Nucleoside derivatives and preparation methods and uses thereof

Abstract

The present invention belongs to the field of biomedicine and relates to a class of nucleoside derivatives and preparation methods and uses thereof. The present invention specifically discloses a sofosbuvir derivative represented by formula (I) or a pharmaceutically acceptable salt, tautomer, mesomer, racemate, stereoisomer or solvate thereof, wherein R 1 Select from: H or C 1‑3 Alkyl; R 2 Select from: H or C 1‑3 Alkyl; X is selected from: S or O; Ar is selected from: phenyl, 1-naphthyl or 2-naphthyl. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a class of sofosbuvir derivatives. The present invention also relates to a preparation method of the class of compounds and their medical use as anti-hepatitis C virus drugs. Background Art

[0002] Nucleoside drugs have become an effective way to treat cancer and viral infectious diseases. This type of drug often needs to be converted into monophosphate, diphosphate and triphosphate metabolites in the body in sequence, and finally exerts its efficacy through triphosphate nucleoside analogs (active metabolites). However, in the above phosphorylation process, the generation of monophosphate nucleosides is often the rate-limiting step, which leads to the low content of active metabolites in the body of nucleoside drugs and affects the efficacy. In order to overcome this problem, Professor McGuigan developed a class of ProTide prodrug technology, which increases the lipophilicity of the compound while increasing the metabolic activation ability of the drug, greatly improving the therapeutic effect of nucleoside prodrugs. ProTide prodrugs have promoted the birth of many blockbuster nucleoside drugs, such as sofosbuvir, tenofovir alafenamide and remdesivir, which have played an irreplaceable role in the treatment of chronic hepatitis C, chronic hepatitis B and COVID-19, respectively.

[0003] ProTide prodrugs have become a key strategy in prodrug design, and further expanding the diversity of this type of prodrug structure has become the key to obtaining more active molecules and structure-activity relationship information. In the most classic ProTide prodrug preparation method, chiral phosphoramide is a key intermediate; expanding chiral phosphoramide intermediates with different structures and functions is one of the main directions to enrich the ProTide prodrug structure.

[0004] Prior art CN110840907 and CN111320650 describe a class of structurally innovative ProTide prodrugs, the synthesis of which is also achieved with the aid of phosphoramide intermediates. However, the two prior art articles do not disclose the specific preparation and structural characterization of chiral phosphoramide intermediates.

[0005] After infection with hepatitis C virus (HCV), 60%-80% of patients will develop chronic hepatitis C. Hepatitis C is very likely to cause liver fibrosis, and 10%-30% of infected people will develop cirrhosis, accompanied by a variety of complications, hepatocellular carcinoma and even the risk of premature death (World. J. Gastroenterol. 2020, 26: 2931). It is reported that in 2015, more than 170 million people were infected with HCV worldwide, of which about 70 million were chronically infected.

[0006] Treatment of chronic hepatitis C was initially with a combination of interferon and ribavirin, a regimen with poor efficacy and severe side effects. In 2001, pegylated interferon was introduced; it has a longer half-life and improved virological response rate (SRV), while most adverse effects remain. In 2011, direct-acting antiviral (DAA) therapy became available, with boceprevir and telaprevir being the first generation of DAAs; they inhibit the viral NS3 / 4A protease and are approved as add-ons to established pegylated interferon and ribavirin therapy. These drugs improve the incidence of SVR, but patients experience a higher incidence of anemia, gastrointestinal side effects, and rash (N. Engl. J. Med. 2011;364:1207). Sofosbuvir (SOF) is the first HCV NS5B polymerase inhibitor approved for clinical use, which has a strong direct antiviral effect and has quickly become the backbone of various anti-HCV combination therapy regimens.

[0007] SOF is a type of prodrug that must undergo a series of metabolic activations in vivo before it can be converted into triphosphate active metabolites to inhibit the viral NS5B polymerase. In the above process, the hydrolysis of the prodrug by cathepsin A (CatA) and carboxylesterase 1 (CES1) in the liver and hepatocytes to release free carboxyl groups is a key step in metabolic activation. Among them, the degree of drug hydrolysis by CatA or CES1 and the changes in the functional activity of such hydrolases under different physiological or pathological conditions are key factors affecting the fate and efficacy of the drug in vivo. Therefore, avoiding or reducing the effects of esterases on drug activation and increasing the metabolic activation efficiency of prodrugs are important directions for the design and research of new prodrugs. Summary of the invention

[0008] In view of the problems existing in the prior art, the first object of the present invention is to provide a novel chiral phosphoramide intermediate. The intermediate has a completely new structure and can be used to enrich the diversity of ProTide prodrug structures, which is conducive to finding better active drug molecules.

[0009] The second object of the present invention is to provide a method for preparing the aforementioned intermediate, which is simple and easy to operate, does not require column chromatography for purification, and has high product diastereoselectivity.

[0010] The third object of the present invention is to provide the use of the aforementioned intermediate.

[0011] Based on the significant accumulation of reactive oxygen species (ROS) in liver tissue and hepatocytes of chronic hepatitis C patients, the fourth object of the present invention is to provide a sofosbuvir derivative that can be activated by ROS.

[0012] The fifth object of the present invention is to provide a method for preparing the aforementioned sofosbuvir derivatives.

[0013] The sixth object of the present invention is to provide the medical use of the aforementioned sofosbuvir.

[0014] In order to achieve the above object, the technical solution adopted by the present invention to solve the technical problem is:

[0015] In a first aspect, the present invention provides a chiral phosphoramide intermediate, the structural formula of which is shown in formula (VI):

[0016]

[0017] Among them, R 1 Select from: H or C 1-3 Alkyl; R 2 Select from: H or C 1-3 Alkyl; X is selected from: S or O; Ar is selected from: phenyl, 1-naphthyl or 2-naphthyl.

[0018] Preferably, R 1 Selected from: H or methyl; R 2 is selected from: H or methyl; X is selected from: S or O; Ar is selected from: phenyl, 1-naphthyl or 2-naphthyl;

[0019] More preferably, Ar is selected from: phenyl.

[0020] In some specific embodiments, the chiral phosphoramide intermediate of the present invention has a structural formula as shown in formula (VI-a):

[0021]

[0022] Wherein, X is selected from: O or S; the phosphorus atom is in S configuration.

[0023] In a second aspect, the present invention provides a method for preparing a chiral phosphoramide intermediate of formula (VI), and the synthetic route thereof is as follows:

[0024]

[0025] The method specifically comprises the following steps:

[0026] Step A: Compound II and Compound III are mixed with a solvent and a base, and then a condensing agent is added to obtain Compound IV through an acylation reaction;

[0027] Step B: Compound IV is deprotected to obtain compound V; optionally, the compound V may be: free base, hydrochloride or trifluoroacetate;

[0028] Step C: Compound V, phenyl dichlorophosphate and a solvent are mixed, and then a base and pentafluorophenol are added in sequence. After phosphorylation reaction, the mixture is cooled, filtered, washed and dried to obtain Compound VI.

[0029] In some specific embodiments, in step A, the condensing agent is pivaloyl chloride (pivaloyl chloride), isobutyl chloroformate, DCC, EDCI, HATU or HBTU;

[0030] Preferably, the condensing agent is pivaloyl chloride.

