Synthesis and evaluation of a novel 2-azabicyclo[2.2.2]octane class of long chain fatty acid elongase 6 (ELOVL6) inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.06.042

A series of novel 2-azabicyclo[2.2.2]octane derivatives was synthesized and evaluated as long chain fatty acid elongase 6 (ELOVL6) inhibitors. Screening of our corporate chemical collections against ELOVL6 resulted in the identification of lead 1. Explora

Full text of DOI:10.1016/j.bmc.2009.06.042

Bioorganic & Medicinal Chemistry 17 (2009) 5639–5647
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of a novel 2-azabicyclo[2.2.2]octane class of long
chain fatty acid elongase 6 (ELOVL6) inhibitors
Takahide Sasaki, Tsuyoshi Nagase, Toshiyuki Takahashi, Akira Nagumo, Ken Shimamura,
Yasuhisa Miyamoto, Hidefumi Kitazawa, Maki Kanesaka, Ryo Yoshimoto,
*
Katsumi Aragane, Shigeru Tokita, Nagaaki Sato
Tsukuba Research Institute, Merck Research Laboratories, Banyu Pharmaceutical Co., Ltd, Okubo 3, Tsukuba, Ibaraki 300-2611, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 2-azabicyclo[2.2.2]octane derivatives was synthesized and evaluated as long chain fatty
acid elongase 6 (ELOVL6) inhibitors. Screening of our corporate chemical collections against ELOVL6
resulted in the identification of lead 1. Exploratory chemistry efforts were applied to lead 1 to identify
the orally available, potent, and selective ELOVL6 inhibitor 28a.
Received 22 April 2009
Revised 6 June 2009
Accepted 11 June 2009
Available online 26 June 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Long chain fatty acid elongase 6 inhibitor
2-Azabicyclo[2.2.2]octane
Anti-diabetic agent
Lipogenesis
1. Introduction
3-enoyl-CoA reductase.4,5 Long chain fatty acid elongase (ELOVL)
enzymes are responsible for the rate-limiting initial condensation
The incidence of type 2 diabetes has dramatically increased
over the past decade. Accumulated evidence suggests a strong cor-
relation between insulin resistance and the development of type 2
diabetes mellitus. An increase in de novo lipid synthesis and fat
storage in tissues such as liver leads to the dysfunction in those tis-
sues, that is, insulin resistance.1 Although it is unclear how in-
creased intracellular lipid content exacerbates tissue and whole
body insulin sensitivity, it has been suggested that increased levels
of long chain fatty acyl-CoA antagonize the metabolic actions of
insulin. Interestingly, recent reports suggested that alteration of
the specific fatty acid component (i.e., palmitoleate) has significant
impact on the insulin sensitivity of liver and whole body.2,3
With regard to de novo lipid synthesis, microsomal enzymes are
responsible for the elongation of long chain fatty acids with chain
length ˜C16 while fatty acid synthase (FAS), a cytosolic enzyme, is
responsible for the de novo synthesis of the fatty acid with chain
lengths up to C16.4 Fatty acid elongation at microsomal fractions
requires four sequential steps; (1) condensation between fatty
acyl-CoA and malonyl-CoA to generate b-ketoacyl-CoA, (2) reduc-
tion by b-ketoacyl-CoA reductase, (3) dehydrogenation by b-
hydroxyacyl-CoA dehydrogenase, and (4) reduction by trans-2,
reaction.6,7
So far, seven ELOVL enzymes have been identified in mammals,
and are designated ELOVL1–7.6–11 Each ELOVL enzyme exhibits dif-
ferent fatty acid substrate preferences and tissue distributions,
suggesting that they play different physiological roles in vivo.12
Among ELOVL enzymes, ELOVL3 and ELOVL6 show the highest
homology to each other (43.7%) and are expressed in the highly lip-
ogenic tissues, such as liver and adipose, that play important roles
in the regulation of lipid metabolism.6,9 ELOVL6 regulates the syn-
thesis of stearoyl-CoA (C18:0) and cis-vaccenoyl-CoA (C18:1).6,7
Regarding the regulatory mechanism for expression, liver ELOVL6
expression is up-regulated by feeding after fasting, high carbohy-
drate diet, and in obese rodents.6,7,13 At the molecular level,
ELOVL6 expression is directly and primarily regulated by sterol
regulatory element-binding protein (SREBP)-1c.14
Recent investigations using gene-deleted mice for ELOVL6 sug-
gest that ELOVL6 regulates the hepatic insulin sensitivity by mod-
ulating the fatty acid components.3 Although the increasing
information on the physiological and pathological roles of ELOVL6
has drawn much attention, a lack of useful chemical tools makes it
difficult to address the pharmacological roles of ELOVLs and their
therapeutic potentials.
Our laboratory recently reported the establishment of a homog-
enous enzyme assay for ELOVL615 and the discovery of an indoledi-
one class of ELOVL6 inhibitors.16 A novel 2-azabicyclo[2.2.2]octane
class of lead 1 was also identified by screening of our corporate
* Corresponding author. Permanent address: Taisho research laboratories, Taisho
pharmaceutical Co., Ltd, 1-403 Yoshinocho, Kitaku, Saitama 331-9530, Japan. Tel.:
+81 48 669 3056; fax: +81 29 877 2029.
E-mail address: nagaaki.sato@po.rd.taisho.co.jp (N. Sato).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.042

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T. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 5639–5647
sulfonyl chloride to furnish target compounds 12–28. Substituting
methyl 8-azabicyclo[3.2.1]octane-2-carboxylate18 for 31, target
compound 11 was prepared following Scheme 2. Compound 28
was resolved by CHIRALPAK AD, eluting with 40% i-PrOH in hex-
anes. The active isomer 28a was obtained as the first-eluted
enantiomer.
H
N
n-Bu
N
S
O O
O
O
1: hELOVL6 (IC50) = 1710 nM
Figure 1. Potency and structure of lead 1.
3. Results and discussion
All the derivatives tested in the present study are racemic mix-
tures of endo-isomers with exception of 28a and 28b. The
corresponding exo-isomers were completely inactive for inhibition
of the ELOVL6 enzyme. Substituent effects on the right-hand
phenyl group of the lead compound 1 were investigated initially
(Table 1). The isopropoxy derivative 2 was 10-fold more potent
than lead 1. The trifluoromethoxy and phenoxy derivatives (3
and 4) were slightly more potent, and the benzyloxy derivative 5
displayed a decreased in activity. The fluoro derivative 6 was de-
void of potency. No noticeable improvement was observed with
methyl, trifluoromethyl, and benzyl substitution as observed with
7, 8, and 9. The 2-azabicyclo[2.2.2]octane ring was modified as
shown in Figure 2. As a result, the corresponding piperidine
derivative 10 (removal of the bridge head) and 8-azabicy-
clo[1.2.3]octane derivative 11 were much less potent than the 2-
azabicyclo[2.2.2]octane derivative 2.
Next, the substituent on the left-hand sulfonyl group was mod-
ified to improve potency using compound 2 as a template (Table 2).
Additionally, metabolic stability of active derivatives was assessed
by percent remaining of the parent after incubation of test com-
pounds with human and mouse microsomes.19 Smaller alkyl sub-
stituents, such as the methyl, ethyl, and propyl groups in
compounds 12–15, resulted only in decreases in potency. Larger al-
kyl groups were not tested since introduction of additional lipo-
chemical collection against human ELOVL6 (Fig. 1). Exploratory
chemistry was applied to this lead to improve intrinsic potency
and metabolic stability with the goal of discovering orally active
tool compounds. In this report, preliminary structure–activity rela-
tionships (SARs) of the novel 2-azabicyclo[2.2.2]octane derivatives
and the discovery of the orally available, potent, selective ELOVL6
inhibitor 28a are reported.
2. Chemistry
Synthesis of compounds 1–10 is illustrated in Scheme 1. Methyl
2-azabicyclo[2.2.2]octane-6-carboxylate hydroiodate (29)17 was
coupled with n-butylsulfonyl chloride followed by hydrolysis of
the ester group under basic conditions to give 30. The carboxylic
acid group of 30 was coupled with the desired aniline to furnish
target compounds 1–9 as a mixture of endo- and exo-isomers.
