WO2020063663A1 - 一类三环类似物、其制备方法和用途 - Google Patents

一类三环类似物、其制备方法和用途 Download PDF

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WO2020063663A1
WO2020063663A1 PCT/CN2019/107792 CN2019107792W WO2020063663A1 WO 2020063663 A1 WO2020063663 A1 WO 2020063663A1 CN 2019107792 W CN2019107792 W CN 2019107792W WO 2020063663 A1 WO2020063663 A1 WO 2020063663A1
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compound
reaction
mmol
alkylene
substituted
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French (fr)
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南发俊
李伯良
刘艺
张晓维
张梅
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中国科学院上海药物研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4433—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4436—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a heterocyclic ring having sulfur as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • 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
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the field of medicinal chemistry, and relates to a series of analogs having the structure of natural product Pyripyropene A, and a preparation method and use thereof. More specifically, the present invention relates to analogs of natural product Pyripyropene A, a preparation method thereof, and an acyl-CoA : Application of cholesterol acyltransferase 2 (ACAT2) inhibitors in the treatment of cardiovascular diseases such as atherosclerosis.
  • ACAT2 cholesterol acyltransferase 2
  • Cholesterol is the main sterol form in vertebrates and is found in cells as free cholesterol or as cholesterol esters. Free cholesterol in cells is an essential component of cell membranes and plays a key role in maintaining cell membrane fluidity and cell signaling. In order to maintain normal life activities of mammals, it is necessary to have a fine regulation mechanism to maintain cholesterol metabolism balance. Intracellular free cholesterol mainly plays a functional role on the plasma membrane of the cell. It is an indispensable component of the cell's life and activities, including the survival of the cell, various functions of the cell such as signal transduction, immunity, infection, etc. All are closely related to the dynamic balance of free cholesterol on the plasma membrane, which involves the membrane life structure of the plasma membrane, the functions of various proteins or complexes, and so on.
  • ACAT Cholesteryl acyltransferase
  • ACAT is the only enzyme in the cell that uses free cholesterol as a substrate to synthesize cholesterol esters. It is a key enzyme that regulates the balance of cholesterol metabolism in the cell. It mainly participates in the absorption, transport and storage of living organisms to maintain Cellular cholesterol homeostasis. Intracellular ACAT activity is directly related to the function of the cell membrane microdomain and the formation of foam cells in early atherosclerosis. ACAT is a membrane-bound protein located on the rough endoplasmic reticulum in tissue cells. So far, two subtypes have been found: ACAT1 and ACAT2. They have different organizational distributions.
  • ACAT1 exists in almost all kinds of tissues and cells, and regulates the cholesterol balance in brain, macrophages and adrenal glands.
  • ACAT2 is only expressed in liver and small intestine cells, and is mainly responsible for the esterification and synthesis of cholesterol in the liver and small intestine. It has long been recognized that ACAT is closely related to the occurrence of atherosclerosis. Therefore, inhibiting ACAT may not only reduce the absorption of cholesterol in the small intestine, but also inhibit the formation of multiple types of foam cells including macrophages, which is a very important target for treating cardiovascular diseases.
  • ACAT inhibitors are roughly classified as follows: a. Synthetic inhibitors: including ureas, amides, and imidazoles; b. Microbial inhibitors; c. Natural plant inhibitors.
  • ACAT inhibitors that have been discovered so far have not become drugs, because the selectivity for the inhibitory activity of the two subtypes of ACAT is ignored.
  • Later studies examining the effects of inhibiting ACAT1 on atherosclerosis had different conclusions.
  • One laboratory believes that the absence of ACAT1 can inhibit the occurrence of atherosclerosis; the results of another laboratory experiment show that mice with ACAT1 deficiency have a significantly increased risk of atherosclerosis.
  • ACAT2 knockout mice found that the ACAT-2 -/- mice's ability to absorb cholesterol was reduced, and they were resistant to stone disease and food-induced hypercholesterolemia. Therefore, it is speculated that the specific inhibition of ACAT1 will destroy the intracellular cholesterol metabolism balance, lead to the cytotoxic effect of cholesterol, and is not conducive to preventing the occurrence of atherosclerosis.
  • ACAT2 may be an effective target for the prevention of hyperlipidemia and atherosclerosis. Specific inhibition of ACAT2 will reduce the absorption and transport of cholesterol and will not affect the intracellular cholesterol metabolism balance. Based on the above conclusions, it is particularly important to develop highly selective inhibitors targeting ACAT2.
  • Pyripyropene A has ACAT2-specific inhibitory activity. Pyripyropenes was obtained in 1993 by Satoshi Omura and others from the fermentation broth of Aspergillus fumigates and FO-1289 microorganisms. Naturally, it is very difficult to obtain Pyripyropenes, the procedure is tedious, and the amount is small. In addition, natural Pyripyropenes has the disadvantage of being difficult to prepare. Starting from carvone, the total synthetic route is as many as nineteen steps, and extremely harsh reaction conditions are used many times, and the yield is extremely low. Dr. Zhanyang of the Institute of Medicine has performed a preliminary simplification of Pyripyropene A.
  • the purpose of the present invention is to provide a simplified class of Pyripyropene A analogs, which can be used as ACAT2 inhibitors to prepare drugs for treating cardiovascular diseases such as atherosclerosis.
  • Another object of the present invention is to provide a method for preparing the aforementioned Pyripyropene A analog.
  • Another object of the present invention is to provide the use of the analogue of Pyripyropene A described above.
  • a compound represented by the general formula I a tautomer, an optical isomer and a stereoisomer thereof, or a pharmaceutically acceptable salt thereof,
  • X is O, S, NH or C 1 -C 6 alkylene
  • R 4 is a 4-8 membered heteroaryl group
  • W is hydrogen or hydroxyl
  • R 1 is hydrogen or C 1 -C 6 alkyl
  • n 0, 1, or 2;
  • R 2 and R 3 are each independently hydrogen, substituted or unsubstituted C 3 -C 8 cycloalkyl, substituted or unsubstituted C 6 -C 12 aryl, substituted or unsubstituted 4-8 membered heteroaryl , Substituted or unsubstituted C 1 -C 6 alkoxy;
  • Each of the above substitutions independently refers to having one or more substituents selected from the group consisting of: C 1 -C 6 alkyl, C 6 -C 12 aryl, 4-8 membered heteroaryl, C 3 -C 8 cycloalkyl , C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, halogen, cyano, hydroxyl, amino, -COC 1 -C 6 alkyl, -COC 3 -C 6 cycloalkyl, -COC 6 -C 12 aryl.
  • X is O.
  • R 4 is a 5- to 7-membered heteroaryl group, preferably pyridine.
  • R 1 is methyl, ethyl or propyl.
  • n 1 or 2.
  • R 2 and R 3 are each independently hydrogen, substituted or substituted C 3 -C 6 cycloalkyl, substituted or substituted C 6 -C 10 aryl, substituted or substituted 4- to 6-membered heteroaryl, substituted or unsubstituted C 1 -C 6 alkoxy;
  • Each of the above substitutions independently refers to having one or more substituents selected from the group consisting of: C 1 -C 6 alkyl, C 6 -C 10 aryl, 4- to 8-membered heteroaryl, C 3 -C 6 cycloalkyl , C 1 -C 4 alkoxy, C 1 -C 4 alkylamino, halogen, cyano, hydroxyl, amino, -COC 1 -C 6 alkyl, -COC 3 -C 6 cycloalkyl, -COC 6 -C 10 aryl.
  • the compound is:
  • the compound of the present invention which is a simplified structural analog of the natural product Pyripyropene A, has a selective inhibitory activity of acyl-CoA: cholesterol acyltransferase 2 and can be used for treating cardiovascular diseases such as atherosclerosis.
  • a method for preparing the compound according to the first aspect is provided.
  • the structure of the compound represented by Formula I is shown by Formula Ia, Formula Ib, Formula Ic, or Formula Id.
  • the preparation method includes the following steps. :
  • n, Z, Y, R 2 , R 3 , and R 4 are the same as before, and PG 1 and PG 2 are hydroxyl protecting groups, and the hydroxyl protecting groups are silyl ether, ester group, benzyl ether, or alkyl ether;
  • Compound 14 is ozonated to obtain compound 15, and then Baeyer-Villiger is oxidized to obtain compound 16;
  • compound 20 is reduced to obtain compound Ib.
  • Compound Ib is deprotected and then reacted with acid anhydride, acid chloride, isocyanate or chloroformate to obtain compound Id.
  • the preparation method has one or more of the following characteristics:
  • the solvent used in the ozonation reaction in step (1) is selected from the group consisting of dichloromethane and methanol;
  • the reaction temperature of the ozonation reaction in step (1) is -78 ° C to room temperature
  • the solvent used for the reduction of DIBAL-H in step (3) is dichloromethane
  • the reaction temperature of the DIBAL-H reduction reaction in step (3) is -78 ° C-0 ° C;
  • the solvent used in the coupling reaction in step (4) is an aprotic solvent, which is selected from ether and the like;
  • the reaction temperature of the coupling reaction in step (4) is -78 ° C to room temperature
  • the solvent used in the solvolysis reaction in step (4) is selected from the group consisting of toluene and methanol;
  • the reaction temperature of the solvolysis reaction in step (4) is 80 ° C;
  • the solvent used for the C-acylation ring closure in step (5) is an aprotic solvent selected from tetrahydrofuran;
  • the temperature of the C-acylation ring-closure reaction in step (5) is from 0 ° C to room temperature;
  • the solvent used for the deprotection reaction and the acyl reaction in step (6) is an aprotic solvent selected from dichloromethane;
  • the reaction temperature of the deprotection reaction and the acyl reaction in step (6) is room temperature
  • the solvent used in the Luche reduction reaction in step (6) is selected from alcohol solvents, preferably methanol or ethanol; the reaction temperature is -78 ° C to room temperature.
  • a pharmaceutical composition comprising the compound described in the first aspect or a pharmaceutically acceptable salt thereof;
  • “Pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must be of sufficient purity and low enough toxicity. "Compatibility” herein means that each component of the composition can be blended with the compound of the present invention and each other without significantly reducing the efficacy of the active ingredient.
  • pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and 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.
