WO2017121188A1 - Hepatitis c virus inhibitor, pharmaceutical composition and application thereof - Google Patents

Abstract

Provided in the present invention are a hepatitis C virus inhibitor, a pharmaceutical composition and an application thereof. The hepatitis C virus inhibitor is a compound as shown in formula (I), or a crystal form, a pharmaceutically acceptable salt, prodrug, hydrate or solvent thereof. The compound of the present invention has better inhibitory activity on hepatitis C viral protein NS5A, as well as better pharmacodynamical / pharmacokinetical performance. The compound is very applicable and safe, can be used to prepare a medicament for treating hepatitis C viral infection, and has good market development potential.

Description

Hepatitis C virus inhibitor, pharmaceutical composition and application thereof Technical field

The invention belongs to the technical field of medicine, and in particular relates to a hepatitis C virus inhibitor, a pharmaceutical composition and the use thereof.

Background technique

HCV (Hepatitis C Virus) is an RNA virus belonging to the genus Hepacivirus genus in the Flaviviridae family. The encapsulated HCV virion contains a positive-stranded RNA genome that encodes all known virus-specific proteins in a single uninterrupted open reading frame. The open reading frame comprises approximately 9500 nucleotides and encodes a single large polyprotein of approximately 3000 amino acids. Polyproteins include core proteins, envelope proteins E1 and E2, membrane-bound protein P7, and non-structural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B.

Sofabru is the first medicine in the world that can completely cure hepatitis C in the short term. It takes the oral route directly to the lesion, and the method has simple side effects and is highly sought after by patients. Sofibuvir is produced by Gilead, USA, and is marketed in the United States in 2013. It has been clinically proven to be effective in treating hepatitis C, type 1, 2, 3, and 4, including liver transplantation, liver cancer, and HCV/HIV-1 co-infection. Clinical Trials. This breakthrough has brought the gospel to hepatitis C patients around the world.

HCV infection is associated with progressive liver disease, including cirrhosis and hepatocellular carcinoma. Odalasvir (Achillion's Hepatitis C Drug, also known as ACH-3102) is a new drug developed by Achillion for the potential treatment of hepatitis C. It works by inhibiting the hepatitis C virus protein NS5A and is currently pre-registered. status. Its hepatitis C cocktail therapy combined with sofosbuvir, in the trial of patients with type I genotype hepatitis C without ribavirin, obtained the ideal phase II clinical data, which can kill the virus found within six weeks. It is the shortest treatment time and highest response rate that can be achieved when all two drugs are combined.

The US Gilead company has extended the authorization of the new hepatitis C drug Sofibuvir to Egypt last year. Egypt is the country with the highest incidence of hepatitis C in the world. Subsequently agreed to produce sofibru generic drugs in India. Up to now, a total of eight Indian pharmaceutical companies have obtained Gilead's authorization to sell Sophie Buwei to 91 developing countries around the world (China is excluded). Sophie Buwei is currently in a blank space in China. On the one hand, there is a large demand, and on the other hand, it is drug-free. In the face of such awkward dilemma, some large hospitals are trying to solve this problem through video remote overseas medical treatment. Some patients who can't wait, have to consider going abroad to see a doctor. Therefore, there is still a need in the art to develop compounds having inhibitory activity or better pharmacodynamic properties against the hepatitis C virus protein NS5A.

Summary of the invention

In view of the above technical problems, the present invention discloses a substituted hepatitis C virus inhibitor, a pharmaceutical composition thereof and use thereof, which have better hepatitis C virus protein NS5A inhibitory activity and/or have better pharmacodynamics/pharmacokinetics. Power Learning performance.

In this regard, the technical solution adopted by the present invention is: a hepatitis C virus inhibitor, such as a hepatitis C virus inhibitor represented by the formula (I), or a crystalline form thereof, a pharmaceutically acceptable salt, a hydrate or Solvent compound,

Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ And R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ , R ⁴⁰ , R ⁴¹ , R ⁴² , R ⁴³ , R ⁴⁴ , R ⁴⁵ , R ⁴⁶ , R ⁴⁷ , R ⁴⁸ , R ⁴⁹ , R ⁵⁰ , R ⁵¹ , R ⁵² , R ⁵³ , R ⁵⁴ , R ⁵⁵ , R ⁵⁶ , R ⁵⁷ , R ⁵⁸ , R ⁵⁹ , R ⁶⁰ , R ⁶¹ , R ⁶² , R ⁶³ , R ⁶⁴ , R ⁶⁵ , R ⁶⁶ , R ⁶⁷ and R ⁶⁸ are each independently hydrogen, deuterium, halogen or trifluoromethyl;

Additional conditions are R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ And R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ , R ⁴⁰ , R ⁴¹ , R ⁴² , R ⁴³ , R ⁴⁴ , R ⁴⁵ , R ⁴⁶ , R ⁴⁷ , R ⁴⁸ , R ⁴⁹ , R ⁵⁰ , R ⁵¹ , R ⁵² , R ⁵³ , R ⁵⁴ , R ⁵⁵ , R ⁵⁶ , R ⁵⁷ , R ⁵⁸ , R ⁵⁹ , R ⁶⁰ , R ⁶¹ , R ⁶² , R ⁶³ , R ⁶⁴ , R ⁶⁵ At least one of R ⁶⁶ , R ⁶⁷ and R ⁶⁸ is deuterated or deuterated.

With this technical solution, the shape and volume of the ruthenium in the drug molecule are substantially the same as those of the hydrogen. If the hydrogen in the drug molecule is selectively replaced with hydrazine, the deuterated drug generally retains the original biological activity and selectivity. At the same time, the inventors have confirmed through experiments that the binding of carbon-germanium bonds is more stable than the combination of carbon-hydrogen bonds, which can directly affect the absorption, distribution, metabolism and excretion of some drugs, thereby improving the efficacy, safety and tolerability of the drugs.

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

Specifically, in the present invention, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ And R ³² , R ³³ , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ , R ⁴⁰ , R ⁴¹ , R ⁴² , R ⁴³ , R ⁴⁴ , R ⁴⁵ , R ⁴⁶ , R ⁴⁷ , R ⁴⁸ , R ⁴⁹ , R ⁵⁰ , R ⁵¹ , R ⁵² , R ⁵³ , R ⁵⁴ , R ⁵⁵ , R ⁵⁶ , R ⁵⁷ , R ⁵⁸ , R ⁵⁹ , R ⁶⁰ , R ⁶¹ , R ⁶² , R ⁶³ , The strontium isotope content of each of the R ⁶⁴ , R ⁶⁵ , R ⁶⁶ , R ⁶⁷ and R ⁶⁸ is at least 5%, preferably more than 10%, more preferably more than 15%, more preferably more than 20%, more preferably More than 25%, more preferably more than 30%, more preferably more than 35%, more preferably more than 40%, more preferably more than 45%, more preferably more than 50%, more preferably more than 55%, more preferably More than 60%, more preferably more than 65%, more preferably more than 70%, more preferably more than 75%, more preferably more than 80%, more preferably more than 85%, more preferably 90%, more preferably greater than 95%, more preferably greater than 99%.

