Synthesis and biological evaluation of geminal disulfones as HIV-1 integrase inhibitors

Article abstract of DOI:10.1021/jm049171v

Integration of HIV-1 viral DNA into the host genome is carried out by HIV-integrase (IN) and is a critical step in viral replication. Although several classes of compounds have been reported to inhibit IN in enzymatic assays, inhibition is not always corr

Full text of DOI:10.1021/jm049171v

4526
J. Med. Chem. 2005, 48, 4526-4534
Synthesis and Biological Evaluation of Geminal Disulfones as HIV-1 Integrase
Inhibitors
D. Christopher Meadows,‡ Timothy B. Mathews,§ Thomas W. North,§ Michael J. Hadd,‡,† Chih Lin Kuo,#
Nouri Neamati,# and Jacquelyn Gervay-Hague*,‡
Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616, Center for
Comparative Medicine and Department of Molecular Biosciences, School of Veterinary Medicine, University of California,
Davis, Davis, California 95616, and Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern
California, 1985 Zonal Avenue, PSC 304, Los Angeles, California 90089
Received October 15, 2004
Integration of HIV-1 viral DNA into the host genome is carried out by HIV-integrase (IN) and
is a critical step in viral replication. Although several classes of compounds have been reported
to inhibit IN in enzymatic assays, inhibition is not always correlated with antiviral activity.
Moreover, potent antiviral IN inhibitors such as the chicoric acids do not act upon the intended
enzymatic target but behave as entry inhibitors instead. The charged nature of the chicoric
acids contributes to poor cellular uptake, and these compounds are further plagued by rapid
ester hydrolysis in vivo. To address these critical deficiencies, we designed neutral, nonhydro-
lyzable analogues of the chicoric acids. Herein, we report the synthesis, enzyme inhibition
studies, and cellular antiviral data for a series of geminal disulfones. Of the 10 compounds
evaluated, 8 showed moderate to high inhibition of IN in purified enzyme assays. The purified
enzyme data correlated with antiviral assays for all but two compounds, suggesting alternative
modes of inhibition. Time-of-addition studies were performed on these analogues, and the results
indicate that they inhibit an early stage in the replication process, perhaps entry. In contrast,
the most potent member of the correlative group shows behavior consistent with IN being the
cellular target.
Introduction
emergence of HIV-1 variants that contain multiple drug-
resistance mutations.5 These multi-drug-resistant vari-
ants of HIV-1 can also be transmitted.6 The problem of
drug resistance is not limited to inhibitors of reverse
transcriptase and protease. In vitro studies have also
identified mutations that confer resistance to drugs
targeted against HIV-1 IN and entry inhibitors,7 sug-
gesting that resistance is likely to occur with any new
agent regardless of the viral target.
There are currently four classes of antiretroviral
drugs that are approved for use in AIDS therapy. Three
of these classes have been extensively used in highly
active antiretroviral therapy (HAART): nucleoside ana-
logues that inhibit reverse transcriptase (RT), non-
nucleoside inhibitors of reverse transcriptase (NNRTI),
and protease inhibitors (PI).1 A fourth class is repre-
sented by T20 (Fuzeon), a fusion/entry inhibitor that
was recently approved for use in humans. Current
HAART is based on using potent combinations of these
drugs, usually three or more drugs from two or more
classes. Major forces leading to development of combi-
nation therapy for AIDS were the inability of individual
drugs (monotherapy) to adequately reduce virus loads
and the emergence of drug-resistant mutants that was
usually rapid with any single drug. Viral drug resistance
was considered the major limitation of antiretroviral
drugs in the pre-HAART era.2-4 The development of
HAART enabled suppression of virus load to undetect-
able levels for prolonged periods in many patients but
has not eliminated problems from viral drug resistance.
The potent combinations used in HAART, when suc-
cessful, decrease the rate of emergence of resistant
variants because of greatly decreased viral load. Nev-
ertheless, treatment failure is usually accompanied by
HIV integrase (IN) is required for integration of viral
DNA into host chromosomes. It is an especially attrac-
tive chemotherapeutic target because no mammalian
counterpart to this enzyme has been identified, and the
region of the HIV pol gene encoding IN shows more
conservation than RT- and PR-encoding regions.8 IN is
active in both the cytoplasm and nucleus of the host cell.
While in the cytoplasm, IN forms a preintegration
complex with viral DNA and selectively cleaves 3′-
terminal dinucleotides from each end.9 This so-called
3′-processing event occurs prior to nuclear translocation
of the complex. In the nucleus, IN catalyzes strand
transfer, which leads to integration of viral DNA into
the host DNA.10 The latter process is essential for viral
replication and thus has become a primary target for
drug discovery.11 Because IN is actively involved in viral
replication for a limited period of time, its viability as
a target was long debated. Skeptics felt the chances
were low that sufficient concentrations of drug would
be available to inhibit strand transfer for the relatively
short time IN is activated. Recent findings by Hazuda
et al. have essentially disproved this argument by
demonstrating sustained suppression of viremia in
* To whom correspondence should be addressed. Phone: 530-754-
9577. Fax: 530-752-8995. E-mail: gervay@chem.ucdavis.edu.
‡ Department of Chemistry, University of California, Davis.
§ School of Veterinary Medicine, University of California, Davis.
† Current Address: Discovery Partners, San Diego, CA.
