Synthesis and biological evaluation of quinuclidine derivatives incorporating phenothiazine moieties as squalene synthase inhibitors

Article abstract of DOI:10.1248/cpb.52.1204

Squalene synthase inhibitors have the potential to be superior hypocholesterolemic agents. A series of quinuclidine derivatives incorporating phenothiazine systems was synthesized in order to investigate the effects of their structure on the inhibition of hamster liver microsomal enzyme. (±)-3-(10-Methyl-10H-phenothiazin-3-ylmethoxy) quinuclidine hydrochloride (19) was the most potent inhibitor in this series with an IC₅₀ value of 0.12 μM. Oral dosing of compound 19 to hamsters demonstrated effective reduction of both plasma total cholesterol levels and plasma triglyceride levels. Compound 19 showed a reduced tendency to elevate plasma transaminase levels, an indicator of hepatotoxicity. Enantiomerically pure (-)-19, YM-53546, was found to be more potent than the corresponding (+)-enantiomer.

Full text of DOI:10.1248/cpb.52.1204

1204
Chem. Pharm. Bull. 52(10) 1204—1209 (2004)
Vol. 52, No. 10
Synthesis and Biological Evaluation of Quinuclidine Derivatives
Incorporating Phenothiazine Moieties as Squalene Synthase Inhibitors
Tsukasa ISHIHARA,* Hirotoshi KAKUTA, Hiroshi MORITANI, Tohru UGAWA, and Isao YANAGISAWA
Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co. Ltd.; 21 Miyukigaoka, Tsukuba, Ibaraki
305–8585, Japan. Received May 25, 2004; accepted July 28, 2004
Squalene synthase inhibitors have the potential to be superior hypocholesterolemic agents. A series of quin-
uclidine derivatives incorporating phenothiazine systems was synthesized in order to investigate the effects of
their structure on the inhibition of hamster liver microsomal enzyme. (ꢀ)-3-(10-Methyl-10H-phenothiazin-3-yl-
methoxy)quinuclidine hydrochloride (19) was the most potent inhibitor in this series with an IC50 value of
0.12 mM. Oral dosing of compound 19 to hamsters demonstrated effective reduction of both plasma total choles-
terol levels and plasma triglyceride levels. Compound 19 showed a reduced tendency to elevate plasma transami-
nase levels, an indicator of hepatotoxicity. Enantiomerically pure (ꢁ)-19, YM-53546, was found to be more
potent than the corresponding (ꢂ)-enantiomer.
Key words squalene synthase; hypocholesterolemic agent; quinuclidine; hepatotoxicity
In spite of major advances in pharmaceutical and surgical
In our previous paper, we disclosed that phenothiazine
treatments, coronary heart disease remains a major cause of derivative 2 was equipotent to 3-(4ꢀ-fluorobiphenyl-4-
death in the industrialized world.1,2) Elevated plasma choles- yl)quinuclidin-3-ol 1, which had been a potent inhibitor of
terol is widely accepted as a risk factor for the disease,3,4) and squalene synthase reported by Brown et al. (Fig. 1),14) in the
thus there have been worldwide research efforts to discover hamster microsomal enzyme assay (IC50 values of 0.38 and
antihypercholesterolemic agents. As over 70% of cholesterol 0.39 mM for compounds 1 and 2, respectively).15) Efforts were
in the body is derived from de novo cholesterol biosynthesis, thus focused on the further modifications of compound 2.
inhibition of this biosynthesis presents an effective approach This paper describes the results of our work on the synthesis,
to reduce plasma cholesterol. In particular, 3-hydroxy-3- structure–activity relationships, and biological activities of
methylglutaryl coenzyme A (HMG-CoA) reductase inhibi- the quinuclidine derivatives containing phenothiazine sys-
tors, the inhibitors of the cholesterol biosynthetic pathway, tems as novel squalene synthase inhibitors.
are currently the most effective therapeutic agents in reduc-
ing the levels of plasma cholesterol.5—7) Treatment of hyper- Chemistry
cholesterolemia with HMG-CoA reductase inhibitors is
reported to reduce the risk of the coronary heart disease by 19—24 is shown in Chart 1. 3-Formyl-10-methyl-10H-pheno
approximately one-third.8)
thiazine (9) was synthesized via the method stated in the lit-
The synthetic pathway for the preparation of compounds
-
However, HMG-CoA reductase inhibitors may also erature,16) and the related derivatives 10—12 were prepared
prevent the formation of biologically important isoprenoids by identical procedures from the known N-alkylated pheno-
such as dolicols, ubiquinones, and isopentenyl t-RNA, thiazines.17) These aldehydes 9—12 were in turn converted
because the inhibitors suppress the cholesterol biosynthetic into the corresponding alcohols 13—16 through reduction
pathway early on. Moreover, HMG-CoA reductase inhibitors with sodium borohydride. Phenothiazine 5-oxide derivative
have little effect on plasma triglyceride levels.9) A study 17 and phenothiazine 5,5-dioxide derivative 18 were pre-
group from the European Atherosclerosis Society has pared by oxidation of the phenothiazine derivative 13 using
proposed that hypertriglyceridemia is another risk factor for 1.4 and 3.0 eq of m-chloroperbenzoic acid, respectively. Cou-
coronary heart disease.10) An agent that could reduce both pling of the phenothiazine moiety with 3-quinuclidinol was
plasma cholesterol levels and plasma triglyceride levels with- achieved through the known method of protecting the quinu-
out interruption of isoprenoid biosyntheses would therefore clidine ring nitrogen atom as a borane complex.18) 3-Quinu-
be highly desirable.
