Design, synthesis of celecoxib-tolmetin drug hybrids as selective and potent COX-2 inhibitors

Bioorganic Chemistry 90 (2019) 103029

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Design, synthesis of celecoxib-tolmetin drug hybrids as selective and potent COX-2 inhibitors

T

Khaled R.A. Abdellatif , Eman K.A. Abdelall, Madlen B. Labib, Wael A.A. Fadaly, Taha H. Zidan ⁎

Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt

ARTICLE INFO

ABSTRACT

Keywords: Tolmetin analogues Triarylpyrazole COX-2 Celecoxib Anti-inflammatory

Three novel series of diarylpyrazole 10b-d and triarylpyrazole derivatives 11a-d &12a-d were synthesized through Vilsmier-Haack condition. The structures of prepared compounds were determined through IR, 1H NMR, 13 C NMR, Mass spectral and elemental analysis. Docking of the synthesized compounds over COX-2 active site ensure their selectivity. Moreover, the target compounds were evaluated for both in vitro and in vivo inhibitory activity. All compounds were more selective for COX-2 isozyme than COX-1 isozyme and with excellent antiinflammatory activity. Compounds 11b, 11d and 12b showed the highest anti-inflammatory activity (67.4%, 62.7%, 61.4% respectively), lower ulcerogenic liability (UI = 2.00, 2.75, 3.25 respectively) than indomethacin (UI = 14) and comparable to celecoxib (UI = 1.75) which were confirmed from the histopatholgical study.

1. Introduction Inflammation is a normal protective process of body system towards various infectious agents and traumatic process [1–4]. These causative agents cause stimulation of various cyclooxygenase (COX) isozymes to produce inflammatory mediators like prostaglandins (PGs) [5,6]. Suppression of COX isozymes via the use of traditional Non steroidal antiinflammatory drugs (NASIDS) leads to reduction of inflammation [7,8]. There are three different COX isozymes COX-1, COX-2 and COX-3 [7]. COX-1 isozyme is responsible for production of PGs important for some physiological process such as renal function, gastric homeostasis and platelet aggregation [8–11]. COX-2 isozyme is produced during inflammatory process to exert PGs inquired in inflammation and pain [12–15]. COX-3 isozyme is mainly produced in brain and responsible for anti-pyretic action of drugs which can penetrate blood brain barrier like paracetamol [7,16]. Non selective COX inhibitors such as indomethacin (1), tolmetin (2) which are COX isozymes treat inflammation and pain and characterized by high potency [17–20] (Fig. 1). But also, they cause kidney dysfunction and serious gastrointestinal side effects like bleeding and ulcer [17–20]. Tolmetin is a pyrrole derivative used in treatment of rheumatoid arthritis but not used for a long term due to undesirable side effects [21,22]. Regarding tolmetin, gastric side effects results from non selective inhibition of COX isozymes and direct effect of carboxylate group on gastric mucosa [23–25]. On the other hand, selective COX-2 inhibitors as coxibs alle-

⁎

viate inflammation with minimal undesirable gastric side effects [26–28]. Structure features of coxibs showed that they have common central rings with vicinal diarylsubstituents, in addition to the aminosulphonyl or methane sulfonylmoities that proved to be amajor definite of COX-2 selectivity as in celecoxib (3) [29–31]. Certain coxibs have been synthesized and withdrawn from the market due to their cardiovascular side effects as rofecoxib (4) and valdecoxib (5) [32]. Due to side effects of both traditional NSAIDs & coxibs drugs, until now, the necessity for safe NSAIDs is directly attached to pyrazole ring but separated by polar methylene hydrazino spacer to increase flexibility of the molecules, increase hydrogen bond and fitting with receptor. Also, trifluroromethyl group is replaced by phenyl ring carrying sulfamoyl moiety. Additionally, methyl group of tolyl moiety is replaced by variant substituents such as methoxy, dimethoxy, ethoxy and isobutyl groups to explore their electronic effects. Tolmetin pyrrole ring is replaced by pyrazole ring to increase selectivity. Furthermore, removal of acetic acid group to decrease the gastric side effect. The designed hybrids have been docked on COX-2 isozyme using molecular operating environment (MOE) to show the binding interaction between designed molecules and COX-2 active site. Also, novel compounds have been evaluated for their in vitro, in vivo anti inflammatory activity. Moreover, the most active in vivo anti inflammatory activities have been evaluated for their effective dose 50%, ulcerogenic liability and histopathological study (see Fig. 2).

Corresponding author. E-mail address: [email protected] (K.R.A. Abdellatif).

https://doi.org/10.1016/j.bioorg.2019.103029 Received 23 December 2018; Received in revised form 1 April 2019; Accepted 1 June 2019 Available online 03 June 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved.

Bioorganic Chemistry 90 (2019) 103029

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COOH O

H2NO2S

O N

N

N

O

N O Cl

(2) OH

(1)

(3)

SO2CH3

SO2NH2

N

O O

CF3

Me O

(5)

(4)

Fig. 1. Chemical structures of indomethacin (1), tolmetin (2), celecoxib (3), rofecoxib (4), and valdecoxib (5).

Fig. 2. Designed compounds 11a-d and 12a-d as celecoxib-tolmetin drug hybrids.

CeH at δ 2835–2870 cm-1and C]O at δ 1662–1678 cm−1. While, NMR spectral analysis of 9b-d and 10b-d confirmed the formation of pyrazole ring via the presence of signal at δ 9.40–9.43 ppm due to pyrazole proton at C-5 in 1H NMR and three peaks at δ 153.3–154.1, 118.2–123.2, 129.9–136.1 ppm in 13C NMR indicating C-3, C-4, C-5 of pyrazole ring respectively. Also, the presence of signal at δ 9.24–10.01 ppm in 1H NMR and at δ 185.1–185.2 ppm in 13C NMR of compounds 9b-d & 10b-d confirmed the presence of aldehyde moiety. Additionally, 1H NMR of compounds 9b-d displayed the presence of signals due to protecting dimethylaminomethylene moiety at 2.93–3.17 ppm and 8.26–8.27 ppm due to N(CH3)2 and N]CH respectively. Disappearance of these signals in 1H NMR of pyrazoles 10bd and presence of D2O exchangeable signal at 7.49–7.53 ppm due to

2. Results and discussion 2.1. Chemistry The synthetic route used to synthesize the key intermediates 8a-d, 9a-d, the target compounds diarylpyrazole 10a-d and pyrazole4-hydrazino derivatives 11a-d and 12a-d is outlined in Scheme 1. Reaction of hydrazones 8a-d applying Vilsmier-Haack condition yields 4-formyl pyrazole 9a-d endowed with 4-dimethyl aminomethylene moiety that was removed adopting basic condition via the use of NaOH methanolic solution and tetrahydrofuran as a co solvent to give diarylpyrazoles 10a-d. IR spectra of new pyrazoles 9b-d and 10b-d displayed the presence of CHO moiety by two stretching vibration bands of aldehydic 2

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Scheme 1. Synthesis of the target compounds.

SO2NH2 confirmed the structure. Condensation of 4-formyl pyrazoles 10a-d with either 4-hydrazinobenzenesulfonamide hydrochloride (6) or 4-methylsufonylbenzene hydrochloride gave target compounds 11ad and 12a-d respectively. IR spectra of triarylpyrazoles 11a-d & 12a-d showed two characteristic stretching bands at 1311–1276 and 1157–1143 cm-1 indicating SO2 groups. Also, The structure of new compounds 11a-d and 12a-d was proved using NMR spectra through the absence of signals due aldehydic protons and presence of others at δ 7.99–8.35 ppm and δ 10.71–11.24 ppm due to olefinic (N]CH) and NH protons respectively. Additionally, 1H NMR spectra of compounds 12ad showed signals due to SO2CH3 in the range of δ 3.10–3.11 ppm. While 13 C NMR showed peaks of SO2CH3 at 44.71–44.85 ppm. Finally, mass spectroscopy support structure elucidation by the presence of molecular ion peaks of different derivatives such as, 11a (M+, 527), 12b (M+, 540), 12d (M+, 552) respectively.

