2'-Deoxythioguanosine and Related Nucleosides'
The conversion of 2-amino-6-chloropurine in t u o steps t u the 2-acetamido derivative V IS described. '1'11~ condensation of a merLury derivative of \- u 1th L: protected %-deoxyribofiimnos4-1vhloride VI1 :ifTorded the blocked nucleosides, :ind p-I?II, \I hose structures \I ere assigned on the basis of n.1n.r. dJt:L. I k a raplareinent uf the 6-chlorines J ielded the thiols, PIS and 8-IX (9'-deosythioguanosine. n hich is of spec*ialintcirr s t for possible nntitunior properties). l'hc conversion of p-IX t o .?'-t3eoxvgumosine Irovided L' definltiw I hemic a1 striictiirr proof for 8-1s (Y-
r ,
I liioguanine (2-amino-~i-niercaptopurinc) ant1 its riboside, thioguaiiosinr, have I)crii studied a8 anticancw eoinpouiids, particulai*l,v against lrukcniia; thry appear t o be equally c~ffcctivc aqciits.' In his studies of the inecliaiiisni of action of thioguaniiw, LePage5 has found a correlation h t n ecii the tuniorinhibitory response aiid thti incorpoi,atioii of the drug into nucleic acids, probably specifically into the DKAI. Revistalice of t urnors to thioguaniiir. iq a s~riouslimitation in its use, and efforts have been made t o circumvent thc. rcsistancr. Oiic possibk mcchaniwi of resistance lies ill the inability of tlie tiiiiioi, r ~ 1 Bto iwlucc the ribonucleotide of thioguanine to its dcoayribonuclcotide, which caii then lie incorporated iiito DS,I.6 To furnish thioguanine as the deoxyriboside might be a way of circumvrnting such a re.istancc nicchanisni ; phosphorylatioii to thc cleos~.riboiiucleotictc might tlicri provide the desircd forni of tlic drug for incorporatioii. Thus the synthesis of 2'-d(,~)xythiogiiaiioyiti(~ (P-IX) w w of iiitrreit for in tigatioii of this hypotlicsi.;. In a griirral mihe d(1oxytliiogunno~iii~~ rould b(>a tlicrapcritically niorc iiscful foim oi' tliioguaiiinc~. Important thcrapcutic diffcrriicer Iwtivcrii tlw ribosiclc aiid the 2'-tlc.oxyribosicl(i of' ~ 1 , cotiiinoii Iic~tcrocyc~lic 1)asc. liavr 1 ) w i i iiottd; tlic effcctivc,iitw of 5-iodo-2'tlrosyui~idiiieagainst herpes simp1c.e I ii*us in roiitraht to the ineffectivcnrss of the r i I ) o d v , ;,-iotlo~iridiiic.. i y an csainplc.' The gciioral instability5 of puriiic deoxyi ibosides (relative to ribosides or to pyriniidine nucleosidea) suggested that the direct thiation3 of 2''-tltwxyguanosine would not provide a useful roiitr, to $-IS; indecd. studies in anothcr laboi-atop ai i i i accord ~i itli tlii. view.9 'l'he most e.;pcdiciit iwtliod for pi*tqmring P-IX thcii seciiied to 11sto 1~ fnifli(~1~ application of thc (1
(1) This work was carried out uniler tlir
of t l x (::rnr,r.r ('1irlimthr.ri t u t r , National Institittias of Health, Public IIealtlr Service. Cuntrac i--43-i~li-l8Y2. T h e winions expressed in this pager a r e those o f t h e aiitliors and not nec~essarilytlwsc of tlie Cancer Chemotherapy h-ationnl Service Pt,nter. Sorllc o f t h i s \I o r k tias been reported in preliminary form: R. 11. TU~LIIIOTO, If. 11. . \ r t o n , :inrl 1.. (;oodman, .Nulure, 198, 288 ( 1 9 6 3 ) . 12) T o whoni reprint requests should b (3) ,J. J. Fox. I. Wompen, A. llainptun. a n d I. 1.. Jlorrr, .i.Ani. Ciir m.
apy National Servire Center, National Ps
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I. 11. Kr;~lioff,IL. R. Ellison,
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(1961). ( 5 ) (;. ;\.I.oPage a n d M. Jones, ibid., 21, l:!l(J (6) G. A . LePape. private cornmuniratiiin.
