WO2019218956A1 - Compound acting as protease-activated receptor 4 (par4) inhibitor for treating platelet aggregation - Google Patents

Compound acting as protease-activated receptor 4 (par4) inhibitor for treating platelet aggregation Download PDF

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WO2019218956A1
WO2019218956A1 PCT/CN2019/086557 CN2019086557W WO2019218956A1 WO 2019218956 A1 WO2019218956 A1 WO 2019218956A1 CN 2019086557 W CN2019086557 W CN 2019086557W WO 2019218956 A1 WO2019218956 A1 WO 2019218956A1
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acid
group
methoxy
mmol
pharmaceutically acceptable
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PCT/CN2019/086557
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French (fr)
Chinese (zh)
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吴俊军
李硕
温晓明
阳华
魏国平
胡允金
钱苏
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深圳信立泰药业股份有限公司
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Priority to CN201980030137.2A priority Critical patent/CN112074521B/en
Publication of WO2019218956A1 publication Critical patent/WO2019218956A1/en

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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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • 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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • 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
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the present invention is in the field of chemical pharmaceutical technology, and particularly provides a series of compounds as protease inhibitory receptor 4 (PAR4) inhibitors for treating platelet aggregation and their medical uses.
  • PAR4 protease inhibitory receptor 4
  • Thromboembolic disease remains one of the leading causes of death, although available anticoagulants such as warfarin, heparin, and antiplatelet agents such as aspirin and clopidogrel, ticagrelor, rivaroxaban and many more.
  • thrombosis The main physiological function of platelets is to participate in thrombosis and hemostasis. Any blood coagulation process involves the activation of platelets, which is a complex signal cascade process in which thrombin plays a central role.
  • Thrombin-activated platelets are mediated primarily by a family of G-protein-coupled protease-activated receptors (PARs). Human platelets express both PAR1 and PAR4 receptors. Thrombin can bind and cleave PAR1 or PAR4, exposing a new N-terminus, which acts as a binding ligand for intramolecular binding to the receptor, thereby stimulating transmembrane signaling. , causing platelet aggregation, release, and a series of changes in membrane glycoproteins.
  • PAR1 G-protein-coupled protease-activated receptors
  • PAR4 inhibitor patent applications disclose, such as CN104640869A and CN104583218A, respectively, disclose a series of uses of the following formulas I and II as protease activated receptor 4 (PAR4) inhibitors for inhibiting or preventing platelet aggregation. .
  • PAR4 protease activated receptor 4
  • the pentad nucleus should contain an oxygen atom to have the corresponding PAR4 activity.
  • the present invention provides a series of compounds as protease activated receptor 4 (PAR4) inhibitors for the treatment of platelet aggregation, the structural nucleus of the compound of the present invention is different from A PAR4 inhibitor has been disclosed.
  • PAR4 protease activated receptor 4
  • the present invention first provides a series of compounds, or a pharmaceutically acceptable salt thereof, as a protease activated receptor 4 (PAR4) inhibitor for the treatment of platelet aggregation, characterized in that the mother nucleus of the compound is as follows:
  • R 0 is selected from the group consisting of hydrogen, halogen, C 1-4 alkyl, C 1-4 amino group, C 1-4 alkoxy group, C 1-4 alkylthio group;
  • X, Z are selected from CH or N;
  • Y is selected from O or CH
  • R 1 is selected from the group consisting of hydrogen, halogen, hydroxy, decyl, C 1-4 alkoxy;
  • the halogen is selected from the group consisting of fluorine, chlorine, bromine, and iodine.
  • the C 1-4 alkyl group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.
  • the amino group of C 1-4 is selected from the group consisting of methylamino, ethylamino, propylamino, isopropylamino, n-butylamino, isobutylamino, sec-butylamino and t-butylamino, and dimethyl Amino group.
  • the C 1-4 alkoxy group is selected from the group consisting of a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a n-butoxy group, an isobutoxy group, and a sec-butyl group. Oxyl, tert-butoxy.
  • the C 1-4 alkylthio group is selected from the group consisting of methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, isobutylthio, and sec Sulfur-based, tert-butylthio.
  • the C 3-6 cycloalkyl group is selected from the group consisting of cyclopropane, cyclopentane, and cyclohexane.
  • the C 3-6 heterocyclic ring is selected from
  • R 0 , R 1 , and X are as defined above;
  • n 0, 1, 2, 3 or 4;
  • A is selected from an oxygen atom
  • B is selected from H, C 1-4 alkyl, or alkoxy substituted C 1-4 alkyl
  • A is selected from a carbonyl group
  • B is selected from a hydroxyl group, an amino group or a C 1-4 alkoxy group
  • A is selected from a methylene group
  • B is selected from a hydroxyl group, a C 3-6 cycloalkyl group, a nitrogen-containing and/or oxygen C 3-6 heterocyclic ring, a C 1-4 alkyl-substituted or unsubstituted amino group.
  • A is selected from a C 1-4 alkyl-substituted or unsubstituted thiazole ring, a substituted or unsubstituted benzene ring, a substituted or unsubstituted pyridine ring
  • B is selected from Wherein R 3 and R 4 are selected from the group consisting of hydrogen, methyl, and hydroxy, For the connection key.
  • X, Y, Z are as defined above;
  • R 0 is selected from the group consisting of hydrogen and methoxy
  • R 1 is selected from the group consisting of hydrogen and methoxy
  • R 2 is selected from the group consisting of hydroxyl, methoxy, Methoxyethoxy, Hydroxyethoxy, Methoxypropoxy, Dimethoxy,
  • the pharmaceutically acceptable salt is a salt of a compound with an inorganic acid or an organic acid selected from the group consisting of 2-acetoxybenzoic acid and 2-hydroxyethyl.
  • the invention is preferably derived from the following compounds:
  • Another object of the present invention is to provide a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the aforementioned compound or a pharmaceutically acceptable salt thereof.
  • Another object of the present invention is to provide a pharmaceutical use comprising the aforementioned compound or a pharmaceutically acceptable salt thereof for use in the preparation of a medicament for the preparation of a thrombosis-related disease.
  • Preferred thrombi-related disorders are selected from the group consisting of arterial cardiovascular thromboembolic disorders, venous cardiovascular thromboembolic disorders, cerebrovascular thromboembolic disorders, and thromboembolic disorders in the heart or peripheral circulation.
  • pharmaceutically acceptable salt refers to a salt of a compound of the invention prepared from a compound having a particular substituent found in the present invention and a relatively non-toxic acid or base.
  • a base addition salt can be obtained by contacting a neutral amount of such a compound with a sufficient amount of a base in a neat solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts.
  • an acid addition salt can be obtained by contacting a neutral form of such a compound with a sufficient amount of an acid in a neat solution or a suitable inert solvent.
  • pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogencarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts including, for example, formic acid, acetic acid, trifluoroacetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, amber Acids, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, cit
  • the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated, thereby regenerating the neutral form of the compound.
  • the parent form of the compound differs from the form of its various salts by certain physical properties, such as differences in solubility in polar solvents.
  • a "pharmaceutically acceptable salt” is a derivative of a compound of the invention wherein the parent compound is modified by salt formation with an acid or with a base.
  • pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of bases such as amines, alkali metal or organic salts of acid groups such as carboxylic acids, and the like.
  • Pharmaceutically acceptable salts include the conventional non-toxic salts or quaternary ammonium salts of the parent compound, for example salts formed from non-toxic inorganic or organic acids.
  • Conventional non-toxic salts include, but are not limited to, those derived from inorganic acids and organic acids selected from the group consisting of 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, formic acid, acetic acid, Ascorbic acid, benzenesulfonic acid, benzoic acid, hydrogen carbonate, carbonic acid, citric acid, edetic acid, ethane disulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, ethanol Acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, dodecylsulfonic acid, maleic acid, mal
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing an acid group or a base by conventional chemical methods.
  • such salts are prepared by reacting these compounds in water or an organic solvent or a mixture of the two via a free acid or base form with a stoichiometric amount of the appropriate base or acid.
  • a nonaqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is preferred.
  • the compounds provided herein also exist in the form of prodrugs.
  • Prodrugs of the compounds described herein are readily chemically altered under physiological conditions to convert to the compounds of the invention.
  • prodrugs can be converted to the compounds of the invention by chemical or biochemical methods in an in vivo setting.
  • Certain compounds of the invention may exist in unsolvated or solvated forms, including hydrated forms.
  • the solvated forms are equivalent to the unsolvated forms and are included within the scope of the invention.
  • the compounds of the invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including the cis and trans isomers, the (-)- and (+)-p-enantiomers, the (R)- and (S)-enantiomers, and the diastereomeric a conformation, a (D)-isomer, a (L)-isomer, and a racemic mixture thereof, and other mixtures, such as enantiomerically or diastereomeric enriched mixtures, all of which belong to It is within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in the substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the invention.
  • optically active (R)- and (S)-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If an enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary wherein the resulting mixture of diastereomers is separated and the auxiliary group cleaved to provide pure The desired enantiomer.
  • a diastereomeric salt is formed with a suitable optically active acid or base, followed by conventional methods well known in the art.
  • the diastereomers are resolved and the pure enantiomer is recovered.
  • the separation of enantiomers and diastereomers is generally accomplished by the use of chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (eg, formation of an amino group from an amine). Formate).
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound.
  • a compound can be labeled with a radioisotope such as hydrazine (3H), iodine-125 (125I) or C-14 (14C). Alterations of all isotopic compositions of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
  • pharmaceutically acceptable carrier refers to any formulation carrier or vehicle that is capable of delivering an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects to the host or patient.
  • Representative carriers include water, oil. , vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, tackifiers, transdermal enhancers and the like. Their formulations are well known to those skilled in the cosmetic or topical pharmaceutical arts. For additional information on vectors, reference is made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are hereby incorporated by reference.
  • excipient generally refers to the carrier, diluent and/or vehicle required to formulate an effective pharmaceutical composition.
  • an "effective amount” or “therapeutically effective amount” with respect to a pharmaceutical or pharmacologically active agent refers to a sufficient amount of a drug or agent that is non-toxic but that achieves the desired effect.
  • an "effective amount” of an active substance in a composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount will vary from person to person, depending on the age and general condition of the recipient, and also on the particular active substance, and a suitable effective amount in a case can be determined by one skilled in the art based on routine experimentation.
  • active ingredient refers to a chemical entity that is effective in treating a target disorder, disease or condition.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, combinations thereof with other chemical synthetic methods, and those well known to those skilled in the art. Equivalent alternatives, preferred embodiments include, but are not limited to, embodiments of the invention.
  • the 2,4,6-trihydroxybenzoic acid was dissolved in acetone.
  • the reaction solution was cooled to 0 ° C, and then trifluoroacetic acid and trifluoroacetic anhydride were slowly added dropwise to the above solution.
  • the reaction solution was heated to 65 ° C and stirred for 16 hours.
  • the reaction solution was cooled to room temperature and concentrated under reduced pressure.
  • the resulting residue was dissolved in a mixed solution of ethyl acetate and water.
  • Saturated sodium bicarbonate was added to the mixture to adjust the pH to about 8.
  • the mixture was extracted three times with ethyl acetate.
  • the organic phase was combined and the organic phase was washed three times with brine, dried over anhydrous sodium sulfate
  • the reaction solution was concentrated under reduced pressure.
  • the residue obtained was dissolved in water, and dilute hydrochloric acid was slowly added dropwise to the above solution to adjust the pH to about 2, and a white solid was precipitated. Filtration, collection of the filter cake, and drying of the filter cake gave 2-(3-(benzyloxy)-2-formyl-5-methoxyphenoxy) acetic acid as a white solid. It is used directly in the next step without purification.
  • reaction solution was cooled to room temperature, and water was added to the reaction mixture to quench the reaction.
  • the mixture was extracted with ethyl acetate.
  • the combined organic layers were washed with saturated brine and dried over anhydrous sodium sulfate.
  • the residue was purified by silica gel column chromatography (EtOAc:EtOAc Obtained 2-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxa as a yellow oil Borane.
  • reaction solution was cooled to room temperature.
  • the mixture was extracted with ethyl acetate.
  • the combined organic layers were washed with saturated brine and dried over anhydrous sodium sulfate.
  • the residue was purified by silica gel column chromatography (EtOAc:EtOAc 2-(4-(Benzyloxy)-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole was obtained as a white solid.
  • the crude product was purified by reverse phase column [reverse phase column: C18; mobile phase A: water (containing 0.05% formic acid), mobile phase B: acetonitrile; gradient: 30% acetonitrile to 90% acetonitrile in 8 min; detection wavelength: 254 nm
  • reverse phase column [reverse phase column: C18; mobile phase A: water (containing 0.05% formic acid), mobile phase B: acetonitrile; gradient: 30% acetonitrile to 90% acetonitrile in 8 min; detection wavelength: 254 nm
  • the fractions were collected and directly lyophilized.
  • the yellow solid 6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol was obtained.
  • 2-Bromo-5-methoxyimidazo[5,4-d]thiazole (110 mg, 0.4 mmol) was dissolved in 1,4-dioxane (10.0 mL). Subsequently, anhydrous sodium carbonate (140 mg, 1.3 mmol), (4,6-dimethoxybenzofuran-2-yl) borate (201 mg, 0.66 mmol) and four were added to the above solution. (Triphenylphosphine) palladium (51 mg, 0.044 mmol) in water (2.0 mL). The reaction solution was heated to 90 ° C and stirred for 2 hours.
  • the reaction solution was cooled to room temperature and concentrated under reduced pressure.
  • the crude product was purified by preparative high performance liquid chromatography.
  • the separation conditions were as follows, column: X select C18 19 mm * 150 mm; mobile phase: water (containing 0.05% trifluoroacetic acid) and acetonitrile; flow rate: 25 ml / min; gradient: acetonitrile increased from 5% in 7 minutes 100%; detection wavelength: 254 nm.
  • freeze-drying to obtain 25.0 mg of pale yellow solid 2-(4,6-dimethoxybenzofuran-2-yl)-5-methoxythiazolo[5,4-d]thiazole (yield : 17.9%).
  • Step B Synthesis of 2-(4,6-dimethoxybenzofuran-2-yl)thiazolo[5,4-d]thiazole
  • the mixture was extracted with ethyl acetate (20 mL EtOAc). The combined organic phases were back-washed with saturated brine (10 mL) then dried over anhydrous sodium sulfate.
  • the crude product was purified by preparative high performance liquid chromatography.
  • the preparation conditions are as follows. Column: Xselect C18 19mm*150mm; mobile phase: water (0.05% formic acid) and acetonitrile; flow rate: 25 ml/min; gradient: acetonitrile increased from 10% to 80% in 7 minutes; detection wavelength: 254 nm.
  • Step B Synthesis of 5-hydroxy-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxan-4-one
  • Step E Synthesis of ethyl 2-(3-(benzyloxy)-2-formyl-5-methoxyphenoxy)acetate
  • Step F Synthesis of 2-(3-(benzyloxy)-2-formyl-5-methoxyphenoxy)acetic acid
  • Step H Synthesis of 2-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxa Borane
  • Step I Synthesis of 2-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole
  • Step B Synthesis of 4-(6-((tert-butyldimethylsilyloxy)methyl)pyridin-2-yl)-tetrahydro-2H-pyran-4-ol
  • a saturated aqueous ammonium chloride solution (5 ml) was added to the mixture and the mixture was evaporated.
  • the mixture was extracted with ethyl acetate (20 mL ⁇ 3 ⁇ ) and the organic phases were combined.
  • the obtained organic phase was back-washed with saturated brine (20 ml ⁇ 3 ⁇ ), then dried over anhydrous sodium sulfate and evaporated.
  • Step D Synthesis of 4-(6-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy) Methyl)pyridin-2-yl)tetrahydro-2H-pyran-4-ol
  • 2-Bromo-3,5-dimethoxypyridine 800 mg, 3.7 mmol was dissolved in acetonitrile (14.0 mL). Subsequently, trimethylsilylacetylene (362 mg, 3.7 mmol), bis(triphenylphosphine)palladium dichloride (53 mg, 0.075 mmol), cuprous iodide (14 mg, 0.07 mmol, triphenylphosphine (39 mg, 0.15 mmol) and N,N-diisopropylethylamine ((1.10 g, 8.59 mmol). stirred at room temperature for 4 hours. Anhydrous potassium carbonate (3.60 g, 26.0 mmol) and methanol (20.0 mL) were then evaporated.
  • Step C Synthesis of 3,5-dimethoxy-2-(2-[5-methoxy-[1,3]thiazolo[5,4-d][1,3]thiazol-2-yl] Ethynyl)pyridine
  • Step D Synthesis of 4,6-dimethoxy-2-[5-methoxy-[1,3]thiazolo[5,4-d][1,3]thiazol-2-yl]pyrazole [1,5-a]pyridine
  • reaction solution was concentrated under reduced pressure.
  • Step A Synthesis of 4-(4-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy) Methyl)-5-methylthiazol-2-yl)morpholine
  • Step A Synthesis of 2-methoxy-5-(6-methoxy-4-(2-methoxyethoxy)benzofuran-2-yl)thiazolo[5,4-d]thiazole
  • Step A Synthesis of ethyl 5-methyl-2-(4-methylpiperazin-1-yl)thiazole-4-carboxylate
  • Step C Synthesis of 2-methoxy-5-(6-methoxy-4-((5-methyl-2-(4-methylpiperazin-1-yl)thiazol-4-yl)methoxy) Benzofuran-2-yl)thiazolo[5,4-d]thiazole
  • Step A Synthesis of 2-((6-methoxy-2-(5-methoxythiazolo[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy)acetate ester
  • Step A Synthesis of 2-methoxy-5-(6-methoxy-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)benzofuran-2 -yl)thiazolo[5,4-d]thiazole
  • Step B Synthesis of 2-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy)e-1- alcohol
  • a saturated sodium bicarbonate solution was added to the mixture to adjust the pH to about 8.
  • the mixture was extracted with ethyl acetate (10 mL ⁇ 3 ⁇ ).
  • the combined organic layers were washed with brine (10 mL ⁇ 3 ⁇ ) and then dried over anhydrous sodium sulfate.
  • the crude product was purified by preparative high performance liquid chromatography. The separation conditions were as follows, column: X select C18 19 mm * 150 mm; mobile phase: water (containing 0.05% TFA) and acetonitrile; flow rate: 25 ml / min; gradient: acetonitrile increased from 5% to 100% in 7 minutes Detection wavelength: 254 nm.
  • Step A Synthesis of 2-methoxy-5-(6-methoxy-4-(2-(pyrrolidin-1-yl)ethoxy)benzofuran-2-yl)thiazolo[5,4 -d]thiazole formate
  • the crude product was purified by preparative high performance liquid chromatography.
  • the separation conditions were as follows, column: X select C18 19 mm * 150 mm; mobile phase: water (containing 0.05% formic acid) and acetonitrile; flow rate: 25 ml / min; gradient: acetonitrile increased from 50% to 60% in 7 minutes Detection wavelength: 254 nm.
  • lyophilization to give a yellow solid 2-methoxy-5-(6-methoxy-4-(2-(pyrrolidin-1-yl)ethoxy)benzofuran-2-yl)thiazole And [5,4-d]thiazole (9 mg, 31.3%).
  • Step A Synthesis of 2-methoxy-5-(6-methoxy-4-(3-methoxypropoxy)benzofuran-2-yl)thiazolo[5,4-d]thiazole
  • Step A Synthesis of 3-(benzyloxy)-5-methoxy-2-((trimethylsilyl)ethynyl)pyridine
  • Step B Synthesis of 3-(benzyloxy)-2-ethynyl-5-methoxypyridine
  • Step C Synthesis of 2-((3-(benzyloxy)-5-methoxypyridin-2-yl)ethynyl)-5-methoxyimidazo[5,4-d]thiazole
  • Step D Synthesis of 2-(4-(benzyloxy)-6-methoxy[1,5-a]pyridin-2-yl)-5-methoxyimidazo[5,4-d]thiazole
  • Step E Synthesis of 6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)pyrazolo[1,5-a]pyridin-4-ol
  • Step F Synthesis of 4-(4-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)pyrazolo[1,5-a]pyridine- 4-yloxy)methyl)-5-methylthiapyridin-2-yl)morpholine
  • Triphenylphosphine (19 mg, 0.072 mmol) was dissolved in tetrahydrofuran (0.5 mL).
  • Diisopropyl azodicarboxylate (9 mg, 0.043 mmol) was added dropwise to the system at 0 °C. Stir at 0 degrees Celsius for 5 minutes.
  • 6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)pyrazolo[1,5-a]pyridin-4-ol (12 mg, A solution of 0.036 mmol) and (5-methyl-2-morpholino-4-yl)methanol (8 mg, 0.036 mmol) in THF (2 mL). Stir at room temperature for 1 hour.
  • Step A Synthesis of 2-methoxy-5-(6-methoxy-4-(2-(2-methoxyethoxy)ethoxy)benzofuran-2-yl)thiazolo[5 ,4-d]thiazole
  • 1-(1H-Pyrrol-2-yl)ethanone (200.0 g, 1.83 mol) was dissolved in dimethyl sulfoxide (1.0 L). Subsequently, a hydrobromic acid aqueous solution (1.0 liter, 48% by weight) was slowly added dropwise to the above solution. The reaction solution was heated to 50 ° C and stirred for 3 hours.
  • Step B Synthesis of ethyl 2-(2-acetyl-4-bromo-1H-pyrrol-1-yl)acetate
  • 2-Bromopyridazine-6,8-diol (1.00 g, 4.39 mmol) was dissolved in dry methanol (50.0 mL). Subsequently, concentrated sulfuric acid (0.5 ml) was added to the above solution. The reaction solution was heated to 60 ° C and stirred for 1 hour.
  • Step E 6,8-Dimethoxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indolizine
  • Step F 2-(6,8-Dimethoxyindolizin-2-yl)-5-methoxythiazolo[5,4-d]thiazole
  • Step A Synthesis of 2-(4-isopropoxy-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole
  • Step A Synthesis of 2-((6-methoxy-2-(5-methoxythiazolo[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy)-N , N-dimethylethyl-1-amine
  • Step A Synthesis of 2-(4-(cyclopropylmethoxy)-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole
  • the mammalian cell expression vector containing the human PAR4 cDNA was separately introduced into Flp-In-TREx-293 cells by conventional transfection method, and the stably high-expression Flp-In-TREx-293-PAR4 cell line was obtained by corresponding antibiotic screening. The function was verified by PAR4FLIPR calcium ion outflow experiment. According to the literature, HEK293 cells express endogenously high expression of PAR4, so Flp-In-TREx-293-PAR4 cell line can be used for screening experiments of PAR4 small molecule inhibitors.
  • the medium was cultured in a 37 ° C, 5% CO 2 cell incubator.
  • the cells were seeded into poly-d-lysine-treated 384-well cell culture plates (Corning, 3845) at a seeding density of 8,000 cells/well/25 ⁇ l of cell seeding matrix (DMEM high glucose, 10% fetal bovine serum, 2 mM GlutaMAX). ) Incubate overnight in a cell culture incubator.
  • DMEM high glucose, 10% fetal bovine serum, 2 mM GlutaMAX cell seeding matrix
  • test compound and the PAR4 agonist were diluted to a 6X concentration using the experimental buffer, 10 ⁇ l of the 6X test compound was added to the 384-well cell culture plate, and after incubation for 30 minutes at room temperature, 10 ⁇ l of the 6X PAR4 agonist was added to the 384-well cell using FLIPR Tetra. The plates were incubated and the data were measured and analyzed. The entire reaction system was 60 ⁇ l, the final concentration of the PAR4 agonist was 1.3 ⁇ M (EC50), and the final concentration of DMSO was 0.3%.
  • platelet-rich plasma samples were centrifuged at 1300 g (Eppendorf Model 5810R centrifuge) for 6 minutes at room temperature; after centrifugation, the supernatant liquid was discarded and carefully resuspended and washed with ACD buffer. Once again.
  • PPP platelet-poor plasma
  • the measurement time was set to 600 s, and the measurement mode was Aggr mode; the washed platelet samples after 280 uL incubation were added to a cuvette (Beijing Shidi Scientific Instrument Co., Ltd.). Join a shocking bead (Beijing Shidi Scientific Instrument Co., Ltd.) and warm up for 1 minute. After the preheating is completed, place the cuvette in the test channel. After pressing the start button for 10 seconds, slowly add 20uL of PAR4-AP agonist or 20ul of lead compound. During the addition process, the tip does not enter the deep surface. Position, get the MAR value.
  • Negative agonist control As in the operation of 2, only the last added PARP-AP agonist was replaced with the vehicle used for the agonist (15% HBSS + 20 mM HEPES physiological saline solution), and the test result was a negative agonist control. .
  • Platelet aggregation inhibition rate (%) 1 - (test compound MAR% - negative agonist MAR%) / (negative control MAR% - negative agonist MAR%) * 100%
  • the test compound is provided by the client.
  • the control compound digoxin was purchased from a local supplier, propranolol was purchased from Sigma, Inc.
  • 1.2 non-essential amino acids (NEAA), HEPES and Hank's equilibration buffer (HBSS) were purchased from Gibco.
  • Fetal bovine serum (FBS) was purchased from Corning.
  • Penicillin/streptomycin mixed solution (100x) and trypsin/EDTA solution were purchased from Beijing Suobao Technology Co., Ltd.
  • HTS 96-well transwell cell culture plates were purchased from Corning.
  • the resistance meter was purchased from World Precision Instruments.
  • the Infinite 200PRO microplate reader was purchased from Tecan.
  • the MTS 2/4 mixer was purchased from IKA.
  • Caco-2 was cultured on Transwell for 14-18 days and then the bidirectional permeation test of the test compound was started.
  • Caco-2 was cultured in a T-75 cell culture flask.
  • the incubator was set to 37 ° C, 5% CO 2 , and the relative humidity was 95%.
  • Transwell can be used to inoculate Transwell when the cell confluence reaches 70-90%.
  • the process of changing the medium is as follows. Separate the Transwell chamber from the receiving plate, discard the medium in the receiving plate and then discard the Transwell chamber medium. Finally, add 100 ⁇ L of fresh medium to each chamber, and add 25 mL of fresh medium to the receiving plate. .
  • Caco-2 After 14 days of incubation, Caco-2 should be completely pooled to complete differentiation. At this time, it can be applied to the penetration test.
  • the hole cannot be used for the penetration test.
  • Verapamil (Cat. No.: V4629-1G; Lot No.: 021M1429V) was purchased from Sigma.
  • Mixed human liver microsomes and mixed male rat liver microsomes were purchased from Corning.
  • Mixed male monkey liver microsomes were purchased from Reid. Store in a refrigerator at -80 ° C for use.
  • Other reagents are purchased from local suppliers.
  • the reaction system was configured, and the NADPH start-up reaction was added, and the cells were incubated at 37 ° C in a water bath, and samples were taken at 0, 15, 30, 45, and 60 minutes, respectively.
  • the test drug and the positive drug were detected by LC-MS/MS, and the remaining percentage was determined. Calculate the half-life of the test compound as well as the intrinsic clearance rate in vitro.
  • reaction system was pre-incubated for 10 minutes in a 37 ° C water bath.
  • the reaction was initiated by adding 40 ⁇ L of 10 mM NADPH solution to the reaction system, and the final concentration of NADPH was 1 mM.
  • 40 ⁇ L of ultrapure water was used instead of NADPH solution as a negative control. The effect of the negative control is to rule out the effects of the chemical stability of the compound itself.
  • reaction sample 50 ⁇ L was taken at 0, 15, 30, 45 and 60 minutes, respectively, using 4 times acetonitrile containing an internal standard (200 nM alprazolam, 200 nM labetalol, 2 ⁇ M ketoprofen, 200 nM caffeine) Quenched.
  • the sample was centrifuged at 4,000 rpm for 40 minutes. After centrifugation, 100 ⁇ L of the supernatant and 100 ⁇ L of ultrapure water were mixed for LC-MS/MS analysis.
  • the reference compound of BMS-986120 has poor water solubility and high protein binding rate, so that the compound has a low plasma free concentration, so that high dose administration produces a drug effect, which greatly reduces the drug safety window.
  • the series of compounds of the invention have high activity in vitro, especially high anticoagulant activity, comparable to, or better than, the activity of the reference compound.
