WO2023160708A1 - 吲哚酮取代-1,3-噻唑烷酮类衍生物及其制备方法和应用 - Google Patents

吲哚酮取代-1,3-噻唑烷酮类衍生物及其制备方法和应用 Download PDF

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WO2023160708A1
WO2023160708A1 PCT/CN2023/078485 CN2023078485W WO2023160708A1 WO 2023160708 A1 WO2023160708 A1 WO 2023160708A1 CN 2023078485 W CN2023078485 W CN 2023078485W WO 2023160708 A1 WO2023160708 A1 WO 2023160708A1
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indole
thiazolidinone
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孟歌
葛维娟
童静
程亚楠
曹慧玲
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复旦大学
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
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    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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  • the invention belongs to the technical field of medicine, and specifically relates to an indolinone-containing substituted-1,3-thiazolidinone derivative, a preparation method thereof, and an application as a PART14 inhibitor.
  • ADP-ribosylation is a reversible and evolutionarily conserved post-transcriptional modification of proteins, which participates in the regulation of various biological processes in the body, and plays an important role in maintaining gene stability and cell apoptosis.
  • ADPr is mainly catalyzed by the ADP-ribosyltransferase (ART) superfamily of proteins.
  • ART ADP-ribosyltransferase
  • ART ADP-ribosyltransferase
  • PARPs Poly ADP-ribose polymerase
  • PARP 1-18 isoforms Based on the homology with the structure of the catalytic domain of PARP1, the initial member of the family, 18 members were found in mammals, and their proteins were named PARP 1-18 isoforms.
  • the PARP family is extremely important in cells, and it plays an important role in maintaining gene stability, maintaining telomere length, and responding to external stimuli.
  • PARP family members According to the respective structural domains and functional characteristics of PARP family members, they can be divided into the following four main types: 1) DNA-dependent PARPs: including PARP1 2 , PARP2 3 and PARP3; 2) Tankyrase: including PARP5a (Tankyrase1) , PARP5b (Tankyrase2); 3) CCCH (ie Cys-Cys-Cys-His) PARPs: including PARP7, PARP12, PARP13; 4) macro PARPs, including PARP9, PARP14 4 , PARP15.
  • DNA-dependent PARPs including PARP1 2 , PARP2 3 and PARP3
  • Tankyrase including PARP5a (Tankyrase1) , PARP5b (Tankyrase2)
  • CCCH ie Cys-Cys-Cys-His
  • PARPs including PARP7, PARP12, PARP13
  • macro PARPs including PARP9, PARP14 4 , PARP15
  • PARP14 Not all members of the PARP family have ADP-ribosyltransferase activity, and some members (such as PARP14) act as single ADP-ribosyltransferases rather than poly-ADP-ribosyltransferases.
  • PARP14 is a DNA damage repair factor that plays a key role in tumor cell damage repair 2 . Based on the key role of PARP14 in the interaction process of tumor proliferation and other inflammatory factors, inhibitors targeting PARP14 have become a new strategy for the development of anti-tumor drugs and therapeutic drugs for allergic inflammation 6 .
  • PARP14 consists of 5 structural domains, and its catalytic domain is responsible for catalyzing the modification of ADP monoribosylation, which is a new target for the design of anti-tumor drugs 7 , these binding domains are closely related to PARP1 inhibitors suggested by structural bioinformatics analysis As shown in Figure 1 1 :
  • H10 and H10 are a kind of selective PARP14 inhibitor developed and researched from the compound library by using the small molecule microarray technology developed by the company. There is a certain interaction between them ( Figure 2).
  • the present invention also designed and synthesized a bismuth containing a 1H-indole ring and a nitrogen- and sulfur-containing five-membered heterocycle linked by a linker. Heterocyclic structural system to accommodate the dual binding sites of PARP14 (Site A and Site B, Figure 3).
  • the purpose of the present invention is to provide a PARP14 inhibitor with novel structure, good action site specificity, high efficiency and low toxicity.
  • the present invention utilizes the combination principle, the pharmacophore binding model and the characteristics of the active site of PARP14, selects aromatic indole and 1,3-thiazolidin-4-one five-membered heterocyclic ring as the basic active skeleton, and links the It is fused into one molecule, and the compound TM is designed, specifically by introducing various substituents at different positions of the basic core of 1H-indol-2-one and 1,3-thiazolidin-4-one to adjust the inhibitor Molecular size, the choice of linking group is to consider that more potential hydrogen bond donors are beneficial to improve the inhibitory activity of the drug enzyme target, and the hydrogen bond acceptor hydrazine group is selected as the linking group to adapt to the PARP14 receptor binding cavity
  • the purpose is to discover PARP14 inhibitor candidate compounds with
  • the novel PARP14 inhibitor provided by the present invention is specifically a heterocyclic substituted-1,3-thiazolidinone derivative containing indole alkaloids, which is denoted as TM, and its general structural formula is:
  • R 1 is a halogen electron-withdrawing group represented by F and Br, an alkyl electron-donating group represented by methyl (Me), R 2 is an electron-withdrawing group represented by halogen, and an electron-withdrawing group represented by tert-butyl Alkyl electron donating group.
  • the present invention also includes the pharmaceutically acceptable salts, hydrates and solvates thereof containing indole-substituted-1,3-thiazolidinone derivatives, Its polymorphs and co-crystals, its equally biologically functional precursors and derivatives.
  • the pharmaceutically acceptable salts of the p-hydroxybenzoylidene-1H-indol-2-one-3-substituted-1,3-thiazolidin-4-one derivatives include hydrochloride, hydrogen Bromate, sulfate, phosphate, acetate, methanesulfonate, p-toluenesulfonate, tartrate, citrate, fumarate, or malate.
  • the present invention also provides a synthetic method for the above-mentioned p-hydroxybenzoyl-1H-indol-2-one-3-substituted-1,3-thiazolidin-4-one derivatives (TM), specifically based on various
  • TM p-hydroxybenzoyl-1H-indol-2-one-3-substituted-1,3-thiazolidin-4-one derivatives
  • the substituted aromatic amines (1) wherein R 1 is CH 3 , F, Br, the corresponding substituted aromatic amines are sequentially denoted as (1a, 1b, 1c), and the corresponding intermediates (2 ), intermediate 5-substituted isatin (3), which are successively recorded as intermediate (2a, 2b, 2c), intermediate 5-substituted isatin (3a, 3b, 3c);
  • disubstituted thiosemicarbazone intermediates 7 which are sequentially recorded as disubstituted thiosemicarbazones 7a, 7b, ..., 7f, and their corresponding R 1 and R 2 are as follows:
  • target compound TM1, TM2, ..., TM6 is:
  • Indole alkaloid-containing heterocyclic substituted-1,3-thiazolidinone derivatives designed and synthesized by the invention are characterized by NMR and mass spectrometry analysis.
  • the activity test at the enzyme level in vitro is to study the thermal drift experiment between small molecules and PARP14 protein macromolecules.
  • the results show that all 6 target compounds and PARP14 will cause large thermal changes to the surface molecules after interaction with PARP14, that is, ⁇ TM All are greater than 3, such a strong positive value generally indicates that the compound has a good interaction with the target protein, and can be a lead compound of PARP14 inhibitor.
  • Figure 1 shows the binding domains closely related to PARP1 inhibitors.
  • Figure 2 presents existing PARP14 inhibitors and potential active sites of PARP14.
  • Figure 3 shows the design idea of the target compound PARP14 inhibitor.
  • FIG. 4 Docking study analysis between TM1 molecules PARP14.
