Discovery and optimization of novel 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides as bifunctional antidiabetic agents stimulating both insulin secretion and glucose uptake

Article abstract of DOI:10.1016/j.ejmech.2021.113325

A novel series of 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides was designed, synthesized and evaluated for their biological activities on glucose-stimulated insulin secretion (GSIS). The cytotoxicity of all 41 novel compounds was screened to assess their

Full text of DOI:10.1016/j.ejmech.2021.113325

European Journal of Medicinal Chemistry 217 (2021) 113325
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery and optimization of novel 3-benzyl-N-phenyl-1H-pyrazole-
5-carboxamides as bifunctional antidiabetic agents stimulating both
insulin secretion and glucose uptake
,
,
Jeyun Jo a y, Dahae Lee b y, Yeong Hye Park a, Hyeonjin Choi a, Jinhee Han a, Do Hwi Park b,
You-Kyung Choi b, Jinsook Kwak a, Min-Kyu Yang c, Jin-Wook Yoo a, Hyung Ryong Moon a,
Dongho Geum d, Ki Sung Kang b *, Hwayoung Yun a
,
, *
a College of Pharmacy, Pusan National University, Busan, 46241, Republic of Korea
b College of Korean Medicine, Gachon University, Seongnam, 13120, Republic of Korea
c Mother’s Pharmaceutical, Seoul, 08506, Republic of Korea
d Department of Biomedical Sciences, Korea University Medical School, Seoul, 02841, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides was designed, synthesized and eval-
uated for their biological activities on glucose-stimulated insulin secretion (GSIS). The cytotoxicity of all
41 novel compounds was screened to assess their pharmacological safety in pancreatic b-cells. A two-
step optimization process was carried out to establish the structure-activity relationship for this class
and subsequently we identified the most active analogue 26. Further modification study of 26 evidenced
the necessity of N-hydrogens in the core architecture. Protein expression analysis suggested that 26
increases insulin secretion via the activation of the upstream effector of pancreatic and duodenal ho-
meobox 1 (PDX-1), which is an important factor promoting GSIS. Moreover, the administration of 26
effectively augmented glucose uptake in C2C12 myotube cells via the suppression of Mitsugumin 53
(MG53), an insulin receptor substrate 1 (IRS-1) ubiquitination E3 ligase.
Received 8 December 2020
Received in revised form
8 February 2021
Accepted 19 February 2021
Available online 6 March 2021
Keywords:
Type 2 diabetes mellitus (T2DM)
3-Benzyl-N-phenyl-1H-pyrazole-5-
carboxamide
© 2021 Elsevier Masson SAS. All rights reserved.
Glucose-stimulated insulin secretion (GSIS)
Glucose stimulation index (GSI)
Structure-activity relationship (SAR)
1. Introduction
T2DM is a decrease in glucose-stimulated insulin secretion (GSIS) in
pancreatic
oral insulinotropic agents in the clinical management of T2DM [11],
increase insulin secretion from -cells by closing K-ATP channels in
b-cells [9,10]. Sulfonylureas, which are commonly used
Type 2 diabetes mellitus (T2DM), which represents approxi-
mately 90% of all cases of diabetes [1], is characterized by persistent
b
hyperglycemia caused by abnormalities in insulin action and
b
-cell
the cell membrane. However, drugs in this class often elicit hypo-
glycemia because they continuously stimulate insulin secretion
independently of blood glucose level [11]. In order to compensate
for the major drawback of sulfonylureas, glucose-dependent insulin
secretagogues, including dipeptidyl peptidase-4 (DPP-4) inhibitors
and ligands that target islet G-protein-coupled receptors (GPCRs),
have recently been developed [12]. Nonetheless, recent studies
have suggested that DPP-4 inhibitors cause severe joint pain and
glucagon-like peptide-1 (GLP-1) receptor agonists increase the risk
of cholelithiasis [13,14]. Moreover, these agents may be associated
with an increased risk of cholangiocarcinoma [15]. Continuous ef-
forts for the development of improved therapies, such as G-protein-
coupled receptor (GPR) 40 and GPR119 agonists, are ongoing to
address the limitations of current treatments; however, these
function [2,3]. T2DM affected more than 400 million people
worldwide in 2018 and this number is projected to rise to about
500 million by 2030 [4,5]. T2DM causes a series of dysfunctions in
multiple organs and tissues and can result in severe chronic com-
plications, such as end-stage renal disease, blindness, arterial dis-
ease, slow wound healing, and limb amputation [6]. Numerous
therapeutic options for the treatment of T2DM have been devel-
oped but only a minority of patients achieve long-term glycaemic
control [7,8].
One of the primary factors associated with the development of
* Corresponding authors.
E-mail addresses: kkang@gachon.ac.kr (K.S. Kang), hyun@pusan.ac.kr (H. Yun).
These authors contributed equally to this work.
y
https://doi.org/10.1016/j.ejmech.2021.113325
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

J. Jo, D. Lee, Y.H. Park et al.
European Journal of Medicinal Chemistry 217 (2021) 113325
options are still not clinically available [16,17]. The identification of
novel molecules and pathways that have the potential to correct
glycaemia via the stimulation of GSIS is, therefore, highly desirable.
In an effort to discover novel antidiabetic agents, an in-house
chemical library containing approximately heterocycle-based 500
compounds was screened using INS-1 cells with a GSIS assay. The
INS-1 cell line has been widely used for studies of glucose-
carboxamides 1 and 6e22.
We subsequently turned our attention to the derivatization of
the potent analogue 9 to find out the anti-diabetic effect of the
methoxy group. With the previously prepared building blocks 5aec
in hand, we manipulated similar EDC couplings with diversely
methoxy-substituted anilines (Scheme 2). The new 3-benzyl-N-
phenyl-1H-pyrazole-5-carboxamides 23e40 were efficiently
obtained.
dependent b-cell function because it secretes insulin in response
to concentrations of glucose within the physiological range [18].
From the preliminary screening, we identified hit compound 1
which exhibited a strong insulin secretion activity, equal to that of a
Lastly, we then executed further optimization of the best
analogue 26, which possessing a 2,4-dimethoxy group, to confirm
SAR analysis. A similar attempt utilizing the 4-step process depicted
in Scheme 1 successfully resulted in the final 3-benzyl-N-phenyl-
1H-pyrazole-5-carboxamides, 41 (R1 ¼ 3-Cl) and 42 (R1 ¼ 2-Cl),
starting from the commercially available phenylacetones 2d and 2e,
respectively (Scheme 3A). The exposure of the building block 5a to
oxalyl chloride and the coupling of the resulting acid chloride with
2,4-dimethoxy-N-methylaniline smoothly yielded 3-benzyl-N-
methyl-N-phenyl-1H-pyrazole-5-carboxamide 43 (Scheme 3B). As
a final entry in our diversification, the common intermediate 4a
was subjected to N-alkylation with iodomethane and benzylic
oxidation with t-butyl hydroperoxide (TBHP) [20] to afford N-
methylpyrazole ester 44 and 4-chlorobenzoylpyrazole ester 47,
respectively (Scheme 3C). Finally, the saponification of esters 44
and 47, followed by the amidation of the resulting carboxylic acids
45 and 48, furnished 3-benzyl-N-phenyl-1H-pyrazole-5-
carboxamides 46 and 49, respectively.
representative sulfonylurea gliclazide, at a concentration of 10
mM
(Fig. 1A). Interestingly, compound consists of core 5-
1
a
carboxamidopyrazole and two para-halo-substituted benzene
rings and has relatively good lead-like properties (MW ꢀ 460,
rings
ꢀ
4, hydrogen-bond donors
ꢀ
5, hydrogen-bond
acceptors ꢀ 9, and ꢁ4 ꢀ LogP ꢀ 4.2) [19]. Herein, we designed
and synthesized a new set of 3-benzyl-N-phenyl-1H-pyrazole-5-
carboxamides by decorating various substituents on both ben-
zene rings (Fig. 1B). Furthermore, we attempted to optimize our
analogues in detail and establish a brief structure-activity rela-
tionship (SAR).
2. Results and discussion
2.1. Chemistry
Based on the structure of hit 1, we initially decided to introduce
various substituents on the para positions of both benzene rings.
The synthetic route for the final analogues 1 and 6e22 is illustrated
in Scheme 1. The commercially available phenylacetones 2aec were
first condensed with diethyl oxalate in the presence of t-BuOK,
2.2. Biological evaluation
2.2.1. GSIS assay
The synthesized compounds were evaluated for their insulin
releasing activities in INS-1 cells by measuring glucose-stimulated
insulin secretion (GSIS). The GSIS data can be normalized as a
glucose stimulation index (GSI), which is calculated as the ratio of
the insulin value under high-glucose divided by that under low-
followed by
a second condensation of the resulting 2,4-
dioxopentanoates 3aec with hydrazine under acidic conditions to
afford the desired pyrazoles 4aec. The key intermediates 5aec
were smoothly produced by the simple saponification of the cor-
glucose conditions [21]. INS-1 cells were exposed to 10 mM of the
responding
pyrazoles
4aec.
Finally,
1-ethyl-3-(3-
compounds for 2 h and stimulated with 3.3 mM and 16.7 mM
glucose for 1 h. Gliclazide was run in parallel as a positive control
drug. Additionally, the viability of INS-1 cells was determined after
dimethylaminopropyl)carbodiimide (EDC) coupling of 5aec with
various anilines provided 3-benzyl-N-phenyl-1H-pyrazole-5-
Fig. 1. (A) Identification of hit compound 1; (B) Optimization of novel 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides.
2

J. Jo, D. Lee, Y.H. Park et al.
European Journal of Medicinal Chemistry 217 (2021) 113325
Scheme 1. Synthesis of 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides 1 and 6e22. Reagents and conditions: (a) (CO2Et)2, t-BuOK, toluene, 0 ꢂC, 67e97%; (b) H2NNH2$H2O,
AcOH, 67e93%; (c) LiOH$H2O, THF/H2O (2:1), 50 ꢂC, 71e89%; (d) R2-C6H4-NH2, EDC$HCl, DMAP, CH2Cl2, 18e79%.
Scheme 2. Synthesis of 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides 23e40. Reagents and conditions: (a) R2-C6H4-NH2 or R2-C6H3-NH2, EDC$HCl, DMAP, CH2Cl2, 8e58%.
treatment with newly synthesized compounds using the 3-(4,5-
dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay.
(compound 7). However, compounds 8 and 10, which possess a 4-
CF3 and a 4-CO2Me at part B, respectively, did not lead to any
improvement in insulin secretion under high-glucose conditions.
Taken together, the GSI value of the first designed series suggests
that the presence of an electron-donating group at part B would be
beneficial for insulin-secreting activity. Gratifyingly, none of syn-
thesized compounds exhibited significant cytotoxicity against INS-
1 cells at the treated concentrations.
The GSI value and cytotoxicity of the initially designed de-
rivatives 6e22 at a concentration of 10
mM are summarized in
Table 1. Our efforts were focused on exploring the best substituents
on both benzene rings of hit compound 1. Generally, the 4-
chlorobenzene (6e10) series at the part A position displayed
improved GSI values compared with the 4-fluorobenzene (11e16)
and 4-methoxybenzene (17e22) series. Among the 4-
chlorobenzylpyrazoles (6e10), compound 9, which containing a
4-methoxy group at the part B position, exhibited the best insulin-
secreting activity, which was more potent than that of hit 1. The
removal of the 4-fluoro substituent of 1 resulted in a slightly lower
potency (compound 6). A similar behavior was observed with the
replacement of the fluoro group of 1 with a tert-butyl group
At the stage of further optimization, 18 new analogues (23e40),
which were structurally derived from compound 9, were tested for
GSIS at a concentration of 5
mM (Table 2). Generally, the 4-
chlorobenzene (23e28) and 4-fluorobenzene (29e34) series at
the part A position seemed to be more effective than the 4-
methoxybenzene (35e40) series. Among them, compounds 26,
27, 29, and 30 were found to be more potent than compound 9 at
the treated concentration. Especially, 4-chlorobenzene analogue
3

J. Jo, D. Lee, Y.H. Park et al.
European Journal of Medicinal Chemistry 217 (2021) 113325
Scheme 3. Synthesis of 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides 41e43, 46, and 49. Reagents and conditions: (a) (CO2Et)2, t-BuOK, toluene, 0 ꢂC, 68e77%; (b) H2NNH2$H2O,
AcOH, 69e79%; (c) LiOH$H2O, THF/H2O (2:1), 50 ꢂC, 71e95%; (d) 2,4-dimethoxyaniline, EDC$HCl, DMAP, CH2Cl2, 60e93%; (e) 2,4-dimethoxy-N-methylaniline, (COCl)2, Et3N, DMF,
CH2Cl2, 54%; (f) MeI, K2CO3, DMF, 44%; (g) LiOH$H2O, THF/H2O (2:1), 50 ꢂC, 95%; (h) 2,4-dimethoxyaniline, (COCl)2, Et3N, DMF, CH2Cl2, 87%; (i) TBHP, 120 ꢂC, 96%; (j) LiOH$H2O, THF/
H2O (2:1), 50 ꢂC, 92%; (k) 2,4-dimethoxyaniline, EDC$HCl, HOBt$H2O, DMAP, DIPEA, DMF, 81%.
26, which containing a 2,4-dimethoxy group at the part B position,
showed the highest insulin-secreting activity, with a GSI value of
14.9, which was approximately six-fold higher than that of 9.
Likewise, 4-fluorobenzene analogue 30, which possessing a 3-
methoxy group at the part B position, also exhibited substantial
insulin-secreting activity. These results suggest that the potential
targets of the 4-chlorobenzene and 4-fluorobenzene series might
be different or do not share the same binding mode. The intro-
duction of a methoxy group to the part A benzene and a methyl-
enedioxy group to the part B benzene were not helpful to enhance
potency. Furthermore, all compounds 23e40 had no cytotoxic ef-
fects on INS-1 cells at a concentration of 10 mM (Table 2).
These results encouraged us to extend the SAR study by
exploring an additional modification of the best analogue 26. As
4

