PCFT为转运靶的新型选择性GARFTase抑制剂的研究

The reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT) are three major uptake systems for reduced folates, and are shared by antifolates. RFC is ubiquitously expressed in tumors and tissues and is the primary uptake mechanism for folates and antifolates. Lack of selectivity for folate transporters is one of the major causes of toxicity and chemotherapy failure with clinically used classical antifolates. Impaired transport by RFC is also a key mechanism of drug resistance for classical antifolates. FRs along with PCFT represent another mode of folate uptake and are considered potential chemotherapeutic targets for selective tumor uptake. FRα is expressed in normal apical membranes of the kidney, choroid plexus, and placenta, whereas FRβ is expressed in placenta, spleen, and thymus. FRβ in normal hematopoietic cells cannot bind folate ligand. FRα is expressed in solid tumors such as nonmucinous adenocarcinomas of the ovary, uterus, and cervix, whereas FRβ is expressed in leukemia blasts in chronic myelogenous leukemia and in acutemyelogenous leukemia. PCFT is most abundant in the duodenum and upper jejunum, where it serves as the primary means of intestinal uptake of dietary folates at the acid pH characterizing the upper small intestine. PCFT is present at high levels in assorted solid tumors including lung cancers, ovarian cancer, and hepatomas. Further,PCFT is maximally active at pHs approximating those attained in the solid tumor microenvironment. Therefore,the central hypothesis we have is that, if a FR and/or PCFT-targeted ligand were itself cytotoxic without RFC activity, selective tumor targeting would ensue. We recently identified 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with selective membrane transport by PCFT and/or FRs over RFC, resulting in selective inhibition of proliferation of cells expressing these systems including human tumors. Cytotoxicity was attributed to the potent inhibition of β-glycinamide ribonucleotide formyltransferase (GARFTase), which catalyzes the first folate-dependent reaction in de novo purine nucleotide biosynthesis. Small molecules that could specifically transported by PCFT have not been reported in the literature. Thus,the research objective of this study is to design, synthesis and evaluate novel GARFTase inhibitors that could selectively transported by PCFT over RFC and FR, and to build a structure activity relationship (SAR) for PCFT.

还原叶酸转运蛋白（RFC），叶酸受体（FR）和质子偶联叶酸转运体（PCFT）是转运叶酸和叶酸拮抗剂的三种转运体。其中RFC为主要转运体，在正常组织和肿瘤中广泛表达。缺乏对叶酸转运体的选择性是传统叶酸拮抗剂产生毒性并导致化疗失败的主要原因。FR和PCFT代表了叶酸拮抗剂抗肿瘤研究的新方向。这两类转运蛋白在正常组织极少表达，但在多种肿瘤细胞表面大量表达，为选择性抑制肿瘤提供了新的有效靶点。通过设计合成新型叶酸拮抗剂，选择性的被FR和/或PCFT所转运，达到选择性杀死肿瘤细胞的目的。我们近期报道的具有抗肿瘤活性的吡咯并[2,3-d]嘧啶类化合物为甘氨酰核苷酸甲基转移酶（GARFTase）抑制剂，可以选择性被FR和PCFT共同转运而不被RFC转运。目前尚未发现特异性结合PCFT的小分子。本研究的目的在于设计、合成并筛选以PCFT为特定转运靶并且具有抗肿瘤活性的新型选择性GARFTase抑制剂。

缺乏对叶酸转运体的选择性是传统叶酸拮抗剂产生毒性并导致化疗失败的主要原因。还原叶酸转运蛋白（the reduce folate carrier, RFC）是转运叶酸和叶酸拮抗剂进入细胞的主要载体，在正常组织和肿瘤中广泛表达。叶酸受体（folate receptors, FR）和质子偶联叶酸转运体（proton coupled folate transporter, PCFT）在正常组织极少表达，但在多种肿瘤细胞表面大量表达，为选择性抑制肿瘤生长提供了新的有效靶点。本研究课题为PCFT为转运靶的新型选择性GARFTase抑制剂的研究。在前期工作基础上，运用计算机辅助药物设计方法，设计并合成了一系列6-取代吡咯并[2,3-d]嘧啶类化合物，通过改变吡咯并[2,3-d]嘧啶母核与谷氨酸或氨甲基吡啶侧链之间的化学键及连接长度，考察该类化合物对抗肿瘤活性和转运选择性的影响。所有新化合物的化学结构经质谱、核磁共振氢谱和碳谱予以确证。生物活性研究表明，所合成的目标化合物能够有效抑制KB，SW620，A549和HepG2肿瘤细胞的生长，部分目标化合物能够改变肿瘤细胞周期并引发细胞凋亡。作用机制研究表明，目标化合物能够抑制叶酸循环中多个重要的酶，如：胸腺嘧啶合成酶（thymidylate synthase, TS）、甘氨酰核苷酸甲基转移酶（glycinamide ribonucleotide formyltransferase, GARFTase）及5-氨基咪唑-4-甲酰胺核苷酸甲基转移酶（5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase, AICARFTase）。另外，在完成化合物设计合成及生物活性评价的基础上，对所合成的新化合物开展分子模拟研究。采用MOE 2009.10软件构建分子对接实验所需的化学结构，实验结果用SYBYL-X 1.1软件进行验证，所得到的分子模拟实验结果与生物活性研究结果一致。本研究在化合物结构设计与合成、反应路线优化、生物活性评价研究与分子模拟实验相结合等方面进行了探索，所合成的6-取代吡咯并[2,3-d]嘧啶类化合物可为开发和应用新型靶向抗肿瘤GARFTase抑制剂提供科学依据。

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