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Pyrimidine C-5 Nucleosides Related t o Pseudouridine Ed., Academic Press, New York, N.Y., 1971, pp 375-473. (6) M. Lazdunski, J. P. Vincent, H. Schweitz, M. Peron-Renner, and J. Pudles in “Proteinase Inhibitors”, H. Fritz, H. Tschesche, L. J. Greene, and E. Truscheit, Ed., Springer-Verlag New York, New York, N.Y., 1974, pp 420-431. (7) B. Kassel and M. Laskowski, Sr., Biochem. Biophys. Res. Commun., 17, 792 (1964). (8) B. Kassell, M. Radicevic, M. J. Ansfield, and M. Laskowski, Sr., Biochem. Biophys. Res. Commun., 18, 255 (1965). (9) 8. Kassell and M. Laskowski, Sr., Biochem. Biophys. Res. Commun., 20, 463 (19651. B. Kassell’and M. Laskowski, Sr., Acta Biochim. Poi., 13, 287 (1966). R. Huber, D. Kukla, A. Ruhlmann, 0. Epp, and H. Formanek. Naturwissenschaffen, 57,389 (1970). R. Huber, D. Kukia, A. Ruhlmann, and W. Steigemann, Cold Sprlng Harbor Symp. Quant. Blol., 36, 141 (1971). J. Deisenhofer and W. Steigemann in ref 6, pp 484-496. F. A. Anderer and S.Hornle, J. Biol. Chem., 241, 1568 (1966). J. Chauvet and R. Acher, Bull. SOC.Chim. Biol., 48, 1248 (1966). D. Posplsilova, B. Meloun, I. Fric, and F. Sorm, Collect. Czech. Chem. Commun., 32, 4108 (1967). J. P. Vincent and M. Lazdunski, Biochemistry, 11, 2967 (1972). B. W. Erickson and R. B. Merrlfield, J. Am. Chem. SOC., 95, 3750 (1973). B. W. Erickson and R. B. Merrifield, J. Am. Chem. Soc., 95, 3757 (1973). D. Yamashiro and C. H. Li, J. Am. Chem. Soc., 95, 1310 (1973). E. Kaiser, R. L. Colescott, C. D. Bossinger, and P. I. Cook, Anal. Biochem., 34, 595 (1970). C. B. Anfinsen, Biochem. J., 128, 737 (1972). R. R. Hantgan, G. G. Hammes, and H. A. Scheraga, Biochemistry, 13,3421 (1974).
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(25) V. Dlouha, D. Pospisilova, B. Meloun, and F. Sorm, Collect. Czech. Chem. Commun., 30, 1311 (1965). (26) B. Jirgensons, M. Kawabata, and S.Capetillo, Makromoi. Chem., 125, 126 (1969). (27) C.-H.-Hirs, J. Biol. Chem., 219, 611 (1956). (28) B. Meloun, I. Fric, and F. Sorm, Eur. J. Biochem., 4, 112 (1968). (29) Professor M. Lazdunski and Dr. J. P. Vincent provided us with 14CRCAM”BPT1 for this experiment. (30) K. Noda, S. Terada, N. Mitsuyasu, M. Waki, T. Kato, and N. Izumiya, Nafurwissenschaften, 58, 147 (1971). (31) H. Yajima, Y. Kiso. Y. Okada, and H. Watanabe, J. Chem., SOC., Chem. Commun., 106 (1974). (32) Y. Kiso and H. Yajima, Chem. fharm. Bull., 22, 1087 (1974). (33) G. R. Stark, W. H. Stein, and S. Moore, J. Biol. Chem., 235, 3177 (1960). (34) D. D. Schroeder and E. Shaw, J. Bioi. Chem., 243,2943 (1968). (35) J. Chauvet and R. Acher, F€BS Lett., 23, 317 (1972). (36) E. Sach, M. Thely, and J. Choay, C.R. Acad. Sci., 260, 3491 (1965). (37) R. Avineri-Goldman, I. Snir, G. Blauer, and M. Rigbi, Arch. Biochem. Biophys., 121, 107 (1967). (38) T. Chase and E. Shaw, Biochem. Biophys. Res. Commun., 29, 508 (1967). (39) J. C. Zahnley and J. G. Davis, Biochemistry, 6, 1428 (1970). (40) R. Repaske, Methods Enzymol., 22, 322-325 (1971). (41) A. Marglin, Tetrahedron Lett., 33, 3145 (1971). (42) These cleavages were performed by Mr. Jim Geever of the Armour Pharmaceutical Co. (43) S. Sakakibara, Y. Shimonishi, Y. Klshida, M. Okada, and H. Sugihara, Bull. Chem. SOC.Jpn., 40, 2164 (1967). (44) W. C.Chan, Biochemistry, 7, 4247 (1968).
