Bis(2-(acylamino)phenyl) disulfides, 2-(acylamino)benzenethiols, and S-(2-(acylamino)phenyl) alkanethioates as novel inhibitors of cholesteryl ester transfer protein

Article abstract of DOI:10.1021/jm000224s

A series of bis(2-(acylamino)phenyl) disulfides, 2-(acylamino)benzenethiols, S-(2-(acylamino)-phenyl) alkanethioates, and related compounds were synthesized, and their inhibitory effect on cholesteryl ester transfer protein activity in human plasma was evaluated. This study elucidated the structural requirements for inhibitory activity and determined that the optimum compound was S-(2-((1-(2-ethylbutyl)cyclohexane)carbonylamino)phenyl) 2-methylpropanethioate (27) (JTT-705). This compound achieved 50% inhibition of CETP activity in human plasma at a concentration of 9 μM and 95% inhibition of CETP activity in male Japanese white rabbits at an oral dose of 30 mg/kg. It increased the plasma HDL cholesterol level by 27% and 54%, respectively, when given at oral doses of 30 or 100 mg/kg once a day for 3 days to male Japanese white rabbits.

Full text of DOI:10.1021/jm000224s

3566
J . Med. Chem. 2000, 43, 3566-3572
Bis(2-(Acyla m in o)p h en yl) Disu lfid es, 2-(Acyla m in o)ben zen eth iols, a n d
S-(2-(Acyla m in o)p h en yl) Alk a n eth ioa tes a s Novel In h ibitor s of Ch olester yl Ester
Tr a n sfer P r otein
Hisashi Shinkai,* Kimiya Maeda, Takahiro Yamasaki, Hiroshi Okamoto, and Itsuo Uchida
Central Pharmaceutical Research Institute, J T Inc., 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, J apan
Received May 30, 2000
A series of bis(2-(acylamino)phenyl) disulfides, 2-(acylamino)benzenethiols, S-(2-(acylamino)-
phenyl) alkanethioates, and related compounds were synthesized, and their inhibitory effect
on cholesteryl ester transfer protein activity in human plasma was evaluated. This study
elucidated the structural requirements for inhibitory activity and determined that the optimum
compound was S-(2-((1-(2-ethylbutyl)cyclohexane)carbonylamino)phenyl) 2-methylpropanethi-
oate (27) (J TT-705). This compound achieved 50% inhibition of CETP activity in human plasma
at a concentration of 9 µM and 95% inhibition of CETP activity in male J apanese white rabbits
at an oral dose of 30 mg/kg. It increased the plasma HDL cholesterol level by 27% and 54%,
respectively, when given at oral doses of 30 or 100 mg/kg once a day for 3 days to male J apanese
white rabbits.
In tr od u ction
action was considered to involve the formation of a
disulfide bond between the thiol form of compound 27
and the cysteine residue at position 13 (Cys13) of CETP.
Cholesteryl ester transfer protein (CETP) is a plasma
protein that transfers neutral lipids among the lipopro-
teins.1,2 Its most important action is the exchange of
cholesteryl esters (CE) in high-density lipoprotein (HDL)
for triglycerides in very low-density lipoprotein (VLDL).1,2
The net effect of this process is to reduce the level of
antiatherogenic HDL cholesterol while increasing pro-
atherogenic VLDL and low-density lipoprotein (LDL).1,2
Thus, CETP is a potentially atherogenic protein, and
its atherogenicity has been supported by many studies.3-6
On the other hand, a possible antiatherogenic role of
CETP and the participation of CETP in reverse choles-
terol transport (RCT) have also been suggested.7-10 RCT
is the movement of cholesterol from the peripheral cells
through the plasma compartment (including HDL) to
the liver for catabolism.11 Thus, the role of CETP in the
progression of atherosclerosis is still not defined. A
powerful CETP inhibitor could be useful in studies to
elucidate the role of CETP in atherosclerosis, so we have
attempted to synthesize such an inhibitor. If the anti-
atherogenicity of CETP inhibition were confirmed, we
should also be able to obtain a novel antiatherogenic
drug from among the synthetic inhibitors of CETP.
Here we describe the structure-activity relationship
(SAR) and optimization studies performed on a series
of bis(2-(acylamino)phenyl) disulfides, 2-(acylamino)-
benzenethiols, S-(2-(acylamino)phenyl) alkanethioates,
and related compounds. These studies determined the
structural requirements for CETP inhibition and even-
tually led to the optimum compound, S-(2-((1-(2-ethyl-
butyl)cyclohexane)carbonylamino)phenyl) 2-methylpro-
panethioate (27) (J TT-705), which achieved marked
inhibition of CETP in human plasma and caused
significant elevation of HDL cholesterol in male J apa-
nese white rabbits (J W rabbits). The mechanism of
Ch em istr y
The routes used for synthesis of the compounds
assessed in this study are shown in Schemes 1-4.
Scheme 1 shows the preparation of disulfides, thiols,
thioesters, and a sulfide. Bis(2-(acylamino)phenyl) di-
sulfides (1-9 and the precursors of 17-24) were syn-
thesized by N-acylation of bis(2-aminophenyl) disulfide
with the corresponding acyl chloride. Reduction of the
disulfide bond with triphenylphosphine gave 2-acylami-
nobenzenethiols (16-24). The benzenethiols were coupled
with the corresponding acyl chloride to give thioesters
(14 and 26-28). The sulfide 25 was synthesized by
methylation of 2-((1-(2-ethylbutyl)cyclohexane)carbonyl-
amino)benzenethiol with iodomethane.
Bis(2-(N-methyl-N-pivaloylamino)phenyl) disulfide (11),
bis(3-(pivaloylamino)phenyl) disulfide (12), or bis(4-
(pivaloylamino)phenyl) disulfide (13) was also prepared
by N-acylation of bis(2-(methylamino)phenyl) disulfide,
bis(3-aminophenyl) disulfide, or bis(4-aminophenyl) di-
sulfide, respectively, with pivaloyl chloride.
