CN108473525A

CN108473525A - Deuterated HCV NS5b inhibitor nucleotide derivatives and application thereof

Info

Publication number: CN108473525A
Application number: CN201680077803.4A
Authority: CN
Inventors: 王喆; 曾志宏; 江荣珍
Original assignee: Shanghai Longwood Biopharmaceuticals Co Ltd
Current assignee: Shanghai Longwood Biopharmaceuticals Co Ltd
Priority date: 2016-01-04
Filing date: 2016-12-30
Publication date: 2018-08-31
Also published as: CN106699828A; WO2017118355A1; CN106699828B

Abstract

This application provides a kind of deuterated HCV (Hepatitis C Virus) NS5b inhibitor nucleotide derivatives or its pharmaceutically acceptable salt, stereoisomer, tautomer inhibitor and its purposes in preparing the drug for treating or preventing HCV infection, the inhibitor to be indicated by general formula I：

Description

Deuterated HCV NS5b inhibitor nucleotide derivative and application thereof

Technical Field

the invention belongs to the field of medicines, and particularly relates to a novel deuterated nucleoside (amino) phosphate compound, a composition containing the compound, preparation of the compound and application of the compound in drugs for HCV (hepatitis C virus) infectious diseases.

Background

Infection with hepatitis c virus causes chronic liver diseases such as cirrhosis and liver cancer. Hepatitis c virus infection is one of the major infectious diseases. According to the statistics of the world health organization, 1.7 hundred million hepatitis C victims exist in the world, and nearly 3900 million hepatitis C infectors exist in China. Various studies to date have shown that Hepatitis C Virus (HCV) is the major pathogen responsible for most non-a, non-b hepatitis. Hepatitis C virus is a positive single-stranded RNA virus of Flaviviridae (F1 aviviridae). Standard regimens for the treatment of HCV have previously been the combination of interferon or PEG interferon a with ribavirin. However, the side effects of interferon are significant, and the combined use of interferon and ribavirin has more side effects, which can cause hemolysis, anemia, fatigue and the like.

In recent years, there has been little progress in drug development in the treatment of hepatitis c. For example, Sofosbuvir (Sofosbuvir) by giride, approved by the FDA in the united states 12/6 in 2013, which is a drug for use in oral HCV treatment regimens, eliminates the need for the traditional injected drug interferon when used in the treatment of chronic hepatitis c of a particular genotype (type 2, type 3).

Although considerable progress has been made in the treatment of hepatitis c, difficulties and frustration have also arisen. BMS-986094 of BMS, for example, is a guanosine nucleoside phosphate prodrug for clinical treatment of hepatitis C. In 8 months of 2012, the patients died of their clinical trials due to heart failure. IDX19368 by Idenix also stopped clinical trials. Thus, there is still a need for effective compounds for the treatment and/or prevention of HCV infection.

Disclosure of Invention

The invention aims to provide a compound represented by the following general formula I and pharmaceutically acceptable salts, stereoisomers and tautomers thereof for treating and/or preventing HCV infection.

Wherein R is₁H or D; r₂H or D; r₃H or D; r₄H or D; r₅＝CH₃Or CD₃；R₆H or D; x is-O or-NH.

According to some embodiments of the present application, the compound is selected from the group consisting of:

according to some embodiments of the present application, the position of the deuterium (D) substitution in the compounds of the present application has at least 50% D, e.g. at least 90% D, 50-90% D.

According to some embodiments of the present application, the position of the deuterium (D) substitution in the compounds of the present application has at least 95% D.

According to some aspects of the present application there is also provided a mixture of isomers of the compounds described herein, wherein the two isomers in the mixture each comprise 50%, the mixture being as described in claim 5.

According to one aspect of the present application, there is provided a compound of formula I (including the specific compounds listed) for use in the treatment or prevention of HCV infection.

According to yet another aspect of the present application, there is provided a pharmaceutical composition comprising a compound or a mixture of isomers of the present application as an active ingredient, and a pharmaceutically acceptable carrier.

According to some embodiments of the present application, the pharmaceutical composition of the present application further comprises one or more additional active ingredients.

According to some embodiments of the present application, the one or more additional active ingredients in the pharmaceutical composition of the present application are selected from (1) an immunomodulator; (2) inhibitors of hepatitis c viral protease (NS 3); (3) hepatitis c virus NS4b inhibitors; (4) hepatitis c virus NS5a inhibitors; (5) hepatitis c virus polymerase (NS5b) inhibitors; (5) nucleosides and nucleoside derivatives not belonging to (2) - (5); (6) hepatitis B Virus (HBV) inhibitors; (7) human Immunodeficiency Virus (HIV) inhibitors; (8) a cancer drug; (9) anti-inflammatory agents; or (10) other compounds not belonging to the above-mentioned (1) to (9). For example, wherein the one or more additional active ingredients are selected from HCV NS3 protease inhibitors, HCV NS5A inhibitors, HCV NS5B polymerase inhibitors, interferons or interferon derivatives, ribavirin. The active ingredients may be combined in a single dosage form or may be in separate dosage forms without being combined with each other.

