WO2020143800A1 - 肝脏特异性ampk激动剂及其制法和应用 - Google Patents

肝脏特异性ampk激动剂及其制法和应用 Download PDF

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WO2020143800A1
WO2020143800A1 PCT/CN2020/071525 CN2020071525W WO2020143800A1 WO 2020143800 A1 WO2020143800 A1 WO 2020143800A1 CN 2020071525 W CN2020071525 W CN 2020071525W WO 2020143800 A1 WO2020143800 A1 WO 2020143800A1
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amino
substituted
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purin
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徐华强
栾林波
张振伟
孙锋
杨生生
高善云
戴金威
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凯思凯迪（上海）医药科技有限公司
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    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
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    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07H1/00—Processes for the preparation of sugar derivatives

Definitions

  • the invention provides an AMPK (AMP-dependent protein kinase) agonist based on a liver-specific method and its medical use.
  • the compounds of the present invention are used to treat non-alcoholic fatty liver disease (NAFLD) including non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH) and related cirrhosis, liver cancer, and can also be used for obesity , Diabetes, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, cardiovascular diseases and other metabolic diseases.
  • AMPK is called AMP Activated Protein Kinase, that is, AMP-dependent protein kinase, which is a heterotrimeric protein kinase composed of catalytic subunit ⁇ , regulatory subunit ⁇ and ⁇ .
  • the protein kinase is an energy kinase that can sense changes in AMP in the body.
  • concentration of AMP in the body rises to a certain level, AMPK is activated to inhibit anabolism and promote catabolism; the net effect of activation is the inhibition of the ATP consumption process and the ATP production pathway Activation and thus regenerate ATP storage.
  • AMPK Activated AMPK can shut down almost all anabolic pathways (including lipid, carbohydrate, protein and ribosomal RNA biosynthesis) through short-term and long-term regulation modes.
  • AMPK can directly regulate downstream substrate activity through phosphorylation at the protein level, thereby regulating cellular energy metabolism in the short term.
  • studies have found that AMPK can down-regulate the expression of downstream substrates, thereby regulating cell energy metabolism for a long time.
  • AMPK plays an important role in regulating cellular energy metabolism, oxidative stress, and tropism.
  • AMPK fatty acid oxidation and synthesis
  • lipid metabolism Due to the important role of the liver in regulating fatty acid oxidation and synthesis, and lipid metabolism, the relationship between AMPK and normal liver function The connection is obvious. Patients with metabolic diseases all have changes in liver metabolism. Through research on AMPK to improve glucose metabolism and lipid metabolism in patients, this may become an effective method for treating such diseases.
  • AMPK can control tumor invasion and metastasis by regulating the activity of tumor suppressor genes and improving energy metabolism disorders.
  • AMPK can be activated by phosphorylating the downstream signal P53 serine residues, thereby negatively regulating the mTOR pathway to interrupt tumor proliferation and reduce tumor invasion and metastasis.
  • Protein kinase B Protein Kinase B, PKB
  • PKB Protein Kinase B
  • Fas receptor that promotes tumor cell apoptosis can be activated in drug-resistant tumor cells by inducing the transcription factor NF-kB (Nuclear factor kB, NF -k)B)activation increases tumor invasiveness, and SNARK in the AMPK family can affect the invasion and metastasis by regulating the CD95-NF-kB pathway.
  • AMPK can also reduce the invasiveness of tumor cells by affecting the MMP-2 and MMP-9 proteins in the matrix metalloproteinase (MMP) family.
  • AMPK Due to the different combinations of AMPK subunits, in theory, AMPK has a total of 12 protein combinations.
  • the ⁇ 1 and ⁇ 2 subunits have different alternative splice variants, which further increases the complexity of the AMPK trimer.
  • the widespread expression of AMPK in vivo currently lacks AMPK agonists with strong tissue specificity, high selectivity, and low toxicity.
  • the present invention synthesizes a class of prodrug molecules of thionucleoside monophosphate (AMPS) and its analogs. After oral administration, the drug is metabolized in liver tissue to obtain the parent molecule.
  • AMPK agonist of the present invention has high selectivity and high Advantages of curative effect and low side effects.
  • R 1 is selected from the group consisting of substituted or unsubstituted C6-C18 aryl, substituted or unsubstituted 5-12 membered heteroaryl;
  • R 2 is selected from the group consisting of hydrogen, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C18 alkyl acyl, substituted or unsubstituted C2 -C18 alkoxycarbonyl, mono- or di-C2-C18 alkylaminocarbonyl: halogen, haloalkyl, nitro, hydroxyl, amino and cyano;
  • R 3 and R 4 are each independently selected from the group consisting of hydrogen, fluorine, C1-C6 alkyl, C1-C6 alkoxy; or R 3 and R 4 together form a group selected from the group consisting of C3-C8 carbon Ring, or 5-12 membered heterocycle;
  • X is O, S, NH, substituted or unsubstituted C1-C4 alkylene
  • the compound of formula I has a structure selected from the group consisting of:
  • the X is selected from the group consisting of O, S, NH, CH 2 , CF 2 , or CD 2 .
  • said R 1 has the structure represented by the following formula II, III, IV, V, VI or VII:
  • the dotted line is chemical bond or none
  • Each of A 1 , A 2 , A 3 , A 5 , A 6 , A 7 , and A 8 is independently O, S, N, NH, CH, or CH 2 ;
  • a 4 and A 9 are independently C or N ;
  • Each B 1 , B 2 , B 3 , B 4 , B 6 , B 7 , B 8 , and B 9 is independently O, S, N, NH, CH, or CH 2 ;
  • B 5 and B 10 are each independently C or N;
  • Each R 5 , R 6 , R 7 and R 8 is independently selected from the group consisting of halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy;
  • i 0, 1, 2, 3, 4 or 5;
  • Y 1 is O, S or NH
  • Y 2 and Y 3 are each independently selected from O, N or CH;
  • j 0, 1, 2, 3 or 4;
  • n 0, 1, 2 or 3;
  • n 0, 1, 2, 3 or 4;
  • the R 2 is selected from the group consisting of acetyl and butyryl.
  • each of R 3 and R 4 is independently a hydrogen atom.
  • the compound of formula (I) is a prodrug of a direct AMPK agonist.
  • the compound of formula (I) may have one or more chiral centers and thus exist in a variety of stereoisomeric forms.
  • isomers include tautomers, cis-trans isomers, Conformation isomers, meso compounds, optical isomers with enantiomeric or diastereomeric relationships, and mixtures of various isomers that may occur.
  • the compound has a structure selected from the group consisting of:
  • the compound is selected from the structures shown in the following group:
  • the compound has the structure shown in Table 1:
  • a pharmaceutical composition comprising (a) a therapeutically effective amount of a compound as described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, Hydrate or solvate; and (b) a pharmaceutically acceptable carrier.
  • the disease or disorder is selected from the group consisting of non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH) and related cirrhosis, liver cancer, non-alcoholic fatty liver disease (NAFLD ), obesity, diabetes, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, cardiovascular diseases, metabolic diseases.
  • NAFL non-alcoholic fatty liver
  • NASH non-alcoholic steatohepatitis
  • NAFLD non-alcoholic fatty liver disease
  • obesity diabetes
  • hypertriglyceridemia hypercholesterolemia
  • atherosclerosis atherosclerosis
  • cardiovascular diseases metabolic diseases.
  • a use of the compound of formula I as described in the first aspect of the present invention characterized in that it is used to prepare a pharmaceutical composition for treating or preventing a disease or disorder associated with AMPK activation.
  • the disease or disorder is selected from the group consisting of non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH) and related cirrhosis, liver cancer, non-alcoholic fatty liver disease (NAFLD ), obesity, diabetes, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, cardiovascular diseases, metabolic diseases.
  • NAFL non-alcoholic fatty liver
  • NASH non-alcoholic steatohepatitis
  • NAFLD non-alcoholic fatty liver disease
  • obesity diabetes
  • hypertriglyceridemia hypercholesterolemia
  • atherosclerosis atherosclerosis
  • cardiovascular diseases metabolic diseases.
  • Figure 1 shows the concentration-time curve of the active molecule AMPS in the liver metabolically released by the rat after intragastric administration of 20 ⁇ mol/kg of liver-targeted AMPS prodrug.
  • Fig. 2 shows the concentration-time curve of the active molecule AMPS in the liver metabolically released by the CS0002 series of AMPS prodrugs after intragastric administration of 20 ⁇ mol/kg.
  • Figure 3 shows the results of HTRF detection after compound treatment of mouse primary hepatocytes.
  • Figure 4 shows the effect of compounds on the phosphorylation level of AMPK in mouse primary hepatocytes.
  • C1-C6 alkyl refers to a linear or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, Sec-butyl, tert-butyl, etc., or similar groups.
  • the alkyl group is a linear or branched saturated chain having 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, or isopropyl.
  • C1-C18 alkyl refers to a linear or branched alkyl group having 1 to 18 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, Sec-butyl, tert-butyl, etc., or similar groups.
  • C3-C8 cycloalkyl refers to a cyclic alkyl group having 1 to 8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl .
  • C1-C6 alkoxy refers to a C1-C6 alkyl group as defined above, which is connected to the rest of the molecule through an oxygen atom.
  • C1-C6 alkoxy may include methoxy, ethoxy, and isopropoxy.
  • C1-C6 alkylamino refers to the C1-C6 alkyl group defined above, which is connected to the rest of the molecule through a nitrogen atom.
  • the alkylamino group may include dimethylamino and diethylamino.
  • C1-C6 carboxyl refers to a substituent shaped like a "straight-chain or branched alkyl-carboxyl group having 1 to 5 carbon atoms" structure, which is connected to the rest of the molecule through an alkyl carbon atom .
  • C1-C6 ester group refers to a substituent shaped like a "straight-chain or branched alkyl-ester group having 1 to 5 carbon atoms" structure, which is connected to the molecule through an alkyl carbon atom The remaining part; wherein, the alkyl part of the ester group is a C1-C6 alkyl group.
  • alkyl part of the ester group is a C1-C6 alkyl group.
  • C2-C6 alkanoyl refers to a substituent shaped like a “straight-chain or branched alkyl-carbonyl having 1 to 5 carbon atoms" structure, which is connected to the rest of the molecule through a carbonyl group. Such as acetyl, propionyl, butyryl, or similar groups.
  • C2-C18 alkanoyl refers to a substituent such as “straight-chain or branched alkyl-carbonyl having 1 to 17 carbon atoms" structure, such as acetyl, propionyl, butyryl, or Similar groups.
  • C2-C6 alkanoylamino refers to a substituent shaped like “having a C2-C6 alkanoyl-amino" structure, which is connected to the rest of the molecule through a nitrogen atom; such as acetamido, propionamido , Butanamide, or similar groups.
  • C2-C18 alkoxycarbonyl refers to a linear or branched alkyl-oxy-carbonyl group having 1 to 17 carbon atoms, which is connected to the rest of the molecule through a carbonyl group.
  • C2-C18 alkylaminocarbonyl refers to a linear or branched alkyl-nitrogen-carbonyl group having 1 to 17 carbon atoms, which is connected to the rest of the molecule through a carbonyl group.
  • halogen refers to F, Cl, Br and I.
  • haloalkyl refers to C1-C3 alkyl substituted with halogen.
  • the haloalkyl group is trifluoromethyl, difluoromethyl, trifluoromethoxy.
  • C1-C3 alkyl refers to a linear or branched alkyl group having 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, and isopropyl.
  • aryl refers to a C6-C18 aromatic group, such as phenyl or naphthyl, which is unsubstituted, substituted with one or more (eg 2, 3, 4 or 5) atoms or groups selected from Aryl: halogen, nitro, hydroxyl, 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 alkylamido.
  • heteroaryl refers to a 5-12 membered aromatic group that contains one or more heteroatoms selected from nitrogen, oxygen, and sulfur.
  • Heteroaryl groups can include pyridine, pyrazine, pyrimidine, thiophene, furan, isoxazole, isothiazole, pyrazole, imidazole.
  • Such groups may be unsubstituted, substituted by one or more (eg 2, 3, 4 or 5) atoms or groups selected from the following heteroaryl groups: halogen, nitro, hydroxyl, amino, cyano , Halogenated alkyl, 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 carboxyl, substituted or unsubstituted C1-C6 ester, substituted or unsubstituted C2-C6 alkanoyl, substituted or unsubstituted C2-C6 alkanoyl.
  • heterocycle refers to a 5-12 membered non-aromatic group (including saturated, partially saturated or unsaturated groups), which contains one or more selected from nitrogen, oxygen and sulfur Heteroatoms have a single ring or fused ring (including bridge ring system and spiro ring system. In the 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 N-oxide, sulfinyl, and sulfonyl moieties.
  • heterocyclic groups examples include pyrrolidinyl , Piperidinyl, piperazinyl, imidazolidinyl, 2,3-dihydrofuran (2,3-b)pyridinyl, benzoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, Indoline, etc.
  • the term also includes non-aromatic, partially unsaturated monocyclic rings, such as 2- or 4-pyridone or N-substituted-(1H, 3H)-pyrimidine-2 connected through a nitrogen atom ,4-Diketones (N-substituted uracils).
  • the terms "containing”, “comprising” or “including” mean that various ingredients can be applied together in the mixture or composition of the present invention. Therefore, the terms “consisting mainly of” and “consisting of” are included in the term “contains”.
  • the term "pharmaceutically acceptable” ingredient refers to a substance that is suitable for humans and/or animals without excessive adverse side effects (such as toxicity, irritation, and allergies), that is, has a reasonable benefit/risk ratio.
  • the term "effective amount" refers to an amount by which a therapeutic agent treats, alleviates or prevents a target disease or condition, or an amount that shows a detectable therapeutic or preventive effect.
  • the precise effective amount for a subject depends on the subject's body type and health condition, the nature and degree of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Therefore, it is useless to specify an accurate effective amount in advance. However, for a given condition, routine experimentation can be used to determine the effective amount, which the clinician can judge.
  • the term "pharmaceutically acceptable salt” refers to a salt of a compound of the present invention formed with an acid or base suitable for use as a medicine.
  • Pharmaceutically acceptable salts include inorganic salts and organic salts.
  • One type of preferred salts are the salts of the compounds of the invention with acids.
  • Acids suitable for salt formation include, but are not limited to: hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid and other inorganic acids, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Organic acids such as maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzylsulfonic acid, benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.
  • Some compounds in the present invention may be crystallized or recrystallized with water or various organic solvents, and in this case, various solvates may be formed.
  • the 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 low-pressure sublimation drying.
