Design and synthesis of 2',3'-dideoxy-2',3'-didehydro-β-L-cytidine (β- L-d4C) and 2',3'-dideoxy-2'-3'-didehydro-β-L-5-fluorocytidine (β-L-Fd4C), two exceptionally potent inhibitors of human hepatitis B virus (HBV) and potent inhibitors of human immunodeficiency virus (HIV) in vitro

Full text of DOI:10.1021/jm950836q

© Copyright 1996 by the American Chemical Society
Volume 39, Number 9
April 26, 1996
Communications to the Editor
Design a n d Syn th esis of 2′,3′-Did eoxy-
2′,3′-d id eh yd r o-â-L-cytid in e (â-L-d 4C) a n d
2′,3′-Did eoxy-2′,3′-d id eh yd r o-â-L-5-
flu or ocytid in e (â-L-F d 4C), Tw o
Excep tion a lly P oten t In h ibitor s of
Hu m a n Hep a titis B Vir u s (HBV) a n d
P oten t In h ibitor s of Hu m a n
Im m u n od eficien cy Vir u s (HIV) in Vitr o
Tai-Shun Lin,† Mei-Zhen Luo, Mao-Chin Liu,
Yong-Lian Zhu, Elizabeth Gullen,
Ginger E. Dutschman, and Yung-Chi Cheng*
Department of Pharmacology and The Comprehensive
Cancer Center, Yale University School of Medicine,
New Haven, Connecticut 06520-8066
F igu r e 1.
Received November 13, 1995
fluoro-5-methyl-â-L-arabinofuranosyluracil (L-FMAU)12
(Figure 1). Since 2′,3′-dideoxy-2′,3′-didehydro-â-D-cyti-
dine (â-D-d4C) has proven to be about as equipotent as
â-L-ddC as an anti-HIV agent,13 it seemed logical to
synthesize and evaluate the antiviral activity of its
L-enantiomer, 2′,3′-dideoxy-2′,3′-didehydro-â-L-cytidine
(â-L-d4C, 1), and its 5-fluoro derivative, 2′,3′-dideoxy-
2′,3′-didehydro-â-L-5-fluorocytidine (â-L-Fd4C, 2). In
this communication, we report the synthesis and bio-
logical evaluation of â-L-d4C and â-L-Fd4C, which show
exceptional potent activity against HBV and significant
activity against HIV. Since patients receiving long-
term, anti-HBV or -HIV nucleoside therapy have expe-
rienced delayed toxicity, which may be linked to the
drugs inhibition of mitochondrial DNA synthesis,14 the
effect of â-L-d4C and â-L-Fd4C in decreasing the mito-
chondrial DNA content in cells upon long-term exposure
to these two drugs was also studied. â-L-d4C (1) was
also independently synthesized by Mansuri et al.;15
however, no physical properties and spectroscopic data
were reported. Furthermore, contrary to our results,
they reported that this compound has no antiviral
activity.
It has been estimated that 300 million people world-
wide are now infected with the hepatitis B virus (HBV),
a causative agent of both acute and chronic forms of
hepatitis.1 Furthermore, chronic infection with HBV
has been associated with a high risk for the development
of primary hepatocellular carcinogen.2 In fact, at the
present time there is no clinically useful drug for the
treatment of HBV infection. Considerable effort has
been directed in the search for novel nucleoside struc-
tures for use as antiviral and anticancer agents. Most
of these analogues are synthesized by modification of
the naturally occurring nucleosides and, therefore,
possess the â-D-configuration. In the past, little atten-
tion has been given to the synthesis and study of the
biological activity of L-nucleosides, the enantiomers of
natural D-nucleosides. Recently, a number of the un-
natural L-configuration nucleoside analogues have
emerged as potent antiviral agents against HBV and
human immunodeficiency virus (HIV), which include
â-L-[2-(hydroxymethyl)-1,3-oxathiolan-4-yl]cytosine [â-L-
SddC, (-)-SddC, 3TC, Lamivudine],3-5 â-L-[2-(hydroxy-
methyl)-1,3-oxathiolan-4-yl]-5-fluorocytosine [(-)-FSd-
dC, (-)-FTC],3,6,7 2′,3′-dideoxy-â-L-cytidine (â-L-ddC),
2′,3′-dideoxy-â-L-5-fluorocytidine (â-L-FddC),8-11 and 2′-
Ch em istr y. The synthesis of 2′,3′-dideoxy-2′,3′-di-
dehydro-â-L-cytidine (â-L-d4C, 1) and 2′,3′-dideoxy-2′,3′-
didehydro-â-L-5-fluorocytidine (â-L-Fd4C, 2) is depicted
† Deceased.
