CN111434671B

CN111434671B - Liver-specific AMPK agonist and preparation method and application thereof

Info

Publication number: CN111434671B
Application number: CN201910027560.9A
Authority: CN
Inventors: 徐华强; 栾林波; 张振伟; 孙锋; 杨生生; 高善云; 戴金威
Original assignee: Kaisi Kaidi Shanghai Pharmaceutical Technology Co ltd
Current assignee: Kaisi Kaidi Shanghai Pharmaceutical Technology Co ltd
Priority date: 2019-01-11
Filing date: 2019-01-11
Publication date: 2023-07-11
Anticipated expiration: 2039-01-11
Also published as: CN111434671A; WO2020143800A1

Abstract

The invention provides liver-specific AMPK agonists, and a preparation method and application thereof. Specifically, the invention provides a compound shown in the following formula I and application thereof in treating non-alcoholic fatty liver disease (NAFLD) including non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH) and related liver cirrhosis and liver cancer, or obesity, diabetes, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, cardiovascular diseases, metabolic diseases and other diseases. Wherein, each group is defined as the specification.

Description

Liver-specific AMPK agonist and preparation method and application thereof

Technical Field

The present invention provides AMPK (AMP-dependent protein kinase) agonists based on liver-specific methods and medical uses thereof. For example, the compounds of the present invention are useful for the treatment of non-alcoholic fatty liver disease (NAFLD) including non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH) and liver cirrhosis related thereto, liver cancer, and also useful for metabolic diseases such as obesity, diabetes, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, cardiovascular diseases, and the like.

Background

AMPK, which is known as AMP Activated Protein Kinase, an AMP-dependent protein kinase, is a heterotrimeric protein kinase composed of a catalytic subunit α, a regulatory subunit β and γ together. The protein kinase is an energy kinase capable of sensing in-vivo AMP change, and when the in-vivo AMP concentration rises to a certain degree, AMPK is activated to inhibit anabolism and promote catabolism; the net effect of activation is the inhibition of the ATP consuming process and the activation of the ATP production pathway, thus regenerating ATP storage. AMPK after activation is able to shut down almost all anabolic pathways (including biosynthesis of lipids, carbohydrates, proteins and ribosomal RNAs) by both short-term and long-term modes of regulation. In one aspect, AMPK is capable of directly regulating downstream substrate activity at the protein level through phosphorylation, thereby regulating cellular energy metabolism in a short period of time. On the other hand, it was found that AMPK was able to down-regulate the expression level of downstream substrates, thereby regulating cellular energy metabolism for a long period of time. AMPK, an energy balancer, plays an important role in regulating cellular energy metabolism, oxidative stress and autophagy, and has a very remarkable link with normal liver function due to the important role of liver in regulating fatty acid oxidation and synthesis, and lipid metabolism. The liver metabolism is changed in patients with metabolic diseases, and the glucose metabolism, lipid metabolism and the like in the patients are improved by researching AMPK, which is possible to be an effective method for treating the diseases.

AMPK can control tumor invasion and metastasis by controlling the activity of cancer suppressor genes and improving energy metabolism disorders. When the energy metabolism of the cells is unbalanced, AMPK can activate downstream signal P53 serine residues through phosphorylation, so that m TOR channels are negatively regulated to interrupt the proliferation of tumors, and invasion and metastasis of the tumors are reduced. Protein Kinase B (PKB) is associated with metastasis of tumors, whereas AMPK can affect metastasis of tumor a through modulation of PKB. In liver cancer patients taking chemotherapy drugs for a long period, fas Receptor (FasR) promoting apoptosis of tumor cells can be activated in drug-resistant tumor cells, increasing tumor invasiveness by inducing activation of transcription factor NF-k B (Nuclear factor kB, NF-k B), while SNARK in AMPK family can affect invasive metastasis by modulating CD95-NF-k B pathway. AMPK can also reduce tumor cell invasiveness by affecting MMP-2 and MMP-9 proteins in the family of metalloproteinases (Matrix Metalloproteinase, MMP).

Since the combinations of the respective subunits of AMPK are different, it is theorized that AMPK has 12 protein combinations in total. Whereas the α1 and γ2 subunits possess different variable cleavages (alternative splice variants), further increasing the complexity of AMPK trimer. The wide expression of AMPK in vivo is lacking at present, and the AMPK agonist with strong tissue specificity, high selectivity and low toxic and side effect is provided.

Disclosure of Invention

The invention synthesizes a kind of prodrug molecules of phosphorothioate nucleoside monophosphate (AMPS) and analogues thereof, and the drugs are metabolized in liver tissues after oral administration to obtain parent molecules.

In a first aspect of the present invention there is provided a compound of formula I, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof,

wherein:

R ₁ selected from the group consisting of: a substituted or unsubstituted C6-C18 aryl, a substituted or unsubstituted 5-12 membered heteroaryl;

R ₂ selected from the group consisting of: hydrogen, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C18 alkanoyl, substituted or unsubstituted C2-C18 alkoxycarbonyl, mono-or di-C2-C18 alkylaminocarbonyl: halo, haloalkyl, nitro, hydroxy, amino and cyano;

R ₃ and R is ₄ Each independently selected from the group consisting of: hydrogen, fluorine, C1-C6 alkyl, C1-C6 alkoxy; or R is ₃ And R is ₄ Together forming a group selected from the group consisting of: a C3-C8 carbocyclic ring, or a 5-12 membered heterocyclic ring;

x is O, S, NH, substituted or unsubstituted C1-C4 alkylene;

wherein the substitution refers to substitution of a hydrogen atom on the group with one or more (e.g., 2, 3, 4, etc.) substituents selected from the group consisting of: halogen, deuterated, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, halogenated C3-C8 cycloalkyl, methylsulfonyl, -S (=O) ₂ NH ₂ Oxo (=o), -CN, hydroxy, -NH ₂ Carboxyl, C1-C6 amido (-C (=O) -N (Rc)) ₂ or-NH-C (=O) (Rc), rc being H or C1-C5 alkyl), C1-C6 alkyl- (C1-C6 amide), or a substituted or unsubstituted group selected from the group consisting of: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 amino, C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-12 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O, - (CH) ₂ ) -C6-C10 aryl, - (CH) ₂ ) - (5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O), and said substituents are selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkoxy, oxo, -CN, -NH ₂ -OH, C6-C10 aryl, C1-C6 amino, C1-C6 amido, 5-to 10-membered heteroaryl having 1-3 heteroatoms selected from N, S and O.

In another preferred embodiment, the compound of formula I has a structure selected from the group consisting of:

in another preferred embodiment, X is selected from the group consisting of: o, S, NH, CH ₂ 、CF ₂ Or CD ₂ 。

In another preferred embodiment, R is ₁ Has a structure represented by the following formula II, III, IV, V, VI or VII:

wherein:

the dotted line is a chemical bond or none;

each A is ₁ 、A ₂ 、A ₃ 、A ₅ 、A ₆ 、A ₇ 、A ₈ Each independently is O, S, N, NH, CH or CH ₂ ；A ₄ And A ₉ Each independently is C or N;

each B is ₁ 、B ₂ 、B ₃ 、B ₄ 、B ₆ 、B ₇ 、B ₈ 、B ₉ Each independently is O, S, N, NH, CH or CH ₂ ；B ₅ And B ₁₀ Each independently is C or N;

each R is ₅ 、R ₆ 、R ₇ And R is ₈ Each independently selected from the group consisting of: halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy;

i is 0, 1, 2, 3, 4 or 5;

Y ₁ o, S or NH;

Y ₂ and Y ₃ Each independently selected from O, N or CH;

j is 0, 1, 2, 3 or 4;

m is 0, 1, 2 or 3;

n is 0, 1, 2, 3 or 4;

in another preferred embodiment, R is ₂ Selected from the group consisting ofGroup: acetyl and butyryl.

In another preferred embodiment, R is ₃ ，R ₄ Each independently is a hydrogen atom.

In another preferred embodiment, the compound of formula (I) is a prodrug of a direct AMPK agonist.

In another preferred embodiment, the compounds of formula (I) may have one or more chiral centers and thus exist in a variety of stereoisomeric forms, including tautomers, cis-trans isomers, conformational isomers, meso compounds, optical isomers having an enantiomeric or diastereomeric relationship, and mixtures of various isomers that may occur.

In another preferred embodiment, the compound has a structure selected from the group consisting of:

In another preferred embodiment, the compound is selected from the structures shown in the following group:

in another preferred embodiment, the compound has the structure shown in table 1:

in a second aspect of the invention there is provided a pharmaceutical composition comprising (a) a therapeutically effective amount of a compound as described in the first aspect of the invention, or a pharmaceutically acceptable salt, hydrate or solvate thereof; and (b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the disease or condition is selected from the group consisting of: non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH) and related liver cirrhosis, liver cancer, non-alcoholic fatty liver disease (NAFLD), obesity, diabetes, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, cardiovascular diseases, metabolic diseases.

In a third aspect the present invention provides the use of a compound of formula I according to the first aspect of the invention for the preparation of a pharmaceutical composition for the treatment or prophylaxis of diseases or conditions associated with AMPK activation.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows a graph of concentration of the in vivo metabolically-released active molecule AMPS in the liver versus time following intragastric administration of 20. Mu. Mol/kg of liver-targeted AMPS prodrug in rats.

FIG. 2 shows a graph of concentration of the in vivo metabolically-released active molecule AMPS in the liver versus time following intragastric administration of 20. Mu. Mol/kg of a CS0002 series AMPS prodrug in rats.

FIG. 3 shows the effect of compounds on AMPK phosphorylation levels in primary hepatocytes of mice.

Detailed Description

Unless explicitly indicated otherwise, the terms used according to the invention and herein have the following meanings:

as used herein, the term "C1-C6 alkyl" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, and the like, or the like. Preferably, the alkyl group is a straight or branched saturated chain having 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl or isopropyl.

As used herein, the term "C1-C18 alkyl" refers to a straight or branched chain alkyl group having 1 to 18 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, and the like, or the like.

As used herein, the term "C3-C8 cycloalkyl" refers to a cyclic alkyl group having 1 to 8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl.

As used herein, the term "C1-C6 alkoxy" refers to a C1-C6 alkyl group as defined above, which is attached to the remainder of the molecule through an oxygen atom. Preferably, the C1-C6 alkoxy groups may include methoxy, ethoxy and isopropoxy.

As used herein, the term "C1-C6 alkylamino" refers to a C1-C6 alkyl group as defined above, which is attached to the remainder of the molecule through a nitrogen atom. Preferably, the alkylamino group may include a dimethylamino group and a diethylamino group.

As used herein, the term "C1-C6 carboxyl" refers to a substituent such as "straight or branched alkyl-carboxyl having 1 to 5 carbon atoms" structure that is attached to the remainder of the molecule through an alkyl carbon atom. Such as formate, acetate, propionate, butyrate, or the like.

As used herein, the term "C1-C6 ester group" refers to a substituent such as a "straight or branched alkyl-ester group having 1 to 5 carbon atoms" structure that is attached to the remainder of the molecule through an alkyl carbon atom; wherein the alkyl portion of the ester group is a C1-C6 alkyl group. Such as methyl formate, ethyl formate, methyl acetate, or the like.

As used herein, the term "C2-C6 alkanoyl" refers to a substituent such as "a straight or branched alkyl-carbonyl group having 1 to 5 carbon atoms" structure that is attached to the remainder of the molecule through a carbonyl group. Such as acetyl, propionyl, butyryl, or the like.

As used herein, the term "C2-C18 alkanoyl" refers to a substituent such as "a straight or branched alkyl-carbonyl group having 1 to 17 carbon atoms" structure, such as acetyl, propionyl, butyryl, or the like.

As used herein, the term "C2-C6 alkanoylamino" refers to a substituent such as "having a C2-C6 alkanoyl-amino" structure that is attached to the remainder of the molecule through a nitrogen atom; such as acetamido, propionamido, butyramido, or the like.

As used herein, the term "C2-C18 alkoxycarbonyl" refers to a straight or branched alkyl-oxy-carbonyl group having, for example, 1 to 17 carbon atoms, which is attached to the remainder of the molecule through a carbonyl group.

As used herein, the term "C2-C18 alkylaminocarbonyl" refers to a straight or branched alkyl-nitrogen-carbonyl group, e.g., having 1 to 17 carbon atoms, which is attached to the remainder of the molecule through a carbonyl group.

The term "halogen" refers to F, cl, br and I.

The term "haloalkyl" refers to a C1-C3 alkyl group substituted with a halogen. Preferably, the haloalkyl is trifluoromethyl, difluoromethyl, trifluoromethoxy. Here, "C1-C3 alkyl" means a straight-chain or branched alkyl group having 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl.

The term "aryl" refers to a C6-C18 aromatic group, such as phenyl or naphthyl, which is unsubstituted, substituted with one or more (e.g., 2, 3, 4, or 5) atoms or groups selected from the group consisting of: halogen, nitro, hydroxy, amino, cyano, haloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylamino, substituted or unsubstituted C1-C6 carboxy, substituted or unsubstituted C1-C6 ester, substituted or unsubstituted C2-C6 alkanoyl, substituted or unsubstituted C2-C6 alkanoylamino.

The term "heteroaryl" refers to a 5-12 membered aromatic group containing one or more heteroatoms selected from nitrogen, oxygen and sulfur. Heteroaryl groups may include pyridine, pyrazine, pyrimidine, thiophene, furan, isoxazole, isothiazole, pyrazole, imidazole. Such groups may be unsubstituted, heteroaryl substituted with one or more (e.g., 2, 3, 4, or 5) atoms or groups selected from: halogen, nitro, hydroxy, amino, cyano, haloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylamino, substituted or unsubstituted C1-C6 carboxy, substituted or unsubstituted C1-C6 ester, substituted or unsubstituted C2-C6 alkanoyl, substituted or unsubstituted C2-C6 alkanoylamino.

The term "heterocycle" or "heterocyclyl" refers to a 5-12 membered non-aromatic group (including saturated, partially saturated or unsaturated groups) containing one or more heteroatoms selected from nitrogen, oxygen and sulfur, having a single or fused ring (including bridged and spiro ring systems: in a fused ring system, one or more rings may be cycloalkyl, aryl or heteroaryl: in one embodiment, the nitrogen and/or sulfur atoms of the heterocyclic group are optionally oxidized to provide an N-oxide, sulfinyl and sulfonyl moiety; examples of "heterocyclyl" and its fused analogs include pyrrolidinyl, piperidinyl, piperazinyl, imidazolidinyl, 2, 3-dihydrofuranyl (2, 3-b) and pyridinyl, benzoxazinyl, tetrahydroquinolinyl, indolinyl, and the like.

