WO2022257831A1 - 一类以pd-1受体为靶点的多肽化合物、其制备方法及应用 - Google Patents

一类以pd-1受体为靶点的多肽化合物、其制备方法及应用 Download PDF

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WO2022257831A1
WO2022257831A1 PCT/CN2022/096623 CN2022096623W WO2022257831A1 WO 2022257831 A1 WO2022257831 A1 WO 2022257831A1 CN 2022096623 W CN2022096623 W CN 2022096623W WO 2022257831 A1 WO2022257831 A1 WO 2022257831A1
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compound
resin
compounds
seq
polypeptide
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陈罡
赵龙
赵雅玉
陈颖
罗浩
张永辉
魏金环
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南通大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • 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 biomedicine, and specifically relates to a class of polypeptide compounds targeting PD-1 receptors, a preparation method and application thereof.
  • opioids have been widely used in the clinical treatment of non-cancer chronic pain, but opioids inevitably cause drug tolerance, physical and mental dependence, gastrointestinal function inhibition, nausea, sedation, irritability, hallucinations, and Side effects such as motor function impairment are the main problems hindering its application in clinical treatment. Therefore, there is an urgent need to develop new effective and safe analgesic drugs.
  • Programmed death ligand (PD-L1 also known as B7-H1 or CD274), a member of the B7 family, is widely expressed in humans with cancer.
  • PD-L1 can combine with PD-1, as a co-inhibitory checkpoint molecule, involved in immune regulation.
  • PD-1/PD-L1 pathway mainly focuses on the tolerance of T-lymphocytes and the activation and polarization of macrophages in tumor immune escape.
  • the functions of PD-L1/PD-1 outside of tumor therapy have gradually been reported, including murine lupus, colitis, collagen-induced arthritis, and brain injury.
  • PD-L1/PD-1 pathway has a good effect on acute and chronic pain after nerve injury, and injection of exogenous PD-L1 can significantly increase the pain threshold of normal mice (Nat.Neurosci.2017 ,20,917-926); and the combined administration of PD-L1 and low-dose morphine can significantly enhance the analgesic effect of morphine, thereby reducing the side effects caused by high-dose morphine (Sci.Transl.Med.2020,12, eaaw6471). Therefore, targeting PD-1 has potential application prospects in the development of new analgesic drugs.
  • the present invention provides a class of polypeptide compounds targeting the PD-1 receptor, its preparation method and application.
  • the embodiments of the present invention provide a class of polypeptide compounds targeting the PD-1 receptor, characterized in that the polypeptide compounds include Compound 1, Compound 2, Compound 3, Compound 4 and Compound 5 ; Wherein, the amino acid sequence of compound 1-5 is as shown in SEQ ID NO: 1-5, and SEQ ID NO: 1-5 is respectively:
  • SEQ ID NO: 1 Val Tyr Arg Cys Met Ile Ser Tyr Gly;
  • SEQ ID NO: 2 Tyr Arg Cys Met Ile Ser Tyr Gly Gly;
  • SEQ ID NO: 3 Met Ile Ser Tyr Gly Gly Ala Asp Tyr;
  • SEQ ID NO: 4 Ser Tyr Gly Gly Ala Asp Tyr Lys Arg
  • SEQ ID NO: 5 Tyr Gly Gly Ala Asp Tyr Lys Arg Ile.
  • the embodiment of the present invention also provides a preparation method of a class of polypeptide compounds targeting the PD-1 receptor, which is characterized in that it includes the following process: (1) using solid-phase synthesis, compound 1 to The amino acid of 5 is coupled to the solid-phase resin, (2) the target compound is obtained by cleavage with a cleavage reagent, freeze-drying, preparation and purification.
  • step (1) specifically includes the following steps:
  • the solid phase carrier in the solid phase synthesis is wang resin, the wang resin is swollen with dichloromethane, compressed with anhydrous methanol, and washed with N,N-dimethylacetamide;
  • step (2) specifically includes the following steps:
  • the embodiment of the present invention also provides the application of a class of polypeptide compounds targeting the PD-1 receptor in the preparation of analgesic drugs.
  • the analgesic drugs include drugs for treating inflammatory pain and visceral pain.
  • the drug is suitable for any one of intrathecal administration, subcutaneous administration, tail vein administration, intracerebroventricular administration, intraperitoneal administration or oral administration.
  • the therapeutic target of the drug is PD-1 receptor.
  • the polypeptide compound includes one of compound 1, compound 2, compound 3, compound 4 and compound 5.
