Identification of a novel hormone sensitive lipase inhibitor with a reduced potential of reactive metabolites formation

Article abstract of DOI:10.1016/j.bmc.2017.02.045

Hormone sensitive lipase (HSL) has emerged as an attractive target for the treatment of dyslipidemia. We previously reported compound 1 as a potent and orally active HSL inhibitor. Although an attractive profile was demonstrated, subsequent studies revealed that compound 1 has a bioactivation liability. The oxygen-carbon linker in compound 1 was identified as being potentially responsible for reactive metabolite formation. By exchanging of this susceptible fragment was feasible, and a benzanilide derivative 6b with a decreased bioactivation liability was obtained. Further modification of the novel benzanilide scaffold resulted in the identification of compound 24b. Compound 24b exhibited potent HSL inhibitory activity (IC₅₀ = 2 nM) with a significantly reduced bioactivation potential. Oral administration of compound 24b exhibited an antilipolytic effect on rats at 3 mg/kg.

Full text of DOI:10.1016/j.bmc.2017.02.045

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of a novel hormone sensitive lipase inhibitor with
a reduced potential of reactive metabolites formation
b
c
d
e
Tomoko Ogiyama a, , Mitsuhiro Yamaguchi , Nobuya Kurikawa , Shoko Honzumi , Yuka Yamamoto ,
⇑
Daisuke Sugiyama f, Hideo Takakusa f, Shin-ichi Inoue f
a Modality Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b R&D Planning & Management Department, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Venture Science Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Rare Disease & LCM Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
e Biological Research Department, Daiichi Sankyo RD Novare Co., Ltd., 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
f Drug Metabolism & Pharmacokinetics 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:
Hormone sensitive lipase (HSL) has emerged as an attractive target for the treatment of dyslipidemia. We
previously reported compound 1 as a potent and orally active HSL inhibitor. Although an attractive profile
was demonstrated, subsequent studies revealed that compound 1 has a bioactivation liability. The oxy-
gen-carbon linker in compound 1 was identified as being potentially responsible for reactive metabolite
formation. By exchanging of this susceptible fragment was feasible, and a benzanilide derivative 6b with
a decreased bioactivation liability was obtained. Further modification of the novel benzanilide scaffold
resulted in the identification of compound 24b. Compound 24b exhibited potent HSL inhibitory activity
(IC50 = 2 nM) with a significantly reduced bioactivation potential. Oral administration of compound 24b
exhibited an antilipolytic effect on rats at 3 mg/kg.
Received 11 January 2017
Revised 20 February 2017
Accepted 21 February 2017
Available online xxxx
Keywords:
Hormone sensitive lipase
Dyslipidemia
Lipolysis
Boronic acid
Ó 2017 Elsevier Ltd. All rights reserved.
Reactive metabolite
1. Introduction
enlargement of AT mass and attenuation of insulin-mediated AT
lipolysis metabolism, which could cause dysregulation of lipolysis.
Hormone sensitive lipase (HSL) is an intracellular neutral lipase
that catalyzes hydrolysis of broad substrates such as tri-, di-, and
monoacylglycerol (TG, DG and MG), cholesterylester, retinyl ester
and numerous water soluble ester substrates.1 HSL contains an
Furthermore, the increased FFA flux could impair lipid profiles by
enhancing hepatic production of very-low-density lipoprotein
(VLDL).7 HSL inhibition could be a promising therapeutic approach
for dyslipidemia, however, the reports of HSL inhibitors have been
limited.8–17
a
/b-hydrolase fold2 and a catalytic triad of serine, aspartic acid,
and histidine,3 which are essential for catalysis.4 The highest
expression of HSL is observed in adipose tissues (ATs) where it cat-
alyzes lipolysis of stored triglyceride (TG); one TG molecule is
sequentially broken down into one glycerol and three free fatty
acids (FFAs). The cellular concentration of FFAs is tightly controlled
by the balance between lipolysis of TG and esterification of FFAs by
several hormones depending on energy demand.5 In the fasted
state, HSL is activated in response to catecholamines and FFAs
are subsequently released into circulation as an energy source for
most tissues.6
We previously reported that compound 1 exerted HSL inhibi-
tory activity with an IC50 value of 7 nM and an in vivo antilipolytic
effect at 3 mg/kg in rats.18 Despite potent in vitro and in vivo activ-
ities, subsequent studies revealed that compound 1 has a potential
to form reactive metabolites. Reactive metabolite formation could
cause organ toxicity and carcinogenesis, since these metabolites
covalently bind to biological macromolecules such as protein and
DNA.19 Therefore, minimizing the potential to form reactive
metabolites could be required.20
The propensity of forming reactive metabolites was generally
assessed by incubation of compounds with glutathione (GSH) in
the presence of liver microsomes.21 In the reactive metabolite trap-
ping assay, a GSH-derived adduct of compound 1 was detected
(Fig. 1).22 Its structural elucidation using LC-MS/MS analysis pro-
vided that the site of GSH conjugation was tentatively assigned
to the benzyl carbon. On the basis of the results, the proposed
FFAs have a pathophysiological role in dyslipidemia as well as a
physiological role in energy production. Elevation of plasma FFA
level is associated with obesity and insulin resistance that induce
⇑
Corresponding author.
E-mail address: yamamoto.tomoko.me@daiichisankyo.co.jp (T. Ogiyama).
http://dx.doi.org/10.1016/j.bmc.2017.02.045
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ogiyama T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.045

2
T. Ogiyama et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
HS
N
1) oxidation of benzyl carbon
2) conjugation with Cys-Gly
H
N
F C
F C
3
CO H
2
3
O
B(OH)
2
O
N
O
N
O
Glu-Cys-Gly
Glu
Schiff base intermediate
1
CO H
2
H
N
S
cyclization
F C
3
N
H
382
O
325
353
N
428
O
267
252
GSH-derived adduct
Fig. 1. Proposed mechanism of the GSH-derived adduct formation of 1.
mechanism of the GSH-derived adduct formation involves initial
oxidation of the benzyl carbon by cytochrome P450, followed by
conjugation with a primary amino group of a cystein moiety that
is released from GSH. The resulting Schiff base intermediate ring-
contracts to a thiazolidine leading to the GSH-derived adduct.23,24
We focused on exchanging from the susceptible fragment, namely,
the oxygen-carbon linker, to an alternative fragment to circumvent
the reactive metabolite liability while maintaining HSL inhibitory
activity.
of 1726 and following condensation. Compounds 22a–c were pre-
pared in a similar manner to that of compound 11.
Scheme 3 illustrates the synthesis of compounds 24a–f. Con-
densation of carboxylic acid 17 with corresponding anilines led
to compounds 23a–f, followed by a two-step deprotection of the
pinacol group via trifluoroborate intermediates27 afforded com-
pounds 24a–f.
3. Results and discussion
Herein, we report the identification of novel benzanilide deriva-
tives that showed potent in vitro and in vivo activities with a
decreased potential for forming reactive metabolites.
The inhibitory activity of the synthesized compounds against
HSL was measured by a colorimetric assay using human HSL frac-
tions and p-nitrophenyl butyrate (PNPB) as a substrate.8
2. Chemistry
In terms of efficient screening, we initially synthesized and
evaluated derivatives as a boronate ester since the inhibitory activ-
ity of a boronic acid was considered to be comparable to that of a
corresponding ester.18 As seen in Table 1, replacement of the oxy-
gen-carbon linker with an amide linker led to compound 6a with
an IC50 value of 0.18 mM. Conversion of the boronate ester 6a to a
boronic acid 6b resulted in a comparable HSL inhibitory activity.
N-substituted amide 6c or the reverse amide 3 significantly deteri-
orated the inhibitory activity compared with 6a. Expanding the
amide linker also resulted in a loss of the inhibitory activity (8).
As expected, GSH-derived adduct formation in the boronic acid
6b was not detected by the reactive metabolite trapping assay with
GSH. Therefore, boronic acid 6b was identified as a lead compound,
and this benzanilide scaffold was selected for further exploration.
We explored the left hand moiety of the novel derivatives as
boronate ester derivatives (Table 2). First, the m-substituted analog
Scheme 1 depicts the synthesis of derivatives 3, 6a–c and 8.
Acylation of 2 furnished 3. Condensation of corresponding acids
(425 or 7) with anilines (5a or 5b) yielded compound 6a, 6c and
8. Deprotection of pinacol group in 6a led to boronic acid derivative
6b.
Scheme 2 describes the synthesis of derivatives 11, 13a–b, 16a–b,
19a–b and 22a–c. Condensation of 2-chloro-5-(trifluoromethyl)
pyridine 9 with corresponding phenols followed by hydrolysis gave
intermediates 10b, 12a and 12b, which were converted to 11, 13a
and 13b in a two-step sequence. The intermediates 15a and 15b
were synthesized from 2-cyano-5-fluoropyridine 14 by condensa-
tion and a following two-step hydrolysis. Compounds 16a and
16b were obtained by acylation of 5a with the intermediates 15a
and 15b. Compounds 19a and 19b were synthesized by hydrolysis
H
N
F C
3
NH2
a
F C
3
N
N
O
O
Bpin
N
O
2
3
O
F C
3
CO H
2
d, e
1
F C
3
R
N
R
+
N
H
1
2
R
O
BPin
N
O
5a R1 = H
6a R1 = H, R2 = Bpin
4
b, c
f
5b R1 = Me
6b R1 = H, R2 = B(OH)2
6c R1 = Me, R2 = Bpin
Bpin
H
F C
3
g
CO H
F C
N
O
2
3
N
O
N
O
7
8
Scheme 1. Reagents and conditions: (a) 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid, EDCÁHCl, HOBT, DMF; (b) 1H-benzotriazole-1-methanol, EtOH;
(c) NaBH4, THF; (d) oxalyl chloride, DMF (cat.), CH2Cl2; (e) 5a or 5b, DIPEA, CH2Cl2; (f) NaIO4, THF, H2O, then 1 M HCl; (g) 5a, EDCÁHCl, HOBT, NMM, CH2Cl2.