Preferably, the compound of the formula (I) contains at least one deuterium atom, and the number of deuterium atoms may be any one of 1 to 68.

As a further improvement of the present invention, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are each independently hydrazine or hydrogen.

In another preferred embodiment, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are 氘.

As a further improvement of the present invention, R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ and R ²² are each independently hydrazine or hydrogen.

In another preferred embodiment, R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² is 氘.

As a further improvement of the present invention, R ⁷ and R ¹⁵ are 氘.

As a further improvement of the present invention, R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ and R ²¹ are 氘.

As a further improvement of the present invention, R ¹⁴ and R ²² are 氘.

As a further improvement of the present invention, R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ , R ³³ , R ³⁴ , R ³⁵ , R ^36. R ³⁷ , R ³⁸ , R ³⁹ , R ⁴⁰ , R ⁴¹ , R ⁴² , R ⁴³ , R ⁴⁴ , R ⁴⁵ , R ⁴⁶ , R ⁴⁷ and R ⁴⁸ are each independently hydrazine or hydrogen.

In another preferred embodiment, R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ , R ³³ , R ³⁴ , R ³⁵ , R ^36. R ³⁷ , R ³⁸ , R ³⁹ , R ⁴⁰ , R ⁴¹ , R ⁴² , R ⁴³ , R ⁴⁴ , R ⁴⁵ , R ⁴⁶ , R ⁴⁷ and R ⁴⁸ are 氘.

As a further improvement of the present invention, R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ , R ⁴⁰ , R ⁴¹ , R ⁴² , R ⁴³ , R ⁴⁴ , and R ⁴⁵ are 氘.

As a further improvement of the present invention, R ³³ , R ³⁴ , R ³⁵ , R ⁴⁶ , R ⁴⁷ and R ⁴⁸ are fluorene.

As a further improvement of the present invention, R ⁴⁹ , R ⁵⁰ , R ⁵¹ , R ⁵² , R ⁵³ and R ⁵⁴ are each independently hydrazine or hydrogen.

In another preferred embodiment, R ⁴⁹ , R ⁵⁰ , R ⁵¹ , R ⁵² , R ⁵³ and R ⁵⁴ are 氘.

As a further improvement of the present invention, R ⁵⁵ , R ⁵⁶ , R ⁵⁷ , R ⁵⁸ , R ⁵⁹ and R ⁶⁰ are each independently hydrazine or hydrogen.

In another preferred embodiment, R ⁵⁵ , R ⁵⁶ , R ⁵⁷ , R ⁵⁸ , R ⁵⁹ , R ⁶⁰ are 氘.

As a further improvement of the present invention, R ⁶¹ , R ⁶² , R ⁶³ , R ⁶⁴ , R ⁶⁵ , R ⁶⁶ , R ⁶⁷ and R ⁶⁸ are each independently hydrazine or hydrogen.

In another preferred embodiment, R ⁶¹ , R ⁶² , R ⁶³ , R ⁶⁴ , R ⁶⁵ , R ⁶⁶ , R ⁶⁷ , R ⁶⁸ are 氘.

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

In another preferred embodiment, the compound does not include a non-deuterated compound.

The present invention also discloses a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the hepatitis C virus inhibitor as described above, or a crystalline form, a pharmaceutically acceptable salt, a hydrate or a solvent thereof A pharmaceutical composition of a compound, stereoisomer, prodrug or isotopic variation.

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

As a further improvement of the present invention, the pharmaceutical composition is a tablet, a pill, a capsule, a powder, a granule, an ointment, an emulsion, a suspension, a solution, a suppository, an injection, an inhalant, a gel, a microsphere or Aerosol.

Typical routes of administration of the pharmaceutical compositions of the invention include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal , intramuscular, subcutaneous, intravenous administration. Oral administration or injection administration is preferred.

The pharmaceutical composition of the present invention can be produced by a method known in the art, such as a conventional mixing method, a dissolution method, a granulation method, a sugar-coating method, a pulverization method, an emulsification method, a freeze-drying method, and the like.

As a further improvement of the invention, it further comprises other active compounds which are immunomodulatory or antiviral pharmaceutical compounds.

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

As a further improvement of the present invention, the antiviral drug compound is ribavirin, amantadine, other inhibitors of NS5A, helicase in the HCV life cycle, protease, polymerase, metalloproteinase or internal ribosome entry. An inhibitor of a site target, wherein the other inhibitor of NS5A is Radipap or Dhakavir At least one.

The present invention also provides a method of preparing a pharmaceutical composition comprising the steps of: administering a pharmaceutically acceptable carrier to a hepatitis C virus inhibitor as described above, or a crystalline form thereof, a pharmaceutically acceptable salt, or a hydrate thereof Or the solvate is mixed to form a pharmaceutical composition.

The invention also discloses the use of a hepatitis C virus inhibitor as described above, characterized in that it is used for the preparation of a medicament for the treatment of hepatitis C virus infection.

The HCV includes a plurality of genotypes thereof and a plurality of gene subtypes, such as 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a, 6a.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

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

As used herein, unless otherwise specified, "deuterated" means that one or more hydrogens in the compound or group are replaced by deuterium; deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted. The terms "one or more deuterated" are used interchangeably with "one or more deuterated".

As used herein, unless otherwise specified, "non-deuterated compound" means a compound containing a proportion of germanium atoms not higher than the natural helium isotope content (0.015%).

Pharmaceutically acceptable salts include inorganic and organic salts. A preferred class of salts are the salts of the compounds of the invention with acids. Suitable acids for forming salts include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid; formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, Organic acids such as fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid; Amino acids such as amino acid, phenylalanine, aspartic acid, and glutamic acid. Another preferred class of salts are the salts of the compounds of the invention with bases, such as alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example magnesium or calcium salts), ammonium salts (for example lower alkanolammonium). Salts and other pharmaceutically acceptable amine salts), such as methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tert-butyl A base amine salt, an ethylenediamine salt, a hydroxyethylamine salt, a dihydroxyethylamine salt, a trihydroxyethylamine salt, and an amine salt formed of morpholine, piperazine, and lysine, respectively.

The term "solvate" refers to a complex of a compound of the invention that is coordinated to a solvent molecule to form a specific ratio. "Hydrate" means a complex formed by the coordination of a compound of the invention with water.

Compared with the prior art, the beneficial effects of the present invention are:

First, the compound of the present invention has excellent inhibitory properties against the hepatitis C virus protein NS5A.

Second, by deuteration this technique changes the metabolism of the compound in the organism, giving the compound better pharmacokinetic parameter characteristics. In this case, the dosage can be changed and a long-acting preparation can be formed to improve the applicability.

Third, replacing the hydrogen atom in the compound with hydrazine increases the drug concentration of the compound in the animal due to its strontium isotope effect, thereby improving the drug efficacy.