  • prodrug means that when administered to a biological system due to one or more spontaneous chemical reactions, one or more enzyme-catalyzed chemical reactions and/or one or more metabolic chemical reactions Any compound of "drug” substance (biologically active compound). It also includes biodegradable polymer derivatives of the compounds of the present 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 invention.
  • Isotopic variants of the compounds of the 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 common in nature.
  • isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 17 respectively O, 18 O, 35 S, 18 F and 36 Cl.
  • isotopic variants of the invention for example, those in which radioisotopes (eg, 3 H or 14 C) are incorporated, are used in drug and/or substrate tissue distribution studies. Tritium, ie, 3 H, and carbon-14, ie, 14 C, isotopes are particularly preferred because they are easy to prepare and detect. In addition, substitution with isotopes (eg, deuterium, ie, 2 H) may provide some therapeutic advantages due to increased metabolic stability, such as increased half-life in vivo or reduced dosage requirements and may therefore be preferred in some cases of. Isotopic variants of the compounds of the present invention can generally be prepared by conventional procedures, for example using appropriate reagents of appropriate isotopic variants, by exemplary methods or by the preparation described in the experimental section below.
  • radioisotopes eg, 3 H or 14 C
  • isotopes are particularly preferred because they are easy to prepare and detect.
  • substitution with isotopes eg, deuterium, ie,
  • the compounds of the present invention can be prepared by many methods well known to those skilled in the art, including but not limited to those described below, or by applying standard techniques known to those skilled in organic synthesis to improve these methods. All methods disclosed in connection with the present invention are carried out on any scale, including milligrams, grams, multigrams, kilograms, multikilograms, or commercial industrial scale. In the following reaction formulas and hereinafter, unless otherwise specified, R 1 to R 4 are as defined in the first aspect. These methods form other aspects of the invention.
  • the general formula is represented by Roman numerals (I), (II), (III), (IV), and the like.
  • the subsets of these general formulas are defined as (Ia), (Ib), (Ic), etc..., (IVa), (IVb), (IVc), etc; or (Ia), (Ib), (Ic), etc... , (IV-a), (IV-b), (IV-c), etc.
  • the compound of the general formula (I') can be prepared by reacting triethylamine trihydrofluoride with the general formula (Ia/b) according to reaction formula one.
  • Typical reaction conditions include triethylamine trihydrofluoride and general formula (Ia/Ib) in anhydrous tetrahydrofuran at room temperature for about 12 hours.
  • the reaction solution was directly subjected to column chromatography using a C18 column and a rapid column passing instrument (the mobile phase was water and acetonitrile). The separated fraction was lyophilized with a freeze dryer to obtain the compound of general formula (I').
  • the compound of the general formula (Ib) can be prepared by performing a condensation reaction of the R 2 substituent and the general formula (Ia) according to reaction formula two to obtain the general formula (Ib).
  • the compound of the general formula (Ia) can be prepared by reacting phophothion, triethylamine and the general formula (Ic) according to reaction formula three. Then, it reacts with diol (Id), or after concentration, the reaction solution is subjected to silica gel column chromatography (mobile phase is petroleum ether and ethyl acetate) and then reacted with diol (Id). Typical reaction conditions include reaction of phosphorous trichloride, triethylamine and the general formula (Ic) in an inert solvent (such as DCM) at room temperature for about 0.5 hours, then diol (ld) is added, and the reaction is performed at room temperature for about 12 hours. After concentration, the reaction solution was purified by column chromatography using a C18 column through a rapid column passer (mobile phase is water and acetonitrile). The separated fraction was lyophilized with a freeze dryer to obtain the compound of general formula (Ia).
  • silica gel column chromatography
  • the compound of general formula (Ic) can be prepared by reacting tert-butyldimethylchlorosilane (TBSCl), general formula (Ie) and imidazole according to reaction formula 4.
  • Typical reaction conditions include reacting tert-butyldimethylchlorosilane, general formula (Ie) and imidazole in a solvent (such as DMF) at room temperature for about 12 hours.
  • a solvent such as DMF
  • the protective group of tert-butyldimethylsilane at the 5′-end can be obtained by removing the 5′-terminal of the general formula (If).
  • Typical reaction conditions include the reaction of general formula (If), trifluoroacetic acid and water in a solvent (such as THF) at 0°C for about 5 hours. The pH value of the reaction solution was adjusted to neutral and then filtered, and the compound of general formula (Ic) was obtained after the cake was collected.
  • the compound of general formula (Ih) can be prepared by reacting general formula (Ig), dichlorosulfoxide and a catalytic amount of DMF.
  • Typical reaction conditions include reacting a compound of general formula (Ig), dichlorosulfoxide, and a catalytic amount of DMF in a solvent (such as DCM) at room temperature for about 2 hours. Then, the reaction solution is concentrated. After adding ethanol, the reaction is carried out at room temperature for about one hour. After completion, the reaction solution is concentrated and passed through silica gel column chromatography (mobile phase is petroleum ether and ethyl acetate). The separated fraction is concentrated to obtain the general formula (Ih) .
  • the intermediate of general formula (Ih) reacts with the ester under the action of lithium hexamethyldisilazide.
  • Typical reaction conditions include: at a temperature of about -60°C, the general formula (Ih), lithium hexamethyldisilazide and ethyl acetate are reacted, and the reaction is performed at this low temperature for about 20 minutes.
  • the pH value of the reaction solution was adjusted to neutral with acetic acid and then concentrated, and purified by silica gel column chromatography (mobile phases were petroleum ether and ethyl acetate). The separated fraction was concentrated to obtain the general formula (Ii).
  • a protic solvent such as MeOH
  • a reducing agent such as NaBH 4
  • a protic solvent such as MeOH
  • a reducing agent such as NaBH 4
  • the chiral 1,3-propanediol derivative (11/lm) of the compound of general formula (ld) is prepared by the following method:
  • a protic solvent such as MeOH
  • a reducing agent such as NaBH 4
  • Ip the general formula (In)
  • the compound of the present invention has excellent AMPK activation activity, the compound of the present invention and its various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and containing the compound of the present invention as the main active ingredient
  • the pharmaceutical composition of can be used to treat, prevent and alleviate diseases caused by hepatitis B virus.
  • the compounds of the present invention can be used to treat the following diseases: non-alcoholic fatty liver disease (NAFL), non-alcoholic fatty liver disease (NASH) and related non-alcoholic fatty liver disease (NAFLD) including liver cirrhosis, liver cancer, It can also be used for obesity, diabetes, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, cardiovascular diseases and other metabolic diseases.
  • the pharmaceutical composition of the present invention contains a compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier in a safe and effective amount.
  • the "safe and effective amount” refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 0.1-1000 mg of the compound/dose of the present invention, and more preferably, 0.5-500 mg of the compound/dose of the present invention.
  • the "one dose” is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility” here means that the components of the composition can be blended with the compound of the present invention and between them without significantly reducing the efficacy of the compound.
  • Examples of pharmaceutically acceptable carrier parts are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid) , Magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting Agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose
  • the mode of administration of the compound or pharmaceutical composition 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.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) filler or compatibilizer, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, For example, glycerin; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) retarding solvents, such as paraffin; (f) Absorption accelerators, for example, quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and
  • Solid dosage forms such as tablets, sugar pills, capsules, pills, and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain an opaque agent, and the release of the active compound or compound in such a composition may be released in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxy substances. If necessary, the active compound can also be formed into a microcapsule form with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures.
  • the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils,
  • composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and flavors.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and flavors.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • composition for parenteral injection may contain a physiologically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion.
  • Suitable aqueous and non-aqueous 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 that may be required if necessary.
  • the compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.
  • a safe and effective amount of the compound of the present invention is suitable for mammals (such as humans) in need of treatment, wherein the dose when administered is a pharmaceutically effective dose, for a person of 60 kg body weight, daily
  • the dose to be administered is usually 0.2 to 1000 mg, preferably 0.5 to 500 mg.
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skills of skilled physicians.
  • the invention prepares thionucleoside monophosphate into a class of cyclic phosphorothioate prodrugs with liver tissue specificity.
  • the drug molecules have good stability to the gastrointestinal tract and plasma, and are not easily used by esters in the body Hydrolytic enzyme hydrolysis; after the drug enters the liver, it is oxidized by CYP3A in the family of cytochrome P450 isozymes in tissue cells, and the drug molecule phosphorothioate ring is opened at the 4 position to form an intermediate with a negative charge of monophosphate, and then passes through Phosphatase catalyzes hydrolysis and ⁇ -elimination reactions, releasing the parent drug thionucleoside monophosphate.
  • the thionucleoside monophosphate is in a protonated state in the cell, and it is not easy to remain in the cell through the cell membrane, so that the drug concentration in the liver cell is higher than that in normal tissue.
  • the aryl ketene a by-product of the oxidation of the prodrug by CYP3A, can be quickly eliminated by binding to glutathione, which is rich in antioxidants and free radicals in liver cells. So far, no side effects have been reported.
  • the main advantages of the present invention include:
  • the liver has strong specificity, and the prodrug of the present invention, that is, the compound of formula (I) is metabolized in hepatocytes and orally produced into the parent drug after oral administration; the drug molecule is highly negatively charged and is not easily discharged from the liver In addition, the concentration in the liver is higher and the liver is targeted.
  • the activity is high and the toxic and side effects are low, because the present invention belongs to liver-targeted drugs, the active drug molecules have good tissue distribution, more drug molecules are stored in liver cells, and the drug efficacy is greatly improved. Because the drug molecules are mainly concentrated in liver cells, and the amount of metabolized into active molecules outside the liver is very small, the side effects on the kidney, heart, etc. will be greatly reduced.
  • Step 1 9-((2R,3R,4R,5R)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl) Oxy)methyl)tetrahydrofuran-2-yl)-9H-purine-6-amino
  • Step 2 ((2R, 3R, 4R, 5R)-5-(6-amino-9H-purin-9-yl)-3,4-bis((tert-butyldimethylsilyl)oxy)tetrahydrofuran -2-yl) methanol
  • the crude product was slurried with ethyl acetate (15 mL) to obtain 2.2 g of white solid in 73% yield.
  • Step 2 Preparation of ethyl 3-(3-chloro-2-fluorobenzene)-3-oxopropionate
  • Step 2 and The two-step yield of Step 3 is 55%.
  • the crude product was further dissolved in dry dichloromethane (30mL), (2S,5R)-2-isopropyl-5-methylcyclohexanone (2.72g, 17.64mmol) was added, the temperature was lowered to -40°C, and trifluoro was added Trimethylsilyl methanesulfonate (391mg, 1.764mmol), the reaction solution was stirred at -40°C overnight, after the reaction was completed, pyridine (2mL) was added to quench, the reaction solution was added dichloromethane (30mL), saturated sodium bicarbonate solution (30mL) Wash twice.
  • Step 1 N-(9-((2R,3R,4R,5R)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-)(((tert-butyldimethyl Preparation of silyl)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, 2.7mmol) was dissolved in N,N-dimethylformamide (20mL), cooled to 0 °C, add imidazole (1.8g, 26.9mmol) and tert-butyl dimethyl chlorosilane (3.2g, 21.5) mmol), the reaction solution was stirred at room temperature overnight. After the reaction was completed, the reaction solution was distilled off under reduced pressure to remove the organic solvent.
  • Step 2 N-(9-((2R,3R,4R,5R)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2- Group)-9H-purin-6-yl)benzamide
  • the ethyl acetate phases were combined and washed with saturated sodium chloride. After drying with anhydrous sodium sulfate, spin dry.
  • the crude product was slurried with ethyl acetate (15 mL) to obtain 300.0 mg of a white solid with a yield of 88%.
  • Step 3 ((2R,3R,4R,5R)-5-(6-benzoyl-9H-purin-9-yl)-3,4-bis((tert-butyldimethylsilyl)oxy ) Tetrahydrofuran-2-yl) methyl methyl sulfonate
  • Step 4 N-(9-((2R,3R,4R,5R)-5-(azidomethyl)-3,4-bis((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2 -Yl)-9H-purin-6-yl)benzamide
  • Step 5 N-(9-((2R,3R,4R,5R)-5-(aminomethyl)-3,4-bis((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2- Group)-9H-purin-6-yl)benzamide
  • Step 6 9-((2R,3R,4R,5R)-5-(aminomethyl)-3,4-bis((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)- Preparation of 9H-purine-6-amino
  • Step 2 2-((((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-bis((tert-butyldimethylsilyl) Oxygen)tetrahydrofuran-2-yl)methyl)amino)-4-(3-chlorophenyl)-1,3,2-dioxaphosphorinane 2-sulfide
  • Example 1 2-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy) -4-(3-chloro-2-fluorophenyl)-1,3,2-dioxaphosphorane 2-sulfide
  • Example 7 2-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy) -4-(3-methoxyphenyl)-1,3,2-dioxaphosphorane 2-sulfide
  • Example 8 2-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy) -4-(3-fluorophenyl)-1,3,2-dioxaphosphorane 2-sulfide
  • Example 10 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-dioxaphosphorane 2-sulfide
  • Example 12 2-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy) -4-(2,5-dichlorophenyl)-1,3,2-dioxaphosphorane 2-sulfide
  • Example 13 2-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy) -4-(2-chloro-4-fluorophenyl)-1,3,2-dioxaphosphorane 2-sulfide
  • Example 14 2-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy) -4-(2,4,5-trifluorophenyl)-1,3,2-dioxaphosphorane 2-sulfide
  • Example 15 2-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy) -4-(2-chloro-4,5-difluorophenyl)-1,3,2-dioxaphosphorane 2-sulfide
  • Example 16 2-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy) -4-(5-chloro-2,4-difluorophenyl)-1,3,2-dioxaphosphorane 2-sulfide
  • Example 17 2-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy) -4-(2,4-dichloro-5-fluorophenyl)-1,3,2-dioxaphosphorane 2-sulfide
  • Example 18 2-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy) -4-(2,3,4,5-tetrafluorophenyl)-1,3,2-dioxaphosphorane 2-sulfide
  • Example 19 2-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy) -4-(3-chloro-2,4,5-trifluorophenyl)-1,3,2-dioxaphosphorane 2-sulfide
  • Example 20 2-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy) -4-(pentafluorophenyl)-1,3,2-dioxaphosphorane 2-sulfide
  • Example 33 2-((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl))methyl )Amino)-4-(3-chlorophenyl)-1,3,2-dioxaphosphorane 2-sulfide
  • each compound has a faster clearance efficiency in human liver microsomes, of which CS0005 and CS0009 have the fastest clearance rate. Moreover, not all compounds were converted into the expected product AMPS, among which CS0008 and CS0009 produced AMPS at the fastest rate. The results show that the compounds of this structure can be effectively converted into AMPS under the action of human liver microsomes and CYP3A4 enzyme, which can be converted into the active substance AMPS after being taken up by the liver as a drug.
