WO2022134395A1 - 一种抑制ptp1b活性的新型bh3模拟肽类似物及其应用 - Google Patents

一种抑制ptp1b活性的新型bh3模拟肽类似物及其应用 Download PDF

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WO2022134395A1
WO2022134395A1 PCT/CN2021/087669 CN2021087669W WO2022134395A1 WO 2022134395 A1 WO2022134395 A1 WO 2022134395A1 CN 2021087669 W CN2021087669 W CN 2021087669W WO 2022134395 A1 WO2022134395 A1 WO 2022134395A1
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novel
peptidomimetic
ptp1b
analog
dmf
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WO2022134395A9 (zh
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张传亮
苏贤斌
黄鼎旻
王振炜
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青岛科技大学
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  • the invention belongs to the field of biomedicine, and in particular relates to a novel BH3 mimetic peptide analog that inhibits the activity of PTP1B and its application.
  • PTP1B Protein tyrosine phosphatase 1B
  • IR insulin receptor
  • IRS-1 insulin receptor substrate
  • PTP1B is also a popular target for the study of T2DM in recent years, and a number of candidate compounds have entered preclinical and clinical phase I and II experiments.
  • the latest research ideas in recent years have shown that PTP1B can be used as a (potential) target for anti-tumor and Alzheimer's disease drug development.
  • Some studies have found that PTP1B overexpression can significantly promote the occurrence and growth of tumors in mice, and inhibiting the expression of PTP1B by inhibitors can produce anti-tumor effects; mechanism studies have found that PTP1B regulates the RNF213 gene to control the non-mitochondrial oxygen consumption of cells, thereby promoting the lack of Tumor cell survival and growth under oxygen conditions.
  • PTP1B is regarded as a target of antitumor drugs.
  • PTP1B has also been regarded as a regulatory role in the physiological processes related to Alzheimer's disease in the central nervous system. It is proposed to inhibit PTP1B and then antagonize the harmful physiological processes related to Alzheimer's disease regulated by PTP1B. Development of Alzheimer's drugs. Therefore, PTP1B has become a potential hot target for the development of anti-diabetic, cancer and Alzheimer's disease drugs, and PTP1B inhibitors are expected to be used in the development of anti-diabetic, cancer and Alzheimer's disease drugs that target PTP1B middle.
  • PTP1B inhibitors mainly include inorganic small molecule compounds, organic compounds and PTP1B inhibitors in natural products.
  • the selectivity of inorganic small molecule compounds is very low, and they have strong inhibitory effect on all PTPs; while organic compounds are mostly screened by organic synthesis and combinatorial chemistry methods, the compounds with inhibitory PTP1B activity are screened first, and then Modify the substituent group of the compound, and finally obtain a better PTP1B inhibitor.
  • Such inhibitors have problems such as poor stability, high charge, and high lipophilic coefficient that restrict the druggability; PTP1B inhibitors in natural products It is a high-throughput screening of natural products isolated and identified in nature. Although it has high selectivity and activity, the action site is not very clear. Therefore, it is very necessary to make up for the defects of the existing PTP1B inhibitory molecules and develop novel PTP1B inhibitors with novel structure, strong selectivity, low toxicity and high efficiency to meet the urgent domestic clinical needs.
  • the present invention provides a novel BH3 mimetic peptide analog that inhibits the activity of PTP1B and its application.
  • the novel BH3 mimetic peptide analog has significant PTP1B inhibitory activity, and can be used for the development of drugs for preventing or treating related diseases targeting PTP1B.
  • the present invention provides a novel BH3 mimetic peptide analog that inhibits the activity of PTP1B, and the structural formula of the novel BH3 mimetic peptide analog is as follows:
  • R1 is a long-chain carboxylic acid
  • R2 is COOH
  • R3 is a carboxylic acid or polycarboxylic acid with different chain lengths.
  • R1 is palmitic acid.
  • the peptide chains of the BH3 peptidomimetic analogs scan-1 to scan-11 adopt the Ala-scanning strategy, and the N-terminal protecting group thereof is modified with palmitic acid.
  • the N-terminus of the peptide chain of other described compounds is modified with carboxylic or polycarboxylic acids of different chain lengths.
