Pyrazolone Derivatives: Synthesis, Anti-inflammatory, Analgesic, Quantitative Structure–Activity Relationship and in Vitro Studies

Abstract

Some 1-(4-chlorophenyl or benzenesulfonamide)-2,3- and/or 4-substituted-1H-pyrazol-5(4H)-one derivatives were synthesized and screened for their anti-inflammatory and analgesic activities, in addition to their ulcerogenic liability. They were found to be active as anti-inflammatory and analgesic agents. Compound 6b was found to be the most active as anti-inflammatory agent and compound 9b was found to be the most active one as anti-inflammatory and analgesic agent. On the other hand, cyclooxygenase-1/-2 (COX-1)/COX-2 isozyme selectivity was also done and the tested compounds showed equal inhibition to both isoforms. Moreover, 2D-quantitative structure–activity relationship (QSAR) studies revealed well predictive and statistically significant and cross validated QSAR model that helps to explore some expectedly potent compounds.

Non steroidal anti-inflammatory drugs (NSAIDs) were known as important class of therapeutic agents for the alleviation of pain and inflammation. The pharmacological activity of NSAIDs is due to the inhibition of prostaglandins biosynthesis from arachidonic acid through inhibiting cyclooxygenases (COXs).^1,2) However, long term use of NSAIDs was associated with several side effects such as gastrointestinal mucosal damage, bleeding, intolerance, and renal toxicity.^3–6) The gastrointestinal (GI) damage from NSAIDs generally attributes to two factors, i.e., local irritation by the carboxylic acid moiety, common to most NSAIDs (topical effect) and decreased tissue prostaglandins production which maintains GI health and homeostasis. Therefore, synthetic approaches based upon chemical modification of NSAIDs were taken with the aim of improving NSAID safety profile.^7–10)

The core pyrazolone structure generally attracted widespread attention because of the diversity of biological activity as they showed anti-inflammatory^11–13) analgesic,^14,15) antitumor,¹⁶⁾ antimicrobial,^17,18) hypoglycemic,¹⁹⁾ and antitubercular²⁰⁾ activities. One of the first synthetic organic compounds that used as an important drug and having a pyrazolone nucleus was antipyrine I.²¹⁾ In addition, many pyrazolones like aminophenazone II, propyphenazone III, and famorofazone IV (Fig. 1) are widely used as anti-inflammatory, analgesic, and antipyretic drugs.^7,22,23) Besides, different pyrazolones found to possess significant anti-inflammatory activity as compounds V,²⁴⁾ VI,¹¹⁾ VII,¹¹⁾ and VIII¹³⁾ (Fig. 2). Thus, our main objective is to design novel pyrazolone derivatives to be further explored as powerful and novel non-ulcerogenic anti-inflammatory lead candidates. Some of these derivatives contain acidic centers with further esterification aiming to decrease the local side effects that might be attained by its acidic moieties.

Fig. 1. Anti-inflammatory, Analgesic and Antipyretic Pyrazolone Derivatives

Fig. 2. Pyrazolone Derivatives Exhibited Significant Anti-inflammatory Activity

The synthesized compounds were evaluated for their anti-inflammatory and analgesic activities. Ulcerogenic liability was also performed; in addition, evaluation for their in vitro COX-1/COX-2 inhibition was done. Two dimensional-quantitative structure–activity relationship (2D-QSAR) studies were also performed to correlate between the structures of the synthesized compounds and their pharmacological activities.

Results and Discussion

Chemistry

Synthesis of the pyrazolone derivatives was illustrated in the Charts 1–3. The starting intermediates 1-(4-substitutedphenyl)-3-methyl-1H-pyrazol-5(4H)-ones 2a and 2c were synthesized by reacting the commercially available 4-chlorophenylhydrazine hydrochloride 1a or the synthesized 4-hydrazinylbenzenesulfonamide hydrochloride 1b²⁵⁾ with ethyl acetoacetate in acetic acid in the presence of sodium acetate to obtain 2a^26–28) or in refluxing methanol to obtain 2c.^29,30) Methylation of 2a and 2c with dimethylsulfate (DMS) in alkaline medium afforded the N-methyl derivatives 3a³¹⁾ and 3d.³²⁾ Similarly, 1-(4-substitutedphenyl)-3-phenyl-1H-pyrazol-5(4H)-ones 2b^33–35) and 2d were obtained by reacting 1a or 1b with ethyl benzoylacetate. Alkylation of 2b with DMS gave a mixture of the O-methyl and N-methyl derivatives 3b and 3c in a ratio of 3 : 1, respectively. The two isomers were separated by preparative TLC using chloroform–methanol (9.5 : 0.5) as a developing solvent. IR spectrum of 3c revealed the appearance of C=O absorbance band at 1762 cm⁻¹, while in case of 3b this band disappeared. ¹H-NMR spectrum of 3b showed a singlet peak at δ 4.01 ppm corresponding to (–O–CH₃), while ¹H-NMR spectrum of 3c showed a singlet peak at δ 3.13 ppm corresponding to (–N–CH₃). Also, the melting points of the two isomers were different. On the contrary, alkylation of 2d under the same conditions gave exclusively the O-methyl derivative 3e (Chart 1).

Chart 1. Synthesis of Target Compounds 1–3

Introduction of dimethylaminomethyl group at 4-position of the pyrazol-5(4H)ones 2a–d was achieved by Mannich reaction using dimethylamine hydrochloride and formaldehyde to obtain the Mannich bases 4a–d. The acetic acid derivatives 6a–d were synthesized through multistep reactions involving quaternarization of Mannich bases with iodomethane, cyanation and finally hydrolysis of the cyanomethyl derivatives 5a–d with sulfuric acid. Reaction of 6a–d with thionyl chloride yielded the unstable acid chlorides derivatives which reacted directly without further purification with methanol in sodium methoxide to yield the corresponding ester derivatives 7a–d (Chart 2).

On the other hand, reaction of 2a–d with N,N-dimethylfomamide/diethylacetal (DMF/DEA) in isopropyl alcohol at room temperature led to the incorporation of the dimethylaminomethylene moiety into the position-4 of the pyrazolone derivatives 2a–d together with protection the sulfamoyl group in case of 2c and 2d with N,N-dimethylformimidoamide moiety to yield 8a–d. It is worthy to mention that the literature survey revealed that 8a was previously synthesized by reaction of 2a with Vilsmeier–Haack reagent (DMF-POCl₃).³⁶⁾ Alkaline hydrolysis of 8a–d afforded the corresponding 4-hydroxymethylene pyrazolone derivatives 9a–d from which 9b and 9d were subjected to Schotten–Baumann reaction³⁷⁾ using acetyl chloride or benzoyl chloride in the presence of sodium hydroxide to give the corresponding ester derivatives 10a–d (Chart 3).

Chart 2. Synthesis of Target Compounds 4–7

Chart 3. Synthesis of Target Compounds 8–10

Pharmacological Screening. Anti-inflammatory Activity

All the targeted compounds were evaluated for their anti-inflammatory activity via carrageenan-induced rat paw edema method of Winter et al.³⁸⁾ using phenylbutazone as a reference drug. The results were recorded in Table 1. IC₅₀ values results of the compounds 3a, b, d, e, 4a–d, 5a–d, 6a–d, 9a–d, and phenylbutazone were calculated and recorded in Table 2.

Table 1. Anti-inflammatory Effect and Percentage Inhibition of Phenylbutazone, and Synthesized Compounds on Carrageenan Induced Edema of the Hind Paw in Rats (n=5)