2.2. Anti-inflammatory activity 2.2.1. In vitro anti-inflammatory activity The target of in vitro testing is to define the ability of synthesized compounds to inhibit ovine COX-1 and human recombinant COX-2 using an enzyme immunoassay (EIA) kit. The efficacy of the tested compounds was expressed as the concentration cause 50% inhibition (IC50). The obtained (IC50) values were listed in Table 1. All tested compounds are weak inhibitors of COX-1 inhibition (IC50 = 3.74–11.21 μM). Compounds 11a, 11band12dshowed the lowest COX-1 (IC50 = 9.88, 10.32, 11.21 μM) in comparison with reference drug celecoxib (IC50 = 8.97 μM). In contrast, all compounds showed good COX-2 isozyme inhibitory activities (IC50 = 0.023–0.125 μM) and selectivity index (SI) values (SI = 85.3–185.2 μM) comparable to reference drug celecoxib 3

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ethoxy 10b (AI = 37.2%) and isobutyl 10d (AI = 48.5%) derivatives showed higher AI activity than dimethoxy analogues 10c (AI = 27.9%). Triarylpyrazoles 11a-d and 12a-d showed better AI activity in range of (AI = 34.8–67.4%) at 6 h than non vicinal diarylpyrazole 10b-d (AI = 27.9–48.8%). The bisaminosulphonyl derivatives 11a-d displayed better anti-inflammatory range (AI = 37.2–67.4%) than aminosulphonyl-methylsulphonyl derivatives 12a-d (AI = 39.5–61.4%) range in comparison with reference drug celecoxib (AI = 72.0%). Within bisaminosulphonyl 11a-d derivatives, the ethoxy11b derivative (AI = 67.4%) and isobutyl derivative 11d (AI = 62.7%) showed higher AI activity than methxoy derivative 11a (AI = 37.2%) and dimethoxyderivative 11c (AI = 34.8%). Compounds 11b, 11d and 12b showed good (AI = 67.4, 62.7, 67.4%) in sequence to standard drug celecoxib (AI = 72.0%). Moreover, Effective dose (ED50) causing 50% edema inhibition was determined for the most potent AI agents 11b, 11d, 12b and celecoxib. Compounds 11b, 11d and12b were more potent (ED50 = 101, 59, 77 µMol/kg respectively) than celecoxib (ED50 = 123 µMol/kg). The isobutyl derivative 11d was the most potent compound possessing twice potency of celecoxib ED50 = 123 µMol/kg). Furthermore, the most active compounds were subjected for determination of ulcer index and histopathlogical study (see Table 3).

Table 1 In vitro COX-1, COX-2, Selectivity Index (SI) of diarylpyrazoles (10b-d) and triarypyrazoles (11a-d and 12a-d) and reference drug celecoxib. Compound

COX-1 (IC50)a

COX-2 (IC50)a

Selectivity Index SI (COX-1/COX-2)b

10b 10c 10d 11a 11b 11c 11d 12a 12b 12c 12d Celecoxib

9.34 5.74 3.74 9.88 10.32 9.23 8.45 4.98 8.11 6.12 11.21 8.97

0.062 0.033 0.023 0.077 0.056 0.09 0.055 0.037 0.095 0.049 0.125 0.063

150.6 173.9 162.6 128.3 185.2 115.3 153.5 134.5 85.3 124.8 89.6 142.3

a The concentration of test compound produce 50% inhibition of COX-1, COX-2 enzyme, the result is the mean of three values obtained by assay of enzyme kits obtained from (Cayman Chemicals Inc., Ann Arbor, MI, USA). b The in vitro COX-2 selectivity index(COX-1/COX-2).

(IC50 = 0.063 μM, SI = 142.2 μM). The bisaminosulfonyl derivatives 11a-d showed higher inhibitory activity against COX-2 isozyme (IC50 = 0.055–0.09 μM) range and accordingly higher SI (SI = 128.3, 185.2, 115.3, 153.5 μM respectively) than aminosulphonyl-methylsulphonyl compound 12a-d with (IC50 = 0.037–0.125 μM) range and SI (SI = 85.3–134.5 μM). The ethoxy11b and isobutyl 11d showed have higher SI values (SI = 185.2, 153.5 μM respectively) than methoxy 11a and dimethoxy 11c analogues (SI = 128.3, 115.3 μM respectively). Compounds 10c, 10d, 11b, 11d and 12a exhibited the highest COX-2 selectivity (SI = 173.9, 162.6, 185.2, 153.5, 134.5 μM respectively).

2.2.3. Ulcerogenic liability The most potent AI compounds 11b, 11d and 12b were subjected to further examination to determine their ulcerogenic effect (Ulcer Index). The results are expressed in Table 4. The results revealed that the three compounds were less ulcerogenic (Ulcer Index) (UI) = 2.75, 2.00, 3.25 respectively) than indomethacin (UI = 14) and comparable to celecoxib (UI = 1.75). The isobutyl derivative 11d that showed superior COX-2 selectivity was the least ulcerogenic compound.

2.2.2. In vivo anti-inflammatory activity The diarylpyrazoles 10b-d and triarylpyrazoles 11a-d and 12a-d were evaluated for the in vivo anti-inflammatory activity (AI). The AI was determined using carrageenan - induced rat paw edema assay using 100 mg/kg of the tested compounds and the% edema inhibition after 1 h, 3 h and 6 h of carrageenan injection was expressed in Table 2. Generally, all compounds showed good AI activity at all time intervals. After 1 h, they showed moderate AI activity with % inhibition range (AI = 10.87–63.04%) in comparison with reference drug celecoxib (AI = 45.65%). After 3 h and 6 h the activity increased gradually. After 6 h, diarylpyrazoles 10b-d showed moderate (AI = 27.9–48.5%) activity compared to celecoxib (AI = 72.0%). Within diarylpyrazoles the

2.2.4. Docking study Molecular Docking is one of the most important techniques in drug discovery [33]. The crystal structure of COX-2 isozyme with its selective inhibitor celecoxib was obtained from PDB (ID.3LN1). Docking calculations were carried out using molecular operating environment (MOE, version 2008). From docking study, it was observed that celecoxib interact with COX-2 active site through sulfamoyl moiety NH– to form hydrogen bonds with amino acid ser339, Glu178, Leu338. While the most active compounds 11b, 11d and 12b showed multiple bond formation with receptor site. The triarylpyrazole derivative 11b interacts with COX-2 receptor through formation of hydrogen bond with Glu510, Tyr371 through sulfamoyl moiety NeH. Also, it form H-bond and hydrophobic interaction with Arg106 indicate the observed potent activity. Moreover, triarylpyrazole 11d forms multiple hydrogen bond through both aminosulphamoyl moieties through both nitrogen and oxygen atoms which may potentiate the high potency due to presence of two sulfamoyl moieties. Furthermore, triaryl pyrazole 12b compounds forms hydrogen bond through SO2NH2 with Leu338, Gln178, His175 and another hydrogen bond with Leu82 through SO2CH3 oxygen atom (see Fig. 3 and Table 5).

Table 2 In vivo anti-inflammatory activity of diarylpyrazoles (10b-d) and triarypyrazoles (11a-d and 12a-d) and reference drug celecoxib. Compound no

10b 10c 10d 11a 11b 11c 11d 12a 12b 12c 12d Celecoxib

% inhibition of rat paw edema 1 ha

3 ha

6 ha

50.32 56.12 32.22 63.04 40.74 26.09 45.46 17.39 10.87 34.78 17.39 59.26

20.37 51.56 55.56 33.33 40.74 46.30 38.89 62.96 72.22 31.48 59.26 59.26

37.21 27.91 48.52 37.21 67.44 34.88 62.79 44.19 61.44 53.49 39.53 72.09

Table 3 ED50 for the most active compound 11b, 11d, 12b and celecoxib. Compound No

11b 11d 12b Celecoxib

a Inhibitory activity of compounds determined at 1 h, 3h and 6 h after carrageenan injection. The results expressed as % inhibition using one way ANOVA followed by post Hook (Tukey′s test) for multiple pear wise comparison between different compounds at P < 0.01.

% inhibition of rat paw edema

ED50

50 mg/kga

100 mg/kga

150 mg/kga

(mg/kg)a

(µMol/kg)a

47.5 53.5 51.2 49.5

67.4 62.7 61.4 72.26

82.3 76.7 72.1 85.3

55 32 43 47

101 59 77 123

a Inhibitory activity in carrageenan-induced rat paw edema assay. The results are expressed at 6 h after oral administration of the test compound.

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CT, USA) at the regional center for mycology and Bio-technology, Alazhar University, Egypt. All data were within ± 0.4% of the theoretical values and were corrected.