  • novel BH3 peptidomimetic analogs are specifically:
  • the preparation method of the novel BH3 peptidomimetic analog comprises the following steps:
  • Fmoc-Phe-Wang (scan-11 is Fmoc-Ala-Wang) resin is placed in manual polypeptide solid-phase synthesizer, activated with dichloromethane, dimethylformamide;
  • step (4) adding the lysate to the product of step (4), stirring at room temperature, filtering, adding anhydrous ether to separate out the solid, washing and drying in vacuo to obtain the crude product of the peptidomimetic analog;
  • the lysing solution includes phenol, water, anisole and trifluoroacetic acid.
  • the excess trifluoroacetic acid is removed by blowing N 2 after filtration.
  • the present invention also provides a medicament or a pharmaceutical composition using the novel BH3 mimetic peptide analog as an active ingredient, comprising any of the novel BH3 mimetic peptide analogs and one or more pharmaceutically acceptable carriers or excipients.
  • the present invention also provides the application of the novel BH3 mimetic peptide analog in the preparation of an inhibitor for inhibiting the activity of PTP1B.
  • the present invention also provides the application of the novel BH3 peptidomimetic analog in the preparation of a medicament for preventing or treating a disease targeting PTP1B.
  • the diseases include diabetes, cancer and Alzheimer's disease.
  • the mode of administration of the medicine or pharmaceutical composition using the novel BH3 peptidomimetic analog as an active ingredient is oral administration or injection.
  • the present invention obtains a novel BimBH3 mimetic peptide analog by a polypeptide solid-phase synthesis method, the novel BH3 mimetic peptide analog is derived from the core region of the Bim-BH3 domain, and the amino acids in the structure are all natural amino acids.
  • the present invention proves through experiments that the novel BH3 peptidomimetic analog exhibits a significant inhibitory effect on protein tyrosine phosphatase 1B (PTP1B), and the obtained peptidomimetic analog has high purity and can be used as an excellent PTP1B inhibitor , and can also be applied in the drug development of related diseases targeting PTP1B, such as diabetes, cancer, Alzheimer's disease, etc. Therefore, the novel BH3 peptidomimetic analogs have potential application value and good development prospects.
  • PTP1B protein tyrosine phosphatase 1B
  • Figures 1 to 17 are peptidomimetics scan-2, scan-3, scan-4, scan-5, scan-6, scan-7, scan-8, scan-11, C13-SM6, C14-SM6, C16 Dose-inhibitory effect curves of diacid-SM6, C18-SM6, C18 diacid-SM6, C20-SM6, C22-SM6, C20 diacid-SM6, C22 diacid-SM6 and Lila-SM6 on the target protein PTP1B.
  • Resin activation Weigh a corresponding amount of Fmoc-Phe-Wang resin and place it in a manual peptide solid-phase synthesizer, wash 4 times with DCM, add 5ml DCM for swelling and activation for 3h, wash 4 times with DMF, add 20% piperidine DMF removed the Fmoc protecting group twice (20min+5min), washed 4 times with 5ml DMF, washed 4 times with 5ml DCM, and detected with Kaiser's reagent.
  • DMF washes 3 times, respectively adds 3 times the resin molar amount of Fmoc-Phe-OH, HBTU, HOBt and 6 times the resin molar amount of DIEA, dissolves in 10ml DMF, and stirs at room temperature for 2h , washed 4 times with DMF, added 20% piperidine DMF to remove Fmoc protecting group twice (20min+5min), washed 4 times with 5ml DMF, washed 4 times with 5ml DCM, and detected by Kaiser's reagent.
  • MES buffer is used as the reaction system
  • human protein tyrosine phosphatase 1B PTP1B
  • pNPP disodium p-nitrophenyl phosphate
  • the lead compound SM-6 is selected as the positive Control
  • DMSO as negative control
  • a screening model based on enzyme reaction rate 96-well microplate as carrier was established to search for PTP1B inhibitor by enzymatic method.
  • the specific implementation method is as follows: using MES buffer system (25mM, pH6.5), 10 ⁇ L pNPP (77mM), 86 ⁇ L MES buffer, 4 ⁇ L compound (2mM), and 100 ⁇ L PTP1B solution (50nM) were sequentially added to the 96-well plate, and the total reaction volume was to 200 ⁇ L.
  • MES buffer system 25mM, pH6.5
  • 10 ⁇ L pNPP 77mM
  • 86 ⁇ L MES buffer 4 ⁇ L compound (2mM)
  • 100 ⁇ L PTP1B solution 50nM
  • the reaction rate in the initial stage of each well is linearly related, and the slope of the linear part of the kinetic curve determines the reaction rate of PTP1B, and the enzyme activity is expressed in rate.
