Discovery of a new 2-aminobenzhydrol template for highly potent squalene synthase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.01.065

To obtain small and efficient squalene synthase inhibitors, a flexible 2-aminobenzhydrol open form structure was designed and showed potent inhibitory activity comparable to 4,1-benzoxazepin compounds. Further chemical modification led to the discovery of a novel template with a strong squalene synthase inhibitory activity, and its basic structure-activity relationship was revealed. The X-ray crystallographic data of compound 12 bound to the active site of squalene synthase provided an important insight into the binding mode of this alternative template that formed 11-membered ring conformations with an intramolecular hydrogen bond.

Full text of DOI:10.1016/j.bmc.2011.01.065

Bioorganic & Medicinal Chemistry 19 (2011) 1930–1949
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of a new 2-aminobenzhydrol template for highly potent
squalene synthase inhibitors
Masanori Ichikawa a, , Aki Yokomizo b, Masao Itoh b, Kazuyuki Sugita f, Hiroyuki Usui b,
⇑
Hironari Shimizu b, Makoto Suzuki c, Koji Terayama d, Akira Kanda e
a Lead Discovery and Optimization Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Lead Discovery and Optimization Research Laboratories II, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
c R&D Planning Department, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Cardiovascular-Metabolics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
e Biological Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
f Institute of Molecular and Cellular Biosciences, The University of Tokyo, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To obtain small and efficient squalene synthase inhibitors, a flexible 2-aminobenzhydrol open form struc-
ture was designed and showed potent inhibitory activity comparable to 4,1-benzoxazepin compounds.
Further chemical modification led to the discovery of a novel template with a strong squalene synthase
inhibitory activity, and its basic structure–activity relationship was revealed. The X-ray crystallographic
data of compound 12 bound to the active site of squalene synthase provided an important insight into the
binding mode of this alternative template that formed 11-membered ring conformations with an intra-
molecular hydrogen bond.
Received 28 December 2010
Revised 28 January 2011
Accepted 29 January 2011
Available online 3 February 2011
Keywords:
Hypercholesterolemia
Squalene synthase inhibitor
2-Aminobenzhydrol
X-ray analysis
Ó 2011 Elsevier Ltd. All rights reserved.
Intramolecular hydrogen bond
1. Introduction
It is known that squalene synthase converts two molecules of far-
nesyl pyrophosphates into a squalene, which is downstream of
The serum level of low-density lipoprotein (LDL) is one of the
important factors associated with coronary heart disease (CHD).
In industrialized countries, CHD remains one of the most common
causes of mortality. Patients who have higher serum levels of LDL
cholesterol are generally treated with statins,1 which inhibit 3-hy-
droxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the
rate-limiting enzyme in hepatic cholesterol biosynthesis.
However, statins also have a potential risk of adverse effects, such
as mytotoxicity, muscle pain and in very rare cases, rhabodmyoly-
sis.2 These adverse effects might be caused from the inhibition of
HMG-CoA reductase not only to block the synthesis of cholesterol,
but also to inhibit the biosynthesis of physiologically essential iso-
prenoid-derived molecules. As a general trend, statins have become
increasingly recognized as an imperfect medicine for all types of
hypercholesterolemia, and an alternative antihyperlipidemic agent
which does not inhibit isoprenoid biosynthesis is desired in medical
practice.
HMG-CoA reductase in the cholesterol biosynthesis cascade.3 This
indicates that an inhibitor of the enzyme does not interfere with
thebiosynthesis of many highly demanded non-steroidal isoprenoid
molecules, such as geranylgeranyl pyrophosphate and ubiquinone
(coenzyme Q).4 Therefore, squalene synthase is an interesting target
for the development of a safer cholesterol-lowering medicine, and
we have started to explore this field.5
Our research group already disclosed a series of novel squalene
synthase inhibitors, as exemplified by compound 1, which has a
sub-nano molar grade of IC50 against rat squalene synthase.6
Although these inhibitors have strong inhibitory activities in vitro,
they failed to show sufficient efficacy in vivo at reasonable doses.
This is presumably because of their extremely high protein binding
affinities, which is due to their high lipophilicity and flat naphtha-
lene rings, to inhibit cholesterol biosynthesis sufficiently in hepatic
cells in vivo.
In 2001, the X-ray crystal structures of squalene synthase solu-
ble domain with inhibitors, compound 1, and Takeda’s compound 2
(Fig. 1)7 were reported by our research group.8
From the analytical data, two aryl rings of compounds 1 and 2
crossed each other. These aromatic rings were finely superimposed
and similarly interacted with the lipophilic pockets of squalene
⇑
Corresponding author. Tel.: +81 3 3492 3131.
E-mail address: ichikawa.masanori.uf@daiichisankyo.co.jp (M. Ichikawa).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.065

M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
1931
O
2. Results
C-D
O
O
N
H
2.1. Chemistry
A-B
HO2C
CO2H
O
A series of the novel open form squalene synthase inhibitors
were prepared from benzophenone as shown in Scheme 2. Com-
mercially available aminobenzophenone 3 was reduced by sodium
borohydrate (NaBH4) to afford 2-aminobenzhydrol 4, followed by
Compound 1
A'
O
O
H
Cl
O
N
Compound 2
CO2H
HO2C
B'
N
O
Figure 1.
synthase (Figs. 2a and b). Ring A–B of compound 1 and Ring B0 of
compound 2 were surrounded by the residues of Val 179, Leu
183, Met 207, Gly 208, Leu 211, Tyr 276, Phe 288, and Pro292. Ring
C–D of compound 1 and Ring A0 of compound 2 were also bound
within a lipophilic cavity formed by the residues of Phe 54, Val
69, Phe 72, Tyr 73, Leu 76, Val 179, Leu 183, and Phe 288.
These two lipophilic cavities are important targets for the inhi-
bition of squalene synthase. Thus, it appears to be a rational
approach to identify alternative inhibitors that directly link these
two aromatic rings (Scheme 1). Therefore, we designed and syn-
thesized a flexible, two aromatic rings linked, open form template
to afford a small and efficacious squalene synthase inhibitor. To
prevent a repeat of the failure of compound 1, lower lipophilic
inhibitors were designed such that phenyl rings were selected in-
stead of naphthalene rings and the exposed hydroxyl group was
incorporated to increase hydrophilicity.
In this article, we will describe in detail the discovery of the
open form template, which possesses a 2-aminobenzhydrol core
structure, as well as its structure–activity relationship (SAR) as a
squalene synthase inhibitor.
Figure 2b. X-ray crystal structure of compound 1 (green carbons, PDB code: 3Q30)
overlaid with the bound crystal structure of 2 (magenta carbons, PDB code: 3Q2Z)
(protein not shown).
Figure 2a. Crystal structure of compound 1 bound in two lipophilic pockets of squalene synthase (PDB code: 3Q30).

1932
M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
O
O
O
Ring A
N
H
HO2C
CO2H
Cl
O
Cl
Compound 1
COR
OH
O
N
O
H
O
Linker
Cl
O
N
Compound 2
CO2H
HO2C
N
Ring B
O
Scheme 1.
Cl
O
Cl
O
Cl
Cl
e
Cl
a
OR
Cl
OH
NHR
4 R = H
R1
c
OH
OH
Cl
Cl
O
N
N
O
O
NH2
6 R = Me
7 R = H
3
b
d
5 R = CH2tBu
O
R2
O
N
N
R2
N
N
H
N
R1 =
R2
13
14 R2 = CO2Et
15 R2 = CO2Bn
16 R2 = H
11 R2 = OEt
12 R2 = OH
8 R2 = OEt
9 R2 = OH
10a R2 = NMe2
10b R2 = NHMe
10c R2 = NH2
d
e
d
f
g
17 R2 = SO2Me
18 R2 = CH2CO2Et
10d R2 = NHEt
10e R2 = NHiPr
10f R2 = NHnBu
Scheme 2. Synthesis of open form compounds 8–18. Reagents and conditions: (a) NaBH4, MeOH; (b) tBuCHO, NaBH4, AcOH; (c) methyl 4-chloro-4-oxobutanoate, NaHCO3,
DCM; (d) K2CO3, MeOH–H2O; (e) amine, EDC, HOBt, DCM; (f) H2, Pd–C, MeOH, rt; (g) MsCl, Et3N, DCM.
reductive aminoalkylation to obtain neopentyl amine 5 in good
O
Cl
OH
Cl
OH
yield. Amide 6 was produced by the treatment of 5 with methyl
succinyl chloride. Saponification of 6 afforded the desired carbox-
ylic acid 7. Succinic diamide 8 was prepared by the condensation
of 7 with isonipecotic ester using N-[3-(dimethylamino)propyl]-
N0-ethylcarbodiimide (EDC), and subsequent hydrolysis provided
acid 9. EDC coupling of 9 with various amines afforded a series
of amides 10a–f. Nipecotic acid derivatives 11 and 12 were also
prepared similarly.
Cl
Cl
a
c
OEt
OR
O
5
N
N
N
n
n
O
O
O
19a R = Et, n = 3
19b R = Et, n = 1
21a n = 3
21b n = 1
b
20a R = H, n = 3
20b R = H, n = 1
Piperazine derivatives 14–18 were prepared from acid 7.
Hydrogenation of benzyl carbamate 15 using 10% Pd/C as a catalyst
furnished amine 16, and the following methanesulfonylation gave
17. Amine 18 was produced by the condensation of acid 7 and
4-ethoxycarbonylmethyl piperazine.
Scheme 3. Synthesis of various length compounds. Reagents and conditions:
(a) acid chloride, NaHCO3, DCM; (b) Na2CO3, MeOH–H2O, rt; (c) amine, EDC, HOBt,
DCM.
Neopentyl amine 5 was coupled with malonyl chloride and
glutaryl chloride to afford amides 19a and 19b, respectively.
Sequential hydrolysis and amidation with ethyl isonipecotate
yielded 21a and 21b (Scheme 3).
N-Methyl and N-propyl derivatives 26a and 26b were prepared
as outlined in Scheme 4. Saponification of 22, followed by conden-
sation gave isonipecotate 24. N-Alkylation of 24 with alkyl iodides
and sodium hydride afforded 25a and 25b, and the following
NaBH4 reduction led to the desired benzhydroles 26a and 26b,
respectively.
Dehydroxy analog 28 was prepared from benzhydrol 5. The key
dehydration was carried out by triphenylphosphine oxide, trifluo-
romethanesulfonic anhydride, and NaBH4 in 87% yield (Scheme 5).9
The upper ring-modified compounds were prepared similarly from
benzophenones 29A–C and aniline 32 as shown in Scheme 6.
Cl
O
Cl
O
O
O
O
Cl
Cl
OR
N
O
O
b
OEt
N
N
H
H
22 R = Et
23 R = H
24
a
Cl
O
O
O
25a X = CO, R = Me
25b X = CO, R =nPr
Cl
X
c
N
b
OEt
26a X = CHOH, R = Me
26b X = CHOH, R =nPr
N
R
Scheme 4. Synthesis of smaller alkyl compounds. Reagents and conditions:
(a) K2CO3, MeOH–H2O, 60 °C; (b) amine, EDC, HOBt, DCM; (c) alkyl iodide, NaH,
DMF, 0 °C; (d) NaBH4, EtOH, 0 °C.

M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
1933
O
ester 11 showed an inverse relationship in which acid 12 had more
potent SSI activity than ester 11. For SSI activity, the lipophilic
substituent, like the ester group, might favor the 4th position of
the piperidine ring; and the hydrophilic substitute, like carboxylic
acid, might favor the 3rd position of the piperidine ring.
N
CO2Et
Cl
Cl
Cl
O
O
O
Cl
Cl
a
b
N
OEt
5
NH
N
O
Surprisingly, the CSI activity of acid 9 was seven-fold stronger
than that of ester 8, even though the SSI activity of ester 8 was
one hundred fifty-fold stronger than that of acid 9. With regard
to the CSI/SSI ratio, carboxylic acid 9 and 12 showed better value.
In our speculation, the carboxylic part of 9 and 12 might be rec-
ognized by a cell surface anion transporter,11 and these compounds
were well ingested into hepatic cells. In contrast, extremely high
lipophilic compound 13 showed good CSI/SSI ratio; however, it
could be due to its high cell permeability. Further investigation
needs to be performed to verify our hypothesis.
27
28
Scheme 5. Synthesis of non-hydroxyl compounds. Reagents and conditions:
(a) Ph3PO, Tf2O, NaBH4, DCM, 87%; (b) acid chloride, NaHCO3, DCM.
2-Aminobenzhydrols 34D–K were prepared from various
aldehydes and pivaloyl amide 33.
2.2. Evaluation and discussion
As a result of the modification of the isonipecotic acid part, all
compounds showed moderate to strong SSI activities. The activities
of mono-substituted isonipecotic amides (10b, d, e, f) were ten-
fold higher than those of non- and di-substituted amides (10a, c).
These data revealed that SSI activity does not depend on the size
of the alkyl substituents of isonipecotic amides.
The biological activities of the new compounds are shown in
Table 1. The initially prepared 2-aminobenzhydrol compound 6
and its acid 7 had very weak squalene synthase inhibitory (SSI)
activities10 (IC50 = 1.7 and 6.5
lM, respectively) compared with
4,1-benzoxazapin compounds (IC50 = 11 nM in Takeda’s patent,7
which might be ascribed to the relative conformational change of
their lipophilic parts.
Piperazine carbamates 14, 15 and ethoxy carbonyl methyl
piperazine 18, which has a basic nitrogen, were more potent than
the non-substituted piperazine 16 and methyl sulfonamide 17.
To identify the essential part of compound 8 for enzyme inhibi-
tion, the following conversions were attempted: adjustment of the
length of the methylene side chain between two amide residues,
replacement of the large neopentyl part to less lipophilic methyl
or propyl groups, and the removal of the hydroxyl group.
Extended three-carbon-linked pentanoic amide 21a and short-
ened one-carbon-linked maronic amide 21b, in addition to the
smaller alkyl compounds 26a, b, showed very weak SSI activities
compared with compound 8. Dehydroxy compound 28 also
showed weak SSI activity (Table 2). It would appear that the
two-carbon-linked succinic amide side chain and the bulky alkyl
part are necessary to maintain the appropriate conformation of
this template.
From the X-ray crystal structure studies,8 the carboxylic acid of
compound 7 seems to face the solvent side through the active
domain pocket of squalene synthase, the shape resembles a valley.
We then attempted to incorporate a cyclic substituent into the car-
boxylic acid unit by an amide bond, which was expected to interact
with the side walls of the pocket.
Isonipecotic ester 8 showed dramatically improved SSI activity
(IC50 = 0.85 nM) even though the precursor acid 7 had less activity.
A novel template with potent activity comparable to 4,1-ben-
zoxazepin compounds has been found.
To obtain the structure–activity relationship (SAR), prepared
derivatives were evaluated in terms of their SSI activities and cho-
lesterol synthesis inhibitory (CSI) activities in rat hepatic cells. CSI
activity was used as a potential parameter to predict in vivo CSI
activity, and was considered more important than SSI activity as
a guide to afford effective cholesterol-lowering medicine. Hence,
the early stage of our research effort was focused on improving
both SSI and CSI activities.
To investigate the effect of substituents on the upper benzene
ring,
a series of compounds were prepared and evaluated
(Table 3). Compound 37A, which has no upper ring, and non-substi-
tuted benzene compound 37B showed no measurable and very
weak SSI activities, respectively. On the other hand, 2-fluorobenzene
isonipecotic ester 37C demonstrated the highest SSI activity with an
Interestingly, the SSI activity of isonipecotic acid 9 was dramat-
ically reduced from its ester 8. In contrast, nipecotic acid 12 and its
35A-K R2 = OMe
R1
OH
NH
R1
g
O
R1
R1
OH
NH2
30A-C
Cl
Cl
36A-K R2 = OH
R2
OH
Cl
Cl
a
b
e
f
O
h
g
N
CO2Et
CO2Et
37A-K R2 =
38C-K R2 =
N
NH2
29B-C
O
N
31A-K
R1
OH
NH
Cl
N
CO2H
CO2H
Cl
39C-K R2 =
40C-K R2 =
d
R
N
H
N
O
32 R = H
33 R = COtBu
c
34D-K
F
F
O
O
O
O
O
F
H
R1 =
F
Cl
Cl
Cl
O
O
A
B
C
D
E
F
G
H
I
J
K
Scheme 6. Synthesis of various upper ring compounds. Reagents and conditions: (a) NaBH4, MeOH, 0 °C; (b) tBuCHO, NaBH4, AcOH; (c) pivaloyl chloride, DMAP, Et3N, DCM;
(d) secBuLi, THF then aldehyde ꢀ78 °C to rt; (e) Red-Al, THF, rt; (f) methyl 4-chloro-4-oxobutanoate, NaHCO3, DCM, rt; (g) K2CO3, MeOH–H2O, 50 °C; (h) amine, EDC, HOBt,
DCM.

1934
M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
Table 1
series E demonstrated drastically decreased inhibitory potency
and 2-chloro-5-fluorobenzene series F showed no SSI activities.
These results suggest that substitution on the 4- and 5-positions
of the upper benzene ring would sterically disfavor the lipophilic
cavity of squalene synthase.
Evaluation of squalene synthase inhibitory (SSI) activities and cholesterol synthesis
inhibitory (CSI) activities (part 1)
Cl
O
OH
Cl
R
Furthermore, 2-methoxy, 2,3-dimethoxybenzene, and 1,4-ben-
zodioxane derivatives (G, J, K) showed strong SSI activities. In par-
ticular, 2-methoxybenzene and 1,4-benzodioxane derivatives (G,
K) had excellent CSI activities as well. In general, there was a ten-
dency for lower C log P compounds to have stronger CSI activities.
As described above, the present results suggest that the upper
ring is crucial for the inhibition of squalene synthase. Also the hy-
droxyl part, succinic amide side chain lengths, and a large alkyl
substituent (e.g., neopentyl part) are very important for maintain-
ing strong SSI activity.
Most benzhydrol derivatives, as represented by compound 9,
were composed of a pair of atrop isomers.12 These two rotamers
are created by a high rotational barrier around the C–N bond be-
tween the amide nitrogen and the aryl ring because the bulky neo-
pentyl group and ortho-substituted aryl ring are located on the
nitrogen of the amide bond.
To elucidate the absolute structure of the active isomer, we car-
ried out X-ray structure analysis of squalene synthase co-crystal-
lized with racemic inhibitor 12. The results showed that only the
(S)-hydroxyl-(aR)-atrop isomer was buried in the squalene syn-
thase lipophilic cavities (Fig. 3).13 Surprisingly, an intramolecular
hydrogen bonding interaction between the proton of the hydroxyl
group and the oxygen atom of the side chain amide was observed.
Thus, it can be predicted that the improvement of SSI activity de-
pends on the formation of this intramolecular hydrogen bond,
which results from the increased dipole moment of the carbonyl
part, due to the formation of an amide bond by the introduction
of the nipecotin and iso-nipecotin parts.
N
O
Compound
R
SSI
(IC50, nM)
CSI
(IC50, nM)
CSI/SSI ratio
6
7
OMe
OH
1700
6500
—
—
—
—
N
N
N
N
N
N
N
N
CO2Et
8
9
0.85
130
97
1700
250
—
200
1.9
—
CO2H
CONMe2
CONHMe
CONH2
CONHEt
CONHiPr
CONHBu
10a
10b
10c
10d
10e
10f
1.3
280
2.4
3.7
1.6
900
—
692
—
—
—
1600
2000
432
1250
CO2Et
11
410
—
—
N
CO2H
12
13
20
270
13.5
11.8
N
N
H
220
2600
N
N
N
N
N CO2Et
14
15
16
17
2.3
—
—
—
—
—
—
—
—
N
CO2Bn
1.1
NH
2100
260
3. Conclusion
N
N
SO2Me
CO2Et
We designed a novel 2-aminobenzhydrol template and revealed
its high potential as a squalene synthase inhibitor. The finding of
compound 8, showing strong inhibitory activity comparable to
4,1-benzoxazepin compounds, was taken as a starting point for
our research. In subsequent investigations to clarify the SAR of
our newly synthesized template, carboxylic acid compounds
showed higher CSI activities in rat hepatic cells. In particular,
2-methoxybenzene and 1,4-benzodioxane derivatives demon-
strated strong SSI and CSI activities. Finally, the unique active bind-
ing formation of our new template was elucidated. The X-ray
analysis of squalene synthase co-crystallized with racemic atrop
mixture 12 showed that the (S)-hydroxyl-(aR)-atrop isomer of 12
was buried in the lipophilic cavity of the enzyme, and formed
11-membered ring conformations with an intramolecular hydro-
gen bond between the hydroxyl group and the side chain amide
carbonyl oxygen. Further structure optimization and biological
evaluations of the new template will be reported in due course.
18
7.9
1400
177
N
SSI: Squalene synthase inhibitory activity. CSI: cholesterol synthase inhibitory
activity in rat hepatic cell. ‘—, not tested’.
Table 2
Evaluation of squalene synthase inhibitory (SSI) activities and cholesterol synthesis
inhibitory (CSI) activities (part 2)
Cl
O
X
N
Cl
OEt
N
n
O
R
O
Compound
X
R
n
SSI (IC50, nM)
8
CHOH
CHOH
CHOH
CHOH
CHOH
CH2
CH2tBu
CH2tBu
CH2tBu
Me
2
3
1
2
2
2
8.5
21a
21b
26a
26b
28
>6000
>600
21,000
600
4. Experimental
4.1. Chemistry
nPr
CH2tBu
650
4.1.1. General
Unless otherwise noted, materials were obtained from commer-
cial suppliers and used without further purification. 1H NMR spec-
tra were recorded on JEOL JNM-EX400 spectrometers, and
chemical shifts are given in ppm from tetramethylsilane as an
internal standard. FAB mass spectra were recorded on a JEOL
JMS-HX110 spectrometer. HR-FAB mass spectra were recorded
IC50 value of 0.45 nM. The other 2-fluorobernzene derivatives
38–40C showed lower SSI activities than 2-chlorobenzene
compounds.
2-Chloro-3-fluorobenzene series D showed higher SSI activities
than 2-chloro compounds. However, 2-chloro-4-fluorobenzene

