Design, synthesis and biological evaluation of novel biphenylsulfonamide derivatives as selective AT2 receptor antagonists.

A novel series of benzenesulfonamide derivatives that selectively act on the AT2 receptor have been designed and synthesized. The binding affinity and functional activity were evaluated by radio-ligand binding analysis and cell neurite outgrowth assay, respectively. The compounds 8d, 8h, 8i, 8j, 8l, and 9h exhibited moderate selectivity and affinity for the AT2 receptor. Among them, 8j exhibited agonist activity and 8l displayed similar selectivity to the AT2 receptor with PD123,319. Molecular docking was carried out to analyze the binding mode and binding site between the compound and the AT2 receptor to provide a reference for further development.

1 Introduction

Benzenesulfonamide derivatives have a variety of biological activities, such as being anti-microbial and anti-tumor, protecting against cardiovascular disease, offering resistance to diabetes, and so on. The AT2 receptor, which is one of the subtypes of Angiotensin II receptor, is rarely expressed in normal tissue. In certain pathological conditions, the expression of the AT2 receptor was significantly up-regulated, such as myocardial infarction, vascular injury, cerebral ischemia, and so on (Carey, 2005; Juillerat-Jeanneret, 2020). A growing number of studies have suggested that AT2 receptor antagonists could alleviate peripheral neuropathic pain by blocking neuronal excitability (Hein et al., 1995).

C21/M024 is the first selective non-peptide AT2 receptor agonist (Wan et al., 2004). It was observed that a migration of the methylene imidazole substituent resulted in the compound with AT2 receptor antagonist activity (Murugaiah et al., 2012; Wallinder et al., 2019) (Figure 1). To develop an AT2 receptor antagonist with novel scaffold, a series of heterocyclic substituted benzenesulfonamide derivatives were designed based on the principle of scaffold hopping and bioelectronic isosteric (as shown in Figure 2). The binding affinity and functional activity were evaluated by the recognized assay.

FIGURE 1

Small changes in the structure of the compound lead to the change of functional activity.

FIGURE 2

The design of target compounds.

2 Results and discussion

2.1 Chemistry

The boric acid intermediates 4a-4c were obtained according to the synthetic route shown in Scheme 1 (Liu et al., 2013). The triazole and pyrazole was substituted to produce 5a and 5b, respectively. Then, the target compounds (8a-8l and 9a-9h) were synthesized by Suzuki coupling, amino deprotection, and esterification (as shown in Scheme 2).

SCHEME 1

Synthesis of intermediates 4a-4c.

SCHEME 2

Synthesis of target compounds 8a-8l, 9a-9h.

2.2 Biological evaluation

2.2.1 Binding assay

The target compounds were evaluated for AT1 receptor binding affinity by displacement of [125I]-Ang II from the AT1 receptor in rat liver membranes. The radio-iodinated AT2 receptor selective ligand [125I]-CGP42112 was used to study AT2 receptor binding affinity in HEK-293 cells (HEK293-hAT2R) (Dudley et al., 1990; Whitebread et al., 1991). All of above methods were widely accepted and used to evaluate the binding activity of AT1 and AT2 receptors. PD-123,319 is often used as a tool compound in the analysis of AT2 receptor antagonists (Blankley et al., 1991). The results are summarized in Table 1.

TABLE 1

The binding affinity and selectivity of the compound to the AT2 receptor.

Compound	R1	R2	R3	Ki (nM)
				AT1R	AT2R
8a	1,2,4-triazole	n-Bu	i-Bu	629.4 ± 1.1	873.7 ± 1.1
8b	1,2,4-triazole	OBu-n	i-Bu	846.1 ± 1.17	273.5 ± 1.2
8c	1,2,4-triazole	OBu-i	i-Bu	1037 ± 1.1	322 ± 1.24
8d	1,2,4-triazole	OPr-i	i-Bu	1786 ± 1.37	115.9 ± 1.18
8e	1,2,4-triazole	n-Bu	n-Bu	505.2 ± 1.4	753.9 ± 1.1
8f	1,2,4-triazole	OBu-n	n-Bu	1425 ± 1.12	252.7 ± 1.2
8g	1,2,4-triazole	OBu-i	n-Bu	945.5 ± 1.22	347.5 ± 1.36
8h	1,2,4-triazole	OPr-i	n-Bu	1505 ± 1.11	82.9 ± 1.19
8i	1,2,4-triazole	n-Bu	OBu-n	1250 ± 1.12	199.6 ± 1.17
8j	1,2,4-triazole	OBu-n	OBu-n	889.3 ± 1.24	620.1 ± 1.27
8k	1,2,4-triazole	OBu-i	OBu-n	775 ± 1.08	712.5 ± 1.17
8l	1,2,4-triazole	OPr-i	OBu-n	2005 ± 1.16	56.59 ± 1.16
9a	pyrazole	n-Bu	i-Bu	1264 ± 1.14	1152 ± 1.1
9b	pyrazole	OBu-n	i-Bu	989.7 ± 1.08	1027 ± 1.15
9c	pyrazole	OBu-i	i-Bu	1027 ± 1.08	916.2 ± 1.3
9d	pyrazole	OPr-i	i-Bu	1549 ± 1.11	74.09 ± 1.16
9e	pyrazole	n-Bu	n-Bu	363.9 ± 1.28	992.9 ± 1.26
9f	pyrazole	OBu-n	n-Bu	1310 ± 1.11	467.9 ± 1.19
9g	pyrazole	OBu-i	n-Bu	1212 ± 1.12	513.3 ± 1.18
9h	pyrazole	OPr-i	n-Bu	1723 ± 1.07	138 ± 1.34
PD-123,319 (Blankley et al., 1991)	-	-	-	2758 ± 1.09	34 ± 1.15

The affinity of 8a, 8e, 8j, 8k, 9a, 9b, and 9c to the AT1 and AT2 receptors was lower and with rare selectivity. The selectivity of 8l to the AT2 receptor was modest, which binding force to the AT2 receptor is equivalent to PD-123,319 [Ki: AT2R = 34 nM (Blankley et al., 1991)].

2.2.2 Function evaluation assay

The compounds (i.e., 8d, 8h, 9h, 8i, 8j, 8k, and 8l) with higher-affinity and which were structurally diverse were selected for further function evaluation by neurite outgrowth assay with NG108-15 cells, which is accepted as an AT2 receptor function assay (Buisson et al., 1992; Gasparo, 1996). The ratio of cell neurite outgrowth was used to evaluate, and the details are shown in the Supplementary Material.