The active endo-isomers were isolated by silica gel chromatogra-
phy without difficulty. Substituting methyl piperidine-3-carboxyl-
ate for 29, target compound 10 was prepared following Scheme 1.
Scheme 2 outlines the synthesis of target compounds 11–28. The
amino group of 29 was protected as its tert-butylcarbamate. The
ester group of 31 was saponified, followed by coupling with 4-iso-
propoxylaniline to give 32 as a mixture of endo- and exo-isomers,
which were separated by silica gel chromatography. The endo-iso-
mer of 32 was exposed to acidic conditions to remove the tert-
butylcarbonyl group followed by coupling with the desired
philic groups is not preferred from
a metabolic stability
perspective. The phenyl derivative 16 was found to have increased
activity; however, the microsomal stability of 16 was extremely
R1
O
a
b
n
-Bu
HN
HI
n
-Bu
N
N
N
H
OH
S
O
S
H
O O
O O
O
O
29
30
1−10
Scheme 1. Reagents and conditions: (a) (i) n-BuSO2Cl, Et3N, CHCl3, rt, 13 h, 55%; (ii) 5 N aq NaOH, MeOH, rt, 17 h, 97%; (b) (i) 4-R1PhCOOH, EDCIꢀHCl, CHCl3, 14–24 h; (ii)
separation of endo- and exo-isomers by silica gel chromatography, 30–55%.
O
O
a
b
HN
HI
N
N
N
H
O
d
O
Boc
Boc
H
O
O
31
32
29
O
O
O
O
c
2
HN
N
N
H
N
H
R
S
H
H
HCl
O O
11−28
33
Scheme 2. Reagents and conditions: (a) Boc2O, Et3N, DMAP, CHCl3, rt, 24 h, 96%; (b) (i) 5 N aqueous NaOH, MeOH, rt, 3 h, 87%, (ii) 4-i-PrPhCOOH, EDCIꢀHCl, pyridine, rt, 14 h,
(iii) separation of the endo- and exo-isomers by silica gel chromatography, 49%; (c) 4 N HCl–EtOAc, rt, 13 h, 100%; (d) R2SO2Cl, Et3N, CHCl3, rt, 12–24 h, 30–70%.

T. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 5639–5647
5641
Table 1
Table 2
Human ELOVL6 inhibitory activity of compounds 1–9a
Human ELOVL6 inhibitory activity of compounds 18–23a
H
H
N
R2
N
N
N
S
S
O O
O O
O
O
R1
O
Compd
R1
Human ELOVL6b (IC50, nM)
Compd
R2
Human ELOVL6b (IC50, nM)
Microsomal stabilityd
Human
Mouse
O
1
1710 640
120 11
CH3
2
n-Bu
Me
120 11
n-Pr
i-Pr
120 11
>10,000
2900 890
210 38
620 190
70 17
610 210
600 220
3500 790
>10,000
190 42
110 13
130 21
320 62
67 17
35
10
c
c
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
c
c
O
O
61
34
14
11
2
3
4
5
6
7
8
Ph
13
0
c
c
4-MeOPh
3-MeOPh
4-CF3Ph
4-t-BuPh
4-FPh
3-FPh
2-FPh
2-Thiophenyl
3-Thiophenyl
2-Pyridyl
CF3
Ph
c
c
c
c
c
c
510 240
710 240
2900 1100
>10,000
9
8
1
6
6
53
1
3
1
0
1
1
9
0
O
O
Ph
120 43
3-Pyridyl
80
5
F
N
N
28
230 65
88
86
2300 610
1300 280
830 280
CH3
N
N
N
N
28a
220 38
800 95
88
91
75
71
CF3
28b
Ph
9
a
The values represent the mean SE for n P 3.
a
The values represent the mean SE for n P 3.
Inhibitory activity of compounds on human ELOVL6 for palmitoyl-CoA
b
Inhibitory activity of compounds on human ELOVL6 for palmitoyl-CoA
elongation.
b
elongation.
c
Not determined.
d
See Ref. 18 for the experimental conditions for the microsomal stability assay.
poor. Among the substituted phenyl derivatives prepared
(17–23), only fluoro substitution was tolerated. Unfortunately,
metabolic stability of the fluoro derivatives 21–23 also was very
poor. Regarding heteroaromatic derivatives, common bioisosters,
pyridine and thiophene rings, were tolerated as in the derivatives
24–27, which again exhibited very poor metabolic stability. A
hydrophilic imidazole ring was introduced to identify moderately
potent but metabolically significantly stable derivative 28. Com-
pound 28 was resolved to give the enantiomers 28a and 28b. The
active isomer 28a displayed an IC50 value of 221 nM at ELOVL6
and retained excellent metabolic stability. Compound 28a was
selective over other human ELOVL sub-types (ELOVL1, 2, 3, and
5) and has potent inhibitory activity for mouse ELOVL6 as well (Ta-
ble 3). Selectivity over human and mouse ELOVL3 is moderate
probably due to the highest sequence homology of ELOVL6 and
ELOVL3 enzymes among the ELOVL sub-types.
The plasma and liver levels 8 h following 30 mg/kg oral admin-
istration of 28a in mice were determined to be 0.66 lM and
3.95 nmol/g, respectively, demonstrating compound 28a to be
highly liver penetrable (liver-to-plasma ratio: 6.2).
Given the potent ELOVL6 activity and appreciable plasma and
liver exposure, compound 28a was evaluated for its effects on
the fatty acid profile in the liver in mice. ELOVL6 is mainly respon-
sible for the elongation of palmitoyl-CoA. Enzymatic reactions of
palmitoyl-CoA involving ELVOL6 and stearoyl-CoA desaturase 1
(SCD-1) are depicted in Scheme 3. When ELOVL6 elongation takes
place first, palmitoyl-CoA is elongated to give strearoyl-CoA fol-
lowed by desaturation by SCD-1 to give oleoyl-CoA. When the
H
H
n-Bu
N
N
n-Bu
N
N
S
S
O O
O O
O
O
O
O
10: hELOVL6 (IC50) : 930 nM
11: hELOVL6 (IC50) : 5950 nM
Figure 2. Potency and structures of compounds 10 and 11.

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T. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 5639–5647
Table 3
Human ELOVL sub-type selectivity and mouse ELOVL3 and 6 activity of compound 28aa
Enzyme
hELOVL1
>10
hELOVL2
>10
hELOVL3
1510 nM
hELOVL5
>10
hELOVL6
221 nM
mELOVL6
73 nM
mELOVL3
717 nM
Activity (IC50
)
l
M
l
M
lM
a
Inhibitory activity of compound 28a on ELOVLs for their respective substrates. The values are the mean for n P 3.
C16 fatty acids
C18 fatty acids
CoA
CoA
CoA
O
ELOVL6
SCD-1
O
O
Palmitoyl-CoA
Stearoyl-CoA
Oleoyl-CoA
SCD-1
CoA
CoA
ELOVL6
O
O
Palmitoleoyl-CoA
cis-Vaccenoyl-CoA
Scheme 3. Enzymatic reactions of palmitoyl-CoA with ELOVL6 and SCD-1.
and the sulfonyl group of the screen hit 1, orally available and
selective inhibitor 28a was identified. Appreciable liver penetra-
tion was observed after oral administration of 28a in mice. After
oral dosing, 28a potently suppressed the elongation index of fatty
acids in the liver in mice, suggesting that 28a has considerable util-
ity to probe the biology of the ELOVL6 enzyme inhibition in vivo.
Further characterization of the biology of 28a will be reported in
due course.
5. Experimental
5.1. Chemistry
5.1.1. General procedures
Unless otherwise noted, all solvents, chemicals, and reagents
were obtained commercially and used without purification. The
1H NMR spectra were obtained at 400 MHz on a MERCURY-400
(Varian) or a JMN-AL400 (JEOL) spectrometer, with chemical
shift (d, ppm) reported relative to TMS as an internal standard.