# University of Southern California.
10.1021/jm049171v CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/14/2005

Disulfone-Containing HIV-1 Integrase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4527
Scheme 1. Synthesis of Symmetrical Geminal Disulfones
Scheme 2. Synthesis of Unsymmetrical Geminal Disulfones
simian-human immunodeficiency virus (SHIV) infected
rhesus macaques treated with IN inhibitors.12 In follow-
up studies, these researchers showed that several
known IN inhibitors have unique resistance profiles
highlighting the importance of continual identification
of new classes of inhibitors that are active at all levels
of viral replication.13
Although several compounds have been reported to
inhibit IN,14,15 enzyme activity is not always correlated
with antiviral activity.16 Even compounds showing
antiviral activity may not act upon the intended intra-
cellular enzymatic target but serve to block viral entry
instead.17,18 Compounds possessing charge at biological
pH appear to be particularly susceptible to this alterna-
tive pathway of viral inhibition, prompting us to develop
synthetic methods for the preparation of neutral IN
inhibitors. In particular, we were inspired by develop-
ments in the Robinson labs indicating that chicoric acids
are potent IN inhibitors.19 Unfortunately, these inhibi-
tors are plagued by poor cellular uptake and rapid
hydrolysis in vivo, but they present an excellent starting
point for drug discovery.
sulfones is 12.5, indicating that it would not be depro-
tonated at biological pH.23 Given that IN is a metal-
loenzyme that makes and breaks phosphate bonds,24 we
felt that metallophillic phosphate isosteres would be
well-suited as structural cores for IN inhibitors.
Previously, we reported the synthesis of 1 and its
utility in synthesizing geminal disulfones via Horner-
Emmons-Wadsworth reaction with a variety of ali-
phatic and aromatic aldehydes.25 In this report, we
describe the synthesis and biological evaluation of a
collection of geminal disulfones having the general
structure 2 as potential HIV-1 IN inhibitors. Neamati
and co-workers have reported using monosulfones for
this purpose,26 but to our knowledge geminal disulfone
analogues have not been previously evaluated.
We have been involved in studies targeting phosphate
mimics for several years with studies centered on using
sulfones as neutral phosphate analogues.20,21 Critical to
the success of these endeavors was the discovery that
highly functionalized geminal disulfones could be ef-
ficiently prepared from simple bis-phosphonate reagents
1 invented in our laboratory.22 The basic design ele-
ments include maintaining the bond distance between
functional groups by substituting a carbon atom for the
oxygen atoms in the phosphate backbone. Similarly, the
phosphorus atoms are substituted with sulfur, which
is oxidized to the sulfone to increase metallophilicity.
Results and Discussion
Synthesis of the biaryl/disulfone library was ac-
complished by coupling a variety of aromatic aldehydes
to disulfone/diphosphonate reagent 1 in a Horner-
Emmons-Wadsworth olefination reaction using either
LiOtBu or Hu¨nig’s base in the presence of LiBr in dry
THF (Scheme 1). This strategy allows for the synthesis
of both symmetric and asymmetric compounds depend-
ent upon reactant stoichiometry. For symmetric com-
pounds, 1 equiv of 1 is reacted with 3 equiv of aldehyde.
Asymmetric compounds are prepared using 3 equiv of
1 with 1 equiv of aldehyde followed by workup to give
the monocoupled product. This adduct (1 equiv) can then
be reacted under the same conditions using 3 equiv of
a different aldehyde (Scheme 2). Table 1 lists the
compounds prepared with the corresponding aldehydes
reacted according to Schemes 1 and 2. Aldehydes
containing phenols or phenols masked as esters were
primarily chosen because catechols are known to be
important functionalities in many IN inhibitors.27-29
Biological Assays with Purified Enzyme. The
compounds shown in Table 1 were first tested against
purified enzyme to determine whether 3′-processing or
The pKa of the methylene proton between the two

4528 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Table 1. Geminal Disulfones Prepared According to Schemes 1 and 2
Meadows et al.
Table 2. HIV-IN Inhibition Data
Table 3. Antiviral Activity, Cytotoxicity, and Antiviral
Selectivity Data for the IN Inhibitors
IC50 (µM)
3′-processing
IC50 (µM)
compd
integration
integrase antiviral activitya cytotoxicityb antiviral selectivity
inhibitor EC50 ( SE (µM)
IC50 (µM)
IC50/EC50
3
4
5
6
7
8
9
70 ( 5
4 ( 1
70 ( 5
5 ( 1
3
8.0 ( 2.5
2.4 ( 0.3
0.4 ( 0.1
0.3 ( 0.03
1.5 ( 0.02
1.3 ( 0.09
3.9 ( 1.2
4.5 ( 0.86
0.78
148 ( 2.7
187 ( 7.6
27 ( 0.8
169 ( 6.4
18 ( 1.2
29 ( 1.5
21 ( 5.6
19 ( 1.9
NDc
19 ( 6.0
78 ( 6.7
69 ( 16
563 ( 35
12 ( 0.7
22 ( 0.4
5.3 ( 0.24
4.3 ( 0.4
NDc
50 ( 6
1.8 ( 0.3
30 ( 3
50 ( 10
80 (10
80 ( 11
>1000
700 ( 40
>1000
5 ( 2
4
0.9 ( 0.1
30 ( 2
5
6
50 ( 10
80 ( 10
80 ( 11
>1000
7
8
10
9
11
12
10
700 ( 70
L-708,906
L-708,906
0.48 ( 0.0830
a EC50 values are the mean ( SE from three separate deter-
minations except for L-708,906, which is from a single experiment.
b IC50 values are the mean ( SE from two separate determinations.
c ND: not determined.
integration would be inhibited. As can be seen in Table
2, five of the compounds, 3, 7, 8, 9, and 10, exhibited
moderate integration activity, while three compounds,
4, 5, and 6 displayed potent integration inhibition.
Replacing a m-acetoxy (as in 3) with an electron-
donating methoxy (as in 11) has deleterious effects,
which can be overcome by substituting the other meta
position with an acetoxy functionality (as in 7). Simi-
larly, substituting acetates with benzoates (compare 3
and 12) results in loss of activity. It is noteworthy that
only 5 shows any selectivity for 3′-processing over
integration while all other compounds show comparable
inhibition for both catalytic functions.
