CN103421094A - Polypeptide compound with EPO-like activity - Google Patents

Polypeptide compound with EPO-like activity Download PDF

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CN103421094A
CN103421094A CN2012101632419A CN201210163241A CN103421094A CN 103421094 A CN103421094 A CN 103421094A CN 2012101632419 A CN2012101632419 A CN 2012101632419A CN 201210163241 A CN201210163241 A CN 201210163241A CN 103421094 A CN103421094 A CN 103421094A
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polypeptide
seq id
selected
sequence
dimer
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CN2012101632419A
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Chinese (zh)
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冯军
张喜全
刘子谦
东圆珍
薛春佳
徐宏江
宋伟
张颖
马艳池
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上海医药工业研究院
正大天晴药业集团股份有限公司
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Priority to CN2012101632419A priority Critical patent/CN103421094A/en
Publication of CN103421094A publication Critical patent/CN103421094A/en

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Abstract

The invention belongs to the field of biochemistry, and particularly relates to a polypeptide compound with EPO-like activity and applications of the compound in treating diseases related to insufficient or faulty red blood cell production. The compound is formed by two polypeptide sequences, wherein a first sequence is X1-G-X2-Y-X3-C-X4-M-G-P-X5-T-W-X6-C-Q-P-L-X7-G-X8-X9, a second sequence is X10-G-X11-Y-X12-C-X13-M-G-P-X14-X15-W-X16-C-X17-X18-X19-X20-G, and the first sequence and the second sequence are connected by a connecting amino acid (X8) of the first sequence to form a dimer.

Description

一种具有EPO类似活性的多肽化合物 One kind of compound EPO polypeptide having similar activity

发明领域 Field of the Invention

[0001] 本发明属于生物化学领域,具体而言涉及一种具有EPO类似活性的多肽化合物。 [0001] The present invention belongs to the field of biochemistry, in particular, to a compound EPO polypeptide having similar activity. 以及该化合物在治疗与血红细胞产生不足或缺陷相关的疾病中的应用。 And insufficient or defective red blood cell production compound related application in the treatment of diseases and blood.

[0002] 发明背景 [0002] Background of the Invention

[0003] 红细胞生成素(EPO)是具有165个氨基酸的糖蛋白激素,其分子量为约34kDa,授予Lin的美国专利4,703,008已描述了促红细胞生成素编码基因的鉴定、克隆和表达,其说明书通过引用以其整体结合到本文中。 [0003] Erythropoietin (EPO) is a glycoprotein hormone of 165 amino acids having a molecular weight of about 34 kDa, Lin U.S. Patent No. 4,703,008 granted already describes the identification of genes encoding erythropoietin, Cloning and Expression , the specification of which is incorporated herein by reference in its entirety.

[0004] EPO通常产生于肾脏(90% )和肝脏,经分泌后进入外周血循环,并主要在肝脏中代谢,只有少量经尿液排出,它是促进骨髓红系祖细胞生长、增生、分化和成熟的主要刺激因子。 [0004] EPO is typically produced in the kidney (90%) and liver, secreted into the peripheral blood, and primarily metabolized in the liver, only a small amount of urine excreted, which is bone marrow erythroid progenitor cells to promote the growth, proliferation, differentiation, and mature major stimulating factor. EPO的生物学效应是通过位于骨髓红系祖细胞表面的特异性EPO受体(EPOR)介导完成的。 Biological effects of EPO on the cell surface by bone marrow erythroid progenitors specific EPO receptor (the EPOR) mediated completed. EPO与EPOR结合后形成二聚体,再通过信号传导途径调节红细胞的增生和分化。 EPO dimers and upon binding to EPOR is formed, and then adjusting the proliferation and differentiation of red blood cells through the signal transduction pathway. EPO信号转导过程有多条途径,其中研究比较透彻的是EPOR-JAK2-STAT5途径,其他已证实的信号传导机制还包括:EP0R-JAK2-PI3K 途径、EPOR-JAK2_ERKs 途径、EPOR-JAK2_NF_KappaB途径、EPOR-JAK2-Ras蛋白-MAPK途径等。 EPO signal transduction pathways a number of which is more thorough study EPOR-JAK2-STAT5 pathway other signal transduction mechanisms has been demonstrated further comprising: EP0R-JAK2-PI3K pathway, EPOR-JAK2_ERKs pathway, EPOR-JAK2_NF_KappaB pathway, EPOR-JAK2-Ras protein -MAPK way and so on. 可见,EPO胞内信号传导不是单一的级联通路,而是多个通路交联、互补、形成网络的复杂传导工过程。 Seen, the EPO intracellular signaling cascade path is not a single, but a plurality of passages crosslinked, complementary, to form a complex network of conducting industrial process.

[0005] 对于慢性肾衰以及放化疗晚期的癌症病人,由于其正常的造血功能受到抑制,导致EPO绝对或相对不足,常伴发有贫血。 [0005] For patients with chronic renal failure and cancer chemotherapy late, due to its normal hematopoiesis is suppressed, resulting in an absolute or relative lack of EPO, often accompanied by anemia. 现阶段临床上多采用输入重组人EPO注射液来改善患者的贫血状况,并取得了显著的效果。 The input stage clinical use of recombinant human EPO injection to improve anemia in patients, and have achieved remarkable results. 然而在实际应用中仍存在以下问题:常常引起心血管并发症,例如高血压、心肌梗塞等;对某些实体性肿瘤有促进发展;发生单纯红细胞再生障碍性贫血;体内半衰期较短,需每周注射1-3次等。 However, there are still problems in the practical application of the following: often caused by cardiovascular complications such as hypertension, myocardial infarction; promote the development of certain solid tumors; the occurrence of pure red cell aplasia; in vivo half-life is short, every need injection 1-3 weeks inferior. 因此,亟需开发治疗贫血的新型药物。 Therefore, the urgent need to develop new drugs for treating anemia. 中国专利ZL94109128.7公开了一种高糖基化的重组红细胞生成刺激蛋白,临床研究表明,体内半衰期相比于重组人EPO显著延长,中国专利ZL00809895.6公开了一种促红细胞生成素与聚乙二醇的偶联物,平均每月注射I次即可相当于每周注射1-3次重组人EPO的效力。 Chinese Patent No. ZL94109128.7 discloses a high-glycosylated recombinant erythropoiesis stimulating protein, clinical studies have shown that in vivo half-life compared to recombinant human EPO significantly prolonged, China patent ZL00809895.6 discloses an erythropoietin with polyethylene glycol conjugates, I mean monthly injections of injections corresponds to 1-3 times the efficacy of recombinant human EPO weekly. 然而,这一类机制的药物在体内均会与EPO抗体发生交叉反应,降低这一类药物的疗效。 However, this type of mechanism of drug are cross-react with antibodies in vivo EPO, reduce the efficacy of this class of drugs. 本发明人通过大量试验,意外发现了一种与EPO序列和结构完全不相关的化合物,该化合物具有显著的EPO类似活性且不会与EPO抗体发生交叉反应,并能够用于治疗与血红细胞产生不足或缺陷相关的疾病,从而完成本发明。 The present invention, through extensive testing, discovered by accident with a compound of the structure and the EPO sequence completely unrelated, the compound has significant activity and similar EPO does not react with the cross-EPO antibodies, and can be used for the treatment of red blood cell production deficiency related diseases or defects, thereby completing the present invention.

发明内容: SUMMARY:

[0006] 本发明涉及一种多肽化合物,由两条多肽序列构成,其中第一序列为:X1-G-X2_Y-X3-C-X4-MGP-X5-TW-X6-CQPL-X7-G-X8-X9 ;第二序列为:X10-G-X11-Y-X12-C-X13_M-GP-X14-X15-W-X16-C-X17-X18-X19-X20-G ;并且第一序列和第二序列通过第一序列中的连接氨基酸(X8)连接形成二聚体。 [0006] The present invention relates to a polypeptide compound, composed of two polypeptide sequences, wherein the first sequence is: X1-G-X2_Y-X3-C-X4-MGP-X5-TW-X6-CQPL-X7-G- X8-X9; second sequence: X10-G-X11-Y-X12-C-X13_M-GP-X14-X15-W-X16-C-X17-X18-X19-X20-G; and a first sequence and amino acid sequence are connected by a second connector (X8) the first sequence to form a dimer.

[0007] 本发明的多肽化合物与EPO受体结合,发挥受体激动作用,藉此治疗与血红细胞产生不足、缺陷或者过度消耗相关的疾病。 [0007] The compounds of the present invention bind to EPO receptor polypeptide, play a role in receptor agonist, whereby the treatment and insufficient red blood cell production, or excessive consumption of defects related diseases.

[0008] 本发明所使用的氨基酸除了包括本领域技术人员所熟知的二十种常见氨基酸外,还包括部分非常见氨基酸。 [0008] The amino acids used in the present invention includes twenty common amino acids in addition to those skilled in the art is, also includes a portion unconventional amino acids. 常见氨基酸的名称和缩写将在表I中概述;非常见氨基酸,名 Common names and abbreviations of amino acids will be outlined in Table I; uncommon amino acids, name

称、缩写以及其结构,绘制于表2中。 Said abbreviation, and its structure, plotted in Table 2.

[0009] 表I常见氨基酸的名称和缩写 [0009] Table I common names and abbreviations of amino acids

Figure CN103421094AD00051

[0011] 表2非常见氨基酸的名称、缩写和结构 [0011] See Table 2 is an amino acid names, abbreviations and structure

[0012] [0012]

Figure CN103421094AD00061
Figure CN103421094AD00071

[0014] 本发明所采用的表I和表2中的氨基酸若不特别限定D或L构型,均为L型氨基酸;若不特别限定,所有氨基酸均为α-氨基酸。 [0014] The present invention is employed in Tables I and 2 unless particularly limited, an amino acid D or L configuration, are L-amino acid; if not otherwise defined, all amino acids are α-. 本发明所有序列的方向若不特别说明,从左向右均为N末端-C末端的方向。 All directions sequence of the invention unless otherwise indicated, are N-terminal -C-terminal direction from left to right.

[0015] 本发明首先提供了一种多肽化合物,由两条多肽序列构成,其中第一序列为:X1-G-X2-Y-X3-C-X4-MGP-X5-TW-X6-CQPL-X7-G-X8-X9 ;第二序列为:X10-G-X11-Y-X12_C-X13-MGP-X14-X15-W-X16-C-X17-X18-X19-X20-G ;并且第一序列和第二序列通过第一序列中的连接氨基酸(X8)连接形成二聚体;X1选自G或AcG ;X2、X5和X6分别独立地选自L、 [0015] The present invention firstly provides a polypeptide compound, composed of two polypeptide sequences, wherein the first sequence is: X1-G-X2-Y-X3-C-X4-MGP-X5-TW-X6-CQPL- X7-G-X8-X9; second sequence: X10-G-X11-Y-X12_C-X13-MGP-X14-X15-W-X16-C-X17-X18-X19-X20-G; and a first and second sequences of amino acids connected by a connector (X8) the first sequence forms dimers; G is selected from the X1 or AcG; X2, X5 and X6 are each independently selected from L,

I 或V ;X3 选自A 或G ;X4 选自H 或D-His ;X7 选自R、HomoArg 或Pal-Lys ;X8 选自Lys 或D-Lys,以其结构中α-氨基参与第一序列合成,以其结构中ε -氨基与第二序列的C末端氨基酸连接形成二聚体,优选使用Lys ;Χ9为赖氨酸或由2-10个氨基酸组成的C末端为赖氨酸的短肽,更优选的,Χ9由3-7个氨基酸组成,特别优选的,Χ9选自GK,GGK, GGARRAGK,GGAGAGK, GADEAGGKK 或GADEGGAK ;Χ10 选自G 或AcG ;Χ11、Χ14、Χ16 或Χ19 分别独立地选自L、1、V 或Nle ;Χ12 选自A、Aib 或G ;Χ13 选自H 或D-His ;Χ15 选自T 或S ;Χ17 选自Q 或N ;Χ18 选自P 或Hyp ;Χ20 选自R、HomoArg> Cit 或Pal-Lys。 I or V; X3 is selected from A or G; X4 is selected from H, or D-His; X7 is selected from R, HomoArg, or Pal-Lys; X8 is selected from Lys or D-Lys, an amino group participating in its first configuration α- sequences of the synthetic, its structure ε - C-terminal amino acid sequence of the second connector to form a dimer, it is preferred to use Lys; Χ9 is lysine or a C-terminal 2-10 amino acids is lysine short peptide, more preferably, Χ9 of 3-7 amino acids, particularly preferred, Χ9 selected GK, GGK, GGARRAGK, GGAGAGK, GADEAGGKK or GADEGGAK; Χ10 selected from G or AcG; Χ11, Χ14, Χ16 each independently or Χ19 selected from L, 1, V or Nle; Χ12 selected from A, Aib or G; Χ13 selected from H or D-His; Χ15 selected from T or S; Χ17 is selected from Q or N; Χ18 selected from P or Hyp; Χ20 selected R, HomoArg> Cit or Pal-Lys.

