CN103435672A

CN103435672A - Structure and synthesis of novel nucleoside phosphate prodrug containing substituted benzyl

Info

Publication number: CN103435672A
Application number: CN2013101464795A
Authority: CN
Inventors: 刘沛
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-04-25
Filing date: 2013-04-25
Publication date: 2013-12-11
Also published as: CN103980318A; CN103980318B

Abstract

The invention describes a phosphate structure containing substituted benzyl, wherein the phosphate structure is shown as a formula (I) and a formula (II). The phosphate structure can be used as prodrugs for various nucleoside analogues including non-cyclic nucleoside, carbon-cyclic nucleoside, furan-cyclic nucleoside and the like, and enhance bioactivity of nucleoside compounds, and thus the structure can be applied in treatment for virus infections and cancers.

Description

Structure and synthesis of novel nucleoside phosphate ester prodrug containing substituted benzyl

Technical Field

The invention relates to a novel nucleoside analogue phosphate prodrug (I) and (II) containing substituted benzyl, isomers or medicinal salts of the nucleoside analogue phosphate prodrug (I) and (II), an equivalent serving as nucleoside monophosphate, structural characteristics of the nucleoside analogue phosphate prodrug and a preparation method of the nucleoside analogue phosphate prodrug.

Background

Nucleosides are structural monomers of deoxyribonucleic acid and ribonucleic acid (DNA) and RNA, which are biogenetic genes, and thus have important functions in all living bodies, and are widely used for treating viral infections and cancers. Since the 1960 s, a number of biologically active nucleoside analogs have been used to treat various viral infections, such as herpes, AIDS, hepatitis B and C. These synthetic nucleoside analogs are able to act as antiviral agents by blocking the growth of viral nucleic acid strands, disrupting viral gene replication (FIG. 1).

As shown in FIG. 1, nucleosides must first be activated into nucleoside triphosphates in three steps to participate in DNA or RNA synthesis and thus exhibit physiological activity. When a nucleoside analog can selectively drive into the genetic gene of a virus or cancer cell to prevent the reproduction of the nucleic acid chain, and has no harm (toxicity) to the host cell gene, the nucleoside analog can be used as an antiviral or anticancer drug.

Nucleoside triphosphates themselves carry multiple negative charges, are very highly polar, and are difficult to enter the interior of cells through cell walls, and therefore cannot be used as antiviral drugs directly. The early nucleoside antiviral drugs are nucleosides with moderate polarity, which are phosphorylated in three steps under the action of host cell kinase (kinase) after entering cells, and finally become nucleoside triphosphates to play the drug effect. With the recent advances in phosphate prodrug technology, the low-polarity equivalent structural unit of the monophosphate is directly introduced into the nucleoside molecule by a chemical method, so that the nucleoside phosphate prodrug is released after entering the interior of the cell without being limited by the selectivity of the nucleoside kinase. Thus, nucleoside phosphate prodrugs have become a popular way to upgrade the performance of nucleoside drugs.

The research on nucleoside prodrugs is a hot spot at present, and particularly, phosphate prodrugs are the most effective way to upgrade nucleoside drugs and can also enable some nucleosides which cannot be phosphorylated due to the limitation of nucleoside kinases to show biological activity, and the structure types of the phosphate prodrugs of the nucleosides reported in the literature are mainly seven (J.Am.chem.Soc., 2004, 126, 5154-5163; WO 2012094248; FIG. 2).

As shown in FIG. 2, (2-1)-(2-5) These five nucleoside prodrugs all contain ester groups and are hydrolyzed by esterases (including phospholipases) to release the nucleoside monophosphate. Because esterases are widely distributed in digestive systems such as gastrointestinal tract and liver, they do not have very strong liver targeting property. A prominent representative of these prodrugs is the aryl phosphoramidate of McGuigan et al (see2-5) Is one of the most widely and successfully applied nucleoside prodrug at present; nucleoside prodrugs (2-6) And (a)2-7) Containing substituted benzyl groups, they can only be cleaved oxidatively with the aid of oxidizing enzymes, such as cytochrome P450 enzymes, to release the nucleoside monophosphates. Since P450 enzyme lines are mainly distributed in the liver, these nucleoside prodrugs are mainly released by lysis in hepatocytes and are called liver-targeted (hepdi ect) prodrugs.