In the present invention, the terms "comprising," "including," or "comprising" mean that the various ingredients may be used together in a mixture or composition of the present invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "containing.

In the present invention, the term "pharmaceutically acceptable" component refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.

In the present invention, the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates, or prevents a disease or condition of interest, or that exhibits a detectable therapeutic or prophylactic effect. The precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Thus, it is not useful to pre-specify an accurate effective amount. However, for a given condition, the effective amount can be determined by routine experimentation and can be determined by a clinician.

As used herein, the term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention with acids or bases that are suitable for use as medicaments. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is the salts of the compounds of the present invention with acids. Suitable salts forming acids include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as 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, benzenesulfonic acid, and the like; acidic amino acids such as aspartic acid and glutamic acid.

Some of the compounds of the present invention may be crystallized or recrystallized from water or various organic solvents, in which case various solvates may be formed. Solvates of the present invention include stoichiometric solvates such as hydrates and the like, as well as compounds containing variable amounts of water formed when prepared by the low pressure sublimation drying process.

The term "prodrug" as used herein refers to any compound that when administered to a biological system produces a "drug" substance (biologically active compound) as a result of one or more spontaneous chemical reactions, one or more enzymatic chemical reactions, and/or one or more metabolic chemical reactions. It also includes biodegradable polymer derivatives of the compounds of the invention, for example as described in int.J.pharm.115,61-67 (1995).

The invention also includes all suitable isotopic variations of the compounds of the present invention. Isotopic variations of compounds of the present invention are defined as those in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, respectively, for example ² H、 ³ H、 ¹¹ C、 ¹³ C、 ¹⁴ C、 ¹⁵ N、 ¹⁷ O、 ¹⁸ O、 ³⁵ S、 ¹⁸ F and F ³⁶ Cl. Some isotopic variants of the present invention, for example,in which radioactive isotopes (e.g ³ H or ¹⁴ C) Are used for drug and/or substrate tissue distribution studies. Tritiated, i.e., ³ h, and carbon-14, i.e., ¹⁴ isotopes of C are particularly preferred because of their ease of preparation and detection. In addition, the use of isotopes (e.g., deuterium, i.e., ² h) May provide some therapeutic advantages resulting from increased metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements and may therefore be preferred in some cases. Isotopic variations of the compounds of the present invention can generally be prepared by conventional procedures, for example, by exemplary methods or by using suitable reagents for the appropriate isotopic variations described in the experimental section below.

The preparation method of the invention is as follows:

the compounds of the present invention may be prepared by a number of methods well known to those skilled in the art, including but not limited to those described below, or by modifying these methods using standard techniques known to those skilled in the art of organic synthesis. All methods disclosed in connection with the present invention are performed on any scale, including milligrams, grams, several grams (multigram), kilograms, several kilograms (multikilogram), or commercial industrial scale. In the following formulae and below, R, unless otherwise indicated ₁ To R ₄ As defined in the first aspect. These methods form other aspects of the invention.

Throughout the specification, the general formulae are represented by roman numerals (I), (II), (III), (IV), and the like. Subsets of these formulae are defined as (Ia), (Ib), (Ic) etc. …, (IVa), (IVb), (IVc) etc.; or (I-a), (I-b), (I-c), etc. …, (IV-a), (IV-b), (IV-c), etc.

The general preparation method of the compound is shown as the following formula:

the following description will be given of the production method by taking the case where X in the general formula (I) is O as an example:

reactive one

The compounds of formula (I') may be prepared by: according to formula one, triethylamine trihydrofluoride is reacted with formula (Ia/b). Typical reaction conditions include reacting triethylamine trihydrofluoride salt and the general formula (Ia/Ib) in anhydrous tetrahydrofuran at room temperature for about 12 hours. After the reaction is finished, the reaction solution is directly subjected to column chromatography (mobile phase is water and acetonitrile) by a C18 column through a rapid column chromatography instrument, and the separated fraction is subjected to freeze-drying by a freeze dryer to obtain the compound of the general formula (I').

Reactive type II

The compounds of formula (Ib) may be prepared by: r is according to reaction II ₂ The substituent and the general formula (Ia) are subjected to condensation reaction to obtain the general formula (Ib).

Reactive type III

The compounds of formula (Ia) may be prepared by: according to reaction formula III, phosphorus oxychloride, triethylamine and formula (Ic) are reacted. Then, the reaction was carried out with diol (Id), or the reaction solution was concentrated and then subjected to silica gel column chromatography (the mobile phase was petroleum ether and ethyl acetate) and then reacted with diol (Id). Typical reaction conditions include reacting phosphorus trichloride, triethylamine and formula (Ic) in an inert solvent such as DCM for about 0.5 hours at room temperature, then adding diol (ld) and reacting for about 12 hours at room temperature. After the reaction solution is concentrated, column chromatography purification (mobile phase is water and acetonitrile) is carried out by a C18 column through a rapid column passing instrument, and the separated fraction is freeze-dried by a freeze dryer to obtain the compound shown in the general formula (Ia).

Reactive type IV

The compounds of formula (Ic) can be prepared by: according to equation four, t-butyldimethylchlorosilane (TBSCl), formula (Ie), and imidazole are reacted. Typical reaction conditions include reacting t-butyldimethylchlorosilane, formula (Ie) and imidazole in a solvent such as DMF at room temperature for about 12 hours. After the reaction, the reaction solution was concentrated and then subjected to silica gel column chromatography (the mobile phase is petroleum ether and ethyl acetate), and the fraction obtained by separation was concentrated to obtain the general formula (If).

ii removing the 5' -terminal tert-butyldimethylsilane protecting group of formula (If) under the action of trifluoroacetic acid and water to give formula Ic). Typical reaction conditions include reacting formula (If), trifluoroacetic acid and water in a solvent such as THF at 0 ℃ for about 5 hours. The reaction solution is filtered after the pH value is regulated to be neutral, and the compound of the general formula (Ic) is obtained after the filter cake is collected.

Reactive type five

The compounds of the general formula (Ih) can be prepared by: reacting the general formula (Ig), thionyl chloride and a catalytic amount of DMF. Typical reaction conditions include reacting a compound of formula (Ig), thionyl chloride and a catalytic amount of DMF in a solvent such as DCM for about 2 hours at room temperature. Then the reaction solution is concentrated, ethanol is added, the reaction is carried out at room temperature for about one hour, after the completion, the reaction solution is concentrated and then is subjected to silica gel column chromatography (the mobile phase is petroleum ether and ethyl acetate), and the fraction obtained by separation is concentrated to obtain the general formula (Ih).

ii reaction of the intermediate of formula (Ih) with the ester at low temperature (about-60 ℃) under the action of lithium hexamethyldisilazide. Typical reaction conditions include: at a temperature of about-60 ℃, the reaction of the general formula (Ih), lithium hexamethyldisilazide and ethyl acetate is carried out at this low temperature for about 20min. The reaction solution was neutralized with acetic acid, concentrated, purified by silica gel column chromatography (petroleum ether and ethyl acetate as mobile phases), and the fraction obtained by separation was concentrated to obtain the general formula (Ii).

iii-in a protic solvent (e.g., meOH) with a reducing agent (e.g., naBH) ₄ ) With the general formula (Ii) to obtain the compound of the general formula (Id).

Reactive six

In a particular embodiment, the chiral 1, 3-propanediol derivative (ll/lm) of the compound of formula (ld) is prepared by the following method:

the method comprises the following steps:

in the presence of an acid (such as TMSOTF), hexamethyldisilazane and trimethyl silicone triflate are reacted in an inert solvent (such as DCM), and the crude product obtained by concentration after washing with water is reacted with (2S, 5R) -2-isopropyl-5-methylcyclohexanone under the action of the acid (such as TMSOTF) to obtain the intermediate compounds of the general formulas (Ij) and (Ik).

And ii, respectively reacting concentrated hydrochloric acid with the general formulas (Ij) and (Ik) at room temperature, concentrating the reaction solution, and separating the obtained fraction by silica gel column chromatography (the mobile phase is petroleum ether and ethyl acetate), thereby obtaining the general formula (Il/Im).

The second method is as follows:

i, at 40-80 ℃, in an inert solvent (such as DMF), formic acid, triethylamine, (S, S) -N- (p-toluenesulfonyl) -1, 2-diphenyl ethylenediamine (dichloro) (p-cymene) ruthenium (II) and an intermediate Ij react to obtain an intermediate of a general formula (Ip).

ii in a protic solvent (e.g., meOH) with a reducing agent (e.g., naBH ₄ ) And reacting with the general formula (Ip) to obtain the compound of the general formula (In).

The preparation of the formulae (VI) and (VII) can be referred to in formulae one to five, where the achiral diols of formula five need to be replaced by the chiral diols obtained in formula six. The compounds of the general formulae (VIII) and (IX) can be obtained further after separation of the compounds of the general formulae ((VI) and (VII) by SFC it is understood that within the scope of the invention the above-described technical features of the invention and the technical features described in detail below (e.g.in the examples) can be combined with one another to form new or preferred technical solutions.

Pharmaceutical compositions and methods of administration

Since the compound of the present invention has excellent activation activity to AMPK, the compound of the present invention and various crystalline forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and a pharmaceutical composition containing the compound of the present invention as a main active ingredient can be used for treating, preventing and alleviating diseases caused by hepatitis b virus. According to the prior art, the compounds of the invention are useful for the treatment of: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and liver cirrhosis related thereto, and non-alcoholic fatty liver disease (NAFLD) including liver cancer, and can be used for treating metabolic diseases such as obesity, diabetes, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, cardiovascular diseases, etc.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical compositions contain 0.1 to 1000mg of the compound of the invention per dose, more preferably 0.5 to 500mg of the compound of the invention per dose. Preferably, the "one dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifying agents (e.g., tween), wetting agents (e.g., sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration. A particularly preferred mode of administration is oral.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, e.g., glycerin; (d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, such as paraffin; (f) an absorption accelerator, e.g., a quaternary amine compound; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) an adsorbent, for example, kaolin; and (i) a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.

In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-agar or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When a pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective dose, and the daily dose is usually 0.2 to 1000mg, preferably 0.5 to 500mg, for a human having a body weight of 60 kg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

The main advantages of the invention include:

the invention prepares the phosphorothioate nucleoside monophosphate into a cyclic phosphorothioate prodrug with liver tissue specificity, and the drug molecules have better stability to gastrointestinal tracts and blood plasma and are not easy to be hydrolyzed by ester hydrolase in vivo; after the medicine enters the liver, the medicine is oxidized by CYP3A in cytochrome P450 isoenzyme family in tissue cells, the 4-position of the medicine molecule phosphorothioate ring is opened to generate an intermediate with monophosphate negative charge, and then the intermediate is subjected to phosphatase catalytic hydrolysis and beta-elimination reaction to release the nucleoside monophosphate of the parent medicine phosphorothioate. The phosphorothioate nucleoside monophosphate is in a protonated state in cells, is not easy to pass through cell membranes and is kept in the cells, so that the concentration of the medicine in liver cells is higher than that of normal tissues. The byproduct aryl ketene obtained after the prodrug is oxidized by CYP3A can be rapidly combined with glutathione with abundant antioxidant and free radical in liver cells to be cleared, and no report on side effects of the byproduct aryl ketene is found up to the present. The main advantages of the invention include:

(1) The liver specificity is strong, and the prodrug in the invention, namely the compound of the general formula (I), is metabolized in liver cells and generates parent drug after being orally taken; the medicine molecule has high negative charge and is not easy to be discharged out of the liver, so that the concentration in the liver is higher, and the liver targeting effect is achieved.

(2) The invention belongs to liver targeting drugs, has good tissue distribution of active drug molecules, and greatly improves the drug effect because more drug molecules exist in liver cells. Because drug molecules are mainly concentrated in liver cells, the amount of active molecules metabolized extrahepatic is small, and thus side effects on kidneys, heart, etc. are greatly reduced.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Intermediate preparation example 1: ((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methanol

The reaction steps are as follows:

step 1: preparation of 9- ((2R, 3R,4R, 5R) -3, 4-di ((tert-butyldimethylsilyl) oxy) -5- (((tert-butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2-yl) -9H-purin-6-amino)

(2R, 3R,4S, 5R) -2- (6-amino-9H-purin-9-yl) -5- (hydroxymethyl) tetrahydrofuran-3, 4-diol (16.0 g,60.0 mmol) was dissolved in N, N-dimethylformamide (120 mL), cooled to 0℃and imidazole (20.4 g,300.0 mmol) and t-butyldimethylchlorosilane (36.2 g,240.0 mmol) were added, respectively, and the reaction mixture was stirred at room temperature. After the completion of the reaction, the organic solvent was distilled off under reduced pressure, and the crude product was dissolved in ethyl acetate (100 mL), washed twice with saturated ammonium chloride (100 mL), and the organic phase was dried over anhydrous sodium sulfate, concentrated, and chromatographed on a silica gel column (petroleum ether: ethyl acetate=2:1) to give 34g of a white solid in 93% yield. MS (ES) ⁺ )m/z 610(M+H ⁺ ).

Step 2: preparation of((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methanol

9- ((2R, 3R,4R, 5R) -3, 4-di ((tert-butyldimethylsilyloxy) oxy) -5- (((tert-butyldimethylsilyloxy) methyl) tetrahydrofuran-2-yl) -9H-purin-6-amino (3.9 g,6.4 mmol) was dissolved in tetrahydrofuran (30 mL) and trifluoroacetic acid/water (12 m) was added at 0deg.CL/12 mL). The reaction solution was reacted at 0 ℃ for 16 hours, after the reaction was completed, the reaction solution was poured into a saturated sodium bicarbonate solution to adjust ph=8, extracted with ethyl acetate (150 ml×2), and the ethyl acetate phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and dried by spin-drying. The crude product was slurried with ethyl acetate (15 mL) to give 2.2g of a white solid in 73% yield. . ¹ H NMR(400MHz,DMSO-d ₆ ):δ8.41(s,1H),8.14(s,1H),7.40(s,2H),5.91(d,J＝4.0Hz,1H),5.76-5.74(m,1H),4.91-4.90(m,1H),4.31(d,J＝4.0Hz,1H),4.00(s,1H),3.77-3.76(m,1H),3.58-3.35(m,1H),0.93(s,9H),0.70(s,9H),0.13-0.12(m,6H),-0.14(s,3H),-0.46(s,3H).

