WO2016058501A1 - 5-甲基-2-(吡啶-2-基氨基)-8H-吡啶并[2,3-d]嘧啶-7-酮化合物 - Google Patents

5-甲基-2-(吡啶-2-基氨基)-8H-吡啶并[2,3-d]嘧啶-7-酮化合物 Download PDF

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WO2016058501A1
WO2016058501A1 PCT/CN2015/091668 CN2015091668W WO2016058501A1 WO 2016058501 A1 WO2016058501 A1 WO 2016058501A1 CN 2015091668 W CN2015091668 W CN 2015091668W WO 2016058501 A1 WO2016058501 A1 WO 2016058501A1
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compound
added
pharmaceutically acceptable
acceptable salt
cancer
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PCT/CN2015/091668
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French (fr)
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江岳恒
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上海页岩科技有限公司
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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
    • A61K31/495—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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/5383—1,4-Oxazines, e.g. morpholine ortho- or peri-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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—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 relates to the field of medical technology, and in particular to 5-methyl-2-(pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one compounds or pharmaceutically acceptable thereof Salts, enantiomers, diastereomers, racemates or mixtures thereof, prodrugs, solvent compounds, and methods for their preparation, pharmaceutical compositions comprising these materials, and uses thereof.
  • G1 phase growth phase
  • S phase DNA synthesis phase
  • G2 and M phase mitotic phase
  • Each phase in the cell cycle is regulated by a cyclin-dependent kinase (CDK).
  • CDK4 The key regulatory protein kinase that determines the G1-S phase transition is CDK4, which forms a complex with cyclin D to phosphorylate retinoblastoma (Rb) proteins.
  • the tumor suppressor protein Rb Once the tumor suppressor protein Rb is phosphorylated, it releases its transcription factor E2F, which binds tightly in an unphosphorylated state, while E2F activates further transcription and pushes the cell cycle through a restriction checkpoint (point R). Once the cell passes through the R point, it irreversibly enters the S phase.
  • CDK1 and CDK2 cooperate with their partners, cyclin A and B, to play an important role in pushing cells into M phase, completing cell division and growth cycle.
  • cell cycle regulation is disrupted, and cancer cells become unregulated, divide, and replicate. Therefore, finding and discovering selective and potent CDK inhibitors in response to cell cycle pathways has become an oncology for many years. The main goal of drug research.
  • CDK4 gene amplification or high protein expression which is frequently found in highly differentiated and dedifferentiated liposarcoma And also in some other solid tumors and hematological malignancies;
  • cyclin D1 which is observed in mantle cell lymphoma, also in various solid tumors;
  • p16INK4A gene The loss of CDKN2A, which is also a common event in many cancers.
  • the p16INK4A protein is a natural inhibitor of CDK4, and cancer with p16 loss is generally susceptible to inhibition of CDK4.
  • CDK4/CDK6 inhibitors that have entered clinical trials: palbociclib (PD0332991), LEE011 and LY2835219. These inhibitors prevent CDK4/6 from forming complexes with Cyclin D, effectively blocking the cell cycle from G1 to S phase.
  • the present invention provides a series of 5-methyl-2-(pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one compounds, pharmaceutically acceptable salts thereof, Enantiomers, diastereomers, racemates, prodrug molecules or solvates useful in the preparation of a medicament for the treatment of diseases associated with the cyclin-dependent kinase signaling pathway.
  • the present invention provides a compound represented by the following formula I, a pharmaceutically acceptable salt thereof, an enantiomer, a diastereomer, a racemate, a prodrug molecule or a solvate thereof,
  • R 1 is selected from the group consisting of:
  • R 2 and R 2 ' are each independently selected from the group consisting of H, hydrazine (D), C1-C4 alkyl, fluorine and cyano, and R 2 and R 2 ' may be on the same or different carbon atoms;
  • n 1 or 2
  • R 3 is selected from the group consisting of:
  • R 4 is selected from the group consisting of H, hydrazine (D) and C1-C4 alkyl,
  • W is selected from CH 2 , CH 2 CH 2 , O and NH
  • X is selected from CH and N
  • Y is selected from CH 2 , NH and O
  • n 0, 1 or 2;
  • R 1 is selected from the group consisting of:
  • R 3 is selected from the group consisting of:
  • R 1 is selected from the group consisting of:
  • R 3 is selected from the group consisting of:
  • R 1 is selected from the group consisting of:
  • R 3 is selected from the group consisting of:
  • the 5-methyl-2-(pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one compound of the present invention a pharmaceutically acceptable salt thereof
  • the enantiomers, diastereomers, racemates, prodrug molecules or solvates include the following compounds:
  • the above compounds include enantiomers, diastereomers, racemates and mixtures thereof, prodrug molecules or solvates thereof, for example, compound 1 has enantiomers 1A and 1B, and compound 5 Having enantiomers 5A and 5B, compound 7 having enantiomers 7A and 7B, compound 24 having diastereomers 24A and 24B, and compound 25 having diastereomers 25A and 25B, compound 26 There are diastereomers 26A and 26B and diastereomers 27A and 27B.
  • the compound is selected from the group consisting of:
  • cis denotes cis and trans denotes trans.
  • Another aspect of the present invention provides the 5-methyl-2-(pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one compound, which is pharmaceutically acceptable Use of accepted salts, enantiomers, diastereomers, racemates, prodrug molecules or solvates for the preparation of CDK4 and/or CDK6 selective inhibitors.
  • Another aspect of the invention also provides the 5-methyl-2-(pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one compound, which is pharmaceutically acceptable Salt, enantiomer, diastereomer, racemate, prodrug molecule or
  • a composition especially as a selective inhibitor of CDK4 and/or CDK6, for the manufacture of a medicament for the treatment of a disease associated with a cyclin-dependent kinase signaling pathway.
  • the diseases associated with the cyclin-dependent kinase signaling pathway include tumors such as breast cancer, glioma, glioblastoma, lung cancer, colorectal cancer, gastric cancer, gastrointestinal stromal tumor (GIST), hepatocytes Cancer, prostate tumor, sarcoma, ovarian cancer, cervical cancer, pancreatic cancer, melanoma, thyroid cancer, cholangiocarcinoma, endometrial cancer, renal cancer, mesothelioma, lymphoma, leukemia, non-Hodgkin's lymphoma, Denaturing large cell lymphoma, acute myeloid leukemia (AML), multiple myeloma.
  • tumors such as breast cancer, glioma, glioblastoma, lung cancer, colorectal cancer, gastric cancer, gastrointestinal stromal tumor (GIST), hepatocytes Cancer, prostate tumor, sarcoma, ovarian cancer, cervical cancer, pancreatic cancer,
  • Another aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a therapeutically effective amount of 5-methyl-2-(pyridin-2-ylamino)-8H- selected from the above-described according to the invention Pyrido[2,3-d]pyrimidin-7-one compounds, pharmaceutically acceptable salts, enantiomers, diastereomers, racemates and mixtures thereof, prodrug molecules And one or more of the solvates; and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is useful for in vivo treatment and is biocompatible.
  • a further object of the invention is to provide a compound of the invention, a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, prodrug molecule or solvate thereof
  • a method of treating cancer in combination with other anti-tumor drugs comprising administering the compound in combination, separately or sequentially, with pharmaceutically acceptable salts, enantiomers, and diastereoisomers thereof. Isomers, racemates, prodrug molecules or solvates with other antitumor drugs.
  • anti-tumor agents can be administered in parallel, simultaneously, sequentially, or separately with the compounds of the invention and their salts or prodrugs, and can include, but are not limited to, the following types of anti-tumor agents One or more of: alkylating agents (eg, molybdenum, oxaliplatin, cisplatin, cyclophosphamide, nitrosourea, nitrogen mustard, melphalan), antimetabolites (eg, gemcitabine), and Antifolates (eg 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytarabine, hydroxyurea), topoisomerase inhibitors (eg etoposide, topotecan, hi Alkaline), anti-mitotic agents (eg, vincristine, vinblastine, vinorelbine, paclitaxel, taxotere), anti-tumor antibiotics (eg, doxorubicin, doxorubicin
  • the pharmaceutically acceptable salt is a conventional pharmaceutically acceptable salt, including a hydrochloride, a hydrobromide, a hydroiodide, a sulfate, a phosphate, a nitrate, an acetate, Trifluoroacetate, p-toluenesulfonate, salicylate, methanesulfonate, oxalate, succinate, citrate, malate, lactate, fumarate, and the like.
  • a compound of the invention also includes polymorphs thereof, ie, chemical drug molecules Crystals of different structures crystallized under different physicochemical conditions.
  • Step 1 Under a nitrogen atmosphere, A, 5-bromo-2,4-dichloropyrimidine, potassium carbonate and 1,4-dioxane were added to a three-necked flask, and the reaction was stirred at room temperature overnight. Ethyl acetate was added and washed with brine. The organic phase was dried over anhydrous sodium sulfate, and then applied to silica gel column chromatography.
  • Step 2 Nitrogen protection, add B, crotonic acid, N, N-diisopropylethylamine in a three-necked flask, stir for 10 minutes, add tris(2-tolyl)phosphine, di(benzonitrile) dichloride Palladium was added, and the nitrogen was replaced three times, and the reaction was heated overnight. After cooling to room temperature, it was diluted with ethyl acetate and backwashed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and dried to give Compound C.
  • Step 3 In a three-necked flask, under a nitrogen atmosphere, C was dissolved in re-distilled tetrahydrofuran, acetic anhydride was added, and the reaction was heated overnight. After cooling to room temperature, it was diluted with ethyl acetate and backwashed with saturated brine. The organic phase was dried over anhydrous sodium sulfate.
  • Step 4 In a three-necked flask, under the protection of nitrogen, the solid D is dissolved in acetic acid, sodium acetate is added, the temperature is lowered to 0 ° C, bromine is added dropwise, and the reaction is heated. After cooling to room temperature, the reaction was quenched by the addition of saturated sodium hydrogensulfite. Extract with dichloromethane and collect the organic phase. The mixture was washed with saturated brine and dried over anhydrous sodium sulfate.
  • Step 5 In a three-necked flask, under nitrogen protection, cool the H 2 NR toluene solution to 0 ° C, add hexamethyldisilazide lithium, stir at room temperature for 30 minutes, cool to 0 ° C, add E toluene dropwise The solution was added and the reaction was allowed to proceed overnight at room temperature. Diluted with dichloromethane and backwashed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness.
  • Step 6 Dissolve F in n-butanol under nitrogen, add vinyl n-butyl ether, add N,N-diisopropylethylamine, add catalytic amount 1,1'-two (Diphenylphosphino)ferrocene palladium (II) dichloride, after completion, the air was replaced three times, and the reaction was heated overnight. After cooling to room temperature, it was diluted with ethyl acetate and backwashed with saturated brine. The organic phase was collected and dried over anhydrous sodium sulfate.
  • 1,1'-two (Diphenylphosphino)ferrocene palladium (II) dichloride after completion, the air was replaced three times, and the reaction was heated overnight. After cooling to room temperature, it was diluted with ethyl acetate and backwashed with saturated brine. The organic phase was collected and dried over anhydrous sodium sulfate.
  • R is R 1 and R 3 are as defined above.
  • the inhibitory activity of the compound of the present invention, its pharmaceutically acceptable salt, enantiomer, diastereomer or racemate, prodrug molecule or solvate for CDK4 and/or CDK6 is not only present
  • the biological inhibitors of the technical inhibitors are comparable or better, and have better in vivo metabolic properties, higher blood levels, and fewer toxic side-effect metabolites than prior art inhibitors.
  • the analytical data of the sample is determined by the following instruments:
  • LC/MS shimadzu LCMS-2020
  • Chiral column CHIRALPAK AD-H, CHIRALPAK IA, CHIRALPAK IB, CHIRALPAK IC, Lux 3um Cellulose-4.
  • Nanjing Yaoshi Drug Research and Development Co., Ltd. Tiexiai (Shanghai) Chemical Industry Development Co., Ltd.; Shanghai Shuiya Pharmaceutical Technology Co., Ltd.; Zhangjiagang Amat Chemical Co., Ltd.; (Jiangsu) Yancheng Jinghua Chemical Co., Ltd.; Chengdu Ester Trading Co., Ltd.; Tianjin Famoxi Pharmaceutical Technology Co., Ltd.; Tianjin Guangfu Technology Development Co., Ltd.; Sichuan Xiqiao Chemical Co., Ltd.; Tianjin Fuchen Chemical Reagent Factory; Sinopharm Chemical Reagent Shaanxi Co., Ltd.; Shaanxi Xintong Chemical Co., Ltd.
  • Step 1 In a 2000 mL three-necked flask, 1-1 (80 g), aqueous ammonia solution (665 g, 25%) was added under nitrogen. The temperature was raised to 60 ° C and allowed to react overnight. The temperature was lowered and nitrogen was passed through the system for 1 hour. Adjust the pH of the system to 4-5 with hydrochloric acid (6N). After the solvent is dried, 1-2 is obtained, and the next step is directly taken.
  • DAST diethylaminosulfur trifluoride
  • Step 5 In a 250 mL three-necked flask, 1-5 (15 g) and dichloromethane (200 mL) were added. After passing hydrogen chloride gas, it was reacted at room temperature for 2 hours. The solvent was dried to give the product 1-6.
  • Step 1 Nitrogen protection. To a 250 mL three-necked flask, 2-1 (10 g), azidotrimethylsilane and toluene (120 mL) were added. After cooling to 20-30 ° C, boron trifluoride diethyl ether (17 g) was added dropwise to react at room temperature for 24 hours. The layers were allowed to stand, and the organic phase was washed with 1 ⁇ 50 mL of 10% aqueous sodium hydrogencarbonate and dried over anhydrous sodium sulfate.
  • Step 2 Under a nitrogen atmosphere, a 2 mL toluene solution, diethyl ether (100 mL) was added to a 500 mL three-necked flask. Then, triethoxyphosphorus (16.3 g) was slowly added, and the reaction was carried out at room temperature for 2 days. It was dried to a colorless transparent oil for the next reaction.
  • Step 3 Nitrogen protection.
  • 2-3 (2 g)
  • ethanol 60 mL
  • water 0.8 g
  • the filter cake was washed with 3 x 30 mL of diethyl ether and dried to give the desired product 2-4.
  • Step 1 500 mL of N,N-dimethyl sulfoxide was added to a 2000 mL four-necked flask under nitrogen. The temperature was lowered to 20-25 ° C, sodium hydride (19 g) was added in portions, and then trimethylsulfoxonium iodide (118 g) was added portionwise at this temperature, and stirred at this temperature for 1 hour, and added dropwise at 20 ° C. 3-1 (40 g) of a solution of N,N-dimethyl sulfoxide (100 mL). The system was stirred at 20-25 ° C for 30 minutes, then warmed to 50 ° C and reacted for 2 hours. After the reaction is completed, the temperature is lowered and poured into ice water to quench. It was extracted with 3 ⁇ 1000 mL of anhydrous diethyl ether. The filtrate was spun dry below 30 ° C to give 3-2 (crude).
  • Step 3 Lithium aluminum hydride (34 g) and 600 mL of tetrahydrofuran were added under a nitrogen atmosphere in a 2000 mL four-necked flask. The temperature was lowered to 0 ° C, and 3-3 was dissolved in 400 mL of tetrahydrofuran, added dropwise to the above system, and returned to room temperature for overnight reaction. Quenched by slowly adding 34 mL of water at 0 °C. Then add 102 mL of aqueous sodium hydroxide (15%) and 34 mL of water. After suction filtration, the filter cake was washed with 4 ⁇ 100 mL of tetrahydrofuran. The resulting filtrate was spun dry to give 3-4.
  • Step 1 Under a nitrogen atmosphere, a 1 L three-necked flask was charged with potassium hydroxide (46 g), water (12 mL), and ethanol (225 mL). 4-1 (130 g) was added dropwise under ice bath. The reaction was completed at room temperature for 10 minutes, and the reaction was completed. Filter and collect the filter cake. Wash the filter cake with 500 mL of ether and dry to obtain 4-2.
