428
Iodination of 2'-Deoxycytidine and Related Substances'
P'-l)eosycpt~idinehas been iodinated in the presenre of iudic acid to yield 5-i[
dro-.5,B-diiododeoxyuridine:the lat,ter, on treatment n-ith base, produced 64 :m estremely useful prerursor of deoxpuridine-€i-H3.
The same conditions 11: idine to yield S-iodo-:!'-deoxycyt,idine 5'-phosphate and preparation of 5-iododeosgcytidine-1125 and the acetylat,ion of thc deoxyrihr)nnnleosides are described. I11 an earlier brief coiii~iiuiiicatioii,~ n e have reported tlie preparation of 5-iododeoxycytidine (II), a conipound that exhibits greater cytotoxicity than its bronio analog3 and, in sonic niammalian systems, compares favorably to 5-iododeoxyuridinej as either a cheniotlierapentic agent or one to reduce the threshold of cell* to radiation injury.6 Biological studies of b i o d o dcoxycytidine with cells in culture and in animals.' :IS well as in r11ar1,~ lo have demonstrated that I1 is not readily converted to 5-iododeoxyuridiiie .j'-phosphatc 1% tlie intermediation of 5-iododeoxycytidine -5'phosphate; however, I1 per sc i:, rapidly deaminatrd hy ai1 eiizynie variably distributed in tissue:, (part icrilarly kidney, liver and intestine), witli the releaw of 5-iododeoxyuridine; this, in turn, is converted intrac~llularlyto phosphorylated forrrib of .i-iododeoxui*icliiic." It is clear, however. that as predicted.'? 11 i i iiot cleaved biologically to 3-iodocytosiiie, in roiitrait t o 5-iododeoxyuridine, froni nliicli 5-iodoiiracil i k toriiied -\.cry rapidly, particularly in tlie 1i-\.er.l3 AItliough the hiological activity of I1 is still under investiSation, its potential utility in man has hecn influenccd adversely by the finding that sonif' individuals convert I 1 into IT-DI1 too slowly to permit IT t o l i p iised effectil-ely as a reservoir-form of 5-iododeoxyiiridine It has been established that .i-iododcoxSuridin(. call .:upprcs- profoundly tlie de\ clopnir1iit of coi.iieal lesionh caiizcd hy herpes hirnplex Lririi*'4- I R ~ v l i c i i appliid topically, and the dernial lwioiis of vaccinia 1-inis i n f w tions. i i i hoth rabbit and iiian, when tlic d n i g i \ acl-
iimii+tercd parenterally. Ii I k e i i t htudicl. i v i t l i huniaii adenovirus. type 12, highly oncogenic i i i iien-born haiu\ters, lmx c denioiistrated that .i-iododeoxyiiridiiit~,111jected in the bani? rcgioii as the. x i i u , almost UJNpletely prevtwts tlic drvelopnieiit of ncoplaznii." ' V l i i l \.olunie of solutiou 10.1 nil.) that caii bc giveii to t l i r ~ w iie~vboriiIianisters lias restricted severely tlic. iiiaxinial aniouiit of 3-iododeor;yuridiiic that can lw aciiiiiiii~tri.c~l (O,,jn i g . ) . Iiow~vc~r, tht. greater solubility of II arid ti rapid deaiiiiiiation 1)y n e ~ b o r i lianistei* i tissiirq p m " t 1argi.r dowi of IT tliaii of 5-iododeoxvuridiiIc arid coniparstircl studio. of the. effect, of t l i c w agelit.: a i ~ xiiou I I I progrcw .Ilthoiigli deoxycytidine (1) I* i ~ ~ l i cl . yh l o i ~ i i a t t 0~1 i hioniiiiated,' i t cannot bo iodiiiated t)y staiidald proc ~ d i i i ~ h Iodiiiation . i l l diluttl iiitric acid, iiiider c+olid~iiiiilar to those used in the preparatioii ot 5-iododeoxyuridiiic,5 prodiiccd 0 1 1 1 ~ 5-iodocytosiiic. '1'11~ standard 1)asic iodination ( i i i t l i ( x presence‘ of potaz4iiiii hydroxide a i d potasbium iodide) similar to that i i s ~ d hy ,Johiisoii and ,JohiislYfor the iodination of cytosliics, led t o the production of deaminated iiiatcrials Tt m:~y 1 ) suggested ~ tliat cytosinc. i i i irliicli the S I positioii i* iiiisubstitutcd, in msily lodiiiatcd iii the lmqic coiiditioii I I I tlic follon iiig i i i ; i n i i ~ i
i,1) T i i s a o r k u-as supported by grants froni the Sational Cnncer Insti-
I-Iealth Service (CT-2817), a n d from t h e Ameriran Cancer Soincluding its Connecticut Division. P. K. Chang a n d A . D. Welch, B i o c h r m . I-'harmnrol., 8 , 327 (19611. I ) . 31. I'riscli and D. n-. Yisser, .I. - 4 m Chrm. S O C . . 81, 1756 (19Rli). P. K. Charig a n d .\. D. K e l r h , Bioche?n. Piiarmaroi.. 6, 50 (1961). IT. If. Prusoff, Biuchetn. B i o p i i y s . Bcin, 32, 295 (1959). J. 17.. Crarner, K. 11. Prosoff. -1.C. Sartorelli. I. F.Delamore. P. Ialabresi.S.S. Cardoso, 9.C. Finch, AI. AI. Kliner-nian. C. 3 . !'on E-sen, XI. T.Chu, and .I. D. Welch. i h i d . , 21, ,550 (1901). (1-1) 11. E. Kauirnan. P r u c . SOC.E.rptl. Bioi. J l r d . , 109, 2 3 1 (1962). (15) 11. E , K a u f m a n , E. I,. ~ I a r t o l n and . C . L)ohliiian, A r c h . Ophtlialmoi.. 68, 253 (19621.
