CN106883280B

CN106883280B - A kind of prodrug, preparation method, medical composition and its use

Info

Publication number: CN106883280B
Application number: CN201611158070.5A
Authority: CN
Inventors: 周星露; 刘兴国; 董晓武
Original assignee: HANGZHOU HERTZ PHARMACEUTICAL Co Ltd
Current assignee: Guangdong HEC Pharmaceutical
Priority date: 2015-12-15
Filing date: 2016-12-15
Publication date: 2019-05-21
Anticipated expiration: 2036-12-15
Also published as: CN106883279A; CN105348345A; CN106883279B; CN106883280A

Abstract

The invention discloses a kind of prodrugs containing class nucleotide structure or its tautomer, stereoisomer shown in formula I, stereoisomer mixture or its pharmaceutically acceptable solvate, preparation method is also disclosed, and the pharmaceutical composition being made from it and its purposes as anti-hepatitis C drug.Such compound or its pharmaceutical composition can be used for the treatment of hepatitis C, while having the effects that protect hepatic tissue, liver cell, improve liver function, reduce aminopherase.

Description

Prodrug, preparation method thereof, pharmaceutical composition and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry. Specifically, the invention relates to a compound shown as the following general formula I, or a tautomer, a stereoisomer mixture, a prodrug, a pharmaceutically acceptable salt or a solvate thereof, a preparation method thereof, a pharmaceutical composition and application thereof as an anti-hepatitis C medicament.

Background

The hepatitis C is a liver disease seriously threatening the health of human beings caused by Hepatitis C Virus (HCV), 1978, the hepatitis C virus is discovered for the first time, 1989, gene sequence determination is completed, HCV is identified as another main pathogen of non-A type and non-B type hepatitis, according to the heterogeneity characteristics of HCV genome, the hepatitis C virus can be divided into 6 types and 30 subtypes, the infection types are strong regionality, China mainly takes type 1, 2011 researches show that type 1 accounts for 58.2% of Chinese HCV infectors, wherein type 1a accounts for 1.4% of the virus genome, type 1b accounts for 56.8% of the virus genome, the HCV infectors in the global range exceed 2 hundred million, the HCV infectors in the U.S. account for 3.3% of the general population in the world have about 320 ten thousand of the virus infectors, the Chinese patients exceed 4000 times, the first in the world, many HCV infectors are hepatitis B patients and even hepatitis B patients, the hepatitis C virus are also clinically approved by HCV polymerase, the hepatitis C virus is a hepatitis C virus-B virus-2 viral polymerase, the hepatitis C virus-B virus-2 viral polymerase gene, the hepatitis C virus is a viral polymerase chain-2 viral polymerase chain-resistant gene-resistant drug, the viral-2 viral-resistant viral-resistant viral-resistant viral-resistant viral-resistant viral-resistant viral-like viral-resistant viral-resistant viral-resistant viral-.

Disclosure of Invention

The invention aims to provide a novel anti-HCV virus and liver-protecting dual-function medicine, which plays the role of the dual-function medicine, has the advantages of high oral bioavailability and good metabolic property, and can be used for treating viral hepatitis and other symptoms.

One object of the present invention is to provide a prodrug represented by the general formula I, or a stereoisomer, a mixture of stereoisomers, or a pharmaceutically acceptable solvate thereof.

Another object of the present invention is to provide a process for the preparation of the compounds provided by the present invention.

The invention also aims to provide the application of the prodrug shown as the general formula I or the stereoisomer, the stereoisomer mixture or the pharmaceutically acceptable salt or the solvate thereof as an NS5B inhibitor, protecting liver cells and liver tissues and improving liver functions, and the application in preparing medicines for treating viral hepatitis.

It is a further object of the present invention to provide pharmaceutical compositions comprising one or more of the prodrugs of formula I or stereoisomers, mixtures thereof or pharmaceutically acceptable solvates thereof.

It is still another object of the present invention to provide a method for treating viral hepatitis while protecting liver cells, liver tissues and improving liver function.

The invention adopts the following technical scheme:

according to one aspect of the invention, the invention protects a compound having a structure represented by general formula I, or a stereoisomer, a mixture of stereoisomers, or a pharmaceutically acceptable salt or solvate thereof, or a prodrug represented by general formula I:

wherein X is hydroxyl or F, Cl or Br; r isLinker is Or is absent, A is from the market alreadyThe medicament for treating or adjunctively treating liver diseases or derivatives thereof; r₁Is H or C₁-C₅An alkyl group; n is an integer of 1 to 19.

In the general formula I, a further preferable compound is a prodrug containing a nucleoside-like structure shown in the general formula II-A, or a stereoisomer, a stereoisomer mixture or a pharmaceutically acceptable salt or a solvate thereof:

wherein X is hydroxyl or F, Cl or Br; linker isOr is absent; r₁Is H or C₁-C₅An alkyl group; n is 1,2, 3, 4 or 5; r₃、R₄Each independently is H or C₁-C₅An alkyl group.

In the general formula II-A, further preferred compounds are structures represented by the general formula II-Aa or formula II-Ab, or tautomers, optical isomers, stereoisomers, mixtures of stereoisomers, prodrugs, pharmaceutically acceptable salts or solvates thereof:

In the general formula I, further preferred compounds are structures represented by general formula II-B, or tautomers, optical isomers, stereoisomers, mixtures of stereoisomers, prodrugs, pharmaceutically acceptable salts, or solvates thereof:

In the general formula II-B, the structure shown in the general formula II-Ba or the general formula II-Bb, or an optical isomer, a pharmaceutically acceptable salt or a solvate thereof is further preferable:

In the general formula I, the prodrug containing a tiopronin structure shown in the general formula II-A-A (1) or a stereoisomer, a stereoisomer mixture or a pharmaceutically acceptable solvate thereof is further preferable:

wherein X is hydroxyl or F, Cl, Br, Linker isR₁Is H or C₁-C₅Alkyl, n is an integer from 1 to 19.

Alternatively, in the general formula II-a (1), a prodrug having a tiopronin structure represented by the following general formula II-a (2) or a stereoisomer, a mixture of stereoisomers, or a pharmaceutically acceptable solvate thereof is more preferable:

in the above formula: x, Linker A, R₁And n is as defined above for formula II-A-A (1).

In the general formula II-A-A (1), the following prodrugs containing tiopronin structure or its stereoisomers, mixture of stereoisomers or its pharmaceutically acceptable solvates are further preferred:

wherein X is hydroxyl or F, Cl or Br; r₁Is H or C₁-C₅Alkyl, n is 1,2, 3, 4 or 5.

Alternatively, in the general formula II-a (1), the following prodrugs containing a tiopronin structure or stereoisomers, mixtures of stereoisomers, or pharmaceutically acceptable solvates thereof are further preferred:

wherein, X, R₁And n is as defined above.

wherein R is₁Is H or C₁-C₅Alkyl, n is 1,2, 3, 4 or 5.

Among the compounds represented by the general formula I, a further preferred specific compound is one of the following compounds:

or a stereoisomer, a mixture of stereoisomers of the above preferred compounds or a pharmaceutically acceptable salt or solvate thereof.

It is another object of the present invention to provide a process for the preparation of a compound of formula II-A, said process comprising: 1) reacting the compound of the formula III with a compound of a formula IV-A to obtain a compound of a formula V-A, and 2) carrying out deprotection reaction on the compound of the formula V-A to obtain a compound of a formula II-A;

wherein X is hydroxyl or halogen (F, Cl, Br); linker is Or is absent; n is 1,2, 3, 4 or 5; r₁Is H or C₁-C₅An alkyl group; r₃、R₄Each independently is H or C₁-C₅An alkyl group; LG is a leaving group, preferably halogen; z is a mercapto-protecting group, preferably trityl, 4-methoxytrityl, 4' -bismethoxytrityl.