  • Step 2 Under a nitrogen atmosphere, 4-2 (140 g), N,N-dimethyl sulfoxide (1000 mL), 1,2-dibromoethane (174 g) were added to a 2 L three-necked flask. The reaction was carried out at room temperature overnight. Pour into 1 L of water to quench the reaction, extract with ether (300 mL x 3), and wash with brine. The organic phase was dried over anhydrous sodium sulfate and concentrated to give 4-3.
  • Step 3 Under a nitrogen atmosphere, 4-3 (210 g), hexamethylphosphoric triamide (1000 mL), and lithium chloride (19 g) were added to a 2 L three-necked flask. The temperature was raised to 140 ° C and allowed to react overnight. The product was distilled under reduced pressure, and the product was collected at 4 ° C to give 4-4.
  • Step 4 Under a nitrogen atmosphere, add 4-4 (16 g), ethanol (150 mL), water (150 mL), hydroxylamine hydrochloride (20 g), sodium carbonate (30 g) to a 500 mL three-necked flask. The reaction was carried out at room temperature overnight. 50 mL of water was added, and ethyl acetate was extracted (100 mL x 3), and the organic phase was collected. Wash with saline (200 mL x 2). The organic phase was dried over anhydrous sodium sulfate and concentrated 4-5.
  • Step 5 Under a nitrogen atmosphere, 4-5 (6 g), tetrahydrofuran (200 mL) was added to a 500 mL three-necked flask. The temperature was lowered to 0 ° C, and lithium aluminum hydride (5.5 g) was added in portions. After the addition, the temperature was raised to 55 ° C for 5 hours. Cool to 0 ° C and add 5.5 mL of water. The pH was adjusted to 7 with a 1N aqueous sodium hydroxide solution. An additional 5.5 mL of water and 200 mL of ethyl acetate were added. The organic phase was dried over anhydrous sodium sulfate, filtered and filtered. After the filtrate was dried, 5.5 g of oxalic acid was added to the filtrate, stirred for 30 minutes, filtered, and the filter cake was collected to give 4-6 of oxalate.
  • Step 1 Under a nitrogen atmosphere, a 5-1 (20 g), ethanol (250 mL), hydroxylamine hydrochloride (16 g), and pyridine (18.6 g) were added to a 500 mL three-necked flask. The temperature was raised to 80 ° C and allowed to react overnight. After cooling to room temperature, 200 mL of water was added, and ethyl acetate was extracted (200 mL x 3), and washed with brine (500 mL x 5). The organic phase was dried over anhydrous sodium sulfate and concentrated to 5-2.
  • Step 1 Under a nitrogen atmosphere, in a 2000 mL three-necked flask, 6-1 (60 g), toluene (600 mL), triethylamine (81 g), and diphenylphosphoryl azide (DPPA, 191 g) were added. The reaction system was reacted at 100 ° C for 1 hour. Then, the temperature was lowered to 40-50 ° C and benzyl alcohol was added dropwise. After the completion of the dropwise addition, the temperature was raised to 100 ° C and the reaction was allowed to proceed overnight. After cooling to room temperature, it was diluted with 500 mL of ethyl acetate, and 500 mL of x 3 was washed with saline. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated.
  • 6-1 60 g
  • toluene 600 mL
  • triethylamine 81 g
  • DPPA diphenylphosphoryl azide
  • Step 3 In a 1000 mL single-mouth bottle, anhydrous methanol (600 mL), 6-3 (45 g) and palladium on water (10 g, 10%) were added under nitrogen. Hydrogenation at room temperature overnight. After suction filtration, the filtrate was collected and the solvent was evaporated to give 6-4.
  • 7-1 80 g
  • triethylamine 107
  • Step 2 In a 2000 mL three-necked flask, 7-2 (50 g), tetrahydrofuran (500 mL) was added under nitrogen. The mixture was cooled to 0 ° C, and a solution of borane tetrahydrofuran (450 mL, 1 M) was added dropwise, and reacted at this temperature for 1 hour, and allowed to react at room temperature overnight. The temperature was lowered to 0 ° C, 100 mL of water was added, sodium hydroxide (350 mL, 10%) was added dropwise, and hydrogen peroxide (250 mL, 30%) was added dropwise at 0 ° C, and reacted at room temperature for 5 hours.
  • tetrahydrofuran 500 mL
  • a solution of borane tetrahydrofuran 450 mL, 1 M
  • the temperature was lowered to 0 ° C 100 mL of water was added, sodium hydroxide (350 mL, 10%) was added
  • PCC pyridinium chlorochromate
  • DAST diethylamine trifluoride
  • Step 5 In a 1000 mL single-mouth bottle, 7-5 (30 g, 0.12 mol) and methanol (500 mL), palladium on water (6 g, 10%) were added under nitrogen. After hydrogen gas was introduced, the reaction was allowed to proceed overnight at room temperature. After suction filtration, the filtrate was added with 10 mL of concentrated hydrochloric acid, and dried to give the hydrochloride salt of product 7-6.
  • Step 1 To a 250 mL round bottom flask was added diethyl ether (150 mL), and then lithium aluminum hydride-d4 (2.15 g), 8-1 (15 g). Stir at 40 ° C overnight. Water (2.15 mL), 15% sodium hydroxide (2.15 mL), water (6.45 mL) was added, and the filtrate was dried over anhydrous sodium sulfate.
  • H-NMR 400MHz, CDCl 3 , ppm
  • ⁇ 1.77-1.75 (m, 4H), 1.55 (d, J 2.8Hz, 4H).
  • Step 2 To a 500 mL round bottom flask was added 8-2 (6 g), dichloromethane (200 mL) and triethylamine (14 g). Methanesulfonyl chloride (11.8 g) was added dropwise and stirred for 1 hour. The organic phase was washed with anhydrous sodium sulfate and evaporated to ethyl ether.
  • H-NMR 400 MHz, CDCl 3 , ppm): ⁇ 2.97 (s, 3H), 1.90-1.83 (m, 4H), 1.82-1.77 (m, 2H), 1.74-1.65 (m, 2H).
  • Step 3 To a 250 mL round bottom flask was added 8-3 (10 g), N,N-dimethylformamide (150 mL), sodium azide (7.4 g). Stir at 65 ° C overnight. 100 mL of water was added, and the mixture was extracted with ethyl acetate. H-NMR (400 MHz, CDCl 3 , ppm): ⁇ 1.83-1.78 (m, 2H), 1.74-1.66 (m, 4H), 1.62-1.59 (m, 2H).
  • Step 4 To a 250 mL round bottom flask was added 8-4 (5 g), methanol (100 mL), palladium carbon (300 mg). Hydrogen was introduced and stirred at 25 ° C overnight. Filtration, the filtrate was added to 5 mL of concentrated hydrochloric acid, and concentrated to give 8-5.
  • H-NMR 400 MHz, d 6 -DMSO, ppm: ⁇ 8.10 (br s, 2H), 1.87-1.80 (m, 2H), 1.68-1.64 (m, 2H), 1.63-1.46 (m, 4H) .
  • Step 1 In a 500 mL single-necked flask, sodium azide (32 g) was dissolved in water (300 mL), and then a solution of ethyl chloroformate (45 g) in diethyl ether (100 mL) was added dropwise. To the reaction mixture was added diethyl ether (300 mL)
  • Step 2 In a 500 mL three-necked flask, to a solution of 10-2 (24 g) of n-pentane (200 mL) was added norbornene (20 g), and stirred at 20 ° C for 4 days. The reaction solution was concentrated to give 10-3.
  • Step 3 In a 500 mL single-mouth bottle, 10-3 (55 g) was dissolved in methanol (300 mL), and the system was reacted at 40 degrees for 2 hours. The reaction solution was concentrated and distilled under reduced pressure (70 °, 10 mmHg vacuum) to afford 10-4.
  • Step 4 Slowly add liquid bromine (28 g) to tetralin (20 g) at 40 °C, and complete the dropwise addition for 2 hours while the hydrogen bromide gas is passed into 10-4 (16 g) of n-pentane (200 mL). The solution was reacted at room temperature for 4 hours. The reaction mixture was concentrated, EtOAc (EtOAc)EtOAc. The organic phase was dried over anhydrous sodium sulfate, filtered andEtOAc
  • Step 5 In a 250 mL three-necked flask, Na (5 g) was slowly added to methanol (100 mL), stirred for 1 hour, and then 10-5 (10 g) was added. The system was heated to 60 degrees and reacted for 16 hours. The reaction mixture was concentrated, and then diethyl ether (300 mL) The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and evaporated to dryness ⁇ RTIgt;
  • Step 6 In a 100 mL single-mouth bottle, 10-6 (4.5 g) was dissolved in ethanol (40 mL), 10% palladium carbon (400 mg) was added, and after hydrogen replacement, the system was reacted overnight at room temperature under a hydrogen balloon. The reaction system was filtered and the filtrate was concentrated to give 10-7.
  • Step 7 In a 100 mL single-mouth bottle, dissolve sodium hydroxide (9 g) in water (40 mL), then add ethylene glycol (20 mL) and 10-7 (7 g), the system was heated to 110 degrees for 3 days. After cooling to room temperature, brine (60 mL) was added and the mixture was extracted with n-pentane (300 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated
  • Step 1 In a 1000 mL single-mouth flask, sodium hydride (15.4 g, 60%) was dissolved in tetrahydrofuran (700 mL), then adiponitrile (37.9 g) was added and the system was heated to reflux overnight. Water (300 mL) was added to the mixture and the mixture was evaporated. The organic phase was dried, filtered and concentrated to give 11-1.
  • Step 3 In a 250 mL single-mouth bottle, 11-2 (3.0 g), N,N-diisopropylethylamine (6.86 g), 5-bromo-2,4-dichloropyrimidine (3.0 g) in acetonitrile (100 mL), the system was heated to 70 ° C overnight. Water (100 mL) was added to the reaction mixture, and ethyl acetate (3 ⁇ 100m) was evaporated. The organic phase is dried, filtered, concentrated and purified with silicagel eluting 15-1 (2g).
  • Step 2 To a 100 mL single-mouth bottle was added 12-2 (2.0 g), 15 mL of methanol, 15 mL of dichloromethane, water palladium on carbon (60%) 2.0 g, and ammonium formate 2.0 g. Add the reaction at room temperature overnight. Filter and collect the filtrate. The filter cake was washed 3 times with 75 mL of dichloromethane. The organic phase was collected and backwashed three times with 100 mL of brine. Dry over anhydrous sodium sulfate and spin dry the organic phase to give 12-3.
  • Step 1 Under a nitrogen atmosphere, add 13-1 (2.0 g), 5-bromo-2-nitropyridine (1.73 g), N,N-dimethyl sulfoxide (20 mL), and triethylbenzene to a 100 mL three-necked flask.
  • Step 2 To a 100 mL single-mouth bottle, 13-2 (1.5 g), 15 mL of methanol, 15 mL of dichloromethane, 1.5 g of palladium on carbon (60%), and 1.5 g of ammonium formate were added. Add the reaction at room temperature overnight. Filter and collect the filtrate. The filter cake was washed 3 times with 75 mL of dichloromethane. The organic phase was collected and backwashed three times with 100 mL of brine. Dry over anhydrous sodium sulfate and spin dry to give 13-3.
  • Step 2 14-2 (1.1 g), 15 mL of methanol, 15 mL of dichloromethane, 1.1 g of palladium on carbon (60%) and 1.1 g of ammonium formate were added to a 100 mL vial. Add the reaction at room temperature overnight. Filter and collect the filtrate. The filter cake was washed 3 times with 75 mL of dichloromethane. The organic phase was collected and backwashed three times with 100 mL of brine. The organic phase was dried over anhydrous sodium sulfate and dried to give 14-3.
  • Step 2 To a 100 mL single-mouth bottle, 16-1 (600 mg), methanol 15 mL, dichloromethane 15 mL, water palladium on carbon (60%) 600 mg, ammonium formate (804 mg) were sequentially added. Add the reaction at room temperature overnight. Filter and collect the filtrate. The filter cake was rinsed 3 times with 75 mL of dichloromethane. 100 mL of saturated saline was backwashed three times. Dry over anhydrous sodium sulfate and spin dry to give 16-2.
  • Step 1 Under a nitrogen atmosphere, a 250 mL three-necked flask, 17-1a (3.0 g) was dissolved in 30 mL of tetrahydrofuran, and the reaction flask was added, and bis-trimethylsilylamino potassium (16 mL, 1 M) was added dropwise at -78 ° C, -78 The reaction was kept at ° C for 3 h. A solution of 17-1b (5.3 g) in tetrahydrofuran (10 mL) was added dropwise at -78 ° C, and the mixture was allowed to react at room temperature for 2 h. Quenched with saturated ammonium chloride 50mL reaction.
  • Step 1 Under a nitrogen atmosphere, a 100 mL round bottom flask was charged with 18-1 (8 g), ethanol (50 mL), hydroxylamine hydrochloride (5.15 g), and sodium acetate (2.92 g). The reaction mixture was stirred at 80 ° C for 5 hours. The reaction mixture was cooled to room temperature. Filter and collect the filtrate. Spin dry. 100 mL of water was added to the mixture, and the mixture was extracted three times with 100 mL of ethyl acetate. 100 mL of saturated saline was backwashed once. Dry over anhydrous sodium sulfate and spin dry to give 18-2.
  • Step 2 Under a nitrogen atmosphere, a 250 mL three-necked flask was charged with 18-2 (8.2 g), acetone (50 mL), water (50 mL) and sodium carbonate (10.86 g). 4-Methylbenzene-1-sulfonyl chloride (9.79 g) was added portionwise over 5 minutes at 10-20 °C. The resulting reaction mixture was stirred at room temperature overnight. Quenched with 50 mL of water. It was extracted three times with 100 mL of ethyl acetate, and the organic phases were combined. 100 mL of saturated saline was backwashed once. Dry over anhydrous sodium sulfate and spin dry. The resulting mixture was added to hexanol (100 mL). The resulting product 18-3 was collected by filtration.
  • Step 3 Under a nitrogen atmosphere, add 18-3 (6.2 g), 5-bromo-2-nitropyridine (5.24 g), 4,5-bisdiphenylphosphine-9,9- to a 100 ml three-necked flask. Xanthos (2.24 g), palladium acetate (579 mg), cesium carbonate (12.6 g). Stirred at 105 ° C for 3 hours. Quenched with 50 mL of water. It was extracted three times with 100 mL of ethyl acetate, and the organic phases were combined. The saturated saline solution was backwashed twice with 75 ml. Dry over anhydrous sodium sulfate and spin dry. The resulting mixture was passed through a column and washed with ethyl acetate / dichloromethane (1:10-1:1). Product 18-4 was obtained.
  • Step 5 Under a nitrogen atmosphere, add 500-5 (500 mg), methanol (100 mL), dichloromethane (100 mL), palladium on carbon (10%) (0.5 g) and ammonium formate (500 mg) in a 500 ml round bottom flask. . Stir at room temperature overnight. Filter and spin dry. Product 18-6 was obtained as a white solid.
  • Step 1 Under nitrogen protection, add 20-1 (2.7 g), acetonitrile 60 mL, triethylamine (3.86 g), and add bromobenzyl (2.61 g) dropwise at 0 °C under a nitrogen atmosphere. . After the reaction was completed, 50 mL of water was added to quench the reaction. The solution was extracted 3 times with 75 mL of dichloromethane. 100 mL of saturated saline was backwashed 4 times. Dry with anhydrous sodium sulfate and spin dry to give 20-2.
  • Step 1 To a 250 mL three-necked flask, 18-5 (2.62 g), methylene chloride (102 mL) was added in vacuo. Trifluoroacetic acid (10.2 mL) was added dropwise at 0 ° C, and the mixture was dropped over 3 minutes. The reaction was carried out for 4 h at room temperature. Spin dry to get 21-1.
  • Step 2 Under a nitrogen atmosphere, 21-1 (2.25 g), methanol (160 mL), and formaldehyde (695 mg) were sequentially added to a 250 mL three-necked flask. Acetic acid (899 mg) and sodium cyanoborohydride (566 mg) were added sequentially at 0 °C. The reaction was carried out at 0 ° C for 5 min. The reaction was quenched by adding 160 mL of ice water. The saturated sodium carbonate solution was adjusted to pH 9, and extracted with ethyl acetate (3 mL). Wash back 3 times with 500 mL of saturated saline. Dry over anhydrous sodium sulfate and spin dry to 21-2.