  • (a) is a panorama of the interaction between PARP14 enzyme chain A holoenzyme and TM1 molecule
  • (b) is a diagram of the interaction between TM1 molecule and PARP14 enzyme catalytic active domain (three hydrogen bonds are represented by blue dotted lines ).
  • Examples include synthesis of related intermediates and target compounds, biological activity screening of PARP14 inhibitory enzyme activity, and related data and structure-activity relationship analysis.
  • Embodiment 5 Synthesis of double-substituted thiosemicarbazone intermediates 7a-f
  • Embodiment 7 the synthesis of target compound (TM1 ⁇ TM6)
  • TSA Thermal shift analysis
  • TSA test is described as follows: protein is sensitive to external temperature, and heating will cause protein denaturation.
  • the thermal stability of protein refers to the ability to maintain biological activity under the influence of temperature rise and other factors, and is an important indicator to measure protein stability.
  • TSA is a method to detect protein thermal stability.
  • the protein gradually heats up with the increase of the external temperature. When the temperature is higher than the critical temperature, the protein denatures, and the structure stretches and unfolds.
  • the denaturation temperature is the denaturation temperature (Tm) or melting temperature. The higher the Tm value, the better the thermal stability.
  • Tm denaturation temperature
  • the denatured protein structure stretches, exposing hydrophobic regions that bind the fluorescent dye in solution.
  • the fluorescence of the fluorescent dye used is weak when it contacts water, it will be excited to generate a fluorescent signal when it contacts a hydrophobic environment, and the change of the fluorescence intensity of the fluorescent dye reflects the denaturation of the protein.
  • the combination of the compound and the protein makes the protein more stable and increases the Tm value.
  • Subtract Tm 1 (without compound) from Tm 2 (with compound added) to obtain the ⁇ Tm value.
  • the higher the ⁇ Tm value the stronger the binding of the protein to the compound.
  • a fluorescent real-time quantitative PCR instrument is used to run a melting curve to screen small molecule ligands.
  • Each target protein has a relatively constant melting temperature (Tm) under certain conditions (buffer, pH, salt ionic strength).
  • the specific operation method of testing TSA is described as follows: First, the protein is dissolved with buffer, and the small molecule ligand is dissolved with DMSO. Proteins were mixed with small molecule ligands and fluorescent dyes as experimental samples, and blank and control samples were set up, with 4 replicates. The molar ratio of protein to small molecule ligand is 1:3-1:5. The samples were added to 96-well plates respectively, the heating strategy was 25-95°C, and the temperature was raised at 1°C/min, and the Tm and ⁇ Tm values were measured. The preliminary evaluation results are summarized in Table 4. The ⁇ Tm values are shown in Table 4. Noteworthy It is noted that the interaction between all compounds and PAPR14 has a strong positive correlation with protein stability, and the ⁇ Tm is greater than 3.
  • Table 4 lists the test results of the primary screening experiment of the activity of the target compound on the enzyme inhibitory activity.
  • the ⁇ TM of the compounds TM1-TM with the highest measured inhibition rate are all greater than 3, which can be considered as having a strong inhibitory effect.
  • the experimental data suggest that there is a strong interaction between these six compounds and PARP14, indicating that these compounds can be used as potent inhibitors of PARP14, thereby inhibiting the further MAPR of the target protein, and performing post-genome transformation in the cell signal transmission pathway. Protein activity regulation, so as to be further used in the development of precision anti-tumor drugs
  • the structural characteristics and activity of the target compound PARP14 inhibitor are as follows: the ⁇ TM of the compound TM1 with the best activity is 3.25, and its molecular structure has methyl and trifluoromethyl or hydroxyl substitutions, and the inhibitory activity of the next two The substituents in the compound structure are similar, and there is also an electron-withdrawing group Cl at the meta-position of the aromatic ring on the 3N- of thiazolidinone.
  • the distance may be the key to inhibiting the ADP of the PARP14 macrostructure domain, and the cooperative adjustment between substituents to jointly affect the electronic cloud arrangement of the entire molecule should play a very important role in the selective interaction between targets.
  • the special and similar substituent arrangement in the target molecule is worthy of reference in the design process of PAPR14 inhibitors in the future.
  • Example 9 The target compound and the catalytic activity structure of PARP14 enzyme and the docking research
  • the crystal 4PY4 which has a ligand (benzothiazole) and a ring similar to it, is selected as the target protein for docking research 9.
  • the SYBYL-X 2.0 software runs the SYBYL-X 2.0 software to build and import a compound library including 6 target compounds, and optimize the energy of the compound with Tripos force field plus charge (Gasteiger Hückel) until the energy no longer decreases.
  • the Surflex-Dock module of the software was used to process and optimize the target protein 4PY4, including deletion of the B chain, hydrogenation treatment, extraction of ligands, etc., and the active pocket of the catalytic domain of PARP14 was used as a docking research site to construct a Protomol binding pocket and carry out compound libraries.
  • the docking study and virtual screening of the system evaluates the biological activity of the target compound based on ten scoring values including the total score. According to the docking results, combined with Pymol software, the molecular mechanism of the interaction between the most active target compound TM1 and PARP414 was analyzed.
  • Fig. 4 is a docking study analysis of TM1 molecule PARP14 enzyme molecules.
  • the analysis of docking results showed that the target compound was closely combined with the catalytic domain of PARP14 mainly through hydrogen bond interaction and aromatic interaction.
  • the specific docking results are analyzed as follows: there is a ⁇ - ⁇ stacking aromatic interaction between the indole ring in the molecular structure of TM1 and the aromatic ring of Tyr1714 in the catalytic domain of PARP14, and a fluorine atom of 3-CF 3 on the aromatic ring interacts with Thr1713 There is a hydrogen bond interaction between the amide-peptide bond backbone NH and Tyr1714...F
  • the fluorine atom also forms a hydrogen bond with the 1-NH of the 1,3-imidazole ring of His1682, which is similar to the hydrogen bond hydrogen bond formed by the fluorine atom and the 1-NH of the 1,3-imidazole ring Not only in one place, another fluorine atom of 3-CF 3 also has a double NH...F interaction with the 1-NH of the 1,3-imidazole ring of His
  • the embodiment of the present invention successfully synthesized 6 target compounds, and all of them have good crystal form and purity. All new compounds The chemical structures have been confirmed by 1 H NMR or 13 C NMR.
  • the PARP14 inhibitory activity of the synthesized target was tested at the enzyme level. All of the six target compounds exhibit potent enzyme inhibitory activity, and the ⁇ TM in thermal drift experiments are all greater than 3, which can be used as lead compounds for the design of PARP14 inhibitors, so as to provide precision therapeutic drugs for the design of specific targeting breast tumors.
  • the present invention is not limited to the above examples.