It is another object of the present invention to provide a process for the preparation of compounds of formula II-B, said process comprising: 1) reacting the compound shown in the formula III with the compound shown in the formula IV-B to obtain a compound shown in the formula V-B; 2) carrying out deprotection reaction on the compound of the formula V-B to obtain a compound of a formula II-B;

wherein, X is hydroxyl or halogen (F, Cl, Br); linker is Or is absent; n is 1,2, 3, 4 or 5; r₁Is H or C₁-C₅An alkyl group; r₃、R₄Each independently is H or C₁-C₅An alkyl group; LG is a leaving group, preferably halogen; z is a mercapto-protecting group, preferably trityl, 4-methoxytrityl, 4' -bismethoxytrityl.

Furthermore, the preparation method of the compound shown in the general formula II-A-Aa can be used for obtaining the compound shown in the general formula V-A by alkylation reaction of the compound shown in the general formula III and the compound shown in the general formula IV and then deprotection reaction.

Wherein X is F, Cl or Br; r₁Is H or C₁-C₅An alkyl group; y is a leaving group and Z is a mercapto-protecting group.

A preparation method of a compound shown as a formula II-A-Ab can be obtained by performing alkylation reaction on a compound shown as a formula III and a compound shown as a formula IV-B to obtain a compound shown as a formula V-B, and then performing deprotection reaction:

wherein X is hydroxyl or halogen (F, Cl, Br); r₁Is H or C₁-C₅An alkyl group; n is an integer of 1 to 19; y is a leaving group; z is a mercapto protecting group.

Another object of the present invention is to provide a process for the preparation of compounds of formula II-A-B, which comprises subjecting a compound of formula III to a protection reaction, then reacting with an aldehyde to obtain a compound of formula VII-1, then subjecting the compound of formula VII-1 to an esterification or acylation reaction with a compound of formula VIII-1A to obtain a compound of formula IX-A-B, and then removing the protecting group to obtain a compound of formula II-1A-B. The reaction active sites of the key intermediates of the preparation method are properly protected, which is beneficial to reducing the occurrence of side reactions, the reaction selectivity is high, and the obtained intermediates and final products have high purity and are easy to purify. The reaction involved in the method has the advantages of simple operation, convenient purification and good process controllability, and is suitable for industrial production.

Wherein X is hydroxyl or halogen (F, Cl, Br); r₁Is H or C₁-C₅An alkyl group; r₃、R₄Each independently is H or C₁-C₅An alkyl group; p₁Is a hydroxyl protecting group, and is preferably trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl or triphenylsilyl; p₂Is an amide NH protecting group, preferably tert-butoxycarbonyl, benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl; p₃The mercapto group-protecting group is preferably trityl, 4-methoxytrityl, or 4, 4' -bismethoxytrityl.

Another object of the present invention is to provide a process for the preparation of compounds of the formula II-B-B, which comprises subjecting a compound of the formula VII-1 to esterification or acylation with a compound of the formula VIII-1B to give a compound of the formula IX-B-B, and then removing the protecting group to give a compound of the formula II-B-B. The reaction active sites of the key intermediates of the preparation method are properly protected, which is beneficial to reducing the occurrence of side reactions, the reaction selectivity is high, and the obtained intermediates and final products have high purity and are easy to purify. The reaction involved in the method has the advantages of simple operation, convenient purification and good process controllability, and is suitable for industrial production.

Wherein, X, R₁，R₃，R₄Is as defined in formula II-B, P₁Is a hydroxyl protecting group, and is preferably trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl or triphenylsilyl; p₂Is an amide NH protecting group, preferably tert-butoxycarbonyl, benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl; p₃The mercapto group-protecting group is preferably trityl, 4-methoxytrityl, or 4, 4' -bismethoxytrityl.

Another object of the present invention is to provide intermediates shown in the following VII and their use for preparing anti-hepatitis c virus drugs:

wherein X is hydroxyl or halogen (F, Cl, Br); r₁Is H or C₁-C₅An alkyl group; p₁As the hydroxyl protecting group, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl and triphenylsilyl are preferable.

It is another object of the present invention to provide intermediates as shown in the following VIII-A or stereoisomers or mixtures thereof:

wherein, P₂Is NH protecting group, preferably tert-butyloxycarbonyl, benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl; p₃As the mercapto group-protecting group, trityl, 4-methoxytrityl, 4' -bismethoxytrityl and the like are preferable.

It is another object of the present invention to provide prodrugs as shown below in IX-A-B or stereoisomers, mixtures thereof:

wherein X is hydroxyl or halogen (F, Cl, Br); r₁Is H or C₁-C₅An alkyl group; p₁Is a hydroxyl protecting group, and is preferably trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl or triphenylsilyl; p₂Is an NH protective group, preferably t-butoxycarbonyl, benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, or the like; p₃As the mercapto group-protecting group, trityl, 4-methoxytrityl, 4' -bismethoxytrityl and the like are preferable.

Another object of the present invention is to provide the following intermediates VIII-B or stereoisomers or mixtures thereof:

wherein R is₃、R₄Each independently isH or C₁-C₅An alkyl group; p₂Is an amide NH protecting group, preferably tert-butoxycarbonyl, benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl; p₃The mercapto group-protecting group is preferably trityl, 4-methoxytrityl, or 4, 4' -bismethoxytrityl.

It is another object of the present invention to provide prodrugs as shown below in IX-B-B or stereoisomers, mixtures thereof:

The compounds of formula I may contain one or more chiral centers, as stereoisomers, i.e. enantiomers, diastereomers or mixtures thereof, may be present. The compounds of the formula I-according to the invention can therefore be present as individual isomers or as mixtures of individual isomers.

The present invention includes any prodrug form of the compounds of formula I.

The invention also includes pharmaceutically acceptable solvates of the compounds of formula I.

The invention also includes pharmaceutically acceptable oxides of the compounds of formula I, and pharmaceutically acceptable salts and pharmaceutically acceptable solvates thereof.

The invention also includes various crystal forms of the compound shown in the general formula I.

According to still another aspect of the present invention, the present invention provides a use of the compound represented by the general formula I, or a tautomer, a stereoisomer, a mixture of stereoisomers, a prodrug, a pharmaceutically acceptable salt or a solvate thereof, as an NS5B inhibitor, for simultaneously protecting liver cells and liver tissues and improving liver functions, and for preparing a medicament for treating diseases such as viral hepatitis.

According to yet another aspect of the present invention, there is also provided a pharmaceutical composition comprising a therapeutically effective amount of one or more of a compound of formula I, or a tautomer, stereoisomer, mixture of stereoisomers, prodrug, pharmaceutically acceptable salt or solvate thereof, which can act as an NS5B inhibitor, and which composition can optionally comprise a pharmaceutically acceptable carrier or excipient.

According to another aspect of the present invention, the present invention also provides an NS5B inhibitor, which contains a therapeutically effective amount of one or more of the compounds represented by the general formula I, or a tautomer, stereoisomer mixture, prodrug, pharmaceutically acceptable salt or solvate thereof, and the inhibitor may optionally contain a pharmaceutically acceptable carrier or excipient.

The composition consists of one or more compounds shown in a general formula I in a treatment effective dose or a tautomer, a stereoisomer mixture, a prodrug, a pharmaceutically acceptable salt or a medicinal solvate thereof and at least one medicinal auxiliary material. The choice of pharmaceutical excipients varies with the route of administration and the nature of action, and is usually fillers, diluents, binders, wetting agents, disintegrants, lubricants, emulsifiers, suspending agents and the like. The proportion of the compound of formula I, stereoisomer mixture or pharmaceutically acceptable solvate thereof in the composition is 0.1-99.9%, preferably 1-99% of the total weight.

The pharmaceutically acceptable carrier refers to a pharmaceutical carrier conventional in the pharmaceutical field, such as: diluents such as water and the like; fillers, such as starch, sucrose, and the like; binders such as cellulose derivatives, alginates, gelatin, polyvinylpyrrolidone; humectants, such as glycerol; disintegrating agents such as agar, calcium carbonate and sodium bicarbonate; absorption promoters, such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite clay; lubricants, such as talc, calcium stearate and magnesium stearate, and polyethylene glycol, and the like. In addition, other adjuvants such as flavoring agents and sweeteners can also be added to the pharmaceutical composition.