The pharmaceutical compositions of the present application include, but are not limited to, oral dosage forms and parenteral dosage forms. In some embodiments, the pharmaceutical composition may be tablets, capsules, pills, powders, sustained release formulations, solutions and suspensions for oral administration, sterile solutions, suspensions or emulsions for parenteral injection. In other embodiments, the pharmaceutical composition is in unit dosage form suitable for single administration of a precise dose. In other embodiments, the amount of the compound ranges from about 0.001mg/kg body weight/day to about 1000mg/kg body weight/day. In other embodiments, the amount of the compound ranges from about 0.5mg/kg body weight/day to about 50mg/kg body weight/day. In some embodiments, the amount of the compound is from about 0.001 g/day to about 7 g/day. In other embodiments, the amount of the compound is from about 0.002 g/day to about 6 g/day. In other embodiments, the amount of the compound is from about 0.005 g/day to about 5 g/day. In other embodiments, the amount of the compound is from about 0.01 g/day to about 5 g/day. In other embodiments, the amount of the compound is from about 0.02 g/day to about 5 g/day. In other embodiments, the amount of the compound is from about 0.05 g/day to about 2.5 g/day. In other embodiments, the amount of the compound is from about 0.1 g/day to about 1 g/day. In other embodiments, dosage levels below the lower limit of the aforesaid range may be more than adequate. In other embodiments, dosage levels above the upper limit of the range recited above may be desired. In some embodiments, the compound is administered in a single dose, once per day. In other embodiments, the compound is administered in multiple doses, more than once per day. In some embodiments, the compound is administered twice daily. In other embodiments, the compound is administered three times per day. In other embodiments, the compound is administered four times per day. In other embodiments, the compound is administered four or more times per day. In some embodiments, the subject to which the pharmaceutical composition is administered is a mammal. In other embodiments, the mammal is a human. In other embodiments, if the pharmaceutical composition comprises a plurality of active ingredients, these active ingredients may be mixed in a single dosage form, or they may be separately in different dosage forms without being mixed with each other.

According to one aspect of the present application, there is provided the use of a compound or mixture of the present application in the manufacture of a medicament for the treatment or prevention of HCV infection.

According to one aspect of the present application, there is provided the use of a compound or mixture of the present application in combination with one or more other active ingredients described herein for the manufacture of a medicament for the treatment or prevention of HCV infection.

According to one aspect of the present application, there is provided a method of treating or preventing HCV infection, comprising administering to a subject in need thereof an effective amount of a compound or mixture or pharmaceutical composition of the present application.

According to some embodiments of the present application, the above-described methods of treating or preventing HCV infection comprise administering to a subject in need thereof an effective amount of a compound or mixture of the present application in combination with one or more additional active ingredients described herein.

According to some embodiments of the present application, the HCV infection described herein comprises chronic hepatitis c.

Definition of terms

Certain pharmaceutical terms as used herein with respect to the terms "subject", "patient" or "individual" refer to an individual suffering from a disease, disorder or condition, and the like, including mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class mammalia: humans, non-human primates (e.g., chimpanzees and other apes and monkeys); livestock, such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs, and the like. Examples of non-human mammals include, but are not limited to, birds, fish, and the like. In one embodiment related to the methods and compositions provided herein, the mammal is a human.

As used herein, the term "treating" and other similar synonyms include alleviating, or ameliorating a symptom of a disease or disorder, preventing other symptoms, ameliorating, or preventing an underlying metabolic cause of a symptom, inhibiting a disease or disorder, e.g., arresting the development of a disease or disorder, alleviating a disease or disorder, ameliorating a disease or disorder, alleviating a symptom of a disease or disorder, or discontinuing a symptom of a disease or disorder, and further, the term encompasses prophylactic purposes. The term also includes obtaining a therapeutic effect and/or a prophylactic effect. The therapeutic effect refers to curing or ameliorating the underlying disease being treated. In addition, a cure or amelioration of one or more physiological symptoms associated with the underlying disease is also a therapeutic effect, e.g., an improvement in the condition of the patient is observed, although the patient may still be affected by the underlying disease. For prophylactic effect, the composition can be administered to a patient at risk of developing a particular disease, or to a patient presenting with one or more physiological symptoms of the disease, even if a diagnosis of the disease has not yet been made.

The terms "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" as used herein, refer to an amount of at least one agent or compound that is sufficient to alleviate one or more symptoms of the disease or disorder being treated to some extent after administration. The result may be a reduction and/or alleviation of signs, symptoms, or causes, or any other desired change in a biological system. For example, an "effective amount" for treatment is the amount of a composition comprising a compound disclosed herein that is clinically necessary to provide a significant remission effect of the condition. An effective amount suitable in any individual case can be determined using techniques such as a dose escalation assay.

The terms "administering," "administration," "administering," and the like as used herein refer to a method capable of delivering a compound or composition to a desired site for biological action. These methods include, but are not limited to, oral routes, via the duodenal route, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical and rectal administration. Administration techniques useful for The compounds and methods described herein are well known to those skilled in The art, for example, in Goodman and Gilman, The pharmaceutical Basis of Therapeutics, current ed.; pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. In preferred embodiments, the compounds and compositions discussed herein are administered orally.

The term "acceptable" as used herein with respect to a formulation, composition or ingredient means that there is no long-term deleterious effect on the general health of the subject being treated.

The term "pharmaceutically acceptable" as used herein refers to a substance (e.g., carrier or diluent) that does not affect the biological activity or properties of the compounds of the present application, and is relatively non-toxic, i.e., the substance can be administered to an individual without causing an adverse biological response or interacting in an adverse manner with any of the components contained in the composition.

The term "pharmaceutical composition" as used herein refers to a biologically active compound optionally admixed with at least one pharmaceutically acceptable chemical ingredient, including but not limited to a carrier. The term "carrier" as used herein refers to a relatively non-toxic chemical compound or agent that facilitates the introduction of the compound into a cell or tissue.

