CN101585854B - A new class of acyclic nucleoside phosphonate compounds and their composition, preparation method and use - Google Patents

Abstract

The invention provides a new acyclic nucleoside phosphonate compound as well as a composition, a preparation method and application thereof, in particular an acyclic nucleoside phosphonate compound presented in a general formula (1), an inorganic salt or an organic salt pharmaceutically accepted and a composition containing the compound of the general formula (1). The invention also provides the preparation method of the compound and the application of the compound of the general formula (1) to the treatment of virus infective diseases, in particular virus infective diseases caused by an HBV virus or an HIV virus.

Description

A new class of acyclic nucleoside phosphonate compounds and their composition, preparation method and use

technical field

The invention belongs to the field of pharmacy, relates to the fields of drug synthesis and pharmacology, more specifically, relates to acyclic nucleoside phosphonate compounds containing amino acid groups, their compositions, preparation methods and their use in the preparation of antiviral drugs use.

Background technique

According to the estimates of the World Health Organization, there are currently about 350 million HBV (hepatitis B virus, HBV) carriers in the world, and there are 120 million HBV carriers in China alone. Chronic HBV infection can cause related diseases such as chronic hepatitis B (CHB), liver cirrhosis, and primary hepatocellular carcinoma (HCC). At present, the main drugs for the treatment of hepatitis B are interferon and nucleoside compounds. α-interferon combined with ribavirin not only has limited efficacy but also has many adverse reactions, so nucleoside compounds are the main field of hepatitis B antiviral drug research in recent years [Current Medicinal Chemistry, 2003, 10, 1825; Expert Opinion on Investigatinal Drugs 2003 , 12, 1269; Current Drug Targets-Infections Disorders 2005, 5, 307].

Nucleoside antiviral drugs are divided into nucleoside analogs and acyclic nucleoside phosphonates. Acyclic nucleoside phosphonate analogs have broad-spectrum antiviral activity. This type of compound is characterized by a stable phosphonomethyl ether structure in the structure, and its O-C-P bond connection makes the acyclic nucleoside phosphonate highly effective. biological (enzyme) and chemical stability. Compared with nucleoside analogs, due to the phosphonate moiety in the non-cyclic nucleoside phosphonate structure, it can effectively bypass the initial phosphorylation process catalyzed by virally encoded kinases, so for those that cannot encode thymidine kinase or thymidine kinase Lower levels of virus produced better inhibitory effects. The currently marketed acyclic nucleoside phosphonate antiviral drugs include three drugs, cidofovir, adefovir dipivoxil and tenofovir disoproxil, among which adefovir dipivoxil is the second lamivudine It is the second small-molecule anti-HBV drug on the market, and it has strong activity against lamivudine-resistant mutant hepatitis B virus (YMDD) (Expert Opinion on Pharmacotherapy 2004, 5(11): 2361-7). Tenofovir diisoproxil fumarate has been marketed as an anti-HIV drug, and is currently entering phase III clinical research as an anti-HBV drug. The PMCG dipyrfurate prodrug LB 80380 developed by LG Life Sciences has entered phase II clinical trials [Current Drug Targets-Infectious Disorders 2005, 5, 307].

Acyclic nucleoside phosphonate compounds dissociate into dianion phosphonic acid groups under physiological pH conditions, making it difficult to absorb through the cell membrane, with low bioavailability and dose-dependent nephrotoxicity, which limits their use as For the application of anti-HBV drugs, phosphonate prodrugs must be prepared for clinical use. Therefore, the listed and clinically researched drugs mentioned above are all phosphonate prodrugs.

Moreover, acyclic nucleoside phosphonate drugs generally have the following disadvantages: (1) chemical instability, which is highly sensitive to serum enzyme-mediated hydrolysis, and cannot effectively increase the drug concentration at the site of action [Pharmaceutical Research.1997, 14 (4): 492-496.]; (2) converting one molecule of nucleic acid requires the release of two equivalents of potentially toxic formaldehyde and pivalic acid [Drugs of the Future 2004, 29(2); 163-177].

In order to overcome the above disadvantages, the present invention provides a new class of acyclic nucleoside phosphonate compounds, their synthesis method and application.

Contents of the invention

An object of the present invention is to provide the acyclic nucleoside phosphonate compound represented by the following general formula (I) or its various optical isomers, various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates.

(I)

Another object of the present invention is to provide a pharmaceutical composition containing one or more of the compounds represented by the general formula (I) as the main active ingredient.

Another object of the present invention is to provide acyclic nucleoside phosphonate compounds represented by general formula (I) or various optical isomers, various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrated Preparation methods of substances or solvates.

Another object of the present invention is to provide acyclic nucleoside phosphonate compounds represented by general formula (I) or various optical isomers, various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrated The application of the compound or solvate, and the pharmaceutical composition containing the compound represented by formula (I) as the main active ingredient in the preparation of drugs for treating viral infectious diseases, especially HBV and infectious diseases caused by HBV virus.

The present invention will be specifically described below.

The present invention provides acyclic nucleoside phosphonate compounds represented by the following general formula (I) or various optical isomers, various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thing:

In general formula (I):

R ₁ represents hydrogen, halogen, amino, hydroxyl, straight or branched or cyclic C ₁ -C ₆ alkylamino, straight or branched C ₁ -C ₆ alkoxy, substituted aryloxy or substituted aromatic Sulfuryl;

_R represents hydrogen or amino;

R ₃ represents hydrogen, cyclopropyl, straight or branched C ₁ -C ₆ alkyl, monofluoromethyl or difluoromethyl;

R ₅ represents: hydrogen, aromatic group, or a five-membered or six-membered aromatic heterocyclic group containing 1 to 2 heteroatoms selected from N, O and S;

R ₄ represents hydrogen, straight chain or branched C ₁ -C ₆ alkyl, straight chain or branched C ₁ -C ₆ haloalkyl, benzyl or formula (II),

In formula (II),

R ₆ and R ₇ independently represent hydrogen, benzyl, straight or branched C ₁ -C ₆ alkyl or the side chain of a natural amino acid or an unnatural amino acid, and the configuration of the amino acid can be L-form or D -type;

R ₈ represents hydrogen, linear or branched C ₁ -C ₆ alkyl, trifluoroethyl or benzyl.

In a preferred embodiment of the present invention, for the above-mentioned compound of general formula (I):

R ₁ represents hydrogen, halogen, amino, hydroxyl or linear or branched C ₁ -C ₄ alkylamino;

_R represents hydrogen or amino;

R ₃ represents hydrogen or straight or branched C ₁ -C ₄ alkyl;

_R represents hydrogen;

R ₄ represents straight chain or branched C ₁ -C ₄ alkyl, straight chain or branched C ₁ -C ₄ haloalkyl, benzyl or formula (II),

In formula (II),

R ₆ and R ₇ independently represent hydrogen, benzyl or linear or branched C ₁ -C ₄ alkyl,

R ₈ represents hydrogen or straight or branched C ₁ -C ₄ alkyl.

The compound of the present invention has one or more asymmetric carbon atoms or phosphorus atoms, so the general formula (I) includes single optical body, single enantiomer, single diastereomer, diastereomeric mixture and racemic mixture.

"Pharmaceutically acceptable inorganic or organic salts" specifically include salts formed between compounds of the present invention and acids, and acids suitable for forming salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; The invention compound can be combined with formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, ethanesulfonic acid, benzene Salt formation of organic acids such as sulfonic acid or acidic amino acids such as aspartic acid and glutamic acid; or salts with alkalis, such as sodium salts, potassium salts, calcium salts, aluminum salts and other inorganic base salts, ammonium salts, methylamine Salts, ethylamine salts, ethanolamine salts, etc., or salts formed with basic amino acids such as lysine, arginine, ornithine.

For the C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylamino defined above, the alkyl can be C ₁ -C ₆ straight chain, branched chain Or cycloalkyl, specifically methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclobutyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, Neopentyl, tert-pentyl, 1-methylbutyl, 2-methylpropyl, hexyl, isohexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2- Methylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 3,3-di Methylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1- Methylpropyl or 1-ethyl-2-methylpropyl. Among these groups, alkyl groups having 1 to 4 carbon atoms such as methyl, ethyl, propyl, cyclopropyl, isopropyl, butyl, isobutyl and sec-butyl are preferable.

In the most preferred embodiment of the present invention, the representative compound of the compound represented by general formula (I) of the present invention can be following compound:

Compound No. P configuration R ₃ R ₆ R ₇ R ₈ 1a R h (S) _-CH3 h _CH3 1b S h (S) _-CH3 h _CH3 2a R _CH3 (S) _-CH3 h _CH3 2b S _CH3 (S) _-CH3 h _CH3 3 RS h (S)-CH ₂ CH(CH ₃ ) ₂ h _CH3 4a R _CH3 (S)-CH ₂ CH(CH ₃ ) ₂ h _CH3 4b S _CH3 (S)-CH ₂ CH(CH ₃ ) ₂ h _CH3 5 RS h (S)- ⁱ Pr h _CH3 6 RS _CH3 (S)- ⁱ Pr h _CH3 7 RS h (S)-Bn h _CH3 8 RS _CH3 (S)-Bn h _CH3 9 RS h h h _CH3 10 RS h (S)-CH(CH ₃ )CH ₂ CH ₃ h _CH3 11 RS _CH3 (S)-CH(CH ₃ )CH ₂ CH ₃ h _CH3 12a R h (S)- ⁱ Pr h Et 12b S h (S)- ⁱ Pr h Et

13 RS h (S)- ⁱ Pr h ⁱ Pr 14a R h (R)- ⁱ Pr h Et 14b S h (R)- ⁱ Pr h Et 15 RS h _{CH3 CH3} Et 16 RS h (S)- ⁱ Pr h h

Compound No. P configuration R ₃ R ₄ 17 RS h Bn 18 RS _CH3 Bn 19 RS h Pr 20 RS h (CH ₂ ) ₄ Cl

The preparation method of the compound of general formula (I) of the present invention is specifically described below, but these specific methods do not constitute any limitation to the present invention.

The compounds of general formula (I) of the present invention can be prepared by the following methods, but the conditions of the following methods, such as reactants, solvents, bases, the amount of the compound used, reaction temperature, reaction time required, etc. are not limited to the following explanations. The compound of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in the specification or known in the art. Such a combination can be easily performed by those skilled in the art to which the present invention belongs.