The invention also provides a preparation method of the compound shown in the general formula I or a medicinal composition of the tautomer, the stereoisomer mixture, the prodrug, the pharmaceutically acceptable salt or the solvate thereof. The tiopronin structure-containing compound shown in the general formula I or a tautomer, a stereoisomer mixture, a prodrug, a pharmaceutically acceptable salt or a solvate thereof is usually mixed with a pharmaceutically acceptable auxiliary material, and is prepared into a form (dosage form) suitable for certain administration by a conventional preparation method. The dosage forms include tablets, capsules, granules, pills, solutions, suspensions, emulsions, ointments, films, creams, aerosols, injections, suppositories and the like. Tablets and capsules are preferred.

The dosage of the compound is generally 1-1000 mg per day, and the compound can be used for one time or multiple times. However, if necessary, the above-mentioned dose may be appropriately deviated. The optimal dosage can be determined by the skilled person according to the specific situation and the professional knowledge. These include the severity of the disease, individual differences among patients, the nature of the formulation and the route of administration, etc.

In addition, the invention also provides the application of the compound shown in the general formula I or a tautomer, a stereoisomer mixture, a prodrug, a pharmaceutically acceptable salt or a solvate thereof, or a medicinal composition thereof as a human medicine.

According to yet another aspect of the present invention, there is provided a method for treating viral hepatitis and the like, the method comprising administering to a patient a therapeutically effective amount of one or more of a compound of formula I, or a tautomer, stereoisomer, mixture of stereoisomers, prodrug, pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of the present invention.

The compounds or compositions provided herein can be administered by oral, injectable (intravenous, intramuscular, subcutaneous, and intracoronary), sublingual, buccal, rectal, urethral, vaginal, nasal, inhalation, or topical routes. The preferred route is oral. For oral administration, it can be made into conventional solid preparations such as tablet, powder, granule, capsule, etc., or liquid preparations such as water or oil suspension, or other liquid preparations such as syrup, etc.; for parenteral administration, it can be formulated into solution for injection, aqueous or oily suspension, etc.

The invention also provides a compound shown in the general formula I, or a tautomer, a stereoisomer mixture, a prodrug, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, and application of the compound in preparation of human medicines of the NS5B inhibitor.

The invention also provides a compound shown as the general formula I or a tautomer, a stereoisomer mixture, a prodrug, a pharmaceutically acceptable salt or a solvate thereof, and a drug for treating liver diseases, wherein at least one of the compounds is selected from interferon, interferon β, interferon gamma and interferon omega, interleukins including interleukin 10 and interleukin 12, ribavirin, interferon α or pegylated interferon α and ribavirin or levovirin for combined use, the treatment comprises the treatment of levovirin, protease inhibitors including NS3 inhibitors, NS3/4A inhibitors, NS5A inhibitors, helicase inhibitors, polymerase inhibitors including HCV RNA polymerase and NS5B polymerase inhibitors, gliotoxin, IRES inhibitors, antisense oligonucleotides, thiazolidine derivatives, N-benzoylaniline, ribozymes, nucleoside prodrugs or derivatives of nucleoside, 1-amino-alkylcyclohexane, antioxidants including vitamin E, squalene, amantadine, bile acid, N- (acetyl) -L-phenylalanine, dicarboxyieldrin, a phosphatidylcholine, a heparin-containing immunomodulator, a silymarin and a silybin.

"pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio, and which are effective for their intended use.

The terms "halogen" and "halo" are used interchangeably herein to refer to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

The term "alkyl" or "alkyl group" as used herein, denotes a saturated, straight or branched chain, monovalent hydrocarbon group containing 1 to 20 carbon atoms, wherein the alkyl group may be optionally substituted with one or more substituents as described herein. In one embodiment, the alkyl group contains 1 to 6 carbon atoms; in another embodiment, the alkyl group contains 1 to 4 carbon atoms; in yet another embodiment, the alkyl group contains 1 to 3 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH)₃) Ethyl group (Et, -CH)₂CH₃) N-propyl (n-Pr, -CH)₂CH₂CH₃) Isopropyl group (i-Pr, -CH (CH)₃)₂) N-butyl (n-Bu, -CH)₂CH₂CH₂CH₃) Isobutyl (i-Bu, -CH)₂CH(CH₃)₂) Sec-butyl (s-Bu, -CH (CH)₃)CH₂CH₃) Tert-butyl (t-Bu, -C (CH)₃)₃) And so on.

As used herein, "pharmaceutically acceptable salts" refer to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described hereinThe document is as follows: berge et al, descriptive acceptable salts in detail in J. pharmaceutical Sciences,1977,66:1-19. Pharmaceutically acceptable non-toxic acid salts include, but are not limited to, salts of inorganic acids formed by reaction with amino groups such as hydrochlorides, hydrobromides, phosphates, sulfates, perchlorates, and salts of organic acids such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, or those obtained by other methods described in the literature above, such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, cyclopentylpropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxy-ethanesulfonates, lactobionates, lactates, laurates, malates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, palmitates, pamoates, pectinates, persulfates, 3-phenylpropionates, picrates, pivalates, propionates, stearates, thiocyanates, p-toluenesulfonate, undecanoate, valerate, and the like. Salts obtained with appropriate bases include alkali metals, alkaline earth metals, ammonium and N⁺(C_1-4Alkyl radical)₄A salt. The present invention also contemplates quaternary ammonium salts formed from compounds containing groups of N. Water-soluble or oil-soluble or dispersion products can be obtained by quaternization. Alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts and amine cations resistant to formation of counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C₁-C₈Sulfonates and aromatic sulfonates.

"solvate" of the present invention refers to an association of one or more solvent molecules with a compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, ethanolamine, or mixtures thereof. The term "hydrate" refers to an association of solvent molecules that is water.

When the solvent is water, the term "hydrate" may be used. In one embodiment, a molecule of a compound of the present invention may be associated with a molecule of water, such as a monohydrate; in another embodiment, one molecule of the compound of the present invention may be associated with more than one molecule of water, such as a dihydrate, and in yet another embodiment, one molecule of the compound of the present invention may be associated with less than one molecule of water, such as a hemihydrate. It should be noted that the hydrates of the present invention retain the biological effectiveness of the compound in its non-hydrated form.

Unless otherwise indicated, all suitable isotopic variations, stereoisomers, tautomers, solvates, metabolites, salts and pharmaceutically acceptable prodrugs of the compounds of the present invention are encompassed within the scope of the present invention.

In the structures disclosed herein, when the stereochemistry of any particular chiral atom is not specified, then all stereoisomers of that structure are contemplated as within this invention and are included as disclosed compounds in this invention. When stereochemistry is indicated by a solid wedge (solid wedge) or dashed line representing a particular configuration, then the stereoisomers of the structure are so well-defined and defined.

The compounds of formula (I) may be present in the form of salts. In one embodiment, the salt refers to a pharmaceutically acceptable salt. The term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal being treated therewith. In another embodiment, the salts need not be pharmaceutically acceptable salts and may be intermediates useful in the preparation and/or purification of compounds of formula (I) and/or in the isolation of enantiomers of compounds of formula (I).

The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, basic or acidic moiety, by conventional chemical methods. In general, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of the appropriate base (e.g., Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, etc.), or by reacting the free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are usually carried out in water or an organic solvent or a mixture of both. Generally, where appropriate, it is desirable to use a non-aqueous medium such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile. In, for example, "Remington's Pharmaceutical Sciences", 20 th edition, Mack Publishing Company, Easton, Pa., (1985); and "handbook of pharmaceutically acceptable salts: properties, Selection and application (Handbook of pharmaceutical salts: Properties, Selection, and Use) ", Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002) may find some additional lists of suitable salts.

