lometrexol 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

A series of 7 human squamous carcinoma cell lines of the head and neck (HNSCC), grown in standard medium containing high folate concentrations and in "folate-conditioned" medium containing nanomolar concentrations of folates, were all found to be sensitive (IC50: less than or equal to 50 nM) in growth-inhibition studies to methotrexate (MTX) following drug exposure for 7 days. However, when MTX exposure was limited to 24 hr, only 2 out of 7 HNSCC cell lines were sensitive to MTX (IC50: less than 500 nM), 2 were moderately sensitive (IC50: 1-2 microM), and 3 exhibited inherent resistance to MTX (IC50: greater than 250 microM). In these last 3 cell lines, the mechanism of resistance was not correlated with altered membrane transport of MTX or changes in dihydrofolate reductase activity, but rather was associated with a 3-fold lower activity of intracellular folylpolyglutamate synthase (FPGS) activity compared to MTX-sensitive HNSCC cells. The 3 cell lines exhibiting inherent resistance to a short exposure to MTX, however, did not show inherent cross-resistance after exposure for 24 hr to one or more of 3 novel antifolate compounds. These compounds, which appear to be more efficiently transported and polyglutamylated than MTX, include: 10-ethyl-10-deazaaminopterin (10-EdAM), 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI-198,583), and 5,10-dideazatetrahydrofolic acid (DDATHF). These results indicate that antifolate membrane transport and intracellular FPGS activity are important factors in determining sensitivity or resistance of HNSCC cells to short-term antifolate compound exposures.

Topics: Aminopterin; Biological Transport; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Membrane; Drug Resistance; Drug Screening Assays, Antitumor; Folic Acid; Folic Acid Antagonists; Head and Neck Neoplasms; Humans; Methotrexate; Tetrahydrofolate Dehydrogenase; Tetrahydrofolates; Tumor Cells, Cultured