M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
1935
Table 3
Evaluation of squalene synthase inhibitory (SSI) activities and cholesterol synthesis inhibitory (CSI) activities (part 3)
R1
O
Cl
OH
R2
N
O
SSI: Squalene synthase inhibitory activity. CSI: Cholesterol synthase inhibitory activity in rat liver cell. ‘— not tested’. C log P was calculated using ACD/labs ver. 11.00. ACD/
log P DB.
on a JEOL JMS-700. ESI mass spectra were recorded on SCIEX
API-150EX and Agilent Technologies Agilent 1100 series LC/MS.
Column chromatography was performed with Merck silica gel 60
(particle size 0.060–0.200 or 0.040–0.063). Flash column chroma-
tography was performed with YAMAZEN cartridge series or ultra
pack series. Thin-layer chromatography (TLC) was performed on
Merck pre-coated TLC glass sheets with silica gel 60F254 or
Whatman Partisil PLK5F with Silica gel 150 Å.
was washed with brine, dried over Na2SO4, and then concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (AcOEt/Hexane = 1:2–1:1 as eluent) to give compound 6
(0.41 g, 0.90 mmol, 61%) as a colorless powder. 1H NMR (CDCl3) d
0.87 (0.88) (9H, s), 2.23–2.39 (3H, m), 2.80–2.90 (1H, m), 3.02
(2.98) (1H, d, J = 13.8 Hz), 3.66 (3.57)(3H, s), 4.44 (4.50) (1H, d,
13.8 Hz), 4.56–4.57 (1H, m), 6.13 (6.33) (1H, br s), 7.03 (1H, br s),
7.21–7.37 (5H, m), 7.73–7.78 (1H, m). IR (ATR) cmꢀ1 3359, 2950,
1745, 1650, 1432, 1168, 1027, 752, 420. Mp 130–132 °C. MS
(FAB) m/z 452 (M+H)+. Anal. Calcd for C23H27NO4Cl2: C, 61.07; H,
6.02; N, 3.10; Cl, 15.67. Found: C, 61.09; H, 6.04; N, 3.00; Cl, 15.78.
4.1.2. Methyl 4-[{4-chloro-2-[(2-chlorophenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoate (6)
An ice-cooled solution of {5-chloro-2-[(2,2-dimethylpro-
pyl)amino]phenyl}(2-chlorophenyl)methanol (0.50 g, 1.5 mmol)
in CH2Cl2 (50 ml) was added ethyl 4-chloro-4-oxobutanoate
(0.23 g, 1.7 mmol) and NaHCO3 (0.37 g, 4.4 mmol). After being stir-
red for 2 h at room temperature, the reaction was quenched with
water. The organic material was extracted with CH2Cl2. The extract
4.1.3. 4-[{4-Chloro-2-[(2-chlorophenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoic acid (7)
Compound 6 (0.20 g, 0.44 mmol) was suspended in a mixture of
MeOH (10 ml) and water (5 ml), and K2CO3 was added at room
temperature, followed by stirring for 12 h at same temperature.

1936
M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
Figure 3. Crystal structure of compound 12 bound in squalene synthase. The intramolecular hydrogen bond between the proton of the hydroxyl group and the oxygen atom
of side chain amide are highlighted (PDB code: 3ASX).
The solvent was removed under reduced pressure and was adding
1 N hydrochloric acid and CH2Cl2. The organics were extracted
with CH2Cl2 (3 times). The extract was washed with brine, dried
over Na2SO4, and then concentrated in vacuo. Then, the residue
was washed with diethyl ether and hexane to give compound 7
as a colorless powder. 1H NMR (DMSO-d6) d 0.76 (0.84) (9H, s),
1.06–1.27 (1H, m), 1.71–2.48 (3H, m), 2.54(3.03) (1H, d,
J = 13.7 Hz), 4.20 (4.40) (1H, d, J = 13.7 Hz), 5.89 (6.09) (1H, br s),
6.27 (1H, br), 7.07 (1H, d, J = 2.4 Hz), 7.28–7.61 (6H, m). IR (ATR)
cmꢀ1 2954, 1710, 1641, 1477, 1396, 1168, 1027, 750. Mp
78–80 °C. MS (ESI) m/z 438 (M+H)+. Anal. Calcd for
4.1.5. 1-{4-[{4-Chloro-2-[(2-chlorophenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}piper-
idine-4-carboxylic acid (9)
Compound 9 was prepared in a similar manner described for 8
in 88% yield as a colorless powder. 1H NMR (DMSO-d6) d 0.73 (0.81)
(9H, s), 1.27–1.46 (2H, m), 1.73–1.81 (2H, m), 1.96–2.17 (1H, m),
2.29–2.69 (5H, m), 2.97–3.06 (2H, m), 4.12–4.38 (3H, m), 5.88
(6.07) (1H, br s), 6.26 (1H, br s), 7.02 (1H, s), 7.26 (1H, s),
7.34–7.60 (5H, m). IR (ATR) cmꢀ1 2952, 1727, 1658, 1621,
1475, 1396, 1170, 1020, 744, 541, 420. Mp 168–170 °C. MS (ESI)
m/z 549 (M+H)+. Anal. Calcd for C28H34N2O5Cl2: C, 61.20; H,
6.24; N, 5.10; Cl, 12.90. Found: C, 60.96; H, 6.29; N, 4.89; Cl,
12.69.
C23H27NO4Cl2ꢁ0.25H2O: C, 59.67; H, 5.80; N, 3.16; Cl, 16.01. Found:
C, 59.36; H, 5.96; N, 2.96; Cl, 16.30.
4.1.6. 1-{4-[{4-Chloro-2-[(2-chlorophenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-N,N-di-
methylpiperidine-4-carboxamide (10a)
4.1.4. Ethyl 1-{4-[{4-chloro-2-[(2-chlorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-4-carboxylate (8)
Compound 10a was prepared from 9 in a similar manner
described for 8 in 57% yield as a colorless powder. 1H NMR (CDCl3)
d 0.91 (9H, s), 1.69–1.76 (3H, m), 2.11–2.24 (2H, m), 2.41–2.49 (1H,
m), 2.66–2.75 (2H, m), 2.93 (3H, s), 3.06 (3H, s), 3.10–3.17 (3H, m),
3.29 (3.27) (1H, d, J = 13.7 Hz), (3.95) 4.50 (1H, d, J = 13.7 Hz),
4.45–4.57 (1H, m), 6.15–6.18 (1H, m), 6.42 (6.25) (1H, d,
J = 5.0 Hz), 6.97–6.99 (1H, m), 7.22–7.42 (5H, m), 7.95 (7.91) (1H,
d, J = 7.7 Hz). IR (ATR) cmꢀ1 3278, 2948, 1639, 1617, 1398, 1276,
1027, 750, 482. Mp 197–199 °C. MS (FAB) m/z 576 (M+H)+.
Anal. Calcd for C30H39N3O4Cl2ꢁ1.5H2O: C, 59.70; H, 7.01; N, 6.96;
Cl, 11.75. Found: C, 59.96; H, 6.76; N, 6.84; Cl, 11.84.
To a solution of compound 7 (0.16 g, 0.37 mmol) and isonipe-
cotic acid ethyl ester (0.086 ml, 0.56 mmol) in CH2Cl2 was added
WSCIꢁHCl (0.11 g) and HOBt (0.085 g), and then the mixture was
stirred at room temperature for 18 h. The reaction mixture was di-
luted with water and the organics were extracted with CH2Cl2. The
extract was washed with brine, dried over Na2SO4, then concen-
trated in vacuo, and then the residue was purified with silica gel
column chromatography (0–5% MeOH–CH2Cl2 as eluent) to give
compound 8 (0.16 g, 0.28 mmol, 76%) as a colorless powder. 1H
NMR (CDCl3) d 0.91 (9H, s), 1.24 (1.26) (3H, t, J = 7.0 Hz), 1.80–
1.97 (3H, m), 2.11–2.22 (2H, m), 2.38–2.52 (2H, m), 2.68–2.89
(2H, m), 3.07–3.19 (2H, m), 3.29 (3.28) (1H, d, J = 13.9 Hz), 3.83–
3.84 (1H, m), 4.12 (4.14) (2H, q, J = 7.3 Hz), 4.26–4.40 (1H, m),
4.50 (4.51) (1H, d, J = 13.7 Hz), 6.16 (1H, s), 6.40 (6.30) (1H, d,
J = 5.0 Hz), 6.98 (1H. d, J = 1.2 Hz), 7.30–7.42 (5H, m), 7.92–7.95
(1H, m). IR (ATR) cmꢀ1 3320, 2950, 1731, 1664, 1625, 1394,
1166, 1041, 746, 478. Mp 138–140 °C. MS (ESI) m/z 577 (M+H)+.
Anal. Calcd for C30H38N2O5Cl2: C, 62.39; H, 6.63; N, 4.85; Cl,
12.28. Found: C, 62.24; H, 6.63; N, 4.79; Cl, 12.05.
4.1.7. 1-{4-[{4-Chloro-2-[(2-chlorophenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-N-methyl-
piperidine-4-carboxamide (10b)
Compound 10b was prepared from 9 in a similar manner de-
scribed for 8 in 61% yield as a colorless powder. 1H NMR (CDCl3)
d 0.90(0.91) (9H, s), 1.61–1.87 (3H, m), 2.11–2.47 (5H, m), 2.63–
2.70 (1H, m), 2.81 (3H, s), 3.03–3.14 (2H, m), 3.28 (3.94) (1H, d,

M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
1937
J = 13.7 Hz), 4.49 (4.50) (1H, d, J = 13.8 Hz), 4.11–4.57 (1H, m), 5.42
(5.58) (1H, br), 6.14–6.17 (1H, m), 6.37 (6.28) (1H, d, J = 5.1 Hz),
6.98 (6.99) (1H, s), 7.22–7.41 (5H, m), 7.91–7.95 (1H, m). IR
(ATR) cmꢀ1 3266, 2948, 1652, 1627, 1475, 1407, 1168, 1027, 833,
746, 534. Mp 130–132 °C. MS (FAB) m/z 562 (M+H)+. Anal. Calcd
for C29H37N3O4Cl2ꢁ0.5H2O: C, 60.94; H, 6.70; N, 7.35; Cl, 12.41.
Found: C, 60.84; H, 6.64; N, 7.21; Cl, 12.42.
(M+H)+. Anal. Calcd for C32H43N3O4Cl2ꢁ0.25H2O: C, 63.10; H, 7.20;
N, 6.90; Cl, 11.64. Found: C, 63.09; H, 7.14; N, 6.88; Cl, 11.87.
4.1.12. Ethyl 1-{4-[{4-chloro-2-[(2-chlorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-3-carboxylate (11)
Compound 11 was prepared from 7 in a similar manner de-
scribed for 8 in 85% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.91 (0.90) (9H, s), 1.25 (1.26) (3H, t, J = 7.08 Hz), 1.35–
2.21 (4H, m), 2.32–3.53 (6H, m), 3.73–4.19 (5H, m), 4.51 (4.50)
(1H, d, J = 13.7 Hz), 4.48–4.75 (1H, m), 6.16–6.18 (1H, m), 6.28–
6.40 (1H, m), 6.98 (1H, d, J = 1.95 Hz), 7.24–7.43 (5H, m), 7.91–
7.96 (1H, m). IR (ATR) cmꢀ1 3345, 2950, 1727, 1627, 1168, 1027,
750, 480. MS (ESI) m/z 577 (M+H)+. Anal. Calcd for C30H38N2O5Cl2:
C, 62.39; H, 6.63; N, 4.85; Cl, 12.28. Found: C, 62.02; H, 6.69; N,
4.71; Cl, 11.99.
4.1.8. 1-{4-[{4-Chloro-2-[(2-chlorophenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}piperidine-
4-carboxamide (10c)
Compound 10c was prepared from 9 in a similar manner de-
scribed for 8 in 34% yield as a colorless powder. 1H NMR (CDCl3)
d 0.91 (9H, s), 1.84–1.95 (3H, m), 2.11–2.22 (2H, m), 2.35–2.48
(2H, m), 2.68–2.74 (2H, m), 3.03–3.17 (2H, m), 3.28 (3.27) (1H, d,
J = 13.8 Hz), (3.94) 4.49 (1H, d, J = 13.8 Hz), 4.48–4.55 (1H, m),
5.28 (1H, br), 5.41 (5.60) (1H, br), 6.15–6.17 (1H, m), 6.34 (6.27)
(1H, d, J = 5.0 Hz), 6.98 (1H, d, J = 1.7 Hz), 7.23–7.52 (4H, m),
7.91–7.94 (1H, m). IR (ATR) cmꢀ1 3324, 2950, 1677, 1654, 1614,
1475, 1402, 1270, 1027, 763, 570. Mp 90–92 °C. MS (ESI) m/z 548
(M+H)+. Anal. Calcd for C28H35N3O4Cl2ꢁ1.0H2O: C, 59.36; H, 6.58;
N, 7.42; Cl, 12.52. Found: C, 59.12; H, 6.64; N, 7.21; Cl, 12.31.
4.1.13. 1-{4-[{4-Chloro-2-[(2-chlorophenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}piperidine-
3-carboxylic acid (12)
Compound 12 was prepared from 11 in a similar manner de-
scribed for 7 in 68% yield as a colorless powder. 1H NMR (DMSO-
d6) d 0.69–0.86 (9H, m), 1.09–3.96 (13H, m), 4.08–4.42 (2H, m),
5.76–6.30 (2H, m), 6.98–7.73 (7H, m). IR (ATR) cmꢀ1 3326, 2952,
1621, 1475, 1394, 1168, 1029, 748, 480, 420. Mp 153–155 °C. MS
(ESI) m/z 549 (M+H)+. Anal. Calcd for C28H34N2O5Cl2ꢁ2.1H2O: C,
57.26; H, 6.56; N, 4.77; Cl, 12.07. Found: C, 56.99; H, 6.32; N,
4.49; Cl, 11.86.
4.1.9. 1-{4-[{4-Chloro-2-[(2-chlorophenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-N-ethyl-
piperidine-4-carboxamide (10d)
Compound 10d was prepared from 9 in a similar manner de-
scribed for 8 in 42% yield as a colorless powder. 1H NMR (CDCl3)
d 0.91 (0.90) (9H, s), 1.12 (1.14) (3H, t, J = 7.1 Hz), 1.78–2.66 (8H,
m), 2.93–3.10 (3H, m), 3.29 (2H, q, J = 7.1 Hz), 3.26–3.31 (1H, m),
3.94 (1H, br d, J = 11.7 Hz), 4.50 (4.51) (1H, d, J = 13.6 Hz), 4.45–
4.53 (1H, m), 5.38 (5.50) (1H, br), 6.15–6.17 (1H, m), 6.37 (6.28)
(1H, d, J = 5.0 Hz), 6.98 (1H, s), 7.23–7.42 (5H, m), 7.91–7.95 (1H,
m). IR (ATR) cmꢀ1 3266, 2954, 1662, 1625, 1475, 1394, 1180,
1029, 744, 576, 480. Mp 148–150 °C. MS (FAB) m/z 576 (M+H)+.
Anal. Calcd for C30H39N3O4Cl2ꢁ0.5H2O: C, 61.53; H, 6.89; N, 7.18;
Cl, 12.11. Found: C, 61.56; H, 6.81; N, 7.12; Cl, 12.22.
4.1.14. N-{4-Chloro-2-[(2-chlorophenyl)(hydroxy)methyl]-
phenyl}-N0-(cyclohexylmethyl)-N-(2,2-dimethylpropyl)succi-
namide (13)
Compound 13 was prepared from 7 in a similar manner de-
scribed for 8 in 65% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.87 (9H, s), 0.88–1.68 (11H, m), 2.09–2.17 (1H, m),
2.23–2.28 (1H, m), 2.46–2.54 (1H, m), 2.63–2.77 (1H, m), 2.94–
3.03 (1H, m), 3.03 (1H, d, J = 13.8 Hz), 3.14–3.21 (1H, m), 4.42
(4.52) (1H, d, J = 13.8 Hz), 5.70 (1H, m), 5.89 (1H, d, J = 5.3 Hz),
6.07 (1H, d, J = 5.3 Hz), 7.11 (1H, d, J = 2.20 Hz), 7.21–7.38 (5H,
m), 7.81–7.83 (1H, m). IR (ATR) cmꢀ1 3330, 2923, 1639, 1475,
1394, 1276, 1168, 1027, 748, 478, 422. MS (ESI) m/z 533 (M+H)+.
Anal. Calcd for C29H38N2O3Cl2ꢁ0.9H2Oꢁ0.3ether: C, 63.42; H, 7.54;
N, 4.90; Cl, 12.40. Found: C, 63.77; H, 7.28; N, 4.81; Cl, 12.07.
4.1.10. 1-{4-[{4-Chloro-2-[(2-chlorophenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-N-isopro-
pylpiperidine-4-carboxamide (10e)
Compound 10e was prepared from 9 in a similar manner de-
scribed for 8 in 38% yield as a colorless powder. 1H NMR (CDCl3)
d 0.91 (9H, s), 1.12–1.16 (6H, m), 1.76–1.87 (3H, m), 2.11–2.66
(5H, m), 3.02–3.13 (3H, m), 3.28 (3.27) (1H, d, J = 13.8 Hz), 3.93
(1H, br d, J = 14.2 Hz), 4.03–4.06 (1H, m), 4.51 (4.50) (1H, d,
J = 13.7 Hz), 4.48–4.56 (1H, m), 5.20 (5.29) (1H, br), 6.16 (1H, br),
6.37 (6.27) (1H, br), 6.98 (1H, s), 7.24–7.52 (5H, m), 7.91–7.95
(1H, m). IR (ATR) cmꢀ1 3282, 2958, 1662, 1623, 1473, 1394,
1166, 1027, 744, 541, 480. Mp 148–150 °C. MS (ESI) m/z 590
(M+H)+. Anal. Calcd for C31H41N3O4Cl2ꢁ0.25H2O: C, 62.57; H, 7.03;
N, 7.06; Cl, 11.91. Found: C, 62.69; H, 6.98; N, 7.04; Cl, 11.53.
4.1.15. Ethyl 4-{4-[{4-chloro-2-[(2-chlorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperazine-1-carboxylate (14)
Compound 14 was prepared from 7 in a similar manner de-
scribed for 8 in quantum yield as a colorless powder. 1H NMR
(CDCl3) d 0.91 (0.89) (9H, s), 1.27 (1.21) (3H, t, J = 7.1 Hz), 2.13–
2.49 (3H, m), 3.05–3.14 (1H, m), 3.28 (2.94) (1H, d, J = 13.7 Hz),
3.33–3.66 (8H, m), 4.15 (2H, q, J = 7.1 Hz), 4.50 (4.46) (1H, d,
J = 13.7 Hz), 6.15–6.18 (2H, m), 6.98 (1H, dd, J = 2.0, 0.73 Hz),
7.21–7.43 (4H, m), 7.92 (1H, dd, J = 7.8, 1.5 Hz). IR (ATR) cmꢀ1
3332, 2948, 1700, 1662, 1635, 1423, 1234, 1184, 1027, 750. Mp
177–179 °C. MS (ESI) m/z 578 (M+H)+. Anal. Calcd for
4.1.11. N-Butyl-1-{4-[{4-chloro-2-[(2-chlorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-4-carboxamide (10f)
C29H37N3O5Cl2: C, 60.21; H, 6.45; N, 7.26; Cl, 12.26. Found: C,
Compound 10f was prepared from 9 in a similar manner de-
scribed for 8 in 45% yield as a colorless powder. 1H NMR (CDCl3)
d 0.91 (9H, s), 1.30–1.55 (7H, m), 1.78–1.88 (3H, m), 2.11–2.45
(4H, m), 2.64–2.70 (1H, m), 3.03–3.30 (6H, m), 3.94 (1H, br d,
J = 13.2 Hz), 4.51 (4.50) (1H, d, J = 13.6 Hz), 4.48–4.51 (1H, m),
5.38 (5.51) (1H, br), 6.16 (1H, t, J = 4.9 Hz), 6.38 (6.28) (1H, d,
J = 4.9 Hz), 6.98 (1H, d, J = 2.0 Hz), 7.22–7.42 (5H, m), 7.91–7.95
(1H, m). IR (ATR) cmꢀ1 3259, 2956, 1662, 1623, 1473, 1394,
1180, 1027, 744, 478, 422. Mp 178–180 °C. MS (ESI) m/z 604
60.07; H, 6.46; N, 7.30; Cl, 12.18.
4.1.16. Benzyl 4-{4-[{4-chloro-2-[(2-chlorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperazine-1-carboxylate (15)
Compound 15 was prepared from 7 in a similar manner de-
scribed for 8 in 69% yield as a colorless powder. 1H NMR (CDCl3)
d 0.91 (9H, s), 2.15–2.45 (2H, m), 3.09–3.62 (11H, m), 4.50 (1H, d,
J = 14.9 Hz), 5.15 (2H, s), 6.15 (2H, m), 6.98 (1H, br s), 7.26–7.36

