WO2020237709A1 - 长效化艾塞那肽衍生物及其盐与制备方法和用途 - Google Patents

长效化艾塞那肽衍生物及其盐与制备方法和用途 Download PDF

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WO2020237709A1
WO2020237709A1 PCT/CN2019/090218 CN2019090218W WO2020237709A1 WO 2020237709 A1 WO2020237709 A1 WO 2020237709A1 CN 2019090218 W CN2019090218 W CN 2019090218W WO 2020237709 A1 WO2020237709 A1 WO 2020237709A1
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acid
exenatide
ser
exenatide derivative
derivative
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PCT/CN2019/090218
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English (en)
French (fr)
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谷海涛
赵呈青
王蔡典
姜建军
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江苏诺泰澳赛诺生物制药股份有限公司
杭州诺泰澳赛诺医药技术开发有限公司
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Publication of WO2020237709A1 publication Critical patent/WO2020237709A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the invention relates to the technical field of polypeptide compounds, in particular to an exenatide derivative, a preparation method thereof, a medicament administration compound and its use as a medicine.
  • the cause of metabolic syndrome is abnormal metabolism of protein, fat and carbohydrates. Overnutrition and reduced physical activity can lead to obesity and obesity-related diseases such as diabetes. In recent years, the incidence of type 2 diabetes and abnormal blood lipid metabolism has been increasing.
  • Glucagon-like peptide-1 (GLP-1) is a glucose-dependent incretin hormone. It can stimulate GLP-1 receptors and exert a hypoglycemic effect. The most significant function is to promote the regeneration and repair of ⁇ cells, increase the number of islet ⁇ cells, and at the same time avoid the risk of hypoglycemia that often occurs in the treatment of diabetes. It has broad application prospects in the treatment of diabetes. Although natural GLP-1 has many advantages in the treatment of diabetes, it is easily degraded by dipeptidyl peptidase IV (DPP-IV) in the body, and its half-life in vivo is only about 3 minutes.
  • DPP-IV dipeptidyl peptidase IV
  • Exenatide is extracted from the salivary glands of the American venomous lizard and was approved for marketing by the FDA in 2005. Its peptide sequence is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH 2 . Exenatide is not a substrate of DPP-4, so it will not be degraded by DPP-4 which is widely present in the body. Its half-life in vivo is longer than that of endogenous GLP-1, about 2.4h. However, Exenatide will still be filtered and eliminated quickly in the kidney, and the degradation of the anti-DPP-IV enzyme can only extend the half-life of GLP-1 to a certain extent.
  • the purpose of the present invention is to provide a long-acting exenatide derivative in view of the deficiencies of the prior art.
  • the derivative On the basis of exenatide, the derivative maintains its highly agonistic activity on GLP-1 receptors.
  • glucagon On the basis of, glucagon is used for site replacement to increase its agonistic activity on glucagon to obtain exenatide-derived sequence with dual target agonistic activity.
  • the present invention innovatively connects two preferred exenatide-derived polypeptide sequences using lysine and conjugated with fatty acid chains of different lengths.
  • the fatty acid chain can increase the binding time of the conjugate and serum albumin.
  • the overall molecular volume is larger than that of exenatide, which can slow down the kidney's rapid filtration, and both work at the same time, which greatly extends the peptide chain Action time in the body. Therefore, this type of dual-effect exenatide analogue can significantly prolong its in vivo action time under the premise of having hypoglycemic and weight loss activities.
  • Another object of the present invention is to provide a method for preparing exenatide derivatives.
  • Another object of the present invention is to provide pharmaceutically acceptable exenatide derivative salts prepared by using exenatide derivatives.
  • Another object of the present invention is to provide an exenatide derivative medicament prepared by the exenatide derivative.
  • Another object of the present invention is to provide a pharmaceutical composition containing exenatide derivatives.
  • Another object of the present invention is to provide the use of exenatide derivatives, the use of exenatide derivative salts and the use of exenatide derivative medicaments.
  • the purpose of the present invention is also achieved through the following technical solutions.
  • the present invention is a long-acting exenatide derivative whose amino acid sequence is:
  • Xaa1 is taken from: Aib or Gly
  • Xaa2 is taken from:
  • n is a natural number of 5-17, preferably 11, 13, or 15.
