技术领域technical field
本发明涉及一种衍生物及其制备方法与应用，具体涉及一种含有吲哚的肉桂酰胺类组蛋白去乙酰化酶抑制剂及其制备方法和应用,属于有机化合物合成与医药应用技术领域。The invention relates to a derivative and its preparation method and application, in particular to a cinnamic amide histone deacetylase inhibitor containing indole, its preparation method and application, and belongs to the technical field of organic compound synthesis and medical application.
背景技术Background technique
恶性肿瘤是威胁人类生命健康的头号杀手。肿瘤的发生和发展不仅与基因序列的改变有关，也与染色体的后生变化有密切关系。组蛋白的可逆性共价修饰是染色体后生变化的重要表现形式，包括磷酸化/去磷酸化、乙酰化/去乙酰化、腺苷酰化/去腺苷酰化、尿苷酰化/去尿苷酰化、甲基化/去甲基化等。组蛋白的乙酰化水平对染色体结构的重塑有着重要作用，乙酰化/去乙酰化是由组蛋白乙酰化酶（HATs）和组蛋白去乙酰化酶（HDACs）这两种相互作用的金属蛋白酶共同催化完成(Shahbazian,M.D.;Grunstein,M.Functions ofSite-Specific Histone Acetylation and Deacetylation.Annu.Rev.Biochem.,2007,76(1),75-100.)。HDACs通过水解核小体组蛋白中的赖氨酸残基末端氨基上的乙酰基，增加了N末端的正电荷密度，增强了其与带有负电荷的DNA的相互作用，使染色体结构变得更加紧实，进而阻碍了转录因子与DNA的结合，最终使基因的转录受到抑制，其中包括抑癌基因。HATs的作用与HDACs恰好相反，最终促进了基因的转录。(Legube G,TroucheD.Regulating histone acetyltransferases and deacetylases[J].Eur mole Biolorg(EMBO)reports.2003,4(10):944-947)在癌细胞中，HDACs的异常过度表达打破了HDACs与HATs之间的动态平衡。除了组蛋白之外，HDACs的作用底物还包括转录因子（p53,E2F,pRb），分子伴侣（HSP90）以及结构蛋白（微管蛋白）等等。(Glozak,M.A.;Sengupta,N.;Zhang,X.H.;Seto,E.Gene,2005,363,15)近年来，HDACs已经成为抗肿瘤研究的一个热点，抑制HDACs的活性已经成为包括肿瘤在内的多种疾病治疗的一个重要策略。Malignant tumor is the number one killer threatening human life and health. The occurrence and development of tumors are not only related to changes in gene sequences, but also closely related to epigenetic changes in chromosomes. Reversible covalent modification of histones is an important manifestation of epigenetic changes in chromosomes, including phosphorylation/dephosphorylation, acetylation/deacetylation, adenylation/deadenylation, uridylylation/deurination Glycylation, methylation/demethylation, etc. The acetylation level of histone plays an important role in the remodeling of chromosome structure. Acetylation/deacetylation is caused by two interacting metalloproteinases, histone acetylases (HATs) and histone deacetylases (HDACs). Co-catalyzed completion (Shahbazian, M.D.; Grunstein, M. Functions of Site-Specific Histone Acetylation and Deacetylation. Annu. Rev. Biochem., 2007, 76(1), 75-100.). HDACs increase the positive charge density at the N-terminus by hydrolyzing the acetyl group on the terminal amino group of lysine residues in nucleosomal histones, enhancing its interaction with negatively charged DNA and making the chromosome structure more It is more compact, which in turn hinders the binding of transcription factors to DNA, and finally inhibits the transcription of genes, including tumor suppressor genes. The effect of HATs is just opposite to that of HDACs, which ultimately promotes the transcription of genes. (Legube G,TroucheD.Regulating histone acetyltransferases and deacetylases[J].Eur mole Biolorg(EMBO)reports.2003,4(10):944-947) In cancer cells, the abnormal overexpression of HDACs breaks the relationship between HDACs and HATs the dynamic balance between. In addition to histones, the substrates of HDACs also include transcription factors (p53, E2F, pRb), molecular chaperones (HSP90) and structural proteins (tubulin) and so on. (Glozak, M.A.; Sengupta, N.; Zhang, X.H.; Seto, E. Gene, 2005, 363, 15) In recent years, HDACs has become a hotspot in anti-tumor research, and the inhibition of HDACs activity has become An important strategy in the treatment of many diseases.
目前，在人类中已经发现18种HDACs，按其结构功能不同可以分为4类，Ⅰ类HDACs包括HDAC1,2,3,8；Ⅱ类HDACs包括HDAC4,5,6,7,9,10；Ⅳ类HDACs包括HDAC11；Ⅲ类HDACs包括sirtuins1-7。其中ⅠⅡⅣ类HDACs为Zn2+依赖性的组蛋白去乙酰化酶，Ⅲ类HDACs为NAD+依赖性组蛋白去乙酰化酶。At present, 18 HDACs have been found in humans, which can be divided into 4 types according to their structure and function. Class I HDACs include HDAC1, 2, 3, and 8; class II HDACs include HDAC4, 5, 6, 7, 9, and 10; Class IV HDACs include HDAC11; class III HDACs include sirtuins1-7. Among them, class Ⅰ, Ⅱ, and Ⅳ HDACs are Zn2+ -dependent histone deacetylases, and class Ⅲ HDACs are NAD+ -dependent histone deacetylases.
许多HDAC抑制剂（HDACi）已经进入临床前或临床研究，研究结果表明HDACi对多种肿瘤具有抑制作用，包括皮肤T细胞瘤（CTCL）、多形性成胶质细胞瘤、B细胞淋巴瘤、甲状腺癌、非小细胞肺癌、恶性肾细胞瘤、恶性间皮细胞瘤、前列腺癌等等。Many HDAC inhibitors (HDACi) have entered preclinical or clinical research, and the results show that HDACi has inhibitory effects on a variety of tumors, including cutaneous T-cell tumor (CTCL), glioblastoma multiforme, B-cell lymphoma, Thyroid cancer, non-small cell lung cancer, malignant renal cell tumor, malignant mesothelioma, prostate cancer, etc.
发明内容Contents of the invention
针对现有技术的不足，本发明提供了一种肉桂酰胺类组蛋白去乙酰化酶抑制剂，本发明还提供了上述化合物的制备方法和用途。Aiming at the deficiencies of the prior art, the present invention provides a cinnamamide histone deacetylase inhibitor, and the present invention also provides a preparation method and application of the above compound.
为解决上述技术问题，本发明的技术方案为：In order to solve the problems of the technologies described above, the technical solution of the present invention is:
一、肉桂酰胺类组蛋白去乙酰化酶抑制剂1. Cinnamamide histone deacetylase inhibitors
本发明的肉桂酰胺类组蛋白去乙酰化酶抑制剂，以及其光学异构体、非对映异构体和消旋体混合物，其药学上可接受的盐，溶剂合物或前药，具有如下通式I所示的结构。The cinnamic amide histone deacetylase inhibitor of the present invention, and its optical isomers, diastereoisomers and racemic mixtures, and its pharmaceutically acceptable salts, solvates or prodrugs, have The structure shown in the following general formula I.
其中：in:
R1是氢、各种氨基酸制备的酰基、芳酰基、杂芳酰基、芳基C1-6烷酰基、杂芳基C1-9烷酰基、C1-6烷酰基、环烷酰基、芳磺酰基、杂磺酰基、芳基C1-6烷磺酰基、杂芳基C1-9烷磺酰基、C1-8烷氧羰基或芳基C1-8烷氧羰基、芳基C1-6烷基；R1 is hydrogen, acyl, aroyl, heteroaroyl, aryl C1-6 alkanoyl, heteroarylC1-9 alkanoyl, C1-6 alkanoyl, cycloalkanoyl, arylsulfonyl prepared from various amino acids, Heterosulfonyl, aryl C1-6 alkylsulfonyl, heteroaryl C1-9 alkylsulfonyl, C1-8 alkoxycarbonyl or aryl C1-8 alkoxycarbonyl, aryl C1-6 alkyl;
R2是氢；卤素原子、硝基、羧基等吸电基团；烷基、氨基、羟基、羟甲基、胺甲基、氨甲酰基等给电基团；R2 is hydrogen; electricity-absorbing groups such as halogen atoms, nitro groups, and carboxyl groups; electricity-donating groups such as alkyl groups, amino groups, hydroxyl groups, hydroxymethyl groups, aminomethyl groups, and carbamoyl groups;
R3是氢、甲基；R3 is hydrogen, methyl;
X是间位和对位O，N原子；X is meta-position and para-position O, N atom;
Y是或Y is or
*是立体构型为S或R光学纯度或其消旋体。* is stereo configuration S or R optical purity or its racemate.
