Discovery of triazolone derivatives as novel, potent stearoyl-CoA desaturase-1 (SCD1) inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.12.014

Stearoyl-CoA desaturase-1 (SCD1) plays an important role in lipid metabolism. Inhibition of SCD1 activity represents a potential novel approach for the treatment of metabolic diseases such as obesity, type 2 diabetes and dyslipidemia, as well as skin diseases, acne and cancer. Herein, we report the synthesis and structure-activity relationships (SAR) of a series of novel triazolone derivatives, culminating in the identification of pyrazolyltriazolone 17a, a potent SCD1 inhibitor, which reduced plasma C16:1/C16:0 triglycerides desaturation index (DI) in an acute Lewis rat model in a dose dependent manner, with an ED₅₀ of 4.6 mg/kg. In preliminary safety studies, compound 17a did not demonstrate adverse effects related to SCD1 inhibition after repeat dosing at 100 mg/kg. Together, these data suggest that sufficient safety margins can be achieved with certain SCD1 inhibitors, thus allowing exploration of clinical utility in metabolic disease settings.

Full text of DOI:10.1016/j.bmc.2014.12.014

Bioorganic & Medicinal Chemistry 23 (2015) 455–465
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of triazolone derivatives as novel, potent stearoyl-CoA
desaturase-1 (SCD1) inhibitors
a
a
a
a
a
Shaoyi Sun a, , Zaihui Zhang , Natalia Pokrovskaia , Sultan Chowdhury , Qi Jia , Elaine Chang ,
⇑
Kuldip Khakh a, Rainbow Kwan a, David G. McLaren a, Chris C. Radomski a, Leslie G. Ratkay a, Jianmin Fu a,
Natalie A. Dales b, , Michael D. Winther
a
⇑
a Xenon Pharmaceuticals Inc., 200-3650 Gilmore Way, Burnaby, BC V5G 4W8, Canada
b Novartis Institute for Biomedical Research, 250 Massachusetts Ave, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Stearoyl-CoA desaturase-1 (SCD1) plays an important role in lipid metabolism. Inhibition of SCD1 activity
represents a potential novel approach for the treatment of metabolic diseases such as obesity, type 2
diabetes and dyslipidemia, as well as skin diseases, acne and cancer. Herein, we report the synthesis
and structure–activity relationships (SAR) of a series of novel triazolone derivatives, culminating in the
identification of pyrazolyltriazolone 17a, a potent SCD1 inhibitor, which reduced plasma C16:1/C16:0
triglycerides desaturation index (DI) in an acute Lewis rat model in a dose dependent manner, with an
ED50 of 4.6 mg/kg. In preliminary safety studies, compound 17a did not demonstrate adverse effects
related to SCD1 inhibition after repeat dosing at 100 mg/kg. Together, these data suggest that sufficient
safety margins can be achieved with certain SCD1 inhibitors, thus allowing exploration of clinical utility
in metabolic disease settings.
Received 30 October 2014
Revised 3 December 2014
Accepted 12 December 2014
Available online 19 December 2014
Keywords:
Stearoyl-CoA desaturase
Thiazolytriazolone
Pyrazolyltriazolone
Desaturation index
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
diet-induced obese (DIO) mice treated with SCD1 antisense oligo-
nucleotides.13,14 In humans, elevated SCD1 activity is positively
It has been well established that lipids play a crucial role in the
etiology of metabolic diseases, therefore, several enzymes regulat-
ing lipid metabolism have recently been proposed as therapeutic
targets.1,2 One such target is stearoyl-CoA desaturase-1 (SCD1).
SCD1, also known as delta-9 desaturase (D9D), is a microsomal
enzyme that catalyzes the de novo synthesis of monounsaturated
fatty acids (MUFA) from saturated fatty acids by introducing a
cis-double bond between carbons 9 and 10. The products, mainly
oleate and palmitoleate, are key substrates for synthesis of triglyc-
erides, wax esters, cholesterol esters and phospholipids.3 To date,
four SCD isoforms (SCD1–4) have been characterized in
rodents,4–7 and two SCD isoforms (SCD1 and SCD5) in humans.
SCD1, with 85% identity across species, is the predominant isoform
expressed in lipogenic tissues including liver and adipose.8,9 SCD1
knockout mice display a beneficial metabolic phenotype character-
ized by increased energy expenditure, reduced adiposity, improved
insulin sensitivity and resistance to high fat diet-induced
obesity.10–12 Similar beneficial effects are observed in high fat
correlated with high triglyceride levels in familial hypertriglyceri-
demia subjects,15 increased body mass index (BMI) and high
plasma insulin levels.16 Cross species studies provide evidence to
support the view that SCD1 is a critical player in the regulation
of skeletal muscle and fat metabolism.17 In addition to the benefi-
cial metabolic effects associated with SCD1 inhibition, studies in
mice further suggested that SCD1 activity is important to main-
taining the normal functioning of the skin as a result of its impor-
tant role in lipid synthesis within sebaceous glands. Mouse strains
deficient in the enzyme SCD1 exhibit severe hypoplasia of seba-
ceous glands.18,19 Small molecule SCD1 inhibition has shown to
reduce sebaceous size and number in animal models.20 SCD1 is
also involved in the regulation of cell proliferation, growth and
apoptosis. A couple of studies have implicated SCD1 expression
and activity in the pathogenesis of cancer.21–24 Therefore inhibition
of SCD1 represents a potential novel approach for the treatment of
metabolic diseases such as obesity, type 2 diabetes and dyslipide-
mia, as well as skin diseases, acne, and cancer.
Small molecule SCD1 inhibitors have been reviewed,25–27 with
several new structures reported recently.28,29 Our efforts continue
to focus on the identification of novel scaffolds, with differing
properties, as SCD1 inhibitors.30–32 We previously described the
⇑
Corresponding authors. Tel.: +1 604 484 3300 (S.S.), +1 617 871 7796 (N.A.D.).
E-mail addresses: ssun@xenon-pharma.com (S. Sun), natalie.dales@novartis.com
(N.A. Dales).
http://dx.doi.org/10.1016/j.bmc.2014.12.014
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

456
S. Sun et al. / Bioorg. Med. Chem. 23 (2015) 455–465
application of bioisostere strategies in the replacement of the
amide bond at the C2-position of compound 1.31 These studies
led us to identify a series of novel thiazolylimidazolidinone SCD1
inhibitors. The advantages of this chemical series are improved
inhibitory potency and metabolic stability, as exemplified by
XEN723 (Fig. 1). This compound is a potent SCD1 inhibitor with
an enzymatic IC50 of 6 nM in mouse SCD1 assay, and good
in vivo efficacy in an acute Lewis rat model with ED50 of 4.5 mg/
kg. Similar to other systemic SCD1 inhibitors,30 undesired skin
and eye adverse effects were observed in XEN723 treated rats in
chronic studies. These adverse events (AEs) are believed to be
due to mechanism-based depletion of essential SCD-derived lubri-
cating lipids.18,19 We wished to identify potent SCD1 inhibitors
with desirable physiochemical properties in alternative chemical
space to mitigate mechanism-related AEs. In this report, we
describe the synthesis and structure–activity relationships (SAR)
of a series of novel triazolone-based SCD1 inhibitors. This work
led to the discovery of the pyrazolyltriazolone analogue 17a, an
efficacious SCD1 inhibitor with an improved AEs profile.
intermediate 10. Reduction of the nitro group in 10 by hydrogenol-
ysis yielded 11. The amine intermediate 11 was then converted
into amides 16a–16f in a similar manner as described above for
preparation of 7a–7w. The 5-step sequence to reach 16a–16f was
followed by Pmb deprotection to finally provide the desired prod-
ucts 17a–17f.
3. Results and discussion
The potency of the prepared compounds was first assessed in a
primary SCD1 biochemical assay, using mouse liver microsomes.
Compounds with significant SCD1 inhibition in this assay were
then advanced for further characterization in a human liver hepa-
tocellular carcinoma (HepG2) cell-based assay. In the mouse liver
microsomal assay, the SCD1 activity was determined by measuring
the decreased production of tritiated water released from
(9,10-3H)-labeled stearoyl coenzyme A substrate mediated by
SCD1.30,33 In the human HepG2 cell-based assay, SCD1 activity
was assessed by determination of the amount of 14C-oleic acid
product formed from 14C stearate substrate.30,34 Besides SCDs,
there are two other fatty acid desaturases in humans: delta-5
desaturases (D5D) and delta-6 desaturases (D6D). These related
enzymes are required for the synthesis of highly unsaturated fatty
acids (HUFAs), which are mainly esterified into phospholipids and
contribute to maintaining membrane fluidity. Therefore, achieving
selectivity against D5D and D6D is essential to avoid undesirable
toxicities.35 Compounds with good potency against SCD1 were also
screened for their selectivity against D5D and D6D. None of the
compounds evaluated (all with IC50 <100 nM in SCD1 mouse liver
microsomes) demonstrated significant reduction in D5D and D6D
2. Chemistry
The synthesis of thiazolyltriazolone analogues 7a–7w is out-
lined in Scheme 1. Ethyl 2-amino-4-methylthiazole-5-carboxylate
2 was treated with 4-nitrophenyl chloroformate in the presence
of pyridine in THF to provide the corresponding nitrophenyl carba-
mate, which was converted to 3 by in situ cleavage with hydrazine.
Upon heating with trimethyl orthoformate in ethanol, 3 was con-
verted to the key triazolone intermediate 4. Alkylation of 4 with
various alkyl halides and subsequent hydrolysis of the ester 5a–
5i gave the corresponding acids 6a–6i. Amide formation of 6a–6i
under standard amide formation conditions with select amines
produced amides 7a–7dd and 7e–7w. The piperidinyl analogue
7d was obtained upon removal of the Boc protecting group of 7dd.
Synthesis of pyrazolyltriazolone analogues 17a–17f (Scheme 2)
started from commercially available 3-nitro-1H-pyrazole-5-
carboxylic acid 8, which was treated with thionyl chloride in
methanol, resulting in methyl ester 9. Subsequent alkylation of 9
with 4-methoxybenzyl bromide afforded the Pmb-protected
activity when tested at a concentration of 10 l
M.30,34 Based on
their in vitro potencies, as well as their overall properties, several
potent SCD1 inhibitors were selected for evaluation of their
in vivo effects on the plasma C16:1n7/C16:0 triglycerides (TG)
desaturation index (DI) in an acute Lewis rat model.31 C16 and
C18 DI have been well-documented as biomarkers for SCD1 target
engagement.14,18
Using XEN723 as the starting point, we replaced the
imidazolidinone ring with a triazolone moiety, while keeping the
F
O
N
H
N
N
H
N
N
N
N
N
H
S
S
O
O
O
1
XEN723
Mouse SCD1 IC50: 6 nM
Mouse SCD1 IC50: 3400 nM
Figure 1. Aminothiazole-based SCD inhibitors.
N
N
NH
N
S
N
N
N
N
a
N
c
b
N
NH
O
R2
NHNH2
NH2
S
EtO2C
S
S
EtO2C
EtO2C
EtO2C
O
O
2
3
4
5a-5i
N
N
N
N
N
N
S
e
f
d
N
H
N
N
N
H
N
N
N
N
O
F
R2
HN
R1
S
7d
S
for
HO2C
O
R2
O
O
O
6a-6i
7d
7a-7dd, 7e-7w
Scheme 1. Reagents and conditions: (a) 4-nitrophenyl chloroformate, pyridine, THF, rt, then NH2NH2ÁH2O, rt; (b) HC(OMe)3, TsOHÁH2O, ethanol, reflux; (c) R2X, K2CO3,
i
acetone, reflux; (d) NaOH, ethanol, H2O, reflux; (e) R1NH2, EDCI or TBTU, HOBt, Pr2NEt, DMF, rt; (f) CF3CO2H, CH2Cl2, rt.

