Synthesis, cytotoxicity and carbonic anhydrase inhibitory activities of new pyrazolines

Figures & data

Table 1. Experimental data of the compounds 1–6.

Compounds	Chalcone mmol	PHBS mmol	Time h	Yield %	Crystallization Solvents	Mp, °C
1	1.68	1.84	5	53	Ethanol/Chloroform	267–269
2	1.68	1.84	18	71	Ethanol/Chloroform	197–199
3	1.68	1.84	4	19	Methanol	200–202
4	1.52	1.67	6	67	Methanol	168–170
5	1.52	1.67	4	85	Methanol/Chloroform	242–244
6	1.52	1.67	1	72	Methanol	238–240

PHBS: 4-Hydrazinobenzensulfonamide hydrochloride.

Table 2. Spectral data of the compounds 1–6.

Compounds	^1H NMR, ^13C NMR, and HRMS
1	4-(5-(2,5-dimethoxyphenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide. ^1H NMR (DMSO-d6, δ ppm): 7.69 (d, J = 8.8 Hz, 2H), 7.57(d, J = 9.2 Hz, 2H), 7.02-6.94 (m, 6H), 6.79 (dd, J = 2.9, 9.0 Hz, 2H), 6.35 (d, J = 2.9 Hz, 1H), 5.62 (dd, J = 11.9, 5.0 Hz, 1H), 3.84 (s, 3H, -OCH3), 3.82 (s, 3H,OCH3), 3.77 (s, 3H, -OCH3), 3.04 (dd, J = 17.6, 5.0 Hz, 1H)(One of the proton peak of the pyrazole ring under the solvent peak). ^13C NMR (DMSO-d6, δ ppm):160.9, 153.8, 150.9, 150.8, 146.6, 133.1, 130.2, 128.4, 127.9, 125.0, 114.8, 113.3, 112.8, 112.1, 57.8, 56.8, 55.9, 55.8, 42.6. HRMS (ESI-MS) Calc. forC24H26N3O5S [M + H]^+, 468.1588; found 468.1582
2	4-(5-(2,4-dimethoxyphenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1- yl)benzenesulfonamide. ^1H NMR (DMSO-d6, δ ppm): 7.69 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H), 6.99-6.93 (m, 6H), 6.73 (d, J = 8.8 Hz, 1H), 6.62 (d, J = 2.2 Hz, 1H), 6.38 (dd, J = 8.4, 2.6 Hz, 1H), 5.58 (dd, J = 11.7, 4.8 Hz, 1H), 3.87 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.69 (s, 3H, -OCH3), 3.01 (dd, J = 17.4, 4.8 Hz, 1H)(One of the proton peak of the pyrazole ring under the solvent peak). ^13C NMR (DMSO-d6, δ ppm): 160.9, 160.7, 157.8, 150.8, 146.6, 132.9, 128.3, 127.9, 127.2, 125.2, 121.2, 114.8, 112.1, 105.7, 99.7, 57.5, 56.5, 55.9, 55.8, 42.8. HRMS (ESI-MS) Calc. for C24H26N3O5S [M + H]^+ 468.1593; found 468.1590
3	4-(5-(3,4-dimethoxyphenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide. ^1H NMR (DMSO-d6) δ (ppm): 7.69 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 9.0 Hz, 2H), 7.04-6.97 (m, 6H), 6.90 (d, J = 2.2 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H, Ar-H), 6.63 (dd, J = 8.2, 2.0 Hz, 1H), 5.45 (dd, J = 11.6, 5.3 Hz, 1H), 3.11 (dd, J = 17.2, 2.9 Hz, 1H) (One of the proton of the pyrazole ring and methoxy peaks under the solvent peak).^13C NMR (DMSO-d6) δ (ppm): 160.9, 150.4, 149.7, 148.7, 146.9, 134.7, 133.1, 128.4, 127.7, 125.0, 118.0, 114.9, 112.7, 112.5, 110.3, 62.8, 56.1, 56.0, 55.9, 43.9. HRMS (ESI-MS) Calc. for C24H26N3O5S [M + H]^+ 468.1593; found 468.1599
4	4-(3-(4-methoxyphenyl)-5-(2,3,4-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide. ^1H NMR (DMSO-d6, δ ppm): 7.71 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 9.1 Hz, 2H), 6.99-6.97 (m, 6H), 6.69-6.65 (m, 2H), 5.57 (dd, J = 12.1, 5.1 Hz, 1H), 3.86 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.75 (s, 3H, -OCH3), 3.71 (s, 3H, -OCH3), 3.09 (dd, J = 17.6, 5.1 Hz, 1H) (One of the proton peak of the pyrazole ring under the methoxy peak). ^13C NMR (DMSO-d6, δ ppm): 160.9, 153.7, 151.2, 150.7, 146.6, 142.6, 133.1, 128.3, 127.9, 127.1, 125.2, 121.4, 114.8, 112.2, 108.6, 61.5, 61.0, 58.3, 56.4, 55.9, 43.1. HRMS (ESI-MS) Calc. for C25H28N3O6S [M + H]^+ 498.1699; found 498.1691
5	4-(3-(4-methoxyphenyl)-5-(2,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide. ^1H-NMR (DMSO-d6, δ ppm): 8.24 (s, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.8 Hz, 2H), 6.98-6.95 (m, 4H), 6.72 (s, 1H), 6.49 (brs, 2H), 5.55 (dd, J = 12.1, 5.6 Hz, 1H), 3.81 (s, 3H, -OCH3), 3.76 (s, 3H, -OCH3), 3.74 (s, 3H, -OCH3), 3.63 (s, 3H, -OCH3), 3.05 (dd, J = 17.4, 5.6 Hz, 1H) (One of the proton peak of the pyrazole ring). ^13C NMR (DMSO-d6, δ ppm): 160.9, 151.4, 150.8, 149.9, 146.9, 143.4, 132.9, 128.3, 127.8, 125.1, 120.4, 114.9, 112.2, 111.9, 99.3, 79.7, 57.2, 57.1, 56.4, 55.9, 42.7. HRMS (ESI-MS) Calc. for C25H28N3O6S [M + H]^+ 498.1699; found 498.1701
6	4-(3-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide. ^1H NMR (DMSO-d6, δ ppm): 8.2 (s, 1H), 7.70 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 9.1 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 7.01 (brs, 2H), 6.98 (d, J = 8.8 Hz, 2H), 6.54 (s, 1H), 5.40 (dd, J = 12.0, 5.0 Hz, 1H), 3.77 (s, 3H, -OCH3), 3.65 (s, 3H, -OCH3), 3.10 (dd, J = 17.6, 5.0 Hz, 1H)(Other methoxy peaks under the solvent peak). ^13C NMR (DMSO-d6, δ ppm): 160.9, 153.9, 150.6, 147.2, 138.3, 137.1, 133.3, 128.4, 127.8, 124.9, 114.9, 112.5, 103.4, 79.2, 63.5, 60.6, 56.5, 55.9, 43.9. HRMS (ESI-MS) Calc. for C25H28N3O6S [M + H]^+ 498.1699; found 498.1698