  • N.D. is the compound's rapid clearance, which was not detected at 5 minutes.
  • the rats were euthanized with carbon dioxide gas.
  • Samples were collected: blood was drawn through the heart, stored in heparin anticoagulation tubes, and centrifuged at 6000 rpm for 5 minutes at 4°C. The supernatant plasma was kept on ice; the liver and heart tissues of the rats were collected, rinsed with 4°C pre-cooled physiological saline, and then weighed after absorbing water, and added 5 times the volume of tissue of 0.5 ⁇ g/mL tenofovir Methanol (pre-cooled at 4°C). After the experiment, the samples were stored in the refrigerator at -80°C.
  • Plasma Take 40 ⁇ L of plasma sample in a centrifuge tube, add 200 ⁇ L of methanol solution containing 0.5 ⁇ g/mL PMPA, vortex for 1 min, centrifuge at 4° C. (15000 rpm) for 5 min, take supernatant and water to mix 1:1 and inject for analysis.
  • Tissue Weigh tissue samples quantitatively, add 5 volumes of 0.5 ⁇ g/mL PMPA methanol solution in a homogenization tube, homogenize at low temperature, ultrasonic for 15min at low temperature, centrifuge (15000rpm) at 4°C for 5min, take supernatant and water 1: 1 Inject and analyze after mixing.
  • LC-MS/MS-AJ Triple Quad 5500, AB SCIEX
  • Mobile phase A 0.1% formic acid in water
  • mobile phase B acetonitrile/methanol/formic acid (900/100/1, v/v). The sample was separated by gradient elution. The procedure is shown in Table 3.
  • Mass spectrometry conditions for corresponding internal standards electrospray ionization (ESI) positive ion mode, multiple reaction monitoring (MRM) monitoring ion pair m/z: 364/136 (AMPS); 288/176 (PMPA), capillary voltage It is 16.0kV; the temperature is 500°C; the solvent removal gas flow is 1000L/h; the scanning time is 0.025 seconds; the collision energy is 25V.
  • ESI electrospray ionization
  • MRM multiple reaction monitoring
  • AMPS multiple reaction monitoring
  • PMPA 288/176
  • the concentration-time curves of AMPS released by each prodrug in plasma, liver and heart were used for fitting calculation to obtain the area under the tissue concentration-time curve of AMPS (AUC0-t), time to peak (T max ) and Peak concentration (C max ) in AMPS tissue.
  • the liver tissue distribution results showed that the exposure and peak concentration of the active molecule AMPS released by CS0002, CS0009 and CS0013 was more than twice that of CS0005, CS0008, CS0012, CS0015 and CS0018 ( Table 4 and Figure 1).
  • the results show that 3 chloro substitution, 2 fluoro-4 chloro substitution and 2,5 dichloro substitution on the benzene ring all contribute to the release and enrichment of prodrugs in the liver.
  • the 3-chloro-substituted CS0002 showed a longer peak time than CS0009 and CS0013 (Table 4 and Figure 1), indicating that CS0002 is more likely to maintain the AMPS in the liver above the effective therapeutic concentration for a long time.
  • the compounds CS0002, CS0009, CS0013, CS0029, CS0033, and CS0037 all showed higher liver exposure.
  • the distribution of AMPS in liver tissue showed that CS0034 was 1.5 times that of CS0035.
  • the chiral resolution of CS0034 resulted in a CS0053 AMPS liver exposure that was 275 times higher than the corresponding CS0054 and 3.7 times higher than CS0002 and CS0034, respectively. Times and 2.8 times, the peak concentration of CS0053 is 4.5 times and 2.2 times higher than that of CS0002 and CS0034, respectively.
  • CS0053 In addition to the higher AMPS liver exposure and peak concentration, CS0053 also retains the 3-hour peak time similar to CS0002. Chiral resolution of CS0035 resulted in a liver exposure of CS0045 that was 44 times higher than the corresponding CS0046, which was 94.6% and 112.3% of CS0002 and CS0035.
  • Candidate prodrugs include CS0053, CS0045, CS0034, CS0035, and CS0002.
  • concentration of AMPS in rat plasma is close to or lower than the detection limit of LC/MS.
  • AMPS exposure and peak concentration in rat heart tissue cannot be determined ( Table 4).
  • AMPK agonists have been reported that sufficient exposure to the heart is the main cause of toxic side effects of myocardial hypertrophy (Science, Myers et al.
  • liver-specific delivery performance of the precursor structure namely 1) CYP3A in the family of cytochrome P450 isozymes in hepatocytes specifically catalyzes the release of AMPS, 2 ) And the strong polar AMPS can not enter the circulatory system and reach the heart effectively from liver cells, so it is expected to reduce the cardiotoxicity caused by the AMPS agonist AMPS.
  • N.D. means that the specific tissue concentration of metabolites is lower than the lower detection limit of LC-MS/MS method during the experimental design detection period, 5ng/mL.
  • mice Male C57BL/6 mice, weighing 18-20g, provided by Shanghai Cypre-Bikai Laboratory Animal Co., Ltd.
  • the two-step in situ liver perfusion method was used to isolate primary mouse liver cells.
  • the specific method is: 37°C water bath to preheat D-Hank's perfusate (add 0.5mM EGTA, 25mM HEPES in 1 ⁇ HBSS) and low sugar DMEM digestion solution (add 100CDU/ml collagenase IV, 15mM HEPES, 100ug/ml Streptomycin and 100IU /ml Penicillin); mice are shaved with Zoletil 50 anesthesia, sterilized with 75% ethanol and fixed on an ultra-clean table on the operating table, the abdominal cavity is opened, the inferior vena cava and portal vein are exposed, and a trocar is used Intubate the inferior vena cava and infuse D-Hank's perfusate.
  • the isolated primary mouse liver cells were seeded into a 96-well cell culture plate at a cell density of 2 ⁇ 10 5 /ml, placed in an incubator (37°C, 5% CO 2 ) for 12 hours, and then added at a final concentration of 200uM compounds (including CS0002, CS0005, CS0008, CS0009, CS0015, CS0018, CS0034, CS0035, CS0045, CS0046, CS0053, and CS0054) were processed.
  • 200uM compounds including CS0002, CS0005, CS0008, CS0009, CS0015, CS0018, CS0034, CS0035, CS0045, CS0046, CS0053, and CS0054
  • HTRF Homogeneous Time Resolved Fluorescence detection was performed according to the Phospho-AMPK (Thr172) Cellular Assay Kit (Cat: 64MPKPEG) manual of Cisbio.
  • the specific method is as follows: discard the culture solution, blot dry on absorbent paper, add 50ul of lysate to each well immediately, and shake at room temperature for 30min; take out 16ul of lysate and transfer it to a 384-well plate, then add 4ul of antibody detection solution, and shake the reaction at room temperature 2h; at PerkinElmer Test on Multilabel Plate Reader and record the signal value.
  • the activation efficiency is used as an index to evaluate the AMPK activation activity of the compound.
  • Example 36 AlphaScreen in vitro kinase activity detection method to detect the activation of AMPK by AMP (adenosine monophosphate) and AMPS (thioadenosine monophosphate (AMPS)
  • AMP adenosine monophosphate
  • AMPS thioadenosine monophosphate
  • AMPK protein is a complex composed of three different subunits of ⁇ , ⁇ and ⁇ .
  • the AMPK ( ⁇ 1/ ⁇ 1/ ⁇ 1) protein used for the detection of kinase activity mainly refers to the method of Neumann (Neumann D, Woods A, Carling D, et al. Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli. Protein Expr Purif, 2003; 30(2): 230-7.).
  • the main steps are as follows: constructing a 6 ⁇ His-tagged H6- ⁇ 1- ⁇ 1- ⁇ 1 tricistronic expression plasmid and introducing it into E.
  • the His6-GST-FHA protein required for the AlphaScreen reaction was prepared according to the aforementioned His6-AMPK ( ⁇ 1/ ⁇ 1/ ⁇ 1) protein expression and purification methods.
  • FHA is a domain protein molecule (NCBI Reference Sequence: NP_015172.1) derived from Saccharomyces cerevisiae, a DNA damage checkpoint kinase Rad53 located at amino acids 22-162, which can selectively bind phosphorylated AMPK substrate peptide (AMPK substrate peptide, ASP).
  • AMPK AlphaSreen kinase activity detection method mainly refers to Yan's method (Yan Y, Gu X, Xu HE, Melcher K, et al. A Highly Sensitive Non-Radioactive Activity Assay for AMP-Activated Protein Kinase (AMPK). Methods Protoc, 2018: 1(1):3.), divided into 2 steps: kinase reaction and alphascreen reaction. The specific method is as follows:
  • AMPK and AMPS were divided into 12 different concentrations from the highest concentration of 100uM by a 3-fold gradient, and added to AMPK substrates containing 10nM AMPK ( ⁇ 1/ ⁇ 1/ ⁇ 1) and 50uM biotin labeling Peptide (biotinylated AMPK substrate, b-ASP) and 100uM ATP kinase buffer (25mM Tris, pH 8.0, 300mM NaCl, 5mM MgCl2, 1mM EDTA, 10% glycerol, 2mM DTT), the final volume is 20uL, room temperature reaction 20min; take 10uL reaction solution, add 490uL kinase buffer to dilute to stop the kinase reaction.
  • the AMPK substrate peptide (b-ASP) sequence used in the kinase reaction is: biotin-GSTKMRRVATLVDLGYKK, synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.
  • AlphaScreen reaction take 5ul of the diluted solution after kinase reaction and add to 50nM His6-GST-FHA, 5ug/mL AlphaScreen Streptavidin-coated Donor beads (PerkinElmer, #6760002) and 5ug/mL Nickel-chelate Acceptor beads (PerkinElmer,# AL108M) in AlphaScreen buffer (50mM MOPS, pH7.4, 50mM NaF, 0.5mM CHAPS, 0.1mg/mL bovine serum albumin), the final volume is 100uL, protected from light, shaking at room temperature for 1.5h; in a dark room, the reaction The solution was transferred to OptiPlate-384 well plate (PerkinElmer, #6007290) with white bottom and opaque white base at 40uL/well; using PerkinElmer’s Multilabel Plate Reader detection at 680nM / 620nM Alpha value of the fluorescence signal; plotted using GraphPad Prism dose - response curves, EC 50 values were calculated.
  • the results of detection of the AMPK AlphaScreen kinase activity of the compounds AMPS and AMP are shown in FIG. 4.
  • the EC 50 biological activity of AMPS is 7 times that of AMP, which indicates that AMPS has better AMPK activates activity.