  • Small molecule polypeptides of the present invention are 5 compounds with Amber scores in the interval [-90,-80] obtained from peptide library screening by molecular docking technology; these five compounds can bind to PD-1 , Intrathecal administration has significant inhibitory effects on inflammatory pain and visceral pain; and the synthesis is simple, easy to purify, and is conducive to large-scale synthesis.
  • the present invention discovers a new type of small molecular polypeptide, which has some similar characteristics to PD-L1, can target PD-1, and treat formalin-induced inflammatory pain and acetic acid-induced pain. Visceral pain has significant analgesic effect and has potential clinical application value.
  • Fig. 1 is the ESI-MS spectrogram of compound 4 in the embodiment of the present invention.
  • Fig. 2 is the inhibitory effect of compound 4 on the increase of free Ca2+ in DRG cells induced by high K+ solution in the examples of the present invention
  • FIG. 3 is an analgesic effect diagram of intrathecal injection of compound 4 on formalin solution-induced acute inflammatory pain in an embodiment of the present invention
  • Fig. 4 is a graph showing the analgesic effect of intrathecal injection of compound 4 on visceral pain induced by intraperitoneal injection of acetic acid solution in an example of the present invention.
  • Compounds 1-5 of the present invention are synthesized by Fmoc-solid-phase synthesis method, and the solid-phase carrier used is wang-resin.
  • the reagent for removing the amino-protecting group 9-fluorenylmethoxycarbonyl (Fmoc) is 20% hexahydropyridine in N,N-dimethylformamide.
  • the condensation reagent required for peptide chain extension is N, N-diisopropylethylamine/1-hydroxybenzotriazole/O-benzotriazole-tetramethyluronium hexafluorophosphate, and the required amount is the resin used 3 to 6 times the amount of the substance.
  • the cleavage agent required for cleavage of the crude peptide from the resin is a mixture of trifluoroacetic acid/triisopropylsilane/double distilled water with a volume ratio of 95:2.5:2.5.
  • the components and proportions of the condensation reagent, the Fmoc removal reagent and the indene detection reagent are well known to those skilled in the art.
  • N-fluorenylmethoxycarbonyl- Glycine N-fluorenylmethoxycarbonyl-O-tert-butyl-L-tyrosine
  • N-fluorenylmethoxycarbonyl-L-serine N-fluorenylmethoxycarbonyl-L-isoleucine
  • N-fluorenylmethyl Oxycarbonyl-L-methionine N-(9-fluorenylmethoxycarbonyl)-S-trityl-L-cysteine
  • N-fluorenylmethoxycarbonyl-Glycine N-fluorenylmethoxycarbonyl-O-tert-butyl-L-tyrosine
  • the crude peptide resin was swollen with dichloromethane, compressed with anhydrous methanol, and dried three times. Use trifluoroacetic acid/triisopropylsilane/double distilled water to cut the crude peptide from the resin, spin dry, extract, and freeze-dry to obtain the crude peptide:
  • the crude peptide resin was swollen with dichloromethane, compressed with anhydrous methanol, and dried three times. Use trifluoroacetic acid/triisopropylsilane/double distilled water to cut the crude peptide from the resin, spin dry, extract, and freeze-dry to obtain the crude peptide:
  • the crude peptide resin was swollen with dichloromethane, compressed with anhydrous methanol, and dried three times. Use trifluoroacetic acid/triisopropylsilane/double distilled water to cut the crude peptide from the resin, spin dry, extract, and freeze-dry to obtain the crude peptide:
  • the crude peptide resin was swollen with dichloromethane, compressed with anhydrous methanol, and dried three times. Use trifluoroacetic acid/triisopropylsilane/double distilled water to cut the crude peptide from the resin, spin dry, extract, and freeze-dry to obtain the crude peptide:
  • the crude peptide resin was swollen with dichloromethane, compressed with anhydrous methanol, and dried three times. Use trifluoroacetic acid/triisopropylsilane/double distilled water to cut the crude peptide from the resin, spin dry, extract, and freeze-dry to obtain the crude peptide:
  • the present invention selects ICR male rats with a body weight range of 25-30g, puts them into an observation room and adapts to them for 15 minutes, injects different doses of compound 4 (5nmol, 10nmol, 30nmol) or normal saline through intrathecal injection, and injects 5% of them subcutaneously through the sole of the foot after 5 minutes.
  • the formalin solution (20 ⁇ l) was put into the observation room immediately, and the cumulative time of the pain behavior (licking, biting, and shaking the injected foot) of the mice in the time period of 0-10min and 10-45min was counted respectively.
