Synthesis and potent anti-HIV activity of L-3′-fluoro-2′,3′-unsaturated cytidine

Article abstract of DOI:10.1021/ol0168059

(Matrix Presented) L-2′,3′-Didehydro-2′,3′-dideoxy-3′-fluorocytidine (L-3′-Fd4C), a novel potent anti-HIV agent (EC₅₀ 0.03 μM in PBM cells), has been synthesized from L-xylose in 14 steps.

Full text of DOI:10.1021/ol0168059

ORGANIC
LETTERS
2001
Vol. 3, No. 26
4177-4180
Synthesis and Potent Anti-HIV Activity
of L-3′-Fluoro-2′,3′-Unsaturated Cytidine
,†
Giuseppe Gumina,† Raymond F. Schinazi,‡ and Chung K. Chu*
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy,
The UniVersity of Georgia, Athens, Georgia 30602, and Emory UniVersity School of
Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033
dchu@rx.uga.edu
Received September 24, 2001
ABSTRACT
L-2′,3′-Didehydro-2′,3′-dideoxy-3′-fluorocytidine (L-3′-Fd4C), a novel potent anti-HIV agent (EC50 0.03 µM in PBM cells), has been synthesized
from L-xylose in 14 steps.
Nucleoside analogues have been the cornerstone of antiviral
therapy over the past thirty years. In the effort to discover
effective antiviral agents against AIDS and viral hepatitis, a
large number of nucleoside analogues have been synthesized
and evaluated. Although structure-activity relationship stud-
ies have not led to a pharmacophore model for the antiviral
activities of nucleosides, some structural features have been
particularly successful. For example, all six of the nucleoside
reverse transcriptase inhibitors approved by the FDA for the
treatment of AIDS1 can be considered as 2′,3′-dideoxy-
nucleosides. In addition, among ring substituents, electron-
withdrawing groups such as azido2 and fluorine3 have often
produced potent antiviral agents. Another structural feature
that is often beneficial for antiviral activity is a 2′,3′-
unsaturated bond. We have extensively explored these
substitutions in nucleoside analogues, particularly with the
synthesis and biological evaluation of D- and L-2′,3′-
didehydro-2′,3′-dideoxy-2′-fluoro nucleosides (Figure 1).3d-f
† The University of Georgia.
Figure 1. Structures of D- and L-2′,3′-didehydro-2′,3′-dideoxy-2′-
fluoro nucleosides.
‡ Emory University School of Medicine/Veterans Affairs Medical Center.
(1) Zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), stavudine
(d4T), lamivudine (3TC), and abacavir (1596U89).
(2) Mitsuya, H.; Weinhold, K. J.; Furman, P. A.; St. Clair, M. H.;
Lehrman, S. N.; Gallo, R. C.; Bolognesi, D.; Barry, D. W.; Broder, S. Proc.
Natl. Acad. Sci. U.S.A. 1985, 82, 7096-7100.
Among them, the cytosine and 5-fluorocytosine derivatives
displayed potent anti-HIV and anti-HBV activity without
significant cytotoxicity. For this reason, we decided to
explore the chemistry and biology of 2′,3′-didehydro-2′,3′-
dideoxy-3′-fluoronucleosides. In this series, the D-cytidine
and D-thymidine analogues were previously synthesized and
shown to have low to moderate anti-HIV-1 activity without
cytotoxicity.4 The D-adenine derivative also showed moderate
anti-HIV activity with some toxicity.4a In view of the fact
(3) (a) Owen, G. R.; Verheyden, J. P. H.; Moffatt, J. G. J. Org. Chem.
1976, 41, 3010-3017. (b) Marquez, V. E.; Tseng, C. K.-H.; Mitsuya, H.;
Aoki, S.; Kelley, J. A.; Ford, H., Jr.; Driscoll, J. S. J. Med. Chem. 1990,
33, 978-985. (c) Pai, S. B.; Liu, S.-H.; Zhu, Y.-L.; Chu, C. K.; Cheng,
Y.-C. Antimicrob. Agents Chemother. 1996, 40, 380-386. (d) Lee, K.; Choi,
Y.; Gullen, E.; Schlueter-Wirtz, S.; Schinazi, R. F.; Cheng, Y.-C.; Chu, C.