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Abstract

本发明提供了肝脏特异性AMPK激动剂及其制法和应用。具体地，本发明提供了一种如下式(I)所示的化合物，以及其用于治疗包含非酒精性脂肪肝(NAFL)、非酒精性脂肪肝炎(NASH)及其相关肝硬化、肝癌在内的非酒精性脂肪肝病(NAFLD)，或肥胖、糖尿病、高甘油三酯血症、高胆固醇血症、动脉粥样硬化、心血管疾病、代谢性疾病等疾病的用途。其中，各基团定义如说明书中所示。

Description

肝脏特异性AMPK激动剂及其制法和应用 技术领域
本发明提供了基于肝脏特异性方法的AMPK(AMP依赖的蛋白激酶)激动剂及其医疗用途。例如，本发明的化合物用于治疗包含非酒精性脂肪肝(NAFL)、非酒精性脂肪肝炎(NASH)及其相关肝硬化、肝癌在内的非酒精性脂肪肝病(NAFLD)，也可用于肥胖、糖尿病、高甘油三酯血症、高胆固醇血症、动脉粥样硬化、心血管疾病等代谢性疾病。
背景技术
AMPK全称为AMP Activated Protein Kinase，即AMP依赖的蛋白激酶，是一个由催化亚基α、调节亚基β和γ共同组成的异源三聚体蛋白激酶。该蛋白激酶是一个能够感受体内AMP变化的能量激酶，当体内AMP浓度上升到一定程度后，AMPK被激活，抑制合成代谢，促进分解代谢；活化的净效果是ATP消耗过程的抑制和ATP产生途径的激活，因而再生ATP存储。激活后的AMPK能够通过短期和长期两种调控模式，关闭几乎所有合成代谢途径(包括脂类、糖类、蛋白质和核糖体RNA的生物合成)。一方面，AMPK能在蛋白水平上通过磷酸化直接调控下游底物活性，从而在短期内调控细胞能量代谢。另一方面，研究发现AMPK能够下调下游底物的表达量，从而长期调节细胞能量代谢。AMPK作为一种能量平衡器,在调节细胞能量代谢、氧化应激及自嗜方面发挥着重要作用,由于肝脏调节脂肪酸氧化和合成,和脂质代谢的重要作用,AMPK与正常肝功能之间的联系非常明显。代谢性疾病患者都存在肝脏代谢的变化，通过对AMPK进行研究来改善患者体内的糖代谢、脂代谢等，这可能成为治疗此类疾病的有效方法。
AMPK可以通过对抑癌基因的活性进行调控，改善能量代谢紊乱来控制肿瘤的侵袭和转移。当细胞能量代谢失衡时,AMPK可通过磷酸化下游信号P53丝氨酸残基而使其激活,进而负性调控m TOR通路中断肿瘤的增殖,降低肿瘤的侵袭转移。蛋白激酶B(Protein Kinase B，PKB)与肿瘤的转移相关,而AMPK可以通过对PKB的调节来影响肿瘤a的转移。在长期服用化疗药物的肝癌患者中,促进肿瘤细胞凋亡的Fas受体(Fas Receptor,FasR)在耐药的肿瘤细胞中可以被激活,通过诱导转录因子NF-k B(Nuclear factor kB，NF-k B)的活化增加肿瘤的侵袭性,而AMPK家族中的SNARK可以通过调节CD95-NF-k B通路来影响的侵袭转移。同时AMPK也可通过影响金属基质蛋白酶(Matrix Metalloproteinase，MMP)家族中的MMP-2和MMP-9蛋白来降低肿瘤细胞的侵袭性。
由于AMPK各亚基的组合形式不同，理论上AMPK共有12种蛋白组合形式。而α1亚基和γ2亚基拥有不同的可变剪切体(alternative splice variants)，进一步增加AMPK三聚体的复杂性。AMPK在体内的广泛表达，目前尚缺乏组织特异性强、选择性高、毒副作用低的AMPK激动剂。
发明内容
本发明合成了一类硫代核苷单磷酸(AMPS)及其类似物的前体药物分子，口服后 该药物在肝脏组织内代谢获得母体分子，该发明的AMPK激动剂具有高选择性、高疗效、低副作用的优点。
本发明的第一方面，提供了一种如下式I所示的化合物，或其立体异构体或互变异构体，或其药学上可接受的盐、水合物或溶剂化物，
Figure PCTCN2020071525-appb-000001
其中：
R 1选自下组：取代或未取代的C6-C18芳基、取代或未取代的5-12元杂芳基；
R 2选自下组：氢、取代或未取代的C1-C18烷基、取代或未取代的C3-C8环烷基、取代或未取代的C2-C18烷基酰基、取代或未取代的C2-C18烷氧基羰基、单-或二-C2-C18烷基氨基羰基：卤素、卤代烷基、硝基、羟基、氨基和氰基；
R 3和R 4各自独立地选自下组：氢、氟、C1-C6烷基、C1-C6烷氧基；或R 3和R 4共同形成选自下组的基团：C3-C8碳环，或5-12元杂环；
X为O、S、NH、取代或未取代的C1-C4亚烷基；
其中，所述的取代指基团上的氢原子被一个或者多个(例如2个、3个、4个等)选自下组的取代基所取代：卤素、氘代、C1-C6烷氧基、卤代的C1-C6烷基、卤代的C1-C6烷氧基、卤代的C3-C8环烷基、甲基砜基、-S(＝O) 2NH 2、氧代(＝O)、-CN、羟基、-NH 2、羧基、C1-C6酰胺基(-C(＝O)-N(Rc) 2或-NH-C(＝O)(Rc)，Rc为H或C1-C5的烷基)、C1-C6烷基-(C1-C6酰胺基)、或取代或未取代的选自下组的基团：C1-C6烷基、C3-C8环烷基、C1-C6胺基、C6-C10芳基、具有1-3个选自N、S和O的杂原子的5-10元杂芳基、具有1-3个选自N、S和O的杂原子的5-12元杂环基、-(CH 2)-C6-C10芳基、-(CH 2)-(具有1-3个选自N、S和O的杂原子的5-10元杂芳基)，且所述的取代基选自下组：卤素、C1-C6烷基、C1-C6烷氧基、氧代、-CN、-NH 2、-OH、C6-C10芳基、C1-C6胺基、C1-C6酰胺基、具有1-3个选自N、S和O的杂原子的5-10元杂芳基。
在另一优选例中，所述的式I化合物具有选自下组的结构：
Figure PCTCN2020071525-appb-000002
在另一优选例中，所述的X选自下组：O、S、NH、CH 2、CF 2、或者CD 2。
在另一优选例中，所述的R 1具有如下式II、III、IV、V、VI或VII所示的结构：
Figure PCTCN2020071525-appb-000003
其中：
虚线为化学键或无；
各A 1、A 2、A 3、A 5、A 6、A 7、A 8各自独立地为O、S、N、NH、CH或CH 2；A 4和A 9各自独立地为C或N；
各B 1、B 2、B 3、B 4、B 6、B 7、B 8、B 9各自独立地为O、S、N、NH、CH或CH 2；B 5和B 10各自独立地为C或N；
各个R 5、R 6、R 7和R 8各自独立地选自下组：卤素、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基；
i为0、1、2、3、4或5；
Y 1为O、S或者NH；
Y 2和Y 3各自独立地选自O、N或者CH；
j为0、1、2、3或4；
m为0、1、2或3；
n为0、1、2、3或4；
在另一优选例中，所述的R 2选自下组：乙酰基、丁酰基。
在另一优选例中，所述的R 3，R 4各自独立地为氢原子。
在另一优选例中，式(I)的化合物为直接AMPK激动剂的前体药物。
在另一优选例中，式(I)的化合物可具有一个或多个手性中心并因此以多种立体异构形式存在，这些异构体包括互变异构体、顺反异构体、构象异构体、内消旋化合物、具有对映或非对映关系的光学异构体，和可能出现的各种不同异构体的混合物。
在另一优选例中，所述的化合物具有选自下组所示的结构：
Figure PCTCN2020071525-appb-000004
在另一优选例中，所述的化合物选自下组所示的结构：
Figure PCTCN2020071525-appb-000005
在另一优选例中，所述的化合物具有如表1所示的结构：
本发明的第二方面，提供了一种药物组合物，所述的药物组合物包括(a)治疗有效量的如本发明第一方面中所述的化合物、或其药学上可接受的盐、水合物或溶剂化物；和(b)药学上可接受的载体。
在另一优选例中，所述的疾病或病症选自下组：非酒精性脂肪肝(NAFL)、非酒精性脂肪肝炎(NASH)及其相关肝硬化、肝癌、非酒精性脂肪肝病(NAFLD)、肥胖、糖尿病、高甘油三酯血症、高胆固醇血症、动脉粥样硬化、心血管疾病、代谢性疾病。
本发明的第三方面，提供了一种如本发明第一方面所述的式I化合物的用途，其特征在于，用于制备治疗或预防与AMPK活化相关的疾病或病症的药物组合物。
在另一优选例中，所述的疾病或病症选自下组：非酒精性脂肪肝(NAFL)、非酒精性脂肪肝炎(NASH)及其相关肝硬化、肝癌、非酒精性脂肪肝病(NAFLD)、肥胖、糖尿病、高甘油三酯血症、高胆固醇血症、动脉粥样硬化、心血管疾病、代谢性疾病。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了大鼠灌胃给予20μmol/kg的肝靶向AMPS前药后，体内代谢释放活性分子AMPS在肝脏中的浓度-时间曲线图。
图2显示了大鼠灌胃给予20μmol/kg的CS0002系列AMPS前药后，体内代谢释放活性分子AMPS在肝脏中的浓度-时间曲线图。
图3显示了化合物处理小鼠原代肝细胞后的HTRF检测结果。
图4显示了化合物对小鼠原代肝细胞中AMPK磷酸化水平的影响。
具体实施方式
除非明确另外指出，根据本发明和本文所用的术语具有以下含义：
如本文所用，术语“C1-C6烷基”指具有1～6个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基等，或类似基团。优选地，烷基为具有1-3个碳原子的直链或支链的饱和链，例如甲基、乙基、正丙基或异丙基。
如本文所用，术语“C1-C18烷基”指具有1～18个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基等，或类似基团。
如本文所用，术语“C3-C8环烷基”指具有1～8个碳原子的环状烷基，例如环丙基、环丁基、环戊基、环己基、环庚基、环辛基。
如本文所用，术语“C1-C6烷氧基”指上文定义的C1-C6烷基，其通过氧原子连接至分子的剩余部分。优选地，C1-C6烷氧基可包括甲氧基、乙氧基和异丙氧基。
如本文所用，术语“C1-C6烷氨基”指上文定义的C1-C6烷基，其通过氮原子连接至分子的剩余部分。优选地，烷基氨基可包括二甲基氨基和二乙基氨基。
如本文所用，术语“C1-C6羧基”指形如“具有1～5个碳原子的直链或支链烷基-羧基”结构的取代基，其通过烷基碳原子连接至分子的剩余部分。如甲酸基、乙酸基、丙酸基、丁酸基，或类似基团。
如本文所用，术语“C1-C6酯基”指形如“具有1～5个碳原子的直链或支链烷基-酯基”结构的取代基，其通过烷基碳原子连接至分子的剩余部分；其中，酯基的烷基部分为C1-C6烷基。如甲酸甲酯基、甲酸乙酯基、乙酸甲酯基、乙酸甲酯基，或类似基团。
如本文所用，术语“C2-C6烷酰基”指形如“具有1～5个碳原子的直链或支链烷基-羰基”结构的取代基，其通过羰基连接至分子的剩余部分。如乙酰基、丙酰基、丁酰基，或类似基团。
如本文所用，术语“C2-C18烷酰基”指形如“具有1～17个碳原子的直链或支链烷基-羰基”结构的取代基，如乙酰基、丙酰基、丁酰基，或类似基团。
如本文所用，术语“C2-C6烷酰胺基”指形如“具有C2-C6烷酰基-氨基”结构的取代基，其通过氮原子连接至分子的剩余部分；如乙酰胺基、丙酰胺基、丁酰胺基，或类似基团。
如本文所用，术语“C2-C18烷氧基羰基”指形如具有1～17个碳原子的直链或支链烷基-氧-羰基,其通过羰基连接至分子的剩余部分。
如本文所用，术语“C2-C18烷基氨基羰基”指形如具有1～17个碳原子的直链或支链烷基-氮-羰基,其通过羰基连接至分子的剩余部分。
术语“卤素”指F、Cl、Br和I。
术语“卤代烷基”指被卤素取代的C1-C3的烷基。优选地，卤代烷基为三氟甲基、二氟甲基、三氟甲氧基。这里“C1-C3烷基”指具有1～3个碳原子的直链或支链烷基，例如甲基、乙基、正丙基、异丙基。
术语“芳基”指C6-C18芳族基团，例如苯基或萘基，未被取代、被一个或者多个(例如2、3、4或5个)选自以下的原子或基团取代的芳基：卤素、硝基、羟基、氨基、氰基、卤代烷基、取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C1-C6烷胺基、取代或未取代的C1-C6羧基、取代或未取代的C1-C6酯基、取代或未取代的C2-C6烷酰基、取代或未取代的C2-C6烷酰胺基。
术语“杂芳基”指5-12元芳族基团，其包含一个或多个选自氮、氧和硫的杂原子。杂芳基可包括吡啶、吡嗪、嘧啶、噻吩、呋喃、异噁唑、异噻唑、吡唑、咪唑。此类基团可以未被取代、被一个或者多个(例如2、3、4或5个)选自以下的原子或基团取代的杂芳基：卤素、硝基、羟基、氨基、氰基、卤代烷基、取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C1-C6烷胺基、取代或未取代的C1-C6羧基、取代或未取代的C1-C6酯基、取代或未取代的C2-C6烷酰基、取代或未取代的C2-C6烷酰胺基。
术语“杂环”或“杂环基”指5-12元非芳族基团(包括饱和的、部分饱和的或不饱和的基团)，其包含一个或多个选自氮、氧和硫的杂原子，具有单环或稠环(包括桥环体系和螺环体系。在稠环体系中，一个或多个环可以是环烷基、芳基或杂芳基。在一实施例中，杂环基团的氮原子和/或硫原子任选地被氧化，以提供N-氧化物、亚磺酰基和磺酰基部分。“杂环基”及其稠和类似物的例子包括吡咯烷基、哌啶基、哌嗪基、咪唑烷基、2,3-二氢呋喃(2,3-b)并吡啶基、苯并噁嗪基、四氢喹啉基、四氢异喹啉基、二氢吲哚基等。该术语也包括非芳香性的部分不饱和的单环，如通过氮原子连接的2-或4-吡啶酮或N-取代的-(1H，3H)-嘧啶-2,4-二酮类(N-取代的尿嘧啶)。
本发明中，术语“含有”、“包含”或“包括”表示各种成分可一起应用于本发明的混合物或组合物中。因此，术语“主要由...组成”和“由...组成”包含在术语“含有”中。
本发明中，术语“药学上可接受的”成分是指适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)，即有合理的效益/风险比的物质。
本发明中，术语“有效量”指治疗剂治疗、缓解或预防目标疾病或状况的量，或是表现出可检测的治疗或预防效果的量。对于某一对象的精确有效量取决于该对象的体型和健康状况、病症的性质和程度、以及选择给予的治疗剂和/或治疗剂的组合。因此，预先指定准确的有效量是没用的。然而，对于某给定的状况而言，可以用常规实验来确定该有效量，临床医师是能够判断出来的。
如本文所用，术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝 酸、磷酸等无机酸，甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等有机酸；以及天冬氨酸、谷氨酸等酸性氨基酸。
本发明中的一些化合物可能用水或各种有机溶剂结晶或重结晶，在这种情况下，可能形成各种溶剂化物。本发明的溶剂合物包括化学计量的溶剂化物如水合物等，也包括在用低压升华干燥法制备时形成的包含可变量水的化合物。
本文所用的术语“前药”指当给药至生物系统时由于一种或多种自发的化学反应、一种或多种酶催化化学反应和/或一种或多种代谢化学反应而产生“药物”物质(生物学活性化合物)的任意化合物。其还包括本发明的化合物的生物可降解的聚合物衍生物，例如如Int.J.Pharm.115,61-67(1995)所述。
本发明还包括本发明化合物的所有合适的同位素变体。本发明化合物的同位素变体被定义为其中至少一个原子被具有相同原子数但原子质量不同于自然界中常见的原子质量的原子替代的那些。可并入本发明化合物的同位素的实例包括氢、碳、氮、氧、磷、硫、氟和氯的同位素，分别例如 2H、 3H、 11C、 13C、 14C、 15N、 17O、 18O、 35S、 18F和 36Cl。本发明的一些同位素变体，例如，其中并入放射性同位素(例如 3H或 14C)的那些，被用于药物和/或底物组织分布研究。氚代的，即， 3H，和碳-14，即， 14C，同位素是特别优选的，因为它们易于制备和检测。此外，用同位素(例如氘，即， 2H)的取代，可提供由增加的代谢稳定性引起的一些治疗优势，例如，增加的体内半衰期或降低的剂量需求并因此在一些情况下可能是优选的。本发明化合物的同位素变体通常可通过常规操作制备，例如使用适当的同位素变体的合适试剂，通过示例性的方法或下文实验部分中描述的制备。
本发明式(I)的制备方法：
本发明的化合物可以通过本领域技术人员熟知的许多方法制备，包括但不限于下文所述的那些方法，或者通过应用有机合成领域的技术人员已知的标准技术改进这些方法而制备。与本发明相关而公开的所有方法以任意规模进行，包括毫克、克、若干克(multigram)、千克、若干千克(multikilogram)或商业工业规模。在以下反应式和下文中，除非另外说明，R 1至R 4如第一方面中所定义。这些方法形成本发明的其他方面。
在整个说明书中，通式用罗马数字(I)、(II)、(III)、(IV)等表示。这些通式的子集被定义为(Ia)、(Ib)、(Ic)等…、(IVa)、(IVb)、(IVc)等；或者(I-a)、(I-b)、(I-c)等…、(IV-a)、(IV-b)、(IV-c)等。
本发明化合物的通用制备方法如下式所示：
Figure PCTCN2020071525-appb-000006
以下以通式(I)中X为O的情况为例对于制备方法进行说明：
反应式一
Figure PCTCN2020071525-appb-000007
通式(I’)化合物可通过以下制备：根据反应式一，将三乙胺三氢氟酸盐和通式(Ia/b)进行反应。典型的反应条件包括将三乙胺三氢氟酸盐和通式(Ia/Ib)在无水四氢呋喃中，室温反应大约12小时。反应结束后，反应液直接用C18柱，通过快速过柱仪进行柱层析(流动相为水和乙腈)，分离得到的馏分利用冷冻干燥机进行冻干后获得通式(I’)化合物。