J. Jo, D. Lee, Y.H. Park et al.
European Journal of Medicinal Chemistry 217 (2021) 113325
Table 1
depicted in Scheme 3, the parent compound 26 was structurally
transformed into 3-chlorobenzene (41), 2-chlorobenzene (42), N-
GSIS activity and cytotoxicity of analogues 6e22 on INS-1 cell line.
Comp
GSIa at 10
m
M
Cell viability at 10 mM
methylamide
(43),
N-methylpyrazole
(46),
and
4-
(% of untreated control)b
chlorobenzoylpyrazole (49). These compounds were then sub-
jected to GSIS and MTT assays with gliclazide as a positive control
(Table 3). Most novel derivatives exhibited slightly less potent ac-
tivity (GSI ¼ 1.29e2.43) than gliclazide. Taken together, the 4-
chloro substituent in the part A benzene was preferred to other
substituents for anti-diabetic activity. The introduction of a methyl
group to the amide backbone or the pyrazole ring resulted in the
loss of activity, which presumably indicates that both N-hydrogen
moieties play a crucial role in the insulin-secreting activity of the
compounds. Additionally, the insertion of a carbonyl group to the
benzylic position, which may have provided a conformationally
restricted environment, resulted in low activity. Based on the brief
SAR of the 41 derivatives, the most potent analogue 26 was finally
selected for detailed biological evaluation. We tested cytotoxicity of
compound 26 on two different normal cell lines, LX-2 human he-
patic stellate cells and 3T3L-1 mouse embryonic fibroblast cells for
further biological evaluation and 26 did not show any toxicity at all
the tested concentrations (Fig. S1 in Supplementary Data).
1
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
6.73 0.50
5.24 2.14
4.89 0.04
1.69 0.12
7.59 0.15
1.48 0.10
1.86 0.08
2.00 0.01
1.96 0.16
2.02 0.01
2.03 0.31
1.42 0.09
2.49 0.19
4.40 0.08
1.64 0.05
2.07 0.11
1.66 0.05
1.85 0.01
5.86 0.46
94.9 5.2
109 2.1
97.5 4.1
102 5.3
101 4.8
101 3.3
100 4.0
100 2.7
98.6 2.3
92.8 2.9
95.6 3.6
102 3.3
105 0.4
105 1.2
99.4 4.3
98.4 1.0
105 0.5
99.2 2.2
98.7 2.6
21
22
Gliclazide
a
Insulin content in 16.7 mM glucose media/insulin content in 3.3 mM glucose
media.
b
Values are the mean standard deviation.
2.2.2. Effect of 26 on protein expression related to pancreatic b-cell
function
To gain insight into the potential mechanism of 26 on insulin
secretion, we investigated the effects of 26 on protein expression
related to pancreatic b-cell function using western blotting (Fig. 2).
Table 2
GSIS activity and cytotoxicity of analogues 9 and 23e40 on INS-1 cell line.
Comp
GSIa at 5
m
M
Cell viability at 10 mM
The activation of serine/threonine kinase Akt, a downstream
effector of phosphoinositide 3-kinase (PI3K), is mainly involved in
(% of untreated control)b
pancreatic b-cell metabolism and survival and the promotion of
9
2.60 0.07
1.56 0.11
1.54 0.04
1.85 0.01
14.9 0.20
3.25 0.08
1.29 0.06
3.67 0.05
7.49 0.63
1.16 0.01
1.93 0.33
1.52 0.29
1.25 0.003
1.66 0.03
1.55 0.01
1.13 0.02
1.58 0.02
2.35 0.15
1.13 0.003
101 4.8
98.1 2.3
96.5 2.1
98.8 3.5
96.3 5.5
97.9 0.8
101 4.3
104 0.6
101 0.7
102 2.7
99.9 2.0
104 1.1
97.6 1.7
101 3.2
102 1.1
98.9 3.3
101 2.8
100 0.3
102 3.6
insulin secretion [22e25]. Previous studies have reported that
PI3K/Akt signaling can be activated using a direct binding ligand for
PI3K or by the regulation of upstream components such as insulin
receptor substrate 2 (IRS-2) [23e26]. Therefore, we first decided to
analyze the cellular levels of IRS-2, p-IRS-2, PI3K, p-PI3K, Akt, and
p-Akt. As demonstrated in Fig. 2AeD, compound 26 dose-
dependently upregulated the expression of p-Akt, p-PI3K, and p-
IRS-2 in INS-1 cells, which suggests that IRS-2 is the upstream
regulator of PI3K-dependent Akt phosphorylation (Ser473) in
response to treatment with 26. In the additional western blot
experiment, we could observe that the expression of peroxisome
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
proliferator-activated receptor-g (PPAR-g) and pancreatic and
duodenal homeobox 1 (PDX-1) was significantly increased when
compound 26 was administered (Fig. 2A, E, and 2F). PDX-1 is an
important regulator of glucose-stimulated insulin gene expression
a
and can be transcriptionally regulated by PPAR-g in INS-1 cells
Insulin content in 16.7 mM glucose media/insulin content in 3.3 mM glucose
[27,28]. Considering that the PI3K/Akt pathway is critical for the
expression and activation of PDX-1 [27,29,30], compound 26
plausibly enhances GSIS by regulating PDX-1 function via the
media.
b
Values are the mean standard deviation.
activation of PPAR-
g and IRS-2-mediated PI3K/Akt signaling in
Table 3
pancreatic -cells [31].
b
GSIS activity and cytotoxicity of analogues 41e43, 46, and 49 on INS-1 cell line.
GSIa at 5
mM
Cell viability at 10 mM
2.2.3. Effect of 26 on glucose uptake in C2C12 myotube cells
Comp
(% of untreated control)b
As compound 26 showed an ability to activate insulin receptor
substrate-associated PI3K activity and Akt phosphorylation, we
next examined whether the compound regulates glucose homeo-
stasis in the C2C12 myotube cell line (Fig. 3). Recent studies have
suggested that compounds with antidiabetic potential, including
several natural products and AMP-activated protein kinase (AMPK)
41
42
43
46
1.29 0.04
2.43 0.03
1.33 0.01
1.37 0.06
1.49 0.08
4.22 0.01
97.7 1.9
102 1.6
93.3 1.1
96.2 3.6
100 2.7
98.7 2.6
49
Gliclazide
activators, that promote insulin secretion in b-cells via PI3K acti-
a
Insulin content in 16.7 mM glucose media/insulin content in 3.3 mM glucose
vation, could dually increase glucose uptake in skeletal muscles
[32e37]. Therefore, compound 26 was first evaluated for its ca-
pacity to augment glucose uptake in the myotube cell line. With the
treatment of 26, glucose uptake was dose-dependently increased in
the absence of insulin (Fig. 3A). To explore the molecular
media.
b
Values are the mean standard deviation.
5

J. Jo, D. Lee, Y.H. Park et al.
European Journal of Medicinal Chemistry 217 (2021) 113325
Fig. 2. Effect of 26 on the protein expression of phospho-insulin receptor substrate-2 (p-IRS-2) (Ser731), IRS-2, phospho-phosphatidylinositol 3-kinase (p-PI3K), PI3K, phospho-Akt
(p-Akt) (Ser473), Akt, peroxisome proliferator-activated receptor- (PPAR ), and pancreatic and duodenal homeobox-1 (PDX-1) in INS-1 cells. (A) Protein expression levels of p-IRS-
2 (Ser731), IRS-2, p-PI3K, PI3K, p-Akt (Ser473), Akt, PPAR- , PDX-1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in INS-1 cells treated with 0 M, 2.5 M, 5 M, and
10 M of 26 for 24 h. (BeF) Bar graph presents the densitometric quantification of western blot bands. The experiment was performed in triplicate, and the error bars represent the
standard deviation of samples (n ¼ 3). *p < 0.05 compared with the control.
g
g
g
m
m
m
m
Fig. 3. Effect of 26 on glucose uptake in C2C12 myotube cells. (A) Basal glucose uptake in 26-treated C2C12 myotubes under normal conditions. (B) Protein expression levels of
MG53, IRS-1, p-PI3K, PI3K, p-Akt, Akt, and GAPDH in C2C12 cells treated with 0 M, 5 M, 10 M, 20 M, and 50 M of 26 for 16 h.
m
m
m
m
m
mechanism underlying glucose transportation regulated by 26, we
further investigated the expression of PI3K/Akt pathway proteins.
As displayed Fig. 3B, treatment with 26 increased the levels of PI3K
and Akt phosphoproteins, which suggests that 26 has a capacity to
stimulate PI3K/Akt signaling in the two different types of cell lines,
muscle [38,39]. In Fig. 3B, the expression of IRS-1 was elevated as
the concentration of the 26 was increased. However, the expression
of Mitsugumin 53 (MG53), which is a ubiquitin E3 ligase that in-
duces IRS-1 degradation in skeletal muscle [39,40], was
concentration-dependently suppressed by 26. Therefore, these re-
sults suggest that compound 26 could decrease the expression of
MG53 and interfere with MG53-induced IRS-1 ubiquitination,
which result in the activation of the IRS-1/PI3K/Akt pathway and
the upregulation of glucose uptake. The bi-functionality of 26 could
myotube cells and pancreatic b-cells. To uncover the upstream of
PI3K/Akt signaling in response to 26, the expression of additional
proteins was investigated, considering that IRS-1 is one of the most
common effectors of the PI3K/Akt signaling pathway in skeletal
6

J. Jo, D. Lee, Y.H. Park et al.
European Journal of Medicinal Chemistry 217 (2021) 113325
provide a novel class of antidiabetic therapy that have a potential to
0.6 Hz, OCH2CH3), 3.74 (s, 2H, ArꢁCH2), 1.35 (td, 3H, J ¼ 7.1, 0.7 Hz,
reduce blood glucose levels through both insulin secretion in
cells and glucose uptake in muscle cells.
b
-
OCH2CH3); 13C NMR (CDCl3, 100 MHz)
d 200.1, 166.9, 161.9, 133.6,
132.1, 130.9, 129.2, 101.7, 62.8, 47.0, 14.1; LR-MS (ESIþ) m/z 269 [M þ
H]þ; HR-MS (ESIþ) calcd for C13H14ClO4 [M þ H]þ 269.0575; found
269.0574.
3. Conclusions
In summary, a novel series of 3-benzyl-N-phenyl-1H-pyrazole-
5-carboxamides was synthesized and evaluated for their glucose-
stimulated insulin secretion activity in INS-1 cell line. The newly
synthesized 41 analogues were screened for their cytotoxicity
4.1.1.2. Ethyl
5-(4-fluorophenyl)-2,4-dioxopentanoate
(3b).
4-Fluorophenylacetone (2b) (1.19 g, 7.82 mmol) afforded 1.38 g
(67%) of 3b as an orange oil. 3b was purified by flash column
chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 7): 1H NMR
against
based optimization led to the discovery of compound 26, which
potently increased GSIS from -cells without cytotoxicity. Western
b-cells to ensure pharmaceutical safety. Stepwise SAR-
(CDCl3, 500 MHz)
d
7.21 (dd, 2H, J ¼ 8.3, 5.4 Hz, ArꢁH), 7.04 (t, 2H,
J ¼ 8.6 Hz, ArꢁH), 6.35 (s, 1H, CH), 4.33 (q, 2H, J ¼ 7.1 Hz, OCH2CH3),
3.75 (s, 2H, ArꢁCH2), 1.36 (t, 3H, J ¼ 7.1 Hz, OCH2CH3); 13C NMR
b
blot assay showed that 26 could augment GSIS via the regulation of
PDX-1 activity. In addition, we found that 26 stimulates glucose
uptake in C2C12 myotube cells through suppression of MG53
expression. The unique combination of pharmacological actions
could provide a novel class of antidiabetic agents that affect the key
pathogenic mechanisms in diabetes. Given the bifunctional anti-
diabetic effects of 26 and its protein expression profiles, further
studies are underway to identify the molecular target of the
compound.
(CDCl3, 125 MHz)
d 200.4, 167.0, 163.3, 162.0, 161.4, 131.2, 131.1,
129.4, 129.4, 116.1, 115.9, 101.6, 62.8, 46.9, 14.2; LR-MS (ESIþ) m/z
253 [M þ H]þ; HR-MS (ESIþ) calcd for C13H14FO4 [M þ H]þ
253.0871; found 253.0874.
4.1.1.3. Ethyl
5-(4-methoxyphenyl)-2,4-dioxopentanoate
(3c).
4-Methoxyphenylacetone (2c) (5.06 g, 30.8 mmol) afforded 5.73 g
(70%) of 3c as a dark orange oil. 3c was purified by flash column
chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 7): 1H NMR
(CDCl3, 400 MHz)
d
7.15 (d, 2H, J ¼ 8.3 Hz, ArꢁH), 6.88 (d, 2H,
4. Materials and methods
J ¼ 8.8 Hz, ArꢁH), 6.35 (s, 1H, CH), 4.31 (q, 2H, J ¼ 7.1 Hz, OCH2CH3),
3.79 (s, 3H, OCH3), 3.70 (s, 2H, ArꢁCH2), 1.34 (t, 3H, J ¼ 7.2 Hz,
4.1. Chemistry
OCH2CH3); 13C NMR (CDCl3, 125 MHz)
d 201.2, 166.8, 162.1, 159.1,
130.6, 125.7, 114.5, 101.7, 62.7, 55.4, 47.0, 14.2; DEPT 90 (CDCl3,
125 MHz) 130.6, 114.5, 101.7; DEPT 135 (CDCl3, 125 MHz) 130.6,
114.5, 101.7, 62.7 (ꢁ), 55.4, 47.0 (ꢁ), 14.2; LR-MS (ESIþ) m/z 287
[M þ Na]þ; HR-MS (ESIþ) calcd for C14H16NaO5 [M þ Na]þ
287.0890; found 287.0879.
Unless noted otherwise, all starting materials and reagents were
obtained from commercial suppliers and were used without further
purification. Reaction flasks were dried at 100 ꢂC. Air- and
moisture-sensitive reactions were performed under an argon at-
mosphere. All solvents used for routine isolation of products and
chromatography were reagent-grade. Flash column chromatog-
raphy was performed using silica gel 60 (230e400 mesh, Merck)
with the indicated solvents. Thin-layer chromatography was per-
formed using 0.25 mm silica gel plates (Merck). 1H and 13C NMR
spectra were recorded on Varian Unity 400, AVANCE NEO 500, and
Unity-Inova 500 as solutions in the indicated solvents. Chemical
d
d
4.1.1.4. Ethyl
5-(3-chlorophenyl)-2,4-dioxopentanoate
(3d).
3-Chlorophenylacetone (2d) (200 mg, 1.54 mmol) afforded 254 mg
(61%) of 3d as a colorless oil. 3d was purified by flash column
chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 7): 1H NMR
(CDCl3, 500 MHz)
d
7.27 (m, 3H, ArꢁH), 7.13 (m, 1H, ArꢁH), 6.36 (s,
1H, CH), 4.34 (q, 2H, J ¼ 7.1 Hz, OCH2CH3), 3.75 (s, 2H, ArꢁCH2), 1.36
shifts were expressed in parts per million (ppm, d) downfield from
(t, 3H, J ¼ 7.1 Hz, OCH2CH3); 13C NMR (CDCl3, 125 MHz)
d 199.7,
tetramethylsilane and were referenced to the deuterated solvent.
1H NMR data were reported in the order of chemical shift, multi-
plicity (s, singlet; brs, broad singlet; d, doublet; dd, doublet of
doublets; t, triplet; td, triplet of doublets; q, quartet; qd, quartet of
doublets; m, multiplet and/or multiple resonance), number of
protons, and coupling constant in hertz (Hz). High-resolution mass
spectra were obtained with an Agilent 6530 Accurate-Mass Q-TOF
and a JEOL JMS-AX 505WA instrument.
167.1, 161.9, 135.6, 134.8, 130.2, 129.7, 127.9, 127.8, 101.7, 62.8, 47.2,
14.1; DEPT 90 (CDCl3, 125 MHz)
d 130.2, 129.7, 127.9, 127.8, 101.7;
DEPT 135 (CDCl3, 125 MHz)
d 130.2, 129.7, 127.9, 127.8, 101.7, 62.8
(ꢁ), 47.2 (ꢁ), 14.1; LR-MS (ESIþ) m/z 269 [M þ H]þ; HR-MS (ESIþ)
calcd for C13H14ClO4 [M þ H]þ 269.0575; found 269.0583.
4.1.1.5. Ethyl
5-(2-chlorophenyl)-2,4-dioxopentanoate
(3e).
2-Chlorophenylacetone (2e) (200 mg, 1.54 mmol) afforded 316 mg
(77%) of 3e as a pale yellow oil. 3e was purified by flash column
chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 4): 1H NMR
4.1.1. General procedures for the preparation of 3a-3e
To a stirred solution of phenylacetone in toluene was added t-
BuOK (1.2 equiv. of a 1.0 M solution in THF) at 0 ꢂC. After stirring for
30 min at the same temperature, to the reaction mixture was added
diethyl oxalate (1.1 equiv.). The resulting mixture was stirred for
12 h at ambient temperature and quenched with saturated aqueous
NH4Cl. The reaction mixture was extracted with EtOAc and the
combined organic layers were dried over MgSO4 and then
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel.
(CDCl3, 500 MHz)
d
7.41 (m, 1H, ArꢁH), 7.26 (m, 3H, ArꢁH), 6.37 (s,
1H, CH), 4.33 (q, 2H, J ¼ 7.1 Hz, OCH2CH3), 3.95 (s, 2H, ArꢁCH2), 1.35
(t, 3H, J ¼ 7.2 Hz, OCH2CH3); 13C NMR (CDCl3, 125 MHz)
d 200.3,
165.8, 162.1, 134.7, 132.1, 131.9, 129.9, 129.3, 127.3, 101.9, 62.7, 45.7,
14.2; LR-MS (ESIþ) m/z 269 [M þ H]þ; HR-MS (ESIþ) calcd for
C
13H14ClO4 [M þ H]þ 269.0575; found 269.0577.
4.1.2. General procedures for the preparation of 4a-4e
To a stirred solution of 2,4-dioxopentanoate in acetic acid was
added hydrazine monohydrate (2.0 equiv.) at ambient temperature.
After stirring for 12 h, the reaction mixture was quenched with
saturated aqueous NaHCO3 and then extracted with EtOAc. The
combined organic layers were dried over MgSO4 and concentrated
in vacuo. The residue was purified by flash column chromatography
on silica gel.
4.1.1.1. Ethyl
5-(4-chlorophenyl)-2,4-dioxopentanoate
(3a).
4-Chlorophenylacetone (2a) (12.1 g, 71.7 mmol) afforded 18.6 g
(97%) of 3a as a dark red oil. 3a was purified by flash column
chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 7 to 1 : 2): 1H
NMR (CDCl3, 400 MHz)
J ¼ 8.2 Hz, ArꢁH), 6.35 (d, 1H, J ¼ 0.7 Hz, CH), 4.33 (qd, 2H, J ¼ 7.1,
d
7.32 (d, 2H, J ¼ 8.1 Hz, ArꢁH), 7.17 (d, 2H,
7