  • Step 3 Under nitrogen protection, add 21-2 (970 mg), methanol 50 mL, dichloromethane (50 mL), Pd/C (10%, 0.97 g), ammonium formate (970 mg) to a 250 mL single-mouth bottle, and react at room temperature. overnight. Filter, concentrate, purify with C18 column and spin dry to give 21-3.
  • Step 1 Under a nitrogen atmosphere, 19-1 (2.21 g) and 121 mL of dichloromethane were sequentially added to a 250 mL three-necked flask. 12.1 mL of trifluoroacetic acid was added at 0 °C. The reaction was carried out for 4 h at room temperature. Spin dry to get 22-1.
  • Step 2 Under a nitrogen atmosphere, 22-1 (2.08 g), methanol 150 mL, and formaldehyde (642 mg) were sequentially added to a 250 mL three-necked flask. Acetic acid (830 mg) and sodium cyanoborohydride (523 mg) were added sequentially at 0 °C. 0 ° C reaction for 5 minutes, plus 150 mL of ice water was quenched. The saturated sodium carbonate solution was adjusted to pH 9, and extracted with ethyl acetate (3 mL). Wash back three times with 500 mL of saturated brine, dry over anhydrous sodium sulfate, and spin dry to give 22-2.
  • Step 3 To a 100 mL single-necked flask, 22-2 (720 mg), methanol 50 mL, dichloromethane (50 mL), palladium carbon 0.72 g, ammonium formate (720 mg), and then reacted at room temperature overnight. Filtered, concentrated, purified by C18 column and dried to give 22-3.
  • Step 1 In a 250 mL three-necked flask, m-chloroperoxybenzoic acid (m-CPBA, 12 g) was added portionwise to a solution of 1-methylcyclopentene (3.8 g) in dichloromethane (100 mL) at 5 °C. The reaction was carried out for 16 hours at room temperature. The reaction solution was filtered, and the filtrate was washed with water (100mL), aqueous sodium hydrogen carbonate (150mL) and aqueous sodium thiosulfate (150mL). The organic phase was dried, filtered and concentrated to give 23-2.
  • m-CPBA m-chloroperoxybenzoic acid
  • Step 2 Ammonia water (8 mL) was added to a solution of 23-3 (2.4 g) in ethanol (8 mL), and then reacted in a glass jar for 6 hours at 85 °C. The reaction solution was cooled to room temperature and concentrated to give 23-3.
  • Step 3 In a 100 mL single-mouth bottle, 5-bromo-2,4-dichloropyrimidine (1.26 g) was dissolved in ethanol (20 mL), then 23-3 (0.9 g), N,N-diisopropyl Ethylamine (2.1 g) was reacted overnight at room temperature. After the reaction mixture was concentrated, water (50 mL) The organic phase is dried, filtered, concentrated and recrystallized from petroleum ether to afford 23-4.
  • Step 4 Diethylamino trifluoride (DAST, 1.5 g) was added to a solution of 23-4 (1.3 g) in dichloromethane (40 mL) at -78 ° C under nitrogen atmosphere, and reacted at -78 ° C for 1 hour. . After quenching with the addition of isopropanol at -78 ° C, the mixture was warmed to room temperature, and the mixture was washed with water (30mL) and aqueous sodium hydrogen carbonate (30mL). The organic phase was dried, filtered, concentrated and purified eluting elut elut
  • 2,2-difluorocyclopentylamine hydrochloride (6.0 g), 70 mL of dioxane, and triethylamine (33 g) were placed under a nitrogen atmosphere in a 250 mL three-necked flask. The temperature was lowered to 10 ° C or lower, and 5-bromo-2,4-dichloropyrimidine (7.5 g) was added in portions and allowed to react at room temperature overnight.
  • Step 8 Separation of chiral preparation
  • Preparation column CHIRALPAK AD-H SFC 5*25 cm, 5 um, mobile phase: phase A: CO 2 : 50, phase B: EtOH (0.2% DEA) - HPLC: 50; cycle time: 12 minutes, detection wavelength: 220 nm.
  • Example 1B (retention time 23.9 minutes) corresponds to the second peak in the chiral separation.
  • Example 2 The compound of Example 2 was synthesized in a similar manner as in Example 1 except that 2-4 was used instead of 1-6 in Step 1.
  • LC-MS (ES, m/z): 462 [M+H] + ;
  • Example 3 The compound of Example 3 was synthesized in a similar manner to that in Example 1 except that 1-6 was used instead of 1-6 in Step 1.
  • LC-MS (ES, m/z): 460 [M+H] + ;
  • Example 7A LC-MS (ES, m/z): 484 [M+H] + ; H-NMR (300 MHz, CDCl 3 , ppm): ⁇ 8.83 (s, 1H), 8.13 - 8.04 (m, 2H), 7.98 (s, 1H), 7.37-7.33 (m, 1H), 6.19-6.12 (m, 1H), 3.18-3.11 (m, 8H), 2.64 (m, 2H), 2.57 (s, 3H) , 2.40 (s, 3H), 2.39-2.15 (s, 3H).
  • Example 8 The compound of Example 8 was synthesized in a similar manner to that in Example 1, except that in Step 1, substituting 8-5 for 1-6.
  • Example 10 The compound of Example 10 was synthesized in a similar manner to that in Example 1, except that in Step 1, substituting 10-8 for 1-6.
  • LC-MS (ES, m/z): 474 [M+H] + ;
  • Example 11 The compound of Example 11 was synthesized in a similar manner to that in Example 1 except that in Step 2, 11-3 was used instead of 1-7.
  • LC-MS (ESI, m/z): 473 [M+H] + ;
  • Example 12 The compound of Example 12 was synthesized in a similar manner to that in Example 1 except that in Step 1, substituting s.
  • Example 15 The compound of Example 15 was synthesized in a similar manner to that in Example 1 except that in Step 2, substituting 15-1 for 1-7.
  • Example 16 The compound of Example 16 was synthesized in a similar manner to that in Example 1 except that in Step 1, substituting 1-6 with cyclopentylamine, and substituting 16-2 for J in step 5.
  • Example 17 The compound of Example 17 was synthesized in a similar manner to that in Example 1 except that in Step 1, substituting s.
  • Example 18 The compound of Example 18 was synthesized in a similar manner to that in Example 1 except that in Step 1, substituting s.
  • Example 19 The compound of Example 19 was synthesized in a similar manner to that in Example 1 except that in Step 1, substituting s.
  • LC-MS (ES, m/z): 503 [M+H] + ;
  • Example 20 The compound of Example 20 was synthesized in a similar manner to that in Example 1 except that in Step 1, substituting s.
  • H-NMR (300 MHz, d 6 - DMSO, ppm): ⁇ 1.48-2.32 (14H, m), 2.43 (3H, s) , 3.04-3.14(4H,m), 4.40-4.49(2H,m), 5.78-5.87(1H,m),7.48-7.50(1H,d,J 5.7Hz),7.86-7.92(1H,m) , 8.08 (1H, s), 8.74 (1H, broad s), 8.90-8.96 (2H, m), 10.19 (1H, s).
  • Example 21 was synthesized in a similar manner to Example 1 except that in Step 1, substituting 1-6 with cyclopentylamine, and substituting 21-3 for J in step 5, which was purified by reverse phase column to give Example 21 The trifluoroacetate salt of Compound 21.
  • Example 23 The compound of Example 23 was synthesized in a similar manner as in Example 1 except that in Step 1, substituting 23-5 for 1-6.
  • Example 25A and Example 25B can then be obtained by reference to Step 7 in the synthetic route of Example 1.
  • the obtained racemic intermediate compound 25-12 was separated by Pre-SFC chiral preparation, and the preparation column was: Chiralpak AS-H5*25 cm, 5 um, mobile phase: phase A: CO2: 50, phase B: MEOH (0.2% DEA) : 50; cycle time: 3.4 minutes, detection wavelength: 220 nm. Two peaks were collected, the first peak 25-12A (peak time: 5.51 minutes) and the second peak 25-12B (peak time: 6.76 minutes).
  • the synthesis from 26-1 to 26-4 and 26-5 can be referred to steps 4, 5 and 6 in the synthetic route of Example 1.
  • the chiral separation method yields 26-4 and 26-5.
  • the synthesis of the final product from 26-4 to 26-5 can be referred to step 7 in the synthetic route of Example 1.
  • Example 26B LC-MS: (ESI, m/z): 513 [M+H] + ; H-NMR: (300 MHz, CDCl3, ppm): ⁇ 0.72-0.74 (m, 1H), 1.35-2.37 (m,7H),2.42(s,3H),2.43-2.52(m,1H),2.54(s,3H),2.97-3.14(m,2H),3.58-3.65(m,1H),3.79-3.83 (m, 2H), 4.19-4.24 (m, 1H), 5.89-5.90 (m, 1H), 7.57-7.61 (m, 1H), 8.20-8.21 (m, 2H), 8.45-8.46 (m, 1H) , 8.83 (s, 1H).
  • Example 27B LC-MS: (ESI, m/z): 513 [M+H] + ; H-NMR: (300 MHz, CDCl 3 , ppm): ⁇ 0.72-0.74 (m, 1H), 1.35-2.10 (m, 5H), 2.42 (s, 3H), 2.54-2.58 (m, 4H), 2.97-2.98 (m, 2H), 3.58-3.65 (m, 1H), 3.79-3.83 (m, 2H), 4.19 -4.24(m,2H), 5.89-5.90(m,1H), 7.57-7.61(m,1H), 8.20-8.21(m,2H), 8.45-8.46(m,1H),8.83(s,1H) .
  • CDK4 and CDK6 kinase in vitro activity screening experiments were carried out using the Caliper Mobility Shift Assay, which is based on the mobility detection technology of microfluidic chip technology.
  • Experimental configuration 1x CDK4 Kinase Reaction Buffer (20 mM HEPES, pH 7.5, 0.01% Triton X-100, 10 mM MgCl 2 , 2 mM DTT); 1x CDK6 Kinase Reaction Buffer (50 mM HEPES, pH 7.5, 0.0015% Brij-35, 10 mM MgCl 2 , 2 mM DTT); kinase reaction stop solution (100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA); 2.5x CDK4 kinase solution [25 nM CDK4 kinase/cyclin D3 (Carna) , Cat#04-105) in 1x CDK4 Kin
  • CDK4 kinase reaction solution final concentration 10 nM CDK4/Cyclin D3, 3 ⁇ M substrate peptide FAM-P8, 221 ⁇ M ATP, 10 mM MgCl 2 , 2 mM DTT
  • CDK6 kinase reaction solution final concentration 20 nM CDK6/Cyclin D3, 3 ⁇ M substrate peptide FAM- P8, 800 ⁇ M ATP, 10 mM MgCl 2 , 2 mM DTT.
  • Experimental procedure first prepare a 5x reaction concentration compound solution in a 96-well plate: mix 50x DMSO with a final concentration of the compound solution, and dilute with 1x kinase reaction buffer to 5x final concentration compound solution; in the oscillator Shake for 10 minutes; then add the following solution to the 384-well test plate: 5 uL of 5x reaction final concentration of compound solution (final 384-well test plate with 1x reaction compound final concentration of 0.1nM-1000nM and 10 ⁇ L of 2.5x CDK4 or CDK6 kinase solution was incubated for 10 minutes at room temperature, then 10 ⁇ L of 2.5x substrate peptide solution was added, and after standing at 28 ° C for one hour, 25 ⁇ L of kinase reaction stop solution was added, and centrifuged at 1200 RPM for one minute.

