GS-4997

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,
Yadong Chen a, b, Tao Lu a, b, ***, 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

Article history:
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
a b s t r a c t

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.

⦁ Introduction

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

* Corresponding author.
** Corresponding author.
*** Corresponding author. School of Sciences, China Pharmaceutical University, 639 Longmian Avenue, Nanjing, 211198, PR China.
E-mail addresses: [email protected] (T. Lu), [email protected] (Q. Hu), [email protected] (D. Du).
1 Shaohua Hou and Xiping Yang contributed equally to this work. The manuscript was written through contributions of all authors.
signiﬁcant global impact on public health. Although the patho- genesis of UC has not been completely revealed, immune dysre- 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

https://doi.org/10.1016/j.ejmech.2020.113114

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- 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
treated with TNF-a [34]. Hence, we focused our effort to investigate the effect of ASK1 inhibitors on UC.
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.

⦁ 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.
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.

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.

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.

⦁ Results and discussion

⦁ Structure-based design strategy

¼
In our previous work, hit compound 7 (Fig. 3) (ASK1 IC50 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
designed with the aim of forming an additional interaction with Val757 in the hinge region (Table 1).

⦁ Inhibitory activity against ASK1

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.)

Fig. 3. Structure-based design and optimization of 1H-indole-2-carboxamide derivatives as ASK1 inhibitors.

Table 1
Inhibitory activity of compounds 7e12 against ASK1.

Cpds Structure ASK1 IC50(nM)a

7 16700 ± 2130

8 1106 ± 24

9 151.6 ± 11

10 506 ± 12

11 41.4 ± 2.17

12 125.1 ± 12.10
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.
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

a 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
with pyridine (24). Compounds 22e24 all showed decreased mo- lecular activity, likely attributed to larger aromatic heterocyclic substituents not well tolerated in this area.
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.)

Table 2
Inhibitory activity of compounds 13e18 against ASK1.

Cpds Posa R1 R2 ASKIC50 (nM)b Cpds Posa R1 R2 ASKIC50 (nM)b
13 1- 743 ± 48 16 4- 19.6 ± 3.05

14
3-
88.2 ± 6.18
17
5-
31.6 ± 1.61

15
3-
142.8 ± 6.55
18
e
H
65.5 ± 3.61

a Substituted position of R1 on the indole ring.
b IC50 values are the mean ± SD from two independent experiments.

Table 3
Table 4
AP1-HEK293 cell activity of select compounds.

Inhibitory activity of compounds 19e29 against ASK1.

6 76.67 ± 1.13 19 95.59 ± 0.24
9 25.33 ± 3.10 20 95.93 ± 1.10
11 56.46 ± 0.29 21 94.39 ± 0.25
12 32.54 ± 1.91 22 94.04 ± 1.36
14 92.48 ± 0.88 23 64.63 ± 1.45
16 72.88 ± 0.14 24 95.66 ± 1.29

Cpds Inhibition rate (%) at 10 mMb Cpds Inhibition rate (%) at 10 mMb

Cpds Posa R ASKIC50 (nM)b Cpds Posa R ASKIC50 (nM)b

19 6- 32.8 ± 1.88 25 7- 240.1 ± 8.05

20 6- 54.3 ± 4.87 26 7- 89.6 ± 4.16

21 6- 37.5 ± 2.79 27 7- 38.9 ± 2.26

17 89.55 ± 2.61 26 90.84 ± 0.02
18 61.26 ± 0.44 27 59.86 ± 1.88

b Values are the mean ± SD from three independent experiments.

Table 5
Permeability assay of compounds 6 and 19 in MDRI-MDCKII cellsa.