Cpd. No.	Dose (mmol/kg)	Edema (mm)±S.E.M.		% Inhibition
		2 h	3 h	2 h	3 h
Control	0	3.24±0.39	3.60±0.24	0	0
Phenylbutazone	0.32	1.38±0.09***	1.06±0.19***	57.28	70.55
2a	0.32	2.39±0.25	2.60±0.38	26.23	27.70
2b	0.32	2.20±0.25	2.33±0.29**	32.09	35.27
2c	0.32	1.14±0.18***	1.52±0.12***	64.80	57.80
2d	0.32	1.33±0.17***	1.81±0.08***	59.04	49.70
3a	0.32	2.16±0.14*	2.50±0.17*	33.18	30.69
3b	0.32	1.83±0.21***	3.06±0.19	43.50	15.00
3d	0.32	1.30±0.21***	1.31±0.18***	60.34	63.69
3e	0.32	2.03±0.19**	3.07±0.17	37.19	14.72
4a	0.32	1.99±0.30**	2.20±0.08***	38.67	38.88
4b	0.32	2.54±0.21	2.08±0.22***	21.73	42.33
4c	0.32	2.60±0.33	3.03±0.26	19.90	15.80
4d	0.32	2.96±00.19	2.76±0.16	8.58	23.44
5a	0.32	1.77±0.19***	2.33±0.18**	45.37	35.28
5b	0.32	1.22±0.24***	1.58±0.18***	62.34	56.11
5c	0.32	1.72±0.24***	2.41±0.16*	46.90	33.05
5d	0.32	1.65±0.03***	2.72±0.19	48.90	24.44
6a	0.32	1.54±0.14***	0.79±0.13***	52.34	78.05
6b	0.32	0.48±0.06***	0.48±0.07***	85.20	86.67
6c	0.32	1.41±0.05***	1.42±0.24***	56.60	60.50
6d	0.32	1.57±0.17***	1.97±0.16***	51.50	45.28
7a	0.32	3.07±0.14	3.07±0.10	5.25	14.58
7b	0.32	0.45±0.07***	1.22±0.12***	86.10	66.19
7c	0.32	2.05±0.28**	2.11±0.28***	36.79	41.40
7d	0.32	2.10±0.20*	2.33±0.22**	35.20	35.30
8a	0.32	1.73±0.08***	2.39±0.26**	46.60	33.70
8b	0.32	1.08±0.04***	1.23±0.07***	66.67	65.80
8c	0.32	1.76±0.08***	2.32±0.12**	45.52	35.40
8d	0.32	1.43±0.16***	1.96±0.23***	55.86	45.56
9a	0.32	0.60±0.11***	1.40±0.17***	81.50	61.11
9b	0.32	0.75±0.05***	0.61±0.13***	76.85	82.90
9c	0.32	1.55±0.18***	2.11±0.16***	52.00	40.38
9d	0.32	1.23±0.17***	2.22±0.20***	62.09	38.33
10a	0.32	1.88±0.06***	2.66±0.08	42.06	25.97
10b	0.32	0.41±0.05***	1.34±0.07***	87.30	62.78
10c	0.32	1.62±0.16***	2.33±0.17**	50.00	35.14
10d	0.32	1.64±0.29***	2.04±0.16***	49.23	43.33

Statistical analysis was carried out by one-way ANOVA test. * Significance difference from the control value at p<0.05. ** Significance difference from the control value at p<0.01. *** Significance difference from the control value at p<0.001.

Table 2. IC₅₀ Values of the Chosen Compounds and Reference Drug Phenylbutazone

Cpd. No.	IC50 (mmol/kg) after 3 h
Phenylbutazone	0.22
3a	0.52
3b	1.08
3d	0.25
3e	1.09
4a	0.41
4b	0.38
4c	1.00
4d	0.69
5a	0.45
5b	0.28
5c	0.49
5d	0.66
6a	0.20
6b	0.19
6c	0.27
6d	0.35
7b	0.23
9a	0.26
9b	0.19
9c	0.50
9d	0.42

Results summarized in Table 1 revealed that all tested compounds showed anti-inflammatory activity. After 2h the activity of compounds 2c, d, 3d, 5b, 6b, 7b, 8b, 9a, b, d, and 10b exhibited higher activity (58.95–87.35% inhibition) than the reference drug phenylbutazone (57.41% inhibition). After 3 h only compounds 6a, b, and 9b showed higher activity (78.06 to 86.67% inhibition) than the reference drug (70.56% inhibition).

Structure–Activity Correlation

Considering the anti-inflammatory activity of the synthesized pyrazolones unsubstituted at the 4-position, compounds 2c,d, and 3d bearing a benzenesulfonamide moiety in the 1-position showed superior activity than their 1-(4-chlorophenyl) analogues 2a, b, and 3a. The only exception was compound 3e which showed lower potency than 3b. Also, N-methylated compounds 3a and 3d showed better activity than O-methylated ones 3b and 3e. In the synthesized compounds, the 3-position was substituted either with CH₃ or phenyl group. Generally, it was observed in derivatives carrying 1-(4-chlorophenyl) group that the 3-phenyl derivatives 2b, 3b, 4b, 5b, 6b, 7b, 8b, and 9b were more active than their corresponding 3-methyl analogues 2a, 3a, 4a, 5a, 6a, 7a, 8a, and 9a. However, the case was different with the series carrying 1-(benzenesulfonamide) moiety i.e., those with 3-methyl group 2c, 3d, 4c, 5c, 6c, 7c, and 9c exhibited higher activity than the 3-phenyl analogues 2d, 3e, 4d, 5d, 6d, 7d, and 9d.

The 4-position of the pyrazolone ring tended to be tolerant of a variety of functionality. Introduction of dimethylaminomethyl moiety 4a–d in this position greatly decreased the anti-inflammatory activity. Conversion of dimethylaminomethyl into cyanomethyl analogues 5a–d improved the activity. Also, rigidification of the dimethylaminomethyl moiety in 4a–d by double bond to give dimethylaminomethylene derivatives 8a–d greatly improved the activity. The presence of acidic center as COOH group in 6a–d or enolic group in 9a–d resulted in a significant increase in the activity. Blocking of these acidic centers by esterification as in 7a–d and 10a–d, respectively led to decrease of the activity at least at one time interval. Also, it was observed specifically for 1-(4-chlorophenyl) derivatives that aryl ester 10b was more active than the corresponding alkyl ester 10a.

Analgesic Activity

Compounds that exhibited good anti-inflammatory activity and phenylbutazone were screened for their analgesic activity using the reported method of p-benzoquinone induced writhing in mice by Okun et al.³⁹⁾ From the obtained results in Table 3, it was noticed that almost all tested compounds which showed good anti-inflammatory activity showed also good analgesic activity equal to or higher than phenylbutazone except compounds 7b, 8b, and 10d that exhibited only good anti-inflammatory activity but couldn’t protect any animal from writhing. Moreover, the acidic pyrazolone 9b found to be the best derivative that showed analgesic activity. Ester formation resulted in decreasing of analgesic activity as shown in compounds 7a, d, and 10a–d when compared with their acidic counterparts 6a, b and 9b, d.

Table 3. Analgesic Activity of Phenylbutazone, and 17 Synthesized Compounds in Mice

Cpd. No.	Dose (mmol/kg)	No. of animals	No. of protected animals	% Protection
Control	0	6	0	0
Phenylbutazone	0.32	6	2	33.33
2c	0.32	6	1	16.67
3b	0.32	6	1	16.67
3d	0.32	6	4	66.67
6a	0.32	6	2	33.33
6b	0.32	6	2	33.33
6c	0.32	6	1	16.67
7a	0.32	6	1	16.67
7b	0.32	6	0	0
7c	0.32	6	1	16.67
8b	0.32	6	0	0
9a	0.32	6	1	16.67
9b	0.32	6	6	100
9d	0.32	6	3	50
10a	0.32	6	4	66.67
10b	0.32	6	1	16.67
10c	0.32	6	1	16.67
10d	0.32	6	0	0

Ulcerogenic Effect

The ulcerogenic effect of six active compounds and phenylbutazone was evaluated by the reported method of Meshali et al.,⁴⁰⁾ and the ulcer index was calculated according to the method of Robert et al.⁴¹⁾ and recorded in Table 4. From the obtained results, it was observed that all the tested compounds for ulcerogenic potential showed better GIT tolerance than phenylbutazone and compound 2c was showed the least GIT side effects with ulcer index 8.54. Ester derivatives 7b and 10b exhibited better GIT tolerance with ulcer indices 10.73 and 10.46, respectively than their corresponding acidic analogues 6b and 9b that having ulcer indices 17.26 and 12.80, respectively.

Table 4. Ulcerogenic Effect of Phenylbutazone and 6 Synthesized Compounds in Rats

Cpd. No.	Dose (mmol/kg)	No. of animals	% Incidence divided by 10	Average No. of ulcer	Average severity	Ulcer index
Control	0	5	0	0	0	0
Phenylbutazone	0.32	5	10	19.0	1.20	30.20
2c	0.32	5	6	1.4	1.14	8.50
3d	0.32	5	8	3.0	1.06	12.06
6b	0.32	5	10	6.2	1.06	17.26
7b	0.32	5	6	3.6	1.13	10.70
9b	0.32	5	8	3.8	1.00	12.80
10b	0.32	5	6	3.4	1.06	10.46

Acute Toxicity

LD₅₀ of some representative compounds 3d, 6b, c, 7b, c, 9b, d, and 10a, b was determined using Finney’s method.⁴²⁾ The tested compounds showed a high safety margin since intraperitoneal injection of doses less than 1.62 mmol/kg body weight of the tested compounds failed to kill any mouse after their observation for 24 h. LD₅₀ of compounds 7b, c, and 9d was 3.25 mmol/kg body weight, while of compounds 6a, c, 9b and 10a, b was 2.44 mmol/kg body weight and of compound 5a was 1.62 mmol/kg body weight.

