Synthesis and biological evaluation of nonionic prenyl, geranyl, and farnesyl diphosphate surrogates

Article abstract of DOI:10.1006/bioo.1996.0022

Prenyl, geranyl, and farnesyl derivatives containing nonionic surrogates for the diphosphate moiety, including disulfones 4-6 and 7-9, methylene disulfonamides 10-12, and carbamyl sulfamides 13-15, have been synthesized and evaluated biologically in an effort to find suitable nonlabile, neutral inhibitors for enzymatic reactions which use these isoprenoid diphosphates as substrates. Farnesyl derivatives 6, 9, 12, and 15 were ineffective as squalene synthase inhibitors in vitro. Compounds 4-15 were screened in human skin fibroblasts for their effects on fatty acid, cholesterol, and DNA synthesis. In general, compounds 10-15 showed more inhibition than 4-9 and had a greater effect on DNA synthesis than on lipid synthesis, with the exception of 15.

Full text of DOI:10.1006/bioo.1996.0022

BIOORGANIC CHEMISTRY 24, 242–250 (1996)
ARTICLE NO. 0022
Synthesis and Biological Evaluation of Nonionic Prenyl,
Geranyl, and Farnesyl Diphosphate Surrogates
ALFREDO
C
ASTRO,* SANDRA K. ERICKSON,† ISHAIAHU
S
HECHTER,†,1 AND
,2
THOMAS A. SPENCER*
*Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755; †Department of
Medicine, University of California, San Francisco and Veterans Administration Medical Center,
San Francisco, California 94121; and ‡Eleanor Roosevelt Institute, Denver, Colorado 80206
Received February 2, 1996
Prenyl, geranyl, and farnesyl derivatives containing nonionic surrogates for the diphosphate
moiety, including disulfones 4–6 and 7–9, methylene disulfonamides 10–12, and carbamyl
sulfamides 13–15, have been synthesized and evaluated biologically in an effort to find suitable
nonlabile, neutral inhibitors for enzymatic reactions which use these isoprenoid diphosphates
as substrates. Farnesyl derivatives 6, 9, 12, and 15 were ineffective as squalene synthase
inhibitors in vitro. Compounds 4–15 were screened in human skin fibroblasts for their effects
on fatty acid, cholesterol, and DNA synthesis. In general, compounds 10–15 showed more
inhibition than 4–9 and had a greater effect on DNA synthesis than on lipid synthesis, with
the exception of 15.
1996 Academic Press, Inc.
This study was undertaken in an effort to find an appropriate surrogate for the
diphosphate group that is nonlabile and neutral and that could be used in prospective
inhibitors of enzymatic reactions which have substituted diphosphates as substrates.
The diphosphate moiety itself is unsuitable as part of inhibitors because it is readily
hydrolyzed by phosphatases in vivo and because, being ionic, it presumably would
have difficulty crossing cell membranes (1). A number of modified diphosphates,
e.g. (phosphinylmethyl)phosphonates (2), have been prepared in order to remove
the hydrolytic lability, but these compounds are still ionic, and in general, with
some recent exceptions (3), have not been effective inhibitors in vivo.
The specific type of diphosphate substrates for which nonionic surrogates are
considered in the present work are the isoprenoid building blocks prenyl (1), geranyl
(2), and farnesyl diphosphates (3). These mevalonate-derived intermediates are of
central biochemical importance (4–6). They play a key role in the biosynthesis of
lipids, including cholesterol, steroid hormones, bile acids, ubiquinone, and dolichols.
In addition, these isoprenoids are intermediates in the biosynthesis of isoprenylated
tRNAs, of protein conjugates involved in cell signaling, such as the RAS family,
and of heme a of cytochrome oxidase. Thus, specific inhibitors of the formation or
further metabolism of prenyl, geranyl, or farnesyl diphosphates have been widely
1 Current address: Department of Biochemistry, Uniformed Services University School of Medicine,
Bethesda, MD 20814.
2 To whom correspondence should be addressed.
242
0045-2068/96 $18.00
Copyright 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.

NONIONIC DIPHOSPHATE SURROGATES
243
sought because they could have a profound impact on a variety of cellular processes
and could serve as therapeutic agents for the prevention or treatment of atheroscle-
rosis, malignancies, or other proliferative disorders.