  • 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
  • a fourth aspect of the present invention provides the use of the compound described in the first aspect or a pharmaceutically acceptable salt thereof for the preparation of: (i) a drug that selectively inhibits ACAT2; or (ii) preventing and / or treating the heart Drugs for vascular diseases.
  • the cardiovascular disease is selected from the group consisting of hyperlipidemia and atherosclerosis.
  • the inventor continued to simplify the structure of Pyripyropenes and removed the ring system that was the most energy-intensive for total synthesis in the mother core of Pyripyropenes. It also eliminated the ortho-dibisacyl structure of the left-most ring system. In order to make the structure simpler and easier to functionalize, the chiral center brought by the o-dibisacyl structure is removed, the structure and the synthetic route are simplified, and the structure of the o-bisbisacyl group is solved in the subsequent research. Poor plasma stability and easy metabolism.
  • the compound of the present invention is not only simple to synthesize, but also has significantly better ACAT2 activity and selective inhibition of ACAT2 than Pyripyropene A. It has better pharmacokinetic properties and is expected to be effective in
  • the target is a new drug for cardiovascular diseases such as atherosclerosis. Based on this, the present invention has been completed.
  • C 1 -C 6 are intended to include corresponding groups having one, two, three, four, five, or six carbon atoms
  • C 1- C 6 alkyl refers to an alkyl group having one, two, three, four, five or six carbon atoms
  • C 3 -C 6 cycloalkyl refers to one having three, four, five Or a cycloalkyl group of 6 carbon atoms.
  • the alkyl group is preferably an aliphatic alkyl group, which may be a linear alkyl group or a branched alkyl group, and includes, without limitation, methyl, ethyl, n-propyl, isopropyl, and n-butyl.
  • Base isobutyl, tert-butyl.
  • An alkylene group is a group formed after an alkane loses two hydrogen atoms, such as -CH 2- , -CH 2 CH 2- , -CH 2 CH 2 CH 2 -or -CH 2 CH 2 CH 2 CH 2- .
  • Alkoxy refers to -O-alkyl
  • alkylamino refers to -NH (alkyl) or -N (alkyl) (alkyl), and alkyl is as defined above.
  • the cycloalkyl group may be a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, including 3 to 20 carbon atoms, preferably including 3 to 12 carbon atoms, and more preferably a cycloalkyl group. Contains 3 to 6 carbon atoms.
  • monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentenyl, cyclohexyl, cyclooctyl, and the like; polycyclic cycloalkyl includes spiro, fused, and bridged cycloalkyl.
  • the aryl group refers to a full-carbon monocyclic or fused polycyclic (that is, a ring sharing a pair of adjacent carbon atoms) group, and the group has a conjugated pi electron system, such as a phenyl group and a naphthyl group.
  • the aryl ring may be fused with a heterocyclic group, a heteroaryl group, or a cycloalkyl ring.
  • Non-limiting examples include benzimidazole, benzothiazole, benzoxazole, benzoisoxazole, and benzopyridine. Azole, quinoline, benzoindole, benzodihydrofuran.
  • the heteroaryl group refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms include oxygen, sulfur, and nitrogen.
  • Heteroaryl is preferably 5- or 6-membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like.
  • the heteroaryl group may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring.
  • the structural formulae described herein are intended to include all tautomeric, optical, and stereoisomeric forms (e.g., enantiomers, diastereomers, geometric isomers, or conformations) (Conformation): For example, R and S configurations containing asymmetric centers, (Z), (E) isomers of double bonds and conformational isomers of (Z), (E).
  • the individual stereochemical isomers, tautomers or enantiomers, diastereomers or geometric isomers or mixtures of tautomers or tautomers of the compounds of the invention All belong to the scope of the present invention.
  • the pharmaceutically acceptable salts are not particularly limited, and preferably include: inorganic acid salts, organic acid salts, alkyl sulfonates, and aryl sulfonates;
  • the inorganic acid salts include hydrochloride , Hydrobromide, nitrate, sulfate, phosphate, etc .
  • the organic acid salts include formate, acetate, propionate, benzoate, maleate, fumarate, amber Acid salts, tartrate salts, citrate salts, etc .
  • the alkyl sulfonates include methane sulfonate, ethyl sulfonate, etc .
  • the aryl sulfonates include benzene sulfonate, p-toluene sulfonate Wait.
  • NMR NMR was measured with a Mercury-Vx 300M instrument manufactured by Varian, and the NMR was calibrated: ⁇ H 7.26 ppm (CDCl 3 ), 2.50 ppm (DMSO-d 6 ), 3.15 ppm (CD 3 OD); Provided by Shanghai Chemical Reagent Company; TLC thin-layer chromatography silica gel plate is produced by Shandong Yantai Huiyou Silica Gel Development Co., Ltd., model HSGF 254; normal phase column chromatography silica gel used for compound purification is produced by Shandong Qingdao Marine Chemical Plant Branch, model zcx -11, 200-300 mesh.
  • Acetyl chloride (37 ⁇ l, 0.52 mmol) was added dropwise to 0.5 ml of methanol, and the mixture was stirred at room temperature for 5 min. Then, a methanol solution of intermediate 6-5 (30 mg, 0.052 mmol) was added and stirred at room temperature for 1 hour. Concentrate and go directly to the next step.
  • the crude product was dissolved in dichloromethane, and a catalytic amount of DMAP, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) (50 mg, 0.26 mmol), p-cyanobenzene was added. Formic acid (38 mg, 0.26 mmol) was stirred at room temperature overnight.
  • DIBAL-H (1.05 ml, 1.05 mmol) was added dropwise to a DCM solution of compound 7-1 (260 mg, 0.70 mmol) in a dry ice acetone bath. After the dropwise addition, the temperature was raised to room temperature and stirred for 2 h. The reaction of the raw materials was monitored by TLC. The reaction was quenched by adding a saturated aqueous solution of potassium sodium tartrate, and the mixture was warmed to room temperature, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and column chromatography (petroleum ether / ethyl acetate).
  • Acetyl chloride (13 ⁇ l, 0.18 mmol) was added dropwise to 0.5 ml of methanol, and the mixture was stirred at room temperature for 5 min, followed by the addition of a methanol solution of intermediate 7-5 (10 mg, 0.018 mmol) and stirred at room temperature for 1 hour. Concentrate and go directly to the next step.
  • the crude product was dissolved in dichloromethane, and a catalytic amount of DMAP, EDCI (10 mg, 0.053 mmol) and p-cyanobenzoic acid (8 mg, 0.053 mmol) were added, followed by stirring at room temperature overnight. The reaction was monitored by TLC.
  • the compound S-carvone in the preparation example was replaced with R-carvone to prepare a compound ALY578R.
  • Acetyl chloride (27 ⁇ l, 0.38 mmol) was added dropwise to 0.5 ml of methanol, and the mixture was stirred at room temperature for 5 min. Then, a methanol solution of intermediate 10-1 (22 mg, 0.038 mmol) was added, and stirred at room temperature for 1 hour. Concentrate and go directly to the next step.
  • the crude product was dissolved in dichloromethane, and a catalytic amount of DMAP, EDCI (36 mg, 0.19 mmol) and p-cyanobenzoic acid (28 mg, 0.19 mmol) were added, followed by stirring at room temperature overnight. The reaction was monitored by TLC.
  • the compound S-carvone in the preparation example was replaced with R-carvone to prepare a compound ALY585R.
  • Acetyl chloride (10 ⁇ l, 0.15 mmol) was added dropwise to 0.5 ml of methanol, and the mixture was stirred at room temperature for 5 min. Then, a methanol solution of intermediate 11-7 (11 mg, 0.038 mmol) was added and stirred at room temperature for 1 hour. Concentrate and go directly to the next step.
  • the crude product was dissolved in dichloromethane, and a catalytic amount of DMAP, EDCI (8 mg, 0.03 mmol), p-cyanobenzoic acid (5 mg, 0.03 mmol) were added, and the mixture was stirred at room temperature overnight. The reaction was monitored by TLC.
  • the method for measuring ACAT2 activity by using a fluorescently labeled sterol was used to detect the inhibitory effect of the analogue of Pyripyropene A structure on ACAT2 activity at the intact cell level.
  • HepG2 cells were cultured in a 96-well plate at a starting density of 1.5 ⁇ 10 4 per well for 24 hours. After the cholesterol mixture was added and mixed, the culture was continued for 24 hours, and then NBD22-fluorescent labeling was added at a final concentration of 0.5 ⁇ g / ml. Sterols and compounds with final concentration gradients of 0, 0.008, 0.04, 0.2, 1, and 5 ⁇ M. Three replicate wells were set for each concentration. After 6 hours of incubation, the fluorescence intensity was measured using a fluorescence analyzer (E488, A535). intensity value and plotted for various concentrations of compound to obtain IC 50.
  • E488, A535 fluorescence analyzer
  • IC 50 is an evaluation of 50% inhibition of ACAT2 activity by sample compounds.
  • IC 50 is obtained to calculate the SI (ACAT1-IC 50 / ACAT2 -IC 50).
  • HepG2 cells were cultured in a 6-well plate at a starting density of 4 ⁇ 10 5 cells per well for 24 hours. The medium was changed and 10 ⁇ g / ml cholesterol and compounds of different concentrations were added. The culture was continued for 9 hours. Amount of cholesterol.