In Vitro COX Inhibition Assay

Compounds 6b, 7b, 9b and indomethacin were evaluated for their selectivity to inhibit COX-1 and/or COX-2 isoenzymes using Cayman’s COX-Activity Assay kit according to the manufacturer instruction.⁴³⁾ The ratio of percentage inhibition of COX-1/COX-2 was calculated and recorded in Table 5. Results revealed that the tested compounds were non-selective as they have almost equal inhibitory effect on both isoform of COX-enzyme ranging from 42–57% inhibition; also it was found that the ester derivative 7b showed COX-2 inhibition which indicates that intact ester was active as acidic analogue 6b.

Table 5. COX-1/COX-2 Percentage Inhibition Ratio of Compounds 6b, 7b, 9b and Indomethacin

Cpd. No.	COX-1/COX-2
Indomethacin	3.94
6b	1.22
7b	0.74
9b	1.14

2D-QSAR Study

2D-QSAR study was performed in order to find a mathematical correlation between the structures of twenty compounds 3a, b, d, e, 4a–d, 5a–d, 6a–d, and 9a–d and their anti-inflammatory activity^44,45) that expressed as −log IC₅₀ values, using Molecular Operating Environment (MOE) software package. The most relevant descriptors derived for modeling the anti-inflammatory activity are listed in Table 6 and the relation between the obtained results of experimentally observed and predicted values of anti-inflammatory activity were presented in Table 7 and illustrated by Fig. 3. 2D-QSAR models were validated with the leave one out (LOO) method. The best derived QSAR model for the twenty pyrazolone derivatives was presented by the following triparametric equation with correlation coefficient (R²)=0.88 and root mean square (RMSE)=0.072.

Fig. 3. Relation between Experimentally Observed and Predicted Activity

  

Table 6. The Most Relevant Discriptors Derived for Modeling the Anti-inflammatory Activity

Cpd. No.	P IC50	Dipole	FASA_H	AM1_IP
3a	0.2790	1.2776	0.7647	9.4780
3b	-0.0330	1.1271	0.8191	11.1909
3d	0.6000	1.5005	0.6058	9.9810
3e	0.0370	1.3553	0.6570	10.2922
4a	0.3790	1.3059	0.5880	9.7443
4b	0.4200	0.9460	0.6700	9.2869
4c	0.0000	1.8596	0.4598	10.4407
4d	0.1610	1.3212	0.5542	9.1167
5a	0.3460	0.2782	0.6384	10.0057
5b	0.5520	1.4235	0.7396	9.4651
5c	0.3090	1.9773	0.5503	9.0965
5d	0.1800	1.7702	0.6011	10.1263
6a	0.6860	0.5800	0.7966	10.3644
6b	0.7210	0.6779	0.8044	10.3734
6c	0.5680	0.9918	0.6206	9.4124
6d	0.4460	1.6806	0.6866	10.5202
9a	0.5760	0.9654	0.7999	10.9451
9b	0.7140	1.3182	0.8936	11.0372
9c	0.2950	1.3242	0.7101	9.9182
9d	0.3730	1.4027	0.7535	10.3089

Table 7. Eperimentally Observed, Predicted Activity of 20 Compounds ($PRED) and Their Residual ($RES)

Cpd. No.	Activity P IC50	$PRED	$RES
3a	0.2790
3b	−0.0330
3d	0.6000
3e	0.0370
4a	0.3790	0.2743	0.1047
4b	0.4200	0.4546	−0.0346
4c	0.0000	−0.0064	0.0064
4d	0.1610	0.2501	−0.0891
5a	0.3460	0.3882	−0.0422
5b	0.5520	0.5445	0.0075
5c	0.3090	0.2178	0.0912
5d	0.1800	0.2566	−0.0766
6a	0.6860	0.6274	0.0586
6b	0.7210	0.6361	0.0849
6c	0.5680
6d	0.4460	0.3851	0.0609
9a	0.5760	0.5853	−0.0093
9b	0.7140	0.7264	−0.02124
9c	0.2950
9d	0.3730	0.5228	−0.1498

From this model, it was observed that FASA_H (i3D fractional hydrophobic surface area) was positively correlated with anti-inflammatory activity but dipole (i3D The dipole moment calculated using the MNDO Hamiltonian [MOPAC]) and AM1_IP (i3D The ionization potential (kcal/mol) calculated using the AM1 Hamiltonian [MOPAC]) were negatively correlated with activity indicated that increasing in the values of FASA_H is beneficial for the anti-inflammatory activity and this matched with the experimental data where the most active compounds 6b and 9b which are having P IC₅₀ 0.72 and 0.71 possessed relatively higher values of FASA_H 0.80 and 0.89, respectively. Also, Compounds with lower activity 4c and 4d with P IC₅₀ 0.00 and 0.16 possessed lower FASA_H values 0.45 and 0.55, respectively.

Conclusion

From this study, we conclude that 1-(4-chlorophenyl or benzenesulfonamide)-2,3- and/or 4-substitiuted-1H-pyrazol-5(4H)-ones could be considered to be useful building blocks for future research to synthesis of potential anti-inflammatory and analgesic agents with minimal side effects. Pharmacological screening and biological evaluation revealed that all the tested compounds have good anti-inflammatory activity with respect to the newly synthesized compounds 6b, 7b, and 9b that showed the best anti-inflammatory activity. Also, they have good analgesic activity except for 7b. Furthermore, they are non-selective towards COX-1 or COX-2 but they exhibited better GIT tolerance than the reference drug phenylbutazone. Analysis of 2D-QSAR model provided details on fine relationship linking structure and activity which indicated that the fractional hydrophobic surface area (FASA_H), the dipole moment (dipole), and the ionization potential (AM1_IP) influence the anti-inflammatory activity of pyrazolone derivatives. From SAR point of view, it was found that the presence of acidic center as COOH or enolic group in pyrazolone derivatives improves anti-inflammtory activity and formation of ester derivatives found to be safer than their acidic counterparts but with lower anti-inflammatory and analgesic activities.

Experimental

Chemistry

All chemicals were purchased from VWR International Merck, Germany or Sigma-Aldrich and used without further purification. Compound (4-chlorophenyl)hydrazine hydrochloride Ia was purchased from Sigma-Aldrich. Melting Points were carried out by open capillary tube method using Stuart SMP3 Melting Point apparatus and they are uncorrected. Elemental Microanalysis was carried out at the Micro Analytical Center, Cairo University or at The Regional Center for Mycology and Biotechnology, Al-Azhar University. Infrared spectra were recorded on Shimadzu Infrared spectrometer IR Affinity-1 (FTIR-8400S, Kyoto, Japan) or Jasco Infrared spectrometer (FTIR-4100, Japan), and expressed in wave number (cm⁻¹), using potassium bromide discs. ¹H- and ¹³C-NMR Spectra were recorded on a Varian Mercury VX- 300 MHz, ¹³C, 70 MHz NMR spectrometer, or JEOL-ECA500, 500 MHz Japan, NMR spectrometer, the spectra were run at 300 MHz or 500 MHz in deuterated chloroform (CDCl₃) or dimethylsulfoxide (DMSO-d₆). Chemical shifts were expressed in δ units and were related to that of the solvents. As for the proton magnetic resonance, D₂O was carried out for NH and OH exchangeable protons. Mass Spectra were recorded using Shimadzu Gas Chromatograph Mass spectrometer-Qp 2010 plus (Japan) or JEOL JMS-AX 500 mass spectrometer (Japan). All the reactions were followed by TLC using silica gel F254 plates (Merck), using chloroform: methanol 9 : 1 or chloroform as eluting system and were visualized by UV-lamp. Compounds 1b,²⁵⁾ and 3a³¹⁾ were prepared according to reported methods, while compounds 8a,³⁶⁾ 8b,⁴⁶⁾ 2a,^26–28) 2c,^29,30) 2b,^33–35) and 3d³²⁾ were prepared according to modified procedures from the reported methods.

General Procedure for the Synthesis of Compounds 2a–d

Method A; A mixture of equimolar amounts of 1a (1.78 g, 10 mmol) and ethyl acetoacetate (1.30 g, 10 mmol) or ethyl benzoylacetate (1.92 g, 10 mmol) in glacial acetic acid (15 mL) and fused sodium acetate (0.08 g, 1 mmol) was stirred at room temperature for 24 h, the reaction mixture was poured onto ice water, the solid was collected, dried and crystallized from ethanol.

Method B; A mixture of 1b (11.15 g, 50 mmol) and ethyl acetoacetate (6.50 g, 50 mmol) or ethyl benzoylacetate (9.60 g, 50 mmol) was refluxed in methanol (75 mL) for 15 h. The resulting solution was then concentrated and poured onto ice water. The separated solid was filtered, washed with water, dried and crystallized from methanol.