Commercially unavailable 1-alkylcyclohexanecarbo-
nyl chlorides were synthesized by dianion coupling
between cyclohexanecarboxylic acid and the correspond-
ing alkyl bromide, followed by treatment with oxalyl
chloride (Scheme 2).
The reversed amide 10 was prepared by coupling
between 2,2′-dithiodibenzoic acid and tert-butylamine
using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (WSCD‚HCl; water-soluble carbodiimide
hydrochloride) and 1-hydroxybenzotriazole (HOBt)
(Scheme 3).
Diacylation of 2-aminophenol with 1-methylcyclohex-
anecarbonyl chloride and subsequent hydrolysis gave
1-((1-methylcyclohexane)carbonylamino)phenol. Cou-
* To whom correspondence should be addressed. Tel: 81 726 81
9700. Fax: 81 726 81 9725. E-mail: hisashi.shinkai@ims.jti.co.jp.
10.1021/jm000224s CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/01/2000

Novel Inhibitors of CETP
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3567
Sch em e 1a
a
Reagents: (a) R-COCl, pyridine, 100 °C; (b) triphenylphosphine; (c) R′-COCl, pyridine; (d) iodomethane, K2CO3.
Sch em e 2a
Sch em e 4a
a
Reagents: (a) 2 equiv of lithium diisopropylamide, R-Br; (b)
oxalyl chloride.
Sch em e 3a
a
Reagents: (a) R-COCl, Et3N; (b) KOH; (c) phenylacetyl
chloride, pyridine.
compounds were further evaluated for their ability to
inhibit CETP and to increase HDL cholesterol in J W
rabbits.
a
Reagents: (a) tert-butylamine, WSCD‚HCl, HOBt.
pling between the phenol and phenylacetyl chloride gave
the ester 15 (Scheme 4).
First, the effect of the acyl groups on the inhibition
of CETP was examined (Table 1). The propionyl com-
pound 2 was about 2.8-fold more potent than the acetyl
compound 1, while the isobutyryl compound 3 was about
12-fold more potent and the valeryl compound 4 was
20-fold more potent. These results indicated that the
improvement of potency was attributable to an increase
in the lipophilicity of the acyl moiety. However, the
potency of the cyclohexanecarbonyl compound 6 and the
octanoyl compound 7 was almost equivalent to that of
the valeryl compound 4. This showed that a further
increase of lipophilicity in the acyl moiety did not lead
to a further improvement in potency. Thus, the effect
of acyl moiety lipophilicity on potency showed a plateau.
On the other hand, the pivaloyl compound 5 and the
1-methylcyclohexanecarbonyl compound 8 with a ter-
Resu lts a n d Discu ssion
The goal of this study was to synthesize a specific
CETP inhibitor that showed activity when administered
orally. We selected bis(2-(acetylamino)phenyl) disulfide
(1) as the initial lead compound in our chemical library,
because it showed 50% inhibition of CETP activity in
human plasma at a concentration of 500 µM. The
structure of this lead compound was systematically
varied in order to elucidate the structural requirements
for inhibition of CETP and to synthesize the optimum
compound. The various compounds synthesized were
primarily evaluated for their ability to inhibit CETP-
mediated CE transfer in human plasma. Selected

3568 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Ta ble 1. Bis(2-(Acylamino)phenyl) Disulfides
Shinkai et al.
Ta ble 2. Analogues of Compounds 5 and 8
a
Concentration achieving 50% inhibition of CETP-mediated CE
transfer from HDL to VLDL and LDL.
tiary alkyl group next to the carbonyl group displayed
higher potency than the other alkyl compounds shown
in Table 1. Replacement of the cyclohexane ring of
compound 6 with a phenyl ring (compound 9) caused a
decrease in potency. Accordingly, not only lipophilicity
but also the shape of the acyl moiety seemed to influence
the potency, and tertiary alkyl carbonyl groups were
found to be specific for high potency.
Next, the amide moiety was examined (Table 2). The
reversed amide 10 showed no activity, and introduction
of a methyl group into the amide nitrogen (11) caused
loss of activity. These results indicated that the benz-
amide structure was important for activity and that the
amide proton may be essential.
Subsequently, the relative position between the di-
sulfide moiety and the amide group on the benzene ring
of compound 5 was changed (Table 2). Regioisomers 12
and 13, in which the disulfide group was moved from
the ortho-position of the pivaloylaminobenzene to the
meta- or para-position, showed loss of activity. Thus,
both the disulfide group and the amide group substi-
tuted at the next position were essential for activity.
On the other hand, the thioester 14 also showed activity,
indicating that the thioester group could play the same
role as the disulfide group and that the dimeric struc-
ture was not essential. Since the ester 15 did not show
activity, the sulfur atom was essential. Because the
thioester was readily hydrolyzed to a thiol, we suspected
a
Concentration achieving 50% inhibition of CETP-mediated CE
transfer from HDL to VLDL and LDL.
that it was the precursor of an active thiol. In fact, thiol
16 showed activity, as anticipated (Table 3). The disul-
fide would also be active because it was not observed to
undergo transformation to the thiol in vitro. Therefore,
the disulfide group and the thiol group were functionally
equivalent. Because both disulfides and thiols have the
ability to form a disulfide bond with another thiol group,
we considered that formation of a disulfide bond be-
tween the test compounds and a cysteine residue of
CETP may have been related to the mechanism of
inhibition. Therefore, we performed a study using point
mutations of recombinant human CETP and showed
that Cys13 of CETP was targeted when the compounds
synthesized in this study exhibited an inhibitory effect.14
We also confirmed that the inhibitory activity was
maintained after washout of the test compounds.