In the current study, four types of nonionic diphosphate mimics were selected
for attachment to these isoprenoid units. These include 1,3-disulfones with two
substitution patterns, namely compounds 4–6 and 7–9. Sulfones have previously
been proposed as nonionic, nonhydrolyzable substitutes for biological phosphodies-
ters (7). In addition, methylene disulfonamide derivatives 10–12 and carbamyl
sulfamide derivatives 13–15 were selected as more polar analogs than 1,3-disulfones
4–6. The carbamyl sulfamide group has previously been used as an isosteric substi-
tute for the diphosphate moiety in uridine diphosphate glucose (8).
To assess their potential as diphosphate surrogates, the farnesyl derivatives 6, 9,
12, and 15 were tested for their ability to act as in vitro inhibitors of squalene
synthase, the enzyme which couples two farnesyl diphosphates in a key step in
cholesterol biosynthesis. All of the synthesized compounds, 4–15, were evaluated
for their effects on lipid and DNA synthesis in human skin fibroblasts in culture.
SYNTHESIS
The syntheses of the two classes of prenyl, geranyl, and farnesyl disulfones 4–6
and 7–9, via alkylation of the trianion and dianion, respectively, of bis(methylsul-
fonyl)methane, have been described previously (9). Preparation of 10–12 was under-
taken via analogous alkylation of methylene disulfonamide (16) (10). Since no
alkylation reactions of 16 had been reported, treatment of 16 with D2O/D3CSO2CD3
was used to establish that the protons on N are much more readily exchanged than

244
CASTRO ET AL.
SCHEME 1
those on C, suggesting that the desired N alkylation might be achieved by use of
the monoanion of 16. This proved to be correct, although alkylation was successful
only when HMPA or DMPU was used as solvent, and then only in modest yield.
Under the most efficient conditions discovered, consisting in treatment of 16 in
DMPU with one equivalent of NaH and then with one equivalent of alkylating agent
at room temperature, prenyl bromide (17) afforded 40% of 10, geranyl bromide (18)
gave 35% of 11, and farnesyl bromide (19) gave 32% of 12 (Scheme 1).
Synthesis of carbamyl sulfamides 13–15 was achieved by a slight modification of
the procedure of Graf (11). Treatment of the appropriate allylic alcohol (20–22)
in CH2Cl2 at Ϫ23ЊC with one equivalent of chlorosulfonyl isocyanate (23) for 45
min followed by addition of a large excess of liquid NH3 led to 80% of 13, 88% of
14, and 84% of 15, respectively (Scheme 1).
BIOLOGICAL ACTIVITIES
The farnesyl derivatives 6, 9, 12, and 15 were tested as substrate analog inhibitors
of the conversion of farnesyl diphosphate (3) to squalene in a rat liver microsomal
squalene synthase assay (12). None of these four compounds caused significant
reduction of squalene synthase activity at concentrations up to 1 m , with each
M
exhibiting at most 5–10% inhibition at that highest concentration tested. These
results suggest that anionic charge is an important component of any diphosphate
surrogate for binding in the active site, consistent with earlier observations (13).
In addition, all twelve compounds 4–15 were screened for biological activity in

NONIONIC DIPHOSPHATE SURROGATES
245
TABLE 1
Effects of Compounds 10–15 on [14C]Acetate
Incorporation into Fatty Acids and Cholesterol
in Confluent Human Skin Fibroblast Cultures
Compound FA (IC50 , m
M)
CH (IC50 , mM)
3
10
10Ϫ
1
5
11a
12a
13
ȁ10Ϫ
ȁ0.1
ȁ0.1
Ͼ1
ȁ0.05
Ͼ1
14
ȁ0.1
ȁ0.01
ȁ0.1
ȁ0.1
15b
a All cells dead at 1 m
b Many cells dead at 1 m
M.
M
.
human skin fibroblasts (HSF). They were evaluated for effects on [3H]thymidine in-
corporation into DNA in growing cultures, and for effects on [14C]acetate incorpora-
tion into fatty acids and cholesterol in both growing and confluent cultures. Com-
pounds 4–9 had little effect on fatty acid or cholesterol synthesis in either confluent
or growing HSF, and 4–8 had no effect on DNA synthesis or cell viability in general.
However, in growing HSF, farnesyl derivative 9 inhibited DNA synthesis by 50% at
ȁ10Ϫ4 m
M
and caused complete cell death at the highest concentration used, 1 m .
