技术领域Technical Field
本发明属药物合成技术领域，涉及新型二氢吡喃酮类化合物，其制备方法和应用，以及含有它们作为活性成分的药物组合物，以及其在制备用以治疗与微管蛋白相关的疾病，特别是微管蛋白相关的癌症的药物中的用途。The present invention belongs to the technical field of drug synthesis, and relates to novel dihydropyrone compounds, preparation methods and applications thereof, as well as pharmaceutical compositions containing them as active ingredients, and applications thereof in the preparation of drugs for treating diseases associated with tubulin, in particular tubulin-related cancers.
背景技术Background Art
文献记载了癌症是威胁人类健康的重大疾病之一。近年来，全球每年有大约1800万新增癌症病例，导致了约1000万人死亡。癌症发病率的居高不下，据信与社会老龄化以及不健康的生活方式密切相关。研究显示，致癌起因既有外部因素，如烟草、化学物质、放射线和传染性生物等，也有内在因素，如遗传易感性、免疫状况等。随着现代生活环境和生活方式的变化，导致癌症的发病率一直在持续增加。肿瘤细胞增长快、易变异，从而产生多药耐药，导致化疗失败。据有关统计，90％以上的化疗失败与肿瘤细胞的多药耐药相关，常规治疗副作用大，治疗效果不佳，肿瘤预后复发，转移等现象迫切需要新的治疗技术来解决这类瓶颈作用。国际医学界将分子基础上的个体化化疗和靶向治疗看作是突破目前肺癌治疗瓶颈的希望所在。Literature records that cancer is one of the major diseases that threaten human health. In recent years, there are about 18 million new cancer cases worldwide each year, resulting in about 10 million deaths. The high incidence of cancer is believed to be closely related to the aging of society and unhealthy lifestyles. Studies have shown that the causes of cancer include both external factors, such as tobacco, chemicals, radiation and infectious organisms, as well as internal factors, such as genetic susceptibility and immune status. With the changes in modern living environment and lifestyle, the incidence of cancer has continued to increase. Tumor cells grow fast and are prone to mutation, thus producing multidrug resistance and leading to chemotherapy failure. According to relevant statistics, more than 90% of chemotherapy failures are related to multidrug resistance of tumor cells. Conventional treatment has large side effects, poor treatment effects, and tumor recurrence and metastasis are urgently needed. New treatment technologies are needed to solve this bottleneck. The international medical community regards molecular-based individualized chemotherapy and targeted therapy as the hope to break through the current bottleneck in lung cancer treatment.
研究表明，微管(microtubule)是广泛存在于各种真核细胞中的一种中空管状结构(内径约15nm，外径约24-26nm，璧厚约5nm)。其基本组成单位是由α和β微管蛋白(tubulin)结合而成的异二聚体(heterodimer)。作为真核细胞的组分，微管具有多种重要的生物学功能。Studies have shown that microtubules are hollow tubular structures (inner diameter of about 15nm, outer diameter of about 24-26nm, wall thickness of about 5nm) that are widely present in various eukaryotic cells. Its basic component is a heterodimer composed of α and β tubulin. As a component of eukaryotic cells, microtubules have many important biological functions.
据对微管参与信号转导的研究证明，微管参与ehog、JNK、Wnt以及ERK蛋白激酶信号转导通路，信号分子可通过直接与微管作用或通过马达蛋白或通过一些支架蛋白来与微管作用。微管的信号转导功能具有重要的生物学作用，它与细胞的极化、微管的不稳定动力学行为、微管的稳定性变化、微管的方向性及微管的组织中心的位置均有关。(Nogales E.Annu.Rev.BioPhyS.Biomol.Struct.，2001，30：397-420，PhiliP E.T.，Clinical CancerResearch 2004，10(2)：415-427，Wittmann T.，Hyman A.，Nat.CellBiof.2001，3(1)：28-34，Gurdersen G.G.，Cook T.，Cell Biol.，1999，11(1)：81-94)由于微管在肿瘤细胞的生长和发育中起到非常关键的作用，使得微管成为比较理想的抗肿瘤药物研究的靶点。According to the research on microtubules involved in signal transduction, microtubules are involved in the signal transduction pathways of ehog, JNK, Wnt and ERK protein kinases. Signal molecules can interact with microtubules directly or through motor proteins or some scaffold proteins. The signal transduction function of microtubules has important biological effects. It is related to cell polarization, unstable dynamic behavior of microtubules, changes in microtubule stability, directionality of microtubules and the location of the organizing center of microtubules. (Nogales E.Annu.Rev.BioPhyS.Biomol.Struct.,2001,30:397-420, PhiliPET.,Clinical CancerResearch 2004,10(2):415-427, Wittmann T., Hyman A.,Nat.CellBiof.2001,3(1):28-34, Gurdersen G.G., Cook T.,Cell Biol.,1999,11(1):81-94) Since microtubules play a very critical role in the growth and development of tumor cells, microtubules have become an ideal target for anti-tumor drug research.
Plocabulin是2013年西班牙PharmaMar公司从马达加斯加海域采集到的海绵Lithoplocamia lithistoides提取得到的一种新型聚酮类化合物，对多种肿瘤细胞增殖抑制活性均为亚纳摩尔级，而且能够克服对P-gp介导的肿瘤耐药，其抗肿瘤作用机制是通过与微管蛋白结合而发挥微管去稳定作用。目前正在进行多种肿瘤治疗的临床研究。(MaríaJ.M et al.，J.Am.Chem.Soc.2013，135，10164-10171，P.Aviles et al.，Eur.J.Cancer，2013，49，A888，M.Hidalgo et al.，Eur.J.Cancer，2013，49，A889)Plocabulin分子具有4个手性中心及多烯等复杂骨架结构，依据现有公开专利，Plocabulin化学全合成需要线性18步(总步骤30步)，这极大提高了应用开发成本(WO2007144423A1，WO2014191578A1)。Plocabulin is a new type of polyketide compound extracted from the sponge Lithoplocamia lithistoides collected from the waters of Madagascar by the Spanish company PharmaMar in 2013. It has sub-nanomolar inhibitory activity against the proliferation of various tumor cells and can overcome P-gp-mediated tumor resistance. Its anti-tumor mechanism is to destabilize microtubules by binding to tubulin. Currently, clinical studies on the treatment of various tumors are underway. (María J.M et al., J.Am.Chem.Soc.2013, 135, 10164-10171, P.Aviles et al., Eur.J.Cancer, 2013, 49, A888, M.Hidalgo et al., Eur.J.Cancer, 2013, 49, A889) The Plocabulin molecule has four chiral centers and a complex skeleton structure such as polyene. According to existing published patents, the total chemical synthesis of Plocabulin requires 18 linear steps (30 steps in total), which greatly increases the cost of application development (WO2007144423A1, WO2014191578A1).
基于现有技术的基础与现状，本申请拟提供新型二氢吡喃酮类化合物，其制备方法和应用。本发明对Plocabulin进行构效关系研究和化学结构改造，获得具有较强抗肿瘤活性的化合物。Based on the foundation and status of the prior art, the present application intends to provide novel dihydropyrone compounds, preparation methods and applications thereof. The present invention studies the structure-activity relationship and chemical structure modification of Plocabulin to obtain a compound with strong anti-tumor activity.
发明内容Summary of the invention
本发明的目的之一在于，提供具有通式I结构的二氢吡喃酮类化合物，具体涉及一类含苯环或吡啶的二氢吡喃酮类化合物，该类化合物对多种肿瘤细胞具有亚纳摩尔抑制增殖活性，从而发挥防治癌症的作用。One of the purposes of the present invention is to provide a dihydropyrone compound having a general structure of formula I, specifically a class of dihydropyrone compounds containing a benzene ring or pyridine, which has subnanomolar inhibitory activity on proliferation of various tumor cells, thereby playing a role in preventing and treating cancer.
其中，当X为NH时，R1为叔丁基，R2为H，R3为H，R4为H、CONH2、邻苯二甲酰基、三苯甲基或者苄氧羰基等常见保护基；Wherein, when X is NH, R1 is tert-butyl, R2 is H, R3 is H, and R4 is a common protecting group such as H, CONH2 , phthaloyl, trityl or benzyloxycarbonyl;
当X为O时，R1为叔丁基，R2为CH3，R3为H，R4为H、CONH2、叔丁基二甲基硅基或者叔丁基二苯硅基等常见保护基；When X is O, R1 is tert-butyl, R2 is CH3 , R3 is H, and R4 is a common protecting group such as H, CONH2 , tert-butyldimethylsilyl or tert-butyldiphenylsilyl;
当X为O时，R1为含杂原子取代基的苄基，R2为H或者为CH3，R3为H或者为CH2-CH＝CH-CH3，R4为H、CONH2、叔丁基二甲基硅基或者叔丁基二苯硅基等常见保护基。When X is O, R1 is a benzyl group containing a heteroatom substituent, R2 is H or CH3 , R3 is H or CH2 -CH=CH-CH3 , and R4 is a common protecting group such as H, CONH2 , tert-butyldimethylsilyl or tert-butyldiphenylsilyl.
具体的，本发明提供具有通式I结构的二氢吡喃酮类化合物。Specifically, the present invention provides dihydropyrone compounds having a structure of general formula I.