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Abstract

本发明提供了式（I）所示的一系列5-甲基-2-(吡啶-2-基氨基)-8H-吡啶并[2,3-d]嘧啶-7-酮类化合物、其药学上可接受的盐、对映异构体、非对映异构体或外消旋体、前药分子和溶剂合物，其作为细胞周期蛋白依赖性激酶CDK4和/或CDK6选择性抑制剂，可用于治疗与细胞周期蛋白依赖性激酶信号通路有关的疾病。更特别地，该类化合物可有效抑制多种肿瘤细胞的生长，可用于制备抗肿瘤药物，用于不同的癌症的治疗、联合治疗或预防。

Description

5-甲基-2-(吡啶-2-基氨基)-8H-吡啶并[2，3-d]嘧啶-7-酮化合物 技术领域
本发明涉及医药技术领域，具体涉及5-甲基-2-(吡啶-2-基氨基)-8H-吡啶并[2，3-d]嘧啶-7-酮类化合物或其药学上可接受的盐、对映异构体、非对映异构体、外消旋体或它们的混合物、前药、溶剂化合物及其制备方法，包含这些物质的药物组合物以及其用途。
背景技术
癌症的一个主要标志就是细胞周期失调引起的的无节制的生长。在正常情况下，细胞必须经过四个阶段的细胞周期进行生长，分裂和复制：G1期(生长期)，S期(DNA合成期)，G2和M期(有丝分裂期)。在细胞周期中每个阶段都由被称为细胞周期蛋白依赖性激酶(cyclin-dependent kinase，CDK)的调控。决定G1-S期过渡的关键调节蛋白激酶就是CDK4，它和细胞周期蛋白D(cyclin D)形成一个复合体，将视网膜母细胞瘤(Rb)的蛋白质磷酸化。肿瘤抑制蛋白Rb一旦发生磷酸化，可释放其在未被磷酸化的状态下紧密结合的转录因子E2F，而E2F激活进一步转录并推动细胞周期通过限制检查点(restriction checkpoint，R点)。细胞一旦通过R点就不可逆的进入S期。在S期的后期和G2期，CDK1和CDK2与他们的合作伙伴细胞周期蛋白A和B一同合作发挥重要的作用，推动细胞进入M期，完成细胞的分裂与生长周期。在癌细胞中，细胞周期调控受到破坏，癌细胞变得毫无节制地生长、分裂和复制，因此针对调节细胞周期的通路来寻找和发现选择性和强效CDK抑制剂，成为多年来肿瘤学药物研究的主要目标。
针对CDK4和/或CDK6的选择性靶向治疗药物可以在以下几种机制中发挥作用：(1)CDK4基因扩增或蛋白的高表达，这种变化经常出现在高分化和去分化的脂肪肉瘤，并且也在一些其它实体瘤和血液恶性肿瘤；(2)细胞周期蛋白D1的扩增或高表达，这在套细胞淋巴瘤，也在多种实体瘤中观察到；(3)p16INK4A基因(CDKN2A)的损失，这也是在许多癌症中常见的事件。在细胞里，p16INK4A蛋白是CDK4的天然抑制剂，有p16损失癌症一般可能对CDK4的抑制敏感。例如在卵巢癌中，这些癌细胞系具有低p16蛋 白水平和高Rb的表达，会对CDK4的抑制剂是比较敏感。因为CDK6和CDK4的蛋白的高度相似性，目前的CDK4抑制剂同时也对CDK6蛋白激酶有类似的抑制作用。目前有三个CDK4/CDK6抑制剂已进入临床试验：palbociclib(PD0332991)，LEE011和LY2835219，这些抑制剂使CDK4/6无法与Cyclin D形成复合物，有效的阻滞了细胞周期自G1期向S期的进程，从而达到抑制肿瘤细胞增殖的目的，并在乳腺癌，神经母细胞瘤，恶性横纹肌瘤，淋巴瘤，肉瘤和其它肿瘤中表现出单剂或与其他靶向治疗药物组合的抗肿瘤活性。
发明内容
本发明提供了一系列5-甲基-2-(吡啶-2-基氨基)-8H-吡啶并[2，3-d]嘧啶-7-酮类化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物，可用于制备治疗与细胞周期蛋白依赖性激酶信号通路有关的疾病的药物。
本发明提供了以下式I所示的化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物，
Figure PCTCN2015091668-appb-000001
其中，
R1选自以下基团：
Figure PCTCN2015091668-appb-000002
其中，R2和R2’各自独立地选自H，氘(D)，C1-C4烷基，氟和氰基，并且，R2和R2’可以在同一个或不同碳原子上；
n＝1或2
R3选自以下基团：
Figure PCTCN2015091668-appb-000003
其中，R4选自H，氘(D)和C1-C4烷基，
W选自CH2，CH2CH2，O和NH
X选自CH和N
Y选自CH2，NH和O
m＝0，1或2
n＝0，1或2；
优选地，
R1选自以下基团：
Figure PCTCN2015091668-appb-000004
R3选自以下基团：
Figure PCTCN2015091668-appb-000005
更优选地，
R1选自以下基团：
Figure PCTCN2015091668-appb-000006
R3选自以下基团：
Figure PCTCN2015091668-appb-000007
更优选地，
R1选自以下基团：
Figure PCTCN2015091668-appb-000008
R3选自以下基团：
Figure PCTCN2015091668-appb-000009
优选地，本发明的5-甲基-2-(吡啶-2-基氨基)-8H-吡啶并[2，3-d]嘧啶-7-酮类化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物包括如下化合物：
Figure PCTCN2015091668-appb-000010
Figure PCTCN2015091668-appb-000011
其中，以上化合物包括其对映异构体、非对映异构体、外消旋体和其混合物、前药分子或溶剂合物，例如化合物1具有对映异构体1A和1B，化合物5具有对映异构体5A和5B，化合物7具有对映异构体7A和7B，化合物24具有非对映异构体24A和24B，化合物25具有非对映异构体25A和25B，化合物26具有非对映异构体26A和26B和非对映异构体27A和27B。
最优选地，所述化合物选自以下化合物：
Figure PCTCN2015091668-appb-000012
Figure PCTCN2015091668-appb-000013
其中，cis表示顺式，trans表示反式。
也即，上述具体化合物为如下化合物：
Figure PCTCN2015091668-appb-000014
Figure PCTCN2015091668-appb-000015
本发明的另一方面还提供了所述5-甲基-2-(吡啶-2-基氨基)-8H-吡啶并[2，3-d]嘧啶-7-酮类化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物在制备CDK4和/或CDK6选择性抑制剂中的应用。
本发明的另一方面还提供了所述5-甲基-2-(吡啶-2-基氨基)-8H-吡啶并[2，3-d]嘧啶-7-酮化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶 剂合物，尤其是作为CDK4和/或CDK6选择性抑制剂用于制备治疗与细胞周期蛋白依赖性激酶信号通路有关的疾病的药物中的用途。
所述与细胞周期蛋白依赖性激酶信号通路有关的疾病包括肿瘤，例如乳腺癌，胶质瘤、胶质母细胞瘤、肺癌、结肠直肠癌、胃癌、胃肠道基质瘤(GIST)、肝细胞癌、前列腺瘤、肉瘤、卵巢癌、宫颈癌、胰腺癌、黑色素瘤、甲状腺癌、胆管癌、子宫内膜癌、肾癌、间皮瘤、淋巴瘤、白血病、非霍奇金淋巴瘤、间变性大细胞淋巴瘤、急性髓细胞白血病(AML)、多发性骨髓瘤。
本发明的另一个方面提供了一种药物组合物，所述药物组合物含有：治疗有效量的选自上述根据本发明的5-甲基-2-(吡啶-2-基氨基)-8H-吡啶并[2，3-d]嘧啶-7-酮类化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体和它们的混合物、前药分子和溶剂合物中的一种或多种；和任选地药学上可接受的载体。所述药物组合物可用于体内治疗并具有生物相容性。
本发明的又一目的是提供一种本发明所述的化合物，其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物与其他的抗肿瘤药物(包括化疗，肿瘤免疫疗法)联合治疗癌症的方法，包括同时、分别或先后联合施用所述的化合物，其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物与其他的抗肿瘤药物。在这种联合疗法中，其他抗肿瘤药物与本发明化合物及其盐或前药可以并列、同时地、序贯地、或分别地给药，并且可包含但不限制于以下类型的抗肿瘤剂的一种或多种：烷化剂(例如卡钼、奥沙利钼、顺钼、环磷酰胺、亚硝基脲类、氮芥、美法仑)，抗代谢药(例如吉西他滨)，和抗叶酸剂(例如5-氟尿嘧啶和替加氟、雷替曲塞、甲氨喋呤、阿糖胞苷、羟基脲)，拓扑异构酶抑制剂(例如依托泊苷、托泊替康、喜树碱)，抗有丝分裂剂(例如长春新碱、长春碱、长春瑞滨，紫杉醇、泰索帝)，抗肿瘤抗生素(例如阿霉素、博来霉素、多柔比星、道诺霉素、丝裂霉素C、放线菌素)，抗雌激素药(例如他莫昔芬、氟维司群、托瑞米芬、雷洛昔芬、屈洛昔芬)，抗雄激素药(例如比卡鲁胺、氟他胺、尼鲁米特)、LHRH拮抗剂或LHRH激动剂(例如戈舍瑞林、亮丙瑞林、和布舍瑞林)，芳香酶抑制剂(例如阿 那曲唑、来曲唑)，CYP17裂解酶抑制剂(例如阿比特龙)，抗erbB2抗体曲妥珠单抗[赫赛汀]，抗EGFR抗体西妥昔单抗[Erbitux]；酪氨酸激酶，丝氨酸/苏氨酸激酶的抑制剂(例如伊马替尼，尼洛替尼，索拉非尼，曲美替尼(trametinib)，达拉非尼(dabrafenib)，拉帕替尼(lapatinib))，抗人血管内皮细胞生长因子抗体贝伐珠单抗(阿瓦斯丁)以及VEGF受体酪氨酸激酶抑制剂(例如阿帕替尼)，免疫肿瘤治疗方法，例如抗PD-1抗体(pembrolizumab，nivolumab)，抗PD-L1抗体，抗LAG-3抗体，抗CTLA-4抗体，抗4-1BB抗体，抗GITR抗体，抗ICOS抗体，抗OX40抗体，白细胞介素2。
根据本发明，所述药学上可接受的盐为常规的药学上可接受的盐，包括盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、磷酸盐、硝酸盐、乙酸盐、三氟乙酸盐、对甲苯磺酸盐、水杨酸盐、甲磺酸盐、草酸盐、琥珀酸盐、柠檬酸盐、苹果酸盐、乳酸盐、富马酸盐等。
本发明的化合物或者其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物，还包括其多晶型，即化学药物分子在不同的物理化学条件下结晶成的结构不同的晶体。
根据本发明的5-甲基-2-(吡啶-2-基氨基)-8H-吡啶并[2，3-d]嘧啶-7-酮类化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物，具体按照如下路线制备：
Figure PCTCN2015091668-appb-000016
步骤1：氮气保护下，在三口瓶中，加入A，5-溴-2，4-二氯嘧啶，碳酸钾和1，4-二氧六环，室温搅拌反应过夜。加入乙酸乙酯，盐水洗。有机相无水硫酸钠干燥，硅胶柱层析，收集产品得化合物B。
步骤2：氮气保护，三口瓶中，加入B，巴豆酸，N，N-二异丙基乙胺，保温搅拌10分钟，加入三(2-甲苯基)膦，二(苯腈)二氯化钯，加毕，氮气置换三次，加热反应过夜。降至室温，加入乙酸乙酯稀释，饱和食盐水反洗。有机相用无水硫酸钠干燥，旋干，得化合物C用于下一步反应。
步骤3：在三口瓶中，氮气保护下，将C溶于重蒸四氢呋喃中，加入醋酸酐，加热反应过夜。降至室温，加入乙酸乙酯稀释，饱和食盐水反洗。有机相用无水硫酸钠干燥，旋干溶剂后直接硅胶柱分离，得到固体D。
步骤4：在三口瓶中，氮气保护下，将固体D溶于醋酸中，加入醋酸钠，降温至0℃滴加溴，加毕，加热反应。降至室温，加入饱和亚硫酸氢钠淬灭反应。二氯甲烷萃取，收集有机相。饱和食盐水反洗，无水硫酸钠干燥，旋干溶剂后直接硅胶柱分离得固体E。
步骤5：在三口瓶中，氮气保护下，将H2N-R的甲苯溶液降温至0℃，加入六甲基二硅基胺基锂，室温搅拌30分钟，降温至0℃，滴加E的甲苯溶液，加毕，室温反应过夜。加入二氯甲烷稀释，饱和食盐水反洗。有机相无水硫酸钠干燥，旋干溶剂后直接硅胶柱分离得固体F。
步骤6：在三口瓶中，氮气保护下，将F溶于正丁醇中，加入乙烯基正丁基醚，加入N，N-二异丙基乙胺，加入催化量1，1′-二(二苯膦基)二茂铁二氯化钯(II)，加毕，置换空气三次，加热反应过夜。降至室温，加入乙酸乙酯稀释，饱和食盐水反洗。收集有机相，无水硫酸钠干燥，旋干溶剂后直接硅胶柱分离得固体G。
步骤7：在三口瓶中，加入G，四氢呋喃。冰浴下加入6N盐酸，室温反应过夜。冰浴下调节反应液PH＝8～9，乙酸乙酯萃取，收集有机相。有机相用无水硫酸钠干燥，旋干得目标产物H。若目标产物H为立体异构体的混合物，可以通过手性分离得到光学纯的对映异构体。若目标产物H用反相柱纯化，可得三氟乙酸盐。目标产物H也可以用盐酸 处理，得到盐酸盐，
其中，R为
Figure PCTCN2015091668-appb-000017
R1和R3的定义同上。
技术效果
本发明所述5-甲基-2-(吡啶-2-基氨基)-8H-吡啶并[2，3-d]嘧啶-7-酮类化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物，作为新型CDK4和/或CDK6选择性抑制剂，可用于制备治疗与细胞周期蛋白依赖性激酶信号通路有关的疾病或由异常细胞增殖引起的障碍或病症的药物。本发明的化合物、其药学上可接受的盐、对映异构体、非对映异构体或外消旋体、前药分子或溶剂合物对于CDK4和/或CDK6的抑制活性不但与现有技术的抑制剂的生物活性相当或更好，而且比现有技术的抑制剂具有更好的体内代谢性质，更高血药浓度，更少的有毒副作用的代谢物。
具体实施方式
在以下的实施例中将进一步举例说明本发明。这些实施例仅用于说明本发明，但不以任何方式限制本发明。
样品的分析数据由以下仪器测定：
液质联用：shimadzu LCMS-2020色谱柱：shimadzu shim-pack XR-ODS 3.0*50mm，2.2um
核磁共振：Bruker AV-III 300MHz；Varian 400MHz
氘代DMSO和MeOH：西格玛奥德里奇(上海)贸易有限公司。氘代氯仿：北京伊诺凯科技有限公司。重水：北京百灵威科技有限公司。
手性分离HPLC
仪器：shimadzu LC20AD
手性柱：CHIRALPAK AD-H，CHIRALPAK IA，CHIRALPAK IB，CHIRALPAK IC，Lux 3um Cellulose-4。
制备高压液相色谱(Prep-HPLC)仪器
仪器：Waters 2545
色谱柱：1)Waters X-bridge RP18，19*150mm，5um.2)sunfire prep C18/OBD，19*150mm，5um.
超临界流体色谱仪器(SFC)
仪器：SFC350
色谱柱：1)ChiralpakAS-H5*25cm，5um.2)CHIRALPAKAD-H SFC5*25cm，5um
除另有说明外，以下实施例中所用常规试剂、药品均购自：
南京药石药物研发有限公司；梯希爱(上海)化成工业发展有限公司；上海书亚医药科技有限公司；张家港爱玛特化学有限公司；(江苏)盐城市晶华化工有限公司；成都爱斯特贸易有限公司；天津法莫西医药科技有限公司；天津市光复科技发展有限公司；四川西陇化工有限公司；天津市福晨化学试剂厂；国药集团化学试剂陕西有限公司；陕西欣通化工有限公司；天津市康科德科技有限公司；西格玛奥德里奇(上海)贸易有限公司；上海星可生化有限公司；西安东冠蒸馏水有限公司，等公司。
具体制备例如下：
中间体1-6的合成
Figure PCTCN2015091668-appb-000018
步骤1：在2000mL三口瓶中，氮气保护下加入1-1(80g)，氨水溶液(665g，25％)。升温至60℃反应过夜。降温，向体系通氮气1小时。将体系pH值用盐酸(6N)调至4-5， 旋干后溶剂后得1-2，直接投下一步。
步骤2：在2000mL三口瓶中，氮气保护下加入1-2(60g)，无水甲醇(1000mL)，三乙胺(132g)。分批加入二叔丁基二碳酸酯，室温反应过夜。旋干甲醇(1000mL)，加入1500mL乙酸乙酯稀释，水3x 500mL反洗，合并有机相，无水硫酸钠干燥，粗品硅胶柱纯化(戊烷∶乙酸乙酯＝50∶1-20∶1)得1-3。
步骤3：在1000mL三口瓶中，氮气保护下加入1-3(40g)和800mL二氯甲烷中。降温到0℃，分批加入戴斯-马丁(Dess-Martin)氧化剂试剂(101g)，回到室温反应过夜。加入1000mL乙酸乙酯稀释，水3x 500mL反洗，合并有机相，无水硫酸钠干燥。粗品硅胶柱纯化(戊烷∶乙酸乙酯＝30∶1-10∶1)得1-4。
步骤4：在1000mL三口瓶中，氮气保护下加入1-4(30g)和600mL二氯甲烷中。降温到0℃，滴加二乙氨基三氟化硫(DAST，121g)，回到室温反应过夜。将体系倒入冰水中，二氯甲烷(600mL)萃取3x 500mL。无水硫酸钠干燥，过滤，滤液浓缩后硅胶柱分离(戊烷∶乙酸乙酯＝30∶1-10∶1)得1-5。
步骤5：在250mL三口瓶中，加入1-5(15g)和二氯甲烷(200mL)。通入氯化氢气体后，室温反应2小时。旋干溶剂后得产品1-6。
中间体2-4的合成：
Figure PCTCN2015091668-appb-000019
步骤1：氮气保护，往250mL三口瓶中，加入2-1(10g)，叠氮基三甲基硅烷和甲苯(120mL)。冷至20-30℃，滴加三氟化硼·乙醚(17g)室温反应24小时。静置分层，1x50mL10％碳酸氢钠水溶液洗有机相，无水硫酸钠干燥，用于下一步反应。
步骤2：在氮气保护下，500mL三口瓶中，加入2-2的甲苯溶液，乙醚(100mL)。然后慢慢加入三乙氧基磷(16.3g)，完毕，室温反应2天。旋干得无色透明油状物，用于下一步反应。
步骤3：氮气保护，在100mL三口瓶中，加入2-3(23g)，对甲基苯磺酸(9.51g)，乙醇(60mL)和水(0.8g)，80℃反应过夜。旋干，残余物加入100mL乙醚，抽滤，收集滤饼。滤饼用3x30mL乙醚洗，烘干得目标产物2-4。
中间体3-4的合成
Figure PCTCN2015091668-appb-000020
步骤1：在2000mL四口瓶中，氮气保护下加入500mL N，N-二甲基亚砜。降温至20-25℃，分批加入氢化钠(19g)，然后在此温度下分批加入三甲基碘化亚砜(118g)，并在该温度下搅拌1小时，在20℃下滴加3-1(40g)的N，N-二甲基亚砜(100mL)溶液。该体系在20-25℃搅拌30分钟后升温至50℃并反应2小时。反应结束后，降温，倒入冰水中淬灭。用3x1000mL无水乙醚萃取，合并有机相，无水硫酸钠干燥，过滤。滤液在低于30℃下旋干，得到3-2(粗品)。
步骤2：在2000mL三口瓶中，氮气保护下加入3-2(90g)，水(900mL)，盐酸羟胺(82.8g)，乙酸钠(153g)。升温至80℃反应2小时。降温后用乙酸乙酯萃取4x500mL，合并有机相，无水硫酸钠干燥，过滤。滤液浓缩后硅胶柱分离(乙酸乙酯/戊烷＝1∶30-1∶10)得3-3。
步骤3：在2000mL四口瓶中，氮气保护下加入氢化铝锂(34g)和600mL四氢呋喃中。降温到0℃，将3-3溶于400mL四氢呋喃中，滴加到上述体系中，回到室温反应过夜。0℃缓慢加入34mL水淬灭。然后再加102mL氢氧化钠(15％)水溶液和34mL水。抽滤，滤饼用四氢呋喃5x100mL洗。所得滤液旋干，得到3-4。
中间体4-6的合成
Figure PCTCN2015091668-appb-000021
步骤1：氮气保护下，1L三口瓶中加入氢氧化钾(46g)，水(12mL)，乙醇(225mL)。冰浴下滴加4-1(130g)。加毕室温反应10分钟，反应结束。过滤，收集滤饼。乙醚500mL淋洗滤饼，烘干，得4-2。
步骤2：氮气保护下，2L三口瓶中加入4-2(140g)，N，N-二甲基亚砜(1000mL)，1，2-二溴乙烷(174g)。室温反应过夜。倒入1L水中淬灭反应，乙醚萃取(300mL x 3)，盐水洗。有机相用无水硫酸钠干燥，浓缩，得4-3。
步骤3：氮气保护下，2L三口瓶中加入4-3(210g)，六甲基磷酰三胺(1000mL)，氯化锂(19g)。升温至140℃反应过夜。减压蒸馏，100℃收集产品得4-4。
步骤4：氮气保护下，500mL三口瓶中加入4-4(16g)，乙醇(150mL)，水(150mL)，盐酸羟胺(20g)，碳酸钠(30g)。室温反应过夜。加入50mL水，乙酸乙酯萃取(100mL x3)，收集有机相。食盐水洗(200mL x 2)。有机相用无水硫酸钠干燥，浓缩得4-5。
步骤5：氮气保护下，500mL三口瓶中加入4-5(6g)，四氢呋喃(200mL)。降温至0℃，分批加入氢化铝锂(5.5g)。加毕，升温至55℃反应5小时。降温至0℃，加入5.5mL水。用1N的氢氧化钠水溶液调pH＝7。再加入5.5mL的水和200mL的乙酸乙酯。有机相用无水硫酸钠干燥，过滤，收集滤液。滤液干燥后在滤液中加入5.5g草酸，搅拌30分钟，过滤，收集滤饼得4-6的草酸盐。
中间体5-3的合成
Figure PCTCN2015091668-appb-000022
步骤1：氮气保护下，500mL三口瓶中加入5-1(20g)，乙醇(250mL)，盐酸羟胺(16g)，吡啶(18.6g)。升温至80℃反应过夜。降至室温，加入200mL水，乙酸乙酯萃取(200mL x 3)，食盐水洗(500mL x 5)。有机相用无水硫酸钠干燥，浓缩得5-2。
步骤2：氮气保护下，500mL三口瓶中加入5-2(18g)，四氢呋喃(500mL)。降温至0℃，分批加入氢化铝锂(16g)。加毕，室温反应过夜。降温至0℃，加入16mL水。用氢氧化钠溶液(1N)调反应液pH＝7。加入水16mL，200mL的二氯甲烷分层。分出有机相用无水硫酸钠干燥，浓缩得5-3。
中间体6-4的合成
Figure PCTCN2015091668-appb-000023
步骤1：氮气保护下，在2000mL三口瓶中，加入6-1(60g)，甲苯(600mL)，三乙胺(81g)，二苯基磷酰基叠氮(DPPA，191g)。反应体系在100℃反应1小时。然后降温至40-50℃下滴加苯甲醇。滴完后升温至100℃，反应过夜。降至室温，加入500mL乙酸乙酯稀释，食盐水洗500mL x 3。有机相用无水硫酸钠干燥，旋干溶剂后硅胶柱分离得6-2。
步骤2：在2000mL三口瓶中，氮气保护下加入6-2(50g)，二氯甲烷(1000mL)。降温至0℃，滴加二乙基锌(276mL，1M)。然后滴加入二碘甲烷(73g)，并在此温度反应1小时后，室温反应过夜。反应液用3x 500mL水反洗，有机相用无水硫酸钠干燥，浓缩后硅胶柱分离(洗脱剂戊烷∶乙酸乙酯＝50∶1-20∶1)得6-3.
