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CN107029243A - A kind of double acylhydrazone connecting keys of polypeptide bridging are applied to the delivering of aldehyde drug derivative - Google Patents

️Fri Aug 11 2017

A kind of double acylhydrazone connecting keys of polypeptide bridging are applied to the delivering of aldehyde drug derivative Download PDF

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Publication number

CN107029243A

CN107029243A CN201710425299.9A CN201710425299A CN107029243A CN 107029243 A CN107029243 A CN 107029243A CN 201710425299 A CN201710425299 A CN 201710425299A CN 107029243 A CN107029243 A CN 107029243A Authority

China

Prior art keywords

acylhydrazone

mmae

prodrug

polypeptide

design

Prior art date

2017-06-08

Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)

Pending

Application number

CN201710425299.9A

Other languages

Chinese (zh)

Inventor

吴川六

郑艺武

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Xiamen University

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Xiamen University

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2017-06-08

Filing date

2017-06-08

Publication date

2017-08-11

2017-06-08 Application filed by Xiamen University filed Critical Xiamen University

2017-06-08 Priority to CN201710425299.9A priority Critical patent/CN107029243A/en

2017-08-11 Publication of CN107029243A publication Critical patent/CN107029243A/en

Status Pending legal-status Critical Current

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IEDXPSOJFSVCKU-HOKPPMCLSA-N [4-[[(2S)-5-(carbamoylamino)-2-[[(2S)-2-[6-(2,5-dioxopyrrolidin-1-yl)hexanoylamino]-3-methylbutanoyl]amino]pentanoyl]amino]phenyl]methyl N-[(2S)-1-[[(2S)-1-[[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-3-[[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]-N-methylcarbamate Chemical compound CC[C@H](C)[C@@H]([C@@H](CC(=O)N1CCC[C@H]1[C@H](OC)[C@@H](C)C(=O)N[C@H](C)[C@@H](O)c1ccccc1)OC)N(C)C(=O)[C@@H](NC(=O)[C@H](C(C)C)N(C)C(=O)OCc1ccc(NC(=O)[C@H](CCCNC(N)=O)NC(=O)[C@@H](NC(=O)CCCCCN2C(=O)CCC2=O)C(C)C)cc1)C(C)C IEDXPSOJFSVCKU-HOKPPMCLSA-N 0.000 claims description 15
ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical compound N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 claims description 14
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IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 claims description 10
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Classifications

- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/06—Tripeptides

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Health & Medical Sciences (AREA)
Life Sciences & Earth Sciences (AREA)
Pharmacology & Pharmacy (AREA)
Proteomics, Peptides & Aminoacids (AREA)
Engineering & Computer Science (AREA)
Bioinformatics & Cheminformatics (AREA)
Immunology (AREA)
Medicinal Chemistry (AREA)
Gastroenterology & Hepatology (AREA)
Chemical & Material Sciences (AREA)
Epidemiology (AREA)
Animal Behavior & Ethology (AREA)
General Health & Medical Sciences (AREA)
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Abstract

一种多肽桥连的双酰腙连接键应用于醛衍生药物的递送，涉及pH响应型双酰腙连接键。该发明涉及一类新的化学连接键，与传统单酰腙键不同，该连接键通过两个酰腙键间的协同稳定作用，使得该类PTA‑linkers在中性(血液循环)或者酸性条件下(肿瘤组织，内含体，溶酶体)都具有超高的稳定性。当桥连双酰腙键的多肽链被特定蛋白酶水解后，两个酰腙键间的协同稳定作用消失，因此在酸性条件又可以具有良好的响应性。以PTA‑linkers作为连接键，设计细胞外和细胞内多肽链定点水解的两种前药分子，两种前药都可以迅速地释放醛衍生药物分子并杀死肿瘤细胞。A polypeptide-bridged diacylhydrazone linkage applied to the delivery of aldehyde-derived drugs involves a pH-responsive diacylhydrazone linkage. The invention relates to a new type of chemical linkage, which is different from the traditional single acylhydrazone linkage. The linkage is through the synergistic stabilization between two acylhydrazone linkages, making this type of PTA-linkers work under neutral (blood circulation) or acidic conditions. Down (tumor tissue, endosomes, lysosomes) have ultra-high stability. When the polypeptide chain bridging diacylhydrazone bonds is hydrolyzed by a specific protease, the synergistic stabilizing effect between the two acylhydrazone bonds disappears, so it can have good responsiveness in acidic conditions. Using PTA-linkers as linkers, two prodrug molecules were designed for site-specific hydrolysis of extracellular and intracellular polypeptide chains. Both prodrugs can rapidly release aldehyde-derived drug molecules and kill tumor cells.

Description

一种多肽桥连的双酰腙连接键应用于醛衍生药物的递送A Polypeptide-Bridged Diacylhydrazone Linker Applied to the Delivery of Aldehyde-Derivative Drugs

技术领域technical field

本发明涉及涉及pH响应型双酰腙连接键，尤其是涉及一种多肽桥连的双酰腙连接键应用于醛衍生药物的递送。The invention relates to a pH-responsive diacylhydrazone linkage, in particular to a polypeptide-bridged diacylhydrazone linkage applied to the delivery of aldehyde-derived drugs.

背景技术Background technique

恶性肿瘤是一种严重威胁人类健康的常见病和多发病，是引起死亡的主要原因之一。目前化疗是临床上治疗肿瘤的主要途径之一,在肿瘤治疗中一直发挥着重要作用，但大多数化疗药物由于无选择性、毒副作用大、体内代谢快、药效低等缺点而限制了临床应用。克服上述缺陷,寻找新的、有效的治疗肿瘤的化疗药物仍然是研究者关注的热点。抗肿瘤靶向前药的设计就是其中一种。Malignant tumor is a common and frequently-occurring disease that seriously threatens human health and is one of the main causes of death. Chemotherapy is currently one of the main ways to treat tumors clinically, and it has been playing an important role in tumor treatment. However, most chemotherapy drugs have limited clinical application due to their shortcomings such as non-selectivity, large toxic and side effects, fast metabolism in the body, and low drug efficacy. application. Overcoming the above defects and finding new and effective chemotherapeutic drugs for the treatment of tumors are still the focus of researchers. The design of anti-tumor targeting prodrugs is one of them.