Table 4 Ulcerogenic liability for the most active triarylpyrazoles 11b, 11d, 12b indomethacin and celecoxib. Compound no

% incidence

Average no of ulcer

Average severity

Ulcer index

11b 11d 12b Indomethacin Celecoxib

0.5 1.5 2.5 9 1.00

0.75 0.5 0.25 2 0.25

0.75 0.75 0.5 3 0.5

2.00a 2.75a 3.25a 14 1.75a

4.1.1. General procedure for synthesis of hydrazones 8a-d To a solution of the appropriate acetophenones 6a-d (0.04 mol) in absolute ethanol (20 mL), p-sulfamoyl phenyl hydrazine hydrochloride (7) (8.9 g, 0.04 mol) and glacial acetic acid (1 mL) were added. The reaction mixture was heated under reflux for 12 h. The solidprecipitated was filtered, washed with ethanol, dried and recrystalized to give 8a [34] and 8b-d compounds. 1-[4-(Ethoxyphenyl)ethylidene]-2-(4-sulfamoylphenyl)hydrazine (8b): Yield (2.29 g, 69%); white solid; mp 220–222 °C; IR (KBr): 3318, 3195 (NH2), 3062 (CeH aromatic), 2975 (CeH aliphatic), 1599 (C]N), 1312, 1145 (SO2) cm−1. 1 H NMR (DMSO-d6, 400 MHz, δ ppm): 1.34 (t, J = 6.8 Hz, 3H, CH2CH3), 2.26 (s, 3H, CH3), 4.04 (q, J = 6.8 Hz, 2H, CH2CH3), 6.93 (d, J = 8.0 Hz, 2H, sulfamoylphenyl H-2, H-6), 7.06 (s, 2H, SO2NH2, D2O exchangeable), 7.30 (d, J = 8.0 Hz, 2H, ethoxyphenyl H-3, H-5), 7.66 (d, J = 8.0 Hz, 2H, ethoxyphenyl H-2, H-6), 7.75 (d, J = 8.0 Hz, 2H, sulfamoylphenylH-3, H-5), 9.60 (s, 1H, NH, D2O exchngeable).13C NMR (DMSO-d6, 100 MHz, δ ppm): 13.5 (CH2CH3), 15.0 (CH3), 63.5 (CH2CH3), 112.2 (CH, ethoxyphenyl C-3, C5), 114.6 (CH, sulfamoylphenyl C-2, C-6), 127.3 (CH, sulfamoylphenyl C-3, C-5), 127.7 (CH, ethoxyphenyl C-2, C-6), 131.6 (C, ethoxyphenyl C-1), 133.6 (C, sulfamoylphenyl C-4), 143.8 (C, sulfamoylphenyl C-1), 149.2 (C, ethoxyphenyl C-4), 159.1 (C, C]N). MS m/z (ES+), 334 (M++1, 20%), 333 (M+, 100%), Anal. Calcd. for: C16H19N3O3S; C, 57.64; H, 5.74; N, 12.60; Found; C, 57.43; H, 5.83; N, 12.87. 1-[(3,4-Dimethoxyphenyl)ethylidene]-2-(4-sulfamophenyl)hydrazine (8c): Yield (2.51 g, 72%); white solid; mp 208–210 °C; IR (KBr): 3323, 3204 (NH2), 3082 (CeH aromatic), 2963 (CeH aliphatic), 1598 (C]N), 1318, 1143 (SO2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.27 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 6.96 (d, J = 8.4 Hz, 1H, dimethoxyphenyl H-5), 7.07 (s, 2H, SO2NH2, D2O exchangeable), 7.31 (m, 3H, dimethoxyphenyl H-6 & sulfamoylphenyl H-2, H-6), 7.45 (d, J = 2.0 Hz, 1H, dimethoxyphenylH-2), 7.67 (d, J = 8.4 Hz, 2H, sulfamoylphenyl H-3, H-5), 9.62 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6 100 MHz, δ, ppm): 13.6 (CH3), 55.94 (OCH3), 55.98 (OCH3), 109.2 (CH, dimethoxyphenyl C-2), 111.7 (CH, dimethoxyphenyl C-5), 112.3 (CH, sulfamoylphenyl C-2, C-6), 119.0 (CH, dimethoxyphenyl C-6), 127.7 (CH, sulfamoylphenyl C-3, C-5), 132.1 (C, dimethoxyphenyl C-1), 133.7 (C, sulfamoylphenyl C-4), 143.8 (C, sulfamoylphenyl C-1), 149.0 (C, dimethoxyphenyl C-3), 149.1 (C, dimethoxyphenyl C-4), 149.7 (C, C]N). MS m/z (ES+) 350 (M+1, 21%), 349 (M+, 100%). Anal. Calcd. for: C16H19N3O4S; C, 55.00; H, 5.48; N, 12.03; Found; C, 54.89; H, 5.64; N, 12.25. 1-[(4-Isobutylphenyl)ethylidene]-2-(4-sulfamoylphenyl)hydrazine (8d): Yield (2.58 g, 75%); white solid; mp 214–216 °C; IR (KBr): 3326, 3225 (NH2), 3098 (CeH aromatic), 2954 (CeH aliphatic), 1597 (C]N), 1316, 1145, (SO2NH2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δ ppm): 0.87 (d, J = 6.4 Hz, 6H, 2CH3, CH2CH(CH3)2), 1.84 (m, 1H, CH2CH (CH3)2), 2.27 (s, 3H, CH3), 2.47 (d, J = 6.8 Hz, 2H, CH2CH(CH3)2), 7.08 (s, 2H, SO2NH2, D2O exchangeable), 7.18 (d, J = 7.6 Hz, 2H, sulfamoylphenylH-2, H-6), 7.32 (d, J = 8.4 Hz, 2H, isobutylphenyl H-3, H-5), 7.67 (d, J = 8.4 Hz, 2H, isobutylphenyl H-2, H-6), 7.73 (d, J = 7.6 Hz, 2H, sulfamoylphenyl H-3, H-5), 9.68 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6 100 MHz, δ ppm): 13.6 (CH3), 22.5 (CH2CH(CH3)2), 30.0 (CH2CH(CH3)2), 44.7 (CH2CH(CH3)2), 112.3 (CH, sulfamoylphenyl C-2, C-6), 125.7 (CH, sulfamoylphenyl C-3, C-5), 127.7 (CH, isobutylphenyl C-2, C-6), 129.4 (CH, isobutylphenyl C-3, C5), 133.8 (C, sulfamoylphenyl C-4), 136.8 (C, isobutylphenyl C-1), 141.6 (C, isobutylphenyl C-4), 143.8 (C, sulfamoylphenyl C-1), 149.1 (C, C]N). MS m/z (ES+), 346 (M+1, 24%), 345 (M+, 100%), Anal. Calcd. for: C18H23N3O2S; C, 62.58; H, 6.71; N, 12.16; Found; C, 62.75; H, 6.89; N, 12.40.

a Significant from indomethacin using one way ANOVA followed by post Hook (Tukey′s test) for multiple pearwise comparison between different compounds at P < 0.01.

2.2.5. Histopathologicalgical examination results Histopathological examination was carried out to explore the effect of the most potent compounds (11b, 11d and 12b), celecoxib, indomethacin and control group. Control group showed normal histological structure of stomach (fundic region) with intact mucosahaving normal glandular epithelium, submucosa, muscular coat and serosal membrane (Fig. 4 Ia and Ib for control). In contrast, indomethacin group showed multiple degenerative changes inculding necrosis in some areas and ulceration in mucosal layer (stars) and submucosal edema with inflammatory cells infiltration like leucocytes, blood vessels were dilated and coagulative necrosis of muscular layer were found (Fig. 4 IIa, IIb for indomethacin). For Celecoxib treated group showed apparent normal histological structure of stomach with intactglandular mucosa, submucosa, muscular coat and serosal membrane. (Fig. 4 IIIa and IIIb for celecoxib). Furthermore, Compound 11b treated group have an intact mucosal epithelium with sever inflammatory cells infiltrations indeep mucosal and submucosal tissue (Fig. 4 IVa, IVb for 11b). Additionally, compound 11d examination group has apparent intact mucosal epithelium with congested mucosal Capillaries, submucosal inflammatory cells infiltrations and congested blood vessels (Fig. 4 Va and Vb for 11d). Furthermore, compound 12b has apparent normal histological structure of mucosal, submucosal, muscular and serosal layer without detected alteration (Fig. 4 VIa and VIb for 12b). 3. Conclusion Three novel diarylpyrazoles and eight novel triarylpyrazoles were synthesized and evaluated for in vitro and in vivo anti-inflammatory activity. The docking study assured COX-2 selectivity of all synthesized compounds. The in vitro testing authenticate that all compounds are more selective for COX-2 isozyme than COX-1 isozyme. Also, the in vivo study corroborate that test compounds have good AI activity. Moreover, compounds 11b, 11d and 12b are more potent than celecoxib and have approximate ulcerogenic liability to celecoxib as obtained from the histopathological examination. The bisaminosulphonyl compounds showed better anti-inflammatory activity than aminosulfonyl-methylsulfonyl compounds and comparable to celecoxib. 4. Experimental 4.1. Chemistry All Melting points were determined using aThomas-Hoover capillary apparatus and were uncorrected. Infrared (IR) spectra were recorded as films on KBr plates using Nicolet 550 seriesII Magna FT-IR spectrometer.1H NMR, 13C NMR were performed on a BurkerAvence III 400 MHz Spectrophotometer, Faculty of Pharmacy, Beni-Suef University, Egypt in CDCl3 or DMSO-d6, where J (coupling constant) values are estimated in Hertz (Hz). Microanalyses for C, H and N were carried out on Perkin–Elmer 2400 analyzer (Perkin–Elmer, Norwalk, 5

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3ia

3ib

3iia

3iib

3iiia

3iiib

3iva

3ivb

Fig. 3. (3ia) 2D, (3ib) 3D interaction between celecoxib and COX-2 active site. (3iia) 2D interaction, (3iib) 3D interaction between compounds 11b and COX-2 active. (3iii) 2D 3iii (B) 3D interaction between compounds 11d and COX-2 active site. (3iva) 2D, (3ivb) 3D interaction between compounds 12b and COX-2 active site.