Please cite this article in press as: Ogiyama T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.045

T. Ogiyama et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
F C
3
Bpin
F C
3
F C
3
c, d
a
H
N
N
O
N
Cl
Cl
N
O
CO R
2
O
O
9
11
10a R = Et
10b R = H
b
1
1
R
R
F C
3
2
2
a, b
e
F C
3
R
CO H
2
F C
3
R
N
H
N
9
Bpin
N
O
N
O
13a R1 = Me, R2 = H
12a R1 = Me, R2 = H
12b R1 = H, R2 = Me
13b R1 = H, R2 = Me
O
N
CN
F C
CO H
2
a, f, b
c, d
3
F C
3
N
N
H
N
F
X
O
Bpin
X
g
O
14
15a X = N
15b X= C
16a X = N
16b X= C
O
CO Me
2
CO H
2
F C
3
F C
b
3
F C
3
N
H
N
N
O
O
R
O
N
19a R = Bpin
19b R = B(OH)2
17
18
O
CO Me
CO H
2
2
c, d
a or h, b
R
N
H
R
Cl
N
X
N
Bpin
X
N
20
21a R = 4- OCF3, X= O
21b R = 3- OCF3, X= O
21c R = 4- OCF3, X= NMe
22a R = 4- OCF3, X= O
22b R = 3- OCF3, X= O
22c R = 4- OCF3, X= NMe
Scheme 2. Reagents and conditions: (a) phenols, K2CO3, DMF; (b) 5 M NaOH, MeOH; (c) oxalyl chloride, DMF (cat.), CH2Cl2; (d) 5a, DIPEA, CH2Cl2; (e) 5a, EDCÁHCl, HOBT,
NMM, CH2Cl2; (f) conc. HCl, conc. H2SO4, MeOH; (g) 5a, HBTU, DIPEA, DMF; (h) N-methyl-p-trifluoromethoxyaniline, AcOH.
O
O
CO H
F C
2
a, b
R
R
3
c, d
F C
F C
3
3
N
H
N
H
O
N
Bpin
B(OH)
2
O
N
O
N
17
23a R = 4- F
23b R = 4- Cl
23c R = 4- Me
23d R = 5- F
23e R = 5- Cl
23f R = 5- OMe
24a R = 4- F
24b R = 4- Cl
24c R = 4- Me
24d R = 5- F
24e R = 5- Cl
24f R = 5- OMe
Scheme 3. Reagents and conditions: (a) oxalyl chloride, DMF (cat.), CH2Cl2; (b) anilines, DIPEA, CH2Cl2; (c) KHF2, MeOH, H2O; (d) TMSCl, CH3CN, H2O.
11 resulted in a loss of HSL inhibitory activity. Next, incorporation
of a methyl group into the central ring on the 2-position led to a
slight decrease in the inhibitory activity (13a), whereas that on
the 3-position led to a significant loss of the inhibitory activity
(13b). By exchanging of the central benzene ring in compound 6a
with a 2-N-pyridine ring led to a decrease in the inhibitory activity
(16a). By exchanging of the left terminal pyridine ring in com-
pound 16a with trifluoromethyl benzene ring led to a 7-fold
increase in HSL inhibitory activity with an IC50 value of 0.13 mM
(16b). Furthermore, 3-N-pyridine 19a showed a 10-fold increase
in HSL inhibitory activity with an IC50 value of 0.014 mM compared
to compound 16b. Replacement of the trifluoromethyl group with
a trifluoromethoxy group also showed potent inhibitory activity
(22a), and the trifluoromethoxy group in the meta position main-
tained the inhibitory activity (22b). By exchanging of the ether lin-
ker in 22a with an N-methyl amino linker was also tolerated (22c).
As a result of this investigation, the 3-N-pyridine was identified
as the most desirable substructure in the central ring to exert
potent HSL inhibitory activity. Moreover, the 3-N-pyridine deriva-
tive 19a, which bears highly hydrophobic 4-trifluoromethyl ben-
zene moiety in the left hand, exhibited the most potent HSL
inhibitory activity in this series.
Encouraged by the results, we further evaluated the effects of
substituents on the right hand benzene ring of compound 19a
(Table 3). Conversion of the boronate ester 19a to a boronic acid
19b resulted in a slight decrease in the inhibitory activity. Intro-
duction of lipophilic substituents such as a halogen atom or a
methyl group on the C-4 position in compound 19b led to an
increase in the inhibitory activity (24a–24c), and especially the
4-chlorine substituent 24b exhibited a 23-fold increase in HSL
inhibitory activity compared to 19b. Interestingly, incorporation
of a 4-chlorine group into the boronate derivative 23b did not
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4
T. Ogiyama et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 1
Table 2
Replacement of the ether linker of compound 1.
SAR of the left hand moiety of the benzanilide derivatives.
F C
3
R
O
R
N
H
N
O
Bpin
Compd
R
IC50 (mM)a
Compd
R
IC50 (mM)a
6a
0.18
O
O
O
6a
0.18
F C
3
N
H
Bpin
N
O
11
33%b
0.38
6b
6c
3
0.091
8%b
F3C
N
H
N
N
O
O
B(OH)
2
13a
2
F C
3
3
N
Bpin
Bpin
8%b
13b
2
3
30%b
F C
3
H
N
N
N
O
O
O
O
16a
16b
0.92
0.13
F C
3
8
12%b
Bpin
H
N
N
N
F3C
F3C
a
Assay results are the average of triplicates.
% inhibition at 1 mM was evaluated as described in Section 5.
2
O
b
3
19a
0.014
2
O
N
3
show a dramatic improvement in the inhibitory activity compared
with the corresponding boronic acid derivative 24b. Substitution at
the C-5 position with a halogen atom resulted in a slight improve-
ment in the inhibitory activity (24d and 24e), whereas incorpora-
tion of a methoxy group did not have any effect on the IC50 value
(24f). Overall, the effect of introducing substituents at the C-4 posi-
tion (24a-c) was more sensitive to HSL inhibitory activity than that
of C-5 (24d–f).
22a
22b
22c
0.024
0.021
0.040
F3CO
O
O
N
N
N
N
F3CO
F CO
3
The reason why 4-chloro substituent 24b exhibited potent HSL
inhibitory activity might be regarded as follows:
a
Assay results are the average of triplicates.
% inhibition at 1 mM was evaluated as described in Section 5.
b
i) An increased Lewis acidity of boronic acid
Table 3
The calculated pKa value of boronic acid in 4-chloro substituent
24b (pKa = 7.4) is much lower than that of the unsubstituted analog
19b (pKa = 8.4). The lower pKa value increases the electrophilicity
of a boron center,28 which enhances both the formation of a dative
bond between the hydroxyl group of a catalytic serine and the
boron center and the stabilization of a formed boron-lipase
complex.29
SAR of the right hand moiety of the benzanilide derivatives.
5
4
O
2
R
F C
3
N
H
1
R
O
N
R1
R2
IC50 (mM)a
pKa
b
ii) Estimated the existence of binding pocket around C-4 moiety
Compd
19a
19b
24a
24b
24c
23b
24d
24e
24f
Bpind
H
H
4-F
4-Cl
4-Me
4-Cl
5-F
5-Cl
5-OMe
0.014
0.046
0.013
0.002
0.020
0.008
0.016
0.024
0.059
NCc
8.4
7.3
7.4
8.5
NCc
8.5
8.2
8.8
4-Me substituent 24c showed an adequate inhibitory activity
comparable to that of a unsubstituted analog 19b despite a nearly
equivalent pKa value of boronic acid (8.5 and 8.4, respectively).
This tendency was also observed in between 4-Cl substituent 24b
and 4-F substituent 24a (the pKa values of boronic acid are 7.4
and 7.3, respectively). This might imply that there was a binding
pocket of the lipase around the C-4 position, which was probably
hydrophobic. Thereby, compounds with substituents at the 4-posi-
tion such as 24c and 24b resulted in enhanced HSL inhibitory
activity.
The potent HSL inhibitor 24b was obtained, and further charac-
terization was performed (Table 4). As expected, in the reactive
metabolite trapping assay, GSH-derived adducts were not
detected. Compound 24b showed high metabolic stability (81%)
in rat liver microsomes, suggesting that 24b could be devoid of
B(OH)2
B(OH)2
B(OH)2
B(OH)2
Bpind
B(OH)2
B(OH)2
B(OH)2
a
b
c
Assay results are the average of triplicates.
Calculated by ACD/Percepta (version 2014).
Not calculated.
d
Bpin: pinacolboronate.
the reactive intermediates formation since the metabolically labile
benzyl group was replaced with the amide group that might be
resistant to oxidative metabolism and further modification did
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T. Ogiyama et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Table 4
4. Conclusion
Pharmaceutical profiles of compound 24b.