反应式二
Figure PCTCN2020071525-appb-000008
通式(Ib)化合物可通过以下制备：根据反应式二，将R 2取代基和通式(Ia)进行缩合反应，获得通式(Ib)。
反应式三
Figure PCTCN2020071525-appb-000009
通式(Ia)化合物可通过以下制备：根据反应式三，将三氯硫磷、三乙胺和通式(Ic)进行反应。然后和二醇(Id)进行反应，或者反应液浓缩后通过硅胶柱层析(流动相为石油醚和乙酸乙酯)后和二醇(Id)进行反应。典型的反应条件包括将三氯硫磷、三乙胺和通式(Ic)在惰性溶剂(如DCM)中，室温反应大约0.5小时，然后二醇(ld)加入，室温反应大约12小时。反应液浓缩后，用C18柱通过快速过柱仪进行柱层析纯化(流动相为水和乙腈)，分离得到的馏分利用冷冻干燥机进行冻干后获得通式(Ia)化合物。
反应式四
Figure PCTCN2020071525-appb-000010
i:通式(Ic)化合物可通过以下制备：根据反应式四，将叔丁基二甲基氯硅烷(TBSCl)、通式(Ie)和咪唑进行反应。典型的反应条件包括将将叔丁基二甲基氯硅烷、通式(Ie)和咪唑进行反应在溶剂(如DMF)中，室温反应大约12小时。反应结束后，反应液浓缩后通过硅胶柱层析(流动相为石油醚和乙酸乙酯)，分离得到的馏 分浓缩后获得通式(If)。
ii:在三氟乙酸和水的作用下，通式(If)脱掉5‘-端的叔丁基二甲基硅烷保护基可以获得通式Ic)。典型的反应条件包括将通式(If)、三氟乙酸和水在溶剂(如THF)中，0℃反应大约5小时。反应液调PH值至中性后过滤，虑饼收集后得到通式(Ic)化合物。
反应式五
Figure PCTCN2020071525-appb-000011
i:通式(Ih)化合物可通过以下制备：将通式(Ig)、二氯亚砜和和催化量的DMF进行反应。典型的反应条件包括将通式(Ig)化合物、二氯亚砜和和催化量的DMF反应在溶剂(如DCM)中，室温反应大约2小时。然后反应液浓缩，加入乙醇后室温反应大约一个小时，结束后，反应液浓缩后通过硅胶柱层析(流动相为石油醚和乙酸乙酯)，分离得到的馏分浓缩后获得通式(Ih)。
ii:低温下(大约-60℃)，通式(Ih)中间体，在六甲基二硅基胺基锂的作用下，和酯进行反应。典型的反应条件包括：大约-60℃温度下，通式(Ih)、六甲基二硅基胺基锂和乙酸乙酯进行反应，反应在此低温下进行大约20min。用乙酸将反应液调PH值至中性后浓缩，通过硅胶柱层析纯化(流动相为石油醚和乙酸乙酯)，分离得到的馏分浓缩后获得通式(Ii)。
iii:在质子溶剂(如MeOH)中，用还原试剂(如NaBH 4)与通式通式(Ii)进行反应，从而获得通式(Id)化合物。
反应式六
在一个特定的实施方案中，所述的通式(ld)化合物的手性1,3-丙二醇衍生物(ll/lm)是通过以下方法制备的：
方法一：
Figure PCTCN2020071525-appb-000012
i:在惰性溶剂(如DCM)中，在酸的(如TMSOTf)存在下,六甲基二硅氮烷和三氟甲磺酸三甲基硅酯进行反应，水洗后浓缩得到的粗品在在酸的(如TMSOTf)的作用下和(2S,5R)-2-异丙基-5-甲基环己酮进行反应，获得中间体通式(Ij)和(Ik)化合物。
ii:室温下，浓盐酸和通式(Ij)和(Ik)分别进行反应，反应液浓缩后通过硅胶柱层析(流动相为石油醚和乙酸乙酯)，分离得到的馏分浓缩后获得通式(Il/Im)。
方法二：
Figure PCTCN2020071525-appb-000013
i:40℃～80℃下，在惰性溶剂(如DMF)中，甲酸、三乙胺、(S,S)-N-(对甲苯磺酰)-1,2-二苯基乙二胺(二氯)(对甲基异丙基苯)钌(II)和中间体Ij进行反应，获得通式(Ip)中间体.
ii:在质子溶剂(如MeOH)中，用还原试剂(如NaBH 4)与通式通式(Ip)进行反应，从而获得通式(In)化合物。
通式(VI)和(VII)的准备可以参考反应式一到反应式五，这里需要用反应式六中获得的手性二醇替代反应式五的非手性二醇。通式((VI)和(VII)化合物通过SFC分离后可以进一步获得通式(VIII)和通式(IX)化合物。应理解，在本发明范围内，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以相互结合，从而构成新的或优选的技术方案。
药物组合物和施用方法
由于本发明化合物具有优异的对AMPK的激活活性，因此本发明化合物及其各种晶型，药学上可接受的无机或有机盐，水合物或溶剂合物，以及含有本发明化合物为主要活性成分的药物组合物可用于治疗、预防以及缓解由乙肝病毒所导致的疾病。根据现有技术，本发明化合物可用于治疗以下疾病：非酒精性脂肪肝(NAFL)、非酒精性脂肪肝炎(NASH)及其相关肝硬化、肝癌在内的非酒精性脂肪肝病(NAFLD)，也可用于肥胖、糖尿病、高甘油三酯血症、高胆固醇血症、动脉粥样硬化、心血管疾病等代谢性疾病。
本发明的药物组合物包含安全有效量范围内的本发明化合物或其药理上可接受的盐及药理上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有0.1-1000mg本发明化合物/剂，更佳地，含有0.5-500mg本发明化合物/剂。较佳地，所述的“一剂”为一个胶囊或药片。
“药学上可以接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。特别优选的 施用方式是口服。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明化合物可以单独给药，或者与其他药学上可接受的化合物联合给药。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为0.2～1000mg，优选0.5～500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点包括：
本发明将硫代的核苷单磷酸制备成一类具有肝组织特异性的环状硫代磷酸酯前体药物，药物分子对胃肠道、血浆具有较好的稳定性，不容易被体内的酯水解酶水解； 在药物进入肝脏后被组织细胞中的细胞色素P450同功酶家族中CYP3A氧化，药物分子硫代磷酸酯环4位开环生成一个带有单磷酸负电荷的中间体，再经过磷酸酶催化水解、β-消除反应，释放出母体药物硫代的核苷单磷酸。硫代的核苷单磷酸在细胞内处于质子化状态，不易通过细胞膜而保留在细胞内，使肝细胞内的药物浓度高于正常组织。前体药物被CYP3A氧化后的副产物芳基乙烯酮，可以快速与肝细胞中含量丰富抗氧化和自由基的谷胱甘肽结合被清除，截至目前还没有发现关于其的副作用报道。本发明的主要优点包括：
(1)肝脏特异性强，本发明中的前体药物，即通式(I)化合物被口服后，在肝细胞中被代谢并生成母体药物；该药物分子带高负电荷，不容易排出肝外，所以在肝中浓度更高，达到肝脏靶向性的效果。
(2)活性高和毒副作用低，因为本发明属于肝脏靶向性药物，活性药物分子组织分布性好，更多的药物分子存在肝脏细胞中，药效也获得了大大的提高。因为药物分子主要集中在肝脏细胞中，在肝外代谢成活性分子的量很少，所以对肾脏、心脏等的副作用会大大的减少。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
中间体制备例1：((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲醇
反应步骤：
步骤1：9-((2R,3R,4R,5R)-3,4-二((叔丁基二甲基硅基)氧基)-5-(((叔丁基二甲基硅基)氧基)甲基)四氢呋喃-2-基)-9H-嘌呤-6-氨基的制备
(2R,3R,4S,5R)-2-(6-氨基-9H-嘌呤-9-基)-5-(羟甲基)四氢呋喃-3,4-二醇(16.0g,60.0mmol)溶于N,N-二甲基甲酰胺(120mL)中，降温至0℃，分别加入咪唑(20.4g,300.0mmol)和叔丁基二甲基氯硅烷(36.2g,240.0mmol)，反应液室温搅拌过。反应完毕后，将反应液减压蒸馏去除有机溶剂，粗产品溶于乙酸乙酯(100mL)，饱和氯化铵(100mL)洗两次，有机相用无水硫酸钠干燥，浓缩，硅胶柱层析(石油醚：乙酸乙酯＝2:1)，得到34g白色固体，收率93％。MS(ES +)m/z 610(M+H +).
步骤2：((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲醇的制备
9-((2R,3R,4R,5R)-3,4-二((叔丁基二甲基硅基)氧基)-5-(((叔丁基二甲基硅基)氧基)甲基)四氢呋喃-2-基)-9H-嘌呤-6-氨基(3.9g,6.4mmol)溶于四氢呋喃(30mL),在0℃条件下加入三氟乙酸/水(12mL/12mL)。反应液在0℃下反应16小时，反应完成后将反应液倒入饱和碳酸氢钠溶液中调节pH＝8,乙酸乙酯萃取(150mL*2)，乙酸乙酯相合并，饱和氯化钠洗，无水硫酸钠干燥后旋干。粗品用乙酸乙酯(15mL)打浆，得到2.2g白色固体,收率73％。. 1H 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).
中间体制备例2：1-(3-氯-2-氟苯基)丙烷-1,3-二醇
反应步骤：
步骤1：乙基 3-氯-2-氟苯甲酸酯的制备
将3-氯-2-氟苯甲酸(4.9g,28.5mmol)和DMF(10滴)溶于二氯甲烷中(40mL)，在0℃下加入草酰氯(4.3g,34.2mmol)，反应搅拌1小时后浓缩旋干，粗品在0℃条件下加入乙醇(40mL)，室温下搅拌1小时后，浓缩后加入乙酸乙酯(50mL)，碳酸氢钠和饱和氯化钠洗，无水硫酸钠干燥后浓缩得5.8g无水油状物，收率100％。 1H 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).
步骤2：乙基 3-(3-氯-2-氟苯)-3-氧代丙酸酯的制备
将乙基3-氯-2-氟苯甲酸酯(5.8g,28.5mmol)和乙酸乙酯(17.6g,199.5mmol)溶于四氢呋喃(60mL)，降温至-60℃后加入LiHMDS(85.5mL,85.5mmol)，反应液在-60℃搅拌30分钟后加入醋酸(9mL)猝灭，加水(50mL)，反应液用乙酸乙酯萃取(40mL*2)，有机相合并，无水硫酸钠干燥浓缩得8.2g棕色油状物粗品。 1H 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).
步骤3：1-(3-氯-2-氟苯基)丙烷-1,3-二醇的制备
乙基3-(3-氯-2-氟苯)-3-氧代丙酸酯(8.2g,28.5mmol)溶于甲醇(70mL)，在0℃下加入NaBH4(5.4g,14.2mmol)，反应室温搅拌3小时后，加入乙酸乙酯(20mL)。反应液浓缩，加入乙酸乙酯/水(50mL/50mL)，乙酸乙酯相浓缩，粗品通过硅胶柱层析(二氯甲烷：甲醇＝10:1)得到3.2g无色油状物,步骤2和步骤3的两步收率55％。 1H 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).
中间体制备例3：1-苯基丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以苯甲酸为起始原料，收率74％， 1H 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).
中间体制备例4：1-(吡啶-4-基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以异烟酸为起始原料，收率50％， 1H 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).
中间体制备例5：1-(3-氯苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以3-氯苯甲酸为起始原料，收率25％， 1H 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).
中间体制备例6：1-(3-(三氟甲基)苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以3-(三氟甲基)苯甲酸为起始原料，收率48％，MS(ES +)m/z 221(M+H +).
中间体制备例7：1-(3-甲基苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以3-甲基苯甲酸为起始原料， 1H 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).
中间体制备例8：1-(3-甲氧基苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以3-甲氧基氯苯甲酸为起始原料， 1H 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).
中间体制备例9:1-(3-氟苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以3-氟苯甲酸为起始原料，收率50％，MS(ES +)m/z 171(M+H +).
中间体制备例10：1-(5-氯-2-氟苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以5-氯-2-氟苯甲酸为起始原料，收率55％, 1H 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).
中间体制备例11：1-(4-氯-2-氟苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以4-氯-2-氟苯甲酸为起始原料，收率64％， 1H 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).
中间体制备例12:1-(2,5-二氟苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以2,5-二氟苯甲酸为起始原料，收率50％, 1H 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).
中间体制备例13：1-(2,5-二氯苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以2,5-二氯苯甲酸为起始原料，收率54％,MS(ES +)m/z 221(M+H +).
中间体制备例14:1-(2-氯-4-氟苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以2-氯-4-氟苯甲酸为起始原料，收率64％， 1H 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).
中间体制备例15:1-(2,4,5-三氟苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以2,4,5-三氟苯甲酸为起始原料，收率71％, 1H 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).
中间体制备例16：1-(2-氯-4,5-二氟苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以2-氯4,5-二氟苯甲酸为起始原料，收率75％, 1HNMR:(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 +)
中间体制备例17：1-(5-氯-2,4-二氟苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以5-氯-2,4-二氟苯甲酸为起始原料，收率74％, 1HNMR:(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 +)
中间体制备例18：1-(2,4-二氯-5-氟苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以2,4-二氯-5-氟苯甲酸为起始原料，收率91％, 1H-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).
中间体制备例19：1-(2,3,4,5-四氟苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以2,3,4,5-四氟苯甲酸为起始原料,收率40％, 1HNMR:(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).
中间体制备例20：1-(3-氯-2,4,5-三氟苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以3-氯-2,4,5-三氟苯甲酸为起始原料，收率78％, 1HNMR:(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).
中间体制备例21：1-(五氟苯基)丙烷-1,3-二醇
参考中间体制备例2的制备方法，不同的是以五氟苯甲酸为起始原料，收率88％， 1HNMR:(400MHz,CD 3OD)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).