Ang II can induce the outgrowth of cell neurite at a ratio of nearly 20% (shown in Figure 3A). When co-incubated with AT2 receptor antagonist PD-123,319, the outgrowth of Ang II induce ratio were decreased. This means the outgrowth of cell neurite might be induced by the AT2 receptor. As shown in Figures 3B–D , the outgrowth of the cell neurite was significantly less than 20% when incubated with the evaluated compounds alone, which means that the compounds inhibit the Ang II effect. The cell neurite outgrowth was slightly increased when the cells were inhibited by PD-123,319, which means that the inhibition of Ang II was induced by the AT2 receptor. Accordingly, 8i, 8k, 8d, 8l, 8h, and 9h had antagonistic activity against the AT2 receptor. The cell outgrowth ability of 8j were blocked by PD-123,319, which means that 8j exerted AT2 receptor agonistic activity (shown in Figure 3A). These results were found to be significant according to a two-way analysis of variance (ANOVA).

FIGURE 3

Effect of compounds 8j, 8i, 8k, 8d, 8l, 8h, and 9h on neurite outgrowth in NG108-15 cells. The number of cells with neurites was expressed as the percentage of the total number in the micrographs (at least 400 cells per well plate according to the experimental requirements). The results are significant according to two-way ANOVA: ***, p < 0.001; **, p < 0.01; *, p < 0.05; no significant difference is not shown.

2.3 Molecular docking studies

The binding affinity with the AT2 receptor and the functional activity of selected compounds (i.e., 8j, 8i, 8k, 8d, 8l, 8h, and 9h) are summarized in Table 2. The pyrazole group may provide an opportunity for binding to the AT1 receptor. Minor changes of the sulfonamide substituents of ligands not only affect the selectivity between compounds and receptors but also affect the functional activity of compounds.

TABLE 2

Differences in structure, affinity, and functional activity of compounds.

Compound	R1	R2	R3	Ki (nM)		Function
				AT1R	AT2R
8d	1,2,4-triazole	OPr-i	i-Bu	1786 ± 1.37	115.9 ± 1.18	Antagonist
8h	1,2,4-triazole	OPr-i	n-Bu	1505 ± 1.11	82.9 ± 1.19	Antagonist
8i	1,2,4-triazole	n-Bu	OBu-n	1250 ± 1.12	199.6 ± 1.17	Antagonist
8j	1,2,4-triazole	OBu-n	OBu-n	889.3 ± 1.24	620.1 ± 1.27	Agonist
8k	1,2,4-triazole	OBu-i	OBu-n	775 ± 1.08	712.5 ± 1.17	Antagonist
8l	1,2,4-triazole	OPr-i	OBu-n	2005 ± 1.16	56.59 ± 1.16	Antagonist
9h	pyrazole	OPr-i	n-Bu	1723 ± 1.07	138 ± 1.34	Antagonist

To better acquire comprehension of the relationship of structure modification and affinity and activity, and to elucidate the key binding sites where compounds retain antagonistic activity, molecular docking studies were implemented by utilizing SYBYL software (Porrello et al., 2009; Sallander et al., 2016; Zhang et al., 2017; Connolly et al., 2019). As shown in Figures 4B,D, the disappearance of hydrogen bond interaction between sulfonamide side chain and receptor protein not only reduced the affinity between the compound and the AT2 receptor by 100 times but also changed the functional activity from antagonist to agonist. As shown in Figures 4A,D, when the isopropoxy side chain of the sulfonamide group of 8l is replaced with n-butyl (compound 8i), the n-butyl of the sulfonamide group will competitively extend to the hydrophobic pocket.

FIGURE 4

The structural formula and docking results of compounds (A) 8i, (B) 8j, (C) 8k, and (D) 8l. The green dotted line indicates hydrogen bond interaction.

We speculated that the Thr 125, Lys 215, Arg 182, and Met 137 residues of the AT2 receptor may be the key binding sites for binding affinity. The additional hydrogen bond formed by residues Lys 215, Arg 182, and Pro 177 with the alkyl chain on the sulfonamide group may be the key binding sites for the antagonistic activity of the compound.

3 Discussion

PD-123,319 is the classical AT2 receptor antagonist. Why then did we design a novel series compound with a benzenesulfonamide structure? As shown in Figure 5, an AT2 receptor antagonist with the tetrahydroisoquinoline structure, especially EMA401, has been reported to exhibit analgesic efficacy in animal models, and finished the phase II clinical trial for neuropathic pain with satisfactory results. However, more recent trials were discontinued due to drug toxicity after most patients had completed enrollment (Smith et al., 2013; Rice et al., 2014; Smith and Muralidharan, 2015; Smith et al., 2016), which indicated that EMA401 was ineffective. Therefore, we speculated that the tetrahydroisoquinoline structure may have had some “off-target” effects or pharmacokinetic problems. Consequently, we focused on the benzenesulfonamide scaffold.

FIGURE 5

AT2 receptor antagonist with the tetrahydroisoquinoline structure.

As shown in Figure 6, both 8l and PD-123,319 can bind to the receptor protein through hydrophobic bonds with amino acid residues, such as Thr 125, Lys 215, Met 128, Tyr 103, and Trp 100, and occupy the cavity of the receptor protein. Furthermore, 81 (Ki: AT2R = 56.59 nM) and PD-123,319 [Ki: AT2R = 34 nM (Blankley et al., 1991)] have almost familiar binding affinity to the AT2 receptor. Therefore, the formation of these hydrophobic bonds is considered to be an important molecular basis for compounds to maintain high affinity for AT2 receptors.

FIGURE 6

The structural formula and docking results of (A) compound 8l and (B) PD-123,319 (Blankley et al., 1991) (Ki: AT2R = 34 nM). The green dotted line indicates hydrogen bond interaction. The purple dotted line indicates hydrophobic interaction.

4 Conclusion

A series of novel AT2 receptor selective compounds with benzenesulfonamide scaffolds have been synthesized and evaluated. Among them, 8j with agonist activity and compounds 8i, 8k, 8d, 8l, and 8h exhibited moderate higher selectivity and antagonist activity. The structure activity relationship and the key binding site of the AT2 receptor were discussed. These results may provide potential compounds and references for further development.

5 Experimental section

5.1 Chemistry

Starting material and solvents were purchased from commercial sources. Reactions were monitored by thin-layer chromatography (TLC) using precoated silica gel plates (silica gel GF/UV 254), and spots were visualized under UV light (254 nm). Melting points (uncorrected) were determined on a Mel-TEMP II melting point apparatus and are uncorrected. 1H NMR and 13C NMR spectra were recorded with a Bruker Avance 300 MHz spectrometer at 300 K, using TMS as an internal standard. MS spectra or high-resolution mass spectra (HRMS) were recorded on a Shimadzu GC-MS 2050 (ESI) or an Agilent 1946AMSD (ESI) Mass Spectrum. MS spectra or high-resolution mass Column chromatography was performed with silica gel (200e300 mesh). Chemical shifts were reported on the d scale and J values were given in Hz. The synthesis of intermediates 4a-4c is shown in the Supplementary Material.