  • cell membrane permeability, protein binding rate is similar to the reference compound.
  • the in vivo pharmacokinetic parameters are also comparable to the reference compound.
  • novel bicyclic core structure in the compounds of the present invention is unique to this patent.
  • the uniqueness of the structure may result in different pharmacological effects and even different indications compared to the reference compound.

Abstract

The invention falls within the technical field of chemical medicine. Specifically provided are a series of compounds acting as protease activated receptor 4 (PAR4) inhibitors for treating platelet aggregation, and a medical use thereof.

Description

作为用于治疗血小板聚集的蛋白酶激活受体4(PAR4)抑制剂的化合物a compound that acts as a protease activated receptor 4 (PAR4) inhibitor for the treatment of platelet aggregation 技术领域Technical field
本发明属于化学药物技术领域，特别提供了一系列作为用于治疗血小板聚集的蛋白酶激活受体4(PAR4)抑制剂的化合物及其医药用途。The present invention is in the field of chemical pharmaceutical technology, and particularly provides a series of compounds as protease inhibitory receptor 4 (PAR4) inhibitors for treating platelet aggregation and their medical uses.
背景技术Background technique
血栓栓塞性疾病仍是目前死亡的主要原因之一，尽管有可利用的抗凝血剂如华法林，肝素，和抗血小板剂如阿司匹林和氯吡格雷，替格瑞洛，利伐沙班等等。Thromboembolic disease remains one of the leading causes of death, although available anticoagulants such as warfarin, heparin, and antiplatelet agents such as aspirin and clopidogrel, ticagrelor, rivaroxaban and many more.
血小板的主要生理功能是参与血栓与止血，任何凝血过程都涉及血小板的活化，这是一个复杂的信号级连过程，凝血酶在其中占有核心地位。凝血酶活化血小板主要通过一族G蛋白偶连的蛋白酶活化受体PARs(protease-activated receptors)介导。人类血小板表面表达PAR1与PAR4两种受体，凝血酶能连接并切割PAR1或PAR4，暴露新的N-末端，后者作为一个固定配基与受体进行分子内结合，从而激发跨膜信号传导，引起血小板聚集、释放以及膜糖蛋白的一系列改变。The main physiological function of platelets is to participate in thrombosis and hemostasis. Any blood coagulation process involves the activation of platelets, which is a complex signal cascade process in which thrombin plays a central role. Thrombin-activated platelets are mediated primarily by a family of G-protein-coupled protease-activated receptors (PARs). Human platelets express both PAR1 and PAR4 receptors. Thrombin can bind and cleave PAR1 or PAR4, exposing a new N-terminus, which acts as a binding ligand for intramolecular binding to the receptor, thereby stimulating transmembrane signaling. , causing platelet aggregation, release, and a series of changes in membrane glycoproteins.
PAR1抑制剂已得到广泛研究，包括vorapaxar(沃拉帕沙)和atopaxar(阿托帕沙)的数种化合物已进入后期临床试验。近来，在ACS患者的示踪III期试验中，vorapaxar没有显著减少心血管事件，而是显著增加严重出血的风险(Tricoci,P.等人，N.Eng.J.Med.,366(1)：20-33(2012)。因此，仍然需要发现新的具有增加功效和减少出血副作用的抗血小板药。PAR1 inhibitors have been extensively studied, and several compounds including vorapaxar (wolapasa) and atopaxar (attopasa) have entered late clinical trials. Recently, in the Phase III trial of ACS patients, vorapaxar did not significantly reduce cardiovascular events, but significantly increased the risk of severe bleeding (Tricoci, P. et al., N. Eng. J. Med., 366(1) :20-33 (2012). Therefore, there is still a need to discover new antiplatelet drugs with increased efficacy and reduced bleeding side effects.
另外，有一些PAR4抑制剂专利申请公开，如CN104640869A和CN104583218A分别公开了下式I和II的一系列作为蛋白酶活化受体4(PAR4)抑制剂，用于抑制或防止血小板聚集的药物中的用途。In addition, some PAR4 inhibitor patent applications disclose, such as CN104640869A and CN104583218A, respectively, disclose a series of uses of the following formulas I and II as protease activated receptor 4 (PAR4) inhibitors for inhibiting or preventing platelet aggregation. .
Figure PCTCN2019086557-appb-000001
Figure PCTCN2019086557-appb-000001
所述化合物的研究还处于临床研究阶段，据专利申请公开资料显示，其五元母核中应当含有氧原子才有相应的PAR4活性。The study of the compound is still in the clinical research stage. According to the patent application publication, the pentad nucleus should contain an oxygen atom to have the corresponding PAR4 activity.
发明内容Summary of the invention
为了寻找具有增加功效和减少出血副作用的抗血小板药，并且本发明提供了一系列作为用于治疗血小板聚集的蛋白酶激活受体4(PAR4)抑制剂的化合物，本发明的化合物结构母核不同于已公开的PAR4抑制剂。In order to find anti-platelet drugs with increased efficacy and reduced bleeding side effects, and the present invention provides a series of compounds as protease activated receptor 4 (PAR4) inhibitors for the treatment of platelet aggregation, the structural nucleus of the compound of the present invention is different from A PAR4 inhibitor has been disclosed.
本发明首先提供了一系列的作为用于治疗血小板聚集的蛋白酶激活受体4(PAR4)抑制剂的化合物或其药学上可接受的盐,其特征在于，所述化合物的母核如下：The present invention first provides a series of compounds, or a pharmaceutically acceptable salt thereof, as a protease activated receptor 4 (PAR4) inhibitor for the treatment of platelet aggregation, characterized in that the mother nucleus of the compound is as follows:
Figure PCTCN2019086557-appb-000002
Figure PCTCN2019086557-appb-000002
其中，R 0选自氢，卤素，C 1-4烷基，C 1-4的氨基，C 1-4的烷氧基，C 1-4的烷硫基； Wherein R 0 is selected from the group consisting of hydrogen, halogen, C 1-4 alkyl, C 1-4 amino group, C 1-4 alkoxy group, C 1-4 alkylthio group;
X、Z选自CH或者N；X, Z are selected from CH or N;
Y选自O或者CH；Y is selected from O or CH;
R 1选自氢，卤素、羟基、巯基、C 1-4的烷氧基； R 1 is selected from the group consisting of hydrogen, halogen, hydroxy, decyl, C 1-4 alkoxy;
R 2选自氢，卤素、羟基、巯基、C 1-4的烷氧基、C 1-4的氨基取代的C 1-4烷氧基、C 3-6的环烷基取代的C 1-4烷氧基、羟基取代的C 1-4烷氧基、C 1-4的烷氧基-W-C 1-4烷氧基、C 3-6杂环-W-C 1-4烷氧基，其中W为键、(CH 2)n-O、羰基、(CH 2)n-S、(CH 2)n-吡啶或(CH 2)n-噻唑，其中C 3-6杂环可进一步被卤素、羟基所取代，噻唑或吡啶可进一步被C 1-4烷基或羟基取代，n＝0、1、2或3。 R 2 is selected from the group consisting of hydrogen, halogen, hydroxy, decyl, C 1-4 alkoxy, C 1-4 amino substituted C 1-4 alkoxy, C 3-6 cycloalkyl substituted C 1- 4 alkoxy, hydroxy-substituted C 1-4 alkoxy, C 1-4 alkoxy -WC 1-4 alkoxy, C 3-6 heterocycloalkyl -WC 1-4 alkoxy, wherein W Is a bond, (CH 2 ) nO, carbonyl, (CH 2 ) nS, (CH 2 ) n-pyridine or (CH 2 ) n-thiazole, wherein the C 3-6 heterocycle can be further substituted by halogen, hydroxy, thiazole Or the pyridine may be further substituted by a C 1-4 alkyl group or a hydroxyl group, n = 0, 1, 2 or 3.
作为本发明的一种优选方案，所述卤素选自氟、氯、溴、碘。As a preferred embodiment of the present invention, the halogen is selected from the group consisting of fluorine, chlorine, bromine, and iodine.
作为本发明的一种优选方案，所述C 1-4的烷基选自甲基、乙基、丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基。 As a preferred embodiment of the present invention, the C 1-4 alkyl group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.
作为本发明的一种优选方案，所述C 1-4的氨基选自甲氨基、乙氨基、丙氨基、异丙胺基、正丁氨基、异丁氨基、仲丁氨基和叔丁氨基、二甲氨基。 As a preferred embodiment of the present invention, the amino group of C 1-4 is selected from the group consisting of methylamino, ethylamino, propylamino, isopropylamino, n-butylamino, isobutylamino, sec-butylamino and t-butylamino, and dimethyl Amino group.
作为本发明的一种优选方案，所述C 1-4的烷氧基选自甲氧基、乙氧基、丙氧基、异丙氧基、正丁氧基、异丁氧基、仲丁氧基、叔丁氧基。 As a preferred embodiment of the present invention, the C 1-4 alkoxy group is selected from the group consisting of a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a n-butoxy group, an isobutoxy group, and a sec-butyl group. Oxyl, tert-butoxy.
作为本发明的一种优选方案，所述C 1-4的烷硫基选自甲硫基、乙硫基、丙硫基、异丙硫基、正丁硫基、异丁硫基、仲丁硫基、叔丁硫基。 As a preferred embodiment of the present invention, the C 1-4 alkylthio group is selected from the group consisting of methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, isobutylthio, and sec Sulfur-based, tert-butylthio.
作为本发明的一种优选方案，C 3-6的环烷基选自环丙烷、环戊烷、环己烷。 As a preferred embodiment of the present invention, the C 3-6 cycloalkyl group is selected from the group consisting of cyclopropane, cyclopentane, and cyclohexane.
作为本发明的一种优选方案，C 3-6杂环选自
Figure PCTCN2019086557-appb-000003
As a preferred embodiment of the present invention, the C 3-6 heterocyclic ring is selected from
Figure PCTCN2019086557-appb-000003
作为本发明的一种优选方案，所述化合物通式结构如下式(II-1，II-2)所示：As a preferred embodiment of the present invention, the general structure of the compound is as shown in the following formula (II-1, II-2):
Figure PCTCN2019086557-appb-000004
Figure PCTCN2019086557-appb-000004
其中，R 0、R 1、X如上定义； Wherein R 0 , R 1 , and X are as defined above;
m等于0，1，2，3或4；m is equal to 0, 1, 2, 3 or 4;
当A选自氧原子，B选自H、C 1-4烷基、或者烷氧基取代的C 1-4的烷基； When A is selected from an oxygen atom, B is selected from H, C 1-4 alkyl, or alkoxy substituted C 1-4 alkyl;
当A选自羰基，B选自羟基、氨基或者C 1-4烷氧基； When A is selected from a carbonyl group, B is selected from a hydroxyl group, an amino group or a C 1-4 alkoxy group;
当A选自亚甲基，B选自羟基、C 3-6的环烷基、含氮和/或氧C 3-6的杂环、C 1-4烷基取代或者非取代氨基。 When A is selected from a methylene group, B is selected from a hydroxyl group, a C 3-6 cycloalkyl group, a nitrogen-containing and/or oxygen C 3-6 heterocyclic ring, a C 1-4 alkyl-substituted or unsubstituted amino group.
当A选自C 1-4烷基取代或非取代的噻唑环、取代或非取代的苯环、取代或非取代的吡啶环，B选自
Figure PCTCN2019086557-appb-000005
其中R 3、R 4选自氢、甲基、羟基，所述
Figure PCTCN2019086557-appb-000006
为连接键。 When A is selected from a C 1-4 alkyl-substituted or unsubstituted thiazole ring, a substituted or unsubstituted benzene ring, a substituted or unsubstituted pyridine ring, B is selected from
Figure PCTCN2019086557-appb-000005
Wherein R 3 and R 4 are selected from the group consisting of hydrogen, methyl, and hydroxy,
Figure PCTCN2019086557-appb-000006
For the connection key.
作为本发明的一种优选方案，其特征在于，As a preferred embodiment of the present invention, it is characterized in that
X、Y、Z如上定义；X, Y, Z are as defined above;
R 0选自氢、甲氧基； R 0 is selected from the group consisting of hydrogen and methoxy;
R 1选自氢、甲氧基； R 1 is selected from the group consisting of hydrogen and methoxy;
R 2选自羟基、甲氧基、
Figure PCTCN2019086557-appb-000007
甲氧乙氧基、
Figure PCTCN2019086557-appb-000008
羟乙氧基、
Figure PCTCN2019086557-appb-000009
甲氧丙氧基、
Figure PCTCN2019086557-appb-000010
二甲氧基、
Figure PCTCN2019086557-appb-000011
R 2 is selected from the group consisting of hydroxyl, methoxy,
Figure PCTCN2019086557-appb-000007
Methoxyethoxy,
Figure PCTCN2019086557-appb-000008
Hydroxyethoxy,
Figure PCTCN2019086557-appb-000009
Methoxypropoxy,
Figure PCTCN2019086557-appb-000010
Dimethoxy,
Figure PCTCN2019086557-appb-000011
作为本发明的一种优选方案，所述药学上可接受的盐是化合物与无机酸或有机酸成盐，所述的无机酸或有机酸选自2-乙酰氧基苯甲酸、2-羟基乙磺酸、乙酸、抗坏血酸、苯磺酸、苯甲酸、氢碳酸、碳酸、柠檬酸、依地酸、乙烷二磺酸、乙烷磺酸、富马酸、葡庚糖酸、葡糖酸、谷氨酸、乙醇酸、氢溴酸、盐酸、氢碘酸、羟萘酸、羟乙磺酸、乳酸、乳糖酸、十二烷基磺酸、马来酸、苹果酸、扁桃酸、甲烷磺酸、硝酸、草酸、双羟萘酸、泛酸、苯乙酸、磷酸、多聚半乳糖醛酸、丙酸、水杨酸、硬脂酸、亚乙酸、琥珀酸、氨基磺酸、对氨基苯磺酸、硫酸、单宁酸、酒石酸和对甲苯磺酸。As a preferred embodiment of the present invention, the pharmaceutically acceptable salt is a salt of a compound with an inorganic acid or an organic acid selected from the group consisting of 2-acetoxybenzoic acid and 2-hydroxyethyl. Sulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, hydrogen carbonate, carbonic acid, citric acid, edetic acid, ethane disulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, Glutamate, glycolic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxynaphthoic acid, isethionethane, lactic acid, lactobionic acid, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonate Acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, sulfamic acid, p-aminobenzenesulfonate Acid, sulfuric acid, tannic acid, tartaric acid and p-toluenesulfonic acid.
本发明优选自以下化合物：The invention is preferably derived from the following compounds:
Figure PCTCN2019086557-appb-000012
Figure PCTCN2019086557-appb-000012
Figure PCTCN2019086557-appb-000013
Figure PCTCN2019086557-appb-000013
本发明的另一目的在于提供一种药物组合物，其包括药学上可接受的载体和前述的化合物或其药学上可接受的盐。Another object of the present invention is to provide a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the aforementioned compound or a pharmaceutically acceptable salt thereof.
本发明的另一目的在于提供一种药物用途，包括前述化合物或其药学上可接受的盐在制备用于制备治疗血栓相关疾病的药物用途。Another object of the present invention is to provide a pharmaceutical use comprising the aforementioned compound or a pharmaceutically acceptable salt thereof for use in the preparation of a medicament for the preparation of a thrombosis-related disease.
优选的所述血栓相关疾病选自动脉心血管血栓栓塞性病症、静脉心血管血栓栓塞性病症、脑血管血栓栓塞性病症、和心脏腔室或外周循环中的血栓栓塞性病症。Preferred thrombi-related disorders are selected from the group consisting of arterial cardiovascular thromboembolic disorders, venous cardiovascular thromboembolic disorders, cerebrovascular thromboembolic disorders, and thromboembolic disorders in the heart or peripheral circulation.
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered undefined or unclear without a particular definition, but should be understood in the ordinary sense. When a trade name appears in this document, it is intended to refer to its corresponding commodity or its active ingredient. The term "pharmaceutically acceptable" as used herein is intended to mean that those compounds, materials, compositions and/or dosage forms are within the scope of sound medical judgment and are suitable for use in contact with human and animal tissues. Without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物的中性形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机氨或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物的中性形式接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如甲酸、乙酸、三氟乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐(参见Berge et al.,"Pharmaceutical Salts",Journal of Pharmaceutical Science 66:1-19(1977))。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。The term "pharmaceutically acceptable salt" refers to a salt of a compound of the invention prepared from a compound having a particular substituent found in the present invention and a relatively non-toxic acid or base. When a relatively acidic functional group is contained in the compound of the present invention, a base addition salt can be obtained by contacting a neutral amount of such a compound with a sufficient amount of a base in a neat solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When a relatively basic functional group is contained in the compound of the present invention, an acid addition salt can be obtained by contacting a neutral form of such a compound with a sufficient amount of an acid in a neat solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogencarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts including, for example, formic acid, acetic acid, trifluoroacetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, amber Acids, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid, etc.; also include amino acids (such as fine Salts of amino acids and the like, and salts of organic acids such as glucuronic acid (see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds of the invention contain both basic and acidic functional groups which can be converted to any base or acid addition salt.
优选地，以常规方式使盐与碱或酸接触，再分离母体化合物，由此再生化合物的中性形式。化合物的母体形式与其各种盐的形式的不同之处在于某些物理性质，例如在极性溶剂中的溶解度不同。本文所用的“药学上可接受的盐”属于本发明化合物的衍生物，其中，通过与酸成盐或与碱成盐的方式修饰所述母体化合物。药学上可接受的盐的实例包括但不限于：碱基比如胺的无机酸或有机酸盐、酸根比如羧酸的碱金属或有机盐等等。药学上可接受的盐包括常规的无毒性的盐或母体化合物的季铵盐，例如无毒的无机酸或有机酸所形成的盐。常规的无毒性的盐包括但不限于那些衍生自无机酸和有机酸的盐，所述的无机酸或有机酸选自2-乙酰氧基苯甲酸、2-羟基乙磺酸、甲酸、乙酸、抗坏血酸、苯磺酸、苯甲酸、氢碳酸、碳酸、柠檬酸、依地酸、乙烷二磺酸、乙烷磺酸、富马酸、葡庚糖酸、葡糖酸、谷氨酸、乙醇酸、氢溴酸、盐酸、氢碘酸、羟萘酸、羟乙磺酸、乳酸、乳糖酸、十二烷基磺酸、马来酸、苹果酸、扁桃酸、甲烷磺酸、硝酸、草酸、双羟萘酸、泛酸、苯乙酸、磷酸、多聚半乳糖醛酸、丙酸、水杨酸、硬脂酸、亚乙酸、琥珀酸、氨基磺酸、对氨基苯磺酸、硫酸、三氟乙酸、单宁酸、酒石酸和对甲苯磺酸。Preferably, the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated, thereby regenerating the neutral form of the compound. The parent form of the compound differs from the form of its various salts by certain physical properties, such as differences in solubility in polar solvents. As used herein, a "pharmaceutically acceptable salt" is a derivative of a compound of the invention wherein the parent compound is modified by salt formation with an acid or with a base. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of bases such as amines, alkali metal or organic salts of acid groups such as carboxylic acids, and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts or quaternary ammonium salts of the parent compound, for example salts formed from non-toxic inorganic or organic acids. Conventional non-toxic salts include, but are not limited to, those derived from inorganic acids and organic acids selected from the group consisting of 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, formic acid, acetic acid, Ascorbic acid, benzenesulfonic acid, benzoic acid, hydrogen carbonate, carbonic acid, citric acid, edetic acid, ethane disulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, ethanol Acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid , pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, sulfamic acid, p-aminobenzenesulfonic acid, sulfuric acid, three Fluoroacetic acid, tannic acid, tartaric acid and p-toluenesulfonic acid.
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与 化学计量的适当的碱或酸反应来制备。一般地，优选醚、乙酸乙酯、乙醇、异丙醇或乙腈等非水介质。The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing an acid group or a base by conventional chemical methods. In general, such salts are prepared by reacting these compounds in water or an organic solvent or a mixture of the two via a free acid or base form with a stoichiometric amount of the appropriate base or acid. Generally, a nonaqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is preferred.
除了盐的形式，本发明所提供的化合物还存在前药形式。本文所描述的化合物的前药容易地在生理条件下发生化学变化从而转化成本发明的化合物。此外，前体药物可以在体内环境中通过化学或生化方法被转换到本发明的化合物。In addition to the form of the salt, the compounds provided herein also exist in the form of prodrugs. Prodrugs of the compounds described herein are readily chemically altered under physiological conditions to convert to the compounds of the invention. Furthermore, prodrugs can be converted to the compounds of the invention by chemical or biochemical methods in an in vivo setting.
本发明的某些化合物可以以非溶剂化形式或者溶剂化形式存在，包括水合物形式。一般而言，溶剂化形式与非溶剂化的形式相当，都包含在本发明的范围之内。Certain compounds of the invention may exist in unsolvated or solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are included within the scope of the invention.
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。The compounds of the invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including the cis and trans isomers, the (-)- and (+)-p-enantiomers, the (R)- and (S)-enantiomers, and the diastereomeric a conformation, a (D)-isomer, a (L)-isomer, and a racemic mixture thereof, and other mixtures, such as enantiomerically or diastereomeric enriched mixtures, all of which belong to It is within the scope of the invention. Additional asymmetric carbon atoms may be present in the substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the invention.
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体，以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。The optically active (R)- and (S)-isomers, as well as the D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If an enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary wherein the resulting mixture of diastereomers is separated and the auxiliary group cleaved to provide pure The desired enantiomer. Alternatively, when a molecule contains a basic functional group (e.g., an amino group) or an acidic functional group (e.g., a carboxyl group), a diastereomeric salt is formed with a suitable optically active acid or base, followed by conventional methods well known in the art. The diastereomers are resolved and the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomers is generally accomplished by the use of chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (eg, formation of an amino group from an amine). Formate).
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚(3H)，碘-125(125I)或C-14(14C)。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。The compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound. For example, a compound can be labeled with a radioisotope such as hydrazine (3H), iodine-125 (125I) or C-14 (14C). Alterations of all isotopic compositions of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
术语“药学上可接受的载体”是指能够递送本发明有效量活性物质、不干扰活性物质的生物活性并且对宿主或者患者无毒副作用的任何制剂载体或介质，代表性的载体包括水、油、蔬菜和矿物质、膏基、洗剂基质、软膏基质等。这些基质包括悬浮剂、增粘剂、透皮促进剂等。它们的制剂为化妆品领域或局部药物领域的技术人员所周知。关于载体的其他信息，可以参考Remington:The Science and Practice of Pharmacy,21st Ed.,Lippincott,Williams&Wilkins(2005)，该文献的内容通过引用的方式并入本文。The term "pharmaceutically acceptable carrier" refers to any formulation carrier or vehicle that is capable of delivering an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects to the host or patient. Representative carriers include water, oil. , vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, tackifiers, transdermal enhancers and the like. Their formulations are well known to those skilled in the cosmetic or topical pharmaceutical arts. For additional information on vectors, reference is made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are hereby incorporated by reference.
术语“赋形剂”通常是指配制有效的药物组合物所需要载体、稀释剂和/或介质。The term "excipient" generally refers to the carrier, diluent and/or vehicle required to formulate an effective pharmaceutical composition.
针对药物或药理学活性剂而言，术语“有效量”或“治疗有效量”是指无毒的但能达到预期效果的药物或药剂的足够用量。对于本发明中的口服剂型，组合物中一种活性物质的“有效量”是指与该组合物中另一种活性物质联用时为了达到预期效果所需要的用量。有效量的确定因人而异，取决于受体的年龄和一般情况，也取决于具体的活性物质，个案中合适的有效量可以由本领域技术人员根据常规试验确定。The term "effective amount" or "therapeutically effective amount" with respect to a pharmaceutical or pharmacologically active agent refers to a sufficient amount of a drug or agent that is non-toxic but that achieves the desired effect. For oral dosage forms in the present invention, an "effective amount" of an active substance in a composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount will vary from person to person, depending on the age and general condition of the recipient, and also on the particular active substance, and a suitable effective amount in a case can be determined by one skilled in the art based on routine experimentation.
术语“活性成分”、“治疗剂”，“活性物质”或“活性剂”是指一种化学实体，它可以有效地治疗目标紊乱、疾病或病症。The term "active ingredient", "therapeutic agent", "active substance" or "active agent" refers to a chemical entity that is effective in treating a target disorder, disease or condition.
“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。"Optional" or "optionally" means that the subsequently described event or condition may, but is not necessarily, to occur, and that the description includes instances in which the event or condition occurs and instances in which the event or condition does not occur.
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, combinations thereof with other chemical synthetic methods, and those well known to those skilled in the art. Equivalent alternatives, preferred embodiments include, but are not limited to, embodiments of the invention.
具体实施方式Detailed ways
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。The invention is described in detail below by the examples, but is not intended to limit the invention. The present invention has been described in detail herein, the embodiments of the present invention are disclosed herein, and various modifications and changes may be made to the embodiments of the present invention without departing from the spirit and scope of the invention. It will be obvious.
通式制备路线:General formula preparation route:
Figure PCTCN2019086557-appb-000014
Figure PCTCN2019086557-appb-000014
通式合成步骤A：合成5,7-二羟基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮General Synthesis Step A: Synthesis of 5,7-Dihydroxy-2,2-dimethyl-4H-benzo[d][1,3]dioxe-4-one
Figure PCTCN2019086557-appb-000015
Figure PCTCN2019086557-appb-000015
将2,4,6-三羟基苯甲酸溶于丙酮中。将反应液冷却到0摄氏度，然后，向上述溶液中依次缓慢滴加三氟乙酸和三氟乙酸酐。将反应液加热至65摄氏度，并搅拌16小时。The 2,4,6-trihydroxybenzoic acid was dissolved in acetone. The reaction solution was cooled to 0 ° C, and then trifluoroacetic acid and trifluoroacetic anhydride were slowly added dropwise to the above solution. The reaction solution was heated to 65 ° C and stirred for 16 hours.
将反应液冷却到室温，并在减压下浓缩。所得残余物溶于乙酸乙酯与水的混合溶液中。向混合液中加入饱和碳酸氢钠调节PH值约为8。混合液用乙酸乙酯萃取3次。合并有机相，有机相先用饱和食盐水洗涤3次，然后用无水硫酸钠干燥，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/5)。得到白色固体5,7-二羟基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮。The reaction solution was cooled to room temperature and concentrated under reduced pressure. The resulting residue was dissolved in a mixed solution of ethyl acetate and water. Saturated sodium bicarbonate was added to the mixture to adjust the pH to about 8. The mixture was extracted three times with ethyl acetate. The organic phase was combined and the organic phase was washed three times with brine, dried over anhydrous sodium sulfate The residue obtained was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1/5). A white solid 5,7-dihydroxy-2,2-dimethyl-4H-benzo[d][1,3]dioxine-4-one was obtained.
通式合成步骤B：合成5-羟基-7-甲氧基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮General Synthesis Step B: Synthesis of 5-Hydroxy-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxan-4-one
Figure PCTCN2019086557-appb-000016
Figure PCTCN2019086557-appb-000016
在冰水浴和氮气保护下，将5,7-二羟基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮溶于四氢呋喃中。随后，向上述溶液中依次加入甲醇和偶氮二甲酸二异丙酯。将反应液升至室温，并搅拌3小时。5,7-dihydroxy-2,2-dimethyl-4H-benzo[d][1,3]dioxan-4-one was dissolved in tetrahydrofuran under ice-water bath and nitrogen atmosphere . Subsequently, methanol and diisopropyl azodicarboxylate were sequentially added to the above solution. The reaction solution was warmed to room temperature and stirred for 3 hours.
向反应液中加入水淬灭反应。混合液用乙酸乙酯萃取3次。合并有机相，有机相先用饱和食盐水洗涤3次，然后用无水硫酸钠干燥，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/10)。得到白色固体5-羟基-7-甲氧基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮。Water was added to the reaction solution to quench the reaction. The mixture was extracted three times with ethyl acetate. The organic phase was combined and the organic phase was washed three times with brine, dried over anhydrous sodium sulfate The residue obtained was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1/10). A white solid 5-hydroxy-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxine-4-one was obtained.
