Correlation of anti-HIV activity with anion spacing in a series of cosalane analogues with extended polycarboxylate pharmacophores

Article abstract of DOI:10.1021/jm000290u

Cosalane and its synthetic derivatives inhibit the binding of gp120 to CD4 as well as the fusion of the viral envelope with the cell membrane. The binding of the cosalanes to CD4 is proposed to involve ionic interactions of the negatively charged carboxylates of the ligands with positively charged arginine and lysine amino acid side chains of the protein. To investigate the effect of anion spacing on anti-HIV activity in the cosalane system, a series of cosalane tetracarboxylates was synthesized in which the two proximal and two distal carboxylates are separated by 6-12 atoms. Maximum activity was observed when the proximal and distal carboxylates are separated by 8 atoms. In a series of cosalane amino acid derivatives containing glutamic acid, glycine, aspartic acid, β-alanine, leucine, and phenylalanine residues, maximum activity was displayed by the di(glutamic acid) analogue. A hypothetical model has been devised for the binding of the cosalane di(glutamic acid) conjugate to CD4. In general, the compounds in this series are more potent against HIV-1_RF in CEM-SS cells than they are vs HIV-1_IIIB in MT-4 cells, and they are least potent vs HIV-2_ROD in MT-4 cells.

Full text of DOI:10.1021/jm000290u

J . Med. Chem. 2001, 44, 703-714
703
Cor r ela tion of An ti-HIV Activity w ith An ion Sp a cin g in a Ser ies of Cosa la n e
An a logu es w ith Exten d ed P olyca r boxyla te P h a r m a cop h or es
Kalpathy C. Santhosh,† Gitendra C. Paul,† Erik De Clercq,‡ Christophe Pannecouque,‡ Myriam Witvrouw,‡
Tracy L. Loftus,§ J im A. Turpin,§ Robert W. Buckheit, J r.,§ and Mark Cushman*,†
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 Infectious Disease Research Department,
Southern Research Institute, 431 Aviation Way, Frederick, Maryland 21701
Received J uly 10, 2000
Cosalane and its synthetic derivatives inhibit the binding of gp120 to CD4 as well as the fusion
of the viral envelope with the cell membrane. The binding of the cosalanes to CD4 is proposed
to involve ionic interactions of the negatively charged carboxylates of the ligands with positively
charged arginine and lysine amino acid side chains of the protein. To investigate the effect of
anion spacing on anti-HIV activity in the cosalane system, a series of cosalane tetracarboxylates
was synthesized in which the two proximal and two distal carboxylates are separated by 6-12
atoms. Maximum activity was observed when the proximal and distal carboxylates are separated
by 8 atoms. In a series of cosalane amino acid derivatives containing glutamic acid, glycine,
aspartic acid, â-alanine, leucine, and phenylalanine residues, maximum activity was displayed
by the di(glutamic acid) analogue. A hypothetical model has been devised for the binding of
the cosalane di(glutamic acid) conjugate to CD4. In general, the compounds in this series are
more potent against HIV-1RF in CEM-SS cells than they are vs HIV-1IIIB in MT-4 cells, and
they are least potent vs HIV-2ROD in MT-4 cells.
In tr od u ction
The currently available chemotherapeutic agents for
the treatment of AIDS include six nucleoside reverse
transcriptase inhibitors (AZT, ddI, ddC, 3TC, d4T, and
ABC), three nonnucleoside reverse transcriptase inhibi-
tors (nevirapine, delavirdine, and efavirenz), and five
protease inhibitors (saquinavir, ritonavir, indinavir,
nelfinavir, and amprenavir). The recent trend toward
early and aggressive intervention with combination
chemotherapy has resulted in a significant decrease in
the rate of disease progression in AIDS patients.1-4
However, major problems remain in the chemotherapy
of AIDS, including viral resistance and drug toxicity.
Therefore, interest remains in the development of new
anti-HIV agents that target additional steps in the rep-
lication of the virus. Low-molecular-weight CD4 ligands
that inhibit the binding of the viral surface glycoprotein
(gp120) to its cellular receptor (CD4) might circumvent
the usual problems with the emergence of resistant viral
strains because CD4 is not expected to mutate.
Cosalane (1), which was derived conceptually by
attaching a membrane-interactive steroid to a dichlo-
rinated disalicylmethane fragment of aurintricarboxylic
acid (ATA, schematic representation shown in structure
2), binds to both gp120 and CD4.5,6 It inhibits both the
attachment of gp120 to CD4 as well as an undefined
postattachment event prior to reverse transcription.7
Attempts to improve the potency of cosalane (EC50: 5.1
* To whom correspondence should be addressed. Tel: 765-494-1465.
Fax: 765-494-6790. E-mail: cushman@pharmacy.purdue.edu.
† Purdue University.
‡ Katholieke Universiteit Leuven.
§ Southern Research Institute.
10.1021/jm000290u CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/19/2001

704 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Santhosh et al.
Sch em e 1a
µM vs HIV-1RF in CEM-SS cells) have resulted in
several series of cosalane analogues, including those
derived from attachment of the disalicylmethane phar-
macophore to various regions of the steroid8 and from
modification of the linker chain.9-11
The available evidence indicates that the disalicyl-
methane moiety of cosalane (1) is the “pharmacophore”
and that the steroid simply functions as a lipophilic
accessory appendage. A hypothetical model for the
binding of the cosalane “pharmacophore” to CD4 has
been proposed.12 According to this model, the two
carboxylate groups of the disalicylmethane moiety of
cosalane bind electrostatically to the Arg58 and Arg59
side chains of the protein. Inspection of the structure
of the surrounding region13,14 reveals several additional
basic functional groups (e.g. the side chains of Lys46
and Lys72) that would be positively charged at physi-
ological pH and could possibly bind to additional anionic
groups that could be attached to the cosalane (1)
“pharmacophore”. An additional line of reasoning in
support of the hypothesis that the incorporation of
additional anionic groups would increase potency comes
from examination of the biological activities of low-
molecular-weight ATA components15 and pharmaco-
phore analogues.16 Specifically, the activities of 3 and
a
Reagents and conditions: (a) PdCl2, aq NaOH; (b) TMSCHN2
in hexane, benzene and methanol; (c) NBS, (BzO)2, CCl4, heat (for
18) or heat and irradiation (for 19 and 20).
prepared by coupling 4-methylbenzeneboronic acid (8)
with the appropriate o-, m-, and p-bromobenzoic acids
9-11 in the presence of palladium chloride and aqueous
sodium hydroxide. The resulting carboxylic acids 12-
14 were converted to the corresponding methyl esters
15-17 with (trimethylsilyl)diazomethane.19 Free radical
bromination of 15-17 in the presence of NBS and
benzoyl peroxide afforded the desired benzyl bromides
18-20. The free radical bromination of 16 and 17 to
yield 19 and 20 was carried out by irradiation with a
200-W tungsten lamp, but the conversion of 15 to 18
was accomplished without irradiation.
As shown in Scheme 2, the two carboxylic acid groups
of cosalane (1) were selectively methylated with (tri-
methylsilyl)diazomethane19,20 to afford the diester 21.
Treatment of diphenol 21 with potassium carbonate in
DMF afforded the diphenoxide dianions, which were
alkylated with the benzyl bromides 18-20 to afford 22-
24. For conversion of the methyl esters of 23 and 24 to
the acids 26 and 27, the hydrolyses were performed
using potassium carbonate in the presence potassium
cyanide in hot aqueous ethanol. However, in the case
of 22, this method gave unsatisfactory yields, so its hy-
drolysis to afford 25 was performed with sodium hydrox-
ide in a refluxing mixture of methanol and ethanol (5:1).
The synthesis of the tetraacid 30, in which the two
terminal acids are closer to the cosalane nucleus than
they are in 25-27, is shown in Scheme 3. The required
benzyl bromide 28 was made by methylation of com-
mercially available 4-(bromomethyl)phenylacetic acid
with (trimethylsilyl)diazomethane.19 Deprotonation of
the phenolic hydroxyl groups of 21 with potassium
carbonate in DMF afforded the dianion, which was
alkylated with 28 to yield intermediate 29. The four
ester groups of 29 were then hydrolyzed with potassium
carbonate in the presence of potassium cyanide in
aqueous ethanol.
4 are higher than that of 5, and 6 is more potent than
7. Recent studies in selected systems have documented
several examples in which moderate increases in anti-
HIV potency resulted from the attachment of additional
substituted benzoic acid rings onto the cosalane “phar-
macophore”.17,18 The purpose of the present study was
to define exactly how the constellation (spatial arrange-
ment) and the constitution of additional anionic groups
attached to the cosalane pharmacophore would affect
the anti-HIV activity.
Ch em istr y
Several substituted biphenyls carrying a benzyl bro-
mide alkylating group in one ring and a methyl ester
group in the other were synthesized for attachment to
the phenolic hydroxyl groups of cosalane (1) (Scheme
1). The tolyl-substituted benzoic acids 12-14 were

Correlation of Anti-HIV Activity with Anion Spacing
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 705
Sch em e 2a
Sch em e 3a
a
Reagents and conditions: (a) K2CO2, DMF, 23 °C (22 h); (b)
K2CO3, KCN, aq EtOH, 80 °C (14 h), then H2O, 80 °C (2 h).
Sch em e 4a
a
Reagents and conditions: (a) TMSCHN2 in hexane, benzene
and methanol, 25 °C (25 min); (b) 18, 19 or 20, K2CO3, DMF; (c)
for 25: aq NaOH, MeOH, EtOH, reflux (14 h); for 26: KCN,
K2CO3, aq EtOH, 95 °C (20 h) followed by H2O, 80 °C (2 h); for
27: KCN, K2CO3, aq EtOH, 90 °C (20 h) followed by H2O, 80 °C
(1 h).
To explore the importance of the geometrical con-
straint afforded by the alkene double bond in the linker
chain connecting the steroid to the “pharmacophore”,
the hydrogenated product 34 was also synthesized
(Scheme 4). Dihydrocosalane9 was converted to the
diester 32 with (trimethylsilyl)diazomethane.19 Alkyla-
tion of the dianion derived from 32 with methyl p-
bromomethylbenzoate afforded the dialkylated product
33. Hydrolysis of the four ester groups yielded the
tetraacid 34.
A new series of cosalane derivatives was also made
in which the two carboxyl groups of cosalane were linked
to amino acid residues through amide bond formation.
We felt that the successful implementation of this
strategy would allow access to a wide variety of poly-
anionic compounds which could be used as a tool to
observe the effects of anion spacing, as well as the effects
of the nearby amino acid side chains, on biological
activity. This in fact resulted in a novel array of
dicarboxylic acids and tetracarboxylic acids including
compounds 36 (derived from glycine), 38 (derived from
â-alanine), 40 (derived from leucine), 42 (derived from
phenylalanine), 44 (derived from aspartic acid), and 46
(derived from glutamic acid). A further variation in the
structure of the glycine derivative 36 and the glutamic
acid derivative 46 involved the corresponding dihydro
a
Reagents and conditions: (a) TMSCHN2 in hexane, benzene
and methanol; (b) K2CO3, methyl 4-(bromomethyl)phenylacetate,
DMF, 23 °C (20 h); (c) K2CO3, KCN, aq EtOH, 80 °C (12 h), then
H2O, 80 °C (2 h).
compounds 48 and 50, in which the alkene in the linker
chain connecting the “pharmacophore” to the steroid
nucleus of 44 and 46 had been hydrogenated.

