Anti-HIV-1 activity of resveratrol derivatives and synergistic inhibition of HIV-1 by the combination of resveratrol and decitabine

Article abstract of DOI:10.1016/j.bmcl.2012.08.108

Ribonucleotide reductase inhibitors enhance the anti-HIV-1 activities of a variety of nucleoside analogs, including those that act as chain terminators and those that increase the HIV-1 mutation rate. However the use of these ribonucleotide reductase inhi

Full text of DOI:10.1016/j.bmcl.2012.08.108

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6642–6646
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anti-HIV-1 activity of resveratrol derivatives and synergistic inhibition
of HIV-1 by the combination of resveratrol and decitabine
Christine L. Clouser a,b, , Jay Chauhan c, Matthew A. Bess a,b, Jessica L. van Oploo a,b,c, Ding Zhou c,
⇑
Sarah Dimick-Gray c, Louis M. Mansky a,b,c,d, Steven E. Patterson a,c,e,
⇑
a Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, United States
b Department of Diagnostic and Biological Sciences, School of Dentistry, University of Minnesota, Minneapolis, MN 55455, United States
c Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455, United States
d Department of Microbiology, University of Minnesota, Minneapolis, MN 55455, United States
e Pharmacology Graduate Program Medical School, University of Minnesota, Minneapolis, MN 55455, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2012
Revised 22 August 2012
Accepted 28 August 2012
Available online 6 September 2012
Ribonucleotide reductase inhibitors enhance the anti-HIV-1 activities of a variety of nucleoside analogs,
including those that act as chain terminators and those that increase the HIV-1 mutation rate. However
the use of these ribonucleotide reductase inhibitors is limited by their associated toxicities. The hydrox-
ylated phytostilbene resveratrol has activity in a host of systems including inhibition of ribonucleotide
reductase and has minimal toxicity. Here we synthesized derivatives of resveratrol and examined them
for anti-HIV-1 activity and their ability to enhance the antiviral activity of decitabine, a nucleoside analog
that decreases viral replication by increasing the HIV-1 mutation rate. The data demonstrates that six of
the derivatives have anti-HIV-1 activity greater than resveratrol. However, only resveratrol acted in syn-
ergy with decitabine to inhibit HIV-1 infectivity. These results reveal novel resveratrol derivatives with
anti-HIV-1 activity that may have mechanisms of action that differ from the drugs currently used to treat
HIV-1.
Keywords:
HIV-1
Resveratrol
Antiviral
Mutagenesis
Decitabine
Ó 2012 Elsevier Ltd. All rights reserved.
Human immunodeficiency virus type 1 (HIV-1), the causative
agent of AIDS, currently infects more than 30 million individuals
worldwide.1 While drug therapy effectively suppresses viral repli-
cation, resistance to these drugs is a significant problem and neces-
sitates the development of novel drug therapies.2 The development
of drug resistance is facilitated by the high mutation rate of HIV-1
as well as the large number of virus particles produced by infected
cells. Although the mutation rate facilitates the emergence of drug
resistance, it also renders HIV-1 particularly susceptible to modest
increases in mutation rate.3 An antiviral strategy termed lethal
mutagenesis aims to take advantage of this susceptibility by inten-
tionally increasing the mutation rate of RNA viruses such that
these viruses are unable to replicate their genome with enough
fidelity to remain infectious.4 A number of RNA viruses have been
shown to be susceptible to lethal mutagenesis including hepatitic C
virus, poliovirus, vesicular stomatitis virus and HIV-1.5–10 In fact,
hepatitis C virus is treated with ribavirin, a drug that has been
shown to inhibit viral replication through lethal mutagenesis in
cell culture.11,12 Whether ribavirin has the same mechanism
in vivo is not yet clear.13,14
Although none of the clinically approved anti-HIV-1 therapies
act through lethal mutagenesis, we have reported the ability of
the cytosine analog decitabine (5-aza-20-deoxycytidine) and its
riboside analog, 5-azacytidine to lethally mutagenize HIV-1 in a
cell culture system.10,15 Additionally, we have demonstrated that
select ribonucleotide reductase inhibitors (RNRIs) such as hydroxy-
urea and gemcitabine dramatically enhance the antiviral activity of
decitabine.15 RNRIs have also been shown to increase the antiviral
activity of nucleoside analogs that act as chain terminators such as
zidovudine and didanosine.10,16,17 RNRIs may increase the anti-
HIV-1 activity of nucleoside analogs by decreasing endogenous
nucleotide concentrations thereby increasing incorporation of the
antiviral nucleoside analog. Although gemcitabine and hydroxy-
urea are clinically approved, their use is limited by the associated
toxicities.18–20
Resveratrol (Fig. 1 1a) is a natural product that is well tolerated
and has a number of biological activities including the ability to in-
hibit HIV-1 replication.21–23 Resveratrol’s anti-HIV-1 activity has
been related to its ability to increase activity of SIRT1, a protein
that may decrease transcription of the proviral genome.23,24 Addi-
tionally, resveratrol synergistically increased the antiviral activity
of nucleoside derivatives and this anti-HIV-1 activity may be
attributed to its ability to inhibit RNRI.25 Besides being a RNRI with
anti-HIV-1 activity, resveratrol has limited toxicity and is relatively
⇑
Corresponding authors.
E-mail addresses: cclouser@umn.edu (C.L. Clouser), patte219@umn.edu
(S.E. Patterson).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.108

