Investigational New Drugs 5: 281-287, 1987 9 Martinus Nijhoff Publishers, Boston - Printed in the Netherlands Enhancement of 1-(2-chloroethyl)-3-cyclohexyi-l-nitrosourea (CCNU) toxicity by acetohydroxamic acid analogues of 3-nitropyrazole in vitro R. Timothy Mulcahy 1, David J. Wustrow 2, Richard R. Hark 3 and Andrew S. Kende 4 1Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison, WI 53792, USA; 2-4Experimental Therapeutics Division; University of Rochester Cancer Center, and the Department of Chemistry, University of Rochester, Rochester, N Y 14642, USA Key words: chemosensitization, misonidazole, hypoxia, CCNU Abstract A series of acetohydroxamic acid derivatives of 3-nitropyrazole were synthesized and evaluated for their ability to potentiate (chemosensitization) the activity of 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU) against EMT-6 mouse mammary tumor cells in vitro. The compounds were designed to test the hypothesis that the chemosensitizing activity of the analogues would be proportional to the rate of isocyanate formation via a Lossen rearrangement, in part a function of the leaving group at the N terminus of each aceto- hydroxamate. Substitution of acetohydroxamic acid side chains at the N-1 position of the parent 3-nitro- pyrazole resulted in compounds which were preferentially toxic to cells treated under hypoxic conditions, and which were capable of enhancing the toxicity of CCNU in hypoxia. As was observed for cytotoxicity, the enhancement of CCNU toxicity by these sensitizing agents was significantly reduced under aerobic treatment conditions. A strong correlation was established between hypoxic toxicity and chemosensitizing potency. The activity of the analogue, however, was not proportional to their excepted rates of Lossen rearrangement. Nevertheless, several potent chemosensitizing compounds were identified; some of which were 10-50x's more potent on a molar basis than Misonidazole, the reference chemosensitizing compound. Introduction In addition to effectively sensitizing hypoxic tumor cells to ionizing radiation, the nitroheterocyclic sen- sitizer Misonidazole (MISO) has been shown to sig- nificantly enhance the activity of several chemo- therapeutic agents in vitro and in vivo; with the largest and most consistent enhancements being observed with cyclophosphamide, L-phenylalanine mustard (L-PAM) and several nitrosoureas, most notably CCNU (reviewed in [1] and [2]). This sensi- tizing effect of MISO and other nitroheterocyclic related compounds has been referred to as chemo- sensitization or chemopotentiation. The rationale for combining radiation sensitizing agents such as MISO with chemotherapeutic agents was first prompted by data suggesting that hypoxic tumor cells were relatively resistant to a variety of anti- tumor drugs [3, 4]. It was hypothesized [5] that MISO and similar compounds could be used to se- lectively target hypoxic cells due to their favorable penetration characteristics and their preferential hypoxic toxicity, expressed even in the absence of radiation. Combination therapy including MISO and certain alkylating agents has been evaluated in a number of experimental systems and found to be superior to treatment with the chemotherapeutic agents alone. Sub-lethal doses of MISO were ob- served to increase drug anti-tumor effectiveness by factors of 1.5 to 2.2 in murine tumor systems, with little or no enhancement of concomitant normal tis- sue toxicity [1]. As a result of its proven effective-