Extension of the polyanionic cosalane pharmacophore as a strategy for increasing anti-HIV potency

Article abstract of DOI:10.1021/jm980727m

The anti-HIV agent cosalane inhibits both the binding of gp120 to CD4 as well as an undefined postattachment event prior to reverse transcription. Several cosalane analogues having an extended polyanionic 'pharmacophore' were designed based on a hypothetical model of the binding of cosalane to CD4. The analogues were synthesized, and a number of them displayed anti-HIV activity. One of the new analogues was found to possess enhanced potency as an anti-HIV agent relative to cosalane itself. Although the new analogues inhibited both HIV-1 and HIV-2, they were more potent as inhibitors of HIV-1 than HIV-2. Mechanism of action studies indicated that the most potent of the new analogues inhibited fusion of the viral envelope with the cell membrane at lower concentrations than it inhibited attachment, suggesting inhibition of fusion as the primary mechanism of action.

Full text of DOI:10.1021/jm980727m

J . Med. Chem. 1999, 42, 1767-1777
1767
Exten sion of th e P olya n ion ic Cosa la n e P h a r m a cop h or e a s a Str a tegy for
In cr ea sin g An ti-HIV P oten cy
,
†
†
†
†
‡
‡
Mark Cushman,* Shabana Insaf, Gitendra Paul, J effrey A. Ruell, Erik De Clercq, Dominique Schols,
‡
‡
§
§
§
Christophe Pannecouque, Myriam Witvrouw, Catherine A. Schaeffer, J im A. Turpin, Karen Williamson, and
William G. Rice§
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal Sciences,
Purdue University, West Lafayette, Indiana 47907, Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium, and Laboratory of Antiviral Drug Mechanisms, SAIC Frederick,
National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201
Received December 23, 1998
The anti-HIV agent cosalane inhibits both the binding of gp120 to CD4 as well as an undefined
postattachment event prior to reverse transcription. Several cosalane analogues having an
extended polyanionic “pharmacophore” were designed based on a hypothetical model of the
binding of cosalane to CD4. The analogues were synthesized, and a number of them displayed
anti-HIV activity. One of the new analogues was found to possess enhanced potency as an
anti-HIV agent relative to cosalane itself. Although the new analogues inhibited both HIV-1
and HIV-2, they were more potent as inhibitors of HIV-1 than HIV-2. Mechanism of action
studies indicated that the most potent of the new analogues inhibited fusion of the viral envelope
with the cell membrane at lower concentrations than it inhibited attachment, suggesting
inhibition of fusion as the primary mechanism of action.
In tr od u ction
The binding of gp120 to CD4, as well as the subse-
quent postbinding fusion events, offers potentially use-
ful targets for the development of new anti-HIV agents.
A number of years ago, we reported the design and
synthesis of cosalane (1), a novel anti-HIV agent which
inhibits both the initial attachment of the virus to the
cell membrane as well as a postattachment event
1
,2
occurring prior to reverse transcription.
Although
cosalane (1) has a number of positive features including
synergy with AZT and activity against a wide spectrum
of laboratory, drug-resistant, and clinical HIV-1 and
HIV-2 isolates, its potency is moderate, ranging from
an EC50 of 3.2 µΜ vs the IIIB strain in CEM-SS cells to
8
0.2 µΜ vs the VIHU (NSI) isolate in CEM-SS cells. A
number of cosalane analogues have been synthesized,
but none of them offer significantly enhanced potencies
relative to cosalane (1) itself.3-7 The present study was
undertaken in order to design and synthesize more
potent cosalane analogues.
Design
Prior studies have shown that the inhibition of gp120
to CD4 binding by cosalane (1) results from its ability
8
to bind to both gp120 and CD4. Since the structure of
9
CD4 has been determined by X-ray crystallography, it
is possible to construct hypothetical models of the
binding of cosalane (1) to CD4 and to use these models
1
0
for designing more effective cosalane analogues. Since
the D1D2 region of CD4 contains residues that have
been implicated in binding to gp120, it is logical to look
†
Purdue University.
Katholieke Universiteit Leuven.
NCI-FCRDC.
‡
§
1
0.1021/jm980727m CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/28/1999

1
768 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Cushman et al.
F igu r e 1. Hypothetical model of the binding of the cosalane “pharmacophore” to CD4 (programmed for walleyed viewing): blue
dots, attraction; red dots, repulsion.
there for potential cosalane binding sites.9,11-13 Residues
Lys29, Lys35, Phe43, Leu44, Lys46, Gly47, and Arg59
of CD4 have been implicated as being directly involved
in binding to gp120. In one study, it was reported that
the alteration of Arg59 “dramatically disrupted the
ability of CD4 to bind to gp120”.12 Prior studies have
indicated that the disalicylmethane moiety of cosalane
is the “pharmacophore”, which makes it reasonable to
look for possible modes of binding of this fragment to
substructures within the D1D2 region of CD4. These
considerations led to the conclusion that the two nega-
tively charged carboxylate residues of cosalane may be
binding to the two adjacent, positively charged Arg58
and Arg59 residues of CD4. A hypothetical model based
on this idea is shown in Figure 1. The model was
constructed by placing the two carboxylates of cosalane
next to the guanidinium residues of Arg58 and Arg59
and “freezing” the structure of the protein while allow-
ing the ligand to move (Sculpt software, Interactive
Simulations, Inc.).
Although the model portrayed in Figure 1 is specula-
tive, it provides a possible starting point for the design
of more potent cosalane analogues. There is another
basic residue, Lys72, in close proximity to the proposed
binding site, and this could be targeted through the
incorporation of additional anionic groups on aromatic
rings that could be added to the structure of cosalane.
Accordingly, in the present investigation, benzyl groups
were attached to the two phenolic oxygens of cosalane
through ether linkages, and carboxylic acid and nitro
groups were attached in the ortho, meta, and para
positions. The resulting compounds were tested for
inhibition of the cytopathic effect of HIV-1RF in CEM
cell cultures and the activities compared with that of
cosalane itself. This would allow an examination of the
effect of the location of the new carboxyl groups and
nitro groups on anti-HIV activity.
aurintricarboxylic acid (ATA), a heterogeneous mixture
of polymers that forms when salicylic acid is treated
with formaldehyde in the presence of sulfuric acid and
1
4-18
sodium nitrite.
The anti-HIV activity of ATA has
been well-documented and results from its ability to
16,19,20
inhibit the attachment of gp120 to CD4.
On the
basis of the isolation and structure elucidation of low-
molecular-weight ATA oligomers, a schematic repre-
sentation (2) of the “structure” of ATA was proposed.
This schematic representation does not depict the
structure of ATA “literally”, since ATA is a very complex
heterogeneous mixture of polymers, and it is therefore
impossible to know its “structure”. Nevertheless, during
the process of investigating the structures and anti-HIV
activities of low-molecular-weight ATA components,
several compounds were isolated which retained some
of the anti-HIV activity of ATA, although their potencies
1
7
were lower than that of ATA. Specifically, some of the
compounds isolated and their anti-HIV-1IIIB activities
in CEM cells were 3 (EC50 >350 µM), 4 (EC50 77 µM), 5
(EC50 110 µM), 6 (EC50 380 µM), and 7 (EC50 92 µM).
Although these EC50 values are significantly higher
than the corresponding value for cosalane (EC50 3.2 µM),
the comparison of the activities of 3 with 4-7 suggests
that the potency of cosalane might be increased through
the incorporation of additional aromatic rings having
carboxylic acid groups. This idea, in conjunction with
the molecular modeling results discussed above, pro-
vided a strong case for extension of the cosalane
polyanionic pharmacophore as a possible method for
increasing potency.
Syn th esis
The syntheses of the three desired cosalane benzyl
ethers 11a -c containing ortho, meta, and para car-
boxylic acid groups, as well as the three nitro com-
pounds 11d -f, are outlined in Scheme 1. Bromination
of methyl 2-methylbenzoate with N-bromosuccinimide
and dibenzoyl peroxide in carbon tetrachloride afforded
The idea to extend the cosalane pharmacophore to
include additional aromatic rings with carboxyl groups
is also supported by a second line of reasoning. Cosalane
was originally designed on the basis of our work with
2
1,22
methyl 2-(bromomethyl)benzoate 8a .
The remaining
benzyl bromides 8b -f were available commercially.

Extension of the Polyanionic Cosalane Pharmacophore
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1769
Ta ble 1. Anti-HIV Activities of Cosalane Analogues
cytotoxicity (µΜ)
EC50a (µM)
CEM-SS
cells
MT-4
cells
compd HIV-1RFc HIV-1IIIBd HIV-2RODd
b
b
1
5.1
39.8
5.7
3.0
4.0
>200
>316
>316
72
>125
37
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
1a
1b
1c
1d
1e
1f
>37
>37
>29
22
>125
>125
>83
>52
>35
55
2.2
1.7
29
88
108
97
83
52
35
95
99
0.5
NAe
>125
>125
>83
16
66
e
NA
NAe
17.9
7.6
>316
>316
>316
76
>316
>316
>316
22.1
2.4
1.4
0.8
12.2
21.5
78.2
>125
>125
48
80
29
33
>125
>125
a
The concentration required to reduce the cytopathic effect of
the virus by 50%. b The concentration required for a 50% reduction
c
in cellular viability in uninfected cells. Determined in CEM-SS
cells. d Determined in MT-4 cells. e No activity was observed up
to a concentration of 125 µM.
both HIV-1RF and HIV-1IIIB. On the other hand, the
corresponding ortho acid and salt 10a and 11a were
significantly less active than cosalane in all of the anti-
HIV assays, and they were the least active of the
regioisomers.
With regard to the nitro acids 10d -f and nitro salts
1d -f, one might expect on the basis of the results with
1
the acids 10a -c and 11a -c that the ortho nitro isomers
0d and 11d would be less active as anti-HIV agents
1
Reaction of cosalane (1) with the benzyl bromides 8a -f
in DMF in the presence of potassium carbonate afforded
the corresponding products 9a -f in which the two
phenolic hydroxyl groups as well as the two carboxyl
groups had been alkylated. The saponifications of the
ester groups of 9a -f were carried out in aqueous
ethanol in the presence of potassium carbonate to afford
than the para nitro isomers 10f and 11f. This expecta-
tion was borne out for the HIV-1IIIB strain in MT-4 and
for HIV-2ROD in MT-4 cells, but not for HIV-1RF in CEM-
SS cells. In addition, the effect on potency resulting from
replacement of the two carboxylic acid moieties on the
benzyl groups with nitro groups is not consistent. For
example, in the case of the para carboxylate 11c versus
HIV-1RF in CEM-SS cells, the EC50 increases substan-
tially from 0.8 to 78.2 µM in 11f when the carboxylate
group is replaced by a nitro group. On the other hand,
replacement of the carboxylate group in 11a with a nitro
group resulted in a small decrease in the EC50 value
from 17.9 µM in 11a to 12.2 µM in 11d .
1
0a -f. In the cases of 9a -c, the hydrolysis reactions
were catalyzed by potassium cyanide. The four acid
groups present in 10a -c and the two acid groups
present in 10d -f were converted to the corresponding
sodium salts 11a -f using sodium carbonate in methanol
or ethanol.