  • the preferred long-acting exenatide derivative of the present invention can be expressed as:
  • the invention also discloses a pharmaceutically acceptable exenatide derivative salt prepared by the exenatide derivative, and the salt is exenatide derivative and hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid , Pyrosulfuric acid, phosphoric acid, nitric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid , Undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentane propionic acid, digluconic acid, 3-hydroxy- 2-Naphthoic acid, Niacin
  • the present invention also provides a pharmaceutical composition, which includes a therapeutically effective amount of at least one of the above-mentioned exenatide derivative compounds or pharmaceutically acceptable salts thereof, or pharmaceutically acceptable carriers or diluents.
  • the present invention further provides the use of the exenatide derivative compound and its pharmaceutically acceptable salt, or a pharmaceutically acceptable carrier or diluent, in the preparation of drugs for the treatment and prevention of diabetes.
  • the present invention also discloses the exenatide derivative medicament prepared by using the exenatide derivative.
  • the dosage form of the exenatide derivative is selected from the group consisting of tablets, capsules, elixirs, syrups, lozenges, inhalants, sprays, injections, and films. Preparations, patches, powders, granules, blocks, emulsions, suppositories or compound preparations.
  • the present invention also discloses the use of the exenatide derivative, which is characterized in that the use is in the preparation of exenatide derivatives for the treatment and/or prevention of diabetes, obesity, hyperlipidemia, non-alcoholic fat Application of liver medicine.
  • the present invention also discloses the use of exenatide derivative salt, which is characterized in that the use is the preparation of exenatide derivative salt for the treatment and/or prevention of diabetes, obesity, hyperlipidemia, and non-alcoholic fatty liver Application of the drug.
  • the present invention also discloses the use of exenatide derivative medicament, which is characterized in that the use is the preparation of exenatide derivative medicament for the treatment and/or prevention of diabetes, obesity, hyperlipidemia, and non-alcoholic fatty liver Application of the drug.
  • the present invention also provides methods for preparing exenatide derivatives and intermediates thereof.
  • the method for preparing exenatide derivative polypeptides provided by the present invention adopts solid-phase synthesis to gradually couple each of the main chains of such hypoglycemic polypeptides.
  • the amino acid is cleaved to obtain the main chain polypeptide protected by the side chain, and the aliphatic chain lysine is reacted with the main chain to obtain the exenatide derivative.
  • This method has simple synthesis steps, high coupling efficiency and easy purification, which is beneficial to the industrial production of such polypeptides.
  • the preparation method of the preferred exenatide derivative of the present invention comprises the following steps:
  • Step 1 Take the resin, and after activation, gradually couple amino acids to obtain the first peptide resin;
  • Step 2 Take the first peptide resin, cleavage and purify to obtain the side chain protected polypeptide main chain;
  • Step 3 The lysine with the fatty chain is reacted with twice the amount of the main chain, and it is obtained after purification;
  • the resin in step 1 is 2-CTC Resin or Wang Resin.
  • the lysis reagent used for lysis in step 2 is a mixture of TFA and DCM.
  • the volume ratio of TFA and DCM in the reagent used for lysis in step 2 is 0.1:99.9.
  • the purification method used in step 2 and step 3 is chromatographic separation.
  • the chromatographic column used for purification in step 2 and step 3 is a C18 column.
  • GLP-1 and glucagon can be obtained after translation and shearing of preproglucagon, and the two have certain sequence homology. Therefore, by introducing part of the glucagon sequence into Exenatide, which has a high agonistic activity to GLP-1, a dual-acting agonist that simultaneously agonizes GLP-1R and GCGR can be obtained, thereby obtaining a polypeptide with excellent hypoglycemic activity and weight loss effect. Compound.
  • the long-acting exenatide derivative with dual-receptor agonism proposed by the present invention can have a weight-reducing effect on the basis of retaining the glucose-lowering activity, and the biological half-life is longer than that of the exenatide prototype ( ⁇ 2.4h ) Significantly prolonged, partially reaching more than 36 hours, greatly prolonging the effect of reducing blood sugar and weight.
  • the method of the present invention adopts the solid-phase synthesis of exenatide derivatives with orthogonal protection strategy to obtain a crude peptide chain with a purity of greater than 85%, which is greatly improved compared with conventional synthesis methods, and facilitates subsequent purification work.
  • the method of the present invention adopts a solid-phase method to synthesize exenatide derivatives with low cost. Due to the high coupling efficiency, the required amount of protected amino acids only needs an average of 2 times excess, while conventional synthetic methods require 4 to 5 times excess amino acids, which greatly saves costs.
