Pronounced anti-proliferative activity and tumor cell selectivity of 5-alkyl-2-amino-3-methylcarboxylate thiophenes

5-(2-(4-Methoxyphenyl)ethyl)-2-amino-3-methylcarboxylate thiophene (TR560) is the prototype drug of a recently discovered novel class of tumor-selective compounds that preferentially inhibit the proliferation of specific tumor cell types (e.g. leukemia/lymphoma). Here, we further increased tumor selectivity by simplification of the molecule through replacing the 4-methoxyphenyl moiety by an alkyl chain. Several 2-amino-3-methylcarboxylate thiophene derivatives containing at C-5 an alkyl group consisting of at least 6 (hexyl) to 9 (nonyl) carbon units showed pronounced anti-proliferative activity in the mid-nanomolar range with 500- to 1000-fold tumor cell selectivity. The compounds preferentially inhibited the proliferation of T-lymphoma CEM and Molt/4, prostate PC-3, kidney Caki-1 and hepatoma Huh-7 tumor cells, but were virtually inactive against Other tumor cell lines including B-lymphoma Raji and cervix carcinoma HeLa cells. The novel prototype drug 3j (containing a 5-heptyl chain) elicited a cytotoxic, rather than cytostatic activity, already after 4 h of exposure. The unusual tumor selectivity could not be explained by a differential uptake (or efflux) of the drug by sensitive versus resistant tumor cells. Exposure of a fluorescent derivative of 3j revealed pronounced uptake of the drug in the cytoplasm, no visible appearance in the nucleus, and a predominant localization in the endoplasmic reticulum. These observations may be helpful to narrow down the intracellular localization and identification of the molecular target of the 5-substituted thiophene derivatives.

5-Alkyl-2-aminothiophenes; Anti-proliferative activity; Gewald reaction; Intracellular uptake; Tumor selectivity.