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Abstract

本发明公开了一种三环类似物，结构如式I所示，各取代基的定义如说明书和权利要求书所述。本发明的化合物，为简化的天然产物Pyripyropene A的结构类似物，具有酰基辅酶A：胆固醇酰基转移酶2的选择性抑制活性，可用于治疗心血管疾病比如动脉粥样硬化等。

Description

一类三环类似物、其制备方法和用途 技术领域
本发明属于药物化学领域，涉及一系列具有天然产物Pyripyropene A结构的类似物及其制备方法和用途，更具体而言，本发明涉及天然产物Pyripyropene A的类似物，其制备方法以及作为酰基辅酶A:胆固醇酰基转移酶2(ACAT2)的抑制剂在治疗心血管疾病比如动脉粥样硬化等的应用。
背景技术
胆固醇作为脊椎动物中主要的固醇形式，以游离胆固醇或作为胆固醇酯存在于细胞内。细胞中的游离胆固醇是细胞膜的必需组分，在维持细胞膜的流动性和细胞的信号传导方面起着关键作用。哺乳类动物为维持其正常的生命活动，必需有精细的调控机制以维持胆固醇代谢平衡。细胞内游离胆固醇主要在细胞质膜上发挥功能作用，是细胞的生命与活动所不可或缺的细胞膜组分，包括细胞的存活与否、细胞的各种功能如信号转导、免疫、感染等，均与细胞质膜上游离胆固醇的动态平衡息息相关，其涉及细胞质膜的膜性生命结构、各种蛋白或复合物功能等。
酰基辅酶A:胆固醇酰基转移酶(ACAT)是细胞内唯一以游离胆固醇为底物合成胆固醇酯的酶、是细胞内胆固醇代谢平衡调控的关键酶，主要参与生命体吸收、转运和贮存，以维持细胞胆固醇动态平衡。细胞内ACAT活性与细胞膜微结构域功能、动脉粥样硬化早期病变泡沫细胞形成等直接相关。ACAT是一种位于组织细胞内粗面内质网上的膜结合蛋白，至今发现有两种亚型：ACAT1和ACAT2。它们二者的组织分布较为不同。ACAT1存在于几乎各种组织和细胞中，调控脑、巨噬细胞和肾上腺等组织中的胆固醇平衡。而ACAT2只在肝脏和小肠细胞中有表达，主要负责肝脏和小肠中的胆固醇的酯化合成。人们很早就认识到ACAT与动脉粥样硬化的发生密切相关。因此，抑制ACAT可能不仅能减弱小肠对胆固醇的吸收，而且可以抑制包括巨噬细胞来源的多类泡沫细胞的形成，是一个非常重要的治疗心血管疾病的靶点。
目前已知的ACAT抑制剂大致分类如下：a.合成类抑制剂：包括脲类，酰胺类，以及咪唑类；b.微生物类抑制剂；c.天然植物类抑制剂。但是，至今为止所有已发现的ACAT抑制剂均未成为药物，原因就是忽略了对ACAT两种亚型抑制活性的选择性。后来考察抑制ACAT1对于动脉粥样硬化的影响有不同的结论。一家实验室认为ACAT1的缺失可抑制动脉粥样硬化的发生；另一实验室实验结果表明ACAT1缺失的小鼠患动脉粥样硬化症的几率大大增加。而对ACAT2基因敲除的小鼠分析则发现，ACAT-2 -/-的小鼠吸收胆固醇的能力下降了，对于结石病和食物诱导的高胆固醇血症有抵御作用。因此，推测特异性抑制ACAT1将破坏细胞内胆固醇代谢平衡，导致胆固醇的细胞毒性作用，并不利于防止动脉粥样硬化的发生。而ACAT2对于预防高血脂症和动脉粥样硬化，可能是一个有效的靶点。特异性抑制ACAT2将减少胆固醇的吸收和运输，并不会影响细胞内胆固醇代谢平衡。综合以上结论，开发靶向ACAT2的高选择性抑制剂则显得尤为重要。
但是重新检测已发现的ACAT抑制剂，只有Pyripyropene A具有ACAT2特异抑制活性。Pyripyropenes是1993年Satoshi Omura等人从Aspergillus fumigates FO-1289微生物的发酵液中提取获得的，天然分离获取Pyripyropenes的难度非常大，程序繁琐，获取量少。而且天然Pyripyropenes具有制备困难的缺点，以香芹酮为起点，全合成路线多达十九步，且多次用到极为苛刻的反应条件，产率极低。药物所湛洋博士已对Pyripyropene A进行了初步的简化，将天然产物中的最左侧的A环上方环结构打开、去除环中的两个亚甲基和一个角甲基得到一系列化合物，当制备仍然复杂，本领域尚需合成简便、抑制活性更好，选择性更高的新型ACAT2抑制剂。
发明内容
本发明的目的在于提供一类简化的Pyripyropene A类似物，其可以作为ACAT2抑制剂，从而制备成治疗心血管疾病比如动脉粥样硬化等药物。
本发明的另一目的在于提供上述的Pyripyropene A的类似物的制备方法。
本发明的又一目的是提供上述的Pyripyropene A的类似物的用途。
本发明的第一方面，提供通式I所示的化合物、其互变异构体、光学异构体和立体异构体或其药学上可接受的盐，
Figure PCTCN2019107792-appb-000001
其中，
X为O、S、NH或C 1-C 6亚烷基；
R 4为4-8元杂芳基；
W为氢或羟基；
Figure PCTCN2019107792-appb-000002
表示单键或双键；
R 1为氢或C 1-C 6烷基；
n＝0、1或2；
Y、Z各自独立为O、NH、S、-OC(＝O)-、-OC(＝O)O-、-OC(＝O)O(C 1-C 6亚烷基)-、-OC(＝O)(C 1-C 6亚烷基)-、-O(C 1-C 6亚烷基)-、-OC(＝O)(C 1-C 6亚烷基)NHC(＝O)-、-OC(＝O)NH(C 1-C 6亚烷基)-或-OC(＝O)NH-；
R 2和R 3各自独立地为氢、取代或未取代的C 3-C 8环烷基、取代或未取代的C 6-C 12芳基、取代或未取代的4-8元杂芳基、取代或未取代的C 1-C 6烷氧基；
上述各取代独立指具有选自下组的一个或多个取代基：C 1-C 6烷基、C 6-C 12芳基、4-8元杂芳基、C 3-C 8环烷基、C 1-C 6烷氧基、C 1-C 6烷胺基、卤素、氰基、羟基、氨基、-COC 1-C 6烷基、-COC 3-C 6环烷基、-COC 6-C 12芳基。
在另一优选例中，X为O。
在另一优选例中，R 4为5-7元杂芳基，优选为吡啶。
在另一优选例中，R 1为甲基、乙基或丙基。
在另一优选例中，n为1或2。
在另一优选例中，Y、Z各自独立为O、NH、S、-OC(＝O)-、-OC(＝O)O-、-OC(＝O)O(C 1-C 4亚烷基)-、-OC(＝O)(C 1-C 4亚烷基)-、-O(C 1-C 4亚烷基)-、-OC(＝O)(C 1-C 4亚烷基)NHC(＝O)-、-OC(＝O)NH(C 1-C 4亚烷基)-或-OC(＝O)NH-。
在另一优选例中，R 2和R 3各自独立地为氢、取代或为取代的C 3-C 6环烷基、取代或为取代的C 6-C 10芳基、取代或为取代的4-6元杂芳基、取代或未取代的C 1-C 6烷氧基；
上述各取代独立指具有选自下组的一个或多个取代基：C 1-C 6烷基、C 6-C 10芳基、4-8元杂芳基、C 3-C 6环烷基、C 1-C 4烷氧基、C 1-C 4烷胺基、卤素、氰基、羟基、氨基、-COC 1-C 6烷基、-COC 3-C 6环烷基、-COC 6-C 10芳基。
在另一优选例中，所述化合物为：
Figure PCTCN2019107792-appb-000003
Figure PCTCN2019107792-appb-000004
本发明的化合物，为简化的天然产物Pyripyropene A的结构类似物，具有酰基辅酶A:胆固醇酰基转移酶2的选择性抑制活性，可用于治疗心血管疾病比如动脉粥样硬化等。
本发明的第二方面，提供第一方面所述的化合物的制备方法，通式I所示的化合物的 结构如式Ia、式Ib、式Ic或式Id所示，所述制备方法包括以下步骤：
Figure PCTCN2019107792-appb-000005
n、Z、Y、R 2、R 3、R 4与前相同，PG 1、PG 2为羟基保护基，所述羟基保护基为硅醚、酯基、苄醚或烷基醚；
(1)化合物14双键臭氧化得到化合物15，再经过Baeyer-Villiger氧化，得到化合物16；
(2)化合物16脱去乙酰基，然后保护基保护羟基得化合物17；
(3)化合物17DIBAL-H还原得到化合物18；
(4)化合物18与化合物267以及正丁基锂试剂发生偶联反应，之后被氧化，经过溶剂解反应得到化合物19；
(5)化合物19被LHMDS拔氢烯醇化，随后和R 4COCl发生C-酰基化关环得到化合物20；
(6)化合物20脱去保护基，再和酸酐、酰氯、异氰酸酯或氯甲酸酯发生反应得到化合物Ia；化合物Ia经过Luche还原反应得到化合物Ic；
或化合物20经过还原得到化合物Ib，化合物Ib经脱保护基、再与酸酐、酰氯、异氰酸酯或氯甲酸酯反应可以得到化合物Id。
在另一优选例中，所述制备方法具有以下一个或多个特征：
步骤(1)中所述臭氧化反应所用溶剂选自：二氯甲烷和甲醇；
步骤(1)中所述臭氧化反应的反应温度为-78℃到室温；
步骤(3)中DIBAL-H还原所用溶剂为二氯甲烷；
步骤(3)中DIBAL-H还原反应的反应温度为-78℃-0℃；
步骤(4)中所述偶联反应所用溶剂为非质子性溶剂，选自乙醚等；
步骤(4)中所述偶联反应的反应温度为-78℃到室温；
步骤(4)中所述溶剂解反应所用溶剂选自：甲苯和甲醇；
步骤(4)中所述溶剂解反应的反应温度为80℃；
步骤(5)中所述C-酰基化关环所用溶剂为非质子性溶剂，选自四氢呋喃；
步骤(5)中所述C-酰基化关环反应温度为0℃到室温；
步骤(6)中所述脱保护基反应及酰基反应所用溶剂为非质子性溶剂，选自二氯甲烷；
步骤(6)中所述脱保护基反应及酰基反应的反应温度为室温；
步骤(6)中所述Luche还原反应所用溶剂选自醇类溶剂，优选为甲醇或乙醇；反应温度为-78℃至室温。
本发明的第三方面，提供一种药物组合物，包含第一方面所述的化合物或其药学上可接受的盐；和
药学上可接受的载体。
“药学上可接受的载体”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，而不明显降低活性成分的药效。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
Figure PCTCN2019107792-appb-000006
)、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。
本发明的第四方面，提供第一方面所述的化合物或其药学上可接受的盐的应用，用于制备：(i)选择性抑制ACAT2的药物；或(ii)预防和/或治疗心血管疾病的药物。
