Sterol synthesis, Synthesis of 3β-hydroxy-25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one and its effects on HMG-CoA reductase activity in Chinese hamster ovary cells, on ACAT activity in rat jejunal microsomes, and serum cholesterol levels in rats

Article abstract of DOI:10.1016/S0009-3084(98)00058-9

3β-Hydroxycholest-5-en-7-one (I; 7-ketocholesterol) is an oxysterol of continuing interest in biology and medicine. In the present study, we have prepared a side-chain fluorinated analog, 3β-hydroxy-25,26,26, 26,27,27,27-heptafluorocholest-5-en-7-one (VI), with the anticipation that the F₇ substitution would block major metabolism of the 7-ketosterol, and thereby enhance its potential in vivo effects on serum cholesterol levels and other parameters. Chromium trioxide/dimethyl pyrazole oxidation of the acetate derivative of the previously described 25,26,26,26,27,27,27-heptafluorocholest-5-en-3β-ol followed by mild alkaline hydrolysis gave VI. The effects of VI on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in Chinese hamster ovary (CHO-K1) cells, on acyl coenzyme A-cholesterol acyltransferase (ACAT) activity in rat jejunal microsomes, and on serum cholesterol levels and other parameters in male Sprague-Dawley rats were determined and compared with those obtained with I and with another α,β-unsaturated ketosterol, i.e. 3β-hydroxy-5α-cholest-8(14)-en-15-one (II). I and VI showed essentially the same potency, considerably less than that of II, in lowering the levels of HMG-CoA reductase activity in CHO-K1 cells. Whereas addition of II to rat jejunal microsomes inhibited ACAT activity (IC₅₀ ~ 3 μM), I and VI had no effect under the conditions studied (from 1 to 16 μM). Dietary administration of I, at levels of 0.1 and 0.15%, had no effect on food consumption, gain in body weight, or serum cholesterol level. At 0.2%, I caused a modest decrease in body weight gain and a slight decrease in serum cholesterol levels (relative to ad libitum but not pair-fed control animals). The F₇-7-ketosterol VI, at 0.26% in diet (the molar equivalent of 0.2% I), had no effect on food consumption, body weight, or serum cholesterol levels. Administration of I (0.1, 0.15 or 0.2% in diet) caused increases in the weight of small intestine. In contrast, no effect of VI (0.26% in diet) on small intestinal weight was observed.

Full text of DOI:10.1016/S0009-3084(98)00058-9

Chemistry and Physics of Lipids
94 (1998) 209–225
Sterol synthesis. Synthesis of
3i-hydroxy-25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one
and its effects on HMG-CoA reductase activity in Chinese
hamster ovary cells, on ACAT activity in rat jejunal
microsomes, and serum cholesterol levels in rats
Jeffery N. Carroll, Frederick D. Pinkerton, Xiangdong Su, Nicolas Gerst,
William K. Wilson, George J. Schroepfer Jr.*
Departments of Chemistry and Biochemistry and Cell Biology, Rice Uni6ersity, Houston, TX 77251-1892, USA
Received 1 May 1998; received in revised form 1 June 1998; accepted 1 June 1998
Abstract
3i-Hydroxycholest-5-en-7-one (I; 7-ketocholesterol) is an oxysterol of continuing interest in biology and medicine.
In the present study, we have prepared a side-chain fluorinated analog, 3i-hydroxy-25,26,26,26,27,27,27-heptafluoro-
cholest-5-en-7-one (VI), with the anticipation that the F7 substitution would block major metabolism of the
7-ketosterol, and thereby enhance its potential in vivo effects on serum cholesterol levels and other parameters.
Chromium trioxide/dimethyl pyrazole oxidation of the acetate derivative of the previously described
25,26,26,26,27,27,27-heptafluorocholest-5-en-3i-ol (Swaminathan et al., 1993. J. Lipid Res. 34, 1805–1823) followed
by mild alkaline hydrolysis gave VI. The effects of VI on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)
reductase activity in Chinese hamster ovary (CHO-K1) cells, on acyl coenzyme A–cholesterol acyltransferase (ACAT)
activity in rat jejunal microsomes, and on serum cholesterol levels and other parameters in male Sprague-Dawley rats
were determined and compared with those obtained with I and with another h,i-unsaturated ketosterol, i.e.
3i-hydroxy-5h-cholest-8(14)-en-15-one (II). I and VI showed essentially the same potency, considerably less than that
of II, in lowering the levels of HMG-CoA reductase activity in CHO-K1 cells. Whereas addition of II to rat jejunal
microsomes inhibited ACAT activity (IC50 ꢀ3 vM), I and VI had no effect under the conditions studied (from 1 to
Abbre6iations: ACAT, acyl coenzyme A–cholesterol acyltransferase; BSTFA, bis(trimethylsilyl)trifluoroacetamide; CHO-K1,
Chinese hamster ovary; GC, gas chromatography; HSQC, heteronuclear single quantum coherence; IR, infrared (spectroscopy);
LDL, low density lipoprotein; m.p., melting point; MPLC, medium pressure liquid chromatography; MS, mass spectrum or mass
spectrometry; NMR, nuclear magnetic resonance (spectroscopy); NZW, New Zealand White (rabbits); TLC, thin-layer chromatog-
raphy; TMS, trimethylsilyl; VLDL, very low density lipoprotein..
* Corresponding author. Tel.: +1 713 5274914; fax: +1 713 2855154.
0009-3084/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved.
PII S0009-3084(98)00058-9