  • the present invention selects ICR male mice in the range of 25-30g body weight, puts them into the observation room and adapts to them for 15 minutes, injects different doses of compound 4 (5nmol, 10nmol, 30nmol) or normal saline intrathecally, and injects 0.6% glacial acetic acid intraperitoneally after 5 minutes solution (10mL/kg). Put them into the observation room, and count the number of writhing times of the mice within 20 minutes.
  • mice In the acetic acid-induced writhing model, mice exhibited behaviors such as writhing, limb extension, and belly pressing.
  • the experimental results are shown in Figure 4, the times of writhing of the mice decreased with the increase of the administered dose. Compound 4 can significantly reduce the number of abdominal twisting in mice in a dose-dependent manner. Intrathecal injection of 30nmol dose, compound 4 inhibited the writhing behavior of mice by 63%. The ED50 of compound 4 is 13.5 (11.88-15.34) nmol.
  • the experimental results show that compound 4 has a good inhibitory effect on acetic acid-induced visceral pain.
  • the compounds 1-5 involved in the present invention can target PD-1 receptors, have a good relieving effect on inflammatory pain and visceral pain, and have potential application value in the preparation of analgesic drugs.
  • the compound 4 is preferably evaluated in vitro and in vivo for drug activity; compound 4 is evaluated for drug analgesic activity by DRG neuron calcium imaging, formalin-induced inflammatory pain model, and acetic acid writhing experiment Evaluation, but the application of the polypeptide compound of the present invention in the preparation of analgesic drugs, including but not limited to inflammatory pain and visceral pain, such as postoperative neuralgia, chemotherapy pain caused by chemotherapy, diabetes pain, and cancer pain, etc. Various pathological pains.
