are also useful, among other things, in tissue distribution experiments for drugs and substrates. Tritium, i.e.³H and carbon-14, i.e.¹⁴C, their preparation and detection are relatively easy, and are the first choice among isotopes. Isotopically labeled compounds can be prepared by conventional methods by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents using the protocols set forth in the examples.

Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, and the like; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, 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 and the like; and amino acids such as proline, phenylalanine, aspartic acid, glutamic acid, etc. Another preferred class of salts are those of the compounds of the invention with bases, for example alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (e.g., lower alkanolammonium salts and other pharmaceutically acceptable amine salts), for example methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, triethanolamine salts, and amine salts formed from morpholine, piperazine, lysine, respectively.

The term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio. "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.

Compared with the prior art, the invention has the beneficial effects that:

first, the compounds of the present invention have excellent inhibitory activity against the hepatitis c virus protein NS 5A.

Secondly, the technical scheme of the invention changes the metabolism of the compound in organisms through deuteration, so that the compound has better pharmacokinetic parameter characteristics. In this case, the dosage can be varied and a long acting formulation formed, improving the applicability.

Thirdly, deuterium is used for replacing hydrogen atoms in the compound, and due to the deuterium isotope effect, the medicine concentration of the compound in an animal body is improved, and the medicine curative effect is improved.

Fourth, replacement of hydrogen atoms in compounds with deuterium improves the safety of the compounds as certain metabolites are inhibited.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The following describes more specifically the processes for the preparation of the compounds of formula (I) according to the invention, but these particular processes do not constitute any limitation of the invention. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.

In general, in the preparation schemes, each reaction is usually carried out in an inert solvent at a temperature ranging from room temperature to reflux temperature (25 ℃ to 100 ℃, preferably 25 ℃ to 80 ℃). The reaction time is usually 0.1 to 60 hours, preferably 0.5 to 24 hours.

Example 1

Preparing a hepatitis C virus inhibitor V-1 with the following molecular formula:

the preparation method comprises the following steps:

step 1, compound 3 is synthesized.

After compound 1(7.11g, 25mmol) and compound 2(4.26g, 26.3mmol) were added to 20mL of Dimethylformamide (DMF), the mixture was stirred at room temperature for 10 minutes and dispersed uniformly, potassium carbonate (3.8g, 27.5mmol) was added thereto, and the mixture was heated in an oil bath at 50 ℃ and stirred for 2 hours, and TLC showed that the reaction was complete. The reaction mixture was poured into 200mL of water, extracted with ethyl acetate, the organic phases combined, washed with 2N aqueous NaOH, then dried over anhydrous sodium sulfate, filtered and evaporated to dryness to afford intermediate 3 as a cream solid (8.83g, 97% yield).¹H NMR(300MHz，CDCl₃)δ7.70-7.40(m，3H)，7.35-7.05(m，3H)，5.10(s，2H)，2.92(s，2H)，2.65(s，2H)，2.13(s，2H)。

And 2, synthesizing a compound 4.

Compound 3(8.83g, 24mmol), palladium pivalate (370mg, 1.2mmol), tris (4-fluoro-phenyl) phosphine (379mg, 1.2mmol), pivalic acid (735mg, 7.2mmol) and potassium carbonate (6.6g, 48mmol) were charged under nitrogen into a 500mL single-neck reaction flask, and dimethylacetamide (DMAC, 120mL) deoxygenated by nitrogen was added. The above reaction system was replaced with nitrogen gas three times with stirring, and then the reaction was stirred in an oil bath at 65 ℃ for 3.5 hours with stirring, and TLC showed that the reaction was complete. The heating was turned off, the reaction system was cooled to room temperature, then DMAC was evaporated to dryness under reduced pressure, 5g of celite was added, 100mL of ethyl acetate and 50mL of water were added, stirring was carried out at room temperature for 2 hours, and the liquid was separated by filtration. The organic phase was evaporated to dryness and column chromatography afforded compound 4 as a yellow particulate solid (3.72g, 54% yield).¹H NMR(300MHz，DMSO-d₆)δ7.96(d，J＝8.4Hz，1H)，7.90(s，1H)，7.51(dd，J＝8.3，2.2Hz，1H)，7.45(d，J＝2.1Hz，1H)，7.35(s，1H)，5.14(s，2H)，2.95(t，J＝5.9Hz，2H)，2.69-2.53(m，2H)，2.05(dd，J＝12.2，6.3Hz，2H)。

And 3, synthesizing a compound 5.

Compound 4(3.72g, 13.07mmol) obtained above, palladium acetate (147mg, 0.65mmol), 2-dicyclohexylphosphine-2 ', 6' -dimethoxybiphenyl (Sphos, 538mg, 1.31mmol), potassium vinyltrifluoroborate (5.25g, 39.21mmol) and potassium carbonate (5.42g, 39.21mmol) were charged under nitrogen into a 250mL single-neck reaction flask, and 130mL of n-propanol deoxygenated by nitrogen were added. Nitrogen gas device of the above reaction systemAnd (3) changing for three times, heating and refluxing in an oil bath at 120 ℃ for 12 hours, monitoring by LC-MS to show that the raw materials disappear, turning off heating, cooling to room temperature, and evaporating the n-propanol to dryness under reduced pressure. To the paste obtained above was added 50mL of ethyl acetate, stirred at room temperature for 30 minutes, filtered, and the filtrate was evaporated under reduced pressure to dryness and column chromatography was performed to obtain compound 5 as a yellow granular solid.¹H NMR(400MHz，CDCl₃)δ7.70(d，J＝8.1Hz，1H)，7.62(s，1H)，7.58(s，1H)，7.43(d，J＝8.2Hz，1H)，7.22(s，1H)，6.73(dd，J＝17.6，10.9Hz，1H)，5.81(d，J＝17.6Hz，1H)，5.32(d，J＝11.3Hz，1H)，5.12(s，2H)，2.97(t，J＝6.0Hz，2H)，2.74-2.57(m，2H)，2.14(dd，J＝12.6，6.3Hz，2H)。

And 4, synthesizing a compound 6.

The intermediate 5 obtained above was dissolved in a mixed solvent of THF-DMSO-water (10mL +10mL +5mL), and N-bromosuccinimide (NBS, 2.4g, 13.76mmol) was added thereto at room temperature to stir the reaction for 1 hour, and TLC showed completion of the reaction. THF in the reaction system is evaporated under reduced pressure, then 100mL of water is added for dilution, ethyl acetate is used for extraction, organic phases are combined, saturated salt solution is used for washing, then anhydrous magnesium sulfate is used for drying, and the solvent is filtered and evaporated to dryness to obtain an intermediate. The crude intermediate was dissolved in 20mL DCM and activated manganese dioxide (24g, 275.2mmol) was added and the reaction stirred at RT overnight, TLC indicated complete reaction. Insoluble solids are removed by filtration, 5mL of methanol is added to the filtrate, DCM is distilled off under reduced pressure until a large amount of orange-yellow solid precipitates, the reduced pressure distillation is stopped, and the solid is collected by filtration. 20mL of DCM is added into the mother liquor, then the mixture is distilled under reduced pressure until orange yellow solid is separated out, the reduced pressure distillation is stopped, and the solid is collected by filtration. The above operations were repeated once, and the solids obtained by combining three times were intermediate 6(1.72g, yield 35%).¹H NMR(300MHz，CDCl₃)δ8.01(d，J＝8.2Hz，1H)，7.91-7.77(m，2H)，7.66(s，2H)，5.18(s，2H)，4.45(s，2H)，2.99(t，J＝6.0Hz，2H)，2.73-2.57(m，2H)，2.28-2.07(m，2H)。

And 5, synthesizing a compound 8.