S. Sun et al. / Bioorg. Med. Chem. 23 (2015) 455–465
457
O
O
O
O
a
c
b
HO
MeO
MeO
Pmb
10
MeO
NO2
NO2
NH2
NO2
HN
HN
N
N
N
N
N
N
Pmb
8
9
11
O
O
O
F
N
N
H
N
H
f
d
N
NH
e
RO
MeO
Pmb
N
MeO
Pmb
N
NH2
N
N
N
N
N
N
N
Pmb
O
N
O
O
14: R = Me
15
13
g
12
: R = H
O
O
F
F
R1
N
N
R1
N
N
h
i
N
H
N
H
N
N
HN
N
N
Pmb
O
O
16a-16f
17a-17f
Scheme 2. Reagents and conditions: (a) methanol, SOCl2, reflux; (b) 4-methoxybenzyl bromide, K2CO3, DMF, 60 °C; (c) H2, 10% Pd-C, methanol; (d) 4-nitrophenyl
chloroformate, pyridine, THF, rt, then NH2NH2ÁH2O, rt; (e) HC(OMe)3, TsOHÁH2O, ethanol, reflux; (f) 4-fluorobenzyl chloride, K2CO3, acetone, reflux; (g) NaOH, ethanol, H2O,
i
reflux; (h) R1NH2, TBTU, HOBt, Pr2NEt, DMF, rt; (i) CF3SO3H, CF3CO2H, CH2Cl2, rt.
Table 1
SAR of thiazolyltriazolones
O
N
H
N
R1
R2
N
N
S
N
O
Compound R1
R2
Mouse SCD1 IC50
(nM)
HepG2 SCD1 IC50
(nM)
Metabolic
stabilityb
Permeabilityc
a
a
(Â10À6 cm/s)
7a
7b
7c
Pyridin-3-ylmethyl
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
20
22
1722
268
67
nd
65
68
nd
22/19
35/30
nd
Pyridin-2-ylmethyl
6-(Trifluoromethyl)pyridin-3-
yl)methyl
7d
7e
7f
7g
7h
7i
Piperidin-3-ylmethyl
(5-Methylpyrazin-2-yl)methyl
Oxazol-4-ylmethyl
Oxazol-2-ylmethyl
Thiazol-2-ylmethyl
(2-Methylthiazol-4-yl)methyl
(1H-Pyrazol-3-yl)methyl
(5-Methyl-1H-pyrazol-3-
yl)methyl
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
>10,000
15
nd
194
39
214
9
32
nd
75
76
90
71
51
83
66
nd
36/28
32/27
24/23
24/20
25/19
25/25
21/20
12
22
12
24
46
9
7j
7k
105
280
7l
1-Methyl-1H-pyrazol-4-
yl)methyl
(1-Methyl-1H-imidazol-4-
yl)methyl
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
(4-Trifluoromethyl)-benzyl
3,5-Difluorobenzyl
5-(Trifluoromethyl)-furan-
2-yl
19
27
477
504
90
96
32/27
41/33
7m
7n
7o
7p
7q
H
65
16
19
7
945
46
125
8
nd
80
51
45
nd
Pyridin-3-ylmethyl
Pyridin-3-ylmethyl
Pyridin-3-ylmethyl
15/15
28/22
39/31
7r
7s
7t
Pyridin-3-ylmethyl
Pyridin-3-ylmethyl
Pyridin-3-ylmethyl
(4-Fluorophenoxy)ethyl
Cyclopropylmethyl
(2,2-Difluorocyclopropyl)-
methyl
15
98
55
9
140
306
22
85
91
40/33
23/22
34/33
7u
7v
7w
Pyridin-3-ylmethyl
Pyridin-2-ylmethyl
Pyridin-2-ylmethyl
4,4,4-Trifluorobutyl
(4-Trifluoromethyl)-benzyl
1-(4-Fluorophenyl)ethyl)
21
14
30
398
227
66
76
100
71
26/20
26/23
27/25
nd: not determined.
a
IC50s are an average of at least two independent determinations; nd: not determined.
Expressed as % of compound remaining after a 30 min incubation with 0.5 mg/mL rat liver microsomes.
Permeability was determined using Caco-2 cells. Data are expressed as Papp (a to b)/Papp (b to a).
b
c
remaining structural elements unchanged. This modification (7a)
resulted in decrease in potency in both assays with an enzymatic
significant loss in cellular potency, compound 7a, at 5 mg/kg,
demonstrated potent in vivo efficacy in an acute Lewis rat model
with 54% plasma C16:1/C16:0 TG DI reduction 4 h post oral
administration.
IC50 of 20 nM in mouse liver microsomal assay, and
a
cellular IC50 of 268 nM in human HepG2 cell assay. Despite the

458
S. Sun et al. / Bioorg. Med. Chem. 23 (2015) 455–465
Based on the encouraging results observed with 7a, we pro-
Based on potency and favorable overall properties, the R1 pyri-
dyl substituents as found in compounds 7a and 7b were held con-
stant while modifications to the right hand side, R2, where
explored. R2 analogues with modified benzyl groups (7o, 7p) and
the 5-(trifluoromethyl)-furan-2-yl group (7q) displayed good
potency. The longer chain, (4-fluorophenoxy)ethyl in 7r was also
tolerated, but was less metabolically stable. Replacement of 4-flu-
orobenzyl with 1-(4-fluorophenyl)ethyl) group (7w) was also well
tolerated and retained good metabolic stability. Replacement of
the aryl group with an alkyl or a cycloalkyl group at R2, as in com-
pounds 7s, 7t and 7u, retained good inhibitory potency in both
assay systems. 7u was evaluated in the acute Lewis rat model
and demonstrated 50% plasma C16:1/C16:0 TG DI reduction at
5 mg/kg, 4 h post oral administration. Compound 7o, one of the
most potent compound R2 modifications, demonstrated similar
in vivo efficacy as compared to 7b, reducing plasma C16:1/C16:0
TG DI by 71% at 5 mg/kg, 4 h post oral administration.
To explore additional scaffold modifications, we replaced the
thiazole core with a pyrazole group. The pyrazolyl core introduces
a hydrogen bond donor, lowers clogP by 1.76 units (1.47 of 17a vs
3.23 of 7a), and increases tPSA to 102 from 90 of 7a, results in
decreased permeability. However, despite the low permeability
as assessed by a Caco2 assay, 17a displayed good in vitro potency,
and demonstrated reduction of the plasma C16:1/C16:0 TG DI by
54% at 5 mg/kg, 4 h post oral administration in Lewis rats. PK anal-
ysis indicated that compound 17a had good plasma exposure
ceeded with a systematic SAR investigation of the thiazolyltriazo-
lone core. Initially, we modified the left-hand side changing the
R1 group, while keeping the right hand side of the molecule, R2,
constant with the 4-fluorobenzyl group. The results of in vitro
activity, metabolic stability and permeability are summarized in
Table 1. In general, the modifications at R1 were well tolerated.
Both six-membered heterocycle analogues 7b, 7e, and five-mem-
bered heterocycle analogues 7f, 7g, 7h and 7i demonstrated better
or comparable inhibitory activity against SCD1. However, the five-
membered analogs 7g, 7h and 7i displayed undesired activity
against P450 CYP3A4 with more than 50% inhibition at 10 lM. A
CF3 substitution at the 6-position on pyridinyl ring was found to
be detrimental to SCD1 inhibition, as the activity of 7c dropped
by almost 80-fold in mouse SCD1 assay. Replacement of the pyrid-
inyl ring with a saturated piperidinyl moiety abolished the activity,
as observed in 7d. Truncation to the primary amide provided 7n
which was only about 3-fold less potent in the mouse SCD1 assay,
however a much more significant loss was observed in the HepG2
assay.
The in vivo efficacy of compounds 7b, 7e, 7f, and 7k on the
plasma C16:1/C16:0 TG DI reductions are shown in Figure 2. Con-
sistent with the potency in both in vitro assays, 7b and 7f were the
most efficacious compounds tested, as they reduced plasma C16:1/
C16:0 TG DI by more than 70% at 5 mg/kg, 4 h post oral administra-
tion. It was later found that 7f displayed undesired activity toward
pregnane X receptor (PXR), which induced CYP3A4 mRNA expres-
(AUC0–24 h = 6.0
lM h). 17a was readily absorbed in Lewis rats with
sion level in human hepatocytes by almost 10-fold at 50
lM.
a Cmax of 2.2 M. The oral terminal half-life of 17a was 4.0 h. Fol-
l
lowing intravenously administration with 1 mg/kg of 17a in Lewis
rats, the plasma concentration of 17a appeared to decline rapidly
with t of 1.3 h, and the plasma exposure was 2.3 lM h. As a result,
½
the oral bioavailability of 17a is 52% at 5 mg/kg in Lewis rats
(Tables 2 and 3).
100
80
Disappointingly, modifications of the left-hand side pyridin-4-
ylmethyl R1 of 17a with other heteroaryl groups resulted in signif-
icant loss in potency. For example, the pyridin-2-ylmethyl ana-
logue 17b is 3-fold less potent against mouse SCD1 enzyme and
7-fold less potent in human HepG2 cellular assay, while pyridin-
4-ylmethyl analogue 17c was more than 380-fold less potent
against mouse SCD1 enzyme. Other analogues generated from
replacement of pyridin-3-ylmethyl at R1 such as oxazol-4-ylmethyl
(17d), 1-methyl-1H-pyrazol-4-yl)methyl (17e), and methyl (17f)
were all less potent against mouse SCD1 enzyme. Thus, the pyri-
din-3-ylmethyl group appears to be essential for maintaining the
in vitro activity in this sub-series.
60
40
20
0
7a
7b
7e
7f
7k
7o
7p
7u
7v
17a
Compound
Figure 2. Effects of compounds on plasma C16:1/C16:0 TG desaturation index 4 h
after a 5 mg/kg oral dose in Lewis rats. Each bar represents the mean SEM (n = 4),
and the error bars represent standard errors of the mean.
To evaluate the mechanism-based AEs associated with SCD1
deficiency of SCD1 inhibitors, we developed a preclinical safety
Table 2
SAR of pyrazolyltriazolones
O
NH
N
N
H
N
N
R1
N
F
O
a
a
Compound
R1
Mouse SCD1 IC50 (nM)
HepG2 SCD1 IC50 (nM)
Metabolic stabilityb
Permeabilityc (Â10À6 cm/s)
17a
17b
17c
17d
17e
17f
Pyridin-3-ylmethyl
Pyridin-2-ylmethyl
Pyridin-4-ylmethyl
Oxazol-4-ylmethyl
1-Methyl-1H-pyrazol-4-yl)methyl
Methyl
7
23
2673
165
147
385
103
720
nd
nd
nd
66
58
nd
nd
nd
nd
12/19
nd
nd
nd
nd
nd
nd
nd: not determined.
a
IC50s are an average of at least two independent determinations; nd: not determined.
Expressed as % of compound remaining after a 30 minute incubation with 0.5 mg/mL rat liver microsomes.
Permeability was determined using Caco-2 cells. Data are expressed as Papp (a to b)/Papp (b to a).
b
c

S. Sun et al. / Bioorg. Med. Chem. 23 (2015) 455–465
459
Table 3
metabolic stability and permeability led to the identification of
17a, a potent, orally active SCD1 inhibitor, which demonstrated
good in vivo efficacy reducing plasma C16:1/C16:0 TG DI in an
acute Lewis rat model in a dose dependent manner, with an ED50
of 4.6 mg/kg. In preliminary safety studies, compound 17a did
not demonstrate adverse effects related to SCD1 inhibition, sug-
gesting that a safety margin was achieved as compared to previ-
ously identified SCD1 inhibitors. Further work including efforts
toward optimization of in vivo efficacy and safety, as well as phar-
macological evaluation in obesity and type 2 diabetes diseases
models, will be reported in due course.