[0031] In some specific embodiments, in step A, an additive may be added, and the additive is lithium chloride, magnesium chloride, magnesium bromide or copper chloride; preferably lithium chloride.

[0032] Preferably, the molar ratio of the condensing agent to the additive is 1:0.5-5, preferably 1:0.5-2, more specifically 1:0.5, 1:0.8, 1:2, etc.

[0033] In some specific embodiments, in step A, the base is triethylamine, diisopropylethylamine, N-methylmorpholine, DMAP or DBU; preferably triethylamine.

[0034] In some specific embodiments, in step A, the solvent includes but is not limited to: dichloromethane, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide or a mixed solvent optionally composed of these solvents, preferably tetrahydrofuran.

[0035] In some specific embodiments, in step A, the reaction temperature is -50°C to 80°C, preferably, the reaction temperature is -20°C to 30°C, more preferably -20°C.

[0036] In some specific embodiments, in step B, the deprotection method is well known to those skilled in the art; examples thereof can be found in Peter GM Wuts, Greene's Protective Groups in Organic Synthesis (5th Edition), John Wiley & Sons, NY (2015), which is hereby incorporated by reference in its entirety.

[0037] In some specific embodiments, in step B, compound V can be a free base, a hydrochloride salt, or a trifluoroacetate salt.

[0038] In some specific embodiments, in step B, the enantiomeric excess (ee value) of compound V can be determined using methods well known to those skilled in the art, such as chiral HPLC, cultured single crystals and single crystal derivatization, and Mosher method.

[0039] In some specific embodiments, in step B, the enantiomeric excess (ee value) of compound V is 90-99%.

[0040] In some specific embodiments, in step C, the base is triethylamine, diisopropylethylamine, DMAP or DBU; preferably triethylamine.

[0041] In some specific embodiments, in step C, the solvent is a combination of one or more of diethyl ether, methyl tert-butyl ether, isopropyl ether, dichloromethane, 1,2-dichloroethane, chloroform, toluene, tetrahydrofuran, acetone, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, and N-methylpyrrolidone, preferably dichloromethane.

[0042] In some specific embodiments, in step C, the reaction temperature is -80°C to 50°C, preferably, the reaction temperature is -50°C to 0°C.

[0043] In some specific embodiments, in step C, the molar ratio of the compound phenyl dichloride phosphate to the base is 1:1 to 1:10; preferably 1:2 to 1:5, more preferably 1:2, 1:3, 1:5, etc.

[0044] In some specific embodiments, in step C, after the reaction is monitored by TLC, the reaction solution is preferably cooled to below 0°C, and more preferably the temperature is -30°C to -10°C.

[0045] In a third aspect, the present invention also protects the use of the chiral phosphoramide intermediates described above in the preparation of sofosbuvir derivatives, or pharmaceutically acceptable salts, tautomers, mesomers, racemates, stereoisomers or solvates thereof.

[0046] Preferably, the sofosbuvir derivative is as shown in formula (I):

[0047]

[0048] Among them, R 1 Select from: H or C 1-3 Alkyl; R 2 Select from: H or C 1-3 Alkyl; X is selected from: S or O; Ar is selected from: phenyl, 1-naphthyl or 2-naphthyl.

[0049] In a fourth aspect, the present invention also protects the sofosbuvir derivative represented by formula (I) or a pharmaceutically acceptable salt, tautomer, mesomer, racemate, stereoisomer or solvate thereof,

[0050]

[0051] Among them, R 1 Select from: H or C 1-3 Alkyl; R 2Select from: H or C 1-3 Alkyl; X is selected from: S or O;

[0052] Ar is selected from: phenyl, 1-naphthyl or 2-naphthyl;

[0053] Preferably, R 1 Selected from: H or methyl; R 2 is selected from: H or methyl; X is selected from: S or O; Ar is selected from: phenyl, 1-naphthyl or 2-naphthyl;

[0054] More preferably, Ar is selected from: phenyl.

[0055] The present invention also protects any of the following sofosbuvir derivatives or pharmaceutically acceptable salts, tautomers, mesomers, racemates, stereoisomers or solvates thereof:

[0056]

[0057] In a fifth aspect, the present invention also protects a method for preparing a sofosbuvir derivative of formula (I), and the synthetic route thereof is as follows:

[0058]

[0059] In the above synthetic route, R 1 , R 2 The definitions of X, Ar and Ar are consistent with those in the derivative of formula (I), and the preparation method comprises the following steps:

[0060] Step A: Compound II and Compound III are mixed with a solvent and a base, and then a condensing agent is added to obtain Compound IV through an acylation reaction; the solvent used includes but is not limited to: dichloromethane, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide or a mixed solvent optionally composed of these solvents; the condensing agent used is pivaloyl chloride, isobutyl chloroformate, DCC or EDCI, and the condensing agent is preferably pivaloyl chloride; the base used is triethylamine, diisopropylethylamine, N-methylmorpholine, DMAP or DBU, and the base is preferably triethylamine; additives can also be added in step A, and the additives are lithium chloride, magnesium chloride, magnesium bromide or copper chloride, and the additives are preferably lithium chloride; the reaction temperature is -50°C to 80°C, preferably, the reaction temperature is -20°C to 30°C.

[0061] Step B: Deprotection of compound IV to generate compound V. Deprotection methods are well known to those skilled in the art; examples thereof can be found in Peter GMWuts, Greene's Protective Groups in Organic Chemistry. Synthesis (5th edition), John Wiley & Sons, NY (2015), which is hereby incorporated by reference in its entirety; Step C: Compound V, phenyl dichlorophosphate and a solvent are mixed, and then a base and pentafluorophenol are added in sequence, and after phosphorylation reaction, Compound VI is obtained; the base used is triethylamine, diisopropylethylamine, DMAP or DBU, and the base is preferably triethylamine; the solvent used is a combination of one or more of diethyl ether, methyl tert-butyl ether, isopropyl ether, dichloromethane, 1,2-dichloroethane, chloroform, toluene, tetrahydrofuran, acetone, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, and N-methylpyrrolidone, and the solvent is further preferably dichloromethane; the solvent used includes but is not limited to: dichloromethane, ethyl acetate, diethyl ether or a mixed solvent optionally composed of these solvents; the reaction temperature is -80°C to 50°C, preferably, the reaction temperature is -50°C to 0°C;

[0062] Step D: Compound VI undergoes a substitution reaction with compound VII to obtain compound I. The solvent used includes, but is not limited to, toluene, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, or a mixed solvent optionally composed of these solvents; the base used is sodium hydrogen hydride, potassium tert-butoxide, sodium tert-butoxide, tert-butyl magnesium chloride, methyl magnesium chloride, phenyl magnesium chloride, DMAP or DBU, and the base is preferably methyl magnesium chloride; the reaction temperature is -50°C to 80°C, preferably, the reaction temperature is -10°C to 30°C.

[0063] In a sixth aspect, the present invention also protects a pharmaceutical composition comprising the sofosbuvir derivative described above or a pharmaceutically acceptable salt, tautomer, mesomer, racemate, stereoisomer or solvate thereof, and a pharmaceutically acceptable carrier.

