J. Org. Chem. 2000, 65, 979-984
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Synthesis of 2-(N-Acetylamino)-2-deoxy-C-glucopyranosyl Nucleosides as Potential Inhibitors of Chitin Synthases† Je´roˆme Grugier, Juan Xie,* Isabelle Duarte, and Jean-Marc Vale´ry Laboratoire de Chimie des Glucides, CNRS UMR 7613, Universite´ Pierre et Marie Curie, 4 Place Jussieu, 75005 Paris, France Received July 28, 1999
The C-glucopyranosyl nucleosides (1-4) containing the N-acetyl glucosaminyl and uridine units have been synthesized as nonhydrolyzable substrate analogues of UDP-GlcNAc aimed to inhibit the chitin synthases. The key intermediate, 4-(2′-(N-acetylamino)-3′,4′,6′-tri-O-benzyl-2′-deoxy-RD-glucopyranosyl)but-2-enoic acid (5), was prepared from the perbenzylated (N-acetylamino)-R-Callylglucoside (7), by successive oxidative cleavage, Wittig olefination, and ester deprotection. The coupling of the acid 5 with the hydroxyl or amine function of the uridine derivatives (6a or 6b) afforded, respectively, the ester 12 and amide 14. The dihydroxylation of the conjugated double bond in ester 12 or amide 14 was better achieved with osmium tetraoxide/barium chlorate, leading to the expected diols 13 and 15 as a mixture of two diastereoisomers. The desired compounds 1-4 were obtained after catalytic hydrogenation of compounds 12-15. Introduction Inhibitors of glycosyltransferases, which are involved in the biosynthesis of glycoproteins, glycolipids, and other glycoconjugates, have proven to be useful for studies on biological function of cell-surface carbohydrates. Such compounds present particular interest in the development of potential pharmaceuticals such as antiviral, antitumor, and immunoregulatory agents.1 For example, tunicamycin, an inhibitor of UDP-GlcNAc dolicyl phosphate GlcNAc-1-phosphotransferase, has been shown to inhibit replication in yeast, fungi, protozoa, enveloped virus, and mammalian cell lines in culture.2 Chitin, the β-1f4-linked homopolymer of N-acetyl-Dglucosamine (GlcNAc), is one of the most abundant natural polymers and one of the major structural components of the cell wall of most fungi. The biosynthesis of chitin is assumed by chitin synthases (UDP-N-acetylD-glucosamine:chitin 4-β-N-acetylglucosaminyl transferase EC 2.4.1.16) which perform polymerization of N-acetyl-D-glucosamine starting from UDP-GlcNAc.3 Inhibition of chitin synthases represents an attractive approach to the design of effective new antifungal agents. Polyoxines and nikkomycines, a group of peptidyl nucleoside antibiotics produced by some species of Streptomyces, have been demonstrated to be competitive inhibitors of chitin synthases and exhibit antifungal, insecticidal, and acarcidal activities.4 To the best of our knowledge, no synthetic inhibitor of chitin synthases has even been * To whom correspondence should be addressed. Telephone: 33-144-27-58-93. Fax: 33- 1-44-27-55-13. E-mail:
[email protected]. † In the memory of Professor Stanislas Czernecki. Deceased October 20, 1997. (1) (a) Schwarz, R. T.; Datema, R. Adv. Carbohydr. Chem. Biochem. 1982, 40, 287-379. (b) Elbein, A. D. Annu. Rev. Biochem. 1987, 56, 497-534. (c) Pan, Y. T.; Elbein, A. D. Glycoproteins; Montreuil, J.; Vliegenthart, J. F. G.; Schachter, H., Ed.; Elsevier Science B. V.: The Netherlands, 1995; pp 415-445. (d) Sears, P.; Wong, C. H. Cell Mol. Life Sci. 1998, 54, 223-252. (2) (a) Takatsuki, A.; Tamura, G. J. Antibiot. 1971, 24, 224-230. (b) Takatsuki, A.; Shimizu, K. T.; Tamura, G. J. Antibiot. 1972, 25, 75-85. (3) Bulawa, C. E. Annu. Rev. Microbiol. 1993, 47, 505-534. (4) Calib, E. Antimicrob. Agents Chemother. 1991, 35, 170-173.