Intermediate 6(1.41g, 3.8mmol), amino acid 7(1.09g, 4.2mmol) were added to acetonitrile (8mL) and stirred to disperse well, diisopropylethylamine (517mg, 4.0mmol) was added and stirred at room temperatureShould be overnight, TLC showed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure, and column chromatography was performed to give intermediate 8 as a gray powdery solid (1.3g, yield 62%).¹H NMR(500MHz，CDCl₃)δ7.92(t，J＝7.6Hz，1H)，7.83(d，J＝8.2Hz，1H)，7.73(s，1H)，7.65(d，J＝3.3Hz，2H)，5.68-5.03(m，4H)，4.50-4.37(m，1H)，3.84-3.60(m，1H)，3.53-3.30(m，5H)，3.24(dd，J＝10.8，7.8Hz，1H)，2.99(t，J＝6.0Hz，2H)，2.73-2.63(m，2H)，2.56(dd，J＝19.8，7.2Hz，2H)，2.19-2.12(m，2H)，1.45(s，9H)。

And 6, synthesizing a compound 9.

Intermediate 8(1.3g, 2.36mmol) was dissolved in DCM-MeOH (17mL +5mL), pyridine trihydrobromide (0.832g, 2.60mmol) was added in one portion and the reaction stirred at room temperature for 2.5 h, TLC indicated complete reaction. The reaction mixture was washed with 4N hydrochloric acid and saturated brine, then dried over anhydrous sodium sulfate, filtered and evaporated to dryness to give intermediate 9 as a yellow-green foamy solid (1.46 g).

Step 7. Synthesis of Compound 11.

Taking pyroglutamic acid ethyl ester 10(3.97g, 15.4mmol), adding 30mL of anhydrous tetrahydrofuran for dissolving under the protection of nitrogen, cooling to-40 ℃ by using a constant-temperature low-temperature reaction bath, slowly dropwise adding a tetrahydrofuran solution (3M, 5.4mL) of methyl magnesium chloride, stirring for reaction after dropwise addition is finished, and naturally heating to room temperature. TLC showed complete disappearance of the starting material, 4N hydrochloric acid was added dropwise to the reaction under ice-cooling until the system pH reached about 2, the liquid was separated, the tetrahydrofuran phase was washed with saturated brine and then column chromatography was evaporated under reduced pressure to dryness to afford intermediate 11 as a colorless oil (3.80g, 91% yield).¹H NMR(400MHz，CDCl₃)δ5.10(d，J＝6.9Hz，1H)，4.36-4.05(m，3H)，2.54(dd，J＝15.4，7.8Hz，2H)，2.21-1.72(m，5H)，1.44(s，9H)，1.28(t，J＝7.1Hz，3H)。

Step 8, compound 12 is synthesized.

The intermediate 11 obtained above is added with DCM (20mL) to be dissolved, then 10mL of trifluoroacetic acid is added, the reaction is stirred at room temperature until no bubbles are generated in the system, and the colorless oily matter is obtained after reduced pressure evaporation. Dissolving the obtained oily substance in 60mL of anhydrous ethanol, adding 5% Pd/C (380mg), introducing 4 atm hydrogen, and reacting at room temperatureAnd (4) at night. LC-MS showed the disappearance of the starting material, hydrogen was removed, Pd/C was removed by filtration and the filtrate was evaporated to dryness to afford intermediate 12(4.92 g).¹H NMR(400MHz，CDCl₃)δ11.29(s，1H)，4.49(dd，J＝9.3，4.3Hz，1H)，4.30(q，J＝7.1Hz，2H)，3.96-3.85(m，1H)，2.53-2.40(m，1H)，2.34-2.17(m，2H)，1.74-1.60(m，1H)，1.51(d，J＝6.5Hz，2H)，1.32(t，J＝7.2Hz，3H)。

Step 9. Synthesis of Compound 13.

Taking intermediate 12(0.84g), N-Moc-L-valine-d₈(0.35g, 1.91mmol) is dissolved in anhydrous DMF (5mL), cooled in an ice bath, then diisopropylethylamine (DIPEA, 2.77g, 21.45mmol) and 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU, 0.91g, 2.39mmol) are added in sequence, and the reaction is stirred at the natural temperature for 2 hours. The reaction solution was poured into 50mL of water, extracted with ethyl acetate, and the organic phase was evaporated to dryness under reduced pressure to give a crude intermediate as an oil. The oily substance is added with 10mL of MeOH to be dissolved, the mixture is cooled in an ice bath, 5mL of 1M lithium hydroxide aqueous solution is added dropwise, the reaction is naturally heated for 3 hours after the dropwise addition, and TLC shows that the intermediate disappears. The MeOH in the reaction mixture was evaporated under reduced pressure, the aqueous phase was extracted with DCM, then 4N hydrochloric acid was added dropwise to the aqueous phase to adjust the pH to about 2, DCM was extracted, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and evaporated to dryness to give intermediate 13 as a colorless foamy solid (0.46 g).¹H NMR(300MHz，CDCl₃)δ5.24(s，1H)，4.65(s，1H)，4.56(t，J＝8.6Hz，1H)，3.67(s，3H)，2.48-2.38(m，2H)，2.09-2.01(m，2H)，1.85-1.75(m，1H)，1.30(d，J＝6.7Hz，3H)。

And step 10, synthesizing a compound V-1.

Intermediate 9(408mg, 0.65mmol) and dipeptide derivative 13(325mg, 1.10mmol) were taken, 3mL of 2-methyl-tetrahydrofuran was added and stirred at room temperature until clear, cesium carbonate (169mg, 0.52mmol) was added all at once and stirred at room temperature for 10 minutes, after which the reaction system was transferred to a 50 ℃ constant temperature oil bath and stirred for reaction for 24 hours, and TLC showed disappearance of intermediate 9. The heating was turned off and the reaction was allowed to cool to room temperature and diluted with 10mL of ethyl acetate before washing with saturated sodium bicarbonate. Organic phase was chromatographed by autoclaving to give the intermediate as a gray foamy solid diester (358 mg).

The diester intermediate (358mg, 0.42mmol) was taken and 10mL of toluene and 1mL of ethylene glycol methyl ether were added, and stirred at room temperature to dissolve it, and 1.64g (21.26mmol) of ammonium acetate was added in one portion, followed by heating and refluxing reaction in a constant temperature oil bath at 110 ℃ for 4 hours, and TLC showed disappearance of the intermediate. And (3) turning off heating, naturally cooling to room temperature, adding 10mL of ethyl acetate and 2M of sodium hydroxide aqueous solution into the reaction, washing, separating liquid, drying an organic phase by anhydrous sodium sulfate, and then concentrating under reduced pressure to obtain a crude intermediate. The crude product was dissolved in 10mL of DCM, and activated manganese dioxide (2.22g, 25.50mmol) was added in one portion with stirring and reacted overnight at room temperature. The reaction system was filtered, and the residue was washed with methanol until the filtrate was colorless. The combined filtrates were evaporated to dryness and subjected to column chromatography to give a compound as a gray solid (109 mg).

The intermediate (109mg, 0.17mmol) was dissolved in 3mL of anhydrous DCM, and 4N HCl-dioxane solution was added at room temperature and the reaction was stirred for 2 hours until the starting material disappeared. DCM, hydrogen chloride and dioxane in the reaction system are evaporated to dryness under reduced pressure to obtain a brown solid as an intermediate, anhydrous DMF (3 mL) is added, diisopropylethylamine (176mg, 1.32mmol) is added, stirring and dissolving are carried out, the solution is cooled in an ice bath, N-Moc-L-phenylglycine (34mg, 0.16mmol) and HATU (62mg, 0.16mmol) are added successively, and stirring and reaction are carried out for 30 minutes. The above reaction solution was added to 30mL of water, extracted with ethyl acetate, combined, evaporated to dryness, and subjected to column chromatography to obtain V-1 as a yellow powdery solid (67 mg). LC-MS (APCI): m/z 889.9(M +1)⁺，¹H NMR(500MHz，MeOD)δ8.46-7.21(m，12H)，5.59(s，1H)，5.34-5.09(m，3H)，4.35-4.02(m，1H)，3.96-3.34(m，8H)，3.29-2.89(m，2H)，2.75-2.54(m，1H)，2.45(s，1H)，2.40-2.11(m，2H)，1.99(s，1H)，1.60(d，J＝6.6Hz，2H)，1.31(s，1H)，1.15(s，1H)。

Example 2

Preparing a hepatitis C virus inhibitor V-2 with the following molecular formula:

the preparation method comprises the following steps:

step 1. Synthesis of Compound 14.