J. Jo, D. Lee, Y.H. Park et al.
European Journal of Medicinal Chemistry 217 (2021) 113325
4.1.2.1. Ethyl 3-(4-chlorobenzyl)-1H-pyrazole-5-carboxylate (4a).
Ethyl 5-(4-chlorophenyl)-2,4-dioxopentanoate (3a) (2.47 g,
9.20 mmol) afforded 2.26 g (93%) of 4a as a dark red oil. 4a was
purified by flash column chromatography on silica gel (EtOAc: n-
organic layers were dried over MgSO4 and concentrated in vacuo.
The residue was purified by flash column chromatography on silica
gel.
hexane ¼ 1 : 3): 1H NMR (CDCl3, 400 MHz)
d
11.31 (brs, 1H, NH),
4.1.3.1. 3-(4-Chlorobenzyl)-1H-pyrazole-5-carboxylic
acid
(5a).
7.27 (d, 2H, J ¼ 8.4 Hz, ArꢁH), 7.15 (d, 2H, J ¼ 8.4 Hz, ArꢁH), 6.56 (s,
1H, pyrazoleꢁH), 4.35 (q, 2H, J ¼ 7.1 Hz, OCH2CH3), 4.01 (s, 2H,
ArꢁCH2), 1.36 (t, 3H, J ¼ 7.1 Hz, OCH2CH3); 13C NMR (CDCl3,
Ethyl 3-(4-chlorobenzyl)-1H-pyrazole-5-carboxylate (4a) (10.3 g,
39.0 mmol) afforded 8.24 g (89%) of 5a as pale yellow solid. 5a was
purified by flash column chromatography on silica gel (EtOAc: n-
hexane ¼ 1 : 2 to MeOH: EtOAc ¼ 1 : 10): 1H NMR (DMSO‑d6,
100 MHz) d 161.3, 148.1, 139.9, 136.9, 132.6, 130.2, 128.9, 107.5, 61.3,
32.6, 14.4; LR-MS (ESIþ) m/z 265 [M þ H]þ; HR-MS (ESIþ) calcd for
400 MHz)
d
7.34 (d, 2H, J ¼ 7.6 Hz, ArꢁH), 7.26 (d, 2H, J ¼ 7.5 Hz,
C
13H14ClN2O2 [M þ H]þ 265.0738; found 265.0736.
ArꢁH), 6.30 (s, 1H, pyrazoleꢁH), 3.92 (s, 2H, ArꢁCH2); 13C NMR
(DMSO‑d6, 100 MHz)
d 162.3, 146.6, 140.3, 138.3, 131.0, 130.4, 128.4,
4.1.2.2. Ethyl 3-(4-fluorobenzyl)-1H-pyrazole-5-carboxylate (4b).
Ethyl 5-(4-fluorophenyl)-2,4-dioxopentanoate (3b) (2.25 g,
8.92 mmol) afforded 1.48 g (67%) of 4b as a yellow oil. 4b was
purified by flash column chromatography on silica gel (EtOAc: n-
106.9, 31.5; LR-MS (ESIþ) m/z 237 [M þ H]þ; HR-MS (ESIþ) calcd for
C
11H10ClN2O2 [M þ H]þ 237.0425; found 237.0424.
4.1.3.2. 3-(4-Fluorobenzyl)-1H-pyrazole-5-carboxylic
acid
(5b).
hexane ¼ 1 : 3): 1H NMR (CDCl3, 400 MHz)
d
7.17 (dd, 2H, J ¼ 8.4,
Ethyl 3-(4-fluorobenzyl)-1H-pyrazole-5-carboxylate (4b) (990 mg,
3.99 mmol) afforded 766 mg (87%) of 5b as white solid. 5b was
purified by flash column chromatography on silica gel (EtOAc: n-
hexane ¼ 1 : 2 to MeOH: EtOAc ¼ 1 : 10): 1H NMR (DMSO‑d6,
5.4 Hz, ArꢁH), 6.97 (t, 2H, J ¼ 8.6 Hz, ArꢁH), 6.54 (s, 1H,
pyrazoleꢁH), 4.33 (q, 2H, J ¼ 7.1 Hz, OCH2CH3), 4.02 (s, 2H, ArꢁCH2),
1.33 (t, 3H, J ¼ 7.1 Hz, OCH2CH3); 13C NMR (CDCl3, 100 MHz)
d 163.0,
161.2, 160.6, 148.6, 139.7, 134.1, 134.0, 130.3, 130.2, 115.7, 115.5, 107.6,
61.3, 32.5, 14.4; LR-MS (ESIþ) m/z 249 [M þ H]þ; HR-MS (ESIþ)
calcd for C13H14FN2O2 [M þ H]þ 249.1034; found 249.1036.
400 MHz)
d
7.28 (t, 2H, J ¼ 6.1 Hz, ArꢁH), 7.12 (t, 2H, J ¼ 8.1 Hz,
ArꢁH), 6.40 (s, 1H, pyrazoleꢁH), 3.94 (s, 2H, ArꢁCH2); 13C NMR
(DMSO‑d6, 100 MHz)
d 162.1, 159.7, 147.2, 141.4, 135.6, 130.4, 130.3,
115.2, 115.0, 106.3, 31.5; LR-MS (ESIþ) m/z 221 [M þ H]þ; HR-MS
4.1.2.3. Ethyl 3-(4-methoxybenzyl)-1H-pyrazole-5-carboxylate (4c).
Ethyl 5-(4-methoxyphenyl)-2,4-dioxopentanoate (3c) (3.47 g,
13.1 mmol) afforded 3.10 g (91%) of 4c as an orange oil. 4c was
purified by flash column chromatography on silica gel (EtOAc: n-
(ESIþ) calcd for C11H10FN2O2 [M þ H]þ 221.0721; found 221.0724.
4.1.3.3. 3-(4-Methoxybenzyl)-1H-pyrazole-5-carboxylic acid (5c).
Ethyl
3-(4-methoxybenzyl)-1H-pyrazole-5-carboxylate
(4c)
hexane ¼ 1 : 3): 1H NMR (CDCl3, 500 MHz)
d
7.13 (d, 2H, J ¼ 8.5 Hz,
(2.28 g, 8.76 mmol) afforded 1.45 g (71%) of 5c as white solid. 5c was
purified by flash column chromatography on silica gel (EtOAc: n-
hexane ¼ 1 : 2 to MeOH: EtOAc ¼ 1 : 10): 1H NMR (DMSO‑d6,
ArꢁH), 6.83 (d, 2H, J ¼ 8.6 Hz, ArꢁH), 6.55 (s, 1H, pyrazoleꢁH), 4.34
(q, 2H, J ¼ 7.1 Hz, OCH2CH3), 3.98 (s, 2H, ArꢁCH2), 3.78 (s, 3H,
OCH3), 1.34 (t, 3H, J ¼ 7.1 Hz, OCH2CH3); 13C NMR (CDCl3, 100 MHz)
400 MHz)
d
7.15 (d, 2H, J ¼ 8.6 Hz, ArꢁH), 6.86 (d, 2H, J ¼ 8.7 Hz,
d
161.2, 158.6, 149.1, 140.0, 130.2, 129.8, 114.3, 107.5, 61.3, 55.4, 32.5,
ArꢁH), 6.40 (s, 1H, pyrazoleꢁH), 3.87 (s, 2H, ArꢁCH2), 3.71 (s, 3H,
14.4; LR-MS (ESIþ) m/z 261 [M þ H]þ; HR-MS (ESIþ) calcd for
OCH3); 13C NMR (DMSO‑d6, 100 MHz)
d 162.6, 157.8, 147.1, 141.0,
C
14H17N2O3 [M þ H]þ 261.1234; found 261.1231.
131.1, 129.5,113.9, 106.5, 55.0, 31.2; LR-MS (ESIþ) m/z 233 [M þ H]þ;
HR-MS (ESIþ) calcd for C12H13N2O3 [M þ H]þ 233.0921; found
233.0923.
4.1.2.4. Ethyl 3-(3-chlorobenzyl)-1H-pyrazole-5-carboxylate (4d).
Ethyl 5-(3-chlorophenyl)-2,4-dioxopentanoate (3d) (2.60 g,
9.68 mmol) afforded 1.78 g (69%) of 4d as red solid. 4d was purified
by flash column chromatography on silica gel (EtOAc: n-hexane ¼ 1
4.1.3.4. 3-(3-Chlorobenzyl)-1H-pyrazole-5-carboxylic acid (5d).
Ethyl 3-(3-chlorobenzyl)-1H-pyrazole-5-carboxylate (4d) (180 mg,
0.680 mmol) afforded 153 mg (95%) of 5d as a colorless oil. 5d was
recrystallized with EtOAc/n-hexane: 1H NMR (DMSO‑d6, 500 MHz)
: 3): 1H NMR (CDCl3, 500 MHz)
d 12.01 (brs, 1H, NH), 7.20 (m, 3H,
ArꢁH), 7.09 (m, 1H, ArꢁH), 6.56 (s, 1H, pyrazoleꢁH), 4.34 (q, 2H,
J ¼ 7.1 Hz, OCH2CH3), 4.03 (s, 2H, ArꢁCH2), 1.33 (t, 3H, J ¼ 7.1 Hz,
d
13.10 (brs,1H, NH), 7.33 (t,1H, J ¼ 7.4 Hz, ArꢁH), 7.32 (s,1H, ArꢁH),
OCH2CH3); 13C NMR (CDCl3, 100 MHz)
d
161.2, 148.2, 140.6, 139.7,
7.27 (d, 1H, J ¼ 8.2 Hz, ArꢁH), 7.21 (d, 1H, J ¼ 7.6 Hz, ArꢁH), 6.51 (s,
134.6, 130.0, 129.0, 127.0, 127.0, 107.7, 61.3, 33.0, 14.4; LR-MS (ESIþ)
m/z 265 [M þ H]þ; HR-MS (ESIþ) calcd for C13H14ClN2O2 [M þ H]þ
265.0738; found 265.0739.
1H, pyrazoleꢁH), 3.97 (s, 2H, ArꢁCH2); 13C NMR (DMSO‑d6,
125 MHz) d 162.2,146.0,141.8,140.3,133.0,130.4,128.4,127.3,126.3,
107.0, 31.7; LR-MS (ESIþ) m/z 237 [M þ H]þ; HR-MS (ESIþ) calcd for
C
11H10ClN2O2 [M þ H]þ 237.0425; found 237.0426.
4.1.2.5. Ethyl 3-(2-chlorobenzyl)-1H-pyrazole-5-carboxylate (4e).
Ethyl 5-(2-chlorophenyl)-2,4-dioxopentanoate (3e) (316 mg,
1.18 mmol) afforded 246 mg (79%) of 4e as an orange oil. 4e was
purified by flash column chromatography on silica gel (EtOAc: n-
4.1.3.5. 3-(2-Chlorobenzyl)-1H-pyrazole-5-carboxylic
acid
(5e).
Ethyl 3-(2-chlorobenzyl)-1H-pyrazole-5-carboxylate (4e) (226 mg,
0.855 mmol) afforded 192 mg (95%) of 5e as pale yellow solid. 5e
was purified by flash column chromatography on silica gel (EtOAc:
n-hexane ¼ 1 : 1 to MeOH: EtOAc ¼ 1 : 10): 1H NMR (DMSO‑d6,
hexane ¼ 1 : 4): 1H NMR (CDCl3, 500 MHz)
d 8.15 (brs, 1H, NH), 7.38
(m, 1H, ArꢁH), 7.25 (m, 1H, ArꢁH), 7.20 (m, 2H, ArꢁH), 6.60 (s, 1H,
pyrazoleꢁH), 4.35 (q, 2H, J ¼ 7.1 Hz, OCH2CH3), 4.19 (s, 2H, ArꢁCH2),
500 MHz)
d
13.12 (brs, 1H, NH), 7.45 (m, 1H, ArꢁH), 7.32 (m, 1H,
1.35 (t, 3H, J ¼ 7.2 Hz, OCH2CH3); 13C NMR (CDCl3, 125 MHz)
d
160.9,
ArꢁH), 7.29 (m, 2H, ArꢁH), 6.40 (s, 1H, pyrazoleꢁH), 4.07 (s, 2H,
147.5, 139.6, 136.1, 134.1, 130.9, 129.8, 128.5, 127.3, 107.9, 61.4, 31.1,
ArꢁCH2); 13C NMR (DMSO‑d6, 125 MHz)
d 162.3, 145.8, 140.4, 136.7,
14.4; LR-MS (ESIþ) m/z 265 [M þ H]þ; HR-MS (ESIþ) calcd for
132.9, 131.0, 129.3, 128.5, 127.4, 106.9, 30.2; LR-MS (ESIþ) m/z 237
[M þ H]þ; HR-MS (ESIþ) calcd for C11H10ClN2O2 [M þ H]þ 237.0425;
found 237.0422.
C
13H14ClN2O2 [M þ H]þ 265.0738; found 265.0739.
4.1.3. General procedures for the preparation of 5a-5e, 45, and 48
To a stirred solution of 1H-pyrazole-5-carboxylate in THF was
added a solution of LiOH$H2O (2.0 equiv.) in H2O at ambient tem-
perature. After stirring for 6 h at 50 ꢂC, the reaction mixture was
acidified with 2 N HCl then extracted with EtOAc. The combined
4.1.3.6. 3-(4-Chlorobenzyl)-1-methyl-1H-pyrazole-5-carboxylic acid
(45). Ethyl
3-(4-chlorobenzyl)-1-methyl-1H-pyrazole-5-
carboxylate (44) (237 mg, 0.850 mmol) afforded 202 mg (95%) of
45 as pale yellow solid. 45 was purified by flash column
8

