.. I i a . 3.= H IIb R - C H
!1
ii!
Synthesis of Some Steroidal [3,2-cl]- and [J 7,16d]-2',6'- Diaminopyrimidines
, /
IT'a. 11 = 14
IVb.R=CH,
May, 19G3 idine VI derived from testosterone absorbed a t 300 mp, and this spectrum was not altered by acid or base. The infrared spectra of the diaminopyrimidines 11, 111, T', and T'I n-ere dominated by three strong absorption bands in the 3 p region, a t 2.87-2.90, 2.97-3.02, and 3.14-3.17 p , typical of diamiiiopyrimidines in general. I'urther complex strong absorption occurred in the G.lc?-G.GD p region, also considered typical of diaiiiiiiopvriiiiidiiies,~ Io The dianiiiiopyrimidiiies IIa 2nd I I b are n eak bases nith pKa values of 7.50 and 7.33, respectively. The estroiie derivatives T7a and T'b, pKa 6 7 0 and 6.43, respectively, are slightly stronger bases. Only one inflection could be determined in the titration curves: hon eyer, 2 ,3-diamiiiopyrimidiiie shon ed only one pKa value (7.-36)." The fusion reaction was conducted in a test tube heated in an oil bath a t 200-250O. Air \vas displaced by nitrogen. The melted steroid and cyanoguanidine were miscible in some cases, but two phascs were formed n ith jcu-cholestaii-3-oiie, estrone n:ethyl ether (IT-b), and dehydroisoandrosteroiie. In these three cases large amounts of unaltered steroid reactant were recovered and oiily relatively small amounts of diaminopyrimidine n-ere formed. Generally agitation of the molten reaction mixture did not improve these cases. The reaction product solidified on cooling and was removed, ground, and '\T. ashed with water to remove melamine TI hich n as also formed in the reaction. The steroidal products possessed a t this stage the characteristic ultraviolet and infrared absorption properties of the purified materials, and despite constant spectral properties satisfactory elemental analyses could not be obtained in certain cases. The preparations were readily solvated and were electrostatic. Chromatographic purity of each preparation was attained, as evidenced by both thin-layer and paper chrornatographic procedures. The two dihydrotestosterone derivatives I I a and I I b had a low order of androgenic activity n ith no anabolic activity.12 The estrone derivative T'a had low order (O.ly0of estrone) estrogenic activity, but neither T'a nor T-b exhibited antilipemic effects.I3 I n view of the variety of antimicrobial activities of other 2,4-diaminopyrimidiiies, including folic acidfolinic acid ailtimalarial activity,8f8g 14a I 5 (9) L. S . Short and H. IT. Thompson, J . Chern. Soc., 168 (1952). (10) T h e two regions of complex absorption have also been reported for some monoaminopyrimidines beaiing t h e fused steroidal nucleus (see references 5 a n d 6). (11) A. Albert, R. Goldacre, and J . Phillips, J. Chem. Soc., 2240 (1948). (12) Using t h e test of L. G. Hershberg, E. G . Shipley, and R . K. Meyer, !'roc. Soc. Ezptl. Biol. M e d . , 83, 175 (1953), I I a was 3-10%.I I b was 0.6% as potent a s testosterone propionate (ventral prostate). Potency of I I a and I I b (lei,ator ani) was less t h a n l . 5 7 0 compared n i t h testosterone propionate. (13) Estrogenic activity was measured by mouse uterine gro\lth, cf. B. I,. Ruhin, -1.Dorfman, L. Black, and R . I. Dorfman, Endorrinology. 49, 429 (1931). The antilipemia test employed was one devised h y Dr. R using intact adult male r8ts o n a normal diet, n i t h test compound administered over 9 days. .ictive compounds reduce t h e Liebermann-Rurcliard positive lipids of whole plasma extracts. Testicular atrophy was also noted as a sign of "feminization." (14) (a) G. 11. Hitcliings. G. B. Clion, H. T-anderWerff, and E. -4, Fzlco, J . Biol. Chem., 174, 765 (1948); (b) E. A . Falco. G . H. Hitchinrs, P. R. Russell, and H. VanderKerff, S a t u r e , 164, 107 (1949); (c) G. H.Hitchings. G . B. Elion. E. -4. Falco, P. B. Russell, .\I. R. Sherxood, and H. I'anderWerff, J . Bzol. Chem., 183, 1 (1950); (d! G . H. Hitchings, T ~ a n sR. o y . Soc. T r o p . .Ired. H u g . , 46, 467 (1952); (e) G. H. Hitchings, E. A. Falco, G. B. Elion, S. Singer, G. B. Waring, D. J. Hutchison, and J. H. Burchenal, A r c h . Biochem. B i o p h y s . , 40, 479 (1952); (f) G. H. Hitchings, Congr. intern. biochim.. R l s u m B s communs., 2e Congr., Paris, 1952, p. 8 3 ; ( 9 ) E. J. Nodest,
SOTES
331
anticoccidial activity,16 antileukemic and antitumor activity,17 antibacterial activity,I8 etc., the dianiinopyrimidiiies IIa, IIb, T'a, and T*b n-ere tested against a variety of bacteria and fungi. hntibacterial activity was found for all four against several Gram-positive organisms, and particularly against Staphylococcus aurezis strains (Table I). TABLEI [:3,2-(!]-A S D [ 1 ;,I 6.d; -2 ',B'-DI.~~~ISOPTRI~LIDISES
.\XTIBACTER1.4L .~CTIVITIESOF STEROI!)
