Design, synthesis and evaluation of benzenesulfonamide-substituted 1,5-diarylpyrazoles containing phenylacetohydrazide derivatives as COX-1/COX-2 agents against solid tumors

Abstract

Novel benzenesulfonamide-substituted 1,5-diarylpyrazoles containing phenylacetohydrazide derivatives have been designed, synthesized and evaluated for their biological activities as selective COX-2 inhibitors with anticancer potential. In vitro the bioassay results revealed that some of them displayed potent inhibitory activity in the enzymatic and cellular assays. Among them, compound 48 showed the most powerful and potent selective inhibitory activity (IC₅₀ = 82.21 μM for COX-1 and IC₅₀ = 0.37 μM for COX-2), comparable to the control positive compound Celecoxib (40.29 μM, 0.15 μM). Antiproliferative assay results indicated that compound 48 possess potent antiproliferative activity against A549 cells in vitro with an IC₅₀ value of 0.78 μM. We then performed a PI staining assay and cell apoptosis analysis for compound 48 and found that it effectively causes A549 cell apoptosis. A docking simulation was further performed to position compound 48 into the COX-2 active site to determine the probable binding model. The 3D-QSAR models were built for reasonable design of selective COX-2 inhibitors in the future.

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1 Introduction

The cyclooxygenase which catalyzes the conversion of arachidonic acid to prostaglandins (PGs) exists in two isoforms: the constitutive COX-1 and the inducible COX-2, and has led to new targets for the design of mechanism based drugs in cancer chemoprevention research.¹ The two isoforms differ in regulation and expression, depending on the tissue, which lead to the formation of various eicosanoids involved in several disease states,² such as inflammation,³ fever,⁴ arthritis,⁵ and more recently, cancer.⁶ COX-1 has been known as a housekeeping enzyme constitutively expressed in most mammalian tissues and responsible for the production of cytoprotective prostaglandins (PGs) in the gastrointestinal (GI) tract; whereas COX-2 can be rapidly expressed during inflammatory conditions by pro-inflammatory stimuli in macrophages and other cells.^7,8 The implications of COXs have been researched in the treatment of a variety of disease states. For example, the treatment of Alzheimer's disease (AD), Parkinson's disease, inflammatory diseases and cancers, so inhibiting these enzymes seems to be even more advantageous in various cardiovascular diseases and cancer therapy.^9,10 Nevertheless, interruption of COX-1 activity may lead to gastrointestinal toxicity such as ulceration, bleeding, and perforation. Selective inhibition of COX-2 might be endowed with improved blocking biosynthesis of PGs and reduced gastrointestinal toxicity profiles.¹¹ Numerous studies have demonstrated that overexpression of COX-2 is sufficient to cause tumorigenesis in animal models.^12–14 Inhibition of the COX-2/PGE2 pathway results in reduction in tumorigenesis via a number of distinct mechanisms: promoting tumour maintenance and progression, encouraging metastatic spread, and perhaps even participating in tumour initiation.^15,16 Epidemiological, clinical, and preclinical investigations also provide compelling evidence that COXs (either COX-2 or both COX-1 and COX-2) inhibitors could act as chemopreventive agents, for example, Celecoxib as well as other coxibs such as Valdecoxib could induce cancer cell growth inhibition and apoptosis.¹⁷ Those COXs inhibitors may hinder biosynthesis of prostaglandins and other pathways related to tumorigenesis and progression.^18,19

Diarylheterocycles is the most widely explored pharmacophore for the development of selective COX-2 inhibitors (Fig. 1), such as Celecoxib (1), Rofecoxib (2), Valdecoxib (3), Etoricoxib (4).²⁰ Extensive structure–activity relationship (SAR) studies for the diarylheterocycle class have shown that a SO₂NH₂ or SO₂Me substituent at the para-position of one of the aryl rings is a requirement for optimum COX-2 selectivity and potency.^9,21 Furthermore, sulfonamides possess many types of biological activities and representatives of this class of pharmacological agents are widely used in clinic as antibacterial,²² anti-carbonic anhydrase,²³ antithyroid²⁴ and antitumor,²⁵ devoid of the side effects of the presently available pharmacological agents.

Fig. 1 Selective COX-2 inhibitors.

Celecoxib belongs to the 1,5-diarylpyrazole class of compounds and a recent study revealed that this class of compounds possesses promising anticancer properties,^26,27 Accordingly, in pursuance of our synthesis of various 1,5-diarylpyrazoles heterocycles, we preferred to possess a diarylheterocyclic ring template with a central five-membered pyrazole ring over others for the development of novel class of COX-2 inhibitors. Acylhydrazones had been selected as pharmacological agents due to their wide-ranging pharmacological properties and their potential applications of anticancer.²⁸ The acylhydrazones group attached to 1,5-diarylpyrazoles as a pharmacophore to explore the possibility of better antitumor potency of COX-2 inhibitors (Fig. 1).

In a preceding work, we have reported the decent inhibitory activity against COX-1/COX-2 of a series of dihydropyrazole sulfonamide derivatives as a part of our studies on the design and synthesis of inhibitors of COXs.²⁹ Here we extend the earlier work of dihydropyrazole incorporating sulfonamide moieties, and now describing the structure-based design and synthesis of novel and potent inhibitors of COX-2 utilizing a 1,5-diarylpyrazoles, benzenesulfonamide and phenylacetohydrazide as new structural classes.

Thus, on the basis of this finding, we designed 44 novel benzenesulfonamide-substituted 1,5-diarylpyrazoles containing phenylacetohydrazide derivatives (21–64) that showed significant selective COX-2 inhibition as a source for new effective and safe antitumor drugs.

2 Results and discussion

2.1 Chemistry

The routes to synthesis the benzenesulfonamide-substituted 1,5-diarylpyrazoles containing phenylacetohydrazide derivatives followed the general pathway outlined in Scheme 1. The starting diverse substituted chalcones (6–10) were synthesized by treating dimethyl oxalate and the substituted acetophenone in the presence of excess sodium methoxide as catalyst in methanol. Then the reaction of cyclization of different chalcones with 4-hydrazinylbenzenesulfonamide in refluxing ethanol, afforded the formation of benzenesulfonamide-substituted 1,5-diarylpyrazoles containing 3-carboxylate skeleton (11–15). Compounds 11–15 and the 85% hydrazine monohydrate were dissolved in anhydrous ethanol and vigorously stirred at 80 °C under oil bath for 7–8 h to obtain compounds 16–20. Finally, Target compounds 21–64 were obtained from compounds 16–20 after reactions with various benzaldehyde in 70–90% yield. All the compounds are reported for the first time. All of the target compounds gave satisfactory analytical and spectroscopic data ¹H NMR, ¹³C NMR, ESI-MS, which are in accordance with their depicted structures. Furthermore, the crystal structures of compounds 22 (CCDC: 1434485) and 40 (CCDC: 1434486) were determined by single crystal X-ray diffraction analysis in Fig. 2 and Table 1.

Scheme 1 General synthesis of compounds (21–64). Reagents and conditions: (i) 2.0 equiv. dimethyl oxalate, MeOH, MeONa, reflux, 6 h; (ii) 1.0 equiv. 4-hydrazinylbenzenesulfonamide, MeOH, reflux, 6 h; (iii) 10.0 equiv hydrazine hydrate, EtOH, reflux, 8 h; (iv) 1.5 equiv. benzaldehyde, EtOH, AcOH, rt, 12 h.

Fig. 2 Crystal structure diagrams of compounds 22 and 40.

Table 1 Crystal data for compounds 22 and 40

Compounds	22	40
Empirical formula	C25H23N5O5S	C24H20BrN5O3S
Formula weight	505.55	538.42
Temperature (K)	273(2)	273(2)
Crystal system	Triclinic	Triclinic
Space group	P1̄	P1̄
a (Å)	10.9102(19)	10.683(2)
b (Å)	11.1286(19)	10.986(2)
c (Å)	16.302(2)	16.0730(19)
α (°)	76.597(3)	91.984(3)
β (°)	87.151(3)	100.304(3)
γ (°)	60.847(3)	117.555(3)
V (Å)	1676.8(5)	1630.4(5)
Z	2	2
Dcalcd/g cm^−3	1.311	1.403
θ range (deg)	25.16	2.32–25.08
F(000)	696	704
Reflections collected	14 307(Rint = 0.0835)	14 915(Rint = 0.0425)
Data/restraints/parameters	5816/38/428	5720/11/401
Absorption coefficient (mm^−1)	0.272	1.496
R1; wR2 [I > 2σ(I)]	0.0884/0.1939	0.0580/0.1405
R1; wR2 (all data)	0.2136/0.2564	0.0991/0.1643
GOF	1.000	1.013
Larg.peak/hole (e Å)	0.593/−0.437	0.887/−0.591

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2.2 Biological activity

2.2.1 Antiproliferation inhibition assay. All synthesized derivatives (21–64) were evaluated for their in vitro antiproliferation activities against four cancer cell lines, which were HepG2, A549, HeLa, F10, with the positive contrast drugs Celecoxib. The outcomes were summarized up in Tables 2 and 3. As shown in Tables 2 and 3, the results revealed indicated that most of the target compounds can effectively inhibit the proliferation of the four tumor cells. Especially on A549 cell antiproliferative activities. The IC₅₀ values of compounds 21–64 against A549 cell lines were between 0.78 and 35.34 μM and GI₅₀ values were range from 0.32 to 21.55 μM. Among them, compound 48 displayed the most potent anti-tumor activity in A549 with IC₅₀ of 0.78 μM and GI₅₀ of 0.32 μM_, compared to the positive control Celecoxib (IC₅₀ = 16.22 μM, GI₅₀ = 10.56 μM). More potent anti-tumor activity for A549 together with virtual screening results both indicated that the COX-2 might be a potential target which these benzenesulfonamide-substituted 1,5-diarylpyrazoles containing phenylacetohydrazide derivatives interacted with.

Table 2 In vitro antiproliferation activities (IC₅₀, μM^a) of target compounds (21–64)

Compounds	R1	R2	R3	R4	R5	IC50^a (μM)
						A549^b	HepG2^b	HeLa^b	F10^b
a Antiproliferation activity was measured using the MTT assay. Values are the average of three independent experiments run in triplicate. Variation was generally 5–10%.b Cancer cells kindly supplied by State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University.c Errors were in the range of 5–10% of the reported values, from three different assays.
21	OCH3	H	H	H	H	30.79	15.87	64.20	84.79
22	OCH3	H	H	OCH3	H	32.12	34.98	47.79	10.49
23	OCH3	OCH3	H	H	H	18.52	23.19	32.13	23.08
24	OCH3	H	OCH3	H	H	24.47	4.39	33.47	44.34
25	CH3	H	H	OCH3	H	13.30	18.46	49.58	170.68
26	F	H	H	OCH3	H	19.75	35.91	46.85	24.21
27	Br	H	H	OCH3	H	35.34	14.64	38.21	17.64
28	OCH3	OCH3	H	OCH3	OCH3	24.79	42.57	37.92	26.18
29	OCH3	H	OCH3	OCH3	OCH3	21.30	34.54	46.35	47.32
30	CH3	OCH3	H	OCH3	OCH3	23.79	35.57	50.54	55.37
31	CH3	H	OCH3	OCH3	OCH3	16.23	26.54	52.31	60.14
32	F	OCH3	H	OCH3	OCH3	9.05	13.01	44.19	5.44
33	F	H	OCH3	OCH3	OCH3	2.92	19.23	43.55	8.09
34	Br	OCH3	H	OCH3	OCH3	35.22	20.02	61.31	38.48
35	Br	H	OCH3	OCH3	OCH3	33.60	21.86	75.74	66.41
36	H	OCH3	H	OCH3	OCH3	20.81	32.52	32.61	66.54
37	OCH3	H	H	CH3	H	30.33	17.99	50.39	10.12
38	CH3	H	H	CH3	H	5.85	11.94	30.51	26.33
39	F	H	H	CH3	H	4.41	17.62	28.23	89.11
40	Br	H	H	CH3	H	21.66	4.49	9.81	8.11
41	OCH3	H	H	OH	H	26.32	40.68	62.08	18.69
42	OCH3	OH	H	H	H	3.13	7.53	34.43	81.96
43	OCH3	H	OH	H	H	19.36	44.68	52.20	28.76
44	CH3	H	H	OH	H	34.60	27.39	77.54	63.46
45	CH3	OH	H	H	H	1.87	4.44	29.78	62.69
46	F	OH	H	H	H	0.86	30.78	34.10	47.74
47	Br	H	H	OH	H	14.47	28.65	72.88	87.49
48	Br	OH	H	H	H	0.78	14.71	6.60	14.48
49	OCH3	H	H	F	H	13.69	16.20	31.02	26.06
50	CH3	H	H	F	H	5.82	4.63	21.20	14.57
51	Br	H	H	F	H	8.51	6.06	27.44	16.91
52	OCH3	H	H	NO2	H	10.86	11.61	75.21	85.21
53	OCH3	NO2	H	H	H	8.65	10.54	20.71	108.50
54	CH3	H	H	NO2	H	3.13	4.49	23.29	23.15
55	F	H	H	NO2	H	3.46	23.03	46.97	76.15
56	Br	H	H	NO2	H	3.35	4.92	7.99	22.97
57	OCH3	H	H	Br	H	16.91	3.88	50.65	116.13
58	CH3	H	H	Br	H	3.11	5.90	25.26	3.46
59	F	H	H	Br	H	1.99	20.81	41.97	66.74
60	Br	H	H	Br	H	1.47	15.54	12.80	12.67
61	H	H	H	Br	H	7.71	32.58	54.15	74.26
62	CH3	H	H	Cl	H	5.69	4.44	20.93	6.59
63	F	H	H	Cl	H	7.75	8.74	31.91	23.25
64	Br	H	H	Cl	H	4.01	17.39	3.57	9.66
Celecoxib^c						16.22	27.03	20.79	26.65

