“两步法”合成基于二硫键连接子的奥曲肽-阿霉素偶联物

doi:10.6023/A25020034

摘要/Abstract

癌症仍然是一个全球性的健康挑战, 开发具有新骨架的新型抗癌药物具有重要意义. 多肽-药物偶联物(PDCs)是新型靶向抗癌药物, 具有对肿瘤组织穿透性强、生产成本低及易于结构改造等优势, 可显著提高细胞毒性药物的靶向性和抗肿瘤活性. 奥曲肽是一种可特异性靶向肿瘤细胞表面生长抑素受体2 (SSTR2)的环肽, 在PDCs药物研发中得到广泛应用. 二硫键是PDCs分子中常用的可裂解连接基团. 以往合成含有二硫键连接子的奥曲肽-小分子药物偶联物一般采用“三步法”策略, 但合成路线繁琐且产率较低. 基于此, 本研究开发了“两步法”合成策略, 即先在固相上合成含有分子内二硫键和巯基末端的奥曲肽衍生物, 再通过液相反应与含有巯基的小分子药物共价偶联. 为了实现含巯基末端奥曲肽的高效合成, 本研究探索了不同的固相缩合路线和切肽体系, 综合考虑色谱纯度和分离收率等, 筛选出最佳合成路线SPPS-2和最优切肽体系. 为了进一步验证SPPS-2合成路线的普适性, 探讨了不同树脂和切肽时间对合成效率的影响. 固相高效合成巯基奥曲肽(OCT-SH)后, 将化疗药物阿霉素(DOX)通过液相反应与OCT-SH共价偶联, 形成奥曲肽-阿霉素偶联物. 体外抗肿瘤实验表明, 奥曲肽-阿霉素偶联物具有较强的抗肿瘤活性, 可以显著降低阿霉素对正常细胞的毒性并实现对肿瘤细胞的高选择性. 细胞摄取实验表明, 阿霉素的共价偶联对奥曲肽的细胞摄取能力没有明显影响. 本研究利用“两步法”合成策略, 建立了最佳的固相缩合路线和切肽体系, 实现了基于二硫键连接子的奥曲肽-阿霉素偶联物的高效合成. 本研究为固相合成含有多对二硫键的PDCs分子提供了重要参考, 为开发新型抗肿瘤药物提供了启发.

关键词: 固相多肽合成, 二硫键, 多肽-药物偶联物, 奥曲肽, 阿霉素, 巯基

Cancer is still a global health challenge and developing novel anticancer drugs with new skeletons is of great importance. Recently, peptide-based agents have become promising candidates for anticancer treatments, especially for the treatment of drug-resistant refractory malignancies. Peptide drug conjugates (PDCs) are novel targeted anticancer drugs with advantages including high permeability to cancer tissues, low production costs, and ease of structural optimization. PDCs could significantly enhance the selectivity and anticancer activity of cytotoxic drugs. Octreotide, which is a cyclic peptide inherently containing one pair of disulfide bonds, could specifically target the somatostatin receptor 2 (SSTR2) on the surface of cancer cells, leading to its widespread clinical applications. The reducible disulfide bonds serve as a commonly utilized cleavable linker in PDCs. However, traditional synthesis of octreotide-drug conjugates based on disulfide bond linkers generally used the “three-step” strategy, including complex synthetic procedure and inducing low isolation yields. To address this issue, we developed a “two-step” synthetic strategy. First, octreotide containing one disulfide bond and the free thiol group (OCT-SH) was synthesized by the straightforward solid-phase peptide synthesis (SPPS). Subsequently, the OCT-SH was conjugated to small molecule drugs through site-specific liquid-phase reaction. To achieve the efficient and economic synthesis of OCT-SH, various SPPS based strategies and peptide cleavage systems were explored. Considering the chromatographic purity and synthetic yield, the optimal synthetic route (SPPS-2) and peptide cleavage system were established. To further validate the robustness of SPPS-2, the influences of different solid resins and peptide cleavage time on synthetic efficiency were investigated. After the efficient manual synthesis of OCT-SH, the promising chemotherapeutic drug doxorubicin (DOX) was covalently coupled to OCT-SH through liquid-phase reaction, affording the octreotide-doxorubicin conjugate. In vitro anticancer studies indicated that the octreotide-doxorubicin conjugate exhibited potent anticancer activity, significantly reducing toxicity to normal cells while achieving selectivity towards cancer cells. The cellular uptake experiment indicated that the covalent conjugation of doxorubicin did not significantly impair the cellular uptake potency of octreotide by cancer cells. Collectively, this study established the “two-step” synthetic strategy, enabling efficient and straightforward preparation of octreotide-doxorubicin conjugates containing the disulfide bond linker. This work also provides valuable references for the SPPS based synthesis of PDCs containing multiple disulfide bonds and inspires the development of novel PDCs for cancer therapy.