J. Jo, D. Lee, Y.H. Park et al.
European Journal of Medicinal Chemistry 217 (2021) 113325
chromatography on silica gel (EtOAc to MeOH: EtOAc ¼ 1 : 1): 1H
31.4, 30.4; LR-MS (ESIþ) m/z 368 [M þ H]þ; HR-MS (ESIþ) calcd for
NMR (DMSO‑d6, 500 MHz)
d
7.34 (d, 2H, J ¼ 8.4 Hz, ArꢁH), 7.26 (d,
C
21H23ClN3O [M þ H]þ 368.1524; found 368.1529.
2H, J ¼ 8.3 Hz, ArꢁH), 6.55 (s, 1H, pyrazoleꢁH), 4.00 (s, 3H, CH3),
3.87 (s, 2H, ArꢁCH2), 3.34 (brs, 1H, COOH); 13C NMR (DMSO‑d6,
4.1.4.4. 3-(4-Chlorobenzyl)-N-(4-(trifluoromethyl)phenyl)-1H-pyr-
azole-5-carboxamide (8).
3-(4-Chlorobenzyl)-1H-pyrazole-5-carboxylic acid (5a) (60.4 mg,
125 MHz) d 160.8, 149.1, 138.9, 134.1, 130.7, 130.5, 128.3, 109.6, 38.8,
32.8; LR-MS (ESIþ) m/z 251 [M þ H]þ; HR-MS (ESIþ) calcd for
C
12H12ClN2O2 [M þ H]þ 251.0582; found 251.0583.
0.255 mmol) and 4-(trifluoromethyl)aniline (32.8 mL, 0.255 mmol)
afforded 17.1 mg (18%) of 8 as pale yellow solid. 8 was purified by
flash column chromatography on silica gel (EtOAc: n-hexane ¼ 1 :
4.1.3.7. 3-(4-Chlorobenzoyl)-1H-pyrazole-5-carboxylic acid (48).
Ethyl 3-(4-chlorobenzoyl)-1H-pyrazole-5-carboxylate (47)
4): 1H NMR (DMSO‑d6, 400 MHz)
d 13.36 (s, 1H, NH), 10.39 (s, 1H,
(645 mg, 2.31 mmol) afforded 534 mg (92%) of 48 as white solid. 48
was purified by flash column chromatography on silica gel (MeOH:
NH), 8.04 (d, 2H, J ¼ 8.4 Hz, ArꢁH), 7.67 (d, 2H, J ¼ 8.4 Hz, ArꢁH),
7.39 (d, 2H, J ¼ 7.5 Hz, ArꢁH), 7.29 (d, 2H, J ¼ 8.2 Hz, ArꢁH), 6.56 (s,
1H, pyrazoleꢁH), 4.04 (s, 2H, ArꢁCH2); 13C NMR (DMSO‑d6,
CH2Cl2 ¼ 1 : 10): 1H NMR (DMSO‑d6, 500 MHz)
8.17 (s, 2H, ArꢁH),
d
7.63 (d, 2H, J ¼ 8.5 Hz, ArꢁH), 7.26 (s, 1H, pyrazoleꢁH); 13C NMR
100 MHz) d 161.1, 146.9, 143.9, 142.8, 137.9, 131.4, 130.8, 130.6, 128.7,
(DMSO‑d6, 125 MHz)
d
185.3, 160.5, 150.6, 138.0, 137.1, 135.3, 131.9,
128.6, 126.3, 126.1, 126.0, 124.0, 123.7, 123.3, 123.0, 120.2, 105.2,
128.6, 111.0; LR-MS (ESIþ) m/z 251 [M þ H]þ; HR-MS (ESIþ) calcd
30.4; LR-MS (ESIþ) m/z 380 [M þ H]þ; HR-MS (ESIþ) calcd for
for C11H8ClN2O3 [M þ H]þ 251.0218; found 251.0219.
C
18H14ClF3N3O [M þ H]þ 380.0772; found 380.0786.
4.1.4. General procedures for the preparation of 1 and 6e42
4.1.4.5. 3-(4-Chlorobenzyl)-N-(4-methoxyphenyl)-1H-pyrazole-5-
carboxamide (9). 3-(4-Chlorobenzyl)-1H-pyrazole-5-carboxylic
To a stirred solution of carboxylic acid and aniline in CH2Cl2
were added EDC$HCl (2.0 equiv.) and DMAP (0.1 equiv.) at ambient
temperature. After stirring for 2 h, the reaction mixture was
quenched with H2O and then extracted with CH2Cl2. The combined
organic layer was dried over MgSO4 and concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel.
acid (5a) (45.1 mg, 0.190 mmol) and 4-methoxyaniline (23.4 mg,
0.190 mmol) afforded 23.9 mg (37%) of 9 as white solid. 9 was
purified by flash column chromatography on silica gel (EtOAc: n-
hexane ¼ 1 : 2 to 1 : 1): 1H NMR (DMSO‑d6, 400 MHz)
d 13.24 (s, 1H,
NH), 9.84 (s, 1H, NH), 7.68 (d, 2H, J ¼ 7.4 Hz, ArꢁH), 7.38 (d, 2H,
J ¼ 7.8 Hz, ArꢁH), 7.29 (d, 2H, J ¼ 7.4 Hz, ArꢁH), 6.88 (d, 2H,
J ¼ 8.1 Hz, ArꢁH), 6.50 (s, 1H, pyrazoleꢁH), 4.02 (s, 2H, ArꢁCH2),
4.1.4.1. 3-(4-chlorobenzyl)-N-(4-fluorophenyl)-1H-pyrazole-5-
carboxamide
(1). 3-(4-Chlorobenzyl)-1H-pyrazole-5-carboxylic
L,
3.73 (s, 3H, OCH3); 13C NMR (DMSO‑d6, 100 MHz)
d 160.2, 155.3,
acid (5a) (45.4 mg, 0.192 mmol) and 4-fluoroaniline (18.2
m
147.2, 143.4, 137.7, 132.0, 131.2, 130.4, 128.5, 121.7, 113.7, 104.5, 55.2,
0.192 mmol) afforded 32.1 mg (51%) of 1 as pale yellow solid. 1 was
purified by flash column chromatography on silica gel (EtOAc: n-
30.3; LR-MS (ESIþ) m/z 342 [M þ H]þ; HR-MS (ESIþ) calcd for
C
18H17ClN3O2 [M þ H]þ 342.1004; found 342.1007.
hexane ¼ 1 : 2 to 1 : 1): 1H NMR (DMSO‑d6, 400 MHz)
d 10.08 (s, 1H,
NH), 7.77 (m, 2H, ArꢁH), 7.38 (d, 2H, J ¼ 8.2 Hz, ArꢁH), 7.29 (d, 2H,
J ¼ 8.4 Hz, ArꢁH), 7.15 (t, 2H, J ¼ 8.8 Hz, ArꢁH), 6.53 (s, 1H,
pyrazoleꢁH), 4.01 (s, 2H, ArꢁCH2); 13C NMR (DMSO‑d6, 100 MHz)
4.1.4.6. Methyl 4-(3-(4-chlorobenzyl)-1H-pyrazole-5-carboxamido)
benzoate (10). 3-(4-Chlorobenzyl)-1H-pyrazole-5-carboxylic acid
(5a) (102 mg, 0.431 mmol) and methyl 4-aminobenzoate (65.1 mg,
0.431 mmol) afforded 70.2 mg (44%) of 10 as pale yellow solid. 10
was purified by flash column chromatography on silica gel (EtOAc:
d
160.6, 159.5, 157.1, 146.9, 143.7, 138.0, 135.2, 131.2, 130.5, 128.6,
122.2, 122.1, 115.4, 115.1, 104.9, 30.4; LR-MS (ESIþ) m/z 330 [M þ
H]þ; HR-MS (ESIþ) calcd for C17H14ClFN3O [M þ H]þ 330.0804;
found 330.0801.
n-hexane ¼ 1 : 2 to 2 : 1): 1H NMR (DMSO‑d6, 400 MHz)
d 13.37 (s,
1H, NH), 10.36 (s, 1H, NH), 7.97 (d, 2H, J ¼ 8.8 Hz, ArꢁH), 7.91 (d, 2H,
J ¼ 8.5 Hz, ArꢁH), 7.39 (d, 2H, J ¼ 7.9 Hz, ArꢁH), 7.30 (d, 2H,
J ¼ 7.9 Hz, ArꢁH), 6.55 (s, 1H, pyrazoleꢁH), 4.04 (s, 2H, ArꢁCH2),
4.1.4.2. 3-(4-Chlorobenzyl)-N-phenyl-1H-pyrazole-5-carboxamide
(6). 3-(4-Chlorobenzyl)-1H-pyrazole-5-carboxylic
acid
(5a)
3.82 (s, 3H, COOCH3); 13C NMR (DMSO‑d6, 100 MHz)
d 165.9, 160.9,
(48.2 mg, 0.204 mmol) and aniline (18.6 L, 0.204 mmol) afforded
m
146.7, 143.7, 143.5, 137.7, 131.2, 130.4, 130.1, 128.6, 124.0, 119.5, 105.0,
52.0, 30.2; LR-MS (ESIþ) m/z 370 [M þ H]þ; HR-MS (ESIþ) calcd for
C19H17ClN3O3 [M þ H]þ 370.0953; found 370.0962.
22.3 mg (35%) of 6 as pale yellow solid. 6 was purified by flash
column chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 2 to 1 :
1): 1H NMR (DMSO‑d6, 400 MHz)
d 13.26 (s, 1H, NH), 9.94 (s, 1H,
NH), 7.78 (d, 2H, J ¼ 8.0 Hz, ArꢁH), 7.38 (d, 2H, J ¼ 8.5 Hz, ArꢁH),
7.30 (d, 2H, J ¼ 6.0 Hz, ArꢁH), 7.28 (d, 2H, J ¼ 8.2 Hz, ArꢁH), 7.04 (t,
1H, J ¼ 7.2 Hz, ArꢁH), 6.51 (s,1H, pyrazoleꢁH), 4.02 (s, 2H, ArꢁCH2);
4.1.4.7. 3-(4-Fluorobenzyl)-N-(4-fluorophenyl)-1H-pyrazole-5-
carboxamide
(11). 3-(4-Fluorobenzyl)-1H-pyrazole-5-carboxylic
L,
acid (5b) (47.7 mg, 0.217 mmol) and 4-fluoroaniline (20.6
m
13C NMR (DMSO‑d6,100 MHz)
d
160.4,147.1,143.5,138.8,137.7, 131.1,
0.217 mmol) afforded 37.7 mg (55%) of 11 as white solid. 11 was
purified by flash column chromatography on silica gel (EtOAc: n-
130.3, 128.5, 128.5, 123.3, 120.1, 104.7, 30.2; LR-MS (ESIþ) m/z 312
[M þ H]þ; HR-MS (ESIþ) calcd for C17H15ClN3O [M þ H]þ 312.0898;
found 312.0897.
hexane ¼ 1 : 2 to 1 : 1): 1H NMR (DMSO‑d6, 400 MHz)
d 13.29 (s, 1H,
NH), 10.10 (s, 1H, NH), 7.83 (dd, 2H, J ¼ 9.0, 5.1 Hz, ArꢁH), 7.32 (dd,
2H, J ¼ 8.4, 5.6 Hz, ArꢁH), 7.17 (dd, 2H, J ¼ 8.9, 3.1 Hz, ArꢁH), 7.15
(dd, 2H, J ¼ 8.9, 3.2 Hz, ArꢁH), 6.52 (s, 1H, pyrazoleꢁH), 4.03 (s, 2H,
4.1.4.3. N-(4-(tert-Butyl)phenyl)-3-(4-chlorobenzyl)-1H-pyrazole-5-
carboxamide
(7). 3-(4-Chlorobenzyl)-1H-pyrazole-5-carboxylic
L,
ArꢁCH2); 13C NMR (DMSO‑d6, 100 MHz)
d 162.2, 160.5, 159.8, 159.3,
acid (5a) (48.9 mg, 0.207 mmol) and 4-t-butylaniline (33.0
m
156.9, 147.0, 143.9, 135.3, 135.3, 134.9, 134.8, 130.4, 130.3, 122.0,
121.9, 115.4, 115.2, 115.0, 104.7, 30.1; LR-MS (ESIþ) m/z 314 [M þ
H]þ; HR-MS (ESIþ) calcd for C17H14F2N3O [M þ H]þ 314.1099; found
314.1099.
0.207 mmol) afforded 20.3 mg (27%) of 7 as pale yellow solid. 7 was
purified by flash column chromatography on silica gel (EtOAc: n-
hexane ¼ 1 : 2 to 1 : 1): 1H NMR (DMSO‑d6, 400 MHz)
d 13.24 (s, 1H,
NH), 9.86 (s, 1H, NH), 7.67 (d, 2H, J ¼ 8.8 Hz, ArꢁH), 7.37 (d, 2H,
J ¼ 8.0 Hz, ArꢁH), 7.30 (d, 2H, J ¼ 7.8 Hz, ArꢁH), 7.28 (d, 2H,
J ¼ 8.2 Hz, ArꢁH), 6.50 (s, 1H, pyrazoleꢁH), 4.01 (s, 2H, ArꢁCH2),
4.1.4.8. 3-(4-Fluorobenzyl)-N-phenyl-1H-pyrazole-5-carboxamide
(12). 3-(4-Fluorobenzyl)-1H-pyrazole-5-carboxylic
(53.6 mg, 0.243 mmol) and aniline (22.1 L, 0.243 mmol) afforded
40.1 mg (56%) of 12 as white solid. 12 was purified by flash column
acid
(5b)
1.24 (s, 9H, (CH3)3); 13C NMR (DMSO‑d6, 100 MHz)
d
160.5, 147.4,
m
145.8, 143.6, 137.9, 136.5, 131.4, 130.5, 128.7, 125.3, 120.1, 104.9, 34.2,
9