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Table 3 In vitro growth inhibition (GI₅₀, μM^a) of target compounds (21–64)

Compounds	GI50^a (μM)				Compounds	GI50^a (μM)
	A549	HepG2	HeLa	F10		A549	HepG2	HeLa	F10
a Growth inhibition was measured using the MTT assay. Values are the average of three independent experiments run in triplicate. Variation was generally 5–10%.
21	21.55	9.36	37.87	58.22	44	19.60	12.87	24.81	37.57
22	15.09	24.13	34.89	4.93	45	0.88	2.62	21.74	36.98
23	10.18	13.68	23.45	16.38	46	0.47	22.47	20.12	28.16
24	11.50	3.11	23.09	26.16	47	10.27	13.46	40.08	48.12
25	6.25	10.15	29.25	121.18	48	0.32	6.91	3.10	6.81
26	10.86	24.77	30.45	11.38	49	8.07	8.91	17.06	12.25
27	19.43	8.63	21.01	10.41	50	4.13	2.73	12.51	8.01
28	17.60	23.41	17.82	15.44	51	5.02	4.18	12.89	9.30
29	12.56	16.24	21.78	27.92	52	5.97	5.45	35.34	17.89
30	9.51	14.22	19.71	42.87	53	2.68	2.95	11.31	92.33
31	6.47	16.39	48.61	46.57	54	0.96	3.68	15.76	14.68
32	4.97	9.23	31.37	3.21	55	1.79	16.35	25.83	14.46
33	2.02	11.15	30.04	5.09	56	1.57	2.63	5.76	12.63
34	19.37	11.81	42.30	22.70	57	7.94	1.82	27.86	87.26
35	18.48	15.08	35.59	13.94	58	1.71	2.30	13.89	1.69
36	12.27	23.09	17.93	20.62	59	1.07	11.44	24.76	56.92
37	16.68	10.61	30.23	2.12	60	1.25	8.08	4.29	7.47
38	1.81	6.56	21.05	18.16	61	4.24	19.47	48.31	56.98
39	2.60	10.39	20.04	49.01	62	3.27	1.55	9.84	2.41
40	12.78	2.65	5.39	4.78	63	4.57	6.20	18.82	10.93
41	14.47	28.87	42.83	10.28	64	1.26	6.26	2.64	6.84
42	1.09	4.44	18.93	38.52	Celecoxib	10.56	23.24	13.10	18.26
43	9.09	30.83	28.71	16.97

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2.2.2 Enzyme inhibition assay. Compounds 21–64 were evaluated for their ability to inhibit COX-1 and COX-2 using human COX-1/COX-2 ELISA Kit and results obtained are presented in Table 3. The results were compared with Celecoxib under identical conditions, which showed that the majority of the synthesized compounds exhibited excellent inhibiting COX-2 activities displaying IC₅₀ values between 0.37 and 23.88 μM, but weak to COX-1.

The following structure–activity relationship (SAR) studies were performed by modification of the parent compound to determine how the substituents of the subunits affect the COX-2 inhibitory activities. Inspection of the chemical structures of compounds 21–64 suggested that they could be divided into two subunits: A and B rings (Scheme 1). As showed in Table 4, compound 48 was the most active and potent selectivity with IC₅₀ value of 0.37 μM, 82.21 μM which had basically similar cyclooxygenase inhibition capability with the positive control drug Celecoxib with IC₅₀ value of 0.15 μM, 40.29 μM. We found that OH at position of B rings substituent –R² (ortho-position) possess potent anti-cancer effect and favorable inhibition of COX-2, when the substituent –R¹ is kind of halogen groups (Br, F), the compounds gained better activity than CH₃, OCH₃, as shown in inhibition of COX-2: 48 (IC₅₀ = 0.37 μM) > 46 (IC₅₀ = 0.40 μM) > 42 (IC₅₀ = 0.56 μM) > 45 (IC₅₀ = 1.29 μM) and in A549 cell antiproliferative activities: 48 (IC₅₀ = 0.78 μM) > 46 (IC₅₀ = 0.86 μM) > 45 (IC₅₀ = 1.87 μM) > 42 (IC₅₀ = 3.13 μM). Based on the obtained data, we also simplified modifying merely substituents on the para-position of A rings and B rings. When substituents B-ring kept immutability, the changes with different substituents –R¹ for COX-2 inhibition activity at para-position of A-ring showed electron-withdrawing groups are preferable than electron-donating groups. For instance, the active gradients for compounds were 26 (IC₅₀ = 8.39 μM) > 27 (IC₅₀ = 9.63 μM) > 22 (IC₅₀ = 14.79 μM), 25 (IC₅₀ = 16.21 μM); 55 (IC₅₀ = 1.17 μM) > 56 (IC₅₀ = 1.62 μM) > 54 (IC₅₀ = 17.46 μM). In addition, we studied the same A ring and different substituents of B ring, it can be deduced that the substituent on the para-position of the A rings is kind of potent electron-withdrawing groups rather than electron-donating groups, the compounds gained better activity, as shown in inhibition of COX-2: 56 (IC₅₀ = 1.62 μM) > 51 (IC₅₀ = 3.20 μM) > 64 (IC₅₀ = 4.58 μM) > 60 (IC₅₀ = 6.33 μM) > 47 (IC₅₀ = 7.30 μM) > 27 (IC₅₀ = 9.63 μM) > 40 (IC₅₀ = 16.10 μM); 55 (IC₅₀ = 1.17 μM) > 63 (IC₅₀ = 3.78 μM) > 59 (IC₅₀ = 5.21 μM) > 26 (IC₅₀ = 8.39 μM) > 39 (IC₅₀ = 16.72 μM); 52 (IC₅₀ = 3.63 μM) > 49 (IC₅₀ = 8.16 μM) > 57 (IC₅₀ = 10.54 μM) > 22 (IC₅₀ = 14.79 μM) > 21 (IC₅₀ = 16.41 μM) > 37 (IC₅₀ = 17.22 μM); and the inhibitory activities increased in the following order: NO₂ > F > Cl > Br > OCH₃ > CH₃.

Table 4 Inhibition activities of compounds (21–64) against COX-1/COX-2

Compounds	IC50,^a μM		COX% inhibition^a at 20 μM
	COX-1	COX-2	COX-1	COX-2
a COX-1/COX-2 inhibitory activity was measured using the human COX-1/COX-2 assay kit. Values are the average of six independent experiments run in triplicate. Variation was generally 5–10%.
21	>100	16.41	18.13	55.25
22	79.22	14.79	23.29	58.11
23	>100	20.10	13.08	49.37
24	40.21	13.52	37.51	59.73
25	44.11	16.21	34.19	58.47
26	>100	8.39	10.11	66.02
27	69.26	9.63	30.24	63.33
28	43.08	9.69	34.16	63.49
29	44.09	19.40	29.24	52.57
30	40.37	16.99	35.44	57.15
31	58.19	14.24	31.73	59.11
32	>100	12.45	13.29	60.21
33	>100	9.63	15.16	67.44
34	39.06	10.35	34.44	66.43
35	27.41	15.34	40.29	59.25
36	>100	9.08	24.76	65.89
37	51.26	17.22	30.21	56.32
38	70.13	11.31	28.24	60.18
39	>100	16.72	16.19	58.35
40	>100	16.10	9.20	58.79
41	53.12	9.22	31.10	61.37
42	>100	0.56	10.42	82.91
43	86.42	10.54	21.75	60.84
44	30.97	23.88	40.99	48.17
45	>100	1.29	14.36	79.61
46	70.64	0.40	28.31	84.24
47	>100	7.30	11.19	64.98
48	82.21	0.37	20.12	86.80
49	64.18	8.16	31.26	63.14
50	>100	7.56	12.05	65.08
51	70.66	3.20	27.56	73.11
52	52.14	3.63	31.09	67.33
53	19.01	12.32	51.04	57.82
54	27.72	17.46	42.19	53.41
55	39.81	1.17	39.25	81.13
56	>100	1.62	9.64	79.05
57	59.09	10.54	32.78	58.94
58	86.26	4.65	22.29	76.29
59	68.35	5.21	27.46	76.31
60	>100	6.33	8.37	70.66
61	59.06	14.21	29.85	56.69
62	31.20	19.47	40.11	53.10
63	>100	3.78	9.20	76.04
64	66.27	4.58	29.11	66.19
Celecoxib	40.29	0.15	36.15	87.13

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From the above mentioned analysis, it could be indicated that hydroxy at ortho-position of B ring play an important role in the COX-2 inhibitory activity which conformed to the docking of the hydroxy in compound 48 bonding to COX-2 through two hydrogen bonds. Moreover, modifying substituents on the para-position of A rings and B rings revealed that bonds A ring and B ring in the benzenesulfonamide-substituted 1,5-diarylpyrazoles containing phenylaceto-hydrazide derivatives also crucial of the COXs inhibitory activity.

In comparison, we found that these compounds with OH at position of B rings substituent –R² (ortho-position) possess remarkable inhibitory activity, with electron-withdrawing groups group on the para-position of the A-ring and B-ring (such as NO₂, F), exhibited more potent anticancer activities than those have electron-donating substituents (such as OCH_3, CH₃). From the above-mentioned analysis, it could be concluded that the compounds with nitro and fluorine substituted para-position of A-ring and B-ring were found to be the most favorable for the anticancer activity.

2.2.3 Apoptosis assay. Subsequently, we investigated the effect of compound 48 on cell apoptosis. We treated A549 cells with various concentrations of compound 48 and for 48 h and analyzed cells for changes in apoptotic markers with a flow cytometer in vitro, as shown in Fig. 3, the percentage of apoptotic cell significantly increased after treatment with high doses of compound 48. The percentages of cell apoptosis 6.55%, 12.95%, 16.23%, 27.70%, 61.46% were responding to the concentration of compound 48 0, 2, 4, 8, 16 μM, respectively. We also measured the effect of other five compounds (compounds 26, 40, 44, 45, 56) causing A549 cells apoptosis and results displayed in Fig. 4, compound 26 inducing the percentage of cell apoptosis 5.67%, 10.55%, 14.33%, 23.77%, 38.2% was corresponding to the concentration 0, 2, 4, 8, 16 μM. Compound 40 (1.58%, 3.76%, 6.94%, 10.04%, 18.14%), compound 44 (5.67%, 11.92%, 16.17%, 21.29%, 25.34%), compound 45 (5.67%, 8.94%, 15.12%, 25.23%, 47.8%), compound 56 (5.67%, 12.15%, 17.41%, 31.5%, 55.87%) separately inducing the cells apoptosis in identical concentration gradient. On the basis of these data, these compounds which possess high-efficiency COX-2 inhibition activity also causing high percentages of cell apoptosis. IC₅₀ of inducing A549 cells apoptosis were obtained for the six compounds on the Y axis versus the corresponding IC₅₀ of COX-2 inhibition on the X axis to create liner regression model: compound 26 (IC₅₀ of COX-2 inhibition 8.39 μM, IC₅₀ of inducing A549 cells apoptosis 30.07 μM); compound 40 (16.10 μM, 104.45 μM); compound 44 (23.88 μM, 174.45 μM); compound 45 (1.29 μM, 18.65 μM); compound 48 (0.37 μM, 13.24 μM); compound 56 (1.62 μM, 14.22 μM). As displayed in Fig. 5, the correlation coefficient R² between six compounds respective IC₅₀ of COX-2 inhibition and IC₅₀ of inducing A549 cells apoptosis was found to be 0.948, which proved that cell apoptosis was closely associated with COX-2 inhibition activity.

