A group of potential alkylating agents have been synthesized that are structurally related to the oligopeptide antiviral antibiotic distamycin. All derivatives form complexes with native calf-thymus DNA but compounds 2, 3, and 6 give rise to covalent adducts. Cytostatic activity against both human and murine tumor cell lines in vitro is displayed by the new compounds. Compounds 3 and 4 are active on melphalan-resistant L1210 leukemia in mice.

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6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) is a powerful inhibitor of the glutathione transferase P1-1 (GSTP1-1) and causes the disruption of the complex between GSTP1-1 and c-Jun N-terminal Kinase (JNK). This induces JNK activation and apoptosis in tumour cells. In the present work we assess the in vitro and in vivo effectiveness of NBDHEX on two human melanoma cell lines, Me501 and A375. NBDHEX shows IC(50) values in the low micromolar range (IC(50) of 1.2+/-0.1microM and 2.0+/-0.2 microM for Me501 and A375, respectively) and is over 100 times more cytotoxic to these cell lines than temozolomide. Apoptosis is observed in Me501 cells within 3h of the addition of NBDHEX, while in A375 cells the apoptotic event is rather late, and is preceded by a G2/M phase arrest. In both melanoma cell lines, JNK activity is required for the ability of NBDHEX to trigger apoptosis, confirming that the JNK pathway is an important therapeutic target for this tumour. NBDHEX is also both effective and well tolerated in in vivo tumour models. A tumour inhibition of 70% is observed in vivo against Me501 human melanoma and a similar result is obtained on A375 model, with 63% of tumour inhibition. These findings indicate that the activation of the JNK pathway, through a selective GSTP1-1 targeting, could prove to be a promising new strategy for treating melanoma, which responds poorly to conventional therapies.

We designed and synthesized two novel nitrobenzoxadiazole (NBD) analogues of the anticancer agent 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX). The new compounds, namely MC3165 and MC3181, bear one and two oxygen atoms within the hydroxy-containing alkyl chain at the C4 position of the NBD scaffold, respectively. This insertion did not alter the chemical reactivity with reduced glutathione, while it conferred a remarkable increase in water solubility. MC3181 was more selective than NBDHEX towards the target protein, glutathione transferase P1-1, and highly effective in vitro against a panel of human melanoma cell lines, with IC50 in the submicromolar-low micromolar range. Interestingly, the cellular response to MC3181 was cell-type-specific; the compound triggered a JNK-dependent apoptosis in the BRAF-V600E-mutated A375 cells, while it induced morphological changes together with an increase in melanogenesis in BRAF wild-type SK23-MEL cells.MC3181 exhibited a remarkable therapeutic activity against BRAF-V600E-mutant xenografts, both after intravenous and oral administration. Outstandingly, no treatment-related signs of toxicity were observed both in healthy and tumor-bearing mice after single and repeated administrations.Taken together, these results indicate that MC3181 may represent a potential novel therapeutic opportunity for BRAF-mutated human melanoma, while being safe and water-soluble and thus overcoming all the critical aspects of NBDHEX in vivo.

Purpose: Nemorubicin (3V-deamino-3V-[2 00 (S)-methoxy-4 00 -morpholinyl]doxorubicin; MMDX) is an investigational drug currently in phase II/III clinical testing in hepatocellular carcinoma. A bioactivation product of MMDX, 3V-deamino-3 00 ,4V-anhydro-[2 00 (S)-methoxy-3 00 (R)-oxy-4 00 -morpholinyl]doxorubicin (PNU-159682), has been recently identified in an incubate of the drug with NADPHsupplemented rat liver microsomes. The aims of this study were to obtain information about MMDX biotransformation to PNU-159682 in humans, and to explore the antitumor activity of PNU-159682.Experimental Design: Human liver microsomes (HLM) and microsomes from genetically engineered cell lines expressing individual human cytochrome P450s (CYP) were used to study MMDX biotransformation. We also examined the cytotoxicity and antitumor activity of PNU-159682 using a panel of in vitro-cultured human tumor cell lines and tumor-bearing mice, respectively.Results: HLMs converted MMDX to a major metabolite, whose retention time in liquid chromatography and ion fragmentation in tandem mass spectrometry were identical to those of synthetic PNU-159682. In a bank of HLMs from 10 donors, rates of PNU-159682 formation correlated significantly with three distinct CYP3A-mediated activities. Troleandomycin and ketoconazole, both inhibitors of CYP3A, markedly reduced PNU-159682 formation by HLMs; the reaction was also concentration-dependently inhibited by a monoclonal antibody to CYP3A4/5. Of the 10 cDNA-expressed CYPs examined, only CYP3A4 formed PNU-159682. In addition, PNU-159682 was remarkably more cytotoxic than MMDX and doxorubicin in vitro, and was effective in the two in vivo tumor models tested, i.e., disseminated murine L1210 leukemia and MX-1 human mammary carcinoma xenografts.Conclusions: CYP3A4, the major CYP in human liver, converts MMDX to a more cytotoxic metabolite, PNU-159682, which retains antitumor activity in vivo.