步骤3：在1000mL单口瓶中，氮气保护下加入无水甲醇(600mL)，6-3(45g)和有水钯碳(10g，10％)。室温加氢反应过夜。抽滤后，收集滤液，旋干溶剂得6-4。
中间体7-6的合成
Figure PCTCN2015091668-appb-000024
步骤1：在2000mL三口瓶中，氮气保护下加入7-1(80g)，三乙胺(107g)，二苯基磷酰基叠氮(DPPA，255g)，无水甲苯(800mL)。100℃反应1小时，然后降温至40-50℃，在该温度下滴加苯甲醇(114g)，滴完后升温至100℃反应过夜。降温，加入500mL乙酸乙酯稀释，食盐水洗500mL洗3次，有机相硫酸钠干燥，过滤，旋干后硅胶柱分离(乙酸乙酯/戊烷＝1∶100-1∶50)得7-2。
步骤2：在2000mL三口瓶中，氮气保护下加入7-2(50g)，四氢呋喃(500mL)。降温至0℃，滴加硼烷四氢呋喃溶液(450mL，1M)，并在此温度反应1小时后，室温反应过夜。降至0℃，加入100mL水，滴加氢氧化钠(350mL，10％)，0℃下滴加双氧水(250mL，30％)，室温反应5小时。降温后乙酸乙酯萃取500mL萃取3次，合并有机相，无水硫酸钠干燥，过滤，浓缩后硅胶柱分离(乙酸乙酯/戊烷＝1∶50-1∶30)得7-3。
步骤3：在3L四口瓶中，氮气保护下，加入7-3(40g)和二氯甲烷(1600mL)。降温到0℃，分批加入氯铬酸吡啶鎓(PCC，54g)，回到室温反应4小时。加入水，二氯甲烷稀释。分离，水相用二氯甲烷萃取3次。无水硫酸钠干燥，过滤，浓缩后硅胶柱分离(乙酸乙酯/戊烷＝1∶20-1∶10)得7-4。
步骤4：在2000mL四口瓶中，氮气保护下加入7-4(33.8g)和三氯甲烷(1500mL)中，降温到0℃，滴加二乙氨基三氟化(DAST，116g)。回到室温反应过夜。将体系倒入1000 mL冰水中淬灭，乙酸乙酯1000mL萃取3次，500mL水反洗4次，无水硫酸钠干燥，过滤，浓缩后硅胶柱分离(乙酸乙酯/戊烷＝1∶50-1∶20)得7-5。
步骤5：在1000mL单口瓶中，氮气保护下加入7-5(30g，0.12mol)和甲醇(500mL)、有水钯碳(6g，10％)。通入氢气后，室温反应过夜。抽滤，滤液加入10mL浓盐酸，旋干后得产品7-6的盐酸盐。
中间体8-5的合成
Figure PCTCN2015091668-appb-000025
步骤1：0℃条件下，向250mL圆底烧瓶中依次加入乙醚(150mL)，氘代氢化铝锂-d4(2.15g)，8-1(15g)。40℃搅拌过夜。依次加入水(2.15mL)，15％氢氧化钠(2.15mL)，水(6.45mL)，过滤，滤液用无水硫酸钠干燥，浓缩，减压蒸馏得到8-2。H-NMR(400MHz，CDCl3，ppm)：δ1.77-1.75(m，4H)，1.55(d，J＝2.8Hz，4H)。
步骤2：0℃条件下，向500mL圆底烧瓶中依次加入8-2(6g)，二氯甲烷(200mL)，三乙胺(14g)。滴加甲基磺酰氯(11.8g)，搅拌1小时。依次用1N盐酸(100mL)和饱和碳酸氢钠(150mL)洗涤，有机相用无水硫酸钠干燥，浓缩得到8-3。H-NMR(400MHz，CDCl3，ppm)：δ2.97(s，3H)，1.90-1.83(m，4H)，1.82-1.77(m，2H)，1.74-1.65(m，2H)。
步骤3：向250mL圆底烧瓶中依次加入8-3(10g)，N，N-二甲基甲酰胺(150mL)，叠氮化钠(7.4g)。65℃搅拌过夜。加水100mL，乙酸乙酯萃取，有机相用无水硫酸钠干燥，浓缩得到8-4。H-NMR(400MHz，CDCl3，ppm)：δ1.83-1.78(m，2H)，1.74-1.66(m，4H)，1.62-1.59(m，2H)。
步骤4：向250mL圆底烧瓶中依次加入8-4(5g)，甲醇(100mL)，钯碳(300mg)。通入氢气，25℃搅拌过夜。过滤，滤液加入浓盐酸5毫升，浓缩得到8-5的盐酸盐。H-NMR(400MHz，d6-DMSO，ppm)：δ8.10(br s，2H)，1.87-1.80(m，2H)，1.68-1.64(m，2H)，1.63-1.46(m，4H)。
中间体10-8的合成
Figure PCTCN2015091668-appb-000026
步骤1：500mL单口瓶中，将叠氮化钠(32g)溶于水(300mL)中，然后冰浴下滴加氯甲酸乙酯(45g)的乙醚(100mL)溶液，体系室温搅拌过夜。向反应液中加入乙醚(300mL)，有机相用无水硫酸钠干燥，浓缩，得到10-2。
步骤2：500mL三口瓶中，向10-2(24g)的正戊烷(200mL)溶液中加入降冰片烯(20g)，20℃搅拌4天。反应液浓缩得到10-3。
步骤3：500mL单口瓶中，将10-3(55g)溶于甲醇(300mL)中，体系在40度反应2小时。反应液浓缩，减压蒸馏(70度，10mmHg真空度)得到10-4。
步骤4：在40度下向四氢化萘(20g)中缓慢滴加液溴(28g)，2小时滴完，同时产生的溴化氢气体通入10-4(16g)的正戊烷(200mL)溶液中，室温反应4小时。反应液浓缩，加入乙醚(300mL)，饱和碳酸氢钠(100mL)，萃洗。有机相用无水硫酸钠干燥，过滤，浓缩得到10-5。
步骤5：250mL三口瓶中，将Na(5g)缓慢加入到甲醇(100mL)中，搅拌1小时，然后加入10-5(10g)。加热体系至60度，反应16h。反应液浓缩，加入乙醚(300mL)，水(200mL)，萃洗。有机相用无水硫酸钠干燥，过滤，浓缩，减压蒸馏(100度，5mmHg真空度)得到10-6。
步骤6：100mL单口瓶中，将10-6(4.5g)溶于乙醇(40mL)中，加入10％钯碳(400mg)，氢气置换后，体系在氢气球下室温反应过夜。反应体系过滤，滤液浓缩得10-7。
步骤7：100mL单口瓶中，将氢氧化钠(9g)溶于水(40mL)中，然后加入乙二醇(20 mL)和10-7(7g)，加热体系至110度反应3天。冷却到室温，向反应液中加入食盐水(60mL)，用正戊烷(300mL)萃取。有机相用无水硫酸钠干燥，过滤，浓缩，得到10-8。
中间体11-3和15-1的合成
Figure PCTCN2015091668-appb-000027
步骤1：1000mL单口瓶中，将氢化钠(15.4g，60％)溶于四氢呋喃(700mL)中，然后加入己二腈(37.9g)，加热体系至回流反应过夜。向反应液中加入水(300mL)，用乙醚(3x 100mL)萃取。有机相干燥，过滤，浓缩，得到11-1。
步骤2：250mL三口瓶中，向11-1(6.0g)的甲醇溶液中加入盐酸至pH＝3。保持pH＝3～4，分批次加入氰基硼氢化钠(10.5g)，室温搅拌2h。反应液浓缩，加入二氯甲烷(100mL)，分别用饱和碳酸氢钠水溶液(100mL)和饱和食盐水(100mL)萃洗。有机相干燥，过滤，浓缩，得到11-2。
步骤3：250mL单口瓶中，将11-2(3.0g)，N，N-二异丙基乙胺(6.86g)，5-溴-2，4-二氯嘧啶(3.0g)溶于乙腈(100mL)中，加热体系至70度反应过夜。向反应液中加入水(100mL)，用乙酸乙酯(3x 100m了)萃取。有机相干燥，过滤，浓缩，硅胶柱层析(乙酸乙酯/石油醚/二氯甲烷＝1∶15∶1-1∶7∶1)纯化，分别得到黄色固体产品11-3(1g)和15-1(2g)。
中间体12-3的合成：
Figure PCTCN2015091668-appb-000028
步骤1：氮气保护下，向100mL三口瓶中依次加入12-1(2.3g)，5-溴-2硝基吡啶(1.99 g)，25mLN，N-二甲基亚砜，三乙胺(996mg)，油浴升温至80℃反应过夜。降温至室温。加水50mL淬灭反应。75mL乙酸乙酯萃取3次。100mL饱和食盐水反洗3次。无水硫酸钠干燥，旋干。粗品硅胶柱层析(洗脱剂乙酸乙酯∶戊烷＝1∶5-2∶1)，旋干后得到12-2。
步骤2：向100mL单口瓶中依次加入12-2(2.0g)，15mL甲醇，二氯甲烷15mL，有水钯炭(60％)2.0g，甲酸铵2.0g。加毕室温反应过夜。过滤，收集滤液。滤饼用75mL二氯甲烷洗3次。收集有机相用100mL饱和食盐水反洗3次。无水硫酸钠干燥，有机相旋干后得12-3。
中间体13-3的合成
Figure PCTCN2015091668-appb-000029
步骤1：氮气保护下，向100mL三口瓶中依次加入13-1(2.0g)，5-溴-2硝基吡啶(1.73g)，N，N-二甲基亚砜(20mL)，三乙胺(865mg)，油浴升温至80℃反应过夜。降温至室温。加水50mL淬灭反应。75mL乙酸乙酯萃取3次。100mL饱和食盐水反洗3次。无水硫酸钠干燥，旋干。粗品硅胶柱层析(洗脱剂乙酸乙酯∶戊烷＝1∶5-2∶1)。旋干得13-2。
步骤2：向100mL单口瓶中依次加入13-2(1.5g)，甲醇15mL，二氯甲烷15mL，有水钯炭(60％)1.5g，甲酸铵1.5g。加毕室温反应过夜。过滤，收集滤液。滤饼用75mL二氯甲烷洗3次。收集有机相用100mL饱和食盐水反洗3次。无水硫酸钠干燥，旋干得13-3。
中间体14-3的合成
Figure PCTCN2015091668-appb-000030
步骤1：氮气保护下，向100mL三口瓶中依次加入14-1(2.0g)，5-溴-2硝基吡啶(1.85g)，N，N-二甲基亚砜20mL，三乙胺(925mg)，油浴升温至80℃反应过夜。降温至室温。 加水50mL淬灭反应。75mL乙酸乙酯萃取3次。100mL饱和食盐水反洗3次。无水硫酸钠干燥，旋干。粗品硅胶柱层析(洗脱剂乙酸乙酯∶戊烷＝1∶5-2∶1)。旋干后得14-2。
步骤2：向100mL单口瓶中依次加入14-2(1.1g)，甲醇15mL，二氯甲烷15mL，有水钯炭(60％)1.1g，甲酸铵1.1g。加毕室温反应过夜。过滤，收集滤液。滤饼用75mL二氯甲烷洗3次。收集有机相用100mL饱和食盐水反洗3次。有机相用无水硫酸钠干燥，旋干得14-3。
中间体16-2的合成
Figure PCTCN2015091668-appb-000031
步骤1：氮气保护下，向100mL三口瓶中依次加入8-氧杂-3-氮杂双环[3.2.1]辛烷盐酸盐(3.29g)，5-溴-2-硝基吡啶(4.06g)，N，N-二甲基亚砜(45mL)，三乙胺(4.06g)，油浴升温至80℃反应过夜。降温至室温。加水50mL淬灭反应。乙酸乙酯75mL萃取3次。饱和食盐水100mL反洗3次。无水硫酸钠干燥，旋干。粗品硅胶柱层析(洗脱剂乙酸乙酯∶戊烷＝1∶5-2∶1)。旋干得16-1。
步骤2：向100mL单口瓶中依次加入16-1(600mg)，甲醇15mL，二氯甲烷15mL，有水钯炭(60％)600mg，甲酸铵(804mg)。加毕室温反应过夜。过滤，收集滤液。二氯甲烷75mL淋洗滤饼3次。饱和食盐水100mL反洗3次。无水硫酸钠干燥，旋干得16-2。
中间体17-5的合成
Figure PCTCN2015091668-appb-000032
步骤1：在氮气保护下，250mL三口瓶，将17-1a(3.0g)溶于四氢呋喃30mL中加入反应瓶，-78℃滴加双三甲基硅基氨基钾(16mL，1M)，-78℃保温反应3h。-78℃滴加17-1b(5.3g)的四氢呋喃10mL溶液，滴毕回室温反应2h。饱和氯化铵50mL淬灭 反应。乙酸乙酯100mL萃取2次，合并有机相。饱和食盐水100mL反洗1次。无水硫酸钠干燥，旋干。粗品硅胶柱层析(乙酸乙酯∶戊烷＝1∶20)。旋干得17-2。
步骤2：氮气保护下，向100mL三口瓶中依次加入17-2(2.6g)，1，4二氧六环26mL，17-2b(2.0g)，1，1′-二(二苯膦基)二茂铁二氯化钯(II)(0.21g)，醋酸钾(2.08g)，加毕油浴升温至80℃反应过夜。降至室温，抽滤，收集滤液。100mL水淬灭反应。乙酸乙酯100mL萃取3次。饱和食盐水100mL反洗3次。无水硫酸钠干燥，旋干。粗品硅胶柱层析(乙酸乙酯∶戊烷＝1∶30)。旋干得17-3。
步骤3：氮气保护下，向100mL三口瓶中依次加入17-3(0.96g)，5-溴-2-硝基吡啶(1.6g)，1，4二氧六环40mL，水1.5mL，1，1′-二(二苯膦基)二茂铁二氯化钯(II)(0.33g)，碳酸铯(3.0g)，加毕油浴升温至85℃反应过夜，降至室温。体系倒入冰水(100mL)中。乙酸乙酯100mL萃取3次。饱和食盐水100mL反洗3次。无水硫酸钠干燥，旋干。粗品硅胶柱层析(乙酸乙酯∶戊烷＝1∶10-1∶3)。旋干得17-4。
步骤4：100mL三口瓶，17-4(900mg)溶于乙醇45mL和醋酸4mL中，升温至70℃，批量加入铁粉(696mg)，70℃反应30分钟。过滤，收集滤液。旋干。加入1M氢氧化钠调pH＝8。用乙酸乙酯100mL萃取3次，合并有机相。饱和食盐水100mL反洗1次。无水硫酸钠干燥，旋干。50mL乙醚洗1次，得17-5。
中间体18-6和19-2的合成
Figure PCTCN2015091668-appb-000033
步骤1：在氮气保护下，100mL的圆底烧瓶中加入18-1(8g)，乙醇(50mL)，盐酸羟胺(5.15g)，醋酸钠(2.92g)。反应混合物在80℃搅拌5小时。反应混合物冷却到室温。过滤，收集滤液。旋干。混合物中加入100mL水，用乙酸乙酯100mL萃取3次，合并有机相。饱和食盐水100mL反洗1次。无水硫酸钠干燥，旋干得18-2。
步骤2：在氮气保护下，250mL三口瓶中，加入18-2(8.2g)，丙酮(50mL)，水(50mL)和碳酸钠(10.86g)。在10-20℃下，5分钟内分批加入4-甲基苯-1-磺酰氯(9.79g)。所得反应混合物在室温下搅拌过夜。用50mL水淬灭。用乙酸乙酯100mL萃取3次，合并有机相。饱和食盐水100mL反洗1次。无水硫酸钠干燥，旋干。所得混合物加入己醇(100mL)。过滤收集所得产品18-3。
步骤3：在氮气保护下，100ml三口瓶中，加入18-3(6.2g)，5-溴-2-硝基吡啶(5.24g)，4，5-双二苯基膦-9，9-二甲基氧杂蒽(Xantphos)(2.24g)，醋酸钯(579mg)，碳酸铯(12.6g).在105℃下搅拌3小时。用50mL水淬灭。用乙酸乙酯100mL萃取3次，合并有机相。饱和食盐水75ml反洗2次。无水硫酸钠干燥，旋干。所得混合物过柱，用乙酸乙酯/二氯甲烷淋洗(1∶10-1∶1).。得到产品18-4。
步骤4：拆分18-4
拆分条件：手性柱，CHIRALPAK IC尺寸20mm*250mm；流动相为乙醇/正己烷＝50％/50％，得到18-5(3.34g，峰一)为白色固体，和19-1(3.66g，峰二)为白色固体。
步骤5：在氮气保护下，500ml圆底瓶中，加入18-5(500mg)，甲醇(100mL)，二氯甲烷(100mL)，钯碳(10％)(0.5g)和甲酸铵(500mg)。在室温下搅拌过夜。过滤，旋干。得到产品18-6为白色固体。
按照步骤5相似的方法，从化合物19-1得到化合物19-2。
中间体20-5的合成
Figure PCTCN2015091668-appb-000034