M
Compounds 10–15 proved in general to be more interesting, although in confluent
HSF only compound 11 showed a significant effect, selectively inhibiting fatty acid
synthesis (Table 1), suggesting a degree of specificity. In the growing cell cultures,
compounds 10–15 exhibited strikingly varied effects (Table 2). Farnesyl methylene
disulfonamide (12) had little effect on either fatty acid or cholesterol synthesis, but
was a potent inhibitor of DNA synthesis, with IC50 in the nanomolar range. This
suggests that 12 might be useful in attenuating reactions involving nonsterol products
of the mevalonate pathway that are known to be critical for cell growth and differen-
TABLE 2
Effects of Compounds 10–15 on [3H]Thymidine Incorporation into
DNA and [14C]Acetate Incorporation into Fatty Acids and Choles-
terol in Growing Human Skin Fibroblasts
Compound DNA (IC50 , m
M
)
FA (IC50 , m
M
)
CH (IC50 , mM)
4
4
10
11
12a
13
14
15
10Ϫ
ȁ10Ϫ
10Ϫ
10Ϫ
Ͼ1
Ͼ1
Ͼ1
5
6
5
0.01
Ͼ1
3
10Ϫ
1
ȁ10Ϫ
6
4
4
5 ϫ 10Ϫ
10Ϫ
ȁ10Ϫ
6
7
ȁ0.1
10Ϫ
5 ϫ 10Ϫ
a All cells dead at 1 m
M.

246
CASTRO ET AL.
tiation. In contrast, farnesyl carbamyl sulfamide (15), unlike any of the other com-
pounds in this group, was very effective at inhibiting cellular cholesterol and fatty
acid synthesis (IC50’s in the nanomolar range) but had little effect on DNA synthesis
in these growing cells. Because 15 had minimal effect on squalene synthase in vitro,
its inhibition of cholesterol synthesis in intact cells is probably at another site.
Compound 15 could prove useful as a lipid synthesis inhibitor with relatively little
effect on cell growth. In contrast, geranyl carbamyl sulfamide (14) was effective at
inhibiting all tested functions in growing cells, although its effect on DNA synthesis
was 100-fold greater than that on lipid synthesis. The mechanism by which any of
these effects in growing HSF takes place is unknown, and it is unclear whether any
of the polar groups in 4–15 is functioning as a diphosphate surrogate, but compounds
10–15 certainly exhibit inhibitory properties deserving further exploration.
EXPERIMENTAL
General synthesis procedures. Melting points were determined in a Thomas-
Hoover apparatus in unsealed capillaries. Infrared (IR) spectra were recorded on
a Perkin–Elmer Model 599 spectrophotometer for neat liquids on NaCl plates or
in KBr pellets for solids. Nuclear magnetic resonance (NMR) spectra were recorded
on a Varian XL-300 spectrometer in acetone-d6 unless otherwise noted. Elemental
Analyses were performed by Atlantic Microlab Inc. (Norcross, GA). Dry flash
chromatography was carried out by the method of Harwood (14) on Kieselgel 60
silica (230–400 mesh). Reaction progress was monitored by thin layer chromatogra-
phy (TLC) using Kieselgel 60 F254 silica plates from EM Science. Visualization was
accomplished by either exposure to iodine vapors or illumination with 254- and
365-nm UV light. All reagents were purchased from Aldrich Chemical Co. (Milwau-
kee, WI) and were used directly or purified according to procedures described
below. Tetrahydrofuran (THF) was distilled from sodium metal with benzophenone
indicator. Titration of n-butyllithium in hexanes (n-BuLi) with Ph2CHCO2H in
THF was performed in duplicate prior to use. Methylene chloride (CH2Cl2) was
distilled from calcium hydride. Diethyl ether (ether) was distilled from sodium
metal. 1,3-Dimethyl-3,4,5,6-tetrahydro-2(1h)-pyrimidinone (DMPU) was distilled
˚
from calcium hydride and stored over molecular sieves (4 A). Buten-1-ol, 3-methyl-
2-buten-1-ol (20), trans-3,7-dimethyl-2,6-octadienol (geraniol, 21), and trans,trans-
3,7,11-trimethyl-2,6,10-dodecatrienol (farnesol, 22) were dried over molecular sieves
˚
(4 A) for at least 12 h prior to use. Sodium hydride as a 60% dispersion in mineral
oil (60% NaH) was washed with hexane and dried by passing N2 or Ar over the
white powder prior to use. Brine refers to saturated aqueous NaCl. Anhydrous
reactions were performed in glassware that had been flame-dried or heated in an
oven overnight at 125ЊC and then cooled in a desiccator containing Ca2SO4 or P2O5 .