优选的，R1是芳酰基；R2是氢、卤素原子；R3是氢；X是对位O原子；Y是Preferably, R1 is an aroyl group; R2 is hydrogen, a halogen atom; R3 is hydrogen; X is a para-position O atom; Y is
更为优选的，上述化合物是下列之一：More preferably, the above-mentioned compound is one of the following:
(S,E)-3-(4-(2-(苯甲酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L1）、(S,E)-3-(4-(2-(benzamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide (L1),
(S,E)-3-(4-(2-(苯乙酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L2）、(S,E)-3-(4-(2-(phenylacetamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide (L2),
(S,E)-3-(4-(2-(3-苯丙酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L3）、(S,E)-3-(4-(2-(3-phenylpropanylamino)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide (L3 ),
(S,E)-3-(4-(2-(4-甲基苯磺酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L4）、(S,E)-3-(4-(2-(4-methylbenzenesulfonamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide (L4),
(S,E)-3-(4-(2-([1,1'-联苯基]-4-酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L5）、(S,E)-3-(4-(2-([1,1'-biphenyl]-4-amido)-3-(1H-indol-3-yl)propoxy)phenyl )-N-hydroxyacrylamide (L5),
(S,E)-3-(4-(2-([1,1'-联苯]-4-磺酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L6）、(S,E)-3-(4-(2-([1,1'-biphenyl]-4-sulfonamido)-3-(1H-indol-3-yl)propoxy)phenyl )-N-hydroxyacrylamide (L6),
(S,E)-3-(4-(2-(4-氯苯甲酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L7）、(S,E)-3-(4-(2-(4-chlorobenzamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide ( L7),
(S,E)-3-(4-(2-(3-氯苯甲酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L8）、(S,E)-3-(4-(2-(3-chlorobenzamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide ( L8),
(S,E)-3-(4-(2-(2-氯苯甲酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L9）、(S,E)-3-(4-(2-(2-chlorobenzamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide ( L9),
(S,E)-3-(4-(2-(2,4-二氯苯甲酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L10）、(S,E)-3-(4-(2-(2,4-dichlorobenzamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxy Acrylamide (L10),
(S,E)-3-(4-(2-(3,5-二氯苯甲酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L11）、(S,E)-3-(4-(2-(3,5-dichlorobenzamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxy Acrylamide (L11),
(S,E)-3-(4-(2-(4-氟苯甲酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L12）、(S,E)-3-(4-(2-(4-fluorobenzamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide ( L12),
(S,E)-3-(4-(2-(4-溴苯甲酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L13）、(S,E)-3-(4-(2-(4-bromobenzamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide ( L13),
(S,E)-3-(4-(2-(4-甲氧基苯甲酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L14）、(S,E)-3-(4-(2-(4-methoxybenzamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxypropene Amide (L14),
(S,E)-3-(4-(2-(2-乙基丁酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L15）、(S,E)-3-(4-(2-(2-Ethylbutyramide)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide ( L15),
(S,E)-3-(4-(2-(2-丙基戊酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L16）、(S,E)-3-(4-(2-(2-Propylpentanoyl)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide ( L16),
(S,E)-3-(4-(2-((叔丁氧羰基)氨基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L17）、(S,E)-3-(4-(2-((tert-butoxycarbonyl)amino)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide ( L17),
(S,E)-3-(4-(2-(2-((叔丁氧羰基)氨基)乙酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L18）、(S,E)-3-(4-(2-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(1H-indol-3-yl)propoxy)phenyl) -N-Hydroxyacrylamide (L18),
(E)-3-(4-((S)-2-((S)-2-((叔丁氧羰基)氨基)-4-(甲硫基)丁酰基-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L19）、(E)-3-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-4-(methylthio)butanoyl-3-(1H-indole -3-yl)propoxy)phenyl)-N-hydroxyacrylamide (L19),
(E)-3-(4-((S)-2-((S)-2-((叔丁氧羰基)氨基)-3-羟基丙酰基-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L20）、(E)-3-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-hydroxypropionyl-3-(1H-indol-3-yl )propoxy)phenyl)-N-hydroxyacrylamide (L20),
(E)-3-(4-((S)-2-((S)-2-((叔丁氧羰基)氨基)-3-羟基丁酰基-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L21）、(E)-3-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-hydroxybutyryl-3-(1H-indol-3-yl )propoxy)phenyl)-N-hydroxyacrylamide (L21),
(S,E)-3-(4-(2-(2-羟基苯甲酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L22）、(S,E)-3-(4-(2-(2-Hydroxybenzamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide ( L22),
(S,E)-3-(4-(2-(2-((2,3-二甲基苯基)氨基)苯甲酸)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L23）、(S,E)-3-(4-(2-(2-((2,3-dimethylphenyl)amino)benzoic acid)-3-(1H-indol-3-yl)propoxy )phenyl)-N-hydroxyacrylamide (L23),
(E)-3-(4-((2S)-2-(2-(4-异丁基苯基)丙酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L24）、(E)-3-(4-((2S)-2-(2-(4-isobutylphenyl)propionyl)-3-(1H-indol-3-yl)propoxy)benzene base)-N-hydroxyacrylamide (L24),
(E)-3-(4-((2S)-2-(2-(4-苯甲酰基苯基)丙酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L25）、(E)-3-(4-((2S)-2-(2-(4-benzoylphenyl)propionyl)-3-(1H-indol-3-yl)propoxy)benzene base)-N-hydroxyacrylamide (L25),
(E)-3-(4-((S)-2-((S)-2-(6-甲氧基萘基-2-基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L26）、(E)-3-(4-((S)-2-((S)-2-(6-methoxynaphthyl-2-yl)-3-(1H-indol-3-yl)propane Oxy)phenyl)-N-hydroxyacrylamide (L26),
(S,E)-3-(3-(2-((叔丁氧羰基)氨基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L27）、(S,E)-3-(3-(2-((tert-butoxycarbonyl)amino)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide ( L27),
(S,E)-3-(3-(2-(苯甲酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L28）、(S,E)-3-(3-(2-(benzamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide (L28),
(S,E)-3-(4-(2-(4-氯苯甲酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L29）、(S,E)-3-(4-(2-(4-chlorobenzamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide ( L29),
(S,E)-3-(4-(2-(苄氨基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L30）、(S,E)-3-(4-(2-(Benzylamino)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide (L30),
(S,E)-3-(4-(2-((4-氟苄基)氨基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L31）、(S,E)-3-(4-(2-((4-fluorobenzyl)amino)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide (L31),
(S,E)-3-(4-(2-((4-氯苄基)氨基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L32）、(S,E)-3-(4-(2-((4-chlorobenzyl)amino)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide (L32),
(S,E)-3-(4-(2-((4-溴苄基)氨基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L33）、(S,E)-3-(4-(2-((4-bromobenzyl)amino)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide (L33),
(S,E)-3-(4-(2-((4-甲氧基苄基)氨基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L34）或(S,E)-3-(4-(2-((4-methoxybenzyl)amino)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxy Acrylamide (L34) or
(S,E)-3-(4-(2-((叔丁氧羰基)氨基)-3-(1-甲基-1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺（L35）。(S,E)-3-(4-(2-((tert-butoxycarbonyl)amino)-3-(1-methyl-1H-indol-3-yl)propoxy)phenyl)-N - Hydroxyacrylamide (L35).
二、肉桂酰胺类组蛋白去乙酰化酶抑制剂的制备方法Two, the preparation method of cinnamamide histone deacetylase inhibitor
本发明的肉桂酰胺类组蛋白去乙酰化酶抑制剂的制备方法为如下之一：The preparation method of the cinnamamide histone deacetylase inhibitor of the present invention is one of the following:
合成路线1：以L-色氨酸为起始原料，在乙酰氯作用下生成色氨酸甲酯盐酸盐2，用(Boc)2O保护α-氨基得到中间体3，继而经过LiAIH4还原甲酯成中间体4，中间体4与4-羟基肉桂酸或3-羟基肉桂酸经Mitsunobu反应成化合物7，7可以直接转化为异羟肟酸目标化合物L17，或者7经EtOAc/HCl脱去Boc保护基团，最后与各种Boc保护的氨基酸或羧酸缩合为酰胺，并将甲酯转化为羟肟酸得到目标化合物；Synthetic route 1: Using L-tryptophan as the starting material, generate tryptophan methyl ester hydrochloride 2 under the action of acetyl chloride, protect the α-amino group with (Boc)2 O to obtain intermediate 3, and then pass through LiAIH4 Reduction of methyl ester to intermediate 4, intermediate 4 reacts with 4-hydroxycinnamic acid or 3-hydroxycinnamic acid via Mitsunobu to form compound 7, 7 can be directly converted to hydroxamic acid target compound L17, or 7 can be desorbed by EtOAc/HCl Remove the Boc protecting group, and finally condense with various Boc-protected amino acids or carboxylic acids to form amides, and convert the methyl ester into hydroxamic acid to obtain the target compound;
合成路线1反应式如下：Synthetic route 1 reaction formula is as follows:
其中R是氢，各种氨基酸制备的酰基，芳酰基，杂芳酰基，芳基C1-6烷酰基，杂芳基C1-9烷酰基，C1-6烷酰基，环烷酰基，芳磺酰基，杂磺酰基，芳基C1-6烷磺酰基，杂芳基C1-9烷磺酰基，C1-8烷氧羰基或芳基C1-8烷氧羰基；Where R is hydrogen, acyl, aroyl, heteroaroyl, aryl C1-6 alkanoyl, heteroaryl C1-9 alkanoyl, C1-6 alkanoyl, cycloalkanoyl, arylsulfonyl prepared from various amino acids, Heterosulfonyl, aryl C1-6 alkanesulfonyl, heteroaryl C1-9 alkanesulfonyl, C1-8 alkoxycarbonyl or aryl C1-8 alkoxycarbonyl;
上述合成路线反应式中的试剂：(a)CH3OH，乙酰氯，加热回流5h；(b)(Boc)2O，三乙胺，二氯甲烷；(c)氢化铝锂，无水四氢呋喃；(d)DEAD，Ph3P，无水四氢呋喃；(e)EtOAc的饱和HCl溶液；(f)TBTU，三乙胺，无水四氢呋喃；(g)NH2OK，无水甲醇。Reagents in the above synthetic route reaction formula: (a) CH3 OH, acetyl chloride, heated to reflux for 5h; (b) (Boc)2 O, triethylamine, dichloromethane; (c) lithium aluminum hydride, anhydrous tetrahydrofuran (d ) DEAD, Ph3P, anhydrous THF; (e) EtOAc in saturated HCl; (f) TBTU, triethylamine, anhydrous THF; (g ) NH2OK, anhydrous methanol.
合成路线1的目标化合物的结构式如下所示：The structural formula of the target compound of synthetic route 1 is as follows:
合成路线2：中间体8与苯环取代的苯甲醛反应，生成希夫碱11，11经硼氢化钠还原生成仲氨12，Boc保护产物13转化为羟肟酸14，最后N脱去保护生成目标化合物（L30-L33）；Synthetic route 2: Intermediate 8 reacts with benzaldehyde substituted with benzene ring to generate Schiff base 11, which is reduced by sodium borohydride to generate secondary ammonia 12, Boc protected product 13 is converted to hydroxamic acid 14, and finally N is deprotected to generate Target compounds (L30-L33);
合成路线2反应式如下：Synthetic route 2 reaction formula is as follows:
其中R为氢；卤素原子，硝基，羧基等吸电基团；烷基，氨基，羟基，羟甲基，胺甲基，氨甲酰基等给电基团；Where R is hydrogen; halogen atom, nitro, carboxyl and other charge-absorbing groups; alkyl, amino, hydroxyl, hydroxymethyl, aminomethyl, carbamoyl and other charge-donating groups;
上述合成路线反应式中的试剂:(a)苯甲醛，三乙胺,CH3OH;(b)NaBH4，无水CH3OH，85%;(c)(Boc)2O，三乙胺，CH3OH，65%;(d)NH2OK，无水CH3OH，30-40%;(e)AcOEt/HCl，70%。Reagents in the above synthetic route reaction formula: (a) benzaldehyde, triethylamine, CH3 OH; (b) NaBH4 , anhydrous CH3 OH, 85%; (c) (Boc)2 O, triethylamine , CH3 OH, 65%; (d) NH2 OK, anhydrous CH3 OH, 30-40%; (e) AcOEt/HCl, 70%.