To determine if the two most active compounds (10c
and 11c) could possibly interact with all three (Arg58,
Arg59, and Lys72) residues of CD4, the structure of the
“pharmacophore” of 10c was docked on the surface of
the protein. The structure of the protein was “frozen”
and the energy of the bound complex was minimized
using a procedure similar to that used to derive the
model in Figure 1. This resulted in the structure shown
in Figure 2. The positively charged guanidinium ions
on the side chains of Arg58 and Arg59 interact by ionic
bonding with two of the negatively charged carboxylates
of the ligand. In addition, the oxygen atom of a third
carboxylate residue is capable of ionic bonding to the
positively charged amino group of Lys72. This model
supports the possibility that three of the four carbox-
ylates of the ligand could bind to the protein as depicted
in Figure 2. In contrast, when the least potent of the
three tetracarboxylate analogues (10a ) was docked in
a similar fashion as 10c, it was calculated to be capable
of binding ionically to Arg58 and Lys72, but the car-
boxylate on the “upper” ring in Figure 3 was clearly not
positioned to bind to Arg59 (Figure 3). On the other
Biologica l Resu lts a n d Discu ssion
All of the new cosalane analogues were tested for their
ability to inhibit the cytopathic effect of HIV-1RF in
CEM-SS cells, of HIV-1IIIB in MT-4 cells, and of HIV-
2
-ROD in MT-4 cells, and the resulting EC50 values are
listed in Table 1. Intermediates 9a -f were all inactive
as anti-HIV agents, and they are not listed in Table 1.
The two most potent analogues proved to be compound
1
0c, having two benzyl ether groups bearing para
carboxylic acid groups, and the corresponding tetraso-
dium salt 11c. These two compounds were more potent
than cosalane itself against both HIV-1RF in CEM-SS
cells and HIV-1IIIB in MT-4 cells. Compounds 10c and
1
1c were also active versus HIV-2-ROD in MT-4 cells,
but they were less potent than they were versus the two
HIV-1 strains tested. The meta acid 10b and its salt
1
1b were only slightly less active versus HIV-1IIIB than
the para acid and salt 10c and 11c, respectively, but
they were approximately 10-fold less active versus HIV-
1
RF. These meta isomers were intermediate in activity
versus the para and ortho isomers when tested versus

1
770 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Cushman et al.
Sch em e 1
hand, an alternative model can be constructed for the
binding of 10a to CD4 in which the four carboxylates
are positioned to have possible attractive interactions
with the side chains of Lys72, Arg58, and Arg59 and
the backbone carbonyl of Arg59 (Figure 4). Therefore,
it cannot be argued that the hypothetical models have
any meaningful quantitative predictive value for in vitro
antiviral potency, since the most potent of the tetracar-
boxylates (10c) as well as the least potent (10a ) can
both be docked favorably in the proposed binding site
of CD4.
Although cosalane inhibits HIV-1 reverse tran-
scriptase, integrase, and protease in cell-free systems,
the available evidence indicates that the primary mech-
anism of the anti-HIV action of cosalane involves
inhibition of gp120-CD4 binding as well as inhibition
of a postattachment event prior to reverse transcrip-
2
tion. To gain some insight into the mechanism of action
of the most potent analogue 10c and its salt 11c, both
compounds were tested as inhibitors of HIV-1 attach-
ment, fusion, reverse transcriptase, integrase, protease,
and infection. The results of these mechanistic studies
are presented in Figures 5 and 6 for analogues 10c and
1
1c, respectively. It is unlikely that inhibition of reverse
transcriptase, integrase, and protease by these com-
pounds would be responsible for their antiviral action,
since 10c and 11c are inactive as inhibitors of protease
and integrase and very weak inhibitors of reverse
transcriptase with the concentration required for 50%
inhibition approaching 100 µM. Both compounds are
more potent inhibitors of attachment and fusion, but
they are clearly more potent as inhibitors of a fusion
event than attachment. The relative potencies of the
compounds as inhibitors of attachment and fusion
suggest that the inhibition of infection is due to inhibi-
tion of fusion rather than attachment. If fusion, rather
than attachment, is the primary target for the cosalane
analogues 10c and 11c, then they should be modeled
on the coreceptors (CXCR4 or CCR5) and/or gp41/gp120.
Additional studies were undertaken in order to pin-
point the mechanism of action of 10c and 11c in more
detail. These involved investigation of the effects of the
compounds on the binding of 12G5 antibody to the
second extracellular loop of CXCR4, inhibition of binding
of the Leu3a monoclonal antibody to the CD4 receptor,
inhibition of the binding of recombinant gp120 to MT-4
cells, and inhibition of the binding of monoclonal
antibody to the V3 loop of gp120 expressed in HIV-1-
infected cells. The results of these investigations are
detailed in Table 2. The results indicate that the
compounds bind to CD4 and consequently inhibit the
binding of gp120 to the cell. They also interact with the
V3 loop of gp120, which is important for fusion, but they
do not interact with the second extracellular loop of
CXCR4. As the CD4 receptor is in close proximity with
a chemokine receptor, an interaction with a chemokine
receptor cannot be excluded when a compound interacts
with CD4. However, a direct interaction with the second
extracellular loop of CXCR4, as measured by the binding
of a mAb (12G5) against CXCR4, was not observed. It
is possible that 10c and 11c could bind to a domain
other than the second extracellular loop of CXCR4,
which would then prevent the proper association of the
gp120-CD4 complex with the CXCR4 receptor. In any

Extension of the Polyanionic Cosalane Pharmacophore
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1771
F igu r e 2. Hypothetical model of the binding of the 10c “pharmacophore” to CD4 (programmed for walleyed viewing): blue dots,
attraction; red dots, repulsion.
F igu r e 3. Hypothetical model of the binding of the 10a “pharmacophore” to CD4 (programmed for walleyed viewing): blue dots,
attraction; red dots, repulsion.
case, it appears likely that multiple mechanisms are
operating. One mechanism may be a direct interference
of the gp120 interaction with CD4, and the other mech-
anism may prevent the proper orientation of the gp120-
CD4 complex with CXCR4 that is required for fusion.
In conclusion, the cosalane analogues 10c and 11c are
the most potent analogues of cosalane synthesized to
date. In contrast to cosalane itself, both compounds are
either very weak or inactive as inhibitors of reverse
transcriptase, integrase, and protease in cell-free sys-
tems, but they resemble cosalane in their ability to
inhibit attachment and fusion. Studies are in progress
on the mechanism of fusion inhibition observed with
these compounds.
spectrometer. The chemical shift values are expressed in ppm
parts per million) relative to tetramethylsilane as internal
standard. Elemental analyses were performed by the Purdue
Microanalytical Laboratory.
Meth yl 2-(Br om om eth yl)ben zoa te (8a ). Methyl 2-meth-
ylbenzoate (1.50 g, 10 mmol) was dissolved in carbon tetra-
chloride (85 mL), and N-bromosuccinimide (1.81 g, 10 mmol)
and dibenzoyl peroxide (52 mg) were added. The resulting
mixture was heated at reflux for 12 h and stirred overnight.
The precipitated succinimide was filtered off, and the filtrate
(
2
was concentrated and flash chromatographed (SiO , 50 g;
hexanes/ether, 9:1) to yield a colorless liquid (1.84 g, 80%)
which turned yellow upon exposure to light: IR (neat) 3067,
3
026, 2998, 2951, 2840, 1767, 1720, 1600, 1578, 1491, 1434,
1289, 1268, 1225, 1202, 1191, 1150, 1115, 1077, 1046, 965, 873,
-1 1
840, 798, 761, 739, 708, 664, 610, 572 cm ; H NMR (300 MHz,
CDCl ) δ 7.97 (d, J ) 7.6 Hz, 1 H), 7.53-7.44 (m, 2 H), 7.38
m, 1 H), 4.96 (s, 2 H), 3.95 (s, 3 H); C NMR (75 MHz, CDCl
δ 166.9, 139.2, 132.5, 131.6, 131.2, 128.5, 52.2, 31.5. Anal.
(C Br) C, H.
3
Exp er im en ta l Section
13
(
3
)
Melting points are uncorrected. Nuclear magnetic resonance
1
spectra for proton ( H NMR) were recorded on a 300-MHz
9 9 2
H O

1
772 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Cushman et al.
F igu r e 4. Hypothetical model of the binding of the 10a “pharmacophore” to CD4 (programmed for walleyed viewing): blue dots,
attraction; red dots, repulsion.
Ta ble 2. Effects of Compounds 10c and 11c on the Binding of the mAb (12G5) to the CXCR4 Receptor, Binding of the mAb (Leu3a)
to the CD4 Receptor, Binding of Recombinant gp120 (strain IIIB) to CD4+ T-Cell Lines, and Binding of the mAb (NEA9305) to the
V3 Loop of gp120 Expressed on HIV-1-Infected MT-4 Cells
CXCR4a
CD4b
gp120b
V3 loop interactionb
%
%
%
%
%
%
%
%
compd
concn (µM)
92
positive cells
inhibn
positive cells
inhibn
positive cells
inhibn
positive cells
inhibn
1
0c
99.8
99.9
99.9
99.4
99.3
99.8
99.9
99.8
99.2
99.1
99.1
2.1
0
0
0
0
0
0
0
0
0
0
0
1.4
1.2
7.8
99.1
99.9
1.2
2.1
3.2
99.9
99.8
99.9
1.5
100
100
94
4
6.8
8.3
25.3
88.2
99
98
79
9
23.8
34.4
58.4
65.9
76
58
16
3
1
3
0
0
8
.7
.73
.15
0
1
1c
81
100
100
99
0
0
0
7.8
6.9
19.9
76.2
98
99
82
22
26.9
33.6
60.9
66.6
69
58
15
5
1
3
0
0
0
6
.2
.65
.13
controls
95.6
6.2
0
70.3
8.4
0
isotype mAb
a
The assay was performed as previously described.29
b
The assay was performed as previously described.16
F igu r e 6. Mechanism of action studies of 11c.
100 g), eluting with hexanes-ethyl acetate (5:2), to give a
white foamy solid (466 mg, 86%): mp (softens at 43 °C) 92-
94 °C; IR (CHCl ) 3022, 2924, 2850, 1766, 1727, 1602, 1580,
1490, 1467, 1437, 1405, 1371, 1267, 1216, 1168, 1138, 1085,
F igu r e 5. Mechanism of action studies of 10c.