[0016] 优选的,本发明的第一序列包括但不限于如下序列: [0016] Preferably, the first sequence of the present invention include, but are not limited to the following sequence:

[0017] SEQ ID NO: I (AcG)GLYACHMGPITffVCQPLRGKGK [0017] SEQ ID NO: I (AcG) GLYACHMGPITffVCQPLRGKGK

[0018] SEQ ID NO:2(AcG)GLYACHMGPITffVCQPLRGKGGK [0018] SEQ ID NO: 2 (AcG) GLYACHMGPITffVCQPLRGKGGK

[0019] SEQ ID NO:3(AcG)GLYACHMGPITffVCQPLRGKGGARRAGK [0019] SEQ ID NO: 3 (AcG) GLYACHMGPITffVCQPLRGKGGARRAGK

[0020] SEQ ID NO:4 (AcG)GLYACHMGPITffVCQPLRGKGGAGAGK [0020] SEQ ID NO: 4 (AcG) GLYACHMGPITffVCQPLRGKGGAGAGK

[0021] SEQ ID NO:5 (AcG)GLYACHMGPITffVCQPLRGKGADEAGGKK [0021] SEQ ID NO: 5 (AcG) GLYACHMGPITffVCQPLRGKGADEAGGKK

[0022] SEQ ID NO:6(AcG)GLYACHMGPITffVCQPLRGKGADEGGAK [0022] SEQ ID NO: 6 (AcG) GLYACHMGPITffVCQPLRGKGADEGGAK

[0023] SEQ ID NO:7 (AcG)GLYACHMGPITffICQPLRGKGK [0023] SEQ ID NO: 7 (AcG) GLYACHMGPITffICQPLRGKGK

[0024] SEQ ID NO:8 (AcG)GLYACHMGPITffICQPLRGKGGK [0024] SEQ ID NO: 8 (AcG) GLYACHMGPITffICQPLRGKGGK

[0025] SEQ ID NO:9 (AcG)GLYACHMGPITffICQPLRGKGGARRAGK [0025] SEQ ID NO: 9 (AcG) GLYACHMGPITffICQPLRGKGGARRAGK

[0026] SEQ ID NO:10(AcG)GLYACHMGPITffICQPLRGKGGAGAGK [0026] SEQ ID NO: 10 (AcG) GLYACHMGPITffICQPLRGKGGAGAGK

[0027] SEQ ID NO: 11 (AcG)GLYACHMGPITffICQPLRGKGADEAGGKK [0027] SEQ ID NO: 11 (AcG) GLYACHMGPITffICQPLRGKGADEAGGKK

[0028] SEQ ID NO: 12 (AcG)GLYACHMGPITffICQPLRGKGADEGGAK [0028] SEQ ID NO: 12 (AcG) GLYACHMGPITffICQPLRGKGADEGGAK

[0029] SEQ ID NO:13 (AcG)GLYAC(D-His)MGPITffVCQPLRGKGK[0030] SEQ ID NO:14(AcG)GLYAC(D-His)MGPITffVCQPLRGKGGK [0029] SEQ ID NO: 13 (AcG) GLYAC (D-His) MGPITffVCQPLRGKGK [0030] SEQ ID NO: 14 (AcG) GLYAC (D-His) MGPITffVCQPLRGKGGK

[0031 ] SEQ ID NO:15 (AcG)GLYAC(D-His)MGPITffVCQPLRGKGGARRAGK [0031] SEQ ID NO: 15 (AcG) GLYAC (D-His) MGPITffVCQPLRGKGGARRAGK

[0032] SEQ ID NO: 16 (AcG)GLYAC(D-His)MGPITffVCQPLRGKGGAGAGK [0032] SEQ ID NO: 16 (AcG) GLYAC (D-His) MGPITffVCQPLRGKGGAGAGK

[0033] SEQ ID NO:17(AcG)GLYAC(D-His)MGPITffVCQPLRGKGADEAGGKK [0033] SEQ ID NO: 17 (AcG) GLYAC (D-His) MGPITffVCQPLRGKGADEAGGKK

[0034] SEQ ID NO:18(AcG)GLYAC(D-His)MGPITffVCQPLRGKGADEGGAK [0034] SEQ ID NO: 18 (AcG) GLYAC (D-His) MGPITffVCQPLRGKGADEGGAK

[0035] SEQ ID NO: 19 (AcG)GLYACHMGPLTffVCQPLRGKGK [0035] SEQ ID NO: 19 (AcG) GLYACHMGPLTffVCQPLRGKGK

[0036] SEQ ID NO:20 (AcG)GLYACHMGPLTffVCQPLRGKGGK [0036] SEQ ID NO: 20 (AcG) GLYACHMGPLTffVCQPLRGKGGK

[0037] SEQ ID NO:21(AcG)GLYACHMGPLTffVCQPLRGKGGARRAGK [0037] SEQ ID NO: 21 (AcG) GLYACHMGPLTffVCQPLRGKGGARRAGK

[0038] SEQ ID NO:22 (AcG)GLYACHMGPLTffVCQPLRGKGGAGAGK [0038] SEQ ID NO: 22 (AcG) GLYACHMGPLTffVCQPLRGKGGAGAGK

[0039] SEQ ID NO:23 (AcG)GLYACHMGPLTffVCQPLRGKGADEAGGKK [0039] SEQ ID NO: 23 (AcG) GLYACHMGPLTffVCQPLRGKGADEAGGKK

[0040] SEQ ID NO:24 (AcG)GLYACHMGPLTffVCQPLRGKGADEGGAK [0040] SEQ ID NO: 24 (AcG) GLYACHMGPLTffVCQPLRGKGADEGGAK

[0041] SEQ ID NO: 25 (AcG)GlYGCHMGPITffVCQPLRGKGK [0041] SEQ ID NO: 25 (AcG) GlYGCHMGPITffVCQPLRGKGK

[0042] SEQ ID NO:26 (AcG)GlYGCHMGPITffVCQPLRGKGGK [0042] SEQ ID NO: 26 (AcG) GlYGCHMGPITffVCQPLRGKGGK

[0043] SEQ ID NO:27 (AcG)GlYGCHMGPITffVCQPLRGKGGARRAGK [0044] SEQ ID NO:28 (AcG)GlYGCHMGPITffVCQPLRGKGGAGAGK [0043] SEQ ID NO: 27 (AcG) GlYGCHMGPITffVCQPLRGKGGARRAGK [0044] SEQ ID NO: 28 (AcG) GlYGCHMGPITffVCQPLRGKGGAGAGK

[0045] SEQ ID NO:29(AcG)GlYGCHMGPITffVCQPLRGKGADEAGGKK [0045] SEQ ID NO: 29 (AcG) GlYGCHMGPITffVCQPLRGKGADEAGGKK

[0046] SEQ ID NO:30(AcG)GlYGCHMGPITffVCQPLRGKGADEGGAK [0046] SEQ ID NO: 30 (AcG) GlYGCHMGPITffVCQPLRGKGADEGGAK

[0047] SEQ ID NO:31(AcG)GIYACHMGPLTffVCQPL(Pal-Lys)GKGK [0047] SEQ ID NO: 31 (AcG) GIYACHMGPLTffVCQPL (Pal-Lys) GKGK

[0048] SEQ ID NO:32 (AcG)GIYACHMGPLTffVCQPL(Pal-Lys)GKGGK [0048] SEQ ID NO: 32 (AcG) GIYACHMGPLTffVCQPL (Pal-Lys) GKGGK

[0049] SEQ ID NO:33 (AcG)GIYACHMGPLTffVCQPL(Pal-Lys)GKGGARRAGK [0049] SEQ ID NO: 33 (AcG) GIYACHMGPLTffVCQPL (Pal-Lys) GKGGARRAGK

[0050] SEQ ID NO:34 (AcG)GIYACHMGPLTffVCQPL(Pal-Lys)GKGGAGAGK[0051 ] SEQ ID NO:35 (AcG)GIYACHMGPLTffVCQPL(Pal-Lys)GKGADEAGGKK [0050] SEQ ID NO: 34 (AcG) GIYACHMGPLTffVCQPL (Pal-Lys) GKGGAGAGK [0051] SEQ ID NO: 35 (AcG) GIYACHMGPLTffVCQPL (Pal-Lys) GKGADEAGGKK

[0052] SEQ ID NO:36 (AcG)GIYACHMGPLTffVCQPL(Pal-Lys)GKGADEGGAK [0052] SEQ ID NO: 36 (AcG) GIYACHMGPLTffVCQPL (Pal-Lys) GKGADEGGAK

[0053] 优选地,本发明的第二序列包括但不限于如下序列: [0053] Preferably, the second sequence of the present invention include, but are not limited to the following sequence:

[0054] SEQ ID NO:37 (AcG)GLYACHMGPITffVCQPLRG [0054] SEQ ID NO: 37 (AcG) GLYACHMGPITffVCQPLRG

[0055] SEQ ID NO:38 (AcG)GLYACHMGPITffICQPLRG [0055] SEQ ID NO: 38 (AcG) GLYACHMGPITffICQPLRG

[0056] SEQ ID NO:39 (AcG)GLYAC(D-His)MGPITffVCQPLRG [0056] SEQ ID NO: 39 (AcG) GLYAC (D-His) MGPITffVCQPLRG

[0057] SEQ ID NO:40 (AcG)GLYACHMGPLTffVCQPLRG [0057] SEQ ID NO: 40 (AcG) GLYACHMGPLTffVCQPLRG

[0058] SEQ ID NO:41 (AcG)GlYGCHMGPITffVCQPLRG [0058] SEQ ID NO: 41 (AcG) GlYGCHMGPITffVCQPLRG

[0059] SEQ ID NO:42 (AcG)GIYACHMGPLTffVCQPL(Pal-Lys)G [0059] SEQ ID NO: 42 (AcG) GIYACHMGPLTffVCQPL (Pal-Lys) G

[0060] SEQ ID NO:43(AcG)GLY(Aib)CHMGPITffVCQ(Hyp)L(Cit)G [0060] SEQ ID NO: 43 (AcG) GLY (Aib) CHMGPITffVCQ (Hyp) L (Cit) G

[0061] SEQ ID NO:44 (AcG)GLYACHMGPISffVCNPLRG [0061] SEQ ID NO: 44 (AcG) GLYACHMGPISffVCNPLRG

[0062] SEQ ID NO:45 (AcG)GLYACHMGPITffVCNPL(HomoArg)G [0062] SEQ ID NO: 45 (AcG) GLYACHMGPITffVCNPL (HomoArg) G

[0063] SEQ ID NO:46(AcG)GLY(Aib)CHMGPISffVCQP(Nle)RG [0063] SEQ ID NO: 46 (AcG) GLY (Aib) CHMGPISffVCQP (Nle) RG

[0064] 本发明多肽化合物可进行修饰,例如N末端乙酰化、形成分子内二硫键、C末端酰胺化等。 Polypeptide compound [0064] The present invention can be modified, for example, N-terminal acetylation, intramolecular disulfide bridge, C-terminal amidation. 优选地,N末端乙酰化和形成分子内二硫键可在第一序列和第二序列中同时存在,并任选在第一序列中进行C末端酰胺化。 Preferably, N-terminal acetylated and disulfide bond formation may be present in the first and second sequences simultaneously in the molecule, and optionally the C-terminal amidated performed in the first sequence. 本文所述的分子内二硫键是指第一序列中第6和第15位半胱氨酸形成的二硫键和第二序列中第6和第15位半胱氨酸形成二硫键。 The intramolecular disulfide bonds this article refers to a disulfide bond in the first sequence and the second 6 and the cysteine ​​at position 15 is formed in the 6th and 15 cysteines form a disulfide bond. 在本发明的一个具体实施方案中,第一序列的氨基酸序列选自SEQ ID NO:1,第6和第15位半胱氨酸形成分子内二硫键,C末端酰胺化;第二序列的氨基酸序列选自SEQ ID N0:37,第6和第15位半胱氨酸形成分子内二硫键。 In one particular embodiment of the invention, the amino acid sequence of the first sequence is selected from SEQ ID NO: 1, 6 and 15 form an intramolecular disulfide bond of cysteine, C-terminal amidated; second sequence an amino acid sequence selected from SEQ ID N0: 37, 6 and 15 cysteine ​​intramolecular disulfide bond formation.

[0065] 在本发明的一个实施方案中,多肽化合物的第一序列的氨基酸序列选自SEQ IDNO:1,第6和第15位半胱氨酸形成分子内二硫键,C末端酰胺化;第二序列的氨基酸序列选自SEQID N0:37,第6和第15位半胱氨酸形成分子内二硫键,并通过第一序列中的第21位L型赖氨酸形成二聚体,下文简称为多肽二聚体1,结构如下所示: [0065] In one embodiment of the invention, the amino acid sequence of the first polypeptide sequence selected from compounds of SEQ IDNO: 1, 6 and 15 form an intramolecular disulfide bond of cysteine, C-terminal amidated; the amino acid sequence of the second selected sequence SEQID N0: 37, 6 and 15 form an intramolecular disulfide bond cysteine, and form a dimer by a first sequence of L-lysine 21, hereinafter simply referred to as a polypeptide dimer structure as shown below:

[0066] [0066]

Figure CN103421094AD00091

[0067] 在本发明的另一实施方案中,多肽化合物的第一序列的氨基酸序列选自SEQ IDNO:2,第6和第15位半胱氨酸形成分子内二硫键,C末端酰胺化;第二序列的氨基酸序列选自SEQID N0:37,第6和第15位半胱氨酸形成分子内二硫键,并通过第一序列中的第21位L型赖氨酸形成二聚体,下文简称为多肽二聚体2。 [0067] In another embodiment of the invention, the amino acid sequence of the first polypeptide sequence selected from compounds of SEQ IDNO: 2, 6 and 15 form an intramolecular disulfide bond of cysteine, C-terminal amidated ; a second amino acid sequence selected from the sequence SEQID N0: 37, 6 and 15 form an intramolecular disulfide bond cysteine, and form a dimer by a first sequence of L-lysine 21 , hereinafter referred to as 2 polypeptide dimer.