Intermediate 12(1.50g), N-Moc- (d) was taken₃) L-valine (0.76g, 4.29mmol) was dissolved in anhydrous DMF (5mL), cooled in an ice bath, diisopropylethylamine (2.77g, 21.45mmol) and HATU (1.96g, 5.15mmol) were added, and the reaction was stirred at room temperature for 2 hours. The reaction solution was poured into 50mL of water, extracted with ethyl acetate, and the organic phase was evaporated to dryness under reduced pressure to give an oily intermediate. To this oil was added THF (23 mL) and dissolved, and then cooled in an ice bath, 12mL of 1M aqueous lithium hydroxide solution was added dropwise, and after the addition, the reaction was naturally warmed up for 3 hours, and TLC showed the disappearance of the starting material. THF in the reaction solution was distilled off under reduced pressure, the aqueous phase was extracted with dichloromethane, then 4N hydrochloric acid was added dropwise to the aqueous phase to adjust pH to about 2, dichloromethane was extracted, the combined organic phases were washed with saturated saline, dried over anhydrous sodium sulfate, filtered and evaporated to dryness to give intermediate 14 as a colorless foamy solid (0.9 g).¹H NMR(500MHz，CDCl₃)δ5.49(d，J＝9.2Hz，1H)，4.67-4.61(m，1H)，4.52(t，J＝8.5Hz，1H)，4.22-4.15(m，1H)，2.27(dd，J＝8.7，4.2Hz，2H)，2.05(d，J＝11.5Hz，2H)，1.77(d，J＝12.4Hz，1H)，1.32(d，J＝6.6Hz，3H)，1.00(d，J＝6.5Hz，3H)，0.97(d，J＝6.5Hz，3H)，0.93(dd，J＝7.7，5.5Hz，1H)。

And 2, synthesizing a compound V-2.

Intermediate 9(373mg, 0.65mmol) and dipeptide derivative 14(292mg, 1.01mmol) were taken, 5mL of 2-methyl-tetrahydrofuran was added and stirred at room temperature until clear, cesium carbonate (154mg, 0.47mmol) was added all at once, and stirred at room temperature for 10 minutes, after which the reaction system was transferred to a 40 ℃ constant temperature oil bath and stirred for reaction for 24 hours, and TLC showed disappearance of intermediate 9. Heating was turned off, filtered and the column chromatography evaporated to dryness to give the diester intermediate as a grey foamy solid (145 mg).

The diester intermediate (145mg, 0.17mmol) was taken, 5mL of toluene and 0.5mL of ethylene glycol methyl ether were added, the mixture was stirred at room temperature to dissolve, ammonium acetate (267mg, 3.46mmol) was added in one portion, and then the mixture was heated under reflux in a constant temperature oil bath at 110 ℃ for 7 hours, and TLC showed that the starting material was completely reacted. And (3) turning off heating, naturally cooling to room temperature, adding 10mL of ethyl acetate into the reaction, washing with saturated sodium bicarbonate aqueous solution, separating liquid, drying an organic phase through anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a crude product of the intermediate. The crude intermediate was dissolved in 5mL of dichloromethane and activated manganese dioxide (839mg, 8.5mmol) was added in one portion with stirring and reacted overnight at room temperature. The reaction system was filtered, and the residue was washed with methanol until the filtrate was colorless. The combined filtrates were evaporated to dryness and subjected to column chromatography to give a grey solid intermediate (42 mg).

The intermediate (42mg, 0.053mmol) was dissolved in 3mL of anhydrous dichloromethane, and 4N HCl-dioxane solution was added at room temperature and the reaction was stirred for 2 hours until the starting material disappeared. Evaporating dichloromethane, hydrogen chloride and dioxane in the reaction system to dryness under reduced pressure to obtain brown solid intermediate, adding anhydrous DMF 3mL, adding diisopropylethylamine (82mg, 0.63mmol), stirring to clear, cooling the solution in ice bath, and sequentially adding N-Moc (d)₃) L-phenylglycine (13mg, 0.063mmol) and HATU (24mg, 0.063mmol), and the reaction was stirred for 30 minutes. The above reaction solution was added to 30mL of water, extracted with ethyl acetate, combined, evaporated to dryness, and subjected to column chromatography to obtain V-2 as a yellow powdery solid (18 mg). LC-MS (APCI): m/z 889.9(M +1)⁺，¹H NMR(400MHz，MeOD)δ8.44-6.89(m，12H)，5.57(s，1H)，5.34-4.98(m，3H)，4.35-3.93(m，2H)，3.72-3.35(m，2H)，3.29-2.80(m，4H)，2.77-2.03(m，5H)，1.96(s，1H)，1.58(d，J＝6.6Hz，2H)，1.28(s，1H)，1.14-1.01(m，4H)，0.98-0.80(m，3H)。

Example 3

Preparing a hepatitis C virus inhibitor V-3 with the following molecular formula:

the preparation method comprises the following steps:

step 1. Synthesis of Compound 15.

Magnesium turnings (1.36g, 57mmol) were taken and added to 40mL of anhydrous ether under nitrogen atmosphere with elemental iodine (50 mg). And dropwise adding 0.9mL of deuterated iodomethane into the system under stirring, heating to slightly boil the diethyl ether, gradually eliminating the reddish brown color of the system to obtain a milky suspension, slowly dropwise adding 2mL of deuterated iodomethane, heating after dropwise adding to keep the diethyl ether slightly boiling for reacting for 2 hours, and cooling to obtain the deuterated methyl magnesium iodide Grignard reagent. Taking pyroglutamic acid ethyl ester 10(4.15g, 16.1mmol), adding 30mL of anhydrous tetrahydrofuran for dissolving under the protection of nitrogen, cooling to-40 ℃ by using a constant-temperature low-temperature reaction bath, slowly dropwise adding the ether solution (15mL) of the deuterated methyl magnesium iodide, stirring for reaction after dropwise addition, and naturally heating to room temperature. After the raw materials completely disappeared, 4N hydrochloric acid was added dropwise to the reaction under ice-bath cooling until the system pH reached about 2, and the mixture was separated, the tetrahydrofuran phase was washed with saturated brine and then column chromatography was evaporated to dryness under reduced pressure to obtain intermediate 15 as a colorless oil (2.63g, yield 59%).¹H NMR(300MHz，CDCl₃)δ5.10(d，J＝7.4Hz，1H)，4.35-4.07(m，3H)，2.53(dd，J＝8.5，6.9Hz，2H)，2.12(dd，J＝13.1，6.3Hz，1H)，1.87(tt，J＝14.3，7.2Hz，1H)，1.44(s，9H)，1.28(t，J＝7.1Hz，3H)。

Step 2. Synthesis of Compound 16.

And (3) dissolving the intermediate 15(0.61g and 2.2mmol) in 6mL of DCM, adding 3mL of deuterated trifluoroacetic acid, stirring at room temperature to react until no bubbles are generated in the system, and evaporating under reduced pressure to obtain a colorless oily substance. The resulting oil was dissolved in 6mL of deuterated absolute ethanol, 5% Pd/C (60mg, heavy water, ethanol-d washed three times each) was added, and the reaction was allowed to proceed overnight at room temperature by passing 4 atmospheres of deuterium gas through the mixture. LC-MS shows that the raw material disappears, deuterium gas is removed, Pd/C is removed by filtration, and the filtrate is evaporated to dryness to obtain an intermediate 16.¹H NMR(300MHz，CDCl₃)δ4.51(dd，J＝9.3，4.2Hz，1H)，4.38-4.24(m，2H)，2.64-2.19(m，2H)，1.59-1.19(m，5H)。

Step 3, compound 17 is synthesized.