1938
M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
(10H, m), 7.93 (1H, m). IR (ATR) cmꢀ1: 3345, 2960, 1695, 1664,
0625, 1427, 1222, 1020, 748, 696, 574. Mp 129–132 °C. MS (ESI)
m/z 640 (M+H)+. Anal. Calcd for C34H39N3O5Cl2: C, 63.75; H, 6.14;
N, 6.56; Cl, 11.07. Found: C, 63.58; H, 6.18; N, 6.33; Cl, 11.22.
4.1.21. 5-[{4-Chloro-2-[(2-chlorophenyl)(hydroxy)methyl]phe-
nyl}(2,2-dimethylpropyl)amino]-5-oxopentanoic acid (20a)
Compound 20a was prepared from 19a in a similar manner de-
scribed for 7 in quantum yield as a colorless amorphous. 1H NMR
(DMSO-d6) d 0.77–0.86 (9H, m), 0.93–3.03 (6H, m), 4.22 (1H, brd,
J = 13.3 Hz), 4.40 (1H, brd, J = 13.3), 5.84–6.20 (2H, m), 6.76–7.62
(7H, m), 11.97 (1H, br). IR (ATR) cmꢀ1 2954, 1706, 1635, 1475,
1396, 1166, 1025, 754, 418. Mp 65–67 °C. MS (ESI) m/z 452
(M+H)+. Anal. Calcd for C23H27NO4Cl2ꢁ0.3H2Oꢁ0.2ether: C, 60.49;
H, 6.31; N, 2.96; Cl, 15.00. Found: C, 60.83; H, 6.27; N, 2.80; Cl,
14.71.
4.1.17. N-{4-Chloro-2-[(2-chlorophenyl)(hydroxy)methyl]-
phenyl}-N-(2,2-dimethylpropyl)-4-oxo-4-piperazin-1-ylbu-
tanamide (16)
A suspension of 15 (0.15 g, 0.23 mmol) and 10% Palladium on
carbon (50% water, 30 mg) in MeOH (30 ml) was hydrogenated
for 2 h. The reaction mixture was filtrated and concentrated in va-
cuo to give 16 (0.12 g, quant.) as colorless amorphous. 1H NMR
(CDCl3) d 0.87 (9H, s), 2.14–2.25 (2H, m), 2.54–3.98 (11H, m),
4.35–4.60 (1H, m), 5.79–6.28 (2H, m), 7.00 (1H, s), 7.24–7.37 (5H,
m), 7.81 (1H, s). IR (ATR) cmꢀ1 3338, 2952, 1637, 1392, 1247,
1024, 700, 561, 509. Mp 148–150 °C. MS (FAB) m/z 506 (M+H)+.
HRMS (FAB) m/z 506.1951 (calcd for C26H34O3N3Cl2: 506.1977).
4.1.22. Ethyl 1-{5-[{4-chloro-2-[(2-chlorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-5-oxopentanoyl}-
piperidine-4-carboxylate (21a)
Compound 21a was prepared from 20a in a similar manner de-
scribed for 8 in 86% yield as a colorless amorphous. 1H NMR (CDCl3)
d 0.81–0.90 (9H, s), 1.26 (3H, t, J = 7.1 Hz), 1.41–2.31 (8H, m), 2.48–
3.21 (6H, m), 3.68–3.81 (1H, m), 4.12–4.18 (2H, m), 4.27–4.53 (2H,
m), 5.84–6.33 (2H, m), 7.17–7.74 (7H, m). IR (ATR) cmꢀ1 2952,
1727, 1639, 1475, 1170, 1037, 755, 418. Mp 58–60 °C. MS (ESI) m/z
591(M+H)+. Anal. Calcd for C31H40N2O5Cl2ꢁ0.4H2Oꢁ0.2ether: C,
62.25; H, 7.03; N, 4.57; Cl, 11.56. Found: C, 62.52; H, 6.97; N, 4.46;
Cl, 11.16.
Anal. Calcd for
C
26H33N3O3Cl2ꢁ1.5H2Oꢁ0.25hexaneꢁ0.20ether: C,
59.65; H, 7.34; N, 7.37; Cl, 12.44. Found: C, 59.77; H, 7.72; N,
7.46; Cl, 12.56.
4.1.18. N-{4-Chloro-2-[(2-chlorophenyl)(hydroxy)methyl]-
phenyl}-N-(2,2-dimethylpropyl)-4-[4-(methylsulfonyl)-
piperazin-1-yl]-4-oxobutanamide (17)
Compound 17 was dissolved in CH2Cl2, followed by the addition
of Et3N (0.028 ml, 0.20 mmol) and methane sulfonyl chloride
(0.013 ml, 0.16 mmol). The reaction mixture was stirred at room
temperature for 18 h. The mixture was diluted with water and
CH2Cl2. The organics were extracted with CH2Cl2, the extract was
washed with brine, and then dried with Na2SO4. The organic
solvent was removed under reduced pressure and the residue
was purified with silica gel column chromatography (5–7%
MeOH–CH2Cl2) to give compound 17 (49 mg, 0.083 mmol, 62%)
as colorless powder. 1H NMR (DMSO-d6) d 0.76–0.86 (9H, m),
2.04–2.71 (5H, m), 2.87 (3H, s), 2.92–3.12 (4H, m), 3.37–3.54
(4H, m), 4.08–4.42 (1H, m), 5.63–6.21 (2H, m), 7.02–7.69 (7H,
m). IR (ATR) cmꢀ1 3388, 2954, 1635, 1324, 1157, 958, 775, 516.
Mp 104–106 °C. MS (FAB) m/z 584 (M+H)+. HRMS (FAB) m/z
584.1714 (calcd for C27H36O5N3Cl2S: 584.1753).
4.1.23. Ethyl 1-{3-[{4-chloro-2-[(2-chlorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-3-oxopropanoyl}-
piperidine-4-carboxylate (21b)
Compound 21b was prepared from 5 in a similar manner de-
scribed for 21a in 78% yield from 5 as a colorless amorphous. 1H
NMR (DMSO-d6) d 0.74–0.84 (9H, m), 1.15 (3H, t, J = 7.1 Hz),
1.32–1.78 (3H, m), 2.23–3.74 (8H, m), 4.04 (2H, q, J = 7.2 Hz),
4.10–4.38 (2H, m), 5.86–6.03 (1H, m), 6.34 (1H, br), 6.98 (1H, dd,
J = 11.2, 2.4 Hz), 7.30–7.53 (6H, m). IR (ATR) cmꢀ1 3345, 2952,
1727, 1623, 1475, 1166, 1029, 748, 586, 418. Mp 90–92 °C. MS
(FAB) m/z 563 (M+H)+. HRMS (FAB) m/z 563.2100 (calcd for
C29H37O5N2Cl2: 563.2080). Anal. Calcd for C29H37N2O5Cl2: C,
61.81; H, 6.44; N, 4.96; Cl, 12.58. Found: C, 61.45; H, 6.46; N,
4.85; Cl, 12.54.
4.1.24. Ethyl 4-({4-chloro-2-[(2-chlorophenyl)carbonyl]-
phenyl}amino)-4-oxobutanoate (22)
4.1.19. Ethyl (4-{4-[{4-chloro-2-[(2-chlorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperazin-1-yl)acetate (18)
Compound 22 was prepared from 3 in a similar manner de-
scribed for 6 as a colorless amorphous. 1H NMR (CDCl3) d 1.27
(3H, t, J = 7.1 Hz), 2.74–2.77 (2H, m), 2.81–2.85 (2H, m), 4.17 (2H,
q, J = 7.1 Hz), 7.29–7.33 (2H, m), 7.39–7.42 (1H, m), 7.48–7.53
(3H, m), 8.78 (1H, d, J = 9.3 Hz), 11.48 (1H, br s). IR (ATR) cmꢀ1
3291, 1735, 1698, 1637, 1577, 1425, 1398. MS (ESI) m/z 396
[(M+H)+, 37Cl], 394 [(M+H)+, 35Cl]. Anal. Calcd for C19H17Cl2NO4:
C, 57.88; H, 4.35; Cl, 17.99; N, 3.55; found: C, 57.84; H, 4.34; Cl,
17.97; N, 3.42.
Compound 18 was prepared from 7 in a similar manner de-
scribed for 8 in 71% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.91 (9H, s), 1.27 (3H, t, J = 7.10 Hz), 2.11–2.22 (2H, m),
2.38–2.66 (5H, m), 3.05–3.12 (1H, m), 3.20 (2H, s), 3.27 (1H, d,
J = 13.7 Hz), 3.48–3.72 (4H, m), 4.18 (2H, q, J = 7.10 Hz), 4.50 (1H,
d, J = 13.7 Hz), 6.15 (1H, d, J = 5.0 Hz), 6.28 (1H, d, J = 5.0 Hz), 6.97
(1H, s), 7.24–7.41 (5H, m), 7.92 (1H, d, J = 7.6 Hz). IR (ATR) cmꢀ1
3266, 2962, 1749, 1664, 1625, 1440, 1180, 1160, 746. MS (ESI)
m/z 592 (M+H)+. Anal. Calcd for C30H39N3O5Cl2ꢁ0.5H2O: C, 59.90;
H, 6.70; N, 6.99; Cl, 11.79. Found: C, 60.04; H, 6.61; N, 6.93; Cl,
12.03.
4.1.25. 4-({4-Chloro-2-[(2-chlorophenyl)carbonyl]phenyl}-
amino)-4-oxobutanoic acid (23)
Compound 23 was prepared from 22 in a similar manner
described for 7 in 67% yield as a colorless crystal. 1H NMR (CDCl3)
d 2.79–2.85 (4H, m), 7.26–7.34 (2H, m), 7.39–7.43 (1H, m),
7.48–7.54 (3H, m), 8.76 (1H, d, J = 9.3 Hz), 11.50 (1H, br s). Mp
152–155 °C. MS (ESI) m/z 366 [(M+H)+, 35Cl].
4.1.20. Ethyl 5-[{4-chloro-2-[(2-chlorophenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-5-oxopentanoate (19a)
Compound 19a was prepared from 5 in a similar manner de-
scribed for 6 in quantum yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.91 (9H, s), 1.17–1.28 (3H, m), 1.77–2.56 (6H, m),
4.00–4.16 (2H, m), 4.35 (1H, d, J = 13.7 Hz), 4.53 (1H, d,
J = 13.7 Hz), 6.11 (1H, br s), 6.34 (1H, s), 7.19–7.76 (7H, m). MS
(FAB) m/z 480 (M+H)+. HRMS (FAB) m/z 480.1682 (calcd for
4.1.26. Ethyl 1-[4-({4-chloro-2-[(2-chlorophenyl)carbonyl]-
phenyl}amino)-4-oxobutanoyl]piperidine-4-carboxylate (24)
Compound 24 was prepared from 23 in a similar manner
described for 8 in 95% yield as a colorless crystal. 1H NMR (CDCl3)
d 1.26 (3H, t, J = 7.1 Hz), 1.59–1.78 (2H, m), 1.89–2.05 (2H, m),
2.51–2.57 (1H, m), 2.72–2.90 (5H, m), 3.14–3.21 (1H, m),
C29H37O5N2Cl2: 480.1708).

M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
1939
3.87–3.94 (1H, m), 4.13 (2H, q, J = 7.1 Hz), 4.37–4.43 (1H, m),
7.26–7.33 (2H, m), 7.37–7.43 (1H, m), 7.47–7.54 (3H, m), 8.78
(1H, d, J = 9.1 Hz), 11.50 (1H, br s). IR (ATR) cmꢀ1 3300, 3200,
1727, 1702, 1641, 1598, 1577, 1504, 1432, 1398, 1375, 1315,
1240, 1162, 1101. Mp 155–157 °C (AcOEt–Hexane). MS (ESI) m/z
507 [(M+H)+, 37Cl], 505 [(M+H)+, 35Cl]. HRMS (FAB) m/z 507.1316
6.23–6.26 (1H, m), 6.51 (0.4H, d, J = 5.6 Hz), 6.59 (0.6H, d,
J = 2.4 Hz), 7.01–7.04 (1H, m), 7.26–7.35 (3H, m), 7.39–7.43 (1H,
m), 7.77–8.00 (1H, m). MS (ESI) m/z 551 [(M+H)+, 37Cl], 549
[(M+H)+, 35Cl]. HRMS (FAB) m/z 549.1901 (calcd for C28H35Cl2N2O5:
549.1923).
(calcd for
C
25H27Cl2N2O5: 507.1297). Anal. Calcd for C25H26
-
4.1.31. 4-Chloro-2-(2-chlorobenzyl)-N-(2,2-dimethylpropyl)-
aniline (27)
A solution of triphenylphosphineoxide (329 mg, 1.18 mmol) in
CH2Cl2 (5.0 ml) was cooled to 0 °C, added trifulic anhydride
Cl2N2O5: C, 59.41; H, 5.19; Cl, 14.03; N, 5.54; found: C, 59.41; H,
5.13; Cl, 13.87; N, 5.61.
4.1.27. Ethyl 1-{4-[{4-chloro-2-[(2-chlorophenyl)carbonyl]-
phenyl}(methyl)amino]-4-oxobutanoyl}piperidine-4-carb-
oxylate (25a)
(109 ll, 0.65 mmol), and stirred for 1 h. At the same temperature,
the mixture was added [5-chloro-2-(2,2-dimethylpropylami-
no)phenyl](2-chlorophenyl)methanol (200 mg, 0.591 mmol) and
stirred for 2 h. Then, the reaction mixture was added sodium boro-
hydrate (89.5 mg, 2.36 mmol) and allowed to warm to room tem-
perature for 2 h. The solution was removed under reduced
pressure, the residue was extracted with AcOEt, and then saturated
NH4Claq. The organic layer was washed with brine and dried over
Na2SO4. The solution was concentrated in vacuo and the residue
was purified by column chromatography (n-hexane/AcOEt = 1:0–
0:1) to give the title compound (166 mg, 87%). 1H NMR (CDCl3) d
0.81 (9H, s), 2.77 (2H, s), 3.34 (1H, br s), 3.94 (2H, s), 6.55 (1H, d,
J = 8.8 Hz), 6.97–7.03 (2H, m), 7.10–7.18 (3H, m), 7.38–7.42 (1H,
m). MS (ESI) m/z 322 (M+H)+.
To the solution of compound 24 (0.52 g, 1.03 mmol) in N,N-
dimethylformamide (10 ml) was added sodium hydride (60%net,
45 mg, 1.13 mmol) at 0 °C, and stirred for 10 min. Then, the reac-
tion mixture was added diazomethane (0.79 ml, 0.73 g, 5.15 mmol)
and stirred for 50 min at the same temperature. The reaction mix-
ture was diluted with AcOEt and organics were washed with brine.
The extract was dried over Na2SO4 and then concentrated in va-
cuo, and the residue was purified with silica gel column chroma-
tography (0–5% MeOH- CH2Cl2 as eluent) to give compound 25a
(0.51 g, 0.98 mmol, 95%) as a colorless oil. 1H NMR (CDCl3) d 1.25
(3H, t, J = 7.1 Hz), 1.55–1.74 (2H, m), 1.86–1.97 (2H, m), 2.16–
2.25 (1H, m), 2.34–2.62 (3H, m), 2.76–2.90 (2H, m), 2.99 (3.28)
(3H, s), 3.82–3.90 (1H, m), 4.14 (2H, q, J = 7.1 Hz), 4.31–4.40 (1H,
m), 7.40–7.59 (7H, m). MS (ESI) m/z 521 [(M+H)+, 37Cl], 519
[(M+H)+, 35Cl].
4.1.32. Ethyl 1-(4-{[4-chloro-2-(2-chlorobenzyl)phenyl](2,2-
dimethylpropyl)amino}-4-oxobutanoyl)piperidine-4-carboxy-
late (28)
A
solution of 1-(3-carboxypropionyl)piperidine-4-carboxylic
acid ethyl ester (100 mg, 0.384 mmol) in CH2Cl2 (3.0 ml) was
added N,N-dimethylformamide (50
l) and cooled to ꢀ15 °C. The
mixture was added oxalyl chloride (40.2 l, 0.471 mmol) and stir-
4.1.28. Ethyl 1-{4-[{4-chloro-2-[(2-chlorophenyl)carbonyl]-
phenyl}(propyl)amino]-4-oxobutanoyl}piperidine-4-carb-
oxylate (25b)
l
l
Compound 25b was prepared from 24 in a similar manner de-
scribed for 25a in 68% yield as a pale yellow amorphous. 1H NMR
(CDCl3) d 0.83 (0.89) (3H, t, J = 7.6 Hz), 1.25 (3H, t, J = 7.1 Hz),
1.40–1.77 (4H, m), 1.86–1.97 (2H, m), 2.14–2.25 (1H, m), 2.36–
2.54 (3H, m), 2.76–2.91 (3H, m), 3.06–3.15 (1H, m), 3.86–3.91
(1H, m), 3.95–4.02 (1H, m), 4.13 (2H, q, J = 7.1 Hz), 4.29–4.37
(1H, m), 7.27–7.47 (6H, m), 7.54 (7.55) (1H, d, J = 8.3 Hz). MS
(ESI) m/z 549 [(M+H)+, 37Cl], 547 [(M+H)+, 35Cl]. HRMS (FAB) m/z
547.1789 (calcd for C28H33Cl2N2O5: 547.1767).
red for 1 h. The solution was concentrated in vacuo. The residue
was dissolved in CH2Cl2 (3.0 ml) and added [4-chloro-2-(2-chloro-
benzyl)phenyl](2,2-dimethylpropyl)amine (82.5 mg, 0.256 mmol)
at 0 °C, 4-dimethylaminopyridine (47.0 mg, 0.384 mmol) and
diisopropylethylamine (66.9 ll, 0.384 mmol). The reaction mixture
was stirred for 12 h and treated with H2O. The organic layer was
washed with brine and dried over Na2SO4. The solution was con-
centrated in vacuo and the residue was purified by column chro-
matography (n-hexane/AcOEt = 1:0–0:1) to give the title
compound (45.3 mg, 32%). 1H NMR (CDCl3) d 0.92 (9H, s), 1.25
(3H, t, J = 7.2 Hz), 1.53–1.75 (2H, m), 1.85–1.95 (2H, m), 1.95–
2.13 (1H, m), 2.40–2.82 (6H, m), 3.05–3.13 (1H, m), 3.79–3.89
(1H, m), 3.95–4.06 (2H, m), 4.14 (2H, q, J = 7.2 Hz), 4.32–4.40
(2H, m), 6.87–6.92 (1H, m), 7.15–7.27 (3H, m), 7.36–7.45 (1H,
m). MS (ESI) m/z 561 (M+H)+. Anal. Calcd for C30H38Cl2N2O4: C,
64.17; H, 6.82; N, 4.99; found: C, 64.10; H, 7.25; N, 4.63.
4.1.29. Ethyl 1-{4-[{4-chloro-2-[(2-chlorophenyl)(hydroxy)-
methyl]phenyl}(methyl)amino]-4-oxobutanoyl}piperidine-4-
carboxylate (26a)
Compound 26a was prepared from 25a in a similar manner de-
scribed for 4 in 83% yield as a colorless amorphous. 1H NMR
(CDCl3) d 1.22–1.27 (3H, m), 1.50–1.72 (2H, m), 1.82–2.04 (3H,
m), 2.20–2.25 (1H, m), 2.48–2.67 (2H, m), 2.80–2.96 (1H, m),
3.08–3.22 (2H, m), 3.37 (3.38) (3H, s), 3.82–3.89 (1H, m),
4.10–4.17 (2H, m), 4.26–4.40 (1H, m), 6.25–6.28 (1H, m), 6.56
(0.45H, d, J = 5.6 Hz, exchangeable with D2O), 6.66 (0.55H, d,
J = 5.9 Hz, exchangeable with D2O), 6.94 (1H, d, J = 2.5 Hz),
7.05–7.08 (1H, m), 7.26–7.32 (3H, m), 7.39–7.43 (1H, m),
7.98–8.01 (1H, m). MS (ESI) m/z 523 [(M+H)+, 37Cl], 521 [(M+H)+,
35Cl]. HRMS (FAB) m/z 521.1636 (calcd for C26H31Cl2N2O5:
521.1610).
4.1.33. {5-Chloro-2-[(2,2-dimethylpropyl)amino]phenyl}-
methanol (31A)
Compound 31A was prepared from 30A in a similar manner de-
scribed for 5 in 73% yield as a colorless oil. 1H NMR (CDCl3) d 1.02
(9H, s), 2.88 (2H, s), 4.64 (2H, d, J = 3.2 Hz), 4.85 (1H, br s), 6.58 (1H,
d, J = 8.5 Hz), 7.02 (1H, d, J = 2.4 Hz), 7.14 (1H, dd, J = 8.5, 2.4 Hz). IR
(ATR) cmꢀ1 3568, 2954, 1604, 1581, 1508, 1475, 1200, 1001, 872,
802. MS (ESI) m/z 228 [(M+H)+, Cl35], 230 [(M+H)+, Cl37].
4.1.30. Ethyl 1-{4-[{4-chloro-2-[(2-chlorophenyl)(hydroxy)-
methyl]phenyl}(propyl)amino]-4-oxobutanoyl}piperidine-4-
carboxylate (26b)
Compound 26b was prepared from 25b in a similar manner de-
scribed for 4 in 79% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.88 (3H, t, J = 7.3 Hz), 1.20–1.28 (4H, m), 1.40–1.75
(3H, m), 1.82–2.03 (5H, m), 2.44–2.55 (2H, m), 2.73–2.90 (1H,
m), 3.05–3.29 (2.4H, m), 3.81–3.89 (0.6H, m), 4.11–4.41 (2H, m),
4.1.34. Methyl 4-{[4-chloro-2-(hydroxymethyl)phenyl](2,2-di-
methylpropyl)amino}-4-oxobutanoate (35A)
Compound 35A was prepared from 31A in a similar manner de-
scribed for 6 in 85% yield as a colorless oil. 1H NMR (CDCl3) d 0.89
(9H, s), 2.09–2.42 and 2.80–2.90 (4H, m), 2.68 (4.26) (1H, d,
J = 13.4 Hz), 3.14–3.20 (1H, m), 3.66 (3H, s), 4.49 (4.51) (1H, d,
J = 12.9 Hz), 4.67 (4.68) (1H, d, J = 12.9 Hz), 7.21 (1H, d, J = 8.3 Hz),
7.29 (1H, dd, J = 8.1, 2.2 Hz), 7.66 (1H, d, J = 2.2 Hz). IR (ATR) cmꢀ1