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J.N. Carroll et al. / Chemistry and Physics of Lipids 94 (1998) 209–225
16 vM). Dietary administration of I, at levels of 0.1 and 0.15%, had no effect on food consumption, gain in body
weight, or serum cholesterol levels. At 0.2%, I caused a modest decrease in body weight gain and a slight decrease
in serum cholesterol levels (relative to ad libitum but not pair-fed control animals). The F7-7-ketosterol VI, at 0.26%
in diet (the molar equivalent of 0.2% I), had no effect on food consumption, body weight, or serum cholesterol levels.
Administration of I (0.1, 0.15 or 0.2% in diet) caused increases in the weight of small intestine. In contrast, no effect
of VI (0.26% in diet) on small intestinal weight was observed. © 1998 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: 7-Ketocholesterol; Fluorinated analog; Oxidation
1. Introduction
terolemic cynomologous monkeys (Hodis et al.,
1994) and from normocholesterolemic subjects
(Sevanian et al., 1997). The levels of I in plasma
of NZW rabbits (Daugherty et al., 1989) and
WHHL rabbits (Stalenhoef et al., 1993) have been
reported to be lower in probucol-treated animals
than in controls. Notably high (4.5 vM) levels of
I have been reported for commercial samples of
fetal calf serum (Pie and Seillan, 1995), which is
routinely used in a wide variety of studies employ-
ing cultured cells. Highly variable levels of I in the
LDL fraction of plasma obtained from normal
human subjects have been reported (Brooks et al.,
1986; Addis et al., 1989; Dyer et al., 1995; Dzele-
tovic et al., 1995a; Mori et al., 1996; Sevanian et
al., 1997), ranging from 0.18 vM (Dzeletovic et
al., 1995a) to 92 vM (Mori et al., 1996). Very
high levels of I, plus its decomposition product,
i.e. cholesta-3,5-dien-7-one, have been reported
(Hodis et al., 1994) for LDL from normocholes-
terolemic (ꢀ12 vM) and hypercholesterolemic
(ꢀ430 vM) cynomologous monkeys. It is impor-
tant to note that the large variation in the re-
ported levels of I in plasma, and in LDL
preparations derived therefrom, may reflect vary-
ing amounts of artifactual generation of I by
auto-oxidation of cholesterol during sample pro-
cessing and/or sample storage. In those experi-
ments involving supplementation with dietary
cholesterol, variable amounts of I in the choles-
terol supplement may also be important.
7-Ketocholesterol (3i-hydroxycholest-5-en-7-
one; I) is an oxysterol of continuing interest in
biology and medicine. I is a product of the auto-
oxidation of cholesterol (Smith, 1981) and is read-
ily formed by thermal decomposition of the 7h-
and 7i-hydroperoxides of cholesterol (Lercker et
al., 1996). Variable levels of I in plasma of normal
human subjects have been reported (Sevanian et
al., 1994; Dzeletovic et al., 1995b; Mol et al.,
1997), ranging from mean levels of 0.055 vM
(Dzeletovic et al., 1995b) to 4.8 vM (Sevanian et
al., 1994). Variable levels of I in plasma have also
been reported for New Zealand White (NZW)
rabbits on chow or cholesterol-supplemented diets
(Mattsson Hulten et al., 1996; Sevanian et al.,
1994), with a mean value for animals on a chow
diet of 1.0 vM (Sevanian et al., 1994) and, for
rabbits on 1% cholesterol diets, mean values of
ꢀ3.6 vM (Mattsson Hulten et al., 1996) and 29.8
vM (Sevanian et al., 1994). A very large number
of studies address the effects of oxidized low
density lipoprotein (LDL) on various cellular pro-
cesses, especially those related to the pathogenesis
of arteriosclerosis. I has been shown to be present
at extraordinarily high levels in human LDL oxi-
dized by incubation with Cu2+ (Zhang et al.,
1990; Jialal et al., 1991; Malavasi et al., 1992;
Kritharides et al., 1993; Maor and Aviram, 1994;
Dzeletovic et al., 1995a; Breuer et al., 1996;
Brown et al., 1996, 1997; Chang et al., 1997; Patel
et al., 1997), lipoxygenase (Dzeletovic et al.,
1995a) or macrophages (Jialal et al., 1991; Brown
et al., 1996). Very high levels of I have been
reported in electronegatively charged LDL
(LDL−) isolated from plasma from hypercholes-
I has been reported to be present in atheroscle-
rotic lesions at significant levels (Brooks et al.,
1966; van Lier and Smith, 1967; Smith and
Pandya, 1973; Bjo¨rkhem et al., 1994; Suarna et
al., 1995; Brown et al., 1997; Crisby et al., 1997).

J.N. Carroll et al. / Chemistry and Physics of Lipids 94 (1998) 209–225
211
The levels of 7-oxygenated derivatives of choles-
terol (combination of 7h-hydroxycholesterol, 7i-
hydroxycholesterol, and I) in atheroma, induced
in NZW rabbits by cholesterol feeding, have been
reported to have been unaffected by ascorbic acid
administration. I has been reported as a major
oxysterol present in arterial macrophage-derived
foam cells from cholesterol-fed NZW rabbits
(Mattsson Hulten et al., 1996).
somes. These in vivo results are in contrast to
reports of inhibitory in vitro effects of I on choles-
terol 7h-hydroxylase activity of liver microsomes
(Shefer et al., 1968; Van Cantfort, 1972; Breuer et
al., 1993) and the inhibitory effect of I on the
binding of cholesterol to the enzyme (Breuer et
al., 1993; Bostro¨m, 1994). I, added as such or in
LDL preparations, has been reported to inhibit
the efflux of cholesterol from mammalian cells
(Maor and Aviram, 1994; Kilsdonk et al., 1995;
Gelissen et al., 1996), an effect that may be due to
an inhibition of the transport of cholesterol to the
surface of the cells (Fielding et al., 1997).
The results of early studies demonstrated that I
inhibited sterol synthesis and lowered the levels of
3-hydroxy-3-methylglutaryl coenzyme A (HMG-
CoA) reductase activity in cultured mammalian
cells (Kandutsch and Chen, 1973; Brown and
Goldstein, 1974; Brown et al., 1975). The potency
of I in the inhibition of sterol synthesis was
essentially the same as that for lowering HMG-
CoA reductase activity (Kandutsch and Chen,
1973). The potency of I (IC50 1.7 vM) in lowering
HMG-CoA reductase activity in mouse L-cells
was considerably less than that (IC50 0.1 vM) of
another h,i-unsaturated ketosterol, i.e. 3i-hy-
droxy-5h-cholest-8(14)-en-15-one (Schroepfer et
al., 1977b; Taylor et al., 1984). A number of other
effects of I have been reported. These include
inhibition of secretion of VLDL from cultured
hepatocytes (Kosykh et al., 1988), inhibition of
cholesterol uptake by carotid and femoral arteries
of intact rabbits (Bing et al., 1979), inhibition of
intercellular communication in human arterial
smooth muscle cells (Zwijsen et al., 1992), de-
creases in Ca2+ influx into erythrocytes (Neyes et
al., 1985), increases in Ca2+ influx into rat hepa-
tocytes (Sevanian and Peterson, 1986), cytotoxic-
ity (Hwang 1992; Bakos et al., 1993; Carpenter et
al., 1993; Chisholm et al., 1994; Guyton et al.,
1995) and induction of apoptosis (Lizzard et al.,
1996; Nishio et al., 1996; Nishio and Watanabe,
1996; Palladini et al., 1996) in various cell lines,
conversion of erythrocytes to echinocytes (Hsu et
al., 1980), changes in morphology of endothelial
and smooth muscle cells in rabbit aortic segments
(Deckert et al., 1997), and in vitro inhibition of
lysosomal sphingomyelinase (Maor et al., 1995).
Intravenous (Breuer et al., 1993) or dietary
(Tamasawa et al., 1994) administration of I has
been reported to result in increased levels of
cholesterol 7h-hydroxylase activity in liver micro-
Except for those noted above, only a few re-
ports have concerned in vivo effects observed
after administration of I. Kandutsch et al. (1977)
reported that administration of I in a basal low
cholesterol diet at levels of 0.25% or 0.5% resulted
in suppression of gain in body weight of immature
male mice. Addition of cholesterol was reported
to reduce the suppressive effect of the 7-ketosterol
on body weight gain. Data on food consumption
were not presented. Erickson et al. (1977) re-
ported that short-term (18 h or 66 h) administra-
tion of I at 0.05, 0.10 or 0.5% in a chow diet had
no effect on body weights of male Sprague-Daw-
ley rats, except for animals fed I at 0.5% for 66 h,
for which a mean loss of 19 g in body weight was
reported. Tamasawa et al. (1994) reported that
dietary administration of I at a level of 0.1% in a
chow diet had no effect on food consumption or
body weight in male Wistar rats, but no data were
presented. Vargas et al. (1986) reported that di-
etary administration of I at a level of 0.025% had
no effect on food consumption or body weight in
laying hens. However, Toda et al. (1981) reported
that administration of I at a level of 1% in diet to
5-day-old chicks resulted in the following: ‘after
12 days on this regimen, chicks started dying, and
survivors gained little or no weight’. Enlarged gall
bladders and intestinal atrophy were reported as
autopsy findings. Osada et al. (1994) reported that
administration of an oxysterol mixture (0.5% in
diet), in which I was the major oxysterol compo-
nent, to male Sprague-Dawley rats resulted in
decreased food consumption and decreased gain
in body weight.

