Correlation of anti-HIV potency with lipophilicity in a series of cosalane analogs having normal alkenyl and phosphodiester chains as cholestane replacements

Article abstract of DOI:10.1021/jm950666h

In order to define the role of the cholestane moiety in the anti-HIV agent cosalane, a series of cosalane analogs was synthesized in which the cholestane ring system was replaced by normal alkenyl and phosphodiester substituents having varied chain lengths and lipophilicities. The compounds containing simple alkenyl substituents were found to be more potent as inhibitors of the cytopathic effect of HIV-1 in cell culture than the phosphodiesters. In addition, the potencies of the alkene congeners correlated positively with chain length and lipophilicity of the alkene. The results indicate that the cholestane moiety of cosalane functions as a lipophilic accessory appendage to escort the dichlorodisalicylmethane pharmacophore to a lipid environment.

Full text of DOI:10.1021/jm950666h

508
J . Med. Chem. 1996, 39, 508-514
Cor r ela tion of An ti-HIV P oten cy w ith Lip op h ilicity in a Ser ies of Cosa la n e
An a logs Ha vin g Nor m a l Alk en yl a n d P h osp h od iester Ch a in s a s Ch olesta n e
Rep la cem en ts
Robert F. Keyes, W. Marek Golebiewski, and Mark Cushman*
Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University,
West Lafayette, Indiana 47907
Received September 8, 1995X
In order to define the role of the cholestane moiety in the anti-HIV agent cosalane, a series of
cosalane analogs was synthesized in which the cholestane ring system was replaced by normal
alkenyl and phosphodiester substituents having varied chain lengths and lipophilicities. The
compounds containing simple alkenyl substituents were found to be more potent as inhibitors
of the cytopathic effect of HIV-1 in cell culture than the phosphodiesters. In addition, the
potencies of the alkene congeners correlated positively with chain length and lipophilicity of
the alkene. The results indicate that the cholestane moiety of cosalane functions as a lipophilic
accessory appendage to escort the dichlorodisalicylmethane pharmacophore to a lipid environ-
ment.
Cosalane (1) is a recently reported anti-HIV agent
displaying a wide range of activity against a variety of
laboratory, drug-resistant, and clinical HIV-1 isolates,
as well as HIV-2.1,2 The mechanism of action of cos-
alane (1) involves inhibition of gp120-CD4 binding as
well as inhibition of a postattachment event prior to
reverse transcription.2 Cosalane (1) was designed con-
ceptually by taking a dichlorinated disalicylmethane
fragment 23 of the polymeric anti-HIV agent aurintri-
carboxylic acid (ATA, represented schematically as 3)4-8
and attaching a cholestane moiety to it through a three-
carbon linker chain. During the cosalane (1) design
process, it was reasoned that the dichlorodisalicyl-
methane would act as the “pharmacophore” and the
cholestane fragment would serve as an accessory mod-
ule to increase potency by directing the molecule to the
lipid environment of the cell membrane and the viral
envelope. Consistent with this idea, the dichlorinated
disalicylmethane fragment 23 does have some, albeit
very low, anti-HIV activity (EC50 329 µM against HIV-1
strain RF in CEM-SS cells) compared to that of cosalane
(1) (EC50 3.0 µM against HIV-1 strain RF in CEM-SS
cells). In order to further test the validity of this design
concept, a series of cosalane (1) analogs was prepared
in which the lipophilicity of the accessory appendage
attached to the proposed pharmacophore (2) was modu-
lated in a systematic fashion. Several n-alkenyl- and
phosphodiester-containing chains of varying lengths and
lipophilicities were attached to the central carbon atom
of the dichlorodisalicylmethane fragment 2, and the
potencies of the resulting compounds 9-12 (Scheme 1)
and 25-29 (Scheme 2) as anti-HIV agents were deter-
mined in an assay which monitors inhibition of HIV-
1RF cytopathicity in CEM-SS.9 If the design concept
were valid, then the potencies of 9-12 and 25-29 might
be expected to correlate in a positive fashion with the
lipophilicities of the substituents attached to the dichlo-
rodisalicylmethane unit.
It has been suggested that the steroid moiety of
cosalane (1) may imbed perpendicularly in the lipid
bilayer of the cell membrane and viral envelope, with
the dichlorodisalicylmethane fragment pointing outward
X Abstract published in Advance ACS Abstracts, December 15, 1995.
0022-2623/96/1839-0508$12.00/0 © 1996 American Chemical Society

Synthesis of Cholestane Replacements
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 509
Sch em e 2a
F igu r e 1. Schematic representation of the anchoring of the
disalicylmethane moiety of cosalane and cosalane analogs to
the cell membrane and viral envelope by the steroid nucleus
of cosalane or the hydrocarbon moieties of the cosalane
analogs.
Sch em e 1a
a
Reagents: (a) Ph3PdCHR; (b) BBr3, CH2Cl2.
in an obstructive mode (Figure 1).2 An interaction of
cosalane (1) with the cell membrane and viral envelope
lipid bilayers may be involved in the inhibition of the
fusion of the viral envelope with the cell membrane.
This hypothesis is consistent with the fact that cosalane
inhibits the cytopathic effect of the Rauscher murine
leukemia virus, which does not depend on gp120-CD4
binding.2 In addition, cosalane (1) inhibits HIV-1 cy-
topathicity if added immediately after attachment of the
virion to the cell membrane, and time of addition
experiments have established that it acts prior to
reverse transcription.2 If viral and cellular membranes
are in fact acting as a cosalane receptor, then biologi-
cally active analogs of cosalane might be obtained by
replacement of the steroid unit with lipophilic alkenyl
or phospholipid moieties that could also enter into
phospholipid membrane bilayers. Various phospholip-
ids have already demonstrated anti-HIV activity, and
although the mechanism of antiviral action is not
completely understood, it is thought that it involves
insertion of the phospholipids into cellular and viral
membranes.10-14 Prior studies of anti-HIV phospholip-
ids have correlated increased lipid chain length with
a
Reagents and conditions: (a) B2H6, THF, reflux (3.5 h); (b)
CH3C(OCH3)2CH3, p-TsOH, DMF, 75 °C (24 h); (c) CrO3, Ac2O,
0-23 °C (18 h); (d) (1) Ph3P+(CH2)3OTBDMSBr-, NaN[Si(CH3)3]2,
THF, 0 °C (30 min); (2) compound 16, THF, 23 °C (24 h); (e)
n-Bu4NF, THF, 0 °C (1 h); (f) (1) alcohol 18, CHCl3, -10 °C (15
min), 23 °C (2.5 h); (2) ROH, -10 to 23 °C (24 h); (g) (1) aqueous
K2CO3, 1,4-dioxane, reflux (3 h); (2) 3 N HCl, 23 °C (1.5 h).
increased potency.11,14 Recently, it has been reported
that certain membrane-interactive phospholipids inhibit
HIV-1-induced membrane fusion by an unidentified
mechanism.15
Ch em istr y
The desired 1,1-diphenyl-1-alkenes 9-12 were syn-
thesized from the known benzophenone 42 in two steps
as outlined in Scheme 1. Reaction of 4 with the
appropriate Wittig reagents derived from n-alkyltri-

510 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Keyes et al.