Mass spectra were recorded with electron-spray ionization
(ESI) or atmospheric pressure chemical ionization (APCI) on a
Waters micromass ZQ, micromass Quattro II or micromass
Q-Tof-2 instrument. Flash chromatography was carried out with
prepacked silica gel columns (KP-SilTM silica) from Biotage. Pre-
parative thin-layer chromatography (TLC) was performed on a
TLC Silica Gel 60 F (Merck KGaA). Preparative HPLC purification
was carried out on a YMC-Pack Pro C18 (YMC, 50 ꢁ 30 mm id),
eluting with a gradient of CH3CN/0.1% aqueous CF3CO2H = 10/
90 to 50/50 over 8 min at a flow rate of 40 mL/min. Purity of
the target compounds was determined by HPLC with the two
different eluting methods as follows. Analytical HPLC was per-
formed on a SPELCO Ascentis Express (150 ꢁ 4.6 mm id), eluting
with a gradient of (A) 0.1% H3PO4/CH3CN = 95/5 to 10/90 over
7 min followed by 10/90 isocratic over 1 min and (B) 10 mM
potassium phosphate buffer (pH 6.6)/CH3CN = 95/5 to 20/80 over
7 min followed by 20/80 isocratic over 1 min (detection at
210 nm). HPLC retention times and purity for the target com-
pounds are available as Supplementary data. High resolution
Figure 3. Effects of compound 28a on ELOVL6 activity in the liver of C57BL/6J mice.
Male C57BL/6J mice (n = 5) were orally administered 10 mg/kg of compound 28a
(dissolved in 0.5% methylcellulose) and 1 h later [1-14C]-palmitic acid was
interperitoneally administered at 10 lCi/body. At 2 h post-dosing of compound
28a, fatty acids were extracted and measured by radio-HPLC to calculate the
elongation index. *P < 0.05 (ANOVA Dunnett) compared with the vehicle control.
desaturation process mediated by SCD-1 takes place first, palmi-
toyl-CoA is converted to palmitoleoyl-CoA, which is elongated by
ELVOL6 to yield cis-vaccenoyl-CoA. We defined the elongation in-
dex as follows: Elongation index = C18 fatty acids/C16 fatty
acids = [Stearoyl acid + Oleic acid + cis-vaccenic acid]/[palmitic
acid + palmitoleic acid]. The elongation index was used as a surro-
gate readout for ELOVL6 inhibitory activity in the liver using [14C]-
palmitic acid as a radiotracer. After oral administration, compound
28a potently suppressed the elongation index in the liver in mice
(Fig. 3).
4. Conclusion
A new series of 2-azabicyclo[2.2.2]octane derivatives was syn-
thesized and evaluated as ELOVL6 inhibitors. After exploratory
modification of the substituents on the right-hand phenyl group

T. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 5639–5647
5643
mass spectra were recorded with electron-spray ionization on a
micromass Q-Tof-2 instrument.
(2H, m), 7.30–7.46 (7H, m), 7.54 (1H, br s). ESI-MS m/z 457.2
(M+H)+. HRMS (M+H)+ calcd for C25H33N2O4S, 457.2161; found,
457.2169.
5.1.2. (1R*,4S*,6R*)-( )-2-(Butylsulfonyl)-N-(4-methoxyphenyl)-
2-azabicyclo[2.2.2]octane-6-carboxamide (1)
5.1.7. (1R*,4S*,6R*)-( )-2-(Butylsulfonyl)-N-(4-fluorophenyl)-2-
azabicyclo[2.2.2]octane-6-carboxamide (6)
To a solution of 30 (28 mg, 0.1 mmol) and 4-methoxyaniline
(18.5 mg, 0.15 mmol) in pyridine (1 mL) was added 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (29.3 mg,
0.15 mmol), and the mixture was stirred at room temperature for
14 h. The resulting mixture was concentrated, and the residue
was partitioned between ethyl acetate and 1 N aqueous NaOH.
The layers were separated, and the organic layer was washed with
brine, dried over magnesium sulfate, and concentrated. The residue
was purified by preparative TLC (hexanes/ethyl acetate = 55/45) to
give 1 (19.7 mg, 51%). 1H NMR (400 MHz, CDCl3): d 0.96 (3H, t,
J = 7.3 Hz), 1.38–1.52 (2H, m), 1.56–1.67 (1H, m), 1.72–2.03 (6H,
m), 2.07–2.16 (1H, m), 2.27–2.36 (1H, m), 2.92–3.13 (3H, m),
3.23–3.31 (1H, m), 3.51–3.59 (1H, m), 3.79 (3H, s), 3.93–3.98
(1H, m), 6.81–6.89 (2H, m), 7.39–7.47 (2H, m), 7.66 (1H, br s).
ESI–MS m/z 381.1 (M+H)+. HRMS (M+H)+ calcd for C19H29N2O4S,
381.1848; found, 381.1851.
Compound 6 was prepared from 30 and 4-fluoroaniline using
the procedure described for 1. 1H NMR (400 MHz, CDCl3): d 0.96
(3H, t, J = 7.3 Hz), 1.38–1.52 (2H, m), 1.55–1.68 (1H, m), 1.73–
1.90 (4H, m), 1.92–2.15 (3H, m), 2.27–2.37 (1H, m), 2.87–3.16
(3H, m), 3.21–3.30 (1H, m), 3.50–3.59 (1H, m), 3.89–4.00 (1H, m),
6.95–7.06 (2H, m), 7.45–7.54 (2H, m), 7.89 (1H, br s). ESI-MS m/z
369.1 (M+H)+. HRMS (M+H)+ calcd for C18H26N2O3SF, 369.1648;
found, 369.1657.
5.1.8. (1R*,4S*,6R*)-( )-2-(Butylsulfonyl)-N-(4-methylphenyl)-2-
azabicyclo[2.2.2]octane-6-carboxamide (7)
Compound 7 was prepared from 30 and 4-methylaniline using
the procedure described for 1. 1H NMR (400 MHz, CDCl3): d 0.96
(3H, t, J = 7.3 Hz), 1.41–1.53 (2H, m), 1.75–1.88 (6H, m), 1.92–
2.02 (1H, m), 2.09–2.15 (1H, m), 2.28–2.36 (4H, m), 2.91–3.12
(3H, m), 3.24–3.29 (1H, m), 3.54–3.60 (1H, m), 3.93–3.97 (1H,
m), 7.09–7.15 (2H, m), 7.38–7.43 (2H, m), 7.54 (1H, br s). ESI-MS
m/z 365.3 (M+H)+. HRMS (M+H)+ calcd for C19H29N2O3S,
365.1899; found, 365.1909.
5.1.3. (1R*,4S*,6R*)-( )-2-(Butylsulfonyl)-N-[4-(propan-2-yloxy)
phenyl]-2-azabicyclo[2.2.2]octane-6-carboxamide (2)
Compound 2 was prepared from 30 and 4-isopropoxyaniline
using the procedure described for 1. 1H NMR (400 MHz, CDCl3):
d 0.96 (3H, t, J = 7.3 Hz), 1.31 (6H, d, J = 6.3 Hz), 1.40–1.53 (2H,
m), 1.58–1.88 (6H, m), 1.93–2.04 (1H, m), 2.07–2.16 (1H, m),
2.27–2.39 (1H, m), 2.89–3.13 (3H, m), 3.22–3.31 (1H, m), 3.51–
3.62 (1H, m), 3.92–3.99 (1H, m), 4.44–4.56 (1H, m), 6.81–6.87
(2H, m), 7.36–7.43 (2H, m), 7.48 (1H, br s). ESI-MS m/z 409.2
(M+H)+. HRMS (M+H)+ calcd for C21H33N2O4S, 409.2161; found,
409.2166.