212
J.N. Carroll et al. / Chemistry and Physics of Lipids 94 (1998) 209–225
Other h,i-unsaturated ketosterols have been
been reported to reduce serum cholesterol levels in
rats (Osada et al., 1994). However, a pair-fed
group was not included as a control for the
reported decrease in food consumption resulting
from the administration of the oxysterol mixture.
Only very limited studies have addressed the
metabolism of I. Results from studies of the fate
of labeled I after administration to rats (Bjo¨rkhem
et al., 1968; Sarma et al., 1976; Erickson et al.,
1977), guinea pigs (Bjo¨rkhem et al., 1968), and
rabbits (Sarma et al., 1976; Erickson et al., 1977)
indicated substantial formation of polar material
(Bjo¨rkhem et al., 1968; Sarma et al., 1976; Erick-
son et al., 1977), some of which appears to repre-
sent acidic metabolites (Bjo¨rkhem et al., 1968;
Erickson et al., 1977). Full characterization of
these metabolites was not provided. In contrast to
the case of I, fairly extensive studies have been
made on the metabolism of the D8(14)-15-ketos-
terol, II. The results of these studies indicated
very rapid and substantial conversion of II to
polar metabolites by processes in which initial
oxidation at C-26 predominates (Schroepfer et al.,
1988a,b,c; St. Pyrek et al., 1989, 1991). Blocking
this metabolism by fluorine substitution, as in
3i-hydroxy-25,26,26,26,27,27,27-heptafluoro-5h-
cholest-8(14)-en-15-one (Swaminathan et al.,
1993) and 3i-hydroxy-7h-methyl-25,26,26,26,
27,27,27-heptafluoro-5h-cholest-8(14)-en-15-one
(Swaminathan et al., 1995) led to increased po-
tency in the reduction of serum cholesterol levels
in rats (Gerst et al., 1994; Swaminathan et al.,
1995). These results provided the stimulus for the
preparation of 3i-hydroxy-25,26,26,26,27,27,27-
heptafluorocholest-5-en-7-one (VI) and studies of
its in vitro and in vivo effects relative to those of
7-ketocholesterol (I) (Fig. 1).
reported to reduce food consumption and body
weight gain in rodents. 3i-Hydroxy-5h-cholest-
8(14)-en-15-one (II) suppressed food consumption
and decreased body weight gain in Sprague-Daw-
ley rats (Schroepfer et al., 1977a; Brabson and
Schroepfer, 1988; Miller et al., 1988; Smith et al.,
1989; Gerst et al., 1994). Dietary administration
of 3i-hydroxy-5h-cholest-8-en-7-one and 3i-hy-
droxy-5h-cholest-8-en-11-one (0.15% in a choles-
terol-free test diet) has also been reported to
suppress food consumption and growth in
Sprague-Dawley rats (Parish et al., 1986); how-
ever, no experimental data were presented.
The results of previous studies have indicated
little or no effect of I on serum cholesterol levels
upon its dietary administration to animals. Erick-
son et al. (1977) reported that administration of I
at levels of 0.05, 0.1 and 0.5% in diet to rats for 18
or 66 h had no effect on serum cholesterol levels
except for the animals receiving 0.5% I for 66 h
for which ‘a decrease of about 50% was found’.
However, a pair-fed control group of animals was
not included to deal with the decreased food
consumption reported for I under these condi-
tions. Kandutsch et al. (1977) reported no hypo-
cholesterolemic action of I upon dietary adminis-
tration (0.25 or 0.5% in diet) to immature male
mice. However, only a small number of animals
were used in this experimentation. Administration
of I (10 mg/day for 2 weeks and 20 mg/day
thereafter) to 5-day-old chicks as an emulsion in
corn oil was reported to have no effect on serum
cholesterol levels in birds fed a basal diet or a
cholesterol-supplemented diet (Toda et al., 1981).
Dietary administration of an oxysterol mixture
(0.5%), containing I as the major oxysterol, has
Fig. 1. Structures of 3i-hydroxycholest-5-en-7-one (I) and 3i-hydroxy-25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one (VI), and
3i-hydroxy-5h-cholest-8(14)-en-15-one (II).

J.N. Carroll et al. / Chemistry and Physics of Lipids 94 (1998) 209–225
213
2. Experimental procedures and results
1993). The effects of the sterols on the level
of acyl coenzyme A–cholesterol acyltransferase
(ACAT) in jejunal microsomes were assayed as
described previously (Gerst et al., 1994). The ef-
fects of dietary administration of 7-ketocholes-
terol I and its F7-analog (VI) were studied in male
Sprague-Dawley rats, which were purchased from
Harlan Sprague-Dawley (Houston, TX) and
housed in pairs for six days on a light–dark cycle
(06:00–18:00 h) and fed a basal diet (Purina For-
mulab 5008) and water ad libitum. During experi-
ments, the animals were housed individually and
were provided the appropriate diets and water ad
libitum. In experiment 1, the animals were divided
into seven groups containing seven animals each,
such that the mean values of serum cholesterol
and body weight were approximately the same.
The seven groups of animals included: an ad
libitum control group; groups which received I at
levels of 0.1, 0.15 and 0.2% in diet; pair-fed
control groups (to the animals receiving I at the
three dietary levels), in which the individual ani-
mals were provided with the amount of food
consumed on the previous day by their respective
counterparts in the experimental groups. In exper-
iment 2, three groups of animals were studied: the
ad libitum control groups (n=8), animals (n=4)
fed VI at a level of 0.26% in diet, and a pair-fed
control group (n=4). On days 6 and 7, one of the
animals in the experimental group turned over its
food dish. Accordingly, the corresponding pair-
fed control animal received no food on the follow-
ing days (days 7 and 8). The body weight and
food consumption of the individual rats were
determined daily. Blood for serum sterol concen-
trations was obtained at 08:00 h from the tail vein
on days 5 and 9. The experiments were terminated
with ketamine anesthesia (0.2 ml; 100 mg/ml)
followed by decapitation. The following organs
were removed and weighed: liver, heart, spleen,
small intestine, kidneys, adrenal glands and testes.
Serum cholesterol was measured using a commer-
cial assay kit (‘Single Vial’; Boehringer Mannheim
diagnostics; catalog number 236691).
2.1. Materials and methods
Melting points (m.p.) were measured with a
Thomas-Hoover apparatus in sealed, evacuated
capillary tubes. Infrared (IR) spectra were ob-
tained on a Mattson Galaxy 6020 Fourier-trans-
form spectrometer; samples were analyzed as KBr
pellets. Thin-layer chromatography (TLC) was
carried out on aluminum-backed silica gel 60
plates (EM Science, Gibbstown, NJ). Compo-
nents of the plates were visualized after spraying
with 5% ammonium molybdate in 10% sulfuric
acid followed by heating. Column chromatogra-
phy was done with 70–230 mesh silica gel, and
medium pressure liquid chromatography (MPLC)
was done in glass columns dry-packed with silica
gel (230–400 mesh). Fraction volumes were 20 ml
unless otherwise specified.
Low-resolution and high-resolution mass spec-
tra (MS) were recorded on a VG ZAB-HF double
sector instrument with an electron energy of 70 eV
and direct inlet sample introduction. Trimethylsi-
lyl (TMS) ether derivatives were prepared by
treating the sterols with a 1:1 mixture of bis(tri-
methylsilyl)trifluoroacetamide (BSTFA) and pyri-
dine for 1 h under nitrogen at room temperature,
followed by evaporation to dryness at 40 C under
nitrogen. Samples for gas chromatography (GC)
were injected in hexane (5 vl, 1 mg/ml). GC
analyses were done on a Shimadzu 9A chro-
matograph (split injection; 30 m×0.25 mm i.d.
DB-5 column at 250 C; 0.1 vm film thickness;
nitrogen carrier gas at 1.1 kg/cm2). Nuclear mag-
netic resonance (NMR) spectra were measured in
1
CDCl3 solutions (510 mM for H, ꢀ50 mM for
13C) on a Bruker AMX500 (500.1 MHz for H,
1
25 C; 125.8 MHz for 13C, 22 C) spectrometer and
referenced to internal tetramethylsilane (1H) or
CDCl3 at 77.0 ppm (13C). NMR experiments,
including heteronuclear single quantum coherence
(HSQC), were performed as described previously
(Wilson et al., 1996). The purity of sterol samples
1
was determined by H NMR and TLC.
Chromium trioxide, 3,5-dimethylpyrazole, and
BSTFA were purchased from Aldrich Chemical
(Milwaukee, WI). 3i-Hydroxy-5h-cholest-8(14)-
en-15-one (II) was prepared as described previ-
The effects of the synthetic sterols on HMG-
CoA reductase activity were studied in CHO-K1
cells as described previously (Swaminathan et al.,