Reactive metabolite formationa
Metabolic stabilityc
NDb
81%
3.35
19.65
Starting from compound 1, by exchanging from the oxygen-car-
bon linker to the amide linker resulted in a promising lead com-
pound 6b with the moderate HSL inhibitory activity and a
reduced bioactivation liability. Further optimization of the left
hand and the right hand moiety of 6b led to the identification of
compound 24b with an IC50 value of 2 nM and a significantly
reduced reactive metabolite liability. Reflecting its in vitro efficacy,
the oral administration of compound 24b at 3 mg/kg in rats led to a
decrease in plasma glycerol level. Further optimization of the
derivatives is currently underway, which will clarify the biological
importance of the HSL inhibition.
d
Cmax (mg/mL)
AUCd (mgÁh/mL)
a
Reactive metabolite assessment was conducted using
isotope labeled glutathione as a trapping agent in NADPH-
supplemented human liver microsomes.
b
Not detected.
c
%
remaining after 30 min in rat liver microsomes
(0.5 mg/mL).
d
3 mg/2 mL/kg of compound solution in propylene
glycol/Tween80 = 4:1 (v/v) solution.
5. Experimental
OH
Cl
O
F C
O
N
F C
3
3
N
H
5.1. Chemistry
O
B(OH)
2
N
O
O
N
Unless otherwise noted, materials were obtained from commer-
cial suppliers and used without further purification. NMR spectra
were recorded on a Varian Mercury 400 or 500 spectrometer with
tetramethylsilane as an internal reference. Mass spectra were
recorded on an Agilent Technologies Agilent 1100 series LC/MS.
TLC analysis was performed on 60F354 plates (Merck). Flash col-
umn chromatography was performed on a Shoko scientific SI series
25
HSL IC50 = 270 nM
24b
HSL IC50 = 2 nM
Fig. 2. Structures of 25 and 24b.
not increase metabolically vulnerable sites that led to
bioactivation. Owing to this metabolic stability, compound
24b showed good oral exposure (Cmax = 3.35 mg/mL and
AUC = 19.65 mgÁh/mL at 3 mg/kg p.o.) in rats.
on a Shoko Scientific Purif-a2. Purities of assayed compounds were
in all cases greater than 90%, as determined by NMR analysis and
After these promising profiles were identified, we examined an
antilipolytic effect of compound 24b in rats. To evaluate in vivo
potency, a head-to-head comparison was performed by measuring
the plasma glycerol level as a lipolytic end point.30 For the compar-
ison, compound 25 (Fig. 2), which was identified by Ebdrup et al. as
a potent orally active HSL inhibitor and exerted the antilipolytic
effect at a dose of 10 mg/kg p.o. due to its good PK profiles,14
was selected.
LC/MS. The following abbreviations are used: EDCÁHCl, 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide
hydrochloride;
HOBT,
1-hydroxybenzotriazole monohydrate; DMF, N,N-dimethylfor-
mamide; AcOEt, ethyl acetate; EtOH, ethanol; THF, tetrahydrofu-
ran; DIPEA, N,N-diisopropylethylamine; MeOH, methanol; DMSO,
dimethylsulfoxide; DMA, N,N-dimethylacetamide; EDTA, ethylene-
diaminetetraacetic acid; DTT, dithiothreitol; PNPB, p-nitrophenyl
butyrate.
As shown in Fig. 3, oral administration of compound 24b rela-
tively lowered plasma glycerol level. The reduced plasma glycerol
AUC value of compound 24b at 3 mg/kg p.o. was higher than that
of 25 at 10 mg/kg p.o., with the value of 17%, due to its potent
HSL inhibitory activity (IC50 = 2 nM) compared with compound
25 (IC50 = 270 nM).18 This indicates that compound 24b is an orally
active HSL inhibitor that has the potential for acting as an
antilipolytic agent.
5.1.1. 2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-N-(4-{[5-
(trifluoromethyl)pyridin-2-yl]oxy}phenyl)benzamide (3)
A mixture of 2 (0.15 g, 0.60 mmol), 2-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzoic acid (0.15 g, 0.60 mmol), EDCÁHCl
(0.13 g, 0.66 mmol), HOBT (0.10 g, 0.66 mmol) and DMF (6.0 mL)
was stirred at room temperature overnight. The reaction mixture
was diluted with water and extracted with AcOEt. The organic
layer was washed with H2O and brine, dried over Na2SO4 and evap-
orated. The resulting residue was purified by silica gel chromatog-
raphy (hexane-AcOEt). The obtained solid was triturated in
hexane/AcOEt and filtered to give 3 (0.011 g, 4%) as a colorless
solid. 1H NMR (CDCl3) d: 8.46 (1H, s), 8.31 (1H, s), 7.90 (1H, dd,
J = 8.6, 2.3 Hz), 7.74–7.70 (4H, m), 7.51–7.48 (2H, m), 7.16 (2H, d,
J = 9.0 Hz), 7.01 (1H, d, J = 9.0 Hz), 1.38 (12H, s). MS (ESI+) m/z:
485 (M+H)+. HRMS (ESI+) m/z: 485.1855 (M+H)+ (calcd for
C25H25BF3N2O4: 485.1854).
5.1.2. N-Methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
aniline (5b)
Step A: To a mixture of 5a (0.44 g, 2.0 mmol) and EtOH (9.0 mL)
was added 1H-benzotriazole-1-methanol (0.30 g, 2.0 mmol). After
stirring at room temperature overnight, the reaction mixture was
concentrated in vacuo and the obtained residue was triturated
with hexane. The resulting precipitate was collected by filtration
to give N-(1H-Benzotriazol-1-ylmethyl)-2-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)aniline (0.59 g, 84%) as a brown solid. 1H
NMR (CDCl3) d: 8.03 (1H, d, J = 8.2 Hz), 7.67 (1H, d, J = 8.2 Hz),
7.62 (1H, dd, J = 7.4, 1.6 Hz), 7.44–7.40 (1H, m), 7.33–7.28 (2H,
m), 7.09 (1H, t, J = 7.2 Hz), 7.02 (1H, d, J = 8.2 Hz), 6.73 (1H, td,
Fig. 3. The acute effect of HSL inhibition in rats treated with 25 at 10 mg/kg (white
square) or 24b at 1 and 3 mg/kg (black square). Data are reported as a percentage of
plasma glycerol reduction of the area under the curve (AUC) (0–7 h), setting at 100%
the reduction measured in compound 25. Data are mean SEM n = 3–4.
Please cite this article in press as: Ogiyama T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.045

6
T. Ogiyama et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
J = 7.4, 0.8 Hz), 6.16 (2H, d, J = 7.4 Hz), 1.37 (12H, s). MS (ESI-) m/z:
349 (MÀH). Step B: To solution of N-(1H-Benzotriazol-1-
5.1.6. N-[2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-2-
(4-{[5-(trifluoromethyl)pyridin-2-yl]oxy}phenyl)acetamide (8)
A mixture of 7 (0.59 g, 2.0 mmol), 5a (0.66 g, 2.6 mmol),
EDCÁHCl (0.50 g, 2.6 mmol), HOBT (0.50 g, 2.6 mmol), 4-methyl-
morpholine (0.33 mL, 3.0 mmol) and CH2Cl2 (12 mL) was stirred
at room temperature overnight. The reaction mixture was diluted
with water and extracted with AcOEt. The organic layer was
washed with brine, dried over Na2SO4 and concentrated in vacuo.
The resulting residue was purified by silica gel chromatography
(hexane-AcOEt). The obtained solid was triturated in hexane/AcOEt
and filtered to give 8 (0.50 g, 50%) as a colorless solid. 1H NMR
(CDCl3) d: 9.41 (1H, s), 8.48 (1H, d, J = 8.2 Hz), 8.42 (1H, s), 7.89
(1H, dd, J = 8.4, 2.9 Hz), 7.76 (1H, dd, J = 7.4, 1.6 Hz), 7.47–7.43
(3H, m), 7.14 (2H, d, J = 8.6 Hz), 7.07 (1H, td, J = 7.3, 0.9 Hz), 7.01
(1H, d, J = 8.6 Hz), 3.72 (2H, s), 1.34 (12H, s). MS (ESI+) m/z: 499
(M+H)+. HRMS (ESI+) m/z: 499.2011 (M+H)+ (calcd for C26H27BF3-
N2O4: 499.2010).
a
ylmethyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
(0.37 g, 1.1 mmol) in THF (10 mL) was added sodium tetrahydrob-
orate (0.040 g, 1.1 mmol). The mixture was stirred at 76 °C for 1 h.
After cooling to room temperature, the reaction mixture was
diluted with water and extracted with AcOEt. The organic layer
was washed with brine, dried over Na2SO4 and concentrated in
vacuo. The resulting residue was purified by silica gel chromatog-
raphy (hexane-AcOEt) to give 5b (0.19 g, 75%) as a colorless solid.
1H NMR (CDCl3) d: 7.63 (1H, dd, J = 7.2, 1.8 Hz), 7.35–7.30 (1H,
m), 6.62 (1H, td, J = 7.2, 1.0 Hz), 6.54 (1H, d, J = 8.2 Hz), 2.85 (3H,
s), 1.33 (12H, s). MS (FAB) m/z: 256 (M+Na)+.
