Synthesis and anti-HIV activity of 2′-deoxy-2′-fluoro-4′-C-ethynyl nucleoside analogs

Article abstract of DOI:10.1016/j.bmcl.2010.05.090

Based on the favorable antiviral profiles of 4′-substituted nucleosides, novel 1-(2′-deoxy-2′-fluoro-4′-C-ethynyl-β-d-arabinofuranosyl)-uracil (1a), -thymine (1b), and -cytosine (2) analogs were synthesized. Compounds 1b and 2 exhibited potent anti-HIV-1 activity with IC₅₀ values of 86 and 1.34 nM, respectively, without significant cytotoxicity. Compound 2 was 35-fold more potent than AZT against wild-type virus, and also retained nanomolar antiviral activity against resistant strains, NL4-3 (K101E) and RTMDR. Thus, 2 merits further development as a novel NRTI drug.

Full text of DOI:10.1016/j.bmcl.2010.05.090

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4053–4056
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-HIV activity of 20-deoxy-20-fluoro-40-C-ethynyl nucleoside
analogs
Qiang Wang a, Yanfeng Li a, Chuanjun Song a, Keduo Qian b, Chin-Ho Chen c, Kuo-Hsiung Lee b,
,
*
Junbiao Chang a,
*
a Department of Chemistry, Zhengzhou University, PR China
b Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA
c Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
a r t i c l e i n f o
a b s t r a c t
Based on the favorable antiviral profiles of 40-substituted nucleosides, novel 1-(20-deoxy-20-fluoro-40-C-
ethynyl-b- -arabinofuranosyl)-uracil (1a), -thymine (1b), and -cytosine (2) analogs were synthesized.
Compounds 1b and 2 exhibited potent anti-HIV-1 activity with IC50 values of 86 and 1.34 nM, respec-
tively, without significant cytotoxicity. Compound 2 was 35-fold more potent than AZT against wild-type
virus, and also retained nanomolar antiviral activity against resistant strains, NL4-3 (K101E) and RTMDR.
Thus, 2 merits further development as a novel NRTI drug.
Article history:
Received 8 April 2010
Revised 20 May 2010
Accepted 21 May 2010
Available online 25 May 2010
D
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
20-Deoxy-20-fluoro-40-C-ethynyl nucleosides
Anti-HIV activity
Human immunodeficiency virus type-1 (HIV-1) infection affects
approximately 40 million individuals worldwide. The HIV-1 re-
verse transcriptase (RT) enzyme is responsible for converting the
genomic single-strand RNA of HIV into double-strand DNA; there-
fore, it is a major target for anti-HIV drug discovery.1 HIV-1 RT
inhibitors fall into two classes: nucleoside RT inhibitors (NRTIs)
and non-nucleoside RT inhibitors (NNRTIs). Since the discovery of
zidovudine (AZT),2 many nucleoside analogs have been designed
and synthesized. Currently, seven NRTIs have been approved by
the US FDA for the treatment of HIV infections, including zidovu-
dine (AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T),
lamivudine (3TC), abacavir (ABC), and emtricitabine [(ꢀ)FTC].3
These drugs block the synthesis of double-strand viral DNA from
the newly made single-strand DNA, and thus terminate or abort
the polymerization process catalyzed by HIV RT.4
ated clinically, NRTIs remain the most potent and efficient antiviral
drugs and are still used as first-line clinical therapies. Therefore,
structurally modified novel nucleosides are needed to overcome
the treatment drawbacks.
From a structure–activity relationship (SAR) standpoint, the
emergence of highly drug-resistant HIV-1 variants suggests that
RT is capable of discriminating physiologic 20-deoxynucleosides
(dNs) from 20,30-dideoxynucleosides (ddNs), at least by recognizing
the difference in the 30-position.6 To overcome the resistance is-
sues, novel nucleoside analogs that retain the 30-
a-OH were de-
signed in our study to exert antiviral activity against HIV-1. RT
might not be able to discriminate such analogs or, if it does, it
may do so less effectively. In addition, a fluorine moiety was also
incorporated into the 20 position of the ribose. Thus, in our
continued research on NRTIs,7–9 we report herein the synthesis of
Emerging drug-resistant viral strains as well as long-term toxic-
ity are the main problems in current antiviral chemotherapy.5
Although there are many RT inhibitor-resistant viral strains gener-
O
NH2
N
O
N
NH
NH
Abbreviations: HIV-1, human immunodeficiency virus type-1; RT, reverse trans-
criptase; NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside
reverse transcriptase inhibitor; dN, 20-deoxynucleoside; ddN, 20,30-dideoxynucleo-
side; AZT, zidovudine; SAR, structure–activity relationship; DMTrCl, 4,40-dimethoxy-
trityl chloride; TBDMSCl, tert-butyldimethylsilyl chloride; TLC, thin-layer
chromatography.
HO
N
O
HO
N
O
HO
O
O
O
O
F
F
F
OH
HO
OH
* Corresponding authors. Tel.: +1 919 962 0066; fax: +1 919 966 3893 (K.-H.L.);
tel./fax: +86 371 67783017 (J.C.).
E-mail addresses: khlee@unc.edu (K.-H. Lee), changjunbiao@zzu.edu.cn (J.
Chang).
1a
1b
2
Figure 1. Structures of 1-(20-deoxy-20-fluoro-40-C-ethynyl-b-
uracil (1a), -thymine (1b), and -cytosine (2).
D-arabinofuranosyl)-
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.090