5.1.9. (1R*,4S*,6R*)-( )-2-(Butylsulfonyl)-N-[4-(trifluoromethyl)
phenyl]-2-azabicyclo[2.2.2]octane-6-carboxamide (8)
Compound 8 was prepared from 30 and 4-trifluoromethylani-
line using the procedure described for 1. 1H NMR (400 MHz,
CDCl3): d 0.96 (3H, t, J = 7.3 Hz), 1.40–1.52 (2H, m), 1.58–2.00
(7H, m), 2.11–2.17 (1H, m), 2.27–2.37 (1H, m), 2.92–3.08 (2H,
m), 3.11–3.18 (1H, m), 3.22–3.30 (1H, m), 3.55–3.64 (1H, m),
3.94–4.00 (1H, m), 7.53–7.61 (2H, m), 7.65–7.73 (2H, m), 8.09
(1H, br s) ESI-MS m/z 419.2 (M+H)+. HRMS (M+H)+ calcd for
C19H26N2O3SF3, 419.1616; found, 419.1625.
5.1.4. (1R*,4S*,6R*)-( )-2-(Butylsulfonyl)-N-[4-(trifluoromethoxy)
phenyl]-2-azabicyclo[2.2.2]octane-6-carboxamide (3)
Compound 3 was prepared from 30 and 4-trifluoromethox-
yaniline using the procedure described for 1. 1H NMR
(400 MHz, CDCl3): d 0.96 (3H, t, J = 7.3 Hz), 1.38–1.52 (2H, m),
1.57–1.69 (1H, m), 1.73–1.88 (5H, m), 1.91–1.99 (1H, m), 2.09–
2.17 (1H, m), 2.28–2.37 (1H, m), 2.90–3.15 (3H, m), 3.23–3.31
(1H, m), 3.56–3.61 (1H, m), 3.93–3.97 (1H, m), 7.14–7.20 (2H,
m), 7.55–7.61 (2H, m), 7.95 (1H, br s). ESI-MS m/z 435.2
(M+H)+. HRMS (M+H)+ calcd for C19H26N2O4SF3, 435.1565; found,
435.1562.
5.1.10. (1R*,4S*,6R*)-( )-N-(4-Benzylphenyl)-2-(butylsulfonyl)-
2-azabicyclo[2.2.2]octane-6-carboxamide (9)
Compound 9 was prepared from 30 and 4-benzylaniline using
the procedure described for 1. 1H NMR (400 MHz, CDCl3): d 0.96
(3H, t, J = 7.2 Hz), 1.40–1.50 (2H, m), 1.55–2.02 (8H, m), 2.09–
2.14 (1H, m), 2.26–2.35 (1H, m), 2.90–3.13 (3H, m), 3.23–3.30
(1H, m), 3.54–3.61 (1H, m), 3.94 (2H, s), 7.10–7.22 (6H, m), 7.41–
7.48 (2H, m), 7.63 (1H, br s). ESI-MS m/z 441.2 (M+H)+. HRMS
(M+H)+ calcd for C25H33N2O3S, 441.2212; found, 441.2212.
5.1.5. (1R*,4S*,6R*)-( )-2-(Butylsulfonyl)-N-(4-phenoxyphenyl)-
2-azabicyclo[2.2.2]octane-6-carboxamide (4)
5.1.11. ( )-1-(Butylsulfonyl)-N-[4-(propan-2-yloxy)phenyl]
piperidine-3-carboxamide (10)
Compound 4 was prepared from 30 and 4-phenoxyaniline using
the procedure described for 1. 1H NMR (400 MHz, CDCl3): d 0.97
(3H, t, J = 7.4 Hz), 1.21–1.29 (1H, m), 1.40–1.51 (2H, m), 1.59–
1.89 (5H, m), 1.92–2.04 (1H, m), 2.09–2.16 (1H, m), 2.25–2.38
(1H, m), 2.90–3.15 (3H, m), 3.22–3.32 (1H, m), 3.55–3.61 (1H,
m), 3.94–3.99 (1H, m), 6.94–7.02 (4H, m), 7.05–7.11 (1H, m),
7.30–7.35 (2H, m), 7.47–7.54 (2H, m), 7.72 (1H, br s). ESI-MS m/z
443.2 (M+H)+. HRMS (M+H)+ calcd for C24H31N2O4S, 443.2005;
found, 443.2012.
Compound 10 was prepared from methyl piperidine-3-carbox-
ylate and 4-isopropoxyaniline using the procedure described for
30 and 1. 1H NMR (400 MHz, CDCl3): d 0.95 (3H, t, J = 7.3 Hz),
1.31 (6H, d, J = 5.9 Hz), 1.40–1.51 (2H, m), 1.65–2.02 (6H, m),
2.54–2.63 (1H, m), 2.90–2.98 (2H, m), 2.99–3.08 (1H, m), 3.21–
3.33 (1H, m), 3.53–3.64 (1H, m), 3.72–3.79 (1H, m), 4.44–4.54
(1H, m), 6.81–6.87 (2H, m), 7.39–7.45 (2H, m), 7.63 (1H, br s).
ESI-MS m/z 383.1 (M+H)+. HRMS (M+H)+ calcd for C19H31N2O4S,
383.2005; found, 383.2013.
5.1.6. (1R*,4S*,6R*)-( )-N-[4-(Benzyloxy)phenyl]-2-(butylsulfo-
nyl)-2-azabicyclo[2.2.2]octane-6-carboxamide (5)
5.1.12. (1R*,2R*,5R*)-( )-8-(Butylsulfonyl)-N-[4-(propan-2-yloxy)
phenyl]-8-azabicyclo[3.2.1]octane-2-carboxamide (11)
Compound 11 was prepared from 8-tert-Butyl 2-methyl
(1R*,2R*,5R*)-( )-8-azabicyclo[3.2.1]octane-2,8-dicarboxylate19
and 1-butanesulfonyl chloride using the procedure described for
31, 32, 33, and 12. 1H NMR (400 MHz, CDCl3): d 0.96 (3H, t,
Compound 5 was prepared from 30 and 4-benzyloxyaniline
using the procedure described for 1. 1H NMR (400 MHz, CDCl3):
d 0.96 (3H, t, J = 7.3 Hz), 1.41–1.50 (2H, m), 1.74–1.88 (6H, m),
1.93–2.01 (1H, m), 2.08–2.15 (1H, m), 2.26–2.37 (1H, m),
2.91–3.04 (2H, m), 3.05–3.11 (1H, m), 3.24–3.30 (1H, m),
3.54–3.60 (1H, m), 3.93–3.97 (1H, m), 5.05 (2H, s), 6.90–6.95

5644
T. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 5639–5647
J = 7.3 Hz), 1.31 (6H, d, J = 6.1 Hz), 1.40–1.52 (2H, m), 1.60–1.70
(1H, m), 1.72–1.85 (5H, m), 1.87–2.14 (4H, m), 2.20–2.31 (1H,
m), 2.77–2.87 (1H, m), 2.93–3.01 (2H, m), 4.13–4.20 (1H, m),
4.31–4.39 (1H, m), 4.45–4.55 (1H, m), 6.80–6.88 (2H, m), 7.35–
7.43 (3H, m). ESI-MS m/z 409.1 (M+H)+. HRMS (M+H)+ calcd for
C21H33N2O4S, 409.2161; found, 409.2169.
m), 2.02–2.08 (1H, m), 2.17–2.27 (1H, m), 2.82–2.91 (1H, m),
3.23–3.28 (1H, m), 3.44–3.50 (1H, m), 4.03–4.08 (1H, m), 4.46–
4.53 (1H, m), 6.82–6.88 (2H, m), 7.34–7.42 (3H, m), 7.52–7.57
(2H, m), 7.59–7.64 (1H, m), 7.86–7.92 (2H, m). ESI-MS m/z 429.2
(M+H)+. HRMS (M+H)+ calcd for C23H29N2O4S, 429.1848; found,
429.1855.