The term "pharmaceutically acceptable salt" as used herein refers to salts that retain the biological potency of the free acid and free base of the specified compound, and that are biologically or otherwise non-adverse. The compounds of the present application also include pharmaceutically acceptable salts. Pharmaceutically acceptable salts refer to the form in which the base group in the parent compound is converted to a salt.

"stereoisomers" as used herein refers to isomers resulting from the different arrangement of atoms in a molecule in space. The compounds of formula I contain asymmetric or chiral centers and, therefore, exist in different stereoisomeric forms. All stereostructures of formula I are as well as mixtures, including racemic mixtures, as part of the present application. Diastereomeric mixtures can be separated into the individual diastereomers, based on their different physicochemical properties, by well-known means, e.g., resolution of the enantiomers can be converted into the diastereomers by reaction with a suitable optically active substance (e.g., a chiral alcohol or Mosher's moylchloride), which can be separated and converted (e.g., hydrolyzed) into the corresponding individual isomers. Some of the compounds of formula 1 may be atropisomers (e.g., substituted aryl) are also part of this application. Enantiomers can also be separated using a chiral chromatography column. The compounds of formula I may exist in different tautomeric forms, all of which are intended to be encompassed by the present application. For example, keto-enol and imine-enamine forms of the compounds.

Detailed Description

The claims hereof set forth the features of novelty that characterize the present application. The following sets forth exemplary embodiments that utilize the principles of the present application. The features and advantages of the present application may be better understood by reference to the following.

Although preferred embodiments of the present application are described herein, these embodiments are provided by way of example only. It is to be understood that variations of the embodiments of the present application described herein may also be used to implement the teachings of the present application. Those of ordinary skill in the art will appreciate that various modifications, changes, and substitutions can be made without departing from the scope of the present application. It should be understood that the scope of the various aspects of the application is defined by the claims and that methods and structures within the scope of these claims and their equivalents are intended to be covered by the claims.

Synthetic route

Methods for the synthesis of compounds of the present application include, but are not limited to, the following equations and reaction steps:

intermediate 1a synthetic route:

synthetic route to compound a:

synthetic route for compound B:

synthetic route to compound C:

synthetic route for compound G:

synthetic route for compound U:

synthetic route for compound M:

synthetic route for compound W:

examples

Example 1: preparation of Compound A

Example 1 a:

compound 10(84g,0.17mol) was dissolved in ethanol (1.2L) and NaBH was added in portions₄(25g,0.69mol), heating to 40-45 ℃ for reaction for 4h, carrying out TLC tracking reaction, cooling to 0 ℃, adding acetone (50ml), stirring for 15min, quenching the reaction, filtering, concentrating the filtrate till the filtrate is completely dried, adding ethyl acetate for extraction, concentrating, and carrying out column chromatography separation, wherein the eluent is DCM/MeOH (20: 1-10: 1), so that 30.5g of white solid is obtained, and the yield is 71%.¹HNMR(400MHz,d6-DMSO)：11.41(s,1H),8.13(d,1H,J＝8.3Hz),6.18(s,1H),5.89(d,1H,J＝5.9Hz),5.60(dd,1H,J＝8.2,2.0Hz),5.34(dd,1H,J＝4.3,3.5Hz),3.85(m,3H),3.62(dd,1H,J＝10.5,4.3Hz),1.40(s,3H)

Example 1 b:

dissolving the compound 1a (26.4g,95mmol) in 520ml of pyridine, distilling off 260ml of solvent by an oil pump under reduced pressure, cooling to room temperature, adding DMAP (4.0g,33mmol) and DMTCl (120g,354mmol), heating to 40-45 ℃, reacting for 4-5 h, tracking the reaction by TLC completely, concentrating pyridine under reduced pressure till dryness, adding DCM for extraction, concentrating an organic phase, and carrying out column chromatography to obtain 53g of a white solid compound with the yield of 96%.

¹HNMR(400MHz,d6-DMSO)：11.47(s,1H),7.95(d,1H,J＝8.0Hz),7.34(m,4H),7.21(m,5H),6.88(m,4H),6.20(s,1H),6.07(d,1H,J＝6.0Hz),4.95(d,1H,J＝7.6Hz),4.18(dd,1H,J＝6.4,3.2Hz),3.70(s,6H),3.43(dd,1H,J＝3.6,11.6Hz),3.33(m,1H),1.45(s,3H).

Example 1 c:

dissolving the compound 1b (6.24g) in 60ml of DCM, adding DMAP (2.9g), cooling to 0-5 ℃, dropwise adding BzCl (1.8g), heating to room temperature for reaction for 2h after the addition, washing with water after the TLC tracking reaction is finished, drying, concentrating a solvent, and performing column chromatography to obtain 6.3g of a product with the yield of 86%.

¹HNMR(400MHz,d6-DMSO)：11.52(s,1H),8.01(m,3H),7.72(t,1H,J＝7.8Hz),7.57(t,2H,J＝7.8Hz),7.29(d,2H,J＝8.0Hz),7.25(m,7H),6.75(m,4H),6.30(s,1H),5.78(d,1H,J＝8.0Hz),5.21(d,1H,J＝6.4Hz),4.43(d,1H,J＝8.8Hz),3.65(s,3H),3.63(s,3H),3.40(m,2H),1.55(s,3H).