22 6- 91.1 ± 1.77 28 7- 23.1 ± 2.19
Cpds P-gp inhibitor Papp
( × 10—6 cm/s) (N ¼ 2)
Efﬂux ratio P-gp substratec

23 6- 73.7 ± 4.62 29 7- 153.3 ± 7.65 A/B B/A
6 without 11.22 26.47 2.4 Yes

24 6- 68.5 ± 9.55

a Substituted position of R on the indole ring.
b IC50 values are the mean ± SD from two independent experiments.
with CsAb 34.49 25.02 0.7
19 without 21.12 15.63 0.7 No
with CsA 36.62 26.05 0.7

a Cells that overexpress P-gp.
b A known P-gp inhibitor ciclosporin A.
c Efﬂux ratio (ER) ≤ 2 and the ER decreases to < 50% of the ER in the absence of 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).

⦁ AP1-HEK293 cell activity

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

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
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.

¼
To better understand why compound 6 showed a stronger ASK1 kinase inhibition (IC50 ¼ 5.9 nM, Fig. 1) compared to compound 19 (IC50 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
Table 7
Pharmacokinetic (PK) proﬁle of compound 6 in ratsa.

Cpd CL (L/h/kg), iv Vss(L/kg), iv T1/2(h), iv F(%)

6 0.11 0.55 5.07 75

19 (Papp(A/B) ¼
21.12 ×
10—6 cm/s) had a better cell membrane
—6
a Referenced to https://www.cortellis.com/drugdiscovery, iv dose 1 mg/kg and po dose 5 mg/kg.

permeability than compound 6 (Papp(A/B) ¼ 11.22 × 10 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.

⦁ Pharmacokinetic properties

¼
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 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
¼ ¼
2 2.31 h). These data fully justiﬁed to assess the efﬁcacy of 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 0.55 L/kg) and long half-life (T1/2 5.07 h), and upon oral administration at an oral dose of 5 mg/kg, compound 6 had a good oral bioavailability (75%).

⦁ 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.
—
þ
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 9.6%, 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 11.2%. 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

Table 6
Pharmacokinetic (PK) proﬁle of compound 19 in ratsa.

Parametersb iv (1 mg/kg) Parametersb po (10 mg/kg)
CL(L/h/kg) 1.38 ± 0.106 Tmax(h) 2.00 ± 0.17
Vss(L/kg) 2.82 ± 0.225 Cmax(ng/mL) 676 ± 101
AUClast(h*ng/mL) 713 ± 51.5 AUClast(h*ng/mL) 4517 ± 1042
AUCINF(h*ng/mL) 727 ± 54.7 AUCINF(h*ng/mL) 4521 ± 1043
T1/2(h) 1.45 ± 0.0885 T1/2(h) 2.31 ± 0.112
MRTINF(h) 2.04 ± 0.109 F(%) 62.2 ± 14.4
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.
b PK parameters are the mean ± SD from three independent experimental rats.

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.

⦁ 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.

⦁ Reduction of inﬂammatory cytokine levels by compound 19 in DSS-induced mouse colon tissues

Increasing literature highlights an abnormal upregulation of pro-inﬂammatory cytokines, such as IL-1b, IL-6 and TNF-a, is 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. As shown in Fig. 6C, the signiﬁcantly enhanced levels of IL-1b, IL-6 and TNF-a in DSS-treated mice were decreased by compound 19.

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, including IL-1b, IL-6 and TNF-a.

⦁ Conclusion

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
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 pro-inﬂammatory cytokines (IL-1b, TNF-a, IL-6) in the colon tis- 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.

⦁ Experimental

⦁ Chemistry

All reagents and solvents are commercially available and were purchased as reagent grade and used without further puriﬁcation.

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-1b, 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, triplet, quartet, doublet of doublets and multiplet, respectively. 13C 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 ≤ 95% pure.
⦁ 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
solid (4.50 g, 29.3 mmol, yield: 89%). 1H NMR (400 MHz, DMSO‑d6)
¼ ¼
d 9.14 (s, 1H), 7.51 (t, J 8.4 Hz, 1H), 7.11 (d, J 7.2 Hz, 1H), 6.60 (d,
¼ þ
J 7.2 Hz, 1H), 6.07 (s, 2H), 4.47 (s, 2H). MS (ESI) [M 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,
þ
¼ ¼
¼
19.1 mmol, yield: 66%) as a white solid. 1H NMR (400 MHz, DMSO‑d6) d 10.69 (s, 1H), 8.86 (s, 1H), 8.06 (s, 1H), 7.68 (t, J 7.7 Hz, 1H), 7.48 (d, J 6.8 Hz, 1H), 6.91 (d, J 8.0 Hz, 1H), 3.14 (s, 3H), 3.02 (s, 3H), 2.88 (s, 6H). MS (ESI) [M 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:

¼ ¼ ¼
65%) as a white solid. 1H NMR (300 MHz, DMSO‑d6) d 8.78 (s, 1H), 7.52 (t, J 7.8 Hz, 1H), 7.16 (d, J 7.4 Hz, 1H), 6.52 (d, J 8.3 Hz, 1H),
¼ þ
6.17 (s, 2H), 5.62e5.45 (m, 1H), 1.43 (d, J 6.7 Hz, 6H). MS (ESI) [M H]þ: 204.1.
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-
isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-3- methyl- benzofuran-2-carboxamide (8) (100 mg, 0.28 mmol, yield: 49%) as a white solid. mp 175e177 ◦C. 1H NMR (300 MHz, DMSO‑d6) d 10.55 (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) 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,
þ
23.75, 9.46. HRMS-EI m/z [M H]þ calcd for C20H20N5Oþ2 : 362.1612,
¼
found: 362.1615. HPLC analysis: retention time 6.835 min; 98.45%.
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)benzo- 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)
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) 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.
¼
þ
HRMS-EI m/z [M H]þ calcd for C19H18N5O2þ: 348.1455, found: 348.1459. HPLC analysis: retention time 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, 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). 13C 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,
⦁ þ
⦁ , 23.79. HRMS-EI m/z [M H]þ calcd for C18H17N6OSþ:
365.1179, found: 365.1179. HPLC analysis: retention
¼
time 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
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, 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,
⦁ þ
⦁ , 23.70. HRMS-EI m/z [M H]þ calcd for C19H19N6Oþ:
347.1615, found: 347.1615. HPLC analysis: retention
¼
time 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, DMSO‑d6) d 160.22, 151.66, 150.47, 149.21, 146.72, 143.65, 140.00,
131.66, 131.05, 119.80, 119.60, 117.23, 115.64, 105.27, 48.36, 23.70.
¼
þ
HRMS-EI m/z [M H]þ calcd for C18H18N7Oþ: 348.1567, found: 348.1570. HPLC analysis: retention time 3.817 min, 95.21%.
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1- methyl-1H-indole-2-carboxamide (13) Compound 13 was pre- pared from 1-methyl-1H-indole-2-carboxylic acid (52 mg,
¼ ¼ ¼
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 229e231 ◦C. 1H NMR (400 MHz, DMSO‑d6) d 10.80 (s, 1H), 8.88 (s, 1H), 8.18 (d, J 8.2 Hz, 1H), 8.03 (t, J 7.9 Hz, 1H), 7.88 (d, J 7.5 Hz,
¼ ¼
1H), 7.73 (d, J 7.9 Hz, 1H), 7.61 (d, J 8.4 Hz, 1H), 7.42 (s, 1H), 7.35
¼
¼ ¼
(t, J 7.6 Hz, 1H), 7.16 (t, J 7.4 Hz, 1H), 5.84e5.68 (m, 1H), 4.04 (s, 3H), 1.45 (d, J 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.
¼
þ
HRMS-EI m/z [M H]þ calcd for C20H21N6Oþ: 361.1771, found: 361.1776. HPLC analysis: retention time 5.442 min, 98.65%.
N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-3- methyl-1H-indole-2-carboxamide (14) Compound 14 was pre- pared from 3-methyl-1H-indole-2-carboxylic acid (87 mg,
¼ ¼
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 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 8.2 Hz, 1H), 8.04 (t, J 8.0 Hz, 1H), 7.86
¼ ¼ ¼
(d, J 7.5 Hz, 1H), 7.69 (d, J 8.0 Hz, 1H), 7.47 (d, J 8.2 Hz, 1H), 7.28
¼
¼ ¼
(t, J 7.4 Hz, 1H), 7.10 (t, J 7.4 Hz, 1H), 5.71e5.57 (m, 1H), 2.61 (s, 3H), 1.47 (d, J 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.
¼
þ
HRMS-EI m/z [M H]þ calcd for C20H21N6Oþ: 361.1771, found: 361.1770. HPLC analysis: retention time 5.460 min, 98.34%.
⦁ 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 (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,