  • the administration method is preferably intrathecal administration.
  • the compound described in this patent can be applied to various administration methods, such as subcutaneous administration , tail vein administration, lateral ventricle administration, intraperitoneal administration, and oral administration, etc.

Abstract

本发明公开了一类以PD-1受体为靶点的多肽化合物，多肽化合物包括化合物1、化合物2、化合物3、化合物4和化合物5；其中，化合物1-5的氨基酸序列如SEQ ID NO：1-5所示。本发明还公开了一类以PD-1受体为靶点的多肽化合物的制备方法，包括以下过程：利用固相合成方式，按照肽序列依次将化合物1-5的氨基酸偶联到固相树脂上，经过切割试剂裂解、冷冻干燥、制备纯化获得目的化合物。发明还公开了一类以PD-1受体为靶点的多肽化合物在制备镇痛药物的应用。本发明的小分子多肽：化合物1～5能够结合PD-1,鞘内给药下对炎症痛和内脏痛均具有显著的抑制效果；且合成简单，易纯化，利于大规模合成。

Description

一类以PD-1受体为靶点的多肽化合物、其制备方法及应用 技术领域
本发明属于生物医药技术领域，具体涉及一类以PD-1受体为靶点的多肽化合物、其制备方法及应用。
背景技术
近年来，包括腰痛、关节炎、术后持久性疼痛、纤维组织肌痛、神经病理性疾病在内的多种慢性疼痛是当今非常普遍的社会问题，发达国家中有超过20％的成年人正遭受慢性痛的折磨(J.Neurosci.2021,41，855-865)。慢性痛虽不致命，但并未得到好的治疗，给社会和经济带来严重的负担。尽管免疫细胞、胶质细胞和神经元之间的信号转导现在被认为是引发和维持慢性痛所不可缺少的，绝大多数的治疗药物依旧仅针对神经元。
当前，阿片类药物已被广泛应用于临床治疗非癌症慢性痛，但阿片药物不可避免的引起药物耐受性、身体和精神依赖性、胃肠道功能抑制、恶心、镇定、烦躁、幻觉、以及运动功能损伤等副作用，是阻碍其在临床治疗中应用的主要问题。因而，迫切需要研发新型的有效且安全镇痛药物。
程序性死亡配体(PD-L1也称为B7-H1或CD274),是B7家族中的成员之一，在患有癌症人类中广泛表达。肿瘤的微环境中，PD-L1能够与PD-1结合，作为一种共抑制检查点分子，参与免疫调节。当前，PD-1/PD-L1通路大量的研究工作主要集中在肿瘤免疫逃逸中T-淋巴细胞的耐受以及与巨噬细胞的活化与极化。近些年来，PD-L1/PD-1在肿瘤治疗之外的功能也逐渐的被报道，包括鼠狼疮、结肠炎、胶原诱导性关节炎、以及脑损伤。
最新的研究发现，PD-L1/PD-1通路对神经损伤后的急性和慢性疼痛具有很好的疗效，注射外源性PD-L1能够明显提高正常小鼠的疼痛阈值(Nat.Neurosci.2017,20,917-926)；且PD-L1和低剂量的吗啡联合给药后，能够显著增强吗啡的镇痛效果，从而降低吗啡高剂量用药所引起的副作用(Sci.Transl.Med.2020,12,eaaw6471)。因此，以PD-1靶点在镇痛新药的研发中具有潜在的应用前景。
发明内容
发明目的：针对现有技术中存在问题或不足，本发明提供一类以PD-1受体为靶点的多肽化合物、其制备方法及应用。
为实现上述发明目的，本发明的实施例提供一类以PD-1受体为靶点的多肽化合物，其特征在于，所述多肽化合物包括化合物1、化合物2、化合物3、化合物4和化合物5；其中，化合物1-5的氨基酸序列如SEQ ID NO：1-5所示，SEQ ID NO：1-5分别为：
SEQ ID NO：1：Val Tyr Arg Cys Met Ile Ser Tyr Gly；
SEQ ID NO：2：Tyr Arg Cys Met Ile Ser Tyr Gly Gly；
SEQ ID NO：3：Met Ile Ser Tyr Gly Gly Ala Asp Tyr；
SEQ ID NO：4：Ser Tyr Gly Gly Ala Asp Tyr Lys Arg；
SEQ ID NO：5：Tyr Gly Gly Ala Asp Tyr Lys Arg Ile。
