Discovery of novel SCD1 inhibitors: 5-Alkyl-4,5-dihydro-3H-spiro[1,5- benzoxazepine-2,4′-piperidine] analogs

Article abstract of DOI:10.1016/j.ejmech.2011.02.002

Expansion of the 6-membered ring and subsequent fine-tuning of the newly obtained 7-membered spiropiperidine structure resulted in the discovery of a series of novel and potent SCD1 inhibitors. Preliminary SAR was explored by modifying an alkyl chain on t

Full text of DOI:10.1016/j.ejmech.2011.02.002

European Journal of Medicinal Chemistry 46 (2011) 1892e1896
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Discovery of novel SCD1 inhibitors: 5-Alkyl-4,5-dihydro-3H-spiro
[1,5-benzoxazepine-2,40-piperidine] analogs
,
a
a
b
a
c
Yoshikazu Uto a , Yuko Ueno , Yohei Kiyotsuka , Yuriko Miyazawa , Hitoshi Kurata , Tsuneaki Ogata ,
*
Toshiyuki Takagi b, Satoko Wakimoto d, Jun Ohsumi d
a Lead Discovery & Optimization Research Laboratories I, Daiichi Sankyo Co., Ltd.,1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Clinical Development Department I, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Global Project Management 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
a r t i c l e i n f o
a b s t r a c t
Article history:
Expansion of the 6-membered ring and subsequent fine-tuning of the newly obtained 7-membered
spiropiperidine structure resulted in the discovery of a series of novel and potent SCD1 inhibitors.
Preliminary SAR was explored by modifying an alkyl chain on the azepine nitrogen and resulted in the
identification of a highly potent SCD1 inhibitor: 6-[5-(cyclopropylmethyl)-4,5-dihydro-10H,3H-spiro[1,5-
benzoxazepine-2,40-piperidin]-10-yl]-N-(2-hydroxy-2-pyridin-3-ylethyl)pyridazine-3-carboxamide (9).
Received 25 January 2011
Received in revised form
29 January 2011
Accepted 3 February 2011
Available online 26 February 2011
Compound 9 exhibited an IC50 value of 0.01
m
M against human SCD1.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
SCD1 inhibitor
Spiropiperidine
Benzoxazepine
Pyridazine
1. Introduction
completely understood, inhibition of SCD1 may represent a novel
approach for the treatment of metabolic syndrome.
In many species, stearoyl-CoA desaturase 1 (SCD1) is a rate-
limiting enzyme in the biosynthesis of monounsaturated fatty acids
from their saturated fatty acid precursors [1,2]. SCD1 introduces
Since Xenon Pharmaceuticals published the first example of
small molecule SCD1 inhibitors (1, Fig. 1) in 2005 [9], numerous
structures of SCD1 inhibitors have been reported in patents and
scientific literature [10e15]. We have previously reported on the
optimization of piperidine-based SCD1 inhibitors, which resulted
in the discovery of highly potent spiropiperidines [16e18]. In
continuing our SCD1 inhibitor project, spiropiperidine (1, Fig. 1)
was chosen as a lead compound for further optimization. Since the
X-ray structure of SCD1 has not been resolved and the structural
information of this enzyme is very limited, our design of the novel
SCD1 inhibitors mainly relied on the results obtained from our own
SAR studies. Here in this article, we would like to disclose our
recent efforts on the optimization of the spiropiperidine structures
and the discovery of novel and highly potent SCD1 inhibitors.
a double bond at the
D9 position (between carbons 9 and 10) of
stearoyl (C18:0) and palmitoyl-CoA (C16:0) [1,2] in conjunction
with NAD(P)H, cytochrome b5 reductase, and cytochrome b5.