reported and little is known about the biochemical nature of these enzymes.5 Accordingly, our initial approach in the design and synthesis of competitive inhibitors of chitin synthases was to synthesize nucleosidic C-glycosides derivatives designed as nonhydrolyzable analogues of the natural substrate UDP-GlcNAc (Figure 1). It is assumed that a nonhydrolyzable substrate analogue featuring a N-acetyl-D-glucosamine residue linked to uridine through a suitable carbon chain could block the glycosyl transfer. A hydroxylated carbon chain is expected to imitate the diphosphate moiety by chelating the divalent cation present at the active site of the enzyme. Furthermore, because of the carbon linker, such compounds would be resistant to acidic and enzymatic hydrolysis. These molecules, which represent the first nonhydrolyzable substrate analogues of UDP-GlcNAc, are also expected to be useful in glycobiology for probing the details of catalytic mechanism of other N-acetylglucosaminyltransferases. Indeed the GlcNAc residue is a common component of natural glycoconjugates that are generated by a large class of N-acetylglucosaminyl transferases processing with UDP-GlcNAc as glycosyl donnor.6 We reported herein the synthesis of compounds 1-4. Results and Discussion The synthesis of the target molecules 1-4 was planned by coupling the (N-acetylamino)-R-C-glucopyranoside 5 and the protected uridine derivative 6 (Scheme 1). Compound 5 was prepared from N-acetyl-D-glucosamine, which was transformed into amino R-C-allylglucopyranoside 7 (containing less than 10% of β anomer) as previously described.7 Zemple´n deacetylation of 7 followed by classical benzylation afforded 8 in 94% yield (5) Kamst, E.; Bakkers, J.; Quaedvlieg, N. E. M.; Pilling, J.; Kijne, J. W.; Lugtenberg, B. J. J.; Spaink, H. P. Biochemistry 1999, 38, 40454052. (6) (a) Schachter, H. Glycobiology 1991, 1, 453-461. (b) Schachter, H. Biochem. Cell Biol. 1986, 64, 163-181. (c) Lu, P. P.; Hindsgaul, O.; Li, H.; Palcic, M. M. Carbohydr. Res. 1997, 303, 283-291. (7) Roe, B. A.; Boojamra, C. G.; Griggs, J. L.; Bertozzi, C. R. J. Org. Chem. 1996, 61, 6442-6445.
10.1021/jo991206p CCC: $19.00 © 2000 American Chemical Society Published on Web 02/03/2000
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J. Org. Chem., Vol. 65, No. 4, 2000
Grugier et al.
Figure 1. (a) Postulated transition structure for chitin synthases. (b) Designed nonhydrolyzable substrate analogues.
Scheme 1
(Scheme 2). Oxidative cleavage of the double bond (OsO4/ NaIO4) of 8 yielded the aldehyde 9 which is in equilibrium with the cyclic form 9′ as demonstrated by 1H and 13C NMR spectra (see Experimental Section). Wittig condensation was performed on the mixture of 9 and 9′ with Ph3PdCHCOOEt in toluene and afforded compound 10a as the sole isomer (69% yield). The E configuration of the so-formed double bond was established by the large coupling constant between the two vinylic protons (J ) 16 Hz) on the 1H NMR spectrum. However, the basic deprotection of 10a to 5 was not effective, leading to less than 50% yield, and was accompanied by partial epimerization to β-anomer (until ∼50%), presumably via a retroMichael pathway (Chart 1). A similar epimerization of R- to β-C-glucopyranosides under basic conditions was observed previously.8 To avoid this side reaction, we decided to use Ph3PdCHCOOtBu as the olefination agent. In this case, we were able to separate (in 2% yield) the small amount of β-anomer 10c (8) (a) Giannis, A.; Sandhoff, K. Carbohydr. Res. 1987, 171, 201210. (b) Allevi, P.; Anastasia, M.; Ciuffreda, P.; Fiecchi, A.; Scala, A. J. Chem. Soc., Perkin Trans. 1 1989, 1275-1280.
Scheme 2a
arising from the allylation reaction which was used to obtain compound 7. The R-tert-butyl ester 10b was obtained in 69% and was easily cleaved (CF3COOH/CH2Cl2) to afford the desired acid 5 in good yield (86%). The required 2′,3′-di-O-benzyl-protected uridine 6a was obtained as previously described.9 The 5′-amino-5′-deoxy derivative 6b was easily prepared from 6a, by a one-pot azidation of the 5′-hydroxyl group (CBr4/PPh3/NaN3 in (9) Michelson, A. M.; Todd, A. J. Chem. Soc. 1956, 3459-3463.
2-(N-Acetylamino)-2-deoxy-C-glucopyranosyl Nucleosides Chart 1
Scheme 3a
DMF), affording 11, and consecutive selective reduction (Scheme 3). The esterification of a primary hydroxyl group is a wellestablished process. However, the coupling between the acid 5 and the alcohol 6a was particularly difficult: the activations of the acid function of 5 with (CF3CO)2O or Bop-Cl were both inefficient. The use of DCC/DMAP or IIDQ as activating agents afforded 12 in low yield (
