MATERIAL AND METHODS:

Chemistry:

All over the chemical materials and solvents were procured from commercial providers and were utilized without further refining. Melting points were estimated and uncorrected by utilizing the open capillary tube procedure using IA 9100MK-Digital melting point apparatus. Elemental analysis was performed at the micro- analytical center at the regional center for mycology and biotechnology, Al-Azhar University. The IR spectra were reported in Bruker FT-IR spectrophotometer Vector 22 and recorded in wave number (cm^-1) using KBr discs at the micro-analytical center, Faculty of Science, Cairo University. The proton/carbon magnetic resonance (¹H NMR and ¹³C NMR) spectra were executed with a Bruker APX400 spectrometer at 400 MHz and 101 MHz, respectively in the specified solvent at the Faculty of Pharmacy, Beni-Suef University. Moreover, δ was outlined on the d scale and J values were recorded in Hz. The molecular weights were outlined on the mass spectra utilizing Finnegan MAT, SSQ 7000, Mass spectrometer, at 70 eV (EI) at the micro-analytical center, Faculty of Science, Cairo University. Macherey-Nagel Alugram Sil G/UV254 silica gel plats were used to perform thin layer chromatography (TLC) in presence of petroleum ether-ethyl acetate (6:4) as the eluting system.

General procedure for synthesis of compounds 13a-13d.

To the suspension of compound 6 or compound 12 (2 mmol) in POCl₃ (8 mL), the corresponding acid (2 mmol) was added and then the reaction mixture was allowed to be heated under reflux for 8 hours at 110 °C. Following up the reaction was performed via using TLC until the disappearance of the starting materials followed by careful pouring onto a cold solution of KHCO₃. Recrystallization of obtained precipitate was carried out from ethanol providing the desired compounds.

4-(3-(5-(adamantan-1-yl)-1,3,4-oxadiazol-2-yl)-5-phenyl-1H-pyrazol-1-yl)benzenesulfonamide

13a.

IR (cm^-1): 3433 (NH₂ of SO₂NH₂), 3062 (CH aromatic), 2908 (CH aliphatic), 1496 (C=N), 1323, 1161 (SO₂NH₂). ¹H NMR (400 MHz, DMSO-d₆): δ ¹H NMR (400 MHz, DMSO-d₆): δ 7.87-7.90 (m, 2H, Ar-H), 7.57-7.55 (m, 2H, Ar-H), 7.43-7.44 (m, 2H, Ar-H), 7.35-7.40 (m, 3H, Ar-H), 7.20-7.22 (d, J = 8 Hz, 2H, SO₂NH₂ exchangeable with D₂O),6.82 (s, 1H, Pyrazolyl-H), 1.99-2.07 (m, 6H, -CCH₂CH-), 1.86 (m, 3H, -CCH₂CH-), 1.67-1.78 (m, 6H, -CHCH₂CH-).¹³C NMR (101MHz, DMSO-d₆): δ 153.54, 143.47, 142.38, 142.24, 130.31, 129.32, 128.96, 127.19, 127.07, 126.35, 124.57, 119.60, 99.97, 53.43, 42.08, 36.74, 29.37. MS (EI): m/z 501 (M⁺). Anal. Calcd. For C₂₇H₂₇N₅O₃S: C, 64.65; H, 5.43; N, 13.96. Found: C, 64.89; H, 5.61; N, 14.23.

4-(3-(5-(adamantan-1-yl)-1,3,4-oxadiazol-2-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)benzenesulfonamide

13b.

IR (cm^-1): 3417 (NH₂ of SO₂NH₂), 3070 (CH aromatic), 2908 (CH aliphatic), 1504 (C=N), 1373, 1172 (SO₂NH₂).¹H NMR (400 MHz, DMSO-d₆): δ 7.76-7.69 (m, 2H, Ar-H), 7.59-7.52 (m, 4H, Ar-H), 7.49 (m, 3H, Ar-H), 7.47 (s, broad, 2H, SO₂NH₂ exchangeable with D₂O), 2.09 (m, 3H,-CH₂CHCH₂-), 1.96-2.04 (m, 6H, -CCH₂-), 1.74-1.81 (m, 6H, -CHCH₂CH-). ¹³C NMR (101MHz, DMSO-d₆): δ 171.80, 129.41, 129.36, 129.31, 129.28, 129.23, 127.36, 127.32, 127.24, 125.86, 125.81, 125.72, 56.50, 47.73, 36.16, 27.58. MS (EI): m/z 502 (M⁺). Anal. Calcd. For C₂₆H₂₆N₆O₃S: C, 62.13; H, 5.21; N, 16.72. Found: C, 61.89; H, 5.43; N, 16.98.