(
3
′′,3′′′-Dich lor o-4′′,4′′′-b is(o-ca r b oxyb en zyloxy)-5′′,5′′′-
bis(o-ca r boxyben zyloxyca r bon yl)-4′,4′-d ip h en yl-3â-[1-(3′-
bu ten yl)]ch olesta n e Tetr a m eth yl Ester (9a ). A mixture
of cosalane (1) (308 mg, 0.4 mmol), potassium carbonate (280
mg, 2.03 mmol), and methyl 2-(bromomethyl)benzoate (8a )
3
-
1
1
1052, 997, 895, 767, 668 cm ; H NMR (300 MHz, CDCl ) δ
3
8.07-7.92 (m, 4 H), 7.89 (dd, J ) 7.4, 1.6 Hz, 2 H), 7.69 (d, J
) 2.4 Hz, 1 H), 7.65 (d, J ) 2.1 Hz, 1 H), 7.54 (dd AB pattern,
J ) 7.6, 6.6 Hz, 2 H), 7.47-7.22 (m, 8 H), 7.40 (d, J ) 2.3 Hz,
1 H), 7.36 (d, J ) 2.3 Hz, 1 H), 6.13 (t, J ) 7.5 Hz, 1 H), 5.67
(s, 2 H), 5.64 (s, 2 H), 5.56 (s, 2 H), 5.48 (s, 2 H), 3.84 (s, 3 H),
3.83 (s, 3 H), 3.83 (s, 3 H), 3.81 (s, 3 H), 2.16 (q, J ) 7.3 Hz, 2
(
404 mg, 1.76 mmol) in DMF (4 mL) was stirred at ambient
temperature for 16 h. The solution was poured into iced water
14 mL) and extracted with EtOAc (4 × 10 mL). The organic
layers were pooled, dried with Na SO , and evaporated, and
the residue was purified by flash chromatography on silica gel
(
2
4

Extension of the Polyanionic Cosalane Pharmacophore
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1773
H), 1.94 (d, J ) 12.0 Hz, 1 H), 1.88-1.71 (m, 1 H), 1.71-0.95
was purified by flash chromatography on silica gel (100 g),
eluent hexane/ethyl acetate, 2:1, to give a pale-yellow foamy
solid (491 mg, 94%): mp (starts softening at 65 °C) 102-103
(m, 32 H), 0.89 (d, J ) 6.6 Hz, 3 H), 0.86 (d, J ) 6.6 Hz, 6 H),
0
1
1
1
5
3
2
.72 (s, 3 H), 0.63 (s, 3 H); 13C NMR (75 MHz, CDCl
3
) δ 167.0,
65.2, 164.8, 153.9, 153.6, 140.0, 139.8, 139.0, 137.3, 136.8,
35.9, 135.3, 134.1, 132.6, 132.3, 131.4, 130.8, 130.4, 130.1,
29.8, 128.4, 128.3, 127.7, 127.2, 127.1, 126.8, 73.9, 73.8, 65.6,
6.6, 56.3, 54.7, 52.1, 51.9, 46.6, 42.6, 40.2, 39.5, 38.6, 37.6,
7.3, 36.2, 36.1, 35.8, 35.6, 32.1, 29.0, 28.9, 28.3, 28.0, 27.4,
°C; IR (CHCl
3
) 3021, 2926, 2851, 1732, 1614, 1579, 1529, 1467,
1373, 1344, 1278, 1247, 1215, 1166, 1094, 1050, 1002, 896, 860,
-
1 1
754, 669 cm ; H NMR (300 MHz, CDCl ) δ 8.20-8.05 (m, 4
3
H), 8.02-7.94 (m, 2 H), 7.68 (tdd, J ) 7.7, 4.5, 1.0 Hz, 2 H),
7.65 (d, J ) 2.2 Hz, 1 H), 7.63 (d, J ) 2.4 Hz, 1 H), 7.55-7.36
(m, 10 H), 6.16 (t, J ) 7.6 Hz, 1 H), 5.63 (s, 2 H), 5.60 (s, 2 H),
5.53 (s, 2 H), 5.45 (s, 2 H), 2.17 (q, J ) 7.4 Hz, 2 H), 1.92 (d,
J ) 12.1 Hz, 1 H), 1.83-1.70 (m, 1 H), 1.70-0.91 (m, 32 H),
0.87 (d, J ) 6.5 Hz, 3 H), 0.84 (d, J ) 6.6 Hz, 6 H), 0.70 (s, 3
2 14
4.2, 23.9, 22.8, 22.6, 21.0, 18.7, 12.3, 12.1. Anal. (C81H92Cl O )
C, H.
3
′′,3′′′-Dich lor o-4′′,4′′′-bis(m -ca r boxyben zyloxy)-5′′,5′′′-
b is(m -ca r b oxyb en zyloxyca r b on yl)-4′,4′-d ip h en yl-3â-[1-
(
(
3′-bu ten yl)]ch olesta n e Tetr a m eth yl Ester (9b). Cosalane
1) (308 mg, 0.4 mmol), potassium carbonate (280 mg), and
H), 0.61 (s, 3 H); 13C NMR (75 MHz, CDCl
3
) δ 164.4, 164.2,
153.2, 146.3, 139.4, 136.4, 135.8, 134.8, 134.0, 133.7, 133.6,
133.0, 131.6, 131.4, 131.2, 130.2, 130.0, 129.4, 129.3, 129.1,
129.0, 128.7, 128.5, 128.0, 126.6, 126.3, 125.0, 124.7, 72.2, 64.1,
56.6, 56.3, 54.7, 46.6, 42.6, 40.1, 39.5, 38.6, 37.6, 37.2, 36.2,
36.1, 35.8, 35.6, 32.2, 29.1, 28.9, 28.3, 28.0, 27.4, 24.2, 23.9,
22.8, 22.6, 21.0, 18.7, 12.3, 12.1. Anal. (C H Cl N O ) C, H,
methyl 3-(bromomethyl)benzoate (8b) (403 mg, 1.76 mmol) in
DMF (4 mL) were stirred at room temperature for 16 h. The
solution was poured into iced water (14 mL) and extracted with
EtOAc (4 × 10 mL). The organic layers were pooled, dried with
2 4
Na SO , and evaporated, and the residue was purified by flash
7
3
80
2
4
14
chromatography on silica gel (40 g), eluting with hexanes-
ethyl acetate (9:4), to give a white foamy solid (456 mg, 84%):
N.
5
r-3â-[4,4-(3′,3′′-Bis(ca r bo-m -n itr oben zyloxy)-5′,5′′-d i-
mp (softens at 43 °C) 92-94 °C; IR (CHCl
3
) 3020, 2951, 2926,
850, 1722, 1593, 1468, 1450, 1437, 1372, 1289, 1212, 1167,
109, 1088, 973, 752, 668 cm-1; 1H NMR (300 MHz, CDCl
) δ
ch lor o-4′,4′′-bis(m -n itr oben zyloxy)d ip h en yl)bu ten -3-yl]-
ch olesta n e (9e). Cosalane (1) (308 mg, 0.4 mmol), potassium
carbonate (280 mg, 2.03 mmol), and m-nitrobenzyl bromide
2
1
8
4
3
.08 (d, J ) 13.4 Hz, 2 H), 8.04-7.91 (m, 6 H), 7.68-7.57 (m,
H), 7.57-7.47 (m, 2 H), 7.47-7.30 (m, 6 H), 6.12 (t, J ) 7.5
(
8e) (380 mg, 1.76 mmol) in DMF (3 mL) were stirred at room
temperature for 16 h. The solution was poured into iced water
14 mL) and extracted with EtOAc (4 × 10 mL). The organic
layers were pooled, dried with Na SO , and evaporated, and
the residue was purified by flash chromatography on silica gel
80 g), eluent hexane/ethyl acetate, 2:1, to give a white foamy
solid (507 mg, 97%): mp (starts softening at 53 °C) 128 °C; IR
CHCl ) 3028, 2927, 2851, 1731, 1532, 1470, 1352, 1247, 1195,
166, 1094, 1013, 980, 896, 806, 760, 753, 731, 691, 668 cm
Hz, 1 H), 5.35 (s, 2 H), 5.33 (s, 2 H), 5.14 (s, 2 H), 5.06 (s, 2 H),
(
3
.92 (s, 3 H), 3.91 (s, 3 H), 3.88 (s, 6 H), 2.11 (q, J ) 7.3 Hz, 2
H), 1.94 (d, J ) 12.0 Hz, 1 H), 1.87-1.70 (m, 1 H), 1.70-0.94
m, 32 H), 0.89 (d, J ) 6.5 Hz, 3 H), 0.85 (d, J ) 6.5 Hz, 6 H),
.71 (s, 3 H), 0.63 (s, 3 H); 13C NMR (75 MHz, CDCl
2
4
(
(
0
1
1
1
1
3
2
3
) δ 166.7,
66.4, 165.1, 164.8, 153.4, 153.0, 138.9, 137.0, 136.5, 136.0,
35.7, 135.3, 134.2, 132.6, 132.2, 131.3, 130.8, 130.5, 130.2,
30.0, 129.7, 129.5, 129.3, 129.2, 128.7, 128.6, 128.4, 128.3,
27.1, 126.9, 75.1, 66.7, 56.5, 56.3, 54.6, 52.0, 46.5, 42.5, 40.0,
9.5, 38.5, 37.5, 37.1, 36.1, 36.0, 35.7, 35.5, 32.1, 28.9, 28.8,
8.2, 27.9, 27.3, 24.1, 23.8, 22.7, 22.5, 21.0, 18.6, 12.2, 12.0.
(
3
-
1
1
;
1
H NMR (300 MHz, CDCl ) δ 8.32 (s, 1 H), 8.28 (s, 1 H), 8.22-
3
8
2
.15 (m, 4 H), 8.15 (s, 1 H), 8.12 (s, 1 H), 7.79 (t, J ) 8.2 Hz,
H), 7.68 (t, J ) 7.8 Hz, 2 H), 7.62 (t apparent, J ) 2.5 Hz, 2
H), 7.59-7.44 (m, 4 H), 7.44 (d, J ) 2.2 Hz, 1 H), 7.42 (d, J )
.4 Hz, 1 H), 6.16 (t, J ) 7.6 Hz, 1 H), 5.41 (s, 2 H), 5.40 (s, 2
H), 5.22 (s, 2 H), 5.14 (s, 2 H), 2.13 (q, J ) 7.5 Hz, 2 H), 1.95
d, J ) 12.1 Hz, 1 H), 1.87-1.70 (m, 1 H), 1.70-0.92 (m, 32
Anal. (C81
2
H92Cl O14) C, H.
2
3
′′,3′′′-Dich lor o-4′′,4′′′-b is(p -ca r b oxyb en zyloxy)-5′′,5′′′-
bis(p-ca r boxyben zyloxyca r bon yl)-4′,4′-d ip h en yl-3â-[1-(3′-
bu ten yl)]ch olesta n e Tetr a m eth yl Ester (9c). Cosalane (1)
(
H), 0.89 (d, J ) 6.5 Hz, 3 H), 0.86 (d, J ) 6.7 Hz, 3 H), 0.86 (d,
(616 mg, 0.8 mmol), potassium carbonate (560 mg), and
J ) 6.6 Hz, 3 H), 0.71 (s, 3 H), 0.63 (s, 3 H); 13C NMR (75
p-carboxymethylbenzyl bromide (8c) (806 mg, 3.52 mmol) in
DMF (8 mL) were stirred at room temperature for 24 h. The
solution was poured into iced water (30 mL) and extracted with
EtOAc (4 × 20 mL). The organic layers were pooled, dried with
MHz, CDCl
3
) δ 164.7, 164.4, 153.3, 153.0, 148.4, 139.3, 138.7,
1
1
1
3
2
37.4, 136.4, 136.3, 135.8, 134.9, 134.0, 133.5, 133.4, 133.0,
31.4, 130.2, 129.9, 129.7, 129.4, 128.5, 126.7, 126.5, 123.4,
23.1, 122.9, 122.5, 74.2, 65.9, 56.6, 56.4, 54.7, 46.6, 42.6, 40.1,
9.5, 38.6, 37.6, 37.2, 36.2, 36.1, 35.8, 35.6, 32.2, 29.1, 28.9,
8.2, 28.0, 27.4, 24.2, 23.9, 22.8, 22.6, 21.0, 18.7, 12.3, 12.1.