Rat PK profiles for compound 17a
Parameter
Cl (L/h/kg)
IVa (1 mg/kg)
P.O.b (5 mg/kg)
1.1
1.3
3.3
t
(h)
4.0
2.3
0.50
½
Cmax
(
l
M)
Tmax (h)
Vss (L/kg)
AUC0–24
1.2
2.3
(
lM h)
6.0
52
h
F (%)
a
Average of two Lewis rats, intravenously dosed with 17a in 10% DMA, 10%
solutol, 50% PG and 30% water.
b
Average of four Lewis rats, orally dosed with 17a in 1% carboxymethyl cellulose
(low viscosity), 0.2% Tween 20 and 98.8% water.
5. Experimental section
5.1. General method
100
80
Chemicals, reagents and solvents were purchased from com-
mercial sources and were either used as supplied or purified using
reported methods. Final compounds reported herein exhibited
spectral data consistent with their proposed structure by nuclear
magnetic resonance spectra (1H NMR and 13C NMR) and mass
spectra data. NMR spectra were recorded on a Bruker Avance 300
spectrometer with chemical shifts (d) reported in parts-per-million
(ppm) relative to the residual signal of the deuterated solvent. 1H
NMR data are tabulated in the following order: multiplicity (s, sin-
glet; d, doublet; t, triplet; m, multiplet; br, broad), coupling con-
stants in Hertz and number of protons. Mass spectra were
obtained using a Waters 2795/ZQ LC/MS system (Waters Corpora-
tion, Milford, MA). Final compounds were greater than 95% pure as
determined by analytical HPLC on Agilent 1200 systems (Agilent
Technologies, Santa Clara, CA) using an EMD Chromolith SpeedROD
RP-18e column (4.6 mm i.d. Â 50 mm length) (Merck KGaA,
Darmstadt, Germany). The mobile phase consisted of a gradient
of component ‘A’ (0.1% v/v aqueous trifluoroacetic acid) and com-
ponent ‘B’ (acetonitrile) at a flow rate of 1 mL/min. The gradient
program used was as follows: initial conditions 5% B, hold at 5%
B for 1 min., linear ramp from 5% to 95% B over 5 min., 100% B
for 3 min., return to initial conditions for 1 min. Peaks were
detected at a wavelength of 254 nm with an Agilent photodiode
array detector. Melting points were determined on a Fisher-Johns
melting point apparatus and are uncorrected. Chemical names
were generated using ChemBioDraw version 12.0 (CambridgeSoft,
Cambridge, MA.).
60
50
40
30
20
ED50 = 4.6 0.9 mg/kg
0
2
4
6
8
10
12
Dose (mg/kg)
Figure 3. Dose response of compound 17a on plasma C16:1/C16:0 TG desaturation
index 4 h after oral dosing in Lewis rats. Each data point represents the mean SEM
(n = 4), and the error bars represent standard errors of the mean.
assessment screening model in rats.30 This model provides an
efficient means to assess the mechanism-based toxicity of SCD1
inhibitors at an early stage. Compounds 7a, 7b 7e, 7f, 7o, 7u, 7v,
and 17a were selected for evaluation in this model. Female Spra-
gue-Dawley rats, which were found to be particularly sensitive to
SCD1 AEs, were administered SCD1 inhibitors orally, once daily
at 100 mg/kg (ꢀ20-fold of the efficacious dose) for 10 consecutive
days. Rats were examined daily for general health and the specific
observations on eyes and skin. Most inhibitors manifested mecha-
nism-based toxicities, such as red eye, dry skin and hair loss as
early as day 3 of dosing with symptoms. We were encouraged by
the observation that 17a did not elicit any abnormalities related
to SCD inhibition or any other general health issue over the course
of this study. The plasma exposure (AUC0–24 h) of compound 17a on
5.1.1. Ethyl 2-(hydrazinecarboxamido)-4-methylthiazole-5-
carboxylate (3)
To a solution of 2 (100.0 g, 0.537 mol) and pyridine (74.0 mL,
0.913 mol) in anhydrous tetrahydrofuran (2.5 L) was added 4-
nitrophenyl chloroformate (140.7 g, 0.698 mol) in anhydrous tetra-
hydrofuran (0.5 L) dropwise at 0 °C over 40 min. The reaction mix-
ture was stirred for 2 h at 0 °C, then a solution of hydrazine
monohydrate (156 mL, 0.322 mol) in anhydrous tetrahydrofuran
(0.5 L) was added to the reaction mixture dropwise at 0 °C over
30 min. The cooling bath was removed and the reaction mixture
was stirred at ambient temperature for 2 h, then cooled to 0 °C.
The solid was collected by filtration, rinsed with methanol
(200 mL) and dried to afford 3 as a pale yellow solid (108.0 g,
82%). 1H NMR (300 MHz, DMSO-d6) d 7.24 (br s, 4H), 4.21 (t,
J = 7.1 Hz, 2H), 2.48 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H); MS (ES+) m/z
245.0 (M+1).
Day 11 was 207
obtained in the Lewis rat model (5 mg/kg, AUC0–24 h = 6.0
l
M h. Comparing this value to the exposure
lM h),
a greater than 34-fold window exists between efficacy and adverse
effects due to SCD1 inhibition in these models. Though sebaceous
and meibomian glands levels of 17a was not determined, the
improved AEs profile might indicate low exposure in these tissues
due to its favorable physicochemical properties.
Compound 17a was evaluated in the acute Lewis rat DI model in
a dose–responsive manner, using doses from 2 mg/kg to 10 mg/kg
and measuring effects at the 4 hour time point. The results
indicated a dose–responsive reduction of plasma TG DI with an
ED50 estimated to be about 4.6 mg/kg (Fig. 3).
4. Conclusion
5.1.2. Ethyl 4-methyl-2-(5-oxo-1H-1,2,4-triazol-4(5H)-
yl)thiazole-5-carboxylate (4)
In summary, we discovered a series of novel, potent triazolone-
based SCD1 inhibitors. SAR investigations addressing potency,
To a suspension of 3 (115.0 g, 0.471 mol) and trimethyl ortho-
formate (155.0 mL, 1.412 mol) in anhydrous ethanol (2.5 L) was

460
S. Sun et al. / Bioorg. Med. Chem. 23 (2015) 455–465
added TsOHÁH2O (1.0 g, 0.005 mol). The reaction mixture was
heated at reflux for 2 h, then cooled to 0 °C. The solid was collected
by filtration, rinsed with methanol (200 mL), and dried to afford 4
as an off-white solid (110.0 g, 91%). 1H NMR (300 MHz, DMSO-d6) d
12.94 (br s, 1H), 9.09 (s, 1H), 4.64 (t, J = 7.1 Hz, 2H), 2.98 (s, 3H),
1.67 (t, J = 7.1 Hz, 3H); MS (ES+) m/z 255.0 (M+1).
122.1, 120.9, 115.4 (d, JCF = 21 Hz), 47.7, 44.7, 16.9; MS (ES+) m/z
424.9 (M+1). Anal. Calcd for C20H17FN6O2S: C, 56.59; H, 4.04; N,
19.80. Found: C, 56.24; H, 4.09; N, 19.74.
5.1.7. 2-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-4-
methyl-N-((6-(trifluoromethyl)pyridin-3-yl)methyl)-thiazole-5-
carboxamide (7c)
To a solution of 6 (0.33 g, 1.00 mmol) and iPr2NEt (0.1 mL,
5.55 mmol) in anhydrous tetrahydrofuran (15 mL) was added
TBTU (0.64 g, 1.99 mmol) and HOBt (0.27 g, 1.99 mmol). After
stirring for 15 min at ambient temperature, a solution of 3-amino-
methyl-6-(trifluoromethyl)pyridine (0.53 g, 3.00 mmol) in anhy-
drous tetrahydrofuran (10 mL) was added to the reaction
mixture. After stirring at ambient temperature for 16 h, the reac-
tion mixture was quenched with a saturated NaHCO3 solution
(20 mL), and extracted with ethyl acetate (30 mL Â 3). The com-
bined organic layers were dried over anhydrous Na2SO4 and fil-
tered. The filtrate was concentrated in vacuo, and the residue
was purified by column chromatography eluting with 5% methanol
in dichloromethane to afford 7c as an off-white solid (0.17 g, 34%).
Mp 168–170 °C (dichloromethane/methanol); 1H NMR (300 MHz,
DMSO-d6) d 8.97 (t, J = 5.8 Hz, 1H), 8.78 (s, 1H), 8.74 (d, J = 1.3 Hz,
1H), 8.01 (dd, J = 8.1, 1.3 Hz, 1H), 7.90 (d, J = 8.1 Hz, 1H),
7.41–7.36 (m, 2H), 7.21–7.17 (m, 2H), 5.00 (s, 2H), 4.56–4.52 (m,
2H), 2.57 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d 162.2 (d,
JCF = 240 Hz), 161.7, 152.1, 152.0, 150.2, 149.9, 145.3 (t, JCF = 38 Hz),
139.4, 137.6, 132.7 (d, JCF = 8 Hz), 132.5, 130.5 (d, JCF = 8 Hz), 124.0,
122.3, 121.1, 115.9 (d, JCF = 23 Hz), 50.7, 48.2, 17.5; MS (ES+)
m/z 492.9 (M+1).
5.1.3. Ethyl 2-(1-(4-fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-
yl)-4-methylthiazole-5-carboxylate (5a)
To a solution of 4 (25.0 g, 98.4 mmol) and K2CO3 (20.4 g,
147.6 mmol) in acetone (800 mL) was added 4-fluorobenzyl benzyl
bromide (15.7 mL, 126.0 mmol). The reaction mixture was heated
at reflux for 5 h, then cooled to ambient temperature and filtered.
The filtrate was concentrated in vacuo and the residue was tritu-
rated in hexanes (200 mL). The solid was collected by filtration,
rinsed with water, and dried to afford 5a as an off-white solid
(30.0 g, 84%): 1H NMR (300 MHz, CDCl3) d 8.27 (s, 1H), 7.47–7.30
(m, 2H), 7.11–6.97 (m, 2H), 4.98 (s, 2H), 4.32 (q, J = 7.1 Hz, 2H),
2.65 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H); MS (ES+) m/z 363.1 (M+1).
5.1.4. 2-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-4-
methylthiazole-5-carboxylic acid (6a)
To a solution of 5 (30.0 g, 82.85 mmol) in ethanol (330 mL), and
water (170 mL) was added NaOH (6.96 g, 174.0 mmol) at ambient
temperature. The resulting reaction mixture was heated at reflux
for 1 h, and then concentrated in vacuo to remove most of the
organic volatiles. The residue was neutralized to pH 4–5 with
10% hydrochloric acid, and the resulting precipitate was collected
by filtration, rinsed with water then diethyl ether and dried to
afford 6a as an off-white solid (20.21 g, 73%). 1H NMR (300 MHz,
DMSO-d6) d 13.40 (br s, 1H), 8.74 (s, 1H), 7.37–7.31 (m, 2H),
7.19–7.11 (m, 2H), 4.96 (s, 2H), 2.57 (s, 3H); MS (ESÀ) m/z 333.0
(MÀ1).