肿瘤靶向治疗基于前药精确地选择致癌位点，使肿瘤细胞特异性死亡，而不会波及肿瘤周围的正常组织细胞。这种方法取决于合理的设计刺激响应的连接键(如通过酶的过量表达，氧化还原微环境变化，pH值变化等)。在这些用于设计前药的连接键中，pH敏感的酰腙连接键一直引起广泛的关注，一方面主要是由于醛基修饰的药物可以在温和条件下直接偶联到含酰肼的配体构建出酰腙键基于的前药，另一方面，醛基是一类低极性的化学官能团(相比于羟基、羧基、氨基)，疏水的抗癌药物修饰上醛基基本没有改变药物的扩散性质。而且，近期的研究表明，醛基在醛脱氢酶(ALDH)的作用下可以转变成羧基，羧基在细胞内(pH＝7.4)带负电荷使得难以从细胞中逃逸出来。因此治疗试剂和显影试剂包含醛基的标签会展现出超高的细胞保留时间从而达到更好的治愈效果。虽然很多研究报告酰腙键在中性条件下水解动力学比较惰性，但是在稀溶液中酰腙键在热力学上是不稳定的，因此很难长时间保存。此外，在生理条件下，酰腙键会缓慢水解导致过早释放药物从而产生毒副作用，使得其很难在生命体系中应用。最近研究报告抗体药物偶联涉及到使用酰腙连接键使得myotarg和inotuzumab ozogamicin分别从市场上和临床第三阶段中撤离(Chari,R.V.；Miller,M.L.；Widdison,W.C.Angew.Chem.Int.Ed.2014,53,3796)。Tumor-targeted therapy is based on prodrugs that precisely select oncogenic sites to specifically kill tumor cells without affecting normal tissue cells around the tumor. This approach depends on the rational design of stimuli-responsive linkages (e.g., through enzyme overexpression, changes in the redox microenvironment, changes in pH, etc.). Among these linkages for the design of prodrugs, pH-sensitive acylhydrazone linkages have attracted extensive attention, mainly because aldehyde-modified drugs can be directly coupled to hydrazide-containing ligands under mild conditions. Construct the prodrug based on the acylhydrazone bond. On the other hand, the aldehyde group is a type of chemical functional group with low polarity (compared to hydroxyl, carboxyl, amino group). The modification of the aldehyde group on the hydrophobic anticancer drug basically does not change the drug's Diffusion properties. Moreover, recent studies have shown that aldehyde groups can be converted into carboxyl groups under the action of aldehyde dehydrogenase (ALDH), and carboxyl groups are negatively charged in cells (pH=7.4), making it difficult to escape from cells. Therefore, labels containing aldehyde groups in therapeutic reagents and imaging reagents will exhibit ultra-high cell retention time to achieve better healing effects. Although many studies have reported that the hydrolysis kinetics of acylhydrazone bonds are relatively inert under neutral conditions, acylhydrazone bonds are thermodynamically unstable in dilute solutions, making it difficult to preserve them for a long time. In addition, under physiological conditions, the slow hydrolysis of the acylhydrazone bond leads to premature release of the drug, resulting in toxic side effects, making it difficult to apply in living systems. Recent reports of antibody-drug conjugation involving the use of an acylhydrazone linkage have led to the withdrawal of myotarg and inotuzumab ozogamicin from the market and Phase III clinical trials, respectively (Chari, R.V.; Miller, M.L.; Widdison, W.C. Angew. Chem. Int. Ed. 2014, 53, 3796).

为了克服酰腙连接键的这种限制。目前，主要有两种方法可应用于调控酰腙键的稳定性：1)调控醛基邻近的化学基团(Hamann,P.R.et al.Bioconjugate Chem.2002,13,47)；2)利用其它的亲核试剂(Casi,G.；Huguenin-Dezot,N.；Zuberbuhler,K.；Scheuermann,J.；Neri,D.J.Am.Chem.Soc.2012,134,5887)。然而，尽管这些方法可以用于调控酰腙键的稳定性，但是这种提高酰腙键稳定性的方法必然要以牺牲其在靶点的响应性为代价。而且在实际应用中，同时获得稳定性和响应性一直无法在传统的单酰腙键(SA-linkers)中实现。To overcome this limitation of the acylhydrazone linkage. At present, there are mainly two methods that can be applied to regulate the stability of the acylhydrazone bond: 1) regulating the chemical groups adjacent to the aldehyde group (Hamann, P.R. et al. Bioconjugate Chem. 2002, 13, 47); 2) using other Nucleophiles (Casi, G.; Huguenin-Dezot, N.; Zuberbuhler, K.; Scheuermann, J.; Neri, D.J. Am. Chem. Soc. 2012, 134, 5887). However, although these methods can be used to regulate the stability of the acylhydrazone bond, this method of improving the stability of the acylhydrazone bond must be at the expense of its responsiveness at the target site. Moreover, in practical applications, simultaneously obtaining stability and responsiveness has been unable to be achieved in traditional single acylhydrazone linkages (SA-linkers).

发明内容Contents of the invention

本发明的第一目的在于提供一类多肽桥连的双酰腙连接键(PTA-linkers)。The first object of the present invention is to provide a kind of polypeptide-bridged bisacylhydrazone linkers (PTA-linkers).

本发明的第二目的在于提供一类多肽桥连的双酰腙连接键(PTA-linkers)的分子骨架通式。The second object of the present invention is to provide a molecular skeleton general formula of a kind of polypeptide-bridged bisacylhydrazone linkers (PTA-linkers).

本发明的第三目的在于提供一类基于双酰腙连接键(PTA-linkers)的前药设计通式。The third object of the present invention is to provide a general formula for designing prodrugs based on diacylhydrazone linkages (PTA-linkers).

本发明的第四目的在于提供基于双酰腙键(PTA-linkers)前药分子(D₁和D₂)的合成方法。The fourth object of the present invention is to provide a synthesis method of prodrug molecules (D ₁ and D ₂ ) based on bisacylhydrazone bonds (PTA-linkers).

所述一类多肽桥连的双酰腙连接键，为多肽链桥连和双酰腙键，具有蛋白酶水解和酸水解双重响应性，在生物环境中的兼具稳定性和响应性，所述生物环境包括血液循环、细胞外环境、细胞内环境等。The diacylhydrazone linkage of the polypeptide bridge is a polypeptide chain bridge and a diacylhydrazone linkage, which has dual responsiveness to protease hydrolysis and acid hydrolysis, and has both stability and responsiveness in a biological environment. The biological environment includes blood circulation, extracellular environment, and intracellular environment.

