Synthesis and anti-HIV activity of cosalane analogues incorporating nitrogen in the linker chain

Article abstract of DOI:10.1016/S0968-0896(99)00269-2

Introduction of an amido group or an amino moiety into the alkenyl linker chain of cosalane (1) provided a new series of analogues 3-8. The new compounds were evaluated as inhibitors of the cytopathic effect of HIV-1 and HIV-2 in cell culture. The replace

Full text of DOI:10.1016/S0968-0896(99)00269-2

Bioorganic & Medicinal Chemistry 8 (2000) 191±200
Synthesis and Anti-HIV Activity of Cosalane Analogues
Incorporating Nitrogen in the Linker Chain
Agustin Casimiro-Garcia, a Erik De Clercq, b Christophe Pannecouque, b
Myriam Witvrouw, b Tracy L. Stup, c Jim A. Turpin, c Robert W. Buckheit, Jr. c
and Mark Cushman a,*
aDepartment of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal Sciences, Purdue University,
West Lafayette, IN 47907, USA
bRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
cInfectious Disease Research Department, Serquest, a Southern Research Institute company, 431 Aviation Way, Frederick,
MD 21701, USA
Received 15 March 1999; accepted 13 September 1999
AbstractÐIntroduction of an amido group or an amino moiety into the alkenyl linker chain of cosalane (1) provided a new series of
analogues 3±8. The new compounds were evaluated as inhibitors of the cytopathic e€ect of HIV-1 and HIV-2 in cell culture. The
replacement of the 10 and 20 carbons in the linker chain of 1 by an amido group was generally tolerated. The length of the linker
chain and the stereochemistry of the substituent at C-3 of the steroidal ring had signi®cant e€ects on the antiviral activity and
potency. Incorporation of an amino moiety into the linker completely abolished the anti-HIV activity. There are several steps in the
HIV replication cycle that have been proposed as targets for the development of therapeutic agents (De Clercq, E. J. Med. Chem.
1995, 38, 2491; De Clercq, E. Pure Appl. Chem. 1998, 70, 567). However, currently approved anti-HIV drugs are only directed
against the viral enzymes reverse transcriptase or protease (Carpenter, C. C. J.; Fischl, M. A.; Hammer, S. M.; Hirsch, M. S.;
Jacobsen, D. M.; Katzenstein, D. A.; Montaner, J. S. G.; Richman, D. D.; Saag, M. S.; Schooley, R. T.; Thompson, M. A.; Vella,
S.; Yeni, P. G.; Volberding, P. A. JAMA 1998, 280, 78). Drugs capable of interfering with other steps of the virus life cycle will be
highly valuable in the antiretroviral therapy of AIDS, as they will have di€erent patterns of resistance mutations than the drugs
currently used clinically. In addition, their utilization in combination with other therapeutic agents could provide more potent drug
`cocktails' capable of completely suppressing virus replication. Consequently, there is an urgent need for the discovery of clinically
useful anti-HIV agents possessing novel mechanisms of action. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
carboxylate groups of cosalane. There are additional
residues near the proposed binding site of 1 to CD4,
including Asp-56 and Lys-72, that could be targeted to
generate analogues having higher anity for this recep-
tor. This strategy has been shown to be successful in
the generation of analogues with improved potency
over cosalane itself.3 Besides alterations in the dis-
alicylmethane `pharmacophore',3,4 the e€ects of chan-
ging the point of attachment of the linker chain to the
steroid nucleus and the replacement of the steroid with
other lipophilic appendages have been investigated.5±7
Aside from the documented detrimental e€ect of phos-
phate incorporation into the linker chain connecting
the steroid to the disalicylmethane `pharmacophore',7
there is little information available regarding either the
biological e€ects of alterations in composition of the
linker chain or the stereochemistry of its attachment at
C-3 of the steroid. We have therefore decided to prepare
a new series of cosalane analogues 3±8 in which an
Cosalane (1) is a unique anti-HIV agent that acts pri-
marily by inhibition of virus fusion and virus attach-
ment.1,2 Cosalane prevents the cytopathic e€ect of HIV-
1(IIIB) in CEM-SS cells with an EC50 of 3.4 mM, and it
has been demonstrated to be active against a wide
variety of HIV laboratory and clinical isolates.2 A hypo-
thetical model of the interaction of the disalicylmethane
cosalane `pharmacophore' with CD4 has been recently
proposed.3 In this model, the binding of 1 to CD4 was
proposed to take place by two electrostatic interactions
between the positively charged Arg-58 and Arg-59
residues of CD4 with the two negatively charged
Keywords: antivirals; cytotoxins; steroids and sterols; chemotherapy.
*Corresponding author. Tel.: +1-765-494-1465; fax: +1-765-494-
1414; e-mail: cushman@pharmacy.purdue.edu
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00269-2

192
A. Casimiro-Garcia et al. / Bioorg. Med. Chem. 8 (2000) 191±200
amido group or an amino moiety is incorporated into
the alkenyl linker chain. The cosalane molecule can
be viewed as a disalicylmethane `pharmacophore'
attached to a membrane-interactive steroid that may
serve to anchor the molecule in a membrane, with the
`pharmacophore' protruding outward in an obstruc-
tive mode that might inhibit the fusion of the viral
envelope with the cell membrane. According to this
model, the incorporation of a positively-charged (at
physiological pH) amino group might facilitate the
anchoring of the molecule in the membrane through
electrostatic interactions with the negatively-charged
phosphates present there on the surface. Both amido
and amino groups also are capable of acting as hydro-
gen bond donors or acceptors, and this might provide
additional sites for interaction with residues on CD4.
The preparation and evaluation of these new analogues
as inhibitors of the cytopathic e€ect of HIV-1 and HIV-2
will be presented in this report.
Chemistry
During the design of the new analogues, we decided to
prepare compounds 3, 4, 7 and 8 having the same alke-
nyl chain length as that of cosalane. Compounds 5 and
6 have an extended alkenyl chain length, and were pre-
pared based on the consideration that cosalane analo-
gue 2 showed very similar anti-HIV activity as that of
the parent compound.7 In addition, we were interested
in investigating the e€ect of the stereochemistry at C-3
of the steroidal ring on the anti-HIV activity. Com-
pounds 3, 5 and 7 have the C-3 substituent with an
equatorial, b-stereochemistry, while in analogues 4, 6
and 8 the substituent has an axial, a-stereochemistry.
For the synthesis of analogues incorporating an amido
group, we planned to couple amines having de®ned
stereochemistry at C-3 of the steroidal ring with appro-
priate carboxylic acids. Reductive amination of these
amines with a suitable aldehyde would provide analo-
gues with amino functionality in the linker chain.