1-(4-Chlorophenyl)-3-methyl-1H-pyrazol-5(4H)-one (2a): Yield 95% (from method A). mp 169–171°C. ¹H-NMR (CDCl₃) δ: 2.19 (3H, s, CH₃), 3.43 (2H, s, CH₂), 7.34 (2H, d, Ar-H, J=7.0 Hz), 7.84 (2H, d, Ar-H, J=7.3 Hz). IR (KBr) cm⁻¹: 3047 (CH aromatic), 2962, 2924 (CH aliphatic), 1680 (C=O). MS m/z: 210 (M⁺+2), 208 (M⁺). Anal. Calcd for C₁₀H₉ClN₂O (208.64): C, 57.57; H, 4.35; N, 13.43. Found: C, 57.74; H, 4.04; N, 13.60.

1-(4-Chlorophenyl)-3-phenyl-1H-pyrazol-5(4H)-one (2b): Yield 91% (from method A). mp 163–164°C. ¹H-NMR (DMSO-d₆) δ: 6.05 (1H, s, CH of pyrazole), 7.3–7.92 (9H, m, Ar-H), 12.02 (1H, s, ex, OH). ¹³C-NMR (DMSO-d₆) δ: 85.3, 122.2, 125.1, 127.9, 128.5, 128.8, 129.6, 133.1, 137.7, 150.0, 154.0. IR (KBr) cm⁻¹: 3050 (CH aromatic), 2962, 2912 (CH aliphatic), 1712 (C=O). MS m/z: 272 (M⁺+2), 270 (M⁺). Anal. Calcd for C₁₅H₁₁ClN₂O (270.71): C, 66.55; H, 4.10; N, 10.35. Found: C, 66.68; H, 4.13; N, 10.26.

4-(3-Methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (2c): Yield 88% (from method B). mp 237–238°C. ¹H-NMR (DMSO-d₆) δ: 2.12 (3H, s, CH₃), 3.42 (2H, s, CH₂), 7.35 (2H, s, ex, NH₂), 7.84 (2H, d, Ar-H, J=7.8 Hz), 7.91 (2H, d, Ar-H, J=7.1 Hz). IR (KBr) cm⁻¹: 3317, 3244 (NH₂), 3043 (CH aromatic), 2981, 2958 (CH aliphatic), 1627 (C=O), 1334, 1161 (SO₂). MS m/z: 253 (M⁺). Anal. Calcd for C₁₀H₁₁N₃O₃S (253.27): C, 47.42; H, 4.38; N, 16.59. Found: C, 47.70; H, 4.63; N, 16.74.

4-(5-Oxo-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (2d): Yield 75% (from method B). mp 211–213°C. ¹H-NMR (DMSO-d₆) δ: 6.05 (1H, s, CH of pyrazole), 6.51 (2H, s, ex, NH₂), 7.36–8.06 (9H, m, Ar-H), 12.00 (1H, s, ex, OH). ¹³C-NMR (DMSO-d₆) δ: 86.0, 120.8, 125.2, 126.7, 128.1, 128.3, 128.5, 140.5, 141.2, 150.5, 154.5. IR (KBr) cm⁻¹: 3329, 3244 (NH₂), 3066 (CH aromatic), 2981, 2958 (CH aliphatic), 1732 (C=O), 1323, 1153 (SO₂). MS m/z: 315 (M⁺). Anal. Calcd for C₁₅H₁₃N₃O₃S (315.34): C, 57.13; H, 4.16; N, 13.33. Found: C, 57.37; H, 4.40; N, 13.08.

General Procedure for the Synthesis of Compounds 3b–e

A solution of sodium hydroxide (0.50 g, 12.50 mmol) in water (1 mL) was added to a solution of 2a–d (5.73 mmol) in methanol (5 mL). The mixture was warmed on a water bath and dimethyl sulfate (0.70 g, 7.50 mmol) was added. The mixture was refluxed for 1 h and allowed to cool, with continuous stirring. Methanol was distilled off, hot water was added to the residue, the mixture was filtered, the filtrate was extracted with benzene, calcium oxide was added to remove excess dimethyl sulfate and filtered. The filtrate was evaporated under vacuum and the residue was recrystallized from benzene.

Separation of the mixture 3b and 3c by preparative TLC. TLC plates are prepared by mixing silica gel, with a small amount of calcium sulfate (gypsum) and water. This mixture is spread as thick slurry on glass plates (20 cm×20 cm). The resultant plate is dried and activated by heating in an oven for thirty minutes at 110°C. The mixture (about 0.5 g was dissolved in chloroform) is applied to the plate as a thin even layer horizontally to and just above the solvent level. When developed with solvent (chloroform–methanol, 9.5 : 0.5), the compounds separate in horizontal bands with light to deep yellow color. Lower band (Rf is 0.18) and upper band (Rf is 0.90) were scraped off the backing material. The backing material is then extracted with chloroform and filtered to give the isolated material upon removal of the solvent. Lower bands yielded compounds 3c (yield 23%) while, upper bands yielded compounds 3b (yield 71%).

1-(4-Chlorophenyl)-5-methoxy-3-phenyl-1H-pyrazole (3b): Yield 71%. mp 56–58°C. ¹H-NMR (CDCl₃) δ: 4.01 (3H, s, OCH₃), 6.02 (1H, s, CH of pyrazole), 7.36–7.89 (9H, m, Ar-H). IR (KBr) cm⁻¹: 3061, 3026 (CH aromatic), 2926, 2854 (CH aliphatic). MS m/z: 286 (M⁺+2), 284 (M⁺). Anal. Calcd for C₁₆H₁₃ClN₂O (284.74): C, 67.49; H, 4.60; N, 9.84. Found: C, 67.49; H, 4.62; N, 9.97.

2-(4-Chlorophenyl)-1-methyl-5-phenyl-1H-pyrazol-3(2H)-one (3c): Yield 23%. mp 211–213°C. ¹H-NMR (CDCl₃) δ: 3.13 (3H, s, N–CH₃), 6.02 (1H, s, CH of pyrazolone), 7.15–7.54 (9H, m, Ar-H). IR (KBr) cm⁻¹: 3000 (CH aromatic), 2926, 2852 (CH aliphatic), 1762 (C=O); MS m/z: 286 (M⁺+2), 284 (M⁺). Anal. Calcd for C₁₆H₁₃ClN₂O (284.74): C, 67.49; H, 4.60; N, 9.84. Found: C, 67.53; H, 4.58; N, 9.97.

4-(2,3-Dimethyl-5-oxo-2,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (3d): Yield 60%. mp 252–253°C. ¹H-NMR (CDCl₃) δ: 2.27 (3H, s, CH₃), 2.85 (3H, s, N–CH₃), 5.00 (1H, s, CH of pyrazolone), 7.18–8.27 (6H, m, Ar-H+ex, NH₂). IR (KBr) cm⁻¹: 3410, 3279 (NH₂), 3100 (CH aromatic), 2924, 2860 (CH aliphatic), 1735 (C=O). MS m/z: 267 (M⁺). Anal. Calcd for C₁₁H₁₃N₃O₃S (267.30): C, 49.43; H, 4.90; N, 15.72. Found: C, 49.51; H, 4.88; H, 15.84.

4-(5-Methoxy-3-phenyl-1H-pyrazol-1-yl)benzenesulfonamide (3e): Yield 48%. mp 143–145°C. ¹H-NMR (DMSO-d₆) δ: 4.07 (3H, s, OCH₃), 6.51 (1H, s, CH of pyrazole), 7.26–8.09 (11H, m, Ar-H+ex, NH₂). ¹³C-NMR (DMSO-d₆) δ: 59.6, 85.0, 120.7, 127.2, 127.8, 128.5, 128.7, 132.5, 141.1, 141.2, 126.7, 156.6. IR (KBr) cm⁻¹: 3410, 3279 (NH₂), 3060 (CH aromatic), 2924, 2854 (CH aliphatic). MS m/z: 329 (M⁺). Anal. Calcd for C₁₆H₁₅N₃O₃S (329.37): C, 58.34; H, 4.59; N, 12.76. Found: C, 58.36; H, 4.50; N, 12.89.

General Procedure for the Synthesis of Compounds 4a–d

A solution of dimethylamine hydrochloride (0.81 g, 10 mmol) in 37% HCHO (0.30 g, 10 mmol) was stirred at room temperature for 30 min; acetic anhydride (3.20 mL) was added dropwise at (70–90°C). After being stirred for 30 min, compound 2a–d (6.70 mmol) was added and the mixture was stirred for 12 h at (70–75°C). After cooling to room temperature, the reaction mixture was poured onto ice water, neutralized with ammonia solution. The solid was collected, dried and crystallized from ethanol.