步骤1：氮气保护下，向100mL三口瓶中依次加入20-1(2.7g)，乙腈60mL，三乙胺(3.86g)，0℃滴加溴苄(2.61g)，滴毕室温反3小时。反应结束后，加水50mL淬灭反应。二氯甲烷75mL萃取3次。饱和食盐水100mL反洗4次。用无水硫酸钠干燥，旋干得20-2。
步骤2：氮气保护下，向100mL单口瓶中依次加入20-2(2.9g)，二氯甲烷30mL，0℃滴加三氟乙酸2mL，滴毕室温反应3小时。反应结束后加水50mL淬灭反应，碳酸氢钠饱和溶液调pH＝8。二氯甲烷50mL萃取3次，饱和食盐水75mL反洗2次，无水硫酸钠干燥，旋干得20-3。
步骤3：氮气保护下，向100mL三口瓶中依次加入20-3(2.0g)，5-溴-2-硝基吡啶(1.82g)，N，N-二甲基亚砜20mL，三乙胺(910mg)，油浴升温至80℃反应过夜，降温至室温。加水50mL淬灭反应，乙酸乙酯75mL萃取3次，饱和食盐水100mL反洗3次，无水硫酸钠干燥，旋干。粗品硅胶柱层析(洗脱剂乙酸乙酯∶戊烷＝1∶5-2∶1)。旋干后得20-4。
步骤4：向100mL三口瓶中依次加入20-4(1.1g)，乙醇50mL，醋酸5mL，升温至70℃，批量加入铁粉(951mg)，70℃反应2小时，降温至室温。过滤，收集滤液，滤液旋干。1N氢氧化钠溶液调节pH＝8-9，乙酸乙酯75mL萃取3次，饱和食盐水100mL反洗2次，无水硫酸钠干燥，旋干得20-5。
中间体21-3的合成
Figure PCTCN2015091668-appb-000035
步骤1：氮气保护下，向250mL三口瓶中依次加入18-5(2.62g)，二氯甲烷(102mL)。0℃滴加三氟乙酸(10.2mL)，3分钟滴完。室温反应4h。旋干得到21-1。
步骤2：氮气保护下，向250mL三口瓶中依次加入21-1(2.25g)，甲醇(160mL)，甲醛(695mg)。0℃依次加入乙酸(899mg)，氰基硼氢化钠(566mg)。0℃反应5min。加冰水160mL淬灭反应。饱和碳酸钠溶液调pH为9，160mL乙酸乙酯萃取3次。500mL饱和食盐水反洗3次。无水硫酸钠干燥，旋干得21-2。
步骤3：氮气保护下，向250mL单口瓶中依次加入21-2(970mg)，甲醇50mL，二氯甲烷(50mL)，Pd/C(10％，0.97g)，甲酸铵(970mg)，室温反应过夜。过滤，浓缩，用C18柱纯化，旋干得21-3。
中间体22-3的合成
Figure PCTCN2015091668-appb-000036
步骤1：氮气保护下，向250mL三口瓶中依次加入19-1(2.21g)，二氯甲烷121mL。0℃加入三氟乙酸12.1mL。室温反应4h。旋干，得到22-1。
步骤2：氮气保护下，向250mL三口瓶中依次加入22-1(2.08g)，甲醇150mL，甲醛(642mg)。0℃依次加入乙酸(830mg)，氰基硼氢化钠(523mg)。0℃反应5分钟，加 冰水150mL淬灭反应。饱和碳酸钠溶液调pH为9，150mL乙酸乙酯萃取3次。500mL饱和食盐水反洗3次，无水硫酸钠干燥，旋干得22-2。
步骤3：氮气保护下，向100mL单口瓶中依次加入22-2(720mg)，甲醇50mL，二氯甲烷(50mL)，钯碳0.72g，甲酸铵(720mg)，室温反应过夜。过滤，浓干，用C18柱纯化，旋干得22-3。
中间体23-5的合成
Figure PCTCN2015091668-appb-000037
步骤1：250mL三口瓶中，5℃下将间氯过氧苯甲酸(m-CPBA，12g)分批加到1-甲基环戊烯(3.8g)的二氯甲烷(100mL)溶液中，室温反应16小时。反应液过滤，滤液用水(100mL)，碳酸氢钠水溶液(150mL)和硫代硫酸钠水溶液(150mL)萃洗。有机相干燥，过滤，浓缩得到23-2。
步骤2：将氨水(8mL)加到23-3(2.4g)的乙醇(8mL)溶液中，85℃下在玻璃闷罐中反应6小时。反应液冷却到室温，浓缩得到23-3。
步骤3：100mL单口瓶中，将5-溴-2，4-二氯嘧啶(1.26g)溶于乙醇(20mL)中，然后加入23-3(0.9g)，N，N-二异丙基乙胺(2.1g)，室温反应过夜。反应液浓缩后加入水(50mL)，用乙酸乙酯(100mL)萃取。有机相干燥，过滤，浓缩，石油醚重结晶得23-4。
步骤4：氮气保护下，-78℃下将二乙氨基三氟化(DAST，1.5g)加到23-4(1.3g)的二氯甲烷(40mL)溶液中，-78℃下反应1小时。-78℃下加入异丙醇淬灭，升到室温，反应液用水(30mL)，碳酸氢钠水溶液(30mL)萃洗。有机相干燥，过滤，浓缩，硅胶柱层析(乙酸乙酯/石油醚＝1∶10)纯化，得到23-5。
中间体26-1(顺式)和27-1(反式)的合成
Figure PCTCN2015091668-appb-000038
步骤1：氮气保护下，在1L四口瓶中，将5-溴-2，4-二氯嘧啶(70g)溶于600mL二氧六环中，降温至11℃，滴加三乙胺(62.5g)，3-4(30g)的二氧六环溶液，加毕，室温反应过夜。将体系倒入1L冰水混合物淬灭反应。用1L乙酸乙酯萃取3次，有机相合并后用0.5L饱和食盐水反洗3次，有机相用无水NaSO4干燥后浓缩干。粗品硅胶柱层析(洗脱剂石油醚/乙酸乙酯＝100∶1-30∶1)，收集产品浓缩得3-5。
步骤2：在1L单口瓶中，将3-5(23g)，(E)-2-丁烯酸(17.3g)，N，N-二异丙基乙胺(104g)溶于500mL四氢呋喃中，用氮气置换空气3次，再加入三(邻甲苯基)膦(2.42g)和二(氰基苯)二氯化钯(1.5g)，再用氮气置换空气3次，升温80℃反应过夜。将反应体系降至室温后浓缩干。粗品硅胶柱层析(洗脱剂石油醚∶乙酸乙酯＝5∶1-1∶1)，收集产品浓缩得3-6。
步骤3：在1L三口瓶中，将3-6(40g)溶于乙酸酐(200mL)中，升温至130℃，反应2h。反应体系降至室温后将乙酸酐旋干。粗品硅胶柱层析(洗脱剂油醚∶乙酸乙酯＝10∶1-4∶1)得混合物。混合物用正相快速柱层析分离纯化，分别收集产品峰浓缩得到第一个峰26-1(顺式)3.6g黄色固体和第二个峰27-1(反式)1.7g黄色固体。
实施例
实施例1A和1B
Figure PCTCN2015091668-appb-000039
步骤1：1-7的合成
在250mL三口瓶中，氮气保护下加入2，2-二氟环戊胺盐酸盐(6.0g)，70mL二氧六环，三乙胺(33g)。降温到10℃以下，分批加入5-溴-2，4-二氯嘧啶(7.5g)，室温反应过夜。加100mL乙酸乙酯稀释，用3x100mL水反洗，无水硫酸钠干燥，过滤后硅胶柱层析(洗脱剂乙酸乙酯∶石油醚＝1∶50-1∶30)得到1-7。
步骤2：1-8的合成
在250mL三口瓶中，氮气保护下加入1-7(5g)，正丁醇(70mL)，(E)-2-丁烯酸(6.86g)，N，N-二异丙基乙胺(20.5g)。置换空气三次，加入三(邻甲苯基)膦(2.42g)和二(氰基苯)二氯化钯(1.5g)，升温至95℃反应过夜。降至室温，旋干溶剂后直接硅胶柱分离(洗脱剂乙酸乙酯∶石油醚＝1∶10-1∶2)得1-8。
步骤3：1-9的合成
在50mL单口瓶中，加入1-8(2.8g)和乙酸酐(15mL)，升至130℃反应2小时。降至室温，旋干溶剂后直接硅胶柱分离(洗脱剂乙酸乙酯∶石油醚＝1∶20-1∶10)得到1-9。
步骤4：1-10的合成
在50mL单口瓶中，加入1-9(1.5g)，20mL乙腈/甲醇(3∶1)，N-溴代丁二酰亚胺(NBS，1.2g)。升至80℃反应2小时。加50mL乙酸乙酯稀释，用3x50mL水反洗，无水硫酸钠干燥，过滤后滤液旋干。硅胶柱层析(洗脱剂乙酸乙酯∶石油醚＝1∶50-1∶20)得1-10。
步骤5：1-11的合成
在100mL三口瓶中，氮气保护下加入J(1.29g)和20mL甲苯。降温至10℃，加入六甲基二硅基胺基锂(1.29g)，并在该温度下搅拌1小时。在10℃下，分批加入1-10(0.8g)，回到室温反应过夜。过滤，收集滤饼，并用3x10mL的水和2x20mL的正己烷洗涤，得到1-11。
步骤6：1-12的合成
在100mL三口瓶中，氮气保护下加入1-11(0.23g)，正丁醇(20mL)，乙烯基正丁基醚(0.18g)，N，N-二异丙基乙胺(0.14g)。置换空气三次，加入1，1′-二(二苯膦基)二茂铁二氯化钯(II)(0.03g)，升温至95℃反应过夜。降至室温，旋干溶剂后直接硅胶柱分离(洗脱剂乙酸乙酯∶石油醚＝1∶20-1∶3)得到1-12。
步骤7：1-13的合成
在50mL的三口瓶中，氮气保护下加入1-12(0.13g)，盐酸(15mL，6N)。室温反应2h。降至室温，旋干溶剂得1-13。
步骤8：手性制备分离
制备柱：CHIRALPAK AD-H SFC5*25cm，5um，流动相：A相：CO2：50，B相：EtOH(0.2％DEA)-HPLC：50；循环时间：12分钟，检测波长：220nm。
手性HPLC分析(Phenomenex Lux Cellulose-4柱)：实施例1A(保留时间＝28.6分钟)对应手性分离中的第一个峰。实施例1B(保留时间＝23.9分钟)对应手性分离中的第二个峰。
实施例1A：LC-MS(ES，m/z)：484[M+H]+；H-NMR(300MHz，CDCl3，ppm)：δ8.86(s，1H)，8.34(s，1H)，8.17(d，J＝9Hz，1H)，8.07(d，J＝3Hz，1H)，7.35-7.27(m，1H)，6.27-6.11(m，1H)，3.50-3.09(m，8H)，2.89-2.63(m，2H)，2.56(s，3H)，2.41(s，3H)，2.31-2.10(m，4H)。
实施例1B：LC-MS(ES，m/z)：484[M+H]+；H-NMR(300MHz，CDCl3，ppm)：δ8.84(s，1H)，8.33(s，1H)，8.16(d，J＝9Hz，1H)，8.06(d，J＝3Hz，1H)，7.35-7.26(m，1H)，6.25-6.11(m，1H)，3.49-3.10(m，8H)，2.86-2.61(m，2H)，2.54(s，3H)，2.40(s，3H)，2.30-2.11(m，4H)。
实施例2
Figure PCTCN2015091668-appb-000040
除了在步骤1中以2-4代替1-6以外，按照实施例1中的相似方法合成实施例2的化合物。LC-MS(ES，m/z)：462[M+H]+；H-NMR(300MHz，DMSO，ppm)：δ10.02(1H，s)，8.87(1H，s)，8.04-8.03(1H，d，J＝3Hz)，7.86-7.83(1H，d，J＝8.7Hz)，7.46-7.42(1H，dd，J＝9.2Hz，J＝3.0Hz)，3.32-3.07(4H，m)，2.94-2.87(4H，m)，2.38(3H，s)，2.28(3H，s)，1.73-1.56(11H，m)。
实施例3
Figure PCTCN2015091668-appb-000041
除了在步骤1中以3-4代替1-6以外，按照实施例1中的相似方法合成实施例3的化合物。LC-MS(ES，m/z)：460[M+H]+；H-NMR(300MHz，CDCl3，ppm)：δ10.12(s，1H)，8.94(s，1H)，8.03(s，1H)，7.87(d，J＝7.5Hz，1H)，7.42(d，J＝6.9Hz，1H)，6.09-6.00(m，1H)，3.06(s，4H)，2.86(s，4H)，2.42(s，3H)，2.30(s，3H)，1.84(m，2H)，1.49(s，1H)，1.32-1.24(m，4H)，0.46(s，1H)。
实施例4
Figure PCTCN2015091668-appb-000042
除了在步骤1中以4-6代替1-6以外，按照实施例1中的相似方法合成化合物4，将其用反相柱纯化，得实施例4的化合物4的三氟乙酸盐。LC-MS(ES，m/z)：474[M+H]+；H-NMR(300MHz，CD3OD，ppm)：δ0.10-0.70(4H，m)，1.32-2.22(4H，m)，2.43(3H，s)，2.51-2.56(5H，m)，3.46-3.50(8H，m)，5.95-6.02(1H，m)，7.78(1H，br s)，7.93-7.96(1H，d，J＝6.6Hz)，8.05(1H，s)，9.02(1H，s)。
实施例5A和5B
Figure PCTCN2015091668-appb-000043
除了在步骤1中以5-3代替1-6以外，按照实施例1中的相似方法合成。手性分离，得到实施例5A和实施例5B的化合物。
手性制备分离，制备柱：CHIRALPAK AD-H SFC5*25cm，5um，流动相：A相：CO2：50，B相：EtOH(0.2％DEA)-HPLC：50；循环时间：6分钟，检测波长：220nm。
手性HPLC分析(CHIRALPAK IC柱)：实施例5A(保留时间＝44.8分钟)对应手性分离中的第一个峰。实施例5B(保留时间＝53.5分钟)对应手性分离中的第二个峰。
实施例5A：LC-MS(ES，m/z)：474[M+H]+；H-NMR(300MHz，CF3COOD，ppm)：δ1.15-1.82(7H，m)，2.39(3H，s)，2.40-2.76(6H，m)，3.64-3.72(8H，m)，4.96-5.24(1H，m)， 7.89-8.16(3H，m)，9.07-9.08(1H，d，J＝3.6Hz)。
实施例5B：LC-MS(ES，m/z)：474[M+H]+；H-NMR(300MHz，CF3COOD，ppm)：δ1.32-1.74(7H，m)，2.40(3H，s)，2.47-2.75(6H，m)，3.66-3.71(8H，m)，5.19-5.23(1H，m)，7.89-7.92(1H，d，J＝9.3Hz)，8.06(1H，s)，8.13-8.16(1H，d，J＝9.6Hz)，9.08(1H，s)。
实施例6
Figure PCTCN2015091668-appb-000044
除了在步骤1中以6-4代替1-6以外，按照实施例1中的相似方法合成化合物6，将其用反相柱纯化，得实施例6的化合物6的三氟乙酸盐。LC-MS(ES，m/z)：460[M+H]+；H-NMR(300MHz，D2O，ppm)：δ8.99(1H，s)，8.06-8.02(1H，dd，J＝9.3Hz，J＝2.7Hz)，7.81(1H，s)，7.48-7.45(1H，m)，6.16-6.07(1H，m)，3.70-3.64(8H，m)，2.45(3H，s)，2.32(3H，s)，2.17-1.90(4H，m)，1.36(2H，m)，0.85(1H，m)，0.54(1H，m)。
实施例7A和7B
Figure PCTCN2015091668-appb-000045
除了在步骤1中以7-6代替1-6以外，按照实施例1中的相似方法合成。手性分离，得到实施例7A和实施例7B的化合物。
手性制备分离，制备柱：CHIRALPAK AD-H SFC5*25cm，5um，流动相：A相：CO2：50，B相：EtOH(0.2％DEA)-HPLC：50；循环时间：12分钟，检测波长：220nm。
手性HPLC分析(CHIRALPAK AD-H柱)：实施例7A(保留时间＝30.7分钟)对应手 性分离中的第一个峰。实施例7B(保留时间＝38.6分钟)对应手性分离中的第二个峰。