Ta ble 1. Anti-HIV Activities of Cosalane Analogs
phenylphosphonium bromides of various chain lengths
afforded the intermediates 5-8. These compounds were
demethylated with boron tribromide in methylene chlo-
ride to afford the cosalane analogs 9-12.
a
b
compd
EC50 (µM)
IC50 (µM)
1
2
9
3.0
329
>300
>364
>95
>49
>35
>220
101
84
NAc
43.2
19.8
13.1
NAd
NAd
NAd
51.8
49.0
NAd
The syntheses of the phospholipid cosalane analogs
25-29, outlined in Scheme 2, was more problematic
because of the instability of phosphodiesters to the
demethylation conditions used in the conversion of 5-8
to 9-12. Therefore, an alternative strategy for the
protection and deprotection of the carboxylic acid and
phenolic hydroxyl groups had to be devised. The
protection of these four groups as two cyclic acetonides
as in 16 seemed appealing, but all attempts to directly
convert 13 to 16 proved fruitless. Accordingly, the two
carboxylic acids present in 132 were reduced to alcohols
with diborane in refluxing THF, and the resulting
product 14 was converted to the diacetonide 15 on
heating with 2,2-dimethoxypropane and p-toluenesulfon-
ic acid in N,N-dimethylformamide. The three benzylic
methylene groups present in 15 were then oxidized with
chromium trioxide in acetic anhydride to give the
desired benzophenone derivative 16. Treatment of 16
with the Wittig reagent derived from deprotonation of
triphenyl(3-((tert-butyldimethylsilyl)oxy)propyl)phos-
phonium bromide provided the alkene 17. The tert-
butyldimethylsilyl protecting group was removed from
17 using tetra-n-butylammonium fluoride in tetrahy-
drofuran to afford the primary alcohol 18. The acetoin
enediol cyclophosphoimidazole reagent (19) was chosen
for the conversion of the primary alcohol 18 to the
desired analogs 25-29 because it is particularly well
suited for the synthesis of unsymmetrical phosphodi-
esters.16 Sequential treatment of 19 with 18, followed
by various primary alcohols, provided intermediate
phosphotriesters 20-24. Both of the acetonide protect-
ing groups as well as the 3-keto-2-butyl group were
removed from the intermediates 20-24 by potassium
carbonate in refluxing 1,4-dioxane to yield the final
products 25-29.
10
11
12
25
26
27
28
29
30
74
86
61.3
105
a
The EC50 is the 50% inhibitory concentration for cytopathicity
of HIV-1RF in CEM-SS cells. The values are the averages of at
least two determinations. All compounds were tested as their
b
ammonium salts. The IC50 is the 50% cytotoxic concentration for
mock-infected CEM-SS cells. The values are the averages of at
least two determinations. All compounds were tested as their
ammonium salts. c No antiviral activity was observed at concen-
d
trations up to 95 µM. No antiviral acticity was observed at the
IC50 concentration.
tively, did show anti-HIV activity. Compound 28 had
an EC50 of 51.8 µM for prevention of the cytopathic effect
of HIV-1 in cell culture, while the corresponding value
of 29 was 49.0 µM. In contrast to cosalane (1) and the
analogs 9-12, the phosphodiesters proved to be more
cytotoxic in uninfected CEM-SS cells, with the IC50
values for 28 and 29 being close to the EC50 values. This
degree of cytotoxicity effectively eliminates these phos-
phodiesters as potential therapeutic agents for the
treatment of AIDS, and it also does not bode well for
the general idea of replacing the cholestane structural
unit of cosalane with phospholipid fragments resembling
membrane components.
The results seen with the phosphodiester analogs 25-
29 is reminiscent of the anti-HIV activities reported for
simple alkylphosphatidylethanolamines, which increased
in potency in the order n-hexyl < n-dodecyl < n-
octadecyl < oleyl.11,14 As a group, the phosphodiester
analogs 25-29 were less potent as anti-HIV agents than
the simpler alkenes 9-12. This difference in activity
is rather intriguing and may reflect the reduced lipo-
philicity expected as a result of introducing a polar
phosphate group into an otherwise very lipophilic side
chain. Another possibility is that the phosphate moiety
restrict the sliding motion of the side chain in the
membrane (Figure 1), thus making it more difficult to
achieve the optimal positioning of the compound in the
membrane for biological activity. Additional phosphodi-
ester analogs in which the location of the phosphate
moiety in the side chain is varied might be useful in
probing this question further. In connection with the
discussion of the inactive analogs, it is interesting to
note that the cosalane congener (30), having an ada-
mantyl-containing side chain, is also inactive as an anti-
Biologica l Resu lts a n d Discu ssion
The new cosalane analogs 9-12 and 25-29 were
tested for inhibition of the cytopathic effect of HIV-1RF
in CEM-SS cell culture as well as for cytotoxicity in
uninfected CEM-SS cells. The results are listed in Table
1. In comparing the EC50 values for prevention of HIV-1
cytopathicity for compounds 9 (not active), 10 (EC50 43.2
µM), 11 (19.8 µM), and 12 (EC50 13.1), it is apparent
that greater potency correlates with longer chain length
and increased lipophilicity. This indicates that the
antiviral activity displayed by cosalane is not structur-
ally specific with regard to the cholestane moiety, since
activity remains after replacement of the steroid moiety
with straight chain hydrocarbons, provided they are
sufficiently long and lipophilic. These results are
consistent with the idea that the role of the cholestane
fragment of cosalane is to escort the dichlorodisilicyl-
methane pharmacophore to a lipid environment.
Unlike the active compounds 10-12 having normal
alkenyl side chains, the phosphodiester cosalane analogs
25-27 were inactive as anti-HIV agents. None of these
compounds prevented the cytopathic effect of HIV-1 in
CEM cells at concentrations lower than the cytotoxic
concentrations. However, the phosphodiesters 28 and
29, containing linoleyl and oleyl substituents, respec-

Synthesis of Cholestane Replacements
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 511
HIV agent.17 In this case, the bulky nature of the
adamantyl substituent may prevent its packing ef-
ficiently between the alkyl chains in the membrane
phospholipid bilayer.
In conclusion, the present studies have helped to
define the cholestane moiety of cosalane (1) as a
lipophilic accessory module involved in ushering the
dichlorodisalicylmethane pharmacophore to a lipid en-
vironment. This concept may prove to be useful in the
design of additional cosalane analogs of potential use
in the treatment of AIDS.
hexane-ethyl acetate mixture (6:1) afforded undecene 6 (1.07
g, 83%): mp 71 °C; IR (KBr) 2927, 2853, 1736, 1476, 1250,
1
1210, 1092, 1000, 843, 744 cm-1; H NMR (500 MHz, CDCl3)
δ 7.48 (d, J ) 2.4 Hz, 1 H), 7.46 (d, J ) 2.2 Hz, 1 H), 7.31 (d,
J ) 2.2 Hz, 1 H), 7.28 (d, J ) 2.4 Hz, 1 H), 6.05 (t, J ) 7.5 Hz,
1 H, H-6), 3.98 (s, 3 H), 3.91 (s, 3 H), 3.90 (s, 3 H), 3.89 (s, 3
H), 2.06 (q, J ) 7.4 Hz, 2 H, H-3), 1.42 (m, 2 H, H-4), 1.26 (m,
2 H, H-5), 1.23 (m, 10 H), 0.85 (t, J ) 6.9 Hz, 3 H, H-11); 13C
NMR (CDCl3, 125.7 MHz) δ 165.81, 165.49, 154.88, 154.53,
138.67, 136.83, 135.51, 135.07, 133.66, 132.44, 131.03, 129.60,
129.36, 128.04, 126.64, 126.56, 62.05, 62.00, 52.55, 52.52,
31.90, 29.83, 29.66, 29.54, 29.46, 29.32, 29.27, 22.70, 14.15;
CIMS m/z (relative intensity) 551 (MH+, 100), 522 (10), 519
(15). Anal. (C29H36Cl2O6) C, H.
