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

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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.ÌFirst 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 (

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RESULTS AND DISCUSSION after the tumor weight had been subtracted. The The first experiment was designed to test figures were obtained by means of the following whether the effect of the malononitriles, if any, expression : was due to the release of cyanide or of some of the A (change in body wt.) most probable intermediates of their catabolism on ( [final wt. —tumorwt. ] —initialbody wt.) the basis of their relative toxicity. For this test number of animals analyzable aqueous solutions of sodium cyanide, malononi- trile, sodium thiocyanate, acetonitrile, cyanaceta- This was abbreviated to read (Jw —tw)—iw mide, malonamide, and malonic acid were injected A Wt. = into both LCS-A- and C3H-S-bearing mice. No N effect on the tumor growth was observed. However, we have found that the LCS tumor Our finding regarding cyanide itself agrees with shows very irregular takes and that very often, Bullock's (3) observation on the growth of the even after it has taken, some tumors of the control Ehrlich sarcoma, on which it also had no effect. group showed regression during the later course of Compounds of groups 1, 2, and 4 showed no effect the experiments. Furthermore, strain A mice, in TABLE2 EFFECTOFETHOXYMETHYLENEMALONONITRILEONTUMORGROWTHDays

between Days Mean tumorTumorLCS- Daily t transplant of 11,- Eip. a wt. gres- Mor BO. gm)400400900400800600uf ment10141009ISRegreswt.(gm.)0.514 (gm.) .-¡1,111*tality l +0.6 1 ControlLCSControlLCSControlS3710(7)15(15) 0.9551.558 0.0.167630 0.0670.160 +0.2 8 8 +0.7 1 15(9)20(19) 3.5680.800 1.0.ISO425 0.2920.098 +0.8 0 4 +0.3 0 20(20)10(6) 1.0561. 0.0.503190 0.1120.077 +0.5 0 t -0.1 4 Control6C34-EDControlCSH-S10(6)10(6) 11640831.008 0.0.165168 0.0680.068 + 1.4 9 +3.3 4 10(6)13(9) 1.147g 001f220.114.972 0.0460.323 +4.3 4 10 +3.2 2 ControlNo.animals*10(7)13(12)dose(mg/100 .147sions4.834 .200included 0.346analysis.2(D)0.0.0.0.0.0106333146103082.473+3.2 1 animals analyzed statistically are inandtreatment688668parentheses.treatare not0. in the ;2nitrile and could not be affected ministered in freshly prepared aqueous solution. any further. When the compound was given in a daily dose of We have also attempted, in view of the relative 50 ¿ig.,asin experiment 4, the effect was not too ly nontoxic character of the compound, to inject it appreciable. A slight loss of body weight was alternately with 8-azaguanine into strain A mice noticeable only in experiment 2, but, in general, bearing the LCS tumor. This combined treatment, the administration did not influence the body when statistically analyzed, did not show any weight, nor had it any effect on the mortality rate. greater effect on the retardation of growth of the The average loss of body weight was computed LCS carcinoma than did 8-azaguanine itself.

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As a consequence of the above-mentioned diffi haps, have possessed growth-retarding effect on culties, C3H and C57 mice have been used almost other tumors. exclusively in the rest of our experiments. The results of substitutions on the benzene ring Since, as Table 1 shows, we did not find any of the aromatic malononitriles were extremely in other nitrile of the aliphatic series to possess any teresting. Although the ortho-substituted malono growth-retarding property, we turned our atten nitriles had no effect on the tumor growth, they tion to the aromatic and heterocyclic substituted were found to be powerful irritants to skin and malononitriles. These malononitriles were screened mucous membranes. Meta-substitution did not TABLE3 RETARDINGEFFECTOFP-NITROBENZALMALONONITHELF,ONTUMORGROWTH

between trans plantDaysofand tumorwt.(gm.)0.1720.8261.0141.990*0.2660.7200.8001.0672(M)0.0840 Exp.DO.4!)515!55550Õ526164626854675957TumorLCSControlS37Control6C3H-EDControlMyeloidLeukemiaControlCSH-SControlCSH-SControlCSH-SControlCSH-SControlCSH-SControlCSH-SControlCSH-SControlCSH-SControlEdose(mg/100 treat- treat Mor gm)1.800.5ml. ment6741757827i24711ment21141511111414142012211«812Mean sions*tality34

S.O.1.500.05ml. 2550.2800.3452(D)0.2670.444No 11062

S.O.1.000.05ml. survivalNoeffect on the tttt2 P.G.0.800.05ml.