K. J. Med. Chem. 1964, 7, 1320-1328. (e) Lee, K.; Choi, Y.; Schinazi, R.
F.; Cheng, Y.-C.; Chu, C. K. Abstracts of Papers, Part 1, 218th National
Meeting of the American Chemical Society; American Chemical Society:
Washington, DC, 1999; MEDI 9. (f) Chun, B. K.; Schinazi, R. F.; Cheng,
Y.-C.; Chu, C. K. Carbohydr. Res. 2000, 328, 49-59.
10.1021/ol0168059 CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/04/2001

Scheme 1. Synthesis of L-2′,3′-Didehydro-2′,3′-dideoxy-3′-fluorocytidine
a Reagents and conditions: (a) ref 5; (b) NaH, THF, from 0 °C to room temperature, 1 h, then BnBr, TBAI, from 0 °C to room temperature,
overnight; (c) 1:2 (4.0 M HCl/dioxane)/MeOH, room temp, 1.5 h; (d) (i) PhOC(S)Cl, DMAP, Tol, 90 °C, 3 h, (ii) Bu3SnH, AIBN, reflux,
1h; (e) H2 (55 psi), 10% Pd/C, EtOH, room temp, 72 h; (f) CrO3, Ac2O, Py, CH2Cl2, room temp, 15 min; (g) DAST, CH2Cl2, reflux, 36
h; (h) concentrated H2SO4, Ac2O, AcOH, 0 °C, 5 min; (i) persilylated N4-benzoylcytosine, TMSOTf, MeCN, from 0 °C to room temperature,
72 h; (j) saturated NH3/MeOH, room temp, 4 h; (k) MeONa, DMF, room temp, overnight.
that among the 2′-fluoro derivatives L-isomers have potent
antiviral activity with no toxicity or less toxicity than their
D-counterparts, it was of interest to synthesize L-2′,3′-
didehydro-2′,3′-dideoxy-3′-fluorocytidine (L-3′-Fd4C) 1
(Scheme 1). Our synthetic method can also provide an entry
to the L-2′,3′-dideoxy-3′,3′-difluoro nucleoside 11.
at 55 psi for 3 days. Although the yield was modest (60%),
most of the unreacted starting material could be recovered
and recycled. Oxidation of the debenzylated product 5 by
chromic anhydride/pyridine/acetic anhydride gave ketone 6
in 88% yield. Treatment with (diethylamino)sulfur trifluoride
(DAST) afforded difluorinated intermediate 78 in 66% yield.
Compound 7 was converted to the acetate 8 by the modifica-
tion of a known literature method.9 Condensation of the
acetate 8 with persilylated N4-benzoylcytosine was effected
under Vorbru¨ggen conditions using trimethylsilyl trifluoro-
methanesulfonate (TMSOTf) as a catalyst. The epimeric
products 9 and 10 were chromatographically separable, and
The starting material of our synthetic approach (Scheme
1) was L-xylose, which was converted to the protected
L-ribose analogue 2 in four steps in 73% overall yield by a
well-known procedure in our laboratory.5 Benzylation of 2
was easily accomplished by treatment with sodium hydride,
followed by benzyl bromide and catalytic tetrabutylammo-
nium iodide. Methanolysis of the resulting benzyl ether gave
1
the â-isomer was more abundant. In fact, H NMR of the
1
the intermediate 3 as the sole isomer. H NMR showed the
crude reaction mixture showed an epimeric ratio of 5:4.
Deprotection of each isomer was accomplished by am-
monolysis to give difluorinated nucleosides 1110 and 12.
Elimination by treatment with sodium methoxide in DMF
signal related to the H-1 as a singlet, which indicates the
â-stereochemistry.6 Comparison of the proton spectrum with
that of the known enantiomer7 confirmed the assignment.