Intermediate preparation example 2:1- (3-chloro-2-fluorophenyl) propane-1, 3-diol

The reaction steps are as follows:

step 1: preparation of ethyl 3-chloro-2-fluorobenzoate

3-chloro-2-fluorobenzoic acid (4.9 g,28.5 mmol) and DMF (10 drops) were dissolved in dichloromethane (40 mL), oxalyl chloride (4.3 g,34.2 mmol) was added at 0deg.C, the reaction stirred for 1 hour, concentrated to dryness, the crude product was added with ethanol (40 mL) at 0deg.C, stirred for 1 hour at room temperature, concentrated to ethyl acetate (50 mL), washed with sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate and concentrated to give 5.8g of anhydrous oil, 100% yield. ¹ H NMR(400MHz,CDCl ₃ ):δ7.84-7.81(m,1H),7.59-7.56(m,1H),7.17-7.13(m,1H),4.41(q,J＝4.0Hz,H),1.40(t,J＝4.0Hz,3H).

Step 2: preparation of ethyl 3- (3-chloro-2-fluorobenzene) -3-oxopropionate

Ethyl 3-chloro-2-fluorobenzoate (5.8 g,28.5 mmol) and ethyl acetate (17.6 g,199.5 mmol) were dissolved in tetrahydrofuran (60 mL), after cooling to-60 ℃ LiHMDS (85.5 mL,85.5 mmol) was added, the reaction mixture was stirred at-60 ℃ for 30 min, then quenched by the addition of acetic acid (9 mL), water (50 mL) was added, the reaction mixture was extracted with ethyl acetate (40 mL x 2), the organic phases combined, dried over anhydrous sodium sulfate and concentrated to give 8.2g crude brown oil. ¹ H NMR(400MHz,CDCl ₃ ):δ12.68(s,0.4H),7.84-7.81(m,1H),7.64-7.61(m,1H),7.23-7.20(m,1H),5.83(s,0.4H),4.29-4.22(m,2H),4.05-3.99(m,2H),1.36-1.29(m,3H).

Step 3: preparation of 1- (3-chloro-2-fluorophenyl) propane-1, 3-diol

Ethyl 3- (3-chloro-2-fluorobenzene) -3-oxopropionate (8.2 g,28.5 mmol) was dissolved in methanol (70 mL), naBH4 (5.4 g,14.2 mmol) was added at 0 ℃ and after stirring at room temperature for 3 hours, ethyl acetate (20 mL) was added. The reaction mixture was concentrated, ethyl acetate/water (50 mL/50 mL) was added, the ethyl acetate phase was concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane: methanol=10:1) to give 3.2g of a colorless oil, and the two-step yields of step 2 and step 3 were 55%. ¹ H NMR(400MHz,CDCl ₃ ):δ7.51-7.41(m,1H),7.36-7.32(m,1H),7.16-7.12(m,1H),5.34-5.30(m,1H),3.96-3.91(m,2H),3.27(d,J＝4.0Hz,1H),2.19-2.16(m,1H),2.07-2.00(m,2H).

Intermediate preparation example 3: 1-phenylpropane-1, 3-diol

Reference to the process for the preparation of intermediate preparation 2, except that benzoic acid was used as starting material, yield 74%, ¹ H NMR(400MHz,CDCl ₃ ):δ7.45-7.35(m,5H),5.17-5.12(m,1H),4.19-4.05(m,2H),2.23-2.16(m,1H),2.07-2.00(m,1H).

intermediate preparation example 4:1- (pyridin-4-yl) propane-1, 3-diol

Reference to the process for the preparation of intermediate preparation 2, except that isonicotinic acid was used as starting material, yield 50%, ¹ H NMR(400MHz,CDCl ₃ ):δ8.56-8.53(m,2H),7.55-7.54(m,2H),5.13-5.11(m,1H),4.11-4.10(m,1H),3.97-3.95(m,2H),2.38(brs,1H),2.01-1.99(m,2H).

intermediate preparation example 5:1- (3-chlorophenyl) propane-1, 3-diol

The preparation method of reference intermediate preparation example 2 is different from that 3-chlorobenzoic acid is taken as a starting material, the yield is 25 percent, ¹ H NMR(400MHz,CDCl ₃ )δ7.39(t,J＝1.8Hz,1H),7.34-7.25(m,3H),4.95(dd,J＝8.4,4.2Hz,1H),3.87(t,J＝5.4Hz,2H),1.99-1.88(m,2H).

intermediate preparation example 6:1- (3- (trifluoromethyl) phenyl) propane-1, 3-diol

Reference to the preparation of intermediate preparation 2, except that 3- (trifluoromethyl) benzoic acid was used as starting material in 48% yield, MS (ES ⁺ )m/z 221(M+H ⁺ ).

Intermediate preparation example 7:1- (3-methylphenyl) propane-1, 3-diol

Reference to the process for the preparation of intermediate preparation 2, except that 3-methylbenzoic acid was used as the starting material, ¹ H NMR(400MHz,CDCl ₃ )δ7.27-7.09(m,4H),4.95-4.92(m,1H),3.88-3.85(m,2H),2.36(s,3H),2.04-1.91(m,2H).

intermediate preparation example 8:1- (3-methoxyphenyl) propane-1, 3-diol

Reference to the process for the preparation of intermediate preparation 2, except that 3-methoxychlorobenzoic acid was used as starting material, ¹ H NMR(400MHz,CDCl ₃ )δ7.29-7.25(m,1H),6.95-6.93(m,2H),6.83-6.81(m,1H),4.97-4.94(m,1H),3.89-3.86(m,2H),3.82(s,3H),1.99-1.92(m,2H).

intermediate preparation example 9:1- (3-fluorophenyl) propane-1, 3-diol

Reference to the process for preparing intermediate preparation 2, except that 3-fluorobenzoic acid was used as the starting material in a yield of 50%, MS (ES) ⁺ )m/z 171(M+H ⁺ ).

Intermediate preparation 10:1- (5-chloro-2-fluorophenyl) propane-1, 3-diol

Reference to the process for preparing intermediate preparation 2, except that 5-chloro-2-fluorobenzoic acid was used as the starting material in 55% yield, ¹ H NMR(400MHz,CDCl ₃ ):7.51(t,J＝8.0Hz,1H),7.18(d,J＝8.0Hz,1H),7.18-7.05(m,1H),5.27-5.24(m,1H),3.93-3.89(m,2H),3.37(brs,1H),2.38(brs,1H),2.00-1.98(m,2H).

Intermediate preparation 11:1- (4-chloro-2-fluorophenyl) propane-1, 3-diol

The preparation method of reference intermediate preparation example 2 is different in that 4-chloro-2-fluorobenzoic acid is taken as a starting material, the yield is 64%, ¹ H NMR(400MHz,CDCl ₃ ):δ7.51(t,J＝8.0Hz,1H),7.18(d,J＝8.0Hz,1H),7.18-7.05(m,1H),5.27-5.24(m,1H),3.93-3.89(m,2H),3.37(brs,1H),2.38(brs,1H),2.00-1.98(m,2H).

intermediate preparation example 12:1- (2, 5-difluorophenyl) propane-1, 3-diol

The preparation method of reference intermediate preparation example 2 is different in that 2, 5-difluorobenzoic acid is taken as a starting material, the yield is 50%, ¹ H NMR(400MHz,CDCl ₃ )δ7.38-7.27(m,1H),7.10-6.88(m,2H),5.48-5.13(m,1H),4.26-4.00(m,1H),3.90-3.94(m,1H),3.34(d,J＝3.6Hz,1H),2.15-1.81(m,2H).

intermediate preparation example 13:1- (2, 5-dichlorophenyl) propane-1, 3-diol

Reference to the preparation of intermediate preparation 2, except that 2, 5-dichlorobenzoic acid was used as the starting material in 54% yield, MS (ES ⁺ )m/z 221(M+H ⁺ ).

Intermediate preparation example 14:1- (2-chloro-4-fluorophenyl) propane-1, 3-diol

The preparation method of reference intermediate preparation example 2 is different in that 2-chloro-4-fluorobenzoic acid is taken as a starting material, the yield is 64%, ¹ H NMR(400MHz,CDCl ₃ )δ7.70-7.45(m,1H),7.09-7.07(m,2H),5.49-5.28(m,1H),4.27-4.01(m,2H),2.34-2.31(m,1H),1.97-1.71(m,1H).

intermediate preparation example 15 1- (2, 4, 5-trifluorophenyl) propane-1, 3-diol

Reference to the process for preparing intermediate preparation 2, except that 2,4, 5-trifluorobenzoic acid was used as the starting material in 71% yield, ¹ H NMR(400MHz,MeOD)δ7.41-7.36(m,1H),7.14-7.07(m,1H),5.09-5.06(m,1H),3.68-3.62(m,2H),1.90-1.85(m,2H).

intermediate preparation example 16:1- (2-chloro-4, 5-difluorophenyl) propane-1, 3-diol

The preparation method of reference intermediate preparation example 2 is different from that 2-chloro-4, 5-difluorobenzoic acid is taken as a starting material, the yield is 75%, ¹ HNMR:(400MHz,CDCl ₃ )δ7.50(dd,J＝11.2,8.6Hz,1H),7.17(dd,J＝9.6,7.0Hz,1H),5.26(d,J＝7.4Hz,1H),3.96-3.91(m,2H),2.02(dd,J＝5.2,2.8Hz,1H),1.89-1.78(m,1H).MS(ES ⁺ )m/z 245.1(M+Na ⁺ )

Intermediate preparation example 17:1- (5-chloro-2, 4-difluorophenyl) propane-1, 3-diol

Reference to the process for preparing intermediate preparation 2, except that 5-chloro-2, 4-difluorobenzoic acid was used as the starting material in 74% yield, ¹ HNMR:(400MHz,CDCl ₃ )δ7.62(t,J＝7.8Hz,1H),6.87(dd,J＝9.6,9.0Hz,1H),5.22(dd,J＝8.0,3.4Hz,1H),3.93-3.90(m,2H),1.97-1.93(m,2H).MS(ES ⁺ )m/z245.1(M+Na ⁺ )

intermediate preparation example 18:1- (2, 4-dichloro-5-fluorophenyl) propane-1, 3-diol

Reference to the process for preparing intermediate preparation 2, except that 2, 4-dichloro-5-fluorobenzoic acid was used as the starting material in 91% yield, ¹ H-NMR:(400MHz CDCl ₃ )δ7.48(d,J＝9.8Hz,1H),7.38(d,J＝6.6Hz,1H),5.25(dd,J＝8.8,2.2Hz,1H),3.99-3.85(m,2H),2.04-2.00(m,1H),1.86-1.79(m,1H).

intermediate preparation example 19:1- (2, 3,4, 5-tetrafluorophenyl) propane-1, 3-diol

The preparation method of reference intermediate preparation example 2 is different in that 2,3,4, 5-tetrafluorobenzoic acid is taken as a starting material, the yield is 40%, ¹ HNMR:(400MHz,CDCl ₃ )δ7.26-7.18(m,1H),5.26(dd,J＝8.4,2.5Hz,1H),4.11-3.88(m,2H),1.97-1.90(m,2H).

intermediate preparation example 20:1- (3-chloro-2, 4, 5-trifluorophenyl) propane-1, 3-diol

Reference to the process for preparing intermediate preparation 2, except that 3-chloro-2, 4, 5-trifluorobenzoic acid was used as the starting material in a yield of 78%, ¹ HNMR:(400MHz,CDCl ₃ )δ7.38(ddd,J＝10.4,8.4,6.6Hz,1H),5.26(dd,J＝8.2,2.4Hz,1H),4.01-3.83(m,2H),1.98-1.93(m,2H).

intermediate preparation example 21:1- (pentafluorophenyl) propane-1, 3-diol

Reference to the process for preparing intermediate preparation 2, except that pentafluorobenzoic acid was used as the starting material in 88% yield, ¹ HNMR:(400MHz,CD ₃ OD)δ5.25(dd,J＝8.6,5.8Hz,1H),3.75-3.66(m,1H),3.66-3.58(m,1H),2.302.16(m,1H),2.04-1.90(m,1H).

intermediate preparation example 22: chiral 1- (4-chloro-2-fluorophenyl) propane-1, 3-diol

Step 1: preparation of (2R, 6S,7S, 10R) -2- (4-chloro-2-fluorophenyl) -7-isopropyl-10-methyl-1, 5-dioxaspiro [5.5] undecane

1- (4-chloro-2-fluorophenyl) propane-1, 3-diol (3.0 g,14.7 mmol) and hexamethyldisilazane (6.73 mL,32.3 mmol) were dissolved in tetrahydrofuran (12 mL), a plurality of drops of trimethylsilicone triflate were added at room temperature, and the reaction was stirred at room temperature for 2 hours,ethyl acetate (50 mL) was added, and the organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated. The crude product was further dissolved in dry dichloromethane (30 mL), added with (2S, 5R) -2-isopropyl-5-methylcyclohexanone (2.72 g,17.64 mmol), cooled to-40 ℃, added with trimethylsilicone triflate (399mg, 1.764 mmol), the reaction was stirred overnight at-40 ℃, pyridine (2 mL) was added to quench after the reaction was completed, dichloromethane (30 mL) was added to the reaction, and saturated sodium bicarbonate solution (30 mL) was washed twice. The organic phases were combined and concentrated and the crude silica gel column chromatographed (petroleum ether: ethyl acetate=100:1) to give 1.9g of (2 r,6s,7s,10 r) -2- (4-chloro-2-fluorophenyl) -7-isopropyl-10-methyl-1, 5-dioxaspiro [5.5 ] as a colorless oil]Undecane, yield 38%, ¹ H NMR(400MHz,CDCl ₃ ) Delta 7.54 (t, j=8.0 hz, 1H), 7.20 (dd, j=8.0, 2.0hz, 1H), 7.07 (dd, j=10.0, 2.0hz, 1H), 5.41-5.37 (m, 1H), 4.10-4.03 (m, 1H), 3.89-3.85 (m, 1H), 2.91-2.86 (m, 1H), 2.63-2.59 (m, 1H), 1.87-1.24 (m, 8H), 1.10-0.71 (m, 12H); and oily (2S, 6S,7S, 10R) -2- (4-chloro-2-fluorophenyl) -7-isopropyl-10-methyl-1, 5-dioxaspiro [5.5 ] ]Undecane, yield 30%, ¹ H NMR(400MHz,CDCl ₃ )δ7.52(t,J＝8.0Hz,1H),7.18(dd,J＝8.0,2.0Hz,1H),7.07(dd,J＝10.0,2.0Hz,1H),5.19-5.16(m,1H),4.32-4.26(m,1H),3.94-3.89(m,1H),2.92-2.88(m,1H),2.55-2.51(m,1H),1.91-1.22(m,8H),1.11-0.65(m,12H).