Cell-Based Assays. After the geminal disulfones
were demonstrated to be capable of inhibiting purified
HIV-1 IN, the next step was to evaluate these com-
pounds for antiviral activity. The North Laboratory has
used a focal infectivity assay (FIA) extensively for
studies of antiviral drugs and viral drug resistance, with
feline immunodeficiency virus (FIV), simian immuno-
deficiency virus (SIV),31-33 and HIV-1. The assay we
currently use for HIV-1 has recently been described.31,32
It is similar to the FIA that was originally developed
by Chesebro34 except that indicator cells are HeLa H1-
JC.37 cells. These are HeLa CD4 cells expressing
human CCR5 (these cells naturally express CXCR4)35
and are permissive for infection by T-cell tropic and
macrophage tropic isolates of HIV-1, SIV, or Env-SHIVs.
With this assay, we have determined that a known IN
inhibitor, L-708,906, inhibits HIV-1 with an EC50 of 0.78
µM, which is consistent with previous reports of this
compound.36 These data validate use of the FIA to
evaluate IN inhibitors (Table 3). As shown in Table 3,
the most potent inhibitor of HIV-1 is compound 6.
Representative dose response curves for compounds 3-6
and are shown in Figure 1.
Comparison of the data in Tables 2 and 3 shows that
diacetate 3 is less active than its hydroxy analogue 4
in both the IN and FIA assays, whereas there is little
difference between 7 and 8 and between 9 and 10,
suggesting that acetate substitution has no effect in the
latter cases. Furthermore, antiviral activities for these
three pairs of compounds strongly correlate with puri-

Disulfone-Containing HIV-1 Integrase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4529
Figure 1. Dose response curves for inhibitors 3-6. Reported data represent the mean ( SE for three independent experiments.
fied enzyme studies, consistent with the hypothesis that
they are targeting IN. To ensure that the inhibitors are
capable of crossing the cell membrane, cells were
incubated for 6 days with 4. On the sixth day, the
supernatant was removed and the cells were lysed via
multiple freeze/thaw cycles. The lysate was removed and
purified on HPLC detecting at the absorption maximum
for 4, which gave a single peak corresponding to the
molecular ion of 4 as determined by mass spectrometry.
These combined studies suggest that the inhibitors are
crossing the cell membrane and entering the cytoplasm
of the cell.
The fact that antiviral activities are generally higher
than purified enzyme activity is not particularly alarm-
ing because similar observations have been reported for
viral neuraminadase activity.37 However, we did take
note of the discrepancy between antiviral activity of 5
and 6 and its lack of correlation with purified enzyme
activity because this result could be indicative of an
alternative mode of action such as entry. To address this
critical issue, we next looked for evidence of early stage
inhibition.
can be delayed only 1 h, RT inhibitors for 4 h, and
protease inhibitors for 18-19 h.38 In our study, we used
dextran sulfate, a known entry inhibitor, T20, a fusion
inhibitor, and 3TC, a reverse transcriptase inhibitor and
compared their activity to compounds 4-6. The inhibi-
tors were each used at 10 times their respective EC50
values and were added at six different time points: -1,
0, 2, 4, 6, and 10 h. The cells were incubated for 4 days,
after which they were fixed and stained and foci were
counted and plotted against a no-drug control. As can
be seen from Figure 2, 5 and 6, like dextran sulfate and
T20, exhibit a sharp decrease in activity if added more
than 2 h after infection. This behavior strongly impli-
cates an early event, such as viral entry, as the primary
mode of action for these compounds. In contrast, 4 shows
behavior similar to that of 3TC, suggesting that its mode
of action is at a later stage in the replication cycle. Given
that 4 is a potent IN inhibitor, it is likely that IN is the
cellular target, but further studies including selecting
for mutant viruses will be required to prove this
hypothesis.
We have used a standard cell proliferation assay
(MTS assay) to evaluate cytotoxicity of these compounds
to HeLa H1-JC.37 cells. Cytotoxicity of all the inhibitors
we tested required concentrations substantially higher
than required for antiviral activity (Table 3). The most
selective inhibitors were compounds 4-6, which had 78-,
69-, and 570-fold selectivity, respectively (Table 3).
Time-of-Addition Studies. Time-of-addition studies
are performed to determine how long after infection the
addition of a drug can be delayed and still retain its
activity. The earlier in the replication cycle the drug
inhibits, the shorter the time its addition can be delayed
and still retain activity. For example, entry inhibitors

4530 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Meadows et al.
Figure 2. Time-of-addition studies for 4-6. The data represent mean values ( SE from five independent experiments.
To rule out the possibility of conjugate addition
contributing to toxicity, active compounds were screened
for their ability to act as Michael acceptors of glu-
tathione. Briefly, 1.5 mL of a 0.5 M solution of glu-
tathione was prepared in PBS, and to this was added
40 µL of a 0.75 mM solution of the vinyl sulfone in
DMSO (a large excess of the nucleophile is used to
ensure first-order kinetics). This mixture was incubated
for 4 days at 37 °C and then subjected to ESI mass
spectrometry. The spectrum of the mixture was then
compared to spectra of glutathione monoaddition and
double-addition compounds prepared synthetically. The
spectrum of the mixture showed only molecular ion
peaks corresponding to unreacted vinyl sulfone, glu-
tathione, and oxidized glutathione, indicating that
under the assay conditions the vinyl sulfones are not
reactive toward biological nucleophiles.
assays. We have also demonstrated that the compounds
are cell-permeable and therefore capable of targeting
IN in vitro. For the most part, there is little difference
in antiviral activity between pairs of substituted ana-
logues differing only in the acetylation pattern, while
replacement of an acetate with either a benzoate or a
methyl ether completely obliterates activity. A trihy-
droxy analogue (6) (EC50 ) 0.3 µM) is the most potent
compound we have identified thus far, and it has >500-
fold antiviral selectivity. However, the purified enzyme
activity of 6 compared to its triacetoxy analogue 5 did
not correlate well with cell-based assays, raising the
possibility of an alternative mode of action. To probe
this possibility, time-of-addition studies were conducted,
and the results indicate that 5 and 6 target an earlier
event in viral replication. This activity contrasts with
the dihydroxy analogue 4, which inhibits at a later
stage, consistent with IN being the cellular target. It is
possible that these compounds actually have dual modes
of inhibition acting both at entry and at viral integra-
tion. Continuing investigations in our laboratory focus
on identifying lead candidates for viral resistance stud-
ies to identify the molecular targets of inhibition.