合成路线2的目标化合物的结构式如下所示：The structural formula of the target compound of synthetic route 2 is as follows:
合成路线3：中间体4吲哚环上的N甲基保护产物16与4香豆酸甲酯反应，生成中间体17，17经过脱Boc，酰胺缩合，最终生成羟肟酸终产物；Synthetic route 3: The N-methyl protected product 16 on the indole ring of the intermediate 4 reacts with methyl 4 coumarate to generate the intermediate 17, which undergoes de-Boc and amide condensation to finally generate the hydroxamic acid final product;
合成路线3反应式如下：Synthetic route 3 reaction formula is as follows:
其中R是氢，各种氨基酸制备的酰基，芳酰基，杂芳酰基，芳基C1-6烷酰基，杂芳基C1-9烷酰基，C1-6烷酰基，环烷酰基，芳磺酰基，杂磺酰基，芳基C1-6烷磺酰基，杂芳基C1-9烷磺酰基，C1-8烷氧羰基或芳基C1-8烷氧羰基；Where R is hydrogen, acyl, aroyl, heteroaroyl, aryl C1-6 alkanoyl, heteroaryl C1-9 alkanoyl, C1-6 alkanoyl, cycloalkanoyl, arylsulfonyl prepared from various amino acids, Heterosulfonyl, aryl C1-6 alkanesulfonyl, heteroaryl C1-9 alkanesulfonyl, C1-8 alkoxycarbonyl or aryl C1-8 alkoxycarbonyl;
上述合成路线反应式中的试剂：（a）CH3I，KOH，TBABr，H2O，THF；（b）PPh3，DEAD，无水THF；(c)EtOAc的饱和HCl溶液；(d)TBTU，三乙胺，无水四氢呋喃；(e)NH2OK，无水CH3OH。Reagents in the above synthetic route reaction formula: (a) CH3 I, KOH, TBABr, H2 O, THF; (b) PPh3 , DEAD, anhydrous THF; (c) saturated HCl solution of EtOAc; (d) TBTU, triethylamine, anhydrous THF; (e)NH2OK , anhydrous CH3OH.
所述化合物的具体操作步骤在实施例中将加以详细说明。The specific operation steps of the compound will be described in detail in the examples.
本领域技术人员可以对上述步骤进行变动以提高收率，他们可据本领域的基本知识确定合成的路线，如选择反应物，溶剂和温度，可以通过使用各种常规保护基以避免副反应的发生从而提高收率。这些常规的保护方法可参见例如T.Greene,Protecting Groups inOrganic Synthesis.Those skilled in the art can change the above-mentioned steps to improve the yield. They can determine the synthetic route according to the basic knowledge in the art, such as selecting reactants, solvent and temperature, and can avoid side reactions by using various conventional protecting groups. occur thereby increasing the yield. These conventional protection methods can be found, for example, in T. Greene, Protecting Groups in Organic Synthesis.
三、含有本发明化合物的药物组合物及应用3. Pharmaceutical compositions and applications containing the compounds of the present invention
本发明还提供了这些化合物在预防或治疗与组蛋白去乙酰化酶活性异常表达相关的哺乳动物疾病的药物中的应用。所述的与组蛋白去乙酰化酶活性异常表达的相关哺乳动物疾病包括：癌症，神经变性疾病，病毒感染，炎症，疟疾和糖尿病等。因此，本发明还涉及含有（I）结构化合物的药物组合物。The present invention also provides the application of these compounds in medicines for preventing or treating mammalian diseases related to abnormal expression of histone deacetylase activity. The mammalian diseases related to abnormal expression of histone deacetylase activity include: cancer, neurodegenerative disease, virus infection, inflammation, malaria and diabetes, etc. Therefore, the present invention also relates to pharmaceutical compositions containing the compound of structure (I).
此外，本发明还包括一种适于口服给予哺乳动物的药物组合物，包含上述通式（I）的任一化合物，和药学上可接受载体，任选包含一种或多种药学上可接受的赋形剂。In addition, the present invention also includes a pharmaceutical composition suitable for oral administration to mammals, comprising any compound of the above general formula (I), and a pharmaceutically acceptable carrier, optionally comprising one or more pharmaceutically acceptable excipients.
此外，本发明还包括一种适于胃肠外给予哺乳动物的药物组合物，包含上述通式（I）的任一化合物，和药学上可接受载体，任选包含一种或多种药学上可接受的赋形剂。In addition, the present invention also includes a pharmaceutical composition suitable for parenteral administration to mammals, comprising any compound of the above general formula (I), and a pharmaceutically acceptable carrier, optionally comprising one or more pharmaceutically acceptable excipients.
发明详述Detailed description of the invention
所用的定义和术语Definitions and terms used
本文中所用的术语和定义含义如下：The terms and definitions used in this document have the following meanings:
“各种氨基酸制备的酰基”是指将各种氨基酸的羧基经酰化后得到的基团，优选疏水性氨基酸，如甘氨酸，丙氨酸，缬氨酸，亮氨酸，异亮氨酸，苯丙氨酸，脯氨酸，丝氨酸，苏氨酸，甲硫氨酸。"Acyl groups prepared from various amino acids" refer to groups obtained by acylation of carboxyl groups of various amino acids, preferably hydrophobic amino acids, such as glycine, alanine, valine, leucine, isoleucine, Phenylalanine, Proline, Serine, Threonine, Methionine.
“芳酰基”是指芳香族碳环末端连有羰基的基团，优选的芳环含有6-10个碳原子。"Aroyl" refers to a group having a carbonyl group at the end of an aromatic carbocyclic ring, preferably the aromatic ring contains 6-10 carbon atoms.
“杂芳酰基”指芳族杂环末端连有羰基的基团，可以是单环或双环基团。较佳的杂芳基包括噻吩基，呋喃基，吡咯基，吡啶基，吡嗪基，噻唑基，嘧啶基，喹啉基以及四氮唑基，苯并噻唑基，苯并呋喃基，吲哚基等。"Heteroaroyl" refers to a group with a carbonyl group attached to the end of an aromatic heterocycle, which may be a monocyclic or bicyclic group. Preferred heteroaryl groups include thienyl, furyl, pyrrolyl, pyridyl, pyrazinyl, thiazolyl, pyrimidinyl, quinolinyl and tetrazolyl, benzothiazolyl, benzofuryl, indole Base etc.
“环烷酰基”指取代或为取代的，饱和或不饱和的环状末端连有羰基的基团，其含有碳原子和/或一个或多个杂原子。该环可以是单环或稠环，桥环或螺环的环系。单环通常有3-9个原子，优选有4-7个原子，多环含有7-17个原子，优选含有7-13个原子。"Cycloalkanoyl" refers to a substituted or substituted, saturated or unsaturated cyclic terminus with a carbonyl group, which contains carbon atoms and/or one or more heteroatoms. The ring may be a monocyclic or fused, bridged or spiro ring system. Monocyclic rings generally contain 3-9 atoms, preferably 4-7 atoms, and polycyclic rings contain 7-17 atoms, preferably 7-13 atoms.
“药学上可接受的盐”是指式(II)化合物具有疗效且无毒的盐形式。其可由任一酸性基团(如羧基)形成阴离子盐，或由任一碱性基团(如氨基)形成阳离子盐。本领域已知许多这样的盐。在任何酸性基团(如羧基)上形成的阳离子盐，或是在任何碱性基团(如氨基)上形成的阴离子盐。这些盐有许多是本领域已知的，如阳离子盐包括碱金属(如钠和钾)和碱土金属(如镁和钙)的盐以及有机盐(如铵盐)。还可通过使用相应的酸处理碱性形式的(II)方便地获得阴离子盐，这样的酸包括无机酸如硫酸、硝酸、磷酸等；或有机酸如乙酸、丙酸、羟基乙酸、2-羟基丙酸、2-氧代丙酸、草酸、丙二酸、琥珀酸、马来酸、富马酸、苹果酸、酒石酸、2-羟基-1,2,3-丙三酸、甲磺酸、乙磺酸、苯甲磺酸、4-甲基苯磺酸、环己基亚磺酸、2-羟基苯甲酸、4-氨基-2-羟基苯甲酸等。这些盐是熟练技术人员熟知的，熟练的技术人员可制备本领域知识所提供的任何盐。此外，熟练技术人员可根据溶解度、稳定性、容易制剂等因素取某种盐而舍另一种盐。这些盐的测定和最优化在熟练技术人员的经验范围内。"Pharmaceutically acceptable salt" refers to a curative and non-toxic salt form of the compound of formula (II). It can form an anionic salt from any acidic group (such as carboxyl group), or form a cationic salt from any basic group (such as amino group). Many such salts are known in the art. A cationic salt formed on any acidic group such as carboxyl, or an anionic salt formed on any basic group such as amino. Many such salts are known in the art, eg cationic salts including alkali metal (such as sodium and potassium) and alkaline earth metal (such as magnesium and calcium) salts and organic salts (such as ammonium salts). Anionic salts are also conveniently obtained by treating the basic form of (II) with the corresponding acid, such acids include mineral acids such as sulfuric acid, nitric acid, phosphoric acid, etc.; or organic acids such as acetic acid, propionic acid, glycolic acid, 2-hydroxy Propionic acid, 2-oxopropionic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, 2-hydroxy-1,2,3-propanetriic acid, methanesulfonic acid, Ethylsulfonic acid, benzenemethanesulfonic acid, 4-methylbenzenesulfonic acid, cyclohexylsulfinic acid, 2-hydroxybenzoic acid, 4-amino-2-hydroxybenzoic acid, etc. Such salts are well known to the skilled artisan and the skilled artisan can prepare any salt given by the knowledge in the art. Furthermore, the skilled artisan may prefer one salt to another based on factors such as solubility, stability, ease of formulation, and the like. Determination and optimization of these salts are within the experience of the skilled artisan.
式(I)化合物还可以是其他被保护的形式或衍生物的形式存在，这些形式对本领域技术人员而言是显而易见的，均应该包含于本发明的范围内。The compound of formula (I) can also exist in other protected forms or derivative forms, which are obvious to those skilled in the art, and should be included within the scope of the present invention.
如上所述的取代基自身还可被一个或多个取代基取代，优选的取代基包括，例如烷基，烯基，烷氧基，羟基，氧基，硝基，氨基，氨基烷基(如氨甲基等)，氰基，卤，羧基，羰基烷氧基(如羰基乙氧基等)，硫基，芳基，环烷基，杂芳基，杂环烷基(如哌啶基，吗啉基，吡咯基等)，亚氨基，羟烷基，芳基氧基，芳基烷基，及其结合。The substituents mentioned above may themselves be substituted by one or more substituents, preferred substituents include, for example, alkyl, alkenyl, alkoxy, hydroxy, oxy, nitro, amino, aminoalkyl (such as Aminomethyl, etc.), cyano, halogen, carboxyl, carbonylalkoxy (such as carbonylethoxy, etc.), thio, aryl, cycloalkyl, heteroaryl, heterocycloalkyl (such as piperidinyl, morpholinyl, pyrrolyl, etc.), imino, hydroxyalkyl, aryloxy, arylalkyl, and combinations thereof.