Conversion of 3 to the phenoxythiocarbonyl derivative
followed by the radical deoxygenation of the latter gave
protected L-2-deoxyribose 4. Compound 4 was rather unre-
active toward catalytic hydrogenation, and its palladium-
catalyzed debenzylation required treatment with hydrogen
(8) Yellow oil: [R]23 40.78° (c 4.28, CHCl3); 1H NMR (CDCl3, 400
D
MHz) δ 8.08 (m, 2H), 7.57 (m, 1H), 7.45 (m, 2H), 5.15 (dt, 1H, H1, J )
5.7, 1.7 Hz), 4.63-4.41 (m, 3H, H4, H5), 3.40 (s, 3H), 2.67 (tdd, 1H, H2R
,
J ) 16.6, 15.0, 5.7 Hz), 2.49 (tdd, 1H, H2â, J ) 15.0, 9.0, 2.0 Hz); 13C
NMR (CDCl3, 100 MHz) δ 166.05, 133.14, 129.71, 128.38, 128.32 (t, JC-F
) 252.8 Hz), 103.70 (dd, JC-F ) 7.1, 4.2 Hz), 79.46 (dd, JC-F ) 32.1,
24.9 Hz), 62.94 (dd, JC-F ) 7.5, 4.5 Hz), 55.38, 42.26 (t, JC-F ) 24.3 Hz);
HRMS (FAB) m/z found 273.0950, calcd for C13H15F2O4 273.0938 (MH+).
Anal. Calcd for C13H14F2O4: C, 57.35; H, 5.18. Found: C, 57.64; H, 5.30.
(9) Mikhailopulo, I. A.; Poopeiko, N. E.; Pricota, T. I.; Sivets, G. G.;
Kvasyuk, E. I.; Balzarini, J.; De Clercq, E. J. Med. Chem. 1991, 34, 2195-
2202.
(4) (a) Koshida, R.; Cox, S.; Harmenberg, H.; Gilljam, G.; Wahren, B.
Antimicrob. Agents Chemother. 1989, 33, 2083-2088. (b) Van Aerschot,
A.; Herdewijn, P.; Balzarini, J.; Pauwels, R.; De Clerq, E. J. Med. Chem.
1989, 32, 1743-1749.
(5) (a) Ma, T.; Pai, S. B.; Zhu, Y. L.; Lin, J. S.; Shanmuganathan, K.;
Du, J.; Wang, C.; Kim, H.; Newton, M. G.; Cheng, Y.-C.; Chu, C. K. J.
Med. Chem. 1996, 39, 2835-2843. (b) Cooperwood, J. S.; Boyd, V.;
Gumina, G.; Chu, C. K. Nucleosides Nucleotides 2000, 19, 219-236.
(6) Binkley, R. W. Modern Carbohydrate Chemistry; Marcel Dekker
Inc.: New York, 1988; pp 53-60.
(10) White solid: mp 194-196 °C (dec); [R]24 -51.89° (c 1.15,
D
MeOH); UV (MeOH) λmax 276.5 (ꢀ 18 160) (pH 2), 268.0 (ꢀ 13 280) (pH
7), 268.5 (ꢀ 13 580) (pH 11); 1H NMR (CD3OD, 400 MHz) δ 7.97 (d, 1H,
H6, J ) 7.3 Hz), 6.27 (t, 1H, H1′, J ) 6.8 Hz), 5.93 (d, 1H, H5, J ) 7.3
Hz), 4.17 (m, 1H, H4′), 3.83 (m, 2H, H5′), 2.90 (m, 1H, H2′), 2.51 (m, 1H,
H2′); 13C NMR (DMSO-d6, 100 MHz) δ 168.19, 158.46, 142.62, 128.71
(dd, JC-F ) 255.1, 247.4 Hz), 97.04, 84.73 (dd, JC-F ) 6.7, 4.9 Hz), 83.74
(7) Kosma, P.; Christian, R.; Schulz, G.; Unger, F. M. Carbohydr. Res.