5.1.18. (1R*,4S*,6R*)-( )-2-[(4-Methoxyphenyl)sulfonyl]-N-[4-
(propan-2-yloxy)phenyl]-2-azabicyclo[2.2.2]octane-6-carbo-
xamide (17)
5.1.13. (1R*,4S*,6R*)-( )-2-(Methylsulfonyl)-N-[4-(propan-2-yloxy)
phenyl]-2-azabicyclo[2.2.2]octane-6-carboxamide (12)
To a solution of 33 (25 mg, 0.077 mmol) and triethylamine
Compound 17 was prepared from 33 and 4-meth-
oxybenzenesulfonyl chloride using the procedure described for
12. 1H NMR (400 MHz, CDCl3): d 1.25–1.40 (7H, m), 1.43–1.53
(1H, m), 1.63–1.82 (3H, m), 1.99–2.06 (1H, m), 2.17–2.27 (1H,
m), 2.84–2.93 (1H, m), 3.17–3.27 (1H, m), 3.39–3.47 (1H, m),
3.88 (3H, s), 3.99–4.05 (1H, m), 4.44–4.55 (0H, m), 6.81–6.88
(2H, m), 6.97–7.03 (2H, m), 7.36–7.42 (3H, m), 7.76–7.85 (2H,
m). ESI-MS m/z 459.2 (M+H)+. HRMS (M+H)+ calcd for C24H31N2O5S,
459.1954; found, 459.1947.
(14.4
chloride (10
l
L, 0.31 mmol) in CHCl3 (2 mL) was added methanesulfonyl
lL, 0.13 mmol) at room temperature, and the mixture
was stirred at room temperature for 15 h. The resulting mixture
was washed with saturated aqueous sodium bicarbonate, dried
over magnesium sulfate, and concentrated. The residue was puri-
fied by preparative TLC (hexanes/ethyl acetate = 40/60) to give 12
(11.2 mg, 40%). 1H NMR (400 MHz, CDCl3):
d 1.31 (6H, d,
J = 5.9 Hz), 1.55–1.66 (1H, m), 1.77–1.90 (3H, m), 1.93–2.03 (1H,
m), 2.10–2.16 (1H, m), 2.29–2.38 (1H, m), 2.91 (3H, s), 3.02–3.10
(1H, m), 3.27–3.32 (1H, m), 3.48–3.54 (1H, m), 3.99–4.03 (1H,
m), 4.44–4.54 (1H, m), 6.81–6.87 (2H, m), 7.37–7.43 (2H, m),
7.55 (1H, br s). ESI-MS m/z 367.2 (M+H)+. HRMS (M+H)+ calcd for
C18H27N2O4S, 367.1692; found, 367.1698.
5.1.19. (1R*,4S*,6R*)-( )-2-[(3-Methoxyphenyl)sulfonyl]-N-[4-
(propan-2-yloxy)phenyl]-2-azabicyclo[2.2.2]octane-6-carbo-
xamide (18)
Compound 18 was prepared from 33 and 3-meth-
oxybenzenesulfonyl chloride using the procedure described for
12. 1H NMR (400 MHz, CDCl3): d 1.26–1.43 (7H, m), 1.46–1.60
(1H, m), 1.65–1.84 (3H, m), 2.01–2.08 (1H, m), 2.17–2.26 (1H,
m), 2.75–2.83 (1H, m), 3.26–3.33 (1H, m), 3.41–3.48 (1H, m),
3.87 (3H, s), 4.02–4.07 (1H, m), 4.44–4.55 (1H, m), 6.82–6.88
(2H, m), 7.11–7.17 (1H, m), 7.30–7.34 (1H, m), 7.37–7.42 (3H,
m), 7.44–7.49 (2H, m). ESI-MS m/z 459.2 (M+H)+. HRMS (M+H)+
calcd for C24H31N2O5S, 459.1954; found, 459.1947.
5.1.14. (1R*,4S*,6R*)-( )-2-(Ethylsulfonyl)-N-[4-(propan-2-yloxy)
phenyl]-2-azabicyclo[2.2.2]octane-6-carboxamide (13)
Compound 13 was prepared from 33 and ethanesulfonyl chlo-
ride using the procedure described for 12. 1H NMR (400 MHz,
CDCl3): d 1.31 (6H, d, J = 5.9 Hz), 1.37 (3H, t, J = 7.3 Hz), 1.55–1.66
(1H, m), 1.75–1.89 (3H, m), 1.94–2.05 (1H, m), 2.08–2.14 (1H,
m), 2.27–2.36 (1H, m), 2.94–3.11 (3H, m), 3.24–3.30 (1H, m),
3.54–3.60 (1H, m), 3.92–3.97 (1H, m), 4.44–4.55 (1H, m), 6.80–
6.88 (2H, m), 7.38–7.45 (2H, m), 7.56 (1H, br s). ESI-MS m/z
381.2 (M+H)+. HRMS (M+H)+ calcd for C19H29N2O4S, 381.1848;
found, 381.1852.
5.1.20. (1R*,4S*,6R*)-( )-N-[4-(Propan-2-yloxy)phenyl]-2-{[4-(tri-
fluoromethyl)phenyl]sulfonyl}-2-azabicyclo[2.2.2]octane-6-car-
boxamide (19)
Compound 19 was prepared from 33 and 4-(trifluoromethyl)
benzenesulfonyl chloride using the procedure described for 12.
1H NMR (400 MHz, CDCl3): d 1.24–1.37 (7H, m), 1.38–1.49 (1H,
m), 1.63–1.88 (3H, m), 2.04–2.11 (1H, m), 2.20–2.29 (1H, m),
2.93–3.01 (1H, m), 3.19–3.24 (1H, m), 3.49–3.55 (1H, m), 4.08–
4.16 (1H, m), 4.45–4.56 (1H, m), 6.82–6.90 (2H, m), 7.35–7.44
(3H, m), 7.76–7.84 (2H, m), 7.96–8.03 (2H, m). ESI-MS m/z 497.2
(M+H)+. HRMS (M+H)+ calcd for C24H28N2O4SF3, 497.1722; found,
497.1722.
5.1.15. (1R*,4S*,6R*)-( )-N-[4-(Propan-2-yloxy)phenyl]-2-(propyl
sulfonyl)-2-azabicyclo[2.2.2]octane-6-carboxamide (14)
Compound 14 was prepared from 33 and 1-propanesulfonyl
chloride using the procedure described for 12. 1H NMR
(400 MHz, CDCl3):
d 1.07 (3H, t, J = 7.4 Hz), 1.31 (6H, d,
J = 6.1 Hz), 1.52–1.66 (1H, m), 1.75–1.91 (5H, m), 1.94–2.04 (1H,
m), 2.08–2.14 (1H, m), 2.27–2.36 (1H, m), 2.88–3.02 (2H, m),
3.04–3.11 (1H, m), 3.24–3.30 (1H, m), 3.53–3.60 (1H, m), 3.92–
3.98 (1H, m), 4.45–4.55 (1H, m), 6.80–6.87 (2H, m), 7.37–7.43
(2H, m), 7.53 (1H, br s). ESI-MS m/z 395.1 (M+H)+. HRMS (M+H)+
calcd for C20H31N2O4S, 395.2005; found, 395.2011.
5.1.21. (1R*,4S*,6R*)-( )-2-[(4-tert-Butylphenyl)sulfonyl]-N-[4-
(propan-2-yloxy)phenyl]-2-azabicyclo[2.2.2]octane-6-carboxa-
mide (20)
5.1.16. (1R*,4S*,6R*)-( )-N-[4-(Propan-2-yloxy)phenyl]-2-(propan-
2-ylsulfonyl)-2-azabicyclo[2.2.2]octane-6-carboxamide (15)
Compound 15 was prepared from 33 and isopropylsulfonyl
chloride using the procedure described for 12. 1H NMR
(400 MHz, CDCl3): d 1.31 (6H, d, J = 6.1 Hz), 1.35–1.41 (6H, m),
1.57–1.66 (1H, m), 1.77–1.91 (3H, m), 1.94–2.04 (1H, m), 2.07–
2.13 (1H, m), 2.25–2.35 (1H, m), 3.05–3.12 (1H, m), 3.14–3.22
(1H, m), 3.23–3.29 (1H, m), 3.64–3.71 (1H, m), 3.86–3.92 (1H,
m), 4.45–4.53 (1H, m), 6.80–6.87 (2H, m), 7.37–7.46 (3H, m) ESI-
MS m/z 395.2 (M+H)+. HRMS (M+H)+ calcd for C20H31N2O4S,
395.2005; found, 395.2009.