所述一类多肽桥连的双酰腙连接键(PTA-linkers)的分子骨架通式为：The general formula of the molecular skeleton of the diacylhydrazone linker (PTA-linkers) bridged by the polypeptide is:

其中：(x)代表任意活性多肽序列，可以被特定蛋白酶降解；(M)代表N端可进一步进行化学修饰的任意氨基酸，所述氨基酸包括赖氨酸、半胱氨酸、谷氨酸、天冬氨酸等；R代表抗癌药物，E*代表侧链是酰肼的氨基酸。Among them: (x) represents any active polypeptide sequence, which can be degraded by specific proteases; (M) represents any amino acid that can be further chemically modified at the N-terminal, and the amino acid includes lysine, cysteine, glutamic acid, natural Partic acid, etc.; R stands for anticancer drugs, and E* stands for amino acids whose side chains are hydrazides.

基于上述通式(I)，本发明使用羟基功能化的双醛基小分子(bis-CHO)和MMP-2酶高特异性识别的酰肼修饰的多肽分子(Peptide 1，Peptide 1活性片段：PLGLA)设计合成了一种PTA-linker，基质金属蛋白酶(matrix metalloprotinase，MMP)是一类依赖金属锌离子的蛋白水解酶，对细胞外基质(ECM)的降解、组织重建以及细胞内多种可溶性因子的调控起重要作用，是一类与肿瘤发生、侵袭和转移密切相关的蛋白水解酶，与许多恶性肿瘤的发生密切相关，因此选择peptide 1作为酶解锁单元。将两个构建单元peptide 1和bis-CHO混合在DMSO溶液中(含0.05％TFA)，色谱表明两个构建单元倾向于形成分子间双酰腙键。此外，双醛基小分子上的羟基可以用于灵活修饰药物分子来构建双醛衍生的药物。Based on the above general formula (I), the present invention uses hydroxyl-functionalized bisaldehyde-based small molecules (bis-CHO) and hydrazide-modified polypeptide molecules (Peptide 1, Peptide 1 active fragments: PLGLA) designed and synthesized a PTA-linker, matrix metalloproteinase (matrix metalloprotinase, MMP) is a class of proteolytic enzymes dependent on metal zinc ions, the degradation of the extracellular matrix (ECM), tissue reconstruction and a variety of soluble intracellular The regulation of factors plays an important role. It is a class of proteolytic enzymes closely related to tumorigenesis, invasion and metastasis, and is closely related to the occurrence of many malignant tumors. Therefore, peptide 1 was selected as the enzyme unlocking unit. The two building blocks peptide 1 and bis-CHO were mixed in DMSO solution (containing 0.05% TFA). Chromatography showed that the two building blocks tended to form intermolecular diacylhydrazone bonds. In addition, the hydroxyl groups on dialdehyde-based small molecules can be used to flexibly modify drug molecules to construct dialdehyde-derived drugs.

作为参照实验，比较双酰腙键和单酰腙键在不同pH条件下(pH 7.0模拟血液循环，pH 6.0模拟肿瘤组织微环境和内含体，pH 4.8模拟溶酶体)的水解断裂动力学。PTA-linker在pH 4.8条件下具有超高的稳定性。相反，SA-linker在pH 4.8(半衰期τ_1/2 2.9h)和pH 6.0(半衰期τ_1/2 19.9h)条件下水解断裂都非常迅速。此外，在pH 7.4条件下，SA-linker也会缓慢的发生水解。这种缓慢水解限制该类连接键应用于白蛋白或者抗体作为载体的前药递送系统，该类配体通常具有极长的血液循环半衰期(19～23天)，所以会导致药物在非靶点释放。PTA-linker这种超高稳定性来源于两个酰腙键的协同作用，当多肽桥连的双酰腙被MMP酶解锁后，在pH 4.8条件下两个酰腙键迅速水解断裂。As a reference experiment, compare the hydrolytic fragmentation kinetics of diacylhydrazone bonds and monoacylhydrazone bonds under different pH conditions (pH 7.0 simulates blood circulation, pH 6.0 simulates tumor tissue microenvironment and inclusion bodies, pH 4.8 simulates lysosomes) . PTA-linker has ultra-high stability at pH 4.8. On the contrary, the hydrolytic cleavage of SA-linker was very rapid at pH 4.8 (half-life τ _1/2 2.9h) and pH 6.0 (half-life τ _1/2 19.9h). In addition, under the condition of pH 7.4, SA-linker will be hydrolyzed slowly. This slow hydrolysis limits the application of this type of linkage to prodrug delivery systems with albumin or antibody as a carrier. This type of ligand usually has a very long blood circulation half-life (19-23 days), so it will cause the drug to be released at the non-target site. freed. The ultra-high stability of PTA-linker comes from the synergistic effect of two acylhydrazone bonds. When the diacylhydrazone bridged by the polypeptide is unlocked by MMP enzyme, the two acylhydrazone bonds are rapidly hydrolyzed and broken at pH 4.8.

本发明对于PTA-liners性质考察结果，提供一类基于PTA-linkers的前药设计通式(II)。该通式在通式(I)的基础上引入配体和药物。The present invention provides a class of prodrug design general formula (II) based on PTA-linkers based on the investigation results of the properties of PTA-liners. The general formula introduces the ligand and the drug on the basis of the general formula (I).

其中：代表任意多肽链，代表间隔子(用于隔离双醛基小分子和药物的空间距离，最小化双醛基小分子对药物活性的影响)。in: represents any polypeptide chain, Represents a spacer (used to isolate the spatial distance between the dialdehyde-based small molecule and the drug, minimizing the impact of the dialdehyde-based small molecule on the activity of the drug).

本发明基于上述的通式(II)，本发明设计两个基于PTA-linkers前药分子(D₁和D₂)，两种前药由三部分组成：The present invention is based on the above general formula (II). The present invention designs two prodrug molecules (D ₁ and D ₂ ) based on PTA-linkers. The two prodrugs are composed of three parts:

I)RGD作为靶向配体，RGD是一类含精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp)序列的短肽,为纤维连接蛋白与其受体的特异性结合位点。RGD肽与整合素受体具有高度亲和力,而受体在肿瘤血管新生过程中的内皮细胞上具有高度的表达。I) RGD as a targeting ligand, RGD is a kind of short peptide containing arginine-glycine-aspartic acid (Arg-Gly-Asp) sequence, which is the specific binding site of fibronectin and its receptor. RGD peptide has a high affinity with integrin receptors, and the receptors are highly expressed on endothelial cells during tumor angiogenesis.