The preparation method of the compound of general formula (I) of the present invention comprises:

method one:

(1) Reaction of N-benzyl amino acid ester with allyl bromide, followed by hydration, debenzylation and other reactions to generate compound (IIIa);

Wherein R ₆ , R ₇ , R ₈ are as defined above;

(2) Compound (IIIa) and 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) or 9-[2-(R)-(phosphonomethoxy)propyl]adenine The phosphonochloride compound of (PMPA) reacts to generate a phosphonate compound (I), and the compound (I) reacts with a corresponding acid in a solvent to generate a salt of the compound (I) as required, and the compound (I) can also be prepared according to a conventional method as required. 1) Make hydrate or solvate. For example, compound (I) is reacted with fumaric acid to obtain fumarate (Ia) of compound (I).

or,

Method Two:

(1) 3-aminopropanol reacts with corresponding aldehyde or 3-chloropropanol reacts with corresponding amine to generate compound (IIIb);

Wherein R ₄ is as defined above, but not formula (II);

(2) Compound (IIIb) and 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) or 9-[2-(R)-(phosphonomethoxy)propyl]adenine The phosphonochloride compound of (PMPA) reacts to generate a phosphonate compound (I); as required, the compound of formula (I) can be prepared into a corresponding pharmaceutically acceptable salt by reacting with a corresponding acid; according to a usual method, the formula ( I) The compound can be prepared into its hydrate or solvate by recrystallization from water or a water-containing solvent. Compound (I) reacts with fumaric acid to obtain the fumarate (Ib) of compound (I)

wherein _R3 is as defined above.

The preparation method of the compound of the present invention is described in more detail below:

For method one, the compound of formula (Ia) of the present invention can be prepared according to the following process:

Process I

a: SOCl ₂ ; b: PhCHO, NaBH ₃ CN, MeOH; c: CH ₂ =CHCH ₂ Br, DIPEA, DMSO; d: 9-BBN, THF; e: Pd/C, H ₂ , MeOH; f: Et ₂ NCHO, (COCl) ₂ , CH ₂ Cl ₂ ; g: Py, TEA, CH ₂ Cl ₂ ; h: AcOH, MeOH; i: HOOCCH=CHCOOH, CH ₃ CN.

Wherein R ₃ , R ₆ , R ₇ and R ₈ are as defined above.

a. Compound (III-1) represents various natural or unnatural α-amino acids. The configuration of the above α-amino acids can be L-type or D-type. In the presence of an acid chloride reagent, R ₈ OH is used as a reaction solvent, React at -30-100°C for 0.5-72 hours to obtain amino acid ester (III-2). The acid chloride reagent can be selected from oxalyl chloride, phosphorus pentachloride, phosphorus oxychloride or thionyl chloride. The optimal reaction conditions are methanol (MeOH), adding thionyl chloride (SOCl ₂ ), reacting at -10°C for 1 hour, and heating to reflux for 24 hours.

b. Compound (III-2) and benzaldehyde (PhCHO), under the action of a reducing agent, react in a protic solvent at -30 to 50°C for 0.5 to 72 hours to obtain the N-benzyl amino acid ester product (III-3 ). The reducing agent can be lithium borohydride, sodium borohydride, potassium borohydride, sodium cyanoborohydride or sodium triacetoxyborohydride. The protic solvent is ethanol, methanol or isopropanol. The optimal reaction condition is to add sodium cyanoborohydride (NaBH ₃ CN) to methanol (MeOH), react at -10°C for 1 hour, and react at room temperature for 24 hours.

c. Compound (III-3) reacts with allyl bromide (CH ₂ =CHCH ₂ Br) in the presence of an organic base in a polar aprotic solvent at -30 to 100°C for 0.5 to 72 hours to form N-Benzyl-N-allylated amino acid ester (III-4). The organic base is triethylamine, N, N'-dicyclohexyl-4-morpholino amidine (DCMC), 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU ), N,N-dimethylaminopyridine (DMAP), N,N-diisopropylethylamine (DIPEA), morpholine, N-methylmorpholine (NMM), pyridine or 2,6-dimethyl base pyridine. The polar aprotic solvent is selected from chloroform, ethyl acetate, tetrahydrofuran, diethyl ether, methyl tert-butyl ether, dimethylformamide, dimethyl sulfoxide or hexamethylphosphoramide. The optimal reaction condition is adding diisopropylethylamine in dimethyl sulfoxide (DMSO) and reacting at 40° C. for 24 hours.

d. Compound (III-4) is reacted in an ether solvent at -30-100° C. for 0.5-72 hours in the presence of an oxidizing agent to generate compound (III-5). The oxidizing agent can be hydrogen peroxide, potassium permanganate, sodium periodate, sodium hypochlorite, chromium trioxide, potassium dichromate, DMSO or 9-boronbicyclo[3,3,1]nonane (9-BBN). The ether solvent can be selected from tetrahydrofuran, diethyl ether, methyl tert-butyl ether, 1,6-dioxane or ethylene glycol dimethyl ether. The best reaction conditions are adding 9-BBN in tetrahydrofuran (THF), reacting at -10°C for 1 hour, and reacting at room temperature for 24 hours.

e. Compound (III-5) is catalytically hydrogenated in a protic solvent under the action of a metal-containing catalyst at -30-50° C. for 0.5-72 hours to generate the debenzylated product (IIIa). A suitable metal-containing catalyst is a palladium, platinum or nickel catalyst, and a suitable protic solvent is ethanol, methanol, isopropanol, a mixed solvent of alcohols and acetic acid or a mixed solvent of alcohols and sulfuric acid. The optimal reaction condition is hydrogenation reaction in 10% palladium carbon (Pd/C) ethanol or methanol at room temperature for 24 hours.

f. PMEA and PMPA were reacted with acid chloride reagent and Et ₂ NCHO in a polar aprotic solvent at -30-100°C for 0.5-72 hours to obtain phosphonyl chloride compound (I-1), and then activated with an organic base spare. The acid chloride reagent can be selected from oxalyl chloride, phosphorus pentachloride, phosphorus oxychloride, phosphorus oxychloride or thionyl chloride. Polar aprotic solvents can be selected from dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, tetrahydrofuran, ether, methyl tert-butyl ether, dioxane, dimethylformamide or dimethylformamide base sulfoxide. The organic base is selected from triethylamine, N, N'-dicyclohexyl-4-morpholino amidine (DCMC), 1,8-diazabicyclo[5,4,0]undecane-7-ene (DBU ), N,N-dimethylaminopyridine (DMAP), N,N-diisopropylethylamine (DIEA), morpholine, N-methylmorpholine (NMM), pyridine or 2,6-dimethyl base pyridine. The optimal reaction condition is to add oxalyl chloride ((COCl) ₂ ) and Et ₂ NCHO in dichloromethane (CH ₂ Cl ₂ ) at room temperature, and heat under reflux for 3 hours to generate the corresponding phosphonyl chloride (I-1), Then add pyridine to activate it for later use.

g. Compound (IIIa) and the above prepared compound (I-1) solution [Bio. & Med. Chem. 2003, 11, 5461], in a polar aprotic solvent, under the action of an organic base, at -80 Reaction at ~25°C for 0.5 to 72 hours produces the corresponding phosphonate (I-2). The organic base is triethylamine, N, N'-dicyclohexyl-4-morpholino amidine (DCMC), 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU ), N,N-dimethylaminopyridine (DMAP), N,N-diisopropylethylamine (DIEA), morpholine, N-methylmorpholine (NMM), pyridine or 2,6-dimethyl base pyridine. Dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, tetrahydrofuran, diethyl ether, methyl tert-butyl ether, 1,6-dioxane, dimethylformamide are selected as polar aprotic solvents or dimethyl sulfoxide. The optimum reaction condition is to react in dichloromethane (CH ₂ Cl ₂ ) with triethylamine (TEA) as a base at -70 to -50°C for 1 to 2 hours.

h. Add acid to phosphonate (I-2) in a protic solvent, react at 25-100° C. for 0.5-72 hours, and generate phosphonate (I-3), the product of removal of the imine protecting group. The acid can be selected from sulfuric acid, hydrochloric acid, glacial acetic acid, trifluoroacetic acid, methanesulfonic acid or phosphoric acid. Suitable protic solvents are ethanol, methanol or isopropanol. The optimal reaction condition is methanol (MeOH), adding glacial acetic acid (AcOH) and refluxing for 2 hours.

i. The phosphonate (I-3) is reacted with the corresponding acid in a polar aprotic solvent at 25-100° C. for 0.5-72 hours to form the salt (Ia). The polar aprotic solvent can be selected from dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, tetrahydrofuran, acetonitrile, diethyl ether, methyl tert-butyl ether or dioxane. The acid can be inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid; formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid , tartaric acid, citric acid, picric acid, organic acids such as methanesulfonic acid, ethanesulfonic acid or benzenesulfonic acid, or acidic amino acids such as aspartic acid and glutamic acid; preferably react with fumaric acid (HOOCCH=CHCOOH) in acetonitrile (CH ₃ CN), reflux for 2 hours.

According to needs, the compound of formula (I-3) can be prepared into corresponding pharmaceutically acceptable salts, hydrates and solvates according to conventional methods.

For method two, the compound of formula (Ib) of the present invention can be prepared according to the following process:

Process II

a: CH(OMe) ₃ , NaBH ₄ , MeOH; b: Et ₂ NCHO, (COCl) ₂ , CH ₂ Cl ₂ ; c: Py, TEA, CH ₂ Cl ₂ ; d: AcOH, MeOH; e: HOOCCH= CHCOOH, _CH3CN .

Wherein, R ₃ and R ₄ are as defined above, but R ₄ is not in formula (II).

