A number of 2,4-diaminopyriniidines and related heteroi~yc~lic~ ronipoiiiid~have heen evdiiated :I> iiiliititur> i J f dihydrofolate redrictaie obtained from Trypanosoma cqiripprdcrtn, rhicken liver, and rat liver. %,+I )ianiini)pyrimidine itoelf (at. 10-4 121)was not, effective ab an inhibitor of dihydrofolate redrict ion i n all t,hr ,tudied hiit >-aryl derivatives of 2~4-diamiriopyrirnidiliewere good inhibitors iII)->o = 10Vk t i l 10-6 enzymatic reaction. ~,,l-l)iarnino-.i-beiizylpyrimidiile~ an -dianiiiio-.i-ar\-lc)xypyriIiiidiiies were cc riiore effective as inhihitors of the trypanosoma1 enzyme em than of the mammalian and avia wa, not acative as an inhibitor of the enzyme> studied, r ..\ltholigh 2,4-diamino-6-pheiiyl-s-t~riaxine tfiamino-l,2-dihydro-s-ti.iaziries were potent inhihitors of the reditctase-. 2,4-1jiamino-6,7\va? folind to be approximately tenfold more effective a:: an inhihitor of the three rediicta.-e , L',4-diamino-6,7-dimethylpteridirie; 2-itmino-6,i-dipherlylpteridirie arid ~-amino-6,7-diphei riot effective as inhibitor+ of these enzymes. 2,4!7-Trianliri0-6-arglpteridine~ bearing an ortho sithstituent i n the &:try1 rnoiety were foiirid t o be 10 -1OO-fold more potent a i inhihiton of the rediirt tenis thaii were t be cy,rre.poiiding para-substitilted derivative>.. The 2-~1ni11o-d-h>-drc hiopteriri, x:~iitIrq)hitoir of the t r y ~ ~ n i i i ~ ~ i ~ r t i a l i'oxarithopterin were f o i i n d to tie effective iieithei, :i> < l i t
Iiiitially dihydrofolate redurtase from 7'. cquiperduni was prep:ii,cd exactly as oiitliried in ref 1; in later studies acetone powtler::, prepared from :1-6 x 1U'" trypanosomes, were extracted
with i nil of p l l 7.0 pho.~phatebuffer (0. I J l ) arid the exti':i(,ts mr'e centrifuged for 10" a t 100,OOOg and 4' in ii 13eckrii:in .\Iode1 L 1,efrigerated wntrifLtge; the resulting supernatant so111tiona served as the soiirce of enzyme. This modifiedprocedirre for obtaining tr>-pnnosomal w e d becaure of the high loss uf enzymr itc during t,he dialyhis itep employed in (nir origii roceditre. It yielded an enzynie preparation m-hich did 1 tly, with rerpevt to sensitivity t,o varioiis inhibitors, from the enzyme preparatioik made by the previous procedure. In thi.: connect,ion, it i h m r t h pointing oiit that Schrecker arid Hitennekens5 found 1 ti:i1 dihpdrofolate rediic,iii.;e in crlide extracts of chicken liver aiitl this enzyme after partid purificntiori showed similar nensitivit). to the diamiiiopteridirie inhibitor, aminopterin. Acetone powd e r , ~were ~ prepared from swtioii; of chiclieii liver, the powder> were extracted as fnr the trypniiohunial preparation, the estracts were dialyzed overnight against 100 vol of p H 5 . 5 biiffer 1 0.U1 .lf), and the dialyzed rn:tmial was used for erizymat.ic :issays. Acetone powders were prepared also from i;ection.: of rat, liver and were estracted a n d dialyzed as described for t h r h chicken liver preparations. .-I similar preparation' of (dihydro i folat,e reductase, obtained hy high-speed ceritrifugation of a rat liver homogenate, W:LS iisrti b)- Hampshire and her colleagiio~~ for evaliiating the inhibitor>-poteric'y of a series of 2,4-diamino-5arylazopyrimidiiies . Iteduced riicotirinmide-ndeniii~, dinucleotide p1ioaph:cte (SADPH) TVXX purchased from P. I,. Biochemicals, Iiic., l l i l waiikee, Wis. : folic acid war piirchased from Calbiochem, Lo. .hgeles, Calif. Dihydrofolate x a s prepared by the method of Futtermang and alw by t,he procedure of Friedkin and hii cdleagues.'o S o differences were observed between the rates of enzymatic rednctiori of d drofolate prepared by the different methods. Reference> t o ithehes of pteridines carried out i i i this laboratory are given in the tables.