实施例7A：LC-MS(ES，m/z)：484[M+H]+；H-NMR(300MHz，CDCl3，ppm)：δ8.83(s，1H)，8.13-8.04(m，2H)，7.98(s，1H)，7.37-7.33(m，1H)，6.19-6.12(m，1H)，3.18-3.11(m，8H)，2.64(m，2H)，2.57(s，3H)，2.40(s，3H)，2.39-2.15(s，3H)。
实施例7B：LC-MS(ES，m/z)：484[M+H]+；H-NMR(300MHz，CDCl3，ppm)：δ8.83(s，1H)，8.13-8.05(m，2H)，7.97(s，1H)，7.6(d，J＝3Hz，1H)，6.21-6.13(m，1H)，3.18-3.11(m，8H)，2.65(s，2H)，2.57(s，3H)，2.40(s，3H)，2.39-2.15(s，3H)。
实施例8
Figure PCTCN2015091668-appb-000046
除了在步骤1中以8-5代替1-6以外，按照实施例1中的相似方法合成实施例8的化合物。LC-MS(ES，m/z)：449[M+H]+；H-NMR(400MHz，CD3OD，ppm)：δ9.14(s，1H)，8.20-8.17(m，1H)，8.01(d，J＝2.8Hz，1H)，7.62(d，J＝9.2Hz，1H)，3.57-3.54(m，4H)，3.45-3.43(m，4H)，2.50(s，3H)，2.43(s，3H)，2.31-2.26(m，2H)，2.10-2.08(m，2H)，2.07-1.90(m，2H)，1.90-1.67(m，2H)。
实施例9
Figure PCTCN2015091668-appb-000047
除了在步骤1中以3，3-二氟环丁胺代替1-6以外，按照实施例1中的相似方法合成化 合物9，将其用盐酸处理，得实施例9的化合物9的盐酸盐。LC-MS(ES，m/z)：470[M+H]+；H-NMR(400MHz，CD3OD，ppm)：δ9.16(s，1H)，8.21(dd，J＝2.4Hz，9.6Hz，1H)，8.01(d，J＝2.4Hz，1H)，7.61(d，J＝9.6Hz，1H)，5.72-5.67(m，1H)，3.57-3.55(m，6H)，3.35-3.25(m，4H)，3.06-2.98(m，2H)，2.52(s，3H)，2.45(s，3H)。
实施例10
Figure PCTCN2015091668-appb-000048
除了在步骤1中以10-8代替1-6以外，按照实施例1中的相似方法合成实施例10的化合物。LC-MS(ES，m/z)：474[M+H]+；H-NMR(400MHz，CD3OD，ppm)：δ9.06(s，1H)，8.15(dd，J＝2.8Hz，9.6Hz，1H)，7.96(d，J＝2.8Hz，1H)，7.61(d，J＝9.6Hz，1H)，4.29(s，1H)，3.42-3.54(m，8H)，3.25(s，2H)，2.46(s，3H)，2.38(s，3H)，1.84(m，2H)，1.54(m，2H)，1.34(m，4H)。
实施例11
Figure PCTCN2015091668-appb-000049
除了在步骤2中以11-3代替1-7以外，按照实施例1中的相似方法合成实施例11的化合物。LC-MS(ES，m/z)：473[M+H]+；H-NMR(400MHz，CD3OD，ppm)：δ9.07(s，1H)，8.02(d，J＝2.8Hz，1H)，7.94(dd，J＝2.4Hz，9.6Hz，1H)，7.82(d，J＝9.6Hz，1H)，6.38-6.30(m，1H)，3.49-3.40(m，9H)，2.61-2.56(m，2H)，2.49(s，3H)，2.43(s，3H)，2.22-2.15(m，2H)，2.06-2.03(m， 1H)，1.70-1.65(m，1H)。
实施例12
Figure PCTCN2015091668-appb-000050
除了在步骤1中以环戊胺代替1-6，在步骤5中以12-3代替J以外，按照实施例1中的相似方法合成实施例12的化合物。LC-MS(ES，m/z)：474[M+H]+；H-NMR(300MHz，CDCl3，ppm)：δ1.69-2.07(10H，m)，2.24-384(5H，m)，2.56(3H，s)，2.98-3.01(2H，d，J＝10.5Hz)，3.42-3.46(2H，d，J＝10.5Hz)，3.72(2H，m)，2.83-5.95(1H，m)，7.19-7.23(1H，dd，J1＝2.4Hz，J2＝9.3Hz)，7.94-7.95(1H，m)，8.03(1H，m)，8.10-8.13(1H，d，J＝9.3Hz)，8.81(1H，s)。
实施例13
Figure PCTCN2015091668-appb-000051
除了在步骤1中以环戊胺代替1-6，在步骤5中以13-3代替J以外，按照实施例1中的相似方法合成化合物13，将其用反相柱纯化，得实施例13的化合物13的三氟乙酸盐。LC-MS(ES，m/z)：474[M+H]+；H-NMR(300MHz，d6-DMSO，ppm)：δ0.96-0.98(2H，m)，1.00-1.09(2H，m)，1.58-1.90(6H，m)，2.21-2.27(2H，m)，2.32(3H，s)，2.43(3H，s)，3.32(2H，s)，3.41-3.43(4H，m)，5.80-5.86(1H，m)，7.56-7.60(1H，dd，J1＝2.7Hz，J2＝9.3Hz)，7.88-7.91(1H，d，J＝9Hz)，8.10-8.11(1H，d，J＝2.7Hz)，8.97(1H，s)，9.21(1H，br s)，10.31(1H，s)。
实施例14
Figure PCTCN2015091668-appb-000052
除了在步骤1中以环戊胺代替1-6，在步骤5中以14-3代替J以外，按照实施例1中的相似方法合成化合物14，将其用反相柱纯化，得实施例14的化合物14的三氟乙酸盐。LC-MS(ES，m/z)：460[M+H]+；H-NMR(300MHz，d6-DMSO，ppm)：δ1.59-1.96(m，6H)，2.14-2.25(m，3H)，2.31(s，3H)，2.43(s，3H)，3.21-3.29(m，3H)，3.64-3.67(m，1H)，4.49(s，1H)，4.70(s，1H)，5.81-5.86(m，1H)，7.27-7.30(d，1H，J＝8.7Hz)，7.81-7.84(m，2H)，8.54-8.57(m，1H)，8.95(br s，1H)，9.02(s，1H)，10.21(s，1H)。
实施例15
Figure PCTCN2015091668-appb-000053
除了在步骤2中以15-1代替1-7以外，按照实施例1中的相似方法合成实施例15的化合物。LC-MS(ES，m/z)：473[M+H]+；H-NMR(400MHz，CDCl3，ppm)：δ9.07(s，1H)，8.02(d，J＝2.8Hz，1H)，7.94(dd，J＝2.4Hz，9.6Hz，1H)，7.82(d，J＝9.6Hz，1H)，6.38-6.30(m，1H)，3.49-3.40(m，9H)，2.61-2.56(m，2H)，2.49(s，3H)，2.43(s，3H)，2.22-2.15(m，2H)，2.06-2.03(m，1H)，1.70-1.65(m，1H)。
实施例16
Figure PCTCN2015091668-appb-000054
除了在步骤1中以环戊胺代替1-6，在步骤5中以16-2代替J以外，按照实施例1中的相似方法合成实施例16的化合物。LC-MS(ES，m/z)：475[M+H]+；H-NMR(300MHz，CDCl3，ppm)：δ1.40-1.87(9H，m)，2.21-2.32(3H，m)，2.33(3H，s)，2.44(3H，s)，2.86-2.89(2H，d，J＝9.9Hz)，3.42-3.46(2H，d，J＝10.8Hz)，4.39-4.47(2H，m)，5.72-5.87(1H，m)，7.69-7.92(3H，m)，8.98(1H，s)，10.74(1H，br s)。
实施例17
Figure PCTCN2015091668-appb-000055
除了在步骤1中以环戊胺代替1-6，在步骤5中以17-5代替J以外，按照实施例1中的相似方法合成实施例17的化合物。LC-MS(ES，m/z)：475[M+H]+；H-NMR(300MHz，CDCl3，ppm)：δ δ1.75-1.91(2H，m)，1.93-2.04(2H，m)，2.08-2.11(3H，m)，2.26-2.41(6H，m)，2.44(3H，s)，2.51(3H，s)，2.67-2.78(1H，m)，3.17-3.32(1H，m)4.38-4.48(2H，m)，6.00-6.06(1H，m)，6.63-6.65(1H，d，J＝5.7Hz)，7.86-7.89(1H，d，J＝9.0Hz)，8.28-8.32(1H，dd，J＝2.1Hz，J＝9.0Hz)，8.44(1H，s)，9.10(1H，s)。
实施例18
Figure PCTCN2015091668-appb-000056
除了在步骤1中以环戊胺代替1-6，在步骤5中以18-6代替J以外，按照实施例1中的相似方法合成实施例18的化合物。LC-MS(ES，m/z)：503[M+H]+；H-NMR(300MHz，CDCl3，ppm)：δ1.60-1.94(m，10H)，2.23-2.29(m，2H)，2.33(s，3H)，2.43(s，3H)，2.60-2.67(m，1H，J＝21.9Hz)，2.81-2.86(d，1H，J＝15.9Hz)，3.48-3.60(m，3H)，3.99-4.04(d，1H，J＝14.1Hz)，5.81-5.92(m，1H)，7.64-7.68(m，1H)，8.07-8.10(d，1H，J＝9Hz)，8.18-8.18(d，1H，J＝2.4Hz)，9.02(s，1H)，10.43(s，1H)。手性HPLC分析(Lux Cellulose-4柱)：实施例18(保留时间＝21.5分钟)。
实施例19
Figure PCTCN2015091668-appb-000057
除了在步骤1中以环戊胺代替1-6，在步骤5中以19-2代替J以外，按照实施例1中的相似方法合成实施例19的化合物。LC-MS(ES，m/z)：503[M+H]+；H-NMR(DMSO，300MHz，ppm)：δ1.59-1.93(m，10H)，2.23-2.33(m，2H)，2.34(s，3H)，2.47(s，3H)，2.59-2.67(m，1H)，2.81-2.86(d，1H，J＝15.3Hz)，3.48-3.58(m，3H)，3.99-4.04(d，1H，J＝14.4Hz)，5.81-5.92(m，1H)，7.64-7.68(m，1H)，8.07-8.09(d，1H，J＝8.7Hz)，8.17-8.18(d，1H，J＝2.4Hz)，9.01(s，1H)，10.43(s，1H)。手性HPLC分析(Lux Cellulose-4柱)：实施例19(保留时间＝15.2分钟)。
实施例20
Figure PCTCN2015091668-appb-000058
除了在步骤1中以环戊胺代替1-6，在步骤5中以20-5代替J以外，按照实施例1中的相似方法合成实施例20的化合物。LC-MS(ES，m/z)：503[M+H]+；H-NMR(300MHz，d6-DMSO，ppm)：δ1.48-2.32(14H，m)，2.43(3H，s)，3.04-3.14(4H，m)，4.40-4.49(2H，m)，5.78-5.87(1H，m)，7.48-7.50(1H，d，J＝5.7Hz)，7.86-7.92(1H，m)，8.08(1H，s)，8.74(1H，broad s)，8.90-8.96(2H，m)，10.19(1H，s)。
实施例21
Figure PCTCN2015091668-appb-000059
除了在步骤1中以环戊胺代替1-6，在步骤5中以21-3代替J以外，按照实施例1中的相似方法合成化合物21，将其用反相柱纯化，得实施例21的化合物21的三氟乙酸盐。LC-MS(ES，m/z)：516[M+H]+；H-NMR(300MHz，d6-DMSO，ppm)：δ1.60-1.67(m，2H)，1.81-1.83(m，6H)，1.96-2.30(m，4H，)，2.33(S，1H)，2.50-2.51(m，3H)，2.83-2.90(d，4H)，3.19-3.25(d，1H)，3.83-3.90(m，1H)，4.02-4.12(m，2H)，4.33-4.37(d，1H)，5.84-5.90(m，1H)，7.77-7.81(m，1H)，8.11-8.29(d，1H)，8.03(s，1H)，9.02(s，1H)，10.09(s，1H)，10.52(s，1H)。手性HPLC分析(Lux Cellulose-4柱)：实施例21(保留时间＝34.7分钟)。
实施例22
Figure PCTCN2015091668-appb-000060
除了在步骤1中以环戊胺代替1-6，在步骤5中以22-3代替J以外，按照实施例1中的相似方法合成化合物22，将其用反相柱纯化，得实施例22的化合物22的三氟乙酸盐。LC-MS(ES，m/z)：516[M+H]+；H-NMR(300MHz，d6-DMSO，ppm)：1.95-2.01(m，2H)，2.14-2.16(m，6H)，2.23-2.30(m，4H)，2.38(s，1H)，2.51(s，3H)，2.83-2.90(m，4H)，3.21-3.26(d，1H，J＝17.1Hz)，3.83-3.90(m，1H，J＝23.1Hz)，4.00-4.13(m，2H)，4.33-4.39(d，1H，J＝17.1Hz)，5.85-5.91(m，1H)，7.78-7.82(m，1H)，8.11-8.14(d，1H，J＝8.7Hz)，8.30-8.31(d，1H，J＝2.4Hz)，9.03(s，1H)，10.14(s，1H)，10.53(s，1H)。手性HPLC分析(Lux Cellulose-4柱)：实施例22(保留时间＝24.2分钟)。
实施例23
Figure PCTCN2015091668-appb-000061
除了在步骤1中以23-5代替1-6以外，按照实施例1中的相似方法合成实施例23的化合物。LC-MS(ES，m/z)：480[M+H]+；H-NMR(400MHz，d6-DMSO，ppm)：δ10.15(s，1H)，8.92(s，1H)，8.07(d，J＝2.4Hz，1H)，7.90(d，J＝8.4Hz，1H)，7.52(dd，J＝2.4，8.8Hz，1H)，5.79(m，1H)，3.17-3.18(m，8H)，3.04(m，1H)，2.38(s，3H)，2.29(s，3H)，2.22(m，1H)，2.00(m，1H)，1.80(m，2H)，1.55(m，1H)，1.32(d，J＝22Hz，3H)。
实施例24A和24B
Figure PCTCN2015091668-appb-000062
除了在步骤5中以18-6代替J以外，按照实施例1中的相似方法合成。手性分离中间体24-11得到24-11A和24-11B，然后可参照实施例1合成线路中的步骤6和7得到实施例24A和实施例24B的化合物。