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( 7 ) E. S. Perkins, R. M. Wood, 11. I,. Sexti s , It., 11. I'rii8ul'f. ?+nil !\-elrti, .Yulzire, 194, Y85 (1962). 18) J. u'. .Jonrs a n d 11. IC. Robins, .I. 1; r n . C h , in. F. ,\lichrrl a n d A . Hresinp. Chrm. Ber.. 94, 181.1 (1!!61). ( ! I : It. IC. Ilohins. priviitc ~ , , i , , i i , ~ i i i i ~ , : ~ t i , ~ i i .
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indirect mcthod1" uicd to prepare thc rt>lntcd t l o o ~ v r il )osi d e of G-nir. re a ptop u ri 1 1r . 2-;iiiiiiio-O-cliloropuriiic ( I ) v a s thc pi opi iatr puriiic preewqoi' to be coiidriised itli 3 1 1 acylatd chloro sugar (sii(1li as T'II) by tlic inei-ciu.i proctdiii~c"; the final steps TI ould he deacylatioii aiid rcplarcnic,iit oi the chloriiir I\ itli the thiol group. l17hcii ckiloi oniercuri-a-ainino-G-chloropuriiie was coiicle~i L'-deoxy-~,.~-di-0-p-toluoyl-u-riboiurallos~l (YII) in refluxing benzene, however, a stable S - ~ ~ J T O sidic bond n-as formed at the frcc 2-aniiiio group, as ne11 as a t X-9. If this condellsation was cai.ricd out at room temperature iii dimethyl sulfoxide,'? 11i(, piviluct appeared to consist of nioiio-0- and bis-2,CJ-fiii aiioaide in roughly equal amounts; 11 2-ien the product 17 deacylated 11 itli methanolic ammonia a t a c~l.ystalline material was isolated iii 7s yield aftrr fiactiorial ~eciytallizatioii11 hich gave spectral and niialytical data espectcdlz for a ~-(2'-d('oxyli~)ofIlrariosiC1(~) of I l'hc anolneric nature of this material ]\-as iiot i i i gatcd, and it n a s roncludcd that u i i ' of iiiipi otcv I\ as impractical. The useful derivative L'-acetaniido-G-chlor~)r~u~inc. (Y)had to be prepared in two steps from I, siricc dirwt nionoacetylat ion of I uiider a variety of conditioiib nfforded only t'-a~iiiiio-G-chloro-~-ncetylpurinc (TI), identical nith an autlientic sample.' I'olyucctylat ion1* of I affordd ~-acetarnido-F-chloro-Y-acetylpuriilc(I 11) apparently miscd with 2-(T,S-diacetamido)-G-chloro-O:ic.ctylpuririr16 (I\r), after 7 hr. i n idiuuing acetic aiiliydride, or after 1; min. if phosphoric n :is use(1 as catalyst.*i Tlic compositioii of niixtiirey of I11 an(1 I\' xm-ird m n i r ~hat j from run to run a i d \vas detci m i n d 1)y c~lcnientalanalyws. Tlirse riiatci.i:il~i\ri IiiistaMc t o hot water or aqiicon-;I5 or ruct1i:uiolic. I J ~ w . TI hich gcnc~ratctltlic. rclatii clg atablr 3-aret:Lniido-Gc~hloropiirinr~(V) in good yicld. I-ndcr biniilar ( * o i i clition5 tlic S-9-acctyl c~onipouncl11 \ins cilca\ o t l t o I . (5
) R IC. U o i . .Linl 11 ( A . I letclict, Jr J . i m . P h t ) u . , q ' c ~ 82, , 3iii 11'3601 (1.3) 1%.I t Raker, I1) -diirt.t\lailiino I,? ~ i i i ~ i c l i \~ i I) c ,J. l i r o i i i i I' j c I'
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:rcylation t t i form 1- was c.:trrietl out in h50 n i l . 1 1 1 ii1etIi:inol i,oiit:tiiiing S 5 nil. i i f viiriretitr:iteil :iniiiioiiiii~ii l i > , -
