Synthesis and biological evaluation of quinoxaline derivatives as ASK1 inhibitors

Abstract

Inhibiting apoptosis signal regulated kinase 1 (ASK1) is an attractive strategy for treating diseases such as non-alcoholic steatohepatitis and multiple sclerosis. Here, we report the discovery of a dibromo substituted quinoxaline fragment containing 26e as an effective small-molecule inhibitor of ASK1, with an IC₅₀ value of 30.17 nM. In addition, the cell survival rate of 26e at different concentrations was greater than 80%, especially at 0.4 μM. Its cell survival rate was significantly higher than GS-4997, indicating its good safety in normal human liver LO2 cells. The Oil Red O staining experiment showed that 26e decreased the lipid droplets in a dose-dependent manner. Further biochemical analyses revealed that 26e could reduce the content of T-CHO, LDL, and TG in FFA-induced LO2 cells, and had the potential to treat non-alcoholic fatty disease. These findings provide a good choice for the future development of ASK1 inhibitors.

Graphical Abstract

Introduction

Apoptosis signal regulated kinase 1 (ASK1, also known as MAP3K5) is a widely expressed member of the serine/threonine kinase, located upstream of Jun N-terminal kinase (JNK) and p38 MAPK, and mediates their signalling pathways^1–3. It is sensitive to oxidative stress, tumour necrosis factor-α, Fas ligand, and lipopolysaccharides^2,4–7. A large number of experiments have shown that ASK1 is a key regulatory factor that activates the p38 and JNK signalling pathways, induces apoptosis, inflammation and fibrosis signalling, increases abnormal cell proliferation, and leads to metabolic dysfunction^8–18. Furthermore, inhibiting ASK1 has been shown to improve inflammation and fibrosis in non-alcoholic steatohepatitis (NASH) animal models^19,20. Importantly, studies suggest that ASK1 knockout mice do not exhibit any apparent phenotype under basal conditions²¹, providing evidence that inhibiting ASK1 could be a safe therapeutic strategy for treating various diseases. Therefore, ASK1 is an important potential target for treating these diseases, and the development of novel ASK1 inhibitors is of great research significance for alleviating or even curing these diseases¹.

MSC2032964A (1, Figure 1)²² is an orally administered ASK1 inhibitor developed by a Japanese scientific and technological institution, with an IC₅₀ value of 93 nM. It has been proven to have therapeutic effects in reducing spinal cord demyelination and inhibiting activation of astrocytes and microglia, significantly preserving the average visual response in experimental autoimmune encephalomyelitis (EAE) mice. Furthermore, MSC2032964A exhibits good oral bioavailability, a moderate clearance rate, and a long half-life. GS-4997 (2, Figure 1) is the first ASK1 inhibitor to enter the clinical phase. It was developed by Gilead in 2013²³ and is currently in the third phase of clinical use for the treatment of non-alcoholic fatty liver (NAFL) disease. In addition, GS-444217 (3, Figure 1), another product of Gilead’s research efforts, exhibits notable efficacy in diminishing inflammation, fibrosis, and cell death in renal tubules, thereby safeguarding renal function^24,25. In a rat model of kidney injury, the pre-administration of GS-444217 significantly mitigated tubular cell death, inflammation, and fibrosis by suppressing the ROS–ASK1–P38/JNK response, underscoring its protective role in renal function^24,25.

Figure 1.

Selected examples of compounds 1 (GS-4997) and 2 as ASK1 inhibitors.

Recently, we have explored and synthesised a series of new skeleton compounds, including hydrogenated indole, indole, benzimidazole, tetrahydroquinoxaline, benzoxazine, tetrahydroquinoline, phenol, nitrobenzene, and pyridine as ASK1 inhibitors²⁶. Among them, compound 4 containing triazolylpyridine fragments showed strong inhibitory activity, with IC₅₀ values of 0.15 μM²⁶. In addition, the cell survival rates of compound 4 was greater than 90% at different concentrations, especially at 0.05, 0.1, and 0.8 μM; their cell survival rate is higher than GS-4997, indicating that they exhibit good safety in normal human liver LO2 cells. In order to further explore and discover novel and efficient ASK1 inhibitors for the treatment of NASH, this study conducted a detailed structure–activity relationship study on compound 4 with the aim of augmenting the inhibitory potential of its derivatives (Figure 2).

Figure 2.

Structural optimisation strategy for quinoline derivatives with compound 2 as the lead compound.

The specific structural optimisation strategy is to first evaluate the structure–activity relationship of moiety A and introduce different nitrogen heteroaromatic or benzene rings; next, replace or modify the triazole of moiety B (R1); finally, we introduced electron withdrawing or donating groups at the R2–R5 position of the benzene ring of quinoxaline (Figure 2). Therefore, a series of quinoxaline derivatives were synthesised and evaluated as ASK1 inhibitors in this article.

Results and discussion

Chemistry

We first synthesised the targeting products 9a–9e, and their synthesis route is shown in Scheme 1. We used benzoate (5a), methyl aminopyridine carboxylate (5b–5d), or pyrazine carboxylate (5e) as starting materials, and reacted with hydrazine hydrates (N₂H₄⋅H₂O) in methanol (MeOH) under reflux to get hydrazide products 6a–6e with high yield. The obtained 6a–6e were then reacted with 1,1-dimethoxy-N,N-dimethylamine in toluene to obtain 7a–7e, which was directly cyclised with isopropylamine without purification in a mixed solvent of acetic acid and acetonitrile (v:v = 1:5) to provide triazole intermediates 8a–8e. The resulting 8a–8e then underwent amidation reaction with quinoxaline-2-carboxylic acid to give the target products 9a–9e.

Scheme 1.

Synthesis of title compounds 9a–9e. Reagents and conditions: (i) N₂H₄⋅H₂O (2.0 equiv.), MeOH, 75 °C, 7 h, 95­100% yield; (ii) 1,1-dimethoxy-N,N-dimethylmethylamine (3.0 equiv.), MeCN, 95 °C, 3 h; (iii) isopropylamine (5.0 equiv.), AcOH (3.0 equiv.), MeCN, 90 °C, 16 h, 30.7–57.9% yield; (iv) (a) for 9a–9d: 2-quinoxalinecarboxylic acid (1.0 equiv.), T₃P (50.0% solution in ethyl acetate, 4.0 equiv.), Et₃N (7.0 equiv.), CH₂Cl₂, r.t., 6–12 h, 24.3–83.2% yield; (b) for 9e: 2-quinoxalinecarboxylic acid (1.0 equiv.), POCl₃ (4.0 equiv.), pyridine, r.t., 5 h, 82.6% yield.

The synthesis of target products 12a–12h is shown in Scheme 2. 2-Amino-bromopyridine raw material 10 was reacted with 1-Isopropylpyrazole-5-boronic acid pinacol ester by Suzuki coupling reaction to obtain 11a, which was further reacted with quinoxaline-2-carboxylic acid in presence of propylphosphonic anhydride solution (T3P, 50.0% solution in ethyl acetate, 4.0 equiv.) as the coupling reactant to provide amide 12a. Compounds 12b–12f share a comparable synthetic procedure. Initially, methyl-6-aminopicolinate 13 serves as the starting material, reacting with hydrazine hydrate to yield acylhydrazide intermediate 14. Subsequently, it undergoes heating with 1,1-dimethoxy-N,N-dimethylmethylamine in toluene as a solvent, producing imine intermediate 15. The obtained 15 is then employed in the synthesis of triazole intermediates 11b–11f. Specifically, the synthesis of 11b–11d involves cyclisation with cyclopropylamine, cyclobutylamine, and cyclopentylamine under acetic acid conditions. On the other hand, the synthesis of 11e–11f is accomplished through cyclisation with (S)-1,1,1-trifluoropropan-2-amine and (R)-1,1,1-trifluoropropan-2-amine in a mixed solvent system of AcOH/MeCN (v:v = 1:2). The synthesis of products 12b–12f involved a condensation reaction between the previously acquired 11b–11f and quinoxaline-2-carboxylic acid.

Scheme 2.