通式合成步骤C：合成5-(苄氧基)-7-甲氧基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮General Procedure Step C: Synthesis of 5-(Benzyloxy)-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxan-4- ketone
Figure PCTCN2019086557-appb-000017
Figure PCTCN2019086557-appb-000017
将5-羟基-7-甲氧基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮溶于N,N-二甲基甲酰胺中。随后，向上述溶液中依次加入无水碳酸钾和溴化苄，在室温下搅拌16小时。Dissolving 5-hydroxy-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxine-4-one in N,N-dimethyl In formamide. Subsequently, anhydrous potassium carbonate and benzyl bromide were sequentially added to the above solution, and the mixture was stirred at room temperature for 16 hours.
向反应液中加入水淬灭反应。混合液用乙酸乙酯萃取，合并有机相，有机相先用饱和食盐水洗涤，然后用无水硫酸钠干燥，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/5)。得到白色固体5-(苄氧基)-7-甲氧基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮。Water was added to the reaction solution to quench the reaction. The mixture was extracted with EtOAc. EtOAc was evaporated. The residue obtained was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1/5). 5-(Benzyloxy)-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxine-4-one was obtained as a white solid.
通式合成步骤D：合成2-(苄氧基)-6-羟基-4-甲氧基苯甲醛General Synthesis Step D: Synthesis of 2-(Benzyloxy)-6-hydroxy-4-methoxybenzaldehyde
Figure PCTCN2019086557-appb-000018
Figure PCTCN2019086557-appb-000018
在氮气保护下，将5-(苄氧基)-7-甲氧基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮溶于二氯甲烷中。在-78摄氏度下，向上述溶液中缓慢滴加二异丁基氢化铝。在-78摄氏度下搅拌3小时。在-78摄氏度，向反应液中缓慢滴加甲醇淬灭反应，然后向反应液中滴加稀盐酸溶液。将反应液升至室温。混合液用二氯甲烷萃取。合并有机相，有机相先用饱和食盐水洗涤，然后用无水硫酸钠干燥，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/7)，得到棕色固体2-(苄氧基)-6-羟基-4-甲氧基苯甲醛。5-(Benzyloxy)-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxine-4-one under nitrogen protection Dissolved in dichloromethane. To the above solution, diisobutylaluminum hydride was slowly added dropwise at -78 °C. Stir at -78 degrees Celsius for 3 hours. At -78 ° C, methanol was slowly added dropwise to the reaction solution to quench the reaction, and then a dilute hydrochloric acid solution was added dropwise to the reaction solution. The reaction was allowed to warm to room temperature. The mixture was extracted with dichloromethane. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate The obtained residue was purified to silicagel elut elut elut elut elut elut elut
通式合成步骤E：合成2-(3-(苄氧基)-2-甲酰基-5-甲氧基苯氧基)乙酸乙酯General Procedure Step E: Synthesis of 2-(3-(Benzyloxy)-2-formyl-5-methoxyphenoxy)acetate
Figure PCTCN2019086557-appb-000019
Figure PCTCN2019086557-appb-000019
将2-(苄氧基)-6-羟基-4-甲氧基苯甲醛，2-溴乙酸乙酯和无水碳酸钾溶于乙腈。将反应液加热至70摄氏度，并搅拌3小时。2-(Benzyloxy)-6-hydroxy-4-methoxybenzaldehyde, ethyl 2-bromoacetate and anhydrous potassium carbonate were dissolved in acetonitrile. The reaction solution was heated to 70 ° C and stirred for 3 hours.
将反应液冷却到室温，过滤，滤饼用乙酸乙酯洗涤。滤液在减压下浓缩。得到黄色油状物2-(3-(苄氧基)-2-甲酰基-5-甲氧基苯氧基)乙酸乙酯。无需纯化，直接用于下步反应。The reaction solution was cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. Ethyl acetate 2-(3-(benzyloxy)-2-formyl-5-methoxyphenoxy) was obtained as a yellow oil. It is used directly in the next step without purification.
通式合成步骤F：合成2-(3-(苄氧基)-2-甲酰基-5-甲氧基苯氧基)乙酸General Scheme Synthesis Step F: Synthesis of 2-(3-(Benzyloxy)-2-formyl-5-methoxyphenoxy)acetic acid
Figure PCTCN2019086557-appb-000020
Figure PCTCN2019086557-appb-000020
将2-(3-(苄氧基)-2-甲酰基-5-甲氧基苯氧基)乙酸乙酯溶于四氢呋喃与水的混合溶液中。随后，向上述溶液中加入氢氧化钠。在室温下搅拌30分钟。Ethyl 2-(3-(benzyloxy)-2-formyl-5-methoxyphenoxy)acetate was dissolved in a mixed solution of tetrahydrofuran and water. Subsequently, sodium hydroxide was added to the above solution. Stir at room temperature for 30 minutes.
将反应液在减压下浓缩。所得残余物溶于水中，向上述溶液中缓慢滴加稀盐酸调节PH值约为2，有白色固体析出。过滤，收集滤饼，滤饼烘干得到白色固体2-(3-(苄氧基)-2-甲酰基-5-甲氧基苯氧基)乙酸。无需纯化，直接用于下步反应。The reaction solution was concentrated under reduced pressure. The residue obtained was dissolved in water, and dilute hydrochloric acid was slowly added dropwise to the above solution to adjust the pH to about 2, and a white solid was precipitated. Filtration, collection of the filter cake, and drying of the filter cake gave 2-(3-(benzyloxy)-2-formyl-5-methoxyphenoxy) acetic acid as a white solid. It is used directly in the next step without purification.
通式合成步骤G：合成4-(苄氧基)-6-甲氧基苯并呋喃General Scheme Synthesis Step G: Synthesis of 4-(Benzyloxy)-6-methoxybenzofuran
Figure PCTCN2019086557-appb-000021
Figure PCTCN2019086557-appb-000021
将2-(3-(苄氧基)-2-甲酰基-5-甲氧基苯氧基)乙酸溶于乙酸酐中。随后，向上述溶液中加入醋 酸钠。将反应液加热至140摄氏度，并搅拌1小时。2-(3-(Benzyloxy)-2-formyl-5-methoxyphenoxy)acetic acid was dissolved in acetic anhydride. Subsequently, sodium acetate was added to the above solution. The reaction solution was heated to 140 ° C and stirred for 1 hour.
将反应液冷却到室温，过滤，滤饼用乙酸乙酯，滤液在减压下浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/20)。得到黄色固体4-(苄氧基)-6-甲氧基苯并呋喃。The reaction solution was cooled to room temperature, filtered, and then filtered and evaporated. The residue obtained was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1 / 20). The yellow solid 4-(benzyloxy)-6-methoxybenzofuran was obtained.
通式合成步骤H：合成2-(4-(苄氧基)-6-甲氧基苯并呋喃-2-基)-4,4,5,5-四甲基-1,3,2-二氧杂硼烷General Scheme I: Synthesis of 2-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-4,4,5,5-tetramethyl-1,3,2- Dioxaborane
Figure PCTCN2019086557-appb-000022
Figure PCTCN2019086557-appb-000022
在氮气保护下，将4-(苄氧基)-6-甲氧基苯并呋喃和4,4,4’，4’，5,5,5’，5’-八甲基-2,2’-二(1,3,2-二氧杂硼烷)溶于正己烷中。随后，向上述溶液中加入[Ir(COD)(OMe)] 2,dtbpy。将反应液加热至60摄氏度，并搅拌1小时。 4-(Benzyloxy)-6-methoxybenzofuran and 4,4,4',4',5,5,5',5'-octamethyl-2,2 under nitrogen protection '-Di(1,3,2-dioxaborane) was dissolved in n-hexane. Subsequently, [Ir(COD)(OMe)] 2 , dtbpy was added to the above solution. The reaction solution was heated to 60 ° C and stirred for 1 hour.
将反应液冷却到室温，向反应液中加入水淬灭反应。混合液用乙酸乙酯萃取。合并有机相，有机相先用饱和食盐水反洗，然后用无水硫酸钠干燥，最后减压浓缩。将所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/10)。得到黄色油状物2-(4-(苄氧基)-6-甲氧基苯并呋喃-2-基)-4,4,5,5-四甲基-1,3,2-二氧杂硼烷。The reaction solution was cooled to room temperature, and water was added to the reaction mixture to quench the reaction. The mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated brine and dried over anhydrous sodium sulfate. The residue was purified by silica gel column chromatography (EtOAc:EtOAc Obtained 2-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxa as a yellow oil Borane.
通式合成步骤I：合成2-(4-(苄氧基)-6-甲氧基苯并呋喃-2-基)-5-甲氧基咪唑并[5,4-d]噻唑General Scheme I: Synthesis of 2-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000023
Figure PCTCN2019086557-appb-000023
在氮气保护下，将2-溴-5-甲氧基咪唑并[5,4-d]噻唑和2-(4-(苄氧基)-6-甲氧基苯并呋喃-2-基)-4,4,5,5-四甲基-1,3,2-二氧杂硼烷溶于1,4-二氧六环中。随后向上述溶液中加入无水碳酸钾，四三苯基膦钯和水。将反应液加热至80摄氏度，并搅拌1小时。2-bromo-5-methoxyimidazo[5,4-d]thiazole and 2-(4-(benzyloxy)-6-methoxybenzofuran-2-yl) under nitrogen atmosphere -4,4,5,5-Tetramethyl-1,3,2-dioxaborane is dissolved in 1,4-dioxane. Anhydrous potassium carbonate, tetrakistriphenylphosphine palladium and water were then added to the above solution. The reaction solution was heated to 80 ° C and stirred for 1 hour.
将反应液冷却到室温。混合液用乙酸乙酯萃取。合并有机相，有机相先用饱和食盐水反洗，然后用无水硫酸钠干燥，最后减压浓缩。将所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/10)。得到白色固体2-(4-(苄氧基)-6-甲氧基苯并呋喃-2-基)-5-甲氧基咪唑并[5,4-d]噻唑。The reaction solution was cooled to room temperature. The mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated brine and dried over anhydrous sodium sulfate. The residue was purified by silica gel column chromatography (EtOAc:EtOAc 2-(4-(Benzyloxy)-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole was obtained as a white solid.
通式合成步骤J：合成6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇General Synthetic Step J: Synthesis of 6-Methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol
Figure PCTCN2019086557-appb-000024
Figure PCTCN2019086557-appb-000024
在氮气保护下，2-(4-(苄氧基)-6-甲氧基苯并呋喃-2-基)-5-甲氧基咪唑并[5,4-d]噻唑和五甲基苯溶于二氯甲烷中。在-78摄氏度下向上述溶液中缓慢滴加三氯化硼。在-78摄氏度下搅拌1小时。向反应体系中加入甲醇淬灭反应。将反应液升至室温，并减压浓缩。粗产品用反相柱纯化[反相柱:C18；流动相A：水(含有0.05％甲酸)，流动相B：乙腈；梯度:30％乙腈到90％乙腈在8分钟内；检测波长：254nm]收集馏分，直接低温冻干。得到黄色固体6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇。2-(4-(Benzyloxy)-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole and pentamethylbenzene under nitrogen protection Dissolved in dichloromethane. Boron trichloride was slowly added dropwise to the above solution at -78 °C. Stir at -78 degrees Celsius for 1 hour. The reaction was quenched by adding methanol to the reaction system. The reaction solution was warmed to room temperature and concentrated. The crude product was purified by reverse phase column [reverse phase column: C18; mobile phase A: water (containing 0.05% formic acid), mobile phase B: acetonitrile; gradient: 30% acetonitrile to 90% acetonitrile in 8 min; detection wavelength: 254 nm The fractions were collected and directly lyophilized. The yellow solid 6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol was obtained.
通式合成步骤K：General synthetic step K:
Figure PCTCN2019086557-appb-000025
Figure PCTCN2019086557-appb-000025
在室温和氮气保护下，将6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇，R 6-醇溶于四氢呋喃(1毫升)中。随后，向上述溶液中依次加入三丁基膦和偶氮二甲酰二哌啶。在室温下搅拌1小时。 6-Methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol, R 6 -alcohol dissolved in tetrahydrofuran at room temperature under nitrogen (1 ml). Subsequently, tributylphosphine and azodiyldipiperidine were sequentially added to the above solution. Stir at room temperature for 1 hour.
向反应液中加入水淬灭反应。混合液用乙酸乙酯萃取。合并有机相。有机相先用饱和食盐水洗涤，然后用无水硫酸钠干燥，最后减压浓缩。粗产品用制备型高效液相色谱纯化。分离条件如下，色谱柱：X select C18 19mm*150mm；流动相：水(含有0.05％的TFA)和乙腈；流速：25毫升/分钟；梯度：在7分钟内，乙腈从5％升到100％；检测波长：254nm。纯化后，低温冻干得到目标化合物。Water was added to the reaction solution to quench the reaction. The mixture was extracted with ethyl acetate. Combine the organic phases. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and evaporated. The crude product was purified by preparative high performance liquid chromatography. The separation conditions were as follows, column: X select C18 19 mm * 150 mm; mobile phase: water (containing 0.05% TFA) and acetonitrile; flow rate: 25 ml / min; gradient: acetonitrile increased from 5% to 100% in 7 minutes Detection wavelength: 254 nm. After purification, the target compound is obtained by lyophilization at low temperature.
通式合成步骤L：General synthetic step L:
Figure PCTCN2019086557-appb-000026
将6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇溶于N,N-二甲基甲酰胺中，加入
Figure PCTCN2019086557-appb-000027
(R＝Cl,或Br)和无水碳酸钾。在室温下搅拌4小时。LCMS监测显示原料消失后，过滤除去不溶物。滤液用制备型高效液相色谱纯化。分离条件如下，色谱柱：X select C18 19mm*150mm；流动相：水(含有0.05％的甲酸)和乙腈；流速：25毫升/分钟；梯度：在7分钟内，乙腈从50％升到60％；检测波长：254nm。减压冻干，得到mm目标化合物。
Figure PCTCN2019086557-appb-000026
6-Methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol is dissolved in N,N-dimethylformamide and added
Figure PCTCN2019086557-appb-000027
(R = Cl, or Br) and anhydrous potassium carbonate. Stir at room temperature for 4 hours. LCMS monitoring showed disappearance of the starting material and filtration to remove insolubles. The filtrate was purified by preparative high performance liquid chromatography. The separation conditions were as follows, column: X select C18 19 mm * 150 mm; mobile phase: water (containing 0.05% formic acid) and acetonitrile; flow rate: 25 ml / min; gradient: acetonitrile increased from 50% to 60% in 7 minutes Detection wavelength: 254 nm. It was lyophilized under reduced pressure to give mm target compound.
实施例1：合成2-(4,6-二甲氧基苯并呋喃-2-基)-5-甲氧基噻唑并[5,4-d]噻唑Example 1: Synthesis of 2-(4,6-dimethoxybenzofuran-2-yl)-5-methoxythiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000028
Figure PCTCN2019086557-appb-000028
步骤A：合成2,5-二溴噻唑并[5,4-d]噻唑Step A: Synthesis of 2,5-dibromothiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000029
Figure PCTCN2019086557-appb-000029
将噻唑并[5,4-d]噻唑(500毫克，3.5毫摩尔)溶于四氯化碳(14.0毫升)中。随后，向上述溶液中加入吡啶(556毫克，7.0毫摩尔)和溴素(5.56克，34.7毫摩尔)。将反应液加热至80摄氏度，并搅拌5小时。Thiazolo[5,4-d]thiazole (500 mg, 3.5 mmol) was dissolved in carbon tetrachloride (14.0 mL). Subsequently, pyridine (556 mg, 7.0 mmol) and bromine (5.56 g, 34.7 mmol) were added to the above solution. The reaction solution was heated to 80 ° C and stirred for 5 hours.
将反应液冷却到室温。将反应液倒入饱和的亚硫酸氢钠溶液(80毫升)中淬灭反应。混合液用二氯甲烷(100毫升×3次)萃取，合并有机相。有机相先用饱和食盐水(50毫升×3次)洗涤，然后用无水硫酸钠干燥，最后减压浓缩。所得残余物通过硅胶柱层析纯化(洗脱剂：石油醚/乙酸乙酯＝10/1)。得到320毫克浅灰色固体2,5-二溴噻唑并[5,4-d]噻唑(收率：30.5％)。MS(ESI)M/Z：301[M+H +]。 The reaction solution was cooled to room temperature. The reaction was poured into a saturated sodium hydrogensulfite solution (80 mL) and the mixture was evaporated. The mixture was extracted with dichloromethane (100 mL×3×) and the organic phases were combined. The organic phase was washed with saturated brine (50 mL×3×) then dried over anhydrous sodium sulfate. The residue was purified by silica gel column chromatography eluting elut 320 mg of light gray solid 2,5-dibromothiazolo[5,4-d]thiazole was obtained (yield: 30.5%). MS (ESI) M / Z: 301 [M+H + ].
步骤B：合成2-溴-5-甲氧基咪唑并[5,4-d]噻唑Step B: Synthesis of 2-bromo-5-methoxyimidazo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000030
Figure PCTCN2019086557-appb-000030
将2,5-二溴噻唑并[5,4-d]噻唑(150毫克，0.5毫摩尔)溶于二氯甲烷(4.0毫升)与甲醇(2.0毫升)的混合溶液中。随后，向上述溶液中加入叔丁醇钾(62毫克，0.6毫摩尔)。在室温下搅拌4小时。向反应液中加水淬灭，混合液用二氯甲烷(30毫升×3次)萃取。合并有机相。有机相先用饱和食盐水(30毫升×3次)洗涤，然后用无水硫酸钠干燥，最后减压浓缩。得到110毫克浅棕色固体2-溴-5-甲氧基咪唑并[5,4-d]噻唑。无需纯化，直接用于下一步反应。MS(ESI)M/Z：251,253[M+H] +。 2,5-Dibromothiazolo[5,4-d]thiazole (150 mg, 0.5 mmol) was dissolved in a mixed solution of dichloromethane (4.0 ml) and methanol (2.0 ml). Subsequently, potassium t-butoxide (62 mg, 0.6 mmol) was added to the above solution. Stir at room temperature for 4 hours. The reaction mixture was quenched with water and the mixture was extracted with dichloromethane (30 ml, 3). Combine the organic phases. The organic phase was washed with saturated brine (30 ml×3×) then dried over anhydrous sodium sulfate 110 mg of light brown solid 2-bromo-5-methoxyimidazo[5,4-d]thiazole was obtained. It was used directly in the next reaction without purification. MS (ESI) M / Z: 251, 253 [M+H] + .
步骤C：合成2-(4,6-二甲氧基苯并呋喃-2-基)-5-甲氧基噻唑并[5,4-d]噻唑Step C: Synthesis of 2-(4,6-dimethoxybenzofuran-2-yl)-5-methoxythiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000031
Figure PCTCN2019086557-appb-000031
在氮气保护下，将2-溴-5-甲氧基咪唑并[5,4-d]噻唑(110毫克，0.4毫摩尔)溶于1,4-二氧六环(10.0毫升)。随后，向上述溶液中加入无水碳酸钠(140毫克，1.3毫摩尔)，(4,6-二甲氧基苯并呋喃-2-基)硼酸酯(201毫克，0.66毫摩尔)和四(三苯基磷)钯(51毫克，0.044毫摩尔)，水(2.0毫升)。将反应液加热至90摄氏度，并搅拌2小时。2-Bromo-5-methoxyimidazo[5,4-d]thiazole (110 mg, 0.4 mmol) was dissolved in 1,4-dioxane (10.0 mL). Subsequently, anhydrous sodium carbonate (140 mg, 1.3 mmol), (4,6-dimethoxybenzofuran-2-yl) borate (201 mg, 0.66 mmol) and four were added to the above solution. (Triphenylphosphine) palladium (51 mg, 0.044 mmol) in water (2.0 mL). The reaction solution was heated to 90 ° C and stirred for 2 hours.
将反应液冷却到室温，并减压浓缩。粗产品用制备型高效液相色谱纯化。分离条件如下，色谱柱：X select C18 19mm*150mm；流动相：水(含有0.05％的三氟乙酸)和乙腈；流速：25毫升/分钟；梯度：在7分钟内，乙腈从5％升到100％；检测波长：254nm。纯化后，低温冻干得25.0毫克浅黄色固体2-(4,6-二甲氧基苯并呋喃-2-基)-5-甲氧基噻唑并[5,4-d]噻唑(收率：17.9％)。MS(ESI)M/Z：349[M+H +]； 1HNMR(300MHz,CDCl 3,ppm)δ7.34(s,1H),6.71(s,1H),6.36(s,1H),4.22(s,3H),3.94(s,3H),3.89(s,3H)。 The reaction solution was cooled to room temperature and concentrated under reduced pressure. The crude product was purified by preparative high performance liquid chromatography. The separation conditions were as follows, column: X select C18 19 mm * 150 mm; mobile phase: water (containing 0.05% trifluoroacetic acid) and acetonitrile; flow rate: 25 ml / min; gradient: acetonitrile increased from 5% in 7 minutes 100%; detection wavelength: 254 nm. After purification, freeze-drying to obtain 25.0 mg of pale yellow solid 2-(4,6-dimethoxybenzofuran-2-yl)-5-methoxythiazolo[5,4-d]thiazole (yield : 17.9%). MS (ESI) M / Z: 349 [M+H + ]; 1 HNMR (300 MHz, CDCl 3 , ppm) δ 7.34 (s, 1H), 6.71 (s, 1H), 6.36 (s, 1H), 4.22 (s, 3H), 3.94 (s, 3H), 3.89 (s, 3H).
实施例2：合成2-(4,6-二甲氧基苯并呋喃-2-基)噻唑并[5,4-d]噻唑Example 2: Synthesis of 2-(4,6-dimethoxybenzofuran-2-yl)thiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000032
Figure PCTCN2019086557-appb-000032
步骤A：合成2-溴噻唑并[5,4-d]噻唑Step A: Synthesis of 2-bromothiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000033
Figure PCTCN2019086557-appb-000033
将噻唑并[5,4-d]噻唑(200毫克，1.41毫摩尔)溶于四氯化碳(10毫升)中。向体系中依次加入吡啶(175毫克，2.22毫摩尔)和溴素(334毫克，2.09毫摩尔)。在80摄氏度下搅拌2小时后，再向体系中依次加入吡啶(55毫克，0.70毫摩尔)和溴素(333毫克，2.08毫摩尔)。在80摄氏度下继续搅拌2小时。TLC监测显示原料消失后，将体系冷却到室温。将混合液倒入饱和亚硫酸氢钠水溶液(100毫升)淬灭反应。混合液用二氯甲烷(100毫升X2)萃取。合并后的有机相先用饱和盐水(100毫升)反洗，然后用无水硫酸钠干燥，最后真空浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚25％)浓缩得浅黄色固体2-溴噻唑并[5,4-d]噻唑(90毫克，28.8％)。MS(ESI)M/Z：221[M+H] + Thiazolo[5,4-d]thiazole (200 mg, 1.41 mmol) was dissolved in carbon tetrachloride (10 mL). Pyridine (175 mg, 2.22 mmol) and bromine (334 mg, 2.09 mmol) were sequentially added to the system. After stirring at 80 ° C for 2 hours, pyridine (55 mg, 0.70 mmol) and bromine (333 mg, 2.08 mmol) were sequentially added to the system. Stirring was continued for 2 hours at 80 degrees Celsius. After TLC monitoring showed disappearance of the starting material, the system was cooled to room temperature. The mixture was poured into a saturated aqueous solution of sodium hydrogensulfite (100 mL) and then evaporated. The mixture was extracted with dichloromethane (100 mL EtOAc). The combined organic phases were back-washed with saturated brine (100 mL) then dried over anhydrous sodium sulfate. The residue was purified with EtOAc EtOAc EtOAc EtOAc (EtOAc: MS (ESI) M/Z: 221 [M+H] +
步骤B：合成2-(4,6-二甲氧基苯并呋喃-2-基)噻唑并[5,4-d]噻唑Step B: Synthesis of 2-(4,6-dimethoxybenzofuran-2-yl)thiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000034
Figure PCTCN2019086557-appb-000034
在室温和氮气保护下，将2-溴噻唑并[5,4-d]噻唑(90毫克，0.407毫摩尔)溶于1,4-二氧六环(6毫升)中。向体系中依次加入无水碳酸钠(130毫克，1.23毫摩尔)，(4,6-二甲氧基苯并呋喃-2-基)硼酸酯(187毫克，0.615毫摩尔)，四(三苯基磷)钯(47毫克，0.041毫摩尔)和水(1毫升)。在90摄氏度下搅拌2小时。TLC监测显示原料消失后，将体系冷却到室温。向体系中加入水(10毫升)淬灭反应。混合液用乙酸乙酯(20毫升X2)萃取。合并后的有机相先用饱和盐水(10毫升)反洗，然后用无水硫酸钠干燥，最后真空浓缩。粗产品用制备型高效液相色谱纯化。制备条件如下。色谱柱：Xselect C18 19mm*150mm；流动相：水(0.05％甲酸)和乙腈；流速：25毫升/分钟；梯度：在7分钟内，乙腈从10％升到80％；检测波长：254nm。纯化后，低温冻干得浅黄色固体2-(4,6-二甲氧基苯并呋喃-2-基)噻唑并[5,4-d]噻唑(53.3毫克，41.0％)。MS(ESI)M/Z：319[M+H] +； 1H NMR(300MHz,CDCl 3,ppm)δ8.87(s,1H),7.48(s,1H),6.73(s,1H),6.37(s,1H),3.96(s,3H),3.90(s,3H)。 2-Bromothiazolo[5,4-d]thiazole (90 mg, 0.407 mmol) was dissolved in 1,4-dioxane (6 mL). Anhydrous sodium carbonate (130 mg, 1.23 mmol), (4,6-dimethoxybenzofuran-2-yl) borate (187 mg, 0.615 mmol), four (three) were sequentially added to the system. Phenylphosphine)palladium (47 mg, 0.041 mmol) and water (1 ml). Stir at 90 degrees Celsius for 2 hours. After TLC monitoring showed disappearance of the starting material, the system was cooled to room temperature. Water (10 mL) was added to the system to quench the reaction. The mixture was extracted with ethyl acetate (20 mL EtOAc). The combined organic phases were back-washed with saturated brine (10 mL) then dried over anhydrous sodium sulfate. The crude product was purified by preparative high performance liquid chromatography. The preparation conditions are as follows. Column: Xselect C18 19mm*150mm; mobile phase: water (0.05% formic acid) and acetonitrile; flow rate: 25 ml/min; gradient: acetonitrile increased from 10% to 80% in 7 minutes; detection wavelength: 254 nm. After purification, it was lyophilized to give a pale yellow solid of 2-(4,6-dimethoxybenzofuran-2-yl)thiazolo[5,4-d]thiazole (53.3 mg, 41.0%). MS (ESI) M / Z: 319 [M+H] + ; 1 H NMR (300 MHz, CDCl 3 , ppm) δ 8.87 (s, 1H), 7.48 (s, 1H), 6.73 (s, 1H), 6.37 (s, 1H), 3.96 (s, 3H), 3.90 (s, 3H).
实施例3：合成6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇Example 3: Synthesis of 6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol
Figure PCTCN2019086557-appb-000035
Figure PCTCN2019086557-appb-000035
步骤A：合成5,7-二羟基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮Step A: Synthesis of 5,7-dihydroxy-2,2-dimethyl-4H-benzo[d][1,3]dioxine-4-one
Figure PCTCN2019086557-appb-000036
Figure PCTCN2019086557-appb-000036
详见通用合成步骤A。2,4,6-三羟基苯甲酸(100.0克，588.2毫摩尔),丙酮(1.0升),三氟乙酸(400毫升),三氟乙酸酐(250毫升),乙酸乙酯(1.0升)与水(1.5升),乙酸乙酯(1.0升×3次),饱和食盐水(1.0升×3次)。得到59.50克白色固体5,7-二羟基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮(收率：48.1％)。MS(ESI)M/Z：211[M+H +]。 See General Synthesis Step A for details. 2,4,6-trihydroxybenzoic acid (100.0 g, 588.2 mmol), acetone (1.0 L), trifluoroacetic acid (400 ml), trifluoroacetic anhydride (250 ml), ethyl acetate (1.0 L) Water (1.5 L), ethyl acetate (1.0 L × 3 times), saturated brine (1.0 L × 3 times). 59.50 g of a white solid 5,7-dihydroxy-2,2-dimethyl-4H-benzo[d][1,3]dioxine-4-one (yield: 48.1%) was obtained. MS (ESI) M / Z: 211 [M+H + ].