5.1.1 General procedure A: Synthesis of intermediates (5a-5b)

+To a 250 ml round-bottom flask was added potassium carbonate (4.98 g, 36.01 mmol) dissolved in acetone (100 ml). The base was activated at room temperature by stirring for 10 min. Afterward, 3-bromobenzyl bromide (6 g, 24.01 mmol), 1,2,4-triazole or pyrazole (36.01 mmol), and potassium iodide (797.03 mg, 4.80 mmol) were added to a round-bottom flask and stirred 15 h at 55°C. The reaction was diluted with water and ethyl acetate. The layers were separated, and the organic layer was washed twice with saturated NaCl solution and dried with Na2SO4. The organic layer was concentrated to give 5a (Yellow oil) or 5b (Yellow oil).

5.1.2 General procedure B: Synthesis of intermediates (6a-6f)

Compounds 5a (2.89 g, 12.13 mmol), 4a (3.80 g, 12.13 mmol), Pd(OAc)2 (54.48 mg, 242.64 umol), PPh3 (254.57 mg, 970.57 umol), toluene (30 ml), ethanol (20 ml), and NaOH (1.6 M, 15 ml) were added to a 250 ml round-bottom flask and stirred for 5 h at 90°C. The reaction was extracted with ethyl acetate. The organic layers were combined, washed with saturated NaCl solution, dried over MgSO4, and concentrated in vacuo to give crude product. The residue was purified on silica gel with PE/EA (5:1) as eluent to get 6a. Yellow oil (3.20 g, 61.83%).

Compounds 6b-6f were prepared by the same synthetic method as 6a.

5.1.3 General procedure C: Synthesis of intermediates (7a-7f)

To a 100 ml round-bottom flask was added 6a (1.80 g, 4.22 mmol). Then, trifluoroacetic acid (6.92 ml, 92.83 mmol) was added slowly to the solution in an ice bath and the mixture was stirred under N2 atmosphere for 12 h at room temperature. The reaction mixture was evaporated and most TFA was removed, leaving oil which was dissolved in ethyl acetate and washed with water and saturated NaCl solution. The crude product was achieved after drying over anhydrous Na2SO4 and evaporating under reduced pressure. Using silica gel column chromatography (PE/EA as eluent), the residue was purified to obtain 7a. White solid (1.0 g, 63.97%). 1H NMR (300 MHz, Chloroform-d) δ 8.20 (s, 1H, triazole-H), 8.05 (d, J = 8.1 Hz, 1H, triazole-H), 7.94 (s, 1H, Ar-H), 7.50 (d, J = 8.3 Hz, 3H, Ar-H), 7.34 (d, J = 6.4 Hz, 1H, Ar-H), 7.30 (d, J = 8.2 Hz, 1H, Ar-H), 7.13 (d, J = 1.4 Hz, 1H, Ar-H), 5.41 (s, 2H, -CH ), 4.74 (s, 2H, -NH ), 2.58 (d, J = 7.2 Hz, 2H, -CH ), 1.94 (m, 1H, -CH), 0.96 (d, J = 6.6 Hz, 6H, -CH ). 13C NMR (75 MHz, CDCl3) δ 152.25, 146.72, 140.18, 139.24, 138.27, 134.43, 132.71, 129.69, 129.52, 129.01, 128.57, 127.84, 53.30, 44.96, 30.07, 22.34. HR-MS: [M + H]+: 371.1542 C H N O S require 371.1542.

Compounds 7b-7f were prepared by the same synthetic method as 7a.

5.1.4 General procedure D: Synthesis of target compounds (8a-8l, 9a-9h)

N-((3'-((1H-1,2,4-triazol-1-yl)methyl)-5-isobutyl-[1,1′-biphenyl]-2-yl)sulfonyl)pentanamide (8a).

Compound 7a (200 mg, 539.85 umol) was dissolved in DCM (5 ml), followed by triethylamine (375.20 mmL, 2.70 mmol) and valeryl chloride (127.63 mmL, 1.08 mmol) added on an ice bath. The reaction mixture was stirred for 4 h at room temperature under N2 atmosphere. The reaction was extracted with ethyl acetate. The organic layers were combined, washed with saturated NaCl solution, dried over MgSO4, and concentrated in vacuo. The crude product was purified by silica gel column chromatography using CH2Cl2/MeOH as an eluent to give 8a. White solid (146 mg, 59.49%). m.p. 138.4–139.7°C. 1H NMR (300 MHz, Chloroform-d) δ 9.88 (s, 1H, -NH), 8.26 (d, J = 8.2 Hz, 1H, Ar-H), 8.22 (s, 1H, triazole-H), 7.96 (s, 1H, triazole-H), 7.63 (s, 1H, Ar-H), 7.49-7.27 (m, 4H, Ar-H), 7.15 (s, 1H, Ar-H), 5.34 (s, 2H, -CH ), 2.59 (d, J = 7.0 Hz, 2H, -CH ), 1.97 (t, J = 6.9 Hz, 3H, -CH , -CH), 1.46 (m, 2H, -CH ), 1.31-1.20 (m, 2H, -CH ), 0.96 (d, J = 6.5 Hz, 6H, -CH ), 0.85 (t, J = 7.2 Hz, 3H, -CH ). 13C NMR (75 MHz, CDCl3) δ 171.15, 147.97, 140.02, 139.50, 134.96, 133.63, 132.71, 130.93, 130.32, 129.59, 128.99, 128.93, 127.79, 53.50, 45.09, 35.46, 30.02, 26.04, 22.39, 22.08, 13.72. ESI-MS: [M + H]+: 455.2107 C H N O S require 455.2111.

((3'-((1H-1,2,4-triazol-1-yl)methyl)-5-isobutyl-[1,1′-biphenyl]-2-yl)sulfonyl)carbamae (8b).

Compound 8b was prepared as described for 8a, replacing valeryl chloride with butyl chloroformate. Yield: 125.60 mg (49.44%). m.p. 148.1-149.3°C; 1H NMR (400 MHz, DMSO-d 6) δ 11.47 (s, 1H, -NH), 8.60 (s, 1H, triazole-H), 8.00 (s, 1H, triazole-H), 7.97 (d, J = 8.2 Hz, 1H, Ar-H), 7.41 (dd, J = 14.5, 7.8 Hz, 2H, Ar-H), 7.29 (d, J = 7.7 Hz, 1H, Ar-H), 7.23 (d, J = 7.9 Hz, 2H, Ar-H), 7.12-7.09 (m, 1H, Ar-H), 5.46 (s, 2H, -CH ), 3.93 (t, J = 6.5 Hz, 2H, -CH ), 2.56 (d, J = 7.1 Hz, 2H, -CH ), 1.89 (m, 1H, -CH), 1.39 (m, 2H, -CH ), 1.15 (m, 2H, -CH ), 0.88 (d, J = 6.6 Hz, 6H, -CH ), 0.81 (t, J = 7.4 Hz, 3H, -CH ). 13C NMR (101 MHz, DMSO) δ 152.11, 151.32, 147.46, 144.78, 140.50, 139.58, 135.93, 135.43, 133.49, 130.17, 128.94, 128.85, 128.66, 128.41, 127.62, 65.88, 52.60, 44.31, 30.49, 29.87, 22.54, 18.73, 13.89. ESI-MS: [M + H]+: 471.2055 C H N O S require 471.2061.