SCD1 has recently been shown to be a crucial factor in lipid
metabolism and body weight control [3e6] because the products of
SCD1, oleic (C18:1 n-9) and palmitoleic acids (C16:1 n-7) are the
most abundant fatty acids found in phospholipids, cholesterol
esters, and triglycerides. In humans, a higher desaturation index
(the ratio of oleate to stearate or 18:1/18:0) strongly correlates with
higher plasma triglyceride levels and (to a lesser extent) with
plasma HDL levels [7]. The 18:1/18:0 ratio in plasma VLDL has
recently been shown to be closely associated with the hepatic SCD1
expression in humans [8]. Even though the detailed mechanism by
which SCD1 deficiency affects body weight and adiposity is not
2. Results and discussion
In the preliminary investigation (Fig. 1), expansion of the 6-
membered ring via Beckmann rearrangement gave 7-membered
spiropiperidine, 3H-spiro[1,5-benzoxazepine-2,40-piperidin]-4(5H)-
one (2). Subsequent reduction of the amide group in the newly
* Corresponding author. Tel.: þ81 3 3492 3131; fax: þ81 3 5436 8563.
E-mail address: uto.yoshikazu.pw@daiichisankyo.co.jp (Y. Uto).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.002

Y. Uto et al. / European Journal of Medicinal Chemistry 46 (2011) 1892e1896
1893
Table 1
O
X
Evaluation of the 7-membered spiropiperidines for enzymatic SCD1 inhibitory
N
H
activity.a
N
OH
N
N
O
O
O
1a: X =CH
1b: X = N
IC50 (mouse) = 0.05 µM
IC50 (human) = 0.25 µM
IC50 (mouse) : Not Tested
IC50 (human) >1 µM (54% at 2 µM)
N
N
H
N
OH
N
N
Ring expansion
O
N
O
N
O
N
H
H
N
OH
N
OH
N
N
N
N
Reduction
Alkylation
R
O
O
HN
N
2
3: R =H
R
CLogPb
O
4: R =Me
#
R
IC50
(m
M) Mouse
IC50 (mM) Human cell
microsomal
D
9
(293A) microsomal D9
2,3:
IC50 (mouse or human) > 1µM
5
6
Me
Et
0.3
0.04
0.7
0.1
1.5
2.0
4: R =Me
IC50 (mouse) = 0.05 µM
IC50 (human) = 0.28 µM
7
8
9
0.04
>2
0.04
2.4
2.0
2.5
0.7
Fig. 1. Ring expansion of 6-membered spiropiperidine to 7-membered spiropiperidine.
Not tested
0.01
0.01
1.8
prepared spiropiperidine generated a secondary amine (3) with the
4,5-dihydro-3H-spiro[1,5-benzoxazepine-2,40-piperidine] structure.
Although the inhibitory activity against SCD1 decreased during the
course of these transformations, methylation of the secondary amine
in 3 successfully led to 4, which demonstrated potencycomparable to
1a. These results prompted us to further investigate optimal struc-
tures of the novel 7-membered spiropiperidines for better inhibitory
activity against SCD1.
10
0.6
HO
a
Values are the arithmetic means of at least two experiments.
The cLogP values were calculated by CLOGP software (Version 4.8.3, Daylight
Chemical Information Systems, Inc.).
b
We prepared a series of spiropiperidine-based SCD1 inhibitors
with 4,5-dihydro-3H-spiro[1,5-benzoxazepine-2,40-piperidine] as
the key structural motif. Synthesis of the novel spiropiperidine-
based SCD1 inhibitors shown in Table 1 is outlined in Schemes 1 and
2. Condensation of the o-hydroxyacetophenone (11) with 1-ben-
zylpiperidin-4-one in the presence of pyrrolidine gave 10-benzyl-
spiro[chromene-2,40-piperidin]-4(3H)-one (12) [19e21]. Treatment
of 12 with hydroxylamine gave oxime that was subjected to
reductive rearrangement (Beckmann rearrangement) to give 10-
benzyl-4,5-dihydro-3H-spiro[1,5-benzoxazepine-2,40-piperidine] (13)
[19e21] with an overall yield of 64% from 11. Introduction of the
various alkyl group on the azepine nitrogen was carried out by
reductive amination (Me (14) and cyclopropyl (19)), alkylation (Et,
iPr and cyclopropylmethyl), or epoxide opening (hydroxyethoxy).