The preparation of the required amines 11 and 14 was
achieved from 3-cholestanol (9) in two, and three steps,
respectively, using the Mitsunobu reaction to establish
the stereochemistry at C-3 of the steroid (Scheme 1). In
this way, azide 10, prepared as previously described,8
was reduced with LAH in re¯uxing ether to a€ord the
amine 11.9,10 No C-3 epimer was detected in the 1H NMR
spectrum of this compound. Cholestane derivative 12
was prepared as described by Loibner and Zbiral.11
Displacement of iodide with sodium azide in DMSO at
Scheme 1. Reagents and conditions: (a) PPh3, DEAD, (PhO)2PON3,
THF, 25 ꢀC, 24 h, 51%; (b) LiAlH4, Et2O, re¯ux, 11: 65%; 14: 69.7%;
(c) PPh3, DEAD, CH3I, THF, 25 ꢀC, 76%; (d) NaN3, DMSO, 90 ꢀC,
5 h, 81%.

A. Casimiro-Garcia et al. / Bioorg. Med. Chem. 8 (2000) 191±200
193
95 ꢀC cleanly provided azide 13 as the sole isomer.12
Reduction of the azido group as indicated above pro-
vided amine 14.13
For the preparation of the new analogues possessing an
amido group, carboxylic acids 15 and 17 were required.
Compound 15 was prepared as described previously
during our work on the synthesis of non-nucleoside
reverse transcriptase inhibitors.14 Compound 17 was
obtained by an inverse-addition Jones oxidation of the
known alcohol 16,14,15 which involved the slow addition
of a solution of 16 in acetone to chromium trioxide-
aqueous sulfuric acid. Having both intermediates on
hand, the coupling of carboxylic acid 15 with amine 14
was performed with EDCI-HCl, HOBt, and Et3N in
DMF. In this way, compound 18 possessing the 3b-
stereochemistry was obtained. A similar protocol, but
starting with amine 11, a€orded compound 19.
Removal of the methyl esters and methyl ethers from 18
and 19 was initially attempted using boron tribromide-
dimethyl sul®de complex in re¯uxing 1,2-dichloro-
ethane, conditions that have been widely used in the
synthesis of other cosalane analogues.1,2,5,7 Unfortu-
nately, these conditions provided only complex mixtures
of products. Boron tribromide in dichloromethane is a
milder agent for this type of transformation.16 Using
this reagent, deprotection of 18 was started at 78 ꢀC,
and continued at room temperature for 2 days, to a€ord
amide 3. Applying these conditions to 19 provided the
expected amide 4.
Similar chemistry was used for the preparation of 5 and
6. Coupling the carboxylic acid 17 with amines 11 or 14
provided the desired amides 20 and 21. Removal of the
methyl ethers and methyl esters was attempted using the
conditions mentioned above. This method proved only
partially successful for the preparation of 5, which was
obtained in low yield. Our initial plan was to remove all
methyl groups from 21 to obtain the fully deprotected
analogue (6, R=H), but all our attempts to obtain this
compound failed. Therefore, it was decided to cleave
only the methyl esters. This transformation was
achieved using potassium carbonate in ethanol-water to
a€ord 6. We were not particularly concerned about
leaving the methyl ethers intact, as analogue 22 showed
similar anti-HIV activity as that of cosalane (unpub-
lished results).
applied to our system, and reacting a mixture of amine
14, aldehyde 25 and magnesium sulfate in dry benzene
for 48 h provided the corresponding imine, which was
reduced with methanolic sodium cyanoborohydride to
a€ord 26. Starting with amine 11, a similar procedure
a€orded amine 27. To ®nish the synthesis of the target
compounds, cyanide-catalyzed ester hydrolysis with
potassium carbonate in THF±MeOH±water a€orded
compounds 7 and 8.21 We also attempted to remove the
methyl ethers from 7 and 8 using boron tribromide, but
only complex mixtures of products were obtained.
We planned to prepare analogues 7 and 8 by reductive
amination of 11 and 14 with an appropriate aldehyde,
which was obtained using Peterson ole®nation.17 The
anion derived from silyl imine 23 and LDA18 was reac-
ted with the known benzophenone 242 at 78 ꢀC to give
an intermediate imine, which was hydrolyzed with aqu-
eous citric acid to a€ord aldehyde 25. The reductive
amination reaction between 25 and amines 11 or 14 was
®rst attempted in methanol±THF, using molecular
sieves as the water scavenger, and sodium cyanobor-
ohydride to reduce the intermediate imine.19 However,
formation of the imine was not observed under these
conditions. Magnesium sulfate has been used to promote
the formation of the imine between cinnamaldehyde and
cyclohexylamine in benzene.20 These conditions were
Biological Results and Discussion
The new cosalane analogues 3±8 were evaluated for
inhibition of the cytopathic e€ect of HIV-1 and HIV-2
in cell culture, as well as for cytotoxicity in uninfected
cells. The results are listed in Table 1. The compounds
were tested against HIV-1 in both CEM-SS and MT-4
cells, while for prevention of the cytophatic e€ect of
HIV-2 only MT-4 cells were used. In general, the amides
3±5 were found to possess anti-HIV-1 or anti-HIV-2
activity, while compounds having an amino group (7
and 8), as well as the amide 6, were completely inactive

194
A. Casimiro-Garcia et al. / Bioorg. Med. Chem. 8 (2000) 191±200
in both systems. The anti-HIV activities of the new
compounds were dependent on the virus strain. The 3-b
amides 3 (EC50=5.2 mM) and 5 (EC50=22.6 mM) were
the only active compounds against HIV-1RF, with com-
pound 3 being equipotent with cosalane (EC50=
5.1 mM). For the IIIB strain in MT-4 cells, the 3-b
amides 3 (EC50=15.1 mM) and 5 (EC50=35.8 mM) were
again the only compounds that displayed activity.
However, both of them were less potent than cosalane
(EC50=1.8 mM). The 3-a amide 4 (EC50=5.1 mM) was
equipotent to cosalane (EC50=5.1 mM) against HIV-2
(ROD) in MT-4 cells, with the 3-b amides 3 (EC50=
9.8 mM) and 5 (EC50=14.2 mM) being slightly less
potent. Compounds 6, 7 and 8, the last two possessing
an amino group in the alkenyl linker chain, did not
inhibit the cytopathic e€ect of HIV-1 and HIV-2 at
concentrations lower than the cytotoxic concentrations.
All of the new compounds were more cytotoxic than
cosalane (CC50=>200 mM) for CEM-SS cells with
CC50 values ranging from 13.5 mM (analogue 6) to
161 mM (analogue 5). The new analogues were also
more cytotoxic than 1 (CC50=>125 mM) against MT-4
cells, with 6 (CC50=6.0 mM) being the most cytotoxic
compound.
In comparing the active analogues against HIV-1RF and
HIV-1IIIB, it seems that there is a decrease in potency
relative to the length of the linker chain. Analogue 3
possesses a three atom linker, which is identical in
length to that of cosalane, while 5 has a four atom
chain. Data from our previous work indicated that
analogue 2 (EC50=3.4 mM), possessing a four atom lin-
ker similar to that present in 5, was equipotent with 1
versus HIV-1RF.5 Therefore, it would be expected that
increasing the length of the linker by one atom would
not produce a signi®cant e€ect on the antiviral potency.
The results observed with these analogues are probably
due to changes in conformation and rigidity introduced
by incorporation of the amido group.