5.1.8. tert-Butyl 3-((2-(1-(4-fluorobenzyl)-5-oxo-1H-1,2,4-
triazol-4(5H)-yl)-4-methylthiazole-5-carboxamido)methyl)-
piperidine-1-carboxylate (7dd)
By a similar procedure as described for 7c, 7dd was obtained as
an off-white solid (0.82 g, 77%), which was used for next step with-
out further purification. MS (ES+) m/z 531.1 (M+1), 431.1 (MÀ100).
5.1.5. 2-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-4-
methyl-N-(pyridin-3-ylmethyl)thiazole-5-carboxamide (7a)
To a solution of 6a (2.00 g, 5.98 mmol), EDCI (1.50 g, 7.82 mmol)
and iPr2NEt (2.67 mL, 15.64 mmol) in N,N-dimethylformamide
(80 mL) was added HOBt (1.00 g, 7.40 mmol). The reaction mixture
was stirred at ambient temperature for 15 min, followed by addi-
tion of 3-(aminomethyl)pyridine (0.73 mL, 7.16 mmol). After stir-
ring for 17 h at ambient temperature, the reaction mixture was
diluted with ethyl acetate (300 mL) and sequentially washed with
water, saturated NaHCO3 solution, water and brine. The organic
solution was dried over anhydrous Na2SO4 and filtered. The filtrate
was concentrated in vacuo, and the residue was crystallized from
ethyl acetate and hexanes to yield 7a as an off-white solid
(1.93 g, 76%), mp 178–180 °C (ethyl acetate/hexanes); 1H NMR
(300 MHz, CDCl3) d 8.59 (br s, 2H), 8.23 (s, 1H), 7.80–7.71 (m,
1H), 7.44–7.27 (m, 2H), 7.08–6.96 (m, 2H), 6.42 (t, J = 5.8 Hz, 1H),
6.46 (t, J = 7.4 Hz, 1H), 4.96 (s, 2H), 4.62 (d, J = 5.8 Hz, 2H), 2.64
(s, 3H); 13C NMR (75 MHz, CDCl3) d 162.7 (d, JCF = 244 Hz), 161.7,
161.0, 159.6, 153.4, 151.0, 149.9, 136.1, 131.0, 130.8 (d, JCF = 3 Hz),
130.3 (d, JCF = 8 Hz), 121.6, 119.1, 115.8 (d, JCF = 22 Hz), 48.9, 41.6,
17.2; MS (ES+) m/z 425.3 (M+1). Anal. Calcd for C20H17FN6O2S: C,
56.59; H, 4.04; N, 19.80. Found: C, 56.24; H, 4.01; N, 19.55.
5.1.9. 2-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-4-
methyl-N-(piperidin-3-ylmethyl)thiazole-5-carboxamide
trifluoroacetic acid salt (7d)
To a solution of 7dd (0.82 g, 1.55 mmol) in dichloromethane
(10 mL) was added trifluoroacetic acid (4.0 mL). The reaction mix-
ture was stirred at ambient temperature for 16 h, then concentrated
in vacuo. The residue was crystallized from ethanol and diethyl
ether to yield 7d as an off-white solid (0.43 g, 65%). Mp 137–
138 °C (ethanol/diethyl ether); 1H NMR (300 MHz, DMSO-d6) d
8.96–8.77 (m, 1H), 8.72 (s, 1H), 8.64–8.46 (m, 1H), 8.41 (t,
J = 5.7 Hz, 1H), 7.37–7.31 (m, 2H), 7.18–7.12 (m, 2H), 4.97 (s, 2H),
3.27–3.03 (m, 4H), 2.64–2.42 (m, 5H), 2.04–1.84 (m, 1H), 1.79–
1.71 (m, 2H), 1.62–1.45 (m, 1H), 1.24–1.11 (m, 1H); MS (ES+) m/z
431.1 (M+1).
5.1.10. 2-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
4-methyl-N-((5-methylpyrazin-2-yl)methyl)thiazole-5-
carboxamide (7e)
By a similar procedure as described for 7a, 7e was obtained as
an off-white solid (1.90 g, 58%). Mp 188–189 °C (ethyl acetate/hex-
anes); 1H NMR (300 MHz, DMSO-d6) d 8.92 (t, J = 5.7 Hz, 1H), 8.77
(s, 1H), 8.50–8.49 (m, 2H), 7.41–7.36 (m, 2H), 7.22–7.16 (m, 2H),
5.00 (s, 2H), 4.53 (d, J = 5.7 Hz, 2H), 2.57 (s, 3H), 2.47 (s, 3H); 13C
NMR (75 MHz, DMSO-d6) d 161.7 (d, JCF = 244 Hz), 161.1, 151.8,
151.4, 151.3, 150.5, 149.7, 143.3, 142.1, 132.2 (d, JCF = 3 Hz),
132.0, 130.0 (d, JCF = 8 Hz), 122.2, 115.5, 115.2, 47.7, 42.5, 20.7,
16.9; MS (ES+) m/z 440.2 (M+1). Anal. Calcd for C20H18FN7O2S: C,
54.66; H, 4.13; N, 22.31. Found: C, 54.47; H, 4.11; N, 22.33.
5.1.6. 2-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-4-
methyl-N-(pyridin-2-ylmethyl)thiazole-5-carboxamide (7b)
By a similar procedure described for 7a, 7b was obtained as an
off-white solid (2.36 g, 46%). Mp 174–175 °C (ethanol); 1H NMR
(300 MHz, DMSO-d6) d 8.89 (t, J = 5.8 Hz, 1H), 8.78 (s, 1H), 8.53–
8.51 (m, 1H), 7.80–7.75 (m, 1H), 7.41–7.16 (m, 6H), 5.01 (s, 2H),
4.53 (d, J = 5.8 Hz, 2H), 2.59 (s, 3H); 13C NMR (75 MHz, DMSO-d6)
d 161.7 (d, JCF = 244 Hz), 161.1, 158.2, 151.4, 151.1, 149.7, 148.8,
136.7, 132.2 (d, JCF = 3 Hz), 132.0, 130.0 (d, JCF = 8 Hz), 122.4,

S. Sun et al. / Bioorg. Med. Chem. 23 (2015) 455–465
461
5.1.11. 2-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
4-methyl-N-(oxazol-4-ylmethyl)thiazole-5-carboxamide (7f)
By a similar procedure as described for 7a, 7f was obtained as
an off-white solid (1.98 g, 51%). Mp 183–184 °C (ethyl acetate/hex-
anes); 1H NMR (300 MHz, DMSO-d6) d 8.79–8.76 (m, 2H), 8.34 (s,
1H), 7.98 (s, 1H), 7.41–7.36 (m, 2H), 7.22–7.17 (m, 2H), 5.00 (s,
2H), 4.33 (d, J = 5.4 Hz, 2H), 2.55 (s, 3H); 13C NMR (75 MHz,
DMSO-d6) d 162.1 (d, JCF = 243 Hz), 160.9, 152.0, 151.3, 151.0,
149.7, 137.4, 136.1, 132.2 (d, JCF = 3 Hz), 132.0, 129.9 (d, JCF = 8 Hz),
122.4, 115.4 (d, JCF = 21 Hz), 47.7, 35.2, 16.9; MS (ES+) m/z 415.3
(M+1). Anal. Calcd for C18H15FN6O3S: C, 52.17; H, 3.65; N, 20.28.
Found: C, 52.12; H, 3.60; N, 20.54.
47.7, 37.3, 16.9, 10.4; MS (ES+) m/z 428.0 (M+1); Anal. Calcd for
C19H18FN7O2S: C, 53.39; H, 4.24; N, 22.94. Found: C, 53.47; H,
4.28; N, 22.82.
5.1.17. 2-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
4-methyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)thiazole-5-
carboxamide (7l)
By a similar procedure as described for 7a, 7l was obtained as an
off-white solid (2.55 g, 62%). Mp 205–206 °C (ethyl acetate/hex-
anes); 1H NMR (300 MHz, DMSO-d6) d 8.76 (s, 1H), 8.66 (t,
J = 5.6 Hz, 1H), 7.60 (s, 1H), 7.40–7.45 (m, 3H), 7.22–7.17 (m, 2H),
5.00 (s, 2H), 4.23 (d, J = 5.6 Hz, 2H), 3.78 (s, 3H), 2.55 (s, 3H); 13C
NMR (75 MHz, DMSO-d6) d 161.7 (d, JCF = 244 Hz), 160.6, 151.2,
150.7, 149.7, 138.0, 132.2 (d, JCF = 3 Hz), 132.0, 130.0 (d, JCF = 8 Hz),
129.5, 122.6, 118.6, 115.4 (d, JCF = 21 Hz), 47.7, 38.4, 33.6, 16.9; MS
(ES+) m/z 428.1 (M+1); Anal. Calcd for C19H18FN7O2S: C, 53.39; H,
4.24; N, 22.94. Found: C, 53.41; H, 4.26; N, 23.28.
5.1.12. 2-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
4-methyl-N-(oxazol-2-ylmethyl)thiazole-5-carboxamide (7g)
By a similar procedure as described for 7a, 7g was obtained as an
off-white solid (0.14 g, 57%). Mp 152–153 °C (ethyl acetate/hex-
anes); 1H NMR (300 MHz, CDCl3) d 8.26 (s, 1H), 7.65 (s, 1H), 7.41–
7.32 (m, 2H), 7.09 (s, 1H), 7.07–6.98 (m, 2H), 6.54 (t, J = 5.2 Hz,
1H), 4.98 (s, 2H), 4.73 (d, J = 5.2 Hz, 2H), 2.67 (s, 3H); MS (ES+) m/z
415.1 (M+1).
5.1.18. 2-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
4-methyl-N-((1-methyl-1H-imidazol-4-yl)methyl)thiazole-5-
carboxamide (7m)
By a similar procedure as described for 7a, 7m was obtained as
an off-white solid (2.01 g, 57%). Mp 225–226 °C (ethyl acetate/hex-
anes); 1H NMR (300 MHz, DMSO-d6) d 8.76 (s, 1H), 8.65–8.60 (m,
1H), 7.49 (s, 1H), 7.41–7.35 (m, 2H), 7.22–7.17 (m, 2H), 6.96 (s,
1H), 5.00 (s, 2H), 4.27 (d, J = 5.6 Hz, 2H), 3.60 (s, 3H), 2.55 (s,
3H); 13C NMR (75 MHz, DMSO-d6) d 161.7 (d, JCF = 244 Hz), 160.7,
151.2, 150.4, 149.7, 138.9, 137.2, 132.2 (d, JCF = 3 Hz), 132.0,
130.0 (d, JCF = 84 Hz), 122.9, 117.6, 115.4 (d, JCF = 22 Hz), 47.7,
37.4, 32.8, 16.9; MS (ES+) m/z 428.2 (M+1). Anal. Calcd for C19H18
FN7O2S: C, 53.39; H, 4.24; N, 22.94. Found: C, 53.50; H, 4.11; N,
22.89.
5.1.13. 2-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
4-methyl-N-(thiazol-2-ylmethyl)thiazole-5-carboxamide (7h)
By a similar procedure as described for 7a, 7h was obtained as
an off-white solid (0.17 g, 66%). Mp 189–190 °C (ethyl acetate/hex-
anes); 1H NMR (300 MHz, CDCl3) d 8.25 (s, 1H), 7.74 (br s, 1H),
7.40–7.31 (m, 3H), 7.06–6.97 (m, 2H), 6.78 (t, J = 5.4 Hz, 1H), 4.97
(s, 2H), 4.92 (d, J = 5.4 Hz, 2H), 2.67 (s, 3H); MS (ES+) m/z 431.1
(M+1).
