raltitrexed and Carcinoma--Squamous-Cell

2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl side chain and four to six carbon bridge lengths (compounds 1-3) were synthesized as substrates for folate receptors (FRs) and the proton-coupled folate transporter (PCFT). Conversion of acetylene carboxylic acids to alpha-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidines. Sonogashira coupling with (S)-2-[(5-bromo-thiophene-2-carbonyl)-amino]-pentanedioic acid diethyl ester, followed by hydrogenation and saponification, afforded 1-3. Compounds 1 and 2 potently inhibited KB and IGROV1 human tumor cells that express FR alpha, reduced folate carrier (RFC), and PCFT. The analogs were selective for FR and PCFT over RFC. Glycinamide ribonucleotide formyltransferase was the principal cellular target. In SCID mice with KB tumors, 1 was highly active against both early (3.5 log kill, 1/5 cures) and advanced (3.7 log kill, 4/5 complete remissions) stage tumors. Our results demonstrate potent in vitro and in vivo antitumor activity for 1 due to selective transport by FRs and PCFT over RFC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Carrier Proteins; Cell Proliferation; Cells, Cultured; CHO Cells; Cricetinae; Cricetulus; Female; Folate Receptors, GPI-Anchored; Folic Acid Antagonists; Humans; Membrane Transport Proteins; Mice; Mice, Inbred ICR; Mice, SCID; Ovarian Neoplasms; Proton-Coupled Folate Transporter; Purines; Pyrimidines; Receptors, Cell Surface; Reduced Folate Carrier Protein