A, 3-aminopropanol reacts with corresponding aldehyde or 3-chloropropanol reacts with corresponding amine to generate compound (IIIb);

b. Reaction of PMEA and PMPA with acid chloride reagent and Et ₂ NCHO in a polar aprotic solvent at -30-100°C for 0.5-72 hours to obtain phosphonyl chloride compound (I-1), which is then activated with an organic base spare. The acid chloride reagent can be selected from oxalyl chloride, phosphorus pentachloride, phosphorus oxychloride, phosphorus oxychloride or thionyl chloride. Polar aprotic solvents can be selected from dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, tetrahydrofuran, ether, methyl tert-butyl ether, dioxane, dimethylformamide or dimethylformamide base sulfoxide. The organic base is triethylamine, N, N'-dicyclohexyl-4-morpholino amidine (DCMC), 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU ), N,N-dimethylaminopyridine (DMAP), N,N-diisopropylethylamine (DIEA), morpholine, N-methylmorpholine (NMM), pyridine or 2,6-dimethyl base pyridine. The optimal reaction condition is to add oxalyl chloride ((COCl) ₂ ) and Et ₂ NCHO in dichloromethane (CH ₂ Cl ₂ ) at room temperature, and heat under reflux for 3 hours to generate the corresponding phosphonyl chloride (I-1), Then add pyridine to activate it for later use.

c, compound (I-1) [Bio. & Med.Chem.2003, 11, 5461] and compound (IIIb) [J.Org.Chem.1998, 63: 6] in polar aprotic solvent, organic Under the action of alkali, react at -80-25°C for 0.5-72 hours to generate the corresponding phosphonate (Ib-1). The organic base is triethylamine, N, N'-dicyclohexyl-4-morpholino amidine (DCMC), 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU ), N,N-dimethylaminopyridine (DMAP), N,N-diisopropylethylamine (DIEA), morpholine, N-methylmorpholine (NMM), pyridine or 2,6-dimethyl base pyridine. Dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, tetrahydrofuran, diethyl ether, methyl tert-butyl ether, 1,6-dioxane, dimethylformamide are selected as polar aprotic solvents or dimethyl sulfoxide. The optimum reaction condition is to react in dichloromethane (CH ₂ Cl ₂ ) with triethylamine (TEA) as a base at -70 to -50°C for 1 to 2 hours.

d. Add acid to phosphonate (Ib-1) in a protic solvent and react at 25-100° C. for 0.5-72 hours to generate phosphonate (Ib-2) which is the product of removal of the imine protecting group. The acid can be selected from sulfuric acid, hydrochloric acid, glacial acetic acid, trifluoroacetic acid, methanesulfonic acid or phosphoric acid. Suitable protic solvents are ethanol, methanol or isopropanol. The optimal reaction condition is methanol (MeOH), adding glacial acetic acid (AcOH) and refluxing for 2 hours.

e. The phosphonate (Ib-2) is reacted with the corresponding acid in a polar aprotic solvent at 25-100° C. for 0.5-72 hours to form the salt (Ib). The polar aprotic solvent can be selected from dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, tetrahydrofuran, acetonitrile, diethyl ether, methyl tert-butyl ether or dioxane. The acid can be inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid; formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid , tartaric acid, citric acid, picric acid, organic acids such as methanesulfonic acid, ethanesulfonic acid or benzenesulfonic acid, or acidic amino acids such as aspartic acid and glutamic acid; preferably react with fumaric acid (HOOCCH=CHCOOH) in acetonitrile (CH ₃ CN), reflux for 2 hours.

The compound of formula (Ib-2) can be prepared into corresponding pharmaceutically acceptable salts, hydrates and solvates as needed.

The present invention provides an acyclic nucleoside phosphonate compound represented by formula (I) or its various optical isomers, various crystal forms, pharmaceutically acceptable salts, hydrates or solvates One or more pharmaceutical compositions as the main active ingredient, which may further contain pharmaceutically acceptable adjuvants, such as excipients, carriers and the like.

The acyclic nucleoside phosphonate compound represented by formula (I) provided by the present invention or its various optical isomers, various crystal forms, pharmaceutically acceptable salts, hydrates or solvates have antiviral activity, so it can be used in the preparation of medicines for treating viral infectious diseases, especially infectious diseases caused by hepatitis B virus HBV or HIV virus.

The acyclic nucleoside phosphonate compounds containing amino acid groups claimed in the present invention and their pharmaceutically acceptable salts have the function of treating viral infectious diseases, especially infectious diseases caused by HBV and HIV viruses.

The acyclic nucleoside compounds containing amino acid groups claimed in the present invention and pharmaceutically acceptable salts thereof have similar or stronger therapeutic effects on viral infections than the nucleoside small molecule anti-HBV drug adefovir in the prior art Disease activity, so these compounds can be used to prepare novel drugs for treating viral infectious diseases, especially infectious diseases caused by HBV and HIV viruses.

Detailed ways

The present invention is explained more specifically in the following Preparations and Examples. It should be understood, however, that these Preparations and Examples are given to illustrate the invention and not to limit the scope of the invention in any way. In all the preparation examples and examples, the melting point was measured with a MEL-TEMP melting point apparatus, and the thermometer was not calibrated; ¹ H-NMR was recorded with a Varian Mercury 400 nuclear magnetic resonance instrument, and the chemical shift was expressed in δ (ppm); the silica gel used for separation was not specified. 200-300 mesh. The ratio of the eluent is volume ratio.

Preparation of compounds of formula (IIIa)

The preparation of embodiment 1L-valine methyl ester hydrochloride

At -10°C, valine (10g, 85.5mmol) was dissolved in methanol (120ml), and thionyl chloride (22.4ml, 307mmol) was slowly added dropwise. Hour. After cooling down to room temperature, the methanol was distilled off under reduced pressure, and the residue was recrystallized from a methanol-ether mixture to obtain the title product (10 g, 70%). ¹ H NMR (CDCl ₃ ): 8.8 (br, 3H), 4.0 (t, 1H), 3.8 (s, 3H), 2.5 (m, 1H), 1.1 (d, 6H).

The compounds listed in the following table can be prepared from various natural amino acids and unnatural amino acids according to the same steps as in Example 1.

Example 2-10

Example R ₆ R ₇ R ₈ ¹ H NMR (CDCl ₃ , ppm) Yield 2 (S)- ⁱ Pr h Et 8.7(br, 3H), 5.0(s, 1H), 3.95(q, 4H), 2.4(m, 1H), 1.3(t, 3H), 1.1(d, 6H) 90% 3 (S)- ⁱ Pr h ⁱ Pr 8.8-8.6(br, 3H), 5.0(m, 1H), 3.8(m, 1H), 2.1(m, 1H), 1.4(d, 6H), 1.0(d, 6H) 98%

4 (R)- ⁱ Pr h Et 8.7(br, 3H), 5.0(s, 1H), 3.95(q, 4H), 2.4(m, 1H), 1.3(t, 3H), 1.1(d, 6H) 88% 5 (S) _-CH3 h _CH3 8.8(br, 3H), 4.0(m, 1H), 3.8(s, 3H), 0.93(d, 3H) 97% 6 (S)-CH ₂ CH(CH ₃ ) ₂ h _CH3 8.8(br, 3H), 4.0(q, 1H), 3.8(s, 3H), 1.8(q, 1H), 1.5(m, 2H), 0.95(d, 3H), 1.1(d, 3H) 88% 7 (S)-Bn h _CH3 8.8(br, 3H), 7.2-7.3(m, 5H), 4.0(s, 2H), 4.0(m, 1H), 3.8(s, 3H) 90% 8 h h _CH3 8.8(br, 3H), 3.8(s, 3H), 3.4(s, 2H) 76% 9 (S)-CH(CH ₃ )CH ₂ CH ₃ h _CH3 8.6(br, 3H), 4.05(q, 1H), 3.8(s, 3H), 2.2(m, 1H), 1.5(m, 2H), 1.05(d, 3H), 0.9(t, 3 96%

H) 10 _{CH3 CH3} Et 8.86(br, 3H), 4.16(q, 2H), 1.47(s, 6H), 1.2(t, 3H) 100%

The preparation of embodiment 11N-benzyl-L-valine methyl ester

At 0°C, L-valine methyl ester hydrochloride (7g, 41.8mmol) was dissolved in methanol (150ml), then sodium cyanoborohydride (2.63g, 41.8mmol) was added, and then benzene was added dropwise Formaldehyde (4.43g, 41.8mmol) was raised to room temperature and reacted overnight. Cool to 0°C, acidify the reaction solution with concentrated hydrochloric acid until pH = 1, stir at the same temperature for 3.5 h, distill under reduced pressure, add water, adjust pH = 9 with saturated sodium carbonate solution, extract twice with ethyl acetate, wash with water, After washing with saturated sodium chloride, it was dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was subjected to column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain the title product (6 g, 64.9%). ¹ H NMR (CDCl ₃ ): 7.2-7.4 (m, 5H), 3.82, 3.6 (dd, 2H), 3.7 (s, 3H), 3.0 (d, 1H), 1.9 (m, 1H), 1.8 (s , 1H), 0.95 (m, 6H).

The compounds listed in the following table can be prepared from the compounds obtained in Examples 2-10 as raw materials according to the same steps as in Example 11.

Example 12-20

Example R ₆ R ₇ R ₈ ¹ H NMR (CDCl ₃ , ppm) Yield 12 (S)- ⁱ Pr h Et 7.3(m, 5H), 4.2(m, 2H), 3.82, 3.6(dd, 2H), 3.0(d, 1H), 1.95(m, 1H), 1.9(s, 1H), 1.3(t, 3H) , 0.95(dd,6H). 53.6% 13 (S)- ⁱ Pr h ⁱ Pr 7.3(m, 5H), 5.1(m, 1H), 3.8, 3.6(dd, 2 55.7%

H), 3.0(d, 1H), 1.95(m, 1H), 1.9(s, 1H), 1.4(m, 6H), 1.0(m, 6H). 14 (R)- ⁱ Pr h Et 7.3(m, 5H), 4.2(m, 2H), 3.82, 3.6(dd, 2H), 3.0(d, 1H), 1.95(m, 1H), 1.9(s, 1H), 1.3(t, 3H) , 0.95(dd,6H). 54% 15 (S) _-CH3 h _CH3 7.3(m, 5H), 3.82, 3.6(dd, 2H), 3.85(s, 3H), 3.4(d, 1H), 2.0(m, 1H), 1.15(d, 3H). 53.8% 16 (S)-CH ₂ CH(CH ₃ ) ₂ h _CH3 7.3(m, 5H), 3.82, 3.6(dd, 2H), 3.77(s, 3H), 3.4(t, 1H), 1.85(m, 1H), 1.8(q, 1H), 1.5(m, 2H) , 0.95(d,3H), 0.93(d,3H). 57.9% 17 (S)-Bn h _CH3 7.1-7.3(m, 10H), 3.9(d, 1H), 3.63(m, 5H), 3.60(t, 1H), 3.0(d, 2H), 1.9(br, 1H). 67% 18 h h _CH3 7.3(m, 5H), 3.9(s, 2H), 3.7(s, 3H), 3.4(s, 2H). 59.3% 19 (S)-CH(CH ₃ )CH ₂ CH ₃ h _CH3 7.2(m, 5H), 3.87, 3.7(dd, 2H), 3.8(s, 3H), 3.1(d, 1H), 1.8(m, 1H), 1.6(m, 2H), 1.2(m, 1H) , 0.9(m, 6H). 48.9% 20 _{CH3 CH3} Et 7.2(m, 5H), 3.87, 3.7(dd, 2H), 4.16(q, 2H), 1.47(s, 6H), 1.2(t, 3H). 55%

The preparation of embodiment 21N-benzyl-N-allyl-L-valine methyl ester

At room temperature, N-benzyl-L-valine methyl ester (5g, 22.6mmol) and allyl bromide (3.93ml, 45.2mmol) were dissolved in dimethyl sulfoxide (20ml), added dropwise Diisopropylethylamine (7.46ml, 4.52mmol), react overnight at 40°C, dilute with 10 times water, extract once with ethyl acetate, wash the organic layer with water, then wash with saturated brine, and dry over anhydrous magnesium sulfate , filtered, concentrated under reduced pressure, and the residue was subjected to column chromatography (petroleum ether: ethyl acetate = 20:1) to obtain the title product (3.6 g, 61%). ¹ H NMR (CDCl ₃ ): 7.2-7.4 (m, 5H), 5.8 (m, 1H), 5.2-5.1 (dd, 2H), 4.0, 3.3 (dd, 2H), 3.8 (s, 3H), 3.4 (d,2H), 2.9(d,1H), 2.1(m,1H), 1.0(d,3H), 0.8(d,3H).