5.1.14. 2-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
4-methyl-N-((2-methylthiazol-5-yl)methyl)thiazole-5-
carboxamide (7i)
By a similar procedure as described for 7a, 7i was obtained as an
off-white solid (0.18 g, 68%). Mp 176–177 °C (ethyl acetate/hex-
anes); 1H NMR (300 MHz, CDCl3) d 8.25 (s, 1H), 7.41–7.31 (m,
2H), 7.07–6.97 (m, 3H), 6.49 (t, J = 5.3 Hz, 1H), 4.97 (s, 2H), 4.62
(d, J = 5.3 Hz, 2H), 2.70 (s, 3H), 2.64 (s, 3H); MS (ES+) m/z 445.1
(M+1).
5.1.19. 2-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
4-methylthiazole-5-carboxamide (7n)
i
To a solution of 6 (0.35 g, 1.05 mmol) and Pr2NEt (1.09 mL,
6.28 mmol) in anhydrous N,N-dimethylformamide (10 mL) was
added HOBt (0.28 g, 2.09 mmol), HATU (0.80 g, 2.09 mmol), and
NH4Cl (0.22 g, 4.19 mmol). The resulting reaction mixture was
stirred at ambient temperature for 72 h then concentrated in
vacuo. The residue was triturated with saturated NaHCO3 solution
(100 mL). The crude product was collected by filtration, rinsed
with water and then recrystallization in ethanol to afford 7n as
an off-white solid (0.27 g, 73%). Mp 232–233 °C (ethanol); 1H
5.1.15. N-((1H-Pyrazol-3-yl)methyl)-2-(1-(4-fluorobenzyl)-5-
oxo-1H-1,2,4-triazol-4(5H)-yl)-4-methylthiazole-5-
carboxamide (7j)
By a similar procedure as described for 7a, 7j was obtained as an
off-white solid (1.77 g, 31%). Mp 181–182 °C (ethyl acetate); 1H
NMR (300 MHz, DMSO-d6) d 12.63 (br s, 1H), 8.76–8.72 (m, 2H),
7.62 (s, 1H), 7.41–7.36 (m, 2H), 7.22–7.17 (m, 2H), 6.17 (d,
J = 1.8 Hz, 1H), 5.00 (s, 2H), 4.42 (d, J = 5.6 Hz, 2H), 2.56 (s, 3H);
13C NMR (75 MHz, DMSO-d6) d 161.7 (d, JCF = 244 Hz), 160.8,
151.3, 150.7, 149.7, 132.2 (d, JCF = 3 Hz), 132.1, 130.0 (d, JCF = 8 Hz),
129.1, 122.8, 115.4 (d, JCF = 22 Hz), 103.1, 47.7, 37.1, 16.9; MS (ES+)
m/z 413.8 (M+1); Anal. Calcd for C18H16FN7O2S: C, 52.29; H, 3.90;
N, 23.72. Found: C, 51.78; H, 3.94; N, 23.23.
NMR (300 MHz, DMSO-d6)
d 8.75 (s, 1H), 7.64 (br s, 2H),
7.40–7.36 (m, 2H), 7.22–7.16 (m, 2H), 5.00 (s, 2H), 2.55
(s, 3H); 13C NMR (75 MHz, DMSO-d6) d 162.7, 161.69 (d, JCF = 244
Hz), 151.3, 150.9, 149.7, 132.2 (d, JCF = 3 Hz), 132.0, 129.9 (d,
JCF = 8 Hz), 123.0, 115.4 (d, JCF = 22 Hz), 47.7, 16.8; MS (ES+) m/z
334.3 (M+1).
5.1.20. Ethyl 4-methyl-2-(5-oxo-1-(4-(trifluoromethyl)-benzyl)-
1H-1,2,4-triazol-4(5H)-yl)thiazole-5-carboxylate (5b)
By a similar procedure as described for 5a, 5b was obtained as
an off-white solid (16.0 g, 99%). 1H NMR (300 MHz, CDCl3) d 8.29
(s, 1H), 7.61 (d, J = 8.3 Hz, 2H), 7.50 (d, J = 8.3 Hz, 2H), 5.07 (s,
2H), 4.32 (q, J = 7.1 Hz, 2H), 2.67 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H);
MS (ES+) m/z 413.2 (M+1).
5.1.16. 2-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
4-methyl-N-((3-methyl-1H-pyrazol-5-yl)methyl)-thiazole-5-
carboxamide (7k)
By a similar procedure as described for 7a, 7k was obtained as
an off-white solid (1.90 g, 35%). Mp 237–238 °C (N,N-dimethyl-
formamide/water); 1H NMR (300 MHz, DMSO-d6) d 12.25 (br s,
1H), 8.76 (s, 1H), 8.67 (t, J = 5.5 Hz, 1H), 7.41–7.16 (m, 4H), 5.91
(s, 1H), 5.00 (s, 2H), 4.33 (d, J = 5.5 Hz, 2H), 2.55 (s, 3H), 2.17
(s, 3H); 13C NMR (75 MHz, DMSO-d6) d 161.7 (d, JCF = 244 Hz),
160.7, 151.2, 150.5, 149.7, 149.6, 138.7, 132.2 (d, JCF = 3 Hz),
132.1, 130.0 (d, JCF = 8 Hz), 122.9, 115.4 (d, JCF = 22 Hz), 102.3,
5.1.21. 4-Methyl-2-(5-oxo-1-(4-(trifluoromethyl)benzyl)-1H-
1,2,4-triazol-4(5H)-yl)thiazole-5-carboxylic acid (6b)
By a similar procedure as described for 6a, 6b was obtained as
an off-white solid (11.8 g, 80%). 1H NMR (300 MHz, DMSO-d6) d
13.43 (br s, 1H), 8.82 (s, 1H), 7.75–7.72 (m, 2H), 7.57–7.54 (m,
2H), 5.13 (s, 2H), 2.61 (s, 3H); MS (ES+) m/z 385.2 (M+1).

462
S. Sun et al. / Bioorg. Med. Chem. 23 (2015) 455–465
5.1.22. 4-Methyl-2-(5-oxo-1-(4-(trifluoromethyl)benzyl)-1H-
1,2,4-triazol-4(5H)-yl)-N-(pyridin-3-ylmethyl)thiazole-5-
carboxamide (7o)
(d, JCF = 41 Hz), 135.7, 131.4, 123.7 (q, JCF = 2 Hz), 122.1(d,
JCF = 244 Hz), 116.9, 122.5 (q, JCF = 3 Hz), 110.4, 42.1, 41.6, 17.2;
MS (ES+) m/z 465.3 (M+1).
By a similar procedure as described for 7a, 7o was obtained as
an off-white solid (2.95 g, 79%). Mp 168–169 °C (ethyl acetate/hex-
anes). 1H NMR (300 MHz, CDCl3) d 8.58 (s, 1H), 8.55 (s, 1H), 8.30–
8.27 (m, 1H), 7.73–7.68 (m, 1H), 7.65–7.58 (m, 2H), 7.53–7.46 (m,
2H), 7.42–7.23 (m, 1H), 6.21 (s, 1H), 5.07 (s, 2H), 4.63–4.60 (m, 2H),
2.65 (s, 3H); 13C NMR (75 MHz, CDCl3) d 161.6, 153.3, 151.0, 149.9,
149.3, 138.9, 135.7, 131.2, 128.7, 125.9 (q, JCF = 112 Hz), 125.7,
123.9 (d, JCF = 271 Hz), 123.8, 122.9, 121.8, 49.1, 41.6, 17.2; MS
(ES+) m/z 475.3 (M+1). Anal. Calcd for C21H17F3N6O2S: C, 53.16;
H, 3.61; N, 17.71. Found: C, 52.94; H, 3.71; N, 17.52.
5.1.29. Ethyl 2-(1-(2-(4-fluorophenoxy)ethyl)-5-oxo-1H-1,2,4-
triazol-4(5H)-yl)-4-methylthiazole-5-carboxylate (5e)
By a similar procedure as described for 5a, 5e was obtained as
an off-white solid (0.11 g, 48%) by column chromatography eluting
with 10–50%
a
gradient ethyl acetate in hexanes. 1H NMR
(300 MHz, CDCl3) d 8.26 (s, 1H), 6.93–6.84 (m, 2H), 6.81–6.75 (m,
2H), 4.32–4.15 (m, 6H), 2.62 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H); MS
(ES+) m/z 393.3 (M+1).
5.1.30. 2-(1-(2-(4-Fluorophenoxy)ethyl)-5-oxo-1H-1,2,4-triazol-
4(5H)-yl)-4-methylthiazole-5-carboxylic acid (6e)
By a similar procedure as described for 6a, 6e was obtained as
an off-white solid (0.08 g, 78%). MS (ESÀ) m/z 363.1 (MÀ1).
5.1.23. Ethyl 2-(1-(3,5-difluorobenzyl)-5-oxo-1H-1,2,4-triazol-
4(5H)-yl)-4-methylthiazole-5-carboxylate (5c)
By a similar procedure as described for 5a, 5c was obtained as
an off-white solid (7.0 g, 93%). 1H NMR (300 MHz, DMSO-d6) d
8.82 (s, 1H), 7.23–7.16 (m, 1H), 7.12–7.05 (m, 2H), 5.06 (s, 2H),
4.30 (q, J = 7.1 Hz, 2H), 2.64 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H).
5.1.31. 2-(1-(2-(4-Fluorophenoxy)ethyl)-5-oxo-1H-1,2,4-triazol-
4(5H)-yl)-4-methyl-N-(pyridin-3-ylmethyl)thiazole-5-
carboxamide (7r)
5.1.24. 2-(1-(3,5-Difluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-
yl)-4-methylthiazole-5-carboxylic acid (6c)
By a similar procedure as described for 6a, 6c was obtained as
an off-white solid (4.66 g, 73%). 1H NMR (300 MHz, DMSO-d6) d
13.42 (br s, 1H), 8.79 (s, 1H), 7.22–7.06 (m, 3H), 5.06 (s, 2H), 2.61
(s, 3H).
By a similar procedure as described for 7a, 7r was obtained as
an off-white solid (0.03 g, 45%). 1H NMR (300 MHz, CDCl3) d 8.58
(br s, 1H), 8.53–8.45 (m, 1H), 8.26 (s, 1H), 7.72–7.65 (m, 1H),
7.30–7.21 (m, 1H), 6.98–6.86 (m, 2H), 6.85–6.75 (m, 2H), 6.61 (t,
J = 5.8 Hz, 1H), 4.59 (d, J = 5.8 Hz, 2H), 4.28–4.15 (m, 4H), 2.63 (s,
3H); 13C NMR (75 MHz, CDCl3) d 161.7, 157.6 (d, JCF = 239 Hz),
154.2, 153.3, 150.6 (d, JCF = 59 Hz), 149.2 (d, JCF = 16 Hz), 135.8,
133.6, 131.0, 123.7, 121.6, 116.0, 115.9, 115.8, 115.7, 65.5, 45.3,
41.6, 17.2; MS (ES+) m/z 455.3 (M+1).
5.1.25. 2-(1-(3,5-Difluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-
yl)-4-methyl-N-(pyridin-3-ylmethyl)thiazole-5-carboxamide
(7p)
By a similar procedure as described for 7a, 7p was obtained as
an off-white solid (0.10 g, 80%). Mp 194–196 °C (ethyl acetate/hex-
anes); 1H NMR (300 MHz, DMSO-d6) d 8.91 (t, J = 5.8 Hz, 1H), 8.79
(s, 1H), 8.55 (d, J = 1.9 Hz, 1H), 8.41–8.38 (m, 1H), 7.76–7.70 (m,
1H), 7.42–7.35 (m, 1H), 7.23–7.07 (m, 3H), 5.06 (s, 2H), 4.44 (d,
J = 5.8 Hz, 2H), 2.58 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d 162.4
(dd, JCF = 246 Hz, 13 Hz), 161.1, 151.5, 151.3, 149.9, 148.9, 148.1,
140.5 (dd, JCF = 9 Hz, 9 Hz), 135.2, 134.7, 132.4, 123.5, 122.0,
110.9–110.6 (m), 103.2 (dd, JCF = 26 Hz, 26 Hz), 47.4, 40.5, 16.9;
MS (ES+) m/z 442.8 (M+1).