4054
Q. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4053–4056
20-deoxy-20-fluoro-40-C-ethynyl nucleoside analogs ( Fig. 1) and
6 in CHCl3 to give the desired dibenzoylated b-nucleoside analogs
7a and 7b in over 80% yield. Deprotection of the benzoyl groups
with saturated methanolic ammonia afforded 20-deoxy-20-fluoro-
their potent anti-HIV-1 activity.
1-(20-Deoxy-20-fluoro-40-C-ethynyl-b-
D-arabinofuranosyl)-ura-
cil (1a) and -thymine (1b) were synthesized from uracil (5a) and
thymine (5b) in 15 steps. Compound 18a, the precursor to 1a,
was converted to the corresponding 40-C-ethynyl-cytosine analog
2 in two steps (Scheme 1).
b-D-arabinofuranosyl-uracil (8a) and -thymine (8b) in 90% and
97% yields, respectively. Compounds 8a and 8b were reacted
sequentially with 4,40-dimethoxytrityl chloride (DMTrCl) and
tert-butyldimethylsilyl chloride (TBDMSCl) to protect the 50- and
30-hydroxy groups, respectively. The 50-DMTr protecting group of
the resulting compounds 10a and 10b was then selectively
removed with trifluoroacetic acid (TFA) in CH2Cl2 to provide the
2-Deoxy-2-fluoro-1,3,5-O-tribenzoyl-d-arabinofuranoside (3)
was first converted to 10-
a-bromide 4 with HBr–HOAc in 45% yield.
Bromide 4 was then glycosylated with silylated pyrimidines 5 and
BzO
BzO
O
O
OBz
O
N
i
O
N
Br
F
F
R
R
NH
NH
OBz
OBz
4
iv
v
3
HO
O
iii
BzO
O
O
O
O
F
OSiMe3
N
F
R
ii
NH
R
OH
8a, R = H
OBz
O
N
H
7a, R = H
7b, R = CH3
N
OSiMe3
8b
, R = CH3
5a
, R = H
6a, R = H
6b, R = CH3
5b, R = CH3
O
N
O
O
O
N
R
R
R
R
NH
NH
NH
NH
vi
ix
vii
viii
HO
O
N
O
O
O
O
DMTrO
N
O
DMTrO
O
O
O
F
F
F
F
TBDMSO
TBDMSO
12a
OH
9a
, R = H
TBDMSO
10a, R = H
11a
, R = H
, R = H
9b, R = CH3
10b
11b, R = CH3
12b, R = CH3
, R = CH3
O
N
O
O
O
R
R
R
R
NH
NH
NH
NH
xii
TBDPSO
HO
O
HO
HO
N
x
O
xi
HO
DMTrO
N
O
TBDPSO
N
O
O
O
O
O
F
F
F
F
DMTrO
TBDMSO
TBDMSO
TBDMSO
15a, R = H
TBDMSO
13a
13b, R = CH3
, R = H
16a
, R = H
16b, R = CH3
14a
, R = H
15b, R = CH3
14b, R = CH3
O
O
N
O
N
R
R
R
NH
NH
NH
O
xiii
xv
xiv
HO
N
O
TBDPSO
Cl
O
TBDPSO
O
O
O
F
F
F
OH
TBDMSO
17a, R = H
TBDMSO
1a, R = H
1b, R = CH3
18a
18b, R = CH3
, R = H
17b
, R = CH3
NH2
NH2
N
N
xvi
xv
TBDPSO
N
F
O
HO
N
O
O
O
F
TBDMSO
HO
19
2
Scheme 1. Reagents and conditions: (i) HBrꢁHOAc, CH2Cl2; (ii) HMDS, (NH4)2SO4; (iii) CHCl3, reflux; (iv) saturated NH3/CH3OH, rt; (v) DMTrCl, Pyr, 0 °C; (vi) imidazole,
TBDMSCl, CH2Cl2; (vii) TFA, CH2Cl2; (viii) Pyr, TFA, EDCꢁHCl, DMSO; (ix) 1—37% HCHO, 2 N NaOH, 1,4-dioxane; 2—HOAc, NaBH4, EtOH; (x) DMTrCl, CH2Cl2, Pyr; (xi) imidazole,
TBDPSCl, CH2Cl2; (xii) TFA, CH2Cl2; (xiii) 1—Pyr, TFA, EDCꢁHCl, DMSO; 2—chloromethyl triphenyl phosphonium chloride, n-BuLi, ꢀ78 °C, THF; (xiv) n-BuLi, ꢀ78 °C, THF; (xv)
NH4F, MeOH, reflux; (xvi) 1—1,2,4-triazole, POCl3, Pyr, CH2Cl2; 2—NH4OH, THF.