The term ‘‘under N2’’ or ‘‘under Ar’’ refers to the maintenance of a positive pressure
of that gas which had been passed through a column of anhydrous Ca2SO4 .
(E)-3,7-Dimethyl-2,6-octadienyl bromide (geranyl bromide, 18). According to a
procedure of Edstrom (15), to a solution of 2.0 ml (12 mmol) of 21 in 10 ml of
hexane at 0ЊC in a flask wrapped in Al foil was added 0.36 ml (3.8 mmol) of PBr3 .
The mixture was allowed to warm to 24ЊC, stirred under N2 for 18 h, and 5.0 ml

NONIONIC DIPHOSPHATE SURROGATES
247
of 5% aqueous NaHCO3 solution was added. The aqueous layer was removed after
gas evolution ceased. Evaporation of solvent and vacuum distillation gave 2.0 g
1
(82%) of 113: bp 72ЊC, 0.6 Torr (lit. (16) bp 110ЊC, 3 Torr); H NMR ͳ 5.35 (t,
J ϭ 7.2 Hz, 1H), 5.08 (br s, 1H), 4.05 (d, J ϭ 8.4 Hz, 2H), 2.20-1.95 (m, 4H), 1.72
1
(s, 3H), 1.66 (s, 3H), 1.59 (s, 3H) (lit. (16) H NMR (CCl4) ͳ 5.46 (br t, J ϭ 8 Hz,
1H), 5.02 (br s, 1H), 3.95 (d, J ϭ 8.0 Hz, 2H), 2.05 (m, 4H), 1.70 (s, 3H), 1.65 (s,
3H), 1.58 (s, 3H)).
(E,E)-3,7,11-Trimethyl-2,6,10-dodecatrienyl bromide (farnesyl bromide, 19). Ex-
actly the same procedure used for 18 was employed with 1.0 ml (4.0 mmol) of 22
to afford 0.92 g (82%) of 114: bp 80ЊC, 0.05 Torr (lit. (17) bp 100–110ЊC, 0.15 Torr);
1H NMR ͳ 5.51 (t, J ϭ 8.0 Hz, 1H), 5.09 (m, 2H), 3.99 (d, J ϭ 9.0 Hz, 2H), 2.20-
1.90 (m, 8H), 1.71 (s, 3H), 1.65 (s, 3H), 1.59 (s, 3H), 1.58 (s, 3H) (lit. (17) 1H NMR
ͳ 5.52 (t, J ϭ 9 Hz, 1H), ȁ5.09 (br, s, 2H), 3.95 (d, J ϭ 9 Hz, 2H), 2.2-2.0 (m, 8H),
1.69 (s), 1.61 (s) (12H)).
Methylene disulfonamide (16). According to the procedure of Fild and Rieck
(18), to a mixture of 23 ml (0.25 mol) of POCl3 and 16 ml (0.24 mol) of ClSO3H
was added 7.5 ml (0.13 mol) of CH3CO2H. The resulting mixture was heated at
105ЊC for 1 h and then at 145ЊC for 8 h. The residue was distilled under vacuum
to give 22 g (85%) of methylenedisulfonylchloride as a clear fuming oil: bp 85ЊC,
0.05 T (lit. (18) 93ЊC, 0.2 T); 1H NMR (CDCl3) ͳ 5.58 (s) (lit. (18) 1H NMR (neat)
ͳ 5.69). According to the procedure of Bauer and Jenkins (10), to a solution of
20.2 g (0.0950 mol) of methylenedisulfonylchloride in 100 ml of benzene was added
a solution of 17.8 g (0.189 mol) of phenol and 30.5 ml (0.370 mol) of pyridine in
100 ml of benzene via cannula, under Ar, with both flasks at 0ЊC. The resulting
mixture was allowed to warm to rt, stirred for 16 h, quenched with 25 g of ice, and
extracted with 2
M
HCl (5 ϫ 50 ml), water (50 ml), and warm (ȁ45ЊC) 2
M
NaOH
HCl
(8 ϫ 50 ml). The combined basic extracts were acidified with 450 ml of 2
M
and cooled in an ice-water bath. The precipitate was collected and recrystallized
from 4 : 1 ethanol-water to give 24.9 g (80%) of diphenyl methylenedisulfonate: mp
1
83–84ЊC (lit. (10) mp 82–83ЊC): H NMR (benzene-d6) ͳ 7.10–7.20 (m, 4H), 6.75–
6.90 (m, 6H), 4.03 (s, 2H); 13C NMR (benzene-d6) ͳ 149.4, 130.2, 127.8, 122.3, 62.1.