5.1.32. Ethyl 2-(1-(cyclopropylmethyl)-5-oxo-1H-1,2,4-triazol-
4(5H)-yl)-4-methylthiazole-5-carboxylate (5f)
By a similar procedure as described for 5a, 5f was obtained as a
off-white solid (2.47 g, 99%). 1H NMR (300 MHz, CDCl3) d 8.27 (s,
1H), 4.31 (q, J = 7.1 Hz, 2H), 3.63 (d, J = 7.1 Hz, 2H), 2.64 (s, 3H),
1.35 (t, J = 7.1 Hz, 3H), 1.19–1.04 (m, 1H), 0.53–0.42 (m, 2H),
0.36–0.27 (m, 2H); MS (ES+) m/z 309.2 (M+1).
5.1.33. 2-(1-(Cyclopropylmethyl)-5-oxo-1H-1,2,4-triazol-4(5H)-
yl)-4-methylthiazole-5-carboxylic acid (6f)
By a similar procedure as described for 6a, 6f was obtained as a
off-white solid (2.1 g, 95%). 1H NMR (300 MHz, DMSO-d6) d 13.39
(br s, 1H), 8.72 (s, 1H), 3.63 (d, J = 7.1 Hz, 2H), 2.57 (s, 3H), 1.19–
1.04 (m, 1H), 0.53–0.42 (m, 2H), 0.36–0.27 (m, 2H); MS (ESÀ)
m/z 279.0 (MÀ1).
5.1.26. Ethyl 4-methyl-2-(5-oxo-1-((5-(trifluoromethyl)-furan-
2-yl)methyl)-1H-1,2,4-triazol-4(5H)-yl)thiazole-5-carboxylate
(5d)
By a similar procedure as described for 5a, 5d was obtained as
an off-white solid (0.31 g, 56%). 1H NMR (300 MHz, CDCl3) d 8.26
(s, 1H), 6.69–6.62 (m, 1H), 6.29–6.20 (m, 1H), 5.03 (s, 2H), 4.32
(q, J = 7.0 Hz, 2H), 2.64 (s, 3H), 1.35 (t, J = 7.0 Hz, 3H); MS (ES+)
m/z 403.3 (M+1).
5.1.34. 2-(1-(Cyclopropylmethyl)-5-oxo-1H-1,2,4-triazol-4(5H)-
yl)-4-methyl-N-(pyridin-3-ylmethyl)thiazole-5-carboxamide
(7s)
By a similar procedure as described for 7a, 7s was obtained as
an off-white solid (2.38 g, 89%). Mp 136–137 °C (ethyl acetate/hex-
anes); 1H NMR (300 MHz, CDCl3) d 8.57 (br s, 1H), 8.47 (br s, 1H),
8.24 (s, 1H), 7.79–7.71 (m, 1H), 7.44–7.27 (m, 1H), 6.38 (t,
J = 5.8 Hz, 1H), 4.62 (d, J = 5.8 Hz, 2H), 3.70 (d, J = 7.2 Hz, 2H),
2.65 (s, 3H), 1.34–1.14 (m, 1H), 0.63–0.52 (m, 2H), 0.46–0.31 (m,
2H); 13C NMR (75 MHz, CDCl3) d 161.8, 153.4, 151.2, 149.9,
149.2, 148.9, 135.8, 133.7, 130.5, 123.8, 121.4, 117.9, 50.6, 41.6,
17.2, 10.2, 3.7; MS (ES+) m/z 371.3 (M+1). Anal. Calcd for C17H18N6
O2S: C, 55.12; H, 4.90; N, 22.69. Found: C, 55.05; H, 4.90; N, 22.99.
5.1.27. 4-Methyl-2-(5-oxo-1-((5-(trifluoromethyl)-furan-2-
yl)methyl)-1H-1,2,4-triazol-4(5H)-yl)thiazole-5-carboxylic acid
(6d)
By a similar procedure as described for 6a, 6d was obtained as
an off-white solid (0.2 g, 84%). MS (ESÀ) m/z 373.0 (MÀ1).
5.1.28. 4-Methyl-2-(5-oxo-1-((5-(trifluoromethyl)furan-2-
yl)methyl)-1H-1,2,4-triazol-4(5H)-yl)-N-(pyridin-3-
ylmethyl)thiazole-5-carboxamide (7q)
By a similar procedure as described for 7a, 7q was obtained as
an off-white solid (0.16 g, 57%). Mp 124–125 °C (ethyl acetate/hex-
anes); 1H NMR (300 MHz, CDCl3) d 8.63 (s, 2H), 8.28 (s, 1H), 7.82–
7.75 (m, 1H), 7.36 (s, 1H), 6.77–6.72 (m, 1H), 6.52 (t, J = 5.5 Hz, 1H),
5.1.35. Ethyl 2-(1-((2,2-difluorocyclopropyl)methyl)-5-oxo-1H-
1,2,4-triazol-4(5H)-yl)-4-methylthiazole-5-carboxylate (5g)
By a similar procedure as described for 5a, 5g was obtained as
an off-white solid (10.8 g, 98%). 1H NMR (300 MHz, DMSO-d6) d
8.74 (s, 1H), 4.23 (t, J = 7.1 Hz, 2H), 3.97–3.77 (m, 2H), 2.54 (s,
6.47–6.42 (m, 1H), 5.04 (s, 2H), 4.66–4.60 (m, 2H), 2.64 (s, 3H); 13
C
NMR (75 MHz, CDCl3) d 161.6, 153.3, 150.8 (d, JCF = 28 Hz), 149.2

S. Sun et al. / Bioorg. Med. Chem. 23 (2015) 455–465
463
3H), 2.20–2.04 (m, 1H), 1.73–1.60 (m, 1H), 1.48–1.37 (m, 1H), 1.26
(t, J = 7.1 Hz, 3H); MS (ES+) m/z 344.9 (M+1).
474.8 (M+1). Anal. Calcd for C21H17F3N6O2S: C, 53.16; H, 3.61; N,
17.71. Found: C, 53.19; H, 3.66; N, 17.67.
5.1.36. 2-(1-((2,2-Difluorocyclopropyl)methyl)-5-oxo-1H-1,2,4-
triazol-4(5H)-yl)-4-methylthiazole-5-carboxylic acid (6g)
By a similar procedure as described for 6a, 6g was obtained as
an off-white solid (1.21 g, 80%). 1H NMR (300 MHz, DMSO-d6) d
13.43 (br s, 1H), 8.74 (s, 1H), 3.97–3.77 (m, 2H), 2.54 (s, 3H),
2.20–2.04 (m, 1H), 1.73–1.60 (m, 1H), 1.48–1.37 (m, 1H); MS
(ES+) m/z 317.1 (M+1).
5.1.42. Ethyl 2-(1-(1-(4-fluorophenyl)ethyl)-5-oxo-1H-1,2,4-
triazol-4(5H)-yl)-4-methylthiazole-5-carboxylate (5i)
By a similar procedure as described for 5a, 5i was obtained as an
off-white solid (6.30 g, 69%). 1H NMR (300 MHz, DMSO-d6) d 8.81
(s, 1H), 7.45–7.40 (m, 2H), 7.21–7.16 (m, 2H), 5.49 (q, J = 7.1 Hz,
1H), 4.28 (q, J = 7.1 Hz, 2H), 2.61 (s, 3H), 1.71 (d, J = 7.1 Hz, 3H),
1.29 (t, J = 7.1 Hz, 3H); MS (ES+) m/z 377.1 (M+1).
5.1.37. 2-(1-((2,2-Difluorocyclopropyl)methyl)-5-oxo-1H-1,2,4-
triazol-4(5H)-yl)-4-methyl-N-(pyridin-3-ylmethyl)thiazole-5-
carboxamide (7t)
5.1.43. 2-(1-(1-(4-Fluorophenyl)ethyl)-5-oxo-1H-1,2,4-triazol-
4(5H)-yl)-4-methylthiazole-5-carboxylic acid (6i)
By a similar procedure as described for 6a, 6i was obtained as an
off-white solid (0.56 g, 78%). 1H NMR (300 MHz, DMSO-d6) d 13.43
(br s, 1H), 8.80 (s, 1H), 7.45–7.40 (m, 2H), 7.21–7.16 (m, 2H), 5.49
(q, J = 7.1 Hz, 1H), 2.60 (s, 3H), 1.70 (d, J = 7.1 Hz, 3H); MS (ES+) m/z
349.0 (M+1).
By a similar procedure as described for 7a, 7t was obtained as
an off-white solid (2.90 g, 90%). Mp 120–122 °C (ethanol/diethyl
ether); 1H NMR (300 MHz, DMSO-d6) d 8.87 (t, J = 5.8 Hz, 1H),
8.75 (s, 1H), 8.51 (s, 1H), 8.43 (d, J = 3.7 Hz, 1H), 7.69 (d,
J = 7.9 Hz, 1H), 7.33 (dd, J = 7.8, 4.8 Hz, 1H), 4.44 (d, J = 5.8 Hz,
2H), 4.01–3.83 (m, 2H), 2.57 (s, 3H), 2.24–2.08 (m, 1H), 1.77–1.63
(m, 1H), 1.52–1.40 (m, 1H); 13C NMR (75 MHz, DMSO-d6) d 160.9,
151.3, 151.2, 149.4, 148.8, 148.0, 135.1, 131.8, 123.4, 122.0, 113.9
(t, JCF = 285 Hz), 42.2, 42.1, 20.2 (t, JCF = 8 Hz), 16.8, 14.4 (t, JCF = 8
Hz); MS (ES+) m/z 406.8 (M+1); Anal. Calcd for C17H16F2N6O2S: C,
50.24; H, 3.97; N, 20.68. Found: C, 50.41; H, 4.03; N, 20.44.
5.1.44. 2-(1-(1-(4-Fluorophenyl)ethyl)-5-oxo-1H-1,2,4-triazol-
4(5H)-yl)-4-methyl-N-(pyridin-2-ylmethyl)thiazole-5-
carboxamide (7w)
By a similar procedure as described for 7a, 7w was obtained as
an off-white solid (2.35 g, 75%). Mp 169–170 °C (tetrahedrofuran/
diethyl ether); 1H NMR (300 MHz, DMSO-d6) d 8.87 (t, J = 5.8 Hz,
1H), 8.80 (s, 1H), 8.50 (d, J = 4.7 Hz, 1H), 7.81–7.75 (m, 1H), 7.45–
7.40 (m, 2H), 7.33 (d, J = 7.9 Hz, 1H), 7.27 (dd, J = 7.2, 5.0 Hz, 1H),
7.22–7.17 (m, 2H), 5.50 (q, J = 6.9 Hz, 1H), 4.52 (d, J = 5.8 Hz, 2H),
2.59 (s, 3H), 1.70 (d, J = 7.1 Hz, 3H); 13C NMR (75 MHz, DMSO-d6)
d 161.4 (d, JCF = 244 Hz), 161.0, 158.1, 151.2, 150.9, 149.1, 148.7,
136.9 (d, JCF = 3 Hz), 136.6, 131.8, 128.6 (d, JCF = 8 Hz), 122.4,
115.2 (d, JCF = 21 Hz), 120.8, 52.9, 44.6, 19.8, 16.8; MS (ES+) m/z
439.1 (M+1).