706 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Santhosh et al.
In general, amide bond formation during the coupling
of cosalane to the amino acid derivatives was performed
by reacting the hydrochloride salts of the amino acids
with cosalane or dihydrocosalane in the presence of BOP
and triethylamine in THF. The dicarboxylic acids 40 and
42 were synthesized by coupling cosalane with the
methyl esters of leucine and phenylalanine to afford the
protected intermediates 39 and 41, respectively. Hy-
drolysis of the methyl esters of 39 and 41 yielded the
desired analogues 40 and 42. The aspartic acid deriva-
tive 44 and the glutamic acid derivative 46 were
synthesized by reaction of cosalane with the correspond-
ing di-tert-butyl esters of the amino acids, followed by
hydrolysis of the four tert-butyl ester groups. The
dicarboxylic acid 36, having two glycine residues, was
prepared similarly by coupling cosalane to the tert-butyl
ester of glycine to afford 35, followed by hydrolysis. The
â-alanine derivative 38 was prepared by coupling cos-
alane to â-alanine ethyl ester, resulting in intermediate
37, followed by hydrolysis. The dihydro analogues 48
and 50 were prepared by coupling dihydrocosalane (31)9
with the corresponding di-tert-butyl esters of glycine and
glutamic acid to afford 47 and 49, followed by depro-
tection.
Biologica l Resu lts a n d Discu ssion
The new cosalane derivatives were evaluated for
inhibition of the cytopathic effect of HIV-1RF in CEM-
SS cells and HIV-1IIIB and HIV-2ROD in MT-4 cells.
Cytotoxicities in uninfected CEM-SS cells and MT-4
cells were also determined. The results are listed in
Table 1.
In general, the new cosalane polycarboxylates syn-
thesized in the present study were more potent against
HIV-1RF in CEM-SS cells than they were vs HIV-1IIIB
in MT-4 cells. This difference in activity was more
pronounced with some of the compounds in the amino
acid series (e.g. 36 and 46) than in the biphenyl series
(25 and 26) or in the benzyl compound 30. All new
cosalane derivatives were also either inactive or dis-
played very low antiviral activity against HIV-2ROD in
Ta ble 1. Anti-HIV Activities of Cosalane Analogues
a
b
EC50 (µM)
CC50 (µM)
compd
HIV-1RF
HIV-1IIIB
HIV-2ROD
4.0
CEM-SS
MT-4
1
25
26
27
30
34c
35
36
38
40
42
44
46
48
50
51
52
53
54c
55c
5.1
5.2 ( 0.3
6.1 ( 0.9
3.0
>200
>125
>125
79 ( 15
10.1 ( 1.6
>125
>200
12.0 ( 3.0
>95
>200
>100
>100
>100
>100
100
>125
>125
>125
>125
>125
>125
62
>125
>125
>125
>125
37
1.8 ( 0.1
4.8 ( 0.6
4.1 ( 2.6
5.1 ( 2.1
83.8 ( 30.4
133 ( 21
>200
70.4 ( 25.6
NTd
>125
>125
>125
120
>125
>62.0
122
>125
3.4 ( 0.4
34.7 ( 0.3
40.0 ( 2.0
57.6 ( 10.7
73.8 ( 3.1
63.4 ( 13.1
>200
>200
>200
>200
>62
>200
10.7 ( 1.2
1.1 ( 0.1
4.5 ( 1.0
11.6 ( 1.0
39.8 ( 3.2
5.7 ( 0.7
0.5 ( 0.1
28.6 ( 4.3
2.9 ( 0.1
16.6 ( 7.2
8.9 ( 2.9
39.4 ( 34
10.3 ( 3.9
90 ( 13
82 ( 11
>200
69.6 ( 32.0
>125
73.0 ( 25.0
34 ( 4
>316
>316
>37
>37.0
>29.0
22.0
2.2 ( 0.1
1.7 ( 0.3
8.4
76 ( 33
72 ( 14
47 ( 6
50 ( 4
89 ( 22
81 ( 17
41 ( 3
>66.0
19.9 ( 3.8
12.5 ( 6.3
a
Concentration required to reduce the cytopathic effect of the virus by 50%. The HIV-1RF assays were performed in CEM-SS cells,
b
while the HIV-1IIIB and HIV-2ROD assays were carried out in MT-4 cells. Concentration required for a 50% reduction in cellular viability
of uninfected cells. c Tested as the tetrasodium salt. Not tested.
d

Correlation of Anti-HIV Activity with Anion Spacing
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 707
MT-4 cells. The low activity of the compounds in the
present series vs HIV-2ROD is not totally unexpected,
because the previously reported polyanions 51-55, also
having extended polyanionic pharmacophores, were less
active vs HIV-2ROD than they were vs HIV-1RF or HIV-
17,18
1IIIB
.
However, the generally greater potency in the
presently reported cosalane derivatives against HIV-
1RF in comparison to HIV-1IIIB was not expected, either
on the basis of the activity of cosalane (1) or in the 51-
55 series.
F igu r e 1. Relationship between anti-HIV activity (1/EC50
,
µM, vs HIV-1RF in CEM-SS cells) and the number of atoms
separating the internal and external carboxylates in the series
of compounds 51 (6 atoms), 52 (7 atoms), 53 (8 atoms), 30 (9
atoms), 25 (10 atoms), 26 (11 atoms), and 27 (12 atoms).
as the two terminal carboxyl groups are moved farther
away from the two internal carboxyl groups. Evidently,
there is an optimal distance between the internal and
external carboxyl groups that is approximated by the
distances in the analogue having structure 53 (Figure
1). Consistent with this hypothesis, the most active of
the new compounds presently being reported is 30,
which is slightly less active than 53 but slightly more
active than 25.
The correlation of the distance separating the internal
and external carboxyl groups and antiviral activity is
apparent for both HIV-1RF in CEM-SS cells (Figure 1)
as well as HIV-1IIIB in MT-4 cells, although it is less
apparent vs HIV-2ROD in MT-4 cells because of the low
activities observed vs HIV-2ROD. The optimal spacing
of the carboxyl groups may reflect a requirement for
interaction with basic amino acid side chains on the
surface of the CD4 molecule. A hypothetical model for
the binding of 53 to CD4, involving ionic bonding of
three of the carboxylates of the ligand with the Arg58,
Arg59, and Lys72 side chains of the protein, has been
proposed.17
Considering the amino acid derivatives 36, 38, 40, 42,
44, and 46, the first hypothesis to be examined was that
the two carboxyl groups of cosalane could be sacrificed
and activity still maintained as long as two new carboxyl
groups were generated in the process. This was first
tested by synthesizing the glycine derivative 36, which
proved to be slightly more active than cosalane (1) itself
when evaluated vs HIV-1RF in CEM-SS cells (EC50: 5.1
µM for 1 vs 3.4 µM for 36). However, the glycine
derivative 36 was significantly less active than cosalane
(1) when tested vs HIV-1IIIB in MT-4 cells (EC50: 3.0
µM for 1 vs 57.6 µM for 36). The requirement for two
free carboxyl groups of 36 for activity was apparent by
the inactivity of the di-tert-butyl ester 35 when evalu-
ated vs HIV-1IIIB. The inactivity of the diester 35 is
consistent with the importance of the proposed hypo-
thetical ionic binding of the cosalane derivitives to the
protein and is in agreement with the general observa-
Turning to the biphenyl series of compounds 25-27,
the activities of 25 and 26 are very close, but in general,
a decline in activity vs both HIV-1 strains is observed
as the two terminal carboxyl groups are moved farther
away from the internal carboxyl groups, eventually
resulting in the inactive compound 27. This effect is in
contrast to the relative activities seen in the benzyl
series containing 51-53, in which the activity increased

708 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Santhosh et al.
F igu r e 2. Hypothetical model of the binding of the 46 “pharmacophore” to CD4 (programmed for walleyed viewing): red, 46
“pharmacophore”; orange, CD4 protein residues involved in hydrogen bonding; yellow, hydrogen bonds.
tion that the ester precursors of the carboxylic acids of
cosalane derivatives are inactive as antiviral agents.
Like many of the polyanions in the present series, 36
was inactive when tested against HIV-2ROD in MT-4 cell
culture (EC50: >125 µM), even though cosalane (1) was
active vs HIV-2ROD (EC50: 4.0 µM).
The effect of extending the distance between the
amide nitrogens of 36 and the two carboxyl groups was
determined by synthesizing the â-alanine derivative 38.
Compound 38 was less active than 36 vs both HIV-1
strains. However, 38 did display low activity against
HIV-2ROD, in contrast to 36, which was inactive.
The effect of attaching various amino acid side chains
to 36 was examined next. The analogue having a leucine
side chain (40) was less active against both HIV-1RF
(EC50: 40.0 µM vs 3.4 µM) and HIV-1IIIB (EC50: 63.4
µM vs 57.6 µM) than 36. Incorporation of the phenyla-
lanine side chain (42) led to an inactive compound. The
compound having the aspartic acid side chain (44) was
more active than either the leucine or phenylalanine
derivatives. Compound 44 proved to be less active than
the glycine derivative 36 against HIV-1RF (EC50: 10.7
µM vs 3.4 µM), but it was more active than 36 vs HIV-
1IIIB (EC50: 16.6 µM vs 57.6 µM). In addition, 44 had
low activity against HIV-2ROD (EC50: 122 µM), in con-
trast to the inactive glycine derivative 36. Finally, the
glutamic acid derivative 46 was more active than the
glycine derivative 36 in all three systems tested (EC50:
1.1 µΜ vs 3.4 µM against in HIV-1RF, 8.9 µM vs 57.6
µM against HIV-1IIIB, and 69.6 µM vs >125 µM against
HIV-2ROD). This indicates that when spaced properly,
the addition of two carboxyl groups to the two already
present in the glycine derivative 36 does offer an
increase in anti-HIV activity. In summary, the rank
order of the di(amino acid) derivatives vs HIV-1RF in
CEM-SS cells was glutamic acid (EC50: 1.1 µM), glycine
(EC50: 3.4 µM), aspartic acid (EC50: 10.7 µM), â-alanine
(EC50: 34.7 µM), leucine (EC50: 40.0 µM), and phenyl-
alanine (EC50: >200 µM). The rank order of these
derivatives vs HIV-1IIIB in MT-4 cells was basically the
same, except the aspartic acid derivative (EC50: 16.6 µM)
was more potent than the glycine derivative (EC50: 57.6
µM) and the leucine derivative (EC50: 63.4 µM) was
slightly more potent than the â-alanine derivative
(EC50: 73.8 µM).
A computer graphics molecular modeling study was
performed using Sybyl software (Tripos, Inc.) in order
to investigate how the most active conjugate 46 might
interact with CD4. Gasteiger-Hu¨ckel charges were
calculated for cosalane, and the charges from the
Kollman united-atom force field were loaded for the
enzyme portion of the complex. The “pharmacophore”
of the di(glutamic acid) conjugate 46 was then docked
on the surface of CD413 in the site previously proposed
for the binding of cosalane.17 During the docking
procedure, the protein was “frozen” and cosalane was
allowed to move. Hydrogen bonds between the ligand
and the protein were then displayed, with the maximum
acceptable distance between the donor and acceptor
atoms for a hydrogen bond specified at 2.8 Å and the
minimum angle at 120°. The results are pictured in
Figure 2. According to the hypothetical model, the
“upper” carboxyl group at the end of the chain of the
cosalane analogue forms a hydrogen bond with the
amide NH of Leu44, while the amide moiety of the
“upper chain” of the ligand bonds through both CdO
and NH groups to the guanidine moiety of Arg59. The
“lower” carboxyl group at the end of the chain of the
cosalane analogue bonds to the amino group of Lys72
of CD4. The Arg58 guanidine group of CD4 is bonded
to the proximal carboxyl group of the “lower” chain of
the ligand, as well as to the amide NH and the phenolic
hydroxyl group of cosalane. The hypothetical model
shown in Figure 2 makes it plausible that 46 could bind
in the site proposed for cosalane, with both of the
glutamic acid moieties of the cosalane analogue partici-
pating in hydrogen bonding to the protein.
To investigate the effect of increased conformational
flexibility of the linker chain attached to the two
substituted benzene rings in the cosalane analogues, the

