5,6-Diaminocytidine, a versatile synthon for pyrimidine-based bicyclic nucleosides

Article abstract of DOI:10.1021/ol0064765

(matrix presented) This communication describes a convenient, facile, and high-yield synthesis of 3-(β-D-ribofuranosyl)isoguanine and its 8-methyl derivative, as well as nucleoside analogues of pteridines, from a common precursor, 5,6-diaminocytidine. 5,6

Full text of DOI:10.1021/ol0064765

ORGANIC
LETTERS
2000
Vol. 2, No. 23
3595-3598
5,6-Diaminocytidine, a Versatile Synthon
for Pyrimidine-Based Bicyclic
Nucleosides†
Kallanthottathil G. Rajeev and Arthur D. Broom*
Department of Medicinal Chemistry, UniVersity of Utah, 30 S. 2000 E,
201 Skaggs Hall, Salt Lake City, Utah 84112
abroom@deans.pharm.utah.edu
Received August 17, 2000
ABSTRACT
This communication describes a convenient, facile, and high-yield synthesis of 3-(â-D-ribofuranosyl)isoguanine and its 8-methyl derivative, as
well as nucleoside analogues of pteridines, from a common precursor, 5,6-diaminocytidine. 5,6-Diamino-2′,3′,5′-tri-O-benzoylcytidine was
synthesized from 4,6-diamino-2-oxopyrimidine in three steps.
Incorporation of 9-(â-D-ribofuranosyl)isoguanine into oligo-
nucleotides and its ability to form parallel duplex,1 tetra-
plex1b-e,2 and quintet3 structures have been well recognized.
It is also known that 9-(â-D-ribofuranosyl)isoguanine by itself
and in combination with berberin is active against various
tumor cell lines.4 It would be interesting to know the effect
of position of glycosylation of isoguanine on its structural
properties and antitumor activity. As a part of an ongoing
project leading to the synthesis of anti-HIV polynucleotides,
we became interested in 3-(â-D-ribofuranosyl)isoguanine (6a)
and its analogues. A synthesis of 6a has been reported by
Schmidt and Townsend.5 However, the effect of 6a on the
structure of oligonucleotides and its biological relevance are
lacking, perhaps because of the synthetic difficulties in
obtaining substantial quantities of the compound. The present
work discusses a convenient, facile, and high-yield synthesis
of 6a and its 8-substituted analogues from a pyrimidine
nucleoside, 5,6-diaminocytidine (4).
Also described is a convenient route to the synthesis of
4-amino-2-oxo-3-(â-D-ribofuranosyl)-2,3-dihydropteridine and
its 6,7-dimethyl derivative from 4. Synthesis of analogues
of pteridine nucleosides starting from pteridines are well-
known in the literature.6 Recently, the fluorescent properties
of pteridine nucleosides have stimulated interest in their use
as fluorophores in nucleic acid interaction studies.7 Glyco-
† An abstract was presented at the XIVth International Roundtable on
Nucleosides, Nucleotides and Their Biological Applications, San Francisco,
September 10-14, 2000.
(1) (a) Seela, F.; He, Y.; Wei, C. Tetrahedron 1999, 55, 9481-9500.
(b) Seela, F.; Wei, C.; Melenewski, A. Leonard, P. Nucleic Acids Symp.
Ser. 1997, 37, 149-150. (c) Yang, X.-L.; Sugiyama, H.; Ikeda, S.; Saito,
I.; Wang, A. H.-J. Biophys. J. 1998, 75(3), 1163-1171. (d) Seela, F.; Wei,
C.; Melenewski, A.; Feiling, E. Nucleosides Nucleotides 1998, 17, 2045-
2052. (e) Sugiyama, H.; Ikeda, S.; Saito, I. J. Am. Chem. Soc. 1996, 118,
9994-9995. (f) Roberts, C.; Bandaru, R.; Switzer, C. J. Am. Chem. Soc.
1997, 119, 4640-4649. (g) Seela, F.; Wei, C. HelV. Chim. Acta 1997, 80,
73-85.
(2) (a) Seela, F.; Wei, C.; Melenewski, A. Origins Life EVol. Biosphere
1997, 27, 597-608. (b) Davis, J. T.; Tirumala, S.; Jenssen, J. R.; Radler,
E.; Fabris, D. J. Org. Chem. 1995, 60, 4167-76.
(3) Chaput, J. C.; Switzer, C. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
10614-10619.
(5) Schmidt, C. L.; Townsend, L. B. J. Chem. Soc., Perkin Trans. 1 1975,
1257-1260.
(4) (a) Liebich, H. M.; Lehmann, R.; Di Stefano, C.; Haring, H. U.; Kim,
J. H.; Kim, K. R. J. Chromatogr., A 1998, 795, 388-393. (b) Kim, J. H.;
Lee, S. J.; Han, Y. B.; Moon, J. J.; Kim, J. B. Arch. Pharmacal Res. 1994,
17, 115-18.
(6) (a) Pfleiderer, F. Chemistry of Nucleosides and Nucleotides; Townsend,
L. B., Ed.; Plenum: New York, 1988; Vol. 3 (rev), pp 214-255 and
references cited therein. (b) Harzer, von K.; Pfleiderer, W. HelV. Chim.
Acta 1973, 56, 1225-1234.
10.1021/ol0064765 CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/25/2000