5.1.38. Ethyl 4-methyl-2-(5-oxo-1-(4,4,4-trifluorobutyl)-1H-
1,2,4-triazol-4(5H)-yl)thiazole-5-carboxylate (5h)
By a similar procedure as described for 5a, 5h was obtained as
an off-white solid (0.09 g, 32%). 1H NMR (300 MHz, CDCl3) d 8.28 (s,
1H), 4.29 (q, J = 7.1 Hz, 2H), 3.92 (t, J = 6.6 Hz, 2H), 2.64 (s, 3H),
2.99–1.96 (m, 4H), 1.35 (t, J = 7.1 Hz, 3H); MS (ES+) m/z 365.3
(M+1).
5.1.39. 4-Methyl-2-(5-oxo-1-(4,4,4-trifluorobutyl)-1H-1,2,4-
triazol-4(5H)-yl)thiazole-5-carboxylic acid (6h)
By a similar procedure as described for 6a, 6h was obtained as
an off-white solid (0.07 g, 84%). MS (ESÀ) m/z 335.2 (MÀ1).
5.1.45. Methyl 3-nitro-1H-pyrazole-5-carboxylate (9)
To a solution of 8 (20.0 g, 127.32 mmol) in anhydrous methanol
(70 mL) was added SOCl2 (10.2 mL, 140.11 mmol) dropwise at 0 °C.
The resulting mixture was heated at reflux for 16 h, and then con-
centrated in vacuo. The residue was dissolved in ethyl acetate
(200 mL) and washed with saturated NaHCO3 solution
(40 mL Â 2), water (40 mL), and brine (40 mL), dried over anhy-
drous Na2SO4, and filtered. The filtrate was concentrated in vacuo,
and the residue was crystallized from ethyl acetate/hexane to
afford 9 as an off-white solid (15.0 g, 69%). 1H NMR (300 MHz,
CDCl3) d 11.52 (br s, 1H), 7.41 (s, 1H), 4.01 (s, 3H); MS (ESÀ) m/z
170.0 (MÀ1).
5.1.40. 4-Methyl-2-(5-oxo-1-(4,4,4-trifluorobutyl)-1H-1,2,4-
triazol-4(5H)-yl)-N-(pyridin-3-ylmethyl)thiazole-5-
carboxamide (7u)
By a similar procedure as described for 7a, 7u was obtained as
an off-white solid (0.04 g, 45%). Mp 192–195 °C (ethyl acetate/hex-
ane); 1H NMR (300 MHz, DMSO-d6) d 8.87 (s, 1H), 8.71 (s, 1H), 8.51
(d, J = 1.7 Hz, 1H), 8.43 (dd, J = 4.7, 1.4 Hz, 1H), 7.69 (d, J = 8.4 Hz,
1H), 7.33 (dd, J = 7.8, 4.7 Hz, 1H), 4.40 (s, 2H), 3.84 (t, J = 6.7 Hz,
2H), 2.53 (s, 3H), 2.41–2.24 (m, 2H), 1.93–1.83 (m, 2H); 13C NMR
(75 MHz, DMSO-d6) d 161.5, 151.9, 151.8, 150.2, 149.3, 148.6,
135.6, 135.2, 132.3, 129.8, 124.0, 122.4, 44.1, 41.0, 30.2 (q,
JCF = 120 Hz), 21.2, 17.4; MS (ES+) m/z 427.2 (M+1).
5.1.46. Methyl 1-(4-methoxybenzyl)-3-nitro-1H-pyrazole-5-
carboxylate (10)
To a mixture of 9 (13.00 g, 76.02 mmol) and K2CO3 (18.30 g,
132.40 mmol) in anhydrous N,N-dimethylformamide (350 mL)
was added 4-methoxybenzyl bromide (15.0 mL, 120.38 mmol).
The resulting mixture was heated at 60 °C for 5 h. The solid was fil-
tered off and the filtrate was concentrated under reduced pressure
at a temperature below 80 °C. The residue was dissolved in ethyl
acetate (500 mL), washed with 10% aqueous NH4Cl solution
(100 mL Â 2) and brine (100 mL), dried over anhydrous Na2SO4,
and filtered. The filtrate was concentrated in vacuo, and the residue
was crystallized from methanol to afford crude 10 as an off-white
solid (23.0 g), which was used in the next step without further
purification. For an analytical sample, a small amount of crude
material was purified by column chromatography eluted with a
gradient of 50–80% dichloromethane in hexanes to afford 10 as
an off-white solid. 1H NMR (300 MHz, CDCl3) d 7.39 (s, 1H), 7.34
5.1.41. 4-Methyl-2-(5-oxo-1-(4-(trifluoromethyl)benzyl)-1H-
1,2,4-triazol-4(5H)-yl)-N-(pyridin-2-ylmethyl)thiazole-5-
carboxamide (7v)
By a similar procedure as described for 7a, 7v was obtained as
an off-white solid (4.80 g, 77%). Mp 168–169 °C (ethanol/water);
1H NMR (300 MHz, DMSO-d6) d 8.89 (t, J = 5.8 Hz, 1H), 8.82 (s,
1H), 8.53–8.51 (m, 1H), 7.80–7.72 (m, 3H), 7.56 (d, J = 8.1 Hz,
2H), 7.34 (d, J = 8.1 Hz, 1H), 7.29–7.25 (m, 1H), 5.14 (s, 2H), 4.53
(d, J = 5.8 Hz, 2H), 2.60 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d
161.1, 158.2, 151.4, 151.1, 149.8, 148.8, 140.7, 136.7, 132.3,
128.5, 128.4 (q, JCF = 32 Hz), 125.5 (q, JCF = 4 Hz), 124.2 (d,
JCF = 272 Hz), 122.5, 122.1, 120.9, 47.9, 44.7, 16.9; MS (ES+) m/z

464
S. Sun et al. / Bioorg. Med. Chem. 23 (2015) 455–465
(d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 5.76 (s, 2H), 3.92 (s, 3H),
2H), 5.64 (s, 2H), 4.94 (s, 2H), 3.71 (s, 3H); MS (ES+) m/z 445.9
3.78 (s, 3H).
(M+23).
5.1.47. Methyl 3-amino-1-(4-methoxybenzyl)-1H-pyrazole-5-
carboxylate (11)
5.1.52. 3-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
1-(4-methoxybenzyl)-N-(pyridin-3-ylmethyl)-1H-pyrazole-5-
carboxamide (16a)
To a solution of crude 10 (23.0 g, 78.96 mmol) in methanol
(500 mL) and tetrahydrofuran (100 mL) was added palladium
(10% Pd on carbon). The reaction mixture was stirred under hydro-
gen atmosphere at ambient temperature for 48 h, then filtered
through a pad of celite. The filtrate was concentrated in vacuo,
and the residue was purified by column chromatography eluted
using a gradient of 3–10% methanol in dichloromethane to afford
11 as a pale yellow solid (17.0 g, 82%). 1H NMR (300 MHz, CDCl3)
d 7.21–7.18 (m, 2H), 6.82–6.79 (m, 2H), 6.15 (s, 1H), 5.48 (s, 2H),
5.28 (s, 2H), 3.81 (s, 3H), 3.75 (s, 3H); MS (ES+) m/z 283.8 (M+23).
To a solution of 15 (2.00 g, 4.72 mmol) and iPr2NEt (5.0 mL
28.70 mmol) in anhydrous tetrahydrofuran (110 mL) was added
HOBt (1.28 g, 9.45 mmol), TBTU (3.03 g, 9.43 mmol), and 3-(amino-
methyl)pyridine (0.73 mL, 7.16 mmol). The reaction mixture was
stirred at ambient temperature for 3 h, then concentrated in vacuo.
The residue was triturated in saturated NaHCO3 solution (400 mL).
The solid was collected by filtration, rinsed with water and dried to
afford 16a as an off-white solid (2.30 g, 95%). 1H NMR (300 MHz,
DMSO-d6) d 9.37 (t, J = 5.9 Hz, 1H), 8.53–8.43 (m, 2H), 8.44 (s,
1H), 7.68–7.64 (m, 1H), 7.37–7.32 (m, 4H), 7.21–7.14 (m, 4H),
6.85–6.82 (m, 2H), 5.65 (s, 2H), 4.95 (s, 2H), 4.45 (d, J = 5.9 Hz,
2H), 3.71 (s, 3H); MS (ES+) m/z 514.0 (M+1).
5.1.48. Methyl 3-(hydrazinecarboxamido)-1-(4-methoxybenzyl)-
1H-pyrazole-5-carboxylate (12)
To a solution of 11 (6.90 g, 26.41 mmol) and pyridine (2.57 mL,
31.77 mmol) in dichloromethane (80 mL) and tetrahydrofuran
(80 mL) was added 4-nitrophenyl chloroformate in dichlorometh-
ane (20 mL) dropwise at 0 °C. The reaction mixture was stirred at
ambient temperature for 2.5 h. Hydrazine monohydrate (7.68 mL,
158.32 mmol) was added and stirring was continued for 3 h, then
the mixture was concentrated in vacuo. The residue was triturated
in dichloromethane (100 mL). The solid was collected by filtration,
rinsed with water (100 mL) and dried to give 12 as an off-white solid
(5.90 g, 70%). 1H NMR (300 MHz, CDCl3) d 8.45 (br s, 1H), 7.21–7.18
(m, 2H), 6.96 (br s, 2H), 6.84–6.81 (m, 2H), 5.57 (s, 2H), 5.28 (s, 2H),
3.84 (s, 3H), 3.76 (s, 3H); MS (ES+) m/z 319.8 (M+1).
5.1.53. 3-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
N-(pyridin-3-ylmethyl)-1H-pyrazole-5-carboxamide (17a)
To a solution of 16a (3.50 g, 6.82 mmol) in dichloromethane
(100 mL) was added trifluoroacetic acid (100 mL) and trifluoro-
methanesulfonic acid (5.30 g, 35.31 mmol). The dark purple reac-
tion mixture was stirred at ambient temperature for 1.5 h and
concentrated in vacuo. The residue was triturated in methanol
(30 mL) and saturated aqueous NaHCO3 solution (100 mL). The
solid was collected by filtration and rinsed with water. The crude
product was then purified by column chromatography eluting with
a gradient of 2–10% methanol in CH2Cl2 then recrystallization from
ethanol to afford 17 as an off-white solid (1.72 g, 64%). Mp 235–
236 °C (ethanol); 1H NMR (300 MHz, DMSO-d6) d 13.84 (br s,
1H), 9.31 (t, J = 5.8 Hz, 1H), 8.56 (d, J = 1.7 Hz, 1H), 8.48 (dd,
J = 4.7, 1.3 Hz, 1H), 8.45 (s, 1H), 7.72 (ddd, J = 7.8, 1.7, 1.7 Hz, 1H),
7.39–7.31 (m, 4H), 7.21–7.16 (m, 2H), 4.96 (s, 2H), 4.49 (d,
J = 5.8 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) d 161.6 (d, JCF = 243
Hz), 158.1, 150.4, 148.9, 148.2, 142.5, 138.0, 135.2, 134.4, 133.5,
133.0 (d, JCF = 3 Hz), 129.8 (d, JCF = 8 Hz), 123.6, 115.4 (d, JCF = 21
Hz), 95.7, 47.5, 39.9; MS (ES+) m/z 394.1 (M+1); Anal. Calcd for
5.1.49. Methyl 1-(4-methoxybenzyl)-3-(5-oxo-1H-1,2,4-triazol-
4(5H)-yl)-1H-pyrazole-5-carboxylate (13)
To a suspension of 12 (5.20 g, 16.28 mmol) and trimethyl ortho-
formate (5.18 g, 48.90 mmol) in ethanol (160 mL) was added
TsOHÁH2O (0.50 g, 2.60 mmol). The reaction mixture was heated at
reflux for 2.5 h then cooled to 0 °C. The solid was recovered by filtra-
tion, rinsed with diethyl ether and dried to afford 13 as an off-white
solid (3.81 g, 71%). 1H NMR (300 MHz, DMSO-d6) d 12.07 (br s, 1H),
8.30 (d, J = 1.2 Hz, 1H), 7.17–7.12 (m, 3H), 6.86–6.83 (m, 2H), 5.59 (s,
2H), 3.82 (s, 3H), 3.68 (s, 3H); MS (ES+) m/z 351.7 (M+23).