Isobutyl((3'-((1H-1,2,4-triazol-1-yl)methyl)-5-isobutyl-[1,1′-biphenyl]-2-yl)sulfonyl)carbamate (8c).

Compound 8c was prepared as described for 8a, replacing valeryl chloride with isobutyl chloroformate. Yield: 111.00 mg (45.23%). M.p. 145.3-146.2°C; 1H NMR (300 MHz, Chloroform-d) δ 9.62 (s, 1H, -NH), 8.24 (d, J = 8.2 Hz, 1H, triazole-H), 8.17 (s, 1H, triazole-H), 7.95 (s, 1H, Ar-H), 7.56 (s, 1H, Ar-H), 7.47-7.28 (m, 4H, Ar-H), 7.16 (d, J = 1.4 Hz, 1H, Ar-H), 5.31 (s, 2H, -CH ), 3.83 (d, J = 6.5 Hz, 2H, -CH ), 2.60 (d, J = 7.1 Hz, 2H, -CH ), 1.96 (m, 1H, -CH), 1.81 (m, 1H, -CH), 0.97 (d, J = 6.6 Hz, 6H, -CH ), 0.81 (d, J = 6.7 Hz, 6H, -CH ). 13C NMR (75 MHz, Chloroform-d) δ 152.03, 150.85, 147.93, 143.22, 139.99, 139.79, 134.89, 133.64, 132.71, 130.96, 130.03, 129.38, 128.93, 128.70, 127.74, 72.58, 53.43, 45.07, 30.05, 27.61, 22.36, 18.76. ESI-MS: [M + H]+: 471.2043 C H N O S require 471.2061.

Isopropyl((3’-((1H-1,2,4-triazol-1-yl)methyl)-5-isobutyl-[1,1′-biphenyl]-2-yl)sulfonyl)carbamate (8d).

`Compound 8d was prepared as described for 8a, replacing valeryl chloride with isopropyl chloroformate. Yield: 145.00 mg (57.08%). m.p. 150.9-151.7°C; 1H NMR (400 MHz, DMSO-d 6) δ 11.34 (s, 1H, -NH), 8.60 (s, 1H, triazole-H), 7.98 (d, J = 9.2 Hz, 2H, triazole-H, Ar-H), 7.47-7.37 (m, 2H, Ar-H), 7.29 (d, J = 7.8 Hz, 1H, Ar-H), 7.26∼7.20 (m, 2H, Ar-H), 7.12 (d, J = 15.9 Hz, 1H, Ar-H), 5.46 (s, 2H, -CH ), 4.69 (m, 1H, -CH), 2.56 (d, J = 7.1 Hz, 2H, -CH ), 1.89 (m, 1H, -CH), 1.05 (d, J = 6.2 Hz, 6H, -CH ), 0.88 (d, J = 6.6 Hz, 6H, -CH ). 13C NMR (101 MHz, DMSO-d 6) δ 152.08, 150.74, 147.39, 144.78, 140.47, 139.53, 135.84, 135.55, 133.45, 130.30, 129.00, 128.97, 128.76, 128.65, 127.62, 70.13, 52.64, 44.31, 29.87, 22.50, 21.83. ESI-MS: [M + H]+: 457.1886 C H N O S require 457.1904.

N-((3'-((1H-1,2,4-triazol-1-yl)methyl)-5-butyl-[1,1′-biphenyl]-2-yl)sulfonyl)pentanamide (8e).

Yield: 153.00 mg (43.29%). m.p. 141.8–142.4°C; 1H NMR (400 MHz, DMSO-d 6) δ 11.44 (s, 1H, -NH), 8.63 (s, 1H, triazole-H), 8.02-7.97 (m, 2H, triazole-H, Ar-H), 7.45 (m, 1H, Ar-H), 7.40 (d, J = 7.6 Hz, 1H, Ar-H), 7.28 (m, 3H, Ar-H), 7.11 (d, J = 1.6 Hz, 1H, Ar-H), 5.46 (s, 2H, -CH ), 2.73-2.64 (m, 2H, -CH ), 1.98 (t, J = 7.4 Hz, 2H, -CH ), 1.64-1.53 (m, 2H, -CH ), 1.38-1.32 (m, 2H, -CH ), 1.30 (m, 2H, -CH ), 1.17 (m, 2H, -CH ), 0.90 (t, J = 7.3 Hz, 3H, -CH ), 0.81 (t, J = 7.3 Hz, 3H, -CH ). 13C NMR (101 MHz, DMSO) δ 171.74, 152.16, 148.64, 144.78, 140.55, 139.68, 136.01, 135.39, 132.77, 130.64, 129.15, 129.02, 128.61, 128.14, 127.66, 52.57, 35.17, 34.82, 33.01, 26.29, 22.28, 21.96, 14.18, 14.08. ESI-MS: [M + H]+: 455.2186 C H N O S require 455.2111.

Butyl((3'-((1H-1,2,4-triazol-1-yl)methyl)-5-butyl-[1,1′-biphenyl]-2-yl)sulfonyl)carbamate (8f).

Yield: 116.00 mg (50.34%). m.p. 150.1-151.4°C; 1H NMR (400 MHz, DMSO-d 6) δ 11.46 (s, 1H, -NH), 8.60 (s, 1H, triazole-H), 8.03∼7.99 (m, 1H, triazole-H), 7.97 (dd, J = 8.3, 1.6 Hz, 1H, Ar-H), 7.46 (d, J = 8.3 Hz, 1H, Ar-H), 7.41 (t, J = 7.5 Hz, 1H, Ar-H), 7.29 (d, J = 7.7 Hz, 1H, Ar-H), 7.23 (d, J = 7.6 Hz, 2H, Ar-H), 7.14 (s, 1H, Ar-H), 5.46 (s, 2H, -CH ), 3.94 (t, J = 6.4 Hz, 2H, -CH ), 2.68 (t, J = 7.6 Hz, 2H, -CH ), 1.59 (m, 2H, -CH ), 1.45-1.37 (m, 2H, -CH ), 1.36-1.28 (m, 2H, -CH ), 1.16 (m, 2H, -CH ), 0.93∼0.87 (m, 3H, CH ), 0.86∼0.76 (m, 3H, -CH ). 13C NMR (101 MHz, DMSO) δ 152.10, 151.36, 148.65, 144.77, 140.65, 139.60, 135.88, 135.39, 132.84, 130.32, 128.95, 128.93, 128.63, 128.17, 127.61, 65.85, 52.62, 34.80, 33.03, 30.50, 22.25, 18.73, 14.18, 13.89. ESI-MS: [M + H]+: 472.2199 C H N O S require 471.2061.