Fourth, replacing the hydrogen atom in the compound with hydrazine can inhibit certain metabolites and improve the safety of the compound.

detailed description

The preparation of the structural compound of the formula (I) of the present invention is more specifically described below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention may also be conveniently prepared by combining various synthetic methods described in the specification or known in the art, and such combinations are readily made by those skilled in the art to which the present invention pertains.

Usually, in the preparation scheme, each reaction is usually carried out in an inert solvent at room temperature to reflux temperature (e.g., 0 ° C to 100 ° C, preferably 0 ° C to 80 ° C). The reaction time is usually from 0.1 to 60 hours, preferably from 0.5 to 24 hours.

Example 1

A hepatitis C virus inhibitor with the chemical name di-d3-methyl ((2S, 2'S)-((2S, 2'S, 3aS, 3a'S, 7aS, 7a'S)-2, 2'-(5, 5' -(tricyclo[8.8.2.24,7]hexa-4,6,10,12,13,15-hexene-5,11-diyl)di(1H-benzimidazole-5,2-di Base)) bis(octahydro-1hydro-indole-2,1-diyl))bis(3-methyl-1-oxobutane-2,1-diyl)dicarbamate, ie Compound T-1, the molecular formula is as follows:

Use the following route for synthesis:

Includes the following steps:

1. Synthesis of (2R, 3aS, 7aS)-1-(tert-butoxycarbonyl)octahydro-1hydro-indole-2-carboxylic acid (Compound 1);

L-octahydroindole-2-carboxylic acid (1.69 g, 10 mmol) was added to the reaction flask, and 30 mL of THF and 30 mL of distilled water were added to stir and dissolve, and a 7 mL aqueous solution of NaOH (0.68 g, 17 mmol) was added dropwise thereto in an ice bath. The reaction was stirred for 10 minutes. (Boc) ₂ O (2.84 g, 13 mmol) was added dropwise, and the mixture was allowed to react at room temperature for 12 hours. After the TLC detection reaction was completed, it was diluted with water, extracted with methyl tert-butyl ether, and the aqueous phase was adjusted to pH with citric acid, extracted with ethyl acetate three times, and the organic phases were combined, washed with saturated brine, Na ₂ SO _4, dried and concentrated by filtration to give 3.21 g of Compound 1 in a yield of 100%. LC-MS (APCI): m / z = 268.3 (M-1) -.

2, (2R, 3aS, 7aS)-tert-butyl-2-((2-amino-4-bromophenyl)carbamoyl) octahydro-1hydro-indole-1-carboxylate (Compound 2) Synthesis;

Compound 1 (2.69 g, 10 mmol) and 4-bromo-o-phenylenediamine (2.244 g, 12 mmol) were added to the reaction flask, dissolved in 60 mL of acetonitrile, and the condensing agent EDCI (1-ethyl-(3-dimethylamino) was added. Propyl)carbodiimide hydrochloride) (2.3 g, 12 mmol), N,N-diisopropylethylamine (1.68 g, 13 mmol) was added dropwise at 0 ° C, and the mixture was stirred at room temperature and stirred for 1 hour. After the completion of the dot plate reaction, half of the acetonitrile was removed by concentration, and 120 mL of water was added thereto, and the mixture was stirred at room temperature overnight. Filtration, the filter cake was washed with water and dried in vacuo to give 3.6 g of product. LC-MS (APCI): m / z = 439.1 (M + 1) +.

3. (2R, 3aS, 7aS)-tert-butyl-2-(6-bromo-1hydro-benzimidazol-2-yl)octahydro-1hydro-indole-1carboxylate (compound 3) synthesis;

Compound 2 (2.19g, 5mmol) was dissolved in 10mL of acetic acid, heated to 65 ° C and stirred for 3 hours. After TLC detection reaction, the solvent was concentrated and removed, diluted with ethyl acetate, and 2 mL of aqueous ammonia was added under stirring to adjust the pH to weakly alkaline. The mixture was washed with water and extracted with ethyl acetate. EtOAc was evaporated. LC-MS (APCI): m / z = 421.1 (M + 1) +.

4, (2R, 3aS, 7aS)-tert-butyl-2-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1 hydrogen Synthesis of -benzimidazol-2-yl)octahydro-1hydro-indole-1carboxylate (Compound 4);

Compound 3 (2.1 g, 5 mmol), pinacol borate (1.9 g, 7.5 mmol), Pd(dppf)Cl ₂ (0.366 g, 1.5 mmol) and potassium acetate (1.47 g, were added to the dried reaction flask. Add 15mL anhydrous dioxane under nitrogen protection, heat to 85 ° C and stir the reaction for 4 hours. After TLC detection, dilute with ethyl acetate, add activated carbon for 1 hour, filter, concentrate to obtain oily liquid, column layer The product was purified to give 3.1 g of Compound 4 in a yield of 100%. LC-MS (APCI): m / z = 468.7 (M + 1) +.

Synthesis of 5,4,16-dibromo[2.2]polyxylene (Compound 5);

Bromine (0.863 g, 20.3 mmol) and iron powder (48 mg, 1.86 mmol) were added to the reaction flask, and 16 ml of dichloromethane was added thereto, and the reaction was stirred at room temperature for 1 hour. Adding a solution of the raw material di-p-xylene (2.08 g, 10 mmol) in 3 mL of dichloromethane, heating to reflux, slowly adding 2.4 g of bromine in 5 ml of dichloromethane solution, adding, refluxing for 3 hours, and reducing to room temperature. overnight. The mixture was diluted with methylene chloride, washed successively with 5% sodium thiosulphate, water, and brine, dried over anhydrous sodium sulfate, and filtered and concentrated to yield 1.56 g of compound 5. ¹ H NMR (300MHz, CDCl ₃ ): δ (ppm): 2.79-3.00 (m, 4H), 3.10-3.21 (m, 2H), 3.44-3.54 (m, 2H), 6.44 (d, J = 8.0 Hz) , 2H), 6.51 (d, J = 2.0 Hz, 2H), 7.14 (dd, J = 8.0 Hz, 2.0 Hz, 2H).

6, (2S, 2'S, 3aS, 3a'S, 7aS, 7a'S)-di-tert-butyl-2,2'-(5,5'-(tricyclo[8.8.2.2 ^4,7 ]hexa-4,6, 10,12,13,15-hexene-5,11-diyl)bis(1H-benzimidazole-5,2-diyl)di(octahydro-1hydro-indole-1-carboxylate (Synthesis of Compound 6);

Compound 4 (2.3 g, 4.92 mmol), compound 5 (0.72 g, 1.97 mmol), cesium carbonate (1.61 g, 4.92 mmol) and Pd(PPh ₃ ) ₄ (1.115 g, 0.1 mmol), nitrogen, were added to the reaction flask. Under the protection, 18 mL of DMF and 1 ml of water were added, and the reaction was carried out at 130 ° C for 3 hours. After the reaction of the spot plate was detected, the catalyst was removed by filtration, distilled water was added to precipitate a white solid, and the mixture was filtered and dried to obtain a crude product. After purification by column chromatography, 460 mg of compound 6 was obtained. The rate is 26.3%. LC-MS (APCI): m / z = 888.7 (M + 1) +.