5.1.3. N-[2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-4-
{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzamide (6a)
Step A: To a solution of 4 (1.1 g, 4.0 mmol) in CH2Cl2 (24 mL)
was added oxalyl chloride (1.0 mL, 12 mmol) followed by DMF (4
drops) at room temperature. The reaction mixture was stirred at
room temperature for 1 h and concentrated in vacuo to give crude
4-{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzoyl chloride, which
was used directly in the next step. Step B: Crude 4-{[5-(trifluo-
romethyl)pyridin-2-yl]oxy}benzoyl chloride was dissolved in
CH2Cl2 (24 mL) at 0 °C. To the solution was added DIPEA (0.75 mL,
4.4 mmol) and 5a (0.88 g, 4.4 mmol). After stirring at room tem-
perature for 1 h, the mixture was diluted with H2O and extracted
with AcOEt. The organic layer was washed with brine, dried over
Na2SO4 and concentrated in vacuo. The resulting residue was puri-
fied by silica gel chromatography (hexane-AcOEt) to give 6a
(0.56 g, 29%) as a colorless solid. 1H NMR (CDCl3) d: 10.28 (1H, s),
8.75 (1H, d, J = 8.2 Hz), 8.47–8.46 (1H, m), 8.13 (2H, d, J = 8.6 Hz),
7.96 (1H, dd, J = 8.8, 2.5 Hz), 7.83 (1H, dd, J = 7.4, 1.6 Hz), 7.54
(1H, td, J = 7.8, 1.4 Hz), 7.28 (2H, d, J = 9.0 Hz), 7.11 (2H, dd,
J = 15.4, 8.0 Hz), 1.41 (12H, s). MS (ESI+) m/z: 485 (M+H)+. HRMS
(ESI-) m/z: 483.1712 (MÀH)À (calcd for C25H23BF3N2O4: 483.1708).
5.1.7. Ethyl 3-{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzoate (10a)
A
mixture of 2-chloro-5-(trifluoromethyl)pyridine (5.45 g,
30 mmol), ethyl 3-hydroxybenzoate (5.5 g, 33 mmol), K2CO3
(4.98 g, 36 mmol) and DMF (20 mL) was stirred at 100 °C for 5 h.
After cooling to room temperature, the reaction mixture was
diluted with water and extracted with AcOEt. The organic layer
was washed with brine, dried over Na2SO4 and concentrated in
vacuo. The resulting residue was purified by silica gel chromatog-
raphy (hexane-AcOEt) to give 10a (7.42 g, 79%) as a colorless oil. 1H
NMR (CDCl3) d: 8.43 (1H, s), 7.94 (2H, tt, J = 8.8, 2.4 Hz), 7.82 (1H, t,
J = 2.0 Hz), 7.51 (1H, t, J = 7.8 Hz), 7.36 (1H, dq, J = 8.0, 1.2 Hz), 7.06
(1H, d, J = 9.0 Hz), 4.38 (2H, q, J = 7.0 Hz), 1.39 (3H, t, J = 7.0 Hz). MS
(ESI+) m/z: 312 (M+H)+.
5.1.8. 3-{[5-(Trifluoromethyl)pyridin-2-yl]oxy}benzoic acid (10b)
A mixture of 10a (0.15 g, 0.48 mmol), 5 M NaOH aq. (0.19 mL,
9.5 mmol) and MeOH (4.8 mL) was stirred at 60 °C for 20 min. After
cooling to room temperature, the reaction mixture was concen-
trated in vacuo and the obtained residue was diluted with 1 M
HCl aq. The resulting precipitate was collected by filtration to give
10b (0.11 g, 78%) as a colorless solid. 1H NMR (DMSO-D6) d: 8.59–
8.59 (1H, m), 8.27 (1H, dd, J = 8.4, 2.9 Hz), 7.84 (1H, dt, J = 7.7,
1.3 Hz), 7.68 (1H, dd, J = 2.3, 1.6 Hz), 7.55 (1H, t, J = 7.8 Hz), 7.43
(1H, dq, J = 8.1, 1.2 Hz), 7.31 (1H, d, J = 8.6 Hz). MS (ESI+) m/z:
284 (M+H)+.
5.1.4. {2-[(4-{[5-(Trifluoromethyl)pyridin-2-yl]oxy}benzoyl)amino]
phenyl}boronic acid (6b)
To a solution of 6a (0.46 g, 0.95 mmol), THF (24 mL) and H2O
(6.0 mL) was added sodium periodate (0.61 g, 2.7 mmol). After stir-
ring at room temperature for 1 h, sodium periodate (0.61 g,
2.7 mmol) was additionally added, and the reaction mixture was
stirred at room temperature for 1 h. 1 M HCl aq. (1.2 mL) was
added, and the mixture was stirred overnight. The reaction mixture
was diluted with water and extracted with AcOEt. The organic
layer was washed with brine, dried over Na2SO4 and concentrated
in vacuo. The resulting residue was purified by silica gel chro-
matography (AcOEt-MeOH). The obtained solid was triturated in
hexane/AcOEt and filtered to give 6b (0.15 g, 39%) as a colorless
solid. 1H NMR (CD3OD) d: 8.45 (1H, s), 8.24 (2H, d, J = 9.0 Hz),
8.14 (1H, dd, J = 8.6, 2.3 Hz), 7.49 (1H, d, J = 7.0 Hz), 7.42 (2H, d,
J = 9.0 Hz), 7.35–7.24 (4H, m). MS (ESI-) m/z: 401 (MÀH)À. HRMS
(ESI-) m/z: 401.0931 (MÀH)À (calcd for C19H13BF3N2O4: 401.0926).
5.1.9. N-[2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-3-
{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzamide (11)
Compound 11 was prepared in a similar manner described for
6a. Yield: 13%. 1H NMR (CDCl3) d: 10.28 (1H, s), 8.74 (1H, d,
J = 8.6 Hz), 8.46 (1H, s), 7.95 (2H, dq, J = 10.5, 2.9 Hz), 7.86–7.82
(2H, m), 7.57–7.52 (2H, m), 7.37–7.35 (1H, m), 7.12 (1H, td,
J = 7.4, 0.8 Hz), 7.08 (1H, d, J = 8.6 Hz), 1.34 (12H, s). MS (ESI+)
m/z: 485 (M+H)+.
5.1.10. 2-Methyl-4-{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzoic acid
(12a)
Step A: methyl 2-methyl-4-{[5-(trifluoromethyl)pyridin-2-yl]
oxy}benzoate was prepared in a similar manner described for
10a. Yield: 67%. 1H NMR (CDCl3) d: 8.45 (1H, d, J = 1.0 Hz), 8.02
(1H, d, J = 7.8 Hz), 7.93 (1H, dd, J = 8.8, 2.4 Hz), 7.06–7.01 (3H, m),
3.90 (3H, s), 2.63 (3H, s). MS (ESI+) m/z: 312 (M+H)+. Step B: Com-
pound 12a was prepared in a similar manner described for 10b.
Yield: 74%. 1H NMR (CDCl3) d: 8.47 (1H, s), 8.16 (1H, d,
J = 9.0 Hz), 7.95 (1H, dd, J = 8.6, 2.7 Hz), 7.09–7.06 (3H, m), 2.68
(3H, s). MS (ESI+) m/z: 298 (M+H)+.
5.1.5. N-Methyl-N-[2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
phenyl]-4-{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzamide (6c)
Compound 6c was prepared in a similar manner described for
6a. Yield: 30%. 1H NMR (CDCl3) d: 8.40 (1H, s), 7.84 (1H, d,
J = 9.0 Hz), 7.74 (1H, d, J = 7.4 Hz), 7.37 (3H, d, J = 8.6 Hz), 7.26–
7.19 (1H, m), 7.07 (1H, d, J = 7.8 Hz), 6.88–6.86 (3H, m), 3.43 (3H,
s), 1.33 (12H, br s). MS (ESI+) m/z: 499 (M+H)+. HRMS (ESI+) m/z:
499.2010 (M+H)+ (calcd for C26H27BF3N2O4: 499.2010).
Please cite this article in press as: Ogiyama T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.045

T. Ogiyama et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
5.1.11. 3-Methyl-4-{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzoic acid
(12b)
Step A: Methyl 3-methyl-4-{[5-(trifluoromethyl)pyridin-2-yl]
5.1.15. 5-[4-(Trifluoromethyl)phenoxy]pyridine-2-carboxylic acid
(15b)
Step A:
A
mixture of 4-(trifluoromethyl)phenol (2.9 g,
oxy}benzoate was prepared in a similar manner described for
10a. Yield: 80%. 1H NMR (CDCl3) d: 8.41 (1H, s), 8.01 (1H, d,
J = 1.2 Hz), 7.95–7.93 (2H, m), 7.12 (1H, d, J = 8.6 Hz), 7.06 (1H, d,
J = 8.6 Hz), 3.92 (3H, s), 2.22 (3H, s). MS (ESI+) m/z: 312 (M+H)+.
Step B: Compound 12b was prepared in a similar manner described
for 10b. Yield: 77%. 1H NMR (CDCl3) d: 8.42 (1H, s), 8.07 (1H, s),
8.02 (1H, dd, J = 8.6, 2.0 Hz), 7.95 (1H, dd, J = 8.6, 2.3 Hz), 7.15
(1H, d, J = 8.2 Hz), 7.09 (1H, d, J = 8.6 Hz), 2.24 (3H, s). MS (ESI+)
m/z: 298 (M+H)+.