在另一优选例中，所述心血管疾病选自下组：高血脂症，动脉粥样硬化。
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。说明书中所揭示的各个特征，可以被任何提供相同、均等或相似目的的替代性特征取代。限于篇幅，在此不再一一赘述。
具体实施方式
本发明人基于长期而深入的研究，继续简化Pyripyropenes A的结构，将Pyripyropenes的母核中全合成耗费精力最多的环系构筑去除之外，还消除最左边环系的邻二双酰基结构，变为更为简单且容易官能团化的结构，去除了邻二双酰基结构所带来的手性中心，简化了结构以及合成路线，且解决了邻二双酰基的结构在后续研究中所带来的血浆稳定性差易代谢的问题。这样获得全新骨架的结构简化了的目标分子，结构复杂程度大大降低，且易于从简单的天然原料香芹酮制备。本发明的化合物，与天然产物Pyripyropene A相比，不仅合成简便，而且其对ACAT2活性的抑制和对ACAT2的选择性抑制均明显优于Pyripyropene A，药代动力学性质更优，有望成为作用于该靶点治疗心血管疾病比如动脉粥样硬化等的新型药物。在此基础上，完成了本发明。
术语
本发明中，形如“C 1-C 6”的表述意在包括具有1个、2个、3个、4个、5个、或6个碳原子的相应基团，例如，“C 1-C 6烷基”指具有1个、2个、3个、4个、5个或6个碳原子的烷基，“C 3-C 6环烷基”指具有3个、4个、5个或6个碳原子的环烷基。
在本文中，所述的烷基优选为脂肪族烷基，可以是直链烷基、支链烷基，非限制性地包括：甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基。亚烷基是烷烃失去两个氢原子后形成的基团，如-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-或-CH 2CH 2CH 2CH 2-。
烷氧基是指-O-烷基，烷胺基是指-NH(烷基)或-N(烷基)(烷基)，烷基定义同上。
在本文中，所述环烷基可以为饱和或者部分不饱和单环或多环环状烃取代基，其中包括3至20个碳原子，优选包括3至12个碳原子，更优选环烷基包含3至6个碳原子。单环环烷基非限制实施例包含环丙基、环丁基、环戊烯基、环己基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
所述芳基指全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，且所述的基团具有共轭的π电子体系，例如苯基和萘基。所述芳基环可以与杂环基、杂芳基或环烷基环稠合，非限制性实施例含苯并咪唑、苯并噻唑、苯并恶唑、苯并异恶唑、苯并吡唑、喹啉、苯并吲哚、苯并二氢呋喃。
所述杂芳基指包含1至4个杂原子，5至14个环原子的杂芳族体系，其中杂原子包括氧、硫和氮。杂芳基优选为是5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述的杂芳基可以稠合于芳基、杂环基或者环烷基环上，其中与母体结构连接在一起的环为杂芳基环。
除非特别说明，本发明所描述的结构式意在包括所有的互变异构、光学异构和立体异构形式(如对映异构体、非对映异构体，几何异构体或构象异构体)：例如含有不对称中心的R、S构型，双键的(Z)、(E)异构体和(Z)、(E)的构象异构体。因此本发明的化合物的单个立体化学异构体、互变异构体或其对映异构体、非对映异构体或几何异构体或构象异构体或互变异构体的混合物都属于本发明的范围。
在本文中，所述的药学上可接受的盐没有特别的限制，优选包括：无机酸盐、有机酸 盐、烷基磺酸盐和芳基磺酸盐；所述无机酸盐包括盐酸盐、氢溴酸盐、硝酸盐、硫酸盐、磷酸盐等；所述有机酸盐包括甲酸盐、乙酸盐、丙酸盐、苯甲酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、柠檬酸盐等；所述烷基磺酸盐包括甲基磺酸盐、乙基磺酸盐等；所述芳基磺酸盐包括苯磺酸盐、对甲苯磺酸盐等。
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(如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件)或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
化合物制备实施例
下述制备实施例中，NMR用Varian生产的Mercury-Vx 300M仪器测定，NMR定标：δH 7.26ppm(CDCl 3)，2.50ppm(DMSO-d 6)，3.15ppm(CD 3OD)；试剂主要由上海化学试剂公司提供；TLC薄层层析硅胶板由山东烟台会友硅胶开发有限公司生产，型号HSGF 254；化合物纯化使用的正相柱层析硅胶为山东青岛海洋化工厂分厂生产，型号zcx-11，200-300目。
制备实施例一(化合物编号ALY603)
Figure PCTCN2019107792-appb-000007
从天然来源易得的原料(S)-香芹酮(20.0g,133.2mmol)出发，和L-脯氨酸的锂盐(1.6g,13.3mmol)放入圆底烧瓶中，再缓慢加入三甲基氰硅烷(35.4ml,266.4mmol)，所得悬浮液室温搅拌24h。TLC显示原料几乎反应完全，加入100mlTHF稀释，和100ml1M盐酸，室 温搅拌1h，随后加入水稀释，乙醚萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩后得到粗品直接下一步。三氧化铬(18.8g，106.6mmol)缓慢加入到乙酸酐(107ml)中，混合物室温搅拌至完全溶解，随后把所得的铬酸试剂滴加到上一步所得粗品的-55℃的二氯甲烷溶液中，保持此温度下搅拌半小时。TLC显示原料反应完全，加入甲醇淬灭，水稀释，二氯甲烷萃取，有机相用饱和碳酸氢钠水溶液洗涤，浓缩。柱层析分离(石油醚/乙酸乙酯＝10/1)，得到产物黄色油状物1(14.0g,两步产率60％): 1H NMR(CDCl3,300MHz)δ4.86(s,1H),4.78(s,1H),2.77-2.32(m,5H),2.05(s,3H),1.68(s,3H).
中间体1(162mg,0.93mmol)和七水氯化亚铈(345mg,0.93mmol)溶于甲醇中，冷至冰水浴中，小心加入硼氢化钠(35.0mg,0.93mmol)，混合物搅拌30mim原料消失。加入丙酮淬灭，乙酸乙酯稀释，有机相用水洗，饱和食盐水洗，干燥，浓缩，柱层析(石油醚/乙酸乙酯＝10/1)分离纯化，得到无色透明油状物为产物2(131.0mg,产率80％) 1H NMR(CDCl3,300MHz)δ4.75(s,1H),4.70(s,1H),4.17(d,J＝5.7Hz,1H),3.01(d,J＝7.2Hz,1H),2.30-2.07(m,4H),2.04(s,3H),1.76(s,3H),1.45(dd,J＝12.6,22.8Hz,1H)。
化合物中间体2(131mg,0.74mmol)溶于N,N-二甲基甲酰胺中加入咪唑(100.7mg,1.48mmol)，二甲基氨基吡啶(cat.),搅拌5min后加入叔丁基二甲基氯硅烷(223.0mg,1.48mmol)，混合物室温搅拌过夜。次日加入水淬灭，乙酸乙酯萃取。有机相用饱和食盐水洗，干燥浓缩。柱层析纯化(石油醚/乙酸乙酯＝25/1)得到中间体3无色透明油状物(213mg,产率98％): 1H NMR(CDCl3,300MHz)δ4.74(s,1H),4.72(s,1H),4.23(brs,1H),2.30-2.00(m,4H),2.00(s,3H),1.69(s,3H),1.49(dd,J＝12.6,22.8Hz,1H),0.89(s,9H),0.09(s,3H),0.07(s,3H)。
中间体3(4.4g,15mmol)溶于二氯甲烷(DCM)(100ml)和MeOH(25ml)中，-78℃下通入臭氧直至反应完全，加入过量二甲硫醚，升至室温搅拌过夜，浓缩后柱层析(石油醚/乙酸乙酯＝10/1)得到中间体4透明油状物(4.2g,14.3mmol,95％): 1H NMR(CDCl 3,300MHz)δ4.23(m,1H),2.74-2.64(m,1H),2.39-2.38(m,2H),2.18(s,3H),2.03(s,3H),1.68-1.57(m,2H),0.89(s,9H),0.10(s,6H)。
中间体4(4.2g,14.3mmol)溶于200ml二氯甲烷中，室温加入间氯过氧苯甲酸(9.9g,42.9mmol)，升温至回流，保持此温度回流12h。硫代硫酸钠饱和溶液淬灭，乙酸乙酯萃取，有机相用饱和碳酸钠溶液洗涤，饱和食盐水洗涤，干燥浓缩。柱层析分离(石油醚/乙酸乙酯(v/v)＝10:1)得到化合物5透明油状物为产物(1.37g,4.4mmol,31％)。 1H NMR(CDCl 3,300MHz)δ4.97-4.87(m,1H),4.28(m,1H),2.60-2.53(m,1H),2.24-2.19(m,2H),2.04(s,3H),2.03(s,3H),1.83-1.72(m,1H),0.90(s,9H),0.10(s,3H),0.09(s,3H)。
中间体5(1.37g,4.4mmol)溶于30ml甲醇中，加入无水碳酸钾(0.92g,6.65mmol),室温搅拌2h，旋去甲醇，乙酸乙酯稀释，水洗涤，饱和食盐水洗涤，干燥浓缩，柱层析分离(石油醚/乙酸乙酯(v/v)＝5/1)得到产物6(1g,3.74mmol,85％)。 1H NMR(CDCl 3,300MHz)δ4.22-4.20(m,1H),4.06(s,1H),2.42(s,2H),2.07(s,3H),2.02-1.92(m,2H),1.99-1.83(m,1H),1.59-1.22(m,1H),1.32-1.24(m,1H)0.90(s,9H),0.10(s,3H),0.09(s,3H)。