The stereochemistry of the substituent at C-3 of the
steroidal ring was important for activity against HIV-1,
but not HIV-2. Both of the active amides 3 and 5 have
the alkenyl linker chain with a C-3 b-stereochemistry
Table 1. Anti-HIV activities of cosalane analogues
a
Compound
EC50 (mM)
Cytotoxicity (mM)
c
d
d
HIV-1RF
HIV-1IIIB
HIV-2ROD
CES-SS cellsb
MT-4 cellsb
1
3
4
5
6
7
8
22
5.1‹1.6
5.2
1.84‹0.07
15.1‹2.08
NAe
5.1‹2.8
9.8‹1.0
5.1‹0.1
14.2‹10.5
NAe
>200
107‹50.2
26.6‹0.7
160.7‹39.2
13.5‹4.5
>50
>125
52.2‹9.4
24.4‹5.9
>60
6.0‹3.2
>150
NAe
22.6‹5.8
NAe
35.8‹5.1
NAe
NAe
NAe
NAe
NAe
13.9‹6.33
NA1ec
Ð
NAe
Ð
61.7‹2.2
>300
87‹27.2
Ð
aConcentration required to reduce the cytopathic e€ect of the virus by 50%.
bConcentration required for 50% reduction in cellular viability of uninfected cells.
cDetermined in CEM-SS cells.
dDetermined in MT-4 cells.
eNo activity was observed up to a concentration of 125 mM.

A. Casimiro-Garcia et al. / Bioorg. Med. Chem. 8 (2000) 191±200
195
(equatorial), while the alkenyl linker chain is axial in 4.
Experimental
It could be argued that in the corresponding C-3 a-epi-
mers 4 and 6 the steroidal moiety is imposing steric
hindrance to the carboxylate groups to interact with the
positively charged residues on CD4. However, this
interpretation is not in agreement with the antiviral
activity of 4 against HIV-2, because the interaction with
CD4 would also be involved in that case as well. Prior
studies demonstrated that cosalane binds to both gp120
and CD4,22 and it can be assumed that the new analo-
gues would interact with both molecules as well. A more
complete understanding of the mechanisms of action
and potencies of the present compounds may therefore
require a model for their interaction with gp120 in
addition to the CD4 binding model. Distinct modes of
interaction of analogues 3, 4 and 5 with gp120 could
explain the di€erences observed in the anti-HIV-1 ver-
sus anti-HIV-2 activity. In this regard, it is interesting
that changing the con®guration of the linker chain from
equatorial in 3 to axial in 4 abolishes antiviral activity
against both strains of HIV-1, but it actually increases
activity against HIV-2ROD (Table 1). This intriguing
result is not easily explained by any simple molecular
model. The relative con®guration of the side chain also
has an e€ect on cytotoxicity in uninfected cells, with the
axial con®guration 4 being more cytotoxic than the
equatorial con®guration 3 in both CEM-SS cells and
MT-4 cell. The lack of antiviral activity in analogues 7
and 8 incorporating an amino group provides new
insights into the functionality tolerated in this part of
the molecule. Previously, we reported a series of cosa-
lane analogues, including 28, that incorporated a phos-
phate group into the linker chain.7 This modi®cation
completely abolished the anti-HIV activity. Under the
assay conditions, the amino groups of compounds 7 and
8 are protonated. The positively charged amino func-
tionality and the phosphate moiety are both polar
groups that reduce the lipophilicity of the alkenyl linker
chain. This factor has been related before to the anti-
viral potency.6 The presence of a polar group next to the
steroid is probably obstructing the imbedding of this
moiety into the membrane. Although the model that we
proposed for the interaction of compounds 7 and 8 with
a membrane is de®nitively not correct, this general
model should still be valid for other analogues, pro-
vided that the lipophilicity of the linker chain is not
disrupted.
General
Melting points (mp) 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; FAB mass spectra and EI mass
spectra on a Kratos MS50 spectrometer; 1H NMR
spectra on Varian VXR-500S and Bruker ARX-300
spectrometers; IR spectra on a Beckman IR-33 spectro-
meter or on a Perkin±Elmer 1600 series FTIR. Micro-
analyses were performed at the Purdue Microanalysis
Laboratory, and all values were within ‹0.4% of the
calculated compositions. Silica gel used for column
chromatography was 230±400 mesh.
5ꢀ,3ꢀ-Azidocholestane (10). This compound was pre-
pared in 51% yield following a literature procedure:8
mp 54±56 ꢀC (lit.8 mp 62.5±63 ꢀC); IR (®lm) 2950, 2866,
1
2100, 1653, 1465, 1381 cm
;
1H NMR (CDCl3,
300 MHz) d 3.88 (t, J=2.5 Hz, 1H), 0.90 (d, J=6.5 Hz,
3H), 0.86 (dd, J=7.5 Hz and J=1.1 Hz, 6H), 0.78 (s,
3H), 0.64 (s, 3H); CIMS m/z (relative intensity) 413
(M+, 24), 387 (MH2+ N2, 100).
3ꢀ-Amino-5ꢀ-cholestane (11). This compound was pre-
pared from 5a,3a-azido cholestane (10) in 65% yield
following the method described by Bose et al.:9 mp 100±
101 ꢀC (lit.9 mp 87±88 ꢀC; lit.10 mp 104.5±105.5 ꢀC); IR
(®lm) 3450, 3350, 2933, 2866, 2850, 1560, 1467, 1381,
844, 757 cm 1; 1H NMR (CDCl3, 300 MHz) d 3.16 (b.s.,
1H), 1.63 (m, 2H, D2O exchange), 0.87 (d, J=6.5 Hz,
3H), 0.83 (dd, J=6.6 Hz and J=1.3 Hz, 6H), 0.75 (s,
3H), 0.62 (s, 3H); CIMS m/z (relative intensity) 388
(MH+, 100), 390 (21), 386 (17).
3ꢀ-Iodo-5ꢀ-cholestane (12). This compound was
obtained in 76% yield as a colorless solid following
the method described by Loibner and Zbiral:11 mp
104.5±106 ꢀC (lit.11 mp 109±112 ꢀC); H NMR (CDCl3,
300 MHz) d 4.95 (s, 1H), 0.90 (d, J=6.5 Hz, 3H), 0.86
(dd, J=6.6 Hz and J=1.2 Hz, 6H), 0.79 (s, 3H), 0.65 (s,
3H); CIMS m/z (relative intensity) 497 (MH+, 8), 371
(M+ ± HI, 100).
1
5ꢀ,3ꢁ-Azidocholestane (13). A solution of compound
12 (1.0 g, 2.00 mmol) and sodium azide (1.3 g,
20 mmol) in dry DMSO (50 mL) was stirred at 90 ꢀC
for 5 h. The reaction mixture was cooled at room tem-
perature and water (50 mL) was added. The product
was extracted with ethyl ether (4Â30 mL). The com-
bined organic extracts were washed with brine
(1Â100 mL), dried over sodium sulfate, ®ltered and the
solvent evaporated to give a yellowish solid (0.82 g,
99%). The product was recrystallized from ether/
methanol to a€ord 13 (0.67 g, 81%) as a white crystal-
line solid: mp 61±62.5 ꢀC (lit.12 mp 65±66 ꢀC); IR (®lm)
In conclusion, the present study reveals that the incor-
poration of an amido group into the linker chain of
cosalane provides analogues exhibiting anti-HIV-1 and
anti-HIV-2 activity. The range of activity and the
potency of these compounds is related to the length of
the linker chain, as well as to the stereochemistry of the
substituent at C-3 of the steroidal ring. Introduction of
an amino group into the linker chain completely abol-
ishes the antiviral activity. We are currently con-
templating the preparation of new analogues in which
the nitrogen atom of the amido functionality in the
present series could be used to attach a second unit of
the cosalane ``pharmacophore'' through an appropriate
linker. Results of these studies will be presented
shortly.