Scheme 1a
a (a) i. HMDS, TMS-Cl (cat.) reflux, 6 h; ii. 1-O-acetyl-2.3.5-(tri-O-benzoyl)-â-D-ribofuranose, Sn(IV)Cl, rt, 24 h (yield 88%). (b)
NaNO2 (2 equiv), HOAc, H2O (10%), < 10 °C, 4 h (yield > 95%). (c) Na2S2O4 (2 equiv), DMF, H2O (10%), 50 °C, 4 h (yield > 95%).
(d) Y-CO-CO-Y, DMF (yield 80-90%, overall). (e) X-CO-N(CH3)2, POCl3 (1.2 equiv) (yield 80-90%). (f) NaOMe-MeOH or NaOH-
H2O/MeOH.
sylation of silylated 4-amino-2-oxo-1,2-dihydropteridine gave
predominantly the N1-glycosylated pteridine nucleoside; it
is difficult to synthesize the corresponding N3 nucleoside
analogue with the existing methodology.6b The present
approach depicts a convenient way of synthesizing N3-
glycosylated 4-amino-2-oxopteridines.
5,6-Diamino-2′,3′,5′-tri-O-benzoylcytidine (4) was syn-
thesized from 4,6-diamino-2-oxopyrimidine (1) as shown in
Scheme 1. Trimethylsilylated 1 was allowed to react with
1-O-acetyl-2,3,5-tri-O-benzoyl-â-D-ribofuranose in 1,2-
dichloroethane under Vorbru¨ggen coupling conditions8 to
yield 6-amino-2′,3′,5′-tri-O-benzoylcytidine (2) in 90% yield.
The synthesis of 6-aminocytidine9 from 5-bromocytidine and
6-amino-2′-O-methylcytidine10 from 1 and the corresponding
2-O-methyl sugar have been reported. Nitrosation of 2 in
acetic acid below 10 °C for 4 h gave the purple 5-nitroso
derivative 3 in quantitative yield.11,12 Quantitative reduction
of nitroso to amine was achieved by the treatment of 3 with
sodium hydrosulfite and water in DMF at 50 °C for 4 h to
yield 4 as a yellowish green solid.
(10) Pudlo, J. S.; Wadwani, S.; Milligan, J. F.; Matteucci, M. D. Bioorg.
Med. Chem. Lett. 1994, 4, 1025-1028.
(11) Rogers, G. T.; Ulbricht, T. L. V. J. Chem. Soc., C 1971, 2364-
2366.
(12) (a) The reaction proceeded more slowly when there was methanol
in the reaction mixture. It is recommended that the reaction be performed
in an acetic acid-water solvent system at a temperature less than 10 °C to
obtain the nitroso derivative in quantitative yield. The analytically pure
product gave unusually complex proton and carbon NMR spectra, with many
signals split into two peaks. This may arise from the presence of tautomers
such as those described by Pfleiderer12b involving the imino-oxime at the
6- and 5-positions, respectively. (b) Pfleiderer, W.; Kempter, F. E. Angew.
Chem., Int. Ed. Engl. 1967, 6, 259-260.
(7) (a) Hawkins, M. PCT Int. Appl. WO 9826093 A2. 18 Jun 1998. (b)
Hawkins, M. E.; Pfleiderer, W.; Balis, F. M.; Porter, D.; Knutson, J. R.
Anal. Biochem. 1997, 244, 86-95.
(8) Niedballa, U.; Vorbruggen, H. J. Org. Chem. 1974, 39, 3654-3660.
(9) Goldman, D.; Kalman, T. I. Nucleosides Nucleotides 1983, 2, 175-
187.
3596
Org. Lett., Vol. 2, No. 23, 2000

