Synthesis and anti-HIV activity of a bile acid analog of cosalane

Article abstract of DOI:10.1016/S0040-4020(01)00955-3

Cosalane is a novel anti-HIV agent that inhibits the attachment of gp120 to CD4. The therapeutic potential of cosalane is limited by poor oral absorption. In an attempt to target the ileal bile acid transporter and thus facilitate oral bioavailability, a cosalane analog was synthesized in which the disalicylmethane pharmacophore is attached to a bile acid through a linker chain appended to C-17 of the steroid nucleus. The resulting bile acid analog of cosalane retained antiviral activity vs. HIV-1_IIIB and HIV-2_ROD in MT-4 cells, but was less potent than cosalane.

Full text of DOI:10.1016/S0040-4020(01)00955-3

TETRAHEDRON
Pergamon
Tetrahedron 57 72001) 9385±9391
Synthesis and anti-HIV activity of a bile acid analog of cosalane
Arunachalam Kannan,a Erik De Clercq,b Christophe Pannecouque,b Myriam Witvrouw,b
c
c
c
Tracy L.Hartman, Jim A.Turpin, Robert W.Buckheit, Jr. and Mark Cushmana,p
aDepartment of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA
bRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
cDepartment of Infectious Disease Research, Southern Research Institute, 431 Aviation Way, Frederick, MD 21701, USA
Received 8 August 2001; accepted 26 September 2001
AbstractÐCosalane is a novel anti-HIV agent that inhibits the attachment of gp120 to CD4.The therapeutic potential of cosalane is limited
by poor oral absorption.In an attempt to target the ileal bile acid transporter and thus facilitate oral bioavailability, a cosalane analog was
synthesized in which the disalicylmethane pharmacophore is attached to a bile acid through a linker chain appended to C-17 of the steroid
nucleus.The resulting bile acid analog of cosalane retained antiviral activity vs.HIV-1 IIIB and HIV-2ROD in MT-4 cells, but was less potent
than cosalane. q 2001 Elsevier Science Ltd.All rights reserved.
1. Introduction
identical.4 In addition to high capacity, the bile acid trans-
porters have been shown to accept and transport a variety of
analogs that are derivatized at the 3- and 24-positions.4,5 For
example, a number of amino acids and peptides, attached to
C-24 of cholic acid 72), have been shown to interact with
the human bile acid transporter in a Caco-2 cell line used
to model intestinal transport.6,7 Peptides attached to the
3-position of cholic acid through an aminoethylene linker
are transported in vitro in rabbit ileal vesicles as well as in
vivo during ileum perfusion in rats, whereas the unconju-
gated peptides are not transported.8 Similarly, the anticancer
drug chlorambucil and the prolyl-4-hydroxylase inhibitor
4-nitrobenzo-2-oxo-1,3-diazol-b-Ala-Phe-5-oxaproline-Gly
have been linked to the 3-position of the cholic acid nucleus
and have been to shown interact with the ileal bile salt
transport system in membrane vesicles prepared from rabbit
ileum, and they also target the liver after intravenous
administration.9 In a related study, bile acid-derived
HMG-CoA reductase inhibitors were demonstrated to inter-
act with the ileal uptake system for bile acids.10 Taken
together, these studies have demonstrated conclusively
that the bile acid transport system can be utilized to facilitate
the intestinal absorption of poorly absorbable compounds.
Cosalane 71) is an inhibitor of HIV replication with activity
against both HIV-1 and HIV-2.1,2 Cosalane acts primarily
by inhibiting the binding of gp120 to CD4, as well as by
hindering a post-binding fusion event.2 Pharmacokinetic
studies in male Sprague-Dawley rats have shown that
cosalane follows a biphasic elimination with a very long
half-life following intravenous administration.3 The drug
is metabolically very stable with low renal and biliary
excretion.The poor oral bioavailability 7 ,1%) of cosalane
is mainly attributed to its poor permeation across the
intestinal epithelium due to its very high lipophilicity and
membrane interacting nature.There is therefore a need for
cosalane analogs that would retain the anti-HIV activity of
cosalane but would have enhanced oral bioavailability.