  • the data obtained The data of each group were analyzed by t test.
  • Inhibition rate (%) (v DMSO -v sample )/v DMSO ⁇ 100%
  • v DMSO and v sample represent the initial average reaction rates of the negative control group and the test compound, respectively
  • the PTP1B inhibition rate is initially screened at a concentration of 10 ⁇ mol/L for the peptidomimetic peptide, and the IC 50 is determined for the compounds whose primary screening inhibition rate is higher than 70%.
  • the inhibition results are shown in Table 2.
  • test results show that the peptidomimetic analogs of the present invention have a significant inhibitory effect on protein tyrosine phosphatase 1B, can be used as an excellent PTP1B inhibitor, and can be used in anti-diabetic, anti-tumor and anti-diabetic drugs targeting PTP1B.

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Abstract

本发明公开了一种抑制PTP1B活性的新型BH3模拟肽类似物及其应用。所述新型BH3模拟肽类似物的结构式(I)如下所示。该类模拟肽化合物衍生自Bim-BH3结构域的核心区域，采用多肽固相合成方法进行制备，其结构中的氨基酸均为天然氨基酸。所述新型BH3模拟肽类似物具有显著的抑制PTP1B的活性，在以PTP1B为靶点的相关疾病如糖尿病、癌症、阿尔兹海默症等的药物开发中具有潜在的应用价值。

Description

一种抑制PTP1B活性的新型BH3模拟肽类似物及其应用 技术领域
本发明属于生物医药领域，具体涉及一种抑制PTP1B活性的新型BH3模拟肽类似物及其应用。
背景技术
蛋白酪氨酸磷酸酶1B(protein tyrosine phosphatase 1B，PTP1B)与2型糖尿病及肥胖症的发病和发展有密切的关系，其是胰岛素信号转导通路中的关键的负调节蛋白。PTP1B异常过量表达，会使胰岛素敏感性降低，形成胰岛素抵抗。而PTP1B抑制剂能够通过阻断胰岛素刺激的胰岛素受体(IR)的酪氨酸磷酸化，进而影响胰岛素受体底物(IRS-1)的磷酸化，使类胰岛素和胰岛素敏感性增强，有效地从源头改善胰岛素抵抗，从而降低血糖，同时不存在胰岛素类药物的低血糖不良反应。因此，PTP1B也是近年来研究T2DM的热门靶点，并且已有多个候选化合物已进入临床前及临床I、II期实验。近年来的最新研究观点表明：PTP1B可以作为抗肿瘤和阿尔兹海默症药物开发的(潜在)靶标。一些研究发现PTP1B过表达能够显著促进小鼠体内肿瘤的发生和生长，通过抑制剂抑制PTP1B的表达能够产生抗肿瘤效果；机制研究发现PTP1B通过调控RNF213基因从而控制细胞非线粒体氧消耗，进而促进缺氧条件下肿瘤细胞的生存和生长。据此，PTP1B被看作是抗肿瘤药物的靶标。近些年PTP1B还被作为中枢神经系统中与阿尔兹海默症相关生理过程中的调控作用，提出通过抑制PTP1B进而拮抗PTP1B调控的与阿尔兹海默症相关的有害生理过程的策略，进行抗阿尔兹海默症药物的研发。因此，PTP1B已成为抗糖尿病、癌症及阿尔兹海默症药物开发的潜在热门靶点，而PTP1B抑制剂有望应用于以PTP1B为靶点的抗糖尿病、癌症和阿尔兹海默症等药物的开发中。