0022-2623/96/1839-1757$12.00/0 © 1996 American Chemical Society

1758 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9
Communications to the Editor
Sch em e 1
in Scheme 1. The key intermediate 3′,5′-dibenzoyl-2′-
deoxy-â-L-uridine (3) was synthesized from L-arabinose
by the methodology of Holy16 with minor modification.
Transglycosylation17 reaction of 3 with silylated 5-fluo-
rouracil using trimethylsilyl trifluoromethanesulfonate
as a catalyst afforded a mixture of the R- and â-anomers
3′,5′-dibenzoyl-2′-deoxy-R-L-5-fluorouridine (4a) and 3′,5′-
dibenzoyl-2′-deoxy-â-L-5-fluorouridine (4b), which were
separated by silica gel chromatography. Treatment of
compounds 3 and 4b with saturated NH3/MeOH solu-
tion overnight produced 2′-deoxy-â-L-uridine (5) and 2′-
deoxy-â-L-5-fluorouridine (6). Reaction of 5 and 6 with
2 M equiv of mesyl chloride in anhydrous pyridine
followed by 1 N NaOH solution18 resulted in the
formation of the respective cyclic ethers 7 and 8.
Treatment19 of compounds 7 and 8 with 4-chlorophenyl
phosphorodichloridate and 1,2,4-triazole in anhydrous
pyridine followed by a mixture of ammonium hydroxide/
dioxane (2:1, v/v) afforded the corresponding cytidine
derivatives 9 and 10, which were then treated with tert-
butoxide in DMSO to furnish the target compounds 2′,3′-
dideoxy-2′,3′-didehydro-â-L-cytidine (â-L-d4C, 1)20 and
2′,3′-dideoxy-2′,3′-didehydro-â-L-5-fluorocytidine (â-L-
Fd4C, 2), respectively.21
Biologica l Eva lu a tion . The synthesized compounds
â-L-d4C (1) and â-L-Fd4C (2), along with the known
antiviral compounds â-D-ddC, â-D-d4C, â-L-FddC, and
â-L-SddC, were tested for their antiviral activities in
vitro, and the results are shown in Table 1. The
procedures used were the same as described previ-
ously.3,9 Among these nucleoside analogues, 2′,3′-
dideoxy-2,3′-didehydro-â-L-5-fluorocytidine (â-L-Fd4C, 2)
was found to be most active against HBV. Compounds
demonstrating significant anti-HBV activity (extracel-
lular and intracellular, respectively) in decreasing activ-
Ta ble 1. Evaluation of 2′,3′-Dideoxycytidine Analogue
Antiviral Activity against Hepatitis B Virus (HBV) and Human
Immunodeficiency Virus (HIV-1), Effects on Mitochondrial DNA
Synthesis, and Cytotoxicity against CEM Cells in Vitro
cytotoxicity
a
HBV ED50 (nM)
ED50c(µM)
extra-
intra-
HIV-1b
cell
mt-
DNA
compd
DddC
cellular cellular ED50(µM) growth
5200
3000
10
6200
1.5
0.7
10
22
20
7
0.022
2
>20
Dd4C
1 (â-L-d4C)
2 (â-L-Fd4C)
â-L-FddC
â-L-SddC
(3TC)
8
2
∼1.0
2
0.09
>100
>100
>50
55
17
36
30
0.5
2.0
67
50
a
Concentration required to inhibit 50% of extracellular circular
and intracellular replicating HBV DNA using 2215 cell line.3
b
Concentration required to inhibit 50% of HIV activity in MT-2
cells.9 c Concentration required to inhibit 50% of cell growth and
to decrease 50% of mt-DNA content in CEM cells.3,9
ity were â-L-Fd4C, â-L-d4C, â-L-SddC, and â-L-FddC. To
the best of our knowledge, â-L-Fd4C appears to be the
most potent and selective compound against HBV
reported to date. In addition, the compounds exhibiting
activity against HIV in decreasing antiviral activity
were â-L-Fd4C, â-L-FddC, â-D-d4C, â-L-d4C, â-D-ddC,
and â-L-SddC. The compounds were also tested in vitro
for their cytotoxicity against CEM cell lines. The
cytotoxity of these nucleotides in decreasing order were
â-L-Fd4C, â-D-ddC, â-L-d4C, â-D-d4C, â-L-SddC, and â-L-
FddC.