  • the method of the present invention adopting the Fmoc/tBu orthogonal protection solid-phase synthesis strategy to synthesize exenatide derivatives is easy to realize automation and large-scale, which makes it more suitable for industrial production.
  • Exenatide derivatives prepared by solid-phase synthesis technology provided by the present invention have good hypoglycemic and weight-reducing activities, long drug effect time, high yield, short synthesis cycle, easy purification of crude products, and low production cost. , Easy to industrial automation production.
  • the prepared exenatide derivative is suitable as an active ingredient of drugs for treating diabetes and obesity.
  • Figure 1 shows the experimental results of abdominal glucose tolerance of exenatide derivatives SEQ.ID NO:1 ⁇ 6;
  • Figure 2 shows the results of the blood glucose stabilization experiment of the exenatide derivative SEQ. ID NO: 1 to 6.
  • Example 7 The following are related pharmacological experiment methods and results of Exenatide derivatives involved in the present invention:
  • HEK293 cells were co-transfected with cDNA encoding GLP-1R or GCGR.
  • cells were seeded in a 96-well plate 2 hours in advance, the compound was dissolved in DMSO, diluted to different multiples with a medium containing 0.1% bovine serum albumin, and added to the co-transfected cells. After the cells were incubated for 20 minutes, the ELISA kit of Cisbo was used to measure the fluorescence readings with a microplate reader, and a standard curve was established to convert the fluorescence readings into the corresponding cAMP values. The EC 50 values of the compounds were calculated using the nonlinear regression of Graphpad Prism 5.0 software.
  • Results are expressed as mean ⁇ SD,**P ⁇ 0.01 vs Glucagon,##P ⁇ 0.01 vs Exenatide.
  • mice Normal Kunming mice were randomly divided into groups with 8 mice in each group. The mice were kept in a standardized animal room. Fasting for 12 hours before the experiment and only drinking water.
  • the initial blood glucose value was measured and set at -30 min, and then 50 nmol/kg exenatide derivatives were injected intraperitoneally. After 30 minutes, 18mmol/kg glucose solution was injected intraperitoneally, set as 0min, and the control group was injected with the same volume of normal saline or 50nmol/kg exenatide. At 0, 15, 30, 45, 60, 120 min, the blood glucose level was measured with a blood glucose meter to detect the hypoglycemic activity of exenatide derivatives.
  • the results of the hypoglycemic experiment show that when the concentration of the exenatide derivative in the present invention is 50 nmol/kg, the hypoglycemic effect is equivalent to the hypoglycemic effect of exenatide.
  • the blood glucose of STZ-induced diabetic model mice was measured, and mice with a value higher than 20mmol/L were selected for random grouping, six in each group, and the mice were free to eat during the experiment.
  • the positive control group was intraperitoneally injected with exenatide or liraglutide at a dose of 50 nmol/kg
  • the negative control group was intraperitoneally injected with normal saline
  • the administration group was injected with 50 nmol/kg of exenatide derivatives.
  • Compounds were administered at 0h, and blood glucose levels were measured using a blood glucose meter at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 24, 36, 48, and 60 h, respectively.
  • the evaluation index is the time when the blood glucose value of the mouse is lower than 8.35 mmol/L after intraperitoneal injection of the compound.
  • the blood glucose stabilization time of exenatide is only 4 hours, and the blood glucose stabilization time of liraglutide is 10 hours.
  • the long-acting hypoglycemic polypeptides involved in the present invention have a blood glucose stabilization time of more than 24 hours. Can exceed 35h. Stabilization of blood sugar experiments showed that Exenatide derivatives have good long-acting hypoglycemic effects, can achieve better long-acting hypoglycemic effects, and have the potential to develop into once-a-day hypoglycemic drugs.
  • mice Male C57bl/6 mice were fed with high-fat diet for 4 weeks, and mice weighing more than 30g were selected for the experiment. Mice were randomly divided into 8 groups, 8 groups in total, exenatide derivatives (50nmol/kg, 10mL/kg) were administered daily for 56 consecutive days, the negative control group was administered physiological saline daily, and the positive control The group was given Liraglutide. The fasting body weight of the mice in each group was tested on the 1st day and the 56th day, and the average weight change of the mice in each group was investigated.