Exp er im en ta l Section
n -Hexa d ecyltr ip h en ylp h osp h on iu m Br om id e. Triphen-
ylphosphine (10.18 g, 38.81 mmol) was dissolved in hot
acetonitrile (15 mL). n-Hexadecyl bromide (11.85 mL, 38.81
mmol), was added, and the solution was heated at reflux with
stirring for 30 h. The solvent was removed in vacuo, and the
resulting oil was triturated three times with n-hexane with
decantation of the solvent each time until crystallization came
to completion. The salt (18.31 g, 83%) was dried in the vacuum
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; FAB mass spectra and EI mass spectra on a
Kratos MS50 spectrometer; 1H NMR spectra on Varian VXR-
500S, XL-200A, and Bru¨ker ARX-300 spectrometers; IR spec-
tra on a Beckman IR-33 spectrometer or on a Perkin-Elmer
1600 series FTIR. Microanalyses were performed at the
Purdue Microanalysis Laboratory.
1
desiccator: mp 103 °C; H NMR (CD3OD, 200 MHz) δ 8.9-
8.7 (m, 15 H), 1.6 (m, 4 H), 1.27 (bs, 26 H), 1.89 (t, J ) 6.7 Hz,
3 H); FABMS m/z (relative intensity) 487 (100). Anal. (C34H48
-
3′,3′′-Dich lor o-4′,4′′-d im eth oxy-5′,5′′-bis(m eth oxyca r bo-
n yl)-1,1-d ip h en yl-1-h ep ten e (5). Sodium hydride (54 mg,
1.35 mol, 60% dispersion in mineral oil) was washed with
n-hexane (3 × 5 mL). Dimethyl sulfoxide (2 mL) was intro-
duced via a syringe, and the mixture was heated at 75 °C until
the evolution of hydrogen ceased. The clear solution was
cooled in an ice-water bath, and a solution of the n-hexyl-
triphenylphosphonium bromide18 (576 mg, 1.35 mmol) in
DMSO (3 mL) was added dropwise. The resulting solution was
stirred at room temperature for 15 min. A solution of the
ketone 42 (0.86 mmol, 369 mg) in warm DMSO (6 mL) was
added dropwise and the reaction mixture heated at 55 °C for
27 h. It was then cooled in an ice bath. A solution of
ammonium chloride (143 mg) in water (5 mL) was added, and
the reaction mixture was extracted with ethyl ether (5 × 5
mL). The organic extracts were washed twice with brine, dried
over sodium sulfate, and evaporated in vacuo. Flash chroma-
tography on silica gel (230-400 mesh), eluting with n-hexane/
ethyl acetate, 4:1, yielded starting ketone (39 mg) and heptene
8 (320 mg, 75%): mp 88 °C; IR (KBr) 3046, 1732, 1595, 1364,
BrP) C, H.
3′,3′′-Dich lor o-4′,4′′-d im eth oxy-5′,5′′-bis(m eth oxyca r bo-
n yl)-1,1-d ip h en yl-1-h ep ta d ecen e (7). Compound 7 was
prepared similarly to the alkene 5 from n-hexadecyltriphen-
ylphosphonium bromide (1.065 g, 1.876 mmol) and benzophen-
one 4 (536 mg, 1.255 mmol) in DMSO. The flash chromatog-
raphy yielded the starting material 4 (65 mg) and the hepta-
decene 7 (210 mg, 58%): mp 75 °C; IR (KBr) 2923, 2852, 1735,
1476, 1435, 1251, 1211, 1092, 1000, 841, 743 cm-1; CIMS m/z
(relative intensity) 635 (MH+, 100), 603 (13); 1H NMR (CDCl3,
500 MHz) δ 7.48 (d, J ) 2.4 Hz, 1 H), 7.46 (d, J ) 2.2 Hz, 1
H), 7.31 (d, J ) 2.2 Hz, 1 H), 7.28 (d, J ) 2.4 Hz, 1 H), 6.05 (t,
J ) 7.5 Hz, 2 H), 3.98 (s, 3 H), 3.91 (s, 3 H), 3.90 (s, 3 H), 3.89
(s, 3 H), 2.06 (q, J ) 6.8 Hz, 2 H), 1.26 (m, 2 H), 1.25 (m, 2 H),
1.23 (m, 22 H), 0.85 (t, J ) 6.8 Hz, 3 H); 13C NMR (125.7 MHz,
CDCl3) δ 165.80, 165.47, 154.87, 154.52, 138.65, 136.82, 135.49,
135.06, 133.65, 132.40, 132.02, 129.59, 129.35, 128.03, 126.62,
126.55, 31.93, 29.83, 29.71, 29.70, 29.67, 29.59, 29.47, 29.38,
29.27, 22.71, 14.16. Anal. (C35H48Cl2O6) C, H.
Tr ia con ta n yl Br om id e. A solution of triacontanol (946
mg, 2.156 mmol) and carbon tetrabromide (1.43 mg, 4.31
mmol) in dry acetonitrile (12 mL) was heated under reflux with
stirring, and a solution of triphenylphosphine (1.696 mg, 6.466
mmol) was added dropwise over 2 min. The mixture was
heated at reflux for 2 h and cooled, the solvent was removed
in vacuo, and the residue was extracted with benzene (3 × 10
mL). The combined extracts were filtered, and the solvent was
removed in vacuo. The residue was flash chromatographed
on silica gel (85 g, hexane-ethyl acetate, 6:1) and the product
(0.936 g, 85.5%) recrystallized from acetone: mp 67 °C; 1H
NMR (CDCl3, 200 MHz) δ 3.41 (t, J ) 6.9 Hz, 2 H), 1.85 (m,
54 H), 0.88 (t, J ) 5.8 Hz, 3 H); CIMS m/z (relative intensity)
499 ((M - H)+, 2), 421 (MH+ - HBr, 31). Anal. (C10H61Br) C,
H.
Tr ia con ta n yltr ip h en ylp h osp h on iu m Br om id e. Bro-
mide 2 (737.3 mg, 1.47 mmol) and triphenylphosphine (385.5
mg, 1.47 mmol) were dissolved in chlorobenzene (5 mL), and
the solution was heated at reflux for 4 days under argon with
stirring. The solvent was removed in vacuo. The residue was
triturated with hexane, filtered, and washed with hexane to
afford a solid (1.049 g, 93.4%). The analytical sample was
prepared by crystallization from acetone: mp 106-107 °C; 1H
NMR (CD4OD, 200 MHz) δ 7.0-7.7 (m, 15 H), 3.38 (m, 2 H),
1.57 (m, 4 H), 1.27 (bs, 52 H), 0.89 (t, 3 H); FABMS m/z
(relative intensity) 683 (M+, 100). Anal. (C48H76BrP) C, H.