( 1 ) A preliminary account of a portion of this work mas presented a t thr. I'all Meeting of t h e American Society for Pharmacology a n d Experimental Therapeutics, Washington. D. C., .\up 1967: J. .I. lIeCormack and J. , J . Jaffe. I'hnrmacologist, 9 , 193 (1468). ( 2 ) J. .J. Jaffe and J. J. McCormack, M o L . f'hurmacol., 3, 359 (1967). ( 3 ) .J. J. Rurchall and G . H. Hitchings, i 6 i d . , 1, 126 (1965). 14) See. for example, (a) 13. R. Baker and U-T. Ho, J . I'hurm. Sci., 63, 1 1 3 i (1964); (b) 13. R. Baker and B-T. Ho, ? b i d l . . 66, 470 (1966); (c) I?. I?. I3akP.r and G. J. Lonrrnr. .I. .?fed. C h e m . , 10, 1113 (1967): Id) t i . R. Baker. t t , u / , , 11, 483 (1968).
t.5) .j. \V, Si,liiecker n n i i 1.. \ I . Ihirnnekens, Biochem. Pliarmacul., 13, i : i I (l'J64). 16) 8. F . Zakrzewski, ./. Hie/. C l i r m . . 256, 1 7 i 6 (1960). ( 7 ) 9. E'. Zakrsenski a n d i'. i. Nirhol, .I. Phnrmueol. B s p l l . Therap.. 157, 162 (1962). (81 J. Haminhire. 1'. IIeljlwin, \ . rll. Triggle, D. J. Triggle, and S. Vi cstablishecl t h:it seemingly inial1 ch:inge\ i t i chemlcal structure can produce niarbed alterations in the abllity of :in agent to inhibit :t particular reductabe. 1T7e have iiiitiated a similar comparative htudy of the relatioiithip between the chemical ttructure of variouq 2,4-di:Imiiiopyrimidines and related heterocyclic byitems, and their ability to inhibit dihydrofolatc reductases from 2'. cyiiipcrrlunz. chickeri liver, and rat liver. It ->IL\ hoped that huch :I htudy would provide information \r-hich might prove useful iii the design of new agelit. for i i w i i i the chemotherapy of trypanosomia.;es > t i i d other protozoal disea
Experimental Section
July 1969
ISHIBITOKS OF DIHTDHOFOLATE REDUCTASE
663
tion to the pyrimidine ring. In this regard it should be noted that o-halophenyl derivatives of 2,4-diaminopyrimidine have been shown to be considerably less effective than the corresponding p-halophenyl derivatives as antimalarial agenkZ0 2,4-Diamino-6-et hyl-5-(4-chlorophenyl)pyrimidine (8, pyrimethamine), a clinically useful antiplasmodial agent, has been found very recentlyz1 to be a highly selective inhibitor (ID50 E 10"O 14) of dihydrofolate reductase isolated from Plasmodium beryhei; this pyrimidine derivative is also a potent inhibitor of dihydrofolate reductase isolated from Erlich ascites carcinoma cells.z2 Pyrimethamine was also found to be an effective inhibitor of dihydrofolate reductase from T . equiperdumz and analogous reductases from other trypanosoma1 species of ilfrican origin.23 As can be seen in Table I, the rat liver enzyme system and the chicken liver system exhibited sensitivities to inhibition by pyrimethamine which were not very different from that of the trypanosomal enzyme. These comparative data are of considerable interest because they indicate Results and Discussion12 that the reductase obtained from the protozoan, T . 2,4-Diaminopyrimidines.-The effects of various eyuiperdum, is more akin to the mammalian and avian derivatives of 2,4-diaminopyrimidine on dihydrofolate enzymes used in this study, with respect to sensitivity reductase activity in ext'racts prepared from T . eyuiperto pyrimethamine, than to the reductase isolated by dum, chicken liver, and rat liver are summarized in Ferone, et al.,zl from the protozoan P. berghei. The Table I. Compounds 1-4 appear to possess no useful IDsovalues obtained for pyrimethamine in the rat liver degree of inhibitory potency against the reduct'ases and chicken liver systems are considerably (ten- to studied; the lack of efficacy of these compounds as twentyfold) higher than those reported24 for pyriminhibit'ors is consistent wit'h previous observations by ethamine as an inhibitor of dihydrofolate reduction other^.