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(6,870), 319 (19,900); :XL: mp ( E ) , 208 (23,500), 225 (13,100), droxide. After 15 min. a t room temperature, the solution was 258 (8,400), 341 (25,700); Rr 0.49 us. 0.26 for thioguanine. The concentrated to form a white residual solid which was washed onto analytical sample (SOYoyield) was recrystallized from hot water a filter with cold water. The solid (12.7 g., 647,) was pure (125 nil./g.); it decomposed on heating above 190". enough for the next step, Rr as described. A sample for analysis Anal. Calcd. for CloH13S503S.H?O: C, 39.9; H, 5.02; S, was reprecipitated on neutralization with JI hydrochloric acid 23.2; S, 10.6. Found: C,39.6;H,5.11; N, 23.0; S, 10.6. of a solution in JI sodium hydroxide; it did not melt below 280"; 2-Amino-9-( 2-deoxy-a-D-ribofuranosyl)purine-6-thiol( a-IX) hO,:,v NsoH mp (e), 236 (20,100), 285 (8040). was obtained by the same procedure from a-VI11 in 76% yield, Anal. Calcd. for C?H&lK,O: C, 39.7; H , 2.86; C1, 16.8; [ a I z 5$58.2 ~ 5 1.0" (0.1 M sodium hydroxide). After 1 reN , 33.1; 0, 7.56. Found: C, 39.9; H, 3.02; C1, 16.6; N, 33.2; crystallization from hot water, the yield was 45%, [CY] 2 4 +68.6 ~ 0,7.74. & 1.0" (0.1 Jl sodium hydroxide); Ai:." laoH mp (E), 220 (14,800), (B) From the Mercury Derivative VI.--il 500-mg. portion of 251 (14,000), 271 (6,930), 319.5 (20,200); Inp (e), 209 ( 2 2 , VI was dissolved in 30Yc aqueous KI to form a solution of pH 800), 227 (14,600), 258.5 (7,920), 341 (25,400); Rr 0.49. The 10-11. Adjustment to pH 5 with acetic acid afforded 186 mg compound decomposed on heating above 170'. (i3Yc) of T' as a white precipitate with properties identical to Anal. Found: C, 39.9; H,4.83; S , 23.2; S, 10.8. those recorded in A. 2-Amin0-6-benzylthio-S-( 2-deoxy-8-~-ribofuranosyl)purine Mercury Derivative VI of V.-A solution of 28.65 g. (105.7 (6-X) was obtained from S I X and benzyl chloride in aqueous mmoles) of HgCL in 1.5 1. of 507, aqueous ethanol was treated dioxane containing KOH.31 The stirred reaction mixture, after with pulverized \- (11.16 g., 52.84 mmoles), and then with 105.7 30 min. a t room temperature, was seeded (seed obtained from a ml. of JI NaOH during ca. 1 hr., according to the procedure for preliminary reaction, by evaporation) and stirred further for 3.5 chloromercuri-6-chloropurine.13 The product (22.09 g., 101.5%) hr. while the product crystallized. After standing 2 hr. a t 3", was mixed with 25.60 g. of Celite to aid filtration. On a 1-g. the product (SOY0)was collected: addition of water to the filtrate scale, the product was collected by centrifugation. The infrared afforded a second crop (total yield 7670). Recrystallization from spectrum disclosed loss of carbonyl absorption a t 5.78 p ; loss of 50% aqueous methanol afforded 54%, m.p. 162-165', [ C Z ] ~ ~ D bands a t 8.31 and 11.0 p (s) and appearance of new bands a t 8.28 -3.9 i 0.1" (methanol); ";:A:: mp ( e ) , 218 (24,500), 246 (16,and 10 53 p (ni) was also characteristic of the transformation. 500), 312.5 (14,200); Ri0.26. Anal. Calcd. for C,H,ClHgS,03: C, 19.0; H, 1.13; C1, 8.00; Anal. Calcd. for C17H19S503S: C, 54.8; HI 5.13; N, 18.8; Hg, 45.3; S , 15.8. Found for two separate preparations: C, S,8 59. Found. C, 54.4; H, 5.00; K, 18.4; S,8.62. 