The utilization of carrier-mediated transport mechanisms
offers a potentially useful strategy to enhance intestinal
absorption.In order for this approach to be successful, the
compound must be recognized and transported by a carrier
system having high capacity and relatively low substrate
speci®city.Both of these speci®cations are satis®ed by the
bile acid transporters, which facilitate the daily absorption
of 10±20 g of bile salts with .95% ef®ciency.Because of
these facts, Ho ®rst proposed using the bile acid carrier
mechanisms to enhance hepatic and small intestinal absorp-
tion of drugs, and demonstrated that 3-tosylcholic acid is
transported in the rat ileum almost as rapidly as cholic acid
itself, and that for 3-benzoylcholic acid, the rates were
The successful targeting of the intestinal bile acid trans-
porter must take advantage of the known structural require-
ments for recognition, which include a negative charge
located near the atom 24-to-29 region in the C-17 side
chain of the bile acid or bile acid conjugate,7,11,12 and at
least one hydroxyl group at C-3, C-7, or C-12 of the steroid
moiety.The rate of transport in the ileum depends on the
number of hydroxyl groups, with trihydroxy.dihydroxy.
monohydroxy5,12±14
Keywords: cosalane; HIV; AIDS; gp120; CD4; bile acid; antiviral therapy.
Corresponding author.Tel:. 11-765-494-1465; fax: 11-765-494-6790;
e-mail: cushman@pharmacy.purdue.edu
p
Cosalane 71) consists of a disalicylmethane pharmacophore
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd.All rights reserved.
PII: S0040-4020701)00955-3

9386
A. Kannan et al. / Tetrahedron 57 32001) 9385±9391
Scheme 1. Reagents and conditions: 7a) Ac2O, Py, DMAP, CH2Cl2; 7b) 7i) Et3N, ClCOOEt, 7ii) NaBH4, H2O; 7c) Dess-Martin reagent, CH2Cl2;
7d) TiCl4´2THF, Zn, benzophenone 1, THF, D; 7e) KOH, MeOH, D, 24 h; 7f) BBr3´7CH3)2S, ClCH2CH2Cl, 708C, 40 h.
attached through a linker chain to a lipophilic steroid moiety
that targets the compound to the cell membrane and viral
envelope.Prior work has demonstrated that the point of
attachment of the linker chain to the steroid can be moved
from C-3 to C-6 or C-17 and antiviral activity maintained,
and that a trans A/B ring fusion in the steroid is not critical
for anti-HIV activity.15,16 This suggests that a bile acid
analog of cosalane with the pharmacophore attached at
C-17 might retain antiviral activity and would also be a
good choice for enhanced intestinal transport.The attach-
ment could be accomplished while maintaining all three
hydroxyls at C-3, C-7, and C-12, and as discussed earlier,
bile acids having all three hydroxyl groups intact are in
general transported faster than those having only two
hydroxyl groups.The purpose of the present investigation
was to determine the feasibility of attaching the cosalane
pharmacophore to C-24 of a bile acid derivative while
retaining the anti-HIV activity.
2. Results and discussion
In order to construct the desired cholic acid analog 9 of
cosalane 71), the commercially available and inexpensive
starting material cholic acid 72)2 was employed as the
starting material in a convergent synthesis in which the

A. Kannan et al. / Tetrahedron 57 32001) 9385±9391
9387
Scheme 2. Reagents and conditions: 7a) aq.CH 2O, H2SO4, MeOH, H2O, D; 7b) TMSCHN2, MeOH, benzene; 7c) Ac2O, Py, DMAP, CH2Cl2; 7d) CrO3, AC2O,
7e) TiCl4´2THF, Zn, aldehyde 5, THF, D; 7f) KOH, MeOH, D, 24 h.