J ¼ 9.3 Hz, 1H), 5.78e5.60 (m, 1H), 1.47 (d, J ¼ 6.6 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6) d 160.39, 151.74, 150.54, 146.69, 143.58, 140.18, 139.90, 131.99, 124.25, 123.99, 119.67, 115.65, 109.97, 106.24, 98.53,
þ
48.30, 23.65. HRMS-EI m/z [M H]þ calcd for C20H21N6Oþ:
365.1521, found: 365.1520. HPLC analysis: retention
¼
time 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 (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
¼
C20H21N6O2þ: 377.1721, found: 377.1720. HPLC analysis: retention time 4.210 min, 99.04%.
⦁ ¼ ¼
⦁ ¼
⦁ 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, DMSO‑d6) d 12.06 (s, 1H), 10.85 (s, 1H), 8.90 (s, 1H), 8.26 (dd, J 6.3,
0.3 Hz, 1H), 8.06 (t, J 8.7 Hz, 1H), 7.84 (dd, J 7.6, 0.6 Hz, 1H), 7.68
¼ ¼ ¼
(d, J 6.0 Hz, 1H), 7.55 (d, J 2.1 Hz, 1H), 7.37 (dd, J 5.7, 0.6 Hz,
¼ ¼
1H), 7.12 (t, J 7.8 Hz, 1H), 5.63e5.49 (m, 1H), 1.48 (d, J 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
¼
C19H18ClN6Oþ: 381.1225, found: 381.1225. HPLC analysis: retention time 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
(300 MHz, DMSO‑d6) d 11.95 (s, 1H), 10.74 (s, 1H), 8.89 (s, 1H), 8.22
¼ ¼ ¼
(dd, J 8.1, 0.6 Hz, 1H), 8.04 (t, J 8.1 Hz, 1H), 7.85 (dd, J 7.5,
¼ ¼
0.6 Hz, 1H), 7.70 (d, J 5.1 Hz, 1H), 7.49 (dd, J 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.
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-
(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-

3-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol -3-yl)pyridin-2-yl)-1H- indole-2-carboxamide (15) (54 mg, 0.13 mmol, yield: 57%) as a white solid. mp 223e225 ◦C. 1H NMR (300 MHz, DMSO‑d6) d 12.10 (s, 1H), 9.56 (s, 1H), 8.88 (s, 1H), 8.31 (d, J 8.0 Hz, 1H), 8.18 (s, 1H),
¼ ¼ ¼
¼
8.04 (t, J 8.0 Hz, 1H), 7.94 (t, J 1.6 Hz, 1H), 7.90 (d, J 7.5 Hz, 1H),
¼ ¼ ¼
7.58 (d, J 8.1 Hz, 1H), 7.53 (d, J 8.3 Hz, 1H), 7.32 (t, J 7.3 Hz, 1H),
¼
7.14 (t, J 7.5 Hz, 1H), 6.89e6.83 (m, 1H), 5.43e5.32 (m, 1H), 1.43 (d,
¼
J 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,
þ
48.63, 23.56. HRMS-EI m/z [M H]þcalcd for C23H21N6Oþ2 : 413.1721,
¼
found: 413.1722. HPLC analysis: retention time 3.917 min, 96.84%.
¼ ¼
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, yield: 75%. 1H NMR (300 MHz, DMSO‑d6) d: 11.95 (s, 1H), 10.74 (s,
1H), 8.89 (s, 1H), 8.22 (dd, J 8.1, 0.6 Hz, 1H), 8.04 (t, J 8.1 Hz, 1H),
¼ ¼
7.85 (dd, J 7.5, 0.6 Hz, 1H), 7.56 (s, 1H), 7.49 (dd, J 8.1, 0.3 Hz, 1H),
7.32e7.21 (m, 1H), 7.08e6.88 (m, 1H), 5.73e5.57 (m, 1H), 1.47 (d,
¼ þ
J 9.6 Hz, 6H). MS (ESI) [M H]þ: 425.1.
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
(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
¼
þ
m/z [M H]þ calcd for C23H23N8Oþ: 427.1989, found: 427.1990. HPLC analysis: retention time 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- (4,4,5,5-tetramethyl-1,3,2- dioxaborolane-2-yl)-1H-pyrazole (115 mg, 0.59 mmol) according to a similar procedure described for 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,
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, 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,
þ
106.08, 48.45, 23.59. HRMS-EI m/z [M H]þ calcd for C22H21N8Oþ:
413.1833, found: 413.1836. HPLC analysis: retention
¼
time 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,