A solution of 10.95 g (0.0334 mol) of diphenyl methylenedisulfonate in 60 ml of
benzene cooled in an ice bath was saturated with dry ammonia gas (from ȁ20 ml
of liq NH3 and sodium) in a bomb flask. The flask was sealed and placed in a pipe
heater for 4 h at 145ЊC (external temperature). The resulting mixture was filtered
to give 5.78 g (99%) of off-white 16. Recrystallization from water gave 4.98 g (86%)
of 16 as scaly white crystals: mp 237–239ЊC (lit. (8) 232–233ЊC): 1H NMR (DMSO-
d6) ͳ 7.24 (s, 4H), 4.66 (s, 2H); 13C NMR (DMSO-d6) ͳ 72.2.
(1-(3-Methyl-2-butenyl)sulfamoyl)methanesulfonamide (10). To 0.047 g (1.2
mmol) of 60% NaH was added a solution of 0.20 g (1.2 mmol) of 16 in 10 ml of
DMPU and the resulting mixture was stirred at rt for 5 h. Then 0.13 ml (1.1 mmol)
of 17 was added via syringe and the reaction mixture was stirred in the dark at rt
under Ar for 16 h, quenched with 1.0 ml of 5% HCl, diluted to 50 ml with water,
and extracted with ethyl acetate (4 ϫ 25 ml). The combined organic layers were
washed with water (2 ϫ 50 ml) and brine (2 ϫ 25 ml), dried over MgSO4 , filtered,
and evaporated to give 0.18 g of oily residue, which was subjected to dry flash

248
CASTRO ET AL.
chromatography (9 : 1 to 1 : 1 hexane-ethyl acetate) to yield 0.11 g (40%) of 10.
Recrystallization from acetone-hexane gave an analytical sample: mp 151.4–152.2ЊC;
1H NMR (DMSO-d6) ͳ 7.45 (t, J ϭ 5.3 Hz, 1H), 7.25 (s, 2H), 5.18 (t, J ϭ 7.1 Hz,
1H), 4.72 (s, 2H), 3.62 (t, J ϭ 6.2 Hz), 1.69 (s, 3H), 1.63 (s, 3H); 13C NMR ͳ 137.2,
120.8, 69.6, 42.0, 25.7, 18.9. Anal. Calcd for C6H14N2O4S2 : C, 29.74; H, 5.82. Found:
C, 29.82; H, 5.84.
(1-(3,7-Dimethyl-2,6-octadienyl)sulfamoyl)methanesulfonamide (11). To a mix-
ture of 0.10 g (2.5 mmol) of 60% NaH and 0.44 g (2.5 mmol) of 16 was added 10.0
ml of DMPU and the resulting mixture was stirred at rt for 1 h. Then 0.57 g (2.6
mmol) of 18 was added via syringe and the reaction mixture was stirred in the dark
at rt under Ar for 41 h, quenched with 3 ml of 5% HCl, diluted to 50 ml with water,
and extracted with ethyl acetate (5 ϫ 25 ml). The combined organic layers were
washed with water (3 ϫ 50 ml), brine (2 ϫ 30 ml), dried over MgSO4 , filtered, and
evaporated to give 0.536 g of oily solid residue, which was subjected to dry flash
chromatography (9 : 1 to 1 : 1 hexane-ethyl acetate) to yield 0.26 g (34%) of 11.