[0068] 在本发明的另一实施方案中,多肽化合物的第一序列的氨基酸序列选自SEQ IDNO:3,第6和第15位半胱氨酸形成分子内二硫键,C末端酰胺化;第二序列的氨基酸序列选自SEQID N0:37,第6和第15位半胱氨酸形成分子内二硫键,并通过第一序列中的第21位L型赖氨酸形成二聚体,下文简称为多肽二聚体3。 [0068] In another embodiment of the invention, the amino acid sequence of the first polypeptide sequence selected from compounds of SEQ IDNO: 3, 6 and 15 form an intramolecular disulfide bond of cysteine, C-terminal amidated ; a second amino acid sequence selected from the sequence SEQID N0: 37, 6 and 15 form an intramolecular disulfide bond cysteine, and form a dimer by a first sequence of L-lysine 21 , hereinafter referred to as 3 polypeptide dimer.

[0069] 在本发明的另一实施方案中,多肽化合物的第一序列的氨基酸序列选自SEQ IDNO:4,第6和第15位半胱氨酸形成分子内二硫键,C末端酰胺化;第二序列的氨基酸序列选自SEQID NO:37,第6和第15位半胱氨酸形成分子内二硫键,并通过第一序列中的第21位L型赖氨酸形成二聚体,下文简称为多肽二聚体4。 [0069] In another embodiment of the invention, the amino acid sequence of the first polypeptide sequence selected from compounds of SEQ IDNO: 4, 6 and 15 form an intramolecular disulfide bond of cysteine, C-terminal amidated ; selected from the amino acid sequence of a second sequence of SEQID NO: 37, 6 and 15 form an intramolecular disulfide bond cysteine, and form a dimer by a first sequence of L-lysine 21 , hereinafter referred to as 4 polypeptide dimer.

[0070] 在本发明的又一实施方案中,多肽化合物的第一序列的氨基酸序列选自SEQ IDNO:5,第6和第15位半胱氨酸形成分子内二硫键,C末端酰胺化;第二序列的氨基酸序列选自SEQID N0:37,第6和第15位半胱氨酸形成分子内二硫键,并通过第一序列中的第21位L型赖氨酸形成二聚体,下文简称为多肽二聚体5。 [0070] In yet another embodiment of the present invention, the amino acid sequence of the first polypeptide sequence selected from compounds of SEQ IDNO: 5, 6 and 15 form an intramolecular disulfide bond of cysteine, C-terminal amidated ; a second amino acid sequence selected from the sequence SEQID N0: 37, 6 and 15 form an intramolecular disulfide bond cysteine, and form a dimer by a first sequence of L-lysine 21 , hereinafter referred to as 5 polypeptide dimer.

[0071] 在本发明的又一实施方案中,多肽化合物的第一序列的氨基酸序列选自SEQ IDNO:6,第6和第15位半胱氨酸形成分子内二硫键,C末端酰胺化;第二序列的氨基酸序列选自SEQID N0:37,第6和第15位半胱氨酸形成分子内二硫键,并通过第一序列中的第21位L型赖氨酸形成二聚体,下文简称为多肽二聚体6。 [0071] In yet another embodiment of the present invention, the amino acid sequence of the first polypeptide sequence selected from compounds of SEQ IDNO: 6, 6 and 15 form an intramolecular disulfide bond of cysteine, C-terminal amidated ; a second amino acid sequence selected from the sequence SEQID N0: 37, 6 and 15 form an intramolecular disulfide bond cysteine, and form a dimer by a first sequence of L-lysine 21 , hereinafter referred to as 6 polypeptide dimer.

[0072] 在本发明的又一实施方案中,多肽化合物的第一序列的氨基酸序列选自SEQ IDNO:26,第6和第15位半胱氨酸形成分子内二硫键,C末端酰胺化;第二序列的氨基酸序列选自SEQID NO:41,第6和第15位半胱氨酸形成分子内二硫键,并通过第一序列中的第21位L型赖氨酸形成二聚体,下文简称为多肽二聚体7。 [0072] In yet another embodiment of the present invention, the amino acid sequence of the first polypeptide sequence selected from compounds of SEQ IDNO: 26, 6 and 15 form an intramolecular disulfide bond of cysteine, C-terminal amidated ; selected from the amino acid sequence of a second sequence of SEQID NO: 41, 6 and 15 form an intramolecular disulfide bond cysteine, and form a dimer by a first sequence of L-lysine 21 , hereinafter referred to as 7 polypeptide dimer.

[0073] 在本发明的又一实施方案中,多肽化合物的第一序列的氨基酸序列选自SEQ IDNO:32,第6和第15位半胱氨酸形成分子内二硫键,C末端酰胺化;第二序列的氨基酸序列选自SEQID NO:42,第6和第15位半胱氨酸形成分子内二硫键,并通过第一序列中的第21位D型赖氨酸形成二聚体,下文简称为多肽二聚体8。 [0073] In yet another embodiment of the present invention, the amino acid sequence of the first polypeptide sequence selected from compounds of SEQ IDNO: 32, 6 and 15 form an intramolecular disulfide bond of cysteine, C-terminal amidated ; selected from the amino acid sequence of a second sequence of SEQID NO: 42, 6 and 15 form an intramolecular disulfide bond cysteine, and form a dimer by a first sequence of 21 D-lysine , hereinafter referred to as polypeptide dimer 8.

[0074] 本发明的多肽化合物,还优选按下表所示的第一序列和第二序列的组合构成的二 [0074] The combination of the first and second sequences of the polypeptide compounds of the invention, shown in the following table are also preferably composed of two

聚体,任选在所述多肽二聚体的第一序列C末端酰胺化: Mer, optionally a first sequence of the C-terminus amidated polypeptide dimer:

[0075] [0075]

Figure CN103421094AD00101

[0077] 本发明所述的多肽化合物还可以包括聚乙二醇(PEG)部分,聚乙二醇可与第一序列的C端X9中的赖氨酸缀合,从而形成与PEG缀合的多肽化合物。 [0077] The polypeptide compound of the present invention may also be part of, a polyethylene glycol with a C-terminal sequence of the first X9 lysine conjugated include polyethylene glycol (PEG), thereby forming conjugated to PEG polypeptide compound. 其中PEG具有约5,000到约60,000道尔顿的分子量(术语“约”指在PEG的制备物中,某些分子将具有大于所陈述的分子量,有些分子具有小于所陈述的分子量),其与第一序列的C末端赖氨酸的ε -氨基共价连接。 Wherein the PEG has a molecular weight from about 5,000 to about 60,000 Daltons (the term "about" refers to a preparation of PEG, some molecules will have a molecular weight greater than stated, some molecules having a molecular weight of less than stated) , the C-terminal lysine of the first sequence of ε - amino group is covalently attached. 本发明的活化的PEG可以通过氨基甲酸酯键或酰胺键连接到第一序列末端赖氨酸的ε -NH2上,从而对本发明的多肽二聚体进行PEG修饰。 Activated PEG according to the present invention may be connected to a first end of the sequence lysine ε -NH2 by an amide bond or a urethane bond, thereby polypeptide dimer of the present invention are PEG-modified. 当第一序列的短肽Χ9含有两个以上赖氨酸时,还可对本发明的多肽二聚体进行双或多PEG修饰。 When the first short peptide sequence containing two or more lysine Χ9, polypeptide dimer of the present invention may be bi- or PEG modification.

[0078] 用于本发明的PEG可以是直链或分枝的PEG,其分子量为约5,000道尔顿(5k)到约60,000道尔顿(60k),优选地,PEG具有约20k到约40k的分子量。 [0078] PEG used in the present invention may be linear or branched PEG, molecular weight of about 5,000 daltons (. 5K) to about 60,000 daltons (60k), preferably, PEG having about a molecular weight of about 40k to 20k. 技术人员将能够基于诸如所希望的剂量、循环时间、对蛋白酶解的抗性和PEG对治疗肽的其他已知的影响等考虑选择适宜聚合物的大小。 Based on such art will be able desired dosage, circulation time, resistance to impact and the known PEG on a therapeutic peptide to proteolysis like other considerations for selecting the right size of the polymer.

[0079] 多种聚乙二醇种类可用于PEG化本发明的多肽二聚体,例如,包括但不限于mPEG2-NHS、mPEG2_ALD、mPEG (MAL) 2, mPEG2 (MAL)、mPEG_NH2、mPEG-SPA、mPEG-SBA、mPEG-BTC、mPE-ACET、mPEG-SPA-NHS 等。 [0079] can be used for various types of polyethylene glycol PEG polypeptide dimer of the present invention, e.g., including but not limited to mPEG2-NHS, mPEG2_ALD, mPEG (MAL) 2, mPEG2 (MAL), mPEG_NH2, mPEG-SPA , mPEG-SBA, mPEG-BTC, mPE-ACET, mPEG-SPA-NHS and so on.

[0080] 在本发明的一个实施方案中,多肽二聚体1、多肽二聚体2、多肽二聚体3、多肽二聚体4、多肽二聚体5、多肽二聚体6、多肽二聚体7、多肽二聚体8、多肽二聚体9、多肽二聚体10、多肽二聚体11、多肽二聚体12、多肽二聚体13、多肽二聚体14、多肽二聚体15、多肽二聚体16、多肽二聚体17、多肽二聚体18、多肽二聚体19、多肽二聚体20、多肽二聚体21、多肽二聚体22、多肽二聚体23通过氨基甲酸酯键与活化的PEG连接。 [0080] In one embodiment of the present invention, a polypeptide dimer, dimer of 2 polypeptides, polypeptide dimer 3, 4 dimeric polypeptide, polypeptide dimer 5, 6 polypeptide dimer, two polypeptide 7-mer, 8 dimeric polypeptide, polypeptide dimer 9, 10 dimeric polypeptide, polypeptide dimer 11, 12 dimeric polypeptide, polypeptide dimer 13, 14 dimeric polypeptide, polypeptide dimer 15. The polypeptide dimer 16, 17 dimeric polypeptide, polypeptide dimer 18, 19 dimeric polypeptide, polypeptide dimer 20, 21 dimeric polypeptide, polypeptide dimer 22, 23 by a polypeptide dimer urethane linkage with the activated PEG.

[0081] 在本发明的另一实施方案中,多肽二聚体1、多肽二聚体2、多肽二聚体3、多肽二聚体4、多肽二聚体5、多肽二聚体6、多肽二聚体7、多肽二聚体8、多肽二聚体9、多肽二聚体10、多肽二聚体11、多肽二聚体12、多肽二聚体13、多肽二聚体14、多肽二聚体15、多肽二聚体16、多肽二聚体17、多肽二聚体18、多肽二聚体19、多肽二聚体20、多肽二聚体21、多肽二聚体22、多肽二聚体23通过酰胺键与活化的PEG连接。 [0081] In another embodiment of the present invention, a polypeptide dimer, dimer of 2 polypeptides, polypeptide dimer 3, 4 dimeric polypeptide, polypeptide dimer 5, 6 dimeric polypeptide, polypeptide 7 dimer, polypeptide dimer 8, 9 dimeric polypeptide, polypeptide dimer 10, 11 dimeric polypeptide, polypeptide dimer 12, 13 dimeric polypeptide, polypeptide dimer 14, dimer polypeptide 15, 16 dimeric polypeptide, polypeptide dimer 17, 18 dimeric polypeptide, polypeptide dimer 19, 20 dimeric polypeptide, polypeptide dimer 21, 22 dimeric polypeptide, polypeptide dimer 23 activated PEG is connected via an amide bond.

[0082] 在本发明的一个具体实施方案中,多肽二聚体I通过氨基甲酸酯键与活化的PEG20k共价连接时,结构如下所示: [0082] In one particular embodiment of the present invention, a polypeptide dimer is connected to the I PEG20k activated via covalent urethane bond, the following structure:

[0083] [0083]

Figure CN103421094AD00111

[0084] 在本发明的一个具体实施方案中,多肽二聚体6通过酰胺键与活化的PEG2tlk共价 [0084] In one particular embodiment of the invention, a polypeptide dimer 6 via an amide bond covalently with activated PEG2tlk

连接时,结构如下所示: When connected, the structure shown below:

[0085] [0085]

Figure CN103421094AD00121

(AcG)GL YACHMGPITWVCQPLRG-NH NH—C——PEG20k (AcG) GL YACHMGPITWVCQPLRG-NH NH-C - PEG20k

[0086] 在本发明的一个具体实施方案中,多肽二聚体5通过氨基甲酸酯键与活化的PEG20k共价连接时,结构如下所示: [0086] In one particular embodiment of the present invention, a polypeptide dimer activated PEG20k 5 connected via covalent urethane bond, the following structure:

[0087] [0087]

Figure CN103421094AD00122

[0088] 再一方面,本发明所述多肽化合物的制备方法为本领域公知的经典固相合成方法,直接制备本发明的多肽二聚体的第一序列和第二序列;使用氧化试剂(例如,DMS0)形成所述分子内二硫键;并任选与活化的PEG(例如mPEG-SPA-NHS)通过氨基甲酸酯键或酰胺键共价连接等。 [0088] In another aspect, the method of preparing the compounds of the present invention is a polypeptide known in the art of classical solid phase synthesis methods, the first and second sequences of the polypeptide dimer of the present invention is prepared directly; using an oxidizing agent (e.g. , DMS0) formed disulfide bonds in the molecule; and optionally connected to the activated PEG (e.g. mPEG-SPA-NHS) through a urethane bond or an amide bond covalently like. 具体而言,第一序列中X8连接氨基酸(赖氨酸)使用Fmoc (9-芴基-甲基羰基)作为α-氨基或氨基的保护基,也可使用Alloc (烯氧丙基羰基)作为该赖氨酸ε -氨基的保护基。 Specifically, the first amino acid sequence is connected X8 (lysine) using Fmoc (9- fluorenyl - methylcarbonyl) as a protecting group of the α- amino group or amino group, may also be used of Alloc (alkylene carbonyl oxopropyl) as the lysine ε - amino-protecting group. 当使用Fmoc作为该赖氨酸的α -氨基和ε -氨基的保护基时,优选第二序列与第一序列的前20个氨基酸的序列相同,此时可同时脱除Fmoc,从而同时进行两条序列的固相合成;当使用Fmoc作为该赖氨酸的α -氨基的保护基,并且使用Alloc作为该赖氨酸的ε -氨基的保护基时,本发明的第一序列和第二序列依次合成,合成时先采用弱碱性脱保护试剂(如25%哌啶/DMF)脱除Fmoc保护基,此时该赖氨酸ε -氨基的Alloc保护基不受影响。 When the Fmoc lysine as α - amino and ε - amino protecting group, the same sequence as the first 20 amino acids preferably the first sequence a second sequence, then Fmoc removal can be simultaneously, thereby simultaneously two solid phase synthesis of sequences; when using Fmoc as the lysine α - amino-protecting group, and using as the Alloc lysine ε - amino protecting group, the first sequence and the second sequence of the present invention synthetic sequence, the first synthesis using weakly basic deprotecting agent (e.g., 25% piperidine / DMF) removing Fmoc protecting group, the case of lysine ε - amino Alloc protective groups are not affected. 当第一序列合成结束后,使用合适的试剂(如Pd(PPh3)4,即四三苯基膦钯)脱除Alloc保护基,再合成第二序列。 After the end of the first synthetic sequence using a suitable reagent (e.g., Pd (PPh3) 4, i.e., tetrakistriphenylphosphine palladium) Alloc protecting group is removed, and then a second synthetic sequence.