II)双醛基衍生的MMAE作为抗癌药物，单甲基奥利他汀E(monomethyl auristainE，MMAE)是一种普遍的抗癌药物，它通过与微管蛋白结合并抑制其聚合从而干扰后续的细胞周期活动，并导致肿瘤细胞凋亡。II) MMAE derived from dialdehyde group as an anticancer drug, monomethyl auristainE (monomethyl auristainE, MMAE) is a common anticancer drug, which interferes with subsequent tubulin by binding to tubulin and inhibiting its polymerization. Cell cycle activity and lead to apoptosis of tumor cells.

III)以PTA-linkers作为连接键。III) Use PTA-linkers as the connection key.

所述基于双酰腙键(PTA-linkers)前药分子(D₁和D₂)的合成方法包括如下步骤：The synthesis method based on the bisacylhydrazone bond (PTA-linkers) prodrug molecules (D ₁ and D ₂ ) comprises the following steps:

步骤1)酰肼修饰多肽的设计：Step 1) Design of hydrazide modified polypeptide:

如结构式(a)和结构式(b)Peptide 2设计针对肿瘤细胞外过表达MMP-2酶特异性水解，peptide 3设计针对细胞内组织蛋白酶B特异性水解。组织蛋白酶是一大类主要存在于溶酶体的半胱氨酸蛋白水解酶，多以无活性的酶原形式存在，蛋白酶酶原通过溶酶体的酸性环境被激活。由于肿瘤细胞能够酸化其周围环境，其分泌的组织蛋白酶也会在此环境中被激活,因此，肿瘤组织及细胞中组织蛋白酶的活性远高于远离肿瘤的其它正常组织和正常细胞的组织蛋白酶活性。因此，基于上述肿瘤微环境的特征，设计了细胞外或者细胞内定点水解的PTA-linkers。此外，赖氨酸的引入设计可用于进一步修饰感兴趣的靶向配体。For example, structural formula (a) and structural formula (b) Peptide 2 is designed to specifically hydrolyze MMP-2 overexpressed outside tumor cells, and peptide 3 is designed to specifically hydrolyze intracellular cathepsin B. Cathepsins are a large class of cysteine proteolytic enzymes that mainly exist in lysosomes, mostly in the form of inactive zymogens, and protease zymogens are activated by the acidic environment of lysosomes. Because tumor cells can acidify their surrounding environment, the cathepsin secreted by them will also be activated in this environment, so the activity of cathepsin in tumor tissues and cells is much higher than that of other normal tissues and normal cells far away from the tumor . Therefore, based on the above characteristics of the tumor microenvironment, PTA-linkers with extracellular or intracellular site-specific hydrolysis were designed. In addition, the introduction of lysine design can be used to further modify the targeting ligand of interest.

步骤2)双醛衍生药物的设计：Step 2) the design of dialdehyde derivative drug:

MMAE是一种常见抗癌药物，其N端一甲基化的氨基可以进行灵活修饰，为了最小化对MMAE活性的影响，采用一段结构灵活且双功能化的聚乙二醇用于偶联MMAE和bis-CHO，得到了一种双醛衍生的MMAE(命名为：bis-CHO-PEG₃-MMAE,其结构式如结构式(C)所示，细胞毒性曲线表明bis-CHO-PEG3-MMAE(IC₅₀＝14.7nM)表现出很强杀死肿瘤细胞的能力，虽然其活性相比于母体药物MMAE(IC₅₀＝0.75nM)有所降低。MMAE is a common anticancer drug, and its N-terminal monomethylated amino group can be flexibly modified. In order to minimize the impact on the activity of MMAE, a flexible and bifunctional polyethylene glycol was used to couple MMAE and bis-CHO, obtained a kind of MMAE derived from dialdehyde (designated as: bis-CHO-PEG ₃ -MMAE, its structural formula is shown in structural formula (C), and the cytotoxicity curve shows that bis-CHO-PEG 3 -MMAE (IC ₅₀ =14.7nM) showed a strong ability to kill tumor cells, although its activity was lower than that of the parent drug MMAE (IC ₅₀ =0.75nM).

步骤3)含RGD配体的前药D1和D2的合成：Step 3) Synthesis of prodrugs D1 and D2 containing RGD ligands:

通过peptides 2/3的两个酰肼基团与bis-CHO-PEG₃-MMAE的两个醛基反应形成两个酰腙键，随后加入NHS-PEG₄-Maleimide，进一步与peptides 2/3的赖氨酸侧链进行反应，最后再加入c(RGDyC)配体；此外，作为参照实验，没有偶联MMAE的前药C₁(对应D₁)和C₂(对应D₂)同时也被设计合成。结果表明前药D₁和D₂具有很强的杀死肿瘤细胞的能力，对照实验中的C₁和C₂基本没有细胞毒性。Two hydrazide groups of peptides 2/3 react with two aldehyde groups of bis-CHO-PEG ₃ -MMAE to form two acylhydrazone bonds, and then NHS-PEG ₄ -Maleimide is added to further react with peptides 2/3 Lysine side chains were reacted, and finally c(RGDyC) ligand was added; in addition, as a reference experiment, prodrugs C ₁ (corresponding to D ₁ ) and C ₂ (corresponding to D ₂ ) without coupling MMAE were also designed synthesis. The results show that the prodrugs D ₁ and D ₂ have a strong ability to kill tumor cells, and C ₁ and C ₂ in the control experiment have basically no cytotoxicity.

本发明的原理：Principle of the present invention:

1)传统的SA-linkers在中性或弱酸性的情况下，会迅速形成正四面体的过渡态，随后发生水解断裂。1) Traditional SA-linkers will rapidly form a tetrahedral transition state under neutral or weakly acidic conditions, followed by hydrolytic cleavage.

2)如图1所示，PTA-liners的水解断裂过程与SA-linkers不同，PTA-linkers利用多肽桥连锁定双酰腙键间的协同效应。在酸性条件下，发生部分断裂的酰腙键(即其中一个酰腙键发生断裂)中间体由于双酰腙键间的协同稳定效应，会发生分子内醛基和酰肼的反应，从而重新形成双酰腙键；因此具有超高稳定性。酶解锁后，酰腙键的协同作用消失，从而导致两个酰腙键相继发生断裂。2) As shown in Figure 1, the hydrolytic cleavage process of PTA-liners is different from that of SA-linkers, and PTA-linkers utilizes the synergistic effect between peptide bridges to lock diacylhydrazone bonds. Under acidic conditions, the partially broken acylhydrazone bond (that is, one of the acylhydrazone bonds is broken) intermediates will react with the aldehyde group and hydrazide in the molecule due to the synergistic stabilization effect between the diacylhydrazone bonds, thereby re-forming Diacylhydrazone bond; thus ultra-high stability. After the enzyme is unlocked, the synergistic effect of the acylhydrazone bond disappears, resulting in the successive cleavage of the two acylhydrazone bonds.