The intermediate 16, N-Moc-L-valine (0.47g, 2.69mmol) obtained above was dissolved in anhydrous DMF (5mL) and cooled in an ice bath, after whichDiisopropylethylamine (2.90g, 22.4mmol) and HATU (1.02g, 2.69mmol) were added successively, and the reaction was stirred at natural temperature for 2 hours. The reaction was poured into 50mL of water, extracted with ethyl acetate, and the organic phase was evaporated to dryness under reduced pressure to give an oil. The oily substance is added with 10mL of anhydrous methanol for dissolution, the mixture is cooled in an ice bath, 5mL of 1M lithium hydroxide aqueous solution is dropwise added, the temperature is naturally raised for reaction for 3 hours after the dropwise addition, and TLC shows that the raw material disappears. The methanol in the reaction solution was evaporated under reduced pressure, the aqueous phase was extracted with dichloromethane, then 4N hydrochloric acid was added dropwise to the aqueous phase to adjust the pH to about 2, dichloromethane was extracted, saturated saline was combined and washed with anhydrous sodium sulfate, dried, filtered and evaporated to dryness to obtain intermediate 17 as a colorless foamy solid (0.45 g).¹H NMR(500MHz，CDCl₃)δ4.56(t，J＝8.7Hz，1H)，4.20(t，J＝8.9Hz，1H)，3.67(s，3H)，2.45-2.36(m，2H)，2.05(d，J＝7.7Hz，2H)，1.01(d，J＝6.7Hz，3H)，0.97(d，J＝6.7Hz，3H)，0.94(d，J＝7.0Hz，1H)。

And 4, synthesizing a compound V-3.

A similar process to that described in step 10 of example 1, except that Compound 17 is used instead of Compound 13, thereby obtaining the objective compound V-3. LC-MS (APCI): m/z 889.9(M +1)⁺，¹H NMR(500MHz，MeOD)δ8.36-7.09(m，12H)，5.61-5.53(m，1H)，5.30-5.01(m，3H)，4.42-4.02(m，2H)，3.83-3.34(m，7H)，3.31-3.07(m，2H)，2.99-2.33(m，3H)，2.08(dd，J＝19.7，12.3Hz，1H)，1.98(s，1H)，1.29(s，1H)，1.14-0.79(m，6H)。

Example 4

Preparing a hepatitis C virus inhibitor V-4 with the following molecular formula:

the preparation method comprises the following steps:

step 1. Synthesis of Compound 18.

Intermediate 12(1.64g), N-Moc-L-valine (0.82g, 4.67mmol) were dissolved in 5mL of anhydrous DMF and cooled in an ice bath, diisopropylethylamine (3.02g, 23.35mmol) and HATU (2.13g, 5.60mmol) were added, and the reaction was stirred at room temperature for 2 hours. The reaction was poured into 50mL of water, extracted with ethyl acetate and the organic phase was evaporated to dryness under reduced pressure to give a red oily intermediate. And (3) adding 23mL of anhydrous methanol to the oily matter for dissolving, cooling in an ice bath, dropwise adding 12mL of 1M lithium hydroxide aqueous solution, naturally heating to react for 3 hours after the dropwise adding is finished, evaporating the methanol in the reaction solution under reduced pressure after the intermediate disappears, extracting the aqueous phase with dichloromethane, dropwise adding 4N hydrochloric acid into the aqueous phase to adjust the pH to about 2, extracting with dichloromethane, combining saturated salt, washing with anhydrous sodium sulfate, drying, filtering and evaporating to dryness to obtain an intermediate 18 which is a colorless foamy solid (1.01 g).¹H NMR(400MHz，CDCl₃)δ5.54(d，J＝9.4Hz，1H)，4.70-4.59(m，1H)，4.51(t，J＝8.8Hz，1H)，4.18(t，J＝9.2Hz，1H)，3.66(s，3H)，2.33-2.22(m，2H)，2.10-1.99(m，2H)，1.77(d，J＝12.0Hz，1H)，1.32(d，J＝6.7Hz，3H)，1.00(d，J＝6.7Hz，3H)，0.97(d，J＝6.7Hz，3H)，0.92(dd，J＝6.0，4.4Hz，1H)。

Step 2. Synthesis of Compound 19.

Taking intermediate 9(654mg, 1.04mmol) and dipeptide derivative 18(200mg, 0.67mmol), adding 4mL of 2-methyl-tetrahydrofuran, stirring at room temperature until the mixture is clear, adding cesium carbonate (114mg, 0.35mmol) at one time, stirring at room temperature for 10 minutes, transferring the reaction system to a 50 ℃ constant-temperature oil bath, stirring and reacting for 24 hours, stopping heating after the intermediate 9 is completely reacted, cooling the reaction system to room temperature, adding 10mL of dichloromethane for dilution, and washing with water. The organic phase was reduced by pressure-less, dry column chromatography to give diester intermediate 19 as a gray foamy solid (433 mg). LC-MS (APCI): m/z 835.4(M +1)⁺。

And 3, synthesizing a compound 20.

Taking diester intermediate 19(433mg, 0.52mmol), adding 10mL of toluene and 1mL of ethylene glycol methyl ether, stirring at room temperature to dissolve, adding ammonium acetate (2.00g, 25.99mmol) at one time, heating and refluxing in a constant-temperature oil bath at 110 ℃ for 4 hours, closing heating,and naturally cooling to room temperature, adding 10mL of ethyl acetate and 2M sodium hydroxide aqueous solution into the reaction, washing, separating, drying an organic phase by anhydrous sodium sulfate, and concentrating under reduced pressure to obtain an intermediate crude product. The crude product was dissolved in 10mL of dichloromethane, and activated manganese dioxide (2.26g, 25.99mmol) was added in one portion with stirring and reacted at room temperature overnight. The reaction system was filtered, and the residue was washed with methanol until the filtrate was colorless. The combined filtrates were evaporated to dryness and subjected to column chromatography to give compound 20 as a gray solid (204 mg). LC-MS (APCI): m/z 792.4(M +1)⁺。

And 4, synthesizing a compound V-4.

Intermediate 20(134mg, 0.17mmol) was dissolved in 3mL of anhydrous dichloromethane, and 4N hydrogen chloride-dioxane solution was added at room temperature and the reaction was stirred for 1.5 hours until the starting material disappeared. Evaporating dichloromethane, hydrogen chloride and dioxane in the reaction system to dryness under reduced pressure to obtain brown solid intermediate, adding anhydrous dimethylformamide 3mL, adding diisopropylethylamine (269mg, 1.69mmol), stirring to dissolve, cooling the solution in ice bath, and sequentially adding N-Moc (d)₃) L-phenylglycine (43mg, 0.20mmol) and HATU (77mg, 0.20mmol), and the reaction was stirred for 30 minutes. Adding the reaction solution into 30mL of water, extracting with ethyl acetate, combining, evaporating and performing column chromatography to obtain a yellow powdery solid V-4. LC-MS (APCI): m/z 886.8(M +1)⁺，¹H NMR(400MHz，MeOD)δ8.47-7.22(m，12H)，5.60(s，1H)，5.33-5.03(m，3H)，4.43-4.03(m，2H)，3.95-3.58(m，3H)，3.56-2.86(m，11H)，2.78-2.07(m，5H)，2.00(d，J＝9.5Hz，1H)，1.76-1.29(m，5H)，1.22-0.81(m，8H)。

Example 5

Preparing a hepatitis C virus inhibitor V-5 with the following molecular formula:

the preparation method comprises the following steps:

step 1. Synthesis of Compound 22.

The diester compound 21(2.08g, 7.22mmol) was dissolved in 15mL of tetrahydrofuran, cooled in an ice bath, and 7.22mL of a 1M aqueous solution of sodium hydroxide was slowly added dropwise thereto, followed by allowing the reaction to warm naturally overnight after completion of the dropwise addition. The tetrahydrofuran was evaporated under reduced pressure, the aqueous phase was washed with dichloromethane, then the pH was adjusted to about 2 with 4N hydrochloric acid, extracted with dichloromethane, the combined organic phases were washed with saturated brine and then dried over anhydrous sodium sulfate. Filtration to dryness afforded intermediate 22 as a colorless oil, 1.71 g.

Step 2. compound 23 was synthesized.

10mL of anhydrous tetrahydrofuran was added to dissolve the monocarboxylic acid 22 obtained above, the resulting solution was cooled in an ice bath, and triethylamine (0.95g, 9.38mmol) and ethyl chloroformate (1.02g, 9.38mmol) were added successively and the reaction was stirred for 2 hours. Insoluble solids in a reaction system are filtered out, filtrate is decompressed and evaporated to dryness to obtain light-colored oily matter, 15mL of tetrahydrofuran is added to be cooled to minus 5 ℃, NaBH4(0.47g, 12.50mmol) is added to 5mL of deionized water to be dissolved, and the dissolved solution is slowly dripped into tetrahydrofuran solution of the light-colored oily matter to be kept at minus 5 ℃ for stirring reaction for 2 hours. 4N hydrochloric acid was added dropwise to the reaction at-5 ℃ to adjust the pH to 2, the mixture was separated, and the organic phase was washed with saturated aqueous sodium bicarbonate solution and dried over anhydrous sodium sulfate to obtain 1.25g of an intermediate 23 as a colorless oil.