Isobutyl((3'-((1H-1,2,4-triazol-1-yl)methyl)-5-butyl-[1,1′-biphenyl]-2-yl)sulfonyl)carbamate (8g).

Yield: 119.00 mg (55.79%). m.p. 168.4–169.1°C; 1H NMR (300 MHz, Chloroform-d) δ 8.24 (d, J = 8.2 Hz, 1H, Ar-H), 8.16 (s, 1H, triazole-H), 7.95 (s, 1H, -NH), 7.54 (s, 1H, triazole-H), 7.40 (d, J = 7.4 Hz, 2H, Ar-H), 7.33 (d, J = 13.0 Hz, 2H, Ar-H), 7.24 (d, J = 7.5 Hz, 1H, Ar-H), 7.18 (d, J = 1.6 Hz, 1H, Ar-H), 5.30 (s, 2H, -CH ), 3.83 (d, J = 6.5 Hz, 2H, -CH ), 2.81-2.66 (m, 2H, -CH ), 1.82 (m, 1H, -CH), 1.74-1.60 (m, 2H, -CH ), 1.42 (m, 2H, -CH ), 0.98 (t, J = 7.3 Hz, 3H, -CH ), 0.81 (d, J = 6.7 Hz, 6H, -CH ). 13C NMR (75 MHz, Chloroform-d) δ 152.05, 150.86, 149.12, 143.23, 140.14, 139.81, 134.77, 133.66, 132.08, 131.09, 129.97, 129.37, 128.89, 128.02, 127.69, 72.57, 53.41, 35.44, 33.06, 27.61, 22.38, 18.74, 13.89. ESI-MS: [M + H]+: 471.2061 C H N O S require 471.2061.

Isopropyl((3'-((1H-1,2,4-triazol-1-yl)methyl)-5-butyl-[1,1′-biphenyl]-2-yl)sulfonyl)carbamate (8h).

Yield: 149.00 mg (59.51%). m.p. 167.7–168.3°C; 1H NMR (300 MHz, Chloroform-d) δ 8.23 (d, J = 8.2 Hz, 1H, Ar-H), 8.14 (s, 1H, triazole-H), 7.93 (s, 1H, -NH), 7.51 (s, 1H, triazole-H), 7.40 (dd, J = 7.8, 2.6 Hz, 2H, Ar-H), 7.35 (s, 1H, Ar-H), 7.32 (d, J = 4.7 Hz, 1H, Ar-H), 7.23 (d, J = 7.4 Hz, 1H, Ar-H), 7.17 (d, J = 1.5 Hz, 1H, Ar-H), 5.31 (s, 2H, -CH ), 4.86 (m, 1H, -CH), 2.81-2.69 (m, 2H, -CH ), 1.67 (m, 2H, -CH ), 1.48-1.36 (m, 2H, -CH ), 1.15 (d, J = 6.3 Hz, 6H, -CH ), 0.97 (t, J = 7.3 Hz, 3H, -CH ). 13C NMR (75 MHz, Chloroform-d) δ 152.04, 150.28, 149.07, 143.23, 140.16, 139.78, 134.82, 133.71, 132.08, 131.19, 129.89, 129.43, 128.81, 127.92, 127.64, 70.74, 53.38, 35.44, 33.06, 22.37, 21.66, 13.89. ESI-MS: [M + H]+: 457.1910 C H N O S require 457.1904.

N-((3'-((1H-1,2,4-triazol-1-yl)methyl)-5-butoxy-[1,1′-biphenyl]-2-yl)sulfonyl)pentanamide (8i).

Yield: 124.00 mg (50.94%). m.p. 167.4–168.8°C; 1H NMR (300 MHz, Chloroform-d) δ 9.72 (s, 1H, -NH), 8.30 (d, J = 9.0 Hz, 1H, triazole-H), 8.21 (s, 1H, triazole-H), 7.96 (s, 1H, Ar-H), 7.61 (s, 1H, Ar-H), 7.43 (t, J = 7.6 Hz, 1H, Ar-H), 7.34 (d, J = 7.7 Hz, 1H, Ar-H), 7.30 (d, J = 4.3 Hz, 1H, Ar-H), 7.05 (dd, J = 9.0, 2.6 Hz, 1H, Ar-H), 6.84 (d, J = 2.6 Hz, 1H, Ar-H), 5.34 (s, 2H, -CH ), 4.07 (t, J = 6.4 Hz, 2H, -CH ), 2.01 (t, J = 7.4 Hz, 2H, -CH ), 1.83 (m, 2H, -CH ), 1.52 (m, 4H, -CH ), 1.28 (m, 2H, -CH ), 1.02 (t, J = 7.4 Hz, 3H, -CH ), 0.86 (t, J = 7.3 Hz, 3H, -CH ). 13C NMR (75 MHz, Chloroform-d) δ 171.08, 162.61, 152.18, 143.48, 141.91, 139.82, 133.63, 129.43, 128.98, 128.94, 127.92, 118.22, 113.06, 93.76, 68.30, 53.50, 35.51, 31.03, 26.07, 22.11, 19.16, 13.80, 13.74. ESI-MS: [M + H]+: 471.2044 C H N O S require 471.2061.

Butyl((3'-((1H-1,2,4-triazol-1-yl)methyl)-5-butoxy-[1,1′-biphenyl]-2-yl)sulfonyl)carbamate (8j).

Yield: 118.00 mg (51.63%). m.p. 158.9–160.4°C; 1H NMR (300 MHz, Chloroform-d) δ 9.46 (s, 1H, -NH), 8.26 (d, J = 8.9 Hz, 1H, triazole-H), 8.15 (s, 1H, triazole-H), 7.94 (s, 1H, Ar-H), 7.51 (s, 1H, Ar-H), 7.41 (t, J = 7.5 Hz, 1H, Ar-H), 7.34 (m, 1H, Ar-H), 7.31 (s, 1H, Ar-H), 7.04 (dd, J = 9.0, 2.6 Hz, 1H, Ar-H), 6.85 (d, J = 2.6 Hz, 1H, Ar-H), 5.31 (s, 2H, -CH ), 4.07 (td, J = 6.5, 2.6 Hz, 4H, -CH ), 1.89-1.77 (m, 2H, -CH ), 1.53 (m, 4H, -CH ), 1.26 (dd, J = 15.3, 7.6 Hz, 2H, -CH ), 1.02 (t, J = 7.4 Hz, 3H, -CH ), 0.90 (t, J = 7.3 Hz, 3H, -CH ). 13C NMR (75 MHz, Chloroform-d) δ 162.58, 152.04, 151.50, 150.92, 143.23, 142.43, 139.62, 133.67, 129.71, 129.23, 128.81, 127.80, 121.66, 118.11, 112.95, 68.32, 66.40, 53.39, 31.04, 30.49, 19.16, 18.81, 13.80, 13.61. ESI-MS: [M + H]+: 487.1998 C H N O S require 487.2010.