step 2:

preparation of (R) -1- (4-chloro-2-fluorophenyl) propane-1, 3-diol

(2R, 6S,7S, 10R) -2- (4-chloro-2-fluorophenyl) -7-isopropyl-10-methyl-1, 5-dioxaspiro [5.5]Undecane (1.9 g,5.59 mmol) was dissolved in methanol (10 mL), concentrated hydrochloric acid (1.5 mL) was added at room temperature, the reaction stirred overnight at room temperature, concentrated, water (30 mL) was added, ethyl acetate was extracted (30 mL x 2), the organic phases were combined and concentrated, the crude product was purified by silica gel column chromatography (dichloromethane: methanol=10:1) to give 0.9g of (R) -1- (4-chloro-2-fluorophenyl) propane-1, 3-diol as a colorless oil in 80% yield, ¹ H NMR(400MHz,CDCl ₃ ):δ7.51(t,J＝8.0Hz,1H),7.18(d,J＝8.0Hz,1H),7.09-7.05(m,1H),5.27-5.26(m,1H),3.93-3.90(m,2H),3.30(brs,1H),2.28-2.26(m,1H),2.06-1.98(m,2H).

step 3:

preparation of (S) -1- (4-chloro-2-fluorophenyl) propane-1, 3-diol

Referring to the process for preparing (R) -1- (4-chloro-2-fluorophenyl) propane-1, 3-diol in step 2, the yield was 78%, ¹ H NMR(400MHz,CDCl ₃ ):δ7.51(t,J＝8.0Hz,1H),7.18(d,J＝8.0Hz,1H),7.09-7.05(m,1H),5.27-5.26(m,1H),3.93-3.90(m,2H),3.31(brs,1H),2.27-2.25(m,1H),2.06-1.98(m,2H).

intermediate preparation example 23 (R) -1- (3-chlorophenyl) propane-1, 3-diol

Reference intermediate preparation 22 step 1 and step 2 except that 1- (4-chloro-2-fluorophenyl) propane-1, 3-diol in reference intermediate preparation 22 was replaced with 1- (3-chloro-phenyl) propane-1, 3-diol. The yield was 9%, ¹ H NMR(400MHz,CDCl ₃ )δ7.38-7.07(m,4H),4.84-4.80(m,1H),4.22-4.20(m,1H),3.75-3.72(m,2H),3.54-3.53(m,1H),1.88-1.82(m,2H).

intermediate preparation example 24: (S) -1- (3-chlorophenyl) propane-1, 3-diol

Reference intermediate preparation 22 step 1 and step 3 except that 1- (4-chloro-2-fluorophenyl) propane-1, 3-diol in reference intermediate preparation 22 was replaced with 1- (3-chloro-phenyl) propane-1, 3-diol. The yield thereof was found to be 17%, ¹ H NMR(400MHz,CDCl ₃ )δ7.37-7.07(m,4H),4.84-4.80(m,1H),4.23-4.20(m,1H),3.75-3.72(m,2H),3.55-3.53(m,1H),1.88-1.82(m,2H).

Intermediate preparation example 25: (R) -1- (2, 5-dichlorophenyl) propane-1, 3-diol

Reference intermediate preparation example 22 step 1 and step 2 except that 1- (4-chloro-2-fluorophenyl) propane-1, 3-diol in reference intermediate preparation example 22 was replaced with 1- (2, 5-dichloro-phenyl) propane-1, 3-diol. The yield thereof was found to be 12%, ¹ H NMR(400MHz,CDCl ₃ ):δ7.66-7.65(m,1H),7.25-7.18(m,2H),5.32-5.29(m,1H),3.95-3.92(m,2H),3.65(brs,1H),2.53(brs,1H),2.09-2.03(m,1H),1.92-1.78(m,1H).

intermediate preparation example 26: (S) -1- (2, 5-dichlorophenyl) propane-1, 3-diol

Reference intermediate preparation example 22 step 1 and step 3 except that 1- (2, 5-dichloro-phenyl) propane-1, 3-diol was used instead of the reference intermediate preparation1- (4-chloro-2-fluorophenyl) propane-1, 3-diol in example 22. The yield thereof was found to be 17%, ¹ H NMR(400MHz,CDCl ₃ ):δ7.66-7.65(m,1H),7.25-7.18(m,2H),5.32-5.29(m,1H),3.95-3.92(m,2H),3.64(brs,1H),2.52(brs,1H),2.09-2.03(m,1H),1.91-1.71(m,1H).

intermediate preparation example 27:2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-chloro-2-fluorobenzene) -1,3, 2-dioxaphosphorinane 2-sulfide

((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methanol (396 mg,0.8 mmol) was dissolved in anhydrous pyridine (4 mL) and PSCl was added at 0deg.C ₃ After reaction at 189mg,1.1 mmol) for 30 min at 0deg.C, a solution of 1- (3-chloro-2-fluorophenyl) propane-1, 3-diol (277 mg,1.4 mmol) in DCM (0.5 mL) was added and the reaction stirred overnight, concentrated and the crude product purified via a C18 prep column to give 220mg of a white solid in 36% yield. MS (ES) ⁺ )m/z 760(M+H ⁺ ).

Intermediate preparation example 28:2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4-phenyl-1, 3, 2-dioxaphosphorinane 2-sulfide

With reference to the preparation method of intermediate preparation example 27, except that 1-phenylpropane-1, 3-diol was used as a raw material, the yield was 25%, ¹ H NMR(400MHz,CDCl ₃ )δ8.37(d,J＝1.4Hz,1H),8.21(d,J＝2.2Hz,1H),7.39-7.36(m,5H),5.75-5.72(m,3H),4.88-4.68(m,3H),4.45-4.31(m,5H),2.49-2.27(m,1H),2.27-2.01(m,1H),0.95(d,J＝2.8Hz,9H),0.80(d,J＝2.4Hz,9H),0.14-0.11(m,6H),-0.02(d,J＝5.0Hz,3H),-0.23(d,J＝1.6Hz,3H).

intermediate preparation 29:2- (((2 r,3r,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (pyridin-4-yl) -1,3, 2-dioxaphosphorinane 2-sulfide

With reference to the preparation method of intermediate preparation example 27, except that 1- (pyridin-4-yl) propane-1, 3-diol was used as a starting material in 40% yield, ¹ H NMR(400MHz,CDCl ₃ ):δ8.66-8.61(m,2H),8.36(s,1H),8.12(s,1H),7.30-7.24(m,2H),6.02-6.00(m,1H),5.76-5.72(m,1H),5.62(s,2H),4.91-4.90(m,1H),4.81-4.72(m,2H),4.46-4.13(m,4H),2.35-2.21(m,1H),2.10-2.07(m,1H),0.96(s,9H),0.83-0.81(s,9H),0.17-0.13(m,6H),0.02(s,3H),-0.18(s,3H).

intermediate preparation example 30:2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

With reference to the preparation method of intermediate preparation example 27, except that 1- (3-chlorophenyl) propane-1, 3-diol was used as the starting material, the yield was 59%, ¹ H NMR(400MHz,CDCl ₃ )δ8.37(d,J＝3.6Hz,1H),8.18-7.99(m,1H),7.58-7.32(m,4H),6.13-5.98(m,1H),5.91(d,J＝5.0Hz,1H),5.59(s,2H),5.18-5.08(m,1H),4.96-4.66(m,2H),4.53-4.29(m,2H),4.09(dd,J＝11.8,6.8Hz,1H),3.77(dd,J＝11.8,4.4Hz,1H),2.33-2.00(m,2H),0.96(d,J＝2.8Hz,9H),0.86-0.80(m,9H),0.19-0.07(m,6H),-0.01(d,J＝1.4Hz,3H),-0.21(d,J＝7.4Hz,3H).

intermediate preparation 31:2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3- (trifluoromethyl) phenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Referring to the preparation method of intermediate preparation example 27, except that 1- (3- (trifluoromethyl) phenyl) propane-1, 3-diol was used as a raw material, the yield was 41%, ¹ H NMR(400MHz,CDCl ₃ )δ8.36(d,J＝2.6Hz,1H),8.14(d,J＝0.8Hz,1H),7.67-7.58(m,2H),7.53(dd,J＝14.8,8.6Hz,2H),6.01(dd,J＝5.2,3.4Hz,1H),4.94-4.67(m,3H),4.59-4.35(m,4H),4.33(s,1H),2.43-2.20(m,2H),0.95(d,J＝2.6Hz,9H),0.81(d,J＝4.8Hz,9H),0.20-0.05(m,6H),0.00--0.06(m,3H),-0.16--0.25(m,3H).

intermediate preparation example 32:2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-methylphenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Reference to the preparation of intermediate preparation 27, except that 1- (3-methylphenyl) was usedPropane-1, 3-diol is used as raw material, the yield is 54 percent, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.55-8.33(m,1H),8.25-8.04(m,1H),7.47-7.03(m,6H),5.97(d,J＝6.8Hz,1H),5.66(d,J＝11.2Hz,1H),5.04-4.88(m,1H),4.65-4.59(m,2H),4.54-4.37(m,3H),4.23-4.17(m,1H),2.31-2.09(m,5H),0.99-0.82(m,9H),0.78-0.60(m,9H),0.20-0.05(m,6H),-0.06--0.12(m,3H),-0.34--0.39(m,3H).

intermediate preparation example 33:2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-trimethoxyphenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Referring to the preparation method of intermediate preparation example 27, except that 1- (3-trimethoxyphenyl) propane-1, 3-diol was used as a raw material, the yield was 47%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.48-8.39(m,1H),8.18-8.14(m,1H),7.61-7.15(m,3H),7.09-6.78(m,3H),5.98-5.96(m,1H),5.68(d,J＝11.0Hz,1H),5.50-4.91(m,1H),4.72-4.00(m,6H),3.76-3.74(m,3H),2.39-2.09(m,2H),1.03-0.80(m,9H),0.80-0.51(m,9H),0.22-0.05(m,6H),-0.04--0.16(m,3H),-0.39(d,J＝4.4Hz,3H).

intermediate preparation example 34:2- (((2 r,3r,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

With reference to the preparation method of intermediate preparation example 27, except that 1- (3-fluorophenyl) propane-1, 3-diol was used as a raw material, the yield was 87%, ¹ H NMR(400MHz,MeOD)δ8.39(d,J＝1.4Hz,1H),8.24(s,1H),7.46-7.24(m,1H),7.24-6.97(m,3H),6.14-6.10(m,1H),5.76-5.67(m,1H),4.73-4.70(m,2H),4.65-4.50(m,2H),4.50-4.31(m,3H),2.35-2.16(m,1H),2.15-2.00(m,1H),0.96(d,J＝1.4Hz,9H),0.82(d,J＝3.2Hz,9H),0.16-0.13(m,6H),0.02-0.01(m,3H),-0.19(d,J＝6.2Hz,3H).

Intermediate preparation example 35:2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (5-chloro-2-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Preparation of reference intermediate preparation example 27The method is characterized in that 1- (5-chloro-2-fluorophenyl) propane-1, 3-diol is taken as a raw material, the yield is 31 percent, ¹ H NMR(400MHz,CDCl ₃ )δ8.37(s,1H),8.16-8.15(m,1H),7.54-7.41(m,2H),7.05-7.01(m,1H),6.01-5.91(m,2H),5.71(s,2H),4.91-4.78(m,3H),4.56-4.33(m,4H),2.32-2.22(m,1H),2.13-2.03(m,1H),0.98-0.79(m,18H),0.17-0.13(m,6H),0.02-0.01(m,3H),-0.07(s,3H).

intermediate preparation example 36:2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (4-chloro-2-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Reference to the preparation method of intermediate preparation example 27, except that 1- (4-chloro-2-fluorophenyl) propane-1, 3-diol was used as a raw material in a yield of 45%, ¹ H NMR(400MHz,MeOD):δ8.36(s,1H),8.17(s,1H),7.41-7.28(m,1H),7.15-7.13(m,2H),6.02-5.93(m,2H),5.64(brs,2H),4.89-4.80(m,3H),4.44-4.32(m,4H),2.31-2.11(m,2H),0.98-0.78(m,18H),0.16-0.12(m,6H),-0.01--0.19(m,3H),-0.21(s,3H).

intermediate preparation example 37:2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2, 5-difluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

With reference to the preparation method of intermediate preparation example 27, except that 1- (2, 5-difluorophenyl) propane-1, 3-diol was used as the starting material, the yield was 68%, ¹ H NMR(400MHz,CDCl ₃ )δ8.37(s,1H),8.16(d,J＝3.6Hz,1H),7.11-6.96(m,3H),6.04-5.93(m,2H),5.56(s,2H),4.94-4.87(m,1H),4.87-4.70(m,2H),4.56-4.41(m,3H),4.34(s,1H),2.32-1.99(m,2H),0.96(t,J＝2.0Hz,9H),0.83(d,J＝3.6Hz,9H),0.19-0.12(m,6H),0.09(s,3H),-0.16(d,J＝4.8Hz,3H).

intermediate preparation example 38:2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2, 5-dichlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

With reference to the preparation method of intermediate preparation example 27, except that 1- (2, 5-dichlorophenyl) propane-1, 3-diol was used as the starting material, the yield was 31%, ¹ H NMR(400MHz,CDCl3)δ8.37(s,1H),8.15(s,1H),7.51(dd,J＝23.8,2.5Hz,1H),7.36–7.28(m,3H),6.02(t,J＝5.0Hz,1H),5.67(s,2H),5.00–4.72(m,3H),4.62–4.30(m,5H),2.19–2.01(m,2H),0.97(s,9H),0.83(d,J＝9.5Hz,9H),0.21–0.11(m,6H),0.02(s,3H),-0.16(d,J＝23.6Hz,3H).

intermediate preparation 39:2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2-chloro-4-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Reference to the preparation method of intermediate preparation example 27, except that 1- (2-chloro-4-fluorophenyl) propane-1, 3-diol was used as a raw material in 33% yield, ¹ H NMR(400MHz,CDCl ₃ )δ8.36(d,J＝2.4Hz,1H),8.14(d,J＝1.8Hz,1H),7.49-7.45(m,1H),7.19-7.12(m,1H),7.12-6.94(m,1H),6.02(t,J＝5.6Hz,1H),5.70(s,2H),4.96-4.89(m,1H),4.89-4.69(m,2H),4.59-4.30(m,5H),2.28-1.91(m,2H),0.96(d,J＝1.6Hz,9H),0.82(d,J＝3.2Hz,9H),0.16-0.14(m,6H),0.01(s,3H),-0.19(s,3H).

intermediate preparation 40:2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2, 4, 5-trifluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Reference to the preparation method of intermediate preparation example 27, except that 1- (2, 4, 5-trifluorophenyl) propane-1, 3-diol was used as the starting material, the yield was 50%, MS (ES ⁺ )m/z 762(M+H ⁺ )。

Intermediate preparation 41:2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2-chloro-4, 5-difluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Reference to the preparation method of intermediate preparation example 27, except that 1- (2-chloro-4, 5-trifluorophenyl) propane-1, 3-diol was used as the starting material in a yield of 50%, ¹ H NMR(400MHz,CDCl ₃ )δ8.37(d,J＝2.0Hz,1H),8.15(d,J＝4.4Hz,1H),7.56-7.33(m,1H),7.28-7.22(m,1H),6.06-5.91(m,2H),5.65(s,2H),4.97-4.71(m,3H),4.63-4.25(m,4H),2.34-2.07(m,2H),1.08-0.59(m,18H),0.20-0.06(m,6H),0.02(d,J＝1.5Hz,3H),-0.08--0.29(m,3H).