[0064] In the seventh aspect, the present invention also protects the use of the sofosbuvir derivatives or their pharmaceutically acceptable salts, tautomers, mesomers, racemates, stereoisomers or solvates described above or the compositions described above in the preparation of anti-hepatitis virus drugs.

[0065] In some specific embodiments, the hepatitis is hepatitis C.

[0066] In the present invention, the following:

[0067] EDCI refers to: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (CAS: 25952-53-8)

[0068] DMAP means: 4-dimethylaminopyridine (CAS: 1122-58-3)

[0069] DCC means: dicyclohexylcarbodiimide (CAS: 538-75-0)

[0070] HATU refers to: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (CAS: 148893-10-1)

[0071] HBTU means: O-Benzotriazole-tetramethyluronium hexafluorophosphate (CAS: 94790-37-1)

[0072] DBU means: 1,8-diazabicyclo[5.4.0]undec-7-ene (CAS: 6674-22-2)

[0073] definition

[0074] Unless otherwise specified, the following terms used in this application have the following meanings. A particular term should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to the common meaning in the art. When a trade name appears in this article, it is intended to refer to the corresponding commodity or its active ingredient.

[0075] In this article, C 1-3 , means that the moiety has 1-3 carbon atoms in the given range. Specifically, it means that the group can have 1 carbon atom, 2 carbon atoms or 3 carbon atoms.

[0076] The term "alkyl" refers to a group of the formula C n H 2n+1 The alkyl group may be straight chain or branched. For example, the term "C 1-3 The term "alkyl" refers to an alkyl group containing 1 to 3 carbon atoms (eg, methyl, ethyl, n-propyl, isopropyl).

[0077] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0078] As the pharmaceutically acceptable salt, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids and the like can be mentioned.

[0079] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application or their salts and pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism.

[0080] The term "pharmaceutically acceptable excipients" refers to those excipients that have no significant irritation to the organism and do not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0081] The present application also includes isotope-labeled compounds of the present application that are identical to those described herein, but one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number commonly found in nature. Examples of isotopes that can be incorporated into the compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 31P, 32P, 35S, 18F, 123I, 125I, and 36Cl, etc.

[0082] The pharmaceutical composition of the present application can be prepared by combining the compound of the present application with suitable pharmaceutically acceptable excipients, for example, it can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols, etc.

[0083] Typical routes of administration of the compounds of the present application or their pharmaceutically acceptable salts or their pharmaceutical compositions include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0084] The pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing methods, dissolution methods, granulation methods, sugar-coated pill making methods, grinding methods, emulsification methods, freeze-drying methods, etc.

[0085] In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be prepared by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of the present application to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to patients.

[0086] Solid oral compositions can be prepared by conventional mixing, filling or tableting methods. For example, they can be obtained by mixing the active compound with a solid excipient, optionally grinding the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into particles to obtain a tablet or sugar-coated core. Suitable excipients include, but are not limited to, adhesives, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, etc.

[0087] The pharmaceutical composition may also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in appropriate unit dosage forms.

[0088] The compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitution methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present application.

[0089] The chemical reactions of the specific embodiments of the present application are carried out in a suitable solvent, which must be suitable for the chemical changes of the present application and the reagents and materials required. In order to obtain the compounds of the present application, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction processes based on the existing embodiments.

[0090] Beneficial Effects

[0091] Compared with the prior art, the present invention creatively provides a class of chiral phosphoramide intermediates with a completely new structure, which has a convenient preparation process, a simple purification method, no column chromatography required throughout the process, and the product has good diastereoselectivity, which provides a technical guarantee for the discovery of further new drug candidate molecules.

[0092] Compared with the prior art, the sofosbuvir derivative prodrug of the present invention has ideal in vitro anti-HCV viral activity; has good stability in plasma, and can be efficiently metabolized in a buffer solution containing hydrogen peroxide and liver S9; and has a more outstanding triphosphate active metabolite level in cells with elevated oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 This is the X-ray single crystal diffraction pattern of compound VI-1.

[0094] Figure 2 This is a comparison chart of the phosphorus spectra of compound VI-2 and its racemization.

[0095] Figure 3 The hydrogen spectrum and phosphorus spectrum of compound I-2.

[0096] Figure 4 It is the accumulation of triphosphate active metabolites in cells. DETAILED DESCRIPTION

[0097] The present invention is specifically described below by way of examples. In the present invention, the following examples are intended to better illustrate the present invention and are not intended to limit the scope of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0098] Example 1 Synthesis of Compound VI-1

[0099]

[0100] 2-Oxazolidinone II-1 (9.6 g, 110 mmol), compound III (18.9 g, 100 mmol), lithium chloride (8.5 g, 200 mmol) were placed in a dry 1000 mL double-necked bottle, 500 mL of anhydrous tetrahydrofuran was added under argon protection, the reaction solution was stirred at room temperature for 60 min and cooled to -20 ° C, then pivaloyl chloride (31 mL, 250 mmol) and triethylamine (36 mL, 260 mmol) were slowly added dropwise, and the reaction was kept at -20 ° C for 1 h. After the reaction was completed, the reaction solution was stirred at room temperature for 30 min. The reaction was quenched with 100 mL of saturated ammonium chloride solution, the reaction solution was extracted with ethyl acetate (100 mL × 3), washed with 10% sodium carbonate solution and saturated brine in turn, and dried over anhydrous sodium sulfate. After vacuum concentration, compound IV-1 was obtained as a white solid (24.5 g, 95 mmol) with a yield of 94.9%. 1H NMR (300MHz, Chloroform-d) δ5.39 (m, 1H), 5.15 (s, 1H), 4.47 (t, J = 8.0Hz, 2H), 4.17–3.95 (m, 2H), 1.45 (s, 9H), 1.41 (d, J = 7.0Hz, 3H).

[0101] In a 250 mL single-necked bottle, the above compound IV-1 was suspended in ethyl acetate (30 mL), 4N HCl-ethyl acetate solution (110 mL, 470 mmol) was slowly added dropwise, and stirred at room temperature overnight. After the reaction was completed by TLC monitoring, the reaction solution was concentrated in vacuo to obtain compound V-1 as a white solid product (18.4 g, 94 mmol), with a yield of 99%. 1 H NMR (300MHz, DMSO-d6) δ8.61 (s, 3H), 4.77 (m, 1H), 4.46 (m, 2H), 3.93 (t, J = 8.0Hz, 2H), 1.43 (d, J = 6.9Hz, 3H).