1940
M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
3450, 2952, 1736, 1645, 1479, 1406, 1365, 1236, 1169, 1045, 847. MS
(CDCl3) d 0.87 (0.92) (9H, s), 1.80–1.89 and 2.00–2.24 and 2.33–
2.47 and 2.91–2.99 (4H, m), 2.48 (2.90) (1H, d, J = 13.4 Hz), 4.39
(4.49) (1H, d, J = 13.4 Hz), 5.79 (5.84) (1H, s), 7.15–7.43 (7H, m),
8.00 (1H, d, J = 2.7 Hz). MS (ESI) m/z 404 [(M+H)+, Cl35], 406
[(M+H)+, Cl37].
(ESI) m/z 342 [(M+H)+, Cl35], 364 [(M+Na)+, Cl35].
4.1.35. 4-{[4-Chloro-2-(hydroxymethyl)phenyl](2,2-dimethyl-
propyl)amino}-4-oxobutanoic acid (36A)
Compound 36A was prepared from 35A in a similar manner de-
scribed for 7 in 98% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.90 (9H, s), 2.15–2.45 and 2.77–2.90 (4H, m), 2.68 (1H,
d, J = 13.5 Hz), 4.29 (1H, d, J = 13.5 Hz), 4.50 (1H, d, J = 13.5 Hz),
4.65 (1H, d, J = 13.5 Hz), 7.20 (1H, d, J = 8.5 Hz), 7.30 (1H, dd,
J = 8.5, 2.4 Hz), 7.64 (1H, d, J = 2.4 Hz). IR (ATR) cmꢀ1 3423, 2954,
1712, 1639, 1477, 1394, 1246, 1171, 1093, 1043, 829. MS (ESI)
m/z 328 (M+H)+.
4.1.41. Ethyl 1-{4-[{4-chloro-2-[hydroxy(phenyl)methyl]phe-
nyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}piperidine-4-
carboxylate (37B)
Compound 37B was prepared from 36B in a similar manner de-
scribed for 8 in 53% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.92 (9H, s), 1.24 (1.25) (3H, t, J = 7.1 Hz), 1.47–1.73
(2H, m), 1.75–1.99 (2H, m), 2.00–2.09 (1H, m), 2.14–2.23 (1H,
m), 2.36–2.55 (2H, m), 2.66 (1H, d, J = 13.7 Hz), 2.70–2.89 (1H,
m), 3.05–3.19 (2H, m), 3.79–3.89 (1H, m), 4.12 (4.14) (2H, q,
J = 7.1 Hz), 4.23–4.40 (1H, m), 4.50 (4.52) (1H, d, J = 13.7 Hz),
5.89–5.95 (1H, m), 6.08–6.19 (1H, m), 7.18–7.43 (8H, m). IR
(ATR) cmꢀ1 3388, 2952, 1728, 1626, 1475, 1392, 1274, 1169,
1038, 700. MS (ESI) m/z 543 [(M+H)+, Cl35], 445 [(M+H)+, Cl37].
Anal. Calcd for C30H39ClN2O5: C, 66.35; H, 7.24; N, 5.16; Cl, 6.53;
found: C, 66.41; H, 7.25; N, 4.96; Cl, 6.83.
4.1.36. 1-(4-{[4-Chloro-2-(hydroxymethyl)phenyl](2,2-di-
methylpropyl)amino}-4-oxobutanoyl)piperidine-4-carboxylate
(37A)
Compound 37A was prepared from 36A in a similar manner de-
scribed for 8 in 81% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.89 (9H, s), 1.24 (1.26) (3H, t, J = 7.1 Hz), 1.70–2.05
(5H, m), 2.06–2.21 (1H, m), 2.29–2.54 (2H, m), 2.60 (2.63) (1H, d,
J = 13.4 Hz), 2.70–2.85 (1H, m), 2.92–3.19 (2H, m), 3.78–3.87 (1H,
m), 4.13 (4.14) (2H, q, J = 7.1 Hz), 4.18–4.40 (2H, m), 4.30 (1H, d,
J = 13.6 Hz), 4.67 (1H, d, J = 13.6 Hz), 4.75–4.94 (1H, m),
7.10–7.31 (2H, m), 7.65 (1H, s). IR (ATR) cmꢀ1 3440, 2952, 1728,
1630, 1477, 1392, 1273, 1173, 1039. MS (ESI) m/z 467 [(M+H)+,
Cl35], 469 [(M+H)+, Cl37]. Anal. Calcd for C24H35ClN2O5ꢁ0.2H2O: C,
61.25; H, 7.58; Cl, 7.53; N, 5.95; found: C, 61.06; H, 7.56; Cl,
7.76; N, 5.65.
4.1.42. (2-Amino-5-chlorophenyl)(2-fluorophenyl)methanol
(30C)
Compound 30C was prepared from 29C in a similar manner de-
scribed for 4 in 88% yield as a colorless crystal. 1H NMR (CDCl3) d
4.10 (1H, br s), 6.10 (1H, s), 6.62 (1H, d, J = 8.5 Hz), 7.00 (1H, s),
7.04–7.13 (2H, m), 7.19 (1H, dd, J = 7.6 Hz), 7.22–7.27 (1H, m),
7.29–7.37 (1H, m), 7.43 (1H, dd, J = 7.6 Hz). IR (ATR) cmꢀ1 3307,
1618, 1585, 1483, 1454, 1279, 1217, 1026, 802, 748. MS (ESI) m/z
252 [(M+H)+, Cl35], 254 [(M+H)+, Cl37]. Anal. Calcd for C13H11ClFNO:
C, 61.32; H, 4.43; Cl, 14.04; F, 7.52; N, 5.55; found: C, 61.69; H,
4.38; Cl, 13.96; F, 7.43; N, 5.47.
4.1.37. (2-Amino-5-chlorophenyl)(phenyl)methanol (30B)
Compound 30B was prepared from 29B in a similar manner
described for 4 in 91% yield as a colorless crystal. 1H NMR (CDCl3)
d 3.94 (1H, br s), 5.81 (1H, s), 6.59 (1H, dd, J = 8.8, 1.0 Hz), 7.02–7.09
(2H, m), 7.27–7.41 (5H, m). IR (ATR) cmꢀ1 3213, 1603, 1485, 1448,
1417, 1257, 1200, 1028, 906, 854, 823, 686. MS (ESI) m/z 234
[(M+H)+, Cl35], 236 [(M+H)+, Cl37]. Anal. Calcd for C13H12ClNOꢁ
0.05H2O: C, 66.56; H, 5.20; Cl, 15.11; N, 5.97; found: C, 66.58; H,
5.15; Cl, 15.03; N, 5.90.
4.1.43. {5-Chloro-2-[(2,2-dimethylpropyl)amino]phenyl}-
(2-fluorophenyl)methanol (31C)
Compound 31C was prepared from 30C in a similar manner de-
scribed for 5 in 62% yield as a pale yellow syrup. 1H NMR (CDCl3) d
0.94 (9H, s), 2.49 (1H, br s), 2.82 (2H, s), 4.59 (1H, br s), 6.07 (1H, s),
6.59 (1H, d, J = 8.8 Hz), 7.00 (1H, d, J = 2.2 Hz), 7.05–7.18 (4H, m),
7.28–7.35 (1H, m), 7.38 (1H, ddd, J = 7.6, 7.6, 1.7 Hz). MS (ESI) m/
z 322 [(M+H)+, Cl35], 324 [(M+H)+, Cl37].
4.1.38. {5-Chloro-2-[(2,2-dimethylpropyl)amino]phenyl}-
(phenyl)methanol (31B)
Compound 31B was prepared from 30B in a similar manner de-
scribed for 5 in 45% yield as a pale yellow syrup. 1H NMR (CDCl3) d
0.81 (9H, s), 2.45 (1H, br s), 2.72 and 2.73 (2H, both s), 4.34 (1H, br
s), 5.78 (1H, br s), 6.55 (1H, d, J = 8.8 Hz), 7.08 (1H, d, J = 2.4 Hz),
7.14 (1H, dd, J = 8.6, 2.4 Hz), 7.25–7.40 (5H, m). IR (ATR) cmꢀ1
3409, 2954, 1600, 1579, 1508, 1475, 1252, 1170, 1018, 804, 700.
MS (ESI) m/z 304 [(M+H)+, Cl35], 306 [(M+H)+, Cl37].
4.1.44. Methyl 4-[{4-chloro-2-[(2-fluorophenyl)(hydroxy)meth-
yl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoate (35C)
Compound 35C was prepared from 31C in a similar manner de-
scribed for 6 in 87% yield as a colorless syrup. 1H NMR (CDCl3) d
0.91 (0.93) (9H, s), (1.18–1.27 and 1.93–2.03) 2.14–2.49 and
2.95–3.04 (4H, m), 2.95 (2.97) (1H, d, J = 13.7 Hz), 3.58 (3.61)
(3H, s), 4.51 (4.52) (1H, d, J = 13.7 Hz), 5.05–5.09 (1H, m), 6.08–
6.18 (1H, m), 6.99–7.06 (2H, m), 7.13–7.17 (1H, m), 7.22–7.39
(3H, m), 7.69–7.78 (1H, m). IR (ATR) cmꢀ1 2954, 1736, 1579,
1508, 1367, 1230, 1144, 985, 806, 756, 696. MS (ESI) m/z 436
[(M+H)+, Cl35], 438 [(M+H)+, Cl37].
4.1.39. Methyl 4-[{4-chloro-2-[hydroxy(phenyl)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoate (35B)
Compound 35B was prepared from 31B in a similar manner de-
scribed for 6 in 85% yield as a colorless syrup. 1H NMR (CDCl3) d
(0.70–0.82 and 1.73–1.82) 2.07–2.19 and 2.23–2.44 and 2.94–
3.04 (4H, m), 0.90 (0.92) (9H, s), 2.55 (2.86) (1H, d, J = 13.3 Hz),
3.58 (3.69) (3H, s), 4.45 (4.48) (1H, d, J = 13.3 Hz), 4.71 (4.72)
(1H, br s), 5.81 (5.88) (1H, s), 7.22–7.43 (7H, m), 7.93 (1H, d,
J = 2.2 Hz). IR (ATR) cmꢀ1 3423, 2952, 1736, 1664, 1475, 1238,
1167, 1024, 835, 700, 501. MS (ESI) m/z 418 [(M+H)+, Cl35], 420
[(M+H)+, Cl37].
4.1.45. 4-[{4-Chloro-2-[(2-fluorophenyl)(hydroxy)methyl]phe-
nyl}(2,2-dimethylpropyl)amino]-4-oxobutanoic acid (36C)
Compound 36C was prepared from 35C in a similar manner de-
scribed for 7 in quantum yield as a light green amorphous. 1H NMR
(CDCl3) d 0.91 (0.92) (9H, s), (1.25–1.35 and 1.95–2.10) 2.20–2.50
and 2.85–2.95 (4H, m), 2.87 (2.98) (1H, d, J = 13.6 Hz), 4.47 (4.53)
(1H, d, J = 13.6 Hz), 6.06 (6.17) (1H, s), 6.96–7.07 (1H, m), 7.09–
7.17 (1H, m), 7.20–7.39 (4H, m), 7.70–7.77 (1H, m). MS (ESI) m/z
422 (M+H)+.
4.1.40. 4-[{4-Chloro-2-[hydroxy(phenyl)methyl]phenyl}(2,2-di-
methylpropyl)amino]-4-oxobutanoic acid (36B)
Compound 36B was prepared from 35B in a similar manner de-
scribed for 7 in quantum yield as a colorless amorphous. 1H NMR

M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
1941
4.1.46. Ethyl 1-{4-[{4-chloro-2-[(2-fluorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-4-carboxylate (37C)
2.23 mmol), triethylamine (3.11 ml, 22.3 mmol), and pivaloyl chlo-
ride (2.63 ml, 251.3 mmol) at 0 °C. The mixture was stirred at room
temperature for 1 h. After water was added, the two layers were
separated. The aqueous layer was extracted with CH2Cl2 and the
combined organic layer was washed with brine and dried over
Na2SO4. The solvent was removed under reduced pressure, and
the residue was recrystallized from n-hexane and CH2Cl2 to afford
the title compound (33, 3.912 g, 18.48 mmol, 91%) as a colorless
crystal. 1H NMR (CDCl3) d 1.31 (9H, s), 7.26–7.30 (2H, m), 7.45–
7.52 (2H, m). IR (ATR) cmꢀ1 3294, 2966, 1655, 1593, 1523, 1491,
1396, 1309, 1244, 1173, 827. MS (ESI) m/z 212 (M+H)+. Anal. Calcd
for C11H14ClNO: C, 62.41; H, 6.67; Cl, 16.75; N, 6.62; found: C,
62.37; H, 6.71; Cl, 16.59; N, 6.55.
Compound 37C was prepared from 36C in a similar manner de-
scribed for 8 in 78% yield as a colorless amorphous. 1H NMR
(CDCl3) d (0.90) 0.93 (9H, s), 1.24 (1.25) (3H, t, J = 6.8 Hz), 1.48–
1.72 (2H, m), 1.80–1.99 (2H, m), 2.04–2.21 (2H, m), 2.34–2.55
(2H, m), 2.74–2.90 (1H, m), 3.03–3.20 (3H, m), 3.79–3.88 (1H,
m), 4.12 (4.14) (2H, q, J = 6.8 Hz), 4.21–4.41 (1H, m), 4.53 (4.56)
(1H, d, J = 13.7 Hz), 6.09–6.16 (1H, m), (6.54) 6.55 (1H, d,
J = 5.1 Hz), 6.95–7.03 (1H, m), 7.10–7.14 (1H, m), 7.19–7.34 (4H,
m), 7.90–7.97 (1H, m). IR (ATR) cmꢀ1 3322, 2952, 1728, 1662,
1624, 1477, 1392, 1167, 1038, 756. MS (ESI) m/z 562 (M+H)+. Anal.
Calcd for C30H38ClFN2O5ꢁ0.1CH2Cl2ꢁ0.2H2O: C, 63.07; H, 6.79; N,
4.89; Cl, 7.42; F, 3.31; found: C, 63.24; H, 6.74; N, 4.70; Cl, 7.15;
F, 3.33.
4.1.51. 2-Chloro-3-fluorobenzaldehyde
To
a solution of 2-chloro-3-fluoro-benzoic acid (1.00 g,
5.73 mmol) in THF (40 ml) was added borane–tetrahydrofuran
complex (1.0 M solution, 20.1 ml, 20.1 mmol) at 0 °C. The mixture
was stirred under reflux overnight and then cooled with crushed
ice. After 1 N HClaq and AcOEt were added to the reaction mixture,
the two layers were separated. The organic layer was dried over
Na2SO4 and concentrated in vacuo, and then alcohol residue was
4.1.47. Ethyl 1-{4-[{4-chloro-2-[(2-fluorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-3-carboxylate (38C)
Compound 38C was prepared from 36C in a similar manner de-
scribed for 8 in 93% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.94 (9H, s), 1.19–1.29 (3H, m), 1.30–1.88 (2H, m),
1.90–2.22 (2H, m), 2.26–2.52 (2H, m), 2.57–2.74 (1H, m),
2.86–3.28 (3H, m), 3.47–4.02 (2H, m), 4.06–4.19 (2H, m),
4.34–4.57 (2H, m), 6.13 (1H, s), 6.48–6.60 (8H, m), 6.96–7.03
(1H, m), 7.06–7.15 (1H, m), 7.19–7.34 (4H, m), 7.92–7.99 (1H,
m). IR (ATR) cmꢀ1 3321, 2951, 1728, 1662, 1624, 1475, 1248,
1169, 1030, 756. MS (ESI) m/z 561 [(M+H)+, Cl35], 563 [(M+H)+,
Cl37]. Anal. Calcd for C30H38ClFN2O5ꢁ0.1H2O: C, 64.01; H, 6.84; N,
4.98; Cl, 6.30; F, 3.38; found: C, 64.10; H, 6.68; N, 4.83; Cl, 6.40;
F, 3.40.
obtained. Separately, to the solution of oxalyl chloride (737 ll,
8.60 mmol) in CH2Cl2 (50 ml) was dropped dimethylsulfoxide
(1.22 ml, 17.2 mmol) at ꢀ78 °C. The mixture was stirred for
5 min at the same temperature. Then, the residue described above
was added with CH2Cl2 (15 ml) to the reaction mixture at ꢀ78 °C.
The solution was stirred for 1 h at ꢀ40 °C, and then triethylamine
(3.97 ml, 28.7 mmol) was added. The mixture was stirred and
warmed to room temperature. After 0.5 N HClaq was added, the
two layers were separated. The organic layer was washed with
brine and dried over Na2SO4. The solvent was removed under re-
duced pressure, and the residue was chromatographed (n-hex-
ane/AcOEt = 50:1) to give the title compound (500 mg, 55%). 1H
NMR (CDCl3) d 7.35–7.43 (2H, m), 7.72–7.78 (1H, m), 10.47 (1H, s).
4.1.48. 1-{4-[{4-Chloro-2-[(2-fluorophenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}piper-
idine-4-carboxylic acid (39C)
Compound 39C was prepared from 37C in a similar manner de-
scribed for 9 in quantum yield as a light green amorphous. 1H NMR
(CDCl3) d 0.90 (0.94) (9H, s), 1.24–1.76 (3H, m), 1.77–2.04 (2H, m),
2.05–2.22 (1H, m), 2.28–47 (1H, m), 2.49–2.65 (1H, m), 2.70–2.97
(1H, m), 2.98–3.23 (2H, m), 3.68–3.88 (1H, m), 4.24–4.39 (1H,
m), 4.43–4.57 (1H, m), 6.13 (6.19) (1H, br s), 6.95–7.06 (1H, m),
7.08–7.18 (1H, m), 7.22–7.45 (7.61–7.67 and 7.90–7.97) (5H, m).
IR (ATR) cmꢀ1 3346, 2952, 1728, 1620, 1477, 1396, 1169, 1030,
756. MS (ESI) m/z 533 [(M+H)+, Cl35], 535 [(M+H)+, Cl37]. Anal.
Calcd for C28H34ClFN2O5ꢁ1.0H2O: C, 61.03; H, 6.58; N, 5.08; found:
C, 60.86; H, 6.28; N, 4.85.
4.1.52. N-{4-Chloro-2-[(2-chloro-3-fluorophenyl)hydroxymethyl]-
phenyl}-2,2-dimethylpropionamide (34D)
N-Pivaloyl-4-chloroaniline (33, 595 mg, 2.81 mmol) was dis-
solved in THF (50 ml), and the solution was added sec-butyl lith-
ium c-hexane, n-hexane solution (0.99 M, 5.96 ml, 6.18 mmol) at
ꢀ78 °C, and then stirred at 0 °C for 2 h. The mixture was added
2-chloro-3-fluoro-benzaldehyde (490 mg, 3.09 mmol) at the same
temperature for 30 min. The reaction mixture was poured satu-
rated with NH4Claq and AcOEt. The organic layer was washed with
brine and dried over Na2SO4. The solvent was removed under re-
duced pressure, and the residue was purified by column chroma-
tography (n-hexane/AcOEt = 9:1) to give the title compound
(676 mg, 65%) as light yellow oil. 1H NMR (CDCl3) d 1.27 (9H, s),
3.85 (1H, d, J = 3.5 Hz), 4.81 (1H, br s), 6.95 (1H, d, J = 3.5 Hz),
7.08–7.17 (1H, m), 7.28–7.32 (3H, m), 7.72 (1H, d, J = 8.8 Hz),
8.39 (1H, br s). MS (FAB) m/z 370 (M+H)+.
4.1.49. 1-{4-[{4-Chloro-2-[(2-fluorophenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}piperi-
dine-3-carboxylic acid (40C)
Compound 40C was prepared from 38C in a similar manner de-
scribed for9 in 96%yieldas a lightgreen amorphous. 1H NMR(CDCl3)
d 0.90 (0.93) (9H, s), 1.19–1.47 (1H, m), 1.50–1.88 (2H, m), 1.98–2.22
(2H, m), 2.25–3.22 (7H, m), 3.40–4.07 (2H, m), 4.40–4.58 (2H, m),
6.00–6.21 (2H, m), 6.90–7.04 (1H, m), 7.06–7.46 (4H, m), 7.53–
7.67 (1H, m), 7.84–7.97 (1H, m). IR (ATR) cmꢀ1 3500–3200, 2952,
1728, 1620, 1477, 1396, 1169, 756. MS (ESI) m/z 531 [(M+H)+,
Cl35], 533 [(M+H)+, Cl37]. HRMS (FAB) m/z 533.2195 (calcd for
C28H35ClFN2O5 533.2219). Anal. Calcd for C28H34ClFN2O5ꢁ0.2CH2-
Cl2ꢁ0.3H2O: C, 60.98; H, 6.35; N, 5.04; found: C, 61.09; H, 5.96; N,
4.85.
4.1.53. [5-Chloro-2-(2,2-dimethylpropylamino)phenyl]-(2-
chloro-3-fluorophenyl)methanol (31D)
A
solution of N-{4-chloro-2-[(2-chloro-3-fluorophenyl)hydr-
oxymethyl]phenyl}-2,2-dimethylpropionamide (34D, 670 mg,
1.81 mmol) was dissolved in THF (30 ml). To the solution was
added dropwise a toluene solution of sodium bis(2-methoxyeth-
oxy)aluminum hydride (35%, 3.37 ml, 10.9 mmol) at 0 °C. The mix-
ture was stirred for 2 h at room temperature. The reaction mixture
was added saturated (+)-tartaric acid sodium potassium solution
and stirred for 5 min. The mixture was added AcOEt and separated
with a funnel. The organic layer was washed with brine and dried
over Na2SO4. The solvent was removed under reduced pressure,
and the residue was purified by column chromatography
4.1.50. N-(4-Chlorophenyl)-2,2-dimethylpropanamide (33)
To a solution of 4-Chloroaniline (32, 2.592 g, 20.32 mmol) in
CH2Cl2 (40 ml) was added 4-(dimethylamino)pyridine (273 mg,