Since we found that the 1-methylcyclohexanecarbonyl
structure of the acyl moiety was specific for high potency
in the series of disulfide compounds (Table 1), synthesis
of various 1-alkylcyclohexanecarbonyl compounds was
carried out for further optimization using a series of
thiol compounds (Table 3). Introduction of alkyl groups

Novel Inhibitors of CETP
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3569
Ta ble 3. 2-Acylaminobenzenethiols
ited by certain cysteine-modifying reagents under serum-
free conditions,12,13 but such compounds have not been
confirmed to be effective in plasma or in vivo. Nonspe-
cific reactions between these compounds and endog-
enous cysteine-containing compounds, such as glu-
tathione, could decrease their inhibitory activity under
in vivo conditions.
Since the disulfides in Tables 1 and 2 were not orally
bioavailable and the thiols in Table 3 were unstable,
orally bioavailable thioesters were used for the in vivo
assays (Table 4). In vitro, the acetyl compound 26 was
more potent than the isobutyryl compound 27, and the
pivaloyl compound 28 was much weaker than 26 or 27.
The differences in the IC50 values of these thioesters
were probably due to differences in the rate of hydroly-
sis. In contrast, the isobutyryl compound 27 showed 95%
inhibition of CETP activity at an oral dose of 30 mg/kg
in J W rabbits and was the most potent of these
compounds invivo (26-28). Thus, compound 27 had a
higher absorption than compound 26. After the in vivo
inhibitory effect of compound 27 on CETP was con-
firmed, its effect on the plasma HDL cholesterol level
was examined in J W rabbits. As a result, a 27% or 54%
increase of plasma HDL cholesterol was respectively
observed when 30 or 100 mg/kg of compound 27 was
administered orally once a day for 3 days.
Con clu sion s
SAR studies on disulfide, thiol, and thioester com-
pounds elucidated the structural requirements for in-
hibition of CETP and led to the optimum compound 27.
This compound showed marked inhibition of CETP in
human plasma and in J W rabbits and caused a signifi-
cant increase of the plasma HDL cholesterol level in J W
rabbits. The mechanism of action was considered to be
formation of a disulfide bond between the inhibitor and
Cys13 of CETP. Since 6 months of treatment with
compound 27 (J TT-705) increased the HDL cholesterol
level, decreased the LDL level, and prevented the
progression of atherosclerosis in rabbits,14 development
of this compound as an antiatherosclerosis drug is
currently underway in clinical trials.
a
Concentration achieving 50% inhibition of CETP-mediated CE
transfer from HDL to VLDL and LDL.
with 2-7 carbon atoms at the 1-position of the cyclo-
hexane ring was performed. Introducing an alkyl group
with 6 carbon atoms was found to be the most effective
modification, and the 2-ethylbutyl compound 23 showed
the highest potency. This compound achieved 50%
inhibition of CETP activity in human plasma at a
concentration of 5 µM and was about 100-fold more
potent than lead compound 1. The S-methyl compound
25, which was unable to form disulfide bonds with Cys
of CETP, showed loss of activity as expected. Several
studies have already demonstrated that CETP is inhib-
Exp er im en ta l Section
Ch em istr y. Melting points were obtained with a Yanagi-
moto micro melting point apparatus or a Mettler-Toledo FP62
melting point instrument and are uncorrected. Elemental
Ta ble 4. S-(2-(Acylamino)phenyl) Alkanethioates
CETP inhibition in J W rabbits (%)b
HDL elevation in J W rabbits (%)c
CETP inhibition in
compd
R
human plasma IC50 (µM)a
10 mg/kg
30 mg/kg
30 mg/kg × 3
100 mg/kg × 3
26
27
28
CH3
(CH3)2CH
(CH3)3C
4
6
41
40
95
32
35
27
54
a
b
Concentration achieving 50% inhibition of CETP-mediated CE transfer from HDL to VLDL and LDL. Percent inhibition of CETP
was measured 3 h after administration of the test compound and was calculated from CETP-mediated CE transfer in the plasma of J W
rabbits administered the test compounds at oral doses of 10 and 30 mg/kg relative to the transfer in plasma from rabbits given the
vehicle. Inhibition was shown to persist for 9 h. c Percent elevation was calculated from the plasma HDL levels in J W rabbits administered
the test compounds at oral doses of 30 and 100 mg/kg once a day for 3 days relative to the levels in rabbits given the vehicle.

3570 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Shinkai et al.
analysis was performed with a Perkin-Elmer 2400 series II
CHNS/O analyzer, and all values were within (0.4% of the
calculated values. FAB mass spectra were recorded on a
Finnigan TSQ 700 spectrometer. High-resolution mass spectra
Bis(2-(N-m eth yl-N-pivaloylam in o)ph en yl) disu lfide (11):
mp 192-193 °C; NMR (300 MHz, CDCl3) δ 1.13 (s, 18H), 3.42
(s, 6H), 7.14-7.33 (m, 6H), 7.44-7.58 (m, 2H). Anal. (C24H32N2-
O2S2) C, H, N.
Bis(3-(p iva loyla m in o)p h en yl) d isu lfid e (12): mp 155-
157 °C; NMR (300 MHz, CDCl3) δ 1.32 (s, 18H), 7.16-7.27
(m, 4H), 7.35-7.48 (m, 4H), 7.75 (s, 2H). Anal. (C22H28N2O2S2)
C, H, N.
Bis(4-(p iva loyla m in o)p h en yl) d isu lfid e (13): mp 200-
201 °C; NMR (300 MHz, DMSO -d6) δ 1.19 (s, 18H), 7.41 (d, J
) 8.7 Hz, 4H), 7.60 (d, J ) 8.7 Hz, 4H), 9.30 (br s, 2H). Anal.
(C22H28N2O2S2‚0.3C6H14) C, H, N.
1
were obtained with a J EOL SX 102A spectrometer. H NMR
spectra were recorded on a J EOL J NM-A300W or Bruker AMX
300 spectrometer in a solution of either CDCl3 or DMSO-d6
using tetramethylsilane as the internal standard. Chemical
shifts are expressed as δ (ppm) values for protons relative to
the internal standard. All compounds gave spectra consistent
with their assigned structures.