key step was the McMurry coupling of the substituted
benzophenone 6 with the intermediate aldehyde 5 7Scheme
1).As the ®rst step, the three hydroxyl groups of cholic acid
72) were protected as acetates by treatment with acetic
anhydride in pyridine in the presence of 4-dimethylamino-
pyridine.The carboxyl group was converted to a mixed
anhydride with ethyl chloroformate, which was then
reduced to the alcohol 4 with sodium borohydride.17 Oxi-
dation of the free alcohol of 4 with Dess-Martin periodinane
afforded the corresponding aldehyde 5.18 McMurry coupling
of the aldehyde 5 with the ketone 62 yielded the expected
alkene 7.19,20 The two methyl esters and three acetates
present in 7 were then hydrolyzed with potassium hydroxide
in methanol to afford the diacid 8, and the remaining two
methyl ethers were cleaved using boron tribromide±
dimethyl sul®de complex in 1,2-dichloroethane at 708C.21
expected to be ideal because of the formation of dimers in
addition to the desired cross-coupling product.In our case,
the isolation of the desired product required careful
chromatography.The ether cleavage reaction required
prolonged heating of intermediate 8 in the presence of a
Lewis acid.In an attempt to improve the overall yield,
the synthesis was modi®ed by replacement of the methyl
ether protection of the phenols with acetyl protection.The
resulting route is shown in Scheme 2.
Reaction of 3-chlorosalicylic acid 710)22 with formaldehyde
under acidic conditions afforded the disalicylmethane
derivative 112.Treatment of 11 with trimethylsilyldiazo-
methane23 resulted in selective methylation of both
carboxylic acids to yield intermediate 12.The two phenolic
hydroxyl groups of 12 were then acetylated with acetic
anhydride in pyridine, resulting in the expected diacetate
13, which was oxidized with chromium trioxide in acetic
anhydride to afford the benzophenone 14.The McMurry
coupling of 14 with aldehyde 5, followed by the usual acidic
The two low-yielding steps in the synthesis outlined in
Scheme 1 were the McMurry reaction 725%) and the ether
cleavage 735%).The yield of the McMurry reaction is not
Table 1. Anti-HIV activities of cosalane 71) and analogs
EC50 7mM)a
CC50 7mM)b
c
d
d
Compound
HIV-1RF
HIV-1IIIB
HIV-2ROD
CEM-SS cells
MT-4 cells
1
8
9
16
5.1^2.1
53.1
.150
28.0
3.0^0.18
.50
62.3^13.9
27.2
4.0^2.1
.54.1
67.2^20.1
.31.3
.200
130
152
.125
.54.1
.125
31.4
69.5
a
Concentration required to reduce the cytopathic effect of the virus by 50%.
Concentration required for a 50% reduction in cellular viability of uninfected cells.
Determined in CEM-SS cells.
b
c
d
Determined in MT-4 cells.

9388
A. Kannan et al. / Tetrahedron 57 32001) 9385±9391
with the cell membrane.Previous studies have demonstrated
a correlation of anti-HIV potency with lipophilicity in a
series of cosalane analogs having normal alkenyl and
phosphodiester chains as cholestane replacements.24 More-
over, recent evidence indicates that cosalane may in fact
accumulate in the lipid bilayer of the hepatocyte
membranes.25 The decrease in lipophilicity of the steroid
fragment of the bile acid analog 9 versus cosalane 1 may
therefore be at least partially responsible for the observed
decrease in antiviral potency.This hypothesis is also in
agreement with the fact that the previously reported
cosalane analog 16, which lacks the three hydroxyl groups
present in 9 but is otherwise identical, is more active than
9 as an anti-HIV agent 7EC50 of 16 against HIV-1RF in
CEM-SS cells, 28.0 mM; against HIV-1IIIB in MT-4 cells,
27.2 mM; and against HIV-1ROD in MT-4 cells,
.31.3 mM).16
Figure 1. Schematic representation of a hypothetical model of the anchor-
ing of the disalicylmethane moiety of cosalane and cosalane analogs to the
cell membrane and viral envelope by the steroid fragment.Reprinted with
permission from 7see Ref.24).Copyright 1996 American Chemical
Society.
workup with hydrochloric acid, resulted in 15, in which the
two phenolic acetates had been selectively hydrolyzed.The
hydrolysis of the phenolic acetates presumably occurred
under the acidic conditions employed in the workup of the
reaction mixture, and not during the McMurry reaction
itself.The remaining three acetates present in 15 were
then hydrolyzed with potassium hydroxide in methanol to
provide the desired compound 9.The overall yield of the
route shown in Scheme 2 was higher than that in Scheme 1,
mainly because it avoided the low-yielding methyl ether
cleavage reaction.