1942
M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
(n-hexane/AcOEt = 20:1) to give the title compound (300 mg, 47%)
as light yellow oil. 1H NMR (CDCl3) d 0.94 (9H, s), 2.85 (2H, s), 6.14
(1H, s), 6.62 (1H, d, J = 8.8 Hz), 6.88 (1H, d, J = 2.4 Hz), 7.10–7.18
(2H, m), 7.24–7.30 (2H, m). MS (FAB) m/z 357 (M+H)+.
4.1.58. 1-{4-[{4-Chloro-2-[(2-chloro-3-fluorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-4-carboxylic acid (39D)
Compound 39D was prepared from 37D in a similar manner de-
scribed for 9 in 87% yield as a colorless amorphous. 1H NMR
(CDCl3) d (0.89) 0.91 (9H, s), 1.36–1.74 (3H, m), 1.75–2.27 (3H,
m), 2.28–2.62 (3H, m), 2.68–3.28 (3H, m), 3.71–3.87 (1H, m),
4.20–4.52 (2H, m), 6.14 (1H, s), (6.31) 6.94 (1H, d, J = 8.6 Hz),
7.00–7.46 (4H, m), (7.53–7.58) 7.67–7.77 (1H, m). IR (ATR) cmꢀ1
2954, 2865, 1727, 1623, 1467, 1444, 1396, 1365, 1263, 1170,
1105, 1031, 948, 929, 902, 875, 833, 808. MS (FAB) m/z 567
(M+H)+. Anal. Calcd for C28H33Cl2FN2O5: C, 59.26; H, 5.86; N,
4.94; found: C, 59.49; H, 6.10; N, 4.58.
4.1.54. Methyl 4-[{4-chloro-2-[(2-chloro-3-fluorophenyl)-
(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-
oxobutanoate (35D)
Compound 35D was prepared from 31D in a similar manner de-
scribed for 6 in 55% yield as a colorless solid. 1H NMR (CDCl3) d 0.90
(0.94) (9H, s), 2.18–2.27 (1H, m), 2.23–2.47 (2H, m), 2.87–2.96 (1H,
m), 3.07 (1H, d, J = 16.3 Hz), 3.66 (3.69) (3H, s), 4.49 (1H, d,
J = 16.3 Hz), 6.14 (1H, s), 7.00–7.06 (1H, m), 7.14–7.21 (1H, m),
7.27–7.32 (1H, m), 7.23–7.39 (2H, m), 7.63 (1H, d, J = 7.8 Hz). IR
(ATR) cmꢀ1 3411, 2956, 2869, 1743, 1648, 1577, 1469, 1434,
1409, 1351, 1263, 1232, 1164, 1114, 1068, 1043, 998, 948, 904,
873, 846, 809. MS (FAB) m/z 470 (M+H)+.
4.1.59. 1-{4-[{4-Chloro-2-[(2-chloro-3-fluorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-3-carboxylic acid (40D)
Compound 40D was prepared from 38D in a similar manner de-
scribed for 9 in 92% yield as a colorless amorphous. 1H NMR
(CDCl3) d (0.88) 0.90 (9H, s), 1.46–1.87 (2H, m), 1.88–2.25 (3H,
m), 2.30–2.70 (1H, m), 2.77–3.22 (3H, m), (3.40–3.49) 3.55–4.15
(4.29–4.44) (4.57–4.64) (4H, m), 4.45–4.56 (1H, m), 6.10–6.14
(6.23–6.31) (1H, m), 6.97 (1H, s), 7.01–7.41 (7.42–7.60) (4H, m),
7.62–7.73 (1H, m). IR (ATR) cmꢀ1 2950, 2867, 1727, 1619, 1579,
1467, 142, 1396, 1365, 1261, 1170, 1116, 1043, 1008, 977, 948,
900, 873, 856, 833, 808. MS (FAB) m/z 567 (M+H)+. Anal. Calcd
for C28H33Cl2FN2O5: C, 59.26; H, 5.86; N, 4.94; found: C, 59.04; H,
5.98; N, 4.63.
4.1.55. 4-[{4-Chloro-2-[(2-chloro-3-fluorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoic
acid (36D)
Compound 36D was prepared from 35D in a similar manner de-
scribed for 7 in 86% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.89 (0.90) (9H, s), (1.56–1.58 and 2.05–2.10) 2.24–2.48
and 2.78–2.86 (4H, m), 2.96 (3.08) (1H, d, J = 13.4 Hz), 4.45 (4.50)
(1H, d, J = 13.4 Hz), 6.05 (6.29) (1H, s), 6.92 (1H, d, J = 2.2 Hz),
7.09–7.17 (1H, m), 7.24–7.32 (4H, m), 7.50–7.59 (1H, m). IR
(ATR) cmꢀ1 2956, 2869, 1708, 1637, 1579, 1467, 1442, 1396,
1365, 1322, 1263, 1168, 1114, 1101, 1043, 968, 946, 902, 875,
833, 808. MS (FAB) m/z 456 (M+H)+. Anal. Calcd for
4.1.60. {5-Chloro-2-[(2,2-dimethylpropyl)amino]phenyl}-
(2-chloro-4-fluorophenyl)methanol (31E)
C
22H24Cl2FNO4ꢁ0.4CHCl3: C, 53.81; H, 4.83; N, 2.75; found: C,
53.90; H, 4.99; N, 2.65.
Compound 31E was prepared from 33 in a similar manner de-
scribed for 31D in 14% yield in two steps as a pale yellow amor-
phous. 1H NMR (400 MHz, CDCl3) d 0.93 (9H, s), 2.84 (2H, s), 6.11
(1H, s), 6.61 (1H, d, J = 8.8 Hz), 6.95 (1H, d, J = 2.4 Hz), 6.96–7.05
(1H, m), 7.13–7.18 (2H, m), 7.38–7.42 (1H, m). MS (FAB) m/z 356
(M+H)+.
4.1.56. Ethyl 1-{4-[{4-chloro-2-[(2-chloro-3-fluorophenyl)-
(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-
oxobutanoyl}piperidine-4-carboxylate (37D)
Compound 37D was prepared from 36D in a similar manner
described for 8 in 58% yield as a colorless amorphous. 1H NMR
(CDCl3) d (0.89) 0.91 (9H, s), 1.24 (1.26) (3H, t, J = 7.1 Hz),
1.75–2.00 and 2.05–2.20 (3H, m), 2.41–2.56 (2H, m), (2.69–2.91)
3.00–3.21 (3H, m), 3.12 (1H, d, J = 13.5 Hz), 3.67–3.82 (1H, m),
4.10–4.18 (3H, m), 4.22–4.50 (1H, m), 4.52 (4.53) (1H, d,
J = 13.5 Hz), 6.13–6.16 (1H, m), 6.45–6.71 (6.51–6.54) (1H, m),
6.96 (1H, s), 7.14–7.20 and 7.33–7.40 (3H, m), 7.73–7.80 (1H, m).
IR (ATR) cmꢀ1 3318, 2954, 2865, 1727, 1625, 1577, 1467, 1442,
1392, 1365, 1313, 1263, 1168, 1097, 1039, 987, 970, 946, 902,
875, 833, 808. MS (FAB) m/z 595 (M+H)+. Anal. Calcd for
4.1.61. Methyl 4-[{4-chloro-2-[(2-chloro-4-fluorophenyl)-
(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-
oxobutanoate (35E)
Compound 35E was prepared from 31E in a similar manner de-
scribed for 6 in 60% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.89 (0.90) (9H, s), 2.18–2.25 (1H, m), 2.32–2.49 (2H,
m), 2.88–2.98 (1H, m), 3.02 (1H, d, J = 13.7 Hz), (3.60) 3.68 (3H,
s), 4.48 (1H, d, J = 13.7 Hz), 4.80 (1H, d, J = 5.8 Hz), 6.10 (6.30)
(1H, d, J = 5.8 Hz), 7.02 (1H, d, J = 2.0 Hz), 7.08–7.16 (2H, m),
7.28–7.37 (2H, m), (7.68–7.70) 7.78–7.83 (1H, m). IR (ATR) cmꢀ1
3392, 2960, 1739, 1646, 1600, 1481, 1434, 1411, 1396, 1351,
1288, 1226, 1164, 1112, 1054, 1031, 998, 962, 937, 912, 863,
835, 804. MS (FAB) m/z 470 (M+H)+.
C
30H37Cl2FN2O5ꢁ0.5H2O: C, 59.60; H, 6.34; N, 4.63; found: C,
59.26; H, 6.20; N, 4.46.
4.1.57. Ethyl 1-{4-[{4-chloro-2-[(2-chloro-3-fluorophenyl)-
(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-
oxobutanoyl}piperidine-3-carboxylate (38D)
4.1.62. 4-[{4-Chloro-2-[(2-chloro-4-fluorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoic
acid (36E)
Compound 38D was prepared from 36D in a similar manner
described for 8 in 80% yield as a colorless amorphous. 1H NMR
(CDCl3) d (0.91) 0.92 (9H, s), 1.23 (1.24) (3H, t, J = 7.3 Hz),
(1.65–1.73) 1.90–2.14 (4H, m), 2.20–2.52 and 2.55–2.70 (3H, m),
(2.79–2.88) 2.93–3.18 (2H, m), 3.19–3.28 (3.49–3.56) (1H, m),
3.70–3.86 (3.92–3.99) (4.35–4.44) (4.70–4.73) (2H, m), 4.07–4.19
(2H, m), 4.52 (1H, d, J = 13.7 Hz), 6.16 (6.32) (1H, d, J = 4.1 Hz),
6.93–7.00 (1H, m), 7.14–7.19 (2H, m), 7.28–7.41 (2H, m),
7.72–7.78 (1H, m). IR (ATR) cmꢀ1 3332, 2950, 2865, 1727, 1625,
1579, 1467, 1442, 1392, 1365, 1309, 1261, 1170, 1114, 1068,
1031, 1006, 946, 900, 875, 856, 833, 808. MS (FAB) m/z 595
(M+H)+. Anal. Calcd for C30H37Cl2FN2O5ꢁ0.5H2O: C, 59.60; H, 6.34;
N, 4.63; found: C, 59.26; H, 6.19; N, 4.46.
Compound 36E was prepared from 35E in a similar manner de-
scribed for 7 in 86% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.88 (0.90) (9H, s), (1.38–1.46) (2.01–2.09) 2.21–2.49
(2.77–2.86) (4H, m), 2.89 (3.03) (1H, d, J = 13.7 Hz), 4.43 (4.51)
(1H, d, J = 13.7 Hz), 6.02 (6.28) (1H, s), 6.93–7.14 and 7.27–7.36
(5H, m), 7.62–7.68 (1H, m). IR (ATR) cmꢀ1 2954, 1710, 1639,
1600, 1477, 1394, 1326, 1259, 1226, 1166, 1114, 1052, 1029,
981, 912, 860, 833. MS (FAB) m/z 456 (M+H)+. Anal. Calcd for
C22H24Cl2FNO4ꢁ0.5H2O: C, 56.78; H, 5.41; N, 3.01; found: C,
56.56; H, 5.25; N, 3.07.

M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
1943
4.1.63. Ethyl 1-{4-[{4-chloro-2-[(2-chloro-4-fluorophenyl)-
(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-
oxobutanoyl}piperidine-4-carboxylate (37E)
(2.62 ml, 27.8 mmol) and stirred for 10 min. The solution was
added sodium borohydrate (2.60 g, 68.9 mmol) and stirred for
5 min. The mixture was added H2O (10 ml) and stirred for
20 min at room temperature. The solution was concentrated in va-
cuo and the residue was added CHCl3 and 1 N HClaq. The organic
layer was washed with 1 N NaOHaq and dried over Na2SO4. The
solution was removed in reduced pressure. The residue was
washed with n-hexane to give alcohol as a colorless solid. To the
CH2Cl2 suspention of dimethylsulfoxide (2.43 ml, 34.2 mmol) was
added a solution of oxalyl chloride (1.47 ml, 17.1 mmol) in 50 ml
of dry CH2Cl2 at ꢀ78 °C, and the resulting mixture was stirred for
20 min. CH2Cl2 (15 ml) solution of the alcohol was added at
ꢀ78 °C. The mixture was allowed to warm to ꢀ40 °C for 1 h. The
mixture was added triethylamine (7.89 ml, 57.0 mmol), allowed
to warm to room temperature, and was treated with 0.1 N HClaq.
The organic layer was washed with saturated Na2CO3aq, dried over
Na2SO4, and concentrated in vacuo to give the title compound
(1.33 g, 37%) as a colorless solid after trituration with n-hexane.
1H NMR (400 MHz, CDCl3) d 7.22–7.28 (1H, m), 7.41–7.47 (1H,
m), 7.58–7.64 (1H, m), 10.42 (1H, d, J = 3.2 Hz). MS (EI) m/z 158 M+.
Compound 37E was prepared from 36E in a similar manner
described for 8 in 76% yield as a colorless amorphous. 1H NMR
(CDCl3) d (0.89) 0.90 (9H, s), 1.23 (1.26) (3H, t, J = 7.1 Hz),
(1.28–1.39) 1.49–1.71 (1H, m), 1.78–1.98 (2H, m), 2.06–2.66 (4H,
m), 2.69–2.90 (1H, m), 2.94–3.21 (3.63–3.85) (3H, m), 4.05–4.13
(2H, m), 4.18–4.51 (2H, m), 6.06–6.09 (6.28–6.30) (6.37–6.40)
(6.42–6.48) (1H, m), 6.95 (1H, d, J = 2.0 Hz), 7.08–7.12 (2H, m),
7.17–7.39 (2H, m), (7.66–7.68) 7.84–7.91 (1H, m). IR (ATR) cmꢀ1
2952, 2867, 1727, 1627, 1475, 1448, 1392, 1365, 1313, 1272,
1220, 1168, 1112, 1033, 946, 912, 858, 833, 804, 752. MS (FAB)
m/z 595 (M+H)+. Anal. Calcd for C30H37Cl2FN2O5ꢁ0.5H2O: C, 59.60;
H, 6.34; N, 4.63; found: C, 59.44; H, 6.18; N, 4.58.
4.1.64. Ethyl 1-{4-[{4-chloro-2-[(2-chloro-4-fluorophenyl)-
(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-
oxobutanoyl}piperidine-3-carboxylate (38E)
Compound 38E was prepared from 36E in a similar manner
described for 8 in 63% yield as a colorless amorphous. 1H NMR
(CDCl3) d (0.89) 0.90 (9H, s), 1.23 (1.25) (3H, t, J = 7.1 Hz),
(1.30–1.46) 1.50–1.85 (3H, m), 1.90–2.26 (3H, m), 2.27–3.15 (5H,
m), 3.16–3.26 (3.46–3.52) (1H, m), 3.62–3.87 (3.94–4.00)
(4.32–4.40) (4.69–4.73) (2H, m), (4.43) 4.48 (1H, d, J = 13.7 Hz),
6.07–6.12 (6.28–6.31) (6.35–6.40) (6.43–6.47) (1H, m), 6.95 (1H,
s), 7.05–7.12 (2H, m), 7.19–7.46 (2H, m), (7.63–7.71) 7.83–7.93
(1H, m). IR (ATR) cmꢀ1 2950, 2865, 1727, 1627, 1475, 1442,
1392, 1309, 1280, 1255, 1220, 1174, 1114, 1031, 912, 856, 833,
804, 754. MS (FAB) m/z 595 (M+H)+. Anal. Calcd for
4.1.68. N-{4-Chloro-2-[(2-chloro-5-fluorophenyl)(hydroxy)-
methyl]phenyl}-2,2-dimethylpropanamide (34F)
Compound 34F was prepared from 33 in a similar manner de-
scribed for 34D in 46% yield as a colorless solid. 1H NMR
(400 MHz, CDCl3) d 1.30 (9H, s), 3.90 (1H, d, J = 3.5 Hz), 5.99 (1H,
d, J = 3.5 Hz), 6.93 (1H, br s), 6.98–7.04 (1H, m), 7.27–7.36 (3H,
m), 7.63 (1H, d, J = 8.8 Hz), 8.21 (1H, br s). MS (FAB) m/z 371
(M+H)+.
C
30H37Cl2FN2O5ꢁ0.5H2O: C, 59.60; H, 6.34; N, 4.63; found: C,
4.1.69. {5-Chloro-2-[(2,2-dimethylpropyl)amino]phenyl}(2-
chloro-5-fluorophenyl)methanol (31F)
59.44; H, 6.18; N, 4.58.
Compound 31F was prepared from 34F in a similar manner de-
scribed for 31D in 87% yield as a pale yellow syrup. 1H NMR
(400 MHz, CDCl3) d 0.95 (9H, s), 2.85 (2H, s), 6.09 (1H, s), 6.63
(1H, d, J = 8.8 Hz), 6.90 (1H, d, J = 2.4 Hz), 6.98–7.07 (1H, m),
7.14–7.21 (2H, m), 7.37 (1H, dd, J = 5.1, 8.8 Hz). MS (FAB) m/z
357 (M+H)+.
4.1.65. 1-{4-[{4-Chloro-2-[(2-chloro-4-fluorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-4-carboxylic acid (39E)
Compound 39E was prepared from 37E in a similar manner de-
scribed for 9 in 83% yield as a colorless amorphous. 1H NMR
(CDCl3) d (0.89) 0.90 (9H, s), (1.20–1.38) 1.50–1.72 (2H, m), 1.75–
2.02 (3H, m), 2.05–2.60 (3H, m), 2.70–2.92 (1H, m), 2.96–3.37
(3H, m), 3.67–3.88 (1H, m), 4.21–4.56 (2H, m), 6.10(6.29) (1H, s),
6.94 (1H, d, J = 7.2 Hz), 7.03–7.16 (2H, m), 7.20–7.34 (7.37–7.43)
(2H, m), (7.62–7.67) 7.82–7.93 (1H, m). IR (ATR) cmꢀ1 2956,
2867, 1724, 1621, 1602, 1477, 1396, 1365, 1265, 1222, 1168,
1112, 1029, 912, 858, 833, 804, 750. MS (FAB) m/z 567 (M+H)+.
Anal. Calcd for C28H33Cl2FN2O5ꢁ0.5H2O: C, 55.01; H, 5.33; N, 4.42;
found: C, 55.35; H, 5.58; N, 4.39.
4.1.70. Methyl 4-[{4-chloro-2-[(2-chloro-5-fluorophenyl)-
(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxo-
butanoate (35F)
Compound 35F was prepared from 31F in a similar manner de-
scribed for 6 in 67% yield as a colorless solid. 1H NMR (CDCl3) d 0.90
(0.92) (9H, s), 2.17–2.24 (1H, m), 2.33–2.45 (2H, m), 2.88–2.98 (1H,
m), 3.16 (1H, d, J = 13.7 Hz), (3.61) 3.69 (3H, s), 4.50 (1H, d,
J = 13.7 Hz), 4.97 (1H, d, J = 5.4 Hz), 6.07 (6.30) (1H, d, J = 5.4 Hz),
6.94 (1H, d, J = 2.0 Hz), 6.97–7.03 (1H, m), 7.27–7.35 (3H, m),
7.60–7.64 (1H, m). IR (ATR) cmꢀ1 3338, 3266, 2950, 1729, 1625,
1587, 1465, 1434, 1396, 1357, 1290, 1263, 1199, 1170, 1114,
1064, 1031, 998, 943, 904, 873, 819. MS (FAB) m/z 470 (M+H)+.
4.1.66. 1-{4-[{4-Chloro-2-[(2-chloro-4-fluorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-3-carboxylic acid (40E)
Compound 40E was prepared from 38E in a similar manner de-
scribed for 9 in 83% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.89 (9H, s), 1.18–1.90 (5H, m), 1.91–2.69 (4H, m),
2.80–3.22 (3.36–3.55) (3H, m), 3.69–3.92 (3.96–4.09) (1H, m),
4.30–4.69 (2H, m), 6.00–6.12 (6.20–6.3) (1H, m), 6.83–7.48 (5H,
m), (7.56–7.70) 7.80–7.95 (1H, m). IR (ATR) cmꢀ1 2952, 2867,
1725, 1623, 1602, 1475, 1396, 1365, 1286, 1255, 1222, 1174,
1114, 1052, 1031, 912, 858, 833. MS (FAB) m/z 567 (M+H)+. Anal.
Calcd for C28H33Cl2FN2O5ꢁ0.4CHCl3: C, 55.79; H, 5.43; N, 4.52;
found: C, 55.59; H, 5.58; N, 4.29.
4.1.71. 4-[{4-Chloro-2-[(2-chloro-5-fluorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoic
acid (36F)
Compound 36F was prepared from 35F in a similar manner de-
scribed for 7 in 97% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.90 (0.91) (9H, s), (1.50–1.58) (2.08–2.15) 2.22–2.49
(2.78–2.88) (4H, m), 3.04 (3.07) (1H, d, J = 13.7 Hz), 4.48 (4.53)
(1H, d, J = 13.7 Hz), 6.01 (6.25) (1H, s), 6.88 (1H, d, J = 2.2 Hz),
6.94–7.07 (7.08–7.14) (2H, m), 7.20–7.33 (2H, m), 7.48–7.51
(7.58–7.60) (1H, m). IR (ATR) cmꢀ1 2954, 1710, 1637, 1587, 1469,
1396, 1324, 1261, 1170, 1112, 1052, 1027, 966, 941, 902, 885,
813. MS (FAB) m/z 456 (M+H)+. Anal. Calcd for C22H24Cl2-
FNO4ꢁ0.3CHCl3: C, 54.75; H, 4.94; N, 2.83; found: C, 54.58; H,
5.02; N, 2.91.
4.1.67. 2-Chloro-5-fluorobenzaldehyde
The solution of 2-chloro-5-fluoro-benzoic acid (4.00 g,
23.0 mmol) in THE (200 ml) was added triethylamine (4.14 ml,
29.9 mmol). At ꢀ30 °C, the mixture was added ethyl chloroformate