P.G.1.750.05ml. survival0.1732.1001.5303.2801effect on the

4010.5260.2800.8810.3990.3930.8380.3870.2920.424AWt.+4.2+5.4-4.4-5.2-0.5-0.2+2.2+1.7+0.20+0.24-s.o-05+0.7+1.4+631 S.O.1 000.05 551 S.O.0.800.1 9003.0002.1003.3202.4503.1801 1266115 ml.S.O.0.800.2ml. 1.8-0.8-0.8— S.O.0.80§0.2ml.

S.O.4.000 .6303 1.9+2.0— 1344 4 ml.S.O.+0.5ml.S2O0.800.05ml. 3001.6002.5772.7004.6001.8003.0000.9001.7700.1291.3900.9982.4000.9400.8700.8251.2300.7251.5000.6701.2100.7902.4000.5801.3700.8951.1300.6650.7550.0530.3980.2660.4540.2200.2050.2120.3170.1870.3430.2520.3020.4300.7200.1630.3520.2100.2030.3320.2830

1.5-0.6-4.2-3.3-5.8-5.8-2.4-1.3Re-gres-40857ell85 P.G.0.800.05ml.

P.G.0.400.1 0771-C57ControlE ml.S.O.0.600.05ml. 0771-C57ControlNo.animals*13(10)13(8)15(7)15(12)2020151515(7)15(12)20(14)20(15)20(18)20(18)20(14)20(15)15(15)20(19)25(7)20(16)11(3)11(11)20(12)20(15)20(18)33(33)12(4)12(7)Daily P.G.Days * No. of animals analyzed statistically are in parentheses. Regressions are not included. t Run until all controls died. §Injected every other day. ! Young animals. ¡IDiedthe day exp. terminated.

on C3H-S-bearing mice, as this tumor showed ex lend any particular biological effect to the com cellent takes and is known to be among the least pounds. Para-substitution of the aromatic malono resistant to chemotherapeutic agents. Most of the nitriles introduced none of the irritating proper malononitriles had no effect on the growth of the ties of ortho-substitution. In their action on the re C3H-S tumor, so they were not expected to have tardation of tumor growth, however, they ranged any effect on other tumors; and also, because of from none to a significant retarding effect. One the necessity of economy of effort, they were not might express the effect of para-substitution some further screened on other tumors. While this ap what loosely in the following order of magnitude : proach seemed to be the most practical, the possi bility exists that some of the nitriles might, per NO2 > HO > Cl > CH3O > CH,.