J. Jo, D. Lee, Y.H. Park et al.
European Journal of Medicinal Chemistry 217 (2021) 113325
chromatography on silica gel (EtOAc: n-hexane ¼ 2 : 3 to 3 : 1): 1H
124.0, 119.4, 115.4, 115.2, 104.9, 51.9, 30.1; LR-MS (ESIþ) m/z 354
[M þ H]þ; HR-MS (ESIþ) calcd for C19H17FN3O3 [M þ H]þ 354.1248;
found 354.1263.
NMR (DMSO‑d6, 400 MHz) d 13.29 (s, 1H, NH), 9.99 (s, 1H, NH), 7.81
(d, 2H, J ¼ 7.8 Hz, ArꢁH), 7.33 (m, 2H, ArꢁH), 7.29 (m, 2H, ArꢁH),
7.15 (t, 2H, J ¼ 8.7 Hz, ArꢁH), 7.05 (t, 1H, J ¼ 7.2 Hz, ArꢁH), 6.53 (s,
1H, pyrazoleꢁH), 4.03 (s, 2H, ArꢁCH2); 13C NMR (DMSO‑d6,
4.1.4.13. N-(4-Fluorophenyl)-3-(4-methoxybenzyl)-1H-pyrazole-5-
carboxamide (17).
3-(4-Methoxybenzyl)-1H-pyrazole-5-carboxylic acid (5c) (100 mg,
100 MHz) d 162.2,160.6,159.8,147.1, 144.0,138.9,134.9,130.4,130.3,
128.7, 128.6, 123.4, 120.2, 115.4, 115.2, 104.7, 30.1; LR-MS (ESIþ) m/z
296 [M þ H]þ; HR-MS (ESIþ) calcd for C17H15FN3O [M þ H]þ
296.1194; found 296.1194.
0.431 mmol) and 4-fluoroaniline (40.8 mL, 0.431 mmol) afforded
71.5 mg (51%) of 17 as white solid. 17 was purified by flash column
chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 2): 1H NMR
4.1.4.9. N-(4-(tert-Butyl)phenyl)-3-(4-fluorobenzyl)-1H-pyrazole-5-
(DMSO‑d6, 400 MHz)
d 13.26 (s, 1H, NH), 10.10 (s, 1H, NH), 7.83 (m,
carboxamide
(13). 3-(4-Fluorobenzyl)-1H-pyrazole-5-carboxylic
L,
2H, ArꢁH), 7.19 (d, 2H, J ¼ 8.4 Hz, ArꢁH), 7.15 (m, 2H, ArꢁH), 6.88 (t,
acid (5b) (62.5 mg, 0.284 mmol) and 4-t-butylaniline (45.2
m
2H, J ¼ 8.9 Hz, ArꢁH), 6.48 (s, 1H, pyrazoleꢁH), 3.95 (s, 2H,
0.284 mmol) afforded 78.8 mg (79%) of 13 as white solid. 13 was
purified by flash column chromatography on silica gel (EtOAc: n-
ArꢁCH2), 3.72 (s, 3H, OCH3); 13C NMR (DMSO‑d6, 100 MHz)
d 160.4,
159.1, 157.7, 156.7, 146.7, 144.3, 135.2, 130.4, 129.3, 121.8, 121.7, 115.0,
114.8, 113.8, 104.4, 54.9, 29.9; LR-MS (ESIþ) m/z 326 [M þ H]þ; HR-
MS (ESIþ) calcd for C18H17FN3O2 [M þ H]þ 326.1299; found
326.1293.
hexane ¼ 1 : 3 to 1 : 1): 1H NMR (CDCl3, 400 MHz)
d 11.60 (brs, 1H,
NH), 8.80 (s, 1H, NH), 7.54 (d, 2H, J ¼ 8.6 Hz, ArꢁH), 7.33 (d, 2H,
J ¼ 8.6 Hz, ArꢁH), 7.09 (dd, 2H, J ¼ 8.4, 5.4 Hz, ArꢁH), 6.91 (t, 2H,
J ¼ 8.6 Hz, ArꢁH), 6.62 (s, 1H, pyrazoleꢁH), 3.94 (s, 2H, ArꢁCH2),
1.30 (s, 9H, (CH3)3); 13C NMR (CDCl3, 100 MHz)
d 163.1, 160.6, 160.3,
147.5, 146.8, 145.2, 135.0, 133.2, 133.1, 130.2, 130.2, 126.0, 120.0,
115.8, 115.6, 105.4, 34.5, 31.4, 31.3; LR-MS (ESIþ) m/z 352 [M þ H]þ;
HR-MS (ESIþ) calcd for C21H23FN3O [M þ H]þ 352.1820; found
352.1823.
4.1.4.14. 3-(4-Methoxybenzyl)-N-phenyl-1H-pyrazole-5-
carboxamide (18).
3-(4-Methoxybenzyl)-1H-pyrazole-5-carboxylic acid (5c) (100 mg,
0.431 mmol) and aniline (39.3 mL, 0.431 mmol) afforded 58.3 mg
(44%) of 18 as white solid. 18 was purified by flash column chro-
4.1.4.10. 3-(4-Fluorobenzyl)-N-(4-(trifluoromethyl)phenyl)-1H-pyr-
azole-5-carboxamide (14).
matography on silica gel (EtOAc: n-hexane ¼ 1 : 2): 1H NMR
(DMSO‑d6, 400 MHz)
d 13.25 (s, 1H, NH), 9.96 (s, 1H, NH), 7.80 (d,
3-(4-Fluorobenzyl)-1H-pyrazole-5-carboxylic acid (5b) (72.0 mg,
0.327 mmol) and 4-(trifluoromethyl)aniline (41.1 mL, 0.327 mmol)
2H, J ¼ 7.9 Hz, ArꢁH), 7.30 (t, 2H, J ¼ 7.4 Hz, ArꢁH), 7.19 (d, 2H,
J ¼ 8.3 Hz, ArꢁH), 7.05 (t, 1H, J ¼ 7.0 Hz, ArꢁH), 6.89 (d, 2H,
J ¼ 8.2 Hz, ArꢁH), 6.48 (s, 1H, pyrazoleꢁH), 3.95 (s, 2H, ArꢁCH2),
afforded 55.9 mg (47%) of 14 as pale yellow solid. 14 was purified by
flash column chromatography on silica gel (EtOAc: n-hexane ¼ 1 :
3.72 (s, 3H, OCH3); 13C NMR (DMSO‑d6, 100 MHz)
d 160.6, 157.9,
4): 1H NMR (DMSO‑d6, 500 MHz)
d
13.35 (s, 1H, NH), 10.37 (s, 1H,
147.0, 144.6, 139.0, 130.6, 129.5, 128.6, 123.3, 120.1, 114.0, 104.5, 55.1,
NH), 8.05 (d, 2H, J ¼ 8.1 Hz, ArꢁH), 7.67 (d, 2H, J ¼ 8.3 Hz, ArꢁH),
7.31 (dd, 2H, J ¼ 8.5, 5.6 Hz, ArꢁH), 7.15 (t, 2H, J ¼ 8.8 Hz, ArꢁH),
6.56 (s, 1H, pyrazoleꢁH), 4.03 (s, 2H, ArꢁCH2); 13C NMR (DMSO‑d6,
30.1; LR-MS (ESIþ) m/z 308 [M þ H]þ; HR-MS (ESIþ) calcd for
C
18H18N3O2 [M þ H]þ 308.1394; found 308.1384.
100 MHz) d 162.2, 161.0, 159.8, 146.7, 144.1, 142.6, 134.8, 130.4,130.3,
4.1.4.15. N-(4-(tert-Butyl)phenyl)-3-(4-methoxybenzyl)-1H-pyr-
azole-5-carboxamide (19).
3-(4-Methoxybenzyl)-1H-pyrazole-5-carboxylic acid (5c) (100 mg,
125.8,123.8,123.5,123.2,123.1, 122.8,120.0,115.4,115.2,104.9, 30.1;
LR-MS (ESIþ) m/z 364 [M
þ
H]þ; HR-MS (ESIþ) calcd for
C
18H14F4N3O [M þ H]þ 364.1068; found 364.1064.
0.431 mmol) and 4-t-butylaniline (68.6 mL, 0.431 mmol) afforded
66.3 mg (42%) of 19 as pale yellow solid. 19 was purified by flash
4.1.4.11. 3-(4-Fluorobenzyl)-N-(4-methoxyphenyl)-1H-pyrazole-5-
carboxamide (15). 3-(4-Fluorobenzyl)-1H-pyrazole-5-carboxylic
column chromatography on silica gel (EtOAc: n-hexane ¼ 2 : 3): 1H
NMR (CDCl3, 400 MHz)
d
8.67 (s, 1H, NH), 7.56 (d, 2H, J ¼ 8.6 Hz,
acid (5b) (52.5 mg, 0.238 mmol) and 4-methoxyaniline (29.3 mg,
0.238 mmol) afforded 54.0 mg (70%) of 15 as white solid. 15 was
purified by flash column chromatography on silica gel (EtOAc: n-
ArꢁH), 7.34 (d, 2H, J ¼ 8.6 Hz, ArꢁH), 7.12 (d, 2H, J ¼ 8.5 Hz, ArꢁH),
6.84 (d, 2H, J ¼ 8.5 Hz, ArꢁH), 6.68 (s, 1H, pyrazoleꢁH), 3.97 (s, 2H,
ArꢁCH2), 3.77 (s, 3H, OCH3), 1.30 (s, 9H, (CH3)3); 13C NMR (CDCl3,
hexane ¼ 1 : 2 to 1 : 1): 1H NMR (DMSO‑d6, 400 MHz)
d 13.25 (s, 1H,
100 MHz) d 160.1, 158.7, 147.5, 147.2, 145.3, 135.2, 129.9, 129.1, 125.9,
NH), 9.88 (s, 1H, NH), 7.69 (d, 2H, J ¼ 8.8 Hz, ArꢁH), 7.31 (m, 2H,
ArꢁH), 7.15 (m, 2H, ArꢁH), 6.88 (d, 2H, J ¼ 6.6 Hz, ArꢁH), 6.49 (s,1H,
pyrazoleꢁH), 4.01 (s, 2H, ArꢁCH2), 3.71 (s, 3H, OCH3); 13C NMR
119.7, 114.4, 105.3, 55.4, 34.5, 31.5, 31.1; LR-MS (ESIþ) m/z 364 [M þ
H]þ; HR-MS (ESIþ) calcd for C22H26N3O2 [M þ H]þ 364.2020; found
364.2023.
(DMSO‑d6, 100 MHz)
d 162.2, 160.2, 159.8, 155.3, 147.2, 143.9, 135.0,
134.9, 132.1, 130.4, 130.3, 121.7, 115.4, 115.2, 113.7, 104.6, 55.2, 30.1;
LR-MS (ESIþ) m/z 326 [M
þ
H]þ; HR-MS (ESIþ) calcd for
4.1.4.16. 3-(4-Methoxybenzyl)-N-(4-(trifluoromethyl)phenyl)-1H-
pyrazole-5-carboxamide (20).
3-(4-Methoxybenzyl)-1H-pyrazole-5-carboxylic acid (5c) (56.3 mg,
C
18H17FN3O2 [M þ H]þ 326.1299; found 326.1296.
4.1.4.12. Methyl 4-(3-(4-fluorobenzyl)-1H-pyrazole-5-carboxamido)
benzoate (16). 3-(4-Fluorobenzyl)-1H-pyrazole-5-carboxylic acid
(5b) (68.9 mg, 0.313 mmol) and methyl 4-aminobenzoate (47.3 mg,
0.313 mmol) afforded 44.2 mg (40%) of 16 as pale yellow solid. 16
was purified by flash column chromatography on silica gel (EtOAc:
0.242 mmol) and 4-(trifluoromethyl)aniline (30.4 mL, 0.242 mmol)
afforded 30.9 mg (34%) of 20 as pale yellow solid. 20 was purified by
flash column chromatography on silica gel (EtOAc: n-hexane ¼ 1 :
4): 1H NMR (DMSO‑d6, 500 MHz)
d 13.31 (s, 1H, NH), 10.36 (s, 1H,
NH), 8.04 (d, 2H, J ¼ 7.8 Hz, ArꢁH), 7.67 (d, 2H, J ¼ 8.4 Hz, ArꢁH),
7.19 (d, 2H, J ¼ 8.7 Hz, ArꢁH), 6.89 (d, 2H, J ¼ 8.7 Hz, ArꢁH), 6.52 (s,
1H, pyrazoleꢁH), 3.96 (s, 2H, ArꢁCH2), 3.72 (s, 3H, OCH3); 13C NMR
n-hexane ¼ 1 : 2): 1H NMR (DMSO‑d6, 400 MHz)
d 13.36 (s, 1H, NH),
10.36 (s, 1H, NH), 7.98 (d, 2H, J ¼ 8.7 Hz, ArꢁH), 7.91 (d, 2H,
J ¼ 8.4 Hz, ArꢁH), 7.31 (dd, 2H, J ¼ 8.4, 5.7 Hz, ArꢁH), 7.15 (t, 2H,
J ¼ 8.8 Hz, ArꢁH), 6.55 (s, 1H, pyrazoleꢁH), 4.03 (s, 2H, ArꢁCH2),
(DMSO‑d6, 100 MHz)
d 161.0, 157.9, 146.6, 144.7, 142.6, 130.5, 129.5,
125.8, 125.8, 123.7, 123.4, 123.1, 122.8, 119.9, 113.9, 104.7, 55.0, 30.1;
3.82 (s, 3H, COOCH3); 13C NMR (DMSO‑d6, 100 MHz)
d
165.9, 162.2,
LR-MS (ESIþ) m/z 376 [M
þ
H]þ; HR-MS (ESIþ) calcd for
160.9, 159.8, 146.7, 144.1, 143.5, 134.8, 134.8, 130.4, 130.3, 130.0,
C
19H17F3N3O2 [M þ H]þ 376.1267; found 376.1284.
10