2932, 2866, 2090, 1653, 1540, 1507, 1465, 1381, 1252
1
cm
; d 3.23 (tt,
1H NMR (CDCl3, 300 MHz)
J=11.7 Hz and J=4.6 Hz, 1H), 0.87 (d, J=6.5 Hz, 3H),
0.84 (dd, J=6.6 Hz and J=1.2 Hz, 6H), 0.77 (s, 3H),
0.62 (s, 3H); CIMS (relative intensity) m/z 414 (MH+,
60), 388 (100).

196
A. Casimiro-Garcia et al. / Bioorg. Med. Chem. 8 (2000) 191±200
3ꢁ-Amino-5ꢀ-cholestane (14). A solution of 3b-azide 13
(0.45 g, 1.09 mmol) in dry ether (20 mL) was placed
under argon. A 1.0 M solution of LiAlH4 in ether
(4.4 mL, 4.4 mmol) was added dropwise. A re¯ux con-
denser was adapted and the reaction mixture was
re¯uxed for 3 h. After cooling the reaction at room
temperature, wet ether (10 mL) was slowly added fol-
lowed by water (20 mL), which was added very slowly.
The layers were separated and the ethereal phase was
washed with brine (1Â20 mL), dried over sodium sulfate
and ®ltered. To the dry solution, a 1.0 M solution of
HCl in ether (2 mL) was added. The precipitate was
separated by vacuum ®ltration, washed with dry ether
and dried. The dry salt was suspended in 10% aqueous
KOH and extracted with ether. After evaporation of the
solvent, free amine 14 (0.29 g, 69.7%) was obtained as a
mixture was stirred at room temperature for 48 h. Water
(135 mL) was added and the mixture stirred for 5 min.
The product was then extracted with dichloromethane
(5Â35 mL). The combined organic extracts were washed
with brine (1Â50 mL), dried over magnesium sulfate,
®ltered, and the solvent removed. Puri®cation was
achieved by ¯ash chromatography on silica gel (ꢁ50 g;
column: 3 cmÂ9.5 inch), eluting with hexanes/ethyl
acetate 2/1, to yield pure 18 as a light-tan solid (0.165 g,
76.3%): mp 137±139 ꢀC; IR (®lm) 3290, 2932, 2866,
1
1737, 1636, 1476, 1260, 1207, 999 cm
;
1H NMR
(300 MHz, CDCl3) d 7.55 (d, J=2.4 Hz, 1H), 7.53 (d,
J=2.2 Hz, 1H), 7.43 (d, J=2.2 Hz, 1H), 7.33 (d,
J=2.3 Hz, 1H), 6.26 (s, 1H), 5.07 (d, J=8.4 Hz, 1H),
3.96 (s, 3H), 3.94 (s, 3H), 3.89 (s, 3H), 3.88 (s, 3H), 3.67
(bm, 1H), 0.67 (s, 3H), 0.60 (s, 3H); FABMS m/z 838
(MH+) and 451 [MH+ ± 387(R-NH)]. Anal. calcd for
C48H65Cl2NO7: C, 68.72; H, 7.81; N, 1.67. Found: C,
68.55; H, 7.63; N, 1.43.
white solid: mp 104±106 ꢀC (lit.13 mp 118 ꢀC); IR (®lm)
1
3400, 3277, 2933, 2848, 1540, 1466, 1382 cm
;
1H
NMR (CDCl3, 300 MHz) d 2.61 (tt, J=10.9 Hz and
J=4.3 Hz, 1H), 1.38 (m, 2H, D2O exchange), 0.86 (d,
J=6.5 Hz, 3H), 0.83 (dd, J=6.6 Hz and J=1.2 Hz, 6H),
0.75 (s, 3H), 0.61 (s, 3H); CIMS m/z (relative intensity)
388 (MH+, 100), 386 (26).
N-(5ꢀ,3ꢀ-Cholestanyl)-3,3-(30300-dicarbomethoxy-50,500 -
dichloro-40400-dimethoxydiphenyl)-2-propenamide (19). A
solution of the acid 1514 (0.203 g, 0.434 mmol) and tri-
ethylamine (0.36 mL, 2.6 mmol) in dry DMF (20 mL)
was stirred at room temperature. HOBt (0.117 g,
0.868 mmol) was then added, followed 5 min later by
3a-aminocholestane (11) (0.252 g, 0.651 mmol). A solu-
30,300 -Dichloro-40,400 -dimethoxy-50,500 -bis(methoxycarb-
onyl)-4,4-diphenyl-3-butenoic acid (17). A solution of
CrO3 (1.073 g, 10,73 mmol) and 0.75 N sulfuric acid
(15.5 mL, 23.2 mmol) was stirred in an ice bath. A
solution of alcohol 1614 (0.837 g, 1.79 mmol) in acetone
(80 mL) was added over 6 h, while maintaining the tem-
perature between 0 and 10 ꢀC. After the addition was
complete, the reaction mixture was stirred at room
temperature for 6 h. Then, the solvents were removed
and the residue diluted with water (100 mL). The aqu-
eous mixture was extracted with ethyl ether (4Â50 mL).
The combined organic fractions were washed with water
(1Â50 mL), brine (1Â50 mL), dried over magnesium
sulfate and ®ltered. The solvent was removed and the
residue was puri®ed by ¯ash chromatography on silica
gel (105 g, column: 4 cmÂ7.5 inch), eluting with hex-
anes/ethyl acetate (3/1 to 1/2). Compound 17 was
obtained as a light-yellow solid (0.365 g, 42.3%). The
analytical sample was recrystallized from acetone/hex-
anes: mp 138±140 ꢀC; IR (®lm) 3500±2500, 2952, 1732,
.
tion of EDCI HCl (0.166 g, 0.868 mmol) in dry DMF
(10 mL) was incorporated. The reaction mixture was
stirred at room temperature for 3 h. Dry dichloro-
methane (5 mL) was added to facilitate the dissolution
of the amine. The resulting cloudy solution was stirred
at room temperature for 48 h. Water (100 mL) was
added and the mixture stirred for 5 min. The product
was then extracted with ethyl ether (4Â45 mL). The
combined organic extracts were washed with brine
(1Â40 mL), dried over magnesium sulfate, ®ltered, and
the solvent removed. Puri®cation was achieved by
¯ash chromatography on silica gel (37 g; column:
3 cmÂ7 inch), eluting with hexanes/ethyl acetate (2/1),
to yield pure 19 as a white solid (0.324 g, 89.1%): mp
86±88 ꢀC; IR (®lm) 3316, 2934, 1734, 1646, 1474, 1436,
1
1263, 1205, 996 cm 1; H NMR (300 MHz, CDCl3) d
7.58 (d, J=2.4 Hz, 1H), 7.56 (d, J=2.3 Hz, 1H), 7.47 (d,
J=2.2 Hz, 1H), 7.33 (d, J=2.4 Hz, 1H), 6.34 (s, 1H),
5.56 (d, J=8.1 Hz, 1H), 4.06 (m, 1H), 3.98 (s, 3H), 3.94
(s, 3H), 3.90 (s, 3H), 3.88 (s, 3H), 0.69 (s, 3H), 0.60 (s,
3H); FABMS m/z: 838 (MH+). Anal. calcd for
C48H65Cl2NO7: C, 68.72; H, 7.81; N, 1.67. Found: C,
68.51; H, 7.85; N, 2.01.
