Novel spiropiperidine-based stearoyl-CoA desaturase-1 inhibitors: Identification of 1′-{6-[5-(pyridin-3-ylmethyl)-1,3,4-oxadiazol-2-yl]pyridazin-3-yl}-5-(trifluoromethyl)-3,4-dihydrospiro[chromene-2,4′-piperidine]

Article abstract of DOI:10.1016/j.bmcl.2009.11.043

Cyclization of the benzoylpiperidine in lead compound 2 generated a series of novel and highly potent spiropiperidine-based stearoyl-CoA desaturase (SCD)-1 inhibitors. Among them, 1′-{6-[5-(pyridin-3-ylmethyl)-1,3,4-oxadiazol-2-yl]pyridazin-3-yl}-5-(trifluoromethyl)-3,4-dihydrospiro[chromene-2,4′-piperidine] (19) demonstrated the most powerful inhibitory activity against SCD-1, not only in vitro but also in vivo (C57BL/6 J mice). With regard to the pharmacological evaluation, 19 showed powerful reduction of the desaturation index in the plasma of C57BL/6 J mice on a non-fat diet after a 7-day oral administration (q.d.) without causing notable abnormalities in the eyes or skin up to the highest dose (3 mg/kg) in our preliminary analysis.

Full text of DOI:10.1016/j.bmcl.2009.11.043

Bioorganic & Medicinal Chemistry Letters 20 (2010) 746–754
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel spiropiperidine-based stearoyl-CoA desaturase-1 inhibitors:
Identification of 10-{6-[5-(pyridin-3-ylmethyl)-1,3,4-oxadiazol-2-yl]-
pyridazin-3-yl}-5-(trifluoromethyl)-3,4-dihydrospiro[chromene-2,40-piperidine]
a
a
a
a
b
Yoshikazu Uto a, , Yohei Kiyotsuka , Yuko Ueno , Yuriko Miyazawa , Hitoshi Kurata , Tsuneaki Ogata ,
Tsuneo Deguchi c, Makiko Yamada c, Nobuaki Watanabe c, Masahiro Konishi d, Nobuya Kurikawa d,
Toshiyuki Takagi e, Satoko Wakimoto d, Keita Kono d, Jun Ohsumi d
*
a Medicinal Chemistry Research Laboratories I, Daiichi Sankyo Co. Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Global Project Management Department, Daiichi Sankyo Co. Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co. Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Biological Research Laboratories II, Daiichi Sankyo Co. Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
e Clinical Development Department I, 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:
Received 23 July 2009
Revised 5 November 2009
Accepted 11 November 2009
Available online 14 November 2009
Cyclization of the benzoylpiperidine in lead compound 2 generated a series of novel and highly potent
spiropiperidine-based stearoyl-CoA desaturase (SCD)-1 inhibitors. Among them, 10-{6-[5-(pyridin-
3-ylmethyl)-1,3,4-oxadiazol-2-yl]pyridazin-3-yl}-5-(trifluoromethyl)-3,4-dihydrospiro[chromene-2,40-
piperidine] (19) demonstrated the most powerful inhibitory activity against SCD-1, not only in vitro but
also in vivo (C57BL/6 J mice). With regard to the pharmacological evaluation, 19 showed powerful reduc-
tion of the desaturation index in the plasma of C57BL/6 J mice on a non-fat diet after a 7-day oral admin-
istration (q.d.) without causing notable abnormalities in the eyes or skin up to the highest dose (3 mg/kg)
in our preliminary analysis.
Keywords:
Stearoyl-CoA desaturase (SCD)
Spiropiperidine
Ó 2009 Elsevier Ltd. All rights reserved.
Desaturation index
As part of our research program to investigate novel treatments
for metabolic disorders, we have recently reported the thiazole-
based (1) and benzoylpiperidine-based (2) stearoyl-CoA desatur-
ase-1 (SCD-1) inhibitors (Fig. 1).1,2 SCD-1 is a rate-limiting enzyme
in the synthesis of monounsaturated fatty acids from their satu-
rated fatty acid precursors.3,4 The products of SCD-1, 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.
While the detailed mechanism by which SCD-1 deficiency affects
body weight and adiposity is not completely understood, inhibi-
tion of SCD-1 may represent a novel approach for the treatment
of metabolic syndrome.5,6
In our continuing investigation of the novel SCD-1 inhibitors as
a new treatment for metabolic disorders such as dyslipidemia,
obesity and diabetes, we tried to improve the potency of ben-
zoylpiperidine-based SCD-1 inhibitors such as 2. We envisioned
that simple cyclization of the benzoylpiperidine to form a spirocy-
clic system, such as in 3 (Fig. 2), would reduce the number of free
rotatable bonds and make the entire molecule more rigid, poten-
tially resulting in a more efficient interaction with the enzyme
and possibly better pharmacokinetic (PK) profile. Herein, we would
like to disclose our efforts to optimize the spiropiperidines in order
to obtain more potent SCD-1 inhibitors.
The synthetic routes to the spiropiperidines (3–21)7 are outlined
in Schemes 1–8. As shown in Scheme 1, the spiropiperidine inter-
mediates for 3 and 4 were synthesized8 via piperidine formation
at the a-carbon of the ketone (indanone (24a) or 3,4-dihydro-2H-
naphthalen-1-one (24b)) with dibromide (23). The intermediate
with a spiro[1-benzofuran-2,40-piperidin]-3-one scaffold (29) was
prepared in accord with the procedures known in literature.9 The
intermediates for the synthesis of 7 and related analogs were pre-
pared10 by way of condensation between various o-hydroxyace-
tophenones and N-protected-4-piperidones in the presence of
pyrrolidine. Although some of the substituted o-hydroxyacetophe-
nones were not commercially available, they were readily accessi-
ble as shown in Scheme 2. The pivotal step for the preparation of
31a–c was the ortho-lithiation of the corresponding substrates with
appropriate directing groups. The methyl substituted o-hydroxy-
acetophenone (31d) was prepared by a transformation from the
ethyl ester. 2-Fluorophenol (34) was acetylated and subjected to
the Fries rearrangement conditions to provide 3-fluoro-2-hydroxy
analog (31f). After spirocyclization, subsequent manipulation
(Scheme 3) was necessary for the appropriate functional group
* Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5436 8563.
E-mail address: uto.yoshikazu.pw@daiichisankyo.co.jp (Y. Uto).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.043

Y. Uto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 746–754
747
O
S
X
N
NH
O
R
H
N
OH
N
N
N
O
CF3
OH
O
(1)
(2)
2a: X = H
2b: X = N
1a: R = OMe
1b: R = NHEt
Figure 1. Structures of previously reported small molecule SCD-1 inhibitors.
O
O
N
H
N
H
N
OH
N
OH
O
N
N
N
N
O
O
Z
N
H
N
OH
N
N
K
L
M
Figure 2. Transition from benzoylpiperidine to spiropiperidine.
a-c
Br
Br
HO
OH
N
N
H
Boc
22
23
O
O
NH
HCl
d, e
n
n
24a: n = 1
24b: n = 2
25a: n = 1
25b: n = 2
O
O
S
F
OH
f
g-h
H
S
N
F
F
Boc
26
27
28
O
O
i, e
NH
HCl
29
Scheme 1. Reagents and conditions: (a) Boc2O, CH2Cl2, rt, 96%; (b) CH3SO2Cl, Et3N, CH2Cl2, ꢀ78 °C; (c) LiBr, THF, rt (61%, two steps); (d) NaH, 23, DMF, 50 °C, 30% from 24a or
14% from 24b (ꢀ25 °C to rt); (e) 4 N HCl in 1,4-dioxane, rt 94% for 25a or 83% for 25b; (f) 1,3-propanedithiol, I2, CHCl3, quantitative; (g) diisopropylamine, n-BuLi, ꢀ78 °C, then
4-oxo-piperidine-1-carboxylic acid tert-butyl ester, 93%; (h) pyridinium tribromide, tetrabutylammonium bromide, pyridine/CH2Cl2/H2O, 84%; (i) tert-BuOK, 1,4-dioxane,
reflux, 63%.
setting to provide the spiropiperidines (39a–j, 41–45). The interme-
diates for 8 and 14 are shown in Scheme 4. The spiropiperidines (49
and 50) are prepared in accord with the procedures reported in lit-
erature.11 Preparation of the chloropyridazines (51) for the final
condensation step (Scheme 5) has already been disclosed in our
previous publication.2

748
Y. Uto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 746–754
Y
X
Y
OH
OH
OH
O
a-c
O
NEt2
d, e
O
30
31a
O
32
CF3
CF3
X
31b: X = Cl, Y = H
31c: X = Y = F
F
OH
OEt
OH
O
F
OH
g, h
f
OH
O
34
31f
O
33
31d
Scheme 2. Reagents and conditions: (a) n-BuLi, tert-BuOK, TMEDA, then dry ice, ꢀ78 °C–0 °C, 61%; (b) trimethylsilyldiazomethane, MeOH–EtOAc–hexane, 0 °C to rt, 93%; (c)
MeMgBr, Et3N, toluene, 0 °C to rt, 43%; (d) n-BuLi, THF, then Ac2O, 41% (X = Y = F) or 37% (X = Cl, Y = H); (e) TiCl4, CH2Cl2, 63% (31) or quantitative yield (31c); (f) MeMgBr, Et3N,
toluene, 0 °C to rt, 75%; (g) AcCl, pyridine, CH2Cl2, 82%; (h) AlCl3, 1,2-dichlorobenzene.
Y
Y
X
Boc
Y
X
Boc
W
V
OH
O
N
N
W
V
O
O
W
V
O
a
b
36j: R = H
37: R = Me
36a, c-j
c
X
31a, c-j
OR
35a, c-j
Y
X
Y
X
Boc
N
NH
d-e
f
W
V
O
W
V
O
36a, c-e, j
HCl
38a, c-e, j
39a, c-e, j
O
N
NH
g
OH
O
h-j
OEt
O
O
O
Cl
31b
35b
Cl
39b
Cl
V = W = H
X = Y = H
a
h: V = F, W = H
i: V = H, W = F
j: V = W = X = Y = H
: X = CF3, Y = H
: X = Cl, Y = H
: X = Y = F
b
c
d: X = Me, Y = H
e: X = F, Y = H
f: X = H, Y = F
g: X = OMe, Y = H
Boc
N
NH
HCl
k-l
m, n
O
O
38j
OH
O
40
41
Y
X
Boc
Y
X
N
NH
n
W
V
O
L
W
O
L
42e-j
: L = C=O, M = CH2
L = CH(OMe), M= CH2
L, M = -(CH=CH)-
HCl
43
44
45
:
:
:
M
M
V
L = CH2, M = CH(OH)
35e-j, 37, 38j, 40
Scheme 3. Reagents and conditions: (a) N-Boc-4-piperidone, pyrrolidine, 1-PrOH or EtOH, rt–reflux; (b) NaBH4, MeOH, rt; (c) NaH, MeI, DMA, rt, 55%; (d) MsCl, Et3N, CH2Cl2;
(e) DBU, NMP, 100 °C; (f) Pd/C (10wt%), H2, 1 N HCl, (if necessary, acidic deprotection (4 N HCl, 1,4-dioxane) was followed.); (g) pyrrolidine, 4-oxo-piperidine-1-carboxylic
acid ethyl ester, EtOH, rt, 34%; (h) NaBH4, MeOH/THF, rt, 68%; (i) Et3SiH, BF3ꢁEt2O, CH2Cl2, 40 °C, 95%; (j) KOH, EtOH/H2O, 100 °C, 95%; (k) m-CPBA, 0.5 N NaHCO3(aq), CH2Cl2,
58%; (l) LiAlH4, THF, 98%; Dess–Martin periodinane, CH2Cl2, 86%; (n) 4 N HCl, 1,4-dioxane, rt, 94% (41).

Y. Uto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 746–754
749
HCl
NH
Bn
Bn
N
N
a
b
c
H
N
HO
O
O
O
O
46
47
48
49
NH
O
O
NH
HCl
d-e, c
47
O
50
Scheme 4. Reagents and conditions: (a) n-BuLi, THF, 0 °C to rt, then 1-benzyl-piperidin-4-one, Et2O, rt; (b) 50% AcOH, concd H2SO4, reflux, 12% (two steps); (c) H2, Pd/C
(10 wt %), 1 N HCl, EtOH, 38% (49) or quantitative (50); (d) LiAlH4, THF, 0 °C; (e) H3PO4, 100 °C, 49% (two steps).
O
(HCl)
Ar
Y
N
NH
O
H
W
V
K
L
Y
X
OH
a
Ar
OH
N
+
N
W
V
K
L
M
H
N
Cl
N
M
X
51
3 15
-
25, 29, 39, 42-45,
49-50
Scheme 5. Reagents and conditions: (a) Et3N (or iPrNEt2), n-BuOH, 100 °C.
NH2
N
H
H
N
N
N
NH
O
N
N
N
O
O
N
N
a
b
O
HCl
CF3
39a
52
16
CF3
CF3
Scheme 6. Reagents and conditions: (a) 6-chloropyridazin-3-ylamine, Et3N, n-BuOH, 120 °C, 68%; (b) 3-isocyanatomethylpyridine, CH2Cl2, reflux, 62%.
N
R
N
N
CN
N
NH
N
O
N
N
N
O
N
N
17
18
: R = H
: R = Me
a
b
O
HCl
CF3
39a
53
CF3
CF3
O
H
54: R = H
55: R = Me
N
N
R
N
H
O
O
c
O
H
+
H2N
N
N
N
N
N
N
EtO
H
56
55
57
Scheme 7. Reagents and conditions: (a) 6-chloro-pyridazine-3-carbonitrile, Et3N, NMP, 100 °C, 96%; (b) 54 or 55, NaOMe, MeOH, rt to reflux, then AcOH, reflux: 80% for 17
and 23% for 18; (c) methylhydrazine, EtOH, 80 °C, 55 (61%), 57 (13%).
Schemes 6–8 describe the synthetic routes for various right-
hand amide modifications. For the urea analog (16, Scheme 6),
the spiropiperidine (39a)12 was condensed with 6-chloropyrid-
azine-3-ylamine to provide 52 that was subsequently reacted
with 3-isocyanatomethylpyridine to give the desired product.
Scheme 7 outlines the synthesis of the triazole analogs (17 and

750
Y. Uto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 746–754
O
O
a
OH
Cl
N
N
O
N
Cl
N
H
58
59
N
N
O
c, d
e, d
OEt
NHNH2
N
b
b
N
N
N
N
O
O
N
60
54
Cl
N
61
N
N
N
O
O
NHNH2
O
OMe
N
63
62
Cl
N
64
N
O
N
n
NH
HCl
O
f
N
N
N
N
O
19:n = 1
20:n = 2
39a
CF3
CF3
N
O
N
O
O
N
g
h, i, d
N
N
N
N
N
N
39a
O
O
65
21
CF3
CF3
Scheme 8. Reagents and conditions: (a) SOCl2, DMF, CHCl3; (b) hydrazine monohydrate, EtOH, quant; (c) 59, Et3N, DMF, 57%; (d) TsCl, DMAP, CH3CN, 32% (61) or 71% (64); (e)
6-chloropyridazine-3-carboxylic acid, T3P, Et3N, DMF, rt, 43%; (f) 61 or 64, Et3N, n-BuOH, 100 °C, 65% (19), 72% (20); (g) 6-chloropyridazine-3-carboxylic acid methyl ester,
Et3N, NMP, 100 °C, quantitative; (h) NaOH (aq), 1,4-dioxane, 60 °C, 92%; (i) BOP reagent, Et3N, nicotinic acid hydrazide, quantitative.
18). The nitrile derivative (53) was separately condensed with
corresponding hydrazides to provide the desired triazoles (17
from 54 and 18 from 55). The hydrazide (57), which was obtained
as a minor regioisomer in the preparation of 55, failed to provide
a corresponding triazole under the same reaction conditions. In
order to synthesize the oxadiazole analog (19), the hydrazide
(54) was acylated with 59 and readily cyclized in the presence
of TsCl and Et3N to provide the pyridazyloxadiazole (61)12 that
was coupled with the spiropipridine (39a) in the analogous con-
ditions shown in Scheme 5.
As shown in Table 1, simple cyclization of the benzoylpiperidine
(2a) generated the five-membered spiro (3) that showed much
weaker inhibitory activity12 against human SCD-1. Ring expansion
to the six-membered ring (4) regained inhibitory activity against
human SCD-1 with sub-micro molar IC50. Replacement of the
methylene linkage in the spiro[indene-2,40-piperidin]-1(3H)-one
of 3 to the ether as shown in 5 did not improve the potency in
SCD-1 inhibition. Insertion of a methylene between the phenyl
and the carbonyl in the five-membered ring of 5 was unproductive
for murine SCD-1 and 0.25 lM for human enzyme. With the posi-
tion of the O-linker of 7 being changed within the six-membered
spirocyclic system, the lactone (8) exhibited sub-micro molar
IC50 against human SCD-1. Among the three spiro-ring systems
(4, 7, and 8) that presented comparable inhibitory activity against
human SCD-1, 7 was chosen for further structural optimization be-
cause this spirocyclic system is much easier to prepare and more
suitable for robust structural transformation.
Further optimization of the spirocyclic system of 7 is displayed
in Table 2. Reduction of the ketone to hydroxyl as in 9j resulted in a
threefold improvement in the potency against murine SCD-1. The
methoxy analog (10) exhibited much weaker inhibitory activity
against murine SCD-1 while the compound showed comparable
activity against human enzyme. Changing the position of the hy-
droxyl of 9j to the adjacent carbon as shown in 11 or introducing
the olefin as shown in 12 did not make significant improvements
in terms of potency.
Significant improvement was actually provided, to our sur-
prise, by simply reducing the olefin of 12. Compound 13, featured
with a 3,4-dihydrospiro[chromene-2,40-piperidine]-motif, presented
more than a 20-fold increase in inhibitory activity against murine
SCD-1 and a 100-fold increase in that against human enzyme.
as shown in 6 (IC50 (human) >10
methylene between the piperidine and the carbonyl of 5 produced
7, which showed reasonable potency, with IC50 values of 0.050
lM). By contrast, insertion of a
lM

Y. Uto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 746–754
751
Table 1
Table 2
Evaluation of the spiropiperidinesa
Evaluation of the spiropiperidines (7 and related compounds)a
O
O
N
H
N
H
N
OH
N
N
OH
R
N
R
N
Enzymatic assayb
IC50 M)
Human cell (293A)
Compound R
Enzymatic assayb
Cellular
assayb
Compound
R
IC50
Mouse liver
microsomal
(lM)
(l
b
IC50
(l
M) IC50
(
l
M)
IC50 (lM)
Human cell
Mouse liver Human cell
microsomal (293A)
9
D
9
microsomal
D9
(293A)
D9
D
microsomal
D9
2a
0.007
0.015
N
O
O
O
7
0.050
0.016
0.25
0.69
O
O
N
O
O
3
4
5
NTc
2.0
N
N
N
9j
0.34
0.48
N
0.020
>10
0.57
NTc
OH
O
N
10
11
0.060
0.049
0.60
0.30
NTc
O
O
O
N
N
O
6
7
NTc
>10
0.56
O
OH
N
N
N
O
O
NTc
O
O
12
13
14
0.061
0.0027
0.076
0.79
0.050
0.25
0.007
>0.40d
0.068
NTc
N
8
0.23
0.32
O
O
O
a
Values are the arithmetic mean of at least two experiments.
See Supplementary data for the experimental procedures for the desaturase
b
a
Values are the arithmetic mean of at least two experiments.
See Supplementary data for the experimental procedures for the desaturase
b
assay.
c
NT = not tested.
assay and cellular assay.
c
NT = not tested.
d
29% inhibition at 0.40 lM.
Compound 13 also showed strong potency in the cellular assay
with an IC50 value of 0.068 M. A similar analog, 14, with the only
l
difference from 13 being the position of the ether in the ring sys-
tem, caused a significant decrease in inhibitory activity.
8-substitution, the 5-substituted 9e was preferred over the 8-
substituted 9f because 9e demonstrated three times better activ-
ity against human enzyme than that of 9f. Even more, in terms of
in vivo potency, as evaluated by inhibitory activity in the liver
SCD-1 of db/db mice and described as ID50 (mg/kg),12 9e pre-
sented a twofold increase in the in vivo potency, with an ID50
value of 7 mg/kg, compared to that of the unsubstituted 9j
(ID50 = 15 mg/kg). As exemplified by the results of 9g, an elec-
tron-donating substituent such as methoxy is not ideal for strong
SCD-1 inhibition; 9g showed almost no inhibitory activity against
human SCD-1 under the standard assay conditions. In the next
round of optimization with the more potent spirosystem of 13,
the east side phenyl was replaced with 3-pyridyl because the
3-pyridyl substitution was proved to be more favorable for
With the optimized spirocyclic system in hand, the substitu-
tion on the west side phenyl was investigated. For synthetic ease,
the spirocyclic structure of 9 was utilized for various substitu-
tions. As exemplified in Table 3, the available positions for substi-
tution on the phenyl were numbered as 5, 6, 7 and 8. Fluorine
was used in the search for a favorable substitution position
because the corresponding starting materials were either com-
mercially available (9e, h, i) or readily accessible by simple syn-
thetic procedures (9f). While the enzymatic inhibitory activity
of the 5- or 8-substituted compounds (9e or 9f) were comparable
to that of the unsubstituted 9j, the 6- or 7-substituted ones (9h or
9i) showed only marginal inhibitory activity. Between the 5- and

752
Y. Uto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 746–754
Table 3
Evaluation of the substituents of the spiropiperidinesa
O
O
N
N
H
N
H
N
OH
N
OH
8
8
N
N
N
N
O
O
7
7
R
R
9
15
6
6
5
5
OH
Compound
R
Enzymatic assayb
Cellular assayb
db/db miceb
ID50 (mg/kg)
IC50 (nM) Mouse liver microsomal
D
9
IC50 (nM) Human cell (293A) microsomal
D
9
IC50 (nM) Human cell (293A) D9
9j
9e
9h
9i
9f
9g
15e
15b
15d
15a
15c
—
5-F
6-F
7-F
8-F
5-OMe
5-F
5-Cl
5-Me
5-CF3
5,8-Di-F
16
17
NTc
156
33
NTc
6
1
1
0.5
2
338
97
481
157
NTc
NTc
NTc
NTc
7
2
6
3
7
15
7
>1000
548
315
>1000
12
0.6
2
0.3
2
NTc
20
NTc
NTc
2
3
4
0.8
5
a
b
c
Values are the arithmetic means of at least two experiments.
See Supplementary data for the experimental procedures for the desaturase assay, cellular assay, and the determination of ID50
NT = not tested.
.
in vivo potency in our previous investigation.2 As showcased in
Table 3, the halogens (5-F in 15e and 5-Cl in 15b), alkyl (5-Me
in 15d), and haloalkyl (5-CF3 in 15a)12 exhibited very strong po-
tency in all of the enzymatic (murine and human), cellular, and
in vivo assay. As expected from the combined results of the
mono-substitution, the 5,8-difluoro substitution (15c) also
showed strong potency with an IC50 value of 7 nM in the cellular
assay. Among the cluster of strong SCD-1 inhibitors, 15a, the most
potent compound, was chosen for further investigation.
For the analysis of the in vivo efficacy of SCD-1 inhibitors, we
took note that Attie and co-workers reported that the hepatic tri-
glyceride levels of mice on a very low-fat diet increased by
240%.13 We assumed that the SCD-1 activity in the liver of these
mice was very high and were interested in the inhibitory effect
of the spiropiperidine-based SCD-1 inhibitors against the liver
SCD-1 in C57BL/6 J mice on a non-fat diet.12,14 In comparison with
the prior model (db/db mice on a chow diet), utilized in the evalu-
ation of the compounds in Table 3, 15a showed strong potency and
an almost identical ID50 value (Table 6) in the C57BL/6 J on a non-
fat diet at the same time-point (ID50 = 0.9 mg/kg at 2–3 h). Simi-
larly, 19 demonstrated very strong potency in liver SCD-1 inhibi-
tion (ID50 (2–3 h) = 0.5 mg/kg) resulting from a combination of
strong enzymatic inhibitory activity (IC50 (mouse) = 0.06 nM) and
Table 4 summarizes the optimization of the right-hand moiety of
15a, particularly the amide bond replacement. The hydroxyl group
was removed in this series for synthetic simplicity and our prior
studies2 indicated that the inhibitory activity against SCD-1 was
stronger without the hydroxyl. The first example, the urea (16)
showed a 20-fold decrease in murine SCD-1 inhibition and more
than a 30-fold decrease in the cellular assay. The triazole analog
(17) demonstrated comparable inhibitory activity against both
SCD-1s to 15a. However, simple methylation of the nitrogen of the
triazole (18) resulted in an evident loss of inhibitory activity,
especially more than a >100-fold loss against murine SCD-1. The
oxadiazole (19)12 was most powerful in this series, presenting about
a 10-fold increase in inhibitory activity against human SCD-1
(IC50 = 0.03 nM). The length of the linker between the oxadiazole
and the terminal pyridine turned out to be critical since elongation
of the linker to the ethylene (20) resulted in a significant loss of inhi-
bition (>200-fold against murine SCD-1) and removal of the linker
(21) resulted in a complete loss of inhibitory activity against SCD-1.
The representative spiropiperidines (15a and 19) were evalu-
ated for PK profiles in C57BL/6 J mice (Table 5). The plasma half-life
of 15a (t1/2 = 0.9 h) was as short as that of lead compound 22 and
15a was quickly eliminated from plasma (Cl = 17 mL/min/kg). After
oral administration, 15a was readily absorbed and detected in the
plasma through the tested time course with fair values of concen-
good oral exposure right after oral administration (Cmax = 2.6
mL and Tmax = 1 h).
lg/
For multiple dosing studies of SCD-1 inhibitors, 19 was tested in
a 7-day efficacy study using C57BL/6 J mice on a non-fat diet.14 The
desaturation index, calculated as the ratio of C18:1/C18:0, was
used as an in vivo biomarker. After once-daily administration for
seven days,12 19 dose-dependently reduced the desaturation in-
dex, with a 75% reduction at 3 mg/kg (Fig. 3). In the preliminary
analysis, we did not observe notable abnormalities in the skin or
eyes of the C57BL/6 J mice at 3 mg/kg (cutaneous abnormalities
and narrow eye fissure have been reported in studies on SCD-1
deficient mice).15 We assume that the balanced combination of
the strong potency and short plasma half-life of 19 resulted in
pharmacological efficacy in vivo and may be beneficial in prevent-
ing adverse events. While this is a preliminary speculation, the rel-
atively short plasma half-life of 19 may help to accomplish
favorable tissue selectivity (liver over eyes or skin). Currently, we
do not know the long term effect of 19. However, some abnormal-
ities15 may be observable with an extended administration period.
Histopathological analysis of the key tissues (eyes, skin, and liver)
of the C57BL/6 J mice after a 7-day treatment with the SCD-1 inhib-
itor 19 is currently in progress and will be reported elsewhere
along with more details about the pharmacological studies of the
spiropiperidine-based SCD-1 inhibitors.
tration (Cmax = 3.5 lg/mL and AUC = 13 lg h/mL) and bioavailabil-
ity (F = 67%). The iv data for the oxadiazole analog 19 was not
taken at this point because we could make sure that the compound
showed enough plasma exposure after oral administration
(Cmax = 2.6 lg/mL and AUC = 7.8 lg h/mL).

Y. Uto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 746–754
753
Table 4
Evaluation of the spiropiperidines (right-hand structure)a
W
N
N
N
O
CF3
Compound
W
Enzymatic assayb
Cellular assayb
IC50 (nM) Mouse liver microsomal
0.45
D
9
IC50 (nM) Human cell (293A) microsomal
D
9
IC50 (nM) Human cell (293A) D9
O
N
N
15d
0.28
3
N
H
OH
H
N
H
N
16
17
10
1
2
106
O
N
N
N
N
N
0.13
4
HN
N
N
N
18
19
65
4
38
0.8
N
O
0.06
0.03
N
N
N
O
N
O
N
N
20
21
16
2
35
N
>10,000
NTc
NTc
a
b
c
Values are the arithmetic means of at least two experiments.
See Supplementary data for the experimental procedures for the desaturase assay and the cellular assay.
NT = not tested.
Table 5
PK parameters of 15a and 19 in C57BL/6 J micea
No
PKb (iv, 5 mg/kg)
Cl (mL/min/kg)
PKb (po, 20 mg/kg)
F (%)
t1/2 (h)
Vd (L/kg)
Cmax
(
lg/mL)
t1/2 (h)
Tmax (h)
AUC(0–8 h) (lg h/mL)
15a
19
0.9
17
1.0
3.5
2.6
2.0
1.4
1.0
1.0
13
7.8
67
NA
NTc
NTc
NTc
a
A dose of each compound was either intravenously (5 mg/kg, DMA/Tween80/saline = 10/10/80) injected into the tail vein of C57BL/6 J mice (n = 2) or orally (20 mg/kg, PG/
Tween = 4/1, n = 3) administered using an intubation tube. Plasma samples (20
lL) were collected up to 8 h after intravenous or oral administration. The plasma concen-
trations of compounds were determined by LC/MS.
b
Values are the arithmetic means of at least two experiments.
Not tested.
c
In summary, cyclization of the benzoylpiperidine in lead com-
pound 2 generated a series of spiropiperidines. Comprehensive
optimization of the spiropiperidines led to a series of powerful
SCD-1 inhibitors. Among them, 10-{6-[5-(pyridin-3-ylmethyl)-1,
3,4-oxadiazol-2-yl]pyridazin-3-yl}-5-(trifluoromethyl)-3,4-dihy-
drospiro[chromene-2,40-piperidine] (19) demonstrated strong
inhibitory activity against SCD-1 not only in vitro but also in vivo
(C57BL/6 J mice). As for pharmacological evaluation, 19 showed
powerful reduction of the desaturation index in the plasma of
C57BL/6 J mice on a non-fat diet after a 7-day oral administration
(q.d.) without causing notable abnormalities in the eyes or skin
up to the highest dose (3 mg/kg). Further evaluation of the novel

754
Y. Uto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 746–754
C18=1(n-9)cis/C18=0
References and notes
7
6
5
4
3
2
1
0
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0.3 mpk
1 mpk
3 mpk
Cont
Compd 19
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Figure 3. Plasma desaturation index lowering effect of the treatment with SCD-1
inhibitor 19 for 7 days (q.d.) in C57BL/6 J mice (n = 4) fed a non-fat diet.
7. All the final compounds were characterized by 1H NMR and mass spectroscopy.
8. Yang, L.; Morriello, G.; Prendergast, K.; Cheng, K.; Jacks, T.; Chan, W. W.-S.;
Schleim, K. D.; Smith, R. G.; Patchett, A. A. Bioorg. Med. Chem. Lett. 1998, 8, 107.
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Table 6
The liver SCD-1 inhibition by 15a and 19 in C57BL/6 J mice on a non-fat dieta
10. (a) Nerenberg, J. B.; Erb, J. M.; Bergman, J. M.; O’Malley, S.; Chang, R. S. L.; Scott,
A. L.; Broten, T. P.; Bock, M. G. Bioorg. Med. Chem. Lett. 1999, 9, 291; (b) Fletcher,
S. F.; Burkamp, F.; Blurton, P.; Cheng, S. K. F.; Clarkson, R.; O’Connor, D.; Spinks,
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S.; Bentley, G.; Cook, G. P.; Bristow, L. J.; Castro, J. L.; Hutsaon, P. H.; MacLeod, A.
M. J. Med. Chem. 2002, 45, 492.
b
Compound
ID50 (mg/kg)
at 2–3 hc
at 6–7 hc
15a
19
0.9
0.5
2.5
0.8
11. Butcher, J. W.; Claremon, D. A. Synthesis 1993, 211.
12. For the experimental procedures, please see Supplementary data.
13. (a) Flowers, M. T.; Groen, A. K.; Oler, A. T.; Keller, M. P.; Choi, YJ.; Schueler,
K. L.; Richards, O. C.; Lan, H.; Miyazaki, M.; Kuipers, F.; Kendziorski, C. M.;
Ntambi, J. M.; Attie, A. E. J. Lipid Res. 2006, 47, 2668; The association of
high-carbohydrate diets with high SCD activity was recently reviewed (b)
Flowers, M. T.; Ntambi, J. M. Biochim. Biophys. Acta. Mol. Cell Biol. Lipids
2009, 1791, 85.
a
b
c
See Supplementary data for the experimental protocols.
Values are the arithmetic means of at least two experiments.
SCD-1 inhibitors were administered to the mice (n = 2) 2 h or 6 h prior to the
administration of [14C] stearate. One hour after the injection of [14C] stearate, the
mice were sacrificed and their livers were removed.
14. The non-fat diet was purchased from Research Diets, Inc. (D05052506). The
diet is composed of protein (22.2 kcal%) and carbohydrate (77.8 kcal%).
15. (a) Miyazaki, M.; Man, W. C.; Ntambi, J. M. J. Nutr. 2001, 131, 2260; (b) Binczek,
E.; Jenke, B.; Holtz, B.; Gunter, R. H.; Thevis, M.; Stoffel, W. Biol. Chem. 2007,
388, 405.
spiropiperidine-based SCD-1 inhibitors reported in this article will
be disclosed in future.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.11.043.