Combining the structural characteristics of McGuigan prodrug and HepDirect prodrug, the present inventors have designed phosphate ester prodrug (I) containing substituted benzyl group and prodrug (II) related to it by replacing the aromatic ring in McGuigan prodrug with substituted benzyl group in the structure of liver targeting prodrug, and have described its structural characteristics and preparation method.

The phosphate derivative can strengthen the biological activity of nucleoside compounds and upgrade the inherent antiviral or anticancer activity of the nucleoside compounds. When the nucleoside is combined with the novel phosphate containing the substituted benzyl, the generated prodrug is stable to esterase, and benzoic acid compounds generated by metabolism are relatively safe, so that the defect that the McGuigan prodrug releases toxic phenol is effectively overcome. In particular, the prodrug described by the invention also has a remarkable liver targeting effect, and is particularly suitable for developing medicaments for treating liver diseases, such as liver cancer, hepatitis and the like.

Disclosure of Invention

The invention provides a structure of a novel nucleoside analogue phosphate prodrug containing substituted benzyl and a synthesis method thereof, and the phosphate can be used as a monophosphate equivalent of nucleoside analogue after being combined with the nucleoside analogue to activate the nucleoside and improve the activity of the nucleoside medicament. The structure of the compound is shown as (I) and (II), and the compound can be isomer or medicinal salt thereof, and is characterized by the following molecular structure:

wherein,

the phosphate ester prodrug structure of the nucleoside comprises at least one substituted benzyl group as shown in formula (I) and formula (II):

a phosphate function of formula (I) comprising a phosphoester bond formed by the participation of an appropriately substituted benzyl alcohol and a phosphoramide bond formed by the participation of an appropriately substituted alpha-amino acid ester; the phosphate function in formula (II) contains two phosphate linkages formed by participation of the same or different substituted benzyl alcohols.

R₁And R₁' independently of one another, may be halogen, carboxyl, nitro, ester, amido, C₁-C₈Straight or branched alkyl of (2), C₁-C₈Linear or branched alkoxy of (C)₁-C₈Amino group of (A), C₂-C₈Linear or branched alkenyl of, C₂-C₈Straight-chain or branched alkynyl of, C₂-C₈An acyl group of (1).

R₂And R₃Independently of each other, can be a (substituted) benzene ring, a (substituted) aryl group, a (substituted) benzyl group or C₁-C₁₂Straight or branched alkyl of (2), C₃-C₆Saturated or unsaturated cycloalkyl of (C)₂-C₈Linear or branched alkenyl of, C₂-C₈Straight or branched alkynyl groups of (a).

Nucleoside refers to various Nucleoside analogs, including common furan cyclic nucleosides, carbocyclic nucleosides and acyclic nucleosides, and has the following structural formula:

wherein,

X₁may be absent, or CH₂Or CHMe.

X₂Selected from O, CH₂、C＝CH₂Or CHCH₃。

X₃May be absent, or methylene CH₂。

T₁、T₂Independently of each other, may be H and CH₂R₄And R is₄H, OH or F; t is₁、T₂Or may be bonded to each other to form the following functional groups

And

here, R₅、R₆、R₇、R₈Independently of one another, may be hydrogen, hydroxy, halogen, cyano, azide, amino or C₁-C₄Alkyl, alkenyl, alkynyl or C₁-C₄Alkoxy group of (2).

Base is a purine or pyrimidine Base, or a chemical and metabolic derivative thereof, of the general formula:

X₄can be selected from hydrogen, halogen (F, Cl, Br, I), hydroxyl, methyl, amino, vinyl, 2-bromovinyl, C_1-8Straight or branched alkyl or (substituted) ethynyl;

X₅、X₆and X₇Each independently selected from hydrogen, halogen, hydroxy, amino, C₁-C₄Alkoxy or C₁-C₄An alkylamino group.

Z is nitrogen, CH or CX₄，X₄The definition is as described above.

More specific are the moieties such as, but not limited to, the following structures:

isomers include tautomers, cis-trans isomers, conformational isomers and optical isomers; the pharmaceutically acceptable salts refer to pharmaceutically acceptable salts formed by the nucleoside prodrugs (I) and (II) and inorganic acids or organic acids.

The nucleoside analogue phosphate derivative prodrug containing the substituted benzyl, or the isomer and the medicinal salt thereof can be used for upgrading various nucleoside antiviral drugs or anticancer drugs, and can be used for preparing drugs for treating and/or preventing virus infection such as HIV, HBV, HCV and the like, or preparing anticancer drugs.