Any formulae given herein are also intended to represent the non-isotopically enriched forms as well as the isotopically enriched forms of these compounds. Isotopically enriched compounds have the structure depicted by the formulae given herein, except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Exemplary isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as²H、³H、¹¹C、¹³C、¹⁴C、¹⁵N、¹⁷O、¹⁸O、¹⁸F、³¹P、³²P、³⁵S、³⁶Cl and¹²⁵I。

in another aspect, the invention relates to intermediates for the preparation of compounds of formula (I).

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention. In one embodiment, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier, excipient, adjuvant, vehicle or combination thereof. In another embodiment, the pharmaceutical composition may be in a liquid, solid, semi-solid, gel, or spray dosage form.

The compound or the pharmaceutical composition thereof can be used for treating hepatitis C, has the advantages of high oral bioavailability and good metabolic property, and simultaneously has the effects of protecting liver tissues and liver cells and improving liver functions.

Detailed Description

To illustrate the invention, the following examples are set forth. It is to be understood that the invention is not limited to these embodiments, but is provided as a means of practicing the invention.

In general, the compounds of the present invention may be prepared by the methods described herein, wherein the substituents are as defined in formula (I), unless otherwise indicated. The following reaction schemes and examples serve to further illustrate the context of the invention.

Those skilled in the art will recognize that: the chemical reactions described herein may be used to suitably prepare a number of other compounds of the invention, and other methods for preparing the compounds of the invention are considered to be within the scope of the invention. For example, the synthesis of those non-exemplified compounds according to the present invention can be successfully accomplished by those skilled in the art by modification, such as appropriate protection of interfering groups, by the use of other known reagents in addition to those described herein, or by some routine modification of reaction conditions. In addition, the reactions disclosed herein or known reaction conditions are also recognized as being applicable to the preparation of other compounds of the present invention.

The examples described below, unless otherwise indicated, are all temperatures set forth in degrees Celsius. Reagents were purchased from commercial suppliers such as Aldrich Chemical Company, Arco Chemical Company and Alfa Chemical Company and were used without further purification unless otherwise indicated. General reagents were purchased from Shantou Wen Long chemical reagent factory, Guangdong Guanghua chemical reagent factory, Guangzhou chemical reagent factory, Tianjin Haojian Yunyu chemical Co., Ltd, Tianjin Shucheng chemical reagent factory, Wuhan Xin Huayuan scientific and technological development Co., Ltd, Qingdao Tenglong chemical reagent Co., Ltd, and Qingdao Kaolingyi factory.

The anhydrous tetrahydrofuran, dioxane, toluene and ether are obtained through reflux drying of metal sodium. The anhydrous dichloromethane and chloroform are obtained by calcium hydride reflux drying. Ethyl acetate, petroleum ether, N-hexane, N, N-dimethylacetamide and N, N-dimethylformamide were used as they were previously dried over anhydrous sodium sulfate.

The following reactions are generally carried out under positive pressure of nitrogen or argon or by sleeving a dry tube over an anhydrous solvent (unless otherwise indicated), the reaction vial being stoppered with a suitable rubber stopper and the substrate being injected by syringe. The glassware was dried.

The column chromatography is performed using a silica gel column. Silica gel (300 and 400 meshes) was purchased from Qingdao oceanic chemical plants.

¹H NMR spectra were recorded using a Bruker 400MHz or 600MHz NMR spectrometer.¹H NMR Spectrum in CDC1₃、DMSO-d₆、CD₃OD or acetone-d₆TMS (0ppm) or chloroform (7.26ppm) was used as a reference standard for the solvent (in ppm). When multiple peaks occur, the following abbreviations will be used: s (singleton), d (doublet), t (triplet), m (multiplet), br (broad), dd (doublet of doublets), dt (doublet of triplets). Coupling constants are expressed in hertz (Hz).

The conditions for determining low resolution Mass Spectrometry (MS) data were: agilent 6120 four-stage rod HPLC-M (column model: Zorbax SB-C18,2.1X 30mm,3.5 micron, 6min, flow rate 0.6 mL/min. mobile phase: 5% -95% (CH with 0.1% formic acid)₃CN) in (H containing 0.1% formic acid)₂O) by electrospray ionization(ESI), detection at 210nm/254nm with UV.

Pure compounds were detected by UV at 210nm/254nm using Agilent 1260pre-HPLC or Calesep pump 250pre-HPLC (column model: NOVASEP 50/80mm DAC).

The following acronyms are used throughout the invention:

CH₂Cl₂DCM dichloromethane; CDC1₃Deuterated chloroform; DMF N, N-dimethylformamide; DIPEA N, N-diisopropylethylamine; DMSO dimethyl sulfoxide; MeOH with methanol; MeCN, CH₃CN acetonitrile; HCl hydrogen chloride; KI potassium iodide; t-BuOK potassium tert-butoxide; NaHCO 2₃Sodium bicarbonate; na (Na)₂S₂O₃Sodium thiosulfate; na (Na)₂SO₄Sodium sulfate; Pd/C10% palladium on carbon; g, g; h hours; mL, mL; PE petroleum ether (60-90 deg.C); RT, RT, r.t. room temperature; rt retention time; TFA trifluoroacetic acid.

Example 1: synthesis of Compound II-A-1

The method A comprises the following steps:

the method comprises the following steps: 529mg (1.0mmol) of III-1 was dissolved in 10mL of anhydrous acetone, and 680mg (1.5mmol) of IV-A-1 and 414mg (3.0mmol) of potassium carbonate were added thereto at room temperature, and the mixture was stirred under reflux for 6 hours under heating to complete the reaction by TLC. After filtration, the filtrate was diluted with dichloromethane (30mL), washed successively with water (10mL) and saturated brine (10mL), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by concentration under reduced pressure. Purifying the residue by silica gel column chromatography to obtain 710mg of white solid V-A-1 with yield of 75%, ESI-MS: M/z [ M + H ]]⁺＝947。

Step two: 200mg (0.21mmol) of V-A-1 was dissolved in 10mL of anhydrous dichloromethane, and 2mL (volume ratio: two) was added thereto at room temperatureChloromethane/triisopropylsilane/trifluoroacetic acid ═ 5/1/1) solution, reacted for about 1h, checked by TLC for completion of the reaction, saturated sodium bicarbonate solution was carefully added, the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 96mg of a white solid II-A-1 in a yield of 65%.¹H NMR(400MHz,DMSO-d₆)δ8.43(t,J＝5.7Hz,1H),7.68(d,J＝7.2Hz,1H),7.39(t,J＝7.7Hz,2H),7.30–7.14(m,3H),6.14–5.96(m,2H),5.90(d,J＝4.9Hz,1H),5.84(d,J＝9.6Hz,1H),5.81(d,J＝9.6Hz,1H),5.74(d,J＝8.2Hz,1H),4.87(dt,J＝12.4,6.2Hz,1H),4.47–4.34(m,1H),4.29–4.20(m,1H),4.08–4.01(m,1H),3.94–3.76(m,4H),3.57–3.46(m,1H),2.82(d,J＝8.3Hz,1H),1.35(d,J＝6.9Hz,3H),1.27(t,J＝15.0Hz,6H),1.16(d,J＝6.2Hz,6H).¹³C NMR(100MHz,DMSO-d₆)δ173.7,173.1,173.0,169.2,161.1,151.2,151.1,150.6,130.1,125.1,120.6,120.5,101.9,101.6,99.8,80.2,72.1,71.9,68.5,65.1,64.9,50.3,41.0,36.3,22.4,22.3,21.9,21.8,20.3,20.2,17.2,16.9.³¹P NMR(162MHz,DMSO)δ3.83(s)ESI-MS:m/z[M+H]⁺＝705。

The method B comprises the following steps:

the method comprises the following steps: 5.0g (9.5mmol) of Compound III-1 are dissolved in 25mL of anhydrous N, N-Dimethylformamide (DMF), and 1.6g (23.5mmol) of imidazole, 0.12g (1.0mmol) of 4-Dimethylaminopyridine (DMAP) and 1.6mL (9.5mmol) of triethylchlorosilane (TESCl) are added in succession under Ar protection. The reaction was stirred at room temperature for about 10 hours and checked by TLC for completion. Then, 7mL of 37% HCHO solution was added to the reaction system, and the reaction was stirred for 1 hour while heating to 80 ℃. Cooling to room temperature, adding 80mL of ethyl acetate, washing with water and saturated brine in sequence, separating the organic phase, drying with anhydrous sodium sulfate, filtering, and concentrating under reduced pressure to obtain colorless oily substance VII-1, which is used in the next step without purification.