Deprotection of the benzyl group by hydrogenolysis proceeded
smoothly for most of the tertiary amines (15e18, 20e21) except for
the cyclopropyl analog (19), which gave decomposed mixtures
under the reaction conditions. Condensation between the
secondary amines (15e18, 20e21) and 6-chloro-N-(2-hydroxy-2-
pyridin-3-ylethyl)pyridazine-3-carboxamide [16] in BuOH provided
the desired SCD1 inhibitors without complication (Scheme 2). For
the preparation of cyclopropyl analog, 22 was subjected to reductive
amination with [(1-ethoxycyclopropyl)oxy]trimethylsilane [22] to
give 8 in 63% yield.
exhibited an IC50 value of 0.28
3-pyridyl analog (5) showed 0.7
m
M against human SCD1 while the
mM in the same assay as shown
in Fig. 1 and Table 1. As for lipophilicity, cLogP value for 4 is 3.0
while that for 5 is 1.5. We thought the less-lipophilic compound
(4) was a good starting point in the structural optimization of
spiropiperidine, in which addition of lipophilic groups was
implemented. Elongation of the alkyl group on the azepine
nitrogen significantly increased inhibitory activity against SCD1
with increase in lipophilicity (CLogP). One carbon elongation from
methyl (5) to ethyl (6) resulted in a 7-fold increase in the
inhibitory activity against human SCD1 with increase of lip-
ophilicity (from 1.5 to 2.0). Secondary alkyl group, such as propyl
(7), did not improve potency in a meaningful scale in spite of the
increase in lipophilicity (from 2.0 to 2.4), indicating that there is
a steric hindrance around the position. In addition, cyclopropyl (8)
exhibited a sharp loss of activity with less than 50% inhibition
even at 2 mM. From these data, it is assumed that the alkyl chain
needs to rotate freely so that it can adapt an optimal interaction
with the enzyme in the binding pocket. To examine this
assumption, a methylene unit was inserted between cyclopropyl
and the azepine nitrogen. This small modification remarkably
increased inhibitory activity: cyclopropylmethyl analog (9)
exhibited an IC50 value of 0.01 mM against human SCD1. This rise
of inhibitory activity was accompanied with increase of lip-
ophilicity as well (CLogP 2.5). Introduction of the hydrophilic
functional group reduced lipophilicity in a considerable scale
(CLogP 0.7) and was rather detrimental in terms of potency.
Hydroxylethyl analog (10) showed 45 times weaker inhibitory
activity against murine SCD1 than the ethyl analog (6).
Some of the potent compounds (6, 7 and 9) were tested in the
cellular assay. In cellular assay, whole transfected 293A cells were
used for the evaluation of our SCD1 inhibitors. All of the
compounds exhibited excellent potency presumably due to their
good cell-membrane permeability. Among the tested compounds,
The results of the biological evaluation of 10-alkyl-4,5-dihydro-
3H-spiro[1,5-benzoxazepine-2,40-piperidine] analogs (5e10) are
shown in Table 1. The inhibitory activity against enzymatic SCD1
was measured for both mice and humans. The sources of the
SCD1 enzyme are the liver microsomes (for murine SCD1) and the
microsome fractions prepared from the 293A cells transiently
transfected with human SCD1 (for human SCD1). While other
isoforms of SCD are known to exist in mouse liver, their expres-
sion levels are low. The right-hand phenyl was replaced with
3-pyridyl in this series of compounds in Table 1 to reduce lip-
ophilicity even though a decrease in the inhibitory activity was
observed by this structural modification. The phenyl analog (4)
9 demonstrated strongest potency with an IC50 value of 0.01 mM
Table 2.