Treatment of 4 with 1 equiv13 of POCl3 in DMF (Vils-
meier-Haack reagent) at ambient temperature gave pre-
dominantly 6-amino-3-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)-
purine-2-one (5a).14 One might expect a mixture of 5a and
its isomer 7 from the reaction, but the major product isolated
was 5a in 85% yield. However, a small amount of a slightly
faster moving component was observed on TLC.15 This could
be the isomer 7, but the very low yield made isolation and
characterization impractical. Debenzoylation of 5a in metha-
nolic sodium methoxide gave the free nucleoside 6a. The
structure of compound 6a has been established by HMQC
and gradient HMBC (GHMBC) NMR experiments and by
the identity of its UV spectra with those reported in the
literature.5 In the reported procedure, the authors took
advantage of the steric directing effect of iodide present at
position 8 of 8-iodoisoguanine to selectively glycosylate at
N3 of the base. This was followed by catalytic hydrogenoly-
sis of the iodide to obtain the target compound.5
Reaction of 4 with 1 equiv of POCl3 and DMAc gave the
corresponding 8-methyl derivative 5b. Synthesis of 8-sub-
stituted 3-(â-D-ribofuranosyl)isoguanine, while possible,
would be challenging using the earlier method.5 The present
methodology thus provides an excellent route to 3-(â-D-
ribofuranosyl)isoguanine and its 8-methyl derivative in high
yield (and fewer steps than in the previous procedure) by
application of Vilsmeier-Haack reagents. The position of
the sugar was defined by its position on the starting
pyrimidine nucleoside.
Reaction of 4 with 1 equiv of glyoxal in DMF at ambient
temperature for 1 h gave a 3:2 mixture of 4-amino-2-
oxo-1-(2,3,5-tri-O-benzoyl-â-D-ribufuranosyl)-1,2-dihydro-
pteridine6b (9a) and 4-amino-2-oxo-3-(2,3,5-tri-O-benzoyl-
â-D-ribufuranosyl)-2,3-dihydropteridine (8a), respectively.
The slower eluting compound on a silica gel column was
debenzoylated and characterized by HMQC and GHMBC
NMR experiments as 11a. Its UV spectrum matched that of
the reported compound.6b The faster eluting compound was
debenzoylated,16 and its structure was established as 10a by
HMQC and GHMBC NMR experiments (vide infra). Reac-
tion of 4 with 1 equiv of butane-2,3-dione in DMF at ambient
temperature gave the 3-(â-D-ribofuranosyl)pteridine deriva-
tive 8b as the major product (80% yield, faster eluting
fraction on silica gel column) and 1-(â-D-ribofuranosyl)-
pteridine17 derivative 9b as the minor product (less than
10%). The predominant formation of 8b rather than 9b is
probably due to steric constraints in the transition state
between the methyl group and the sugar in the case of 9b.
Preliminary studies on the fluorescent properties of the N3-
glycosylated pteridines revealed that the fluorescence inten-
sity of these analogues is greater than that of the correspond-
ing N1-glycosylated derivatives.7
1
In the H NMR spectra in CDCl3, the H1′ of 8a and 8b
appeared as broad singlets at δ 6.98 and 6.94, and one of
the amino protons appeared as a sharp peak at δ 9.70 and
9.46, respectively. The appearance of the sharp NH peak in
CDCl3 at around 9.5 ppm probably results from intramo-
lecular H-bonding between the NH (of 8a/8b) and the
carbonyl of the 5′-O-benzoate. On the other hand, the amino
protons were not detected in CDCl3 for 9a, a more typical
finding when specific hydrogen bonding is not present. These
observations are consistent with the assignment of 8a and
8b as 3-ribosylated pteridines, but additional proof was
