Temozolomide (TMZ) is the standard of care chemotherapeutic agent used in the treatment of glioblastoma multiforme. Cytotoxic O6 -methylguaine lesions formed by TMZ are repaired by O6 -methyl-guanine DNA methyltransferase (MGMT), a DNA repair protein that removes alkyl groups located at the O6 -position of guanine. Response to TMZ requires low MGMT expression and functional mismatch repair. Resistance to TMZ conferred by MGMT, and tolerance to O6 -methylguanine lesions conferred by deficient MMR severely limit TMZ clinical applications. Therefore, development of new TMZ derivatives that can overcome TMZ-resistance is urgent. In this study, we investigated the anti-tumor mechanism of action of two novel TMZ analogs: C8-imidazolyl (377) and C8-methylimidazole (465) tetrazines. We found that analogs 377 and 465 display good anticancer activity against MGMT-overexpressing glioma T98G and MMR deficient colorectal carcinoma HCT116 cell lines with IC 50 value of 62.50, 44.23, 33.09, and 25.37 μM, respectively. Analogs induce cell cycle arrest at G2/M, DNA double strand break damage and apoptosis irrespective of MGMT and MMR status. It was established that analog 377, similar to TMZ, is able to ring-open and hydrolyze under physiological conditions, and its intermediate product is more stable than MTIC. Moreover, DNA adducts of 377 with calf thymus DNA were identified: N7 -methylguanine, O6 -methylguanine, N3 -methyladenine, N3 -methylthymine, and N3 -methylcytidine deoxynucleotides. We conclude that C8 analogs of TMZ share a mechanism of action similar to TMZ and are able to methylate DNA generating O6 -methylguanine adducts, but unlike TMZ are able at least in part to thwart MGMT- and MMR-mediated resistance.

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The efficacy of temozolomide (TMZ) treatment for cancers is currently limited by inherent or the development of resistance, particularly, but not exclusively, due to the expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) in a significant proportion of tumors. We have found that TMZ analog C8-methyl imidazole tetrazine (PMX 465) displayed good anticancer activity against the colorectal carcinoma HCT116 cells which are MGMT-overexpressing and mismatch repair (MMR)-deficient. In this study, we found that PMX 465 could downregulate the expression of MGMT in HCT116 cells at the protein and mRNA levels. We found that PMX 465 could reduce MGMT expression by increasing the binding of wild-type p53 to the MGMT promoter and reducing the binding of Sp1 to the MGMT promoter.