19.2, 19.0; H , 1.05, 1.26; C1,8 09,7.92; Hg,-, 45.3; K, 15.8,15 7 . 2-Amin0-6-benzylthio-S-( 2'-deoxy-~~-~-ribofuranosyl) purine 2-Acetamido-6-chloro-S-( 2-deoxy-3,5-di-O-p-toluoyl-~-~(a-X) was obtained by the same procedure from a-IX in 507, ribofuranosy1)purine ( 8-VIII).-Using a previously described yield after recrystallization from acetonitrile, m.p. 163-166", procedure,Io 31.8 g (0.0718 mmole calcd. as C17HaC1HgS603) [ a ]$48.1 ~ f 0.9" (methanol); At,:H mp (e), 220 (24,100), 246 of VI suspended in 1.9 1. of refluxing benzece was treated with 30.1 g. (0.0773 mole) of 2-deoxy-3,5-di-O-p-toluoyl-~-ribofurano- (16,500), 312.5 (13,900); Rf 0.26. A m.m.p. with p-X was 153-158'. syl chloridez8(VII). The chloroform solution of the crude prodAnal. Found: C, 55.0; H, 5.09; N , 18.8; S, 8.68. uct, which had been washed with aqueous KI,concentrated to 2-Amino-6-methylthio-9-( 2-deoxy-a-~-ribofuranosyl)purine 350 ml., and chilled overnight a t 5', afforded 12.75 g. (31.57,) of ( CY-XIV) was prepared from PIX in aqueous NaOH solution with crystalline 6-anomer, m.p. 180-187'. Recrystallization from methyl iodide.3 The product (78% yield) was purified by rechloroform (ca. 36 ml./g ) afforded 11.11 g. (27.47,), m.p. 189crystallization from water (53y0yield) rather than by treatment 190", [a]z4n -27.2 ==! 0.4" (chloroform). An infrared band a t with boiling ethan01,~m.p.104-107" with softening a t 96", [ a ]2 4 ~ 13.52 p (m), indicative of a chloroform solvate, was removed upon $57.7 f 0.8" (methanol);2 : : :A mp (e). 220.5 (18,300), 245 recrystallization from methanol (250 ml./g.) of a sample for anal(15,600), 310 (11,900); Rr 0.37. ysis, m.p. unchanged; [aIz4D-28 7 f 0.4" (chloroform); Anal. Calcd. for C1,H1jN,03S.O 5H20: C, 43.1; H, 5 20; mp ( e ) 231 (48,900), 243 (sh, 38,800), 285 (10,400). S,22.9; S,10.5. Found: C,42.6; H , 5.31; S,22.8; S, 10.4. Anal. Calcd. for C25H&:C106: C, 59.6; H, 4.65, C1, 6.29; 2'-Deoxyguanosine ( p-XIII).-2-Amino-R-( 2-hydrouyethylN, 12.4. Found: C, 59.7; HI 4.59; C1,6.14; N, 12.2. (8-XII)was prepared 2-Acetamido-6-chloro-9-( 2-deoxy-3,5-di-O-p-toluoyl-a-~- thio)-9-(2-deoxy-~-~-ribofuranosyl)purine as a sirupy intermediate from P-IX,by the procedure for 6-(2ribofuranosy1)purine (a-VIII).-The mother liquor (350 ml. of hydroxyethy1thio)purine.25 The isolation involved filtering the chloroform, plus washings) remaining after initial separation of dimethylformamide reaction solution to remove inorganic solids the crude (12.75 g.) 8-VI11 was diluted to 1 1. with ethyl ether. and concentrating it 2% vacuo. A 0.2 JI NaOH solution (10 ml.) After standing a t 3' overnight, a small amount of gum (to be disof the sirup was stirred for 3 hr. at room temperature; a white solid carded; some @-VI11 was present) had separated, and the superseparated but was discarded, since a paper chromatogram showed natant was decanted and diluted further with 2 1. of petroleum no trace of deoxyguanosine. The filtrate Bas neutralized with ether (b.p. 30-60'). Upon standing a t 