1985, 141, 239-253.
4178
Org. Lett., Vol. 3, No. 26, 2001

afforded the target unsaturated nucleosides 111 and 13 in 53
and 51% yields, respectively.
Direct reaction of intermediate 7 with N4-benzoylcytosine
in the standard Vorbru¨ggen conditions did not give the
expected protected nucleosides. Instead, the inseparable
epimeric mixture 1614 (Scheme 3) was isolated in 60% yield.
Intermediate 7 could have been obtained from 2 through
a more straightforward route (Scheme 2) involving oxidation
Scheme 3. Ring-Opening of 7 in Vorbru¨ggen Conditions
Scheme 2. Alternative Route to 7
The stereochemistry of condensation products 9 and 10
has been established by 2D NMR NOESY experiments.
Thus, the NOESY spectrum of epimer 9 showed clear
correlations between protons H1′ and H4′. Correlations
between H4′ and H2′R and between the latter and H1′ were
also observed. In the case of 10, the H4′ proton strongly
correlated with one of the H2′ protons (which could thus be
identified as R), while the other H2′ (â) correlated with H1′.
of 2 to the known ketone 14, difluorination of 14 to the
difluororibose derivative 15, followed by methanolysis and
deoxygenation. Since 14 can also be obtained from L-xylose
in three steps,5 this route would have saved four steps in
our synthetic scheme. Unfortunately, ketone 14 proved to
be unreactive in the classic fluorinating conditions. Even
when the reaction was run in neat DAST, no products could
be detected, and attempts to increase the reactivity by
increasing the temperature only caused decomposition of 14.
It is known that the steric hindrance as well as electronic
effects of the isopropylidene moiety of 14 (Scheme 2) can
prevent nucleophilic attack on position 3 of the furanose
ring.12 Besides, literature examples show that high temper-
atures are required to difluorinate sterically hindered ketones.
Furthermore, in the only example of difluorination of an R,R′-
trans-disubstituted five-membered cyclic ketone found in the
literature, the carbonyl group is relatively unhindered and
the yield is still low (25%).13
Figure 2. NOE correlations and stereochemistry of 9 and 10.
In summary, L-2′,3′-dideoxy-3′,3′-difluoro- and L-2′,3′-
didehydro-2′,3′-dideoxy-3′-fluoro nucleosides have been
synthesized from L-xylose in 13 and 14 steps, respectively.
The target compounds were obtained by condensation of a
difluorinated intermediate 8 with a nucleobase in a process
of general applicability. The key difluorination step of ketone
6 proceeded with a 66% yield. Our approach seems to be
more convenient and versatile than the reported synthesis
of the D-thymidine analogue.15
(dd, JC-F ) 29.5, 25.0 Hz), 60.73 (t, JC-F ) 4.8 Hz), 42.14 (t, JC-F ) 23.4
Hz), 12.29; HRMS (FAB) m/z found 248.0850, calcd for C9H12F2N3O3
248.0847 (MH+). Anal. Calcd for C9H11F2N3O3‚0.1H2O: C, 43.41; H, 4.53;
N, 16.88. Found: C, 43.63; H, 4.50; N, 16.48.
(11) White solid: mp 182-183 °C (dec); [R]22D 6.67° (c 0.54, MeOH);
UV (MeOH) λmax 276.0 (ꢀ 11 990) (pH 2), 267.5 (ꢀ 8010) (pH 7), 264.5 (ꢀ
1
8060) (pH 11); H NMR (CD3OD, 400 MHz) δ 8.14 (d, 1H, H6, J ) 7.5
Hz), 7.01 (m, 1H, H1′), 5.90 (d, 1H, H5 J ) 7.5 Hz), 5.46 (m, 1H, H2′),
4.76 (m, 1H, H4′), 3.80 (m, 1H, H5′), 3.79 (m, 1H, H5′); 13C NMR (CD3-
OD, 100 MHz) δ 168.31, 163.40 (d, JC-F ) 284.8 Hz), 159.05, 144.05,
102.34 (d, JC-F ) 9.7 Hz), 95.68, 88.42 (d, JC-F ) 14.9 Hz), 81.96 (d,
JC-F ) 24.7 Hz), 61.82 (d, JC-F ) 2.2 Hz); HRMS (FAB) m/z found
228.0778, calcd for C9H11FN3O3 228.0784 (MH+). Anal. Calcd for C9H10-
FN3O3: C, 47.58; H, 4.44; N, 18.50. Found: C, 47.47; H, 4.44; N, 18.17.