sought using HMQC and GHMBC NMR spectroscopy.
Debenzoylation of 8a afforded nucleoside 10a. An HMQC
experiment in DMSO-d6 readily established all the expected
1-bond correlations, of which those of interest were H1′ to
C1′ (δ 6.44 to 88.9) and the two aromatic protons (δ 8.42 to
138.6 and 8.56 to 147.6). GHMBC NMR experiment in
DMSO-d6 unequivocally established the structure of 10a by
means of the following connectivities: (1) two three-bond
correlations involving H1′(δ 6.44) to C2(δ 156.7) and C4(δ
150.3); (2) a one-bond correlation between H6(δ 8.42) and
C6(δ 138.6); a two-bond correlation between H6 and C7(δ
147.6) and a three-bond correlation between H6 and C4a(δ
125.8); and (3) a one-bond correlation between H7(δ 8.56)
and C7(δ 147.), a two-bond correlation between H7 and C6(δ
138.6), and a three-bond correlation between H7 and C8a
(δ 147.1). The critical three-bond couplings are illustrated
by arrows in the structure of 10a (Figure 1).
Figure 1. Important connectivities observed in the GHMBC NMR
spectra.
(13) Addition of excess POCl3 led to the formation of multiple products.
It is recommended that only enough POCl3 be added to the reaction mixture
to force the reactant 4 to react completely to avoid a very difficult
chromatographic separation.
(14) Anderson, G. L.; Rizkalla, B. H.; Broom, A. D. J. Org. Chem. 1974,
39, 937-939.
(15) The reaction was performed at 0 and 50 °C, but no noticeable change
was observed.
(16) The debenzoylation of 8a in methanolic sodium methoxide was very
slow and the 5′-mono-O-benzoate of 8a was isolated as a major product.
Prolonged treatment of 8a with sodium methoxide in methanol led to partial
deglycosylation. The debenzoylation of 9a was complete within 3 h. Details
of the successful debenzoylation procedure are provided in the Supporting
Information.
There was no cross-peak seen between H6 and/or H7 with
C4 (which gave a cross-peak with H1′). Also, no cross-peak
was seen between H7 and C4a or between H6 and C8a. At
the same time, the strong cross-peak between H6 and C4a
and between H7 and C8a along with the cross-peak of H1′
with C2 and C4 clearly support the structure 10a.
In conclusion, 5,6-diaminocytidine can be used as a general
synthon for the synthesis of a variety of 8-substituted 3-(â-
D-ribofuranosyl)isoguanines and N3-glycosylated 4-amino-
2-oxopteridines. The incorporation of 3-(â-D-ribofuranosyl)-
(17) Angelika, R.; Pfleiderer, W. Collect. Czech. Chem. Commun. 1996,
61, S230-S233.
Org. Lett., Vol. 2, No. 23, 2000
3597

isoguanine, which has H-bond acceptor/donor switches at
N1 and N7, into oligonucleotides will be interesting for
comparison with the corresponding 9-(â-D-ribofuranosyl)-
isoguanine.1e,18 Further studies on these aspects and the evalu-
ation of the anticancer properties of all the isoguanine and
pteridine derivatives are underway. The fluorescent properties
of the pteridine derivatives will also be evaluated in detail.
Acknowledgment. The authors gratefully acknowledge
the support of these studies by grant R01 AI 27692 from
the National Institute for Allergy and Infectious Disease,
NIH, PHS.
Supporting Information Available: Experimental detail
and characterization data for all compounds are provided.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(18) (a) Robinson, H.; Gao, Y.-G.; Bauer, C.; Roberts, C.; Switzer, C.;
Wang, A. H.-J. Biochemistry 1998, 37, 10897-10905. (b) Liaw, Y- C.;
Chern, J.-W.; Lin, G.-S.; Wang, A. H.-J. FEBS Lett. 1992, 297, 4-8.
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