中间体6(1g,3.74mmol)溶于DMF中加入咪唑(509mg,7.48mmol)，DMAP(cat.),搅 拌5min后加入TBSCl(1.13mg,7.48mmol)，混合物室温搅拌过夜。次日加入水淬灭，乙酸乙酯萃取。有机相用饱和食盐水洗，干燥浓缩。柱层析纯化(石油醚:乙酸乙酯(v/v)＝25:1)得到中间体6-1(1.14g,2.99mmol,产率80％) 1H NMR(CDCl 3,300MHz)δ4.26-4.21(m,1H),3.86-3.76(m,1H),2.45-2.37(m,1H),2.28-2.11(m,2H),2.00(s,3H),1.70-1.59(m,1H),0.90(s,9H),0.88(s,9H),0.10-0.06(m,12H)。
干冰丙酮浴下中间体6-1(1.14g,2.99mmol)的溶液中滴加二异丁基氢化铝(DIBAL-H)(4.5ml,4.49mmol)，滴毕后升至室温搅拌2h。TLC监测原料反应尽，加入饱和酒石酸钾钠水溶液淬灭，乙酸乙酯萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩，柱层析(正己烷/乙酸乙酯(v/v)＝25/1)得到产物6-2(1.03g,2.69mmol,产率90％)。 1H NMR(CDCl 3,300MHz)δ10.13(s,1H),4.34-4.29(m,1H),3.77-3.73(m,1H),2.66(dd,J＝18.0Hz,3.0Hz,1H),2.23-2.16(m,1H),2.14(s,3H),2.01-1.92(m,1H)1.71-1.50(m,1H)0.93-0.88(m,18H),0.14-0.06(m,12H)。
中间体6-2(1.03g,2.69mmol)及碘代物267(1.08g,4.04mmol)溶于干燥的乙醚中，冷至-78℃，滴加正丁基锂(4.1ml,4.04mmol)，此温下搅拌30分钟，随后加入饱和氯化铵溶液淬灭。乙酸乙酯萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩，直接下一步。粗品溶于二氯甲烷中，0℃加入戴斯-马丁氧化剂(DMP)(1.71g,4.04mmol)，升至室温搅拌过夜。次日加入饱和硫代硫酸钠溶液/饱和碳酸氢钠溶液＝1/1淬灭，二氯甲烷萃取，饱和食盐水洗涤，浓缩，柱层析纯化(石油醚/乙酸乙酯(v/v)＝10/1),得到中间体6-3(0.48g,产率33％)。中间体6-3(0.48g,0.89mmol)溶于甲苯(40ml)和甲醇(10ml)中，反应混合物加热至外温80℃下回流过夜。次日浓缩柱层析分离(石油醚/乙酸乙酯(v/v)＝10/1)，得到中间体6-4(0.45g,0.88mmol,产率99％)。 1H NMR(CDCl 3,300MHz)δ3.87–3.79(m,4H),3.72–3.63(m,1H),2.46–2.41(m,1H),2.22(s,3H),1.99–1.93(m,1H),1.55-1.42(m,2H),1.37–1.24(m,1H),1.24(s,3H),0.91–0.87(m,18H),0.12–0.05(m,12H)。
向0℃下LHMDS(8.8ml,8.8mmol)的THF溶液中滴加中间体6-4(0.45g,0.88mmol)的THF溶液，升至室温搅拌4h,快速加入烟酰氯盐酸盐，室温搅拌2h。TLC检测原料反应彻底，加入乙酸淬灭，水稀释，二氯甲烷萃取，无水硫酸钠干燥，浓缩。柱层析纯化(石油醚/丙酮(v/v)＝3/1)，得到中间体6-5(195mg,0.41mmol,产率47％)。 1H NMR(CDCl 3,300MHz)δ9.04(s,1H),8.75(d,J＝3.0Hz,1H),8.19–8.16(m,1H),7.44(dd,J＝9.0,6.0Hz,1H),6.48(s,0.3H),6.42(s,0.7H),3.96(dd,J＝12.0,6.0Hz,1H),3.77–3.70(m,1H),2.60(dd,J＝12.0,3.0Hz,1H),2.37(d,J＝15.0Hz,1H),2.03–1.94(m,1H),1.53(d,J＝9.0Hz,1H),1.35(s,3H),1.21–1.14(m,1H),0.96–0.89(m,18H),0.19–0.07(m,12H)。
乙酰氯(37μl,0.52mmol)滴加到0.5ml甲醇中，混合物在室温搅拌5min，随后加入中间体6-5(30mg,0.052mmol)的甲醇溶液，室温下搅拌1小时。浓缩，直接下一步。粗品溶于二氯甲烷中，加入催化量DMAP，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)(50mg,0.26mmol)，对氰基苯甲酸(38mg,0.26mmol)，室温搅拌过夜。TLC监测反应完全，加入水淬灭，乙酸乙酯萃取，无水硫酸钠干燥，柱层析(二氯甲烷/甲醇(v/v)＝25/1)分离纯化，得到淡黄固体中间体6-6(20mg,0.033mmol,产率63％)。 1H NMR(CDCl 3,300 MHz)δ9.08(s,1H),8.82(s,1H),8.23–7.99(m,4H),7.83–7.49(m,6H),6.53(s,0.5H),6.51(s,0.5H),5.70–5.59(m,1H),5.37–5.25(m,1H),3.19(dd,J＝9.0,3.0Hz,1H),2.99(dd,J＝12.0,3.0Hz,,1H),2.83–2.68(m,1H),2.22–2.13(m,1H),1.99–1.86(m,1H),1.74(s,2H),1.68(s,1H)。
中间体6-6(20mg,0.033mmol)和七水氯化亚铈(86mg,0.23mmol)溶于甲醇中，冷至-78℃小心加入硼氢化钠(9mg,0.23mmol)，混合物搅拌30分钟原料消失。加入丙酮淬灭，乙酸乙酯稀释，有机相用水洗，饱和食盐水洗，干燥，浓缩，柱层析(二氯甲烷/甲醇(v/v)＝25/1)分离纯化得到终产物ALY603(18mg,90％): 1H NMR(CDCl 3,300MHz)δ9.03(s,1H),8.72(d,J＝3.0Hz,1H),8.13(d,J＝9.0Hz,1H),8.02–7.94(m,4H),7.62–7.54(m,4H),7.46–7.42(m,1H),6.52(s,1H),5.55(s,1H),5.46(s,1H),5.14(d,J＝6.0Hz,1H),2.89(s,1H),2.58–2.35(m,4H),1.26(s,3H)。
用同样方法合成以下化合物：
将制备实施例一中的化合物S-香芹酮替换为R-香芹酮，制备得到化合物ALY603R。
Figure PCTCN2019107792-appb-000008
Figure PCTCN2019107792-appb-000009
Figure PCTCN2019107792-appb-000010
制备实施例二(化合物编号ALY578)
由制备实施例一得到的中间体6(270mg,1.01mmol)出发，NaH(121mg,3.03mmol)悬浮于干燥四氢呋喃中，滴加中间体6的干燥THF溶液，室温搅拌30min，滴加对甲基溴苄(374mg,2.02mmol)的干燥THF溶液，升至75℃回流2h。TLC检测反应完全，水淬灭，乙酸乙酯萃取，有机相用饱和食盐水洗，干燥，浓缩，柱层析(石油醚/乙酸乙酯(v/v)＝25/1)分离纯化，得到透明油状物为中间体7-1(260mg,0.70mmol,产率70％): 1H NMR(CDCl 3,300MHz)δ7.24–7.15(m,4H),4.57–4.47(m,2H),4.21(s,1H),3.63–3.54(m,1H),2.64–2.56(m,1H),2.35–2.22(m,5H),2.00(s,3H),1.71–1.60(m,1H),0.91–0.89(m,9H),0.10(d,J＝3.0Hz,6H)。
Figure PCTCN2019107792-appb-000011
干冰丙酮浴下化合物7-1(260mg,0.70mmol)的DCM溶液中滴加DIBAL-H(1.05ml,1.05mmol)，滴毕后升至室温搅拌2h。TLC监测原料反应尽，加入饱和酒石酸钾钠水溶液淬灭，混合物升至室温，乙酸乙酯萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩，柱层析(石油醚/乙酸乙酯(v/v)＝25/1)，得到产物7-2(160mg,0.43mmol,产率61％)。 1H NMR(CDCl 3,300MHz)δ10.15(s,1H),7.26–7.14(m,4H),4.55(dd,J＝15.0,12.0Hz,2H),4.30(s,1H),3.56–3.50(m,1H),2.92(d,J＝18.0Hz,1H),2.34(s,4H),2.15(s,3H),2.06–1.98(m,1H),1.73–1.62(m,1H),0.92(s,9H),0.13(d,J＝3.0Hz,6H)。
中间体7-2(160mg,0.43mmol)及碘代物267(172mg,0.64mmol)溶于干燥的乙醚中，冷至-78℃，滴加正丁基锂(0.64ml,0.64mmol)，此温下搅拌30分钟，随后加入饱和氯化铵溶液淬灭。乙酸乙酯萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩，直接下一步。粗品溶于二氯甲烷中，0℃加入DMP(272mg,0.64mmol)，升至室温搅拌过夜。次日加入饱和硫代硫酸钠溶液/饱和碳酸氢钠溶液＝1/1淬灭，二氯甲烷萃取，饱和食盐水洗涤，浓缩，柱层析纯化(石油醚/乙酸乙酯(v/v)＝10/1),得到中间体7-3(73mg,产率33％)。中间体7-3(73mg,0.14mmol)溶于甲苯(10ml)和甲醇(2ml)中，反应混合物加热至外温80℃下回流过夜。次日浓缩柱层析分离(石油醚/乙酸乙酯(v/v)＝10/1)，得到中间体7-4(41mg,0.084mmol,产率60％)。 1H NMR(CDCl 3,300MHz)δ7.23–7.13(m,4H),4.60–4.44(m,2H),3.86–3.80(m,4H),3.49–3.39(m,1H),2.55(d,J＝15.0Hz,1H),2.38(dd,J＝15.0,6.0Hz,1H),2.34(s,3H),2.22(s,3H),2.15–2.09(m,1H),1.55–1.43(m,1H),1.25(s,3H),0.90(s,9H),0.10(d,J＝6.0Hz,6H)。