Correlation of Anti-HIV Activity with Anion Spacing
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 709
double bond in the linker chain was reduced. The three
cases studied were the glycine derivatives 36 vs 48, the
glutamic acid derivatives 46 vs 50, and the benzyl
derivatives 53 vs 34. When investigated in the HIV-
1IIIB and HIV-2ROD systems, the activity decreased in
two cases when the double bond was reduced (46 vs 50
and 53 vs 34), but it increased in one case (36 vs 48).
In the HIV-1RF system, the activity consistently de-
creased in all three cases when the double bond was
reduced. The general decrease in antiviral activity
accompanying the reduction of the double bond in the
linker chain is consistent with the previously reported
activity of cosalane (1) vs HIV-1RF in CEM-SS cells
(EC50: 5.1 µM) in comparison with the activity of
dihydrocosalane (31) (EC50: 20 µM) in the same sys-
tem.9 Overall, the data show that the conformational
restriction provided by the double bond does contribute
to the antiviral activity.
inhibitors. This can be seen, for example, in the pro-
posed use and initial success of the pentafuside (T-20),
a 36-amino acid fragment of gp41 that inhibits mem-
brane fusion.21-23 The cosalanes may offer some of the
same advantages resulting from an alternative antiviral
target. Although the cosalanes are not as potent as anti-
HIV agents that inhibit reverse transcriptase or pro-
tease, their antiviral potencies are consistent with
current inhibitors of HIV-cell fusion.
The potential therapeutic use of cosalane is compro-
mised by its poor oral bioavailability, which may be
ascribed to poor enterohepatic transport and sequestra-
tion in liver parenchymal cells.24 However, many drugs
that are short peptides, are peptide-derived, or are
structurally related to peptides are able to utilize the
peptide transport system in the gastrointestinal tract
for absorption.25 The cosalane amino acid conjugates
synthesized here therefore also offer the prospect of
enhanced oral bioavailability through utilization of
peptide transporters.
Mechanism of action studies have been performed on
both cosalane (1) as well as the most potent of the
tetraanions, compound 53.7,17 These investigations have
established that both inhibition of binding of gp120 to
CD4 and inhibition of an unidentified postbinding fusion
event play a role in the inhibition of the cytopathic effect
of the virus by cosalane and related compounds. In the
case of 53, inhibition of fusion occurs at a lower
concentration than inhibition of attachment.17 More
detailed studies have shown that 53 binds to both CD4
as well as gp120, but a direct interaction with CXCR4
was not observed.17 It is possible that 53 prevents the
proper orientation of the gp120-CD4 complex with
CXCR4 that is required for binding. We assume that
the additional polyanions in the present series have a
similar mechanism of action, involving inhibition of both
viral attachment and fusion.
Exp er im en ta l Section
Gen er a l. Melting points were determined in capillary tubes
on a Mel-Temp apparatus and are uncorrected. Spectra were
obtained as follows: CI mass spectra on a Finnegan 4000
spectrometer; liquid SIMS mass spectra and EI mass spectra
on a MAT 95 XL spectrometer; 1H NMR spectra on Varian
VXR-500S and Bruker ARX-300 spectrometers; IR spectra on
a Perkin-Elmer 1600 series FTIR. Microanalyses were per-
formed 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.
Meth yl 4′-(Br om om eth yl)bip h en yl-2-ca r boxyla te (18).
A solution of 2-(p-tolyl)benzoic acid (12)26,27 (250 mg, 1.18
mmol) in benzene (8 mL) and methanol (4 mL) was esterified
with excess (trimethylsilyl)diazomethane19 (2.0 M solution in
hexane) to afford the corresponding methyl ester 15:28,29 1H
NMR (300 MHz, CDCl3) δ 7.79 (bd, J ) 7.6 Hz, 1 H), 7.51 (dt,
J ) 1.6 and 7.6 Hz, 1 H), 7.38 (t, J ) 7.5 Hz, 2 H), 7.21 (bs, 4
H), 3.66 (s, 3 H), 2.39 (s, 3 H). A solution of ester 15 (301 mg,
1.33 mmol), N-bromosuccinimide (260 mg, 1.46 mmol) and
benzoyl peroxide (20 mg, 0.083 mmol) in CCl4 (5 mL) was
heated under reflux for 3 h. After cooling, the mixture was
filtered. The filtrate was concentrated under vacuum to obtain
a liquid residue that was used for the next step without further
purification. From proton NMR the conversion to bromide 1828
was estimated to be greater than 90%: 1H NMR (300 MHz,
CDCl3) δ 7.82 (d, J ) 8.2 Hz, 1 H), 7.58 (d, J ) 8.2 Hz, 1 H),
7.50-7.26 (m, 6 H), 4.54 (s, 2 H), 3.64 (s, 3 H).
Meth yl 4′-(Br om om eth yl)bip h en yl-3-ca r boxyla te (19).
A solution of 3-(p-tolyl)benzoic acid (13)30 (250 mg, 1.18 mmol)
in benzene (8 mL) and methanol (4 mL) was esterified with
excess (trimethylsilyl)diazomethane19 (2.0 M solution in hex-
ane) to afford the corresponding methyl ester 16:29,31,32 1H NMR
(300 MHz, CDCl3) δ 8.26 (t, J ) 1.6 Hz, 1 H), 8.00 (td, J ) 1.5
and 7.6 Hz, 1 H), 7.77 (td, J ) 1.6 and 7.6 Hz, 1 H), 7.52 (d, J
) 7.5 Hz, 1 H), 7.50 (AB quartet, J ) 7.0 Hz, 2 H), 7.26 (d, J
) 7.5 Hz, 2 H), 3.94 (s, 3 H), 2.40 (s, 3 H). A solution of ester
16 (301 mg, 1.33 mmol), N-bromosuccinimide (260 mg, 1.46
mmol) and benzoyl peroxide (20 mg, 0.083) in CCl4 (5 mL) was
irradiated with a 200-W tungsten lamp while being heated
under reflux for 3 h. After cooling, the mixture was filtered.
The filtrate was concentrated under vacuum to obtain a liquid
residue that was used for the next step without further
purification. From proton NMR, the conversion to bromide 1931
was estimated to be greater than 90%: 1H NMR (300 MHz,
CDCl3) δ 8.26 (t, J ) 1.2 Hz, 1 H), 8.04 (td, J ) 1.1 and 8.2
Hz, 1 H), 7.78 (td, J ) 1.1 and 8.2 Hz, 1 H), 7.60 (d, J ) 8.2
Hz, 2 H), 7.54-7.46 (m, 3 H), 4.54 (s, 2 H), 3.94 (s, 3 H).
Meth yl 4′-(Br om om eth yl)bip h en yl-4-ca r boxyla te (20).
A solution of 4-(p-tolyl)benzoic acid30,33,34 (14) (250 mg, 1.18
It is clear from the data presented in Table 1 that
there are differences in the potencies of the cosalane
analogues when they are examined vs HIV-1RF in CEM-
SS cells compared with HIV-1IIIB in MT4 cells. In
general, the compounds appear to be less potent in the
HIV-1IIIB system than they are in the HIV-1RF system,
although in four cases (1, 50, 52, and 54), the com-
pounds were more active vs HIV-1IIIB. These differences
in potency may be ascribed to both differences in the
cell types as well as differences between the viral
strains. Whereas the MT-4 cells are co-infected with
HTLV-1, CEM-SS cells are not. This can affect potency,
since in the MT-4 cells, there is a second source of
antiviral targets in addition to HIV-1. Since the cos-
alanes are both attachment and fusion inhibitors, their
potencies may also be affected by differences in the end
sequences between HIV-1RF and HIV-1IIIB. The main
point to notice from the data presented in Table 1 is
that some of the compounds (e.g. 1, 30, 34, 46, 52, and
53) are active against both HIV-1 strains, while others
(e.g. 36, 48, and 54) are more selective and are therefore
more questionable as anti-HIV agents.
In the cosalane series we have created low to submi-
cromolar inhibitors of HIV-1 replication (e.g. 53). Since
the current paradigm for antiviral therapy is the use of
antiviral agents in combination, one of the current goals
for the development of new antivirals is to obtain new
compounds with novel mechanisms of action that comple-
ment the existing reverse transcriptase and protease