[0102] Compound V-1 (9.75 g, 50 mmol) was placed in a 500 mL double-necked flask, 150 mL of dichloromethane was added under argon protection, cooled to -30 ° C, and compound VI (7.5 mL, 50 mmol) and triethylamine (14 mL, 100 mmol) were slowly added dropwise in sequence. The reaction solution was stirred at -30 ° C for 30 min, and then heated to 0 ° C and continued to react for 3 h until the reaction was complete as monitored by TLC. At 0 ° C, a dichloromethane solution of pentafluorophenol (9.2 g, 50 mmol) and triethylamine (14 mL, 100 mmol) were added to the reaction solution, and the reaction was stirred at 10 ° C overnight. After the reaction was completed as monitored by TLC, the mixture was filtered, and the filter cake was washed with cooled ethyl acetate. After vacuum concentration, compound VI-1 was obtained as a white solid (17 g, 38.5 mmol), with a yield of 77% and a total yield of 72% in three steps. 1 H NMR(300MHz,Chloroform-d)δ7.38(t,J=7.7Hz,2H),7.32–7.12(m,3H),5.49–5.23(m,1H), 4.47(t,J=7.9Hz,2H), 4.24(t,J=11.9Hz,1H), 3.98(t,J=8.1Hz,2H), 1.51(d,J=6.8Hz,3H). 31 P NMR (162 MHz, DMSO-d6) δ 0.04.

[0103] Example 2 Synthesis of Compound VI-2

[0104]

[0105] 2-thiazolidinone II-2 (2.83 g, 27.5 mmol), compound III (4.73 g, 25 mmol), lithium chloride (2.12 g, 100 mmol) were placed in a dry 250 mL double-necked bottle, 125 mL of anhydrous methyltetrahydrofuran was added under argon protection, cooled to -10 ° C and stirred for 30 min, then pivaloyl chloride (6.2 mL, 50 mmol) and N-methylmorpholine (7.3 mL, 65 mmol) were slowly added dropwise, and the mixture was moved to room temperature (25 ° C) for 2 hours. The reaction was quenched with 35 mL of saturated ammonium chloride solution, the reaction solution was extracted with ethyl acetate (40 mL × 3), washed with saturated sodium carbonate solution and saturated brine in turn, and dried over anhydrous sodium sulfate. The organic phase was concentrated to obtain IV-2 semi-solid (6.07 g, 22 mmol) with a yield of 88%. 1H NMR (300MHz, Chloroform-d) δ5.35–5.22(m,1H),5.18(s,1H),4.31–4.07(m,2H),3.43–3.25(m,2H),1.45(s,9H),1.37(d,J=6.8Hz,3H).

[0106] In a 100 mL single-necked bottle, the above compound IV-2 was dissolved in ethyl acetate (15 mL), 4N HCl-ethyl acetate solution (25 mL, 100 mmol) was slowly added dropwise, and stirred at room temperature overnight. After the reaction was completed by TLC monitoring, the reaction solution was concentrated in vacuo to obtain compound V-2 as a white solid product (1.0 g, 4.74 mmol), with a yield of 95%. 1 H NMR (300MHz, DMSO-d6) δ8.54 (s, 3H), 4.69 (s, 1H), 4.11 (m, 2H), 3.46 (m, 2H), 1.39 (d, J = 6.4Hz, 3H).

[0107] Compound V-2 (2.1 g, 10 mmol) was placed in a 100 mL double-necked flask, anhydrous dichloromethane (33 mL) was added under argon protection, cooled to -80 ° C, and compound dichlorophenyl phosphate (1.5 mL, 10 mmol) and triethylamine (7 mL, 50 mmol) were added dropwise. The reaction solution was stirred at -80 ° C for 30 min, and then heated to 0 ° C for 3 h. At 0 ° C, a dichloromethane solution of pentafluorophenol (1.84 g, 10 mmol) and triethylamine (2.94 mL, 21 mmol) were added to the reaction solution, and after reacting for 5 hours, the temperature was lowered to -10 ° C and stirred overnight. Filtered by suction, the filter cake was dissolved in ethyl acetate, washed with water, dried, and then concentrated in vacuo, and slurried with isopropyl ether to obtain a white solid (3.1 g, 6.1 mmol) with a yield of 62% and a three-step yield of 52%. 1 H NMR(300MHz,Chloroform-d)δ7.36((t,J=7.4,7.0Hz,2H),7.31–7.18(m,3H),5.26( m,1H),4.38–4.22(m,1H),4.11(t,J=7.2Hz,2H),3.31(m,2H),1.46(d,J=6.8Hz,3H). 31 P NMR (162 MHz, DMSO-d6) δ 0.05.

[0108] Example 3 Determination of ee value of compound V-2

[0109]

[0110] In a 10 mL double-necked bottle, V-2 (21 mg, 0.1 mmol), triethylamine (20 μL) and anhydrous DCM (2 mL) were added, and the mixture was cooled to -15 °C and stirred under nitrogen protection. A DCM solution (2 mL) of MTPA-Cl (25 mg, 0.1 mmol, 98% ee) was slowly added dropwise to the reaction solution and the mixture was naturally warmed to room temperature for 1 h. After the reaction was completed, the reaction was quenched with water, extracted with ether (5 mL × 2), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was concentrated in vacuo. The white solid VII (31 mg) was obtained by preparative TLC purification (petroleum ether: ethyl acetate = 5:1). 19 F NMR (376MHz, CDCl3) δ-69.09 (95%), -69.12 (5%).

[0111] application 19 The F-NMR Mosher method can determine that the diastereoisomer excess of compound VII is 95%, and further infer that the ee value of compound V-2 is greater than 90%.

[0112] Example 4 Application of Compound VI-2 in the Preparation of ProTide Prodrug

[0113]

[0114] In a 50mL double-necked flask, add 5-fluorodeoxyuridine VIII (246mg, 1mmol) and anhydrous THF (15mL), and stir under nitrogen protection after cooling to -15°C. Slowly add tert-butylmagnesium chloride (1.7M, 1.5mL, 2.5mmol) to the reaction solution and continue to keep warm for 1h. Then add a tetrahydrofuran suspension (10mL) of compound I-2 (500mg, 1mmol) in batches, and react at room temperature for 24 hours after the addition. After the reaction is completed, quench the reaction with saturated ammonium chloride solution, extract with ethyl acetate (40mL×3), combine the organic phases, wash with saturated brine, dry with anhydrous sodium sulfate, concentrate the solvent in vacuo, and purify by silica gel column chromatography (dichloromethane: methanol = 100: 1-20: 1) to obtain a white glassy solid IX (227mg, 0.41mmol) with a yield of 41%. [M+H] + :559.1067.

[0115] This example shows that the intermediate of the present invention can effectively prepare a novel ProTide prodrug. The reaction product VIII has good anti-tumor activity in the prior art CN 110840907.

[0116] Comparative Example VI-2 Phosphorus Atom Racemization Treatment

[0117] In a 10 mL sealed tube, compound VI-2 (50 mg, 0.1 mmol), phenol (5 mg), triethylamine (20 μL) and tetrahydrofuran (2 mL) were added; after the addition, nitrogen was blown and the sealed tube was tightened. After heating at 40°C for 8 hours, ethyl acetate (5 mL) and water (5 mL) were added to the reaction solution, stirred for 30 minutes, and then allowed to stand for separation. The supernatant was concentrated and purified by 31 P NMR confirms the change of chiral phosphorus atom. 31 PNMR(162MHz,DMSO-d6)δ0.05,-0.10.

[0118] The results showed that after the above treatment, there were two obvious single peaks in the phosphorus spectrum of compound VI-2 (see Appendix Figure 2 ). This indicates that the chiral phosphorus atom of compound VI-2 undergoes racemization during the treatment process, and products with two configurations, S and R, are obtained. Comparing the phosphorus spectrum with the phosphorus spectrum of Example 2, it can be seen that the phosphorus atom of compound VI-2 obtained according to Example 2 has a single configuration, and it also indicates that the present invention can well provide a method for preparing a chiral phosphoramide intermediate.