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It was not possible to ascertain any such approxi nyliden-malononitrile(5-NTMN)caused a retarda mate order of activation with di- or tri-substitu tion of tumor growth. It is to be noted that the 5-ni- tions on the benzene ring. tro substitution on the above heterocyclic rings is Table 3 summarizes the experiments with p-ni- indeed essentially equivalent to the para position with respect to the malononitrile. Our results with trobenzalmalononitrile (PNBMN) (N02 5-NFMN are summarized in Table 4. The results on 447 animals are totaled and analyzed in Table CH:C[CN]2) carried out after it was found in ex 4. The mortality of the treated animals reached periments 49 and 50 that this compound slowed 59.5 per cent, while that of the controls was 36.3 down the growth of the carcinoma. per cent. The mortality was high, even though in the When the volume measurements are plotted treated animals there was no loss of body weight. against time, they appeared to indicate a trend This was attributed to the fact that, in experiment which implied that the tumor growth was slowed 50, young animals were used. In the experiments down but the tumors became somewhat resistant that followed, the daily dose of the nitrile was re to the treatment. duced, and older animals with an average initial The rate of growth (f) can be calculated from body weight of 25 gm. were employed. The results the volume (V) according to the equation were encouraging with respect to the retardation of the tumor growth, the body weight changes, and 3"\-——= / y r(t —/o).which can be arrived at from the mortality, in contrast to those in the control the two equations d = r(t - t0) and V = 0.523d3 animals. All attempts failed in an effort to increase the effectiveness of the compound either by change described by Schrek (15), when d is the mean di of the vehicle from sesame oil to asymmetrical- ameter, t is the period of treatment, and /0 the propylene glycol or by administering higher doses latent period. of the nitrile. Administration in propylene glycol Plots of the measured volumes of the tumors increased the mortality rate and made use of against time indicate the changing trends in the smaller doses of the compound imperative at the rates of tumor growth with the progress of the expense of its effectiveness. treatment plotted. Analysis of the rate of growth When PNBMN was administered every other of the animals treated with 5-NFMN and of the day, as in experiment 62, no growth-retarding ef controls gave r values of 0.78 and 1.23 mm., re fect was observed. In experiment 68 (Table 3) high spectively, at the termination of the experiment. doses of PNBMN were given with 4 times the The mean diameter, d, was between 0.3 and 0.4 equivalent doses of sodium thiosulfate. It was ex cm. at the time of the beginning of treatment for pected that, as in the nitrile detoxications de each group. scribed by Hunt (11), the effect of cyanide, if any, As an example of the growth retardation that is in causing the high mortality could be reduced by obtained, the tumors of experiment 72 have been the administration of sodium thiosulfate. How photographed, and the difference in size is shown ever, it did not give any protection, and, on the in Figure 1 between the controls (1) and the contrary, the mortality rate was increased. 5-NFMN-treated (2). Illustrative and more sig PNBMN had no effect on either the 6C3H-ED nificant, perhaps, is an analysis of the distribution (Gardner lymphosarcoma) or on the myeloid leu of the tumor weights of the treated animals versus kemia C1498 (Bar Harbor). Its effect on E 0771- the controls. In Chart 1 there is plotted the per C57 is questionable. In Table 3 it is shown that, centage distribution of the size of 308 individual out of a total of 495 animals, the mortality was C3H-S tumors; 58 from 5-NFMN-treated, 90 42.8 per cent in the treated animals against 31.5 from PNBMN-treated, and 160 from control per cent for the controls. None of the other aro animals. matic malononitriles showed nearly so strong an At first glance one would be inclined to ascribe effect as PNBMN. the effect of the malononitriles to their nitrile We were led to believe that the orientation of group or to the action of cyanide resulting there the nitrile group para to the malononitrile might from. However, the effect of these compounds on be a requirement for its action. According the tumors can hardly be attributed to cyanide re ly, the investigation was extended to the oxy- lease, because the injection of equivalent amounts and thioheterocyclic malononitriles, and it was of cyanide had no effect on the tumor growth. found that 5-nitrofuranalmalononitrile (5-NFMN) Favorable to this deduction is the observation that no appreciable thiocyanate appears in the urine N02- -CH:C[CN]2) and also 5-nitro-2-the- after 5-NFMN and PNBMN treatment, while a

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1952 American Association for Cancer Research. 570 Cancer Research considerable amount of thiocyanate appears after not the causative factor, as 90 per cent of the the administration of malononitrile or of other malononitriles tested exhibited no effect on tumor substituted benzalmalononitriles, such as the growth. benzalmalononitrile itself. Furthemore, the tox- We believe that one of the most important fea icity of malononitriles or of nitriles that yield a tures of modern chemotherapeutic research on high percentage of thiocyanate can be counteract cancer ought to be the correlation of the growth- ed by thiosulfate, whereas the PNBMN and 5- retarding effect of a compound with its structural NFMN-treated animals responded very poorly, if make-up and the establishment of its interplay on at all, to thiosulfate administration. the chemistry of cellular processes. It was such a The nitrile group itself in these compounds is consideration that led to the systematic, although TABLE4 RETARDINGEFFECTOFS-NITROFURFUHALMALONONITRILEONTUMORGROWTH