1
1477, 1258, 1210, 996, 744 cm 1; H NMR (300 MHz,
CDCl3) d 7.49 (d, J=2.3 Hz, 1H), 7.46 (d, J=2.2 Hz,
1H), 7.32 (d, J=2.2 Hz, 1H), 7.31 (d, J=2.5 Hz, 1H),
6.20 (t, J=7.4 Hz, 1H), 3.95 (s, 3H), 3.89 (s, 3H), 3.87
(s, 3H), 3.86 (s, 3H), 3.14 (d, J=7.4 Hz, 2H); FABMS
m/z (relative intensity): 483 (MH+, 10) and 451 (MH+
± CH3OH, 15). Anal. calcd for C22H20Cl2O8: C: 54.67,
H: 4.17. Found: C: 54.45, H: 4.06.
N-(5ꢀ,3ꢁ-Cholestanyl)-3,3-(30,300-dicarboxy-50,500-dichloro-
40,400-dihydroxydiphenyl)-2-propenamide (3). A 1.0 M
solution of BBr3 in dichloromethane (0.36 mL, 0.36mmol)
was stirred under argon in a dry ice±acetone bath. Dry
dichloromethane (4 mL) was added. A solution of
amide 18 (0.050 g, 0.059 mmol) in dry dichloromethane
(4.5 mL) was added dropwise. The reaction mixture was
stirred at 78 ꢀC for 3 h. The dry ice-acetone bath was
removed, allowing the temperature to reach room tem-
perature, and the mixture was stirred at this tempera-
ture for 2 days. Additional 1.0 M solution of BBr3
(0.2 mL) was added on the second day. Water (10 mL) was
N-(5ꢀ,3ꢁ-Cholestanyl)-3,3-(30,300-dicarbomethoxy-50,500-
dichloro-40,400-dimethoxydiphenyl)-2-propenamide (18). A
solution of the acid 1514 (0.121 g, 0.259 mmol), triethyl-
amine (0.22 mL, 1.6 mmol) in dry DMF (12 mL)
was stirred at room temperature. HOBt (0.070 g,
0.52 mmol) was then added, followed 5 min later by 3b-
aminocholestane (14) (0.150 g, 0.388 mmol). A solution
.
of EDCI HCl (0.099 g, 0.52 mmol) in dry DMF (5 mL)
was incorporated. Dry dichloromethane (3 mL) was
added to facilitate dissolution of amine. The reaction

A. Casimiro-Garcia et al. / Bioorg. Med. Chem. 8 (2000) 191±200
197
added, followed by 10% NaOH (20 mL), and the mix-
ture was stirred overnight. The basic solution was
washed with Et2O (2Â20 mL), followed by acidi®cation
with conc HCl. The product was then extracted with
Et2O (3Â25 mL) and the organic extracts were washed
with brine (1Â30 mL), dried over magnesium sulfate
and ®ltered. The solvent was removed in vacuo to give a
pale brown residue. Puri®cation was achieved by column
chromatography on silica gel (10 g, column: 1.2 cm
Â8.5 inch), eluting with CHCl3:MeOH:formic acid (95:
5:0.5), to a€ord 3 as a pale-tan solid (32.2 mg, 69%): mp
100±102.5 ꢀC; IR (®lm) 3500±2500, 2928, 1667, 1463,
stirred at room temperature under Ar. HOBt (0.039 g,
0.290 mmol) was then added, followed by aminocholes-
.
tane 14 (0.084 g, 0.22 mmol). A solution of EDCI HCl
(0.056 g, 0.29 mmol) in dry DMF (4.0 mL) was incorpo-
rated. The reaction mixture was stirred at room tem-
perature for 48 h. Water (70 mL) was added and the
mixture stirred for 5 min. Ethyl ether was added (30
mL) and the layers were separated. The aqueous frac-
tion was then extracted with ethyl ether (3Â30 mL). The
combined organic extracts were washed with brine
(1Â50 mL), dried over magnesium sulfate, ®ltered, and
the solvent removed. Puri®cation was achieved by ¯ash
chromatography on silica gel (30 g, column dimensions:
2 cmÂ8.25 inch), eluting with hexanes:ethyl acetate (3:1
to 2:1). Compound 20 was obtained as a light tan solid
1
1233, 1191, 978 cm 1; H NMR (300 MHz, acetone-d6)
d 7.58 (d, J=2.4 Hz, 1H), 7.56 (d, J=2.3 Hz, 1H), 7.47
(d, J=2.2 Hz, 1H), 7.33 (d, J=2.4 Hz, 1H), 6.34 (s, 1H),
5.56 (d, J=8.1 Hz, 1H), 4.06 (m, 1H), 3.98 (s, 3H), 3.94
(s, 3H), 3.90 (s, 3H), 3.88 (s, 3H), 0.69 (s, 3H), 0.60 (s,
3H); FABMS m/z 782 (MH+). Anal. calcd for
(92 mg, 75%): mp 87±95 ꢀC; IR (®lm) 3302, 2933, 1736,
1
1636, 1476, 1257, 1209, 999, 737 cm
;
1H NMR
(300 MHz, CDCl3) d 7.51 (d, J=2.1 Hz, 1H), 7.46 (d,
J=2.0 Hz, 1H), 7.34 (d, J=2.2 Hz, 1H), 7.31 (d, J=
2.5 Hz, 1H), 6.29 (t, J=7.5 Hz, 1H), 5.25 (d, J=8.1 Hz,
1H), 3.97 (s, 3H), 3.90 (s, 3H), 3.89 (s, 3H), 3.88 (s, 3H),
3.73 (m, 1H), 2.93 (d, J=7.5 Hz, 2H), 0.75 (s, 3H), 0.61
(s, 3H); FABMS m/z (relative intensity): 852 (MH+,
90). Anal. calcd for C49H67Cl2NO7: C: 69.00, H: 7.92,
N: 1.64. Found: C: 69.23, H: 8.02, N: 1.65.
.
C44H57Cl2NO7 0.7 H2O: C, 66.44; H, 7.40; N, 1.76.
Found: C, 66.11; H, 7.30; N, 1.73.