C. L. Clouser et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6642–6646
6643
Figure 1. Resveratrol and analogues.
Scheme 1. Reagents and conditions: (i) (a) NH2OHꢀHCl, NaHCO3, (b) NCS; (ii) (a) 1-ethynyl-3,5-dimethoxybenzene, NEt3, (b) 1M BBr3, DCM, rt, 20 h; (iii) NaN3, cat. CuSO4; (iv)
(a) 1-ethynyl-3,5-dimethoxybenzene, sodium ascorbate, (b) 1M BBr3, DCM, rt, 20 h; (v) 3,5-dimethoxy benzaldehyde, NEt3, triazolium salt; (vi) (a) NH2MeꢀHCl, AcOH, p-
TsOH; (b) 1M BBr3, DCM, rt, 20 h.
inexpensive, making it a strong lead compound for the develop-
ment of new anti-HIV-1 compounds that could be used alone or
in combination with nucleoside analogs such as decitabine.
Using resveratrol as a lead compound, 15 derivatives were
screened against HIV-1 alone and in combination with decitabine
using an assay that quantifies their ability to inhibit HIV-1 infectiv-
ity within a single round of replication. We report here the anti-
HIV-1 activity of six of the resveratrol derivatives and demonstrate
that resveratrol potentiates the antiviral activity of decitabine.
Compounds 1a and 1b were purchased from Sigma–Aldrich and
used without further purification. Compound 2 was purchased from
Tokyo Chemical Industry. Compounds 6,2 and 82 were prepared fol-
lowing literature procedures. The synthesis of 5-membered hetero-
cyclic compounds 3, 4 and 5 are as follows (Scheme 1). Aldehyde 13
was converted to the corresponding imidoyl chloride before cycliza-
tion with 1-ethynyl-3,5-dimethoxybenzene. Deprotection with bor-
on trifluoride afforded the isoxazole 3. The 1, 4-substituted triazole
4, was accessed through the corresponding azide precursor by cop-
per catalyzed Huisgen [3 + 2] cycloaddition with 1-ethynyl-3,5-
dimethoxybenzene, followed by deprotection. The pyrrole analogue
5 was synthesized by condensation of 1,3-dicarbonyl intermediate
with methyl amine followed by treatment with boron tribromide.