Synthesis of title compounds 12a–12f. Reagents and conditions: (i) 1-isopropyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (2. 0 equiv.), Xphos-Pd-G₂ (0.1 equiv.), Na₂CO₃ (10.0 equiv.), 1,4-dioxane/H₂O (v:v = 5:1), 100 °C, 2 h, 51.3% yield; (ii) 2-quinoxalinecarboxylic acid (1.0 equiv.), T₃P (50.0% solution in ethyl acetate, 4.0 equiv.), Et₃N (7.0 equiv.), CH₂Cl₂, r.t., 8 h, 24.0% yield; (iii) N₂H₄⋅H₂O, (2.0 equiv.), MeOH, 75 °C, 7 h, 100.0% yield; (iv) 1,1-dimethoxy-N,N-dimethylmethylamine (3.0 equiv.), toluene, 95 °C; (v) (a) for 11b–11d: corresponding amines (5.0 equiv.), AcOH (4.0 equiv.), toluene, 95 °C, 20 h, 17.6–65.1% yield; (b) for 11e–11f: corresponding amines (4.0 equiv.), AcOH/MeCN (v: v = 1:2), 75 °C, 10–12 h, 25.0–38.4% yield; (vi) (a) for 12b–12c: 2-quinoxalinecarboxylic acid (1.0 equiv.), T₃P (50.0% solution in ethyl acetate, 4.0 equiv.), Et₃N (7.0 equiv.), CH₂Cl₂, r.t., 6–10 h, 40.3–44.9% yield; (b) for 12d–12f: 2-quinoxalinecarboxylic acid (1.0 equiv.), POCl₃ (4.0 equiv.), pyridine, r.t., 5 h, 31.3–59.6% yield.

Next, we synthesised product 22 using the synthesis method shown in Scheme 3. First, we used a similar preparation method as 8a–8e in Scheme 1 to synthesise the key intermediate 16²⁶. The 2-quinoxaline carboxylic acid 21 substituted with a single nitro group was synthesised using 17 as the starting material. Compound 17 and prop-2-yn-1-amine were refluxed in ethanol (EtOH) under alkaline conditions of triethylamine (Et₃N) to obtain intermediate 18. Then, using the method reported in the literature²⁷, the adjacent nitro group was selectively reduced to give compound 19 under the catalysis of metal Ni. The obtained 19 was then cyclised with copper (I) tetra (acetonitrile) tetrafluoroborate to obtain 20. The resulting 20 was further oxidised by SeO₂ and 30% H₂O₂ to produce pyrazine carboxylic acid 21. Finally, 21 and 16 were further stirred at room temperature (r.t.) for 4 h under POCl₃ and pyridine conditions to obtain the targeting product 22.

Scheme 3.

Synthesis of title compound 22. Reagents and conditions: (i) isopropylamine (5.0 equiv.), AcOH (3.0 equiv.), toluene, 95 °C, 20 h, 95.0% yield; (ii) propargylamine (4.5 equiv.), Et₃N (4.5 equiv.), EtOH, 80 °C, 6 h, 41.9% yield; (iii) N₂H₄⋅H₂O (2.4 equiv.), Ni (2.1 equiv.), EtOH/DCM (v: v = 1:1), 60 °C, 9 h, 31.7% yield; (iv) (CH₃CN)₄CuBF₄ (1.2 equiv.), toluene, 85 °C, 20 h, 38.7% yield; (v) SeO₂ (2.0 equiv.), 30.0 wt.% H₂O₂ (1.2 equiv.), THF, 60 °C to 90 °C, 9 h, 72.7% yield; (vi) 6-(4-isopropyl-4H-1,2,4-triazol-3-yl) pyridine-2-amine (1.0 equiv.), POCl₃ (4.0 equiv.), pyridine, r.t., 4 h, 61.9% yield.

The synthesis route for products 26a–26f and 30 is shown in Scheme 4. The bisubstituted phenylenediamine derivatives 23a–23f were reacted with methylglyoxal at r.t. in THF to obtain bisubstituted 2-methylquinoxaline derivatives 24a–24f. The resulting 24a–24f is further oxidised to provide the key intermediate 25a–25f, which directly reacted with intermediate 16 without purification to obtain the targeting product 26a–26f. The synthesis of 30 started with naphthalene-2,3-diamine and went through a similar preparation process as 26a–26f.

Scheme 4.

Synthesis of title compounds 26a–26f, 30. Reagents and conditions: (i) methylglyoxal (40.0 wt.% in H₂O, 2.0 equiv.), THF, r.t., 3–8 h, 42.4–88.5% yield; (ii) SeO₂ (2.0 equiv.), 30.0 wt.% H₂O₂ (1.2 equiv.), THF, 60 °C to 90 °C, 24 h, 7.5–99.8% yield; (iii) 16 (1.0 equiv.), POCl₃ (4.0 equiv.), pyridine, r.t., 3–8 h, 40.1–68.0% yield.

Biological evaluation

Inhibitory activity against ASK1

In order to explore the structure–activity relationship of the newly synthesised quinoxaline derivatives as ASK1 inhibitors in this study, we comprehensively modified the structure of the inhibitor’s pyridine fragment (moiety A), triazole fragment (moiety B), and quinoxaline ring (moiety C). The in vitro ASK1 kinase inhibitory activities of all synthesised products are shown in Tables 1–3. First, we investigated the inhibitory effect of modification of pyridine ring moieties with different N atom positions or numbers. Unfortunately, none of the products 9a–9e obtained from this modification showed significant inhibitory activity (Table 1). Although 9a containing a benzene ring fragment possessed a 40.67% inhibition rate at a concentration of 10 µM, its IC₅₀ was still greater than 10000 nM. Therefore, the pyridine moiety could not be arbitrarily modified, and it significantly contributed to the maintenance of the inhibitory activity.

Table 1.

Inhibitory activity of compounds 9a–9e against ASK1.

Comp.a	R	Inhibition rateb (10 µM)	IC50c (nM)
4 26		97.80 ± 1.10	147
9a		40.67 ± 1.24	>10 000
9b		6.68 ± 4.22	>10 000
9c		30.00 ± 2.17	>10 000
9d		2.12 ± 0.53	>10 000
9e		21.65 ± 1.66	>10 000
GS-4997	–d	–	6.0

^a Compounds.

^b Values are the means ± SD from two independent experiments.

^c Values are the means from single independent assay.

^d The dash "–" represents no relevant information or untested.

Table 2.

Inhibitory activity of compounds 12a–12f against ASK1.

Comp.a	R1	Inhibition rateb (10 µM)	IC50c (nM)
4 26		97.80 ± 1.10	147
12a		1.47 ± 0.79	>10 000
12b		92.31 ± 0.17	502.46
12c		97.24 ± 0.16	117.61
12d		94.85 ± 0.11	49.63
12e		97.73 ± 0.21	46.32
12f		72.30 ± 0.24	2469.68
GS-4997	–d	–	6.0

^a Compounds.

^b Values are the means ± SD from two independent experiments.

^c Values are the means from single independent assay.

^d The dash "–" represents no relevant information or untested.

Table 3.

Inhibitory activity of compounds 22, 26a–26f, and 30 against ASK1.

Comp.a	R2	R3	R4	R5	Inhibition rateb (10 µM)	IC50c (nM)
426	H	H	H	H	97.80 ± 1.1	147
22	H	NO2	H	H	90.15 ± 0.05	619.46
26a	H	F	F	H	95.36 ± 0.02	264.09
26b	H	CH3	CH3	H	94.79 ± 1.31	73.48
26c	H	Cl	Cl	H	88.01 ± 0.50	299.87
26d	H	Br	Br	H	79.00 ± 0.16	249.53
26e	Br	H	H	Br	69.50 ± 1.27	30.17
26f	OCH3	H	H	OCH3	91.54 ± 0.23	543.17
30	H	R3 and R4 were cyclised into benzene ring		H	93.62 ± 0.38	69.24
GS-4997	–d	–d		–d	–d	6.0

^a Compounds.

^b Values are the means ± SD from two independent experiments.

^c Values are the means from single independent assay.

^d The dash "–" represents no relevant information or untested.

Next, we kept the pyridine portion unchanged and focused on the substitution and modification of the triazole portion (moiety B). Therefore, we synthesised isopropylpyrazole substituted products (12a), as well as 1,2,4-triazole products substituted with cyclopropyl (12b), cyclobutyl (12c), cyclopentyl (12d), and (R) or (S)-4-(1,1,1-trifluoropropane-2-yl) (12e and 12f). Through in vitro enzyme inhibition activity testing, it was found that the pyrazole product 12a did not exhibit inhibitory activity. Among the substituted products of alicyclic hydrocarbons, the order of ASK1 inhibitory activity from highest to lowest is 12d (IC₅₀ = 49.63 nM) > 12c (IC₅₀ = 117.61 nM) > 12b (IC₅₀ = 502.46 nM). Interestingly, the two enantiomers of trifluoropropyl substituted products 12e and 12f showed significant differences in inhibitory activity. The inhibitory activity of (S)-4-(1,1,1-trifluoropropane-2-yl) product 12e (IC₅₀ = 46.32 nM) is much higher than that of (R)-4-(1,1,1-trifluoropropane-2-yl) product 12f (IC₅₀ = 2469.68 nM), with an IC₅₀ value difference of more than 50 folds.