步骤B：合成5-羟基-7-甲氧基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮Step B: Synthesis of 5-hydroxy-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxan-4-one
Figure PCTCN2019086557-appb-000037
Figure PCTCN2019086557-appb-000037
详见通用合成步骤B。5,7-二羟基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮(59.50克，283毫摩尔)，四氢呋喃(952毫升)，甲醇(9.70克，303毫摩尔)和偶氮二甲酸二异丙酯(61.24克，303毫摩尔)，水(100毫升)，乙酸乙酯(1.0升×3次)，饱和食盐水(1.0升×3次)。得到60.05克白色固体5-羟基-7-甲氧基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮(收率：94.7％)。MS(ESI)M/Z：225[M+H +]。 See General Synthesis Step B for details. 5,7-Dihydroxy-2,2-dimethyl-4H-benzo[d][1,3]dioxine-4-one (59.50 g, 283 mmol), tetrahydrofuran (952 ml ), methanol (9.70 g, 303 mmol) and diisopropyl azodicarboxylate (61.24 g, 303 mmol), water (100 ml), ethyl acetate (1.0 L × 3 times), saturated brine ( 1.0 liter × 3 times). 60.05 g of white solid 5-hydroxy-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxine-4-one was obtained (yield: 94.7 %). MS (ESI) M/Z: 225 [M+H + ].
步骤C：合成5-(苄氧基)-7-甲氧基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮Step C: Synthesis of 5-(benzyloxy)-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxan-4-one
Figure PCTCN2019086557-appb-000038
Figure PCTCN2019086557-appb-000038
详见通用合成步骤C。5-羟基-7-甲氧基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮(60.05克，268毫摩尔)，N,N-二甲基甲酰胺(780毫升)，无水碳酸钾(38.91，281毫摩尔)，溴化苄(48.05克，281毫摩尔)，水(200毫升)，乙酸乙酯(500毫升×3次)，饱和食盐水(100毫升×3次)。得到66.07克白色固体5-(苄氧基)-7-甲氧基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮(收率： 78.5％)。MS(ESI)M/Z：315[M+H +]。 See General Synthesis Step C for details. 5-hydroxy-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxine-4-one (60.05 g, 268 mmol), N , N-dimethylformamide (780 ml), anhydrous potassium carbonate (38.91, 281 mmol), benzyl bromide (48.05 g, 281 mmol), water (200 ml), ethyl acetate (500 ml × 3 times), saturated saline solution (100 ml × 3 times). Yield 66.07 g of white solid 5-(benzyloxy)-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxine-4-one ( Yield: 78.5%). MS (ESI) M / Z: 315 [M+H + ].
步骤D：合成2-(苄氧基)-6-羟基-4-甲氧基苯甲醛Step D: Synthesis of 2-(benzyloxy)-6-hydroxy-4-methoxybenzaldehyde
Figure PCTCN2019086557-appb-000039
Figure PCTCN2019086557-appb-000039
详见通用合成步骤D。5-(苄氧基)-7-甲氧基-2,2-二甲基-4H-苯并[d][1,3]二氧杂环己烯-4-酮(35.00克，111毫摩尔)，二氯甲烷(525毫升)，二异丁基氢化铝(222毫升，333毫摩尔，1.5摩尔/升)，甲醇(30毫升)，稀盐酸溶液(35毫升，6.0摩尔/升)，二氯甲烷(500毫升×3次)，饱和食盐水(500毫升×3次)。得到17.14克棕色固体2-(苄氧基)-6-羟基-4-甲氧基苯甲醛(收率：59.9％)。MS(ESI)M/Z：259[M+H +]。 See General Synthesis Step D for details. 5-(Benzyloxy)-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxine-4-one (35.00 g, 111 m Mole), dichloromethane (525 ml), diisobutylaluminum hydride (222 ml, 333 mmol, 1.5 mol/l), methanol (30 ml), dilute hydrochloric acid (35 ml, 6.0 mol/l), Dichloromethane (500 ml × 3 times), saturated brine (500 ml × 3 times). 17.14 g of a brown solid 2-(benzyloxy)-6-hydroxy-4-methoxybenzaldehyde (yield: 59.9%) was obtained. MS (ESI) M / Z: 259 [M+H + ].
步骤E：合成2-(3-(苄氧基)-2-甲酰基-5-甲氧基苯氧基)乙酸乙酯Step E: Synthesis of ethyl 2-(3-(benzyloxy)-2-formyl-5-methoxyphenoxy)acetate
Figure PCTCN2019086557-appb-000040
Figure PCTCN2019086557-appb-000040
详见通用合成步骤E。2-(苄氧基)-6-羟基-4-甲氧基苯甲醛(17.14克，66.43毫摩尔)，2-溴乙酸乙酯(13.30克，79.63毫摩尔)，无水碳酸钾(27.47克，199毫摩尔)，乙腈(514毫升)，乙酸乙酯(50毫升×3次)。得到19.02毫克黄色油状物2-(3-(苄氧基)-2-甲酰基-5-甲氧基苯氧基)乙酸乙酯。无需纯化，直接用于下步反应。MS(ESI)M/Z：345[M+H +]。 See General Synthesis Step E for details. 2-(Benzyloxy)-6-hydroxy-4-methoxybenzaldehyde (17.14 g, 66.43 mmol), ethyl 2-bromoacetate (13.30 g, 79.63 mmol), anhydrous potassium carbonate (27.47 g) , 199 mmol, acetonitrile (514 ml), ethyl acetate (50 ml x 3 times). There was obtained 19.02 mg of ethyl 2-(3-(benzyloxy)-2-formyl-5-methoxyphenoxy)acetate as a yellow oil. It is used directly in the next step without purification. MS (ESI) M / Z: 345 [M+H + ].
步骤F：合成2-(3-(苄氧基)-2-甲酰基-5-甲氧基苯氧基)乙酸Step F: Synthesis of 2-(3-(benzyloxy)-2-formyl-5-methoxyphenoxy)acetic acid
Figure PCTCN2019086557-appb-000041
Figure PCTCN2019086557-appb-000041
将2-(3-(苄氧基)-2-甲酰基-5-甲氧基苯氧基)乙酸乙酯(19.02克，55.2毫摩尔)溶于四氢呋喃(95毫升)与水(38毫升)的混合溶液中。随后，向上述溶液中加入氢氧化钠(8.84克，221毫摩尔)。在室温下搅拌30分钟。Ethyl 2-(3-(benzyloxy)-2-formyl-5-methoxyphenoxy)acetate (19.02 g, 55.2 mmol) was dissolved in tetrahydrofuran (95 ml) and water (38 ml) In a mixed solution. Subsequently, sodium hydroxide (8.84 g, 221 mmol) was added to the above solution. Stir at room temperature for 30 minutes.
将反应液在减压下浓缩。所得残余物溶于水(50毫升)中，向上述溶液中缓慢滴加稀盐酸(1.0摩尔/升)调节PH值约为2，有白色固体析出。过滤，收集滤饼，滤饼烘干得到14.52克白色固体2-(3-(苄氧基)-2-甲酰基-5-甲氧基苯氧基)乙酸。无需纯化，直接用于下步反应。MS(ESI)M/Z：317[M+H +]。 The reaction solution was concentrated under reduced pressure. The obtained residue was dissolved in water (50 ml), and dilute hydrochloric acid (1.0 mol/liter) was slowly added dropwise to the above solution to adjust the pH to about 2, and a white solid was precipitated. Filtration, collection of the filter cake, and drying of the filter cake gave 14.52 g of white solid 2-(3-(benzyloxy)-2-formyl-5-methoxyphenoxy) acetic acid. It is used directly in the next step without purification. MS (ESI) M/Z: 381 [M+H + ].
步骤G：合成4-(苄氧基)-6-甲氧基苯并呋喃Step G: Synthesis of 4-(benzyloxy)-6-methoxybenzofuran
Figure PCTCN2019086557-appb-000042
Figure PCTCN2019086557-appb-000042
将2-(3-(苄氧基)-2-甲酰基-5-甲氧基苯氧基)乙酸(14.52克，45.95毫摩尔)溶于乙酸酐(145毫升)中。随后，向上述溶液中加入醋酸钠(38.8克，473毫摩尔)。将反应液加热至140摄氏度，并搅拌1小时。2-(3-(Benzyloxy)-2-formyl-5-methoxyphenoxy)acetic acid (14.52 g, 45.95 mmol) was dissolved in EtOAc (EtOAc) Subsequently, sodium acetate (38.8 g, 473 mmol) was added to the above solution. The reaction solution was heated to 140 ° C and stirred for 1 hour.
将反应液冷却到室温，过滤，滤饼用乙酸乙酯(50毫升×3次)，滤液在减压下浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/20)。得到9.50克黄色固体4-(苄氧基)-6-甲氧基苯并呋喃(收率：81.4％)。MS(ESI)M/Z：255[M+H +]。 The reaction solution was cooled to room temperature, filtered, and then filtered and evaporated. The residue obtained was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1 / 20). 9.50 g of a yellow solid 4-(benzyloxy)-6-methoxybenzofuran was obtained (yield: 81.4%). MS (ESI) M / Z: 495 [M+H + ].
步骤H：合成2-(4-(苄氧基)-6-甲氧基苯并呋喃-2-基)-4,4,5,5-四甲基-1,3,2-二氧杂硼烷Step H: Synthesis of 2-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxa Borane
Figure PCTCN2019086557-appb-000043
Figure PCTCN2019086557-appb-000043
在氮气保护下，将4-(苄氧基)-6-甲氧基苯并呋喃(2.50克，9.84毫摩尔)和4,4,4’，4’，5,5,5’，5’-八甲基-2,2’-二(1,3,2-二氧杂硼烷)(2.75克，10.82毫摩尔)溶于正己烷(55.0毫升)中。随后，向上述溶液中加入[Ir(COD)(OMe)] 2(100毫克，0.15毫摩尔),dtbpy(79毫克，0.29毫摩尔)。将反应液加热至60摄氏度，并搅拌1小时。 4-(Benzyloxy)-6-methoxybenzofuran (2.50 g, 9.84 mmol) and 4,4,4',4',5,5,5',5' under nitrogen. Octamethyl-2,2'-bis(1,3,2-dioxaborane) (2.75 g, 10.82 mmol) was dissolved in n-hexane (55.0 mL). Subsequently, [Ir(COD)(OMe)] 2 (100 mg, 0.15 mmol), dtbpy (79 mg, 0.29 mmol) was added to the above solution. The reaction solution was heated to 60 ° C and stirred for 1 hour.
将反应液冷却到室温，向反应液中加入水(20毫升)淬灭反应。混合液用乙酸乙酯(50毫升×3次)萃取。合并有机相，有机相先用饱和食盐水(50毫升×3次)反洗，然后用无水硫酸钠干燥，最后减压浓缩。将所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/10)。得到3.50克黄色油状物2-(4-(苄氧基)-6-甲氧基苯并呋喃-2-基)-4,4,5,5-四甲基-1,3,2-二氧杂硼烷(收率：93.6％)。MS(ESI)M/Z：381[M+H +]。 The reaction solution was cooled to room temperature, and water (20 ml) was added to the reaction mixture to quench the reaction. The mixture was extracted with ethyl acetate (50 mL×3×). The combined organic layers were washed with brine (50 mL×3×) and then dried over anhydrous sodium sulfate. The residue was purified by silica gel column chromatography (EtOAc:EtOAc Yield 3.50 g of a yellow oil as 2-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-4,4,5,5-tetramethyl-1,3,2-di Oxaborane (yield: 93.6%). MS (ESI) M / Z: 381 [M+H + ].
步骤I：合成2-(4-(苄氧基)-6-甲氧基苯并呋喃-2-基)-5-甲氧基咪唑并[5,4-d]噻唑Step I: Synthesis of 2-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000044
Figure PCTCN2019086557-appb-000044
在氮气保护下，将2-溴-5-甲氧基咪唑并[5,4-d]噻唑(330毫克，1.31毫摩尔)和2-(4-(苄氧基)-6-甲氧基苯并呋喃-2-基)-4,4,5,5-四甲基-1,3,2-二氧杂硼烷(765毫克，2.01毫摩尔)溶于1,4-二氧六环(20毫升)中。随后向上述溶液中加入无水碳酸钾(361毫克，2.62毫摩尔)，四三苯基膦钯(132毫克，0.11毫摩尔)和水(2毫升)。将反应液加热至80摄氏度，并搅拌1小时。2-Bromo-5-methoxyimidazo[5,4-d]thiazole (330 mg, 1.31 mmol) and 2-(4-(benzyloxy)-6-methoxyl under N2 Benzofuran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (765 mg, 2.01 mmol) is dissolved in 1,4-dioxane (20 ml). Anhydrous potassium carbonate (361 mg, 2.62 mmol), tetrakistriphenylphosphine palladium (132 mg, 0.11 mmol) and water (2 mL) were then added to the solution. The reaction solution was heated to 80 ° C and stirred for 1 hour.
将反应液冷却到室温。混合液用乙酸乙酯(20毫升×3次)萃取。合并有机相，有机相先用饱和食盐水(20毫升×3次)反洗，然后用无水硫酸钠干燥，最后减压浓缩。将所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/10)。得到400毫克白色固体2-(4-(苄氧基)-6-甲氧基苯并呋喃-2-基)-5-甲氧基咪唑并[5,4-d]噻唑(收率：72.0％)。MS(ESI)M/Z：425[M+H +]。 The reaction solution was cooled to room temperature. The mixture was extracted with ethyl acetate (20 mL×3×). The combined organic layers were washed with brine (20 mL×3×) and then dried over anhydrous sodium sulfate. The residue was purified by silica gel column chromatography (EtOAc:EtOAc Yield 400 mg of white 2-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole (yield: 72.0) %). MS (ESI) M/Z: 425 [M+H + ].
步骤J：合成6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇Step J: Synthesis of 6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol
Figure PCTCN2019086557-appb-000045
Figure PCTCN2019086557-appb-000045
在氮气保护下，2-(4-(苄氧基)-6-甲氧基苯并呋喃-2-基)-5-甲氧基咪唑并[5,4-d]噻唑(425毫克，1.0毫摩尔)和五甲基苯(1.04克，7.0毫摩尔)溶于二氯甲烷(80毫升)中。在-78摄氏度下向上述溶液中缓慢滴加三氯化硼(2.5毫升，2.5毫摩尔，1.0摩尔/升)。在-78摄氏度下搅拌1小时。向反应体系中加入甲醇(10毫升)淬灭反应。将反应液升至室温，并减压浓缩。粗产品用反相柱纯化[反相柱:C18；流动相A：水(含有0.05％甲酸)，流动相B：乙腈；梯度:30％乙腈到90％乙腈在8分钟内；检测波长：254nm]收集馏分，直接低温冻干。得到130毫克黄色固体6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇(收率：38.9％)。MS(ESI)M/Z：335[M+H +]。 1H NMR(300MHz,DMSO-d 6,ppm)δ10.4(s,1H),7.48(s,1H),6.80(s,1H),6.32(s,1H),4.20(s,3H),3.78(s,3H)。 2-(4-(Benzyloxy)-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole (425 mg, 1.0 under nitrogen) Millimol) and pentamethylbenzene (1.04 g, 7.0 mmol) were dissolved in dichloromethane (80 mL). Boron trichloride (2.5 ml, 2.5 mmol, 1.0 mol/l) was slowly added dropwise to the above solution at -78 °C. Stir at -78 degrees Celsius for 1 hour. The reaction was quenched by the addition of methanol (10 mL). The reaction solution was warmed to room temperature and concentrated. The crude product was purified by reverse phase column [reverse phase column: C18; mobile phase A: water (containing 0.05% formic acid), mobile phase B: acetonitrile; gradient: 30% acetonitrile to 90% acetonitrile in 8 min; detection wavelength: 254 nm The fractions were collected and directly lyophilized. There was obtained 130 mg of a yellow solid 6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol (yield: 38.9%). MS (ESI) M/Z: 335 [M+H + ]. 1 H NMR (300MHz, DMSO- d 6, ppm) δ10.4 (s, 1H), 7.48 (s, 1H), 6.80 (s, 1H), 6.32 (s, 1H), 4.20 (s, 3H), 3.78 (s, 3H).
实施例4：合成4-(6-(((6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)甲基)吡啶-2-基)四氢-2H-吡喃-4-醇Example 4: Synthesis of 4-(6-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy) )methyl)pyridin-2-yl)tetrahydro-2H-pyran-4-ol
Figure PCTCN2019086557-appb-000046
Figure PCTCN2019086557-appb-000046
步骤A：合成2-溴-6-((叔丁基二甲硅氧基)甲基)吡啶Step A: Synthesis of 2-bromo-6-((tert-butyldimethylsilyloxy)methyl)pyridine
Figure PCTCN2019086557-appb-000047
Figure PCTCN2019086557-appb-000047
将(6-溴吡啶-2-基)甲醇(1.88克，10.0毫摩尔),4-二甲氨基吡啶(122毫克，1.00毫摩尔)和1H-咪唑(3.40克，50.0毫摩尔)溶于四氢呋喃(60.0毫升)中。随后,向上述溶液中缓慢滴加叔丁基二甲基氯硅烷(7.55克，50.0毫摩尔)的四氢呋喃溶液(10.0毫升)。在室温下搅拌6小时。(6-Bromopyridin-2-yl)methanol (1.88 g, 10.0 mmol), 4-dimethylaminopyridine (122 mg, 1.00 mmol) and 1H-imidazole (3.40 g, 50.0 mmol) were dissolved in tetrahydrofuran (60.0 ml). Subsequently, a solution of t-butyldimethylsilyl chloride (7.55 g, 50.0 mmol) in tetrahydrofuran (10.0 ml) was slowly added dropwise to the above solution. Stir at room temperature for 6 hours.
向反应液中加入饱和碳酸氢钠溶液(50.0毫升)淬灭反应。混合液用乙酸乙酯(50毫升×3次)萃取。合并有机相，有机相先用饱和食盐水(50毫升×3次)洗涤，然后用无水硫酸钠干燥，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂:石油醚)。得到2.98克无色油状物2-溴-6-((叔丁基二甲硅氧基)甲基)吡啶(2.98克，98.7％)。MS(ESI)M/Z：302,304[M+H +]。 A saturated sodium bicarbonate solution (50.0 ml) was added to the reaction mixture to quench. The mixture was extracted with ethyl acetate (50 mL×3×). The combined organic layers were washed with brine (50 mL×3×) and dried over anhydrous sodium sulfate. The residue obtained was purified by silica gel column chromatography (eluent: petroleum ether). 2.98 g of 2-bromo-6-((tert-butyldimethylsilyloxy)methyl)pyridine (2.98 g, 98.7%) was obtained as a colourless oil. MS (ESI) M/Z: 302,304 [M+H + ].
步骤B：合成4-(6-((叔丁基二甲硅氧基)甲基)吡啶-2-基)-四氢-2H-吡喃-4-醇Step B: Synthesis of 4-(6-((tert-butyldimethylsilyloxy)methyl)pyridin-2-yl)-tetrahydro-2H-pyran-4-ol
Figure PCTCN2019086557-appb-000048
Figure PCTCN2019086557-appb-000048
将2-溴-6-((叔丁基二甲硅氧基)甲基)吡啶(2.98克，9.87毫摩尔)和四氢吡喃-4-酮(1.18克，11.8毫摩尔)溶于四氢呋喃(50毫升)中。在-78摄氏度下，向上述溶液中缓慢滴加正丁基锂的四氢呋喃溶液(12.0毫升，12.0毫摩尔，1.0摩尔/升)。在-78摄氏度下搅拌1小时。2-Bromo-6-((tert-butyldimethylsilyloxy)methyl)pyridine (2.98 g, 9.87 mmol) and tetrahydropyran-4-one (1.18 g, 11.8 mmol) were dissolved in tetrahydrofuran (50 ml). A solution of n-butyllithium in tetrahydrofuran (12.0 ml, 12.0 mmol, 1.0 mol/liter) was slowly added dropwise to the above solution at -78 °C. Stir at -78 degrees Celsius for 1 hour.
向反应液中加入氯化铵水溶液(20毫升)淬灭反应，混合液用乙酸乙酯(50毫升×3)萃取，合并有机相。有机相先用饱和食盐水(50毫升×3次)反洗，然后用无水硫酸钠干燥，最后真空浓缩至干。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚60％-90％)。得到1.20克黄色油状物4-(6-((叔丁基二甲硅氧基)甲基)吡啶-2-基)-四氢-2H-吡喃-4-醇(收率：37.7％)。MS(ESI)M/Z：324[M+H +]。 The reaction mixture was quenched with EtOAc EtOAc (EtOAc) The organic phase was backwashed with saturated brine (50 mL×3×) then dried over anhydrous sodium sulfate. The residue obtained was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether 60% - 90%). 1.20 g of a yellow oil 4-(6-((tert-butyldimethylsilyloxy)methyl)pyridin-2-yl)-tetrahydro-2H-pyran-4-ol (yield: 37.7%) . MS (ESI) M / Z: 324 [M+H + ].
步骤C：合成4-(6-(羟基甲基)吡啶-2-基)-四氢-2H-吡喃-4-醇Step C: Synthesis of 4-(6-(hydroxymethyl)pyridin-2-yl)-tetrahydro-2H-pyran-4-ol
Figure PCTCN2019086557-appb-000049
Figure PCTCN2019086557-appb-000049
将4-(6-((叔丁基二甲硅氧基)甲基)吡啶-2-基)-四氢-2H-吡喃-4-醇(1.20克，3.72毫摩尔)溶于四氢呋喃(20.0毫升)中。随后，向上述溶液中滴加四丁基氟化铵(1.16克，4.44毫摩尔)的四氢呋喃溶液(5毫升)。在室温下搅拌1小时。4-(6-((tert-Butyldimethylsilyloxy)methyl)pyridin-2-yl)-tetrahydro-2H-pyran-4-ol (1.20 g, 3.72 mmol) was dissolved in tetrahydrofuran ( 20.0 ml). Subsequently, a solution of tetrabutylammonium fluoride (1.16 g, 4.44 mmol) in tetrahydrofuran (5 ml) was added dropwise to the solution. Stir at room temperature for 1 hour.
向反应液中加入饱和氯化铵水溶液(5毫升)淬灭反应。混合液用乙酸乙酯(20毫升×3次)萃取，合并有机相。得到的有机相先用饱和食盐水(20毫升×3次)反洗，然后用无水硫酸钠干燥，最后 减压浓缩。粗产品用反相柱纯化[反相柱:C18；流动相A：水(含有0.05％甲酸)，流动相B：乙腈；梯度:30％乙腈到90％乙腈在8分钟内；检测波长：254nm]，低温冻干得到600毫克无色油状物4-(6-(羟基甲基)吡啶-2-基)-四氢-2H-吡喃-4-醇(收率：77.2％)。MS(ESI)M/Z：210[M+H +]。 A saturated aqueous ammonium chloride solution (5 ml) was added to the mixture and the mixture was evaporated. The mixture was extracted with ethyl acetate (20 mL×3×) and the organic phases were combined. The obtained organic phase was back-washed with saturated brine (20 ml×3×), then dried over anhydrous sodium sulfate and evaporated. The crude product was purified by reverse phase column [reverse phase column: C18; mobile phase A: water (containing 0.05% formic acid), mobile phase B: acetonitrile; gradient: 30% acetonitrile to 90% acetonitrile in 8 min; detection wavelength: 254 nm ], lyophilized to give 600 mg of 4-(6-(hydroxymethyl)pyridin-2-yl)-tetrahydro-2H-pyran-4-ol as a colorless oil (yield: 77.2%). MS (ESI) M / Z: 210 [M+H + ].
步骤D：合成4-(6-(((6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)甲基)吡啶-2-基)四氢-2H-吡喃-4-醇Step D: Synthesis of 4-(6-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy) Methyl)pyridin-2-yl)tetrahydro-2H-pyran-4-ol
Figure PCTCN2019086557-appb-000050
详见通用合成步骤K。6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇(22毫克，0.065毫摩尔)，4-(6-(羟基甲基)吡啶-2-基)-四氢-2H-吡喃-4-醇(26毫克，0.124毫摩尔)，四氢呋喃(1毫升)，三丁基膦(60毫克，0.297毫摩尔)，偶氮二甲酰二哌啶(75毫克，0.285毫摩尔)，水(1毫升)，偶氮二甲酰二哌啶(74毫克，0.293毫摩尔)，乙酸乙酯(5毫升×3次)，饱和食盐水(10毫升×3次)。得到2.3毫克4-(6-(((6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)甲基)吡啶-2-基)四氢-2H-吡喃-4-醇(收率：6.7％)。MS(ESI)M/Z：526[M+H +]。 1H NMR(300MHz,CDCl 3，ppm)δ7.89(t,J＝7.8Hz,1H),7.61(t,J＝6.0Hz,1H)，7.44(s,1H),7.40(d,J＝8.1Hz,1H),6.76(s,1H),6.44(s,1H),5.40(s,2H),4.23(s,3H),4.09-3.95(m,4H),3.87(s,3H),2.23-2.13(m,2H),1.66(d,J＝13.2Hz,2H)。
Figure PCTCN2019086557-appb-000050
See General Synthesis Step K for details. 6-Methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol (22 mg, 0.065 mmol), 4-(6-(hydroxyl) Methyl)pyridin-2-yl)-tetrahydro-2H-pyran-4-ol (26 mg, 0.124 mmol), tetrahydrofuran (1 ml), tributylphosphine (60 mg, 0.297 mmol), Nitrodiyl dipiperidine (75 mg, 0.285 mmol), water (1 ml), azodiyldipiperidine (74 mg, 0.293 mmol), ethyl acetate (5 mL x 3) Saturated saline solution (10 ml × 3 times). Yield 2.3 mg of 4-(6-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy)) Pyridin-2-yl)tetrahydro-2H-pyran-4-ol (yield: 6.7%). MS (ESI) M/Z: 564 [M+H + ]. 1 H NMR (300 MHz, CDCl 3 , ppm) δ 7.89 (t, J = 7.8 Hz, 1H), 7.61 (t, J = 6.0 Hz, 1H), 7.44 (s, 1H), 7.40 (d, J = 8.1 Hz, 1H), 6.76 (s, 1H), 6.44 (s, 1H), 5.40 (s, 2H), 4.23 (s, 3H), 4.09-3.95 (m, 4H), 3.87 (s, 3H), 2.23-2.13 (m, 2H), 1.66 (d, J = 13.2 Hz, 2H).
实施例5：合成4,6-二甲氧基-2-[5-甲氧基-[1,3]噻唑并[5,4-d][1,3]噻唑-2-基]吡唑并[1,5-a]吡啶Example 5: Synthesis of 4,6-dimethoxy-2-[5-methoxy-[1,3]thiazolo[5,4-d][1,3]thiazol-2-yl]pyrazole And [1,5-a]pyridine
Figure PCTCN2019086557-appb-000051
Figure PCTCN2019086557-appb-000051
步骤A：合成2-溴-3,5-二甲氧基吡啶Step A: Synthesis of 2-bromo-3,5-dimethoxypyridine
Figure PCTCN2019086557-appb-000052
Figure PCTCN2019086557-appb-000052
将3,5-二甲氧基吡啶(1.00克,7.2毫摩尔)溶于乙腈溶液(30.0毫升)中。随后，向上述溶液加入N-溴代琥珀酰亚胺(1.30克，7.2毫摩尔)。将反应液加热至80摄氏度，并搅拌1小时。3,5-Dimethoxypyridine (1.00 g, 7.2 mmol) was dissolved in acetonitrile (30.0 mL). Subsequently, N-bromosuccinimide (1.30 g, 7.2 mmol) was added to the above solution. The reaction solution was heated to 80 ° C and stirred for 1 hour.