214
J.N. Carroll et al. / Chemistry and Physics of Lipids 94 (1998) 209–225
ously (Wilson et al., 1988). 25,26,26,26,27,27,27-
Heptafluoro-5i-cholestane-3h,6h-diol bis(p-tolue-
nesulfonate) (III) was prepared as described
previously (Swaminathan et al., 1993), with the
following improvements. As described previously
(Ochi et al., 1980), hyodeoxycholic acid (89.7 g)
was refluxed in acetic anhydride/acetic acid (270
ml/540 ml) for 1 h followed by evaporation of the
solvent. Mixed anhydride material (1H NMR sig-
nals at l 2.50 (ddd, 16.5, 10.0, 5.2 Hz) and 2.22
(s)) was hydrolyzed by refluxing in pyridine/water
(600 ml/120 ml) for 1 h, followed by evaporation
of the solvent. The resulting oily residue was
dissolved in chloroform and washed with 5% HCl
(2×150 ml) and water (3×200 ml), dried over
anhydrous sodium sulfate, and evaporated to a
residue that was subjected in several batches to
MPLC purification (elution with ethyl ac-
Hz, H-12i), 1.991† (td, 12.8, 11.3 Hz, H-4h),
1.972 (s, Ac-CH3), 1.961† (dddd, ꢀ13, 4.7, 4.0,
2.2 Hz, H-4i), 1.886 (dtd, 14.4, 3.5, 0.6 Hz,
H-1h), 1.816 (br dtd, ꢀ13, 9, 6.1 Hz, H-16h),
1.796 (td, ꢀ12, 4.7 Hz, H-9h), 1.746 (m, H-2i),
1.707 (dt, ꢀ13, 5 Hz, H-5i), 1.692 (td, 11.2, 3.1
Hz, H-8i), 1.633 (m, H-23S), 1.497 (dq, 13.6, 3.7
Hz, H-11h), 1.465 (m, H-2h), 1.446† (m, H-23R),
1.429† (m, H-22R), 1.419† (m, H-20), 1.353 (dddd,
12.2, 9.2, 7.0, 2.8 Hz, H-15h), 1.280 (tdd, ꢀ13.6,
12, 3.9 Hz, H-11i), 1.270 (br ddd, ꢀ12, 11, 7 Hz,
H-14h), 1.262 (br dtd, ꢀ13, 9, 2.9 Hz, H-16i),
1.151 (br dddd, ꢀ14, 13, 4, 0.7 Hz, H-12h), 1.140
(td, ꢀ14.4, 3.6 Hz, H-1i), 1.125 (br q, ꢀ9.8 Hz,
H-17h), 1.080 (qd, ꢀ11.9, 6.5 Hz, H-15i), 1.073
(m, H-22S), 1.000 (s, H-19), 0.933 (d, 6.6 Hz,
H-21), 0.651 (br d, ꢀ0.3 Hz, H-18); 13C NMR,
C-1 to C-24, l 34.91, 26.69, 73.70, 28.64, 45.34,
69.89, 70.68, 37.15, 33.92, 36.05, 20.51, 39.21,
42.73, 50.16, 23.42, 27.98, 55.78, 11.66, 22.99,
35.39, 18.59, 31.67, 29.18, 63.42; acetate signals,
21.44, 170.54, 20.94, 170.28, 21.15, 170.19. 5i-
Cholane-3h,6h,24-triol 3,6-diacetate was con-
verted to ditosylate III, as described previously
(Swaminathan et al., 1993)2.
etate:hexane
3:7).
Recrystallization
from
methanol/water gave 3h,6h-diacetoxy-5i-cholan-
24-oic acid as a white solid (79.6 g; 73% yield),
m.p. 102.5–104.5 C. Reduction of the acid (6.76
g) with borane methyl sulfide as described previ-
ously (Swaminathan et al., 1993) gave an oil that
was subjected to MPLC (500×25 mm i.d.
column; elution with ethyl acetate:hexane 2:8).
Recrystallization from methanol gave 5i-cholane-
3h,6h,24-triol-3,6-diacetate (4.42 g, 67% yield),
m.p. 173–173.5 C (literature, 80.5–83 C, Swami-
2.2. 25,26,26,26,27,27,27-Heptafluorocholest-
5-en-3i-ol acetate (IV)
1
nathan et al., 1993)1, ]99% purity by H NMR.
A solution of ditosylate III (7.89 g, 9.4 mmol)
and potassium acetate (58.5 g, 596 mmol) in
dimethylformamide (185 ml) and water (27 ml)
was refluxed with stirring for 5 h. The cooled
solution was poured into cold 5% sulfuric acid
(350 ml) and extracted with ether (3×175 ml).
The combined organic layers were washed with
water (2×200 ml), dried over anhydrous sodium
sulfate, and evaporated to dryness to give an
off-white solid (6.72 g). The crude product was
dissolved in pyridine (12 ml) and acetic anhydride
(10 ml) and left overnight at room temperature.
The reaction was then poured into water (200 ml),
Material derived from hyodeoxycholic acid from
Eastern Chemical (Hauppauge, NY) contained an
impurity that was removed by MPLC (500 mg
sterol; 500×10 mm i.d. column; elution with
ethyl acetate:hexane 3:7); fractions 13–23 gave the
desired 3h,6h-diacetate (440 mg), and fractions
26–37 gave the impurity, which was identified by
NMR as 5i-cholane-3h,6h,7h,24-tetraol-3,6,7-tri-
1
acetate. H NMR (superscript † indicates 90.003
ppm precision), l 5.214 (td, 3.4, 0.9 Hz, H-7i),
5.117 (dd, 5.5, 3.5 Hz, H-6i), 4.579 (tt, 11.3, 4.6
Hz, H-3i), 3.624† (ddd, 10.3, 7.2, 6.0 Hz, H-24),
3.595† (dt, 10.3, 6.7 Hz, H-24), 2.087 (s, 3h-Ac-
CH3), 2.050 (s, Ac-CH3), 1.996 (dt, ꢀ12.9, 3.3
2 As noted previously (Schroepfer, 1996), the reagent for
saponification of 25,26,26,26,27,27,27-heptafluoro-5i-choles-
tane-3h,6h-diol diacetate in Swaminathan et al. (1993) should
be potassium carbonate, not potassium acetate.
1 Similar melting points obtained in 14 other multigram
preparations of this sterol indicate that our previously re-
ported (Swaminathan et al., 1993) m.p. was in error.

