Anti-leukemic Assays on Certain Pyrimidines, Purines, Benzimidazolcs, and Related Compounds* HOWARDE. SKIPPER,PH.D., LEONARDL. BENNETT,JR., PH.D., PRADISTHC. EDWARDS,PH.D., CARL E. BRYAN,PH.D., OLIVIAS. HUTCHISON,B.S., JUANITAB. CHAPMAN,A.B., ANDMARTELIABELL, B.S. (From the Organic and Biochemistry Division, Southern Research institute, Birmingham, Alabama) Much of the fundamental biochemical effort in inhibitors in the nucleic acid metabolic system. the cancer field has been directed toward gaining Such findings as are mentioned above not only a better understanding of the role of nucleic acids offer compounds worthy of testing against various in the normal and neoplastic cell. The rationale be types of human neoplasia but also aid in our under hind such studies may be partially summarized in standing of the differences in normal and neoplas the words of Spiegelman and Kamen (l) : "... it is tic cells. supposed that genes control ultimately all cellular Woolley (4) has reported that benzirnidazole is functions, including protein synthesis. From the antagonistic to amino-purines in certain micro close association of genes with nucleoproteins, it is organisms and that its growth inhibition can be natural to assume that nucleic acids may be in overcome by adenine or guanine. volved in protein synthesis. Such an assumption The present study involves the screening of a can be supported by the fact that nucleic acids are number of pyrimidines, purines, benzimidazoles, always found associated with self-duplicating units and related compounds against a strain of mouse (e.g., genes, viruses, plastids, transforming prin leukemia known to respond to aminopterin (4-am- cipals of pneumococci and coli)." Since, from a inopteroylglutamic acid) and to 2,6-diamino genetic standpoint, cancer is a mutant strain of purine. cells with one or more genes or systems excessively EXPERIMENTAL promoting (or failing to inhibit) growth, a logical The synthesis of the compounds involved in the point for chemotherapeutic attack might be the present study will be described elsewhere. Com metabolic system utilizing the constituents of pounds Nos. 88, 122, 124, and 82 were commercial which the normal and cancer chromosomes are samples. All others were prepared in this laboratory. largely composed, e.g., nucleic acids and histones. The procedure used for screening compounds If there is reason to believe that the nucleic acid for toxicity and anti-leukemic action has been de metabolism (and in turn purine or pyrimidine scribed in an earlier report (o). In all experiments, metabolism) of the normal and the mutant cancer candidate compounds have been injected inIra- cell differ in certain respects, then a search for peritoneally at the maximum tolerated dose, every compounds which might inhibit that part of the other day, for a total of 10 injections, starting on system peculiar to the cancer cell is a worth-while the second day after leukemic inoculation. Results endeavor. are reported in terms of average survival times of The recent reports of the anti-leukemic activ groups of ten mice as compared to concurrently ity of 2,6-diaminopurine, an antagonist of pteroyl- run untreated controls and aminopterin-treated glutamic acid and adenine in the metabolism of controls. These data are summarized in Table 1 Lactobacillus casei (2), and the tumor-inhibiting and are further compared in Figure 1. activity of 5-amino-7-hydroxy-l//-r-triazolo [d] pyrimidine, a powerful purine inhibitor (3), have DISCUSSION emphasized the importance of searching for other It is interesting to note that 2,4,.5,(>-tetramino- * This work was supported by grants from the American pyrimidine, which contains the 2,4-diaminopyri- Cancer Society on recommendation of the Committee on mido portion common to most anti-folic acid com Growth of the National Research Council; Mr. Ben May, Mo bile, Alabama; and the Institute-Sponsored Research Fund, pounds, and which is the pyrimidine used in in Southern Research Institute. vitro synthesis of 4-amino-pteroylglutamic acid, Received for publication, November 18, 1949. showed no anti-leukemic activity. Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1950 American Association for Cancer Research. SKIPPER et al.