其中，当X为NH时，R1为叔丁基，R2为H，R3为H，R4为H、CONH2、邻苯二甲酰基、三苯甲基或者苄氧羰基等常见保护基；Wherein, when X is NH, R1 is tert-butyl, R2 is H, R3 is H, and R4 is a common protecting group such as H, CONH2 , phthaloyl, trityl or benzyloxycarbonyl;
当X为O时，R1为叔丁基，R2为CH3，R3为H，R4为H、CONH2、叔丁基二甲基硅基或者叔丁基二苯硅基等常见保护基；When X is O, R1 is tert-butyl, R2 is CH3 , R3 is H, and R4 is a common protecting group such as H, CONH2 , tert-butyldimethylsilyl or tert-butyldiphenylsilyl;
当X为O时，R1为含杂原子取代基的苄基，R2为H或者为CH3，R3为H或者为CH2-CH＝CH-CH3，R4为H、CONH2、叔丁基二甲基硅基或者叔丁基二苯硅基等常见保护基；When X is O, R1 is a benzyl group containing a heteroatom substituent, R2 is H or CH3 , R3 is H or CH2 -CH＝CH-CH3 , and R4 is a common protecting group such as H, CONH2 , tert-butyldimethylsilyl or tert-butyldiphenylsilyl;
更具体的，本发明提供的化合物，其中优选的化合物为：More specifically, the compounds provided by the present invention are preferably:
第二方面，本发明提供了一种制备所述的化合物的方法，所述方法包括如下步骤：In a second aspect, the present invention provides a method for preparing the compound, the method comprising the steps of:
(1)中间体B在合适的反应溶剂和温度条件下经脱Boc保护基制备中间体C，所述反应溶剂为乙二醇或者乙二醇二甲醚等，所述反应温度为0～250℃；(1) intermediate B is subjected to deprotection of the Boc protecting group to prepare intermediate C under suitable reaction solvent and temperature conditions, wherein the reaction solvent is ethylene glycol or ethylene glycol dimethyl ether, and the reaction temperature is 0 to 250° C.;
(2)中间体C在合适的反应溶剂、温度和缩合剂条件下经缩合反应制得片段D，所述反应溶剂为二氯甲烷、二氯乙烷、四氢呋喃或N，N-二甲基甲酰胺等常用溶剂，所述缩合剂为2-(7-氮杂苯并三氮唑)-N，N，N′，N′-四甲基脲六氟磷酸酯、二环己基碳二亚胺、二异丙基碳二亚胺或1-乙基-3(3-二甲基丙胺)碳二亚胺)等常用试剂，所述反应温度为0～50℃；(2) Intermediate C is subjected to a condensation reaction under appropriate reaction solvent, temperature and condensation agent conditions to obtain fragment D, wherein the reaction solvent is a common solvent such as dichloromethane, dichloroethane, tetrahydrofuran or N,N-dimethylformamide, and the condensation agent is a common reagent such as 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, dicyclohexylcarbodiimide, diisopropylcarbodiimide or 1-ethyl-3(3-dimethylpropylamine)carbodiimide), and the reaction temperature is 0 to 50° C.;
(3)片段D与片段A在合适的反应溶剂、温度和催化剂条件下发生偶联反应制备中间体E，所述反应溶剂为四氢呋喃、N，N-二甲基甲酰胺或N-甲基吡咯烷酮等常用溶剂，所述催化剂为双(乙腈)二氯化钯(II)或噻吩-2-甲酸亚铜等试剂，所述反应温度为0～50℃；(3) Fragment D and fragment A undergo coupling reaction under appropriate reaction solvent, temperature and catalyst conditions to prepare intermediate E, wherein the reaction solvent is a common solvent such as tetrahydrofuran, N,N-dimethylformamide or N-methylpyrrolidone, the catalyst is a reagent such as bis(acetonitrile)palladium(II) chloride or cuprous thiophene-2-carboxylate, and the reaction temperature is 0 to 50° C.;
(4)当X为NH时，将合适的中间体E在合适的反应溶剂、温度和试剂条件下进行脱保护基反应制备相应中间体F；所述反应溶剂为四氢呋喃、甲醇、乙醇或二氯甲烷等常用溶剂，所述试剂为水合肼、无水肼或甲胺等试剂，所述反应温度为-30～50℃；(4) When X is NH, a suitable intermediate E is subjected to a deprotection reaction under suitable reaction solvent, temperature and reagent conditions to prepare a corresponding intermediate F; the reaction solvent is a common solvent such as tetrahydrofuran, methanol, ethanol or dichloromethane, the reagent is a reagent such as hydrazine hydrate, anhydrous hydrazine or methylamine, and the reaction temperature is -30 to 50°C;
或者当X为O时，将合适的中间体E在合适的反应溶剂、温度和试剂条件下进行脱保护基反应制备相应中间体F；所述反应溶剂为四氢呋喃、甲醇、乙醇、二氯甲烷或者水等常用溶剂，所述试剂为四丁基氟化铵、氟化氢、吡啶氟化氢或者三乙胺氟化氢等试剂，所述反应温度为-30～100℃；Alternatively, when X is O, a suitable intermediate E is subjected to a deprotection reaction under suitable reaction solvent, temperature and reagent conditions to prepare a corresponding intermediate F; the reaction solvent is a common solvent such as tetrahydrofuran, methanol, ethanol, dichloromethane or water, the reagent is a reagent such as tetrabutylammonium fluoride, hydrogen fluoride, pyridine hydrogen fluoride or triethylamine hydrogen fluoride, and the reaction temperature is -30 to 100°C;
(5)当X为NH时，将合适的中间体F在合适的反应溶剂、温度和试剂条件下进行官能团转换制备通式II所示化合物，所述反应溶剂为甲醇、乙醇或水等常用溶剂，所述试剂氰酸钾或者氰酸钠等试剂，所述反应温度为0～100℃；(5) When X is NH, a suitable intermediate F is subjected to functional group conversion under suitable reaction solvent, temperature and reagent conditions to prepare a compound represented by formula II, wherein the reaction solvent is a common solvent such as methanol, ethanol or water, the reagent is a reagent such as potassium cyanate or sodium cyanate, and the reaction temperature is 0 to 100° C.;
当X为O时，将合适的中间体F在合适的反应溶剂、温度条件下、经合适的试剂处理官能团转换制备通式II所示化合物，所述反应溶剂为二氯甲烷、二氯乙烷或四氢呋喃等常用溶剂，所述试剂三氯乙酰基异氰酸酯或者三氧化二铝等试剂，所述反应温度为-30～50℃；When X is O, a suitable intermediate F is treated with a suitable reagent under a suitable reaction solvent and temperature conditions to convert the functional group to prepare a compound represented by the general formula II, wherein the reaction solvent is a common solvent such as dichloromethane, dichloroethane or tetrahydrofuran, the reagent is trichloroacetyl isocyanate or aluminum oxide, and the reaction temperature is -30 to 50°C;
第三方面，本发明提供了所述的化合物在制备防治肿瘤药物中的应用。In a third aspect, the present invention provides the use of the compound in the preparation of drugs for preventing and treating tumors.
本发明以化合物1-1为例、但不限于化合物1-1，其分子结构如下：The present invention takes compound 1-1 as an example, but is not limited to compound 1-1, and its molecular structure is as follows:
其制备过程如下：The preparation process is as follows:
中间体B-1后脱去Boc保护基得到中间体C-1；再与羧酸进行缩合得到中间体D-1。将中间体A与D-1其进行Stille偶联反应得到中间体E-1；再脱去中间体E-1上的邻苯二甲酰保护基得到中间体F-1。最后将F-1在酸性条件下与氰酸钾反应制备得到终产物1-1。The intermediate B-1 is then deprotected from the Boc protecting group to obtain the intermediate C-1; it is then condensed with carboxylic acid to obtain the intermediate D-1. The intermediate A and D-1 are subjected to Stille coupling reaction to obtain the intermediate E-1; the phthaloyl protecting group on the intermediate E-1 is then removed to obtain the intermediate F-1. Finally, F-1 is reacted with potassium cyanate under acidic conditions to obtain the final product 1-1.
测试了本发明的化合物对人肺癌细胞A549，人结肠癌细胞HT-29，人肝癌细胞HepG2和人乳腺癌细胞MDA-MB-231的体外增殖抑制作用，所采用的药效学试验方法，是本领域技术人员所熟知的方法。结果显示，本发明的化合物显示出较好的癌细胞增殖抑制作用，可以进一步研制开发新型的微管蛋白抑制剂和抗肿瘤药物。The compounds of the present invention were tested for their in vitro proliferation inhibition effects on human lung cancer cells A549, human colon cancer cells HT-29, human liver cancer cells HepG2 and human breast cancer cells MDA-MB-231. The pharmacodynamic test methods used are well known to those skilled in the art. The results show that the compounds of the present invention exhibit good cancer cell proliferation inhibition effects and can be further developed into novel microtubule inhibitors and anti-tumor drugs.
本发明中，所述药物的组合物中含有治疗有效量的权利要求1-3中任一项所述的化合物。In the present invention, the pharmaceutical composition contains a therapeutically effective amount of the compound according to any one of claims 1 to 3.
本发明中，所述化合物占所述药物组合物总重量的20％～99％。In the present invention, the compound accounts for 20% to 99% of the total weight of the pharmaceutical composition.
由于微管蛋白在细胞分裂中有极其重要的作用，而抑制微管蛋白可以抑制多种恶性肿瘤增殖，因此，本发明所述的新型二氢吡喃酮类化合物可用于制备微管蛋白抑制剂或抗肿瘤药物，可用于恶性肿瘤包括肺癌、胃癌、食管癌、乳腺癌、卵巢癌、头颈部肿瘤等多种肿瘤的治疗。Since tubulin plays an extremely important role in cell division, and inhibiting tubulin can inhibit the proliferation of various malignant tumors, the novel dihydropyrone compounds described in the present invention can be used to prepare tubulin inhibitors or anti-tumor drugs, and can be used to treat malignant tumors including lung cancer, gastric cancer, esophageal cancer, breast cancer, ovarian cancer, head and neck tumors, and other tumors.
具体实施方式DETAILED DESCRIPTION
实施例1 制备二氢吡喃酮类化合物Example 1 Preparation of dihydropyrone compounds
(S，2Z，4Z，6E)-N-((S)-3，3-二甲基-1-氧代-1-((Z)-4-脲基-1-基)氨基)丁烷-2-基)-8-((S)-5-甲氧基-6-氧代-3，6-二氢-2H-吡喃-2-基)-6-甲基壬-2，4，6-三酰胺(1-1)(S,2Z,4Z,6E)-N-((S)-3,3-dimethyl-1-oxo-1-((Z)-4-ureido-1-yl)amino)butan-2-yl)-8-((S)-5-methoxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)-6-methylnonane-2,4,6-triamide (1-1)
(S，Z)-2-氨基-N-(4-(1，3-二氧异吲哚-2-基)丁基-1-烯-1-基)-3，3-二甲基丁酰胺(C-1)(S,Z)-2-amino-N-(4-(1,3-dioxoisoindol-2-yl)butyl-1-en-1-yl)-3,3-dimethylbutyramide (C-1)
中间体B-1(600mg，1.40mmol)悬浮于于乙二醇(30mL)中，油浴设置200℃，待达到目标温度后，将反应液放入油浴，保温搅拌15分钟。到达时间后立即将反应体系移出油浴，用冷水降至室温。加入乙酸乙酯(300mL)，有机相用水(3 x 150mL)洗涤。浓缩有机相，柱层析得380mg中间体C-1呈黄色油状液体，收率83％。1H NMR(600MHz，CDCl3)δ：8.79(d，1H，J＝11.4Hz)，7.84(dd，2H，J＝5.4，3.0Hz)，7.72(dd，2H，J＝5.4，3.0Hz)，6.77(m，1H)，4.77(dt，1H，J＝8.4，7.8Hz)，3.75(t，1H，J＝7.8Hz)，3.18(s，1H)，2.49-2.41(m，2H)，0.99(s，9H).13CNMR(150MHz，CDCl3)δ：171.27，168.42，134.07，131.95，123.30，106.15，64.18，37.11，34.35，26.65，24.97.HRMS(m/z)：330.1821[M+1]+(Calcd.for C18H23N3O3 330.1812).Intermediate B-1 (600 mg, 1.40 mmol) was suspended in ethylene glycol (30 mL), and the oil bath was set at 200 ° C. After reaching the target temperature, the reaction solution was placed in the oil bath and stirred for 15 minutes. After reaching the time, the reaction system was immediately removed from the oil bath and cooled to room temperature with cold water. Ethyl acetate (300 mL) was added, and the organic phase was washed with water (3 x 150 mL). The organic phase was concentrated and column chromatography was performed to obtain 380 mg of intermediate C-1 as a yellow oily liquid with a yield of 83%.1 H NMR (600MHz, CDCl3 ) δ: 8.79 (d, 1H, J=11.4Hz), 7.84 (dd, 2H, J=5.4, 3.0Hz), 7.72 (dd, 2H, J=5.4, 3.0Hz), 6.77 (m, 1H), 4.77 (dt, 1H, J=8.4, 7.8Hz), 3.75 (t , 1H, J=7.8Hz), 3.18 (s, 1H), 2.49-2.41 (m, 2H), 0.99 (s, 9H).13 CNMR (150MHz, CDCl3 )δ: 171.27, 168.42, 134.07, 131.95, 123.30, 106.15, 64.18, 37.11, 34.35, 26.65, 24.97.HRMS (m/z): 330.1821[M+1]+ (Calcd.for C18 H23 N3 O3 330.1 812).
