Journal of
MEDICINAL AND PHARMACEUTICAL CHEMISTRY Volume 5 , Number 6
@ Copyright 2080 b :he American Chmical k!ociety
November 27, 1962
The Synthesis of 6-Fluoro-9-methylpurine* XLDEKG. BEAMAX and ROLAND I 6-fluoropurine-S-oxide, and that heating of purine-fi-sulfonyl fluoride5 failrd to give 6-fluoropiirine I t has k e n iiiggrsted? that the inabiiity CJ prepare ti-fluoropurine may t i e diie to it5 instabiliti~ Hendic*h6ha. predieted that 6-fluoropiirinrx TI ould he iinstahle to avid. in addition. thermal instahility might be mentioned The latter may cvplain the l was failure to obtain 0-fluoropurint. 11heii piiririr-fj-bulf o n ~ fluoride heated at ahoiit 200' in tetralin. under which conditions sulfur dioxide a a s evolved caopiouslj . Thus, a c*ornplrtely different approticah \\ah sought iza fluoropyrimidines. which it was hoped would avoid the difficulties inherent in the previous synthetic. methods aimed at 6-fluoropurine. In addition. appropriate I-substituted aminopyrimidii1r.j caould serve as intermediates for various S-sut)stitiited-~-fliioropurinee of interest. I'iit was felt nally, t)ecause of thc antitumor activity of .S-fl~iorouracil.~ that certain fluoropjrrimidine, might t w of intereht in themsehrs. Schroeder8 found that heating L'.1,0-trichloropyrimidine with silver fluoride gave 2,1,&trifluoropyrimidine. We fouiid that 1,G-dichloroz-nitropyrimidine (I)9and silver fluoride gave the corresponding dlfluoronitropyrimidine (11) I\ hich tipon monoaminatiori and reduction gave the important iiitermediatc l.j-diamino-(j-fluoropyrimidinc (IT'). The standard reagents for cyclizing &c*hloro-I,.',-diaminopyrimidirlc to 6-chloropurine. I iz., ethyl orthoformate-acetic anhydride,"' I ' diethoxymethyl acetate [(C,H50),CHOCOCH,11~ and ethyl orthoformate plus an acidic ratalyst all gave hypoxanthine nhen applied ( 2 ) 4 Bendl(1i i ( i n ~ * ~ - ~ u i o and I l a 1 I I . o \ i n 1 h i C l i L i i i i b t i ) and Biolod\ of l'uiinc. Wolstoniioline a n d 0 Cunnw (eds ) Littk BI O U I I nnd C B m t o n l 9 i 7 / ) I ) 7-8 (3) 4 Ginei-horolla and A B e n d d i J 4 m C h r m \ a i 80, 57-14 ( l q j 8 ) (4) Unpublished obseri ations of the autlioib ( 5 ) .4 L Beaiiisn and 1% I< Robin5 J I m C'hrln >uc 83,4038 (11Jiil) (IJ) Ref 2 11 l i i (7) %e Canccr C h e m o t / ~ e , a pl l~i p l u \ ( I i i i, W ilUCi0) f c i i I, wmpilatiun of t i i t rtftlt III on 5-fluo1ouiaril (8) H f3chrodr.i .I A n i C h u m Sa, i l l i (1WLJ) (4) \I R Boon \\ C 31 Jiinrr itndC: It Raiiiari I C h r f r i b u r O ~ i ~ l ~ l ~ I l (10) 1 1 h l o n t y o n ~ r i J~ l i n Chrni hor 7 8 , 192R i l l t i l ~ ) ( 1 1) L C~oldman,J 1% Alarsiw dnd I, ( r a / d u l a I O r u ( ' h r m 21, j l ! ( J i 1'1 ) O l anrlC' T L i i i l r l L 11 I t m Ctirni Su( 79, i 2 3 8 I l ~ l 5 7 ) and T c i u p l c 1 1 I O r v C i r m 26, JOB ( l W i ( l ~ i j
t
-
~-FLUORU-~-METHYLPLTRINE 1069
November, 1962
F
F .%NO,
2 H
:;:&N iv IV
y$, N
kN
Raney NI
I11
N$
is;;2CHOC2H,
V
NH2
6N
N= C H N (C H3) z h’=CHN(CHs)z VI