2 4
Na SO , and evaporated, and the residue was purified by flash
chromatography on silica gel (120 g), eluting with hexanes-
ethyl acetate (2:1), to give a white foamy solid (1.05 g, 96%):
Anal. (C73
2 4
H80Cl N O14) C, H, N.
mp (starts softening at 59 °C) 132 °C; IR (CHCl
3
) 3027, 2953,
5
r-3â-[4,4-(3′,3′′-Bis(car bo-p-n itr oben zyloxy)-5′,5′′-dich lo-
2
1
929, 2850, 1722, 1616, 1470, 1437, 1417, 1374, 1283, 1194,
179, 1168, 1111, 1021, 970 cm-1; H NMR (300 MHz, CDCl
1
r o-4′,4′′-bis(p-n itr oben zyloxy)diph en yl)bu ten -3-yl]ch oles-
ta n e (9f). Cosalane (1) (230 mg, 0.3 mmol), potassium
carbonate (210 mg), and p-nitrobenzyl bromide (8f) (285 mg,
1.32 mmol) in DMF (2 mL) were stirred at room temperature
for 16 h. The solution was poured into iced water (10 mL) and
extracted with EtOAc (3 × 10 mL). The organic layers were
3
)
δ 8.02 (d, J ) 6.5 Hz, 2 H), 7.99 (d, J ) 6.5 Hz, 2 H), 7.94 (d,
J ) 7.8 Hz, 4 H), 7.61-7.56 (m, 2 H), 7.52-7.42 (m, 4 H), 7.42-
7
2
3
.33 (m, 6 H), 6.13 (t, J ) 7.6 Hz, 1 H), 5.34 (s, 2 H), 5.32 (s,
H), 5.15 (s, 2 H), 5.07 (s, 2 H), 3.94 (s, 3 H), 3.93 (s, 3 H),
.91 (s, 3 H), 3.89 (s, 3 H), 2.09 (q, J ) 7.6 Hz, 2 H), 1.95 (d,
J ) 12.0 Hz, 1 H), 1.88-1.70 (m, 1 H), 1.70-0.92 (m, 32 H),
0
H), 0.64 (s, 3 H); C NMR (75 MHz, CDCl
1
1
1
4
3
1
2 4
pooled, dried with Na SO , and evaporated, and the residue
.90 (d, J ) 6.5 Hz, 3 H), 0.86 (d, J ) 6.6 Hz, 6 H), 0.71 (s, 3
was purified by flash chromatography on silica gel (60 g),
eluent hexane/ethyl acetate, 2:1, to give a white foamy solid
(389 mg, 99%): mp (starts softening at 71 °C) 106-109 °C; IR
1
3
3
) δ 166.7, 166.4,
65.0, 164.7, 153.4, 153.1, 141.6, 140.2, 139.0, 136.5, 136.0,
35.4, 134.3, 132.6, 131.3, 130.1, 129.8, 129.7, 129.6, 128.3,
27.8, 127.4, 127.0, 126.8, 75.1, 66.6, 56.6, 56.3, 54.6, 52.1, 52.0,
6.5, 42.6, 40.1, 39.5, 38.5, 37.5, 37.2, 36.2, 36.0, 35.8, 35.5,
2.1, 29.0, 28.8, 28.2, 28.0, 27.3, 24.2, 23.8, 22.7, 22.5, 21.0,
(CHCl
3
) 3113, 3082, 3031, 2926, 2852, 1732, 1608, 1523, 1496,
1470, 1404, 1348, 1281, 1248, 1195, 1166, 1110, 1092, 1014,
-
1 1
857 cm ; H NMR (300 MHz, CDCl ) δ 8.22 (d, J ) 7.8 Hz, 2
3
H), 8.20 (d, J ) 7.8 Hz, 2 H), 8.13 (d, J ) 8.4 Hz, 4 H), 7.69-
7.40 (m, 12 H), 6.18 (t, J ) 7.6 Hz, 1 H), 5.40 (s, 2 H), 5.37 (s,
2 H), 5.24 (s, 2 H), 5.15 (s, 2 H), 2.14 (q, J ) 7.2 Hz, 2 H), 1.94
(d, J ) 12.1 Hz, 1 H), 1.87-1.70 (m, 1 H), 1.70-0.92 (m, 32
H), 0.88 (d, J ) 6.5 Hz, 3 H), 0.85 (d, J ) 6.5 Hz, 6 H), 0.71 (s,
8.6, 12.3, 12.0. Anal. (C81
2
H92Cl O14) C, H.
5
r-3â-[4,4-(3′,3′′-Bis(car bo-o-n itr oben zyloxy)-5′,5′′-dich lo-
r o-4′,4′′-bis(o-n itr oben zyloxy)diph en yl)bu ten -3-yl]ch oles-
ta n e (9d ). Cosalane (1) (308 mg, 0.4 mmol), potassium
carbonate (280 mg), and o-nitrobenzyl bromide (8d ) (380 mg,
3 H), 0.63 (s, 3 H); 13C NMR (75 MHz, CDCl
) δ 164.3, 164.1,
3
1
.76 mmol) in DMF (3 mL) were stirred at room temperature
153.3, 153.0, 147.7, 147.6, 143.7, 142.4, 142.3, 139.2, 136.3,
136.1, 135.8, 134.7, 132.8, 131.3, 130.2, 129.9, 128.4, 128.3,
128.0, 126.5, 126.2, 123.8, 123.6, 123.5, 74.3, 65.8, 65.7, 56.5,
56.2, 54.6, 46.5, 42.5, 40.0, 39.4, 38.5, 37.5, 37.2, 36.1, 36.0,
for 16 h. The solution was poured into iced water (20 mL) and
extracted with EtOAc (3 × 20 mL). The organic layers were
2 4
pooled, dried with Na SO , and evaporated, and the residue

1
774 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Cushman et al.
3
2
N.
5.7, 35.5, 35.4, 32.1, 29.0, 28.8, 28.2, 27.9, 27.4, 24.1, 23.8,
96%): mp 223-225 °C; IR (CHCl
3
) 3373, 3019, 2918, 2849,
2660, 2556, 1694, 1615, 1597, 1580, 1556, 1514, 1471, 1422,
1381, 1314, 1288, 1250, 1214, 1179, 1127, 1103, 1045, 973, 959,
2.8, 22.5, 21.0, 18.6, 12.2, 12.0. Anal. (C73
H
80Cl
2
N
4
O
14) C, H,
-
1 1
7
8
79, 746, 669 cm ; H NMR (300 MHz, CD
3 3
COCD ) δ 8.13-
3
′′,3′′′-Dich lor o-4′′,4′′′-b is(o-ca r b oxyb en zyloxy)-5′′,5′′′-
d ica r b oxy-4′,4′-d ip h en yl-3â-[1-(3′-b u t en yl)]ch olest a n e
10a ). Hexaester 9a (402 mg, 0.3 mmol), K CO (1.55 g), and
KCN (12 mg) were suspended in ethanol (10 mL) and water
2 mL). The mixture was heated on an oil bath (80 °C) for 8 h.
.02 (m, 4 H), 7.75-7.62 (m, 6 H), 7.59-7.54 (m, 2 H), 6.35 (t,
J ) 7.6 Hz, 1 H), 5.29 (s, 2 H), 5.22 (s, 2 H), 2.22 (q, J ) 7.2
(
2
3
Hz, 2 H), 1.98 (d, J ) 13.7 Hz, 1 H), 1.89-1.74 (m, 1 H), 1.74-
0
6
.96 (m, 32 H), 0.93 (d, J ) 8.3 Hz, 3 H), 0.87 (d, J ) 6.7 Hz,
H), 0.76 (s, 3 H), 0.68 (s, 3 H); 13C NMR (75 MHz, CD
COCD )
3
(
Ethanol was distilled off, water (10 mL) was added, and the
mixture was stirred at 80 °C for 2 h. After cooling to room
temperature, the aqueous solution was washed with ethyl
acetate (2 × 10 mL), acidified with 1 N HCl, and extracted
with ethyl acetate (3 × 15 mL). The organic layers were pooled,
3
δ 167.5, 166.3, 154.2, 153.8, 143.1, 139.9, 137.8, 137.1, 135.8,
1
7
3
2
3
34.8, 132.7, 132.4, 131.2, 130.5, 130.2, 129.3, 129.0, 128.8,
5.9, 62.4, 57.5, 57.2, 55.6, 47.4, 43.4, 41.0, 40.2, 39.4, 38.2,
7.7, 37.0, 36.8, 36.6, 36.4, 36.3, 32.9, 28.9, 28.6, 27.9, 24.8,
4.5, 23.0, 22.8, 21.7, 19.1, 12.7, 12.4. Anal. (C61
O) C, H.
5r-3â-[4,4-(3′,3′′-Dica r boxy-5′,5′′-d ich lor o-4′,4′′-bis(o-n i-
tr oben zyloxy)d ip h en yl)bu ten -3-yl]ch olesta n e (10d ). Di-
ester 9d (392 mg, 0.3 mmol) and K CO (720 mg, 5.22 mmol)
2 10
H72Cl O ‚
2 4
dried over Na SO , concentrated by evaporation, and purified
H
2
by precipitation from acetone to afford a white solid (98 mg,
3
2
1
3
2%): mp 217-220 °C; IR (CHCl ) 3021, 2917, 2840, 2657,
549, 1683, 1598, 1580, 1468, 1444, 1424, 1369, 1268, 1216,
151, 1106, 1004, 928, 906, 854, 771, 670 cm-1; 1H NMR (300
2
3
MHz, C
5
D
5
N) δ 8.54 (apparent t, J ) 6.5 Hz, 2 H), 8.41 (dd, J
were suspended in ethanol (6 mL) and water (1.2 mL). The
mixture was heated on an oil bath (105-110 °C) for 8 h. The
reaction mixture was concentrated to remove ethanol. KOH
(500 mg) was added, and the alkaline solution was washed
)
6.6, 5.4 Hz, 2 H), 8.26 (d, J ) 2.2 Hz, 1 H), 8.24 (d, J ) 2.1
Hz, 1 H), 7.82 (d, J ) 2.2 Hz, 1 H), 7.73 (d, J ) 2.3 Hz, 1 H),
7
2
.68-7.60 (m, 2 H), 7.40 (t apparent, J ) 7.6 Hz, 2 H), 6.39 (s,
H), 6.36 (s, 2 H), 6.34 (t, J ) 7.2 Hz, 1 H), 2.29 (q, J ) 7.2
2 2
with CH Cl (5 mL) and acidified with concentrated HCl. The
Hz, 2 H), 1.91 (d, J ) 12.1 Hz, 1 H), 1.86-1.69 (m, 1 H), 1.69-
acidified suspension was extracted with ethyl acetate (3 × 20
0
6
1
1
1
1
3
2
.98 (m, 32 H), 0.94 (d, J ) 6.4 Hz, 3 H), 0.88 (d, J ) 6.6 Hz,
mL). The organic layer was separated, dried over Na SO , and
2
4
H), 0.71 (s, 3 H), 0.62 (s, 3 H); 13C NMR (75 MHz, C
D
N) δ
5
5
2 2 2
chromatographed (SiO , 80 g; hexanes:ethyl acetate:CH Cl ,
69.8, 169.7, 168.0, 154.5, 154.2, 149.2, 142.3, 140.9, 139.4,
38.5, 137.9, 136.5, 136.2, 134.8, 134.2, 133.4, 132.4, 132.1,
31.9, 131.2, 130.4, 130.3, 130.2, 130.0, 129.7, 128.9, 127.8,
27.2, 124.2, 122.8, 75.1, 75.0, 56.7, 56.6, 54.8, 46.7, 42.8, 40.4,
9.8, 38.8, 37.6, 36.5, 36.3, 36.1, 35.9, 35.7, 32.4, 29.3, 28.5,
8.3, 27.7, 24.4, 24.2, 23.0, 22.7, 21.3, 18.9, 12.5, 12.3. Anal.