1944
M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
4.1.72. Ethyl 1-{4-[{4-chloro-2-[(2-chloro-5-fluorophenyl)-
(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-
oxobutanoyl}piperidine-4-carboxylate (37F)
(2H, m), 7.28–7.35 (2H, m), 8.20 (1H, d, J = 8.8 Hz), 9.20 (1H, br).
IR (ATR) cmꢀ1 3313, 2958, 1645, 1510, 1394, 1254, 1039, 818,
746, 654. MS (ESI) m/z 348 (M+H)+.
Compound 37F was prepared from 36F in a similar manner de-
scribed for 8 in 71% yield as a colorless amorphous. 1H NMR
(CDCl3) d (0.89) 0.92 (9H, s), 1.24 (1.26) (3H, t, J = 7.1 Hz), (1.48–
1.74) 1.78–1.99 (3H, m), 2.07–2.23 (2H, m), 2.33–2.54 (2H, m),
(2.55–2.65) 2.71–2.96 (2H, m), 3.00–3.19 (2H, m), 3.33 (1H, d,
J = 13.4 Hz), 3.72–3.87 (1H, m), 4.07–4.16 (2H, m), 4.18–4.43 (1H,
m), 4.52 (4.54) (1H, d, J = 13.4 Hz), 6.04–6.09 (6.22–6.26) (1H, m),
(6.55) 6.62 (1H, d, J = 5.2 Hz), 6.90 (1H, s), 6.92–7.11 (1H, m),
(7.05–7.17) 7.20–7.32 (7.40–7.52) (2H, m), 7.69–7.75 (1H, m). IR
(ATR) cmꢀ1 3330, 2952, 2863, 1727, 1625, 1467, 1392, 1365,
1313, 1261, 1170, 1112, 1035, 946, 902, 875, 813. MS (FAB) m/z
595 (M+H)+. Anal. Calcd for C30H37Cl2FN2O5: C, 60.37; H, 6.27; N,
4.71; found: C, 60.50; H, 6.26; N, 4.70.
4.1.77. {5-Chloro-2-[(2,2-dimethylpropyl)amino]phenyl}-
(2-methoxyphenyl)methanol (31G)
Compound 31G was prepared from 34G in a similar manner de-
scribed for 31D in 49% yield as a colorless solid. 1H NMR (CDCl3) d
0.92 (9H, s), 2.83 (2H, s), 3.27 (1H, br s), 3.89 (3H, s), 4.86 (1H, br s),
5.99 (1H, s), 6.58 (1H, d, J = 8.5 Hz), 6.91–7.00 (3H, m), 7.09–7.15
(2H, m), 7.28–7.34 (1H, m). IR (ATR) cmꢀ1 3421, 2954, 1601,
1508, 1464, 1240, 1026, 752. MS (ESI) m/z 334 (M+H)+.
4.1.78. Methyl 4-[{4-chloro-2-[hydroxy(2-methoxyphenyl)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoate
(35G)
Compound 35G was prepared from 31G in a similar manner de-
scribed for 6 in 79% yield as a colorless solid. 1H NMR (CDCl3) d
(0.91) 0.93 (9H, s), (0.95–1.10) (1.76–1.88) 2.15–2.47 (3H, m),
(2.64–2.70) 2.78–2.94 (1H, m), (2.95) 3.05 (1H, d, J = 13.4 Hz),
(3.58) 3.68 (3H, s), 3.73 (3.82) (3H, s), 4.41–4.47 (1H, m), 4.60
(1H, d, J = 5.1 Hz), 6.09 (6.23) (1H, d, J = 5.1 Hz), 6.84–6.91 (2H,
m), 7.05–7.11 (2H, m), 7.16–7.18 (1H, m), 7.21–7.35 (1H, m),
7.65–7.69 (7.82–7.85) (1H, m). IR (ATR) cmꢀ1 3373, 2949, 1738,
1635, 1242, 1167, 1038, 748. MS (ESI) m/z 448 (M+H)+. Anal. Calcd
for C24H30ClNO5: C, 64.35; H, 6.75; Cl, 7.91; N, 3.13; found: C,
64.27; H, 6.76; Cl, 8.02; N, 3.13.
4.1.73. Ethyl 1-{4-[{4-chloro-2-[(2-chloro-5-fluorophenyl)-
(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-
oxobutanoyl}piperidine-3-carboxylate (38F)
Compound 38F was prepared from 36F in a similar manner
described for 8 in 73% yield as a colorless amorphous. 1H NMR
(CDCl3) d (0.89) 0.92 (9H, s), 1.18–1.28 (3H, m), (1.30–1.45)
1.47–1.86 (3H, m), 1.96–2.23 (3H, m), 2.28–2.51 (2.55–2.72) (2H,
m), 2.83–3.24 (2H, m), 3.27–3.36 (3.46–3.53) (1H, m), 3.68–3.87
(3.94–4.02) (4.33–4.46) (4.68–4.73) (2H, m), 4.48–4.54 (2H, m),
6.04–6.12 (6.24–6.29) (1H, m), 6.54–6.65 (1H, m), 6.90 (1H, s),
6.92–7.02 (1H, m), (7.03–7.14) 7.18–7.30 (7.36–7.57) (2H, m),
7.69–7.77 (1H, m). IR (ATR) cmꢀ1 3320, 2950, 2865, 1727, 1625,
1467, 1392, 1365, 1309, 1278, 1249, 1172, 1112, 1029, 960, 902,
813. MS (FAB) m/z 595 (M+H)+. Anal. Calcd for C30H37Cl2FN2O5:
C, 60.50; H, 6.26; N, 4.70; found: C, 60.48; H, 6.33; N, 4.64.
4.1.79. 4-[{4-Chloro-2-[hydroxy(2-methoxyphenyl)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoic acid (36G)
Compound 36G was prepared from 35G in a similar manner de-
scribed for 7 in 93% yield as a colorless amorphous. 1H NMR
(DMSO-d6) d 0.85 (9H, s), (0.90–1.05) (1.73–1.86) 2.07–2.40 (4H,
m), 2.81 (3.01) (1H, d, J = 14.0 Hz), 3.65 (3.66) (3H, s), 4.27 (4.34)
(1H, d, J = 13.5 Hz), 5.75 (5.81) (1H, s), 5.85 (6.06) (1H, d,
J = 4.9 Hz), 6.86–7.08 (3H, m), 7.17–7.61 (4H, m). IR (ATR) cmꢀ1
3421, 2964, 1734, 1643, 1475, 1394, 1246, 1169, 1028, 756. MS
(ESI) m/z 434 (M+H)+. Anal. Calcd for C23H28ClNO5ꢁ0.1CH2Cl2: C,
62.71; H, 6.42; Cl, 9.62; N, 3.17; found: C, 63.03; H, 6.44; Cl,
9.37; N, 3.12.
4.1.74. 1-{4-[{4-Chloro-2-[(2-chloro-5-fluorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-4-carboxylic acid (39F)
Compound 39F was prepared from 37F in a similar manner de-
scribed for 9 in 64% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.92 (9H, s), 1.50–1.75 (2H, m), 1.77–2.04 (3H, m),
2.07–2.29 (2H, m), 2.30–2.70 (2H, m), 2.72–3.40 (3H, m), 3.71–
3.90 (1H, m), 4.18–4.58 (2H, m), 5.99–6.27 (1H, m), 6.46–7.09
(3H, m), 7.11–7.54 (2H, m), 7.60–7.83 (1H, m). IR (ATR) cmꢀ1
2950, 2865, 1725, 1619, 1587, 1469, 1407, 1365, 1265, 1170,
1112, 1029, 964, 929, 902, 875, 813. MS (FAB) m/z 567 (M+H)+.
Anal. Calcd for C28H33Cl2FN2O5ꢁ0.3CHCl3: C, 56.60; H, 5.53; N,
4.62; found: C, 56.85; H, 5.72; N, 4.57.
4.1.80. Ethyl 1-{4-[{4-chloro-2-[hydroxy(2-methoxyphenyl)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-4-carboxylate (37G)
Compound 37G was prepared from 36G in a similar manner de-
scribed for 8 in 91% yield as a colorless amorphous. 1H NMR
(CDCl3) d (0.91) 0.97 (9H, s), 1.14–1.28 (4H, m), 1.55–1.71 (1H,
m), 1.74–1.98 (2H, m), 2.02–2.23 (2H, m), 2.34–2.53 (2H, m),
2.66–2.90 (2H, m), 2.96–3.19 (2H, m), 3.21–3.34 (1H, m), 3.69
(3H, s), 3.73–3.89 (1H, m), 4.12 (2H, q, J = 7.0 Hz), 4.19–4.55 (2H,
m), 6.04–6.41 (1H, m), 6.85 (1H, d, J = 8.3 Hz), 7.03–7.13 (2H, m),
7.15–7.34 (2H, m), 7.79–7.92 (1H, m).
4.1.75. 1-{4-[{4-Chloro-2-[(2-chloro-5-fluorophenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-3-carboxylic acid (40F)
Compound 40F was prepared from 38F in a similar manner de-
scribed for 9 in 80% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.91 (9H, s), 1.18–1.90 (5H, m), 1.91–3.55 (7H, m),
3.71–4.07 (1H, m), 4.34–4.70 (2H, m), 6.01–6.16 (6.19–6.28) (1H,
m), 6.85–7.10 (2H, m), 7.13–7.54 (3H, m), 7.63–7.79 (1H, m). IR
(ATR) cmꢀ1 2952, 2865, 1725, 1619, 1587, 1469, 1409, 1365,
1249, 1172, 1112, 1052, 1029, 960, 902, 875, 813. MS (FAB) m/z
567 (M+H)+. Anal. Calcd for C28H33Cl2FN2O5ꢁ0.4CHCl3: C, 55.79;
H, 5.43; N, 4.52; found: C, 55.41; H, 5.57; N, 4.43.
IR (ATR) cmꢀ1 3309, 2964, 1730, 1660, 1624, 1444, 1394, 1240,
1176, 1028, 750. MS (ESI) m/z 573 (M+H)+. Anal. Calcd for
C31H41ClN2O6: C, 64.97; H, 7.21; Cl, 6.19; N, 4.89; found: C,
64.71; H, 7.22; Cl, 6.59; N, 4.90.
4.1.81. Ethyl 1-{4-[{4-chloro-2-[hydroxy(2-methoxyphenyl)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-3-carboxylate (38G)
4.1.76. N-{4-Chloro-2-[hydroxy(2-methoxyphenyl)methyl]-
phenyl}-2,2-dimethylpropanamide (34G)
Compound 34G was prepared from 33 in a similar manner de-
scribed for 34D in 76% yield as a colorless crystal. 1H NMR
(400 MHz, CDCl3) d 1.13 (9H, s), 3.93 (3H, s), 4.10 (1H, d,
J = 4.4 Hz), 5.99 (1H, d, J = 4.6 Hz), 6.82–6.93 (2H, m), 6.96–7.00
Compound 38G was prepared from 36G in a similar manner de-
scribed for 8 in 69% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.97 (9H, s), 1.14–1.39 (4H, m), 1.55–1.84 (2H, m),
1.89–2.88 (6H, m), 2.94–3.35 (3H, m), 3.40–3.90 (2H, m), 3.69
(3H, s), 3.94–4.19 (2H, m), 4.33–4.58 (2H, m), 6.05–6.41 (2H, m),
6.85 (1H, d, J = 8.8 Hz), 7.02–7.13 (2H, m), 7.17–7.35 (3H, m),