[0119] Example 5 Synthesis of Compound I-1

[0120] Synthesis route:

[0121]

[0122] Specific steps:

[0123] Compound II-1 (1.9 g, 10 mmol), III-1 (1.13 g, 11 mmol), and LiCl (848 mg, 20 mmol) were placed in a dry 250 mL double-necked bottle, 100 mL of anhydrous tetrahydrofuran was added under argon protection, and the mixture was cooled to -15 °C; pivaloyl chloride (PivCl, 3.08 mL, 25 mmol) and triethylamine (3.61 mL, 26 mmol) were slowly added dropwise in sequence, and the mixture was kept warm for 1 h; the reaction solution was then stirred at room temperature for 30 min. The reaction was quenched with 50 mL of cooled saturated ammonium chloride solution, the reaction solution was extracted with ethyl acetate (40 mL × 3), and the organic phases were combined. The mixture was washed with saturated sodium bicarbonate solution and saturated brine, respectively, and dried over anhydrous sodium sulfate. The solvent was removed by vacuum concentration, and the mixture was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1-1:1) to obtain a white solid IV-1 (2.49 g, 9.06 mmol) with a yield of 90.6%. 1H NMR (300MHz, Chloroform-d) δ5.28 (p, J=7.2Hz, 1H), 5.17 (s, 1H), 4.32–4.05 (m, 2H), 3.34 (td, J=7.1, 4.0Hz, 2H), 1.45 (s, 9H), 1.37 (d, J=6.8Hz, 3H).

[0124] The above compound IV-1 (2.49 g, 9 mmol) was placed in a 50 mL single-necked bottle and dissolved with 6 mL of ethyl acetate; 4N hydrochloric acid-ethyl acetate solution (11 mL, 4.5 mmol) was slowly added dropwise at 0°C and stirred overnight. The reaction solution was filtered and the filter cake was vacuum dried to obtain a white solid product V-1 (1.80 g, 8.55 mmol) with a yield of 95%. 1 H NMR (300MHz, DMSO-d6) δ8.58 (s, 3H), 4.69 (d, J = 8.9Hz, 1H), 4.12 (q, J = 9.0, 7.9Hz, 2H), 3.46 (d, J = 7.2Hz, 2H), 1.40 (d, J = 6.7Hz, 3H).

[0125] Compound V-1 (1.80 g, 8.55 mmol) was placed in a 100 mL double-necked flask, and anhydrous dichloromethane (30 mL) was added under argon protection. The solution was cooled to -50 ° C; dichlorophenyl phosphate (1.5 mL, 10 mmol) and triethylamine (2.8 mL, 20 mmol) were slowly added dropwise, and the reaction solution was reacted at low temperature for 30 min and then heated to 0 ° C and continued to react for 4 h. The temperature of the reaction solution was lowered to -50 ° C again, and a dichloromethane solution of pentafluorophenol (1.84 g, 10 mmol) and triethylamine (2.94 mL, 21 mmol) were added thereto in sequence, and the mixture was stirred overnight. The reaction solution was filtered, and the filter cake was washed with ethyl acetate to obtain a white solid VI-1 (2.00 g, 4 mmol) with a yield of 40%. 1 H NMR(300MHz,Chloroform-d)δ7.38(t,J=7.7Hz,2H),7.32–7.17(m,3H),5.38–5.18(m,1H),4. 26(t,J=12.2Hz,1H), 4.11(t,J=7.1Hz,2H), 3.32(t,J=7.4,3.6Hz,2H), 1.46(d,J=6.7Hz,3H).

[0126] Compound VI-1 (0.240 g, 0.48 mmol) and nucleoside VII (0.104 g, 0.40 mmol) were placed in a 50 mL double-necked bottle, anhydrous tetrahydrofuran 20 mL was added, and the mixture was cooled to 0 ° C. Methylmagnesium bromide (334 μL, 1 mmol) was slowly added dropwise and reacted for 24 h. The reaction was quenched with saturated ammonium chloride solution (10 mL), extracted with ethyl acetate (40 mL × 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, the solvent was concentrated in vacuo, and purified by silica gel column chromatography (dichloromethane: methanol = 100: 1-20: 1) to obtain a white solid I-1 (64 mg, 0.11 mmol) with a yield of 28%. 1 H NMR (300MHz, Methanol-d4) δ7.54 (d, J=8.1Hz, 1H), 7.41 (q, J=7.3, 6.7Hz, 2H), 7. 35–7.15(m,3H),6.20(d,J=19.3Hz,1H),5.69(d,J=8.1Hz,1H),5.12–4.93(m,1H) ,4.51(ddd,J=11.7,4.8,2.2Hz,1H),4.32(ddd,J=11.7,4.1,2.4Hz,1H),4.24–4. 00(m,3H),3.83(dd,J=23.5,9.7Hz,1H),3.52–3.19(m,3H),1.27(d,J=6.3Hz,6H); 31 P NMR(121MHz,Methanol-d4)δ3.13; HRMS[M+Na] + :calcd for C 22 H 26 FN4NaO9PS, 595.1034; found, 595.1031.

[0127] Example 6 Synthesis of Compound I-2

[0128] Synthesis route:

[0129]

[0130] Specific steps:

[0131] Referring to the method of Example 5, III-1 in Example 5 was replaced by III-2 to obtain compound IV-2: 1HNMR (300MHz, Chloroform-d) δ5.38–5.21 (m, 1H), 5.21–5.08 (m, 1H), 4.37–3.99 (m, 2H), 3.52–3.16 (m, 2H), 1.45 (s, 9H), 1.37 (d, J=6.8 Hz, 3H), Compound V-2: 1 H NMR (300 MHz, DMSO-d6) δ8.54 (s, 3H), 4.69 (s, 1H), 4.12 (t, 2H), 3.47 (t, 2H), 1.39 (d, J = 6.5 Hz, 3H), compound VI-2: 1 H NMR(300MHz,Chloroform-d)δ7.49–7.30(m,2H),7.33–7.17(m,3H),5.45–5.12(m,1H),4.2 9(t,J=13.7,10.6Hz,1H),4.11(t,J=7.2Hz,2H),3.50–3.20(m,2H),1.46(d,J=6.8Hz,3H).

[0132] Referring to the method of Example 5, VI-2 was reacted with nucleoside VII to obtain compound I-2: 1 H NMR (300MHz, Methanol-d4) δ7.63(d,J=8.1Hz,1H),7.40(t,J=7.9Hz,2H),7.24(dd,J=1 6.4,7.9Hz,3H),6.15(d,J=19.4Hz,1H),5.62(d,J=8.0Hz,1H),5.11(dq,J=9.6,6.9Hz, 1H),4.53(ddd,J=12.0,5.9,2.0Hz,1H),4.34(ddd,J=11.9,6.1,3.5Hz,1H),4.12(tq,J =8.2,4.6Hz,3H),3.91(dt,J=23.5,12.6Hz,1H),3.55–3.15(m,3H),1.55–1.12(m,6H); 31 P NMR(121MHz,Methanol-d4)δ3.62; HRMS[M+Na] + :calcd for C 22 H 26 FN4NaO9PS, 595.1034; found, 595.1033.