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Abstract

一种芳甲叉基-吲哚酮-3-取代噻唑烷-酮类衍生物及其制备方法和应用，该类化合物可作为PARP14抑制剂的先导化合物。

Description

吲哚酮取代-1,3-噻唑烷酮类衍生物及其制备方法和应用 技术领域
本发明属于医药技术领域，具体涉及一种含吲哚酮取代-1,3-噻唑烷酮类衍生物及其制备方法和作为PART14抑制剂的用途。
背景技术
ADP核糖基化(ADPr)是蛋白质的一种可逆的、进化保守的转录后修饰过程，参与调节体内多种生物过程，在维持基因的稳定性和细胞凋亡等方面发挥着重要的作用。ADPr主要由ADP-核糖基转移酶(ART)蛋白超家族催化。目前，研究最多的ART家族是聚腺苷二磷酸核糖聚合酶家族(Poly ADP-ribose polymerase,PARPs)，其广泛存在于真核细胞，均包含序列高度保守的催化结构域1。依据与家族初始成员PARP1催化域结构的同源性，有18个成员在哺乳动物体内发现，其蛋白质被命名为PARP 1-18亚型。PARP家族在细胞中极其重要，在维持基因稳定性、保持端粒长度及细胞应对外界刺激响应等方面功不可没。
依据PARP家族成员各自结构域和功能特征可分为以下四种主要类型：1)DNA依赖性PARPs：包括PARP12，PARP23和PARP3；2)端锚聚合酶(Tankyrase)：包括PARP5a(Tankyrase1)，PARP5b(Tankyrase2)；3)CCCH(即Cys-Cys-Cys-His)PARPs：包括PARP7，PARP12，PARP13；4)macro PARPs，包括PARP9，PARP144，PARP15。并非所有PARP家族成员都有ADP核糖基转移酶活性，有些成员(如PARP14)则是作为单ADP核糖基转移酶而非聚ADP核糖基转移酶。
因此，基于PARP家族成员间结构和功能的差异来设计合成具有高度亚型选择性小分子PARP抑制剂是目前主要问题5。现有PARP抑制剂主要是针对PAPR1设计的，其代表性的药物根据基本结构特征包括苯并咪唑甲酰胺类(1)、酞嗪酮类(2)、三环吲哚内酰胺类(3)、吲唑甲酰胺类(4)、咔唑二酰亚胺类(5)和烟酰胺类似物(6)，其作用靶点主要为DNA依赖性的结构域和不同的其它结构与(图2)，这些抑制剂虽然对不同的亚型具有不同抑制作用，有些具有一定的选择性，但对于具有MARP功能的PARP14却无能为力2。这些已上市的代表性PARP1抑制剂临床药物的化学结构分别如下式所示：
PARP14是DNA损伤修复因子，在肿瘤细胞损伤修复中发挥关键作用2。基于PARP14在肿瘤增殖和其它炎症因子互作過程中的关键作用，靶向PARP14的抑制剂已成为抗肿瘤药物和過敏性炎症治療藥物研发的新策略6。PARP14由5个结构域组成，其催化结构域负责催化ADP单核糖基化修饰，是设计抗肿瘤药物的新靶点7，这些由结构生物信息学分析提示的与PARP1抑制剂密切相关的结合域如图1所示1：
虽然已有临床用药都是针对PAPR1，选择性针对PARP14的抑制剂还鲜为报道，因此寻找结构新颖，作用位点特异性好，高效低毒的新型PARP14抑制剂已成为研究热点。H10和H10是人们采用公司开发的小分子微数组技术，从化合物库中开发研究得到的一种选择性PARP14抑制剂8，其作用特征是和PARP14的主要结合位点和第二结合位点之间都有一定相互作用(图2)。
含有稠环吲哚结构的化合物GeA-69是一种选择性的PARP14变构抑制剂，靶向作用于宏结构域(Macrodomain 2,MD2)，Kd＝2.1μM。受上述将不同结构片段之间通过链接基链接起来的分子设计策略的启发，本发明也设计合成了含有通过连接基相连的含有1H-吲哚环和含氮和含硫五元杂环的双杂环结构体系，以适应PARP14的双结合位点(Site A和Site B,图3)。
发明内容
本发明的目的在于提供一种结构新颖、作用位点特异性好、高效低毒的PARP14抑制剂。
为现有PARP抑制剂选择性较低的普遍问题，希望通过拼合原理，将不同活性片段通过合适连接基，设计出能同时作用于A，B双位点的抑制剂(图2，图3)。
本发明利用拼合原理、药效团结合模型及PARP14活性位点的特点，选取芳香性吲哚和1,3-噻唑烷-4-酮五元杂环作为基本活性骨架，通过不同长度连接基将其融合到一个分子中，设计化合物TM，具体是通过在1H-吲哚-2-酮和1,3-噻唑烷-4-酮的基本母核不同位置引入各种取代基来调节抑制剂的分子大小，连接基的选择是考虑到更多潜在氢键供体有利于提高对药物酶靶标的抑制活性，而选用具有氢键受体亚肼基作为连接基团，以适应PARP14受体结合腔各结合位点之间的空间位置与距离要求，并通过在这些小分子中引入更多取代基，以便能伸展至靶标蛋白多个位点(Site A和Site B)，而产生较为充分的相互作用，旨在发现理化性质好，活性高，选择性更好的PARP14抑制剂候选化合物，目标化合物设计思路参见上图3。
根据上述设计思想，本发明提供的新型PARP14抑制剂，具体为含吲哚生物碱杂环取代-1,3-噻唑烷酮类衍生物，记为TM，其结构通式为：
其中，R1为以F,Br为代表的卤素吸电子基，以甲基(Me)为代表的烷基供电子基，R2为以卤素为代表的吸电子，以叔丁基为代表的烷基供电子基。
典型的，化合物TM有6种，依次记为TM1，TM2，…，TM6；其与R1，R2的对应关系如下：
本发明还包括所述含吲哚-取代-1,3-噻唑烷酮类衍生物的药用盐，其水合物和溶剂化物， 其多晶和共晶，其同样生物功能的前体和衍生物。
本发明中，所述对羟基苯甲叉基-1H-吲哚-2-酮-3-取代-1,3-噻唑烷-4-酮类衍生物的药用盐，包括盐酸盐、氢溴酸盐、硫酸盐、磷酸盐、醋酸盐、甲磺酸盐、对甲苯磺酸盐、酒石酸盐、柠檬酸盐、富马酸盐或苹果酸盐。
本发明还提供上述对羟基苯甲叉基-1H-吲哚-2-酮-3-取代-1,3-噻唑烷-4-酮类衍生物(TM)的合成方法，具体是以各种的廉价易得的取代芳香胺类化合物为起始原料，经过多步骤反应制备得到目标化合物，其合成路线为：
合成的具体步骤为：
(1)首先以各种取代的芳香胺(1,1.0～1.1equiv.)为起始原料，在水合氯醛(1,1.1～1.2equiv.)，盐酸羟氨(1,3.0～3.3equiv.)的作用进行反应，经过简单后处理后，分别经过硅胶柱层析分离，制备得到中间体(2),产率介于62.6～87.6％之间；
(2)中间体(2)在浓硫酸的作用下环合得到重要中间体各种5-取代的靛红(3),产率介于70.8～98.3％之间)；
(3)与上述合成步骤的同时，以各种取代的苯胺(1.1,1.0～1.1equiv.)和CS2(4,1.8～2.0equiv.)为原料，在碱性条件下合成不稳定的硫代乙酸氨基盐类中间体，稍加分离纯化后，继而采用氯甲酸甲酯(1.0～1.1equiv.)进脱硫反应，简单后处理经硅胶柱层析分离，即可得到各种取代的芳 基异硫氰酸酯(5),产率介于40.6～85.5％之间；
(4)将异硫氰酸酯(5)在水合肼(80％)作用下肼解得到各种N-取代氨基硫脲(6),产率介于58.2～87.8％之间，为另一种重要中间体。
(5)将上述两种重要中间体5-取代的靛红(3,1.0～1.1equiv.)和N-取代氨基硫脲类中间体(6,1.0～1.1equiv.)在浓硫酸催化下在乙醇中进行分子间脱水缩合反应，得到各种双取代缩氨基硫脲中间体7,产率介于58.3～87.8％之间，