本发明的实施例还提供一类以PD-1受体为靶点的多肽化合物的制备方法，其特征在于，包括以下过程：(1)利用固相合成方式，按照肽序列依次将化合物1～5的氨基酸偶联到固相树脂上，(2)经过切割试剂裂解、冷冻干燥、制备纯化获得目的化合物。
进一步的，所述步骤(1)具体包括以下步骤：
(1-1)固相合成中的固相载体为wang树脂，wang树脂经二氯甲烷溶胀，无水甲醇压缩，N,N-二甲基乙酰胺洗涤；
(1-2)将经过预处理后的树脂，加入适量的Fmoc基团脱除试剂使树脂氨基保护，所使用的氨基保护基团9-芴甲氧羰基的脱除试剂为20％六氢吡啶的N,N-二甲基甲酰胺溶液，洗脱次数为3次，每次5min，洗脱结束后，用N,N-二甲基甲酰胺洗涤，树脂/肽树脂茚检；
(1-3)在固相合成缩合剂的参与下，从化合物1～5肽序列的C端第一个氨基酸开始，依次缩合至肽树脂上，氨基酸的量为肽树脂的物质的量的3倍，氨基酸缩合所用时间为1～1.5h，缩合反应应在惰性气体氛围下进行，得到粗肽树脂；其中，固相合成缩合剂为N,N-二异丙基乙胺/1-羟基苯并三唑/O-苯并三氮唑-四甲基脲六氟磷酸盐，所用量为树脂的3～6倍。
进一步的，所述步骤(2)具体包括以下步骤：
(2-1)将步骤(1)所得到的粗肽树脂经由二氯甲烷/无水甲醇反复溶胀压缩处理；
(2-2)采用切割试剂将粗肽从粗肽树脂上切割、旋干、萃取、冷冻干燥得到所需粗肽；粗肽树脂的裂解所需切割试剂包括三氟乙酸、三异丙基硅烷和双蒸水，其中，三氟乙酸:三异丙基硅烷：双蒸水的体积比＝95:2.5:2.5；
(2-3)粗肽树脂经过冷冻干燥、反向-HPLC制备柱纯化，得到白色固体化合物1～5，产率35％～45％；制得一类以PD-1受体为靶点的多肽化合物。
本发明的实施例还提供一类以PD-1受体为靶点的多肽化合物在制备镇痛药物的应用。
优选的，所述镇痛药物包括治疗炎症痛和内脏痛的药物。
优选的，所述药物适合鞘内给药、皮下给药、尾静脉给药、侧脑室给药、腹腔给药或口服给药中的任一种方式。
优选的，所述药物的治疗靶点为PD-1受体。
优选的，所述多肽化合物包括化合物1、化合物2、化合物3、化合物4和化合物5中的一种。
本发明的上述技术方案的有益效果如下：
(1)本发明的小分子多肽：化合物1～5为利用分子对接技术从肽库筛选得到的5条Amber得分在[-90,-80]区间内的化合物；这五条化合物能够结合PD-1,鞘内给药下对炎症痛和内脏痛均具有显著的抑制效果；且合成简单，易纯化，利于大规模合成。
(2)本发明的化合物1～5，其简短的肽序列相比于PD-L1更容易做为化学模板进行修饰改造，以提高其药理学特性和药代动力学特性。
(3)本发明基于之前的研究基础，发现一类新型小分子多肽，其与PD-L1具有部分相似的特征，能够靶向结合PD-1，对福尔马林诱发炎症痛与醋酸诱发的内脏痛具有显著地镇痛作用，具有潜在的临床应用价值。
(4)本发明的化合物1～5，相比于常规阿片类药物，其作用靶点为PD-1受体，能够更好的规避由激活阿片受体所诱发的阿片相关的副作用，在药物的安全性上，相对更具优势，具有临床应用的潜力。
附图说明
图1为本发明的实施例中化合物4的ESI-MS谱图；
图2为本发明的实施例中化合物4对高K+溶液诱导DRG细胞内游离Ca2+增加的抑制作用图；
图3为本发明的实施例中鞘内注射化合物4对福尔马林溶液诱发的急性炎症痛的镇痛作用图；
图4为本发明的实施例中鞘内注射化合物4对腹腔注射醋酸溶液诱导的内脏痛的镇痛作用图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合具体实施例进行详细描述。
实施例1化合物1～5的固相合成
本发明的化合物1～5的合成采用Fmoc-固相合成方法，所用固相载体为wang-树脂。氨基保护基团9-芴甲氧羰基(Fmoc)的脱除试剂为20％六氢吡啶的N,N-二甲基甲酰胺溶液。肽链延伸所需缩合试剂为N，N-二异丙基乙胺/1-羟基苯并三唑/O-苯并三氮唑-四甲基脲六氟磷酸盐，所需量为所用树脂物质的量的3～6倍。粗肽从树脂上切割所需切割剂为体积比为95:2.5:2.5的三氟乙酸/三异丙基硅烷/双蒸水的混合液。本发明中缩合试剂、Fmoc脱除试剂、茚检试剂的组分及比例为本领域技术人员公知的技术。
1)化合物1的合成