3′,3′′-Dich lor o-4′,4′′-d im eth oxy-5′,5′′-bis(m eth oxyca r bo-
n yl)-1,1-d ip h en yl-1-u n t r ia con t en e (8). Triacontanyltri-
phenylphosphonium bromide (857 g, 1.12 mmol) was sus-
pended in dry THF (25 mL), the mixture was cooled in an ice
bath, and sodium bis(trimethylsilyl)amide (1 M solution in
THF, 1.12 mL) was added dropwise. The mixture was stirred
1
1250, 1211 cm-1; H NMR (CDCl3, 500 MHz) δ 7.49 (d, J )
2.4 Hz, 1 H), 7.48 (d, J ) 2.2 Hz, 1 H), 7.32 (d, J ) 2.2 Hz, 1
H), 7.30 (d, J ) 2.4 Hz, 1 H), 6.07 (t, J ) 7.6 Hz, 1 H), 4.00 (s,
3 H), 3.93 (s, 3 H), 3.92 (s, 3 H), 3.91 (s, 3 H), 2.08 (m, 2 H),
1.44 (m, 2 H), 1.27 (m, 4 H), 0.88 (t, J ) 6.9 Hz, 3 H); CIMS
m/z (relative intensity) 495 (MH+, 100), 463 (12). Anal.
(C25H28Cl2O6) C, H.
3′,3′′-Dich lor o-4′,4′′-d im eth oxy-5′,5′′-bis(m eth oxyca r bo-
n yl)-1,1-diph en yl-1-u n decen e (6). Finely ground and freshly
dried decyltriphenylphosphonium bromide19 (1.14 g, 2.60
mmol) was placed in a dry 50 mL, two-necked, round-bottomed
flask equipped with a Teflon-coated magnetic stirring bar, a
reflux condenser connected to an argon flow line, and a rubber
septum. The apparatus was flushed with argon, and the argon
atmosphere was maintained throughout the reaction. THF
(18 mL, freshly distilled from sodium benzophenone) was
added via the septum, the suspension of the phosphonium salt
was cooled in an ice bath, and a 1 M solution of sodium bis-
(trimethylsilyl)amide in THF (2.6 mL) was added dropwise.
The ice bath was removed and the mixture stirred at room
temperature until a homogeneous solution was obtained (15
min). The orange solution was cooled again in the ice bath,
and a solution of the ketone 4 (1.00 g, 2.34 mmol) in THF (5
mL) was added dropwise through the septum via the syringe.
The bath was removed, and the reaction mixture was stirred
at room temperature overnight. The reaction mixture was
quenched with a solution of ammonium chloride (0.25 g) in
water (10 mL). The organic layer was separated and the
aqueous layer extracted with ether (1 × 5 mL). The combined
organic extracts were dried (sodium sulfate), and the solvent
was removed in vacuo to yield a crystallizing oil (2.05 g) which
was flash chromatographed on silica gel (85 g). Elution with

512 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Keyes et al.
for 15 min, and a solution of ketone 4 (0.479 g, 1.12 mmol) in
THF (7 mL) was added dropwise. The ice bath was removed,
and the reaction mixture was stirred 9 h at 70 °C. The
reaction was quenched with ammonium chloride solution, the
THF phase was separated, and aqueous phase was extracted
with ether. The combined organic extracts were dried (sodium
sulfate) and evaporated to dryness, and the residue was
subjected to flash chromatography on silica gel (hexane-ethyl
acetate, 9:1) followed by recrystallization from ethanol to yield
the product as a solid (0.73 g, 78%): mp 88 °C; IR (KBr) 2919,
acetone to afford a solid (150 mg, 65%): mp 173-174 °C; IR
(KBr) 3500-2600, 2919, 2851, 1672, 1604, 1468, 1236, 1181,
900, 798, 720 cm-1
; 1H NMR (acetone-d6, 200 MHz) δ 7.70 (d,
J ) 2 Hz, 1 H), 7.67 (d, J ) 2.2 Hz, 1 H), 7.56 (d, J ) 2.2 Hz,
1 H), 7.48 (d, J ) 2 Hz, 1 H), 6.18 (t, J ) 7.4 Hz, 1 H), 2.13 (m,
2 H), 1.48 (m, 2 H), 1.27 (m, 54 H), 0.86 (t, J ) 6.4 Hz, 3 H);
FABMS m/z (relative intensity) 797 (MNa+, 7), 774 (M+, 11).
Anal. (C45H68Cl2O6‚2H2O) C, H.
3′,3′′-Dich lor o-4′,4′′-dih ydr oxy-5′,5′′-bis(h ydr oxym eth yl)-
1,1-d ip h en ylm eth a n e (14). Compound 132 (0.807 g, 2.30
mmol) was dissolved in anhydrous THF (40 mL) and placed
under an argon atmosphere. A 1 M solution of borane-
tetrahydrofuran complex (9.1 mL, 9.10 mmol) was added
dropwise. The reaction mixture was heated at reflux for 3.5
h and then allowed to cool. Brine (20 mL) was slowly added,
and stirring was continued for 30 min. The layers were then
separated, and the aqueous layer was extracted with diethyl
ether (3 × 30 mL). The extracts were combined and washed
with saturated sodium bicarbonate (2 × 100 mL). The extracts
were dried over sodium sulfate and filtered, and the solvent
was removed in vacuo. The material was purified by flash
chromatography (silica gel 230-400 mesh, ethyl acetate) to
2851, 1735, 1475, 1435, 1362, 1287, 1251, 1209, 797, 742, cm-1
;
1H NMR (CDCl3, 200 MHz) δ 7.49 (d, J ) 2.3 Hz, 1 H), 7.48
(d, J ) 2.1 Hz, 1 H), 7.32 (d, J ) 2.1 Hz, 1 H), 7.30 (d, J ) 2.3
Hz, 1 H), 6.07 (t, J ) 7.8 Hz, 1 H), 4.00 (s, 3 H), 3.93 (s, 3 H),
3.92 (s, 6 H), 2.08 (m, 2 H), 1.42 (m, 2 H), 1.25 (m, 52 H), 0.88
(t, J ) 5.7 Hz, 3 H); FABMS m/z relative intensity 831 (M+,
9), 799 (M+ - MeOH, 16). Anal. (C49H76Cl2O6) C, H.
3′,3′′-Dich lor o-5′,5′′-dicar boxy-4′,4′′-dih ydr oxy-1,1-diph e-
n yl-1-h ep ten e (9). A solution of methoxy ester 5 (148.6 mg,
0.3 mmol) in dry methylene chloride (2.5 mL) was added by
syringe through a septum under an argon atmosphere to a
stirred solution of boron tribromide (1 M) in methylene chloride
(1.6 mL) cooled in a dry ice-acetone bath. The cooling bath
was removed after 2 h and stirring continued at ambient
temperature for 2 days. More BBr3 (0.8 mL) was added on
the second day. The reaction was quenched with water (2 mL),
stirring was continued for 30 min, and the product was
extracted with 20% aqueous KOH. The alkaline solution was
acidified on cooling with concentrated hydrochloric acid and
the product extracted with ethyl acetate. The organic extracts
were washed with brine, dried (sodium sulfate), and concen-
trated in vacuo. The product was crystallized from methylene
chloride: mp 234-236 °C; IR (KBr) 3500-2500, 2926, 2856,
1
give 14 (0.555 g, 74%) as a white solid: mp 143-145 °C; H
NMR (acetone-d6, 300 MHz) δ 7.10 (s, 2 H), 7.09 (s, 2 H), 4.75
(s, 4 H), 3.80 (s, 2 H); IR (KBr) 3286, 2927, 1581, 1481, 1311,
1250, 1184, 1115, 1008, 891, 789, 696, 634 cm-1; EIMS m/z
(relative intensity) 328 (M+, 17), 310 (35), 292 (100), 257 (35),
229 (32), 194 (24), 165 (51). Anal. (C15H14Cl2O4) C, H.
