Studies on the Biological Action of Malononitriles* I. The Effect of Substituted Malononitriles on the Growth of Transplanted Tumors in Mice EMERYM. GAL,| FUNG-ÃœAANFUNG,ANDDAVIDM. GREENBERG ASSISTEDBYHARRIETEZRAANDSHERBURNEF.COOK,JB. (Department of Biochemistry, School of Medicine, Unirersity of California, Berkeley, Calif.') This laboratory has been engaged for a number MATERIALS of years on a program of cancer chemotherapy re Malononitrile, by virtue of its active méthylènegroup, search guided mainly by the concept of metabolite readily condenses with carbonyl compounds to yield dinitriles. The condensation products show different degrees of saturation antagonism (9) in the selection of compounds to be and can be divided into the following groups: tested for chemotherapeutic activity. The impor 1. R-CH,CH(CN), tance attributed to the components of the nucleic 2. R-CH (CH[CN]S), acids in the genesis of neoplasms and the control of 3. R-CH:C(CN)j cellular functions has, with some measure of suc R.-. cess, oriented extensive investigations on experi 4. R2-C:C(CN)S 5. R-N:N-CH(CN)2 mental tumor chemotherapy. An outstanding ex ample is the work of Kidder and his associates (13) With a few exceptions the compounds were prepared in our on the growth-inhibiting properties of the purine laboratory according to known procedures (1, 4, 14). The car analog, 8-azaguanine. bonyl compounds used in the syntheses were, for the most part, purchased from Distillation Products Industries. The prepared Our efforts have been considerably influenced compounds were checked for purity and the elementary compo by the work of Hyden and Hartelius (12), who re sition of those which were not described in the literature deter ported that administration of malononitrile mined by micro-analysis. The choice of substitution was sys (CH2[CN]s) specifically increased the polynucleo- tematically determined by steric relations, as well as by the re semblance to the active groups in some of the known experi tide and protein content of the nerve cells, both in mental antitumorigenic agents, as, for example, the methoxy experimental animals and human beings. This ef groups of colchicine or podophyllin. fect was described as specific for the neurons. Al Because of the instability of the compounds in alkaline or in though this seeming selective action was apparent acid solutions, they were administered intraperitoneally either in sesame oil (S.O.) or in asymmetrical propylene glycol (P.G.) ly limited to nervous tissue, while the contrary —the latter because of its toxicity to mice never exceeded a effect was desired from the point of view of cancer daily dose of 0.05 ml. therapy, the results suggested possibilities for un To determine the toxicity of the compounds, solutions were dertaking a program for the synthesis of test com injected intraperitoneally into the mice to establish the mini pounds. It was hoped that the introduction of dif mum lethal concentration. With this as the upper level, three dilutions of each compound were injected daily into two normal ferent substituents into the méthylènegroupof mice, each for 5 days. The final concentration of the compound malononitrile would lead to a more generalized and chosen for therapy trial was one which caused no mortality and desirable effect without critically altering the re no significant change in weight. The toxicity studies did not re ported biological characteristics of the nitriles. veal any influence of either the sex or the strain of the mouse. Certain of the results of this work have been The symptoms of toxicity of the malononitriles were much the published in brief (6-8). A detailed account of the same as those described for the parent compound by Heymans and Masoin (10). work is presented in this series of papers. The In the course of these studies, about 500 strain A, 500 C57, mode of action and the fate of the malononitriles, and over 1,500 C3H mice were used. The number of animals in both in normal and tumor-bearing animals, have a group, whenever possible, was between 10 and 20. The weights of the animals selected before the start of the experi been studied. ment were between 20 and 25 gm., and the sex ratio was 1:1. * This work was aided by grants from the National Cancer At times, young animals were employed in the study, and in Institute, U.S. Public Health Service. each instance this will be specified. The animals received a diet t U.S. Public Health Special Fellow, 1948-50. of Purina Laboratory Chow and water ad libitum. They were kept six to eight in a cage, with the sexes separated. The Received for publication February 12, 1952. weights of the animals were checked every third day and the 565 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1952 American Association for Cancer Research. 