Conclusion
We have prepared a series of neutral nonhydrolyzable
geminal disulfone analogues of the chicoric acids using
a Horner-Wadsworth-Emmons condensation reaction
between aromatic aldehydes and a geminal disulfone
bisphosphonate reagent developed in our laboratory.
Biological evaluation of these compounds reveals potent
activity in purified HIV-IN enzyme assays that is
correlated with antiviral activity using focal infectivity
Experimental Section
Elemental analyses were obtained from Desert Analytics
Laboratory, Tucson, AZ. All materials were obtained from
commercial sources and used without additional purification.

Disulfone-Containing HIV-1 Integrase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4531
The aromatic aldehydes used were bought from Aldrich
Chemical Co., Inc. The THF used for reaction was 99.9%
anhydrous, inhibitor-free in sureseal bottles also obtained from
Aldrich Chemical Co., Inc. All glassware for reactions under
anhydrous conditions was flame-dried prior to use. Flash
chromatography was performed on silica gel Geduran (40-63
um) from Merck and using EM Science ACS grade solvents.
For TLC, silica gel 60 F254 plates from Merck were used with
145.9, 168.4, 168.6. HRFABMS calcd for C25H25O12S2 (M +
H): 581.0787. Found: 581.0795 (M + H). Anal. (C25H24O12S2)
C, H.
Bis(trans-â-3,4-dihydroxystyrenesulfonyl)methane (4).
To a solution of 3 in 3 mL of methanol was added a catalytic
amount of sodium methoxide, and the solution was stirred for
20 min. Dowex 50WX8-100 strongly acidic resin was then
added, and the solution was stirred for 5 min. The solution
was filtered and the solvent was removed in vacuo to give a
quantitative yield (NMR analysis) of the target compound. 1H
NMR (acetone-d6, 250 MHz) δ 5.07 (s, 2H), 6.93 (d, 2H, J ) 8
Hz), 7.10 (m, 4H), 7.20 (d, 2H, J ) 2 Hz), 7.47 (d, 2H, J ) 15
Hz), 8.39 (s, 2H), 8.71 (s, 2H); 13C NMR (acetone-d6, 62.5 MHz)
δ 73.9, 115.8, 116.5, 123.2, 123.8, 146.5. Electrospray (-) MS
calcd for C17H15O8S2: 411.0 (M - H). Found: 411.0 (M - H).
Anal. Calcd for C17H16O8S2 (2 H2O): C, 45.53; H, 4.50. Found:
C, 46.00; H, 3.70.
detection by UV light and/or iodine chamber. 1H NMR and 13
C
NMR spectra were recorded on Bruker DRX-500, Varian
Mercury 300 MHz, or Varian Inova 400 MHz spectrometers
at 25 °C. Chemical shifts in ppm were referenced to CDCl3
(7.26 ppm, 77.16 ppm), DMSO-d6 (2.50 ppm, 39.52 ppm), and
acetone-d6 (2.05 ppm, 29.84 ppm) as internal standards. IR
data were recorded on a Galaxy series FT-IR 3000 instrument
at 25 °C. Melting points were determined on a Fisher-Johns
melting point apparatus.
AceticAcid2,6-Diacetoxy-4-{2-[2-(3,4,5-triacetoxyphenyl)-
ethenesulfonylmethanesulfonyl]vinyl}phenyl Ester (5).
To a solution of 1 (144 mg, 0.28 mmol) in THF (2 mL) was
added LiBr (88 mg, 1 mmol) followed by DIEA (175 µL, 1
mmol). 3,4,5-Tri-O-acetylbenzaldehyde (202 mg, 7.2 mmol) was
then added, and the reaction mixture was allowed to sit for 7
days. Then approximately 100 mL of acetic acid was added,
the solvent was removed in vacuo, and the resulting oil was
subjected to column chromatography (6:4 hexanes/ethyl ac-
etate) to yield 70 mg (35% yield) of the title compound. 1H
NMR (acetone-d6, 600 MHz) δ 2.30 (s, 12H), 2.32 (s, 6H), 5.61
(s, 2H), 7.44 (d, 2H, J ) 15.5 Hz), 7.58 (d, 2H, J ) 15.5 Hz),
7.66 (s, 4H); 13C NMR (acetone-d6, 150 MHz) δ 19.8, 20.3, 71.5,
121.6, 128.2, 130.5, 136.8, 142.4, 143.6, 167.0, 168.0. HR-
FABMS (+) calcd C29H29O16S2 (M + H): 697.0897. Found:
697.0906 (M + H). Anal. (C29H29O16S2) C, H.
General Procedure for the Synthesis of Symmetrical
Geminal Disulfones. In a flame-dried flask, 1.0 equiv of the
disulfone reagent (1), 3.0 equiv of aldehyde, and 3.3 equiv of
LiBr were dissolved in 4 mL of dry THF. Once in solution, 3.3
equiv of Hunig’s base was added. The mixture was stirred
overnight before the reaction was quenched by the addition
of 5% HCl until pH 3-4 was attained. The solution was
partitioned between ethyl acetate (80 mL) and water (50 mL)
and extracted three times (50 mL). The organic phase was
collected and dried over sodium sulfate. The solvent was
evaporated to yield a solid material, which was purified as
indicated for the individual compounds.
(Diisopropoxyphosphorylmethanesulfonylmethane-
sulfonylmethyl)phosphonic Acid Diisopropyl Ester (1).