(S)-N-((Z)-4-(1，3-二氧异吲哚-2-基)丁基-1-烯-1-基)-3，3-二甲基-2-((Z)-3-(三丁基锡基)丙烯酰胺基)丁胺(D-1)(S)-N-((Z)-4-(1,3-dioxoisoindol-2-yl)butyl-1-en-1-yl)-3,3-dimethyl-2-((Z)-3-(tributyltinyl)acrylamide)butylamine (D-1)
中间体C-1(380mg，1.15mmol，1.0eq.)溶于DCM(32mL)和DMF(8mL)中，将反应体系置于冰水浴中，控制反应温度0-10℃依次加入(Z)-3-(三丁基)丙烯酸(500mg，1.39mmol，1.2eq.)、DIPEA(302μL，1.73mmol，1.5eq.)、HOAT(173mg，1.27mmol，1.1eq.)和HATU(570mg，1.50mmol，1.3eq.)，加完保温搅拌30分钟，点板显示反应完全。控制反应温度0-10℃加入半饱和氯化铵水溶液(50mL)淬灭反应，搅拌5分钟后加入MTBE(100mL)。搅拌后分层，有机相用水(50mL)洗涤，浓缩有机相，柱层析得497mg中间体D-1呈无色油状液体，收率64％。1H NMR(600MHz，CDCl3)δ：7.86(dd，2H，J＝5.4，3.0Hz)，7.81(d，1H，J＝10.8Hz)，7.73(dd，2H，J＝5.4，3.0Hz)，7.01(d，1H，J＝12.0Hz)，6.81-6.74(m，2H)，6.34(d，1H，J＝9.6Hz)，4.83(dt，1H，J＝8.4，7.8Hz)，4.49(d，1H，J＝9.6Hz)，3.74(t，1H，J＝7.2Hz)，2.55-2.36(m，2H)，1.53-1.39(m，6H)，1.31-1.21(m，6H)，1.01(s，9H)，0.95-0.82(m，15H).13C NMR(150MHz，CDCl3)δ：168.50，168.22，166.18，153.53，136.19，134.15，131.91，123.43，123.28，107.06，60.71，37.07，35.26，29.24，27.41，26.56，24.88，13.77，11.52.HRMS(m/z)：696.2788[M+Na]+(Calcd.for C33H51N3O4Sn 696.2794).Intermediate C-1 (380 mg, 1.15 mmol, 1.0 eq.) was dissolved in DCM (32 mL) and DMF (8 mL), and the reaction system was placed in an ice-water bath, and the reaction temperature was controlled at 0-10°C. (Z)-3-(tributyl)acrylic acid (500 mg, 1.39 mmol, 1.2 eq.), DIPEA (302 μL, 1.73 mmol, 1.5 eq.), HOAT (173 mg, 1.27 mmol, 1.1 eq.) and HATU (570 mg, 1.50 mmol, 1.3 eq.) were added in sequence, and the mixture was stirred for 30 minutes after addition. The reaction was complete when the plate was dotted. The reaction temperature was controlled at 0-10°C, and a half-saturated aqueous ammonium chloride solution (50 mL) was added to quench the reaction. After stirring for 5 minutes, MTBE (100 mL) was added. After stirring, the mixture was separated into layers, and the organic phase was washed with water (50 mL). The organic phase was concentrated and subjected to column chromatography to obtain 497 mg of intermediate D-1 as a colorless oily liquid with a yield of 64%.1 H NMR (600 MHz, CDCl3 ) δ: 7.86 (dd, 2H, J=5.4, 3.0 Hz), 7.81 (d, 1H, J=10.8 Hz), 7.73 (dd, 2H, J=5.4, 3.0 Hz), 7.01 (d, 1H, J=12.0 Hz), 6.81-6.74 (m, 2H), 6.34 (d, 1H, J=9.6 Hz), 4.83 (d t, 1H, J=8.4, 7.8Hz), 4.49 (d, 1H, J=9.6Hz), 3.74 (t, 1H, J=7.2Hz), 2.55-2.36 (m, 2H), 1.53-1.39 (m, 6H), 1.31-1.21 (m, 6H), 1.01 (s, 9H), 0.95-0.82 ( m, 15H).13 C NMR (150MHz, CDCl3 ) δ: 168.50, 168.22, 166.18, 153.53, 136.19, 134.15, 131.91, 123.43, 123.28, 107.06, 60.71, 37.07, 35.26, 29.24, 27.41, 26 .56, 24.88, 13.77, 11.52. HRMS (m/z): 696.2788[M+Na]+ (Calcd. for C33 H51 N3 O4 Sn 696.2794).
(S，2Z，4Z，6E)-N-((S)-1-((Z)-4-(1，3-二氧异吲哚-2-基)丁基-1-烯-1-基)胺-O)-3，3-二甲基-1-氧代丁烷-2-基)-8-((S)-5-甲氧基-6-氧代-3，6-二氢-2H-吡喃-2-基)-6-甲基壬-2，4，6-三酰胺(E-1)(S,2Z,4Z,6E)-N-((S)-1-((Z)-4-(1,3-dioxoisoindol-2-yl)butyl-1-en-1-yl)amine-O)-3,3-dimethyl-1-oxobutan-2-yl)-8-((S)-5-methoxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)-6-methylnonane-2,4,6-triamide (E-1)
中间体D-1(274mg，0.407mmol，1.0eq.)和A(170mg，0.488mmol，1.2eq.)溶于NMP(20mL)中，将反应体系置于冰水浴中，控制反应液温度0-10℃加入CuTC(116mg，0.611mmol，1.5eq.)，加完保温搅拌45分钟。将反应体系移出冰水浴，自然升至室温，并在室温搅拌过夜，点板显示反应完全。在漏斗内铺中性氧化铝过滤，滤渣用MTBE∶EA＝1∶1混合溶剂(75mL)洗涤。合并有机相，用0.5M稀盐酸(75，2 x 45mL)洗涤三次，再用水(2 x 45mL)洗涤两次。浓缩有机相，先用制备板纯化，再用半制备色谱纯化得140mg中间体E-1呈白色固体，收率57％。[α]20D+33.0(c＝0.1，CHCl3).1H NMR(600MHz，CDCl3)δ：8.17(d，1H，J＝10.8Hz)，7.87(dd，2H，J＝5.4，3.0Hz)，7.73(dd，2H，J＝5.4，3.0Hz)，7.30(dd，1H，J＝12.0，11.4Hz)，6.88(dd，1H，J＝11.4，11.4Hz)，6.79(dd，1H，J＝10.2，9.6Hz)，6.38(d，1H，J＝9.6Hz)，6.16(d，1H，J＝11.4Hz)，5.69(d，1H，J＝11.4Hz)，5.62(dd，1H，J＝6.6，3.6Hz)，5.28(d，1H，J＝10.2Hz)，4.84(dt，1H，J＝8.4，7.8Hz)，4.53(d，1H，J＝9.6Hz)，4.21(ddd，1H，J＝12.0，7.8，4.8Hz)，3.72(t，2H，J＝7.8Hz)，3.66(s，3H)，2.85(m，1H)，2.50-2.33(m，4H)，1.84(s，3H)，1.15(d，3H，J＝6.6Hz)，1.04(s，9H).13C NMR(150MHz，CDCl3)δ：168.58，168.47，166.11，161.57，145.26，140.07，137.33，134.19，134.12，131.92，124.25，123.50，120.84，108.15，106.95，81.83，60.51，55.45，37.34，37.13，35.03，26.67，26.31，24.88，17.19，16.74.HRMS(m/z)：626.2845[M+Na]+(Calcd.for C34H41N3O7 626.2837).Intermediate D-1 (274 mg, 0.407 mmol, 1.0 eq.) and A (170 mg, 0.488 mmol, 1.2 eq.) were dissolved in NMP (20 mL), and the reaction system was placed in an ice-water bath. The temperature of the reaction solution was controlled to 0-10°C, and CuTC (116 mg, 0.611 mmol, 1.5 eq.) was added. After addition, the mixture was stirred for 45 minutes. The reaction system was removed from the ice-water bath, naturally warmed to room temperature, and stirred at room temperature overnight. The plate showed that the reaction was complete. Neutral alumina was spread in the funnel for filtration, and the filter residue was washed with a MTBE: EA = 1: 1 mixed solvent (75 mL). The organic phases were combined and washed three times with 0.5 M dilute hydrochloric acid (75, 2 x 45 mL), and then washed twice with water (2 x 45 mL). The organic phase was concentrated, first purified by preparative plate, and then purified by semi-preparative chromatography to obtain 140 mg of intermediate E-1 as a white solid with a yield of 57%. [α]20D +33.0 (c=0.1, CHCl3 ).1 H NMR (600MHz, CDCl3 ) δ: 8.17 (d, 1H, J=10.8Hz), 7.87 (dd, 2H, J=5.4, 3.0Hz), 7.73 (dd, 2H, J=5.4, 3.0Hz), 7.30 (dd, 1H, J=12.0, 11.4Hz), 6.88 (dd, 1H, J=11.4, 11.4Hz), 6.79 (dd, 1H, J=10.2, 9.6Hz), 6.38 (d, 1H, J=9.6Hz), 6.16 (d, 1H, J=11.4Hz), 5.69 (d, 1H, J=11.4Hz), 5.62 (dd, 1H, J=6.6, 3.6Hz), 5.28 (d, 1H, J=10.2Hz), 4.84 (dt, 1H, J=8.4, 7.8Hz), 4.53 (d, 1H, J=9.6Hz), 4.21 (ddd, 1H, J=12.0, 7.8, 4.8Hz), 3.72 (t, 2H, J=7.8Hz ), 3.66 (s, 3H), 2.85 (m, 1H), 2.50-2.33 (m, 4H), 1.84 (s, 3H), 1.15 (d, 3H, J=6.6Hz), 1.04 (s, 9H).13 C NMR (150MHz, CDCl3 ) δ: 168.58, 168.47, 166.11, 161.57, 145.26, 140.07, 137.33, 134.19, 134.12, 131.92, 124.25, 123.50, 120.84, 108.15, 106. 95, 81.83, 60.51, 55.45, 37.34, 37.13, 35.03, 26.67, 26.31, 24.88, 17.19, 16.74. HRMS (m/z): 626.2845[M+Na]+ (Calcd. for C34 H41 N3 O7 626.2837).
(S，2Z，4Z，6E)-N-((S)-1-(((Z)-4-氨基-1-烯-1-基)氨基)-3，3-二甲基-1-氧代丁烷-2-基)-8-((S)-5-甲氧基-6-氧代-3，6-二氢-2H-吡喃-2-基)-6-甲基-2，4，6-三烯酰胺(F-1)(S,2Z,4Z,6E)-N-((S)-1-(((Z)-4-amino-1-en-1-yl)amino)-3,3-dimethyl-1-oxobutan-2-yl)-8-((S)-5-methoxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)-6-methyl-2,4,6-trienamide (F-1)
中间体E-1(106mg，0.176mmol，1.0eq.)溶于THF(8mL)中，将反应体系置于冰水浴中，控制反应温度0-5℃加入80％水合肼的水溶液(132mg，2.11mmol，12.0eq.)，加完保温搅拌12小时。用制备板纯化，得到20mg粗品F-1呈淡黄色固体，收率24％。HRMS(m/z)：496.2783[M+Na]+(Calcd.for C26H39N3O5 496.2782).Intermediate E-1 (106 mg, 0.176 mmol, 1.0 eq.) was dissolved in THF (8 mL), and the reaction system was placed in an ice-water bath. The reaction temperature was controlled at 0-5 °C, and an aqueous solution of 80% hydrazine hydrate (132 mg, 2.11 mmol, 12.0 eq.) was added. After addition, the mixture was stirred for 12 hours. Purification was performed using a preparative plate to obtain 20 mg of crude F-1 as a light yellow solid, with a yield of 24%. HRMS (m/z): 496.2783 [M+Na]+ (Calcd. for C26 H39 N3 O5 496.2782).