对化合物的抑酶活性的评价，我们以hela细胞提取物（含HDAC1,2,3,8的混合酶），用HDACs荧光分析方法进行测试，分为两步，第一步，HDAC荧光底物（含一个乙酰化的赖氨酸侧链-Boc-Lys（acetyl）-AMC）用含HDAC活性的样本孵育（如Hela细胞核提取液,表达的HDAC8等），使底物去乙酰基化，激活底物。第二步，用胰酶水解Boc-Lys-AMC，产生AMC这一荧光基团（或发色团），在发射波长/激发波长（390nm/460nm）测定荧光强度。见下面的反应式III：For the evaluation of the anti-enzyme activity of the compound, we use the hela cell extract (mixed enzymes containing HDAC1, 2, 3, 8) to test with the HDACs fluorescence analysis method, which is divided into two steps. The first step is the HDAC fluorescent substrate (Containing an acetylated lysine side chain-Boc-Lys(acetyl)-AMC) Incubate with samples containing HDAC activity (such as Hela cell nucleus extract, expressed HDAC8, etc.) to deacetylate the substrate and activate substrate. In the second step, trypsin is used to hydrolyze Boc-Lys-AMC to generate AMC as a fluorescent group (or chromophore), and the fluorescence intensity is measured at the emission wavelength/excitation wavelength (390nm/460nm). See Reaction III below:
反应式III中Histone deacetylase为组蛋白去乙酰化酶，Trypsin为胰蛋白酶，4-amino-7-methylcoumarin为4-氨基-7-甲基香豆素。In Reaction Formula III, Histone deacetylase is histone deacetylase, Trypsin is trypsin, and 4-amino-7-methylcoumarin is 4-amino-7-methylcoumarin.
化合物的体外抗肿瘤细胞活性的测试我们采用MTT比色法，MTT全称为3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide，汉语化学名为3-(4,5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐，商品名为噻唑蓝。是一种黄颜色的染料。MTT比色法是一种检测细胞存活和生长的方法。其检测原理为活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT还原为水不溶性的蓝紫色结晶甲臜(Formazan)并沉积在细胞中，而死细胞无此功能。二甲基亚砜(DMSO)能溶解细胞中的甲瓒，用酶标仪在570nm波长处测定其光吸收值 可间接反映活细胞数量。在一定细胞数范围内，MTT结晶形成的量与细胞数成正比，进而测得目标化合物对肿瘤细胞的抑制率。We used the MTT colorimetric method to test the anti-tumor cell activity of the compound in vitro. The full name of MTT is 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenyltetrazolium romide, and the Chinese chemical name is 3-( 4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide salt, trade name is thiazolium blue. It is a yellow dye. MTT colorimetry is a method to detect cell survival and growth. The detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystal formazan (Formazan) and deposit in the cells, while dead cells have no such function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value is measured at a wavelength of 570nm with a microplate reader, which can indirectly reflect the number of living cells. Within a certain cell number range, the amount of MTT crystal formation is proportional to the cell number, and then the inhibitory rate of the target compound on tumor cells is measured.
目标化合物体内抗肿瘤细胞活性采用裸鼠异种移植模型。将裸鼠皮下接种肿瘤细胞，给药20天，测量肿瘤体积，绘制肿瘤曲线，称出肿瘤重量并计算抑瘤率和相对肿瘤增殖率The in vivo anti-tumor cell activity of the target compound was determined by nude mouse xenograft model. Inoculate nude mice with tumor cells subcutaneously, administer the drug for 20 days, measure the tumor volume, draw a tumor curve, weigh the tumor weight and calculate the tumor inhibition rate and relative tumor proliferation rate
相对肿瘤体积（RTV）=V t/V oRelative tumor volume (RTV) = V t/V o
抗肿瘤活性的评价指标为相对肿瘤增殖率T/C（%），The evaluation index of anti-tumor activity is the relative tumor proliferation rate T/C (%),
附图说明Description of drawings
图1为裸鼠肿瘤体积曲线图。Figure 1 is a graph of tumor volume in nude mice.
图2为U937裸鼠肿瘤重量对比图。Figure 2 is a comparison chart of tumor weight in U937 nude mice.
图3为U937肿瘤组织照片。Figure 3 is a photo of U937 tumor tissue.
具体实施方式detailed description
下面结合实施例对本发明做进一步的说明，但不限于此。The present invention will be further described below in conjunction with the examples, but not limited thereto.
实施例1：L1-L30的合成，以L7为例。Example 1: Synthesis of L1-L30, taking L7 as an example.
1)色氨酸甲酯盐酸盐的制备（2）1) Preparation of tryptophan methyl ester hydrochloride (2)
将色氨酸（20.4g,100mmol）溶于甲醇（200mL）中，冰浴下滴加乙酰氯（24g,300mmol），加完后继续冰浴0.5h，75℃加热回流5h，TLC检测反应完全。减压蒸除大部分甲醇，再加少量甲醇减压蒸除（将HCl蒸除），重复2-3次。加入无水乙醚，抽滤得白色固体2粗产品。Dissolve tryptophan (20.4g, 100mmol) in methanol (200mL), add acetyl chloride (24g, 300mmol) dropwise under ice-cooling, continue ice-bathing for 0.5h after the addition, heat and reflux at 75°C for 5h, TLC detects that the reaction is complete . Most of the methanol was evaporated under reduced pressure, and a small amount of methanol was evaporated under reduced pressure (to remove HCl), and repeated 2-3 times. Anhydrous diethyl ether was added, and the crude product 2 was obtained by suction filtration as a white solid.
2)(S)-2-((叔丁氧羰基)氨基)-3-(1H-吲哚-3-基)丙酸甲酯的制备（3）2) Preparation of (S)-2-((tert-butoxycarbonyl)amino)-3-(1H-indol-3-yl)propionic acid methyl ester (3)
将上一步粗产品2，加入300mL DCM中，再加入TEA（30g,300mmol）使其溶解。将(BOC)2O（26.2g,120mmol）溶于DCM中，然后将(BOC)2O的DCM溶液逐次加入反应液中，反应4-5h，TLC监测反应。反应液用1M柠檬酸，饱和NaHCO3(3×100ml)，饱和NaCl溶液(3×100ml)洗涤。有机相用无水MgSO4干燥过夜，抽滤，减压蒸除溶剂，得到粗产物3，然后用石油醚少量DCM洗涤滤饼得产物3，为白色固体25.8g(81mmol,81%)Mp138-139℃。3)(S)-2-((叔丁氧羰基)氨基)-3-(1H-吲哚-3-基)丙醇的制备（4）The crude product 2 from the previous step was added to 300 mL of DCM, and TEA (30 g, 300 mmol) was added to dissolve it. (BOC)2 O (26.2 g, 120 mmol) was dissolved in DCM, and then the DCM solution of (BOC) 2 O was gradually added to the reaction liquid, and reacted for 4-5 h, and the reaction was monitored by TLC. The reaction solution was washed with 1M citric acid, saturated NaHCO3 (3×100ml), saturated NaCl solution (3×100ml). The organic phase was dried overnight with anhydrous MgSO4 , suction filtered, and the solvent was evaporated under reduced pressure to obtain the crude product 3, and then the filter cake was washed with a small amount of petroleum ether DCM to obtain the product 3 as a white solid 25.8g (81mmol, 81%) Mp138- 139°C. 3) Preparation of (S)-2-((tert-butoxycarbonyl)amino)-3-(1H-indol-3-yl)propanol (4)
将中间体3（9.5g,30mmol）加入无水THF中，冰浴下逐次加入LiAIH4（2.25g,60mmol），室温反应3-4h，TLC监测反应。反应完全后加入冰水猝灭，减压蒸除THF，用EtOAc（200mL×2）提取，有机相用饱和NaHCO3(3×100mL)，饱和NaCl溶液(3×100mL)洗涤，无水MgSO4干燥，抽滤，减压蒸除溶剂，得到产物4，为淡黄色固体7.5g(25.8mmol,86%)。1H-NMR(600MHz,DMSO-d6):δ1.52(m,9H),δ2.68-2.89(m,2H),δ3.28-3.37(m,2H),δ3.65(m,1H),4.62(t,J=5.4,1H),7.08(s,1H),6.50-7.30(m,4H),7.57(d,J=7.8,1H),δ10.7(s,1H)。Intermediate 3 (9.5g, 30mmol) was added to anhydrous THF, and LiAIH4 (2.25g, 60mmol) was added gradually under ice-cooling, and reacted at room temperature for 3-4h, and the reaction was monitored by TLC. After the reaction was complete, add ice water to quench, evaporate THF under reduced pressure, extract with EtOAc (200mL×2), wash the organic phase with saturated NaHCO3 (3×100mL), saturated NaCl solution (3×100mL), and anhydrous MgSO4 After drying, suction filtration, and evaporation of the solvent under reduced pressure, the product 4 was obtained as 7.5 g (25.8 mmol, 86%) of light yellow solid.1 H-NMR(600MHz,DMSO-d6 ):δ1.52(m,9H),δ2.68-2.89(m,2H),δ3.28-3.37(m,2H),δ3.65(m, 1H), 4.62(t, J=5.4, 1H), 7.08(s, 1H), 6.50-7.30(m, 4H), 7.57(d, J=7.8, 1H), δ10.7(s, 1H).
4)4-羟基肉桂酸甲酯的制备(6)4) Preparation of methyl 4-hydroxycinnamate (6)
将4-羟基肉桂酸（16.4g,100mmol）溶于甲醇（200mL）中，冰浴下滴加乙酰氯（24g,300mmol），75℃加热回流5h，TLC检测反应完全。减压蒸除甲醇，产物溶于EtOAc（300mL），用饱和NaHCO3溶液（2×100mL），1M柠檬酸溶液（2×100mL），饱和NaCl溶液(2×100mL)。有机相用无水MgSO4干燥，抽滤，减压除掉溶剂，得产物6，为白色固体15.6g(87.5mmol,87.5%)。Mp140-142℃。1H NMR(300MHz,DMSO-d6)δ3.69(s,3H),6.43(d,J=16.2Hz,1H),6.81(d,J=8.7Hz,2H),7.54-7.59(m,3H),10.01(s,1H).ESI-MS m/z:179.2[M+H]+。4-Hydroxycinnamic acid (16.4g, 100mmol) was dissolved in methanol (200mL), and acetyl chloride (24g, 300mmol) was added dropwise under ice-cooling, heated to reflux at 75°C for 5h, and the reaction was complete as detected by TLC. Methanol was distilled off under reduced pressure, the product was dissolved in EtOAc (300mL), saturated NaHCO3 solution (2×100mL), 1M citric acid solution (2×100mL), saturated NaCl solution (2×100mL). The organic phase was dried with anhydrous MgSO4 , filtered with suction, and the solvent was removed under reduced pressure to obtain 15.6 g (87.5 mmol, 87.5%) of product 6 as a white solid. Mp140-142°C.1 H NMR (300MHz,DMSO-d6 )δ3.69(s,3H),6.43(d,J=16.2Hz,1H),6.81(d,J=8.7Hz,2H),7.54-7.59(m, 3H), 10.01(s, 1H). ESI-MS m/z: 179.2[M+H]+ .