Then, we modified the benzene ring of the quinoxaline fragment by introducing nitro group (22), difluoro group (26a), dimethyl group (26b), dichloro group (26c), dibromo group (26d and 26e), dimethoxy group (26f), and benzene ring (30). Among these products, we found that product 26e, which was replaced by dibromides, possessed the strongest inhibitory activity in this series, with an IC₅₀ of 30.17 nM. As a comparison, the IC₅₀ values of 30 substituted with benzene ring and 26b substituted with dimethyl ring are at the level of 70 nM. Other compounds such as 22, 26a, 26c, 26d, and 26f have moderate inhibitory activity, with IC₅₀ values in the range of 200–700 nM.

Cytotoxicity assessment

To further elucidate the advantages of compound 26e, we evaluated its cytotoxic effects on LO2 cells (Figure 3). LO2 cells were incubated with varying concentrations (0.5, 1, 5, 10, and 20 μM) of compound 26e, with GS-4997 used as a positive control. Noteworthy is that, within the concentration range of 0.5–10 μM, compound 26e maintained a cell survival rate above 80%, particularly evident at the elevated concentration of 10 μM, where its cell survival rate surpassed that of GS-4997 significantly. Although at 20 μM, 26e displayed some cytotoxicity with a cell survival rate dropping below 70%, it still exhibited superior safety compared to GS-4997, which recorded a cell survival rate below 60%. In summation, these observations proposed that 26e exhibited a wide safety range (0.5–10 μM), demonstrating robust safety even at high concentrations in normal human liver cells (10 μM).drug

Figure 3.

Cell viability assessed by the CCK-8 assay in 24-h human normal liver LO2 cells cultures. The columns represent the mean ± standard deviation (n = 6). 26e had better cell viability at high concentrations of 10 µM, with a slight difference from GS-4997.

Oil Red O staining

To establish a NASH cell model, LO2 cells were subjected to a 24-h treatment with free fatty acids (FFAs). The results from Oil Red O staining revealed that within the normal group, LO2 cells demonstrated a polygonal morphology characterised by distinct cell boundaries, a blue nucleus, and an intact nuclear membrane. The cytoplasm appeared transparent, with nearly absent red lipid droplets. In contrast to the normal control group, cells treated with FFA exhibited a significant presence of dense red lipid droplets, signifying the inducement of lipid droplet formation in LO2 cells by FFA (Figure 4(A,B)). In comparison to the FFA-treated group, cells cultured with 26e and FFA exhibited intracellular lipid droplets that were lighter in colour and more diffusely distributed. Furthermore, 26e exhibited a dose-dependent reduction in lipid droplet accumulation. This implies that 26e has the potential to diminish both the size and quantity of lipid droplets (Figure 4(C–E)).

Figure 4.

Effect of 26e on the content of lipid droplets under light microscopy in LO2 cells (A: normal control group; B: oleic acid-induced fatty liver model group; C: 26e group of 1 µM; D: 26e group of 3 µM; E: 26e group of 6 µM) (10 × 20).

Effects of 26e on HDL-C, T-CHO, LDL, and TG content in LO2 cells

Compared with the control group, the FFA group showed significant increases in high-density lipoprotein-cholesterol (HDL-C), total-cholesterol (T-CHO), low-density lipoprotein (LDL), and triglyceride (TG) levels, which indicated that our cell model of NAFL was successful (Figure 5(A–D)). Meanwhile, the T-CHO, LDL, and TG contents of the 26e group with different concentrations were all reduced compared with the FFA group. Especially, the T-CHO content of 26e groups with different concentrations (Figure 5(C), 1, 3, and 6 μM) was significantly decreased in a dose-dependent manner (p < 0.05, 6 µM). Therefore, it is worth noting that 26e could reduce the content of T-CHO, LDL, and TG in FFA-induced LO2 cells, and had the potential to treat non-alcoholic fatty disease.

Figure 5.

The effects of 26e on HDL-C (A), LDL (B), T-CHO (C), and TG (D) content in LO2 cells. *p < 0.05 vs. the FFA group.

Molecular docking

In order to better understand the possible molecular interaction modes between inhibitors and ASK1 protein (PDB code: 5UOX)²⁸, the representative molecule 26e and control drug GS-4997 were docked into ASK1 protein, using Discovery Studio version 4.5, respectively, and docked into ASK1 protein. As shown in Figure 6, the docking results show that 26e and GS-4997 possess similar H-bonding modes (Figure 6(A,B)). The amide carbonyl group in the ligand structure forms a hydrogen bond with the main chain NH of Val757, while the N1 of triazole nitrogen forms another H-bonding interaction with the catalytic Lys709. Among them, Lys709 residue is located at the catalytic active centre of ASK1 protein, forming hydrogen bonds with the triazole fragment of the ligand, generating inhibitory activity. Val757 is located in the hinge region of ASK1 protein, forming hydrogen bonds with the amide portion of the ligand, thereby limiting the binding conformation of the ligand and stabilising the binding mode between the ligand and the active pocket. In addition, the bromobenzene fragment of 26e is similar to the imidazole ring of GS-4997 and is located in the solvent-exposed region (Figure 6(C,D)).

Figure 6.

Receptor–ligand interactions. (A) Binding mode of 26e with ASK1 (PDB code: 5UOX). (B) Surface representation of 26e binding to active pocket of ASK1 protein. (C) Binding mode of GS-4997 with ASK1 (PDB code: 5UOX). (D) Surface representation of 26e binding to active pocket of ASK1 protein. The ligands and important residues are represented as stick structures. The hydrogen bonds are depicted as green dashed lines. The 26e is shown in white, GS-4997 is shown in orange, and key amino acid residues are shown in red or yellow.

Conclusions

In this article, we synthesised a series of quinoxaline derivatives as ASK1 inhibitors, and found a potent inhibitor 26e with an IC₅₀ value of up to 30.17 nM. Through further cytotoxicity experiments, Oil Red O Staining experiments, and biochemical analyses, the potential of 26e as a treatment for NAFL disease has been confirmed. These findings provide a good choice for the future development of ASK1 inhibitors.

Experimental

Chemistry

All chemicals, reagents, and solvents were purchased from Anhui Zesheng Technology Co., Ltd. (Anqing, China) and Bide Pharmatech Co., Ltd. (Shanghai, China). The high resolution mass spectra (ESI-HRMS) were operated on a Bruker microOTOF-Q II mass spectrometer (Bruker Daltonik, Bremen, Germany) equipped with an electrospray ionisation source. The ¹H NMR and ¹³C NMR spectrums were measured on a Bruker Avance 400 MHz NMR spectrometer. The chemical shift value is in Hertz. The split mode is expressed as s, singlet; d, doublet; t, triplets; m, multiplet; dd, doublet of ¹H NMR data.

Synthesis of aryl triazole derivatives (8a–8e)

The detailed synthesis procedures and NMR data of 8a–8e have been reported in the literature²⁹. Simply put, 8a–8e starts from the raw material ester 5a–5e, undergoes a hydrazide reaction to obtain 6a–6e, then reacts with 1,1-dimethoxy-N,N-dimethylmethylamine to prepare imine product 7a–7e, and finally cyclises with isopropylamine to obtain triazole intermediate 8a–8e.

Synthesis of quinoxaline derivatives (9a–9d)

A solution of 2-quinoxalinecarboxylic acid (1.0 equiv.) and 8a–8d (1.0 equiv.) in DCM (3.0 mL) was stirred at 0 °C under dry argon, then added Et₃N (7.0 equiv.) and T₃P (50.0% solution in ethyl acetate, 4.0 equiv.) to the above solution, stirred at r.t. for 6–12 h. The reaction progress was monitored by TLC. After completion of the reaction, the solution was diluted with DCM; mixed solution was extracted with water (3 × 20 ml) and NaHCO₃ solution (3 × 20 ml). The organic layer was separated, dried by anhydrous Na₂SO₄, filtered and condensed to get compounds 9a–9d.

N-(3-(4-isopropyl-4H-1,2,4-triazol-3-yl) phenyl) quinoxaline-2-carboxamide (9a)

Yellow solid, 16.0 mg, 26.0% yield. ¹H NMR (400 MHz, DMSO-d₆): δ 11.13 (s, 1H), 9.57 (s, 1H), 8.91 (s, 1H), 8.31–8.34 (m, 1H), 8.22–8.26 (m, 2H), 8.12–8.15 (m, 1H), 8.01–8.06 (m, 2H), 7.61 (t, J = 8.0 Hz, 1H), 7.36–7.45 (dt, J = 8.0, 1.2 Hz, 1H), 4.49–4.55 (m, 1H), 1.46 (d, J = 6.8 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆): δ 162.45, 152.30, 144.65, 144.08, 143.02, 139.71, 138.71, 132.28, 131.51, 131.37, 129.63, 129.55, 129.20, 127.97, 124.75, 121.84, 120.72, 47.52, 23.34. ESI-HRMS (m/z) calcd for C₂₀H₁₈N₆O, [M + Na]⁺: 381.1440, found 381.1434.