Fig. 3 Compound 48 induced apoptosis in A549 cells with the density of 0, 2, 4, 8, 16 μM. A549 cells were treated with for 48 h. Values represent the mean ± S.D, n = 3, P < 0.05 versus control. The percentage of cells in each part was indicated.

Fig. 4 Compounds 26, 40, 44, 45, 56 inducing the percentage of cell apoptosis with the density of 0, 2, 4, 8, 16 μM, A549 cells were treated with for 48 h. The percentages of cell apoptosis of compound 26 (5.67%, 10.55%, 14.33%, 23.77%, 38.2%), compound 40 (1.58%, 3.76%, 6.94%, 10.04%, 18.14%), compound 44 (5.67%, 11.92%, 16.17%, 21.29%, 25.34%), compound 45 (5.67%, 8.94%, 15.12%, 25.23%, 47.8%), compound 56 (5.67%, 12.15%, 17.41%, 31.5%, 55.87%) in identical concentration gradient. Values represent the mean ± S.D, n = 3, P < 0.05 versus control.

Fig. 5 Plot of IC₅₀ for COX-2 inhibition versus IC₅₀ of inducing A549 cells apoptosis for the six compounds: compound 26 (8.39 μM, 30.07 μM), compound 40 (16.10 μM, 104.45 μM), compound 44 (23.88 μM, 174.45 μM), compound 45 (1.29 μM, 18.65 μM), compound 48 (0.37 μM, 13.24 μM), compound 56 (1.62 μM, 14.22 μM). The correlation coefficient R² between six compounds respective IC₅₀ of COX-2 inhibition and IC₅₀ of inducing A549 cells apoptosis was found to be 0.948, which proved that cell apoptosis was closely associated with COX-2 inhibition activity.

2.2.4 Cytotoxicity test. All the target compounds were evaluated for their toxicity against human kidney epithelial cell 293T and mouse primary hepatocytes with the median cytotoxic concentration (CC₅₀) data of tested compounds by the MTT assay. As shown in Table 5, 44 compounds with the minimum CC₅₀ of 293T cells and mouse primary hepatocytes were 24.81 μM and 43.29 μM that demonstrated almost no cytotoxic activities in vitro against human kidney epithelial cell 293T and mouse primary hepatocytes.

Table 5 The median cytotoxic concentration (CC₅₀) data of all compounds against 293T cell and mouse primary hepatocytes

Compounds	CC50^a (μM)		Compounds	CC50^a (μM)
	293T	Primary hepatocytes		293T	Primary hepatocytes
a The cytotoxicity of each compound was expressed as the concentration of compound that reduced cell viability to 50% (CC50).
21	100.32	84.42	44	>300	197.37
22	57.76	100.26	45	118.45	124.05
23	133.76	89.27	46	83.53	64.30
24	42.22	61.17	47	156.32	>300
25	232.64	>300	48	27.58	46.72
26	126.97	114.86	49	160.38	206.41
27	196.63	86.27	50	56.49	71.27
28	65.63	87.89	51	94.53	67.48
29	131.64	176.53	52	30.77	43.29
30	102.86	>300	53	24.81	68.15
31	31.73	59.30	54	31.85	125.32
32	>300	>300	55	>300	>300
33	>300	69.21	56	85.35	64.24
34	106.15	66.47	57	175.90	>300
35	89.68	104.39	58	42.36	46.92
36	261.53	>300	59	179.62	106.54
37	>300	>300	60	47.06	61.29
38	>300	129.33	61	198.54	>300
39	112.14	69.28	62	63.85	71.57
40	125.42	44.51	63	214.43	>300
41	133.21	85.84	64	168.57	95.53
42	42.89	51.26	Celecoxib	93.43	87.33
43	189.42	>300

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2.3 Molecular docking

To better understand the potency of compound 48 and further guide structure–activity relationship (SAR) studies, we examined the interaction of compound 48 and Celecoxib with COX-2 (PDB code: 3PGH). All amino acid residues of COX-2 that interacted with compound 48 and Celecoxib were exhibited in pictures Fig. 6–9. The estimated interaction energies of synthesized compounds were ranging from −41.78 to −56.84 kcal mol⁻¹, as displayed in Fig. 6 and 7. In the binding mode, Celecoxib binds to COX-2 with binding free energy of −49.72 kcal mol⁻¹ via two hydrogen bonds with TRP387, two Pi bonds and a Sigma-Pi bond with HIS207. However, compound 48 had a best estimated binding free energy of −56.84 kcal mol⁻¹ and bound well to COX-2 through four hydrogen bonds, a Sigma-Pi bond and Pi bonds. As shown, five amino acids, ALA199, His386, GLN454, GLN203 and HIS207 were of significance in the binding of ligand with enzyme. The ALA199 formed two hydrogen bonds with 48 (angle O⋯H–N = 105.17°, distance = 2.11 Å, angle O⋯H–N = 100.96°, distance = 2.17 Å). Furthermore, compound 48 was also bonded with His386 (angle H⋯O–H = 147.68°, distance = 2.02 Å) and GLN454 (angle O⋯H–O = 151.20°, distance = 2.02 Å) by a hydrogen and GLN203 (distance = 2.49 Å) and HIS207 (distance = 3.36 Å) by a Sigma-Pi and Pi bond. Thus it can be concluded the sulfanilamide, pyrazole ring and hydroxyl group are vital for their activities. According to the above, these molecular docking results along with the biological assay data suggested that compound 48 might be a potential inhibitor of the COX-2.

Fig. 6 The histogram about CDOCKER_INTERACTION_ENERGY (kcal mol⁻¹) of compounds (21–64) for COX-2, in the binding mode, compound 48 had a best estimated binding free energy of −56.84 kcal mol⁻¹, Celecoxib binds to COX-2 with binding free energy of −49.72 kcal mol⁻¹.

Fig. 7 Molecular docking 2D modeling of compound 48 with COX-2: the ALA199 formed two hydrogen bonds with 48. Compound 48 was also bonded with His386 and GLN454 by a hydrogen and GLN203 and HIS207 by a Sigma-Pi and Pi bond.

Fig. 8 Molecular docking 2D modeling of Celecoxib with COX-2: for clarity, only interacting residues are displayed. Each amino acid residue of different colors represented different residue interaction. Three amino acids HIS207, HIS386, TRP387 were significant in the binding of ligand with enzyme with two Pi bonds, a Sigma-Pi bonds and two hydrogen bonds.

Fig. 9 Molecular docking 3D modeling of compound 48 with COX-2: for clarity, only interacting residues are displayed.

2.4 3D-QSAR model

In order to evaluate the synthesized compounds as COX-2 inhibitors comprehensively and explore more powerful and selective inhibitors, 44 compounds with definite IC₅₀ values against COX-2 were selected as the model dataset by using the Create 3D QSAR protocol of Discovery Studio 3.5, all the molecular converted to the active conformation and corresponding pIC₅₀ (μM) values, which were converted from the obtained IC₅₀ (μM) values of COX-2 inhibition. 33 compounds were chosen as training set and rest of compounds comprising a test set as shown in Table 6.

Table 6 The experimental and predicted inhibitory activity of compounds (21–64) by 3D-QSAR models based upon active conformation achieved by molecular docking^a

Compounds	COX-2		Residual error
	Actual pIC50	Predicted pIC50
a The underlined for the test set, and the rest for training set.
21	4.78	4.71	0.07
22	4.83	4.78	0.05
2̲3̲	4.69	5.01	−0.32
24	4.87	4.90	−0.03
2̲5̲	4.79	5.05	−0.26
26	5.08	5.17	−0.09
27	5.02	5.08	−0.06
28	4.90	4.94	−0.04
2̲9̲	4.71	4.84	−0.13
30	4.77	4.79	−0.02
31	4.85	4.84	0.01
32	5.01	5.02	−0.01
3̲3̲	5.02	5.24	−0.22
3̲4̲	4.99	4.90	0.09
35	4.81	4.81	0.00
36	5.04	5.02	0.02
37	4.76	4.70	0.06
38	4.95	4.91	0.04
39	4.78	4.75	0.03
40	4.79	4.89	−0.10
41	5.04	5.01	0.03
42	6.25	6.19	0.06
4̲3̲	4.98	5.13	−0.15
44	4.62	4.62	0.00
45	5.89	5.93	−0.04
4̲6̲	6.39	5.91	0.48
4̲7̲	5.14	4.98	0.16
48	6.43	6.38	0.05
49	5.09	5.11	−0.02
50	5.12	5.10	0.02
51	5.49	5.50	−0.01
52	5.44	5.43	0.01
53	4.91	4.87	0.04
54	4.76	4.76	0.00
55	5.93	5.78	0.15
5̲6̲	5.79	5.52	0.27
5̲7̲	4.98	5.07	−0.09
58	5.33	5.35	−0.02
59	5.28	5.32	−0.04
60	5.20	5.22	−0.02
61	4.85	4.86	−0.01
62	4.71	4.69	0.02
6̲3̲	5.42	5.23	0.19
64	5.34	5.37	−0.03

---

By default, the alignment conformation of each molecule possessed the lowest CDOCKER_INTERACTION_ENENGY among all of the docked poses. The critical regions (steric or electrostatic) affecting the binding affinity was gained by this 3D-QSAR model. Exerting CHARMM force filed and PLS regression. The model was set up with correlation coefficient R² of 0.926, which indicated that this model possessed pretty good predicting capability. Predicted pIC₅₀ values and residual errors for the 44 compounds by this QSAR model are given in Table 6. The good agreement between predicted and experimental pIC₅₀ values for both test sets and training sets were shown in Fig. 10. The molecules aligned with the iso-surfaces of the 3D-QSAR model coefficients on van der Waals grids and electrostatic potential grids were also listed in Fig. 11. Electrostatic map indicates red contours around regions where high electron density (negative charge) is expected to increase activity, and blue contours represent areas where low electron density (partial positive charge) is expected to increase activity. Similarly, steric map indicates areas here steric bulk is predicted to increase (green) or decrease (yellow) activity. According to the maps, A rings and B rings were mainly surrounded by red contours, it implied the compound with high negative charged would show higher activity, validating that most NO₂, Br, F substituent being a better choice than CH₃, OCH₃. Big steric bulk substituents were unfit for A rings and B rings (surrounded by multiple yellow contours), demonstrating that most compounds with small substituents possess better activity. This model was accordant with the actual situation for potent compounds. Thus, this promising model would provide a direction to design and optimize more effective COX-2 inhibitors and pave the way for the further study. It can be predicted that greater electronegativity substituents and decreasing steric bulk might enhance the COX-2 inhibitory activity.

Fig. 10 Plot for experimental vs. predicted COX-2 inhibitory activities of training set and test set. The model was set up with correlation coefficient R² of 0.926, which indicated that this model possessed pretty good predicting capability.

Fig. 11 (a) Isosurface of the 3D-QSAR model coefficients on electrostatic potential grids. Blue represents positive coefficients; red represents negative coefficients. (b) Isosurface of the 3D-QSAR model coefficients on Van der Waals grids. Green represents positive coefficients; yellow represents negative coefficients.