Since anti-HIV, D-nucleoside analogues produce de-
layed toxicity in patients from long-term treatment with
these drugs, and this delayed toxicity caused by these
drugs may relate to their ability to inhibit mitochondrial
DNA synthesis,14 the ability of â-L-Fd4C and â-L-d4C

Communications to the Editor
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9 1759
Schinazi, R. F.; Painter, G. R. The Anti-Hepatitis B Virus
Activities, Cytotoxicities, and Anabolic Profiles of the (-) and
(+) Enantiomers of cis-5-Fluoro-1-[2-(hydroxymethyl)-1,3-ox-
athiolan-5-yl]cytosine. Antimicrob. Agents Chemother. 1992, 36,
2686-2692.
to decrease the mitochondrial DNA content in cells upon
long-term exposure to these two drugs was also evalu-
ated. Both compounds showed no effect on mitochon-
drial DNA content of CEM cells after a 6 day exposure
at 10 µM, which is a much higher concentration required
to inhibit HBV in culture. The spectrum of biological
activity of â-L-Fd4C and â-L-d4C in culture is similar
to â-L-SddC (3TC), which has just been approved by the
FDA for use in combination drug therapy against HIV
and HBV.
In conclusion, we present the synthesis and prelimi-
nary biological profile of 2′,3′-dideoxy-2′,3′-didehydro-
â-L-cytidine (â-L-d4C, 1) and 2′,3′-dideoxy-2′,3′-didehydro-
â-L-5-fluorocytidine (â-L-Fd4C, 2), which demonstrated
exceptional potent activity against HBV and significant
activity against HIV. Therefore, these two compounds
merit further development as potential anti-HBV and
HIV agents.
(8) Lin, T. S.; Luo, M. Z.; Liu, M. C.; Pai, S. B.; Dutschman, G. E.;
Cheng, Y. C. Antiviral Activity of 2′,3′-Dideoxy-â-L-5-fluorocy-
tidine (â-L-FddC) and 2′,3′-Dideoxy-â-L-cytidine (â-L-ddC) against
Hepatitis B Virus and Human Immunodeficiency Virus Type 1
in Vitro. Biochem. Pharmacol. 1994, 47, 171-174.
(9) Lin, T. S.; Luo, M. Z.; Liu, M. C.; Pai, S. B.; Dutschman, G. E.;
Cheng, Y. C. Synthesis and Biological Evaluation of 2′,3′-
Dideoxy-L-pyrimidine Nucleosides as Potential Antiviral Agents
against Human Immunodeficiency Virus (HIV) and Hepatitis
B Virus (HBV). J . Med. Chem. 1994, 37, 798-803.
(10) Gosselin, G.; Schinazi, R. F.; Sommadossi, J . P.; Mathe´, C.;
Bergogne, M. C.; Aubertin, A. M.; Kirn, A.; Imbach, J . L. Anti-
Human Immunodeficiency Virus Activities of the â-L-Enanti-
omer of 2′,3′-Dideoxycytidine and Its 5-Fluoro Derivative in
Vitro. Antimicrob. Agents Chemother. 1994, 38, 1292-1297.
(11) Van Draanen, N. A.; Tisdale, M.; Parry, N. R.; J ansen, R.;
Dornsife, R. E.; Tuttle, J . V.; Averett, D. R.; Koszalka, G. W.
Influence of Stereochemistry on Antiviral Activities and Resis-
tance Profiles of Dideoxycytidine Nucleosides. Antimicrob. Agents
Chemother. 1994, 38, 868-871.