Bis(2-a cyla m in op h en yl) Disu lfid es 1-9. A typical run
(8 in Table 1) was as follows. A mixture of 1-methylcyclohex-
anecarboxylic acid (680 mg, 4.78 mmol) and oxalyl chloride
(0.5 mL, 5.73 mmol) in methylene chloride (5 mL) was stirred
at room temperature for 1 h and concentrated to a crude acid
chloride. This crude 1-methylcyclohexanecarbonyl chloride in
methylene chloride (5 mL) was added dropwise to a solution
of bis(2-aminophenyl) disulfide (500 mg, 2.01 mmol) and
pyridine (0.4 mL, 4.95 mmol) in methylene chloride (5 mL) at
room temperature for 1 h. The mixture was diluted with
chloroform (50 mL), washed with water (30 mL) and with brine
(30 mL), dried over sodium sulfate, and concentrated. The
crude product was subjected to silica gel chromatography and
eluted with ethyl acetate/n-hexane (1:5) to give bis(2-((1-
methylcyclohexane)carbonylamino)phenyl) disulfide (8) (980
mg, 98%) as an amorphous powder: 1H NMR (300 MHz,
CDCl3) δ 1.18 (s, 6H), 1.25-1.70 (m, 16H), 1.80-2.00 (m, 4H),
6.95 (dt, J ) 1.5 and 7.8 Hz, 2H), 7.22 (dd, J ) 1.5 and 7.8 Hz,
2H), 7.40 (ddd, J ) 1.5, 7.8, and 8.4 Hz, 2H), 8.48 (dd, J ) 1.5
and 8.4 Hz, 2H), 8.51 (br s, 2H); MS (FAB) m/z 497 (M + H)+;
HRMS calcd for C28H37N2O2S2, 497.2296; found, 497.2288.
Bis(2-(a cetyla m in o)p h en yl) d isu lfid e (1): mp 162-163
°C; NMR (300 MHz, CDCl3) δ 1.98 (s, 6H), 7.03 (dt, J ) 1.5
and 7.8 Hz, 2H), 7.37-7.45 (m, 4H), 7.90 (br s, 2H), 8.35 (d, J
) 8.1 Hz, 2H). Anal. (C16H16N2O2S2‚0.5H2O) C, H, N.
Bis(2-(p r op ion yla m in o)p h en yl) d isu lfid e (2): mp 139-
140 °C; NMR (300 MHz, CDCl3) δ 1.15 (t, J ) 7.5 Hz, 6H),
2.19 (q, J ) 7.5 Hz, 4H), 7.00 (dt, J ) 1.5 and 7.8 Hz, 2H),
7.37-7.42 (m, 4H), 7.96 (br s, 2H), 8.38 (dd, J ) 1.5 and 8.4
Hz, 2H). Anal. (C18H20N2O2S2) C, H, N.
2-((1-(2-Eth ylbu tyl)cycloh exa n e)ca r bon yla m in o)ben -
zen eth iol (23). Lithium diisopropylamide (2.0 M) in tetrahy-
drofuran (100 mL, 200 mmol) was added dropwise to a solution
of cyclohexanecarboxylic acid (10.3 g, 80 mmol) in tetrahydro-
furan (100 mL) at 0 °C. After stirring for 1.5 h at room
temperature, a solution of 1-bromo-2-ethylbutane (21.1 g, 128
mmol) in tetrahydrofuran (20 mL) was added dropwise at 0
°C. Then the mixture was stirred overnight at room temper-
ature. The solution was poured into water (50 mL), acidified
with 2 N HCl (150 mL), and extracted with ethyl acetate (200
mL). The combined extracts were washed with water (100 mL),
dried over sodium sulfate, and concentrated to a crude 1-(2-
ethylbutyl)cyclohexanecarboxylic acid. A mixture of this crude
acid and oxalyl chloride (8.0 mL, 91.7 mmol) in methylene
chloride (100 mL) was stirred for 1 h at room temperature.
After the solvent was evaporated under a vacuum, the residue
was distilled to give the acid chloride (6.97 g, 38%) as a
colorless liquid (bp 135 °C, 10 mmHg). The acid chloride (2.72
g, 11.8 mmol) was added dropwise to a solution of bis(2-
aminophenyl) disulfide (1.46 g, 5.88 mmol) in pyridine (15 mL).
Then the mixture was heated overnight at 100 °C. After
evaporation of the solvent, water (50 mL) was added to the
residue. The aqueous solution was extracted with ethyl acetate
(2 × 100 mL), after which the extract was washed with brine
(50 mL), dried over sodium sulfate, and concentrated. A
mixture of this disulfide, triphenylphosphine (3.10 g, 11.8
mmol) in dioxane (25 mL) and water (25 mL) was stirred for
1 h at 50 °C. After cooling to room temperature, 1 N aqueous
sodium hydroxide (50 mL) was added to the mixture. This
aqueous solution was washed with n-henxane (30 mL), acidi-
fied to pH 3-4 with 10% hydrochloric acid, and extracted with
ethyl acetate (2 × 100 mL). The organic layer was washed with
brine (50 mL), dried over sodium sulfate, and concentrated.
Chromatography of the residue on a silica gel column eluted
with ethyl acetate/n-hexane (1:10) gave 23 as a colorless solid
Bis(2-(isobu tyr yla m in o)p h en yl) d isu lfid e (3): mp 147-
148 °C; NMR (300 MHz, CDCl3) δ 1.18 (d, J ) 6.9 Hz, 12H),
2.32 (sep, J ) 6.9 Hz, 2H), 6.96 (dt, J ) 1.5 and 7.8 Hz, 2H),
7.31 (dd, J ) 1.5 and 7.8 Hz, 2H), 7.40 (ddd, J ) 1.5, 7.8, and
8.4 Hz, 2H), 8.07 (br s, 2H), 8.40 (dd, J ) 1.5 and 8.4 Hz, 2H).
Anal. (C20H24N2O2S2) C, H, N.
