NOTES
1'45
by a procedure analogous to that used for selective N 4-acylation of cytidine." For comparative studies with deoxycytidine deaininase16several S4-hydroxycytosine nucleosides were prepared in which the sugar moiety was n-ribose or 2-deoxy-n-erythroperitose (L'i2-d~ox\.-i)-rihose11) instead of n-arabinose. The compounds selected for these studies were the S4-hydroxy derivatives (5) of cytidine, 2'-deoxycytidine, 2'-deoxy-5-fluorocytidine1 and 2'deoxy-5-methylcytidine. N4-Hydroxycytidine and 2'-deoxy-S4-hydroxy-5iiiethylcytidine had been synthesized previously8 by hydroxylamination of 4-thiouridine and 4-thiothymidine. The reported procedures was modified by the use of milder conditions and the yields have been improved. I n the case of 2'-deoxy-5-fluoro-~4-hydroxycytidine (5b), it was advantageous to S-methylate 4-thio-5-fluorouridine3 with diazomethane (quantitaHO HO tive yield) and to treat the resulting 4-methylthio derivative with hydroxylamine. During the treatment of 4-thiouridine (4.5 mmoles) with a tenfold excess of anhydrous hydroxylamine in 40 ml of methanol containing 1ml of water, a "bishydroxylamine" derivative formed as indicated by the disappearance of uv absorption at 328 mp and the concomitant rise of a strong peak a t 222 nip. The white solid obtained from this reaction gave elemental analH d R"' H O OH yses consistent with 6 obtained by Brown and Schell12 from cytidine. Acid treatment of 6 gave the known 0 series, R ' = H R=ocetyl, benzoyl, or H S4-hydroxycytidine (5aJR"' = OH). The formation R"=OH, NHz,CH3 or LOCH3 b series, R ' = F of a bishydroxylamino derivative (6) in the reaction of R " ' = Hor OH hydroxylamine with a 4-thio precursor has not been noted previ~usly.'~ (known to be more stable under thiating conditions) For practical considerations, more facile preparations would give higher yields of pure 2b (R = COPh). The of N4-hydroxycytidine were made when considerably available 1-(2,3,5-tri-0-benzoy~-~-~-arabinosyl)-5-fluohigher dilutions (4 mmoles of the thione/250 nil of rouracilg (1) was employed. Better yields of 2b (R methanol) were employed and hydroxylamine was = COPh) were obtained in the thiation step, alregenerated in situ from the hydrochloride salt. Under though in this case also, more than one PzSj treatment these conditions, the "bis" intermediate (6) was not was required.10 Conipound 2b (R = COPh) was deisolated. That 6 was an intermediate in those highesterified in alkali and S-alkylated to 3b. Thismethyladilution reactions was shown by spectral monitoring of tion step (2b + 3b) was used because, as reported prethe reaction mixture (appearance of a strong maximum viously,j the methylthio group is more easily replaced at 222 mp which is converted rapidly in acid to the S4by nucleophiles than a 4-thione group. Thus, milder hydroxycytidine spectrum with a maximum a t 280 conditions may be employed for the conversion of 3b + mp). 