1894
Y. Uto et al. / European Journal of Medicinal Chemistry 46 (2011) 1892e1896
Bn
N
Bn
N
OH
O
O
O
O
a
b, c
N
H
11
13
12
Bn
NH
HCl
N
O
N
d
O
N
e
13
15
14
NH
16: R = Et
f, e
O
17: R = iPr (HCl salt)
13
18: R = CH2(cyclopropyl)
N
R
Bn
N
g
e
O
N
decomposition
13
19
NH
NH
HCl
e
h, e
O
N
O
13
13
N
H
20
21
OH
Scheme 1. Reagents and conditions: (a) 1-benzylpiperidin-4-one, pyrrolidine, EtOH, reflux, 90%; (b) NH2OH$HCl, pyridine, EtOH, 88%; (c) DIBAL, CH2Cl2, 81%; (d) HCHO (aq),
NaBH3CN, CH3CO2H/THF, rt, 93%. (e) Pd/C, H2, 1 N HCl, MeOH; (f) R-X, K2CO3, DMF or DMA, 50e70 ꢀC; (g) (1-ethoxycyclopropoxy)trimethylsilane, NaBH3CN, CH3CO2H/MeOH, reflux,
quantitative yield; (h) oxirane, CH3CO2H/THF, 78%.
O
alkyl chain on the azepine nitrogen, which resulted in the identi-
fication of a highly potent SCD1 inhibitor: 5-(cyclopropylmethyl)-
10-{6-[5-(pyridin-3-ylmethyl)-1,3,4-oxadiazol-2-yl]pyridazin-3-yl}-
4,5-dihydro-3H-spiro[1,5-benzoxazepine-2,40-piperidine] (9). Fur-
ther structural optimization and biological evaluation of the novel
spiropiperidine-based SCD1 inhibitors will be our future work.
N
NH
N
H
a
N
OH
O
N
N
N
O
HCl
N
R
15-18, 20-21
R
5-7, 9-10, 22
4. Experimental
O
O
N
N
N
H
N
H
4.1. Chemistry
b
N
OH
N
OH
N
N
N
N
O
O
4.1.1. General procedures
8
HN
N
22
1H NMR spectra were recorded on a Varian Mercury 400 or 500
spectrometer with tetramethylsilane as an internal reference. The
mass spectra (Low- or High-resolution mass) spectroscopy was
Scheme 2. Reagents and conditions: (a) 6-chloro-N-(2-hydroxy-2-pyridin-3-ylethyl)
pyridazine-3-carboxamide, Et3N, n-BuOH, 100 ꢀC; (b) (1-ethoxycyclopropoxy)trime-
thylsilane, NaBH3CN, CH3CO2H, MeOH.
Table 2
Evaluation of the 7-membered spiropiperidines for cellular SCD1 inhibitory
activity.a
3. Conclusion
Compound
IC50 (mM) Human cell (293A) D9
In summary, expansion of the 6-membered ring and subsequent
fine-tuning of the newly obtained 7-membered spiropiperidine
structure (benzoxazepine) resulted in the discovery of novel and
potent SCD1 inhibitors. Preliminary SAR was explored by modifying
6
7
9
0.10
0.14
0.01
a
Values are the arithmetic means of at least two experiments.

Y. Uto et al. / European Journal of Medicinal Chemistry 46 (2011) 1892e1896
1895
carried out with a JEOL GCmate, JEOL JMS-700 mass spectrometer
or JEOL T100LC (AccuTOF). Thin-layer chromatography (TLC) was
used routinely to monitor the progress and the purity of the
compounds and was performed on Merck Kieselgel 60 F254 plates
(0.25 mm thickness). For flash column chromatography, silica gel
(Kieselgel 60, 230e400 mesh or Chromatorex NH Fuji Silysia
Chemical Ltd. 200e300 mesh) or pre-packed silica gel column (KP-
SilÔ silica) from Biotage was employed. The experimental proce-
dures for the preparation of the intermediates in Schemes 1 and 2
are described in the supplementary information.