3 Conclusion

In this study, a series of novel COX-2 inhibitors has been designed and synthesized; their biological activities have been evaluated which suggest these compounds possess moderate to potent antiproliferative activities against A549 cells, HepG2, HeLa, F10 cells and COX-2 inhibitory activities. According to analyzed the data of COX-2 inhibition and A549 cell apoptosis, we could find cell apoptosis was closely associated with COX-2 inhibition activity. Among these compounds, compound 48 showed the most powerful antiproliferative activity (IC₅₀ = 0.78 μM GI₅₀ = 0.32 μM for A549 cell) and COX-2 inhibitory activity (IC₅₀ = 0.37 μM). In addition, compound 48 can induce cancer cell apoptosis effectively and employing mouse primary hepatocytes and human kidney epithelial cell 293T also indicates high safety. The probable binding mode proposed by the docking simulation may be a good explanation of the impressive performance of compound 48, in which 48 binds well with COX-2 through four hydrogen bonds, Sigma-Pi and Pi bond with best estimated binding free energy of −56.84 kcal mol⁻¹. It can be concluded that compound 48 as well as the other benzenesulfonamide-substituted 1,5-diarylpyrazoles containing phenylacetohydrazide derivatives are promising leads for further study as potential anticancer agents. Moreover, A QSAR model was built to provide a reliable tool for the rational design of novel COX-2 inhibitors in the future.

4 Experiments

4.1 Materials and measurements

All chemicals and reagents used in current study were analytical grade. All the ¹H NMR spectra were recorded on a Bruker DPX 400 model Spectrometer in DMSO-d₆ and chemical shifts (δ) were reported as parts per million (ppm) elemental analyses were performed using a CHN-O-Rapid instrument and were within 0.4% of the theoretical values. ESI-MS spectra were recorded by Mariner System 5304 Mass spectrometer. Melting points (uncorrected) were determined on an XT4 MP apparatus (Taike Corp, Beijing, China). Thin layer chromatography (TLC) was performed on silica gel plates (Silica Gel 60 GF254) and visualized in UV light (254 nm).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) were purchased from Beyotime Institute of Biotechnology (Haimen, China). AnnexcinV-FITC cell apoptosis assay kit (#BA11100) was purchased from BIO-BOX (Nanjing, China). Human COX-1/COX-2 ELISA Kits were purchased from Cayman Chemical (catalog number 701070/701080). SD mouse were provided by Laboratory Animal Center in School of Life Science, Nanjing University.

4.2 General procedure for the synthesis of compounds (6–10)

Dimethyl oxalate (100 mmol) was added to a stirred suspension of sodium methoxide (200 mmol) in methanol (100 mL) at 0 °C. Then, a solution of acetophenone (50 mmol) in methanol (50 mL) was added dropwise over 5 min, the mixture was heated at reflux for 6 h. After cooling to room temperature, the solvent was removed and the residue was taken up in water and acidified with concentrated hydrochloric acid (1 mol L⁻¹) to pH = 3. The heavy precipitate formed was filtered off, washed with cold enthanol (50 mL) for three times, and dried in vacuo. The crude products were purified by recrystallization with ethanol, ethyl acetate and petroleum ether (1 : 1 : 0.5) washed by ice-water for three times to give pure intermediate products (6–10).

4.3 General procedure for the synthesis of compounds (11–15)

A mixture of 6–10 (15 mmol) and 4-hydrazinylbenzenesulfonamide (15 mmol) in methanol (40 mL) was heated at reflux for 6 h. After cooling to room temperature, the precipitate was filtered and washed with enthanol, the solid compounds (11–15) were crystallized from ethyl acetate, filtered and dried with Na₂SO₄.

4.4 General procedure for the synthesis of compounds (16–20)

Hydrazine hydrate (120 mmol) were successively added to a solution of compounds 11–15 (12 mmol) in enthanol and the mixture was stirred at 80 °C from several hours to overnight. After being cooled, the mixture put into brine to obtain the target compounds. Then precipitated solid was collected by filtration and washed with H₂O and cold enthanol to afford desired compounds (16–20).

4.5 General procedure for the synthesis of compounds (21–64)

Compounds 16–20 (1 mmol), various benzaldehyde (1.5 mmol) and acetic acid (0.5 mL) were dissolved in enthanol and stirred at room temperature for 12 h. The precipitate solid was collected by filtration and washed with enthanol and H₂O. The crude products were purified by recrystallization to obtain the target compounds (21–64).

4.5.1 4-(3-(2-Benzylidenehydrazine-1-carbonyl)-5-(4-methoxyphenyl)-1H-pyrazol-1-yl)benzenesulfonamide (21). White solid, yield 71.0%, mp 251–253 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.81 (s, 1H, CONH), 8.55 (s, 1H, CHN), 7.90 (d, J = 8.4 Hz, 2H, ArH), 7.72 (d, J = 6.8 Hz, 2H, ArH), 7.60 (d, J = 8.4 Hz, 2H, ArH), 7.48 (t, J = 8.4 Hz, 5H, ArH and SO₂NH₂), 7.27 (d, J = 8.6 Hz, 2H, ArH), 7.12 (s, 1H, CH), 6.98 (d, J = 8.5 Hz, 2H, ArH), 3.78 (s, 3H, OCH₃). ESI-MS: 476.1 [M + H]⁺. ¹³C NMR δ: 160.20, 157.91, 148.56, 147.02, 145.09, 144.06, 142.13, 134.81, 130.67, 130.57, 129.34, 127.57, 127.17, 126.41, 121.50, 114.78, 108.86, 55.73. Anal. calcd for C₂₄H₂₁N₅O₄S: C, 60.62; H, 4.45; N, 14.73. Found: 60.43; H, 4.46; N, 14.76.

4.5.2 4-(3-(2-(4-Methoxybenzylidene)hydrazine-1-carbonyl)-5-(4-methoxyphenyl)-1H-pyrazol-1-yl)benzenesulfonamide (22). White solid, yield 70.6%, mp 169–171 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.66 (s, 1H, CONH), 8.48 (s, 1H, CHN), 7.89 (t, J = 8.5 Hz, 2H, ArH), 7.66 (d, J = 8.6 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.49 (s, 2H, SO₂NH₂), 7.26 (d, J = 8.6 Hz, 2H, ArH), 7.10 (s, 1H, CH), 7.03 (d, J = 8.6 Hz, 2H, ArH), 6.98 (d, J = 8.6 Hz, 2H, ArH), 3.82 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃). ¹³C NMR δ: 160.20, 157.95, 148.56, 147.02, 145.13, 144.06, 142.13, 134.81, 130.66, 129.34, 127.57, 127.17, 126.41, 121.50, 119.74, 114.78, 108.86, 55.73. ¹³C NMR δ: 160.18, 157.89, 148.57, 147.12, 145.16, 144.06, 143.30, 134.82, 129.37, 127.47, 127.17, 126.46, 121.50, 119.76, 114.77, 108.86, 58.69, 55.76. ESI-MS: 506.1 [M + H]⁺. Anal. calcd for C₂₅H₂₃N₅O₅S: C, 59.40; H, 4.59; N, 13.85. Found: C, 59.26; H, 4.60; N, 13.89.

4.5.3 4-(3-(2-(2-Methoxybenzylidene)hydrazine-1-carbonyl)-5-(4-methoxyphenyl)-1H-pyrazol-1-yl)benzenesulfonamide (23). White solid, yield 82.6%, mp 157–160 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.86 (s, 1H, CONH), 8.89 (s, 1H, CHN), 7.89 (t, J = 8.5 Hz, 3H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.43 (t, J = 8.5 Hz, 1H, ArH), 7.27 (d, J = 8.6 Hz, 2H, ArH), 7.12 (d, 2H, CH and ArH), 7.04 (t, J = 7.4 Hz, 1H, ArH), 6.99 (d, J = 8.6 Hz, 2H, ArH), 3.87 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃). ¹³C NMR δ: 160.12, 157.87, 148.57, 147.16, 145.15, 144.07, 143.31, 134.83, 129.38, 127.46, 127.14, 126.465, 121.51, 119.73, 114.87, 108.89, 58.67, 55.72. ESI-MS: 506.1 [M + H]⁺. Anal. calcd for C₂₅H₂₃N₅O₅S: C, 59.40; H, 4.59; N, 13.85. Found: C, 59.52; H, 4.62; N, 13.87.

4.5.4 4-(3-(2-(3-Methoxybenzylidene)hydrazine-1-carbonyl)-5-(4-methoxyphenyl)-1H-pyrazol-1-yl)benzenesulfonamide (24). Yellow solid, yield 78.0%, mp 150–152 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.81 (s, 1H, CONH), 8.52 (s, 1H, CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.39 (t, J = 8.0 Hz, 1H, ArH), 7.27 (d, J = 8.4 Hz, 4H, ArH), 7.11 (s, 1H, CH), 7.04–7.01 (m, 1H, ArH), 6.98 (d, J = 8.6 Hz, 2H, ArH), 3.82 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃). ¹³C NMR δ: 160.21, 158.86, 148.52, 147.32, 145.18, 144.09, 143.30, 134.80, 129.39, 127.48, 127.18, 126.47, 121.52, 119.77, 114.74, 108.86, 57.63, 55.74. ESI-MS: 506.1 [M + H]⁺. Anal. calcd for C₂₅H₂₃N₅O₅S: C, 59.40; H, 4.59; N, 13.85. Found: C, 59.32; H, 4.60; N, 13.87.

4.5.5 4-(3-(2-(4-Methoxybenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H-pyrazol-1-yl)benzenesulfonamide (25). White solid, yield 75.3%, mp 267–268 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.68 (s, 1H, CONH), 8.48 (s, 1H, CHN), 7.89 (d, J = 8.5 Hz, 2H, ArH), 7.67 (d, J = 8.6 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.23 (t, J = 8.8 Hz, 4H, ArH), 7.13 (s, 1H, CH), 7.04 (d, J = 8.6 Hz, 2H, ArH), 3.82 (s, 3H, OCH₃), 2.33 (s, 3H, CH₃). ¹³C NMR δ: 159.92, 157.60, 148.88, 147.30, 145.17, 144.10, 142.11, 139.19, 129.92, 129.36, 129.12, 128.15, 126.31, 126.32, 125.72, 116.29, 109.06, 55.73, 21.29. ESI-MS: 490.1 [M + H]⁺. Anal. calcd for C₂₅H₂₃N₅O₅S: C, 61.34; H, 4.74; N, 14.31. Found: C, 61.19; H, 4.75; N, 14.34.

4.5.6 4-(5-(4-Fluorophenyl)-3-(2-(4-methoxybenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (26). White solid, yield 73.8%, mp 185–187 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.70 (s, 1H, CONH), 8.48 (s, 1H, CHN), 7.91–7.87 (m, 2H, ArH), 7.67 (d, J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.42–7.39 (m, 2H, ArH), 7.28 (t, J = 8.5 Hz, 2H, ArH), 7.18 (s, 1H, CH), 7.04 (d, J = 8.6 Hz, 2H, ArH), 3.82 (s, 3H, OCH₃). ¹³C NMR δ: 158.85, 147.43, 147.10, 145.24, 144.14, 142.08, 139.26, 131.41, 129.92, 129.82, 129.71, 128.51, 127.20, 126.43, 116.62, 116.32, 109.16, 54.93. ESI-MS: 494.1 [M + H]⁺. Anal. calcd for C₂₅H₂₃N₅O₄S: C, 58.41; H, 4.08; N, 14.19. Found: C, 58.36; H, 4.12; N, 14.20.

4.5.7 4-(5-(4-Bromophenyl)-3-(2-(4-methoxybenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (27). White solid, yield 80.3%, mp 264–267 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.75 (s, 1H, CONH), 8.48 (s, 1H, CHN), 7.91 (t, J = 8.5 Hz, 2H, ArH), 7.67–7.60 (m, 6H, ArH), 7.52 (s, 2H, SO₂NH₂), 7.29 (d, J = 8.5 Hz, 2H, ArH), 7.23 (s, 1H, CH), 7.04 (d, J = 8.6 Hz, 2H, ArH), 3.82 (s, 3H, OCH₃). ¹³C NMR δ: 159.76, 157.92, 148.26, 147.02, 145.14, 144.09, 142.13, 134.89, 130.67, 129.36, 127.56, 127.19, 126.43, 121.53, 119.76, 114.79, 108.85, 55.74. ESI-MS: 555.0 [M + H]⁺. Anal. calcd for C₂₄H₂₀BrN₅O₄S: C, 51.99; H, 3.64; N, 12.63. Found: C, 51.89; H, 3.62; N, 12.68.