18 mmol), 2-cyano-5-fluoropyridine (2.0 g, 9.0 mmol), K2CO3
(2.7 g, 20 mmol) and DMA (20 mL) was stirred at 100 °C for 6 h.
After cooling to room temperature, the reaction mixture was
diluted with water and extracted with AcOEt. The organic layer
was washed with brine, dried over Na2SO4 and concentrated in
vacuo. The resulting residue was purified by silica gel chromatog-
raphy (hexane-AcOEt) to give 5-[4-(trifluoromethyl)phenoxy]pyri-
dine-2-carbonitrile (4.0 g, 92%) as a colorless solid. 1H NMR (CDCl3)
d: 8.50 (1H, d, J = 2.4 Hz), 7.71–7.69 (3H, m), 7.35 (1H, dd, J = 8.8,
2.9 Hz), 7.19 (2H, d, J = 8.8 Hz). MS (ESI+) m/z: 264 (M+H)+. Step
B:
A mixture of 5-[4-(trifluoromethyl)phenoxy]pyridine-2-car-
5.1.12. 2-Methyl-N-[2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
phenyl]-4-{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzamide (13a)
Compound 13a was prepared in a similar manner described for
8. Yield: 12%. 1H NMR (CDCl3) d: 9.84 (1H, s), 8.67 (1H, d,
J = 8.2 Hz), 8.44 (1H, s), 7.91 (1H, dd, J = 9.0, 2.3 Hz), 7.79 (1H, dd,
J = 7.4, 1.6 Hz), 7.70 (1H, d, J = 8.6 Hz), 7.51 (1H, td, J = 7.8,
1.8 Hz), 7.12–7.02 (4H, m), 2.59 (3H, s), 1.32 (12H, s). MS (ESI+)
m/z: 499 (M+H)+. HRMS (ESI+) m/z: 499.2010 (M+H)+ (calcd for
bonitrile (1.0 g, 3.8 mmol), conc. hydrochloric acid (3.0 mL), conc.
sulfuric acid (1.5 mL) and MeOH (10 mL) was stirred at 85 °C for
3 h. After cooling to room temperature, the reaction mixture was
concentrated in vacuo. The residue was diluted with 5 M NaOH
aq. and extracted with AcOEt. The resulting residue was purified
by silica gel chromatography (hexane-AcOEt) to give methyl
5-[4-(trifluoromethyl)phenoxy]pyridine-2-carboxylate
(0.74 g,
2.5 mmol) as a colorless solid. Yield: 65%. 1H NMR (CDCl3) d: 8.54
(1H, d, J = 2.4 Hz), 8.16 (1H, d, J = 8.8 Hz), 7.68 (2H, d, J = 8.3 Hz),
7.39 (1H, dd, J = 8.5, 2.7 Hz), 7.16 (2H, d, J = 8.3 Hz), 4.02 (3H, s).
MS (ESI+) m/z: 298 (M+H)+. Step C: Compound 15b was prepared
in a similar manner described for 10b. Yield: 82%. 1H NMR (CDCl3)
d: 8.39 (1H, d, J = 2.7 Hz), 8.24 (1H, d, J = 8.6 Hz), 7.72 (2H, d,
J = 8.6 Hz), 7.48 (1H, dd, J = 8.6, 2.7 Hz), 7.20 (2H, d, J = 9.0 Hz).
MS (ESI+) m/z: 284 (M+H)+.
C26H27BF3N2O4: 499.2010).
5.1.13. 3-Methyl-N-[2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
phenyl]-4-{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzamide (13b)
Compound 13b was prepared in a similar manner described for
8. Yield: 17%. 1H NMR (CDCl3) d: 10.22 (1H, s), 8.76 (1H, d,
J = 8.3 Hz), 8.44 (1H, s), 7.99 (1H, d, J = 2.0 Hz), 7.95 (2H, td,
J = 5.7, 2.8 Hz), 7.83 (1H, dd, J = 7.6, 1.7 Hz), 7.53 (1H, td, J = 7.8,
1.8 Hz), 7.18 (1H, d, J = 8.3 Hz), 7.12 (1H, td, J = 7.3, 1.0 Hz), 7.07
(1H, d, J = 8.8 Hz), 2.28 (3H, s), 1.41 (12H, s). MS (ESI+) m/z:
499 (M+H)+. HRMS (ESI+) m/z: 499.2015 (M+H)+ (calcd for
5.1.16. N-[2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-5-
{[5-(trifluoromethyl)pyridin-2-yl]oxy}pyridine-2-carboxamide (16a)
Compound 16a was prepared in a similar manner described for
6a. Yield: 32%. 1H NMR (CDCl3) d: 11.83 (1H, s), 8.82 (1H, d,
J = 8.3 Hz), 8.75 (1H, d, J = 2.4 Hz), 8.52 (1H, d, J = 8.3 Hz), 7.99
(1H, dd, J = 8.3, 2.4 Hz), 7.86 (1H, dd, J = 7.3, 1.5 Hz), 7.80 (1H, d,
J = 1.0 Hz), 7.61–7.57 (2H, m), 7.18 (1H, td, J = 7.3, 1.0 Hz), 6.82
(1H, d, J = 9.8 Hz), 1.46 (12H, s). MS (ESI+) m/z: 486 (M+H)+. HRMS
(ESI+) m/z: 486.1804 (M+H)+ (calcd for C24H24BF3N3O4: 486.1806).
C26H27BF3N2O4: 499.2010).
5.1.14. 5-{[5-(Trifluoromethyl)pyridin-2-yl]oxy}pyridine-2-carboxylic
acid (15a)
Step A: A mixture of 5-(trifluoromethyl)pyridin-2-ol (1.0 g,
8.2 mmol), 2-cyano-5-fluoropyridine (1.5 g, 9.0 mmol), K2CO3
(1.4 g, 9.8 mmol) and DMA (3.0 mL) was stirred at 100 °C for 3 h.
After cooling to room temperature, the reaction mixture was
diluted with water and extracted with AcOEt. The organic layer
was washed with brine, dried over Na2SO4 and concentrated in
vacuo. The resulting residue was purified by silica gel chromatog-
raphy (hexane-AcOEt) to give 5-{[5-(trifluoromethyl)pyridin-2-yl]
oxy}pyridine-2-carbonitrile (1.9 g, 86%) as a colorless solid. 1H
NMR (CDCl3) d: 8.80 (1H, d, J = 2.0 Hz), 8.02 (1H, dd, J = 8.4,
2.5 Hz), 7.89 (1H, d, J = 9.0 Hz), 7.72 (1H, s), 7.59 (1H, dd, J = 9.8,
2.7 Hz), 6.78 (1H, d, J = 9.8 Hz). MS (ESI+) m/z: 266 (M+H)+. Step
B: A mixture of 5-{[5-(trifluoromethyl)pyridin-2-yl]oxy}pyridine-
2-carbonitrile (1.9 g, 7.0 mmol), conc. hydrochloric acid (6.0 mL),
conc. sulfuric acid (3.0 mL) and MeOH (20 mL) was stirred at
85 °C for 4 h. After cooling to room temperature, the reaction mix-
ture was concentrated in vacuo. The residue was diluted with 5 M
NaOH aq. and extracted with AcOEt. The resulting residue was
purified by silica gel chromatography (hexane-AcOEt) to give
5.1.17. N-[2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-5-
[4-(trifluoromethyl)phenoxy]pyridine-2-carboxamide (16b)
Compound 16b was prepared in a similar manner described for
6a. Yield: 11%. 1H NMR (CDCl3) d: 11.71 (1H, s), 8.75 (1H, d,
J = 7.8 Hz), 8.44 (1H, d, J = 2.4 Hz), 8.32 (1H, d, J = 8.8 Hz), 7.82
(1H, dd, J = 7.3, 1.5 Hz), 7.68 (2H, d, J = 8.8 Hz), 7.54–7.50 (1H, m),
7.46 (1H, dd, J = 8.5, 2.7 Hz), 7.17–7.11 (3H, m), 1.42 (12H, s). MS
(ESI+) m/z: 485 (M+H)+. HRMS (ESI+) m/z: 485.1855 (M+H)+ (calcd
for C25H25BF3N2O4: 485.1854).
