Targeted antifolates with heteroatom replacements of the carbon vicinal to the phenyl ring in 1 by N (4), O (8), or S (9), or with N-substituted formyl (5), acetyl (6), or trifluoroacetyl (7) moieties, were synthesized and tested for selective cellular uptake by folate receptor (FR) α and β or the proton-coupled folate transporter. Results show increased in vitro anti-proliferative activity toward engineered Chinese hamster ovary cells expressing FRs by 4–9 over the CH2 analog 1. Compounds 4–9 inhibited de novo purine biosynthesis and glycinamide ribonucleotide formyltransferase (GARFTase). X-ray crystal structures for 4 with FRα and GARFTase showed that the bound conformations of 4 required flexibility for attachment to both FRα and GARFTase. In mice bearing IGROV1 ovarian tumor xenografts, 4 was highly efficacious. Our results establish that heteroatom substitutions in the 3-atom bridge region of 6-substituted pyrrolo[2,3-d]pyrimidines related to 1 provide targeted antifolates that warrant further evaluation as anticancer agents.

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Structure–activity relationships for cellular uptake and inhibition of cell proliferation were studied for 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates in which the terminal l-glutamate of the parent structure (7) was replaced by natural or unnatural amino acids. Compounds 7 and 10–13 were selectively inhibitory toward folate receptor (FR) α-expressing Chinese hamster ovary (CHO) cells. Antiproliferative effects of compounds 7 and 9–13 toward FRα- and FRβ-expressing CHO cells were only partly reflected in binding affinities to FRα and FRβ or in the docking scores with molecular models of FRα and FRβ. Compounds 7 and 11 were potent inhibitors of glycinamide ribonucleotide formyltransferase in de novo purine biosynthesis in KB human tumor cells. These studies establish for the first time the importance of the α- and γ-carboxylic acid groups, the length of the amino acid, and the conformation of the side chain for transporter binding and biological activity of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates.

The α2,3selective potentiator of GABAA receptors, KRM-II-81, reduces nociceptive-associated behaviors induced by formalin and spinal nerve ligation in rats. Pharmacology Biochemistry and Behavior.

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