566 Cancer Research 1TOXICITY TABLE ContinuedToleranceSubstitutionI— MALONONITRILESToleranceSubstitutionANDBIOLOGICALEFFECTSOK doseof on CH:-group doseof on CHt-group VehicleGroup malononitrile (mg/100 gm) gm)Group malononitrile (mg/100 5:Phenylazo-|| 1:•y-Dicyano-methylbutyrate S.O.p-Dimethylamino-phenylazo-tH 1.6 0 .80 12.0 S.O. •y-Dicyano-ethylbutyrate72•Y-Dicyano-butyronitrile 0 . P.G.P.G.S.O. p-NitrophenyIazo-j|S.O.p-Chlorophenylazo-H 1.80 60Group 0 . S.O.Miscellaneous 1.0 2:Methy t1,3-Dimethyl-cyclobutane Iene-bis- 2.0 S.O.tetranitrile 2,2,4,4- 4.0 Ethylidene-bis-2Heptylidene-bis- 8 . S.O.S.O.HS0H2OH2OH2OH20S.O.S.O.P.G.P.G.P.G.S.O.P.G.S.O. 0Group 4 . 2-Cyano-l,4-endomethyleneP.G.hexeneí cyclo- 0.4 3:Ethoxymethylene-* (¿4,5)Fumaronitrile§ 0.60Aminomethylene- S.O.* 3.0 20.0Glyceral-t 40.0Citral-t inhibition.tInduced tumor-growth 40.0Tiglal-f laboratory.ÃŒFirst synthesized in our 40.0Furanal- membranes.§Irritant of skin and mucous 05-Nitrofuranal-*t 8 . ofDrs. Obtained from Monsanto Chemical Co., through the courtesy 0.800.402-Thenyliden-§ Schlesinger.||F. C. Meyer and A. H. effect.P.G.Slight tumor growth-promoting 0.805-Nitro-2-thenyliden-§ glycol.S.O.=»Asym.propylcne 0.40Benzal-*î oil.diameters = Sesame 1.00.80o-Hydroxy-benzal- of the tumors every fifth day. No caliper measure 6 .0 ments were taken on the Gardner lymphosarcoma-bearing ani m-Hydroxy-benzal- 4 .0 S.O. mals. At the end of the experiment, which usually took 2-3 p-Hydroxy-benzal- *{ 4.0 S.O. 1.62,4-Dihydroxy-benzal- P.GP.G.P.G.P.G.P.G.P.G.P.G.weeks, the animals were sacrificed, the tumors carefully re 0.803,5-Dihydroxy-benzal- recorded.Somemoved, weighed, and photographed, and the results 0o-Methylbenzal-t 1. of the tumors were preserved for histológica!examina 0.80m-Methylbenzal- tion.16CSH-ED The mouse tumors employed were Sarcoma 37, 1.0p-Methylbenzal- C57-E0771(lymphosarcoma), the carcinomas LCS-A, CSH-S,1 1.0o-Chlorobenzal-í leukemia).Theand C57-C1498 (Bar Harbor myeloid 0.40 p-Chlorobenzal-* 0.40 transplantation of the tumors was carried out by inject P.G. ing, by means of a trocar, uniform small pieces of non-necrotic 2-Hydroxy-5-chlorobenzaI- 1.0 S.O. 2,4-Dichloro-benzal-0.802,6-Dichloro-benzal- P.G.P.G.S.O.S.O.s.o.P.G.P.G.tumor tissue, obtained under aseptic conditions, into the axil 0.80o-Nitrobenzal- larygrownfor region of one side. In transplanting, donor tumors 1.0m-Nitrobenzal-} eachtumorthe same time period were used, arid the pieces from 2p-Nitrobenzal-* 1. experimentalgroups.were evenly distributed among each of the 1.80.82,4-Dinitro-benzal- take.AfterTwo to 6 days were allowed for the tumors to wereeliminated,6 days, all the animals without palpable tumors 1.6 and those with tumors made up the final groups for 2,6-Dinitro-benzal- 1.6 P.G. the injection of the compounds. In the grouping of these ani 2-Chloro-5-nitrobenzaI-î 1.0 P.G. p-Methoxy-benzal- 0 .8 P.G. mals, the range of size of the tumors and the weight of the ani 2,4-Dimethoxy-benzal-0.82,5-Dimethoxy-benzal- P.G.P.G.P.G.S.OP.G.S.O.s.o.Cthefoodmals were kept as uniform as feasible. In some experiments 03,4-Dimethoxy-benzal- 1. checked.Statisticalintake was also 23,4,5-Trihydroxy-benzal-t 1. thatsurvived analysis was made only on those animals 4.81.63,4,5-Trimethoxy-benzal- ofthe or died within a day or two before the termination oftheexperiment. The standard deviation (<r),standard error f 8.0 mean (S[Mj) and standard error of difference of the means 3-Methoxy-4-hydroxybenzal-0Piperonal-*§ 2 . 0.4' f\s.o. (S[D)) were calculated. The calculation of the <values for cer tainthesignificance curves were at times a necessity in order to appraise 2p-Acetylamino-benzal-i :M 111ii i,¡I 1 . s.o.s.o. 0 .60 of the difference. p-Dimethylamino-benzal-80p-Diethylamino-benzal- 0 . s.o.s.o.P.G.P.G.S.O.P.G.Because of the great number of the compounds tested de 21 1. tailedcompoundswhich experimental data will be restricted to those 729-Anthral--Ethoxy-2-naphthal- 0 . growth.Theshowed a marked effect on the tumor 0.80Chinchoninal- list of compounds tested, the tolerated dosage, and cer 00Group 1. tain1.1 physiological effects are reported in Table 4:Methyl-benzylidene- 0.80 We gratefully acknowledge our great indebtedness to Dr. Methyl-(p-chlorophenyl)ethyl¡dene- 0 .72 P.G.