J.N. Carroll et al. / Chemistry and Physics of Lipids 94 (1998) 209–225
215
extracted with ether (3×100 ml), and evapo-
rated to an oily residue. Water (200 ml) was
added to the residue, and the resulting precipi-
tate was collected by filtration. The precipitate
was purified by MPLC (elution with hexane
(500 ml) and ethyl acetate:hexane 8:92). Evapo-
ration of fractions 15–35 furnished IV as a
ꢀ90% purity, containing 3% of the 5h,6h-epoxy
isomer). Evaporation of fractions 83–218 fur-
nished V as a white solid (2.84 g): m.p. 141.5–
143 C; single component by TLC (solvent, ethyl
acetate:hexane 1:1); 92% purity by 1H NMR;
partial 1H NMR, l 5.707 (d, 1.7 Hz, H-6),
4.717 (tdd, 11.5, 5.1, 4.3 Hz, H-3h), 2.052 (s,
Ac-CH3), 1.212 (d, 0.6 Hz, H-19), 0.948 (d, 6.6
Hz, H-21), 0.691 (d, 0.4 Hz, H-18); partial 13C
NMR, l 201.83 (C-7), 163.90 (C-5), 126.65 (C-
6), 72.15 (C-3), 29.35 (d, JCF 20.8 Hz, C-24),
18.12 (br s, C-23), 17.22 (C-19), 11.93 (C-18).
Samples of V contained a 7% component show-
ing the following NMR signals: lH 4.931 (tt,
11.7, 4.8 Hz, H-3h), 3.042 (s, H-6), 2.277 (dd,
13.2, 11.8 Hz, H-4i), 2.032 (s, Ac-CH3), 1.93 (t,
11 Hz, H-8i), 1.821 (br dt, 13.5, 3.7 Hz, H-1i),
1.50 (td, 14, 3.5 Hz, H-1h), 1.466 (ddd, 14, 5, 2
Hz, H-4h), 1.040 (d, 0.5 Hz, H-19), 0.926 (d,
6.5 Hz, H-21), 0.670 (s, H-18), lC (from HSQC
spectrum) 46.9 (C-8), 35.1 (C-4), 32.6 (C-1), 21.2
(Ac-CH3), 18.4 (C-21), 15.5 (C-19), 11.9 (C-18).
This byproduct was identified as the 5h,6h-
epoxy derivative of V based on a comparison
with 1H and 13C NMR data reported for 3i-
acetoxy-5,6h-epoxy-5h-cholestan-7-one (Boto et
al., 1997).
1
white solid (3.05 g, ꢀ80% purity by H NMR).
Recrystallization of a 1-g portion of this mate-
rial from acetone provided an analytical sample
of IV (0.39 g): m.p. 137–139 C; single compo-
nent by TLC (solvent, ethyl acetate:hexane 1:1);
98% purity by GC; 96% purity by 1H NMR
(containing 3% 25,26,26,26,27,27,27 - heptafluoro-
1
cholest-4-en-3i-ol acetate); partial H NMR, l
5.375 (m, H-6), 4.604 (m, H-3h), 2.032 (s, Ac-
CH3), 1.020 (d, 0.6 Hz, H-19), 0.940 (d, 6.6 Hz,
H-21), 0.684 (d, 0.5 Hz, H-18); partial 13C
NMR, l 139.63 (C-5), 122.56 (C-6), 121.11 (qd,
285, 28 Hz, C-26 and C-27), 73.93 (C-3), 29.38
(d, 21 Hz, C-24), 18.09 (br d, 4 Hz, C-23), 19.29
(C-19), 11.82 (C-18).
2.3. 3i-Acetoxy-25,26,26,26,27,27,27-
heptafluorocholest-5-en-7-one (V)
Chromium trioxide (12.3 g, 123 mmol) was
suspended in dry dichloromethane (200 ml) and
stirred for 5 min at −25 C. Dimethylpyrazole
(11.8 g, 123 mmol) was added in one por-
tion, and the reaction was stirred for 30 min at
−20 C. F7-cholesteryl acetate (IV) (4.26 g; 7.5
mmol) was added, and the mixture was stirred
at −15 C for 1 h and at 0–5 C for 2 h. Ether
(400 ml) was added and the mixture was filtered
through Celite. The filtrate was evaporated to a
brown solid that was filtered through silica gel
(300×40 mm i.d. column; elution with ethyl ac-
etate:hexane 15:85; 100-ml fraction volumes).
Evaporation of fractions 4–8 gave a white solid
(3.57 g; ꢀ85% purity) that was subjected to
MPLC (1000×25 mm i.d. column; elution with
ethyl acetate:hexane 8:92). Fractions 25–35 gave
a yellowish solid (210 mg) consisting mainly of
25,26,26,26,27,27,27-heptafluorocholest-4-en-3i-
ol acetate, and fractions 55–75 afforded 5,6i-
epoxy-25,26,26,26,27,27,27-heptafluoro-5i-cho-
lestan-3i-ol acetate as a white solid (130 mg;
2.4. 3i-Hydroxy-25,26,26,26,27,27,27-
heptafluorocholest-5-en-7-one (VI)
To a suspension of acetate V (2.80 g, 4.92
mmol, 92% purity) in methanol (50 ml) potas-
sium carbonate (680 mg, 4.92 mmol) in water (8
ml) was added. The reaction was stirred for 20
h, diluted with water (300 ml), and extracted
with ether (4×200 ml). The combined organic
extracts were washed with water (2×200 ml)
and dried over anhydrous sodium sulfate. Evap-
oration afforded a white solid that was purified
with MPLC (1000×25 mm i.d. column; elution
with ethyl acetate:hexane 15:85 (2000 ml), ethyl
acetate:hexane 3:7 (2000 ml), and ethyl ac-
etate:hexane 85:15) (see Fig. 2). Fractions 200–
230 gave a white solid (2.18 g; 98:2 mixture of
1
VI and its 5h,6h-epoxy derivative by H NMR
analysis) that was subjected to further MPLC
(500×25 mm i.d. column; elution with ethyl