**^'*'^*'^ 1nt)roductionof a suitably substituted by reductases obtained from guinea pig liver and small aromatic function into position 5 of the pyrimidine intestine. nucleus can greatly enhance the ability of t'he 2,4-diThe introduction of a second C1 in the 3' position of aminopyrimidine system to inhibit enzymatic reduction pyrimethamine resulted in little alteration of inhibitory of dihydr~folate,'~ and 5 with a 4-chlorophenyl subpotency against the trypanosomal reductase, camstituent in the 5 position was an effective inhibit>orof pared with that of pyrimethamine. On the other hand, dihydrofolate reductase in the trypanosomal system as the 3',4'-dichloro derivative (9) proved to be coneiderwell as in the chicken liver and rat liver systems. The ably more effective than pyrimethamine as an inhibitor ID5ovalue for 5 is approximately tenfold lower for bhe of reductases from chicken liver and rat liver. Ret,rypanosomal reductase than for bhe avian and mamplacement of 6-Et of pyrimethamine and its 3'-C1 malian enzymes, but it should be stressed that this difanalog (9) by CH3 mas found to produce a tenfold deference may simply reflect the order of magnitude difcrease in the ability of the resulting compounds (6, 10) ference in K , values for dihydrofolate which exist's to inhibit the trypanosomal reductase, but against the between the trypanosomal reductase ( K , = 4.3 X avian and mammalian enzymes the decrease in acL1.l)zand those from chicken liver (1.7 X lo-' ~ ! ! f ) ' ~tivity was not so marked. Baker and H o have ~ ~shown, and rat liver (2 X lo-' M ) . I 6 Introduction of CH3 using dihydrofolate reductase systems from both pigeon into the 6 posit,ion of 5 results in increased inhibitory liver and Escherichia coli B, that there was relatively potency against the rat liver and chicken liver enlittle difference between the inhibitory potencies of 9 zymes, but no significant alteration in activity was oband its 6-Me analog 10. It is noteworthy that the deserved in t,he trypanosomal system. 2,4-Diamino-5crease in ability to inhibit the trypanosomal reductase, (2-bromopheny1)pyrimidine (7) was a much poorer inwhich is a consequence of the replacement of the ethyl hibitor of dihydrofolate reductase from all three sources group of pyrimethamine by a methyl group, correlates than was the 4-chlorophenyl analog; it may be that the roughly with the decrease in antiplasmodial activity poor inhibition observed with the 2-bromophenyl observedz0for the 6-Me analog relative to pyrimethderivative is due, at least in part, to st'eric complicaamine. Compound 6 was considerably less effective tion~''-'~arising from the proximity of the bromo functhan 10 as an inhibitor of dihydrofolate reductase from (11) H. R. Baker and H. S. Sachdev. J . Pharm. Sci., 62, 933 (1963). pigeon liverla and guinea pig tissue\24and, as seen in
Assay of dihydrofolate reductase activity was carried out spectrophotometrically by following the decrease in optical density a t 340 mp which occurs on reduction of dihydrofolate and concomitant oxidation of NADPH. The assay solution contained 0.16 pmole of dihydrofolat,e, 0.24 pmole of NADPH, 33 pmoles of mercaptoethanol, the appropriate enzyme extract (sufficient extract to give a change in absorption of at least 0.01 OD unit/min), and p H 7.0 phosphate buffer (0.1 M ) in a total volume of 3 ml. Spectrophotometric measurements weye made using a Beckman model DU spectrophotometer equipped with thermospacers to maintain the temperature of the cell compartment at 37'. All drugs were preincubated a t 37" for 10 min in the presence of enzyme, mercaptoethanol, and buffer, then NADPH and dihydrofolate were added t o the cuvette, and reductase activity was measured a t 1-min intervals over a 10-min period. The level of drug required to produce 50% inhibition was estimated graphically from a plot of Va / VI us. increasing inhibitor concentration where V , = reaction rate in absence of inhibitor and VI = reaction rate in presence of inhibitor.ll Stock solut,ions of diaminopteridine derivatives were made up in DMSO and appropriate amounts of DbISO were added t o control cuvettes where required.