中间体制备例22：手性1-(4-氯-2-氟苯基)丙烷-1,3-二醇
步骤1：(2R,6S,7S,10R)-2-(4-氯-2-氟苯基)-7-异丙基-10-甲基-1,5-二氧杂螺[5.5]十一烷的制备
将1-(4-氯-2-氟苯基)丙烷-1,3-二醇(3.0g,14.7mmol)和六甲基二硅氮烷(6.73mL,32.3mmol)溶于四氢呋喃(12mL)中，室温下加入多滴三氟甲磺酸三甲基硅酯，反应室温搅拌2小时，加入乙酸乙酯(50mL)，有机相用饱和氯化钠洗，无水硫酸钠干燥，浓缩。粗品进一步溶解于干燥二氯甲烷中(30mL)，加入(2S,5R)-2-异丙基-5-甲基环己酮(2.72g,17.64mmol)，降温至-40℃，加入三氟甲磺酸三甲基硅酯(391mg,1.764mmol)，反应液在-40℃搅拌过夜，反应完成后加入吡啶(2mL)猝灭，反应液加入二氯甲烷(30mL)，饱和碳酸氢钠溶液(30mL)洗两次。有机相合并浓缩，粗品硅胶柱层析(石油醚：乙酸乙酯＝100:1)，得到1.9g无色油状物(2R,6S,7S,10R)-2-(4-氯-2-氟苯基)-7-异丙基-10-甲基-1,5-二氧杂螺[5.5]十一烷，收率38％， 1H NMR(400MHz,CDCl 3)δ7.54(t,J＝8.0Hz,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)；和油状物(2S,6S,7S,10R)-2-(4-氯-2-氟苯基)-7-异丙基-10-甲基-1,5-二氧杂螺[5.5]十一烷,收率30％， 1H 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).
步骤2：
(R)-1-(4-氯-2-氟苯基)丙烷-1,3-二醇的制备
将(2R,6S,7S,10R)-2-(4-氯-2-氟苯基)-7-异丙基-10-甲基-1,5-二氧杂螺[5.5]十一烷(1.9g,5.59mmol)溶于甲醇(10mL)，室温下加入浓盐酸(1.5mL),反应液室温搅拌过夜，浓缩，加入水(30mL)，乙酸乙酯萃取(30mL*2)，有机相合并浓缩，粗品通过硅胶柱层析(二氯甲烷：甲醇＝10:1)得到0.9g无色油状物(R)-1-(4-氯-2-氟苯基)丙烷-1,3-二醇,收率80％， 1H 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).
步骤3：
(S)-1-(4-氯-2-氟苯基)丙烷-1,3-二醇的制备
参考步骤2中(R)-1-(4-氯-2-氟苯基)丙烷-1,3-二醇的制备方法，收率78％， 1H 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).
中间体制备例23:(R)-1-(3-氯苯基)丙烷-1,3-二醇
参考中间体制备例22步骤1和步骤2，不同的是以1-(3-氯-苯基)丙烷-1,3-二醇代替参考中间体制备例22中的1-(4-氯-2-氟苯基)丙烷-1,3-二醇。收率9％， 1H 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).
中间体制备例24：(S)-1-(3-氯苯基)丙烷-1,3-二醇
参考中间体制备例22步骤1和步骤3，不同的是以1-(3-氯-苯基)丙烷-1,3-二醇代替参考中间体制备例22中的1-(4-氯-2-氟苯基)丙烷-1,3-二醇。收率17％， 1H 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).
中间体制备例25：(R)-1-(2,5-二氯苯基)丙烷-1,3-二醇
参考中间体制备例22步骤1和步骤2，不同的是以1-(2,5-二氯-苯基)丙烷-1,3-二醇代替参考中间体制备例22中的1-(4-氯-2-氟苯基)丙烷-1,3-二醇。收率12％， 1H 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).
中间体制备例26：(S)-1-(2,5-二氯苯基)丙烷-1,3-二醇
参考中间体制备例22步骤1和步骤3，不同的是以1-(2,5-二氯-苯基)丙烷-1,3-二醇代替参考中间体制备例22中的1-(4-氯-2-氟苯基)丙烷-1,3-二醇。收率17％， 1H 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).
中间体制备例27:2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-氯-2-氟苯)-1,3,2-二氧杂磷杂环己烷 2-硫化物
((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲醇(396mg,0.8mmol)溶于无水吡啶(4mL)，在0℃下加入PSCl 3(189mg,1.1mmol).0℃反应30分钟后,加入1-(3-氯-2-氟苯基)丙烷-1,3-二醇(277mg,1.4mmol)的DCM(0.5mL)溶液，反应搅拌过夜，浓缩，粗品通过C18制备柱纯化得到220mg白色固体，收率36％。MS(ES +)m/z 760(M+H +).
中间体制备例28：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-苯基-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-苯基丙烷-1,3-二醇为原料，收率25％， 1H 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).
中间体制备例29：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(吡啶-4-基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(吡啶-4-基)丙烷-1,3-二醇为原料，收率40％， 1H 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).
中间体制备例30：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(3-氯苯基)丙烷-1,3-二醇为原料，收率59％， 1H 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).
中间体制备例31：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-(三氟甲基)苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(3-(三氟甲基)苯基)丙烷-1,3-二醇为原料，收率41％， 1H 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).
中间体制备例32：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-甲基苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(3-甲基苯基)丙烷-1,3-二醇为原料，收率54％， 1H 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).
中间体制备例33：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-三甲氧基苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(3-三甲氧基苯基)丙烷-1,3-二醇为原料，收率47％， 1H 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).
中间体制备例34：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(3-氟苯基)丙烷-1,3-二醇为原料，收率87％, 1H 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).
中间体制备例35：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(5-氯-2-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(5-氯-2-氟苯基)丙烷-1,3-二醇为原料，收率31％， 1H 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).
中间体制备例36：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(4-氯-2-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(4-氯-2-氟苯基)丙烷-1,3-二醇为原料，收率45％， 1H 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).
中间体制备例37：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2,5-二氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(2,5-二氟苯基)丙烷-1,3-二醇为原料，收率68％， 1H 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).
中间体制备例38：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2,5-二氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(2,5-二氯苯基)丙烷-1,3-二醇为原料，收率31％， 1H 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).
中间体制备例39：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2-氯-4-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(2-氯-4-氟苯基)丙烷-1,3-二醇为原料，收率33％, 1H 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).
中间体制备例40：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2,4,5-三氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(2,4,5-三氟苯基)丙烷-1,3-二醇为原料，收率50％,MS(ES +)m/z 762(M+H +)。
中间体制备例41：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2-氯-4,5-二氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(2-氯-4,5-三氟苯基)丙烷-1,3-二醇为原料，收率50％, 1H 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).
中间体制备例42：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(5-氯-2,4-二氟苯基)-1,3,2-二氧杂磷杂环己烷 2- 硫化物
参考中间体制备例27的制备方法，不同的是以1-(5-氯-2,4-二氟苯基)丙烷-1,3-二醇为原料，收率56％, 1H 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).
中间体制备例43：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2,4-二氯-5-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(2,4-二氯-5-氟苯基苯基)丙烷-1,3-二醇为原料，收率50％, 1H 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).
中间体制备例44：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2,3,4,5-四氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(2,3,4,5-四氟苯基)丙烷-1,3-二醇为原料，收率51％, 1H 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).
中间体制备例45：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-氯-2,4,5-三氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(3-氯-2,4,5-三氟苯基)丙烷-1,3-二醇为原料，收率86％, 1H 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).
中间体制备例46：2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(五氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以1-(五氟苯基)丙烷-1,3-二醇为原料，收率55％, 1H 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).
中间体制备例47：N-(9-(3,4-二((叔丁基二甲基硅基)氧基)-5-(((4-(3-氯苯基)-2-硫基-1,3,2-二氧杂磷杂环己烷-2-基)氧基)甲基)四氢呋喃-2-基)-9H-嘌呤-6-基)乙酰胺
2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物(1.1g,1.6mmol)溶于无水吡啶(5mL)中，在0℃条件下，加入乙酰氯(187mg,2.39mmol)，反应室温搅拌1小时，浓缩，粗品硅胶柱层析(石油醚:乙酸乙酯＝1:1)得到380mg白色固体,收率30％， 1H 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).
中间体制备例48：N-(9-(3,4-二((叔丁基二甲基硅基)氧基)-5-(((4-(3-氯苯基)-2-硫基-1,3,2-二氧杂磷杂环己烷-2-基)氧基)甲基)四氢呋喃-2-基)-9H-嘌呤-6-基)丁酰胺
参考中间体制备例47的制备方法，不同的是2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物和丁酰氯反应，收率33％, 1H 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).
中间体制备例49：(4R)-2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(4-氯-2-氟苯)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以(R)-1-(4-氯-2-氟苯基)丙烷-1,3-二醇原料，收率39％。MS(ES +)m/z 760(M+H +).
中间体制备例50：(4S)-2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(4-氯-2-氟苯)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以(S)-1-(4-氯-2-氟苯基)丙烷-1,3-二醇原料，收率21.8％， 1H 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).
中间体制备例51：(4R)-2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-氯苯)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以(R)-1-(3-氯-苯基)丙烷-1,3-二醇原料，收率42％， 1H 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).
中间体制备例52：(4S)-2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-氯苯)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以(S)-1-(3-氯-苯基)丙烷-1,3-二醇原料，收率52.7％, 1H 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).
中间体制备例53：(4R)-2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2,5-二氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以(R)-1-(2,5-二氯-苯基)丙烷-1,3-二醇原料，收率42％, 1H 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).
中间体制备例54：(4S)-2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2,5-二氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考中间体制备例27的制备方法，不同的是以(S)-1-(2,5-二氯-苯基)丙烷-1,3-二醇原料，收率41％, 1H 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).
中间体制备例55：9-((2R,3R,4R,5R)-5-(氨基甲基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)-9H-嘌呤-6-氨基
反应步骤：
步骤1：N-(9-((2R,3R,4R,5R)-3,4-二((叔丁基二甲基硅基)氧基)-5-(((叔丁基二甲基硅基)氧基)甲基)四氢呋喃-2-基)-9H-嘌呤-6-基)苯甲酰胺的制备
N-(9-((2R,3R,4S,5R)-3,4-二羟基-5-(羟甲基)四氢呋喃-2-基)-9H-嘌呤-6-基)苯甲酰胺(1.0g,2.7mmol)溶于N,N-二甲基甲酰胺(20mL)中，降温至0℃，分别加入咪唑(1.8g,26.9mmol)和叔丁基二甲基氯硅烷(3.2g,21.5mmol),反应液室温搅拌过夜。反应完毕后，将反应液减压蒸馏去除有机溶剂，粗产品溶于乙酸乙酯(50mL)，饱和氯化铵(20mL)洗两次，有机相用无水硫酸钠干燥，浓缩，硅胶柱层析(石油醚：乙酸乙酯＝3:1)，得到1.6g白色固体，收率84％。 1H 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)。
步骤2：N-(9-((2R,3R,4R,5R)-3,4-二((叔丁基二甲基硅基)氧基)-5-(羟甲基)四氢呋 喃-2-基)-9H-嘌呤-6-基)苯甲酰胺的制备
N-(9-((2R,3R,4R,5R)-3,4-二((叔丁基二甲基硅基)氧基)-5-(((叔丁基二甲基硅基)氧基)甲基)四氢呋喃-2-基)-9H-嘌呤-6-基)苯甲酰胺(400.0mg,0.6mmol)溶于四氢呋喃(3mL),在0℃条件下加入三氟乙酸/水(1.2mL/1.2mL)。反应液在0℃下反应6小时，反应完成后将反应液倒入饱和碳酸氢钠溶液中调节pH＝8,乙酸乙酯萃取(50mL*2)，乙酸乙酯相合并，饱和氯化钠洗，无水硫酸钠干燥后旋干。粗品用乙酸乙酯(15mL)打浆，得到300.0mg白色固体,收率88％， 1H 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)。
步骤3：((2R,3R,4R,5R)-5-(6-苯甲酰基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲基 磺酸甲酯的制备
冰浴下，将甲磺酰氯(152.0mg,1.3mmol)加入到N-(9-((2R,3R,4R,5R)-3,4-二((叔丁基二甲基硅基)氧基)-5-(羟甲基)四氢呋喃-2-基)-9H-嘌呤-6-基)苯甲酰胺(200.0mg,0.3mmol)和三乙胺(202.0mg,2.0mmol)的二氯甲烷(5mL)溶液中。反应室温搅拌12小时完成后，加水(20mL)猝灭，二氯甲烷萃取(20mL x 2)，有机相合并后浓缩，粗品用硅胶柱层析(石油醚：乙酸乙酯＝2:1)得到140mg淡黄色油状物，收率87％， 1H 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 +)。
步骤4：N-(9-((2R,3R,4R,5R)-5-(叠氮甲基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)-9H-嘌呤-6-基)苯甲酰胺的制备
叠氮化钠(56.0mg,0.9mmol)在室温下，加入到((2R,3R,4R,5R)-5-(6-苯甲酰基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲基 磺酸甲酯(140.0mg,0.2mmol)的DMF(2mL)溶液中。反应液室温搅拌12小时后，加水(30mL)猝灭，乙酸乙酯(30.0mL x 2)萃取，有机相合并后浓缩，粗品用硅胶柱层析(石油醚：乙酸乙酯＝2:1)得到淡黄色油状物80mg，收率54％， 1H 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)。
步骤5：N-(9-((2R,3R,4R,5R)-5-(氨基甲基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)-9H-嘌呤-6-基)苯甲酰胺的制备
N-(9-((2R,3R,4R,5R)-5-(叠氮甲基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)-9H-嘌呤-6-基)苯甲酰胺(80.0mg,0.1mmol.)溶解在甲醇(3.0mL)，加入Pd(OH) 2/C(30.0mg)，在氢气环境下(1atm)室温搅拌2小时，反应液过滤，滤液浓缩得到50mg淡黄色固体，收率65％， 1H 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 +)。
步骤6：9-((2R,3R,4R,5R)-5-(氨基甲基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)-9H-嘌呤-6-氨基的制备
N-(9-((2R,3R,4R,5R)-5-(氨基甲基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)-9H-嘌呤-6-基)苯甲酰胺(1.0g,1.7mmol)溶解在NH 3/MeOH(30.0mL，7.0N)溶液中，室温搅拌12小时后浓缩，粗品用硅胶柱层析(二氯甲烷：甲醇＝10:1)得到500mg淡黄色固体，收率60％， 1H NMR(400MHz,CD 3OD)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 +)。
中间体制备例56：2-((((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲基)氨基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
反应步骤：
步骤1：2-氯-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物的制备
将三乙胺(19.5mg,0.2mmol)和1-(3-氯苯基)丙基-1,3-二醇(30.0mg,0.16mmol)溶解在四氢呋喃(3mL)中，冰浴下，加入三氯硫磷(27.0mg,0.2mmol)，反应室温搅拌12小时后浓缩，粗品通过硅胶柱层析(石油醚：乙酸乙酯＝3:1)得到20.0mg油状物,收率47％， 1H 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)。
步骤2：2-((((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲基)氨基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物的制备
9-((2R,3R,4R,5R)-5-(氨基甲基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)-9H-嘌呤-6-氨基(216.0mg,0.4mmol)和三乙胺(64.0mg,0.6mmol)溶解于二氯甲烷(6mL)中，冰浴下加入2-氯-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物(120mg,0.42mmol)，反应液室温搅拌12小时，浓缩，粗品用硅胶柱层析(二氯甲烷：甲醇＝10:1)得到160mg浅黄色固体,收率51％， 1H 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 +)。
实施例1：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯-2-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-氯-2-氟苯)-1,3,2-二氧杂磷杂环己烷 2-硫化物(200mg,0.3mmol)溶于THF(3mL)，室温下加入三乙胺三氟化氢(838mg,5.2mmol),反应室温下搅拌过夜。反应完成后，加入浓氨水调节pH＝8，浓缩，粗品通过C18制备柱(CH 3CN/H 2O＝2-50％)纯化得到75mg白色固体，收率54％。 1H 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 +).