(S，2Z，4Z，6E)-N-((S)-3，3-二甲基-1-氧代-1-((Z)-4-脲基-1-基)胺-O)丁烷-2-基)-8-((S)-5-甲氧基-6-氧代-3，6-二氢-2H-吡喃-2-基)-6-甲基-壬-2，4，6-三烯酰胺(1-1)(S,2Z,4Z,6E)-N-((S)-3,3-dimethyl-1-oxo-1-((Z)-4-ureido-1-yl)amine-O)butan-2-yl)-8-((S)-5-methoxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)-6-methyl-nona-2,4,6-trienamide (1-1)
中间体F-1(20mg，0.042mmol，1.0eq.)和氰酸钾(5mg，0.063mmol，1.5eq.)溶于甲醇(5mL)、水(5mL)以及盐酸(1M，5mL)的混合溶液中，将反应体系置于油浴中，设定油浴温度45℃，保温搅拌过夜，点板显示反应完全。直接浓缩，残留物快速过柱后用半制备液相进行纯化得到3mg化合物1-1呈白色固体，收率14％。[α]20D-9.0(c＝0.1，CHCl3).1H NMR(600MHz，CDCl3)δ：9.25(d，1H，J＝10.8Hz)，7.24(dd，1H，J＝12.0，11.4Hz)，6.97(d，1H，J＝6.0Hz)，6.88(dd，1H，J＝12.0，11.4Hz)，6.75(dd，1H，J＝10.2，9.6Hz)，6.14(d，1H，J＝12.0Hz)，5.73(d，1H，J＝11.4Hz)，5.65(dd，1H，J＝6.6，3.0Hz)，5.46(t，1H，J＝5.4Hz)，5.33(d，1H，J＝9.6Hz)，4.78(dt，1H，J＝9.0，7.2Hz)，4.52(d，1H，J＝9.6Hz)，4.27(ddd，1H，J＝11.4，6.6，3.6Hz)，3.66(s，3H)，3.21(m，1H)，3.02(m，1H)，2.83(m，1H)，2.47(m，1H)，2.40(m，1H)，2.31(m，1H)，2.24(m，1H)，1.83(s，3H)，1.16(d，3H，J＝6.6Hz)，1.06(s，9H).13C NMR(150MHz，CDCl3)δ：168.94，166.84，161.81，160.32，145.16，140.05，137.13，134.24，133.79，124.19，123.55，120.97，108.87，108.38，81.77，60.99，55.48，40.34，37.12，34.83，26.77，26.58，26.19，17.15，16.34.HRMS(m/z)：539.2844[M+Na]+(Calcd.forC27H40N4O6 539.2840).Intermediate F-1 (20 mg, 0.042 mmol, 1.0 eq.) and potassium cyanate (5 mg, 0.063 mmol, 1.5 eq.) were dissolved in a mixed solution of methanol (5 mL), water (5 mL) and hydrochloric acid (1 M, 5 mL). The reaction system was placed in an oil bath, the oil bath temperature was set to 45°C, and the mixture was stirred overnight. The plate showed that the reaction was complete. The mixture was directly concentrated, and the residue was quickly passed through a column and purified by semi-preparative liquid phase to obtain 3 mg of compound 1-1 as a white solid, with a yield of 14%. [α]20D -9.0 (c = 0.1, CHCl3 ).1 H NMR (600 MHz, CDCl3 )δ: 9.25 (d, 1H, J = 10.8Hz), 7.24 (dd, 1H, J = 12.0, 11.4Hz), 6.97 (d, 1H, J = 6.0Hz), 6.88 (dd, 1H, J = 12.0, 11.4Hz), 6.75 (dd, 1H, J = 10.2, 9.6Hz), 6.14 (d, 1H, J = 12.0Hz), 5.73 (d, 1H, J=11.4Hz), 5.65 (dd, 1H, J=6.6, 3.0Hz), 5.46 (t, 1H, J=5.4Hz), 5.33 (d, 1H , J=9.6Hz), 4.78 (dt, 1H, J=9.0, 7.2Hz), 4.52 (d, 1H, J=9.6Hz), 4.27 (ddd, 1H, J=11.4, 6.6, 3.6Hz), 3.66 (s, 3H), 3.21 (m, 1H), 3.02 (m, 1H), 2.83 (m, 1H) , 2.47 (m, 1H), 2.40 (m, 1H), 2.31 (m, 1H), 2.24 (m, 1H), 1.83 (s, 3H), 1.16 (d, 3H, J=6.6Hz), 1.06 (s, 9H).13 C NMR (150MHz, CDCl3 ) δ: 168.94, 166.84, 161.81, 160.32, 145.16, 140.05, 137.13, 134.24, 133.79, 124.19, 123.55, 120.97, 108.87, 108.38, 81.7 7, 60.99, 55.48, 40.34, 37.12, 34.83, 26.77, 26.58, 26.19, 17.15, 16.34. HRMS (m/z): 539.2844[M+Na]+ (Calcd.forC27 H40 N4 O6 539.2840).
实施例2 制备二氢吡喃酮类化合物Example 2 Preparation of dihydropyrone compounds
(Z)-4-((S)-2-((S，2Z，4Z，6E)-8-((S)-5-甲氧基-6-氧代-3，6-二氢-2H-吡喃-2-基)-6-甲基壬-2，4，6-三胺基)-3，3-二甲基丁胺基)-3-甲基-3-烯-1-基氨基甲酸酯(2-1)(Z)-4-((S)-2-((S,2Z,4Z,6E)-8-((S)-5-methoxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)-6-methylnon-2,4,6-triamino)-3,3-dimethylbutylamino)-3-methyl-3-en-1-ylcarbamate (2-1)
(S，Z)-2-氨基-N-(4-((叔丁基二甲基硅基)氧基)-2-甲基-1-烯-1-基)-3，3-二甲基丁酰胺(C-2)(S,Z)-2-amino-N-(4-((tert-butyldimethylsilyl)oxy)-2-methyl-1-en-1-yl)-3,3-dimethylbutanamide (C-2)
中间体B-2(622mg，1.45mmol)悬浮于乙二醇(35mL)中，油浴设置200℃，待达到目标温度后，将反应液放入油浴，保温搅拌15分钟。到达时间后立即将反应体系移出油浴，用冷水降至室温。加入乙酸乙酯(350mL)，有机相用水(3 x 175mL)洗涤后用无水硫酸钠干燥。浓缩有机相得363mg中间体C-2呈黄色油状液体，不经纯化直接用于下一步，收率76％。Intermediate B-2 (622 mg, 1.45 mmol) was suspended in ethylene glycol (35 mL), and the oil bath was set at 200 ° C. After reaching the target temperature, the reaction solution was placed in the oil bath and stirred for 15 minutes. Immediately after the time was reached, the reaction system was removed from the oil bath and cooled to room temperature with cold water. Ethyl acetate (350 mL) was added, and the organic phase was washed with water (3 x 175 mL) and dried over anhydrous sodium sulfate. The organic phase was concentrated to obtain 363 mg of intermediate C-2 as a yellow oily liquid, which was used directly in the next step without purification, with a yield of 76%.
(S)-N-((Z)-4-((叔丁基二甲基硅基)氧基)-2-甲基-1-烯-1-基)-3，3-二甲基-2-((Z)-3-(三丁基三烷基)丙烯酰胺基)丁胺(D-2)(S)-N-((Z)-4-((tert-butyldimethylsilyl)oxy)-2-methyl-1-en-1-yl)-3,3-dimethyl-2-((Z)-3-(tributyltrialkyl)acrylamide)butylamine (D-2)
中间体C-2(362mg，1.10mmol，1.0eq.)溶于DCM(12mL)和DMF(3mL)中，将反应体系置于冰水浴中，控制反应温度0-10℃依次加入(Z)-3-(三丁基)丙烯酸(477mg，1.32mmol，1.2eq.)、DIPEA(288μL，1.65mmol，1.5eq.)、HOAT(165mg，1.21mmol，1.1eq.)和HATU(545mg，1.43mmol，1.3eq.)，加完保温搅拌30分钟，点板显示反应完全。控制反应温度0-10℃加入半饱和氯化铵水溶液(50mL)淬灭反应，搅拌5分钟后向反应液内加入MTBE(100mL)，搅拌后分层，有机相用水(50mL)洗涤。浓缩有机相，柱层析得549mg中间体D-2呈无色油状液体，收率74％。1H NMR(600MHz，CDCl3)δ：8.03(d，1H，J＝9.6Hz)，6.98(d，1H，J＝12.0Hz)，6.75(d，1H，J＝12.0Hz)，6.54(d，1H，J＝9.0Hz)，6.24(d，1H，J＝9.0Hz)，4.35(d，1H，J＝9.6Hz)，3.79(m，1H)，3.71(m，1H)，2.33(ddd，1H，J＝13.8，8.4，4.2Hz)，2.13(ddd，1H，J＝14.4，5.4，3.6Hz)，1.72(d，3H，J＝1.2Hz)，1.52-1.41(m，6H)，1.32-1.21(m，6H)，1.00(s，9H)，0.91(s，9H)，0.91-0.83(m，15H)，0.11(s，3H)，0.09(s，3H).13C NMR(150MHz，CDCl3)δ：167.88，165.78，153.17，136.17，119.76，118.66，62.13，60.54，35.34，35.03，29.29，27.43，26.64，26.06，21.03，18.64，13.77，11.68，-5.20，-5.27.HRMS(m/z)：695.3603[M+Na]+(Calcd.forC32H64N2O3SiSn 695.3600).Intermediate C-2 (362 mg, 1.10 mmol, 1.0 eq.) was dissolved in DCM (12 mL) and DMF (3 mL), and the reaction system was placed in an ice-water bath, and the reaction temperature was controlled at 0-10 ° C. (Z)-3-(tributyl)acrylic acid (477 mg, 1.32 mmol, 1.2 eq.), DIPEA (288 μL, 1.65 mmol, 1.5 eq.), HOAT (165 mg, 1.21 mmol, 1.1 eq.) and HATU (545 mg, 1.43 mmol, 1.3 eq.) were added in sequence, and the mixture was stirred for 30 minutes after addition. The plate showed that the reaction was complete. The reaction temperature was controlled at 0-10 ° C. and half-saturated aqueous ammonium chloride solution (50 mL) was added to quench the reaction. After stirring for 5 minutes, MTBE (100 mL) was added to the reaction solution, and the layers were separated after stirring. The organic phase was washed with water (50 mL). The organic phase was concentrated and column chromatography was performed to obtain 549 mg of intermediate D-2 as a colorless oily liquid with a yield of 74%.1 H NMR (600 MHz, CDCl3 ) δ: 8.03 (d, 1H, J=9.6 Hz), 6.98 (d, 1H, J=12.0 Hz), 6.75 (d, 1H, J=12.0 Hz), 6.54 (d, 1H, J=9.0 Hz), 6.24 (d, 1H, J=9.0 Hz), 4.35 (d, 1H, J=9.6 Hz), 3.79 (m, 1H), 3.71 (m, 1H), 2.33 (ddd, 1H, J=1 3.8, 8.4, 4.2Hz), 2.13 (ddd, 1H, J=14.4, 5.4, 3.6Hz), 1.72 (d, 3H, J=1.2Hz), 1.52-1.41 (m, 6H), 1.32-1.21 (m, 6H), 1.00 (s, 9H), 0.91 (s, 9H), 0.91-0.8 3(m, 15H), 0.11(s, 3H), 0.09(s, 3H).13 C NMR (150MHz, CDCl3 ) δ: 167.88, 165.78, 153.17, 136.17, 119.76, 118.66, 62.13, 60.54, 35.34, 35.03, 29.29, 27.43, 26.64, 26.06, 21.03, 18.64, 13.77, 11.68, -5.20, -5.27. HRMS (m/z): 695.3603[M+Na]+ (Calcd.forC32 H64 N2 O3 SiSn 695.3600).