Intermediate preparation example 42:2- (((2 r,3r,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (5-chloro-2, 4-difluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Reference to the preparation method of intermediate preparation 27, except that 1- (5-chloro-2, 4-difluorophenyl) propane-1, 3-diol was used as the starting material in a yield of 56%, ¹ H NMR(400MHz,CDCl ₃ )δ8.37(s,1H),8.14(s,1H),7.59-7.47(m,1H),6.99-6.92(m,1H),6.03–6.02(m,1H),5.96-5.92(m,1H),5.72(s,2H),4.90-4.71(m,3H),4.55-4.33(m,4H),2.33-2.19(m,2H),0.98-0.78(m,18H),0.16-0.12(m,6H),0.02(s,3H),-0.08--0.39(m,3H).

intermediate preparation 43:2- (((2 r,3r,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2, 4-dichloro-5-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

With reference to the preparation method of intermediate preparation example 27, except that 1- (2, 4-dichloro-5-fluorophenyl) propane-1, 3-diol was used as a raw material, the yield was 50%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.49-8.30(m,1H),8.24-8.11(m,1H),8.05-7.87(m,1H),7.73-7.53(m,1H),7.42-7.38(m,2H),6.03-5.78(m,2H),4.98-4.86(m,1H),4.72-4.33(m,5H),4.17-4.14(m,1H),2.39-2.25(m,1H),2.18-2.08(m,1H),1.01-0.79(m,9H),0.79-0.53(m,9H),0.20-0.04(m,6H),-0.05--0.18(m,3H),-0.33--0.53(m,3H).

intermediate preparation example 44:2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2, 3,4, 5-tetrafluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Referring to the preparation method of intermediate preparation example 27, except that 1- (2, 3,4, 5-tetrafluorophenyl) propane-1, 3-diol was used as the starting material, the yield was 51%, ¹ H NMR(400MHz,CDCl ₃ )δ8.36(d,J＝1.8Hz,1H),8.24-7.92(m,1H),7.27-7.06(m,1H),6.05-5.86(m,2H),5.66-5.59(m,2H),4.97-4.69(m,3H),4.58-4.32(m,4H),2.25-2.18(m,1H),2.11-2.08(m,1H),0.98-0.78(m,18H),0.23-0.07(m,6H),0.03-0.01(m,3H),-0.08--0.29(m,3H).

intermediate preparation example 45:2- (((2 r,3r,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-chloro-2, 4, 5-trifluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Referring to the preparation method of intermediate preparation example 27, except that 1- (3-chloro-2, 4, 5-trifluorophenyl) propane-1, 3-diol was used as the starting material, the yield was 86%, ¹ H NMR(400MHz,CDCl ₃ )δ8.37-8.36(m,1H),8.14-8.13(m,1H),7.45-7.20(m,1H),6.01(t,J＝4.0Hz,1H),5.95(t,J＝12.0Hz,1H),5.61(brs,2H),4.90(q,J＝9.4Hz,1H),4.84-4.72(m,2H),4.55-4.30(m,4H),2.28-2.15(m,1H),2.15-2.05(m,1H),0.96-0.92(m,9H),0.88-0.80(m,9H),0.18-0.10(m,6H),0.02(s,3H),-0.02(s,3H),-0.12--0.16(m,3H).

intermediate preparation example 46:2- (((2 r,3r,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (pentafluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

With reference to the preparation method of intermediate preparation example 27, except that 1- (pentafluorophenyl) propane-1, 3-diol was used as a starting material, in 55% yield, ¹ H NMR(400MHz,CDCl ₃ )δ8.37(d,J＝1.4Hz,1H),8.11(d,J＝5.0Hz,1H),6.12-5.97(m,2H),5.63(s,2H),4.88-4.65(m,3H),4.53-4.30(m,4H),2.79-2.71(m,1H),1.96-1.90(m,1H),1.03-0.72(m,18H),0.21-0.06(m,6H),-0.01--0.04(m,3H),-0.19--0.21(m,3H).

intermediate preparation example 47: n- (9- (3, 4-di ((tert-butyldimethylsilyl) oxy) -5- (((4- (3-chlorophenyl) -2-thio-1, 3, 2-dioxaphosphorinan-2-yl) oxy) methyl) tetrahydrofuran-2-yl) -9H-purin-6-yl) acetamide

2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide (1.1 g,1.6 mmol) is dissolved in anhydrous pyridine (5 mL), acetyl chloride (187 mg,2.39 mmol) is added at 0deg.C, the reaction is stirred at room temperature for 1 hour, concentrated, crude silica gel column chromatography (petroleum ether: ethyl acetate=1:1) gives 380mg white silica gel column chromatography (petroleum ether: ethyl acetate=1:1:1)The color solid, the yield is 30%, ¹ H NMR(400MHz,DMSO-d ₆ )δ10.73-10.74(m,1H),8.69-8.70(m,1H),8.64-8.65(m,1H),7.38-7.50(m,4H),6.08-6.09(m,1H),5.72-5.74(m,1H),4.94-4.98(m,1H),4.40-4.69(m,6H),2.24-2.28(m,3H),1.96–2.04(m,1H),0.92-0.93(m,9H),0.83-0.88(m,1H),0.68-0.69(m,9H),0.09-0.15(m,6H),(-0.11)-(-0.08)(m,3H),(-0.40)-(-0.38)(m,3H).

Intermediate preparation 48: n- (9- (3, 4-di ((tert-butyldimethylsilyl) oxy) -5- (((4- (3-chlorophenyl) -2-thio-1, 3, 2-dioxaphosphorinan-2-yl) oxy) methyl) tetrahydrofuran-2-yl) -9H-purin-6-yl) butanamide

Referring to the preparation method of intermediate preparation 47, except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was reacted with butyryl chloride in a yield of 33%, ¹ H NMR(CDCl ₃ ,400MHz)δ8.34(s,1H),8.23(s,1H),7.18-7.42(m,4H),6.07-6.10(m,1H),5.65-5.75(m,1H),4.68-4.85(m,4H),4.33-4.53(m,3H),2.85-2.88(m,2H),2.25-2.41(m,2H),1.57-1.963(m,2H),1.04-1.08(m,3H),0.95(s,9H),0.80(s,9H),0.11-0.14(m,6H),(-0.2)-0.02(m,3H),(-0.22)–(-0.20)(m,3H).

intermediate preparation example 49: (4R) -2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (4-chloro-2-fluorobenzene) -1,3, 2-dioxaphosphorinane 2-sulfide

Reference was made to the procedure for the preparation of intermediate preparation 27, except that (R) -1- (4-chloro-2-fluorophenyl) propane-1, 3-diol was used as starting material in 39% yield. MS (ES) ⁺ )m/z 760(M+H ⁺ ).

Intermediate preparation example 50: (4S) -2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (4-chloro-2-fluorobenzene) -1,3, 2-dioxaphosphorinane 2-sulfide

Reference to the preparation method of intermediate preparation 27, except that (S) -1- (4-chloro-2-fluorophenyl) propane-1, 3-diol was used as the starting material in 21.8% yield, ¹ H NMR(400MHz,MeOD)δ8.37(s,1H),8.13(s,1H),7.41(t,J＝8.0Hz,1H),7.27-7.12(m,2H),6.03-5.94(m,2H),5.67(m,2H),4.91-4.72(m,3H),4.52-4.33(m,4H),2.33-2.24(m,1H),2.10-2.07(m,1H),0.98-0.95(m,9H),0.83-0.78(m,9H),0.17-0.12(m,6H),-0.02(s,3H),-0.20(s,3H).

Intermediate preparation example 51: (4R) -2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Reference to the preparation method of intermediate preparation 27, except that (R) -1- (3-chloro-phenyl) propane-1, 3-diol was used as the starting material in 42% yield, ¹ H NMR(400MHz,CDCl ₃ )δ8.39-8.38(m,1H),8.18(s,1H),7.41-7.29(m,3H),7.23-7.20(m,1H),6.02(d,J＝5.0Hz,1H),5.84(s,2H),5.70(d,J＝9.4Hz,1H),4.93-4.66(m,3H),4.55-4.24(m,4H),2.43-2.21(m,1H),2.05-2.01(m,1H),0.96-0.94(m,9H),0.83-0.80(m,9H),0.21-0.10(m,6H),0.02(s,1H),-0.02(s,2H),-0.20(s,3H).

intermediate preparation example 52: (4S) -2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Reference to the preparation method of intermediate preparation 27, except that (S) -1- (3-chloro-phenyl) propane-1, 3-diol was used as the starting material in a yield of 52.7%, ¹ H NMR(400MHz,CDCl ₃ )δ8.40(s,1H),8.25(s,1H),7.41-7.29(m,3H),7.28-7.26(m,1H),6.20(s,2H),6.03-6.01(m,1H),5.73-5.69(m,1H),4.86-4.31(m,7H),2.41-2.34(m,1H),2.05-2.01(m,1H),0.99-0.75(m,18H),0.20-0.07(m,6H),-0.01(s,3H),-0.20(d,J＝16.0Hz,3H).

intermediate preparation example 53: (4R) -2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2, 5-dichlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Reference to the preparation method of intermediate preparation 27, except that (R) -1- (2, 5-dichloro-phenyl) propane-1, 3-diol was used as the starting material in 42% yield, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.43-8.41(m,1H),8.16-8.15(m,1H),7.58-7.29(m,5H),6.04-5.76(m,2H),5.50(d,J＝4.8Hz,1H),4.97-4.94(m,1H),4.66-4.45(m,4H),4.35-4.18(m,1H),2.33-2.16(m,2H),0.94-0.91(m,6H),0.70(s,9H),0.19-0.08(m,6H),-0.09(d,J＝5.4Hz,3H),-0.39(d,J＝11.0Hz,3H).

intermediate preparation example 54: (4S) -2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2, 5-dichlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Reference to the preparation method of intermediate preparation 27, except that (S) -1- (2, 5-dichloro-phenyl) propane-1, 3-diol was used as the starting material in a yield of 41%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.39-8.34(m,1H),8.16-8.02(m,1H),7.63-7.31(m,5H),5.98-5.89(m,2H),5.01-4.98(m,1H),4.73-4.38(m,5H),4.40-4.18(m,1H),2.38-2.14(m,2H),0.94-0.90(m,9H),0.71-0.68(m,9H),0.22-0.03(m,6H),-0.12(s,3H),-0.43(s,3H).

intermediate preparation example 55:9- ((2R, 3R,4R, 5R) -5- (aminomethyl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) -9H-purin-6-amino

The reaction steps are as follows:

step 1: preparation of N- (9- ((2R, 3R,4R, 5R) -3, 4-di ((tert-butyldimethylsilyl) oxy) -5- (((tert-butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2-yl) -9H-purin-6-yl) benzamide

N- (9- ((2R, 3R,4S, 5R) -3, 4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -9H-purin-6-yl) benzamide (1.0 g,2.7 mmol) was dissolved in N, N-dimethylformamide (20 mL), cooled to 0℃and imidazole (1.8 g,26.9 mmol) and t-butyldimethylchlorosilane (3.2 g,21.5 mmol) were added, respectively, and the reaction stirred at room temperature overnight. After the completion of the reaction, the organic solvent was distilled off under reduced pressure, and the crude product was dissolved in ethyl acetate (50 mL), washed twice with saturated ammonium chloride (20 mL), and the organic phase was dried over anhydrous sodium sulfate, concentrated, and chromatographed on a silica gel column (petroleum ether: ethyl acetate=3:1) to give 1.6g of a white solid in 84% yield. ¹ H NMR(400MHz,CDCl ₃ )δ8.83(s,1H),8.45(s,1H),8.08-8.06(m,2H),7.61(d,J＝7.4Hz,1H),7.55-7.52(m,2H),6.13(d,J＝5.0Hz,1H),4.67(t,J＝4.6Hz,1H),4.31(t,J＝3.8Hz,1H),4.17(d,J＝2.8Hz,1H),4.07-4.00(m,1H),3.81(dd,J＝11.4,2.6Hz,1H),0.96(s,9H),0.95(s,9H),0.80(s,9H),0.15(s,3H),0.14(s,3H),0.11(s,3H),0.10(s,3H),-0.02(s,3H),-0.23(s,3H)。

Step 2: preparation of N- (9- ((2R, 3R,4R, 5R) -3, 4-di ((tert-butyldimethylsilyl) oxy) -5- (hydroxymethyl) tetrahydrofuran-2-yl) -9H-purin-6-yl) benzamide

N- (9- ((2R, 3R,4R, 5R) -3, 4-di ((tert-butyldimethylsilyl) oxy) -5- (((tert-butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2-yl) -9H-purin-6-yl) benzamide (400.0 mg,0.6 mmol) was dissolved in tetrahydrofuran (3 mL) and trifluoroacetic acid/water (1.2 mL/1.2 mL) was added at 0deg.C. The reaction solution was reacted at 0 ℃ for 6 hours, after the reaction was completed, the reaction solution was poured into a saturated sodium bicarbonate solution to adjust ph=8, extracted with ethyl acetate (50 ml×2), and the ethyl acetate phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and dried by spin-drying. The crude product was slurried with ethyl acetate (15 mL) to give 300.0mg of a white solid in 88% yield, ¹ H NMR(400MHz,CDCl ₃ )δ8.83(s,1H),8.08(s,1H),8.06-8.01(m,2H),7.66-7.59(m,1H),7.54(t,J＝7.6Hz,2H),5.87(d,J＝7.8Hz,1H),5.04(dd,J＝7.8,4.4Hz,1H),4.36(d,J＝4.4Hz,1H),4.20(s,1H),3.98(dd,J＝13.2,1.8Hz,1H),3.74(dd,J＝13.2,1.8Hz,1H),0.94(s,9H),0.75(s,9H),0.14(s,3H),0.13(s,3H),-0.12(s,3H),-0.63(s,3H)。

step 3: preparation of methyl (((2R, 3R,4R, 5R) -5- (6-benzoyl-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methylsulfonate