目前，PTP1B的抑制剂主要包括无机小分子类化合物、有机化合物和天然产物中PTP1B抑制剂。但无机小分子类化合物的选择性非常低，对所有的PTPs都有较强的抑制性；而有机化合物大多通过有机合成和组合化学的方法进行筛选，先筛选到具有抑制PTP1B活性的化合物，再对化合物的取代基团进行修饰，最后得到一种较好的PTP1B抑制剂，此类抑制剂存在稳定性差、带电荷较高、亲脂系数过高等制约成药性的问题；天然产物中PTP1B抑制剂是通过对自然界 中分离鉴定的天然产物进行高通量筛选，虽然其具有高选择性和活性，但作用位点并不是很明确。因此，弥补现有PTP1B抑制分子的缺陷，开发结构新颖、选择性强、低毒且高效的新型PTP1B抑制剂以满足国内临床上的迫切需求，就显得十分必要。
发明内容
本发明提供了一种抑制PTP1B活性的新型BH3模拟肽类似物及其应用。所述新型BH3模拟肽类似物具有显著的PTP1B的抑制活性，可用于制备预防或治疗以PTP1B为靶点的相关疾病的药物开发。
为实现上述发明目的，本发明采用以下技术方案予以实现：
本发明提供了一种抑制PTP1B活性的新型BH3模拟肽类似物，所述新型BH3模拟肽类似物的结构式如下：
Figure PCTCN2021087669-appb-000001
其中，R1为长链羧酸，R2为COOH，R3为不同链长度的羧酸或多羧酸。
进一步的，所述R1为棕榈酸。
进一步的，所述BH3模拟肽类似物scan-1～scan-11的肽链采用Ala-scanning策略，其N端保护基采用棕榈酸修饰。其他所述化合物的肽链N端采用不同链 长度的羧酸或多羧酸修饰。
进一步的，所述新型BH3模拟肽类似物具体为：
Figure PCTCN2021087669-appb-000002
进一步的，所述新型BH3模拟肽类似物的制备方法包括以下步骤：
(1)室温下，将Fmoc-Phe-Wang(scan-11为Fmoc-Ala-Wang)树脂置于手动 多肽固相合成器中，用二氯甲烷、二甲基甲酰胺活化；
(2)加入哌啶/二甲基甲酰胺混合液脱除Fmoc保护基；
(3)加入3-4倍树脂摩尔量的N-Fmoc保护氨基酸或羧酸、HOBT、HBTU以及5-6倍树脂摩尔量的DIEA，室温振荡反应2～4h；
(4)重复步骤(2)和(3)，直至完成整个模拟肽序列的合成；
(5)将步骤(4)的产物中加入裂解液，室温搅拌，过滤，加入无水乙醚析出固体后，经洗涤、真空干燥得模拟肽类似物粗产物；
(6)所述肽类似物粗产物使用反相制备液相色谱纯化，收集目标峰流动相溶液脱去乙腈后，冷冻干燥得到絮状或粉末状固体，即得BH3模拟肽类似物纯品。
进一步的，所述裂解液包括苯酚、水、苯甲硫醚和三氟乙酸。
进一步的，所述步骤(5)中过滤后鼓吹N 2去除多余的三氟乙酸。
本发明还提供了以权所述的新型BH3模拟肽类似物为活性成分的药物或药物组合物，包括任一所述新型BH3模拟肽类似物和一种或多种药学上可接受的载体或赋型剂。
本发明还提供了所述的新型BH3模拟肽类似物在制备用于抑制PTP1B活性的抑制剂中的应用。
本发明还提供了所述的新型BH3模拟肽类似物在制备用于预防或治疗以PTP1B为靶点的疾病的药物中的应用。
进一步的，所述疾病包括糖尿病、癌症和阿尔兹海默症。
进一步的，以新型BH3模拟肽类似物为活性成分的药物或药物组合物的给药方式为口服或注射。
与现有技术相比，本发明的优点和技术效果是：
本发明通过多肽固相合成方法得到一种新型BimBH3模拟肽类似物，该新型BH3模拟肽类似物衍生自Bim-BH3结构域的核心区域，其结构中的氨基酸均为天然氨基酸。本发明通过实验证明，所述新型BH3模拟肽类似物对蛋白质酪氨酸磷酸酯酶1B(PTP1B)表现出显著的抑制作用，且得到的模拟肽类似物纯度高，能够作为优异的PTP1B抑制剂，也能够应用在以PTP1B为靶点的相关疾病如糖尿病、癌症、阿尔兹海默症等的药物开发中。因此，所述新型BH3模拟肽类似物具有潜在的应用价值及很好的开发前景。
附图说明