710 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Santhosh et al.
mmol) in benzene (8 mL) and methanol (4 mL) was esterified
with excess (trimethylsilyl)diazomethane19 (2.0 M solution in
hexane) to afford the corresponding methyl ester 17:29,35 1H
NMR (300 MHz, CDCl3) δ 8.09 (d, J ) 8.4 Hz, 2 H), 7.64 (d, J
) 8.4 Hz, 2 H), 7.52 (d, J ) 8.1 Hz, 2 H), 7.27 (d, J ) 8.1 Hz,
2 H), 3.93 (s, 3 H), 2.40 (s, 3 H). A solution of ester 17 (301
mg, 1.33 mmol), N-bromosuccinimide (260 mg, 1.46 mmol) and
benzoyl peroxide (20 mg) in CCl4 (5 mL) was irradiated with
a 200-W tungsten lamp while being heated under reflux for 3
h. After cooling, the mixture was filtered. The filtrate was
evaporated off to leave a solid residue that was purified by
crystallization from hexane to give white crystals of 20 (0.34
g, 84%): mp 113-114 °C; IR (CHCl3) 3011, 2952, 2842, 1732,
1614, 1435, 1341, 1260, 1229, 1203 cm-1; 1H NMR (300 MHz,
CDCl3) δ 8.11 (d, J ) 8.2 Hz, 2 H), 7.64 (d, J ) 8.2 Hz, 2 H),
7.59 (d, J ) 8.2 Hz, 2 H), 7.48 (d, J ) 8.2 Hz, 2 H), 4.54 (s, 2
H), 3.93 (s, 3 H); HRMS for C15H13BrO2 calcd 305.0177, found
305.0166.
3.86 (s, 3 H), 2.11 (q, J ) 7.6 Hz, 2 H), 1.95 (bd, J ) 12.0 Hz,
1 H), 0.88 (d, J ) 6.5 Hz, 3 H), 0.85 (d, J ) 6.6 Hz, 3 H), 0.84
(d, J ) 6.5 Hz, 3 H), 0.71 (s, 3 H), 0.62 (s, 3 H). Anal. (C77H88
-
Cl2O10) C, H.
3′′,3′′′-Dich lor o-5′′,5′′′-d i(m eth oxyca r bon yl)-4′′,4′′′-d i[p-
(4-m eth oxyca r bon ylp h en yl)ben zyloxy]-4′,4′-d ip h en yl-3â-
(3′-bu ten -1′-yl)ch olesta n e (24). A mixture of cosalane di-
methyl ester (21) (239 mg, 0.30 mmol), potassium carbonate
(200 mg) and methyl 4′-(bromomethyl)biphenyl-4-carboxylate
(20; 215 mg, 0.70 mmol) in DMF (5 mL) was stirred at room
temperature for 36 h. The solution was poured into ice-water
(10 mL) and extracted with EtOAc (4 × 15 mL). The organic
layers were combined, dried over Na2SO4, evaporated and the
residue purified by flash chromatography on silica gel (45 g),
eluting with hexanes-ethyl acetate (5:1), to give a white foamy
solid (0.173 g, 46%): mp 89-91 °C; IR (CHCl3) 3027, 2925,
2849, 1725, 1608, 1463, 1278, 1106 cm-1; 1H NMR (300 MHz,
CDCl3) δ 8.11 (d, J ) 8.3 Hz, 2 H), 8.10 (d, J ) 8.3 Hz, 2 H),
7.71-7.65 (m, 12 H), 7.55 (d, J ) 2.3 Hz, 1 H), 7.52 (d, J ) 2.1
Hz, 1 H), 7.38 (d, J ) 2.1 Hz, 1 H), 7.36 (d, J ) 2.3 Hz, 1 H),
6.10 (t, J ) 7.6 Hz, 1 H), 5.22 (s, 2 H), 5.13 (s, 2 H), 3.94 (s, 3
H), 3.94 (s, 3 H), 3.87 (s, 3 H), 3.86 (s, 3 H), 2.08 (q, J ) 7.6
Hz, 2 H), 1.95 (bd, J ) 12.0 Hz, 1 H), 0.88 (d, J ) 6.5 Hz, 3 H),
0.86 (d, J ) 6.6 Hz, 3 H), 0.85 (d, J ) 6.5 Hz, 3 H), 0.71 (s, 3
H), 0.62 (s, 3 H). Anal. (C77H88Cl2O10‚H2O) C, H.
Cosa la n e Dim eth yl Ester (21). Cosalane (1) (384 mg, 0.5
mmol) was dissolved under nitrogen in a mixture of methanol
(4.0 mL) and benzene (8.0 mL). (Trimethylsilyl)diazomethane19
(2.0 M in hexane, 0.8 mL, 1.6 mmol) was added dropwise. After
stirring for 25 min, the solvent was evaporated off to leave a
white foamy solid of the desired diester in quantitative yield:
mp (softens at 88 °C) 161-162 °C; IR (CHCl3) 3160, 2926,
2852, 2362, 1682, 1606, 1465, 1443, 1324, 1286, 1243, 1198,
1175 cm-1; 1H NMR (300 MHz, CDCl3) δ 11.31 (s, 1 H), 11.23
(s, 1 H), 7.47 (d, J ) 2.1 Hz, 1 H), 7.44 (d, J ) 2.3 Hz, 1 H),
7.33 (d, J ) 2.2 Hz, 1 H), 7.27 (d, J ) 2.1 Hz, 1 H), 5.90 (t, J
) 7.6 Hz, 1 H), 3.89 (s, 3 H), 3.87 (s, 3 H), 1.99 (q, J ) 7.4 Hz,
2 H), 1.88 (d, J ) 12.2 Hz, 1 H), 1.82-1.65 (m, 1 H), 1.65-
0.88 (m, 32 H), 0.82 (d, J ) 6.6 Hz, 3 H), 0.79 (d, J ) 6.6 Hz,
6 H), 0.65 (s, 3 H), 0.57 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ
170.2, 156.5, 137.0, 134.4, 134.1, 132.1, 130.7, 129.4, 126.7,
122.3, 122.2, 113.5, 113.2, 56.6, 56.3, 54.7, 52.8, 46.6, 42.6, 40.1,
39.5, 38.5, 37.5, 37.4, 36.2, 36.1, 35.8, 35.5, 32.1, 29.0, 28.9,
28.2, 28.0, 27.2, 24.2, 23.8, 22.8, 22.5, 21.0, 18.7, 12.3, 12.1.
Anal. (C47H64Cl2O6) C, H.
3′′,3′′′-Dich lor o-5′′,5′′′-d i(m eth oxyca r bon yl)-4′′,4′′′-d i[p-
(2-m eth oxyca r bon ylp h en yl)ben zyloxy]-4′,4′-d ip h en yl-3â-
(3′-bu ten -1′-yl)ch olesta n e (22). A mixture of cosalane di-
methyl ester (21) (360 mg, 0.45 mmol), potassium carbonate
(700 mg) and methyl 4′-(bromomethyl)biphenyl-2-carboxylate
(18; 368 mg, 1.2 mmol) in DMF (7 mL) was stirred at 80 °C
for 24 h. The solution was cooled, poured into ice-water (15
mL) and extracted with EtOAc (4 × 20 mL). The organic layers
were combined, dried over Na2SO4, evaporated and the residue
purified by flash chromatography on silica gel (80 g), eluting
with hexanes-ethyl acetate (5:1), to give a white foamy solid
(0.336 g, 60%): mp 70-72 °C; IR (CHCl3) 3027, 2925, 2849,
1725, 1608, 1463, 1278, 1106 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.82 (bd, J ) 7.7 Hz, 2 H), 7.64-7.50 (m, 8 H), 7.45-7.32
(m, 10 H), 6.10 (t, J ) 7.6 Hz, 1 H), 5.20 (s, 2 H), 5.12 (s, 2 H),
3.87 (s, 3 H), 3.86 (s, 3 H), 3.67 (s, 3 H), 3.66 (s, 3 H), 2.11 (q,
J ) 7.6 Hz, 2 H), 1.95 (bd, J ) 12.0 Hz, 1 H), 0.88 (d, J ) 6.5
Hz, 3 H), 0.85 (d, J ) 6.6 Hz, 6 H), 0.72 (s, 3 H), 0.63 (s, 3 H).
Anal. (C77H88Cl2O10) C, H.
3′′,3′′′-Dich lor o-5′′,5′′′-d i(m eth oxyca r bon yl)-4′′,4′′′-d i[p-
(3-m eth oxyca r bon ylp h en yl)ben zyloxy]-4′,4′-d ip h en yl-3â-
(3′-bu ten -1′-yl)ch olesta n e (23). A mixture of cosalane di-
methyl ester (21) (360 mg, 0.45 mmol), potassium carbonate
(700 mg) and methyl 4′-(bromomethyl)biphenyl-3-carboxylate
(19; 368 mg, 1.2 mmol) in DMF (7 mL) was stirred at 80 °C
for 24 h. The solution was cooled, poured into ice-water (15
mL) and extracted with EtOAc (4 × 20 mL). The organic layers
were combined, dried over Na2SO4, evaporated and the residue
purified by flash chromatography on silica gel (80 g), eluting
with hexanes-ethyl acetate (5:1), to give a white foamy solid
(0.336 g, 60%): mp 83-85 °C; IR (CHCl3) 3027, 2925, 2849,
1725, 1608, 1463, 1278, 1106 cm-1; 1H NMR (300 MHz, CDCl3)
δ 8.32-8.28 (m, 2 H), 8.02 (dd, J ) 1.4 and 7.7 Hz, 2 H), 7.82-
7.77 (m, 2 H), 7.70-7.61 (m, 8 H), 7.55-7.49 (m, 4 H), 7.37
(dd, J ) 2.3 and 8.5 Hz, 2 H), 6.11 (t, J ) 7.6 Hz, 1 H), 5.22 (s,
2 H), 5.13 (s, 2 H), 3.95 (s, 3 H), 3.95 (s, 3 H), 3.87 (s, 3 H),
3′′,3′′′-Dich lor o-5′′,5′′′-d ica r boxy-4′′,4′′′-d i[p-(2-ca r boxy-
ph en yl)ben zyloxy]-4′,4′-diph en yl-3â-(3′-bu ten -1′-yl)ch oles-
ta n e (25). Tetraester 22 (210 mg, 0.16 mmol) was suspended
in a mixture (5:1) of methanol-ethanol (5 mL) and aqueous
NaOH (5 M, 2 mL) was added. The mixture was heated at
reflux for 14 h to obtain a clear solution. The solvent was
evaporated and water (10 mL) was added. The solution was
extracted with ethyl acetate (2 × 6 mL). The aqueous layer
was acidified with dilute HCl. The precipitate was collected
by filtration, washed with water and dried under vacuum to
afford the acid 25 (120 mg, 60%): mp 152-154 °C; IR (CHCl3)
1
2925, 2849, 1697, 1469, 1245 cm-1; H NMR (300 MHz, CD3-
SOCD3) δ 7.74 (d, J ) 8.0 Hz, 2 H), 7.62-7.50 (m, 8 H), 7.50-
7.32 (m 10 H), 6.27 (t, J ) 7.6 Hz, 1 H), 5.12 (s, 2 H), 5.04 (s,
2 H), 0.86 (d, J ) 7.0 Hz, 3 H), 0.82 (d, J ) 7.5 Hz, 6 H), 0.67
(s, 3 H), 0.59 (s, 3 H). Anal. (C73H80Cl2O10‚H2O) C, H.
3′′,3′′′-Dich lor o-5′′,5′′′-d ica r boxy-4′′,4′′′-d i[p-(3-ca r boxy-
ph en yl)ben zyloxy]-4′,4′-diph en yl-3â-(3′-bu ten -1′-yl)ch oles-
ta n e (26). Tetraester 23 (230 mg, 0.18 mmol), KCN (7.0 mg)
and K2CO3 (400 mg) were suspended in ethanol (7 mL) and
water (1.5 mL). The mixture was heated on an oil bath (95
°C) for 20 h. Ethanol was evaporated off and water (10 mL)
was added. The aqueous solution was stirred at 80 °C for 2 h.
After cooling to room temperature, the solution was extracted
with ethyl acetate (2 × 12 mL). The aqueous layer was
acidified with concentrated HCl and diluted with water. The
precipitate was collected by filtration and dried under vacuum
to yield the acid (82 mg, 38%): mp (softens at 182 °C) 196-
198 °C; IR (CHCl3) 3400, 2923, 2854, 1696, 1606, 1453, 1250
1
cm-1; H NMR (300 MHz, DMSO-d6) δ 8.33 (td, J ) 1.5 and
5.3 Hz, 2 H), 8.03 (dd, J ) 1.5 and 7.5 Hz, 2 H), 7.95 (bd, J )
7.9 Hz, 2 H), 7.78-7.59 (m, 12 H), 7.57 (d, J ) 2.5 Hz, 2 H),
6.34 (t, J ) 7.6 Hz, 1 H), 5.28 (s, 2 H), 5.20 (s, 2 H), 0.91 (d, J
) 7.0 Hz, 3 H), 0.85 (d, J ) 7.5 Hz, 6 H), 0.75 (s, 3 H), 0.66 (s,
3 H). Anal. (C73H80Cl2O10‚3H2O) C, H.
3′′,3′′′-Dich lor o-5′′,5′′′-d ica r boxy-4′′,4′′′-d i[p-(4-ca r boxy-
ph en yl)ben zyloxy]-4′,4′-diph en yl-3â-(3′-bu ten -1′-yl)ch oles-
ta n e (27). Tetraester 24 (200 mg, 0.16 mmol), KCN (5.4 mg)
and K2CO3 (300 mg) were suspended in ethanol (6 mL) and
water (1.5 mL). The mixture was heated on an oil bath (90
°C) for 20 h. Ethanol was distilled off and water (10 mL) was
added. The aqueous solution was stirred at 80 °C for 1 h. After
cooling to room temperature, the solution was extracted with
ethyl acetate (3 × 10 mL). The aqueous layer was acidified
with concentrated HCl and diluted with water. The precipitate
was collected by filtration and dried under vacuum to afford
an almost pure acid (100 mg). It was further purified by
crystallization from ethyl acetate-hexane mixture to obtain
the pure sample (70 mg, 42%): mp 210-212 °C; IR (CHCl3)