(12) Binkley, R. W. Modern Carbohydrate Chemistry; Marcel Dekker
Inc.: New York, 1988; pp 171-173.
(14) Yellow oil: 1H NMR (CDCl3, 400 MHz) δ 8.75 (bs, 2H), 8.06-
7.45 (m, 24H), 6.14-6.10 (m, 2H), 7.57 (m, 2H), 4.56 (dd, 1H, J ) 11.5,
3.2 Hz), 4.51 (dd, 1H, J ) 11.7, 4.2 Hz), 4.37 (m, 2H), 4.27 (m, 2H), 3.40
(s, 3H), 3.38 (s, 3H), 2.57 (m, 2H), 2.42 (m, 2H), 0.17 (s, 9H), 0.16 (s,
9H); 13C NMR (CDCl3, 100 MHz) δ 166.13, 166.07, 154.90, 154.81, 143.12,
133.20, 133.16, 129.60, 129.56, 128.97, 128.45, 128.41, 127.60, 121.33 (t,
JC-F ) 248.2 Hz), 121.10 (t, JC-F ) 248.2 Hz), 97.69, 97.53, 83.91, (dd,
JC-F ) 5.1, 3.1), 83.76 (dd, JC-F ) 7.1, 3.1), 72.13 (t, JC-F ) 29.3 Hz),
(13) (a) Biggadike, K.; Borthwick, A. D.; Evans, D.; Exall, A. M.; Kirk,
B. E.; Roberts, S. M.; Stephenson, L.; Youds, P.; Slawin, A. M. Z.; Williams,
D. J. J. Chem. Soc., Chem. Commun. 1987, 4, 251-254. (b) Borthwick, A.
D.; Evans, D. N.; Kirk, B. E.; Biggadike, K.; Exall, A. M.; Youds, P.;
Roberts, S. M.; Knight, D. J.; Coates, J. A. V. J. Med. Chem. 1990, 33,
179-186.
72.08 (t, JC-F ) 28.0 Hz), 64.20, 64.16, 57.08, 57.01, 37.92 (t, JC-F
)
23.5 Hz), 37.81 (t, JC-F ) 23.6 Hz), 0.11; MS (FAB) m/z 560 (MH+).
(15) Bergstrom, D.; Romo, E.; Shum, P. Nucleosides Nucleotides 1987,
6, 53-63.
Org. Lett., Vol. 3, No. 26, 2001
4179

Preliminary biological evaluation of the synthesized
compounds showed that L-3′-Fd4C 1 has potent anti-HIV
activity (EC50 0.03 µM in PBM cells) with little or no
significant toxicity (IC50 ) 86.9 µM in PBM cells and IC50
> 100 µM in CEM cells).16 The difluorinated analogue 11
was inactive. These promising results prompt us to synthesize
other pyrimidine and purine analogues in order to study the
full structure-activity relationships, which is in progress in
our laboratory.
Acknowledgment. This research was supported by the
U.S. Public Health Service Research Grant AI 32351 from
the National Institute of Allergy and Infectious Diseases,
NIH. We thank Dr. Michael Bartlett of the College of
Pharmacy, The University of Georgia, for performing the
high-resolution mass spectra.
(16) Whereas AZT showed EC50 of 0.004 µM, with IC50 > 100 in PBM
cells and IC50 ) 14.3 in CEM cells (ref 3d).
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