( )bviously, t l l l h callllot occur 111 the C a s oi dcoaycyt Idiiie (I); furthermore, the amino group in I shoidd t ) ( ~ more labile because of the decreased aromaticity of t l i v pyrimidine moiety. By caarrying out tlit. iodiiiatioii of dtwxycytidiiicx i i i tliv presencr' oi iodic acid. uiiclcr coiiditions similar to thosc used by Wirtli, rt ( t i f o i . tlie iodinatioii of aromatic coinpound*, (' obtuiiwti .S-iododeoxycytidiw (11) in 68C;, yield, together u ith :L 9-10?, yield of 5,ti-diliydro-3,Ci-cliiodo-2'-deor;yuI id~w (IT). It appears that two modes of iodiiiatioii : ~ I P taking place simultaneously, ie., substitiitioii aiid addition. with the former predominating. ,?'I
July, 1963
429
5-IODO-2 '-DEOXYCYTIDIXE
TABLEI ULTRAVIOLET ABSORPTIONSPECTRA* 0.1 .V HC1
0.1 N NaOH
Ha0
95% E t O H
b" (emsx) 292 5-Iodocytosineb 289 (3980) 305 (7000) 297 (5770) 293 (6020) 5-Iodo-2'-deoxycytidine 294 309 (8150) 294 (5930) 29T 294 (844G) 294 (9430) 5-1odo-3',5'-di-O-acetylS'-deoxycytidine .HCl 309 (9700) 5-Iodo-3 ',5'-di-O-acetyl-2 '-deoxycytidine 309 (9510) 294 (6790) 297 294 (6530) 291 (51601 293 (5130) 297 5-Iodo-2'-deoxycytidine 5'-phosphate 309 (7060) 6-Iodocytidine 295 2993 (5660) 309 (8280) 294 (5960) 324 (5950) N4-Acetyl-5-iodo-2'-deoxy-5'-O-acety~-cytidine" 329 (5560) 316 (9960) 323 5-Iodouracilbad 284 (6170) 296 (6950) 283 284 (6050) 5-Iodo-2 '-deoxyuridine 289 (7830) 280 (5790) 287 289 (7690) 289 287 (7690) 5-Iodo-3 ',5'-di-O-acetyl-2 '-deoxyuridine 288 (7976) 280 (5980) 5-Iodouridine 289 (8020) 280 (6170) 289 288 (7960) 6-Iodo-2 '-deoxyuridinec 285 (9030) "83 285 (9190) 278 (7250) a Spectra were determined with a Model 505 Bausch 6: Lomb Spectronic recording spec.trophotorneter. Commercial Unstable on standing in solution for several days. Xmax3a4 in 0.01 S S a 0 H . j Pyrimidine
dR
Amax
dR I1
dR Ia
I
(emax)
Xdition
NH
T x
0
0 deamination I
dR
I11
dR V
dR TV
dR
I n this case, iodic acid is the fortunate choice as the oxidizing agent, since, unlike nitric acid, it does not attack the glycosidic linkage, which apparently is chemically more labile in deoxycytidine than in deoxyuridine. It is of interest to note that the addition of iodine to the polarized double bond must occur before the deamination, since (a) the imino group in I11 is nom more labile than before addition of iodine and (b) deoxyuridine, under the same conditions, produces only the substitution product, with no trace of IV. This circumstance implies that in an acidic medium, the 5,6-double bond in I a is more reactive than its counterpart in deoxyuridine. It is also observed that deoxycytidine, a s the base, gives a higher yield of I1 and lower yield of IV, but requires a longer reaction time than does its hydrochloride; this finding is in accordance with the vital role of the protonation of the imino group. The structure of IV is established by its lack of absorption in the region of 250-320 mp and its dehydroiodination by base to form 6-iododeoxyuridine (V), a compound less stable than its 5-iodo isomer.5 The elimination from IV of the iodine in position-& rather than that in position-6, is contrary to the known examples of dehydrohalogenation of 5,6-dihydro-5,6dihalouracils, in which the N' position is unsubstituted.
