[CANCER RESEARCH 46, 744-756, February 1986] Comparison of Triazines as Inhibitors of L1210 Dihydrofolate ReducÃ-aseand of L1210 Cells Sensitive and Resistant to Methotrexate1 Cynthia Dias Selassie, Cynthia D. Strong, Corwin Mansch,2 Tavner J. Delcamp, J. H. Freisheim, and Tasneem A. Khwaja Department oÃ-Chemistry, Pomona College, Claremont, California 97777 [C. D. S., C. D. S., C. H.]; Department of Biochemistry, Medical College of Ohio, C.S. 10008, Toledo, Ohio 43699 [T. J. D., J. H. F.]; and Department of Pathology and Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles, California 90033 ¡T.A. K.] ABSTRACT The inhibition of dihydrofolate redactase from L1210 leukemia cells as well as the inhibition of intact L1210 cells, both sensitive and resistant, to methotrexate by over 100, 4,6-diamino-2,2- dihydro - 2,2 - dimethyl -1 - (X - phenyl) - s - triazines was studied. Quantitative structure-activity relationships were derived for the three systems. These equations, based on a set of congeners having a range in lipophilicity of about 700,000,000 on the octanol-water scale, delineate the inhibitory potency of the tria zines in relation to their hydrophobicity. The data demonstrate that there is a close parallel between the way isolated dihydro folate reducÃ-aseand methotrexate sensitive cells respond to the triazines. However, the resistant L1210 cells behave in an entirely different manner, which suggests that the passive diffusion of triazines into the cells dominates the structure-activity relation ship. The optimum lipophilicity (ir0) of triazine substituents for purified L1210 dihydrofolate reducÃ-aseis 1.76 to 2.11; for sen sitive cells, it is 1.45 to 1.83, and for resistant cells, it is ~6. INTRODUCTION A general trend in recent years in drug research has been to begin drug development at the molecular level when possible. More recently another step toward more fundamental levels has been made in the use of X-ray crystallography to define the three-dimensional structures of various drugs and receptor pro teins (1-4). Since the pioneering work of Hitchings and Roth (5) and Baker (6), we have become adept at the design of enzyme inhibitors once some biochemical information about their substrates has been elucidated. However, success in finding clinically useful drugs starting with studies of purified enzymes has been limited. There are formidable obstacles to overcome in designing inhibi tors which can reach their sites of action in vivo with minimal toxicity and without undergoing extensive metabolism, elimina tion, or distribution in the wrong compartments. The develop ment of effective in vitro inhibitors into useful drugs is a multi- variate problem of huge proportions in which computerized structure-activity analysis offers some hope of disentangling the many variables involved (2-4, 7, 8). However, while it is clear that mathematical SAR3 can be Received 4/3/85; revised 8/16/85; accepted 10/16/85. 'This research was supported by Grants GM-30362 (C. H.), CA-11666 (J. H. F.), and CA-14089 (T. A. K.) from the National Institutes of Health. 2 To whom requests for reprints should be addressed. 3The abbreviations used are: SAR, structure activity relationships; DHFR, dihydrofolate reducÃ-ase;MIX, methotrexate; OSAR, quantitative structure activity relationships; MR. molar refraction; IDW concentration of drug that halves the growth rate relative to the untreated controls; CMS, central nervous system. formulated for inhibitors acting on isolated enzymes (9), it is not readily apparent what such relationships portend for the action of inhibitors in vivo. Thus, jt seems that the first step toward a better understanding of drug SAR in vivo might be to make systematic and extensive comparative structure-activity studies of the action of inhibitors on isolated, purified enzymes with their SAR on cell cultures. There is a surprising shortage of such comprehensive studies in the literature. Preliminary work on bacterial (10, 11) and mammalian cell systems (12-14), which involved comparison of inhibition of purified, isolated DHFR with that of cell growth, has been encouraging. QSAR has revealed similarities as well as distinct differences in drug action on the two distinct types of systems. A problem which limits the effectiveness of cancer chemother apy is the spontaneous selection and overgrowth of permanent drug resistant, neoplastia populations (15). Insight into this mat ter can also be obtained by comparing the action of a well designed set of inhibitors on isolated, purified, DHFR enzyme with the action on intact cells resistant to methotrexate (12,13). The toxic effect of MTX in mammalian cells is attributed to an inhibition of DHFR. DHFR catalyzes the NADPH dependent reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate. Re duced folate cofactors are required for the transfer of one-carbon units in both thymidylate, purine, and amino acid biosynthesis (16). Decreases in the levels of thymidylate lead to diminished cellular levels of thymidine triphosphate (17). It is of interest to note that inhibition of DNA synthesis by MTX and related antag onists is also accompanied by inhibition of RNA and protein synthesis (18). While remarkably similar SAR have been found for triazines I inhibiting DHFR from L5178Y tumor cells and sensitive L5178Y cell culture, the SAR for L5178Y cells resistant to MTX were extremely different. The intriguing results obtained from the L5178Y system prompted the present study on the clinically much more important L1210 mouse leukemia. In this report, a comparison is made of the potency of a large number of triazines I (>100) acting on purified DHFR from L1210 leukemia cells (L1210/R71 ) with the inhibition of growth of L1210 cells sensitive to MTX (L1210/0) and L1210 cells resistant to MTX (L1210/ R71). MATERIALS AND METHODS Chemicals. The triazines used in this study have been synthesized previously (13). Dihydrofolic acid (90%) and /3-NADPH (95-97%) were obtained from Sigma Chemical Company (St. Louis, MO). MTX was a generous gift from the National Cancer Institute, as were Bakers Antifols I and II. Enzymes. DHFR from L1210/R71 cells was utilized for the enzyme CANCER RESEARCH VOL. 46 FEBRUARY 1986 744 Research. on January 20, 2022. © 1986 American Association for Cancer cancerres.aacrjournals.org Downloaded from