本发明中所述酶解锁响应的双酰腙连接键，其中解锁方式有多种选择，比如可以设计成光解锁，H₂O₂解锁等方式。 _The enzyme in the present invention unlocks the corresponding _{diacylhydrazone} linkage, and there are many options for unlocking, such as light unlocking, H2O2 unlocking and other ways.

本发明的优点在于：The advantages of the present invention are:

1)采用多肽锁定双酰腙键间的协同效应，使得酰腙键具有超高的稳定性。1) Polypeptides are used to lock the synergistic effect between the diacylhydrazone bonds, so that the acylhydrazone bonds have ultra-high stability.

2)本发明突破了以往的连接键稳定性和响应性难以兼具的缺陷，其具有超高的水解稳定性，而在酶解锁后又可以发生迅速的水解断裂。这种独特的响应性并不以牺牲其在靶点的响应性为代价。2) The present invention breaks through the previous defect that it is difficult to have both the stability and responsiveness of the linkage, and it has ultra-high hydrolytic stability, and rapid hydrolytic cleavage can occur after the enzyme is unlocked. This unique responsiveness does not come at the expense of its responsiveness at the target site.

3)只需将两个构建单元放置一起就可以直接形成分子间的二聚体，因此PTA-linkers非常容易被设计合成出来。3) Intermolecular dimers can be directly formed by simply placing two building blocks together, so PTA-linkers are very easy to design and synthesize.

附图说明Description of drawings

图1为PTA-linkes的还原断裂机理。Figure 1 shows the reduction cleavage mechanism of PTA-links.

图2为PTA-linker形成色谱图。Figure 2 is a chromatogram formed by PTA-linker.

图3为SA-linker和PTA-linker水解动力学。Figure 3 is the hydrolysis kinetics of SA-linker and PTA-linker.

图4为PTA-linker酶解锁后水解色谱图。Figure 4 is the hydrolysis chromatogram after unlocking the PTA-linker enzyme.

图5为bis-CHO-PEG3-MMAE和MMAE细胞毒性曲线。Figure 5 is the cytotoxicity curves of bis-CHO-PEG3-MMAE and MMAE.

图6为前药D₁、D₂、C₁、C₂的细胞毒性曲线。Fig. 6 is the cytotoxicity curves of prodrugs D ₁ , D ₂ , C ₁ , and C ₂ .

图7为bis-CHO-PEG3-MMAE质谱表征图。Figure 7 is a mass spectrogram of bis-CHO-PEG3-MMAE.

图8为前药D₁质谱表征图。Figure 8 is _a mass spectrum characterization diagram of prodrug D1.

图9为前药D₂质谱表征图。Fig. ₉ is a mass spectrum characterization diagram of prodrug D2.

图10为前药C₁质谱表征图。Figure 10 is a mass spectrum characterization diagram of prodrug _C1 .

图11为前药C₂质谱表征图。Figure 11 is a prodrug _C2 mass spectrum characterization diagram.

具体实施方式detailed description

以下实施例将结合附图对本发明进行进一步说明。The following embodiments will further illustrate the present invention in conjunction with the accompanying drawings.

实施例1Example 1

色谱监测PTA-linker的形成过程：Chromatographic monitoring of the formation of PTA-linker:

取500μL(2mM，溶解在含0.5％DMSO)peptide 1和500μL(2mM，溶解在含0.5％DMSO)bis-CHO。到预定的时间点，取样50μL到色谱进样瓶中的内插管，将每个样品进行高效液相色谱(HPLC)分析(进样体积20μL)，并在280nm处监测反应产物峰(如图2)。Take 500 μL (2 mM, dissolved in 0.5% DMSO) peptide 1 and 500 μL (2 mM, dissolved in 0.5% DMSO) bis-CHO. At the predetermined time point, 50 μL was sampled into the inner insert tube in the chromatographic sampling bottle, and each sample was subjected to high-performance liquid chromatography (HPLC) analysis (injection volume 20 μL), and the peak of the reaction product was monitored at 280 nm (as shown in Fig. 2).

实施例2Example 2

分析SA-linker和PTA-linker水解动力学：Analysis of SA-linker and PTA-linker hydrolysis kinetics:

分别取1mL磷酸缓冲液(100mM,pH 7.4)、1mL磷酸缓冲液(100mM,pH 6)、1mL醋酸盐缓冲液(100mM,pH 4.8)于2.0mL色谱进样瓶中，分别加入50μL SA-linker和PTA-linker的溶液(最终浓度为10μM)。每隔一定时间，色谱自动取样进行分析(进样体积80μL)。并在280nm处监测反应产物峰。记录SA-linker和PTA-linker在不同反应时间点的峰面积并绘制连接键断裂动力学曲线(如图3)。Take 1mL of phosphate buffer (100mM, pH 7.4), 1mL of phosphate buffer (100mM, pH 6), and 1mL of acetate buffer (100mM, pH 4.8) in a 2.0mL chromatographic injection bottle, add 50μL of SA- Linker and PTA-linker solutions (final concentration 10 μM). At regular intervals, the chromatograph automatically samples for analysis (injection volume 80 μL). And monitor the reaction product peak at 280nm. Record the peak areas of SA-linker and PTA-linker at different reaction time points and draw the bond breaking kinetic curve (as shown in Figure 3).

实施例3Example 3

色谱监测PTA-linker酶解锁后水解过程：Chromatographic monitoring of the hydrolysis process after unlocking the PTA-linker enzyme:

取180μL醋酸盐缓冲液(100mM,pH 4.8)于0.5mL低蛋白吸附离心管中，分别加入20μL PTA-linker酶解锁产物(最终浓度为10μM)，到预定的时间点，取样100μL到色谱进样瓶中的内插管，对样品进行高效液相色谱(HPLC)分析(进样体积70μL)，并在280nm处监测反应产物峰(如图4)。Take 180 μL of acetate buffer (100 mM, pH 4.8) in a 0.5 mL low-protein adsorption centrifuge tube, add 20 μL of PTA-linker enzyme unlocking product (final concentration: 10 μM) respectively, and sample 100 μL at the predetermined time point for chromatographic analysis. The inner tube in the sample bottle was used to analyze the sample by high performance liquid chromatography (HPLC) (injection volume 70 μL), and the peak of the reaction product was monitored at 280 nm (as shown in Figure 4).