To commercially available diisopropyl bromomethylphospho-
nate (Lancaster) (5.24 g, 20.2 mmol) in 15 mL of DMF, 3.46 g
(30.3 mmol) of potassium thioacetate and 373 mg of tetrabu-
tylamonium iodide were added, and the mixture was heated
to 80 °C with stirring for 2 h. The solution was cooled and
then partitioned between water and ethyl acetate. The ethyl
acetate layer was collected and dried over sodium sulfate and
then evaporated to dryness. To the crude oil was added
acetonitrile (15 mL), 3 M NaOH (7.4 mL), and methanol (7.4
mL), and the reaction mixture was stirred for 30 min. The flask
was then cooled to 0 °C. Diiodomethane (797 µL, 9.90 mmol)
was then added, and the reaction mixture was stirred and
allowed to warm to room temperature overnight. The reaction
mixture was then partitioned between water and ethyl acetate,
and the ethyl acetate layer was collected and dried over sodium
sulfate. After evaporation, the crude oil was oxidized using
24.86 g (40.44 mmol) of oxone in methanol/water (∼100 mL
1:1) overnight to give a crude solid after ether/bicarbonate
extraction. The crude solid was dissolved in ether and hexanes
were added dropwise until crystals formed, yielding 3.15 g of
1 (69% yield). 1H NMR (CDCl3, 500 MHz) δ 1.33 (d, 24H, J )
6.2 Hz), 3.91 (d, 4H, J ) 16.0 Hz), 4.80 (m, 4H), 5.58 (s, 2H);
13C NMR (CDCl3, 125 MHz) δ 23.7, 24.1, 50.7, 51.8, 68.1, 73.2,
73.3. HRFABMS calcd for C15H35O10P2S2 (M + H): 501.1147.
Found: 501.1151 (M + H).
Bis(trans-â-3,4-diacetoxystyrenesulfonyl)methane (3).
To a solution of disulfone 1 (98 mg, 0.20 mmol) in 3 mL of
THF was added 0.59 mL of a 1 M solution of lithium
tert-butoxide in THF. After 10 min 165 mg (0.74 mmol) of 3,4-
diacetoxybenzaldehyde in 1 mL of THF was added and the
reaction proceeded for an additional 30 min. Approximately
100 µL of acetic acid was then added, and the solvents were
removed to yield a crude oil that was subjected to column
chromatography (1:1 hexanes/ethyl acetate) to yield a mixture
of the monosubstituted and bis-substitutued products. This
mixture is resolved by letting the mixture sit in ether in the
freezer overnight to give 83 mg (60%) of the disubstituted as
a white powder and 14% monosubstituted product in solution.
1H NMR (acetone-d6, 250 MHz) δ 2.29 (s, 12H), 5.21 (s, 2H),
7.36 (m, 4H), 7.62 (m, 6H); 13C NMR (acetone-d6, 62.5 MHz) δ
20.4, 20.5, 73.3, 124.7, 125.3, 127.7, 128.2, 131.8, 143.9, 144.7,
Bis(trans-â-3,4,5-trihydroxystyrenesulfonyl)meth-
ane (6). The disulfone reagent 1 (153 mg, 0.31 mmol) and
3,4,5-tri(tert-butyldimethylsilyloxy)benzaldehyde (530 mg, 1.07
mmol) were reacted according to general procedure A. Follow-
ing removal of solvent, 350 mg of the crude coupled product
was isolated (55%). The crude product (356 mg, 0.32 mmol)
was dissolved in 4 mL of THF to which was added acetic acid
(120 µL, 2.08 mmol) and a 1 M solution of tetrabutylammo-
nium fluoride (2.08 mL, 2.08 mmol). This was allowed to stir
for 1 h, at which time the solvent was removed. The crude oil
was extracted with ethyl acetate (3 × 100 mL), and the organic
phases were collected and dried over sodium sulfate. The
solvent was removed and the crude oil was run through a short
plug of silica, eluting with 15% methanol/85% ethyl acetate
1
(Rf ) 0.41) to give 142 mg of pure title compound (56%). H
NMR (acetone-d6, 400 MHz) δ 5.05 (s, 2H), 6.78 (s, 4H), 7.03
(d, 2H, J ) 15.2 Hz), 7.39 (d, 2H, J ) 15.2 Hz), 8.27 (br s, 6H);
13C NMR (acetone-d6, 100 MHz) δ 74.0, 109.3, 123.4, 124.4,
137.8, 146.7, 146.8; FT-IR (KBr) ν 3526 (OH str), 1651 (conj
alkene), 1323, 1152 (SO2). Anal. Calcd for C17H16O10S2: C,
45.94; H, 3.63. Found: C, 46.72; H, 3.40.
Bis(trans-â-3,4-diacetoxy-5-methoxy-styrenesulfonyl)-
methane (7). The disulfone reagent 1 (245 mg, 0.49 mmol)
was reacted with 3,4-diacetoxy-5-methoxybenzaldehyde (407
mg, 1.61 mmol) according to general procedure A. After
removal of solvent, the crude product was subjected to column
chromatography (hexanes/ethyl acetate 1:1, Rf ) 0.33) to yield
207 mg (66%) of the title compound. 1H NMR (acetone-d6, 250
MHz) δ 2.29 (s, 6H), 2.30 (s, 6H), 3.92 (s, 6H) 5.22 (s, 2H),
7.24 (d, 2H, J ) 1.9 Hz), 7.36-7.44 (m, 4H), 7.60 (d, 2H, J )
15.5 Hz); 13C NMR (acetone-d6, 125 MHz) δ 20.2, 20.5, 57.1,
73.5, 111.2, 117.1, 128.0, 131.7, 135.7, 145.1, 145.4, 154.1,
167.9, 168.7. HRFABMS calcd for C27H29O14S2 (M + H):
641.0999. Found: 641.1013 (M + H). Anal. (C27H28O14S2) C,
H.