to -I,.j-diamino-ti-fluoropyrimidine. With ethyl orthoformate alone -I,bdiamino-6-fluoropyrimidine gave 4-amino-5-ethoxymethyleneimino-ti-fluoropyrimidine (V), which is analogous to the findings of Montgomery13 for the corresponding chloro compound. When 4,5diamino-6-fluoropyrimidine was treated with phosphorus oxychloride and N,N-dimethylformamide according to the method of Clark and Lister.” 4,5-bis(dim~thylaminomethyleneimino)-6-fluoropyrimidine (TI)was obtained. I t was found that this method with 6-chloro4,5-diaminopyrimidine l5 gave a mixture of 6-chloropurine and 4,5bis(dimethylaminomethyleneamino)-ti-chloropyrimidine. The very reactive and unstable 4,ti-difluoro-5-nitropyrimidine (11) was best reduced with ferrous hydroxide to S-arnino-4,6-difluoropyrimidine (VII) by the method of Brown.I6 This compound reacted readily with methylamine and with cyclopentylamine in refluxing ethanol to give 5-amino-Bfluoro-4me thylaminopyrimidine (VIII) and 5-amino-6-fluoro-4-cyclopentylaminopyrimidine(IX), respectively. Refluxing VI11 with ethyl orthoformate-acetic anhydride gave ti-fluoro-9-methylpurine (X). Similarly, IX gave g-cyclopentylti-fluoropurine (XI). 6-E’luoro-9-methylpurine (X) was stable to storage and recrystallization from organic solvents such as benzene. When boiled with concentrated aqueous ammonia, X gave 9-methyl.I. C1arkand.J. €I. Lister. J . Chem. S u c . . W-18 ( 1 9 I i l ) . ( 1 5 ) R. K. Robins, K. J. Dille. C. 11. Willitb. and B. E. Christuiisun, J . A m . Chem. Soc.. 76, 2tiY ( I U x 3 ) . (16) D. J. Brown. J . d p p l . Chem., 4 , 72 (IKj-L), (1.1)
c'ol. 5
1070
F
IX
F
F
adenine indicating that the fluorine atom is more readily replaced than the chlorine atom in 6-chloro-9-methylpurine.1b G-Fluoro-9methylpurine (X) was changed slowly to 9-methylhypoxanthinc in aqueous solution over a period of one week. These reactions provide evidence that the fluorine atom is at position 8 and that no unexpected rearrangement has occurred. 6-Fluoro-9-methylpurine (X) was negative when screened againbt .Idenocarcinoma 7.53 a t dosages up to G8 mg./kg. per day. At this dosage no toxicity was evident. 1;urther testing of X arid related derivatives against a wider spectrum of tumors is in progress.
Experimental" 4,6-Difluoro-5-nitropyrimidine(II).-A mixture of 200 g. of 4,6-dic~lilorc1-5tiitropyrimidine,g 1000 g. of silver fluoride (Harshaw Chem. Co.), and 200 ml. of riitrobenzerie was st,irred and refluxed a t 170-180" for 45 min. The 4,8-difl11oro5-nitropyrimidine which formed (about 150 m1.j \\-as dist,illed at atmospheric. pressure, h.p. 185-195'. This compound was unstablr and was uscd \\-it,hout' purification. 4-Amino-6-fluoro-5-nitropyrimidine (III).-4.6-Difliioro-5-nitrop~rimidili~~ (150 ml.) %as diluted with 2.5 1. of anhydrous ether, filt.ered and into the stirred filtrate (at, -55 t,o -65") was bubbled anhydrous ammonia unt'il a. drop of tlic. reaction mixture gave a basic reaction with pH "Hydrion" paper. The yellonsolid was filtered, washed with ether, boiled rvith 4 1. of anhydrous benzenz, ctntl filtcred. From thc filtrate crystallized 38 g. (24%,) [iiwluding a second crop) of 111, m.p. 161-163" (sublimation). Anal. Calcd. for C4HaFX402: C, 30.4; H. 1.9; F, 12.0; S , 35.4, Fourid: C, 30.2; H,1.2; F,12.0; N,35.6. 4,5-Diamino-6-fluoropyrimidine(IV). Method A.-A ;;olut,ion ~ I E I O K . of -i-amino-G-fluoro-5-nitrop~rimidi1i~~ iii met,h:uiol \\-as li~'lrogc'li:.cl,c,tf :it rooli) tcmpcrat~urcin a Parr shaker with 2.46-2.8 kg. of liydrogcti pcr ( m . 2 using Rancay