(12) Reeaiise many of t h e compounds used in this study were a t or near d u b i l i t y limits a t 10-4 M ,we chose this concentration as a "oiit-ofF point," and compounds which did not produce appreciable inhibition a t this le\,el were classified a s "inert." (13) S. F. Zakraewski, J . B i d . Chem., 7.38, 1489 (1963). (14) B. R. Baker, B-T. Ho, and D. V. Santi, J . Pharm. Sci., 64, 1415 (1966).
(15) 13. T. ICaiifmao and It. G . Gardiner, J . B i d . Chem., 241, l 3 l Y ( 1 Y W ) . (16) 1). \Vang and \V. C. Werkheiser. Fed. l'roc., 23, 324 (1964). (17) I. M .Rollo, Trans. Roy. SOC.Trap. M e d . H y g . , 46, 474 (1952). (18) P. R. Russell, J . Chem. Soc., 2951 (1954). (19) 13. R. Baker, J . M e d . Chem., 10, 912 (1967).
(20) E. A . Falco, L. G. Guudwin. 0.11. liitcliings, I. h1. Kullu, and 1'. U . Russell, Brit.J. Phurmucol., 6 , 185 (1951). (21) R. Ferone, J. J. Burchall, and G. H. Hitchings. Mol. Phurmacol., 6, 49 (1968). (22) J. R. Bertino, J . P. Perkiiis, and D. G. Johns. Biochemistru, 4, 839 (1965).
(23) J. J. Jaffe, J. J. McCormaak, and I\-, E. Gutteridye, t ' z p t l . Purcisitoi.. in press. (24) J. R . Bertino, .I T. . Iannotti. J. P. Perkins. and D. G. Johns, Biochem. Phurmacol., 16, 663 (1966).
1cs of thebe cilllydroThe differential itbility to penetrate various cel1ul:w membrane3 betwtvi niethotresate :iiirl L'sn~aIl-mole~uic~' dihydrofolate wductabe inhibitorb 3uch a h pyrimethamiiic has h i i clearly dem~nstrated.~'The observation that th(s 3'-SHJ and 3'-KOL derivative, of pyrimethamine iirv roughly equiwtive iis inhibitors of the t,rypaiiosomnl reductase but differ considerably in their inhibitory potency against the rat liver and chicken liver cnzymc5 m:ty indicate :L coiisiderable difference in the electroiiic requirement.; for tlinding of inhibitors t o the protozod emynie on the one hand, and to the avian and m:iiiimtlian enzynics o n the other. L',~,G-Triai~iiiio->-phenylazopyrlmidirl~ (15) w:i< found8 capable of inhibiting rat liver folic x i d retlucatase, arid the yuggehtion has been made that the >-aryl group contributes heavily to the "binding" of thi. agent to the reductase eiizynieb. We found it to Ine I I L inhibitor of trypanosoma1 dihydrofolate reductase awell as reductase from chicken arid rat liver; the k'l value for inhihitioli of r:tt liver dihydrofolate reductase
by 15, estimated from the relationship,E K I = Kar (Iso/S50), where Is,,and Sso represent, respectively, the inhibitor and substrate concentrations present at 50% inhibition was found to be 2 X X,a value which is essentially the same as that reported for this substance as an inhibitor of folic acid reduction by rat liver reductase. The trimethoxybenzylpyrimidine, trimethoprim (16), mas found to be several-thousand-fold more effective as an inhibitor of dihydrofolate reductase from bacterial sources than as an inhibitor of reductase preparations from mammalian liver.3 Recently Bakerlg made the iriteresting observation that trimethoprim was much less active as an inhibitor of dihydrofolate reductase from Tz bacteriophage than of bacterial reductases per se. We have found that dihydrofolate reductase in extracts prepared from chicken liver was only