Key words: solid-phase peptide synthesis (SPPS), disulfide bond, peptide-drug conjugates, octreotide, doxorubicin, thiol

引用此文

于千尧, 孟铭, 姚景方, 杜姗姗, 齐昀坤. “两步法”合成基于二硫键连接子的奥曲肽-阿霉素偶联物[J]. 化学学报, 2025, 83(4): 341-353.

Qianyao Yu, Ming Meng, Jingfang Yao, Shanshan Du, Yunkun Qi. Two Step Synthesis of Octreotide-doxorubicin Conjugates Based on Disulfide Bond Linker[J]. Acta Chimica Sinica, 2025, 83(4): 341-353.

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图/表 11

图1. Pepaxto, Lutathera和PEN-221的结构式

Figure 1. The structures of Pepaxto, Lutathera and PEN-221

图2. (A)基于“三步法”策略合成含有二硫键连接子的奥曲肽-小分子偶联物; (B)基于“两步法”策略合成含有二硫键连接子的奥曲肽-小分子偶联物. I.固相氧化折叠, II.引入巯基末端, III.缀合小分子药物

Figure 2. The three-step strategy (A) and the two-step strategy (B) for the synthesis of octreotide-drug conjugates. I. Solid phase based oxidative folding. II. Introduction of terminal thiol group. III. Conjugation of small molecule drugs

Peptides	Resin	SPPS strategy	Peptide cleavage cocktails	Peptide cleavage time/h	RP-HPLC purity/%	Isolated yield/%	Yield/mg
1	Wang resin	SPPS-1	TFA/phenol/water/thioanisole/EDT^a	2	16.4	6.3	4.1
2	Wang resin	SPPS-1	TFA/phenol/water/thioanisole/ DODT^b	2	Not detected	Not detected	Not detected
3	Wang resin	SPPS-1	TFA/TIPS/water^c	2	23.4	8.3	5.4
4	Wang resin	SPPS-2	TFA/phenol/water/thioanisole/EDT	2	24.2	9.4	6.1
5	Wang resin	SPPS-2	TFA/phenol/water/thioanisole/DODT	2	61.3	18.5	12.0
6	Wang resin	SPPS-2	TFA/TIPS/water	2	77.6	28.3	18.4
7	Wang resin	SPPS-2	TFA/TIPS/water	3	71.3	21.6	14.0
8	2-Chlorotrityl chloride resin	SPPS-2	TFA/TIPS/water	2	74.6	24.4	15.8

表1. 多肽1~8的合成

Table 1. The synthetic strategies for peptides 1~8

图3. (A)基于SPPS-1的固相多肽合成路线; (B)基于SPPS-2的固相多肽合成路线; (C) Fmoc-Cys(Trt)-OH, Fmoc-Cys(Acm)-OH和三氟乙酸铊的结构式. 大写字母和小写字母分别代表L-型和D-型氨基酸

Figure 3. (A) The synthetic route of SPPS-1; (B) The synthetic route of SPPS-2; (C) The structures of Fmoc-Cys(Trt)-OH, Fmoc-Cys(Acm)-OH and Tl(TFA)3. The capital first letters and small first letters represent L-type and D-type amino acid residues respectively

图4. (A)多肽1; (B)多肽2; (C)多肽3; (D)多肽4; (E)多肽5; (F)多肽6; (G)多肽7; (H)多肽8的分析型RP-HPLC色谱图; (I)多肽7和多肽8共进样的分析型RP-HPLC色谱图和ESI-MS质谱图. 测定分子量为1297.236 Da(理论分子量为 1297.526 Da)