The compounds listed in the following table can be prepared from the compounds obtained in Examples 12-20 as raw materials according to the same steps as in Example 21.

Examples 22-30

Example R ₆ R ₇ R ₈ ¹ H NMR (CDCl ₃ , ppm) Yield twenty two (S)- ⁱ Pr h Et 7.3(m, 5H), 5.8(m, 1H), 5.3(dd, 2H), 4.2(m, 2H), 4.0, 3.4(dd, 2H), 3.3(d, 1H), 2.8(m, 2H) , 2.2(m, 1H), 1.3(t, 3H), 1.0, 0.8(dd, 6H). 43.8% twenty three (S)- ⁱ Pr h ⁱ Pr 7.3(m, 5H), 5.8(m, 1H), 5.3(dd, 2H), 5.1(m, 1H), 4#0, 3.8(dd, 2H), 3.4(d, 1H), 2.9(dd, 2H), 2.05(m, 1H), 1.3(dd, 6H), 1.0(m, 3H), 0.8(m, 3H). 58.8% twenty four (R)- ⁱ Pr h Et 7.3(m, 5H), 5.8(m, 1H), 5.3(dd, 2H), 4.2(m, 2H), 4.0, 3.4(dd, 2H), 3.3(d, 1H), 2.8(m, 2H) , 2.2(m, 1H), 1.3(t, 3H), 1.0, 0.8(dd, 6H). 44% 25 (S) _-CH3 h _CH3 7.3(m, 5H), 5.8(m, 1H), 5.05-5.3(dd, 2H), 3.82, 3.6(dd, 2H), 3.8(q, 1H), 3.78(s, 3H), 3.05-3.4( m, 2H), 1.3(d, 3H). 72.6% 26 (S)-CH ₂ CH(CH ₃ ) ₂ h _CH3 7.2(m, 5H), 5.8(m, 1H), 5.2(m, 2H), 4.0, 3.5(dd, 2H), 3.77(s, 3H), 3.4(t, 1H), 3.4, 3.05(m, 2H), 1.6(m, 1H), 1.3(m, 2H), 0.95(d, 3H), 0.8(d, 3H). 96.4%

27 (S)-Bn h _CH3 7.1-7.3(m, 10H), 5.8(m, 1H), 5.05-5.3(dd, 2H), 4.0, 3.6(dd, 2H), 3.7(s, 3H), 3.7, 3.4(m, 2H), 3.05(m, 2H), 2.9(m, 1H). 86.9% 28 h h _CH3 7.3(m, 5H), 5.8(m, 1H), 5.2(m, 2H), 3.8(s, 2H), 3.7(s, 3H), 3.4(s, 2H), 3.3(d, 2H). 59.5% 29 (S)-CH(CH ₃ )CH ₂ CH ₃ h _CH3 7.3(m, 5H), 5.8(m, 1H), 5.1(m, 2H), 4.0, 3.05(dd, 2H), 3.8(s, 3H), 3.4(m, 1H), 3.0, 2.9(d, 2H), 1.9(m, 2H), 1.2(m, 1H), 0.9(m, 6H). 71.3% 30 _{CH3 CH3} Et 7.2(m, 5H), 5.8(m, 1H), 5.1(m, 2H), 3.87, 3.7(dd, 2H), 4.16(q, 2H), 3.0, 2.9(d, 2H), 1.47(s, 6H), 1.2(t, 3H). 70%

The preparation of embodiment 31N-benzyl-N-(3-hydroxypropyl)-L-valine methyl ester

Under the protection of _N2 , N-benzyl-N-allyl-L-valine methyl ester (2.6g, 10mmol) was dissolved in anhydrous tetrahydrofuran (10ml), and the solution was added dropwise and cooled with ice. 9-boronbicyclo[3,3,1]nonane (30ml, 15mmol) tetrahydrofuran solution, stirred at room temperature for 4.5h, the reaction solution was cooled to 0°C, added water (8ml), stirred for 15min, and then added 20% hydrogen Sodium oxide (6ml), 30% hydrogen peroxide (5ml), the reaction solution was stirred at 0°C for 1.5h, neutralized with 4N hydrochloric acid to pH=7, concentrated under reduced pressure, added water, extracted 3 times with ethyl acetate, anhydrous sulfuric acid It was dried over magnesium, filtered, concentrated under reduced pressure, and the residue was chromatographed (petroleum ether: ethyl acetate = 2:1) to obtain the title product (2.5 g, 90%). ¹ H NMR (CDCl ₃ ): 7.2-7.4 (m, 5H), 3.82, 3.6 (dd, 2H), 3.8 (t, 2H), 3.75 (s, 3H), 3.05 (d, 1H), 3.0, 2.6 (m, 2H), 1.98(m, 1H), 1.63(m, 2H), 0.98(m, 6H).

The compounds listed in the following table can be prepared from the compounds obtained in Examples 22-30 by the same steps as in Example 31.

Examples 32-40

Example R ₆ R ₇ R ₈ ¹ H NMR (CDCl ₃ , ppm) Yield 32 (S)- ⁱ Pr h Et 7.2-7.4(m, 5H), 3.82, 3.6(dd, 2H), 3.8(t, 2H), 4.2(s, 2H), 3.05(d, 1H), 3.0, 2.6(m, 2H), 1.98( m, 1H), 1.63(m, 2H), 1.3(t, 3H), 0.98(m, 6H). 93.9% 33 (S)- ⁱ Pr h ⁱ Pr 7.2-7.4(m, 5H), 3.82, 3.6(dd, 2H), 5.0(m, 1H), 3.8(t, 2H), 3.05(d, 1H), 3.0, 2.6(m, 2H), 1.95( m, 1H), 1.7(m, 2H), 1.3(m, 6H), 0.98(m, 6H). 81.6%

34 (R)- ⁱ Pr h Et 7.2-7.4(m, 5H), 3.82, 3.6(dd, 2H), 3.8(t, 2H), 4.2(s, 2H), 3.05(d, 1H), 3.0, 2.6(m, 2H), 1.98( m, 1H), 1.63(m, 2H), 1.3(t, 3H), 0.98(m, 6H). 92% 35 (S) _-CH3 h _CH3 7.3(m, 5H), 4.3(s, 1H), 3.6-3.8(m, 8H), 2.8(m, 2H), 1.75(m, 2H), 1.3(d, 3H). 41.6% 36 (S)-CH ₂ CH(CH ₃ ) ₂ h _CH3 7.2-7.4(m, 5H), 4.05, 3.6(dd, 2H), 3.8(t, 2H), 3.85(s, 3H), 3.05(d, 1H), 3.0, 2.6(m, 2H), 1.7( m, 2H), 1.5(m, 2H), 1.04(m, 1H), 0.98(m, 6H). 83.4% 37 (S)-Bn h _CH3 7.4(m, 8H), 7.1(m, 2H), 3.9, 3.7(dd, 2H), 3.7(s, 3H), 3.6(t, 2H), 3.2, 2.7(m, 2H), 2.9(m, 2H), 1.7(m, 2H). 99% 38 h h _CH3 7.3(m, 5H), 3.8(t, 2H), 3.7(m, 5H), 3.3(s, 2H), 3.8(t, 2H), 1.8(m, 2H). 38.5% 39 (S)-CH(CH ₃ )CH ₂ CH ₃ h _CH3 7.3(m, 5H), 4.05, 3.6(dd, 2H), 3.85(s, 3H), 3.3(m, 2H), 2.9, 2.5(m, 2H), 1.8(m, 2H), 1.6(m, 2H), 1.04(m, 1H), 0.95(m, 6H). 83.6%

40 _{CH3 CH3} Et 7.2(m, 5H), 4.16(q, 2H), 3.87, 3.7(dd, 2H), 3.6(t, 2H), 3.0, 2.9(d, 2H), 1.6(m, 2H), 1.47(s, 6H), 1.2(t, 3H). 50%

The preparation of embodiment 41N-(3-hydroxypropyl)-L-valine methyl ester

N-benzyl-N-(3-hydroxypropyl)-L-valine methyl ester (2.8g, 10mmol) and 10% Pd/C (280mg) were added to absolute ethanol (20ml) and catalyzed Hydrogenation reaction 2.5h. Concentration under reduced pressure, column chromatography of the residue (dichloromethane:methanol=30:1) gave the title product (1.6 g, 84.6%). ¹ HNMR (CDCl ₃ ): 3.8(t, 2H), 3.75(s, 3H), 3.05(d, 1H), 3.0, 2.6(m, 2H), 1.98(m, 1H), 1.75(m, 2H) , 0.98 (m, 6H).

The compounds listed in the following table can be prepared from the compounds obtained in Examples 32-40 by the same steps as in Example 41.