5)(S,E)-3-(4-(2-((叔丁氧羰基)氨基)-3-(1H-吲哚-3-基)丙氧基)苯基)丙烯酸甲酯的制备（7）5) Preparation of (S,E)-3-(4-(2-((tert-butoxycarbonyl)amino)-3-(1H-indol-3-yl)propoxy)phenyl)methyl acrylate (7)
将4（5.8g,20mmol），6（4.2g,24mmol），PPh3（7.9g,30mmol）溶于THF，冰浴下滴加DEAD（5.2g,30mmol），反应1-2h，TLC监测反应。蒸除THF，flash柱层析纯化，得产物7，为白色固体5.4g(12mmol,60%)。Mp109-110℃。1H NMR(300MHz,DMSO-d6)δ1.36(s,9H),2.85-3.00(m,2H),3.70(s,3H),3.95-4.07(m,3H),6.51(d,J=15.9Hz,1H),6.93-6.99(m,4H),7.08(t,J=7.4Hz,1H),7.13(d,J=2.1Hz,1H),7.34(d,J=7.8Hz,1H),7.56(d,J=7.8Hz,1H),7.63(d,J=15.9Hz,1H),7.66(d,J=8.4Hz,2H),10.82(d,J=1.5Hz,1H).ESI-MS m/z:451.5[M+H]+。Dissolve 4 (5.8g, 20mmol), 6 (4.2g, 24mmol), PPh3 (7.9g, 30mmol) in THF, add DEAD (5.2g, 30mmol) dropwise under ice bath, react for 1-2h, monitor the reaction by TLC . THF was evaporated and purified by flash column chromatography to obtain product 7 as 5.4 g (12 mmol, 60%) of white solid. Mp109-110°C.1 H NMR (300MHz,DMSO-d6 )δ1.36(s,9H),2.85-3.00(m,2H),3.70(s,3H),3.95-4.07(m,3H),6.51(d,J =15.9Hz,1H),6.93-6.99(m,4H),7.08(t,J=7.4Hz,1H),7.13(d,J=2.1Hz,1H),7.34(d,J=7.8Hz,1H ),7.56(d,J=7.8Hz,1H),7.63(d,J=15.9Hz,1H),7.66(d,J=8.4Hz,2H),10.82(d,J=1.5Hz,1H). ESI-MS m/z: 451.5 [M+H]+ .
6)(S,E)-3-(4-(2-氨基-3-(1H-吲哚-3-基)丙氧基)苯基)丙烯酸甲酯盐酸盐的制备（8）6) Preparation of (S,E)-3-(4-(2-amino-3-(1H-indol-3-yl)propoxy)phenyl)methyl acrylate hydrochloride (8)
将7溶于EtOAc的饱和HCl（50mL）溶液中，反应过夜，析出固体，过滤，得到产物8，为白色固体3.8g（10mmol,83%）。Mp220-222℃。δ3.10-3.24(m,2H),3.71(s,3H),3.71-3.75(m,1H),4.07(dd,J=5.7Hz,J=10.5Hz,1H),4.21(dd,J=5.7Hz,J=10.5Hz,1H),6.53(d,J=15.9Hz,1H),6.97-7.01(m,3H),7.11(t,J=6.9Hz,1H),7.27(d,J=2.4Hz,1H),7.38(d,J=8.1Hz,1H),7.59-7.64(m,2H),7.70(d,J=8.7Hz,2H),8.40(s,2H),11.05(s,1H).ESI-MS m/z:351.5[M+H]+。7 was dissolved in EtOAc in saturated HCl (50 mL) and reacted overnight, a solid precipitated out and was filtered to give product 8 as a white solid 3.8 g (10 mmol, 83%). Mp220-222°C. δ3.10-3.24(m,2H),3.71(s,3H),3.71-3.75(m,1H),4.07(dd,J=5.7Hz,J=10.5Hz,1H),4.21(dd,J= 5.7Hz,J=10.5Hz,1H),6.53(d,J=15.9Hz,1H),6.97-7.01(m,3H),7.11(t,J=6.9Hz,1H),7.27(d,J= 2.4Hz,1H),7.38(d,J=8.1Hz,1H),7.59-7.64(m,2H),7.70(d,J=8.7Hz,2H),8.40(s,2H),11.05(s, 1H). ESI-MS m/z: 351.5 [M+H]+ .
7)(S,E)-3-(4-(2-(4-氯苯甲酰胺基))-3-(1H-吲哚-3-基)丙氧基)苯基)丙烯酸甲酯的制备（9）7) (S,E)-3-(4-(2-(4-chlorobenzamido))-3-(1H-indol-3-yl)propoxy)phenyl)methyl acrylate preparation (9)
将4-氯苯甲酸（0.31g，2mmol)溶于THF中，冰浴下加入TBTU（0.71g，2.2mmol）和TEA（0.3g，3mmol）。0.5h后加入化合物8（0.85g，2.2mmol），室温反应过夜，蒸除THF，产物溶于DCM中，1M盐酸（2×100mL）饱和Na2CO3(2×100mL)，饱和NaCl溶液(2×100mL)洗涤，无水MgSO4干燥过夜，抽滤，减压除掉溶剂，乙酸乙酯石油醚重结晶得到产物9,为白色固体0.5g（1.1mmol,51%）。4-Chlorobenzoic acid (0.31g, 2mmol) was dissolved in THF, and TBTU (0.71g, 2.2mmol) and TEA (0.3g, 3mmol) were added under ice cooling. After 0.5h, compound 8 (0.85g, 2.2mmol) was added, reacted at room temperature overnight, THF was distilled off, the product was dissolved in DCM, 1M hydrochloric acid (2×100mL) was saturated with Na2 CO3 (2×100mL), saturated NaCl solution ( 2 × 100 mL), dried over anhydrous MgSO4 overnight, filtered with suction, removed the solvent under reduced pressure, and recrystallized from ethyl acetate petroleum ether to obtain product 9 as a white solid 0.5 g (1.1 mmol, 51%).
8)(S,E)-3-(4-(2-(4-氯苯甲酰胺基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺的制备（L7）8) (S,E)-3-(4-(2-(4-chlorobenzamido)-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxypropene Preparation of amides (L7)
将9（0.5g，1.1mmol）溶于NH2OK的甲醇溶液（10mL）中，室温反应2h，TLC监测反应。减压蒸除甲醇，加入1M盐酸调节PH至酸性，EtOAc（2×30mL）萃取，饱和NaCl溶液（3×100mL）洗涤，无水Na2SO4干燥，抽滤，减压除掉溶剂，flash柱层析进行纯化，得到目标产物L7。白色固体0.12g(0.88mmol，24%)。Mp182-184℃。1H NMR(300MHz,DMSO-d6)δ3.11(d,J=6.6Hz,2H),4.06-4.11(m,1H),4.14-4.20(m,1H),4.54-4.61(m,1H),6.32(d,J=15.6Hz,1H),6.93-6.98(m,3H),7.08(t,J=6.9Hz,1H),7.18(d,J=2.1Hz,1H),7.34(d,J=7.8Hz,1H),7.42(d,J=15.9Hz,1H),7.49(d,J=8.4Hz,2H),7.55(d,J=8.7Hz,2H),7.63(d,J=7.8Hz,1H),7.88(d,J=8.7Hz,2H),8.63(d,J=7.8Hz,1H),8.98(s,1H),10.66(s,1H),10.83(d,J=1.5Hz,1H).HRMS(AP-ESI)m/z calcd for C27H24ClN3O4[M+H]+490.1526,found490.1528。9 (0.5 g, 1.1 mmol) was dissolved in NH2 OK methanol solution (10 mL), reacted at room temperature for 2 h, and the reaction was monitored by TLC. Evaporate methanol under reduced pressure, add 1M hydrochloric acid to adjust the pH to acidic, extract with EtOAc (2×30mL), wash with saturated NaCl solution (3×100mL), dry with anhydrous Na2 SO4 , filter with suction, remove the solvent under reduced pressure, flash Purified by column chromatography to obtain the target product L7. White solid 0.12g (0.88mmol, 24%). Mp182-184°C.1 H NMR (300MHz,DMSO-d6 )δ3.11(d,J=6.6Hz,2H),4.06-4.11(m,1H),4.14-4.20(m,1H),4.54-4.61(m,1H ),6.32(d,J=15.6Hz,1H),6.93-6.98(m,3H),7.08(t,J=6.9Hz,1H),7.18(d,J=2.1Hz,1H),7.34(d ,J=7.8Hz,1H),7.42(d,J=15.9Hz,1H),7.49(d,J=8.4Hz,2H),7.55(d,J=8.7Hz,2H),7.63(d,J =7.8Hz,1H),7.88(d,J=8.7Hz,2H),8.63(d,J=7.8Hz,1H),8.98(s,1H),10.66(s,1H),10.83(d,J =1.5Hz,1H). HRMS (AP-ESI) m/z calcd for C27 H24 ClN3 O4 [M+H]+ 490.1526, found 490.1528.
实施例2：目标化合物L31-L34的合成，以L31为例。Example 2: Synthesis of target compounds L31-L34, taking L31 as an example.
1）(E)-3-(4-((S)-2-((Z)-苄亚甲基氨基)--3-(1H-吲哚-3-基)丙氧基)苯基)丙烯酸甲酯的制备。（11）1) (E)-3-(4-((S)-2-((Z)-benzylideneamino)--3-(1H-indol-3-yl)propoxy)phenyl) Preparation of methyl acrylate. (11)
将8（0.76g，2mmol）溶于甲醇中，加入TEA（0.22g，2.2mmol），后加入苯甲醛（0.21g，2mmol），室温反应2h后TLC监测，反应完全后蒸干溶剂，真空干燥箱干燥的产物11的粗品。Dissolve 8 (0.76g, 2mmol) in methanol, add TEA (0.22g, 2.2mmol), then add benzaldehyde (0.21g, 2mmol), react at room temperature for 2h, monitor by TLC, evaporate the solvent to dryness after the reaction is complete, and dry in vacuo Crude product 11 was oven dried.
2）(S,E)-3-(4-(2-(苄氨基))-3-(1H-吲哚-3-基)丙氧基)苯基)丙烯酸甲酯的制备。（12）2) Preparation of (S,E)-3-(4-(2-(benzylamino))-3-(1H-indol-3-yl)propoxy)phenyl)methyl acrylate. (12)
将上步所得溶于重蒸甲醇中，冰浴下加入NaBH4(0.15g，4mmol)，室温反应4h后TLC监测反应。反应完全后加入冰水猝灭，蒸除有机溶剂，加入EtOAc（3×30mL）萃取，无水Na2SO4干燥过夜。蒸除溶剂，得黄色油状物，即为产物12。1H NMR(300MHz,DMSO-d6)δ2.95(d,J=6.3Hz,2H),3.14-3.18(m,1H),3.70(s,3H),3.87(s,2H),3.94(d,J=5.1Hz,2H),6.50(d,J=15.9Hz,1H),6.92(t,J=7.5Hz,1H),6.95(d,J=8.7Hz,2H),7.06(t,J=7.4Hz,1H),7.14(d,J=2.4Hz,1H),7.16-7.22(m,1H),7.26-7.33(m,5H),7.43(d,J=7.8Hz,1H),7.63(d,J=15.9Hz,1H),7.65(d,J=8.7Hz,2H),8.99(s,1H),10.83(s,1H).ESI-MS m/z:441.5[M+H]+。The obtained product from the previous step was dissolved in redistilled methanol, NaBH4 (0.15 g, 4 mmol) was added under ice-cooling, and the reaction was monitored by TLC after reacting at room temperature for 4 h. After the reaction was complete, it was quenched by adding ice water, the organic solvent was evaporated, extracted by adding EtOAc (3×30 mL), and dried over anhydrous Na2 SO4 overnight. The solvent was distilled off to obtain a yellow oily substance, namely the product 12.1 H NMR (300MHz,DMSO-d6 )δ2.95(d,J=6.3Hz,2H),3.14-3.18(m,1H),3.70(s,3H),3.87(s,2H),3.94( d,J=5.1Hz,2H),6.50(d,J=15.9Hz,1H),6.92(t,J=7.5Hz,1H),6.95(d,J=8.7Hz,2H),7.06(t, J=7.4Hz,1H),7.14(d,J=2.4Hz,1H),7.16-7.22(m,1H),7.26-7.33(m,5H),7.43(d,J=7.8Hz,1H), 7.63(d,J=15.9Hz,1H),7.65(d,J=8.7Hz,2H),8.99(s,1H),10.83(s,1H).ESI-MS m/z:441.5[M+H ]+ .