clidinol-N-borane (4) was allowed to react with chlorides,
Squalene synthase (EC 2.5.1.21), which catalyzes the which were prepared from the alcohols 13—18, in the pres-
reductive dimerization of two molecules of farnesyl diphos- ence of sodium hydride. Deprotection of the borane com-
phate to form squalene via intermediate presqualene diphos- plexes with ethanolic hydrogen chloride solution afforded
phate, is involved in the first committed step in cholesterol target compounds 19—24. Enantiomerically pure (ꢁ)- and
biosynthesis. Inhibition of squalene synthase does not inter- (ꢂ)-19 were obtained from the known (ꢁ)- and (ꢂ)-3-quinu-
fere with the biosyntheses of the above-mentioned isopren-
oids, because the enzymatic step occurs after the branches
leading to the isoprenoids. Furthermore, squalene synthase
inhibitors have been reported to lower plasma triglyceride
levels as well as plasma cholesterol levels in vivo.11—13) Con-
sequently, squalene synthase is an attractive therapeutic
target for the treatment of not only hyperlipidemia but
coronary heart disease.
Fig. 1
* To whom correspondence should be addressed. e-mail: ishihat@yamanouchi.co.jp
© 2004 Pharmaceutical Society of Japan

October 2004
1205
Table 1. In Vitro Activities of Compounds 19—24
Compd.
R
n
IC50 (mM)a)
2
19
20
21
22
23
24
1
0.39
0.12
ꢅ1
ꢅ1
0.44
0.56
0.37
0.38
Me
Me
Me
Et
n-Bu
i-Pr
0
1
2
0
0
0
a) Compounds were tested for their ability to inhibit the conversion of [3H]farnesyl
diphosphate to [3H]squalene by squalene synthase derived from hamster liver. IC50 val-
ues were determined by a single experimental run in duplicate.
Reagents: (a) BH3–THF complex, THF; (b) POCl3, N-methylformanilide, 1,2-
dichlorobenzene; (c) NaBH4, MeOH; (d) 1.4 eq m-CPBA, CHCl3; (e) 3.0 eq m-CPBA,
CH2Cl2; (f) MeSO2Cl, Et3N, CH2Cl2; (g) SOCl2, cat. DMF, CHCl3; (h) NaH, 4, DMF;
(i) HCl, EtOH, acetone.
Chart 1. Preparation of Compounds 19—24
clidinol,19) respectively, in the same manner as the racemic
mixture of compound 19.
Results and Discussion
All compounds were evaluated by their IC50 values for the
inhibition of squalene synthase prepared from hamster liver.
The inhibitory activities were measured according to the
method of Amin et al. with a slight modification.20) The
selected compounds were evaluated for lipid lowering effects
Fig. 2. Effects of Compounds 1 and 19 on Plasma Total Cholesterol Lev-
els after Oral Administration in Hamsters at a Dose of 50 mg/kg/d for 5 d
(nꢃ7 and 6, Respectively)
*** pꢄ0.001 versus control using the Student’s t-test.
after oral dosing in hamsters, and their inhibitory activity azine 5-oxide, and 10-methyl-10H-phenothiazine 5,5-dioxide
against squalene synthase derived from human hepatoma were 4.49, 1.50, and 1.99, respectively,22) suggesting that a
(HepG2) cells was also investigated.
lipophilic ring system is beneficial for interacting with the
The introduction of an ether linkage into the molecule was enzyme. We therefore explored an incorporation of a bulky
known to improve its inhibitory activity against squalene substituent into the phenothiazine system in order to increase
synthase for phosphonate-based squalene synthase inhib- its lipophilicity. Rather unexpectedly, 10-ethyl-10H-phenothi-
itors.21) Also, we had previously reported that a hydroxyl azine derivative 22, 10-butyl-10H-phenothiazine derivative
group at the 3-position of the quinuclidine nucleus was 23, and 10-(1-methyl)ethyl-10H-phenothiazine derivative 24
unnecessary for the ability to inhibit the enzyme.15) These were less potent inhibitors compared to 10-methyl-10H-phe-
two facts were the impetus for our work on an O-alkylated nothiazine derivative 19.23) These results demonstrated that
3-quinuclidinol derivative that had an ether linker between bulky substituents on the phenothiazine system were undesir-
the quinuclidine moiety and the tricyclic system without a able for the inhibition of squalene synthase. The most potent
hydroxy group on the quinuclidine nucleus. Our earlier study inhibitor in this series was compound 19.
also demonstrated that the 10-methyl-10H-phenothiazine de-
Because of the potent inhibitory activity against squalene
rivative 2 was a potent inhibitor of squalene synthase,15) synthase, compound 19 was evaluated for its lipid lowering
which showed that a 10-methyl-10H-phenothiazine system is effects; the results are shown in Figs. 2 and 3. Hamsters have
an attractive moiety for a squalene synthase inhibitor. Using plasma lipid compositions very similar to humans,24) and
a combination of the results described above, we designed therefore the animals are suitable for assessments of the
3-(10-methyl-10H-phenothiazin-3-ylmethoxy)quinuclidine potential efficacy of lipid-lowering agents. Compound 19
(19). Compound 19 exhibited a 3-fold enhancement in lowered plasma total cholesterol levels by 46% compared to
inhibitory activity over compound 2, as expected (Table 1).