J. Jo, D. Lee, Y.H. Park et al.
European Journal of Medicinal Chemistry 217 (2021) 113325
4.1.4.17. 3-(4-Methoxybenzyl)-N-(4-methoxyphenyl)-1H-pyrazole-5-
carboxamide (21).
column chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 3): 1H
NMR (DMSO‑d6, 400 MHz) d 13.34 (s, 1H, NH), 9.38 (s, 1H, NH), 7.92
3-(4-Methoxybenzyl)-1H-pyrazole-5-carboxylic acid (5c) (100 mg,
0.431 mmol) and 4-methoxyaniline (53.1 mg, 0.431 mmol) afforded
60.7 mg (42%) of 21 as white solid. 21 was purified by flash column
chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 1): 1H NMR
(d, 1H, J ¼ 8.2 Hz, ArꢁH), 7.38 (d, 2H, J ¼ 8.4 Hz, ArꢁH), 7.29 (d, 2H,
J ¼ 8.3 Hz, ArꢁH), 7.05 (t, 1H, J ¼ 8.3 Hz, ArꢁH), 6.81 (d, 1H,
J ¼ 8.6 Hz, ArꢁH), 6.54 (s, 1H, pyrazoleꢁH), 4.03 (s, 2H, ArꢁCH2),
3.82 (s, 3H, OCH3), 3.80 (s, 3H, OCH3); 13C NMR (DMSO‑d6,100 MHz)
(CDCl3, 400 MHz)
d
10.59 (brs, 1H, NH), 8.62 (s, 1H, NH), 7.53 (d, 2H,
d 159.5, 152.0, 146.7, 144.2, 137.6, 137.3, 131.8, 131.2, 130.4, 128.6,
J ¼ 8.8 Hz, ArꢁH), 7.11 (d, 2H, J ¼ 8.4 Hz, ArꢁH), 6.86 (d, 2H,
J ¼ 9.0 Hz, ArꢁH), 6.84 (d, 2H, J ¼ 8.6 Hz, ArꢁH), 6.66 (s, 1H,
pyrazoleꢁH), 3.96 (s, 2H, ArꢁCH2), 3.78 (s, 3H, OCH3), 3.77 (s, 3H,
124.1,111.7,107.9, 104.5, 60.4, 55.8, 30.2; LR-MS (ESIþ) m/z 372 [M þ
H]þ; HR-MS (ESIþ) calcd for C19H19ClN3O3 [M þ H]þ 372.1109;
found 372.1110.
OCH3); 13C NMR (CDCl3, 100 MHz)
d 160.0, 158.7, 156.4, 147.3, 145.4,
130.9, 129.8, 129.2, 121.8, 114.4, 114.3, 105.2, 55.6, 55.4, 31.4; LR-MS
(ESIþ) m/z 338 [M þ H]þ; HR-MS (ESIþ) calcd for C19H20N3O3 [M þ
H]þ 338.1499; found 338.1502.
4.1.4.22. 3-(4-Chlorobenzyl)-N-(2,4-dimethoxyphenyl)-1H-pyrazole-
5-carboxamide (26).
3-(4-Chlorobenzyl)-1H-pyrazole-5-carboxylic acid (5a) (599 mg,
2.53 mmol) and 2,4-dimethoxyaniline (433 mL, 3.04 mmol) afforded
4.1.4.18. Methyl 4-(3-(4-methoxybenzyl)-1H-pyrazole-5-
carboxamido)benzoate (22).
514 mg (55%) of 26 as brown solid. 26 was purified by flash column
chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 4): 1H NMR
3-(4-Methoxybenzyl)-1H-pyrazole-5-carboxylic acid (5c) (57.5 mg,
0.248 mmol) and methyl 4-aminobenzoate (37.5 mg, 0.248 mmol)
afforded 20.3 mg (22%) of 22 as pale yellow solid. 22 was purified by
flash column chromatography on silica gel (EtOAc: n-hexane ¼ 1 :
(CDCl3, 400 MHz)
d
9.01 (s, 1H, NH), 8.31 (d, 1H, J ¼ 8.4 Hz, ArꢁH),
7.22 (d, 2H, J ¼ 8.3 Hz, ArꢁH), 7.08 (d, 2H, J ¼ 8.4 Hz, ArꢁH), 6.61 (s,
1H, pyrazoleꢁH), 6.48 (s, 1H, ArꢁH), 6.46 (d, 1H, J ¼ 2.6 Hz, ArꢁH),
3.97 (s, 2H, ArꢁCH2), 3.82 (s, 3H, OCH3), 3.79 (s, 3H, OCH3); 13C NMR
2): 1H NMR (DMSO‑d6, 400 MHz)
d
13.32 (s, 1H, NH), 10.34 (s, 1H,
(CDCl3, 100 MHz) d 160.0, 157.0, 150.2, 147.5, 145.0, 136.4, 133.2,
NH), 7.97 (d, 2H, J ¼ 8.8 Hz, ArꢁH), 7.91 (d, 2H, J ¼ 8.6 Hz, ArꢁH),
7.19 (d, 2H, J ¼ 8.6 Hz, ArꢁH), 6.89 (d, 2H, J ¼ 8.6 Hz, ArꢁH), 6.51 (s,
1H, pyrazoleꢁH), 3.96 (s, 2H, ArꢁCH2), 3.82 (s, 3H, COOCH3), 3.72 (s,
130.3, 129.3, 121.3, 121.2, 105.5, 104.2, 99.1, 56.2, 56.0, 32.0; LR-MS
(ESIþ) m/z 372 [M þ H]þ; HR-MS (ESIþ) calcd for C19H19ClN3O3
[M þ H]þ 372.1109; found 372.1114.
3H, OCH3); 13C NMR (DMSO‑d6, 100 MHz)
d 165.9, 160.9, 157.9,
146.6, 144.7, 143.5, 130.5, 130.0, 129.5, 123.9, 119.4, 114.0, 104.7, 55.1,
4.1.4.23. 3-(4-Chlorobenzyl)-N-(2,5-dimethoxyphenyl)-1H-pyrazole-
5-carboxamide (27).
51.9, 30.1; LR-MS (ESIþ) m/z 366 [M þ H]þ; HR-MS (ESIþ) calcd for
C
20H20N3O4 [M þ H]þ 366.1448; found 366.1458.
3-(4-Chlorobenzyl)-1H-pyrazole-5-carboxylic acid (5a) (613 mg,
2.59 mmol) and 2,5-dimethoxyaniline (476 mg, 3.11 mmol) affor-
ded 422 mg (44%) of 27 as pale yellow solid. 27 was purified by flash
column chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 4): 1H
4.1.4.19. 3-(4-Chlorobenzyl)-N-(2-methoxyphenyl)-1H-pyrazole-5-
carboxamide (23).
3-(4-Chlorobenzyl)-1H-pyrazole-5-carboxylic acid (5a) (45.5 mg,
NMR (CDCl3, 400 MHz)
d
9.22 (s, 1H, NH), 8.24 (d, 1H, J ¼ 2.8 Hz,
0.192 mmol) and 2-methoxyaniline (21.7
mL, 0.192 mmol) afforded
ArꢁH), 7.28 (d, 2H, J ¼ 8.4 Hz, ArꢁH), 7.14 (d, 2H, J ¼ 8.2 Hz, ArꢁH),
6.81 (d, 1H, J ¼ 9.0 Hz, ArꢁH), 6.66 (s, 1H, pyrazoleꢁH), 6.59 (dd, 1H,
J ¼ 8.9, 3.0 Hz, ArꢁH), 4.03 (s, 2H, ArꢁCH2), 3.86 (s, 3H, OCH3), 3.80
20.2 mg (31%) of 23 as pale yellow solid. 23 was purified by flash
column chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 4 to 1 :
1): 1H NMR (DMSO‑d6, 400 MHz)
d
13.33 (s, 1H, NH), 9.31 (s, 1H,
(s, 3H, OCH3); 13C NMR (CDCl3, 100 MHz)
d 159.8, 153.9, 147.4, 144.6,
NH), 8.28 (d, 1H, J ¼ 7.9 Hz, ArꢁH), 7.38 (d, 2H, J ¼ 7.1 Hz, ArꢁH),
7.29 (d, 2H, J ¼ 7.3 Hz, ArꢁH), 7.08 (m, 2H, ArꢁH), 6.95 (t, 1H,
J ¼ 6.8 Hz, ArꢁH), 6.53 (s, 1H, pyrazoleꢁH), 4.03 (s, 2H, ArꢁCH2),
142.6, 135.7, 133.1, 130.1, 129.1, 128.2, 111.0, 108.8, 106.1, 105.3, 56.5,
55.9, 31.7; LR-MS (ESIþ) m/z 372 [M þ H]þ; HR-MS (ESIþ) calcd for
C
19H19ClN3O3 [M þ H]þ 372.1109; found 372.1111.
3.89 (s, 3H, OCH3); 13C NMR (DMSO‑d6, 100 MHz)
d 159.5, 148.1,
146.7, 144.2,137.6,131.2, 130.4,128.6,127.2, 123.8,120.7,119.0, 110.9,
104.5, 56.0, 30.3; LR-MS (ESIþ) m/z 342 [M þ H]þ; HR-MS (ESIþ)
calcd for C18H17ClN3O2 [M þ H]þ 342.1004; found 342.1006.
4.1.4.24. N-(Benzo[d][1,3]dioxol-5-yl)-3-(4-chlorobenzyl)-1H-pyr-
azole-5-carboxamide (28).
3-(4-Chlorobenzyl)-1H-pyrazole-5-carboxylic acid (5a) (48.8 mg,
0.206 mmol) and 3,4-(methylenedioxy)aniline (28.3 mg,
0.206 mmol) afforded 33.6 mg (46%) of 28 as pale yellow solid. 28
was purified by flash column chromatography on silica gel (EtOAc:
4.1.4.20. 3-(4-Chlorobenzyl)-N-(3-methoxyphenyl)-1H-pyrazole-5-
carboxamide (24).
3-(4-Chlorobenzyl)-1H-pyrazole-5-carboxylic acid (5a) (45.3 mg,
n-hexane ¼ 1 : 3): 1H NMR (DMSO‑d6, 400 MHz)
d 13.26 (s, 1H, NH),
0.191 mmol) and 3-methoxyaniline (21.5
mL, 0.191 mmol) afforded
9.92 (s, 1H, NH), 7.45 (s, 1H, ArꢁH), 7.38 (d, 2H, J ¼ 7.8 Hz, ArꢁH),
7.28 (d, 2H, J ¼ 8.2 Hz, ArꢁH), 7.26 (m, 1H, ArꢁH), 6.85 (d, 1H,
J ¼ 7.8 Hz, ArꢁH), 6.49 (s,1H, pyrazoleꢁH), 5.98 (s, 2H, OCH2O), 4.02
30.3 mg (46%) of 24 as a brown oil. 24 was purified by flash column
chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 2 to 1 : 1): 1H
NMR (DMSO‑d6, 400 MHz)
d
13.28 (s, 1H, NH), 9.92 (s, 1H, NH), 7.49
(s, 2H, ArꢁCH2); 13C NMR (DMSO‑d6, 100 MHz)
d 160.2, 147.1, 146.9,
(s, 1H, ArꢁH), 7.40 (s, 1H, ArꢁH), 7.38 (d, 2H, J ¼ 7.4 Hz, ArꢁH), 7.30
(d, 2H, J ¼ 8.4 Hz, ArꢁH), 7.20 (t, 1H, J ¼ 8.1 Hz, ArꢁH), 6.64 (d, 1H,
J ¼ 7.4 Hz, ArꢁH), 6.52 (s, 1H, pyrazoleꢁH), 4.03 (s, 2H, ArꢁCH2),
143.5, 143.0, 137.7, 133.3, 131.2, 130.4, 128.5, 113.1, 107.9, 104.7, 102.3,
100.9, 30.2; LR-MS (ESIþ) m/z 356 [M þ H]þ; HR-MS (ESIþ) calcd
for C18H15ClN3O3 [M þ H]þ 356.0796; found 356.0806.
3.73 (s, 3H, OCH3); 13C NMR (DMSO‑d6, 100 MHz)
d 160.6, 159.5,
147.1, 143.6, 140.1, 137.7, 131.2, 130.4, 129.4, 128.6, 112.4, 108.9, 105.9,
104.8, 55.0, 30.3; LR-MS (ESIþ) m/z 342 [M þ H]þ; HR-MS (ESIþ)
calcd for C18H17ClN3O2 [M þ H]þ 342.1004; found 342.1001.
4.1.4.25. 3-(4-Fluorobenzyl)-N-(2-methoxyphenyl)-1H-pyrazole-5-
carboxamide (29). 3-(4-Fluorobenzyl)-1H-pyrazole-5-carboxylic
acid (5b) (78.2 mg, 0.355 mmol) and 2-methoxyaniline (40.0 mL,
0.355 mmol) afforded 61.7 mg (53%) of 29 as pale yellow solid. 29
was purified by flash column chromatography on silica gel (EtOAc:
4.1.4.21. 3-(4-Chlorobenzyl)-N-(2,3-dimethoxyphenyl)-1H-pyrazole-
5-carboxamide (25).
3-(4-Chlorobenzyl)-1H-pyrazole-5-carboxylic acid (5a) (45.1 mg,
n-hexane ¼ 1 : 2 to 1 : 1): 1H NMR (DMSO‑d6, 400 MHz)
d 13.31 (s,
1H, NH), 9.32 (s, 1H, NH), 8.30 (d, 1H, J ¼ 7.6 Hz, ArꢁH), 7.31 (dd, 2H,
J ¼ 8.4, 5.6 Hz, ArꢁH), 7.15 (t, 2H, J ¼ 8.9 Hz, ArꢁH), 7.08 (m, 1H,
ArꢁH), 7.06 (m, 1H, ArꢁH), 6.95 (m, 1H, ArꢁH), 6.53 (s, 1H,
0.190 mmol) and 2,3-dimethoxyaniline (30.1
mL, 0.224 mmol)
afforded 20.7 mg (25%) of 25 as white solid. 25 was purified by flash
11

J. Jo, D. Lee, Y.H. Park et al.
European Journal of Medicinal Chemistry 217 (2021) 113325
pyrazoleꢁH), 4.03 (s, 2H, ArꢁCH2), 3.89 (s, 3H, OCH3); 13C NMR
132.8, 130.3, 130.3, 128.3, 116.0, 115.8, 110.9, 108.8, 106.0, 105.3, 56.5,
(DMSO‑d6, 100 MHz)
d
162.2, 159.8, 159.5, 148.1, 146.7, 144.6, 134.7,
55.9, 31.6; LR-MS (ESIþ) m/z 356 [M þ H]þ; HR-MS (ESIþ) calcd for
134.7, 130.4, 130.3, 127.2, 123.7, 120.7, 119.0, 115.4, 115.2, 110.9, 104.4,
C
19H19FN3O3 [M þ H]þ 356.1405; found 356.1413.
56.0, 30.1; LR-MS (ESIþ) m/z 326 [M þ H]þ; HR-MS (ESIþ) calcd for
C
18H17FN3O2 [M þ H]þ 326.1299; found 326.1295.
4.1.4.30. N-(Benzo[d][1,3]dioxol-5-yl)-3-(4-fluorobenzyl)-1H-pyr-
azole-5-carboxamide (34).
4.1.4.26. 3-(4-Fluorobenzyl)-N-(3-methoxyphenyl)-1H-pyrazole-5-
carboxamide (30). 3-(4-Fluorobenzyl)-1H-pyrazole-5-carboxylic
acid (5b) (53.3 mg, 0.242 mmol) and 3-methoxyaniline (27.2 mL,
0.242 mmol) afforded 39.9 mg (51%) of 30 as pale yellow solid. 30
was purified by flash column chromatography on silica gel (EtOAc:
3-(4-Fluorobenzyl)-1H-pyrazole-5-carboxylic acid (5b) (78.0 mg,
0.354 mmol) and 3,4-(methylenedioxy)aniline (48.5 mg,
0.354 mmol) afforded 61.5 mg (51%) of 34 as brown solid. 34 was
purified by flash column chromatography on silica gel (EtOAc: n-
hexane ¼ 1 : 2): 1H NMR (DMSO‑d6, 400 MHz)
d 13.25 (s, 1H, NH),
n-hexane ¼ 1 : 2 to 1 : 1): 1H NMR (DMSO‑d6, 400 MHz)
d
13.27 (s,
9.91 (s, 1H, NH), 7.46 (s, 1H, ArꢁH), 7.30 (m, 2H, ArꢁH), 7.26 (d, 1H,
J ¼ 8.6 Hz, ArꢁH), 7.15 (t, 2H, J ¼ 8.7 Hz, ArꢁH), 6.85 (d, 1H,
J ¼ 8.4 Hz, ArꢁH), 6.49 (s,1H, pyrazoleꢁH), 5.98 (s, 2H, OCH2O), 4.01
1H, NH), 9.92 (s, 1H, NH), 7.50 (s, 1H, ArꢁH), 7.41 (d, 1H, J ¼ 8.3 Hz,
ArꢁH), 7.31 (dd, 2H, J ¼ 8.3, 5.6 Hz, ArꢁH), 7.21 (d, 1H, J ¼ 8.2 Hz,
ArꢁH), 7.15 (t, 2H, J ¼ 8.9 Hz, ArꢁH), 6.63 (dd, 1H, J ¼ 8.2, 2.1 Hz,
ArꢁH), 6.51 (s, 1H, pyrazoleꢁH), 4.02 (s, 2H, ArꢁCH2), 3.73 (s, 3H,
(s, 2H, ArꢁCH2); 13C NMR (DMSO‑d6, 100 MHz)
d 162.2, 160.3, 159.8,
147.1, 146.9, 143.9, 143.0, 134.9, 134.9, 133.3, 130.4, 130.3, 115.4,
115.2, 113.1, 107.9, 104.6, 102.3, 100.9, 30.1; LR-MS (ESIþ) m/z 340
[M þ H]þ; HR-MS (ESIþ) calcd for C18H15FN3O3 [M þ H]þ 340.1092;
found 340.1105.
OCH3); 13C NMR (DMSO‑d6, 100 MHz)
d 162.2, 160.5, 159.8, 159.4,
147.1, 144.0, 140.1, 134.9, 134.8, 130.4, 130.3, 129.3, 115.4, 115.2, 112.3,
108.8, 105.9, 104.7, 55.0, 30.1; LR-MS (ESIþ) m/z 326 [M þ H]þ; HR-
MS (ESIþ) calcd for C18H17FN3O2 [M þ H]þ 326.1299; found
326.1301.
4.1.4.31. 3-(4-Methoxybenzyl)-N-(2-methoxyphenyl)-1H-pyrazole-5-
carboxamide (35).
4.1.4.27. N-(2,3-Dimethoxyphenyl)-3-(4-fluorobenzyl)-1H-pyrazole-
5-carboxamide (31).
3-(4-Methoxybenzyl)-1H-pyrazole-5-carboxylic acid (5c) (70.1 mg,
0.302 mmol) and 2-methoxyaniline (34.1 mL, 0.302 mmol) afforded
3-(4-Fluorobenzyl)-1H-pyrazole-5-carboxylic acid (5b) (67.9 mg,
44.9 mg (44%) of 35 as pale yellow solid. 35 was purified by flash
0.308 mmol) and 2,3-dimethoxyaniline (41.4
mL, 0.308 mmol)
column chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 4): 1H
afforded 58.4 mg (52%) of 31 as pale yellow solid. 31 was purified by
flash column chromatography on silica gel (EtOAc: n-hexane ¼ 1 :
NMR (DMSO‑d6, 400 MHz) d 13.29 (s, 1H, NH), 9.32 (s, 1H, NH), 8.31
(d, 1H, J ¼ 8.4 Hz, ArꢁH), 7.18 (d, 2H, J ¼ 7.0 Hz, ArꢁH), 7.07 (m, 1H,
ArꢁH), 7.05 (m, 1H, ArꢁH), 6.95 (t,1H, J ¼ 7.1 Hz, ArꢁH), 6.88 (d, 2H,
J ¼ 6.9 Hz, ArꢁH), 6.49 (s, 1H, pyrazoleꢁH), 3.95 (s, 2H, ArꢁCH2),
3.89 (s, 3H, OCH3), 3.72 (s, 3H, OCH3); 13C NMR (DMSO‑d6,100 MHz)
3): 1H NMR (DMSO‑d6, 400 MHz)
d 13.37 (s, 1H, NH), 9.40 (s, 1H,
NH), 7.94 (d, 1H, J ¼ 8.2 Hz, ArꢁH), 7.31 (m, 2H, ArꢁH), 7.15 (t, 2H,
J ¼ 8.8 Hz, ArꢁH), 7.06 (t, 1H, J ¼ 8.3 Hz, ArꢁH), 6.81 (d, 1H,
J ¼ 8.4 Hz, ArꢁH), 6.54 (s, 1H, pyrazoleꢁH), 4.03 (s, 2H, ArꢁCH2),
3.82 (s, 3H, OCH3), 3.81 (s, 3H, OCH3); 13C NMR (DMSO‑d6,100 MHz)
d
159.6, 158.0, 148.0, 146.6, 145.2, 130.4, 129.5, 127.2, 123.7, 120.7,
118.9,114.0,110.9,104.2, 56.0, 55.1, 30.1; LR-MS (ESIþ) m/z 338 [M þ
H]þ; HR-MS (ESIþ) calcd for C19H20N3O3 [M þ H]þ 338.1499; found
338.1513.
d
162.2, 159.8, 159.6, 152.0, 146.7, 144.7, 137.3, 134.7, 134.7, 131.8,
130.4, 130.3, 124.1, 115.5, 115.2, 111.7, 107.9, 104.4, 60.4, 55.8, 30.1;
LR-MS (ESIþ) m/z 356 [M
þ
H]þ; HR-MS (ESIþ) calcd for
C
19H19FN3O3 [M þ H]þ 356.1405; found 356.1419.
4.1.4.32. 3-(4-Methoxybenzyl)-N-(3-methoxyphenyl)-1H-pyrazole-
5-carboxamide (36).
4.1.4.28. N-(2,4-Dimethoxyphenyl)-3-(4-fluorobenzyl)-1H-pyrazole-
5-carboxamide (32).
3-(4-Methoxybenzyl)-1H-pyrazole-5-carboxylic acid (5c) (70.3 mg,
0.303 mmol) and 3-methoxyaniline (34.0 mL, 0.303 mmol) afforded
3-(4-Fluorobenzyl)-1H-pyrazole-5-carboxylic acid (5b) (70.6 mg,
59.3 mg (58%) of 36 as pale yellow solid. 36 was purified by flash
0.321 mmol) and 2,4-dimethoxyaniline (45.7
mL, 0.321 mmol)
column chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 4): 1H
afforded 24.9 mg (22%) of 32 as a light brown oil. 32 was purified by
flash column chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 2
NMR (CDCl3, 400 MHz)
d
8.76 (s, 1H, NH), 7.43 (s, 1H, ArꢁH), 7.21 (t,
1H, J ¼ 8.1 Hz, ArꢁH), 7.09 (m, 3H, ArꢁH), 6.82 (d, 2H, J ¼ 7.8 Hz,
ArꢁH), 6.66 (m, 1H, ArꢁH), 6.65 (s, 1H, pyrazoleꢁH), 3.95 (s, 2H,
ArꢁCH2), 3.77 (s, 3H, OCH3), 3.75 (s, 3H, OCH3); 13C NMR (DMSO‑d6,
to 1 : 1): 1H NMR (CDCl3, 400 MHz)
d 10.88 (brs, 1H, NH), 9.03 (s, 1H,
NH), 8.33 (d, 1H, J ¼ 7.6 Hz, ArꢁH), 7.13 (m, 2H, ArꢁH), 6.95 (t, 2H,
J ¼ 8.6 Hz, ArꢁH), 6.63 (s, 1H, pyrazoleꢁH), 6.49 (s, 1H, ArꢁH), 6.47
(m, 1H, ArꢁH), 3.99 (s, 2H, ArꢁCH2), 3.83 (s, 3H, OCH3), 3.79 (s, 3H,
100 MHz) d 160.6, 159.4, 157.9, 147.0, 144.6, 140.1, 130.6, 129.5, 129.4,
114.0, 112.4, 108.8, 105.9, 104.5, 55.1, 55.0, 30.1; LR-MS (ESIþ) m/z
338 [M þ H]þ; HR-MS (ESIþ) calcd for C19H20N3O3 [M þ H]þ
338.1499; found 338.1512.
OCH3); 13C NMR (CDCl3, 100 MHz)
d 163.1, 160.7, 159.6, 156.6, 149.8,
147.3, 145.0, 133.2, 133.1, 130.3, 130.2, 121.0, 120.8, 115.9, 115.7, 105.1,
103.8, 98.8, 55.9, 55.7, 31.5; LR-MS (ESIþ) m/z 356 [M þ H]þ; HR-MS
(ESIþ) calcd for C19H19FN3O3 [M þ H]þ 356.1405; found 356.1405.
4.1.4.33. N-(2,3-Dimethoxyphenyl)-3-(4-methoxybenzyl)-1H-pyr-
azole-5-carboxamide (37).
4.1.4.29. N-(2,5-Dimethoxyphenyl)-3-(4-fluorobenzyl)-1H-pyrazole-
5-carboxamide (33).
3-(4-Methoxybenzyl)-1H-pyrazole-5-carboxylic acid (5c) (70.0 mg,
0.301 mmol) and 2,3-dimethoxyaniline (40.4
mL, 0.301 mmol)
3-(4-Fluorobenzyl)-1H-pyrazole-5-carboxylic acid (5b) (74.6 mg,
0.339 mmol) and 2,5-dimethoxyaniline (52.0 mg, 0.339 mmol)
afforded 11.0 mg (9%) of 33 as a brown oil. 33 was purified by flash
column chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 2 to 1 :
afforded 43.3 mg (39%) of 37 as a yellow oil. 37 was purified by flash
column chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 4): 1H
NMR (DMSO‑d6, 400 MHz) d 13.33 (s, 1H, NH), 9.39 (s, 1H, NH), 7.93
(d, 1H, J ¼ 8.2 Hz, ArꢁH), 7.19 (d, 2H, J ¼ 8.5 Hz, ArꢁH), 7.05 (t, 1H,
J ¼ 8.4 Hz, ArꢁH), 6.88 (d, 2H, J ¼ 8.3 Hz, ArꢁH), 6.81 (d, 1H,
J ¼ 8.4 Hz, ArꢁH), 6.49 (s, 1H, pyrazoleꢁH), 3.95 (s, 2H, ArꢁCH2),
3.82 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.71 (s, 3H, OCH3); 13C NMR
1): 1H NMR (CDCl3, 400 MHz)
d 9.25 (s, 1H, NH), 8.24 (d, 1H,
J ¼ 2.7 Hz, ArꢁH), 7.17 (dd, 2H, J ¼ 7.9, 5.6 Hz, ArꢁH), 7.00 (t, 2H,
J ¼ 8.2 Hz, ArꢁH), 6.81 (d, 1H, J ¼ 8.8 Hz, ArꢁH), 6.67 (s, 1H,
pyrazoleꢁH), 6.59 (dd, 1H, J ¼ 8.8, 2.4 Hz, ArꢁH), 4.03 (s, 2H,
ArꢁCH2), 3.86 (s, 3H, OCH3), 3.80 (s, 3H, OCH3); 13C NMR (CDCl3,
(DMSO‑d6, 100 MHz)
d 159.6, 157.9, 152.0, 146.6, 145.3, 137.3, 131.9,
130.4, 129.5, 124.1, 114.0, 111.7, 107.9, 104.2, 60.4, 55.8, 55.1, 30.1; LR-
100 MHz)
d
163.2, 160.8, 159.8, 153.9, 147.6, 144.9, 142.6, 132.9,
MS (ESIþ) m/z 368 [M þ H]þ; HR-MS (ESIþ) calcd for C20H22N3O4
12