Example 1 d:

dissolving the compound 1c (0.486g,0.71mmol) in 80% acetic acid (5ml), reacting for 4h at 20-25 ℃, following the completion of the TLC reaction, adding water, extracting with ethyl acetate, washing the organic phase with a saturated aqueous sodium bicarbonate solution and a saturated brine in sequence, drying over anhydrous magnesium sulfate, filtering, concentrating the filtrate, and performing column chromatography to obtain 0.25g of a white foamy solid with a eluent of n-heptane/ethyl acetate of 1:1, wherein the yield is 93%.

¹HNMR(400MHz,d6-DMSO)：11.52(s,1H),8.18(d,1H,J＝8.0Hz),8.00(d,2H,J＝8.4Hz),7.70(t,1H,J＝8.4Hz),7.54(t,2H,J＝8.4Hz),6.32(s,1H),5.73(d,1H,8.0Hz),5.49(m,2H),4.33(dt,1H,J＝2.0,8.4Hz),3.85(m,1H),3.63(m,1H),1.51(s,3H).

Example 1 e:

compound 1d (1.10g,2.88mmol) was added to acetonitrile (3ml) and water (3ml) and the solid was not completely dissolved, then TEMPO (46mg,0.288mmol) and BAIB (2.04g,6.34mmol) were added and the reaction was allowed to proceed at 10 ℃ for 18h, followed by TLC to complete the reaction, DCM (50ml) and saturated aqueous sodium bicarbonate (50ml) were added and the bulk of the solid (sodium salt of the product) precipitated, filtered, the solid washed with DCM, the organic phases combined, extracted with saturated aqueous sodium bicarbonate (20ml), the aqueous and solid combined, ethyl acetate was added, 4NHCl aqueous solution was added dropwise with stirring to adjust the pH <2, the organic phase separated, dried and concentrated to give 1.19g of a red foamy solid in quantitative yield.

¹HNMR(400MHz,d6-DMSO)：13.0(br,1H),11.56(d,1H,J＝2.0Hz),8.00(m,3H),7.70(m,1H),7.57(t,2H,J＝8.0Hz),6.34(s,1H),5.78(dd,1H,J＝8.0,2.0Hz),5.72(br,1H),4.74(d,1H,J＝8.0Hz),1.55(s,3H).

Example 1 f:

dissolving compound 1e (66mg,0.168mmol) in THF (1ml), adding triethylamine (26mg,0.252mmol) at 0-5 ℃, then adding ethyl chloroformate (22mg,0.202mmol), reacting for 2h at 15 ℃, tracking the reaction by TLC to be almost complete, filtering, adding NaBD into filtrate₄(21mg,0.504mmol) and D was added dropwise at 15 deg.C₂O (0.5g,25mmol), reacting for 30min, TLC tracing reaction completely, adding ethyl acetate, washing with water, separating organic phase, drying, concentrating, and separating by column chromatography to obtain solid 30mg with 47% yield.

¹HNMR(400MHz,d6-DMSO):11.54(s,1H),8.19(d,1H,J＝8.0Hz),8.00(d,2H,J＝7.2Hz),7.69(t,1H,J＝7.2Hz),7.55(t,2H,J＝7.2Hz),6.33(s,1H),5.73(d,1H,J＝8.0Hz),5.48(d,1H,J＝8.4Hz),5.47(s,1H),4.32(d,1H,J＝8.4Hz),1.51(s,3H).

Example 1 g:

dissolving a compound 1f (93mg,0.261mmol) in 1.0ml of THF, dropwise adding a THF solution of t-BuMgCl (1M,0.784ml) at 0-5 ℃, reacting for 30min at 0-5 ℃ after dropwise adding, separating out a large amount of solid, adding a THF (1.0ml) solution of a compound 11(183mg,0.418mmol) at 0-5 ℃, completely dissolving the solid, heating to room temperature, stirring overnight, tracking the reaction by TLC, and adding NH after the reaction is finished₄Saturated aqueous solution of Cl, EA extraction, washing of the organic phase successively with saturated aqueous sodium bicarbonate solution and saturated brine, drying over anhydrous magnesium sulfate, filtration, concentration of the filtrate, column chromatography separation with n-heptane/ethyl acetate 1:1 to 1:2 to 0:1 to yield 95mg of product a in 67% yield.

¹HNMR(400MHz,d4-MeOD):7.73(d,1H,J＝8.2Hz),7.36(t,2H,J＝7.9Hz),7.25(d,2H,J＝7.9Hz),7.19(t,1H,J＝7.9Hz),6.37(s,1H),5.59(d,1H,J＝8.2Hz),4.94(m,1H),4.16(dd,1H,J＝9.4,2.3Hz),3.99(d,1H,J＝9.4Hz),3.90(dq,1H,J＝10.2,7.4Hz),1.51(s,3H),1.33(dd,3H,J＝7.1,0.8Hz),1.20(d,3H,J＝6.3Hz),1.19(d,3H,J＝5.9Hz).

¹³CNMR(400MHz,d4-MeOD):172.91,172.86,164.19,150.80,150.68,150.61,129.50,124.87,119.90,119.85,101.65,80.17,80.09,76.57,68.77,50.23,50.21,21.50,20.55,20.46,19.24,19.18.

MS positive ion 548[ M + H]⁺，570[M+Na]⁺

Anion 546[ M-H]^-，582[M+Cl]^-

Example 2: preparation of Compound B

Example 2a

Adding the compound 1b (500mg,0.863mmol) and sodium bicarbonate (363mg,4.32mmol) into DCM (10ml), adding DMP (732mg,1.73mmol), reacting at 10-15 ℃ for 3h, tracking the reaction by TLC to be almost complete, adding DCM (50ml) and saturated aqueous sodium bicarbonate solution (30ml), separating out an organic phase, drying and concentrating to obtain 0.6g of red foamy solid which is directly used for the next reaction.