According to the above structural features, one specific example compound has the following structure:

where R is₁、R₂And R₃As mentioned above, a more specific example Is (IV)

Where R is₂And R₃Independently of one another, as defined above.

In addition to the benzylphosphoramidate derivative (III) of the above acyclic nucleoside tenofovir, another furan ring nucleoside, i.e., 2' -methyl ribonucleoside, whose substituted benzyl phosphate compound has the following structure:

where R is₁、R₂、R₃And Base independently of each other, as defined above, more specific examples being (VI)

Where R is₂And R₃Independently of one another, as defined above. The Base definition is as previously described and refers to the pyrimidine, purine bases of various nucleosides or chemical and metabolic derivatives thereof.

The phosphate function of the above nucleoside analogue prodrug may also contain two appropriately substituted benzyloxy groups, and example compounds are of the following structure:

where R is₁And R₁' independently of one another, the definitions of which are as described above, more specific examples being (VI)

The substituted benzyl phosphate derivative (I) of the present invention may be prepared from a phosphate intermediate having a phenol leaving group having a strong electron-withdrawing group (I)1) Or (a)3) With 5' -hydroxy-unprotected nucleoside analogs: (2) Prepared in organic solvent in the presence of proper base catalyst. The reaction formula is as follows:

in the formula R₁、R₁’、R₂、R₃X is a strongly electron-withdrawing group and may be one or more, and Y is an oxygen atom, a sulfur atom, a methylene group or C ═ CH₂. The organic base is mainly tert-butyl magnesium chloride. Nucleoside substrates (2) Phosphate with a phenol leaving group containing a strong electron-withdrawing group: (1) Or (a)3) And the organic base reagent in a molar ratio of 1: 1 to 5: 1 to 10, and usually the amount of the organic base used is increased so as not to hinder the reaction.

With the preparation method of the present invention, the reaction temperature is from-78 ℃ to the reflux temperature of the solvent, preferably from 0 ℃ to room temperature, and the reaction time is monitored by TLC, usually from 5 to 100 hours. The organic solvent for the reaction is dichloromethane, tetrahydrofuran, chloroform, dioxane, benzene, toluene, diethyl ether, acetonitrile, DMF, etc., preferably dichloromethane, dioxane, tetrahydrofuran.

Monobenzyl phenol phosphate intermediate (A)1) Phosphorus oxychloride can be respectively mixed with equivalent substituted phenol(s) ((R))4) Substituted benzyl alcohol(s) ((s))6) Andalpha-amino acid esters of (A)7) Reacting in proper organic solvent in the presence of proper alkaline acid-binding agent. The basic acid-binding agent can be triethylamine or pyridine. The reaction solvent may be benzene, toluene, chloroform, methylene chloride, diethyl ether, tetrahydrofuran, dioxane, ethyl acetate, acetonitrile, etc., and methylene chloride, benzene and tetrahydrofuran are recommended. The reaction temperature is-78 ℃ to the reflux temperature of the solvent, preferably-78 ℃ to room temperature, the reaction time is 3-12 hours, and the reaction is not influenced by the prolonged reaction time. The reaction formula is as follows:

formula (III) X, R₁、R₂、R₃The definition is as described above. Under certain conditions, phosphorus oxychloride and substituted phenol (C)4) Reaction product of (A), (B), (C)5) Can be used in the next reaction without isolation, i.e. with benzyl alcohol (A), (B), (C), (6) And alpha-amino acids (a)7) Stepwise condensation to produce the product of monobenzyl phenol phosphate (1). The multi-step reaction can be processed in one pot, the synthesis method is simple, the yield is high, and the method can be used for industrial production. Benzylphenol phosphate ester (A)1) Has high stability, and can be purified by column chromatography.

