Structure-based discovery of 1H-indole-2-carboxamide derivatives as potent ASK1 inhibitors for potential treatment of ulcerative colitis

Article abstract of DOI:10.1016/j.ejmech.2020.113114

Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase (MAPK) family, is implicated in many human diseases. Here, we describe the structural optimization of hit compound 7 and conduct further structure-activity relationship (SAR) studies that result in the development of compound 19 with a novel indole-2-carboxamide hinge scaffold. Compound 19 displays potent anti-ASK1 kinase activity and stronger inhibitory effect on ASK1 in AP1-HEK293 cells than previously described ASK1 inhibitor GS-4997. Besides improved in vitro activity, compound 19 also exhibits an appropriate in vivo PK profile. In a dextran sulfate sodium (DSS)-induced mouse model of ulcerative colitis (UC), compound 19 shows significant anti-UC efficacy and markedly attenuates DSS-induced body weight loss, colonic shortening, elevation in disease activity index (DAI) and inflammatory cell infiltration in colon tissues. Mechanistically, compound 19 represses the phosphorylation of ASK1-p38/JNK signaling pathways and suppresses the overexpression of inflammatory cytokines. Together, these findings suggest that ASK1 inhibitors can potentially be used as a therapeutic strategy for UC.

Full text of DOI:10.1016/j.ejmech.2020.113114

European Journal of Medicinal Chemistry 211 (2021) 113114
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Structure-based discovery of 1H-indole-2-carboxamide derivatives as
potent ASK1 inhibitors for potential treatment of ulcerative colitis
,
,
Shaohua Hou a 1, Xiping Yang c 1, Yu Tong a, Yuejing Yang a, Quanwei Chen a,
Boheng Wan a, Ran Wei a, Yuchen Wang a, Yanmin Zhang a, Bo Kong a, Jianhang Huang a,
,
,
b,
,
, *
Yadong Chen a b, Tao Lu a ***, Qinghua Hu c **, Ding Du a
a School of Sciences, China Pharmaceutical University, 639 Longmian Avenue, Nanjing, 211198, PR China
b State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, PR China
c School of Pharmacy, China Pharmaceutical University, 639 Longmian Avenue, Nanjing, 211198, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase (MAPK)
family, is implicated in many human diseases. Here, we describe the structural optimization of hit
compound 7 and conduct further structure-activity relationship (SAR) studies that result in the devel-
opment of compound 19 with a novel indole-2-carboxamide hinge scaffold. Compound 19 displays
potent anti-ASK1 kinase activity and stronger inhibitory effect on ASK1 in AP1-HEK293 cells than pre-
viously described ASK1 inhibitor GS-4997. Besides improved in vitro activity, compound 19 also exhibits
an appropriate in vivo PK profile. In a dextran sulfate sodium (DSS)-induced mouse model of ulcerative
colitis (UC), compound 19 shows significant anti-UC efficacy and markedly attenuates DSS-induced body
weight loss, colonic shortening, elevation in disease activity index (DAI) and inflammatory cell infiltra-
tion in colon tissues. Mechanistically, compound 19 represses the phosphorylation of ASK1-p38/JNK
signaling pathways and suppresses the overexpression of inflammatory cytokines. Together, these
findings suggest that ASK1 inhibitors can potentially be used as a therapeutic strategy for UC.
© 2020 Elsevier Masson SAS. All rights reserved.
Received 26 October 2020
Received in revised form
16 December 2020
Accepted 16 December 2020
Available online 25 December 2020
Keywords:
ASK1 inhibitor
1H-indole-2-carboxamide derivatives
Ulcerative colitis
1. Introduction
significant global impact on public health. Although the patho-
genesis of UC has not been completely revealed, immune dysre-
Ulcerative colitis (UC), a major clinical type of inflammatory
bowel disease (IBD), is characterized by an uncontrolled inflam-
matory response, mucosal inflammation and ulceration. UC usu-
ally involves a combination of severe diarrhea, abdominal pain
and weight loss [1e3]. The morbidity of the UC tends to be
increasing in recent years in the Middle East, South America, Asia,
and much of the developing world. Moreover, UC greatly impairs
the quality of life of patients and UC is also associated with an
increased risk of colorectal cancer (CRC) [4,5], which has a
gulation caused by various genetic and environmental factors has
been considered as a major cause [6,7]. Emerging literature sug-
gests that excessive pro-inflammatory cytokines play a key role in
the occurrence of UC, such as TNF-a, IL-1b, IL-6, and IFN-g [8e12].
Currently, therapeutic medications for UC include amino-
salicylate, glucocorticoids, immunosuppressive agents and bi-
ologics [13]. However, severe side effects, economic burdens,
limited efficacy and administration compliance of these drugs
greatly limit their clinical applications [14,15]. These limitations
dictate an urgent need to develop new anti-UC drugs with better
therapeutic efficacy and better safety.
Apoptosis signal-regulating kinase 1 (ASK1) is a member of the
mitogen-activated protein kinase (MAPK) family that activates the
c-Jun N-terminal kinase (JNK) and p38 MAPK signaling pathways
[16]. ASK1 is activated in response to various forms of cellular
stress including reactive oxygen species (ROS), endoplasmic re-
ticulum (ER) stress, bacterial and viral infections, calcium influx
and inflammatory signals [17]. ASK1 has been reported to be
* Corresponding author.
** Corresponding author.
*** Corresponding author. School of Sciences, China Pharmaceutical University,
639 Longmian Avenue, Nanjing, 211198, PR China.
E-mail addresses: lutao@cpu.edu.cn (T. Lu), huqh@cpu.edu.cn (Q. Hu),
ddmn9999@cpu.edu.cn (D. Du).
1
Shaohua Hou and Xiping Yang contributed equally to this work. The manuscript
was written through contributions of all authors.
https://doi.org/10.1016/j.ejmech.2020.113114
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
involved in cell death, survival, differentiation or apoptosis [18].
Recent studies reveal that ASK1 is related to a number of human
pathological conditions including inflammatory, tumorigenesis,
neurodegenerative disorders, cardiovascular and metabolic dis-
orders [19,20]. Consequently, pharmacological inhibition of ASK1
by small molecules represents a promising strategy to ameliorate
these human diseases [21]. For instance, compound 1 (K811, Fig. 1)
was reported to be effective in a SOD1G93A transgenic mouse
model of amyotrophic lateral sclerosis (ALS) [22]. In addition,
K811 presented a therapeutic potential for gastric cancer in in vitro
and in vivo assays [23]. Compound 2 was shown to be protective in
an isolated heart perfusion model of heart failure [24]. Compound
3, a preclinical candidate compound from Gilead, demonstrated
significant efficacy in several rodent models of acute and chronic
kidney diseases [25]. In a rat collagen-induced arthritis (CIA)
model, compound 4 could improve the joint damage in a dose-
dependent manner [26]. Moreover, a novel imidazopyridine-
treated with TNF-
the effect of ASK1 inhibitors on UC.
a
[34]. Hence, we focused our effort to investigate
Here, we report our structure-guided discovery of small mole-
cule ASK1 inhibitors based on the 1H-indole-2-carboxamide de-
rivatives. Our efforts have yielded the discovery of compound 19 as
a potent and orally active small molecule inhibitor of ASK1, which
exhibits good efficacy in a DSS-induced mouse model of UC, sug-
gesting that ASK1 inhibitors may have potential therapeutic utility
for the treatment of UC disease.
2. Chemistry
As presented in Schemes 1e5, a total of 22 target compounds
were prepared. The synthesis of compounds 8e14, 19, 20, 26 is
highlighted in Scheme 1. Commercially available methyl 6-
aminopicolinate reacted with hydrazine hydrate in EtOH to give
6-aminopicolinohydrazide 8-b, which was treated with DMF-DMA
to provide 8-c. The key intermediate 8-d was prepared by reacting
8-c with isopropylamine in the presence of acetic acid in acetoni-
trile. Treatment of appropriate acids with oxalyl chloride provided
acid chloride, which underwent amide formation with 8-d to
generate the target compounds in DCM.
Compound 15 was generated via the synthetic route illustrated
in Scheme 2. Bromination of compound 11 with NBS followed by a
Pd-catalyzed Suzuki cross-coupling reaction of 15-a with 2-(furan-
3-yl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane gave the target
compound.
The preparation of compounds 16, 17, 21e24, 27e29 is depicted
in Scheme 3. Conversion of the appropriate acid 16-a, 17-a, 21-a,
27-a to the acid chloride followed by amide formation with inter-
mediate 8-d furnished 16-b, 17-b, 21-b, 27-b. Suzuki cross-coupling
between the prepared bromine intermediates and appropriate ar-
omatic borates gave the desired products.
The synthesis of compound 18 is depicted in Scheme 4. (R)-2-(3-
(6-aminopyridin-2-yl)-4H-1,2,4-triazol-4-yl)propan-1-ol 18-a was
produced by a cyclization of 8-c with (R)-2-aminopropan-1-ol. O-
TBS protection of 18-a and subsequent amide coupling between 18-
b and 1H-indole-2-carbonyl chloride gave 18-c, which underwent
O-TBS deprotection to afford the target compound 18.
based selective ASK1 inhibitor
5 was obtained through
structure-based design and optimization [27]. The development
of ASK1 small molecule inhibitors with different structural types
may have a potential clinical application value. However, in recent
years, the therapeutic application of ASK1 small molecule in-
hibitors on ASK1-related diseases progresses slowly. Only GS-
4997 (6, selonsertib) from Gilead Sciences has been evaluated in
clinical trials, but failed to reach clinical endpoints in two phase III
clinical trials of nonalcoholic steatohepatitis (NASH) [28].
Considering ASK1 as an important therapeutic drug target, it is
reasonable to further explore the potential benefits of ASK1 in-
hibition in various diseases.
ASK1 is involved in the regulation of many inflammatory re-
sponses. Specifically, markedly elevated mRNA and protein levels of
ASK1 is observed in sodium fluoride (NaF)-induced mouse liver
inflammatory responses [29]. MiR-23b, an endogenous short non-
coding single-stranded RNA molecule, has been reported to sup-
press the inflammatory responses in the process of cutaneous
wound healing by targeting ASK1 [30]. Atsushi and his colleagues
found that ASK1 is required for LPS-induced p38 activation,
resulting in the production of pro-inflammatory cytokines
including IL-6, TNF-a and IL-1b [31]. Gurung et al. reported that RIF-
ASK1-MAPK signaling is activated in 2,4,6-trinitrobenzene sulfonic
acid (TNBS)-induced UC [32]. The Ca2þ/ASK1/MKK7/JNK2/cSrc
signaling pathway is related to DSS-induced tight junction
disruption and barrier dysfunction, which may lead to an inflam-
matory process and colitis [33]. Moreover, Baregamian et al.
demonstrated that ASK1-JNK/p38 stress signaling and mitochon-
drial apoptotic cascade are activated in rat intestinal epithelial cells
The preparation of compound 25 is shown in Scheme 5. Con-
version of 2-nitrophenylhydrazine 25-a to 25-b was followed by
Fischer Indole Synthesis to furnish 25-c. 25-e was obtained by
reduction of 25-c with Fe and NH4Cl in ethanol followed by amide
coupling of 25-d with acetyl chloride in DCM. Hydrolysis of 25-e
with sodium hydroxide and amide coupling of 25-f with 8-d gave
the desired compound 25.
Fig. 1. Representative ASK1 small molecule inhibitors.
2

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
Scheme 1. Reagents and conditions: (a) hydrazine hydrate, EtOH, 90 ꢀC, 12 h; (b) DMF-DMA, ref, 3 h; (c) isopropylamine, CH3CN/CH3COOH 5:1, 75 ꢀC, 4 h; (d) oxalyl chloride, DMF,
DCM, 0 ꢀC to rt, 1 h; (e) pyridine, DCM, 0 ꢀC to rt, 2 h.
Scheme 2. Reagents and conditions: (a) NBS, THF, rt, 2 h; (b) Pd(dppf)Cl2, K2CO3, 2-(furan-3-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, 1,4-dioxane/H2O 5:1, 100 ꢀC, N2, 6 h.
Scheme 3. Reagents and conditions: (a) oxalyl chloride, DMF, DCM, 0 ꢀC to rt, 1 h; (b) pyridine, DCM, 0 ꢀC to rt, 2 h; (c) Pd(dppf)Cl2, K3PO4, corresponding pinacol ester, 1,4-dioxane/
H2O 5:1, 110 ꢀC, 3 h.
Scheme 4. Reagents and conditions: (a) isopropylamine, CH3CN/CH3COOH 5:1, 75 ꢀC, 4 h; (b) tert-butyldimethylsilyl chloride, DIPEA, DME, rt, 2 h; (c) 1H-indole-2-carbonyl chloride,
pyridine, DCM, 0 ꢀC to rt, 2 h; (d) TBAF, THF, rt, 2 h.
3