向0℃下LHMDS(0.6ml,0.59mmol)的THF溶液中滴加中间体7-4(41mg,0.084mmol)的THF溶液，升至室温搅拌4h，快速加入烟酰氯盐酸盐，室温搅拌2h。TLC检测原料反应彻底，加入乙酸淬灭，水稀释，二氯甲烷萃取，无水硫酸钠干燥，浓缩。柱层析纯化(石 油醚/丙酮(v/v)＝3/1)，得到中间体7-5(10mg,0.018mmol,产率23％)。 1H NMR(CDCl 3,300MHz)δ9.05(s,1H),8.75(d,J＝3.0Hz,1H),8.18(d,J＝9.0Hz,1H),7.47–7.43(m,1H),7.25–7.14(m,4H),6.42(s,1H),4.63–4.46(m,2H),3.95(dd,J＝12.0,6.0Hz,1H),3.53–3.44(m,2H),2.66(d,J＝15.0Hz,1H),2.55(dd,J＝12.0,3.0Hz,1H),2.34(s,3H),2.19(d,J＝12.0Hz,1H),1.60–1.52(m,2H),1.25(s,3H),0.95–0.88(m,9H),0.16(d,J＝12.0Hz,6H)。
乙酰氯(13μl,0.18mmol)滴加到0.5ml甲醇中，混合物在室温搅拌5min，随后加入中间体7-5(10mg,0.018mmol)的甲醇溶液，室温下搅拌1小时。浓缩，直接下一步。粗品溶于二氯甲烷中，加入催化量DMAP，EDCI(10mg,0.053mmol)，对氰基苯甲酸(8mg,0.053mmol)，室温搅拌过夜。TLC监测反应完全，加入水淬灭，乙酸乙酯萃取，无水硫酸钠干燥，柱层析(二氯甲烷/甲醇(v/v)＝25/1)分离纯化，得到淡黄固体中间体7-6(5mg,0.009mmol,产率50％)。 1H NMR(CDCl 3,300MHz)δ9.04(s,1H),8.74(s,1H),8.21–8.00(m,3H),7.82–7.79(m,1H),7.60(d,J＝9.0Hz,1H),7.44(s,1H),7.23–7.08(m,4H),6.48(s,0.3H),6.48(s,0.7H),5.50(dd,J＝12.0,6.0Hz,1H),4.63–4.35(m,2H),3.82–3.67(m,1H),3.11(dd,J＝9.0,3.0Hz,1H),2.76(d,J＝9.0Hz,1H),2.59–2.52(m,1H),2.34(s,3H),2.24–2.17(m,1H),2.04–1.94(m,1H),1.60(s,3H)。
化合物7-6(5mg,0.009mmol)和七水氯化亚铈(20mg,0.063mmol)溶于甲醇中，冷至-78℃小心加入硼氢化钠(2mg,0.063mmol)，混合物搅拌30分钟原料消失。加入丙酮淬灭，乙酸乙酯稀释，有机相用水洗，饱和食盐水洗，干燥，浓缩，柱层析(二氯甲烷/甲醇＝50/1)分离纯化，得到淡黄固体为最终产物ALY578(4mg,产率77％): 1HNMR(500MHz,CDCl3)δ9.02–8.95(m,1H),8.71–8.66(m,1H),8.20–7.79(m,4H),7.47–7.347(m,2H),7.23–7.01(m,4H),6.51(s,0.3H),6.40(s,0.7H),5.30(dd,J＝10.0,5.0Hz,1H),4.61–4.44(m,2H),4.28(d,J＝10.0Hz,1H),3.96(s,1H),3.74–3.67(m,1H),2.79(d,J＝15.0Hz,1H),2.56–2.53(m,1H),2.35(d,J＝5.0Hz,3H),2.11–2.08(m,1H),1.92–1.88(m,1H),1.79–1.71(m,1H),1.48–1.40(m,3H)。
将制备实施例中的化合物S-香芹酮替换为R-香芹酮，制备得到化合物ALY578R。
用同样方法合成以下化合物：
Figure PCTCN2019107792-appb-000012
Figure PCTCN2019107792-appb-000013
制备实施例三(化合物编号ALY515)
Figure PCTCN2019107792-appb-000014
由制备实施例一得到的化合物6-5出发，乙酰氯(19μl,0.26mmol)滴加到0.5ml甲醇中，混合物在室温搅拌5min，随后加入中间体6-5(15mg,0.026mmol)的甲醇溶液，室温下搅拌1小时。浓缩，直接下一步。粗品溶于干燥DMF中，加入催化量DBU，干燥三乙胺(15μl,0.104mmol)，异氰酸丙酯(15μl,0.156mmol)，80℃搅拌过夜。TLC监测反应完全，加入水淬灭，乙酸乙酯萃取，无水硫酸钠干燥，柱层析(二氯甲烷/甲醇(v/v)＝25/1)分离纯化，得到透明油状物中间体8-1(5mg,0.01mmol,产率39％)。 1H NMR(CDCl 3,300MHz)δ9.06(s,1H),8.75(d,J＝3.0Hz,1H),8.19(d,J＝6.0Hz,1H),7.48–7.43(m,1H),6.55(s,0.5H),6.53(s,0.5H),5.26-5.17(m,1H),4.93–4.65(m,3H),3.25–3.09(m,4H),2.97-2.91(m,1H),2.82-2.78(m,1H),2.64-2.40(m,2H),2.17-2.14(m,1H),1.62-1.43(m,7H),1.00-0.85(m,6H)。
中间体8-1(5mg,0.01mmol)和七水氯化亚铈(25mg,0.07mmol)溶于甲醇中，冷至-78℃小心加入硼氢化钠(3mg,0.07mmol)，混合物搅拌30分钟原料消失。加入丙酮淬灭，乙酸 乙酯稀释，有机相用水洗，饱和食盐水洗，干燥，浓缩，柱层析(二氯甲烷/甲醇(v/v)＝25/1)分离纯化得到终产物ALY515(4mg,78％): 1H NMR(500MHz,CDCl 3)δ9.01(dd,J＝10.0,5.0Hz,1H),8.78–8.59(m,1H),8.11-8.08(m,1H),7.43-7.39(m,1H),6.49(s,1H),5.10–5.02(m,1H),4.83-4.75(m,2H),4.48(d,J＝10.0Hz,1H),4.61(s,1H)4.34(s,1H),3.22–3.12(m,4H),2.66-2.60(m,1H),2.45(s,1H),2.23(s,1H),2.04(s,1H),1.93–1.89(m,1H),1.59-1.49(m,4H),1.38(s,1.5H),1.31(s,1.5H),0.98-0.90(m,6H)。
用同样方法合成以下化合物：
Figure PCTCN2019107792-appb-000015
制备实施例四(化合物编号ALY585-1,ALY585-2)
Figure PCTCN2019107792-appb-000016
由制备实施例一得到的化合物6-5出发，乙酰氯(37μl,0.52mmol)滴加到0.5ml甲醇中，混合物在室温搅拌5min，随后加入中间体6-5(30mg,0.052mmol)的甲醇溶液，室温下搅拌1小时。浓缩，直接下一步。粗品溶于二氯甲烷中，加入吡啶(42μl,0.52mmol)室温搅拌30min，加入氯甲酸苯酯(32μl,0.26mmol)，室温搅拌过夜。TLC监测反应完全，加入水淬灭，乙酸乙酯萃取，无水硫酸钠干燥，柱层析(二氯甲烷/甲醇(v/v)＝25/1)分离纯化，得到透明油状物中间体9-1(15mg,0.026mmol,产率50％)。 1H NMR(500MHz,CDCl3)δ 9.09(s,1H),8.76(d,J＝5.0Hz,1H),8.21–8.18(m,1H),7.45–7.17(m,11H),6.61(s,1H),5.24(dd,J＝15.0,5.0Hz,1H),4.95–4.89(m,1H),2.85(dd,J＝10.0,5.0Hz,1H),2.79–2.73(m,2H),1.90(q,J＝15.0Hz,1H),1.65(q,J＝15.0,1H),1.56(s,3H)。中间体9-2(8mg,0.014mmol,产率27％)。 1H NMR(500MHz,CDCl3)δ 1H NMR(500MHz,CDCl 3)δ9.04(s,1H),8.76(s,1H),8.19–8.16(m,1H),7.46–7.14(m,11H),6.53(s,1H),5.21–5.19(m,1H),5.05–5.00(m,1H),3.13(dd,J＝10.0,5.0Hz,1H),2.48-2.44(m,3H),2.13–2.08(m,1H),1.74(s,3H)。
中间体9-1(15mg,0.026mmol)和七水氯化亚铈(67mg,0.18mmol)溶于甲醇中，冷至-78℃小心加入硼氢化钠(7mg,0.18mmol)，混合物搅拌30分钟原料消失。加入丙酮淬灭，乙酸乙酯稀释，有机相用水洗，饱和食盐水洗，干燥，浓缩，柱层析(二氯甲烷/甲醇(v/v)＝25/1)分离纯化得到终产物ALY585-1(14mg,92％): 1H NMR(CDCl 3,500MHz)δ9.03(S,1H),8.70(d,J＝5.0Hz,1H),8.16–8.06(m,1H),7.47–7.13(m,11H),6.54(s,1H),5.04(dd,J＝10.0,5.0Hz,1H),4.97–4.80(m,1H),4.56(d,J＝10.0Hz,1H),4.41(s,1H),2.85-2.82(m,1H),2.76–2.72(m,1H),2.00–1.90(m,2H),1.59-1.52(m,1H),1.45(s,3H).。
中间体9-2(8mg,0.014mmol)和七水氯化亚铈(36mg,0.096mmol)溶于甲醇中，冷至-78℃小心加入硼氢化钠(4mg,0.096mmol)，混合物搅拌30分钟原料消失。加入丙酮淬灭，乙酸乙酯稀释，有机相用水洗，饱和食盐水洗，干燥，浓缩，柱层析(二氯甲烷/甲醇(v/v)＝25/1)分离纯化得到终产物ALY585-2(7mg,85％): 1H NMR(CDCl 3,500MHz)δ9.01(s,1H),8.71(d,J＝5.0Hz,1H),8.17–8.06(m,1H),7.43–7.16(m,11H),6.49(s,1H),5.28(s,1H),5.09–5.05(m,2H),4.30(s,1H),2.79(s,1H),2.61(s,1H),2.44(d,J＝15.0Hz,1H),2.28(s,1H),2.00–1.90(m,1H),1.54(s,3H).。
用同样方法合成以下化合物：
Figure PCTCN2019107792-appb-000017
