2'-Fluoro-4'-thio-2',3'-unsaturated nucleosides: anti-HIV activity, resistance profile, and molecular modeling studies.

Article abstract of DOI:10.1081/NCN-120021965

Both D- and L-2'-fluoro-4'-thio-2',3'-unsaturated nucleosides were synthesized and their anti-HIV activity against the drug sensitive virus and lamivudine-resistant mutant (M184V) were evaluated. In vitro antiviral evaluation indicated that the L-isomers

Full text of DOI:10.1081/NCN-120021965

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2′-Fluoro-4′-thio-2′,3′-unsaturated Nucleosides: Anti-
HIV Activity, Resistance Profile, and Molecular Modeling
Studies
Youhoon Chong a , Hyunah Choo a , Raymond F. Schinazi b & Chung K. Chu a c
a Department of Pharmaceutical and Biomedical Sciences , College of Pharmacy, The
University of Georgia , Athens, Georgia, USA
b Department of Pediatrics , Emory University School of Medicine/Veterans Affairs Medical
Center , Decatur, Georgia, USA
c Department of Pharmaceutical and Biomedical Sciences , College of Pharmacy, The
University of Georgia , Athens, GA, 30602, USA
Published online: 31 Aug 2006.
To cite this article: Youhoon Chong , Hyunah Choo , Raymond F. Schinazi & Chung K. Chu (2003) 2′-Fluoro-4′-thio-2′,3′-
unsaturated Nucleosides: Anti-HIV Activity, Resistance Profile, and Molecular Modeling Studies, Nucleosides, Nucleotides and
Nucleic Acids, 22:5-8, 611-615, DOI: 10.1081/NCN-120021965
To link to this article: http://dx.doi.org/10.1081/NCN-120021965
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 611–615, 2003
20-Fluoro-40-thio-20,30-unsaturated Nucleosides:
Anti-HIV Activity, Resistance Profile, and
Molecular Modeling Studies
Youhoon Chong,1 Hyunah Choo,1 Raymond F. Schinazi,2
and Chung K. Chu1,
*
1Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy,
The University of Georgia, Athens, Georgia, USA
2Department of Pediatrics, Emory University School of Medicine=Veterans
Affairs Medical Center, Decatur, Georgia, USA
ABSTRACT
Both d- and l-20-fluoro-40-thio-20,30-unsaturated nucleosides were synthesized and
their anti-HIV activity against the drug sensitive virus and lamivudine-resistant
mutant (M184V) were evaluated. In vitro antiviral evaluation indicated that the
l-isomers are more potent than the d-isomers, but unfortunately all were cross-
resistant with 3TC. Molecular modeling studies revealed that the unnatural sugar
moiety of the l-nucleosides as well as 40-sulfur atom of the d-isomer has a steric
conflict with the bulky side chain of valine 184, resulting in cross-resistance.
*Correspondence: Chung K. Chu, Department of Pharmaceutical and Biomedical Sciences,
College of Pharmacy, The University of Georgia, Athens, GA 30602, USA; E-mail: dchu@
rx.uga.edu.
611
DOI: 10.1081/NCN-120021965
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

612
Chong et al.
The past decade has witnessed the discovery of a class of 20,30-unsaturated
nucleosides such as d4T,[1,2] L-d4C,[3,4] L-d4FC[3,4] and abacavir[5,6] as interesting
therapeutic agents for anti-HIV therapy because of their potent antiviral activity.
However, these unsaturated nucleosides are inheritably unstable in acidic environ-
ment, which reduce the bioavailability and antiviral efficacy. In order to alleviate
these drawbacks, 20-F moiety was introduced to increase acid stability while main-
taining the antiviral potency of several purine and pyrimidine nucleosides.[7] There-
fore, agents containing the 20,30-unsaturated sugar moiety with 20-fluoro substitution
has become one of the rational targets in search for safe, effective and chemically
stable antiviral agents. Thus, it was of interest to further modify the structure to
20F-40S-20,30-unsaturated nucleosides with isosteric replacement of the 40-oxygen by
a sulfur atom.
Both d- and l-isomers were synthesized starting from d- and l-glyceraldehyde
derivatives, respectively (Sch. 1 and 2). The 2-fluorobutenolactone, which can be
readily prepared from glyceraldehyde derivative, was converted to 2-fluoro-4-thio-
lactone in four steps, from which the key intermediate, 2-fluoro-4-thio-1-acetate
was prepared for condensation with various pyrimidine and purine bases.
The antiviral activity of the synthesized compounds were evaluated against
HIV-1 in human peripheral blood mononuclear (PBM) cells, among which the cyti-
dine, 5-fluorocytidine, adenosine and 2-fluoroadenosine analogues showed moder-
ate to potent anti-HIV activities. The isosteric substitution of the 40-oxygen with
a 40-sulfur, however, resulted in cross-resistance with the M184V mutant. The cyti-
dine and 5-fluorocytidine analogues showed significantly decreased antiviral activity
against the clinically important lamivudine-resistant variants (HIV-1M184V),
whereas the corresponding d-20-Fd4 nucleosides showed only limited cross-
resistance.
According to our molecular modeling studies using the crystal structure of HIV-1
RT, we found that the M184V mutation in HIV-1 RT causes a serious problem
in positioning the unnatural l-configured nucleoside triphosphates at the active
site because the side chain of Val184 tend to occupy the space where the sugar moiety
Scheme 1. Synthesis of the key intermediate 2-fluoro-4-thio-2-phenylselenyl-1-acetate 8.