[0133] Example 7 Synthesis of Compound I-3

[0134] Synthesis route:

[0135]

[0136] Specific steps:

[0137] Compound II-1 (1.9 g, 10 mmol) and III-3 (1.02 g, 11 mmol) were placed in a 250 mL double-necked flask, and 100 mL of dichloromethane was added under argon protection; DMAP (0.37 g, 3 mmol) and triethylamine (2 mL, 15 mmol) were added under stirring at room temperature. EDCI (2.9 g, 15 mmol) was then added in several portions, and the reaction solution was stirred at room temperature overnight after the addition. The reaction was quenched with 50 mL of cooled saturated ammonium chloride solution, and the reaction solution was extracted with ethyl acetate (40 mL × 3), and the organic phases were combined. The mixture was washed with saturated sodium bicarbonate solution and saturated brine, respectively, and dried over anhydrous sodium sulfate. The solvent was removed by vacuum concentration, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10: 1-1: 1) to obtain a white solid IV-3 (1.07 g, 4.1 mmol) with a yield of 41%. 1 H NMR (300MHz, Chloroform-d) δ5.14 (s, 1H), 4.43 (s, 2H), 4.19 (t, J = 7.3Hz, 2H), 3.36 (t, J = 7.3Hz, 2H), 1.47 (s, 9H);

[0138] Referring to the method of Example 5, IV-1 in Example 5 was replaced by IV-3 to obtain compound V-3: 1 H NMR (300MHz, DMSO-d6) δ8.53 (s, 3H), 4.34–3.86 (m, 4H), 3.46 (t, J = 7.3Hz, 2H);

[0139] Referring to the method of Example 5, V-1 in Example 5 was replaced by V-3, and the reaction temperature was adjusted to 0° C. to obtain compound VI-3: 1 H NMR (300MHz, Chloroform-d) δ7.48–7.34(m,2H),7.35–7.19(m,3H),4.67–4.37(m,2H),4.39–4.24(m,2H),4.21(t,J=7.4Hz,2H),3.40(t,J=7.3Hz,2H);

[0140] Referring to the method of Example 5, VI-3 was reacted with nucleoside VII to obtain compound I-3, wherein the reaction temperature was 40° C.: 1H NMR (300MHz, Methanol-d4) δ7.67(dd,1H),7.48–7.31(m,2H),7.24(dd,J=18.8,7.9Hz,3H),6.17(d,J=19.4Hz,1H),5.69(dd,J=8.9Hz,1H),4. 72–4.50(m,1H),4.53–4.36(m,1H),4.34–4.22(m,2H),4.21–4.04(m,3 H),4.00–3.82(m,1H),3.47–3.33(m,2H),1.36(dd,J=22.4,9.4Hz,3H); 31 P NMR(121MHz,Methanol-d4)δ5.34,4.80; HRMS[M+Na] + :calcd for C 21 H 24 FN4NaO9PS, 581.0878; found, 581.0876.

[0141] Example 8 Synthesis of Compound I-4

[0142] Synthesis route:

[0143]

[0144] Specific steps:

[0145] Referring to the method of Example 5, II-1 in Example 5 was replaced by II-2, and III-1 was replaced by III-2 to obtain compound IV-4: 1 H NMR (300 MHz, Chloroform-d) δ 5.57–5.25 (m, 1H), 5.14 (s, 1H), 4.47 (t, J = 8.0 Hz, 2H), 4.24–3.77 (m, 2H), 1.45 (s, 9H), 1.41 (d, J = 7.1 Hz, 4H); Compound V-4: 1 H NMR (300 MHz, DMSO-d6) δ8.61 (s, 3H), 4.77 (q, J = 7.0 Hz, 1H), 4.59–4.33 (m, 2H), 3.93 (t, J = 8.0 Hz, 2H), 1.43 (d, J = 6.9 Hz, 3H); Compound VI-4: 1H NMR(300MHz,Chloroform-d)δ7.38(t,J=7.9Hz,2H),7.32–7.16(m,3H),5.51–5.27(m,1H), 4.47(t,J=8.5Hz,2H), 4.23(t,J=12.0Hz,1H), 3.98(t,J=8.1Hz,2H), 1.51(d,J=6.8Hz,3H);

[0146] Referring to the method of Example 5, VI-4 reacts with nucleoside VII to obtain compound I-4: 1 H NMR (300MHz, Methanol-d4) δ7.62(d,J=8.1Hz,1H),7.40(t,J=7.9Hz,2H),7.34–7.17(m,3H),6.15(d,J=19.4Hz,1H),5.59(d,J=8.1Hz,1H) ,5.35–5.11(m,1H),4.63–4.48(m,1H),4.44(t,J=8.1Hz,2H),4.37–4.25(m,1H),4.19–4.05(m,1H),4.06–3.80(m,3H),1.39–1.26(m,6H); 31 PNMR(121MHz,Methanol-d4)δ3.52; HRMS[M+Na] + :calcd for C 22 H 26 FN4NaO 10 P, 579.1263; found, 579.1253.

[0147] Example 9 In vitro stability of the compound

[0148] Preparation of biological matrix:

[0149] Preparation of liver S9 and intestinal S9 of SD rats: Rats were fasted for 12 hours in advance, and the liver and small intestine were immediately removed after being killed by cervical dislocation, washed with 4°C PBS buffer, chopped and homogenized in PBS buffer. After homogenization, centrifuged at 9000g for 20 minutes (4°C), the upper fat layer and the lower sediment were discarded, the intermediate liquid was collected and divided into 50mL centrifuge tubes, and stored at -40°C for later use. The protein concentration was determined using the BCA method.

[0150] Preparation of SD rat plasma: Blood was collected from the venous plexus of the retina of the rat. The whole blood was pre-treated with EDTA 2K + Store in the treated centrifuge tube, centrifuge at 8000rpm for 5min, take the upper light yellow clear liquid as plasma, and store at -40℃ until use.

[0151] Preparation of simulated biological matrix:

[0152] Artificial gastric fluid (SGF) and artificial intestinal fluid (SIF) were USP sterile (Zhejiang Lianshuo Biotechnology Co., Ltd.);

[0153] Preparation of H2O2 reaction system: 30% hydrogen peroxide (Nanjing Chemical Reagent Co., Ltd., analytical grade) was diluted to 13.2 M with PBS.

[0154] Preparation of working samples:

[0155] Compounds I-1, I-2, I-3, and I-4 (400 μg / mL, final concentration 200 μg / mL) were mixed with H2O2 (13.2 mM, final concentration 6.6 mM) at a ratio of 1 / 1 (V / V) and incubated in a 37 °C water bath for 0 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, and 4 h;

[0156] The stock solutions of compounds Ⅰ-1, Ⅰ-2, Ⅰ-3, and Ⅰ-4 (10 mg / mL, final concentration 200 μg / mL) were mixed with PBS, artificial gastrointestinal fluid, liver and intestine S9 (final protein concentration 200 μg / mL), and plasma at a ratio of 1 / 50 (V / V) and incubated in a 37°C water bath for 0 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, and 4 h;

[0157] Sample processing:

[0158] Take 100 μL of sample, add 100 μL of MeOH (containing internal standard ZL-01 100 μg / mL) to terminate the reaction, vortex for 5 min, centrifuge twice at 12000 rpm for 5 min, and take 70 μL of supernatant for injection; parallel experiments were performed in triplicate (n=3).