7, (2S, 2'S, 3aS, 3a'S, 7aS, 7a'S)-2,2'-(5,5'-(tricyclic [8.8.2.2 ^4,7 ] sixteen-4,6,10,12,13 Synthesis of 15-(hexene-5,11-diyl)bis(1H-benzimidazole-5,2-diyl)bis(octahydro-1hydro-indole) (Compound 7);

The compound 6 (460 mg, 0.518 mmol) was dissolved in a mixed solvent of 4 mL of dichloromethane and 1 mL of methanol, and 2.33 ml of hydrogen chloride dioxane was added at 0 ° C, and the mixture was allowed to react at room temperature for 1 hour. After the TLC test was completed, the solvent was removed by concentration. The crude compound 7 was obtained and was directly put to the next step without purification.

8. Synthesis of chloroformic acid-d3-methyl ester (Compound 8);

The triphosgene (2.67 g, 9 mmol) was dissolved in 20 ml of toluene, and deuterated methanol (1.08 g, 30) was added dropwise at 0 °C. A solution of mmol and triethylamine (3.34 g, 33 mmol) in 20 ml of toluene was added and then allowed to react to room temperature for 1 hour. The reaction liquid was washed three times with ice water, dried over anhydrous sodium sulfate, and filtered to give a toluene solution of compound 8 and directly to the next reaction.

9. Synthesis of d3-methoxycarbonyl-L-proline (Compound 9);

L-valine (2.81 g, 24 mmol) was dissolved in 20 mL of dioxane, and a 1N aqueous solution of sodium hydroxide (2.9 g, 72 mmol) was added, and a toluene solution of the compound 8 obtained in the step was added dropwise and allowed to react at room temperature overnight. After the TLC reaction was completed, the mixture was diluted with hydrochloric acid to give aq. EtOAc. EtOAc (EtOAc) LC-MS (APCI): m / z = 179.1 (M + 1) +.

10, di-d3-methyl ((2S, 2'S)-((2S, 2'S, 3aS, 3a'S, 7aS, 7a'S)-2,2'-(5,5'-(tricyclic [8.8.2.2 ^{4, 7} ] hexa-4,6,10,12,13,15-hexene-5,11-diyl)bis(1H-benzimidazole-5,2-diyl))bis(octahydro-1) Hydrogen-hydrazine-2,1-diyl))bis(3-methyl-1-oxobutane-2,1-diyl)dicarbamate, ie the synthesis of compound T-1;

Compound 9 (98.53 mg, 0.553 mmol), HOBT (1-hydroxybenzotriazole, 74.7 mg, 0.553 mmol) and EDCI (106.1 mg, 0.553 mmol) were dissolved in a reaction flask, dissolved in 3 mL of acetonitrile, and reacted at room temperature 5 The activated solution 1 was obtained in minutes. Compound 7 (200 mg, 0.24 mmol) was dissolved in 5 mL DMF and DIPEA (223.3 mg, 1.. The solution 1 was added to the solution 2, and the reaction was stirred at room temperature for 3 hours. After the completion of the TLC reaction, distilled water was added to precipitate a white solid, which was filtered with suction, and then filtered, and then purified by column chromatography to obtain 120 mg of compound T-1, yield 49.8%. .

_{^{1 H NMR (300MHz, CDCl 3}} ) δ10.87 (s, 2H), 7.94 (dd, J = 31.7,9.3Hz, 2H), 7.61-7.30 (m, 4H), 6.82-6.54 (m, 6H), 5.54-5.37 (m, 4H), 4.27 (dd, J = 20.0, 10.6 Hz, 4H), 3.60-3.23 (m, 4H), 3.01 (d, J = 5.7 Hz, 2H), 2.95 (s, 2H) , 2.88 (s, 2H), 2.77 (s, 4H), 2.51 (s, 2H), 2.41-2.28 (m, 2H), 2.02 (ddd, J = 35.3, 20.2, 9.3 Hz, 4H), 1.76 (s , 10H), 0.97 (dt, J = 11.6, 5.7 Hz, 6H), 0.85 (dd, J = 6.9, 3.6 Hz, 6H). LC-MS (APCI): m / z = 1008.3 (M + 1) +.

Example 2

Dimethyl ((2S, 2'S)-((2S, 2'S, 3aS, 3a'S, 7aS, 7a'S)-2,2'-(5,5'-(tricyclo[8.8.2.2 ^4,7 ]hexa- 4,6,10,12,13,15-hexene-5,11-diyl)bis(1H-benzimidazole-5,2-diyl))di(octahydro-1hydro-indole- 2,1-diyl))bis(3-d3-methyl-1-oxo-d5-butane-2,1-diyl)dicarbamate, compound T-2, having the formula

Synthesize using the following routes:

On the basis of Embodiment 1, the following steps are specifically included:

1. Synthesis of methyl chloroformate (Compound 10);

The triphosgene (0.3 g, 1 mmol) was dissolved in 2 mL of toluene, and a solution of methanol (96.12 mg, 3 mmol) and triethylamine (334 mg, 3.3 mmol) in 2 mL of toluene was added dropwise at 0 ° C. hour. The reaction liquid was washed three times with ice water, dried over anhydrous sodium sulfate, and filtered to give a toluene solution of compound 10, which was directly applied to the next reaction.

2. Synthesis of methoxycarbonyl-d8-L-proline (Compound 11);

The deuterated L-valine (300 mg, 2.4 mmol) was dissolved in 3 mL of dioxane, and a 1 N aqueous solution of sodium hydroxide (288 mg, 7.2 mmol) was added thereto, and the toluene solution of the compound 8 obtained in the step was added dropwise, and the reaction was carried out at room temperature. overnight. After the TLC reaction was completed, the mixture was diluted with hydrochloric acid to adjust to pH, and extracted with ethyl acetate. The organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate and filtered to afford 280 mg of Compound 11. LC-MS (APCI): m / z = 184.1 (M + 1) +.