216
J.N. Carroll et al. / Chemistry and Physics of Lipids 94 (1998) 209–225
Fig. 2. Synthesis of 3i-hydroxy-25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one (VI).
acetate:hexane 4:96 (500 ml) and ethyl acetate:
hexane 15:85). Fractions 285–370 furnished VI as
a white solid (1.63 g): m.p. 165–166 C; single
component by TLC (solvent, ethyl acetate:hexane
1:1) and by GC-MS of the TMS derivative;
\99% purity by 1H NMR; MS, Table 1; high
resolution MS, calc. for C27H37O2F7, 526.2682,
found, 526.2675; MS of TMS derivative, Table 1;
acetoxy-5,6h-epoxy-5h-cholestan-7-one and 1.5%
5,6i-epoxy-5i-cholestan-3i-ol acetate); partial
1H NMR, l 5.702 (d, 1.7 Hz, H-6), 4.716 (tdd,
11.5, 5.1, 4.3 Hz, H-3h), 2.052 (s, Ac-CH3), 1.209
(d, 0.6 Hz, H-19), 0.922 (d, 6.6 Hz, H-21), 0.866
(d, 6.6 Hz, H-27), 0.862 (d, 6.6 Hz, H-26), 0.682
(br d, ꢀ0.3 Hz, H-18); partial 13C NMR, l
201.96 (C-7), 163.82 (C-5), 126.68 (C-6), 72.17
(C-3), 39.43 (C-24), 23.78 (C-23), 17.22 (C-19),
11.93 (C-18). NMR signals of the two major
impurities: 3i-acetoxy-5,6h-epoxy-5h-cholestan-
7-one, lH 4.930 (tt, 11.7, 4.8 Hz, H-3h), 3.040 (s,
H-6), 2.276 (dd, 13.2, 11.8 Hz, H-4i), 2.032 (s,
Ac-CH3), 1.93 (t, 11 Hz, H-8i), 1.82 (m, H-1i),
1.47 (m, H-4h), 1.036 (d, 0.5 Hz, H-19), 0.899 (d,
6.6 Hz, H-21), 0.862 (d, ꢀ6 Hz, H-27), 0.857 (d,
ꢀ6 Hz, H-26), 0.661 (s, H-18), lC (from HSQC
spectrum) 46.8 (C-8), 35.1 (C-4), 32.6 (C-1), 21.2
(Ac-CH3), 18.6 (C-21), 15.4 (C-19), 12.0 (C-18);
5,6i-epoxy-5i-cholestan-3i-ol acetate, lH 3.072
(br d, 3.0 Hz), 1.002 (s), 0.637 (s). These byprod-
ucts were identified by comparisons with NMR
data reported for the epoxyketone (Boto et al.,
1997) or with the NMR spectrum of an authentic
sample of the i-epoxide prepared as described
previously (Kudo et al., 1989). Saponification of
the 3i-acetate derivative of the D5-7-ketosterol as
1
partial H NMR, l 5.696 (d, 1.7 Hz, H-6), 3.680
(tq, 11.4, 4.4 Hz, H-3h), 1.202 (d, 0.7 Hz, H-19),
0.948 (d, 6.7 Hz, H-21), 0.691 (br d, 0.5 Hz,
H-18); partial 13C NMR, l 202.26 (C-7), 165.30
(C-5), 126.00 (C-6), 121.09 (qd, 285, 28 Hz, C-26
and C-27), 91.79 (d of quintet, 202, 32 Hz, C-25),
70.43 (C-3), 29.34 (d, 21 Hz, C-24), 18.10 (br s,
C-23), 17.26 (C-19), 11.92 (C-18).
2.5. 3i-Hydroxycholest-5-en-7-one (I)
Oxidation of cholesteryl acetate (7.0 g)
analogously to the method of Salmond et al.
(1978) for preparing 3i-benzoyloxycholest-5-en-
7-one gave, after MPLC purification, crude 3i-
acetoxycholest-5-en-7-one as a white solid (4.2 g):
m.p. 151–153.5 C (literature 156 C, Milburn and
Truter, 1956; 153–154 C, Kumar et al., 1987);
1
ꢀ90% purity by H NMR (containing 6% 3i-

J.N. Carroll et al. / Chemistry and Physics of Lipids 94 (1998) 209–225
217
Table 1
MS data for 3i-hydroxycholest-5-en-7-one (I) and 3i-hydroxy-25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one (VI) and the
trimethylsilyl ether derivatives of I and VI
Suggested assignment
Free sterol
TMS ether
I
VI
I
VI
M+
M-CH3
400 (100)
385 (5)
382 (5)
367 (14)
343 (3)
287 (13)
269 (3)
526 (100)
511 (4)
508 (11)
493 (18)
469 (3)
287 (7)
269 (3)
472 (25)
457 (4)
382 (31)
367 (8)
598 (28)
583 (4)
508 (33)
493 (9)
M-H2O (or M-TMSOH)
M-H2O-CH3 (or M-TMSOH-CH3)
M-SC
359 (1)
269 (7)
75 (100)
359 (1)
269 (5)
75 (100)
M-SC-H2O (or M-SC-TMSOH)
SC, side chain.
described for V gave, after MPLC purification, I
as a white solid (1.27 g); m.p. 171.5–172.5 C
(literature: 170 C, Milburn and Truter, 1956;
171–172 C, Parish and Scott, 1983; 172–174 C,
Suginome and Yonebayashi, 1982); single compo-
nent on TLC (solvent, ethyl acetate:hexane 1:1)
and by GC-MS of the TMS derivative; 99% pu-
rity by 1H NMR; MS, Table 1; MS of TMS
contrast, the D8(14)-15-ketosterol (II) caused sig-
nificant inhibition of cholesterol ester formation,
with IC50 values of 3.0, 3.3 and 2.8 vM in three
experiments.
2.7. Effects of dietary administration of
3i-hydroxycholest-5-en-7-one (I) and
3i-hydroxy-25,26,26,26,27,27,27-heptafluoro-
cholest-5-en-7-one (VI) in male Sprague-Dawley
rats
1
derivative, Table 1; partial H NMR, l 5.692 (d,
1.8 Hz, H-6), 3.678 (tt, 11.3, 4.5 Hz, H-3h), 1.199
(d, 0.7 Hz, H-19), 0.922 (d, 6.6 Hz, H-21), 0.866
(d, 6.6 Hz, H-27), 0.862 (d, 6.6 Hz, H-26), 0.683
(d, 0.5 Hz, H-18); partial 13C NMR, l 202.44
(C-7), 165.28 (C-5), 126.02 (C-6), 70.43 (C-3),
39.43 (C-24), 23.78 (C-23), 17.27 (C-19), 11.93
(C-18).
Administration of 7-ketocholesterol (I) at levels
of 0.1 and 0.15% in diet had no effect on food
Table 2
Effects of 3i-hydroxycholest-5-en-7-one (I) and 3i-hydroxy-
25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one (VI) on the
levels of HMG-CoA reductase activity in CHO-K1 cells
2.6. Effects of 3i-hydroxycholest-5-en-7-one (I)
and 3i-hydroxy-25,26,26,26,27,27,27-heptafluoro-
cholest-5-en-7-one (VI) on HMG-CoA reductase
acti6ity in CHO-K1 cells and on ACAT acti6ity
in rat jejunal microsomes
Concentration
(vM)
HMG-CoA reductase activitya % of
control (mean9S.D.)
I
VI
0
100
100
0.1
0.25
0.5
1.0
2.5
104.9910.0
103.3914.6
87.794.0
80.1912.2
59.798.2
97.9912.5
The effects of I and VI on the levels of HMG-
CoA reductase activity in CHO-K1 cells are
shown in Table 2. At a concentration of 2.5 vM,
both I and VI produced significant and roughly
comparable lowering of the levels of HMG-CoA
reductase activity. The D5-7-ketosterol I and the
F7-D5-7-ketosterol VI had no effect on ACAT
activity in jejunal microsomes (Fig. 3) at any of
the concentrations tested (from 1 to 16 vM). In
86.599.3
93.9916.6
71.796.4
52.692.6
a Mean of three separate experiments in which triplicate assays
of enzyme activity were made for each concentration of sterol.
Mean values for control cells (no added sterol) in the three
experiments were 1974954, 1955941 and 1954989 pmol/
min per mg protein.