5.1.18. 6-[4-(Trifluoromethyl)phenoxy]pyridine-3-carboxylic acid (18)
Compound 18 was prepared in a similar manner described for
10b. Yield: 80%. 1H NMR (CDCl3) d: 8.90 (1H, d, J = 2.0 Hz), 8.39
(1H, dd, J = 8.6, 2.7 Hz), 7.72 (2H, d, J = 8.6 Hz), 7.31 (2H, d,
J = 8.6 Hz), 7.08 (1H, dd, J = 8.6, 0.8 Hz). MS (ESI+) m/z: 284 (M+H)+.
methyl
5-{[5-(trifluoromethyl)pyridin-2-yl]oxy}pyridine-2-car-
5.1.19. N-[2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-6-
[4-(trifluoromethyl)phenoxy]pyridine-3-carboxamide (19a) and {2-
[({6-[4-(trifluoromethyl)phenoxy]pyridin-3-yl}carbonyl)amino]
phenyl}boronic acid (19b)
A mixture of 18 (0.68 g, 2.4 mmol), 5a (0.44 g, 2.0 mmol), DIPEA
(1.4 mL, 6 mmol), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethy-
luronium hexafluorophosphate (0.91 g, 2.4 mmol), and DMF
(12 mL) was stirred at room temperature overnight. The reaction
mixture was diluted with water and extracted with AcOEt. The
organic layer was washed with H2O and brine, dried over Na2SO4
boxylate (1.1 g, 3.8 mmol) as a pale yellow solid. 1H NMR (CDCl3)
d: 8.79 (1H, d, J = 2.9 Hz), 8.31 (1H, d, J = 8.3 Hz), 7.99 (1H, dd,
J = 8.3, 2.4 Hz), 7.73 (1H, s), 7.57 (1H, dd, J = 9.5, 2.7 Hz), 6.77
(1H, d, J = 9.8 Hz), 4.06 (3H, s). MS (ESI+) m/z: 299 (M+H)+. Step
C: Compound 15a was prepared in a similar manner described
for 10b. Yield: 91%. 1H NMR (CDCl3) d: 8.81 (1H, d, J = 2.0 Hz),
8.43 (1H, d, J = 8.2 Hz), 8.09 (1H, dd, J = 8.4, 2.5 Hz), 7.78 (1H, s),
7.61 (1H, dd, J = 9.6, 2.5 Hz), 6.82 (1H, d, J = 9.8 Hz). MS (ESI+)
m/z: 285 (M+H)+.
Please cite this article in press as: Ogiyama T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.045

8
T. Ogiyama et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
and evaporated. The resulting residue was purified by silica gel
chromatography (AcOEt-MeOH) to give 19a (0.094 g, 10%) as a col-
orless solid and 19b (0.18 g, 23%) as a colorless solid.
7.29–7.27 (2H, m), 7.21 (2H, d, J = 9.0 Hz), 7.13 (1H, td, J = 7.4,
0.8 Hz), 7.01 (1H, dd, J = 8.6, 0.8 Hz), 1.41 (12H, s). MS (ESI+) m/z:
501 (M+H)+. HRMS (ESI+) m/z: 501.1807 (M+H)+ (calcd for
19a: 1H NMR (CDCl3) d: 10.23 (1H, s), 8.84 (1H, d, J = 2.3 Hz),
8.67 (1H, d, J = 8.2 Hz), 8.36 (1H, dd, J = 8.6, 2.7 Hz), 7.83 (1H, dd,
J = 7.4, 1.6 Hz), 7.70 (2H, d, J = 8.6 Hz), 7.53 (1H, td, J = 7.8,
1.4 Hz), 7.30 (2H, d, J = 8.2 Hz), 7.14 (1H, td, J = 7.4, 0.8 Hz), 7.06
(1H, d, J = 8.6 Hz), 1.41 (12H, s). MS (ESI+) m/z: 485 (M+H)+. HRMS
(ESI+) m/z: 485.1848 (M+H)+ (calcd for C25H25BF3N2O4: 485.1854).
19b: 1H NMR (CD3OD) d: 8.92 (1H, d, J = 2.3 Hz), 8.58 (1H, dd,
J = 8.8, 2.5 Hz), 7.77 (2H, d, J = 9.0 Hz), 7.51 (1H, t, J = 3.9 Hz), 7.39
(2H, d, J = 8.6 Hz), 7.31–7.28 (4H, m). MS (ESI-) m/z: 401 (MÀH)À.
HRMS (ESI-) m/z: 401.0913 (MÀH)À (calcd for C19H13BF3N2O4:
401.0926).
C25H25BF3N2O5: 501.1803).
5.1.24. N-[2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-6-
[3-(trifluoromethoxy)phenoxy]pyridine-3-carboxamide (22b)
Compound 22b was prepared in a similar manner described for
6a. Yield: 39%. 1H NMR (CDCl3) d: 10.23 (1H, s), 8.84 (1H, d,
J = 2.4 Hz), 8.67 (1H, d, J = 8.3 Hz), 8.36 (1H, dd, J = 8.3, 2.4 Hz),
7.83 (1H, dd, J = 7.3, 1.5 Hz), 7.53 (1H, td, J = 7.9, 1.6 Hz), 7.45
(1H, dt, J = 10.3, 3.2 Hz), 7.14–7.12 (4H, m), 7.03 (1H, d,
J = 8.8 Hz), 1.40 (12H, s). MS (ESI+) m/z: 501 (M+H)+. HRMS (ESI+)
m/z: 501.1806 (M+H)+ (calcd for C25H25BF3N2O5: 501.1803).
5.1.20. 6-[4-(Trifluoromethoxy)phenoxy]pyridine-3-carboxylic acid
(21a)
5.1.25. 6-{Methyl[4-(trifluoromethoxy)phenyl]amino}-N-[2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pyridine-3-carboxamide
(22c)
Compound 22c was prepared in a similar manner described for
6a. Yield: 32%. 1H NMR (CDCl3) d: 10.11 (1H, s), 8.90 (1H, d,
J = 2.3 Hz), 8.67 (1H, d, J = 8.2 Hz), 7.96 (1H, dd, J = 8.8, 2.5 Hz),
7.80 (1H, dd, J = 7.4, 1.6 Hz), 7.50 (1H, td, J = 7.9, 1.6 Hz), 7.34–
7.30 (4H, m), 7.09 (1H, td, J = 7.3, 1.0 Hz), 6.52 (1H, d, J = 9.0 Hz),
3.55 (3H, s), 1.41 (12H, s). MS (ESI+) m/z: 514 (M+H)+.
Step A: Methyl 6-[4-(trifluoromethoxy)phenoxy]pyridine-3-
carboxylate was prepared in a similar manner described for 10a.
Yield: 71%. 1H NMR (CDCl3) d: 8.81 (1H, dd, J = 2.3, 0.8 Hz), 8.31
(1H, dd, J = 8.6, 2.3 Hz), 7.29–7.26 (2H, m), 7.21–7.17 (2H, m),
6.98 (1H, dd, J = 8.6, 0.8 Hz), 3.93 (3H, s). MS (ESI+) m/z: 314 (M
+H)+. Step B: Compound 21a was prepared in a similar manner
described for 10b. Yield: 83%. 1H NMR (CDCl3) d: 8.88 (1H, dd,
J = 2.3, 0.8 Hz), 8.35 (1H, dd, J = 8.6, 2.3 Hz), 7.30–7.27 (2H, m),
7.22–7.19 (2H, m), 7.02 (1H, dd, J = 8.6, 0.8 Hz). MS (ESI+) m/z:
300 (M+H)+.
5.1.26. N-[4-Fluoro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
phenyl]-6-[4-(trifluoromethyl)phenoxy]pyridine-3-carboxamide
(23a)
Compound 23a was prepared in a similar manner described for
6a. Yield: 51%. 1H NMR (CD3OD) d: 8.86 (1H, dd, J = 2.7, 0.8 Hz),
8.50 (1H, dd, J = 8.6, 2.7 Hz), 7.76 (2H, d, J = 8.6 Hz), 7.38 (2H, d,
J = 9.0 Hz), 7.33–7.21 (2H, m), 7.15 (1H, dd, J = 8.6, 3.1 Hz), 7.03
(1H, td, J = 8.6, 3.1 Hz), 1.36 (12H, s). MS (ESI+) m/z: 503 (M+H)+.
5.1.21. 6-[3-(Trifluoromethoxy)phenoxy]pyridine-3-carboxylic acid
(21b)
Step A: Methyl 6-[3-(trifluoromethoxy)phenoxy]pyridine-3-
carboxylate was prepared in a similar manner described for 10a.
Yield: 78%. 1H NMR (CDCl3) d: 8.82 (1H, dd, J = 2.3, 0.8 Hz), 8.32
(1H, dd, J = 8.6, 2.3 Hz), 7.44 (1H, t, J = 8.2 Hz), 7.12–7.09 (3H, m),
6.99 (1H, d, J = 8.6 Hz), 3.93 (3H, s). MS (ESI+) m/z: 314 (M+H)+. Step
B: Compound 21b was prepared in a similar manner described for
10b. Yield: 76%. 1H NMR (CDCl3) d: 8.90 (1H, d, J = 2.3 Hz), 8.37 (1H,
dd, J = 8.6, 2.3 Hz), 7.46 (1H, t, J = 8.4 Hz), 7.14 (2H, dd, J = 8.4,
2.2 Hz), 7.09–7.08 (1H, m), 7.03 (1H, dd, J = 8.6, 0.8 Hz). MS (ESI+)
m/z: 300 (M+H)+.