(S，2Z，4Z，6E)-N-((S)-1-((Z)-4-((叔丁基二甲基硅氧基)-2-甲基-1-烯-1-基)氨基)-3，3-二甲基-1-氧代丁烷-2-基)-8-((S)-5-甲氧基-6-氧代-3，6-二二氢-2H-吡喃-2-基)-6-甲基壬-2，4，6-三酰胺(E-2)(S,2Z,4Z,6E)-N-((S)-1-((Z)-4-((tert-butyldimethylsilyloxy)-2-methyl-1-en-1-yl)amino)-3,3-dimethyl-1-oxobutan-2-yl)-8-((S)-5-methoxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)-6-methylnonane-2,4,6-triamide (E-2)
中间体D-2(94mg，0.140mmol，1.0eq.)和A(58mg，0.168mmol，1.2eq.)溶于NMP(10mL)中，将反应体系置于冰水浴中，控制反应温度0-10℃加入CuTC(40mg，0.210mmol，1.5eq.)，加完保温搅拌45分钟。然后将反应体系移出冰水浴，自然升至室温，并在室温搅拌过夜，点板显示反应完全。在漏斗内铺中性氧化铝过滤，滤渣用MTBE∶EA＝1∶1混合溶剂(75mL)洗涤。合并有机相，用0.5M稀盐酸(75，2 x 45mL)洗涤三次，再用水(2 x 45mL)洗涤两次。浓缩有机相，制备板纯化得63mg中间体E-2呈无色油状液体，收率75％。[α]20D-34.8(c＝0.5，CHCl3).1H NMR(600MHz，CDCl3)δ：8.02(d，1H，J＝9.0Hz)，7.24(dd，1H，J＝12.0，11.4Hz)，6.87(dd，1H，J＝11.4，11.4Hz)，6.53(d，1H，J＝9.0Hz)，6.33(d，1H，J＝9.0Hz)，6.15(d，1H，J＝11.4Hz)，5.68(d，1H，J＝11.4Hz)，5.63(dd，1H，J＝6.0，3.6Hz)，5.28(d，1H，J＝10.2Hz)，4.31(d，1H，J＝9.6Hz)，4.21(ddd，1H，J＝12.6，7.8，4.8Hz)，3.79(m，1H)，3.71(m，1H)，3.66(s，3H)，2.86(m，1H)，2.46-2.36(m，2H)，2.32(ddd，1H，J＝13.8，7.8，4.2Hz)，2.16(ddd，1H，J＝14.4，6.0，4.2Hz)，1.85(d，3H，J＝1.2Hz)，1.73(d，3H，J＝1.2Hz)，1.16(d，3H，J＝6.6Hz)，1.03(s，9H)，0.91(s，9H)，0.12(s，3H)，0.10(s，3H).13C NMR(150MHz，CDCl3)δ：168.10，165.89，161.61，145.42，139.85，136.82，134.28，134.04，124.39，121.49，120.00，118.74，108.27，81.96，62.15，60.54，55.55，37.49，35.26，35.07，26.86，26.48，26.18，21.11，18.74，17.30，16.90，-5.06，-5.12.HRMS(m/z)：625.3643[M+Na]+(Calcd.for C33H54N2O6Si 625.3643).Intermediate D-2 (94 mg, 0.140 mmol, 1.0 eq.) and A (58 mg, 0.168 mmol, 1.2 eq.) were dissolved in NMP (10 mL), and the reaction system was placed in an ice-water bath. The reaction temperature was controlled to 0-10°C, and CuTC (40 mg, 0.210 mmol, 1.5 eq.) was added. After addition, the mixture was stirred for 45 minutes. Then the reaction system was removed from the ice-water bath, naturally warmed to room temperature, and stirred at room temperature overnight. The plate showed that the reaction was complete. Neutral alumina was spread in the funnel for filtration, and the filter residue was washed with a MTBE: EA = 1: 1 mixed solvent (75 mL). The organic phases were combined and washed three times with 0.5 M dilute hydrochloric acid (75, 2 x 45 mL), and then washed twice with water (2 x 45 mL). The organic phase was concentrated and purified by preparative plate to obtain 63 mg of intermediate E-2 as a colorless oily liquid with a yield of 75%. [α]20D -34.8 (c=0.5, CHCl3 ).1 H NMR (600MHz, CDCl3 )δ: 8.02 (d, 1H, J = 9.0Hz), 7.24 (dd, 1H, J = 12.0, 11.4Hz), 6.87 (dd, 1H, J = 11.4, 11.4Hz), 6.53 (d, 1H, J = 9.0Hz), 6.33 (d, 1H, J = 9.0Hz), 6.15 (d, 1H, J = 11.4Hz) , 5.68 (d, 1H, J = 11.4Hz), 5.63 (dd, 1H, J = 6.0, 3.6Hz), 5.28 (d, 1H, J = 10.2Hz), 4.31 (d, 1H, J = 9.6Hz), 4.21 (ddd, 1H, J = 12.6 , 7.8, 4.8Hz), 3.79 (m, 1H), 3.71 (m, 1H), 3.66 (s, 3H), 2.86 (m, 1H), 2.46-2.36 (m, 2H), 2.32 (ddd, 1H, J=13.8, 7.8, 4.2Hz), 2.16 (ddd, 1H, J=14.4, 6.0, 4.2Hz), 1.85 (d, 3H, J=1.2Hz), 1.73 (d, 3H, J=1.2Hz), 1.16 (d, 3H, J=6.6Hz), 1.03 (s, 9H), 0.91 (s, 9H), 0.12 (s, 3H), 0.10 (s, 3H).13 C NMR (150MHz, CDCl3 ) δ: 168.10, 165.89, 161.61, 145.42, 139.85, 136.82, 134.28, 134.04, 124.39, 121.49, 120.00, 118.74, 108.27, 81.96, 62.15, 6 0.54, 55.55, 37.49, 35.26, 35.07, 26.86, 26.48, 26.18, 21.11, 18.74, 17.30, 16.90, -5.06, -5.12.HRMS (m/z): 625.3643[M+Na]+ (Calcd. for C33 H54 N2 O6 Si 625.3643).
(S，2Z，4Z，6E)-N-((S)-1-(((Z)-4-羟基-2-甲基-1-烯-1-基)氨基)-3，3-二甲基-1-氧代丁烷-2-基)-8-((S)-5-甲氧基-6-氧代-3，6-二氢-2H-吡喃-2-基)-6-甲基壬-2，4，6-三酰胺(F-2)(S,2Z,4Z,6E)-N-((S)-1-(((Z)-4-hydroxy-2-methyl-1-en-1-yl)amino)-3,3-dimethyl-1-oxobutan-2-yl)-8-((S)-5-methoxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)-6-methylnonane-2,4,6-triamide (F-2)
中间体E-2(63mg，0.104mmol，1.0eq.)溶于THF(20mL)中，将反应体系置于冰水浴中，控制反应温度0-10℃加入TBAF的THF溶液(1.0M，416μL，0.416mmol，4.0eq.)，加完保温搅拌10分钟。然后将反应体系移出冰水浴，自然升至室温，并在室温搅拌1小时，点板显示反应完全。再次将反应体系置于冰水浴中，控制反应温度0-10℃加入饱和氯化铵水溶液(5mL)淬灭反应，搅拌5分钟后加入水(20mL)进行稀释。加入乙酸乙酯(50mL)萃取，水相再次用乙酸乙酯(20mL)萃取。合并有机相后浓缩，快速过柱后用半制备液相进行纯化得到28mg中间体F-2呈白色固体，收率55％。[α]20D-33.0(c＝0.1，MeOH).1H NMR(600MHz，CDCl3)δ：8.66(d，1H，J＝9.6Hz)，7.27(dd，1H，J＝11.4，11.4Hz)，6.89(ddd，1H，J＝11.4，11.4，0.6Hz)，6.59(d，1H，J＝9.0Hz)，6.47(d，1H，J＝9.0Hz)，6.17(d，1H，J＝11.4Hz)，5.70(d，1H，J＝11.4Hz)，5.64(dd，1H，J＝6.0，3.0Hz)，5.30(d，1H，J＝9.6Hz)，4.35(d，1H，J＝9.0Hz)，4.23(ddd，1H，J＝12.0，7.8，4.8Hz)，3.79(t，2H，J＝5.4Hz)，3.68(s，3H)，2.86(m，1H)，2.67(brs，1H)，2.48-2.37(m，2H)，2.27(t，2H，J＝5.4Hz)，1.86(d，3H，J＝0.6Hz)，1.75(d，3H，J＝1.2Hz)，1.18(d，3H，J＝6.6Hz)，1.05(s，9H).13C NMR(150MHz，CDCl3)δ：168.05，166.33，161.60，145.24，140.08，137.21，134.18，134.08，124.12，120.93，119.31，118.95，108.15，81.82，61.30，60.71，55.45，37.35，34.87，34.47，26.71，26.33，21.32，17.20，16.74.HRMS(m/z)：489.2961[M+1]+(Calcd.for C27H40N2O6 489.2959).Intermediate E-2 (63 mg, 0.104 mmol, 1.0 eq.) was dissolved in THF (20 mL), and the reaction system was placed in an ice-water bath. The reaction temperature was controlled at 0-10 ° C. A THF solution of TBAF (1.0 M, 416 μL, 0.416 mmol, 4.0 eq.) was added. After adding, the mixture was stirred for 10 minutes. The reaction system was then removed from the ice-water bath, naturally warmed to room temperature, and stirred at room temperature for 1 hour. The plate showed that the reaction was complete. The reaction system was placed in an ice-water bath again, and the reaction temperature was controlled at 0-10 ° C. A saturated aqueous solution of ammonium chloride (5 mL) was added to quench the reaction. After stirring for 5 minutes, water (20 mL) was added for dilution. Ethyl acetate (50 mL) was added for extraction, and the aqueous phase was extracted with ethyl acetate (20 mL) again. After the organic phases were combined, they were concentrated, and after rapid column purification was performed using a semi-preparative liquid phase to obtain 28 mg of intermediate F-2 as a white solid with a yield of 55%. [α]20D -33.0 (c=0.1, MeOH).1 H NMR (600MHz, CDCl3 ) δ: 8.66 (d, 1H, J=9.6Hz), 7.27 (dd, 1H, J=11.4, 11.4Hz), 6.89 (ddd, 1H, J=11.4, 11.4, 0.6Hz), 6.59 (d, 1H, J =9.0Hz), 6.47 (d, 1H, J = 9.0Hz), 6.17 (d, 1H, J = 11.4Hz), 5.70 (d, 1H, J = 11.4Hz), 5.64 (dd, 1H, J = 6.0, 3.0Hz), 5.30 (d, 1H, J = 9.6Hz), 4.3 5 (d, 1H, J=9.0Hz), 4.23 (ddd, 1H, J=12.0, 7.8, 4.8Hz), 3.79 (t, 2H, J=5.4Hz), 3.68 (s, 3H), 2.86 (m, 1H), 2.67 (brs, 1H), 2.48-2.37 (m, 2H), 2.27 (t, 2H, J=5.4Hz), 1.86 (d, 3H, J=0.6Hz), 1.75 (d, 3H, J=1.2Hz), 1.18 (d, 3H, J=6.6Hz), 1.05 (s, 9H).13 C NMR (150MHz, CDCl3 ) δ: 168.05, 166.33, 161.60, 145.24, 140.08, 137.21, 134.18, 134.08, 124.12, 120.93, 119.31, 118.95, 108.15, 81.82, 61.30 , 60.71, 55.45, 37.35, 34.87, 34.47, 26.71, 26.33, 21.32, 17.20, 16.74.HRMS(m/z): 489.2961[M+1]+ (Calcd.for C27 H40 N2 O6 489.2959).