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Abstract

公开了一种长效化艾塞那肽衍生物，其中将两倍量的艾塞那肽与单倍量的脂肪酸链缀合，得到具有更长药理作用时间的艾塞那肽衍生物。还公开了艾塞那肽衍生物的制备方法、其药学上可接受的艾塞那肽衍生物盐、艾塞那肽衍生物药剂、药物组合物和用途，该用途为艾塞那肽衍生物在制备治疗和/或预防糖尿病、肥胖症、高血脂症、非酒精性脂肪肝的药物中的应用。

Description

长效化艾塞那肽衍生物及其盐与制备方法和用途 技术领域
本发明涉及多肽化合物技术领域，具体涉及一种艾塞那肽衍生物、其制备方法、药剂给合物及其作为药物的用途。
背景技术
代谢综合征的病因是蛋白质、脂肪及碳水化合物等多种物质的代谢异常。营养过剩、体力活动减少等会导致肥胖以及肥胖相关疾病，如糖尿病等。近年来，2型糖尿病、血脂代谢异常的发病率日益增高。
胰高血糖素样肽-1(GLP-1)是一种葡萄糖依赖性肠促胰岛素激素。它可以激动GLP-1受体，发挥降糖作用。最显著的功能是促进β细胞的再生和修复，增加胰岛β细胞的数量，同时还能避免糖尿病治疗中经常发生的低血糖风险，在糖尿病治疗领域有广阔的应用前景。尽管天然GLP-1在治疗糖尿病上有诸多优点，但它在体内易被二肽基肽酶Ⅳ(DPP-Ⅳ)快速降解，其体内半衰期仅3分钟左右。艾塞那肽(Exenatide)提取自美洲毒蜥蜴唾液腺，2005年被FDA批准上市，其多肽序列为HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH 2。Exenatide不是DPP-4的底物，因此不会被体内广泛存在的DPP-4降解，其体内半衰期比内源性GLP-1延长，约为2.4h。但艾塞那肽仍然会在肾脏中快速滤过消除，抗DPP-IV酶的降解只能一定程度的延长GLP-1的半衰期。
发明内容
本发明的目的是针对现有技术的不足，提供一种长效化艾塞那肽衍生物，该衍生物在艾塞那肽的基础上，在保持其对GLP-1受体的高度激动活性的基础上，用胰高血糖素对其进行位点置换，以增加其对胰高血糖素的激动活性得到具有双靶点激动活性艾塞那肽衍生序列。
本发明在上述目的基础上，创新性地将两条优选的艾塞那肽衍生多肽序列利用赖氨酸连接，并与不同长度的脂肪酸链缀合。脂肪酸链可增加缀合物与血清白蛋白的结合时间，同时整体分子体积比艾塞那肽增大，可减慢肾脏快速滤过，两方面同时起效，极大的延长了肽链的在体内作用时间。因而该类双效艾塞那肽类似物在具备降糖及减重活性的前提下，显著延长其体内作用时间。
本发明的另一个目的是提供了一种艾塞那肽衍生物的制备方法。
本发明的再一个目的是提供使用艾塞那肽衍生物制备得到的药学上可接受的艾塞那肽衍生物盐。
本发明的又一个目的是提供了艾塞那肽衍生物制备得到的艾塞那肽衍生物药剂。
本发明的又一个目的是提供了一种含有艾塞那肽衍生物的药物组合物。
本发明的又一个目的是提供了艾塞那肽衍生物的用途、艾塞那肽衍生物盐的用途和艾塞那肽衍生物药剂的用途。
本发明的目的是通过以下的技术方案还实现的。本发明是一种长效化艾塞那肽衍生物，其氨基酸序列为：
His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Xaa2-Ser-Pro-Pro-Pro-Ala-Gly-Ser-Ser-Pro-Gly-Gly-Asn-Lys-Leu-Trp-Glu-Ile-Phe-Asp-Gln-Ala-Arg-Arg-Ser-Asp-Met-Gln-Lys-Ser-Leu-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Xaa1-His
其中：
Xaa1取自：Aib或Gly
Xaa2取自：
Figure PCTCN2019090218-appb-000001
n为自然数5-17，优选11，13或15。
优选的本发明长效化艾塞那肽衍生物可以表示为：
Figure PCTCN2019090218-appb-000002
Figure PCTCN2019090218-appb-000003
Figure PCTCN2019090218-appb-000004