1-(4-Chlorophenyl)-4-((dimethylamino)methyl)-3-methyl-1H-pyrazol-5(4H)-one (4a): Yield 82%. mp 203–204°C. ¹H-NMR (CDCl₃) δ: 2.09 (3H, s, CH₃), 2.24 (6H, s, N(CH₃)₂), 2.31–2.35 (1H, m, CH), 2.42–2.45 (1H, m, CH), 3.40–3.50 (1H, m, CH), 7.37–7.45 (4H, m, Ar-H). IR (KBr) cm⁻¹: 3062 (CH aromatic), 2924, 2854 (CH aliphatic), 1712 (C=O). MS m/z: 266 (M⁺+1), 264 (M−1). Anal. Calcd for C₁₃H₁₆ClN₃O (265.73): C, 58.76; H, 6.07; N, 15.81. Found: C, 58.81; H, 6.12; N, 16.04.

1-(4-Chlorophenyl)-4-((dimethylamino)methyl)-3-phenyl-1H-pyrazol-5(4H)-one (4b): Yield 81%. mp 112–114°C. ¹H-NMR (CDCl₃) δ: 1.92 (6H, s, N(CH₃)₂), 2.79–2.85 (1H, m, CH), 2.89–3.05 (1H, m, CH), 3.30–3.40 (1H, m, CH), 7.10–7.92 (9H, m, Ar-H); ¹³C NMR (CDCl₃): δ 29.6, 30.2, 61.2, 126.1, 128.1, 128.8, 129.1, 130.3, 133.0, 136.0, 142.6, 151.8, 166.3. IR (KBr) cm⁻¹: 3059, 3032 (CH aromatic), 2924, 2850 (CH aliphatic), 1718 (C=O). MS m/z: 329 (M⁺+2), 327 (M⁺). Anal. Calcd for C₁₈H₁₈ClN₃O (327.80): C, 65.95; H, 5.53; N, 12.82. Found: C, 66.03; H, 5.51; N, 12.98.

4-(4-((Dimethylamino)methyl)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (4c): Yield 80%. mp 194–196°C. ¹H-NMR (DMSO-d₆) δ: 1.89 (3H, s, CH₃), 2.07 (6H, s, N(CH₃)₂), 2.70–2.80 (1H, m, CH), 2.88–3.00 (1H, m, CH), 3.38–3.44 (1H, m, CH), 7.37 (2H, s, ex, NH₂), 7.83–7.67 (4H, m, Ar-H). IR (KBr) cm⁻¹: 3414, 3232 (NH₂), 3082 (CH aromatic), 2920, 2850 (CH aliphatic), 1716 (C=O), 1334, 1161 (SO₂). MS, m/z: 310 (M⁺). Anal. Calcd for C₁₃H₁₈N₄O₃S (310.37): C, 50.31; H, 5.85; N, 18.05. Found: C, 50.28; H, 5.91; N, 18.24.

4-(4-((Dimethylamino)methyl)-5-oxo-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (4d): Yield 93%. mp 128–130°C. ¹H-NMR (DMSO-d₆) δ: 1.95 (6H, s, N(CH₃)₂), 2.75–2.78 (1H, m, CH), 2.90–2.94 (1H, m, CH), 3.38–3.44 (1H, m, CH), 6.99 (2H, s, ex, NH₂), 7.30–8.12 (9H, m, Ar-H). IR (KBr) cm⁻¹: 3425, 3221 (NH₂), 3062 (CH aromatic), 2924, 2854 (CH aliphatic), 1720 (C=O), 1369, 1161 (SO₂); MS m/z: 372 (M⁺). Anal. Calcd for C₁₈H₂₀N₄O₃S (372.44): C, 58.05; H, 5.41; N, 15.04. Found: C, 58.12; H, 5.38; N, 15.22.

General Procedure for the Synthesis of Compounds 5a–d

To a solution of 4a–d (10 mmol) in acetone (50 mL), iodomethane (2.31 g, 16.30 mmol) was added below 5°C in an ice bath. Then the mixture was stirred at room temperature over night. The product was concentrated under vacuum and the obtained residue was dissolved in methanol then a solution of KCN (1.57 g, 24.20 mmol) in water (1 mL) was added. The solution was stirred at room temperature over night and poured onto ice water. The resulting mixture was extracted with CHCl₃. The extract was washed with water, dried over anhydrous Na₂SO₄, and concentrated under vacuum.

2-(1-(4-Chlorophenyl)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-yl)acetonitrile (5a): Yield 65% as oil. ¹H-NMR (CDCl₃) δ: 2.06 (3H, s, CH₃), 2.19–2.24 (1H, m, CH), 2.71–2.74 (1H, m, CH), 2.89–2.92 (1H, m, CH), 7.80–7.95 (4H, m, Ar-H). IR (KBr) cm⁻¹: 3060 (CH aromatic), 2926, 2856 (CH aliphatic), 2343 (CN), 1762 (C=O). MS m/z: 251 (M⁺+4), 249 (M⁺+2). Anal. Calcd for C₁₂H₁₀ClN₃O (247.68): C, 58.19; H, 4.07; N, 16.97. Found: C, 58.27; H, 4.12; N, 17.26.

2-(1-(4-Chlorophenyl)-5-oxo-3-phenyl-4,5-dihydro-1H-pyrazol-4-yl)acetonitrile (5b): Yield 88% as oil. ¹H-NMR (CDCl₃) δ: 2.02–2.19 (1H, m, CH), 2.71–2.74 (1H, m, CH), 2.92–2.96 (1H, m, CH), 7.19–7.90 (9H, m, Ar-H). ¹³C-NMR (CDCL₃) δ: 25.8, 43.8, 119.9, 126.9, 128.4, 128.5, 128.6, 128.7, 129.7, 136.5, 138.0, 148.9, 162.5. IR (KBr) cm⁻¹: 3060 (CH aromatic), 2921, 2853 (CH aliphatic), 2253 (CN), 1716 (C=O). MS m/z: 311 (M⁺+2), 309 (M⁺). Anal. Calcd for C₁₇H₁₂ClN₃O (309.74): C, 65.92; H, 3.90; N, 13.57. Found: C, 65.97; H, 3.88; N, 13.71.

4-(4-(Cyanomethyl)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (5c): Yield 88% as oil. ¹H-NMR (CDCl₃) δ: 2.06 (3H, s, CH₃), 2.16–2.22 (1H, m, CH), 2.68–2.72 (1H, m, CH), 2.88–2.92 (1H, m, CH), 7.68–8.06 (6H, m, Ar-H+ex, NH₂). IR (KBr) cm⁻¹: 3421, 3201 (NH₂), 3066, 3047 (CH aromatic), 2924, 2845 (CH aliphatic), 2260 (CN), 1705 (C=O), 1338, 1161 (SO₂). MS m/z: 294 (M⁺+2). Anal. Calcd for C₁₂H₁₂N₄O₃S (292.31): C, 49.31; H, 4.14; N, 19.17. Found: C, 49.29; H, 4.31; N, 19.43.

4-(4-(Cyanomethyl)-5-oxo-3-phenyl-4,5-dihydro-1H-pyrazol-yl)benzenesulfonamide (5d): Yield 61% as oil. ¹H-NMR (CDCl₃) δ: 2.23–2.29 (1H, m, CH), 2.52–2.57 (1H, m, CH), 2.90–2.93 (1H, m, CH), 7.35–8.00 (11H, m, Ar-H+ex, NH₂). IR (KBr) cm⁻¹: 3050 (CH aromatic), 2900, 2850 (CH aliphatic), 2260 (CN), 1707 (C=O), 1371, 1159 (SO₂). MS m/z: 356 (M⁺+2). Anal. Calcd for C₁₇H₁₄N₄O₃S (354.38): C, 57.62; H, 3.98; N, 15.81. Found: C, 57.71; H, 4.07; N, 16.12.

General Procedure for the Synthesis of Compounds 6a–d

A solution of 5a–d (10 mmol) in 65% sulfuric acid (40 mL) was refluxed 3 h and poured into ice water. The solid was filtered, washed with water, dried and crystallized from ethanol.

2-(1-(4-Chlorophenyl)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-yl)acetic Acid (6a): Yield 47%. mp 168–170°C. ¹H-NMR (DMSO-d₆) δ: 2.18–2.34 (4H, m, CH₃+CH), 2.72–2.77 (1H, m, CH), 2.90–2.95 (1H, m, CH), 7.48–7.65 (4H, m, Ar-H), 7.27 (1H, s, ex, OH). IR (KBr) cm⁻¹: 3406–2503 (OH carboxylic), 2924, 2850 (CH aliphatic), 1716 (C=O). MS m/z: 268 (M⁺+2), 266 (M⁺). Anal. Calcd for C₁₂H₁₁ClN₂O₃ (266.68): C, 54.05; H, 4.16; N, 10.50. Found: C, 54.12; H, 4.19; N, 10.77.