[0089] 具体地,制备本发明化合物的步骤如下所示: [0089] Specifically, the step of preparation of the compounds of the present invention are shown below:

[0090] (I)固相合成第一序列和第二序列 [0090] (I) the solid phase synthesis of the first sequence and the second sequence

[0091] 根据第一序列C末端是否需要进行酰胺化修饰,可选择合适的树脂用于本发明的多肽化合物的制备。 [0091] whether the first sequence amidated C-terminus according to need, select the appropriate polypeptide compounds of the resins prepared according to the present invention. 当第一序列的C末端进行酰胺化修饰时,可选择Rink Amide MBHA树脂;当第一序列的C末端不需要进行酰胺化修饰时,可选择偶联C末端氨基酸的Wang树脂。 When the C-terminus of the first sequence is amidated, optionally Rink Amide MBHA resin; when the C-terminus of the first sequence does not need to be amidated, Wang resin selectively coupling the C-terminal amino acid.

[0092] 固相合成的方向为C末端-N末端,以第一序列中X9部分C端赖氨酸起始,延长序列至第一序列的连接氨基酸(赖氨酸),然后同时或先后脱除该连接体赖氨酸的α-氨基保护基和ε-氨基保护基,进行第一序列的继续合成和第二序列的合成。 [0092] Solid phase synthesis is terminated -N direction C-terminus, the C-terminal portion of X9 to a first lysine starting sequence, the amino acid sequence is connected to a first extended sequence (lysine), then simultaneous or sequential removal in addition to the linker lysine ε- and α- amino protecting groups amino-protecting group, and the synthesis continued synthesis of the second sequence of the first sequence. 当同时脱除连接体氨基酸的α-氨基保护基和ε_氨基保护基时,步骤如下:浸泡树脂,脱除树脂的氨基保护基,洗涤并监测,偶联第一序列中Χ9部分的第一个氨基酸(即用于连接PEG的赖氨酸),洗涤并监测,脱除α-氨基保护基并依次偶联X9部分的其他氨基酸,脱除连接体氨基酸的α -氨基保护基和ε -氨基的保护基,进行第一序列剩余氨基酸的继续合成和第二序列氨基酸序列的合成,任选对各序列中最后一个氨基酸进行乙酰化修饰。 When both α- amino protecting group, and removal of amino protecting group ε_ linker amino acid, the following steps: soaking the resin, removal of the amino protective group resin, washed and monitoring a first sequence of a first coupling portion in Χ9 amino acids (i.e. lysine for attachment of PEG), washed and monitoring, removal of α- amino protecting group the other amino acid sequence and X9 coupling portion, removal of the linker amino acid α - amino-protecting group, and [epsilon] - amino protecting group, the synthesis continued synthesis of the first sequence and the remaining amino acid sequence of a second amino acid sequence, optionally in each of the last amino acid sequence for acetylation. 当先后脱除连接体氨基酸的α -氨基保护基和ε -氨基保护基时,步骤如下:浸泡树脂,脱除树脂的氨基保护基,洗涤并监测,偶联第一序列中Χ9部分的第一个氨基酸(即用于连接PEG的赖氨酸),洗涤并监测,脱除α-氨基保护基并依次偶联Χ9部分的其他氨基酸,脱除连接体氨基酸的α-氨基保护基并进行第一序列剩余氨基酸的继续合成,脱除连接体氨基酸的ε -氨基保护基并进行第二序列氨基酸序列的合成,任选对各序列中最后一个氨基酸进行乙酰化修饰。 When the linker has amino acids removed α - amino-protecting group, and [epsilon] - amino protecting group, the following steps: soaking the resin, removal of the amino protective group resin, washed and monitoring a first sequence of a first coupling portion in Χ9 amino acids (i.e. lysine for attachment of PEG), washed and monitoring, removal of α- amino protecting group the other amino acid sequence and Χ9 coupling portion, removal of α- amino protecting group of the linker and the first amino acid continue remaining synthetic sequence of amino acids, removal of amino acids connected ε - synthesis of amino-protecting group and a second amino acid sequence, and optionally the respective last amino acid sequence for acetylation.

[0093] 本发明所使用的“氨基保护基”、“ α -氨基保护基”和“ ε -氨基保护基”是指为保护参与缩合反应的氨基而引入的化学基团。 [0093] "amino protecting group" used in the present invention, "α - amino protecting group" and "ε - amino-protecting group" refers to an amino group participating in the condensation reaction is protected by a chemical group introduced. 所述的氨基保护基包括但不限于:叔丁氧羰基(Boc)、节氧羰基(cbz)、三氯乙氧羰基(Troc)、荷基甲氧基羰基(Fmoc)、烯丙氧基羰基(Alloc)等。 The amino protecting groups include, but are not limited to: t-butyloxycarbonyl (Boc), Section oxycarbonyl group (cbz), trichloroethoxycarbonyl (Troc), menthyl methoxycarbonyl (Fmoc), allyloxycarbonyl group (of Alloc) and the like. 优选使用Fmoc作为α -氨基保护基,优选使用Fmoc或Alloc作为ε -氨基保护基。 Fmoc is preferred to use as α - amino protecting group, preferably using Fmoc or Alloc as ε - amino protecting group.

[0094] α -氨基保护基和树脂氨基保护基Fmoc的脱除选用哌啶(PIP),浓度20_25 %(PIP:DMF),时间为20-50min。 [0094] α - amino protecting group and the amino protecting group Fmoc resin selected removal piperidine (PIP), concentration 20_25% (PIP: DMF), time 20-50min. ε -氨基的Alloc保护基的脱除选用,例如Pd(PPh3)4,浓度10-15% (Pd(PPh3)4:氯仿),时间为30-60min。 ε - Alloc removal of amino-protective group selected, for example, Pd (PPh3) 4, a concentration of 10-15% (Pd (PPh3) 4: Chloroform), time 30-60min.

[0095] 作为固相多肽合成技术的一个优势,对部分氨基酸的侧链可以通过引入化学基团进行保护,例如Arg可以采用五甲基苯并呋喃-5-磺酰基(Pbf) ;His、Cys、Gln、Asn可以采用三苯甲基(Trt) ;Lys可以采用叔丁氧羰基(Boc) ;Thr、Tyr、Ser可以采用叔丁基(tBu);Asp、Glu可以采用叔丁酯(Otbu)。 [0095] The solid phase synthesis of a polypeptide advantage of an amino acid side chain moiety can be protected by the introduction of chemical groups, for example, may be employed Arg-pentamethyl-benzofuran-5-sulfonyl (Pbf); His, Cys , Gln, Asn may be employed trityl (Trt); Lys t-butyloxycarbonyl (Boc) may be used; Thr, Tyr, Ser may be used t-butyl (tBu); Asp, Glu may be used t-butyl ester (OtBu) . 所述的保护基团不限于此,可以根据本领域常规方案进行合理选择。 The protecting group is not limited thereto, and may be selected in accordance with a reasonable program conventional in the art.

[0096] 合成过程中所用溶剂选自二甲基甲酰胺(DMF)或二氯甲烷(DCM)。 [0096] The synthesis process solvent is selected from dimethylformamide (DMF) or dichloromethane (DCM) with.

[0097] 偶联试剂选自:碳二亚胺型试剂或苯并三氮唑鎗盐型试剂中的一种,与1-羟基苯并三唑(HOBt)或N,N-二异丙基乙胺(DIEA)中的一种的组合。 [0097] The coupling reagent is selected from: one kind of carbodiimide type reagent or benzotriazole type gun salt reagent, and 1-hydroxybenzotriazole (HOBt), or N, N- diisopropylethylamine combination ethylamine (DIEA) in one. 其中,碳二亚胺型试剂包括二环己基碳二亚胺(DCC)、二异丙基碳二亚胺(DIC)及N-二氨基丙基-N-乙基碳二亚胺(EDC)。 Wherein the carbodiimide type reagent comprises dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC) and N- diaminopropyl -N- ethylcarbodiimide (EDC) . 苯并三氮唑鎗盐型试剂包括2-(1Η-苯并三偶氮L-1-基)-1,1,3,3_四甲基脲四氟硼酸酯(TBTU)、O-苯并三唑-N,N,K , N1-四甲基脲六氟磷酸盐(HBTU)、六氟磷酸苯并三唑-1-氧基三(二甲氨基)磷(BOP)、六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBOP)等。 Benzotriazole type reagent gun salts include 2- (1Η- benzo trisazo L-1- yl) -1,1,3,3_ tetramethyluronium tetrafluoroborate (TBTU), O- benzotriazole -N, N, K, N1- tetramethyluronium hexafluorophosphate (HBTU), hexafluorophosphate, benzotriazol-1-yloxytris (dimethylamino) phosphonium (BOP), hexafluoro benzo-triazol-1-yl phosphate - yloxy tripyrrolidinophosphonium (PyBOP) and the like.

[0098] 偶联试剂优选DCC与HOBt的组合,DCC与DIEA的组合,TBTU与HOBt的组合,TBTU与DIEA的组合,最优选DCC与HOBt的组合。 [0098] Preferred coupling reagents combination of DCC and HOBt, and DIEA in a combination of DCC, TBTU and HOBt in combination, TBTU and DIEA, and most preferably a combination of DCC and HOBt.

[0099] 乙酰化试剂可选择使用乙酸、乙酸酐或者其卤化衍生物(如α -氯乙酸等),同时选用吡啶或DIEA等营造出碱性反应环境。 [0099] Alternatively acetylation agent acetic acid, acetic anhydride or halogenated derivative thereof (e.g., α - acid, etc.), pyridine or DIEA and the like also selected to create a basic reaction environment. 优选的,本发明中选择吡啶和乙酸酐。 Preferably, the present invention is selected in pyridine and acetic anhydride. 备选的,本发明中选择乙酸酐和DIEA。 Alternatively, the present invention is selected in acetic anhydride and DIEA.

[0100] 监测方法选用茚三酮检测法。 [0100] Monitoring Methods ninhydrin detection.

[0101] (2)从树脂上裂解多肽二聚体 [0101] (2) a polypeptide dimer cleaved from the resin

[0102] 裂解可在三氟乙酸或其它强酸介质中进行,加入5-20% V/V侧链保护基清除剂,如茴香硫醚、三异丙基硅烷、苯酚、水、乙二硫醇、间甲酚等。 [0102] can be cleaved in trifluoroacetic acid or other strong acid medium, the addition of 5-20% V / V side chain protecting group scavengers, such as thioanisole, triisopropylsilane, phenol, water, ethanedithiol , m-cresol and so on. 侧链保护基清除剂的选择和配比则根据序列中侧链保护基的类别和数量来灵活确定。 Selection and matching side-chain protecting groups scavenger is flexibly determined according to the type and the number of sequences in the side chain protecting groups. 裂解得到的多肽化合物由冰乙醚进行沉淀得到。 Polypeptide compound obtained by cleavage of ether to precipitate the ice.

[0103] (3)纯化多肽二聚体并干燥 [0103] (3) dried and purified polypeptide dimer

[0104] 将所得多肽二聚体使用反相色谱法或离子交换色谱法进行纯化,优选使用反相色谱法。 [0104] The resulting polypeptide dimer using reverse phase chromatography or ion exchange chromatography purification, preferably using reverse phase chromatography. 将纯化后的多肽二聚体真空冷冻干燥后贮存。 The purified polypeptide dimer stored frozen and dried in vacuo.

[0105] 特别有益的是为了能获得分子内二硫键修饰的本发明多肽,可以通过氧化的方法达到。 [0105] It is particularly advantageous in order to obtain the intramolecular disulfide bonds of the modified polypeptides of the invention can be achieved by means of oxidation. 氧化试剂选自DMS0、氰化钾、双氧水、碘等。 Oxidizing agent is selected from DMSO, potassium ferricyanide, hydrogen peroxide, iodine and the like. 优选采用DMSO作为氧化试剂。 DMSO is preferably used as the oxidizing agent. 据报道,其倾向于形成分子内二硫键,从而可获得高纯度的分子内二硫键修饰的本发明多肽。 According to reports, which tend to form intramolecular disulfide bonds, thereby obtain a high-purity intermolecular disulfide modified polypeptides of the present invention. DMSO的浓度为10-40 %,优选20-30 %。 DMSO concentration of 10-40%, preferably 20-30%.