(Z)-4-((S)-2-((S，2Z，4Z，6E)-8-((S)-5-甲氧基-6-氧代-3，6-二氢-2H-吡喃-2-基)-6-甲基壬-2，4，6-三胺基)-3，3-二甲基丁胺基)-3-甲基-3-烯-1-基氨基甲酸酯(2-1)(Z)-4-((S)-2-((S,2Z,4Z,6E)-8-((S)-5-methoxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)-6-methylnon-2,4,6-triamino)-3,3-dimethylbutylamino)-3-methyl-3-en-1-ylcarbamate (2-1)
中间体F-2(25mg，0.051mmol，1.0eq.)溶于DCM(5mL)中，将反应体系置于冰水浴中，控制反应温度0-10℃加入TCAI(10μL，0.084mmol，1.6eq.)，加完保温搅拌30分钟，点板显示反应完全。然后将反应体系移出冰水浴，自然升至室温，加入中性氧化铝(125mg)，搅拌30分钟后点板显示反应完全。在漏斗内铺中性氧化铝过滤，滤渣用DCM∶MeOH＝50∶1混合溶剂(30mL)洗涤。合并有机相后浓缩，快速过柱后用半制备液相进行纯化得到12mg化合物2-1呈白色固体，收率44％。[α]20D-45.0(c＝0.1，CHCl3).1H NMR(600MHz，CDCl3)δ：8.17(d，1H，J＝10.2Hz)，7.21(dd，1H，J＝12.0，11.4Hz)，6.89(ddd，1H，J＝11.4，11.4，1.2Hz)，6.63(d，1H，J＝10.2，1.2Hz)，6.37(d，1H，J＝9.6Hz)，6.16(d，1H，J＝12.0Hz)，5.67(d，1H，J＝11.4Hz)，5.63(dd，1H，J＝6.0，2.4Hz)，5.29(d，1H，J＝9.6Hz)，4.31(d，1H，J＝9.6Hz)，4.23(ddd，1H，J＝12.0，7.8，4.2Hz)，4.14(m，1H)，4.07(m，1H)，3.66(s，3H)，2.86(m，1H)，2.53(m，1H)，2.48-2.35(m，2H)，2.22(m，1H)，1.84(d，3H，J＝1.2Hz)，1.74(d，3H，J＝1.2Hz)，1.16(d，3H，J＝6.6Hz)，1.05(s，9H).13C NMR(150MHz，CDCl3)δ：167.75，166.46，161.60，157.63，145.23，140.30，137.57，134.29，133.93，124.13，120.63，119.21，116.18，108.15，81.82，63.45，60.85，55.46，37.26，34.45，31.15，26.61，26.23，20.95，17.16，16.60.HRMS(m/z)：532.3002[M+1]+(Calcd.for C28H41N3O7 532.3017).Intermediate F-2 (25 mg, 0.051 mmol, 1.0 eq.) was dissolved in DCM (5 mL), and the reaction system was placed in an ice-water bath. The reaction temperature was controlled at 0-10°C and TCAI (10 μL, 0.084 mmol, 1.6 eq.) was added. After addition, the mixture was stirred for 30 minutes under the condition of heat preservation. The reaction was then completed as indicated by the plate. The reaction system was then removed from the ice-water bath, naturally warmed to room temperature, and neutral alumina (125 mg) was added. After stirring for 30 minutes, the plate indicated that the reaction was complete. Neutral alumina was spread in a funnel and filtered, and the residue was washed with a mixed solvent of DCM: MeOH = 50: 1 (30 mL). The organic phases were combined and concentrated, and then quickly passed through a column and purified using a semi-preparative liquid phase to obtain 12 mg of compound 2-1 as a white solid with a yield of 44%. [α]20D -45.0 (c = 0.1, CHCl3 ).1 H NMR (600 MHz, CDCl3 )δ: 8.17 (d, 1H, J = 10.2Hz), 7.21 (dd, 1H, J = 12.0, 11.4Hz), 6.89 (ddd, 1H, J = 11.4, 11.4, 1.2Hz), 6.63 (d, 1H, J = 10.2, 1.2Hz), 6.37 (d, 1H, J = 9.6Hz), 6.16 (d , 1H, J=12.0Hz), 5.67 (d, 1H, J=11.4Hz), 5.63 (dd, 1H, J=6.0, 2.4Hz), 5.29 (d, 1H, J=9.6 Hz), 4.31 (d, 1H, J=9.6Hz), 4.23 (ddd, 1H, J=12.0, 7.8, 4.2Hz), 4.14 (m, 1H), 4.07 (m, 1H), 3.66 (s, 3H), 2.86 (m, 1H), 2.53 (m, 1H), 2.48-2.35 (m, 2H), 2. 22 (m, 1H), 1.84 (d, 3H, J = 1.2Hz), 1.74 (d, 3H, J = 1.2Hz), 1.16 (d, 3H, J = 6.6Hz), 1.05 (s, 9H).13 C NMR (150MHz, CDCl3 ) δ: 167.75, 166.46, 161.60, 157.63, 145.23, 140.30, 137.57, 134.29, 133.93, 124.13, 120.63, 119.21, 116.18, 108.15, 81.8 2, 63.45, 60.85, 55.46, 37.26, 34.45, 31.15, 26.61, 26.23, 20.95, 17.16, 16.60. HRMS (m/z): 532.3002[M+1]+ (Calcd.for C28 H41 N3O7 532.3017).
实施例 制备二氢吡喃酮类化合物Example Preparation of dihydropyrone compounds
(S，1Z，6Z)-1-((S)-3-(4-氯苯基)-2-((S，2Z，4Z，6E)-8-((S)-5-甲氧基-6-氧代-3，6-二氢-2H-吡喃-2-基)-6-甲基壬-2，4，6-三胺基)丙酰胺)辛-1，6-二烯-4-基氨基甲酸酯(3-1)(S,1Z,6Z)-1-((S)-3-(4-chlorophenyl)-2-((S,2Z,4Z,6E)-8-((S)-5-methoxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)-6-methylnona-2,4,6-triamino)propionamide)oct-1,6-dien-4-ylcarbamate (3-1)
(S)-2-氨基-N-((S，1Z，6Z)-4-((叔丁基二苯基硅)氧基)辛-1，6-二烯-1-基)-3-(4-氯苯基)丙胺(C-3)(S)-2-amino-N-((S,1Z,6Z)-4-((tert-butyldiphenylsilyl)oxy)oct-1,6-dien-1-yl)-3-(4-chlorophenyl)propylamine (C-3)
中间体B-3(567mg，0.857mmol)悬浮于于乙二醇(35mL)中，油浴设置200℃，待达到目标温度后，将反应液放入油浴，保温搅拌15分钟。到达时间后立即将反应体系移出油浴，用冷水降至室温。加入乙酸乙酯(350mL)，有机相用水(3 x 175mL)洗涤。浓缩有机相，柱层析得388mg中间体C-3呈黄色油状液体，收率81％。1H NMR(600MHz，CDCl3)δ：8.99(d，1H，J＝10.8Hz)，7.72-7.63(m，4H)，7.45-7.33(m，6H)，7.31-7.33(m，6H)，7.29(d，2H，J＝8.4Hz)，7.13(d，2H，J＝8.4Hz)，6.72(dd，1H，J＝10.2，10.2Hz)，5.44(m，1H)，5.31(m，1H)，4.83(dt，1H，J＝8.4，7.8Hz)，3.85(m，1H)，3.55(m，1H)，3.22(m，1H)，2.67(m，1H)，2.24-2.05(m，4H)，1.41(d，3H，J＝6.6Hz)，1.05(s，9H).13C NMR(150MHz，CDCl3)δ：171.06，136.14，135.91，134.22，132.82，130.60，129.61，128.91，127.53，126.12，125.88，121.92，108.30，72.43，56.13，39.96，33.89，32.51，27.02，19.36，12.91.HRMS(m/z)：561.2704[M+1]+(Calcd.forC33H41ClN2O2Si 561.2699).Intermediate B-3 (567 mg, 0.857 mmol) was suspended in ethylene glycol (35 mL), and the oil bath was set at 200 °C. After reaching the target temperature, the reaction solution was placed in the oil bath and stirred for 15 minutes. After reaching the time, the reaction system was immediately removed from the oil bath and cooled to room temperature with cold water. Ethyl acetate (350 mL) was added, and the organic phase was washed with water (3 x 175 mL). The organic phase was concentrated and column chromatography was performed to obtain 388 mg of intermediate C-3 as a yellow oily liquid, with a yield of 81%.1 H NMR (600 MHz, CDCl3 )δ: 8.99 (d, 1H, J=10.8Hz), 7.72-7.63 (m, 4H), 7.45-7.33 (m, 6H), 7.31-7.33 (m, 6H), 7.29 (d, 2H, J=8.4Hz), 7.13 (d, 2H, J=8.4Hz), 6.72 (dd, 1H, J=10.2 ,10.2Hz), 5.44 (m, 1H), 5.31 (m, 1H), 4.83 (dt, 1H, J=8.4, 7.8Hz), 3.85 (m, 1H), 3.55 (m, 1H), 3.22 (m, 1H), 2.67 (m, 1H), 2.24-2.05 (m, 4H), 1.41 (d, 3H, J=6.6Hz ), 1.05 (s, 9H).13 C NMR (150MHz, CDCl3 ) δ: 171.06, 136.14, 135.91, 134.22, 132.82, 130.60, 129.61, 128.91, 127.53, 126.12, 125.88, 121.92, 108.30, 72.43, 56.13 , 39.96, 33.89, 32.51, 27.02, 19.36, 12.91.HRMS (m/z): 561.2704[M+1]+ (Calcd.forC33 H41 ClN2 O2 Si 561.2699).
