nil. of csthrr, 3 x 200 ml. of nwtonc, and 3 x 200 ml. of boiling water. T l i ~ product \vas t , I i r ~ ~ recrystsllizcd i from tlimethylforma,mide and water t o yield 27 g. (46%) of 5-(2,4dinitroanilino)-2,4pyrirnidinediol, as golden needles, m.p. 285 m p ( e lO,OOO), 340 mp ( e 246 mp ( e 15,800); 312-313" dec.; 286 mp ( e 12,400), 346 mp ( e 10,000). 5,800); Anal. Calcd. for C10H,21Tb0B.H$3: C, 38.6; H, 2.9; S , 22.5. Found: C, 38.2; H, 3.1; N, 22.4. The water of hydration can be removed by drying a t 130" in a vacuum oven for 24hr. ( A n a l . Calcd.: C, 41.0; H, 2.4. Found: C, 41.2; H, 2.8.)
Acknowledgment.-The
authors wish to express their appreciation
t o Mr. Wayne H. Nyberg, Mrs. Margaret L. Rounds, Miss Phyllis G. Shaul, Mrs. Beverly Ann Smith, Mrs. Carol R. Tuttle and Mr. Hal P. Van Fossen for their valuable assistance in performing nnal~Jtical, instrumental and paper chromatographic measurements.
5-Benzyl-2,4-diaminopyrimidinesas Antibacterial Agents. I. Synthesis and Antibacterial Activity in vitro BARBARA ROTH,ELVIRA A. FALCO, AED GEORGE H. HITCHINGS Burroughs Wellcome and Co. (U.S. A . ) Inc., The Wellcome Research Laboratories, Tuckahoe, New York AND
S.R. M. BUSHBY
7'he Wellcome Research Laboratories, Reckenham, Kent, England
Received Mag 31, 1962
A series of 5-benzyl-2,4-diaminopyrimidines has been synthesized and tested for antibacterial activity. Maximal activity occurs among those compounds which are unsubstituted in the pyrimidine 6-position, possess unsubstituted amino groups in the 2- and 4-positions and bear one or more alkoxy1 groups in the meta and para positions of the benzene nucleus. These compounds have high activity against Gram positive microorganisms and significant activity against a variety of Gram negative bacteria. Trimethoprini, 2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine, has been selected for further study on the hasis of the magnitude and lmadth of its antibacterial activities.
The discovery that many 5-benzyl-2,4-diarninopyrimidines1 possess a high degree of antibacterial, as well as antimalarial, a c t i ~ i t y l -has ~
I c d lo
1 iyirtht. 01 ii I n i - g ~t i i i i n l ) w (11 ntlt1iiioii:il ~ i i h i i t i i i ( ~ d tic~tixylpyrimitli~i~~s, \vith t ht. ol)jec*iivc ( i f \wbhig tlrri\ a1 with t \ i t > widest antibacterial spectrum arid optimum oflect i\ ity, particular1)against Gram negative organisms. It is the purpose of this paper t o describe the nen- substituted benzyl derilxtives 11 hich have beeii prepared in our laboratories during t he y c a r ~interlwiing since the original publication' by 1;alco arid (so-workers in 1931, to show striicture--actii.ity relationships among the \ x i o m 5-benzylpyrimidines synthesized to date and the spectrum of activity of thc inore actil(' members of the series against bacterial pathogens in in ~ i t r otesti. Results of in w o tests and biochemical studies will he dercrit)ed iii separate communications to folloiv. It was observed at a 1 ery early date that the d-benzyl-.',4-diaminopyrimidines which had the highest antimalarial activity containrtl (\-alkyl groiips (optimally methyl) in the pyrimidine ring, ant1 p-h:ilo 01- nitro substituents in tho henzence ring.3 Removal of the O-alLyl groiip had a rather remarkahle effect on the activities against microorganisms. in that the antimalarial activity was considerably tlcpressed, hlit :mtihac>tcrial :wtix it\- \ \ a s T t ~ ymarkedly incrcased. .Itnorig the ~ a r l yronipouinds, highest activit y agaimt (;rani ntlgat ivv organimis, siirh a h /'rotcus I u i g c i m , [vas found among thow c~oniairiing p- and m-methoxyl and halo groupi. Attention therefore \vas focusctl particularly on the prcparation of compoiinds containing varioiib romhinsiions of alkoxy1 and halo groups in the henzeiie ring, in a n ($fortto find derivatives of minimum toxicity arid masiniiini activity. The synthetic method:, iised for the preparation of many of tho new pyrimidines \\-ere essentially those of Falco uiid w-ivorkers. .lrylcinnamic acids were prepared from the corresponding aldchytlch 1)y reaction with malonic a d ac~ordinpt o the Iloehner reaciioii " I 11umber of ncw alkouyhenzaldehydes werc prepared for this purpow, iisiiig known techniclurs. 'l'hcl cinnamic acids ~ v e r f ~e-terified. r+ tliiced, and formylated n-ith (.thy1 formate. The resultant c.rritlr formyl derivatives were condensed directly with giianidinc to prodiirc %-amino-,j-benzyl-4-hydroxypyrimidines,which iipon chlorination and amination yielded the %,4-diamino derivatives. T n a few inh c 5
ii,(b-
s.