Figure 4. Analytical RP-HPLC spectra of (A) peptide 1; (B) peptide 2; (C) peptide 3; (D) peptide 4; (E) peptide 5; (F) peptide 6; (G) peptide 7; (H) peptide 8; (I) Analytical RP-HPLC and ESI-MS spectra for the co-injection of peptide 7 and peptide 8. The ESI-MS gave an observed mass of 1297.236 Da (calculated 1297.526 Da, average isotopes) RP-HPLC condition: a linear gradient of 20%~60% buffer B (0.1% TFA in CH3CN) in buffer A (0.1% TFA in water) over 30 min (20% for 2 min, then 20%~60% for 30 min) on a Agilent C18 column, λ=214 nm

图5. Py-SS-DOX和OCT-SS-DOX的合成路线

Figure 5. The synthetic routes of Py-SS-DOX and OCT-SS-DOX

图6. (A)反应0.5 h的RP-HPLC色谱图; (B)反应12 h的RP-HPLC色谱图; (C)反应24 h的RP-HPLC色谱图和OCT-SS-DOX的ESI-MS质谱图. OCT-SS-DOX的测定分子量为1927.786 Da(理论分子量为1927.159 Da)

Figure 6. The RP-HPLC spectra of the reaction at 0.5 h (A) and at 12 h (B). (C) The RP-HPLC spectrum of the reaction at 24 h and the ESI-MS spectrum of OCT-SS-DOX. The ESI-MS gave an observed mass of 1927.786 Da (calculated 1927.159 Da, average isotopes) RP-HPLC condition: a linear gradient of 20%~80% buffer B (0.1% TFA in CH3CN) in buffer A (0.1% TFA in water) over 30 min (20% for 2 min, the 20%~80% for 30 min) on a Agilent C18 column, λ=214 nm

图7. (A) OCT-SS-DOX, OCT-SH和DOX在不同细胞的IC50曲线; (B) OCT-SH和DOX在MCF-7细胞上联合用药的IC50曲线; (C) OCT-SH和DOX在MCF-7细胞上联合用药的联合指数(CI), CI值0~0.5: 蓝色; CI值0.5~1: 红色; CI值>1: 绿色

Figure 7. (A) IC50 curves of OCT-SS-DOX, OCT-SH and DOX in different cells; (B) IC50 curves for combination drugs of OCT-SH and DOX on the MCF-7 cells; (C) The combination indices (CIs) of OCT-SH plus DOX on MCF-7 cells. CI values 0~0.5: blue; 0.5~1: red; >1: green

Combined agents	Agents	MCF-7	HepG2	PC-3	Panc-1	HEK-293	SI^a	SI^b
—	OCT-SH	>100	>100	>100	>100	>100	—	—
—	OCT-SS-DOX	12.56±1.23	13.26±2.11	27.64±2.35	28.82±3.44	48.79±3.98	3.88	1.77
—	DOX	0.25±0.02	0.32±0.06	0.26±0.04	0.36±0.08	0.15±0.04	0.60	0.58
50 μmol/L OCT-SH	DOX	0.31±0.08	—	—	—	—	—	—
0.2 μmol/L DOX	OCT-SH	>100	—	—	—	—	—	—
DOX/OCT-SH (1∶1, molar ratio)		0.28±0.06	—	—	—	—	—	—

表2. OCT-SH, DOX和OCT-SS-DOX的IC50数值(μmol/L)

Table 2. The IC50 (μmol/L) for OCT-SH, DOX and OCT-SS-DOX

图8. OCT-C-DOX-7-DCCA的结构式

Figure 8. The structure of OCT-C-DOX-7-DCCA

图9. (A)多肽11, 12和13在MCF-7细胞72 h的IC50曲线; (B)多肽11, 12和13与MCF-7细胞共孵育48 h的细胞荧光图像; (C)摄取细胞数的定量统计图, n=3, mean±SD. ns: 无显著性差异

Figure 9. (A) IC50 curves of peptides 11, 12 and 13 in MCF-7 cells at 72 h; (B) Fluorescence images of MCF-7 cells treated with peptides 11, 12 and 13 at 48 h. (C) Quantification of uptake ratios. n=3, mean±SD. ns: not significant

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