Step 3, compound 24 is synthesized.

Dissolving the compound 23 in 20mL of anhydrous dichloromethane, adding 2, 6-di-tert-butylpyridine (1.84g, 9.62mmol) and silver trifluoromethanesulfonate (1.85g, 7.22mmol) at room temperature, stirring and dispersing uniformly, then adding deuterated iodomethane (1.39g, 9.62mmol) dropwise, reacting at room temperature overnight after the dropwise addition is finished, and filtering to remove insoluble solids after the reaction is finished. The filtrate was evaporated to dryness with salt to give the crude 24 as a pale red oil.

Step 4. Synthesis of Compound 25.

The crude 24 is dissolved in 10mL of tetrahydrofuran and 5mL of methanol, cooled in an ice bath, and then 5mL of 2.5M aqueous lithium hydroxide solution is added dropwise, and the mixture is stirred and naturally warmed to react for 4 hours. The methanol and tetrahydrofuran in the system were evaporated to dryness under reduced pressure, and the residue was diluted with 10mL of water. The resulting aqueous solution was washed with dichloromethane and then withThe pH value is adjusted to 2 by 4N hydrochloric acid, dichloromethane is used for extraction, and the combined organic phase is dried by anhydrous sodium sulfate, decompressed and evaporated to dryness to obtain 25 which is colorless oily matter 664 mg.¹H NMR(400MHz，CDCl₃)δ4.44-4.21(m，1H)，3.70-3.62(m，1H)，3.48-3.10(m，3H)，2.45-2.20(3，2H)，2.18-1.75(m，1H)，1.49(s，5H)，1.43(s，4H)。

Step 5. Synthesis of Compound 26.

Taking intermediate 6(442mg, 1.19mmol), amino acid 25(312mg, 1.19mmol), adding 5mL of 2-methyltetrahydrofuran, stirring and dispersing uniformly, adding cesium carbonate (213mg, 0.65mmol), stirring and reacting at room temperature overnight, after the reaction is finished, evaporating the reaction solution under reduced pressure, and performing column chromatography to obtain intermediate 26 as a yellow powdery solid (519 mg).¹H NMR(500MHz，CDCl₃)δ7.91(d，J＝7.3Hz，1H)，7.83(dd，J＝8.0，3.5Hz，1H)，7.73(s，1H)，7.65(s，2H)，5.62-5.19(m，2H)，5.16(s，2H)，4.44(dd，J＝33.4，8.1Hz，1H)，3.86-3.68(m，1H)，3.52-3.34(m，2H)，3.23(dd，J＝10.7，7.7Hz，1H)，2.99(t，J＝5.9Hz，2H)，2.75-2.63(m，2H)，2.54(dd，J＝17.5，10.4Hz，2H)，2.17(dd，J＝12.7，6.1Hz，2H)，2.04(s，1H)，1.44(9H)。

Step 6, compound 27 is synthesized.

Dissolving the intermediate 26(517mg, 0.94mmol) in a dichloromethane-methanol (5mL-2mL) mixed solvent, adding pyridine trihydrobromide (314g, 0.98mmol) once, stirring at room temperature for reacting for 4.5 hours, washing the reaction solution with 4N hydrochloric acid and saturated common salt water successively after the reaction is completed, drying with anhydrous sodium sulfate, filtering and evaporating to obtain a yellow-green foamy solid intermediate, adding the yellow-green intermediate and the compound 18(150mg, 0.52mmol) into 5mL 2-methyltetrahydrofuran, stirring and dispersing uniformly, adding cesium carbonate (86mg, 0.26mmol) once, stirring at room temperature for 10 minutes, transferring the reaction into a 50 ℃ constant temperature oil bath, and heating and stirring for reacting for 24 hours. The reaction was diluted with 10mL of ethyl acetate, washed with water, concentrated and chromatographed to give 232mg of a brown solid diester intermediate. LC-MS (APCI): m/z 837.7(M +1)⁺。

To the diester intermediate (232mg, 0.277mmol) was added toluene-ethylene glycol methyl ether (5/1(V/V), 6mL) and stirred until it became clear, and ammonium acetate (1.068g, 13.86mmol) was added in one portion, followed by heating and stirring in a constant temperature oil bath at 100 ℃ overnight. And after the reaction is finished, cooling to room temperature, adding 10mL of ethyl acetate for dilution, dropwise adding 2M NaOH aqueous solution under ice bath to adjust the pH to 8-9, separating liquid, drying the organic phase through anhydrous sodium sulfate, concentrating and evaporating to dryness to obtain an intermediate crude product.

The crude product was dissolved in 10mL of dichloromethane, manganese dioxide (1.205g, 13.86mmol) was added, and the reaction was stirred at room temperature overnight. After the reaction is finished, filtering is carried out, and filter residues are washed by methanol until filtrate is colorless. The filtrates were combined and evaporated to dryness, and subjected to column chromatography to give compound 27(78 mg). LC-MS (APCI): m/z 795.7(M +1)⁺。

And 7, synthesizing a compound V-5.

Intermediate 27(78mg, 0.0981mmol) was dissolved in 3mL of anhydrous dichloromethane, and 4N hydrogen chloride-dioxane solution was added thereto at room temperature to stir the reaction for 1 hour. Evaporating the reaction solvent to dryness under reduced pressure to obtain brown solid intermediate, adding anhydrous DMF 3mL, adding diisopropylethylamine (142mg, 1.10mmol), stirring to dissolve, cooling the solution in ice bath, and adding N-Moc (d) successively₃) L-phenylglycine (127mg, 0.981mmol) and HATU (45mg, 0.118mmol), and the reaction was stirred for 30 min. The above reaction solution was added to 30mL of water, extracted with ethyl acetate, combined, evaporated to dryness, and subjected to column chromatography to obtain V-5 as a yellow powdery solid (41 mg). LC-MS (APCI): m/z 886.9(M +1)⁺，¹H NMR(400MHz，MeOD)δ8.39-6.91(m，12H)，5.66-5.50(m，1H)，5.29-4.97(m，3H)，4.42-3.96(m，2H)，3.86-3.54(m，5H)，3.27-2.81(m，2H)，2.67-2.46(m，1H)，2.46-2.05(m，4H)，1.95-1.90(m，1H)，1.60(t，J＝13.8Hz，2H)，1.35(td，J＝7.6，4.0Hz，2H)，1.28(s，1H)，1.07-0.76(m，7H)。

Example 6

Preparing a hepatitis C virus inhibitor V-6, the molecular formula is as follows:

the preparation method comprises the following steps:

step 1. compound 30 was synthesized.

Compound 28(4.0g, 16.99mmol) was dissolved in 30mL Tetrahydrofuran (THF), triethylamine (TEA, 2.58g, 25.48mmol) was added to the solution in an ice-water bath, chloroethyl chloroformate (2.77g, 25.48mmol) was added dropwise slowly, and the solution was stirred at room temperature for 30 minutes after dropping. Filtering, spin-drying the filtrate to obtain 29, adding 20ml of THF for dissolving, and dropwise adding NaBD in ice water bath₄4mL of D (1.42g, 33.98mmol)₂The O solution was stirred at room temperature for 30 minutes. 7mL of 4N HCl solution, 10mL of saturated saline and 10mL of ethyl acetate were added to the reaction mixture in this order, the mixture was separated, the organic phase was washed successively with saturated sodium bicarbonate and saturated saline, dried over anhydrous sodium sulfate, filtered and concentrated to give 30g of a pale yellow solid (2.69 g).¹H NMR(500MHz，CDCl₃)δ7.51(t，J＝4.1Hz，1H)，7.46(d，J＝8.5Hz，1H)，7.14(dd，J＝8.5，2.6Hz，1H)。

Step 2, compound 33 is synthesized.