the control at an oral dose of 50 mg/kg/d for 5 d in hamsters
The improved inhibitory activity observed with compound (Fig. 2). Plasma triglyceride levels were also reduced upon
19 led us to examine the sensitivity of the structure–activity oral dosing of compound 19; a 64% decrease in plasma
relationships to structural variations of the phenothiazine triglyceride levels was observed (Fig. 3). These results indi-
moiety. The results are summarized in Table 1. Phenothiazine cated that compound 19 was a potent and orally active squa-
5-oxide derivative 20 and phenothiazine 5,5-dioxide deriva- lene synthase inhibitor.
tive 21 sustained at least 8-fold losses in inhibitory activity
Further investigation of compound 19 revealed an impor-
relative to the phenothiazine derivative 19. The c-logP values tant improvement relative to compound 1. Compound 1
of 10-methyl-10H-phenothiazine, 10-methyl-10H-phenothi- caused an approximately 3-fold elevation in plasma transami-

1206
Vol. 52, No. 10
is important for activity; replacement of the phenothiazine
nucleus with phenothiazine 5-oxide or phenothiazine 5,5-
dioxide caused a reduction in in vitro potency, and a smaller
substituent at the 10-position of the phenothiazine system
was preferred for binding to the enzyme. The most potent
inhibitor was 10-methyl-10H-phenothiazine derivative 19
with an IC50 value of 0.12 mM in this series. Compound 19
reduced plasma total cholesterol levels and plasma triglyce-
ride levels by 46% and 64%, respectively, compared to the
control, at an oral dose of 50 mg/kg/d for 5 d in hamsters.
This compound did not affect plasma transaminase levels
despite its good lipid-lowering effects. The (ꢂ)-enantiomer
of compound 19, (ꢂ)-3-(10-methyl-10H-phenothiazin-3-
ylmethoxy)quinuclidine hydrochloride (YM-53546), was
found to be more potent than the corresponding (ꢁ)-enan-
tiomer.
Fig. 3. Effects of Compounds 1 and 19 on Plasma Triglyceride Levels
after Oral Administration in Hamsters at a Dose of 50 mg/kg/d for 5 d (nꢃ7
and 6, Respectively)
* pꢄ0.05 versus control using the Student’s t-test.
Table 2. Effects of Compounds 1 and 19 on Biochemical Parameters in
F344 Ratsa)
We propose that YM-53546 will be a useful lipid-lowering
agent with the potential for the treatment of coronary heart
disease.
Compd.
AST (IU/l)b)
ALT (IU/l)c)
Vehicle
19
1
106
104
319*
38
39
95
Experimental
1H-NMR spectra were measured with a JEOL EX90, LA300, or GX500
spectrometer. Chemical shifts are expressed in d units using tetramethylsi-
lane as the standard (in NMR description, sꢃsinglet, dꢃdoublet, tꢃtriplet,
mꢃmultiplet, and brꢃbroad peak). Mass spectra were recorded with a
Hitachi M-80 or JEOL JMS-DX300 spectrometer. Melting points were mea-
sured with a Yanaco MP-500D melting point apparatus without correction.
The optical purity of the optically active compounds were examined using
on analytical chiral column (Daicel Chemical Industries. Ltd. CHIRALCEL
OJ-H, i.d.ꢃ0.46 cm, lꢃ25 cm). The HPLC conditions were as follows:
mobile phase, EtOH/n-hexane/Et2NHꢃ500/500/1; flow rate, 0.5 ml/min;
detection wavelength, 254 nm. Optical rotation measurements were obtained
using a Horiba SEPA-200 polarimeter. All materials and reagents purchased
were used without further purification.
a) Animals were dosed via oral gavage at 250 mg/kg/d for 3 d. Data are mean values,
and standard errors were less than 30% of the mean (nꢃ3). * pꢄ0.05 versus control
using the Student’s t-test. b) Aspartate aminotransferase. c) Alanine aminotrans-
ferase.
Table 3. In Vitro Activities of Enantiomers of Compound 19
IC50 (mM)
Compd.
Hamstera)
Humanb)
(ꢁ)-19
(ꢂ)-19
0.20
0.081
0.17
0.10
(ꢀ)-3-Quinuclidinol-N-borane (4) A solution of borane–tetrahydrofu-
ran complex in tetrahydrofuran (710 ml, 1.0 M, 710 mmol) was added drop-
wise to (ꢆ)-3-quinuclidinol (3) (86.4 g, 680 mmol) in tetrahydrofuran
(400 ml) at 0 °C. The reaction mixture was allowed to warm to ambient tem-
perature and stirred for 1 h. The solvent was removed in vacuo and the
resulting residue was diluted with chloroform. The organic layer was washed
with H2O and then brine, dried over magnesium sulfate, and then concen-
trated in vacuo. The residue was dissolved in diethyl ether (100 ml). To this
solution n-hexane (400 ml) was added, and the resulting precipitate was
filtered to yield the title compound as a colorless solid (60.9 g, 64%).
1H-NMR (300 MHz, CDCl3) d: 1.55—1.72 (2H, m), 1.81—1.91 (1H, m),
2.02—2.21 (2H, m), 2.81—3.14 (5H, m), 3.21—3.29 (1H, m), 4.06—4.12
(1H, m). EI-MS m/z: 141 [M]ꢁ.
a) Refer to Table 1. b) Compounds were tested for their ability to inhibit the con-
version of [3H]farnesyl diphosphate to [3H]squalene by squalene synthase from human
hepatoma cells. IC50 values were determined by a single experimental run in duplicate.
nase levels (AST, ALT) after administration in rats, whereas
compound 19 did not show pronounced effects on plasma
transaminase levels. Compound 19 did exhibit a reduced
acute effect on plasma transaminase levels, an indicator of
hepatotoxicity.