Bis-benzylphenol phosphate intermediate (A)3) Phosphorus oxychloride can be respectively mixed with equivalent substituted phenol(s) ((R))4) Substituted benzyl alcohol(s) ((s))6) And substituted benzyl alcohol (8) Reacting in proper organic solvent in the presence of proper alkaline acid-binding agent. Substituted benzyl alcohol (6) And (a)8) Independent of each other, do not exclude the possibility of having exactly the same molecular structure. The basic acid-binding agent can be triethylamine or pyridine. The reaction solvent may be benzene, toluene, chloroform, methylene chloride, diethyl ether, tetrahydrofuran, dioxane, ethyl acetate, acetonitrile, etc., and methylene chloride, benzene and tetrahydrofuran are recommended. The reaction temperature is-78 ℃ to the reflux temperature of the solvent, preferably-78 ℃ to room temperature, the reaction time is 3-12 hours, and the reaction is not influenced by the prolonged reaction time. The reaction formula is as follows:

formula (III) X, R₁、R₁’、R₂、R₃The definition is as described above. Under certain conditions, phosphorus oxychloride and substituted phenol (C)4) Reaction product of (A), (B), (C)5) Can be used in the next reaction without isolation, namely with substituted benzyl alcohol(s) ((R))6) And (a)8) Condensation step by step to produce the product of dibenzyl phenol phosphate (3). The multi-step reaction can be processed in one pot, the synthesis method is simple, the yield is high, and the industrial production can be realized. Bis-benzylphenol phosphate ester(s) ((s))3) Has high stability, and can be purified by column chromatography.

The nucleoside compound can be prepared by a method reported in the known literature.

Drawings

FIG. 1 is a process for the polymerization of nucleosides, nucleotides and nucleic acids;

figure 2 is a common nucleoside prodrug.

Detailed Description

All water sensitive reactions were performed under dry conditions. Benzene, tetrahydrofuran or dichloromethane are refluxed, dried and distilled in the presence of metal sodium, and then are stored for later use. Nucleoside substrates are synthesized according to the literature methods, and are preferably dried under vacuum at about 50 ℃ before use. The substituted benzyl phosphate derivative of the nucleoside compound is separated by a silica gel column chromatography method to obtain an epimer mixture of the mono-substituted benzyl phosphate (I) or the di-substituted benzyl phosphate (II).

The following examples are given to aid in the understanding of the invention, but are not intended to limit the scope of the invention.

Example 1

A compound of (A), (B), (C912.8g, 50mmol) are dissolved in dichloromethane (100mL) and cooled to-78 deg.C, and a solution of o-methylbenzyl alcohol (6.1g, 50mmol) and triethylamine (7.7mL, 55mmol) in dichloromethane (100mL) is slowly added dropwise over 20 minutes. The reaction was stirred at-78 ℃ for 30 minutes and then transferred to another reaction vessel containing a solution of dry L-alanine hydrochloride (7.68g, 50mmol) in dichloromethane (100mL) at 0 ℃. Triethylamine (14.7mL, 105mmol) was slowly added dropwise to the reaction mixture over 20 minutes, and the reaction mixture was allowed to stir at zero degrees for an additional hour. Removing solvent by rotary evaporation, adding ethyl acetate, grinding, filtering, concentrating filtrate, and separating and purifying residue with silica gel column chromatography (petroleum ether: ethyl acetate: 7: 3) to obtain colorless oily product11) (17.7g, 84%) and cured slowly with prolonged standing.

¹H NMR(CDCl₃，400MHz)δ8.15-8.17(m，2H)，7.16-7.34(m，6H)，5.15(t，J＝8.0Hz，2H)，4.08-4.15(m，2H)，3.92-3.96(m，1H)，3.70-3.82(m，1H)，2.32(s，1.5H)，2.31(s，1.5H)，1.35(d，J＝7.2Hz，1.5H)，1.32(d，J＝7.2Hz，1.5H)，1.18-1.23(m，3H)；³¹P NMR(CDCl₃)δ3.29，3.21；MS(m/z)423(M+H)。

Example 2

Compound (A) to (B)12，296mg1mmol) was dissolved in 20mL of anhydrous tetrahydrofuran and t-butylmagnesium chloride Grignard reagent (1.0M, 4mL, 4mmol) was added at room temperature. After stirring the reaction mixture for 30 minutes, slowly adding dropwise a compound (A)11844mg, 2mmol) in tetrahydrofuran (4mL), the reaction mixture was stirred at room temperature for 24 h, TLC monitored for completion and quenched by addition of saturated ammonium chloride solution (20mL), extracted with ethyl acetate (20mLx3), the organic phases were combined, dried, concentrated and the residue was purified by column chromatography on silica gel (dichloromethane: methanol 20: 1) to give the product as a white powder: (dichloromethane: methanol ═ 20: 1)13)(370mg，64％)。¹H NMR(CD₃OD，400MHz)δ8.50(s，0.5H)，8.47(s，0.5H)，8.39(s，0.5H)，8.35(s，0.5H)，7.28-7.30(m，1H)，7.09-7.17(m，3H)，6.15(s，0.5H)，6.14(s，0.5H)，5.04-5.09(m，2H)，4.22-4.60(m，4H)，4.16(s，3H)，4.02-4.07(m，2H)，3.78-3.81(m，1H)，2.31(s，1.5H)，2.30(s，1.5H)，1.27-1.31(m，3H)，1.13-1.18(m，3H)，0.88-0.91(m，3H)；³¹P NMR(CD₃OD)δ9.72，9.62；MS(m/z)580(M+H)。