(6)分别将双取代缩氨基硫脲(7)(1.0～1.1equiv.)与2-氯乙酸乙酯(1.0～1.1equiv.)在无水乙酸钠催化下进行等摩尔比的环合反应得到双取代1,3-噻唑烷酮类重要中间体8，产率介于72.1～97.7％之间。
(7)将中间体8(1.0～1.1equiv.)与对羟基苯甲醛(9)1.0～1.1equiv.)，在无水哌啶(1％，催化用量)的催化下，通过Knoevenagel缩合反应，经分离纯化后可得到目标化合物TM；
典型的，所述取代的芳香胺(1)，其中，R1取为CH3，F，Br，对应的取代的芳香胺依次记为(1a,1b,1c)，与其对应的中间体(2)、中间体5-取代的靛红(3)，依次记为中间体(2a，2b，2c)，中间体5-取代的靛红(3a，3b，3c)；
所述取代的苯胺(1.1)，其中，R2取为3-CF3，3-Cl，4-C(CH3)3，对应的取代的苯胺依次记为(1.1a，1.1b，1.1c)，与其对应的中间体芳基异硫氰酸酯(5)依次记为芳基异硫氰酸酯(5a，5b，5c)；与其对应的中间体N-取代氨基硫脲类中间体(5)依次记为N-取代氨基硫脲(6a，6b，6c)；
所述双取代缩氨基硫脲类中间体7共有6种，依次记为双取代缩氨基硫脲7a，7b，…，7f，其对应的R1，R2如下：
所述的双取代1,3-噻唑烷酮类重要中间体8也共有6种，依次记为中间体8a，8b，…，8f，其对应的R1，R2如下：
目标化合物TM也为6种，依次记为TM1，TM2，…，TM6；与其对应R1，R2如下：
目标化合物TM1，TM2，…，TM6的具体结构式为：
本发明设计合成的结构新颖的含吲哚生物碱杂环取代-1,3-噻唑烷酮类衍生物，通过NMR和质谱分析表征。在体外酶水平的活性测试是研究小分子与PARP14蛋白大分子之间的热漂移实验，结果显示所有6个目标化合物与PARP14相互在拥有之后都会使把表分子发生较大热改变，即ΔTM都大于3，这种强的正值一般表明化合物与靶蛋白有较好的相互作用，可成为PARP14抑制剂的先导化合物。
附图说明
图1为与PARP1抑制剂密切相关的结合域。
图2为现有PARP14抑制剂和PARP14的潜在活性位点。
图3为目标化合物PARP14抑制剂的设计思路。
图4.TM1分子PARP14之间的对接研究分析。其中，(a)是PARP14酶A链全酶和TM1分子相互作用的全景图，(b)是TM1分子与PARP14酶催化活性结构域之间相互作用的图(三个氢键用蓝色虚线表示)。
具体实施方式
下面通过具体实施例进一步介绍本发明。
实施例包括相关中间体和目标化合物的合成、PARP14抑制酶活的生物活性筛选及相关数据和构效关系分析。
实施例1.N-羟肟乙酰取代苯胺类中间体2a-2c合成
在一干净的500mL单口瓶中，加入水合氯醛(9.0g,55.0mmol)，水(240mL)，搅拌均匀后依次加入无水硫酸钠(130g)，取代苯胺(1a-1d,50.0mmol)，盐酸溶液(2.2mL HCl+10.0mL水)，盐酸羟氨(10.4g,150.0mmol)，加毕，逐渐升温至65℃，反应2h，停止加热，趁热过滤分别得各固体粗品，分别经柱纯化(P:E＝5:1～3:1)得黄色固体(2a,60.5％,m.p.54.9～155.7℃)；柱纯化(P:E＝3:1～2:1)得淡黄色固体(2b,85.6，m.p.158.5～160.1℃)；柱纯化(P:E＝5:1～3:1)得黄色固体(2c,78.3％,m.p.166.8～168.3℃)；
实施例2. 5-取代靛红类中间体3a-3c合成
在一干净的150mL三口瓶中，加入浓硫酸(24.0mL)，升温至50℃，慢慢加入上述合成中间体(2a-2c,30.0mmol)，随着量的加入，溶液颜色慢慢加深，变黑，加毕，温度调至80℃，反应20min，取碎冰(100g)慢慢加入反应体系，冰水颜色为红棕色，静置，抽滤，水洗至中性，将该固体溶于90mL10％NaOH中，用浓盐酸调节pH至4，抽滤，滤液继续用浓盐酸调pH至2，有大量砖红色固体析出，抽滤干燥得红棕色固体3a(85.9％,185.3～186.8℃，3b(88.6％,219.1～220.8℃)，3c(80.3％,225.8～227.1℃。
实施例3.芳基异硫氰酸酯类中间体5a-5c的合成
称取各取代苯胺(1.1a-1.1c(50.0mmol)置于一干净100mL三颈瓶，依次加入乙醚(15.0mL)、CS2(4,3.6mL,90.0mmol)以及三乙胺(7.2mL)，体系于25～30℃下反应12h。体系中产生大量固体，抽滤，滤饼用无水乙醚(30.0mL)洗涤，得粉末状固体。将该固体置空气中自然干燥10min，挥去其中残存的乙醚后，将其转移至100mL干净三颈瓶中，加氯仿(50.0mL)，体 系成均相，加入三乙胺(7.2mL)，冰盐浴冷却至0℃以下，搅拌下向体系中滴加氯甲酸甲酯(3.9mL,50.0mmol)，滴加过程中，体系温度控制在5℃以下。滴加完毕，水浴29～30℃反应1h，TLC监测反应进行程度，待反应完毕，停止反应，向体系中加入硅胶拌样后，柱层析分离(石油醚体系)，得无色油状液体或白色固体，即得中间体5a～5c(表1)。
表1.中间体5a～5c总结表
实施例4.N-取代氨基硫脲类中间体6a-6c的合成
在一干净的50mL单口瓶中，加入芳基异硫氰酸酯(5a-5c,2.00mmol)，加入20mL异丙醇溶解，搅拌下滴加水合肼(85％,2.40mmol)，立刻有大量白色沉淀生成，将该体系在室温下继续搅拌30min，过滤，滤饼用异丙醇洗涤3次，所得产物分别为N-取代氨基硫脲类中间体6a-6c(表2)。
表2.中间体6a-6c总结表
实施例5.双取代缩氨基硫脲类中间体7a-f的合成
在一干净的100mL单口瓶中，分别加入各种5-取代靛红(3a-3c,3.50mmol)，95％乙醇(30.0mL)，搅拌下加入各种N-取代氨基硫脲(6a-6c,3.50mmol)，待混合均匀，向体系中滴加一滴浓硫酸，逐渐升温至回流，反应5h，TLC监测，待原料反应完全，停止加热，冷却至室温，析出固体，抽滤，滤饼用冷的无水乙醇洗涤，分别得橘红色固体为各种所需中间体(表3)。
表3.双取代缩氨基硫脲类中间体7a-7f总结表
实施例6.双取代-1,3-噻唑烷-2-酮类中间体8a～8f的合成
(1)5-甲基-3-(2-(3-(3-氯苯基)-4-氧代噻唑烷-2-叉基)亚肼基)-1H-吲哚-2-酮(8a)的合成
称取化合物7a(0.38g,1.00mmol)置于一干净的50mL单口瓶中，加入95％乙醇(20.0mL)，搅拌下加入无水乙酸钠(0.34g,4.00mmol)，滴加氯乙酸乙酯(0.24mL,2.00mmol)，逐渐升温至78℃，回流反应约5h，却至室温，加入适量水稀释，析出固体，抽滤，滤饼用冷的无水乙醇洗涤得黄色固体，干燥得红色固体，干燥得纯品(0.32g，76.1％)，m.p.>300℃。1H NMR(400MHz,DMSO-d6)δ:10.60(s,1H,indole-NH),7.99(m,2H,3-N-Ar-H),7.89(d,J＝8.2Hz,2H,3-N-Ar-H),7.06(m,1H,indole-6-H),6.94(s,1H,indole-4-H),6.69(d,J＝7.9Hz,1H,indole-7-H),4.21(s,2H,thiazolidine-CH2-),1.91(s,3H,indole-5-CH3).