Bis(2-(va ler yla m in o)p h en yl) d isu lfid e (4): mp 68-70 °C;
NMR (300 MHz, CDCl3) δ 0.94 (t, J ) 7.2 Hz, 6H), 1.30-1.75
(m, 8H), 2.17 (t, J ) 7.2 Hz, 4H), 7.00 (dt, J ) 1.5 and 7.8 Hz,
2H), 7.36-7.48 (m, 4H), 7.94 (br s, 2H), 8.40 (dd, J ) 1.5 and
8.4 Hz, 2H). Anal. (C22H28N2O2S2‚0.15C6H14) C, H, N.
1
(2.66 g, 71%): mp 68.5-74 °C; H NMR (300 MHz, CDCl3) δ
0.79 (t, J ) 6.9 Hz, 6H), 1.20-1.80 (m, 15H), 2.05-2.25 (m,
2H), 3.07 (s, 1H), 7.00 (dt, J ) 1.5 and 7.8 Hz, 1H), 7.31 (ddd,
J ) 1.5, 7.8, and 8.4 Hz, 1H), 7.51 (dd, J ) 1.5 and 7.8 Hz,
1H), 8.33 (dd, J ) 1.5 and 8.4 Hz, 1H), 8.45 (br s, 1H). Anal.
(C19H29NOS) C, H, N.
Bis(2-(p iva loyla m in o)p h en yl) d isu lfid e (5): mp 86-87
2-((1-Me t h ylcycloh e xa n e )ca r b on yla m in o)b e n ze n e -
1
°C; H NMR (300 MHz, CDCl3) δ 1.25 (s, 18H), 6.94 (dt, J )
1
th iol (16): mp 82-83 °C; H NMR (300 MHz, CDCl3) δ 1.30
1.5 and 7.8 Hz, 2H), 7.21 (dd, J ) 1.5 and 7.8 Hz, 2H), 7.40
(ddd, J ) 1.5, 7.8, and 8.4 Hz, 2H), 8.46 (dd, J ) 1.5 and 8.4
Hz, 2H), 8.52 (br s, 2H). Anal. (C22H28N2O2S2) C, H, N.
(s, 3H), 1.25-1.75 (m, 8H), 2.04-2.20 (m, 2H), 3.07 (s, 1H),
7.00 (dt, J ) 1.5 and 7.8 Hz, 1H), 7.31 (ddd, J ) 1.5, 7.8, and
8.4 Hz, 1H), 7.50 (dd, J ) 1.5 and 7.8 Hz, 1H), 8.31 (dd, J )
1.5 and 8.4 Hz, 1H), 8.42 (br s, 1H). Anal. (C14H19NOS) C, H,
N.
Bis(2-(cycloh exa n eca r b on yla m in o)p h en yl) d isu lfid e
(6): mp 167-170 °C; NMR (300 MHz, CDCl3) δ 1.10-2.10 (m,
22H), 6.99 (dt, J ) 1.5 and 7.8 Hz, 2H), 7.36-7.45 (m, 4H),
8.09 (br s, 2H), 8.44 (dd, J ) 1.5 and 8.4 Hz, 2H). Anal.
(C26H32N2O2S2) C, H, N.
Bis(2-(octa n oyla m in o)p h en yl) d isu lfid e (7): mp 79-81
°C; NMR (300 MHz, CDCl3) δ 0.89 (t, J ) 6.9 Hz, 6H), 1.20-
1.40 (m, 16H), 1.50-1.70 (m, 4H), 2.16 (t, J ) 7.2 Hz, 4H),
7.00 (dt, J ) 1.5 and 7.8 Hz, 2H), 7.35-7.43 (m, 4H), 7.97 (br
s, 2H), 8.40 (dd, J ) 1.5 and 8.4 Hz, 2H). Anal. (C28H40N2O2S2)
C, H, N.
2-((1-E t h y lc y c lo h e x a n e )c a r b o n y la m in o )b e n ze n e -
1
th iol (17): mp 84-85 °C; H NMR (300 MHz, CDCl3) δ 0.90
(t, J ) 7.2 Hz, 3H), 1.20-1.70 (m, 10H), 2.05-2.19 (m, 2H),
3.07 (s, 1H), 7.00 (dt, J ) 1.5 and 7.8 Hz, 1H), 7.31 (ddd, J )
1.5, 7.8, and 8.4 Hz, 1H), 7.50 (dd, J ) 1.5 and 7.8 Hz, 1H),
8.32 (dd, J ) 1.5 and 8.4 Hz, 1H), 8.38 (br s, 1H). Anal. (C15H21
NOS) C, H, N.
-
2-((1-P r op ylcycloh e xa n e )ca r b on yla m in o)b e n ze n e -
1
th iol (18): mp 93-94 °C; H NMR (300 MHz, CDCl3) δ 0.88
Bis(2-(ben zoyla m in o)p h en yl) d isu lfid e (9): mp 144-145
°C; 1H NMR (300 MHz, CDCl3) δ 6.95 (dt, J ) 1.5 and 7.8 Hz,
2H), 7.31 (dt, J ) 1.5 and 8.4 Hz, 2H), 7.40-7.60 (m, 8H), 7.69
(dd, J ) 1.5 and 8.4 Hz, 4H), 8.50 (dd, J ) 1.5 and 8.4 Hz,
2H), 8.93 (br s, 2H). Anal. (C26H20N2O2S2) C, H, N.