3）(S,E)-3-(4-(2-(苄基(叔丁基羰基)氨基)-3-(1H-吲哚-3-基)丙氧基)苯基)丙烯酸甲3) (S,E)-3-(4-(2-(benzyl(tert-butylcarbonyl)amino)-3-(1H-indol-3-yl)propoxy)phenyl)methacrylate
酯的制备。（13）Preparation of esters. (13)
上步所得油状物溶于DCM，先后加入TEA（0.61g，6mmol）和(Boc)2O(0.87g),室温反应过夜，TLC监测反应，反应完全后反应液分别用1M柠檬酸溶液（2×100mL）,饱和NaHCO3溶液（2×100mL），饱和NaCl溶液（2×100mL）洗涤，无水Na2SO4干燥过夜，蒸干后得白色油状物，即为产物13。真空干燥箱干燥备下步使用。Dissolve the oil obtained in the previous step in DCM, add TEA (0.61g, 6mmol) and (Boc)2 O (0.87g) successively, react at room temperature overnight, monitor the reaction by TLC, after the reaction is complete, the reaction solution is respectively washed with 1M citric acid solution (2 ×100mL), washed with saturated NaHCO3 solution (2×100mL), saturated NaCl solution (2×100mL), dried overnight with anhydrous Na2 SO4 , and evaporated to dryness to obtain a white oily product, namely product 13. Dry in a vacuum oven for use in the next step.
4）(S,E)-3-(4-(2-(苄基(叔丁基羰基)氨基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺的制备。（14）4) (S,E)-3-(4-(2-(benzyl(tert-butylcarbonyl)amino)-3-(1H-indol-3-yl)propoxy)phenyl)-N- Preparation of hydroxyacrylamide. (14)
上步所得于真空干燥箱干燥过夜后溶于NH2OK的甲醇溶液（10ml）中，室温反应2h。减压蒸除甲醇，加入1M盐酸调节PH至酸性，EtOAc（2×30mL）萃取，饱和NaCl溶液（3×100mL）洗涤，无水Na2SO4干燥，抽滤，蒸干溶剂，真空干燥箱干燥过夜的白色固体，即为产物14。The obtained product in the previous step was dried overnight in a vacuum oven, then dissolved in NH2 OK methanol solution (10 ml), and reacted at room temperature for 2 h. Evaporate methanol under reduced pressure, add 1M hydrochloric acid to adjust the pH to acidic, extract with EtOAc (2×30mL), wash with saturated NaCl solution (3×100mL), dry with anhydrous Na2 SO4 , filter with suction, evaporate the solvent to dryness, and vacuum oven The white solid, product 14, dried overnight.
5）(S,E)-3-(4-(2-(苄基(叔丁基羰基)氨基)-3-(1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺盐酸盐的制备。（L31）5) (S,E)-3-(4-(2-(benzyl(tert-butylcarbonyl)amino)-3-(1H-indol-3-yl)propoxy)phenyl)-N- Preparation of Hydroxyacrylamide Hydrochloride. (L31)
将14溶于EtOAc的饱和HCl（10mL）溶液中，反应过夜，析出固体，过滤，得到产物L31，为白色固体0.32g（0.67mmol，34%）。Mp:136-138℃1H NMR(300MHz,DMSO-d6)δ3.17-3.27(m,1H),3.41-3.47(m,1H),3.67(s,1H),4.12-4.17(m,1H),4.25-4.27(m,1H),4.36(s,2H),6.38(d,J=15.6Hz,1H),6.91-6.99(m,3H),7.10(t,J=7.2Hz,1H),7.22(d,J=2.4Hz,1H),7.37(d,J=7.8Hz,2H),7.42-7.50(m,6H),7.64-7.68(m,2H),9.72(s,1H),9.92(s,1H),10.31(s,1H),11.05(d,J=1.8Hz,1H).HRMS(AP-ESI)m/z calcd for C27H27N3O3[M+H]+442.2125,found442.2125。14 was dissolved in EtOAc in saturated HCl (10 mL) and reacted overnight, a solid precipitated out and was filtered to give the product L31 as a white solid 0.32 g (0.67 mmol, 34%). Mp:136-138℃1 H NMR(300MHz,DMSO-d6 )δ3.17-3.27(m,1H),3.41-3.47(m,1H),3.67(s,1H),4.12-4.17(m, 1H),4.25-4.27(m,1H),4.36(s,2H),6.38(d,J=15.6Hz,1H),6.91-6.99(m,3H),7.10(t,J=7.2Hz,1H ),7.22(d,J=2.4Hz,1H),7.37(d,J=7.8Hz,2H),7.42-7.50(m,6H),7.64-7.68(m,2H),9.72(s,1H) ,9.92(s,1H),10.31(s,1H),11.05(d,J=1.8Hz,1H).HRMS(AP-ESI)m/z calcd for C27 H27 N3 O3 [M+H ]+ 442.2125, found 442.2125.
实施例3：目标化合物L36的合成Embodiment 3: the synthesis of target compound L36
1）(S)-2-((叔丁氧羰基)氨基)-3-(1-甲基-1H-吲哚-3-基)丙醇的制备（16）1) Preparation of (S)-2-((tert-butoxycarbonyl)amino)-3-(1-methyl-1H-indol-3-yl)propanol (16)
将化合物4（2.9g，10mmol）溶于50mLTHF中，依次加入四丁基溴化铵0.5g，CH3I（3.5g，25mmol），搅拌均匀后加入15mL50%的KOH溶液，反应过夜，TLC监测，待反应完全后反应液蒸除THF，加入EtOAc（50mL×2）萃取，，有机相用1M柠檬酸溶液（100mL×2），饱和NaHCO3溶液（2×100mL），饱和NaCl溶液（2×100mL）洗涤，无水Na2SO4干燥，抽滤，减压除掉溶剂，乙酸乙酯石油醚重结晶，得白色固体16，1.6g（5.4mmol，54%）。Dissolve compound 4 (2.9g, 10mmol) in 50mLTHF, add 0.5g tetrabutylammonium bromide, CH3 I (3.5g, 25mmol) successively, stir well, add 15mL 50% KOH solution, react overnight, monitor by TLC , After the reaction was complete, the reaction solution was evaporated to remove THF, and EtOAc (50mL×2) was added for extraction. The organic phase was extracted with 1M citric acid solution (100mL×2), saturated NaHCO3 solution (2×100mL), saturated NaCl solution (2× 100 mL), washed with anhydrous Na2 SO4 , filtered with suction, and the solvent was removed under reduced pressure, and recrystallized from ethyl acetate and petroleum ether to give 16 as a white solid, 1.6 g (5.4 mmol, 54%).
2）(S,E)-3-(4-(2-((叔丁氧羰基)氨基)-3-(1-甲基-1H-吲哚-3-基)丙氧基)苯基)丙烯酸甲酯的制备（17）2) (S,E)-3-(4-(2-((tert-butoxycarbonyl)amino)-3-(1-methyl-1H-indol-3-yl)propoxy)phenyl) Preparation of Methyl Acrylate (17)
将化合物16（1.5g，5mmol）,4-香豆酸甲酯（1.1g，6mmol），PPh3(2.0g，7.5mmol)溶于重蒸THF中，冰浴下滴加DEAD（1.3g，7.5mmol），室温反应，2h后TLC监测，反应完全后，flash柱层析纯化，得产物17，为白色固体1.4g（3mmol，60%）。Compound 16 (1.5g, 5mmol), methyl 4-coumarate (1.1g, 6mmol), PPh3 (2.0g, 7.5mmol) were dissolved in redistilled THF, DEAD (1.3g, 7.5 mmol), reacted at room temperature, monitored by TLC after 2 h, and purified by flash column chromatography to obtain product 17 as a white solid 1.4 g (3 mmol, 60%) after the reaction was complete.
3）(S,E)-3-(4-(2-((叔丁氧羰基)氨基)-3-(1-甲基-1H-吲哚-3-基)丙氧基)苯基)-N-羟基丙烯酰胺的制备（L35）3) (S,E)-3-(4-(2-((tert-butoxycarbonyl)amino)-3-(1-methyl-1H-indol-3-yl)propoxy)phenyl) - Preparation of N-hydroxyacrylamide (L35)
将17（0.46g，1mmol）溶于NH2OK的甲醇溶液（10mL）中，室温反应2h，TLC监测反应。减压蒸除甲醇，加入1M盐酸调节PH至酸性，EtOAc（2×30mL）萃取，饱和NaCl溶液（3×100mL）洗涤，无水Na2SO4干燥，抽滤，减压除掉溶剂，flash柱层析进行纯化，得到目标产物L35。白色固体0.13g(0.28mmol，28%)。Mp:146-148℃1H NMR(300MHz,DMSO-d6)δ1.36(s,9H),2.83-2.99(m,2H),3.72(s,3H),3.93-4.04(m,3H),6.33(dd,J=4.5Hz,J=15.9Hz,1H),6.95(d,J=8.4Hz,2H),6.97-7.02(m,2H),7.10-7.15(m,2H),7.36-7.42(m,2H),7.49(d,J=8.4Hz,2H),7.58(d,J=7.8Hz,1H),8.97(s,1H),10.66(s,1H).HRMS(AP-ESI)m/z calcd for C26H31N3O5[M+H]+466.2336,found466.2332。17 (0.46 g, 1 mmol) was dissolved in NH2 OK methanol solution (10 mL), reacted at room temperature for 2 h, and monitored by TLC. Evaporate methanol under reduced pressure, add 1M hydrochloric acid to adjust the pH to acidic, extract with EtOAc (2×30mL), wash with saturated NaCl solution (3×100mL), dry with anhydrous Na2 SO4 , filter with suction, remove the solvent under reduced pressure, flash Purified by column chromatography to obtain the target product L35. White solid 0.13g (0.28mmol, 28%). Mp:146-148℃1 H NMR(300MHz,DMSO-d6 )δ1.36(s,9H),2.83-2.99(m,2H),3.72(s,3H),3.93-4.04(m,3H) ,6.33(dd,J=4.5Hz,J=15.9Hz,1H),6.95(d,J=8.4Hz,2H),6.97-7.02(m,2H),7.10-7.15(m,2H),7.36- 7.42(m,2H),7.49(d,J=8.4Hz,2H),7.58(d,J=7.8Hz,1H),8.97(s,1H),10.66(s,1H).HRMS(AP-ESI ) m/z calcd for C26 H31 N3 O5 [M+H]+ 466.2336, found 466.2332.