2-(1-(4-Chlorophenyl)-5-oxo-3-phenyl-4,5-dihydro-1H-pyrazol-4-yl)acetic Acid (6b): Yield 71%. mp 220–223°C. ¹H-NMR (DMSO-d₆) δ: 2.72–2.77 (1H, m, CH), 2.85–2.88 (1H, m, CH), 3.29–3.33 (1H, m, CH), 7.41–7.99 (10H, m, Ar-H+ex, OH). IR (KBr) cm⁻¹: 3402–2495 (OH carboxylic), 2927 (CH aliphatic), 1716 (C=O). MS m/z: 330 (M⁺+2), 328 (M⁺). Anal. Calcd for C₁₇H₁₃ClN₂O₃ (328.74): C, 62.11; H, 3.99; N, 8.52. Found: C, 62.19; H, 4.04; N, 8.68.

2-(3-Methyl-5-oxo-1-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazol-4-yl)acetic Acid (6c): Yield 65%. mp above 350°C. ¹H-NMR (DMSO-d₆) δ: 2.04 – 2.30 (6H, m, CH₃+CH₂+CH), 7.30–7.87 (6H, m, Ar-H+ex, NH₂), 8.08 (1H, s, ex, OH). IR (KBr) cm⁻¹: 3298–2669 (OH carboxylic), 3298, 3240 (NH₂), 3101 (CH aromatic), 2920, 2850 (CH aliphatic), 1720 (C=O), 1330, 1157 (SO₂). MS m/z: 312 (M⁺+1); Anal. Calcd for C₁₂H₁₃N₃O₅S (311.31): C, 46.30; H, 4.21; N, 13.50. Found: C, 46.71; H, 4.42; N, 13.69.

2-(5-Oxo-3-phenyl-1-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazol-4-yl)acetic Acid (6d): Yield 69%. mp 235–236°C. ¹H-NMR (DMSO-d₆) δ: 2.05–2.31 (3H, m, CH₂+CH), 7.32 (2H, s, ex, NH₂), 7.42–8.06 (9H, m, Ar-H), 8.08 (1H, s, ex, OH). IR (KBr) cm⁻¹: 3352–2550 (OH carboxylic), 3352, 3251 (NH₂), 3066 (CH aromatic), 2920, 2850 (CH aliphatic), 1720 (C=O), 1330, 1161 (SO₂). MS m/z: 373 (M⁺). Anal. Calcd for C₁₇H₁₅N₃O₅S (373.38): C, 54.68; H, 4.05; N, 11.25. Found: C, 54.97; H, 4.18; N, 11.38.

General Procedure for the Synthesis of Compounds 7a–d

A suspension of 6a–d (5 mmol) in thionyl chloride (5 mL) was heated gently under reflux until a homogenous solution was obtained then for further 45 min. The solution was then evaporated to dryness under vacuum in a water bath to remove excess thionyl chloride. The residue was azeotroped three times with dry benzene (5 mL) each, where the last traces of thionyl chloride were removed. The residue was then refluxed in sodium methoxide (10 mL) for 3 h. The reaction mixture poured onto ice water, then extracted with chloroform. The extract was washed with water, dried over anhydrous Na₂SO₄, and concentrated under vaccum, dried and crystallized from ethanol.

Methyl 2-(1-(4-Chlorophenyl)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-yl)acetate (7a): Yield 40%. mp 115–116°C (decompose). ¹H-NMR (CDCl₃) δ: 1.84 (3H, s, CH₃), 2.38–2.52 (1H, m, CH), 2.67–2.72 (1H, m, CH), 3.10–3.16 (1H, m, CH), 3.88 (3H, s, OCH₃), 7.42–7.79 (4H, m, Ar-H). IR (KBr) cm⁻¹: 2924, 2850 (CH aliphatic), 1770, 1631 (2C=O). MS m/z: 282 (M⁺+2), 280 (M⁺). Anal. Calcd for C₁₃H₁₃ClN₂O₃ (280.70): C, 55.62; H, 4.67; N, 9.98. Found: C, 55.67; H, 4.72; N, 10.13.

Methyl 2-(1-(4-Chlorophenyl)-5-oxo-3-phenyl-4,5-dihydro-1H-pyrazol-4-yl)acetate (7b): Yield 45%. mp above 350°C. ¹H-NMR (CDCl₃) δ: 2.05–2.08 (1H, m, CH), 2.86–2.88 (1H, m, CH), 2.94–2.96 (1H, m, CH), 3.71 (3H, s, OCH₃), 7.44–8.04 (9H, m, Ar-H). IR (KBr) cm⁻¹: 3059 (CH aromatic), 2924, 2854 (CH aliphatic), 1774, 1716 (2 C=O). MS m/z: 343 (M⁺+1), 341 (M−1). Anal. Calcd for C₁₈H₁₅ClN₂O₃ (342.77): C, 63.07; H, 4.41; N, 8.17. Found: C, 63.16; H, 4.47; N, 8.31.

Methyl 2-(3-Methyl-5-oxo-1-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazol-4-yl)acetate (7c): Yield 70%. mp 222–223°C. ¹H-NMR (DMSO-d₆) δ: 2.14 (3H, s, CH₃), 2.05–2.11 (1H, m, CH), 2.40–2.43 (1H, m, CH), 2.65–2.70 (1H, m, CH), 3.16 (3H, s, OCH₃), 7.32–8.08 (6H, m, Ar-H+ex, NH₂). IR (KBr) cm⁻¹: 3344, 3251 (NH₂), 3105, 3078 (CH aromatic), 2924, 2854 (CH aliphatic), 1724, 1620 (2 C=O), 1327, 1157 (SO₂). MS m/z: 327 (M⁺+2). Anal. Calcd for C₁₃H₁₅N₃O₅S (325.34): C, 47.99; H, 4.65; N, 12.92. Found: C, 48.13; H, 4.42; N, 13.16.

Methyl 2-(5-Oxo-3-phenyl-1-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazol-4-yl)acetate (7d): Yield 67%. mp 229–230°C. ¹H-NMR (DMSO-d₆) δ: 2.22–2.44 (2H, m, CH₂), 2.65–2.70 (1H, m, CH), 3.16 (3H, s, OCH₃), 7.29–8.20 (11H, m, Ar-H+ex, NH₂). IR (KBr) cm⁻¹: 3363, 3259 (NH₂), 3105, 3066 (CH aromatic), 2924, 2854 (CH aliphatic), 1728, 1620 (2 C=O), 1330, 1161 (SO₂). MS m/z: 387 (M⁺). Anal. Calcd for C₁₈H₁₇N₃O₅S (387.40): C, 55.80; H, 4.42; N, 10.85. Found: C, 56.24; H, 4.63; N, 11.22.

General Procedure for the Synthesis of Compounds 8a–d

To a suspension of 2a–d (6 mmol) in isopropanol (12 mL), N,N-dimethylformamide diethylacetal (2.87 mL, 19.50 mmol) was added and stirred for 2 h at room temperature. An additional amount of N,N-dimethylformamide diethylacetal (0.95 mL, 6.50 mmol) was further added and the reaction mixture was stirred at room temperature for another 20 h. The formed precipitate was filtered off, washed with isopropanol, dried and recrystallized from isopropanol.

1-(4-Chlorophenyl)-4-((dimethylamino)methylene)-3-methyl-1H-pyrazol-5(4H)-one (8a): Yield 77%. mp 203–204°C. ¹H-NMR (CDCl₃) δ: 2.14 (3H, s, CH₃), 3.21 (3H, s, N(CH₃)), 3.78 (3H, s, N(CH₃)), 6.95 (1H, s, CHN(CH₃)₂), 7.28 (2H, d, Ar-H, J=8.7 Hz), 7.98 (2H, d, Ar-H, J=9.0 Hz). ¹³C-NMR (CDCl₃) δ: 13.7, 43.3, 48.0, 99.2, 120.1, 128.4, 128.6, 138.2, 152.2, 162.0. IR (KBr) cm⁻¹: 3032 (CH aromatic), 2920 (CH aliphatic), 1674 (C=O). MS m/z: 265 (M⁺+2), 263 (M⁺). Anal. Calcd for C₁₃H₁₄ClN₃O (263.72): C, 59.21; H, 5.35; N, 15.93. Found: C, 59.36; H, 5.48; N, 16.26.

1-(4-Chlorophenyl)-4-((dimethylmino)methylene)-3-phenyl-1H-pyrazol-5(4H)-one (8b): Yield 81%. mp 180–182°C. ¹H-NMR (CDCl₃) δ: 3.29 (3H, s, N(CH₃)), 3.81 (3H, s, N(CH₃)), 7.26 (1H, s, CHN(CH₃)₂), 7.31–8.07 (9H, m, Ar-H). IR (KBr) cm⁻¹: 3055 (CH aromatic), 2924 (CH aliphatic), 1670 (C=O). MS m/z: 327 (M⁺+2), 325 (M⁺). Anal. Calcd for C₁₈H₁₆ClN₃O (325.79): C, 66.36; H, 4.95; N, 12.90. Found: C, 66.45; H, 4.89; N, 13.12.

