Nitric oxide (NO) is an important messenger molecule with multiple biological activities in blood vessels, neurons, macrophages, and other cells. NO is a ubiquitous modulator of both physiological and pathophysiological functions that is generated endogenous by the enzyme nitric oxide synthase of which 3 isoforms are known. NO appears critical for the tumoricidal activity of the immune system, modulates apoptosis of tumors and has other effects on tumor biology, including angiogenesis and metestasis. On the basis of the multifaceted roles of NO in cancer and the fact that methotrexate (MTX) is a very active anticancer drug and one of the nitric oxide synthase (NOS) inhibitors, new conjugates of MTX with NO donors or NOS inhibitors have been synthesized hoping that MTX functions as an active carrier. Methotrexate was coupled with 3-hydroxymethyl-4-phenyl-1,2,5-oxadiazole-N-oxide (an NO donor) at the α- and γ-carboxyl groups to give 1a and 2a. Analogously reacted MTX with N-nitro-L-arginine to 1b and 2b and with N-nitro-L-arginine methyl ester to 1c and 2c. In the biological assay the MTX-conjugates were tested against K-562 human leukemia cells which are resistant to MTX. Increasing in lipiphobicity as shown by 1b and 2b reduced the activity of K-562 cell-killing in culture and contrarily, compounds 1a, 2a, 1c and 2c inhibited the growth of K-562 cells superior to MTX due to their higher lipiphilicity. In general, the MTX-NO-donors enhance tumoricidal activity significantly and the γ-substituted conjugates are more effective than the α-isomers.

Key words: methotrexate derivatives, nitric oxide donor, inhibitor of nitric oxide synthase, antineoplastic