称取wang树脂，二氯甲烷溶胀，无水甲醇压缩，N,N-二甲基乙酰胺洗涤。经过预处理后的树脂，加入适量的Fmoc基团脱除试剂使树脂氨基去保护。在N，N-二异丙基乙胺/1-羟基苯并三唑/O-苯并三氮唑-四甲基脲六氟磷酸盐缩合试剂参与下，依次将N-芴甲氧羰基-甘氨酸、N-芴甲氧羰基-O-叔丁基-L-酪氨酸、N-芴甲氧羰基-L-丝氨酸、N-芴甲氧羰基-L-异亮氨酸、N-芴甲氧羰基-L-蛋氨酸、N-(9-芴甲氧羰基)-S-三苯甲基-L-半胱氨酸、N-(9-芴甲氧羰基)-NG-2,2,4,6,7-五甲基苯并呋喃-5-磺酰基-L-精氨酸、N-芴甲氧羰基-O-叔丁 基-L-酪氨酸、N-芴甲氧羰基-L-缬氨酸缩合到肽树脂上。粗肽树脂经过二氯甲烷溶胀、无水甲醇压缩，反复三次后抽干。采用三氟乙酸/三异丙基硅烷/双蒸水将粗肽从树脂上切割、旋干、萃取、冷冻干燥得到粗肽：
H-Val-Tyr-Arg-Cys-Met-Ile-Ser-Tyr-Gly-OH，经由RP-HPLC制备柱纯化，产率38％。
质谱和纯度分析鉴定结果如表1所示。
2)化合物2的合成
同化合物1合成过程中的树脂预处理和Fmoc基团移除的操作。在N，N-二异丙基乙胺/1-羟基苯并三唑/O-苯并三氮唑-四甲基脲六氟磷酸盐缩合试剂参与下，依次将N-芴甲氧羰基-甘氨酸、N-芴甲氧羰基-甘氨酸、N-芴甲氧羰基-O-叔丁基-L-酪氨酸、N-芴甲氧羰基-L-丝氨酸、N-芴甲氧羰基-L-异亮氨酸、N-芴甲氧羰基-L-蛋氨酸、N-(9-芴甲氧羰基)-S-三苯甲基-L-半胱氨酸、N-(9-芴甲氧羰基)-NG-2,2,4,6,7-五甲基苯并呋喃-5-磺酰基-L-精氨酸、N-芴甲氧羰基-O-叔丁基-L-酪氨酸缩合到肽树脂上。粗肽树脂经过二氯甲烷溶胀、无水甲醇压缩，反复三次后抽干。采用三氟乙酸/三异丙基硅烷/双蒸水将粗肽从树脂上切割、旋干、萃取、冷冻干燥得到粗肽：
H-Tyr-Arg-Cys-Met-Ile-Ser-Tyr-Gly-Gly-OH，经由RP-HPLC制备柱纯化，产率35％。
质谱和纯度分析鉴定结果如表1所示。
3)化合物3的合成
同化合物1合成过程中的树脂预处理和Fmoc基团移除的操作。在N,N-二异丙基乙胺/1-羟基苯并三唑/O-苯并三氮唑-四甲基脲六氟磷酸盐缩合试剂参与下，依次将N-芴甲氧羰基-O-叔丁基-L-酪氨酸、N-芴甲氧羰基-L-天冬氨酸-4-叔丁酯、N-芴甲氧羰基-丙氨酸、N-芴甲氧羰基-甘氨酸、N-芴甲氧羰基-甘氨酸、N-芴甲氧羰基-O-叔丁基-L-酪氨酸、N-芴甲氧羰基-L-丝氨酸、N-芴甲氧羰基-L-异亮氨酸、N-芴甲氧羰基-L-蛋氨酸缩合到肽树脂上。粗肽树脂经过二氯甲烷溶胀、无水甲醇压缩，反复三次后抽干。采用三氟乙酸/三异丙基硅烷/双蒸水将粗肽从树脂上切割、旋干、萃取、冷冻干燥得到粗肽：
H-Met-Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-OH，经由RP-HPLC制备柱纯化，产率45％。
质谱和纯度分析鉴定结果如表1所示。
4)化合物4的合成
同化合物1合成过程中的树脂预处理和Fmoc基团移除的操作。在N，N-二异丙基乙胺/1-羟基苯并三唑/O-苯并三氮唑-四甲基脲六氟磷酸盐缩合试剂参与下，依次将N'-[(2,3-二氢-2,2,4,6,7-五甲基苯并呋喃-5-基)磺酰基]-N-芴甲氧羰基-L-精氨酸、N-alpha-芴甲氧羰基-N-epsilon-叔丁氧羰基-L-赖氨酸、N-芴甲氧羰基-O-叔丁基-L-酪氨酸、N-芴甲氧羰基-L-天冬氨酸-4-叔丁酯、N-芴甲氧羰基-丙氨酸、N-芴甲氧羰基-甘氨酸、N-芴甲氧羰基-甘氨酸、N-芴甲氧羰基-O-叔丁基-L-酪氨酸、N-芴甲氧羰基-L-丝氨酸缩合到肽树脂上。粗肽树脂经过二氯甲烷溶胀、无水甲醇压缩，反复三次后抽干。采用三氟乙酸/三异丙基硅烷/双蒸水将粗肽从树脂上切割、旋干、萃取、冷冻干燥得到粗肽：
H-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-Arg-OH，经由RP-HPLC制备柱纯化，产率40％。
质谱和纯度分析鉴定结果如表1所示，质谱图如附图1所示。
5)化合物5的合成
同化合物1合成过程中的树脂预处理和Fmoc基团移除的操作。在N，N-二异丙基乙胺/1-羟基苯并三唑/O-苯并三氮唑-四甲基脲六氟磷酸盐缩合试剂参与下，依次将N-芴甲氧羰基-L-异亮氨酸、N'-[(2,3-二氢-2,2,4,6,7-五甲基苯并呋喃-5-基)磺酰基]-N-芴甲氧羰基-L-精氨酸、N-alpha-芴甲氧羰基-N-epsilon-叔丁氧羰基-L-赖氨酸、N-芴甲氧羰基-O-叔丁基-L-酪氨酸、N-芴甲氧羰基-L-天冬氨酸-4-叔丁酯、N-芴甲氧羰基-丙氨酸、N-芴甲氧羰基-甘氨酸、N-芴甲氧羰基-甘氨酸、N-芴甲氧羰基-O-叔丁基-L-酪氨酸缩合到肽树脂上。粗肽树脂经过二氯甲烷溶胀、无水甲醇压缩，反复三次后抽干。采用三氟乙酸/三异丙基硅烷/双蒸水将粗肽从树脂上切割、旋干、萃取、冷冻干燥得到粗肽：