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C. L. Clouser et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6642–6646
Scheme 2. Reagents and conditions: (i) Pd(PPh3)4, 2 M Na2CO3, toluene/EtOH 3:1, relux, 6 h; (ii) 1 M BBr3, DCM, rt, 20 h.
Derivatives 7, 9, 10 and 12 were prepared (Scheme 2) by employ-
mentary data. The data show that 1b and 6 had an approximate
fivefold or greater selectivity index relative to the other derivatives.
We next evaluated the ability of resveratrol and three deriva-
tives (1b, 11b, and 6) to inhibit HIV-1 replication when used in
combination with decitabine, a mutagenic nucleoside with anti-
HIV-1 activity. Decitabine and each derivative was used at a con-
centration that would have minimal anti-HIV-1 activity when used
individually (Table 2) and examined for the ability of the combina-
tion to (1) enhance the antiviral activity of decitabine, and (2) in-
crease the mutant frequency of HIV-1 using the single cycle
assay we described previously.10,15
Mutant frequencies of these compounds were calculated as de-
scribed in the Supplementary data. The data, (Table 2), indicate
that resveratrol was the only compound that potentiated the anti-
viral activity of decitabine while increasing HIV-1 mutant fre-
quency. While the average mutant frequency increased when
either resveratrol or 11b was used in combination with decitabine
compared to the no drug control, this was not statistically different
than the mutant frequency seen with decitabine alone.
ing the palladium catalyzed Suzuki–Miyauru cross coupling reac-
tion. Thus, 3,5-dimethoxyphenylboronic acid 16 was reacted with
4-bromoiodobenzene followed by a second coupling reaction with
4-pyridylboronic acid to afford polyaromatic 17. Treatment of the
boronic acid 16 with 6-bromoiosoquinoline generated intermediate
6-phenylisoquinoline 18. Benzofuran 19a and benzothiophene 19b
were synthesized in a similar manner from 2,3-dibromoheteroaro-
matics respectively. Mono-coupling with 3,5-dimethoxyphenylbo-
ronic acid was highly selective for the 2-position, followed by
coupling with second boronic acid to give oxygen and sulfur deriva-
tives, respectively. 2,5-Aryl substituted pyrimidine 20, was synthe-
sized from 2,5-bromochloro pyrimidine, first by coupling to the
bromide, then chloride with dimethoxy- and methoxy- boronic
acids, respectively. Finally, the demethylation of the methoxy deriv-
atives 17–20 was achieved with boron tribromide to afford com-
pounds 7, 9, 10a,b and 12a. In a similar fashion (Scheme 3), the
regioisomers of compounds 10a,b and 12a were synthesized by
inverting the order of the boronic acid coupling partner followed
by deprotection to give compounds 11a,b and 12b.
To further examine the ability of resveratrol to act in synergy
with decitabine to inhibit HIV-1 activity, we used the MacSynergy
II program.26 MacSynergy II determines if the interaction between
two compounds can be described as additive, antagonistic, or syn-
ergistic. A 10 by 7 matrix was used to assess the anti-HIV-1 activity
The antiviral activity of resveratrol and its derivatives was
examined using the single-round replication assay10,15 where mar-
ker genes present in the HIV-1 vector were used to assess infectivity
and mutant frequency in the presence or absence of compounds. Six
compounds demonstrated concentration-dependent inhibition of
of decitabine (0–800 nM) and resveratrol (0–100 lM) alone and in
HIV-1 infectivity with EC50s below 75
l
M (Table 1). Consistent with
combination. The MacSynergy II program calculates theoretical
additive interactions from the individual dose responses. This cal-
culated additivity which represents predicted anti-HIV-1 activity
of the drug combinations was subtracted from the experimental
data to reveal regions of synergy. Interactions that were additive
are expected to lie on a zero plane of additivity (since predicted
a previous report,23 resveratrol showed anti-HIV-1 activity which
was concentration-dependent.
(Fig 2). However the EC50 of resveratrol was relatively high
(>75
lM). Resveratrol and the six compounds showing improved
potency were examined for cytotoxicity as described the Supple-