¼
0.69 mmol), key intermediate 8-d (140 mg, 0.69 mmol) and 4- (4,4,5,5-tetramethyl-1,3,2- dioxaborolane-2-yl)-1H-pyrazole (134 mg, 0.69 mmol) according to a similar procedure described for the synthesis of compound 16. White solid, 99 mg, yield: 35%. mp 159e161 ◦C. 1H NMR (300 MHz, DMSO‑d6) d: 12.92 (s, 1H), 11.88 (s,
1H), 10.66 (s, 1H), 8.89 (s, 1H), 8.23 (d, J 8.2 Hz, 1H), 8.04 (t,
¼ ¼ ¼
J 8.0 Hz, 2H), 7.85 (d, J 7.6 Hz, 1H), 7.68 (d, J 8.3 Hz, 1H), 7.62 (s,
¼
1H), 7.54 (s, 1H), 7.38 (d, J 8.3 Hz, 1H), 5.72e5.53 (m, 1H), 1.47 (d,
¼
J 6.6 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6) d 160.53, 151.85,
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
þ
[M H]þ calcd for C22H21N8Oþ: 413.1833, found: 413.1834. HPLC
¼
analysis: retention time 3.465 min, 97.54%.
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 11.97 (s, 1H), 10.70 (s, 1H), 8.89 (s, 1H), 8.39 (s, 1H), 8.23
(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,
þ
108.62, 106.66, 86.87, 63.51, 48.39, 23.69, 21.63, 15.20. HRMS-EI m/z [M H]þ calcd for C26H29N8O2þ: 485.2408, found: 485.2408. HPLC
¼
analysis: retention time 4.616 min, 96.72%.
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)- 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
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,
¼
1H), 8.90 (s, 1H), 8.27 (s, 1H), 8.23 (d, J 8.1 Hz, 1H), 8.04 (t,
¼ ¼ ¼
J 8.0 Hz, 1H), 7.89 (s, 1H), 7.85 (d, J 7.6 Hz, 1H), 7.69 (d, J 8.4 Hz,
¼
1H), 7.60 (s, 1H), 7.55e7.52 (m, 1H), 7.36 (dd, J 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). 13C NMR (100 MHz, DMSO‑d6) d 160.62, 151.87, 150.59, 146.69, 143.60,
139.89, 138.36, 136.23, 131.22, 129.57, 126.01, 125.37, 122.92, 119.40,
þ
115.64, 108.38, 106.41, 66.42, 57.80, 48.36, 33.43, 23.66. HRMS-EI m/ z [M H]þ calcd for C27H29N8O2þ: 497.2408, found: 497.2408. HPLC
¼
analysis: retention time 3.932 min, 96.98%.
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,
¼ ¼ ¼
¼ ¼
0.64 mmol) according to a similar procedure described for the synthesis of compound 16. White solid, 78 mg, yield: 29%. mp 280e283 ◦C. 1H NMR (300 MHz, DMSO‑d6) d 12.07 (s, 1H), 10.74 (s, 1H), 8.91 (d, J 11.9 Hz, 2H), 8.59 (d, J 4.3 Hz, 1H), 8.24 (d, J 8.1 Hz, 1H), 8.13 (d, J 8.0 Hz, 1H), 8.05 (t, J 7.9 Hz, 1H), 7.86
¼
(dd, J 8.0, 2.3 Hz, 2H), 7.76 (s, 1H), 7.64e7.60 (m, 1H), 7.54 (dd,
¼