H-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-Arg-Ile-OH，经由RP-HPLC制备柱纯化，产率42％。
质谱和纯度分析鉴定结果如表1所示。
本发明的化合物1～5的肽序列、质谱鉴定、Amber打分及纯度分析结果如表1所示：
表1化合物理化性质、Amber打分及纯度分析表
Figure PCTCN2022096623-appb-000001
本发明的氨基酸缩写如表2所示：
简写 英文缩写 全称
V Val 缬氨酸
Y Tyr 酪氨酸
R Arg 精氨酸
C Cys 半胱氨酸
M Met 蛋氨酸
I Ile 异亮氨酸
S Ser 丝氨酸
G Gly 甘氨酸
A Ala 丙氨酸
D Asp 天冬氨酸
K Lys 赖氨酸
实施例2 DRG神经元钙成像实验
通过DRG神经元钙成像技术鉴定化合物1～5对疼痛信号传导的抑制效果，具体包括以下步骤：
3周雌性ICR小鼠，通过急性分离，在无菌操作台提取DRG，胶原酶A和胰酶依次裂解。含有血清的完全培养基终止胰酶裂解，800r离心,弃上清，加Neurobasal培养基(含2％B27，Clutanine 1％，以及1％青霉素-链霉素双抗溶液)吹打均匀后，在提前铺好PDL的小皿中培养48h。实验前采用Flou-4/AM标记DRG神经元，经过不同剂量化合物1～5/等体积HBSS预处理后，高K+溶液刺激DRG神经元，激光共聚焦下检测DRG神经元胞内游离钙离子浓度的变化。
钙成像的实验结果如图2所示：化合物4预处理(1nM)DRG神经元对高K+诱发胞内Ca2+浓度的增加具有显著性的抑制作用,说明化合物4能够调控疼痛信号的传递。
实施例3福尔马林诱发的炎症痛实验
本发明选用体重范围为25-30g的ICR雄鼠，放入观察室适应15min,通过鞘内注射不同剂量的化合物4(5nmol、10nmol、30nmol)或生理盐水，5min后通过足底皮下注射5％的福尔马林溶液(20μl)，立即放入观测室，分别统计小鼠0-10min、10-45min时间段内的疼痛行为(舔、咬、甩注射足)的累积时间。
在福尔马林诱导的小鼠疼痛模型中，经小鼠足底皮下注射福尔马林后，小鼠表现出舔、咬、甩爪子及伸缩腿等疼痛行为。注射福尔马林后0-10min为I相，主要由伤害性神经元的直接激活所引发；10-35min为II相，主要由福尔马林诱发的炎症反应所引发。实验结果如图3所示，与对照组相比，化合物4显著缩短了两相中小鼠舔咬甩注射足的疼痛行为，说明化合物4可以减轻由伤害神经元激活和炎症引起的疼痛行为。化合物4对福尔马林炎症因子引发的疼痛行为的抑制效果比伤害性神经元直接激活引发的伤害行为更显著。其中II相痛中，化合物4的ED50(半数有效量,median effective dose)为6.28(5.34-7.39)nmol。
实施例4醋酸诱发的内脏痛实验
本发明选用25-30g体重范围内的ICR雄鼠，放入观察室适应15min，通过鞘内注射不同剂量的化合物4(5nmol，10nmol,30nmol)或生理盐水，5min 后腹腔注射0.6％的冰乙酸溶液(10mL/kg)。放入观察室，统计20min内的小鼠扭体次数。
在醋酸诱导的扭体模型中，小鼠表现出扭动、肢体伸展、腹部贴地等行为。实验结果如图4所示，小鼠的扭体次数随着给药剂量的增加而减少。化合物4能够剂量依赖性的显著降低小鼠的腹部扭动次数。鞘内注射30nmol剂量，化合物4对小鼠扭体行为的抑制率为63％。化合物4的ED50为13.5(11.88-15.34)nmol。实验结果说明，化合物4对醋酸诱发的内脏痛具有很好地抑制作用。
综上，本发明中所涉及的化合物1～5，能够靶向结合PD-1受体,对炎症痛和内脏痛具有很好的缓解作用，在制备镇痛药物方面具有潜在的应用价值。
本发明的实施例中优选所述化合物4进行体外和在体药物活性评估；化合物4通过DRG神经元钙成像、福尔马林诱导的炎症痛模型、和醋酸扭体实验进行药物镇痛活性的评估，但本发明的多肽化合物在制备镇痛药物中的应用，包括但不应局限于炎症痛和内脏痛，例如术后神经痛、化学药物治疗引发的化疗痛、糖尿病痛、和癌痛等各种病理性疼痛。