Methanesulfonyl chloride (152.0 mg,1.3 mmol) was added to a solution of N- (9- ((2R, 3R,4R, 5R) -3, 4-di ((tert-butyldimethylsilyl) oxy) -5- (hydroxymethyl) tetrahydrofuran-2-yl) -9H-purin-6-yl) benzamide (200.0 mg,0.3 mmol) and triethylamine (202.0 mg,2.0 mmol) in dichloromethane (5 mL) under ice-bath. After stirring at room temperature for 12 hours, the reaction mixture was quenched by adding water (20 mL), extracted with dichloromethane (20 mL. Times.2), the organic phases were combined and concentrated, and the crude product was chromatographed on silica gel (petroleum ether: ethyl acetate=2:1) to give 140mg of a pale yellow oil in 87% yield, ¹ H NMR(400MHz,CDCl ₃ )δ8.81(s,1H),8.23(s,1H),8.05-8.03(m,2H),7.66-7.58(m,1H),7.53(t,J＝7.4Hz,2H),6.00(d,J＝4.8Hz,1H),4.97(t,J＝4.2Hz,1H),4.61(dd,J＝11.0,3.8Hz,1H),4.49(dd,J＝11.2,4.4Hz,1H),4.41-4.32(m,2H),3.03(s,3H),0.94(s,9H),0.82(s,9H),0.14(s,3H),0.13(s,3H),-0.00(s,3H),-0.21(s,3H).MS(ES ⁺ )m/z 678(M+H ⁺ )。

Step 4: preparation of N- (9- ((2R, 3R,4R, 5R) -5- (azidomethyl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) -9H-purin-6-yl) benzamide

Sodium azide (56.0 mg,0.9 mmol) was added to a solution of methyl ((2R, 3R,4R, 5R) -5- (6-benzoyl-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methylsulfonate (140.0 mg,0.2 mmol) in DMF (2 mL) at room temperature. After stirring the reaction at room temperature for 12 hours, adding water (30 mL) for quenching, extracting with ethyl acetate (30.0 mL x 2), merging organic phases, concentrating, subjecting the crude product to silica gel column chromatography (petroleum ether: ethyl acetate=2:1) to obtain 80mg of light yellow oily substance, the yield is 54%, ¹ H NMR(400MHz,CDCl ₃ )δ8.82(s,1H),8.39(s,1H),8.07-8.04(m,2H),7.62(d,J＝7.4Hz,1H),7.54(t,J＝7.4Hz,2H),6.01(d,J＝3.8Hz,1H),4.83(t,J＝3.8Hz,1H),4.33-4.21(m,2H),3.80(d,J＝4.0Hz,1H),3.76-3.67(m,1H),0.94(s,9H),0.84(s,9H),0.13(s,3H),0.12(s,3H),0.03(s,3H),-0.10(s,3H)。

step 5: preparation of N- (9- ((2R, 3R,4R, 5R) -5- (aminomethyl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) -9H-purin-6-yl) benzamide

N- (9- ((2R, 3R,4R, 5R) -5- (azidomethyl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) -9H-purin-6-yl) benzamide (80.0 mg,0.1 mmol) was dissolved in methanol (3.0 mL) and Pd (OH) was added ₂ Stirring at room temperature under hydrogen atmosphere (1 atm) for 2 hr/C (30.0 mg), filtering the reaction solution, concentrating the filtrate to obtain 50mg pale yellow solid with a yield of 65%, ¹ H NMR(400MHz,CDCl ₃ )δ8.83(s,1H),8.21(s,1H),8.04(d,J＝7.2Hz,2H),7.65-7.58(m,1H),7.53(t,J＝7.4Hz,2H),5.92(d,J＝6.2Hz,1H),4.95(dd,J＝6.0,4.6Hz,1H),4.40(d,J＝3.2Hz,1H),4.22(s,1H),3.20-3.19(m,2H),0.94(s,9H),0.78(s,9H),0.11(s,6H),-0.03(s,3H),-0.41(s,3H).MS(ES ⁺ )m/z 599(M+H ⁺ )。

Step 6: preparation of 9- ((2R, 3R,4R, 5R) -5- (aminomethyl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) -9H-purin-6-amino

N- (9- ((2R, 3R,4R, 5R) -5- (aminomethyl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) -9H-purin-6-yl) benzamide (1.0 g,1.7 mmol) was dissolved in NH ₃ In MeOH (30.0 mL, 7.0N) solution, stirred at room temperature for 12 hours, concentrated, and the crude product was chromatographed on silica gel (dichloromethane: methanol=10:1) to give 500mg of a pale yellow solid in 60% yield, ¹ H NMR(400MHz,CD ₃ OD)δ8.30(s,1H),8.20(s,1H),5.96(d,J＝7.0Hz,1H),5.06(dd,J＝6.8,4.6Hz,1H),4.32(d,J＝4.6Hz,1H),4.15-4.11(m,1H),3.15-3.11(m,1H),3.00(dd,J＝13.4,3.6Hz,1H),0.98(s,9H),0.76(s,9H),0.17(d,J＝3.6Hz,6H),-0.05(s,3H),-0.45(s,3H).MS(ES ⁺ )m/z 495(M+H ⁺ )。

intermediate preparation 56:2- ((((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methyl) amino) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The reaction steps are as follows:

step 1: preparation of 2-chloro-4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Triethylamine (19.5 mg,0.2 mmol) and 1- (3-chlorophenyl) propyl-1, 3-diol (30.0 mg,0.16 mmol) were dissolved in tetrahydrofuran (3 mL), phosphorus trichloride (27.0 mg,0.2 mmol) was added under ice-bath, the reaction mixture was stirred at room temperature for 12 hours and then concentrated, the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate=3:1) to give 20.0mg of an oil in 47% yield, ¹ H NMR(400MHz,CDCl ₃ )δ7.41-7.33(m,1H),7.29-7.26(m,3H),5.00-4.87(m,1H),4.62-4.59(m,1H),4.45-4.41(m,1H),2.18-2.16(m,1H),1.29-1.24(m,1H)。

Step 2: preparation of 2- ((((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methyl) amino) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

9- ((2R, 3R,4R, 5R) -5- (aminomethyl) -3, 4-di ((tert-butyldimethylsilyl) silicon)Oxy) tetrahydrofuran-2-yl) -9H-purin-6-amino (216.0 mg,0.4 mmol) and triethylamine (64.0 mg,0.6 mmol) were dissolved in dichloromethane (6 mL), 2-chloro-4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide (120 mg,0.42 mmol) was added under ice-bath, the reaction mixture was stirred at room temperature for 12 hours, concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane: methanol=10:1) to give 160mg of pale yellow solid in 51% yield, ¹ H NMR(400MHz,MeOD)δ8.41-8.17(m,2H),7.58-7.20(m,4H),5.92-5.86(m,1H),5.81-5.54(m,1H),5.16-4.99(m,1H),4.78-4.74(m,1H),4.60-4.26(m,3H),3.67-3.42(m,1H),3.40-3.32(m,1H),2.78-2.43(m,1H),2.19-2.08(m,1H),1.04-0.84(m,9H),0.78-0.58(m,9H),0.24-0.10(m,4H),0.07-0.03(m,1H),-0.02(d,J＝1.6Hz,1H),-0.07--0.25(m,3H),-0.51--0.75(m,3H).MS(ES ⁺ )m/z 741(M+H ⁺ )。

example 1:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-chloro-2-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-chloro-2-fluorobenzene) -1,3, 2-dioxaphosphorinane 2-sulfide (200 mg,0.3 mmol) is dissolved in THF (3 mL), triethylamine hydrogen trifluoride (838 mg,5.2 mmol) is added at room temperature and the reaction is stirred overnight at room temperature. After completion of the reaction, concentrated aqueous ammonia was added to adjust ph=8, concentrated, and the crude product was passed through C18 preparative column (CH ₃ CN/H ₂ O=2-50%) to yield 75mg of white solid in 54% yield. ¹ H NMR(400MHz,MeOD):δ8.36-8.35(m,1H),8.22(s,1H),7.38-7.35(m,2H),7.19-7.04(m,1H),6.12-6.11(m,1H),5.96-5.91(m,1H),4.89-4.33(m,7H),2.37-2.27(m,1H),2.14-2.09(m,1H).MS(ES ⁺ )m/z 532(M+H ⁺ ).

Example 2:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4-phenyl-1, 3, 2-dioxaphosphorinane 2-sulfide

Reference example 1 was followed except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((t-butyldimethylsilyloxy) oxy) tetrahydrofuran-2-yl) methoxy) -4-phenyl was used-1,3, 2-dioxaphosphorinane 2-sulfide is taken as a raw material, the yield is 87 percent, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.32(d,J＝3.2Hz,1H),8.15(s,1H),7.43-7.31(m,5H),5.97-5.95(m,1H),5.68-5.62(m,2H),5.45(t,J＝4.8Hz,1H),4.65-4.41(m,7H),2.29-2.17(m,1H),2.16-2.06(m,1H).MS(ES ⁺ )m/z480(M+H ⁺ ).

example 3:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (pyridin-4-yl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (pyridin-4-yl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a starting material in 67% yield, ¹ H NMR(400MHz,DMSO-d ₆ ):δ8.58-8.54(m,2H),8.33(d,J＝4.0Hz,1H),8.15(s,1H),7.38-7.25(m,4H),5.97(d,J＝4.0Hz,1H),5.74-5.65(m,2H),5.64-5.46(m,1H),4.68-4.31(m,6H),4.27-4.16(m,1H),2.21-2.08(m,2H).MS(ES ⁺ )m/z 481(M+H ⁺ ).

example 4:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a starting material in a yield of 59%, ¹ H NMR(400MHz,MeOD)δ8.37(d,J＝2.0Hz,1H),8.22(s,1H),7.42(d,J＝2.2Hz,1H),7.39-7.23(m,3H),6.11(t,J＝4.6Hz,1H),4.80-4.67(m,2H),4.67-4.51(m,2H),4.51-4.30(m,4H),2.38-2.15(m,1H),2.11(dt,J＝14.6,2.4Hz,1H).MS(ES ⁺ )m/z 514(M+H ⁺ ).

example 5:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3- (trifluoromethyl) phenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Preparation method of reference example 1Except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3- (trifluoromethyl) phenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a raw material, the yield was 31%, ¹ H NMR(400MHz,MeOD)δ8.37(d,J＝1.2Hz,1H),8.22(s,1H),7.75-7.48(m,4H),6.16-6.08(m,1H),5.36(t,J＝4.6Hz,1H),4.73-4.45(m,5H),3.28-3.19(m,2H),2.05(d,J＝5.4Hz,2H).MS(ES ⁺ )m/z 545(M+H ⁺ )。

example 6:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-methylphenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-methylphenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a raw material in a yield of 72%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.56-8.29(m,1H),8.25-8.07(m,1H),7.52–6.79(m,6H),6.03–5.88(m,1H),5.73–5.57(m,1H),5.53–5.39(m,1H),5.39–5.21(m,1H),4.68–3.87(m,7H),2.39–1.98(m,4H),1.89–1.68(m,1H).MS(ES ⁺ )m/z 494(M+H ⁺ )。

Example 7:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-methoxyphenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-methoxyphenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a raw material in a yield of 62%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.41–8.24(m,1H),8.24–8.10(m,1H),7.41–7.23(m,2H),7.03–6.87(m,2H),5.95(d,J＝5.3Hz,1H),5.64(d,J＝11.0Hz,1H),4.70–4.12(m,7H),3.74(d,J＝8.0Hz,3H),2.30–2.02(m,2H).MS(ES ⁺ )m/z 510(M+H ⁺ )。

example 8:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a starting material in a yield of 41%, ¹ H NMR(400MHz,MeOD)δ8.39(d,J＝1.4Hz,1H),8.24(s,1H),7.44-7.29(m,1H),7.24-6.97(m,3H),6.14-6.11(m,1H),5.76-5.67(m,1H),4.74-4.31(m,7H),2.36-2.10(m,2H).MS(ES ⁺ )m/z 498(M+H ⁺ )。

example 9:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (5-chloro-2-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (5-chloro-2-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a starting material in a yield of 51%, ¹ H NMR(400MHz,DMSO-d ₆ ):δ8.31-8.29(m,1H),8.15-8.09(m,1H),7.57-7.49(m,2H),7.36-7.30(m,3H),5.97-5.95(m,1H),5.85-5.82(m,1H),5.63-5.62(m,1H),5.46-5.44(m,1H),4.62-4.14(m,7H),2.37-2.27(m,1H),2.10-2.09(m,1H).MS(ES ⁺ )m/z 532(M+H ⁺ )。

Example 10:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (4-chloro-2-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (4-chloro-2-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a raw material, the yield was 53%, ¹ H NMR(400MHz,MeOD):δ8.36-8.34(m,1H),8.22(s,1H),7.46-7.40(m,1H),7.27-7.07(m,2H),6.12-6.10(m,1H),5.90-5.89(m,1H),4.89-4.33(m,7H),2.38-2.26(m,1H),2.11-2.06(m,1H).MS(ES ⁺ )m/z 532(M+H ⁺ )。

example 11:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (2, 5-difluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2, 5-difluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a raw material, the yield was 41%, ¹ H NMR(400MHz,MeOD)δ8.36(d,J＝4.4Hz,1H),8.23(d,J＝1.6Hz,1H),7.27-7.02(m,3H),6.12(t,J＝5.0Hz,1H),5.94-5.84(m,1H),4.68-4.54(m,3H),4.53-4.30(m,4H),2.40-2.19(m,1H),2.19-1.98(m,1H).MS(ES ⁺ )m/z 516(M+H ⁺ ).

example 12:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (2, 5-dichlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2, 5-dichlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a raw material, the yield was 29%, ¹ H NMR(400MHz,MeOD)δ8.37(d,J＝11.0Hz,1H),8.23(d,J＝1.6Hz,1H),7.51-7.26(m,3H),6.13(dd,J＝10.8,5.0Hz,1H),4.77-4.57(m,4H),4.57-4.34(m,4H),2.26-2.02(m,2H).MS(ES ⁺ )m/z 548(M+H ⁺ ).