Correlation of Anti-HIV Activity with Anion Spacing
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 711
3390, 3039, 2923, 2852, 1690, 1606, 1454, 1277 cm-1; 1H NMR
(300 MHz, CD3COCD3) δ 8.14 (d, J ) 2.5 Hz, 2 H), 8.12 (d, J
) 2.5 Hz, 2 H), 7.88-7.66 (m, 14 H), 7.58 (d, J ) 2.5 Hz, 2 H),
6.35 (t, J ) 7.6 Hz, 1 H), 5.27 (s, 2 H), 5.20 (s, 2 H), 0.91 (d, J
) 7.0 Hz, 3 H), 0.85 (d, J ) 7.5 Hz, 6 H), 0.75 (s, 3 H), 0.66 (s,
3 H). Anal. (C73H80Cl2O10‚1.5H2O) C, H.
of the solution was added (a light yellow color should persist).
After 20-30 min of stirring, the solvent was evaporated off to
leave a white residue that was purified by flash chromatog-
raphy on silica gel (40 g silica, 10:1 hexanes-ethyl acetate) to
obtain a white foamy solid (145 mg, 70% overall yield): mp
144-146 °C; IR (CHCl3) 3160, 2930, 2852, 1681, 1611, 1462,
1443, 1382, 1335, 1283, 1249, 1198, 1176 cm-1; 1H NMR (300
MHz, CDCl3) δ 11.14 (s, 2 H), 7.50 (d, J ) 2.1 Hz, 2 H), 7.29
(d, J ) 2.2 Hz, 2 H), 3.90 (s, 6 H), 3.66 (t, J ) 7.6 Hz, 1 H),
1.90-1.78 (m, 3 H), 1.78-1.62 (m, 1 H), 1.62-0.86 (m, 34 H),
0.82 (d, J ) 6.6 Hz, 3 H), 0.79 (d, J ) 6.5 Hz, 6 H), 0.65 (s, 3
H), 0.57 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ 170.1, 155.9,
135.7, 135.0, 127.0, 122.4, 113.4, 56.6, 56.3, 54.7, 52.7, 48.9,
46.6, 42.6, 40.1, 39.5, 38.6, 37.7, 37.1, 36.2, 36.1, 35.8, 35.7,
35.5, 35.4, 32.1, 31.6, 29.7, 29.0, 28.2, 28.0, 24.9, 24.2, 23.8,
22.8, 22.5, 21.0, 18.6, 12.3, 12.1. Anal. (C47H66Cl2O6) C, H.
Meth yl 4-(Br om om eth yl)p h en yla ceta te (28). 4-(Bro-
momethyl)phenylacetic acid (0.50 g, 2.19 mmol) was dissolved
in a mixture of methanol (4 mL) and benzene (6 mL). A hexane
solution of (trimethylsilyl)diazomethane19 (2.0 M, 1.5 mL) was
added dropwise to the acid solution until the solution turned
light yellow. After 20 min of stirring, glacial acetic acid was
added in small portions until the yellow color disappeared. The
colorless solution was concentrated by evaporation and dried
in vacuo to yield 2836 as a colorless oily liquid (0.51 g): IR
(CHCl3) 3011, 2952, 2842, 1732, 1614, 1435, 1341, 1260, 1229,
1203, 1069, 1036, 1014 cm-1 1H NMR (300 MHz, CDCl3) δ
;
3′′,3′′′-Dich lor o-5′′,5′′′-d i(m eth oxyca r bon yl)-4′′,4′′′-d i[p-
(m et h oxyca r bon yl)ben zyloxy]-4′,4′-d ip h en yl-3â-(1′-b u -
tyl)ch olesta n e (33). Dimethyl dihydrocosalane (32) (181 mg,
0.23 mmol), potassium carbonate (138 mg, 1 mmol) and methyl
p-bromomethylbenzoate (114 mg, 0.50 mmol) in DMF (4 mL)
were stirred at room temperature for 20 h. The solution was
poured into iced water (10 mL) and extracted with EtOAc (4
× 15 mL). The organic layers were combined, dried over Na2-
SO4, evaporated and the residue purified by flash chromatog-
raphy on silica gel (75 g), eluting with hexanes-ethyl acetate
(7:1 to 9:4), to give a white foamy solid (0.194 g, 78%): mp
70-72 °C; IR (CHCl3) 3026, 2953, 2928, 1724, 1455, 1277,
7.35 (d, J ) 8.2 Hz, 2 H), 7.25 (d, J ) 8.2 Hz, 2 H), 4.47 (s, 2
H), 3.68 (s, 3 H), 3.61 (s, 2 H); 13C NMR (75 MHz, CDCl3) δ
171.6, 136.6, 134.2, 129.7, 129.2, 52.0, 40.8, 33.1. Anal. (C10H11
BrO2) C, H.
-
3′′,3′′′-Dich lor o-5′′,5′′′-d i(m eth oxyca r bon yl)-4′′,4′′′-d i[p-
[(m eth oxyca r bon yl)m eth yl]ben zyloxy]-4′,4′-d ip h en yl-3â-
(3′-bu ten -1′-yl)ch olesta n e (29). Cosalane dimethyl ester (21)
(180 mg, 0.23 mmol), potassium carbonate (140 mg) and
methyl 4-(bromomethyl)phenylacetate (28) (190 mg, 0.78
mmol) in DMF (5 mL) were stirred at room temperature for
22 h. The solution was poured into ice-water (8 mL) and
extracted with EtOAc (4 × 15 mL). The organic layers were
combined, dried over Na2SO4, evaporated and the residue
purified by flash chromatography on silica gel (70 g), eluting
with hexanes-ethyl acetate (4:1), to give a white foamy solid
(0.112 g, 44%): mp 56-58 °C; IR (CHCl3) 3027, 2953, 2926,
1
1194, 1105 cm-1; H NMR (300 MHz, CDCl3) δ 8.06 (d, J )
8.2 Hz, 4 H), 7.58 (d, J ) 8.2 Hz, 4 H), 7.56 (d, J ) 2.2 Hz, 2
H), 7.40 (d, J ) 2.2 Hz, 2 H), 5.12 (s, 4 H), 3.92 (s, 6 H), 3.83
(s, 6 H), 3.90 [t, (burried under singlets), 1 H], 0.88 (d, J ) 6.6
Hz, 3 H), 0.85 (d, J ) 6.6 Hz, 3 H), 0.84 (d, J ) 6.6 Hz, 3 H),
0.71 (s, 3 H), 0.62 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ 168.8,
165.6, 152.6, 141.9, 141.1, 133.2, 130.0, 129.7, 129.1, 127.6,
127.1, 75.1, 56.5, 56.2, 54.6, 52.5, 52.1, 49.5, 46.5, 42.5, 40.0,
39.4, 38.5, 37.6, 37.0, 36.1, 36.0, 35.7, 35.6, 35.5, 35.3, 32.1,
29.0, 28.9, 28.2, 27.9, 24.9, 24.1, 23.8, 22.8, 22.5, 20.9, 18.6,
12.3, 12.0. Anal. (C65H82Cl2O10) C, H.
1
2850, 1736, 1465, 1251, 1161, 1021, 975 cm-1; H NMR (300
MHz, CDCl3) δ 7.58-7.46 (m, 6 H), 7.40-7.30 (m, 6 H), 7.42-
7.33 (m, 6 H), 6.09 (t, J ) 7.6 Hz, 1 H), 5.13 (s, 2 H), 5.05 (s,
2 H), 3.85 (s, 3 H), 3.83 (s, 3 H), 3.69 (s, 3 H), 3.68 (s, 3 H),
3.65 (s, 2 H), 3.64 (s, 2 H), 2.08 (q, J ) 7.6 Hz, 2 H), 1.95 (bd,
J ) 12.0 Hz, 1 H), 0.88 (d, J ) 6.5 Hz, 3 H), 0.86 (d, J ) 6.6
Hz, 3 H), 0.85 (d, J ) 6.5 Hz, 3 H), 0.71 (s, 3 H), 0.63 (s, 3 H);
13C NMR (75 MHz, CDCl3) δ 171.9, 165.8, 165.5, 153.5, 153.1,
138.9, 136.8, 135.8, 135.6, 135.2, 134.0, 132.4, 131.2, 130.0,
129.8, 129.4, 128.7, 128.6, 128.1, 127.3, 127.2, 77.2, 75.7, 56.6,
56.3, 54.6, 52.5, 52.0, 46.5, 42.6, 41.0, 40.0, 39.5, 38.5, 37.4,
37.2, 36.1, 36.0, 35.8, 35.5, 32.1, 29.0, 28.8, 28.2, 28.0, 27.2,
24.2, 23.8, 22.8, 22.5, 18.6, 12.3, 12.0. Anal. (C67H84Cl2O10) C,
H.
3′′,3′′′-Dich lor o-5′′,5′′′-d ica r b oxy-4′′,4′′′-d i(p -ca r b oxy-
ben zyloxy)-4′,4′-diph en yl-3â-(1′-bu tyl)ch olestan e (34). Tet-
raester 33 (0.227 g, 0.207 mmol), KCN (5.4 mg) and K2CO3
(0.23 g) were suspended in ethanol (7 mL) and water (1.5 mL).
The mixture was heated on an oil bath (80 °C) for 12 h. Ethanol
was distilled off and water (8 mL) was added. The aqueous
solution was stirred at 80 °C for 2 h. After cooling to room
temperature, the solution was washed with ethyl acetate (2
× 6 mL). The aqueous layer was acidified with concentrated
HCl and extracted with ethyl acetate (3 × 20 mL). The organic
layers were combined, dried over Na2SO4, and concentrated
by evaporation to obtain the desired acid 34 (150 mg, 70%) as
a white solid: mp 146-148 °C; IR (CHCl3) 3373, 3019, 2924,
1685, 1615, 1471, 1420, 1293 cm-1; 1H NMR (300 MHz, DMSO-
d6) δ 8.06 (d, J ) 8.2 Hz, 4 H), 7.84 (d, J ) 2.2 Hz, 2 H), 7.77
(d, J ) 2.2 Hz, 2 H), 7.68 (d, J ) 8.2 Hz, 4 H), 5.19 (s, 4 H),
4.20 (t, J ) 7.71 Hz, 1 H), 2.15 (q, J ) 7.2 Hz, 2 H), 0.91 (d, J
) 6.4 Hz, 3 H), 0.85 (d, J ) 6.7 Hz, 6 H), 0.75 (s, 3 H), 0.66 (s,
3 H); 13C NMR (75 MHz, DMSO-d6) δ 172.1, 167.4, 166.4,
153.3, 143.1, 142.9, 133.9, 132.7, 130.9, 130.4, 130.3, 128.7,
75.8, 60.5, 57.4, 57.1, 55.5, 50.0, 47.4, 43.3, 40.9, 40.2, 39.4,
38.4, 37.7, 36.9, 36.8, 36.6, 36.4, 36.3, 35.7, 32.9, 25.6, 24.8,
24.5, 23.0, 22.8, 21.7, 20.8, 19.0, 14.4, 12.6, 12.4; MS (electro-
spray, negative ion mode) m/z 1035 (M - H, 100), 991 (10),
947 (8), 901 (45), 765 (10). Anal. (C61H74Cl2O10) C, H.
5′′,5′′′-Di[(ter t-bu toxycar bon ylm eth yl)car bam oyl]-3′′,3′′′-
d ich lor o-4′′,4′′′-d ih yd r oxy-4′,4′-d ip h en yl-3â-(3′-b u t en -1′-
yl)ch olesta n e (35). Cosalane (77 mg, 0.1 mmol), glycine tert-
butyl ester hydrochloride (38 mg, 0.22 mmol) and BOP (90 mg,
0.2 mmol) were taken in a flask and purged with argon for 15
min. The mixture was dissolved in THF (4 mL). To this
solution was added Et3N (0.22 mL, 0.3 mmol) and the reaction
mixture was stirred at ambient temperature for 16 h. The
reaction mixture was diluted with ethyl acetate (25 mL) and
was washed with 5% aqueous HCl (2 × 10 mL), water (2 × 10
3′′,3′′′-Dich lor o-5′′,5′′′-d ica r b oxy-4′′,4′′′-d i[p -(ca r b oxy-
methyl)benzyloxy]-4′,4′-diphenyl-3â-(3′-buten-1′-yl)cholestane
(30). Tetraester 29 (175 mg, 0.156 mmol), KCN (4.2 mg) and
K2CO3 (240 mg) were suspended in ethanol (5 mL) and water
(1.2 mL). The mixture was heated on an oil bath (80 °C) for
14 h. Ethanol was distilled off and water (8 mL) was added.
The aqueous solution was stirred at 80 °C for 2 h. After cooling
to room temperature, the solution was washed with ethyl
acetate (2 × 5 mL). The aqueous layer was acidified with 1 N
HCl and extracted with ethyl acetate (3 × 20 mL). The organic
layers were combined, dried over Na2SO4, and concentrated
by evaporation to afford a white solid (120 mg). It was further
purified by crystallization from ethyl acetate-hexane mixture
to give pure 30 (70 mg, 42%): mp 142-144 °C; IR (CHCl3)
3390, 3039, 2923, 2854, 1580, 1380 cm-1; 1H NMR (500 MHz,
CD3COCD3) δ 7.68 (d, J ) 2.5 Hz, 1 H), 7.67 (d, J ) 2.5 Hz, 1
H), 7.60-7.53 (m, 6 H), 7.40-7.35 (m, 4 H), 6.35 (t, J ) 7.6
Hz, 1 H), 5.20 (s, 2 H), 5.13 (s, 2 H), 3.68 (s, 2 H), 3.67 (s, 2 H),
0.93 (d, J ) 7.0 Hz, 3 H), 0.88 (d, J ) 7.5 Hz, 3 H), 0.87 (d, J
) 6.5 Hz, 3 H), 0.77 (s, 3 H), 0.68 (s, 3 H). Anal. (C63H76Cl2O10
H2O) C, H.
‚
Dih yd r ocosa la n e Dim eth yl Ester (32). Dihydrocosalane
(31) (200 mg, 0.26 mmol) was dissolved under nitrogen
atmosphere in a mixture of methanol (4.0 mL) and benzene
(8.0 mL). (Trimethylsilyl)diazomethane19 (Aldrich, 2.0 M in
hexane) (0.4 mL, ∼0.8 mmol) was added dropwise to the above
solution. The reaction was instantaneous and a slight excess