8,8′-Dich lor o-2,2,2′,2′-tetr a m eth yl-4,4′-d ioxo-6,6′-d i(1,3-
ben zod ioxyl)m eth a n e (15). Compound 14 (0.100 g, 0.305
mmol) was dissolved in anhydrous DMF (0.50 mL) and placed
under an argon atmosphere. 2,2-Dimethoxypropane (0.15 mL,
1.23 mmol) and a catalytic amount of p-toluenesulfonic acid
monohydrate (0.0015 g, 0.0079 mmol) were added. The
reaction mixture was then heated to 75 °C for 24 h. Distilled
water (5 mL) was added, and the resulting solution was
extracted with diethyl ether (3 × 15 mL). The combined
organic extracts were washed with distilled water (5 × 20 mL),
followed by brine (20 mL). The extracts were dried over
sodium sulfate and filtered, and the solvent was removed in
vacuo. Purification by flash chromatography (silica gel 230-
400 mesh, CHCl3) gave 15 (0.110 g, 88%) as a white solid: mp
1670, 1600, 1443, 1232, 1179, 901, 799, 715 cm-1 1H NMR
;
(CDCl3, 200 MHz) δ 7.68 (m, 2 H), 7.50 (d, J ) 1.6 Hz, 1 H),
7.41 (d, J ) 1.6 Hz, 1 H), 6.13 (t, J ) 7.2 Hz, 1 H), 2.11 (m, 2
H), 1.48 (m, 2 H), 1.28 (m, 14 H), 0.85 (t, J ) 6.3 Hz, 3 H);
FABMS m/e (relative intensity) 438 (M+, 2), 307 (10). Anal.
(C21H20Cl2O6‚1/2H2O) C, H.
3′,3′′-Dicar boxy-5′,5′′-dich lor o-4′,4′′-dih ydr oxy-1,1-diph e-
n yl-1-u n d ecen e (10). Boron tribromide (1 M solution in
methylene chloride, 1.75 mL) was cooled in a dry ice-acetone
bath, and a solution of compound 6 (181 mg, 0.328 mmol) in
dry methylene chloride (3 mL) was added via septum in an
argon atmosphere with magnetic stirring. The cooling bath
was removed after 1 h and stirring continued in an ambient
temperature for 16 h. The reaction was quenched with water
(3 mL), stirring was continued for 30 min, and the product
was extracted with 10% KOH (3 × 3 mL). The alkaline
solution was washed with methylene chloride and acidified on
cooling with concentrated HCl (1.2 mL), and the product was
extracted with ethyl acetate (3 × 3 mL). The organic extracts
were washed with brine, dried (sodium sulfate), and concen-
trated in vacuo to afford an oil (93 mg, 57%), which crystallized
on trituration with methylene chloride: mp 207 °C; IR (KBr)
3500-2500, 2925, 2855, 1665, 1445, 1232, 1177, 899, 799, 719
cm-1; FABMS m/z (relative intensity) 494 (M+, 18), 477 (MH+
1
135-137 °C; H NMR (CHCl3, 300 MHz) δ 7.03 (s, 2 H), 6.63
(s, 2 H), 4.78 (s, 4 H), 3.72 (s, 2 H), 1.56 (s, 12 H); IR (KBr)
2999, 2944, 2873, 1480, 1385, 1353, 1309, 1278, 1206, 1122,
1059, 954, 878, 800, 724, 657, 604, 519 cm-1; FABMS m/z
(relative intensity) 409 (MH+, 25), 350 (55), 292 (50).
8,8′-Dich lor o-2,2,2′,2′-tetr a m eth yl-4,4′-d ioxo-6,6′-d i(1,3-
ben zod ioxyl) Keton e (16). Compound 15 (2.43 g, 6.00 mmol)
was suspended in acetic anhydride (55 mL) and the mixture
cooled to 0 °C (internal monitoring) in an ice bath. Chromium-
(VI) oxide (7.86 g, 78.6 mmol) was added in small portions.
Once the addition was complete, the temperature was gradu-
ally allowed to rise to room temperature. CAUTION: in order
to avoid a violent exothermic reaction, do not allow the
temperature to exceed 35 °C. Stirring was continued at room
temperature for 18 h. The acetic anhydride was removed in
vacuo, and the residue was taken up in a mixture of ethyl
acetate (200 mL) and distilled water (200 mL) and vacuum-
filtered through a pad of Celite. The layers were separated,
and the aqueous layer was washed with ethyl acetate (3 ×
200 mL). The combined extracts were washed with distilled
water (3 × 200 mL), followed by brine (200 mL). The extracts
were dried (Na2SO4), and filtered, and the solvent was removed
in vacuo. Purification by flash chromatography (ethyl acetate/
hexanes, 50/50) gave pure 16 (1.51 g, 56%): mp 189-191 °C:
1H NMR (CDCl3, 300 MHz) δ 8.23 (d, J ) 2.1 Hz, 2 H), 8.13
(d, J ) 2.1 Hz, 2 H), 1.86 (s, 12 H); IR (KBr) 3080, 3006, 1749,
1655, 1603, 1482, 1388, 1285, 1236, 1196, 1062, 928, 873, 717
1
- H2O, 20); H NMR (CDCl3, 200 MHz) δ 7.60 (bs, 2 H), 7.42
(bs, 1 H), 7.34 (bs, 1 H), 6.06 (t, 1 H), 2.08 (m, 2 H), 1.46 (m,
2 H), 1.25 (m, 12 H), 0.88 (t, 3 H). Anal. (C25H28Cl2O6) C, H.
3′,3′′-Dicar boxy-5′,5′′-dich lor o-4′,4′′-dih ydr oxy-1,1-diph e-
n yl-1-u n tr ia con ten e (11). A stirred solution of boron tri-
bromide (1 M in methylene chloride, 3.47 mL) under argon
was cooled in a dry ice-acetone bath, and a solution of
compound 8 (247 mg, 0.296 mmol) in dry methylene chloride
(6 mL) was added by syringe through a septum. The cooling
bath was removed after 1 h and stirring continued at an
ambient temperature for 2 days. The reaction was quenched
with water (3 mL), stirring was continued for 30 min, and the
product was extracted with 10% NaHCO3 (3 × 3 mL). The
alkaline solution was washed with methylene chloride and
acidified on cooling with concentrated HCl, and the product
was extracted with ethyl acetate (3 × 3 mL). The organic
extracts were washed with brine, dried (sodium sulfate),
concentrated in vacuo, and recrystallized from chloroform-
cm-1
.