Test orcanisin S t a p h ~ i c o c c u uaureun 209P StaphrJococcus aureus RH- 180 Staphdococcus auwus C H P Stap/,y!ococrus aureus Sn ith Staphylococcus aureus .I 144 Sfrepmcoccus p k o q e n e s G r . , u p A Dipiococcus pneumoniae 37 Sarcina lutea C:aff":pa ietraypnn Snlnioiirlla p a r a t u p h i Brucella bronchiseptica Seisseria ratarTha;is Laciobacilius casei Bacillus subfilzs 6633 Bacillus suhiilis SR N?icc+acieiium sp. P s e u d o m o n a s aeruoinosa Eschevichia coli 6830 Escherichia coli PR
llininluin inhibitory copcentration, ~ g . / r i ~ l . (.izar serial dilution, Potassium Eenzi-l IIa Ilb Vi Vh Penicil.in
31.3 250 230 31.3
..
31.3 31.3 31.3 313 250 500 250 50
25 230 25
1000
loco 1000
100 100
..
..
10 10 10
..
..
..
..
.,
,.
..
..
.. 100 100 500 50 50
1000
..
10
..
10
..
..
.. BO 30 50 25 25
250 >loo0
>loo0 >lo00 1000 1000
10 10
500
500
10
25 25 25 10 25 LOO0 1000 1000 1000
0.05 >IO0 100
..
..
.. .. 0 10 5 0
.. .,
0 026
>loo >loo >lo0 10 50
However, neither IIa nor T7a protected mice challenged intraperitoneally n-ith a penicillin resistant strain of X.a w e u s CHP. Experimental'Q
17P-Hydroxy-5~t-androstano-[3,2-d]-2 ',6'-diaminopyrimidine (IIa).-4,5~~-Dihydrotestosterone (1 g.) was mixed with 400 mg. of cyanoguanidine and placed in an oil bath preheated to 230". The material melted in a few minutes and Tvas heated a t 230250' for 30 min., during which time bubbles evolved. The cooled mass was pon-dered, washed with water, and dried, yielding 1 . 2 i 5 g.,, , A, 254 nip ( E 5000). The solids were dissolved in ethanol, precipitated with water, and the product dried over phosphorus pentoxide, yielding 990 mg. of material Tvith A, 254 m p ( e 5160), n-hich was redissolved in tetrahydrofuran and reprecipitated n-ith water. The n-hite solids were extracted n-ith boiling benzene, and the filtered extracts evaporated under vacuum. The pure product v a s dried thoroughly over phosphrous pentoxide under vacuum, m.p. 190", and a t 272-278" dec. (with shrinking and coloration from 243"); A,., 251 m p ( e 4830), G. E. Foley, and 5. Farher, Acia LTnio Intern. Contra Cancrum, 16, 702 (1960). (15) (a) L. G. Goodain, S a t u r e . 164, 1133 (1949); (b) E . A. Falco, P. B. Russell, and G. H. Hitchings. J . -4m.Chem. Sor., 73. 3733 (1931): (e) E. A . Falco, L. G. Goodain, G. H . Hitchings, I. 11. Rollo, and P. B. Russell, Brzt. J . Pharm. Chemofher.. 6, 185 (19.511: ((1) I. h l . Rollo, Trans. R o y . Sor. T r o p . M e d . Hug., 46, 474 (19.52): (e) L. G. Goodwin, ibid., 46, 483 (1952); (E) G . R. Coatney, ihid., 46, 496 (19.52). (16) R . E. Lox, .4ntibiotzcs and Chemotherapy. 