(2)5-氟-3-(2-(3-(3-氯苯基)-4-氧代噻唑烷-2-叉基)亚肼基)-1H-吲哚-2-酮(8b)的合成
称取化合物7b(1.04g,3.00mmol)置于一干净250mL单口瓶中，加入95％乙醇(60.0mL)，搅拌下加入无水乙酸钠(0.98g,12.0mmol)，滴加氯乙酸乙酯(0.7mL,6.00mmol)，逐渐升温至78℃，回流反应约5h，停止加热，冷却至室温，加入适量水稀释，析出固体，抽滤，滤饼用冷的无水乙醇洗涤得黄色固体，干燥得纯品(1.13g,97.4％)，m.p.>300℃。1H NMR(400MHz,DMSO-d6)δ:10.74(s,1H,indole-NH),7.70(d,J＝1.4Hz,1H,3-N-Ar-4-H),7.66(d,J＝1.2Hz,1H,3-N-Ar-2-H),7.64(t,J＝5.2Hz,1H,3-N-Ar-6-H),7.52(m,1H,3-N-Ar-5-H),7.14(td,J＝9.1,2.8Hz,1H,indole-6-H),6.98(dd,J＝8.7,2.8Hz,1H,indole-4-H),6.81(dd,J＝8.6,4.3Hz,1H,indole-7-H),4.22(s,2H,thiazolidine-CH2-).
(3)5-甲基-3-(2-(3-(3-氯苯基)-4-氧代噻唑烷-2-叉基)亚肼基)-1H-吲哚-2-酮(8c)的合成
称取化合物8c(0.45g,1.30mmol)置于一干净的50mL单口瓶中，加入95％乙醇(25.0mL)，搅拌下加入无水乙酸钠(0.43g,5.20mmol)，滴加氯乙酸乙酯(0.32mL,2.60mmol)，逐渐升温至 78℃，回流反应约5h，后续操作同化合物8a，得橘红色固体，干燥得纯品(0.48g，96.0％)，m.p.>300℃。1H NMR(400MHz,DMSO-d6)δ:10.60(s,1H,indole-NH),7.70(s,1H,3-N-2-Ar-H),7.67(dd,J＝4.0,1.3Hz,2H,3-N-Ar-4,6-H),7.52(m,1H,3-N-5-Ar-H),7.08(d,J＝5.0Hz,2H,indole-4,6-H),6.70(d,J＝8.4Hz,1H,indole-7-H),4.21(s,2H,thiazolidine-CH2-),2.02(s,3H,indole-5-CH3).
(4)5-氟-3-(2-(3-(3-三氟甲基苯基)-4-氧代噻唑烷-2-叉基)亚肼基)-1H-吲哚-2-酮(8d)的合成
称取化合物7d(0.38g,1.00mmol)置于一干净50mL单口瓶中，加入95％乙醇(20.0mL)，搅拌下加入无水乙酸钠(0.34g,4.00mmol)，滴加氯乙酸乙酯(0.24mL,2.00mmol)，逐渐升温至78℃，回流反应约5h，停止加热，冷却至室温，加入适量水稀释，析出固体，抽滤，滤饼用冷的无水乙醇洗涤得土黄色固体，干燥得纯品(0.28g,66.7％)，m.p.>300℃。1H NMR(400MHz,DMSO-d6)δ:10.74(s,1H,indole-NH),8.01(s,1H,3-N-Ar-2-H),7.96(m,1H,3-N-Ar-4-H),7.86(dd,J＝4.9,1.4Hz,2H,3-N-Ar-5,6-H),7.12(td,J＝9.1,2.8Hz,1H,indole-6-H),6.82(ddd,J＝12.9,8.6,3.5Hz,2H,indole-4,7-H),4.23(s,2H,thiazolidine-CH2-).
(5)5-氯-3-(2-(3-(4-氟苯基)-4-氧代噻唑烷-2-叉基)亚肼基)-1H-吲哚-2-酮(8e)的合成
称取化合物7e(0.52g,1.50mmol)置于一干净100mL单口瓶中，加入95％乙醇(50.0mL)，搅拌下加入无水乙酸钠(0.50g,6.00mmol)，滴加氯乙酸乙酯(0.36mL,3.00mmol)，逐渐升温至78℃，回流反应约5h，停止加热，冷却至室温，加入适量水稀释，放置过夜，抽滤，滤饼用冷的无水乙醇洗涤得橘红色固体，干燥得纯品(0.54g,93.1％)，m.p.>300℃。1H NMR(400MHz,DMSO-d6)δ:10.82(s,1H,indole-NH),7.57(dd,J＝8.9,5.0Hz,2H,3-N-Ar-3,5-H),7.45(t,J＝8.8Hz,2H,3-N-Ar-2,6-H),7.31(dd,J＝8.3,2.3Hz,1H,indole-6-H),7.20(d,J＝2.2Hz,1H,indole-4-H),6.81(d,J＝8.3Hz,1H,indole-7-H),4.23(s,2H,thiazolidine-CH2-).
(6)5-溴-3-(2-(3-(4-叔丁基苯基)-4-氧代噻唑烷-2-叉基)亚肼基)-1H-吲哚-2-酮(8f)的合成
称取化合物7f(1.61g,3.60mmol)置于一干净100mL单口瓶中，加入无水乙醇(50.0mL)，搅拌下加入无水乙酸钠(1.18g,14.4mmol)，滴加氯乙酸乙酯(0.49mL,4.00mmol)，逐渐升温至78℃，回流反应8h，后续操作同化合物8a，得橙红色固体，干燥得纯品(1.68g,98.8％)，m.p.>300℃，(eluent:PE:EA＝1:2，Rf＝0.2)。1H NMR(400MHz,DMSO-d6)δ:11.00(s,1H,indole-NH),7.62(d,J＝8.6Hz,2H,3-N-Ar-2,6-H),7.55(d,J＝2.1Hz,1H,3-N-Ar-5-H),7.43(dd,J＝8.3,2.1Hz,1H,3-N-Ar-3-H),7.41(s,1H,3-N-Ar-4-H),7.39(s,1H,indole-Ar-4-H),6.80(d, J＝8.3Hz,1H,indole-Ar-6-H),4.22(s,2H,thiazolidine-CH2-),1.35(s,9H,3-N-Ar-4-C(CH3)3).13C NMR(100MHz,DMSO-d6)δ:174.54(s),172.70(s),164.70(s),151.90(s),148.34(s),143.70(s),135.42(s),132.51(s),130.65(s),127.70(s),126.64(s),118.74(s),113.64(s),112.92(s),35.06(s),33.38(s),31.60(s).