制备实施例五(化合物编号ALY585)
Figure PCTCN2019107792-appb-000018
由制备实施例一得到的化合物6-5出发，中间体6-5(25mg,0.044mmol)和七水氯化亚铈(114mg,0.306mmol)溶于甲醇中，冷至-78℃小心加入硼氢化钠(12mg,0.306mmol)，混合物搅拌30分钟原料消失。加入丙酮淬灭，乙酸乙酯稀释，有机相用水洗，饱和食盐水洗，干燥，浓缩，柱层析(二氯甲烷/甲醇(v/v)＝25/1)分离纯化得到中间体10-1(22mg,0.038mmol,86％): 1H NMR(CDCl 3,300MHz)δ9.00(s,1H),8.70(s,1H),8.10(d,J＝6.0Hz,1H),7.41(dd,J＝9.0,6.0Hz,1H),6.46(s,0.4H),6.37(s,0.6H),4.47(d,J＝12.0Hz,1H),4.39(s,1H),3.77–3.71(m,2H),2.40(d,J＝15.0Hz,1H),1.99(d,J＝12.0Hz,1H),1.59–1.50(m,1H),1.39–1.14(m,4H),0.94–0.88(m,18H),0.15–0.08(m,12H)。
乙酰氯(27μl,0.38mmol)滴加到0.5ml甲醇中，混合物在室温搅拌5min，随后加入中间体10-1(22mg,0.038mmol)的甲醇溶液，室温下搅拌1小时。浓缩，直接下一步。粗品溶于二氯甲烷中，加入催化量DMAP，EDCI(36mg,0.19mmol)，对氰基苯甲酸(28mg,0.19mmol)，室温搅拌过夜。TLC监测反应完全，加入水淬灭，乙酸乙酯萃取，无水硫酸钠干燥，柱层析(二氯甲烷/甲醇(v/v)＝25/1)分离纯化，得到黄色固体终产物ALY585(10mg,0.017mmol,产率45％)。 1H NMR(CDCl 3,500MHz)δ8.97(s,1H),8.67(s,1H),8.21–8.08(m,5H),7.82–7.75(m,4H),7.38(s,1H),6.50(s,1H),6.41(s,1H),5.70(dd,J＝15.0,5.0Hz,1H),5.22–5.18(m,1H),3.02–2.98(m,1H),2.72–2.68(m,1H),2.08–2.01(m,1H),1.25(s,3H)。
将制备实施例中的化合物S-香芹酮替换为R-香芹酮，制备得到化合物ALY585R。
用同样方法合成以下化合物：
Figure PCTCN2019107792-appb-000019
Figure PCTCN2019107792-appb-000020
制备实施例六(化合物编号ALY665)
Figure PCTCN2019107792-appb-000021
由制备实施例一得到的中间体6(200mg,0.75mmol)出发,溶于0.5ml乙基乙烯基醚中，加入催化量的对甲苯磺酸吡啶盐，室温搅拌2h。TLC监测反应完全，加入水淬灭，乙酸乙酯萃取，无水硫酸钠干燥，柱层析(石油醚/乙酸乙酯(v/v)＝25/1)分离纯化，得到透明油状物中间体11-1(251mg,0.74mmol,产率99％)。 1H NMR(CDCl 3,300MHz)δ4.82–4.75(m,1H),4.30–4.25(m,1H),3.87–3.74(m,1H),3.66–3.42(m,2H),2.56-2.49(m,1H),2.34-2.17(m,2H),2.00(d,J＝3.0Hz,3H),1.73–1.57(m,1H),1.31(d,J＝6.0Hz,3H),1.22–1.17(m,3H),0.89(s,9H),0.10(d,J＝3.0Hz,6H).。
干冰丙酮浴下化合物11-1(265mg,0.78mmol)的DCM溶液中滴加DIBAL-H(1.2ml,1.17mmol)，滴毕后升至室温搅拌2h。TLC监测原料反应尽，加入饱和酒石酸钾钠水溶液淬灭，混合物升至室温，乙酸乙酯萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩，柱层析(石油醚/乙酸乙酯(v/v)＝25/1)，得到产物11-2(210mg,0.61mmol,产率 78％)。
中间体11-2(210mg,0.61mmol)及碘代物267(247mg,0.92mmol)溶于干燥的乙醚中，冷至-78℃，滴加正丁基锂(0.92ml,0.92mmol)，此温下搅拌30分钟，随后加入饱和氯化铵溶液淬灭。乙酸乙酯萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩，直接下一步。粗品溶于二氯甲烷中，0℃加入DMP(390mg,0.92mmol)，升至室温搅拌过夜。次日加入饱和硫代硫酸钠溶液/饱和碳酸氢钠溶液＝1/1淬灭，二氯甲烷萃取，饱和食盐水洗涤，浓缩，柱层析纯化(石油醚/乙酸乙酯(v/v)＝10/1),得到中间体11-3(170mg,产率58％)。中间体11-3(170mg,0.35mmol)溶于甲苯(10ml)和甲醇(2ml)中，反应混合物加热至外温80℃下回流过夜。次日浓缩柱层析分离(石油醚/乙酸乙酯(v/v)＝10/1)，得到中间体11-4(120mg,0.26mmol,产率74％)。 1H NMR(CDCl 3,300MHz)δ4.80(dd,J＝9.0,6.0Hz,1H),3.90–3.79(m,1H),3.78(s,3H),3.64–3.58(m,3H),2.47–2.41(m,1H),2.22(s,3H),1.54–1.38(m,4H),1.30(d,J＝6.0Hz,3H),1.24(s,3H),1.21–1.16(m,3H),0.89–0.87(m,9H),0.10(d,J＝6.0Hz,6H)。
向0℃下LHMDS(1.8ml,1.82mmol)的THF溶液中滴加中间体11-4(120mg,0.26mmol)的THF溶液，升至室温搅拌4h,快速加入烟酰氯盐酸盐，室温搅拌2h。TLC检测原料反应彻底，加入乙酸淬灭，水稀释，二氯甲烷萃取，无水硫酸钠干燥，浓缩。柱层析纯化(石油醚/丙酮(v/v)＝3/1)，得到中间体11-5(51mg,0.096mmol,产率37％)。 1H NMR(CDCl 3,300MHz)δ9.04(s,1H),8.74(d,J＝3.0Hz,1H),8.16(d,J＝9.0Hz,1H),7.44(dd,J＝9.0,6.0Hz,1H),6.48(s,0.2H),6.42(s,0.8H),4.83–4.80(m,1H),4.01–3.93(m,1H),3.81–3.38(m,3H),2.61–2.48(m,2H),2.09(s,1H),1.58-1.41(m,2H),1.34–1.17(m,9H),0.94-0.88(m,9H),0.18-0.13(m,5H).。
0℃下，中间体11-5(51mg,0.096mmol)溶于2ml四氢呋喃中，滴加2ml 0.5N HCl,保持此温度搅拌4h。TLC检测原料反应彻底，加入碳酸钠饱和溶液淬灭，乙酸乙酯萃取，无水硫酸钠干燥，浓缩。柱层析纯化(二氯甲烷/甲醇(v/v)＝25/1)，得到中间体11-6(30mg,0.065mmol,产率68％)。 1H NMR(CDCl 3,300MHz)δ9.06(s,1H),8.77(s,1H),8.18(d,J＝6.0Hz,1H),7.46(s,1H),6.43(s,1H),4.00(dd,J＝12.0,3.0Hz,1H),3.89-3.78(m,1H),2.64(dd,J＝12.0,3.0Hz,1H),2.49(d,J＝15.0Hz,1H),2.18(d,J＝15.0Hz,1H),1.81(s,1H),1.55–1.51(m,1H),1.37(d,J＝3.0Hz,3H),0.95(d,J＝3.0Hz,9H),0.20-0.14(m,6H)。
中间体11-6(10mg,0.022mmol)溶于二氯甲烷中，加入催化量DMAP，EDCI(9mg,0.044mmol)，Z-甘氨酸(9mg,0.044mmol)，室温搅拌过夜。TLC监测反应完全，加入水淬灭，乙酸乙酯萃取，无水硫酸钠干燥，柱层析(二氯甲烷/甲醇(v/v)＝25/1)分离纯化，得到中间体11-7(10mg,0.015mmol,产率71％)。 1H NMR(CDCl 3,300MHz)δ9.06(d,J＝3.0Hz,1H),8.76(dd,J＝6.0,3.0Hz,1H),8.20–8.16(m,1H),7.48-7.43(m,1H),7.36(s,5H),6.50(s,0.3H),6.43(s,0.7H),5.24(s,1H),5.14(s,2H),4.97–4.87(m,1H),4.06–3.97(m,3H),2.68(dd,J＝12.0,3.0Hz,1H),2.56-2.51(m,1H),2.22-2.17(m,1H),1.62(d,J＝6.0Hz,2H),1.38(s,3H),0.96–0.90(m,9H),0.21-0.25(m,5H).。
乙酰氯(10μl,0.15mmol)滴加到0.5ml甲醇中，混合物在室温搅拌5min，随后加入 中间体11-7(11mg,0.038mmol)的甲醇溶液，室温下搅拌1小时。浓缩，直接下一步。粗品溶于二氯甲烷中，加入催化量DMAP，EDCI(8mg,0.03mmol)，对氰基苯甲酸(5mg,0.03mmol)，室温搅拌过夜。TLC监测反应完全，加入水淬灭，乙酸乙酯萃取，无水硫酸钠干燥，柱层析(二氯甲烷/甲醇(v/v)＝25/1)分离纯化，得到无色油状中间体11-8(3mg,0.004mmol,产率11％)。 1H NMR(CDCl 3,300MHz)δ9.04(dd,J＝9.0,3.0Hz,1H),8.78-8.73(m,1H),8.20-8.12(m,3H),7.83–7.78(m,2H),7.49–7.35(m,6H),6.51(s,0.5H),6.48(s,0.5H),5.63–5.56(m,1H),5.25–5.10(m,3H),3.99(t,J＝6.0Hz,1H),3.84-3.78(m,1H),3.12–3.08(m,1H),2.92-2.88(m,1H),2.68-2.55(m,2H),2.37(s,1H),1.25(s,3H).。