Bis(trans-â-3,4-hydroxy-5-methoxystyrenesulfonyl)-
methane (8). To a solution of 7 (20 mg, 0.031 mmol) in DMSO-
d6 (1 mL) was added 10 µL of hydrazine hydrate. After
approximately 10 min the solution was subjected to reverse-
phase HPLC (C18) to yield 7.5 mg (51%) of the title compound.
1H NMR (acetone-d6, 500 MHz) δ 3.87 (s, 6H), 5.04 (s, 2H),

4532 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Meadows et al.
6.90 (appdd, 4H, J ) 13.3, 1.9 Hz), 7.10 (d, 2H, J ) 15.3 Hz),
7.45 (d, 2H, J ) 15.3 Hz); 13C NMR (acetone-d6, 125 MHz) δ
56.7, 74.1, 105.6, 111.2, 123.7, 124.4, 138.7, 146.6, 147.2, 149.3.
HRFABMS (+) calcd C19H21O10S2 (M + H): 473.0576. Found:
473.0573 (M + H). Anal. (C19H21O10S2) C, H.
MHz) δ 20.4, 56.0, 71.1, 112.8, 122.3, 123.5, 126.6, 131.0, 141.8,
144.3, 151.3, 168.3; FT-IR (KBr) ν 3074, 1618, 1514 (CdC str),
1763 (CdO str), 1466 (-CH2- str), 1329, 1126 (SO2 str), 1273,
1028 (Ph-O-C str), 1207 (C-O str), 970 (trans CdC str).
Anal. (C23H24O10S2) C, H.
Bis(trans-â-3,4-di-O-benzoyl-styrenesulfonyl)meth-
ane (12). To a solution of 1 (150 mg, 0.3 mmol) in THF (2
mL) was added LiBr (78 mg, 0.9 mmol) followed by DIEA (157
µL, 0.9 mmol). 3,4-Di-O-benzoylbenzaldehyde (387 mg, 0.9
mmol) was then added, and the reaction mixture was allowed
to sit overnight. Then approximately 100 µL of acetic acid was
added followed by water and ethyl acetate. The whole solution
is then filtered to give 185 mg (93% yield) of a crude product.
An analytical sample was acquired by reverse-phase (C18)
HPLC (40% acetonitrile in water (1%TFA) up to 100% aceto-
Acetic Acid 2-Acetoxy-6-methoxy-4-[2-(2-trans-phe-
nylethenesulfonylmethanesulfonyl)-trans-vinyl]phe-
nyl Ester (9). To a solution of 1 (443 mg, 0.88 mmol) in 3 mL
of THF are added DIEA (513 µL, 2.95 mmol) and LiBr (256
mg), and the solution is agitated until solvation of the LiBr
occurs. Then benzaldehyde is added (30 µL, 0.29 mmol) and
the solution is allowed to sit overnight. Approximately 500 µL
of acetic acid is then added, and the solution was diluted with
ethyl acetate and washed with water. The organic layer is then
dried over sodium sulfate and then evaporated. The resulting
oily solid is then subjected to HPLC (7:3 up to 1:2, hexanes/
ethyl acetate) to give 104.3 mg (83%) of the desired monosul-
fone as a yellow oil. 1H NMR (CDCl3, 500 MHz) δ 1.38 (d, 12H,
J ) 6.2 Hz), 4.03 (d, 2H, J ) 15.9 Hz), 4.86 (m, 2H), 5.12 (s,
2H), 7.17 (d, 1H, J ) 15.5 Hz), 7.41-7.47 (m, 3H), 7.54-7.55
(m, 2H), 7.69 (d, 1H, J ) 15.5 Hz); 13C NMR (CDCl3, 125 MHz)
δ 23.6 (d, J(C-P) ) 5 Hz), 24.1, 51.4 (d, J(C-P) ) 138 Hz), 76.9
(d, J(C-P) ) 32 Hz), 77.2, 124.4, 128.3, 129.0, 129.2, 131.7, 146.8.
HRFABMS calcd for C16H26O7PS2 (M + H): 425.0858. Found:
425.0862 (M + H).
To a solution of the monosubstituted disulfone (37.7 mg,
0.088 mmol) in 1 mL of THF are added DIEA (47 µL, 0.26
mmol) and LiBr (23 mg, 0.26 mmol), and the solution was
agitated until solvation of the LiBr occurs. Then 82 mg (0.34
mmol) of crude 3,4-O-acetoxy-5-methoxybenzaldehyde in 1 mL
of THF is added, and the solution continues overnight. The
reaction is quenched by the addition of 250 µL of acetic acid
followed by dilution with ethyl acetate. The resulting mixture
is washed with water and the organic layer is dried over
sodium sulfate to give a crude oil that is subjected to HPLC
(2:1 up to 1:3, hexanes/ethyl acetate) to give 34.5 mg (80%) of
the desired product. 1H NMR (CDCl3, 500 MHz) δ 2.31 (s, 3H),
2.32 (s, 3H), 3.88 (s, 3H), 4.64 (s, 2H), 6.99 (d, 1H, J ) 1.8
Hz), 7.03 (d, 1H, J ) 1.8 Hz), 7.18 (d, 1H, J ) 12.9 Hz), 7.21
(d, 1H, J ) 12.9 Hz), 7.26 (s, 2H), 7.43-7.50 (m, 3H), 7.55-
7.57 (m, 2H), 7.61 (d, 1H, J ) 15.4 Hz), 7.70 (d, 1H, J ) 15.5
Hz); 13C NMR (CDCl3, 125 MHz) δ 20.2, 20.6, 56.5, 74.2, 109.7,
116.3, 124.1, 125.5, 128.3, 129.1, 129.3, 130.0, 131.6, 132.1,
144.0, 145.5, 147.1, 153.0, 167.3, 167.9. HRFABMS calcd for
C22H23O9S2 (M + H): 495.0784. Found: 495.0789 (M + H).
Anal. (C22H22O9S2) C, H.