Example 2 b:

compound 2a (600mg crude, 0.863mmol) was dissolved in EtOD (6ml) and NaBD was added at 10 deg.C₄(83mg, 1.99mmol), reaction for 2h, TLC following reaction substantially complete, addition of heavy water (1ml) to quench the reaction, extraction with ethyl acetate, drying concentration, column chromatography separation, eluent n-heptane/ethyl acetate 2: 1 to 1:1, 400mg of white solid are obtained in 80% yield in two steps.

¹HNMR(400MHz,d6-DMSO):11.49(s,1H),7.96(d,1H,J＝8.0Hz),7.34(m,4H),7.21(m,5H),6.88(m,4H),6.21(s,1H),6.06(s,1H),4.94(d,1H,J＝7.6Hz),3.71(s,6H),3.43(dd,1H,J＝3.6,11.6Hz),3.33(m,1H),1.45(s,3H).

Example 2 c:

compound 2b (400mg, 0.69mmol) was dissolved in DCM (8ml) and CF was added₃COOH (1ml) reacts for 3 hours at 10-16 ℃, TLC tracking reaction is basically complete, column chromatography separation is carried out after concentration, eluent is ethyl acetate, 156mg of solid is obtained, and the yield is 82%.

Example 2 d:

compound 2c (130mg, 0.47mmol) was addedDissolving in 1.5ml of THF, dropwise adding a THF solution (1.41ml) of t-BuMgCl (1M) at 0-5 ℃, reacting for 30min at 0-5 ℃ after dropwise adding, separating out a large amount of solid, adding a THF (1.5ml) solution of a compound 11(340mg, 0.75mmol) at 0-5 ℃, completely dissolving the solid, heating to room temperature, stirring overnight, tracking the reaction by TLC, and adding NH after the TLC is finished₄Saturated aqueous solution of Cl, extraction with ethyl acetate, washing the organic phase with saturated aqueous sodium bicarbonate solution and saturated brine in this order, drying over anhydrous magnesium sulfate, filtration, concentration of the filtrate, and column chromatography separation with n-heptane/ethyl acetate of 1:1 to 1:2 to 0:1 to obtain 60mg of compound B.

¹HNMR(400MHz,d4-MeOD):7.73(d,1H,J＝8.3Hz),7.36(t,2H,J＝8.2Hz),7.25(d,2H,J＝7.9Hz),7.19(t,1H,J＝7.9Hz),6.36(s,1H),5.59(d,1H,J＝8.3Hz),4.94(m,1H),4.52(ddd,1H,J＝12.2,5.9,2.0Hz),4.38(ddd,1H,J＝11.8,5.5,3.2Hz),4.17(dd,1H,J＝4.7,2.3Hz),3.90(dq,1H,J＝10.2,7.0Hz),1.50(s,3H),1.33(dd,3H,J＝7.1,0.8Hz),1.20(d,3H,J＝6.3Hz),1.19(d,3H,J＝5.9Hz).

¹³CNMR(400MHz,d4-MeOD):172.91,172.86,164.19,150.80,150.61,129.50,124.87,119.89,119.85,101.65,80.17,80.09,76.57,68.77,50.23,50.21,21.46,20.54,20.45,19.23,19.17.

MS positive ion 569[ M + Na ]]⁺

Negative ion 545M-H]^-，581[M+Cl]^-

Example 3: preparation of Compound C

Example 3 a:

dissolving 2b (2.66g,4.6mmol) in DM (26ml), adding DMAP (1.12g,9.2mmol), cooling to 0-5 ℃, dropwise adding BzCl (0.78g,5.6mmol), heating to room temperature for reaction for 2h after the addition is finished, adding water for washing after the TLC tracking reaction is finished, drying, concentrating the solvent to obtain 3.4g of crude product, and directly using the crude product in the next step of reaction.

Example 3 b:

dissolving 3a (3.4 g of crude product in the previous step, 4.6mmol) in DCM (30ml), adding trifluoroacetic acid (5ml), reacting at 20-25 ℃ for 4h, after TLC tracking reaction is finished, concentrating the solvent, adding DCM, stirring silica gel, and performing column chromatography, wherein the eluent is n-heptane/ethyl acetate 1:1, so as to obtain 1.0g of white foamy solid with the yield of 57.2% in two steps.

Example 3 c:

adding 3b (1.0g,2.88mmol) into acetonitrile (3ml) and water (3ml), wherein the solid can not be completely dissolved, then adding TEMPO (45mg,0.288mmol) and BAIB (2.04g,6.34mmol), reacting at 10 ℃ for 18h, tracing by TLC to be basically complete, adding DCM (50ml) and saturated sodium bicarbonate water solution (50ml), separating out a large amount of solid (sodium salt of the product), filtering, washing the solid with DM, combining organic phases, extracting with saturated sodium bicarbonate water solution (20ml), combining the water phase and the solid, adding ethyl acetate, dropwise adding 4NHCl water solution under stirring to adjust the pH to be less than 2, separating out the organic phase, drying and concentrating to obtain red foamy solid, and quantifying the yield.