Compound 20 was prepared from 33 and 4-tert-butylbenzene-
sulfonyl chloride using the procedure described for 12. 1H NMR
(400 MHz, CDCl3): d 1.31 (6H, d, J = 5.9 Hz), 1.34 (9H, s), 1.44–
1.55 (1H, m), 1.62–1.83 (3H, m), 1.94–2.05 (2H, m), 2.17–2.27
(1H, m), 2.86–2.94 (1H, m), 3.22–3.28 (1H, m), 3.42–3.48 (1H,
m), 4.05–4.09 (1H, m), 4.44–4.54 (1H, m), 6.78–6.86 (2H, m),
7.36–7.42 (2H, m), 7.51–7.56 (3H, m), 7.76–7.83 (2H, m). ESI-MS
m/z 485.2 (M+H)+. HRMS (M+H)+ calcd for C27H37N2O4S,
485.2474; found, 485.2480.
5.1.22. (1R*,4S*,6R*)-( )-2-[(4-Fluorophenyl)sulfonyl]-N-[4-(pro-
pan-2-yloxy)phenyl]-2-azabicyclo[2.2.2]octane-6-carboxamide
(21)
Compound 21 was prepared from 33 and 4-fluorobenzenesulfo-
nyl chloride using the procedure described for 12. 1H NMR
(400 MHz, CDCl3): d 1.23–1.38 (7H, m), 1.39–1.49 (1H, m), 1.63–
1.85 (3H, m), 2.02–2.09 (1H, m), 2.19–2.28 (1H, m), 2.90–2.99
5.1.17. (1R*,4S*,6R*)-( )-2-(Phenylsulfonyl)-N-[4-(propan-2-yloxy)
phenyl]-2-azabicyclo[2.2.2]octane-6-carboxamide (16)
Compound 16 was prepared from 33 and benzenesulfonyl chlo-
ride using the procedure described for 12. 1H NMR (400 MHz,
CDCl3): d 1.28–1.38 (7H, m), 1.42–1.52 (1H, m), 1.62–1.82 (3H,

T. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 5639–5647
5645
(1H, m), 3.17–3.24 (1H, m), 3.44–3.51 (1H, m), 4.03–4.10 (1H, m),
4.44–4.57 (1H, m), 6.82–6.88 (2H, m), 7.18–7.24 (2H, m), 7.37–7.45
(3H, m), 7.85–7.92 (2H, m). ESI-MS m/z 447.1 (M+H)+. HRMS
(M+H)+ calcd for C23H28N2O4SF, 447.1754; found, 447.1759.
5.1.28. (1R*,4S*,6R*)-( )-N-[4-(Propan-2-yloxy)phenyl]-2-(pyri-
din-3-ylsulfonyl)-2-azabicyclo[2.2.2]octane-6-carboxamide (27)
Compound 27 was prepared from 33 and 3-pyridinesulfonyl
chloride hydrochloride using the procedure described for 12. 1H
NMR (400 MHz, CDCl3): d 1.26–1.39 (7H, m), 1.41–1.51 (1H, m),
1.66–1.90 (3H, m), 2.04–2.11 (1H, m), 2.21–2.28 (1H, m), 2.89–
2.97 (1H, m), 3.20–3.27 (1H, m), 3.47–3.52 (1H, m), 4.09–4.16
(1H, m), 4.44–4.55 (1H, m), 6.80–6.88 (2H, m), 7.37–7.43 (2H,
m), 7.45–7.53 (2H, m), 8.12–8.18 (1H, m), 8.80–8.87 (1H, m),
9.07–9.12 (1H, m). ESI-MS m/z 430.1 (M+H)+. HRMS (M+H)+ calcd
for C22H28N3O4S, 430.1801; found, 430.1808.
5.1.23. (1R*,4S*,6R*)-( )-2-[(3-Fluorophenyl)sulfonyl]-N-[4-(pro-
pan-2-yloxy)phenyl]-2-azabicyclo[2.2.2]octane-6-carboxamide
(22)
Compound 22 was prepared from 33 and 3-fluorobenzenesulfo-
nyl chloride using the procedure described for 12. 1H NMR
(400 MHz, CDCl3): d 1.26–1.39 (7H, m), 1.41–1.52 (1H, m), 1.63–
1.85 (3H, m), 2.03–2.10 (1H, m), 2.20–2.29 (1H, m), 2.87–2.97
(1H, m), 3.21–3.28 (1H, m), 3.45–3.53 (1H, m), 4.04–4.10 (1H,
m), 4.45–4.55 (1H, m), 6.82–6.89 (2H, m), 7.28–7.34 (1H, m),
7.36–7.43 (3H, m), 7.50–7.60 (2H, m), 7.64–7.70 (1H, m). ESI-MS
m/z 447.1 (M+H)+. HRMS (M+H)+ calcd for C23H28N2O4SF,
447.1754; found, 447.1759.
5.1.29. (1R*,4S*,6R*)-( )-2-[(1-Methyl-1H-imidazol-4-yl)
sulfonyl]-N-[4-(propan-2-yloxy)phenyl]-2-azabicyclo[2.2.2]
octane-6-carboxamide (28)
Compound 28 was prepared from 33 and 1-methylimidazole-4-
sulfonyl chloride hydrochloride using the procedure described for
12. 1H NMR (400 MHz, CDCl3): d 1.32 (6H, d, J = 6.3 Hz), 1.41–
1.52 (1H, m), 1.56–1.66 (1H, m), 1.70–1.98 (3H, m), 2.00–2.05
(1H, m), 2.15–2.24 (1H, m), 2.94–3.02 (2H, m), 3.43–3.48 (1H,
m), 3.80 (3H, s), 4.28–4.31 (1H, m), 4.46–4.54 (1H, m), 6.82–6.88
(2H, m), 7.46–7.52 (3H, m), 7.59–7.63 (1H, m), 8.62 (1H, br s).
ESI-MS m/z 433.3 (M+H)+. HRMS (M+H)+ calcd for C21H29N4O4S,
433.1910; found, 433.1909.
5.1.24. (1R*,4S*,6R*)-( )-2-[(2-Fluorophenyl)sulfonyl]-N-[4-(pro-
pan-2-yloxy)phenyl]-2-azabicyclo[2.2.2]octane-6-carboxamide
(23)
Compound 23 was prepared from 33 and 2-fluorobenzenesulfo-
nyl chloride using the procedure described for 12. 1H NMR
(400 MHz, CDCl3): d 1.32 (6H, d, J = 6.3 Hz), 1.46–1.56 (1H, m),
1.62–1.88 (4H, m), 2.05–2.12 (1H, m), 2.24–2.34 (1H, m), 2.92–
3.01 (1H, m), 3.36–3.47 (2H, m), 4.12–4.16 (1H, m), 4.44–4.55
(1H, m), 6.80–6.90 (2H, m), 7.21–7.32 (2H, m), 7.36–7.44 (3H,
m), 7.53–7.62 (1H, m), 7.89–7.97 (1H, m). ESI-MS m/z 447.1
(M+H)+. HRMS (M+H)+ calcd for C23H28N2O4SF, 447.1754; found,
447.1755.