J. Jo, D. Lee, Y.H. Park et al.
European Journal of Medicinal Chemistry 217 (2021) 113325
[M þ H]þ 368.1605; found 368.1618.
4.1.4.38. 3-(2-chlorobenzyl)-N-(2,4-dimethoxyphenyl)-1H-pyrazole-
5-carboxamide (42).
3-(2-Chlorobenzyl)-1H-pyrazole-5-carboxylic acid (5e) (71.6 mg,
0.303 mmol) afforded 67.4 mg (60%) of 42 as ivory solid. 42 was
purified by flash column chromatography on silica gel (EtOAc: n-
4.1.4.34. N-(2,4-Dimethoxyphenyl)-3-(4-methoxybenzyl)-1H-pyr-
azole-5-carboxamide (38).
3-(4-Methoxybenzyl)-1H-pyrazole-5-carboxylic acid (5c) (100 mg,
0.431 mmol) and 2,4-dimethoxyaniline (66.0 mg, 0.431 mmol)
afforded 48.3 mg (31%) of 38 as a dark brown oil. 38 was purified by
flash column chromatography on silica gel (EtOAc: n-hexane ¼ 1 :
hexane ¼ 1 : 2): 1H NMR (DMSO‑d6, 400 MHz)
d 13.32 (s, 1H, NH),
9.13 (s, 1H, NH), 8.11 (d, 1H, J ¼ 8.1 Hz, ArꢁH), 7.47 (d, 1H, J ¼ 6.6 Hz,
ArꢁH), 7.32 (m, 3H, ArꢁH), 6.67 (s, 1H, pyrazoleꢁH), 6.53 (d, 1H,
J ¼ 8.8 Hz, ArꢁH), 6.41 (s, 1H, ArꢁH), 4.14 (s, 2H, ArꢁCH2), 3.87 (s,
3H, OCH3), 3.75 (s, 3H, OCH3); 13C NMR (DMSO‑d6, 100 MHz)
1): 1H NMR (CDCl3, 400 MHz)
d 9.04 (s, 1H, NH), 8.36 (d, 1H,
J ¼ 9.5 Hz, ArꢁH), 7.10 (d, 2H, J ¼ 8.5 Hz, ArꢁH), 6.82 (d, 2H,
J ¼ 8.6 Hz, ArꢁH), 6.65 (s, 1H, pyrazoleꢁH), 6.49 (m, 1H, ArꢁH), 6.48
(m, 1H, ArꢁH), 3.97 (s, 2H, ArꢁCH2), 3.83 (s, 3H, OCH3), 3.79 (s, 3H,
d
159.2, 156.2, 149.7, 146.9, 142.8, 136.0, 133.0, 130.8, 129.5, 128.8,
127.6, 120.5, 120.4, 104.5, 104.2, 98.9, 56.0, 55.4, 29.0; LR-MS (ESIþ)
m/z 372 [M þ H]þ; HR-MS (ESIþ) calcd for C19H19ClN3O3 [M þ H]þ
372.1109; found 372.1107.
OCH3), 3.77 (s, 3H, OCH3); 13C NMR (CDCl3, 100 MHz)
d 159.7, 158.7,
156.5, 149.8, 147.6, 145.4, 129.8, 129.3, 121.2, 120.8, 114.4, 105.0,
103.8, 98.8, 55.9, 55.7, 55.4, 31.4; LR-MS (ESIþ) m/z 368 [M þ H]þ;
HR-MS (ESIþ) calcd for C20H22N3O4 [M þ H]þ 368.1605; found
368.1608.
4.1.5. General procedures for the preparation of 43 and 46
To a stirred solution of carboxylic acid in CH2Cl2 were added
oxalyl chloride (3.0 equiv.) and 2 drops of DMF at ambient tem-
perature. After stirring for 30 min, the reaction mixture was
concentrated in vacuo and then diluted with CH2Cl2. To the reaction
mixture were added aniline (1.0 equiv.) and triethylamine (2.0
equiv.) at ambient temperature. After stirring for 1 h, the mixture
was quenched with saturated aqueous NH4Cl and then extracted
with CH2Cl2. The combined organic layer was dried over MgSO4 and
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel.
4.1.4.35. N-(2,5-Dimethoxyphenyl)-3-(4-methoxybenzyl)-1H-pyr-
azole-5-carboxamide (39).
3-(4-Methoxybenzyl)-1H-pyrazole-5-carboxylic acid (5c) (104 mg,
0.448 mmol) and 2,5-dimethoxyaniline (68.6 mg, 0.448 mmol)
afforded 13.9 mg (8%) of 39 as a brown oil. 39 was purified by flash
column chromatography on silica gel (EtOAc: n-hexane ¼ 2 : 3): 1H
NMR (CDCl3, 400 MHz)
d
9.27 (s, 1H, NH), 8.26 (d, 1H, J ¼ 3.0 Hz,
ArꢁH), 7.14 (d, 2H, J ¼ 8.4 Hz, ArꢁH), 6.87 (d, 2H, J ¼ 8.6 Hz, ArꢁH),
6.81 (d, 1H, J ¼ 8.8 Hz, ArꢁH), 6.69 (s, 1H, pyrazoleꢁH), 6.59 (dd, 1H,
J ¼ 8.8, 3.1 Hz, ArꢁH), 4.01 (s, 2H, ArꢁCH2), 3.86 (s, 3H, OCH3), 3.81
4.1.5.1. 3-(4-Chlorobenzyl)-N-(2,4-dimethoxyphenyl)-N-methyl-
1H-pyrazole-5-carboxamide (43).
(s, 3H, OCH3), 3.80 (s, 3H, OCH3); 13C NMR (CDCl3, 100 MHz)
d 159.9,
3-(4-Chlorobenzyl)-1H-pyrazole-5-carboxylic acid (5a) (86.0 mg,
0.363 mmol) and 2,4-dimethoxy-N-methylaniline (60.7 mg,
0.363 mmol) afforded 76.1 mg (54%) of 43 as pale yellow solid. 43
was purified by flash column chromatography on silica gel (EtOAc:
158.9, 154.0, 147.9, 145.3, 142.6, 129.9, 128.9, 128.4, 114.5, 111.0,
108.8, 105.9, 105.1, 56.5, 55.9, 55.5, 31.5; LR-MS (ESIþ) m/z 368 [M þ
H]þ; HR-MS (ESIþ) calcd for C20H22N3O4 [M þ H]þ 368.1605; found
368.1598.
n-hexane ¼ 1 : 1 to 3 : 1): 1H NMR (CDCl3, 400 MHz)
d 7.18 (d, 2H,
J ¼ 8.4 Hz, ArꢁH), 7.02 (dd, 1H, J ¼ 7.9, 0.8 Hz, ArꢁH), 6.97 (d, 2H,
J ¼ 8.3 Hz, ArꢁH), 6.43 (s, 1H, ArꢁH), 6.41 (d, 1H, J ¼ 2.6 Hz, ArꢁH),
4.68 (s, 1H, pyrazoleꢁH), 3.83 (s, 3H, OCH3), 3.78 (s, 2H, ArꢁCH2),
3.62 (s, 3H, OCH3), 3.30 (s, 3H, OCH3); 13C NMR (CDCl3, 100 MHz)
4.1.4.36. N-(Benzo[d][1,3]dioxol-5-yl)-3-(4-methoxybenzyl)-1H-pyr-
azole-5-carboxamide (40).
3-(4-Methoxybenzyl)-1H-pyrazole-5-carboxylic acid (5c) (63.7 mg,
0.274 mmol) and 3,4-(methylenedioxy)aniline (37.6 mg,
0.274 mmol) afforded 44.6 mg (46%) of 40 as brown solid. 40 was
purified by flash column chromatography on silica gel (EtOAc: n-
d
161.2, 160.9, 156.4, 151.8, 138.1, 136.9, 131.9, 130.2, 129.8, 128.4,
125.0, 106.1, 104.6, 99.8, 55.7, 55.7, 37.3, 33.8; LR-MS (ESIþ) m/z 386
[M þ H]þ; HR-MS (ESIþ) calcd for C20H21ClN3O3 [M þ H]þ
386.1266; found 386.1278.
hexane ¼ 1 : 3): 1H NMR (DMSO‑d6, 400 MHz)
d 13.21 (s, 1H, NH),
9.90 (s, 1H, NH), 7.46 (s, 1H, ArꢁH), 7.26 (d, 1H, J ¼ 7.4 Hz, ArꢁH),
7.18 (d, 2H, J ¼ 8.5 Hz, ArꢁH), 6.88 (d, 2H, J ¼ 8.6 Hz, ArꢁH), 6.86 (m,
1H, ArꢁH), 6.45 (s, 1H, pyrazoleꢁH), 5.98 (s, 2H, OCH2O), 3.94 (s,
2H, ArꢁCH2), 3.72 (s, 3H, OCH3); 13C NMR (DMSO‑d6, 100 MHz)
4.1.5.2. 3-(4-Chlorobenzyl)-N-(2,4-dimethoxyphenyl)-1-methyl-
1H-pyrazole-5-carboxamide (46).
3-(4-Chlorobenzyl)-1-methyl-1H-pyrazole-5-carboxylic acid (45)
d
160.3, 157.9, 147.0, 146.9, 144.5, 143.0, 133.3, 130.6, 129.5, 113.9,
(74.7 mg, 0.298 mmol) and 2,4-dimethoxyaniline (42.5
mL,
113.0, 107.9, 104.4, 102.3, 100.9, 55.1, 30.1; LR-MS (ESIþ) m/z 352
[M þ H]þ; HR-MS (ESIþ) calcd for C19H18N3O4 [M þ H]þ 352.1292;
found 352.1304.
0.298 mmol) afforded 100 mg (87%) of 46 as ivory solid. 46 was
purified by flash column chromatography on silica gel (CH2Cl2:
EtOAc ¼ 3 : 1 to EtOAc: n-hexane ¼ 1 : 1): 1H NMR (CDCl3, 400 MHz)
d
8.21 (m, 1H, ArꢁH), 8.02 (s, 1H, NH), 7.27 (d, 2H, J ¼ 8.6 Hz, ArꢁH),
4.1.4.37. 3-(3-Chlorobenzyl)-N-(2,4-dimethoxyphenyl)-1H-pyrazole-
5-carboxamide (41).
3-(3-Chlorobenzyl)-1H-pyrazole-5-carboxylic acid (5d) (61.8 mg,
0.261 mmol) afforded 90.3 mg (93%) of 41 as brown solid. 41 was
purified by flash column chromatography on silica gel (EtOAc: n-
7.19 (d, 2H, J ¼ 8.7 Hz, ArꢁH), 6.49 (m, 2H, ArꢁH), 6.33 (s, 1H,
pyrazoleꢁH), 4.17 (s, 3H, CH3), 3.95 (s, 2H, ArꢁCH2), 3.85 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3); 13C NMR (CDCl3, 100 MHz)
d 157.5, 157.0,
149.8, 149.6, 138.1, 136.8, 132.3, 130.2, 128.8, 121.0, 120.6, 105.3,
104.0, 98.8, 55.9, 55.7, 39.2, 34.0; LR-MS (ESIþ) m/z 386 [M þ H]þ;
HR-MS (ESIþ) calcd for C20H21ClN3O3 [M þ H]þ 386.1266; found
386.1283.
hexane ¼ 1 : 2): 1H NMR (DMSO‑d6, 400 MHz)
d 13.29 (s, 1H, NH),
9.12 (s, 1H, NH), 8.13 (d, 1H, J ¼ 8.6 Hz, ArꢁH), 7.34 (m, 2H, ArꢁH),
7.30 (d, 1H, J ¼ 7.6 Hz, ArꢁH), 7.23 (d, 1H, J ¼ 7.2 Hz, ArꢁH), 6.67 (s,
1H, pyrazoleꢁH), 6.55 (s, 1H, ArꢁH), 6.52 (m, 1H, ArꢁH), 4.05 (s, 2H,
ArꢁCH2), 3.87 (s, 3H, OCH3), 3.75 (s, 3H, OCH3); 13C NMR (DMSO‑d6,
4.1.6. Ethyl 3-(4-chlorobenzyl)-1-methyl-1H-pyrazole-5-
carboxylate (44).
To a stirred solution of ethyl 3-(4-chlorobenzyl)-1H-pyrazole-5-
carboxylate (4a) (278 mg, 1.05 mmol) in DMF (3.00 mL) were
100 MHz)
d
159.2, 156.1, 149.7, 146.9, 143.7, 141.1, 133.2, 130.5, 128.3,
127.2, 126.6, 120.5, 120.4, 104.5, 104.2, 98.8, 56.0, 55.3, 30.5; LR-MS
(ESIþ) m/z 372 [M þ H]þ; HR-MS (ESIþ) calcd for C19H19ClN3O3
[M þ H]þ 372.1109; found 372.1118.
added K2CO3 (218 mg, 1.58 mmol) and iodomethane (98.4
mL,
1.58 mmol) at ambient temperature. After stirring for 10 h, the
13