N-(4-isopropyl-4H-1,2,4-triazol-3-yl) pyridine-2-yl) quinoxaline-2-carboxamide (9b)

Pale yellow solid, 52.0 mg, 83.2% yield. ¹H NMR (400 MHz, CDCl₃): δ 10.58 (s, 1H), 9.74 (s, 1H), 8.72 (s, 1H), 8.58 (d, J = 5.2 Hz, 1H), 8.45 (s, 1H), 8.08–8.23 (m, 2H), 7.88–7.96 (m, 2H), 7.57 (dd, J = 5.2, 1.6 Hz, 1H), 4.71–4.81 (m, 1H), 1.63 (d, J = 6.8 Hz, 6H); ¹³C NMR (101 MHz, DMSO-d₆: CDCl₃ = 1:1): δ 160.42, 150.01, 147.97, 142.71, 142.11, 141.28, 138.63, 136.05, 131.13, 130.18, 128.53, 128.06, 118.54, 111.38, 47.01, 22.46. ESI-HRMS (m/z) calcd for C₁₉H₁₇N₇O, [M + Na]⁺: 382.1393, found 382.1387.

N-(5-(4-isopropyl-4H-1,2,4-triazol-3-yl) pyridine-3-yl) quinoxaline-2-carboxamide (9c)

Pale yellow solid, 15.0 mg, yield 24.0%. ¹H NMR (400 MHz, CDCl₃): δ 10.16 (s, 1H), 9.75 (s, 1H), 9.08 (s, 1H), 8.76 (s, 1H), 8.71 (s, 1H), 8.45 (s, 1H), 8.22–8.26 (m, 2H), 7.90–7.97 (m, 2H), 4.57–4.67 (m, 1H), 1.60 (d, J = 6.8 Hz, 6H); ¹³C NMR (101 MHz, DMSO-d₆): δ 162.92, 149.86, 144.39, 144.14, 144.07, 143.28, 143.09, 142.86, 139.69, 134.99, 132.40, 131.57, 129.58, 129.23, 127.43, 123.57, 47.78, 23.45. ESI-HRMS (m/z) calcd for C₁₉H₁₇N₇O, [M + Na]⁺: 382.1393, found 382.1387.

N-(2-isopropyl-4H-1,2,4-triazol-3-yl) pyridine-4-yl) quinoxaline-2-carboxamide (9d)

Pale yellow solid, 15.0 mg, 24.0% yield. ¹H NMR (400 MHz, CDCl₃): δ 10.27 (s, 1H), 9.76 (s, 1H), 8.67 (d, J = 5.2 Hz, 1H), 8.48 (s, 1H), 8.40 (d, J = 4.8 Hz, 1H), 8.33 (s, 1H), 8.21–8.27 (m, 2H), 7.92–7.98 (m, 2H), 5.81–5.92 (m, 1H), 1.57 (d, J = 6.7 Hz, 6H); ¹³C NMR (101 MHz, DMSO-d₆): δ 163.38, 150.42, 150.13, 148.76, 146.20, 144.05, 143.32, 143.09, 139.63, 132.49, 131.56, 129.64, 129.18, 114.63, 113.94, 48.09, 23.13. ESI-HRMS (m/z) calcd for C₁₈H₁₈N₈O, [M + Na]⁺: 382.1393, found 382.1387.

Synthesis of 6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl) pyridine-2-amine (9e)

A solution of 2-quinoxalinecarboxylic acid (1.0 equiv.) and 8e (1.0 equiv.) in anhydrous pyridine (3.0 mL) was stirred at r.t. under dry argon. Then, POCl₃ (4.0 equiv.) was slowly added to the solution, and stirred at r.t. for 4 h. The reaction progress was monitored by TLC. After the reaction, it was diluted with water, causing a large number of solid to separated out. The solid was filtered, washed three times with water, and dried to get compound 9e. Yellow solid, 25.0 mg, 82.6% yield. FT-IR: 3435 (br, ^νN–H), 2957, 2924, and 2854 (s, ^νC–H of C-CH₃), 1699 (s, ^νC═O), 1533 (s, ^νN–H), 1505, 1425 (m, ^νC═C) cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 10.32 (s, 1H), 9.83 (d, J = 10.0 Hz, 2H), 9.34 (s, 1H), 8.46 (s, 1H), 8.22–8.29 (m, 2H), 7.92–8.00 (m, 2H), 5.44–5.54 (m, 1H), 1.65 (d, J = 6.4 Hz, 6H); ESI-HRMS (m/z) calcd for C₁₈H₁₈N₈O, [M + H]⁺: 363.1682 found 363.1676.

Synthesis of 6-(1-isopropyl-1H-pyrazole-5-yl) pyridine-2-amine (11a)

The comprehensive synthetic methodologies and ¹H NMR data for 11a have been documented in existing literature²⁹. To summarise, the synthesis involved the Suzuki cross-coupling reaction between 6-bromopyridine-2-amine (10) and 1-isopro-pyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1a-pyrazole, resulting in the formation of intermediate 11a.

Synthesis of (6-(4-substituted-4H-1,2,4-triazol-3-yl) pyridine-2-amine derivatives (11b–11d)

The extensive synthesis protocols and ¹H NMR data for 11b–11e are available in the existing literature²⁹. In brief, the synthetic pathway for 11b–11e commences with the utilisation of raw material ester 13. This compound undergoes a hydrazide reaction to yield 14, which further reacts with 1,1-dimethoxy-N,N-dimethylmethanamine to form the imine product 15. The subsequent cyclisation with isopropylamine results in the formation of triazole intermediates 11b–11d.

Synthesis of aminopyridine derivatives (11e–11f)

Compound 15 (1.0 equiv.) was dissolved in AcOH/MeCN = 1:2 (6.0 ml), cooled to 0 °C, then added corresponding amines (4.0 equiv.) to the above solution, stirred for 8 h at 75 °C. The reaction progress was monitored by TLC. After the reaction, the solution was concentrated by a rotary evaporator and purified by flash chromatography to obtain intermediates 11e–11f.

(R)-6-(4-(1,1,1-trifluoropropane-2-yl)-4H-1,2,4-triazol-3-yl) pyridine-2-amine (11e)

Pure white oil, 94.0 mg, 38.4% yield. ¹H NMR (400 MHz, CD₃OD): δ 8.90 (s, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.31 (dd, J = 7.2, 0.4 Hz, 1H), 6.94–7.02 (m, 1H), 6.63 (dd, J = 8.0, 0.4 Hz, 1H), 1.81 (d, J_F-H = 7.2 Hz, 3H); ¹³C NMR (101 MHz, CD₃OD): δ 160.87, 153.06, 145.53, 139.73, 125.91 (q, J_F-C = 280.70 Hz), 113.60, 111.23, 53.76 (q, J_F-C = 32.40 Hz), 14.18; ESI-HRMS (m/z) calcd for C₁₀H₁₀F₃N₅, [M + H]⁺: 258.0966, found 258.0961.

(S)-6-(4-(1,1,1-trifluoropropane-2-yl)-4H-1,2,4-triazol-3-yl) pyridine-2-amine (11f)

Pure white oil, 94.0 mg, 25.0% yield. ¹H NMR (400 MHz, CD₃OD): δ 8.90 (s, 1H), 7.55 (dd, J = 8.4, 7.6 Hz, 1H), 7.31 (dd, J = 7.6, 0.4 Hz, 1H), 6.93–7.04 (m, 1H), 6.62 (dd, J = 8.4, 0.4 Hz, 1H), 1.81 (d, J _F-H = 7.2 Hz, 3H); ¹³C NMR (101 MHz, CD₃OD): δ 160.86, 153.07, 145.53, 139.73, 125.91 (q, J_F-C = 280.90 Hz), 113.62, 111.24, 53.77 (q, J_F-C = 32.60 Hz), 14.18; ESI-HRMS (m/z) calcd for C₁₀H₁₀F₃N₅, [M + H]⁺: 258.0966, found 258.0961.