218
J.N. Carroll et al. / Chemistry and Physics of Lipids 94 (1998) 209–225
ad libitum controls, and the magnitude of the
increases appeared related to dosage (Table 3).
The mean values for small intestinal weight of the
animals treated with I at levels of 0.15 or 0.2%
(but not at 0.1%) were higher than those of the
pair-fed control animals. Marginal effects of I on
the weight of adrenals were observed. Mean val-
ues of adrenal weights were slightly increased in
rats receiving I at levels of 0.1 or 0.15% relative to
ad libitum controls. However, no significant dif-
ference was observed at a higher dose, i.e. 0.2% I
(Table 4). Moreover, mean values of adrenal
weights expressed as a percentage of total body
weight in rats treated with I at levels of 0.1 or
0.2% were not significantly different from that of
ad libitum control animals. Administration of I,
at levels of 0.1 or 0.15%, had no effect on serum
cholesterol levels (Table 5). Feeding of I, at a level
of 0.2%, was associated with slightly lower levels
of serum cholesterol on days 5 and 9, relative to
ad libitum control animals (but not pair-fed con-
trol rats).
Fig. 3. Effects of 3i-hydroxy-5h-cholest-8(14)-en-15-one (II;
ꢀ—ꢀ), 3i-hydroxycholest-5-en-7-one (I; “—“) and 3i-hy-
droxy-25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one (VI;
ꢁ—ꢁ) on ACAT activity in rat jejunal microsomes. The
results are presented as mean values9S.D. for three separate
experiments in which quadruplicate determinations of activity
were made at each sterol concentration. Mean values for
control incubations (no added sterol) in the three experiments
were 21909317, 1624944 and 1400925 pmol/min per mg
protein.
consumption or on body weight. Dietary adminis-
tration of I at a level of 0.2% was associated with
significantly lower mean values for food consump-
tion (relative to ad libitum controls) on day 1
(−23%; P=0.035), day 2 (−26%; P=0.003),
day 3 (−18%; P=0.0001), day 4 (−23%; P=
0.0001), and day 5 (−11%; P=0.0001). There-
after, the food consumption of the animals treated
with I did not differ from the ad libitum control
animals. The mean values of the body weights of
the 7-ketosterol-treated rats at levels of 0.1, 0.15
and 0.2% did not differ significantly from those of
the ad libitum controls on any day of the experi-
ment. However, when the changes in body weight
were expressed as a percentage of the day 0
values, a slight reduction of the growth of the
animals fed I at a level of 0.2% was noted (Fig. 4).
Administration of I, at levels of 0.1, 0.15 and
0.2% for 10 days, had no significant effect on the
weights of liver, spleen, heart, kidneys, or testis.
The mean values of the weights of small intestine
were higher in the animals treated with I at levels
of 0.1, 0.15 or 0.2% than the mean value for the
Fig. 4. Effect of dietary administration of 3i-hydroxycholest-
5-en-7-one (I; 0.2% in diet) on changes in body weight (ex-
pressed as
a percentage of day 0 values). Experimental,
ꢁ—ꢁ; ad libitum controls, “—“. The results are presented
as mean values 9S.E.M. (n=7). The differences between
mean values for experimental and ad libitum control animals
were significant (PB0.05) on all days except day 7. The mean
values of experimental and pair-fed control animals (not
shown) did not differ significantly on any day of the experi-
ment.

J.N. Carroll et al. / Chemistry and Physics of Lipids 94 (1998) 209–225
219
Table 3
3. Discussion
Effect of dietary administration of 3i-hydroxycholest-5-en-7-
one (I) for 10 days on the weight of small intestine (mean9
S.E.M.; n=7)
Stimulated by our demonstration that the intro-
duction of the 25,26,26,26,27,27,27-heptafluoro
substitution into 3i-hydroxy-5h-cholest-8(14)-en-
15-one (Swaminathan et al., 1993) and into 3i-
hydroxy-7h-methyl-5h-cholest-8(14)-en-15-one
(Swaminathan et al., 1995) provides increased po-
tency with respect to lowering of serum choles-
terol levels as well as other potentially favorable
actions (Gerst et al., 1994; Swaminathan et al.,
1995), we prepared the 25,26,26,26,27,27,27-hep-
tafluoro analog (VI) of 3i-hydroxycholest-5-
en-7-one (I). The ditosylate derivative of
25,26,26,26,27,27,27 - heptafluoro - 5i - cholestane-
3h,6h-diol, prepared as previously described
(Swaminathan et al., 1993), was converted to the
acetate derivative (IV) of the F7 analog of choles-
terol. Chromium trioxide/dimethyl pyrazole oxi-
dation of the F7-cholesteryl acetate IV gave, after
chromatography, the corresponding 7-keto
Group
Weight (g)
% of total body
weight
A. Ad libitum con- 6.2290.13
trol
B. 0.1% I
C. Pair-fed to
group B
D. 0.15% I
E. Pair-fed to
group D
F. 0.20% I
G. Pair-fed to
group F
2.7390.03
6.6790.18
6.5890.15
2.8490.07
2.9190.04
7.3890.18
6.3890.24
3.2090.10
2.7890.06
7.9690.19
6.2390.14
3.5990.06
2.9190.09
B vs. A
D vs. A
F vs. A
+7% (P=
0.046)
+19% (P=
0.0009)
+28% (P=
0.0005)
+4% (n.s.)
+17% (P=
0.0065)
+32% (P=
0.0001)
B vs. C
D vs. E
+1% (n.s.)
+16% (P=
0.017)
+28% (P=
0.0002)
−3% (n.s.)
+15% (P=0.005)
Table 4
Effect of dietary administration of 3i-hydroxycholest-5-en-7-
one (I) for 10 days to male Sprague-Dawley rats on the weight
of adrenal glands (mean9S.E.M.; n=7)
F vs. G
+23% (P=
0.0005)
Group
Weight (mg)
% of total body
weight
A. Ad libitum con- 40.691.3
0.017890.0004
trol
B. 0.1% I
C. Pair-fed to
group B
D. 0.15% I
E. Pair-fed to group 41.892.9
D
F. 0.20% I
G. Pair-fed to
group F
Administration of the F7-D5-7-ketosterol VI at
a level of 0.26% in diet (the molar equivalent of
0.2% of the D5-7-ketosterol I) had little or no
effect on food consumption. A lower (−14%;
P=0.0004) mean value of food consumption was
observed in the treated rats on only one day of
the experiment (day 10) relative to ad libitum
controls. The mean values of body weight of the
treated rats did not differ from those of the ad
libitum control animals. Administration of VI had
no significant effects on the weights of heart, liver,
spleen, adrenals, testis, or small intestine. The
mean weight of kidneys was slightly decreased in
the rats treated with VI, relative to ad libitum
control animals (−10%; P=0.014) or pair-fed
control animals (−13%; P=0.014). Administra-
tion of VI, at a level of 0.26%, had no significant
effect on serum cholesterol levels (Table 5).
46.791.6
38.891.6
0.019990.0006
0.017190.0007
44.891.0
0.019490.0004
0.018390.0011
43.091.5
35.291.6
0.019290.0005
0.016490.0008
B vs. A
D vs. A
+15% (P=
0.032)
+11% (P=
0.021)
+12% (n.s.)
+9% (P=0.011)
F vs. A
B vs. C
+6% (n.s.)
+8% (n.s.)
+20% (P=
0.023)
+7% (n.s.)
+22% (P=
0.005)
+16% (P=0.041)
D vs. E
F vs. G
+6% (n.s.)
+17% (n.s.)