1,1-Bis[8′,8′′-d ich lor o-2′,2′,2′′,2′′-tetr a m eth yl-4′,4′′-d ioxo-
6′,6′′-(1,3-ben zod ioxyl)]-4-((ter t-bu tyld im eth ylsilyl)oxy)-
1-b u t en e (17). Triphenyl(3-((tert-butyldimethylsilyl)oxy)-
propyl)phosphonium bromide2 (1.73 g, 3.35 mmol) was sus-
pended in anhydrous THF (55 mL) under argon. The suspen-
sion was cooled to 0 °C in an ice bath, and a 1 M solution of

Synthesis of Cholestane Replacements
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 513
sodium bis(trimethylsilyl)amide (3.66 mL, 3.66 mmol) was
added dropwise. Stirring was continued at 0 °C for 30 min,
and then a solution of benzophenone 16 dissolved in anhydrous
THF (18 mL) was added slowly. The cooling bath was
removed, and the reaction mixture was allowed to stir at room
temperature for 24 h. Saturated NH4Cl (50 mL) was added,
and the layers were separated. The aqueous layer was
extracted with ethyl acetate (3 × 30 mL). The combined
organic extracts were washed with water (75 mL), followed
by brine (2 × 50 mL). The extracts were dried (Na2SO4) and
filtered, and the solvent was removed in vacuo. Purification
by flash chromatography (ethyl acetate/hexanes, 50/50) gave
pure 17 (1.32 g, 98%): mp 56-59 °C; 1H NMR (CDCl3, 300
MHz) δ 7.65 (d, J ) 1.8 Hz, 2 H), 7.49 (d, J ) 2.0 Hz, 1 H),
7.41 (d, J ) 2.2 Hz, 1 H), 6.10 (t, J ) 7.0 Hz, 1 H), 3.69 (t, J
) 6.4 Hz, 2 H), 2.30 (q, J ) 7.3 Hz, 2 H), 1.81 (s, 6 H), 1.77 (s,
6 H), 0.87 (s, 9 H), 0.03 (s, 6 H). Anal. (C30H36Cl2O7Si) C, H.
7.41 (t, J ) 1.8 Hz, 1 H), 6.10 (m, 1 H), 4.75 (q, J ) 7.8 Hz, 1
H), 4.07 (m, 4 H), 2.50 (m, 2 H), 2.20 (d, J ) 3.2 Hz, 3 H), 1.82
(s, 6 H), 1.77 (s, 6 H), 1.66 (m, 2 H), 1.45 (m, 3 H), 1.23 (s, 26
H), 0.86 (t, J ) 6.4 Hz, 3 H); IR (neat) 2925, 2854, 1749, 1608,
1484, 1380, 1282, 1199, 1062, 1005, 876, 836, 777 cm-1
;
FABMS m/ z (relative intensity) 867 (MH+, 10), 617 (35), 475
(20), 417 (95), 359 (100). Anal. (C44H61Cl2PO11‚H2O) C, H.
4,4-Bis[8′,8′′-d ich lor o-2′,2′,2′′,2′′-tetr a m eth yl-4′,4′′-d ioxo-
6′,6′′-(1,3-ben zod ioxyl)]-3-bu ten yl 2-Keto-1-m eth ylp r op yl
Octa d ecyl P h osp h a te (22). Compound 22 (169.2 mg, 31%)
was obtained as an amorphous mixture of diastereomers: 1H
NMR (CDCl3, 300 MHz) δ 7.69 (d, J ) 2.1 Hz, 1 H), 7.67 (d, J
) 2.2 Hz, 1 H), 7.48 (d, J ) 2.2 Hz, 0.5 H), 7.47 (d, J ) 2.2 Hz,
0.5 H), 7.44 (t, J ) 1.8 Hz, 1 H), 6.08 (m, 1 H), 4.81 (m, 1 H),
4.09 (m, 4 H), 2.52. (m, 2 H), 2.24 (d, J ) 3.2 Hz, 3 H), 1.84 (s,
6 H), 1.80 (s, 6 H), 1.66 (m, 2 H), 1.48 (m, 3 H), 1.26 (s, 30 H),
0.88 (t, J ) 6.4 Hz, 3 H); IR (neat) 2925, 2854, 1747, 1608,
1484, 1391, 1380, 1283, 1200, 1063, 1008, 912, 734 cm-1
;
4,4-Bis[8′,8′′-d ich lor o-2′,2′,2′′,2′′-tetr a m eth yl-4′,4′′-d ioxo-
6′,6′′-(1,3-ben zod ioxyl)]-3-bu ten -1-ol (18). Silyl ether 17
(1.05 g, 1.70 mmol) was dissolved in anhydrous THF (20 mL)
and placed under an argon atmosphere. The solution was
cooled to 0 °C in an ice bath, and a 1 M solution of tetrabu-
tylammonium fluoride (3.47 mL, 3.47 mmol) was added
dropwise. Stirring was continued at 0 °C for 1 h, and then
brine was added. The layers were separated, and the aqueous
layer was extracted with ethyl acetate (3 × 30 mL). The
organic layers were combined and washed with distilled water
(2 × 50 mL), followed by brine (50 mL). The extracts were
dried (Na2SO4) and filtered, and the solvent was removed in
vacuo. Purification by flash chromatography (silica gel, 230-
400 mesh, ethyl acetate/hexanes, 50/50) gave pure 18 (0.524
FABMS m/ z (relative intensity) 895 (MH+, 15), 475 (30), 416
(100), 359 (80). Anal. (C46H65C12PO11) C, H.
4,4-Bis[8′,8′′-d ich lor o-2′,2′,2′′,2′′-tetr a m eth yl-4′,4′′-d ioxo-
6′,6′′-(1,3-ben zod ioxyl)]-3-bu ten yl 2-Keto-1-m eth ylp r op yl
Lin oleyl P h osp h a te (23). Compound 23 (179.1 mg, 40%)
was obtained as an amorphous mixture of diastereomers: 1H
NMR (CDCl3, 300 MHz) δ 7.68 (d, J ) 1.7 Hz, 1 H), 7.67 (d, J
) 2.0 Hz, 1 H), 7.48 (d, J ) 2.0 Hz, 0.5 H), 7.47 (d, J ) 2.5 Hz,
0.5 H), 7.43 (t, J ) 1.6 Hz, 1 H), 6.15 (m, 1 H), 5.34 (m, 4 H),
4.79 (m, 1 H), 4.14 (m, 4 H), 2.77 (t, J ) 5.8 Hz, 2 H), 2.52 (m,
2 H), 2.23 (d, J ) 3.0 Hz, 3 H), 2.03 (m, 8 H), 1.84 (s, 6 H),
1.80 (s, 6 H), 1.68 (m, 2 H), 1.48 (m, 3 H), 1.26 (m, 12 H), 0.89
(t, J ) 6.3 Hz, 3 H); IR (neat) 2927, 2855, 1748, 1608, 1484,
1380, 1282, 1199, 1062, 1005, 876 cm-1; FABMS m/ z (relative
intensity) 913 (MNa+, 45), 855 (10), 797 (5), 417 (50), 359 (100).
Anal. Calcd for C46H61Cl2PO11: C, 61.95; H, 6.89. Found: C,
61.81; H, 6.93.
1
g, 63%): mp 185-188 °C; H NMR (CDCl3, 300 MHz) δ 7.72
(d, J ) 2.0 Hz, 1 H), 7.70 (d, J ) 2.2 Hz, 1 H), 7.47 (d, J ) 2.0
Hz, 1 H), 7.45 (d, J ) 2.2 Hz, 1 H), 6.17 (t, J ) 7.5 Hz, 1 H),
3.77 (t, J ) 6.4 Hz, 2 H), 2.40 (q, J ) 6.4 Hz, 2 H), 1.84 (s, 6
H), 1.80 (s, 6 H).
4,4-Bis[8′,8′′-d ich lor o-2′,2′,2′′,2′′-tetr a m eth yl-4′,4′′-d ioxo-
6′,6′′-(1,3-ben zod ioxyl)]-3-bu ten yl 2-Keto-1-m eth ylp r op yl
Oleyl P h osp h a te (24). Compound 24 (272.0 mg, 61%) was
obtained as an amorphous mixture of diastereomers. This
mixture was used directly in the next step without being
characterized.