将反应液冷却到室温，并减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/10)。得到800毫克黄色固体2-溴-3,5-二甲氧基吡啶(收率：51.0％)。MS(ESI)M/Z：218,220[M+H +]。 The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1/10). There was obtained 800 mg of a yellow solid 2-bromo-3,5-dimethoxypyridine (yield: 51.0%). MS (ESI) M/Z: 218, 220 [M+H + ].
步骤B：合成2-乙炔基-3,5-二甲氧基吡啶Step B: Synthesis of 2-ethynyl-3,5-dimethoxypyridine
Figure PCTCN2019086557-appb-000053
Figure PCTCN2019086557-appb-000053
在室温和氮气气氛下，将2-溴-3,5-二甲氧基吡啶(800毫克,3.7毫摩尔)溶于乙腈(14.0毫升)中。随后，向上述溶液依次加入三甲基硅基乙炔(362毫克,3.7毫摩尔),二(三苯基膦)二氯化钯(53毫克，0.075毫摩尔)，碘化亚铜(14毫克，0.07毫摩尔)，三苯基膦(39毫克，0.15毫摩尔)和N,N-二异丙基乙胺((1.10克，8.59毫摩尔)。在室温下搅拌4小时。向反应液中加入无水碳酸钾(3.60克，26.0毫摩尔)和甲醇(20.0毫升)。在室温下继续搅拌1小时。2-Bromo-3,5-dimethoxypyridine (800 mg, 3.7 mmol) was dissolved in acetonitrile (14.0 mL). Subsequently, trimethylsilylacetylene (362 mg, 3.7 mmol), bis(triphenylphosphine)palladium dichloride (53 mg, 0.075 mmol), cuprous iodide (14 mg, 0.07 mmol, triphenylphosphine (39 mg, 0.15 mmol) and N,N-diisopropylethylamine ((1.10 g, 8.59 mmol). stirred at room temperature for 4 hours. Anhydrous potassium carbonate (3.60 g, 26.0 mmol) and methanol (20.0 mL) were then evaporated.
将反应液在减压下浓缩。所得残余物溶于二氯甲烷(50毫升)与水(50毫升)的混合溶液中。混合液用二氯甲烷(50毫升×3次)萃取，合并有机相。有机相先用饱和食盐水(50毫升×3次)洗涤，然后用无水硫酸钠干燥，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝2/8)。得到130毫克2-乙炔基-3,5-二甲氧基吡啶(收率：21.6％)。MS(ESI)M/Z:164[M+H +]。 The reaction solution was concentrated under reduced pressure. The residue obtained was dissolved in a mixture of dichloromethane (50 ml) and water (50 ml). The mixture was extracted with dichloromethane (50 mL×3×) and the organic phases were combined. The organic phase was washed with saturated brine (50 mL×3×) then dried over anhydrous sodium sulfate. The obtained residue was purified to silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 2/8). 130 mg of 2-ethynyl-3,5-dimethoxypyridine were obtained (yield: 21.6%). MS (ESI) M / Z: 164 [M+H + ].
步骤C：合成3,5-二甲氧基-2-(2-[5-甲氧基-[1,3]噻唑并[5,4-d][1,3]噻唑-2-基]乙炔基)吡啶Step C: Synthesis of 3,5-dimethoxy-2-(2-[5-methoxy-[1,3]thiazolo[5,4-d][1,3]thiazol-2-yl] Ethynyl)pyridine
Figure PCTCN2019086557-appb-000054
Figure PCTCN2019086557-appb-000054
在室温和氮气保护下，将2-乙炔基-3,5-二甲氧基吡啶(130毫克,0.8毫摩尔)溶于乙腈(10.0毫升)中。随后，向上述溶液中加入2-溴-5-甲氧基-[1,3]噻唑并[5,4-d][1,3]噻唑(240毫克，0.96毫摩尔),二(三苯基膦)二氯化钯(28毫克，0.04毫摩尔)，碘化亚铜(6毫克，0.03毫摩尔)，三苯基膦(21毫克，0.08毫摩尔)和N,N-二异丙基乙胺((237毫克，1.84毫摩尔)。在室温下搅拌4小时。2-Ethynyl-3,5-dimethoxypyridine (130 mg, 0.8 mmol) was dissolved in acetonitrile (10.0 mL). Subsequently, 2-bromo-5-methoxy-[1,3]thiazolo[5,4-d][1,3]thiazole (240 mg, 0.96 mmol), bis(triphenyl) was added to the above solution. Palladium dichloride (28 mg, 0.04 mmol), cuprous iodide (6 mg, 0.03 mmol), triphenylphosphine (21 mg, 0.08 mmol) and N,N-diisopropyl Ethylamine ((237 mg, 1.84 mmol) was stirred at room temperature for 4 hours.
将反应液在减压下浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝2/8)。得到125毫克白色固体3,5-二甲氧基-2-(2-[5-甲氧基-[1,3]噻唑并[5,4-d][1,3]噻唑-2-基]乙炔基)吡啶(收率：47.0％)。MS(ESI)M/Z:334[M+H +]。 The reaction solution was concentrated under reduced pressure. The obtained residue was purified to silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 2/8). Obtained 125 mg of white solid 3,5-dimethoxy-2-(2-[5-methoxy-[1,3]thiazolo[5,4-d][1,3]thiazol-2-yl Acetylene)pyridine (yield: 47.0%). MS (ESI) M / Z: 334 [M+H + ].
步骤D：合成4,6-二甲氧基-2-[5-甲氧基-[1,3]噻唑并[5,4-d][1,3]噻唑-2-基]吡唑并[1,5-a]吡啶Step D: Synthesis of 4,6-dimethoxy-2-[5-methoxy-[1,3]thiazolo[5,4-d][1,3]thiazol-2-yl]pyrazole [1,5-a]pyridine
Figure PCTCN2019086557-appb-000055
将3,5-二甲氧基-2-(2-[5-甲氧基-[1,3]噻唑并[5,4-d][1,3]噻唑-2-基]乙炔基)吡啶(100毫克，0.30毫摩尔)溶于二氯甲烷(10.0毫升)中。冰水浴下，向上述溶液中缓慢滴加2-[(氨基氧基)磺酰]-1,3,5-三甲基苯(71毫克，0.33毫摩尔)的二氯甲烷(80.0毫升)溶液。将反应液升至室温，并搅拌过夜。
Figure PCTCN2019086557-appb-000055
3,5-Dimethoxy-2-(2-[5-methoxy-[1,3]thiazolo[5,4-d][1,3]thiazol-2-yl]ethynyl) Pyridine (100 mg, 0.30 mmol) was dissolved in dichloromethane (10.0 mL). A solution of 2-[(aminooxy)sulfonyl]-1,3,5-trimethylbenzene (71 mg, 0.33 mmol) in dichloromethane (80.0 mL) was slowly added dropwise to the above solution. . The reaction was allowed to warm to room temperature and stirred overnight.
将反应液在减压下浓缩。将所得残余物的N,N-二甲基甲酰胺溶液(20.0毫升)溶液缓慢滴加到无水碳酸钾(36毫克，0.26毫摩尔)的N,N-二甲基甲酰胺(1.4升)溶液中。在室温下搅拌过夜。The reaction solution was concentrated under reduced pressure. A solution of the obtained residue in N,N-dimethylformamide (20.0 mL) In solution. Stir at room temperature overnight.
将反应液在减压下浓缩除去N,N-二甲基甲酰胺。所得残余物用硅胶柱层析纯化(洗脱剂：石油醚/乙酸乙酯＝1/1)。得到5.3毫克白色固体4,6-二甲氧基-2-[5-甲氧基-[1,3]噻唑并[5,4-d][1,3]噻唑-2-基]吡唑并[1,5-a]吡啶(收率：8.35％)。MS(ESI)M/Z:334[M+H +]。 1H-NMR(300MHz,CDCl 3,ppm)δ7.78(s,1H),7.12(s,1H),6.27(d,J＝1.8Hz,1H),4.22(s,3H),3.98(s,3H),3.86(s,3H)。 The reaction solution was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue obtained was purified by silica gel column chromatography (EtOAc:EtOAc 5.3 mg of white solid 4,6-dimethoxy-2-[5-methoxy-[1,3]thiazolo[5,4-d][1,3]thiazol-2-yl]pyrazole And [1,5-a]pyridine (yield: 8.35%). MS (ESI) M / Z: 334 [M+H + ]. 1 H-NMR (300MHz, CDCl 3 , ppm) δ 7.78 (s, 1H), 7.12 (s, 1H), 6.27 (d, J = 1.8 Hz, 1H), 4.22 (s, 3H), 3.98 (s) , 3H), 3.86 (s, 3H).
实施例6：合成4-(4-(((6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)甲基)-5-甲基噻唑-2-基)吗啉Example 6: Synthesis of 4-(4-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy) )methyl)-5-methylthiazol-2-yl)morpholine
Figure PCTCN2019086557-appb-000056
Figure PCTCN2019086557-appb-000056
步骤A：合成4-(4-(((6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)甲基)-5-甲基噻唑-2-基)吗啉Step A: Synthesis of 4-(4-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy) Methyl)-5-methylthiazol-2-yl)morpholine
Figure PCTCN2019086557-appb-000057
详见通用合成步骤K。6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇(18毫克，0.054毫摩尔)，(5-甲基-2-吗啉代-4-基)甲醇(16毫克，0.07毫摩尔)，三丁基膦(200毫克，1.0毫摩尔)，偶氮二甲酰二哌啶(50毫克，0.19毫摩尔)，四氢呋喃(1.0毫升)。得到4.6毫克白色固体4-(4-(((6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)甲基)-5-甲基噻唑-2-基)吗啉(收率：16.1％)。MS(ESI)M/Z：531[M+H +]。 1H NMR(300MHz,CDCl 3,ppm)δ7.63(s,1H)，6.74(s,1H),6.62(s,1H),5.30(s,2H),4.22(s,3H)，3.89-3.87(m,7H),3.71(s,4H),2.40(s,3H)。
Figure PCTCN2019086557-appb-000057
See General Synthesis Step K for details. 6-Methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol (18 mg, 0.054 mmol), (5-methyl-2) -morpholino-4-yl)methanol (16 mg, 0.07 mmol), tributylphosphine (200 mg, 1.0 mmol), azodiyldipiperidine (50 mg, 0.19 mmol), tetrahydrofuran (1.0 ml). 4.6 mg of 4-(4-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy) )methyl)-5-methylthiazol-2-yl)morpholine (yield: 16.1%). MS (ESI) M / Z: 531 [M+H + ]. 1 H NMR (300MHz, CDCl 3 , ppm) δ7.63 (s, 1H), 6.74 (s, 1H), 6.62 (s, 1H), 5.30 (s, 2H), 4.22 (s, 3H), 3.89- 3.87 (m, 7H), 3.71 (s, 4H), 2.40 (s, 3H).
实施例7：合成2-甲氧基-5-(6-甲氧基-4-(2-甲氧基乙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑Example 7: Synthesis of 2-methoxy-5-(6-methoxy-4-(2-methoxyethoxy)benzofuran-2-yl)thiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000058
Figure PCTCN2019086557-appb-000058
步骤A：合成2-甲氧基-5-(6-甲氧基-4-(2-甲氧基乙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑Step A: Synthesis of 2-methoxy-5-(6-methoxy-4-(2-methoxyethoxy)benzofuran-2-yl)thiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000059
详见通用合成步骤L。6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇(55毫克，0.17毫摩尔)，N,N-二甲基甲酰胺(6.0毫升)，2-溴乙基甲醚(46毫克，0.33毫摩尔)，无水碳酸钾(46毫克，0.33毫摩尔)。得到32.0毫克黄色固体2-甲氧基-5-(6-甲氧基-4-(2-甲氧基乙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑(收率：48.0％)。MS(ESI)M/Z:393[M+H +]。 1H NMR(300MHz,CDCl 3,ppm)δ7.63(s,1H),6.69(s,1H),6.37(s,1H),4.26(t,J＝4.8Hz,2H),4.22(s,3H),3.88(s,3H),3.85(t,J＝4.8Hz,2H),3.51(s,3H)。
Figure PCTCN2019086557-appb-000059
See General Synthesis Step L for details. 6-Methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol (55 mg, 0.17 mmol), N,N-dimethyl Formamide (6.0 ml), 2-bromoethyl methyl ether (46 mg, 0.33 mmol), anhydrous potassium carbonate (46 mg, 0.33 mmol). 32.0 mg of yellow solid 2-methoxy-5-(6-methoxy-4-(2-methoxyethoxy)benzofuran-2-yl)thiazolo[5,4-d]thiazole was obtained. (Yield: 48.0%). MS (ESI) M / Z: 393 [M+H + ]. 1 H NMR (300MHz, CDCl 3 , ppm) δ7.63 (s, 1H), 6.69 (s, 1H), 6.37 (s, 1H), 4.26 (t, J = 4.8Hz, 2H), 4.22 (s, 3H), 3.88 (s, 3H), 3.85 (t, J = 4.8 Hz, 2H), 3.51 (s, 3H).
实施例8：合成2-甲氧基-5-(6-甲氧基-4-((5-甲基-2-(4-甲基哌嗪-1-基)噻唑-4-基)甲氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑Example 8: Synthesis of 2-methoxy-5-(6-methoxy-4-((5-methyl-2-(4-methylpiperazin-1-yl)thiazol-4-yl)-) Oxy)benzophenan-2-yl)thiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000060
Figure PCTCN2019086557-appb-000060
步骤A：合成5-甲基-2-(4-甲基哌嗪-1-基)噻唑-4-羧酸乙酯Step A: Synthesis of ethyl 5-methyl-2-(4-methylpiperazin-1-yl)thiazole-4-carboxylate
Figure PCTCN2019086557-appb-000061
Figure PCTCN2019086557-appb-000061
将2-溴-5-甲基噻唑-4-羧酸乙酯(1.00克，4.00毫摩尔)，1-甲基哌嗪(2.01克，20.1毫摩尔)和三乙胺 (2.03克，20.1毫摩尔)加入1,4-二氧六环(25.0毫升)中。将反应液加热至120摄氏度，并搅拌24小时。Ethyl 2-bromo-5-methylthiazole-4-carboxylate (1.00 g, 4.00 mmol), 1-methylpiperazine (2.01 g, 20.1 mmol) and triethylamine (2.03 g, 20.1 Mole) was added to 1,4-dioxane (25.0 ml). The reaction solution was heated to 120 ° C and stirred for 24 hours.
将反应液冷却至室温。向反应液中加入水(25.0毫升)淬灭反应。混合液用乙酸乙酯(25毫升×3次)萃取。合并有机相，有机相先用饱和食盐水(25毫升×3次)洗涤，然后用无水硫酸钠干燥，最后减压浓缩。残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝3/2)。得到1.06克黄色固体5-甲基-2-(4-甲基哌嗪-1-基)噻唑-4-羧酸乙酯(收率：98.1％)。MS(ESI)M/Z:270[M+H +]。 The reaction solution was cooled to room temperature. Water (25.0 ml) was added to the reaction mixture to quench the reaction. The mixture was extracted with ethyl acetate (25 mL×3×). The combined organic layers were washed with brine (25 mL×3×) then dried over anhydrous sodium sulfate. The residue was purified with EtOAc EtOAc EtOAcEtOAc 1.06 g of ethyl 5-methyl-2-(4-methylpiperazin-1-yl)thiazole-4-carboxylate as a yellow solid was obtained (yield: 98.1%). MS (ESI) M/Z: 270 [M+H + ].
步骤B：合成(5-甲基-2-(4-甲基哌嗪-1-基)噻唑-4-基)甲醇Step B: Synthesis of (5-methyl-2-(4-methylpiperazin-1-yl)thiazol-4-yl)methanol
Figure PCTCN2019086557-appb-000062
Figure PCTCN2019086557-appb-000062
冰水浴下，将5-甲基-2-(4-甲基哌嗪-1-基)噻唑-4-羧酸乙酯(1.06克,3.94毫摩尔)溶于四氢呋喃溶液(10.0毫升)中。随后，向上述溶液中分批次加入四氢锂铝(448毫克，11.8毫摩尔)。在常温下搅拌1小时。Ethyl 5-methyl-2-(4-methylpiperazin-1-yl)thiazole-4-carboxylate (1.06 g, 3.94 mmol) was dissolved in tetrahydrofurane (10.0 mL). Subsequently, lithium aluminum hydride (448 mg, 11.8 mmol) was added in portions to the above solution. Stir at room temperature for 1 hour.
向反应液中缓慢加入饱和氯化铵溶液(5.0毫升)淬灭反应。混合液用乙酸乙酯(10毫升×3次)萃取。合并有机相，有机相先用饱和食盐水(5毫升×3次)洗涤，然后用无水硫酸钠干燥，最后减压浓缩。粗产品用反相柱纯化[反相柱:C18；流动相A：水(含有0.05％的甲酸)，流动相B：乙腈；梯度:30％乙腈到90％乙腈在8分钟内；检测波长：254nm]浓缩得650毫克黄色油状物(5-甲基-2-(4-甲基哌嗪-1-基)噻唑-4-基)甲醇(收率：72.7％)。MS(ESI)M/Z:228[M+H +]。 A saturated ammonium chloride solution (5.0 ml) was slowly added to the reaction mixture to quench the reaction. The mixture was extracted with ethyl acetate (10 mL×3×). The combined organic layers were washed with brine (5 mL×3×) and dried over anhydrous sodium sulfate. The crude product was purified by reverse phase column [reverse phase column: C18; mobile phase A: water (containing 0.05% formic acid), mobile phase B: acetonitrile; gradient: 30% acetonitrile to 90% acetonitrile in 8 min; detection wavelength: 254 nm] was concentrated to give 650 mg (yield:yield: 72.7%) as a yellow oil (5-methyl-2-(4-methylpiperazin-1-yl)thiazol-4-yl)methanol. MS (ESI) M / Z: 228 [M+H + ].
步骤C：合成2-甲氧基-5-(6-甲氧基-4-((5-甲基-2-(4-甲基哌嗪-1-基)噻唑-4-基)甲氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑Step C: Synthesis of 2-methoxy-5-(6-methoxy-4-((5-methyl-2-(4-methylpiperazin-1-yl)thiazol-4-yl)methoxy) Benzofuran-2-yl)thiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000063
详见通用合成步骤K。在室温和氮气保护下，向四氢呋喃溶液(8.0毫升)中依次加入偶氮二甲酰二哌啶(363毫克，1.44毫摩尔)和三丁基膦(454毫克，2.25毫摩尔)。在室温下搅拌20分钟。随后，向上述溶液中缓慢滴加6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇(50毫克，0.15毫摩尔)和(5-甲基-2-(4-甲基哌嗪-1-基)噻唑-4-基)甲醇(68毫克，0.30毫摩尔)的四氢呋喃溶液(2.0毫升)。在室温下搅拌3小时。
Figure PCTCN2019086557-appb-000063
See General Synthesis Step K for details. To a tetrahydrofuran solution (8.0 ml) was added azodiyldipiperidine (363 mg, 1.44 mmol) and tributylphosphine (454 mg, 2.25 mmol). Stir at room temperature for 20 minutes. Subsequently, 6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol (50 mg, 0.15 mmol) was slowly added dropwise to the above solution. And a solution of (5-methyl-2-(4-methylpiperazin-1-yl)thiazol-4-yl)methanol (68 mg, 0.30 mmol) in THF (MeOH). Stir at room temperature for 3 hours.
将反应液在减压下浓缩。残余物用硅胶柱层析纯化(洗脱剂：甲醇/二氯甲烷＝3/97)。收集产品用N,N-二甲基甲酰胺(4.0毫升)溶至澄清。粗产品用高效液相制备，制备条件：色谱柱：X select C18 19mm*150mm；流动相：水(含有0.05％的碳酸氢铵)和乙腈；流速：25毫升/分钟；梯度：在25分钟内，乙腈从25％升到50％；检测波长：254nm。减压冻干，得到12.8毫克2-甲氧基-5-(6-甲氧基-4-((5-甲基-2-(4-甲基哌嗪-1-基)噻唑-4-基)甲氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑(收率：15.7％)MS(ESI)M/Z：544[M+H +]。 1H NMR(300MHz,DMSO-d 6,ppm)δ7.42(s,1H),6.96(s,1H),6.68(s,1H),5.04(s,2H),4.19(s,3H),3.84(s,3H),3.35–3.34(m,2H),3.33–3.32(m,2H),2.41(t,J＝5.1Hz,4H),2.33(s,3H),2.22(s,3H)。 The reaction solution was concentrated under reduced pressure. The residue was purified with EtOAc EtOAc EtOAcEtOAc The product was dissolved in clarified with N,N-dimethylformamide (4.0 mL). The crude product was prepared by high performance liquid phase. Preparation conditions: column: X select C18 19mm*150mm; mobile phase: water (containing 0.05% ammonium hydrogencarbonate) and acetonitrile; flow rate: 25 ml/min; gradient: within 25 minutes , acetonitrile rose from 25% to 50%; detection wavelength: 254nm. Lyophilized under reduced pressure to give 12.8 mg of 2-methoxy-5-(6-methoxy-4-((5-methyl-2-(4-methylpiperazin-1-yl)thiazole-4- Methoxy)benzofuran-2-yl)thiazolo[5,4-d]thiazole (yield: 15.7%) MS (ESI) M/Z: 544 [M+H + ]. 1 H NMR (300MHz, DMSO- d 6, ppm) δ7.42 (s, 1H), 6.96 (s, 1H), 6.68 (s, 1H), 5.04 (s, 2H), 4.19 (s, 3H), 3.84(s,3H),3.35–3.34(m,2H),3.33–3.32(m,2H),2.41(t,J=5.1Hz,4H),2.33(s,3H),2.22(s,3H) .
实施例9：合成2-((6-甲氧基-2-(5-甲氧基噻唑并[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)乙酸甲酯Example 9: Synthesis of 2-((6-methoxy-2-(5-methoxythiazolo[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy)acetic acid Methyl ester
Figure PCTCN2019086557-appb-000064
Figure PCTCN2019086557-appb-000064
步骤A：合成2-((6-甲氧基-2-(5-甲氧基噻唑并[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)乙酸甲酯Step A: Synthesis of 2-((6-methoxy-2-(5-methoxythiazolo[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy)acetate ester
Figure PCTCN2019086557-appb-000065
Figure PCTCN2019086557-appb-000065
详见通用合成步骤L。将6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇(35毫克，0.105毫摩尔)溶于N,N-二甲基甲酰胺(10.0毫升)中，加入溴代乙酸甲酯(32毫克，0.207毫摩尔)和无水碳酸钾(28毫克，0.203毫摩尔)。将反应液加热至40摄氏度，并搅拌3小时。LCMS监测显示原料消失后，过滤除去不溶物。滤液用制备型高效液相色谱纯化。分离条件如下，色谱柱：X select C18 19mm*150mm；流动相：水(含有0.05％的甲酸)和乙腈；流速：25毫升/分钟；梯度：在7分钟内，乙腈从50％升到60％；检测波长：254nm。减压冻干，得到18.2毫克黄色固体2-((6-甲氧基-2-(5-甲氧基噻唑并[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)乙酸甲酯(收率：42.7％)。MS(ESI)M/Z：407[M+H +]。 1H NMR(300MHz,CDCl 3,ppm):δ7.41(s,1H),6.76(s,1H),6.26(s,1H),4.77(s,2H),4.22(s,3H),3.87(s,3H),3.85(s,3H)。 See General Synthesis Step L for details. Dissolving 6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol (35 mg, 0.105 mmol) in N,N-di Methylformamide (10.0 ml) was added with methyl bromoacetate (32 mg, 0.207 mmol) and anhydrous potassium carbonate (28 mg, 0.203 mmol). The reaction solution was heated to 40 ° C and stirred for 3 hours. LCMS monitoring showed disappearance of the starting material and filtration to remove insolubles. The filtrate was purified by preparative high performance liquid chromatography. The separation conditions were as follows, column: X select C18 19 mm * 150 mm; mobile phase: water (containing 0.05% formic acid) and acetonitrile; flow rate: 25 ml / min; gradient: acetonitrile increased from 50% to 60% in 7 minutes Detection wavelength: 254 nm. It was lyophilized under reduced pressure to give 18.2 mg (yield: 6-methoxy-2-(5-methoxythiazolo[5,4-d]thiazol-2-yl)benzofuran-4-yl. Ethyloxyacetate (yield: 42.7%). MS (ESI) M / Z: 407 [M+H + ]. 1 H NMR (300MHz, CDCl 3 , ppm): δ7.41 (s, 1H), 6.76 (s, 1H), 6.26 (s, 1H), 4.77 (s, 2H), 4.22 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H).
实施例10：合成2-((6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)乙-1-醇Example 10: Synthesis of 2-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy)ethyl-1 -alcohol
Figure PCTCN2019086557-appb-000066
Figure PCTCN2019086557-appb-000066
步骤A：合成2-甲氧基-5-(6-甲氧基-4-(2-((四氢-2H-吡喃-2-基)氧基)乙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑Step A: Synthesis of 2-methoxy-5-(6-methoxy-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)benzofuran-2 -yl)thiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000067
详见通用合成步骤L。6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇(30毫克，0.09毫摩尔)，N,N-二甲基甲酰胺(2.0毫升)2-(2-溴乙氧基)四氢-2H-吡喃(38毫克，0.18毫摩尔)和无水碳酸钾(25毫克，0.18毫摩尔)。得到25毫克黄色固体2-甲氧基-5-(6-甲氧基-4-(2-((四氢-2H-吡喃-2-基)氧基)乙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑。无需纯化，直接用于下步反应。MS(ESI)M/Z:463[M+H +]。
Figure PCTCN2019086557-appb-000067
See General Synthesis Step L for details. 6-Methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol (30 mg, 0.09 mmol), N,N-dimethyl Formamide (2.0 ml) 2-(2-bromoethoxy)tetrahydro-2H-pyran (38 mg, 0.18 mmol) and anhydrous potassium carbonate (25 mg, 0.18 mmol). Yield 25 mg of yellow solid 2-methoxy-5-(6-methoxy-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)benzofuran- 2-yl)thiazolo[5,4-d]thiazole. It is used directly in the next step without purification. MS (ESI) M / Z: 463 [M+H + ].
步骤B：合成2-((6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)乙-1-醇Step B: Synthesis of 2-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy)e-1- alcohol
Figure PCTCN2019086557-appb-000068
Figure PCTCN2019086557-appb-000068
将2-甲氧基-5-(6-甲氧基-4-(2-((四氢-2H-吡喃-2-基)氧基)乙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑(25毫克，0.054毫摩尔)溶于四氢呋喃溶液(2.0毫升)中。随后，向上述溶液中加入稀盐酸溶液(0.5毫升，1.0摩尔/升)。在室温下搅拌1小时。2-methoxy-5-(6-methoxy-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)benzofuran-2-yl) Thiazolo[5,4-d]thiazole (25 mg, 0.054 mmol) was dissolved in tetrahydrofuran (2.0 mL). Subsequently, a dilute hydrochloric acid solution (0.5 ml, 1.0 mol/liter) was added to the above solution. Stir at room temperature for 1 hour.
向混合液中加入饱和碳酸氢钠溶液调节PH值约为8。混合液用乙酸乙酯(10毫升×3次)萃取。合并有机相，有机相先用饱和食盐水(10毫升×3次)洗涤，然后用无水硫酸钠干燥，最后减压浓缩。粗产品用制备型高效液相色谱纯化。分离条件如下，色谱柱：X select C18 19mm*150mm；流动相：水(含有0.05％的TFA)和乙腈；流速：25毫升/分钟；梯度：在7分钟内，乙腈从5％升到100％；检测波长：254nm。纯化后，低温冻干得6.0毫克黄色固体2-((6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)乙-1-醇(收率：29.4％)。MS(ESI)M/Z：379[M+H +]。 1H NMR(300MHz,CDCl 3,ppm):δ7.48(s,1H),6.75(s,1H),6.39(s,1H),4.47–4.32(m,5H),4.08(s,2H),3.89(s,3H)。 A saturated sodium bicarbonate solution was added to the mixture to adjust the pH to about 8. The mixture was extracted with ethyl acetate (10 mL×3×). The combined organic layers were washed with brine (10 mL×3×) and then dried over anhydrous sodium sulfate. The crude product was purified by preparative high performance liquid chromatography. The separation conditions were as follows, column: X select C18 19 mm * 150 mm; mobile phase: water (containing 0.05% TFA) and acetonitrile; flow rate: 25 ml / min; gradient: acetonitrile increased from 5% to 100% in 7 minutes Detection wavelength: 254 nm. After purification, lyophilization at low temperature gave 6.0 mg of a yellow solid 2-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-yl) Oxy)ethan-1-ol (yield: 29.4%). MS (ESI) M / Z: 379 [M+H + ]. 1 H NMR (300MHz, CDCl 3 , ppm): δ7.48 (s, 1H), 6.75 (s, 1H), 6.39 (s, 1H), 4.47-4.32 (m, 5H), 4.08 (s, 2H) , 3.89 (s, 3H).