[0106] 特别有益的是为了能获得PEG修饰的本发明多肽,可以选用针对氨基进行修饰的PEG试剂进行修饰。 [0106] It is particularly advantageous in order to obtain PEG-modified polypeptide of the invention, PEG reagents can be selected for modified amino group modified. 如甲氧基聚乙二醇丙酸NHS酯(mPEG-SPA-NHS,北京键凯),PEG修饰采用的反应体系为DMF,多肽化合物与PEG的摩尔比控制为1: 1.2〜1.5,反应浓度可根据多肽和所用PEG的分子量灵活确定。 Polyethylene glycol such as methoxy propionic acid NHS ester (mPEG-SPA-NHS, Beijing JenKem), PEG-modified reaction system employed for controlling the molar ratio of DMF, polypeptide with PEG compound is 1: 1.2~1.5, reaction concentration It can be flexibly determined according to the molecular weight of PEG and the polypeptide. PEG修饰的产物可使用反相、大孔吸附或离子交换树脂进行纯化,优选使用反相色谱法。 PEG modified products can be used inverted, or macroporous ion exchange resin purification, preferably using reverse phase chromatography. 将纯化后的多肽化合物真空冷冻干燥后贮存。 The polypeptide purified compound was dried in vacuo stored frozen.

[0107] 在本发明的另一方面,本发明提供多肽化合物作为活性成分的药物组合物,其还可包含药学上可接受的合适赋形剂,本领域技术人员可根据给药途径选自合适的赋形剂。 [0107] In another aspect of the present invention, the present invention provides polypeptide compounds as an active ingredient of a pharmaceutical composition, which may also comprise a suitable pharmaceutically acceptable excipients skilled in the art may be suitably selected according to the route of administration excipients. 本发明的药物组合物可以以任何合适的方式给予,包括肠胃外、静脉内、肌内、腹膜内、皮下、经皮、直接输注等。 The pharmaceutical compositions of the invention may be administered in any suitable manner, including parenteral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, direct infusion the like. 本发明多肽化合物优选以冻干粉针的形式存在,可在给药前以适当的剂量与合适的等渗溶液混合后给予所需要的受试者。 Polypeptide compounds of the invention is preferably present in the form of freeze-dried powder, may be mixed after appropriate dose and a suitable isotonic solution is administered to a subject in need prior to administration. 给药剂量和频率将取决于年龄、性别和患者的病况、同时给予的其他药物、禁忌和由临床医生考虑的其他参数等,例如给药剂量可为lug/kg-80mg/kg,给药频率可为每天I次、每周I次或每月I次等,给药剂量和给药频率不特别限制,本领域技术人员可综合考虑上述因素合适地选择。 The dosage and frequency of administration will depend on the age, sex and condition of the patient, and other medications administered, contraindications and other parameters considered by the clinician, the dosage may be, for example, lug / kg-80mg / kg, the frequency of administration I may be times a week or once a month I inferior I, the dosage and frequency of administration is not particularly limited, those skilled in the art considering the above factors may be suitably selected day.

[0108] 本发明的另一方面提供了所述多肽化合物对于血红细胞产生不足、缺陷或者过度消耗相关的疾病的用途。 [0108] Another aspect of the present invention provides a compound for the polypeptide insufficient red blood cell production, the use of defective or excessive consumption related diseases. 例如,本发明的多肽化合物可用于治疗肾机能不全和/或末期肾衰竭/透析导致的贫血、肾移植后的贫血、肿瘤治疗中放化疗导致的贫血、与艾滋病相关的贫血、与慢性炎性疾病(例如,类风湿性关节炎和慢性肠炎)相关的贫血以及用于手术前增加患者的红细胞数。 For example, the polypeptide compounds of the invention may be useful in the treatment of renal insufficiency and / or end stage renal failure / dialysis anemia, anemia following renal transplantation, tumor therapy of anemia caused by chemotherapy, AIDS-related anemia, caused by the chronic inflammatory diseases (e.g., rheumatoid arthritis and chronic bowel disease) and anemia associated with red cell count for increasing patient before surgery. 本发明的多肽化合物还可以用于β_地中海贫血、囊性纤维化、妊娠和绝经病症、早熟性早期贫血、脊髓损伤、太空飞行、急性失血、衰老、中风、局部缺血(CNS和心脏)和伴随异常红细胞生成的多种肿瘤病况等。 Polypeptide compounds of the invention may also be used β_ thalassemia, cystic fibrosis, pregnancy and menopause disorders, early anemia of prematurity, spinal cord injury, space flight, acute blood loss, aging, stroke, ischemia (CNS and cardiac) and various neoplastic conditions associated abnormal erythropoiesis like. 优选地,本发明的多肽化合物特别适用于肾机能不全和/或末期肾衰竭/透析导致的贫血、肾移植后的贫血、肿瘤治疗中放化疗导致的贫血。 Preferably, the polypeptide compounds of the invention are particularly suitable for renal insufficiency and / or end stage renal failure / dialysis anemia, anemia after kidney transplantation, the treatment of anemia caused by cancer chemotherapy caused.

[0109] 本发明所述血红细胞产生不足、缺陷或者过度消耗相关的疾病其共同并且最为直接的病理表现为红细胞数量相对或者绝对不足和或红细胞功能的降低。 [0109] The present invention, blood cell production is insufficient, defective or excessive consumption of its most direct and common pathology related disease is the number of relative or absolute deficiency of red blood cells and red blood cell function or reduced. 非常有益的是本发明的多肽化合物的活性通过网织红细胞法的原理来进行测定并获得确认,具体方法如下:给小鼠皮下注射本发明的多肽化合物,采用全自动网织红细胞分析仪计数每只小鼠血液中的网织红细胞数对红细胞总数的比值(Ret% )。 Active polypeptide is a very useful compound of the present invention is measured and confirmed by network principles erythrocytes ODF, specifically as follows: subcutaneous injection of mice polypeptide compounds of the invention, the use of automatic analyzers reticulocyte counts per only the number of red blood woven mesh ratio of the total number of erythrocytes (Ret%) mice. 以Ret%的增加率来表示实验样品的促红细胞生成的活性。 Ret% increasing rate in the test sample indicates the activity of erythropoietin.

具体实施方式 Detailed ways

[0110] 通过下面的实施例描述本发明。 The present invention is [0110] described by the following examples. 然而,本发明中这些和其他实施例仅用于阐明并不限制本发明或者实施例的范围。 However, the present invention, these and other examples are intended to illustrate embodiments do not limit the present invention or the scope of embodiments. 同样,本发明不限于本文描述的任何具体优选的实施方案。 Similarly, the present invention is not limited to any particular preferred embodiments described herein. 实际上,阅读本说明书时本发明的许多修改和变通方案对于本领域技术人员是显而易见的,并且可以做出所属修改和变通方案而不背离本发明的权利和范围。 Indeed, many modifications and variations of the present invention upon reading the present specification, the skilled person will be apparent to, and may make modifications and variations relevant to programs and without departing from the scope of the invention as claimed.

[0111] 实施例1制备多肽二聚体I [0111] Example 1 Preparation of embodiment I polypeptide dimer

[0112] (I)材料及试剂 [0112] (I) Materials and Reagents

[0113] Rink Amide MBHA 树脂,取代值0.29mmol/g。 [0113] Rink Amide MBHA resin, the value substituted 0.29mmol / g.

[0114]所需的保护氨基酸 Fmoc-L-Ala-OH、Fmoc-Gly-OH、Fmoc-L-Arg (Pbf)-0H、Fmoc-L-Gln(Trt)-OH、 Fmoc-L-His(Trt)-OH> Fmoc-L-Leu-OH、 Fmoc-L-1Ie_0H、Fmoc-L-Cys(Trt)-OH、 Fmoc-L-Lys(Boc)-0H> Fmoc-L-Pro-OH、 Fmoc-L-Met-OH、Fmoc-L-Thr (tBu)-OH、Fmoc-L-Tyr (tBu) -0H> Fmoc-L-Val-OH、Fmoc-L-Lys (Fmoc) -OH 和Fmoc-L-Trp-OH0 [0114] the desired protected amino acid Fmoc-L-Ala-OH, Fmoc-Gly-OH, Fmoc-L-Arg (Pbf) -0H, Fmoc-L-Gln (Trt) -OH, Fmoc-L-His ( Trt) -OH> Fmoc-L-Leu-OH, Fmoc-L-1Ie_0H, Fmoc-L-Cys (Trt) -OH, Fmoc-L-Lys (Boc) -0H> Fmoc-L-Pro-OH, Fmoc -L-Met-OH, Fmoc-L-Thr (tBu) -OH, Fmoc-L-Tyr (tBu) -0H> Fmoc-L-Val-OH, Fmoc-L-Lys (Fmoc) -OH and Fmoc- L-Trp-OH0

[0115] 试剂:HOBt、DIC、DMF、哌啶、乙酸酐、吡啶 [0115] Reagents: HOBt, DIC, DMF, piperidine, acetic anhydride, pyridine

[0116] (2)仪器 [0116] (2) Instruments

.[0117] PSI300型多肽合成仪、Waters高效液相色谱仪、磁力搅拌器 . [0117] PSI300 polypeptide synthesizer, a Waters HPLC, magnetic stirrer

[0118] (3)操作步骤 [0118] (3) Procedure

[0119]以 0.15mmol 为例: [0119] to 0.15mmol example:

[0120] a.称取Rink Amide MBHA树脂0.52g,置于多肽合成仪的反应器中,加入15mLDMF,浸泡2h。 [0120] a. Rink Amide MBHA resin was weighed 0.52g, placed peptide synthesizer reactor, 15 ml of DMF was added, soaking 2h.

[0121] b.脱除树脂氨基保护基 [0121] b. Removal of the amino-protecting group the resin

[0122] 加入20% PIP (DMF)溶液15mL,混合30min,用DMF洗涤树脂7次。 [0122] Add 20% PIP (DMF) solution of 15mL, mixed 30min, the resin was washed 7 times with DMF.

[0123] c.偶联反应 [0123] c. The coupling reaction

[0124]混合反应器中加入 21Img Fmoc-L-Lys (Boc) -OH(0.45mmol)、偶联试剂0.2mol/LHOBt和0.2mol/L DIC各2.5ml进行反应,反应温度为室温,以茚三酮反应检测反应进程,确保Lys偶联到树脂上,用DMF洗涤树脂7次。 [0124] mixing reactor was added 21Img Fmoc-L-Lys (Boc) -OH (0.45mmol), coupling reagent 0.2mol / LHOBt and 0.2mol / L DIC 2.5ml of each reaction, the reaction temperature is room temperature to indene trione reaction detection reaction process to ensure Lys coupled to the resin, the resin was washed 7 times with DMF.

[0125] d.肽链的延长 [0125] d. Extended peptide chain

[0126] 当Lys连接到树脂上后,加入20% PIP (DMF)溶液15mL,混合30min,脱除Lys的α -氨基保护剂Fmoc,用DMF洗涤树脂7次,加入134mg Fmoc-Gly-OH (0.45mmol)、偶联试剂0.2mol/L HOBt和0.2mol/L DIC各2.5ml进行反应,反应温度为室温,以茚三酮反应检测反应进程,确保Gly偶联到树脂上,用DMF洗涤树脂7次。 [0126] When the Lys attached to the resin, was added 20% PIP (DMF) solution of 15mL, mixed 30min, removal of Lys α - amino protecting agent Fmoc, the resin was washed 7 times with DMF, was added 134mg Fmoc-Gly-OH ( 0.45 mmol), coupling reagent 0.2mol / L HOBt and 0.2mol / L DIC 2.5ml of each reaction, the reaction temperature is room temperature, the reaction with ninhydrin detection reaction process to ensure Gly coupled to the resin, the resin was washed with DMF 7 times.

[0127] 当Gly连接到树脂上后,加入20% PIP(DMF)溶液15mL,混合30min,脱除Gly的α -氨基保护剂Fmoc,用DMF洗漆树脂7 次,加入266mg Fmoc-L-Lys (Fmoc) -OH(0.45mmol)、偶联试剂0.2mol/L HOBt和0.2mol/L DIC各2.5ml进行反应,反应温度为室温,以茚三酮反应检测反应进程,确保Gly偶联到树脂上,用DMF洗涤树脂7次。 [0127] When the Gly attached to the resin, was added 20% PIP (DMF) solution of 15mL, mixed 30min, removal of Gly α - amino protecting agent Fmoc, washed with DMF the resin varnish 7, was added 266mg Fmoc-L-Lys (Fmoc) -OH (0.45mmol), coupling reagent 0.2mol / L HOBt and 0.2mol / L DIC 2.5ml of each reaction, the reaction temperature is room temperature, the reaction with ninhydrin detection progress of the reaction, the resin coupled to ensure Gly , the resin was washed 7 times with DMF.

[0128] 当连接氨基酸Lys连接到树脂上后,加入20% PIP(DMF)溶液30mL,混合40min,同时脱除Lys的α-氨基和ε-氨基保护基Fmoc,用DMF洗涤树脂7次,加入268mgFmoc-Gly-OH (0.90mmol)、偶联试剂0.2mol/L HOBt 和0.2mol/L DIC 各5.0ml 进行反应,反应温度为室温,以茚三酮反应检测反应进程,确保Gly偶联到树脂上,用DMF洗涤树脂7次。 [0128] When the connection Lys amino acid attached to the resin, was added 20% PIP (DMF) solution of 30mL, mixed 40min, the simultaneous removal of an amino group of Lys α- and ε- amino protecting group Fmoc, the resin was washed 7 times with DMF, was added 268mgFmoc-Gly-OH (0.90mmol), coupling reagent 0.2mol / L HOBt and 0.2mol / L DIC 5.0ml of each reaction, the reaction temperature is room temperature, the reaction with ninhydrin detection progress of the reaction, the resin coupled to ensure Gly , the resin was washed 7 times with DMF. 接着按相同的步骤继续进行第一序列和第二序列后续氨基酸的合成,直至第一序列和第二序列的所有氨基酸都链接到树脂上。 Followed by the same steps to continue the synthesis of the first and second sequences of subsequent amino acids until all amino acids are the first and second sequences are linked to the resin.