5.1.27. N-[4-Chloro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
phenyl]-6-[4-(trifluoromethyl)phenoxy]pyridine-3-carboxamide
(23b)
Compound 23b was prepared in a similar manner described for
6a. Yield: 62%. 1H NMR (CD3OD) d: 8.86 (1H, t, J = 1.6 Hz), 8.50 (1H,
dd, J = 8.8, 2.5 Hz), 7.77 (2H, d, J = 9.0 Hz), 7.56 (1H, d, J = 2.3 Hz),
7.40 (2H, d, J = 8.6 Hz), 7.33–7.29 (3H, m), 1.37 (12H, s). MS (ESI+)
m/z: 519 (M+H)+. HRMS (ESI-) m/z: 517.1334 (MÀH)À (calcd for
5.1.22. 6-{Methyl[4-(trifluoromethoxy)phenyl]amino}pyridine-3-
carboxylic acid (21c)
Step A:
A mixture of methyl 6-chloronicotinate (0.34 g,
2.0 mmol), N-methyl-p-trifluoromethoxyaniline (0.38 g, 2.0 mmol)
and acetic acid (3.0 mL) was heated at 180 °C with microwave-
assistance for 30 min with stirring. After cooling to room tempera-
ture, the mixture was diluted with 1 M NaOH aq. and extracted
with AcOEt. The organic layer was washed with brine, dried over
Na2SO4 and concentrated in vacuo. The resulting residue was puri-
fied by silica gel chromatography (hexane-AcOEt) to give methyl
6-{methyl[4-(trifluoromethoxy)phenyl]amino}pyridine-3-carboxy-
late (0.066 g, 10%) as a colorless solid. 1H NMR (CDCl3) d: 8.88 (1H,
d, J = 2.3 Hz), 7.91 (1H, dd, J = 9.0, 2.3 Hz), 7.31 (4H, s), 6.44 (1H, dd,
J = 9.0, 0.8 Hz), 3.88 (3H, s), 3.53 (3H, s). MS (ESI+) m/z: 327 (M+H)+.
Step B: Compound 21c was prepared in a similar manner described
for 10b. Yield: 58%. 1H NMR (CDCl3) d: 8.94 (1H, d, J = 2.3 Hz), 7.93
(1H, dd, J = 9.0, 2.3 Hz), 7.31–7.30 (4H, m), 6.44 (1H, dd, J = 9.0,
0.8 Hz), 3.54 (3H, s). MS (ESI+) m/z: 313 (M+H)+.
C25H22BClF3N2O4: 517.1319).
5.1.28. N-[4-Methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
phenyl]-6-[4-(trifluoromethyl)phenoxy]pyridine-3-carboxamide (23c)
Compound 23c was prepared in a similar manner described for
6a. Yield: 37%. 1H NMR (CDCl3) d: 10.17 (1H, s), 8.83 (1H, d,
J = 2.0 Hz), 8.55 (1H, d, J = 8.2 Hz), 8.35 (1H, dd, J = 8.6, 2.3 Hz),
7.69 (2H, d, J = 8.6 Hz), 7.63 (1H, d, J = 2.0 Hz), 7.34 (1H, dd,
J = 8.6, 2.0 Hz), 7.30 (2H, d, J = 8.6 Hz), 7.05 (1H, d, J = 8.6 Hz),
2.33 (3H, s), 1.40 (12H, s). MS (ESI+) m/z: 499 (M+H)+.
5.1.29. N-[5-Fluoro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
phenyl]-6-[4-(trifluoromethyl)phenoxy]pyridine-3-carboxamide
(23d)
Compound 23d was prepared in a similar manner described for
6a. Yield: 61%. 1H NMR (CDCl3) d: 10.33 (1H, s), 8.83 (1H, dd, J = 2.7,
0.8 Hz), 8.49 (1H, dd, J = 12.1, 2.3 Hz), 8.35 (1H, dd, J = 8.6, 2.7 Hz),
7.80 (1H, dd, J = 8.6, 7.0 Hz), 7.70 (2H, d, J = 8.2 Hz), 7.30 (2H, d,
J = 9.0 Hz), 7.06 (1H, d, J = 8.6 Hz), 6.83 (1H, td, J = 8.2, 2.3 Hz),
5.1.23. N-[2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-6-
[4-(trifluoromethoxy)phenoxy]pyridine-3-carboxamide (22a)
Compound 22a was prepared in a similar manner described for
6a. Yield: 74%. 1H NMR (CDCl3) d: 10.22 (1H, s), 8.84 (1H, d,
J = 2.7 Hz), 8.67 (1H, d, J = 8.6 Hz), 8.34 (1H, dd, J = 8.6, 2.7 Hz),
7.83 (1H, dd, J = 7.4, 1.2 Hz), 7.53 (1H, td, J = 7.9, 1.6 Hz),
1.40 (12H, s). HRMS (ESI-) m/z: 501.1595 (MÀH)À (calcd for C25H22
-
BF4N2O4: 501.1614).
Please cite this article in press as: Ogiyama T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.045

T. Ogiyama et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
5.1.30. N-[5-Chloro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
phenyl]-6-[4-(trifluoromethyl)phenoxy]pyridine-3-carboxamide (23e)
Compound 23e was prepared in a similar manner described for
6a. Yield: 37%. 1H NMR (CD3OD) d: 8.86 (1H, dd, J = 2.7, 0.8 Hz),
8.50 (1H, dd, J = 8.8, 2.5 Hz), 7.77 (2H, d, J = 9.0 Hz), 7.62 (1H, d,
J = 7.8 Hz), 7.42–7.40 (3H, m), 7.30–7.26 (2H, m), 1.37 (12H, s).
MS (ESI+) m/z: 519 (M+H)+.
(1H, dd, J = 8.8, 2.4 Hz), 7.78 (2H, d, J = 8.3 Hz), 7.50 (1H, dd,
J = 8.3, 6.8 Hz), 7.40 (2H, d, J = 8.8 Hz), 7.30 (1H, d, J = 8.8 Hz),
7.06–7.05 (2H, m). MS (ESI-) m/z: 419 (MÀH)À. HRMS (ESI-) m/z:
419.0825 (MÀH)À (calcd for C19H12BF4N2O4: 419.0832).
5.1.36. {4-Chloro-2-[({6-[4-(trifluoromethyl)phenoxy]pyridin-3-
yl}carbonyl)amino]phenyl}boronic acid (24e)
Compound 24e was prepared in a similar manner described for
24a. Yield: 66%. 1H NMR (CD3OD) d: 8.91 (1H, br s), 8.57–8.55 (1H,
m), 7.75 (2H, d, J = 8.2 Hz), 7.43 (1H, d, J = 7.8 Hz), 7.37 (2H, d,
J = 8.2 Hz), 7.22 (1H, br s), 7.17 (1H, d, J = 8.2 Hz), 7.13 (1H, br s).
MS (ESI-) m/z: 435 (MÀH)À. HRMS (ESI-) m/z: 435.0561 (MÀH)À
(calcd for C19H12BClF3N2O4: 435.0536).
5.1.31. N-[5-Methoxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl]-6-[4-(trifluoromethyl)phenoxy]pyridine-3-carboxamide
(23f)
Compound 23f was prepared in a similar manner described for
6a. Yield: 42%. 1H NMR (CD3OD) d: 8.83 (1H, d, J = 2.7 Hz), 8.48 (1H,
dd, J = 8.6, 2.3 Hz), 7.77 (2H, d, J = 9.0 Hz), 7.58 (1H, d, J = 8.2 Hz),
7.40 (2H, d, J = 8.6 Hz), 7.27 (1H, d, J = 9.0 Hz), 7.22 (1H, d,
J = 2.3 Hz), 6.85 (1H, dd, J = 8.2, 2.3 Hz), 3.82 (3H, s), 1.37 (12H,
s). MS (ESI+) m/z: 515 (M+H)+.
5.1.37. {4-Methoxy-2-[({6-[4-(trifluoromethyl)phenoxy]pyridin-3-
yl}carbonyl)amino]phenyl}boronic acid (24f)
Compound 24f was prepared in a similar manner described for
24a. Yield: 25%. 1H NMR (CD3OD) d: 8.92 (1H, d, J = 2.4 Hz), 8.57
(1H, dd, J = 8.8, 2.4 Hz), 7.78 (2H, d, J = 8.8 Hz), 7.41 (3H, d,
J = 7.8 Hz), 7.30 (1H, d, J = 8.8 Hz), 6.92 (1H, dd, J = 8.1, 2.2 Hz),
6.88 (1H, d, J = 2.4 Hz), 3.83 (3H, s). MS (ESI-) m/z: 431 (MÀH)À.
HRMS (ESI-) m/z: 432.1084 (MÀH)À (calcd for C20H15BF3N2O5:
431.1032).
5.1.32. {5-Fluoro-2-[({6-[4-(trifluoromethyl)phenoxy]pyridin-3-
yl}carbonyl)amino]phenyl}boronic acid (24a)
Step A: To a solution of 23a (0.51 g, 1.0 mmol) in MeOH (50 mL)
was added 5.6 M KHF2 aq. (1.26 mL). After stirring at room temper-
ature for 15 min, the reaction mixture was concentrated in vacuo.
The residue was dissolved in hot acetone and filtered. The filtrate
was concentrated in vacuo to give crude potassium {5-fluoro-
2-[({6-[4-(trifluoromethyl)phenoxy]pyridin-3-yl}carbonyl)amino]-
phenyl}(trifluoro)borate(1-), which was used directly in the next
step. Step B: Crude potassium {5-fluoro-2-[({6-[4-(trifluo-
romethyl)phenoxy]pyridin-3-yl}carbonyl)amino]phenyl}(trifluoro)-
borate(1-) was dissolved in CH3CN (50 mL) and H2O (0.11 mL) and
chlorotrimethylsilane (0.77 ml, 6.1 mmol) was added. After stirring
overnight, the resulting mixture was evaporated. The resulting
residue was diluted with saturated NaHCO3 solution and extracted
with AcOEt. The organic layer was washed with H2O (twice) and
brine, dried over Na2SO4, filtered and evaporated. The resulting
residue was purified by silica gel chromatography (hexane-AcOEt).