N-(5ꢀ,3ꢀ-Cholestanyl)-3,3-(30,300-dicarboxy-50,500-dichloro-
40,400-dihydroxydiphenyl)-2-propenamide (4). A 1.0 M
solution of BBr3 in dichloromethane (1.07 mL,
1.07 mmol) was stirred under argon in an dry ice-ace-
tone bath. Additional dry dichloromethane (4 mL) was
added. A solution of amide 19 (0.150 g, 0.179 mmol) in
dry dichloromethane (8.0 mL) was added dropwise. The
reaction mixture was stirred at 78 ꢀC for 3 h. The dry
ice-acetone bath was removed, allowing the temperature
to reach room temperature, and the mixture was stirred
at this temperature for 2 days. Additional 1.0 M solu-
tion of BBr3 (0.6 mL, 0.6 mmol) was added on the sec-
ond day. Water (10 mL) was added, followed by 10%
KOH (30 mL), and the mixture was stirred for 48 h. The
basic solution was washed with Et2O (2Â20 mL), fol-
lowed by acidi®cation with conc HCl. The product was
then extracted with Et2O (4Â30 mL) and the organic
extracts were washed with brine (1Â30 mL), dried over
magnesium sulfate and ®ltered. The solvent was
removed in vacuo to give a brownish residue. Puri®ca-
tion was achieved by column chromatography on silica
gel (26 g, column: 2.0 cmÂ8.25 inch), eluting with 100%
CHCl3, followed by CHCl3:MeOH:formic acid (95:5:
0.5). The fractions containing pure product were pooled
and the solvent removed. The residue was then tritu-
rated with dichloromethane. The solid obtained in this
way was separated by vacuum ®ltration and washed
with additional dichloromethane. Compound 4 was
obtained as a white solid in moderate yield (93.7 mg,
67.4%): mp 206±211 ꢀC; IR (®lm) 3500±2500, 2928,
N-(5ꢀ,3ꢀ-Cholestanyl)-4,4-(30,300-dicarbomethoxy-50,500-
dichloro-40,400-dimethoxydiphenyl)-3-butenamide (21). A
solution of the acid 17 (0.120 g, 0.249 mmol), triethyl-
amine (0.21 mL, 1.5 mmol) in dry DMF (13 mL) was
stirred at room temperature under Ar. HOBt (0.067 g,
0.5 mmol) was then added, followed by aminocholes-
.
tane 11 (0.134 g, 0.346 mmol). A solution of EDCI HCl
(0.095 g, 0.5 mmol) in dry DMF (7.0 mL) was incorpo-
rated. The reaction mixture was stirred at room tem-
perature for 2.5 h. Dry CH2Cl2 (5 mL) was added and
the mixture stirred at room temperature for 38 h. Water
(100 mL) was added and the mixture stirred for 5 min.
Ethyl ether was added (35 mL) and the layers were
separated. The aqueous fraction was then extracted with
ethyl ether (3Â35 mL). The combined organic extracts
were washed with brine (1Â60 mL), dried over magne-
sium sulfate, ®ltered, and the solvent removed. Pur-
i®cation was achieved by ¯ash chromatography on silica
gel (45 g, column dimensions: 2 cmÂ12.5 inch), eluting
with hexanes:ethyl acetate (3:1 to 2:1). Compound 21
was obtained as a white solid (0.19 g, 89.6%): mp
167.5±169.5 ꢀC; IR (®lm) 3330, 2932, 1737, 1477, 1259,
1
1208, 999, 742 cm
;
1H NMR (300 MHz, CDCl3) d
7.52 (d, J=2.3 Hz, 1H), 7.47 (d, J=2.2 Hz, 1H), 7.36 (d,
J=2.1 Hz, 1H), 7.33 (d, J=2.3 Hz, 1H), 6.33 (t, J=
7.4 Hz, 1H), 5.67 (d, J=7.6 Hz, 1H), 4.11 (bs, 1H), 3.98
(s, 3H), 3.91 (s, 3H), 3.90 (s, 3H), 3.88 (s, 3H), 3.00 (d,
J=7.4 Hz, 2H), 0.77 (s, 3H), 0.62 (s, 3H); FABMS m/z
(relative intensity): 852 (MH+, 40). Anal. calcd for
C49H67Cl2NO7: C: 69.00, H: 7.92, N: 1.64. Found: C:
69.22, H: 7.89, N: 1.68.
1
1667, 1463, 1233, 1191, 978 cm 1; H NMR (300 MHz,
acetone-d6) d 7.80 (d, J=2.2 Hz, 1H), 7.79 (d, J=
2.2 Hz, 1H), 7.58 (d, J=2.0 Hz, 1H), 7.57 (d, J=2.2 Hz,
1H), 7.56 (d, J=7.3 Hz, 1H), 6.52 (s, 1H), 4.02 (m, 1H),
0.76 (s, 3H), 0.66 (s, 3H); FABMS m/z 782 (MH)+.
.
Anal. calcd for C44H57Cl2NO7 0.3 H2O: C, 67.05; H,
7.37; N, 1.78. Found: C, 66.68; H, 7.42; N, 1.65.
N-(5ꢀ,3ꢁ-Cholestanyl)-4,4-(30,300-dicarboxy-50,500-dichloro-
40,400-dihydroxydiphenyl)-3-butenamide (5). A 1.0 M
solution of BBr3 in dichloromethane (0.65 mL,
0.65 mmol) was stirred under argon. Dry dichloro-
methane (3 mL) was added, and the solution was cooled
N-(5ꢀ,3ꢁ-Cholestanyl)-4,4-(30,300-dicarbomethoxy-50,500-
dichloro-40400-dimethoxydiphenyl)-3-butenamide (20). A
solution of the acid 17 (0.070 g, 0.14 mmol) and tri-
ethylamine (0.12 mL, 0.87mmol) in dry DMF (8mL) was

198
A. Casimiro-Garcia et al. / Bioorg. Med. Chem. 8 (2000) 191±200
at 78 ꢀC. A solution of amide 20 (0.092 g, 0.11 mmol)
in dry dichloromethane (6 mL) was added dropwise.