Recrystallization from acetone-hexane gave an analytical sample: mp 127.5–128.3ЊC;
1H NMR ͳ 6.53 (bs, 2H), 6.40 (bs, 1H), 5.32 (t, J ϭ 7.0 Hz, 1H), 5.11 (t, J ϭ 6.8
Hz, 1H), 3.81 (d, J ϭ 6.8 Hz, 2H), 2.14–2.00 (m, 2H), 1.70 (s, 3H), 1.66 (s, 3H),
1.60 (s, 3H); 13C NMR ͳ 140.7, 132.1, 124.7, 120.7, 69.6, 41.9, 40.2, 27.0, 25.8, 17.7,
16.3. Anal. Calcd for C11H22N2O4S2 : C, 42.56; H, 7.14. Found: C, 42.50; H, 7.13.
(1-(3,7,11-Trimethyl-2,6,10-dodecatrienyl)sulfamoyl)methanesulfonamide 12). To
a mixture of 0.048 g (1.2 mmol) of 60% NaH and 0.20 g (1.2 mmol) of 16 was added
2.5 ml of DMPU and the resulting mixture was stirred at rt for 4 h. Then 0.31 g
(1.1 mmol) of 19 was added via syringe and the reaction mixture was stirred in the
dark at rt under Ar for 23.5 h, quenched with 1.5 ml of 5% HCl, diluted to 100 ml
with water, and extracted with ethyl acetate (1 ϫ 75 ml, 4 ϫ 25 ml). The combined
organic layers were washed with water (2 ϫ 50 ml), brine (2 ϫ 50 ml), dried over
MgSO4 , filtered, and evaporated to give 0.31 g of oily solid residue, which was
subjected to dry flash chromatography (9 : 1 to 1 : 1 hexane-ethyl acetate) to yield
0.13 g (32%) of 12. Recrystallization from ethanol-water gave an analytical sample:
1
mp 91–93ЊC; H NMR ͳ 6.46 (bs, 2H), 6.32 (bs, 1H), 5.33 (t, J ϭ 6.4 Hz, 1H),
5.19–5.01 (m, 2H), 4.77 (s, 2H), 3.81 (d, J ϭ 6.4 Hz, 2H), 2.19–1.84 (m, 8H), 1.70
(s, 3H), 1.63 (s, 3H), 1.61 (s, 3H), 1.59 (s, 3H); 13C NMR ͳ 140.7, 131.5, 130.8, 125.0,
124.6, 120.5, 69.5, 41.9, 40.3, 40.1, 27.3, 26.9, 25.8, 17.7, 16.3, 16.1. Anal. Calcd for
C16H30N2O4S2 : C, 50.77; H, 7.99. Found: C, 50.85; H. 7.98.
3-Methyl-2-butenyl sulfamoylcarbamate (13). To a cold (Ϫ23ЊC) solution of 0.31
ml (3.0 mmol) of 99% 20 in 30 ml of CH2Cl2 was added 0.27 ml (3.1 mmol) of 23
all at once. The resulting mixture was stirred for 45 min, and 5 ml of ammonia was
added dropwise from a CO2-acetone-filled condenser over 45 min. The resulting
mixture was allowed to warm to rt, stirred under Ar for 4 h, and evaporated, and
the residue (0.86 g) was subjected to dry flash chromatography (4 : 1 to 1 : 9 hexane-
1
ethyl acetate) to give 0.50 g (80%) of 13: mp 113.0–114.0ЊC; H NMR (DMSO-d6)
ͳ 11.07 (s, 1H), 7.39 (s, 2H), 5.31 (t, J ϭ 7.3 Hz, 1H), 4.56 (d, J ϭ 7.3 Hz, 2H), 1.72
(s, 3H), 1.68 (s, 3H); 13C NMR (DMSO-d6) ͳ 151.9, 138.6, 118.6, 61.6, 25.3, 17.7.
Anal. Calcd for C6H12N2O4S: C, 34.61; H, 5.81. Found: C, 34.34; H, 5.83.
3,7-Dimethyl-2,6-nonadienyl sulfamoylcarbamate (14). To a cold (Ϫ23ЊC) solu-

NONIONIC DIPHOSPHATE SURROGATES
249
tion of 0.53 ml (3.0 mmol) of 98% 21 in 30 ml of CH2Cl2 was added 0.27 ml (3.1
mmol) of 23 all at once. The resulting mixture was stirred for 1 h and 5 ml of
ammonia was added dropwise from a CO2-acetone-filled condenser over 1 h. The
resulting mixture was allowed to warm to rt, stirred under Ar for 12 h, and evapo-
rated, and the residue (1.2 g) was subjected to dry flash chromatography (9 : 1 to
1
1 : 9 hexane-ethyl acetate) to give 0.73 g (88%) of 14: mp 99.8–101.3ЊC; H NMR
ͳ 9.88 (bs, 1H), 6.64 (s, 2H), 5.36 (t, J ϭ 7.1 Hz, 1H), 5.11 (t, J ϭ 6.8 Hz, 1H), 4.67
(d, J ϭ 7.1 Hz, 2H), 2.16–1.94 (m, 4H), 1.73 (s, 3H), 1.66 (s, 3H), 1.60 (s, 3H); 13C
NMR ͳ 153.0, 143.2, 132.1, 124.5, 119.1, 63.1, 40.1, 26.9, 25.8, 17.7, 16.5. Anal. Calcd
for C11H20N2O4S: C, 47.81; H, 7.29. Found: C, 48.25; H, 7.50.