5.1.30. Optical resolution of ( )-28
Optical resolution of ( )-28 (60 mg) was performed by chiral
HPLC using Daicel CHIRALPAK AD (250 ꢁ 20 mm id), eluting with
40% 2-propanol in hexanes at flow rate of 1.0 mL/min. 26.6 mg of
28a was obtained from the first eluate (tR: 14.2 min) and 29.4 mg
of 28b was obtained from the second eluate (tR: 30.7 min). ½a D25
ꢂ
5.1.25. (1R*,4S*,6R*)-( )-N-[4-(Propan-2-yloxy)phenyl]-2-(thio-
phen-2-ylsulfonyl)-2-azabicyclo[2.2.2]octane-6-carboxamide
(24)
Compound 24 was prepared from 33 and 2-thiophenesulfonyl
chloride using the procedure described for 12. 1H NMR
(400 MHz, CDCl3): d 1.32 (6H, d, J = 6.3 Hz), 1.39–1.47 (1H, m),
1.51–1.84 (4H, m), 2.05–2.11 (1H, m), 2.19–2.27 (1H, m), 2.75–
2.83 (1H, m), 3.34–3.48 (2H, m), 4.04–4.09 (1H, m), 4.45–4.56
(1H, m), 6.82–6.88 (2H, m), 7.13–7.16 (1H, m), 7.20 (1H, br s),
7.35–7.42 (2H, m), 7.59–7.65 (2H, m). ESI-MS m/z 435.3 (M+H)+.
HRMS (M+H)+ calcd for C21H27N2O4S2, 435.1412; found, 435.1411.
for 28a = +282 (c 1.0, CHCl3).
5.1.31. (1R*,4S*,6R*)-( )-2-(Butylsulfonyl)-2-azabicyclo[2.2.2]
octane-6-carboxylic acid (30)
To a solution of 29 (298 mg, 0.989 mmol) and triethylamine
(0.51 mL, 3.7 mmol) in CHCl3 (5 mL) was added 1-butanesulfonyl
chloride (0.23 mL, 1.84 mmol), and the mixture was stirred at
room temperature for 13 h. The mixture was washed with water
and saturated aqueous sodium bicarbonate, dried over magnesium
sulfate, and concentrated. The residue was purified by silica gel
column chromatography with 0?40% ethyl acetate in hexanes to
give the corresponding sulfonamide (157 mg, 55%) as a 3:7 mixture
of diastereomers. 1H NMR (400 MHz, CDCl3): d 0.96 (3H, t,
J = 7.6 Hz), 1.38–1.52 (2H, m), 1.58–1.93 (5H, m), 2.03–2.29 (2H,
m), 2.69–2.75 (1H ꢁ 3/10, m), 2.85–3.00 (2H, m), 3.04–3.12
(1H ꢁ 7/10, m), 3.28–3.51 (2H, m), 3.71 (3H ꢁ 7/10, s), 3.73
(3H ꢁ 3/10, s), 4.05–4.10 (1H ꢁ 7/10, m), 4.14–4.18 (1H ꢁ 3/10,
m). ESI-MS m/z 290.2 (M+H)+. The sulfonamide obtained above
(145 mg, 0.5 mmol) was dissolved in a mixture of 1.5 mL of 5 N
aqueous NaOH and 5 mL of methanol, and the resultant mixture
was stirred at room temperature for 17 h. The mixture was neu-
tralized with 5 N aqueous HCl, and extracted with CHCl3 three
times. The combined organic extracts were dried over magnesium
sulfate, and concentrated to give 30 (134 mg, 97%) as a 3:7 mixture
of diastereomers. 1H NMR (400 MHz, CDCl3): d 0.88–0.99 (3H, m),
1.36–1.50 (2H, m), 1.60–1.86 (4H, m), 1.87–1.97 (1H, m), 2.06–
2.24 (2H, m), 2.74–2.81 (1H ꢁ 3/10, m), 2.87–3.01 (2H, m), 3.10–
3.18 (1H ꢁ 7/10, m), 3.28–3.53 (2H, m), 4.10–4.13 (1H ꢁ 7/10,H,
m), 4.18–4.21 (1H ꢁ 3/10, m). ESI-MS m/z 276.1 (M+H)+.
5.1.26. (1R*,4S*,6R*)-( )-N-[4-(Propan-2-yloxy)phenyl]-2-(thio-
phen-3-ylsulfonyl)-2-azabicyclo[2.2.2]octane-6-carboxamide (25)
Compound 25 was prepared from 33 and 3-thiophenesulfonyl
chloride using the procedure described for 12. 1H NMR
(400 MHz, CDCl3): d 1.25–1.42 (7H, m), 1.46–1.58 (1H, m), 1.66–
1.85 (3H, m), 2.04–2.10 (1H, m), 2.19–2.27 (1H, m), 2.83–2.91
(1H, m), 3.25–3.32 (1H, m), 3.42–3.49 (1H, m), 4.04–4.09 (1H,
m), 4.44–4.55 (1H, m), 6.82–6.88 (2H, m), 7.29 (1H, br s), 7.35–
7.43 (3H, m), 7.44–7.48 (1H, m), 7.96–7.99 (1H, m) ESI-MS m/z
435.1 (M+H)+. HRMS (M+H)+ calcd for C21H27N2O4S2, 435.1412;
found, 435.1413.
5.1.27. (1R*,4S*,6R*)-( )-N-[4-(Propan-2-yloxy)phenyl]-2-(pyri-
din-2-ylsulfonyl)-2-azabicyclo[2.2.2]octane-6-carboxamide (26)
Compound 26 was prepared from 33 and 2-pyridinesulfonyl
chloride hydrochloride using the procedure described for 12. 1H
NMR (400 MHz, CDCl3): d 1.32 (6H, d, J = 5.9 Hz), 1.47–1.64 (2H,
m), 1.69–1.85 (3H, m), 2.01–2.08 (1H, m), 2.24–2.33 (1H, m),
3.03–3.12 (1H, m), 3.25–3.32 (1H, m), 3.46–3.53 (1H, m), 4.28–
4.32 (1H, m), 4.46–4.54 (1H, m), 6.82–6.89 (2H, m), 7.38–7.47
(2H, m), 7.52–7.57 (1H, m), 7.73 (1H, br s), 7.91–7.99 (1H, m),
8.02–8.07 (1H, m), 8.70–8.78 (1H, m). ESI-MS m/z 430.2 (M+H)+.
HRMS (M+H)+ calcd for C22H28N3O4S, 430.1801; found, 430.1804.
5.1.32. 2-tert-Butyl 6-methyl (1R*,4S*)-2-azabicyclo[2.2.2]
octane-2,6-dicarboxylate (31)
To a solution of 29 (2.36 g, 7.94 mmol), N,N-dimethyl-4-amino-
pyridine (97 mg, 0.79 mmol), and triethylamine (4.4 mL,
31.8 mmol) in CHCl3 (30 mL) was added di-tert-butyl carbonate

5646
T. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 5639–5647
(3.47 g, 15.9 mmol), and the mixture was stirred at room temper-
ature for 24 h. The mixture was washed with saturated aqueous
sodium bicarbonate, dried over magnesium sulfate, and concen-
trated. The residue was purified by silica gel column chromatogra-
phy with 0?40% ethyl acetate in hexanes to give to give 31 (2.05 g,
96%) as a 3:7 mixture of diastereomers. 1H NMR (400 MHz, CDCl3):
d 1.43 (9H ꢁ 3/10, s), 1.48 (9H ꢁ 7/10, s), 1.61 (3H, s), 1.90–2.20
(2H, m), 2.61–2.97 (1H, m), 3.20–3.42 (2H, m), 3.65–3.74 (2H,
m), 4.06–4.42 (2H, m). ESI-MS m/z 270.3 (M+H)+.