3. Experimental
3.1. General procedures
Melting points were determined in capillary tubes on a Mel-
Temp apparatus and were uncorrected.Spectra were
obtained as follows: CI mass spectra on a Finnegan 4000
spectrometer; FAB mass spectra and EI mass spectra on a
Kratos MS50 spectrometer; and 1H NMR spectra on a
Varian VXR-500S or Bruker ARX-300 spectrometers.
Microanalyses were performed at the Purdue Microanalysis
Laboratory, and all values were within ^0.4% of the
calculated compositions.Silica gel used for column
chromatography was 230±400 mesh.
The bile acid cosalane analog 9 was evaluated for inhibition
of the cytopathic effect of HIV-1RF in CEM-SS cells, and of
HIV-1IIIB and HIV-2ROD in MT-4 cells 7Table 1).The
compound proved to be inactive against HIV-1RF in
CEM-SS cells 7EC50.150 mM), and was also cytotoxic in
uninfected CEM-SS cells 7CC50: 152 mM).However,
compound 9 did display some activity against HIV-1IIIB in
MT-4 cells 7EC50: 62. 3^13.9 mM), and was also active vs.
HIV-2ROD in MT-4 cells 7EC50: 67.2^20.0 mM).It was not
cytotoxic in uninfected MT-4 cells at a concentration of
125 mM 7CC50.125 mM).In contrast to these results, the
di7methyl ether) 8 proved to be active against HIV-1RF in
CEM-SS cells 7EC50: 53.1 mM), and it was also cytotoxic in
uninfected CEM-SS cells 7CC50: 130.0 mM).Compound 8
was not active against HIV-1IIIB 7EC50.50 mM) or
HIV-2ROD in MT-4 cells 7EC50.54.1 mM), and it was not
cytotoxic in uninfected MT-4 cells at a concentration of
54.1 mM 7CC50.54.1 mM).
3.1.1. 3a,7a,12a-Triacetoxy-5b-cholane-24-al ,5). A
solution of 3a,7a,12a-triacetoxy-5b-cholane-24-ol 74)17
7520 mg, 1 mmol) in methylene chloride 75 mL) was
added to a solution of Dess-Martin reagent 7466 mg,
1.1 mmol) in methylene chloride 710 mL) with stirring.
After stirring for 2 h, the reaction mixture was diluted
with ether 750 mL) and the resulting suspension was
added to a solution of sodium bicarbonate 72 g) and sodium
thiosulfate 77 g) in water 750 mL).The resulting mixture
was stirred for 20 min and the layers were separated.The
organic layer was washed with aq.sodium bicarbonate
750 mL) and brine 750 mL).The organic layer was dried
with Na2SO4, and evaporated, and the residue was puri®ed
by ¯ash chromatography on silica gel 720 g), eluting with
hexanes±EtOAc 73:1), to give a white solid 526±28 7414 mg,
80%): mp 558C.IR 7KBr) 2949, 2874, 1734, 1444, 1377,
Both analogs 8 and 9 were less potent than cosalane 1 7EC50:
5.1^2.1 mM against HIV-1RF in CEM-SS cells, EC50:
3.0^0.18 mM against HIV-1IIIB in MT-4 cells, and EC50:
4.0^2.1 mM vs.HIV-2
in MT-4 cells).It is thought
ROD
that the antiviral activity of cosalane might result, in part,
from the insertion of the steroid fragment in viral and
cellular membranes, with the disalicylmethane moiety
pointing outward in an obstructive mode 7Fig.1).This
could result in inhibition of the fusion of the viral envelope
1248, 1025 cm21 1H NMR 7500 MHz, CDCl3) d 9.74
;
7s, 1H), 5.07 7s, 1H), 4.89 7bs, 1H), 4.58±4.52 7m, 1H),

A. Kannan et al. / Tetrahedron 57 32001) 9385±9391
9389
2.43±2.30 7m, 2H), 2.12 7s, 3H), 2.07 7s, 3H), 2.02 7s, 3H),
2.01±1.04 7m, 22H), 0.90 7s, 3H), 0.80 7d, Jˆ6.5 Hz, 3H),
0.67 7s, 3H). ESIMS m/z 541 7MNa1).Anal.calcd for
C30H46O7: C, 69.45; H, 8.94. Found: C, 69.53; H, 8.96.