实施例11：合成2-甲氧基-5-(6-甲氧基-4-(2-(吡咯烷-1-基)乙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑甲酸盐Example 11: Synthesis of 2-methoxy-5-(6-methoxy-4-(2-(pyrrolidin-1-yl)ethoxy)benzofuran-2-yl)thiazolo[5, 4-d]thiazolylate
Figure PCTCN2019086557-appb-000069
Figure PCTCN2019086557-appb-000069
步骤A：合成2-甲氧基-5-(6-甲氧基-4-(2-(吡咯烷-1-基)乙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑甲酸盐Step A: Synthesis of 2-methoxy-5-(6-methoxy-4-(2-(pyrrolidin-1-yl)ethoxy)benzofuran-2-yl)thiazolo[5,4 -d]thiazole formate
Figure PCTCN2019086557-appb-000070
详见通用合成步骤L。将6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇(20毫克,0.060毫摩尔)溶于N,N-二甲基甲酰胺(8.0毫升)中。向体系中加入碳酸钾(17毫克，0.120毫摩尔)和1-(2-氯乙基)吡咯烷(20毫克，0.120毫摩尔)。在60摄氏度下搅拌1小时。TLC监测显示原料消失后，将体系冷却到室温。向体系中加入水(10毫升)淬灭反应。混合液用乙酸乙酯(20毫升X2)萃取，合并后的有机相先用饱和盐水(20毫升)反洗，然后用无水硫酸钠干燥，最后真空浓缩。粗产品用制备型高效液相色谱纯化。分离条件如下，色谱柱：X select C18 19mm*150mm；流动相：水(含有0.05％的甲酸)和乙腈；流速：25毫升/分钟；梯度：在7分钟内，乙腈从50％升到60％；检测波长：254nm。纯化后，低温冻干得到黄色固体2-甲氧基-5-(6-甲氧基-4-(2-(吡咯烷-1-基)乙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑(9毫克，31.3％)。MS(ESI)M/Z：432[M+H +]。1H NMR(300MHz,CDCl3,ppm):δ7.30(s,1H),6.75(s,1H),6.38(s,1H),4.53(t,J＝4.8Hz,2H),4.23(s,3H),3.88(s,3H),3.45(t,J＝4.8Hz,2H),3.35-3.25(m,4H),2.14-2.07(m,4H)。
Figure PCTCN2019086557-appb-000070
See General Synthesis Step L for details. Dissolving 6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol (20 mg, 0.060 mmol) in N,N-di Methylformamide (8.0 ml). Potassium carbonate (17 mg, 0.120 mmol) and 1-(2-chloroethyl)pyrrolidine (20 mg, 0.120 mmol) were added to the system. Stir at 60 degrees Celsius for 1 hour. After TLC monitoring showed disappearance of the starting material, the system was cooled to room temperature. Water (10 mL) was added to the system to quench the reaction. The mixture was extracted with EtOAc (EtOAc (EtOAc)EtOAc. The crude product was purified by preparative high performance liquid chromatography. The separation conditions were as follows, column: X select C18 19 mm * 150 mm; mobile phase: water (containing 0.05% formic acid) and acetonitrile; flow rate: 25 ml / min; gradient: acetonitrile increased from 50% to 60% in 7 minutes Detection wavelength: 254 nm. After purification, lyophilization to give a yellow solid 2-methoxy-5-(6-methoxy-4-(2-(pyrrolidin-1-yl)ethoxy)benzofuran-2-yl)thiazole And [5,4-d]thiazole (9 mg, 31.3%). MS (ESI) M / Z: 422 [M+H + ]. 1H NMR (300MHz, CDCl3, ppm): δ 7.30 (s, 1H), 6.75 (s, 1H), 6.38 (s, 1H), 4.53 (t, J = 4.8 Hz, 2H), 4.23 (s, 3H) ), 3.88 (s, 3H), 3.45 (t, J = 4.8 Hz, 2H), 3.35-3.25 (m, 4H), 2.14 - 2.07 (m, 4H).
实施例12：合成2-甲氧基-5-(6-甲氧基-4-(3-甲氧基丙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑Example 12: Synthesis of 2-methoxy-5-(6-methoxy-4-(3-methoxypropoxy)benzofuran-2-yl)thiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000071
Figure PCTCN2019086557-appb-000071
步骤A：合成2-甲氧基-5-(6-甲氧基-4-(3-甲氧基丙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑Step A: Synthesis of 2-methoxy-5-(6-methoxy-4-(3-methoxypropoxy)benzofuran-2-yl)thiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000072
Figure PCTCN2019086557-appb-000072
详见通用合成步骤L。6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇(20毫克，0.060毫摩尔)，N,N-二甲基甲酰胺(8毫升)，碳酸钾(16毫克，0.120毫摩尔)和1-溴-3-甲氧基丙烷(18.4毫克，0.120毫摩尔)。得到黄色固体2-甲氧基-5-(6-甲氧基-4-(3-甲氧基丙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑(13.2毫克，54.2％)。MS(ESI)M/Z：407[M+H +]。 1H NMR(300MHz,CDCl 3,ppm):δ7.37(s,1H),6.71(s,1H),6.38(s，1H),4.28(s,3H),4.17(t,J＝6.3Hz,2H),3.88(s,3H),3.60(t,J＝6.3Hz,2H),3.37(s,3H),2.16–2.09(m,2H)。 See General Synthesis Step L for details. 6-Methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol (20 mg, 0.060 mmol), N,N-dimethyl Formamide (8 ml), potassium carbonate (16 mg, 0.120 mmol) and 1-bromo-3-methoxypropane (18.4 mg, 0.120 mmol). Obtained the yellow solid 2-methoxy-5-(6-methoxy-4-(3-methoxypropoxy)benzofuran-2-yl)thiazolo[5,4-d]thiazole (13.2 Mg, 54.2%). MS (ESI) M / Z: 407 [M+H + ]. 1 H NMR (300MHz, CDCl 3 , ppm): δ7.37 (s, 1H), 6.71 (s, 1H), 6.38 (s, 1H), 4.28 (s, 3H), 4.17 (t, J = 6.3Hz , 2H), 3.88 (s, 3H), 3.60 (t, J = 6.3 Hz, 2H), 3.37 (s, 3H), 2.16 - 2.09 (m, 2H).
实施例13：合成4-(4-((6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)吡唑并[1,5-a]吡啶-4-基氧基)甲基)-5-甲基噻吡啶-2-基)吗啉Example 13: Synthesis of 4-(4-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)pyrazolo[1,5-a]pyridine -4-yloxy)methyl)-5-methylthiapyridin-2-yl)morpholine
Figure PCTCN2019086557-appb-000073
Figure PCTCN2019086557-appb-000073
步骤A：合成3-(苄氧基)-5-甲氧基-2-((三甲基甲硅烷基)乙炔基)吡啶Step A: Synthesis of 3-(benzyloxy)-5-methoxy-2-((trimethylsilyl)ethynyl)pyridine
Figure PCTCN2019086557-appb-000074
Figure PCTCN2019086557-appb-000074
在室温和氮气保护下，将3-(苄氧基)-2-碘-5-甲氧基吡啶(1.20克，3.52毫摩尔)溶于三乙胺(25毫升)中。向体系中依次加入三甲基乙炔基硅(520毫克，5.30毫摩尔)、碘化亚铜(130毫克，0.684毫摩尔),双三苯基膦二氯化钯(250毫克，0.350毫摩尔)。在室温下搅拌过夜。LCMS监测显示原料消失后，混合液在真空下浓缩。将所得残余物溶于乙酸乙酯/水(20毫升/20毫升)中。混合液用乙酸乙酯(50毫升X2)萃取。合并后的有机相先用饱和盐水(50毫升)反洗，然后用无水硫酸钠干燥，最后真空浓缩。粗产品用硅胶柱层析法纯化(洗脱剂：乙酸乙酯/石油醚10％)浓缩得黄色固体3-(苄氧基)-5-甲氧基-2-((三甲基甲硅烷基)乙炔基)吡啶(970毫克，89％)。MS(ESI)M/Z：312[M+H +]。 3-(Benzyloxy)-2-iodo-5-methoxypyridine (1.20 g, 3.52 mmol) was dissolved in triethylamine (25 mL). Trimethylethynyl silicon (520 mg, 5.30 mmol), cuprous iodide (130 mg, 0.684 mmol), bistriphenylphosphine palladium dichloride (250 mg, 0.350 mmol) were added to the system. . Stir at room temperature overnight. After LCMS monitoring showed disappearance of the starting material, the mixture was concentrated under vacuum. The residue obtained was dissolved in ethyl acetate / water (20 mL / 20 mL). The mixture was extracted with ethyl acetate (50 mL EtOAc). The combined organic phases were back-washed with saturated brine (50 mL) then dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (EtOAc:EtOAcEtOAc Ethyl acetyl)pyridine (970 mg, 89%). MS (ESI) M / Z: 312 [M+H + ].
步骤B：合成3-(苄氧基)-2-乙炔基-5-甲氧基吡啶Step B: Synthesis of 3-(benzyloxy)-2-ethynyl-5-methoxypyridine
Figure PCTCN2019086557-appb-000075
Figure PCTCN2019086557-appb-000075
将3-(苄氧基)-5-甲氧基-2-((三甲基甲硅烷基)乙炔基)吡啶(970毫克，3.12毫摩尔)溶于甲醇(20毫升)中。向体系中加入碳酸钾(1.30克，9.42毫摩尔)。在室温下搅拌1小时。LCMS监测 显示原料消失后。向体系中加入水(20毫升)淬灭反应。混合液用乙酸乙酯(30毫升X3)萃取。合并后的有机相先用饱和盐水(30毫升)反洗，然后用无水硫酸钠干燥，最后真空浓缩。粗产品经柱层析法分离纯化(洗脱剂：乙酸乙酯/石油醚30％)浓缩得白色固体3-(苄氧基)-2-乙炔基-5-甲氧基吡啶(650毫克，86.8％),无需纯化直接用于下一步。MS(ESI)M/Z：240[M+H] +. 3-(Benzyloxy)-5-methoxy-2-((trimethylsilyl)ethynyl)pyridine (970 mg, 3.12 mmol) was dissolved in methanol (20 mL). Potassium carbonate (1.30 g, 9.42 mmol) was added to the system. Stir at room temperature for 1 hour. LCMS monitoring showed disappearance of the starting material. Water (20 mL) was added to the system to quench the reaction. The mixture was extracted with ethyl acetate (30 mL EtOAc). The combined organics were washed with saturated brine (30 mL) then dried over anhydrous sodium sulfate The crude product was purified by column chromatography (eluent: ethyl acetate / petroleum ether 30%) to yield white crystals of 3-(benzyloxy)-2-ethynyl-5-methoxypyridine (650 mg, 86.8%), used directly in the next step without purification. MS (ESI) M/Z: 240 [M+H] + .
步骤C：合成2-((3-(苄氧基)-5-甲氧基吡啶-2-基)乙炔基)-5-甲氧基咪唑并[5,4-d]噻唑Step C: Synthesis of 2-((3-(benzyloxy)-5-methoxypyridin-2-yl)ethynyl)-5-methoxyimidazo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000076
Figure PCTCN2019086557-appb-000076
在室温和氮气保护下，将3-(苄氧基)-2-乙炔基-5-甲氧基吡啶(453毫克，1.89毫摩尔)溶于甲苯/二异丙基胺(25毫升/25毫升)中。向体系中依次加入3-(苄氧基)-2-乙炔基-5-甲氧基吡啶(683毫克，2.72毫摩尔),碘化亚铜(2毫克，0.01毫摩尔)和四三苯基膦钯(46毫克，0.04毫摩尔)。在室温下搅拌过夜。LCMS监测显示原料消失后，向体系中加入水(20毫升)淬灭反应。混合液用乙酸乙酯(50毫升X2)萃取。合并后的有机相先用饱和盐水(20毫升)反洗，然后用无水硫酸钠干燥，最后真空浓缩。粗产品用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚20％)浓缩得黄色固体2-((3-(苄氧基)-5-甲氧基吡啶-2-基)乙炔基)-5-甲氧基咪唑并[5,4-d]噻唑(510毫克，65.8％)。MS(ESI)M/Z:410[M+H +]。 3-(Benzyloxy)-2-ethynyl-5-methoxypyridine (453 mg, 1.89 mmol) was dissolved in toluene / diisopropylamine (25 mL / 25 mL). )in. To the system was added 3-(benzyloxy)-2-ethynyl-5-methoxypyridine (683 mg, 2.72 mmol), cuprous iodide (2 mg, 0.01 mmol) and tetratriphenyl. Palladium phosphine (46 mg, 0.04 mmol). Stir at room temperature overnight. After LCMS monitoring showed the disappearance of the starting material, water (20 mL) was added to the system to quench the reaction. The mixture was extracted with ethyl acetate (50 mL EtOAc). The combined organic phases were washed with saturated brine (20 mL) then dried over anhydrous sodium sulfate. The crude product was purified by silica gel chromatography eluting eluting 5-)-methoxyimidazo[5,4-d]thiazole (510 mg, 65.8%). MS (ESI) M / Z: 410 [M+H + ].
步骤D：合成2-(4-(苄氧基)-6-甲氧基[1,5-a]吡啶-2-基)-5-甲氧基咪唑并[5,4-d]噻唑Step D: Synthesis of 2-(4-(benzyloxy)-6-methoxy[1,5-a]pyridin-2-yl)-5-methoxyimidazo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000077
Figure PCTCN2019086557-appb-000077
将2-((3-(苄氧基)-5-甲氧基吡啶-2-基)乙炔基)-5-甲氧基咪唑并[5,4-d]噻唑(510毫克，1.24毫摩尔)溶于二氯甲烷(25毫升)中。在室温下向体系中缓慢滴加2,4,6-三甲基苯磺酰羟胺(752毫克，3.72毫摩尔)的二氯甲烷(5毫升)溶液。在室温下搅拌4小时。LCMS监测显示原料消失后，将体系在真空下浓缩得到黄色油状物。将所得到的油状物溶于N，N-二甲基甲酰胺(20毫升)缓慢滴加到碳酸钾(517毫克，3.72毫摩尔)的N，N-二甲基甲酰胺(500毫升)溶液中。在室温下搅拌1小时。LCMS监测显示原料消失后，真空浓缩。粗产品用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚25％)浓缩得棕黄色固体2-(4-(苄氧基)-6-甲氧基[1,5-a]吡啶-2-基)-5-甲氧基咪唑并[5,4-d]噻唑(340毫克，65％)。MS(ESI)M/Z:425[M+H +]。 2-((3-(Benzyloxy)-5-methoxypyridin-2-yl)ethynyl)-5-methoxyimidazo[5,4-d]thiazole (510 mg, 1.24 mmol) ) dissolved in dichloromethane (25 ml). A solution of 2,4,6-trimethylbenzenesulfonylhydroxylamine (752 mg, 3.72 mmol) in dichloromethane (5 mL) was slowly added dropwise to the mixture. Stir at room temperature for 4 hours. After LCMS monitoring showed the disappearance of the material, the system was concentrated in vacuo to afford a yellow oil. The obtained oil was dissolved in N,N-dimethylformamide (20 ml), and slowly added dropwise to a solution of potassium carbonate (517 mg, 3.72 mmol) of N,N-dimethylformamide (500 ml) in. Stir at room temperature for 1 hour. LCMS monitoring indicated disappearance of the starting material and concentration in vacuo. The crude product was purified with EtOAc EtOAc elut elut elut elut elut Pyridin-2-yl)-5-methoxyimidazo[5,4-d]thiazole (340 mg, 65%). MS (ESI) M/Z: 425 [M+H + ].
步骤E：合成6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)吡唑并[1,5-a]吡啶-4-醇Step E: Synthesis of 6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)pyrazolo[1,5-a]pyridin-4-ol
Figure PCTCN2019086557-appb-000078
Figure PCTCN2019086557-appb-000078
在室温和氮气保护下，将2-(4-(苄氧基)-6-甲氧基[1,5-a]吡啶-2-基)-5-甲氧基咪唑并[5,4-d]噻唑(100毫克，0.24毫摩尔)和五甲基苯(244毫克，1.65毫摩尔)溶于二氯甲烷(5毫升)中。在-78摄氏度下向体系中加入三氯化硼的二氯甲烷溶液(0.60毫升，0.61毫摩尔，1.0摩尔/升),在-78摄氏度下搅拌1小时。LCMS监测显示原料消失后，向体系中加入甲醇(10毫升)淬灭反应。将体系缓慢升至室温并真空浓缩。粗产品用反相柱纯化[反相柱:C18；流动相A：水(含有0.05％甲酸)， 流动相B：乙腈；梯度:30％乙腈到90％乙腈在8分钟内；检测波长：254nm]浓缩得棕黄色固体6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)吡唑并[1,5-a]吡啶-4-醇(60毫克，74.6％)。MS(ESI)M/Z:335[M+H +]。 2-(4-(Benzyloxy)-6-methoxy[1,5-a]pyridin-2-yl)-5-methoxyimidazo[5,4- at room temperature under nitrogen. d] Thiazole (100 mg, 0.24 mmol) and pentamethylbenzene (244 mg, 1.65 mmol) were dissolved in dichloromethane (5 mL). A solution of boron trichloride in dichloromethane (0.60 ml, 0.61 mmol, 1.0 mol/L) was added to the system at -78 ° C and stirred at -78 ° C for 1 hour. After LCMS monitoring showed disappearance of the starting material, methanol (10 mL) was added to the system to quench the reaction. The system was slowly warmed to room temperature and concentrated in vacuo. The crude product was purified by reverse phase column [reverse phase column: C18; mobile phase A: water (containing 0.05% formic acid), mobile phase B: acetonitrile; gradient: 30% acetonitrile to 90% acetonitrile in 8 min; detection wavelength: 254 nm Concentrated to a brownish-yellow solid 6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)pyrazolo[1,5-a]pyridin-4-ol (60 Mg, 74.6%). MS (ESI) M/Z: 335 [M+H + ].
步骤F：合成4-(4-((6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)吡唑并[1,5-a]吡啶-4-基氧基)甲基)-5-甲基噻吡啶-2-基)吗啉Step F: Synthesis of 4-(4-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)pyrazolo[1,5-a]pyridine- 4-yloxy)methyl)-5-methylthiapyridin-2-yl)morpholine
Figure PCTCN2019086557-appb-000079
在室温和氮气保护下，将三苯基膦(19毫克，0.072毫摩尔)溶于四氢呋喃(0.5毫升)中。在0摄氏度下向体系中逐滴加入偶氮二甲酸二异丙酯(9毫克，0.043毫摩尔)。在0摄氏度下搅拌5分钟。向体系中加入6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)吡唑并[1,5-a]吡啶-4-醇(12毫克，0.036毫摩尔)和(5-甲基-2-吗啉代-4-基)甲醇(8毫克，0.036毫摩尔)的四氢呋喃(2毫升)溶液。在室温下搅拌1小时。LCMS监测显示原料消失后。向体系中加入水(5毫升)淬灭反应。混合液用乙酸乙酯(5毫升X2)萃取。合并后的有机相先用饱和盐水(10毫升)反洗，然后用无水硫酸钠干燥，最后真空浓缩。粗产品用制备型高效液相色谱纯化。分离条件如下，色谱柱：X select C18 19mm*150mm；流动相：水(含有0.05％的甲酸)和乙腈；流速：25毫升/分钟；梯度：在7分钟内，乙腈从5％升到100％；检测波长：254nm。减压冻干白色固体4-(4-((6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)吡唑并[1,5-a]吡啶-4-基氧基)甲基)-5-甲基噻吡啶-2-基)吗啉(3.1毫克，16.3％)。MS(ESI)M/Z:531[M+H +]。 1H NMR(300MHz,CDCl 3,ppm)δ7.78(s,1H),7.14(s,1H),6.51(s,1H),5.08(s,2H),4.21(s,3H),3.96–3.82(m,7H),3.43–3.57(t,J＝9.6Hz,4H),2.39(s,3H)。
Figure PCTCN2019086557-appb-000079
Triphenylphosphine (19 mg, 0.072 mmol) was dissolved in tetrahydrofuran (0.5 mL). Diisopropyl azodicarboxylate (9 mg, 0.043 mmol) was added dropwise to the system at 0 °C. Stir at 0 degrees Celsius for 5 minutes. To the system was added 6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)pyrazolo[1,5-a]pyridin-4-ol (12 mg, A solution of 0.036 mmol) and (5-methyl-2-morpholino-4-yl)methanol (8 mg, 0.036 mmol) in THF (2 mL). Stir at room temperature for 1 hour. LCMS monitoring showed disappearance of the starting material. Water (5 mL) was added to the system to quench the reaction. The mixture was extracted with ethyl acetate (5 mL EtOAc). The combined organic phases were back-washed with saturated brine (10 mL) then dried over anhydrous sodium sulfate. The crude product was purified by preparative high performance liquid chromatography. The separation conditions were as follows, column: X select C18 19 mm * 150 mm; mobile phase: water (containing 0.05% formic acid) and acetonitrile; flow rate: 25 ml / min; gradient: acetonitrile increased from 5% to 100% in 7 minutes Detection wavelength: 254 nm. Lyophilized white solid 4-(4-((6-methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)pyrazolo[1,5-a] Pyridin-4-yloxy)methyl)-5-methylthiapyridin-2-yl)morpholine (3.1 mg, 16.3%). MS (ESI) M / Z: 531 [M+H + ]. 1 H NMR (300MHz, CDCl 3 , ppm) δ7.78 (s, 1H), 7.14 (s, 1H), 6.51 (s, 1H), 5.08 (s, 2H), 4.21 (s, 3H), 3.96- 3.82 (m, 7H), 3.43 - 3.57 (t, J = 9.6 Hz, 4H), 2.39 (s, 3H).
实施例14：合成2-甲氧基-5-(6-甲氧基-4-(2-(2-甲氧基乙氧基)乙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑Example 14: Synthesis of 2-methoxy-5-(6-methoxy-4-(2-(2-methoxyethoxy)ethoxy)benzofuran-2-yl)thiazolo[ 5,4-d]thiazole
Figure PCTCN2019086557-appb-000080
Figure PCTCN2019086557-appb-000080
步骤A：合成2-甲氧基-5-(6-甲氧基-4-(2-(2-甲氧基乙氧基)乙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑Step A: Synthesis of 2-methoxy-5-(6-methoxy-4-(2-(2-methoxyethoxy)ethoxy)benzofuran-2-yl)thiazolo[5 ,4-d]thiazole
Figure PCTCN2019086557-appb-000081
Figure PCTCN2019086557-appb-000081
详见通用合成步骤L。6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇(20毫克，0.060毫摩尔)，N,N-二甲基甲酰胺(8毫升)1-溴-2-(2-甲氧基乙氧基)乙烷(22毫克，0.120毫摩尔)和碳酸钾(16毫克，0.120毫摩尔)。得到白色固体2-甲氧基-5-(6-甲氧基-4-(2-(2-甲氧基乙氧基)乙氧基)苯并呋喃-2-基)噻唑并[5,4-d]噻唑(11.2毫克，42.9％)。MS(ESI)M/Z：437[M+H +]。 1H NMR(300MHz,CDCl 3,ppm):δ7.38(s,1H),6.72(s,1H),6.36(s,1H),4.28(t,J＝4.8Hz,2H),4.22(s,3H),3.96(t,J＝4.8Hz,2H),3.88(s,3H),3.81–3.78(m,2H),3.64–3.60(m,2H),3.43(s,3H)。 See General Synthesis Step L for details. 6-Methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol (20 mg, 0.060 mmol), N,N-dimethyl Formamide (8 ml) 1-bromo-2-(2-methoxyethoxy)ethane (22 mg, 0.120 mmol) and potassium carbonate (16 mg, 0.120 mmol). Obtained the white solid 2-methoxy-5-(6-methoxy-4-(2-(2-methoxyethoxy)ethoxy)benzofuran-2-yl)thiazolo[5, 4-d]thiazole (11.2 mg, 42.9%). MS (ESI) M / Z: 437 [M+H + ]. 1 H NMR (300MHz, CDCl 3 , ppm): δ7.38 (s, 1H), 6.72 (s, 1H), 6.36 (s, 1H), 4.28 (t, J = 4.8Hz, 2H), 4.22 (s , 3H), 3.96 (t, J = 4.8 Hz, 2H), 3.88 (s, 3H), 3.81 - 3.78 (m, 2H), 3.64 - 3.60 (m, 2H), 3.43 (s, 3H).
实施例15：合成2-(6,8-二甲氧基中氮茚-2-基)-5-甲氧基噻唑并[5,4-d]噻唑Example 15: Synthesis of 2-(6,8-dimethoxyindolizin-2-yl)-5-methoxythiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000082
Figure PCTCN2019086557-appb-000082
步骤A：合成1-(4-溴-1H-吡咯-2-基)乙酮Step A: Synthesis of 1-(4-bromo-1H-pyrrol-2-yl)ethanone
Figure PCTCN2019086557-appb-000083
Figure PCTCN2019086557-appb-000083
将1-(1H-吡咯-2-基)乙酮(200.0克，1.83摩尔)溶于二甲基亚砜(1.0升)中。随后，向上述溶液中缓慢滴加氢溴酸水溶液(1.0升，48％wt)。将反应液加热至50摄氏度，并搅拌3小时。1-(1H-Pyrrol-2-yl)ethanone (200.0 g, 1.83 mol) was dissolved in dimethyl sulfoxide (1.0 L). Subsequently, a hydrobromic acid aqueous solution (1.0 liter, 48% by weight) was slowly added dropwise to the above solution. The reaction solution was heated to 50 ° C and stirred for 3 hours.
将反应液冷却到室温。向反应液中滴加氢氧化钠水溶液(4.0摩尔/升)调节PH值约为7-8。混合液用乙酸乙酯(500毫升×3次)萃取。合并有机相，有机相先用饱和食盐水(500毫升×3次)洗涤，然后用无水硫酸钠干燥，最后减压浓缩。得到300.0克棕色固体1-(4-溴-1H-吡咯-2-基)乙酮(收率：87.2％)。MS(ESI)M/Z：188,190[M+H +]。 The reaction solution was cooled to room temperature. Aqueous sodium hydroxide solution (4.0 mol/liter) was added dropwise to the reaction solution to adjust the pH to about 7-8. The mixture was extracted with ethyl acetate (500 mL×3×). The combined organic layers were washed with brine (500 mL×3×) and then dried over anhydrous sodium sulfate. 300.0 g of a brown solid 1-(4-bromo-1H-pyrrol-2-yl)ethanone (yield: 87.2%) was obtained. MS (ESI) M/Z: 188, 190 [M+H + ].
步骤B：合成乙基2-(2-乙酰基-4-溴-1H-吡咯-1-基)乙酸乙酯Step B: Synthesis of ethyl 2-(2-acetyl-4-bromo-1H-pyrrol-1-yl)acetate
Figure PCTCN2019086557-appb-000084
Figure PCTCN2019086557-appb-000084
将1-(4-溴-1H-吡咯-2-基)乙酮(100.0克，532毫摩尔)溶于乙腈(2.0升)中。随后，向上述溶液中依次加入2-溴乙酸乙酯(178.7克，1.07摩尔)和无水碳酸钾(147.7克，1.07摩尔)。将反应液加热至40摄氏度，并搅拌24小时。1-(4-Bromo-1H-pyrrol-2-yl)ethanone (100.0 g, 532 mmol) was dissolved in acetonitrile (2.0 L). Subsequently, 2-bromoacetic acid ethyl ester (178.7 g, 1.07 mol) and anhydrous potassium carbonate (147.7 g, 1.07 mol) were successively added to the above solution. The reaction solution was heated to 40 ° C and stirred for 24 hours.