Examples 42-50

Example R ₆ R ₇ R ₈ ¹ H NMR (CDCl ₃ , ppm) Yield 42 (S)- ⁱ Pr h Et 3.8(t, 2H), 3.75(s, 3H), 3.05(d, 1H), 2.9, 2.6(m, 2H), 1.98(m, 1H), 1.75(t, 2H), 1.3(t, 3H) , 0.95(m, 6H). 91% 43 (S)- ⁱ Pr h ⁱ Pr 5.0(m, 1H), 3.8(t, 2H), 3.05(d, 1H), 3.0, 2.6(m, 2H), 1.95(m, 1H), 1.7(m, 2H), 1.3(m, 6H) , 0.98(m, 6H). 89% 44 (R)- ⁱ Pr h Et 3.8(t, 2H), 3.75(s, 3H), 3.05(d, 1H), 2.9, 2.6(m, 2H), 1.98(m, 1H), 1.75(t, 2H), 1.3(t, 3H) , 0.95(m, 6H). 90% 45 (S) _-CH3 h _CH3 3.8(t, 2H), 3.7(s, 3H), 3.2(m, 1H), 2.8, 2.6(m, 2H), 1.8(m, 2H), 1.2(d, 3H). 88% 46 (S)-CH ₂ CH(CH ₃ ) ₂ h _CH3 3.8(t, 2H), 3.75(s, 3H), 3.05(d, 1H), 3.0, 2.6(m, 2H), 1.7(m, 2H), 1.5(m, 2H), 1.04(m, 1H) , 0.98(m, 6H). 84%

47 (S)-Bn h _CH3 7.2(m, 5H), 3.7(t, 2H), 3.65(s, 3H), 3.58(m, 2H), 2.8-3.0(m, 3H), 2.6(m, 1H), 1.6(m, 2H) . 90% 48 h h _CH3 3.8(t, 2H), 3.77(s, 3H), 3.4(s, 2H), 2.8(t, 2H), 1.7(t, 2H). 88% 49 (S)-CH(CH ₃ )CH ₂ CH ₃ h _CH3 3.85(s, 3H), 3.8(t, 2H), 3.05(d, 1H), 2.98, 2.6(m, 2H), 1.7(m, 3H), 1.5(m, 1H), 1.4(m, 1H) , 0.95(m, 6H). 89% 50 _{CH3 CH3} Et 4.16(q, 2H), 3.6(t, 2H), 3.0, 2.9(d, 2H), 1.6(m, 2H), 1.47(s, 6H), 1.2(t, 3H). 90%

Preparation of compounds of formula (IIIb)

The preparation of embodiment 51N-benzyl-3-aminopropanol

Benzaldehyde (10g, 94.2mmol) and trimethyl orthoformate (15.5ml, 141mmol) were dissolved in methanol (300ml), and 3-aminopropanol (7.2ml, 94.2mmol) was added at room temperature, stirred at room temperature for 5h, Cooled with ice to 0°C, sodium borohydride (3.6g, 94.2mmol) was added in two batches, when no gas was released, concentrated under reduced pressure, added water (300ml), extracted twice with ethyl acetate, then washed with saturated salt Washed with water, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain the title product (14.82 g, 95.3%). ¹ H NMR (CDCl ₃ ): 7.3 (m, 5H), 3.8 (dd, 2H), 3.0 (s, 2H), 2.9 (t, 2H), 1.63 (t, 2H).

The preparation of embodiment 52N-propyl group-3-aminopropanol

0°C, add n-propylamine (16.4ml, 0.2mol) dropwise to 3-chloropropanol (4.18ml, 0.05mol), react at room temperature for 2-3d, then reflux for 3-5h, wash with water (50ml), anhydrous Dry over magnesium sulfate, filter, concentrate to dryness under reduced pressure, and distill under reduced pressure to obtain the title product (3.427g, 58.6%). ¹ H NMR (CDCl ₃ ): 3.8(t, 2H), 2.9(t, 2H), 2.6(t, 2H), 1.7(m, 2H), 1.5(m, 2H), 0.9(t, 3H).

Example 539-{2-[3-(R)-[1-(S)-methoxycarbonylethyl]-2-oxa-1,3,2-oxazaphosphorylhexyl-2- Methyleneoxy]-Eth-1-yl}adenine fumarate (compound 1a); 9-{2-[3-(S)-[1-(S)-methoxycarbonylethyl]- Preparation of 2-oxa-1,3,2-oxazaphosphorylhexyl-2-methyleneoxy]-eth-1-yl}adenine fumarate (compound 1b)

Under the protection of _N2 , 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) (273mg, 1mmol) was suspended in dichloromethane (20ml), and N,N-diethyl Formamide (0.121ml, 1.08mmol), oxalyl chloride (0.305ml, 3.5mmol) was refluxed for 3h, cooled to room temperature, concentrated under reduced pressure, then added dichloromethane (20ml), concentrated under reduced pressure again, and then added dichloromethane (20ml), cooled to 0°C with ice, added pyridine (0.162ml, 2mmol), and the phosphonyl chloride solution was cooled for later use.

Dissolve N-(3-hydroxypropyl)-L-alanine methyl ester (160mg, 1mmol), triethylamine (0.878ml, 6.3mmol) in dichloromethane (20ml), and cool the reaction solution to -78 ℃. Add the prepared phosphorus oxychloride solution dropwise to the reaction solution, the internal temperature shall not be higher than -50°C, after the drop is completed, slowly rise to room temperature, wash twice with water (20ml), dry over anhydrous magnesium sulfate, and concentrate under reduced pressure , Column chromatography (dichloromethane:methanol=20:1) of the residue gave the phosphonate compound (210 mg).

The phosphonate compound was dissolved in methanol (30ml), added glacial acetic acid (0.25ml, 4.38mmol), heated to reflux for 2h, concentrated under reduced pressure, and the residue was eluted by column chromatography (dichloromethane:methanol=20:1 ~15:1) afforded two diastereomers 1a (100 mg) and 1b (50 mg), respectively.

The obtained two diastereoisomers were respectively dissolved in acetonitrile (20ml), 1 times the amount of fumaric acid was added, refluxed for 2h, and left at room temperature for two hours, a white solid was precipitated, filtered to obtain the title compound 1a ( 100 mg, 29.1%) and 1b (50 mg, 14.5%). Compound 1a ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.8 (s, 2H), 4.6 (m, 2H), 4.4 (m, 3H), 4.0 (m, 4H), 3.6 (s, 3H), 3.1, 2.9 (m, 2H), 1.9, 1.7 (m, 2H), 1.4 (d, 3H). MS (ESI) m/z (%): 399 (MH ⁺ , 100). Compound 1b ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.8 (s, 2H), 4.6 (m, 2H), 4.4 (m, 3H), 4.0 (m, 4H), 3.65 (s, 3H), 3.1, 2.9 (m, 2H), 1.9, 1.7 (m, 2H), 1.15 (d, 3H). MS (ESI) m/z (%): 399 (MH ⁺ , 100).

Example 549-{2-(R)-[3-(R)-[1-(S)-methoxycarbonylethyl]-2-oxa-1,3,2-oxazaphosphorylhexane yl-2-methyleneoxy]-prop-1-yl}adenine fumarate (compound 2a);

9-{2-(R)-[3-(S)-[1-(S)-methoxycarbonylethyl]-2-oxa-1,3,2-oxazaphosphorylhexyl- Preparation of 2-methyleneoxy]-prop-1-yl}adenine fumarate (compound 2b)

According to the operation of Example 53, 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) (287mg, 1mmol) and the compound (161mg, 1mmol) generated in the above-mentioned Example 45 were The starting materials were reacted to yield the title products 2a (30 mg, 5.6%) and 2b (45 mg, 8.4%). Compound 2a ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.8 (s, 2H), 4.4-4.2 (m, 4H), 4.0 (m, 2H), 3.6-4.0 (m, 2H), 3.62 (s, 3H), 3.2, 2.8 (m, 2H), 2.0, 1.7 (m, 2H), 1.4 (d, 3H), 1.22 (d, 3H). MS (ESI) m/z (%): 413 (MH ⁺ , 100). Compound 2b ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.8 (s, 2H), 4.4-4.2 (m, 4H), 4.0 (m, 2H), 3.6-4.0 (m, 2H), 3.66 (s, 3H), 3.2, 2.8 (m, 2H), 2.0, 1.7 (m, 2H), 1.38 (d, 3H), 1.25 (d, 3H). MS (ESI) m/z (%): 413 (MH ⁺ , 100)

Example 559-{2-[3-[1-(S)-methoxycarbonyl-3-methylbutyl]-2-oxa-1,3,2-oxazaphosphorylhexyl-2 Preparation of -Methyleneoxy]-Eth-1-yl}adenine fumarate (compound 3)

According to the operation of Example 53, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) (273mg, 1mmol) and the compound (203mg, 1mmol) generated in the above-mentioned Example 46 were reacted as raw materials to generate the title Product (170 mg, 30.5%). ¹ H NMR (CD ₃ OD): 8.05 (d, 2H), 6.6 (s, 2H), 4.4 (m, 2H), 4.1 (m, 3H), 3.8 (m, 4H), 3.5 (d, 3H) , 3.0-2.8 (m, 2H), 1.7-1.3 (m, 5H), 0.9 (t, 3H), 0.8 (d, 3H). MS (ESI) m/z (%): 441 (MH ⁺ , 100 )

Example 569-{2-(R)-[3-(R)-[1-(S)-methoxycarbonyl-3-methylbutyl]-2-oxa-1,3,2-oxaza Heterophosphorylhexyl-2-methyleneoxy]-propan-1-yl}adenine fumarate (compound 4a);

9-{2-(R)-[3-(S)-[1-(S)-methoxycarbonyl-3-methylbutyl]-2-oxa-1,3,2-oxazaphosphine Preparation of acylhexyl-2-methyleneoxy]-propan-1-yl}adenine fumarate (compound 4b)

According to the operation of Example 53, 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) (430mg, 1.5mmol) and the compound generated in the above-mentioned Example 46 (305mg, 1.5mmol ) as starting material to yield the title products 4a (120 mg, 14%) and 4b (150 mg, 17.5%). Compound 4a ¹ H NMR (CD ₃ OD): 8.05 (d, 2H), 6.75 (s, 2H), 4.4-4.4 (m, 4H), 4.2-4.0 (m, 2H), 3.9-3.8 (m, 2H ), 3.62(s, 3H), 3.1-3.2(m, 2H), 2.0-2.1(m, 1H), 1.6-1.8(m, 4H), 1.2(d, 3H), 1.98(dd, 6H). MS (ESI) m/z (%): 455 (MH ⁺ , 100). Compound 4b ¹ H NMR (CD ₃ OD): 8.05 (d, 2H), 6.75 (s, 2H), 4.4-4.4 (m, 4H), 4.2-4.0 (m, 2H), 3.9-3.8 (m, 2H ), 3.66(s, 3H), 3.1-3.2(m, 2H), 2.0-2.1(m, 1H), 1.6-1.8(m, 4H), 1.2(d, 3H), 1.98(dd, 6H). MS (ESI) m/z (%): 455 (MH ⁺ , 100).