化合物4在福尔马林实验和醋酸扭体实验中，给药方式优选鞘内给药，本领域的技术人员可以理解，本专利所述化合物可适用于多种给药方式，如皮下给药、尾静脉给药、侧脑室给药、腹腔给药、以及口服给药等。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (8)

  1. 一种多肽化合物，其特征在于，所述多肽化合物包括包括化合物1、化合物2、化合物3、化合物4和化合物5；其中，化合物1-5的氨基酸序列如SEQ ID NO：1-5所示，SEQ ID NO：1-5分别为：
    SEQ ID NO：1：Val Tyr Arg Cys Met Ile Ser Tyr Gly；
    SEQ ID NO：2：Tyr Arg Cys Met Ile Ser Tyr Gly Gly；
    SEQ ID NO：3：Met Ile Ser Tyr Gly Gly Ala Asp Tyr；
    SEQ ID NO：4：Ser Tyr Gly Gly Ala Asp Tyr Lys Arg；
    SEQ ID NO：5：Tyr Gly Gly Ala Asp Tyr Lys Arg Ile。
  2. 一种根据权利要求1所述的多肽化合物的制备方法，其特征在于，包括以下过程：(1)利用固相合成方式，按照肽序列依次将氨基酸偶联到固相树脂上，(2)经过切割试剂裂解、冷冻干燥、制备纯化获得目的化合物。
  3. 根据权利要求2所述的一种多肽化合物的制备方法，其特征在于，步骤(1)具体包括以下步骤：
    (1-1)固相合成中的固相载体为wang树脂，wang树脂经二氯甲烷溶胀，无水甲醇压缩，N,N-二甲基乙酰胺洗涤；
    (1-2)将经过预处理后的树脂，加入适量的Fmoc基团脱除试剂使树脂氨基去保护，所使用的氨基保护基团9-芴甲氧羰基的脱除试剂为20％六氢吡啶的N,N-二甲基甲酰胺溶液，洗脱次数为3次，每次5min，洗脱结束后，用N,N-二甲基甲酰胺洗涤，树脂/肽树脂茚检；
    (1-3)在固相合成缩合剂的参与下，从化合物1～5肽序列的C端第一个氨基酸开始，依次缩合至肽树脂上，氨基酸的量为肽树脂的物质的量的3倍，氨基酸缩合所用时间为1～1.5h，缩合反应应在惰性气体氛围下进行，得到粗肽树脂；其中，固相合成缩合剂为N,N-二异丙基乙胺/1-羟基苯并三唑/O-苯并三氮唑-四甲基脲六氟磷酸盐，所用量为树脂的3～6倍。
  4. 根据权利要求2所述的一种多肽化合物的制备方法，其特征在于，步骤(2)具体包括以下步骤：
    (2-1)将步骤(1)所得到的粗肽树脂经由二氯甲烷/无水甲醇反复溶胀压缩处理；
    (2-2)采用切割试剂将粗肽从粗肽树脂上切割、旋干、萃取、冷冻干燥得到所需粗肽；粗肽树脂的裂解所需切割试剂包括三氟乙酸、三异丙基硅烷和双蒸水，其中，三氟乙酸:三异丙基硅烷：双蒸水的体积比＝95:2.5:2.5；
    (2-3)粗肽树脂经过冷冻干燥、反向-HPLC制备柱纯化，得到白色固体化合物1～5，产率35％～45％；制得一类以PD-1受体为靶点的多肽化合物。
  5. 一种根据权利要求1所述的多肽化合物在制备镇痛药物的应用。
  6. 根据权利要求5所述的一种多肽化合物在制备镇痛药物的应用，其特征在于，所述镇痛药物包括治疗炎症痛和内脏痛的药物。
  7. 根据权利要求5所述的一种多肽化合物在制备镇痛药物的应用，其特征在于，所述药物适合鞘内给药、皮下给药、尾静脉给药、侧脑室给药、腹腔给药或口服给药中的任一种方式。
  8. 根据权利要求5所述的一种多肽化合物在制备镇痛药物的应用，其特征在于，所述药物的治疗靶点为PD-1受体。
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CN110183514A (zh) * 2016-04-21 2019-08-30 兰州大学 一类阿片和神经肽ff受体的多靶点肽类分子及其制备和应用
CN111606976A (zh) * 2020-05-26 2020-09-01 中国人民解放军军事科学院军事医学研究院 一种小肽及其在抑制阿片成瘾性和耐受性上的应用
CN113416232A (zh) * 2021-06-10 2021-09-21 南通大学 一类以pd-1受体为靶点的多肽化合物、其制备方法及应用

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