实施例7.目标化合物(TM1～TM6)的合成
(1)3-(2-(5-(4-羟基苯甲叉基)-3-(3-三氟甲基苯基)-4-氧代噻唑烷-2-叉基)亚肼基)-5-甲基-1H-吲哚-2-酮(TM1)的合成
称取化合物8a(0.21g,0.50mmol)，化合物4-羟基苯甲醛(9,0.067g,0.55mmol)置于一干净50mL烧瓶中，加入无水乙醇(15mL)，搅拌下，加入无水哌啶(0.1mL)，加热回流反应6h，停止加热，待体系冷至室温，有大量红色物质析出，抽滤，少量乙醇洗涤，干燥得红色粉末状固体(0.237g，90.8％)，m.p.>300℃。1H NMR(400MHz,DMSO-d6)δ:10.64(s,1H,indole-NH),10.42(s,1H,thiazolidine-5-Ar-OH),8.18(s,1H,3-N-Ar-2-H),8.01(d,J＝7.7Hz,2H,3-N-Ar-H),7.92(m,1H,3-N-Ar-H),7.79(s,1H,thiazolidine-5-＝CH),7.62(d,J＝8.1Hz,2H,thiazolidine-5-Ar-2,6-H),7.03(dd,J＝24.6,8.4Hz,4H,thiazolidine-5-Ar-3,5-H,indole-4,6-H),6.69(d,J＝7.8Hz,1H,indole-7-H),1.91(s,3H,indole-5-CH3).
(2)3-(2-(5-(4-羟基苯甲叉基)-3-(3-氯苯基)-4-氧代噻唑烷-2-叉基)亚肼基)-5-氟-1H-吲哚-2-酮(TM2)的合成
称取化合物8b(0.23g,0.60mmol)，化合物4-羟基苯甲醛(9,0.08g,0.66mmol)置于一干净50mL烧瓶中，加入无水乙醇(10.0mL)，搅拌下，加入无水哌啶(0.12mL)，加热回流反应5h，停止加热，待体系冷至室温，有大量红色物质析出，抽滤，少量乙醇洗涤，干燥得红色粉末状固体(0.245g,82.8％)，m.p.>300℃。1H NMR(400MHz,DMSO-d6)δ:10.77(s,1H,indole-NH),10.41(s,1H,thiazolidine-5-Ar-OH),7.85(s,1H,thiazolidine-5-＝CH),7.82(s,1H,3-N-Ar-2-H),7.68(m,2H,thiazolidine-5-Ar-2,6-H),7.63(m,3H,3-N-Ar-H),7.13(m,1H,indole-4-H),7.05(dd,J＝8.6,2.6Hz,1H,indole-6-H),7.00(d,J＝8.6Hz,2H,thiazolidine-5-Ar-3,5-H),6.81(dd,J＝8.5,4.3Hz,1H,indole-7-H).
(3)3-(2-(5-(4-羟基苯甲叉基)-3-(3-氯苯基)-4-氧代噻唑烷-2-叉基)亚肼基)-5-甲基-1H-吲哚-2-酮(TM3)的合成
称取化合物8c(0.23g,0.60mmol)，化合物4-羟基苯甲醛(9,0.08g,0.66mmol)置于一干净50mL烧瓶中，加入无水乙醇(15.0mL)，搅拌下，加入无水哌啶(0.15mL)，加热回流反应6h， 停止加热，待体系冷至室温，有大量红色物质析出，抽滤，少量乙醇洗涤，干燥得红色粉末状固体(0.278g,94.9％)，m.p.>300℃。1H NMR(400MHz,DMSO-d6)δ:10.65(s,1H,indole-NH),10.39(s,1H,thiazolidine-5-Ar-OH),7.86(s,1H,3-N-Ar-2-H),7.79(s,1H,thiazolidine-5-＝CH),7.70(d,J＝5.2Hz,2H,3-N-Ar-4,6-H),7.65(m,1H,3-N-Ar-5-H),7.62(d,J＝8.7Hz,2H,thiazolidine-5-Ar-2,6-H),7.15(s,1H,indole-4-H),7.09(d,J＝8.0Hz,1H,indole-6-H),7.00(d,J＝8.6Hz,2H,thiazolidine-5-Ar-2,6-H),6.70(d,J＝7.9Hz,1H,indole-7-H),2.01(s,3H,indole-5-CH3).
(4)3-(2-(5-(4-羟基苯甲叉基)-3-(3-三氟甲基苯基)-4-氧代噻唑烷-2-叉基)亚肼基)-5-氟-1H-吲哚-2-酮(TM4)的合成
称取化合物8d(0.19g,0.45mmol)，化合物4-羟基苯甲醛(9,0.06g,0.50mmol)置于一干净50mL烧瓶中，加入无水乙醇(15.0mL)，搅拌下，加入无水哌啶(0.1mL)，加热回流反应6h，停止加热，待体系冷至室温，有大量红色物质析出，抽滤，少量乙醇洗涤，干燥得红色粉末状固体(0.193g,81.4％)，m.p.>300℃。1H NMR(400MHz,DMSO-d6)δ:10.78(s,1H),7.53(tdd,12H).
(5)3-(2-(5-(4-羟基苯甲叉基)-3-(4-氟苯基)-4-氧代噻唑烷-2-叉基)亚肼基)-5-氯-1H-吲哚-2-酮(TM5)的合成
称取化合物8e(0.272g,0.70mmol)，化合物4-羟基苯甲醛(9,0.095g,0.77mmol)置于一干净50mL烧瓶中，加入无水乙醇(15.0mL)，搅拌下，加入无水哌啶(0.15mL)，加热回流反应6h，停止加热，待体系冷至室温，有大量红色物质析出，抽滤，少量乙醇洗涤，干燥得红色粉末状固体(0.294g,85.2％)，m.p.>300℃。1H NMR(400MHz,DMSO-d6)δ:10.86(s,1H,indole-NH),10.40(s,1H,thiazolidine-5-Ar-OH),7.81(s,1H,thiazolidine-5-＝CH),7.70(dd,J＝8.8,5.0Hz,2H,3-N-Ar-3,5-H),7.62(d,J＝8.7Hz,2H,thiazolidine-5-Ar-2,6-H),7.48(t,J＝8.8Hz,2H,3-N-Ar-2,6-H),7.31(dd,J＝8.3,2.2Hz,1H,indole-6-H),7.26(d,J＝2.1Hz,1H,indole-4-H),6.99(d,J＝8.6Hz,2H,thiazolidine-5-Ar-3,5-H),6.81(d,J＝8.3Hz,1H,indole-7-H).
(6)3-(2-(5-(4-羟基苯甲叉基)-3-(4-叔丁基苯基)-4-氧代噻唑烷-2-叉基)亚肼基)-5-溴-1H-吲哚-2-酮(TM6)的合成
称取化合物8f(0.471g,1.00mmol)，化合物4-羟基苯甲醛(9,0.122g,1.00mmol)置于一干净50mL烧瓶中，加入无水乙醇(15.0mL)，搅拌下，加入无水哌啶(0.2mL)，加热回流反应7h，停止加热，待体系冷至室温，有大量红色物质析出，抽滤，少量乙醇洗涤，干燥得橙黄色粉末 状固体(0.456g,79.3％)，m.p.>300℃，(eluent:PE:EA＝1:3，Rf＝0.2)。1H NMR(400MHz,DMSO-d6)δ:10.95(s,1H,indole-NH),7.78(s,1H,thiazolidine-5＝CH),7.64(s,1H,3-N-Ar-2-H),7.62(s,1H,3-N-Ar-6-H),7.61(s,1H,3-N-Ar-3-H),7.59(d,J＝1.9Hz,1H,3-N-Ar-5-H),7.50(d,J＝8.5Hz,2H,thiazolidine-5-Ar-3,6-H),7.42(dd,J＝8.3,2.1Hz,2H,thiazolidine-5-Ar-3,5-H),7.01(d,J＝8.6Hz,2H),6.78(d,J＝8.3Hz,1H,indole-6-H),1.36(s,9H,3-N-Ar-4-C(CH3)2).13C NMR(100MHz,DMSO-d6)δ:168.78(s),166.36(s),164.58(s),160.67(s),152.00(s),148.82(s),143.90(s),135.67(s),133.54(s),133.13(s),132.35(s),130.71(s),127.76(s),126.59(s),124.63(s),118.72(s),116.99(s),116.46(s),113.74(s),112.96(s),35.07(s),31.60(s).