20-Fluoro-40-thio-20,30-unsaturated Nucleosides
613
Scheme 2. Synthesis of various pyrimidine and purine nucleosides.
of l-20F-20,30-unsaturated nucleosides projected (Fig. 1). The resulting steric
hindrance destabilized the l-20F-20,30-unsaturated nucleoside triphosphate=RT
complex. On the other hand, the structure of d-20F-d4CTP=RT complex does not
show any significant steric crash between Val184 and the sugar moiety of nucleoside
triphosphate because the natural d-configured sugar moiety was located at the
opposite side (Fig. 1).
The potent anti-HIV-1M184V activity of the cytidine analog, d-20F-d4C, sup-
ports this result. In this context, it is interesting that M184V RT is cross-resistant
to the natural d-20F-40S-d4C. Molecular modeling studies demonstrated that the
larger van der Waals radius as well as the close proximity to Met184 of the 40-
sulfur atom of d-20-F-40-Sd4C may be the reasons for the decreased antiviral
potency of synthesized 40-thio nucleosides against the lamivudine-resistant variants
(HIV-1M184V). The energy-minimized structures[8] of d-20-Fd4C and d-20-F-40-Sd4C
bound to HIV-1 RT shows that nucleoside inhibitors are located in a well-defined
binding pocket formed by Arg72, Met184 and 30-OH pocket residues. However,
because of its out-of-plane location and large van der Waals radius, the 40-sulfur
atom in the d-20-F-40-Sd4N sugar moiety is in close contact to Met184 (Fig. 2).
As a result, the mutation of Met184 to Val184, which has a bulky side chain,
results in a significant steric hindrance between the 40-sulfur atom in the d-20-F-
40-Sd4N sugar moiety and the side chain of Val184, which might be one of the
reasons for the high cross-resistance of HIV-1M184V to d-20-F-40-Sd4C and d-20-
F-40-Sd4FC.
Figure 1. l-20-F-4’-S-d4CTP=RT complex after mutation from M184 to V184 showing a
steric hindrance between the sugar moiety of l-20-F-40-S-d4CTP and the side chain of V184
(left). d-20-F-d4CTP=RT complex after mutation showing no steric hindrance (right).

614
Chong et al.
Figure 2. Energy minimized structure of D-20-F-40-Sd4C, complexed with the wild-type HIV-
1 RT (left) and M184V mutant RT (right).
Table 1. Activity of selected nucleosides against lamivudine-resistant virus (HIV-1M184V) in
human PBM cells.
Compound
xxBRU (EC90, mM)
M184V (EC90, mM)
FIa
25
>100
0.2
>100
0.3
6,688
d-20-F-40-Sd4Cb
l-20-F-40-Sd4Cb
d-20-F-d4Cb
l-20-F-d4Cb
AZT
5.0
1.4
8.0
2.1
0.01
0.08
ꢀ125
>100
1.8
>100
0.003
535
3TC
In summary, the synthesized 20-fluoro-40-thio-20,30-unsaturated nucleosides
showed potent anti-HIV activity. In vitro antiviral evaluation indicated that the l-iso-
mers are more potent than the d-isomers, but unfortunately all were cross-resistant
with 3TC. Molecular modeling studies showed that the steric hindrance of the bulky
side chain of valine 184 with unnatural sugar moiety of the l-nucleoside as well as
40-sulfur atom of the d-isomer can be one of the reasons for the crossresistance.
ACKNOWLEDGMENTS
This research was supported by the U.S. Public Health Service Grant (AI32351
& AI25899) from the National Institutes of Health and the Department of Veterans
Affairs.
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