1:1:1 containing 1% acetic acid) to afford a pale-yellow solid
(228 mg, 73%): mp (starts softening at 109 °C) 128-136 °C;
IR (KBr) 3071, 2924, 2851, 2656, 1701, 1611, 1596, 1570, 1528,
1466, 1448, 1376, 1341, 1275, 1239, 1167, 1102, 1049, 1000,
-1 1
896, 859, 789, 727, 679 cm ; H NMR (300 MHz, C D ) δ 8.10-
6
6
7.95 (m, 2 H), 7.90 (d, J ) 1.8 Hz, 1 H), 7.84 (dd, J ) 4.2, 2.2
Hz, 1 H), 7.75 (td, J ) 9.3, 4.2 Hz, 2 H), 7.49 (d, J ) 2.1 Hz,
1 H), 7.43 (dd, J ) 3.3, 2.0 Hz, 1 H), 7.11 (t, J ) 8.4 Hz, 2 H),
6.79 (q apparent, J ) 7.4 Hz, 2 H), 5.96 (q apparent, J ) 7.3
Hz, 1 H), 5.62 (s, 2 H), 5.59 (s, 2 H), 2.15-1.96 (m, 2 H), 1.94-
1.78 (m, 1 H), 1.78-1.65 (m, 2 H), 1.65-0.83 (m, 31 H), 1.02
(d, J ) 6.2 Hz, 3 H), 0.93 (d, J ) 6.6 Hz, 6 H), 0.79 (s, 3 H),
(C
61
H
72Cl
2
O
10‚4H
2
O) C, H.
′′,3′′′-Dich lor o-4′′,4′′′-bis(m -ca r boxyben zyloxy)-5′′,5′′′-
d ica r b oxy-4′,4′-d ip h en yl-3â-[1-(3′-b u t en yl)]ch olest a n e
10b). Hexaester 9b (276 mg, 0.203 mmol), KCN (7.9 mg), and
CO (1.06 g) were suspended in ethanol (7 mL) and water
1.4 mL). The mixture was heated on an oil bath at 80 °C for
h. Ethanol was distilled off, water (7 mL) was added, and
3
(
K
2
3
(
8
0.71 (s, 3 H); 13C NMR (75 MHz, C
6 6
D ) δ 169.5, 169.4, 154.7,
the mixture was stirred at 80 °C for 2 h. After cooling to room
temperature, the aqueous solution was washed with ethyl
acetate (2 × 10 mL), acidified with 1 N HCl, and extracted
with ethyl acetate (3 × 30 mL). The organic layers were pooled,
154.3, 147.0, 139.3, 136.8, 136.6, 136.5, 134.8, 133.8, 133.5,
132.7, 130.7, 130.5, 129.7, 129.1, 129.0, 126.2, 125.9, 124.7,
72.6, 56.9, 56.8, 55.0, 46.8, 43.0, 40.5, 39.9, 38.9, 37.8, 37.5,
36.7, 36.4, 36.3, 36.0, 35.9, 32.5, 30.2, 29.4, 28.7, 28.4, 27.6,
dried over Na
2
SO
4
, concentrated by evaporation, and purified
24.6, 24.4, 23.0 22.8, 21.5, 19.0, 12.6, 12.4. Anal. (C59
Cl 10) C, H, N.
70
H -
by precipitation from acetone to afford a white solid (125 mg,
2 2
N O
6
2
1
0%): mp (softens at 201-203 °C) 217-219 °C; IR (CHCl
3
)
5r-3â-[4,4-(3′,3′′-Dica r boxy-5′,5′′-d ich lor o-4′,4′′-bis(m -n i-
923, 2849, 2643, 1693, 1594, 1552, 1466, 1418, 1380, 1283,
tr oben zyloxy)d ip h en yl)bu ten -3-yl]ch olesta n e (10e). Di-
ester 9e (392 mg, 0.3 mmol) and K CO (720 mg, 5.2 mmol)
-1 1
238, 1216, 1170, 1104, 980, 934, 912, 816, 753 cm ; H NMR
N) δ 8.95-8.88 (m, 2 H), 8.40 (d, J ) 7.0 Hz,
2
3
(300 MHz, C
5
D
5
were suspended in ethanol (6 mL) and water (1.2 mL). The
mixture was heated on an oil bath (105-110 °C) for 8 h. KOH
(450 mg) was added, and the alkaline solution was washed
with CH Cl (15 mL) and acidified with concentrated HCl (3
2
H), 8.26 (d, J ) 2.1 Hz, 1 H), 8.25 (d, J ) 2.0 Hz, 1 H), 8.02
(
(
1
t apparent, J ) 6.9 Hz, 2 H), 7.82 (d, J ) 2.0 Hz, 1 H), 7.79
d, J ) 2.3 Hz, 1 H), 7.52-7.43 (m, 2 H), 6.35 (t, J ) 7.5 Hz,
2
2
H), 5.52 (s, 2 H), 5.49 (s, 2 H), 2.31 (q, J ) 7.4 Hz, 2 H), 1.93
mL). The acidified suspension was extracted with ethyl acetate
(
d, J ) 12.0 Hz, 1 H), 1.88-1.69 (m, 1 H), 1.69-0.98 (m, 32
(2 × 40 mL). The organic layer was separated, dried over Na -
2
H), 0.95 (d, J ) 6.3 Hz, 3 H), 0.88 (d, J ) 6.6 Hz, 6 H), 0.73 (s,
SO , and chromatographed (SiO , 150 g; hexanes:ethyl acetate:
4
2
1
3
3
1
1
7
3
2
2
5 5
H), 0.63 (s, 3 H); C NMR (75 MHz, C D N) δ 169.0, 168.3,
CH
2
Cl
2
, 1:1:1 containing 1% acetic acid) to afford a white foamy
68.0, 139.6, 138.4, 137.8, 136.7, 134.9, 134.3, 132.9, 132.7,
32.2, 132.0, 130.3, 130.2, 129.9, 129.1, 128.8, 122.8, 75.9, 75.8,
5.6, 56.7, 56.5, 54.8, 46.6, 42.8, 40.3, 39.7, 38.8, 37.7, 37.5,
6.5, 36.3, 36.1, 35.8, 35.7, 32.4, 29.2, 28.5, 28.3, 27.7, 24.4,
solid (280 mg, 90%): mp (starts softening at 102 °C) 126-128
C; IR (CHCl ) 3076, 3022, 2926, 2851, 2644, 2565, 1698, 1621,
595, 1556, 1531, 1470, 1444, 1380, 1352, 1248, 1221, 1098,
°
1
9
3
-1
1
80, 930, 898, 859, 807, 775, 732, 690, 669 cm ; H NMR (300
) δ 8.43 (d, J ) 8.1 Hz, 2 H), 8.19 (d, J ) 8.0 Hz,
H), 7.86 (dd, J ) 7.67, 4.2 Hz, 2 H), 7.72 (d, J ) 2.1 Hz, 1
4.2, 22.9, 22.7, 21.3, 18.9, 12.5, 12.3. Anal. (C61
O) C, H.
′′,3′′′-Dich lor o-4′′,4′′′-b is(p -ca r b oxyb en zyloxy)-5′′,5′′′-
d ica r b oxy-4′,4′-d ip h en yl-3â-[1-(3′-b u t en yl)]ch olest a n e
10c). Hexaester 9c (1.05 g, 0.77 mmol), KCN (30 mg), and
CO (4.02 g) were suspended in ethanol (28 mL) and water
6 mL). The mixture was heated on an oil bath (80 °C) for 8 h.
2 10
H72Cl O ‚
MHz, CDCl
3
H
2
2
3
H), 7.69 (d, J ) 2.4 Hz, 1 H), 7.55 (td, J ) 8.0, 2.8 Hz, 2 H),
7.51-7.44 (m, 2 H), 6.18 (t, J ) 7.6 Hz, 1 H), 5.29 (s, 2 H),
5.21 (s, 2 H), 2.14 (q, J ) 7.2 Hz, 2 H), 1.92 (d, J ) 11.9 Hz, 1
H), 1.85-1.68 (m, 1 H), 1.68-0.91 (m, 32 H), 0.88 (d over-
lapped, 3 H), 0.86 (d, J ) 6.5 Hz, 3 H), 0.85 (d, J ) 6.5 Hz, 3
(
K
(
2
3
Ethanol was distilled off, and water (20 mL) was added. The
aqueous solution was stirred at 80 °C for 2 h. After cooling to
room temperature and washing with ethyl acetate (2 × 15 mL),
the mixture was acidified with 1 N HCl and extracted with
ethyl acetate (3 × 15 mL). The organic layers were pooled,
H), 0.71 (s, 3 H), 0.62 (s, 3 H); 13C NMR (75 MHz, CDCl
3
) δ
168.3, 168.2, 153.7, 153.4, 148.6, 139.6, 138.4, 138.3, 136.7,
136.6, 136.4, 135.3, 134.3, 134.2, 133.8, 132.5, 130.2, 129.9,
129.7, 129.5, 125.9, 125.5, 123.5, 123.4, 123.3, 74.9, 56.8, 56.6,
54.9, 46.8, 42.8, 40.3, 39.7, 38.7, 37.7, 37.3, 36.4, 36.3, 36.0,
35.8, 32.3, 29.2, 29.1, 28.4, 28.2, 27.5, 24.4, 24.1, 22.9, 22.7,
2 4
dried over Na SO , concentrated by evaporation, and purified
by precipitation from acetone to afford a white solid (762 mg,
2 2
21.2, 18.8, 12.5, 12.2. Anal. (C59H70Cl N O10) C, H, N.

