Tumor-Targeting with Novel Non-Benzoyl 6-Substituted Straight Chain Pyrrolo[2,3-d]pyrimidine Antifolates via Cellular Uptake by Folate Receptor α and Inhibition of de Novo Purine Nucleotide Biosynthesis

Abstract

A new series of 6-substituted straight side chain pyrrolo[2,3-d]pyrimidines 3a–d with varying chain lengths (n = 5–8) was designed and synthesized as part of our program to provide targeted antitumor agents with folate receptor (FR) cellular uptake specificity and glycinamide ribonucleotide formyltransferase (GARFTase) inhibition. Carboxylic acids 4a–d were converted to the acid chlorides and reacted with diazomethane, followed by 48% HBr to generate the α-bromomethylketones 5a–d. Condensation of 2,4-diamino-6-hydroxypyrimidine 6 with 5a–d afforded the 6-substituted pyrrolo[2,3-d]pyrimidines 7a–d. Hydrolysis and subsequent coupling with diethyl L-glutamate and saponification afforded target compounds 3a–d. Compounds 3b–d showed selective cellular uptake via FRα and -β, associated with high affinity binding and inhibition of de novo purine nucleotide biosynthesis via GARFTase, resulting in potent inhibition against FR-expressing Chinese hamster cells and human KB tumor cells in culture. Our studies establish, for the first time, that a side chain benzoyl group is not essential for tumor-selective drug uptake by FRα.

Structural and Enzymatic Analysis of Tumor-Targeted Antifolates That Inhibit Glycinamide Ribonucleotide Formyltransferase

“…22–27 All contain a terminal l -glutamate separated from the bicyclic ring system by a bridge region composed of three (AGF94) or four (AGF23, AGF50, AGF71, AG117, and AGF118) carbons, followed by benzoyl (AGF23 and AGF50) or thienoyl (AGF71, AGF94, AGF117, and AGF118) ring moieties (Figure 1). AGF117 and AGF118 are 3′,5′- and 2′,4′-thiophene regioisomers of AGF71, respectively.…”

“…The structurally diverse 6-substituted pyrrolo- and thieno-[2,3- d ]pyrimidine antifolates all elicit low nanomolar to subnanomolar IC 50 cytotoxic values in cell proliferation assays with KB human tumor cells in vitro 22–27 (Table 1). For AGF71 and AGF94, in vivo antitumor efficacies were established toward FR-expressing KB and IGROV1 tumors.…”

“…22–27 In this report, we used the isolated formyltransferase domain of human GARFTase to compare in vitro inhibitor potencies with crystallographic structures of antifolates to improve our understanding of the molecular basis for variations in inhibitor potencies at GARFTase.…”

Enhanced Receptor-Mediated Endocytosis and Cytotoxicity of a Folic Acid-Desacetylvinblastine Monohydrazide Conjugate in a Pemetrexed-Resistant Cell Line Lacking Folate-Specific Facilitative Carriers but with Increased Folate Receptor Expression

“…Lack of PCFT markedly impaired but did not completely eliminate transport of 5-methytetrahydrofolate or (6S)-5-CHO-THF out of endosomes, consistent with PCFT-dependent and independent processes. The presence of an alternative folate endosomal export mechanism seems likely, based upon the observation that an FR-a-targeted antifolate is active even in the absence of PCFT (Wang et al, , 2013.…”

“…FR-a is expressed in epithelia (proximal renal tubule, choroid plexus, retinal pigment epithelium) (Elnakat and Ratnam, 2004;Kamen and Smith, 2004) and is widely expressed in epithelial cancers (Weitman et al, 1992;Parker et al, 2005); FR-b is expressed in hematopoietic malignancies (Ross et al, 1999). Their selective expression in human cancers is the basis for the development of agents that are transported primarily by this route: 1) folate analogs with high affinity for FRs but very low affinity for RFC (Gibbs et al, 2005;Wang et al, 2011Wang et al, , 2013; 2) folic acid conjugated to cytotoxics that are endocytosed, released within, and diffuse out of endosomes to inhibit their intracellular targets (Xia and Low, 2010).…”