Step two: the last step is freshThe product VII-1 thus obtained was dissolved in 40mL of anhydrous Dichloromethane (DCM), cooled in an ice bath under argon protection, and 62mg (0.5mmol) of 4-Dimethylaminopyridine (DMAP) and 2.36g (12.3mmol) of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDCHCl) and 6.1g (11.4mmol) of the compound VIII-A-1 were added successively, slowly warmed to room temperature and stirred for about 12 hours, and the reaction was checked by TLC to completion. 100mL of methylene chloride was added, and the mixture was washed with a saturated aqueous sodium bicarbonate solution and a saturated brine in this order, and the separated organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was recrystallized from dichloromethane-n-heptane to give 8.5g of the product IX-A-1 as a white solid in 70% yield (two steps).¹H NMR(400MHz,CDCl₃)δ7.57(d,J＝8.1Hz,1H),7.35(dd,J＝17.2,7.6Hz,6H),7.31–7.12(m,12H),6.77(d,J＝8.5Hz,2H),6.17(d,J＝18.4Hz,1H),6.04–5.87(m,2H),5.68(d,J＝8.2Hz,1H),5.00(dt,J＝12.3,6.2Hz,1H),4.60(dd,J＝11.4,6.4Hz,1H),4.35(q,J＝6.9Hz,1H),4.29–4.20(m,2H),4.15–4.08(m,1H),4.02–3.85(m,3H),3.78(s,3H),3.71(t,J＝10.0Hz,1H),1.39–1.32(m,6H),1.35(s,9H),1.27(d,J＝11.9Hz,3H),1.23(d,J＝6.2Hz,6H),0.92(s,9H),0.15(s,6H).ESI-MS:m/z[M+H]⁺＝1191。

Step three: 7.5g (6.3mmol) of Compound IX-A-1 was dissolved in a 50% water-acetone (20mL) mixed solution, 30mL of glacial acetic acid (HOAc) and 6mL of trifluoroacetic acid (TFA) were added, and the reaction was stirred at room temperature for about 6 hours until no residual starting material was detected by TLC. Acetone and water were removed by reduced pressure rotary evaporation, and then 60mL of methylene chloride, 9mL of trifluoroacetic acid (TFA) and 6.5mL of triisopropylsilane (Triisopropylsilane)^i-Pr₃SiH) at room temperature for about 15 minutes, TLC detecting completion of the reaction, and carefully adding saturated aqueous sodium bicarbonate to quench the reaction. 100mL of dichloromethane was added, the mixture was washed with water and saturated brine in this order, the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. Recrystallization from ethyl acetate-petroleum ether gave 3.9g of white solid II-A-1, yield 88%.¹H NMR(400MHz,DMSO)δ8.43(t,J＝5.7Hz,1H),7.68(d,J＝7.2Hz,1H),7.39(t,J＝7.7Hz,2H),7.30–7.14(m,3H),6.14–5.96(m,2H),5.90(d,J＝4.9Hz,1H),5.84(d,J＝9.6Hz,1H),5.81(d,J＝9.6Hz,1H),5.74(d,J＝8.2Hz,1H),4.87(dt,J＝12.4,6.2Hz,1H),4.47–4.34(m,1H),4.29–4.20(m,1H),4.08–4.01(m,1H),3.94–3.76(m,4H),3.57–3.46(m,1H),2.82(d,J＝8.3Hz,1H),1.35(d,J＝6.9Hz,3H),1.27(t,J＝15.0Hz,6H),1.16(d,J＝6.2Hz,6H).ESI-MS:m/z[M+H]⁺＝705。

Concentrating the recrystallization mother liquor, purifying the residue by using silica gel column chromatography to obtain a small amount of white solid II-A-9,¹HNMR(400MHz,DMSO)¹H NMR(400MHz,CDCl₃)δ7.51(d,J＝7.9Hz,1H),7.35(t,J＝7.8Hz,2H),7.20(dd,J＝14.5,7.5Hz,3H),6.19(brs,1H),6.07(d,J＝9.4Hz,1H),5.99(d,J＝9.4Hz,1H),5.72(d,J＝8.2Hz,1H),5.00(dt,J＝12.5,6.2Hz,1H),4.59–4.39(m,2H),4.37–4.22(m,2H),4.14(d,J＝8.6Hz,1H),3.99–3.85(m,3H),3.65–3.58(m,1H),1.48(d,J＝7.0Hz,1H),1.42–1.36(m,3H),1.38–1.26(m,6H),1.24(d,J＝6.2Hz,6H).ESI-MS:m/z[M+H]⁺＝705。ESI-MS:m/z[M+H]⁺＝705。

example 2: synthesis of Compound II-A-1a

Compound VII-1(3mmol of Compound III-1 as starting material) was freshly prepared according to method B, step one of example 1, dissolved in 12mL of anhydrous Dichloromethane (DCM), after cooling in an ice bath under the protection of argon, 18mg (0.15mmol) of 4-Dimethylaminopyridine (DMAP) and 0.75g (3.9mmol) of 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDC HCl) are added in sequence, and 1.93g (3.6mmol) of the compound VIII-A-1a (prepared from chiral tiopronin and having a chiral purity of 95% in R configuration according to the analytical method of AD-H column temperature of 25C, a detection wavelength of 210nm, a flow rate of 1.0ml/min, a mobile phase of n-Hex, EtOH, TFA of 90:10:0.1, and the elution is carried out in an isocratic manner, then the temperature is slowly raised to room temperature, the reaction is stirred for about 12 hours, and the reaction is detected to be complete by TLC. 30mL of methylene chloride was added, and the mixture was washed with a saturated aqueous sodium bicarbonate solution and a saturated brine in this order, and the separated organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was recrystallized from dichloromethane-n-heptane to yield 8.7g of the product IX-A-1a as a white solid in 73% yield.

2.1g (1.76mmol) of Compound IX-A-1a was dissolved in a 50% water-acetone (5mL) mixed solution, and 8mL of glacial acetic acid (HOAc) and 2mL of trifluoroacetic acid (TFA) were added, and the reaction was stirred at room temperature for about 6 hours until no residual starting material was detected by TLC. Acetone and water were removed by rotary evaporation under reduced pressure, and then 15mL of methylene chloride, 3mL of trifluoroacetic acid (TFA) and 2.2mL of triisopropylsilane (i-Pr)₃SiH) at room temperature for about 15 minutes, TLC detecting completion of the reaction, and carefully adding saturated aqueous sodium bicarbonate to quench the reaction. 30mL of dichloromethane was added, the mixture was washed with water and saturated brine in this order, the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. Recrystallization from ethyl acetate-petroleum ether gave 1.7g of a white solid II-A-1a with a yield of 86%. ESI-MS of M/z [ M + H ]]⁺＝705。

Example 3: synthesis of Compound II-A-1b

2.57g of intermediate IX-A-1B, in 72% yield (two steps), were prepared according to method B, from VIII-A-1B (prepared from chiral tiopronin, chiral purity of S configuration 97%, analytical method: AD-H column temperature: 25C; detection wavelength: 210 nm; flow rate: 1.0 ml/min; mobile phase: n-Hex: EtOH: TFA ═ 90:10:0.1 isocratic elution) instead of VIII-A-1a, using 5.3g (10mmol) of compound III-1, in accordance with method B, step one, step two, example 1.