3-Formyl-10-methyl-10H-phenothiazine (9) A mixture of 10-methyl-
10H-phenothiazine (5) (9.61 g, 45.1 mmol), N-methylformanilide (7.03 g,
52.0 mmol), phosphoryl chloride (7.05 g, 46.0 mmol), and 1,2-dichloroben-
zene (10 ml) was stirred at 90 °C for 4 h. After cooling, a solution of sodium
acetate (36 g) in H2O (80 ml) was added dropwise to the reaction mixture.
The mixture was extracted with ethyl acetate, and the extract was dried over
magnesium sulfate and then concentrated in vacuo. The residue was
subjected to chromatography over silica gel eluting with n-hexane–ethyl
acetate (3 : 1 by volume) to give the title compound as a yellow solid (9.19 g,
Throughout this work, compound 19 was prepared and
analyzed as a racemic mixture. It can be assumed that the
biological activity of the pure enantiomer could potentially
be twice that of the racemic mixture. Thus we performed
asymmetric syntheses of both enantiomers of compound 19
and evaluated their inhibitory activities. The (ꢂ)-enantiomer
of 19 was found to be approximately 2-fold more potent than
the corresponding (ꢁ)-enantiomer of 19. The IC50 values
obtained for the (ꢂ)-enantiomer of 19 against hamster and
human HepG2 enzymes were 0.081 and 0.10 mM, respec-
tively (Table 3).
1
84%). H-NMR (90 MHz, CDCl3) d: 3.41 (3H, s), 6.83 (1H, d, Jꢃ5.9 Hz),
6.93—7.29 (4H, m), 7.58—7.70 (2H, m), 9.79 (1H, s). EI-MS m/z: 241
[M]ꢁ.
3-Hydroxymethyl-10-methyl-10H-phenothiazine (13) To a stirred so-
lution of 3-formyl-10-methyl-10H-phenothiazine (9) (908 mg, 3.76 mmol) in
methanol (38 ml) was added sodium borohydride (285 mg, 7.52 mmol) at
0 °C. The reaction mixture was allowed to warm to ambient temperature and
stirred for 1 h. The mixture was concentrated in vacuo, and the residue was
diluted with ethyl acetate. The organic layer was washed with H2O and then
brine, dried over magnesium sulfate, and concentrated in vacuo to yield the
title compound as a colorless solid (830 mg, 91%). 1H-NMR (90 MHz,
CDCl3) d: 3.37 (3H, s), 4.57 (2H, d, Jꢃ5.9 Hz), 6.72—7.00 (3H, m), 7.09—
7.25 (4H, m). EI-MS m/z: 243 [M]ꢁ.
Conclusions
A novel series of quinuclidine derivatives containing phe-
nothiazine moieties was synthesized and evaluated for their
ability to inhibit squalene synthase. The structure–activity
relationships of the prepared compounds provided useful
information on the structural requirements for the inhibition
of the enzyme. The steric factor of the phenothiazine system
(ꢀ)-3-(10-Methyl-10H-phenothiazin-3-ylmethoxy)quinuclidine
Hy-

October 2004
1207
[MꢁH]ꢁ.
drochloride (19) To a stirred solution of 3-hydroxymethyl-10-methyl-
10H-phenothiazine (13) (11.7 g, 48.1 mmol) and triethylamine (8.71 ml,
62.5 mmol) in dichloromethane (80 ml) was added methanesulfonyl chloride
(4.10 ml, 53.0 mmol) at 0 °C. The reaction mixture was allowed to warm to
ambient temperature and stirred for 1 h. After the addition of H2O (4.0 ml),
the reaction mixture was extracted with chloroform. The organic layer was
washed with saturated aqueous sodium bicarbonate and then brine, dried
over magnesium sulfate, and then concentrated in vacuo to yield 3-chloro-
To a stirred solution of (ꢆ)-3-quinuclidinol-N-borane (4) (917 mg,
6.50 mmol) in N,N-dimethylformamide (13 ml) was added sodium hydride
(310 mg, 6.50 mmol, 60% dispersion in mineral oil) at 0 °C. The reaction
mixture was allowed to warm to ambient temperature and stirred for 1 h. To
this mixture 3-chloromethyl-10-methyl-10H-phenothiazine 5-oxide (1.80 g,
6.48 mmol) in N,N-dimethylformamide (10 ml) was added at 0 °C. The reac-
tion mixture was stirred for 0.5 h, allowed to warm to ambient temperature,
and stirred for a further 3 h. The mixture was concentrated in vacuo, and the
resulting residue was diluted with chloroform. The organic layer was washed
with H2O and then brine, dried over magnesium sulfate, and then concen-
trated in vacuo. The residue was subjected to chromatography over silica gel
eluting with chloroform–methanol (100 : 1 by volume) to yield a colorless
solid. The resulting solid in acetone (5.0 ml) was treated with hydrogen chlo-
ride in ethanol (5 M, 8.0 ml) at 0 °C. The reaction mixture was stirred for
0.5 h, and then allowed to warm to ambient temperature, and stirred for a
further 0.5 h. The mixture was concentrated in vacuo. The residue was di-
luted with chloroform and the organic layer was washed with 10% aqueous
potassium carbonate. The organic layer was dried over magnesium sulfate
and concentrated in vacuo. The residue was subjected to chromatography
over silica gel eluting with chloroform–methanol–c. ammonium hydroxide
(90 : 10 : 1 by volume) to yield a colorless solid. The resulting solid was
recrystallized from chloroform–ethyl acetate–diethyl ether to yield the title
compound as a colorless crystalline solid (920 mg, 48%). mp 164—166 °C.