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Table 5 Docking scores and Number of H-bond of 10b-d, 11a-d and 12a-d compounds and reference drug celecoxib. Compound no

E-Score

Amino acid R

No of H-bond

Groups H-bond

10b 10c 10d 11a 11b 11c 11d 12a 12b 12c 12d Celecoxib

−14.6 −12.8 −13.9 −18.5 −19.0 −18.6 −18.9 −17.7 −18.7 −17.5 −17.8 −18.5

Tyr341, Tyr 371, Ser516 Tyr371, Ser516 Tyr371, Ser516 Tyr371, Ser516, Arg499 Arg106, Tyr371 Tyr341, Tyr 108, Arg106, Lys68 His 60, Tyr341, Arg499 Val102, Ser105, Ser516, Lys68 Arg106, Ser516, Lys68 Ser105, Tyr 516, Tyr101, Tyr 341, Ser 371 Arg106, Ser516, Lys68 Ser339, Lys338, Glu106

3 2 2 3 2 4 3 4 4 6 4 3

SO2NH2, SO2NH2 SO2NH2 SO2NH2, SO2NH2 SO2NH2, SO2NH2, SO2NH2, SO2NH2, SO2NH2, SO2NH2 SO2NH2

CHO N NH NH SO2CH3, NH SO2CH3, NH SO2CH3, NH, OCH3

Inhibitory activity (%) = (1 − At/Ac) × 100, where, “At” is the absorbance in the presence of test substance and “Ac” is the absorbance of control. Data was obtained from three independent experiments and each experiment was performed in triplicates.

Ia

Ib

IIa

IIb

IIIa

IIIb

IVa

IVb

Va

Vb

VIa

VIb

Fig. 4. Gastric mucosa examination, Ia and Ib for control. IIa, IIb and IIc for indomethacin. IIIa and IIIb for celecoxib, IVa, IVb and IVc for 11b. Va and Vb for 11d, VIa and VIb for 12b.

4.1.2. General procedure for synthesis of pyrazole-4-carbaldehydes 9a-d Phosphorusoxycholride (9 g, 0.06 mol) was added dropwise to a well stirred ice-cooled solution of the appropriate hydrazone 8a-d (0.02 mol) in dry DMF (15 mL). After complete addition ofPOCl3, the mixture was allowed to attain room temperature and then heated under reflux to 60–70 °C for 12 h. The resulting mixture was poured onto crushed ice, neutralized with 10% sodium carbonate solution. The

precipitate obtained was filtered, washed with water and recrystalized from aqueous ethanol to give compounds 9a [34] and 9b-d. 3-(4-Ethoxyphenyl)-1-(4-N-dimethylamino-methylenebenzenesulfonamide)-1H-pyrazole-4-carbaldehyde (9b): Yield (3.32 g, 78%); pale yellow solid; mp 170–172 °C; IR (KBr: 3078 (CeH aromatic), 2978 (CeH aliphatic), 2870 (CeH aldehyde), 1678 (C]O), 1577 (C]N), 1342, 1149 (SO2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δ ppm): 1.36 (t, J = 6.8 Hz, 7

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3H, CH2CH3), 2.93 (s, 3H, NCH3), 3.17 (s, 3H, NCH3), 4.11 (q, J = 6.8 Hz, 2H, CH2CH3), 7.06 (d, J = 8.4 Hz, 2H, ethoxyphenyl H-3, H-5), 7.90 (d, J = 8.4 Hz, 2H, ethoxyphenyl H-2, H-6), 7.95 (d, J = 8.0 Hz, 2H, sulfamoylphenyl H-2, H-6), 8.15 (d, J = 8.0 Hz, 2H, sulfamoylphenyl H-3, H-5), 8.27 (s, 1H, N]CH), 9.41 (s, 1H, pyrazole H-5), 9.99 (s, 1H, CHO).13C NMR (DMSO-d6 100 MHz, δ ppm): 15.0 (CH2CH3), 35.5 (NCH3), 41.4 (NCH3), 63.6 (CH2CH3), 114.9 (CH, ethoxyphenyl C-3, C-5), 119.7 (CH, sulfamoylphenyl C-2, C-6), 122.8 (C, ethoxyphenyl C-1), 123.6 (C, pyrazole C-4), 128.1 (CH, ethoxyphenyl C-2, C-6), 130.5 (CH, sulfamoylphenyl C-3, C-5,), 136.1 (CH, pyrazole C-5), 141.1 (C, sulfamoylphenyl C-4), 142.1 (C, sulfamoylphenyl C-1), 153.3 (C, pyrazole C-3), 159.9 (C, ethoxyphenyl C-4), 160.1 (N]CH), 185.1 (CHO). MS m/z (ES+) 329 (M+3, 9%), 426 (M+, 3%), 352 (100%). Anal. Calcd. for: C21H22N4O4S; C, 59.14, H, 5.20, N, 13.14; Found; C, 59.39; H, 5.37; N, 13.50. 3-(3,4-Dimethoxyphenyl)-1-(4-N-dimethylamino-methylenebenzenesulfonamide)-1H-pyrazole-4-carbaldehyde (9c): Yield (3.1 g, 69%); brown solid; mp 285–287 °C; IR (KBr): 3074 (CeH aromatic), 2935 (CeH aliphatic), 2835 (CeH aldehyde), 1662 (C]O), 1573 (C]N), 1346, 1153, (SO2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.93 (s, 3H, NCH3), 3.16 (s, 3H, NCH3), 3.82 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 7.08 (d, J = 8.4 Hz, 1H, dimethoxyphenyl H-5), 7.53 (dd, J = 8.4, 2.0 Hz, 1H, dimethoxyphenyl H-6), 7.56 (d, J = 2.0 Hz, 1H, dimethoxyphenyl H-2), 7.95 (d, J = 8.4 Hz, 2H, sulfamoylphenyl H-2, H6), 8.15 (d, J = 8.4 Hz, 2H, sulfamoylphenyl H-3, H-5), 8.26 (s, 1H, N]CH), 9.42 (s, 1H, pyrazole H-5), 10.00 (s, 1H, CHO). 13C NMR (DMSO-d6 100 MHz, δ ppm): 35.3 (NCH3), 40.5 (NCH3), 56.0 (OCH3), 56.0 (OCH3), 112.0 (CH, dimethoxyphenyl C-2), 112.5 (CH, dimethoxyphenyl C-5), 119.8 (CH, dimethoxyphenyl C-6), 122.0 (CH, sulfamoylphenyl C-2, C-6), 123.0 (C, pyrazole C-4), 123.8 (C, dimethoxyphenyl C-1), 128.1 (CH, sulfamoylphenyl C-3, C-5), 136.3 (CH, pyrazole C-5), 141.0 (C, sulfamoylphenyl C-4), 142.1 (C, sulfamoylphenyl C-4), 149.0 (C, dimethoxyphenyl C-3), 150.3 (C, dimethoxyphenyl C-4), 153.4 (C, pyrazole C-3), 160.3 (N]CH), 185.2 (CHO). MS m/z (ES+), M+1 (443, 23%), 442 (M+, 100%). Anal. Calcd. for: C21H22N4O5S: C, 57.00, H, 5.01, N, 12.66; Found; C, 56.58; H, 5.13; N, 13.02. 3-(4-Isobutylphenyl)-1-(4-N-dimethylamino-methylenebenzenesulfonamide)-1H-pyrazole-4-carbaldehyde (9d): Yield (2.4 g, 55%); light yellow solid; mp 170–172 °C; IR (KBr): 3062 (CeH aromatic), 2954 (CeH aliphatic), 2870 (CeH aldehyde), 1678 (C]O), 1597 (C]N), 1338, 1145 (SO2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δ ppm): 0.90 (d, J = 6.4 Hz, 6H, CH2CH(CH3)2), 1.91 (m, 1H, CH2CH(CH3)2), 2.53 (d, J = 6.8 Hz, 2H, CH2CH(CH3)2), 2.93 (s, 3H, NCH3), 3.17 (s, 3H, NCH3), 7.30 (d, J = 8.0 Hz, 2H, isobutylphenylH-3, H-5), 7.85 (d, J = 8.0 Hz, 2H, isobutylphenyl H-2, H-6), 7.95 (d, J = 8.4 Hz, 2H, sulfamoylphenyl H-2, H-6), 8.16 (d, J = 8.4 Hz, 2H, sulfamoylphenyl H-3, H-5), 8.27 (s, 1H, N]CH), 9.44 (s, 1H, pyrazole H-5), 10.00 (s, 1H, CHO).13C NMR (DMSO-d6 100 MHz, δ ppm): 22.6 (CH2CH(CH3)2), 30.0 (CH2CH (CH3)2), 35.6 (NCH3), 41.4 (NCH3), 44.8 (CH2CH(CH3)2), 119.8 (CH, sulfamoylphenyl C-2, C-6), 123.0 (C, Pyrazole C-4), 128.1 (CH, isobutylphenyl C-2, C-6), 128.9 (C, isobutylphenyl C-1), 128.9 (CH, sulfamoylphenyl C-3, C-5), 129.6 (CH, isobutylphenyl, C-3, C-5), 135.8 (CH, pyrazole C-5), 141.1 (C, isobutylphenyl C-4), 142.1 (C, sulfamoylphenyl C-4), 143.1 (C, sulfamoylphenyl C-1), 153.6 (C, pyrazole C3), 160.3 (N]CH), 185.1 (CHO). MS m/z (ES+) 439 (M+1, 26%), 438 (M+, 1%), 348 (100%) Anal. Calcd. for: C23H26N4O3S: C, 62.99; H, 5.98; N, 12.78; Found; C, 62.64; H, 6.04; N, 12.85.