实施例4Example 4

细胞毒性实验：Cytotoxicity test:

将U87细胞以每孔85000个/mL的密度接种于96孔板中，每孔添加100μL DMEM培养基(含5％牛血清，1‰双抗)，置于37℃、5％CO₂的培养箱中孵育24h。吸弃培养液，向每孔加入100μL使用无血清培养基和药物配制的不同浓度的溶液然后放入37℃、5％CO₂的培养箱中，每个浓度设置3个复孔。当细胞与药物共同孵育48h后，在每孔中加入10μL CCK-8溶液，并将其放入培养箱中孵育1h后通过酶标仪检测450nm下的吸光度值。实验重复三次。CCK-8是一种具有高灵敏性、重现性好、操作简便等优点的细胞毒性试验方法。CCK-8含有的活性物能在电子载体的作用下被细胞线粒体中的脱氢酶还原，并生成具有高度水溶性的黄色甲臜产物。根据生成的甲臜物与活细胞数量的正比关系可以反映活细胞的数量(毒性结果如图5和图6)。Seed U87 cells in a 96-well plate at a density of 85,000 cells/mL per well, add 100 μL of DMEM medium (containing 5% bovine serum, 1‰ double antibody) to each well, and culture at 37°C and 5% CO ₂ Incubate for 24 hours in the box. Aspirate the culture solution, add 100 μL of solutions of different concentrations prepared using serum-free medium and drugs to each well, and then put them in an incubator at 37°C, 5% CO ₂ , and set 3 replicate wells for each concentration. After the cells were co-incubated with the drug for 48 h, 10 μL of CCK-8 solution was added to each well, placed in an incubator for 1 h, and then the absorbance value at 450 nm was detected by a microplate reader. Experiments were repeated three times. CCK-8 is a cytotoxicity test method with high sensitivity, good reproducibility and easy operation. The active substance contained in CCK-8 can be reduced by the dehydrogenase in the mitochondria of the cell under the action of the electron carrier, and a highly water-soluble yellow formazan product is produced. According to the proportional relationship between the generated formazan and the number of living cells, the number of living cells can be reflected (the toxicity results are shown in Figures 5 and 6).

实施例5Example 5

化合物bis-CHO的合成Synthesis of compound bis-CHO

称取1.31g(32.7mmol)的氢氧化钠溶解于60mL纯水中。向溶液中加入4.0g(32.7mmol)对羟基苯甲醛，充分混匀后在60℃下缓慢滴加1.1mL的环氧氯丙烷，滴加过程持续2h。随后在60℃的温度下将反应液搅拌3h。反应结束后，用甲醇和水混合比为1︰1的混合溶剂对产物进行重结晶得到产率为85％的终产物，所得产物使用核磁进行表征。Weigh 1.31g (32.7mmol) of sodium hydroxide and dissolve it in 60mL of pure water. Add 4.0g (32.7mmol) of p-hydroxybenzaldehyde to the solution, mix well and then slowly add 1.1mL of epichlorohydrin dropwise at 60°C for 2 hours. The reaction solution was then stirred at a temperature of 60 °C for 3 h. After the reaction, the product was recrystallized with a mixed solvent of methanol and water with a mixing ratio of 1:1 to obtain a final product with a yield of 85%, which was characterized by NMR.

目标产物的结构表征：Structural characterization of the target product:

¹H NMR(400MHz,CDCl₃,δ,ppm):9.91(d,J＝1.7Hz,2H),7.85～7.87(t,J＝1.8Hz,4H),7.05～7.07(s,4H),4.26～4.28(s,3H)，2.61(s,1H)。 ¹ H NMR (400MHz, CDCl ₃ , δ, ppm): 9.91(d, J＝1.7Hz, 2H), 7.85～7.87(t, J＝1.8Hz, 4H), 7.05～7.07(s, 4H), 4.26 ~4.28(s,3H), 2.61(s,1H).

实施例6Example 6

Peptide 1的合成Synthesis of Peptide 1

称取5.0mg(5.29μmol)的多肽(序列：Ac-C-G-G-P-L-G-L-A-G-G-C-NH2)溶解于400μL(100mM,pH 7.4)的磷酸盐缓冲溶液中。向上述溶液中逐滴添加溶解于400μL DMSO中的含7.86mg(32.0μmol)3-马来酰亚胺丙酸酰肼的三氟乙酸盐(BMPH)溶液。待上述混合液反应0.5h后，用制备色谱(HPLC)进行纯化。ESI-MS m/z(M)⁺calcd.1383.56,found(M)⁺1383.7。5.0 mg (5.29 μmol) of the polypeptide (sequence: Ac-CGGPLGLLAGGC-NH2) was weighed and dissolved in 400 μL (100 mM, pH 7.4) of phosphate buffered saline solution. To the above solution was added dropwise a solution of trifluoroacetate (BMPH) containing 7.86 mg (32.0 μmol) of 3-maleimide propionic acid hydrazide dissolved in 400 μL of DMSO. After the above mixture was reacted for 0.5 h, it was purified by preparative chromatography (HPLC). ESI-MS m/z(M) ⁺ calcd.1383.56, found(M) ^+1383.7 .

实施例7Example 7

PTA-linker的合成:Synthesis of PTA-linker:

将上述合成的500μL(2.0mM)的peptide 1与500μL(2.0mM)的双醛分子混合于1mL含0.05％三氟乙酸的DMSO中。将混合溶液置于室温下搅拌反应3h，随后，通过色谱对产物进行纯化。ESI-MS m/z(M)⁺calcd.1647.58(M)⁺,Found 1647.3(M)⁺,824.6(M+2H)²⁺。500 μL (2.0 mM) of peptide 1 synthesized above and 500 μL (2.0 mM) of dialdehyde molecules were mixed in 1 mL of DMSO containing 0.05% trifluoroacetic acid. The mixed solution was stirred at room temperature for 3 h, and then the product was purified by chromatography. ESI-MS m/z(M) ⁺ calcd. 1647.58(M) ⁺ , Found 1647.3(M) ⁺ , 824.6(M+2H) ²⁺ .