实施例2：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-苯基-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是以2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-苯基-1,3,2-二氧杂磷杂环己烷2-硫化物为原料，收率87％, 1H 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/z 480(M+H +).
实施例3：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(吡啶-4-基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是以2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(吡啶-4-基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率67％, 1H 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 +).
实施例4：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率59％， 1H 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 +).
实施例5：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-(三氟甲基)苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-(三氟甲基)苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率31％， 1H 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 +)。
实施例6：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-甲基苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-甲基苯基)-1,3,2-二氧杂磷 杂环己烷 2-硫化物为原料，收率72％， 1H 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 +)。
实施例7：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-甲氧基苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-甲氧基苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率62％， 1H 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 +)。
实施例8：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是以2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率41％， 1H 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 +)。
实施例9：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(5-氯-2-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是以2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(5-氯-2-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料,收率51％， 1H 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 +)。
实施例10：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(4-氯-2-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是以2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(4-氯-2-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料,收率53％， 1H 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 +)。
实施例11：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(2,5-二氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是以2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2,5-二氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率41％， 1H NMR(400MHz,MeOD)δ8.36(d,J＝4.4 Hz,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 +).
实施例12：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(2,5-二氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是以2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2,5-二氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料,收率29％， 1H 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 +).
实施例13：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(2-氯-4-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是以2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2-氯-4-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率56％， 1H 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 +).
实施例14：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(2,4,5-三氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是以2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2,4,5-三氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率65％， 1H 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 +).
实施例15：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(2-氯-4,5-二氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是以2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2-氯-4,5-二氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率65％， 1H 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 +).
实施例16：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(5-氯-2,4-二氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(5-氯-2,4-二氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率61％， 1H 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/z550(M+H +).
实施例17：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(2,4-二氯-5-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2,4-二氯-5-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料， 1H 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 +).
实施例18：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(2,3,4,5-四氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2,3,4,5-四氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率49％， 1H 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 +).
实施例19：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯-2,4,5-三氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-氯-2,4,5-三氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率86％， 1H 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 +).
实施例20：2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(五氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(五氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率59％， 1H 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 +).
实施例21：N-(9-((2R,3R,4S,5R)-5-(((4-(3-氯苯基)-2-硫基-1,3,2-二氧杂磷杂环己烷-2-基)氧基)甲基)-3,4-二羟基四氢呋喃-2-基)-9H-嘌呤-6-基)乙酰胺
参考实施例1的制备方法，不同的是以N-(9-(3,4-二((叔丁基二甲基硅基)氧基)-5-(((4-(3-氯苯基)-2-硫基-1,3,2-二氧杂磷杂环己烷-2-基)氧基)甲基)四氢呋喃-2-基)-9H-嘌 呤-6-基)乙酰胺为原料，收率11％， 1H 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/z 556(M+H +).
实施例22：N-(9-((2R,3R,4S,5R)-5-(((4-(3-氯苯基)-2-硫基-1,3,2-二氧杂磷杂环己烷-2-基)氧基)甲基)-3,4-二羟基四氢呋喃-2-基)-9H-嘌呤-6-基)丁酰胺
参考实施例1的制备方法，不同的是以N-(9-(3,4-二((叔丁基二甲基硅基)氧基)-5-(((4-(3-氯苯基)-2-硫基-1,3,2-二氧杂磷杂环己烷-2-基)氧基)甲基)四氢呋喃-2-基)-9H-嘌呤-6-基)丁酰胺为原料，收率26％， 1H 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 +).
实施例23：(4R)-2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(4-氯-2-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是(4R)-2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(4-氯-2-氟苯)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，白色固体，收率61％。 1H 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). 31P NMR(MeOD):δ＝64.86,61.84ppm.MS(ES +)m/z 532(M+H +).
实施例24：(4S)-2-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(4-氯-2-氟苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是(4S)-2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(4-氯-2-氟苯)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率62％， 1H 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). 31P NMR(MeOD):δ＝64.99,61.92ppm.MS(ES +)m/z 532(M+H +).
实施例25：(4R)-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是(4R)-2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-氯苯)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率67％， 1H 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). 31P NMR(MeOD):δ＝63.84,61.21ppm.MS(ES +)m/z 514(M+H +).
实施例26：(4S)-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是(4S)-2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9- 基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(3-氯苯)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率72％， 1H 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). 31P NMR(MeOD):δ＝63.62,61.25ppm.MS(ES +)m/z 514(M+H +).
实施例27：(4R)-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(2,5-二氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是(4R)-2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2,5-二氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率73％, 1H 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). 31P NMR(MeOD):δ＝64.00,60.98ppm.MS(ES +)m/z 548(M+H +).
实施例28：(4S)-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(2,5-二氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是(4S)-2-(((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲氧基)-4-(2,5-二氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率73％, 1H 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). 31P NMR(MeOD):δ＝63.87,61.08ppm.MS(ES +)m/z 548(M+H +).
实施例29和实施例30：
实施例29(4S)-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物 异构体 1
实施例30(4S)-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物 异构体 2
将(4S)-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物(200mg,0.39mmol)通过手性制备HPLC(乙腈/水＝1/10–1/1)获得(4S)-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物 异构体 1，105mg白色固体，收率53％， 1H 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). 31P NMR(DMSO-d 6):δ＝63.64ppm.MS(ES +)m/z 514(M+H +).和(4S)-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷2-硫化物 异构体 2，8mg白色固体，收率4％， 1H 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)。 31P NMR(Methanol-d 4):δ＝62.07ppm.MS(ES +)m/z  514(M+H +).
实施例31和实施例32：
实施例31(4R)-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物 异构体 1
实施例32(4R)-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物 异构体 2
将(4R)-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物(200mg,0.39mmol)通过手性制备HPLC(乙腈/水＝1/10–1/1)获得(4R)-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物 异构体 1，75mg白色固体，收率38％， 1H 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). 31P NMR(DMSO-d 6):δ＝63.86ppm.MS(ES +)m/z 514(M+H +)；和(4R)-(((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物 异构体 2，6mg白色固体，收率3％， 1H 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). 31P NMR(DMSO-d 6):δ＝61.20ppm.MS(ES +)m/z 514(M+H +)。
实施例33：2-((((2R,3S,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基))甲基)氨基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物
参考实施例1的制备方法，不同的是以2-((((2R,3R,4R,5R)-5-(6-氨基-9H-嘌呤-9-基)-3,4-二((叔丁基二甲基硅基)氧基)四氢呋喃-2-基)甲基)氨基)-4-(3-氯苯基)-1,3,2-二氧杂磷杂环己烷 2-硫化物为原料，收率35％， 1H 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 +)。
表1 各个实例中所制备的化合物如下所示：
Figure PCTCN2020071525-appb-000014
Figure PCTCN2020071525-appb-000015
Figure PCTCN2020071525-appb-000016
Figure PCTCN2020071525-appb-000017
Figure PCTCN2020071525-appb-000018
实施例33 体外代谢稳定性实验
1.方法