Synthesis of N-(6-(1-isopropyl-1H-pyrazole-5-yl) pyridine-2-yl) quinoxaline-2-carboxamide (12a)

The synthesis procedure of amide 12a was similar to that of compounds 9a–9d. Yellow solid, 15.0 mg, 24.0% yield. ¹H NMR (400 MHz, CDCl₃): δ 10.42 (s, 1H), 9.78 (s, 1H), 8.45 (dd, J = 8.4, 0.8 Hz, 1H), 8.18–8.25 (m, 2H), 7.87–7.95 (m, 3H), 7.60 (d, J = 1.6 Hz, 1H), 7.36 (dd, J = 7.6, 0.8 Hz, 1H), 6.54 (d, J = 2.0 Hz, 1H), 5.25–5.35 (m, 1H), 1.59 (d, J = 6.8 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃): δ 161.77, 150.46, 149.07, 144.34, 143.83, 142.81, 140.25, 139.54, 138.43, 132.40, 131.41, 130.02, 129.71, 120.20, 113.00, 106.87, 51.27, 22.95. ESI-HRMS (m/z) calcd for [M + Na]⁺: C₂₀H₁₈N₆O, 381.1440, found 381.1434.

Synthesis of quinoxaline derivatives (12b–12f)

The synthesis procedure of amides 12b–12f was similar to that of compound 9e.

N-(5-(4-cyclopropyl-4H-1,2,4-triazol-3-yl) pyridine-3-yl) quinoxaline-2-carboxamide (12b)

White solid, 28.0 mg, 45.0% yield. ¹H NMR (400 MHz, CDCl₃): δ 10.39 (s, 1H), 9.80 (s, 1H), 8.56 (d, J = 8.4 Hz, 1H), 8.32 (s, 1H), 8.25 (dd, J = 8.0, 1.6 Hz, 1H), 8.20 (dd, J = 8.0, 1.6 Hz, 1H), 7.99–8.03 (m, 2H), 7.89–7.97 (m, 2H), 3.92–3.98 (m, 1H), 1.20–1.25 (m, 2H), 0.99–1.03 (m, 2H).¹³C NMR (101 MHz, CDCl₃): δ 161.72, 150.28, 144.38, 143.89, 142.75, 140.18, 139.69, 132.42, 131.40, 129.92, 129.79, 120.44, 114.70, 29.82, 28.77, 8.24, 1.15. ESI-HRMS (m/z) calcd for C₁₉H₁₅N₇O, [M + Na]⁺: 380.1236, found 380.1230.

N-(5-(4-cyclobutyl-4H-1,2,4-triazol-3-yl) pyridine-3-yl) quinoxaline-2-carboxamide (12c)

Pale yellow solid, 26.0 mg, 40.3% yield. ¹H NMR (400 MHz, CDCl₃): δ 10.40 (s, 1H), 9.80 (s, 1H), 8.51 (s, 1H), 8.50 (d, J = 8.4 Hz, 1H), 8.24–8.26 (m, 1H),8.17–8.20 (m, 1H), 8.04 (dd, J = 7.6, 0.4 Hz, 1H), 7.90–7.98 (m, 3H), 5.49–5.57 (m, 1H), 2.67–2.74 (m, 2H), 2.38–2.48 (m, 2H), 1.94–2.07 (m, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 161.59, 150.14, 146.22, 144.43, 143.86, 142.69, 140.17, 139.85, 132.46, 131.47, 129.88, 129.82, 120.53, 114.50, 50.91, 31.46, 29.82, 15.23, 1.15; ESI-HRMS (m/z) calcd for C₂₀H₁₇N₇O, [M + Na]⁺: 394.1393, found 394.1387.

N-(6-(4-cyclopentyl-4H-1,2,4-triazol-3-yl) pyridine-2-yl) quinoxaline-2-carboxamide (12d)

Pale yellow solid, 25.0 mg, 59.6% yield. ¹H NMR (400 MHz, CDCl₃): δ 10.36 (s, 1H), 9.79 (s, 1H), 8.51 (d, J = 8.0 Hz, 1H), 8.38 (s, 1H), 8.25 (dd, J = 7.6, 1.6 Hz,1H), 8.16 (dd, J = 7.2, 2.0 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H), 7.90–7.99 (m, 3H), 5.56–5.63 (m, 1H), 2.36–2.41 (m, 2H), 1.87–1.97 (m, 6H); ¹³C NMR (101 MHz, CDCl₃): δ 161.56, 150.07, 144.40, 143.84, 142.66, 140.15, 139.91, 132.45, 131.47, 129.83, 129.80, 120.90, 114.52, 58.25, 34.02, 29.81, 23.90, 1.13; ESI-HRMS (m/z) calcd for C₂₁H₁₉N₇O, [M + H]⁺: 386.1729, found 386.1724.

(R)-N-(6-(4-(3,3-dimethylbutyl)-4H-1,2,4-triazol-3-yl) pyridine-2-yl) quinoxaline-2-carboxamide (12e)

White solid, 15.0 mg, 45.6% yield. ¹H NMR (400 MHz, CDCl₃): δ 10.37 (s, 1H), 9.79 (s, 1H), 8.53 (s, 1H), 8.50 (d, J = 8.0 Hz, 1H), 8.24–8.27 (m, 1H), 8.16–8.19 (m, 2H), 8.01 (t, J = 8.0 Hz, 1H), 7.90–7.98 (m, 2H), 6.70–6.77 (m, 1H), 1.90 (d, J_F-H = 8.0 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃): δ 161.47, 149.91, 145.66, 144.36, 143.71, 142.43, 140.23, 132.47, 131.45, 129.82, 129.70, 120.77, 115.02, 52.71 (J_F-C = 16.60 Hz), 29.76, 15.13. ESI-HRMS (m/z) calcd for C₁₉H₁₄F₃N₇O, [M + H]⁺: 414.1290, found 414.1285.

(S)-N-(6-(4-(3,3-dimethylbutyl)-4H-1,2,4-triazol-3-yl) pyridine-2-yl) quinoxaline-2-carboxamide (12f)

White solid, 10.0 mg, 31.1% yield. FT-IR: 3338 (br, ^νN–H), 2962, 2924, and 2852 (s, ^νC–H of C-CH₃), 1694 (vs, ^νC═O), 1579, 1515, 1451 (s, ^νC═C), 1548 (vs, ^νN–H), cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 10.37 (s, 1H), 9.79 (s, 1H), 8.51 (s, 1H), 8.50 (d, J = 8.0 Hz, 1H), 8.25–8.27 (m, 1H), 8.16–8.19 (m, 2H), 8.01 (t, J = 8.0 Hz, 1H), 7.91–7.98 (m, 2H), 6.70–6.77 (m, 1H), 1.90 (d, J_F-H = 8.0 Hz, 3H); ESI-HRMS (m/z) calcd for C₁₉H₁₄F₃N₇O, [M + H]⁺: 414.1290, found 414.1285.

6-(4-Isopropyl-4H-1,2,4-triazol-3-yl) pyridine-2-amine (16)

The synthesis method of 16 is similar to that of 8a–8e. Simply put, 16 starts from the raw material ester 13, undergoes a hydrazide reaction to obtain 14, then reacts with 1,1-dimethoxy-N,N-dimethylmethylamine to prepare imine product 15, and finally cyclises with isopropylamine to obtain triazole intermediate 16. The detailed synthesis procedures and NMR data have been reported in the literature²⁶.

Synthesis of 2,4-dinitro-N-propyl-2-alkylenaniline (18)

A solution of 1-chloro-2,4-dinitrobenzene (1.0 equiv.), propargylamine (4.5 equiv.), and Et₃N (4.5 equiv.) in absolute EtOH (8.0 ml) was heated at 80 °C for 6 h. The reaction progress was monitored by TLC. After the reaction, the solution was allowed to cool to r.t., whereupon the product crystallised, filtered with little cold EtOH washing three times, and dried to obtain intermediate 18 which was used directly for next step. Yellow crystal, 458.0 mg, 41.9% yield. ¹H NMR (400 MHz, DMSO-d₆): δ 9.07 (t, J = 5.6 Hz, 1H), 8.85 (d, J = 2.8 Hz, 1H), 8.37 (ddd, J = 9.2, 2.4, 0.4 Hz, 1H), 7.24 (d, J = 9.6 Hz, 1H), 4.34 (dd, J = 6.0, 2.8 Hz, 2H), 3.32 (t, J = 2.8 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 147.31, 135.56, 130.44, 130.00, 123.41, 115.72, 79.37, 74.84, 32.38; ESI-HRMS (m/z) calcd for C₉H₇N₃O₄, [M + H]⁺: 222.0515, found 222.0509.