Gen er a l P r oced u r e for th e Syn th esis of P h osp h otr i-
ester s 20-24. Acetoin enediol cyclophosphoimidazole (19)16
(0.146 g, 0.728 mmol) was dissolved in anhydrous CHCl3 (1.2
mL) and placed under an argon atmosphere. The solution was
cooled to -10 °C in an ice/salt bath, and a solution of alcohol
18 (0.275 g, 0.560 mmol) dissolved in anhydrous CHCl3 (1 mL)
was added dropwise over a 40-50 min period. Once the
addition was complete, the reaction mixture was stirred at -10
°C for 15 min and at room temperature for 2.5 h. The reaction
mixture was cooled back to -10 °C, and a solution of the second
required primary alcohol (ROH, Scheme 2) (0.560 mmol)
dissolved in anhydrous CHCl3 (1.0 mL) was added slowly. The
cooling bath was removed, and stirring was continued for 24
h. Saturated NH4Cl (5 mL) was added. The solution was
extracted with ethyl acetate (3 × 25 mL). The combined
extracts were washed with distilled water (50 mL), followed
by brine (50 mL). The extracts were dried (Na2SO4) and
filtered, and the solvent was removed in vacuo. Purification
by flash chromatography (silica gel, 230-400 mesh, ethyl
acetate/hexanes, 50/50) gave pure phosphodiesters 20-24.
4,4-Bis[8′,8′′-d ich lor o-2′,2′,2′′,2′′-tetr a m eth yl-4′,4′′-d ioxo-
6′,6′′-(1,3-ben zod ioxyl)]-3-bu ten yl 2-Keto-1-m eth ylp r op yl
Tetr a d ecyl P h osp h a te (20). Compound 20 (272 mg, 60.8%)
was obtained as an amorphous mixture of diastereomers: 1H
NMR (CDCl3, 300 MHz) δ 7.68 (d, J ) 2.1 Hz, 1 H), 7.67 (d, J
) 2.2 Hz, 1 H), 7.48 (d, J ) 2.2 Hz, 0.5 H), 7.47 (d, J ) 2.2 Hz,
0.5 H), 7.43 (t, J ) 1.8 Hz, 1 H), 6.12 (m, 1 H), 4.77 (m, 1 H),
4.12 (m, 4 H), 2.52 (m, 2 H), 2.23 (d, J ) 4.5 Hz, 3 H), 1.84 (s,
6 H), 1.80 (s, 6 H), 1.68 (m, 2 H), 1.47 (m, 3 H), 1.25 (s, 22 H),
0.88 (t, J ) 6.3 Hz, 3 H); IR (neat) 2925, 2853, 1748, 1608,
1483, 1380, 1282, 1199, 1004, 876, 836, 777 cm-1; FABMS m/ z
(relative intensity) 839 (MH+, 40), 475 (45), 417 (98), 359 (100).
Anal. (C42H57Cl2PO11) C, H.
Gen er a l P r oced u r e for th e Syn th esis of P h osp h a tes
25-29. Phosphotriesters 20-24 (0.293 mmol) were dissolved
in 1,4-dioxane (4 mL). A 0.74 M solution of aqueous K2CO3
(0.242 g, 1.75 mmol) was added. The yellow solution was
heated at reflux for 3 h. Distilled water (10 mL) was added,
and the solution was acidified with 3 N HCl. The solution
was extracted with ethyl acetate (5 × 25 mL). The combined
extracts were washed with brine (1 × 50 mL). The extracts
were dried (MgSO4) and filtered, and the solvent was removed
in vacuo. Purification by preparative TLC, eluting with
n-BuOH, H2O, and HOAc (4:1:1), gave a salt. Distilled water
(10 mL) was added. The solution was acidified with 3 N HCl
and stirred at room temperature for 1.5 h. The solution was
extracted with EtOAc (5 × 25 mL). The combined extracts
were washed with brine (50 mL), dried (MgSO4), and filtered.
Concentration of the filtrate gave the desired phosphodiesters
25-29.
4,4-(3′,3′′-Dicar boxy-5′,5′′-dich lor o-4′,4′′-dih ydr oxydiph e-
n yl)-3-bu ten -1-yl Tetr a d ecyl P h osp h a te (25). Phosphate
25 was obtained as an amorphous solid in 33% yield: 1H NMR
(CD3OD, 300 MHz) δ 7.48 (d, J ) 2.1 Hz, 2 H), 7.31 (d, J )
1.7 Hz, 1 H), 7.25 (d, J ) 1.5 Hz, 1 H), 5.98 (t, J ) 7.2 Hz, 1
H), 3.93 (q, J ) 6.1 Hz, 2 H), 3.80 (q, J ) 6.6 Hz, 2 H), 2.33 (q,
J ) 6.5 Hz, 2 H), 1.47 (m, 2 H), 1.06 (m, 22 H), 0.70 (t, J ) 6.8
Hz, 3 H); IR (KBr) 2923, 2852, 1665, 1595, 1460, 1233, 1181,
1024, 800 cm-1; FABMS m/ z (relative intensity) 733 [(MNa2
- H)+, 20], 711 (100), 395 (60), 333 (45), 217 (40), 149 (65).
Anal. (C32H43Cl2PO10) C, H.
4,4-Bis[8′,8′′-d ich lor o-2′,2′,2′′,2′′-tetr a m eth yl-4′,4′′-d ioxo-
6′,6′′-(1,3-ben zod ioxyl)]-3-bu ten yl Hexa d ecyl 2-Keto-1-
m eth ylp r op yl P h osp h a te (21). Compound 21 was obtained
as an amorphous mixture of diastereomers: 1H NMR (CDCl3,
300 MHz) δ 7.66 (d, J ) 2.1 Hz, 1 H), 7.65 (d, J ) 2.2 Hz, 1
H), 7.46 (d, J ) 2.2 Hz, 0.5 H), 7.45 (d, J ) 2.2 Hz, 0.5 H),
4,4-(3′,3′′-Dicar boxy-5′,5′′-dich lor o-4′,4′′-dih ydr oxydiph e-
n yl)-3-bu ten -1-yl Hexa d ecyl P h osp h a te (26). Phosphate
21 was obtained as an amorphous solid in 62% yield: 1H NMR
(CD3OD, 300 MHz) δ 7.54 (t, J ) 1.1 Hz, 2 H), 7.38 (d, J ) 2.2
Hz, 1 H), 7.32 (d, J ) 2.1 Hz, 1 H), 6.04 (t, J ) 7.2 Hz, 1 H),

514 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Keyes et al.
3.99 (q, J ) 6.1 Hz, 2 H), 3.86 (q, J ) 6.6 Hz, 2 H), 2.39 (q, J
) 6.1 Hz, 2 H), 1.52 (m, 2 H), 1.13 (m, 26 H), 0.79 (t, J ) 6.3
Hz, 3 H); IR (KBr) 2923, 2853, 1665, 1460, 1234, 1182, 1024,
799 cm-1; FABMS m/ z (relative intensity) 739 (MNa+, 90),
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533 (15), 415 (30), 395 (100) 359 (80). Anal. (C34H47Cl2PO10
C, H.