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
Scheme 5. Reagents and conditions: (a) ethyl pyruvate, EtOH, rt, 12 h; (b) polyphosphoric acid, 80 ꢀC, 3 h; (c) Fe, NH4Cl, EtOH/H2O (6:1), 80 ꢀC, 4 h; (d) acetyl chloride, DIPEA, DCM,
rt, 3 h; (e) NaOH, THF/H2O 5:1, rt, 3 h; (f) oxalyl chloride, DMF, DCM, 0 ꢀC to rt, 1 h; (g) pyridine, DCM, 0 ꢀC to rt, 2 h.
3. Results and discussion
designed with the aim of forming an additional interaction with
Val757 in the hinge region (Table 1).
3.1. Structure-based design strategy
3.2. Inhibitory activity against ASK1
In our previous work, hit compound
7
(Fig. 3) (ASK1
IC50 ¼ 16700 2130 nM) was identified from our in-house com-
pound library. To improve its kinase potency, first, publicly available
crystallographic data and some pharmacophore models were
studied to better understand the molecular interactions between
ligands and ASK1 protein [35e40]. We found that almost all ASK1
inhibitors interact with the hinge region in ASK1 active site by
forming crucial hydrogen bond interaction with Val757. Typically,
the binding modes of compounds 5 and 6 are as drawn in Fig. 2.
Cocrystal structure 3VW6 reveals that compound 5 interacts with
hinge region through the hydrogen bond donor and receptor motif
of 2-N-acylimidazopyridine core, and the imidazole ring of com-
pound 5 also interacts with Lys709 deeper in the kinase active site.
The t-butyl phenyl group is sandwiched between Arg705 and
Lys769, pointing to the solvent-exposed region and the phenyl ring
stacking over Gly759. Moreover, docking analysis of 6 showed that
the amide carbonyl core of 6 forms a key hydrogen bond with the
backbone NH of Val757 acting as a single-point hinge binder.
Meanwhile, the triazole interacts with Lys709 through an impor-
tant hydrogen bond interaction, and a cyclopropyl-imidazole group
reaches the solvent exposed region around the protein [24,27,41].
Then, based on hit compound 7, we further developed potent
ASK1 small molecule inhibitors with a novel hinge-binder scaffold.
As outlined in Fig. 3, we initially preserved the core amine and
triazole ring of 6 to maintain the key hydrogen bonds with Val757
and Lys709, replacing the benzene group with 3-methylbenzofuran
from hit 7 affording compound 8. Meanwhile, various fused aro-
matic heterocycles, including benzofuran ring and benzothiazole
ring, were incorporated into compounds 9e10. Moreover, com-
pounds 11e12 with an indole ring and azaindole, respectively, were
Compounds 8e12 were prepared from key intermediate 8-
d (Scheme 1) and submitted for kinase potency studies. The re-
sults of the kinase inhibition assay are listed in Table 1. As expected,
compound 8 showed a remarkable increase in ASK1 potency
compared to hit 7, with ASK1 IC50 of 1106 24 nM. Unexpectedly,
benzofuran derivative 9 had significantly enhanced potency in
ASK1 kinase assay compared to 3-methylbenzofuran 8. We specu-
lated that the introduction of methyl is detrimental to the benzo-
furan ring to remain coplanar with the rest of the core, which is
pivotal for suitable binding of compound 8 to the ATP-binging site
[24,41,42]. Moreover, benzothiazole analogue 10 exhibited a greater
than 3-fold loss in potency in comparison with compound 9.
Interestingly, an indole ring replacement (11) yielded a more than
3-fold increase in potency compared to initial compound 9. To
better understand the results, a docking model derived from PDB
5VIO was used to predict the binding modes of compounds 9 and 11
in the ASK1 active site. As seen from Fig. 4, docking studies
confirmed two key hydrogen-bond interactions, that is, the
carbonyl group of core amine forming a H bond with backbone NH
of Val757 and N1 triazole nitrogen participating in a H bond with
catalytic Lys709. Nevertheless, the indole NH of compound 11,
distinct from compound 9, had an additional H bond with the
backbone carbonyl of Val757 in the hinge region, resulting in a
dramatic increase of potency observed in compound 11. However,
azaindole 12 showed 3-fold reduced ASK1 potency than 11 and an
equipotent effect to compound 9, indicating that an electron-
deficient pyridine ring is not tolerated well in this area. There-
fore, we focused further optimization and modification on indole
derivative 11.
Fig. 2. (A) Cocrystal of compound 5 in ASK1 (PDB code: 3VW6). (B) Compound 6 modeled into ASK1 (PDB code: 5VIO). The ligands and important residues are shown in stick form.
The hydrogen bonds are depicted as yellow dashed lines. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
4

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
Fig. 3. Structure-based design and optimization of 1H-indole-2-carboxamide derivatives as ASK1 inhibitors.
Table 1
containing substituents at the N1 nitrogen atom and 3-, 4-, 5-, 6-, 7-
positions of the 1H-indole ring were designed and prepared. The
in vitro ASK1 potency of these compounds is shown in Table 2e3.
First, 1-methyl-1H-indole derivative 13 showed a dramatically
decreased ASK1 kinase inhibition compared to 1H-indole derivative
11 as expected (see Table 2), confirming the importance of the H
bond between Val757 and 1H-indole. Similar to compound 8,
installation of a methyl (14) or furan appendage (15) to the 3-
position of indole also resulted in decreased activity compared to
the unsubstituted indole 11. Specifically, the ASK1 potency of furan
analogue 15 is inferior to that of methyl analogue 14, perhaps due to
the steric bulk of the furan ring being greater than that of methyl
and thus more likely to disturb the planarity of molecule. We also
explored the 4-position and 5-position of the indole ring by
introduction of N-methylpyrazole (16) and pyrazole (17) respec-
tively, in which both showed slightly improved kinase activity. We
speculated that the 4-position and 5-position of the indole ring
point to the solvent-accessible area and have no significant impact
on ASK1 potency. Moreover, replacing isopropyl (11) with (R)-1-
hydroxypropan-2-yl (18) resulted in comparable ASK1 kinase
activity.
Inhibitory activity of compounds 7e12 against ASK1.
Cpds
Structure
ASK1 IC50(nM)a
16700 2130
7
8
1106 24
151.6 11
506 12
9
10
Inspired by the overlay of compounds 11 and 6 in the ASK1
active site (Fig. 4B), we further explored the 6-position and 7-
position of the indole ring. As shown in Table 3, installation of an
electron-withdrawing group F (19) at the 6-position of the
unsubstituted indole slightly improved ASK1 kinase potency, and
the electron-donating group OCH3-substituted derivative (20)
exhibited slightly diminished potency. Moreover, placing a pyrazole
moiety at the 6-position (21) restored the binding affinity relative
to parent indole 11. We further explored the SAR of the solvent-
accessible region by substituting the pyrazole group with 1-
ethoxyethyl (22) and tetrahydropyran (23) and replacing pyrazole
with pyridine (24). Compounds 22e24 all showed decreased mo-
lecular activity, likely attributed to larger aromatic heterocyclic
substituents not well tolerated in this area.
11
12
a
41.4 2.17
125.1 12.10
IC50 values are the mean SD from two independent experiments.
Starting from the promising compound 11, we expanded the
chemical space of ASK1 inhibitors for further SAR exploration and
identified a potent ASK1 inhibitor (compound 19) with an accept-
able ADME profile (Fig. 3). 1H-indole-2-carboxamide analogues
Next, the introduction of an acetamido group to the 7-position
of the indole ring yielded compound 25, leading to a significant
decrease in ASK1 inhibition. The addition of an electron-
withdrawing group Cl (26) to the 7-position also resulted in a
Fig. 4. (A) Compound 9 modeled into ASK1 (PDB code: 5VIO). (B) Compound 11 overlaid 6 modeled into ASK1 (PDB code: 5VIO). The ligands and important residues are shown in
stick form. The hydrogen bonds are depicted as yellow dashed lines. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of
this article.)
5

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
Table 2
Inhibitory activity of compounds 13e18 against ASK1.
Cpds
Posa
1-
R1
R2
ASKIC50 (nM)b
743 48
Cpds
Posa
4-
R1
R2
ASKIC50 (nM)b
19.6 3.05
13
16
14
15
a
3-
3-
88.2 6.18
17
18
5-
31.6 1.61
65.5 3.61
142.8 6.55
e
H
Substituted position of R1 on the indole ring.
b
IC50 values are the mean SD from two independent experiments.
Table 4
Table 3
AP1-HEK293 cell activity of select compounds.
Inhibitory activity of compounds 19e29 against ASK1.
Cpds
Inhibition rate (%) at 10
m
Mb
Cpds
Inhibition rate (%) at 10 m
Mb
6
9
76.67 1.13
25.33 3.10
56.46 0.29
32.54 1.91
92.48 0.88
72.88 0.14
89.55 2.61
61.26 0.44
19
20
21
22
23
24
26
27
95.59 0.24
95.93 1.10
94.39 0.25
94.04 1.36
64.63 1.45
95.66 1.29
90.84 0.02
59.86 1.88
11
12
14
16
17
18
Cpds Posa
R
ASKIC50 (nM)b Cpds Posa
R
ASKIC50 (nM)b
240.1 8.05
19
6-
32.8 1.88
25
7-
b
Values are the mean SD from three independent experiments.
20
21
6-
6-
54.3 4.87
37.5 2.79
26
27
7-
7-
89.6 4.16
38.9 2.26
Table 5
Permeability assay of compounds 6 and 19 in MDRI-MDCKII cellsa.
Cpds
P-gp inhibitor
Papp
Efflux ratio
P-gp substratec
22
23
6-
6-
91.1 1.77
73.7 4.62
28
29
7-
7-
23.1 2.19
( ꢁ 10ꢂ6 cm/s)
(N ¼ 2)
153.3 7.65
A/B
B/A
6
without
with CsAb
without
with CsA
11.22
34.49
21.12
36.62
26.47
25.02
15.63
26.05
2.4
0.7
0.7
0.7
Yes
No
24
6-
68.5 9.55
19
a
a
Substituted position of R on the indole ring.
IC50 values are the mean SD from two independent experiments.
Cells that overexpress P-gp.
A known P-gp inhibitor ciclosporin A.
Efflux ratio (ER) ꢃ 2 and the ER decreases to < 50% of the ER in the absence of
b
b
c
inhibitor.
slightly decreased affinity to ASK1 when compared with compound
11. Furthermore, the five-membered aromatic heterocyclic
pyrazole-substituted analogue 27 and six-membered aromatic
heterocyclic pyridine-substituted analogue 28 showed a slightly
increased inhibition against ASK1. However, the thiophene ring
(29) is not well tolerated in this area, with a diminished ASK1 po-
tency (IC50 ¼ 153.3 7.65 nM).
activate transcription factors such as c-Jun, which binds to the
activator protein-1 (AP1) response element and induces AP1-
driven luciferase reporter activity that can be reduced by the
treatment of ASK1 inhibitors.
A total of 15 compounds were selected for AP1-HEK293 cell
assay. As illustrated in Table 4, the majority of these compounds
showed good inhibition of reporter activity in AP1-HEK293 cells at
10
mM, with inhibition rate typically greater than 50% except
3.3. AP1-HEK293 cell activity
benzofuran 9 and azaindole 12 that were less potent with inhibi-
tion rate of 25.33 3.10% and 32.54 1.91%, respectively. This is
consistent with their in vitro ASK1 kinase potency. In addition, eight
1H-indole-2-carboxamide derivatives had better AP1-HEK293
cellular activity than 6, with inhibition rate of compounds 19, 20,
and 24 all greater than 95%. These results indicated that this series
of compounds might have appropriate physicochemical properties
supporting our rational design for further evaluation.
To evaluate the drug likeness and cell membrane permeability
of these compounds, we analyzed the cellular potency of select
compounds in AP1-HEK293 cells that contain a firefly luciferase
gene under the control of AP1-responsive elements stably inte-
grated into HEK293 cells. Stress-induced activation of ASK1 leads to
phosphorylation and activation of JNKs via MMK4/7. Activated JNKs
then translocate to the nucleus where they phosphorylate and
6