The obtained solid was triturated in hexane/AcOEt and filtered to
give 24a (0.16 g, 38%) as a colorless solid. 1H NMR (CD3OD) d:
8.91 (1H, s), 8.56 (1H, dd, J = 8.6, 2.0 Hz), 7.77 (2H, d, J = 9.0 Hz),
7.38 (2H, d, J = 9.0 Hz), 7.30 (1H, br s), 7.24 (1H, d, J = 8.6 Hz),
7.16 (1H, dd, J = 8.6, 2.7 Hz), 7.03–7.01 (1H, br s). MS (ESIÀ) m/z:
419 (MÀH)À. HRMS (ESI-) m/z: 419.0843 (MÀH)À (calcd for
C19H12BF4N2O4: 419.0832).
5.1.38. 4-{[5-(trifluoromethyl)pyridin-2-yl]oxy}phenyl 4-
hydroxypiperidine-1-carboxylate (25)
Compound 25 was prepared in the same manner previously
reported by Ebdrup et al.14 1H NMR (DMSO-D6) d: 8.58–8.57 (1H,
m), 8.24 (1H, dd, J = 8.8, 2.5 Hz), 7.25 (1H, d, J = 9.0 Hz), 7.21–7.18
(4H, m), 3.89–3.86 (1H, m), 3.76–3.70 (2H, m), 3.31–3.28 (1H,
m), 3.17–3.14 (1H, m), 1.81–1.77 (2H, m), 1.42–1.38 (2H, m). HRMS
(ESI+) m/z: 383.1207 (M+H)+ (calcd for C18H18F3N2O4: 383.1213).
5.2. Pharmacology
5.2.1. In vitro HSL assay
Recombinant human HSL was expressed in Spodoptera frugi-
perda Sf9 insect cells using the Bac-to-BacÒ Baculovirus Expression
System (Invitrogen, Inc.). The infected Sf9 cells were suspended in
3 pellet volumes with a homogenization buffer (25 mM sucrose pH
7.5, 1 mM EDTA, 1 mM DTT and protease inhibitors) to the whole
volume of 30 mL. The homogenate was sonicated on an ice bath.
Insoluble material was removed by centrifugation at 18,000 rpm
for 20 min. The obtained 20 mL of clear supernatant was utilized
for the in vitro assay. The solution of human HSL extract diluted
by 6-fold (10 mL) and various concentrations of compounds in
DMSO (10 mL) were added to a solution of 1.0 mM PNPB in a phos-
phate buffer (0.1 M NaH2PO4 pH 7.25, 0.15 M NaCl, and 1 mM DTT)
(180 mL). The enzymatic reactions were carried out at 37 °C for
5 min. The solution absorbances were measured spectrophotomet-
rically at a k of 405 nm at 0 and 5 min. Each assay was carried out
in triplicate. IC50 values were obtained by fitting data to a nonlin-
ear curve fitting program (GraphPad Software, Inc., La Jolla CA).
5.1.33. {5-Chloro-2-[({6-[4-(trifluoromethyl)phenoxy]pyridin-3-
yl}carbonyl)amino]phenyl}boronic acid (24b)
Compound 24b was prepared in a similar manner described for
24a. Yield: 47%. 1H NMR (CD3OD) d: 8.91 (1H, s), 8.56 (1H, d,
J = 8.6 Hz), 7.77 (2H, d, J = 8.6 Hz), 7.44 (1H, s), 7.39 (2H, d,
J = 8.2 Hz), 7.29–7.26 (3H, m). MS (ESI-) m/z: 435 (MÀH)À. HRMS
(ESI-) m/z: 435.0529 (MÀH)À (calcd for C19H12BClF3N2O4:
435.0536).
5.1.34. {5-Methyl-2-[({6-[4-(trifluoromethyl)phenoxy]pyridin-3-
yl}carbonyl)amino]phenyl}boronic acid (24c)
5.2.2. In vivo inhibition of lipolysis
Compound 24c was prepared in a similar manner described for
24a. Yield: 41%. 1H NMR (CD3OD) d: 8.91 (1H, dd, J = 2.7, 0.8 Hz),
8.56 (1H, dd, J = 8.6, 2.7 Hz), 7.78 (2H, d, J = 8.2 Hz), 7.40 (2H, d,
J = 8.2 Hz), 7.32–7.29 (2H, m), 7.21–7.18 (2H, m), 2.37 (3H, s). MS
(ESI-) m/z: 415 (MÀH)À. HRMS (ESI-) m/z: 415.1100 (MÀH)À (calcd
for C20H15BF3N2O4: 415.1082).
Male Wistar rats 8 weeks of age were purchased from Japan
SLC, Inc. All animals were held under standard laboratory condi-
tions (12 h light per day, lights on at 7:00 AM, 23 2 °C, 55 10%
humidity) with food and water available ad libitum. Overnight-
fasted rats received vehicle (propylene glycol/Tween80 = 4:1
(v/v)) (n = 4) or HSL inhibitor in the vehicle (1 mg/kg, 3 mg/kg
and 10 mg/kg body weight p.o.; n = 3 per group). EDTA-treated
blood samples were collected just prior to and after dosing at
0.5, 1, 3, 5, and 7 h from tail veins. The blood was centrifuged at
12,000 rpm for 5 min at 4 °C to obtain the plasma. The plasma sam-
ples were stored at -20 °C before analysis. Plasma glycerol levels
5.1.35. {4-Fluoro-2-[({6-[4-(trifluoromethyl)phenoxy]pyridin-3-
yl}carbonyl)amino]phenyl}boronic acid (24d)
Compound 24d was prepared in a similar manner described for
24a. Yield: 40%. 1H NMR (CD3OD) d: 8.93 (1H, d, J = 2.4 Hz), 8.58
Please cite this article in press as: Ogiyama T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.045

10
T. Ogiyama et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
were determined using a Free Glycerol Determination Kit (Sigma-
Aldrich). All experimental procedures were reviewed and
approved by the Institutional Animal Care and Use Committee at
Daiichi Sankyo Co., Ltd. The relative value was calculated as the
following formula:
Acknowledgments
We would like to thank Dr. Satoshi Shibuya and Dr. Koichi Oda
for their helpful advice. We would also like to thank Dr. Naoki
Tanaka for careful reading of the manuscript and valuable
suggestions.
Plasma glycerol reduction of the AUC0-7
(%) relative to
h
compound 25 = {[Plasma glycerol AUC0-7 h value (vehicle group) À
Plasma glycerol AUC0-7 value (test group)]/[Plasma glycerol
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AUC0-7 value (vehicle group) À Plasma glycerol AUC0-7 value
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5.2.3. Pharmacokinetic evaluation in rats
Each plasma sample was prepared as described above, followed
by mixing with water, acetonitrile, and IS (internal standard) ace-
tonitrile solution (20 mM of niflumic acid). For each standard sam-
ple, each standard solution of compound 24b (0.02, 0.05, 0.2, 0.5, 2,
5, 20 and 50 mM) was added to blank plasma and mixed with
water, acetonitrile and IS acetonitrile solution. After vortex mixing
for 5 min at room temperature, the mixtures were added to a Cap-
tiva plate (Agilent Technologies) and filtrated. The filtrates were
subjected to LC-MS/MS analysis using an API 4000QTRAP (Applied
Biosystems/MDS SCIEX). Cmax and AUC0-7h were determined by a
non-compartmental model using the BioBook function of E-Work-
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5.2.4. GSH trapping experiments
For the GSH trapping experiments, each test compound
(500 lM) was incubated with human liver microsomes (2 mg of
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22. LC–MS analysis of the incubation mixture of compound 1a with isotope labeled
glutathione ([13C2, 15N-Gly]GSH) in the presence of microsomes led to the
detection of the sulfhydryl adduct m/z 428 with unambiguous doublet isotopic
peaks with a mass difference of 3 Da (m/z 431). The product ion spectrum
obtained by higher-energy collision-induced dissociation (HCD) of the MH+ at
m/z 428 produced identical fragment ions at m/z 382, 353, 325, 267, and 252.
23. It was recently reported that the formation of Cys-Gly-thiazolidine adducts
possibly occurred after the degradation by hydrolysis of the glutamic acid
moiety. See Ref. 23.
protein/mL) supplemented with an NADPH generating system
and an equimolar mixture of GSH and stable isotope-labeled GSH
([13C2, 15N-Gly]GSH). After 60-min incubation, the reaction was
terminated with acetonitrile containing propranolol, followed by
centrifugation and concentration of the supernatant. The incuba-
tion without NADP or a substrate was performed to obtain control
samples. The analytical samples were subjected to LC-MS/MS anal-
ysis in a full scan MSE mode using a Q-Tof Xevo mass spectrometer
(Waters). The data were analyzed by Metabolynx software
(Waters). The structure elucidation of GSH adducts of compound
1 was performed by LC/MS and LC-MS/MS analysis using an LTQ
Orbitrap XL (Thermo Fisher Scientific).
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30. The plasma glycerol level was employed as a lipolytic end point in rats on the
basis of the species difference in the mechanism of TG lipolysis. See the Refs.
13,14.
5.2.5. Metabolic stability
Compound 24b (1 lM) and rat liver microsomes (0.5 mg/mL)
were incubated in sodium phosphate buffer (pH 7.4) at 37 °C with
an NADPH generating system. After 20-min incubation, the reac-
tion was terminated with MeOH. After centrifuging each solution
separately at 3500 rpm for 10 min at 4 °C, the remaining parent
compound was determined by LC-MS/MS.
Please cite this article in press as: Ogiyama T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.045