将反应液冷却到室温，然后，减压除去乙腈。残余物加入水稀释。混合液用乙酸乙酯(500毫升×3次)萃取。合并有机相，有机相先用饱和食盐水(500毫升×3次)洗涤，然后用无水硫酸钠干燥，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/10)。得到105克类白色固体乙基2-(2-乙酰基-4-溴-1H-吡咯-1-基)乙酸乙酯(收率：72.1％)。MS(ESI)M/Z：274,276[M+H +]。 The reaction solution was cooled to room temperature, and then acetonitrile was removed under reduced pressure. The residue was diluted with water. The mixture was extracted with ethyl acetate (500 mL×3×). The combined organic layers were washed with brine (500 mL×3×) and then dried over anhydrous sodium sulfate. The residue obtained was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1/10). There was obtained 105 g of an ethyl 2-ethyl 2-(2-acetyl-4-bromo-1H-pyrrol-1-yl)acetate (yield: 72.1%). MS (ESI) M/Z: 274, 276 [M+H + ].
步骤C：合成2-溴吲哚嗪-6,8-二醇Step C: Synthesis of 2-bromopyridazine-6,8-diol
Figure PCTCN2019086557-appb-000085
Figure PCTCN2019086557-appb-000085
在冰水浴下，于250毫升高压反应釜中，将乙基2-(2-乙酰基-4-溴-1H-吡咯-1-基)乙酸乙酯(20.0克，73.0毫摩尔)溶于干燥的四氢呋喃(200.0毫升)中。随后，向上述溶液中分批次加入氢化钠(17.48克，60％wt，437毫摩尔)。加料完成后，将反应釜置于90摄氏度油浴中搅拌过夜。Ethyl 2-(2-acetyl-4-bromo-1H-pyrrol-1-yl)acetate (20.0 g, 73.0 mmol) was dissolved in dry water in a 250 mL autoclave. In tetrahydrofuran (200.0 ml). Subsequently, sodium hydride (17.48 g, 60% by weight, 437 mmol) was added in portions to the above solution. After the addition was completed, the reaction kettle was placed in an oil bath at 90 ° C and stirred overnight.
将反应液冷却到室温，向反应液中缓慢滴加水(500毫升)淬灭反应。混合液用乙酸乙酯(200毫升×3次)萃取。合并有机相，有机相先用饱和食盐水(100毫升×2次)洗涤，然后用无水硫酸钠干燥，最后减压浓缩。得到17.6克黄色固体2-溴吲哚嗪-6,8-二醇。无需纯化，直接用于下步反应。MS(ESI)M/Z：228,230[M+H +]。 The reaction solution was cooled to room temperature, and water (500 ml) was slowly added dropwise to the reaction mixture to quench the reaction. The mixture was extracted with ethyl acetate (200 mL×3×). The combined organic layers were washed with brine (100 mL×2×) then dried over anhydrous sodium sulfate. 17.6 g of 2-bromopyridazine-6,8-diol were obtained as a yellow solid. It is used directly in the next step without purification. MS (ESI) M/Z: 228, 230 [M+H + ].
步骤D：2-溴-6,8-二甲基中氮茚Step D: 2-Bromo-6,8-dimethylindolizine
Figure PCTCN2019086557-appb-000086
Figure PCTCN2019086557-appb-000086
将2-溴吲哚嗪-6,8-二醇(1.00克，4.39毫摩尔)溶于无水甲醇(50.0毫升)。随后，向上述溶液中加入浓硫酸(0.5毫升)。将反应液加热至60摄氏度，并搅拌1小时。2-Bromopyridazine-6,8-diol (1.00 g, 4.39 mmol) was dissolved in dry methanol (50.0 mL). Subsequently, concentrated sulfuric acid (0.5 ml) was added to the above solution. The reaction solution was heated to 60 ° C and stirred for 1 hour.
将反应液冷却到室温。向反应液中加入饱和碳酸氢钠溶液调节PH值约为7。混合液用乙酸乙酯(100毫升×3次)萃取。合并有机相，有机相先用饱和食盐水100毫升×3次)洗涤，然后用无水硫酸钠干燥，最后减压浓缩。残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/3)。得到350毫克黄色固体2-溴-6,8-二甲基中氮茚(收率：31.3％)。MS(ESI)M/Z：256,258[M+H +]。 The reaction solution was cooled to room temperature. Saturated sodium bicarbonate solution was added to the reaction mixture to adjust the pH to about 7. The mixture was extracted with ethyl acetate (100 mL×3×). The combined organic phases were washed with brine (100 mL×3×) and then dried over anhydrous sodium sulfate. The residue was purified with EtOAc EtOAc EtOAc EtOAc There was obtained 350 mg of a yellow solid 2-bromo-6,8-dimethylindole (yield: 31.3%). MS (ESI) M/Z: 256, 258 [M+H + ].
步骤E：6,8-二甲氧基-2-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)中氮茚Step E: 6,8-Dimethoxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indolizine
Figure PCTCN2019086557-appb-000087
Figure PCTCN2019086557-appb-000087
在氮气保护下，将6,8-二甲氧基-2-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)中氮茚(300毫克，1.17毫摩尔)溶于1,4-二氧六环(30.0毫升)。随后，向上述溶液中加入氯(2-二环己基膦基-2',4',6'-三异丙基-1,1'-联苯基)[2-(2'-氨基-1,1'-联苯)]钯(II)(69毫克，0.088毫摩尔)，2-二环己基磷-2,4,6-三异丙基联苯(84毫克，0.176毫摩尔)，双联频哪醇硼酸酯(1.34克，5.27毫摩尔)。将反应液加热至60摄氏度，并搅拌3小时。Nitrogen in 6,8-dimethoxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) under nitrogen Indole (300 mg, 1.17 mmol) was dissolved in 1,4-dioxane (30.0 mL). Subsequently, chlorine (2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1) was added to the above solution. ,1'-biphenyl)]palladium(II) (69 mg, 0.088 mmol), 2-dicyclohexylphosphine-2,4,6-triisopropylbiphenyl (84 mg, 0.176 mmol), double Bis-trienyl borate (1.34 g, 5.27 mmol). The reaction solution was heated to 60 ° C and stirred for 3 hours.
将反应液冷却到室温，并减压浓缩。残余物通过硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/3)。得到70毫克黄色固体6,8-二甲氧基-2-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)中氮茚(收率：19.7％)。MS(ESI)M/Z：304[M+H +]。 The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc:EtOAc 70 mg of yellow solid 6,8-dimethoxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-indolizine (Yield: 19.7%). MS (ESI) M / Z: 304 [M+H + ].
步骤F：2-(6,8-二甲氧基中氮茚-2-基)-5-甲氧基噻唑并[5,4-d]噻唑Step F: 2-(6,8-Dimethoxyindolizin-2-yl)-5-methoxythiazolo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000088
Figure PCTCN2019086557-appb-000088
在室温和氮气保护下，将6,8-二甲氧基-2-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)中氮茚(70毫克，0.231毫摩尔)和2-溴-5-甲氧基咪唑并[5,4-d]噻唑(63毫克，0.251毫摩尔)溶于1,4-二氧六环(5.0毫升)中。向上述溶液中依次加入无水碳酸钾(63毫克，0.457毫摩尔)，四三苯基膦钯(26.8毫克，0.023毫摩尔)和水(1.0毫升)。在80摄氏度下搅拌1.5小时。6,8-Dimethoxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) at room temperature under nitrogen The indolizine (70 mg, 0.231 mmol) and 2-bromo-5-methoxyimidazo[5,4-d]thiazole (63 mg, 0.251 mmol) are dissolved in 1,4-dioxane ( 5.0 ml). Anhydrous potassium carbonate (63 mg, 0.457 mmol), tetrakistriphenylphosphine palladium (26.8 mg, 0.023 mmol) and water (1.0 ml) were sequentially added to the above solution. Stir at 80 degrees Celsius for 1.5 hours.
将反应体系冷却到室温，在减压下浓缩。残留物用N,N-二甲基甲酰胺(3.0毫升)溶至澄清，经制备型高效液相色谱纯化。纯化条件如下，色谱柱：X select C18 19mm*150mm；流动相：水(含有0.05％的碳酸氢铵)和乙腈；流速:25毫升/分钟；梯度：在8分钟内，乙腈从22％升到48％；检测波长：254nm。收集产品，减压冻干。得到最终产物14.7毫克棕色固体2-(6,8-二甲氧基中氮茚-2-基)-5-甲氧基噻唑并[5,4-d]噻唑(收率：18.3％)。MS(ESI)M/Z：348[M+H +]。 1H NMR(300MHz,CDCl 3,ppm):δ7.74(s,1H),7.15(s，1H),6.88(s,1H),5.91(s,1H),4.19(s,3H),3.94(s,3H),3.81(s,3H)。 The reaction system was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in EtOAc (3 mL) eluting with EtOAc. Purification conditions were as follows, column: X select C18 19 mm * 150 mm; mobile phase: water (containing 0.05% ammonium bicarbonate) and acetonitrile; flow rate: 25 ml / min; gradient: acetonitrile increased from 22% in 8 minutes 48%; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure. The final product 14.7 mg of a brown solid of 2-(6,8-dimethoxyindolizin-2-yl)-5-methoxythiazolo[5,4-d]thiazole was obtained (yield: 18.3%). MS (ESI) M/Z: 348[M+H + ]. 1 H NMR (300MHz, CDCl 3 , ppm): δ7.74 (s, 1H), 7.15 (s, 1H), 6.88 (s, 1H), 5.91 (s, 1H), 4.19 (s, 3H), 3.94 (s, 3H), 3.81 (s, 3H).
实施例16：合成2-(4-异丙氧基-6-甲氧基苯并呋喃-2-基)-5-甲氧基咪唑并[5,4-d]噻唑Example 16: Synthesis of 2-(4-isopropoxy-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000089
Figure PCTCN2019086557-appb-000089
步骤A：合成2-(4-异丙氧基-6-甲氧基苯并呋喃-2-基)-5-甲氧基咪唑并[5,4-d]噻唑Step A: Synthesis of 2-(4-isopropoxy-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000090
Figure PCTCN2019086557-appb-000090
详见通用合成步骤L。6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇(55毫克，0.16毫摩尔)，N,N-二甲基甲酰胺(6.0毫升)，2-溴丙烷(41毫克，0.33毫摩尔)和无水碳酸钾(46毫克，0.33毫摩尔)。得到6.6毫克淡黄色固体2-(4-异丙氧基-6-甲氧基苯并呋喃-2-基)-5-甲氧基咪唑并[5,4-d]噻唑(收率：11.0％)。MS(ESI)M/Z:377[M+H] +； 1H NMR(300MHz,CDCl 3,ppm)δ7.35(s,1H),6.70(s,1H),6.37(s,1H),4.72-4.63(m,1H),4.26(s,3H),3.99(s,3H),1.44(t,J＝6.0Hz,6H)。 See General Synthesis Step L for details. 6-Methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol (55 mg, 0.16 mmol), N,N-dimethyl Formamide (6.0 ml), 2-bromopropane (41 mg, 0.33 mmol) and anhydrous potassium carbonate (46 mg, 0.33 mmol). 6.6 mg of pale yellow solid 2-(4-isopropoxy-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole was obtained (yield: 11.0) %). MS (ESI) M / Z: 377 [M+H] + ; 1 H NMR (300 MHz, CDCl 3 , ppm) δ 7.35 (s, 1H), 6.70 (s, 1H), 6.37 (s, 1H), 4.72-4.63 (m, 1H), 4.26 (s, 3H), 3.99 (s, 3H), 1.44 (t, J = 6.0 Hz, 6H).
实施例17：合成2-((6-甲氧基-2-(5-甲氧基噻唑并[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)-N，N-二甲基乙-1-胺Example 17: Synthesis of 2-((6-methoxy-2-(5-methoxythiazolo[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy)- N,N-dimethylethyl-1-amine
Figure PCTCN2019086557-appb-000091
Figure PCTCN2019086557-appb-000091
步骤A：合成2-((6-甲氧基-2-(5-甲氧基噻唑并[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)-N，N-二甲基乙-1-胺Step A: Synthesis of 2-((6-methoxy-2-(5-methoxythiazolo[5,4-d]thiazol-2-yl)benzofuran-4-yl)oxy)-N , N-dimethylethyl-1-amine
Figure PCTCN2019086557-appb-000092
Figure PCTCN2019086557-appb-000092
详见通用合成步骤K。四氢呋喃(1.0毫升)，偶氮二甲酰二哌啶(181毫克，0.72毫摩尔)和三丁基膦(228毫克，1.13毫摩尔)，6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇(25毫克，0.075毫摩尔)和(2-(二甲基氨基)乙醇(14毫克，0.15毫摩尔)的四氢呋喃溶液(2.0毫升)。得到5.2毫克淡黄色固体2-((6-甲氧基-2-(5-甲氧基噻唑并[5,4-d]噻唑-2-基)苯并呋喃-4-基)氧基)-N，N-二甲基乙-1-胺(收率：17.2％)。MS(ESI)M/Z:406[M+H +]； 1H NMR(300MHz,CDCl 3,ppm)δ7.33(s,1H),6.75(s,1H),6.38(s,1H),4.44–4.32(m,2H),4.22(s,3H),3.89(s,3H),3.22–3.06(m,2H),2.64(s,6H)。 See General Synthesis Step K for details. Tetrahydrofuran (1.0 ml), azodiyldipiperidine (181 mg, 0.72 mmol) and tributylphosphine (228 mg, 1.13 mmol), 6-methoxy-2-(5-methoxy) [5,4-d]thiazol-2-yl)benzofuran-4-ol (25 mg, 0.075 mmol) and (2-(dimethylamino)ethanol (14 mg, 0.15 mmol) in tetrahydrofuran (2.0 ml). 5.2 mg of pale yellow solid 2-((6-methoxy-2-(5-methoxythiazolo[5,4-d]thiazol-2-yl)benzofuran-4- (Ethyloxy)-N,N-dimethylethyl-1-amine (yield: 17.2%). MS (ESI) M/Z: 406 [M+H + ]; 1 H NMR (300 MHz, CDCl 3 , ppm) δ7.33 (s, 1H), 6.75 (s, 1H), 6.38 (s, 1H), 4.44 - 4.32 (m, 2H), 4.22 (s, 3H), 3.89 (s, 3H), 3.22 –3.06 (m, 2H), 2.64 (s, 6H).
实施例18：合成2-(4-(环丙基甲氧基)-6-甲氧基苯并呋喃-2-基)-5-甲氧基咪唑并[5,4-d]噻唑Example 18: Synthesis of 2-(4-(cyclopropylmethoxy)-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000093
Figure PCTCN2019086557-appb-000093
步骤A：合成2-(4-(环丙基甲氧基)-6-甲氧基苯并呋喃-2-基)-5-甲氧基咪唑并[5,4-d]噻唑Step A: Synthesis of 2-(4-(cyclopropylmethoxy)-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole
Figure PCTCN2019086557-appb-000094
Figure PCTCN2019086557-appb-000094
详见通用合成步骤L。6-甲氧基-2-(5-甲氧基[5,4-d]噻唑-2-基)苯并呋喃-4-醇(40毫克,0.12毫摩尔)，N,N-二甲基甲酰胺(6毫升)，2-溴甲基环丙烷(64毫克,0.48毫摩尔)和碳酸钾(66毫克，0.48毫摩尔)。-得到7.7毫克淡黄色固体2-(4-(环丙基甲氧基)-6-甲氧基苯并呋喃-2-基)-5-甲氧基咪唑并[5,4-d]噻唑(收率：16.5％)。MS(ESI)M/Z:389[M+H] +。 1H NMR(300MHz,CDCl3，ppm)δ7.28(s,1H),6.70(s,1H),6.32(s,1H),4.22(s,3H),3.95(d,J＝6.6Hz,2H),3.88(s,3H),1.40-1.30(m,1H),0.72-0.66(m,2H),0.44-0.40(m,2H)。 See General Synthesis Step L for details. 6-Methoxy-2-(5-methoxy[5,4-d]thiazol-2-yl)benzofuran-4-ol (40 mg, 0.12 mmol), N,N-dimethyl Formamide (6 ml), 2-bromomethylcyclopropane (64 mg, 0.48 mmol) and potassium carbonate (66 mg, 0.48 mmol). - 7.7 mg of pale yellow solid 2-(4-(cyclopropylmethoxy)-6-methoxybenzofuran-2-yl)-5-methoxyimidazo[5,4-d]thiazole (Yield: 16.5%). MS (ESI) M / Z: 381 [M+H] + . 1 H NMR (300MHz, CDCl3, ppm) δ7.28 (s, 1H), 6.70 (s, 1H), 6.32 (s, 1H), 4.22 (s, 3H), 3.95 (d, J = 6.6Hz, 2H ), 3.88 (s, 3H), 1.40-1.30 (m, 1H), 0.72-0.66 (m, 2H), 0.44-0.40 (m, 2H).
实施例19：Example 19
体外筛选实验-FLIPR方法检测化合物抑制PAR4活性测试In vitro screening test-FLIPR method for detecting compounds inhibiting PAR4 activity test
1、准备试剂：1. Prepare reagents:
实验材料Experimental Materials
1)PAR4的激动剂及抑制剂1) Agonists and inhibitors of PAR4
PAR4的选择性激动剂TFLLR-NH 2、2-Furoyl-LIGRLO-NH 2和AYPGKF-NH 2由Sangon Biotech合成并购买或从Sigma-Aldrich直接购买。在FLIPR钙离子外流试验中它们对PAR4的EC 50分别为2.0μM、0.2μM和1.3μM。SAL02-001-00和SAL02-002-00为高活性的PAR4选择性抑制剂，在FLIPR钙离子外流试验中其对PAR4的IC 50值为5-10nM。 Selective PAR4 agonist TFLLR-NH 2, 2-Furoyl -LIGRLO-NH 2 and AYPGKF-NH 2 and later synthesized by Sangon Biotech or purchased directly from Sigma-Aldrich. In the FLIPR calcium efflux assay of PAR4 on their EC 50 values of 2.0μM, 0.2μM and 1.3μM. SAL02-001-00 SAL02-002-00 highly active and selective PAR4 inhibitor, calcium ion efflux in the FLIPR assay with an IC 50 value of PAR4 5-10nM.
2)PAR4表达细胞系2) PAR4 expression cell line
利用常规转染的方法将包含人源PAR4 cDNA的哺乳动物细胞表达载体分别导入Flp-In-TREx-293细胞，经相应的抗生素筛选获得稳定高表达的Flp-In-TREx-293-PAR4细胞系并利用PAR4FLIPR钙离子外流实验进行功能验证。据文献报道HEK293细胞本身内源性高表达PAR4，因此Flp-In-TREx-293-PAR4细胞系可以用来进行PAR4小分子抑制剂的筛选实验。The mammalian cell expression vector containing the human PAR4 cDNA was separately introduced into Flp-In-TREx-293 cells by conventional transfection method, and the stably high-expression Flp-In-TREx-293-PAR4 cell line was obtained by corresponding antibiotic screening. The function was verified by PAR4FLIPR calcium ion outflow experiment. According to the literature, HEK293 cells express endogenously high expression of PAR4, so Flp-In-TREx-293-PAR4 cell line can be used for screening experiments of PAR4 small molecule inhibitors.
实施例20Example 20
利用FLIPR钙离子外流实验检测化合物对人源PAR4的抑制活性Detection of the inhibitory activity of compounds on human PAR4 by FLIPR calcium ion outflow assay
Flp-In-TREx-293-PAR4细胞培养于DEME高糖(Gibco)、10％胎牛血清、2mM GlutaMAX、1％青霉素-链霉素、15μg/ml blasticidin和200μg/ml潮霉素的培养基中，置于37℃、5％CO2细胞培养箱中培养。细胞接种至经poly-d-lysine处理过的384孔细胞培养板(Corning,3845)中，接种密度为8,000个细胞/孔/25μl细胞接种基质(DMEM高糖、10％胎牛血清、2mM GlutaMAX)于细胞培养箱培养过夜。加入25μl等体积的2X(400ng/ml)四环素诱导基质继续培养24h。实验当天，将培养板中的诱导基质更换为实验缓冲液(HBSS+20mM HEPES)，随后加入20μl等体积的2X Calcium 6dye，于37℃孵育2小时后置于室温待用。利用实验缓冲液将待测化合物和PAR4激动剂稀释至6X浓度，加入10μl 6X待测化合物至384孔细胞培养板，于室温孵育30分钟后，利用FLIPR Tetra将10μl 6X PAR4激动剂加入384孔细胞培养板，进行数据的测定和分析。整个反应体系为60μl，PAR4激动剂的终浓度为1.3μM(EC50)，DMSO的终浓度为0.3％。Flp-In-TREx-293-PAR4 cells cultured in DEME high glucose (Gibco), 10% fetal bovine serum, 2 mM GlutaMAX, 1% penicillin-streptomycin, 15 μg/ml blasticidin and 200 μg/ml hygromycin The medium was cultured in a 37 ° C, 5% CO 2 cell incubator. The cells were seeded into poly-d-lysine-treated 384-well cell culture plates (Corning, 3845) at a seeding density of 8,000 cells/well/25 μl of cell seeding matrix (DMEM high glucose, 10% fetal bovine serum, 2 mM GlutaMAX). ) Incubate overnight in a cell culture incubator. A 25 μl volume of 2X (400 ng/ml) tetracycline was added to induce the substrate to continue to culture for 24 h. On the day of the experiment, the induction matrix in the culture plate was changed to the experimental buffer (HBSS + 20 mM HEPES), followed by the addition of 20 μl of an equal volume of 2X Calcium 6dye, incubated at 37 ° C for 2 hours and then left at room temperature for use. The test compound and the PAR4 agonist were diluted to a 6X concentration using the experimental buffer, 10 μl of the 6X test compound was added to the 384-well cell culture plate, and after incubation for 30 minutes at room temperature, 10 μl of the 6X PAR4 agonist was added to the 384-well cell using FLIPR Tetra. The plates were incubated and the data were measured and analyzed. The entire reaction system was 60 μl, the final concentration of the PAR4 agonist was 1.3 μM (EC50), and the final concentration of DMSO was 0.3%.
实验结果如下：The experimental results are as follows:
Figure PCTCN2019086557-appb-000095
Figure PCTCN2019086557-appb-000095
Figure PCTCN2019086557-appb-000096
Figure PCTCN2019086557-appb-000096
注：A:0.1-20nM；B:20-100nM。Note: A: 0.1-20nM; B: 20-100nM.
结果显示：本发明所述化合物具有高活性The results show that the compound of the invention has high activity
实施例21 PAR4拮抗剂抗血小板凝聚试验Example 21 Anti-platelet aggregation test of PAR4 antagonist
人洗涤血小板样品的制备：Preparation of human washed platelet samples:
1.健康人全血样品的采集：使用5mL离心管(海门市昊瑞实验器材经营部)，每管加入600uL体积的ACD缓冲液(北京百奥莱博科技有限公司，ACD抗凝剂)。每管采集全血样品至4mL刻度(约采集全血3.4mL)，温和颠倒4-5次混匀，样品采集后20分钟内离心，离心前处于常温保存。1. Collection of whole blood samples from healthy people: Using a 5 mL centrifuge tube (Haimen City Ruirui Experimental Equipment Business Department), 600 uL volume of ACD buffer (Beijing Baiao Laibo Technology Co., Ltd., ACD anticoagulant) was added to each tube. Whole blood samples were taken from each tube to a 4 mL scale (about 3.4 mL of whole blood was collected), gently mixed 4-5 times to mix, centrifuged within 20 minutes after sample collection, and stored at room temperature before centrifugation.
2.富血小板血浆(PRP)的制备：全血样品在170g，常温条件下离心(Eppendorf 5810R型号离心机)14分钟，离心后收集上层较澄清液体至另一洁净空5mL离心管(海门市昊瑞实验器材经营部)，作为富血小板血浆样品。2. Preparation of platelet-rich plasma (PRP): Whole blood samples were centrifuged at 170 g (Eppendorf 5810R centrifuge) for 14 minutes at normal temperature. After centrifugation, the upper clear liquid was collected to another clean empty 5 mL centrifuge tube (Haimen City 昊Rui Experimental Equipment Business Department), as a platelet-rich plasma sample.
3.血小板初次富集及洗涤：富血小板血浆样品在1300g(Eppendorf 5810R型号离心机)，常温条件下离心6分钟；离心后弃去上层澄清液体，使用ACD缓冲液小心缓慢地重悬、洗涤沉淀物一次。3. Primary enrichment and washing of platelets: platelet-rich plasma samples were centrifuged at 1300 g (Eppendorf Model 5810R centrifuge) for 6 minutes at room temperature; after centrifugation, the supernatant liquid was discarded and carefully resuspended and washed with ACD buffer. Once again.
4.人洗涤血小板样品的制备：将重悬后的血小板样品在1300g，常温条件下离心6分钟；离心后弃去上层澄清液体，使用台式液(北京百奥莱博科技有限公司)小心缓慢地重悬沉淀，至洗涤后血小板终浓度为2-4X10 8/mL，常温保存待当天用。 4. Preparation of human washed platelet samples: The resuspended platelet samples were centrifuged at 1300 g for 6 minutes at room temperature; after centrifugation, the supernatant liquid was discarded, using a benchtop liquid (Beijing Biolebo Technology Co., Ltd.) carefully and slowly Resuspend the pellet until the final concentration of platelets after washing is 2-4× 10 8 /mL, and store at room temperature for use on the same day.
5.(选做)乏血小板血浆(PPP)的制备：#2步骤后的下层血液样品，在1800g(Eppendorf 5810R型号离心机)、常温条件下离心10分钟；离心后，收集上清至另一洁净空5mL离心管(海门市昊瑞实验器材经营部)，作为乏血小板血浆样品，常温保存待用。5. (Optional) Preparation of platelet-poor plasma (PPP): The lower blood sample after step #2 was centrifuged at 1800 g (Eppendorf Model 5810R centrifuge) at room temperature for 10 minutes; after centrifugation, the supernatant was collected to another Clean empty 5mL centrifuge tube (Haimen City, Rui Rui experimental equipment business department), as a sample of platelet-poor plasma, stored at room temperature for use.
受试物与人洗涤血小板样品的孵育：Incubation of test substance with human washed platelet samples:
1.孵育前准备：37℃水浴锅(常州国华电器有限公司，HH-4数显恒温水浴锅)，血小板聚集分析仪(北京世帝科学仪器有限责任公司，LG-PABER-I半自动凝血分析仪)开机预热。1. Preparation before incubation: 37 ° C water bath (Changzhou Guohua Electric Co., Ltd., HH-4 digital thermostat water bath), platelet aggregation analyzer (Beijing Shidi Scientific Instrument Co., Ltd., LG-PABER-I semi-automatic coagulation analysis Instrument) start warm up.
2.受试物与人洗涤血小板样品的孵育：取一洁净500uL规格的离心管，标明待测受试物名称及浓度；取276uL人洗涤血小板样品加入其中，再加入60uL待测受试物，温和混匀4-6次后，放置于水浴锅内孵育，并开始计时。水浴孵育20分钟。2. Incubation of the test substance with the human washed platelet sample: Take a clean 500uL centrifuge tube to indicate the name and concentration of the test object to be tested; take 276uL of human washed platelet sample and add 60uL of the test object to be tested. After gently mixing for 4-6 times, place in a water bath and incubate and start timing. Incubate for 20 minutes in a water bath.
血小板最大聚集率(MAR％)的测定：Determination of maximum platelet aggregation rate (MAR%):
1.测定基线：使用台式液或乏血小板血浆(PPP)作为基线物质，取300uL，加入比色杯(北京世帝科学仪器有限责任公司)进行基线值的测定，该数值仅用于仪器自动计算最大聚集率的测定。1. Determination of baseline: using benchtop fluid or platelet-poor plasma (PPP) as the baseline material, take 300uL, add cuvette (Beijing Shidi Scientific Instrument Co., Ltd.) to determine the baseline value, which is only used for automatic calculation of the instrument. Determination of the maximum aggregation rate.