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
Table 7
Pharmacokinetic (PK) profile of compound 6 in ratsa.
To better understand why compound 6 showed a stronger ASK1
kinase inhibition (IC50 ¼ 5.9 nM, Fig. 1) compared to compound 19
(IC50 ¼ 32.8 1.88 nM, Table 3) while being less active in the AP1-
HEK293 cellular assay, we compared their membrane permeability
in MDRI-MDCKII cell-based assay. As shown in Table 5, compound
19 (Papp(A/B) ¼ 21.12 ꢁ 10ꢂ6 cm/s) had a better cell membrane
permeability than compound 6 (Papp(A/B) ¼ 11.22 ꢁ 10ꢂ6 cm/s).
Moreover, unlike compound 19, compound 6 was identified to be a
P-gp substrate, which is subject to removal from the cells by an
ATP-dependent efflux pump. That could explain its lesser activity in
the AP1-HEK293 cell activity assay.
Cpd
CL (L/h/kg), iv
0.11
Vss(L/kg), iv
0.55
T1/2(h), iv
5.07
F(%)
75
6
a
Referenced to https://www.cortellis.com/drugdiscovery, iv dose 1 mg/kg and po
dose 5 mg/kg.
index (DAI) score of the control group remained nearly unchanged
throughout the experiment. Treatment with DSS induced a 2.83
unit increase in DAI score. In contrast to the DSS model group, the
DAI score of the compound 19 group showed about a 2 unit
decrease, with the compound 6 (i.e., GS-4997) group showing a
0.83 unit decrease. These results suggested that compounds 19 and
6 could improve symptoms of the UC mice, in which compound 19
exhibited a stronger alleviation than compound 6.
Given colon shortening is also an obvious symptom in DSS-
induced UC mice, measurement of the colon length in all the
experimental mice showed that compound 19 significantly pre-
vented colon shortening induced by DSS. As shown in Fig. 5C and D,
the colon length of the normal control group was 9.63 cm, the
percentage of colon shortening of DSS model group was 26.7%
(colon length: 7.05 cm). In contrast, the percentage of colon
shortening of compound 19 group was 14.8% (colon length:
8.20 cm), while the percentage of colon shortening of compound 6
group was 23.4% (colon length: 7.37 cm). To further confirm the
therapeutic effect of compound 19, we performed a histopatho-
logical analysis of colon tissues. As shown in Fig. 5E, DSS treatment
induced a severe colonic tissue damage and infiltration of inflam-
matory cells. Consistent with previous results, compound 19
significantly attenuated these pathological changes of colon tissues.
Compound 6 also produced a certain protective effect. Taken
together, compound 19 showed a significant protective effect in the
DSS-induced colitis mouse model.
3.4. Pharmacokinetic properties
With the aim of investigating the pharmacokinetic (PK) profile
of these 1H-indole-2-carboxamides and based on the favorable
in vitro kinase and AP1-HEK293 cell activity, compound 19 was
subsequently evaluated in a rat PK study. As shown in Table 6,
compound 19 had low in vivo clearance (CL ¼ 1.38 L/h/kg) and
moderate half-life (T1/2 ¼ 1.45 h) after iv administration at a dose of
1 mg/kg. Moreover, following oral administration at 10 mg/kg,
compound 19 showed high oral exposure (AUClast ¼ 4517 h*ng/mL),
62.2% oral bioavailability and acceptable terminal half-life (T1/
¼ 2.31 h). These data fully justified to assess the efficacy of
2
compound 19 in in vivo murine animal models. Judging from
published data (Table 7), compound 6 also exhibited a favorable PK
profile in rats. Upon a single iv dose (1 mg/kg), it showed low
clearance rate (CL ¼ 0.11 L/h/kg), modest volume of distribution
(Vss ¼ 0.55 L/kg) and long half-life (T1/2 ¼ 5.07 h), and upon oral
administration at an oral dose of 5 mg/kg, compound 6 had a good
oral bioavailability (75%).
3.5. Compound 19 improved DSS-induced UC
Due to the favorable PK profile and good in vitro potency,
compounds 19 and 6 was further evaluated in a DSS-induced
mouse UC model. In this model, ICR mice were given 3% DSS
drinking water from day 1 to day 7. Weight loss, diarrhea, and rectal
bleeding were observed from day 3, indicating that the UC model
was successfully established. Compounds 19 and 6 were adminis-
tered orally at a dose of 25 mg/kg per day from day 1 to day 7.
As demonstrated in Fig. 5A, the body weights of all experimental
mice increased slightly in the first two days. However, compared
with the vehicle-treated normal control group, the body weights of
DSS-treated model group mice decreased significantly by ꢂ9.6%,
unlike a continued increase seen in the control group. Oral
administration of compound 19 at a dose of 25 mg/kg to the DSS-
treated experimental mice induced a significant recovery of body
weight loss, with an increase of þ11.2%. Although compound 6
treatment also exhibited a certain therapeutic effect, it failed to
prevent body weight loss. As shown in Fig. 5B, the disease activity
3.6. Compound 19 blocked ASK1-p38/JNK signaling pathways in
DSS-induced mouse colon tissues
To better understand the molecular mechanism underlying the
anti-inflammatory activity of compounds 19 and 6, we performed
Western blotting to investigate the inhibitory effect of compounds
19 and 6 on ASK1-p38/JNK signaling pathways involved in the
regulation of various inflammatory responses. As shown in Fig. 6A
and B, DSS treatment resulted in an upregulation of phosphoryla-
tion of ASK1, MKK3/6, MKK4/7, and downstream p38 and JNK in
colon tissues when compared with the control group. However,
abnormally elevated phosphorylated proteins of ASK1-p38/JNK
signaling pathways were effectively reversed by compound 19,
with compound 6 showing weaker effects than that of compound
19. These data suggested that ASK1-p38/JNK signaling is important
for compounds 19- and 6-mediated protection in DSS-induced
acute colitis mice.
Table 6
Pharmacokinetic (PK) profile of compound 19 in ratsa.
3.7. Reduction of inflammatory cytokine levels by compound 19 in
DSS-induced mouse colon tissues
Parametersb
iv (1 mg/kg)
Parametersb
po (10 mg/kg)
CL(L/h/kg)
1.38 0.106
2.82 0.225
713 51.5
727 54.7
1.45 0.0885
2.04 0.109
Tmax(h)
2.00 0.17
676 101
4517 1042
4521 1043
2.31 0.112
62.2 14.4
V
ss(L/kg)
Cmax(ng/mL)
AUClast(h*ng/mL)
AUCINF(h*ng/mL)
T1/2(h)
Increasing literature highlights an abnormal upregulation of
pro-inflammatory cytokines, such as IL-1b, IL-6 and TNF-a, is
AUClast(h*ng/mL)
AUCINF(h*ng/mL)
important in the pathogenesis of UC. To further define the anti-
inflammatory mechanism of compounds 19 and 6, enzyme-linked
immunosorbent assay (ELISA) was conducted to examine the
expression levels of these inflammatory cytokines in colon tissues.
T1/2(h)
MRTINF(h)
F(%)
a
Male Sprague Dawley rats iv at 1 mg/kg and the dosing solution was prepared in
10% DMAC þ 10% (30% Solutol HS 15 in water) þ 80% Saline, po at 10 mg/kg and the
dosing suspension was prepared in 0.5% CMC and 0.5% Tween 80 in water.
As shown in Fig. 6C, the significantly enhanced levels of IL-1
b, IL-6
b
PK parameters are the mean SD from three independent experimental rats.
and TNF- in DSS-treated mice were decreased by compound 19.
a
7

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
Fig. 5. Effect of compounds 19 and 6 in DSS-induced UC mice. Mice were given 3% DSS drinking water ad libitum to induce colitis. Control mice were fed with distilled water only. In
addition to DSS, compounds 19 and 6, dissolved in CMC-Na aqueous solution, were orally administered to compound 19 and compound 6 group mice, respectively. Meanwhile,
CMC-Na aqueous solution were orally administered to the control group and DSS model group mice. (A) The body weight changes of each group during the experiment (n ¼ 5e6 per
group). (B) DAI scores of each group during the experiment (n ¼ 5e6 per group). (C) Representative images of mouse colon from each group. Scale bar, 200
mm. (D) Colon length of
each group (n ¼ 4e6 per group). (E) Representative H&E staining images of the colon tissues from each group. Data are expressed as mean SEM. Compared with the control group:
#
##
###
P < 0.05,
P < 0.01,
P < 0.001; Compared with the DSS þ Veh group: * P < 0.05, ** P < 0.01, *** P < 0.001.
Compound 6 was also helpful but with a weaker effect. These re-
sults showed that compounds 19 and 6 could attenuated DSS-
induced UC via inhibition of pro-inflammatory cytokines,
shortening of UC mice was significantly improved by compound 19
treatment. Furthermore, histopathological examination showed
that compound 19 remarkably relieved the colon tissue damage
induced by DSS treatment. Compound 6 also showed a moderate
protective effect in these tests. Mechanistic studies revealed that
compounds 19 and 6 suppressed ASK1-MKK3/6-p38 and ASK1-
MKK4/7-JNK signaling activation and reduced the up-regulated
including IL-1b, IL-6 and TNF-a.
4. Conclusion
pro-inflammatory cytokines (IL-1b, TNF-a, IL-6) in the colon tis-
In this paper, starting from our previously identified hit com-
pound 7 containing a 3-methylbenzofuran-2-carbonyl fragment,
we designed and synthesized several compounds consisting of
various fused aromatic heterocycles. Interestingly, indole 11
showed a 3-fold more potent ASK1 kinase inhibitory activity than
benzofuran 9. Besides the known hydrogen bond between carbonyl
of 11 and Val757, the docking study identified an additional H bond
between indole NH of 11 and Val757. Subsequent structure-based
optimization of compound 11 led to the discovery of compound
19 with potent anti-ASK1 kinase activity, stronger AP1-HEK293 cell
inhibitory activity than compound 6, and excellent in vivo PK
properties.
Studies showed that the ASK1 signaling pathway is activated in
the development and occurrence of UC [32], and the cellular
function of ASK1 is related to inflammatory responses [43]. For
these reasons, compounds 19 and 6 were subjected to the research
on UC. In the DSS-induced mouse model of UC, oral administration
of compound 19 could ameliorate the inflammatory symptoms of
UC mice caused by DSS administration. Body weights of the com-
pound 19 group mice are generally close to that of the control group
and the DAI score of the compound 19 group also exhibited an
evident decline compared to the DSS model group. Notably, colon
sues of DSS-exposed UC mice. Compound 6 produced weaker anti-
UC effect compared to compound 19, which agrees with its rela-
tively lower activity in the AP1-HEK293 cell-based assay. The better
anti-UC effect of compound 19 could be due to its higher membrane
permeability and a lack of susceptibility to the efflux through the
ATP-dependent efflux pump, so that compound 19 may exert
stronger therapeutic effect in the colon tissues.
In conclusion, we have discovered a series of potent ASK1 small
molecule inhibitors with a novel 1H-indole-2-carboxamide hinge
scaffold. The most promising compound 19 demonstrated signifi-
cant therapeutic effects on DSS-induced UC as an ASK1 small
molecule inhibitor with an anti-inflammatory action. Altogether,
this research unfolds the possibility of developing orally available
ASK1 inhibitors as therapeutic agents for the treatment of UC.
5. Experimental
5.1. Chemistry
All reagents and solvents are commercially available and were
purchased as reagent grade and used without further purification.
8

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
Fig. 6. Inhibitory effect of compounds 19 and 6 on activated ASK1-p38/JNK signaling and up-regulated inflammatory cytokines in colon tissues of DSS-induced UC mice. (A) Western
blot analysis of the protein levels of ASK1-MKK3/6-p38 and ASK1-MKK4/7-JNK signaling pathways as well as corresponding phosphorylated proteins. GAPDH was used as the
loading control protein. (B) Quantitative analysis of the ratios of phosphorylated proteins normalized to their unphosphorylated form. (C) The levels of inflammatory cytokines IL-1
b
,
#
##
###
IL-6, TNF-
a
were detected using ELISA kits. Data are expressed as mean SEM (n ¼ 5e6 per group). Compared with the control group: P < 0.05,
P < 0.01,
P < 0.001;
Compared with the DSS þ Veh group: * P < 0.05, ** P < 0.01, *** P < 0.001.
1H NMR spectra were obtained with Bruker AV-300 or Bruker AV-
400. The abbreviations s, d, t, q, dd and m signify singlet, doublet,
solid (4.50 g, 29.3 mmol, yield: 89%). 1H NMR (400 MHz, DMSO‑d6)
d
9.14 (s, 1H), 7.51 (t, J ¼ 8.4 Hz, 1H), 7.11 (d, J ¼ 7.2 Hz, 1H), 6.60 (d,
triplet, quartet, doublet of doublets and multiplet, respectively. 13
C
J ¼ 7.2 Hz, 1H), 6.07 (s, 2H), 4.47 (s, 2H). MS (ESI) [M þ H]þ: 153.1.
Step B: A mixture of 8-b (4.50 g, 29.0 mmol) in DMF-DMA was
heated under reflux for 3 h, the reaction was monitored by TLC.
Upon completion, the reaction mixture was cooled to rt and then
concentrated under reduced pressure. The residue was taken up in
EA (100 mL) and heated at 50 ꢀC for 30 min. After being cooled to rt,
the solid was collected by filtration and dried in vacuo to give (E)-
N’-(6-(2-((E)-(dimethylamino)methylene)hydrazine-1-carbonyl)
pyridin-2-yl)-N,N-dim ethylformimidamide (8-c) (5.00 g,
19.1 mmol, yield: 66%) as a white solid. 1H NMR (400 MHz,
NMR spectra were obtained with Bruker AV-300 or Bruker AV-400.
Low-Resolution Mass Spectrometry analyses were conducted on
Agilent 1100 LC/MSD mass spectrometer (Agilent, USA). High-
Resolution Mass Spectrometry analyses were conducted on a Q-
Tof micro spectrometer (Micromass Company). Melting points
were determined with a X-4 digital-display melting-point appa-
ratus (Beijing Tech Instrument Co., Ltd.). All reactions were moni-
tored by thin-layer chromatography (TLC) on silica gel plates
(GF254) and spots were visualized with UV light. Flash column
chromatography was performed over silica gel using a mixture of
ethyl acetate (EA), petroleum ether (PE), dichloromethane (DCM)
and MeOH. The purity of the final compounds was assessed by
high-pressure liquid chromatography (HPLC) and determined to
be ꢃ 95% pure.
DMSO‑d6)
d
10.69 (s, 1H), 8.86 (s, 1H), 8.06 (s, 1H), 7.68 (t, J ¼ 7.7 Hz,
1H), 7.48 (d, J ¼ 6.8 Hz, 1H), 6.91 (d, J ¼ 8.0 Hz, 1H), 3.14 (s, 3H), 3.02
(s, 3H), 2.88 (s, 6H). MS (ESI) [M þ H]þ: 263.2.
Step C: To a solution of compound 8-c (5.00 g, 19.0 mmol) in a
mixture of CH3CN/AcOH (30 mL, 5:1) was added isopropylamine
(5.60 g, 95.0 mmol, 5.0 eq). The resulting mixture was heated at
75 ꢀC for 4 h, the reaction was monitored by TLC. Upon completion,
the reaction mixture was cooled to rt, and the solvent was removed
under reduced pressure. The residue was dissolved in water
(100 mL) and 2 M NaOH was added to a pH of 8.0. The water solvent
was extracted with EA (100 mL x 3). The combined organic layers
were dried over anhydrous Na2SO4, filtered, and concentrated un-
der reduced pressure. The resulting residue was chromatographed
over silica gel (3e10% MeOH in DCM) to give 6-(4-isopropyl-4H-
1,2,4-triazol-3-yl)pyridin-2-amine (8-d) (2.52 g, 12.3 mmol, yield:
5.1.1. N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-3-
methylbenzofuran-2-carboxamide (8)
Step A: To a solution of methyl 6-aminopicolinate 8-a (5.00 g,
33.0 mmol) in EtOH (10 mL) was added hydrazine hydrate (4.85 g,
99.0 mmol, 3.0 eq), the mixture was stirred at 90 ꢀC for 12 h in
pressure tube, the reaction was monitored by TLC. Upon comple-
tion, the reaction mixture was cooled to rt. The precipitate formed
in the mixture was collected by filtration, washed with EA and then
dried in vacuo to give 6-aminopicolinohydrazide (8-b) as a white
9