Nucleosides. 100. General Synthesis of Pyrimidine C-5 Nucleosides Related to Pseudouridine. Synthesis of 5-(~-~-Ribofuranosyl)isocytosine (Pseudoisocytidine), 5-(~-~-Ribofuranosyl)-2-thiouracil(2-Thiopseudouridine) and 5-(~-~-Ribofuranosyl)uracil (Pseudouridine) C. K. Chu, I. Wempen, K. A. Watanabe,* and J. J. Fox Laboratory of Organic Chemistry, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Institute, Sloan-Kettering Division of Graduate School of Medical Sciences, Cornel1 University, New York, New York 10021 Received March 12,1976 A general procedure for t h e synthesis o f p y r i m i d i n e C - 5 nucleosides related t o pseudouridine was developed. 5(P-1)-Ribofuranosy1)isocytosine(7, pseudoisocytidine), t h e f i r s t chemotherapeutically active synthetic C nucleoside, was prepared f r o m readily available e t h y l 2-(2,3-0-~sopropylidene-5-~-tr~tyl-D-r~bofuranosyl)acetate (3). Compound 3 was formylated w i t h e t h y l formate a n d s o d i u m h y d r i d e t o t h e corresponding formylacetate sodium enolate 4 a n d m e t h y l a t e d w i t h m e t h y l iodide in DMF t o give 3-methoxy-2-(2,3-~-isopropylidene-5-O~trityl-~-ribofuranosy1)acrylate ( 5 ) . Cyclization of 5 w i t h guanidine afforded t h e protected isocytosine C-5 nucleoside 6. T r e a t m e n t o f 6 w i t h methanolic hydrogen chloride gave t h e desired crystalline p nucleoside, pseudoisocytidine (7). F r o m t h e m o t h e r liquor, t h e 01 isomer 8 was obtained. C o m p o u n d 8 can b e epimerized effectively t o 7 in methanolic hydrogen chloride so t h a t a very h i g h y i e l d o f t h e desired isomer 7 f r o m 6 i s readily achieved. T h e general applicability o f t h i s m e t h o d t o t h e syntheses o f other C nucleosides was demonstrated by t h e synthesis o f 2-thiopseudourid i n e (10) a n d pseudouridine (13). Condensation o f t h e acrylate derivative 5 w i t h thiourea followed by deblocking o f t h e p r o d u c t afforded 10. W h e n 5 was treated w i t h urea, t h e protected pseudouridine derivatives (11a n d 12) were obtained. A f t e r deprotection o f 11, pseudouridine (13) was obtained.