November, 1962
c
1071
6-FLUORO-CJ-METHY LPURlNY
i
1N
nickel catalyst. After 30 niin. the iiickd was filterd and the filtrate evaporatcd to give 7.4 g. 190yo)of crude IV. This waw rccr (charcoal) to give 6.4 g. of pale-yellow needles, m p . A n d . Calcd. for CdHjFY4: C, 37.5; H j 3.9: F. 14,s: S.43.7. Found: C, 37.i; H,4.0; F, 15.0: 5 . 4 3 . 5 . Method B.-5-Amino-4,6-difluoropyri1nidi1ir (VI1, 0.9 g.j was dissolved in 100 ml. of absolute ethanol. Ammonia gas !vas hiibbled into t h e solution for 10 min., and the solution was refluxed \vit,h ammonia hubbling through it for 4 hr. The soliit,ioii wtis cwqiorated and the solid stispendd i n n sniall amount of water t o give, 0.4 g. of c n d e product, which a a s sublimed and recrystallized froni water to give csolorlrs:e nredles of IV, in.13. 215-219". iindrpremed Kith material prepared hy method A. The ultraviolrt absorption spectrum of t,hc product was identical with that of t,he niat#erialpreparrd bj- reduction of 111. 4-Amino-5-ethoxymethyleneamino-6-fluoropyrimidine j V ) .-Two grains of 4,5-dianiino-6-fluorop~rimidine was hoilrd with 30 ml. of ethj-1 orthoformatc until the temperature reached 145' and for 15 niin. thereaft,er. The ethyl ort,hoformate was reincived under vacuum t o give 2.3 g. ( S O ~ ~of o )crude V. T w o recrystallizations from benzene gave colorless needles, m.p. 122-129". Anal. Calcd. for GHsFn'rO: (1. 45.7: H, 4.9: F; 10.3: S. 50.4. Found: C?4 5 . i ; H, 4.8; F, 10.4; X, 30.3. 4,5-Bis(dimethylaminomethyleneamino)-6-fluoropyrimidine(VI).-4,5-Dianii in 1 ml. of ?r',S-dimethylforniamide rio-6-fluoropvrimidiiie (0.20 g.) was dis at room temperature. Phosphorus o ride (0.25 rnl.) was added dropwisc over a period of 10 niin. at 25-35". A hr. at room temperature the reactioii mixt'ure was pourcbd on ice and the pH adjusted to 10 with ammonia and then to 6.5 with aretic acid. The solutioii was extracted with ethyl acetate to give 0.19 g. of cryatdlitie solid. Retc,r!-stallizat,ion froni I)enxene-petroleiim ether gavc needles, m.p. 116-118". .Anal. (hlcd. for C'IOHI~E'S,: C, 50.4; H. 6.3: F, S.0: S . :35.3. Found: C, 50.3; H, 5 . 2 ; F,S.2: S , 35.9. Reaction of 6-Chloro-4,5-diaminopyrimidine with Phosphorus Oxychloride and N,N-Dimethylf0rmamide.~-Chloro-4,5-diaminop~rimidine~~ (5.0 9.1 w:ts dissolved in 50 nil. of ~ , ~ - d i n i e t h y l f o r i i i a r n i date room temperaturtc,, arid 10 nil. of phosphorus oxychloride w:ts added slowly so that, a reaction t,rinperature of 35-90" \vas maintaiiied. Thil mixture wiis a l l o ~ c dto st:md for 1 h r . : the c'x( were removed under vacuum and the sl-rup n-ns tlccomposd \vit,h ice. The 1111 \vas adjusted to 10 with concd. potassiuni hydroxide solution and t,litT solid collected and recryst'allized from acet,one t,o give 3.1 g. of 6-chloro-4,5-bis(dinietli?.larninoniet~hy1eneamino)pyrimidine. m.p. 95-100". A n d . Calctl. for GOH1:ClSG: C, 4i.1; H, 5 . 