(Z)-N-((S)-1-((S，1Z，6Z)-4-((叔丁基二苯基硅氧基)辛-1，6-二烯-1-基)氨基)-3-(4-氯苯基)-1-氧代丙烷-2-基)-3-(三丁基)丙烯酰胺(D-3)(Z)-N-((S)-1-((S,1Z,6Z)-4-((tert-butyldiphenylsilyloxy)oct-1,6-dien-1-yl)amino)-3-(4-chlorophenyl)-1-oxopropane-2-yl)-3-(tributyl)acrylamide (D-3)
中间体C-3(388mg，0.691mmol，1.0eq.)溶于DCM(20mL)和DMF(5mL)中，将反应体系置于冰水浴中，控制反应液温度0-10℃依次加入(Z)-3-(三丁基)丙烯酸(300mg，0.831mmol，1.2eq.)、DIPEA(181μL，1.04mmol，1.5eq.)、HOAT(104mg，0.764mmol，1.1eq.)和HATU(342mg，0.899mmol，1.3eq.)，加完保温搅拌30分钟，点板显示反应完全。控制反应温度0-10℃加入半饱和氯化铵水溶液(50mL)淬灭反应，搅拌5分钟后加入MTBE(100mL)，搅拌后分层，有机相用水(50mL)洗涤。浓缩有机相，柱层析得535mg中间体D-3呈无色油状液体，收率86％。1H NMR(600MHz，CDCl3)：δ：7.70-7.62(m，4H)，7.46-7.34(m，6H)，7.25(d，1H，J＝10.2Hz)，7.22(d，2H，J＝8.4Hz)，7.08(d，2H，J＝8.4Hz)，7.03(d，1H，J＝12.0Hz)，6.67(d，1H，J＝12.6Hz)，6.61(dd，1H，J＝10.8，9.0Hz)，6.23(d，1H，J＝8.4Hz)，5.45(m，1H)，5.26(m，1H)，4.83(m，1H)，4.63(m，1H)，3.81(m，1H)，3.04-2.91(m，2H)，2.20-2.09(m，2H)，2.02(m，1H)，1.83(m，1H)，1.54-1.43(m，6H)，1.38(d，3H，J＝7.8Hz)，1.30-1.24(m，6H)，1.04(s，9H)，0.99-0.83(m，15H).13C NMR(150MHz，CDCl3)δ：167.79，166.18，154.46，135.85，135.63，135.54，134.80，134.15，134.00，132.99，130.61，129.74，129.72，128.81，127.64，127.58，126.43，126.35，125.82，121.52，109.24，72.31，54.40，37.73，33.79，32.17，29.30，29.23，29.17，27.84，27.37，27.00，26.85，19.31，17.52，13.81，13.62，12.90，11.57.HRMS(m/z)：905.3868[M+1]+(Calcd.for C48H69ClN2O3SiSn 905.3861).Intermediate C-3 (388 mg, 0.691 mmol, 1.0 eq.) was dissolved in DCM (20 mL) and DMF (5 mL), and the reaction system was placed in an ice-water bath. The reaction liquid temperature was controlled at 0-10 ° C. (Z)-3-(tributyl)acrylic acid (300 mg, 0.831 mmol, 1.2 eq.), DIPEA (181 μL, 1.04 mmol, 1.5 eq.), HOAT (104 mg, 0.764 mmol, 1.1 eq.) and HATU (342 mg, 0.899 mmol, 1.3 eq.) were added in sequence. After adding, the mixture was stirred for 30 minutes. The plate showed that the reaction was complete. The reaction temperature was controlled at 0-10 ° C. and half-saturated aqueous ammonium chloride solution (50 mL) was added to quench the reaction. After stirring for 5 minutes, MTBE (100 mL) was added. After stirring, the layers were separated and the organic phase was washed with water (50 mL). The organic phase was concentrated and column chromatography was performed to obtain 535 mg of intermediate D-3 as a colorless oily liquid, with a yield of 86%.1 H NMR (600 MHz, CDCl3): δ: 7.70-7.62 (m, 4H), 7.46-7.34 (m, 6H), 7.25 (d, 1H, J = 10.2 Hz), 7.22 (d, 2H, J = 8.4 Hz), 7.08 (d, 2H, J = 8.4 Hz), 7.03 (d, 1H, J = 12.0 Hz), 6.67 (d, 1H, J = 12.6 Hz), 6.61 (dd, 1H, J = 10.8, 9.0 Hz), 6.23 (d, 1H, J = 8.4 Hz). z), 5.45 (m, 1H), 5.26 (m, 1H), 4.83 (m, 1H), 4.63 (m, 1H), 3.81 (m, 1H), 3.04-2.91 (m, 2H), 2.20-2.09 (m, 2H), 2.02 (m, 1H), 1.83 (m, 1H), 1.54-1.43 (m , 6H), 1.38 (d, 3H, J=7.8Hz), 1.30-1.24 (m, 6H), 1.04 (s, 9H), 0.99-0.83 (m, 15H).13 C NMR (150MHz, CDCl3 )δ: 167.79, 166.18, 154.46, 135.85, 135.63, 135.54, 134.80, 134.15, 134.00, 132.99, 130.61, 129.74, 129.72, 128.81, 127.64, 127.58, 126.43, 126.35, 125.82, 121. 52, 109.24, 72.31, 54.40, 37.73, 33.79, 32.17, 29.30, 29.23, 29.17, 27.84, 27.37, 27.00, 26.85, 19.31, 17.52, 13.81, 13.62, 12.90, 11.57.HRMS ( m/z): 905.3868[M+1]+ (Calcd. for C48 H69 ClN2 O3 SiSn 905.3861).
(S，2Z，4Z，6E)-N-((S)-1-((S，1Z，6Z)-4-((叔丁基二苯基硅氧基)辛-1，6-二烯-1-基)氨基)-3-(4-氯苯基)-1-氧代丙烷-2-基)-8-((S)-5-甲氧基-6-氧代-3，6-二氢-2H-吡喃-2-基)-6-甲基壬-2，4，6-三酰胺(E-3)(S,2Z,4Z,6E)-N-((S)-1-((S,1Z,6Z)-4-((tert-butyldiphenylsilyloxy)oct-1,6-dien-1-yl)amino)-3-(4-chlorophenyl)-1-oxopropan-2-yl)-8-((S)-5-methoxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)-6-methylnonane-2,4,6-triamide (E-3)
中间体D-3(120mg，0.133mmol，1.0eq.)和A(55mg，0.159mmol，1.2eq.)溶于NMP(10mL)中，将反应体系置于冰水浴中，控制反应温度0-10℃加入CuTC(38mg，0.200mmol，1.5eq.)，加完保温搅拌45分钟。然后将反应体系移出冰水浴，自然升至室温，并在室温搅拌过夜，点板显示反应完全。在漏斗内铺中性氧化铝过滤，滤渣用MTBE∶EA＝1∶1混合溶剂(75mL)洗涤。合并有机相，用0.5M稀盐酸(75，2 x 45mL)洗涤三次，再用水(2 x 45mL)洗涤两次。浓缩有机相，制备板纯化得79mg中间体E-3呈无色油状液体，收率71％。1H NMR(600MHz，CDCl3)δ：7.70-7.62(m，4H)，7.49-7.35(m，7H)，7.19-7.26(m，3H)，7.13(d，2H，J＝7.8Hz)，6.90(dd，1H，J＝12.0，11.4Hz)，6.61(dd，1H，J＝10.2，9.6Hz)，6.21(d，1H，J＝7.8Hz)，6.16(d，1H，J＝11.4Hz)，5.61(dd，1H，J＝6.6，3.0Hz)，5.61(dd，1H，J＝11.4，4.2Hz)，5.44(m，1H)，5.28(d，1H，J＝9.6Hz)，5.24(m，1H)，4.80(m，1H)，4.60(m，1H)，4.20(m，1H)，3.80(m，1H)，3.65(s，3H)，3.13-2.97(m，2H)，2.86(m，1H)，2.46-2.31(m，2H)，2.27-1.96(m，4H)，1.84(s，3H)，1.39(d，3H，J＝6.6Hz)，1.16(d，3H，J＝7.2Hz)，1.04(s，9H).13CNMR(150MHz，CDCl3)δ：167.83，166.27，161.52，145.27，140.69，138.05，135.89，135.02，134.36，134.19，134.07，133.01，130.64，129.70，128.90，127.61，127.56，126.29，125.90，123.97，121.58，119.97，109.25，108.08，81.77，72.34，55.45，54.38，37.31，33.94，32.25，27.02，26.25，19.32，17.17，16.68，12.92.HRMS(m/z)：857.3735[M+Na]+(Calcd.forC49H59ClN2O6Si 857.3723).Intermediate D-3 (120 mg, 0.133 mmol, 1.0 eq.) and A (55 mg, 0.159 mmol, 1.2 eq.) were dissolved in NMP (10 mL). The reaction system was placed in an ice-water bath. The reaction temperature was controlled at 0-10°C and CuTC (38 mg, 0.200 mmol, 1.5 eq.) was added. After the addition, the mixture was stirred for 45 minutes. The reaction system was then removed from the ice-water bath, naturally warmed to room temperature, and stirred overnight at room temperature. The plate showed that the reaction was complete. Neutral alumina was spread in the funnel and filtered. The filter residue was washed with a MTBE:EA=1:1 mixed solvent (75 mL). The organic phases were combined and washed three times with 0.5 M dilute hydrochloric acid (75, 2 x 45 mL), and then washed twice with water (2 x 45 mL). The organic phase was concentrated and purified by preparative plate to obtain 79 mg of intermediate E-3 as a colorless oily liquid with a yield of 71%.1 H NMR (600 MHz, CDCl3 )δ: 7.70-7.62 (m, 4H), 7.49-7.35 (m, 7H), 7.19-7.26 (m, 3H), 7.13 (d, 2H, J=7.8Hz), 6.90 (dd, 1H, J=12.0, 11.4Hz), 6.61 (dd, 1H, J=10.2, 9.6Hz), 6.21 (d , 1H, J=7.8Hz), 6.16 (d, 1H, J=11.4Hz), 5.61 (dd, 1H, J=6.6, 3.0Hz), 5.61 (dd, 1H, J=11.4, 4.2Hz), 5.4 4 (m, 1H), 5.28 (d, 1H, J=9.6Hz), 5.24 (m, 1H), 4.80 (m, 1H), 4.60 (m, 1H), 4.20 (m, 1H), 3.80 (m, 1H), 3.65 (s, 3H), 3.13-2.97 (m, 2H), 2.86 (m, 1H), 2.4 6-2.31 (m, 2H), 2.27-1.96 (m, 4H), 1.84 (s, 3H), 1.39 (d, 3H, J=6.6Hz), 1.16 (d, 3H, J=7.2Hz), 1.04 (s, 9H).13 CNMR (150MHz, CDCl3 )δ: 167.83, 166.27, 161.52, 145.27, 140.69, 138.05, 135.89, 135.02, 134.36, 134.19, 134.07, 133.01, 130.64, 129.70, 128.90, 127.61, 127.56, 126.29, 125.90, 123.97, 121.58, 119.97, 109.25, 108.08, 81.77, 72.34, 55.45, 54.38, 37.31, 33.94, 32.25, 27.02, 26.25, 19.32, 17.17, 16.68, 12.92.HRMS (m/z): 857.3735[M+Na]+ (Calcd.forC49 H59 ClN2 O6 Si 857.3723).