4b without concomitant replacement3 of the 5-fluorine When a dilute solution of 4-thiothymidines was atom. Treatment of 3b with hydroxylamine in treated with hydroxylamine in anhydrous methanol, a ethanol or with anhydrous hydrazine (99%) at room high yield of 5 (R' = CH3, R"' = H) was obtained. temperature yielded 4b (R" = OH or NH,, respectively) in good yield. The methylamino analog 4b (11) IC. A . Watanahe and J. J . Fox. Angeiu. Chem. Intern. Ed. E n d . , 6, (R" = CH3) was obtained by treatment of 3b with 579 (1966). (12) D. 31. Brown a n d P. Schell, J . Cliem. Soc., 208 (1965). liquid methylamine in a sealed bomb at room tempera(13) These d a t a do not prove t h a t t h e conversion of 4-thiouridine t o ture. Na-hydroxycytidine proceeded esclusiuelg via intermediate 6 . Kinetic exSimilarly, the N4-hydroxy7 -amino, and -methyl periments b y Lawley" showed t h a t a significant portion of t h e reaction of cytosine and 2'-deoxycytidine with NHIOH under aqueous conditions proderivatives of 1-p-n-arabinofuranosylcytosine(4a, R" ceeds by direct displacement of t h e exocyclic amino group b y NHIOH. = OH, hT2, and CH3, respectively) were synthesized This latter mechanism had heen postulated b y Janion and Shugar's for t h e reaction of aqueous ljH20H a t p H 6.5 with 5- and 6-alkylated cytosines and from l a (R = COCH3) by a series of reactions essenwith certain 4-alkoxy-2-pyrimidinones. T h e conversion of t h e S-methylated tially similar to those described in the conversion of arabino nucleosides (3) t o their Nd-hydroxylamino analogs (4, R" = OH) Ib + 4b. The ?J4-acetylderivative of 4a was prepared reported in t h e present paper ( v i d e supra) a e r e performed a t 70-80' in SCHEME I
HoHzcsI HoH2cd,
(9) T h e authors are indebted t o Dr. A. Nusshaum of Hoffmann-La Roche, Inc., Nutley, N. J., for a generous gift of this compound. (10) Apparently t h e difficulty in thiating t h e 4-oxo group of lb (R = COCHi or COPh) is encountered only u i t h t h e 5-fluoro-arahzno nucleosides. mho analogs of 1 thiate normally. This unexpected characteristic of lb is not clearly understood and is worthy of Investigation.
anhydrojis E t O H , conditions which differ from those of t h e other imestigstors.'2.!~~16 I t is known t h a t t h e 4-methylmercapto groups of 2-pyrimidinones and their nucleosides are easily displaced b y nucleophiles,'6 which suggests that t h e conversion of 3 t o 4 (R" OH) probably proceeded cia direct displacement of t h e 4 substituent b y KHzOH. (11) P. D. Lawley, J . 3101. B i d . . 24, 75 (1967). (15) C . Janion and D. Shugar, Acta Biochim. Polun., l a , 337 (1965). (16) Y. hlizuno, hI. Ikehara, and K. A. Watanabe, C h e n . Pharm. BUZZ. (Tokyo), 10, 653 (1962). I
Spectrophotoiiietric motiitoririg of the reaction mixture did tiot reveal the pre-eiicc of :my :in\. "bishj-drox! 1:miiiio" intermediate nor could such a i i iriteriiiediate lw isolated froiii the rcactiori mixture The results u ith I-thiouridiiic and 4-thiothymidine described above point to the iiiarlted effect which 5-alkylation exerts oil the courbe taken for the diiplacciiieiit of' the 3-thiolic by hydroxylaiiiiiie in these riucleohideb. These effects are probubl\. altiii to observntioiisi2I 4 li iiinde for the rcactioii of C,I tosiiies aiid .j-niethylcvtoqiries with hydroxylamiiie. The ultraviolet absorptioii data for the cationic arid iic~itr:il qpcciei'7 of the S4-suhitituteti derivatives 4a :tiid 4b, :ire -howii iri T:iblc T. togcther with the corre-poiidiiig dat:t for the un~ubstituted :trialogs. Thr fluoriiiated compounds (11' = 17) exhibit a bathochroinic i h i f t of ca. S nip it1 the -pectral patterri relative to their .-,-iiiisiil)htitutedarialogs (€1' = H ) . Yiiiiilai. shift hatxwi I cported previouily ! for l-,,j'-i)-ribofurmoy 1-5fluorocytohine, ~'-deoxy-;-,-fluorocytidiiie. arid wiiie of coinpared with CJ tidiric. L"-deoxycyt ih e , aiid the corresporiding deriv:ttive-. 111 :iddition, the preserice of R fluorine atoiii in the 5 positioii of thcie l-p-D-ari~biiiofur~~iio~ylcStosirie nucleo-ides h:is I iii:irked base-neakeiiirig effect (ca. 1 \ iiiiits) oii the cationic pK, V:IIUCX~ of the corresponding .hiisiihbtituted arialogt (5ee thc l a y t coluniri of t h r