Example 579-{2-[3-[1-(S)-methoxycarbonyl-2-methylpropyl]-2-oxa-1,3,2-oxazaphosphorylhexyl-2 Preparation of -methyleneoxy]-eth-1-yl}adenine fumarate (compound 5)

According to the operation of Example 53, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) (722mg, 2.6mmol) and the compound (500mg, 2.6mmol) generated in the above-mentioned Example 41 were reacted as raw materials The title product 5 (200 mg, 14.2%) was produced. ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.78 (s, 2H), 4.5 (m, 2H), 4.2 (m, 2H), 4.0-3.8 (m, 4H), 3.62 (d, 3H), 3.0 (m, 2H), 2.0-1.8 (m, 2H), 1.6-1.8 (m, 1H), 2.0 (dd, 3H), 0.8 (dd, 3H). MS (ESI) m/z (%): 427 (MH ⁺ , 100).

Example 589-{2-(R)-[3-[1-(S)-methoxycarbonyl-2-methylpropyl]-2-oxa-1,3,2-oxazaphosphorylhexyl Preparation of Alkyl-2-methyleneoxy]-propan-1-yl}adenine fumarate (Compound 6)

According to the operation of Example 53, 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) (287mg, 1mmol) and the compound (190mg, 1mmol) generated in the above-mentioned Example 41 were The starting materials were reacted to give the title product (45 mg, 8.1%). ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.88 (s, 2H), 4.5-4.2 (m, 4H), 4.0 (m, 2H), 3.8 (m, 2H), 3.75 (d, 3H), 3.2-3.0 (m, 2H), 2.2 (m, 1H), 2.0 (m, 1H), 1.6-1.8 (m, 1H), 1.2 (dd, 3H), 0.9 (dd, 6H). MS (ESI) m/z (%): 442 (MH ⁺ , 100).

Example 599-{2-[3-[1-(S)-methoxycarbonylphenethyl]-2-oxa-1,3,2-oxazaphosphorylhexyl-2-methylene Preparation of Oxy]-Eth-1-yl}Adenine Fumarate (Compound 7)

According to the operation of Example 53, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) (273mg, 1mmol) and the compound (250mg, 1mmol) generated in the above-mentioned Example 47 were reacted as raw materials to generate the title Product (140 mg, 23.7%). ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 7.0-7.3 (m, 5H), 6.78 (s, 2H), 4.5-4.4 (m, 4H), 4.0-4.4 (m, 2H), 4.0-3.8 (m, 3H), 3.8 (d, 3H), 3.0-3.3 (m, 2H), 2.0-1.8 (m, 2H). MS (ESI) m/z (%): 475 (MH ⁺ , 100).

Example 609-{2-(R)-[3-[1-(S)-methoxycarbonylphenethyl]-2-oxa-1,3,2-oxazaphosphorylhexyl-2 Preparation of -methyleneoxy]-prop-1-yl}adenine fumarate (compound 8)

According to the operation of Example 53, 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) (419mg, 1.46mmol) and the compound generated in the above-mentioned Example 47 (346mg, 1.46mmol ) was reacted as starting material to yield the title product (113 mg, 18.7%). ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 7.0-7.3 (m, 5H), 6.78 (s, 2H), 4.5-4.4 (m, 1H), 4.0-4.4 (m, 6H), 3.8(m, 4H), 3.3(d, 2H), 3.0(m, 2H), 1.8(m, 2H), 1.0(d, 3H). MS (ESI) m/z (%): 489 (MH ⁺ , 100).

Example 619-{2-[3-methoxycarbonylmethylene]-2-oxa-1,3,2-oxazaphosphorylhexyl-2-methyleneoxy]-B-1 Preparation of -yl}adenine fumarate (compound 9)

According to the operation of Example 53, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) (273mg, 1mmol) and the compound (150mg, 1mmol) generated in the above-mentioned Example 48 were reacted as raw materials to generate the title Product (30 mg, 15.4%). ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.78 (s, 2H), 4.5 (m, 2H), 4.3 (m, 2H), 3.96 (m, 2H), 3.9 (d, 2H) , 3.6(s, 3H), 3.3(s, 2H), 3.1, 3.0(m, 2H), 2.0, 1.8(m, 2H). MS (ESI) m/z (%): 385 (MH ⁺ , 100).

Example 629-{2-[3-[1-(S)-methoxycarbonyl-2-methylbutyl]-2-oxa-1,3,2-oxazaphosphorylhexyl-2 Preparation of -Methyleneoxy]-Eth-1-yl}adenine fumarate (compound 10)

According to the operation of Example 53, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) (273mg, 1mmol) and the compound (203mg, 1mmol) generated in the above-mentioned Example 49 were reacted as raw materials to generate the title Product (185 mg, 33.2%). ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.75 (s, 2H), 4.4-4.5 (m, 2H), 4.2-4.0 (m, 2H), 3.9-3.8 (m, 5H), 3.72 (s, 3H), 3.0 (m, 2H), 2.0-2.1 (m, 2H), 1.6-1.8 (m, 2H), 1.4 (m, 1H), 0.8 (dd, 6H). MS (ESI) m/z (%): 441 (MH ⁺ , 100).

Example 639-{2-(R)-[3-[1-(S)-methoxycarbonyl-3-methylbutyl]-2-oxa-1,3,2-oxazaphosphorylhexyl Preparation of Alkyl-2-methyleneoxy]-propan-1-yl}adenine fumarate (compound 11)

According to the operation of Example 53, 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) (287mg, 1.0mmol) and the compound generated in the above-mentioned Example 49 (203mg, 1.0mmol ) was reacted as starting material to yield the title product (50 mg, 8.7%). ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.75 (s, 2H), 4.4-4.5 (m, 3H), 4.2-4.0 (m, 4H), 3.6 (d, 3H), 3.0 ( m, 2H), 2.0(m, 3H), 1.6(m, 2H), 1.2(t, 3H), 1.0(m, 6H). MS (ESI) m/z (%): 455 (MH ⁺ , 100).

Example 649-{2-[3-(R)-[1-(S)-ethoxycarbonyl-2-methylpropyl]-2-oxa-1,3,2-oxazaphosphorylhexyl Alkyl-2-methyleneoxy]-eth-1-yl}adenine fumarate (compound 12a);

9-{2-[3-(S)-[1-(S)-ethoxycarbonyl-2-methylpropyl]-2-oxa-1,3,2-oxazaphosphorylhexyl Preparation of -2-methyleneoxy]-ethyl-1-yl}adenine fumarate (compound 12b)

According to the operation of Example 53, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) (403mg, 1.5mmol) and the compound (300mg, 1.5mmol) generated in the above-mentioned Example 42 were reacted as raw materials The title products 12a (130 mg, 18.5%) and 12b (100 mg, 11.9%) were produced. Compound 12a ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.78 (s, 2H), 4.5 (m, 2H), 4.2 (m, 2H), 4.15 (m, 2H), 4.0-3.8 (q , 2H), 3.62(d, 3H), 3.0(m, 2H), 2.0-1.8(m, 2H), 1.6-1.8(m, 1H), 1.1(t, 3H), 2.0(dd, 3H), 0.8(dd,3H). MS (ESI) m/z (%): 441 (MH ⁺ , 100). Compound 12b ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.78 (s, 2H), 4.5 (m, 2H), 4.2 (m, 2H), 4.15 (m, 2H), 4.0-3.8 ( q, 2H), 3.66(d, 3H), 3.0(m, 2H), 2.0-1.8(m, 2H), 1.6-1.8(m, 1H), 1.1(t, 3H), 1.9(dd, 3H) , 0.7(dd,3H). MS (ESI) m/z (%): 441 (MH ⁺ , 100).

Example 659-{2-[3-[1-(S)-isopropoxycarbonyl-2-methylpropyl]-2-oxa-1,3,2-oxazaphosphorylhexyl- Preparation of 2-methyleneoxy]-eth-1-yl}adenine fumarate (compound 13)

According to the operation of Example 53, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) (403mg, 1.48mmol) and the compound (321mg, 1.47mmol) generated in the above-mentioned Example 43 were reacted as raw materials The title product was produced (210 mg, 24.6%). ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.78 (s, 2H), 5.0 (m, 1H), 4.5 (m, 2H), 4.2 (m, 2H), 4.0-3.8 (m, 4H), 3.6(d, 1H), 3.0(m, 2H), 2.0-1.8(m, 2H), 1.6-1.8(m, 1H), 1.2(m, 6H), 1.0-1.9(dd, 3H) , 0.8-0.7 (dd, 3H). MS (ESI) m/z (%): 455 (MH ⁺ , 100).

Example 669-{2-[3-(R)-[1-(R)-ethoxycarbonyl-2-methylpropyl]-2-oxa-1,3,2-oxazaphosphorylhexyl Alkyl-2-methyleneoxy]-eth-1-yl}adenine fumarate (compound 14a);

9-{2-[3-(S)-[1-(R)-ethoxycarbonyl-2-methylpropyl]-2-oxa-1,3,2-oxazaphosphorylhexyl Preparation of -2-methyleneoxy]-ethyl-1-yl}adenine fumarate (compound 14b)

According to the operation of Example 53, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) (403mg, 1.5mmol) and the compound (300mg, 1.5mmol) generated in the above-mentioned Example 44 were reacted as raw materials The title products 14a (110 mg, 13.5%) and 14b (100 mg, 12.2%) were produced. Compound 14a ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.78 (s, 2H), 4.5 (m, 2H), 4.2 (m, 2H), 4.15 (m, 2H), 4.0-3.8 ( q, 2H), 3.62(d, 3H), 3.0(m, 2H), 2.0-1.8(m, 2H), 1.6-1.8(m, 1H), 1.1(t, 3H), 2.0(dd, 3H) , 0.8 (dd, 3H). MS (ESI) m/z (%): 441 (MH ⁺ , 100). Compound 14b ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.78 (s, 2H), 4.5 (m, 2H), 4.2 (m, 2H), 4.15 (m, 2H), 4.0-3.8 ( q, 2H), 3.66(d, 3H), 3.0(m, 2H), 2.0-1.8(m, 2H), 1.6-1.8(m, 1H), 1.1(t, 3H), 1.9(dd, 3H) , 0.7(dd,3H). MS (ESI) m/z (%): 441 (MH ⁺ , 100).