1H-NMR (500 MHz, CDCl3) d: 1.32—1.46 (2H, m), 1.82—1.86 (1H, m),
1.90—1.96 (1H, m), 2.06—2.12 (1H, m), 2.66—2.74 (1H, m), 2.76—2.86
(3H, m), 2.92—3.00 (1H, m), 3.10—3.16 (1H, m), 3.46—3.60 (1H, m), 3.77
(3H, s), 4.49—4.54 (1H, m), 4.58—4.63 (1H, m), 7.24—7.26 (1H, m),
7.36—7.39 (2H, m), 7.59—7.64 (2H, m), 7.90—7.94 (2H, m). FAB-MS
m/z: 369 [MꢁH]ꢁ. Anal. Calcd for C21H24N2O2S·0.2H2O: C, 67.79; H,
6.61; N, 7.53; S, 8.62. Found: C, 67.78; H, 6.47; N, 7.40; S, 8.85.
1
methyl-10-methyl-10H-phenothiazine as a yellow solid (7.98 g, 63%). H-
NMR (500 MHz, CDCl3) d: 3.36 (3H, s), 4.47 (2H, s), 6.71 (1H, d,
Jꢃ5.9 Hz), 6.77 (1H, d, Jꢃ5.9 Hz), 6.91—6.94 (1H, m), 7.10—7.20 (4H,
m). FAB-MS m/z: 262 [MꢁH]ꢁ.
To a stirred solution of (ꢆ)-3-quinuclidinol-N-borane (4) (3.38 g, 24.0
mmol) in N,N-dimethylformamide (35 ml) was added sodium hydride
(1.16 g, 29.0 mmol, 60% dispersion in mineral oil) at 0 °C. The reaction
mixture was stirred for 1 h at 0 °C. To this mixture 3-chloromethyl-10-
methyl-10H-phenothiazine (7.98 g, 30.5 mmol) in N,N-dimethylformamide
(30 ml) was added at 0 °C. The reaction mixture was stirred for 0.5 h, and
then allowed to warm to ambient temperature, and stirred for a further 1 h.
After the addition of H2O (200 ml), the reaction mixture was extracted with
ethyl acetate. The organic layer was washed with H2O and then brine, dried
over magnesium sulfate, and then concentrated in vacuo. The residue was
subjected to chromatography over silica gel eluting with n-hexane–
dichloromethane–ethyl acetate (70 : 15 : 15 by volume) to yield a yellow
solid (5.09 g). The resulting solid (5.09 g) in acetone (20 ml) was treated
with hydrogen chloride in ethanol (5 M, 10 ml) at 0 °C. The reaction mixture
was stirred for 5 min, and then allowed to warm to ambient temperature, and
stirred for a further 15 min. The mixture was diluted with diethyl ether
(40 ml). The resulting precipitate was filtered, and washed with ethyl acetate
and then diethyl ether to yield the title compound as a light green solid
1
(4.48 g, 83%). mp 220—222 °C. H-NMR (500 MHz, DMSO-d6) d: 1.65—
1.70 (2H, m), 1.81—1.88 (1H, m), 1.91—2.01 (1H, m), 2.28—2.32 (1H, m),
2.95—3.15 (5H, m), 3.31 (3H, s), 3.45—3.49 (1H, m), 3.85—3.88 (1H, m),
4.39 (1H, d, Jꢃ11.5 Hz), 4.44 (1H, d, Jꢃ11.5 Hz), 6.90—7.00 (3H, m),
7.15—7.25 (4H, m) 10.44 (1H, br s). EI-MS m/z: 352 [M]ꢁ. Anal. Calcd for
C21H24N2OS·HCl: C, 64.85; H, 6.48; N, 7.20; S, 8.24; Cl, 9.11. Found: C,
64.57; H, 6.43; N, 7.06; S, 8.22; Cl, 9.21.
3-Hydroxymethyl-10-methyl-10H-phenothiazine 5,5-Dioxide (18) m-
Chloroperbenzoic acid (2.59 g, 15.0 mmol) was added to a stirred suspen-
sion of 3-hydroxymethyl-10-methyl-10H-phenothiazine (13) (1.22 g,
5.00 mmol) in dichloromethane (15 ml) at 0 °C. The mixture was allowed to
warm to ambient temperature and stirred for 18 h. The mixture was diluted
with ethyl acetate and washed with saturated aqueous sodium bicarbonate.
The organic layer was washed with brine, dried over magnesium sulfate, and
concentrated in vacuo. The residue was subjected to chromatography over
silica gel eluting with ethyl acetate to yield the title compound as a yellow
Identical procedures provided the following two compounds:
(ꢁ)-3-(10-Methyl-10H-phenothiazin-3-ylmethoxy)quinuclidine Hydro-
chloride ((ꢁ)-19) The title compound was prepared from (ꢂ)-3-quinu-
clidinol19) as a colorless solid (tRꢃ21.8 min, ꢅ99.5% ee). mp 220—221 °C.
[a]D25ꢃꢂ29.2° (cꢃ0.50, DMSO). The 1H-NMR and mass spectra of the title
compound were identical to those observed for (ꢆ)-19. Anal. Calcd for
C21H24N2OS·HCl: C, 64.85; H, 6.48; N, 7.20; S, 8.24; Cl, 9.11. Found: C,
64.51; H, 6.51; N, 6.93; S, 8.23; Cl, 9.16.