4.5.8 4-(5-(4-Methoxyphenyl)-3-(2-(2,4,5-trimethoxybenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (28). Yellow solid, yield 71.3%, mp 254–256 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.71 (s, 1H, CONH), 8.81 (s, 1H, CHN), 7.89 (d, J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.4 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.37 (s, 1H, ArH), 7.27 (d, J = 8.5 Hz, 2H, ArH), 7.09 (s, 1H, CH), 6.98 (d, J = 8.5 Hz, 2H, ArH), 6.76 (s, 1H, ArH), 3.87 (s, 6H, OCH₃), 3.78 (s, 6H, OCH₃). ¹³C NMR δ: 160.19, 157.60, 153.91, 152.50, 147.19, 144.88, 144.22, 143.88, 143.69, 142.15, 130.64, 127.12, 126.15, 121.61, 114.78, 114.12, 108.84, 108.11, 98.41, 57.05, 56.38, 56.26, 55.72. ESI-MS: 566.1 [M + H]⁺. Anal. calcd for C₂₇H₂₇N₅O₇S: C, 57.34; H, 4.81; N, 12.38. Found: C, 57.46; H, 4.82; N, 12.42.

4.5.9 4-(5-(4-Methoxyphenyl)-3-(2-(3,4,5-trimethoxybenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (29). White solid, yield 70.8%, mp 274–276 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.79 (s, 1H, CONH), 8.47 (s, 1H, CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.60 (d, J = 8.4 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.27 (d, J = 8.5 Hz, 2H, ArH), 7.11 (s, 1H, CH), 6.99 (t, J = 6.4 Hz, 4H, ArH), 3.85 (s, 6H, OCH₃), 3.78 (s, 3H, OCH₃), 3.72 (s, 3H, OCH₃). ¹³C NMR δ: ¹³C NMR δ: 157.70, 153.68, 148.73, 147.23, 144.28, 144.03, 141.75, 139.74, 132.31, 131.30, 130.27, 128.55, 127.28, 126.51, 123.17, 109.67, 104.77, 60.61, 58.47, 56.45. ESI-MS: 566.1 [M + H]⁺. Anal. calcd for C₂₇H₂₇N₅O₇S: C, 57.34; H, 4.81; N, 12.38. Found: C, 57.47; H, 4.83; N, 12.40.

4.5.10 4-(5-(p-Tolyl)-3-(2-(2,4,5-trimethoxybenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (30). White solid, yield 80.9%, mp 171–173 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.72 (s, 1H, CONH), 8.81 (s, 1H, CHN), 7.89 (d, J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.37 (s, 1H, ArH), 7.23 (t, J = 8.8 Hz, 4H, ArH), 7.12 (s, 1H, CH), 6.76 (s, 1H, ArH), 3.87 (s, 6H, OCH₃), 3.78 (s, 3H, OCH₃), 2.33 (s, 3H, CH₃). ¹³C NMR δ: 159.67, 157.62, 153.78, 152.51, 147.21, 144.89, 144.24, 143.89, 142.89, 142.17, 130.66, 127.14, 126.16, 121.64, 114.79, 114.06, 108.86, 106.22, 98.46, 56.40, 56.28, 55.73, 21.27. ESI-MS: 550.1 [M + H]⁺. Anal. calcd for C₂₇H₂₇N₅O₆S: C, 59.01; H, 4.95; N, 12.74. Found: C, 58.89; H, 4.93; N, 12.72.

4.5.11 4-(5-(p-Tolyl)-3-(2-(3,4,5-trimethoxybenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (31). White solid, yield 74.9%, mp 304–307 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.80 (s, 1H, CONH), 8.47 (s, 1H, CHN), 7.89 (d, J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.23 (t, J = 8.8 Hz, 4H, ArH), 7.14 (s, 1H, CH), 7.00 (s, 2H, ArH), 3.85 (s, 6H, OCH₃), 3.72 (s, 3H, OCH₃), 2.33 (s, 3H, CH₃). ¹³C NMR δ: 159.41, 154.63, 148.76, 147.43, 144.29, 144.13, 141.79, 139.73, 132.51, 131.32, 130.47, 128.59, 126.98, 126.53, 123.19, 109.66, 104.78, 60.63, 56.46, 21.28. ESI-MS: 550.1 [M + H]⁺. Anal. calcd for C₂₇H₂₇N₅O₆S: C, 59.01; H, 4.95; N, 12.74. Found: C, 58.96; H, 4.93; N, 12.70.

4.5.12 4-(5-(4-Fluorophenyl)-3-(2-(2,4,5-trimethoxybenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (32). Yellow solid, yield 80.5%, mp 269–270 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.74 (s, 1H, CONH), 8.81 (s, 1H, CHN), 7.89 (d, J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.42–7.32 (m, 3H, ArH), 7.31–7.26 (m, 2H, ArH), 7.17 (s, 1H, CH), 6.76 (s, 1H, ArH), 3.87 (s, 6H, OCH₃), 3.78 (s, 3H, OCH₃). ¹³C NMR δ: 159.68, 157.42, 153.61, 152.30, 147.17, 144.84, 144.24, 143.56, 143.61, 142.02, 130.04, 127.11, 126.09, 120.67, 114.76, 114.10, 108.82, 108.02, 98.37, 56.32, 56.24, 55.73. ESI-MS: 554.1 [M + H]⁺. Anal. calcd for C₂₆H₂₄FN₅O₆S: C, 56.41; H, 4.37; N, 12.65. Found: C, 56.38; H, 4.40; N, 12.42.

4.5.13 4-(5-(4-Fluorophenyl)-3-(2-(3,4,5-trimethoxybenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (33). White solid, yield 73.5%, mp 259–262 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.82 (s, 1H, CONH), 8.47 (s, 1H, CHN), 7.90 (d, J = 8.6 Hz, 2H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.43–7.39 (m, 2H, ArH), 7.29 (t, J = 8.5 Hz, 2H, ArH), 7.19 (s, 1H, CH), 7.01 (s, 2H, ArH), 3.86 (s, 6H, OCH₃), 3.72 (s, 3H, OCH₃). ¹³C NMR δ: 158.83, 154.25, 148.76, 147.26, 144.48, 144.05, 142.85, 139.76, 132.21, 131.36, 130.27, 128.57, 127.26, 126.53, 123.18, 109.67, 104.78, 60.62, 56.46. ESI-MS: 554.1 [M + H]⁺. Anal. calcd for C₂₆H₂₄FN₅O₆S: C, 56.41; H, 4.37; N, 12.65. Found: C, 56.28; H, 4.36; N, 12.57.

4.5.14 4-(5-(4-Bromophenyl)-3-(2-(2,4,5-trimethoxybenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (34). White solid, yield 84.1%, mp 180–182 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.78 (s, 1H, CONH), 8.81 (s, 1H, CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.65–7.59 (m, 4H, ArH), 7.52 (s, 2H, SO₂NH₂), 7.36 (s, 1H, ArH), 7.29 (d, J = 8.5 Hz, 2H, ArH), 7.22 (s, 1H, CH), 6.76 (s, 1H, ArH), 3.86 (s, 6H, OCH₃), 3.78 (s, 3H, OCH₃). ¹³C NMR δ: 159.64, 158.26, 153.60, 152.31, 147.18, 145.04, 144.16, 143.52, 143.60, 142.01, 130.02, 127.06, 126.07, 120.60, 114.73, 114.12, 108.82, 108.43, 97.67, 56.31, 56.18, 55.72. ESI-MS: 615.1 [M + H]⁺. Anal. calcd for C₂₆H₂₄BrN₅O₆S: C, 50.82; H, 3.94; N, 11.40. Found: C, 50.72; H, 3.92; N, 11.36.

4.5.15 4-(5-(4-Bromophenyl)-3-(2-(3,4,5-trimethoxybenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (35). White solid, yield 75.4%, mp 286–288 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.87 (s, 1H, CONH), 8.46 (s, 1H, CHN), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.65–7.60 (m, 4H, ArH), 7.52 (s, 2H, SO₂NH₂), 7.30 (d, J = 8.5 Hz, 2H, ArH), 7.24 (s, 1H, CH), 7.00 (s, 2H, ArH), 3.85 (s, 6H, OCH₃), 3.72 (s, 3H, OCH₃). ¹³C NMR δ: 157.70, 153.68, 148.73, 147.23, 144.28, 144.03, 141.75, 139.74, 132.31, 131.30, 130.27, 128.55, 127.28, 126.51, 123.17, 109.67, 104.77, 60.61, 56.43. ESI-MS: 615.1 [M + H]⁺. Anal. calcd for C₂₆H₂₄BrN₅O₆S: C, 50.82; H, 3.94; N, 11.40. Found: C, 50.78; H, 3.92; N, 11.36.

4.5.16 4-(5-Phenyl-3-(2-(2,4,5-trimethoxybenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (36). White solid, yield 81.4%, mp 243–246 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.77 (s, 1H, CONH), 8.82 (s, 1H, CHN), 7.89 (d, J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.52 (s, 2H, SO₂NH₂), 7.43 (t, J = 6.0 Hz, 3H, ArH), 7.37–7.34 (m, 3H, ArH), 7.18 (s, 1H, CH), 6.76 (s, 1H, ArH), 3.87 (s, 6H, OCH₃), 3.78 (s, 3H, OCH₃). ¹³C NMR δ: 160.06, 159.23, 153.69, 152.42, 147.35, 145.16, 144.32, 143.87, 143.66, 142.06, 130.04, 127.19, 126.08, 120.62, 114.74, 114.14, 108.83, 108.46, 98.96, 56.34, 56.19, 55.74. ESI-MS: 536.1 [M + H]⁺. Anal. calcd for C₂₆H₂₅N₅O₆S: C, 58.31; H, 4.71; N, 13.08. Found: C, 58.16; H, 4.70; N, 13.12.

4.5.17 4-(5-(4-Methoxyphenyl)-3-(2-(4-methylbenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (37). White solid, yield 76.7%, mp 272–274 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.75 (s, 1H, CONH), 8.51 (s, 1H, CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.61 (t, J = 7.6 Hz, 4H, ArH), 7.51 (s, 2H, SO₂NH₂), 7.28 (t, J = 7.6 Hz, 4H, ArH), 7.11 (s, 1H, CH), 6.98 (d, J = 8.6 Hz, 2H, ArH), 3.78 (s, 3H, OCH₃), 2.36 (s, 3H, CH₃). ¹³C NMR δ: 159.61, 157.62, 148.68, 147.09, 145.07, 144.11, 142.17, 134.61, 130.64, 130.53, 129.35, 127.56, 127.14, 126.46, 121.52, 114.73, 108.89, 55.73, 23.26. ESI-MS: 490.1 [M + H]⁺. Anal. calcd for C₂₅H₂₃N₅O₄S: C, 61.34; H, 4.74; N, 14.31. Found: C, 61.20; H, 4.75; N, 14.34.

4.5.18 4-(3-(2-(4-Methylbenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H-pyrazol-1-yl)benzenesulfonamide (38). White solid, yield 81.9%, mp 300–301 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.75 (s, 1H, CONH), 8.51 (s, 1H, CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.63–7.58 (m, 4H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.29 (d, J = 8.0 Hz, 2H, ArH), 7.23 (t, J = 8.8 Hz, 4H, ArH), 7.14 (s, 1H, CH), 2.36 (s, 3H, CH₃), 2.33 (s, 3H, CH₃). ¹³C NMR δ: 159.82, 157.64, 148.74, 147.25, 145.20, 144.09, 142.05, 139.16, 129.92, 129.33, 129.14, 127.13, 126.52, 126.41, 125.78, 116.19, 109.04, 23.47, 21.23. ESI-MS: 474.1 [M + H]⁺. Anal. calcd for C₂₅H₂₃N₅O₃S: C, 63.41; H, 4.90; N, 14.79. Found: C, 63.28; H, 4.91; N, 14.82.

