Two Step Synthesis of Octreotide-doxorubicin Conjugates Based on Disulfide Bond Linker

doi:10.6023/A25020034

Abstract

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

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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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Fig. & Tab. 11

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

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

Table 1. The synthetic strategies for peptides 1~8

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

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

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

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

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	—	—	—	—	—	—

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

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

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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