实施例8Example 8

SA-linker的合成：Synthesis of SA-linker:

将10.0mg(0.060mmol)的4-(2-羟基乙氧基)苯甲醛与70.9mg(0.60mmol)的4-羟基丁酸肼混合于5mL的DMSO溶液中。将混合溶液置于室温下搅拌反应24h，随后，通过色谱将产物进行纯化。ESI-MS m/z(M)⁺calcd.266.29(M)⁺,Found 266.7(M)⁺。10.0 mg (0.060 mmol) of 4-(2-hydroxyethoxy)benzaldehyde and 70.9 mg (0.60 mmol) of 4-hydroxybutyric acid hydrazine were mixed in 5 mL of DMSO solution. The mixed solution was stirred at room temperature for 24 h, and then the product was purified by chromatography. ESI-MS m/z(M) ⁺ calcd.266.29(M) ⁺ , Found 266.7(M) ⁺ .

实施例9Example 9

合成bis-CHO-PEG3-MMAE实施如下步骤：The synthesis of bis-CHO-PEG3-MMAE is carried out as follows:

1)化合物1的合成：1) Synthesis of compound 1:

称取1.0g(3.3mmol)的bis-CHO溶解于20mL的二氯甲烷溶液中，随后向溶液中添加937μL(6.6mmol)的三乙胺和0.66g(1.5mmol)的对硝基苯氯甲酸酯。将混合溶液置于室温下反应4h后向溶液中缓慢添加20mL纯水以猝灭反应。用10mL饱和食盐水洗涤反应液，二氯甲烷萃取分液后，用无水硫酸钠对有机相进行干燥。旋干溶剂，随后采用硅胶柱对产物进行分离纯化，最终得到1.10g的白色粉末状化合物1，产率为75％。¹H NMR(400MHz,Chloroform-d)δ9.93(s,2H),8.35～8.29(m,2H),7.92～7.87(m,4H),7.47～7.41(m,2H),7.12～7.07(m,4H),5.56(p,J＝4.9Hz,1H),4.51(d,J＝4.9Hz,4H)。ESI-MS m/z(M)⁺calcd.465.41(M)⁺,Found 465.88(M)⁺。Weigh 1.0g (3.3mmol) of bis-CHO and dissolve it in 20mL of dichloromethane solution, then add 937μL (6.6mmol) of triethylamine and 0.66g (1.5mmol) of p-nitrobenzochloromethane to the solution esters. The mixed solution was reacted at room temperature for 4 h, and then 20 mL of pure water was slowly added to the solution to quench the reaction. The reaction solution was washed with 10 mL of saturated brine, extracted and separated with dichloromethane, and the organic phase was dried with anhydrous sodium sulfate. The solvent was spin-dried, and then the product was separated and purified using a silica gel column to finally obtain 1.10 g of white powder compound 1 with a yield of 75%. ¹ H NMR (400MHz, Chloroform-d) δ9.93(s,2H), 8.35～8.29(m,2H), 7.92～7.87(m,4H), 7.47～7.41(m,2H), 7.12～7.07( m, 4H), 5.56 (p, J=4.9Hz, 1H), 4.51 (d, J=4.9Hz, 4H). ESI-MS m/z(M) ⁺ calcd. 465.41(M) ⁺ , Found 465.88(M) ⁺ .

2)合成Fmoc-NH-PEG₃-MMAE:2) Synthesis of Fmoc-NH-PEG ₃ -MMAE:

将10mg(14.0μmol)的MMAE、12.4mg(28μmol)的Fmoc-NH-PEG₃-CH₂CH₂COOH、10.6mg(28μmol)的HATU、9.8μL(56μmol)DIEA加入到5ml圆底烧瓶中，用DMSO(1mL)为溶剂，室温下反应1h。随后，用半制备色谱(HPLC)对产物进行纯化，冻干后得到12.8mg的白色固体，产率为80％。ESI-MS m/z(M)⁺calcd.1143.45(M)⁺,Found 1144.6(M+H)⁺。Add 10 mg (14.0 μmol) of MMAE, 12.4 mg (28 μmol) of Fmoc-NH-PEG ₃ -CH ₂ CH ₂ COOH, 10.6 mg (28 μmol) of HATU, 9.8 μL (56 μmol) of DIEA into a 5 ml round bottom flask, Using DMSO (1 mL) as solvent, react at room temperature for 1 h. Subsequently, the product was purified by semi-preparative chromatography (HPLC), and 12.8 mg of a white solid was obtained after lyophilization, with a yield of 80%. ESI-MS m/z(M) ⁺ calcd. 1143.45(M) ⁺ , Found 1144.6(M+H) ⁺ .

3)合成bis-CHO-PEG3-MMAE：3) Synthesis of bis-CHO-PEG3-MMAE:

向10mg(8.7μmol)的Fmoc-NH-PEG3-MMAE中加入二乙胺与二氯甲烷混合比为1︰1的混合溶剂。反应搅拌30min后，旋去溶剂。然后用1mL DMSO溶解粗产物，向溶液中加入6.1μL(34.8μmol)的DIEA和4.0mg(8.7μmol)的化合物1。将混合溶液在室温下搅拌1小时后用半制备色谱(HPLC)对产物进行纯化。ESI-MS m/z(M)⁺calcd.1247.51(M)⁺,Found1248.5(M+H)⁺。To 10 mg (8.7 μmol) of Fmoc-NH-PEG3-MMAE was added a mixed solvent of diethylamine and dichloromethane with a mixing ratio of 1:1. After the reaction was stirred for 30 min, the solvent was spun off. The crude product was then dissolved with 1 mL of DMSO, and 6.1 μL (34.8 μmol) of DIEA and 4.0 mg (8.7 μmol) of compound 1 were added to the solution. After the mixed solution was stirred at room temperature for 1 hour, the product was purified by semi-preparative chromatography (HPLC). ESI-MS m/z(M) ⁺ calcd.1247.51(M) ⁺ , Found 1248.5(M+H) ⁺ .

实施例10Example 10

合成D₁：Synthesis of D ₁ :

将100μL浓度为10mM的peptide 2与100μL浓度为10mM上述合成的bis-CHO-PEG₃-MMAE混合于1mL含有0.05％三氟乙酸的DMSO中，在室温下搅拌反应5h。随后，加入4mM的NHS-PEG₄-Maleimide和100μL浓度为10mM的DIEA混合于1mL的DMSO中，在室温下搅拌反应1h。最后，向溶液中加入100μL浓度为4mM的c(RGDyC)，反应搅拌1h。用半制备色谱(HPLC)对产物进行纯化。ESI-MS m/z(M⁺)calcd.3738.31(M)⁺,Found 1246.5(M+3H)³⁺,935.2(M+4H)⁴⁺,748.7(M+5H)⁵⁺。100 μL of 10 mM peptide 2 and 100 μL of 10 mM bis-CHO-PEG ₃ -MMAE synthesized above were mixed in 1 mL of DMSO containing 0.05% trifluoroacetic acid, and stirred at room temperature for 5 h. Subsequently, 4 mM NHS-PEG ₄ -Maleimide and 100 μL of 10 mM DIEA were added and mixed in 1 mL of DMSO, and the reaction was stirred at room temperature for 1 h. Finally, 100 μL of c(RGDyC) at a concentration of 4 mM was added to the solution, and the reaction was stirred for 1 h. The product was purified by semi-preparative chromatography (HPLC). ESI-MS m/z(M ⁺ ) calcd. 3738.31(M) ⁺ , Found 1246.5(M+3H) ³⁺ , 935.2(M+4H) ⁴⁺ , 748.7(M+5H) ⁵⁺ .