Pseudouridine, the first C nucleoside found in nature, has attracted the interest of organic chemists and biochemists since its discovery in 1957.2Recently, other C nucleosides have-. also been isolated as nucleoside antibiotics from the culture filtrates of various Streptomycetes. T h e unique structural characteristic of C nucleosides which distinguishes them from the ordinary nucleosides is the presence of a carbon to carbon linkage instead of a carbon to nitrogen bond between the aglycon and sugar moieties. This structural feature renders traditional approaches4 for nucleoside synthesis of limited value. Although several reports have appeared on the synthesis of p ~ e u d o u r i d i n eand ~ pseudocytidine,6 these methods involve the condensation of a suitably protected sugar with a pre-
formed pyrimidine-5-yllithium derivative. These procedures are difficult to perform and are not suitable for large-scale preparations. More importantly these methods are specific for each C nucleoside, i.e., for the synthesis of a modified base analogue preparation of a particular pyrimidine 5-lithio derivative is required individually. As a part of our efforts to develop a general method for the synthesis of pseudouridine and analogues thereof we reported in a recent communication7 a synthesis of B-(@-D-ribofuranosy1)isocytosine (7, pseudoisocytidine) in four steps from 2,3-O-isopropylidene-5-O-trityl-D-ribofuranose (1) via in4 6 (see Chart I). termediates 3 Owing to the potential clinical importances ofpseudoisocytidine as an antileukemic agent, we now report our synthetic
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2794 J. Org. Chem., Vol. 41, No. 17,1976
Chu, Wempen, Watanabe, and Fox
chart I
1
2
4
I
\
5
3
6
I HN
AN
“Y procedures including modifications which result in improved yield of pseudoisocytidine. Further, the general applicability of our methods for the synthesis of pyrimidine C nucleosides is exemplified by the synthesis of 2-thio-pseudouridine (10) and pseudouridine (13). Treatment of 1 with (ethoxycarbonylmethy1ene)triphenylphosphorane according t o Ohrui et al.9 afforded ethyl 2(2,3-0-isopropylidene-5-0-trityl-~-ribofuranosyl)acetate (3). This reaction is often accompanied by the formation of significant amounts of a more polar by-product as observed by thin layer chromatography (TLC) in addition t o t h e desired product 3. The by-product was isolated by silica gel column chromatography and was shown by l H N M R t o be a mixture of cis and trans olefins 2 (see Experimental Section). Buchanan e t al.1° had reported t h e isolation of a n olefin analogous to 2 after treatment of tri-0-benzyl-D-ribofuranose with the same Wittig reagent and showed t h a t their olefin could be converted into a cyclic derivative analogous t o 3 by base catalysis. Thus, after the reaction of 1 with the Wittig reagent, the product which contained t h e open-chain intermediate 2 was treated with alkoxide and the desired intermediate 3 was obtained in high yield. The epimeric configuration a t C-1 of the “ribosyl” derivative 3 was not determined, and indeed is not crucial because epimerization occurs in subsequent steps (vide infra). The key step in the synthesis of pseudoisocytidine and related C nucleosides is the formylation of 3. Compound 3 was treated with ethyl formate and sodium hydride in a mixture of anhydrous ether and absolute ethanol. Without purification, the product sodium enolate 4 was treated with guanidine
in ethanol in the presence of sodium ethoxide. Protected pseudoisocytidine (6) was obtained in 5% yield as colorless crystals after silica gel column chromatography. The reproducibility of the yield of 6, however, was inconsistent. Owing to the low acidity of the a hydrogens of the ester 3, formylation did not go t o completion resulting in a n intractable mixture. Base-catalyzed cyclization of crude 4 with guanidine to the protected nucleoside 6 proceeded poorly owing t o the preponderance of the enolate form 4 in base. T h e attack by t h e nitrogen nucleophile on t h e aldehydic (enolic) carbon atom would be electrostatically hindered by the adjacent negatively charged oxygen. Removal of the negative charge from the enolic oxygen by alkylation should therefore favor the cyclization reaction. Thus, the crude sodium enolate 4 was methylated with methyl iodide in D M F and the desired 0methoxyacrylate derivative [ethyl 3-methoxy-2-(2,3-0-isopropylidene-5-0-trityl-D-ribofuranosyl)acrylate]( 5 ) was isolated in crystalline form after column chromatography in -25% yield from 1. Attempts t o separate 5 on a large scale were found not practical owing to appreciable decomposition of this product on the silica gel column. Although the protected C nucleoside 6 could be obtained in -90% yield from crystalline pmethoxyacrylate 5, it was found more practical t o use crude 5 directly for cyclization with guanidine. Under these conditions, pure compound 6 was isolated in crystalline form in -15% overall yield from 1. The a configuration is assigned to crystalline 6 on the basis of Imbach’s rulell and deblocking experiments. The difference in chemical shifts of the methyl signals of the isopropylidene group (11Hz) falls into the a-nucleoside range (
