May 1969
NOTES
583
to the corresponding anomers of thymidine,1° and, as might have been anticipated, they were found to correspond to those previously determined from their ability to support bacterial and phage growth. 11.& \ I E R K O ~ S K I LSD D. S F I U G IR As was to be expected, the uv spectra of 5-ethyldeoxyDepartment of Radzobzology, Instztute of Oncology, and uridine were very similar to those of thymidine." Of Instztute of Bzochemzstry and Bzophyszcs, special interest was the fact that the pK, (9.9) for disA c a d e m y of Sczences, 11 arsaw, Poland sociation of the ring X3hydrogen, determined spectroKecezced February 4,1969 photometrically," was pracitcally identical with that for thymidine." It has been previously shown that 5-ethyluracil may The possible mutagenic activity of 5-ethyldeoxyuriundergo incorporation into bacterial DSX,2 and that dine mas investigated by Dr. Irena Pietrzykowska, with 5-ethyldeoxyuridine readily replaces thymidine (55-bromodeoxyuridine as a control, by means of the spot methyldeoxyuridine) in bacteriophage D N A 3 Partictest of Freese,I2 using 16 1-11mutants of phage T4 and ular interest attaches to 5-ethyluracil and its glycosides, looking for reversions to r+. Gnder conditions where since these would be expected to exhibit the same base5-bromodeoxyuridine gave the expected frequency of pairing properties as the parent compounds, i.e., uracil, reversions, 5-ethyldeoxyuridine was quite inactive. thymine, and their glycoside^.^ Analogous results were obtained by Professor C. HeidelThe syntheses of S - e t h y l ~ r i d i n e , its ~ , ~5'-mono and berger with a sample of V supplied to him. pyrophosphates, and poly-5ethyluridylic acid4s6have The potential antiviral activity of T' was examined by already been reported. The present communication deProfessor Heidelberger, who found that the a anomer scribes the preparation of 5ethyIdeoxyuridiiie (V) and was inactive, as expected, while the p anomer was alsome of its properties; preparation of its 5'-triphosphate most as active as 5-iododeoxyuridine and 5-bromodeis under way with a view to its subsequent polymerizaoxyuridine in inhibition of vaccinia viral replication in tion with either D polymerase or deoxynucleotidyl HeLa cells. I t is of particular interest in this connection transferase. The photochemical behavior of the nuclethat Guari and Ma10rny'~have reported V to be an oside in aqueous medium is also of special interest.' effective inhibitor of herpes simplex virus, although the The deoxynucleoside has been synthesized according preparation and properties of their compound were not to standard procedures8 by the condensation of the described. The simultaneous presence of antiviral acmonomercury salt of 5-ethyluracil (11) with 3,5-di-O-ptivity, and absence of mutagenic activity, is rather chlorobenzoyl- 2-deoxy-a, p-D- ribofuraiios) 1 chloride striking and suggests the advisability of synthesizing (111) to give I- (3,5-di-0-p-chlorobenzoy1-2-deoxy-u,~-~additional 5-alkyl-substituted pyrimidine nucleoside ribofuranosyl)-5-ethyluracil(IV), in about 507; jield analogs. The preparation and properties of 5-ethylwith respect to 5-ethyluracil. cytidine and 5-ethyldeoxy cytidine will he reported The mixture of the anomers of IT- was initiallj desh0rt1y.I~ benzoylated to give a mixture of the anomers of 5-ethyldeoxyuridine, which was deposited on a strongly basic Experimental Section Dowex 1 X 2 coluinn as described by Dekker,Y and 5-Ethyluracil was prepared according to the method of Burckeluted with T\",HC03 to give two reasonably wellhalter and Scarborough16 as modified by Shapira.5 defined peaks. Slow evaporation of these two fractions N-1-Acetyl-5-ethyluracil (I) was obtained as described by gave crystalline residues. One of these, but not the Spector and Kellerlo for other diketopyrimidines. 