Following procedure three of method B of example 1, compound IX-A-1B (2.9mmol) was further deprotected to afford 1.04g of the product II-A-1B as a white solid in 84% yield. ESI-MS of M/z [ M + H ]]⁺＝705。

Example 4: synthesis of Compound II-A-2

The method A comprises the following steps:

the method comprises the following steps: 529mg (1.0mmol) of III-1 was dissolved in 10mL of anhydrous acetone, and 680mg (1.5mmol) of IV-A-2 and 414mg (3.0mmol) of potassium carbonate were added thereto at room temperature, and the mixture was stirred under reflux for 6 hours under heating to complete the reaction by TLC. After filtration, the filtrate was diluted with dichloromethane (30mL), washed successively with water (10mL) and saturated brine (10mL), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by concentration under reduced pressure. The residue was purified by silica gel column chromatography to give 306mg of a white solid V-A-2. ESI-MS of M/z [ M + H ]]⁺＝961。

Step two: 200mg (0.21mmol) of V-A-2 was dissolved in 10mL of anhydrous dichloromethane, 2mL (volume ratio: dichloromethane/triisopropylsilane/trifluoroacetic acid ═ 5/1/1) of the solution was added at room temperature, the reaction was carried out for about 1 hour, the reaction was checked by TLC, a saturated sodium bicarbonate solution was carefully added, the separated organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 100mg of a white solid II-A-2 in 67% yield.¹H NMR(400MHz,CDCl₃)δ9.64(s,1H),7.50(dd,J＝8.1,2.3Hz,1H),7.36(t,J＝7.8Hz,2H),7.30–7.22(m,3H),7.19(d,J＝7.6Hz,1H),6.65(q,J＝6.5Hz,1H),6.19(d,J＝17.9Hz,1H),5.10–4.94(m,1H),4.52(dd,J＝11.4,5.7Hz,1H),4.39–4.21(m,3H),4.22–4.05(m,2H),3.98(tt,J＝11.5,5.8Hz,1H),3.57–3.45(m,1H),2.17(dd,J＝8.5,2.2Hz,1H),1.86(d,J＝6.5Hz,3H),1.56(dd,J＝7.1,1.0Hz,3H),1.37(dd,J＝14.8,7.5Hz,6H),1.27(s,1H),1.25(d,J＝6.3Hz,6H).ESI-MS:m/z[M+H]⁺＝719。

The method B comprises the following steps:

4.6g of intermediate IX-A-2 were prepared according to method B, step one, step two of example 1, using acetaldehyde instead of formaldehyde, and 5.3g (10mmol) of compound III-1, in a yield of 38% (two steps). ESI-MS of M/z [ M + H ]]⁺＝1205。

Following method B, step three of example 1, compound IX-A-2(2.9mmol) continued deprotection to give 1.7g of the white solid product II-A-2 in 82% yield ESI-MS: M/z [ M + H ]]⁺＝719。

Example 5: synthesis of Compound II-A-3

2g of intermediate IX-A-3 were prepared in 33% yield from 2.65g (5mmol) of compound III-1 according to method B, step one, step two of example 1, using propionaldehyde instead of formaldehyde. ESI-MS of M/z [ M + H ]]⁺＝1219。

Continuing deprotection of compound IX-A-3(0.8mmol) according to method B, step three of example 1, 445mg of white solid product II-A-3 were prepared in 76% yield ESI-MS: M/z [ M + H ],]⁺＝733。

example 6: synthesis of Compound II-A-4

The method comprises the following steps: 529mg (1.0mmol) of III-1 was dissolved in 10mL of anhydrous acetone, 690mg of IV-A-3 and 414mg of potassium carbonate (3.0mmol) were added at room temperature, and the mixture was stirred under reflux for 6 hours, and the reaction was detected by TLC to be complete. After filtration, the filtrate was diluted with dichloromethane (30mL), washed successively with water (10mL) and saturated brine (10mL), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by concentration under reduced pressure. The residue was purified by silica gel column chromatography to give 300mg of V-A-4, which was used directly in the next reaction.

Step two: 200mg (0.21mmol) of V-A-4 was dissolved in 10mL of anhydrous dichloromethane, 2mL (volume ratio: dichloromethane/triisopropylsilane/trifluoroacetic acid ═ 5/1/1) of the solution was added at room temperature, the reaction was carried out for about 1 hour, the reaction was checked by TLC, a saturated sodium bicarbonate solution was carefully added, the separated organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 100mg of a white solid II-A-4.¹H NMR(400MHz,CDCl3)δ9.62(s,1H),7.46(dd,J＝8.1,2.2Hz,1H),7.34(t,J＝7.8Hz,2H),7.29-7.22(m,3H),7.19(d,J＝7.4Hz,1H),6.16(d,J＝18.1Hz,1H),5.64-5.53(m,1H),5.37-5.17(m,1H),5.08-4.93(m,1H),4.53(dd,J＝11.4,5.5Hz,1H),4.38-4.20(m,3H),4.40-4.08(m,6H),3.97(tt,J＝11.7,5.9Hz,1H),3.57-3.43(m,1H),2.17(dd,J＝8.4,2.2Hz,1H),1.55(dd,J＝7.1,1.0Hz,3H),1.38(dd,J＝14.8,7.7Hz,6H),1.27(s,1H),1.24(d,J＝6.3Hz,6H).ESI-MS:749(M+1)。

Example 7: synthesis of Compound II-A-5

The method comprises the following steps: 529mg (1.0mmol) of III-1 was dissolved in 10mL of anhydrous acetone, 800mg of IV-A-4 and 414mg of potassium carbonate (3.0mmol) were added thereto at room temperature, and the mixture was stirred under reflux for 12 hours, followed by completion of the reaction by TLC. After filtration, the filtrate was diluted with dichloromethane (30mL), washed successively with water (10mL) and saturated brine (10mL), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by concentration under reduced pressure. The residue was purified by silica gel column chromatography to give 400mg of V-A-5, which was used directly in the next reaction.

Step two: 200mg (0.21mmol) of V-A-5 was dissolved in 10mL of anhydrous dichloromethane, and 2 was added thereto at room temperaturemL (volume ratio: dichloromethane/triisopropylsilane/trifluoroacetic acid ═ 5/1/1) solution, reacted for about 1h, the reaction was checked by TLC, saturated sodium bicarbonate solution was carefully added, the separated organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 100mg of a white solid II-A-5.¹H NMR(400MHz,CDCl₃)δ9.66(s,1H),7.44(dd,J＝7.9,2.1Hz,1H),7.32(t,J＝7.8Hz,2H),7.28–7.20(m,3H),7.16(d,J＝7.5Hz,1H),6.13(d,J＝17.6Hz,1H),5.15–5.03(m,1H),4.60(t,J＝7.5Hz,2H),4.55(dd,J＝11.5,5.7Hz,1H),4.48–4.25(m,3H),4.23–4.09(m,2H),3.95(tt,J＝11.8,5.8Hz,1H),3.59–3.42(m,1H),3.25(t,J＝7.5Hz,2H),2.16(dd,J＝8.4,2.2Hz,1H),1.54(dd,J＝7.1,1.0Hz,3H),1.39(dd,J＝14.7,7.6Hz,6H),1.28(s,1H),1.25(d,J＝6.5Hz,6H).ESI-MS:m/z[M+H]⁺＝719。

Example 8: synthesis of Compound II-A-7

According to example 1, method B, step one, step two, replacing III-1 with III-2 only, and 1.1g (2mmol) of compound III-2 gave IX-A-7 as a colorless oil in 71% yield (two steps). ESI-MS of M/z [ M + H ]]⁺＝1207。

The product II-A-7 was prepared as a white solid in 83% yield by continued deprotection of 0.79g of compound IX-A-7(0.65mmol) according to method B, step three of example 1. ESI-MS of M/z [ M + H ]]⁺＝721。

Example 9: synthesis of Compound II-A-8

1.29g of intermediate IX-A-8 were prepared in 65% yield from 1.6g (3mmol) of compound III-1 according to example 1, method B, step one, step two, replacing VIII-A-1 with VIII-A-2. ESI-MS of M/z [ M + H ]]⁺＝1219.