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
65%) as a white solid. 1H NMR (300 MHz, DMSO‑d6)
7.52 (t, J ¼ 7.8 Hz, 1H), 7.16 (d, J ¼ 7.4 Hz, 1H), 6.52 (d, J ¼ 8.3 Hz, 1H),
6.17 (s, 2H), 5.62e5.45 (m,1H),1.43 (d, J ¼ 6.7 Hz, 6H). MS (ESI) [M þ
H]þ: 204.1.
d
8.78 (s, 1H),
for the synthesis of compound 8 (step D). White solid, 58 mg, yield:
50%. mp 259e262 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d 11.89 (s, 1H),
10.67 (s, 1H), 8.90 (s, 1H), 8.23 (d, J ¼ 8.2 Hz, 1H), 8.04 (t, J ¼ 8.0 Hz,
1H), 7.86 (d, J ¼ 7.5 Hz, 1H), 7.71 (d, J ¼ 8.0 Hz, 1H), 7.60e7.55 (m,
1H), 7.50 (d, J ¼ 8.2 Hz, 1H), 7.26 (t, J ¼ 7.2 Hz, 1H), 7.10 (t, J ¼ 7.4 Hz,
1H), 5.76e5.66 (m, 1H), 1.48 (d, J ¼ 6.7 Hz, 6H). 13C NMR (100 MHz,
Step D: To a 0 ꢀC cooled solution of 3-methylbenzofuran-2-
carboxylic acid (100 mg, 0.57 mmol) and DMF (2 d) in DCM
(10 mL) was added oxalyl chloride (108 mg, 0.85 mmol, 1.5 eq), the
mixture was stirred at 0 ꢀC for 10 min and then rt for 1 h. The
resulting mixture was concentrated under reduced pressure to give
3-methylbenzofuran-2-carbonyl chloride as a white solid, which
was used for further reaction without purification. To a 0 ꢀC cooled
solution of 8-d (116 mg, 0.57 mmol) and pyridine (68 mg,
0.86 mmol, 1.5 eq) in DCM (10 mL) was added the prepared 3-
methylbenzofuran-2-carbonyl chloride, the mixture was stirred at
0 ꢀC for 10 min and then rt for 2 h, the reaction was monitored by
TLC. Upon completion, the resulting mixture was concentrated
under reduced pressure, the residue was dissolved in water
(100 mL) and extracted with EA (100 mL x 3). The combined organic
layers were dried over anhydrous Na2SO4, filtered, and concen-
trated under reduced pressure. The resulting residue was chro-
matographed over silica gel (3e15% MeOH in DCM) to give N-(6-(4-
DMSO‑d6)
d 160.64, 151.80, 150.49, 146.70, 143.64, 139.92, 137.63,
131.19, 127.34, 124.70, 122.43, 120.56, 119.59, 115.66, 112.97, 106.12,
48.32, 23.70. HRMS-EI m/z [M þ H]þ calcd for C19H19N6Oþ:
347.1615,
found:
347.1615.
HPLC
analysis:
retention
time ¼ 4.238 min, 99.56%.
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-pyr-
rolo[2,3-b]pyridine-2-carboxamide (12) Compound 12 was pre-
pared from 1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (64 mg,
0.39 mmol) and key intermediate 8-d (80 mg, 0.39 mmol) ac-
cording to a similar procedure described for the synthesis of
compound 8 (step D). White solid, 60 mg, yield: 45%. mp
279e282 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d 12.47 (s, 1H), 10.68 (s,
1H), 8.88 (s, 1H), 8.41 (dd, J ¼ 4.6, 1.6 Hz, 1H), 8.24 (dd, J ¼ 9.0,
3.0 Hz, 1H), 8.16 (dd, J ¼ 9.0, 1.6 Hz, 1H), 8.05 (t, J ¼ 8.0 Hz, 1H),
7.86(dd, J ¼ 8.0, 1.4 Hz, 1H), 7.51 (s, 1H), 7.18 (q, J ¼ 4.6 Hz, 1H),
5.75e5.62 (m, 1H), 1.47 (d, J ¼ 9.0 Hz, 6H). 13C NMR (100 MHz,
isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-3-
benzofuran-2-carboxamide (8) (100 mg, 0.28 mmol, yield: 49%) as
a white solid. mp 175e177 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
10.55
methyl-
DMSO‑d6)
d 160.22, 151.66, 150.47, 149.21, 146.72, 143.65, 140.00,
d
131.66, 131.05, 119.80, 119.60, 117.23, 115.64, 105.27, 48.36, 23.70.
HRMS-EI m/z [M þ H]þ calcd for C18H18N7Oþ: 348.1567, found:
348.1570. HPLC analysis: retention time ¼ 3.817 min, 95.21%.
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-
(s, 1H), 8.89 (s, 1H), 8.17 (d, J ¼ 8.1 Hz, 1H), 8.03 (t, J ¼ 9.0 Hz, 1H),
7.89 (d, J ¼ 7.5 Hz, 1H),7.82 (d, J ¼ 7.8 Hz, 1H), 7.71 (d, J ¼ 8.1 Hz, 1H),
7.54 (t, J ¼ 7.6 Hz, 1H), 7.39 (t, J ¼ 7.4 Hz, 1H), 2.62 (s, 3H), 5.82e5.65
(m, 1H), 1.47 (d, J ¼ 6.5 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6)
methyl-1H-indole-2-carboxamide (13) Compound 13 was pre-
pared from 1-methyl-1H-indole-2-carboxylic acid (52 mg,
0.30 mmol) and key intermediate 8-d (60 mg, 0.30 mmol) ac-
cording to a similar procedure described for the synthesis of
compound 8 (step D). White solid, 53 mg, yield: 49%. mp
d
158.86, 153.42, 151.18, 150.40, 146.76, 143.74, 142.81, 139.98,
129.54, 128.31, 123.99, 123.95, 121.84, 119.92, 115.74, 112.46, 48.29,
23.75, 9.46. HRMS-EI m/z [M þ H]þ calcd for C20H20N5Oþ2 : 362.1612,
found: 362.1615. HPLC analysis: retention time ¼ 6.835 min;
98.45%.
229e231 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 10.80 (s, 1H), 8.88 (s,
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)benzo-
1H), 8.18 (d, J ¼ 8.2 Hz,1H), 8.03 (t, J ¼ 7.9 Hz,1H), 7.88 (d, J ¼ 7.5 Hz,
1H), 7.73 (d, J ¼ 7.9 Hz, 1H), 7.61 (d, J ¼ 8.4 Hz, 1H), 7.42 (s, 1H), 7.35
(t, J ¼ 7.6 Hz, 1H), 7.16 (t, J ¼ 7.4 Hz, 1H), 5.84e5.68 (m, 1H), 4.04 (s,
furan-2-carboxamide (9) Compound
9 was prepared from
benzofuran-2-carboxylic acid (100 mg, 0.37 mmol) and key inter-
mediate 8-d (75 mg, 0.37 mmol) according to a similar procedure
described for the synthesis of compound 8 (step D). White solid,
77 mg, yield: 60%. mp 224e226 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
3H), 1.45 (d, J ¼ 6.6 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6)
d 161.52,
151.75, 150.39, 146.72, 143.69, 139.87, 139.44, 131.81, 125.85, 124.83,
122.45, 120.92, 119.53, 115.52, 111.14, 107.49, 48.33, 31.97, 23.71.
HRMS-EI m/z [M þ H]þ calcd for C20H21N6Oþ: 361.1771, found:
361.1776. HPLC analysis: retention time ¼ 5.442 min, 98.65%.
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-3-
d
10.93 (s, 1H), 8.90 (s, 1H), 8.17 (dd, J ¼ 8.3, 0.7 Hz, 1H), 8.05 (t,
J ¼ 8.0 Hz, 1H), 7.96 (d, J ¼ 0.8 Hz, 1H), 7.91e7.84 (m, 2H), 7.77 (dd,
J ¼ 8.4, 0.6 Hz, 1H), 7.56e7.51 (m, 1H), 7.42e7.36 (m, 1H), 5.77e5.68
(m, 1H), 1.47 (d, J ¼ 6.7 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6)
methyl-1H-indole-2-carboxamide (14) Compound 14 was pre-
pared from 3-methyl-1H-indole-2-carboxylic acid (87 mg,
0.49 mmol) and key intermediate 8-d (100 mg, 0.49 mmol) ac-
cording to a similar procedure described for the synthesis of
compound 8 (step D). White solid, 127 mg, yield: 72%. mp
d
157.71,155.10,151.30,150.42,148.50,146.75,143.71,140.02,127.97,
127.47, 124.44, 123.59, 120.02, 115.92, 112.57, 112.33, 48.29, 23.72.
HRMS-EI m/z [M þ H]þ calcd for C19H18N5Oþ2 : 348.1455, found:
348.1459. HPLC analysis: retention time ¼ 5.020 min, 99.25%.
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)benzo
[d]thiazole-2-carboxamide (10) Compound 10 was prepared from
benzo[d]thiazole-2-carboxylic acid (75 mg, 0.42 mmol) and key
intermediate 8-d (85 mg, 0.42 mmol) according to a similar pro-
cedure described for the synthesis of compound 8 (step D). White
solid, 96 mg, yield: 63%. mp 263e266 ꢀC. 1H NMR (400 MHz,
269e271 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d 11.65 (s, 1H), 10.24 (s,
1H), 8.90 (s, 1H), 8.26 (d, J ¼ 8.2 Hz, 1H), 8.04 (t, J ¼ 8.0 Hz, 1H), 7.86
(d, J ¼ 7.5 Hz,1H), 7.69 (d, J ¼ 8.0 Hz,1H), 7.47 (d, J ¼ 8.2 Hz,1H), 7.28
(t, J ¼ 7.4 Hz, 1H), 7.10 (t, J ¼ 7.4 Hz, 1H), 5.71e5.57 (m, 1H), 2.61 (s,
3H), 1.47 (d, J ¼ 6.7 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6)
d 161.46,
151.77, 150.41, 146.65, 143.67, 140.06, 136.28, 128.42, 127.42, 125.02,
120.52, 119.90, 119.44, 116.82, 114.89, 112.61, 48.46, 23.67, 10.36.
HRMS-EI m/z [M þ H]þ calcd for C20H21N6Oþ: 361.1771, found:
361.1770. HPLC analysis: retention time ¼ 5.460 min, 98.34%.
6-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-
yl)-1H-indole-2-carboxamide (19) Compound 19 was prepared
from 6-fluoro-1H-indole-2-carboxylic acid (176 mg, 0.98 mmol)
and key intermediate 8-d (200 mg, 0.98 mmol) according to a
similar procedure described for the synthesis of compound 8 (step
D). White solid, 292 mg, yield: 82%. mp 249e252 ꢀC. 1H NMR
DMSO‑d6)
d
11.20 (s, 1H), 8.91 (s, 1H), 8.29 (dd, J ¼ 12.2, 8.1 Hz, 2H),
8.17 (d, J ¼ 8.1 Hz, 1H), 8.09 (t, J ¼ 7.9 Hz, 1H), 7.96 (d, J ¼ 7.5 Hz, 1H),
7.72e7.63 (m, 2H), 5.75e5.65 (m, 1H), 1.49 (d, J ¼ 6.7 Hz, 6H). 13
C
NMR (100 MHz, DMSO‑d6)
d 164.25, 159.08, 153.00, 150.49, 150.27,
146.91, 143.86, 140.23, 137.01, 127.90, 124.91, 123.67, 120.47, 115.96,
48.31, 23.79. HRMS-EI m/z [M þ H]þ calcd for C18H17N6OSþ:
365.1179,
found:
365.1179.
HPLC
analysis:
retention
time ¼ 6.790 min, 97.88%.
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-
indole-2-carboxamide (11) Compound 11 was prepared from 1H-
indole-2-carboxylic acid (55 mg, 0.34 mmol) and key intermediate
8-d (70 mg, 0.34 mmol) according to a similar procedure described
(300 MHz, DMSO‑d6)
d 11.95 (s, 1H), 10.72 (s, 1H), 8.88 (s, 1H), 8.21
(d, J ¼ 8.3 Hz, 1H), 8.03 (t, J ¼ 7.9 Hz, 1H), 7.85 (d, J ¼ 7.6 Hz, 1H),
7.78e7.69 (m, 1H), 7.59 (s, 1H), 7.20 (d, J ¼ 9.9 Hz, 1H), 6.97 (t,
10