The reaction mixture was stirred at 78 ꢀC for 3 h. The
dry ice±acetone bath was removed, allowing the tem-
perature to reach 25 ꢀC, and the mixture was stirred at
this temperature for 2 days. Additional 1.0 M solution
of BBr3 was added (0.49 mL, 0.49 mmol) after 24 h. An
1H NMR of the crude product run after 48 h indicated
incomplete reaction. More 1.0 M BBr3 was added
(0.5 mL, 0.5 mmol) and the mixture was stirred at 25 ꢀC
for another 24 h. Water (10 mL) was added, followed by
10% KOH (10 mL), and the mixture stirred for 7 h. The
basic solution was washed with Et2O (2Â15 mL), fol-
lowed by acidi®cation with conc HCl. The product was
then extracted with Et2O (4Â25 mL) and the organic
extracts were washed with brine (1Â20 mL), dried over
magnesium sulfate and ®ltered. The solvent was
removed in vacuo to give a pale brown residue. Pur-
i®cation was achieved by double column chromato-
graphy on silica gel (40 g, column: 2 cmÂ12 inch),
eluting with a gradient of CHCl3:MeOH:formic acid
(100:0:0 to 90:10:0.1), to a€ord 5 as a pale beige solid
(0.020 g, 23.3%): mp 188±190 ꢀC; IR (®lm) 3500±2500,
THF (2 mL) was added. The red mixture was stirred at
0 ꢀC for 15 min. Then, it was cooled to 78 ꢀC and a
solution of ketone 242 (1.00 g, 2.35 mmol) in THF
(8 mL) was added. The reaction mixture was warmed to
room temperature over 3 h. A 20% aq solution of citric
acid was added to adjust the pH to 4. The mixture was
then stirred at room temperature overnight. The mix-
ture was poured into brine (100 mL) and the layers were
separated. The aqueous fraction was extracted with
ethyl ether (3Â40 mL). The combined organic extracts
were washed with sat NaHCO3 (1Â40 mL) and dried
over MgSO4. The solvent was removed and the residue
was puri®ed by ¯ash chromatography on silica gel
(ꢁ120 g; column: 4Â24 cm), eluting with hexanes:ethyl
acetate 3:1. Compound 25 was obtained as a pale yellow
solid in moderate yield (0.476g, 44.9%): mp 101±103 ꢀC;
IR (®lm) 2953, 1732, 1668, 1477, 1282, 1209, 994,
1
745 cm
;
1H NMR (300 MHz, CDCl3) d 9.50 (d,
J=7.8 Hz, 1H), 7.65 (d, J=2.3 Hz, 1H), 7.61 (d, J=
2.2 Hz, 1H), 7.45 (d, J=2.6 Hz, 1H), 7.44 (d, J=2.7 Hz,
1H), 6.52 (d, J=7.8 Hz, 1H), 4.01 (s, 3H), 3.96 (s, 3H),
3.92 (s, 3H), 3.90 (s, 3H); FABMS m/z 453 (MH+) and
421 (MH+-CH3OH). Anal. calcd for C21H18Cl2O7: C,
55.65; H, 4.00. Found: C, 55.80; H, 4.08.
1
2929, 1677, 1610, 1461, 1235, 1183, 801 cm 1; H NMR
(300 MHz, acetone-d6) d 7.74 (d, J=1.6 Hz, 1H), 7.72
(d, J=2.1 Hz, 1H), 7.50 (d, J=1.7 Hz, 1H), 7.46 (d,
J=2.0 Hz, 1H), 7.27 (d, J=7.9 Hz, 1H), 6.33 (t, J=
7.5 Hz, 1H), 3.73 (m, 1H), 3.09 (d, J=7.5 Hz, 2H), 0.91
(d, J=6.5 Hz, 3H), 0.85 (d, J=6.5 Hz, 6H), 0.77 (s, 3H),
0.66 (s, 3H); FABMS m/z: 796 (MH+). Anal. calcd for
N-(5ꢀ,3ꢁ-Cholestanyl)-3,3-(30,300-dichloro-50,500-dimethoxy-
40,400-dimethoxydiphenyl)-2-propenylamine (26). A mix-
ture of 3b-aminocholestane hydrochloride (14) (0.135 g,
0.320 mmol), aldehyde 25 (0.100 g, 0.221 mmol) and
Et3N (0.045 mL, 0.32 mmol) was partially dissolved in
benzene:THF (3:3 mL). Anhydrous magnesium sulfate
(0.053 g, 0.44 mmol) was added and the mixture was
stirred at room temperature under Ar for 24 h. Addi-
tional magnesium sulfate (0.053 g, 0.44 mmol) was
added at this time, and the mixture was stirred for 24 h
at room temperature. A solution of NaCNBH3 (0.042 g,
0.66 mmol) in MeOH (2 mL) was added. The mixture
was stirred for 6 h at room temperature. More solid
NaCNBH3 (0.014 g) was added and the mixture was
stirred overnight. The solvents were removed and the
residue was partitioned between water (15 mL) and
EtOAc (20 mL). The phases were separated and the
aqueous phase was extracted with EtOAc (4Â15 mL).
The combined organic extracts were washed with brine
(1Â30 mL), dried over magnesium sulfate, ®ltered and
the solvent removed. Puri®cation was achieved by ¯ash
chromatography on silica gel (ꢁ38 g; column: 2Â26 cm),
eluting with hexanes:ethyl acetate 2:1 containing 1%
Et3N, followed by hexanes:ethyl acetate 1:1 containing
1% Et3N. Compound 26 was obtained as a white solid
.
C45H59Cl2NO7 H2O: C, 66.39; H, 7.56; N, 1.72. Found:
C, 66.45; H, 7.69; N, 1.52.
N-(5ꢀ,3ꢀ-Cholestanyl)-4,4-(30,300-dicarboxy-50,500-dichloro-
40,400-dimethoxydiphenyl)-3-butenamide (6). A mixture of
21 (0.065 g, 0.076 mmol), K2CO3 (0.182 g, 1.32 mmol),
EtOH (2 mL) and water (0.5 mL) was stirred and heated
at 60 ꢀC for 4 days. At the end of this time, the solvent
was removed, and the residue was diluted with water
(15 mL) and washed with ethyl acetate (10 mL). The
aqueous phase was acidi®ed with conc HCl and extrac-
ted with EtOAc (4Â10 mL). The combined organic
extracts were washed with brine (1Â15 mL), dried over
Na2SO4, ®ltered and the solvent removed. The crude
product was recrystallized from acetone:acetonitrile to
provide 6 as an o€-white solid (0.052 g, 82.5%): mp
164±166 ꢀC; IR (®lm) 3500±2500, 2931, 1697, 1477,
1
1254, 999 cm 1; H NMR (300 MHz, CDCl3) d 7.76 (d,
J=2.2 Hz, 1H), 7.74 (d, J=2.0 Hz, 1H), 7.46 (d, J=
2.0 Hz, 1H), 7.43 (d, J=2.1 Hz, 1H), 6.36 (t, J=7.3 Hz,
1H), 5.79 (d, J=7.5 Hz, 1H), 4.12 (m, 1H), 4.09 (s, 3H),
4.02 (s, 3H), 3.05 (d, J=7.3 Hz, 2H), 0.85 (s, 6H), 0.82
(s, 3H), 0.77 (s, 3H), 0.61 (s, 3H); FABMS m/z 824
(MH+). Anal. calcd for C47H63Cl2NO7: C, 68.43; H,
7.70; N, 1.70. Found: C, 68.60; H, 7.93; N, 1.61.
in good yield (0.149 g, 82%): mp 152±154 ꢀC; IR (®lm)
1
2930, 2865, 1736, 1475, 1435, 1264, 1206, 999, 742 cm
;
1H NMR (300 MHz, CDCl3) d 7.52 (d, J=1.8 Hz, 1H),
7.46 (d, J=1.7 Hz, 1H), 7.33 (d, J=2.0 Hz, 1H), 7.30 (d,
J=1.8 Hz, 1H), 6.14 (t, J=6.7 Hz, 1H), 3.97 (s, 3H),
3.91 (s, 3H), 3.89 (s, 3H), 3.88 (s, 3H), 3.28 (d, J=
6.9 Hz, 2H), 2.40 (m, 1H), 0.74 (s, 3H), 0.61 (s, 3H);
FABMS m/z 824 (MH+). Anal. calcd for C48H67
Cl2NO6: C, 69.88; H, 8.19; N, 1.70. Found: C, 70.04; H,
8.27; N, 1.76.