)
4,4-(3′,3′′-Dicar boxy-5′,5′′-dich lor o-4′,4′′-dih ydr oxydiph e-
n yl)-3-bu ten -1-yl Octa d ecyl P h osp h a te (27). Phosphate
27 was obtained as an amorphous solid in 40% yield: 1H NMR
(CD3OD, 300 MHz) δ 7.63 (t, J ) 1.9 Hz, 2 H), 7.47 (d, J ) 2.2
Hz, 1 H), 7.41 (d, J ) 2.1 Hz, 1 H), 6.13 (t, J ) 7.3 Hz, 1 H),
4.08 (q, J ) 6.2 Hz, 2 H), 3.95 (q, J ) 6.5 Hz, 2 H), 2.48 (q, J
) 6.4 Hz, 2 H), 1.62 (t, J ) 6.9 Hz, 2 H), 1.23 (m, 30 H), 0.88
(t, J ) 6.1 Hz, 3 H). FABMS m/ z (relative intensity) 767
(MNa+, 50), 395 (40), 205 (30), 149 (100). Anal. (C36H51Cl2-
PO10) C, H.
4,4-(3′,3′′-Dicar boxy-5′,5′′-dich lor o-4′,4′′-dih ydr oxydiph e-
n yl)-3-bu ten -1-yl Lin oleyl P h osp h a te (28). Phosphate 28
was obtained as an amorphous solid in 57% yield: 1H NMR
(CD3OD, 300 MHz) δ 7.53 (t, J ) 1.8 Hz, 2 H), 7.38 (d, J ) 2.4
Hz, 1 H), 7.32 (d, J ) 1.7 Hz, 1 H), 6.03 (t, J ) 7.2 Hz, 1 H),
5.20 (m, 4 H), 3.99 (q, J ) 5.8 Hz, 2 H), 3.85 (q, J ) 6.5 Hz, 2
H), 2.64 (t, J ) 5.4 Hz, 2 H), 2.39 (q, J ) 6.5 Hz, 2 H), 1.92 (m,
8 H), 1.52 (m, 2 H), 1.17 (m, 12 H), 0.78 (t, J ) 6.7 Hz, 3 H);
IR (KBr) 2924, 2854, 1667, 1600, 1454, 1233, 1181, 1023, 800
cm-1; FABMS m/ z (relative intensity) 763 (MNa+, 18), 136
(100). Anal. (C36H47Cl2PO10‚1/2H2O) C, H.
4,4-(3′,3′′-Dicar boxy-5′,5′′-dich lor o-4′,4′′-dih ydr oxydiph e-
n yl)-3-bu ten -1-yl Oleyl P h osp h a te (29). Phosphate 29 was
obtained as an amorphous solid in 65% yield: 1H NMR (CD3-
OD, 300 MHz) δ 7.64 (t, J ) 2.0 Hz, 2 H), 7.48 (d, J ) 2.2 Hz,
1 H), 7.42 (d, J ) 2.1 Hz, 1 H), 6.14 (t, J ) 7.3 Hz, 1 H), 5.30
(t, J ) 5.5 Hz, 2 H), 4.08 (q, J ) 6.2 Hz, 2 H), 3.95 (q, J ) 6.6
Hz, 2 H), 2.49 (q, J ) 6.1 Hz, 2 H), 1.99 (m, 4 H), 1.62 (m, 2
H), 1.27 (m, 22 H), 0.88 (t, J ) 7.0 Hz, 3 H); IR (KBr) 2925,
(12) Meyer, K. L.; Marasco, C. J ., J r.; Morris-Natschke, S. L.; Ishaq,
K. S.; Piantadosi, C. In Vitro Evaluation of Phosphocholine and
Quaternary Ammonium Containing Lipids as Novel Anti-HIV
Agents. J . Med. Chem. 1991, 34, 1377-1383.
(13) Pidgeon, C.; Markovich, R. J .; Liu, M. D.; Holzer, T. J .; Novak,
R. M.; Keyer, K. A. Antiviral Phospholipids. J . Biol. Chem. 1993,
268, 7773-7778.
(14) Cooper, D. L.; Mort, K. A.; Allan, N. L.; Kinchington, D.;
McGuigan, C. Molecular Similarity of Anti-HIV Phospholipids.
J . Am. Chem. Soc. 1993, 115, 12615-12616.
2854, 1667, 1603, 1455, 1232, 1182, 1025, 901, 800, 709 cm-1
;
FABMS m/ z (relative intensity) 764 (MNa+, 80), 704 (20), 395
(100), 376 (70). Anal. (C36H49Cl2PO10) C, H.
An ti-HIV Scr een in g. The anti-HIV screening was per-
formed as previously described.9 This microtiter assay quan-
titates drug-induced protection from the killing of CD4(+)-
lymphoid cells by HIV-1RF
. Briefly, cosalane or 3′-azido-3′-
(15) Krugner-Higby, L.; Goff, D.; Edwards, T.; Iyer, N.; Neufeld, J .;
Kute, T.; Morris-Natschke, S.; Ishaq, K.; Piantadosi, C.; Kucera,
L. S. Membrane-Interactive Phospholipids Inhibit HIV Type
1-Induced Cell Fusion and Surface gp160/gp120 Binding to
Monoclonal Antibody. AIDS Res. Human Retroviruses 1995, 11,
705-712.
deoxythymidine (AZT, NSC 602670, used as a reference
compound) were serially diluted in complete medium and
added to 96-well test plates. Exponentially growing target
cells were pelleted, suspended in complete medium, and added
at 5000 cells/well. Frozen virus stock solutions were thawed
immediately before use, suspended in complete medium to
yield the desired multiplicity of infection (MOI ) 0.01), and
added in the microtiter wells. Test plates were incubated at
37 °C in 5% CO2 for 6 days. On day 6 aliquots of cell-free
supernatant were removed from each well and analyzed for
reverse transcriptase (RT) activity, p24 antigen, and/or infec-
tious virions. Cellular growth or viability was estimated with
the remaining contents of each well by the XTT assay.20
Effective antiviral concentrations (EC50) and cellular growth
inhibitory concentrations (IC50) were calculated.
(16) Ramirez, F.; Marecek, J . F. Synthesis of Phosphotriesters: The
Cyclic Enediol Phosphoryl (CEP) Method. Synthesis 1985, 449-
488.
(17) Cushman, M.; Golebiewski, W. M.; Pommier, Y.; Mazumder, A.;
Reymen, D.; De Clercq, E.; Graham, L.; Rice, W. G. Cosalane
Analogs with Enhanced Potencies as Inhibitors of HIV-1 Pro-
tease and Integrase. J . Med. Chem. 1995, 38, 443-452.
(18) Hauser, C. F.; Brooks, T. W.; Miles, M. L.; Raymond, M. A.;
Butler, G. B. The Wittig Synthesis. I. Use of Certain Aliphatic
Aldehydes as Intermediates in the Synthesis of Olefins. J . Org.
Chem. 1963, 28, 372-379.
(19) Kodato, S.; Nakagawa, M.; Nakayama, K.; Hino, T. Synthesis
of Cerebroside B1b with Antiulcerogenic Activity. I. Synthesis
of Ceramides with Optically Active R-Hydroxypalmitic Acids.
Tetrahedron 1989, 45, 7247-7262.
(20) Gulakowski, R. J .; McMahon, J . B.; Staley, P. G.; Moran, R. A.;
Boyd, M. R. A Semiautomated Multiparameter Approach for
anti-HIV Drug Screening. J . Virol. Methods 1991, 33, 87-100.
Ack n ow led gm en t. This research was made possible
by NIH Grant RO1-AI-36624 and Contract NO1-CM-
17513.
J M950666H