0 : C1, 1:3.9: S , 33.0. Found: Cj 47.3; H, 6.2; C1, 13.7; S,,32.i. Thc aqueous filtrate from the crudc 6-chloru-4,5-bis(diiiicth~laniiriciiii(~tliyl~~ric!:itnino)pyriniidiIie was adjusted to pH :5 with hydrochloric acid arid the solid which formed rocryst,allized from acc:tcint: to give 0.7 g. of 6-chloropurint: idcntificd hy its charactmeristicnltmviolet absorption spt:ctrum. l i 5-Amino-4,6-difluoropyrimidine(VIIj.-4,fi-Difliioro-5-nitr~~~yri1iiitIi11~~ 'I I )(lilt 65 in1. j from 67 g. of 4,6-dichloro-5-nitropyriinidineQ and t,c+mlin, in licu of nitrobc~rizoneas the solvcnt, w:is redured wit,h hot aqucous ferrous hyclroxidc. iisitig t,he method descrihed hy Brown"; for thtx roduc.tion of 4,6-dic.hloro-5-riitropyriiiiittino, t o givv 11.5 I$. (25%,) of crudc product (oxtrartc'd with (!thy1 atc ( s
November, 1962
fbFI,YnRO-%METHYI,PURINE
1078
This was recrystallized from absolute ethanol to give VI1 as flakelets, m.p. 157 -159". Anal. Calcd. for C4H3F2N3: C, 36.i; H, 2.3; F, 29.0; N, 32.1. Found: C, 37.0; H, 2.0; F, 28.8; K , 32.3. 5-Amino-4-eyelopentylamino-6-fluoropyrimidine(IX).-5-Amino-4,6-difluoropyrimidine (0.55 g,), ethanol (25 ml.), and cyclopentylamine (2 ml.) (Aldrich Chemical Go.) were refluxed 2 hr. and evaporated to a gum which was triturated with water. A yellow solid formed, wt. 0.70 g. (857,). Rerrystallization from water gave flat needles, m.p. 125-127". Anal. Calcd. for CgHlzFN4: C, 55.1; H, 6.i; F, 9.7; S, 28.5. Found: C, 55.2; H, 6.5; F, 10.0; N, 28.4. 9-Cyelopentyl-6-fluoropurine(XI).-A solution of 0.47 g. of 5. amino-4-cyclopentylamino-6-fluoropyrimidine (IX) in 7 ml. of 2 : 1 (v./v.) ethyl orthoformate: acetic anhydride (aged one year) was boiled in an open flask for 20 min. The excess solvent was removed an vacuo, and the residue was dissolved in benzeneheptane and allowed to evaporate to give a low-melting solid which was recrystallized from low-boiling petroleum ether to give 150 mg. of a crystalline solid, m.p. 55". This material was further purified by dissolving it in benzene and passing it over a small column of Florisil to give a crystalline product, m.p. 55-58', A qualitative test for fluorine was positive. Anal. Calcd. for CloH,,FN4:C, 58.2; H, 5.4. Found: C, 57.6; H, 6.1. U1traviolet absorption in methanol: A, 243, 275 (shoulder) mp, e 16,480,3,090. 5-Amino-6-fluoro-4-methylaminopyrimidine(VIII).-5-Amino-4,6-difluoropyrimidine (5.0 9.) was dissolved in 500 ml. of absolute ethanol, and the solution was refluxed with a stream of anhydrous methylamine bubbling through it for 1.5 hr. The solution was evaporated to dryness and the solid leached with a little water to give 4.5 g. (83%) of crude VIII. Sublimation gave colorless needles, m.p. 142-144'. Anal. Calcd. for C5H?FNa: C, 42.3; H, 5.0; F, 13.4; N, 39.4. Found: C, 42.5; H, 5.4; F, 13.7; N, 39.6. 6-Fluoro-9-methylpurine(X).