220
J.N. Carroll et al. / Chemistry and Physics of Lipids 94 (1998) 209–225
Table 5
Effect of dietary administration of 3i-hydroxycholest-5-en-7-one (I) at levels of 0.1, 0.15 and 0.2% and of 3i-hydroxy-
25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one (VI) at a level of 0.26% on serum cholesterol levels in male Sprague-Dawley rats
Group
Serum cholesterol (mg/dl; mean9S.E.M.)
Day 0
Day 5
Day 9
Experiment 1
A. Ad libitum control (n=7)
B. 0.1% I (n=7)
C. Pair-fed to group B (n=7)
D. 0.15% II (n=7)
E. Pair-fed to group D (n=7)
F. 0.20% II (n=7)
G. Pair-fed to group F (n=7)
127.293.5
127.093.3
127.193.3
127.693.2
128.093.3
128.493.3
128.693.4
110.294.6
115.693.7
110.994.8
110.495.0
108.394.7
96.293.6
123.993.1
125.895.5
122.493.8
115.194.4
120.294.9
105.494.0
107.896.0
101.993.6
Experiment 2
A. Ad libitum control (n=8)
B. 0.26% VI (n=4)
C. Pair-fed to group B (n=4)
132.592.3
133.091.9
133.792.2
118.694.1
104.394.4
111.995.1
114.695.5
105.495.4
120.895.8
Significant findings for experiment 1: day 5, group F vs. group A, −12.8% (P=0.03); day 9, group F vs. group A, −14.9%
(P=0.0026); day 9, group A vs. group G, −13% (P=0.01).
derivative (V)3. Mild alkaline hydrolysis of V gave
the desired 3i-hydroxy-25,26,26,26,27,27,27-hep-
tafluorocholest-5-en-7-one (VI).
tion of cholesterol by rat jejunal microsomes (with
an IC50 value of ꢀ3 vM), a finding in agreement
with those reported previously (Miller et al., 1987;
Schroepfer et al., 1988c; Kim et al., 1989; Swami-
nathan et al., 1995). In contrast, 7-ketocholesterol
(I) and its F7-analog (VI) had no effect on ACAT
activity in jejunal microsomes at the concentra-
tions studied (from 1 to 16 vM).
Our in vivo results with 7-ketocholesterol (I)
represent an extension of the very limited, previ-
ously published studies (Erickson et al., 1977;
Kandutsch et al., 1977; Toda et al., 1981; Vargas
et al., 1986; Tamasawa et al., 1994) on the effect
of its oral administration to intact animals. In
contrast to the D8(14)-15-ketosterol II, dietary ad-
ministration of I or its F7 analog VI had little
effect on food consumption or on body weight.
The 15-ketosterol II, at a level of 0.1%, caused a
marked decrease in food consumption and a clear
suppression of gain in body weight in male
Sprague-Dawley rats (Schroepfer et al., 1977a;
Miller et al., 1988; Smith et al., 1989; Gerst et al.,
1994). At the same level in the diet (and at
0.15%), I had no effect on food consumption or
on body weight. At a level of 0.2%, I caused a
modest decrease in mean values of food consump-
tion on days 1–5 of the 10-day experiment.
7-Ketocholesterol (I) and its F7 analog VI low-
ered the levels of HMG-CoA reductase activity in
CHO-K1 cells. I and VI showed essentially the
same potency, with ꢀ40 and 47% lowering of
reductase activity at 2.5 vM I and VI, respec-
tively. It should be noted that the corresponding
D
8(14)-15-ketosterol II is considerably more potent
than I or VI in lowering reductase activity, with II
showing IC50 values of ꢀ0.2 vM in CHO-K1
cells (Swaminathan et al., 1995). The difference in
potency between I and II in CHO cells is in
accord with a similar difference in potency ob-
served in mouse L-cells (Schroepfer et al., 1977b;
Taylor et al., 1984).
I and VI differed markedly from II in their
effects on ACAT activity. The D8(14)-15-ketosterol
II inhibited the oleoyl-CoA-dependent esterifica-
3 Purification of the 7-keto acetate V and its nonfluorinated
analog by MPLC on silica gel removed most byproducts,
including 5,6i-epoxy-5i-cholestan-3i-ol acetate (or its F7
analog). However, it proved more expedient to delay separa-
tion from the 3i-acetoxy-5,6h-epoxy-5h-cholestan-7-one
byproduct (or its F7 analog) until after saponification to free
the sterol.

J.N. Carroll et al. / Chemistry and Physics of Lipids 94 (1998) 209–225
221
Whereas mean values of body weight in rats
treated with I at a level of 0.2% did not differ
from those of ad libitum controls, a slight de-
crease in body weight gain (expressed as a per-
centage of body weight on day 0) was observed
relative to ad libitum controls. The F7 analog (VI)
of 7-ketocholesterol, at 0.26% in diet (the molar
equivalent of 0.2% I) had no effect on food con-
sumption or body weight.
Dietary administration of I (at levels of 0.1,
0.15 or 0.2%) caused a significant increase in the
weight of small intestine, which appeared to be
dose-dependent. The magnitude of the increase in
intestinal weight was considerably less than that
observed previously with II (Gerst et al., 1994). It
is also noteworthy that introduction of the F7
substitution into 7-ketocholesterol (I) eliminated
the increase in the weight of small intestine caused
by I. This finding is in accord with the previously
observed (Gerst et al., 1994) reduction of the
increase in intestinal weight caused by introduc-
tion of the F7 substitution into the D8(14)-15-ketos-
terol II. Whereas the morphology of the small
intestine after administration of II to rats has
been studied in considerable detail (Smith et al.,
1989), the mechanisms involved in the increase in
the weight of small intestine caused by I and II
have not been established.
tivity in cultured mammalian cells. Further, in
contrast to the 15-ketosterol II, I had no effect
upon ACAT activity of rat jejunal microsomes in
vitro and has no effect on serum cholesterol levels
in the intact rat at concentrations of I in the diet
at which II causes appreciable lowering of serum
cholesterol levels.
The F7-7-ketosterol VI was similar to I in its
effects on HMG-CoA reductase activity in CHO
cells and on ACAT activity in rat jejunal micro-
somes. However, when VI was administered to
intact rats at a level of 0.26% in diet (the molar
equivalent of 0.2% I), it did not cause an increase
in the weight of small intestine, as seen with I.
Under the conditions studied, VI had no effect on
the level of serum cholesterol. The availability of
VI may be helpful in future studies of the
metabolism of I. It may also find use as an
internal standard in GC-MS studies of the levels
of I in blood, tissues, and other samples.
Acknowledgements
This work was supported in part by grants
from the National Institutes of Health (HL-
49122), the Texas Advanced Technology Program
(3604026), and the Robert A. Welch Foundation
(C-583).
The D8(14)-15-ketosterol II has been shown con-
sistently to lower serum cholesterol levels in rats
receiving the sterol at a level of 0.1% in diet
(Schroepfer et al., 1977a; Miller et al., 1987; Brab-
son and Schroepfer, 1988; Smith et al., 1989;
Gerst et al., 1994). In contrast, the D5-7-ketosterol
I had no effect on serum cholesterol levels of rats
receiving I at levels of 0.1 or 0.15%. Administra-
tion of I at a level of 0.2% caused a very slight
lowering of serum cholesterol relative to ad libi-
tum control rats, but not pair-fed control rats.
Administration of the F7 analog (VI) of the 7-ke-
tosterol at a level of 0.26% in diet (the molar
equivalent of I at 0.2% in diet) had no effect on
serum cholesterol.
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