Example 3 d:

dissolving the compound 3c (508mg,1.28mmol) in THF (10ml), adding triethylamine (194.2mg,1.92mmol) at 0-5 ℃, then adding ethyl chloroformate (250mg,1.54mmol), reacting for 2h at 15 ℃, tracking the reaction by TLC to be almost complete, filtering, adding NaBD into the filtrate₄(160.7mg,3.84mmol) D was added dropwise at 15 deg.C₂O (2.0g,100mmol), reacting for 30min, TLC tracing reaction completely, adding ethyl acetate, washing with water, separating organic phase, drying, concentrating, and separating by column chromatography to obtain solid 160mg with yield 32.6%.

¹HNMR(400MHz,d4-CD₃OD):7.29(d,1H,J＝8.0Hz),6.42(s,1H),5.72(d,1H,J＝8.0Hz),4.61(s,1H),1.56(s,3H).

Example 3 e:

dissolving a compound 3d (150mg, 0.39mmol) in 1.5ml of THF, dropwise adding a t-BuMgCl (1M) THF solution (1.2ml) at 0-5 ℃, reacting for 30min at 0-5 ℃ after dropwise adding, separating out a large amount of solid, adding a compound 11(354mg, 0.78mmol) THF (1.5ml) solution at 0-5 ℃, completely dissolving the solid, heating to room temperature, stirring overnight, tracking the reaction by TLC, and adding NH after the reaction is finished₄Saturated aqueous solution of Cl, extraction with ethyl acetate, washing of the organic phase successively with saturated aqueous sodium bicarbonate solution and saturated brine, drying over anhydrous magnesium sulfate, filtration, concentration of the filtrate, column chromatography separation with n-heptane/ethyl acetate 1:1 to 1:2 to 0:1 to give 40mg of compound C in 19% yield.

¹HNMR(400MHz,d4-MeOD):7.73(d,1H,J＝8.3Hz),7.36(t,2H,J＝8.2Hz),7.25(d,2H,J＝7.9Hz),7.19(t,1H,J＝7.9Hz),6.36(s,1H),5.59(d,1H,J＝8.0Hz),4.94(m,1H),4.16(d,1H,J＝2.3Hz),3.90(dq,1H,J＝10.2,7.0Hz),1.50(s,3H),1.33(d,3H,J＝7.1Hz),1.20(d,3H,J＝6.3Hz),1.19(d,3H,J＝5.9Hz).

MS positive ion 571[ M + Na]⁺

Anion 547[ M-H]^-，583[M+Cl]^-

Example 4: preparation of Compound G

Example 4 a:

compound 12(20g,9.5mmol) was suspended in heavy water (200ml), 10% Pd/C (1.0g) was added, and after 3 times replacement with nitrogen, 3 times replacement with hydrogen was performed, and the mixture was heated under reflux for 96 hours under hydrogen 1atm, and concentrated to give a crude product, which was directly subjected to the next reaction.

Example 4 b:

adding a crude product of the compound 4a (20g) into pyridine (200ml), evaporating the solvent under reduced pressure, adding the pyridine (200ml) again, cooling to 0-5 ℃, dropwise adding BzCl (49.5g), heating to room temperature for reacting for 18h after the addition, evaporating the solvent under reduced pressure, pouring into 1.17L solution containing NaOH (92g), stirring for 1h to dissolve the product, filtering to remove palladium carbon, adjusting the pH value of the filtrate to be about 4.0 by using concentrated hydrochloric acid, filtering, pulping the obtained solid for 30min at the reflux temperature by using 1.2L ethanol, cooling to room temperature, filtering, and drying the solid to obtain 20g of the product.

¹HNMR(400MHz,d6-DMSO):7.97(d,2H,J＝7.2Hz),7.58(t,1H,J＝7.2Hz),7.47(t,2H,J＝7.2Hz).

Example 4 c:

adding 4b (21g,0.097mol) into chlorobenzene (200ml), adding 0.1g ammonium sulfate and HMDS (46.9g,0.29mol), heating and refluxing for 2h, dissolving all solids, cooling, concentrating under reduced pressure by an oil pump, removing HMDS by using chlorobenzene once, adding chlorobenzene (150ml) into residual liquid, dropwise adding a chlorobenzene (100ml) solution of LW1006-7(21g,0.049mol), and adding SnCl₄(37.8g,0.145mol), reacting for 20h at 70 ℃ under the protection of Ar, precipitating a large amount of solid in the reaction solution, extracting with DM, and performing NaHCO extraction₃Washing and concentrating to obtain a crude product, and directly putting the crude product into the next step for reaction.

Example 4 d:

and (3) adding the 4c crude product into acetic acid (300ml) and water (75ml), heating and refluxing for 3h, tracking the reaction by TLC, concentrating to be dry completely, adding ethyl acetate, concentrating again, adding DM and silica gel, concentrating, and performing column chromatography to obtain 16.9g of foamy solid with the yield of two steps of 71%.

Example 4 e:

compound 4d (8.4g,0.017mol) was dissolved in ethanol (120ml), and NaBH was added in portions₄(2.5g,0.069mol), heating to 40-45 ℃ for reaction for 4h, tracking by TLC, cooling to 0 ℃, adding acetone (5ml), stirring for 15min, quenching, filtering, concentrating the filtrate until the filtrate is dried, adding ethyl acetate for extraction, concentrating, and performing column chromatography separation to obtain 3.05g of white solid with the yield of 71%, wherein the eluent is DCM/MeOH (20: 1-10: 1).