[0159] Data processing:

[0160] Using the established HPLC analysis method, a standard curve was drawn to detect the residual concentration of the drug in the incubation system and a concentration-time curve was drawn. Nonlinear regression was performed with time (X, min) as the abscissa and the residual concentration of the obtained compound (Y, μM) as the ordinate. The exponential decay equation was obtained by integrating both sides to calculate the half-life. The results are shown in the following table:

[0161] Table 1. In vitro stability of compounds

[0162]

[0163] The above experimental results show that SOF can be metabolized in liver S9 and rapidly degraded in plasma; while the metabolic rates of compounds I-1, I-2, and I-4 in the present invention in liver S9 are comparable to those of SOF; surprisingly, the stability of compounds I-1, I-2, and I-4 in plasma are much higher than that of SOF, which indicates that the compounds have better stability in the portal vein and are more conducive to the prodrug entering the target tissue liver without degradation. On the other hand, in hydrogen peroxide solution, compounds I-1, I-2, I-3, and I-4 can all be rapidly metabolized to release target metabolites, which indicates that the compounds of the present invention can be better released in tissues with increased oxidative stress; in summary, the in vitro stability data suggest that the compounds of the present invention may have better pharmacokinetic properties.

[0164] Example 10 Anti-HCV in vitro pharmacodynamics test

[0165] Drugs: test compound (prepared in the above examples), control drug SOF.

[0166] Cell model: The in vitro activity of the compound was determined using the GT 1b HCV replicon cell system. The GT1b HCV genome was reconstructed, a portion of the fragments were cut out, and new fragments were added, including antibiotic resistance genes and luciferase genes. The newly constructed recombinant gene was transfected into the human liver cancer cell line Huh-7. After culturing in a culture medium containing neomycin sulfate (G418), G418-tolerant cell clones can be obtained. These cell clones can continuously express HCV replicon RNA, thereby establishing an effectively replicating HCV cell culture system. The replication level of the hepatitis C virus can be determined by detecting the expression level of the luciferase gene.

[0167] Experimental steps:

[0168] 1. Compounds were co-incubated with cells: GT 1b replicon cells were seeded into 96-well plates, with 8,000 cells per well; each compound concentration was tested in duplicate, with 10 points of 3-fold serial dilution, and the final DMSO concentration was 0.5%; cells were cultured in a carbon dioxide incubator for 72 hours;

[0169] 2. Add cell viability detection reagent to each well of cells to detect cell viability. Then add luciferase luminescent substrate to the cells and wait for 5 minutes before detecting using a chemiluminescence detection system. Analyze the data and calculate the compound's cytotoxicity and anti-hepatitis C virus GT 1b genotype replicon activity.

[0170] 3. Perform nonlinear fitting on the data and calculate the CC of the compound 50 or EC 50 .

[0171] Results: The inhibitory activity of the tested compounds against HCV was detected by chemiluminescence detection system, and some experimental results are shown in Table 2. The results showed that the tested compounds showed different degrees of anti-HCV activity and low cytotoxicity. Compared with SOF activity, some compounds had better antiviral effect against HCV.

[0172] Table 2. Anti-HCV activity of compounds

[0173]

[0174] Example 11 Metabolic Activation Study of Compounds

[0175] 1.LC-MS / MS conditions

[0176] ①Chromatographic conditions

[0177] The chromatographic column used was an XSelect HSS T3 column (150 mm × 4.6 mm, 3.5 μm), the mobile phase consisted of 5 mmol ammonium acetate + 80 μL ammonia water (A) and methanol: acetonitrile = 1:1 (V / V) (B), the column temperature was 40°C, the flow rate was 0.6 ml / min, and the gradient elution program was: 0-0.5 min, 1% B; 0.5-0.6 min, 1-12.5% ​​B; 0.6-2.5 min, 12.5% ​​B; 2.5-3.5 min, 12.5-90% B; 3.5-6.5 min, 90% B; 6.5-7 min, 90-1% B; 7-10 min, 1% B. The injection volume was 5 μl.

[0178] ②Mass spectrometry conditions

[0179] The mass spectrometer selected the electrospray ion source (ESI), and the source parameters were set as follows: Curtain Gas / CUR 30Arb, Collision Gas / CAD, N2 Medium, IonSpray Voltage / IS 5500V, Auxiliary Gas Heating Temperature (Temperature / TEM) 550°C, Auxiliary Gas 1 (Ion Source Gas 1, N2) 50Arb, Auxiliary Gas 2 (IonSource Gas 2, N2) 50Arb. The analytes were scanned in positive ion mode. The samples were detected in multiple reaction monitoring (MRM). The Q0 entrance voltage (Entrance Potential / EP) was set to 15V, and the Q2 exit voltage (Collision Cell Exit Potential / CXP) was set to 10V. The mass spectrometry parameters of the triphosphate active metabolite AM and the internal standard TFV (tenofovir) are shown in Table 3.

[0180] Table 3. MRM mode quantitative parameters

[0181]

[0182] 2. Cell Metabolism Experiment

[0183] HepG2 cells were collected at 5×10 5 The cells were inoculated at a density of 1 / well in a 12-well cell culture plate and cultured in a cell culture incubator. After the cells adhered to the wall, the culture medium was discarded and the cells were rinsed twice with PBS. A total of three groups (blank group, H2O2 group and APAP group) were set up, each with 4 replicates. In the blank group, 1 mL of culture medium containing SOF, I-1 or I-2 (final concentration 50 μM) was added to each well, 1 mL of H2O2 with a final concentration of 500 μM and SOF, I-1 or I-2 with a final concentration of 50 μM was added to each well in the H2O2 group, and 1 mL of APAP with a final concentration of 1 mM and SOF, I-1 or I-2 with a final concentration of 50 μM was added to each well in the APAP group. After incubation for 6 h, 12 h and 24 h, the drug-containing culture medium was removed, the cells were rinsed twice with PBS, 300 μL of ultrapure water was added, the cells were blown and transferred to a 1.5 mL centrifuge tube, and the cells were repeatedly frozen and thawed 3 times to break the cells and repeatedly blown. The protein concentration of one part of the cell sample was determined by the BCA method, and the other part was quantitatively taken, 50 μL of the cell sample was precipitated with 250 μL of acetonitrile containing 83.6 nM internal standard, oscillated for 5 min, and centrifuged twice at 18000 rpm × 5 min at 4°C. 50 μL of the supernatant was taken and 5 μL was injected.

[0184] 3. Experimental results

[0185] The drug was co-incubated with the cells, and the content of the intracellular triphosphate active metabolite AM was measured at 6h, 12h and 24h (corrected by protein quantitative data). The results are shown in the attached manual. Figure 4 As shown in A. The test compounds SOF, I-1 or I-2 can all be metabolized into active metabolites AM in cells, and as the co-incubation time increases, AM gradually accumulates in the cells; it is worth noting that the intracellular AM accumulation of compound I-1 is much higher than that of SOF, which suggests that the compound has better antiviral activity.