本发明还公开了艾塞那肽衍生物制备得到的药学上可接受的艾塞那肽衍生物盐，与所述盐为艾塞那肽衍生物与盐酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸、硝酸、甲磺酸、乙磺酸、苯磺酸、对甲苯磺酸、甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、肥酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷酸、天冬氨酸、磺基水杨酸、半硫酸或硫氰酸所成的盐。
本发明还提供了一种药物组合物，包括治疗有效量的至少一种上述艾塞那肽衍生物化合物或者其药学上可接受的盐，或药学上可接受的载体或稀释剂。同时，本发明进一步提供了上述艾塞那肽衍生物化合物和其药学上可接受的盐，或药学上可接受的载体或稀释剂在制备用于治疗和预防糖尿病的药物中的运用。
本发明还公开了用艾塞那肽衍生物制备得到的艾塞那肽衍生物药剂，其药剂剂型选自片剂、胶囊、酏剂、糖浆、锭剂、吸入剂、喷雾剂、注射剂、膜剂、贴剂、散剂、颗粒剂、块剂、乳剂、栓剂或者复方制剂。
本发明还公开了所述的艾塞那肽衍生物的用途，其特点是，该用途为艾塞那肽衍生物在制备治疗和/或预防糖尿病、肥胖症、高血脂症、非酒精性脂肪肝的药物中的应用。
本发明还公开了艾塞那肽衍生物盐的用途，其特点是，该用途为艾塞那肽衍生物盐在制备治疗和/或预防糖尿病、肥胖症、高血脂症、非酒精性脂肪肝的药物中的应用。
本发明还公开了艾塞那肽衍生物药剂的用途，其特点是，该用途为艾塞那肽 衍生物药剂在制备治疗和/或预防糖尿病、肥胖症、高血脂症、非酒精性脂肪肝的药物中的应用。
本发明还提供了艾塞那肽衍生物的制备方法及其中间体，本发明提供的艾塞那肽衍生物多肽的制备方法采用固相合成法逐步偶联此类降糖多肽主链的各个氨基酸，裂解得到侧链保护的主链多肽，将连有脂肪链的赖氨酸与主链反应得到艾塞那肽衍生物。此方法合成步骤简便，耦合效率高，易于纯化，有利于此类多肽的工业化生产。
本发明优选的艾塞那肽衍生物的制备方法包括以下步骤:
步骤1：取树脂，活化后，逐步偶联氨基酸，得到第一肽树脂；
步骤2：取所述第一肽树脂，经裂解、纯化，得到侧链保护的多肽主链；
步骤3：将连有脂肪链的赖氨酸与两倍量主链反应，纯化后即得；
优选地，本发明提供的制备方法中，步骤1中的树脂为2-CTC Resin或Wang Resin。
优选地，本发明提供的制备方法中，步骤2中裂解所用裂解试剂为TFA和DCM的混合物。在本发明的一些实施例中，本发明提供的制备方法中，步骤2中裂解所用试剂中TFA、DCM的体积之比为0.1：99.9。
在本发明的另外一些实施例中，本发明提供的制备方法中，步骤2和步骤3中纯化所用方法为色谱分离法。在本发明的另外一些实施例中，本发明提供的制备方法中，步骤2和步骤3中纯化所用色谱柱为C18柱。
发明人研究认为，前胰高血糖素原翻译剪切后可得到GLP-1与胰高血糖素，且二者具有一定的序列同源性。因此在对GLP-1具有高度激动活性的Exenatide中引入部分胰高血糖素的序列，可得到同时激动GLP-1R和GCGR的双效激动剂，从而得到降糖活性和减重效果俱佳的多肽化合物。
与现有技术相比，本发明的有益效果：
1、本发明提出的一种双受体激动的长效化艾塞那肽衍生物可以在保留降糖活性的基础上，具有减重作用，且生物半衰期较艾塞那肽原型(～2.4h)显著延长，部分达到了36小时以上，极大的延长了降糖减重作用时间。
2、本发明方法采用正交保护策略的固相合成艾塞那肽衍生物得到肽链的粗品的纯度大于85％，与常规合成方法相比大大提高，方便后续的纯化工作。
3、本发明方法采用固相方法合成艾塞那肽衍生物的成本低。由于偶合效率较高，所需要保护氨基酸平均只需要2倍过量，而常规合成方法中需要4到5倍过量的氨基酸，极大的节约了成本。
4、本发明采用Fmoc/tBu正交保护固相合成策略合成艾塞那肽衍生物的方法易于实现自动化、大规模化，这使其更适合工业化生产。
5、用本发明提供的通过固相合成技术制备的艾塞那肽衍生物，降糖和减缓体重增加活性好，药效时间长，收率高、合成周期短、粗品纯化容易，生产成本低、易于工业自动化生产。制备得到的艾塞那肽衍生物，适合作为治疗糖尿病、肥胖药物的活性成分。
附图说明
图1为艾塞那肽衍生物SEQ.ID NO:1～6的腹腔糖耐量实验结果；
图2为艾塞那肽衍生物SEQ.ID NO:1～6的稳定血糖实验结果。
具体实施方式
在本说明书全文中采用以下缩写：
英文缩写 中文
DCM 二氯甲烷
NMP N-甲基吡咯烷酮
HBTU 苯并三氮唑-N，N，N'，N'-四甲基脲六氟磷酸酯
HOBt 1-羟基-苯并三氮唑
DIEA/DIPEA N，N'-二异丙基乙胺
Fmoc N-9-芴甲氧羰基