Example 3

By the same synthesis method as in example 15, compound (b) was prepared14) And (a)11) Coupling under the action of a tert-butyl magnesium chloride Grignard reagent to obtain a white powdery product (A)15)。¹H NMR(CD₃OD，400MHz)δ7.93(s，0.5H)，7.90(s，0.5H)，7.28-7.31(m，1H)，7.10-7.17(m，3H)，5.95(s，0.5H)，5.94(s，0.5H)，5.04-5.09(m，2H)，4.00-4.43(m，6H)，4.02(s，3H)，3.78-3.82(m，1H)，2.27-2.31(m，3H)，1.27-1.31(m，3H)，1.13-1.16(m，3H)，0.90-0.95(m，3H)；³¹P NMR(CD₃OD)δ9.76，9.67；MS(m/z)595(M+H)。

Example 4

By the same synthesis method as in example 2, compound (b)11) With 2' -methyluracils (16) Condensation coupling to obtain white powdery product (17)；³¹P NMR(CD₃OD)δ9.70，9.55；¹H NMR(CD₃OD)δ7.66(d，J＝8.0Hz，0.5H)，7.61(d，J＝8.0Hz，0.5H)，7.31-7.36(m，1H)，7.16-7.22(m，3H)，5.94(s，0.5H)，5.93(s，0.5H)，5.52(d，J＝8.0Hz，0.5H)，5.39(d，J＝8.0Hz，0.5H)，5.06-5.12(m，2H)，3.73-4.44(m，7H)，2.36(s，1.5H)，2.35(s，1.5H)，1.36(s，1.5H)，1.34(s，1.5H)，1.17-1.27(m，3H)，1.12(s，1.5H)，1.11(s，1.5H)；MS(m/z)542(M+H)。

Example 5

2-methylbenzyl alcohol (b)181.22g, 11mmol) and triethylamine (2.1mL, 15mmol) in dichloromethane (10mL) at-78 deg.C over 20 minutes91.285g, 5mmol) in 10mL of dichloromethane, and the reaction mixture was stirred at this temperature for 30 minutes, then warmed to room temperature and stirred overnight. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography (hexane: ethyl acetate 10: 1) to give a colorless oily product19(60％)。³¹P NMR(CDCl₃)δ-5.60；¹H NMR(CDCl₃)δ8.10(d，J＝8.8Hz，2H)，7.14-7.26(m，10H)，5.13-5.16(m，4H)，2.30(s，6H)；¹³C NMR(CDCl₃)δ137.36，133.19，130.76，129.67，129.53，126.37，125.70，120.76，120.70，69.25，69.19，18.92。

Example 6

By the same synthesis method as in example 2, compound (b)19) With 2' -methyluracils (16) Condensation coupling to obtain white powdery product (20)；MS(m/z)547(M+H)。

Example 7

Adding (R) -9- [2- (phosphonomethoxy) propyl group into 80ml of N-dimethylformamide solvent]-adenine (A)213.5g, 8.9mmol), 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (1.9g, 10mmol), triethylamine (3.8mL, 27mmol), 4- (N, N-dimethylamino) pyridine (320mg, 2.6mmol) and 2-methylbenzyl alcohol (1.8g, 14.7mmol) were stirred at room temperature for 30 minutes, then the reaction was heated at 100 ℃ for 24 hours, after completion of the reaction, the solvent was removed by concentration under reduced pressure, the residue was dissolved in water and purified by reverse phase C-18 column chromatography (methanol: water ═ 1: 9) to give the product (C22，69％)。³¹P NMR(MeOH-d₄)δ-30.98；¹H NMR(MeOH-d₄)δ8.32(s，1H)，8.25(s，1H)，7.07-7.23(m，4H)，4.88-4.96(m，2H)，4.39-4.43(m，1H)，4.20-4.25(m，1H)，3.92-3.96(m，1H)，3.80-3.85(m，1H)，3.64-3.69(m，1H)，2.27(s，3H)，1.16(d，J＝6Hz，3H)；¹³C NMR(MeOH-d₄)δ15.59，17.66，62.29，63.92，65.50，65.55，75.84，75.86，118.03，125.73，128.09，128.20，130.03，136.49，143.91，145.23，149.19，150.46。