Q. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4053–4056
4055
30-silyated analogs 11a and 11b. The 50-hydroxymethyl group of
Table 2
Anti-HIV activity of 2 against wild-type virus and resistant strains
11a and 11b was oxidized to an aldehyde by Pfitzner–Moffatt oxi-
dation. The resulting compounds 12a and 12b were then treated
with formaldehyde under basic conditions in 1,4-dioxane, followed
Viral strain
Compound 2 (IC50, l
M)a
NL4-3 (wild-type)
NL4-3 (K101E)
RTMDRb
0.00046
0.00152
0.00145
by sodium borohydride, to yield the corresponding 40-
a
-C-
hydroxymethyl analogs 13a and 13b. To differentiate the two
hydroxymethyl groups of 13a and 13b, the 40-
-hydroxymethyl
a
a
IC50
(
l
M) is the concentration that inhibits HIV by 50%.
b
group was selectively protected with DMTr, and the remaining
b-hydroxymethyl group was then protected with TBDMS.10 Com-
pounds 14a and 14b were obtained in high yields of 71% and
95%. Selective removal of the DMTr group with TFA afforded 16a
RTMDR is a multiple RT inhibitor-resistant strain, has RT mutations—M41L,
L74V, V106A, and T215Y, and is resistant to AZT, ddI, nevirapine, and other NNRTIs.
activity. In the further evaluation of 2, we discovered that it re-
tained its nanomolar activity against drug-resistant HIV strains
including NL4-3 (K101E) and RTMDR (Table 2). K101E tends to de-
crease viral susceptibility to all nucleoside RT inhibitors, while
RTMDR is a multiple RT inhibitor-resistant strain, which is insensi-
tive to AZT, ddI, nevirapine, and other NNRTIs. In our screening, 2
exhibited extremely potent anti-HIV activity against NL4-3 (wild-
type), NL4-3 (K101E), and RTMDR, with IC50 values of 0.46, 1.52,
and 1.45 nM, respectively. These findings indicate that 2 has a
great potential to be developed as a novel NRTI that could over-
come drug-resistance issues.
and 16b, which now had one
a
-C-hydroxymethyl group open for
-hydroxymethyl group
further modification. Oxidation of the 40-
a
of 16a and 16b to the formyl derivatives, followed by Wittig olef-
ination with chloromethyl triphenyl phosphonium chloride, affor-
ded chlorovinyl derivatives 17a and 17b. The chlorovinyl group of
these compounds was directly converted into an ethynyl group by
treatment with n-butyllithium in THF to provide 4-ethynyl analogs
18a and 18b. Finally, removal of the protecting groups with ammo-
nium fluoride in refluxing MeOH provided the target compounds
1a (R = H) and 1b (R = CH3). The uridine analog 18a was converted
to the cytidine derivative 19 by a traditional approach. Deprotec-
tion of 19 with the same method as for 1 yielded compound 2.
The chlorovinyl compound 17 from the classical Wittig
olefination was predominantly in a Z-configuration. In the 1H
NMR spectrum of 17a, d 5.95 (1H, d, J = 8.05 Hz) was assigned to