[0129] e.乙酰化修饰 [0129] e. Acetylation

[0130] 肽序列合成结束后,,加入5ml哌啶和5ml乙酸酐反应30分钟,脱除第一序列和第二序列的α -氨基保护基Fmoc。 [0130] After the sequence of the synthetic peptide was added ,, piperidine 5ml and 5ml of acetic anhydride for 30 minutes to remove α of the first and second sequences - amino protecting group Fmoc.

[0131] f.裂解及沉淀 [0131] f. Lysis and precipitation

[0132] 乙酰化结束后,真空干燥树脂,称重。 After [0132] acetylated resin was dried in vacuo and weighed. 按Ig树脂IOmL裂解试剂的比例加入裂解试剂(试剂配比-JFA/茴香硫醚/三异丙基硅烷/苯酚/水/TIS = 85/5/5/4/1),室温搅拌反应3小时,抽滤。 Ig resin proportion IOmL lytic reagent lysis reagent (ratio -JFA / thioanisole / triisopropylsilane / phenol / water / TIS = 85/5/5/4/1), stirred at room temperature for 3 hours , leaching. 向裂解抽滤液中,加入冰乙醚,沉淀多肽,离心,弃上清,真空干燥,称重粗肽。 Pumping the filtrate into the pyrolysis, ice was added diethyl ether, polypeptide precipitation, centrifugation, supernatant was discarded, and dried in vacuo, the crude peptide weighed.

[0133] g.氧化形成二硫键 [0133] g. Oxidized to form a disulfide bond

[0134] 以5mg/ml的浓度将粗肽溶解于DMSO中,然后加入4倍体积的水。 [0134] at a concentration of 5mg / ml of the crude peptide was dissolved in DMSO and then added to 4 volumes of water. 室温下(彡IO0C )静止36h。 At room temperature (San IO0C) stationary 36h.

[0135] h.反相色谱纯化 Purification [0135] h. Reverse phase chromatography

[0136] 用制备型HPLC,采用反相色谱法,纯化上述粗肽。 [0136] by preparative HPLC, using reverse phase chromatography, purified crude peptide.

[0137] HPLC 柱:C18 制备柱 [0137] HPLC Column: C18 preparative column

[0138]流速:10mT ,/mi η [0138] flow rate: 10mT, / mi η

[0139] A:含0.1 % TFA的水溶液 [0139] A: 0.1% TFA aqueous solution of

[0140] B:含0.1% TFA的乙腈溶液 [0140] B: acetonitrile containing 0.1% TFA,

[0141]用 18-38%的B 相,洗脱90min。 [0141] B phase with 18-38% of eluted 90min.

[0142] 所纯化的产物是均一的,其纯度为95%,总得率为18%。 [0142] The purified product is a homogeneous, having a purity of 95%, 18% overall yield. MS:m/z = 4693.3 [M+H]。 MS: m / z = 4693.3 [M + H].

[0143] 实施例2确定DMSO氧化形成二硫键的反应时间 [0143] Example 2 to determine oxidation DMSO embodiment disulfide bond formation reaction time

[0144] 根据实施例1的方法,类似合成多肽二聚体6,其中另外加入的保护氨基酸为Fmoc-L-Asp(Otbu)-OH和Fmoc-L-Glu(Otbu)-0H。 [0144] According to the method of Example 1 analogously to the synthetic polypeptide dimer 6, wherein the addition of further protected amino acid is Fmoc-L-Asp (Otbu) -OH and Fmoc-L-Glu (Otbu) -0H.

[0145] (I)材料及试剂 [0145] (I) Materials and Reagents

[0146] 多肽二聚体6氧化前的粗肽,DMSO, /K,乙腈,TFA [0146] The crude peptide before 6 polypeptide dimer oxidation, DMSO, / K, acetonitrile, TFA

[0147] (2)仪器 [0147] (2) Instruments

[0148] Waters高效液相色谱仪、25 °C恒温箱。 [0148] Waters HPLC, 25 ° C incubator.

[0149] (3)氧化方法 [0149] (3) the oxidation method

[0150] 称取9份多肽二聚体6氧化前的粗肽样品,每份约2mg。 [0150] 9 parts of crude peptide weighed sample before oxidation polypeptide dimer 6, each about 2mg. 各加入Iml 20% DMSO溶液,置于25°C恒温箱中,取反应0h,lh,2h,4h,8h,12h,24h,30h,36h的样品进行液相分析。 Iml 20% DMSO was added to each solution and placed in 25 ° C incubator, the reaction taking 0h, lh, 2h, 4h, 8h, 12h, 24h, 30h, 36h by liquid samples analyzed.

[0151] (4)分析方法 [0151] (4) Analysis Method

[0152] 应用RP-HPLC样品的氧化过程和趋势,采用色谱柱-Boston PHlex ODSC18 (5 μ m) 4.6X 250mm,流动相(A) ^0.05% TFA 水溶液,(B) ^0.05% TFA 乙腈溶液。 [0152] Oxidation and trends RP-HPLC sample application, the column using -Boston PHlex ODSC18 (5 μ m) 4.6X 250mm, mobile phase (A) ^ 0.05% TFA aqueous solution, (B) ^ 0.05% TFA in acetonitrile . 洗脱条件是B相梯度从20%到30%洗脱10min,30%到45%洗脱25min,检测波长215nm。 Gradient elution condition from 20% B to 30% elution 10min, 30% to 45% elution 25min, detection wavelength 215nm.

[0153] (5)氧化结果: [0153] (5) Oxidation Results:

[0154] 氧化反应前的样品主峰为26.5min,氧化反应后样品主峰为18.7min,考虑杂质峰 [0154] Samples of the oxidation reaction before the main peak 26.5min, the oxidation peak for the sample 18.7min, considering impurity peak

和基线噪音的影响,可认为,36h已反应完全。 And the effect of baseline noise, can be considered, 36h reacted completely.

[0155] [0155]

Figure CN103421094AD00171

[0156] [0156]

[0157] *(已将样品浓度均换算为2mg/ml) [0157] * (sample concentrations were already converted to 2mg / ml)

[0158] 实施例3制备多肽二聚体9 [0158] Example 3 Preparation of embodiment 9 polypeptide dimer

[0159] (I)材料及试剂 [0159] (I) Materials and Reagents

[0160] Rink Amide MBHA 树脂,取代值0.29mmol/g。 [0160] Rink Amide MBHA resin, the value substituted 0.29mmol / g.

[0161]所需的保护氨基酸 Fmoc-L-Ala-OH、Fmoc-Gly-OH、Fmoc-L-Arg (Pbf)-0H、 [0161] the desired protected amino acid Fmoc-L-Ala-OH, Fmoc-Gly-OH, Fmoc-L-Arg (Pbf) -0H,

Fmoc-L-Gln (Trt)-OH、Fmoc-L-His (Trt)-0H> Fmoc-L-Leu-OH> Fmoc-L-1le_0H、 Fmoc-L-Gln (Trt) -OH, Fmoc-L-His (Trt) -0H> Fmoc-L-Leu-OH> Fmoc-L-1le_0H,

Fmoc-L-Cys (Trt)-OH、Fmoc-L-Lys (Boc)-0H> Fmoc-L-Pro-OH、Fmoc-L-Met-OH、 Fmoc-L-Cys (Trt) -OH, Fmoc-L-Lys (Boc) -0H> Fmoc-L-Pro-OH, Fmoc-L-Met-OH,

Fmoc-L-Thr (tBu)-OH、Fmoc-L-Tyr (tBu) -0H> Fmoc-L-Val-OH、Fmoc-L-Lys (Al loc) -OH 和 Fmoc-L-Thr (tBu) -OH, Fmoc-L-Tyr (tBu) -0H> Fmoc-L-Val-OH, Fmoc-L-Lys (Al loc) -OH, and

Fmoc-L-Trp-OH0 Fmoc-L-Trp-OH0

[0162] 试剂:H0Bt、DIC、DMF、哌啶、乙酸酐、吡啶、氯仿、Pd (PPh3)4 [0162] Reagents: H0Bt, DIC, DMF, piperidine, acetic anhydride, pyridine, chloroform, Pd (PPh3) 4

[0163] (2)仪器 [0163] (2) Instruments

[0164] PSI300型多肽合成仪、Waters高效液相色谱仪、磁力搅拌器 [0164] PSI300 polypeptide synthesizer, a Waters HPLC, magnetic stirrer

[0165] (3)操作步骤 [0165] (3) Procedure

[0166]以 0.15mmol 为例: [0166] to 0.15mmol example:

[0167] a.称取Rink Amide MBHA树脂0.52g,置于多肽合成仪的反应器中,加入15mLDMF, [0167] a. Rink Amide MBHA resin was weighed 0.52g, placed peptide synthesizer reactor, 15 ml of DMF was added,

浸泡2h。 Soak 2h.

[0168] b.脱除树脂氨基保护基 [0168] b. Removal of the amino-protecting group the resin

[0169] 加入20% PIP (DMF)溶液15mL,混合30min,用DMF洗涤树脂7次。 [0169] Add 20% PIP (DMF) solution of 15mL, mixed 30min, the resin was washed 7 times with DMF.

[0170] c.偶联反应[0171]混合反应器中加入 21Img Fmoc-L-Lys (Boc) -OH(0.45mmol)、偶联试剂0.2mol/LHOBt和0.2mol/L DIC各2.5ml进行反应,反应温度为室温,以茚三酮反应检测反应进程,确保Lys偶联到树脂上,用DMF洗涤树脂7次。 [0170] c. The coupling reaction [0171] mixing reactor was added 21Img Fmoc-L-Lys (Boc) -OH (0.45mmol), coupling reagent 0.2mol / LHOBt and 0.2mol / L DIC 2.5ml of each reaction the reaction temperature is room temperature, the reaction with ninhydrin detection reaction process to ensure Lys coupled to the resin, the resin was washed 7 times with DMF.

[0172] d.肽链的延长 [0172] d. Extended peptide chain

[0173] 当Lys连接到树脂上后,加入20% PIP(DMF)溶液15mL,混合30min,脱除Lys的α -氨基保护剂Fmoc,用DMF洗涤树脂7次,加入134mg Fmoc-Gly-OH (0.45mmol)、偶联试剂0.2mol/L HOBt和0.2mol/L DIC各2.5ml进行反应,反应温度为室温,以茚三酮反应检测反应进程,确保Gly偶联到树脂上,用DMF洗涤树脂7次。 [0173] When the Lys attached to the resin, was added 20% PIP (DMF) solution of 15mL, mixed 30min, removal of Lys α - amino protecting agent Fmoc, the resin was washed 7 times with DMF, was added 134mg Fmoc-Gly-OH ( 0.45 mmol), coupling reagent 0.2mol / L HOBt and 0.2mol / L DIC 2.5ml of each reaction, the reaction temperature is room temperature, the reaction with ninhydrin detection reaction process to ensure Gly coupled to the resin, the resin was washed with DMF 7 times.

[0174] 当Gly连接到树脂上后,加入20 % PIP(DMF)溶液15mL,混合30min,脱除Gly的α -氨基保护剂Fmoc,用DMF洗涤树脂7次,加入204mgFmoc-L-Lys (Al loc)-OH (0.45mmol)、偶联试剂0.2mol/L HOBt 和0.2mol/L DIC 各2.5ml 进行反应,反应温度为室温,以茚三酮反应检测反应进程,确保Gly偶联到树脂上,用DMF洗涤树脂7次。 [0174] When the Gly attached to the resin, was added 20% PIP (DMF) solution of 15mL, mixed 30min, removal of Gly α - amino protecting agent Fmoc, washed 7 times with DMF the resin was added 204mgFmoc-L-Lys (Al loc) -OH (0.45mmol), coupling reagent 0.2mol / L HOBt and 0.2mol / L DIC 2.5ml of each reaction, the reaction temperature is room temperature, the reaction with ninhydrin detection reaction process and ensure that the resin was coupled to Gly , the resin was washed 7 times with DMF.

[0175] 当连接氨基酸Lys连接到树脂上后,加入20% PIP (DMF)溶液15mL,混合30min,脱除Lys的α -氨基保护基Fmoc,用DMF洗漆树脂7次,加入134mg Fmoc-Gly-OH(0.45mmol)、偶联试剂0.2mol/L HOBt和0.2mol/L DIC各2.5ml进行反应,反应温度为室温,以茚三酮反应检测反应进程,确保Gly偶联到树脂上,用DMF洗涤树脂7次。 [0175] When the connection Lys amino acid attached to the resin, was added 20% PIP (DMF) solution of 15mL, mixed 30min, removal of Lys α - amino protecting group Fmoc, washed with DMF the resin varnish 7, was added 134mg Fmoc-Gly -OH (0.45mmol), coupling reagent 0.2mol / L HOBt and 0.2mol / L DIC 2.5ml of each reaction, the reaction temperature is room temperature, the reaction with ninhydrin detection reaction process to ensure Gly coupled to the resin, with DMF the resin was washed 7 times. 接着按相同的步骤继续进行第一序列后续氨基酸的合成,直至第一序列的所有氨基酸都连接到树脂上。 Then the same procedure continues for subsequent synthesis of a first sequence of amino acids until all amino acid sequence are first attached to the resin.