Extension of the Polyanionic Cosalane Pharmacophore
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1775
5
r-3â-[4,4-(3′,3′′-Dica r boxy-5′,5′′-d ich lor o-4′,4′′-bis(p-n i-
tr oben zyloxy)d ip h en yl)bu ten -3-yl]ch olesta n e (10f). Di-
ester 9f (130 mg, 0.1 mmol) and K CO (240 mg, 1.74 mmol)
r a sod iu m Sa lt (11c). Tetraacid 10c (100 mg, 0.097 mmol)
was dissolved in a mixture of methanol (2 mL) and a 1.016 N
2
3
solution of Na
concentrated and dried in vacuo to yield a white solid (112
mg, 93%): mp > 300 °C; IR (CHCl ) 3390, 3020, 2924, 2849,
2400, 1600, 1564, 1556, 1463, 1455, 1384, 1284, 1214, 1102,
2 3
CO (0.38 mL). The resulting solution was
were suspended in ethanol (2 mL) and water (0.4 mL). The
mixture was heated on an oil bath (105-110 °C) for 6-8 h.
The reaction mixture was concentrated to remove ethanol.
KOH (150 mg) was added, and the alkaline solution was
washed with CH
HCl (1 mL). The acidified suspension was extracted with ethyl
3
-
1
1
972, 957, 887, 844, 780, 741, 669 cm ; H NMR (300 MHz,
CD OD) δ 7.96 (d, J ) 8.1 Hz, 2 H), 7.94 (d, J ) 8.2 Hz, 2 H),
2
Cl
2
(5 mL) and acidified with concentrated
3
7.58 (d, J ) 8.4 Hz, 2 H), 7.55 (d, J ) 8.3 Hz, 2 H), 7.36 (d, J
) 2.3 Hz, 1 H), 7.21 (d, J ) 2.1 Hz, 1 H), 7.09 (d, J ) 2.1 Hz,
1 H), 7.04 (d, J ) 2.3 Hz, 1 H), 6.08 (t, J ) 7.5 Hz, 1 H), 5.20
(s, 2 H), 5.13 (s, 2 H), 2.17 (dd, J ) 7.4, 7.0 Hz, 2 H), 1.98 (d,
J ) 12.2 Hz, 1 H), 1.90-1.73 (m, 1 H), 1.73-0.94 (m, 32 H),
acetate (2 × 10 mL). The organic layer was separated, dried
2 4 2
over Na SO , and chromatographed (SiO , 15 g; hexanes:ethyl
acetate:CH
white foamy solid (80 mg, 77%): mp (starts softening at 110
) 3032, 2926, 2852, 1698, 1608,
2 2
Cl , 1:1:1 containing 1% acetic acid) to afford a
°
1
C) 179-182 °C; IR (CHCl
3
0.91 (d, J ) 6.4 Hz, 3 H), 0.87 (d, J ) 6.6 Hz, 6 H), 0.75 (s, 3
-
1
1
13
558, 1524, 1470, 1348, 1249, 1178, 1109, 1013, 856 cm ; H
3
H), 0.67 (s, 3 H); C NMR (75 MHz, CD OD) δ 175.4, 175.0,
NMR (300 MHz, CDCl
3
) δ 8.26-8.17 (m, 4 H), 7.74-7.65 (m,
174.8, 151.8, 151.6, 140.9, 140.4, 139.9, 138.8, 138.6, 138.3,
137.4, 133.2, 131.8, 130.2, 129.7, 129.3, 129.1, 128.9, 128.6,
126.5, 76.6, 76.5, 58.0, 57.7, 56.2, 48.0, 43.8, 41.5, 40.7, 39.9,
38.8, 38.5, 37.4, 37.2, 37.1, 37.0, 36.8, 33.4, 30.2, 30.1, 29.3,
29.1, 28.2, 25.2, 25.0, 23.2, 22.9, 22.1, 19.2, 12.8, 12.5. Anal.
6
H), 7.48 (d, J ) 2.3 Hz, 1 H), 7.47 (d, J ) 2.5 Hz, 1 H), 6.18
t, J ) 7.6 Hz, 1 H), 5.29 (s, 2 H), 5.21 (s, 2 H), 2.14 (q, J ) 7.2
Hz, 2 H), 1.93 (d, J ) 12.0 Hz, 1 H), 1.85-1.70 (m, 1 H), 1.70-
.92 (m, 32 H), 0.87 (d, J ) 6.5 Hz, 3 H), 0.85 (d, J ) 6.5 Hz,
H), 0.72 (s, 3 H), 0.62 (s, 3 H); C NMR (75 MHz, CDCl
69.3, 153.8, 153.4, 147.8, 143.5, 143.4, 139.4, 136.5, 136.4,
36.1, 135.1, 133.7, 132.2, 130.2, 129.9, 129.2, 128.5, 128.4,
25.6, 125.3, 123.7, 74.6, 56.6, 56.3, 54.7, 46.6, 42.6, 40.1, 39.5,
8.5, 37.5, 37.1, 36.2, 36.1, 35.8, 35.5, 32.1, 29.7, 29.0, 28.9,
8.2, 28.0, 27.4, 24.2, 23.9 22.8, 22.5, 21.0, 18.7, 12.3, 12.1.
(
0
6
1
1
1
3
2
13
3
) δ
(C61
H
68Cl
2
Na
4
O
2
10‚7H O) C, H.
5
r-3â-[4,4-(3′,3′′-Dica r boxy-5′,5′′-d ich lor o-4′,4′′-bis(o-n i-
tr oben zyloxy)d ip h en yl)bu ten -3-yl]ch olesta n e Disod iu m
Sa lt (11d ). Diacid 5d (44 mg, 0.042 mmol) was dissolved in a
mixture of ethanol (2 mL) and a 1.016 N solution of Na
2 3
CO
(0.083 mL). The resulting solution was concentrated and dried
Anal. (C59
H
70Cl
2
N
2
O
10‚2H
2
O) C, H. N.
in vacuo to yield a pale-yellow foam (47 mg, 98%): mp (darkens
above 196 °C) 241 °C with dec; IR (KBr) 3423, 2925, 2851,
1608, 1580, 1527, 1462, 1410, 1376, 1344, 1304, 1287, 1228,
3
′′,3′′′-Dich lor o-4′′,4′′′-b is(o-ca r b oxyb en zyloxy)-5′′,5′′′-
d i-o-ca r b oxy-4′,4′-d ip h e n yl-3â-[1-(3′-b u t e n yl)]ch ole s-
ta n e Tetr a sod iu m Sa lt (11a ). Tetraacid 10a (36 mg, 0.034
mmol) was dissolved in a mixture of ethanol (2 mL) and a 1.016
N solution of Na
concentrated and dried in vacuo to yield a white solid (39 mg,
-1 1
1102, 1049, 1003, 886, 861, 816, 789 cm ; H NMR (300 MHz,
CD OD) δ 8.26 (d, J ) 7.9 Hz, 1 H), 8.22 (d, J ) 7.9 Hz, 1 H),
3
2
CO
3
(0.135 mL). The resulting solution was
8.12 (dd, J ) 8.1, 1.1 Hz, 1 H), 8.11 (dd, J ) 8.1, 1.1 Hz, 1 H),
7.76 (ddd ABX pattern, J AB ) 7.6 Hz, J AB ) 5.2 Hz, J AX ) 1.1
Hz, 2 H), 7.53 (tdd, J ) 7.8, 3.5, 1.2 Hz, 2 H), 7.42 (d, J ) 2.2
Hz, 1 H), 7.25 (d, J ) 2.1 Hz, 1 H), 7.11 (d, J ) 2.1 Hz, 1 H),
7.04 (d, J ) 2.3 Hz, 1 H), 6.12 (t, J ) 7.5 Hz, 1 H), 5.58 (s, 2
H), 5.51 (s, 2 H), 2.18 (dd, J ) 7.2, 6.7 Hz, 2 H), 1.97 (d, J )
12.1 Hz, 1 H), 1.88-1.72 (m, 1 H), 1.72-0.94 (m, 32 H), 0.91
(d, J ) 6.4 Hz, 3 H), 0.87 (d, J ) 6.5 Hz, 3 H), 0.87 (d, J ) 6.6
1
2
9
7
00%): mp (darkens above 200 °C) >300 °C; IR (KBr) 3418,
924, 2845, 1570, 1462, 1381, 1281, 1264, 1241, 1212, 1097,
46, 888, 810, 744, 664 cm-1; H NMR (300 MHz, CD
1
OD) δ
3
.93-7.80 (m, 2 H), 7.63 (dd, J ) 7.4, 6.6 Hz, 2 H), 7.42-7.16
(
6
)
m, 6 H), 7.08 (d, J ) 1.7 Hz, 1 H), 7.36 (d, J ) 2.1 Hz, 1 H),
.07 (t, J ) 7.4 Hz, 1 H), 5.49 (s, 2 H), 5.43 (s, 2 H), 2.17 (q, J
7.0 Hz, 2 H), 1.97 (d, J ) 12.2 Hz, 1 H), 1.90-1.73 (m, 1 H),
.73-0.94 (m, 32 H), 0.91 (d, J ) 6.4 Hz, 3 H), 0.87 (d, J ) 6.6
Hz, 3 H), 0.75 (s, 3 H), 0.66 (s, 3 H); 13C NMR (75 MHz, CD
-
3
1
OD) δ 174.7, 174.4, 151.6, 151.3, 148.3, 140.7, 139.7, 137.8,
135.5, 134.9, 133.5, 131.9, 130.5, 129.9, 129.5, 129.2, 128.7,
126.5, 125.4, 73.0, 58.0, 57.7, 56.2, 48.0, 43.8, 41.5, 40.7, 39.9,
38.8, 38.4, 37.4, 37.2, 37.1, 37.0, 36.8, 33.4, 30.2, 30.1, 29.3,
29.1, 28.2, 25.2, 25.0, 23.2, 22.9, 22.1, 19.2, 12.8, 12.5. Anal.
1
3
Hz, 6 H), 0.75 (s, 3 H), 0.66 (s, 3 H); C NMR (75 MHz, CD
OD) δ 177.7, 177.0, 174.9, 174.7, 152.4, 152.2, 140.2, 140.0,
38.5, 138.2, 137.3, 137.1, 134.9, 133.0, 132.6, 132.2, 131.9,
31.7, 129.9, 129.8, 129.6, 129.4, 129.1, 129.0, 127.9, 126.9,
26.7, 75.8, 75.7, 58.0, 57.7, 56.2, 43.8, 41.5, 40.7, 39.9, 38.9,
8.6, 38.5, 37.4, 37.2, 37.1, 37.0, 36.8, 36.7, 33.4, 30.2, 30.0,
9.3, 29.1, 28.2, 25.2, 24.9, 23.2, 22.9, 22.1, 19.2, 12.8, 12.5.
3
-
1
1
1
3
2
(C59
H
68Cl
2
N
2
Na
2
O
2
10‚3H O) C, H, N.
5
r-3â-[4,4-(3′,3′′-Dica r boxy-5′,5′′-d ich lor o-4′,4′′-bis(m -n i-
tr oben zyloxy)d ip h en yl)bu ten -3-yl]ch olesta n e Disod iu m
Anal. (C61
H
68Cl
2
Na
4
O
10‚5H
2
O) C, H.