712 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Santhosh et al.
mL) and brine (1 × 10 mL). The organic layer was dried
(anhydrous MgSO4). Concentration and rapid purification by
flash chromatography using hexanes-ethyl acetate (4:1) as
eluent to give the product 35 (65 mg, 65%): mp 124-132 °C
ch olesta n e (40). Ester 39 (51 mg, 0.05 mmol) and sodium
hydroxide solution (1 N, 0.3 mL) in a similar procedure as
described for 38 gave pure 40 (32 mg, 75%): mp 224-229 °C;
1H NMR (300 MHz, acetone-d6) δ 13.42 (s, 1 H), 13.32 (s, 1
H), 8.47-8.38 (m, 2 H), 7.79 (d, J ) 2 Hz, 1 H), 7.68 (d, J ) 2
Hz, 1 H), 7.39 (bs, 2 H), 6.04 (t, J ) 7.5 Hz, 1 H), 4.76-4.69
(m, 4 H), 2.14-0.80 (m, 63 H), 0.75 (s, 3 H), 0.66 (s, 3 H); IR
(KBr) 3328, 2936, 1714, 1639, 1588, cm-1; PDMS m/z 994
(MH+). Anal. (C57H82O8N2Cl2‚H2O) C, H, N.
3′′,3′′′-Dich lor o-4′′,4′′′-d ih yd r oxy-5′′,5′′′-d i[(1-m eth oxy-
ca r bon yl-2-p h en yleth yl)ca r ba m oyl]-4′,4′-d ip h en yl-3â-(3′-
bu t en -1′-yl)ch olest a n e (41). Cosalane (77 mg, 0.1 mmol),
L-phenylalanine methyl ester hydrochloride (54 mg, 0.22
mmol), BOP (90 mg, 0.2 mmol), and Et3N (0.22 mL, 0.3 mmol)
in a similar procedure as described for 35 gave pure 41 (66
mg, 62%): mp 110-118 °C; 1H NMR (300 MHz, acetone-d6) δ
13.15 (s, 1 H), 12.98 (s, 1 H), 8.50 (d, J ) 9 Hz, 1 H) 8.42 (d,
J ) 9 Hz, 1 H), 7.60 (d, J ) 2 Hz, 1 H), 7.56 (d, J ) 2 Hz, 1 H),
7.41 (d, J ) 2 Hz, 1 H), 7.38 (d, 1 H), 7.28-7.17 (m, 10 H),
6.03 (t, J ) 7.5 Hz, 1 H), 4.98-4.84 (m, 2 H), 3.69 (s, 3 H),
3.66 (s, 3 H), 3.28-3.22 (m, 2 H), 3.10-2.97 (m, 2 H), 2.00-
0.8 (m, 45 H), 0.75 (s, 3 H), 0.66 (s, 3 H); IR (film) 3385, 2932,
1750, 1723, 1644, 1591 cm-1; PDMS m/z 1090 (MH+).
1
dec; H NMR (300 MHz, CDCl3) δ 13.43 (s, 1 H), 13.34 (s, 1
H), 8.62 (bs, 2 H), 7.67 (d, J ) 2 Hz, 1 H), 7.62 (d, J ) 2 Hz,
1 H), 7.50 (d, J ) 2 Hz, 1 H), 7.15 (d, J ) 2 Hz, 1 H), 7,43 (d,
J ) 2 Hz, 1 H), 6.12 (t, J ) 7.5 Hz, 1 H), 4.03-4.01 (dd, J )
6 Hz, 1.5 Hz, 2 H), 3.96 (dd, J ) 6 Hz, 1.5 Hz, 2 H), 2.19-1.50
(m, 10 H), 1.43 (s, 9 H), 1.42 (s, 9 H), 1.39-0.8 (m, 35 H), 0.74
(s, 3 H), 0.66 (s, 3 H); IR (KBr) 3358, 2916, 1734, 1644, 1548
cm-1; PDMS m/z 994 (MH+). Anal. (C57H82Cl2N2O8) C, H, N.
5′′,5′′′-Di[(ca r boxym eth yl)ca r ba m oyl]-3′′,3′′′-d ich lor o-
4′′,4′′′-d ih yd r oxy-4′,4′-d ip h en yl-3â-(3′-bu ten -1′-yl)ch oles-
ta n e (36). Ester 35 (50 mg, 0.05 mmol) was dissolved in
CF3COOH (2 mL). The mixture was stirred at room temper-
ature for 5-10 min. A white precipitate was formed. After the
reaction was complete, the solvent was evaporated and the
solid obtained was dissolved in ethyl acetate (20 mL). The
organic layer was washed with water (2 × 20 mL) and brine
(1 × 20 mL) and dried (anhydrous MgSO4). Concentration
under vacuum furnished the product (43 mg, 98%): mp 220-
1
226 °C dec; H NMR (300 MHz, acetone-d6) δ 13.42 (s, 1 H),
13.35 (s, 1 H), 8.72-8.66 (m, 2 H), 7.68 (d, J ) 2 Hz, 1 H),
7.63 (d, J ) 2 Hz, 1 H), 7.50 (d, J ) 2 Hz, 1 H), 7.44 (d, J ) 2
Hz, 1 H), 6.12 (t, J ) 7.5 Hz, 1 H), 4.12 (d, J ) 6 Hz, 2 H),
4.06 (d, J ) 6 Hz, 2 H), 2.20-0.8 (m, 45 H), 0.74 (s, 3 H), 0.66
(s, 3 H); IR 3386, 2927, 2849, 1735, 1648, 1593. cm-1; PDMS
m/z 882 (MH+). Anal. (C49H66O8N2Cl2‚0.7CF3COOH) C, H, N.
5′′,5′′′-Di[[2-(et h oxyca r b on yl)et h yl]ca r ba m oyl]-3′′,3′′′-
d ich lor o-4′′,4′′′-d ih yd r oxy-4′,4′-d ip h en yl-3â-(3′-b u t en -1′-
yl)ch olesta n e (37). Cosalane (77 mg, 0.1 mmol), â-alanine
ethyl ester hydrochloride (35 mg, 0.22 mmol), BOP (90 mg,
5′′,5′′′-Di[(1-ca r boxy-2-p h en yleth yl)ca r ba m oyl]-3′′,3′′′-
d ich lor o-4′′,4′′′-d ih yd r oxy-4′,4′-d ip h en yl-3â-(3′-b u t en -1′-
yl)ch olesta n e (42). Ester 41 (55 mg, 0.05 mmol) and sodium
hydroxide solution (1 N, 0.3 mL) in a similar procedure as that
described for 38 gave pure 42 (63 mg, 60%): mp 240-246 °C
dec; 1H NMR (300 MHz, acetone-d6) δ 13.20 (s, 1 H), 12.95 (s,
1 H), 8.5-83 (m, 2 H) 7.60 (d, J ) 2 Hz, 1 H), 7.64 (d, J ) 2
Hz, 1 H), 7.48 (d, J ) 2 Hz, 1 H), 7.40-7.22 (m, 12 H), 6.04 (t,
J ) 7.5 Hz, 1 H), 4.84-4.60 (m, 2 H), 3.44-2.80 (m, 4 H), 2.00-
0.8 (m, 45 H), 0.76 (s, 3 H), 0.67 (s, 3 H); IR (KBr) 3367, 2924,
1719, 1643, 1589 cm-1; PDMS m/z 1062 (MH+). Anal.
(C63H78O8N2Cl2‚H2O) C, H, N.
5′′,5′′′-Di[(1,2-d i-ter t-bu toxyca r bon yleth yl)ca r ba m oyl]-
3′′,3′′′-dich lor o-4′′,4′′′-dih ydr oxy-4′,4′-diph en yl-3â-(3′-bu ten -
1′-yl)ch olesta n e (43). Cosalane (77 mg, 0.1 mmol), L-aspartic
acid di-tert-butyl ester hydrochloride (62 mg, 0.22 mmol), BOP
(90 mg, 0.2 mmol), and Et3N (0.22 mL, 0.3 mmol) in a similar
procedure as described for 35 gave pure 43 (92 mg, 75%): mp
148-152 °C dec; 1H NMR (300 MHz, acetone-d6) δ 13.3 (bs, 2
H), 8.45 (t, J ) 6 Hz, 2 H), 7.64 (bs, 1 H), 7.58 (bs, 1 H), 7.41
(bs, 2 H), 6.07 (t, J ) 7.5 Hz, 1 H), 4.89-4.78 (m, 2 H), 2.92-
2.69 (m, 4 H), 2.14-0.80 (m, 81 H), 0.74 (s, 3 H), 0.66 (s, 3 H);
IR (KBr) 3350, 2929, 2837, 1737, 1639, 1547 cm-1; PDMS m/z
1223 (MH+).
5′′,5′′′-Di[(1,2-dicar boxyeth yl)car bam oyl]-3′′,3′′′-dich lor o-
4′′,4′′′-d ih yd r oxy-4′,4′-d ip h en yl-3â-(3′-bu ten -1′-yl)ch oles-
ta n e (44). Ester 43 (62 mg, 0.05 mmol) and CF3COOH (2 mL)
in a similar procedure as that described for 36 gave pure 44
(94 mg, 95%): mp 242-246 °C dec; 1H NMR (300 MHz, acetone-
d6) δ 13.27 (s, 1 H), 13.18 (s, 1 H), 8.64 (m, 2 H), 7.76 (bs, 1
H), 7.63 (bs, 1 H), 7.42 (d, J ) 2 Hz, 1 H), 7.39 (d, J ) 2 Hz,
1 H), 6.08 (t, J ) 7.5 Hz, 1 H), 5.10-4.96 (m, 2 H), 3.07-2.83
(m, 4 H), 2.20-0.81 (m, 45 H), 0.74 (s, 3 H), 0.67 (s, 3 H); IR
(KBr) 3350, 2929, 1716, 1644, 1542 cm-1; PDMS m/z 998
(MH+). Anal. (C53H70O12N2Cl2‚0.8CF3COOH) C, H, N.
0.2 mmol), and Et3N (0.22 mL, 0.3 mmol) in
a similar
procedure as that described for 35 gave pure 37 (60 mg, 62%):
1
mp 134-138 °C dec; H NMR (300 MHz, acetone-d6) δ 13.88
(s, 1 H), 13.56 (s, 1 H), 8.47-8.41 (m, 2 H), 7.61 (d, J ) 2 Hz,
1 H), 7.53 (d, J ) 2 Hz, 1 H), 7.42 (d, J ) 2 Hz, 1 H), 7.38 (d,
J ) 2 Hz, 1 H), 6.08 (t, J ) 7.5 Hz, 1 H), 4.11-4.03 (q, J ) 6
Hz, 4 H), 3.67-3.55 (m, 4 H), 2.65-2.56 (m, 4 H), 2.20-0.84
(m, 51 H), 0.73 (s, 3 H), 0.66 (s, 3 H); IR 3348, 2926, 1729,
1639, 1588 cm-1; PDMS m/z 965 (MH+).
5′′,5′′′-Di[(2-ca r b oxyet h yl)ca r ba m oyl]-3′′,3′′′-d ich lor o-
4′′,4′′′-d ih yd r oxy-4′,4′-d ip h en yl-3â-(3′-bu ten -1′-yl)ch oles-
ta n e (38). Ester 37 (48 mg, 0.05 mmol) was dissolved in
ethanol (4 mL). Sodium hydroxide solution (1 N, 0.3 mL) was
added. The reaction mixture was stirred at ambient temper-
ature for 16-20 h. The solvent was removed under vacuum
and HCl (1 N, 0.5 mL) was added. The precipitated product
was dissolved in ethyl acetate (20 mL) and the organic solution
was briefly washed with brine (1 × 10 mL) and dried
(anhydrous MgSO4). Concentration and recrystallization from
the mixture of ethanol-ether-hexane (5:3:2) gave the product
38 (32 mg, 72%): mp 224-229 °C; 1H NMR (300 MHz, acetone-
d6) δ 13.70 (s, 1 H), 13.62 (s, 1 H), 8.45-8.40 (m, 2 H), 7.65 (d,
J ) 2 Hz, 1 H), 7.55 (d, J ) 2 Hz, 1 H), 7.40 (d, J ) 2 Hz, 1 H),
7.38 (d, J ) 2 Hz, 1 H), 6.07 (t, J ) 7.5 Hz, 1 H), 3.66-3.55
(m, 4 H), 2.66-2.58 (m, 4 H), 2.14-0.77 (m, 45 H), 0.73 (s, 3
H), 0.66 (s, 3 H); IR (KBr) 3363, 2922, 1712, 1639, 1589 cm-1
;
5′′,5′′′-Di[[1,2-d i(ter t-b u t oxyca r b on yl)p r op yl]ca r b a m -
oyl]-3′′,3′′′-d ich lor o-4′′,4′′′-d ih yd r oxy-4′,4′-d ip h en yl-3â-(3′-
bu t en -1′-yl)ch olest a n e (45). Cosalane (77 mg, 0.1 mmol),
l-glutamic acid di-tert-butyl ester hydrochloride (66 mg, 0.22
mmol), BOP (90 mg, 0.2 mmol), and Et3N (0.22 mL, 0.3 mmol)
in a similar procedure as that described for 35 gave pure 45
PDMS m/z 909 (M + H)+. Anal. (C51H70O8N2Cl2‚0.5H2O) C, H,
N.
3′′,3′′′-Dich lor o-4′′,4′′′-d ih yd r oxy-5′′,5′′′-d i[[1-(m eth oxy-
car bon yl)isopen tyl]car bam oyl]-4′,4′-diph en yl-3â-(3′-bu ten -
1′-yl)ch olesta n e (39). Cosalane (77 mg, 0.1 mmol), L-leucine
methyl ester hydrochloride (40 mg, 0.22 mmol), BOP (90 mg,
1
(95 mg, 76%): mp 154-159 °C; H NMR (300 MHz, acetone-
0.2 mmol), and Et3N (0.22 mL, 0.3 mmol) in
a similar
d6) δ 13.31 (bs, 2 H), 8.50 (d, J ) 9 Hz, 1 H) 8.45 (d, J ) 9 Hz,
1 H), 7.83 (d, J ) 2 Hz, 1 H), 7.67 (bs, 1 H), 7.40 (d, J ) 2 Hz,
1 H), 7.36 (d, J ) 2 Hz, 1 H) 6.10 (t, J ) 7.5 Hz, 1 H), 4.60-
4.52 (m, 2 H), 2.21-2.37 (m, 4 H), 2.2-0.84 (m, 85 H), 0.74 (s,
procedure as that described for 35 gave pure 39 (72 mg, 70%):
mp 112-118 °C; 1H NMR (300 MHz, acetone-d6) δ 13.37 (s, 1
H) 13.25 (1 H), 8.49 (m, 2 H), 7.80 (bs, 1 H), 7.69 (bs, 1 H),
7.43-7.41 (m, 2 H), 6.09 (t, J ) 7.5 Hz, 1 H), 4.70 (m, 2 H),
2.01-0.8 (m, 63 H), 0.77 (s, 3 H), 0.68 (s, 3 H); IR (KBr) 3367,
2957, 1751, 1723, 1640, 1545 cm-1; PDMS m/z 1022 (MH+) .
3 H), 0.66 (s, 3 H); IR (KBr) 3338, 2926, 1729, 1644, 1542 cm-1
;
PDMS m/z 1248 (MH+).
5′′,5′′′-Di[(1,2-d i-ter t-ca r boxyp r op yl)ca r ba m oyl]-3′′,3′′′-
d ich lor o-4′′,4′′′-d ih yd r oxy-4′,4′-d ip h en yl-3â-(3′-b u t en -1′-
yl)ch olesta n e (46). Ester 45 (63 mg, 0.05 mmol) and CF3-
3′′,3′′′-Dich lor o-4′′,4′′′-d ih yd r oxy-5′′,5′′′-d i[(1-ca r b oxy-
isop en t yl)ca r b a m oyl]-4′,4′-d ip h en yl-3â-(3′-b u t en -1′-yl)-