Synthesis of 4-nitro-N1-propyl-2-alkylene-1,2-diamine (19)

Nickel powder (2.1 equiv.) was added in three portions over 40 min to a stirred solution of 18 (1.0 equiv.) and hydrazine hydrate (2.4 equiv.) in EtOH (5.0 ml) and 1, 2-dichloroethane (5.0 ml). The temperature was kept below 60 °C during the addition. After 9 h of additional stirring at 60 °C, the reaction was cooled, filtered, and condensed, then purified by flash chromatography to get intermediate 19. Reddish-brown solid, 132.0 mg, 31.7% yield. ¹H NMR (400 MHz, DMSO-d₆): δ 7.54 (dd, J = 8.8, 2.8 Hz, 1H), 7.44 (d, J = 2.8 Hz, 1H), 6.56 (d, J = 8.8 Hz, 1H), 6.34 (t, J = 5.6 Hz, 1H), 5.18 (s, 2H), 4.07 (dd, J = 5.6, 2.4 Hz, 2H), 3.21 (t, J = 2.4 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆): δ 141.26, 137.62, 135.14, 115.16, 108.23, 107.47, 80.95, 73.89, 32.00; ESI-HRMS (m/z) calcd for C₉H₉N₃O₂, [M + H]⁺: 192.0773, found 192.0768.

Synthesis of 2-methyl-7-nitroquinoxaline (20)

A mixture of 19 (1.0 equiv.) and copper (I) tetra (acetonitrile) tetrafluoroborate (1.2 equiv.) in toluene (5.0 ml) was heated at 90 °C for 20 h, during which time a black precipitate formed. The solution was cooled and decanted, and the precipitate was ground with a spatula and extracted three times with DCM (60.0 ml). The combined toluene and dichloromethane extracts were concentrated and purified by flash chromatography to obtain intermediate 20. Yellow solid, 50.0 mg, 38.7% yield. ¹H NMR (400 MHz, CDCl₃): δ 9.06 (s, 1H), 8.79 (d, J = 2.4 Hz, 1H), 8.48 (dd, J = 9.2, 2.4 Hz, 1H), 8.28 (d, J = 9.2 Hz, 1H), 2.78 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆): δ 157.11, 149.98, 147.50, 142.85, 140.23, 130.84, 124.35, 122.37, 22.36; ESI-HRMS (m/z) calcd for C₉H₇N₃O₂, [M + H]⁺: 190.0616, found 190.0611.

Synthesis of N-(6-isopropyl-1H-1,2,4-triazol-5-yl) pyridine-2-yl)-7-nitroquinoxaline-2-carboxamide (22)

Intermediate 20 (1.0 equiv.) was dissolved in 1,4-dioxane (4.0 ml), then SeO₂ (2.0 equiv.) was added to the mixture. The mixture was stirred for 5–8 h at 60 °C. After that, 30.0 wt.% H₂O₂ (1.2 equiv.) was added to the solution, then the mixture was stirred for 8 h at 90 °C. The precipitation was filtered, and the solution was concentrated. Then, 5.0 wt.% NaOH (20.0 ml) was added to the concentrated solution; the mixed solution was washed successively with DCM (2 × 50 ml) and ethyl acetate (30.0 ml). The pH of the water phase was adjusted to 1–2 by adding 3 M hydrochloric acid, and the solid was separated from the solution by filtration. Brown solid, 160.0 mg, 61.9% yield. FT-IR: 3361 (br, ^νN–H), 2962, 2924, and 2854 (m, ^νC–H of C-CH₃), 1702 (vs, ^νC═O), 1573, 1512, 1446 (ms, ^νC═C), 1530 (s, ^νN–H), 1342 (vs, ^νNO₂) cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆): δ 10.95 (s, 1H), 9.75 (s, 1H), 9.12 (d, J = 2.4 Hz, 1H), 8.92 (s, 1H), 8.71 (dd, J = 9.2, 2.8 Hz, 1H), 8.48 (d, J = 9.2 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 8.14 (t, J = 8.0 Hz, 1H), 7.95 (d, J = 7.6 Hz,1H), 5.57–5.67 (m, 1H), 1.51 (d, J = 6.8 Hz, 6H); ESI-HRMS (m/z) calcd for C₁₉H₁₆N₈O₃, [M + K]⁺: 443.0982, found 443.0977.

Synthesis of 2-methylquinoxaline derivatives (24a–24f)

The differently substituted o-phenylenediamine (1.0 equiv.) was dissolved in THF (4.0 ml) and methylglyoxal (40.0 wt.% in H₂O, 2.0 equiv.) was added dropwise to the above reaction solution under argon protection. Then, the reaction mixture was stirred at r.t. for 4–9 h, followed by concentration and removal of THF. The resulting mixture was purified by flash chromatography to obtain intermediates 24a–24f.

6,7-Difluoro-2-methylquinoxaline (24a)

Pure white solid, 53.0 mg, 42.4% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.72 (s, 1H), 7.81 (dd, J_F-H = 10.4, 8.4 Hz, 1H), 7.76 (dd, J_F-H = 10.4, 8.4 Hz, 1H), 2.76 (s, 3H); ¹³C NMR (101 MHz, CDCl₃): δ 154.28 (d, J_F-C = 3.19 Hz), 153.46 (dd, J_F-C = 72.84, 16.00 Hz), 150.92 (dd, J_F-C = 72.14, 15.90 Hz), 146.25 (d, J_F-C = 3.12 Hz), 139.53 (d, J_F-C = 10.87 Hz), 138.24 (d, J_F-C = 10.56 Hz), 115.04 (d, J_F-C = 17.20 Hz), 114.55 (dd, J_F-C = 17.30, 1.31 Hz), 22.60; ESI-HRMS (m/z) calcd for C₉H₆F₂N₂, [M + K]⁺: 219.0136, found 219.0131.

2,6,7-Trimethylquinoxaline (24b)

Pure white solid, 150.0 mg, 59.2% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.64 (s, 1H), 7.80 (s, 1H), 7.76 (s, 1H), 2.74 (s, 3H), 2.48 (s, 6H); ¹³C NMR (101 MHz, CDCl₃): δ 152.70, 145.05, 140.99, 140.46, 139.93, 139.26, 128.23, 127.77, 22.50, 20.42, 20.25; ESI-HRMS (m/z) calcd for C₁₁H₁₂N₂, [M + Na]⁺: 195.0898, found 195.0893.

6,7-Dichloro-2-methylquinoxaline (24c)

Pure white solid, 280.0 mg, 78.5% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.72 (s, 1H), 8.17 (s, 1H), 8.12 (s, 1H), 2.76 (s, 3H); ¹³C NMR (101 MHz, CDCl₃): δ 155.20, 147.10, 140.88, 139.75, 134.62, 133.51, 129.91, 129.49, 22.76; ESI-HRMS (m/z) calcd for C₉H₆Cl₂N₂, [M + H]⁺: 212.9986, found 212.9981.

6,7-Dibromo-2-methylquinoxaline (24d)

Pure white solid, 300.0 mg, 88.5% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.72 (s, 1H), 8.35 (s, 1H), 8.31 (s, 1H), 2.75 (s, 3H); ¹³C NMR (101 MHz, CDCl₃): δ 155.32, 147.22, 141.36, 140.29, 133.33, 132.94, 126.73, 125.48, 22.83; ESI-HRMS (m/z) calcd for C₉H₆Br₂N₂₂, [M + Na]⁺: 322.8796, found 322.8790.

5,8-Dibromo-2-methylquinoxaline (24e)

Pure white solid, 208.0 mg, 73.7% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.84 (s, 1H), 7.91 (dd, J = 19.2, 8.0 Hz, 2H), 2.89 (s, 3H); ¹³C NMR (101 MHz, CDCl₃): δ 155.96, 147.22, 140.86, 139.70, 133.69, 132.66, 123.84, 123.44, 22.80; ESI-HRMS (m/z) calcd for C₉H₆Br₂N₂₂, [M + Na]⁺: 322.8796, found 322.8790.

5,8-Dimethoxy-2-methylquinoxaline (24f)

Pure white solid, 130.0 mg, 67.5% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.76 (s, 1H), 6.96 (dd, J = 21.6, 8.8 Hz, 2H), 4.04 (s, 6H), 2.81 (s, 3H); ¹³C NMR (101 MHz, CDCl₃): δ 153.27, 149.03, 148.39, 144.99, 134.81, 133.70, 107.62, 106.46, 56.31, 56.25, 22.80; ESI-HRMS (m/z) calcd for C₁₁H₁₂N₂O₂, [M + Na]⁺: 227.0797, found 227.0791.

Synthesis of quinoxaline derivatives (26a–26f)

The synthesis of quinoxaline derivatives (26a–26f) started from 24a–24f, underwent oxidation to obtain quinoxaline-2-carboxylic acid derivatives 25a–25f, which were then directly acylated with compound 16 without purification. The preparation procedure for 26a–26f was similar to the synthesis of compound 22.