Example 679-{2-[3-[1-methyl-1-ethoxycarbonylethyl]-2-oxa-1,3,2-oxazaphosphorylhexyl-2-methylene Preparation of Oxy]-Eth-1-yl}Adenine Fumarate (Compound 15)

According to the operation of Example 53, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) (273mg, 1mmol) and the compound (189mg, 1mmol) generated in the above-mentioned Example 50 were reacted as raw materials to generate the title Product (100 mg, 18.4%). ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.8 (s, 2H), 4.6 (m, 2H), 4.4 (m, 2H), 4.2 (q, 2H), 4.0 (m, 4H) , 3.1, 2.9 (m, 2H), 1.9, 1.7 (m, 2H), 1.4 (s, 6H), 1.0 (t, 3H). MS (ESI) m/z (%): 427 (MH ⁺ , 100).

Example 689-{2-[3-[1-(S)-hydroxycarbonyloxy-2-methylpropyl]-2-oxa-1,3,2-oxazaphosphorylhexyl- Preparation of 2-methyleneoxy]-eth-1-yl}adenine (compound 16)

Compound 5 (110mg, 26mmol) obtained in Example 57 was dissolved in dry methanol (2ml), added with aqueous sodium hydroxide (2M, 2ml), stirred at room temperature overnight, acidified with 4M glacial acetic acid, extracted with ethyl acetate, The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed (chloroform:methanol:glacial acetic acid=80:20:1) to obtain the title product (90 mg, 83.2%). ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 4.5 (m, 2H), 4.2 (m, 2H), 4.0-3.8 (m, 4H), 3.0 (m, 2H), 2.0-1.8 ( m, 2H), 1.6-1.8 (m, 1H), 1.0-1.9 (dd, 3H), 0.8-0.7 (dd, 3H). MS (ESI) m/z (%): 413 (MH ⁺ , 100).

Example 699-{2-[3-Benzyl-2-oxa-1,3,2-oxazaphosphorylhexyl-2-methyleneoxy]-eth-1-yl}adenine Preparation of fumarate (compound 17)

According to the operation of Example 53, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) (819mg, 3mmol) and the compound (495mg, 3mmol) generated in the above-mentioned Example 51 were reacted as raw materials to generate the title Product (227mg, 14.6%). ¹ H NMR (CD ₃ OD): 7.25 (m, 5H), 6.78 (s, 2H), 4.4 (m, 2H), 4.2 (m, 2H), 3.8 (m, 4H), 3.6 (d, 2H) , 2.8(m, 2H), 1.6(m, 2H). MS (ESI) m/z (%): 403 (MH ⁺ , 100).

Example 709-{2-(R)-[3-benzyl-2-oxa-1,3,2-oxazaphosphorylhexyl-2-methyleneoxy]-propan-1- Preparation of base} adenine fumarate (compound 18)

According to the operation of Example 53, 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) (287mg, 1.0mmol) and the compound generated in the above-mentioned Example 51 (165mg, 1.0mmol ) was reacted as starting material to give the title product (50 mg, 11.6%). ¹ H NMR (CD ₃ OD): 8.2 (dd, 2H), 7.5 (m, 5H), 6.78 (s, 2H), 4.4 (m, 2H), 4.2 (m, 2H), 3.8 (m, 3H) , 3.6(d, 2H), 2.8(m, 2H), 2.0(m, 2H), 1.3(d, 3H). MS (ESI) m/z (%): 417 (MH ⁺ , 100).

Example 719-{2-[3-n-propyl-2-oxa-1,3,2-oxazaphosphorylhexyl-2-methyleneoxy]-eth-1-yl}adeno Preparation of purine fumarate (compound 19)

According to the operation of Example 53, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) (546mg, 2mmol) and the compound (234mg, 2mmol) generated in the above-mentioned Example 52 were reacted as raw materials to generate the title Product (152 mg, 16.1%). ¹ H NMR (CD ₃ OD): 8.1 (s, 2H), 6.6 (s, 2H), 4.4 (t, 2H), 4.2 (m, 2H), 3.8 (m, 4H), 2.8 (m, 2H) , 1.8(m, 1H), 1.6(m, 1H), 1.4(m, 2H), 1.2(m, 2H), 0.6(t, 3H). MS (ESI) m/z (%): 255 (MH ⁺ , 100).

Example 729-{2-[3-(1-chloro-n-butyl)-2-oxa-1,3,2-oxazaphosphorylhexyl-2-methyleneoxy]-B Preparation of -1-yl}adenine fumarate (compound 20)

According to the operation of Example 53, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) (273 mg, 1 mmol) and 3-hydroxy n-propyl-1-chloro-n-butylamine (165 mg, 1 mmol) was reacted as starting material to give the title product (100 mg, 24.8%). ¹ H NMR (CD ₃ OD): 8.2 (d, 2H), 6.8 (s, 2H), 4.6 (m, 2H), 4.4 (m, 2H), 4.2 (q, 2H), 4.0 (m, 4H) , 3.1, 2.9(m, 4H), 1.9, 1.7(m, 2H), 1.4(m, 4H). MS (ESI) m/z (%): 403 (MH ⁺ , 100).

Embodiment 73 antiviral experiment

1. Cytotoxicity test

The inhibitory effect of compounds on the growth of HepG 2215 was detected by tetrazolium reduction method. A bottle of HepG 2215 cells was taken, digested with trypsin to prepare a single cell suspension, counted and adjusted the cell concentration to 2×10 ⁴ cell/ml, and added to a 96-well culture plate. Place in a cell culture incubator at 37°C, 5% _CO2 overnight. After aspirating the supernatant, DMEM medium (5% fetal calf serum) containing different concentrations of compounds was added. After 9 days of action, 10 ml of MTT was added to each well and culture was continued for 4 hours. Carefully suck off the supernatant, add 150ml DMSO to each well, shake gently, and detect the OD value at 570nm with a microplate reader.

2. Cell compound treatment

A bottle of HepG 2215 cells was taken, digested with trypsin to prepare a single cell suspension, adjusted the cell concentration to 2×10 ⁴ cell/ml, and added to a 24-well cell culture plate (1ml/well). Incubate overnight at 37°C, 5% CO ₂ . Take out the 24-well cell culture plate, suck out the supernatant, and then add five 5-fold diluted compounds and positive control drugs in sequence. The culture was continued, and the medium containing the drug was replaced every 3 days, and the supernatant of each well was collected on the 9th day and stored in a centrifuge tube at -20°C for future use.

3. Fluorescent quantitative PCR reaction

Serum samples (100 μl) were placed in lysate (1mmol/L Tris-HCl, pH 8.0, 10mmol/L NaCl, 0.1mmol/L EDTA, 0.5% SDS, 0.8mg/ml proteinase K), incubated at 37°C for 5h, Then it was extracted twice with phenol chloroform and chloroform respectively, and then precipitated with ethanol. Dissolved in 50μl water for PCR amplification.

Put 2ml of viral DNA into 48μl of reaction mixture, including 5mmol/L MgCl ₂ , 0.2mmol/L of each dNTP, 2.5U of Gold Taq polymerase, 0.2U of UNG enzyme and 0.4mmol/L of primers, and 0.15mmol of probe /L. After 5 minutes at 37°C, denature at 95°C for 3 minutes for 30s, cycle 41 times at 94°C for 20s, and 60°C for 40s (fluorescent signal detection), and finally store at 4°C. The Taqman probe sequence used for detection: 5'FAM-CCAGCAGCGCCTCCTCCTGC-3'TAMARA; primer sequence: forward primer: 5'-CCCTCAGGCTCAGGGCATA-3'; reverse primer: 5'-CTTCCTGACTGCCGATTGGT-3'.

4. Experimental cell line

HepG 2215 cells were obtained from the Laboratory of Molecular Virology, Fudan University. The cell line is formed by transfecting recipient cell HepG2 with two head-to-tail HBV DNA whole gene recombinant plasmids, which can stably secrete HBsAg, HBeAg and complete Dane particles in vitro, and can also produce a large number of replication intermediates ( RI). Cells were cultured in DMEM containing 10% fetal calf serum.

5. The positive control drug is Adefovir Dipivoxil (ADV).

Antiviral results are as follows:

a. EC ₅₀ : half effective concentration; b. CC ₅₀ : half cytotoxic concentration; c. SI: action selectivity index (CC ₅₀ /EC ₅₀ ).

As can be seen from the above table, the compounds of the present invention all show similar or better anti-HBV activity than the listed drug ADV (Adefovir dipivalate), and the toxicity is generally significantly reduced. Therefore, the compound of the present invention Compared with the marketed drug ADV, the selectivity index (SI) has been greatly improved. In particular, the anti-HBV activity of compound 5 is 2.6 times higher than that of the positive control drug ADV, and the SI value is about 8 times that of ADV. Therefore, the compound of the present invention can become a new type of more effective antiviral drug, especially for treating infectious diseases caused by HBV and HIV viruses.

Claims (10)

1. Acyclic nucleoside phosphonate compounds represented by general formula (I) or various optical isomers, pharmaceutically acceptable inorganic or organic salts thereof:

in the general formula (I):

R₁represents an amino group;

R₂represents hydrogen;

R₃represents hydrogen, straight or branched chain C₁-C₆Alkyl, monofluoromethyl or difluoromethyl;

R₅represents hydrogen;

R₄represents hydrogen, straight or branched chain C₁-C₆Alkyl, straight or branched C₁-C₆Haloalkyl, benzyl or formula (II),

in the formula (II), the compound represented by the formula (II),

R₆and R₇Independently of one another, represents hydrogen, benzyl, straight-chain or branched C₁-C₆Alkyl or the side chain of a natural or unnatural amino acid, the configuration of which is L-or D-form;

R₈represents hydrogen, straight or branched chain C₁-C₆Alkyl or trifluoroethyl.

2. The acyclic nucleoside phosphonate compound according to claim 1, or various optical isomers, pharmaceutically acceptable inorganic or organic salts thereof, wherein in the compound of formula (I):

R₁represents an amino group;

R₂represents hydrogen;

R₃represents hydrogen or straight or branched C₁-C₄An alkyl group;

R₅represents hydrogen;

R₄represents a straight or branched chain C₁-C₄Alkyl, straight or branched C₁-C₄Haloalkyl, benzyl or formula (II),

in the formula (II), the compound represented by the formula (II),

R₆and R₇Independently of one another, represents hydrogen, benzyl or linear or branched C₁-C₄An alkyl group, a carboxyl group,

R₈represents hydrogen or straight or branched C₁-C₄An alkyl group.

3. The acyclic nucleoside phosphonate compound according to claim 1 or 2, or optical isomers, pharmaceutically acceptable inorganic or organic salts thereof, wherein the pharmaceutically acceptable inorganic or organic salt is a salt of the compound of formula (I) with an acid selected from hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, aspartic acid or glutamic acid; or the sodium, potassium, calcium, aluminum, ammonium, methylamine, ethylamine or ethanolamine salt of a compound of formula (I) with a base; or salts of the compounds of the general formula (I) with basic amino acids, the basic amino acids being lysine, arginine or ornithine.