图1-图17分别为模拟肽scan-2、scan-3、scan-4、scan-5、scan-6、scan-7、scan-8、scan-11、C13-SM6、C14-SM6、C16 diacid-SM6、C18-SM6、C18 diacid-SM6、C20-SM6、C22-SM6、C20 diacid-SM6、C22 diacid-SM6和Lila-SM6的对靶蛋白PTP1B的剂量-抑制效应曲线。
具体实施方式
以下结合具体实施例对本发明的技术方案做进一步详细的说明。下述实施例中的方法，如无特别说明，均为常规方法。
实施例1
以scan-1为例的合成路线，具体制备过程如下：
(1)树脂活化：称取相应量的Fmoc-Phe-Wang树脂置于手动多肽固相合成器中，DCM洗4次，加入5ml DCM溶涨活化3h，DMF洗4次，加入20％哌啶DMF脱去Fmoc保护基两次(20min+5min)，5ml DMF洗4次，5ml DCM洗4次，Kaiser’s试剂检测。
(2)连接Phe(F)：DMF洗涤3次，分别加入3倍树脂摩尔量的Fmoc-Phe-OH、HBTU、HOBt和6倍树脂摩尔量的DIEA，溶于10ml DMF中，室温搅拌反应2h，DMF洗4次，加入20％哌啶DMF脱去Fmoc保护基两次(20min+5min)，5ml DMF洗4次，5ml DCM洗4次，Kaiser’s试剂检测。
(3)连接Glu(E)：DMF洗涤3次，分别加入3倍树脂摩尔量的Fmoc-Glu(OtBu)-OH、HBTU、HOBt和6倍树脂摩尔量的DIEA，溶于10ml DMF中，室温搅拌反应2h，DMF洗4次，加入20％哌啶DMF脱去Fmoc保护基两次(20min+5min)，5ml DMF洗4次，5ml DCM洗4次，Kaiser’s试剂检测。
(4)连接Asp(D)：DMF洗涤3次，分别加入3倍树脂摩尔量的Fmoc-Asp(OtBu)-OH、HBTU、HOBt和6倍树脂摩尔量的DIEA，溶于10ml DMF中，室温搅拌反应2h，DMF洗4次，加入20％哌啶DMF脱去Fmoc保护基两次(20min+5min)，5ml DMF洗4次，5ml DCM洗4次，Kaiser’s试剂检测。
(5)连接Gly(G)：DMF洗涤3次，分别加入3倍树脂摩尔量的Fmoc-Gly-OH、HBTU、HOBt和6倍树脂摩尔量的DIEA，溶于10ml DMF中，室温搅拌反应2h，DMF洗4次，加入20％哌啶DMF脱去Fmoc保护基两次(20min+5min)，5ml DMF洗4次，5ml DCM洗4次，Kaiser’s试剂检测。
(6)连接Ile(I)：DMF洗涤3次，分别加入3倍树脂摩尔量的Fmoc-Ile-OH、HBTU、HOBt和6倍树脂摩尔量的DIEA，溶于10ml DMF中，室温搅拌反应2h，DMF洗4次，加入20％哌啶DMF脱去Fmoc保护基两次(20min+5min)，5ml DMF洗4次，5ml DCM洗4次，Kaiser’s试剂检测。
(7)连接Arg(R)：DMF洗涤3次，分别加入3倍树脂摩尔量的Fmoc-Arg(Mtr)-OH、HBTU、HOBt和6倍树脂摩尔量的DIEA，溶于10ml DMF中，室温搅拌反应2h，DMF洗4次，加入20％哌啶DMF脱去Fmoc保护基两次(20min+5min)，5ml DMF洗4次，5ml DCM洗4次，Kaiser’s试剂检测。重复此步骤1次。
(8)连接Arg(R)：DMF洗涤3次，分别加入3倍树脂摩尔量的Fmoc-Arg(Mtr)-OH、HBTU、HOBt和6倍树脂摩尔量的DIEA，溶于10ml DMF中，室温搅拌反应2h，DMF洗4次，加入20％哌啶DMF脱去Fmoc保护基两次(20min+5min)，5ml DMF洗4次，5ml DCM洗4次，Kaiser’s试剂检测。重复此步骤1次。
(9)连接Leu(L)：DMF洗涤3次，分别加入3倍树脂摩尔量的Fmoc-Leu-OH、HBTU、HOBt和6倍树脂摩尔量的DIEA，溶于10ml DMF中，室温搅拌反应2h，DMF洗4次，加入20％哌啶DMF脱去Fmoc保护基两次(20min+5min)，5ml DMF洗4次，5ml DCM洗4次，Kaiser’s试剂检测。
(10)连接Glu(E)：DMF洗涤3次，分别加入3倍树脂摩尔量的Fmoc-Glu(OtBu)-OH、HBTU、HOBt和6倍树脂摩尔量的DIEA，溶于10ml DMF中，室温搅拌反应2h，DMF洗4次，加入20％哌啶DMF脱去Fmoc保护基两次(20min+5min)，5ml DMF洗4次，5ml DCM洗4次，Kaiser’s试剂检测。