所得消旋体中间体化合物24-11用Pre-SFC手性制备分离，制备柱：DAICEL CHIRALPAKAD-H 19*250mm，5um；流动相：A相：HEX(0.1％DEA)，B相：ETOH；梯度：PhaseA/Phase B＝70/30；检测波长：220nm。分别收集两个峰，第一个峰24-11A(出峰时间：8.1分钟)和第二个峰24-11B(出峰时间：11.2分钟)
手性HPLC分析(CHIRALPAK IC柱)：实施例24A(保留时间＝8.53分钟)对应手性分离中的第一个峰。实施例24B(保留时间＝8.73分钟)对应手性分离中的第二个峰。
实施例24A：LC-MS(ES，m/z)：538[M+H]+；H-NMR(300MHz，CDCl3，ppm)：δ1.75-2.34(6H)，2.42(s，3H)，2.50(3H)，2.54-2.99(m，4H)，3.57-3.65(m，1H)，3.72-3.84(m，2H)，4.19-4.24(m，1H)，6.16-6.21(m，1H)，7.57-7.61(m，1H)，8.24-8.25(d，1H，J＝2.7Hz)，8.34-8.37(d，1H，J＝9.0Hz)，8.56(br s，1H)，8.89(s，1H)。
实施例24B：LC-MS(ES，m/z)：538[M+H]+；H-NMR(300MHz，CDCl3，ppm)：δ1.76-1.88(m，2H)，2.03-2.04(m，2H)，2.08-2.33(m，4H)，2.42(s，3H)，2.54(s，3H)，2.68-2.83(m 2H)，2.96-2.99(m，2H)，3.58-3.66(m，1H)，3.76-3.81(m，2H)，4.17-4.22(d，1H，J＝14.7Hz)，6.14-6.22(m，1H)，7.59-7.63(m，1H)，8.24-8.25(d，1H，J＝2.4Hz)，8.36-8.39(d，1H，J＝9.0Hz)，8.86(br s，1H)，8.92(s，1H)
实施例25A和25B
Figure PCTCN2015091668-appb-000063
除了在步骤5中以19-2代替J以外，按照实施例1中的相似方法合成。手性分离中间体25-12得到25-12A和25-12B，然后可参照实施例1合成线路中的步骤7得到实施例25A和实施例25B的化合物。
所得消旋体中间体化合物25-12用Pre-SFC手性制备分离，制备柱：Chiralpak AS-H5*25cm，5um，流动相：A相：CO2：50，B相：MEOH(0.2％DEA)：50；循环时间：3.4分钟，检测波长：220nm。分别收集两个峰，第一个峰25-12A(出峰时间：5.51分钟)和第二个峰25-12B(出峰时间：6.76分钟)。
实施例25A：LC-MS：(ES，m/z)：538[M+H]+；H-NMR(300MHz，CDCl3，ppm)：δ 1.78-2.33(m，6H)，2.42(s，3H)，2.53(s，3H)，2.73-2.83(m，2H)，2.95-2.96(m，2H)，3.58-3.65(m，1H)，3.76-3.81(m，2H)，4.15-4.20(d，1H，J＝14.7Hz)，6.14-6.22(m，1H)，7.59-7.63(m，1H)，8.23-8.24(d，1H，J＝2.4Hz)，8.35-8.38(d，1H，J＝9.0Hz)，8.66(br s，1H)，8.90(s，1H).
实施例25B：LC-MS：(ES，m/z)：538[M+H]+；H-NMR：(300MHz，CDCl3，ppm)：δ1.74-2.27(m，6H)，2.42(s，3H)，2.54(s，3H)，2.73-2.82(m，2H)，2.95-2.96(m，2H)，3.57-3.64(m，1H)，3.77-3.79(m，2H)，4.13-4.18(d，1H，J＝14.7Hz)，6.13-6.21(m，1H)，7.57-7.61(m，1H)，8.25-8.26(d，1H，J＝2.4Hz)，8.34-8.37(d，1H，J＝9.0Hz)，8.62(br s，1H)，8.90(s，1H).
实施例26A和26B
Figure PCTCN2015091668-appb-000064
从26-1到26-4和26-5的合成可参照实施例1合成线路中的步骤4，5和步骤6。手性分离方法得到26-4和26-5。制备柱：CHIRALPAK AD-H SFC5*25cm，5um，流动相：A相：CO2：50，B相：EtOH(0.2％DEA)-HPLC：50；循环时间：6分钟，检测波长：220nm。26-4对应手性分离中的第一个峰(保留时间＝8.69分钟)。26-5对应手性分离中的第二个峰(保留时间＝10.98分钟)。从26-4到26-5到最终产物的合成可参照实施例1合成线路中的步骤7。
实施例26A：LC-MS：(ES，m/z)：513[M+H]+；H-NMR：(300MHz，CDCl3，ppm)：δ0.63-0.66(m，1H)，1.34-2.34(m，7H)，2.38(s，3H)，2.44-2.50(m，1H)，2.55(s，3H)，3.01-3.09 (m，1H)，3.38-3.44(m，1H)，3.67-3.74(m，2H)，4.12-4.19(m，2H)，4.61-4.65(m，1H)，6.01-6.08(m，1H)，7.57-7.61(m，1H)，8.14-8.21(m，2H)，8.42-8.45(d，1H，J＝8.7Hz)，8.82(s，1H)。
实施例26B：LC-MS：(ES，m/z)：513[M+H]+；H-NMR：(300MHz，CDCl3，ppm)：δ0.72-0.74(m，1H)，1.35-2.37(m，7H)，2.42(s，3H)，2.43-2.52(m，1H)，2.54(s，3H)，2.97-3.14(m，2H)，3.58-3.65(m，1H)，3.79-3.83(m，2H)，4.19-4.24(m，1H)，5.89-5.90(m，1H)，7.57-7.61(m，1H)，8.20-8.21(m，2H)，8.45-8.46(m，1H)，8.83(s，1H)。
实施例27A和27B
Figure PCTCN2015091668-appb-000065
从27-1到27-4和27-5的合成可参照实施例1合成线路中的步骤4，5和步骤6。手性分离方法得到27-4和27-5。制备柱：CHIRALPAK AD-H SFC5*25cm，5um，流动相：A相：CO2：50，B相：EtOH(0.2％DEA)-HPLC：50；循环时间：6分钟，检测波长：220nm。27-4对应手性分离中的第一个峰(保留时间＝20.2分钟)。27-5对应手性分离中的第二个峰(保留时间＝26.5分钟)。从27-4到27-5到最终产物的合成可参照实施例1合成线路中的步骤7。
实施例27A：LC-MS：(ES，m/z)：513[M+H]+；H-NMR：(300MHz，CDCl3，ppm)：δ0.72-0.75(m，1H)，1.35-2.07(m，5H)，2.38(s，3H)，2.55-2.98(m，4H)，2.97-2.98(m，2H)，3.58-3.65(m，1H)，3.78-3.83(m，2H)，4.18-4.23(m，2H)，5.88-5.89(m，1H)，7.57-7.61(m，1H)，8.18-8.20(m，2H)，8.42-8.47(d，1H，J＝9.0Hz)，8.82(s，1H)。
实施例27B：LC-MS：(ES，m/z)：513[M+H]+；H-NMR：(300MHz，CDCl3，ppm)：δ 0.72-0.74(m，1H)，1.35-2.10(m，5H)，2.42(s，3H)，2.54-2.58(m，4H)，2.97-2.98(m，2H)，3.58-3.65(m，1H)，3.79-3.83(m，2H)，4.19-4.24(m，2H)，5.89-5.90(m，1H)，7.57-7.61(m，1H)，8.20-8.21(m，2H)，8.45-8.46(m，1H)，8.83(s，1H)。
实验实施例
CDK4和CDK6激酶体外活性实验方法
CDK4和CDK6激酶体外活性筛选实验采用方法为Caliper Mobility Shift Assay，该方法是以微流体芯片技术的迁移率检测技术为核心的检测平台。实验配置：1x CDK4激酶反应缓冲液(20mM HEPES，pH 7.5，0.01％Triton X-100，10mM MgCl2，2mM DTT)；1x CDK6激酶反应缓冲液(50mM HEPES，pH 7.5，0.0015％Brij-35，10mM MgCl2，2mM DTT)；激酶反应终止液(100mM HEPES，pH 7.5，0.015％Brij-35，0.2％Coating Reagent#3，50mM EDTA)；2.5x CDK4激酶溶液[25nM CDK4激酶/cyclin D3(Carna，Cat#04-105)在1x CDK4激酶溶液]；2.5x CDK6激酶溶液[50nM CDK6激酶/cyclin D3(Carna，Cat#04-107)在1x CDK6激酶溶液]；2.5x底物肽溶液[7.5μM底物肽FAM-P8(GL Biochem，Cat.#112396)和552.5μM ATP在1x CDK4激酶溶液中，或7.5uM FAM-P8和2mM ATP在1x CDK6激酶溶液中]。CDK4激酶反应溶液最终浓度：10nM CDK4/Cyclin D3，3μM底物肽FAM-P8，221μM ATP，10mM MgCl2，2mM DTT；CDK6激酶反应溶液最终浓度：20nM CDK6/Cyclin D3，3μM底物肽FAM-P8，800μM ATP，10mM MgCl2，2mM DTT。
实验步骤：先在96孔板中准备5x反应终浓度的化合物溶液：用100％DMSO配好50x反应终浓度的化合物溶液，用1x激酶反应缓冲液稀释至5x反应终浓度化合物溶液；在震荡器上震动10分钟；然后在384孔测试板中加入以下溶液：5uL的5x反应终浓度的化合物溶液(最终384孔测试板中1x反应化合物终浓度为0.1nM-1000nM和10μL的2.5x的CDK4或CDK6激酶溶液，室温孵育10分钟后再加入10μL的2.5x底物肽溶液，在28℃下放置一小时后加入25μL激酶反应终止液，1200RPM离心一分钟。最后将384孔测试板放入Caliper EZ Reader上收集测试数据(转化率)，通过GraphPad Prism  V5.0软件计算出激酶的抑制曲线，并基于此数据计算出获得50％抑制效果所需的化合物浓度，即化合物的IC50。
Figure PCTCN2015091668-appb-000066

Claims (10)

  1. 下式I所示的化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物：
    Figure PCTCN2015091668-appb-100001
    其中，
    R1选自以下基团：
    Figure PCTCN2015091668-appb-100002
    R2和R2’各自独立地选自H，氘(D)，C1-C4烷基，氟和氰基，并且，R2和R2’可以在同一个或不同碳原子上；
    n＝1或2
    R3选自以下基团：
    Figure PCTCN2015091668-appb-100003
    R4选自H，氘(D)和C1-C4烷基，
    W选自CH2，CH2CH2，O和NH
    X选自CH和N
    Y选自CH2，NH和O
    m＝0，1或2
    n＝0，1或2。
  2. 根据权利要求1所述的化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物，其中，
    R1选自以下基团：
    Figure PCTCN2015091668-appb-100004
    R3选自以下基团：
    Figure PCTCN2015091668-appb-100005
  3. 根据权利要求1中式I所示的化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物，其中，
    R1选自以下基团：
    Figure PCTCN2015091668-appb-100006
    R3选自以下基团：
    Figure PCTCN2015091668-appb-100007
  4. 根据权利要求1中式I所示的化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物，其中，
    R1选自以下基团：
    Figure PCTCN2015091668-appb-100008
    R3选自以下基团：
    Figure PCTCN2015091668-appb-100009
  5. 根据权利要求1所述的式I所示化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物，其中，所述化合物选自以下化合物：
    Figure PCTCN2015091668-appb-100010
    Figure PCTCN2015091668-appb-100011
  6. 根据权利要求1-5中任一项所述的化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物，其中，所述药学上可接受的盐包括盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、磷酸盐、硝酸盐、乙酸盐、三氟乙酸盐、对甲苯磺酸盐、水杨酸盐、甲磺酸盐、草酸盐、琥珀酸盐、柠檬酸盐、苹果酸盐、乳酸盐、富马酸盐。
  7. 根据权利要求1-6中任一项所述的化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物用于制备治疗与细胞周期蛋白依赖性激酶信号通路有关的疾病的药物中的用途。
  8. 根据权利要求7所述的用途，其中，所述的化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体、前药分子或溶剂合物作为细胞周期蛋白依赖性激酶CDK4和/或CDK6选择性抑制剂。
  9. 根据权利要求7或8所述的用途，其中，所述与细胞周期蛋白依赖性激酶信号通路有关的疾病包括乳腺癌，神经母细胞瘤、恶性横纹肌瘤、胶质瘤、肺癌、结肠直肠癌、胃癌、胃肠道基质瘤(GIST)、肝细胞癌、前列腺瘤、肉瘤、卵巢癌、宫颈癌、胰腺癌、黑色素瘤、甲状腺癌、胆管癌、子宫内膜癌、肾癌、间皮瘤、淋巴瘤、白血病、非霍奇金淋巴瘤、间变性大细胞淋巴瘤、急性髓细胞白血病(AML)、多发性骨髓瘤。
  10. 一种药物组合物，所述药物组合物含有：治疗有效量的选自根据权利要求1-6中任一项所述的化合物、其药学上可接受的盐、对映异构体、非对映异构体、外消旋体前药分子、溶剂合物和它们的混合物中的一种；和任选地药学上可接受的载体。
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