C19H16FN7O2 1.5H2O: C, 54.28; H, 4.56; N, 23.32. Found: C, 54.20;
H, 4.19; N, 23.24.
5.1.50. Methyl 3-(1-(4-fluorobenzyl)-5-oxo-1H-1,2,4-triazol-
4(5H)-yl)-1-(4-methoxybenzyl)-1H-pyrazole-5-carboxylate (14)
To a mixture of 13 (4.48 g, 13.60 mmol) and K2CO3 (2.82 g,
20.40 mmol) in acetone (450 mL) was added 4-fluorobenzyl bro-
mide (2.1 mL, 16.85 mmol). The reaction mixture was heated at
reflux for 3.5 h. The hot reaction mixture was filtered and washed
with acetone. The filtrate was concentrated in vacuo, and the res-
idue was crystallized from acetone and diethyl ether to afford 14
as an off-white solid (5.90 g, 98%). 1H NMR (300 MHz, CDCl3) d
7.79 (s, 1H), 7.33–7.29 (m, 2H), 7.20–7.17 (m, 3H), 6.99–6.94 (m,
2H), 6.78–6.75 (m, 2H), 5.58 (s, 2H), 4.91 (s, 2H), 3.82 (s, 3H),
3.81 (s, 3H); MS (ES+) m/z 459.9 (M+23).
5.1.54. 3-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
1-(4-methoxybenzyl)-N-(pyridin-2-ylmethyl)-1H-pyrazole-5-
carboxamide (16b)
By a similar procedure as described for 16a, 16b was obtained
as an off-white solid (0.11 g, 51%). MS (ES+) m/z 513.7 (M+1).
5.1.55. 3-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
N-(pyridin-2-ylmethyl)-1H-pyrazole-5-carboxamide (17b)
By a similar procedure as described for 17a, 17b was obtained
as an off-white solid (0.09 g, 58%). Mp 224–225 °C (N,N-dimethyl-
formamide/water); 1H NMR (300 MHz, DMSO-d6) d 13.83 (br s,
1H), 9.36 (t, J = 5.8 Hz, 1H), 8.52 (d, J = 4.2 Hz, 1H), 8.46 (s, 1H),
7.77 (ddd, J = 7.7, 7.7, 1.6 Hz, 1H), 7.38–7.26 (m, 5H), 7.22–7.16
(m, 2H), 4.97 (s, 2H), 4.56 (d, J = 5.8 Hz, 2H); 13C NMR (75 MHz,
DMSO-d6) d 161.6 (d, JCF = 243 Hz), 158.2, 158.1, 150.4, 148.9,
142.4, 138.1, 136.8, 133.6, 132.9 (d, JCF = 3 Hz), 129.8 (d, JCF = 8 Hz),
122.2, 121.1, 115.4 (d, JCF = 21 Hz), 95.9, 47.5, 44.1; MS (ES+) m/z
393.8 (M+1).
5.1.51. 3-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
1-(4-methoxybenzyl)-1H-pyrazole-5-carboxylic acid (15)
To a solution of 14 (3.80 g, 8.69 mmol) in ethanol (100 mL) and
water (20 mL) was added NaOH (0.728 g, 18.20 mmol). The reac-
tion mixture was heated at reflux for 2 h, then concentrated in
vacuo to remove most of the organic volatiles. The residue was
neutralized to pH 4 ꢀ 5 with 10% hydrochloric acid solution. The
solid was collected by filtration, rinsed with water and diethyl
ether, then dried to afford 15 as an off-white solid (3.50 g, 95%).
1H NMR (300 MHz, DMSO-d6) d 13.83 (br s, 1H), 8.44 (s, 1H),
7.38–7.23 (m, 2H), 7.21–7.16 (m, 4H), 7.11 (s, 1H), 6.89–6.86 (m,
5.1.56. 3-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
1-(4-methoxybenzyl)-N-(pyridin-4-ylmethyl)-1H-pyrazole-5-
carboxamide (16c)
By a similar procedure as described for 16a, 16c was obtained as
an off-white solid (0.14 g, 63%). MS (ES+) m/z 513.7 (M+1).

S. Sun et al. / Bioorg. Med. Chem. 23 (2015) 455–465
465
5.1.57. 3-(1-(4-fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
Acknowledgments
N-(pyridin-4-ylmethyl)-1H-pyrazole-5-carboxamide (17c)
By a similar procedure as described for 17a, 17c was obtained as
an off-white solid (0.10 g, 77%). Mp 262–263 °C (N,N-dimethyl-
formamide/water); 1H NMR (300 MHz, DMSO-d6) d 13.86 (br s,
1H), 9.36 (t, J = 5.8 Hz, 1H), 8.53–8.51 (m, 2H), 8.46 (s, 1H), 7.38–
7.30 (m, 5H), 7.22–7.16 (m, 2H), 4.97 (s, 2H), 4.49 (d, J = 5.8 Hz,
2H); 13C NMR (75 MHz, DMSO-d6) d 161.6 (d, JCF = 243 Hz), 158.3,
150.4, 149.6, 147.9, 133.6, 132.9 (d, JCF = 3 Hz), 129.8 (d, JCF = 8 Hz),
122.1, 115.4 (d, JCF = 21 Hz), 95.8, 47.5, 41.2; MS (ES+) m/z 393.8
(M+1).
We thank Caroline Hall, Annick Legendre, Monica Mork, Rostam
Namdari, Pritpaul Samra, Joseph Sangara, Wendy Young, and Jing
Zhong for their technical assistance.
References and notes
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2. Chavez, J. A.; Summers, S. A. Biochim. Biophys. Acta 2010, 1801, 252.
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5.1.58. 3-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
1-(4-methoxybenzyl)-N-(oxazol-4-ylmethyl)-1H-pyrazole-5-
carboxamide (16d)
By a similar procedure as described for 16a, 16d was obtained
as an off-white solid (0.09 g, 39%). MS (ES+) m/z 503.9 (M+1).
5.1.59. 3-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
N-(oxazol-4-ylmethyl)-1H-pyrazole-5-carboxamide (17d)
By a similar procedure as described for 17a, 17d was obtained
as an off-white solid (0.06 g, 66%). Mp 267–268 °C (N,N-dimethyl-
formamide/water); 1H NMR (300 MHz, DMSO-d6) d 13.80 (br s,
1H), 9.18 (t, J = 5.6 Hz, 1H), 8.44 (s, 1H), 8.34 (s, 1H), 8.00 (s, 1H),
7.38–7.32 (m, 3H), 7.22–7.16 (m, 2H), 4.96 (s, 2H), 4.36 (d,
J = 5.6 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) d 161.6 (d, JCF = 243
Hz), 158.0, 152.1, 150.4, 142.4, 138.0, 137.1, 136.2, 133.6, 132.9
(d, JCF = 3 Hz), 129.8 (d, JCF = 8 Hz), 115.4 (d, JCF = 21 Hz), 95.9,
47.5, 34.6; MS (ES+) m/z 383.8 (M+1).
10. Ntambi, J. M.; Miyazaki, M.; Stoehr, J. P.; Lan, H.; Kendziorski, C. M.; Yandell, B.
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2002, 99, 11482.
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Kastelein, J. J.; Lusis, A. J.; Stalenhoef, A. F.; Stoehr, J. P.; Hayden, M. R.; Ntambi,
J. M. J. Lipid Res. 1899, 2002, 43.
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5.1.60. 3-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
1-(4-methoxybenzyl)-N-((1-methyl-1H-pyrazol-4-yl)methyl)-
1H-pyrazole-5-carboxamide (16e)
By a similar procedure as described for 16a, 16e was obtained
as an off-white solid (0.15 g, 68%). MS (ES+) m/z 516.9 (M+1).
18. Miyazaki, M.; Man, W. C.; Ntambi, J. M. J. Nutr. 2001, 131, 2260.
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5.1.61. 3-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
N-((1-methyl-1H-pyrazol-4-yl)methyl)-1H-pyrazole-5-
carboxamide (17e)
20. Meingassner, J. G.; Aschauer, H.; Winiski, A. P.; Dales, N.; Yowe, D.; Winther, M.
D.; Zhang, Z.; Stutz, A.; Billich, A. J. Invest. Dermatol. 2013, 133, 2091.
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By a similar procedure as described for 17a, 17e was obtained
as an off-white solid (0.10 g, 53%). Mp 239–241 °C (N,N-dimethyl-
formamide/water); 1H NMR (300 MHz, DMSO-d6) d 13.77 (br s,
1H), 9.04 (t, J = 5.6 Hz, 1H), 8.44 (s, 1H), 7.61 (s, 1H), 7.37–7.27
(m, 4H), 7.22–7.16 (m, 2H), 4.95 (s, 2H), 4.27 (d, J = 5.6 Hz, 2H),
3.78 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d 161.6 (d, JCF = 243 Hz),
157.7, 150.4, 142.4, 138.3, 138.0, 133.6, 132.9 (d, JCF = 3 Hz), 129.8
(d, JCF = 8 Hz), 129.5, 118.2, 115.4 (d, JCF = 21 Hz), 95.7, 47.5, 38.3,
33.0; MS (ES+) m/z 396.9 (M+1).
5.1.62. 3-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
1-(4-methoxybenzyl)-N-methyl-1H-pyrazole-5-carboxamide
(16f)
By a similar procedure as described for 16a, 16g was obtained
as an off-white solid (0.2 g, 99%). MS (ES+) m/z 437.3 (M+1).
30. Zhang, Z.; Sun, S.; Kodumuru, V.; Hou, D.; Liu, S.; Chakka, N.; Sviridov, S.;
Chowdhury, S.; McLaren, D. G.; Ratkay, L. G.; Khakh, K.; Cheng, X.; Gschwend,
H. W.; Kamboj, R.; Fu, J.; Winther, W. D. J. Med. Chem. 2013, 56, 568.
31. Sun, S.; Zhang, Z.; Kodumuru, V.; Pokrovskaia, N.; Fonarev, J.; Jia, Q.; Leung, P.;
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5.1.63. 3-(1-(4-Fluorobenzyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)-
N-methyl-1H-pyrazole-5-carboxamide (17f)
By a similar procedure as described for 17a, 17f was obtained as
an off-white solid (0.04 g, 29%). Mp 268–269 °C (ethanol); 1H NMR
(300 MHz, DMSO-d6) d 13.72 (br s, 1H), 8.63 (t, J = 4.6 Hz, 1H), 8.40
(s, 1H), 7.36–7.28 (m, 2H), 7.21–7.10 (m, 3H), 4.92 (s, 2H), 2.73 (d,
J = 4.6 Hz, 3H); MS (ES+) m/z 317.1 (M+1).
33. Talamo, B. R.; Bloch, K. Anal. Biochem. 1969, 29, 300.
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