J 7.9, 4.9 Hz, 1H), 7.49e7.43 (m, 1H), 5.78e5.65 (m, 1H), 1.48 (d,
¼
J 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,
133.78, 132.30, 127.25, 124.40, 123.26, 120.11, 119.65, 115.70, 111.09,
þ
106.12, 48.34, 23.71. HRMS-EI m/z [M H]þ calcd for C24H22N7Oþ:
424.1880, found: 424.1882. HPLC analysis: retention
¼
time 4.597 min, 98.75%.
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- (4,4,5,5-tetramethyl-1,3,2-dioxa borolan-2-yl)-1H-pyrazole (143 mg, 0.74 mmol) according to a similar procedure described for 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 1¼3 5.7 Hz, 1H), 6.85 (s,
1H), 5.40 (s, 1H), 1.45 (d, J ¼ 5.4 Hz, 6H). C NMR (100 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,
þ
108.77, 48.35, 23.65. HRMS-EI m/z [M H]þ calcd for C22H21N8Oþ:
413.1833, found: 413.1830. HPLC analysis: retention
¼
time 4.040 min, 95.24%.
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,
¼
0.66 mmol) according to a similar procedure described for the synthesis of compound 16. White solid, 100 mg, yield: 36%, mp 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 8.6 Hz, 1H), 8.06 (d,
¼ ¼
J 7.7 Hz, 1H), 7.82 (d, J 7.4 Hz, 2H), 7.72 (s, 2H), 7.59 (s, 1H), 7.39
¼ ¼
(d, J 6.9 Hz, 1H), 7.28 (d, J 7.6 Hz, 1H), 5.57 (s, 1H), 1.46 (d,
¼
J 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,
¼
þ
23.65. HRMS-EI m/z [M H]þ calcd for C24H22N7Oþ: 424.1880, found: 424.1880. HPLC analysis: retention time 5.000 min, 95.04%.
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,
¼ ¼ ¼
¼ ¼
0.69 mmol) according to a similar procedure described for the synthesis of compound 16. White solid, 91 mg, yield: 31%. mp 178e181 ◦C. 1H NMR (300 MHz, DMSO‑d6) d 11.13 (s, 1H), 10.90 (s, 1H), 8.89 (s, 1H), 8.26 (d, J 7.8 Hz, 1H), 8.05 (t, J 8.0 Hz, 1H), 7.98 (dd, J 2.8, 1.3 Hz, 1H), 7.82 (d, J 7.0 Hz, 1H), 7.76 (dd, J 5.0,
¼
2.9 Hz, 1H), 7.70 (d, J 7.8 Hz, 1H), 7.59e7.52 (m, 2H), 7.41 (dd,
¼
J 7.2, 0.9 Hz, 1H), 7.24e7.15 (m, 1H), 5.58e5.52 (m, 1H), 1.46 (d,
¼
J 6.7 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6) d 160.11, 151.89,
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,
þ
108.53, 48.33, 23.66. HRMS-EI m/z [M H]þ calcd for C23H21N6OSþ:
429.1492, found: 429.1492. HPLC analysis: retention
¼
time 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 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:

220.1.
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- yl)-4H-1,2,4-triazol-3-yl)pyridin-2-amine (18-b) (160 mg,
0.48 mmol, yield: 85%) as a white solid. 1H NMR (300 MHz, CDCl3)
¼ ¼
d 8.43 (s, 1H), 7.59 (d, J 3.3 Hz, 1H), 7.28 (d, J 5.3 Hz, 1H), 6.58
¼
(dd, J 7.1, 1.9 Hz, 1H), 5.68e5.59 (m, 2H), 4.59 (s, 2H), 3.96 (s, 1H),
þ
¼
1.55 (d, J 7.0 Hz, 3H), 0.96e0.90 (m, 6H), 0.87 (s, 9H). MS (ESI) [M H]þ: 334.2.
¼
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 8.3, 0.6 Hz, 1H), 8.03 (t,
¼ ¼ ¼
J 8.0 Hz, 1H), 7.84 (dd, J 7.6, 0.3 Hz, 1H), 7.71 (d, J 7.9 Hz, 1H),
¼ ¼
7.55 (d, J 1.5 Hz, 1H), 7.49 (d, J 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,
¼ þ
J 6.9 Hz, 3H), 0.90e0.86 (m, 6H), 0.83 (s, 9H). MS (ESI) [M 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, DMSO‑d6) d 11.89 (s, 1H), 10.65 (s, 1H), 8.80 (s, 1H), 8.21 (d,
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 (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
¼
C19H19N6O2þ: 363.1564, found: 363.1567. HPLC analysis: retention time 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 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.
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
¼
(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 (300 MHz, DMSO‑d6) d: 11.38 (s, 1H), 8.29 (dd, J 8.0, 2.3 Hz, 1H),
¼ ¼
8.23 (dd, J 7.7 Hz, 2.4 Hz, 1H), 7.47(t, J 2.0 Hz, 1H), 7.43e7.35 (m,
¼ ¼ þ
1H), 4.39 (q, J 7.1 Hz, 2H), 1.37 (t, J 11.6 Hz, 3H). MS (ESI) [M H]þ: 235.1.
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,
¼ ¼
57%) as a white solid. 1H NMR (300 MHz, DMSO‑d6) d: 11.42 (s, 1H), 7.02 (d, J 2.1 Hz, 1H), 6.85e6.76 (m, 2H), 6.42 (dd, J 7.0, 1.2 Hz,
þ
¼ ¼
1H), 5.41 (s, 2H), 4.34 (q, J 7.1 Hz, 2H), 1.34 (t, J 7.1 Hz, 3H). MS (ESI) [M 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 solid. 1H NMR (300 MHz, DMSO‑d6) d: 11.65 (s, 1H), 9.80 (s, 1H), 7.93
¼ ¼
(s, 1H), 7.39 (d, J 7.8 Hz, 1H), 7.25e6.93 (m, 2H), 2.38 (d, J 6.9 Hz,
¼ þ
3H), 2.19 (d, J 13.7 Hz, 3H), 1.49e1.25 (m, 3H). MS (ESI) [M 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,
yield: 75%) as a white solid. 1H NMR (300 MHz, DMSO‑d6) d: 12.07
¼
(s, 1H), 11.65 (s, 1H), 9.80 (s, 1H), 7.93 (s, 1H), 7.39 (d, J 7.8 Hz, 1H),
¼ þ
7.25e6.93 (m, 2H), 2.38 (d, J 6.9 Hz, 3H). MS (ESI) [M 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)
d 11.82 (s, 1H), 10.80 (s, 1H), 9.94 (s, 1H), 8.89 (s, 1H), 8.22 (d,