30,300 -Dichloro-40,400 -dimethoxy-50,500 -bis(methoxycarb-
onyl)-3,3-diphenyl-2-propenal (25). A solution of diiso-
propylamine (0.43 mL, 3.1 mmol) in dry THF (5 mL)
was cooled to 0 ꢀC and put under Ar. A 1.6 M solution
of BuLi in hexanes (1.8 mL, 2.9 mmol) was added
slowly. The mixture was stirred at 0 ꢀC for 10 min. A
solution of silyl imine 2318 (0.625 g, 2.93 mmol) in dry
N-(5ꢀ,3ꢀ-Cholestanyl)-3,3-(30,300-dichloro-50,500-dimethoxy-
40,400-dimethoxydiphenyl)-2-propenylamine (27). A mixture

A. Casimiro-Garcia et al. / Bioorg. Med. Chem. 8 (2000) 191±200
199
of 3a-aminocholestane hydrochloride (11) (0.205 g,
0.484 mmol), aldehyde 25 (0.151 g, 0.334 mmol) and
Et3N (0.07 mL, 0.5 mmol) was partially dissolved in
dry benzene (7 mL). Anhydrous magnesium sulfate
(0.080 g, 0.67 mmol) was added and the mixture was
stirred at room temperature under Ar for 24 h. Addi-
tional magnesium sulfate (0.040 g, 0.33 mmol) was
added at this time, and the mixture was stirred for 24 h
at room temperature. The solvent was partially removed,
and a solution of NaCNBH3 (0.063 g, 1.002 mmol) in
MeOH (2 mL) was added. The cloudy mixture was stir-
red for 1 h at room temperature. The solvents were
removed and the residue was partitioned between water
(15 mL) and EtOAc (20 mL). The phases were separated
and the aqueous phase was extracted with EtOAc
(4Â15 mL). The combined organic extracts were washed
with brine (1Â30 mL), dried over magnesium sulfate,
®ltered and the solvent removed. Puri®cation was
achieved by ¯ash chromatography on silica gel (ꢁ38 g;
column: 2Â29 cm), eluting with hexanes:ethyl acetate
4:1 containing 1% Et3N, followed by hexanes:ethyl
acetate 3:1 containing 1% Et3N. Compound 27 was
obtained as an o€-white solid in good yield (0.238 g,
86.5%): mp 76±77 ꢀC; IR (®lm) 2930, 2867, 1737, 1476,
aqueous solution was then heated at 75ꢀC for 1 h. It was
allowed to cool and washed with EtOAc (2Â15 mL).
Then, it was acidi®ed with conc HCl and extracted with
EtOAc (6Â15 mL). The combined organic extracts were
washed with brine (1Â30 mL), dried over Na2SO4, ®l-
tered and the solvent removed. Drying under high vac-
cum a€orded 8 (0.174 g, 98.1%) in excellent yield. The
analytical sample was obtained by recrystallization from
THF±acetonitrile: mp 205±210 ꢀC; IR (®lm) 3500±2500,
1
2934, 2867, 1704, 1477, 1255, 999 cm
;
1H NMR
(300 MHz, acetone-d6±CDCl3) d 7.58 (d, J=2.2 Hz,
1H), 7.56 (d, J=2.2 Hz, 1H), 7.47 (d, J=2.2 Hz, 1H),
7.45 (d, J=2.1 Hz, 1H), 6.64 (t, J=7.1 Hz, 1H), 3.95 (s,
3H), 3.87 (s, 3H), 3.71 (m, 2H), 3.38 (bs, 1H), 0.73 (s,
3H), 0.57 (s, 3H); FABMS m/z 796 (MH+). Anal. calcd
.
for C46H63Cl2NO6 1.0 H2O: C, 67.80; H, 8.04; N, 1.72.
Found: C, 67.68; H, 8.31; N, 1.53.
In vitro anti-HIV assay. Evaluations of the antiviral
activity of compounds against HIV-1RF, HIV-1IIIB, and
HIV-2ROD infection in CEM-SS and MT-4 cells were
performed as previously described.24,25
1
1434, 1264, 1206, 1000, 743 cm 1; H NMR (300 MHz,
Acknowledgements
CDCl3) d 7.52 (d, J=1.9 Hz, 1H), 7.45 (d, J=1.8 Hz,
1H), 7.36 (d, J=1.8 Hz, 1H), 7.31 (d, J=1.5 Hz, 1H),
6.18 (m, 1H), 3.97 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H), 3.88
(s, 3H), 3.21 (d, J=4.8 Hz, 2H), 2.80 (m, 1H), 0.74 (s,
3H), 0.61 (s, 3H); FABMS m/z 824 (MH+). Anal. calcd
This research was made possible by NIH Grant
NO1-AI-36624, and by grants from the Fonds voor
Wetenschappelijk Onderzoek (FWO) Vlaanderen and
the Vlaamse Gemeenschap. Investigations were sup-
ported in part by the Biomedical Research Programme
of the European Commission. A.C.-G. wishes to thank
Consejo Nacional de Ciencia y Tecnologia (Mexico)
for a scholarship and Cytel Corporation for ®nancial
support.
.
for C48H67Cl2NO6 0.3 H2O: C, 69.43; H, 8.21; N, 1.69.
Found: C, 69.08; H, 8.27; N, 1.57.
N-(5ꢀ,3ꢁ-Cholestanyl)-3,3-(30,300-dicarboxy-50,500-dichloro-
40,400-dimethoxydiphenyl)-2-propenylamine (7). Amine
26 (0.108 g, 0.131 mmol) was partially dissolved in
THF:MeOH:water (4:3:1.5 mL). K2CO3 (0.326 g,
2.36 mmol) was added, followed by KCN (ꢁ2.0 mg,
0.026 mmol). The mixture was heated at 75 ꢀC for 5 h.
The solvents were removed and more water was added
(10 mL). The aqueous solution was then heated at 75 ꢀC
for 1 h. It was allowed to cool and washed with EtOAc
(2Â15 mL). Then, it was acidi®ed with conc HCl, and
extracted with EtOAc (5Â15 mL). The combined
organic extracts were washed with brine (1Â30 mL),
dried over Na2SO4, ®ltered and the solvent removed.
Compound 7 was obtained as an o€-white solid in
excellent yield (0.102 g, 97.9%): mp 205±210 ꢀC; IR
(®lm) 3500±2500, 2934, 1714, 1477, 1257, 1000 cm 1; 1H
NMR (300 MHz, acetone-d6) d 7.72 (s, 1H), 7.71 (s,
1H), 7.70 (s, 1H), 7.51 (s, 1H), 6.68 (t, J=7.0 Hz, 1H),
3.95 (s, 3H), 3.88 (s, 3H), 3.84 (m, 2H), 3.16 (m, 1H),
0.75 (s, 3H), 0.65 (s, 3H); FABMS m/z 796 (MH+).
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