-A solution of 9.8 g. of 5-amino-6-fluoro-4methylaminopyrimidine in 125 ml. of 2 :1 (v./v.) ethyl orthoformate:acetic anhydride was boiled for 12 min. and the excess solvent removed in uacuo. The resulting solid was recrystallized from benzene-heptane to give 7 g. (67%) of colorlessneedles, m.p. 135-136". Anal. Calcd. for CeHsFN4: C, 47.4; H, 3.3; F, 12.5; S, 36.8. Found: C, 47.2; H, 3.1; F, 12.8; N, 36.9. Ultraviolet absorption spectra of 6-fluoro-9-methylpurine: in methanol, Xmar 248 mp, e 8,350; a t pH 1, A,, 249 mp, e 9,150; a t pH 11, Amax 259 and 264-267 (shoulder) mp, e 11,800 and 11,100. The 264 to 267 mp shoulder a t pH 11 appears to be due to a product with base since it increases to a sharp peak on standing a t pH 11. S-Methyladenine.4-Fluoro-9-methylpurine(0.28 g.) and 10 ml. of aqueous ammonia solution (297, ammonia) were heated in a boiling water bath for 45 min. Every 6-8 min. an additional 5 ml. of concd. aqueous ammonia was added. The hot solution was cooled to give tiny rods, m.p. 310' (preceded by sublimation). A mixture melting point of this product with an authentic sample of 9-methyladenine'b showed no depression. The ultraviolet and infrared absorption spectra of the product were identical with those of 9-methyladenineJ as were the R, values in two different solvent,systems.
1 074
C. Vi. NOELL4NI) R. K. RORISS
1-01. 5
9-Methylhypoxanthine.4-Fluoro-4methylpurine(X, 0.15 9.) was dissolved in 5 ml. of distilled water at room trmperaturc. After 7 clays crystals formed. l)y its chnrarteristic u1tr:iThe produrt was idrirtified as 9-m~~thylh\posantliir~c violct sp(vtrum. 1" I n n / . C:ilrd. f(ir CHHCX\lT40: C, 48.0; H, 4.0. Found: 0, 37.7; H, 4.6.
The Antitumor Activity of 2-Amino-6-alkylthio9-(p-D-ribofuranosylpurinesand related Derivatives of 2-Amino-6-purinethiol (Thioguanine)'
A number of 6-alkvlthio-2-aminopurines and their ribosides have been prepared nnd tested against Adenocarcinoma 7 5 5 . Alkylation of 2-aniino-9-P-o-ril)ofuranosyl-6-purinethiol with the appropriate alkyl halide gave the desired riboside derivatives. Many of these compounds exhibit excellent activity against Adenor:rrcinoni:i i s 5 and significant wtivity against Sarcom:i 180 and 1,eukemin 1210.
The antitumor activity of a number of 9-alky1-2-amino-B-purinethiols2 and 6-alkylthio-2-aminopiirinesYagainst .4denocarcinoma 73.5 suggested the synthesis of the corresponding 6-alkylthio-2-aminopurine-9-ribosides. The possibility of the increased antitumor artivity of t he 6-alkylthio-2-amino-9-/3-~-ribofuranosylpur~nes was suggested by the fact that 9-/3-D-ribofuranosyl-6-purinethiol possesses a therapeutic index4 of 200 as compared to 30 for 6-mercaptopurine against Adenocarcinoma 7 5 5 . G Mercaptopurine ribonucleoside is also inhibitory at a lower dosage4 than 6-mercaptopurine in Adenocarcinoma 755. I n addition, 2-amino-9-/3-~-ribofuranosy~-6-purinethiol (thioguanosine) given orally in the clinic is more active on a ( 1 ) This investigation was supported by Contract No. SA-43-ph-1928 with the Cancer Chemotherapy National Service Center of the National Cancer Institute, National Institutes of Health, U. S. Public Health Service. (2) C. W. Noell and R . K. Robins, J . .Wed. Pharm. Chem.. 6 , 558 (1962). (3) G . D. Daves, Jr., C . W.Noell, R. K. Robins, H. C . Koppel, and A . 0. Beaman, J. A m . Cliem. Soc., 82, 2633 (1960). (4) H. E. Skipper, J . .i. Montgornerp. .I. R. ThornRon, and F. h l . Rchabel, ,Jr,, Cnncrr HPnrarch. 1 9 , 4 2 5 (1959).