2.孵育后的目标洗涤血小板的测定：测定时间设定为600s，测定模式为Aggr模式；取280uL孵育后的洗涤血小板样品，加入至比色杯(北京世帝科学仪器有限责任公司)中，加入一枚震荡珠(北京世帝科学仪器有限责任公司)并预热1分钟。预热完成后，将比色杯置于测试通道，按开始按钮10秒中后，缓缓加入20uL的PAR4-AP激动剂或20ul先导化合物，加入过程中枪头不进入液面下很深的位置，得到MAR值。2. Determination of target washed platelets after incubation: the measurement time was set to 600 s, and the measurement mode was Aggr mode; the washed platelet samples after 280 uL incubation were added to a cuvette (Beijing Shidi Scientific Instrument Co., Ltd.). Join a shocking bead (Beijing Shidi Scientific Instrument Co., Ltd.) and warm up for 1 minute. After the preheating is completed, place the cuvette in the test channel. After pressing the start button for 10 seconds, slowly add 20uL of PAR4-AP agonist or 20ul of lead compound. During the addition process, the tip does not enter the deep surface. Position, get the MAR value.
3.阴性激动剂对照：如2的操作，仅将最后加入的PARP-AP激动剂更换为激动剂所用的溶媒(15％HBSS+20mM HEPES的生理盐水溶液)，试验结果作用为阴性激动剂对照。3. Negative agonist control: As in the operation of 2, only the last added PARP-AP agonist was replaced with the vehicle used for the agonist (15% HBSS + 20 mM HEPES physiological saline solution), and the test result was a negative agonist control. .
血小板最大聚集率及抑制率的计算：Calculation of maximum platelet aggregation rate and inhibition rate:
1.血小板最大聚集率＝(600s时比浊度值-基线值)/(10s内比浊度值-基线值)1. Maximum platelet aggregation rate = (turbidity value at baseline - baseline value at 600s) / (turbidity value within 10s - baseline value)
2.血小板聚集抑制率(％)＝1-(待测化合物MAR％-阴性激动剂MAR％)/(阴性对照MAR％-阴性激动剂MAR％)*100％2. Platelet aggregation inhibition rate (%) = 1 - (test compound MAR% - negative agonist MAR%) / (negative control MAR% - negative agonist MAR%) * 100%
体外血小板凝聚试验数据如下：The data of in vitro platelet aggregation test are as follows:
化合物编号Compound number IC50 in PAgT(nM)IC50 in PAgT(nM)
BMS-986120BMS-986120 2.6,n＝112.6, n=11
SAL02-119SAL02-119 1.79,n＝11.79, n=1
结果显示：本发明所述化合物抗血小板凝聚作用优于参比化合物BMS986120。The results show that the anti-platelet aggregation effect of the compound of the present invention is superior to the reference compound BMS986120.
实施例22Example 22
Caco-2细胞渗透性试验Caco-2 cell permeability test
1.试验材料Test material
1.1待测化合物由委托方提供。对照组化合物地高辛购自当地供应商，***购自Sigma公司.1.2非必需氨基酸(NEAA)，HEPES和Hank’s平衡缓冲液(HBSS)购自Gibco公司。胎牛血清(FBS)购自Corning公司。青霉素/链霉素混合溶液(100x)和胰蛋白酶/EDTA溶液购自北京索莱宝科技有限公司。1.1 The test compound is provided by the client. The control compound digoxin was purchased from a local supplier, propranolol was purchased from Sigma, Inc. 1.2 non-essential amino acids (NEAA), HEPES and Hank's equilibration buffer (HBSS) were purchased from Gibco. Fetal bovine serum (FBS) was purchased from Corning. Penicillin/streptomycin mixed solution (100x) and trypsin/EDTA solution were purchased from Beijing Suobao Technology Co., Ltd.
1.3 HTS 96-well transwell细胞培养板购自Corning公司。电阻仪购自World Precision Instruments公司。Infinite 200PRO酶标仪购自Tecan。MTS2/4混合器购自IKA公司。1.3 HTS 96-well transwell cell culture plates were purchased from Corning. The resistance meter was purchased from World Precision Instruments. The Infinite 200PRO microplate reader was purchased from Tecan. The MTS 2/4 mixer was purchased from IKA.
2.试验方案2. Test plan
Caco-2于Transwell上培养14-18天，然后开始待测化合物双向渗透试验。测定化合物由顶端到基底端的转运速率，以及基底端到顶端的转运速率，共孵育2小时。使用LC-MS/MS测定顶端和基底端的化合物浓度，并计算化合物的表观渗透系数。Caco-2 was cultured on Transwell for 14-18 days and then the bidirectional permeation test of the test compound was started. The rate of transport of the compound from the apical to the basal end, as well as the rate of transport from the basal end to the apex, was measured for a total of 2 hours. The concentration of the compound at the apical and basal ends was determined using LC-MS/MS and the apparent permeability coefficient of the compound was calculated.
3.试验步骤3. Test procedure
细胞培养Cell culture
1)使用含L-谷氨酰胺的高糖DMEM培养基，添加10％胎牛血清、1×青霉素/链霉素混合溶液以及1×非必需氨基酸用于细胞培养。1) Using a high glucose DMEM medium containing L-glutamine, 10% fetal bovine serum, 1 x penicillin/streptomycin mixed solution, and 1 x non-essential amino acids were added for cell culture.
2)Caco-2培养于T-75细胞培养瓶。培养箱设置为37℃、5％CO 2、保证相对湿度95％。细胞汇合度达到70‐90％时可用于接种Transwell。 2) Caco-2 was cultured in a T-75 cell culture flask. The incubator was set to 37 ° C, 5% CO 2 , and the relative humidity was 95%. Transwell can be used to inoculate Transwell when the cell confluence reaches 70-90%.
3)细胞接种前，向Transwell上室每孔中加入50μL细胞培养基，下层培养板内加入25mL细胞培养基。将培养板置于37℃,5％CO 2培养箱内孵育1小时后可用于接种细胞。 3) Before cell seeding, 50 μL of cell culture medium was added to each well of Transwell upper chamber, and 25 mL of cell culture medium was added to the lower culture plate. The plate was incubated at 37 ° C in a 5% CO 2 incubator for 1 hour and was used to inoculate cells.
4)使用5mL PBS轻轻清洗细胞。弃掉PBS，加入1.5mL含EDTA的胰酶,于37℃孵育5到10分钟至细胞完全脱落。加入含血清的培养基终止消化。4) Gently wash the cells with 5 mL PBS. Discard the PBS, add 1.5 mL of EDTA-containing trypsin, and incubate at 37 ° C for 5 to 10 minutes until the cells are completely detached. The digestion was terminated by the addition of serum-containing medium.
5)吸取细胞混悬液转移至圆底离心管，于120×g离心10分钟。5) Pipette the cell suspension to a round bottom centrifuge tube and centrifuge at 120 xg for 10 minutes.
6)使用培养基重悬细胞，终浓度为6.86×10 5cells/mL。 6) Resuspend the cells in medium at a final concentration of 6.86 x 10 5 cells/mL.
Caco-2细胞接种Caco-2 cell inoculation
1)将上述细胞悬液以50μL每孔加入到96孔Transwell培养板上室中，最终接种密度为2.4×10 5cells/cm 2。 1) The above cell suspension was added to a 96-well Transwell plate in a 50 μL per well, and the final seeding density was 2.4 × 10 5 cells/cm 2 .
2)接种后48小时开始换液，培养14-18天，隔一天换一次培养基。2) Change the medium 48 hours after inoculation, culture for 14-18 days, and change the medium every other day.
3)更换培养基过程如下，将Transwell小室与接收板分开，先弃掉接收板中培养基然后再弃掉Transwell小室培养基，最后每个小室加入100μL新鲜培养基，接收板加入25mL新鲜培养基。3) The process of changing the medium is as follows. Separate the Transwell chamber from the receiving plate, discard the medium in the receiving plate and then discard the Transwell chamber medium. Finally, add 100 μL of fresh medium to each chamber, and add 25 mL of fresh medium to the receiving plate. .
细胞单层膜完整性的评价Evaluation of cell monolayer membrane integrity
1)Caco-2经过14天培养后，应完全汇合并完成分化。此时，可应用于穿透试验。1) After 14 days of incubation, Caco-2 should be completely pooled to complete differentiation. At this time, it can be applied to the penetration test.
2)用电阻仪测量单层膜电阻，记录每孔电阻。2) Measure the resistance of the single layer film with a resistance meter and record the resistance of each hole.
3)测定结束后，将Transwell培养板放回培养箱。3) After the measurement is completed, the Transwell plate is returned to the incubator.
4)电阻值的计算：4) Calculation of resistance value:
测定电阻值(ohms)x膜面积(cm 2)＝TEER值(ohm·cm 2) Determination of resistance (ohms) x membrane area (cm 2 ) = TEER value (ohm·cm 2 )
若TEER值<230ohms·cm 2，则该孔不能用于穿透试验。 If the TEER value is <230 ohms.cm 2 , the hole cannot be used for the penetration test.
溶液配制Solution preparation
1)配制1L HBSS(10mM HEPES，pH 7.4)，分别称取2.38g HEPES，0.35g碳酸氢钠，加900mL纯水让其溶解，然后加100mL 10×HBSS搅拌均匀，调PH至7.4，最后过滤。1) Prepare 1L HBSS (10mM HEPES, pH 7.4), weigh 2.38g HEPES, 0.35g sodium bicarbonate, add 900mL pure water to dissolve it, then add 100mL 10×HBSS to stir evenly, adjust PH to 7.4, finally filter .
2)配制待测药和对照药的10mM DMSO储备液，用HBSS(10mM HEPES,pH 7.4)稀释得到5μM工作液。对于亲脂性或低溶解度的化合物，通过向HBSS缓冲液中加3％BSA来提高化合物的回收率。2) A 10 mM DMSO stock solution of the test drug and the control drug was prepared and diluted with HBSS (10 mM HEPES, pH 7.4) to obtain a 5 μM working solution. For lipophilic or low solubility compounds, the recovery of the compound is increased by adding 3% BSA to the HBSS buffer.
药物穿透试验Drug penetration test
1)从培养箱中取出Transwell培养板。使用HBSS(10mM HEPES,pH 7.4)缓冲液润洗细胞单层膜两次，37℃条件下孵育30分钟。1) Remove the Transwell plate from the incubator. The cell monolayer membrane was incubated twice with HBSS (10 mM HEPES, pH 7.4) buffer and incubated for 30 minutes at 37 °C.
2)测定化合物由顶端到基底端的转运速率。向上层小室(顶端)每孔加入75μL含待测化合物的HBSS(10mM HEPES,pH 7.4)缓冲液，下层小室(基底端)每孔加入235μL HBSS(10mM HEPES,pH 7.4)缓冲液。2) Determine the rate of transport of the compound from the apical to the basal end. 75 μL of HBSS (10 mM HEPES, pH 7.4) buffer containing the test compound was added to each well of the upper chamber (top), and 235 μL of HBSS (10 mM HEPES, pH 7.4) buffer was added to each well of the lower chamber (basal end).
3)测定化合物由基底端到顶端的转运速率。向上层小室(顶端)每孔加入75μL HBSS(10mM HEPES,pH 7.4)缓冲液，下层小室(基底端)每孔加入235μL含待测化合物的HBSS(10mM HEPES,pH 7.4)缓冲液。转运体系置于涡旋器上于150转/分钟、37℃条件下孵育2小时。3) Determine the rate of transport of the compound from the basal end to the apex. 75 μL of HBSS (10 mM HEPES, pH 7.4) buffer was added to each well of the upper chamber (top), and 235 μL of HBSS (10 mM HEPES, pH 7.4) buffer containing the test compound was added to each well of the lower chamber (basal end). The transport system was incubated on a vortexer at 150 rpm for 2 hours at 37 °C.
4)孵育完成后，分别从Transwell培养板上室和下室每孔取样50μL加入到新的样品管中。向样品管内加入4倍体积含内标的乙腈(100nM阿普***，200nM咖啡因，200nM拉贝洛尔，2μM酮洛芬)，涡旋10分钟后，于3,220g离心30分钟。吸取上清液100μL，与等体积水稀释之后进行LC-MS/MS分析。各样品均采用双平行孵育。4) After the incubation was completed, 50 μL of each well from the Transwell plate and the lower chamber were separately added to the new sample tube. Four volumes of internal standard acetonitrile (100 nM alprazolam, 200 nM caffeine, 200 nM labetalol, 2 μM ketoprofen) were added to the sample tube, vortexed for 10 minutes, and centrifuged at 3,220 g for 30 minutes. 100 μL of the supernatant was aspirated and diluted with an equal volume of water and subjected to LC-MS/MS analysis. Each sample was incubated in double parallel.
实施例23Example 23
肝微粒体稳定性试验Liver microsome stability test
1.试验材料Test material
维拉帕米(货号：V4629-1G；批号：021M1429V)购于Sigma。混合的人肝微粒体和混合雄性大鼠肝微粒体购于康宁。混合雄性猴肝微粒体购于瑞德。贮存在-80℃冰箱备用。其他试剂购买于当地供应商。Verapamil (Cat. No.: V4629-1G; Lot No.: 021M1429V) was purchased from Sigma. Mixed human liver microsomes and mixed male rat liver microsomes were purchased from Corning. Mixed male monkey liver microsomes were purchased from Reid. Store in a refrigerator at -80 ° C for use. Other reagents are purchased from local suppliers.
2.试验方案2. Test plan
配置反应体系，加入NADPH启动反应，并于37℃进行水浴孵育，分别在0、15、30、45和60分钟进行取样。用LC-MS/MS检测待测药和阳性药，测定剩余百分比。计算待测化合物的半衰期以及体外固有清除率。The reaction system was configured, and the NADPH start-up reaction was added, and the cells were incubated at 37 ° C in a water bath, and samples were taken at 0, 15, 30, 45, and 60 minutes, respectively. The test drug and the positive drug were detected by LC-MS/MS, and the remaining percentage was determined. Calculate the half-life of the test compound as well as the intrinsic clearance rate in vitro.
3.试验步骤3. Test procedure
1)主要反应体系的配置见表.1) The configuration of the main reaction system is shown in the table.
表.主要反应体系的配置Table. Configuration of the main reaction system
Figure PCTCN2019086557-appb-000097
Figure PCTCN2019086557-appb-000097
Figure PCTCN2019086557-appb-000098
Figure PCTCN2019086557-appb-000098
2)将该反应体系放在37℃水浴中预孵育10分钟。向反应体系中加入40μL 10mM NADPH溶液来启动反应，NADPH的最终浓度为1mM。用40μL超纯水代替NADPH溶液作为阴性对照。阴性对照的作用是排除化合物自身化学稳定性的影响。2) The reaction system was pre-incubated for 10 minutes in a 37 ° C water bath. The reaction was initiated by adding 40 μL of 10 mM NADPH solution to the reaction system, and the final concentration of NADPH was 1 mM. 40 μL of ultrapure water was used instead of NADPH solution as a negative control. The effect of the negative control is to rule out the effects of the chemical stability of the compound itself.
3)在0,15,30,45和60分钟分别取出50μL反应样品，用4倍的含有内标(200nM阿普***,200nM拉贝洛尔,2μM酮洛芬,200nM咖啡因)的乙腈淬灭。样品在4,000转/分钟转速下离心40分钟。离心完成后取100μL上清液和100μL超纯水混匀用于LC-MS/MS分析检测。3) 50 μL of the reaction sample was taken at 0, 15, 30, 45 and 60 minutes, respectively, using 4 times acetonitrile containing an internal standard (200 nM alprazolam, 200 nM labetalol, 2 μM ketoprofen, 200 nM caffeine) Quenched. The sample was centrifuged at 4,000 rpm for 40 minutes. After centrifugation, 100 μL of the supernatant and 100 μL of ultrapure water were mixed for LC-MS/MS analysis.
体外相关结果统计如下：The in vitro correlation results are as follows:
Figure PCTCN2019086557-appb-000099
Figure PCTCN2019086557-appb-000099
结果显示：本发明所述化合细胞膜渗透好，肝微粒体内稳定性提高。The results showed that the conjugated cell membrane of the present invention penetrated well and the stability of the liver microparticles was improved.
根据已有文献报道，BMS-986120参比化合物水溶性很差，蛋白结合率高，使化合物在血浆游离浓度很低，从而要高剂量给药产生药效，大大降低了药物安全性窗口。According to the literature reports, the reference compound of BMS-986120 has poor water solubility and high protein binding rate, so that the compound has a low plasma free concentration, so that high dose administration produces a drug effect, which greatly reduces the drug safety window.
通过体外活性测试，本发明系列化合物体外活性高，尤其抗凝血活性高，与参比化合物活性相当，或者更优。另外，细胞膜渗透性，蛋白结合率，与参比化合物相似。体内药代药动力学参数也与参比化合物相当。By in vitro activity testing, the series of compounds of the invention have high activity in vitro, especially high anticoagulant activity, comparable to, or better than, the activity of the reference compound. In addition, cell membrane permeability, protein binding rate, is similar to the reference compound. The in vivo pharmacokinetic parameters are also comparable to the reference compound.
本发明化合物中的双环核心结构新颖是本专利所特有的。结构的独特性可能带来具有与参比化合物相比不同的药理药效，甚至不同的适应症。The novel bicyclic core structure in the compounds of the present invention is unique to this patent. The uniqueness of the structure may result in different pharmacological effects and even different indications compared to the reference compound.

Claims (15)

  1. 作为用于治疗血小板聚集的蛋白酶激活受体4(PAR4)抑制剂的化合物或其药学上可接受的盐,其特征在于，所述化合物的母核如下：A compound or a pharmaceutically acceptable salt thereof, which is a protease activated receptor 4 (PAR4) inhibitor for treating platelet aggregation, characterized in that the mother nucleus of the compound is as follows:
    Figure PCTCN2019086557-appb-100001
    Figure PCTCN2019086557-appb-100001
    其中，R 0选自氢，卤素，C 1-4烷基，C 1-4的氨基，C 1-4的烷氧基，C 1-4的烷硫基； Wherein R 0 is selected from the group consisting of hydrogen, halogen, C 1-4 alkyl, C 1-4 amino group, C 1-4 alkoxy group, C 1-4 alkylthio group;
    X、Z选自CH或者N；X, Z are selected from CH or N;
    Y选自O或者CH；Y is selected from O or CH;
    R 1选自氢，卤素、羟基、巯基、C 1-4的烷氧基； R 1 is selected from the group consisting of hydrogen, halogen, hydroxy, decyl, C 1-4 alkoxy;
    R 2选自氢，卤素、羟基、巯基、C 1-4的烷氧基、C 1-4的氨基取代的C 1-4烷氧基、C 3-6的环烷基取代的C 1-4烷氧基、羟基取代的C 1-4烷氧基、C 1-4的烷氧基-W-C 1-4烷氧基、C 3-6杂环-W-C 1-4烷氧基，其中W为键、(CH 2)n-O、羰基、(CH 2)n-S、(CH 2)n-吡啶或(CH 2)n-噻唑，其中C 3-6杂环可进一步被卤素、羟基所取代，噻唑或吡啶可进一步被C 1-4烷基或羟基取代，n＝0、1、2或3。 R 2 is selected from the group consisting of hydrogen, halogen, hydroxy, decyl, C 1-4 alkoxy, C 1-4 amino substituted C 1-4 alkoxy, C 3-6 cycloalkyl substituted C 1- 4 alkoxy, hydroxy-substituted C 1-4 alkoxy, C 1-4 alkoxy -WC 1-4 alkoxy, C 3-6 heterocycloalkyl -WC 1-4 alkoxy, wherein W Is a bond, (CH 2 ) nO, carbonyl, (CH 2 ) nS, (CH 2 ) n-pyridine or (CH 2 ) n-thiazole, wherein the C 3-6 heterocycle can be further substituted by halogen, hydroxy, thiazole Or the pyridine may be further substituted by a C 1-4 alkyl group or a hydroxyl group, n = 0, 1, 2 or 3.
  2. 根据权利要求1所述化合物或其药学上可接受的盐，其特征在于，其中，所述卤素选自氟、氯、溴、碘。A compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein said halogen is selected from the group consisting of fluorine, chlorine, bromine and iodine.
  3. 根据权利要求1所述化合物或其药学上可接受的盐，其特征在于，其中，所述C 1-4的烷基选自甲基、乙基、丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基。 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the C 1-4 alkyl group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, Isobutyl, sec-butyl, tert-butyl.
  4. 根据权利要求1所述化合物或其药学上可接受的盐，其特征在于，其中，所述C 1-4的氨基选自甲氨基、乙氨基、丙氨基、异丙胺基、正丁氨基、异丁氨基、仲丁氨基和叔丁氨基、二甲氨基。 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the amino group of C 1-4 is selected from the group consisting of methylamino, ethylamino, propylamino, isopropylamino, n-butylamino, and iso Butylamino, sec-butylamino and tert-butylamino, dimethylamino.
  5. 根据权利要求1所述化合物或其药学上可接受的盐，其特征在于，其中，所述C 1-4的烷氧基选自甲氧基、乙氧基、丙氧基、异丙氧基、正丁氧基、异丁氧基、仲丁氧基、叔丁氧基。 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the C 1-4 alkoxy group is selected from the group consisting of methoxy, ethoxy, propoxy and isopropoxy. , n-butoxy, isobutoxy, sec-butoxy, tert-butoxy.
  6. 根据权利要求1所述化合物或其药学上可接受的盐，其特征在于，其中，所述C 1-4的烷硫基选自甲硫基、乙硫基、丙硫基、异丙硫基、正丁硫基、异丁硫基、仲丁硫基、叔丁硫基。 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the C 1-4 alkylthio group is selected from the group consisting of methylthio, ethylthio, propylthio and isopropylthio. , n-butylthio, isobutylthio, sec-butylthio, tert-butylthio.
  7. 根据权利要求1所述化合物或其药学上可接受的盐，其特征在于，其中，C 3-6的环烷基选自环丙烷、环戊烷、环己烷。 The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the C 3-6 cycloalkyl group is selected from the group consisting of cyclopropane, cyclopentane, and cyclohexane.
  8. 根据权利要求1所述化合物或其药学上可接受的盐，其特征在于，其中，其中C 3-6杂环选自
    Figure PCTCN2019086557-appb-100002
    Figure PCTCN2019086557-appb-100003
    The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the C 3-6 heterocyclic ring is selected from the group consisting of
    Figure PCTCN2019086557-appb-100002
    Figure PCTCN2019086557-appb-100003
  9. 根据权利要求1所述化合物或其药学上可接受的盐，其特征在于，所述化合物通式结构如下式(II-1，II-2)所示：The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound has a general structure as shown in the following formula (II-1, II-2):
    Figure PCTCN2019086557-appb-100004
    Figure PCTCN2019086557-appb-100004
    其中，R 0、R 1、X如上定义； Wherein R 0 , R 1 , and X are as defined above;
    m等于0，1，2，3或4；m is equal to 0, 1, 2, 3 or 4;
    当A选自氧原子，B选自H、C 1-4烷基、或者烷氧基取代的C 1-4的烷基； When A is selected from an oxygen atom, B is selected from H, C 1-4 alkyl, or alkoxy substituted C 1-4 alkyl;
    当A选自羰基，B选自羟基、氨基或者C 1-4烷氧基； When A is selected from a carbonyl group, B is selected from a hydroxyl group, an amino group or a C 1-4 alkoxy group;
    当A选自亚甲基，B选自羟基、C 3-6的环烷基、含氮和/或氧C 3-6的杂环、C 1-4烷基取代或者非取代氨基。 When A is selected from a methylene group, B is selected from a hydroxyl group, a C 3-6 cycloalkyl group, a nitrogen-containing and/or oxygen C 3-6 heterocyclic ring, a C 1-4 alkyl-substituted or unsubstituted amino group.
    当A选自C 1-4烷基取代或非取代的噻唑环、取代或非取代的苯环、取代或非取代的吡啶环，B选自
    Figure PCTCN2019086557-appb-100005
    其中R 3、R 4选自氢、甲基、羟基，所述
    Figure PCTCN2019086557-appb-100006
    为连接键。     When A is selected from a C 1-4 alkyl-substituted or unsubstituted thiazole ring, a substituted or unsubstituted benzene ring, a substituted or unsubstituted pyridine ring, B is selected from
    Figure PCTCN2019086557-appb-100005
    Wherein R 3 and R 4 are selected from the group consisting of hydrogen, methyl, and hydroxy,
    Figure PCTCN2019086557-appb-100006
    For the connection key.
  10. 根据权利要求1所述化合物或其药学上可接受的盐，其特征在于，The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that
    X、Y、Z如上定义；X, Y, Z are as defined above;
    R 0选自氢、甲氧基； R 0 is selected from the group consisting of hydrogen and methoxy;
    R 1选自氢、甲氧基； R 1 is selected from the group consisting of hydrogen and methoxy;
    R 2选自羟基、甲氧基、
    Figure PCTCN2019086557-appb-100007
    甲氧乙氧基、
    Figure PCTCN2019086557-appb-100008
    羟乙氧基、
    Figure PCTCN2019086557-appb-100009
    甲氧丙氧基、
    Figure PCTCN2019086557-appb-100010
    二甲氧基、
    Figure PCTCN2019086557-appb-100011
    R 2 is selected from the group consisting of hydroxyl, methoxy,
    Figure PCTCN2019086557-appb-100007
    Methoxyethoxy,
    Figure PCTCN2019086557-appb-100008
    Hydroxyethoxy,
    Figure PCTCN2019086557-appb-100009
    Methoxypropoxy,
    Figure PCTCN2019086557-appb-100010
    Dimethoxy,
    Figure PCTCN2019086557-appb-100011
  11. 根据权利要求1所述化合物或其药学上可接受的盐，其特征在于，所述权利要求1化合物与无机酸或有机酸成盐，所述的无机酸或有机酸选自2-乙酰氧基苯甲酸、2-羟基乙磺酸、乙酸、抗坏血酸、苯磺酸、苯甲酸、氢碳酸、碳酸、柠檬酸、依地酸、乙烷二磺酸、乙烷磺酸、富马酸、葡庚糖酸、葡糖酸、谷氨酸、乙醇酸、氢溴酸、盐酸、氢碘酸、羟萘酸、羟乙磺酸、乳酸、乳糖酸、十二烷基磺酸、马来酸、苹果酸、扁桃酸、甲烷磺酸、硝酸、草酸、双羟萘酸、泛酸、苯乙酸、磷酸、多聚半乳糖醛酸、丙酸、水杨酸、硬脂酸、亚乙酸、琥珀酸、氨基磺酸、对氨基苯磺酸、硫酸、单宁酸、酒石酸和对甲苯磺酸。The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of claim 1 is formed with a mineral acid or an organic acid selected from the group consisting of 2-acetoxy groups. Benzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, hydrogen carbonate, carbonic acid, citric acid, edetic acid, ethane disulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptane Sugar acid, gluconic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, dodecyl sulfonic acid, maleic acid, apple Acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, amino Sulfonic acid, p-aminobenzenesulfonic acid, sulfuric acid, tannic acid, tartaric acid and p-toluenesulfonic acid.
  12. 根据权利要求1所述化合物，其特征在于，选自以下化合物：A compound according to claim 1 which is selected from the group consisting of:
    Figure PCTCN2019086557-appb-100012
    Figure PCTCN2019086557-appb-100012
    Figure PCTCN2019086557-appb-100013
    Figure PCTCN2019086557-appb-100013
  13. 一种药物组合物，其特征在于，包括如权利要求1-12中任一项所述的化合物或其药学上可接受的盐和一种以上药学上可接受的载体。A pharmaceutical composition comprising a compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.
  14. 如权利要求1-12任一项所述的化合物或其药学上可接受的盐在制备用于制备治疗血栓相关疾病的药物用途。A pharmaceutical use according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment of a thrombosis-related disease.
  15. 根据权利要求14的用途，其特征在于，所述血栓相关疾病选自动脉心血管血栓栓塞性病症、静脉心血管血栓栓塞性病症、脑血管血栓栓塞性病症、和心脏腔室或外周循环中的血栓栓塞性病症。The use according to claim 14, wherein said thrombotic-related disease is selected from the group consisting of an arterial cardiovascular thromboembolic disorder, an intravenous cardiovascular thromboembolic disorder, a cerebrovascular thromboembolic disorder, and a cardiac chamber or peripheral circulation. A thromboembolic disorder.
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