CHARACTERIZATION OF THE BIOCHEMICAL MECHANISM OF ACTION OF tx-(N)- HETEROCYCLIC CARBOXALDEHYDE THIOSEMICARBAZONES ALAN C. SARTORELLI, KRISHNA C. AGRAWAL, ASTERIOS S. TSIFTSOGLOUand E. COLLEEN MOORE Department of Pharmacology and Section of Developmental Therapeutics, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06510 and Department of Biochemistry, University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77025 INTRODUCTION The a-(N)-heterocyclic carboxaldehyde thiosemicarbazones as a class possess both antineoplastic and antiviral activity. 2-Formylpyridine thiosemicarbazone (PT) was the first agent of this series reported to have such biological activity in that it produced an increase in the life span of mice bearing the L1210 leukemia (1); this compound was abandoned as a potential cancer chemo- therapeutic agent, however, because of its relatively low therapeutic index. Several years later, French and Blanz(2,3) described the synthesis of 1-formylisoquinoline thiosemicarbazone (IQ-1) and a variety of other oe-(N)-heterocyclic carboxaldehyde thiosemicarbazones. These agents formed strong coordination compounds with transition metals such as iron, cobalt, nickel, copper, zinc and manganese (3), with exceptionally strong affinity for iron (4-6). We have carried out extensive modification of the formyl thiosemicarbazone side chain of IQ-1 to ascertain the importance of this part of the molecule for anticancer activity (7). A variety of substitutions and alterations of the various positions of the side chain was made; these alterations, which uniformly led to complete loss or marked decrease in tumor-inhibitory potency, and the structure of IQ-1 are shown in Figure 1. In addition, replacement of the heterocyclic ring N with C also resulted in a biologically inactive compound. These findings indicated the essentiality of this portion of the molecule and supported the initial suggestion of French and Blanz (3) that a conjugate N*-N*-S* tridentate ligand system was a requisite for tumor inhibitory activity. Further support for this concept was provided from the findings of these investigators (2, 3) that other heterocyclic ring systems (i.e., pyridine, quinazoline, phthalazine, pyrazine, pyridazine and purine) possessed significant antineoplastic activity when the carbonyl attachment of the side chain was located at a position tx to 117