4.5.19 4-(5-(4-Fluorophenyl)-3-(2-(4-methylbenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (39). White solid, yield 78.6%, mp 253–256 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.77 (s, 1H, CONH), 8.51 (s, 1H, CHN), 7.91–7.89 (m, 2H, ArH), 7.61 (t, J = 8.0 Hz, 4H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.42–7.39 (m, 2H, ArH), 7.31–7.26 (m, 4H, ArH), 7.19 (s, 1H, CH), 2.36 (s, 3H, CH₃). ¹³C NMR δ: 159.03, 147.42, 146.72, 146.27, 144.19, 142.04, 138.26, 132.43, 129.90, 129.76, 129.67, 129.14, 127.16, 126.47, 116.53, 116.30, 109.12, 21.26. ESI-MS: 478.1 [M + H]⁺. Anal. calcd for C₂₄H₂₀FN₅O₃S: C, 60.37; H, 4.22; N, 14.67. Found: C, 60.26; H, 4.24; N, 14.69.

4.5.20 4-(5-(4-Bromophenyl)-3-(2-(4-methylbenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (40). White solid, yield 87.4%, mp 310–312 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.82 (s, 1H, CONH), 8.51 (s, 1H, CHN), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.65–7.60 (m, 6H, ArH), 7.52 (s, 2H, SO₂NH₂), 7.31–7.28 (m, 4H, ArH), 7.24 (s, 1H, CH), 2.36 (s, 3H, CH₃). ¹³C NMR δ: 159.14, 147.46, 146.73, 146.29, 143.17, 142.08, 138.27, 132.47, 129.92, 129.77, 129.68, 129.15, 126.86, 126.23, 116.53, 116.31, 109.14, 21.24. ESI-MS: 539.0 [M + H]⁺. Anal. calcd for C₂₄H₂₀BrN₅O₃S: C, 53.54; H, 3.74; N, 13.01. Found: C, 53.56; H, 3.73; N, 13.04.

4.5.21 4-(3-(2-(4-Hydroxybenzylidene)hydrazine-1-carbonyl)-5-(4-methoxyphenyl)-1H-pyrazol-1-yl)benzenesulfonamide (41). White solid, yield 88.2%, mp 298–301 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.59 (s, 1H, CONH), 9.92 (s, 1H, OH), 8.43 (s, 1H, CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.60–7.54 (m, 4H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.26 (d, J = 8.6 Hz, 2H, ArH), 7.09 (s, 1H, CH), 6.98 (d, J = 8.6 Hz, 2H, ArH), 6.85 (d, J = 8.4 Hz, 2H, ArH), 3.78 (s, 3H, OCH₃). ¹³C NMR δ: 159.67, 157.89, 157.71, 149.14, 146.63, 145.19, 144.20, 142.11, 131.88, 130.36, 127.17, 126.44, 121.45, 119.78, 116.92, 114.78, 108.93, 55.76. ESI-MS: 492.1 [M + H]⁺. Anal. calcd for C₂₄H₂₁N₅O₅S: C, 58.65; H, 4.31; N, 14.25. Found: C, 58.52; H, 4.32; N, 14.27.

4.5.22 4-(3-(2-(2-Hydroxybenzylidene)hydrazine-1-carbonyl)-5-(4-methoxyphenyl)-1H-pyrazol-1-yl)benzenesulfonamide (42). White solid, yield 79.5%, mp 298–301 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.14 (s, 1H, CONH), 11.28 (s, 1H, OH), 8.74 (s, 1H, CHN), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.61 (d, J = 8.5 Hz, 4H, ArH), 7.50 (d, J = 4.5 Hz, 3H, ArH and SO₂NH₂), 7.33–7.26 (m, 3H, ArH), 7.13 (s, 1H, CH), 6.99–6.91 (m, 4H, ArH), 3.78 (s, 3H, OCH₃). ¹³C NMR δ: 160.23, 157.93, 157.76, 149.14, 146.61, 145.17, 144.11, 142.10, 131.88, 130.68, 130.00, 127.18, 126.43, 121.45, 119.84, 119.20, 116.90, 114.79, 108.92, 55.74. ESI-MS: 492.1 [M + H]⁺ anal. calcd for C₂₄H₂₁N₅O₅S: C, 58.65; H, 4.31; N, 14.25. Found: C, 58.54; H, 4.32 N, 14.26.

4.5.23 4-(3-(2-(3-Hydroxybenzylidene)hydrazine-1-carbonyl)-5-(4-methoxyphenyl)-1H-pyrazol-1-yl)benzenesulfonamide (43). White solid, yield 71.4%, mp 292–294 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.75 (s, 1H, CONH), 9.62 (s, 1H, OH), 8.45 (s, 1H, CHN), 7.90 (t, J = 8.5 Hz, 2H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.26 (t, J = 8.6 Hz, 3H, ArH), 7.20 (s, 1H, CH), 7.09 (t, J = 5.4 Hz, 2H, ArH), 6.98 (d, J = 8.6 Hz, 2H, ArH), 6.84 (d, J = 8.6 Hz, 1H, ArH), 3.78 (s, 3H, OCH₃). ¹³C NMR δ: 160.24, 157.93, 157.77, 149.16, 146.63, 145.20, 143.87, 142.12, 131.88, 130.69, 130.04, 127.18, 126.46, 120.63, 119.82, 119.21, 116.93, 114.73, 108.92, 55.75. ESI-MS: 492.1 [M + H]⁺. Anal. calcd for C₂₄H₂₁N₅O₅S: C, 58.65; H, 4.31; N, 14.25. Found: C, 58.58; H, 4.32; N, 14.27.

4.5.24 4-(3-(2-(4-Hydroxybenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H-pyrazol-1-yl)benzenesulfonamide (44). White solid, yield 70.5%, mp 332–336 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.60 (s, 1H, CONH), 9.92 (s, 1H, OH), 8.43 (s, 1H, CHN), 7.89 (d, J = 8.5 Hz, 2H, ArH), 7.59–7.54 (m, 4H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.23 (t, J = 8.8 Hz, 4H, ArH), 7.12 (s, 1H, CH), 6.85 (d, J = 8.6 Hz, 2H, ArH), 2.33 (s, 3H, CH₃). ¹³C NMR δ: 159.90, 157.59, 148.86, 147.29, 145.15, 144.07, 142.09, 139.18, 129.90, 129.35, 129.10, 127.15, 126.50, 126.40, 125.78, 116.19, 109.02, 21.28. ESI-MS: 476.1 [M + H]⁺. Anal. calcd for C₂₄H₂₁N₅O₄S: C, 60.62; H, 4.45; N, 14.73. Found: C, 60.52; H, 4.45; N, 14.74.

4.5.25 4-(3-(2-(2-Hydroxybenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H-pyrazol-1-yl)benzenesulfonamide (45). White solid, yield 82.3%, mp 304–307 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.16 (s, 1H, CONH), 11.27 (s, 1H, OH), 8.74 (s, 1H, CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.60 (d, J = 8.6 Hz, 2H, ArH), 7.52 (s, 1H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.34–7.29 (m, 1H, ArH), 7.23 (t, J = 8.8 Hz, 4H, ArH), 7.16 (s, 1H, CH), 6.94 (t, J = 8.6 Hz, 2H, ArH), 2.33 (s, 3H, CH₃). ¹³C NMR δ: 159.87, 157.57, 148.84, 147.29, 145.16, 144.08, 143.18, 142.08, 139.19, 139.06, 129.92, 129.34, 129.12, 127.16, 126.50, 126.31, 125.78, 116.19, 108.62, 21.29. ESI-MS: 476.1 [M + H]⁺. Anal. calcd for C₂₄H₂₁N₅O₄S: C, 60.62; H, 4.45; N, 14.73. Found: C, 60.47; H, 4.46; N, 14.76.

4.5.26 4-(5-(4-Fluorophenyl)-3-(2-(2-hydroxybenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (46). White solid, yield 72.9%, mp 256–257 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.17 (s, 1H, CONH), 11.26 (s, 1H, OH), 8.75 (s, 1H, CHN), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.61 (d, J = 8.5 Hz, 2H, ArH), 7.51 (d, J = 6.0 Hz, 3H, ArH and SO₂NH₂), 7.43–7.39 (m, 2H, ArH), 7.32–7.27 (m, 3H, ArH), 7.21 (s, 1H, CH), 6.94 (t, J = 8.6 Hz, 2H, ArH). ¹³C NMR δ: 157.94, 157.57, 149.25, 146.80, 144.34, 144.12, 141.73, 132.32, 131.92, 131.32, 129.96, 128.46, 127.31, 126.53, 123.21, 119.86, 119.19, 116.91, 109.74. ESI-MS: 480.1 [M + H]⁺. Anal. calcd for C₂₃H₁₈FN₅O₄S: C, 57.61; H, 3.78; N, 14.61. Found: C, 57.71; H, 3.80; N, 14.64.

4.5.27 4-(5-(4-Bromophenyl)-3-(2-(4-hydroxybenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (47). White solid, yield 76.6%, mp 304–306 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.67 (s, 1H, CONH), 9.96 (s, 1H, OH), 8.43 (s, 1H, CHN), 7.90 (t, J = 8.5 Hz, 2H, ArH), 7.65–7.59 (m, 4H, ArH), 7.56–7.50 (m, 4H, ArH and SO₂NH₂), 7.29 (d, J = 8.6 Hz, 2H, ArH), 7.22 (s, 1H, CH), 6.85 (d, J = 8.5 Hz, 2H, ArH). ¹³C NMR δ: 159.24, 157.61, 149.24, 146.73, 144.34, 144.12, 140.65, 132.31, 131.92, 131.33, 129.98, 128.48, 127.28, 126.54, 119.18, 116.91, 109.74. ESI-MS: 541.0 [M + H]⁺. Anal. calcd for C₂₃H₁₈BrN₅O₄S: C, 51.12; H, 3.36; N, 12.96. Found: C, 51.06; H, 3.37; N, 13.01.

4.5.28 4-(5-(4-Bromophenyl)-3-(2-(2-hydroxybenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (48). White solid, yield 76.3%, mp 289–291 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.21 (s, 1H, CONH), 11.26 (s, 1H, OH), 8.74 (s, 1H, CHN), 7.92 (d, J = 8.5 Hz, 2H, ArH), 7.66–7.61 (m, 4H, ArH), 7.52 (t, J = 6.6 Hz, 3H, ArH and SO₂NH₂), 7.34–7.29 (m, 3H, ArH), 7.26 (s, 1H, CH), 6.93 (t, J = 6.4 Hz, 2H, ArH). ¹³C NMR δ: 157.93, 157.60, 149.24, 146.78, 144.33, 144.11, 141.73, 132.32, 131.91, 131.32, 129.99, 128.49, 127.29, 126.52, 123.20, 119.85, 119.19, 116.91, 109.73. ESI-MS: 541.0 [M + H]⁺. Anal. calcd for C₂₃H₁₈BrN₅O₄S: C, 51.12; H, 3.36; N, 12.96. Found: C, 51.03; H, 3.38; N, 12.97.

4.5.29 4-(3-(2-(4-Fluorobenzylidene)hydrazine-1-carbonyl)-5-(4-methoxyphenyl)-1H-pyrazol-1-yl)benzenesulfonamide (49). White solid, yield 81.5%, mp 269–272 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.82 (s, 1H, CONH), 8.55 (s, 1H, CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.80–7.76 (m, 2H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.29 (m, 4H, ArH), 7.11 (s, 1H, CH), 6.98 (d, J = 8.6 Hz, 2H, ArH), 3.78 (s, 3H, OCH₃). ¹³C NMR δ: 159.74, 157.93, 148.58, 147.04, 145.10, 144.08, 142.14, 134.82, 130.68, 130.54, 129.36, 127.56, 127.18, 126.42, 121.52, 114.81, 108.87, 55.74. ESI-MS: 494.1 [M + H]⁺. Anal. calcd for C₂₄H₂₀FN₅O₄S: C, 58.41; H, 4.08; N, 14.19. Found: C, 58.26; H, 4.09; N, 14.17.