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
J ¼ 9.3 Hz, 1H), 5.78e5.60 (m, 1H), 1.47 (d, J ¼ 6.6 Hz, 6H). 13C NMR
3-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol
-3-yl)pyridin-2-yl)-1H-
(100 MHz, DMSO‑d6)
d
160.39, 151.74, 150.54, 146.69, 143.58, 140.18,
indole-2-carboxamide (15) (54 mg, 0.13 mmol, yield: 57%) as a
139.90, 131.99, 124.25, 123.99, 119.67, 115.65, 109.97, 106.24, 98.53,
white solid. mp 223e225 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d 12.10
48.30, 23.65. HRMS-EI m/z [M þ H]þ calcd for C20H21N6Oþ:
(s, 1H), 9.56 (s, 1H), 8.88 (s, 1H), 8.31 (d, J ¼ 8.0 Hz, 1H), 8.18 (s, 1H),
8.04 (t, J ¼ 8.0 Hz, 1H), 7.94 (t, J ¼ 1.6 Hz, 1H), 7.90 (d, J ¼ 7.5 Hz, 1H),
7.58 (d, J ¼ 8.1 Hz, 1H), 7.53 (d, J ¼ 8.3 Hz, 1H), 7.32 (t, J ¼ 7.3 Hz, 1H),
7.14 (t, J ¼ 7.5 Hz, 1H), 6.89e6.83 (m, 1H), 5.43e5.32 (m, 1H), 1.43 (d,
365.1521,
found:
365.1520.
HPLC
analysis:
retention
time ¼ 4.366 min, 99.54%.
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-
methoxy-1H-indole-2-carboxamide (20) Compound 20 was pre-
pared from 6-methoxy-1H-indole-2-carboxylic acid (71 mg,
0.37 mmol) and key intermediate 8-d (75 mg, 0.37 mmol) ac-
cording to a similar procedure described for the synthesis of 8 (step
D). White solid, 97 mg, yield: 70%. mp 242e245 ꢀC. 1H NMR
J ¼ 6.7 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6)
d 160.52, 151.11,
150.03, 146.68, 144.58, 143.83, 142.08, 140.26, 136.51, 127.55, 127.39,
125.22, 121.02, 120.94, 119.47, 117.72, 114.44, 113.02, 112.86, 110.13,
48.63, 23.56. HRMS-EI m/z [M þ H]þcalcd for C23H21N6Oþ2 : 413.1721,
found: 413.1722. HPLC analysis: retention time ¼ 3.917 min, 96.84%.
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(1-
methyl-1H-pyrazol-4-yl)-1H-indole-2-carboxamide (16). Step A:
Compound 16-b was prepared from 4-bromo-1H-indole-2- car-
boxylic acid 16-a (177 mg, 0.74 mmol) and key intermediate 8-
d (150 mg, 0.74 mmol) according to a similar procedure described
for the synthesis of compound 8 (step D). Yellow solid, 235 mg,
(300 MHz, DMSO‑d6)
d 11.70 (s, 1H), 10.56 (s, 1H), 8.88 (s, 1H), 8.23
(d, J ¼ 8.3 Hz, 1H), 8.02 (t, J ¼ 7.9 Hz,1H), 7.84 (d, J ¼ 7.5 Hz, 1H), 7.58
(d, J ¼ 8.7 Hz, 1H), 7.52(s, 1H), 6.93 (s, 1H), 6.75 (d, J ¼ 8.7 Hz, 1H),
5.78e5.62 (m, 1H), 3.80 (s, 3H), 1.47 (d, J ¼ 6.5 Hz, 6H). 13C NMR
(100 MHz, DMSO‑d6) d 160.67,158.23,152.00,150.63,146.69,143.57,
139.79, 138.81, 130.08, 123.27, 121.74, 119.46, 115.56, 112.15, 106.69,
94.57, 55.54, 48.38, 23.64. HRMS-EI m/z [M þ H]þ calcd for
yield: 75%. 1H NMR (300 MHz, DMSO‑d6)
d: 11.95 (s, 1H), 10.74 (s,
C
20H21N6Oþ2 : 377.1721, found: 377.1720. HPLC analysis: retention
1H), 8.89 (s, 1H), 8.22 (dd, J ¼ 8.1, 0.6 Hz, 1H), 8.04 (t, J ¼ 8.1 Hz, 1H),
7.85 (dd, J ¼ 7.5, 0.6 Hz, 1H), 7.56 (s, 1H), 7.49 (dd, J ¼ 8.1, 0.3 Hz,1H),
7.32e7.21 (m, 1H), 7.08e6.88 (m, 1H), 5.73e5.57 (m, 1H), 1.47 (d,
J ¼ 9.6 Hz, 6H). MS (ESI) [M þ H]þ: 425.1.
time ¼ 4.210 min, 99.04%.
7-chloro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-
yl)-1H-indole-2-carboxamide (26) Compound 26 was prepared
from 7-chloro-1H-indole-2-carboxylic acid (92 mg, 0.47 mmol) and
key intermediate 8-d (95 mg, 0.47 mmol) according to a similar
procedure described for the synthesis of compound 8 (step D).
White solid, 120 mg, yield: 67%, mp 208e211 ꢀC. 1H NMR (300 MHz,
Step B: A mixture solution of 16-b (100 mg, 0.24 mmol),
Pd(dppf)Cl2 (18 mg, 0.024 mmol, 0.1 eq), K3PO4 (102 mg,
0.48 mmol, 2.0 eq), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan- 2-yl)-1H-pyrazole (75 mg, 0.36 mmol, 1.5 eq) in
1,4-dioxane/H2O (10 mL, 2 mL) was heated at 110 ꢀC for 3 h, the
reaction was monitored by TLC. Upon completion, the mixture was
allowed to cool to rt, filtered, the filtrate was diluted with water
(50 mL) and the water solvent was extracted with EA (50 mL x 3).
The combined organic layers were dried over anhydrous Na2SO4,
filtered, and concentrated under reduced pressure. The resulting
residue was chromatographed over silica gel (3e8% MtOH in DCM)
to give N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(1-
methyl-1H-pyrazol-4-yl)-1H-indole-2-carboxamide (16) (50 mg,
0.12 mmol, yield: 51%) as a white solid. mp 235e237 ꢀC. 1H NMR
DMSO‑d6)
d
12.06 (s, 1H), 10.85 (s, 1H), 8.90 (s, 1H), 8.26 (dd, J ¼ 6.3,
0.3 Hz, 1H), 8.06 (t, J ¼ 8.7 Hz, 1H), 7.84 (dd, J ¼ 7.6, 0.6 Hz, 1H), 7.68
(d, J ¼ 6.0 Hz, 1H), 7.55 (d, J ¼ 2.1 Hz, 1H), 7.37 (dd, J ¼ 5.7, 0.6 Hz,
1H), 7.12 (t, J ¼ 7.8 Hz, 1H), 5.63e5.49 (m, 1H), 1.48 (d, J ¼ 5.1 Hz,
6H). 13C NMR (100 MHz, DMSO‑d6)
d 159.92, 151.83, 150.68, 146.72,
143.52, 139.96, 134.71, 132.87, 129.08, 124.34, 121.59, 121.38, 119.98,
117.20, 115.73, 108.54, 48.38, 23.63. HRMS-EI m/z [M þ H]þ calcd for
C
19H18ClN6Oþ: 381.1225, found: 381.1225. HPLC analysis: retention
time ¼ 5.617 min, 95.52%.
3-(furan-3-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyr-
idin-2-yl)-1H-indole-2-carboxamide (15) Step A: To a solution of
compound 11 (200 mg, 0.57 mmol) in THF was added NBS (102 mg,
0.57 mmol, 1.0 eq), and the mixture was stirred for 2 h at rt, the
reaction was monitored by TLC. Upon completion, the reaction
solution was concentrated under reduced pressure. The residue
was partitioned between saturated aqueous Na2SO3 (50 mL) and EA
(50 mL). The organic layer was separated and washed with brine
(50 mL), dried over Na2SO4, filtered, and concentrated under
reduced pressure. The resulting residue was chromatographed over
silica gel (3e5% MtOH in DCM) to give 3-bromo-N-(6-(4-isopropyl-
4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2- carboxamide (15-
a) (198 mg, 0.46 mmol, yield: 82%) as a yellow solid. 1H NMR
(400 MHz, DMSO‑d6)
d
12.00 (s, 1H), 10.78 (d, J ¼ 7.1 Hz, 1H), 8.89 (s,
1H), 8.27 (s, 1H), 8.22 (d, J ¼ 8.3 Hz, 1H), 8.10e8.00 (m, 2H), 7.96 (s,
1H), 7.80 (d, J ¼ 7.6 Hz, 1H), 7.39e7.33 (m, 1H), 7.29e7.20 (m, 2H),
5.62e5.50 (m, 1H), 3.95 (s, 3H), 1.46 (d, J ¼ 6.7 Hz, 6H). 13C NMR
(100 MHz, DMSO‑d6)
d 160.60, 151.92, 150.80, 146.63, 143.49,
139.90, 138.28, 137.65, 131.19, 129.25, 126.87, 125.08, 124.54, 121.40,
119.85, 117.81, 115.97, 110.96, 105.95, 48.35, 40.50, 23.64. HRMS-EI
m/z [M þ H]þ calcd for C23H23N8Oþ: 427.1989, found: 427.1990.
HPLC analysis: retention time ¼ 4.046 min, 97.48%.
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-(1H-
pyrazol-4-yl)-1H-indole-2-carboxamide (17) Compound 17 was
prepared from 5-bromo-1H-indole-2-carboxylic acid 17-a (142 mg,
0.59 mmol), key intermediate 8-d (120 mg, 0.59 mmol) and 4-
(300 MHz, DMSO‑d6)
d 11.95 (s, 1H), 10.74 (s, 1H), 8.89 (s, 1H), 8.22
(dd, J ¼ 8.1, 0.6 Hz, 1H), 8.04 (t, J ¼ 8.1 Hz, 1H), 7.85 (dd, J ¼ 7.5,
0.6 Hz, 1H), 7.70 (d, J ¼ 5.1 Hz, 1H), 7.49 (dd, J ¼ 8.1, 0.3 Hz, 1H),
7.25e7.10 (m, 1H), 7.08e6.78 (m, 1H), 5.73e5.53 (m, 1H), 1.47 (d,
J ¼ 9.6 Hz, 6H). MS (ESI) [M þ H]þ: 425.1.
(4,4,5,5-tetramethyl-1,3,2-
(115 mg, 0.59 mmol) according to a similar procedure described for
dioxaborolane-2-yl)-1H-pyrazole
the synthesis of compound 16. White solid, 80 mg, yield: 33%. mp
295e297 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d 12.89 (s, 1H), 12.06 (s,
Step B: A mixture of 15-a (100 mg, 0.23 mmol), Pd(dppf)Cl2
(17 mg, 0.023 mmol, 0.1 eq), K2CO3 (64 mg, 0.46 mmol, 2.0 eq), 2-
1H), 10.83 (s, 1H), 8.89 (s, 1H), 8.22 (d, J ¼ 8.0 Hz, 1H), 8.04 (t,
J ¼ 7.9 Hz, 3H), 7.90 (s, 1H), 7.85 (d, J ¼ 7.3 Hz, 1H), 7.57e7.43 (m,
3H), 5.81e5.66 (m, 1H), 1.47 (d, J ¼ 6.7 Hz, 6H). 13C NMR (100 MHz,
(furan-3-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
(67
mg,
0.35 mmol, 1.5 eq) in 1,4-dioxane/H2O 5:1 (10 mL, 2 mL) was heated
at 100 ꢀC for 6 h in nitrogen atmosphere, the reaction was moni-
tored by TLC. Upon completion, the mixture was allowed to cool to
rt, filtered, the filtrate was diluted with water (50 mL) and the water
solvent was extracted with EA (50 mL x 3). The combined organic
layers were dried over anhydrous Na2SO4, filtered, and concen-
trated under reduced pressure. The resulting residue was chro-
matographed over silica gel (3e8% MtOH in DCM) to give 3-(furan-
DMSO‑d6) d 160.62, 151.74, 150.60, 146.43, 143.63, 140.00, 136.46,
131.44, 127.89, 125.54, 123.58, 122.58, 119.74, 118.05, 115.84, 113.35,
106.08, 48.45, 23.59. HRMS-EI m/z [M þ H]þ calcd for C22H21N8Oþ:
413.1833,
found:
413.1836.
HPLC
analysis:
retention
time ¼ 3.389 min, 96.31%.
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-(1H-
pyrazol-4-yl)-1H-indole-2-carboxamide (21) Compound 21 was
prepared from 6-bromo-1H-indole-2-carboxylic acid 21-a (165 mg,
11