81.8 mg of 7 (81%) as a beige solid. 1H NMR (400 MHz, DMSO-d6):
8.71 (1H, dd, J ¼ 5.7 and 5.7 Hz), 8.55 (1H, d, J ¼ 1.6 Hz), 8.46 (1H,
d
dd, J ¼ 4.5 and 1.8 Hz), 7.79 (1H, d, J ¼ 9.8 Hz), 7.79e7.76 (1H, m),
7.38 (1H, d, J ¼ 9.8 Hz), 7.37e7.34 (1H, m), 6.97e6.90 (2H, m),
6.82e6.80 (1H, m), 6.65 (1H, dd, J ¼ 7.6 and 7.6 Hz), 5.77 (1H, d,
J ¼ 5.1 Hz), 4.87 (1H, q, J ¼ 5.2 Hz), 4.23 (2H, d, J ¼ 12.9 Hz),
3.80e3.68 (1H, m), 3.61e3.49 (4H, m), 3.33e3.21 (2H, m), 1.92 (2H,
d, J ¼ 12.9 Hz), 1.79e1.72 (2H, m), 1.62e1.55 (2H, m), 1.16 (6H, d,
J ¼ 6.6 Hz). MS (ESI) m/z: 503 (M þ H)þ; HRMS (ESI) m/z: 503.2773
(calcd for C28H35N6O3 503.2771).
4.1.2. General synthetic procedures to obtain spiropiperidine-based
SCD1 inhibitor (5e10) from benzoxazepine (15e18, 20e21) and 6-
chloro-N-(2-hydroxy-2-pyridin-3-ylethyl)pyridazine-3-
carboxamide [16]
A mixture of benzoxazepine (15e18, 20e21, 1 equivalent), 6-
chloro-N-(2-hydroxy-2-pyridin-3-ylethyl)pyridazine-3-carboxamide
(1 equivalent), and triethylamine (2 equivalents) in n-BuOH was
heated at 100 ꢀC for 2 days, cooled to room temperature, and
concentrated. Chromatography of the residue on SiO2 (Chromatorex
NH Fuji Silysia Chemical Ltd. 200e300 mesh, CH2Cl2/EtOAc) gave the
desired product (5e10). The product was further purified by recrys-
tallization when necessary.
4.1.6. 6-(4,5-Dihydro-10H,3H-spiro[1,5-benzoxazepine-2,40-
piperidin]-10-yl)-N-(2-hydroxy-2-pyridin-3-ylethyl)pyridazine-3-
carboxamide (22)
In accordance with the general procedures (Section 4.1.2), 21
(62 mg, 0.24 mmol) and 6-chloro-N-(2-hydroxy-2-pyridin-3-
ylethyl)pyridazine-3-carboxamide (55.9 mg, 0.200 mmol) provided
88 mg of 22 (96%) as a pale beige solid. 1H NMR(400M Hz, DMSO-
d6):
d
8.71 (1H, dd, J ¼ 6.0 and 6.0 Hz), 8.55 (1H, d, J ¼ 2.4 Hz), 8.45
(1H, d, J ¼ 4.7 Hz), 7.81e7.76 (2H, m), 7.38e7.34 (2H, m), 6.92 (1H, d,
J ¼ 9.0 Hz), 6.81 (1H, dd, J ¼ 7.5 and 7.5 Hz), 6.71 (1H, d, J ¼ 9.8 Hz),
6.58 (1H, dd, J ¼ 4.4 and 4.5 Hz), 5.77 (1H, s), 5.44 (1H, t, J ¼ 3.9 Hz),
4.87 (1H, dd, J ¼ 11.8, 5.9 Hz), 4.24 (2H, d, J ¼ 14.4 Hz), 3.62e3.45
(4H, m), 3.22e3.16 (2H, m), 1.95 (2H, d, J ¼ 13.7 Hz), 1.85 (2H, t,
J ¼ 5.1 Hz), 1.64e1.53 (2H, m).