Isobutyl((3'-((1H-1,2,4-triazol-1-yl)methyl)-5-butoxy-[1,1′-biphenyl]-2-yl)sulfonyl)carbamate (8k).

Yield: 109.00 mg (42.38%). m.p. 172.3–173.1°C; 1H NMR (300 MHz, Chloroform-d) δ 9.48 (s, 1H, -NH), 8.27 (d, J = 8.9 Hz, 1H, triazole-H), 8.15 (s, 1H, triazole-H), 7.95 (s, 1H, Ar-H), 7.53 (s, 1H, Ar-H), 7.41 (t, J = 7.5 Hz, 1H, Ar-H), 7.34 (d, J = 7.7 Hz, 1H, Ar-H), 7.31 (s, 1H, Ar-H), 7.04 (dd, J = 9.0, 2.6 Hz, 1H, Ar-H), 6.85 (d, J = 2.5 Hz, 1H, Ar-H), 5.30 (s, 2H, -CH ), 4.07 (t, J = 6.4 Hz, 2H, -CH ), 3.84 (d, J = 6.6 Hz, 2H, -CH ), 1.88-1.77 (m, 3H, -CH , -CH), 1.53 (m, 2H, -CH ), 1.02 (t, J = 7.4 Hz, 3H, -CH ), 0.84 (d, J = 6.7 Hz, 6H, -CH ). 13C NMR (75 MHz, Chloroform-d) δ 162.58, 152.13, 150.93, 143.22, 142.40, 139.63, 133.66, 133.62, 129.81, 129.23, 128.90, 128.84, 127.82, 118.13, 112.97, 72.51, 68.32, 53.39, 31.02, 27.64, 19.15, 18.78, 13.79. ESI-MS: [M + H]+: 487.1995 C H N O S require 487.2010.

Isopropyl((3'-((1H-1,2,4-triazol-1-yl)methyl)-5-butoxy-[1,1′-biphenyl]-2-yl)sulfonyl)carbamate (8l).

Yield: 163.00 mg (61.82%). m.p. 166.7-168.1°C; 1H NMR (300 MHz, Chloroform-d) δ 9.26 (s, 1H, -NH), 8.25 (d, J = 8.9 Hz, 1H, triazole-H), 8.14 (s, 1H, triazole-H), 7.94 (s, 1H, Ar-H), 7.50 (s, 1H, Ar-H), 7.39 (d, J = 7.5 Hz, 1H, Ar-H), 7.33 (d, J = 13.1 Hz, 2H, Ar-H), 7.03 (dd, J = 9.0, 2.5 Hz, 1H, Ar-H), 6.84 (d, J = 2.5 Hz, 1H, Ar-H), 5.30 (s, 2H, -CH ), 4.87 (m, 1H, -CH), 4.07 (t, J = 6.4 Hz, 2H, -CH ), 1.82 (m, 2H, -CH ), 1.53 (m, 2H, -CH ), 1.18 (d, J = 6.2 Hz, 6H, -CH ), 1.01 (t, J = 7.4 Hz, 3H, -CH ). 13C NMR (75 MHz, Chloroform-d) δ 162.53, 152.04, 150.32, 143.20, 142.38, 139.57, 133.68, 129.72, 129.26, 128.88, 128.83, 127.79, 118.09, 116.27, 112.91, 70.70, 68.31, 53.39, 31.03, 21.72, 19.15, 13.80. ESI-MS: [M + H]+: 473.1846 C H N O S require 473.1853.

N-((3'-((1H-pyrazol-1-yl)methyl)-5-isobutyl-[1,1′-biphenyl]-2-yl)sulfonyl)pentanamide (9a).

Yield: 121.00 mg (47.96%). m.p. 125.8-126.7°C; 1H NMR (400 MHz, DMSO-d 6) δ 11.46 (s, 1H, -NH), 8.00 (d, J = 8.2 Hz, 1H, Ar-H), 7.82 (d, J = 2.1 Hz, 1H, pyrazole-H), 7.48 (d, J = 1.5 Hz, 1H, Ar-H), 7.40 (m, 2H, Ar-H), 7.26-7.17 (m, 3H, Ar-H, pyrazole-H), 7.07 (d, J = 1.6 Hz, 1H, Ar-H), 6.28 (t, J = 2.0 Hz, 1H, pyrazole-H), 5.39 (s, 2H, -CH ), 2.55 (d, J = 7.1 Hz, 2H, -CH ), 1.99 (t, J = 7.4 Hz, 2H, -CH ), 1.88 (m, 1H, -CH), 1.39-1.29 (m, 2H, -CH ), 1.17 (m, 2H, -CH ), 0.88 (d, J = 6.6 Hz, 6H, -CH ), 0.81 (t, J = 7.3 Hz, 3H, -CH ). 13C NMR (101 MHz, DMSO-d 6) δ 171.71, 147.38, 140.54, 139.51, 139.43, 137.50, 135.46, 133.37, 130.70, 130.52, 128.75, 128.72, 128.67, 128.48, 127.25, 105.93, 55.08, 44.34, 35.17, 29.84, 26.29, 22.55, 21.95, 14.06. ESI-MS: [M + H]+: 454.2142 C H N O S require 454.2159.

Butyl((3'-((1H-pyrazol-1-yl)methyl)-5-isobutyl-[1,1′-biphenyl]-2-yl)sulfonyl)carbamate (9b).

Yield: 120.00 mg (48.39%). m.p. 129.1-131.4°C; 1H NMR (400 MHz, DMSO-d 6) δ 11.47 (s, 1H, -NH), 7.97 (d, J = 8.2 Hz, 1H, Ar-H), 7.80 (d, J = 1.8 Hz, 1H, pyrazole-H), 7.47 (d, J = 1.6 Hz, 1H, Ar-H), 7.42 (m, 1H, Ar-H), 7.37 (t, J = 7.8 Hz, 1H, Ar-H), 7.22-7.16 (m, 3H, Ar-H, pyrazole-H), 7.09 (d, J = 1.6 Hz, 1H, Ar-H), 6.27 (t, J = 2.0 Hz, 1H, pyrazole-H), 5.38 (s, 2H, -CH ), 3.93 (t, J = 6.5 Hz, 2H, -CH ), 2.56 (d, J = 7.1 Hz, 2H, -CH ), 1.89 (m, 1H, -CH ), 1.39 (m, 2H, -CH ), 1.15 (m, 2H, -CH ), 0.88 (d, J = 6.6 Hz, 6H, -CH ), 0.81 (t, J = 7.4 Hz, 3H, -CH ). 13C NMR (101 MHz, DMSO-d 6) δ 151.35, 147.37, 140.67, 139.45, 139.35, 137.44, 135.50, 133.47, 130.71, 130.64, 130.21, 128.74, 128.56, 128.46, 127.17, 105.91, 65.85, 55.12, 44.34, 30.52, 29.86, 22.52, 18.74, 13.87. ESI-MS: [M + H]+: 470.2107 C H N O S require 470.2108.