[0176] 接着加入10 % Pd (PPh3) 4 (氯仿)IOmL,混合45min,脱除ε -氨基保护基Alloc,用DMF 洗涤树脂7 次,加入134mg Fmoc-Gly-OH(0.45mmol)、偶联试剂0.2mol/L HOBt 和0.2mol/LDIC各5.0ml进行反应,反应温度为室温,以茚三酮反应检测反应进程,确保Gly偶联到树脂上,用DMF洗涤树脂7次。 [0176] Next was added 4 (chloroform) IOmL, mixed 45min 10% Pd (PPh3), removal of ε - amino protecting group of Alloc, the resin was washed 7 times with DMF, was added 134mg Fmoc-Gly-OH (0.45mmol), coupling Reagents 0.2mol / L HOBt and 0.2mol / LDIC 5.0ml of each reaction, the reaction temperature is room temperature, the reaction with ninhydrin detection reaction process to ensure Gly coupled to the resin, the resin was washed with DMF 7 times. 接着按相同的步骤继续进行第二序列后续氨基酸的合成,直至第二序列的所有氨基酸都连接到树脂上。 Then the same procedure continues for subsequent synthesis of the second sequence of amino acids until all amino acid sequence are connected to the second resin.

[0177] e.乙酰化修饰 [0177] e. Acetylation

[0178] 肽序列合成结束后,加入5ml哌啶和5ml乙酸酐反应30分钟,脱除第一序列和第二序列的α -氨基保护基Fmoc。 [0178] After completion of the synthesis the peptide sequence, and 5ml of piperidine was added 5ml of acetic anhydride for 30 minutes to remove α of the first and second sequences - amino protecting group Fmoc.

[0179] f.裂解及沉淀 [0179] f. Lysis and precipitation

[0180] 乙酰化结束后,真空干燥树脂,称重。 After [0180] acetylated resin was dried in vacuo and weighed. 按Ig树脂IOmL裂解试剂的比例加入裂解试剂(试剂配比-JFA/茴香硫醚/三异丙基硅烷/苯酚/水/TIS = 85/5/5/4/1),室温搅拌反应3小时,抽滤。 Ig resin proportion IOmL lytic reagent lysis reagent (ratio -JFA / thioanisole / triisopropylsilane / phenol / water / TIS = 85/5/5/4/1), stirred at room temperature for 3 hours , leaching. 向裂解抽滤液中,加入冰乙醚,沉淀多肽,离心,弃上清,真空干燥,称重粗肽。 Pumping the filtrate into the pyrolysis, ice was added diethyl ether, polypeptide precipitation, centrifugation, supernatant was discarded, and dried in vacuo, the crude peptide weighed.

[0181] g.氧化形成二硫键 [0181] g. Oxidized to form a disulfide bond

[0182] 以5mg/ml的浓度将粗肽溶解于DMSO中,然后加入4倍体积的水。 [0182] at a concentration of 5mg / ml of the crude peptide was dissolved in DMSO and then added to 4 volumes of water. 室温下(彡IO0C )静止36h。 At room temperature (San IO0C) stationary 36h.

[0183] h.反相色谱纯化 Purification [0183] h. Reverse phase chromatography

[0184] 用制备型HPLC,采用反相色谱法,纯化上述粗肽。 [0184] by preparative HPLC, using reverse phase chromatography, purified crude peptide.

[0185] HPLC 柱:C18 制备柱 [0185] HPLC Column: C18 preparative column

[0186]流速:10mT ,/mi η [0186] flow rate: 10mT, / mi η

[0187] A:含0.1 % TFA的水溶液[0188] B:含0.1% TFA的乙腈溶液 [0187] A: 0.1% TFA aqueous solution of [0188] B: acetonitrile containing 0.1% TFA,

[0189]用 18-38%的B 相,洗脱90min。 [0189] B phase with 18-38% of eluted 90min.

[0190] 所纯化的产物是均一的,其纯度为96%,总得率为16%。 [0190] The purified product is a homogeneous, having a purity of 96%, 16% overall yield. MS:m/z = 4706.2 [M+H]。 MS: m / z = 4706.2 [M + H]. [0191 ] 实施例4多肽二聚体I的PEG (分子量为约20K)修饰 [0191] Example I 4 polypeptide dimer of PEG (molecular weight of about 20K) modified

[0192] 对实施例1的化合物(即多肽二聚体I)进行PEG (分子量为约20K)修饰,分别考察温度、时间、浓度对修饰反应的影响。 [0192] (molecular weight of about 20K) modified were investigated effect of temperature, time, concentration of the modification reaction of the compound of Example 1 (i.e., a polypeptide dimer I) embodiment for PEG.

[0193] (I)材料和试剂 [0193] (I) Materials and Reagents

[0194] DMF, DIEA,甲氧基聚乙二醇丙酸NHS酯(约20k,北京键凯科技有限公司)。 [0194] DMF, DIEA, methoxy polyethylene glycol-propionic acid NHS ester (approximately 20k, Beijing JenKem Ltd.).

[0195] (2)仪器 [0195] (2) Instruments

[0196] Waters高效液相色谱仪、28 °C恒温箱、37 °C恒温箱。 [0196] Waters HPLC, 28 ° C incubator, 37 ° C incubator.

[0197] (3)操作过程 [0197] (3) during operation

[0198] a.按照1: 1.5 (摩尔比)分别称取一定质量的多肽和PEG,溶于DMF中,得到澄 [0198] a 1: 1.5 (molar ratio) were weighed and a mass of polypeptide PEG, dissolved in DMF, to give Cheng

清溶液。 Clear solution.

[0199] b.多肽与PEG完全溶解后加入10 μ I DIEA,每隔30min进样分析。 [0199] b. After complete dissolution polypeptide with PEG was added 10 μ I DIEA, sample analysis every 30min. 分别考察温度、 Temperature were investigated,

时间、浓度对修饰反应的影响,结果以修饰率表示。 Time, concentration, results are expressed modifications affect the reaction rate modification.

[0200] [0200]

Figure CN103421094AD00191

[0201] (4)实验结果 [0201] (4) Test Results

[0202] a.当反应温度为28°C,反应浓度为3mg/ml时,结果如下: . [0202] a When the reaction temperature is 28 ° C, the reaction at a concentration of 3mg / ml, the following results:

Figure CN103421094AD00192

[0204] b.当反应温度为28°C,反应浓度为2mg/ml时,结果如下: . [0204] b When the reaction temperature is 28 ° C, the reaction at a concentration of 2mg / ml, the following results:

Figure CN103421094AD00193

[0206] c.当反应温度为28°C,反应浓度为lmg/ml时,结果如下: . [0206] c When the reaction temperature is 28 ° C, at the concentration of lmg / ml, the following results:

Figure CN103421094AD00194

[0208] d.当反应温度为37 C,反应浓度为2mg/ml时,结果如下: . [0208] d When the reaction temperature is 37 C, the reaction at a concentration of 2mg / ml, the following results:

Figure CN103421094AD00195

[0210] e.当反应温度为4°C,反应浓度为2mg/ml时,结果如下: . [0210] e When the reaction temperature is 4 ° C, the reaction at a concentration of 2mg / ml, the following results:

Figure CN103421094AD00201

[0212] 从以上结果可以看出,在本实施例所选取的条件范围内,修饰反应与温度、浓度和反应时呈正相关。 [0212] As can be seen from the above results within the scope of the conditions selected in the present embodiment, the modification reaction temperature, concentration and correlated the reaction. 当温度为28°C,浓度为2mg/ml或3mg/ml时,2小时已反应完全。 When the temperature is 28 ° C, at a concentration of 2mg / ml or 3mg / ml, 2 HOURS reaction was complete. 当温度为37°C,浓度为2mg/ml时,1.5小时已反应完全。 When the temperature is 37 ° C, at a concentration of 2mg / ml, 1.5 HOURS reaction was complete.

[0213] 可类似进行温度、反应浓度和反应时间对PEG (约5k或约40k)修饰多肽二聚体的影响。 [0213] Similarly the temperature may affect polypeptide dimer reaction concentration and reaction time on PEG (or from about 5k to about 40K) modified.

[0214] 实施例5采用分子量为约20k的PEG对多肽二聚体I进行修饰 [0214] Example 5 PEG molecular weight from about 20k to be modified polypeptide dimer I

[0215] (I)材料和试剂。 [0215] (I) Materials and reagents.

[0216] 甲氧基聚乙二醇丙酸NHS酯(约20k,mPEG-SPA-NHS,北京键凯科技有限公司),DMF,DIEA,其中mPEG-SPA-NHS (约20k)为直链型,其结构如下: [0216] Methoxy polyethyleneglycol acid NHS ester (approximately 20k, mPEG-SPA-NHS, Beijing JenKem Ltd.), DMF, DIEA, wherein mPEG-SPA-NHS (approximately 2OK) is a linear , the following structure:

Figure CN103421094AD00202

[0219] Waters高效液相色谱仪、超滤器、恒温箱 [0219] Waters high performance liquid chromatography, ultrafiltration, incubators

[0220] (3)操作步骤 [0220] (3) Procedure

[0221 ] a.称取约20k的mPEG-SPA-NHS 114mg,溶于5.7ml DMF中,制成反应母液A [0221] a. Weigh about 20k of mPEG-SPA-NHS 114mg, dissolved in 5.7ml DMF in the reaction liquor made of A

[0222] b.称取多肽化合物约6.0mg,加入母液A 2ml。 [0222] b. Weigh polypeptide compound of about 6.0mg, was added liquor A 2ml. 5min后加入IOul DIEA。 After 5min join IOul DIEA. 置于28°C恒温箱中。 Placed in 28 ° C incubator. 每间隔半小时取样,使用HPLC监测反应进程。 Sampled every half-hour intervals, progress of the reaction was monitored using HPLC.

[0223] c.2小时后,反应已基本完全。 After [0223] c.2 h, the reaction was essentially complete. 使用10倍体积的纯净水稀释反应溶液,然后使用反相色谱纯化修饰后产物。 Using 10 volumes of reaction solution was diluted with purified water, and then purified using reverse phase chromatography after the product was modified.

[0224] 反相色谱条件如下: [0224] reverse phase chromatography conditions were as follows:

[0225] HPLC 柱:YMC-Pack C4 (10 μ m) 10 X 250mm [0225] HPLC Column: YMC-Pack C4 (10 μ m) 10 X 250mm

[0226]流速:2mL/min [0226] flow rate: 2mL / min

[0227] A:含0.1 % TFA的水溶液 [0227] A: 0.1% TFA aqueous solution of

[0228] B:含0.1 % TFA的乙腈溶液 [0228] B: acetonitrile containing 0.1% TFA,

[0229] 将稀释后的反应溶液通过C4制备柱,接着使用流动相A/B(95/5)冲洗10个柱体积,然后再用流动相A/B(30/70)将目标产物分离下来,得到含目标产物的收集液。 [0229] The reaction solution was prepared by diluting the C4 column, using a mobile phase followed by A / B (95/5) 10 column volumes of rinse and then with mobile phase A / B (30/70) down to separate the desired product to give a solution containing the objective product were collected.

[0230] d.减压蒸馏除去收集液中的TFA和乙腈,再使用截留分子量为3000的超滤膜超滤除去残余的DMF。 [0230] d. Eluate was evaporated under reduced pressure and TFA in acetonitrile, again using molecular weight cut off ultrafiltration membrane 3000 to remove residual DMF.

[0231] e.对得到的目标产物进行冻干,冻干后化合物纯度为99.14%。 [0231] e. Of the desired product were lyophilized to give, after lyophilization compound purity 99.14%. 通过MALD1-T0F测定分子量。 Molecular weights were determined by MALD1-T0F. 目标产物的分子量应为以24692为中心,在一定范围内成正态分布。 Molecular weight of the desired product should be centered at 24692, normally distributed within a certain range. 理论值与质谱结果相符。 Mass spectrometry results are consistent with the theoretical value.

[0232] 实施例6采用分子量为约40k的PEG对多肽二聚体I进行修饰[0233] (I)材料和试剂。 [0232] Example 6 PEG molecular weight from about 40k to be modified polypeptide dimer I [0233] (I) Materials and reagents.

[0234] 甲氧基聚乙二醇丙酸NHS酯(约40k,mPEG-SPA-NHS,北京键凯科技有限公司),DMF, DIEA,其中mPEG-SPA-NHS (约40k)为支链型,每条支链的分子量为约20k,其结构如 [0234] Methoxy polyethyleneglycol acid NHS ester (approximately 40k, mPEG-SPA-NHS, Beijing JenKem Ltd.), DMF, DIEA, wherein mPEG-SPA-NHS (about 40K) is a branched , each molecular weight branched chain of about 20k, the structure such as

下: under:

[0235] [0235]

Figure CN103421094AD00211

[0236] 仪器和操作步骤参照实施例5,但操作步骤c中的反应时间为2.5小时。 [0236] equipment and procedure of Reference Example 5, but the reaction time of the operation in step c was 2.5 hours.

[0237] 对得到的目标产物进行冻干,冻干后化合物纯度为99.96 %。 [0237] lyophilization of the desired product obtained after lyophilization compound purity was 99.96%. 通过MALD1-T0F验证分子量。 Verify molecular weight by MALD1-T0F. 目标产物的分子量应为以44692为中心,在一定范围内成正态分布,理论值与质谱结果相符。 Molecular weight of the desired product should be centered at 44692, normally distributed within a certain range, consistent with the mass spectrometry results of theory.