ethanol to give compound 10a [34] and 10b-d. 3-(4-Ethoxyphenyl)-1-(4-sulfamoylphenyl)-1H-pyrazole-4-carbaldehyde (10b): Yield (2.52 g, 68%); pale yellow solid; mp 135–137 °C; IR (KBr): 3329, 3255 (NH2), 3078 (CeH aromatic), 2978 (CeH aliphatic), 2881 (CeH aldehyde), 1670 (C]O), 1577 (C]N), 1311, 1161, (SO2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δ ppm): 1.36 (t, J = 6.0 Hz, 3H, CH2CH3), 4.11 (q, J = 6.0 Hz, 2H, CH2CH3), 7.06 (d, J = 7.6 Hz, 2H, ethoxyphenylH-3, H-5), 7.49 (s, 2H, SO2NH2, D2O exchangeable), 7.91 (d, J = 7.6 Hz, 2H, ethoxyphenyl H-2, H-6), 8.00 (d, J = 8.0 Hz, 2H, sulfamoylphenylH-2, H-6), 8.20 (d, J = 8.0 Hz, 2H, sulfamoylphenyl H3, H-5), 9.42 (s, pyrazole H-5), 10.00 (s, 1H, CHO).13C NMR (DMSO-d6 100 MHz, δ ppm): 15.0 (CH2CH3), 63.6 (CH2CH3), 114.9 (CH, ethoxyphenyl C-3, C-5), 119.7 (CH, sulfamoylphenyl C-2, C-6), 122.8 (C, pyrazole C-4), 123.6 (C, ethoxyphenyl C-1), 127.8 (CH, sulfamoylphenyl C-3, C-5), 130.5 (CH, ethoxyphenyl C-2, C-6), 136.1 (CH, pyrazole C-5), 141.1 (C, sulfamoylphenyl C-4), 143.1 (C, sulfamoylphenyl C-1), 153.3 (C, pyrazole C-3), 159.9 (C, ethoxyphenyl C-4), 185.2 (CH, CHO). MS m/z (ES+) 373 (M+2, 100%) (M+). Anal. Calcd. for: C18H17N3O4S; C, 58.21, H, 4.61, N, 11.31; Found; C, 58.47; H, 4.79; N, 11.46. 4.1.4. 3-(3,4-Dimethoxyphenyl)-1-(4-sulfamoylphenyl)-1H-pyrazole-4carbaldehyde (10c): Yield (2.9 g, 75%); brown solid; mp 275–277 °C; IR (KBr): 3340, 3244 (NH2), 3082 (CeH aromatic), 2935 (CeH aliphatic), 2831 (CeH aldehyde), 1674 (C]O), 1597 (C]N), 1330, 1161 (SO2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δ ppm): 3.85 (s, 6H, 2OCH3), 7.11 (d, J = 8.0 Hz, 1H, dimethoxyphenyl H-5), 7.29 (d, J = 7.6 Hz, 1H, dimethoxyphenyl H-6), 7.43 (s, 1H, dimethoxyphenyl H-2), 7.51 (s, 2H, SO2NH2, D2O exchangeable), 8.00 (d, J = 7.6 Hz, 2H, sulfamoylphenyl H-2, H-6), 8.22 (d, J = 8.0 Hz, 2H, sulfamoylphenyl H-3, H-5), 8.70 (s, 1H, pyrazole H-5), 9.24 (s, 1H, CHO). 13C NMR (DMSO-d6 100 MHz, δ ppm): 55.9 (OCH3), 56.0 (OCH3), 112.2 (CH, dimethoxyphenyl C-2), 112.6 (CH, dimethoxyphenyl C-5), 118.2 (C, Pyrazole C-4), 119.3 (CH, sulfamoylphenyl C-2, C-6), 121.8 (CH, dimethoxyphenyl C-6), 124.5 (C, dimethoxyphenyl C-1), 127.8 (CH, sulfamoylphenyl C-3, C-5), 129.9 (CH, pyrazole C-5), 141.4 (C, sulfamoylphenyl C-4), 142.6 (C, sulfamoylphenyl C-1), 149.2 (C, dimethoxyphenyl C-4), 151.1 (C, dimethoxyphenyl C-3), 153.7 (C, pyrazole C-3) 154.1 (CHO). MS m/z (ES+), 389 (M + 2, 4%), 387 (M+, 2%), 93 (100%). Anal. Calcd. for: C18H17N3O5S: C, 55.80, H, 4.40, N, 10.85; Found; C, 55.62; H, 4.69; N, 10.98. 3-(4-Isobutylphenyl)-1-(4-sulfamoylphenyl)-1H-pyrazole-4-carbaldehyde (10d): Yield (2.83 g, 74%); pale yellow solid; mp 140–142 °C; IR (KBr): 3356, 3259 (NH2), 3078 (CeH aromatic), 2954 (CeH aliphatic), 2846 (CeH aldehyde), 1678 (C]O), 1597 (C]N), 1338, 1161, (SO2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δ ppm): 0.88 (d, J = 6.8 Hz, 6H, CH2CH(CH3)2), 1.89 (m, 1H CH2CH(CH3)2), 2.51 (d, J = 7.2 Hz, 2H, CH2CH(CH3)2), 7.30 (d, J = 8.0 Hz, 2H, isobutylphenylH-3, H-5), 7.53 (s, 2H, SO2NH2, D2O exchangeable), 7.86 (d, J = 8.0 Hz, 2H, isobutylphenylH-2, H-6), 8.02 (d, J = 8.8 Hz, 2H, sulfamoylphenyl H-2, H-6), 8.21 (d, J = 8.8 Hz, 2H, sulfamoylphenylH-3, H-5), 9.45 (s, 1H, pyrazole H-5), 10.01 (s, 1H, CHO).13C NMR (DMSO-d6, 100 MHz, δ, ppm): 22.6 (CH2CH(CH3)2), 30.0 (CH2CH(CH3)2), 44.8 (CH2CH(CH3)2), 119.7 (CH, sulfamoylphenyl C-2, C-6), 123.0 (C, Pyrazole C-4), 127.8 (CH, isobutylphenyl C-2, C-6), 128.9 (C, isobutylphenylC-1), 128.9 (CH, sulfamoylphenylC-3, C-5), 129.6 (CH, isobutylphenyl C-3, C-5), 135.9 (CH, pyrazole C-5), 141.1 (C, sulfamoylphenyl C-4), 143.0 (C, isobutylphenyl C-4), 143.2 (C, sulfamoylphenyl C-1), 153.6 (C, pyrazole C3), 185.1 (CH, CHO). MS m/z (ES+) 384 (M + 1, 10%), 383 (M+, 20%), 340 (100%). Anal. Calcd. for: C20H21N3O3S: C, 62.64; H, 5.52; N.