Xmsx
(emax)
(emax)
(3160) (5750) (6110) (6010) (4080) (5500) (5720) (5750) (8050) (6820) (6660) (7310) materiai.
Catalytic hydrogenation of 1-produced deoxyuridine in excellent yield, a procedure that provides a useful route to deoxyuridiiie-6-H3, from which 3-iodo-deoxyuridine-6-H3 of very high specific activity can be prepared. A slight modification of the iodination profrom sodium cedure afforded 3-iododeoxycytidine-1125 iodide-112band deoxycytidine. Deoxycytidine 5'-phosphate and cytidine also can be iodinated in the presence of iodic acid, but with lower vield and longer reaction time. Because of their greater solubility than the deoxyribonucleosides in polar solvents, the isolation of these products is best accomplished by chromatography. 5-Iododeoxycytidine 5'phosphate and 5-iodocytidine were required in studies of the mechanism of action of the nucleosides of 5-iodopyrimidines and of the enzymatic deamination of a large series of substituted cytosine derivatives." I n order to increase the lipid solubility of these iodinated compounds in biological systems, the preparation of the acetyl derivatives mas investigated. The acetylation of 5-iododeoxyuridine proceeds smoothly to give the 3',5'-di-O-acetyl derivative in excellent yield; on the other hand, 5-iododeoxycytidine, depending on the conditions, gives different products. When acetyl chloride is used, the generation of hydrogen chloride apparently prevents the acetylation of the amino group and the 3',5'-di-O-acetyl derivative is first isolated as the salt. However, when the acetylating agent is acetic anhydride, acetylation occurs on the amino group and on only the 5'-position of the sugar. The product, 2 - oxo - 4 - acetamido - 5 - iodo - 1 - ( 2 - deoxy - 3,s - di0-acetyl-p-D-ribofuranosyl) pyrimidine,21 separates out a t this stage with no further acetylation of the 3'hydroxyl, because of its insolubility in the highly polar medium.
Experimental 5-Iodo-2'-deoxycytidine (11)and 5,6-Dihydro-5,6-diiodo-2'-deoxyuridine (IV).-A mixture of deo~ycytidine2~ (3.4 g., 0.015 (21) Shortened t o N~-acetyl-5-iodo-2'-deoxy-5,-O-acetylcytidine~in subsequent usage. (22) Melting points were determined in a capillary tube in a copper block and are corrected. Analyses by Schwarzkopf hlicroanalytical Laboratories, and by Midwest Microlab, Inc., Indianapolis, Ind. Woodside. N. Y., (23) Purchased from California Corp. for Biochemical Research. Los Angeles, Cal., and from Nutritional Biochemicals Corp.. Cleveland. Ohio; deoxycytidine hydrochloride may be used in place of t h e free base, b u t a slightly lower yield of the iodo compound is obtained.2
mole), glacial acetic acid (12 nil.), iodic: acid (1.35 g.j, iodine ( 2 . 2 5 g.), carbon tetrachloride (3 ml.), and water (4.5ml.) \vas stirred :it 40' for 95 min. To the now clear solution, n-ater (45 m1.j WIS added to yield 5,6-dihydro-5,6-diiodo-2 '-deoqwridiiie (IV) :0.66 g., 9.3%). Tho analytical sample was prepared by dissolving the crude material (IV) in tetrahydrofuran and reprecipitating G\addition of water, m.p. 130-134" dec. Anal. Calcd. for CgH121,S,0s: C, 22.42; H, 2.50; S , 5.S 1 ; I, 52.65. Found: C , 22.6Y; 11, 2.37; ?;, 5 . 3 7 ; I, 52.21. The filtrate from I V was extracted several times with carbon tetrachloride (600 1111.i . 'I'he aqueous layer, after concentration to dryness in z " x u a t 4U0, w:ts dissolved in niethanol (60 ml.) and again concentrated to dryness in z"xo :it 40"; this operation was repeated 5 time8 in order to reiiiove most of the acetic acid. The crystalline residue obtained wits taken up in water (45 ml.) and the solution was adjusted to pH 1 0 with sodium hydroxide (10 ,V); 5-iododeoxycytiditie (11) began to precipitate and refrigeration of dfter the mixture produced the rrudc product (3.6 g., li8:;). one recrystallization from water, 11 (3.2 g . ) melted a t 1 i 6 - & ? o dec.; the analytical sample wtts recryst,allized once niore from water, m.p. 179-182" dec. Anal. C:ilcd. for C9H121S301: C, 30.61; H, 3.43; S , 11.90: J , 35.94. Found: C, 30.68; H, 3.32; S , 11.95; I, 35.82. Additional ariiounts of 11 may be isolated froiii the inother liquors by use of :I charcoal column.' 