3" overnight, 10.53 g. 50% acetic acid and concentrated to form a white solid residue 126.07c) of amorphous solid separated, [aIz4n-51.1 j= 0.9" which did contain deoxyguanosine. This was dissolved in hot (chloroform). Further purification (ca. 607c recovery) was water (70 ml./g.) and stored for several weeks a t 5-20' to convert achieved by reprecipitation of a sample (1 g.) from chloroform the resultant gel to a crystalline solidz3(20'5 yield). Two further solution (20 ml., treated with decolorizing carbon) with petroleum recrystallizations from water occurred with increasing ease, ether (250 ml.); just as in the isolation procedure,'O a benzene and the material exhibited physical properties identical with solution (30 ml.) of the precipitate was washed with aqueous KI, those of a sample of recrystallized natural 2'-deoxyguanosine dried, and diluted with petroleum ether 1500 ml.). The resultant f 0.7" us. -44.0 when compared as f o ~ ~ o w[a]24D ~ ~ ~-42.7 : amorphous precipitate, [CY] 2 1 ~ -54.8 & 0.8" (chloroform), f 0 7" ( c 0.3 in water); [ a ] " ~-39.2 j= 0.6' us. -40 1 i 0 6' mp ( e ) 232.5 (49,800), softened to a clear glass a t ca. 92'; (0.1 M sodium hydroxide); A:i mp (e), 252.5 (14,100 us. 14,245 (sh, 36,900), 284 (10,800) Easy solubility in chloroform loo),: ! :A mp (e) 222.5 (3590 us. 5850), Ai:: 0.69 us. 0.69, Ai:: was in marked contrast to 8-VIII. 1.15 us. 1.16; X~:,'vN"oH mp (E) 265 broad (11,300 us. 11,600); Anal. Found: C,60.1; H,4.58; C1,5.87; N, 11 7 . Rr 0.60 us. 0.60. X-Ray powder diffraction patterns were iden2-Amino-S-(2-deoxy-p-n-ribofuranosyl)purine-6-thiol (p-IX).tical in spacing and intensity. A 5.08-g. (9.00 mmoles) portion of &VI11 was dissolved a t reflux 2,6-Diamino-9-( 2-deoxy-a-~-ribofuranosyl)purine ( wXI) .in 540 ml. of anhydrous methanolic H,S,z9 and the hot solution Use of method A in ref. 10 with 400 nig. (0.709 mmole) of a-VI11 was treated with 27.0 ml. of 31 methanolic KaHSZQ,30 and refluxed in methanolic ammonia in a sealed bomb a t 100" for 5 hr. afforded for 2 hr. Following removal of the HqS source and additional 245 mg. (1127,) of crude product, Rr 0.26, containing a conreflux for 15 min., 13.5 ml. of M methanolic SaOCH3 was added, taminating nucleoside of Rr 0 5 0 which was removed by two not ether and the solution refluxed for 1 hr. In the is~!ation,*~ recrystallizations from ethanol-methanol (9/1). The amorphous but chloroform was used to wash the aqueous layer. The crystalsolid (15y0), m.p. 167" dec , was homogeneous, Rr 0.26; [aIz1o line product weighed 2.24 g (S7.570); [a]% -32.0 f 0.5" (0.1 JI sodium hydroxide); A~:;vhao" mp ( E ) , 251 (13,600), 270 (28) M. Hoffer, Chem. Ber., 93, 2777 (1960). (29) Method C in ref 10. (30) J. -4.Johnson, Jr. a n d H. J. Thomas, J . Am. Chem. Soc., 78, 3863 (1956); H. J. Schaeffer and H. J. Thomas, zbzd., 80, 4896 (1958).
(31) Ref. 20b, method A at bottom of p. 586. (32) Values for t h e natural material a r e given second a n d t h e numbers italicized. Ultraviolet d a t a agree with those of Venner*s; extinctions are calculated for t h e monohydrate.