Example 13:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (2-chloro-4-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2-chloro-4-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a raw material in a yield of 56%, ¹ H NMR(400MHz,CDCl ₃ )δ8.30-8.27(m,1H),8.09-8.07(m,1H),7.37-6.84(m,3H),6.12-5.74(m,3H),4.92-4.13(m,6H),2.17-1.98(m,2H).MS(ES ⁺ )m/z 532(M+H ⁺ ).

example 14:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (2, 4, 5-trifluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2, 4, 5-trifluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a raw material in a yield of 65%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.29(d,J＝6.4Hz,1H),8.14-8.05(m,1H),7.77-7.49(m,2H),7.30-7.25(m,2H),5.95(d,J＝5.4Hz,1H),5.88-5.74(m,1H),5.63(d,J＝4.8Hz,1H),5.44(t,J＝5.0Hz,1H),4.69-4.09(m,6H),2.47-2.30(m,1H),2.06-2.04(m,1H).MS(ES ⁺ )m/z 534(M+H ⁺ ).

example 15:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (2-chloro-4, 5-difluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2-chloro-4, 5-difluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a raw material in a yield of 65%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.35-8.25(m,1H),8.14-8.05(m,1H),7.92-7.73(m,1H),7.72-7.57(m,1H),7.31-7.23(m,2H),5.96(d,J＝5.4Hz,1H),5.88-5.76(m,1H),5.65-5.63(m,1H),5.48-5.45(m,1H),4.65-4.15(m,7H),2.38-2.21(m,1H),2.20-2.03(m,1H).MS(ES ⁺ )m/z 550(M+H ⁺ ).

example 16:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (5-chloro-2, 4-difluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Reference example 1 was followed except for 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((t-butyldimethylsilyloxy) tetrahydrofuran-2-yl) methoxy) -4- (5-chloro-2, 4-difluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide is taken as a raw material, the yield is 61 percent, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.39-8.23(m,1H),8.21-8.03(m,1H),7.84-7.71(m,1H),7.66-7.59(m,1H),7.38-7.21(m,2H),5.96-5.95(d,J＝5.2Hz,1H),5.84-5.81(m,1H),5.64-5.62(m,1H),5.45(t,J＝5.2Hz,1H),4.63-4.20(m,6H),4.16-4.13(m,1H),2.47-2.32(m,1H),2.11-2.05(m,1H).MS(ES ⁺ )m/z 550(M+H ⁺ ).

example 17:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (2, 4-dichloro-5-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The procedure of reference example 1 was followed except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2, 4-dichloro-5-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as the starting material, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.33-8.21(m,1H),8.14(s,1H),8.01–7.86(m,1H),7.60(dd,J＝16.2,9.8Hz,1H),7.30(d,J＝8.4Hz,2H),5.96(d,J＝5.4Hz,1H),5.82(s,1H),5.75–5.40(m,2H),4.73–4.09(m,7H),2.31–2.10(m,2H).MS(ES ⁺ )m/z 566(M+H ⁺ ).

Example 18:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (2, 3,4, 5-tetrafluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2, 3,4, 5-tetrafluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a starting material in a yield of 49%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.28(d,J＝11.0Hz,1H),8.14(s,1H),7.58-7.48(m,1H),7.29-7.22(m,2H),5.95(d,J＝5.4Hz,1H),5.85(t,J＝9.4Hz,1H),5.67-5.62(m,1H),5.46-5.38(m,1H),4.64-4.36(m,4H),4.33-3.93(m,3H),2.45-2.32(m,1H),2.13-2.08(m,1H).MS(ES ⁺ )m/z 552(M+H ⁺ ).

example 19:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-chloro-2, 4, 5-trifluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The procedure of example 1 was followed except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-chloro-2, 4, 5-trifluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a starting material in 86% yield, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.30-8.22(m,1H),8.14(s,1H),7.77-7.60(m,1H),7.30-7.22(m，2H),5.95(d,J＝4.0Hz,1H),5.85(t,J＝10.0Hz,1H),5.66-5.60(m,1H),5.46-5.40(m,1H),4.67-4.56(m,2H),4.55-4.35(m,3H),4.27-4.22(m,1H),4.16-4.10(m,1H),2.45-2.32(m,1H),2.15-2.05(m,1H).MS(ES ⁺ )m/z 568(M+H ⁺ ).

example 20:2- (((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (pentafluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (pentafluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a starting material in a yield of 59%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.34-8.25(m,1H),8.14-8.07(m,1H),7.29-7.22(m,2H),5.99-5.92(m,2H),5.63-5.61(m,1H),5.44-5.43(m,1H),4.66-4.09(m,7H),2.66-2.55(m,1H),2.22-2.10(m,1H).MS(ES ⁺ )m/z 570(M+H ⁺ ).

example 21: n- (9- ((2R, 3R,4S, 5R) -5- (((4- (3-chlorophenyl) -2-thio-1, 3, 2-dioxaphosphorinan-2-yl) oxy) methyl) -3, 4-dihydroxytetrahydrofuran-2-yl) -9H-purin-6-yl) acetamide

The preparation method of reference example 1 was conducted except that N- (9- (3, 4-di ((tert-butyldimethylsilyl) oxy) -5- (((4- (3-chlorophenyl) -2-thio-1, 3, 2-dioxaphosphorinane-2-yl) oxy) methyl) tetrahydrofuran-2-yl) -9H-purin-6-yl) acetamide was used as a starting material in an yield of 11%, ¹ H NMR(400MHz,MeOD)δ8.64(s,1H),8.58(d,J＝4.8Hz,1H),7.23-7.39(m,4H),6.20(t,J＝5.2Hz,1H),5.61-5.70(m,1H),4.75-4.80(m,1H),4.66-4.73(m,1H),4.53-4.65(m,2H),4.49-4.51(m,1H),4.31-4.48(m,2H),2.38(d,J＝4Hz,3H),2.17–2.27(m,1H),2.05-2.13(m,1H).MS(ES ⁺ )m/z556(M+H ⁺ ).

example 22: n- (9- ((2R, 3R,4S, 5R) -5- (((4- (3-chlorophenyl) -2-sulfanyl-1, 3, 2-dioxaphosphorinan-2-yl) oxy) methyl) -3, 4-dihydroxytetrahydrofuran-2-yl) -9H-purin-6-yl) butanamide

The preparation method of reference example 1 was repeated except that N- (9- (3, 4-di ((tert-butyldimethylsilyl) oxy) -5- (((4- (3-chlorophenyl) -2-thio-1, 3, 2-dioxaphosphorinane-2-yl) oxy) methyl) tetrahydrofuran-2-yl) -9H-purin-6-yl) butanamide was used as a starting material in a yield of 26%, ¹ H NMR(400MHz,CDCl ₃ )δ8.59-8.63(m,1H),8.32-8.33(m,1H),7.09-7.31(m,4H),6.01-6.13(m,1H),5.48-5.66(m,1H),4.46-4.90(m,6H),4.17-4.40(m,2H),2.79-2.83(m,2H),2.12-2.26(m,2H),1.95-1.99(m,2H),1.04-1.09(m,3H).MS(ES ⁺ )m/z 580(M+H ⁺ ).

Example 23: (4R) -2- (((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (4-chloro-2-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The procedure of example 1 was followed except that (4R) -2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (4-chloro-2-fluorobenzene) -1,3, 2-dioxaphosphorinane 2-sulfide was used as the starting material in 61% yield as a white solid. ¹ H NMR(400MHz,MeOD)δ8.34-8.31(m,1H),8.22-8.21(m,1H),7.37(t,J＝8.0Hz,1H),7.24-7.20(m,1H),7.09-7.06(m,1H),6.12-6.11(m,1H),5.90-5.86(m,1H),4.80-4.23(m,7H),2.42-2.15(m,1H),2.14-2.05(m,1H). ³¹ P NMR(MeOD):δ＝64.86,61.84ppm.MS(ES ⁺ )m/z 532(M+H ⁺ ).

Example 24: (4S) -2- (((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (4-chloro-2-fluorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

Reference example 1 was followed except for (4S) -2- (((2R, 3R, 4)R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (4-chloro-2-fluorobenzene) -1,3, 2-dioxaphosphorinane 2-sulfide is taken as a raw material, the yield is 62 percent, ¹ H NMR(400MHz,MeOD)δ8.36-8.34(m,1H),8.22(s,1H),7.44(t,J＝8.0Hz,1H),7.27-7.19(m,2H),6.12-6.10(m,1H),5.92-5.89(m,1H),4.88-4.32(m,7H),2.36-2.33(m,1H),2.10-2.07(m,1H). ³¹ P NMR(MeOD):δ＝64.99,61.92ppm.MS(ES ⁺ )m/z 532(M+H ⁺ ).

example 25: (4R) - (((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that (4R) -2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a starting material in 67% yield, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.39-8.32(m,1H),8.15(s,1H),7.48-7.21(m,6H),5.97(d,J＝4.0Hz,1H),5.72-5.63(m,2H),5.45-5.34(m,1H),4.74-4.15(m,7H),2.24-1.98(m,2H). ³¹ P NMR(MeOD):δ＝63.84,61.21ppm.MS(ES ⁺ )m/z 514(M+H ⁺ ).

example 26: (4S) - (((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that (4S) -2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a starting material in a yield of 72%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.35-8.32(m,1H),8.14-8.13(m,1H),7.49-7.21(m,6H),5.97(d,J＝4.0Hz,1H),5.71-5.62(m,2H),5.45-5.34(m,1H),4.65-4.15(m,7H),2.23-2.08(m,2H). ³¹ P NMR(MeOD):δ＝63.62,61.25ppm.MS(ES ⁺ )m/z 514(M+H ⁺ ).

example 27: (4R) - (((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (2, 5-dichlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that (4R) -2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2, 5-dichlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a starting material in 73% yield, ¹ H NMR(400MHz,MeOD)δ8.35-8.29(m,1H),8.15-8.14(m,1H),7.58-7.46(m,3H),7.28(s,2H),5.98-5.83(m,2H),5.63-5.61(m,1H),5.46-5.45(m,1H),4.74-4.16(m,7H),2.33-2.28(m,1H),2.18-2.14(m,1H). ³¹ P NMR(MeOD):δ＝64.00,60.98ppm.MS(ES ⁺ )m/z 548(M+H ⁺ ).

Example 28: (4S) - (((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (2, 5-dichlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that (4S) -2- (((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methoxy) -4- (2, 5-dichlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a starting material in 73% yield, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.31-8.25(m,1H),8.14-8.06(m,1H),7.59-7.22(m,5H),5.97-5.85(m,2H),5.64-5.57(m,1H),5.45-5.43(m,1H),4.65-4.08(m,7H),2.33-2.02(m,2H). ³¹ P NMR(MeOD):δ＝63.87,61.08ppm.MS(ES ⁺ )m/z 548(M+H ⁺ ).

example 29 and example 30:

example 29 (4S) - ((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide isomer 1

Example 30 (4S) - ((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide isomer 2

(4S) - (((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4-(3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide (200 mg,0.39 mmol) obtained by chiral preparative HPLC (acetonitrile/water=1/10-1/1) (4S) - (((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide isomer 1, 105mg white solid, yield 53%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.32(s,1H),8.13(s,1H),7.49-7.31(m,6H),5.97(d,J＝4.0Hz,1H),5.72-5.60(m,2H),5.44(d,J＝4.0Hz,1H),4.65-4.63(m,5H),4.28-4.27(m,1H),4.16-4.14(m,1H),2.20-2.10(m,2H). ³¹ P NMR(DMSO-d ₆ ):δ＝63.64ppm.MS(ES ⁺ )m/z 514(M+H ⁺ ) And (4S) - (((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide isomer 2,8mg of white solid in 4% yield, ¹ H NMR(400MHz,Methanol-d ₄ )δ8.25(s,1H),8.15(s,1H),7.22-7.31(m,3H),7.04-7.06(m,1H),6.05(d,J＝4.6Hz,1H),5.49(d,J＝11.6Hz,1H),4.78(t,J＝5.0Hz,1H),4.29-4.60(m,6H),2.88-3.01(m,1H),210-2.21(m,1H),1.90-1.92(m,1H)。 ³¹ P NMR(Methanol-d ₄ ):δ＝62.07ppm.MS(ES ⁺ )m/z 514(M+H ⁺ ).

example 31 and example 32:

example 31 (4R) - (((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide isomer 1

Example 32 (4R) - (((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide isomer 2

(4R) - (((2R, 3s,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide (200 mg,0.39 mmol) is obtained by chiral preparative HPLC (acetonitrile/water=1/10-1/1) ((2R, 3s,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide isomer1,75 mg of white solid, yield 38%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.31(s,1H),8.15(s,1H),7.48(s,1H),7.45-7.23(m,5H),5.96(d,J＝5.2Hz,1H),5.76-5.62(m,2H),5.46(d,J＝5.2Hz,1H),4.69-4.35(m,5H),4.27-4.15(m,2H),2.32-2.08(m,2H). ³¹ P NMR(DMSO-d ₆ ):δ＝63.86ppm.MS(ES ⁺ )m/z 514(M+H ⁺ ) The method comprises the steps of carrying out a first treatment on the surface of the And (4R) - (((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide isomer 2,6mg of white solid in 3% yield, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.38(s,1H),8.15(s,1H),7.41–7.42(m,2H),7.31(s,2H),7.21-7.23(m,1H),5.96(d,J＝4.5Hz,1H),5.63(d,J＝5.6Hz,1H),5.34-5.42(m,2H),4.74(d,J＝5.1Hz,1H),4.39-4.49(m,2H),4.14-4.38(m,5H),2.28–2.12(m,1H),1.83-1.85(m,1H). ³¹ P NMR(DMSO-d ₆ ):δ＝61.20ppm.MS(ES ⁺ )m/z 514(M+H ⁺ )。

Example 33:2- ((((2 r,3s,4r,5 r) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl)) methyl) amino) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide

The preparation method of reference example 1 was repeated except that 2- ((((2R, 3R,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-di ((tert-butyldimethylsilyl) oxy) tetrahydrofuran-2-yl) methyl) amino) -4- (3-chlorophenyl) -1,3, 2-dioxaphosphorinane 2-sulfide was used as a raw material in a yield of 35%, ¹ H NMR(400MHz,MeOD)δ8.38-8.29(m,1H),8.22-8.13(m,1H),7.46-7.29(m,4H),5.95-5.83(m,1H),5.71-5.55(m,1H),5.05-4.93(m,1H),4.82-4.69(m,1H),4.67-4.51(m,1H),4.42-4.26(m,2H),3.68-3.52(m,1H),3.43-3.34(m,1H),2.46-2.28(m,1H),2.21-2.04(m,1H).MS(ES ⁺ )m/z 513.0(M+H ⁺ )。

the compounds prepared in the examples of table 1 are shown below:

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example 33 in vitro Metabolic stability experiment

1. Method of

To understand the possible metabolic profile of compounds in vivo, metabolic stability assays of human liver microsomes (X008070, RILD) and CYP3A4 enzyme (C3 A4R046B, CYPEX) were performed on different compounds in vitro. The specific method comprises the following steps: adding each compound (including CS0002, CS0005, CS0008, CS0009, CS0012, CS0013 and CS 0018) into a human liver microsome or CYP3A4 enzyme reaction system to react, wherein the reaction system is 0.1M Tris-HCl buffer (pH 7.4) containing 200nM compound, 5mM MgCl2, 1mM NADPH, 0.33mg/ml human liver microsome (or 0.136mg/ml CYP3A 4) and the total volume is 500ul; placing in a water bath kettle at 37 ℃ for shaking incubation; taking out 50ul of reaction liquid at 0min, 5min, 15min and 30min respectively, adding into 200ul of pre-cooled anhydrous methanol for precipitation and stopping reaction; after vortex mixing, centrifuging at 16 000rpm and 4 ℃ for 15min, taking 100ul of supernatant, and carrying out LC-MS/MS detection analysis on samples to determine the content of each compound and expected product (AMPS), wherein the conditions of chromatographic mass spectrometry are the same as in example XXX; according to formula (1): intrinsic clearance (Clint) =0.693×reaction volume/(T) _1/2 X protein mass) and formula (2): rate of metabolite production (V _AMPS ) Calculation was performed = concentration of metabolite/reaction time.