实施例4化合物的体外抑酶活性的测试The test of the anti-enzyme activity of embodiment 4 compound in vitro
1.HDAC buffer:15mM Tris-HCl（PH8.0），250μM EDTA，250mM NaCl,10%甘油。(HDACbuffer的配制方法：首先配制1M的Tris-HCl储备液500mL，即1. HDAC buffer: 15mM Tris-HCl (PH8.0), 250μM EDTA, 250mM NaCl, 10% glycerol. (The preparation method of HDACbuffer: first prepare 500mL of 1M Tris-HCl stock solution, namely
1M*500mL*121.14g/mol=60.57g Tris溶于略少于500mL的蒸馏水中，然后用浓盐酸调节pH至8，再用蒸馏水补齐至500mL。取7.5mL1M的Tris-HCl储备液与250μM*500mL*292.25g/mol=0.03654Gedta,250mM*500mL*58.44g/mol=7.31g NaCl混合后加蒸馏水至450mL，再加入50mL甘油，即得500mLHDAC buffer。)1M*500mL*121.14g/mol=60.57g Tris was dissolved in slightly less than 500mL of distilled water, then adjusted to pH 8 with concentrated hydrochloric acid, and then made up to 500mL with distilled water. Take 7.5mL of 1M Tris-HCl stock solution and mix it with 250μM*500mL*292.25g/mol=0.03654Gedta, 250mM*500mL*58.44g/mol=7.31g NaCl, add distilled water to 450mL, and then add 50mL glycerol to obtain 500mL HDAC buffer . )
2.Trypsin solution：10mg/ml胰酶，50mM Tris-HCL（PH8.0）,100mM NaCl，2μM TSA（首先配制不含胰酶和TSA的储备液500mL，即取25mL1M的Tris-HCl储备液和100mM*500mL*58.44g/mol=2.92gNaCl混合后加蒸馏水至500mL，临用前再取适量的储备液加入相应量的胰酶和TSA）。2. Trypsin solution: 10mg/ml trypsin, 50mM Tris-HCL (PH8.0), 100mM NaCl, 2μM TSA (first prepare 500mL of stock solution without trypsin and TSA, that is, take 25mL of 1M Tris-HCl stock solution and 100mM*500mL*58.44g/mol=2.92gNaCl mixed, add distilled water to 500mL, take an appropriate amount of stock solution and add corresponding amount of pancreatin and TSA before use).
3.底物：DMSO溶解配成30mM的储液，用HDAC buffer稀释至300uM，使得DMSO含量为1%左右。3. Substrate: Dissolve DMSO to make a 30mM stock solution, dilute to 300uM with HDAC buffer, so that the DMSO content is about 1%.
4.酶液：Hela细胞提取物，按1:20用HDAC buffer稀释。4. Enzyme solution: Hela cell extract, diluted 1:20 with HDAC buffer.
5.步骤：5. Steps:
a）100%溶液的配制：50μL HDAC buffer与10μL酶液混合，5min后加入40μL底物在37℃下反应30min，然后加入100μL Trypsin solution终止上述反应，并在37℃下反应20min，于390nm/460nm测定荧光强度，即得100%吸收。用AMC作为标准品做标准曲线，计算酶活。a) Preparation of 100% solution: mix 50 μL HDAC buffer with 10 μL enzyme solution, add 40 μL substrate after 5 minutes and react at 37°C for 30 minutes, then add 100 μL Trypsin solution to terminate the above reaction, and react at 37°C for 20 minutes, and react at 390nm/ Measure the fluorescence intensity at 460nm, which means 100% absorption. Use AMC as a standard to make a standard curve and calculate the enzyme activity.
b）空白溶液的配制：60μL HDAC buffer加入40μL底物后在37℃下反应30min，然后加入100μL Trypsin solution，并在37℃下反应20min，于390nm/460nm测定荧光强度，即得空白吸收。b) Preparation of blank solution: add 40 μL substrate to 60 μL HDAC buffer and react at 37 °C for 30 min, then add 100 μL Trypsin solution and react at 37 °C for 20 min, measure the fluorescence intensity at 390 nm/460 nm to obtain the blank absorption.
6.药物抑制HDAC酶活的测定步骤：50μL含有药物的HDAC buffer与10μL酶液混合预先孵育5min，加入40μL底物后，在37℃下反应30min，然后加入100μL Trypsin solution终止上述反应，并在37℃下反应20min，于390nm/460nm测定荧光强度。6. Determination of drug-inhibited HDAC enzyme activity: 50 μL of drug-containing HDAC buffer was mixed with 10 μL of enzyme solution and incubated for 5 minutes in advance, after adding 40 μL of substrate, reacted at 37°C for 30 minutes, then added 100 μL of Trypsin solution to terminate the above reaction, and React at 37°C for 20 minutes, and measure the fluorescence intensity at 390nm/460nm.
最后将化合物的抑制率（%）和其相应浓度进行S曲线拟合，计算出IC50值。Finally, S-curve fitting was performed on the inhibitory rate (%) of the compound and its corresponding concentration, and the IC50 value was calculated.
本发明所述结构通式(I)所示的部分化合物和阳性对照药SAHA对组蛋白去乙酰化酶的抑制活性结果见下面表1：The results of the inhibitory activity of some compounds shown in the general structural formula (I) of the present invention and the positive control drug SAHA to histone deacetylase are shown in the following table 1:
表1部分化合物和阳性对照药SAHA对组蛋白去乙酰化酶的抑制活性结果Table 1 The results of the inhibitory activity of some compounds and the positive control drug SAHA on histone deacetylase
实验结果表明：化合物对Hela细胞提取物有明显的抑制作用，其中化合物L1,L2,L7,L20，L22的抑酶活性要远远好于阳性对照药SAHA。The experimental results show that the compounds have obvious inhibitory effect on Hela cell extracts, and the enzyme inhibitory activity of compounds L1, L2, L7, L20, and L22 is far better than that of the positive control drug SAHA.
实施例5化合物的体外抗肿瘤细胞活性的测试（MTT实验）The test (MTT experiment) of the in vitro anti-tumor cell activity of the compound of embodiment 5
1.实验材料1. Experimental materials
MTT，RPMI1640培养基，胎牛血清，96孔板，CO2恒温培养箱，美国BIO-RAD680型酶标仪人体乳腺癌细胞株（MDA-MB-231），前列腺癌细胞株（PC-3），人结肠癌细胞株（HCT116）,人类红白血病细胞株（K562），人红白血病细胞株（HEL）和急性原始粒细胞白血病细胞株(KG1)，阳性对照药SAHA。MTT, RPMI1640 medium, fetal bovine serum, 96-well plate, CO2 constant temperature incubator, American BIO-RAD680 microplate reader human breast cancer cell line (MDA-MB-231), prostate cancer cell line (PC-3) , human colon cancer cell line (HCT116), human erythroleukemia cell line (K562), human erythroleukemia cell line (HEL) and acute myeloid leukemia cell line (KG1), positive control drug SAHA.
2.实验步骤2. Experimental steps
（1）接种细胞，用含10%胎牛血清的培养液配成单个细胞悬液，以每孔5000个细胞接种到96孔板，每孔体积100uL，培养过夜。(1) Inoculate cells, prepare a single cell suspension with culture medium containing 10% fetal bovine serum, inoculate 5000 cells per well into a 96-well plate with a volume of 100uL per well, and culture overnight.
（2）化合物溶液的配制，在无菌台中，将化合物的DMSO储备液以培养液稀释成待测5个浓度，相邻浓度之间为两倍稀释。(2) Preparation of the compound solution. In a sterile bench, dilute the DMSO stock solution of the compound with the culture medium to 5 concentrations to be tested, and two-fold dilution between adjacent concentrations.
（3）将不同浓度的化合物溶液加入已经培养过夜的95孔板中，每孔中加入100μL，每个浓度加三个副孔。周围由于具有边缘效应，易染菌，因此不加细胞，不加化合物，而加100μL的培养液用作空白。另设置100%孔，即加入细胞和不含化合物的培养液100uL,在37℃恒温培养箱中孵育48h。(3) Add different concentrations of compound solutions into 95-well plates that have been cultured overnight, add 100 μL to each well, and add three auxiliary wells for each concentration. Due to the edge effect and easy contamination of bacteria, no cells or compounds were added, and 100 μL of culture solution was added as a blank. Another 100% well was set, that is, 100 uL of cells and compound-free culture solution were added, and incubated in a constant temperature incubator at 37°C for 48 hours.
（4）染色，向96孔板加入10μL MTT溶液（5mg/mL用PBS配制）染色，孵育4h后，2500rps离心10min，然后用排枪将培养液从孔中吸出，注意枪尖朝下不要将细胞吸出，加150μL DMSO，在振荡板震荡5-10min钟，使甲瓒充分溶解，用酶标仪测定570nm测定每孔的OD值。(4) For staining, add 10 μL of MTT solution (5mg/mL prepared in PBS) to the 96-well plate for staining, incubate for 4 hours, centrifuge at 2500rps for 10 minutes, and then use a row gun to suck out the culture solution from the well, pay attention to the tip of the gun not to drop the cells Aspirate, add 150 μL DMSO, shake on the shaking plate for 5-10 minutes to fully dissolve the formazan, measure the OD value of each well at 570 nm with a microplate reader.
部分化合物MTT实验结果如下表2所示：The results of MTT experiments for some compounds are shown in Table 2 below:
表2化合物MTT实验结果Table 2 Compound MTT experimental results
实验结果表明：化合物L1，L7，L9，L10,L12，L22，L31，L32在以上五株细胞中均有较强的体外抗增殖活性，其中化合物L7，L12抗增殖活性最为明显，IC50值远远小于SAHA。The experimental results show that compounds L1, L7, L9, L10, L12, L22, L31, and L32 all have strong anti-proliferation activities in the above five cell lines, among which compounds L7 and L12 have the most obvious anti-proliferation activities, with IC50 values Much smaller than SAHA.