5-Iod0-2'-deoxycytidine-I~~~.-Tothe shipping vial contairiing :ipprosiniately 10 inc. of sodium i0dide-1'~~ in 0.7 1111. of sodium 1iic:trbonute soluticin, 15.76 iiic./ni1.2' (-,-emitter: half-life, 60 ti:i>,s) arid sodium iudide-II", U.iYY iiic./ml. (p- and -,-emitter: )tit1 solids 0 . 3 ~ i i ~ . / i i i I .w:is , added deosycytiides I, iodine (300 ing.i, iodic: acid (180 nig. i , (0.4 nil,), and gluc-i:rl nwticacid (1.6 ml.) :in11 the mixture wvas atirrctl (stoppered) :it 45' for I05 inin. -4siii;iIl :iincirint of \viis renloved hy fi1tr:ition and washed thoroughljwith water. The filtrate and waehings were collected in a T24/ 40 125-nil. erleniiieyer flask and shaken srver:il times with cxrlion tetrachloride, which was reiii,iveii liy means of a pipet each time. The aqueous layer, after xincentrution to dryness it( zac(m a t do", \WS dissolved in meth:inol ( 5 nil. j and a g : h C O n crntrated in caciio a t 40" to dryness (4times). The cqst:illine residue rrits taken up in water ( 5 x I-inl. portions fiir complete transference to a 10-nil. beaker), the soliition :~djustecito 1)EI IO with sodium hydroxide (12.5 2 drops'l :ind refrigerated overnight to yield tlie crude product (380 ing., 54:; ); this WV:ISrecrystallized once more froni Jv:tter to yield j-ii~dotlei~syc~-tidiiit!1 1 2 j (372 ing.; sp. act. 4.24 nir./mmolc; containing a sin:ill :LlllOUnt of 6-Iodo-2'-deoxyuridine (V).-A suspension of 5,6-d;hydru-5,& diiododeosyuridine (IV, 4.8 g., 0.001 mole) in n-ater (50 m1.j \r:is :idjusted t,o pH 11 with sodium hydroxide (12.5 S ) dropxise until :i calear solution resulted. The solut,ion was stirred for 90 min. :tnd the pH IIUIV adjusted to 7 \vitli coned. hydrochloric wid. Crude 0-iododeosyiiridiiie separated as a colorless solid (2.4 g. 1 . The analytical s:rmple was recryst,allized carefully from ethanc.il, 11i.p. 155-160° tltrc.; the coinpound decomposes on standing in :iqueous solution for several days. r2naZ. Calcd. for CUHlzINa06: C, 30.51; H, 3.11; S , i.91; I, 35.88. Found: C, 81.08; H, 2.73; S , 8.05; I, 35.34. Hydrogenation of 6-Iodo-2'-deoxyuridine (V).-To 360 rng. (0.001 mole) of 6-iododeoxyuridine in SOToethanol (100 ml.) were added 10% palladium-on-charcoal (0.2 g. j , and magnesium oxide (0.3 g.), and the niixt,ure was hydrogenated in :t Parr apparatus for 21 hr. at 3 atin. pressure. The catalyst and some magnesium oxide were removed by filtratiori :md the filtrate. shown by ultraviolet :hsorption t u writitin essentially deoxyuridine, \vas first tre:tted ~vitlia .q;Lfur:ited solution uf sudiuiii carbonate t o remove t h e IniLgriesiurii ion : ~ n dtheu nith a little Doives-50 (hydrogen forni) to remove the sotliiini ion. The solution (now pH 6) M concentrated in Z ' O C I ~ Oto give deoxyuridine (206.2 mg., 90 which behaved chrom:ttographically identically with an authentic sample. 5-Iodo-3',5'-di-0-acetyl-2'-deoxycytidine.--To a solution of 5iododeoxycytidine (5 g., 0.014 mole) in glacial acetic acid (100 ml.) was added acetyl chloride (150 m1J; some solid material :tppeared, but this redissolved on stirring. The mixture was stirred at room temperature for 17 hr. Some of the crude diacetyl procluct 12.47 g., hydrochloride salt. n1.p. 140-15Oo de(..') \ r u s wiriovrd 1)y filtr:itioii. The filtrirte w u cvncent,rated in mciut at
, 1 I,
I ' U ~ ~ I I : L IiCt u~i Iu Oak Itidgt: h~atiunvll . t ~ l w u t u r y , Ouk Ili I I .:tx.