4.1.3. General procedure for synthesis of diarylpyrazole 10a-d To a solution of the appropriate pyrazole 4-carbaldehyde9a-d (0.01 mol) intetrahydrofuran (50 mL), methanolic solution of sodium hydroxide (0.4 g, 0.01 mol, 5 mL) was added and the reaction mixture was stirred at room temperature for 24 h. The mixture was then concentrated and neutralized by drop wise addition of concHCl. The formed precipitated was filtered, dried and crystallized from aqueous

4.1.5. General procedure for synthesis of triarylpyrazoles 11a-d and 12a-d To asolution of 3-(substituted phenyl)-1-(4-sulfamoylphenyl)-1Hpyrazole-4-carbaldehydes 10a-d (0.01 mol) in absolute ethanol (15 mL), p-sulfamoylphenylhydrazine hydrochloride (7), (0.01 mol, 8

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2.23 g) or p-methylsulfonylphenylhydrazine hydrochloride (0.01 mol, 2.24 g) was added. The reaction mixture was heated under reflux for 24 h. The formed precipitated was filtered, dried and recrystalized from ethanol to give triarylpyrazoles 11a-d or 12a-d respectively. 1-[3-(4-Methoxyphenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-4-yl-methylene]-2-(4-sulfamoylphenyl)hydrazine (11a); Yield (3.78 g, 72%); pale yellow solid; mp 206–208 °C; IR (KBr): 3363, 3278 (NH2), 3116 (CeH aromatic), 2974 (CeH aliphatic), 1597 (C]N), 1311, 1149 (SO2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δ ppm): 3.84 (s, 3H, OCH3), 7.01 (s, 2H, SO2NH2, D2O exchangeable), 7.07–7.12 (m, 4H, sulfamoylphenylhydrazine H-2, H-6 & methoxyphenyl H-3, H-5), 7.45 (s, 2H, SO2NH2, D2O exchangeable), 7.60 (d, J = 8.0 Hz, 2H, methoxyphenyl H-2, H-6), 7.70 (d, J = 7.6 Hz, 2H, sulfamoylphenyl H-2, H-6), 7.93 (d, J = 7.6 Hz, 2H, sulfamoylphenylhydrazine H-3, H-5), 8.35 (s, 1H, N]CH), 8.20 (d, J = 7.6 Hz, 2H, sulfamoylphenyl H-3, H-5), 9.06 (s, 1H, pyrazole H-5), 10.71 (s, 1H, NH, D2O exchangeable).13C NMR (DMSO-d6 100 MHz, δ ppm): 55.7 (OCH3), 111.3 (CH, methoxyphenyl C-3, C-5), 114.5 (CH, sulfamoylphenylhydrazine C-2, C-6), 118.81 (CH, sulfamoylphenyl C-2, C-6), 118.89 (C, pyrazole C-4), 124.9 (C, methoxyphenyl C-1), 127.3 (CH, pyrazole C-5), 127.7 (CH, sulfamoylphenyl C-3, C-5), 127.8 (CH, sulfamoylphenylhydrazineC-3, C-5), 130.4 (CH, methoxyphenyl C-2, C-6), 132.0 (N]CH), 133.6 (C, sulfamoylphenylhydrazine C-4), 141.6 (C, sulfamoylphenylC-4), 142.0 (C, sulfamoylphenyl C-1), 148.2 (C, sulfamoylphenylhydrazineC-1), 151.8 (C, pyrazole C-3), 160.1 (C, methoxyphenyl C-4). MS m/z (ES+) 526, 373 (100%). Anal. Calcd. For C23H22N6O5S2C, 52.46; H, 4.21; N, 15.96; Found; C, 52.79; H, 4.08; N, 16.28. 1-[3-(4-Ethoxyphenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-4-yl-methylene]-2-(4-sulfamoylphenyl)hydrazine (11b); Yield (4.05 g, 75%); pale yellow solid; mp 220–222 °C; IR (KBr): 3371, 3282 (NH2), 3074 (CeH aromatic), 2935 (CeH aliphatic), 1597 (C]N), 1311, 1149 (SO2NH2) cm−1. 1H NMR (DMSO-d6 , 400 MHz, δ ppm): 1.37 (t, J = 6.8 Hz, 3H, CH2CH3), 4.11 (q, J = 6.8 Hz, 2H, CH2CH3), 7.07–7.14 (m, 6H, sulfamoylphenylhydrazine H-2, H-6, ethoxyphenyl H-3, H-5 & SO2NH2, D2O exchangeable), 7.48 (s, 2H, SO2NH2), 7.67 (d, J = 8.0 Hz, 2H, ethoxyphenyl H-2, H-6), 7.71 (d, J = 8.0 Hz, 2H, sulfamoylphenyl H-2, H-6), 7.98 (d, J = 8.0 Hz, 2H, sulfamoylphenylhydrazine H-3, H-5), 8.10 (s, 1H, N]CH), 8.19 (d, J = 8.0 Hz, 2H, sulfamoylphenylH-3, H-5), 9.05 (s, 1H, pyrazole H-5), 10.97 (s, 1H, NH, D2O). 13C NMR (DMSO-d6, 100 MHz, δ, ppm): 15.1 (CH2CH3), 63.6 (CH2CH3), 111.3 (CH, ethoxyphenyl C-3, C-5), 114.9 (CH, sulfamoylphenylhydrazine C-2, C-6), 118.7 (CH, sulfamoylphenyl C-2, C-6), 118.9 (C, pyrazole C-4), 124.8 (C, ethoxyphenyl C-1), 127.4 (CH, pyrazole C-5), 127.8 (CH, sulfamoylphenyl C-3, C-5), 127.8 (CH, ethoxyphenyl C-2, C-6), 130.1 (CH, sulfamoylphenylhydrazine C-3, C-5), 132.0 (N]CH), 133.4 (C, sulfamoylphenylhydrazine C-4), 141.6 (C, sulfamoylphenyl C-4), 141.9 (C, sulfamoylphenyl C-1), 148.2 (C, sulfamoylphenylhydrazine C-1), 151.8 (C, pyrazole C-3), 159.3 (C, ethoxyphenyl C-4). Anal. Calcd. For C24H24N6O5S2: C, 53.32; H, 4.47; N, 15.55; Found; C, 53.64; H, 4.59; N, 15.90. 1-[3-(3,4-Dimethoxyphenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-4-ylmethylene]-2-(4-sulfamoylphenyl)hydrazine (11c): Yield (3.94 g, 71%); brown solid; mp 213–215 °C; IR (KBr): 3294, 3224 (NH2), 3082 (CeH aromatic), 2931 (CeH aliphatic), 1593 (C]N), 1311, 1153, (SO2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δppm): 3.86 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 7.08 (s, 2H, SO2NH2, D2O exchangeable), 7.11–7.13 (m, 3H, sulfamoylphenylhydrazine H-2, H-6 & dimethoxyphenyl H-5), 7.27 (d, J = 8.4 Hz, 1H, dimethoxyphenyl H-6) 7.31 (s, 1H, dimethoxyphenyl H-2), 7.46 (s, 2H, SO2NH2, D2O exchangeable), 7.66 (d, J = 8.4 Hz, 2H, sulfamoylpheny H-2, H-6), 7.98 (d, J = 8.8 Hz, 2H, sulfamoylphenylhydrazine H-3, H-5), 8.03 (s, 1H, N]CH), 8.21 (d, J = 8.8 Hz, 2H, sulfamoylphenyl H-3, H-5), 9.06 (s, 1H, pyrazole H-3), 10.71 (s, 1H, NH, D2O exchangeable).13C NMR (DMSO-d6 100 MHz, δ, ppm): 56.1 (2OCH3), 111.6 (CH, dimethoxyphenyl C-2), 112.2 (CH, dimethoxyphenyl C-5), 112.4 (CH, sulfamoylphenylhydrazine C-2, C6), 118.8 (CH, sulfamoylphenyl C-2, C-6), 119.2 (C, pyrazole C-4),