ELOVL1, 10
ELOVL3, 10
ELOVL6, 40
l
l
l
M stearoyl-CoA; ELOVL2, 10
M stearoyl-CoA; ELOVL5, 40
M palmitoyl-CoA . To start reaction, 50 lL of the
l
l
M arachidonoyl-CoA;
M arachidonoyl-CoA;
ELOVL microsomal fraction was added to the substrate mixture,
and then incubated for 1 h at 37 °C with gentle shaking. This
reaction step was performed in a 96-well plate. After 1 h incuba-
tion, 100 lL of 5 N HCl was added for the hydrolysis of acyl-
CoAs, and then the reaction mixture was filtered through a Uni-
filter-96, GF/C plate (PerkinElmer, Waltham, MA) using a Filter-
Mate cell harvester (PerkinElmer, Waltham, MA). The 96-well
GF/C filter plate was subsequently washed with distilled water
5.1.33. tert-Butyl (1R*,4S*,6R*)-( )-6-{[4-(propan-2-yloxy)phe
nyl]carbamoyl}-2-azabicyclo[2.2.2]octane-2-carboxylate (32)
To a solution of 31 (2.05 g, 7.53 mmol) in 27 mL of methanol
was added 5 N aqueous NaOH (15 mL), and the mixture was stirred
at room temperature for 3 h. The mixture was neutralized with 5 N
aqueous HCl, and extracted with CHCl3 three times. The combined
organic extracts were dried over magnesium sulfate, and concen-
trated to give the corresponding carboxylic acid (1.68 g, 87%) as a
3:7 mixture of diastereomers. 1H NMR (400 MHz, CDCl3): d 1.38–
1.49 (9H, m), 1.52–1.87 (3H, m), 1.91–2.17 (2H, m), 2.67–2.79
(1H ꢁ 3/10, m), 2.91–3.03 (1H ꢁ 7/10, m), 3.21–3.45 (2H, m),
4.23–4.46 (1H, m). ESI-MS m/z 256.3 (M+H)+. To a solution of the
carboxylic acid obtained above (1.68 g, 6.58 mmol) and 4-isopro-
poxyaniline (1.49 g, 9.87 mmol) in pyridine (20 mL) was added 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(1.89 g, 9.87 mmol), and the mixture was stirred at room temper-
ature for 14 h. The resulting mixture was concentrated, and the
residue was partitioned between ethyl acetate and water. The lay-
ers were separated, and the organic layer was washed with brine,
dried over magnesium sulfate, and concentrated. The residue was
purified by silica gel column chromatography with 0?25% ethyl
acetate in hexanes to give 32 (1.26 g, 49%). 1H NMR (400 MHz,
CDCl3): d 1.31 (6H, d, J = 5.9 Hz), 1.49 (9H, s), 1.54–2.05 (3H, m),
2.33–2.43 (1H, m), 2.86–2.96 (1H, m), 3.29–3.39 (2H, m), 4.21–
4.28 (1H, m), 4.43–4.55 (1H, m), 6.78–6.90 (2H, m), 7.36–7.46
(2H, m), 7.94 (1H, br s).
to remove excess
25 of MICROSCINT
radioactivity.
[
14C] malonyl-CoA and dried, after which
was added to each well and
lL
0
5.2.2. Plasma and liver exposure in mice
Plasma and liver concentrations: Pharmacokinetic characteriza-
tions of compound 28a were conducted in male C57BL/6J mice
following single oral administration. Single doses of compound
28a at 30 mg/kg body weight were administered orally by ga-
vage in a vehicle of 0.5% methylcellulose aqueous suspension.
Blood samples from the abdominal vein and liver samples were
obtained 8 h after the oral administration. Blood samples were
centrifuged to separate the plasma. Liver samples were homog-
enized with phosphate-buffered saline (pH 7.4). Each sample
was deproteinized with ethanol containing an internal standard.
The respective compound and the internal standard were de-
tected by liquid chromatography mass spectrometry/mass spec-
trometry (LC–MS/MS) in positive ionization mode using an
electrospray ionization probe, and their precursor to production
combinations were monitored using the Multiple Reaction Mon-
itoring mode.
5.2.3. Effect of compound 28a on liver fatty acid elongation
index
Male C57BL/6J mice (CLEA Japan) and SD rats (Charles River Ja-
pan) were individually housed in plastic cages with ad libitum ac-
cess to normal rodent chow (CE2, CLEA Japan) and water. Mice
were orally administered compound 28a (dissolved in 0.5% meth-
ylcellulose) and 1 h later [1-14C]-palmitic acid was interperitoneal-
5.1.34. tert-Butyl (1S*,4S*,6R*)-( )-N-(4-isopropoxyphenyl)-2-
azabicyclo[2.2.2]octane-6-carboxamide hydrochloride (33)
To a solution of compound 32 (1.26 g, 3.24 mmol) in CHCl3
(10 mL) was added 4 N aqueous HCl in ethyl acetate (8.1 mL),
and the mixture was stirred at room temperature for 13 h and con-
centrated to give 33 (1.15 g, 100%). 1H NMR (400 MHz, CDCl3): d
1.29 (6H, d, J = 5.9 Hz), 1.61–1.72 (1H, m), 1.81–1.96 (2H, m),
2.00–2.10 (1H, m), 2.40–2.50 (1H, m), 3.17–3.26 (1H, m), 3.29–
3.48 (2H, m), 4.08–4.14 (1H, m), 4.39–4.50 (1H, m), 6.74–6.80
(2H, m), 7.49–7.54 (2H, m), 8.92 (1H, br s), 9.23 (1H, br s), 9.47
(1H, br s). ESI-MS m/z 289.3 (M+H)+.
ly administered at 10 lCi/body. At 2 h post-dosing of compound
28a, animals were anesthetized with isoflurane (4%) and killed
by blood collection from the vena cava. The liver was harvested,
incubated in potassium hydroxide/ethanol at 70 °C for 1 h and
the lipid fraction then obtained by collecting the ether phase after
the addition of petroleum ether. After acidification with hydrochlo-
ric acid, the mixture was extracted with ether. Radio labeled pal-
mitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), and
vaccenic and oleic acids (18:1) were quantified by reversed-phase
HPLC with a Packard Flow Scintillation Analyzer 500TR (radio-
HPLC) (Amersham Biosciences, Piscataway, NJ). The identity of
the labeled fatty acids was determined by comparing the retention
times with known fatty acid standards. Elongation activity was
monitored as the elongation index (EI), which was the ratio of
radio labeled C18 (C18:0 + C18:1) to C16 (C16:0 + C16:1).All ani-
mal procedures were conducted according to protocols and guide-
lines approved by the Banyu Institutional Animal Care and Use
Committee.
5.2. Biological protocols
5.2.1. Enzyme assay
The full-length ELOVLs cDNAs were amplified from human or
mouse liver cDNA library by polymerase chain reaction using the
gene specific primers. Then COS-7 cells were transfected with the
expression vector encoding the C-terminally V5-epitope tagged
ELOVLs cDNA. After 48 h transfection, the cells were harvested
and the microsome fractions were prepared as enzyme resources.
The expression of each ELOVL was confirmed by immunoblot
analysis using anti-V5-epitope antibody. For elongation reactions,
Acknowledgments
50
DMSO, 100 mM potassium phosphate buffer (pH 6.5), 200
BSA (fatty acid free), 500 M NADPH, 1 M rotenone, 20
malonyl-CoA, 833 kBq/mL [14C] malonyl-CoA and acyl-CoA),
was used as the substrate mixture. The following long-chain
acyl-CoAs were used as a preferential substrate for each ELOVL;
lL of the reaction mixture (1 lL of a diluted test compound in
lM
We thank Mr. Hirokazu Ohsawa for collecting the high-resolu-
tion mass spectral data. We also thank Dr. Peter T. Meinke (Merck
Research Laboratories, Rahway, NJ) for the editing of this
manuscript.
l
l
lM

T. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 5639–5647
5647
10. Zhang, K.; Kniazeva, M.; Han, M.; Li, W.; Yu, Z.; Yang, Z.; Li, Y.; Metzker, M. L.;
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Supplementary data
Supplementary data (HPLC retention times and purity for the
target compounds) associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.06.042.
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19. Metabolic stability in liver microsomes: 1 lM compound was incubated at
37 °C in 0.25 mg/mL of human and mouse liver microsomes supplemented
with 10 mM glucose-6-phospate, 1 mM b-nicotinamide-adenine dinucleotide
phosphate, 1 U/mL glucose-6-phosphate dehydrogenase, 100 mM phosphate
buffer and 3 mM magnesium chloride. Concentrations of respective compound
were determined by LC–MS/MS. Metabolic stability was calculated from the
ratio of respective compound concentration at 0 min to that at 30 min after the
initiation of incubation.