dried over Na2SO4 and evaporated to dryness.The solid
was recrystallized from a mixture of methylene chloride
and acetone to yield diacid 9 750 mg, 35%): mp 1528C
dec.IR 7KBr) 3500±2600, 2930, 2865, 1682, 1459, 1360,
1
1233, 1187 cm21; H NMR 7500 MHz, acetone-d6) d 7.79
3.1.2. 24-[Bis[50-chloro-40-methoxy-30-,methoxycarbonyl)-
phenyl]methylene]3a,7a,12a-triacetoxycholane ,7). THF
720 mL) was added to a mixture of titanium7IV) chloride
tetrahydrofuran complex 71:2) 71.32 g, 4 mmol) and zinc
dust 70.45 g, 6 mmol) kept under argon atmosphere and
the mixture was heated at re¯ux for 3 h.A solution of
benzophenone 6 7427 mg, 1 mmol) and the aldehyde 5
7518 mg, 1 mmol) in THF 715 mL) was added to the
reaction mixture, which was heated at re¯ux for 12 h.The
reaction mixture was cooled to room temperature, 1N HCl
710 mL) was added, and the reaction mixture was extracted
with EtOAc 73£40 mL) and washed with brine 750 mL).
The organic layer was dried with Na2SO4, and evaporated,
and the residue was puri®ed by ¯ash chromatography on
silica gel 720 g), eluting with hexanes±EtOAc 73:1), to
give a white solid 7 7228 mg, 25%): mp 7starts to soften at
558C) 908C.IR 7KBr) 2951, 2872, 1735, 1477, 1438, 1373,
7d, Jˆ1.9 Hz, 1H), 7.76 7d, Jˆ2.1 Hz, 1H), 7.61 7d, Jˆ
2.1 Hz, 1H), 7.55 7d, Jˆ1.9 Hz, 1H), 6.24 7t, Jˆ7.6 Hz,
1H), 4.00 7s, 1H), 3.87 7s, 1H), 3.44±3.41 7m, 1H), 2.35±
1.06 7m, 29H), 1.02 7d, Jˆ6.5 Hz, 3H), 0.95 7s, 3H), 0.75 7s,
3H).ESIMS
m/z 733/7317MH1), 715/713, 697/699,
679/677.Anal.calcd for C 39H48Cl2O9´2.5H2O: C, 60.37;
H, 6.89. Found: C, 60.51; H, 7.03.
3.1.5. 3,30-Dicarbomethoxy-5,50-dichloro-4,40-dihydroxy-
diphenylmethane ,12). 3,30-Dicarboxy-5,50-dichloro-4,40-
dihydroxydiphenylmethane 711,2 0.5 g, 1.40 mmol) was
taken up in methanol 710 mL) and benzene 725 mL).
TMSCHN2 72.5 mL, 5.0 mL, 2.0 M solution in hexanes)
was added dropwise to the stirring solution.The solution
was allowed to stir for 10 h after addition was completed.