Experimental Sectionz" I-( 2,3,5-T~i-O-benzoyl-~-i~-arabinosyl)-5-fluoro-4-thiouracil (2b, R = COPh).--C~i~~~I,t~iiiiti lb ( R = COPh,' 20 g, 0.0:3.i mole), i i i SO0 nil of p > ~ i d i i i wiw e treated with 15.3 g (0.07 molej of P&, arid the viell-.lirred -uspeiisioii was heated t o reflrix. As solrition o c ~ i i r r e t l 11.0 , m l of HyO -,YEAS added cautiously iiritil it semiturbid, oily qii>p ol)t:iitied. The reaction mixtiire
NOTES
was refluxed for 4 hr. Progress of the thiation was monitored by examination of small aliquots. Repeated evaporation of these samples with 5Oc;C EtOH removed all the pyridine. The residual syrups were dissolved in EtOH and the spectrophotometric ratio 2701333 mp was obtained. This ratio should be 0.45-0.55 for completely thiated product. A higher ratio indicates incomplete thiation and the necessity for the addition of a second charge of PpSj (ca. 10 9). The reaction mixture was cooled somewhat, arid the addition was made cautiously to prevent foaming. The thiation was usually completed in ea. 10 hr and with the addition of a t least one extra portion of thiating agent. The black reaction mixture was cooled, and the pyridine was decanted from a solid which was washed well with CHpClp and discarded. The combined solvents were evaporated in t'ucuo to a thin syrrip which was poured into a well-stirred HpO-ire sliirry. Stirring was continiied for 0.5 hr iintil the precipitate had become completely granular. The somewhat colloidal suspension was filtered, and the collected precipitate was washed throroughly with H2O. The precipitate was then dissolved in CHf& and the solution was dried (NapS04). The solvent was removed in vacuo, and the residual, thin syrup was poured with stirring into 600 ml of hot EtOII. The solution was allowed to cool slowly and finally refrigerated overnight. The bright yellow precipkate was filtered, washed with cold EtOH, and finally with EtpO. The yield of piire product was 17 g (83Yc), mp 187-189". Evaporation of the mother liqiior yielded a further crop which was recrystallized from EtOH, 1.9 g, mp 182-186'. Anal. Calcd for Cp,I123FNpO&: N, 4.74; 8, 5.43. Found: N , 5.03: S,5.23. l-~-~-Arabinofuranosyl-5-fluor0-4-thiouracil (2b,R = H).*lCompomid 2b ( R = COPh, 30 g, 0.05 mole), 400 ml of hIeOH, 200 ml of I€&, and 200 ml of 1 NaOII were stirred at room temperatrire for 3 hr. The reaction mixtiire was treated with portion.: of Ilowex 50 ( H + ) resin iintil a neiitral pII was attained. The resiii was filtered and washed with two 100-ml portions of lIeOI1. The combined filtrated were evaporated in vacuo to a thick syrup which was dissolved in hot H 2 0 and cooled slowly. The granular precipitate was filt,ered, trihrated thoroughly with CHZC1, and air-dried; 13 g (937&), mp 172-173'. A portion, recrystallized from hot EtOH, melted at 177-179". Anal. (C,iHiiK,FOjS) C, H, X, F, S. 1-~-~-Arabinofuranosyl-5-fluoro-4-hydroxylamino-2( 1H)pyrimidinone (4b, R" = OH).