[0186] SOF, I-1 or I-2 were co-incubated with cells, and H2O2 or acetaminophen (APAP) was used to intervene to increase the level of cellular oxidative stress. The content of the triphosphate active metabolite AM in the cells was measured 24 hours later (corrected by protein quantitative data). The results are shown in the attached manual. Figure 4 As shown in B. Both I-1 and I-2 can show more ideal metabolic activation in cells with increased oxidative stress, and the accumulation of active metabolites AM increased significantly; while in the cells of the SOF group, the accumulation of AM did not change significantly before and after the intervention. This result shows that under conditions of increased oxidative stress, the compounds described in this patent can more effectively improve the metabolic activation efficiency; this also suggests that under the condition of chronic hepatitis C inflammation, the compounds may have higher active metabolite exposure levels, and thus better antiviral activity.

[0187] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that for ordinary technicians in this technical field, improvements or changes can be made based on the above description, and these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A chiral phosphoramide intermediate, the structural formula of which is shown in formula (VI): in, R 1 Select from: H or C 1-3 Alkyl; R 2 Select from: H or C 1-3 Alkyl; X is selected from: S or O; Ar is selected from: phenyl, 1-naphthyl or 2-naphthyl.

2. The chiral phosphoramide intermediate according to claim 1, whose structural formula is shown in formula (VI-1): in, X is selected from: O or S; the phosphorus atom is in S configuration.

3. The method for preparing the chiral phosphoramide intermediate according to claim 1, wherein the synthetic route is as follows: The method comprises the following steps: Step A: Compound II and Compound III are mixed with a solvent and a base, and then a condensing agent is added to obtain Compound IV through an acylation reaction; Step B: Compound IV is deprotected to obtain compound V; Step C: Compound V, phenyl dichlorophosphate and a solvent are mixed, and then a base and pentafluorophenol are added in sequence to obtain compound VI after phosphorylation reaction.

4. The preparation method according to claim 3, characterized in that: In step A, the condensing agent is pivaloyl chloride, isobutyl chloroformate, DCC, EDCI, HATU or HBTU.

5. The preparation method according to claim 4, characterized in that: In step A, the condensing agent is pivaloyl chloride.

6. The preparation method according to claim 4, characterized in that: In step A, an additive is also added, and the additive is lithium chloride, magnesium chloride, magnesium bromide or copper chloride.

7. The preparation method according to claim 6, characterized in that: The additive is lithium chloride.

8. The preparation method according to claim 6, characterized in that: The ratio of the condensing agent to the additive is 1:0.5-5.

9. The preparation method according to claim 8, characterized in that: The ratio of the condensing agent to the additive is 1:0.5-2.

10. The preparation method according to claim 3, characterized in that: In step A, the base is triethylamine, diisopropylethylamine, N-methylmorpholine, DMAP or DBU.

11. The preparation method according to claim 3, characterized in that: The base is triethylamine.

12. The preparation method according to claim 10, characterized in that: In step C, the base is triethylamine, diisopropylethylamine, DMAP or DBU.

13. The preparation method according to claim 10, characterized in that: In step C, the base is triethylamine.

14. The preparation method according to claim 3, characterized in that: In step A, the solvent is selected from dichloromethane, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide or a mixed solvent optionally composed of these solvents.

15. The preparation method according to claim 14, characterized in that: In step A, the solvent is tetrahydrofuran.

16. The preparation method according to claim 14, characterized in that: In step C, the solvent is a combination of one or more of diethyl ether, methyl tert-butyl ether, isopropyl ether, dichloromethane, 1,2-dichloroethane, chloroform, toluene, tetrahydrofuran, acetone, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, and N-methylpyrrolidone.

17. The preparation method according to claim 16, characterized in that: In step C, the solvent is dichloromethane.

18. The preparation method according to claim 3, characterized in that: In step A, the reaction temperature is -50°C to 80°C.

19. The preparation method according to claim 18, characterized in that: In step A, the reaction temperature is -20°C to 30°C.

20. The preparation method according to claim 18, characterized in that: In step C, the reaction temperature is -80°C to 50°C.

21. The preparation method according to claim 20, characterized in that: In step C, the reaction temperature is -50°C to 0°C.

22. The preparation method according to claim 3, characterized in that: In step C, the ratio of the compound phenyl dichloride phosphate to the base is 1:1 to 1:

10.

23. The preparation method according to claim 22, characterized in that: In step C, the ratio of the compound phenyl dichloride phosphate to the base is 1:2 to 1:

5.

24. Use of the chiral phosphoramide intermediate according to claim 1 in the preparation of a sofosbuvir derivative or a pharmaceutically acceptable salt thereof; the sofosbuvir derivative is represented by formula (I): in, R 1 Select from: H or C 1-3 Alkyl; R 2 Select from: H or C 1-3 Alkyl; X is selected from: S or O; Ar is selected from: phenyl, 1-naphthyl or 2-naphthyl.

25. A sofosbuvir derivative represented by formula (I) or a pharmaceutically acceptable salt thereof: in, R 1 Select from: H or C 1-3 Alkyl; R 2 Select from: H or C 1-3 Alkyl; X is selected from: S or O; Ar is selected from: phenyl, 1-naphthyl or 2-naphthyl.

26. The sofosbuvir derivative or a pharmaceutically acceptable salt thereof according to claim 25, characterized in that: R 1 Selected from: H or methyl; R 2 is selected from: H or methyl; X is selected from: S or O; Ar is selected from: phenyl, 1-naphthyl or 2-naphthyl.

27. The sofosbuvir derivative or a pharmaceutically acceptable salt thereof according to claim 26, characterized in that: Ar is selected from: phenyl.

28. A sofosbuvir derivative or a pharmaceutically acceptable salt thereof as described in any one of the following:

29. A method for preparing the sofosbuvir derivative according to claim 25, characterized in that: Its synthetic route is as follows: In the above synthetic route, R 1 , R 2 The definitions of X, Ar and Ar are consistent with those in the derivative of formula (I), and the preparation method comprises the following steps: Step A: Compound II and Compound III are mixed with a solvent and a base, and then a condensing agent is added to obtain Compound IV through an acylation reaction; Step B: deprotection of compound IV to generate compound V; Step C: Compound V, phenyl dichlorophosphate and a solvent are mixed, and then a base and pentafluorophenol are added in sequence to obtain compound VI after phosphorylation reaction; Step D: Compound VI undergoes substitution reaction with compound VII to obtain compound I.

30. A pharmaceutical composition, characterized in that Comprising the sofosbuvir derivative or a pharmaceutically acceptable salt thereof according to any one of claims 25 to 28 and a pharmaceutically acceptable carrier.

31. Use of the sofosbuvir derivative or a pharmaceutically acceptable salt thereof according to any one of claims 25 to 28 or the composition according to claim 30 in the preparation of an anti-hepatitis virus drug.

32. The use according to claim 31, characterized in that The hepatitis is hepatitis C.

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