The reaction mixture was concentrated and the crude
product was puri®ed by column chromatography on silica
gel 750 g) using ethyl acetate±hexanes 71:4) as the eluent to
give a white solid 12 70.25 g, 45%): mp 1758C.IR 7KBr)
1
1248, 1024 cm21; H NMR 7500 MHz, CDCl3) d 7.26 7d,
Jˆ2.3 Hz, 1H), 7.22 7d, Jˆ2.3 Hz, 1H), 7.07 7d, Jˆ2.1 Hz,
1H), 7.02 7d, Jˆ2.3 Hz, 1H), 5.78 7t, Jˆ7.6 Hz, 1H), 4.79 7s,
1H), 4.64 7bs, 1H), 4.31±4.27 7m, 1H), 3.72 7s, 3H), 3.65 7s,
3H), 3.64 7s, 3H), 3.63 7s, 3H), 1.84 7s, 3H), 1.82 7s, 3H),
1.77 7s, 3H), 1.74±0.77 7m, 24H), 0.77 7s, 3H), 0.48 7d,
Jˆ6.4 Hz, 3H), 0.45 7s, 3H). ESIMS m/z 935/937 7MNa1).
1
3093, 2950, 1735, 1676, 1445, 1344, 1294, 1198 cm21; H
NMR 7300 MHz, CDCl3) d 11.23 7s, 2H), 7.55 7d, Jˆ
2.3 Hz, 2H), 7.35 7d, Jˆ2.2 Hz, 2H), 3.96 7s, 6H), 3.81 7s,
2H).Anal.calcd for C 17H14Cl2O6: C, 53.01; H, 3.66. Found:
C, 52.81; H, 3.77.
3.1.3. 24-[Bis[50-chloro-40-methoxy-30-,carbonyl)phenyl]-
methylene]3a,7a,12a-trihydroxycholane ,8). 24-[Bis[50-
chloro-40-methoxy-30-7methoxycarbonyl)-phenyl]methyl-
ene]-3a,7a,12a-triacetoxy cholane 77, 93.5 mg, 0.1 mmol)
was taken up with stirring in methanol 730 mL).KOH
7200 mg) was added, and the resulting solution was heated
under re¯ux for 24 h.The reaction mixture was poured into
water 775 mL) and acidi®ed to pH 1 by dropwise addition of
1N HCl.The resulting solution was extracted with EtOAc
73£50 mL).The organic extracts were combined and dried
over Na2SO4 and concentrated to give the triol 8 768 mg,
90%): mp 7starts to soften at 358C) 1758C.IR 7KBr) 3507±
3389, 2936, 2861, 1711, 1475, 1425, 1374, 1254, 1079,
3.1.6. 4,40-Diacetoxy-3,30-dicarbomethoxy-5,50-dichloro-
diphenylmethane ,13). 3,30-Dicarbomethoxy-5,50-dichloro-
4,40-dihydroxydiphenylmethane 712, 0.5 g, 1.30 mmol) was
taken up in acetic anhydride 78 mL), pyridine 74 mL) and
DMAP 725 mg) with stirring.Stirring was continued for
24 h.The reaction mixture was diluted with ethyl acetate
740 mL) and the resulting mixture was washed successively
with 5% aqueous HCl solution 72£25 mL), 5% aqueous
NaHCO3 solution 720 mL) and water 720 mL).The organic
layer was dried with Na2SO4, and concentrated, and the
residue was ¯ash chromatographed on silica gel 720 g),
eluting with hexanes±EtOAc 74:1), to give a white powder
13 70.35 g, 58%): mp 1558C.IR 7KBr) 2953, 1765, 1723,
1576, 1467, 1364, 1277 cm21; 1H NMR 7300 MHz, CDCl3)
d 7.71 7d, Jˆ2.2 Hz, 2H), 7.41 7d, Jˆ2.2 Hz, 2H), 3.96 7s,
2H), 3.87 7s, 6H), 2.38 7s, 6H). Found: C, 52.81; H, 3.77.
Anal.calcd for C 21H18Cl2O8: C, 53.75; H, 3.87. Found: C,
54.00; H, 3.20.