M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
1945
7.80–7.94 (1H, m). IR (ATR) cmꢀ1 3354, 2954, 1732, 1664, 1628,
4.1.87. 4-[{4-Chloro-2-[hydroxy(3-methoxyphenyl)methyl]-
1473, 1406, 1242, 1163, 1030, 752. MS (ESI) m/z 573 (M+H)+. Anal.
Calcd for C31H41ClN2O6: C, 64.97; H, 7.21; Cl, 6.19; N, 4.89; found:
C, 64.93; H, 7.26; Cl, 6.37; N, 4.87.
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoic acid (36H)
Compound 36H was prepared from 35H in a similar manner de-
scribed for 7 in quantum yield. 1H NMR (CDCl3) d (0.88) 0.91 (9H,
s), 2.06 (1H, d, J = 12.5 Hz), (1.88–1.98) 2.08–2.25 (2H, m), 2.30–
2.45 (2.63–2.76) (2H, m), (2.51) 2.90 (1H, d, J = 13.5 Hz), 3.76
(3H, s), (3.80) 3.82 (3H, s), (4.39) 4.48 (1H, d, J = 13.5 Hz), 4.67
(1H, s), (5.78) 5.74 (1H, s), 6.75–6.87 (3H, m), 6.91–6.98 (1H, m),
7.32–7.17 (2H, m), (7.39) 7.90 (1H, d, J = 2.5 Hz). MS (ESI) m/z
434 (M+H)+. Anal. Calcd for C23H28ClNO5: C, 63.66; H, 6.50; N,
3.23; found: C, 63.47; H, 6.70; N, 2.76.
4.1.82. 1-{4-[{4-Chloro-2-[hydroxy(2-methoxyphenyl)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}piperidine-
4-carboxylic acid (39G)
Compound 39G was prepared from 37G in a similar manner de-
scribed for 9 in 92% yield as a colorless amorphous. 1H NMR
(DMSO-d6) d (0.85) 0.86 (5H, s), 0.88–1.04 (1H, m), 1.20–1.55
(2H, m), 1.69–1.89 (2H, m), 2.03–2.20 (1H, m), 2.26–2.76 (4H,
m), 2.84–3.14 (2H, m), 3.53–3.86 (1H, m), (3.66) 3.67 (3H, s),
4.05–4.20 (1H, m), 4.29 (4.34) (1H, d, J = 13.7 Hz), 5.79–6.09 (2H,
m), 6.86–7.10 (2H, m), 7.17–7.27 (1H, m), 7.29–7.43 (2H, m),
7.49–7.60 (2H, m). IR (ATR) cmꢀ1 2952, 1726, 1620, 1483, 1396,
1284, 1242, 1171, 1030, 750. MS (ESI) m/z 545 (M+H)+. Anal. Calcd
for C29H37ClN2O6: C, 63.90; H, 6.84; Cl, 6.50; N, 5.14; found: C,
63.84; H, 6.91; Cl, 6.63; N, 5.03.
4.1.88. Ethyl 1-{4-[{4-chloro-2-[hydroxy(3-methoxyphenyl)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-4-carboxylate (37H)
Compound 37H was prepared from 36H in a similar manner de-
scribed for 8 in 98% yield. 1H NMR (CDCl3) d (0.89) 0.93 (9H, s),
(1.21–1.29) 1.48–1.62 (2H, m), 1.77–2.10 (3H, m), 2.13–2.28 (1H,
m), 2.36–2.60 (2H, m), 2.69 (1H, d, J = 13.0 Hz), (2.95–3.21) 2.72–
2.91 (2H, m), (3.78) 3.82 (3H, s), 4.08–4.19 (2H, m), 4.22–4.47
(1H, m), 4.51 (4.53) (1H, d, J = 13.5 Hz), 5.80–5.93 (1H, m), 6.12
(1H, d, J = 5.6 Hz), 6.17 (1H, d, J = 5.1 Hz), 6.75–6.92 (2H, m),
7.07–7.11 (1H, m), 7.17–7.38 (7.83–7.74) (3H, m). MS (ESI) m/z
573 (M+H)+. Anal. Calcd for C31H41ClN2O6: C, 64.97; H, 7.21; N,
4.89; found: C, 64.78; H, 7.26; N, 4.83.
4.1.83. 1-{4-[{4-Chloro-2-[hydroxy(2-methoxyphenyl)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}piperidine-
3-carboxylic acid (40G)
Compound 40G was prepared from 38G in a similar manner de-
scribed for 9 in 90% yield as a colorless amorphous. 1H NMR
(DMSO-d6) d (0.85) 0.86 (7H, s), 0.94–1.34 (1H, m), 1.45–1.72
(2H, m), 1.83–2.24 (3H, m), 2.26–2.71 (3H, m), 2.84–3.09 (2H,
m), 3.15–3.88 (2H, m), 3.67 (3H, s), 4.24–4.42 (2H, m), 5.80–6.09
(2H, m), 6.86–7.10 (2H, m), 7.17–7.28 (1H, m), 7.29–7.42 (2H,
m), 7.47–7.60 (2H, m). IR (ATR) cmꢀ1 2951, 1728, 1622, 1475,
1396, 1240, 1167, 1115, 1030, 756. MS (ESI) m/z 545 (M+H)+. Anal.
Calcd for C29H37ClN2O6: C, 63.90; H, 6.84; Cl, 6.50; N, 5.14; found:
C, 64.03; H, 7.09; Cl, 6.49; N, 4.90.
4.1.89. Ethyl 1-{4-[{4-chloro-2-[hydroxy(3-methoxyphenyl)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-3-carboxylate (38H)
Compound 38H was prepared from 36H in a similar manner de-
scribed for 8 in 95% yield. 1H NMR (CDCl3) d (0.89) 0.93 (9H, s),
1.20–1.31 (1H, m), 1.32–1.88 (3H, m), 1.92–2.12 (3H, m), 2.13–
2.52 (2H, m), 2.54–2.86 (2H, m), 2.89–3.29 (3.47–3.56) (2H, m),
(3.77) 3.82 (3H, s), 4.06–4.20 (2H, m), (4.64–4.72) 4.29–4.60 (2H,
m), 5.80–5.92 (1H, m), 6.08–6.21 (1H, m), 6.75–6.91 (3H, m),
7.06–7.12 (1H, m), 7.19–7.30 (2H, m), 7.31–7.40 (7.81–7.72) (1H,
m). MS (ESI) m/z 573 (M+H)+. Anal. Calcd for C31H41ClN2O6: C,
64.97; H, 7.21; N, 4.89; found: C, 64.75; H, 7.25; N, 4.71.
4.1.84. N-{4-Chloro-2-[hydroxy(3-methoxyphenyl)methyl]-
phenyl}-2,2-dimethylpropanamide (34H)
Compound 34H was prepared from 33 in a similar manner de-
scribed for 34D in quantum yield. 1H NMR (CDCl3) d 1.09 (9H, s),
3.35–3.38 (1H, m), 3.76 (3H, s), 5.79 (1H, br s), 6.80–6.94 (1H,
m), 7.08 (1H, d, J = 2.5 Hz), 7.22–7.30 (2H, m), 8.11 (1H, d,
J = 8.8 Hz), 8.72 (1H, br s). MS (ESI) m/z 348 (M+H)+.
4.1.90. 1-{4-[{4-Chloro-2-[hydroxy(3-methoxyphenyl)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}piperidine-
4-carboxylic acid (39H)
Compound 39H was prepared from 37H in a similar manner de-
scribed for 9 in quantum yield. 1H NMR (CDCl3) d (0.89) 0.92 (9H,
s), (1.07–1.17) 1.48–1.74 (2H, m), 1.77–2.11 (3H, m), 2.14–2.30
(1H, m), 2.37–2.61 (2H, m), 2.65–2.90 (2H, m), 2.97–3.21 (2H,
m), (3.77) 3.82 (3H, s), 4.23–4.40 (2H, m), (4.51) 4.53 (1H, d,
J = 13.2 Hz), 5.83 (1H, s), 5.90 (1H, d, J = 4.2 Hz), 6.75–6.91 (3H,
m), 7.06–7.10 (1H, m), 7.18–7.30 (2H, m), 7.32–7.39 (7.81–7.77)
(1H, m). MS (ESI) m/z 545 (M+H)+. Anal. Calcd for C29H37ClN2O6:
C, 63.90; H, 6.84; N, 5.14; found: C, 63.64; H, 7.16; N, 4.79.
4.1.85. {5-Chloro-2-[(2,2-dimethylpropyl)amino]phenyl}-
(3-methoxyphenyl)methanol (31H)
Compound 31H was prepared from 34H in a similar manner de-
scribed for 31D in quantum yield. 1H NMR (CDCl3) d 0.83 (9H, s),
2.58 (1H, br s), 2.73 (2H, s), 3.78 (3H, s), 4.37 (1H, br s), 5.72 (1H,
s), 6.55 (1H, d, J = 8.6 Hz), 6.82–6.85 (1H, m), 6.82–6.85 (1H, m),
7.06 (1H, d, J = 2.5 Hz), 7.06 (1H, d, J = 2.5 Hz), 7.13 (1H, dd,
J = 8.6, 2.7 Hz,), 7.24–7.28 (1H, m). MS (ESI) m/z 334 (M+H)+.
4.1.86. Methyl 4-[{4-chloro-2-[hydroxy(3-methoxyphenyl)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoate
(35H)
4.1.91. 1-{4-[{4-Chloro-2-[hydroxy(3-methoxyphenyl)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}piperidine-
3-carboxylic acid (40H)
Compound 35H was prepared from 31H in a similar manner de-
scribed for 6 in 86% yield. 1H NMR (CDCl3) d (0.90) 0.91 (9H, s),
(1.81–1.91) 2.09–2.22 and 2.23–2.44 (2.63–2.78) (2.93–3.05) (4H,
m), 2.60 (2.86) (1H, d, J = 13.5 Hz), 3.59 (3.69) (3H, s), (3.78) 3.82
(3H, s), (4.47) 4.49 (1H, d, J = 13.5 Hz), (4.66–4.75), 5.76–5.88
(1H, m), 6.77–6.90 (6.92–6.97), (7.03–7.06) (2H, m), 7.32–7.19
(4H, m), (7.40) 7.89 (1H, d, J = 2.0 Hz). MS (ESI) m/z 448 (M+H)+.
Anal. Calcd for C24H30ClNO5ꢁ0.25H2O: C, 63.71; H, 6.79; N, 3.09;
found: C, 63.83; H, 6.77; N, 2.66.
Compound 40H was prepared from 38H in a similar manner de-
scribed for 9 in quantum yield. 1H NMR (CDCl3) d 0.89 (0.93) (9H,
s), 1.02–1.58 (2H, m), 1.60–1.90 (1H, m), 1.96–2.59 (5H, m), 2.62–
2.91 (1H, m), 2.93–3.27 (2H, m), 3.30–3.94 (1H, m), (3.78) 3.82 (3H,
s), 4.31–4.55 (2H, m), 5.78–5.92 (1H, m), 6.75–6.92 (3H, m), 7.04–
7.10 (1H, m), 7.17–7.31 (7.70–7.82) (2H, m), 7.32–7.41 (1H, m). MS
(ESI) m/z 545 (M+H)+. Anal. Calcd for C29H37ClN2O6: C, 63.90; H,
6.84; N, 5.14; found: C, 64.03; H, 7.15; N, 4.87.

1946
M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
4.1.92. N-{4-Chloro-2-[(2-ethoxyphenyl)(hydroxy)methyl]-
phenyl}-2,2-dimethylpropanamide (34I)
m), 4.47 (4.49) (1H, d, J = 13.5 Hz), (5.92) 6.01 (1H, d, J = 5.1 Hz),
(6.09) 6.12–6.20 (1H, m), 6.81–6.89 (1H, m), 6.97–7.09 (1H, m),
7.11–7.40 (4H, m), 7.74–7.83 (7.92–7.89) (1H, m). MS (ESI) m/z
587 (M+H)+. Anal. Calcd for C32H43ClN2O6: C, 65.46; H, 7.38; N,
4.77; found: C, 65.33; H, 7.48; N, 4.55.
Compound 34I was prepared from 33 in a similar manner
described for 34D in quantum yield. 1H NMR (CDCl3) d 1.10 (9H,
s), 1.44 (3H, d, J = 7.2 Hz), 4.05–4.25 (2H, m), 4.29 (1H, d,
J = 4.4 Hz), 5.99 (1H, d, J = 3.7 Hz), 6.80–6.93 (1H, m), 6.96
(1H, d, J = 8.1 Hz), 7.01 (1H, d, J = 2.5 Hz), 7.23–7.33 (2H, m), 8.20
(1H, d, J = 8.8 Hz), 9.19 (1H, br s). MS (ESI) m/z 362 (M+H)+.
4.1.98. 1-{4-[{4-Chloro-2-[(2-ethoxyphenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}piper-
idine-4-carboxylic acid (39I)
4.1.93. {5-Chloro-2-[(2,2-dimethylpropyl)amino]phenyl}-
(2-ethoxyphenyl)methanol (31I)
Compound 39I was prepared from 37I in a similar manner
described for 9 in quantum yield. 1H NMR (CDCl3) d (0.90) 0.94
(9H, s), 1.20 (1.47) (3H, t, J = 7.1 Hz), 1.51–1.72 (2H, m),
1.76–2.02 (3H, m), 2.13–2.31 (2H, m), 2.36–2.59 (2H, m),
2.68–2.85 (1H, m), 2.89 (3.13) (1H, d, J = 13.5 Hz), 2.94–3.11
(3.63–3.73) (1H, m), 3.78–3.90 (1H, m), 4.23–4.43 (1H, m),
(3.42–3.48) 4.00–4.12 (2H, m), 4.48 (4.49) (1H, d, J = 13.5 Hz),
6.16 (1H, d, J = 7.4 Hz), 6.80–6.89 (2H, m), 6.98–7.09 (1H, m),
7.11–7.39 (3H, m), 7.74–7.80 (7.92–7.88) (1H, m). MS (ESI) m/z
559 (M+H)+. Anal. Calcd for C30H39ClN2O6: C, 64.45; H, 7.03; N,
5.01; found: C, 64.49; H, 7.17; N, 4.80.
Compound 31I was prepared from 34I in a similar manner de-
scribed for 31D in quantum yield. 1H NMR (CDCl3) d 0.91 (9H, s),
1.40 (3H, t, J = 6.9 Hz), 2.82 (2H, s), 3.37 (1H, br s), 4.06–4.16 (2H,
m), 4.78 (1H, s), 5.98 (1H, s), 6.57 (1H, d, J = 8.6 Hz), 6.85–6.96
(2H, m), 6.99 (1H, d, J = 2.7 Hz,), 7.09–7.14 (1H, m), 7.23–7.30
(1H, m). MS (ESI) m/z 348 (M+H)+.
4.1.94. Methyl 4-[{4-chloro-2-[(2-ethoxyphenyl)(hydroxy)meth-
yl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoate (35I)
Compound 35I was prepared from 34I in a similar manner de-
scribed for 6 in 91% yield. 1H NMR (CDCl3) d 0.89 (9H, s), 0.90
(10H, s), 1.29 (3H, t, J = 7.4 Hz), 1.47 (10H, t, J = 7.1 Hz), (0.72–
0.83) 1.66–1.76 (1H, m), (2.08–2.18) 2.23–2.34 and 2.36–2.52
(2.73–2.83) (3H, m), 2.80 (2.89) (1H, d, J = 13.7 Hz), 2.93 (4.15)
(1H, d, J = 5.4 Hz), (3.57) 3.68 (3H, s), 3.99–4.17 (2H, m), 4.37
(4.45) (1H, d, J = 13.5 Hz), (6.11) 6.19 (1H, d, J = 5.1 Hz), 6.81–6.91
(2H, m), 6.98–7.04 (1H, m), 7.17–7.33 (7.45–7.51) (7.96–7.94)
(4H, m). MS (ESI) m/z 462 (M+H)+. Anal. Calcd for C25H32ClNO5:
C, 65.00; H, 6.98; N, 3.03; found: C, 64.65; H, 7.05; N, 2.84.
4.1.99. 1-{4-[{4-Chloro-2-[(2-ethoxyphenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}piperidine-
3-carboxylic acid (40I)
Compound 40I was prepared from 38I in a similar manner de-
scribed for 9 in quantum yield. 1H NMR (CDCl3) d (0.90) 0.92
(9H, s), 1.16–1.25 (1.42–1.49) (3H, m), 1.57–1.87 (2H, m), 1.96–
2.13 (1H, m), 2.14–2.32 (1H, m), 2.32–2.58 (2H, m), 2.61–3.27
(4H, m), 3.39–3.67 (1H, m), 3.69–3.92 (1H, m), 3.96–4.12 (2H,
m), 4.32–4.56 (4.61–4.69) (1H, m), 6.09–6.19 (1H, m), 6.79–6.89
(2H, m), 6.97–7.08 (1H, m), 7.10–7.42 (3H, m), 7.67–7.81 (7.93–
7.87) (1H, m). MS (ESI) m/z 559 (M+H)+. Anal. Calcd for
4.1.95. 4-[{4-Chloro-2-[(2-ethoxyphenyl)(hydroxy)methyl]-
phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoic acid (36I)
Compound 35I was prepared from 34I in a similar manner de-
scribed for 7 in quantum yield. 1H NMR (CDCl3) d 0.88 (0.92)
(9H, s), 1.30 (1.48) (3H, t, J = 7.1 Hz), (1.76–1.86) 1.95–2.14 (1H,
m), 2.31–2.47 (2H, m), 2.51–2.60 (2.62–2.72) (2H, m), 2.74 (2.92)
(1H, d, J = 13.7 Hz), 3.97–4.14 (2H, m), 4.36 (4.48) (1H, d,
J = 13.7 Hz), 6.06 (6.23) (1H, s), 6.83–6.92 (2H, m), 6.97–7.03 (2H,
m), 7.13–7.40 (8.03–7.99) (4H, m). MS (ESI) m/z 448 (M+H)+. Anal.
Calcd for C24H30ClNO5ꢁ0.25H2O: C, 63.71; H, 6.79; N, 3.09; found: C,
63.78; H, 6.73; N, 2.96.
C30H39ClN2O6: C, 64.45; H, 7.03; N, 5.01; found: C, 64.48; H, 7.41;
N, 4.68.
4.1.100. [5-Chloro-2-(2,2-dimethylpropylamino)phenyl]-
(2,3-dimethoxyphenyl)methanol (31J)
Compound 31J was prepared from 32 in a similar manner de-
scribed for 31D. 1H NMR (CDCl3) d 0.92 (9H, s), 2.83 (2H, s), 3.83
(3H, s), 3.88 (3H, s), 5.99 (1H, s), 6.57 (1H, d, J = 8.5 Hz), 6.80 (1H,
dd, J = 7.8, 1.5 Hz), 6.90 (1H, dd, J = 8.3, 1.5 Hz), 6.99 (1H, d,
J = 2.4 Hz), 7.03 (1H, dd, J = 8.1, 7.8 Hz), 7.10 (1H, dd, J = 8.5,
2.4 Hz). Mp 118–119 °C (AcOEt–Hexane). MS (ESI) m/z 362
(M+H)+.
4.1.96. Ethyl 1-{4-[{4-chloro-2-[(2-ethoxyphenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-4-carboxylate (37I)
4.1.101. N-{4-Chloro-2-[(2,3-dimethoxyphenyl)hydroxymethyl]-
phenyl}-N-(2,2-dimethylpropyl)succinamic acid methyl ester
(35J)
Compound 35J was prepared from 31J in a similar manner de-
scribed for 6 in 96% yield. 1H NMR (CDCl3) d 0.91 (0.94) (9H, s),
(1.25–1.35) (1.85–1.95) 2.10–2.25 (3H, m), 2.82–2.93 (1H, m),
2.97 (3.08) (1H, d, J = 13.7 Hz), 3.59 (3.60) (3H, s), 3.68 (3.78)
(3H, s), 3.83 (3.88) (3H, s), 4.46 (4.47) (1H, d, J = 13.7 Hz), 4.64
(1H, d, J = 5.1 Hz), 6.04 (6.09) (1H, d, J = 5.1 Hz), 6.73–7.74 (6H,
m). MS (ESI) m/z 478 (M+H)+.
Compound 37I was prepared from 36I in a similar manner de-
scribed for 8 in 99% yield. 1H NMR (CDCl3) d (0.94) 0.94 (9H, s),
1.17–1.28 (6H, m), (1.44–1.72) 1.76–1.99 (3H, m), 2.10–2.29 (2H,
m), 2.37–2.54 (2H, m), 2.72–2.92 (1H, m), 2.95–3.19 (3H, m),
3.78–3.89 (1H, m), 4.02–4.18 (4H, m), 4.22–4.32 (1H, m), 4.36–
4.45 (1H, m), 4.48 (1H, d, J = 13.7 Hz), (5.89) 6.08 (1H, d,
J = 5.1 Hz), 6.12–6.18 (1H, m), 6.86 (1H, d, J = 8.3 Hz), 6.98–7.09
(1H, m), 7.12–7.16 (1H, m), 7.19–7.31 (7.92–7.73) (4H, m). MS
(ESI) m/z 587 (M+H)+. Anal. Calcd for C32H43ClN2O6: C, 65.46; H,
7.38; N, 4.77; found: C, 65.37; H, 7.47; N, 4.66.
4.1.102. N-{4-Chloro-2-[(2,3-dimethoxyphenyl)hydroxymethyl]-
phenyl}-N-(2,2-dimethylpropyl)succinamic acid (36J)
4.1.97. Ethyl 1-{4-[{4-chloro-2-[(2-ethoxyphenyl)(hydroxy)-
methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxobutanoyl}-
piperidine-3-carboxylate (38I)
Compound 38I was prepared from 36I in a similar manner de-
scribed for 8 in 93% yield. 1H NMR (CDCl3) d (0.90) 0.94 (9H, s),
1.17–1.31 (6H, m), 1.33–1.86 (3H, m), 1.93–2.53 (4H, m), 2.55–
2.93 (2H, m), 2.95–3.29 (3H, m), (3.47) 3.49 (1H, d, J = 13.5 Hz),
3.56–3.90 (1H, m), 4.02–4.21 (4H, m), 4.32–4.60 (4.70–4.78) (1H,
Compound 36J was prepared from 35J in a similar manner de-
scribed for 7 in quantum yield. 1H NMR (CDCl3) d (0.91) 0.92
(9H, s), 1.18–1.39 (3.44–3.52) (1H, m), (1.91–2.01) 2.19–2.34 (2H,
m), 2.38–2.53 (2.73–2.84) (2H, m), (2.96) 2.98 (1H, d, J = 13.9 Hz),
3.62 (3.78) (3H, s), (3.83) 3.87 (3H, s), (4.43) 4.47 (1H, d,
J = 24.7 Hz), (6.05) 6.08 (1H, s), 6.70–7.23 (7.83–7.79) (6H, m).
MS (ESI) m/z 464 (M+H)+.