(12) Chu, C. K.; Ma, T. W.; Shanmuganathan, K.; Wang, C. G.; Xiang,
Y. J .; Pai, S. B.; Yao, G. Q.; Sommadossi, J .-P.; Cheng, Y.-C.
Use of 2′-Fluoro-5-methyl-â-L-arabinosyluracil as a Novel Anti-
viral Agent for Hepatitis B Virus and Epstein-Barr Virus.
Antimicrob. Agents Chemother. 1995, 39, 979-981.
(13) Balzarini, J .; Pauwels, R.; Herdewijn, P.; De Clercq, E.; Cooney,
D. A.; Kang, G.-J .; Dalal, M.; J ohns, D. G.; Broder, S. Potent
and Selective Anti-HTLV-III/LAV Activity of 2′,3′-Dideoxycy-
tidinene, The 2′,3′-Unsaturated Derivative of 2′,3′-Dideoxycyti-
dine. Biochem. Biophys. Res. Commun. 1986, 140, 735-742.
(14) Parker, W. B.; Cheng, Y. C. Mitochondrial Toxicity of Antiviral
Nucleoside Analogs. J . NIH Res. 1994, 6, 57-61.
(15) Mansuri, M. M.; Farina, V.; Starrett, J . E., J r.; Benigni, D. A.;
Brankovan, V.; Martin, J . C. Preparation of the Geometric
Isomers of DDC, DDA, D4C, and D4T as Potential Anti-HIV
Agents. Bioorg. Med. Chem. Lett. 1991, 1, 65-68.
(16) Holy, A. Nucleic Acid Components and Their Analogues. CLIII.
Preparation of 2′-Deoxy-L-ribonucleosides of the Series. Collect.
Czech. Chem. Commun. 1972, 37, 4072-4086.
Ack n ow led gm en t . This investigation was sup-
ported by PHS grants AI-29430 (formerly to T.S.L.), AI-
33655, and CA-44358 (to Y.C.C.), awarded by the
National Institutes of Health, DHHS. We thank Ms.
Diane Mozdziesz for her excellent technical assistance.
We also acknowledge the support of the Instruments
Center of the Department of Chemistry at Yale Uni-
versity for the high-resolution NMR spectra.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for compounds 1-4a ,b and 7-10 and elemental
analyses for compounds 1, 2, 4b, and 7-10 (5 pages). Ordering
information is given on any current masthead page.
Refer en ces
(1) Dusheiko, G.; Hoofnagle, J . H. Hepatitis B. In Oxford Textbook
of Clinical Hepatology; McIntyre, N., Benhamou, J . P., Bircher,
J ., Rizzetto, M., Rodes, J ., Eds.; Oxford University Press: New
York, 1991; pp 571-592.
(2) Di Bisceglei, A. M.; Rustgi, V. K.; Hoofnagle, J . H.; Dusheiko,
G. M.; Lotze, M. T. Hepatocellular Carcinoma. Ann. Intern. Med.
1988, 108, 390-401.
(3) Chang, C. N.; Doong, S. L.; Zhou, J . H.; Beach, J . W.; J eong, L.
S.; Chu, C. K.; Tsai, C. H.; Cheng, Y. C. Deoxycytidine Deami-
nase-Resistant Stereoisomer Is the Active Form of (()-2′,3′-
Dideoxy-3′-thiacytidine in the Inhibition of Hepatitis B Virus
Replication. J . Biol. Chem. 1992, 27, 13938-13942.
(4) Beach, J . W.; J eong, L. S.; Alves, A. J .; Pohl, D.; Kim, H. O.;
Chang, C. N.; Doong, S. L.; Schinazi, R. F.; Cheng, Y. C.; Chu,
C. K. Synthesis of Enantiomerically Pure (2′R,5′S)-(-)-1-[2-
(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine as a Potent Anti-
(17) Imazawa, M.; Eckstein, F. Synthesis of 3′-Azido-2′,3′-dideoxyri-
bofuranosylpurines. J . Org. Chem. 1978, 43, 3044-3048.
(18) Lin, T. S.; Yang, J . H.; Gao, Y. S. Synthesis of 2′,3′-Unsaturated
and 2′,3′-Dideoxy Analogs of 6-Azapyrimidine Nucleosides as
Potential Anti-HIV Agents. Nucleosides Nucleotides 1990, 9, 97-
108.