(ꢂ)-3-(10-Methyl-10H-phenothiazin-3-ylmethoxy)quinuclidine
Hydrochloride ((ꢂ)-19) The title compound was prepared from (ꢁ)-3-
quinuclidinol19) as a colorless solid (tRꢃ26.4 min, ꢅ99.5% ee). mp 215—
217 °C. [a]D25ꢃꢁ28.6° (cꢃ0.50, DMSO). The 1H-NMR and mass spectra of
the title compound were identical to those observed for (ꢆ)-19. Anal. Calcd
for C21H24N2OS·HCl: C, 64.85; H, 6.48; N, 7.20; S, 8.24; Cl, 9.11. Found:
C, 64.45; H, 6.47; N, 6.95; S, 8.23; Cl, 9.44.
1
solid (1.23 g, 89%). H-NMR (500 MHz, CDCl3) d: 3.70 (3H, s), 4.75 (2H,
s), 7.27—7.31 (3H, m), 7.61—7.66 (2H, m), 8.07 (1H, s), 8.11 (1H, d,
Jꢃ8.0 Hz). FAB-MS m/z: 276 [MꢁH]ꢁ.
(ꢀ)-10-Methyl-3-[(3-quinuclidinyloxy)methyl]-10H-phenothiazine 5,5-
Dioxide Hydrochloride (21) The title compound was prepared in a proce-
dure similar to that described for the synthesis of compound 19 from 3-hy-
droxymethyl-10-methyl-10H-phenothiazine 5,5-dioxide (18) as a colorless
1
solid (62%). mp 274—276 °C. H-NMR (500 MHz, DMSO-d6) d: 1.64—
1.74 (2H, m), 1.86—1.94 (1H, m), 1.98—2.06 (1H, m), 2.36—2.40 (1H, m),
3.04—3.22 (5H, m), 3.50—3.54 (1H, m), 3.74 (3H, s), 3.93—3.96 (1H, m),
4.61 (1H, d, Jꢃ11.5 Hz), 4.65 (1H, d, Jꢃ11.5 Hz), 7.35—7.39 (1H, m),
7.61—7.63 (2H, m), 7.74—7.79 (2H, m), 7.98—8.00 (2H, m), 10.34 (1H,
br s). FAB-MS m/z: 385 [MꢁH]ꢁ. Anal. Calcd for C21H24N2O3S·HCl·
0.4H2O: C, 58.91; H, 6.07; N, 6.54; S, 7.49; Cl, 8.28. Found: C, 58.93; H,
5.97; N, 6.70; S, 7.31; Cl, 8.42.
3-Hydroxymethyl-10-methyl-10H-phenothiazine 5-Oxide (17) m-Chl-
o
roperbenzoic acid (1.66 g, 9.60 mmol) was added to a stirred suspension of
3-hydroxymethyl-10-methyl-10H-phenothiazine (13) (1.94 g, 8.00 mmol) in
chloroform (30 ml) at 0 °C. The mixture was allowed to warm to ambient
temperature and stirred for 1 h. Additional m-chloroperbenzoic acid
(280 mg, 1.60 mmol) was added at ambient temperature, and the reaction
mixture was stirred for 0.5 h. The mixture was washed with saturated aque-
ous sodium bicarbonate, and the organic layer was dried over magnesium
sulfate, and concentrated in vacuo. The residue was subjected to chromatog-
raphy over silica gel eluting with chloroform–methanol (100 : 3 by volume)
to yield the title compound as a colorless solid (1.95 g, 94%). 1H-NMR
(500 MHz, CDCl3) d: 2.33 (1H, t, Jꢃ5.5 Hz), 3.76 (3H, s), 4.71 (2H, d,
Jꢃ5.5 Hz), 7.24—7.27 (1H, m), 7.35—7.39 (2H, m), 7.60—7.64 (2H, m),
7.89—7.93 (2H, m). FAB-MS m/z: 260 [MꢁH]ꢁ.
(ꢀ)-10-Methyl-3-[(3-quinuclidinyloxy)methyl]-10H-phenothiazine 5-
Oxide (20) In a similar procedure to the one described above, 3-
chloromethyl-10-methyl-10H-phenothiazine 5-oxide was prepared from 3-
hydroxymethyl-10-methyl-10H-phenothiazine 5-oxide (17) as a colorless
solid (87%). 1H-NMR (500 MHz, CDCl3) d: 3.77 (3H, s), 4.66 (1H, d,
Jꢃ12.0 Hz), 4.68 (1H, d, Jꢃ12.0 Hz), 7.28 (1H, d, Jꢃ8.0 Hz), 7.37—7.40
(2H, m), 7.61—7.65 (2H, m), 7.92—7.95 (2H, m). FAB-MS m/z: 278
The following compounds were prepared in a similar manner described
above:
10-Ethyl-3-formyl-10H-phenothiazine (10) The title compound was
obtained from 10-ethyl-10H-phenothiazine (6)17) as a yellow solid (69%).
1H-NMR (90 MHz, CDCl3) d: 1.44 (3H, t, Jꢃ7.1 Hz), 3.96 (2H, q,
Jꢃ7.1 Hz), 6.84—7.25 (5H, m), 7.56—7.68 (2H, m), 9.78 (1H, s). EI-MS
m/z: 255 [M]ꢁ.