(11)连接Glu(Q)：DMF洗涤3次，分别加入3倍树脂摩尔量的Fmoc-Glu(OtBu)-OH、HBTU、HOBt和6倍树脂摩尔量的DIEA，溶于10ml DMF中，室温搅拌反应2h，DMF洗4次，加入20％哌啶DMF脱去Fmoc保护基两次(20min+5min)，5ml DMF洗4次，5ml DCM洗4次，Kaiser’s试剂检测。
(12)连接Ala(A)：DMF洗涤3次，分别加入3倍树脂摩尔量的Fmoc-Ala-OH、HBTU、HOBt和6倍树脂摩尔量的DIEA，溶于10ml DMF中，室温搅拌反应2h，DMF洗4次，加入20％哌啶DMF脱去Fmoc保护基两次(20min+5min)，5ml DMF洗4次，5ml DCM洗4次，Kaiser’s试剂检测。
(13)连接Ala(A)：DMF洗涤3次，分别加入3倍树脂摩尔量的 Fmoc-Ala-OH、HBTU、HOBt和6倍树脂摩尔量的DIEA，溶于10ml DMF中，室温搅拌反应2h，DMF洗4次，加入20％哌啶DMF脱去Fmoc保护基两次(20min+5min)，5ml DMF洗4次，5ml DCM洗4次，Kaiser’s试剂检测。
(14)连接棕榈酸(Pal)：DMF洗涤3次，分别加入6倍树脂摩尔量的棕榈酸、HBTU、HOBt和10倍树脂摩尔量的DIEA，溶于10ml DMF中，室温搅拌反应4h，5ml DMF洗4次，5ml DCM洗4次，Kaiser’s试剂检测。
(15)切割，解侧链保护基：产物加入250mg苯酚、0.5ml水、0.5ml苯甲硫醚、9.0ml三氟乙酸，室温搅拌2.5h，过滤，N 2吹去三氟乙酸，加入30ml冷的无水乙醚，5000rpm离心5min，得到白色沉淀，用冷的无水乙醚重复洗涤3次，真空干燥，得粗产物。
(16)粗产物使用反相制备液相色谱(RP-HPLC)纯化、收集目标峰流动相溶液脱去乙腈后，冷冻干燥得到絮状或粉末状固体，即BH3模拟肽类似物纯品，通过质谱和高效液相色谱分析进行结构确证。27个BH3模拟肽类似物的质谱数据和HPLC纯度分析数据见表1。
表1BH3模拟肽类似物的质谱数据和HPLC纯度分析数据
Figure PCTCN2021087669-appb-000003
Figure PCTCN2021087669-appb-000004
实施例2：蛋白酪氨酸磷脂酶1B(PTP1B)抑制活性测定
本发明中采用MES缓冲液为反应体系，利用人源蛋白酪氨酸磷酸酶1B(PTP1B)，以对硝基苯磷酸二钠(pNPP)为特异性底物，选择先导化合物SM-6作为阳性对照、以DMSO为阴性对照，建立了基于酶反应速率的96孔微板为载体的筛选模型，通过酶学方法寻找PTP1B抑制剂。
具体实施方法为：采用MES缓冲体系(25mM，pH6.5)，在96孔板内依次加入10μLpNPP(77mM)、86μL MES缓冲液、4μL化合物(2mM)、100μL PTP1B溶液(50nM)，反应总体积为200μL。每组3个平行，以DMSO为阴性对照，原钒酸钠(2mM)为阳性对照，25℃下，在摇床上摇动1min，酶标仪上每隔60s读数一次，动态测定5min，测其OD 405的变化(OD/min)。每个孔的初始阶段反应速率呈线性相关，动力学曲线线性部分的斜率决定PTP1B的反应速度，以速度表示酶活。所得数据用
Figure PCTCN2021087669-appb-000005
表示，各组数据运用t检验分析。化合物对PTP1B的抑制率计算公式：
抑制率(％)＝(v DMSO-v 样本)/v DMSO×100％
其中，v DMSO、v 样本分别表示阴性对照组和受试化合物的初始平均反应速率
本发明对模拟肽在10μmol/L浓度下进行PTP1B抑制率初筛，对初筛抑制率高于70％的化合物进行IC 50测定，抑制结果参见表2。
表2受试模拟肽类似物对PTP1B活性的抑制结果
Figure PCTCN2021087669-appb-000006
Figure PCTCN2021087669-appb-000007
*：初筛抑制率低于50％的化合物未进行IC 50的测定。