中间体11-8(3mg,0.004mmol)和七水氯化亚铈(12mg,0.032mmol)溶于甲醇中，冷至-78℃小心加入硼氢化钠(2mg,0.032mmol)，混合物搅拌30分钟原料消失。加入丙酮淬灭，乙酸乙酯稀释，有机相用水洗，饱和食盐水洗，干燥，浓缩，柱层析(二氯甲烷/甲醇(v/v)＝25/1)分离纯化得到终产物ALY665(2mg,75％): 1H NMR(CDCl 3,300MHz)δ8.99(d,J＝18.0Hz,1H),8.70(dd,J＝12.0,6.0Hz,1H),8.20–8.05(m,3H),7.81(d,J＝9.0Hz,2H),7.46–7.36(m,6H),6.50(s,0.5H),6.41(s,0.5H),5.41-5.36(m,2H),5.14–5.09(m,2H),4.55(d,J＝9.0Hz,1H),4.39(s,1H),3.98(d,J＝6.0Hz,1H),3.88–3.58(m,2H),2.78-2.68(m,1H),2.57-2.53(m,1H),2.28(s,1H),2.00(t,J＝12.0Hz,1H),1.87–1.75(m,1H),1.26(s,3H)。
用同样方法合成以下化合物：
Figure PCTCN2019107792-appb-000022
试验实施例1
对ACAT2活性的抑制作用试验实施例
1.试验目的：
通过利用荧光标记固醇测定ACAT2活性方法，检测所述的Pyripyropene A结构的类似物在完整细胞水平对ACAT2活性的抑制作用。
2.测试原理：
用不同浓度的化合物，对含NBD22-荧光标记固醇的酯合成的抑制，导致荧光强度差异变化，从而绘制抑制曲线，计算IC 50。
3.实验过程：
HepG2细胞以1.5×10 4个每孔的起始密度在96孔板中培养24小时后，加入胆固醇混合物混匀后，继续培养24小时，然后加入终浓度为0.5μg/ml的NBD22-荧光标记固醇，以 及终浓度梯度为0、0.008、0.04、0.2、1和5μM的化合物，每一浓度设三重复孔，再培养6小时后使用荧光分析仪(E488，A535)测定荧光强度，将荧光强度值对化合物不同浓度作图并获得IC 50。
4.实验结果：(以下列化合物为例，但不局限于这些化合物)
表1.化合物对ACAT2活性的抑制作用
化合物编号 IC 50(μM)
Pyripyropene A 0.179
ALY603 0.115
ALY578 0.148
ALY564 0.224
ALY621 0.303
ALY727 0.146
ALY585R 0.442
ALY515 0.383
ALY619 0.281
ALY585-1 0.136
ALY665 0.478
注：IC 50为样品化合物对ACAT2活性的50％抑制评价。
结果表明该类化合物具有对ACAT2的抑制活性，而且该类ACAT2抑制剂与已发现的唯一一个ACAT2的特异抑制剂Pyripyropene A相比，抑制活性有明显提高。
试验实施例2
对ACAT2活性的抑制作用的选择性系数试验实施例
1.试验目的：
通过利用胆固醇氧化酶法测定ACAT活性，检测所述的Pyripyropene A结构的类似物在完整细胞水平对ACAT2和ACAT1的抑制作用，而获得对ACAT2具有高选择性的化合物。
2.测试原理：
通过利用HepG2细胞测定不同浓度的化合物对ACAT1或ACAT2活性的抑制作用，获得IC 50来计算SI(ACAT1-IC 50/ACAT2-IC 50)。
3.实验过程：
HepG2细胞以4×10 5个每孔的起始密度在6孔板中培养24小时后，更换培养基并加入10μg/ml胆固醇以及不同浓度的化合物，继续培养9小时，利用Cholesterol Assay kit测定细胞胆固醇量。
4.实验结果：(以下列化合物为例，但不局限于这些化合物)
表2化合物对ACAT1抑制活性及SI:
化合物编号 IC 50(μM) SI
PPPA >20 200
ALY603 56.31 477
ALY578 8.747 61
ALY564 >10 -
ALY621 >10 -
ALY727 12.90 88
ALY515 >10 -
ALY619 >10 -
ALY582 13.80 76
ALY585-1 34.53 254
结果表明该类化合物对ACAT2活性抑制具有高选择性。
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. 通式I所示的化合物、其互变异构体、光学异构体和立体异构体或其药学上可接受的盐，
    Figure PCTCN2019107792-appb-100001
    其中，
    X为O、S、NH或C 1-C 6亚烷基；
    R 4为4-8元杂芳基；
    W为氢或羟基；
    Figure PCTCN2019107792-appb-100002
    表示单键或双键；
    R 1为氢或C 1-C 6烷基；
    n＝0、1或2；
    Y、Z各自独立为O、NH、S、-OC(＝O)-、-OC(＝O)O-、-OC(＝O)O(C 1-C 6亚烷基)-、-OC(＝O)(C 1-C 6亚烷基)-、-O(C 1-C 6亚烷基)-、-OC(＝O)(C 1-C 6亚烷基)NHC(＝O)-、-OC(＝O)NH(C 1-C 6亚烷基)-或-OC(＝O)NH-；
    R 2和R 3各自独立地为氢、取代或未取代的C 3-C 8环烷基、取代或未取代的C 6-C 12芳基、取代或未取代的4-8元杂芳基、取代或未取代的C 1-C 6烷氧基；
    上述各取代独立指具有选自下组的一个或多个取代基：C 1-C 6烷基、C 6-C 12芳基、4-8元杂芳基、C 3-C 8环烷基、C 1-C 6烷氧基、C 1-C 6烷胺基、卤素、氰基、羟基、氨基、-COC 1-C 6烷基、-COC 3-C 6环烷基、-COC 6-C 12芳基。
  2. 如权利要求1所述的化合物，其特征在于：所述化合物具有以下一个或多个特征：
    (1)X为O；
    (2)R 4为5-7元杂芳基，优选为吡啶；
    (3)R 1为甲基、乙基或丙基；
    (4)n为1或2。
  3. 如权利要求1所述的化合物，其特征在于：Y、Z各自独立为O、NH、S、-OC(＝O)-、-OC(＝O)O-、-OC(＝O)O(C 1-C 4亚烷基)-、-OC(＝O)(C 1-C 4亚烷基)-、-O(C 1-C 4亚烷基)-、-OC(＝O)(C 1-C 4亚烷基)NHC(＝O)-、-OC(＝O)NH(C 1-C 4亚烷基)-或-OC(＝O)NH-。
  4. 如权利要求1所述的化合物，其特征在于：R 2和R 3各自独立地为氢、取代或为取代的C 3-C 6环烷基、取代或为取代的C 6-C 10芳基、取代或为取代的4-6元杂芳基、取代或未取代的C 1-C 6烷氧基；
    上述各取代独立指具有选自下组的一个或多个取代基：C 1-C 6烷基、C 6-C 10芳基、4-8元杂芳基、C 3-C 6环烷基、C 1-C 4烷氧基、C 1-C 4烷胺基、卤素、氰基、羟基、氨基、-COC 1-C 6 烷基、-COC 3-C 6环烷基、-COC 6-C 10芳基。
  5. 如权利要求1所述的化合物，其特征在于，所述化合物为：
    Figure PCTCN2019107792-appb-100003
    Figure PCTCN2019107792-appb-100004
  6. 如权利要求1所述的化合物的制备方法，其特征在于，通式I所示的化合物的结构如式Ia、式Ib、式Ic或式Id所示，所述制备方法包括以下步骤：
    Figure PCTCN2019107792-appb-100005
    Figure PCTCN2019107792-appb-100006
    n、Z、Y、R 2、R 3、R 4与权利要求1相同，PG 1、PG 2为羟基保护基，所述羟基保护基为硅醚、酯基、苄醚或烷基醚；
    (1)化合物14双键臭氧化得到化合物15，再经过Baeyer-Villiger氧化，得到化合物16；
    (2)化合物16脱去乙酰基，然后保护基保护羟基得化合物17；
    (3)化合物17 DIBAL-H还原得到化合物18；
    (4)化合物18与化合物267以及正丁基锂试剂发生偶联反应，之后被氧化，经过溶剂解反应得到化合物19；
    (5)化合物19被LHMDS拔氢烯醇化，随后和R 4COCl发生C-酰基化关环得到化合物20；
    (6)化合物20脱去保护基，再和酸酐、酰氯、异氰酸酯或氯甲酸酯发生反应得到化合物Ia；化合物Ia经过Luche还原反应得到化合物Ic；
    或化合物20经过还原得到化合物Ib，化合物Ib经脱保护基、再与酸酐、酰氯、异氰酸酯或氯甲酸酯反应可以得到化合物Id。
  7. 如权利要求6所述的制备方法，其特征在于，所述制备方法具有以下一个或多个特征：
    步骤(1)中所述臭氧化反应所用溶剂选自：二氯甲烷和甲醇；
    步骤(1)中所述臭氧化反应的反应温度为-78℃到室温；
    步骤(3)中DIBAL-H还原所用溶剂为二氯甲烷；
    步骤(3)中DIBAL-H还原反应的反应温度为-78℃-0℃；
    步骤(4)中所述偶联反应所用溶剂为非质子性溶剂，选自乙醚等；
    步骤(4)中所述偶联反应的反应温度为-78℃到室温；
    步骤(4)中所述溶剂解反应所用溶剂选自：甲苯和甲醇；
    步骤(4)中所述溶剂解反应的反应温度为80℃；
    步骤(5)中所述C-酰基化关环所用溶剂为非质子性溶剂，选自四氢呋喃；
    步骤(5)中所述C-酰基化关环反应温度为0℃到室温；
    步骤(6)中所述脱保护基反应及酰基反应所用溶剂为非质子性溶剂，选自二氯甲烷；
    步骤(6)中所述脱保护基反应及酰基反应的反应温度为室温；
    步骤(6)中所述Luche还原反应所用溶剂选自醇类溶剂，优选为甲醇或乙醇；反应温度为-78℃至室温。
  8. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1所述的化合物或其药学上可接受的盐；和
    药学上可接受的载体。
  9. 一种如权利要求1所述的化合物或其药学上可接受的盐的应用，其特征在于，用于制备：(i)选择性抑制ACAT2的药物；或(ii)预防和/或治疗心血管疾病的药物。
  10. 如权利要求9所述的应用，其特征在于，所述心血管疾病选自下组：高血脂症，动脉粥样硬化。
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