10-Ethyl-3-hydroxymethyl-10H-phenothiazine (14) The title com-
pound was obtained from 10-ethyl-3-formyl-10H-phenothiazine (10) as a
1
yellow oil (quant.). H-NMR (90 MHz, CDCl3) d: 1.40 (3H, t, Jꢃ7.0 Hz),
3.91 (2H, t, Jꢃ7.0 Hz), 4.55 (2H, d, Jꢃ4.1 Hz), 6.76—6.96 (3H, m), 7.06—
7.16 (4H, m). EI-MS m/z: 257 [M]ꢁ.
(ꢀ)-3-(10-Ethyl-10H-phenothiazin-3-ylmethoxy)quinuclidine
(22)
The title compound was prepared from 10-ethyl-3-hydroxymethyl-10H-phe-
nothiazine (14) as a yellow oil (53%). 1H-NMR (500 MHz, CDCl3)
d: 1.36—1.44 (5H, m), 1.66—1.72 (1H, m), 1.89—2.06 (2H, m), 2.66—
2.82 (4H, m), 2.91—2.96 (1H, m), 3.06—3.11 (1H, m), 3.53—3.55 (1H, m),
3.92 (2H, q, Jꢃ7.0 Hz), 4.32 (1H, d, Jꢃ11.5 Hz), 4.42 (1H, d, Jꢃ11.5 Hz),

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Vol. 52, No. 10
6.82—6.91 (3H, m), 7.10—7.15 (4H, m). EI-MS m/z: 366 [M]ꢁ. Anal.
Calcd for C24H26N2OS·0.3H2O: C, 71.05; H, 7.21; N, 7.53; S, 8.62. Found:
C, 70.99; H, 7.26; N, 7.40; S, 8.86.
0.5% methylcellulose vehicle solution. Blood specimens were obtained 2 h
after the last dose from animals that had been fasted for 18 h. All plasma
samples were analyzed for total cholesterol and triglyceride using a Hitachi
7250 Automatic Analyzer (Tokyo, Japan).
10-Butyl-3-formyl-10H-phenothiazine (11) The title compound was
1
obtained from 10-butyl-10H-phenothiazine (7)17) as a yellow oil (68%). H-
Acute Toxic Study in Rats Five-week-old male F344 rats (from SLC,
Shizuoka, Japan) were fed CE-2 diet (CLEA Japan, Inc., Tokyo, Japan) and
water ad libitum. Rats were given test compounds orally at a dose of
250 mg/kg of body weight once a day for 3 d. The test compound was sus-
pended in a 0.5% methylcellulose vehicle solution. The no-treatment control
group was given an equal volume of the 0.5% methylcellulose vehicle solu-
tion. In all experiments, blood specimens were obtained from the animals
24 h after the last dose. All plasma samples were analyzed for aspartate
aminotransferase and alanine aminotransferase using a Hitachi 7250 Auto-
matic Analyzer (Tokyo, Japan).
NMR (90 MHz, CDCl3) d: 0.95 (3H, t, Jꢃ6.7 Hz), 1.26—1.89 (4H, m), 3.89
(2H, t, Jꢃ7.3 Hz), 6.84—7.60 (7H, m), 9.78 (1H, s). EI-MS m/z: 283 [M]ꢁ.
10-Butyl-3-hydroxymethyl-10H-phenothiazine (15) The title compo-
und was obtained from 10-butyl-3-formyl-10H-phenothiazine (11) as a yel-
low oil (quant.). 1H-NMR (90 MHz, CDCl3) d: 0.93 (3H, t, Jꢃ6.7 Hz),
1.20—1.87 (4H, m), 3.84 (2H, t, Jꢃ7.1 Hz), 4.56 (2H, s), 6.57—7.34 (7H,
m). EI-MS m/z: 285 [M]ꢁ.
(ꢀ)-3-(10-Butyl-10H-phenothiazin-3-ylmethoxy)quinuclidine (23) The
title compound was prepared from 10-butyl-3-hydroxymethyl-10H-phenothi-
azine (11) as a yellow oil (18%). 1H-NMR (500 MHz, CDCl3) d: 0.93 (3H, t,
Jꢃ7.3 Hz), 1.37—1.49 (4H, m), 1.67—1.81 (3H, m), 1.90—1.98 (1H, m),
2.06—2.08 (1H, m), 2.70—2.83 (4H, m), 2.92—2.95 (1H, m), 3.08—3.12
(1H, m), 3.54—3.56 (1H, m), 3.84 (2H, t, Jꢃ7.3 Hz), 4.33 (1H, d,
Jꢃ11.5 Hz), 4.42 (1H, d, Jꢃ11.5 Hz), 6.81—6.91 (3H, m), 7.10—7.15 (4H,
m). EI-MS m/z: 394 [M]ꢁ. Anal. Calcd for C24H30N2OS·1.5H2O: C, 68.37;
H, 7.89; N, 6.64; S, 7.61. Found: C, 68.61; H, 7.77; N, 6.62; S, 7.21.
3-Formyl-10-(1-methylethyl)-10H-phenothiazine (12) The title com-
pound was obtained from 10-(1-methylethyl)-10H-phenothiazine (8)17) as a
yellow oil (29%). 1H-NMR (90 MHz, CDCl3) d: 1.68 (6H, d, Jꢃ6.9 Hz),
4.22—4.54 (1H, m), 6.57—7.66 (7H, m), 9.79 (1H, s). EI-MS m/z: 269
[M]ꢁ.
Acknowledgments The authors wish to acknowledge and thank Drs.
Koyo Matsuda and Masahiko Isaka for their important input on the study.
We would like to express our gratitude to Drs. Minoru Okada and Fukushi
Hirayama for helpful support in the preparation of this manuscript, and we
are also grateful to the staff of the Division of Analytical Science Laborato-
ries for the elemental analysis and spectral measurements.