5-Ethyluracll other, proved capahle of sustaining growth of the (1.4 g, 10 mmoles) in 3 ml of XcaO containing 25-50 mg of pyridine was heated for 15-20 min under reflux. Several minutes of heating thymine-less strain I$scherichin coli 15T- and of some sufficed to completely dissolve the ethyluracil. The solution was of the T coliphage^^^^ and was therefore assigned the brought to room temperature and the crystalline precipitate v,as configuration p. filtered off and washed (AclO) to give 1.41 g (7Sc,) of I, mp However, subsequent attempts to effect the above 142-145". Monomercury Salt of 5-Ethyluracil (II).-To a solution of separation on a preparative scale, with the aid of larger 2.46 g of Hg(0Ac)g in 77.5 ml of anhydrous AIeOH, heated columns, were unsuccessful. Preparative thin layer under reflux and constantly stirred, was added 1.41 g of I. chromatography was then tried and the tlyo benzoylated Heating was continued for 2 hr and the mixture then was left anomers of IV, but not the free nucleosides, were readily overnight. The resulting precipitate was filtered off and dried to separated and obtained in highly purified, erg stalline give 2.65 g (100%) of 11, mp >360." 3,5-Di-O-p-chlorobenzoyl-2-deoxy-or,~-~-ribofuranosyl chloform. S m r spectroscopy was then applied to establish ride (111) was prepared according to Fox, et u Z . , ~ by chlorination of the configurations of each of the anomers, by reference A N o n m u t a g e n i c T h y m i d i n e Analog w i t h Antiviral Activity. 5-Ethyldeoxyuridinel
(I) This investigation was supported in part by the IVellcome Trust, the World Health Organization, and the dgricultural Research Service, U. S. Department of Agriculture [UR-E21-(30)-32]. (2) 121. Piechowska and D. Shugar, Biochem. Biophys. Res. C o m m u n . , 20, 768 (1965). (3) (a) I. Pietrzykowska and D. Shugar, ibid., 25, 267 (1966); (b) I. Pietrzykowska and D. Shugar, d c t a Biochim. Polon., 14, 165 (1967). (4) D . Shugar, h l . &ierkonski, A I . Fikus, and D. Barszcz, 7th International Congress of Biochemistry, Tokyo, 1967, Vol. I, Sj-mposium 1, pp 59-60. ( 5 ) J. Shapira, J . O w . Chem., 27, 1918 (1962). (6) AI. Swierkowski and D. Shugar, in preparation (see also ref 4). ( 7 ) I. Pietrzykowska and D . Shugar, Science. 161, 1248 (1968). ( 8 ) J. J. Fox, S . I-ung, I . \$-empen. and 11.Hoffer, J . Am. Chem. Soc., 83, 4066 (1961). (9) C. .1.Dekker, ibid., 87, 4027 (1965).
methyl a,P-2-deosy-~-ribofuranoside.~~ The product, following prolonged washing with Et,O, had nip 113", as compared t o a literatures value of 118-120". Other observers's have also
( I O ) R. U. Lernieux. Can. J . Ckem.. 39, 116 (1961). (11) J. J. FOXand D . Shugar, Biockim. B i o p h y s . Acta, 9. 369 (1952). (12) E. Freese, Proc. N d . d e a d . Sci. U . S . , 45, 622 (1959). (13) K. K. Gauri and G . Zlalorny, Naunyn-Schmiedebeiys . I rch. P h a r m a k o / . E z p . Pathor., 257, 21 (1967). (14) T. Kulikowski and D. Shugar. in preparation. (15) J. H. Burckhalter and H. C. Scarhorough. J . . l m . Phaf'm. .Issoc., 44, 54.5 (1955). (16) L. B. Spector and E. B. Kellei, J. B i d . Ciieni., 232. I83 (1958). ( 1 7 R . E. Deriaz, JT.G. Overend. XI. Stacey, and L. b'. V'iggiris, J . C h r m . S o c , 2836 (1949). (I81 H . J. hliunemeyer, P. B. Clarke. aud H . Tieckelmatin, J. M e d . Chem., 7, 567 (1964).