4.1.3. N-(2-Hydroxy-2-pyridin-3-ylethyl)-6-(5-methyl-4,5-
dihydro-10H,3H-spiro[1,5-benzoxazepine-2,40-piperidin]-10-yl)
pyridazine-3-carboxamide (5)
In accordance with the general procedures (Section 4.1.2), 15
(34.9 mg, 0.130 mmol) and 6-chloro-N-(2-hydroxy-2-pyridin-3-
ylethyl)pyridazine-3-carboxamide (33.4 mg, 0.120 mmol) provided
32.2 mg of 5 (57%) as a brown amorphous. 1H NMR (400M Hz,
4.1.7. 6-(5-Cyclopropyl-4,5-dihydro-10H,3H-spiro[1,5-
benzoxazepine-2,40-piperidin]-10-yl)-N-(2-hydroxy-2-pyridin-3-
ylethyl)pyridazine-3-carboxamide (8)
To a solution of 22 (88.3 mg, 0.192 mmol) in MeOH (2 mL) were
added acetic acid (0.11 mL, 1.9 mmol), powered molecular sieves
(3A, 0.1 g), [(1-ethoxycyclopropyl)oxy]trimethylsilane (0.15 mL,
0.77 mmol), and sodium cyanoborohydride (36 mg, 0.57 mmol) at
room temperature. The reaction mixture was heated to reflux for
2 h, filtered, and concentrated. The residue was diluted with 1 N
HCl and extracted with EtOAc. The organic layer was washed with
brine, dried (Na2SO4), and concentrated. The residue was
successively purified by chromatography on SiO2 (CH2Cl2/MeOH)
and preparative TLC (CH2Cl2/MeOH 10:1). The resulting material
was triturated in hexane, collected by filtration and dried in vacuo
to give 61 mg (63%) of 8 as a white amorphous. 1H NMR (400 MHz,
DMSO-d6):
d
8.68 (1H, dd, J ¼ 5.9 and 5.8 Hz), 8.53 (1H, s), 8.43 (1H,
d, J ¼ 4.7 Hz), 7.77 (1H, d, J ¼ 9.8 Hz), 7.75 (1H, d, J ¼ 6.2 Hz),
7.39e7.31 (2H, m), 6.97 (1H, dd, J ¼ 7.7 and 7.7 Hz), 6.90 (1H, d,
J ¼ 9.0 Hz), 6.76 (1H, d, J ¼ 9.4 Hz), 6.71 (1H, dd, J ¼ 8.2 and 8.2 Hz),
5.75 (1H, d, J ¼ 4.7 Hz), 4.89e4.83 (1H, m), 4.22 (1H, d, J ¼ 13.3 Hz),
3.61e3.47 (4H, m), 3.17 (1H, t, J ¼ 5.5 Hz), 2.77 (3H, s), 2.48 (2H, t,
J ¼ 1.9 Hz), 1.89 (2H, d, J ¼ 13.3 Hz), 1.78 (2H, t, J ¼ 5.5 Hz), 1.57 (2H,
dt, J ¼ 4.3 and 13.2 Hz). MS (ESI) m/z: 475 (M þ H)þ; HRMS (ESI) m/
z: 475.2450 (calcd for C26H31N6O3 475.2458).
4.1.4. 6-(5-Ethyl-4,5-dihydro-10H,3H-spiro[1,5-benzoxazepine-2,40-
piperidin]-10-yl)-N-(2-hydroxy-2-pyridin-3-ylethyl)pyridazine-3-
carboxamide (6)
In accordance with the general procedures (Section 4.1.2), 16
(81.3 mg, 0.330 mmol) and 6-chloro-N-(2-hydroxy-2-pyridin-3-
ylethyl)pyridazine-3-carboxamide (69.6 mg, 0.250 mmol) provided
88.4 mg of 6 (73%) as a pale brown solid. 1H NMR (400 MHz, DMSO-
DMSO-d6):
d
8.70 (1H, dd, J ¼ 5.6 and 5.7 Hz), 8.55 (1H, s), 8.45
(1H, d, J ¼ 4.7 Hz), 7.82e7.74 (2H, m), 7.40e7.32 (2H, m), 7.13 (1H,
d, J ¼ 8.2 Hz), 7.00 (1H, dd, J ¼ 7.9 and 7.9 Hz), 6.94 (1H, d,
J ¼ 7.5 Hz), 6.77 (1H, d, J ¼ 6.6 Hz), 5.77 (1H, d, J ¼ 4.7 Hz), 4.86
(1H, dd, J ¼ 11.9 and 5.7 Hz), 4.23 (2H, d, J ¼ 12.9 Hz), 3.62e3.48
(5H, m), 3.39e3.23 (2H, m), 1.88 (2H, d, J ¼ 13.7 Hz), 1.83e1.77
(2H, m), 1.61e1.52 (2H, m), 0.74 (2H, d, J ¼ 5.1 Hz), 0.39 (2H, s). MS
(ESI) m/z: 501 (M þ H)þ; HRMS (ESI) m/z: 501.2615 (calcd for
C28H33N6O3 501.2614).