3, dimethyl ((2S, 2'S)-((2S, 2'S, 3aS, 3a'S, 7aS, 7a'S)-2,2'-(5,5'-(tricyclo[8.8.2.2 ^4,7 ]10 Hexafluorobenzene-1-hydrogen-oxime Synthesis of bis-2,1-diyl))bis(3-d3-methyl-1-oxo-d5-butane-2,1-diyl)dicarbamate (Compound T-2);

Compound 11 (140 mg, 0.764 mmol), HOBT (103.2 mg, 0.764 mmol) and EDCI (146.1 mg, 0.764 mmol) were added to the reaction flask, and the mixture was dissolved in 2 mL of acetonitrile, and reacted at room temperature for 5 minutes to obtain a solution 1 after activation. Compound 7 (276.53 mg, 0.332 mmol) was dissolved in 5 mL DMF and DIPEA (309 mg, 2. The solution 1 was added to the solution 2, and the reaction was stirred at room temperature for 3 hours. After the completion of the TLC reaction, distilled water was added to precipitate a white solid, which was filtered with suction, and then filtered, and then purified by column chromatography to obtain 90 mg of compound T-2, yield 26.9%. . _{^{1 H NMR (300MHz, CDCl 3}} ) δ10.86 (d, J = 13.5Hz, 2H), 8.02-7.83 (m, 2H), 7.42 (ddd, J = 31.6,25.6,9.6Hz, 4H), 6.68 ( t, J = 18.7 Hz, 6H), 5.52 (d, J = 11.7 Hz, 2H), 5.46 - 5.31 (m, 2H), 4.31 (d, J = 5.9 Hz, 2H), 3.74 (d, J = 15.1) Hz,6H), 3.59-3.25(m,4H),3.02(s,2H), 2.95(s,2H),2.88(s,2H),2.77(s,4H),2.51(s,2H),2.36 (s, 2H), 1.93 (d, J = 12.7 Hz, 2H), 1.85-1.69 (m, 10H). LC-MS (APCI): m / z = 1018.4 (M + 1) +.

Example 3

Dimethyl ((2S, 2'S)-((2S, 2'S, 3aS, 3a'S, 7aS, 7a'S)-2,2'-(5,5'-(tricyclo[8.8.2.2 ^4,7 ]hexa- 4,6,10,12,13,15-hexene-5,11-diyl)bis(1hydro-d3-benzimidazole-5,2-diyl))di(octahydro-1hydro-indole) Indole-2,1-diyl))bis(3-methyl-1-oxo-butane-2,1-diyl)dicarbamate, compound T-3, has the formula:

Synthesized by the following route:

Based on the embodiment 1, the following steps are included:

Synthesis of 4-bromo-3,5,6-tridecan o-phenylenediamine (Compound 12);

4-bromo-o-phenylenediamine (374 mg, 2 mmol), concentrated hydrochloric acid (0.33 mL, 4 mmol) and 5 mL of heavy water were added to the reaction flask, stirred and dissolved, placed in a microwave reactor, and reacted at 140 ° C for 0.5 hour. The pH was adjusted to basic with 1N sodium hydroxide and extracted three times with ethyl acetate. EtOAc was evaporated. LC-MS (APCI): m / z = 191.4 (M + 1) +.

2, (2R, 3aS, 7aS)-tert-butyl-2-((2-amino-4-bromo-3,5,6-tridecylphenyl)carbamoyl) octahydro-1 hydrogen-hydrazine Synthesis of 1-carboxylic acid ester (compound 13);

Compound 1 (1.62 g, 6 mmol) and compound 12 (1.37 g, 7.2 mmol) were added to the reaction flask, dissolved in 36 mL of acetonitrile, condensed reagent EDCI (1.38 g, 7.2 mmol) was added, and DIPEA (N, N-diisopropylethylamine, 1.01 g, 7.8 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed by TLC, half of acetonitrile was removed by concentration, and 72 mL of water was added thereto, and the mixture was stirred at room temperature overnight. Filtration, the filter cake was washed with water and dried in vacuo to give 2.19 g of product. LC-MS (APCI): m / z = 442.1 (M + 1) +.

3, (2R, 3aS, 7aS)-tert-butyl-2-(6-bromo-5,7,8-trisino-1hydro-benzimidazol-2-yl) octahydro-1 hydrogen-hydrazine -1 synthesis of a carboxylate (compound 14);

Compound 13 (2.19g, 5mmol) was dissolved in 10mL of acetic acid, heated to 65 ° C and stirred for 3 hours. After TLC detection, the solvent was concentrated and concentrated, diluted with ethyl acetate, and added with 2 mL of ammonia water under stirring to adjust the pH to weakly alkaline. The extract was washed with water, and the organic layer was combined, evaporated, evaporated, evaporated LC-MS (APCI): m / z = 424.2 (M + 1) +.

4. (2R, 3aS, 7aS)-tert-butyl-2-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5, Synthesis of 7,8-tridesin-1hydrogen-benzimidazol-2-yl)octahydro-1hydro-indole-1carboxylate (Compound 15);

Compound 14 (2.1 g, 5 mmol), pinacol borate (1.9 g, 7.5 mmol), Pd(dppf)Cl ₂ (0.366 g, 1.5 mmol) and potassium acetate (1.47 g, were added to the dried reaction flask. Add 15mL anhydrous dioxane under nitrogen protection, heat to 85 ° C and stir the reaction for 4 hours. After TLC detection, dilute with ethyl acetate, add activated carbon for 1 hour, filter, concentrate to obtain oily liquid, column layer Purification afforded 3.1 g of Compound 15 in a yield of 100%. LC-MS (APCI): m / z = 471.2 (M + 1) +.

5, (2S, 2'S, 3aS, 3a'S, 7aS, 7a'S)-di-tert-butyl-2,2'-(5,5'-(tricyclo[8.8.2.2 ^4,7 ]hexa-4,6, 10,12,13,15-hexene-5,11-diyl)bis(5,7,8-trideutero-1hydro-benzimidazole-5,2-diyl)di(octahydro-1) Synthesis of hydrogen-indole-1-carboxylate) (Compound 16);

Compound 15 (1.85 g, 3.93 mmol), compound 5 (0.58 g, 1.57 mmol), cesium carbonate (1.28 g, 3.92 mmol) and Pd(PPh ₃ ) ₄ (90.7 mg, 0.08 mmol), nitrogen, were added to the reaction flask. Under the protection, 15 mL of DMF and 1 mL of water were added, and the reaction was carried out at 130 ° C for 3 hours. After the TLC reaction was completed, the catalyst was removed by filtration, distilled water was added to precipitate a white solid, which was filtered and dried to give a crude product. 34.3%. LC-MS (APCI): m / z = 894.5 (M + 1) +.

6, (2S, 2'S, 3aS, 3a'S, 7aS, 7a'S)-2,2'-(5,5'-(tricyclic [8.8.2.2 ^4,7 ] sixteen-4,6,10,12,13 ,15-hexene-5,11-diyl)di(5,7,8-trideutero-1 hydrogen-benzimidazole-5,2-diyl)di(octahydro-1hydro-indole) Synthesis of (Compound 17);

The compound 16 (480 mg, 0.537 mmol) was dissolved in a mixed solvent of 4 mL of dichloromethane and 1 mL of methanol, and 2.0 mL of hydrogen chloride dioxane was added at 0 ° C, and the mixture was allowed to react at room temperature for 1 hour. After the TLC was detected, the solvent was concentrated. The crude compound 17 was obtained and was directly taken to the next step without purification.