1
1002 cm21; H NMR 7500 MHz, CDCl3) d 7.61 7d, Jˆ
2.1 Hz, 2H), 7.51 7d, Jˆ2.1 Hz, 1H), 7.49 7d, Jˆ2.3 Hz,
1H), 6.30 7t, Jˆ7.7 Hz, 1H), 3.96 7s, 3H), 3.90 7s, 4H),
3.78 7s, 1H), 3.37±3.26 7m, 1H), 2.35±1.06 7m, 29H),
0.96 7s, Jˆ6.5 Hz, 3H), 0.88 7s, 3H), 0.68 7s, 3H). ESIMS
m/z 759/7617MH1), 743/741, 725/723, 707/705.Anal.calcd
for C41H52Cl2O9´0.5H2O: C, 64.09; H,6.95. Found: C, 64.07;
H, 7.18.
3.1.7. Di[4-acetoxy-3-chloro-5-,methoxycarbonyl)phenyl]
ketone ,14). 3,30-Dicarbomethoxy-5,50-dichloro-4,40-di-
hydroxydiphenylmethane 713, 0.40 g, 0.85 mmol) was
3.1.4. 24-[Bis[50-chloro-40-hydroxy-30-,carbonyl)-phenyl]-
methylene]3a,7a,12a-trihydroxycholane ,9). Boron-
tribromide±dimethyl sul®de complex 71 M solution in
methylene chloride, 0.61 mL) was added dropwise to a
stirred solution of diacid 8 70.15 g, 0.12 mmol) in dichloro-
ethane 725 mL), and the mixture was stirred at 708C for 40 h
and at room temperature overnight.It was cooled in ice, the
reaction quenched with water 710 mL), and the mixture was
stirred for 1 h.The organic layer was separated, and the
aqueous layer was extracted with ethyl acetate 73£20 mL).
The combined organic extracts were washed with water,
taken up in acetic anhydride 720 mL) with stirring.CrO
3
70.357 g, 3.57 mmol) was added in one portion to the
stirring mixture, and the resulting mixture was stirred
under argon at room temperature for 12 h.The reaction
mixture was ®ltered through a pad of celite in a sintered
glass funnel.The celite pad was washed with ethyl acetate
740 mL).The ®ltrate and washes were combined, and the
solvent was removed on a rotary evaporator.The crude
product was ¯ash chromatographed on silica gel 720 g),
eluting with hexanes±EtOAc 74:1), to give a white solid
14 70.40 g, 97%): mp 1608C.IR 7KBr) 3080, 2955, 1780,

9390
A. Kannan et al. / Tetrahedron 57 32001) 9385±9391
1732, 1657, 1597, 1460, 1368, 1270 cm21
7300 MHz, CDCl3) d 8.28 7d, Jˆ2.0 Hz, 2H), 8.07 7d,
Jˆ2.0 Hz, 2H), 3.90 7s, 6H), 2.44 7s, 6H). Anal. calcd for
C21H16Cl2O9: C, 52.19; H, 3.34. Found: C, 52.11; H, 3.20.
;
1H NMR
test compounds were made directly in ¯at bottom 96-well
plastic microtiter trays using a Biomek 2000 robot 7Beck-
man Instruments, Fullerton, CA).Untreated control HIV-
and mock-infected cell samples were included for each
sample.
3.1.8. 24-[Bis[50-chloro-40-hydroxy-30-,methoxycarbonyl)-
phenyl]methylene]3a,7a,12a-triacetoxycholane ,15). THF
720 mL) was added to a mixture of titanium7IV) chloride
tetrahydrofuran complex 71:2) 71.32 g, 4 mmol) and zinc
dust 70.45 g, 6 mmol) kept under argon atmosphere and
the mixture was heated at re¯ux for 3 h.A solution of
di[4-acetoxy-3-chloro-5-7methoxycarbonyl)phenyl] ketone
714, 350 mg, 0.73 mmol) and the aldehyde 5 7375 mg,
0.73 mmol) in THF 715 mL) was added to the reaction
mixture, which was heated at re¯ux for 24 h.The reaction
mixture was cooled to room temperature, 1N HCl 710 mL)
was added, and the reaction mixture was extracted with
EtOAc 73£40 mL) and washed with brine 750 mL).The
organic layer was dried with Na2SO4 and evaporated to
give the crude product.The crude product was ¯ash
chromatographed on silica gel 720 g), eluting with
hexane±EtOAc 74:1), to give a sticky material 15 70.16 g,
25%): mp 828C dec.IR 7KBr) 3500±3400, 2936, 2861,
30
31
HIV-1IIIB or HIV-2ROD stock 750 mL) at 100±300
CCID50 7cell culture infectious dose) or culture medium
was added to either the infected or mock-infected well of
the microtiter tray.Uninfected 7mock-infected) cells were
used to evaluate the cytotoxic effect of the test compounds.