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
0.69 mmol), key intermediate 8-d (140 mg, 0.69 mmol) and 4-
133.78, 132.30, 127.25, 124.40, 123.26, 120.11, 119.65, 115.70, 111.09,
(4,4,5,5-tetramethyl-1,3,2-
dioxaborolane-2-yl)-1H-pyrazole
106.12, 48.34, 23.71. HRMS-EI m/z [M þ H]þ calcd for C24H22N7Oþ:
(134 mg, 0.69 mmol) according to a similar procedure described for
424.1880,
found:
424.1882.
HPLC
analysis:
retention
the synthesis of compound 16. White solid, 99 mg, yield: 35%. mp
time ¼ 4.597 min, 98.75%.
159e161 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d: 12.92 (s, 1H), 11.88 (s,
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(1H-
pyrazol-4-yl)-1H-indole-2-carboxamide (27) Compound 27 was
prepared from 7-bromo-1H-indole-2-carboxylic acid 27-a (177 mg,
0.74 mmol), key intermediate 8-d (150 mg, 0.74 mmol) and 4-
1H), 10.66 (s, 1H), 8.89 (s, 1H), 8.23 (d, J ¼ 8.2 Hz, 1H), 8.04 (t,
J ¼ 8.0 Hz, 2H), 7.85 (d, J ¼ 7.6 Hz,1H), 7.68 (d, J ¼ 8.3 Hz,1H), 7.62 (s,
1H), 7.54 (s, 1H), 7.38 (d, J ¼ 8.3 Hz, 1H), 5.72e5.53 (m, 1H), 1.47 (d,
J ¼ 6.6 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6)
d
160.53, 151.85,
(4,4,5,5-tetramethyl-1,3,2-dioxa
(143 mg, 0.74 mmol) according to a similar procedure described for
borolan-2-yl)-1H-pyrazole
150.52, 146.67, 143.66, 139.88, 138.36, 131.18, 129.80, 125.88, 122.80,
122.51, 119.57, 115.65, 108.48, 106.60, 48.37, 23.70. HRMS-EI m/z
[M þ H]þ calcd for C22H21N8Oþ: 413.1833, found: 413.1834. HPLC
analysis: retention time ¼ 3.465 min, 97.54%.
the synthesis of compound 16. White solid, 94 mg, yield: 31%. mp
170e173 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d: 13.12 (s, 1H), 11.04 (s,
1H), 10.93 (s, 1H), 8.89 (s, 1H), 8.27 (d, J ¼ 11.1 Hz, 1H), 8.04 (t,
J ¼ 7.8 Hz,1H), 7.81 (d, J ¼ 6.9 Hz, 1H), 7.61 (d, J ¼ 8.1 Hz, 1H), 7.49 (s,
1H),7.41 (d, J ¼ 7.2 Hz,1H), 7.15 (s, 1H), 7.13 (t, J ¼ 5.7 Hz,1H), 6.85 (s,
1H), 5.40 (s, 1H), 1.45 (d, J ¼ 5.4 Hz, 6H). 13C NMR (100 MHz,
6-(1-(1-ethoxyethyl)-1H-pyrazol-4-yl)-N-(6-(4-isopropyl-4H-
1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide (22)
Compound 22 was prepared from 6-bromo-1H-indole-2-carboxylic
acid 22-a (188 mg, 0.79 mmol), key intermediate 8-d (160 mg,
0.79 mmol) and 1-(1-ethoxyethyl)-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (210 mg, 0.79 mmol) according
to a similar procedure described for the synthesis of compound 16.
White solid,130 mg, yield: 34%. mp 230e232 ꢀC .1H NMR (400 MHz,
DMSO‑d6)
d 160.20, 151.94, 150.64, 146.64, 143.53, 139.97, 134.90,
132.26, 128.22, 123.59, 121.29, 120.48, 119.82, 118.92, 117.92, 115.70,
108.77, 48.35, 23.65. HRMS-EI m/z [M þ H]þ calcd for C22H21N8Oþ:
413.1833,
found:
413.1830.
HPLC
analysis:
retention
time ¼ 4.040 min, 95.24%.
DMSO‑d6)
d
11.97 (s, 1H), 10.70 (s, 1H), 8.89 (s, 1H), 8.39 (s, 1H), 8.23
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(pyr-
idin-4-yl)-1H-indole-2-carboxamide (28) compound 28 was
prepared from 7-bromo-1H-indole-2-carboxylic acid 28-a (159 mg,
0.66 mmol), key intermediate 8-d (134 mg, 0.66 mmol) and 4-
(4,4,5,5-tetramethyl-1,3,2-dioxa borolan-2-yl)pyridine (135 mg,
0.66 mmol) according to a similar procedure described for the
synthesis of compound 16. White solid, 100 mg, yield: 36%, mp
(d, J ¼ 8.3 Hz, 1H), 8.04 (t, J ¼ 8.0 Hz, 1H), 7.94 (s, 1H), 7.85 (d,
J ¼ 7.6 Hz, 1H), 7.69 (q, J ¼ 6.0 Hz, 1H), 7.63 (s, 1H), 7.54 (s, 1H), 7.39
(dd, J ¼ 8.3, 1.2 Hz, 1H), 5.79e5.66 (m, 1H), 5.58 (q, J ¼ 5.9 Hz, 1H),
3.55e3.41 (m, 1H), 3.28e3.20 (m, 1H), 1.65 (d, J ¼ 6.0 Hz, 3H), 1.47
(d, J ¼ 6.8 Hz, 6H), 1.06 (t, J ¼ 7.0 Hz, 3H). 13C NMR (100 MHz,
DMSO‑d6)
d 160.58, 151.91, 150.55, 146.69, 143.63, 139.85, 138.35,
136.71, 131.46, 129.18, 126.13, 125.34, 123.71, 122.86, 119.36, 115.70,
108.62, 106.66, 86.87, 63.51, 48.39, 23.69, 21.63, 15.20. HRMS-EI m/z
[M þ H]þ calcd for C26H29N8Oþ2 : 485.2408, found: 485.2408. HPLC
analysis: retention time ¼ 4.616 min, 96.72%.
278e280 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d 11.58 (s, 1H), 10.84 (s,
1H), 8.90 (s, 1H), 8.73 (s, 2H), 8.25 (d, J ¼ 8.6 Hz, 1H), 8.06 (d,
J ¼ 7.7 Hz, 1H), 7.82 (d, J ¼ 7.4 Hz, 2H), 7.72 (s, 2H), 7.59 (s, 1H), 7.39
(d, J ¼ 6.9 Hz, 1H), 7.28 (d, J ¼ 7.6 Hz, 1H), 5.57 (s, 1H), 1.46 (d,
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-(1-
(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)-1H-indole-2-
carboxamide (23) Compound 23 was prepared from 6-bromo-1H-
indole-2-carboxylic acid 23-a (141 mg, 0.59 mmol), key interme-
diate 8-d (120 mg, 0.59 mmol) and 1-(tetrahydro-2H-pyran-4-yl)-
J ¼ 6.2 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6)
d 160.07, 151.85,
150.59, 150.55, 150.52, 146.67, 146.08, 143.54, 140.04, 134.78, 132.74,
128.50, 125.38, 124.30, 123.98, 121.35, 119.88, 115.62, 108.28, 48.33,
23.65. HRMS-EI m/z [M þ H]þ calcd for C24H22N7Oþ: 424.1880,
found: 424.1880. HPLC analysis: retention time ¼ 5.000 min,
95.04%.
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
1H-pyrazole
(164 mg, 0.59 mmol) according to a similar procedure described for
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(thi-
ophen-3-yl)-1H-indole-2-carboxamide (29) Compound 29 was
prepared from 7-bromo-1H-indole-2-carboxylic acid 29-a (165 mg,
0.69 mmol), key intermediate 8-d (140 mg, 0.69 mmol) and 4,4,5,5-
tetramethyl-2-(thiophen-3- yl)-1,3,2-dioxaborolane (145 mg,
0.69 mmol) according to a similar procedure described for the
synthesis of compound 16. White solid, 91 mg, yield: 31%. mp
the synthesis of compound 16. White solid, 79 mg, yield: 27%. mp
269e272 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 11.87 (s, 1H), 10.64 (s,
1H), 8.90 (s, 1H), 8.27 (s, 1H), 8.23 (d, J ¼ 8.1 Hz, 1H), 8.04 (t,
J ¼ 8.0 Hz,1H), 7.89 (s, 1H), 7.85 (d, J ¼ 7.6 Hz,1H), 7.69 (d, J ¼ 8.4 Hz,
1H), 7.60 (s, 1H), 7.55e7.52 (m, 1H), 7.36 (dd, J ¼ 8.4, 1.3 Hz, 1H),
5.75e5.65 (m, 1H), 4.48e4.36 (m, 1H), 4.02e3.96 (m, 2H),
3.49e3.42 (m, 2H), 2.05e1.97 (m, 4H), 1.47 (d, J ¼ 6.7 Hz, 6H). 13
C
178e181 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d 11.13 (s, 1H), 10.90 (s,
NMR (100 MHz, DMSO‑d6)
d
160.62, 151.87, 150.59, 146.69, 143.60,
1H), 8.89 (s, 1H), 8.26 (d, J ¼ 7.8 Hz, 1H), 8.05 (t, J ¼ 8.0 Hz, 1H), 7.98
(dd, J ¼ 2.8, 1.3 Hz, 1H), 7.82 (d, J ¼ 7.0 Hz, 1H), 7.76 (dd, J ¼ 5.0,
2.9 Hz, 1H), 7.70 (d, J ¼ 7.8 Hz, 1H), 7.59e7.52 (m, 2H), 7.41 (dd,
J ¼ 7.2, 0.9 Hz, 1H), 7.24e7.15 (m, 1H), 5.58e5.52 (m, 1H), 1.46 (d,
139.89, 138.36, 136.23, 131.22, 129.57, 126.01, 125.37, 122.92, 119.40,
115.64, 108.38, 106.41, 66.42, 57.80, 48.36, 33.43, 23.66. HRMS-EI m/
z [M þ H]þ calcd for C27H29N8O2þ: 497.2408, found: 497.2408. HPLC
analysis: retention time ¼ 3.932 min, 96.98%.
J ¼ 6.7 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6)
d 160.11, 151.89,
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-(pyr-
idin-3-yl)-1H-indole-2-carboxamide (24) Compound 24 was
prepared from 6-bromo-1H-indole-2-carboxylic acid 24-a (153 mg,
0.64 mmol), key intermediate 8-d (130 mg, 0.64 mmol) and 3-
(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)pyridine (131 mg,
0.64 mmol) according to a similar procedure described for the
synthesis of compound 16. White solid, 78 mg, yield: 29%. mp
150.65, 146.68, 143.53, 140.00, 138.88, 134.95, 132.35, 128.39,
128.36, 127.18, 124.64, 123.51, 121.85, 121.64, 121.28, 119.88, 115.61,
108.53, 48.33, 23.66. HRMS-EI m/z [M þ H]þ calcd for C23H21N6OSþ:
429.1492,
found:
429.1492.
HPLC
analysis:
retention
time ¼ 7.333 min, 96.70%.
(R)-N-(6-(4-(1-hydroxypropan-2-yl)-4H-1,2,4-triazol-3-yl)
pyridin-2-yl)-1H-indole-2-carboxamide (18) Step A: Compound
18-a was prepared from compound 8-c (500 mg, 1.90 mmol) and
(R)-2-aminopropan-1-ol (712 mg, 9.5 mmol, 5.0 eq) according to a
similar procedure described for the synthesis of compound 8 (step
280e283 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d 12.07 (s, 1H), 10.74 (s,
1H), 8.91 (d, J ¼ 11.9 Hz, 2H), 8.59 (d, J ¼ 4.3 Hz, 1H), 8.24 (d,
J ¼ 8.1 Hz, 1H), 8.13 (d, J ¼ 8.0 Hz, 1H), 8.05 (t, J ¼ 7.9 Hz, 1H), 7.86
(dd, J ¼ 8.0, 2.3 Hz, 2H), 7.76 (s, 1H), 7.64e7.60 (m, 1H), 7.54 (dd,
J ¼ 7.9, 4.9 Hz, 1H), 7.49e7.43 (m, 1H), 5.78e5.65 (m, 1H), 1.48 (d,
C). White solid, 255 mg, yield: 61%. 1H NMR (300 MHz, CDCl3)
d 8.36
(s, 1H), 7.54 (t, J ¼ 8.1 Hz, 1H), 7.42 (d, J ¼ 7.4 Hz, 1H), 6.57 (d,
J ¼ 8.2 Hz, 1H), 5.32e5.21 (m, 1H), 4.68 (s, 2H), 4.01 (d, J ¼ 11.4 Hz,
1H), 3.86e3.40 (m, 2H), 1.52 (d, J ¼ 6.8 Hz, 3H). MS (ESI) [M þ H]þ:
J ¼ 6.7 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6)
d 160.47, 151.78,
150.50, 148.57, 148.26, 146.73, 143.64, 139.93, 138.15, 137.03, 134.72,
12