N′-(4-(4-((Dimethylamino)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)phenylsulfonyl)-N,N-dimethylformimidamide (8c): Yield 79%. mp 234–236°C. ¹H-NMR (CDCl₃) δ: 2.20 (3H, s, CH₃), 3.00 (3H, s, NCHN(CH₃)), 3.11 (3H, s, NCHN(CH₃)), 3.34 (3H, s, N(CH₃)), 3.83 (3H, s, N(CH₃)), 7.05 (1H, s, CHN(CH₃)₂), 7.85 (2H, d, Ar-H, J=8.7 Hz), 8.09 (1H, s, (NCHN(CH₃)₂), 8.13 (2H, d, Ar-H, J=8.7 Hz). IR (KBr) cm⁻¹: 3078, 3032 (CH aromatic), 2924, 2854 (CH aliphatic), 1674 (C=O), 1330, 1145 (SO₂). MS m/z: 363 (M⁺). Anal. Calcd for C₁₆H₂₁N₅O₃S (363.43): C, 52.88; H, 5.82; N, 19.27. Found: C, 52.91; H, 5.87; N, 19.35.

N′-(4-(4-((Dimethylamino)methylene)-5-oxo-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)phenylsulfonyl)-N,N-dimethylformimidamide (8d): Yield 77%. mp 183–185°C. ¹H-NMR (CDCl₃) δ: 3.01 (3H, s, NCHN(CH₃)), 3.11 (3H, s, NCHN(CH₃)), 3.33 (3H, s, N(CH₃)), 3.78 (3H, s, N(CH₃)), 6.01 (1H, s, CHN(CH₃)₂), 7.42–8.26 (10H, m, Ar-H+NCHN(CH₃)₂). IR (KBr) cm⁻¹: 3055, 3028 (CH aromatic), 2927 (CH aliphatic), 1678 (C=O), 1330, 1141 (SO₂). MS m/z: 425 (M⁺). Anal. Calcd for C₂₁H₂₃N₅O₃S (425.50): C, 59.28; H, 5.45; N, 16.46. Found: C, 59.32; H, 5.48; N, 16.63.

General Procedure for the Synthesis of Compounds 9a–d

A mixture of 8a–d (3.80 mmol), NaOH (0.56 g, 14 mmol), isopropanol (15 mL) and water (15 mL) was refluxed 7 h. The isopropanol was removed under vaccum, ice water (10 mL) was added to the residue, then acidified with 10% hydrochloric acid till pH=1, the solid formed was collected by filteration, washed with water, dried and crystallized from isopropanol.

1-(4-Chlorophenyl)-4-(hydroxymethylene)-3-methyl-1H-pyrazol-5(4H)-one (9a): Yield 67%. mp 316–318°C. ¹H-NMR (CDCl₃) δ: 2.42 (3H, s, CH₃), 4.70 (1H, s, ex, OH), 7.42 (2H, d, Ar-H, J=9.0 Hz), 7.79 (2H, d, Ar-H, J=9.0 Hz), 9.43 (1H, s, CH–OH). ¹³C-NMR (CDCl₃) δ: 29.6, 106.0, 121.0, 126.0, 129.0, 132.0, 149.0, 159.0, 183.4. IR (KBr) cm⁻¹: 3414 (OH), 3050 (CH aromatic), 2924, 2850 (CH aliphatic), 1658 (C=O). MS m/z: 238 (M⁺+2), 236 (M⁺). Anal. Calcd for C₁₁H₉ClN₂O₂ (236.65): C, 55.83; H, 3.83; N, 11.84. Found: C, 55.91; H, 3.87; N, 12.15.

1-(4-Chlorophenyl)-4-(hydroxymethylene)-3-phenyl-1H-pyrazol-5(4H)-one (9b): Yield 76%. mp 126–127°C. ¹H-NMR (CDCl₃) δ: 5.00 (1H, s, ex, OH), 7.45–7.92 (9H, m, Ar-H), 9.72 (1H, s, CH–OH). IR (KBr) cm⁻¹: 3251 (OH), 3062 (CH aromatic), 2924, 2850 (CH aliphatic), 1643 (C=O). MS m/z: 300 (M⁺+2), 298 (M⁺). Anal. Calcd for C₁₆H₁₁ClN₂O₂ (298.72): C, 64.33; H, 3.71; N, 9.38. Found: C, 64.48; H, 3.82; N, 9.62.

4-(4-(Hydroxymethylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (9c): Yield 57%. mp 144–146°C. ¹H-NMR (DMSO-d₆) δ: 2.34 (3H, s, CH₃), 4.93 (1H, s, ex, OH), 7.38 (2H, s, ex, NH₂), 7.90 (2H, d, Ar-H, J=8.7 Hz), 7.98 (2H, d, Ar-H, J=9.0 Hz), 9.28 (1H, s, CH–OH). IR (KBr) cm⁻¹: 3468 (OH), 3309, 3205 (NH₂), 3039 (CH aromatic), 2800 (CH aliphatic), 1701 (C=O), 1323, 1157 (SO₂). MS m/z: 281 (M⁺). Anal. Calcd for C₁₁H₁₁N₃O₄S (281.28): C, 46.97; H, 3.94; N, 14.94. Found: C, 47.11; H, 4.03; N, 15.18.

4-(4-(Hydroxymethylene)-5-oxo-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (9d): Yield 58%. mp 237–239°C (decomposed). ¹H-NMR (DMSO-d₆) δ: 6.10 (s, 1H, ex, OH), 7.43 (2H, s, ex, NH₂), 7.44–8.16 (9H, m, Ar-H), 9.35 (1H, s, CH–OH). IR (KBr) cm⁻¹: 3564 (OH), 3356, 3267 (NH₂), 3062 (CH aromatic), 2924, 2854 (CH aliphatic), 1627 (C=O), 1307, 1161 (SO₂). MS m/z: 343 (M⁺). Anal. Calcd for C₁₆H₁₃N₃O₄S (343.35): C, 55.97; H, 3.82; N, 12.24. Found: C, 56.34; H, 4.08; N, 12.68.

General Procedure for the Synthesis of Compounds 10a–d

To a cold stirred solution of 9b or 9d (2.50 mmol) and NaOH (0.20 g, 5 mmol) in distilled water, acetyl chloride or benzoyl chloride (4 mmol) was added at 5°C portion wise. The reaction mixture was stirred at room temperature for 2h, and then acidified with 10% hydrochloric acid till neutralization; the solid formed was filtered, washed with water, dried and crystallized from ethanol.

(1-(4-Chlorophenyl)-5-oxo-3-phenyl-1H-pyrazol-4(5H)-ylidene)methyl Acetate (10a): Yield 58%. mp 290–291°C. ¹H-NMR (DMSO-d₆) δ: 2.49 (3H, s, CH₃), 7.44–8.00 (10H, m, Ar-H+C=CH). IR (KBr) cm⁻¹: 3055 (CH aromatic), 2924, 2850 (CH aliphatic), 1700 (2 C=O). MS m/z: 340 (M⁺), 338 (M-2). Anal. Calcd for C₁₈H₁₃ClN₂O₃ (340.76): C, 63.44; H, 3.85; N, 8.22. Found: C, 63.42; H, 3.89; N, 8.45.

(1-(4-Chlorophenyl)-5-oxo-3-phenyl-1H-pyrazol-4(5H)-ylidene)methyl Benzoate (10b): Yield 65%. mp 286–288°C. ¹H-NMR (CDCl₃) δ: 7.42–8.06 (15H, m, Ar-H+C=CH). IR (KBr) cm⁻¹: 3055 (CH aromatic), 1693 (2C=O). MS m/z: 404 (M⁺+2), 402 (M⁺). Anal. Calcd for C₂₃H₁₅ClN₂O₃ (402.82): C, 68.58; H, 3.75; N, 6.95. Found: C, 68.63; H, 3.72; N, 7.08.

(5-Oxo-3-phenyl-1-(4-sulfamoylphenyl)-1H-pyrazol-4(5H)-ylidene)methyl Acetate (10c): Yield 66%. mp 336–337°C. ¹H-NMR (DMSO-d₆) δ: 2.46 (3H, s, CH₃), 7.32–8.04 (12H, m, Ar-H+C=CH+ex, NH₂). IR (KBr) cm⁻¹: 3329, 3271 (NH₂), 3070 (CH aromatic), 2981, 2924 (CH aliphatic), 1732 (2 C=O). MS m/z: 385 (M⁺). Anal. Calcd for C₁₈H₁₅N₃O₅S (385.39): C, 56.10; H, 3.92; N, 10.90. Found: C, 56.18; H, 4.03; N, 11.04.