121.5 (CH, dimethoxyphenyl C-6), 125.1 (C, dimethoxyphenyl C-1), 127.2 (CH, pyrazole C-5), 127.7 (CH, sulfamoylphenyl C-3, C-5), 127.8 (CH, sulfamoylphenylhydrazine C-3, C-5), 132.1 (N]CH), 133.6 (C, sulfamoylphenyhydrazine C-4), 141.6 (C, sulfamoylphenyl C-4), 142.0 (C, sulfamoylphenyl C-1), 148.2 (C, sulfamoylphenylhydrazine C-1), 149.2 (C, dimethoxyphenyl C-4), 149.8 (C, dimethoxyphenyl C-3), 152.0 (C, pyrazole, C-3) MS m/z (ES+) 559 (M+2, 4%), 557 (M+, 2%), 417 (100%). Anal. Calcd. For: C24H24N6O6S2: C, 51.79; H, 4.35; N, 15.10; Found; C, 51.92; H, 4.43; N, 14.89. 1-[3-(4-Isobutylphenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-4-yl-methylene]-2-(4-sulfamoylphenyl)hydrazine (11d); Yield (4.02 g, 73%); light yellow solid; mp 260–262 °C; IR (KBr): 3363, 3267 (NH2), 3074 (CeH aromatic), 2924 (CeH aliphatic), 1597 (C]N), 1311, 1149 (SO2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δ, ppm): 0.92 (d, J = 6.4 Hz, , 6H, CH2CH(CH3)2), 1.90 (m, 1H, CH2CH(CH3)2), 2.54 (d, J = 6.4 Hz, 2H, CH2CH(CH3)2), 7.07 (s, 2H, SO2NH2, D2O exchangeable), 7.10 (d, J = 8.8 Hz, 2H, sulfamoylphenylhydrazineH-2, H-6), 7.33 (d, J = 7.6 Hz, 2H, isobutylphenyl H-3, H-5), 7.45 (s, 2H, SO2NH2, D2O exchangeable), 7.68–7.73 (m, 2H, isobutylphenyl H-2, H-6 & sulfamoylphenyl H-2, H-6), 7.98 (d, J = 8.4 Hz, 2H, sulfamoylphenylhydrazineH-3, H-5), 8.05 (s, 1H, N]CH), 8.20 (d, J = 8.4 Hz, 2H, sulfamoylphenyl H-3, H-5), 9.08 (s, 1H, pyrazole H-5), 10.74 (s, 1H, NH, D2O Exchangeable) 13C NMR (DMSO-d6100MHz, δ ppm): 22.6 (CH2CH(CH3)2), 30.1 (CH2CH(CH3)2), 44.8 (CH2CH(CH3)2), 111.3 (CH, sulfamoylphenylhydrazineC-2, C-6), 118.8 (CH, sulfamoylphenyl C-2, C-6), 119.0 (C, pyrazole C-4), 127.4 (CH, pyrazole C-5), 127.8 (CH, isobutylphenyl C-2, C-6), 128.6 (CH, sulfamoylphenyl C-3, C-5), 128.9 (CH, sulfamoylphenylhydrazine C-3, C-5), 129.6 (CH, isobutylphenyl C-3, C-5), 130.1 (isobutylphenyl C-4), 131.9 (C, sulfamoylphenylhydrazine C-4), 133.6 (N]CH), 141.6 (C, isobutylphenyl C1), 142.1 (C, sulfamoylphenyl C-4), 142.2 (C, sulfamoylphenyl C-1), 148.2 (C, sulfamoylphenylhydrazine C-1) 152.0 (C, pyrazole C-3). MS m/z (ES+), 554 (M+1, 8%), 552 (M+, 12%), 349 (100%). Anal. Calcd. For C26H28N6O4S2: C, 56.50; H, 5.11; N, 15.21; Found; C, 56.79; H, 5.18; N, 15.38. 1-[3-(4-Methoxyphenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-4-yl-methylene]-2-(4-methylsulfonylphenyl)hydrazine(12a): Yield (3.46 g, 66%); yellow solid; mp 195–197 °C; IR (KBr): 3275–3113 (NH2), 3074 (CeH aromatic), 2927 (CeH aliphatic), 1597 (C]N), 1315, 1157; 1276, 1134, (2SO2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δ ppm): 3.11 (s, 3H, SO2CH3), 3.84 (s, 3H, OCH3), 7.10–7.19 (m, 4H, methylsulfonylphenyl H-2, H-6 & methoxyphenyl H-3, H-5), 7.50 (s, 2H, SO2NH2, D2O exchangeable) 7.70–7.74 (m, 4H, methoxyphenyl H-2, H-6 & sulfamoylphenyl H-2, H-6), 8.00 (d, 2H, J = 8.4 Hz, methylsulfonylphenylH-2, H6), 8.08 (m, 3H, sulfamoylphenyl H-3, H-5 & N]CeH), 9.07 (s, 1H, pyrazole H-5), 11.26 (s, 1H, NH, D2O exchangeable).13C NMR (DMSOd6100MHz, δ ppm): 44.7 (SO2CH3), 55.9 (OCH3), 111.7 (CH, methoxyphenyl C-3, C-5), 114.5 (CH, methylsulfonylphenyl C-2, C-6), 118.7 (C, pyrazole C-4), 118.8 (CH, sulfamoylphenyl C-2, C-6), 124.9 (C, methoxyphenyl C-1), 127.6 (CH, pyrazole C-5), 127.8 (CH, sulfamoylphenyl C-3, C-5), 129.2 (CH, methoxyphenyl C-2, C-6), 129.3 (C, methylsulfonylphenyl C-4), 130.2 (CH, methylsulfonylphenyl C-3, C-5), 133.0 (N]CH), 141.6 (C, sulfamoylphenyl C-4), 142.0 (C, sulfamoylphenyl C-1), 149.6 (C, methylsulfonylphenyl C-1), 151.8 (C, pyrazole C3), 160.1 (C, methoxyphenyl C-4). MS m/z (ES+), 525 (M+). Anal. Calcd. For C24H23N5O5S2: C, 54.84; H, 4.41; N, 13.32; Found; C, 54.76; H, 4.50; N, 13.59. 1-[3-(4-Ethoxyphenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-4-yl-methylene]-2-(4-methylsulfonylphenyl)hydrazine (12b); Yield (3.93 g, 73%); pale yellow solid; mp 208–210 °C; IR (KBr): 3336, 3275 (NH2), 3074 (CeH aromatic), 2927 (CeH aliphatic), 1597 (C]N), 1276, 1149; 1149, 1138 (2SO2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δ, ppm): 1.38 (t, J = 6.8 Hz, 3H, CH2CH3), 3.10 (s, 3H, SO2CH3), 4.13 (q, J = 6.8 Hz, 2H, CH2CH3), 7.08 (d, J = 8.4 Hz, 2H, methylsulfonylphenyl H-2, H-6), 7.16 (d, J = 8.4 Hz, 2H, ethoxyphenyl H-3, H-5), 7.39 (s, 2H, SO2NH2, D2O exchangeable), 7.70–7.72 (m, 4H, ethoxyphenyl H-2, H-6 & 9