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
220.1.
Step B: A solution of 25-b (1.20 g, 4.78 mmol) in polyphosphoric
acid (20 mL) was heated at 80 ꢀC for 3 h, the reaction was moni-
tored by TLC. Upon completion, the mixture was then allowed to
cooled to rt. The reaction was diluted with water, followed by
carefully acidified to pH 8 by slow addition of saturated aqueous
NaOH solution, the resulting aqueous mixture was extracted with
EA (100 mL ꢁ 3). The mixture was washed with H2O (100 mL), brine
(100 mL), dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was chromatographed over silica gel
(0e30% EA in PE) to give ethyl 7-nitro-1H-indole-2-carboxylate
(25-c) (794 mg, 3.39 mmol, yield: 71%) as a yellow solid. 1H NMR
Step B: To a solution of compound 18-a (200 mg, 0.57 mmol) in
DME (10 mL) was added DIPEA (147 mg,1.14 mmol, 2.0 eq) and tert-
butyldimethylsilyl chloride (103 mg, 0.68 mmol, 1.2 eq), the
mixture was stirred for 2 h at rt and the reaction was monitored by
TLC. Upon completion, the reaction solution was concentrated
under reduced pressure. The residue was partitioned between
saturated aqueous NaHCO3 (50 mL) and EA (50 mL). The organic
layer was separated and washed with brine (50 mL), dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
resulting residue was chromatographed over silica gel (10e35% EA
in PE) to give (R)-6-(4-(1-((tert-butyldimethylsilyl)oxy)propan-2-
(300 MHz, DMSO‑d6)
d
: 11.38 (s, 1H), 8.29 (dd, J ¼ 8.0, 2.3 Hz, 1H),
yl)-4H-1,2,4-triazol-3-yl)pyridin-2-amine
(18-b)
(160
mg,
8.23 (dd, J ¼ 7.7 Hz, 2.4 Hz, 1H), 7.47(t, J ¼ 2.0 Hz, 1H), 7.43e7.35 (m,
1H), 4.39 (q, J ¼ 7.1 Hz, 2H), 1.37 (t, J ¼ 11.6 Hz, 3H). MS (ESI) [M þ
H]þ: 235.1.
0.48 mmol, yield: 85%) as a white solid. 1H NMR (300 MHz, CDCl3)
d
8.43 (s, 1H), 7.59 (d, J ¼ 3.3 Hz, 1H), 7.28 (d, J ¼ 5.3 Hz, 1H), 6.58
(dd, J ¼ 7.1, 1.9 Hz, 1H), 5.68e5.59 (m, 2H), 4.59 (s, 2H), 3.96 (s, 1H),
1.55 (d, J ¼ 7.0 Hz, 3H), 0.96e0.90 (m, 6H), 0.87 (s, 9H). MS (ESI)
[M þ H]þ: 334.2.
Step C: To a solution of 25-c (794 mg, 3.39 mmol) in EtOH/H2O
(12 mL, 2 mL) was added Fe (957 mg, 17.0 mmol, 5 eq) followed by
NH4Cl (1.10 g, 20.0 mmol, 6.0 eq). The mixture was stirred at 80 ꢀC
for 4 h, the reaction was monitored by TLC. Upon completion, the
reaction mixture was filtered through Celite. The filter cake was
washed with 10% MeOH in DCM (30 mL), and the biphasic mother
liquor was separated. The aqueous phase was further extracted
with DCM (50 mL ꢁ 3), and the combined organic extracts were
washed with H2O (50 mL), brine (50 mL), dried over Na2SO4,
filtered, and concentrated under reduced pressure. The residue was
chromatographed over silica gel (0e5% MeOH in DCM) to give ethyl
7-amino-1H-indole-2- carboxylate (25-d) (394 mg, 1.93 mmol,
Step C: Compound 18-c was prepared from 1H-indole-2-
carboxylic acid (193 mg, 1.20 mmol) and (R)-6-(4-(1-((tert-butyl-
dimethylsilyl)oxy)propan-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-
amine 18-b (400 mg, 1.20 mmol) according to a similar procedure
described for the synthesis of compound 8 (step D). White solid,
359 mg, yield: 63%. 1H NMR (300 MHz, DMSO‑d6)
d 11.89 (s, 1H),
10.65 (s, 1H), 8.80 (s, 1H), 8.21 (dd, J ¼ 8.3, 0.6 Hz, 1H), 8.03 (t,
J ¼ 8.0 Hz, 1H), 7.84 (dd, J ¼ 7.6, 0.3 Hz, 1H), 7.71 (d, J ¼ 7.9 Hz, 1H),
7.55 (d, J ¼ 1.5 Hz, 1H), 7.49 (d, J ¼ 8.1 Hz, 1H), 7.32e7.23 (m, 1H),
7.15e7.04 (m, 1H), 5.65e5.57 (m, 1H), 3.69e3.59 (m, 2H), 1.48 (d,
J ¼ 6.9 Hz, 3H), 0.90e0.86 (m, 6H), 0.83 (s, 9H). MS (ESI) [M þ H]þ:
477.2.
Step D: To a solution of compound 18-c (200 mg, 0.42 mmol) in
THF (10 mL) was added TBAF (220 mg, 0.84 mmol, 2.0 eq). The
resulting mixture was stirred for 2 h at rt, the reaction was moni-
tored by TLC. Upon completion, and the solvent was removed under
reduced pressure. The residue was diluted with water (100 mL) and
the water solvent was extracted with EA (100 mL x 3). The com-
bined organic layers were dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The resulting residue
was chromatographed over silica gel (20e40% EA in PE) to give (R)-
N-(6-(4-(1-hydroxypropan-2-yl)-4H-1,2,4-triazol-3-yl)pyridin- 2-
yl)-1H-indole-2- carboxamide (18) (100 mg, 0.28 mmol, yield:
66%) as a white solid. mp 255e257 ꢀC. 1H NMR (300 MHz,
57%) as a white solid. 1H NMR (300 MHz, DMSO‑d6)
d: 11.42 (s, 1H),
7.02 (d, J ¼ 2.1 Hz, 1H), 6.85e6.76 (m, 2H), 6.42 (dd, J ¼ 7.0, 1.2 Hz,
1H), 5.41 (s, 2H), 4.34 (q, J ¼ 7.1 Hz, 2H), 1.34 (t, J ¼ 7.1 Hz, 3H). MS
(ESI) [M þ H]þ: 205.1.
Step D: To a solution of 25-d (200 mg, 0.98 mmol) in DCM
(10 mL) was added DIPEA (253 mg, 1.96 mmol, 2.0 eq) followed by
acetyl chloride (115 mg, 1.47 mmol, 1.5 eq). The mixture was stirred
at rt for 3 h, the reaction was monitored by TLC. Upon completion,
the mixture was concentrated under reduced pressure and then
poured into H2O (50 mL) and extracted with EA (50 mL ꢁ 3). The
combined organic extracts were washed with H2O (50 mL), brine
(50 mL), dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was chromatographed over silica gel
(0e5% MeOH in DCM) to give ethyl 7-acetamido-1H-indole-2-
carboxylate (25-e) (150 mg, 0.61 mmol, yield: 62%) as a white
DMSO‑d6)
d
11.89 (s, 1H), 10.65 (s, 1H), 8.80 (s, 1H), 8.21 (d,
solid. 1H NMR (300 MHz, DMSO‑d6)
d: 11.65 (s, 1H), 9.80 (s, 1H), 7.93
J ¼ 8.3 Hz, 1H), 8.03 (t, J ¼ 8.0 Hz,1H), 7.84 (d, J ¼ 7.6 Hz,1H), 7.71 (d,
J ¼ 7.9 Hz, 1H), 7.56 (d, J ¼ 1.5 Hz, 1H), 7.49 (d, J ¼ 8.2 Hz, 1H), 7.26 (t,
J ¼ 7.6 Hz, 1H), 7.09 (t, J ¼ 7.5 Hz, 1H), 5.61 (q, J ¼ 5.5 Hz, 1H), 4.99 (t,
J ¼ 5.4 Hz, 1H), 3.64 (d, J ¼ 5.9 Hz, 2H), 1.49 (s, 3H). 13C NMR
(s, 1H), 7.39 (d, J ¼ 7.8 Hz, 1H), 7.25e6.93 (m, 2H), 2.38 (d, J ¼ 6.9 Hz,
3H), 2.19 (d, J ¼ 13.7 Hz, 3H), 1.49e1.25 (m, 3H). MS (ESI) [M þ H]þ:
247.1.
Step E: To a solution of 25-e (150 mg, 0.61 mmol) in THF (10 mL)
was added a solution of NaOH (49 mg, 1.22 mmol, 2.0 eq) in H2O
(2 mL). The mixture was stirred at rt for 3 h, the reaction was
monitored by TLC. Upon completion, the resulting reaction was
then extracted with EA (30 mL ꢁ 2). The aqueous phase was sub-
sequently neutralized to pH 2 with 2 N aqueous HCl and then
extracted with EA (30 mL ꢁ 3), and the combined organic extracts
were concentrated under reduced pressure. The residue was
chromatographed over silica gel (0e20% MeOH in DCM) to give 7-
acetamido-1H-indole-2-carboxylic acid (25-f) (100 mg, 0.46 mmol,
(100 MHz, DMSO‑d6) d 160.70, 151.70, 151.08, 146.84, 144.22, 139.88,
137.64, 131.17, 127.38, 124.77, 122.46, 120.62, 119.79, 115.58, 112.99,
106.04, 65.03, 53.79, 18.02. HRMS-EI m/z [M þ H]þ calcd for
C
19H19N6Oþ2 : 363.1564, found: 363.1567. HPLC analysis: retention
time ¼ 3.730 min, 96.04%.
7-acetamido-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyr-
idin-2-yl)-1H-indole-2-carboxamide (25) Step A: To a solution of
2-nitrophenylhydrazine hydrochloride 25-a (1.00 g, 5.27 mmol) in
EtOH (20 mL) was added ethyl pyruvate (731 mg, 6.3 mmol, 1.2 eq)
and the mixture was stirred at rt for 12 h, the reaction was moni-
tored by TLC. Upon completion, the resulting mixture was
concentrated under reduced pressure to give ethyl (E)-2-(2-(2-
nitrophenyl)hydrazono)propanoate (25-b) (1.20 g, 4.80 mmol,
yield: 91%) as a yellow solid, which was used for further reactions
yield: 75%) as a white solid. 1H NMR (300 MHz, DMSO‑d6)
d: 12.07
(s, 1H), 11.65 (s, 1H), 9.80 (s, 1H), 7.93 (s, 1H), 7.39 (d, J ¼ 7.8 Hz, 1H),
7.25e6.93 (m, 2H), 2.38 (d, J ¼ 6.9 Hz, 3H). MS (ESI) [M þ H]þ: 219.1.
Step F: Compound 25 was prepared from 7-acetamido-1H-
indole-2-carboxylic acid 25-f (161 mg, 0.73 mmol) and key inter-
mediate 8-d (150 mg, 0.73 mmol) according to a similar procedure
described for the synthesis of compound 8 (step D). White solid,
114 mg, yield: 39%. mp 269e272 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
without purification. 1H NMR (400 MHz, DMSO‑d6)
d 10.27 (s, 1H),
8.08 (t, J ¼ 2.2 Hz, 1H), 7.78e7.70 (m, 1H), 7.68e7.63 (m, 1H), 7.56 (t,
J ¼ 8.1 Hz, 1H), 4.22 (q, J ¼ 7.1 Hz, 2H), 2.09 (d, J ¼ 10.6 Hz, 3H), 1.28
(t, J ¼ 7.1 Hz, 3H). MS (ESI) [M þ H]þ: 252.1.
d
11.82 (s, 1H), 10.80 (s, 1H), 9.94 (s, 1H), 8.89 (s, 1H), 8.22 (d,
13