实施例8.目标化合物对PARP14酶的抑制活性评价研究
以经典热漂移试验分析(Thermal shift analysis,TSA)方法对22个缩氨基脲取代的双芳基嘧啶目标化合物，针对靶点聚腺苷二磷酸核糖聚合酶-1(PARP-1)进行了PARP-1酶水平的结合测试，初步评鉴其潜在的生物活性。相关空白(Control)样品：荧光染料+DMSO+缓冲液，对照(Reference)样品：蛋白+荧光染料+DMSO+缓冲液，实验(Sample)样品：蛋白+荧光染料+抑制剂+缓冲液。仪器采用的是实时定量荧光PCR。数据分析软件软件为蛋白热熔软件。
TSA测试原理描述如下：蛋白质对外界温度敏感，加热会引起蛋白质变性。蛋白质的热稳定性是指在温度升高和其它因素的影响下保持生物活性的能力，是衡量蛋白稳定性的重要指标。TSA是检测蛋白热稳定性的一种方法。在TSA中，蛋白质随外界温度升高而渐渐升温，当温度大于临界温度时，蛋白质变性，结构发生伸展、去折叠，发生变性的温度即变性温度(Tm)或熔化温度。Tm值越高，热稳定性越好。变性后的蛋白质结构伸展，暴露出疏水区域，与溶液中的荧光染料结合。由于采用的荧光染料在接触水时荧光很弱，而在接触疏水环境时会被激发产生荧光信号，荧光染料的荧光强度的变化反应了蛋白质的变性情况。化合物与蛋白质的结合后使蛋白质更加稳定，使Tm值升高。以Tm2(加入化合物)减去Tm1(未加入化合物)，得到△Tm值，△Tm值越高，蛋白与化合物结合越强。该方法利用荧光实时定量PCR仪运行熔解曲线，对小分子配体进行筛选。每个目的蛋白在一定条件下(缓冲液、pH、盐离子强度)有一个相对恒定的熔解温度(Tm)。
测试TSA的具体操作方法描述如下：是首先蛋白质加缓冲液溶解，小分子配体用DMSO溶解。蛋白质分别与小分子配体、荧光染料混合作为实验样品，并设置空白及对照样品，4个重复。蛋白质与小分子配体的摩尔数之比为1:3～1:5。样品分别加入96孔板，升温策略为25～95℃，升温1℃/min，测定Tm与△Tm值。初步评价结果总结在表4之中。△Tm值如表4所示。值得注 意的是所有化合物与PAPR14之间的相互作用与蛋白稳定性呈较强的正相关，△Tm均大于3。
表4. PARP-14与目标化合物(TM1～TM6)相互作用的△Tm变化图.
表4列出了目标化合物的活性对酶抑制活性的初筛实验的测试结果。所测得抑制率最高的化合物TM1～TM的ΔTM均大于3，可认为是具有较强的抑制作用。实验数据提示这6个化合物与PARP14之间存在着较强的相互作用，表明这些化合物可作为PARP14的强效抑制剂，从而抑制靶标蛋白的进一步MAPR，在细胞信号传到通路里边进行后基因组的蛋白活性调控，从而进一步用于精准抗肿瘤药物的开发
通过对目标化合物PARP14的抑制剂的结构特点和活性分如下：活性最好的化合物TM1的ΔTM＝3.25，其分子结构存在甲基和三氟甲基或羟基取代，抑制活性次之的2个化合物结构中的取代基与之类似，也是在噻唑烷酮的3N-上的芳环间位有一吸电子基Cl。1,3-噻唑烷-4-酮的3-位苄基上的3-Cl或三氟甲基和1,3-噻唑烷-4-酮的5-位苄基上的4-OH之间的距离可能是抑制PARP14宏观结构域ADP的关键，取代基之间的相互协作调节从而共同影响整个分子的电子云排布应该在于靶标之间的选择性作用方面起着非常重要的作用。目标分子中的特殊而类似取代基排布，在日后的PAPR14抑制剂设计过程中值得借鉴。
实施例9.目标化合物与PARP14酶的催化活性结构与之间的对接研究
虽然PDB数据库已发布20多个关于PARP14晶体结构6，而鉴于本专利化合物结构中具有的噻唑环的结构，则选取配体(苯并噻唑)与之类似环的晶体4PY4为对接研究的靶标蛋白9，首先运行SYBYL-X 2.0软件，构建导入包括6个目标化合物的化合物库，对化合物用Tripos力场加电荷(Gasteiger Hückel)优化进行能量优化，至能量不再降低为止。同时采用该软件Surflex-Dock模块，处理优化靶标蛋白4PY4，包括删除B链，加氢处理，提取配体等，将PARP14催化结构域活性口袋作为对接研究位点，构建Protomol结合口袋，进行化合物库的对接研究和虚拟筛选，基于包括总分在内的十种打分值来评价目标化合物的生物活性。 并根据对接结果，结合Pymol软件分析活性最好的目标化合物TM1与PARP414相互作用的分子机制。
表1. DOCK对接打分结果
图4为TM1分子PARP14酶分子之间的对接研究分析。
对接结果分析表明目标化合物主要是通过氢键相互作用和芳香性相互作用与PARP14催化结构域紧密结合。具体对接结果分析如下：TM1分子结构中的吲哚环和PARP14催化结构域的Tyr1714芳香环之间存在π-π堆积芳香性相互作用，同时其芳环上3-CF3的一个氟原子与Thr1713和Tyr1714之间酰胺肽键骨架NH之间…F存在氢键相互作用 同时该氟原子还与His1682的1,3-咪唑环的1-NH之间形成了氢键，这种类似的由氟原子和1,3-咪唑环的1-NH参与形成的氢键氢键还不只在一处，3-CF3的另一个个氟原子还同样与与His1682的1,3-咪唑环的1-NH之间也存在氢键双重NH…F相互作用，而且这两个氢键的键长完全相等由于该两个氢键的形成，在PARP14的催化结构域中形成了由两个C-F键和两个分子间氢键构建而成的四边形结构，其中两两边长几乎相等，从侧面看四个原子几乎在一个平面上。由Thr1713和Tyr1714之间的酰胺肽键骨架NH和其中一个氟原子F形成的第一个氢键相互链接，另外与此平面四边形的一個頂點F原子直接相连的还有一个氢键(NH…F)，由此构建了一种由氢键形成的类似北斗七星的勺形布阵，这种由分子对接结果分析提供小分子与靶标之间的相互特征可从分子水平上初步证实该化合物生物活性较高的原因，因为该化合物不同于其它分子结构的显著热漂移结果对应著其潜在的抑酶活性，这种活性差异可能就是因为TM1分子结构中芳环上的3-CF3与PAPP14的催化部位氨基酸残基Y1714和His1682之间存在芳香性相互作用的和多重氢键相互作用所共同所导致的结果。上述这些分析和结论同时也可为进一步设计PARP14小分子抑制劑的解構骨架和取代基的种类分布提供借鉴。
总之，本发明实施例成功合成6个目标化合物，且都有很好的晶型和纯度，所有新化合物 的化学结构均已通过1H NMR或13C NMR确证。对所合成的目标物进行了酶水平的PARP14抑制活性测试。6个目标化合物中都表现出强效抑酶活性，热漂移实验中的ΔTM均大于3，可作为设计PARP14抑制剂的先导化合物进行研究，从而为设计特异性靶向乳腺肿瘤提供精准治疗药物提供理论参考和物质基础。
本发明不限于上述实例。
参考文献:
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2.Lord,C.J.；Ashworth,A.,PARP inhibitors:Synthetic lethality in the clinic.Science(Washington,DC,U.S.)2017,355(6330),1152-1158.