7, dimethyl ((2S, 2'S)-((2S, 2'S, 3aS, 3a'S, 7aS, 7a'S)-2,2'-(5,5'-(tricyclo[8.8.2.2 ^4,7 ]10 Hexa(1,6,10,12,13,15-hexene-5,11-diyl)bis(1hydro-d3-benzimidazole-5,2-diyl))bis(octahydro-1H) -吲哚-2,1-diyl))bis(3-methyl-1-oxo-butane-2,1-diyl)dicarbamate, ie the synthesis of compound T-3;

Moc-L-proline (388.9 mg, 2.22 mmol), HOBT (300 mg, 2.22 mmol) and EDCI (425.6 mg, 2.22 mmol) were added to the reaction flask, dissolved in 5 mL of acetonitrile, and reacted at room temperature for 5 minutes to obtain activation. Solution 1. Compound 17 (802.2 mg, 0.965 mmol) was dissolved in 10 mL DMF and DIPEA (898.3 mg, 6. The solution 1 was added to the solution 2, and the reaction was stirred at room temperature for 3 hours. After the completion of the TLC reaction, distilled water was added to precipitate a white solid, which was filtered with suction. The filter cake was dried and purified by column chromatography to yield 400 mg of compound T-3, yield 41.2%. . ¹ H NMR (300MHz, CDCl ₃ ) δ 10.85 (d, J = 13.0 Hz, 2H), 6.77 - 6.60 (m, 6H), 5.53 - 5.37 (m, 4H), 4.44 - 4.13 (m, 4H), 3.77 (d, J = 21.4 Hz, 7H), 3.45 (dd, J = 21.0, 8.0 Hz, 4H), 3.03 (s, 2H), 2.96 (s, 2H), 2.86 (d, J = 18.3 Hz, 2H) ), 2.71 (d, J = 42.0 Hz, 4H), 2.52 (s, 2H), 2.37 (s, 2H), 2.08 - 1.90 (m, 4H), 1.72 (s, 12H), 0.98 (dt, J = 10.6, 5.5 Hz, 6H), 0.89 - 0.83 (m, 6H). LC-MS (APCI): m / z = 1008.3 (M + 1) +.

Example 4

Dimethyl ((2S, 2'S)-((2S, 2'S, 3aS, 3a'S, 7aS, 7a'S)-2,2'-(5,5'-(tricyclo[8.8.2.2 ^4,7 ]hexa- 5,7,8,12,13,15-d6-4,6,10,12,13,15-hexene-5,11-diyl)di(1H-d3-benzimidazole-5,2 -diyl)) bis(octahydro-1hydro-indole-2,1-diyl))bis(3-methyl-1-oxo-butane-2,1-diyl)dicarbamic acid The ester, compound T-4, has the following formula:

Synthesized by the following route:

Includes the following steps:

Synthesis of 1,4,16-dibromo-5,7,8,12,13,15-d6[2.2]polyxylene (Compound 18);

Raw material compound 5 (109.8 mg, 0.3 mmol), MoCl ₅ (8.2 mg, 0.03 mmol) and 2 mL of deuterated benzene were added to the reaction flask, and the reaction was carried out in a microwave at 80 ° C for 1.5 hours. After the temperature was lowered to room temperature, the catalyst was removed by filtration and concentrated. The crude product was purified by column chromatography to yield compound (100 g, Compound 18). LC-MS (APCI): m / z = 373.3 (M + 1) +.

2, (2S, 2'S, 3aS, 3a'S, 7aS, 7a'S)-di-tert-butyl-2,2'-(5,5'-(tricyclo[8.8.2.2 ^4,7 ]hexa-5,7, 8,12,13,15-d6-4,6,10,12,13,15-hexene-5,11-diyl)bis(1H-benzimidazole-5,2-diyl)di Synthesis of octahydro-1hydro-indole-1-carboxylate) (Compound 19);

Compound 4 (1.85 g, 3.93 mmol), compound 18 (0.58 g, 1.57 mmol), cesium carbonate (1.28 g, 3.92 mmol) and Pd(PPh ₃ ) ₄ (90.7 mg, 0.08 mmol), nitrogen, were added to the reaction flask. Under the protection, 15 mL of DMF and 1 mL of water were added, and the reaction was carried out at 130 ° C for 3 hours. After the TLC reaction was completed, the catalyst was removed by filtration, distilled water was added to precipitate a white solid, which was filtered and dried to give a crude product. 34.1%. LC-MS (APCI): m / z = 894.5 (M + 1) +.

3, (2S, 2'S, 3aS, 3a'S, 7aS, 7a'S)-2,2'-(5,5'-(tricyclic [8.8.2.2 ^4,7 ] sixteen-5,7,8,12,13 ,15-d6-4,6,10,12,13,15-hexene-5,11-diyl)bis(1H-benzimidazole-5,2-diyl)di(octahydro-1H) -吲哚) (Synthesis of Compound 20);

The compound 16 (480 mg, 0.537 mmol) was dissolved in a mixed solvent of 4 mL of dichloromethane and 1 mL of methanol, and 2.0 mL of hydrogen chloride dioxane was added at 0 ° C, and the mixture was allowed to react at room temperature for 1 hour. After the TLC was detected, the solvent was concentrated. The crude compound 20 was obtained and was directly taken to the next step without purification.

4, dimethyl ((2S, 2'S)-((2S, 2'S, 3aS, 3a'S, 7aS, 7a'S)-2,2'-(5,5'-(tricyclic [8.8.2.2 ^4,7 ]10 Hexa-5,7,8,12,13,15-d6-4,6,10,12,13,15-hexene-5,11-diyl)di(1H-d3-benzimidazole-5 ,2-diyl))bis(octahydro-1hydro-indole-2,1-diyl))bis(3-methyl-1-oxo-butane-2,1-diyl)diamino Synthesis of formate, compound T-4);

Moc-L-proline (98.53 mg, 0.553 mmol), HOBT (74.7 mg, 0.553 mmol) and EDCI (106.1 mg, 0.553 mmol) were added to the reaction flask, dissolved in 3 mL of acetonitrile, and reacted at room temperature for 5 minutes to obtain activation. After solution 1. Compound 20 (200 mg, 0.24 mmol) was dissolved in 5 mL DMF and DIPEA (223.3 mg, 1. The solution 1 was added to the solution 2, and the reaction was stirred at room temperature for 3 hours. After the completion of the TLC reaction, distilled water was added to precipitate a white solid, which was filtered under suction, and then filtered, and then purified by column chromatography to yield 78 mg of compound T-4, yield: 32.4%. . _{^{1 H NMR (300MHz, CDCl 3}} ) δ10.88 (s, 2H), 8.00 (d, J = 9.8Hz, 1H), 7.88 (s, 1H), 7.62-7.33 (m, 4H), 5.54-5.38 ( m, 4H), 4.41 - 4.22 (m, 4H), 3.75 (d, J = 16.2 Hz, 6H), 3.44 (dd, J = 24.4, 10.4 Hz, 4H), 3.03 (s, 2H), 2.96 (s) , 2H), 2.87 (d, J = 9.3 Hz, 2H), 2.79 (d, J = 6.6 Hz, 4H), 2.52 (s, 2H), 2.36 (d, J = 11.9 Hz, 2H), 1.94 (d , J = 14.3 Hz, 4H), 1.76 (d, J = 10.7 Hz, 11H), 1.02 - 0.94 (m, 6H), 0.89 - 0.81 (m, 6H). LC-MS (APCI): m / z = 1008.7 (M + 1) +.