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
J ¼ 8.2 Hz, 1H), 8.05 (t, J ¼ 7.9 Hz, 1H), 7.96 (d, J ¼ 7.5 Hz, 1H), 7.86 (d,
J ¼ 7.4 Hz, 1H), 7.62 (s, 1H), 7.44 (d, J ¼ 7.8 Hz, 1H), 7.05 (t, J ¼ 7.8 Hz,
1H), 5.80e5.63 (m, 1H), 2.18 (s, 3H), 1.47 (d, J ¼ 6.5 Hz, 6H). 13C NMR
treatment for determining the basal activity). Add 10 mL of assay
medium with 0.1% DMSO to cell-free control wells. (6) Incubate the
plate at 37 ꢀC in a CO2 incubator for 6 h (7) Perform luciferase assay
using the ONE-Step™ Luciferase Assay System following the pro-
(100 MHz, DMSO‑d6) d 169.07, 160.55, 151.74, 150.52, 146.72, 143.62,
139.93, 130.88, 128.71, 128.69, 125.24, 120.96, 119.70, 117.73, 115.77,
tocol provided: Add 100 mL of ONE-Step™ Luciferase reagent per
114.80, 106.59, 48.35, 24.45, 23.69. HRMS-EI m/z [M þ H]þ calcd for
well and rock at rt for 15 min. Measuring luminescence using a
luminometer. (8) Data Analysis: Obtain background-subtracted
luminescence by subtracting average background luminescence
(cell-free control wells) from luminescence reading of all wells.
C
21H22N7Oþ2 : 404.1829, found: 404.1835. HPLC analysis: retention
time ¼ 3.703 min, 99.38%.
5.2. ASK1 kinase assay
5.4. Cell membrane permeability assay
ASK1 kinase activity of target compounds was measured using
the Homogeneous Time Resolved Fluorescence (HTRF) assay kit
(Cisbio, Catalog #62ST3PEB) with full-length recombinant human
ASK1 (MAP3K5) (Carna, Catalog #07e107). The assay procedure is
as follows: (1) Dilution series of test compounds were prepared in
100% DMSO and assay buffer (1 X Enzymatic, 5 mM MgCl2, 1 mM
The membrane permeability of compounds 6 and 19 was eval-
uated using MDRI-MDCKII cells by Sundia MediTech Co., Ltd
(Shanghai, China). The assay procedure is as follows. (1) While
80e90% of cell culture bottle was adhered with cells, the cell cul-
ture medium was discarded. (2) The cell culture bottle was washed
with 5 mL of 10 mM PBS buffer (37 ꢀC) and then the PBS was dis-
carded. (3) The adhered cells were dissolved with 5 mL of 0.05%
Trypsin-EDTA, then 10 mL of cell culture medium (37 ꢀC) was added
to terminate reaction, centrifuged (900 rpm, 3 min). (4) Superna-
tant of cell culture medium was discarded and 10 mL of new cell
culture medium (37 ꢀC) was added to calculate. The cells were
diluted to 2 ꢁ 105 cells/mL. (5) Cells were seeded into 24-multiwell
Insert Systems with PET (polyethylene terephthalate) membranes
DTT), and 2.0
dividual wells of a white optiplate-384. (2) 4
L) in assay buffer and 4 L of STK-Substrate 3-biotin (5
(250 M) in assay buffer were added to the assay plates containing
test compounds. (3) Assay plates were incubated for 2 h at rt and
L of STK S3 Antibody-Eu (500 nM) in assay buffer was added. (4)
m
L of diluted compounds were dispensed to the in-
L of MAP3K5 (5 ng/
M)/ATP
m
m
m
m
m
5
m
The assay plates were incubated for an additional 1 h at rt and then
read on an EnVision plate reader using a 340 nm excitation wave-
length and 665 nm emission wavelength for fluorescence detec-
tion. The final amount of enzyme in the assay was 2 nM ASK1, the
final concentration of STK-Substrate 3-biotin was 2 mM and the final
concentration of DMSO was 2%. IC50 values were obtained using
Prism 8.0 (GraphPad Software).
(1 m
m pore size and 0.3 cm2 surface area) at an optimized density of
2 ꢁ 105 cells/ml in cell culture medium. (6) Before transport study,
all the apical sides and basolateral sides were washed with 0.3 mL
and 1 mL of PBS buffer (pH 7.4) for 30 min at 37 ꢀC, in 5% CO2
incubator. (7) MDRI-MDCKII cell monolayers were preincubated (at
37 ꢀC, in 5% CO2 incubator) in transport media, all the apical sides
and basolateral sides were preincubated with 0.2 mL and 0.7 mL of
the transport media with or without specific P-gp inhibitor cyclo-
sporin A for 40 min (8) The test compounds were diluted with
5.3. AP1-HEK293 cell assay
AP1-HEK293 cell activity of select compounds was measured
using AP1 Reporter-HEK293 cells according to the manufacturer’s
instructions. Reagents: (1) Thaw Medium 1 (BPS Catalog #60187):
MEM medium (Hyclone Catalog #SH30024.01) supplemented with
10% FBS (Life technologies Catalog #26140-079), 1% non-essential
amino acids (Hyclone Catalog #SH30238.01), 1 mM Na-pyruvate
(Hyclone Catalog #SH30239.01), 1% Penicillin/Streptomycin
(Hyclone Catalog #SV30010.01). (2) Growth Medium 1B (BPS Cat-
DMEM to the final incubation conc. of 10
compound was diluted by DMEM to the final incubation conc. of
M. (9) For A to B directional transport, 0.2 mL of donor working
mM, While the control
3
m
solution with test articles or positive control was added to the A
compartment and 0.7 mL of the transport media as receiver
working solution then to the B compartment. For B to A directional
transport, 0.7 mL of donor working solution with test articles or
positive control was added to the B compartment and 0.2 mL of the
transport media as receiver working solution then to the A
compartment. The cells were incubated (37 ꢀC, in 5% CO2 incubator)
alog #79531): Thaw Medium 1 (BPS Catalog #60187) and 400 mg/
mL of Geneticin (Life Technologies Catalog #11811031). (3) AP1
Reporter-HEK293 cells (BPS Catalog #60405). (4) Phorbol-12-
Myristate-13-Acetate (PMA) (LC Laboratories Catalog #P-1680).
(5) Assay medium: Opti-MEMI (Life Technologies Catalog #31985-
062), 0.5% FBS, 1% nonessential amino acids, 1 mM Na pyruvate, and
1% Pen/Strep. (6) ONE-Step™ Luciferase Assay Reagent (BPS Catalog
#60690). The assay procedure is as follows. (1) Harvest AP1
ReportereHEK293 cells from culture in Growth Medium 1B and
seed cells at a density of 35,000 cells per well into a white clear-
for 90 min (10) 80
receiver compartments into 96-well assay plates, which pre-added
with 240 L internal standard (IS) solution of acetonitrile each well,
and centrifuged (5000 rpm, 10 min). (11) 80 L of supernatant were
mL of samples were taken from both donor and
m
m
taken into 96-well assay plates pre-added with 160
water and then analyzed by LC-MS/MS.
mL ultrapure
bottom 96-well microplate in 100
m
L of Thaw Medium 1. (2) Incu-
5.5. In vivo rat pharmacokinetic study
bate the cells at 37 ꢀC in a CO2 incubator for overnight. (3) The next
day, the compounds stock were diluted in assay medium. Carefully
The pharmacokinetic study of compound 19 was performed by
Shanghai ChemPartner Co., Ltd. (Shanghai, China). The assay pro-
cedure is as follows. (1) 6 male SD rats were obtained from JH
Laboratory Animal Co., Ltd. (Shanghai, China), Fasted 24 h, divided
into two groups randomly (n ¼ 3 per group). The first group SD rats
by 1 mg/kg dose intravenous to give solution of compound 19 in
10% DMAC þ10% (30% Solutol HS 15 in water) þ 80% Saline. The
second group SD rats by 10 mg/kg dose lavage to give suspension of
compound 19 in 0.5% CMC and 0.5% Tween 80 in water. (2) Took
remove the medium from wells and add 90
pounds in assay medium to the wells. The final concentration of
DMSO in assay medium can be up to 0.5%. Add 90 L of assay
medium with same concentration of DMSO without compound to
compound control wells. Add 90 L of assay medium with DMSO to
mL of diluted com-
m
m
cell-free control wells (for determining background luminescence).
(4) Incubate the plate at 37 ꢀC in a CO2 incubator for 1 h (5) Add
10
concentration of PMA was 10 nM). Final DMSO concentration
should be 0.1%. Add 10 L of assay medium with 0.1% DMSO to the
unstimulated control wells (cells without inhibitor and PMA
mL of diluted PMA in assay medium to stimulated wells (final
blank blood before giving medicine, took about 150 mL of venous
blood at different time points after the treatment of compound 19
in Eppendorf tube with heparin, centrifuged, took plasma about
m
14

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
50
m
L, ꢂ70 ꢀC saved for testing. The point time of blood collection, iv
5.9. Hematoxylin-eosin (H&E) staining
injection: 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h oral
administration: 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h (3) Pro-
cessing and determination of the plasma samples: An aliquot of
The colon tissues were fixed in 4% formaldehyde and embedded
in paraffin for sectioning. Then the colon tissues were cut into 4-
mM
50
mL in ACN). The mixture was vortexed for 10 min at 750 rpm and
centrifuged at 6000 rpm for 10 min. An aliquot of 0.5 L superna-
m
L sample was added with 200
m
L IS (Dexamethasone, 300 ng/
thick sections and stained with hematoxylin and eosin. Histo-
pathological changes were observed using a computer-assisted
light microscope (CX23, Olympus, Japan).
m
tant was injected for LCeMS/MS analysis. (4) The calculation
method of pharmacokinetic parameters: the blood drug
concentration-time data to the DAS 2.1.1 program using the sta-
tistical method to calculate pharmacokinetic parameters.
5.10. Western blotting
The colon tissues were weighed and homogenized in ice-cold
RIPA buffer. The lysates were centrifuged and the supernatants
were collected. Bicinchoninic acid (BCA) protein assay kit was used
for quantification of protein content. The protein samples were
separated using sodium salt-polyacrylamide gel electrophoresis
(SDS-PAGE) and then transferred to PVDF membrane. After block-
ing with 5% Bovine Serum Albumin at rt for 2 h, the membrane was
incubated with primary antibodies overnight at 4 ꢀC. Then the
membrane was washed three times with TBST buffer, incubated
with the horseradish peroxidase (HRP)-conjugated anti-rabbit
antibody for 2 h at rt, then displayed using an ECL detection kit
and quantified by gel analysis software. Primary antibodies: anti-
ASK1 (Catalog #AF6477), anti-MKK3/6 (Catalog #AF6327), anti-
MKK4/7 (Catalog #AF4734), anti-p38 (Catalog #AF6456), anti-JNK
(Catalog #AF6318), anti-p-ASK1 (Catalog #AF8096), anti-p-MKK3/
6 (Catalog #AF3805), anti-p-MKK4/7 (Catalog #AF4434), anti-p-
p38 (Catalog #AF4001), anti-p-JNK (Catalog #AF3318) antibodies
were purchased from Affinity Biosciences, Inc. (Jiangsu, China), and
anti-GAPDH (Catalog #bs-0755R) antibody was obtained from
Bioss Antibodies, Inc. (Beijing, China).
5.6. Docking study
The X-ray crystal structures of ASK1 (PDB codes 3VW6 and
5VIO) were downloaded from the Protein Data Bank (www.rcsb.
org). Protein crystal structure 5VIO was prepared using Protein
Preparation Wizard and Grid generation in Schrodinger suite.
Compounds 6, 9 and 11 were prepared using Ligand Preparation
module in Schrodinger. Then, compounds 6, 9 and 11 were docked
into ASK1 (5VIO) by Glide Docking in Schrodinger. The images files
were generated using Pymol software.
5.7. DSS-induced mouse UC model and compounds treatment
Male ICR mice (18e22 g, 6e8 weeks old) were purchased from
Comparative Medical Center of Yangzhou University (Yangzhou,
China). Before starting the experiments, the mice were housed in a
constant room, with a maintained temperature (22
2
ꢀC), hu-
midity (55 5%), and lighting (12 h light/dark cycle) for 5 days to
acclimatize the lab environment. All animals were allowed free
access to water and food. Animal experiments were organized and
conducted in accordance with the guidelines of the National In-
stitutes of Health Guide and Care of Laboratory Animals. The
methods were approved by the Animal Care and Use Committee of
China Pharmaceutical University. ICR mice were randomly divided
into four groups (n ¼ 6 per group): control group, model group,
compound 19 group and compound 6 group. The model group,
compound 19 group and compound 6 group mice were given 3%
DSS (w/v) orally in drinking water to induce UC and the control
group mice were received distilled water for 7 days. The drinking
water were changed every two days. Compounds 19 and 6 were
administered (po, qd) at a dose of 25 mg/kg during the DSS treat-
ment for 7 days, meanwhile, control group and model group mice
were treated with vehicle. The mice were observed and the changes
in body weight, stool consistency and rectal bleeding were recor-
ded once a day. All experimental mice were sacrificed at the end of
experiment, the colons were harvested and colon length was
measured. The colon tissues were collected immediately, fixed in
4% formaldehyde and preserved for H&E staining analysis; the
remaining colon tissues were stored at ꢂ80 ꢀC for further
examination.
5.11. Enzyme-linked immunosorbent assay (ELISA)
The homogenates of mouse colon tissues were centrifuged and
the supernatants were collected. The expression levels of inflam-
matory cytokines (IL-1
ELISA kits following the manufacturer’s instructions. Mouse IL-1
ELISA kit (Catalog #EMC001b.48), IL-6 ELISA kit (Catalog #K4144-
100) and TNF- ELISA kit (Catalog #ADI-900-047) were provided by
b, IL-6 and TNF-a) were measured using
b
a
Neobioscience Technology Co., Ltd. (Shanghai, China).
Declaration of competing interest
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.
Acknowledgment
This work was financially supported by the National Natural
Science Foundation of China (Program Nos. 81973182 and
81673301) and “Double First-Class” University Project from China
Pharmaceutical University (Program No. CPU2018GF02). We thank
Prof. Cheng-Ming Chiang of UT Southwestern Medical Center for
revising this manuscript.
5.8. Disease activity index (DAI) scores and body weight changes
Abbreviations
DAI scores and body weight changes were calculated according
to the following standards. Body weight loss: 0 ¼ none, 1 ¼ 1e5%,
2 ¼ 5e10%, 3 ¼ 1015%, 4 ¼ over 15%; stool consistency: 0 ¼ normal,
1 ¼ loose stools, 2 ¼ diarrhea; blood in stool: 0 ¼ normal, 1 ¼ fecal
occult blood, 2 ¼ gross bleeding. Mean of the above three factors
was calculated as DAI scores. Body weight changes (%) were
calculated on the basis of body weight loss [44,45].
ASK1
MAPK
DSS
UC
DAI
IBD
apoptosis signal-regulating kinase 1
mitogen-activated protein kinase
dextran sulfate sodium
ulcerative colitis
disease activity index
inflammatory bowel disease
colorectal cancer
CRC
15

S. Hou, X. Yang, Y. Tong et al.
European Journal of Medicinal Chemistry 211 (2021) 113114
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ALS
CIA
NASH
NaF
TNBS
DIPEA
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collagen-induced arthritis
nonalcoholic steatohepatitis
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2,4,6-trinitrobenzene sulfonic acid
N,N-Diisopropylethylamine
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Supplementary data to this article can be found online at
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