d6):
d
8.71 (1H, dd, J ¼ 5.9 and 5.9 Hz), 8.55 (1H, d, J ¼ 2.0 Hz), 8.46
(1H, dd, J ¼ 4.9 and 1.8 Hz), 7.79 (1H, d, J ¼ 9.3 Hz), 7.78e7.77 (1H,
m), 7.39 (1H, d, J ¼ 9.8 Hz), 7.36 (1H, dd, J ¼ 7.8 and 5.1 Hz), 6.96 (1H,
dt, J ¼ 10.4 and 3.8 Hz), 6.90 (1H, dd, J ¼ 7.8 and 1.5 Hz), 6.80 (1H,
dd, J ¼ 8.0 and 1.4 Hz), 6.68 (1H, dt, J ¼ 10.6 and 3.9 Hz), 5.78 (1H, s),
4.87 (1H, t, J ¼ 5.3 Hz), 4.24 (2H, d, J ¼ 12.2 Hz), 3.61e3.50 (3H, m),
3.33e3.27 (3H, m), 3.19 (2H, q, J ¼ 7.0 Hz), 1.92 (2H, d, J ¼ 14.1 Hz),
1.81e1.79 (2H, m), 1.63e1.55 (2H, m), 1.12 (3H, t, J ¼ 6.8 Hz). MS
(ESI) m/z: 489 (M þ H)þ; HRMS (ESI) m/z: 489.2608 (calcd for
C27H33N6O3 489.2614).
4.1.8. 6-[5-(Cyclopropylmethyl)-4,5-dihydro-10H,3H-spiro[1,5-
benzoxazepine-2,40-piperidin]-10-yl]-N-(2-hydroxy-2-pyridin-3-
ylethyl)pyridazine-3-carboxamide (9)
In accordance with the general procedures (Section 4.1.2), 18
(48.0 mg, 0.176 mmol) and 6-chloro-N-(2-hydroxy-2-pyridin-3-
ylethyl)pyridazine-3-carboxamide (41.8 mg, 0.150 mmol) provided
66.7 mg (87%) of 9 as a white solid. 1H NMR (400 MHz, DMSO-d6):
d
8.70 (1H, dd, J ¼ 6.0 and 6.0 Hz), 8.55 (1H, d, J ¼ 1.9 Hz), 8.45 (1H,
4.1.5. N-(2-Hydroxy-2-pyridin-3-ylethyl)-6-(5-isopropyl-4,5-
dihydro-10H,3H-spiro[1,5-benzoxazepine-2,40-piperidin]-10-yl)
pyridazine-3-carboxamide (7)
In accordance with the general procedures (Section 4.1.2), 17
(65.3 mg, 0.220 mmol) and 6-chloro-N-(2-hydroxy-2-pyridin-3-
ylethyl)pyridazine-3-carboxamide (55.7 mg, 0.200 mmol) provided
dd, J ¼ 4.7 and 1.5 Hz), 7.79 (1H, d, J ¼ 9.4 Hz), 7.77 (1H, d,
J ¼ 6.7 Hz), 7.38 (1H, d, J ¼ 9.7 Hz), 7.35 (1H, dd, J ¼ 7.7 and 4.9 Hz),
6.95 (1H, dd, J ¼ 7.7 and 7.7 Hz), 6.90 (1H, dd, J ¼ 7.8 and 1.6 Hz),
6.83 (1H, dd, J ¼ 8.0 and 1.4 Hz), 6.68 (1H, dd, J ¼ 11.9 and 4.5 Hz),
5.77 (1H, d, J ¼ 4.7 Hz), 4.89e4.85 (1H, m), 4.24 (2H, d, J ¼ 13.3 Hz),
3.59e3.52 (4H, m), 3.39 (2H, dd, J ¼ 5.3 and 5.3 Hz), 3.02 (2H, d,

1896
Y. Uto et al. / European Journal of Medicinal Chemistry 46 (2011) 1892e1896
J ¼ 6.2 Hz), 1.93 (2H, d, J ¼ 13.7 Hz), 1.82 (2H, t, J ¼ 5.1 Hz), 1.64e1.57
(2H, m), 1.04e0.98 (1H, m), 0.53e0.48 (2H, m), 0.22e0.19 (2H, m).