Compound 30(2.68g, 12.02mmol) was dissolved in 24mL of THF, and 60% NaH (577mg, 13mmol) was added in portions in an ice-water bath, and p-toluenesulfonyl chloride (2.29g, 12.02mmol) was added in portions, followed by stirring at room temperature for 30 minutes. After quenching with 1mL of water, 5mL of saturated brine was added, and the organic phase was separated and concentrated to give a crude compound 31 as a pale yellow solid (4.50 g). Compound 31(4.50g, 11.91mmol) and compound 32(1.93g, 11.91mmol) were dissolved in 15mL DMF, potassium carbonate (2.47g, 17.87mmol) was added, and the mixture was stirred at 50 ℃ for 2 hours. The reaction solution was poured into 100mL of water. The aqueous phase was extracted with ethyl acetate/petroleum ether (1: 1), and the organic phases were combined, washed successively with 1N NaOH solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a white solid powder as compound (333.86 g).

Step 3. Synthesis of Compound 34.

Take compound 33(3.86g, 10)50mmol), palladium pivalate (149mg, 0.53mmol), tris (4-fluoro-phenyl) phosphine (166mg, 0.53mmol), pivalic acid (322mg, 3.15mmol) and potassium carbonate (2.90g, 21mmol) were added under nitrogen protection to a 500mL single-neck reaction flask, and dimethylacetamide (DMAC, 60mL) deoxygenated with nitrogen was added. The reaction system is stirred and replaced by nitrogen for three times, then the reaction system is heated and stirred in a 70 ℃ oil bath for 3.5 hours, the heating is closed after the reaction is finished, the reaction system is cooled to the room temperature, then DMAC (dimethylacetamide) is evaporated to dryness under reduced pressure, 5g of diatomite is added, 50mL of ethyl acetate is added, 50mL of water is added, the mixture is stirred at the room temperature for 2 hours, and the liquid is filtered and separated. The organic phase was evaporated to dryness and column chromatography afforded compound 34 as a yellow particulate solid (1.75 mg).¹H NMR(400MHz，CDCl₃)δ7.66(d，J＝8.4Hz，1H)，7.62-7.60(m，1H)，7.55-7.53(m，1H)，7.37(dd，J＝8.4，2.2Hz，1H)，7.18(d，J＝2.1Hz，1H)，3.00-2.94(m，2H)，2.69-2.60(m，2H)，2.20-2.10(m，2H)。

Step 4. Synthesis of Compound 35.

Compound 34(1.75g, 6.10mmol) obtained above, palladium acetate (69mg, 0.31mmol), 2-dicyclohexylphosphine-2 ', 6' -dimethoxybiphenyl (Sphos, 251mg, 0.61mmol), potassium vinyltrifluoroborate (2.45g, 18.31mmol) and potassium carbonate (2.52g, 18.31mmol) were charged under nitrogen into a 100mL single-neck reaction flask, and 61mL of n-propanol deoxygenated by nitrogen was added. The nitrogen in the reaction system is replaced for three times, the reaction system is heated and refluxed in an oil bath at the temperature of 120 ℃ for 12 hours, LC-MS monitors that the raw materials disappear, the heating is closed, the reaction system is cooled to room temperature in the ice bath, and the n-propanol is evaporated to dryness under reduced pressure. To the paste obtained above was added 50mL of ethyl acetate, stirred at room temperature for 30 minutes, filtered, and the filtrate was evaporated under reduced pressure to dryness and column chromatography was performed to obtain compound 35 as a yellow granular solid (1.54 g).¹H NMR(500MHz，CDCl₃)δ7.70(d，J＝8.1Hz，1H)，7.62(s，1H)，7.58(s，1H)，7.43(d，J＝8.1Hz，1H)，7.22(d，J＝1.8Hz，1H)，6.73(dd，J＝17.6，10.9Hz，1H)，5.81(d，J＝17.5Hz，1H)，5.32(d，J＝10.9Hz，1H)，3.01-2.93(m，2H)，2.69-2.60(m，2H)，2.20-2.10(m，2H)。

Step 5. Synthesis of Compound 36.

Intermediates obtained aboveAdding a THF-DMSO-water mixed solvent (6mL +6mL +3mL) for dissolving, adding N-bromosuccinimide (NBS, 1.03g and 5.79mmol) at room temperature, stirring for reacting for 1 hour, evaporating THF in a reaction system under reduced pressure after the reaction is finished, adding 60mL of water for diluting, extracting with ethyl acetate, combining organic phases, washing with saturated salt water, drying with anhydrous magnesium sulfate, filtering and evaporating the solvent to obtain a crude product of the intermediate. Dissolving the crude intermediate in 20mL of dichloromethane, adding active manganese dioxide (9.60g, 86.95mmol), stirring at room temperature for reacting overnight, filtering to remove insoluble solid, adding 5mL of methanol into the filtrate, evaporating dichloromethane under reduced pressure until a large amount of orange yellow solid is separated out, stopping the reduced pressure distillation, and filtering to collect the solid. And adding 20mL of dichloromethane into the mother liquor, then distilling under reduced pressure until an orange yellow solid is separated out, stopping distilling under reduced pressure, and filtering to collect the solid. The above operations were repeated once, and the solids obtained by combining three times were intermediate 36(1.72g, yield 35%).¹H NMR(400MHz，CDCl₃)δ8.03(dd，J＝8.2，1.9Hz，1H)，7.89-7.81(m，2H)，7.67(s，2H)，4.47(s，2H)，3.01(dd，J＝7.0，5.2Hz，2H)，2.69(dd，J＝7.2，5.9Hz，2H)，2.18(p，J＝6.4Hz，2H)。

Step 6, compound 37 is synthesized.

Taking intermediate 36(500mg, 1.34mmol), amino acid 7(347mg, 1.34mmol), adding 6mL of 2-methyltetrahydrofuran, stirring and dispersing uniformly, adding cesium carbonate (240mg, 0.74mmol), stirring and reacting at room temperature overnight, evaporating the reaction solution under reduced pressure, and performing column chromatography to obtain intermediate 37 which is 598mg of yellow foamy solid.¹H NMR(400MHz，CDCl₃)7.91(t，J＝6.4Hz，1H)，7.82(dd，J＝8.1，3.2Hz，1H)，7.72(s，1H)，7.63(s，2H)，5.46-5.16(m，2H)，4.48-4.37(m，1H)，3.80-3.68(m，1H)，3.48-3.29(m，5H)，3.22(dd，J＝10.7，7.7Hz，1H)，2.97(t，J＝6.0Hz，2H)，2.70-2.60(m，2H)，2.55-2.45(m，2H)，2.15-2.09(m，2H)，1.48-1.40(9H)。

Step 7. Synthesis of Compound 38.

Dissolving intermediate 37(598mg, 1.08mmol) in a mixed solvent of dichloromethane-methanol (8mL +3mL), adding pyridine trihydrobromide (0.382g, 1.19mmol) at one time, stirring at room temperature, reacting for 2.5 hr, and adding 4N hydrochloric acid successivelyThe reaction mixture was washed with brine, dried over anhydrous sodium sulfate, filtered and evaporated to dryness to give intermediate 38 as a yellow-green foamy solid (684 mg). LC-MS (APCI): m/z 632.5(M +1)⁺。

Step 8, compound 39 is synthesized.

Taking intermediate 38(684mg, 1.08mmol) and dipeptide derivative 18(208mg, 0.73mmol), adding 5mL of 2-methyl-tetrahydrofuran, stirring at room temperature until the mixture is clear, adding cesium carbonate (118mg, 0.36mmol) in one step, stirring at room temperature for 10 minutes, transferring the reaction system to a 50 ℃ constant-temperature oil bath, stirring and reacting for 24 hours, closing heating, filtering, evaporating to dryness, and performing column chromatography to obtain diester intermediate 39 as a gray foamy solid (374 mg). LC-MS (APCI): m/z 836.7(M +1)⁺。

Step 9. Synthesis of Compound 40.