为了解化合物在体内可能的代谢情况，对不同化合物进行了体外人肝微粒体(X008070，RILD公司)和CYP3A4酶(C3A4R046B，CYPEX公司)的代谢稳定性检测。具体方法为：先将各化合物(包括CS0002、CS0005、CS0008、CS0009、CS0012、CS0013、CS0018)加入到人肝微粒体或CYP3A4酶反应体系中进行反应，反应体系为含200nM化合物、5mM MgCl2、1mM NADPH、0.33mg/ml人肝微粒体(或0.136mg/ml CYP3A4)的0.1M Tris-HCl缓冲液(pH 7.4)，总体积为500ul；置于37℃水浴锅中振荡孵育；分别于0min、5min、15min和30min取出50ul反应液，加入到200ul预冷无水甲醇中沉淀并终止反应；涡旋混匀后，16 000rpm、4℃离心15min，取上清100ul，样品进行LC-MS/MS检测分析测定各化合物及预期产物(AMPS)的含量，色谱质谱的条件同实施例XXX；根据公式(1)：固有清除率(Clint)＝0.693×反应体积/(T 1/2×蛋白质量)和公式(2)：代谢产物的生成速率(V AMPS)＝代谢产物的浓度/反应时间来进行计算。
2.结果
如表2所示，各化合物在人肝微粒体中均有较快的清除效率，其中以CS0005和CS0009的清除率为最快。而且化合物并未全部转化为预期产物AMPS，其中CS0008和CS0009产生AMPS的速率最快。结果表明，此类结构的化合物均能在人肝微粒体和CYP3A4酶的作用下有效的转化为AMPS，可作为药物在体内被肝脏摄取后转化为活性物质AMPS发挥效应。
表2 化合物在人肝微粒体和CYP3A4酶反应体系中的固有清除率和代谢产物生成速率
Figure PCTCN2020071525-appb-000019
Figure PCTCN2020071525-appb-000020
N.D.为化合物快速清除，在5min时未测出。
实施例34 肝递送化合物实验
1.方法
1.1.动物实验
雄性SD大鼠，体重180～300g，上海西普尔-必凯实验动物有限公司提供。雄性动物适应环境3天以上，实验前一天晚上禁食12小时，不禁水。制备AMPS前药的溶液剂(Cremophor EL:乙醇：PEG400：生理盐水＝1：1：4：4)，包括CS0002、CS0005、CS0008、CS0009、CS0012、CS0013、CS0015、CS0018、CS0028、CS0029、CS0033、CS0034、CS0035、CS0036、CS0037、CS0038、CS0045、CS0046、CS0053和CS0054。给药前查看动物体重是否与实验要求相符，选取14只大鼠进行分组，每组2只大鼠，灌胃给予20μmol/kg的药液。分别于0.167、0.5、1、3、6、12和24小时，用二氧化碳气体将大鼠安乐死后采集样品：通过心脏抽取血液，贮存于肝素抗凝管中，4℃下以6000rpm离心5min，取上清血浆于冰中保存；收取大鼠的肝脏和心脏组织，并用4℃预冷的生理盐水润洗干净，吸干水分后称重，加入组织5倍体积0.5μg/mL的替诺福韦甲醇(4℃预冷)。实验后，样本保存于-80℃冰箱中。
1.2.AMPS前药活性代谢产物在生物样品中含量测定
样品前处理
血浆：取血浆样品40μL于离心管中，加入200μL含0.5μg/mL PMPA甲醇溶液，涡流1min，4℃离心(15000rpm)5min，取上清液与水1:1混合后进样分析。
组织：定量称取组织样品，加入5倍体积含0.5μg/mL PMPA甲醇溶液于匀浆管中，低温匀浆，低温超声15min，4℃离心(15000rpm)5min，取上清液与水1:1混合后进样分析。
色谱质谱条件
LC-MS/MS-AJ(Triple Quad 5500,AB SCIEX)用于样品的分析。色谱柱：Acquity UPLC HSS T3(2.1×50mm，1.8μm)；柱温：40 OC；流速：0.5mL/min。流动相A：0.1％甲酸水溶液，流动相B：乙腈/甲醇/甲酸(900/100/1,v/v)。样品分离采用梯度洗脱，程序如表3。和对应内标物的质谱条件：电喷雾离子化(ESI)正离子模式，多重反应监测(MRM)的监测离子对m/z：364/136(AMPS)；288/176(PMPA)，毛细管电压为16.0kV；温度为500℃；去溶剂气流1000L/h；扫描时间0.025秒；碰撞能量25V。
表3 AMPS液相洗脱梯度条件
Figure PCTCN2020071525-appb-000021
Figure PCTCN2020071525-appb-000022
1.3数据分析
做各前药释放出AMPS在血浆、肝脏和心脏中的浓度-时间曲线图。使用WinNonlin 8.0(Pharsight,CA)的非房室模型中的对数-线性梯形法进行拟合计算，得到AMPS的组织浓度-时间曲线下面积(AUC0-t)、达峰时间(T max)和AMPS组织中峰浓度(C max)。
2.结果
大鼠灌胃给予20μmol/kg的药液后，肝脏组织分布的结果显示CS0002、CS0009和CS0013释放的活性分子AMPS的暴露量与峰浓度高于CS0005、CS0008、CS0012、CS0015和CS0018两倍以上(表4和图1)，该结果表明苯环上3氯取代、2氟-4氯取代和2，5双氯取代都有助于前药在肝脏部位的释放和富集。3氯取代的CS0002显示出比CS0009与CS0013更长的达峰时间(表4和图1)，表明CS0002更可能将肝脏中的AMPS长久的维持在有效治疗浓度之上。
如表4和图1所示，大鼠灌胃给予20μmol/kg的药液后，化合物CS0002,CS0009,CS0013,CS0029,CS0033,CS0037均显示较高的肝暴露量。其中，肝脏组织AMPS分布的结果显示CS0034为CS0035的1.5倍，对CS0034进行手性拆分，得到的CS0053的AMPS肝脏暴露量又比对应的CS0054高出275倍，比CS0002和CS0034分别高出3.7倍和2.8倍，CS0053的峰浓度比CS0002和CS0034分别高出4.5倍和2.2倍。除了较高的AMPS肝脏暴露量和峰浓度，CS0053也保留了和CS0002相似的3小时达峰时间。对CS0035进行手性拆分，得到的CS0045的AMPS肝脏暴露量又比对应的CS0046高出44倍，为CS0002和CS0035的94.6％和112.3％。
候选前药包括CS0053、CS0045、CS0034、CS0035和CS0002，在大鼠血浆中的AMPS的浓度接近或低于液质联用的检测限，在大鼠心脏组织中AMPS暴露量和峰浓度无法测定(表4)。AMPK激动剂被报道在心脏足够的暴露量是引发心肌肥大的毒副作用的主要原因(Science,Myers et al.2017)，因此本课题通过环状磷酸酯(4-芳基-2-氧代-1,3,2-二氧杂磷杂环己烷)前体结构的肝脏特异性递送性能，即1)利用肝细胞中的细胞色素P450同功酶家族中的CYP3A特异性催化释放AMPS，2)而强极性的AMPS无法从肝细胞中有效进入循环系统和到达心脏，从而有望降低AMPK激动剂AMPS导致的心脏毒性。上述结果表明，由于本发明式CS0045和式CS0053化合物具有更高的肝递送性，这导致治疗时所需的用量更低，因此具有更高的安全性或更低的毒副作用，因而大大提高了AMPS在临床上的治疗指数(图2)。
表4.大鼠灌胃给予各个AMPS前体化合物20μmol/kg后，24小时以内，代谢产物AMPS在肝脏、心脏和血浆中的暴露量(h*nmol/mL，浓度/组织体积)、达峰浓度(nmol/mL)和达峰时间(h)。
Figure PCTCN2020071525-appb-000023
Figure PCTCN2020071525-appb-000024
N.D.表示代谢产物特定组织浓度在实验设计检测时段全部低于LC-MS/MS方法的检测下限,5ng/mL。
实施例35 小鼠肝原代细胞水平的化合物AMPK激活实验
1.方法
1.1小鼠肝原代细胞分离及培养
雄性C57BL/6小鼠，体重18～20g，上海西普尔-必凯实验动物有限公司提供。采用二步法原位肝脏灌流方法进行原代小鼠肝脏细胞分离。具体方法为：37℃水浴预热D-Hank's灌流液(添加0.5mM EGTA，25mM HEPES in 1×HBSS)和低糖DMEM消化液(添加100CDU/ml胶原酶IV，15mM HEPES，100ug/ml Streptomycin和100IU/ml Penicillin)；将小鼠用舒泰50(Zoletil 50)麻醉后剃毛，75％乙醇消毒后固定于超净台内操作台上，打开腹腔，暴露下腔静脉及门静脉，利用套管针进行下腔静脉插管，灌注D-Hank's灌流液，待肝脏稍鼓起后，剪破门静脉以以10ml/min流速灌流约80ml；替换低糖DMEM消化液，继续以10ml/min流速灌流约60ml；小心游离摘除肝脏，转移至含有低糖DMEM消化液的10cm培养皿中；撕破肝脏被膜收集肝脏细胞悬液，用70μm滤网过滤，所得细胞滤液进行离心，4℃，50×g，2min；弃上清，加入20ml预冷完全培养液(含10％胎牛血清的低糖DMEM)重悬细胞后4℃，50×g，2min离心，并重复一次；用20ml预冷完全培养液重悬细胞，进行台盼兰计数，调整至合适细胞密度后接种于细胞培养板中，置于培养箱(37℃，5％CO 2)中培养。
1.2化合物AMPK激活活性的HTRF磷酸化检测
将分离获得的小鼠肝原代细胞以2×10 5/ml细胞密度接种至96孔细胞培养板中，置 于培养箱(37℃，5％CO 2)中培养12h，然后加入终浓度为200uM的化合物(包括CS0002、CS0005、CS0008、CS0009、CS0015、CS0018、CS0034、CS0035、CS0045、CS0046、CS0053和CS0054)进行处理。化合物作用1h后，按Cisbio公司的Phospho-AMPK(Thr172)Cellular Assay Kit(Cat:64MPKPEG)说明书进行HTRF(Homogeneous Time Resolved Fluorescence)检测。具体方法为：甩弃培养液，在吸水纸上吸干，每孔立即加入50ul裂解液，室温振荡30min；取出16ul裂解液转移至384孔板中，再加入4ul的抗体检测液，室温振荡反应2h；在PerkinElmer公司的
Figure PCTCN2020071525-appb-000025
Multilabel Plate Reader上进行检测，记录信号值。以激活效率作为评价化合物的AMPK激活活性的指标。
2.结果
化合物处理小鼠原代肝细胞后，HTRF检测结果显示CS0002、CS0008、CS0009、CS0015和CS0018具有较强的AMPK激活活性，只有CS0005没有活性，其中以CS0015和CS0018的激活活性最为显著(图3)。结果表明此类结构化合物在体外细胞水平可以进入到细胞内发挥AMPK激活效应。
化合物CS0034、CS0035、CS0045、CS0046、CS0053和CS0054为化合物CS0002的结构拆分体。结果显示化合物CS0034的激活效应约为化合物CS0035的2倍，并且CS0034手性拆分体中的CS0053的激活效应约为CS0054的三倍，具有更好的AMPK激活效应。
实施例36 基于AlphaScreen体外激酶活性检测方法检测AMP(腺苷单磷酸)和AMPS(硫代腺苷单磷酸(AMPS)对AMPK的激活作用
化合物AMPS：
Figure PCTCN2020071525-appb-000026
1.方法
1.1AMPK(α1β1γ1)蛋白和His6-GST-FHA蛋白的制备
AMPK蛋白是由α、β和γ三种不同亚基组成的复合物，激酶活性检测所用的AMPK(α1/β1/γ1)蛋白主要是参照Neumann的方法(Neumann D,Woods A,Carling D,et al.Mammalian AMP-activated protein kinase:functional,heterotrimeric complexes by co-expression of subunits in Escherichia coli.Protein Expr Purif,2003；30(2):230-7.)进行表达。主要步骤为：构建带6×His标签的H6-α1-β1-γ1三顺反子表达质粒，并导入大肠杆菌BL21(DE3)细胞中；用LB培养基在28℃恒温振荡培养箱中将工程菌培养至OD600≈1，再加入0.1mM IPTG，16℃诱导培养16h；收集菌体并重悬于裂解液(25mM Tris,pH 8.0,300mM NaCl,25mM imidazole,10％glycerol,5mMβ-mercaptoethanol)利用高压均质破菌方法裂菌；4℃，20000rpm离心30min收集上清后利用Ni
Figure PCTCN2020071525-appb-000027
6Fast Flow beads(GE Healthcare，#17-5318-03)亲和纯化目的蛋白；再使用AKTA蛋白纯化系统利用HiLoad 26/60Superdex 200column(GE Healthcare，#28-9893-36)进行分子筛色谱层析纯化(流动相：25mM Tris,pH 8.0,300 mM NaCl,5mM MgCl2,1mM EDTA,10％glycerol,2mM DTT)；利用Amicon Ultra-15(Millipore，#UFC910008)离心过滤器浓缩，分装保存于-80℃。AlphaScreen反应所需的His6-GST-FHA蛋白按前述His6-AMPK(α1/β1/γ1)蛋白表达及纯化的方法制备。FHA为源自酿酒酵母(Saccharomyces cerevisiae)的DNA损伤检查点激酶Rad53位于22‐162位氨基酸的结构域蛋白分子(NCBI Reference Sequence:NP_015172.1)，可以选择性的结合磷酸化的AMPK底物肽(AMPK substrate peptide，ASP)。
1.2 AMPK AlphaScreen激酶活性检测
AMPK AlphaSreen激酶活性检测方法主要参照Yan的方法(Yan Y,Gu X,Xu HE,Melcher K,et al.A Highly Sensitive Non-Radioactive Activity Assay for AMP-Activated Protein Kinase(AMPK).Methods Protoc,2018:1(1):3.)，分为2个步骤：激酶反应和alphascreen反应。具体方法如下：
激酶反应：将将化合物AMP和AMPS从最高浓度100uM起以3倍梯度稀释的方式共配置成12个不同浓度，分别加入到含10nM AMPK(α1/β1/γ1)、50uM biotin标记的AMPK底物肽(biotinylated AMPK substrate peptide，b-ASP)和100uM ATP的激酶缓冲液(25mM Tris,pH 8.0,300mM NaCl,5mM MgCl2,1mM EDTA,10％glycerol,2mM DTT)中，终体积为20uL，室温反应20min；取10uL反应液，加入490uL激酶缓冲液稀释终止激酶反应。激酶反应中选用的AMPK底物肽(b-ASP)序列为:biotin-GSTKMRRVATLVDLGYKK，由上海强耀生物科技有限公司合成。
AlphaScreen反应：取5ul激酶反应后的稀释液，加入到含50nM His6-GST-FHA、5ug/mL AlphaScreen Streptavidin-coated Donor beads(PerkinElmer,#6760002)和5ug/mL Nickel-chelate Acceptor beads(PerkinElmer,#AL108M)的AlphaScreen缓冲液(50mM MOPS,pH7.4,50mM NaF,0.5mM CHAPS,0.1mg/mL bovine serum albumin)中，终体积为100uL，避光、室温振荡反应1.5h；暗室中，将反应液按40uL/孔的量转移至白色底部不透光的OptiPlate-384孔板(PerkinElmer,#6007290)；利用PerkinElmer公司的
Figure PCTCN2020071525-appb-000028
Multilabel Plate Reader以680nM/620nM检测alpha荧光信号值；用GraphPad Prism绘制剂量-反应曲线，计算EC 50值。
2.结果
化合物AMPS和AMP的AMPK AlphaScreen激酶活性检测结果如图4中所示。结果显示，二者的EC 50生物活性分别为0.69±0.061uM(AMPS)和4.85±0.996uM(AMP)，AMPS的EC 50生物活性是AMP的7倍，该结果说明AMPS较AMP具有更好的AMPK激活活性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种如下式I所示的化合物，或其立体异构体或互变异构体，或其药学上可接受的盐、水合物或溶剂化物，
    Figure PCTCN2020071525-appb-100001
    其中：
    R 1选自下组：取代或未取代的C6-C18芳基、取代或未取代的5-12元杂芳基；
    R 2选自下组：氢、取代或未取代的C1-C18烷基、取代或未取代的C3-C8环烷基、取代或未取代的C2-C18烷基酰基、取代或未取代的C2-C18烷氧基羰基、单-或二-C2-C18烷基氨基羰基：卤素、卤代烷基、硝基、羟基、氨基和氰基；
    R 3和R 4各自独立地选自下组：氢、氟、C1-C6烷基、C1-C6烷氧基；或R 3和R 4共同形成选自下组的基团：C3-C8碳环，或5-12元杂环；
    X为O、S、NH、取代或未取代的C1-C4亚烷基；
    其中，所述的取代指基团上的氢原子被一个或者多个(例如2个、3个、4个等)选自下组的取代基所取代：卤素、氘代、C1-C6烷氧基、卤代的C1-C6烷基、卤代的C1-C6烷氧基、卤代的C3-C8环烷基、甲基砜基、-S(＝O) 2NH 2、氧代(＝O)、-CN、羟基、-NH 2、羧基、C1-C6酰胺基(-C(＝O)-N(Rc) 2或-NH-C(＝O)(Rc)，Rc为H或C1-C5的烷基)、C1-C6烷基-(C1-C6酰胺基)、或取代或未取代的选自下组的基团：C1-C6烷基、C3-C8环烷基、C1-C6胺基、C6-C10芳基、具有1-3个选自N、S和O的杂原子的5-10元杂芳基、具有1-3个选自N、S和O的杂原子的5-12元杂环基、-(CH 2)-C6-C10芳基、-(CH 2)-(具有1-3个选自N、S和O的杂原子的5-10元杂芳基)，且所述的取代基选自下组：卤素、C1-C6烷基、C1-C6烷氧基、氧代、-CN、-NH 2、-OH、C6-C10芳基、C1-C6胺基、C1-C6酰胺基、具有1-3个选自N、S和O的杂原子的5-10元杂芳基。
  2. 如权利要求1所述的化合物，或其立体异构体或互变异构体，或其药学上可接受的盐、水合物或溶剂化物，其特征在于，所述的式I化合物具有选自下组的结构：
    Figure PCTCN2020071525-appb-100002
  3. 如权利要求1所述的化合物，或其立体异构体或互变异构体，或其药学上可接受的盐、水合物或溶剂化物，其特征在于，所述的R 1具有如下式II、III、IV、V、VI或VII所示的结构：
    Figure PCTCN2020071525-appb-100003
    其中：
    虚线为化学键或无；
    各A 1、A 2、A 3、A 5、A 6、A 7、A 8各自独立地为O、S、N、NH、CH或CH 2；A 4和A 9各自独立地为C或N；
    各B 1、B 2、B 3、B 4、B 6、B 7、B 8、B 9各自独立地为O、S、N、NH、CH或CH 2；B 5和B 10各自独立地为C或N；
    各个R 5、R 6、R 7和R 8各自独立地选自下组：卤素、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基；
    i为0、1、2、3、4或5；
    Y 1为O、S或者NH；
    Y 2和Y 3各自独立地选自O、N或者CH；
    j为0、1、2、3或4；
    m为0、1、2或3；
    n为0、1、2、3或4。
  4. 如权利要求1所述的化合物，或其立体异构体或互变异构体，或其药学上可接受的盐、水合物或溶剂化物，其特征在于，所述的化合物具有选自下组所示的结构：
    Figure PCTCN2020071525-appb-100004
  5. 如权利要求1所述的化合物，或其立体异构体或互变异构体，或其药学上可接受的盐、水合物或溶剂化物，其特征在于，所述的化合物选自下组所示的结构：
    Figure PCTCN2020071525-appb-100005
  6. 如权利要求1所述的化合物，或其立体异构体或互变异构体，或其药学上可接受的盐、水合物或溶剂化物，其特征在于，所述的化合物选自下组所示的结构：
    Figure PCTCN2020071525-appb-100006
    Figure PCTCN2020071525-appb-100007
    Figure PCTCN2020071525-appb-100008
    Figure PCTCN2020071525-appb-100009
    Figure PCTCN2020071525-appb-100010
  7. 一种药物组合物，其特征在于，所述的药物组合物包括(a)治疗有效量的如权利要求1中所述的化合物、或其药学上可接受的盐、水合物或溶剂化物；和(b)药学上可接受的载体。
  8. 如权利要求7所述的药物组合物，其特征在于，所述的疾病或病症选自下组：非酒精性脂肪肝(NAFL)、非酒精性脂肪肝炎(NASH)及其相关肝硬化、肝癌、非酒精性脂肪肝病(NAFLD)、肥胖、糖尿病、高甘油三酯血症、高胆固醇血症、动脉粥样硬化、心血管疾病、代谢性疾病。
  9. 如权利要求1所述的式I化合物的用途，其特征在于，用于制备治疗或预防与AMPK活化相关的疾病或病症的药物组合物。
  10. 如权利要求9所述的用途，其特征在于，所述的疾病或病症选自下组：非酒精性脂肪肝(NAFL)、非酒精性脂肪肝炎(NASH)及其相关肝硬化、肝癌、非酒精性脂肪肝病(NAFLD)、肥胖、糖尿病、高甘油三酯血症、高胆固醇血症、动脉粥样硬化、心血管疾病、代谢性疾病。
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