Sa lt (11e). Diacid 10e (10 mg, 0.0096 mmol) was dissolved in
3
′′,3′′′-Dich lor o-4′′,4′′′-bis(m -ca r boxyben zyloxy)-5′′,5′′′-
a mixture of ethanol (0.5 mL) and a 1.016 N solution of Na
2
-
dicar boxy-4′,4′-diph en yl-3â-[1-(3′-bu ten yl)]ch olestan e Tet-
r a sod iu m Sa lt (11b). Tetraacid 10b (48 mg, 0.046 mmol) was
dissolved in a mixture of ethanol (2 mL) and a 1.016 N solution
CO (0.019 mL, 0.019 mmol). The resulting solution was
3
concentrated and dried in vacuo to yield a white solid (10.6
mg, 98%): mp (starts softening at 184 °C) dec at 252 °C; IR
(KBr) 3448, 2925, 2850, 1604, 1579, 1531, 1462, 1411, 1377,
of Na
and dried in vacuo to yield a white solid (56 mg, 98%): mp
) 3415, 2925, 2855,
719, 1600, 1570, 1459, 1450, 1404, 1377, 1286, 1232, 1212,
121, 1103, 966, 934, 905, 881, 813, 767, 742, 674, 645 cm-1
2 3
CO (0.18 mL). The resulting solution was concentrated
-
1
1352, 1285, 1237, 1100, 1002, 894, 807, 734, 693, 672 cm
;
1
(
1
darkens above 220 °C) >300 °C; IR (CHCl
3
3
H NMR (300 MHz, CD OD) δ 8.47 (s, 1 H), 8.44 (s, 1 H), 8.23-
8.13 (m, 2 H), 7.99 (d, J ) 8.2 Hz, 1 H), 7.96 (d, J ) 9.1 Hz, 1
H), 7.62 (t, J ) 7.9 Hz, 1 H), 7.60 (t, J ) 7.9 Hz, 1 H), 7.43 (d,
J ) 2.2 Hz, 1 H), 7.25 (d, J ) 2.0 Hz, 1 H), 7.09 (d, J ) 2.1 Hz,
1 H), 7.01 (d, J ) 2.2 Hz, 1 H), 6.11 (t, J ) 7.5 Hz, 1 H), 5.30
(s, 2 H), 5.22 (s, 2 H), 2.16 (dd, J ) 7.5, 7.1 Hz, 2 H), 1.97 (d,
J ) 12.2 Hz, 1 H), 1.88-1.72 (m, 1 H), 1.72-0.97 (m, 32 H),
0.91 (d, J ) 6.4 Hz, 3 H), 0.87 (d, J ) 6.6 Hz, 6 H), 0.75 (s, 3
1
;
1
H NMR (300 MHz, CD OD) δ 8.14 (s, 1 H), 8.10 (s, 1 H), 8.00-
7
7
)
2
3
.79 (m, 2 H), 7.70 (t, J ) 7.5 Hz, 2 H), 7.44-7.18 (m, 3 H),
.20 (d, J ) 2.0 Hz, 1 H), 7.08 (d, J ) 2.1 Hz, 1 H), 7.03 (d, J
2.2 Hz, 1 H), 6.08 (t, J ) 7.5 Hz, 1 H), 5.20 (s, 2 H), 5.13 (s,
H), 2.17 (q, J ) 6.7 Hz, 2 H), 1.97 (d, J ) 12.0 Hz, 1 H),
1
3
.90-1.74 (m, 1 H), 1.74-0.97 (m, 32 H), 0.91 (d, J ) 6.4 Hz,
H), 0.66 (s, 3 H); 13C NMR (75 MHz, CD
3
OD) δ 174.6, 174.4,
H), 0.87 (d, J ) 6.6 Hz, 6 H), 0.75 (s, 3 H), 0.66 (s, 3 H); 13
OD) δ 175.4, 175.0, 174.8, 174.7, 151.8,
C
151.5, 151.3, 141.3, 140.6, 139.7, 138.4, 138.2, 137.7, 135.4,
135.3, 133.5, 131.9, 130.5, 130.0, 129.5, 129.0, 128.8, 126.6,
123.8, 123.7, 123.6, 75.3, 58.0, 57.7, 56.2, 48.0, 43.8, 41.5, 40.7,
39.9, 38.8, 38.4, 37.4, 37.2, 37.1, 37.0, 36.7, 33.4, 30.2, 30.0,
29.3, 29.1, 28.2, 25.2, 24.9, 23.1, 22.9, 22.1, 19.2, 12.8, 12.5.
NMR (75 MHz, CD
3
1
1
1
3
2
51.6, 140.4, 139.9, 139.3, 138.7, 138.4, 138.3, 137.5, 133.2,
31.8, 131.6, 130.4, 130.3, 129.9, 129.7, 129.5, 129.4, 129.2,
29.0, 128.6, 126.5, 77.0, 58.0, 57.7, 56.2, 48.1, 43.8, 41.5, 40.7,
9.9, 38.9, 38.5, 37.4, 37.2, 37.1, 37.0, 36.8, 33.4, 30.2, 30.1,
9.3, 29.1, 28.2, 25.2, 25.0, 23.2, 22.9, 22.1, 19.2, 12.8, 12.5.
Anal. (C59
H
68Cl
2
N
2
Na
2
O
2
10‚2H O) C, H, N.
5
r-3â-[4,4-(3′,3′′-Dica r boxy-5′,5′′-d ich lor o-4′,4′′-bis(p-n i-
Anal. (C61
′′,3′′′-Dich lor o-4′′,4′′′-b is(p -ca r b oxyb en zyloxy)-5′′,5′′′-
dicar boxy-4′,4′-diph en yl-3â-[1-(3′-bu ten yl)]ch olestan e Tet-
H
68Cl
2
Na
4
O
10‚6H
2
O) C, N.
tr oben zyloxy)d ip h en yl)bu ten -3-yl]ch olesta n e Disod iu m
3
Sa lt (11f). Diacid (10f) (56 mg, 0.054 mmol) was dissolved in
a mixture of ethanol (2 mL) and a 1.016 N solution of Na CO
2 3

1
776 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Cushman et al.
(
0.106 mL). The resulting solution was concentrated and dried
in vacuo to yield a pale-yellow solid (60 mg, 97%): mp (starts
softening at 224 °C) 230 °C with dec; IR (CHCl ) 3419, 2926,
854, 1606, 1581, 1525, 1463, 1408, 1375, 1348, 1284, 1236,
Claes, Kristien Erven, and Erik Fonteyn for excellent
technical assistance.
3
2
1
Refer en ces
105, 1010, 887, 854, 739 cm-1; H NMR (300 MHz, CD
1
OD)
3
δ 8.24 (d, J ) 8.8 Hz, 2 H), 8.23 (d, J ) 8.8 Hz, 2 H), 7.81 (t,
apparent, J ) 8.4 Hz, 4 H), 7.37 (s br, 1H), 7.23 (d, J ) 2.1
Hz, 1 H), 7.08 (d, J ) 2.0 Hz, 1 H), 7.02 (s br, 1 H), 6.10 (t, J
(1) Golebiewski, W. M.; Bader, J . P.; Cushman, M. Design and
Synthesis of Cosalane, a Novel Anti-HIV Agent. Bioorg. Med.
Chem. Lett. 1993, 3, 1739-1742.
2) Cushman, M.; Golebiewski, W. M.; McMahon, J . B.; Buckheit,
R. W. J .; Clanton, D. J .; Weislow, O.; Haugwitz, R. D.; Bader,
J .; Graham, L.; Rice, W. G. Design, Synthesis, and Biological
Evaluation of Cosalane, a Novel anti-HIV Agent which Inhibits
Multiple Features of Virus Replication. J . Med. Chem. 1994, 37,
(
)
7.5 Hz, 1 H), 5.30 (s, 2 H), 5.23 (s, 2 H), 2.16 (q, J ) 7.2 Hz,
H), 1.97 (d, J 12.2 Hz, 1 H), 1.90-1.73 (m, 1 H), 1.73-0.94
2
(
0
(
m, 32 H), 0.91 (d, J ) 6.4 Hz, 3 H), 0.87 (d, J ) 6.5 Hz, 3 H),
.87 (d, J ) 6.6 Hz, 3 H), 0.74 (s, 3 H), 0.66 (s, 3 H); 1 C NMR
75 MHz, CD OD) δ 174.7, 174.4, 151.5, 151.3, 148.9, 146.6,
3
3
040-3050.
3
(
3) Cushman, M.; Golebiewski, W. M.; Pommier, Y.; Mazumder, A.;
Reymen, D.; De Clercq, E.; Graham, L.; Rice, W. G. Cosalane
Analogues with Enhanced Potencies as Inhibitors of HIV-1
Protease and Integrase. J . Med. Chem. 1995, 38, 443-452.
4) Keyes, R. F.; Golebiewski, W. M.; Cushman, M. Correlation of
Anti-HIV Potency with Lipophilicity in a Series of Cosalane
Analogues Having Normal Alkenyl and Phosphodiester Chains
as Cholestane Replacements. J . Med. Chem. 1996, 39, 508-514.
1
1
4
3
1
40.6, 139.7, 138.5, 138.3, 137.7, 133.4, 131.8, 129.8, 129.7,
29.4, 129.0, 128.7, 126.6, 124.3, 75.3, 58.0, 57.7, 56.2, 48.0,
3.8, 41.5, 40.7, 39.9, 38.8, 38.4, 37.4, 37.2, 37.1, 37.0, 36.8,
3.4, 30.2, 30.0, 29.3, 29.1, 28.2, 25.2, 25.0, 23.2, 22.9, 22.1,
(
9.2, 12.8, 12.5. Anal. (C59
H
68Cl
2
N
2
Na
2
O
10‚4H
2
O) C, H, N.
In Vitr o An ti-HIV Assa y. Evaluation of the antiviral
activity of compounds against HIV-1RF infection in CEM-SS
cells was performed using the XTT cytoprotection assay as
(5) Keyes, R. F.; Cushman, M. Studies Directed Toward a More
Potent Cosalane Pharmacophore: Synthesis of a Substituted
Tetraphenylethylene which Inhibits the Cytopathic Effect of
HIV-1. Med. Chem. Res. 1996, 372-376.
2
3
previously described. This cell-based microtiter assay quan-
titates the drug-induced protection from the cytopathic effect
of HIV-1. Data are presented as the percent control of XTT
values for the uninfected, drug-free control. EC50 values reflect
the drug concentration that provides 50% protection from the
cytopathic effect of HIV-1 in infected cultures, while the CC50
reflects the concentration of drug that causes 50% cell death
in the uninfected cultures. XTT-based results were confirmed
by measurement of cell-free supernatant reverse transcriptase
and p24 levels. All XTT cytoprotection data were derived from
triplicate tests on each plate, with two separate sister plates.