4.5.30 4-(3-(2-(4-Fluorobenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H-pyrazol-1-yl)benzenesulfonamide (50). White solid, yield 72.8%, mp 302–304 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.84 (s, 1H, CONH), 8.55 (s, 1H, CHN), 7.91–7.89 (m, 2H, ArH), 7.80–7.76 (m, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.31 (t, J = 8.8 Hz, 2H, ArH), 7.23 (t, J = 8.8 Hz, 4H, ArH), 7.15 (s, 1H, CH), 2.33 (s, 3H, CH₃). ¹³C NMR δ: 157.88, 147.45, 147.06, 145.25, 144.13, 142.06, 139.22, 131.43, 129.90, 129.80, 129.72, 129.11, 127.17, 126.44, 116.52, 116.31, 109.11, 21.28. ESI-MS: 478.1 [M + H]⁺. Anal. calcd for C₂₄H₂₀FN₅O₃S: C, 60.37; H, 4.22; F, 3.98; N, 14.67. Found: C, 60.22; H, 4.24; N, 14.68.

4.5.31 4-(5-(4-Bromophenyl)-3-(2-(4-fluorobenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (51). White solid, yield 75.3%, mp 310–312 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.90 (s, 1H, CONH), 8.55 (s, 1H, CHN), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.80–7.76 (m, 2H, ArH), 7.65–7.60 (m, 4H, ArH), 7.52 (s, 2H, SO₂NH₂), 7.34–7.29 (m, 4H, ArH), 7.24 (s, 1H, CH). ¹³C NMR δ: 159.91, 157.63, 148.85, 147.29, 145.17, 144.23, 142.06, 139.17, 129.90, 129.36, 129.12, 127.16, 126.52, 126.43, 125.78, 116.21, 109.04. ESI-MS: 543.0 [M + H]⁺. Anal. calcd for C₂₃H₁₇BrFN₅O₃S: C, 50.93; H, 3.16; N, 12.91. Found: C, 50.80; H, 3.17; N, 12.94.

4.5.32 4-(5-(4-Methoxyphenyl)-3-(2-(4-nitrobenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (52). White solid, yield 78.2%, mp 294–298 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.14 (s, 1H, CONH), 8.66 (s, 1H, CHN), 8.32 (d, J = 8.6 Hz, 2H, ArH), 7.98 (d, J = 8.6 Hz, 2H, ArH), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.61 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.27 (d, J = 8.6 Hz, 2H, ArH), 7.15 (s, 1H, CH), 6.98 (d, J = 8.6 Hz, 2H, ArH), 3.78 (s, 3H, OCH₃). ¹³C NMR δ: 160.21, 157.92, 148.56, 147.04, 145.08, 143.16, 142.05, 134.82, 130.63, 130.52, 129.32, 127.37, 127.04, 126.21, 116.58, 115.76, 106.36, 55.70. ESI-MS: 521.1 [M + H]⁺. Anal. calcd for C₂₄H₂₀N₆O₆S: C, 55.38; H, 3.87; N, 16.15. Found: C, 55.22; H, 3.86; N, 16.20.

4.5.33 4-(5-(4-Methoxyphenyl)-3-(2-(2-nitrobenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (53). Yellow solid, yield 85.6%, mp 262–264 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.24 (s, 1H, CONH), 8.96 (s, 1H, CHN), 8.15 (d, J = 8.0 Hz, 1H, ArH), 8.08 (d, J = 8.4 Hz, 1H, ArH), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.84 (t, J = 8.0 Hz, 1H, ArH), 7.70 (t, J = 8.0 Hz, 1H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.27 (d, J = 8.6 Hz, 2H, ArH), 7.14 (s, 1H, CH), 6.98 (d, J = 8.6 Hz, 2H, ArH), 3.78 (s, 3H, OCH₃). ¹³C NMR δ: 160.20, 157.87, 157.63, 149.12, 146.52, 145.16, 144.12, 142.08, 133.82, 130.60, 130.02, 127.12, 126.42, 120.42, 117.82, 117.31, 116.72, 114.75, 108.92, 55.73. ESI-MS: 521.1 [M + H]⁺. Anal. calcd for C₂₄H₂₀N₆O₆S: C, 55.38; H, 3.87; N, 16.15. Found: C, 55.22; H, 3.88; N, 16.19.

4.5.34 4-(3-(2-(4-Nitrobenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H-pyrazol-1-yl)benzenesulfonamide (54). Yellow solid, yield 79.7%, mp 297–300 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.15 (s, 1H, CONH), 8.66 (s, 1H, CHN), 8.32 (d, J = 8.8 Hz, 2H, ArH), 7.98 (d, J = 8.8 Hz, 2H, ArH), 7.91–7.89 (m, 2H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.23 (s, 4H, ArH), 7.18 (s, 1H, CH), 2.33 (s, 3H, CH₃). ¹³C NMR δ: 159.86, 157.57, 148.84, 147.28, 145.14, 144.08, 142.10, 139.16, 129.92, 129.36, 129.12, 127.03, 126.52, 121.42, 120.08, 116.17, 109.02, 21.26. ESI-MS: 505.1 [M + H]⁺. Anal. calcd for C₂₄H₂₀N₆O₅S: C, 57.14; H, 4.00; N, 16.66. Found: C, 57.06; H, 4.01; N, 16.68.

4.5.35 4-(5-(4-Fluorophenyl)-3-(2-(4-nitrobenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (55). White solid, yield 73.7%, mp 297–299 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.17 (s, 1H, CONH), 8.66 (s, 1H, CHN), 8.32 (d, J = 8.8 Hz, 2H, ArH), 7.99 (d, J = 8.8 Hz, 2H, ArH), 7.91 (t, J = 8.8 Hz, 2H, ArH), 7.61 (d, J = 8.5 Hz, 2H, ArH), 7.51 (s, 2H, SO₂NH₂), 7.43–7.39 (m, 2H, ArH), 7.31–7.24 (m, 3H, ArH). ¹³C NMR δ: 157.96, 157.32, 148.84, 147.26, 145.13, 144.07, 142.13, 138.72, 129.82, 128.86, 128.12, 127.04, 126.54, 121.43, 119.08, 116.18, 109.02. ESI-MS: 509.1 [M + H]⁺. Anal. calcd for C₂₃H₁₇FN₆O₅S: C, 54.33; H, 3.37; N, 16.53. Found: C, 54.13; H, 3.39; N, 16.55.

4.5.36 4-(5-(4-Bromophenyl)-3-(2-(4-nitrobenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (56). Yellow solid, yield 74.4%, mp 306–308 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.21 (s, 1H, CONH), 8.66 (s, 1H, CHN), 8.32 (d, J = 8.8 Hz, 2H, ArH), 7.98 (d, J = 8.8 Hz, 2H, ArH), 7.92 (d, J = 8.8 Hz, 2H, ArH), 7.63 (t, J = 8.5 Hz, 4H, ArH), 7.53 (s, 2H, SO₂NH₂), 7.31–7.26 (m, 3H, ArH and CH). ¹³C NMR δ: 159.46, 156.52, 148.82, 146.28, 145.11, 143.06, 142.12, 136.81, 129.62, 128.85, 128.15, 127.06, 126.06, 121.23, 119.06, 116.14, 109.02. ESI-MS: 570.1 [M + H]⁺. Anal. calcd for C₂₃H₁₇BrN₆O₅S: C, 48.52; H, 3.01; N, 14.76. Found: C, 48.46; H, 3.02; N, 14.79.

4.5.37 4-(3-(2-(4-Bromobenzylidene)hydrazine-1-carbonyl)-5-(4-methoxyphenyl)-1H-pyrazol-1-yl)benzenesulfonamide (57). White solid, yield 82.3%, mp 284–286 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.89 (s, 1H, CONH), 8.52 (s, 1H, CHN), 7.91–7.85 (m, 3H, ArH), 7.67 (s, 4H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.27 (d, J = 8.6 Hz, 2H, ArH), 7.12 (s, 1H, CH), 6.98 (d, J = 8.6 Hz, 2H, ArH), 3.78 (s, 3H, OCH₃). ¹³C NMR δ: 159.68, 157.84, 148.52, 146.42, 145.06, 144.04, 142.10, 134.80, 130.62, 130.54, 127.32, 126.40, 121.50, 120.27, 119.48, 114.72, 106.82, 55.72. ESI-MS: 550.0 [M + H]⁺. Anal. calcd for C₂₄H₂₀BrN₅O₄S: C, 51.99; H, 3.64; N, 12.63. Found: C, 51.87; H, 3.65; N, 12.66.

4.5.38 4-(3-(2-(4-Bromobenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H-pyrazol-1-yl)benzenesulfonamide (58). White solid, yield 71.7%, mp 290–292 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.90 (s, 1H, CONH), 8.53 (s, 1H, CHN), 7.91–7.85 (m, 2H, ArH), 7.67 (s, 4H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.23 (s, 4H, ArH), 7.15 (s, 1H, CH), 2.33 (s, 3H, CH₃). ¹³C NMR δ: 157.86, 148.42, 147.26, 145.24, 144.12, 142.08, 139.12, 131.42, 129.82, 129.71, 129.62, 129.12, 127.12, 126.41, 116.41, 116.30, 109.09, 21.28. ESI-MS: 539.0 [M + H]⁺. Anal. calcd for C₂₄H₂₀BrN₅O₃S: C, 53.54; H, 3.74; N, 13.01; found: C, 53.42; H, 3.75; N, 13.05.

4.5.39 4-(3-(2-(4-Bromobenzylidene)hydrazine-1-carbonyl)-5-(4-fluorophenyl)-1H-pyrazol-1-yl)benzenesulfonamide (59). White solid, yield 79.3%, mp 173–175 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.92 (s, 1H, CONH), 8.53 (s, 1H, CHN), 7.91–7.89 (m, 2H, ArH), 7.67 (s, 4H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.42–7.38 (m, 2H, ArH), 7.31–7.26 (m, 2H, ArH), 7.20 (s, 1H, CH). ¹³C NMR δ: 157.94, 157.30, 148.76, 147.22, 145.10, 144.04, 142.12, 138.64, 129.80, 128.82, 128.10, 127.02, 126.52, 121.41, 119.06, 116.14, 109.03. ESI-MS: 543.0 [M + H]⁺. Anal. calcd for C₂₃H₁₇BrFN₅O₃S: C, 50.93; H, 3.16; N, 12.91. Found: C, 50.84; H, 3.18; N, 12.95.

4.5.40 4-(3-(2-(4-Bromobenzylidene)hydrazine-1-carbonyl)-5-(4-bromophenyl)-1H-pyrazol-1-yl)benzenesulfonamide (60). White solid, yield 77.4%, mp 278–281 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.97 (s, 1H, CONH), 8.52 (s, 1H, CHN), 8.32 (d, J = 8.8 Hz, 2H, ArH), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.85 (s, 1H, ArH), 7.67–7.60 (m, 8H, ArH), 7.52 (s, 2H, SO₂NH₂), 7.30 (d, J = 8.5 Hz, 2H, ArH), 7.25 (s, 1H, CH). ¹³C NMR δ: 159.24, 157.16, 148.64, 147.24, 145.06, 144.02, 142.08, 138.42, 129.64, 128.82, 128.06, 127.14, 125.42, 121.40, 119.04, 116.12, 109.02. ESI-MS: 600.9 [M + H]⁺. Anal. calcd for C₂₃H₁₇Br₂N₅O₃S: C, 45.79; H, 2.84; N, 11.61. Found: C, 45.68; H, 2.85; N, 11.65.

4.5.41 4-(3-(2-(4-Bromobenzylidene)hydrazine-1-carbonyl)-5-phenyl-1H-pyrazol-1-yl)benzenesulfonamide (61). White solid, yield 70.5%, mp 200–203 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.96 (s, 1H, CONH), 8.53 (s, 1H, CHN), 7.89 (d, J = 8.5 Hz, 2H, ArH), 7.68 (s, 4H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.52 (s, 2H, SO₂NH₂), 7.43 (t, J = 3.6 Hz, 4H, ArH), 7.36–7.33 (m, 2H, ArH), 7.21 (s, 1H, CH). ¹³C NMR δ: 157.84, 157.12, 147.64, 147.21, 145.02, 144.42, 142.16, 137.40, 127.64, 127.42, 127.06, 126.14, 125.32, 121.30, 119.02, 116.10, 109.02. ESI-MS: 525.0 [M + H]⁺. Anal. calcd for C₂₃H₁₈BrN₅O₃S: C, 52.68; H, 3.46; N, 13.36. Found: C, 52.60; H, 3.48; N, 13.39.