(t, J ) 7.2 Hz, 3H), 1.20-1.70 (m, 12H), 2.05-2.20 (m, 2H),
3.07 (s, 1H), 7.00 (dt, J ) 1.5 and 7.8 Hz, 1H), 7.30 (ddd, J )
1.5, 7.8, and 8.4 Hz, 1H), 7.50 (dd, J ) 1.5 and 7.8 Hz, 1H),
8.32 (dd, J ) 1.5 and 8.4 Hz, 1H), 8.38 (br s, 1H). Anal. (C16H23
-
NOS) C, H, N.

Novel Inhibitors of CETP
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3571
and 8.4 Hz, 1H), 8.13 (br s, 1H), 8.38 (dd, J ) 1.5 and 8.4 Hz,
2-((1-B u t y lc y c lo h e x a n e )c a r b o n y la m in o )b e n ze n e -
th iol (19): mp 97-98 °C; H NMR (300 MHz, CDCl3) δ 0.87
1
1H). Anal. (C21
H31NO2S) C, H, N.
(t, J ) 7.2 Hz, 3H), 1.20-1.70 (m, 14H), 2.05-2.20 (m, 2H),
3.07 (s, 1H), 7.00 (dt, J ) 1.5 and 7.8 Hz, 1H), 7.30 (ddd, J )
1.5, 7.8, and 8.4 Hz, 1H), 7.50 (dd, J ) 1.5 and 7.8 Hz, 1H),
S-(2-((1-(2-E t h ylb u t yl)cycloh exa n e)ca r b on yla m in o)-
p h en yl) 2,2-d im eth ylp r op a n eth ioa te (28): mp 64.5-66.5
°C; 1H NMR (300 MHz, CDCl3) δ 0.79 (t, J ) 6.9 Hz, 6H), 1.10-
1.75 (m, 15H), 1.36 (s, 9H), 1.95-2.15 (m, 2H), 7.10 (dt, J )
1.5 and 7.8 Hz, 1H), 7.36 (dd, J ) 1.5 and 7.8 Hz, 1H), 7.45
(ddd, J ) 1.5, 7.8, and 8.4 Hz, 1H), 8.11 (br s, 1H), 8.42 (dd, J
) 1.5 and 8.4 Hz, 1H). Anal. (C24H37NO2S) C, H, N.
8.31 (dd, J ) 1.5 and 8.4 Hz, 1H), 8.37 (br s, 1H). Anal. (C17H25
-
NOS) C, H, N.
2-((1-P e n t ylcycloh e xa n e )ca r b on yla m in o)b e n ze n e -
th iol (20): NMR (300 MHz, CDCl3) δ 0.85 (t, J ) 6.7 Hz, 3H),
1.20-1.70 (m, 16H), 2.05-2.20 (m, 2H), 3.07 (s, 1H), 7.00 (dt,
J ) 1.5 and 7.8 Hz, 1H), 7.31 (ddd, J ) 1.5, 7.8, and 8.4 Hz,
1H), 7.51 (dd, J ) 1.5 and 7.8 Hz, 1H), 8.31 (dd, J ) 1.5 and
8.4 Hz, 1H), 8.36 (br s, 1H). Anal. (C18H27NOS) C, H, N.
2-((1-H e x y lc y c lo h e x a n e )c a r b o n y la m in o )b e n ze n e -
th iol (21): NMR (300 MHz, CDCl3) δ 0.85 (t, J ) 6.9 Hz, 3H),
1.17-1.70 (m, 18H), 2.05-2.18 (m, 2H), 3.07 (s, 1H), 7.01 (dt,
J ) 1.5 and 7.8 Hz, 1H), 7.31 (ddd, J ) 1.5, 7.8, and 8.4 Hz,
1H), 7.51 (dd, J ) 1.5 and 7.8 Hz, 1H), 8.31 (dd, J ) 1.5 and
8.4 Hz, 1H), 8.37 (br s, 1H). Anal. (C19H29NOS) C, H, N.
2-((1-Isoh exylcycloh exa n e)ca r b on yla m in o)b en zen e-
th iol (22): mp 76-79 °C; NMR (300 MHz, CDCl3) δ 0.83 (d,
J ) 6.6 Hz, 6H), 1.07-1.73 (m, 15H), 2.05-2.20 (m, 2H), 3.07
(s, 1H), 7.01 (dt, J ) 1.5 and 7.8 Hz, 1H), 7.31 (ddd, J ) 1.5,
7.8, and 8.4 Hz, 1H), 7.51 (dd, J ) 1.5 and 7.8 Hz, 1H), 8.29
2-((1-Meth ylcycloh exan e)car bon ylam in o)ph en yl P h en -
yla ceta te (15). 1-Methylcyclohexanecarbonyl chloride (6.48
g, 40.3 mmol) was added to a solution of 2-aminophenol (2.00
g, 18.3 mmol) and triethylamine (8 mL, 57.4 mmol) in
chloroform (30 mL) at room temperature. Then the mixture
was stirred at room temperature for 2 h and washed with
water (50 mL), after which the solvent was evaporated under
a vacuum. The residue was added to a solution of potassium
hydroxide (3.0 g, 45.5 mmol) in tetrahydrofuran (10 mL),
methanol (10 mL), and water (2 mL). This mixture was stirred
at room temperature for 1 h, diluted with water (50 mL),
acidified with 10% hydrochloric acid, and extracted with ethyl
acetate (2 × 100 mL). The combined extracts were washed with
saturated aqueous sodium bicarbonate (30 mL), water (30 mL),
and brine (30 mL). After evaporation of the solvent, the residue
was crystallized from ether/n-hexane to give 2-((1-methylcy-
clohexane)carbonylamino)phenol as an off-white solid (3.98 g,
93%). Phenylacetyl chloride (0.48 mL, 3.62 mmol) was added
to a solution of the phenol (0.8 g, 3.43 mmol) and pyridine (0.85
mL, 10.5 mmol) in chloroform (10 mL). Then the mixture was
stirred overnight at room temperature. After evaporation of
the solvent, the residue was subjected to silica gel chroma-
tography and eluted with ethyl acetate/n-henxane (1:10), after
which it was recrystallized from ether/n-henxane to give 15
as a colorless solid (576 mg, 48%): mp 104-106 °C; NMR (300
MHz, CDCl3) δ 1.03 (s, 3H), 1.15-1.60 (m, 8H), 1.66-1.78 (m,
2H), 3.90 (s, 2H), 7.02-7.43 (m, 9H), 8.24 (dd, J ) 1.2 and 7.8
Hz, 1H). Anal. (C22H25NO3‚0.75H2O) C, H, N.