trans plantandtreat

Exp.no.6172«4«75575896968TumorCSH-SControlCSH-SControlC3H-SControlCSH-SControlMyeloidLeukemiaControl6CSH-EDControlEdose(mg/100 Wt.(gm.)-3.0+1.4-2.5+1.3-2.40.0-3.3+0.1-0.4+Mor gm)0.600.1 ment618312722Daysoftreatment14141214112581421Meantumorwt.(gm.;0.8903.0000.9003.2002.2393.3200.9801.650a0.5380.8700.4700.9650.5651.2300.3520.447Nosions*tality9212S«61

ml.S.O.0.600.1

ml.S.O.0.200.1

ml.S.O.0.200.05 81 ml.P.G.0.300.05 0ttt46Î2 theNoeffect on ml.P.G.0.300.05ml. 10—

effect2.5203.0001.5002.2201.5772.220onthe1.0901.1300.5200.4950.6200.9502(M)0.1380.2050.1130.1930.151O.S170.1050.120survivalsurvival0.2710.2030.1730.1360.2360.2372(D)0.2490.2230.3470.1600.3380.3610.334A1.5+2.6-3.1-5.8-1.1-0.4-1.9-2.7Re-gres- P.G.0.400.1 0771-C57ControlE ml.S.O.O.S20.1 0771-C57ControlE 973 ml.S.O.0.320.1 0771-C57ControlNo.animals*20(15)20(18)30(18)30(25)20(14)20(15)20(11)15(14)15Iff666620(16)S320(9)80(16)19(7)30(16)Daily96 ml. S.O.Daysbetween 8 * No. of animals analyzed statistically are in parentheses. Regressions are not included in the analysis. t Run until all controls died. } Died at the termination day of the experiment.

unsuccessful, exploration of the effect of substitu tion on the malononitriles. In the three most active compounds, substitution by the nitro group in the para position was established as being desirable. The effect, however, was not due solely to the para nitro position, as none of the parent aldehydes of the reactive malononitriles exhibited any retard ing effect on tumor growth, although it is known from the work of Boyland and Mawson (2) that certain aldehydes do inhibit the growth of Crocker Sarcoma 180 in mice. The difference in response is no doubt due to the dissimilarity of the tumors. Furthermore, if the malononitrile moiety of the Tumor Weight in gm. molecule was replaced by thiosemicarbazide, the CHART1.—The effect of M 5-NFMN and QU PNBMN growth-retarding action was, interestingly enough, on the percentage distribution of tumor weights in CSH-S bear replaced by that of a growth-accelerating effect. ing mice. (D Control.) That the specificity of action seems to be related

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1952 American Association for Cancer Research. GALet al.—MalononitrüeEffecton Tumor Growth 571 uniquely to a set structural pattern is shown in in the tables attest, many of our experimental ob Table 5. servations are statistically significant, some of The great toxicity of the compounds at their them are probably significant, and, again, many most effective levels of administration and the fre are not significant. It is now clear to us that any quent loss of weight by the animals, as a conse retardation of growth caused by a chemical that is quence, raise the question whether the growth re below 50 per cent of the control growth, or is above tardation is not merely a result of the consequences 50 per cent, but is statistically attributable to of anorexia and malnutrition. If this is true, then, chance, should be considered negative. for all intents and purposes, the growth retarda- In this sense we may state that PNBMN,