4 , 971 (1934). (17; (a) J. H. Burchenal, S.K. Gottchius. C. C. Stock, and G. H. Hitchings, Cancer Res. 12, 231 (1952): (b! D. A . Clark?. S. AI. Buckley, 9. S. Sternberg, C. C. Stock, C. P. Rlioads. a n d G . €€, Hitchings, i b i d . , 12, 25.5 (1Y52); (c) K. Sugiura, i b i d . , 13, 431 (1953). (18) E. F. Rogers, W . J. Leanra, a n d L. H. Sarett J . Org. Ciiem., 22, 1492 (1957). (19) Melting points nere taken on a Kofler block nnder a microscope and are corrected. Infrared spectra %ere determined on gressed potassium bromide disks using the Perkin-Elmer Model 21 instrument. Ultraviolet absorption spectra nere determined on solutions in 95Y0 ethanol. The p K values were determined on 50% aqueous ethanol solutions using 0.1 A- HCI.
2:HJ nip ( t S340); A,i, 256 nip ( 6 1520), 224 inp ( 6 8240): A,">::'"-"' 27:3 niM ( E 4520), A,,,i, 256 m p ( E 3740);':::A 2.90 (shoulder ), i3.01, :$.17,6.21,6.32,6.38,6.95,9.50~ 9.75,12.20p, etv. d n c d . Calcd. for C21H,nX,0: C, 7 0 . 7 5 ; H, 9.05: X, 15.72, Found: C, 70.20, H, 9.07: S , 13.41.
17~-Hydroxy-17a-methyl-5~-androstano[3,2-dj -2',6'-diaminopyrimidine (IIb).-Tn.o grams o f 1701-1riethyl-4,5cu-dihy(ir~itestosterone itnd 1.0 g. of c3yanoguanidine lvere mixed in a lest tulic and placed i n ;in oil tmth preheated to 175'. Heating \vas i ~ c i n h u e t ithe , miterials melting a t 187-200". . i t 220° hubblt~s c~v~ilvetl froin t,he reaction melt, s r i d heating \vas then viintinncd fiir :30 i i i i n . The cooled melt (2.(il g.) was grilund and PStracted \vith benzene in it Soxhlet estr:iibtor firr several d:~ys. Six separate extracts were t,aken, w1iic.h yielded on ev:ipiiratiiiri thc purified pyrimidine, 1.885 g., .I). 2-17--250" del.. (shrinking :ind c~)lorationfrom 172'); , X,,,.,, 4 nip ( e 3000-5300'). After tliescilving in ethanol, precipitatiiJr .it11 \v:iier, dr>-irig,reextr:tvt i i m i n t i 1 Imizenc, ev:ilioration, :tnd tlitrrougli drying over 111 iihorris pentiixidv the product nielted :it 170-175" and at 2 2.X' d t ~ [: a ] " +16" (ly?,KtOH); A,,.,,, 284 nip ( E 5440), 630, shoulder); A,,i, 25-1 ]rip ( c 1970); A ~ ~ ~ ~ " 27s ~"" ), A,,, 257 nip ( e 4280); 2.87 (shnulder), 3.00, 3.17, .52 (shoiilder), 6.19, 6.29, 6.39, 6.95, 9.19, 10.72, 12.69 p , t.tr. . I n c t l . CaIcd. for (,'T2&?i40: (', 7 l . : < l : I f , 9.24: N , 15.12. Vound: C , 7 1 . 6 i ; H,8.5i; S , 13.57. Solvated forms x e r e :ilso obt:tinect, :I hydrate, i i i . p . 17O-lX2" :tnd 252--2(iO0 d e r . . 1 7 ~ n i . Calcd. for C?2H31~40.111'pII?O: 6 i . N,14.26. Fuund: C, 67.08; H,8.T0; S , 14.63. ja-Cholestano- [3,2-d]-2',6'-diaminopyrimidine(1II~.--il niisture i i f 1.0 g. of 5ol-cholestan-3-one and 0.25 g. of cyanoguanidine \\':is heated a t 250" under nitrogen n.it,h vigiirous stirring. After iiielting, two phases were formed. After 15 min. an additional 0 . 