MS (ESI) m/z: 515 (M þ H)þ; HRMS (ESI) m/z: 515.2778 (calcd for
C29H35N6O3 515.2771).
FBS) were seeded to each well of a 96-well plate and grown over-
night to be confluent. The cells were preincubated with the test
compound in fresh media for 30 min, after which 10
mL media
containing 0.1
m
Ci [14C] stearate was added to each well and incu-
bated for another 4 h. Then the cells in each well were washed with
cold PBS and the cellular lipids were saponified directly by adding
100 mL of 5% KOH in methanol:H2O (1:1). The samples were pro-
cessed as described for the SCD1 enzymatic assay to determine the
SCD1 activity by quantifying the ratio of [14C] oleate to [14C]
stearate.
4.1.9. 6-[5-(2-Hydroxyethyl)-4,5-dihydro-10H,3H-spiro[1,5-
benzoxazepine-2,40-piperidin]-10-yl]-N-(2-hydroxy-2-pyridin-3-
ylethyl)pyridazine-3-carboxamide (10)
In accordance with the general procedures (Section 4.1.2), 20
(102 mg, 0.389 mmol) and 6-chloro-N-(2-hydroxy-2-pyridin-3-
ylethyl)pyridazine-3-carboxamide (89.0 mg, 0.319 mmol) provided
86.4 mg (54%) of 10 as a pale brown solid. 1H NMR (400M Hz,
Appendix. Supplementary material
DMSO-d6):
d
8.68 (1H, dd, J ¼ 5.7 and 5.7 Hz), 8.53 (1H, s), 8.43 (1H,
d, J ¼ 3.9 Hz), 7.77 (1H, d, J ¼ 9.4 Hz), 7.75 (1H, d, J ¼ 5.8 Hz), 7.35
(1H, d, J ¼ 9.0 Hz), 7.33 (1H, d, J ¼ 11.3 Hz), 6.92 (1H, dd, J ¼ 7.7 and
7.7 Hz), 6.86 (1H, d, J ¼ 7.4 Hz), 6.81 (1H, d, J ¼ 7.4 Hz), 6.62 (1H, dd,
J ¼ 7.5 and 7.5 Hz), 5.74 (1H, s), 4.85 (1H, dd, J ¼ 9.8 and 4.7 Hz), 4.63
(1H, t, J ¼ 4.5 Hz), 4.20 (2H, d, J ¼ 12.9 Hz), 3.60e3.46 (5H, m), 3.38
(2H, t, J ¼ 4.5 Hz), 3.30 (1H, s), 3.22 (2H, t, J ¼ 5.9 Hz), 1.90 (2H, d,
J ¼ 14.0 Hz), 1.83e1.77 (2H, m), 1.56 (2H, t, J ¼ 10.6 Hz). MS (ESI) m/
z: 505 (M þ H)þ; HRMS (ESI) m/z: 505.2569 (calcd for C27H33N6O4
505.2563).
Supplementary data associated with this article can be found in
online version at doi:10.1016/j.ejmech.2011.02.002.
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preincubated with 10
mL microsomes for 10 min at room temper-
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m
L of
a mixture containing 250 mM sucrose, 150 mM KCl, 40 mM NaF,
5 mM MgCl2, 100 mM sodium phosphate, pH7.4, 1 mM ATP, 1.5 mM
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m
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m
m
m
m
ꢀ
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m
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a
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linear regression using the straight line portions of the concen-
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4.2.2. Cellular assay
A 293A cell-based desaturase assay was performed in a 96-well
plate. Human SCD1 gene was cloned into the expression vector
pCMV-script (Stratagene). 293A cells were transfected with the
expression vector. The cells stably expressing human SCD1 were
selected with G418. The 293A cells in 100
m
L media (DMEM þ 10%