TABLE5 SUMMARYOFTHEEFFECTOFTHEACTIVEMALONONITRILESANDTHEIRCONGENERSONTRANSPLANTEDTUMORS TDMORS* C H8S DAILYDOSE(MO/100 myeloid COIIFOUND OM)0.161.000.801.801.000.80!(no/100 GM) VEHICLE* I.CS-A S37-A C3H-S 8CSH-ED leukemia E 0771 Malononitrile Benzalmalononitrile p-Nitrobenzalmalononitrile + p-Nitrobenzaldehyde p-Nitrobenzalthiosemicarbazone 0.804.000.800.800.321.20uracin) X Furanalmalononitrile 5-Nitrofuranal ± 5-Nitrofurfurylcyanoacetic acid 5-Nitrofuranalsemicarbazone (Furacin)2.00e 5-Nitrofuranalthiosemicarbazone 0.400.600.300.800.40VEHICLE*waterS.O.ÌP.GJs.o.iP.GJP.G.P.G.S.O.IP.GJs.o.!P.GJP.G.P.G.P.G.S.O.IP.GJP.G.P.G.•X 5-NitrofuranalmaIononitrile 2-Thenylidenmalononitrile 5-Nitrothenylidenmalononitrile ++

* Designations represent values of >2.0 —±;•-•»»A» >2.5 "•+î>3.0 » ++. 2 (D) Growth promoting effect »=X;no effect =* —. t S.O. ™sesame oil; P.G. •»asym.propylene glycol. tion of the tumors should tend to become a func TABLE 6 tion of the loss of weight of the experimental ani mals. This did not appear to be the case, as Table THE EFFECT OF ADMINISTRATIONOF 6 demonstrates. MALONONITRILESONBODYWEIGHT OFANIMALS It is apparent from Table 6 that several aro matic malononitriles, such as the p-acetylamino- cally ana- Av. and the 2,4-dinitrobenzal derivatives, which were lyzable initial Abody E*p. no. of body wt. wt. wt. administered in far higher doses than either (gm.)TumorGroups animals* PNBMN or 5-NFMN, did not affect the tumor No.61M0051Statisti (gm.)-2,-0+0-3+(gm.)22.1.03s12220115,'i409089.00.0080.705a.10.17.99.01 C3H-S:p-acetylaminobenzal2,4-nitrobenzalp-nitrobenzal5-nitrofuranalControlTumor growth, yet, nevertheless, caused a loss of body weight. In experiments where the food intake was controlled, the weight changes of the treated ani mals did not show any significant difference from 1-5—5-5-3+0+0-4-Õ917.0,4.8.8.1.1.80.84.4.8Av.tumor that of the controls, when totaled for the entire 0771:Controlp-nitrobenzalp-acetylaminobenzal5-nitrofuranalTumorC57-E duration of the treatment. We cannot sufficiently emphasize that, although some of the malononitriles slowed down the rate of growth, none of them produced complete arrest of C3H-S:Controlp-nitrobenzalTumor growth nor regression of the tumors. Statistical analysis of the material helped to establish wheth S37-A:Controlp-nitrobenzal101918Iff1888181918127ie723.626.8488ti,2ff24¿5252017899»17289857.0.81.8Av. er the differences between the mean tumor weights of the treated and of the control animals belonged to the same population or not. As the £(/))values * Only animala that survived for duration of the experiment.