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 ¼13 7.8 Hz,
1H), 5.80e5.63 (m, 1H), 2.18 (s, 3H), 1.47 (d, J ¼ 6.5 Hz, 6H). C NMR
(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,
114.80, 106.59, 48.35, 24.45, 23.69. HRMS-EI m/z [M þ H]þ calcd for
C21H22N7O2þ: 404.1829, found: 404.1835. HPLC analysis: retention time ¼ 3.703 min, 99.38%.
⦁ ASK1 kinase 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 DTT), and 2.0 mL of diluted compounds were dispensed to the in- dividual wells of a white optiplate-384. (2) 4 mL of MAP3K5 (5 ng/ mL) in assay buffer and 4 mL of STK-Substrate 3-biotin (5 mM)/ATP (250 mM) in assay buffer were added to the assay plates containing test compounds. (3) Assay plates were incubated for 2 h at rt and 5 mL of STK S3 Antibody-Eu (500 nM) in assay buffer was added. (4) 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).

⦁ 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- 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- bottom 96-well microplate in 100 mL of Thaw Medium 1. (2) Incu- bate the cells at 37 ◦C ina CO2 incubator for overnight. (3) The next
day, the compounds stock were diluted in assay medium. Carefully remove the medium from wells and add 90 mL of diluted com- pounds in assay medium to the wells. The ﬁnal concentration of DMSO in assay medium can be up to 0.5%. Add 90 mL of assay medium with same concentration of DMSO without compound to compound control wells. Add 90 mL of assay medium with DMSO to 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 mL of diluted PMA in assay medium to stimulated wells (ﬁnal concentration of PMA was 10 nM). Final DMSO concentration should be 0.1%. Add 10 mL of assay medium with 0.1% DMSO to the unstimulated control wells (cells without inhibitor and PMA
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 ina CO2 incubator for 6 h (7) Perform luciferase assay using the ONE-Step™ Luciferase Assay System following the pro- tocol provided: Add 100 mL of ONE-Step™ Luciferase reagent per 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.

⦁ Cell membrane permeability assay

×
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 (1 mm 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 DMEM to the ﬁnal incubation conc. of 10 mM, While the control compound was diluted by DMEM to the ﬁnal incubation conc. of 3 mM. (9) For A to B directional transport, 0.2 mL of donor working 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) for 90 min (10) 80 mL of samples were taken from both donor and receiver compartments into 96-well assay plates, which pre-added with 240 mL internal standard (IS) solution of acetonitrile each well, and centrifuged (5000 rpm, 10 min). (11) 80 mL of supernatant were taken into 96-well assay plates pre-added with 160 mL ultrapure water and then analyzed by LC-MS/MS.

⦁ In vivo rat pharmacokinetic study

¼
þ þ
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 3 per group). The ﬁrst group SD rats by 1 mg/kg dose intravenous to give solution of compound 19 in 10% DMAC 10% (30% Solutol HS 15 in water) 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 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

—
50 mL, 70 ◦C saved for testing. The point time of blood collection, iv
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 50 mL sample was added with 200 mL IS (Dexamethasone, 300 ng/ 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 mL superna- 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.

⦁ 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.

⦁ 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
¼
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 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 80 ◦C for further
examination.

⦁ Disease activity index (DAI) scores and body weight changes

¼ ¼
DAI scores and body weight changes were calculated according to the following standards. Body weight loss: 0 none, 1 1e5%,
¼ ¼ ¼ ¼
2 5e10%, 3 1015%, 4 over 15%; stool consistency: 0 normal,
¼
¼ ¼ ¼ ¼
1 loose stools, 2 diarrhea; blood in stool: 0 normal, 1 fecal occult blood, 2 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].
⦁ Hematoxylin-eosin (H&E) staining

The colon tissues were ﬁxed in 4% formaldehyde and embedded in parafﬁn for sectioning. Then the colon tissues were cut into 4-mM thick sections and stained with hematoxylin and eosin. Histo- pathological changes were observed using a computer-assisted light microscope (CX23, Olympus, Japan).

⦁ 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).

⦁ 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-1b, IL-6 and TNF-a) were measured using ELISA kits following the manufacturer’s instructions. Mouse IL-1b ELISA kit (Catalog #EMC001b.48), IL-6 ELISA kit (Catalog #K4144- 100) and TNF-a ELISA kit (Catalog #ADI-900-047) were provided by 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.

Abbreviations

ASK1 apoptosis signal-regulating kinase 1 MAPK mitogen-activated protein kinase DSS dextran sulfate sodium
UC ulcerative colitis
DAI disease activity index
IBD inﬂammatory bowel disease CRC colorectal cancer

ALS amyotrophic lateral sclerosis CIA collagen-induced arthritis NASH nonalcoholic steatohepatitis NaF sodium ﬂuoride
TNBS 2,4,6-trinitrobenzene sulfonic acid DIPEA N,N-Diisopropylethylamine

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2020.113114.

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FAK signal

FAK is activated by focal adhesion complex

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