Exponentially growing MT-4 cells32 were centrifuged for
5 min at 1000 rpm and the supernatant was discarded.The
MT-4 cells were resuspended at 6£105 cells/mL, using
slight magnetic stirring, and 50 mL volumes were trans-
ferred to the microtiter tray wells.Five days after infection,
the viability of mock- and HIV-infected cells were
examined spectrophotometrically by the MTT assay.
The MTT assay is based on the reduction of 3-74,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 7MTT)
7Acros Organics, Geel, Belgium) by mitochondrial
dehydrogenase of metabolically active cells to a formazan
that can be measured spectrophotometrically.The absorb-
ances were read in Multiskan Ascent Reader 7Labsystems,
Helsinki, Finland), at a wavelengths 540 nm, with a
reference wavelength of 690 nm.The EC 50 and CC50 values
were then calculated.
1
1710, 1475, 1425, 1374, 1254, 1079, 1002 cm21; H NMR
7500 MHz, CDCl3) d 11.38 7s, 1H), 11.29 7s, 1H), 7.51 7d,
Jˆ2.0 Hz, 1H), 7.49 7d, Jˆ2.1 Hz, 1H), 7.37 7d, Jˆ2.1 Hz,
1H), 7.33 7d, Jˆ2.0 Hz, 1H), 5.92 7t, Jˆ7.5 Hz, 1H), 5.05 7s,
1H), 4.90 7s, 1H), 4.48±4.44 7m, 1H), 3.92 7s, 3H), 3.90 7s,
3H), 2.09 7s, 3H), 2.07 7s, 3H), 2.03 7s, 3H), 1.95±0.90 7m,
29 H), 0.89 7s, 3H), 0.73 7d, Jˆ6.5 Hz, 3H), 0.70 7s, 3H).
ESIMS m/z 885/8877MH1).Anal.calcd for C 47H58Cl2O12:
C, 63.78; H, 6.61. Found: C, 63.96; H, 6.50.
Acknowledgements
This research was made possible by NIH grant AI 36624,
awarded by the National Institutes of Health, DHHS, and by
grants from the 7Belgian) Fonds voor Wetenschappelijk
Onderzoek 7FWO Grant G.0104.98) and the 7Flemish)
Geconcerteerde Onderzoeksacties 7GOA 00/12).We thank
ISEP/FORTIS for ®nancial support.We also thank Kristien
Erven for excellent technical assistance.
3.1.9. 24-[Bis[50-chloro-40-hydroxy-30-,carbonyl)phenyl]-
methylene]3a,7a,12a-trihydroxycholane ,9). 24-[Bis[50-
chloro-40-hydroxy-30-7methoxycarbonyl)phenyl]methylene]-
3a,7a,12a-triacetoxycholane 715, 88 mg, 0.1 mmol) was
taken up with stirring in methanol 730 mL).KOH 7200
mg) was added, and the resulting solution was heated
under re¯ux for 24 h.The reaction mixture was poured
into water 775 mL) and was acidi®ed to pH 1 by dropwise
addition of 1N HCl.The resulting solution was extracted
with EtOAc 73£50 mL).The organic extracts were
combined and dried over Na2SO4 and concentrated to give
the diacid 9 758 mg, 80%).
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