Taking diester intermediate 39(374mg, 0.45mmol), adding 10mL of toluene and 1mL of ethylene glycol methyl ether, stirring at room temperature to dissolve, adding ammonium acetate (1.72g, 22.37mmol) at one time, heating in a constant-temperature oil bath at 110 ℃, refluxing for reaction for 4 hours, closing to heat, naturally cooling to room temperature, adding 10mL of ethyl acetate and 2M sodium hydroxide aqueous solution into the reaction, washing, separating, drying an organic phase with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a crude product of the intermediate. The crude product was dissolved in 10mL of dichloromethane, and activated manganese dioxide (1.94mg, 22.37mmol) was added in one portion with stirring and reacted at room temperature overnight. The reaction system was filtered, and the residue was washed with methanol until the filtrate was colorless. The filtrates were combined and evaporated to dryness, and column chromatography gave compound 40 as a grey solid (145 mg). LC-MS (APCI): m/z 794.8(M +1)⁺。

And step 10, synthesizing a compound V-6.

Intermediate 40(145mg, 0.18mmol) was dissolved in 3mL of anhydrous dichloromethane, and 4N hydrogen chloride-dioxane solution was added thereto at room temperature to stir the reaction for 1 hour. The reaction solvent was evaporated to dryness under reduced pressure to give a brown solid intermediate, which was added to 3mL of anhydrous DMF, then diisopropylethylamine (302mg, 1.83mmol) was added and stirred to dissolve the solution, the solution was cooled in an ice bath, N-Moc-L-phenylglycine (46mg, 0.22mmol) and HATU (84mg, 0.22mmol) were added successively, and the reaction was stirred for 30 minutes. Adding the above reaction solution into 30mL of water, extracting with ethyl acetate, mixing, evaporating to dryness, and performing column chromatographyV-6 was obtained as a yellow powdery solid (115 mg). LC-MS (APCI): m/z 885.9(M +1)⁺，¹H NMR(500MHz，MeOD)δ8.45-7.16(m，12H)，5.60(s，1H)，5.23(dt，J＝16.8，7.9Hz，1H)，4.40-4.02(m，2H)，3.92-3.60(m，5H)，3.60-3.39(m，2H)，3.36-3.24(m，7H)，3.16-2.89(m，1H)，2.68(s，1H)，2.54-2.09(m，3H)，1.96(d，J＝26.0Hz，1H)，1.73-1.55(m，2H)，1.31(s，1H)，1.18-0.80(m，7H)。

Example 7

Preparing a hepatitis C virus inhibitor V-7 with the following molecular formula:

the preparation method comprises the following steps:

step 1. Synthesis of Compound 41.

Intermediate 36(427mg, 1.14mmol), amino acid 24(300mg, 1.34mmol) were taken, 6mL of 2-methyltetrahydrofuran was added, stirring and dispersing were carried out uniformly, cesium carbonate (205mg, 0.63mmol) was added, stirring was carried out at room temperature overnight, the reaction solution was evaporated under reduced pressure, and column chromatography was carried out to obtain intermediate 41 as a yellow foamy solid (440 mg).¹H NMR(400MHz，CDCl₃)δ8.00-7.88(m，1H)，7.83(dd，J＝8.2，3.0Hz，1H)，7.72(s，1H)，7.64(s，2H)，5.46-5.16(m，2H)，4.43(dt，J＝21.4，8.1Hz，1H)，3.82-3.61(m，3H)，3.45-3.33(m，2H)，3.23(dd，J＝10.8，7.7Hz，1H)，2.98(t，J＝6.0Hz，2H)，2.69-2.61(m，2H)，2.57(dd，J＝17.7，10.8Hz，2H)，2.21-2.10(m，3H)，1.50-1.38(9H)。

Step 2. compound 42 was synthesized.

Dissolving intermediate 41(440mg, 0.79mmol) in a mixed solvent of dichloromethane-methanol (6mL +2mL), adding pyridine trihydrobromide (279mg, 0.87mmol) at a time, stirring at room temperature for reaction for 2.5 hours, washing the reaction solution with 4N hydrochloric acid and saturated brine successivelyThen dried over anhydrous sodium sulfate, filtered and evaporated to dryness to give intermediate 42 as a yellow-green foamy solid (491 mg). LC-MS (APCI): m/z 635.5(M +1)⁺。

Step 3. Synthesis of Compound 43.

Taking intermediate 42(491mg, 0.78mmol) and dipeptide derivative 25(150mg, 0.52mmol), adding 2-methyl-tetrahydrofuran 5mL, stirring at room temperature until the mixture is clear, adding cesium carbonate (86mg, 0.26mmol) in one step, stirring at room temperature for 10 minutes, transferring the reaction system to a 50 ℃ constant temperature oil bath, stirring for reaction for 24 hours, turning off heating, filtering, evaporating to dryness, and performing column chromatography to obtain diester intermediate 43 as a gray foamy solid (285 mg). LC-MS (APCI): m/z 846.6(M +1)⁺。

Step 4. Synthesis of Compound 44.

Taking diester intermediate 43(285mg, 0.34mmol), adding 10mL of toluene and 1mL of ethylene glycol methyl ether, stirring at room temperature to dissolve, adding ammonium acetate (1.30g, 16.90mmol) at one time, heating in a constant-temperature oil bath at 100 ℃ for reaction overnight, refluxing at 110 ℃ for 1 hour, closing the heating, naturally cooling to room temperature, adding 10mL of ethyl acetate and 2M of sodium hydroxide aqueous solution into the reaction, washing, separating, drying an organic phase by anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a crude product of the intermediate. The crude product was dissolved in 10mL of dichloromethane, and activated manganese dioxide (1.47mg, 16.90mmol) was added in one portion with stirring to react overnight at room temperature. The reaction system was filtered, and the residue was washed with methanol until the filtrate was colorless. The filtrates were combined and evaporated to dryness, and column chromatography gave compound 44 as a grey solid (145 mg). LC-MS (APCI): m/z 804.8(M +1)⁺。

And 5, synthesizing a compound V-7.

Intermediate 44(88mg, 0.11mmol) was dissolved in 3mL of anhydrous dichloromethane, and 4N hydrogen chloride-dioxane solution was added thereto at room temperature to stir the reaction for 1 hour. Evaporating the reaction solvent to dryness under reduced pressure to obtain brown solid intermediate, adding anhydrous DMF 3mL, adding diisopropylethylamine (142mg, 1.10mmol), stirring to dissolve, cooling the solution in ice bath, and adding N-Moc (d) successively₃) L-phenylglycine (28mg, 0.13mmol) and HATU (50mg, 0.13mmol), and the reaction was stirred for 30 minutes. Adding the above reaction solution into 30mL of water, extracting with ethyl acetate, mixing and steamingDry column chromatography gave V-7 as a yellow powdery solid (39 mg). LC-MS (APCI): m/z 896.3(M +1)⁺。¹H NMR(400MHz，MeOD)δ8.40-6.88(m，12H)，5.55(d，J＝19.3Hz，1H)，5.42-5.04(m，2H)，4.43-3.97(m，2H)，3.88-3.59(m，3H)，3.52-3.33(m，2H)，3.18-2.83(m，1H)，2.74-1.86(m，7H)，1.69-1.47(m，1H)，1.08(dd，J＝22.4，6.4Hz，3H)，0.95(s，1H)，0.89(dd，J＝14.3，6.5Hz，3H)。

And (4) evaluating the biological activity.

To verify the effect of the compounds described herein on HCV, the inventors used the HCV Replicon System (HCV replication System) as an evaluation model. Since the first report in Science1999, the HCV replicon system has become one of the most important tools for studying HCV RNA replication, pathogenicity, and virus persistence, for example, the minimal 5' -NCR region necessary for HCV RNA replication has been successfully demonstrated using the replicon, and the HCV replicon system has been successfully used as an evaluation model for antiviral drugs. The inventors of the present invention performed the verification according to the methods described in Science, 1999, 285(5424), 110-3, and j.virol, 2003, 77(5), 3007-19.

(1) Compounds were tested for activity against HCV 1a and 1b genotypic replicons.

HCV-1a and HCV-1b stably transfected replicon cells were used to detect the inhibitory activity of the compounds of hepatitis C virus genotype 1a and 1b replicons. This experiment will use the NS5A inhibitor GS-5816 as a positive control compound.

The method comprises the following steps: compounds were serially diluted 1: 3 for 8 concentration points, double-plated and added to 96-well plates. DMSO was set as no compound added control. The final concentration of DMSO in the cell culture broth was 0.5%.