References and Notes
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3-Hydroxymethyl-10-(1-methylethyl)-10H-phenothiazine (16) The title
compound was obtained from 3-formyl-10-(1-methylethyl)-10H-phenothi-
1
azine (12) as a yellow oil (quant.). H-NMR (90 MHz, CDCl3) d: 1.61 (6H,
d, Jꢃ6.8 Hz), 4.12—4.43 (1H, m), 4.56 (2H, s), 6.57—7.25 (7H, m). EI-MS
m/z: 271 [M]ꢁ.
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(ꢀ)-3-{10-(1-Methylethyl)-10H-phenothiazin-3-ylmethoxy}quinucli-
dine (24) The title compound was prepared from 3-hydroxymethyl-10-(1-
methylethyl)-10H-phenothiazine (16) as a yellow oil (7.1%). 1H-NMR
(500 MHz, CDCl3) d: 1.34—1.48 (2H, m), 1.62 (6H, d, Jꢃ6.5 Hz), 1.66—
1.76 (1H, m), 1.89—1.95 (1H, m), 2.06—2.10 (1H, m), 2.70—2.86 (4H, m),
2.94—3.00 (1H, m), 3.10—3.16 (1H, m), 3.35—3.57 (1H, m), 4.24—4.30
(1H, m), 4.33 (1H, d, Jꢃ11.5 Hz), 4.43 (1H, d, Jꢃ11.5 Hz), 6.89—6.92 (1H,
m), 7.00—7.04 (2H, m), 7.08—7.14 (4H, m); EI-MS m/z: 380 [M]ꢁ; Anal.
Calcd for C23H28N2OS·1.4H2O: C, 68.08; H, 7.65; N, 6.90; S, 7.90. Found:
C, 68.00; H, 7.28; N, 6.84; S, 7.93.
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Preparation of Microsomes from Hamster Liver and HepG2 Cells
Microsomes were prepared from the livers of hamsters and from HepG2
cells, a human hepatoma cell line described previously.25) The tissues or
harvested cells were homogenized in HEPES buffer (50 mM) using a glass
homogenizer. Homogenates were centrifuged at 500ꢇg for 5 min at 4 °C,
and the resulting supernatants were further centrifuged at 8000ꢇg for
15 min at 4 °C. Microsomes were then isolated from this second supernatant
by ultra-centrifugation at 100000ꢇg for 60 min at 4 °C. The microsome
precipitates were suspended in HEPES buffer (1—5 mg/ml). Protein was
assayed by the Lowry method.26)
14) Brown G. R., Clarke D. S., Foubister A. J., Freeman S., Harrison P. J.,
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F., Minter D. E., Heterocycles, 25, 251—258 (1987).
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(1979).
Assay of Squalene Synthase Inhibitory Activity The squalene syn-
thase activities of these microsomes were assayed using the Amin technique
with modifications. The test compounds were dissolved in DMSO and the
assay was carried out in HEPES buffer (50 mM, pH 7.5) containing: NaF
(11 mM), MgCl2 (5.5 mM), dithiothreitol (3 mM), NADPH (1 mM), FPP
(5 mM), [3H]-FPP (0.017 mM, 15 Ci/mmol), NB-598 (10 mM), and sodium
pyrophosphate decahydrate (1 mM). After pre-incubation of these compo-
nents at 30 °C for 5 min, the reaction was initiated by the addition of micro-
somes (10 mg protein). The reaction was carried out at 30 °C for 20 min and
then terminated by the addition of 40% KOH–ethanol solution (100 ml, 1 : 1
by volume). Synthesized [3H]-squalene was extracted in petroleum ether
after saponification at 60 °C for 30 min and counted in Aquasol-2 using a
Beckman liquid scintillation counter.
20) Amin D., Cornell S. A., Gustafson S. K., Needle S. J., Ullrich J. W.,
Bilder G. E., Perrone M. H., J. Lipid. Res., 33, 1657—1663 (1992).
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rity T., Rich L. C., Ciosek C. P., Jr., J. Am. Chem. Soc., 113, 8522—
8524 (1991).
22) The c-logP values were calculated using the ACD/logP data base (Ad-
vanced Chemistry Development, Inc.).
23) The c-logP values of 10-methyl-10H-phenothiazine, 10-ethyl-10H-
phenothiazine, 10-butyl-10H-phenothiazine, and 10-(1-methyl)ethyl-
Plasma Total Cholesterol and Triglyceride Lowering Effects in Ham-
sters Male Syrian golden hamsters were purchased from Hamri (Ibaraki,
Japan). At the start of the study, the 8-week-old animals weighed approxi-
mately 140 g. They were kept for a week under reverse diurnal light cycles
with the lights off from 07:30 to 20:30. The animals were fed a standard low
cholesterol diet (CE-2), and water was provided ad libitum. Animals were
given test compound orally at a dose of 50 mg/kg of body weight once a day
for 5 d. The test compound was suspended in a 0.5% methylcellulose vehicle
solution. The no-treatment control group was given an equal volume of the

October 2004
1209
10H-phenothiazine were 4.49, 5.02, 6.09, and 5.37, respectively.
(1995).
24) Spady D. K., Dietschy J. M., Proc. Natl. Acad. Sci. U.S.A., 82, 4526— 26) Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J., J. Biol.
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Chem., 193, 265—275 (1951).
25) Lindsey S., Harwood H. J., Jr., J. Biol. Chem., 270, 9083—9096