J. Jo, D. Lee, Y.H. Park et al.
European Journal of Medicinal Chemistry 217 (2021) 113325
reaction mixture was quenched with H2O and then extracted with
EtOAc. The combined organic layer was dried over MgSO4 and
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 5) to afford
viability ¼ (Treatment A value ꢁ Blank A value)/(Control A
value ꢁ Blank A value) ꢃ 100%. Culture medium mixed with Ez-
Cytox reagent was used as a Blank (background control). The con-
trol was the cells treated with 0.5% DMSO, set at 100% cell viability.
The experiment was performed in triplicate.
129 mg (44%) of 44 as yellow solid: 1H NMR (CDCl3, 500 MHz)
d 7.26
(d, 2H, J ¼ 8.3 Hz, ArꢁH), 7.17 (d, 2H, J ¼ 8.3 Hz, ArꢁH), 6.53 (s, 1H,
pyrazoleꢁH), 4.30 (q, 2H, J ¼ 7.1 Hz, OCH2CH3), 4.13 (s, 3H, CH3),
3.92 (s, 2H, ArꢁCH2), 1.34 (t, 3H, J ¼ 7.1 Hz, OCH2CH3); 13C NMR
4.4. GSIS assays
(CDCl3, 125 MHz)
d 160.0, 150.1, 138.2, 133.5, 132.3, 130.2, 128.8,
The INS-1 cells were seeded in a 12-well plate at a density of
2 ꢃ 105 cells/mL and incubated. After incubation for 24 h, each well
was washed twice with Krebs-Ringer bicarbonate buffer (KRB, pH
7.4) containing 3.3 mM glucose (basal condition). After starvation in
fresh KRB for 2 h, the cells were treated with compounds at the
110.1, 61.1, 39.4, 34.0, 14.4; LR-MS (ESIþ) m/z 279 [M þ H]þ; HR-MS
(ESIþ) calcd for C14H16ClN2O2 [M þ H]þ 279.0895; found 279.0893.
4.1.7. Ethyl 3-(4-chlorobenzoyl)-1H-pyrazole-5-carboxylate (47).
To an ethyl 3-(4-chlorobenzyl)-1H-pyrazole-5-carboxylate (4a)
(641 mg, 2.42 mmol) was added TBHP (5.81 mL of a 5.0 M solution
in decane) at ambient temperature. After stirring 22 h at 120 ꢂC, the
reaction mixture was quenched with H2O and then extracted with
EtOAc. The combined organic layer was dried over MgSO4 and
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (EtOAc: n-hexane ¼ 1 : 4) to afford
645 mg (96%) of 47 as white solid.
concentration of 2.5, 5, 10 mM. In the assay, gliclazide at the same
concentration is used as the positive control. After incubation for
30 min, 3.3 mM glucose as basal or 16.7 mM glucose as stimulant
was respectively added to each well and further incubated for
additional for 1 h. According to the manufacturer’s instructions,
GSIS was measured using a rat insulin ELISA kit (Gentaur, Shibayagi
Co. Ltd., Gunma, Shibukaw, Japan). Subsequently, the absorbance at
450 nm of each well was measured using a microplate reader
(PowerWave XS, Bio-Tek Instruments, Winooski, VT, USA).
GSI ¼ stimulatory insulin level/basal insulin level. The experiment
was performed in triplicate.
4.1.8. 3-(4-Chlorobenzoyl)-N-(2,4-dimethoxyphenyl)-1H-pyrazole-
5-carboxamide (49).
To
carboxylic acid (48) (93.7 mg, 0.374 mmol) and 2,4-
dimethoxyaniline (58.6 L, 0.411 mmol) in DMF (2.00 mL) were
added EDC$HCl (108 mg), HOBt$H2O (85.9 mg), DMAP (4.57 mg)
and DIPEA (97.7 L) at ambient temperature. After stirring for 24 h,
a stirred solution of 3-(4-chlorobenzoyl)-1H-pyrazole-5-
4.5. Western blotting assays
m
The INS-1 cells were seeded in a 6-well plate at a density of
4.75 ꢃ 105 cells/mL and incubated for 24 h. The cells were treated
m
the reaction mixture was quenched with H2O and diluted with
EtOAc. The organic layer was washed with 1 N HCl and dried over
MgSO4. The resulting residue was concentrated in vacuo and pu-
rified by flash column chromatography on silica gel (EtOAc: n-
hexane ¼ 1 : 3 to 1 : 1) to afford 117 mg (81%) of 49 as pale red solid:
with 26 at the concentration of 2.5, 5, 10 mM. The control was the
cells treated with 0.5% DMSO. After incubation for 24 h, the cells
were lysed with RIPA buffer (Cell Signaling, Danvers, MA, USA)
containing 1 mM phenylmethylsulfonyl fluoride for 20 min at 4 ꢂC.
The protein concentration was determined using the Pierce™ BCA
1H NMR (CDCl3, 400 MHz)
d 11.91 (brs, 1H, NH), 9.09 (s, 1H, NH),
protein assay kit (Thermo Scientific, Carlsbad, CA, USA). A 20 mg
8.39 (d, 1H, J ¼ 8.2 Hz, ArꢁH), 8.02 (d, 2H, J ¼ 7.8 Hz, ArꢁH), 7.50 (d,
2H, J ¼ 6.8 Hz, ArꢁH), 7.46 (s, 1H, pyrazoleꢁH), 6.53 (m, 2H, ArꢁH),
3.88 (s, 3H, OCH3), 3.81 (s, 3H, OCH3); 13C NMR (CDCl3, 100 MHz)
concentration of protein per lane was separated using 10% sodium
dodecyl sulfate polyacrylamide gel, the proteins were transferred
onto polyvinylidene difluoride membranes and incubated with
d
183.5, 158.2, 156.9, 149.8, 140.5, 134.7, 131.0, 129.3, 121.8, 120.9,
primary antibodies against PPARg, PDX-1, phospho-IRS2 (p-IRS2)
120.8, 114.4, 110.4, 104.0, 98.9, 56.0, 55.7; LR-MS (ESIþ) m/z 386
[M þ H]þ; HR-MS (ESIþ) calcd for C19H17ClN3O4 [M þ H]þ
386.0902; found 386.0915.
(Ser731), IRS-2, phospho-PI3K (p-PI3K), PI3K, phospho-Akt (p-Akt)
(Ser473), Akt, and glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) at room temperature for 1 h. And proteins were incubated
with horseradish peroxidase (HRP)-conjugated anti-rabbit sec-
ondary antibodies at room temperature for 1 h. All antibodies were
purchased from Cell Signaling (Boston, MA, USA). The proteins were
visualized using a chemiluminescence system (FUSION Solo, PEQ-
LAB Biotechnologie GmbH, Erlangen, Germany) after 5 min of in-
cubation with ECL Plus western blotting detection reagents (GE
Healthcare, Little Chalfont, UK). The experiment was performed in
triplicate and representative data presented.
4.2. Cell culture
An insulin secreting pancreatic b-cell line derived from a rat
insulinoma, INS cells (Biohermes, Shanghai, China), were cultured
in a Roswell Park Memorial Institute (RPMI) 1640 medium (Cellgro,
Manassas, VA, USA), supplemented with 2 mM L-glutamine,
0.05 mM 2-mercaptoethanol, 11 mM
D-glucose, 1% penicillin/
streptomycin (Invitrogen Co., Grand Island, NY, USA), 10% fetal
bovine serum (FBS), 10 mM HEPES, and 1 mM sodium pyruvate in a
humidified atmosphere at 37 ꢂC containing 5% CO2.
4.6. Myotube formation, glucose uptake and immunoblotting
C2C12 mouse myoblast cell line was obtained from ATCC and
cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invi-
trogen, Carlsbad, CA, USA) supplemented with 10% FBS (HyClone
Laboratories, Logan, UT, USA) and 1% penicillin/streptomycin
(Invitrogen) at 37 ꢂC with 5% CO2 in air. C2C12 myoblast were
cultured in DMEM until 90% of confluence. Cells were differentiated
into myotubes with DMEM containing 2% horse serum for 4 days
and then incubated for 16 h in DMEM containing 2% BSA and 10%
FBS in the absence or presence of compounds. Glucose uptake was
4.3. Cell viability
The INS-1 cells were seeded in a 96-well plate at a density of
1.5 ꢃ 104 cells/mL and incubated for 24 h. The cells were treated
with compounds at the concentration of 2.5, 5, 10 mM. After incu-
bation for 24 h, 10% (v/v) Ez-Cytox reagent (Daeil Lab Service Co.,
Seoul, Korea) was added to each well and incubated for 1 h in the
incubator. Subsequently, the absorbance (A value) at 450 nm of
each well was measured using a microplate reader (PowerWave XS,
Bio-Tek Instruments, Winooski, VT, USA). Percentage of cell
determined using
a
2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
amino)-2-deoxyglucose (2-NBDG) uptake assay kit according to the
14

J. Jo, D. Lee, Y.H. Park et al.
European Journal of Medicinal Chemistry 217 (2021) 113325
fasiglifam (TAK-875) in patients with type 2 diabetes: review of the global
clinical trial experience, Drug Saf. 41 (2018) 625e640.
[17] T.M.A. Belete, Recent achievement in the discovery and development of novel
targets for the treatment of type-2 diabetes mellitus, J. Exp. Pharmacol. 12
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manufacture’s protocol (Sigma-Aldrich, St Louis, MO, USA). For
Western blotting, antibodies for MG53, IRS-1, PI3K, p-PI3K, Akt, p-
Akt, and GAPDH were used.
[18] H.E. Hohmeier, H. Mulder, G. Chen, R. Henkel-Rieger, M. Prentki,
C.B. Newgard, Isolation of INS-1eDerived cell lines with robust ATP-sensitive
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[19] M.M. Hann, T.I. Oprea, Pursuing the leadlikeness concept in pharmaceutical
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4.7. Statistical analysis
All analyses were determined using SPSS Statistics ver. 19.0
(SPSS Inc., Chicago, IL, USA). Non-parametric comparisons of sam-
ples were conducted by the KruskaleWallis test to analyze the
results. A value of p < 0.05 was considered to be statistically
significant.
Declaration of competing interest
[22] X. Huang, G. Liu, J. Guo, Z. Su, The PI3K/AKT pathway in obesity and type 2
diabetes, Int. J. Biol. Sci. 14 (2018) 1483e1496.
[23] A.M. Hennige, D.J. Burks, U. Ozcan, R.N. Kulkarni, J. Ye, S. Park, M. Schubert,
T.L. Fisher, M.A. Dow, R. Leshan, M. Zakaria, M. Mossa-Basha, M.F. White,
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Upregulation of insulin receptor substrate-2 in pancreatic
b cells prevents
diabetes, J. Clin. Invest. 112 (2003) 1521e1532.
[24] T. Du, L. Yang, X. Xu, X. Shi, X. Xu, J. Lu, J. Lv, X. Huang, J. Chen, H. Wang, J. Ye,
L. Hu, X. Shen, Vincamine as a GPR40 agonist improves glucose homeostasis in
type 2 diabetic mice, J. Endocrinol. 240 (2019) 195e214.
Acknowledgments
[25] J. Wang, X. Lv, J. Xu, X. Liu, T. Du, G. Sun, J. Chen, X. Shen, J. Wang, L. Hu,
Design, synthesis and biological evaluation of vincamine derivatives as po-
This research was supported by the Basic Science Research
Program through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education (NRF-2018R1D1A
1B07045101).
tential pancreatic b-cells protective agents for the treatment of type 2 dia-
betes mellitus, Eur. J. Med. Chem. 188 (2020) 111976.
[26] J.M. Oliveira, S.A. Rebuffat, R. Gasa, R. Gomis, Targeting type 2 diabetes: les-
sons from a knockout model of insulin receptor substrate 2, Can. J. Physiol.
Pharmacol. 92 (2014) 613e620.
€
Appendix A. Supplementary data
[27] S.S. Andrali, M.L. Sampley, N.L. Vanderford, S. Ozcan, Glucose regulation of
insulin gene expression in pancreatic
[28] J.A. Moibi, D. Gupta, T.L. Jetton, M. Peshavaria, R. Desai, J.L. Leahy, Peroxisome
proliferatoreactivated receptor- regulates expression of PDX-1 and NKX6.1
b-cells, Biochem. J. 415 (2008) 1e10.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113325.
g
in INS-1 cells, Diabetes 56 (2007) 88e95.
[29] H. Watanabe, H. Saito, H. Nishimura, J. Ueda, B.M. Evers, Activation of
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