the Z-configured vinyl-H, and d 6.01 (1H, d, J = 13.54 Hz) to the
E-configured vinyl-H, based on the coupling constants. The integra-
tion values of the two peaks were 0.73 (d 5.95) and 0.27 (d 6.01),
indicating that the ratio of Z- to E-isomers was approximately
2.7:1. Both isomers could be converted to 18.
In summary, new 20-deoxy-20-fluoro-40-C-ethynyl nucleoside
analogs were designed, synthesized, and evaluated for in vitro anti-
viral activity in this study. Compound 2 was extremely potent
against HIV-1 wild-type strain without obvious cytotoxicity. It re-
tained nanomolar activity against NRTI-resistant and multi-resis-
tant HIV strains, and merits further development as an anti-AIDS
clinical trial candidate.
Acknowledgments
Compounds 1a, 1b, and 2 were evaluated in an anti-HIV (wild-
type) replication assay and the in vitro anti-HIV activity results are
listed in Table 1. Cytotoxicity was evaluated by MTT assay. All
three compounds did not exhibit significant cytotoxicity at concen-
J. Chang thanks the National Natural Science Foundation of Chi-
na (#20672030) and the Outstanding Young Scholarship of NSFC
(#30825043) for financial support. The research was also partially
supported by Grant AI-33066 from the National Institute of Aller-
gies and Infectious Diseases awarded to K. H. Lee.
trations up to 10 lM.
Compound 1b exhibited potent anti-HIV-1 replication activity
with an IC50 value of 86 nM, and thus, was 10-fold more potent than
1-(20-deoxy-40-C-ethynyl-b-
D
-arabinofuranosyl)-thymine without
Supplementary data
a fluorine atom at the 20-b-position, which had an IC50 value of
830 nM11 (equivalent to that of AZT). This result confirmed that
insertion of an electron-withdrawing atom, such as fluorine, into
the nucleoside deoxyribose moiety can lead to dramatically
improved anti-HIV activity. Such a modification can greatly affect
the electronic properties and conformational shape of the nucleo-
side,12–15 which often results in better biological activity.
Supplementary data (synthesis, 1H NMR data, bioassay meth-
ods,16–20 and HPLC/mass spectral purity analyses of final com-
pounds). Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2010.05.090.
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has a moderate influence on activity, and the anti-HIV-1 activity
of our compounds followed the rank order of cytidine > thymi-
dine > uridine. The results with 1b and 2 also confirmed that com-
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Table 1
Anti-HIV-1 replication activity in MT-2 lymphocytes
a
Compound
IC50
(l
M)
CC50 (lM)
1a
1b
2
6.53
0.086
0.00134
0.047
>10
>10
>10
>200
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IC50 (lM) is the concentration that inhibits HIV by 50%.

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