6,7-Difluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl) pyridine-2-yl) quinoxaline-2-carboxamide (26a)

White solid, 25.0 mg, 44.2% yield. ¹H NMR (400 MHz, CDCl₃): δ 10.19 (s, 1H), 9.77 (s, 1H), 8.51 (dd, J = 8.0, 0.8 Hz, 1H), 8.43 (s, 1H), 8.00 (dd, J_F-H = 16.8, 8.8 Hz, 2H), 7.95 (dd, J = 8.4, 6.4 Hz, 1H), 5.56 (hept, J = 6.8 Hz, 1H), 1.64 (d, J = 6.4 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 161.04, 154.99 (dd, J_F-C = 54.33, 16.10 Hz), 150.81, 149.92, 146.58, 144.12 (d, J_F-C = 3.02 Hz), 142.97 (d, J_F-C = 4.02 Hz), 142.08, 141.96, 139.93, 137.54 (d, J_F-C = 12.07 Hz), 115.47 (td, J_F-C = 18.11, 2.01 Hz), 114.61, 48.77, 23.80; ESI-HRMS (m/z) calcd for C₁₉H₁₅F₂N₇O, [M + H]⁺: 396.1384, found 396.1379.

N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl) pyridine-2-yl)-6,7-dimethylquinoxaline-2-carboxamide (26b)

White solid, 25.0 mg, 44.2% yield. ¹H NMR (400 MHz, CDCl₃): δ 10.32 (s, 1H), 9.68 (s, 1H), 8.52 (dd, J = 8.4, 0.8 Hz, 1H), 8.43 (s, 1H), 8.05 (dd, J = 7.6, 0.8 Hz, 1H), 7.95–7.99 (m, 2H), 7.90 (s, 1H), 5.60 (hept, J = 6.8 Hz, 1H), 2.57 (s, 6H), 1.65 (d, J = 6.8 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 162.02, 150.95, 150.28, 146.50, 143.73, 143.47, 143.00, 142.37, 142.08, 141.84, 139.87, 139.15, 128.72, 128.65, 120.80, 114.59, 48.84, 23.86, 20.84, 20.62. ESI-HRMS (m/z) calcd for C₂₁H₂₁N₇O, [M + Na]⁺: 410.1706, found 410.1700.

6,7-Dichloro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl) pyridine-2-yl) quinoxaline-2-carboxamide (26c)

Purple solid, 30.0 mg, 56.6% yield. ¹H NMR (400 MHz, CDCl₃): δ 10.15 (s, 1H), 9.77 (s, 1H), 8.50 (dd, J = 8.0, 0.8 Hz, 1H), 8.41 (s, 1H), 8.37 (s, 1H), 8.31 (s, 1H), 8.08 (dd, J = 7.6, 0.8 Hz, 1H), 7.98 (t, J = 8.0 Hz, 1H), 5.56 (hept, J = 6.8 Hz, 1H), 1.64 (d, J = 6.8 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 160.92, 149.89, 146.61, 144.93, 143.51, 142.95, 142.13, 139.97, 138.79, 137.45, 136.48, 130.40, 130.19, 121.17, 114.67, 48.83, 23.84. ESI-HRMS (m/z) calcd for C₁₉H₁₅Cl₂N₇O, [M + H]⁺: 428.0793, found 428.0788.

6,7-Dibromo-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl) pyridine-2-yl) quinoxaline-2-carboxamide (26d)

Orange solid, 30.0 mg, 40.1% yield. ¹H NMR (400 MHz, CDCl₃): δ 10.16 (s, 1H), 9.77 (s, 1H), 8.75 (s, 1H), 8.57 (s, 1H), 8.54 (dd, J = 4.8,4.0 Hz, 1H), 8.51 (s, 1H), 8.04 (dd, J = 7.6, 6.8 Hz, 1H), 7.98 (t, J = 8.0 Hz, 1H), 5.56 (hept, J = 6.6 Hz, 1H), 1.66 (d, J = 6.8 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃): δ 160.94, 149.91, 146.67, 145.06, 143.60, 143.35, 139.97, 139.26, 133.85, 133.60, 129.77, 128.66, 121.22, 114.70, 48.83, 23.86. ESI-HRMS (m/z) calcd for C₁₉H₁₅Br₂N₇O, [M + H]⁺: 515.9783, found 515.9778.

5,8-Dibromo-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl) pyridine-2-yl) quinoxaline-2-carboxamide (26e)

White solid, 30.0 mg, 68.0% yield. FT-IR: 3352 (br, ^νN–H), 2962, 2925, and 2854 (m, ^νC–H of C-CH₃), 1694 (vs, ^νC═O), 1536 (s, ^νN–H), 1499, 1443 (s, ^νC═C) cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆): δ 10.57 (s, 1H), 9.74 (s, 1H), 8.95 (s, 1H), 8.39 (dd, J = 8.1,0.36 Hz, 1H), 8.35 (d, J = 1.2 Hz, 2H), 8.17 (t, J = 7.6 Hz, 1H), 7.98 (dd, J = 7.6, 0.8 Hz, 1H), 5.49 (p, J = 6.8 Hz, 1H), 1.56 (d, J = 6.8 Hz, 6H); ESI-HRMS (m/z) calcd for C₁₉H₁₅Br₂N₇O, [M + H]⁺: 515.9783, found 515.9778.

N-(6-isopropyl-4H-1,2,4-triazol-3-yl) pyridine-2-yl)-5,8-dimethoxyquinoxaline-2-carboxamide (26f)

Yellow solid, 28.0 mg, yellow solid, 52.2% yield. ¹H NMR (400 MHz, CDCl₃): δ 10.51 (s, 1H), 9.77 (s, 1H), 8.47–8.52 (m, 2H), 8.08 (d, J = 7.2 Hz, 1H), 7.97 (t, J = 8.0 Hz, 1H), 7.15 (q, J = 8.8 Hz, 2H), 5.65 (hept, J = 6.6 Hz, 1H), 4.09 (d, J = 10.4 Hz, 6H), 1.68 (d, J = 6.8 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 161.52, 150.09, 149.06, 149.01, 146.42, 142.65, 141.57, 139.85, 136.74, 132.99, 120.56, 114.29, 110.14, 109.18, 56.56, 56.54, 48.95, 23.80. ESI-HRMS (m/z) calcd for C₂₁H₂₁N₇O₃, [M + Na]⁺: 442.1604, found 442.1598.

Synthesis of 2-methylbenzo[g]quinoxaline (28)

This compound was synthesised from 27 (1.0 equiv.) in a manner similar to that described for intermediates 24a–24f. Pale orange solid, 200.0 mg, 54.2% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.76 (s, 1H), 8.62 (s, 1H), 8.55 (s, 1H), 8.05–8.11 (m, 2H), 7.52–7.59 (m, 2H), 2.81 (s, 3H); ¹³C NMR (101 MHz, CDCl₃): δ 154.39, 147.32, 138.61, 137.80, 134.01, 133.28, 128.57, 128.48, 127.80, 126.94, 126.84, 126.60, 23.23; ESI-HRMS (m/z) calcd for C₁₃H₁₀N₂, [M + H]⁺: 195.0922, found 195.0917.

Synthesis of N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl) pyridine-2-yl) benzo[g]quinoxaline-2-carboxamide (30)

The synthesis process of amide 30 was similar to that of compound 22. Orange solid, 30.0 mg, 45.6% yield. ¹H NMR (400 MHz, CDCl₃): δ 10.35 (s, 1H), 9.80 (s, 1H), 8.79 (d, J = 11.6 Hz, 2H), 8.54 (d, J = 8.4 Hz, 1H), 8.45 (s, 1H), 8.19 (dd, J = 7.2, 4.8 Hz, 2H), 8.08 (d, J = 7.2 Hz, 1H), 7.99 (t, J = 8.0 Hz, 1H), 7.65–7.70 (m, 2H), 5.63 (hept, J = 6.8 Hz, 1H), 1.68 (d, J = 6.8 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 161.57, 150.90, 150.10, 146.54, 144.00, 142.90, 142.10, 139.88, 139.72, 136.32, 135.24, 134.47, 128.90, 128.79, 128.66, 128.25, 127.82, 120.90, 114.56, 48.82, 23.85; ESI-HRMS (m/z) calcd for C₂₃H₁₉N₇O, [M + Na]⁺: 432.1549, found 432.1543.

