Research on the Process of Synthesizing Antitumor Peptide Drug Acetate Buserelin via Solid-Liquid Combination Method

Abstract

This study employs an innovative solid-liquid combination synthesis strategy to systematically develop a high-efficiency preparation process for the antitumor peptide drug acetate buserelin. Specifically, we first synthesized the key intermediate pyr-his(trt)-trp-ser(trt)-tyr(bzl)-d-ser(tbu)-leu-oh (compound 2) using fmoc solid-phase peptide synthesis technology, while also preparing the dipeptide fragment h-arg-pro-nhet·2hcl (compound 4) through a liquid-phase synthetic route. Subsequently, under optimized liquid-phase condensation conditions, we efficiently coupled both fragments and successfully obtained a target product with over 98% purity through catalytic hydrogenation deprotection and preparative high-performance liquid chromatography purification. The entire synthetic route is elegantly designed with an overall yield of 38.7%, significantly outperforming traditional synthetic methods reported in literature.

Introduction

Pharmacological Properties and Clinical Applications of Buserelin

Buserelin, as a synthetically produced gonadotropin-releasing hormone (GnRH) analog developed by Sanofi-Aventis in France under the brand name Suprefact, consists of nine amino acids with a sequence pGlu1-His2-Trp3-Ser4-Tyr5-D-Ser(tBu)6-Leu7-Arg8-Pro9-NHEt; notably, D-serine at position six is protected by tert-butyl group which characterizes its structure. Since its market launch in Germany in 1984, this drug has been widely used across Europe, Asia and parts of Latin America and Africa; it is currently undergoing Phase III clinical trials in the United States.

In clinical applications, buserelin exhibits significant antitumor activity primarily for treating hormone-dependent malignancies such as prostate cancer and breast cancer. Its mechanism involves continuous stimulation of pituitary GnRH receptors leading to inhibition of gonadotropin secretion and reduction in sex hormone levels. Existing formulations include various dosage forms such as injections, implants and sprays providing flexible options for clinical use.

Limitations of Existing Synthesis Methods

Reported synthesis methods for buserelin face four main technical bottlenecks: First, when employing 5+4 solid-phase fragment coupling method due to exposure during side chain protection leads to significantly reduced crude peptide purity and yield; second although avoiding this issue using 5+4 liquid phase fragment method requires excessive protective groups making operations cumbersome with prolonged synthesis cycles; thirdly full protection strategies utilizing HMPB-MBHA resin encounter economic issues due to expensive resin prices while unprotected hydroxyls on tyrosine residues can easily trigger esterification side reactions; finally conventional solid-phase syntheses often utilize Fmoc-His(Fmoc)-OH that exposes imidazole after deprotection leading to N-acylated byproducts generation.

Experimental Design & Synthetic Route

Synthetic Route Design Principles Based on thorough analysis regarding buserelin's molecular structure we identified unique challenges posed by C-terminal ethylamine structure along with tert-butyl protected D-serine side chain against traditional Fmoc/tBu solid phase strategies. Following systematic literature reviews alongside preliminary experimental validations several critical design principles were established: First considering proline tends towards diketopiperazine formation when positioned at C-terminal resulting loss from resin attachment thus Pro was avoided as terminal amino acid choice within our solid phase fragments assembly process secondly arginine’s large steric hindrance yields unsatisfactory couplings directly onto resins hence incurring higher costs consequently leucine was selected instead establishing our combined approach between solids/liquids synthesizing strategy effectively.

Specific Synthetic Route Details and so forth... in conclusion... etc.