¹HNMR(400MHz,d6-DMSO)：11.41(s,1H),6.18(s,1H),5.93(d,1H,J＝5.9Hz),5.40(dd,1H,J＝4.3,3.5Hz),3.85(m,3H),3.61(dd,1H,J＝10.5,4.3Hz),1.39(s,3H)

Experiment 4 f:

dissolving compound 4e (150mg, 0.39mmol) in 1.5ml THF, dropwise adding t-BuMgCl (1M) THF solution (1.2ml) at 0-5 ℃, reacting for 30min at 0-5 ℃ after dropwise adding, separating out a large amount of solid, adding compound 11(354mg, 0.78mmol) THF (1.5ml) at 0-5 ℃, completely dissolving the solid, heating to room temperature, stirring overnight, tracking the reaction by TLC, adding NH after finishing the reaction₄Saturated aqueous solution of Cl, extraction with ethyl acetate, washing of the organic phase successively with saturated aqueous sodium bicarbonate solution and saturated brine, drying over anhydrous magnesium sulfate, filtration, concentration of the filtrate, and column chromatography separation with n-heptane/ethyl acetate 1:1 to 1:2 to 0:1 to give 40mg of compound G in 19% yield.

¹HNMR:¹HNMR(400MHz,d4-MeOD):7.36(t,2H,J＝8.2Hz),7.25(d,2H,J＝7.9Hz),7.19(t,1H,J＝7.9Hz),6.36(s,1H),4.94(m,1H),4.52(m,1H,4.38(m,1H),4.18(dd,1H,J＝4.7,2.3Hz),4.00(m,1H),3.90(m,1H),1.50(s,3H),1.33(dd,3H,J＝7.1,0.8Hz),1.20(d,3H,J＝6.3Hz),1.19(d,3H,J＝5.9Hz).

MS: positive ion 570[ M + Na ]]⁺

Anion 546[ M-H]^-,582[M+Cl]^-

Example 5: preparation of Compound U

Synthesis of Compound U the procedures of example 1b to example 1g in example 1 were followed.

Compound 5a:¹HNMR(400MHz,d6-DMSO)：11.51(s,1H),7.34(m,4H),7.21(m,5H),6.88(m,4H),6.20(s,1H),6.10(d,1H,J＝6.0Hz),4.18(dd,1H,J＝6.0,9.6Hz),3.71(s,6H),3.43(dd,1H,J＝3.6,11.6Hz),3.32(m,1H),1.45(s,3H).

compound 5c:¹HNMR(400MHz,d6-DMSO)：11.52(s,1H),8.00(d,2H,J＝8.0Hz),7.70(t,1H,J＝8.0Hz),7.55(t,2H,J＝8.0Hz),6.32(s,1H),5.49(d,2H,J＝8.4Hz),4.33(d,1H,J＝8.4Hz),3.86(m,1H),3.63(m,1H),1.51(s,3H).

compound U:¹HNMR(400MHz,d4-MeOD):7.36(t,2H,J＝7.9Hz),7.25(d,2H,J＝7.9Hz),7.19(t,1H,J＝7.9Hz),6.36(s,1H),4.94(m,1H),4.16(dd,1H,J＝9.6,2.4Hz),3.99(d,1H,J＝9.6Hz),3.90(dq,1H,J＝10.2,7.4Hz),1.51(s,3H),1.33(d,3H,J＝7.2Hz),1.20(d,3H,J＝6.3Hz),1.19(d,3H,J＝5.9Hz).

MS: positive ion 572[ M + Na]⁺

Anion 548[ M-H ]]^-，584[M+Cl]^-

Example 6: preparation of Compound M

Synthesis of compound M the procedure of example 2a to example 2d in example 2 was referenced.

Compound 6 b:¹HNMR(400MHz,d6-DMSO):11.50(s,1H),7.33(m,4H),7.21(m,5H),6.89(m,4H),6.20(s,1H),6.07(s,1H),3.70(s,6H),3.43(dd,1H,J＝3.6,11.6Hz),1.45(s,3H).

compound M:¹HNMR(400MHz,d4-MeOD):7.36(t,2H,J＝8.4Hz),7.25(d,2H,J＝8.4Hz),7.19(t,1H,J＝7.9Hz),6.36(s,1H),4.94(m,1H),4.52(dd,1H,J＝12.2,5.9Hz),4.38(m,1H),4.16(d,1H,J＝2.0Hz),3.90(m,1H),1.50(s,3H),1.33(d,3H,J＝7.1Hz),1.20(d,3H,J＝6.0Hz),1.19(d,3H,J＝6.0Hz).

MS：

positive ion 549[ M +1]⁺,571[M+Na]⁺

Example 7: preparation of Compound W

Synthesis of Compound W reference is made to examples 3a to 3e of example 3

A compound W:¹HNMR(400MHz,d4-MeOD):7.36(t,2H,J＝8.2Hz),7.25(d,2H,J＝7.9Hz),7.19(t,1H,J＝7.9Hz),6.36(s,1H),4.94(m,1H),4.16(d,1H,J＝2.3Hz),3.90(m,1H),1.50(s,3H),1.33(d,3H,J＝7.1Hz),1.20(d,3H,J＝6.3Hz),1.19(d,3H,J＝5.9Hz).

MS：

positive ion 573[ M + Na ]]⁺

Anion 549[ M-H [ ]]^-，585[M+Cl]^-

Biological Activity test experiment

Cytological experimental method of HCV GT1a and GT1b

GT1a and GT1b replicon cells:

stable transgenic hepatitis C virus replicon GT1a and GT1b cell lines were established by in vitro transcription of the hepatitis C virus subgenomic GT1a (H77) and GT1b (Con1) replicon plasmid DNA into RNA, followed by transfer into Huh-7 cells and selection by G418.