Thus, the EC50 value from each plate represents the average
of triplicates, and the two EC50 values from sister plates were
averaged. The variation from the mean averaged less than
(
6) Golebiewski, W. M.; Keyes, R. F.; Cushman, M. Exploration of
the Effects of Linker Chain Modification on Anti-HIV Activities
of a Series of Cosalane Analogues. Bioorg. Med. Chem. 1996, 4,
1637-1648.
(7) Patch, R. J .; Roberts, J . C.; Gao, H.; Shi, Z.; Gopalsamy, A.;
Kongsjahju, A.; Daniels, K.; Kowalczyk, P. J .; van Schravendijk,
M.-R.; Gordon, K. A.; Pallai, P. V. Lipophilic Bis-arylsulfonates
as Inhibitors of the CD4-gp120 Interaction. Bioorg. Med. Chem.
Lett. 1996, 6, 2983-2988.
(8) Reymen, D.; Witvrouw, M.; Est e´ , J . A.; Neyts, J .; Schols, D.;
Andrei, G.; Snoeck, R.; Cushman, M.; Hejchman, E.; De Clercq,
E. Mechanism of the Antiviral Activity of New Aurintricarboxylic
Acid Analogues. Antiviral Chem. Chemother. 1996, 7, 142-152.
(
9) Ryu, S.-E.; Truneh, A.; Sweet, R. W.; Hendrickson, W. A.
Structures of an HIV Binding Fragment from Human CD4 as
Refined in Two Crystal Lattices. Structure 1994, 2, 59-74.
1
0%. The methodology used to monitor antiviral activity in
2
4
the remaining assay systems was also described previously.
(10) Cushman, M.; Insaf, S.; Ruell, J . A.; Schaeffer, W. G.; Rice, W.
G. Synthesis of a Cosalane Analogue with an Extended Poly-
anionic Pharmacophore Conferring Enhanced Potency as an
Anti-HIV Agent. Bioorg. Med. Chem. Lett. 1998, 8, 833-836.
Evaluation of the antiviral activity of compounds against HIV-
1
IIIB or HIV-2ROD infection in MT-4 cells was performed using
2
5
the MTT cytoprotection assay as previously described.
(
11) Moebius, U.; Clayton, L. K.; Abraham, S.; Harrison, S.; Rein-
hertz, E. L. The Human Immunodeficiency Virus gp120 Binding
Site on CD4: Delineation by Quantitative Equilibrium and
Kinetic Binding Studies of Mutants in Conjunction with a High-
resolution CD4 Atomic Structure. J . Exp. Med. 1992, 176, 507-
Mech a n ism of An tivir a l Action Stu d ies. Binding of HIV-
1
RF to CEM-SS cells was measured by a p24-based assay as
2
3
previously based. The ability of compounds to block fusion
was measured using a surrogate fusion assay as previously
5
17.
2
3
described. This assay employs the HeLa-CD4-LTR-â-gal
(
12) Choe, H.; Sodrosky, J . Contribution of Charged Amino Acids in
the CDR2 Region of CD4 to HIV-1 Binding. J . Acquired Immune
Defic. Syndr. 1992, 5, 204-210.
(13) Tsui, P.; Sweet, R. W.; Sathe, G.; Rosenberg, M. An Efficient
Phage Plaque Screen for the Random Mutational Analysis of
the Interaction of HIV-1 gp120 with Human CD4. J . Biol. Chem.
(MAGI) cells and the HIV-1 Tat- and Env-expressing HL2/3
cells. The HL2/3 cells, which express HIV-1 Env protein on
the cell surface and Tat protein in the cytoplasm, can fuse with
the MAGI cells and activate the â-galactosidase expression in
the syncytium. Cell monolayers were fixed for 5 min with 2%
formaldehyde-2% glutaraldehyde, washed twice with cold
phosphate-buffered saline, and then stained with 5-bromo-4-
chloro-3-indolyl-â-D-galactopyranoside (X-Gal) substrates for
1
992, 267, 9361-9367.
(
14) Cushman, M.; Kanamathareddy, S. Synthesis of the Covalent
Hydrate of the Incorrectly Assumed Structure of Aurintricar-
boxylic Acid (ATA). Tetrahedron 1990, 46, 1491-1498.
(
15) Cushman, M.; Kanamathareddy, S.; De Clercq, E.; Schols, D.;
Goldman, M.; Bowen, J . A. Synthesis and Anti-HIV Activities
of Low Molecular Weight Aurintricarboxylic Acid Fragments and
Related Compounds. J . Med. Chem. 1991, 34, 337-342.
5
0 min at 37 °C. The blue-stained cells in each well were then
counted as an indicator of the relative levels of fusion events.
The effects of the compounds on the in vitro activity of purified
HIV-1 p66/51 RT (a kind gift of S. Hughes, ABL Basic
Research, NCI-FCRDC, Frederick, MD) were determined by
(16) Cushman, M.; Wang, P.; Chang, S. H.; Wild, C.; De Clercq, E.;
Schols, D.; Goldman, M. E.; Bowen, J . A. Preparation and Anti-
HIV Activities of Aurintricarboxylic Acid Fractions and Ana-
logues: Direct Correlation of Antiviral Potency with Molecular
Weight. J . Med. Chem. 1991, 34, 329-336.
3
2
measurement of incorporation of [ P]TTP onto the poly(rA):
3
2
oligo(dT) (rAdT) or [ P]GTP onto the poly(rC):oligo(dG) ho-
2
6
mopolymer template systems. HIV-1 protease activity was
quantitated by a reversed-phase HPLC assay utilizing the Ala-
Ser-Glu-Asn-Tyr-Pro-Ile-Val-Glu-amide artificial protease sub-
(
17) Wang, P.; Kozlowski, J .; Cushman, M. Isolation and Structure
Elucidation of Low Molecular Weight Components of Aurintri-
carboxylic Acid (ATA). J . Org. Chem. 1992, 57, 3861-3866.
2
7
(18) Cushman, M.; Wang, P.; Stowell, J . G.; Schols, D.; De Clercq,
E. Structural Investigation and Anti-HIV Activities of High
Molecular Weight ATA Polymers. J . Org. Chem. 1992, 57, 7241-
strate as previously described. The in vitro effects of com-
pounds on HIV-1 integrase (a kind gift of S. Hughes, ABL
Basic Research, NCI-FCRDC, Frederick, MD) activity were
determined as previously described.28
7
248.
(
19) Balzarini, J .; Mitsuya, H.; De Clercq, E.; Broder, S. Aurintri-
carboxylic Acid and Evans Blue Represent Two Different Classes
of Anionic Compounds which Selectively Inhibit the Cytopath-
icity of Human T-Cell Lymphotropic Virus Type III/Lymph-
adenopathy-Associated Virus. Biochem. Biophys. Res. Commun.
1986, 136, 64-71.
20) Baba, M.; Schols, D.; Pauwels, R.; Balzarini, J .; De Clercq, E.
Fuchsin Acid Selectively Inhibits Human Immunodeficiency
Virus (HIV) Replication In Vitro. Biochem. Biophys. Res. Com-
mun. 1988, 155, 1404-1411.
Ack n ow led gm en t. This research was made possible
by NIH Grant NO1-AI-36624 and by grants from the
Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaan-
deren, the Belgian Geconcerteerde Onderzoekacties, and
the Belgiun Fonds voor Geneeskundig Wetenschappelijk
Onderzoek (FGWO). We would also like to thank Sandra
(

Extension of the Polyanionic Cosalane Pharmacophore
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1777
(
21) Eliel, E. L.; Rivard, D. E. Photobromination of Substituted
Toluenes as a Route to Substituted Benzyl Alcohols and Benz-
aldehydes. J . Org. Chem. 1952, 17, 1252-1256.
Phases of Human Immunodeficiency Virus Type 1 Replication
by a Dithiane Compound that Attacks the Conserved Zinc
Fingers of Retroviral Nucleocapsid Proteins. Antimicrob. Agents
Chemother. 1997, 41, 419-426.
(
22) Norman, M. H.; Minick, D. J .; Rigdon, G. C. Effect of Linking
Bridge Modifications on the Antiphychotic Profile of Some
Phthalimide and Isoindolinone Derivatives. J . Med. Chem. 1996,
(
27) Rice, W. G.; Schaeffer, C. A.; Graham, L.; Bu, M.; McDougal, J .
S.; Orloff, S. L.; Villiger, F.; Young, M.; Oroszlan, S.; Fesen, M.
R.; Pommier, Y.; Mendelev, J .; Kun, E. The Site of Action of
3
9, 149-157.
(
23) Rice, W. G.; Bader, J . P. Discovery and in Vitro Development of
3
-Nitrosobenzamide on the Infectivity Process of Human Im-
AIDS Antiviral Drugs as Biopharmaceuticals. Adv. Pharmacol.
munodeficiency Virus in Human Lymphocytes. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 9721-9724.
(San Diego) 1995, 6, 389-438.
(
24) Schols, D.; De Clercq, E.; Balzarini, J .; Baba, M.; Witvrouw, M.;
Hosoya, M.; Andrei, G.; Snoeck, R.; Neyts, J .; Pauwels, R.; Nagy,
M.; Gy o¨ rgyi-Edel e´ nyi, J .; Machovich, R.; Horv a´ th, I.; L o¨ w, M.;
G o¨ r o¨ g, S. Sulphated Polymers are Potent and Selective Inhibi-
tors of Various Enveloped Viruses, Including Herpes Simplex
Virus, Cytomegalovirus, Vesicular Stomatitis Virus, Respiratory
Syncytial Virus, and Toga-, Arena-, and Retroviruses. Antiviral.
Chem. Chemother. 1990, 1, 233-240.
(
28) Turpin, J . A.; Buckheit, R. W., J r.; Derse, D.; Hollingshead, M.;
Williamson, K.; Palamone, C.; Osterling, M. C.; Hill, S. A.;
Graham, L.; Schaeffer, C. A.; Bu, M.; Huang, M.; Cholody, W.
M.; Michejda, C. J .; Rice, W. G. Inhibition of Acute-, Latent-,
and Chronic-phase Human Immunodeficiency Virus Type 1
(HIV-1) Replication by a Bistriazoloacridone Analogue that
Selectively Inhibits HIV-1 Transcription. Antimicrob. Agents
Chemother. 1998, 42, 487-494.
(
25) Pauwels, R.; Balzarini, J .; Baba, M.; Snoeck, R.; Schols, D.;
Herdewijn, P.; Desmyter, J .; De Clercq, E. Rapid and Automated
Tetrazolium-based Colorimetric Assay for the Detection of Anti-
HIV Compounds. J . Virol. Methods 1988, 20, 309-321.
26) Rice, W. G.; Baker, D. C.; Schaeffer, C. A.; Graham, L.; Bu, M.;
Terpening, S.; Clanton, D.; Schultz, R.; Bader, J . P.; Buckheit,
R. W.; Field, L.; Singh, P. K.; Turpin, J . A. Inhibition of Multiple
(29) Schols, D.; Struyf, S.; Van Damme, J .; Este, J . A.; Henson, G.;
De Clercq, E. Inhibition of T-Tropic HIV Strains by Selective
Antagonization of the Chemokine Receptor CXCR4. J . Exp. Med.
1997, 186, 1383-1388.
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