(S，2Z，4Z，6E)-N-((S)-3-(4-氯苯基)-1-((S，1Z，6Z)-4-羟基八-1，6-二烯-1-基)氨基)-1-氧代丙烷-2-基)-8-((S)-5-甲氧基-6-氧代-3，6-二氢-2H-吡喃-2-基)-6-甲基壬-2，4，6-三酰胺(F-3)(S,2Z,4Z,6E)-N-((S)-3-(4-chlorophenyl)-1-((S,1Z,6Z)-4-hydroxyocta-1,6-dien-1-yl)amino)-1-oxopropan-2-yl)-8-((S)-5-methoxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)-6-methylnonane-2,4,6-triamide (F-3)
中间体E-3(79mg，0.095mmol，1.0eq.)溶于THF(20mL)中，将反应体系置于冰水浴中，控制反应温度0-10℃加入TBAF的THF溶液(1.0M，378μL，0.378mmol，4.0eq.)，加完保温搅拌10分钟。将反应体系移出冰水浴，自然升至室温，并在室温搅拌1小时，点板显示反应完全。将反应体系置于冰水浴中，控制反应液温度0-10℃加入饱和氯化铵水溶液(5mL)淬灭反应，搅拌5分钟后加入水(20mL)进行稀释。加入乙酸乙酯(50mL)萃取，分层后水相再次用乙酸乙酯(20mL)萃取。合并有机相后浓缩，快速过柱后用半制备液相进行纯化得到29mg中间体F-3呈白色固体，收率52％。[α]20D-45.0(c＝0.1，CHCl3).1H NMR(600MHz，CDCl3)δ：8.74(d，1H，J＝10.2Hz)，7.27(d，2H，J＝7.8Hz)，7.21(dd，1H，J＝12.0，11.4Hz)，7.17(d，2H，J＝8.4Hz)，6.89(dd，1H，J＝11.4，11.4Hz)，6.70(dd，1H，J＝9.6，9.6Hz)，6.41(d，1H，J＝7.2Hz)，6.17(d，1H，J＝12.0Hz)，5.70-5.56(m，3H)，5.36(m，1H)，5.29(d，1H，J＝9.6Hz)，4.86(dt，1H，J＝8.4，7.8Hz)，4.70(m，1H)，4.22(m，1H)，3.66(s，3H)，3.65(m，1H)，3.11(m，1H)，3.05(m，1H)，2.86(m，1H)，2.48-2.32(m，2H)，2.27-2.05(m，4H)，1.84(s，3H)，1.64(d，3H，J＝7.2Hz)，1.16(d，3H，J＝6.6Hz).13C NMR(150MHz，CDCl3)δ：168.16，166.25，161.57，145.25，140.56，137.87，135.12，134.37，134.06，132.89，130.80，128.81，127.84，125.50，123.98，123.42，120.19，109.08，108.14，81.79，71.86，55.46，54.60，37.84，37.30，34.61，32.68，26.26，17.17，16.66，13.09.HRMS(m/z)：597.2713[M+1]+(Calcd.for C33H41ClN2O6597.2726).Intermediate E-3 (79 mg, 0.095 mmol, 1.0 eq.) was dissolved in THF (20 mL), and the reaction system was placed in an ice-water bath. The reaction temperature was controlled at 0-10 ° C. A THF solution of TBAF (1.0 M, 378 μL, 0.378 mmol, 4.0 eq.) was added. After adding, the mixture was stirred for 10 minutes. The reaction system was removed from the ice-water bath, naturally warmed to room temperature, and stirred at room temperature for 1 hour. The plate showed that the reaction was complete. The reaction system was placed in an ice-water bath, and the temperature of the reaction solution was controlled at 0-10 ° C. A saturated aqueous solution of ammonium chloride (5 mL) was added to quench the reaction. After stirring for 5 minutes, water (20 mL) was added for dilution. Ethyl acetate (50 mL) was added for extraction. After separation, the aqueous phase was extracted again with ethyl acetate (20 mL). After the organic phases were combined, they were concentrated, and after rapid column purification was performed using a semi-preparative liquid phase to obtain 29 mg of intermediate F-3 as a white solid with a yield of 52%. [α]20D -45.0 (c=0.1, CHCl3 ).1 H NMR (600MHz, CDCl3 ) δ: 8.74 (d, 1H, J=10.2Hz), 7.27 (d, 2H, J=7.8Hz), 7.21 (dd, 1H, J=12.0, 11.4Hz), 7.17 (d, 2H, J=8.4Hz), 6 .89 (dd, 1H, J=11.4, 11.4Hz), 6.70 (dd, 1H, J=9.6, 9.6Hz), 6.41 (d, 1H, J=7.2Hz), 6.17 (d, 1H, J=12.0Hz), 5.70-5.56 (m, 3H), 5.36 (m, 1H) , 5.29 (d, 1H, J = 9.6Hz), 4.86 (dt, 1H, J = 8.4, 7.8Hz), 4.70 (m, 1H), 4.22 (m, 1H), 3.66 (s, 3H), 3.65 (m, 1H), 3.11 (m, 1H), 3.05 (m, 1H), 2.86 (m, 1H), 2. 48-2.32 (m, 2H), 2.27-2.05 (m, 4H), 1.84 (s, 3H), 1.64 (d, 3H, J=7.2Hz), 1.16 (d, 3H, J=6.6Hz).13 C NMR (150MHz, CDCl3) δ: 168.16, 166.25, 161.57, 145.25, 140.56, 137.87, 135.12, 134.37, 134.06, 132.89, 130.80, 128.81, 127.84, 125.50, 123.98 , 123.42, 120.19, 109.08, 108.14, 81.79, 71.86, 55.46, 54.60, 37.84, 37.30, 34.61, 32.68, 26.26, 17.17, 16.66, 13.09.HRMS (m/z): 597.2713[M+1]+ (Calcd. for C33 H41 ClN2 O6 597.2726).
(S，1Z，6Z)-1-((S)-3-(4-氯苯基)-2-((S，2Z，4Z，6E)-8-((S)-5-甲氧基-6-氧代-3，6-二氢-2H-吡喃-2-基)-6-甲基壬-2，4，6-三胺基)丙酰胺)辛-1，6-二烯-4-基氨基甲酸酯(3-1)(S,1Z,6Z)-1-((S)-3-(4-chlorophenyl)-2-((S,2Z,4Z,6E)-8-((S)-5-methoxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)-6-methylnona-2,4,6-triamino)propionamide)oct-1,6-dien-4-ylcarbamate (3-1)
中间体F-3(25mg，0.042mmol，1.0eq.)溶于DCM(10mL)中，将反应体系置于冰水浴中，控制反应液温度0-10℃加入TCAI(10μL，0.084mmol，2.0eq.)，加完保温搅拌30分钟，点板显示反应完全。将反应体系移出冰水浴，自然升至室温，往反应液内加入中性氧化铝(125mg)，搅拌30分钟后点板显示反应完全。在漏斗内铺中性氧化铝过滤，滤渣用DCM∶MeOH＝50∶1混合溶剂(30mL)洗涤。合并有机相后浓缩，快速过柱后用半制备液相进行纯化得到阳性化合物3-1呈白色固体。质量14mg，收率52％。[α]20D-63.0(c＝0.1，CHCl3).1H NMR(600MHz，CDCl3)δ：8.70(d，1H，J＝10.8Hz)，7.25(m，1H)，7.24(d，2H，J＝8.4Hz)，7.13(d，2H，J＝8.4Hz)，6.91(dd，1H，J＝12.0，11.4Hz)，6.76(dd，1H，J＝9.6，9.6Hz)，6.63(d，1H，J＝8.4Hz)，6.16(d，1H，J＝11.4Hz)，5.70-5.57(m，3H)，5.37(m，1H)，5.31(m，1H)，4.90-4.76(m，2H)，4.36-4.14(m，2H)，3.66(s，3H)，3.18-3.04(m，2H)，2.86(m，1H)，2.47(m，1H)，2.38(m，1H)，2.20(m，1H)，2.05(m，1H)，1.82(s，3H)，1.62(d，3H，J＝6.6Hz)，1.15(d，3H，J＝6.6Hz).13C NMR(150MHz，CDCl3)δ：168.38，166.18，161.66，157.73，145.18，140.42，137.80，134.78，134.21，133.75，132.83，130.90，128.56，127.06，124.90，124.36，124.02，120.36，108.28，106.46，81.87，75.56，55.46，54.11，38.14，37.03，31.25，30.63，26.05，17.10，16.26，13.00.HRMS(m/z)：640.2788[M+1]+(Calcd.for C34H42ClN3O7 640.2784).Intermediate F-3 (25 mg, 0.042 mmol, 1.0 eq.) was dissolved in DCM (10 mL), and the reaction system was placed in an ice-water bath. The temperature of the reaction solution was controlled at 0-10 °C, and TCAI (10 μL, 0.084 mmol, 2.0 eq.) was added. After addition, the mixture was stirred for 30 minutes under the condition of heat preservation. The reaction was completed when the plate was dotted. The reaction system was removed from the ice-water bath and naturally warmed to room temperature. Neutral alumina (125 mg) was added to the reaction solution. After stirring for 30 minutes, the plate was dotted to show that the reaction was complete. Neutral alumina was spread in a funnel and filtered, and the filter residue was washed with a mixed solvent of DCM: MeOH = 50: 1 (30 mL). The organic phases were combined and concentrated, and then quickly passed through a column and purified with a semi-preparative liquid phase to obtain the positive compound 3-1 as a white solid. The mass was 14 mg, and the yield was 52%. [α]20D -63.0 (c = 0.1, CHCl3 ).1 H NMR (600 MHz, CDCl3 )δ: 8.70 (d, 1H, J = 10.8Hz), 7.25 (m, 1H), 7.24 (d, 2H, J = 8.4Hz), 7.13 (d, 2H, J = 8.4Hz), 6.91 (dd, 1H, J = 12.0, 11.4Hz), 6.76 (dd, 1H, J = 9.6, 9.6Hz), 6.63 (d, 1H, J=8.4Hz), 6.16 (d, 1H, J=11.4Hz), 5.70-5.57 (m, 3H), 5.37 (m, 1H), 5.31 (m, 1H), 4.90-4.76 (m, 2H), 4.36-4.14 (m, 2H), 3.66 (s, 3H), 3.18-3.04 (m, 2H), 2.86 (m, 1H), 2.47 (m, 1H), 2.38 (m, 1H), 2.20 (m, 1H), 2 .05 (m, 1H), 1.82 (s, 3H), 1.62 (d, 3H, J=6.6Hz), 1.15 (d, 3H, J=6.6Hz).13 C NMR (150MHz, CDCl3 )δ: 168.38, 166.18, 161.66, 157.73, 145.18, 140.42, 137.80, 134.78, 134.21, 133.75, 132.83, 130.90, 128.56, 127.06, 124.90, 124.36, 124.02, 120.36, 108.28, 106.46, 81.87, 75.56, 55.46, 54.11, 38.14, 37.03, 31.25, 30.63, 26.05, 17.10, 16.26, 13.00.HRMS(m/z): 640.2788[M+1]+ (Calcd.for C34 H42 ClN3 O7 640.2784).
实施例4：体外肿瘤细胞增殖抑制活性测试Example 4: In vitro tumor cell proliferation inhibition activity test
试验方法：Test method:
A549、HT-29、HepG2和MDA-MB-231肿瘤细胞的生长抑制检测采用CCK-8方法。具体步骤如下：处于对数生长期的细胞按合适密度接种至96孔培养板，每孔90μL，培养过夜后，加入不同浓度的药物作用72h，每个浓度设三复孔，并设相应浓度的溶媒对照及无细胞调零孔。作用结束后，每孔加入10μL CCK-8，培养箱中孵育4h后，SpectraMax 190酶标仪测定450nm波长下的光密度(OD值)。The growth inhibition test of A549, HT-29, HepG2 and MDA-MB-231 tumor cells adopts CCK-8 method. The specific steps are as follows: cells in the logarithmic growth phase are inoculated into 96-well culture plates at an appropriate density, 90 μL per well, and cultured overnight. Different concentrations of drugs are added for 72 hours. Three replicate wells are set for each concentration, and solvent controls and cell-free zero wells of corresponding concentrations are set. After the action is over, 10 μL CCK-8 is added to each well. After incubation in the incubator for 4 hours, the optical density (OD value) at a wavelength of 450 nm is measured by SpectraMax 190 microplate reader.
IC50值采用酶标仪随机附带软件以四参数法回归求得。TheIC50 value was obtained by four-parameter regression using the software provided with the microplate reader.
结果表明(如表1所示)，本发明的化合物显示出较好的肿瘤细胞增殖抑制活性，化合物1-1和化合物2-1对于细胞增殖抑制活性IC50值均小于10nM。The results show (as shown in Table 1) that the compounds of the present invention exhibit good tumor cell proliferation inhibitory activity, and the IC50 values of compound 1-1 and compound 2-1 for cell proliferation inhibitory activity are both less than 10 nM.
表1：目标化合物对肿瘤细胞增殖抑制活性Table 1: Inhibitory activity of target compounds on tumor cell proliferation
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