Example 5

The compounds of the above examples were evaluated for biological activity.

To verify the effect of the compounds described herein on HCV, the inventors used the HCV Replicon System as an evaluation model. Since its first report in Science in 1999, the HCV replication system has become one of the most important tools for studying HCV RNA replication, pathogenicity and viral persistence, for example, the use of replicons has successfully demonstrated the 5' required for HCV RNA replication. - NCR minimum region, and the HCV replication subsystem has been successfully used as an evaluation model for antiviral drugs. The inventors of the present invention verified according to the methods described in Science, 1999, 285 (5424), 110-3, and J. Virol, 2003, 77(5), 3007-19.

(1) Detection of compound anti-HCV 1a and 1b genotype replicon activity

The inhibitory activities of the recombinant hepatitis C virus genotype 1a and 1b replicons were detected by stable transfection of replicon cells with HCV-1a and HCV-1b. This experiment will use the NS5A inhibitor BMS-790052 as a positive control compound.

Step 1: The compound was diluted 1:3 in 8 series points, double-replicated, and added to a 96-well plate. The DMSO was set to no compound control. The final concentration of DMSO in the cell culture was 0.5%.

Step 2: HCV-1a and 1b cells were separately suspended in a culture medium containing 10% FBS, and seeded into a 96-well plate containing the compound at a density of 8,000 cells per well. The cells were cultured for 3 days at 5% CO ₂ at 37 °C.

Step 3: The cytotoxicity of the compound against GT1b replicon was determined using CellTiter-Fluor (Promega).

Step 4: Detection of luciferase assay by Bright-Glo (Promega) for anti-hepatitis C virus activity.

Step Five: using GraphPad Prism data analysis software, the curve fitting and EC ₅₀ values _were calculated and the ₅₀ value CC.

The compounds of Examples 1 to 4 were analyzed in the above procedure to calculate the EC50 value and the CC50 value, and the results are shown in Table 1.

(2) Determination of compound anti-infectious disease hepatitis virus (GT2a) activity

This infection model is referred to as A novel luciferase and GFP dual reporter virus for rapid and convenient evaluation of HCV replication. Virus Res 2011. 155: 406. After Huh-7.5.1 cells were infected with HCVcc (MOI = 0.2), the compounds were treated for 3 days (8 concentrations, 3 fold dilution, double duplicate wells). Antiviral activity will be determined by the luciferase method. The activity of the compound on Huh7.5.1 cells was also determined. This experiment will use the NS5A inhibitor BMS-790052 as a positive control compound. Using GraphPad software to calculate EC _50.

The compounds of Examples 1 to 4 were analyzed for the EC ₅₀ according to the above procedure, and the results are shown in Table 1.

Table 1 Biological Activity Evaluation Analysis Tables of Examples 1 to 4

As shown in Table 1, it was confirmed that the compound of the present invention can inhibit multiple genotypes of HCV and exerts superior anti-HCV action by inhibiting the mechanism of HCV NS5A protein.

(3) Metabolic stability evaluation

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

Preparation of the stock solution: A certain amount of the powder of the compound examples 1-4 was accurately weighed and dissolved to 5 mM with DMSO, respectively.

Preparation of phosphate buffer (100 mM, pH 7.4): Mix 150 mL of pre-formed 0.5 M potassium dihydrogen phosphate and 700 mL of 0.5 M potassium dihydrogen phosphate solution, and adjust the mixture with 0.5 M potassium dihydrogen phosphate solution. The pH was adjusted to 7.4, diluted 5 times with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100 mM) containing 100 mM potassium phosphate, 3.3 mM magnesium chloride, and a pH of 7.4.

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

Formulation stop solution: acetonitrile solution containing 50 ng/mL propranolol hydrochloride and 200 ng/mL tolbutamide (internal standard). Take 25057.5 μL of phosphate buffer (pH 7.4) into a 50 mL centrifuge tube, add 812.5 μL of human liver microsomes, and mix to obtain a liver microsome dilution with a protein concentration of 0.625 mg/mL. 25057.5 μL of phosphate buffer (pH 7.4) was taken into a 50 mL centrifuge tube, and 812.5 μL of SD rat liver microsomes were added and mixed to obtain a liver microsome dilution having a protein concentration of 0.625 mg/mL.

Incubation of the sample: The stock solution of the corresponding compound was diluted to 0.25 mM with an aqueous solution containing 70% acetonitrile as a working solution, and was used. 398 μL of human liver microsomes or rat liver microsome dilutions were added to 96-well incubation plates (N=2), and 2 μL of 0.25 mM working solution was added and mixed.

Determination of metabolic stability: 300 μL of pre-cooled stop solution was added to each well of a 96-well deep well plate and placed on ice as a stop plate. The 96-well incubation plate and the NADPH regeneration system were placed in a 37 ° C water bath, shaken at 100 rpm, and pre-incubated for 5 min. 80 μL of the incubation solution was taken from each well of the incubation plate, added to the stopper plate, and mixed, and 20 μL of the NADPH regeneration system solution was added as a sample of 0 min. Then, 80 μL of the NADPH regeneration system solution was added to each well of the incubation plate to start the reaction and start timing. The corresponding compound had a reaction concentration of 1 μM and a protein concentration of 0.5 mg/mL. 100 μL of the reaction solution was taken at 10, 30, and 90 min, respectively, and added to the stopper, and the reaction was terminated by vortexing for 3 min. The plate was centrifuged at 5000 x g for 10 min at 4 °C. 100 μL of the supernatant was taken into a 96-well plate to which 100 μL of distilled water was previously added, mixed, and sample analysis was performed by LC-MS/MS.

Data analysis: The peak area of the corresponding compound and the internal standard was detected by LC-MS/MS system, and the ratio of the peak area of the compound to the internal standard was calculated. The slope is measured by the natural logarithm of the percentage of the remaining amount of the compound versus time, and t _1/2 and CL _{int are} calculated according to the following formula, where V/M is equal to 1/protein concentration.

The compounds of Examples 1-4 were analyzed according to the above procedure, and the results are shown in Table 2.

Table 2 Table of results of determination of metabolic stability of the compounds of Examples 1-4

As shown in Table 2, the compounds of the present invention exhibited excellent metabolic stability in both human liver microsomes and rat liver microsome experiments.

(4) Rat pharmacokinetic experiments

OBJECTIVE: To investigate the pharmacokinetic behavior of the compounds of the present invention after administration of Odalasvir and Compound T-1 to rats.

Experimental animals:

Type and strain: SD rat grade: SPF grade

Gender and quantity: male, 6

Weight range: 180 ~ 220g (actual weight range is 187 ~ 197g)

Source: Shanghai Xipuer Bikai Experimental Animal Co., Ltd.

Experimental and animal certificate number: SCXK (Shanghai) 2013-0016

experiment procedure:

Before the blood sample was collected, 20 L of 2M sodium fluoride solution (esterase inhibitor) was previously added to the EDTA-K2 anticoagulation tube, dried in an 80 degree oven, and stored in a 4 degree refrigerator.