实施例6目标化合物体内抗人白血病细胞U937细胞的活性Example 6 Anti-human leukemia cell U937 cell activity of the target compound in vivo
于裸鼠右肩皮下接种肿瘤细胞U937，每只100uL(细胞计数：1.8*108个/mL)，一周后开始分组给药，将荷瘤鼠分成3组，分别为对照组，SAHA组，待测组。给药剂量100mg/kg/d，给药体积：每次每只200uL/20g，给药方式；灌胃给药，以后每天给药一次，每隔两天测量肿瘤体积,取各组平均值，绘制肿瘤生长曲线（见附图1）,给药第十六天，处死裸鼠，解剖肿瘤及内脏，实验结束，称出瘤块的重量（见附图2和3），并按照公式计算抑瘤率。测量肿瘤最大径(a)和最小径(b)，计算肿瘤体积(V)：V=ab2/2，并计算相对肿瘤增殖率T/C（%）。Inoculate tumor cell U937 subcutaneously in the right shoulder of nude mice, each 100uL (cell count: 1.8*108 cells/mL), start administration in groups one week later, divide tumor-bearing mice into 3 groups, control group, SAHA group, and treat test group. Dosage: 100mg/kg/d, administration volume: 200uL/20g each time, administration method; intragastric administration, after that, administration once a day, measure tumor volume every two days, and take the average value of each group, Draw the tumor growth curve (see attached drawing 1). On the sixteenth day of administration, the nude mice were killed, and the tumor and internal organs were dissected. Tumor rate. The largest diameter (a) and smallest diameter (b) of the tumor were measured, the tumor volume (V) was calculated: V=ab2 /2, and the relative tumor proliferation rate T/C (%) was calculated.
相对肿瘤体积（RTV）=V t/V oRelative tumor volume (RTV) = V t/V o
抗肿瘤活性的评价指标为相对肿瘤增殖率T/C（%），The evaluation index of anti-tumor activity is the relative tumor proliferation rate T/C (%),
选取化合物L7进行体内抗肿瘤细胞活性的筛选，采用人白血病细胞U937异嫁接裸鼠进行实验，观察L7对荷瘤裸鼠的肿瘤抑制效果。如下表3所示：The compound L7 was selected for the screening of anti-tumor cell activity in vivo, and human leukemia cell U937 heterografted nude mice were used for experiments to observe the tumor inhibitory effect of L7 on tumor-bearing nude mice. As shown in Table 3 below:
表3对荷瘤裸鼠的肿瘤抑制效果Table 3 Tumor inhibitory effect on tumor-bearing nude mice
实验结果表明：在100mg/kg/d给药剂量下，L7能明显抑制肿瘤生长，抑瘤率分别为46.91%，阳性对照药SAHA也能明显抑制肿瘤细胞生长，抑瘤率36.24%。从抑瘤率结果中我们可以看出，L7化合物的抑瘤能力要略高于SAHA，也未发现明显的毒副作用。从一系列的活性筛选结果来看化合物L7在体内也具有很好的抗肿瘤活性，能够明显抑制白血病细胞生长，会是一个潜在的治疗白血病的HDAC抑制剂。The experimental results showed that: at a dose of 100mg/kg/d, L7 could significantly inhibit tumor growth, with a tumor inhibition rate of 46.91%, and the positive control drug SAHA could also significantly inhibit tumor cell growth, with a tumor inhibition rate of 36.24%. From the results of the tumor inhibition rate, we can see that the tumor inhibition ability of the L7 compound is slightly higher than that of SAHA, and no obvious toxic side effects have been found. From a series of activity screening results, it can be seen that compound L7 also has good anti-tumor activity in vivo, can significantly inhibit the growth of leukemia cells, and will be a potential HDAC inhibitor for the treatment of leukemia.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310452667.0ACN103467359B (en) | 2013-09-27 | 2013-09-27 | Cinnamon amides histone deacetylase inhibitor with benzpyrole and preparation method and application of same |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310452667.0ACN103467359B (en) | 2013-09-27 | 2013-09-27 | Cinnamon amides histone deacetylase inhibitor with benzpyrole and preparation method and application of same |
| Publication Number | Publication Date |
|---|---|
| CN103467359A CN103467359A (en) | 2013-12-25 |
| CN103467359Btrue CN103467359B (en) | 2015-04-22 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310452667.0AExpired - Fee RelatedCN103467359B (en) | 2013-09-27 | 2013-09-27 | Cinnamon amides histone deacetylase inhibitor with benzpyrole and preparation method and application of same |
| Country | Link |
|---|---|
| CN (1) | CN103467359B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105732459B (en)* | 2014-12-29 | 2019-05-31 | 成都先导药物开发有限公司 | Pyrrole amides class compound and preparation method thereof and purposes |
| CN105801464B (en) | 2014-12-29 | 2019-05-28 | 成都先导药物开发有限公司 | Pyrrole amides class compound and preparation method thereof and purposes |
| CN105732458B (en)* | 2014-12-29 | 2019-06-07 | 成都先导药物开发有限公司 | Pyrrole amides class compound and preparation method thereof and purposes |
| US9993459B2 (en)* | 2015-01-08 | 2018-06-12 | Universiteit Gent | Selective HDAC6 inhibitors and uses thereof |
| CN105198789B (en)* | 2015-10-26 | 2018-06-05 | 山东大学 | Substitute 3- indoles Bcl-2 protein inhibitors and preparation method and application |
| CN105367479A (en)* | 2015-11-16 | 2016-03-02 | 青岛大学 | Histone deacetylase inhibitor 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1 hydro-indole-3-yl-N-(2-(hydroxyamino)-2-ketoacyl) acetamide and preparation method and application thereof |
| CN105669520B (en)* | 2016-01-12 | 2018-10-30 | 山东大学 | O-phenylenediamine class selectivity histon deacetylase (HDAC) inhibitor containing tryptophan basic framework and its preparation method and application |
| CN106896076B (en)* | 2017-04-26 | 2019-08-20 | 江南大学 | A method for high-throughput detection of p-coumaric acid by ultraviolet spectroscopy |
| CN107141244B (en)* | 2017-05-08 | 2019-11-19 | 潍坊医学院 | Indole butyric acid histone deacetylase inhibitor and its preparation method and application |
| CN109879790B (en)* | 2017-12-06 | 2022-09-20 | 华东师范大学 | Amide micromolecule organic compound with indole or indole analogue as mother nucleus structure, application and preparation method thereof |
| CN116924959B (en)* | 2022-03-30 | 2025-08-19 | 山东大学 | HDAC11 subtype selective inhibitor and preparation method and application thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1450991A (en)* | 2000-09-01 | 2003-10-22 | 诺瓦提斯公司 | Hydroxamate derives useful as deacetylase inhibitors |
| CN101255124A (en)* | 2008-03-26 | 2008-09-03 | 山东大学 | Cinnamamide histone deacetylase inhibitor and preparation method thereof |
| WO2012106343A2 (en)* | 2011-02-01 | 2012-08-09 | The Board Of Trustees Of The University Of Illinois | Hdac inhibitors and therapeutic methods using the same |
| CN102746213A (en)* | 2012-05-18 | 2012-10-24 | 潍坊博创国际生物医药研究院 | Cinnamamide histone deacetylase inhibitor, preparation method thereof, and application thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1450991A (en)* | 2000-09-01 | 2003-10-22 | 诺瓦提斯公司 | Hydroxamate derives useful as deacetylase inhibitors |
| US20060189674A1 (en)* | 2000-09-01 | 2006-08-24 | Remiszewski Stacy W | Deacetylase inhibitors |
| CN101255124A (en)* | 2008-03-26 | 2008-09-03 | 山东大学 | Cinnamamide histone deacetylase inhibitor and preparation method thereof |
| WO2012106343A2 (en)* | 2011-02-01 | 2012-08-09 | The Board Of Trustees Of The University Of Illinois | Hdac inhibitors and therapeutic methods using the same |
| CN102746213A (en)* | 2012-05-18 | 2012-10-24 | 潍坊博创国际生物医药研究院 | Cinnamamide histone deacetylase inhibitor, preparation method thereof, and application thereof |
| Title |
|---|
| Development of N-Hydroxycinnamamide-Based Histone Deacetylase Inhibitors with an Indole-Containing Cap Group;Yingjie Zhang等;《Medicinal Chemistry Letters》;20130108;第2013卷(第4期);第235-238页* |
| 新型组蛋白去乙酰化酶抑制剂的设计、合成及抗肿瘤活性研究;张颖杰;《中国博士学位论文全文数据库 医药卫生科技辑》;20121215;全文* |
| 苯甲酰胺类及肉桂酰胺类组蛋白去乙酰化酶抑制剂的设计、合成及活性研究;杨鹏辉;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20130115;第30-61页* |
| Publication number | Publication date |
|---|---|
| CN103467359A (en) | 2013-12-25 |
| Publication | Publication Date | Title |
|---|---|---|
| CN103467359B (en) | Cinnamon amides histone deacetylase inhibitor with benzpyrole and preparation method and application of same | |
| CN105669520B (en) | O-phenylenediamine class selectivity histon deacetylase (HDAC) inhibitor containing tryptophan basic framework and its preparation method and application | |
| CN101723896B (en) | Tyrosine Derivative Histone Deacetylase Inhibitor and Its Application | |
| CN103864793B (en) | Substituted purin-9-acetylamino hydroxamic acid histone deacetylases inhibitor and preparation method and application | |
| CN105142642A (en) | cancer treatment | |
| CN102803246A (en) | HEDGEHOG pathway antagonists and therapeutic applications thereof | |
| CN101255124A (en) | Cinnamamide histone deacetylase inhibitor and preparation method thereof | |
| ES2428539T9 (en) | New histone deacetylase inhibitors | |
| CN107033087A (en) | 1H- indazole -4- aminated compounds and its purposes as IDO inhibitor | |
| US20200331882A1 (en) | Compound for simultaneously inhibiting lsd1 and hdac targets and application thereof | |
| WO2008087514A2 (en) | Hdac inhibitors | |
| CN102746213B (en) | Cinnamamide histone deacetylase inhibitor, preparation method thereof, and application thereof | |
| CN102180826A (en) | Histone deacetylase inhibitor containing alpha amino acid structure and application thereof | |
| CN113880772B (en) | CDK kinase inhibitors and application thereof | |
| CN115572244B (en) | 2' -Aryl chalcone compound, preparation method and application thereof in pharmacy | |
| CN108409608B (en) | Aromatic nitrogen mustard histone deacetylase inhibitor and preparation method and application thereof | |
| CN102149678A (en) | Novel ortho-aminoanilides for the treatment of cancer | |
| CN112608302B (en) | Quinazoline derivative for activating target ubiquitination degradation of EGFR protein through low-oxygen reduction and application thereof | |
| KR100696139B1 (en) | Alkyl carbamoyl naphthalenyloxy octenoyl hydroxyamide derivative having histone deacetylase inhibitory activity and preparation method thereof | |
| TW201934547A (en) | A pyrimidine compound and the preparation method and medical use thereof | |
| CN114831977B (en) | Application of benzoic acid derivative as TRPM2 protein inhibitor | |
| CN109438347B (en) | Cyanoquinoline IDO1 inhibitor, and preparation method and application thereof | |
| KR100820039B1 (en) | Alkylaminonaphthalenyloxymethyl propenyl hydroxybenzamide derivatives having histone deacetylase inhibitory activity, preparation method thereof, and pharmaceutical composition for anticancer agent comprising the same as an active ingredient | |
| CN106496132B (en) | N- (4-substituted phenyl) -2-substituted acetamide compound and application thereof as SIRT2 protein inhibitor | |
| CN115572250B (en) | Diaryl-beta-lactam compound, preparation method and application thereof in pharmacy |
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | Granted publication date:20150422 Termination date:20170927 | |
| CF01 | Termination of patent right due to non-payment of annual fee |