Following method B, step three of example 1, compound IX-A-8(0.8mmol) was further deprotected to give 0.5g of the product II-A-8 as a white solid in 83% yield ESI-MS: M/z [ M + H ]]⁺＝733。

Example 10: synthesis of Compound II-B-1

According to method B of example 1, using VIII-B-1 (prepared from (DL) -N-acetylcysteine) instead of VIII-1 in step two, 3.38g of intermediate IX-B-1 was prepared in 71% yield from 2.1g (4mmol) of compound III-1 (two steps). ESI-MS of M/z [ M + H ]]⁺＝1191。

Continuing deprotection of Compound IX-B-1(2mmol) according to method B, step three of example 1, 1.17g of the white solid product II-B-1 was prepared in 83% yield ESI-MS: M/z [ M + H ],]⁺＝705。

example 11: synthesis of Compound II-B-1a

According to example 1, method B, step two, VIII-B-1a (prepared from L-acetylcysteine, chiral purity)>99%) instead of VIII-1, 2.65g (5mmol) of compound III-1 were used to prepare intermediate IX-B-1a in 67% yield (two steps). ESI-MS:m/z[M+H]⁺＝1191。

following method B, step three of example 1, compound IX-B-1a (2.1mmol) was further deprotected to give 1.2g of the product II-B-1a as a white solid in 85% yield ESI-MS: M/z [ M + H ],]⁺＝705。

example 12: synthesis of Compound II-B-1B

According to method B of example 1, replacing VIII-1 with VIII-B-1B (prepared from D-cysteine, with a chiral purity of 95%) in step two, 4.2g of intermediate IX-B-1B were prepared in 70% yield from 2.65g (5mmol) of compound III-1 (two steps). ESI-MS of M/z [ M + H ]]⁺＝1191。

Following method B, step three of example 1, compound IX-B-1B (2.1mmol) was further deprotected to afford 1.2g of product II-B-1B as a white solid in 81% yield. ESI-MS of M/z [ M + H ]]⁺＝705。

Example 13: synthesis of Compound II-B-2

According to example 1, method B, step one, acetaldehyde was used instead of formaldehyde, according to example 1, method B, step two, VIII-B-1a (obtained from L-acetylcysteine, chiral purity)>99%) instead of VIII-1, 1.3g of intermediate IX-B-2 were prepared in 36% yield from 1.6g (3mmol) of compound III-1 (two steps). ESI-MS of M/z [ M + H ]]⁺＝1205。

According to method B, step three of example 1, compound IX-B-2(1mmol) was followedSubsequently, the protecting group was removed to obtain 0.57g of a white solid product II-B-2 in a yield of 79%. ESI-MS of M/z [ M + H ]]⁺＝719。

Example 14: synthesis of Compound II-B-3

According to example 1, method B, step one, step two, starting with VIII-B-2 (prepared from L-acetylcysteine, chiral purity)>99%) instead of VIII-1, 1.6g (3mmol) of compound III-1 gave intermediate IX-B-3 in 76% yield (two steps). ESI-MS of M/z [ M + H ]]⁺＝1205。

Following method B, step three of example 1, compound IX-B-3(1mmol) was further deprotected to afford product II-B-3 as a white solid in 0.57g, 82% yield. ESI-MS of M/z [ M + H ]]⁺＝719。

Example 15: synthesis of Compound II-B-4

Method B according to example 1, step one, step two, replacing III-1 with III-2 and preparing VIII-B-1a (from L-acetylcysteine, chiral purity)>99%) instead of VIII-1, 2.64g of intermediate IX-B-4 were prepared in 72% yield from 1.6g (3mmol) of compound III-2 (two steps). ESI-MS of M/z [ M + H ]]⁺＝1207。

According to the third step of example 6, compound IX-B-4(1.5mmol) was further deprotected to give 0.93g of the product II-B-4 as a white solid in 84% yield ESI-MS: M/z [ M + H ]]⁺＝721。

Example 16 anti-hepatitis C Virus Activity (HCV, EC)₅₀) And Cytotoxicity (CC)₅₀)

Due to the lack of ideal HCV infection in vitro cells and animal models, HCV viral activity assessment will generally employ enzymes that play a key role in HCV RNA replication to model extracellular molecular replication.

The experimental method comprises the following steps:

the Huh7 cells containing the HCV-replicon (1b genotype) were cultured in DMEM medium containing 10% fetal bovine serum, 1X non-essential amino acids, Pen-Strep-Glu, G418. The antiviral screening test was performed in different media without G418. Cells were seeded in 96-well plates and compounds tested were added immediately after seeding and incubated in an incubator at 37 ℃. The medium is then removed and the cells are used for whole nucleic acid extraction (including replicon RNA and host RNA). Replicon RNA can be amplified in the Q-RT-PCR protocol and quantified accordingly. The observed differences in the levels of replicon HCV RNA from the untreated control group are shown in table 1 as a means of testing the antiviral efficacy of the compounds.

Anti-hepatitis C Virus Activity of the Compounds of Table 1 (HCV, EC)₅₀) And Cytotoxicity (CC)₅₀) Results

Inhibitory Activity of Compounds on intracellular HCV replicon 1b (EC)₅₀) The results of (A) were evaluated as an anti-hepatitis C virus activity, while simultaneously exhibiting a cytotoxic activity (CC)₅₀) The simultaneous test of (2) is used to exclude false positive results due to cytotoxicity. The higher the therapeutic index, the more promising the compound has application prospect. The results show that the compound provided by the invention has similar or better activity and therapeutic index with Sofosbuvir, and takes II-A-1, II-A-1a, II-A-1b and II-A-2 as examples, the compound inhibits HCV replicon in cellsPreparation Activity (EC)₅₀) The compound is stronger than a positive control Sofosbuvir, which shows that the compound can be rapidly degraded into an active metabolite in a liver cell to play a role in resisting HCV in the same way as the Sofosbuvir, in addition, another seed of tiopronin obtained by molecular degradation has a certain gain effect on antiviral activity, and the synergistic effect ensures that the compound provided by the invention has a better application prospect in the aspect of resisting HCV diseases.

Example 17 study of protective Effect of mouse CCl4 liver Damage model

The experimental method comprises the following steps: taking male mice with the weight of 24-28 g before experiment, fasting for 6 hours without water prohibition, randomly dividing the male mice into 3 groups according to the weight, 5 mice in each group, and respectively arranging a blank control group and CCL₄Group and compound group (tiopronin 50mg/kg, test compound 200 mg/kg). The administration is carried out 1 time per 12h, the volume of each intragastric administration is 10mL/kg, and the administration is carried out 4 times in total. The blank control group and the model control group were simultaneously infused with physiological saline of equal volume. 1h after the third dose, two groups of mice were treated with 2mg/kg b.w CCl, except for the placebo group of mice₄Intraperitoneal injection of 50% CCl₄Corn oil solution. After 12 hours, each group was administered 1 more time. After 24 hours, each mouse was bled, serum ALT and AST (table 2) were measured, livers were cut into small pieces, fixed with formaldehyde, paraffin sections were cut, stained with hematoxylin and eosin, and the condition of hepatocytes was observed under a microscope.

Table 2 results of the study of the protective effect of the CCl4 liver damage model of the compounds of the present invention

The results show that CCl₄The ALT and AST of the model group mice are abnormally increased, the tiopronin can remarkably reverse the effect of the altase, and compared with the situation that the Sofosbuvir does not have the activity. The compound provided by the invention has an obvious enzyme reducing effect, and the activity of the compound is equivalent to or better than that of tiopronin. The reason for this is probably because the carboxyl group of tiopronin is made into prodrug, which can improve the stability and the oral absorption capability of tiopronin, thereby enhancing the activity of tiopronin. Taking II-A-1, II-A-a, II-A-2 and II-A-5 as examples, the compound has obvious inhibitory activity on ALT and AST elevation caused by CCl4 induced liver injury, and is superior to the positive control tiopronin. In addition, the pathological section of liver tissue was observed to have CCl₄Model group, hepatic central venous congestion, lobular central or peripheral necrosis around, hepatic cell swelling, balloon-like deformation, administration groups of II-A-1, II-A-a, II-A-2, II-A-5, etc. showed little punctate and fragmented necrosis, and most of them showed balloon-like deformation, as shown by CCl₄The toxicity caused by the compound is greatly reduced, which shows that the compound related to the invention really has the protection CCl₄The induced liver damage.