ESI-MS 电喷雾质谱
EDT 乙二硫醇
HPLC 高效液相色谱
TFA 三氟乙酸
tBu 叔丁基
DMSO 二甲基亚砜
本发明是通过下列实施例来进行说明的，但这些实施例不做任何限制本发明 的解释。
实施例1：
Figure PCTCN2019090218-appb-000005
的合成
1.肽链的合成
1.1树脂的溶胀
称取2-CTC Resin 1g(取代度0.4mmol/g)，经DCM 10mL溶胀30min，抽滤去DCM，再用NMP 10mL溶胀30min，分别用NMP，DCM 10mL冲洗干净。
1.2 Fmoc-Ser(tBu)-2-CTC Resin的合成
将Fmoc-Ser(tBu)-OH(0.8mmol)，DIPEA(1.6mmol)溶于NMP 10mL中，再将此溶液加入上一步得到的树脂中反应2小时，结束后滤去反应液，用DCM和NMP各10mL洗涤树脂3次。
1.3 Fmoc保护基的脱除
向洗涤后的树脂中加入含0.1M HOBt的25％哌啶/NMP(V/V)溶液脱除Fmoc，反应结束后用DCM和NMP各10mL洗涤树脂3次。
1.4肽链的延长
按照艾塞那肽衍生物肽链的序列，重复上述脱保护和偶合的步骤依次连接上相应的氨基酸，依次连接上相应的氨基酸直至肽链合成完毕，得到连有艾塞那肽衍生物的肽树脂。
1.5树脂上多肽的裂解
将上述得到的连有艾塞那肽衍生物肽链的树脂放入反应瓶中，各加入裂解剂(TFA/DCM,0.1/99.9,V/V)10mL，先在0℃下振摇30min，再在常温下反应1h。反应结束后抽滤，合并滤液，减压蒸馏。将浓缩液加入大量的冰乙醚中析出白色絮状沉淀，冷冻离心得到侧链保护的多肽粗品。采用制备液相色谱进行纯化，色谱条件为：C18柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)， 流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～90％，20min；流速为6mL/min检测波长为214nm。
2.(S)-2,6-二氨基-N-十二烷基己酰胺的合成
将Boc-Lys(Boc)-NH-12烷(0.8mmol)溶于7mL DCM中，缓慢滴加1mL TFA，反应3h，减压浓缩，加入5mL 1M NaOH水溶液，析出白色固体，离心收集沉淀，得白色粉状固体。
3.艾塞那肽衍生物的合成
将艾塞那肽衍生物(0.02mmol)，HBTU(0.02mmol)，HOBt(0.08mmol)和DIPEA(0.16mmol)溶于NMP 10mL中，再将此溶液加入(S)-2,6-二氨基-N-十二烷基己酰胺。使用HPLC监测反应，色谱条件为：C18柱(150mm×4.6mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 35％～85％，20min；流速1mL/min；柱温40℃；检测波长214nm。反应结束后，采用制备液相色谱进行纯化，色谱条件为：C18柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～90％，20min；流速为6mL/min检测波长为214nm。收集的溶液冻干得纯品23.8mg。理论相对分子质量为8710.7。ESI-MS m/z:Calcd.[M+5H] 5+1743.1,[M+6H] 6+1452.8；Found[M+5H] 5+1743.5,[M+6H] 6+1453.1。
实施例2：
Figure PCTCN2019090218-appb-000006
合成方法同实施例1，收集的溶液冻干得纯品24.9mg。理论相对分子质量为8654.6。ESI-MS m/z:Calcd.[M+5H] 5+1731.9,[M+6H] 6+1443.4；Found[M+5H] 5+1732.1,[M+6H] 6+1443.7。
实施例3：
Figure PCTCN2019090218-appb-000007
合成方法同实施例1，收集的溶液冻干得纯品26.5mg。理论相对分子质量为8738.8。ESI-MS m/z:Calcd.[M+5H] 5+1748.8,[M+6H] 6+1457.5；Found[M+5H] 5+1748.9,[M+6H] 6+1458.0。
实施例4：
Figure PCTCN2019090218-appb-000008