poorly inhibited by trimethoprim, a concentration in the order of iZI being required for 50% inhibition. The low susceptibility of the chicken liver enzyme to inhibition by trimethoprim parallels the low sensitivities of pigeon liver reductase" and reductase from guinea pig tissuesz4 to this inhibitor. We found earlier2 that dihydrofolate reductase from T . equiperdum is intermediate, in its sensitivity to inhibition by trimethoprim, between the high sensitivity of bacterial reductases and the rather extreme insensitivity of mammalian liver reductases. As can be seen in Table I, several analogs of trimethoprim (17-25) have also been found to be considerably more effective as inhibitors of dihydrofolate reductase from T . eyuiperclum than of the analogous enzymes extracted from mammalian o r avian liver. It is possible that the difference in susceptibility of trypanosomal and mammalian reductases to inhibition by pyrimidines of the trimethoprim type might be used to advantage in the strategy of chemotherapy of certain trypanosomiases. -Although certain 5-benzylpyrimidines and 5-phenylpyrimidines were found to exhibit similar potencies as inhibitors of the protozoal reductase obtained from T eyuzperclum, these classes of compounds differed markedly with respect to antimalarial activity against Plasmodium galliriaceum and P . berqheiZa Thus, the IDLovalues for 6 and the related 5-benzylpyrimidine (24) were essentially the same with regard to the trypanosomal reductase, but in antiplasmodial tests, the &phenyl analog proved to be approximately 50 times more potent than the 5-benzyl derivative. A large series of 5-aryloxy derivatives of 2,4-diaminopyrimidine mas synthesized at the Wellcome Research Laboratories during the course of an intensive research program aimed a t developing new antimalarial agents. I n general, these aryloxy derivatives showed relatively poor antiplasmodial activity.*O The efficacies of the several 5-aryloxy derivatives (26-29) of 2,4-diaminopyrimidine which we have investigated as inhibitors of the trypanosomal, rat liver, and chicken liver systems rather closely resembled those observed for analogous 5-benzyl derivatives since in both the :-benzyl and the 5-aryloxy series inhibitory potency was higher against the trypanosoin:d reduct:ibe th:in agninst the other t w o systems. 4,6-Diamino-1,2-dihydrotriazines.-A number of 4,6diamino-1,2-dih?-drotriaxiries show high antiprotozoal
a ~ t i v i t y , ~ and ~ - ~such l substances have been found also to be potent antagonists of the growth-promoting action of folic acid for certain microorganism^^^ and to possess potentially useful anthelmintic activity.33 Compounds of this class are very effective inhibitors of dihydrofolate reductases from mammalian3 and avian The dihydrotriazine derivatives (Table 11) TABLE I1 INHIBITION O F DIHYDROFOLATE 1 t E D U C T I O N BY ~,6-~1.~~11~O-~,2-D1METHYL-~-~RY~~,~-D1HYDR0-~-TR1~~21N~S
NHz
T
A.5 N'
K-R CH,
-___-CHJ
T . equi-
Compd
30 31 32 33 34 35
R
perdum
IDsu (10-8 .U)-----. Chicken Rat liver liver Sourceu
3,4-C12C& 350 2.5 4-BuCsHc 2000 30 3-ClCsH4 210 5.5 3-BrC& 340 4 4-CICsH4 100 50 2-Naphthyl 400 100 See footnote a, Table I. * Lit.2 14.