ASK1 kinase inhibition assay

ASK1 inhibition activity was tested using a multifunctional enzyme marker (2104 Multilabel Reader, Perkin Elmer, Waltham, MA), and the ultrasonic nanolitre liquid treatment system was derived from Echo. The ASK1 inhibitory activity was evaluated using the ADP-Glo Luminescent Assay²⁶. ASK1 kinase (Eurofins, Brussels, Belgium) and substrate were diluted with HTRF kinase buffer solution (1× kinase buffer, 25 mM MgCl₂, 4 mM DTT, 20 mM HEPES, pH = 7.5, 0.01% Triton X-100). The inhibitor was diluted with 100% DMSO to the final required maximum inhibitor concentration of 100× in the reaction. Diluted 100 µl of the compounds into wells in a 96-well plate and 40 μl compounds were transferred from the source plate to a new 384-well plate as an intermediate plate, and 40 nl of compounds were transferred to the assay plate with the Echo liquid handler. ASK1 solution was prepared in 1× kinase buffer, with a concentration of twice the final concentration of each reagent in the assay, and 2.5 μl of kinase solution was added to each well of the assay plate, except for control wells without enzymes (add 2.5 μl of 1× kinase buffer instead).

In 1× kinase reaction buffer, prepared a substrate solution of MBP substrate and ATP at a fourfold of the desired final concentration of each reagent for the assay. Then, a 2.5 μl of substrate solution was added to each well, and shaked the plate, incubated at 37 °C for 1 h, and added 5 μl ADP-Glo reagent (Promega, Madison, WI), incubated at 37 °C for 3 h, then added 10 μl kinase detection reagent, equilibrated at rt for 30 min, converts ADP into ATP and introduces luciferase and fluorescence to detect ATP. The data were collected from Envision. The RLU values were copied and converted to percentage inhibition using the following formula: Percent inhibition = (max − sample RLU)/(max − min) × 100 (“min” means the RLU of no enzyme control and “max” means the RLU of DMSO control). The data were then fitted using XLFit Excel add-in version 5.4.0.8 to obtain the IC₅₀ value. The formula used for fitting the data was: Y = Bottom + (Top − Bottom)/(1 + (IC₅₀/X)^HillSlope).

Cell culture

The LO₂ human hepatocyte cell line was acquired from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured using RPMI-1640 medium supplemented with 10% foetal bovine serum (FBS) and 1% penicillin–streptomycin (P/S) and cultured in 5% CO₂ at a temperature of 37 °C²⁶. The cells’ morphology and growth were assessed using a light microscope.

In vitro cytotoxicity assays

To assess the cytotoxicity of the highly inhibitory compound 26e, we employed the CCK-8 assay²⁶. Initially, logarithmic stage LO2 human normal liver cells were seeded into 96-well plates at a density of 5000 cells per well and incubated overnight in 100 μl of 1640 culture medium. Subsequently, the culture medium was replaced with fresh medium containing varying concentrations of compound 26e, while GS-4997 served as the positive control drug. After another 24-h incubation, 10 μl of CCK-8 solution was added to each well and incubated for 1 h. Following that, the absorbance of the formazan product was measured at 450 nm using a microplate reader (Multiskan FC; Thermo Fisher Scientific, Boston, MA). The untreated cells in media were used as the blank control. This process was repeated three times for all treatment concentrations. The cytotoxicity was quantified as the percentage of cell viability compared to the blank control.

Oil Red O staining

The lipid droplet formation in LO2 cells was assessed using Oil Red O staining²⁶. To establish the in vitro NASH cell model, LO2 cells were cultured with or without 1 mM FFA containing a 2:1 volume ratio of oleic acid (OA) and palmitic acid (PA) for 24 h.

The LO2 cells in logarithmic growth stage were seeded into a 12-well plate at a cell density of 5 × 10⁴ cells per well with 1 ml of 1640 culture medium and incubated overnight. They were then treated with 1 mM FFA and cultured with the previously indicated concentrations (1, 3, and 6 μM) of 26e for another 12 h. The cells were washed three times with PBS and fixed with a fixing solution for 30 min. After fixation, the cells were washed three times with double distilled water and immersed in a 60% isopropyl alcohol solution for 30 s. Subsequently, the cells were stained with Oil Red O staining solution (Solarbio, G1262, Beijing, China) in the dark for 20 min at rt. After staining, the cells were thoroughly washed with a 60% isopropyl alcohol solution, counterstained with Mayer’s haematoxylin stain solution, and observed under a microscope to determine the degree of fat accumulation.

Biochemical analyses

The TG, LDL-C, HDL-C, and T-CHO assay kits were supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The biochemical analysis procedure was as follows: LO2 cells in the logarithmic growth phase were seeded in six-well plates at a density of 800 000 cells per well and incubated at 37 °C with 5% CO₂ for 24 h. Subsequently, a 1 mM FFA modelling solution was prepared by dissolving OA (11.1672 mg, 0.0396 mmol) and PA (5.0688 mg, 0.0198 mmol) in 3 ml PBS containing 20% BSA, followed by dilution with complete medium to a concentration of 20 mM. A 1.5 ml aliquot of the 20 mM FFA solution was further diluted with 1640 medium containing 1% FBS to a final concentration of 1 mM. The cells were then divided into five groups: control group, FFA group, compound 24e (6 µM) group, compound 24e (3 µM) group, and compound 24e (1 µM) group, with three replicate wells per group. The control group received 1 ml of 1640 medium containing 1% FBS without any drug treatment; the FFA group was treated with 1 ml of medium containing 1 mM FFA (OA:PA in a 2:1 ratio), and the compound 24e groups were treated with 1 ml of medium containing 1 mM FFA and compound 24e at concentrations of 6 µM, 3 µM, or 1 µM, respectively. All groups were incubated at 37 °C for 24 h. After incubation, the culture medium was removed, and the cells were washed twice with PBS. Subsequently, 500 μl of trypsin was added to each well for cell digestion, which was terminated by adding 1 ml of complete medium. The cells were then transferred to centrifuge tubes and centrifuged at 1000 r/min for 10 min. The supernatant was discarded, and the cell pellet was retained. The cells were washed 1–2 times with saline, centrifuged at 1000 r/min for 10 min each time, and the supernatant was discarded, leaving the cell pellet. Next, 0.2–0.3 ml of saline was added to the pellet for homogenisation, followed by ultrasonic treatment in an ice bath for 3–5 s per pulse, with 30-s intervals, repeated 3–5 times. The samples were centrifuged and analysed using an automated biochemical analyser. Data were statistically analysed with SPSS 17.0 software (SPSS Inc., Chicago, IL).

Molecular docking

The molecular docking study was conducted using Discovery Studio (DS, version 4.5), and the DS − "CDOCKER" protocol was implemented through a graphical user interface. The PDB file of the three-dimensional structure of ASK1 protein (PDB code: 5UOX)²⁸ was downloaded from the Protein Data Bank (https://www.rcsb.org/). The energy minimisation of ASK1 protein co-crystalline ligand 26e and GS-4997 was performed using a CHARM force field for 2000 iterations, with a minimum RMS gradient of 0.01. The preparation of docking proteins involved adding hydrogen atoms and removed water and impurities. After molecular docking, the output results were obtained and the interaction types between the ASK1 protein and 26e and GS-4997 were analysed.

Funding Statement

This work was supported by the National Natural Science Foundation of China (NSFC) (No. 22067010), Jiangxi Province Traditional Chinese Medicine Young and Middle-aged Backbone Talents Training Program (Fourth Batch) (Document No. 7 of Gan Traditional Chinese Medicine Science and Education [2022]), Jiangxi University of Chinese Medicine School-level Science and Technology Innovation Team Development Program (No. CXTD22005), the General Program of Jiangxi Natural Science Foundation (No. 20224BAB206117), the PhD Start-Up Fund of Jiangxi University of Chinese Medicine (No. 2021BSZR024), and the Jiangxi Provincial Key Laboratory of TCM Female Reproductive Health and Related Diseases Research and transformation (No. 2024SSY06311).

Author contributions

X.H., P.L., and Q.C. contributed equally to this work. X.H. was responsible for all cell experiments, and wrote the content of cell experiments in the manuscript, as well as the revision of the paper. P.L. synthesised and identified the structure of the target compounds. Q.C. provided the funding, revised the manuscript, guided the preliminary work, and supplemented the relevant data in the revision. H.L. and Z.C. provided guidance in kinase evaluation experiments, cell experiments, laboratory equipment facilities, reagent consumable resources, etc. T.W. provided the funding, resources, supervision of cell experiments, and writing of the cell experiments section of manuscript. Z.W. provided the funding, experimental design, supervision, and manuscript writing. All authors agree to the final approval of the published version and are responsible for all aspects of the work.

Disclosure statement

The authors report no conflicts of interest.

Data availability statement

Data will be made available on request.