Example 8

Adding (R) -9- [2- (phosphonomethoxy) propyl ] in 20mL of N, N-dimethylformamide solvent]-adenine (A)210.57g, 2mmol), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.38g, 2mmol), 1, 8-diazabicyclo [5.4.0]Undec-7-ene (0.46g, 3mmol) and 2-methylbenzyl alcohol (180mg, 1.5mmol) were stirred at room temperature for 30 minutes, then heated at 100 ℃ for 24 hours, after completion of the reaction, the solvent was removed by concentration under reduced pressure, the residue was dissolved in water, and purified by reverse phase C-18 column chromatography (methanol: water: 1: 9) to give the product (a22)。

Example 9

The compound22(45mg, 0.11mmol) was suspended in acetonitrile (1mL), and thionyl chloride (33. mu.L, 0.25mmol) was added with stirring at 50 ℃ followed by reaction at 75-80 ℃ for two hours. The solvent was then distilled off under nitrogen and the residue was dissolved in dry dichloromethane (2mL) and cooled to-30 ℃. A solution of L-alanine ethyl ester hydrochloride (30mg, 0.2mmol) and triethylamine (30. mu.L, 0.22mmol) in methylene chloride (0.5mL) was slowly added over one hour, and the reaction was slowly warmed to room temperature overnight. After the reaction was completed, 10% sodium dihydrogen phosphate solution was added to quench the reaction, dichloromethane (10mL) was added to dilute, extract, the organic phase was washed with saturated saline solution, dried (sodium sulfate), filtered and then concentrated, and the residue was purified by column chromatography to obtain a white solid product: (a)23，48％)。³¹P NMR(MeOH-d₄)δ-23.35，-24.24；¹H NMR(MeOH-d₄)δ8.16(s， 0.5H)，8.15(s，0.5H)，8.10(s，1H)，7.12-7.24(m，4H)，4.95-4.99(m，2H)，4.33(t-d，J₁＝13.6Hz，J₂＝2.4Hz，1H)，4.01-4.20(m，3H)，3.80-3.93(m，3H)，3.64-3.70(m，1H)，2.28(s，1.5H)，2.27(s，1.5H)，1.12-1.30(m，9H)；MS(m/z)491(M+H)。

Example 10

Using the synthesis of example 9, compound (b)22) Condensing with alanine isopropyl ester hydrochloride to obtain the product (A)24)。¹H NMR(MeOH-d₄)δ8.17(s，0.5H)，8.16(s，0.5H)，8.10(s，1H)，7.10-7.24(m，4H)，4.94-4.99(m，2H)，3.80-4.20(m，6H)，3.64-3.70(m，1H)，2.29(s，1.5H)，2.28(s，1.5H)，1.10-1.32(m，12H)；MS(m/z)505(M+H)。

Example 11

A compound of (A), (B), (C220.78g, 2mmol), L-phenylalanine isopropyl ester hydrochloride (1.46g, 6mmol) and triethylamine (0.84mL, 6mmol) in pyridine (8mL) was heated to 60 ℃ for 5 minutes, followed by reacting triphenylphosphine (1.84g, 7mmol) with compound (C)251.54g, 7mmol) of freshly prepared bright yellow solution in pyridine (8mL) was added to the above nucleoside(s) ((II)22) The reaction mixture was reacted with an amino acid at 60 ℃ overnight. Concentrating under reduced pressure after reaction, distributing the residue between ethyl acetate and sodium bicarbonate water solution, and preparing organicThe phases are dried over anhydrous sodium sulfate, filtered, concentrated and the residue is separated by column chromatography (dichloromethane: methanol 20: 1) to give the product (b: (b))26)。¹H NMR(MeOH-d₄)δ8.17(s，0.5H)，8.16(s，0.5H)，8.11(s，1H)，6.98-7.24(m，9H)，4.94-4.99(m，2H)，3.52-4.42(m，9H)，2.29(s，1.5H)，2.28(s，1.5H)，0.92-1.30(m，9H)；MS(m/z)581(M+H)。