reported difficulties in obtaining a product with nip 118-120 '. I t is therefore of interest that, during chlorination, the resultiiig precipitate of I11 ronsisted initially of needles, followed hy tiny platelets; prior to extensive washing with I.;trO, the precipit tit(' melted in t x o steps under a microscope hot stage, mp 94 (p1att.lets) and LO:! (needles). These are obviously the t K O moniers Of I11 :tnd the nielting point of the final product will clearly depend on the ratio of the two anomer!: Ivhich, in turn, determine.< t i i r s ratio of the two anomeric nucleosides in llre roiiderisation rex('tion.19 Because of the lihility of 111, the freshly prepared mist urc of aiiomers was used :is such im1nedi:itely ill tlie subsequeni cwiidensation step. 1-( 3,5-Di-O-p-chlorobenzoyl-2-deoly-cu,P-D-ethyluracil (IF').--11 ( 1.49 g, 4.4 mriioles) was suspended in about I30 ml of anhydrous PhMe, vigorously stirred, and dried azeotropically by removal of about one-third of the solvent; :$.ti g of 111 (8.S Illmoles), previously dried, vas added rapidly, and the rnisture \vas heated 3 min, then cooled and filtered through glass wool. The precipitate was dissolved (CHCI,), ihe solution w i s washed (30(, KI, HZO), and the orgnic phase was dried (S:iZSO, 1. The s d t was filtered off, the CHCla solution was hrriuglit t o dryness, and the residue was dissolved in hot anhydrous CtOH. Crystallization occurred on cooling to give 1.1 g ( 4 7 ' , i ) of IT., melting at 154-178 and eshihiting urider a niicroscwpe hot s t a p
t,4',6,6'-Te trabromo-X,2'-biphenyldiol JIono(dihydrogen phosphate). .4 Neh Agent for Combating Distomatosis'
Attempted Separation of tlie Anomers of V by Ion-Exchange Br W \ B\ r Chromatography:---.I Dowex I - S 2 (200-400 mesh) (OH- I cdumn, 23 X 2 cni, WBS washed with 500 nil of 30( XleOH, and 15 nig of l' in the same solvent, deposi Br Br cwluniii. The latter was then xashed with 250 nil of GO' I aqueous hIeOIl mid 250 nil of !I0 aqueous 1IeOH. Tlie nucleositfe was then eluted with 0.05 SH4HCOI :it :t f l ~ r~~ ; ~ t e OND/OH of ahout I .5 ml/min and fractions uf 13 nil were collected. The nucleoside appeared in two fairly-nell-definetl pecks : fractioii 1 3 o'o l' (4.9 mg, mp 172-171.5") and fraction 16 (5.2 mg, nip 14:3-14iz\. I I Fraction 16, but not fraction 13, supported liacterial u i t l p l i a ~ e multiplicai.ion and is therefore the p :tnomer of \-. Separation of anomers of IV was avliieve deposited as 1.0-mrn layers on 20 X 15 c m I I solvent, system CHC1,-I;* 4.62 X I O 3 ) ; p K , = 9.9b. S m r spectra were determined in I)20 for earh of the momers a t 30" with a T'arian HA-100 spectrometer and .\lerSi its esterrial standard. Except for the added signals due tu tlie presence of :i 5-Et in place of a 5-LIe substituent, tlie spectra were similar t o those of the corresponding anomers of t l i ? m i t i i ~ i e :for ~ ~a-5-echyIIn (Hi, quartet!. = S.O cps, . / F ~ , ~ - , l ~ ~ ~ anoiner! 6.58 ppin (€11, triplet I = ~/11,,-1121
./ll,l..ll,l
Acknowledgments.- -\Ye are indebted to Professor X. Xriteunis (Service tle C:hiniie Orgnniquc, Viiiwrsitc tle Gent,, Belgium) for the iinir spectra. t o Professor C . Heitlell.)erger (1:iiiversit'y of \\-isconsin) for the tests of antirirul activity, uiid to Dr. I. Pietrzykowslrit aiitl Dr. A I . Piccliowska for tli n the gronth-supportiiig properties of the anom d tlic possihle niiitag,~iir
activity of @-5-ethyltleox> I. 1'. Josryh, E. I:. Schaub, nrid J. H. JYilliani?, J . Oru. rhem., 19. 1736 (1054). ( 2 0 ) 11. I'rystas arid 1:. garin, Co~~ectio71 Czech. (:/rem. ~ o ? t r n i u l i . ,31, 1U:
