Discovery of potent and liver-selective stearoyl-CoA desaturase (SCD) inhibitors in an acyclic linker series

Article abstract of DOI:10.1016/j.bmcl.2011.10.070

Elevated levels of stearoyl-CoA desaturase (SCD) activity have been implicated in metabolic disorders such as obesity and type II diabetes. To circumvent skin and eye adverse events observed in rodents with systemically-distributed inhibitors, our research efforts have been focused on the search for new liver-targeting compounds. This work has led to the discovery of novel, potent and liver-selective acyclic linker SCD inhibitors. These compounds possess suitable cellular activity and pharmacokinetic properties to inhibit liver SCD activity in a mouse pharmacodynamic model.

Full text of DOI:10.1016/j.bmcl.2011.10.070

Bioorganic & Medicinal Chemistry Letters 22 (2012) 623–627
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of potent and liver-selective stearoyl-CoA desaturase (SCD)
inhibitors in an acyclic linker series
Nicolas Lachance a, , Sébastien Guiral b, Zheng Huang b, Jean-Philippe Leclerc a, Chun Sing Li a,
⇑
Renata M. Oballa a, Yeeman K. Ramtohul a, Hao Wang b, Jin Wu c, Lei Zhang b
a Department of Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research, 16711 TransCanada Hwy, Kirkland, Quebec, Canada H9H 3L1
b Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, 16711 TransCanada Hwy, Kirkland, Quebec, Canada H9H 3L1
c Department of Drug Metabolism and Pharmacokinetics, Merck Frosst Centre for Therapeutic Research, 16711 TransCanada Hwy, Kirkland, Quebec, Canada H9H 3L1
a r t i c l e i n f o
a b s t r a c t
Article history:
Elevated levels of stearoyl-CoA desaturase (SCD) activity have been implicated in metabolic disorders
such as obesity and type II diabetes. To circumvent skin and eye adverse events observed in rodents with
systemically-distributed inhibitors, our research efforts have been focused on the search for new liver-
targeting compounds. This work has led to the discovery of novel, potent and liver-selective acyclic linker
SCD inhibitors. These compounds possess suitable cellular activity and pharmacokinetic properties to
inhibit liver SCD activity in a mouse pharmacodynamic model.
Received 26 September 2011
Revised 18 October 2011
Accepted 20 October 2011
Available online 28 October 2011
Keywords:
Stearoyl-CoA desaturase inhibitors
SCD inhibitors
Ó 2011 Elsevier Ltd. All rights reserved.
Desaturation index
Liver-selective
MK-8245
Inhibition of stearoyl-CoA desaturase (SCD) represents a poten-
tial novel mechanism of action for the treatment of obesity and
type II diabetes, which continue to expand at epidemic rates.1
The SCD enzyme is present in two isoforms in humans (SCD1
and SCD5) and four in rodents (SCD1-4) where the SCD1 isoform
is predominantly found in liver (target organ for efficacy). In the
literature, adverse events (AEs),2 such as partial eye closure and
progressive alopecia, were observed in mice after ꢀ7 days of treat-
ment with systemically-distributed SCD inhibitors. These AEs are
likely due to the depletion of SCD-derived lubricating lipids in skin
and eye, and the development of liver-targeted SCD inhibitors
should circumvent these issues.3
The initial strategy employed in the design of liver-selective
compounds centers on exploring the addition of polar acidic
moieties which are recognized by organic anionic transporters
(OATPs),4 such as tetrazoles or carboxylic acids, on SCD inhibitors
to obtain the desired in vivo properties: a high liver concentration
(target organ for efficacy) and a low systemic concentration to
minimize exposures in off-target tissues and cells associated with
adverse events (skin and eye).
addition, MK-8245 possesses good in vivo potency in a mouse liver
pharmacodynamic model (mLPD, ED50 ꢀ1 mg/kg) and showed no
AEs after 4 weeks of chronic dosing in mice.5,6 Following the dis-
covery of MK-8245, our group continued our efforts on the identi-
fication of structurally diverse liver-targeted SCD inhibitors for the
selection of a back-up compound to our lead MK-8245 to support
the development of SCD inhibitors in preclinical species and even-
tually in humans.
In vitro studies have demonstrated that the liver-targeted tissue
distribution profile of MK-8245 is likely the result of substrate rec-
ognition by organic anionic transporter proteins (OATPs) which are
highly expressed in hepatocytes.4,5 An important feature in the
structure of MK-8245 is the tetrazole acetic acid moiety, which is
the key functionality for OATPs recognition and liver-targeting. In
a search for new structural classes, we intentionally kept this
group in place for active transporter recognition and modified
other parts of the molecule. We envisioned that the replacement
of the piperidine core present in MK-8245, and also frequently
Br
MK-8245 (Fig. 1), a phenoxy piperidine isoxazole derivative, has
been identified as a potent and liver-selective SCD inhibitor with
an enzymatic potency (IC50) against the rat SCD of 3 nM.5 In
O
HO
N
N
N
N
O
N
N
F
O
MK-8245
⇑
Corresponding author.
E-mail address: nicolas.lachance21@gmail.com (N. Lachance).
Figure 1. MK-8245: a potent SCD inhibitor (Rat SCD IC50 = 3 nM).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.070

624
N. Lachance et al. / Bioorg. Med. Chem. Lett. 22 (2012) 623–627
Br
HO
HO
avoiding the repetitive tedious synthesis of the tetrazole acetic acid
R
N
N
O
Z
n
N
N
N
N
O
n
Z
moiety for each compound, the coupling of 15 with an appropri-
ately substituted phenoxy linker 5 represented an expedient meth-
od that allows the preparation of acyclic linker targets 16 in only
two steps (Scheme 2).
O
O
HetAr
N
N
N
O
1
2
F
n = 2-4
The SCD inhibitors prepared were tested against the SCD1 en-
zyme in an SCD-induced rat liver microsomal assay.10 Their cellu-
lar potencies were evaluated in a human HepG2-based whole cell
assay which was devoid of OATPs,11 and the ability to cross the cell
membrane through active transporters was assessed in a rat hepa-
tocyte (Rat Hep) assay which contains OATPs.5 The goal was to
qualitatively determine if an SCD inhibitor was actively trans-
ported into hepatocytes (potent Rat Hep IC50) while maintaining
poor cell permeability (poor HepG2 IC50).
As shown in Table 1, the optimal replacement of the piperidine
core in MK-8245 was with a 5-atom tether 16b, whereas the pres-
ence of a shorter tether 16a or longer one 16c resulted in loss of
potency. Interestingly, the position of the oxygen atom next to
the phenyl group remains critical as illustrated with the loss of po-
tency observed with the benzyl analog 16d. The acyclic linker 16b
displayed a ꢀ600-fold shift of potency in the HepG2 assay
(IC50 = 7740 nM) and improved to 16-fold shift in the rat hepato-
cyte assay (IC50 = 213 nM), which highly suggests the involvement
of active transporter systems. Overall, the acyclic linker 16b
showed 4 to 7-fold lower in vitro potency (Rat SCD, HepG2 and
Rat Hep) when compared to MK-8245 (Table 1).
Figure 2. Core structure of acyclic linker series 1 and initial tether optimization in
compound 2.
found in many SCD-inhibitors,2a,7 with linkers such as an acyclic
tether would provide a distinct and structurally diverse series of
SCD1 inhibitors 1 (Fig. 2).8
In the absence of an SCD1 enzyme X-ray crystal structure, we
chose to proceed with a systematic SAR study to guide our optimi-
zation efforts. Initially, we turned our attention to the synthesis of
4–6 atom linkers 2 (Fig. 2) while keeping the remaining structural
elements of MK-8245 unchanged.
As depicted in Scheme 1, the construction of 4–6 atom linker
counterparts involved ring opening of ethylene carbonate 4 with
phenol 3, Mitsunobu reaction between phenol 3 and alcohol chains
6 and SN2 alkylation of the phenol or the ethylene glycol 8 with the
alkyl halide 6 or 7 in presence of a base such as K2CO3.
Having in hand a suitable collection of phenoxy linkers 5 with
different chain lengths, we next turned our attention to link these
molecules to the isoxazole tetrazole acetic acid moiety. As illus-
trated in Scheme 2, the initial route consisted of a stepwise con-
struction of the tetrazole acetic acid moiety contained in 13.9 To
this end, reaction of hydroxyisoxazole 9 with 5 under Mitsunobu
conditions or SN2 alkylation with K2CO3 gave exclusively the
O-alkylation product 10. The ester compound 10 thus obtained
was transformed to the nitrile 11 by conversion of the ester group
to the corresponding primary amide with ammonia followed by
dehydration with TFAA. The preparation of the tetrazole 12 was
completed by reaction of the nitrile 11 with NaN3 under slightly
acidic conditions. Introduction of the acetate ester group on the
tetrazole 12 gave two regioisomers 13 and 14. Under several con-
ditions evaluated (NaH in DMF, K2CO3 in DMF, Mitsunobu, Et3N in
THF, Hünig’s base in 1,4-dioxane) the best ratio in favor of 13 was
obtained with tertiary amine bases and ethereal solvents. Final
hydrolysis of 13 with NaOH afforded 16.
The enzymatic potency against the human SCD was also mea-
sured. In a human SCD1 enzyme assay (hSCD1) (delta-9 desatur-
ase), compound 16b displayed an IC50 of 41 nM compared to
1 nM for MK-8245.5 The linker 16b is almost equipotent at inhib-
iting the other isoform found in human (hSCD5 IC50 = 32 nM). In
addition, it is selective against two other desaturase enzymes (del-
ta-5 and delta-6 desaturases) present in human, with IC50s >20
lM
in a whole cell assay.11
Before pursuing further SAR, we decided to evaluate the in vivo
potency of 16b in a mouse liver pharmacodynamic model (mLPD).6
To support this model, the activity for the linker 16b was measured
in a mouse SCD1 enzyme assay (IC50 = 10 nM) which showed a
similar potency compared to the rat SCD enzyme. Also, the dose
selection was based on the pharmacokinetic (PK) profile deter-
mined in C57BL6 mice following oral dosing at 10 mg/kg in 0.5%
methocel as the vehicle. Under these conditions, the linker 16b
Subsequently, a simplified route was realized through the use of
a highly functionalized intermediate 15. Employing p-methoxy-
benzyl (PMB) alcohol 5 under the reaction conditions outlined in
Scheme 2 and subsequent cleavage of the PMB under acidic condi-
tions, then afforded the versatile synthetic intermediate 15. By
had a low liver concentration at a 6 h time-point (0.23 l
M).12 Con-
sequently, a high dose (60 mg/kg) of 16b was required to effi-
ciently suppress liver SCD activity in the mLPD assay (85%
inhibition at a liver concentration of 7.8
MK-8245 required only a dose of 2 mg/kg in the mLPD model to af-
ford 89% liver SCD inhibition at a liver exposure of 4.9 M.
To further understand the underlying cause of the poor PK pro-
file of 16b, we performed in vitro metabolism studies. In standard
mouse and rat hepatocyte incubations, compound 16b showed two
major metabolites: the oxidative defluorination followed by gluta-
thione (GSH) conjugation on the phenyl ring and the reductive ring
opening of the isoxazole heterocycle.13,14
lM of 16b). In contrast,
General schemes to access linker intermediates (5)
l
Route to 4 atom linker R-X intermediates (5a)
O
a
O
Ar OH
O
O
+
Ar
OH
3
4
5a
Route to 5 or 6 atom linker R-X intermediates (5, 5b)
b or c
O
Y
X
Y
These data oriented our synthetic efforts on improving the
in vivo potency by increasing the metabolic stability of this class
of inhibitors.
+
3
Ar
n
n
5
6
n = 3,4
X = Y = Cl, Br, or OH
5b: Y = OH
The identification of more metabolically stable heterocycles
that may not suffer from the ring opening observed with the isox-
azole core became our first priority. The method used to prepare
isoxazole compounds 16 under O-alkylation conditions was unsuc-
cessful for other heteroaromatic rings delivering exclusively the N-
alkylation product. To circumvent this problem, compounds 22–24
were prepared via displacement of a halo-heteroaromatic ring 17
with the alkoxy compounds 5a–b as reported in Scheme 3. Further
elaboration to the tetrazole acetic acid group from ester 18a, amide
Second route to 5 atom linker R-X intermediates (5c)
c
+
OH
OH
Ar
Br
HO
Ar
O
7
8
5c
Scheme 1. Preparation of phenoxy linker moieties 5. Reagents and conditions: (a)
imidazole (cat.), neat, 150 °C for 5 h; (b) (Y = OH); di-tert-butyl azodicarboxylate,
PPh3, CH2Cl2–THF, À78 °C to r.t. for 1–24 h; (c) (Y = Cl, Br); K2CO3, DMF, 60 °C or
100 °C for 1–5 h.

N. Lachance et al. / Bioorg. Med. Chem. Lett. 22 (2012) 623–627
Initial route to isoxazole compounds (16)
625
OH
O
O
O
a or b
c or d,e
R
R
R
R
Y
MeO2C
+
MeO2C
NC
N
N
N
N
O
9
O
10
O
O
5
11
Y = Cl, Br, OH
f
EtO2C
g
N
N
O
O
N
N
N
N
N
N
+
R
R
N
H
N
N
N
N
O
N
O
14: Minor
13: Major
12
EtO2C
R = PMB
h
i
EtO2C
HO2C
N
N
N
OH
O
N
N
R
N
N
N
N
N
O
O
15
16
Simplified route to isoxazole compounds (13)
a or b
R = PMB, ArO-(CH2)n, ArCH2-O-(CH2)2
15
5
13
+
Scheme 2. Preparation of analogs in Table 1. Reagents and conditions: (a) (Y = OH); di-tert-butyl azodicarboxylate, PPh3, CH2Cl2–THF, À78 °C to r.t. for 1À24 h; (b) (Y = Cl, Br);
K2CO3, DMF, 60 °C for 1–5 h; (c) NH4OH (conc.), THF 0 °C to r.t. for 2 days; (d) NH3, MeOH, sealed tube, 125 °C for 30–60 min; (e) TFAA, Hünig’s base, CH2Cl2, À78 to 0 °C for 5–
45 min; (f) NaN3, PyÁHCl, NMP, 125–140 °C for 30–90 min; (g) ethyl bromoacetate, Hünig’s base, 1,4-dioxane, 90 °C for 1 h; (h) (PMB: p-methoxybenzyl); TFA, Me2S, H2O,
CH2Cl2, r.t. for 3 h; (i) 1 N NaOH, MeOH, THF, r.t. for 5 min.
Table 1
Exploration of the piperidine replacement
Br
F
HO
Linker
N
N
N
O
N
N
O
Compound
MK-8245
Linker
IC50 (nM)a
HepG2
Rat SCD
3
Rat Hep
68
O
O
1070
N
O
O
16a
16b
16c
16d
226
13
>40,000
7740
n.d.
213
n.d.
n.d.
O
O
O
248
305
>40,000
>100,000
O
O
a
IC50s are an average of at least two independent titrations; n.d.—not determined.
18b or nitrile 18c employed similar conditions as described for the
isoxazoles 16 (see Scheme 2).
metabolism observed in hepatocyte incubations with 23a. Among
the fluorine replacements explored,16 we found that the phenyl ring
substituted with an OCF3 group instead of the fluorine was metabol-
ically more stable under hepatocyte incubation conditions. In addi-
tion to the superior metabolic stability over the fluorine atom, the
in vitro potency of 23b was also slightly improved when compared
to 23a (Table 2). In the mLPD experiment, 81% inhibition of the
SCD activity was measured following an oral dose of 2 mg/kg of
The activities on the SCD-induced rat liver microsomal assay for
the heterocycles 22–24 prepared are reported in Table 2. Interest-
ingly, SCD inhibitory activity was slightly improved with the
5-membered heterocyclic thiazole 23a.15 Also, this thiazole hetero-
cyclic ring displayed an improved in vitro metabolic profile.
Compound 23a, when subjected to a standard rat hepatocyte incu-
bation for 2 h, was metabolically stable, with 94% parent remain-
ing.13 The only metabolites observed resulted from the oxidative
defluorination and GSH conjugation on the phenyl ring.
23b with a liver exposure of 4.2 lM (Table 3). In summary, the acy-
clic linker 23b possesses a comparable in vitro potency to MK-8245
(Table 1) and excellent in vivo efficacy in the mLPD at 2 mg/kg
(Table 3).
In comparison to 16b, the data from the in vitro metabolism
experiment was in agreement with a better PK profile measured
for the thiazole 23a in C57BL6 mice following typical 10 mg/kg oral
As expected, 23b displayed a liver-selective tissue distribution
profile. However, the liver/plasma and liver/Harderian glands ra-
tios were slightly reduced when compared to MK-8245 (Table 3).
The concentration of 23b in the Harderian glands was in the mi-
cro-molar level, which would not be desirable, given the connec-
tion that inhibiting SCD in this tissue is linked to eye AEs.5
In conclusion, we have identified an acyclic linker series as a
new structural class of SCD inhibitors. In this series, the piperidine
core present in MK-8245, and in typical SCD-inhibitors,7 is
dosing (F = 32%, AUC0–6 h = 11.4
6 h time-point (8.07 M) was also improved. Evaluation of this
compound 23a in the mLPD model showed a 49% inhibition at a
lM h). The liver concentration at a
l
lower dose of 2 mg/kg with a liver exposure of 7.6
concentration, see Table 3).
lM (total drug
Having in hand a good replacement for the central isoxazole ring,
we turned our attention to minimize the oxidative defluorination

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N. Lachance et al. / Bioorg. Med. Chem. Lett. 22 (2012) 623–627
General route to heterocyclic compounds (22-24)
S
S
a
X
O
EWG
EWG
R
OH
18a: EWG = CO2R'
18b: EWG = CONH2
18c: EWG = CN
+
R
b
c
N
N
A
A
5a-b
17
18a-c
EWG = CO2R', CONH2, CN
X = Cl, Br
A = N, CH, CMe
O
N
S
O
O
R
N
N
S
A
N
N
S
A
N
d
e
R
R
R
N
N
N
N
+
18c
N
N
N
N
N
A
H
19
20: Major
21: Minor
CO2Et
EtO2C
f
O
S
N
N
N
N
A
N
22-24
CO2H
Scheme 3. Preparation of analogs in Table 2. Reagents and conditions: (a) NaH, DMF, r.t. or 60 °C for 1 h; (b) NH3, MeOH–THF, sealed tube, 125 °C for 5 h; (c) TFAA, Hünig’s
base, CH2Cl2, À78 to 0 °C for 5 min; (d) NaN3, NH4Cl, DMF, 100 °C for 1–2 h; (e) ethyl bromoacetate, Hünig’s base, 1,4-dioxane, 90 °C for 1 h; (f) 1 N NaOH, THF, r.t. for 15–
30 min.
Table 2
replaced by an alkyl chain connecting the heterocycle ring via an
SAR on the heteroaromatic ring
oxygen atom. SAR and metabolism studies have led to the identifi-
HO
Br
cation of the thiazole heterocycle and the 2-bromo-5-trifluoro-
methoxyphenol in 23b as the optimal combination for in vitro
potency and in vivo efficacy. Presently, the exploration of the acy-
clic linker series is suspended but any future work will need to fo-
cus on improving liver-selectivity of this series and reducing the
Harderian gland drug exposure that may be linked to potential
eye AEs.
N
N
N
O
O
O
HetAr
N
R
Compound
HetAr
R
IC50 (nM)a
HepG2
Rat SCD
Rat Hep
n.d.
S
22
F
26
5
>60,000
3870
Acknowledgments
N
N
S
S
S
The authors thank Dan Sørensen for NMR spectroscopic assis-
tance on gHMBC, 15N gHMBC and 1D NOESY experiments and
David A. Powell for proofreading the manuscript.
23a
23b
F
176
91
N
N
N
OCF3
2
969
References and notes
24
F
22
20,500
945
1. Reviews: (a) Dobrzyn, P.; Dobrzyn, A. Expert Opin. Ther. Patents 2010, 20, 849
and references therein; (b) Liu, G. Expert Opin. Ther. Patents 2009, 19, 1169 and
references therein.
Me
a
IC50
s are an average of at least two independent titrations; n.d.—not
2. Leading references: (Mice AEs): (a) Leger, S.; Black, C.; Deschenes, D.; Dolman,
S.; Falgueyret, J.-P.; Gagnon, M.; Guiral, S.; Huang, Z.; Guay, J.; Leblanc, Y.; Li, C.-
S.; Masse, F.; Oballa, R.; Zhang, L. Bioorg. Med. Chem. Lett. 2010, 20, 499; (b)
Ramtohul, Y. K.; Black, C.; Chan, C.-C.; Crane, S.; Guay, J.; Guiral, S.; Huang, Z.;
Oballa, R.; Xu, L.-J.; Zhang, L.; Li, C. S. Bioorg. Med. Chem. Lett. 2010, 20, 1593; (c)
Li, C. S.; Belair, L.; Guay, J.; Murgasva, R.; Sturkenboom, W.; Ramtohul, Y. K.;
Zhang, L.; Huang, Z. Bioorg. Med. Chem. Lett. 2009, 19, 5214. (Rats AEs): (d)
Atkinson, K. A.; Beretta, E. E.; Brown, J. A.; Castrodad, M.; Chen, Y.; Cosgrove, J.
M.; Du, P.; Litchfield, J.; Makowski, M.; Martin, K.; McLellan, T. J.; Neagu, C.;
Perry, D. A.; Piotrowski, D. W.; Steppan, C. M.; Trilles, R. Bioorg. Med. Chem. Lett.
2011, 21, 1621.
determined.
Table 3
In vitro and in vivo profiles of MK-8245, 23a and 23b in mice
Mouse SCD
IC50 (nM)a
In vivo
TD (l
M)b
mLPDc
3. (a) Ramtohul, Y. K.; Powell, D.; Leclerc, J.-P.; Leger, S.; Oballa, R.; Black, C.;
Isabel, E.; Li, C. S.; Crane, S.; Robichaud, J.; Guay, J.; Guiral, S.; Zhang, L.; Huang,
Z. Bioorg. Med. Chem. Lett. 2011, 21, 5692; (b) Uto, Y.; Ueno, Y.; Kiyotsuka, Y.;
Miyazawa, Y.; Kurata, H.; Ogata, T.; Yamada, M.; Deguchi, T.; Konishi, M.;
Takagi, T.; Wakimoto, S.; Ohsumi, J. Eur. J. Med. Chem. 2010, 45, 4788 and
references therein; (c) Uto, Y.; Ogata, T.; Kiyotsuka, Y.; Ueno, Y.; Miyazawa, Y.;
Kurata, H.; Deguchi, T.; Watanabe, N.; Konishi, M.; Okuyama, R.; Kurikawa, N.;
Takagi, T.; Wakimoto, S.; Ohsumi, J. Bioorg. Med. Chem. Lett. 2010, 20, 341; (d)
Koltun, D. O.; Vasilevich, N. I.; Parkhill, E. Q.; Glushkov, A. I.; Zilbershtein, T. M.;
Mayboroda, E. I.; Boze, M. A.; Cole, A. G.; Henderson, I.; Zautke, N. A.; Brunn, S.
A.; Chu, N.; Hao, J.; Mollova, N.; Leung, K.; Chisholm, J. W.; Zablocki, J. Bioorg.
Med. Chem. Lett. 2009, 19, 3050 and references therein.
MK-8245
23a
3
[Liver] = 2.70
[Plasma] = 0.03
[skin (shaved)] = 0.07
89% inh. at 2 mg/kg
[Liver] = 4.9
49% inh. at 0.4 mg/kg
[Liver] = 1.6
lM
[Harderian glands] = 0.13
lM
13
6
[Liver] = 8.07
[Plasma] = 1.08
[skin (shaved)] = 0.24
[Harderian glands] = 0.59
79% inh. at 10 mg/kg
[Liver] = 28.6
49% inh. at 2 mg/kg
[Liver] = 7.6
lM
l
M
23b
[Liver] = 13.7
[Plasma] = 0.77
[skin (shaved)] = 0.30
[Harderian glands] = 1.85
91% inh. at 10 mg/kg
[Liver] = 18.3
81% inh. at 2 mg/kg
[Liver] = 4.2
lM
4. Review on OATPs: Niemi, M. Pharmacogenomics 2007, 8, 787.
5. Oballa, R. M.; Belair, L.; Black, W. C.; Bleasby, K.; Chan, C. C.; Desroches, C.; Du,
X.; Gordon, R.; Guay, J.; Guiral, S.; Hafey, M. J.; Hamelin, E.; Huang, Z.; Kennedy,
B.; Lachance, N.; Landry, F.; Li, C. S.; Mancini, J.; Normandin, D.; Pocai, A.;
Powell, D. A.; Ramtohul, Y. K.; Skorey, K.; Sørensen, D.; Sturkenboom, W.;
Styhler, A.; Waddleton, D. M.; Wang, H.; Wong, S.; Xu, L.; Zhang, L. J. Med. Chem.
2011, 54, 5082.
l
M
a
b
c
IC50s are an average of at least two independent titrations.
TD—tissue distribution (PO, mouse (n = 2), 10 mg/kg; 6 h post dose).
mLPD (PO, mouse (n = 5), 3 h post dose). inh.—inhibition.

N. Lachance et al. / Bioorg. Med. Chem. Lett. 22 (2012) 623–627
627
6. Mouse liver pharmacodynamic model (mLPD) is expressed in percentage (%)
inhibition and is used to assess the in vivo potency. In the mLPD experiment,
mice were fed on a high carbohydrate diet and the SCD activity was indexed 3 h
post oral dose of SCD inhibitors by following the conversion of intravenously
administered [1–14C]-stearic acid tracer to the SCD-derived [1–14C]-oleic acid
in liver lipids. The percentage (%) of inhibition of an SCD inhibitor is calculated
from the liver SCD activity index (ratio of 14C-oleic acid/14C-stearic acid) from
drug treated animals compared to a vehicle group.
buffer) for 2 h under 95% O2/5% CO2 atmosphere. There were two major
metabolites observed but not fully characterized. In mouse, the main
metabolite is the ring opening of the isoxazole. However in rat, the major
metabolite involves oxidation of the phenyl ring, defluorination and
glutathione (GSH) adduct, in addition to the minor reductive ring-opened
metabolite.
7. Leading references: (a) Zhao, H.; Serby, M. D.; Smith, H. T.; Cao, N.; Suhar, T. S.;
Surowy, T. K.; Camp, H. S.; Collins, C. A.; Sham, H. L.; Liu, G. Bioorg. Med. Chem.
Lett. 2007, 17, 3388; (b) Liu, G.; Lynch, J. K.; Freeman, J.; Liu, B.; Xin, Z.; Zhao, H.;
Serby, M. D.; Kym, P. R.; Suhar, T. S.; Smith, H. T.; Cao, N.; Yang, R.; Janis, R. S.;
Krauser, J. A.; Cepa, S. P.; Beno, D. W. A.; Sham, H. L.; Collins, C. A.; Surowy, T. K.;
Camp, H. S. J. Med. Chem. 2007, 50, 3086.
8. Lachance, N.; Li, C. S.; Leclerc, J.-P.; Ramtohul, Y. WO 2008/128335.
9. The regiochemistry of the acetic acid side chain on the tetrazole was confirmed
by evaluation of the structures through NMR experiments: gHMBC, 15N gHMBC
and 1D NOESY.
Proposed structures
Br
O
OH
GS
O
NH2
Reductive ring opening
Oxidative defluorination
and GSH conjugation
10. Rat microsomal assay conditions: Li, C. S.; Ramtohul, Y.; Huang, Z.; Lachance, N.
WO 2006/130986.
11. Zhang, L.; Ramtohul, Y.; Gagné, S.; Styhler, A.; Guay, J.; Huang, Z. J. Biomol.
Screen. 2010, 15, 169.
14. For comparison, the isoxazole ring-opened metabolite was observed at low
level (4%) in vitro in rat hepatocytes with MK-8245.
15. Compounds containing a 6-membered heterocycle ring pyridazine or pyrazine
were less potent. Data unpublished.
16. Several substituents (H, Cl, Br, CF3, SO2Me, OCyp, NHCH2CF3, Ar) were evaluated
across numerous combinations of different tether lengths and various 5-
membered ring heterocycles. Unpublished results.
12. PK data of compound 16b in C57BL6 mice following oral dosing at 10 mg/kg in
0.5% methocel: F = 10% and AUC0–6 h = 1.0 lM h.
13. For compound 16b, extensive metabolism was observed (mouse: 75% parent
remaining; rat: 55% parent remaining) after incubation at 37 °C with fresh
mouse hepatocytes and rat hepatocytes (2 million cells/mL of Krebs-Henseleit