0.4 T.2b 10 3 120 65
NSC BW
NSC NSC NSC Aldrich
were, with the exception of 34, less active as inhibitors of trypanosomal dihydrofolate reductase than of reductases from chicken liver or rat liver. For example, the 3',4'-dichlorophenyl derivative 30 was approximately 200-fold more effective as an inhibitor of the avian reductase than of the trypanosomal reductase, and this observation is consistent with the finding, previously reported,2that the related 4'-butylphenyl derivative 31 was nearly 200 times less potent an inhibitor of trypanosomal reductase than of rat liver reductase. The increased efficacies, as inhibitors of chicken liver reductase, of the 3'-chlorophenyl- and 3',4'-dichlorophenyldihydrotriazines over that of the 4'-butylphenyl analog parallel o b ~ e r v a t i o n s ~on~ ~the '~ relative effectiveness of these compounds as inhibitors of pigeon liver dihydrofolate reductase. Furthermore, Baker and H o found ~ ~ that replacement of the 3'-C1 of 32 by Br resulted in little change in inhibitory activity against dihydrofolate reductases from E. coli and pigeon liver, and we also found a similar degree of activity for these halogenated derivatives in the systems studied by us. The 3'-chlorophenyldihydrotriazine 32 and the 3',4'-dichloro analog 30 showed comparable inhibitory potencies against the chicken liver enzyme, but we found that the 3',4'-dichloro derivative was about 20-fold more effective than the 3'-monochloro compound in the rat liver system. Similar observations (28) R. D. Powell. R . L. DeGowin, and R . El. Eppes, A m . J. TTOP. Med. Hyg., 14, 913 (1965). (29) B. C. Walton. D. A. Person, hI. H. Ellman, and R. Bernstein, i b i d . , 17, 814 (1968). (30) C. ICI. Johnson, h i d . , 17, 819 (1968). (31) R. I. Hewitt, W. S. Wallace, A. Gumble. E . White, a n d J. H. Williams, ibid.,8, 225 (1954). (32) E. J. Modest, G. E. Foley, RI. M. Rechet. and S. Farber, J . A m . Chem. Soc., 74. 855 (1952). (33) R. R n t l i , R . B. Hiirron.n, and 0. H. Hitchings, .T. .Wed. Che?n.-6, 370 (1963). (34) For a summary of a series of studies on dihydrofolate reductase inhibitors of the dihydrotriaeine class see B. R. Baker, "Design of hctive-siteDirected Irreversible Inhibitors. T h e Organic Chemistry of the Enzymic Active-Site," John JViley and Sons, Inc., New York, N. Y., 1967.
ISHIBITOM OF DIHYDI~OFOLATE REDUCTASE
July 1961)
667
TABLE I11 I N H I B I l'lON O F I)IHYDROFOLATE l?,EDUCTION
Compd
A
36
NH,
n NH, NH?
RI
Rz
H
H H
BY SELECTED L ~ M ~ N O P T E R I D I N E S
T. epuzperdum
IDsa (10-8 U)-------Chicken liver R a t li>er
Source'
Inert Iuert 2.5,000 b 37 NH, hIe 4000 2 5 , 000d 7,000 C 38 NH2 NH, hIe hIe 800 7,300 1 7 , 000e b 39 NHl NHI Ph Ph 40 1,000 400 b 40 H NH, Ph Ph Inert Iller t Inert C 41 NH? H Ph Ph Inert Inert Irier t C a See footnote a, Table I. 31. F. Mallette, E. C. Taylor, and C. K. Cain, J . A m . Chem. SOC.,69,1814 (1947). AI. D. Potter and T. Henshall, J . Chem. SOC.,2000 (1966), for the 2-amino derivative the liteiatiire procedure wab modified by disolving the pyrimidine at pH 7 rather than in dilute acetic acid before the condensation wlth benzil. d K,, calculated8 from IDSo= 5 X lO-'?.I; lit.13 4.7 X IO-' Y. e K I , calculated from IDoo= 1 X 10-6 M; lit.472 x 10-6,11.