合成方法同实施例1，收集的溶液冻干得纯品22.8mg。理论相对分子质量为8682.7。ESI-MS m/z:Calcd.[M+5H] 5+1737.5,[M+6H] 6+1448.1；Found[M+5H] 5+1737.8,[M+6H] 6+1448.7。
实施例5：
Figure PCTCN2019090218-appb-000009
合成方法同实施例1，收集的溶液冻干得纯品24.5mg。理论相对分子质量为8766.9。ESI-MS m/z:Calcd.[M+5H] 5+1754.4,[M+6H] 6+1462.2；Found[M+5H] 5+1754.9,[M+6H] 6+1462.3。
实施例6：
Figure PCTCN2019090218-appb-000010
合成方法同实施例1，收集的溶液冻干得纯品23.9mg。理论相对分子质量为8710.7。ESI-MS m/z:Calcd.[M+5H] 5+1743.1,[M+6H] 6+1452.8；Found[M+5H] 5+1743.8,[M+6H] 6+1453.0。
实施例7：以下是本发明中涉及的艾塞那肽衍生物的相关药理实验方法以及结果：
1、艾塞那肽衍生物的GLP-1受体激动活性筛选
HEK293细胞共转染编码GLP-1R或GCGR的cDNA。测定化合物的试验中，提前2h将细胞种于96孔板中，化合物用DMSO溶解，使用含有0.1％牛血清蛋白的培养基稀释至不同倍数，加入共转染的细胞中。细胞孵化20min后，使用Cisbo公司的ELISA试剂盒，使用酶标仪测定荧光读数，建立标准曲线将荧光读数转化为相应的cAMP数值，使用Graphpad Prism 5.0软件的非线性回归计算化合物的EC 50数值。
表1艾塞那肽衍生物对GLP-1R和GCGR激动活性
Figure PCTCN2019090218-appb-000011
Results are expressed as mean±SD,**P<0.01 vs Glucagon,##P<0.01 vs Exenatide.
如表1所示，与Exenatide原型相比，所有化合物对GLP-1R的激动活性依然保持了相当高程度的激动活性。同时对GCGR的激动活性与艾塞那肽原型相 比有了很大提高，缀合脂肪酸链并没有对激动活性造成太大影响。
2、艾塞那肽衍生物的腹腔葡萄糖耐量实验
正常昆明小鼠，随机分组，每组8只，小鼠饲养在标准化动物房中。实验前12小时禁食，只给予饮水。每组小鼠在给药艾塞那肽衍生物之前，测初始血糖值，定为-30min，然后腹腔注射50nmol/kg的艾塞那肽衍生物。30min后，腹腔注射18mmol/kg的葡萄糖溶液，定为0min，对照组注射同体积的生理盐水或50nmol/kg的艾塞那肽。在0，15，30，45，60，120min用血糖仪测定血糖水平，检测艾塞那肽衍生物的降糖活性。
如图1所示，降血糖实验结果表明，本发明中涉及的艾塞那肽衍生物给药浓度为50nmol/kg时，降血糖效果与艾塞那肽的降糖效果相当。
3、艾塞那肽衍生物的稳定血糖实验
测定STZ诱导的糖尿病模型小鼠的血糖，选择数值高于20mmol/L的小鼠进行随机分组，每组六只，实验期间小鼠自由采食。阳性对照组腹腔注射艾塞那肽或利拉鲁肽，剂量为50nmol/kg，阴性对照组腹腔注射生理盐水，给药组分别注射50nmol/kg的艾塞那肽衍生物。0h给予化合物，分别在0、0.5、1、2、3、4、6、8、10、12、16、24、36、48和60h使用血糖仪测定血糖水平。评价指标为腹腔注射化合物后，小鼠血糖数值低于8.35mmol/L的时间。
由图2可见，艾塞那肽的稳定血糖的时间仅为4h，利拉鲁肽的稳定血糖时间为10h，本发明中涉及的长效化降糖多肽的稳定血糖时间均在24h以上，部分可超过35h。稳定血糖实验表明，艾塞那肽衍生物具有良好的长效化降糖效果，可以达到更优的长效化降糖效果，具有开发成为每一天给药一次的降糖药物的潜力。
4、OXM杂合肽的减缓体重增加实验
雄性C57bl/6小鼠，高脂饲料喂养4周，选体重大于30g的小鼠进行实验。小鼠随机分组，8只为一组，共8组，连续56天每日给药艾塞那肽衍生物(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药Liraglutide。 测试第1天和第56天各组小鼠的空腹体重，考察各组小鼠的平均体重变化。
表2艾塞那肽衍生物的减重效应
Figure PCTCN2019090218-appb-000012
Results are expressed as mean±SD.