3-Methoxy-5-[2-(2-phenylethenesulfonylmethane-
sulfonyl)vinyl]benzene-1,2-diol (10). To a solution of 9 (12.4
mg, 0.025 mmol) in approximately 1 mL of methanol is added
240 µL of 2 M NH3 in methanol, and the reaction proceeded
for 1 h. The solvent was removed and the resulting oil was
subjected to reverse-phase (C18) HPLC (40% gradient aceto-
nitrile in water (1%TFA) up to 70% acetonitrile over 40 min)
to give the 3.0 mg (29%) of 10 as a white powder. 1H NMR
(acetone-d6, 500 MHz) δ 3.87 (s, 3H), 5.17 (s, 2H), 6.91 (d, 2H,
J ) 15.4 Hz), 7.11 (d, 1H, J ) 15.4 Hz), 7.35 (d, 1H, J ) 15.0
Hz), 7.45-7.51 (m, 4H), 7.62 (d, 1H, J ) 15.7 Hz), 7.70 (d, 2H,
J ) 7.3 Hz), 8.06 (broad s, 1H), 8.23 (broad s, 1H); 13C NMR
(acetone-d6, 125 MHz) δ 56.7, 73.8, 105.6, 111.4, 123.5, 127.2,
129.9, 130.1, 132.4, 138.3, 146.2, 147.4. HRFABMS (-) calcd
C18H17O7S2 (M - H): 409.0416. Found: 409.0418 (M - H).
Anal. Calcd for C18H18O7S2 (1 H2O): C, 50.46; H, 4.70. Found:
C, 50.08; H, 4.12.
1
nitrile). H NMR (DMSO-d6, 600 MHz) δ 5.59 (s,2H), 7.42 (d,
4H, J ) 8.1 Hz), 7.45 (d, 4H, J ) 8.1 Hz), 7.48 (d, 2H, J )
15.5 Hz), 7.64-7.65 (m, 6H), 7.74 (dd, 2H, J ) 8.5, 1.9 Hz),
7.91-7.96 (m, 5H); 13C NMR (DMSO-d6, 150 MHz) δ 124.0,
124.4, 127.3, 127.7, 128.0, 128.91, 128.94, 129.6, 131.1, 134.3,
142.4, 143.2, 144.3, 163.2, 163.4. HRFABMS (+) calcd
C45H33O12S2 (M + H): 829.1413. Found: 829.1418 (M + H).
Anal. (C45H33O12S2) C, H.
Purified Enzyme Assays. Biological Materials, Chemi-
cals, and Enzymes. All compounds were dissolved in DMSO,
and the stock solutions were stored at -20 °C. The γ-[32P]-
ATP was purchased from either Amersham Biosciences or
ICN. The expression systems for the wild-type IN and soluble
mutant INF185KC280S were generous gifts of Dr. Robert Craigie,
Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD.
Preparation of Oligonucleotide Substrates. The oligo-
nucleotides 21top, 5′-GTGTGGAAAATCTCTAGCAGT-3′, and
21bot, 5′-ACTGCTAGAGATTTTCCACAC-3′, were purchased
from Norris Cancer Center Microsequencing Core Facility
(University of Southern California) and purified by UV shad-
owing on polyacrylamide gel. To analyze the extent of 3′-
processing and strand transfer using 5′-end labeled substrates,
21top was 5′-end labeled using T4 polynucleotide kinase
(Epicentre, Madison, WI) and γ-[32P]-ATP (Amersham Bio-
sciences or ICN). The kinase was heat-inactivated, and 21bot
was added in 1.5 molar excess. The mixture was heated at 95
°C, allowed to cool slowly to room temperature, and run
through a spin 25 minicolumn (USA Scientific) to separate
annealed double-stranded oligonucleotide from unincorporated
material.
Integrase Assays. To determine the extent of 3′-processing
and strand transfer, wild-type IN was preincubated at a final
concentration of 200 nM with the inhibitor in reaction buffer
(50 mM NaCl, 1 mM HEPES, pH 7.5, 50 µM EDTA, 50 µM
dithiothreitol, 10% glycerol (w/v), 7.5 mM MnCl2, 0.1 mg/mL
bovine serum albumin, 10 mM 2-mercaptoethanol, 10% DMSO,
and 25 mM MOPS, pH 7.2) at 30 °C for 30 min. Then, 20 nM
of the 5′-end 32P-labeled linear oligonucleotide substrate was
added, and incubation was continued for an additional 1 h.
Reactions were quenched by the addition of an equal volume
(16 µL) of loading dye (98% deionized formamide, 10 mM
EDTA, 0.025% xylene cyanol, and 0.025% bromophenol blue).
An aliquot (5 µL) was electrophoresed on a denaturing 20%
polyacrylamide gel (0.09 M tris-borate, pH 8.3, 2 mM EDTA,
20% acrylamide, 8 M urea). Gels were dried, exposed in a
phosphorimager cassette, analyzed using a Typhoon 8610
variable mode imager (Amersham Biosciences), and quanti-
tated using ImageQuant 5.2. Percent inhibition (% I) was
calculated using the following equation:
Bis(trans-acetic acid 4-(2-methanesulfonyl-vinyl)-2-
methoxyphenyl ester) (11). The disulfone reagent 1 (212
mg, 0.42 mmol) and 4-acetoxy-3-methoxybenzaldehyde (271
mg, 1.40 mmol) were used according to general procedure A.
Following removal of the solvent, 5 mL of acetone was added,
from which a white solid precipitated. This was filtered off and
rinsed with cold acetone to give 145 mg (66%) of the title
compound (mp 202-203 °C). 1H NMR (DMSO-d6, 300 MHz) δ
2.27 (s, 6H), 3.81 (s, 6H), 5.54 (s, 2H), 7.15 (d, 2H, J ) 7.8
Hz), 7.27 (d, 2H, J ) 8.1 Hz), 7.43 (d, 2H, J ) 15.6 Hz), 7.49
(s, 2H), 7.55 (d, 2H, J ) 15.3 Hz); 13C NMR (DMSO-d6, 125
1 - (D - C)
% I ) 100 ×
N - C
where C, N, and D are the fractions of a 21-mer substrate
converted to a 19-mer (3′-processing product) or strand transfer
products for DNA alone, DNA plus IN, and IN plus drug,
respectively. The IC50 values were determined by plotting the
logarithm of drug concentration versus percent inhibition to
obtain concentration that produced 50% inhibition.