Isobutyl((3'-((1H-pyrazol-1-yl)methyl)-5-isobutyl-[1,1′-biphenyl]-2-yl)sulfonyl)carbamate (9c).

Yield: 114.00 mg (45.27%). m.p. 149.6-151.3°C; 1H NMR (400 MHz, DMSO-d 6) δ 11.47 (s, 1H, -NH), 7.98 (d, J = 8.2 Hz, 1H, Ar-H), 7.82-7.77 (m, 1H, pyrazole-H), 7.51-7.45 (m, 1H, Ar-H), 7.44-7.36 (m, 2H, Ar-H), 7.23-7.17 (m, 3H, pyrazole-H, Ar-H), 7.09 (s, 1H, Ar-H), 6.27 (t, J = 2.1 Hz, 1H, pyrazole-H), 5.38 (s, 2H, -CH ), 3.73 (d, J = 6.4 Hz, 2H, -CH ), 2.55 (d, J = 7.1 Hz, 2H, -CH ), 1.88 (m, 1H, -CH), 1.70 (m, 1H, -CH), 0.88 (d, J = 6.5 Hz, 6H, -CH ), 0.73 (d, J = 6.7 Hz, 6H, -CH ). 13C NMR (101 MHz, DMSO-d 6) δ 151.37, 147.35, 140.63, 139.46, 139.36, 137.45, 135.59, 133.50, 130.72, 130.07, 128.79, 128.56, 128.53, 128.49, 127.16, 105.92, 71.92, 55.11, 44.31, 29.86, 27.65, 22.50, 18.96. ESI-MS: [M + H]+: 470.2104 C H N O S require 470.2108.

Isopropyl((3'-((1H-pyrazol-1-yl)methyl)-5-isobutyl-[1,1′-biphenyl]-2-yl)sulfonyl)carbamate (9d).

Yield: 132.00 mg (51.27%). m.p. 146.6-147.5°C; 1H NMR (400 MHz, DMSO-d 6) δ 11.35 (s, 1H, -NH), 7.98 (d, J = 8.2 Hz, 1H, Ar-H), 7.80 (d, J = 2.1 Hz, 1H, pyrazole-H), 7.47 (d, J = 1.4 Hz, 1H, Ar-H), 7.42 (dd, J = 8.3, 1.4 Hz, 1H, Ar-H), 7.37 (t, J = 7.8 Hz, 1H, Ar-H), 7.23-7.16 (m, 3H, pyrazole-H, Ar-H), 7.09 (d, J = 1.5 Hz, 1H, Ar-H), 6.27 (t, J = 2.0 Hz, 1H, pyrazole-H), 5.38 (s, 2H, -CH ), 4.70 (t, J = 6.2 Hz, 1H, -CH), 2.56 (d, J = 7.1 Hz, 2H, -CH ), 1.88 (m, 1H, -CH), 1.06 (d, J = 6.2 Hz, 6H, -CH ), 0.88 (d, J = 6.6 Hz, 6H, -CH ). 13C NMR (101 MHz, DMSO-d 6) δ 150.74, 147.35, 140.63, 139.39, 139.36, 137.42, 135.55, 133.46, 130.73, 130.32, 128.70, 128.60, 128.57, 128.48, 127.18, 105.93, 70.12, 55.13, 44.32, 29.87, 22.50, 21.85. ESI-MS: [M + H]+: 456.1945 C H N O S require 456.1952.

N- ((3'-((1H-pyrazol-1-yl)methyl)-5-butyl-[1,1′-biphenyl]-2-yl)sulfonyl)pentanamide (9e).

Yield: 149.00 mg (56.19%). m.p. 140.1-141.3°C; 1H NMR (300 MHz, DMSO-d 6) δ 11.53 (s, 1H, -NH), 8.02 (d, J = 8.2 Hz, 1H, Ar-H), 7.88-7.84 (m, 1H, pyrazole-H), 7.53-7.49 (m, 1H, Ar-H), 7.47 (dd, J = 8.3, 1.4 Hz, 1H, Ar-H), 7.41 (t, J = 7.9 Hz, 1H, Ar-H), 7.29-7.20 (m, 3H, pyrazole-H, Ar-H), 7.15-7.10 (m, 1H, Ar-H), 6.30 (t, J = 2.0 Hz, 1H, pyrazole-H), 5.42 (s, 2H, -CH ), 2.70 (t, J = 7.7 Hz, 2H, -CH ), 2.02 (t, J = 7.3 Hz, 2H, -CH ), 1.60 (m, 2H, -CH ), 1.37 (m, 2H, -CH ), 1.32 (dd, J = 7.2, 3.3 Hz, 2H, -CH ), 1.18 (m, 2H, -CH ), 0.92 (t, J = 7.3 Hz, 3H, -CH ), 0.83 (t, J = 7.2 Hz, 3H, -CH ). 13C NMR (75 MHz, DMSO-d 6) δ 171.78, 148.63, 140.67, 139.51, 139.46, 137.53, 135.37, 133.46, 132.76, 130.74, 128.74, 128.66, 128.50, 128.10, 127.27, 105.95, 55.06, 35.14, 34.81, 33.03, 26.27, 22.30, 21.98, 14.21, 14.11. ESI-MS: [M + H]+: 454.2144 C H N O S require 454.2159.

Butyl((3'-((1H-pyrazol-1-yl)methyl)-5-butyl-[1,1′-biphenyl]-2-yl)sulfonyl)carbamate (9f).