4.5.42 4-(3-(2-(4-Chlorobenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H-pyrazol-1-yl)benzenesulfonamide (62). White solid, yield 80.4%, mp 293–294 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.90 (s, 1H, CONH), 8.54 (s, 1H, CHN), 7.91–7.89 (m, 2H, ArH), 7.74 (d, J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.54 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.23 (s, 4H, ArH), 7.15 (s, 1H, CH), 2.33 (s, 3H, CH₃). ¹³C NMR δ: 159.82, 147.42, 147.06, 145.21, 144.12, 142.07, 139.23, 130.23, 129.93, 129.45, 129.31, 129.14, 127.12, 126.42, 116.54, 116.32, 109.12, 21.26. ESI-MS: 495.0 [M + H]⁺. Anal. calcd for C₂₄H₂₀ClN₅O₃S: C, 58.36; H, 4.08; N, 14.18. Found: C, 58.22; H, 4.10; N, 14.20.

4.5.43 4-(3-(2-(4-Chlorobenzylidene)hydrazine-1-carbonyl)-5-(4-fluorophenyl)-1H-pyrazol-1-yl)benzenesulfonamide (63). White solid, yield 76.3%, mp 185–187 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.92 (s, 1H, CONH), 8.55 (s, 1H, CHN), 7.90 (d, J = 8.6 Hz, 2H, ArH), 7.75 (d, J = 8.5 Hz, 2H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH), 7.54 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO₂NH₂), 7.42–7.39 (m, 2H, ArH), 7.31–7.26 (m, 2H, ArH), 7.20 (s, 1H, CH). ¹³C NMR δ: 157.92, 157.20, 148.56, 147.20, 145.12, 144.02, 142.08, 138.62, 129.72, 128.62, 128.08, 127.21, 126.42, 121.42, 119.04, 116.12, 109.02. ESI-MS: 499.0 [M + H]⁺. Anal. calcd for C₂₃H₁₇ClFN₅O₃S: C, 55.48; H, 3.44; N, 14.07. Found: C, 55.39; H, 3.45; N, 14.10.

4.5.44 4-(5-(4-Bromophenyl)-3-(2-(4-chlorobenzylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (64). White solid, yield 86.5%, mp 314–316 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.96 (s, 1H, CONH), 8.54 (s, 1H, CHN), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.74 (d, J = 8.5 Hz, 2H, ArH), 7.65–7.60 (m, 4H, ArH), 7.53 (t, J = 8.5 Hz, 4H, SO₂NH₂ and ArH), 7.30 (d, J = 8.5 Hz, 2H, ArH), 7.25 (s, 1H, CH). ¹³C NMR δ: 159.56, 157.24, 148.52, 147.23, 145.14, 144.04, 142.06, 138.64, 129.70, 128.60, 128.06, 127.10, 126.41, 121.37, 119.024, 116.10, 109.02. ESI-MS: 558.9 [M + H]⁺. Anal. calcd for C₂₃H₁₇BrClN₅O₃S: C, 49.43; H, 3.07; N, 12.53. Found: C, 49.26; H, 3.08; N, 12.54.

4.6 Anti-proliferation assay

Target tumor cell lines were grown to log phase in DMEM medium supplemented with 10% fetal bovine serum. After diluting to 5 × 10⁴ cells per mL with the complete medium, 100 μL of the obtained cell suspension was added to each well of 96-well culture plates and the number of viable cells was determined at time zero (control wells) then allowed to adhere for 12 h at 37 °C, 5% CO₂ atmosphere. Tested samples at pre-set concentrations (0.1, 1.0, 10, and 100 μM) were added to 96 wells with Celecoxib as positive reference. After 48 h exposure period, 20 μL of PBS containing 5 mg mL⁻¹ of MTT was added to each well. Plates were then incubated for further 4 h, and then 150 μL of DMSO was added to each well for coloration. The plates were shaken vigorously to ensure complete solubilization for 10 min at room temperature. The absorbance was measured and recorded on an ELISA reader (ELx800, BioTek, USA) at a test wavelength of 570 nm. In all the experiments three replicate wells were used for each drug concentration. Each assay was carried out at least three times. The results are shown in Tables 2 and 3.

4.7 Cyclooxygenase inhibition assay

The ability of the test compounds (21–64) to inhibit COX-1 and COX-2 (IC₅₀ values, μM) was determined using a human COX-1/COX-2 ELISA Kit (catalog number 701070 and 701080, Cayman Chemical) by a modification of the method described in the ref. 10. According to the manufacturer's instructions, cyclooxygenase catalyzes the first step in the biosynthesis of arachidonic acid (AA) to PGH₂. PGF_2α, produced from PGH₂ by reduction with stannous chloride, is measured by enzyme immunoassay. Stock solutions of test compounds were dissolved in a minimum volume of DMSO. Briefly, to a series of supplied reaction buffer solutions (960 μL, 0.1 M Tris–HCl pH 8.0 containing 5 mM EDTA and 2 mM phenol) with either COX-1 or COX-2 (10 μL) enzyme in the presence of heme (10 μL) was added 10 μL of various concentrations of test drug solutions (0.1, 1, 10, and 100 μM in a final volume of 1 mL). These solutions were incubated for a period of 5 min at 37 °C after which 10 μL of AA (100 μM) solution was added and the COX reaction was stopped by the addition of 50 μL of 1 M HCl after 2 min. PGF_2α, produced from PGH₂ by reduction with stannous chloride, was measured by enzyme immunoassay. This assay is based on the competition between PGs and a PG–acetylcholinesterase conjugate (PG tracer) for a limited amount of PG antiserum. The amount of PG tracer that is able to bind to the PG antiserum is inversely proportional to the concentration of PGs in the wells since the concentration of PG tracer is held constant while the concentration of PGs varies. The plate is washed to remove any unbound reagents and then Ellman's reagent, which contains the substrate to acetylcholine esterase, is added to the well. The product of this enzymatic reaction produces a distinct yellow color that absorbs at 405 nm. The intensity of this color, determined spectrophotometrically, is proportional to the amount of PG tracer bound to the well, which is inversely proportional to the amount of PGs present in the well during the incubation: absorbance α [Bound PG Tracer] α1/PGs. Percent inhibition was calculated by the comparison of compound-treated to various control incubations. The concentration of the test compound causing 50% inhibition (IC₅₀, μM) was calculated from the concentration-inhibition response curve (duplicate determinations).

4.8 Cell apoptosis assay

For Annexin V/PI assays, A549 cells were stained with Annexin V-FITC and PI and then monitored for apoptosis by flow cytometry. Briefly, 1 × 10⁵ cells were seeded in 6-well plates for 24 h and then were treated with compound 48 (0, 2, 4, 8, and 16 μM) for 48 h. After 48 h, they were trypsinized, washed in PBS and centrifuged at 2000 rpm for 5 min twice and stained with 5 μL of Annexin V-FITC and 5 μL of PI (5 mg mL⁻¹) in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaOH, 2.5 mM CaCl₂) for 30 min at room temperature in the dark. Apoptotic cells were quantified using BD Accuri C6 Flow Cytometer (BD, USA). Statistical analysis was done using Flowjo 7.6.1 software. Both early apoptotic (Annexin V-positive, PI-negative) and late apoptotic (double positive of Annexin V and PI) cells were detected.

4.9 Cytotoxicity test

4.9.1 Primary mouse hepatocyte isolation. SD Mice were anesthetized (200 mg ketamine per kg and 16 mg xylazine per kg) by subcutaneous injection, and the livers harvested. Hepatocytes were isolated from the livers by a modification of the method described previously.³⁰ Briefly, the liver was perfused, through a needle aligned along the inferior vena cava, with buffer (pH 7.5) containing 137 mmol L⁻¹ NaCl, 5.4 mmol L⁻¹ KCl, 0.5 mmol L⁻¹ NaH₂PO₄, 0.42 mmol L⁻¹ Na₂HPO₄, 10 mmol L⁻¹ HEPES, 0.5 mmol L⁻¹ EGTA, 4.2 mmol L⁻¹ NaHCO₃, and 5 mmol L⁻¹ glucose; collagenase buffer, pH 7.5, contained 137 mmol L⁻¹ NaCl, 5.4 mmol L⁻¹ KCl, 5 mmol L⁻¹ CaCl₂, 0.5 mmol L⁻¹ NaH₂PO₄, 0.42 mmol L⁻¹ Na₂HPO₄, 10 mmol L⁻¹ HEPES, 0.15 g L⁻¹ collagenase B, 0.05 g L⁻¹ trypsin inhibitor, 4.2 mmol L⁻¹ NaHCO₃, and 0.016 mmol L⁻¹ phenol red. The collagenase-perfused liver was then dissected, suspended in Hanks' solution (30 mL), and filtered through cheesecloth and a 100 μM nylon membrane to remove connective tissue debris and cell clumps. Hepatocytes were subjected to centrifugation (42 g, 2 min at 4 °C) and resuspended in Hanks solution; this was repeated 4 times. Then hepatocytes were purified using density gradient centrifugation (45% Percoll solution, 42 g for 10 min at 4 °C). Cell viability, measured by trypan blue exclusion, was more than 90%. Primary mouse hepatocytes were maintained in DMEM/F₁₂ medium, supplemented with 10% fetal bovine serum and antibiotics (100 U mL⁻¹ penicillin and 100 μg mL⁻¹ of streptomycin) at 37 °C in humidified air containing 5% CO₂.

4.9.2 Cytotoxicity test of 293T and primary mouse hepatocyte. 293T Cell lines and primary mouse hepatocyte were grown to log phase in DMEM supplemented with 10% fetal bovine serum, under a humidified atmosphere of 5% CO₂ at 37 °C. After diluting to 1 × 10⁴ cells per mL with the complete medium, 100 μL of the obtained cell suspension was added to each well of 96-well culture plates and then allowed to adhere for 12 h at 37 °C, 5% CO₂ atmosphere. Tested samples at pre-set concentrations (0.1, 1.0, 10, and 100 μM) were added to 96 wells with Celecoxib as positive drug. For the cytotoxicity assay, 20 μL of MTT (5 mg mL⁻¹) was added per well 4 h before the end of the incubation. After removing the supernatant, 200 μL DMSO was added to dissolve the formazan crystals. The absorbance at λ 570 nm was read on an ELISA reader (Tecan, Austria).

4.10 Experimental protocol of docking study

Molecular docking of compound 48 into the three dimensional X-ray structure of Matrix COX-2 (PDB code: 3PGH) was carried out using the Discovery Studio (version 3.5) as implemented through the graphical user interface DS-CDOCKER protocol. The crystal structure was obtained from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). All bound water and ligands were eliminated from the protein and the hydrogen was added, the molecular docking was performed by inserting compound 48 into the binding pocket of COX-2 based on the binding mode. Types of interactions of the docked protein with ligand were analyzed after the end of molecular docking.

4.11 3D-QSAR

Among all the 44 compounds, 75% (33 compounds) were utilized as a training set for QSAR modeling and the remaining 25% (11 compounds) were chosen as an external test subset for validating the reliability of the QSAR model by the software of DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd). The training sets were composed of inhibitors with the corresponding pIC₅₀ values which were converted from the obtained IC₅₀ (μM), and test sets comprised compounds of data sets as list in Table 6. All the definition of the descriptors can be seen in the “Help” of DS 3.5 software and they were calculated by QSAR protocol of DS 3.5. The alignment conformation of each molecule was the one with lowest interaction energy in the docked results of CDOCKER. The predictive ability of 3D-QSAR modeling can be evaluated based on the cross-validated correlation coefficient, which qualifies the predictive ability of the models. Scrambled test (Y scrambling) was performed to investigate the risk of chance correlations. The inhibitory potencies of compounds were randomLy reordered for 30 times and subject to leave-one-out validation test, respectively. The models were also validated by test sets, in which the compounds are not included in the training sets.

Acknowledgements

The work was financed by a grant (No. J1103512) from National Natural Science Foundation of China and the Fundamental Research Funds for the Central Universities (No. 020814380011) and the Projects (No. CXY1409 & CG1305) from the Science & Technology Burea of Lianyungang City of Jiangsu Province.

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Footnotes

† Electronic supplementary information (ESI) available. CCDC 1434485 and 1434486. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra02168a

‡ These two authors equally contributed to this paper.

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