3.Chen,Q.；Kassab,M.A.；Yu,X.；Dantzer,F.,PARP2mediates branched poly ADP-ribosylation in response to DNA damage.Nat Commun 2018,9(1),3233.
4.Iwata,H.；Goettsch,C.；Sharma,A.；Ricchiuto,P.；Goh,W.W.B.；Halu,A.；Yamada,I.；Yoshida,H.；Hara,T.；Wei,M.；Inoue,N.；Fukuda,D.；Mojcher,A.；Mattson,P.C.；Barabasi,A.-L.；Boothby,M.；Aikawa,E.；Singh,S.A.；Aikawa,M.,PARP9and PARP14cross-regulate macrophage activation via STAT1ADP-ribosylation.Nat.Commun.2016,7,12849.
5.王世瑞；薛晓文,PARP家族及临床使用的PARP抑制剂.广东化工2019,46(9),3.
6.Qin,W.；Wu,H.-J.；Cao,L.-Q.；Li,H.-J.；He,C.-X.；Zhao,D.；Xing,L.；Li,P.-Q.；Jin,X.；Cao,H.-L.,Research progress on PARP14as a drug target.Front.Pharmacol.2019,10,172.
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Claims (7)

  1. 一种芳甲叉基-吲哚酮-3-取代噻唑烷-酮类衍生物，其特征在于，结构通式为下式(TM)所示：
    其中，R1为以F，Br为代表的卤素吸电子基，以甲基(Me)为代表的烷基供电子基，R2为3-取代或4-取代的官能团，主要是指以卤素，三氟甲基为代表的吸电子基，以叔丁基为代表的各种供电子基。
  2. 根据权利要求1所述的芳甲叉基-吲哚酮-3-取代噻唑烷-酮类衍生物，其特征在于，化合物TM为6种，依次记为TM1，TM2，…，TM6；其与R1，R2，X的对应关系如下：
  3. 根据权利要求1所述的芳甲叉基-吲哚酮-3-取代噻唑烷-酮类衍生物，其特征在于，还包括所述含吲哚生物碱杂环取代-1,3-噻唑烷酮类衍生物的药用盐，其水合物和溶剂化物，其多晶和共晶，其同样生物功能的前体和衍生物。
  4. 根据权利要求3所述的芳甲叉基-吲哚酮-3-取代噻唑烷-酮类衍生物，其特征在于，所述含吲哚生物碱杂环取代-1,3-噻唑烷酮类衍生物的药用盐，包括盐酸盐、氢溴酸盐、硫酸盐、磷酸盐、醋酸盐、甲磺酸盐、对甲苯磺酸盐、酒石酸盐、柠檬酸盐、富马酸盐或苹果酸盐。
  5. 如权利要求1-4之一所述的芳甲叉基-吲哚酮-3-取代噻唑烷-酮类衍生物的制备方法，其特征在于，以取代芳香胺类化合物为起始原料，经过多步骤反应制备得到目标化合物，其合成路线为：
    合成的具体步骤为：
    (1)首先以各种取代的芳香胺(1)为起始原料，在水合氯醛、盐酸羟氨的作用下进行反应，经过后处理后，再经过硅胶柱层析分离，制备得到中间体(2)；这里，取代的芳香胺(1)为1.0～1.1equiv.，水合氯醛为1.1～1.2equiv.，盐酸羟氨为3.0～3.3equiv.；
    (2)中间体(2)在浓硫酸的作用下环合，得到中间体5-取代的靛红(3)；
    (3)与上述合成步骤的同时，以取代的苯胺(1.1)和CS2(4)为原料，在碱性条件下合成不稳定的硫代乙酸氨基盐类中间体，分离纯化后，采用氯甲酸甲酯进脱硫反应，经过硅胶柱层析分离，即得到取代的芳基异硫氰酸酯(5)；这里，取代的苯胺(1.1)为,1.0～1.1equiv.，CS2为1.8～2.0equiv.，氯甲酸甲酯为1.0～1.1equiv.；
    (4)将异硫氰酸酯(5)在水合肼作用下肼解得到各种N-芳基取代氨基硫脲类中间体(6)；
    (5)将上述两种中间体5-取代的靛红(3)和N-芳基取代氨基硫脲类中间体(6)在浓硫酸催化下在乙醇中进行分子间脱水缩合反应，得到各种双取代缩氨基硫脲中间体(7)；将双取代缩氨基硫脲7与2-氯乙酸乙酯在无水乙酸钠催化下进行等摩尔比的环合反应，即得到中间体8；这里，5-取代的靛红(3)为1.0～1.1equiv.，芳基异硫氰酸酯(6)为1.0～1.1equiv.，双取代缩氨基硫脲(7)为1.0～1.1equiv.，2-氯乙酸乙酯为1.0～1.1equiv.；
    (6)将中间体8与对羟基苯甲醛(9)，在无水哌啶(1％，催化用量)的催化下，通过Knoevenagel缩合反应，经分离纯化后可得到1H-吲哚-2-酮取代-1,3-噻唑烷酮类目标化合物TM；这里，中间体8为1.0～1.1equiv.，对羟基苯甲醛(9)为1.0～1.1equiv.。
  6. 根据权利要求5所述的制备方法，其特征在于，所述取代的芳香胺(1)，其中，R1取为CH3，F，Br，对应的取代的芳香胺依次记为1a,1b,1c，与其对应的中间体(2)、中间体5-取代的靛红(3)，依次记为中间体2a，2b，2c，中间体5-取代的靛红3a，3b，3c；
    所述取代的苯胺(1.1)，其中，R2取为3-CF3，3-Cl，4-C(CH3)3，对应的取代的苯胺依次记为1.1a，1.1b，1.1c，与其对应的中间体芳基异硫氰酸酯(5)，依次记为芳基异硫氰酸酯5a，5b，5c，与其对应的中间体N-取代氨基硫脲类中间体(6)，依次记为N-取代氨基硫脲6a，6b，6c；
    双取代缩氨基硫脲类中间体(7)中，R1分别取为CH3，F，Br，R2分别取为3-CF3，3-Cl，4-C(CH3)3，双取代缩氨基硫脲类中间体(7)有6种，依次记为双取代缩氨基硫脲类7a，7b，7c，7d,7e,7f；其对应的R1，R2如下：
    所述的双取代1,3-噻唑烷酮类重要中间体8也共有6种，依次记为中间体8a，8b，…，8f，其对应的R1，R2如下：
    目标化合物TM共有6种，依次记为TM1，TM2，…，TM6；其与R1，R2，X的对应关系如下：
  7. 如权利要求1-4之一所述的芳甲叉基-吲哚酮-3-取代噻唑烷-酮类衍生物在制备PARP14抑制剂中的用途。
PCT/CN2023/078485 2022-02-27 2023-02-27 吲哚酮取代-1,3-噻唑烷酮类衍生物及其制备方法和应用 WO2023160708A1 (zh)

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