[0238] 实施例7采用分子量为约5k的PEG对多肽二聚体I进行修饰 [0238] Example 7 A molecular weight of about 5k PEG-modified polypeptide dimer I

[0239] (I)材料和试剂。 [0239] (I) Materials and reagents.

[0240] 甲氧基聚乙二醇丙酸NHS酯(约5k,mPEG-SPA-NHS,北京键凯科技有限公司),DMF, DIEA,其中mPEG-SPA-NHS (约5k)为直链型,其结构如下: [0240] Methoxy polyethyleneglycol acid NHS ester (approximately 5k, mPEG-SPA-NHS, Beijing JenKem Ltd.), DMF, DIEA, wherein mPEG-SPA-NHS (approximately 5k) is a linear , the following structure:

[0241] [0241]

Figure CN103421094AD00212

[0242] 仪器和操作步骤参照实施例5,但操作步骤c中的反应时间为1.5小时。 [0242] equipment and procedure of Reference Example 5, but the reaction time steps c is 1.5 hours.

[0243] 对得到的目标产物进行冻干,冻干后化合物纯度为99.96 %。 [0243] lyophilization of the desired product obtained after lyophilization compound purity was 99.96%. 通过MALD1-T0F验证分子量。 Verify molecular weight by MALD1-T0F. 目标产物的分子量应为以9692为中心,在一定范围内成正态分布,理论值与质谱结果相符。 Molecular weight of the desired product should be in 9692 as the center, normally distributed within a certain range, consistent with the mass spectrometry results of theory.

[0244] 实施例8本发明多肽化合物的体内活性测定 Determination of in vivo activity of the compounds of the invention polypeptide eight cases of [0244] Embodiment

[0245] (I)试剂 [0245] (I) reagent

[0246] a.乙二胺四乙酸二钾抗凝剂 [0246] a. Anticoagulant dipotassium edetate

[0247] 称取乙二胺四乙酸二钾lOOmg,加生理氯化钠溶液IOml溶解,混匀,使用前新鲜配制。 [0247] Weigh ethylenediaminetetraacetic acid dipotassium lOOmg, IOml physiological sodium chloride solution was added to dissolve, mix, freshly prepared before use.

[0248] b.稀释液 [0248] b. Dilutions

[0249] 称取0.1g牛血清白蛋白,加生理氯化钠溶液溶解并稀释至100ml。 [0249] Weigh 0.1g of bovine serum albumin, physiological sodium chloride solution was added dissolved and diluted to 100ml.

[0250] (2)步骤 [0250] (2) Step

[0251] 选取近郊系6-8周龄雌性BALB/c小鼠(体重约16_18g,来源于中国人民解放军军事医学科学院实验动物中心)68只,分成17组,每组动物4只。 [0251] Select suburban lines 6-8 week old female BALB / c mice (body weight about 16_18g, PLA from Experimental Animal Center of Military Medical Sciences) 68, divided into 17 groups, 4 animals per group. 其中阴性对照I组,给予0.2ml的稀释液;其中阳性对照3组,给予重组人促红素注射液(北京四环生物制药有限公司,3000IU/0.6ml/支),剂量分别为10、20、40IU/鼠;本发明的化合物组13组,分别给予多肽二聚体1、多肽二聚体2、多肽二聚体3、多肽二聚体4、多肽二聚体5、多肽二聚体6、多肽二聚体7、多肽二聚体8、多肽二聚体9、多肽二聚体10、实施例5的化合物、实施例6的化合物、实施例7的化合物的冻干样,剂量为20μ g/鼠。 I wherein the negative control group, 0.2ml of the dilutions given; 3 wherein the positive control group received injection of recombinant human erythropoietin (Ring biological Pharmaceutical Co., Ltd. Beijing, 3000IU / 0.6ml / branch), at doses of 10, 20, , 40IU / mouse; group of compounds of the present invention 13 groups, were given a dimeric polypeptide, polypeptide dimer 2, 3 dimeric polypeptide, polypeptide dimer 4, 5 polypeptide dimer, polypeptide dimer 6 , 7 polypeptide dimer, polypeptide dimer 8, 9 dimeric polypeptide, polypeptide dimer 10, the compound of Example 5, the compound of Example 6, lyophilized sample of the compound of Example 7, a dose of 20μ g / mouse.

[0252] 注射后第3天起从小鼠眼眶采血3-4滴,置于预先加入200 μ I EDTA-K2抗凝剂的采血管中。 From [0252] 3 days after injection mice were bled from the orbital 3-4 drops, placed in a pre added 200 μ I EDTA-K2 anticoagulant in the blood collection tube. 对于阴性对照,取血天数为第3、4、5、6、7、8、11、22、28天;对于阳性对照和多肽二聚体1-10,取血天数为第3、4、5、6、7天;对于实施例5、6、7的化合物,取血的天数为第4、5、8、11、22、28天。 For negative controls, the number of days for the first blood 3,4,5,6,7,8,11,22,28 days; polypeptide dimer positive control and 1-10, the number of days of blood 3,4,5 6, 7 days; for the compound of Example 5,6,7 embodiment, the number of days for the first blood 4,5,8,11,22,28 days. 取抗凝血,用全自动网织血红细胞分析仪(XT-2000IV)计数每只小鼠血液中的网织红细胞对红细胞总数的比值(Ret% )。 Taking anticoagulant, the red blood cells with automated reticulocyte analyzers (XT-2000IV) per mouse blood count reticulocytes on the ratio of the total number of red blood cells (Ret%).

[0253] 以Ret %的增加率来表示本发明多肽化合物的促红细胞生成活性。 [0253] In Ret% increasing rate expressed erythropoietin polypeptide of the present invention produce the active compound.

Figure CN103421094AD00221

,其中样品包括阴性对照、阳性对照和本发明的化 Wherein the sample comprises a negative control, and positive controls of the present invention

合物。 Compounds. 多肽二聚体1-10的结果如表3所示,实施例5-7的化合物的结果如表4所示: 1-10 polypeptide dimer results as shown in Table 3, the results of the embodiment of the compound of Example 5-7 shown in Table 4:

[0254]表 3 [0254] TABLE 3

[0255] [0255]

Figure CN103421094AD00222

[0256][0257] 注:表示未测。 [0256] [0257] Note: indicates not determined.

[0258]表 4 [0258] TABLE 4

[0259] [0259]

Figure CN103421094AD00231

[0260] 本发明的化合物通过PEG修饰后,活性与修饰前相比并未下降,但作用时间显著延长。 [0260] Compounds of the invention by the PEG modification with the activity before modification has not declined in comparison, but the effect was significantly prolonged.

Claims (10)

1.一种多肽化合物,其由两条多肽序列构成,其中第一序列为: X1-G-X2-Y-X3-C-X4-MGP-X5-TW-X6-CQPL-X7-G-X8-X9 ;第二序列为:X10-G_X11-Y-X12-C-X13-MGP-X14-X15-W-X16-C-X17-X18-X19-X20-G ;并且第一序列和第二序列通过第一序列中的连接氨基酸X8连接形成二聚体,其中Xl选自G或AcG ;X2、X5和X6分别独立地选自L、I或V ;X3选自A或G ;X4选自H或D-His ;X7选自R、HomoArg或Pal-Lys ;X8选自Lys或D-Lys,以其结构中α -氨基参与第一序列合成,以其结构中ε-氨基与第二序列的C末端氨基酸连接形成二聚体;Χ9为赖氨酸或由2-10个氨基酸组成的C末端为赖氨酸的短肽;Χ10选自G或AcG ;Χ11、Χ14、Χ16或Χ19分别独立地选自L、1、V或Nle ;X12选自A、Aib 或G ;X13 选自H 或D-His ;X15 选自T 或S ;X17 选自Q 或N ;X18 选自P 或Hyp ;X20选自R、HomoArg> Cit 或Pal-Lys。 1. A polypeptide compound, which consists of two polypeptide sequences, wherein the first sequence is: X1-G-X2-Y-X3-C-X4-MGP-X5-TW-X6-CQPL-X7-G-X8 -X9; second sequence: X10-G_X11-Y-X12-C-X13-MGP-X14-X15-W-X16-C-X17-X18-X19-X20-G; and the first and second sequences X8 connection is formed by connecting a first amino acid sequence dimer, wherein Xl is selected from G or AcG; X2, X5 and X6 are each independently selected from L, I or V; X3 is selected from a or G; X4 is selected from H or D-His; X7 is selected from R, HomoArg, or Pal-Lys; X8 is selected from Lys or D-Lys, its structure α - amino involved in the synthesis of the first sequence, its structure and ε- amino group of a second sequence C-terminal amino acid linked to form a dimer; Χ9 is lysine or 2-10 amino acids of the C-terminal lysine of the peptide; of x10, which is selected from G or AcG; Χ11, Χ14, Χ16 independently or Χ19 is selected from L, 1, V or Nle; X12 is selected from A, Aib, or G; X13 is selected from H, or D-His; X15 is selected from T or S; X17 is selected from Q or N; X18 is selected from P or Hyp; X20 is selected from R, HomoArg> Cit or Pal-Lys.
2.权利要求1的多肽化合物,其中X9选自GK、GGK、GGARRAGK、GGAGAGK、GADEAGGKK或GADEGGAK。 2. A polypeptide compound as claimed in claim 1, wherein X9 is selected from GK, GGK, GGARRAGK, GGAGAGK, GADEAGGKK or GADEGGAK.
3.权利要求1或2的多肽化合物,其中第一序列选自SEQ ID NO:USEQ ID NO:2、SEQID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ IDN0:9、SEQ ID NO: 10,SEQ ID NO:1USEQ ID NO: 12,SEQ ID NO: 13,SEQ ID NO: 14,SEQ IDNO:15, SEQ ID NO:1 6、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQID NO:21、SEQ IDNO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQID NO:27, SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 或SEQ ID NO:36 ;第二序列选自SEQ IDNO:37, SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQID NO:43, SEQ ID NO:44、SEQ IDNO:45 或SEQ ID NO:46。 Polypeptide compound of claim 1 or claim 2, wherein the first sequence is selected from SEQ ID NO: USEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDN0: 9, SEQ ID NO: 10, SEQ ID NO: 1USEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 1 6, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO : 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36; the second sequence is selected from SEQ IDNO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQID NO: 43, SEQ ID NO: 44, SEQ IDNO: 45 or SEQ ID NO: 46.
4.权利要求1-3中任一项的多肽化合物,其中第一序列和第二序列进行N末端乙酰化和形成分子内二硫键修饰,并任选在第一序列中进行C末端酰胺化修饰。 Polypeptide compound according to any one of claim 1, wherein the first and second sequences for N-terminal acetylation and modifications intramolecular disulfide bond formation, and optionally are amidated at the C-terminus of the first sequence modification.
5.权利要求1-4中任一项的多肽化合物,其为多肽二聚体1、多肽二聚体2、多肽二聚体3、多肽二聚体4、多肽二聚体5、多肽二聚体6、多肽二聚体7、多肽二聚体8、多肽二聚体9、多肽二聚体10、多肽二聚体11、多肽二聚体12、多肽二聚体13、多肽二聚体14、多肽二聚体15、多肽二聚体16、多肽二聚体17、多肽二聚体18、多肽二聚体19、多肽二聚体20、多肽二聚体21、多肽二聚体22或多肽二聚体23。 Polypeptide compound according to any one of claims 1 to 4, which is a polypeptide dimer, polypeptide dimer 2, 3 dimeric polypeptide, polypeptide dimer 4, 5 polypeptide dimer, dimeric polypeptide 6, 7 polypeptide dimer, polypeptide dimer 8, 9 dimeric polypeptide, polypeptide dimer 10, 11 dimeric polypeptide, polypeptide dimer 12, 13 dimeric polypeptide, polypeptide dimer 14 , 15 dimeric polypeptide, polypeptide dimer 16, 17 dimeric polypeptide, polypeptide dimer 18, 19 dimeric polypeptide, polypeptide dimer 20, 21 dimeric polypeptide, polypeptide or polypeptide dimer 22 dimer 23.
6.权利要求1-5中任一项的多肽化合物,其进一步包括聚乙二醇部分,其中聚二乙醇与第一序列中C端X9中的赖氨酸缀合。 Polypeptide compound according to any one of claim 1-5, which further comprises a polyethylene glycol moiety, wherein the first polyethylene diethanol X9 C-terminal sequence lysine conjugation.
7.权利要求6的多肽化合物,其中聚乙二醇为直链或分枝的PEG,其分子量为约5,000道尔顿-约60,000道尔顿。 Polypeptide compound of claim 6, wherein the polyethylene glycol is a straight-chain or branched PEG, molecular weight of about 5,000 daltons - about 60,000 daltons.
8.一种用于制备多肽化合物的方法,其包括如下步骤: (1)固相合成第一序列和第二序列; (2)从树脂上裂解多肽二聚体; (3)纯化多肽二聚体并干燥; (4)任选进行权利要求4所述的修饰并纯化;和(5)任选与PEG缀合并纯化。 8. A method for preparing a polypeptide compound, comprising the steps of: (1) solid-phase synthesis of the first and second sequences; (2) a polypeptide dimer cleavage from the resin; (3) purified polypeptide dimer and drying; (4) optionally performed according to claim 4 and purified modified; and (5) optionally purified and conjugated to PEG.
9.一种药物组合物,其包括权利要求1-7的多肽化合物以及药学上可接受的载体。 9. A pharmaceutical composition comprising a polypeptide compound and a pharmaceutically acceptable carrier as claimed in claim 1-7.
10.权利要求1-7的多肽化合物在制备治疗与血红细胞产生不足、缺陷或者过度消耗相关的疾病的药物中的用途。 10. A polypeptide compound as claimed in claim 1-7 in the manufacture of red blood cell production and treating inadequate, defective or excessive consumption of medicament associated diseases.
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