C. L. Clouser et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6642–6646
6645
Scheme 3. Reagents and conditions: (i) Pd(PPh3)4, 2M Na2CO3, toluene/EtOH 3:1, relux, 6 h; (ii) 1M BBr3, DCM, rt, 20 h.
Table 1
Anti-HIV-1 activity (EC50) and toxicity (TC50) of resveratrol and derivatives
Table 2
Effect of compounds on infectivity and HIV-1 mutant frequency
Compound
EC50
(lM)
TC50 (lM)
SIc
a
b
Compound (concentration)
% Infected cellsa
Mutant frequencyb
1a
1b
2
3
4
5
6
7
8
>75
>300
>400
ND
ND
>18.7
ND
ND
ND
ND
20.3
ND
ND
ND
2.4
3.8
1.5
2.6
ND
ND
ND
Dec (70 nM)
1a (20
1a (20
1b (6
1b (6
6 (6
6 (6
11b (30
11b (30
100
1.00
21.4 (17.8–25.1)
>75
>75
>75
>75
8.8 (7.2–10.9)
>100
>75
>75
35.0 (25.9–47.3)
34.4 (28.3–41.9)
65.1 (56.4–75.2)
45.1 (31.1–65.5)
>75
93 18
91 15
36 18c
88 12
71 15
96 14
74 16
99 14
80 34
1.26 0.23
1.08 0.22
1.96 0.78
1.16 0.22
1.54 0.53
1.10 0.09
1.72 0.72
1.08 0.12
1.94 0.69
l
M)
NDd
ND
lM) + Dec (70 nM)
l
M)
ND
l
M) + Dec (70 nM)
179 (107–298)
ND
ND
ND
84.8 (75.1–95.7)
131 (109–158)
108 (95.2–123)
118 (93.7–148)
ND
l
l
M)
M) + Dec (70 nM)
l
l
M)
9
M) + Dec (70 nM)
10a
10b
11a
11b
12a
12b
a
ND (No Drug) was set to 100% and all others converted as described in the
Supplementary data. The data show the mean standard deviation.
ND (No Drug) was set to 1.00 and all others converted as described in Sup-
plementary data. The data show the mean standard deviation.
b
c
>75
ND
Statistically significant difference from individual drug treatment by One-Way
ANOVA followed by Tukey’s post-test with a p <0.05 considered significant.
a
The concentration of compound that inhibits 50% of HIV-1 infection. The 95%
confidence intervals are shown in parentheses after the EC50 value.
b
Concentration of compound that induces toxicity in 50% of the host cells.
c
Selectivity index: TC50/EC50
.
d
additivity represent areas of antagonism. The data was assessed
statistically by using the 95% confidence intervals around the
experimental dose-responses. Synergy was considered to be signif-
icant if the lower 95% confidence limit of the experimental data
was still greater than the calculated additive surface. Figure 3
shows that all combinations of resveratrol and decitabine lie above
the plane of additivity with the highest peaks found at combina-
Not determined.
tions containing between 10 and 60
50 and 200 nM of decitabine.
lM of resveratrol and between
These results indicate that the combination of resveratrol and
decitabine are highly synergistic. The ability of resveratrol and
decitabine to synergistically decrease HIV-1 infectivity could corre-
late with their ability to potentiate cellular toxicity. Therefore, the
MacSynergy II program was used to assess the possibility that deci-
tabine and resveratrol act synergistically to induce cell toxicity. As
shown in Figure 3, the combinations of resveratrol and decitabine
that showed the highest antiviral synergy, did not act synergisti-
cally to induce cellular toxicity.
In summary, we have synthesized novel resveratrol derivatives
and discovered six with anti-HIV-1 activity superior to that of
resveratrol. More importantly we have demonstrated that the
combination of resveratrol and decitabine act synergistically to
inhibit HIV-1 infectivity without a corresponding synergistic in-
crease in cellular toxicity. Future studies will address why resve-
ratrol, but not its derivatives potentiate the antiviral activity of
decitabine.
Figure 2. Resveratrol inhibits HIV-1 in a concentration-dependent manner. The
data shows the average standard deviation of 3 independent experiments.
additive subtracted from experimental data would equal zero in
cases of additivity), while any peaks above the plane of additivity
indicate areas of synergy. Similarly, and peaks below the plane of

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C. L. Clouser et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6642–6646
Figure 3. Resveratrol and decitabine act synergistically to inhibit HIV-1 infectivity in the absence of cellular toxicity.
7. Loeb, L. A.; Essigmann, J. M.; Kazazi, F.; Zhang, J.; Rose, K. D.; Mullins, J. I. Proc.
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