-A solution of NH20H22 (0.05 mole) in 100 ml of absolute EtOH was added to a stirred solution of 3b (1.5 g, 0.005 mole)g in 20 ml of EtOH. The clear solution was stirred and heated at 70-7.5" for 2 hr. The solvent was evaporated in vacuo. Hot absolute EtOH was added to the residue, some insolubles were filtered, and the filtrate was reduced to ca. 20 ml and cooled. The crystalline precipitate (1.3 g, mp 15301 effervescence) was recrystallized from a minimum of EtOH, and the pure product (0.9 g, mp 157-15S0, effervescence) was obtained as a hemialcoholate, [ a I z 3 D +101" (c 0.4, H&). Anal. (CQHIPFNaO6.0.5CzHjOH) C, HI N. 1-p-~-Arabinofuranosyl-5-fluoro-4-hydrazino-2( 1H)-pyrimidinone (4b, 11" = NHp).-A stirred solution of 3b (274 mg, 0.001 mole) in 5 ml of EtOH was treated with 1 ml of EtOH containing 0.0.5 g of anhydrous NH2NH2 (99.9%), and the reaction mixture was left at room temperature for 20 hr. The solvents were removed in z " m (bath -25"). The residual orange syriip was dissolved in 5 ml of JIeOH and treated with IO ml of EtpO previously saturated a t 0" with HC1. The HC1 salt precipitated as yellon- needles, 350 mg, der pt 178". The prodtict was recrystallized by solution in hIeOH and addition of Et20 t,o incipient turbidity. An analytical sample of the product had mp 178-179" (effervescence), [ c Y ] * ~ D +140" (c 0.3, HzO). Anal. (CgHipFN4Oj.HCl) C, H, N. 1 -p-~-ArabinofuranosyI-5-fluoro-4-methyIamino-2( la)-pyrimidinone (4b,R" = CHa).-Compound 3b (500 mg) was placed in a glass liner of a bomb tube and chilled in a Dry Ice bath. l I e N H z gas %vasintroduced into the chilled liner until ea. 15 ml of the liquid amine had condensed. The bomb was sealed, left a t 7 \ L
(21) This compound was isolated as a syrup in ref 6 . ( 2 2 ) T h e KH?OH was prepared i n situ a s follows. T h e correct molar amount of NH?OH.HCl x-as dissolved in a minimum of hot MeOH and allowed t o cool t o room temperature. One drop of phenolphthalein was
added t o the solution which was stirred and treated with a freshly prepared solution of NaOSIe in a minimum of MeOH until a faint pink color persisted. (Over-neutralization must be avoided since t h e 4-hydroxylamino nucleo3ides are rather unstable t o alkali.) T h e precipitated NaCl was r+ m o v d , and t h frep ~ NII?O€I solution was used immediately.
147
room temperature for 20 hr, cooled, vented, and opened, and the excess RIeNH, was evaporated under a stream of air. The residue was dissolved in hot EtOH, and the solvent was evaporated in vacuo. This process was repeated several times to remove any unreacted RIeNH?. During this repetitive evaporation, ,some crystallization occurred. The small amount of granular precipitate was filtered and washed with EtOH: mp 218-219". The filtrate was evaporated to a syrnp which resisted further crystallization. Therefore, the solid and syrup were dissolved in MeOH and treated with EtOH-HC1. The precipitate was crystallized from MeOH-Et20. T h e yield of the hydrochloride was 0.7 g, mp 18&181" (effervescent), [a1230 + l S l " (c 0.5, HYO). Anal. (CloHi,FY,O,.HCl) H, F, ?;; C : calcd, 38.53; found, 39.09. l-~-~-Arabinofuranosyl-4-methylthio-2( 1H)-pyrimidinone (3a). -Compoiind (R = COCHa, 12.5 g, 0.0034 mole) was dissolved in 200 ml of reagent grade pyridine and the efficiently stirred solution was treated with 14.3 g (0.064 mole) of P&. The temperature of the reaction mixture was raised until reflux began. Water ( 5 drops) was added cautiously, and the amber reaction mixt,ure was stirred and refluxed for 3 