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5-NFMN, and 5-NTMN have a statistically sig REFERENCES nificant growth-retarding effect on C3H-S, a prob 1. BADDILEY,D.;LYTHGOE,B.;and TODD,A. R. Experiments able effect on E 0771-C57, and none on S 37, on the Synthesis of Furine Nucleotides. Part II. A New and myeloid leukemia C1498, and 6C3H-ED. This Convenient Synthesis of Adenine. J. Chem. Soc., pp. 386— 87, 1943. latter tumor was also used to test the effect of the 2. BOYLAND,E., and MAWSON,E. H. Experiments on the compounds on the survival time of the animals. Chemotherapy of Cancer. The Effect of Aldehydes and None of the compounds showed a significant effect Glucosides. Biochem. J., 32:1982-87, 1938. on the survival of the animals. We would like to 3. BULLOCK,F. D. The Influence of Sodium Cyanide upon mention that we have also tested the 5-nitrofur- Ehrlich Sarcoma. Proc. Soc. Exper. Biol. & Med., 14:122, analsemicarbazone (Furacin) and could not con 1916. 4. CORSON,B. B., and STOUGHTON,R.W. Reactions of a, firm the reported beneficial effect (5) on the sur 0-Unsaturated Nitriles. J. Am. Chem. Soc., 60:2825-37, vival of 6C3H-ED bearing mice. We have also at 1928. tempted combined treatments employing col- 5. FRIEDGOOD,C.E., and GEBEN,M. N. The Effect of Nitro- chicine, podophyllin, aminopterin, and desoxy- furazone on Growth of Fibrosarcoma in Mice. Cancer Re search, 10:613-15, 1950. pyridoxine in combination with the effective 6. GAL,E. M.; EZRA,H.; FUNG,F. H.; and GREENBERG,D. malononitriles. We hoped for a reciprocal enhanc M. The Effect of Substituted Malononitriles on the Growth ing effect, but the results showed that the com of Transplant Tumors in Mice. Cancer Research, 11:249- bined effect on the tumor growth was not additive. 50, 1951. It is hoped that the systematic studies on the 7. GAL, E. M., and GREENBERG,D. M. Growth-Retarding relation of structure of the chemical agent to the Effect of Substituted Malononitriles on Transplant Tu growth retardation of tumors now in progress in mors in Mice. J. Am. Chem. Soc., 73:502, 1951. 8. GREENBERG,D.M.; IRISH,A. J.; and GAL,E. M. Effect of our laboratory may shed some interesting informa Metabolite Analogs on Growth of Transplantable Tumors. tion on this difficult and time-consuming problem Cancer Research, 10:221-22, 1950. of cancer chemotherapy. 9. GREENBEHG,D.M., and SCHULMAN,M.P. Application of "Metabolite Antagonism" to Cancer Research. Science, SUMMARY 106:271-72, 1947. 1. Systematic studies on the different substitut 10. HEYMANS,J.F., and MASOIN,P. Étudephysiologiquesur ed malononitriles showed that ethoxymethylene- les dinitriles normaux. Arch, de Pharmacodynamie et de Thérap.,iii:77-172, 1897. malononitrile of the aliphatic series had some ef 11. HUNT,R. Zur Kenntnis der Toxikologie einiger Nitrile und fect on the retardation of growth of carcinoma. deren Antidote. Arch. Internat, de Pharmacodynamie, p-Nitrobenzal-, 5-nitrofuranal-, and 5-nitro-2- 12:447-79, 1904. thenylidenmalononitriles retarded the growth of 12. HYDEN,H., and HAHTELICS,H. Stimulation of the Nu- the transplantable carcinomas, C3H-S and E 0771 cleoprotein Production in the Nerve Cells by Malononitrile and Its Effect on Psychic Functions in Mental Disorders. to an even greater degree. Arch. Psych. & Neurol. (Suppl.), XLVIII:1-117, 1948. 2. This effect is attributable to the structural 18. KIDDEH,G. W.; DEWEY,V. C.; and PABKS,R. E., JR. properties of the particular compounds rather Furine Metabolism in Tetrahymena and Its Relation to than to the release of cyanide at different rates. Malignant Cells in Mice. Science, 109:511-14, 1949. Neither sodium cyanide nor malononitrile itself 14. MOWHY,D. T. The Knoevenagel Condensation of Acyl- alkylketones with Malononitrile. J. Am. Chem. Soc., showed any growth retardation on the tumors 67:1050-51, 1945. tested. 15. SCHREK,R. A Quantitative Study of the Growth of the 3. The significance of the observations was sta Walker Rat Tumor and the Flexner-Jobling Rat Carcino tistically appraised and the results discussed. ma. Am. J. Cancer, 24:807-22, 1935.

FIG. 1.—Photographshowing average size of tumors (C3H-S) in controls (1) contrasted to 5-nitrofuranalmalononitrile treated (2) animals (Experiment No. 72).

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1952 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1952 American Association for Cancer Research. Studies on the Biological Action of Malononitriles: I. The Effect of Substituted Malononitriles on the Growth of Transplanted Tumors in Mice

Emery M. Gal, Fung-Haan Fung, David M. Greenberg, et al.

Cancer Res 1952;12:565-572.

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