2,2′-Dith iobis(N-ter t-bu tylben za m id e) (10). A mixture
of 2,2′-dithiodibenzoic acid (1 g, 3.26 mmol), WSCD‚HCl (1.5
g, 7.82 mmol), HOBt (1.06 g, 6.92 mmol), and tert-butylamine
(1.05 mL, 9.99 mmol) in dimethylformamide (15 mL) was
stirred overnight at room temperature. Then the mixture was
diluted with ethyl acetate (100 mL) and washed with 5%
hydrochloric acid (20 mL), 1 N aqueous sodium hydroxide (20
mL), water (20 mL), and brine (20 mL). The resulting organic
solution was dried over sodium sulfate and concentrated.
Chromatography of the residue on a silica gel column eluted
with ethyl acetate/n-hexane (1:1) gave 10 as a slightly yellow
solid (193 mg, 14%): mp 203-204 °C; NMR (300 MHz, CDCl3)
δ 1.49 (s, 18H), 5.87 (br s, 2H), 7.21 (dt, J ) 1.5 and 7.8 Hz,
2H), 7.33 (ddd, J ) 1.5, 7.8, and 8.1 Hz, 2H), 7.43 (dd, J ) 1.5
and 7.8 Hz, 2H), 7.73 (dd, J ) 1.5 and 8.1 Hz, 2H). Anal.
(C22H28N2O2S2‚0.15C6H14) C, H, N.
Bioa ssa ys. 1. In Vitr o CETP Activity. In vitro CETP
activity was determined form the rate of [3H]cholesteryl ester
transfer from HDL to apoprotein B-containing lipoproteins.13,14
Blood was collected from normolipidemic volunteers into tubes
containing heparin (VT-100H, Terumo Inc., J apan). Plasma
was isolated by centrifugation at 1500g for 15-30 min at 4
°C. An appropriate amount of each test compound, dissolved
in a 1:1 mixture of N-methyl-2-pyrrolidinone and poly(ethylene
glycol) (with an average molecular weight of 400), was added
to the plasma. The final concentration of the organic solvent
was 2%. After the reaction mixture with HDL containing [3H]-
CE was incubated at 37 °C for 4 h, apoprotein B-containing
lipoproteins were precipitated using dextran sulfate and
magnesium chloride (at final concentrations of 0.075% and 37.5
mM, respectively), and 100 µL of the supernatant containing
HDL was used for counting the radioactivity. CETP-mediated
transfer was determined by the decrease in radioactivity of
the supernatant, and the concentration achieving 50% inhibi-
tion of CETP activity was estimated from the dose-response
curves obtained with five doses.
(dd, J ) 1.5 and 8.4 Hz, 1H), 8.35 (br s, 1H). Anal. (C19H29
NOS) C, H, N.
-
2-((1-H e p t ylcycloh e xa n e )ca r b on yla m in o)b e n ze n e -
th iol (24): NMR (300 MHz, CDCl3) δ 0.85 (t, J ) 6.9 Hz, 3H),
1.10-1.70 (m, 20H), 2.05-2.18 (m, 2H), 3.07 (s, 1H), 7.01 (dt,
J ) 1.5 and 7.8 Hz, 1H), 7.31 (ddd, J ) 1.5, 7.8, and 8.4 Hz,
1H), 7.51 (dd, J ) 1.5 and 7.8 Hz, 1H), 8.31 (dd, J ) 1.5 and
8.4 Hz, 1H), 8.37 (br s, 1H); MS (FAB) m/z 334 (M + H)+;
HRMS calcd for C20H32NOS, 334.2204; found, 334.2195.
1-Meth ylth io-2-((1-(2-eth ylbu tyl)cycloh exa n e)ca r bon -
yla m in o)ben zen e (25). Iodomethane (0.12 mL, 1.88 mmol)
was added to a mixture of 23 (100 mg, 1.88 mmol) and
potassium carbonate (260 mg, 1.88 mmol) in dimethylforma-
mide (10 mL). The mixture was stirred at room temperature
for 15 min, after which the solution was poured into water
(20 mL) and extracted with ethyl acetate (2 × 50 mL). The
combined extracts were washed with water (20 mL), dried over
sodium sulfate, and concentrated to give 25 as a colorless oil
(585 mg, 93%): NMR (300 MHz, CDCl3) δ 0.78 (t, J ) 7.2 Hz,
6H), 1.15-1.70 (m, 15H), 2.05-2.25 (m, 2H), 2.38 (s, 3H), 7.04
(dt, J ) 1.5 and 7.8 Hz, 1H), 7.30 (ddd, J ) 1.5, 7.8, and 8.4
Hz, 1H), 7.49 (dd, J ) 1.5 and 7.8 Hz, 1H), 8.42 (dd, J ) 1.5
and 8.4 Hz, 1H), 8.78 (br s, 1H); MS (FAB) m/z 334 (M + H)+;
HRMS calcd for C20H32NOS, 334.2204; found, 334.2206.