Step two: HCV-1a and 1b cells were suspended in culture medium containing 10% FBS, respectively, and seeded into compound-containing 96-well plates at a density of 8,000 cells per well. Cells were in 5% CO₂And cultured at 37 ℃ for 3 days.

Step three: cytotoxicity of compounds against GT1b replicon was determined using CellTiter-Fluor (Promega).

Step four: luciferase assay Compounds were assayed for anti-hepatitis C virus activity using Bright-glo (Promega).

Step five: data were analyzed using GraphPadPrism software, curves were fitted and EC calculated₅₀Value sum CC₅₀The value is obtained.

The compounds of examples 1 to 7 were analyzed according to the above procedure to calculate EC50 values and CC50 values, and the results are shown in table 1.

TABLE 1 comparison of anti-HCV genotype replicon activity of examples 0-1 and control Ombitasvir

Numbering	GT1a EC50(nM)	GT1b EC50(nM)	CC50(nM)
				GS-5816	0.022	0.017	＞2
Example 1	0.010	0.012	＞2
				Example 2	0.016	0.009	＞2
Examples3	0.011	0.008	＞2
				Example 4	0.009	0.008	＞2
Example 5	0.017	0.012	＞2
				Example 6	0.010	0.009	＞2
Example 7	0.011	0.013	＞2

The experimental results show that the compounds of the present invention can inhibit multiple genotypes of HCV. Compared with the non-deuterated compound GS-5816, the compound of the invention has better inhibitory activity on GT1a and GT1b replicons, and plays a more excellent anti-hepatitis C virus role through a mechanism of inhibiting HCV NS5A protein.

(3) And (5) evaluating the metabolic stability.

Microsome experiment: human liver microsomes: 0.5mg/mL, Xenotech; rat liver microsomes: 0.5mg/mL, Xenotech; coenzyme (NADPH/NADH): 1mM, Sigma Life Science; magnesium chloride: 5mM, 100mM phosphate buffer (pH 7.4).

Preparing a stock solution: a defined amount of the powders of GS-5816, the test example compound and the control compound, were weighed out finely and dissolved in DMSO to 5mM each.

Preparation of phosphate buffer (100mM, pH 7.4): 150mL of 0.5M potassium dihydrogenphosphate and 700mL of a 0.5M dipotassium hydrogenphosphate solution prepared in advance were mixed, the pH of the mixture was adjusted to 7.4 with the 0.5M dipotassium hydrogenphosphate solution, the mixture was diluted 5-fold with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100mM) containing 100mM potassium phosphate and 3.3mM magnesium chloride at a pH of 7.4.

NADPH regenerating system solution (containing 6.5mM NADP, 16.5mM G-6-P, 3U/mL G-6-P D, 3.3mM magnesium chloride) was prepared and placed on wet ice before use.

Preparing a stop solution: acetonitrile solution containing 50ng/mL propranolol hydrochloride and 200ng/mL tolbutamide (internal standard). 25057.5 mu L of phosphate buffer solution (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of human liver microsome is respectively added and mixed evenly, and liver microsome dilution liquid with the protein concentration of 0.625mg/mL is obtained. 25057.5 mu L of phosphate buffer (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of SD rat liver microsome is respectively added, and the mixture is mixed evenly to obtain liver microsome dilution with the protein concentration of 0.625 mg/mL.

Incubation of the samples: the stock solutions of the corresponding compounds were diluted to 0.25mM each with an aqueous solution containing 70% acetonitrile, and used as working solutions. 398. mu.L of human liver microsome or rat liver microsome dilutions were added to a 96-well plate (N2), 2. mu.L of 0.25mM working solution was added, and mixed well.

Determination of metabolic stability: 300. mu.L 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 are placed in a 37 ℃ water bath box, shaken at 100 rpm and pre-incubated for 5 min. 80. mu.L of the incubation solution was taken out of each well of the incubation plate, added to the stop plate, mixed well, and supplemented with 20. mu.L of NADPH regenerating system solution as a 0min sample. Then 80. mu.L of NADPH regenerating system solution was added to each well of the incubation plate, the reaction was started, and the timer was started. The reaction concentration of the corresponding compound was 1. mu.M, and the protein concentration was 0.5 mg/mL. When the reaction was carried out for 10min, 30min and 90min, 100. mu.L of each reaction solution was added to the stop plate and vortexed for 3min to terminate the reaction. The stop plates were centrifuged at 5000 Xg for 10min at 4 ℃. And (3) taking 100 mu L of supernatant to a 96-well plate in which 100 mu L of distilled water is added in advance, mixing uniformly, and performing sample analysis by adopting LC-MS/MS.

And (3) data analysis: and detecting peak areas of the corresponding compound and the internal standard through an LC-MS/MS system, and calculating the peak area ratio of the compound to the internal standard. The slope is determined by plotting the natural logarithm of the percentage of compound remaining against time and calculating t according to the following formula_1/2And CL_intWhere V/M is equal to 1/protein concentration.

The compounds of the invention and compounds without deuteration were tested simultaneously and compared to evaluate their metabolic stability in human and rat liver microsomes. The half-life and intrinsic hepatic clearance as indicators of metabolic stability are shown in table 2. The non-deuterated compound GS-5816 was used as a control sample in Table 2. As shown in table 2, the compound of the present invention can significantly improve metabolic stability by comparison with the non-deuterated compound GS-5816, and thus is more suitable as a hepatitis c virus inhibitor.

TABLE 2 comparison of metabolic stability of example D-1 with the GS-5816 control

(4) Rat pharmacokinetic experiments.

Purpose of the experiment: study of the pharmacokinetic behavior of the compounds of the invention after administration of GS-5816, the example compounds, in rats was examined.

Experimental animals:

species and strain: SD rat grade: SPF stage

Sex and amount: male, 6

Body weight range: 180 to 220g (actual weight range 187 to 197g)

The source is as follows: shanghai Xipulbikai laboratory animals Co., Ltd

Experimental and animal certification numbers: SCXK (Shanghai) 2013-0016

The experimental process comprises the following steps:

before blood collection, 20L of 2M sodium fluoride solution (esterase inhibitor) was added to an EDTA-K2 anticoagulation tube, dried in an oven at 80 ℃ and stored in a refrigerator at 4 ℃.

Rats, males, weighing 187-197 g, were randomized into 2 groups, fasted overnight but with free access to water starting the afternoon of the day before the experiment, and given food 4h after administration. GS 58163 mg/kg is given to the group A, and 3mg/kg of the compound of the formula A-1 is given to the group B, about 100L of blood is taken from orbital veins of rats after 15min, 30min, 1, 2, 3, 5, 8 and 10h after administration, the blood is placed in an Eppendorf tube of 0.5mL anticoagulated by EDTA-K2 and is immediately mixed, after anticoagulation, the tube is gently inverted and mixed for 5 to 6 times as soon as possible, the blood is taken and placed in an ice box, a blood sample is centrifuged at 4000rpm, 10min and 4 ℃ within 30min to separate plasma, and the whole plasma is collected and immediately stored at-20 ℃. Plasma concentrations were determined in plasma at each time point after sample collection at all time points.

According to the obtained mean plasma concentration-time data after administration, relevant pharmacokinetic parameters of male SD rats after i.g. GS-5816(3mg/kg) and the compound of the example (3mg/kg) are respectively administered are calculated by adopting Winnonin software according to a non-atrioventricular statistical moment theory.

The experimental result shows that compared with GS-5816, the compound of the invention has better activity and excellent pharmacokinetic property, so the compound is more suitable to be used as a compound for inhibiting the hepatitis C virus protein NS5A, and is further suitable to be used for preparing a medicament for treating hepatitis C virus infection.

It is to be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention, and that experimental procedures not specifically identified in the examples will generally be performed under conventional conditions, or under conditions recommended by the manufacturer. Parts and percentages are parts and percentages by weight unless otherwise indicated.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims (4)

1. A compound, wherein the compound is selected from the following compounds or a pharmaceutically acceptable salt thereof:

2. a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of claim 1, or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition of claim 2, further comprising an active compound that is an immunomodulator or antiviral drug compound.

4. Use of a compound of claim 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating a hepatitis c virus infection.