Disulfone-Containing HIV-1 Integrase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4533
(11) Sakai, H.; Kawamura, M.; Sakugari, J.; Sakugari, S.; Shibata,
R.; Ishimoto, A.; Ono, N.; Ueda, S.; Adachi, A. Integration is
essential for efficient gene expression of human immunodefi-
ciency virus type 1. J. Virol. 1993, 67, 1169-1174.
(12) Hazuda, D. J.; Young, S. D.; Guare, J. P.; Anthony, N. J.; Gomez,
R. P.; Wai, J. S.; Vacca, J. P.; Handt, L.; Motzel, S. L.; Klein, H.
J.; Dornadula, G.; Danovich, R. M.; Witmer, M. V.; Wilson, K.
A. A.; Tussey, L.; Schleif, W. A.; Gabryelski, L. S.; Jin, L.; Miller,
M. D.; Casimiro, D. R.; Emini, E. A.; Shiver, J. W. Integrase
inhibitors and cellular immunity suppress retroviral replication
in rhesus macaques. Science 2004, 305, 528-532.
(13) Hazuda, D. J.; Anthony, N. J.; Gomez, R. P.; Jolly, S. M.; Wai,
J. S.; Zhuang, L.; Fisher, T. E.; Embrey, M.; Guare, J. P., Jr.;
Egbertson, M. S.; Vacca, J. P.; Huff, J. R.; Felock, P. J.; Witmer,
M. V.; Stillmock, K. A.; Danovich, R.; Grobler, J.; Miller, M. D.;
Espeseth, A. S.; Jin, L.; Chen, I.-W.; Lin, J. H.; Kassahun, K.;
Ellis, J. D.; Wong, B. K.; Xu, W.; Pearson, P. G.; Schleif, W. A.;
Cortese, R.; Emini, E.; Summa, V.; Holloway, M. K.; Young, S.
D. A naphthyridine carboxamide provides evidence for discor-
dant resistance between mechanistically identical inhibitors of
HIV-1 integrase. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (31),
11233-11238.
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grase: a ten year saga. Expert Opin. Ther. Pat. 2002, 12, 709-
724.
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tors: the 2001-2002 Update. Curr. Pharm. Des. 2003, 9, 1789-
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A.; Witvrouw, M.; Pannecouque, C.; Debyser, Z.; Rando, R. F.;
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Marchand, C.; Burke, T. R., Jr.; Pommier, Y.; Schols, D.; De
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Cell-Based Assays. Drug susceptibility of HIV-1 to IN
inhibitors was determined with a focal infectivity assay (FIA)
as previously described.31,32 Immunostaining was performed
1
using the monoclonal antibody 22-613 at a /800 dilution. Foci
were counted under a dissecting microscope at 30× to 100×
magnification. Data for drug-susceptibility assays were plotted
as a percentage of control foci (no drug) versus inhibitor
concentrations. The concentrations required to inhibit focus
formation by 50% (EC50) were obtained from a best-fit line of
the linear portions of those plots. EC50 values for each drug
were determined from at least three separate experiments.
Cytotoxicity was determined with the Promega CellTiter 96
Aqueous One Solution Cell Proliferation Assay (MTS assay)
using the manufacturer’s recommended conditions. Data for
cell proliferation assays were plotted as a percentage of control
foci (no drug) versus inhibitor concentrations. The concentra-
tions required to inhibit cell proliferation by 50% (IC50) were
obtained from a best-fit line of the linear portions of those plots.
IC50 values for each drug were determined from at least two
separate experiments.
Time of Addition Assays. The time of addition studies
were performed similarly to the focal infectivity assays except
that the virus was incubated with the cells for 2 h at 37 °C,
after which unadsorbed virus was removed from the wells and
replaced with fresh virus-free medium. Dextran sulfate, T20,
3TC, and test compounds were used at 10 times their respec-
tive EC50 values and were added at seven different time points
before or after infection: -1, 0, 2, 4, 6, 10, and 24 h. The cells
were incubated for 4 days at 37 °C when they were fixed and
stained, and foci were counted and plotted against a no-drug
control.
Acknowledgment. This work was supported by the
National Institutes of Health (Grant GM60917), Uni-
versity of California AIDS Research Program (Grant
D02D400), and Richard Donovan and Marianne Gesner
as part of the Northern California Center for Aids
Research (NCCFAR). We are grateful to Yen Duong for
her assistance in the time-of-addition studies and to
Professor Dean J. Tantillo for helpful discussions.
(19) Robinson, W. E., Jr.; Reinecke, M. G.; Abdel-Malek, S.; Jia, Q.;
Chow, S. A. Inhibitors of HIV-1 replication that inhibit HIV
integrase. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6326-
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(20) Gervay, J.; Flaherty, T. M.; Holmes, D. Studies on the stereo-
selective synthesis of C-glycoside sulfones as potential glycosyl
transferase inhibitors. Tetrahedron 1997, 16355-64.
(21) Hadd, M. J.; Smith, M. A.; Gervay-Hague, J. A Novel reagent
for the synthesis of geminal disulfones. Tetrahedron Lett. 2001,
42, 5137-5140.
(22) Gervay-Hague, J.; Hadd, M. J. Divisional application based on
U.S. 09/595,048. Filed Nov. 2002.
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in carbanion formation by bis(methylsulfonyl)methane. J. Org.
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human immunodeficiency virus type 1 integrase inhibitors.
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Chem. 2000, 43, 2031-2039.
Supporting Information Available: Results from el-
emental analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
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