Example 12

Under the protection of nitrogen, the compound (A) is mixed with21287mg, 1mmol) and 1-methyl-2-pyrrolidone (281mL) and triethylamine (300. mu.L) were mixed and stirred for 30 minutes, followed by addition of compound (D)27400mg, 2.5mmol) and reacting the reaction mixture at 55-60 ℃. After the reaction is monitored by TIC, the reaction solution is cooled to room temperature, ethyl acetate (10mL) is added for dilution, the mixture is washed by water, dried (anhydrous sodium sulfate), filtered and concentrated, and the residue is separated by column chromatography to obtain a product (the product can be obtained)29)，MS(m/z)：536(M+H)。

Example 13(HCV activity)

Methods for testing anti-HCV activity of compounds references (WO2007/027248) were reported in human hepatocytes Huh-7, and the activity of test compounds was evaluated by replication of HCV replicons harboring luciferase genes. Here, the signal intensity of luciferase directly corresponds to the amount of viral RNA replicated. The nucleoside phosphate ester prodrugs were formulated in DMSO solutions at varying concentrations ranging from 0.14 to 300 μ M and then applied to 96-well plates followed by the addition of replicon cells (6000 cells per well). Cells were incubated in the presence of nucleoside prodrugs for 48 hours and then luciferase intensity was measured. Reduction of luciferase Signal indicates reduction of HCV replicon RNA in cells, which may proceedTo calculate the antiviral activity index EC₅₀。

Tested, compound (A)13)、(14)、(17) And (a)20) All show better anti-hepatitis C virus activity, such as compound (A), (B), (C), (17) EC of (1)₅₀1.2 mu M, and has no toxicity to PBM, CEM and other cells at 100 mu M.

Example 14(HIV Activity)

Methods for testing anti-HIV-1 activity of compounds references (antimicrob. ingredients chemicother., 1992, 36, 2423) were performed in PBM lymphocytes. The nucleoside phosphate ester prodrug is prepared into DMSO (20-40mM) solution, and then diluted into a series of different concentrations to react with HIV-1_LAIVirus-infected PBM cells were co-cultured. HIV-1_LAIThe number ratio of virus to PBM cells MOI is 0.01, DMSO solvent has no influence on virus propagation, AZT is used as reference, and antiviral activity index EC is used as reference₅₀Calculated from inhibition-concentration curves (adv. enzyme Regul., 1984, 22, 27).

Tested, compound (A)22)、(24)、(26) And (a)29) All exhibit certain anti-HIV activity, e.g. compounds (A), (B), (C), (22) EC of (1)₅₀0.02 μ M, and no toxicity to PBM, CEM and other cells at 100 μ M.

Claims

1. A novel nucleoside phosphate ester prodrug containing substituted benzyl, or isomers and pharmaceutically acceptable salts thereof, is characterized by the following molecular structure:

wherein,

a phosphate function of formula (I) comprising a phosphoester bond formed by the participation of an appropriately substituted benzyl alcohol and a phosphoramide bond formed by the participation of an appropriately substituted alpha-amino acid ester;

the phosphate function in formula (II) contains two phosphate linkages formed by participation of the same or different substituted benzyl alcohols.

2. The substituted benzyl group-containing phosphate derivative according to claim 1, or an isomer, a pharmaceutically acceptable salt thereof,

R₁and R₁' independently of one another, may be halogen, carboxyl, nitro, ester, amido, C₁-C₈Straight or branched alkyl of (2), C₁-C₈Alkoxy group of (C)₁-C₈Amino group of (A), C₂-C₈Linear or branched alkenyl of, C₂-C₈Straight-chain or branched alkynyl of, C₂-C₈An acyl group of (1).

3. The substituted benzyl group-containing phosphate derivative according to claim 1, or an isomer, a pharmaceutically acceptable salt thereof,

nucleoside refers to a variety of Nucleoside analogs, including common furan ring nucleosides, carbocyclic nucleosides and acyclic nucleosides. Such as, but not limited to, groups of the following structures:

4. the substituted benzyl group-containing phosphate derivative according to claim 1, or an isomer, a pharmaceutically acceptable salt thereof,

5. The substituted benzyl group-containing phosphate derivative according to claim 1, or an isomer, a pharmaceutically acceptable salt thereof,

can be used for preparing medicines for treating and/or preventing virus infection such as HIV, HBV, HCV and the like, or can be used for preparing anticancer medicines.