(5-Oxo-3-phenyl-1-(4-sulfamoylphenyl)-1H-pyrazol-4(5H)-ylidene)methyl Benzoate (10d): Yield 78%. mp 274–276°C. ¹H-NMR (DMSO-d₆) δ: 7.23–8.16 (17H, m, Ar-H+C=CH+ex, NH₂). IR (KBr) cm⁻¹: 3344, 3251 (NH₂), 3070, 3028 (CH aromatic), 2962, 2924 (CH aliphatic), 1685 (2C=O). MS m/z: 447 (M⁺). Anal. Calcd for C₂₃H₁₇N₃O₅S (447.46): C, 61.74; H, 3.83; N, 9.39. Found: C, 61.81; H, 3.79; N, 9.52.

Pharmacological Screening

The experimental tests on animals have been performed in accordance with the Institutional Ethical Committee approval, Faculty of Pharmacy, Cairo University.

Anti-inflammatory (in Vivo Screening)

All the targeted compounds were evaluated for their anti-inflammatory activity using carrageenan-induced rat paw edema method of Winter et al.³⁸⁾ The employed technique is based on the ability of the tested compounds to inhibit the edema produced in the hind paw of the rat after injection of carrageenan. Male Wister albino rats (obtained from the animal house of Faculty of Pharmacy, Cairo University) weighing 120–180 g were used to investigate the carrageenan-induced rat paw edema. The rats were kept in animal house under standard conditions of light and temperature with free access to food and water. The animals were randomly divided into 38 groups of five rats each. The initial hind paw volume of rats was determined volumetrically by means of plethysmometer 7150 (UGO, Basile, Italy). Phenylbutazone (reference standard) and the tested compounds suspended in 2% Tween 80 were administered intraperitoneally at a dose of 0.32 mmol/kg body weight, while the control group received only 2% Tween 80, 1 h before induction of inflammation. The paw edema was induced by subplantar injection of (0.1 mL) of 1% carrageenan solution in saline (0.9%). Paw edema volume was measured after 2 and 3 h using the plethysmometer and compared with the initial hind paw volume of each rat. The difference of average values between treated and control group is calculated for each time interval and evaluated statistically. Quantitative variables from normal distribution were expressed as means±standard error (S.E.M.). The anti-inflammatory activity was expressed as percentage inhibition of edema volume in treated animals in comparison with the control group according to the following equation:

  

Where V_c is the mean of edema volume of rat paw after administration of carrageenan in the control group, V_t is the mean of edema volume of rat paw after administration of the tested compounds or the reference drugs.

The IC₅₀ values of the compounds 3a, b, d, e, 4a–d, 5a–d, 6a–d, 9a–d, and phenylbutazone were determined according to the previous reported procedure³⁸⁾ using different doses ranged from 0.08 to 1.30 mmol/kg body weight

Analgesic Activity

Compounds that exhibited good anti-inflammatory activity and phenylbutazone were screened for their analgesic activity using the reported method of p-benzoquinone-induced writhing in mice by Okun et al.³⁹⁾ Adult male albino mice weighing 20–25 g were used in this study. Phenylbutazone (reference standard) and the tested compounds were prepared as suspension in 2% Tween 80. A sensitivity test was carried out one day before drug administration,⁴⁷⁾ were the animals were injected intraperitoneally with 0.20–0.25 mL of 0.02% freshly prepared solution of p-benzoquinone in distilled water. Animals showing writhing to p-benzoquinone within 30 min were chosen for studying the analgesic activity. On the next day, mice were divided into 19 groups of six animals each. The control group received only 2% Tween 80 while the rest of the groups received the reference standards and the tested compounds at a dose of 0.32 mmol/kg body weight. After one hour, 0.02% solution of p-benzoquinone was administered intraperitoneally and the animals were observed for 30 min after injection of the irritant, the animals showing writhing were counted in each group. Writhing is known as stretch, torsion to one side, drawing up of hind leg, retraction of the abdomen, so that the belly of mouse touches the floor. The mice showing any of the previous signs are counted as positive responses and this method depend on the ability of tested compounds to protect the animals from writhing signs made by p-benzoquinone. The analgesic activity was evaluated as the percentage protection of tested animals against irritant p-benzoquinone induced writhing response compared with the control group according the following equation:

  

Ulcerogenic Effect

The ulcerogenic effect of the six most active compounds as anti-inflammatory agents and phenylbutazone was evaluated by the reported method of Meshali et al.⁴⁰⁾ Adult male albino rats weighing 120–180 g were used in this study. Animals were fasted eighteen hours before the drug administration, then divided into 8 groups each of five animals and received the drug orally. The first group received 2% tween 80 and kept as control, the second group received phenylbutazone in a dose of 0.32 mmol/kg body weight and the rest of the groups were received 6 tested compounds in the same dose.

Food was allowed two hours after administration of the drugs and rats were received the same dose orally for three successive days. Two hours after the last dose, rats were sacrificed, the stomach of each rat were removed, opened along greater curvature and cleaned by washing with cold saline. The stomach was stretched on a corkboard using pins and examined with a magnifying lens (10×) for the presence of ulcers and erosions. Ulcer index was calculated according to the method of Robert et al.⁴¹⁾ The degree of ulcerogenic effect was expressed in term of percentage incidence of ulcer in each group of animals divided by 10, the average number of ulcers per stomach and the average severity of ulcers by visual observation. The ulcer index was expressed as summation value of the above three values.

Acute Toxicity

LD₅₀ of some representative compounds was determined using Finney’s method.⁴²⁾ Adult male mice weighing 20–25g were divided into groups each of six animals. Minimal dose that killed all animals and the maximal dose that failed to kill any animal were determined via several increasing intraperitoneal doses. Animals were kept under observation for 24 h during which any mortality in each group were recorded.

In Vitro COX Inhibition Study

The most active compounds as anti-inflammatory 6b, 7b, 9b, and indomethacin were tested for their ability to inhibit COX-1 and/or COX-2 using Cayman’s COX-Activity Assay kit (catalogue No. 760151, Cayman Chemicals, Ann Arbor, MI, U.S.A.) which measure the peroxidase activity of COX by the method of Kulmacz and Lands.⁴³⁾ COX-1/COX-2 percentage inhibition activity ratio of the tested compounds and indomethacin was calculated and recorded.

2D-QSAR Study. Data Set

A data set of twenty compounds was used for the present QSAR study. All the molecular modeling calculations and docking simulation studies were performed using Molecular Operating Environment (MOE®) version 10.2010, Chemical Computing Group Inc., Montreal, Canada. The computational software operated under “Windows XP” installed on an Intel Pentium IV PC with a 1.6 GHz processor and 512 MB memory. All the interaction energies and different calculations were automatically calculated. The biological activity values IC₅₀ were converted to negative logarithmic (P IC₅₀) and used as the dependant variable for the QSAR analysis. Thirteen different molecular descriptors (independent variables) were selected and calculated for the submitted structures aiming cover a wide range of different electronic, hydrophobic and topological characters. The correlation matrix was calculated to avoid multicolinearity between the calculated descriptors. The correlation matrix indicated that some of the descriptors used are highly correlated which suggests avoiding the combinations between such intercorrelated descriptors (|r|≥0.80, where r is the simple linear coefficient). QSAR model was then constructed after ensuring reasonable correlation of anti-inflammatory activity with individual descriptor and minimum inter-correlation among the descriptors used in derived model.

Statistical Analysis

A mathematical structure–activity equation established a statistical relationship between a dependent variable (biological activity) and a set of independent variables (descriptors).^48,49) Stepwise linear regression analysis (SLRA) technique was used to test the best structural predictors for activity. For the current dataset of 20 compounds, the QSAR model development was restricted to a maximum of three variables in accordance to the general accepted rule for the compounds: discrirptors ratio to be around 5 : 1. The developed QSAR models are evaluated using the following statistical measures as root mean square error (RMSE) and correlation coefficient (R²) and validated by the leave-out technique (LOO technique), where each object of the data set is taken away, one at a time. In this case, given n objects, n reduced models are developed. The predicted activities (P IC₅₀) for the tested compounds calculated using multi-linear regression ($PRED) technique.

Acknowledgment

The Authors would like to thank Prof. Dr. Ashraf B. El-Den, Department of Pharmacoclogy, Faculty of Pharmacy, Ain-Shams University for carrying out the in vitro screening and Prof. Dr. Nadia Mahfouz, Department of Medicinal Chemistry, Faculty of Pharmacy, Assuit University for carrying out the 2D-QSAR study. Also, thank Dr. Walaa Wadie, Department of Pharmacology, Faculty of Pharmacy, Cairo University for her helping during carrying the in vivo study.

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