2. Results

As shown in Table 2, each compound had a faster clearance rate in human liver microsomes, with clearance rates of CS0005 and CS0009 being the fastest. Moreover, the compounds are not all converted to the desired product AMPS, with CS0008 and CS0009 producing AMPS at the fastest rate. The results show that the compounds with the structure can be effectively converted into AMPS under the action of human liver microsomes and CYP3A4 enzyme, and can be used as medicines to be converted into active substances AMPS after being taken by the liver in vivo to play a role.

Intrinsic clearance and metabolite production of compounds of Table 2 in human liver microsomes and CYP3A4 enzyme reaction systems

Rate of formation

N.d. is a rapid clearance of the compound, which is undetectable at 5 min.

Example 34 liver delivery Compound experiment

1. Method of

1.1. Animal experiment

Male SD rats weighing 180-300 g, supplied by Shanghai Sipuler-BiKai laboratory animal Co. The male animals are adapted to the environment for more than 3 days, and are fasted for 12 hours at night on the day before the experiment, and are not forbidden. Solutions of AMPS prodrugs (Cremophor EL: ethanol: PEG400: saline = 1:1:4:4) were prepared, including CS0002, CS0005, CS0008, CS0009, CS0012, CS0013, CS0015, CS0018, CS0028, CS0029, CS0033, CS0034, CS0035, CS0036, CS0037, CS0038, CS0045, CS0046, CS0053, and CS0054. Before administration, whether the animal body weight accords with the experimental requirement is checked, 14 rats are selected for grouping, 2 rats in each group are subjected to intragastric administration, and 20 mu mol/kg of liquid medicine is administered. Samples were collected after euthanizing rats with carbon dioxide gas at 0.167, 0.5, 1, 3, 6, 12 and 24 hours, respectively: blood is extracted through the heart and stored in a heparin anticoagulation tube, and is centrifuged at 6000rpm for 5min at 4 ℃, and supernatant plasma is taken and stored in ice; liver and heart tissue of rats was harvested and rinsed with 4 ℃ pre-chilled normal saline, weighed after water was removed, and 5 volumes of 0.5 μg/mL tenofovir Wei Jiachun (4 ℃ pre-chilled) were added to the tissue. After the experiment, the samples were stored in a-80 ℃ refrigerator.

Determination of the content of the active metabolite of AMPS prodrugs in biological samples

Sample pretreatment

Plasma: 40. Mu.L of plasma sample is taken in a centrifuge tube, 200. Mu.L of a methanol solution containing 0.5. Mu.g/mL PMPA is added, vortex 1min, centrifuge (15000 rpm) at 4 ℃ for 5min, and the supernatant is taken and mixed with water 1:1 for sample injection analysis.

Tissue: the tissue sample is quantitatively weighed, 5 times of the volume of the methanol solution containing 0.5 mu g/mL PMPA is added into a homogenizing tube, the homogenization is carried out at low temperature, the ultrasonic treatment is carried out at low temperature for 15min, the centrifugation (15000 rpm) is carried out at 4 ℃ for 5min, the supernatant is taken and mixed with water at a ratio of 1:1, and then the sample injection analysis is carried out.

Chromatographic mass spectrometry conditions

LC-MS/MS-AJ (Triple Quad 5500, AB SCIEX) was used for analysis of the samples. Chromatographic column: acquity UPLC HSS T3 (2.1X105 mm,1.8 μm); column temperature: 40 ℃; flow rate: 0.5mL/min. Mobile phase a:0.1% formic acid in water, mobile phase B: acetonitrile/methanol/formic acid (900/100/1, v/v). Sample separation uses gradient elution and the procedure is as in table 3. And mass spectrometry conditions corresponding to the internal standard: electrospray ionization (ESI) positive ion mode, monitoring ion pair m/z for Multiple Reaction Monitoring (MRM): 364/136 (AMPS); 288/176 (PMPA) with a capillary voltage of 16.0kV; the temperature is 500 ℃; the solvent-removed air flow is 1000L/h; scanning time 0.025 seconds; the collision energy is 25V.

TABLE 3AMPS liquid phase elution gradient conditions

1.3 data analysis

Concentration of each prodrug released AMPS in plasma, liver and heart was plotted versus time. Fitting calculation was performed using the log-linear trapezoidal method in a non-compartmental model of WinNonlin8.0 (Pharsight, calif.), resulting in tissue concentration versus time area under the curve (AUC 0-T) and time to peak (T) for AMPS _max ) And peak concentration in AMPS tissue (C _max )。

2. Results

After gastric lavage of rats with 20. Mu. Mol/kg of the drug solution, the results of liver tissue distribution showed that the exposure and peak concentration of active molecule AMPS released by CS0002, CS0009 and CS0013 were more than twice as high as those of CS0005, CS0008, CS0012, CS0015 and CS0018 (Table 4 and FIG. 1), which showed that 3 chloro substitution, 2 fluoro-4 chloro substitution and 2,5 dichloro substitution on benzene ring all contributed to release and enrichment of prodrug at liver site. 3-chloro substituted CS0002 showed longer peak times than CS0009 and CS0013 (table 4 and fig. 1), indicating that CS0002 is more likely to maintain AMPS in the liver above an effective therapeutic concentration for long periods of time.

As shown in Table 4 and FIG. 1, after 20. Mu. Mol/kg of the drug solution was administered by gavage to rats, the compounds CS0002, CS0009, CS0013, CS0029, CS0033, and CS0037 showed a high liver exposure. The result of the AMPS distribution of the liver tissue shows that CS0034 is 1.5 times of CS0035, chiral resolution is carried out on CS0034, the obtained AMPS liver exposure of CS0053 is 275 times higher than that of corresponding CS0054, the liver exposure is 3.7 times and 2.8 times higher than that of CS0002 and CS0034 respectively, and the peak concentration of CS0053 is 4.5 times and 2.2 times higher than that of CS0002 and CS0034 respectively. In addition to higher AMPS liver exposure and peak concentrations, CS0053 also retained a peak time of 3 hours similar to CS 0002. Chiral resolution of CS0035 resulted in a 44-fold higher AMPS liver exposure of CS0045 than the corresponding CS0046, 94.6% and 112.3% of CS0002 and CS 0035.

Candidate prodrugs include CS0053, CS0045, CS0034, CS0035 and CS0002, where AMPS concentrations in rat plasma are near or below the detection limit of the liquid chromatography, and AMPS exposure and peak concentrations in rat heart tissue are undetectable (table 4). AMPK agonists are reported to be the main cause of toxic side effects causing myocardial hypertrophy in the heart (Science, myers et al 2017), and therefore the subject is expected to reduce cardiac toxicity caused by AMPS by liver-specific delivery properties of cyclic phosphate (4-aryl-2-oxo-1, 3, 2-dioxaphosphorinane) precursor structures, i.e. 1) by means of CYP 3A-specific catalytic release AMPS in cytochrome P450 isozymes family in hepatocytes, 2) while AMPS of strong polarity cannot enter the circulatory system and reach the heart from hepatocytes. The above results indicate that the compounds of formulas CS0045 and CS0053 according to the present invention have higher liver delivery, which results in lower amounts required for treatment and thus have higher safety or lower toxic side effects, thus greatly increasing the clinical therapeutic index of AMPS (fig. 2).

Table 4. Exposure of metabolite AMPS to liver, heart and plasma (h. Times. Nmol/mL, concentration/tissue volume), peak concentration (nmol/mL) and peak time (h) within 24 hours after gavage administration of each AMPS precursor compound to rats.

N.D. shows that the specific tissue concentration of the metabolite was all below the lower limit of detection of the LC-MS/MS method, 5ng/mL, during the experimental design detection period.

EXAMPLE 35 Compound AMPK activation assay at Primary liver cell level in mice

1. Method of

1.1 isolation and culture of Primary cells from mouse liver

Male C57BL/6 mice weighing 18-20 g, supplied by Shanghai Sipulel-BiKai laboratory animal Co. And (3) performing primary mouse liver cell separation by adopting a two-step in-situ liver perfusion method. The specific method comprises the following steps: D-Hank's perfusate (adding 0.5mM EGTA,25mM HEPES in 1 ×HBSS) and low sugar DMEM digests (adding 100CDU/ml collagenase IV,15mM HEPES,100ug/ml Streptomycin and 100IU/ml Penicillin) were pre-warmed in a 37℃water bath; shaving the mice after anesthesia with Shutai 50 (Zoletil 50), sterilizing with 75% ethanol, fixing on an operation table in a super clean bench, opening abdominal cavity, exposing inferior vena cava and portal vein, performing inferior vena cava cannula by using a trocar, pouring D-Hank's perfusion liquid, and after the liver slightly swells, shearing the portal vein to perfuse about 80ml at a flow rate of 10 ml/min; replacing the low-sugar DMEM digestive juice, and continuing to perfuse about 60ml at a flow rate of 10 ml/min; carefully removing the liver freely, transferring to a 10cm dish containing low-sugar DMEM digest; tearing the liver envelope to collect liver cell suspension, filtering with 70 μm filter screen, centrifuging the obtained cell filtrate at 4deg.C, 50Xg, and 2min; the supernatant was discarded, and after resuspension of the cells with 20ml of pre-chilled complete broth (low-sugar DMEM containing 10% fetal bovine serum), centrifugation was performed at 4 ℃,50×g,2min, and repeated once; resuspension of cells with 20ml pre-chilled complete culture medium, trypan blue counting, adjusting to proper cell density, inoculating to cell culture plate, placing in incubator (37deg.C, 5% CO) ₂ ) Is cultured.

1.2 detection of HTRF phosphorylation of AMPK Activity of Compounds

The isolated mouse liver primary cells were then cultured at 2X 10 ⁵ Cell density/ml was inoculated into 96-well cell culture plates and placed in an incubator (37 ℃,5% CO) ₂ ) After 12 hours, compounds (including CS0002, CS0005, CS0008, CS0009, CS0015, CS0018, CS0034, CS0035, CS0045, CS0046, CS0053 and CS 0054) were added to the culture at a final concentration of 200. Mu.M for treatment. After 1h of action, HTRF (Homogeneous Time Resolved Fluorescence) was measured according to the instructions of Cisbio's Phospho-AMPK (Thr 172) Cellular Assay Kit (Cat: 64 MPKPEG). The specific method comprises the following steps: throwing away the culture solution, sucking the culture solution on water-absorbing paper, immediately adding 50ul of lysis solution into each hole, and oscillating for 30min at room temperature; taking out 16ul of lysate, transferring to 384-well plate, adding 4ul of antibody detection solution, and oscillating at room temperature for 2h; in Perkinelmer Corp

Multilabel Plate Reader, and signal values are recorded. The activation efficiency was used as an index for evaluating AMPK activation activity of the compound.

2. Results

After the compound treatment of primary hepatocytes of mice, HTRF detection results showed that CS0002, CS0008, CS0009, CS0015 and CS0018 had strong AMPK activation activity, but only CS0005 was inactive, with the activation activity with CS0015 and CS0018 being most remarkable (fig. 3). The results show that the structural compound can enter cells at the in vitro cell level to exert the AMPK activating effect.

Compounds CS0034, CS0035, CS0045, CS0046, CS0053 and CS0054 are structural resolved bodies of compound CS 0002. The results showed that the activation effect of compound CS0034 was about 2 times that of compound CS0035, and that the activation effect of CS0053 in the chiral resolution of CS0034 was about three times that of CS0054, with better AMPK activation effect.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. A compound of the formula I as shown in the following,

wherein:

R ₁ selected from the group consisting of: a substituted or unsubstituted phenyl group;

R ₂ selected from the group consisting of: hydrogen;

R ₃ and R is ₄ Each independently selected from the group consisting of: hydrogen;

x is O;

wherein the substitution means that a hydrogen atom on the group is substituted with one or more substituents selected from the group consisting of: halogen, C1 alkyl, halogenated C1 alkyl.

2. The compound of claim 1, wherein the compound of formula I has a structure selected from the group consisting of:

3. The compound of claim 1, wherein R ₁ Has a structure shown in the following formula II:

wherein:

each R is ₅ Each independently selected from the group consisting of: halogen, C1 alkyl;

i is 0, 1, 2 or 3.

4. The compound of claim 1, wherein said compound has a structure selected from the group consisting of:

5. the compound of claim 1, wherein said compound is selected from the group consisting of the structures shown in:

6. the compound of claim 1, wherein said compound is selected from the group consisting of the structures shown in:

。

7. a pharmaceutical composition comprising (a) a therapeutically effective amount of a compound of claim 1; and (b) a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is for use in treating a disease or disorder selected from the group consisting of: non-alcoholic fatty liver NAFL, liver cancer, obesity, cardiovascular diseases, and metabolic diseases.

9. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is for use in treating a disease or disorder selected from the group consisting of: non-alcoholic fatty liver disease NAFLD, diabetes, hypertriglyceridemia, hypercholesterolemia, and atherosclerosis.

10. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is for use in treating a disease or disorder selected from the group consisting of: non-alcoholic steatohepatitis NASH.

11. The use of a compound of formula I according to claim 1 for the preparation of a pharmaceutical composition for the treatment or prophylaxis of a disease or condition selected from the group consisting of: non-alcoholic fatty liver NAFL, liver cancer, obesity, cardiovascular diseases, and metabolic diseases.

12. The use of a compound of formula I according to claim 1 for the preparation of a pharmaceutical composition for the treatment or prophylaxis of a disease or condition selected from the group consisting of: non-alcoholic fatty liver disease NAFLD, atherosclerosis, diabetes, hypertriglyceridemia and hypercholesterolemia.

13. The use of a compound of formula I according to claim 1 for the preparation of a pharmaceutical composition for the treatment or prophylaxis of a disease or condition selected from the group consisting of: non-alcoholic steatohepatitis NASH and liver cirrhosis associated therewith.