(1) E. -1.Falco, DuHreuil, and G. H . Hitchings, .J. A m . Cliern. Soe.. 73,3758 (1931). ('2) E. 9. Falco. L. G. Goodain, G . H. Hitchinga, I. M. Rollo, and P. R . Russell. Brit. .I. Pharmacol.. 6, 185 (1951). i : $ ) G. H. Hitchings. A m . .J. Clin. Sutrition, 3, 321 (1953). f 4 ) G . 13. Elion. R. Singpr, and G . If. Hitching.. Antibiotics and Chemotherapy. 10, 556 (1960). fa) C:, H. Hitchings a n d S. R . 11. Ijuchby, V t h Internnt. Congress of Dioclirm., Mosrow. 1,. 183 (1961). ( 6 ) 0. Doebner. Ber., 33, 2140 (1800): 36, 1136 (1902)).
stances thc 4-chloro compounds reacted with alkylamincs to produce the 4-alkylamino-2-amino analogs. Many of the pyrimidines which contain bromo or nitro groups in the benzene ring were prepared by bromination or nitration of the 5benzyl-2,3-diaminopyrimidines. I n the majority of cases, 3,4dialkoxybenzyl derivatives were used for this purpose. It was found that 2,4-diamino-3- (3,4-dimet hoxybenzyl) -pyrimidine gal-e 5-(% bromo-5,j-dimethoxybenzyl)-~,4-diaminopyrimidineexclusively upon bromination. Its structure was proved by independent synthesis, starting from 2-bromo-4,.5-dimethoxybenzaldehyde. It was assumed that the related alkoxy1 derivatives behaved similarly upon bromination; the structures were not proved. Since it had not been demonstrated prer iously that 2,4-diamino substituents were required for activity against microtjrganisms in the 3-benzylpyrimidine series, a number of pyrimidines were prepared containing mercapto or hydroxy substituents in place of one or both amino groups. 'l'hese were prepared by condensing the sodium salts of (.thy1 a-formyl-hydrocinnamates with thiourea in ethanol, whirh yielded 3-benzyl-4-hydroxy-2-mercaptopyrimidine~.7-g The 2- anci 4-sitbstituents were transformed to methylthio, hydroxy, and amino derivatives by well known techniques which are described in the experimental section. The in vitro antibacterial assays were carried out for the most part by determining the minimum concentration of benzylpyrimidine which was required to inhibit growth in cultures of various organisms in nutrient agar and other media. Ahsayswith Streptococcus pyogenes were carried out also in whole blood, because experience has shown that an unknown substance, or substances, is present in blood which reverses the antibacterial activity of' many agents which are active in nutrient agar. Although most of the benzylpyrimidines which were active in nutrient agar were also active in whole blood, there were a few- cases where the activity was considerably diminished, as seen in Table IX (see, for example, compounds LXII', XLT711, and LXXI), In some cases it was found desirable to have a quick screening procedure available for determination of antibacterial potentialities. lcor such purposes, an agar plate was employed. Paper discs, impregnated with solutions of the compound, were applied to the plate which had been inoculated with the bacteria. Zones of inhibition then were determined. This method was used chiefly to spot activity (7) H. L. Wheeler a n d D. F. McFarland, Am. Chem. J . , 42, 101 (1909). (8) T. U. Johnson and J. C. Ambelang, J. Am. Chem. S o c . , 6 0 , 2941 [ l $ l 3 8 ) . (9) E. A . Falco, P. R.Russell, and G . H. Hitchings, ibzd., 73,4466 [lY.il),
nrtlyaris, nlthoiigli otlici, orgaiiim* slho \ v ( w i (*(I arc found 111 ‘l’nbh. X11 Tables Y arid XI1 show the ailtibacterial acti\ itics of ljcnqdpyrimidineh other than ~,4-di~rniino-Ci-iiiisul)st it utetl derivatit es. I t nil1 be seen that the “4-diaminu-ti-methyl derivatil inactit against P. vulgaris and have considerably lower acti\ ity 1s S . uureus than the corresponding 6-unsubstituted derivati1-es. ’lctivity 11s. S‘f. pyogenes is retained, however. Introduction of a (i-hydroxyl substituent almost completely abolishes the activity. C’onversioii of a 4-amino to a methylamino or other alkylamino group gives products which are devoid of interest as antibacterial agents. .I hiinilnr resrilt is o1)served when the 4-ainino group is replaced by hytlro~yl oi’ rhloro. ‘I’he 4-amino-%-liytlrosp, %,-l-dihydroxp, 1hydrosy-L‘-n~c~r(~apto, :tiid ~ - a i ~ i i i i o - ~ - n i c r t ~pyrimidinw ~~i)to arc likcis(’ iriarl i\,cl. ‘I’h(1 interest then f r w i i v < solely o i i .i-t)cnzyl-2*4tIinniiiio-(i-inih~it~stit u t ed pyrimidines. 1)at a pertaining to the relation h c t w e n htruct [ire and antihscterinl activity ot derivatives \vi{h various substituents in the henzene ring are presented in Tables IX and XII. It is to he noted first that very few of the closely related compounds which are listed here have high activity against P. vulgaris, and that variations which may increase the activity against S.(nupus or St. pyogencs often result in a lowering of the actiiity against P . vulgaris 1;ortunately the reverse is not iiecessarilp true, for a few of the caompounds have acti\-ity against n \t idr variety of inicroorganismb. I t will be seen that the most active compounds contain meta alkoxyl o r halo siihstitution, and preferably 4-alkoxy1 or hydroxyl substitritioii Iydroxj-1 substitution later was found to be inadvisablr s, so only one such compound is listed here.) The best stitlition seems to be methoxyl. The 3-ethoxyl derii,a’e us. Z’. vulgaris, although the 3-methoxyl derivative i i \w>- active, In the .l-position, a methoxyl group is best again