从表2可以得出，长期给药后，所有的化合物都表现出了较好的体重控制效果。

Claims (10)

  1. 一种长效化艾塞那肽衍生物，其特征在于，其氨基酸序列为：
    His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Asp-Ser-Arg-Arg-Ala-Gln-Asp-
    Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Xaa2-Ser-Pro-Pro-Pro-Ala-Gly-Ser-Ser-Pro-Gly-Gly-Asn-Lys-Leu-Trp-Glu-Ile-Phe-Asp-Gln-Ala-Arg-Arg-Ser-Asp-Met-Gln-Lys-Ser-Leu-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Xaa1-His；
    其中：
    Xaa1取自Aib或Gly；
    Xaa2取自
    Figure PCTCN2019090218-appb-100001
    n为自然数5-17。
  2. 根据权利要求1所述的艾塞那肽衍生物，其特征在于：Xaa2中n为自然数11，13或15。
  3. 根据权利要求1所述的艾塞那肽衍生物，其特征在于：该艾塞那肽衍生物为下述化合物中的一种：
    Figure PCTCN2019090218-appb-100002
    Figure PCTCN2019090218-appb-100003
  4. 一种如权利要求1所述的艾塞那肽衍生物的制备方法，其特征在于，包括以下步骤：
    (1)取树脂，活化后，逐步偶联氨基酸，得到第一肽树脂；
    (2)取所述第一肽树脂，经裂解、纯化，得到侧链保护的多肽主链；
    (3)将连有脂肪链的赖氨酸与两倍量主链反应，纯化后即得。
  5. 一种如权利要求1－3中任意一项所述的艾塞那肽衍生物制备得到的药学上可接受的艾塞那肽衍生物盐，其特征在于，所述的药学上可以接受的艾塞那肽衍生物盐是艾塞那肽衍生物盐与酸形成的盐，所述的酸选自盐酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸、硝酸、甲磺酸、乙磺酸、苯磺酸、对甲苯磺酸、甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、肥酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷酸、天冬氨酸、磺基水杨酸、半硫酸或硫氰酸。
  6. 一种如权利要求1－3中任意一项所述的艾塞那肽衍生物制备得到的艾塞那肽衍生物药剂，其特征在于，所述的药剂剂型选自片剂、胶囊、酏剂、糖浆、锭剂、吸入剂、喷雾剂、注射剂、膜剂、贴剂、散剂、颗粒剂、块剂、乳剂、栓剂或者复方制剂。
  7. 一种含有如权利要求1－3中任意一项所述的艾塞那肽衍生物的药物组合物，其特征在于，该组合物包括治疗有效量的权利要求1－3中任何一所述的艾塞那肽衍生物和其药学上可接受的载体或稀释剂。
  8. 一种如权利要求1－3中任何一项所述的艾塞那肽衍生物的用途，其特征在于，该用途为艾塞那肽衍生物在制备治疗和/或预防糖尿病、肥胖症、高血脂症、非酒精性脂肪肝的药物中的应用。
  9. 一种如权利要求5所述的药学上可接受的艾塞那肽衍生物盐的用途，其特征在于，该用途为艾塞那肽衍生物盐在制备治疗和/或预防糖尿病、肥胖症、高血脂症、非酒精性脂肪肝的药物中的应用。
  10. 一种如权利要求6所述的艾塞那肽衍生物药剂的用途，其特征在于，该用途为艾塞那肽衍生物药剂在制备治疗和/或预防糖尿病、肥胖症、高血脂症、非酒精性脂肪肝的药物中的应用。
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CN110551203B (zh) * 2019-09-25 2023-02-10 成都奥达生物科技有限公司 一种艾塞那肽类似物
CN116970062B (zh) * 2022-04-29 2024-04-09 南京知和医药科技有限公司 一种超长效glp-1多肽衍生物及其制备方法和用途

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