—Anti-leukemia Chemotherapy Screening 167 We have confirmed the finding of the anti- active over a slightly greater dosage range than was leukemic activity of 2,6-diainiuopurine in strain 2,6-diaminopurine. It appears that substitution at Ak 4 mouse leukemia reported by Burchenal, et ai. the 8-position of 3,6-diaminopurine lowers or ne (2). However, in our limited experiments, this gates the activity of this compound with regard lo purine has not been as active as 4-aminopteroyl- anti-leukemic action. The 2,6-di;'inino-8-hydroxy- irlulainic acid. This difference is probably due to and the 2,(i-diamino-8-mercaptopurine, as well as our failure to use the most effective dose of 2,6-di- the 2,6-diamino-8-iodopurine, showed no signifi aminopurine. The Sloan-Kettering group (2) stated cant activity. that 4-amino-NID-methylpteroylglutamic acid was The benzimidazoles, 5-nitrobenzotriazole, in- TABLK1 THERESULTSOFANTI-LEUKEMICASSAYSONCERTAIN PYRIMIDINES,PURINES,BENZIMIDAZOLES,ANDRELATEDCOMPOUNDS Maximum Average Per cent tolerated survival increase S.K.I. dose time in survival 90-day No. Compound Slruclurc (mg/kg)* Way») time survival R.C.I.t 108 2,4,5,6-Tetranìinopyriiiiiclino 97 11 0 -12.7 0/10 -0 «4 sulfite 116 2,ö-Dihydroxy-4,5-diamino- - 4.7 0/10 -0.09 pyrimidine sulfate 117 2,5-Diamino-4,(i-diliydroxy- 78 11 !) - 63 0/10 -0 13 pyrimidine sulfate 118 2-Mercapto-4-amino-(> liy- 125 ni; - 8.7 1/10 -0.17 droxypyrímicline 135 2,6-Diaminopurine sulfate 100 12.« +29 9 0/10 +0 73 125 li » +29 (I 0/10 +0 48 Hj» 149 2,6-Diamino-S-h.vdroxy- 185 99 + 0/10 +0.05 purine sulfate! 150 2,(i-Diamino-8-meroapto- 125 S !» 8.2 0/9 0.20 purinc sulfate 62.5 !l 1 6.0 0/5 O 10 152 2,6-Diamino-8-i<xlopurine 9.5 2.1 0/10 0.05 sulfate !) r, 5.0 0/10 0.08 88 Caffeine 0-2.5 10.5 -16.7 0/10 -0.31 * Maximum tolera tvii dosi', an approximate value. t Relative chemotlier;i|>eutH-index =Per - cent increase —¿T. in life T-TTÕ span of group treated with a given compound—¿ . Per cent inerease in ufe si>itnof aminoptenn-treated group t Impure sample. NOTE: Animals surviving 00 day«not iwinded in nilcnlntions of survival time. Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1950 American Association for Cancer Research. TABLE l—Continuai Maximum Average Per cent tolerated survival increase S.R.I. dose time in survival 90-day No. Compound Structure (mg/kg)* (days) time survival R.C.I.t 122 Benzimidazole 11.8 + 7.3 0/10 +0 12 98 2-Chloromethylbenzimidazo!e 4* 11.2 -11.8 1/10 -0.23 154 5-Nitrobenzotriazolc 02.5 97 - 3.0 0/10 -0.05 124 Indole 125 10.3 - 4.0 0/10 -0.09 129 Benzoxazole 3.9 9.3 - 4.1 0/10 -0 10 130 Benzothiazole 31.3 10.9 + ().( 1/10 +0.02 113 2-Methyl-4-hydroxyquinazo- 250 10.4 -1S.1 1/10 -0.3(5 line 82 2,3,5-Triphenyltetrazolium 7.8 11.3 -13.7 1/9 -0.40 chloride 114 4-Aminopteroylglntaniir CHCOOH*** 20.419.117.516.113.719.116.0with acid (aminopterin) Hjx-y¡mates!2COOKï * Maximum tolerated dose, an approximatevalue.Per value. cent increa»1in life span of group treated0.23 a given compound+«5.8+50.4+59.1+49.1+41.2+59.2+60.01/100/100/102/100/90/100/10 t HHativf chemotherapeutic index Per cent increase m life span of aimnnptmn-trented group NOTE: Animali surviving 90 days not included in calculations of survival time. Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1950 American Association for Cancer Research. SKIPPERet al.—Anti-leukemia Chemotherapy Screening 169 dole, benzoxazole, benzol hiazole, 2-methyl-4-hy- 2,3,5-triphenyltetrazolium chloride were screened droxyqiiinazoliiK'. und 2,3,5-triphenyltetrazolium but showed no anti-leukemic activity. chloride were all negative when assayed against transplanted Ak 4 leukemia in mice. REFERENCES SUMMARY 1. SPIEGELMAN,S.,and KAMEN,M. D. Cold Spring Harbor Symposia on Quantitative Biology, Vol. XII, Nucleic Acids A number of compounds of interest as possible and Nucleoproteins, pp. 211-23. Menasha, Wis: George purine or pyrimidine antagonists have been as Banta Publishing Co. 1947. sayed for anti-leukemic action against transplant 2. BuRCHENAL,J.H.;BENDicH,A.;BROwN,G.B.;ELioN,G.B.; ed lymphoid leukemia (Ak 4) in mice. HITCHINGS,G.H.; RROADS,C. P.; and STOCK,C. C. Pre liminary Studies on the Effect of 2,6-Diaminopurine on The pyrimidines studied were uniformly in Transplanted Mouse Leukemia. Cancer, 2:119-20, 1949. active. 3. KIDDER, G. W.; DEWEY, V. ('.; and PARKS, R. E., JR. Of the 2,6-diaminopurine derivatives assayed, Purine Metabolism in Tetrahymena and Its Relation to only the parent compound definitely increased sur Malignant Cells in Mice. Science, 109:511-14, 1949. vival time. Introduction of a hydroxy, mercapto, 4. WOOLLEY,D.W. Some Biological Effects Produced by Ben- or iodo group at the 8-position of 2,(>-diamino- zimiduzole and Their Reversal by Purines. J. Biol. Chem., purine apparently inactivates this anti-leukemic 162:225-32, 1944. molecule. 5. SKIPPER, H. E., and BRYAN-,C. E. Carbamates in the Benzimidazole, 2-chloromethylbenzimida/ole, Chemotherapy of Leukemia. III. The Relationship Be tween Chemical Structure and Anti-leukemic Action of a 5-nitrobenzotriazole, indole, benzoxazole, benzo- Series of I'rethan Derivatives.