Yield: 134.00 mg (50.75%). m.p. 142.5-143.1°C; 1H NMR (300 MHz, DMSO-d 6) δ 11.54 (s, 1H, -NH), 7.99 (d, J = 8.2 Hz, 1H, Ar-H), 7.83 (d, J = 2.1 Hz, 1H, pyrazole-H), 7.53-7.44 (m, 2H, Ar-H), 7.43-7.35 (m, 1H, Ar-H), 7.22 (d, J = 8.4 Hz, 3H, pyrazole-H, Ar-H), 7.14 (d, J = 1.3 Hz, 1H, Ar-H), 6.30 (t, J = 2.0 Hz, 1H, pyrazole-H), 5.40 (s, 2H, -CH ), 3.95 (t, J = 6.5 Hz, 2H, -CH ), 2.70 (t, J = 7.6 Hz, 2H, -CH ), 1.60 (m, 2H, -CH ), 1.43 (dd, J = 14.1, 7.4 Hz, 2H, -CH ), 1.37-1.30 (m, 2H, -CH ), 1.17 (m, 2H, -CH ), 0.92 (t, J = 7.3 Hz, 3H, -CH ), 0.83 (t, J = 7.3 Hz, 3H, -CH ). 13C NMR (75 MHz, DMSO-d 6) δ 151.59, 148.58, 147.22, 140.78, 139.48, 139.39, 137.45, 135.46, 132.85, 130.76, 130.37, 130.14, 128.92, 128.55, 127.17, 105.94, 65.81, 55.09, 34.80, 33.07, 30.52, 22.26, 18.75, 14.21, 13.93. ESI-MS: [M + H]+: 470.2103 C H N O S require 470.2108.

Isobutyl((3'-((1H-pyrazol-1-yl)methyl)-5-butyl-[1,1′-biphenyl]-2-yl)sulfonyl)carbamate (9g).`

Yield: 107.00 mg (39.27%). m.p. 149.3-151.2°C; 1H NMR(400 MHz, DMSO-d 6) δ 11.47 (s, 1H, -NH), 7.97 (d, J = 8.2 Hz, 1H, Ar-H), 7.84-7.76 (m, 1H, pyrazole-H), 7.45 (d, J = 11.4 Hz, 2H, Ar-H), 7.41-7.31 (m, 1H, Ar-H), 7.20 (d, J = 6.9 Hz, 3H, Ar-H, pyrazole-H), 7.11 (d, J = 9.5 Hz, 1H, Ar-H), 6.27 (s, 1H, pyrazole-H), 5.38 (s, 2H, -CH ), 3.73 (d, J = 6.4 Hz, 2H, CH ), 2.67 (t, J = 7.5 Hz, 2H, -CH ), 1.71 (m, 1H, -CH), 1.58 (m, 2H, -CH ), 1.33 (dd, J = 14.6, 7.2 Hz, 2H, -CH ), 0.89 (t, J = 7.3 Hz, 3H, -CH ), 0.74 (d, J = 6.7 Hz, 6H, -CH ). 13C NMR (101 MHz, DMSO-d 6) δ 151.35, 148.62, 140.80, 139.45, 139.36, 137.42, 135.44, 132.87, 130.72, 130.26, 128.57, 128.51, 128.49, 128.15, 127.18, 105.92, 71.93, 55.11, 34.78, 33.02, 27.65, 22.19, 18.94, 14.17. ESI-MS: [M + H]+: 470.2093 C H N O S require 470.2108.

Isopropyl((3'-((1H-pyrazol-1-yl)methyl)-5-butyl-[1,1′-biphenyl]-2-yl)sulfonyl)carbamate (9h).

Yield: 114.00 mg (45.19%). m.p. 169.8-171.3°C; 1H NMR(400 MHz, DMSO-d 6) δ 11.36 (s, 1H, -NH), 7.97 (d, J = 8.2 Hz, 1H, Ar-H), 7.80 (d, J = 2.1 Hz, 1H, pyrazole-H), 7.49-7.43 (m, 2H, Ar-H), 7.40-7.34 (m, 1H, Ar-H), 7.23-7.16 (m, 3H, Ar-H, pyrazole-H), 7.12 (d, J = 1.6 Hz, 1H, Ar-H), 6.27 (t, J = 2.0 Hz, 1H, pyrazole-H), 5.38 (s, 2H, -CH ), 4.70 (m, 1H, -CH), 2.73-2.64 (m, 2H, -CH ), 1.58 (m, 2H, -CH ), 1.37-1.29 (m, 2H, -CH ), 1.06 (d, J = 6.3 Hz, 6H, -CH ), 0.89 (t, J = 7.3 Hz, 3H, -CH ). 13C NMR (101 MHz, DMSO-d 6) δ 150.77, 148.60, 140.78, 139.39, 139.36, 137.39, 135.45, 132.83, 130.72, 130.46, 128.62, 128.56, 128.48, 128.06, 127.19, 105.93, 70.11, 55.13, 34.78, 33.02, 22.19, 21.86, 14.16. ESI-MS: [M + H]+: 456.1937 C H N O S require 456.1952.

5.2 Radioligand binding assay

Rat liver membranes were prepared according to the method of Dudley et al (Dudley et al., 1990). After HEK-293 cells were transfected with the AT2 receptor, a lysis buffer was used to separate the cell membrane of HEK-293 cells (Grieger et al., 2016), using 27-G Resuspend. The lysis solution was mixed and centrifuged at 12,000 g for 10 min at 4°C. Details are shown in the Supplementary Material.

5.3 In vitro morphological effects studies

NG108-15 cells (China Center for Type Culture Collection CCTCC) were used to study the in vitro morphological effects. In their undifferentiated state, neuroblastoma × glioma hybrid NG108-15 cells havea rounded shape and divide actively. The cells were cultured from passage 18–25 in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Thermo Fisher Technology (China) Co., Ltd, China) with 10% fetal bovine serum (FBS, Gibco), HAT supplement and 50 mg L−1 gentamycin at 37°C in 75 cm2 Nunclon Delta flasks in a humidified atmosphere of 95% air and 5% CO2, as previously described (Buisson et al., 1992). Subcultures were performed at subconfluency. Under these conditions, cells express mainly the AT2 receptor subtype (Whitebread et al., 1991). Cells were treated during 3 days, once a day (first treatment 24 h after plating), and micrographs were taken on the fourth day. The details of experimental grouping and administration method are shown in the Supplementary Material.

5.4 Molecular docking simulation

The AT2 receptor protein (PDB ID: 5UNF) was obtained from the RSCB protein bank database, the water molecules of theprotein were removed, the endogenous ligands were extracted, the C and D chains were selected, and a series of treatments such as energy optimization, hydrogenation, and side chain repair were carried out on the protein. Using SYBYL-2.1.1, the 2D structure of docking ligand is drawn by software and the energy is optimized. Using the docking suite module in SYBYL, the active pocket is automatically generated based on the endogenous ligand. The processed protein and ligand molecules are semi-flexibly docked in SFXC mode, and the endogenous ligand 8 ES is used as the template molecule to evaluate the similarity between the docked ligand and the endogenous ligand. Combined with the scoring functions, such as Crash, Ploar, and Similarity, the docking results are comprehensively analyzed to obtain the total score and the conformation of the compound with the highest score is taken as the docking result. Finally, the analysis results module in SYBYL is used to judge the action mode of compounds and receptors, and the sequence viewer module is used to determine the key amino acid residues.

5.5 Statistical analysis

Biological results are reported as means ± SE. Statistical analysis was performed by using one-way analysis of variance. p value of less than 0.05 was considered to be statistically significant.