3,7,11-Trimethyl-2,6,10-dodecatrienyl sulfamoylcarbamate (15). To
a cold
(Ϫ23ЊC) solution of 2.0 ml (7.8 mmol) of 98% 22 in 30 ml of CH2Cl2 was added
0.70 ml (7.9 mmol) of 23 all at once. The resulting mixture was stirred for 45 min
and 5 ml of ammonia was added dropwise from a CO2-acetone-filled condenser
over 15 min. The resulting mixture was allowed to warm to rt, stirred under Ar for
20 h, and evaporated, and the residue (3.0 g) was subjected to flash chromatography
1
(1 : 1 hexane-ethyl acetate) to give 2.2 g (84%) of 15: mp 91.0–91.6ЊC; H NMR ͳ
9.83 (bs, 1H), 6.63 (bs, 2H), 5.36 (t, J ϭ 7.0 Hz, 1H), 5.20–5.01 (m, 2H), 4.66 (d, J
ϭ 7.0 Hz, 2H); 2.18–1.86 (m, 8H), 1.74 (s, 3H), 1.66 (s, 3H), 1.61 (s, 3H), 1.59 (s,
3H); 13C NMR ͳ 153.0, 143.2, 135.9, 131.6, 125.1, 124.5, 119.1, 63.1, 40.4, 40.2, 27.4,
26.9, 25.8, 17.7, 16.5, 16.1. Anal. Calcd for C16H28N2O4S: C, 55.79; H, 8.19. Found:
C, 55.87; H, 8.23.
Biochemical methods. The rat liver microsomal squalene synthase assays were
conducted as previously described (12). The screening for effects in cell cultures
was conducted as follows. Human skin fibroblasts (HSF) obtained from the UCSF
Cell Culture Facility were grown in DME H21 medium containing 4.5 g glucose,
0.58 Ȑg -glutamine, and 3.7 g NaHCO3/liter of 10% fetal calf serum, and penicillin/
L
streptomycin. The cells were grown in six well plates to sparse density (growing
cells) or to confluency. The medium was changed to fresh medium containing 0–1
m
M
of each compound and incubated further at 37ЊC overnight (ȁ18 h). Five ȐCi
of [3H]thymidine (82 Ci/mmol) for determination of effects on DNA synthesis or
2 ȐCi of [14C]acetate (57 mCi/mmol) for determination of effects on cholesterol
and fatty acid synthesis was added to each well and the cells were incubated for a
further 2 h. The media were removed and the cells were washed twice with phos-
phate-buffered saline containing Ca2 and Mg . The cells were then taken up in
1 ml of 0.1
2
ϩ
ϩ
N
NaOH/well. Aliquots were taken for determination of [3H]incorpora-
tion into DNA, 100 Ȑg of calf thymus DNA was added as carrier, and DNA was
extracted (19) and analyzed. [14C] Incorporation into cholesterol was assessed after
addition of 100 Ȑg of cholesterol as carrier to these aliquots, saponification, and
extraction, followed by thin layer chromatography. [14C] Incorporation into fatty
acids was assessed after acidification of the remainder following extraction of the
nonsaponifiable fraction at basic pH (20). Samples were counted in a liquid scintilla-
tion counter and the results expressed graphically as a percentage of control values
(wells with no compound added). The IC50 (concentration required for 50% inhibi-
tion of function studied) was then estimated. Cells were also examined microscopi-
cally for gross morphological changes and changes in cell number and/or viability.

250
CASTRO ET AL.
ACKNOWLEDGMENTS
Partial support for this research was provided by NIH Grant HL23083 (T.A.S.), by NIH Grant
DK38553 and the Department of Veterans Affairs (S.K.E.), and by a grant from the Frost Foundation
(I.S.). The authors are grateful to Drs. J. A. Watson and L. Pulliam for assistance in obtaining materials,
including HSF cells, and to Steven R. Lear for technical assistance with some of the experiments.
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