S-(2-((1-(2-E t h ylb u t yl)cycloh exa n e)ca r b on yla m in o)-
p h en yl) 2-Meth ylp r op a n eth ioa te (27). Isobutyryl chloride
(904 mg, 8.48 mmol) was added dropwise to a solution of 23
(2.66 g, 8.33 mmol) and pyridine (1.7 mL, 21 mmol) in
chloroform (30 mL). The mixture was stirred at room temper-
ature for 1 h. After evaporation of the solvent, the residue was
chromatographed on silica gel eluted with ethyl acetate/n-
hexane (1:15) to give 27 as a colorless solid (3.15 g, 97%): mp
63-63.5 °C; 1H NMR (300 MHz, CDCl3) δ 0.78 (t, J ) 6.9 Hz,
6H), 1.15-1.75 (m, 15H), 1.30 (d, J ) 6.9 Hz, 6H), 1.95-2.20
(m, 2H), 2.94 (sep, J ) 6.9 Hz, 1H), 7.11 (dt, J ) 1.5 and 7.8
Hz, 1H), 7.38 (dd, J ) 1.5 and 7.8 Hz, 1H), 7.45 (ddd, J ) 1.5,
7.8, and 8.4 Hz, 1H), 8.12 (br s, 1H), 8.40 (dd, J ) 1.5 and 8.4
Hz, 1H). Anal. (C23H35NO2S) C, H, N.
S-(2-((1-Met h ylcycloh exa n e)ca r b on yla m in o)p h en yl)
2-p h en yleth a n eth ioa te (14): NMR (300 MHz, CDCl3) δ 1.07
(s, 3H), 1.21-1.64 (m, 8H), 1.73-1.88 (m, 2H), 3.94 (s, 2H),
7.10 (dt, J ) 1.2 and 7.8 Hz, 1H), 7.30-7.50 (m, 7H), 7.89 (br
s, 1H), 8.35 (dd, J ) 1.2 and 8.4 Hz, 1H); MS (FAB) m/z 368
(M + H)+; HRMS calcd for C22H26NO2S, 368.1684; found,
368.1683. Anal. C, H, N.
S-(2-((1-(2-E t h ylb u t yl)cycloh exa n e)ca r b on yla m in o)-
1
p h en yl) eth a n eth ioa te (26): mp 76.5-79 °C; H NMR (300
MHz, CDCl3) δ 0.79 (t, J ) 6.9 Hz, 6H), 1.15-1.70 (m, 15H),
2.00-2.15 (m, 2H), 2.46 (s, 3H), 7.12 (dt, J ) 1.5 and 7.8 Hz,
1H), 7.40 (dd, J ) 1.5 and 7.8 Hz, 1H), 7.47 (ddd, J ) 1.5, 7.8,

3572 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Shinkai et al.
Monkey Plasma Lipoproteins. J . Clin. Invest. 1991, 87, 1559-
1566.
2. Ex Vivo CETP Activity. Ex vivo CETP activity was
measured in whole plasma obtained from male J W rabbits
(11-15 weeks old, Kitayama Labes Co., Ltd., J apan), which
were orally administered the test compounds (10 or 30 mg/
kg).15 Plasma was isolated by centrifugation at 11000g for 5
min at 4 °C. Whole plasma (1.8 µL) from control or treated
rabbits was diluted to 600 µL with Tris-buffered saline (pH
7.4) containing 0.1 mg/mL of bovine serum albumin and was
incubated at 37 °C for 15 h in the presence of HDL containing
[3H]CE and VLDL/LDL. After precipitation of VLDL/LDL
using dextran sulfate and magnesium chloride, the radioactiv-
ity of the supernatant containing HDL was counted. CETP
activity was determined by the decrease in radioactivity versus
that of the blank sample without plasma.
3. In Vivo P la sm a HDL Ch olester ol Level. HDL choles-
terol levels were measured in the plasma after precipitation
of apoprotein B-containing lipoproteins with 13% poly(ethylene
glycol) (at an average molecular weight of 6000) by an enzyme
assay (Liquitec-TC, Boehringer Mannheim, Germany). Plasma
was obtained from male J W rabbits (11-15 weeks old, N )
5), which were orally administered compound 27 (30 or 100
mg/kg) once a day for 3 days.
(5) Bhatnagar, D.; Durrington, P. N.; Chennon, K. M.; Prais, H.;
Mackness, M. I. Increased Transfer of Cholesteryl Esters from
High-Density Lipoproteins to Low Density and Very Low-
Density Lipoproteins in Patients with Angiographic Evidence
of Coronary Artery Disease. Atherosclerosis 1993, 98, 25-32.
(6) Foger, B.; Luef, G.; Ritsch, A. Relationship of High-Density
Lipoprotein Subfractions and Cholesteryl Ester Transfer Protein
in Plasma to Carotid Artery Wall Thickness. J . Mol. Med. 1995,
73, 369-372.
(7) Bruce, C.; Chouinard, R. A., J r.; Tall, A. R. Plasma Lipid
Transfer Proteins, High-Density Lipoproteins, and Reverse
Cholesterol Transport. Annu. Rev. Nutr. 1998, 18, 297-330.
(8) Fielding, C. J .; Havel, R. J . Cholesteryl Ester Transfer Protein:
Friend or Foe? J . Clin. Invest. 1996, 97, 2687-2688.
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Atherosclerotic Lesions in Hypertriglyceridemic Mice Expressing
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(10) Zhong, S.; Sharp, D. S.; Grove, J . S.; Bruce, C.; Yano, K.; Curb,
J . D.; Tall, A. R. Increased Coronary Heart Disease in J apanese-
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Protein Gene Despite Increased HDL levels. J . Clin. Invest.
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(11) Fielding, C. J .; Fielding, P. E. Molecular Physiology of Reverse
Cholesterol Transport. J . Lipid Res. 1995, 36, 211-228.
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Biochem. Biophys. Res. Commun. 1996, 223, 42-47.
(13) Morton, R. E.; Zilversmit, D. B. Inter-Relationship of Lipid
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(14) Okamoto, H.; Yonemori, F,; Wakitani, K,; Minowa, T,; Maeda,
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Ack n ow led gm en t. We gratefully acknowledge Drs.
Masami Tsuboshima, Yasuo Urata, and Atsushi Mi-
zushima for their encouragement and suggestions.
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