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sulfamoylphenyl H-2, H-6), 7.98 (d, J = 8.4 Hz, 2H, methylsulfonylphenyl H-2, H-6), 8.09 (s, 1H, N]CH), 8.18 (d, J = 8.4 Hz, 2H, sulfamoylphenyl H-3, H-5), 9.03 (s, 1H, pyrazole H-5), 10.85 (s, 1H, NH, D2O exchangeable).13C NMR (DMSO-d6 100 MHz, δ ppm): 15.1 (CH2CH3), 44.7 (SO2CH3), 63.7 (CH2CH3), 111.7 (CH, ethoxyphenyl C3, C-5), 115.0 (CH, methylsulfonylphenyl C-2, C-6), 118.7 (C, pyrazole C-4), 118.8 (CH, sulfamoylphenyl C-2, C-6), 124.7 (C, ethoxyphenyl C1), 127.2 (CH, pyrazole C-5), 127.3 (CH, ethoxyphenyl C-2, C-6), 129.1 (CH, sulfamoylphenyl C-3, C-5), 129.7 (C, methylsulfonylphenyl C-4), 130.2 (CH, methylsulfonylphenyl C-3, C-5), 133.0 (N]CH), 141.6 (C, sulfamoylphenyl, C-4), 142.0 (C, sulfamoylphenyl C-1), 149.5 (C, methylsulfonylphenyl C-1), 151.9 (C, pyrazole C-3), 159.4 (C, ethoxyphenyl C-4). MS m/z (ES+), 541 (M + 1, 3%), 540 (M+, 2%), 367 (100%). Anal. Calcd. For C25H25N5O5S2, C, 55.64; H, 4.67; N, 12.98; Found; C, 55.96; H, 4.60; N, 13.21. 1-[3-(3,4-Dimethoxyphenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-4-ylmethylene]-2-(4-methylsulfonylphenyl)hydrazine (12c); Yield (3.50 g, 63%); pale yellow solid; mp 250–252 °C; IR (KBr): 3336, 3232 (NH2), 3086 (CeH aromatic), 2993 (CeH aliphatic), 1589 (C]N), 1346, 1288, 1165, 1134 (2SO2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δ ppm): 3.10 (s, 3H, SO2CH3), 3.86 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 7.12 (d, J = 7.4 Hz, 1H, dimethoxyphenyl H-5), 7.19 (d, J = 6.8 Hz, 2H, methylsulfonylphenyl H-2, H-6), 7.31 (m, 2H, dimethoxyphenyl H-6, H-2), 7.43 (s, 2H, SO2NH2, D2O exchangeable), 7.70 (d, J = 6.8 Hz, 2H, methylsulfonylphenyl H-3, H-5), 7.98 (d, J = 6.4 Hz, 2H, sulfamoylphenyl H-2, H-6), 8.10–8.19 (m, 3H, N]CH & sulfamoylphenyl H-3, H5), 9.03 (s, 1H, pyrazole H-5), 11.12 (s, 1H, NH, D2O exchangeable). 13 C NMR (DMSO-d6 100 MHz, δ ppm): 44.7 (SO2CH3), 56.12 (OCH3), 56.15 (OCH3), 111.7 (CH, dimethoxyphenyl C-2), 112.2 (CH, dimethoxyphenyl C-5), 112.3 (CH, methylsulfonylphenyl C-2, C-6), 118.8 (C, pyrazole C-4), 118.9 (CH, sulfamoylphenyl, C-2, C-6), 121.5 (CH, dimethoxyphenyl C-6), 125.0 (C, dimethoxyphenyl C-1), 127.4 (CH, pyrazole C-5), 127.7 (CH, sulfamoylphenyl C-3, C-5), 129.2 (CH, methylsulfonylphenyl C-3, C-5), 129.3 (C, methylsulfonylphenylC-4), 133.0 (N]CH), 141.6 (C, sulfamoylphenyl C-4), 142.1 (C, sulfamoylphenyl C-1), 149.2 (C, methylsulfonylphenyl C-1), 149.6 (C, dimethoxyphenyl C-4), 149.8 (C, dimethoxyphenyl C-3), 152.0 (C, pyrazole C-3). MS m/z (ES+), 557 (M + 2, 6%), 556 (M+, 13%), 358 (100%). Anal. Calcd. For: C25H25N5O6S2; C, 54.04; H, 4.54; N, 12.60: Found; C, 54.28; H, 4.67; N, 12.37. 1-[3-(4-Isobutylphenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-4-yl-methylene]-2-(4-methylsulfonylphenyl) hydrazine (12d): Yield (3.20 g, 58%); brown solid; mp 216–218 °C; IR (KBr): 3383, 3275 (NH2), 3074 (CeH aromatic), 2943 (CeH aliphatic), 1589 (C]N), 1311, 1161; 1276, 1134 (2SO2) cm−1. 1H NMR (DMSO-d6, 400 MHz, δ, ppm): 0.92 (d, J = 4 Hz, 6H, CH2CH(CH3)2), 1.9 (m, 1H, CH2CH(CH3)2), 2.53 (d, J = 6.8 Hz, 2H, CH2CH(CH3)2), 3.11 (s, 3H, SO2CH3), 7.18 (d, J = 6.4 Hz, 2H, methylsulfonylphenyl H-2, H-6), 7.32 (d, J = 5.2 Hz, 2H, isobutylphenyl H-3, H-5), 7.49 (s, 2H, SO2NH2, D2O exchangeable), 7.48–7.71 (m, 4H, isobutylphenyl H-2, H-6 & sulfamoylphenyl H-2, H-6), 8.00 (d, J = 6.4 Hz, 2H, methylsulfonylphenyl H-3, H-5), 8.14–8.21 (m, 3H, sulfamoylphenyl H-3, H-5 & N]CH), 9.10 (s, 1H, pyrazole H-5), 11.24 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6 100 MHz, δ ppm): 22.6 (CH2CH(CH3)2), 30.1 (CH2CH(CH3)2), 44.81 (SO2CH3), 44.84 (CHCH2(CH3)2), 111.6 (CH, methylsulfonylphenyl C-2, C-6), 118.8 (CH, sulfamoylphenyl C-2, C-6), 118.9 (C, pyrazole C-4), 127.8 (CH, isobutylphenyl C-2, C-6), 128.6 (CH, sulfamoylphenyl C-3, C-5), 129.2 (CH, methylsulfonylphenyl C-3, C-5), 129.3 (C, methylsulfonylphenyl C-4), 129.6 (CH, isobutylphenyl C-3, C-5), 130.1 (C, isobutylphenyl C1), 132.9 (N]CH), 141.6 (C, sulfamoylphenyl C-4), 142.1 (C, isobutylphenyl C-4), 142.2 (C, sulfamoylphenyl C-1), 149.6 (C, methylsulfonylphenyl C-1), 152.0 (C, pyrazole C-3). MS m/z (ES+)0.554 (M + 2, 10%), 552 (M+, 16%), 444 (100%). Anal. Calcd. For C27H29N5O4S2: C, 58.78; H, 5.30; N, 12.69; Found; C, 59.01; H, 5.17; N, 13.02.

4.2. Biological activity 4.2.1. Cyclooxygenase inhibition assays The designed compounds efficacy to inhibit ovineCOX-1 and human recombinant COX-2 (IC50 value, µM) was measured using an enzyme immuno assay (EIA) kit (catalog no. 560131, Cayman Chemical, Ann Arbor, MI, USA) according to the previously reported method [35]. 4.2.2. In vivo anti-inflammatory assay The newly synthesized compounds 10b-d, 11a-d and 12a-d and lead drug celecoxib were evaluated for their in vivo anti-inflammatory activity using carrageenan-induced rat foot paw edema model using (100 mg/kg). Measurement of paw thickness was performed at 1 h, 3 h and 6 h after carrageenan injection according to previously reported method [36]. Moreover, ED50 values were determined for the most active compounds 11b, 11d and12b in comparison with reference drug celecoxib. 4.2.3. Ulcerogenic liability The ulcerogenic effect of indomethacin, celecoxib and (11b, 11d and 12b) that showed superior in-vivo anti-inflammatory activity better than celecoxib, determined. Rats were scarified at 4 h after drug administration (150 mg/kg) according to previously described method [17]. 4.2.4. Histopathlogical study Tissue samples were flushed and fixed in neutral buffered formalin 10% remain for 72 h. Samples were trimmed, processed and dehydrated by serial grades of alcohol, clearing in Xylene, synthetic wax infiltration and blocking out into paraplast tissue embedding media. 5µ sections were cut by rotatory microtome. The sections were stained with Harris hematoxylin and Eosin as a general examination staining method as outlined by Bancroft and Stevens [35]. Tissue samples were flushed and fixed in 10% neutral buffered 4.2.5. Docking study The crystal structure of COX-2 isozyme with celecoxib as lead drug was obtained from protein data bank (PDB; ID: 3LN1). For the purpose of studying Scoring interaction, binding interaction with different amino acid, docking of crystallized ligand should be done. Docking was attained using London DG forces. The synthesized compounds were prepared for docking through protonation, obtaining the lowest energy conformer of 3D structure build by Molecular Operating Environment (MOE). The celecoxib was docked first then removed from site of interaction. All molecules were docked using the same condition as standard drug. The binding interaction and docking score was studied using 2D and 3D pictures. Declaration of Competing Interest The authors have declared no conflict of interest. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bioorg.2019.103029. References [1] R. Kumar, S. Bawa, G. Chawla, G. Singh, S. Kumar, V. Rathore, N. Mulakayala, A. Rajaram, A.M. Kalle, O. Afzal, Eur. J. Med. Chem. 57 (2012) 176–184. [2] J.M. Pelletier, D. Lajeunesse, P. Reboul, J.P. Pelletier, 62 (2003) pp. 501–509. [3] M. Parente, L. Perretti, Biochem. Pharmacol. 65 (2003) 153–159. [4] G. Kulkarni, R.G. Achaiah, G. Narahari sastry, Curr. Pharm. Des. 2 (2006) 2437–2454. [5] M.A. Abdelgawad, M.B. Labib, W.A.M. Ali, G. Kamel, A.A. Azouz, E. El-nahass, Bioorg. Chem. 78 (2018) 103–114.

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