hr. The cooled reaction mixture was filtered, and t,he precipitate was washed with a lit,tle pyridine and discarded. The filtrate was evaporated in vacuo t,o a thick syrup which was treated with SOYc EtOH and the solvent was evaporated. This addition and reevaporation process was repeated several times, thereby removing most of the pyridine. The graniilar, amber residue was extracted with CHCl3, and t,he extract was filtered to remove insolubles. The CHC13 filtrate was evaporated to a syriip which was dissolved in 50%, EtOH and filtered and the filtrate was evaporated. The residue was crystallized from hot 7.i% EtOH. The product was obtained in two crops, 12.3 g, mp 110-115". Recrystallization from hot CaH6-EtOH gave platelets, mp 113-117" (sintered at ra. 100'). This prodiict, withoiit fiirt,her piirification, was used directly for the methylation reaction. Comporind 2a (R = COCII,, 12.3 g, 0.032 mole) was stirred and heated with 14.2 g (0.1 mole) of Me1 in a mixture of 100 ml of MeOH and 30 ml of HpO. NaOH (1 S,32 ml) was added dropwise. After the addition was complete, the solution was stirred for 1 hr and then neutralized to pH 5 with dilute AcOH. The JIeOH was evaporated in vacuo, and the aqueous soltition was chilled. A precipitate of long needles, 7 . i g (84% based on la), was obtained. A portion, recrystallized from HSO, gave pure 3a, mp 123-124". ilnal. (CloH14Y20jS) N , S. 1-P-~-Arabinofuranosyl-4-hydroxylamino-2( 1H)-pyrimidinone (4a,R" = OH).-A stirred solution of 3a ( 2 g, 0.0073 mole) in 50 ml of EtOH was treated with 100 ml of EtOH containing 0.05 mole of iYH20H.*Z The mixture was stirred overnight at room temperature. A4second 100-ml portion of EtOH containing 0.05 mole of NHsOH was added, and the reaction mixture was refluxed for 3 hr. The solvent was removed in vacuo, and the residue was dissolved in a little HI0 and applied to a column of Dowex 50 (H+),previously washed free of uv-absorbing mat,erial. The column was washed with HzO until all starting material was reNH,OH, and the moved. The column was eluted wit,h 1 product-containing fractions were combined and evaporated in vacuo to dryness. The residue was crystallized from hot, EtOH; yield 0.8 g, mp 130-131" (effervescent). One recrystallization from EtOH afforded an analytical sample of the hemialcoholate, ~D l 0 l o ( e 0.3, H 2 0 ) . Anal. mp 131-132" (effervescence), [ L Y ] ~ + (C8Hi,S3Oa.O.5C*HjOH) C, H, S . l-p-o-Arabinofuranosyl-4-hydrazino-2( la)-pyrimidinone (4a, R" = NHs).-To a solution of 3a (1.32 g, 0.0048 mole) in 30 ml of EtOH was added slowly 0.35 ml of N H z S H ~ . H Z O The . reaction mixture was refluxed for 3 hr and cooled. Colorless needles, precipitated; 1.15 g, mp 219-220". Recrystallization from 80% EtOH yielded a pure sample, mp 219-220", [ a ] " D +15S0 (C 0.4, H,O). Anal. (CgHiaNiOj) C,H, 1. 1-p-o-ArabinofuranosyI-4-methylamin0-2( 1H)-pyrimidinone (4a,R" = CH~).-Compound 3a (1.0 g, 0.0036mole) was placed in a well-cooled] glass-lined bomb and ea. 10 ml of liquid JleiYH2 was added. The bomb was sealed and left at room temperature for 20 hr. The excess JleNH2 was evaporated in a stream of air. Upon addition of EtOH to the residue, crystallization occurred. T h e crude product, 1.1 g, m p 258-259' (effervescence), was recryst,allized from 60% EtOH; colorless needles, 0.9 g, mp 264-
(23) D. 11. B r o n n , A . Todd, and S. Varadarajan, J . Chrm. Soc., 2388 11056).