Rats, males, weighing 187-197 g, were randomly divided into 2 groups. They were fasted overnight in the afternoon before the experiment but were free to drink water. Food was given 4 h after administration. Group A was given Odalasvir 3 mg/kg, and group B was given 3 mg/kg of compound of formula T-1, respectively, from the eyes of rats at 15 min, 30 min, 1, 2, 3, 5, 8, 10 h after administration. The blood is taken from 100-200L, placed in a 0.5mL Eppendorf tube that is anticoagulated with EDTA-K2, and immediately mixed. After anticoagulation, gently invert the tube 5-6 times as soon as possible, after the blood is taken. Place in an ice box, centrifuge the blood sample at 4000 rpm, 10 min, 4 ° C for 30 min, collect all plasma and store at -20 ° C immediately. Plasma concentrations in plasma at each time point were determined after sample collection at all time points.

According to the average blood drug concentration-time data obtained after the above, the male SD rats were treated with Winnonin software according to the non-compartmental statistical moment theory to give Odalasvir (3 mg/kg) and the compound of formula T-1 (3 mg/). The pharmacokinetic related parameters after kg) are detailed in Table 3.

Table 3 Pharmacokinetic parameters of rats given Odalasvir and compound T-1

PK parameters	Odalasvir	T-1
Cmax(ng/mL)	53865.5±1814.2	58141.1±8516.1
t 1/2 (hr)	7.64±1.31	7.98±0.31
AUC 0-t (hr*ng/mL)	62819.7±3778.6	64653.8±3976.9
AUC 0-∞ (hr*ng/mL)	65019.9±3217.5	67373.6±4116.9
MRT(hr)	4.01±0.37	4.30±0.12

As shown in Table 3, the present inventors found that the compound T-1 has superior activity to Odalasvir and has excellent pharmacokinetic properties as compared with Odalasvir, and thus is more suitable as a compound which inhibits the hepatitis C virus protein NS5A. It is further suitable for the preparation of a medicament for treating hepatitis C virus infection.

It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention, and the experimental methods in which the specific conditions are not indicated in the examples, usually in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer. Parts and percentages are parts by weight and percentage by weight unless otherwise stated.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims (15)

1. A substituted hepatitis C virus inhibitor characterized by a compound of formula (I), or a crystalline form thereof, a pharmaceutically acceptable salt, a prodrug, a hydrate or a solvent compound,

   

   Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ And R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ , R ⁴⁰ , R ⁴¹ , R ⁴² , R ⁴³ , R ⁴⁴ , R ⁴⁵ , R ⁴⁶ , R ⁴⁷ , R ⁴⁸ , R ⁴⁹ , R ⁵⁰ , R ⁵¹ , R ⁵² , R ⁵³ , R ⁵⁴ , R ⁵⁵ , R ⁵⁶ , R ⁵⁷ , R ⁵⁸ , R ⁵⁹ , R ⁶⁰ , R ⁶¹ , R ⁶² , R ⁶³ , R ⁶⁴ , R ⁶⁵ , R ⁶⁶ , R ⁶⁷ and R ⁶⁸ are each independently hydrogen, deuterium, halogen or trifluoromethyl;

   Additional conditions are R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ And R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ , R ⁴⁰ , R ⁴¹ , R ⁴² , R ⁴³ , R ⁴⁴ , R ⁴⁵ , R ⁴⁶ , R ⁴⁷ , R ⁴⁸ , R ⁴⁹ , R ⁵⁰ , R ⁵¹ , R ⁵² , R ⁵³ , R ⁵⁴ , R ⁵⁵ , R ⁵⁶ , R ⁵⁷ , R ⁵⁸ , R ⁵⁹ , R ⁶⁰ , R ⁶¹ , R ⁶² , R ⁶³ , R ⁶⁴ , R ⁶⁵ At least one of R ⁶⁶ , R ⁶⁷ and R ⁶⁸ is deuterated or deuterated.
2. The hepatitis C virus inhibitor according to claim 1, wherein each of R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ^{6 is} independently hydrazine or hydrogen.
3. The hepatitis C virus inhibitor according to claim 1, wherein R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ and R ¹⁷ And R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ and R ²² are each independently hydrazine or hydrogen.
4. The hepatitis C virus inhibitor according to claim 1, wherein R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ and R ²⁰ R ²¹ is 氘.
5. The hepatitis C virus inhibitor according to claim 1, wherein R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ And R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ , R ⁴⁰ , R ⁴¹ , R ⁴² , R ⁴³ , R ⁴⁴ , R ⁴⁵ , R ⁴⁶ , R ⁴⁷ and R ⁴⁸ are each independently The ground is helium or hydrogen.
6. The hepatitis C virus inhibitor according to claim 1, wherein R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³⁶ R ³⁷ , R ³⁸ , R ³⁹ , R ⁴⁰ , R ⁴¹ , R ⁴² , R ⁴³ , R ⁴⁴ and R ⁴⁵ are 氘.
7. The hepatitis C virus inhibitor according to claim 1, wherein R ⁴⁹ , R ⁵⁰ , R ⁵¹ , R ⁵² , R ⁵³ and R ⁵⁴ are each independently hydrazine or hydrogen.
8. The hepatitis C virus inhibitor according to claim 1, wherein each of R ⁵⁵ , R ⁵⁶ , R ⁵⁷ , R ⁵⁸ , R ⁵⁹ and R ^{60 is} independently hydrazine or hydrogen.
9. The hepatitis C virus inhibitor according to claim 1, wherein R ⁶¹ , R ⁶² , R ⁶³ , R ⁶⁴ , R ⁶⁵ , R ⁶⁶ , R ⁶⁷ and R ⁶⁸ are each independently hydrazine or hydrogen.
10. The hepatitis C virus inhibitor according to claim 1, wherein the compound is selected from the group consisting of the following compounds or a pharmaceutically acceptable salt thereof:

    

    
11. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a hepatitis C virus inhibitor according to any one of claims 1 to 10, or a crystalline form thereof, a pharmaceutically acceptable salt thereof A pharmaceutical composition of a hydrate or solvate, stereoisomer, prodrug or isotopic variation.
12. The pharmaceutical composition according to claim 11, which further comprises other active compounds which are immunomodulators or antiviral compounds.
13. Use of a hepatitis C virus inhibitor according to any one of claims 1 to 10, which is for use in the preparation of a medicament for treating hepatitis C virus infection.
14. A method of treating and/or preventing a hepatitis C virus-related disease in a subject, the method comprising administering to the subject the formula (I) according to any one of claims 1 to 10. a compound or a polymorphic form thereof, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variation, a hydrate or a solvent compound, or a pharmaceutical composition according to any one of claims 11 or 12.
15. A compound of formula (I), or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotope, hydrate or solvent thereof, according to any one of claims 1 to 10, or a right A pharmaceutical composition according to any one of claims 11 or 12 for use in the treatment and/or prevention of a protein kinase-associated disease.