TABLE IV IIVHIBITION O F L)IHYDROFOLITE R h D C C T I O X B Y 2,4,7-TRIA~~INO-6-.iRYLPTERIDINES
(
ompd
s
-1Dso 2'. equzperdum
A{-)\
Chicken liver
Rat liver
Source"
42 H 250 150 160e Aldrich 2dIe 43 400 300 130 SKF 3-Me 44 800 330 190 IVY 4-Me 45 10,000 12,000 1,700 WY 2-c1 46 400 250 150 SKF 3-c1 47 130 100 25 WY 4-C1 48 15,000 10,000 1,800 WY 49 2,6-C1? 180 300 60 WY 2,4-C1, 50 5,000 5,700 740 WY 51 3,4-C1, 50,000 2,200 200 WY 2-Br 52 90 260 100 WY 33 4-BI 8,200 29,000 7,200 WY 2-1 54 180 330 120 WY 55 4-1 12,000 11,000 5,000 IVY 2-F 56 500 400 210 W Y 5i 4-F 4,500 3,300 1,400 WY 2-Ph 58 110 9,000 1,400 WY 59 l-Xaphthylb 120 790 100 W Y 4-Ph 60 20,000 8,500 23,000 WY 4,5-Mez 61 9,000 3,000 8,500 WY 62 Phc Inert Inert Inert SKF 63 Phd Inert Inert Inert SKF a See footnote a, Table I. * Xaphthyl fiinction in place of the phenyl group of 42. 4-Amino group replaced by a p-chlorophenylamiiio groiip. 2-Amino group replaced by a phenylamino group. e K I , calculated from IDso = 6 X AI; lit.47 1 x 10-8 M ,
of ID,, values obtained in the trypanosomal system for 2,4,7-triamino-6-o-chlorophenylpteridine (46), which has marked antimalarial activity, and the related 6-m-chlorophengl compound (47), which shows little antimalarial potency, indicates quite clearly that no necessary correlation can be demonstrated between in vitro inhibition of trypanosomal reductase and in vivo antimalarial activity of these pteridines. The report42 that 2,4,7-triamino-6-o-tolylpteridine (43) has activity against leishmania in vitro as well as
the recent demonstration by C o r b a ~ c i oof~ the ~ ability of this compound to antagonize arrhythmias induced by cardiac glycosides further extend the range of pharmacological activity shown by pteridines of this type; it mould be of considerable interest to determine what effect alteration of the position of substituents in the 6-aryl function exerts on the sntileishmanial and antiarrhythmic activity of such triamterene derivatives. When the primary amino group in either the 2 or the 4 position of triamterene was replaced by an aromatic amino function, ability t o inhibit the reductase systems studied was found to decrease substantially. Comparable decreases in ability to inhibit dihydrofolate reductase were obtainedz4 when the amino group in position 2 or 4 of analogous pteridine derivatives was replaced by an alkylamino function. The potency of triamterene as an inhibitor of rat liver reductase is comparable to its potency as an inhibitor of reductase preparations from Ehrlich ascites tumor cellsZ2 and mouse leukemia cells.44 Doctor, in 1958, reported that 2,4,7-triamino-G-phenylpteridines were able to inhibit conversion of folic acid to &formyltetrahydrofolic acid by an extract prepared from chicken liver;45the order of inhibitory activity which Doctor found for certain 6-arylpteridines was similar to that found for inhibition of dihydrofolate reduction by the crude extract from chicken liver which we have used. Thus in Doctor's experiments the inhibitory potencies of 2,4,7-triamino-6-phenylpteridineand its o-methyl and m-methyl derivatives differed by a factor of only three- to fourfold, whereas the p-methyl analog was about 150-fold less potent than the o-methyl derivative. As summarized in Table IV, the same qualitative relationship exists among these compounds as inhibitors of dihydrofolate reductases. It should be noted that addition of p-C1 to either the 0- or the m-chlorophenyl derivatives of 2,4,7-triaminopteridine resulted in a reduction of inhibitory potency, indicating that the simple presence of ortho or meta substituents per se is not sufficient to produce optimal inhibitory activity of such pteridine derivatives against the reductase systems. (43) .i.N . Corbascio, Fed. Proc., 27, 348 (1968). (44) A. R. hlaass, V. D. Wiebelhaus, G. Sosnowski, B. Jenkins, and G. Gessner, Tozicol. A p p l . Pharmacal., 10, 413 (1967). (46) V. >I. Doctor, J . Bid. Chem.. 232, 617 (1968).