Correlation of Anti-HIV Activity with Anion Spacing
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 713
COOH (2 mL) in a similar procedure as that described for 36
gave pure 46 (49 mg, 98%): mp 236-242 °C; 1H NMR (300
MHz, acetone-d6) δ 7.84 (d, J ) 2 Hz, 1 H), 7.71 (bs, 1 H), 7.40
(bs, 2 H), 6.11 (t, J ) 7.5 Hz, 1 H), 4.74-4.67 (m, 2 H), 2.60-
0.84 (m, 49 H), 0.74 (s, 3 H), 0.66 (s, 3 H); IR (KBr) 3360, 2930,
2847, 1716, 1639, 1542 cm-1; PDMS m/z 1026 (MH+). Anal.
(C55H74O12N2Cl2‚CF3COOH) C, H, N.
5′′,5′′′-Di[(1,2-d i-ter t-bu toxyca r bon yleth yl)ca r ba m oyl]-
3′′,3′′′-d ich lor o-4′′,4′′′-d ih yd r oxy-4′,4′-d ip h en yl-3â-(1′-bu -
tyl)ch olesta n e (47). Dihydrocosalane (78 mg, 0.1 mmol),
glycine tert-butyl ester hydrochloride (34 mg, 0.22 mmol), BOP
(90 mg, 0.2 mmol), and Et3N (0.22 mL, 0.3 mmol) in a similar
procedure as that described for 35 gave pure 47 (32 mg, 31%):
1H NMR (500 MHz, acetone-d6) δ 8.60 (bs, 2 H), 7.2 (bs, 2 H),
7.46 (bs, 2 H), 4.05-4.04 (m, 4 H), 3.62 (t, J ) 6 Hz, 1 H),
2.20-0.93 (m, 54 H), 0.92 (d, J ) 4 Hz, 3 H), 0.84 (d, J ) 4
Hz, 6 H), 0.72 (s, 3 H), 0.62 (s, 3 H); IR (CHCl3) 3369, 2923,
1736, 1648, 1591 cm-1; PDMS m/z 995 (MH+).
5′′,5′′′-Di[(1,2-dicar boxyeth yl)car bam oyl]-3′′,3′′′-dich lor o-
4′′,4′′′-dih ydr oxy-4′,4′-diph en yl-3â-(1′-bu tyl)ch olestan e (48).
Ester 47 (50 mg, 0.05 mmol) and CF3COOH (2 mL) in a similar
procedure as that described for 36 gave pure 48 (39 mg, 90%):
1H NMR (300 MHz, acetone-d6) δ 13.3 (s, 2 H), 8.68 (bs, 2 H),
7.8 (bs, 2 H), 7.52 (bs, 2 H), 4.20-4.22 (m, 4 H), 3.80 (t, J ) 6
Hz, 1 H), 2.10-0.90 (m, 36 H), 0.88 (d, J ) 4 Hz, 3 H), 0.84 (d,
J ) 4 Hz, 6 H), 0.72 (s, 3 H), 0.62 (s, 3 H); IR (CHCl3) 3378,
2932, 1725, 1644, 1595 cm-1; PDMS m/z 886 (MH+). Anal.
(C55H74O12N2Cl2‚1.3CF3COOH) C, H, N.
5′′,5′′′-Di[[1,2-d i(ter t-b u t oxyca r b on yl)p r op yl]ca r b a m -
oyl]-3′′,3′′′-d ich lor o-4′′,4′′′-d ih yd r oxy-4′,4′-d ip h en yl-3â-(1′-
bu tyl)ch olesta n e (49). Dihydrocosalane (78 mg, 0.1 mmol),
L-glutamic acid di-tert-butyl ester hydrochloride (66 mg, 0.22
mmol), BOP (90 mg, 0.2 mmol), and Et3N (0.22 mL, 0.3 mmol)
in a similar procedure as described for 35 gave pure 49 (45
mg, 36%): 1H NMR (300 MHz, acetone-d6) 8.30 (bs, 2 H), 7.70
(d, J ) 2 Hz, 2 H), 7.32 (d, J ) 2 Hz, 2 H), 4.48-4.44 (m, 2 H),
3.81(t, J ) 7.5 Hz, 1 H), 2.24-2.22 (m, 4 H), 2.10-0.84 (m, 86
H), 0.76 (s, 3 H), 0.65 (s, 3 H); IR (film) 3369, 2923, 1736, 1648,
1591 cm-1; PDMS m/z 1249 (MH+).
confirmed by both macroscopic and microscopic observation
of the assay. Data are presented as the percent control of MTS
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. All MTS 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.
Evaluation of the antiviral activity of the compounds against
HIV-1IIIB and HIV-2ROD in MT-4 cells was performed using the
MTT cytoprotection assay as previously described.38 Stock
solutions (10× final concentration) of test compounds were
added in 25-µL volumes to two series of triplicate wells so as
to allow simultaneous evaluation of their effects on mock- and
HIV-infected cells at the beginning of each experiment. Serial
5-fold dilutions of test compounds were made directly in flat-
bottom 96-well plastic microtiter trays using a Biomek 2000
robot (Beckman Instruments, Fullerton, CA). Untreated con-
trol HIV- and mock-infected cell samples were included for
each sample.
39
40
HIV-1IIIB or HIV-2ROD stock (50 µL) at 100-300 CCID50
(cell culture infectious dose) or culture medium was added to
either the infected or mock-infected wells of the microtiter tray.
Mock-infected cells were used to evaluate the effect of test
compounds on uninfected cells and to determine the concen-
tration at which the test compounds were cytotoxic. Exponen-
tially growing MT-4 cells41 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-µL volumes were transferred to the microtiter
tray wells. Five days after infection, the viability of mock- and
HIV-infected cells was examined spectrophotometrically by the
MTT assay.
The MTT assay is based on the reduction of yellow colored
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (Acros Organics, Geel, Belgium) by mitochondrial
dehydrogenase of metabolically active cells to a blue formazan
that can be measured spectrophotometrically. The absorbances
were read in an eight-channel computer-controlled photometer
(Multiskan Ascent Reader, Labsystems, Helsinki, Finland), at
two wavelengths (540 and 690 nm). All data were calculated
using the median OD (optical density) value of three wells.
The 50% cytotoxic concentration (CC50) was defined as the
5′′,5′′′-Di[(1,2-d i-ter t-ca r boxyp r op yl)ca r ba m oyl]-3′′,3′′′-
d ich lor o-4′′,4′′′-d ih yd r oxy-4′,4′-d ip h e n yl-3â-(1′-b u t yl)-
ch olesta n e (50). Ester 49 (50 mg) and CF3COOH (2 mL) in
a similar procedure as that described for 36 gave pure 50 (34
mg, 88%): 1H NMR (300 MHz, acetone-d6) δ 13.00 (s, 2 H)
8.53 (bs, 2 H), 7.87 (bs, 1 H), 7.84 (bs, 1 H), 7.51 (d, J ) 1.5
Hz, 1 H), 7.50 (bs, 1 H) 4.72-4.68 (m, 2 H), 3.89 (t, J ) 7.5
Hz, 1 H), 2.60 (m, 4 H) 2.20-0.90 (m, 36 H), 0.92 (d, J ) 4 Hz,
3 H), 0.85 (d, J ) 4 Hz, 6 H), 0.74 (s, 3 H), 0.66 (s, 3 H); IR
3376, 2930, 1722, 1648, 1597 cm-1; PDMS m/z 1029 (MH+).
Anal. (C55H76Cl2N2O12‚0.25CF3COOH) C, H, N.
concentration of extract that reduced the absorbance (OD540
)
of the mock-infected control sample by 50%. The concentration
achieving 50% protection from the cytopathic effect of the virus
in infected cells was defined as the 50% effective concentration
(EC50).
In Vitr o An ti-HIV Assa ys. Evaluation of the antiviral
activity of compounds against HIV-1RF infection in CEM-SS
cells was performed using the MTS cytoprotection assay as
previously described.37 This cell-based microtiter assay quan-
titates the drug-induced protection from the cytopathic effect
of HIV-1. Briefly, exponentially growing CEM-SS cells (ob-
tained from the AIDS Research and Reference Reagent
Repository, Bethesda, MD) were collected by centrifugation
and resuspended in fresh tissue culture medium. A pretitered
aliquot of HIV-1RF (AIDS Research and Reference Reagent
Repository, Bethesda, MD), 5 × 103 cells and serially diluted
compounds were placed into 0.2-cm round-bottomed microtiter
plates (final volume 200 µL). Each plate contained cell control
wells (cells only), virus control wells (cells plus virus), drug
toxicity control wells (cells plus drug only), drug colorimetric
control wells (drug only) as well as experimental wells (drug
plus cells plus virus). Cultures were incubated for 6 days at
37 °C, 5% CO2. At assay termination, the assay plates were
stained with the soluble tetrazolium-based dye MTS (CellTiter
Reagent, Promega) to determine HIV cytoprotection and
quantify compound toxicity. 20 µL of MTS reagent was added
per well and allowed to develop overnight at 37 °C. Prior to
quantitation, the formazan product was mixed and the plate
was read spectrophotometrically at 490 nm. Activity was
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 also thank Kristien Erven for
excellent technical assistance.
Refer en ces
(1) Barry, M.; Mulcahy, F.; Back, D. J . Antiretroviral Therapy for
Patients with HIV Disease. Br. J . Clin. Pharmacol. 1998, 45,
221-228.
(2) Fischl, M. A. Antiretroviral Therapy in 1999 for Antiretroviral-
Naive Individuals with HIV Infection. AIDS 1999, 13 (Suppl.
1), S49-S59.
(3) Vajpayee, M.; Dar, L. Antiretroviral Drugs in HIV Infection.
Indian J . Pharmacol. 1999, 31, 96-103.
(4) Beach, J . W. Chemotherapeutic Agents for Human Immunode-
ficiency Virus Infection: Mechanism of Action, Pharmacokinet-
ics, Metabolism, and Adverse Reactions. Clin. Ther. 1998, 20,
2-25.
(5) 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.