实施例11Example 11

合成D₂：Synthesis of D2 _:

D₂的合成步骤与D₁合成步骤相同。 _The synthesis procedure _of D2 is the same as that of D1.

实施例12Example 12

合成C₁：Synthesis of C ₁ :

C₁的合成步骤与D₁合成步骤相同。 _The synthesis procedure of _C1 is the same as that of D1.

实施例13Example 13

合成C₂：Synthesis of C ₂ :

C₂的合成步骤与D₁合成步骤相同。 _The synthesis procedure _of C2 is the same as that of D1.

Claims (7)

1. double acylhydrazone connecting keys of a class polypeptide bridging, it is characterised in that polypeptide chain bridging and double acylhydrazone keys, with protease water Solution and sour water solution dual responsiveness, have stability and response concurrently, the biotic environment is followed including blood in biotic environment Ring, extracellular environment, intracellular environment.

2. double acylhydrazone connecting keys of class polypeptide bridging as claimed in claim 1, it is characterised in that its molecular skeleton formula is：

Wherein：(x) any active peptides sequence is represented, is degraded by specific proteases；(M) represent N-terminal and further carry out chemistry and repair The arbitrary amino acid of decorations, the amino acid includes lysine, cysteine, glutamic acid, aspartic acid；R represents cancer therapy drug, E* Represent the amino acid that side chain is hydrazides.

3. double acylhydrazone connecting keys of class polypeptide bridging as claimed in claim 2, it is characterised in that the logical formula (I) uses hydroxyl The dialdehyde base small molecule bis-CHO of functionalization and the base containing bishydrazide that can be hydrolyzed by matrix metalloproteinase MMP-2 high specifics Peptide molecule design a kind of pair of acylhydrazone connecting key of synthesis；Matrix metalloproteinase be a class in tumor tissues overexpression, and with Generation, transfer and the invasin protein hydrolase of tumour, will contain bishydrazide Quito peptide molecule and be mixed with bis-CHO in DMSO solution Close, add 0.05%TFA, two construction units of chromatogram tend to form intermolecular pair of acylhydrazone key.

4. double acylhydrazone connecting keys of class polypeptide bridging as claimed in claim 2, it is characterised in that in the dialdehyde base small molecule Hydroxyl be used for modified medicaments molecule and build medicine derived from dialdehyde, structural formula is：

5. prodrug design formula of the class based on double acylhydrazone connecting keys, it is characterised in that the formula draws on the basis of logical formula (I) Enter part and medicine：

Wherein：Any peptide sequence is represented,Represent connection unit between medicine and bis-CHO.

6. prodrug design formula of the class as claimed in claim 5 based on double acylhydrazone connecting keys, it is characterised in that the class base Lead to formula (II) in the prodrug design of double acylhydrazone connecting keys and design two based on double acylhydrazone connecting key prodrugs D₁And D₂, before two kinds Medicine is made up of three parts：

I) there is the targeting ligand of tumour or cancer cell identification function：RGD is as targeting ligand, and RGD is a class containing arginine-sweet The small peptide of propylhomoserin-aspartic acid sequence, it is the specific binding site of fibronectin and its acceptor；RGD peptide is with integrating Plain acceptor has high-affinity, and acceptor altimeter on the endothelial cell during neonate tumour blood vessel reaches；

II) the anti-tumor drug molecule of strength：MMAE derived from dialdehyde base is as cancer therapy drug, and monomethyl Austria profit statin E is a kind of General type cancer therapy drug, it is by with tubulin binding and suppressing its polymerization methodses interference cell cycle events, causing tumour Apoptosis；

III) it is used as connecting key using double acylhydrazone connecting keys.

7. based on double acylhydrazone key prodrugs D₁And D₂Synthetic method, it is characterised in that comprise the following steps：

Step 1) bishydrazide base group modification peptide molecule design：

Structural formula is (a) and (b), and Peptide 2 design is directed to the MMP-2 enzymes being overexpressed outside tumour cell, peptide's 3 Design is directed to intracellular cathepsin B；Cathepsin is the cysteine proteinase that a class is primarily present in lysosome Enzyme, exists with inactive zymogen forms more, and protease zymogens can be activated under lysosomal acid environment；Due to tumour cell Its surrounding environment can be acidified, its cathepsin secreted can also be activated in extracellular environment, tumor tissues microenvironment In and tumour cell in cathepsin active be far above away from tumor tissues other normal structures in and normal cell in Cathepsin active；Based on the feature of above-mentioned tumor microenvironment, the double acylhydrazones for designing extracellular and intracellular fixed point hydrolysis connect Connect key；The introducing of lysine is designed for further modifying targeting ligand interested；

Step 2) dialdehyde drug derivative design：

MMAE is a kind of common cancer therapy drug, and the amino that its N-terminal methylates flexibly is modified, in order to minimize chemical modification pair The influence of MMAE activity, using a segment structure is flexible and polyethylene glycol conjugation MMAE and bis-CHO of difunctionalization, has obtained one Plant MMAE, its structural formula (C) derived from dialdehyde；

Step 3) part containing RGD prodrug D1 and D2 synthesis：

Pass through peptides 2/3 two hydrazides groups and bis-CHO-PEG₃- MMAE two aldehyde radicals react to form two acyls Hydrazone key, is subsequently added NHS-PEG₄- Maleimide, further the lysine side-chain with peptides 2/3 reacted, finally Add c (RGDyC) part；In addition, being used as reference, prodrug C of the design synthesis without coupling MMAE₁With prodrug C₂, wherein prodrug C₁Correspondence D₁, prodrug C₂Correspondence D₂。

CN201710425299.9A 2017-06-08 2017-06-08 A kind of double acylhydrazone connecting keys of polypeptide bridging are applied to the delivering of aldehyde drug derivative Pending CN107029243A (en)

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