2 5 g. of cyaniiguanidine \\-as tidded and heating was continued for 1 5 niin. T h e cooled reaction mixture (1.0 g.) \vas washed with 100 i n l . of hot water t\vice, yielding 0.91 g. of yellow solids, ii1.i). 200-208" (siiftening ~111 100";); A:',":'""''"""""" 298 nip ( e lii:3O); ::,A: :3.05, 3.20, i (residual .ia-c.holestanone), 6.17, (i.39, 6.97 p , e t r . Thin-lay hroni:itography ( 5 5 , ethyl acetate i n hexane) using 257; antimoul- trivhloride in chloroform for detei~tiilnindic.:rtecl the single 1)riduc.t i l l f 0.48) together \\-ith iin:ilteretl ja-chli)eStnnonc. N ( i further purific*:ition was :I ttempted on this preliaratioii. 3-Hydroxy-1,3,5(10) -estratrieno- [ 17,16-dJ-2',6'-diaminopyrimidine (Va).-Estri)ne ( 6 g.) and 4.0 g. ( ~ fcj-anoguanidine \vert \vel1 mixed in a test tube, from which air was excluded by a stream of nitrogen. The mixture was placed in a preheated oil bath (260'). \Tithin 3-5 niin. t h e reactants melted. The niolteri mass \vas stirred under nitrogen a t 260" until bubbling ceased and the mass liecame viscous (5-7 niin.). The cooled, powdered inass \vas slurry-washed with ( 7 0 " ) Ivater twice, then dissolved in 400 1111. of ~ v a r u i( T O " ) water acidified t o pH 1 with concentrated 1iyclr~~chli)ric:wid. -1fter fi1tr:iticiri of insolubles the filtrate was i.oolrd : t i i d neutralized ivith concentr:tted :irnnionium hydroxide, and tlie precipitated product filtered. Solution and precipitwt i i i i i \vere r e l ) e e t d :3 more times, yielding :I rrude product weighing :{.?I g. The pyrimidine ~ v n scxtr:ic.ted with boiling ethyl :Icct:it,c, :md precipitated witti petroleum ether, affording 536 rug. -:3:3oo tirca.: [all, +100" ( l C ' I , Q-
f' 1'.
,Lracc/. Chlcd. for ( ' ? a H ? ~ S ? i ) . ' , ' a H r O(') : 70.19: H , 7 . 2 5 ; 3 , L6.:37. Found: C , i O . l l : H , 7 . 2 4 : S , 16.60. 3-Methoxy-1,3,5 (IO)-estratrieno-j17,16-d] -2 ',6 '-diaminopyrimidine (Vb).--A scih~ticin of 1 .OO p. of 3-h~droxy-1,3,5(10)cxstr:ttrienri-[ 17,1G-d] -2',fi'-di:tniincrI)yriniidiIie,10 g. of potassium hydrcixide, 25 nil. of w:itc.r, a n d 25 nil. of methanol was held :it i3.5' \\-ithstirring while 10 nil. of dimethyl sulfate was added over IO niin. Stirring \vas cwntinued for 2 h r . , at nhich time 50 ml. o f \vatt,r \viis :idtied. .ifter stirring for :tn additional 15 min. tlir 1)riidui.t (41 7 nix, I \vas filtered. liecrystallization from Ix~iliiigiiiethani~lyielded : 3 . 0 2 , 3.14, 6.15, 0.33, 6.93 p , rti..
:Inal. Calcd. for CnlHesS40H 2 0 : C, 68.10; H, 7 . 6 s ; S , 75.18. F o u n d : C , 68,08; H, 7,61; S , 1-1.63,
Acknowledgment.-- The authors are indebted t o G. Warren. S. Rosenman. and J . Yurchenko for the microbiological assays, to Ilr. 11. Edgren for the endocrii~ologicalassaw, and to Dr. T. S.Osdene for csalling to our attention the cynoguanidine fusion synthwi5 of pyrimidines.