采用GraphPad Prism软件进行统计学处理，绘制出抑制剂量效应曲线，见图1-17，并计算得到模拟肽类似物scan-2、scan-3、scan-4、scan-5、scan-6、scan-7、scan-8、scan-11、C13-SM6、C14-SM6、C16 diacid-SM6、C18-SM6、C18 diacid-SM6、C20-SM6、C20 diacid-SM6、C22-SM6、C22 diacid-SM6和Lila-SM6的PTP1B抑制中浓度IC 50分别为91.6nmol/L、703.0nmol/L、580.9nmol/L、1208.0nmol/L、56.5nmol/L、45.4nmol/L、63.7nmol/L、511.9nmol/L、835.4nmol/L、262.7nmol/L、2875nmol/L、120.2nmol/L、384.6nmol/L、3887nmol/L、443.5nmol/L、337.9nmol/L、199.6nmol/L、4345nmol/L。
试验结果表明：本发明的模拟肽类似物对蛋白质酪氨酸磷酸酯酶1B表现出显著的抑制作用，能够作为优异的PTP1B抑制剂，并应用于以PTP1B为靶点的抗糖尿病、抗肿瘤及抗阿尔兹海默症症药物的开发中，因此具有很好的开发前景。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。

Claims (10)

  1. 一种抑制PTP1B活性的新型BH3模拟肽类似物，其特征在于，所述新型BH3模拟肽类似物的结构式如下：
    Figure PCTCN2021087669-appb-100001
    其中，R1为长链羧酸，R2为COOH，R3为不同链长度的羧酸或多羧酸。
  2. 根据权利要求1所述的新型BH3模拟肽类似物，其特征在于，所述新型BH3模拟肽类似物具体为：
    Figure PCTCN2021087669-appb-100002
    Figure PCTCN2021087669-appb-100003
  3. 根据权利要求1所述的新型BH3模拟肽类似物，其特征在于，所述新型BH3模拟肽类似物的制备方法包括以下步骤：
    (1)室温下，将Fmoc-Phe-Wang(scan-11为Fmoc-Ala-Wang)树脂置于手动多肽固相合成器中，用二氯甲烷、二甲基甲酰胺活化；
    (2)加入哌啶/二甲基甲酰胺混合液脱除Fmoc保护基；
    (3)加入3-4倍树脂摩尔量的N-Fmoc保护氨基酸或羧酸、HOBT、HBTU以及5-6倍树脂摩尔量的DIEA，室温振荡反应2～4h；
    (4)重复步骤(2)和(3)，直至完成整个模拟肽序列的合成；
    (5)将步骤(4)的产物中加入裂解液，室温搅拌，过滤，加入无水乙醚析出固体后，经洗涤、真空干燥得模拟肽类似物粗产物；
    (6)所述肽类似物粗产物使用反相制备液相色谱纯化，收集目标峰流动相溶液脱去乙腈后，冷冻干燥得到絮状或粉末状固体，即得BH3模拟肽类似物纯 品。
  4. 根据权利要求3所述的新型BH3模拟肽类似物，其特征在于，所述裂解液包括苯酚、水、苯甲硫醚和三氟乙酸。
  5. 根据权利要求3所述的新型BH3模拟肽类似物，其特征在于，所述步骤(5)中过滤后鼓吹N 2去除多余的三氟乙酸。
  6. 以权利要求1-5任一项所述的新型BH3模拟肽类似物为活性成分的药物或药物组合物，其特征在于，所述药物或药物组合物包括任一所述新型BH3模拟肽类似物和一种或多种药学上可接受的载体或赋型剂。
  7. 权利要求1-5任一项所述的新型BH3模拟肽类似物在制备用于抑制PTP1B活性的抑制剂中的应用。
  8. 权利要求1-5任一项所述的新型BH3模拟肽类似物在制备用于预防或治疗以PTP1B为靶点的疾病的药物中的应用。
  9. 根据权利要求8所述的应用，其特征在于：所述疾病包括糖尿病、癌症和阿尔兹海默症。
  10. 根据权利要求8所述的应用，其特征在于：以新型BH3模拟肽类似物为活性成分的药物或药物组合物的给药方式为口服或注射。
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