Scheme 1. Synthetic pathway of the pyrazoline bearing benzenesulfonamides 1–6. Reagents and conditions. i: EtOH, aq. NaOH (10%), rt. ii: 4-Hydrazinobenzensulfonamide.HCl, EtOH, glacial CH₃COOH, reflux. 1: (R₁ = R₄ = OCH_3, R₂ = R₃ = H), 2: (R₁ = R₃ = OCH_3, R₂ = R₄ = H), 3: (R₂ = R₃ = OCH_3, R₁ = R₄ = H), 4: (R₁ = R₂ = R₃ = OCH_3, R₄ = H), 5: (R₁ = R₃ = R₄ = OCH_3, R₂ = H), 6: (R₂ = R₃ = R₄ = OCH_3, R₁ = H).

Table 3. Cytotoxicities of the synthesized compounds towards human oral squamous carcinoma cell lines and human normal oral cells.

CC50 (μM)
	Human oral squamous carcinoma cell lines						Human normal oral cells
Compounds	Ca9-22 (A)	HSC-2	HSC-3	HSC-4	Mean (B)	SD	HGF (C)	HPLF	HPC	Mean (D)	SD	TS
1	Not Tested											D/B	C/A
2	22	200	200	200	155.5	89.0	22	119	46	62.3	50.5	0.4	1.0
3	200	200	200	200	200.0	0.0	63	120	181	121.3	59.0	0.6	0.3
4	22	200	200	200	155.5	89.0	75	135	115	108.3	30.6	0.7	3.4
5	7.7	41	31	20	24.9	14.3	11	12	12	11.7	0.6	0.5	1.4
6	10	11	14	22	14.3	5.4	20	21	23	21.3	1.5	1.5	2.0
5-FU	55.1	24.4	72.7	38.9	47.8	–	>200	>200	>200	>200	–	>4.2	>3.6
Melphalan	40.3	13.6	11	10.3	18.80	–	>200	151.0	>175.5	175.2	–	>9.3	>4.9

CC₅₀ values refer to the concentrations of the compounds in micromoles which reduce the viable cell number by 50%. Oral squamous cell carcinoma (OSCC) cell lines used are Ca9–22 (derived from gingiva), HSC-2, HSC-3, HSC-4 (derived from tongue). Normal oral cells used are human gingival fibroblasts (HGF), human periodontal ligament fibroblasts (HPLF), and human pulp cells (HPC). Tumor-specificity (TS) value is calculated by dividing the mean CC₅₀ value of each compound against normal cells to mean CC₅₀ value against OSCC. CC₅₀ value was determined from the growth curves plotted at different concentrations of each compounds in triplicate wells. SD: standard deviation, 5-FU: 5-fluorouracil, μM: micromolar.

Table 4. Carbonic anhydrase inhibitory activities of the compounds on hCA I and II isoenzymes.

	IC50 (nM)				KI (nM)
Compounds	hCA I	r^2	hCA II	r^2	hCA I	hCA II
1	30.1	0.99	24.8	0.96	26.5 ± 4.6	18.9 ± 9.0
2	40.8	0.98	30.1	0.98	39.7 ± 8.3	20.5 ± 3.7
3	46.2	0.92	26.7	0.97	55.5 ± 19.4	28.8 ± 6.5
4	38.5	0.98	23.8	0.98	31.4 ± 7.3	19.9 ± 2.7
5	49.2	0.93	25.7	0.98	29.9 ± 5.5	25.9 ± 7.0
6	36.5	0.94	23.9	0.98	34.1 ± 7.3	20.2 ± 3.0
AZA	293.0	0.98	146.5	0.98	276.3 ± 98.1	117.8 ± 14.1

Acetazolamide (AZA) was used as a standard inhibitor for all hCA I and II isoenzymes.