Because of the lack of ideal HCV infection animal models, it is not feasible to directly evaluate the protective effect of the compound provided by the invention on the liver injury caused by HCV, but theoretically and from the clinical application perspective, the liver protective effect of the compound provided by the invention also has the protective effect on the inflammatory response caused by HCV.

Example 18 pharmacokinetic Property Studies

1 materials of the experiment

1.1 Compounds

Compounds II-A-1, II-A-1a, II-A-1B, II-A-2, II-B-1, III-2A-1a (30mg/kg, amount of nucleoside moiety corresponding to Sophia group) of the present invention prepared using the above examples, and a positive control drug is Sophia buvir (25mg/kg), each compound was added to 5% DMSO + 60% PEG400+ 35% physiological saline, and after vortexing, 10mg/ml suspension was prepared for intragastric administration.

GS331007 was purchased from yao shoo technologies (shanghai) limited.

1.2 Experimental animals and sampling

SD rats were used as experimental animals (body weight 180-220g), and 6 SD rats per test compound were randomly divided into 2 groups (gavage group and intravenous group) of 3 animals each. The tail vein blood sampling time points of the intragastric administration are 0.17,0.33,0.5,1,1.5,2,4,6,8,12 and 24 hours; blood was taken at 0.05,0.1,0.17,0.5,1,2,4,6,8,12,24 hours for intravenous administration. 0.3ml of whole blood was collected, centrifuged, and 0.1ml of plasma was analyzed by LC-MS.

1.3 instruments

Agilent 1200 liquid chromatography system, API4000QTRAP triple quadrupole tandem mass spectrometer

1.4 sample treatment

Adding 30 μ l liver homogenate sample or standard curve sample into 100 μ l acetonitrile containing internal standard (100ng/ml), centrifuging for 10min (6000 rpm) after 2min of vortex, and transferring the supernatant into a sample injection bottle;

and 5 mu L of supernatant is taken, injected and the concentration of GS331007 in the sample is detected by adopting LC-MS/MS. From the obtained liver homogenate drug concentration-time data, pharmacokinetic parameters of the metabolite GS331007 of each test compound were calculated using the pharmacokinetic calculation software winnonlin6.2.1 non-compartmental model, respectively, and the results are shown in table 3.

TABLE 3 summary of the major pharmacokinetic parameters after oral dosing in SD rats

According to literature reports, the metabolic pathway in vivo of the compound is a final metabolite, and the compound is a marker for researching the bioavailability and metabolic behavior of Sofosbuvir in vivo. The GS331007 plasma concentration and metabolic behavior of the compounds II-A-1, II-A-1a, II-A-1B, II-A-2 and II-B-1 in the rat body show that the plasma concentration and the area under the curve of the metabolite GS331007 of the tested compound are obviously improved compared with the plasma concentration of the GS331007 in the rat body of the Sofosbuvir group, in other words, the compounds related to the invention are obviously improved in the aspect of oral bioavailability. Taking the compounds II-A-1, II-A-2 and II-B-1 as examples, the oral bioavailability is respectively 3.38 times, 2.75 times and 2.25 times of sofosbuvir. In addition, the half-life periods of II-A-1 and II-A-2 reach 6.14 and 6.23 hours respectively, which are 2.95 and 2.51 times of those of sofosbuvir respectively, and the time curve is more moderate. Therefore, the compound has obvious advantages in pharmacokinetic property, and can be further used for treating the hepatitis C by oral administration.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are included in the scope of the present invention.

Claims

1. The compound has a structure shown as formula II-A or a tautomer, an optical isomer, and a pharmaceutically acceptable salt thereof:

wherein X is hydroxyl or F, Cl or Br;

linker isOr is absent;

R₁is H or C₁-C₅An alkyl group; n is 1,2, 3, 4 or 5; r₃、R₄Each independently is H or C₁-C₅An alkyl group.

2. The compound of claim 1, having a structure represented by formula II-Aa or formula II-Ab, or a tautomer, optical isomer, pharmaceutically acceptable salt thereof:

wherein X is hydroxyl or F, Cl or Br;

linker isOr is absent;

3. The compound of claim 1, which is of the structure shown in formula II-a:

wherein,

x is hydroxyl or F, Cl or Br,

linker is

R₁Is H or C₁-C₅Alkyl, n is an integer from 1 to 19.

4. The compound of claim 3, which is a structure of formula II-A-Aa, formula II-A-Ab, formula II-A-Ac, formula II-A-Ad, respectively:

5. The compound of claim 4, which is a structure of formula II-A-Aa1, formula II-A-Ab1, formula II-A-Ac1, formula II-A-Ad1, respectively:

wherein R is₁Is H or C₁-C₅Alkyl, n is 1,2, 3, 4 or 5.

6. The compound of claim 1, having the structure shown below or a tautomer, pharmaceutically acceptable salt of the compound of the structure shown below:

7. a method for preparing a compound shown as a formula II-A comprises the following steps:

1) reacting the compound of the formula III with the compound of the formula IV-A to obtain a compound of the formula V-A,

2) carrying out deprotection reaction on the compound of the formula V-A to obtain a compound of a formula II-A;

wherein X is hydroxyl or F, Cl or Br;

linker isOr is absent;

n is 1,2, 3, 4 or 5; r₁Is H or C₁-C₅An alkyl group; r₃、R₄Each independently is a hydrogen atom or C₁-C₅An alkyl group; LG is a leaving group; z is a mercapto protecting group.

8. A preparation method of a compound shown as a formula II-A-Aa can be used for obtaining a compound shown as a formula V-A through alkylation reaction of a compound shown as a formula III and a compound shown as a formula IV and then through deprotection reaction.

Wherein X is F, Cl or Br, R₁Is H or C₁-C₅An alkyl group; y is a leaving group and Z is a mercapto-protecting group.

9. A preparation method of a compound shown as a formula II-A-Ab can be obtained by performing alkylation reaction on a compound shown as a formula III and a compound shown as a formula IV-B to obtain a compound shown as a formula V-B, and then performing deprotection reaction:

wherein X is hydroxyl or F, Cl or Br; r₁Is H or C₁-C₅An alkyl group; n is an integer of 1 to 19; y is a leaving group;

z is a mercapto protecting group.

10. A process for preparing a compound of formula II-a-B, comprising the steps of:

1) carrying out a protection reaction on the compound of the formula III, and then reacting the compound of the formula III with aldehyde to obtain a compound of a formula VII;

2) reacting the compound of the formula VII with a compound of VIII-A to obtain a compound of a formula IX-A-B;

3) removing a protecting group from the compound of the formula IX-A-B to obtain a compound of a formula II-A-B;

wherein X is hydroxyl or F, Cl or Br; r₁Is H or C₁-C₅An alkyl group; r₃、R₄Each independently is a hydrogen atom or C₁-C₅An alkyl group; p₁Is a hydroxyl protecting group; p₂Is an amide NH protecting group; p₃Is a mercapto protecting group.

11. A compound having a structure or salt represented by formula IX-a-B:

wherein X is hydroxyl or F, Cl or Br; r₁Is H or C₁-C₅An alkyl group; p₁Is a hydroxyl protecting group; p₂Is an NH protecting group; p₃Is a mercapto protecting group.

12. A pharmaceutical composition comprising one or more of the compounds of any one of claims 1-6.

13. The pharmaceutical composition according to claim 12, wherein the dosage of the pharmaceutical composition containing one or more compounds of formula II-a or tautomers and pharmaceutically acceptable salts thereof in the pharmaceutical composition is 1-1000 mg/day.

14. Use of a compound according to any one of claims 1 to 6 or a composition according to claim 12 in the manufacture of a medicament for the treatment of hepatitis c.