M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
1947
4.1.103. 1-{3-[{4-Chloro-2-[(2,3-dimethoxyphenyl)hydroxy-
methyl]phenyl}-(2,2-dimethylpropyl)carbamoyl]propionyl}-
piperidine-4-carboxylic acid ethyl ester (37J)
4.1.108. {5-Chloro-2-[(2,2-dimethylpropyl)amino]phenyl}-
(2,3-dihydro-1,4-benzodioxin-5-yl)methanol (31K)
Compound 31K was prepared from 34K in a similar manner
described for 31D in 78% yield as a colorless syrup. 1H NMR (CDCl3)
d 0.95 (9H, s), 2.84 (2H, s), 4.27–4.36 (4H, m), 5.95 (1H, s), 6.59 (1H,
d, J = 8.5 Hz), 6.70–6.78 (1H, m), 6.80–6.89 (2H, m), 6.93 (1H, d,
J = 2.4 Hz), 7.10–7.14 (1H, m). MS (CI) m/z 361 M+.
Compound 37J was prepared from 36J in a similar manner
described for 8 in 62% yield. 1H NMR (CDCl3) d 0.94 (9H, s),
1.21–1.25 (3H, m), 1.47–1.97 (4H, m), 2.09–2.22 (2H, m),
2.37–2.51 (2H, m), 2.73–2.92 (1H, m), 3.02–3.18 (2H, m), 3.28
(3.29) (1H, d, J = 13.7 Hz), 3.56 (3.57) (3H, s), 3.79–3.86 (1H, m),
3.87 (3H, s), 4.10–4.16 (2H, m), 4.22–4.28 (4.37–4.43) (1H, m),
4.51 (4.53) (1H, d, J = 13.7 Hz), 6.11 (6.12) (1H, d, J = 5.1 Hz), 6.28
(6.39) (1H, d, J = 5.1 Hz), 6.91–6.94 (1H, m), 7.09–7.24 (4H, m),
7.48–7.52 (1H, m). IR (ATR) cmꢀ1 3316 (br), 3000–2850 (br),
1733, 1662, 1625, 1477, 1442, 1394, 1317. Mp 152–153 °C
(AcOEt–Hexane). MS (ESI) m/z 606, 604 (M+H)+. Anal. Calcd for
4.1.109. Methyl 4-[{4-chloro-2-[2,3-dihydro-1,4-benzodioxin-5-
yl(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxo-
butanoate (35K)
Compound 35K was prepared from 31K in a similar manner
described for 6 in 89% yield as a colorless solid. 1H NMR (CDCl3)
d
0.89 (0.90) (9H, s), (1.80–1.88) 2.15–2.28 and 2.31–2.45
C
32H43ClN2O7: C, 63.72; H, 7.19; Cl, 5.88; N, 4.64; found: C,
(2.82–2.90) (4H, m), 2.90 (2.99) (1H, d, J = 13.7 Hz), (3.59) 3.67
(3H, s), (4.08–4.15) 4.17–4.29 (4.29–4.38) (4H, m), 4.42 (4.44)
(1H, d, J = 13.7 Hz), 4.50–4.53 (1H, m), 6.05 (6.23) (1H, d,
J = 4.9 Hz), (6.63–6.69) 6.70–6.77 (1H, m), 6.83–6.92 (1H, m),
7.18–7.23 (2H, m), 7.87 (1H, s). IR (ATR) cmꢀ1 3409, 2952, 2875,
1735, 1644, 1600, 1473, 1407, 1363, 1324, 1280, 1241, 1193,
1166, 1087, 1039, 993, 956, 921, 889, 817. MS (FAB) m/z 476
(M+H)+.
63.55; H, 7.26; Cl, 5.96; N, 4.51.
4.1.104. 1-{3-[{4-Chloro-2-[(2,3-dimethoxyphenyl)hydroxy-
methyl]phenyl}-(2,2-dimethylpropyl)carbamoyl]propionyl}-
piperidine-3-carboxylic acid ethyl ester (38J)
Compound 38J was prepared from 36J in a similar manner
described for 8 in 51% yield. 1H NMR (CDCl3) d 0.97 (9H, s),
1.2–1.33 (3H, m), 1.5–3.8 (13H, m), 3.56 (3.57) (3H, s), 3.87 (3H,
s), 4.0–4.8 (4H, m), 6.12–6.13 (1H, m), 6.27–6.30, 6.35–6.39 (1H,
m), 6.91 (6.93) (1H, s), 7.08–7.09 (1H, m), 7.15–7.25 (3H, m),
7.48–7.51 (1H, m). IR (ATR) cmꢀ1 3345, 3000–2800 (br), 1731,
1654, 1625, 1475, 1438, 1411, 1367, 1303, 1274. Mp 127–129 °C
(AcOEt–Hexane). MS (ESI) m/z 606, 604 (M+H)+. HRMS (FAB) m/z
603.2834 (calcd for C32H44ClN2O7: 603.2837). Anal. Calcd for
4.1.110. 4-[{4-Chloro-2-[2,3-dihydro-1,4-benzodioxin-5-yl-
(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-oxo-
butanoic acid (36K)
Compound 36K was prepared from 35K in a similar manner
described for 7 in 88% yield as a colorless amorphous. 1H NMR
(CDCl3) d (0.87) 0.91 (9H, s), (1.05–1.14) (1.87–1.96) 2.12–2.52
(2.76–2.83) (4H, m), 2.86 (2.94) (1H, d, J = 13.7 Hz), 4.10–4.33
(4H, m), (4.36) 4.47 (1H, d, J = 13.7 Hz), (6.00) 6.24 (1H, s),
(6.61–6.64) 6.72–6.79 (1H, m), 6.82–6.90 (6.97–7.01) (1H, m),
7.15–7.33 (3H, m), 7.89 (1H, d, J = 2.4 Hz). IR (ATR) cmꢀ1 2950,
2875, 1710, 1637, 1602, 1473, 1396, 1280, 1257, 1193, 1168,
1087, 1051, 956, 921, 889, 817. MS (FAB) m/z 462 (M+H)+. Anal.
Calcd for C23H27ClNO5ꢁ0.3CHCl3: C, 58.93; H, 5.69; N, 2.79; found:
C, 59.38; H, 5.87; N, 2.83.
C
32H43ClN2O7ꢁ0.33C6H14: C, 64.63; H, 7.60; Cl, 5.61; N, 4.43; found:
C, 64.46; H, 7.70; Cl, 5.64; N, 4.26.
4.1.105. 1-{3-[{4-Chloro-2-[(2,3-dimethoxyphenyl)hydroxy-
methyl]phenyl}-(2,2-dimethylpropyl)carbamoyl]propionyl}-
piperidine-4-carboxylic acid (39J)
Compound 39J was prepared from 37J in a similar manner
described for 9 in 91% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.90 (0.97) (9H, s), 1.3–3.3 (13H, m), 3.562 (3.76) (3H,
s), 3.83 (3.87) (3H, s), 4.25–4.6 (2H, m), 6.06 (6.12) (1H, s),
6.7–7.7 (6H, m). IR (ATR) cmꢀ1 2952 (br), 1727, 1625, 1477,
1270. MS (ESI) m/z 578, 576 (M+H)+. Anal. Calcd for
4.1.111. Ethyl 1-{4-[{4-chloro-2-[2,3-dihydro-1,4-benzodioxin-
5-yl(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-
oxobutanoyl}piperidine-4-carboxylate (37K)
Compound 37K was prepared from 36K in a similar manner
described for 8 in 57% yield as a colorless amorphous. 1H NMR
(CDCl3) d (0.90) 0.93 (9H, s), 1.23 (1.25) (3H, t, J = 7.1 Hz),
(1.59–1.80) 1.80–1.97 (3H, m), 2.07–2.32 (1H, m), 2.35–2.52 (3H,
m), 2.68–3.30 (3H, m), 3.66–3.87 (1H, m), 4.02–4.52 (8H, m),
6.05–6.10 (1H, m), 6.18–6.21 (6.30–3.33)(1H, m), (6.61–6.66)
6.70–6.76 (1H, m), (6.82–6.84) 6.88–6.97 (1H, m), 7.12–7.42 (3H,
m), 7.79–7.82 (7.97–8.00) (1H, m). IR (ATR) cmꢀ1 3340, 2950,
2873, 1727, 1625, 1473, 1392, 1363, 1313, 1280, 1259, 1170,
1089, 1039, 958, 921, 887, 817. MS (FAB) m/z 601 (M+H)+. Anal.
Calcd for C32H41ClN2O7: C, 63.94; H, 6.87; N, 4.66; found: C,
63.61; H, 6.86; N, 4.93.
C
30H39ClN2O7ꢁ0.3H2O: C, 62.07; H, 6.88; Cl, 6.11; N, 4.83; found:
C, 62.06; H, 6.87; Cl, 6.10; N, 4.64.
4.1.106. 1-{3-[{4-Chloro-2-[(2,3-dimethoxyphenyl)hydroxy-
methyl]phenyl}-(2,2-dimethylpropyl)carbamoyl]propionyl}-
piperidine-3-carboxylic acid (40J)
Compound 40J was prepared from 40J in a similar manner de-
scribed for 9 in 89% yield. 1H NMR (CDCl3) d 0.90, 0.91, 0.95, 0.96
(total 9H, s each), 1.3–4.9 (20H, m), 4.3–4.7 (1H, m), 6.04–6.14
(1H, m), 6.7–7.7 (6H, m). IR (ATR) cmꢀ1 3050–2850 (br), 1727,
1623, 1477, 1272. MS (ESI) m/z 578, 576 (M+H)+. Anal. Calcd for
4.1.112. Ethyl 1-{4-[{4-chloro-2-[2,3-dihydro-1,4-benzodioxin-
5-yl(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-
oxobutanoyl}piperidine-3-carboxylate (38K)
C
30H39ClN2O7ꢁ0.3H2O: C, 62.07; H, 6.88; Cl, 6.11; N, 4.83; found:
C, 62.12; H, 6.87; Cl, 6.03; N, 4.65.
Compound 38K was prepared from 36K in a similar manner de-
scribed for 8 in 84% yield as a colorless amorphous. 1H NMR
(CDCl3) d (0.90) 0.93 (9H, s), 1.12–1.28 (3H, m), (1.32–1.45)
1.55–1.90 (3H, m), 1.92–2.52 (4H, m), 2.54–3.30 (3H, m),
(3.42–3.52) 3.60–3.89 (1H, m), 4.10–4.37 (7H, m), 4.40–4.59
(4.68–4.4.74) (1H, m), 6.03–6.10 (1H, m), 6.16–6.34 (1H, m),
6.61–6.77 (1H, m), 6.78–6.977 (1H, m), 7.09–7.46 (7.78–7.82)
(4H, m). IR (ATR) cmꢀ1 3322, 2948, 2867, 1727, 1625, 1473,
1392, 1363, 1311, 1280, 1257, 1174, 1114, 1087, 1051, 956, 921,
4.1.107. N-{4-Chloro-2-[2,3-dihydro-1,4-benzodioxin-5-yl-
(hydroxy)methyl]phenyl}-2,2-dimethylpropanamide (34K)
Compound 34K was prepared from 33 in a similar manner
described for 34D in 77% yield as a pale yellow amorphous. 1H
NMR (CDCl3) d 1.16 (9H, s), 3.82 (1H, br s), 4.25–4.40 (4H, m),
5.97 (1H, s), 6.48–6.53 (1H, m), 6.78–6.82 (1H, m), 6.88–6.90
(1H, m), 6.97 (1H, s), 7.28–7.33 (1H, m), 8.17 (1H, d, J = 8.8 Hz),
9.15 (1H, br s). MS (CI) m/z 375 M+.

1948
M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
887, 817. MS (FAB) m/z 601 (M+H)+. Anal. Calcd for C32H41ClN2O7:
C, 63.94; H, 6.87; N, 4.66; found: C, 63.60; H, 6.91; N, 4.76.
dissolved in 1 mL of 0.1 M NaOH. Ten micro liters of the cell lysates
were transferred to a 96-well plate to determine the protein con-
centration in duplicate. Eight hundred microliters of the remains
were saponified for 1 h at 75 °C by adding 2 mL of ethanol and
4.1.113. 1-{4-[{4-Chloro-2-[2,3-dihydro-1,4-benzodioxin-5-
yl(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-
oxobutanoyl}piperidine-4-carboxylic acid (39K)
0.5 mL of 50 (w/v)% KOH. After the addition of 50 or 100
ll of
[3H]cholesterol (0.45
lCi/mL) as an internal standard, the nonsa-
Compound 39K was prepared from 37K in a similar manner
described for 9 in 87% yield as a colorless amorphous. 1H NMR
(CDCl3) d 0.90 (0.93) (9H, s), 1.39–2.00 (4H, m), 2.06–2.59 (4H,
m), 2.65–3.41 (4H, m), 3.62–3.89 (1H, m), 3.96–4.52 (6H, m),
6.10 (6.22) (1H, s), 6.53–6.78 (1H, m), 6.79–6.99 (1H, m),
7.00–7.43 (3H, m), 7.76–7.83 (1H, m). IR (ATR) cmꢀ1 2950, 2933,
2869, 1725, 1621, 1473, 1396, 1365, 1311, 1280, 1259, 1170,
1087, 1031, 956, 921, 889, 815. MS (FAB) m/z 573 (M+H)+. Anal.
Calcd for C30H37ClN2O7ꢁ0.4CHCl3: C, 59.13; H, 6.03; N, 4.48; found:
C, 59.48; H, 6.27; N, 4.50.
ponifiable lipids were extracted with 4.5 mL of petroleum ether.
Water layer was frozen in dry ice and ethanol, and the upper layer
was transferred to another tube. The extracts in the tubes were
dried under N2 gas at 40 °C. The residue was dissolved in 50 ll of
dichloromethane–methanol (2:1) solution including 10 mg/mL
cholesterol, applied onto TLC plastic sheets (Silica gel 60), and
developed with a solvent (toluene–ethyl acetate, 3:1). The radio
activities incorporated into the cholesterol fractions in Aquasol-2
were counted with a liquid scintillation counter.
The protein concentration was determined using a BCA Protein
Assay Kit.
4.1.114. 1-{4-[{4-Chloro-2-[2,3-dihydro-1,4-benzodioxin-5-
yl(hydroxy)methyl]phenyl}(2,2-dimethylpropyl)amino]-4-
oxobutanoyl}piperidine-3-carboxylic acid (40K)
The radioactivities incorporated into the cholesterol fractions
were corrected from the formula as follows:
Radioactivities incorporated (dpm/lg protein) = Radioactivities
Compound 40K was prepared from 38K in a similar manner
described for 9 in quantum yield as a colorless amorphous. 1H
NMR (CDCl3) d 0.90 (0.98) (9H, s), 1.18–1.92 (4H, m), 1.95–2.69
(4H, m), 2.71–3.34 (3H, m), 3.36–3.89 (2H, m), 3.90–4.62 (6H,
m), 6.07 (6.20) (1H, s), 6.60–6.98 (2H, m), 6.99–7.43 (3H, m),
7.72–7.83 (1H, m). IR (ATR) cmꢀ1 2948, 2867, 1725, 1619, 1473,
1396, 1363, 1280, 1255, 1170, 1116, 1087, 1051, 1008, 956, 921,
889, 815. MS (FAB) m/z 573 (M+H)+. Anal. Calcd for
of
[
14C]cholesterol (dpm) ꢂ 50,000 (dpm)/radioactivities of
[3H]cholesterol (dpm)/protein content (
lg)
Referring to the mean radioactivity of the cells in the three wells
treated with 0 nM of inhibitors, inhibition (%) of cholesterol syn-
thesis at each concentration was calculated by the following
equation:
Inhibition (%) = (1 ꢀ arithmetic mean radioactivity incorporated
of three wells at each concentration/arithmetic mean radioactivity
incorporated of three wells at 0 nM) ꢂ 100.
C
30H37ClN2O7ꢁ0.6CHCl3: C, 57.48; H, 5.81; N, 4.30; found: C,
57.80; H, 6.10; N, 4.35.
Acknowledgments
4.2. Biological evaluation procedure of inhibitory effects on
hepatic cholesterol synthesis
We thank the members of Cardiovascular-Metabolics Research
Laboratories for the biological assays, and Daiichi Sankyo RD Asso-
cie Co., Ltd for the elemental analysis. Also, we would like to
acknowledge Dr. Masami Ohtsuka for his helpful scientific guid-
ance and support in the preparation of this manuscript.
4.2.1. Preparation of rat primary hepatocytes
Shechter’s method10 was slightly modified. This study consisted
of three experiments, and the effects of inhibitors at each concen-
tration were evaluated in triplicate. One animal was used to pre-
pare the hepatocytes for each experiment.
A. Supplementary data
Under anesthesia by thiopental sodium (0.1 g/kg, ip), a plastic
catheter was introduced through the portal vein. The liver was per-
fused with Ca2+, Mg2+ free Hanks’ balanced salts solution (pH 7.2)
containing 2% albumin, 0.5 mM EGTA, 10 mM HEPES, and
41.7 mM NaHCO3 at 37 °C for 10 min at 19–21 mL/min; and then
with Ca2+, Mg2+ free Hanks’ balanced salts solution (pH 7.5) con-
taining 0.05% collagenase, 4 mM CaCl2, 10 mM HEPES, and
41.7 mM NaHCO3 for another 15 min. Liver cells were dispersed
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.01.065. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
References and notes
in DMEM supplemented with 100 U/mL penicillin and 100
streptomycin by dissection and gentle pipetting. After filtration
through a 70 m nylon mesh filter (Cell Strainer, BD Falcon), hepa-
lg/mL
1. (a) Tobert, J. A. Nat. Rev. Drug. Disc. 2003, 2, 517; (b) Havel, R. J.; Rapaport, E. N.
Eng. J. Med. 1995, 332, 1491; (c) Shepherd, J. et al N. Eng. J. Med. 1995, 333, 1301.
2. (a) Hodel, C. Toxicol. Lett. 2002, 128, 159; (b) Thompson, P. D.; Clarkson, P.;
Karas, R. H. JAMA 2003, 289, 1681.
3. (a) Abe, I.; Tomesch, J. C.; Wattanasin, S.; Prestwich, G. D. Nat. Prod. Rep. 1994,
11, 279; (b) Rosenberg, S. H. Expert Opin. Ther. Pat. 1998, 8, 521.
l
tocytes were obtained by repeated centrifugation (3 times) at
600 rpm (centrifuge; 5930, swinging bucket rotor: RS-3011M) for
1 min at 4 °C. After the last centrifugation, the medium was chan-
ged to DMEM supplemented with 10% LPDS, 100 U/mL penicillin,
4. One of the original squalene synthase inhibitors, zaragozic acid A, caused
acidosis in rats and dogs; however, the adverse effect was not shown in
monkeys. It might be species-dependent toxicity. Please refer the following
articles: (a) Biller, S. A.; Neuenschwander, K.; Ponpipom, M. M.; Poulter, C. D.
Curr. Pharm. Des. 1996, 2, 1; (b) Gonzalez-Pacanowska, D.; Arison, B.; Havel, C.
M.; Watson, J. A. J. Biol. Chem. 1988, 263, 1301; (c) Bergstrom, J. D.; Kurtz, M. M.;
Rew, D. J.; Amend, A. M.; Karakas, J. D.; Bostedor, R. G.; Bansal, V. S.; Dufresne,
C.; VanMiddlesworth, F. L.; Hensens, O. D. Proc. Natl. Acad. Sci. U.S.A. 1993, 90,
80; (d) Bostedor, R. G.; Karkas, J. D.; Arison, B. H.; Bansal, V. S.; Vaidya, S.;
Germershausen, J. I.; Kurtz, M. M.; Bergstrom, J. D. J. Biol.Chem. 1997, 272, 9197.
5. (a) Hiyoshi, H.; Yanagimachi, M.; Ito, M.; Saeki, T.; Yoshida, I.; Okada, T.; Ikuta,
H.; Shinmyo, D.; Tanaka, K.; Kurusu, N.; Tanaka, H. Eur. J. Pharmacol. 2001, 431,
345; (b) Ishihara, T.; Kakuta, H.; Moritani, H.; Ugawa, T.; Yanagisawa, I. Bioorg.
Med. Chem. 2004, 12, 5899; (c) Ishihara, T.; Kakuta, H.; Moritani, H.; Ugawa, T.;
Sakamoto, S.; Tsukamoto, S.; Yanagisawa, I. Bioorg. Med. Chem. 2003, 11, 2403;
(d) Miki, T.; Kori, M.; Mabuchi, H.; Tozawa, R.; Nishimoto, T.; Sugiyama, Y.;
Teshima, K.; Yukimasa, H. J. Med. Chem. 2002, 45, 4571; (e) Miki, T.; Kori, M.;
and 100 lg/mL streptomycin. Then, viability was determined by
staining with trypan blue. Hepatocytes with over 80% viability
were cultured in 6-well cell culture plates (106 cells/well).
4.2.2. Measurement of cholesterol biosynthetic activity of rat
hepatocytes
One day later, the medium was replaced with media supple-
mented with 5% LPDS, 25 mM HEPES, and inhibitors (final concen-
trations: 0, 1, 3, 10, 30, 100, 300, 1000, and 3000 nM). After
incubation for 1 h at 37 °C, 10 l lCi/
l of [14C]mevalonolactone (5
mL) was added into the media, and the incubation was continued
for another 1 h. The cells were washed with D-PBS (3 times) and

M. Ichikawa et al. / Bioorg. Med. Chem. 19 (2011) 1930–1949
1949
Mabuchi, H.; Banno, H.; Tozawa, R.; Nakamura, M.; Itokawa, S.; Sugiyama, Y.;
Yukimasa, H. Bioorg. Med. Chem. 2002, 10, 401.
6. Usui, H.; Kagechika, K.; Nagashima, H. WO1998/029380.
11. (a) Ieiri, I.; Sugiyama, Y.; Higuchi, S. Expert Opin. Drug Metab. Toxicol.
2009, 5, 703; (b) Shitara, Y.; Horie, T.; Sugiyama, Y. Eur. J. Pharm. Sci.
2006, 27, 425.
7. Yukimasa, H.; Sugiyama, Y.; Tozawa, R. WO1997/010224.
8. Usui, H.; Katakura, S.; Suzuki, M. Jpn. Kokai Tokkyo Koho. 2001, 131 (JP 2001/
354587). The atomic coordinates have been deposited with Protein Data Bank
(PDB code: 3Q30 and 3Q2Z).
9. Hendrickson, J. B.; Singer, M.; Hussoin, M. S. J. Org. Chem. 1993, 58, 6913.
10. Shechter, I.; Klinger, E.; Rucker, M. L.; Engstrom, R. G.; Spirito, J. A.; Islam, M. A.;
Boettcher, B. R.; Weinstein, D. B. J. Biol. Chem. 1992, 267, 8628.
12. (a) Clayden, J.; Moran, W. J.; Edwards, P. J.; LaPlante, S. R. Angew. Chem., Int. Ed.
2009, 48, 6398; (b) Oki, M. Top. Stereochem. 1983, 14, 1; (c) Kitagawa, O.;
Taguchi, T. Yuki Gosei Kagaku Kyokaishi. 2001, 59, 680.
13. The atomic coordinates have been deposited with Protein Data Bank (PDB
code: 3ASX).