714 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Santhosh et al.
(6) Reymen, D.; Witvrouw, M.; Este´, 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.
(7) 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,
3040-3050.
(8) 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.
(9) 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.
(10) 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.
(11) Casimiro-Garcia, A.; De Clercq, E.; Pannecouque, C.; Witvrouw,
M.; Stup, T. L.; Turpin, J . A.; Buckheit, R. W. J .; Cushman, M.
Synthesis and Anti-HIV Activity of Cosalane Analogues Incor-
porating Nitrogen in the Linker Chain. Bioorg. Med. Chem.
2000, 8, 191-200.
(12) Cushman, M.; Insaf, S.; Ruell, J . A.; Schaeffer, W. G.; Rice, W.
G. Synthesis of a Cosalane Analogue with an Extended Polya-
nionic Pharmacophore Conferring Enhanced Potency as an Anti-
HIV Agent. Bioorg. Med. Chem. Lett. 1998, 8, 833-836.
(13) 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.
(14) Kwong, P. D.; Wyatt, R.; Robinson, J .; Sweet, R. W.; Sodroski,
J .; Hendrickson, W. A. Structure of an HIV gp120 Envelope
Glycoprotein in Complex with the CD4 Receptor and a Neutral-
izing Human Antibody. Nature 1998, 393, 648-659.
(15) 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.
(16) 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.
(17) Cushman, M.; Insaf, S.; Paul, G.; Ruell, J .; De Clercq, E.; Schols,
D.; Pannecouque, C.; Witvrouw, M.; Schaeffer, C. A.; Turpin, J .
A.; Williamson, K.; Rice, W. G. Extension of the Cosalane
Pharmacophore as a Strategy for Increasing Anti-HIV Potency.
J . Med. Chem. 1999, 42, 1767-1777.
(18) Ruell, J . A.; De Clercq, E.; Pannecouque, C.; Witvrouw, M.; Stup,
T.; Turpin, J . A.; Buckheit, R. W. J .; Cushman, M. Synthesis
and Anti-HIV Activity of Cosalane Analogues with Substituted
Benzoic Acid Rings Attached to the Pharmacophore through
Methylene and Amide Linkers. J . Org. Chem. 1999, 64, 5858-
5866.
(25) Tsuji, A.; Tamai, I. Carrier-Mediated Intestinal Transport of
Drugs. Pharm. Res. 1996, 13, 963-977.
(26) Bumagin, N.; Bykov, V. V. Ligandless Palladium Catalyzed
Reactions of Arylboronic Acids and Sodium Tetraphenylborate
with Aryl Halides in Aqueous Media. Tetrahedron 1997, 53,
14437-14450.
(27) Hattori, T.; Hayashizaka, N.; Miyano, S. Practical Synthesis of
4′-Methylbiphenyl-2-carboxylic Acid. Synthesis 1995, 41-43.
(28) Carini, D. J .; Duncia, J . V.; Aldrich, P. E.; Chiu, A. T.; J ohnson,
A. L.; Pierce, M. E.; Price, W. A.; Santella, J . B. I.; Wells, G. J .;
Wexler, R. R.; Wong, P. C.; Yoo, S.-E.; Timmermans, P. B. M.
W. M. Nonpeptide Angiotensin II Receptor Antagonists: The
Discovery of a Series of N-(Biphenylmethyl)imidazoles as Potent,
Orally Active Antihypertensives. J . Med. Chem. 1991, 34, 2525-
2547.
(29) Frenette, R.; Friesen, R. W. Biaryl Synthesis via Suzuki Coupling
on a Solid Support. Tetrahedron Lett. 1994, 35, 9177-9180.
(30) Bumagin, N. A.; Bykov, V. V. Reaction of Organoboron Com-
pounds with Organic Halides Catalyzed by Ligand-Free Pal-
ladium in Aqueous Media. Zh. Obshch. Khim. 1996, 66, 1981-
1994.
(31) Carini, D. J .; Duncia, J . V.; J ohnson, A. L.; Chiu, A. T.; Price,
W. A.; Wong, P. C.; Timmermans, P. B. M. W. Nonpeptide
Antiotensin II Receptor Antagonists: N-[(Benzyloxy)benzyl]-
imidazoles and Related Compounds as Potent Antihypertensives.
J . Med. Chem. 1990, 33, 1330-1336.
(32) Kobayashi, Y.; Mizojiri, R. Nickel-Catalyzed Coupling Reaction
of Lithium Organoborates and Aryl Mesylates Possessing an
Electron Withdrawing Group. Tetrahedron Lett. 1996, 37, 8531-
8534.
(33) Hajduk, P. J .; Dinges, J .; Miknis, G. F.; Merlock, M.; Middleton,
T.; Kempf, D. J .; Egan, D. A.; Walter, K. A.; Robins, T. S.;
Shuker, S. B.; Holzman, T.; Fesik, S. W. NMR-Based Discovery
of Lead Inhibitors That Block DNA Binding of the Human
Papillomavirus E2 Protein. J . Med. Chem. 1997, 40, 3144-
3150.
(34) Klein, S. I.; Czekaj, M.; Gardner, C. J .; Guertin, K. R.; Cheney,
D. J .; Spada, A. P.; Bolton, S. A.; Brown, K.; Colussi, D.; Heran,
C. L.; Morgan, S. R.; Leadley, R. J .; Dunwiddie, C. T.; Perrone,
M. H.; Chu, V. Identification and Initial Structure-Activity
Relationships of a Novel Class of Nonpeptide Inhibitors of Blood
Coagulation Factor Xa. J . Med. Chem. 1998, 41, 437-450.
(35) Matsushita, Y.-i.; Sakamoto, K.; Murakami, T.; Matsui, T. A
Convenient Synthesis of Methyl 4-Substituted Benzoates Via
Diels-Alder Reaction in the Presence of Palladium on Activated
Carbon. Synth. Commun. 1994, 24, 3307-3313.
(36) Chauffe, L.; Andrews, L. J .; Keefer, R. M. The Effect of Carboxyl
Groups in Neighboring Side Chains on the Hydrolysis of
Substituted Benzyl and Fluorenyl Bromides. J . Org. Chem. 1966,
31, 3758-3764.
(37) Rice, W. G.; Bader, J . P. Discovery and in Vitro Development of
AIDS Antiviral Drugs as Biopharmaceuticals. Adv. Pharmacol.
(San Diego) 1995, 6, 389-438.
(38) 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 Detection of Anti-HIV
Compounds. J . Virol. Methods 1988, 20, 309-321.
(39) Popovic, M.; Sarngadharan, M. G.; Read, E.; Gallo, R. C.
Detection, Isolation and Continuous Production of Cytopathic
Retroviruses (HTLV-III) from Patients with AIDS and pre-AIDS.
Science 1984, 224, 497-500.
(40) Barre´-Sinoussi, F.; Chermann, J . C.; Nugeyre, M. T.; Chamaret,
S.; Gruest, J .; Dauguet, C.; Axler-Blin, C.; Ve´zinet-Brun, F.;
Rouzioux, C.; Rozenbaum, W.; Montagnier, L. Isolation of a
T-Lymphotropic Retrovirus from a Patient at Risk for AIDS.
Science 1983, 220, 868-871.
(41) Miyoshi, I.; Taguchi, H.; Kobonishi, I.; Yoshimoto, S.; Ohtsuki,
Y.; Shiraishi, Y.; Akagi, T. Type C Virus-producing Cell Lines
Derived from Adult T Cell Leukemia. Gann. Monogr. 1982, 28,
219-228.
(19) Hashimoto, N.; Aoyama, T.; Shioiri, T. New Methods and
Reagents in Organic Synthesis. 14. A Simple Efficient Prepara-
tion of Methyl Esters with Trimethylsilyldiazomethane (TM-
SCHN2) and Its Application to Gas Chromatographic Analysis
of Fatty Acids. Chem. Pharm. Bull. 1981, 29, 1475-1478.
(20) Martin, M. A Convenient, High Yield Synthesis of Trimethylsilyl
Diazomethane. Synth. Commun. 1983, 13, 809-811.
(21) Press, N.; Hedberg, B.; Conway, B. Pentafuside (Trimeris). Drugs
1999, 2, 702-710.
(22) Dong, B. Efavirenz DuPont. Curr. Opin. Anti-Infect. Invest.
Drugs 1999, 1, 196-212.
(23) Press, N.; Hedberg, B.; Conway, B. Pentafuside Trimeris. Curr.
Opin. Anti-Infect. Invest. Drugs 1999, 1, 171-178.
(24) Kuchimanchi, K. R.; Udata, C.; J ohnson, T. P.; Mitra, A. K.
Pharmacokinetics, Biliary Excretion, and Tissue Distribution of
Novel Anti-HIV Agents, Cosalane and Dihydrocosalane, in
Sprague-Dawley Rats. Drug Metab. Dispos. 2000, 28, 403-408.
J M000290U