(19) Lin, T. S.; Yang, J . H.; Liu, M. C.; Shen, Z. Y.; Cheng, Y. C.;
Prusoff, W. H.; Birnbaum, G. I.; Giziewicz, J .; Ghazzouli, I.;
Brankovan, V.; Feng, J . S.; Hsiung, G. D. Synthesis and
Anticancer Activity of Various 3′-Deoxy Pyrimidine Nucleoside
Analogues and Crystal Structure of 1-(3-Deoxy-â-D-threo-pento-
furanosyl)cytosine. J . Med. Chem. 1991, 34, 693-701.
(20) Isolated as a white solid: mp 148-150 °C dec; TLC Rf 0.54 (CH2-
Cl2/MeOH, 2:1, v/v); [R]D -23.2° (c ) 0.11, MeOH); UV (MeOH)
λmax 268 nm (ꢀ 5795), λmin 250 nm; UV (0.01 N HCl) λmax 278 nm
(ꢀ 9711), λmin 240 nm; UV (0.01 N NaOH) λmax 270 nm (ꢀ 6735),
λmin 250 nm; 1H NMR (Me2SO-d6) δ 3.52-3.56 (dd, 2 H, 5′-H),
4.74 (m, 1 H, 4′-H), 4.94-4.98 (t, 1 H, 5′-OH, D2O exchangeable),
5.68 (d, 1 H, 5-H, J ) 7.6 Hz), 5.86 (m, 1 H, 3′-H), 6.32 (m, 1 H,
2-H′), 6.87 (d, 1 H, 1′-H), 7.12 and 7.22 (2 s, 2 H, 4-NH2, D2O
exchangeable), 7.67 (d, 1 H, 6-H, J ) 7.6 Hz).
viral Agent Against Hepatitis
B Virus (HBV) and Human
Immunodeficiency Virus (HIV). J . Org. Chem. 1992, 57, 2217-
2219.
(5) Schinazi, R. F.; Chu, C. K.; Peck, A.; McMillian, A.; Mathis, R.;
Cannon, D. L.; J eong, L. S.; Beach, J . W.; Choi, W. B.; Yeola, S.;
Liotta, D. C. Activities of the Four Optical Isomers of 2′,3′-
Dideoxy-3′-thiacytidine (BCH-189) Against Human Immunode-
ficiency Virus Type 1 in Human Lymphocytes. Antimicrob.
Agents Chemother. 1992, 36, 672-676.
(6) Hoong, L. K.; Strange, L. E.; Liotta, D. C.; Koszalka, G. W.;
Burns, C. L.; Schinazi, R. F. Enzyme-Mediated Enantioselective
Preparation of Pure Enantiomers of the Antiviral Agent 2′,3′-
Dideoxy-5-fluoro-3′-thiacytidine [FTC] and Related Compounds.
J . Org. Chem. 1992, 57, 5563-5565.
(7) Furman, P. A.; Davis, M.; Liotta, D. C.; Pafe, M.; Frick, L. W.;
Nelson, D. J .; Dornsife, R. E.; Wurster, J . A.; Wilson, L. J .; Fyfe,
J . A.; Tuttle, J . V.; Miller, W. H.; Condreay, L.; Averett, D. R.;
(21) Isolated as a white solid: mp 118 °C dec; TLC Rf 0.46 (AcOEt/
EtOH, 5:1, v/v); [R]D -23.4° (c ) 0.11, MeOH); UV (MeOH) λmax
280 nm (ꢀ 5966), λmin 262 nm; UV (0.01 N HCl) λmax 285 nm (ꢀ
7192), λmin 247 nm; UV (0.01 N NaOH) λmax 281 nm (ꢀ 6735),
λmin 262 nm; 1H NMR (Me2SO-d6) δ 3.31-3.70 (dd, 2 H, 5′-H),
4.45-4.48 (t, 1 H, 5′-OH, D2O exchangeable), 4.77 (m, 1 H, 4′-
H), 5.87 (m, 1 H, 3′-H), 6.31 (m, 1 H, 2′-H), 6.82 (d, 1 H, 1′-H),
7.55 and 7.78 (2 br s, 2 H, 4-NH2, D2O exchangeable), 8.03 (dd,
1 H, 6-H, J ) 7.1 Hz).
J M950836Q