4. The acyclic nucleoside phosphonate compound according to any one of claims 1-2, or its various optical isomers, pharmaceutically acceptable inorganic or organic salts, comprising the following compounds:

5. the process for preparing acyclic nucleoside phosphonate compounds of claim 1, or various optical isomers, pharmaceutically acceptable inorganic or organic salts thereof, comprising:

the method comprises the following steps:

(1) reacting N-benzyl amino acid ester with allyl bromide, and then carrying out hydration and debenzylation to generate a compound (IIIa);

wherein R is₆、R₇、R₈The definition is the same as in claim 1;

(2) reacting the compound (IIIa) with a phosphonic chloride compound of 9- [2- (phosphonomethoxy) ethyl ] adenine or 9- [2- (R) - (phosphonomethoxy) propyl ] adenine to produce a phosphonate compound (I), and reacting the compound (I) with a corresponding acid in a solvent as required to produce a salt of the compound (I);

or,

the second method comprises the following steps:

(1) reacting 3-aminopropanol with corresponding aldehyde or reacting 3-chloropropanol with corresponding amine to generate a compound (IIIb);

wherein R is₄The same as in claim 1, but not of formula (II);

(2) reacting the compound (IIIb) with a phosphonic chloride compound of 9- [2- (phosphonomethoxy) ethyl ] adenine or 9- [2- (R) - (phosphonomethoxy) propyl ] adenine to produce a phosphonate compound (I); the compounds of formula (I) may be prepared as the corresponding pharmaceutically acceptable salts by reaction with the corresponding acids, as required.

6. The process for preparing acyclic nucleoside phosphonate compounds according to claim 5, or various optical isomers, pharmaceutically acceptable inorganic or organic salts thereof, wherein the first process is:

wherein R is₃、R₆、R₇、R₈The definition is the same as that of claim 1;

a. the compound (III-1) represents a natural or unnatural alpha-amino acid having the L-or D-configuration in the presence of an acid chloride reagent in the form of R₈OH is used as a reaction solvent to react for 0.5 to 72 hours at the temperature of minus 30 to 100 ℃ to obtain amino acid ester (III-2); the acyl chloride reagent is selected from oxalyl chloride, phosphorus pentachloride, phosphorus oxychloride or thionyl chloride;

b. reacting the compound (III-2) with benzaldehyde in a protic solvent under the action of a reducing agent at the temperature of-30-50 ℃ for 0.5-72 hours to obtain an N-benzyl amino acid ester product (III-3); the reducing agent is selected from lithium borohydride, sodium borohydride, potassium borohydride, sodium cyanoborohydride or sodium triacetoxyborohydride; the protic solvent is selected from ethanol, methanol or isopropanol;

c. reacting the compound (III-3) with allyl bromide in the presence of organic base in a polar aprotic solvent at-30-100 ℃ for 0.5-72 hours to generate N-benzyl-N-allylated amino acid ester (III-4); the organic base is selected from triethylamine, N' -dicyclohexyl-4-morpholinylamidine, 1, 8-diazabicyclo [5, 4, 0] undec-7-ene, N-dimethylaminopyridine, N-diisopropylethylamine, morpholine, N-methylmorpholine, pyridine or 2, 6-dimethylpyridine; the polar aprotic solvent is selected from chloroform, ethyl acetate, tetrahydrofuran, diethyl ether, methyl tert-butyl ether, dimethylformamide, dimethyl sulfoxide or hexamethylphosphoramide;

d. reacting the compound (III-4) in an ether solvent in the presence of an oxidant at-30-100 ℃ for 0.5-72 hours to generate a compound (III-5); the oxidant is selected from hydrogen peroxide, potassium permanganate, sodium periodate, sodium hypochlorite, chromium trioxide, potassium dichromate, DMSO or 9-boron bicyclo [3, 3, 1] nonane; the ether solvent is selected from tetrahydrofuran, diethyl ether, methyl tertiary butyl ether, 1, 6-dioxane or ethylene glycol dimethyl ether;

e. carrying out catalytic hydrogenation reaction on the compound (III-5) in a protic solvent at-30-50 ℃ for 0.5-72 hours under the action of a metal-containing catalyst to generate a debenzylation product (IIIa); the metal-containing catalyst is a palladium catalyst, a platinum catalyst or a nickel catalyst, and the protonic solvent is a mixed solvent of ethanol, methanol, isopropanol, alcohol and acetic acid or a mixed solvent of alcohol and sulfuric acid;

f. PMEA and PMPA are respectively reacted with acyl chloride reagent and Et₂NCHO, reacting in polar aprotic solvent at-30-100 ℃ for 0.5-72 hours to obtain a phosphonyl chloride compound (I-1), and activating with organic base for later use; the acyl chloride reagent is selected from oxalyl chloride, phosphorus pentachloride, phosphorus oxychloride or thionyl chloride; the polar aprotic solvent is selected from dichloromethane, chloroform, 1, 2-dichloroethane, ethyl acetate, tetrahydrofuran, diethyl ether, methyl tert-butyl ether, dioxane, dimethylformamide or dimethyl sulfoxide; the organic base is selected from triethylamine, N' -dicyclohexyl-4-morpholinylamidine, 1, 8-diazabicyclo [5, 4, 0]Undecane-7-ene, N-dimethylaminopyridine, N-diisopropylethylamine, morpholine, N-methylmorpholine, pyridine or 2, 6-dimethylpyridine;

g. reacting the compound (IIIa) with the prepared compound (I-1) solution in a polar aprotic solvent for 0.5-72 hours at-80-25 ℃ under the action of organic base to generate corresponding phosphonate (I-2); the organic base is selected from triethylamine, N' -dicyclohexyl-4-morpholinylamidine, 1, 8-diazabicyclo [5, 4, 0] undec-7-ene, N-dimethylaminopyridine, N-diisopropylethylamine, morpholine, N-methylmorpholine, pyridine or 2, 6-dimethylpyridine; the polar aprotic solvent is selected from dichloromethane, chloroform, 1, 2-dichloroethane, ethyl acetate, tetrahydrofuran, diethyl ether, methyl tert-butyl ether, 1, 6-dioxane, dimethylformamide or dimethyl sulfoxide;

h. adding acid into phosphonate ester (I-2) in a protic solvent, and reacting at 25-100 ℃ for 0.5-72 hours to generate a product phosphonate ester (I-3) with the imine protecting group removed; the acid is selected from sulfuric acid, hydrochloric acid, glacial acetic acid, trifluoroacetic acid, methanesulfonic acid or phosphoric acid; suitable protic solvents are ethanol, methanol or isopropanol;

i. the phosphonate (I-3) reacts with corresponding acid in a polar aprotic solvent at 25-100 ℃ for 0.5-72 hours to generate a salt (Ia); the polar aprotic solvent is selected from dichloromethane, chloroform, 1, 2-dichloroethane, ethyl acetate, tetrahydrofuran, acetonitrile, diethyl ether, methyl tert-butyl ether or dioxane.

7. The method for preparing acyclic nucleoside phosphonate compounds according to claim 5, or various optical isomers, pharmaceutically acceptable inorganic or organic salts thereof, wherein the second method is:

wherein R is₃、R₄Defined as in claim 1, except that R₄Is not of formula (II);

a. reacting 3-aminopropanol with corresponding aldehyde or reacting 3-chloropropanol with corresponding amine to generate a compound (IIIb);

b. PMEA and PMPA are respectively reacted with acyl chloride reagent and Et₂NCHO, reacting in polar aprotic solvent at-30-100 ℃ for 0.5-72 hours to obtain a phosphonyl chloride compound (I-1), and activating with organic base for later use; the acyl chloride reagent is selected from oxalyl chloride, phosphorus pentachloride, phosphorus oxychloride or thionyl chloride; the polar aprotic solvent is selected from dichloromethane, chloroform, 1, 2-dichloroethane, ethyl acetate, tetrahydrofuran, diethyl ether, methyl tert-butyl ether, dioxane, dimethylformamide or dimethyl sulfoxide; the organic base is selected from triethylamine, N' -dicyclohexyl-4-morpholinylamidine, 1, 8-diazabicyclo [5, 4, 0]Undecane-7-ene, N-dimethylaminopyridine, N-diisopropylethylamine, morpholine, N-methylmorpholine, pyridine or 2, 6-dimethylpyridine;

c. reacting the compound (I-1) and the compound (IIIb) in a polar aprotic solvent for 0.5 to 72 hours at a temperature of between 80 ℃ below zero and 25 ℃ under the action of organic base to generate corresponding phosphonate (Ib-1); the organic base is selected from triethylamine, N' -dicyclohexyl-4-morpholinylamidine, 1, 8-diazabicyclo [5, 4, 0] undec-7-ene, N-dimethylaminopyridine, N-diisopropylethylamine, morpholine, N-methylmorpholine, pyridine or 2, 6-dimethylpyridine; the polar aprotic solvent is selected from dichloromethane, chloroform, 1, 2-dichloroethane, ethyl acetate, tetrahydrofuran, diethyl ether, methyl tert-butyl ether, 1, 6-dioxane, dimethylformamide or dimethyl sulfoxide;

d. adding acid into phosphonate (Ib-1) in a protic solvent, and reacting at 25-100 ℃ for 0.5-72 hours to generate a product phosphonate (Ib-2) with an imine protecting group removed; the acid is selected from sulfuric acid, hydrochloric acid, glacial acetic acid, trifluoroacetic acid, methanesulfonic acid or phosphoric acid; suitable protic solvents are ethanol, methanol or isopropanol;

e. the phosphonate (Ib-2) reacts with corresponding acid in a polar aprotic solvent at 25-100 ℃ for 0.5-72 hours to generate a salt (Ib); the polar aprotic solvent is selected from dichloromethane, chloroform, 1, 2-dichloroethane, ethyl acetate, tetrahydrofuran, acetonitrile, diethyl ether, methyl tert-butyl ether or dioxane.

8. A pharmaceutical composition having antiviral activity, comprising a therapeutically effective amount of one or more acyclic nucleoside phosphonate compounds of claims 1-4 or various optical isomers, pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients.

9. The use of the acyclic nucleoside phosphonate compounds of claims 1-4 or various optical isomers, pharmaceutically acceptable salts thereof in the preparation of a medicament for treating viral infectious diseases.

10. The use according to claim 9, wherein the disease is an infectious disease caused by HBV and HIV viruses.