Experimental materials:

GT1a and GT1b stable transcellular lines were constructed from the drug Mingknid (Wuxi ApTech Huh7-GT1a (H77) and Huh7-GT1b (con 1)).

Reagents or consumables: DMEM culture, fetal bovine serum, streptomycin, MEM nonessential amino acids, glutamine, G418, 0.05% pancreatic enzyme, DMSO, 96-well black cell culture plate, CellTiter-flour, Bright-Glo.

The instrument comprises the following steps: ViCell cell counter (Beckman), ECHO (Labcyte), EnVision (PerkinElmer).

The experimental method comprises the following steps:

the compounds were dissolved in DMSO to 10 mM stock and stored in a nitrogen cabinet. Gradient dilution compound: compounds were added to 96-well plates using Echo, and 8 concentration spots were serially diluted 1:3 for each compound, duplicate wells, and added to columns 3-10 of the 96-well plates. Column 11 DMSO alone was added as a null control well. The final volume of DMSO in each well was 607.5 nanoliters. See typesetting below.

Cell plating: HCV GT1a or GT1b cells were digested from T150 cell culture flasks and cell density adjusted to 65,843 cells/ml with cell culture broth, and the cell suspension was added to 96-well plates to which compounds had been added at 121.5 μ l per well. The final density of cells in the 96-well plate was 8,000 cells/well and the final concentration of DMSO in each well was 0.5%. column 2, 100% active control wells had no cells added. Place 96-well plate containing cells and compounds in 5% CO₂And cultured at 37 ℃ for 3 days. After 3 days of culture, the activity of cells per well in a 96-well plate was measured with CellTiter-Flour: add 40. mu.l of CellTitter-flour to each well, then add 5% CO at 37 ℃₂After incubation for 1 hour under conditions, fluorescence signals were detected using an EnVision read plate (excitation light 405nm, emission light 515 nm). The anti-hepatitis C virus activity of the compounds was measured with Bright-Glo: supernatants from 96-well plates were removed after cell viability assay, and 100. mu.l of the prepared 1 XBright-Glo reagent was added to each well and chemiluminescent signal was detected within 5 minutes.

Data analysis

Cell viability: the raw data (RFU) was converted to% Viability of cells per well relative to DMSO control using the following formula

Anti-hepatitis c virus activity of the compounds: the raw data (RLU) was converted to the percent inhibition of hepatitis C Virus activity% per well relative to the 100% effective control using the following formula

CPD (CPD): signal value of compound pore

ZPE: mean value of signal from control wells with DMSO null effect

HPE: mean signal from 100% active control wells (no cell control wells)

RFU: relative fluorescence unit

RLU: relative chemiluminescent unit

Analysis of the EC of Compounds on HCV GT1a or GT1b stable transgenic cell lines using the Sigmodal dose-response (variable slope) program in GraphPad Prism 5 software₅₀Value sum CC₅₀The value is obtained.

II, HCV GT1a activity and GTA1b activity

Representative compounds of formula (I) of the present application showed EC50 (50% inhibition, sofosbuvir drug reference compound) for HCV GT1a and GT1b subgenomic replicon inhibition assays as determined according to the above method as shown in the table below.

TABLE 1 results of activity test of partial compounds for inhibiting hepatitis C virus

Furthermore, the compounds in table 1 all show a selectivity index (CC) of more than 1000₅₀/EC₅₀)。

Claims

A compound represented by formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer thereof:

wherein,

R₁h or D;

R₂h or D;

R₃h or D;

R₄h or D;

R₅＝CH₃or CD₃；

R₆H or D; and

x is-O or-NH.
The compound according to claim 1, selected from the following compounds:
the compound of claim 1 or 2, wherein the deuterated positions have a D of at least 50%.
The compound of claim 3, wherein the deuterated positions have a D of at least 90 or at least 95%.
An isomeric mixture of the compound of claim 2, wherein each of the two compounds in the mixture comprises 50%:

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A pharmaceutical composition comprising a compound according to any one of claims 1 to 4 or a mixture according to claim 5 as active ingredient, together with a pharmaceutically acceptable carrier.
The pharmaceutical composition of claim 6, wherein the composition further comprises one or more additional active ingredients selected from the group consisting of (1) an immunomodulator; (2) hepatitis C virus protease inhibitors (NS3, NS3/4 a); (3) hepatitis c virus NS4b inhibitor, (4) hepatitis c virus NS5a inhibitor, (5) hepatitis c virus polymerase inhibitor; (6) nucleosides and nucleoside derivatives not belonging to (2) - (3); (7) hepatitis B Virus (HBV) inhibitors; (7) human Immunodeficiency Virus (HIV) inhibitors; (9) a cancer drug; (10) anti-inflammatory agents; or (11) other compounds not belonging to the above-mentioned (1) to (10).
The pharmaceutical composition of claim 7, wherein the one or more additional active ingredients are selected from HCV NS3(NS3/4a) protease inhibitor, HCV NS4b inhibitor, HCV NS5A inhibitor, HCV NS5B polymerase inhibitor, interferon or interferon derivative, or ribavirin.
Use of a compound according to any one of claims 1 to 4 or a mixture according to claim 5 in the manufacture of a medicament for the treatment or prophylaxis of HCV infection.
Use of a compound according to any one of claims 1 to 4 or a mixture according to claim 5 in combination with one or more further active ingredients according to claim 7 or 8 for the preparation of a medicament for the treatment or prophylaxis of HCV infections.