4-(1-phenyl-3-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)-1H-pyrazol-5-yl)benzenesulfonamide 13c.

IR (cm^-1): 3435 (NH₂ of SO₂NH₂), 3062 (CH aromatic), 2993, 1496 (C=N), 1327, 1172 (SO₂NH₂).¹H NMR (400 MHz, DMSO-d₆): δ 8.29-8.31 (d, J = 8 Hz, 1H, Ar-H), 8.11-8.13 (d, J = 8 Hz, 1H, Ar-H), 7.98-8.00 (d, J = 8 Hz, 1H, Ar-H), 7.68-7.70 (d, J = 8 Hz, 1H, Ar-H), 7.52-7.50 (d, J = 8Hz, 2H, Ar-H), 7.41-7.43 (m, 5H, Ar-H), 7.33-7.35 (m, 2H, SO₂NH₂ exchangeable with D₂O), 7.29 (m, 3H, Ar-H). ¹³C NMR (101MHz, DMSO-d₆): δ 166.62, 163.31, 160.47, 148.44, 145.49, 139.46, 137.79, 133.92, 130.56, 129.29, 129.18, 128.16, 128.04, 127.03, 126.90, 126.04, 125.49, 108.62. MS (EI): m/z 511 (M⁺).Anal. Calcd. For C₂₄H₁₆F₃N₅O₃S: C, 56.36; H, 3.15; N, 13.96. Found: C, 56.53; H, 3.42; N, 14.20.

4-(1-phenyl-3-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)-1H-1,2,4-triazol-5-yl)benzenesulfonamide

13d.

IR (cm^-1): 3441 (NH₂ of SO₂NH₂), 3070 (CH aromatic), 2993, 1496 (C=N), 1327, 1171 (SO₂NH₂). ¹H NMR (400 MHz, DMSO-d₆): δ 8.39-8.41 (d, J = 8 Hz, 1H, Ar-H ), 8.13-8.15 (d, J = 8 Hz, 1H, Ar-H), 8.03-8.05 (d, J = 8 Hz, 1H, Ar-H), 7.88-7.90 (d, J = 8 Hz, 1H, Ar-H), 7.71-7.77 (m, 2H, Ar-H), 7.55-7.58 (m, 2H, Ar-H), 7.50-7.54 (m, 5H, Ar-H), 7.46-7.48 (d, J = 8 Hz, 2H, SO₂NH₂ exchangeable with D₂O ). ¹³C NMR (101MHz, DMSO-d₆): δ 166.70, 131.10, 130.60, 129.41, 129.40, 129.37, 129.25, 128.31, 128.23, 127.37, 127.24, 126.95, 126.15, 126.13, 126.10, 125.87, 125.82. MS (EI): m/z 512 (M⁺). Anal. Calcd. For C₂₃H₁₅F₃N₆O₃S: C, 53.91; H, 2.95; N, 16.40. Found: C, 54.18; H, 3.22; N, 16.63.

Biological screening:

In vitro cyclooxygenase (COX) inhibition assay:

The inhibitory activity of the new synthesized compounds against both COX-1 and COX-2 was monitored using enzyme immunoassay (EIA) kits from Cayman Chemical Company (catalogue number 701070 and 701080, Ann Arbor, MI) by using N, N, N^/, N^/-tetramethyl-p-phenylenediamine at 590 nm according to the manufacturer’s directions, as reported⁴⁴. Briefly, 10µL of the tested compounds were added to a solution of 0.1M tris-HCl buffer and 10µL of arachidonic acid. Termination of COX reaction was carried out after 5 mins from the incubation of the solution at 37ºC. The yellow color of PGF₂, generated from PGH₂, were determined spectrophotometrically at λ = 405nm. The inhibitory activity and IC₅₀ were determined by performing the comparison between the tested compounds to various control incubations.

In vivo assay:

Anti-inflammatory screening:

All the synthesized compounds were assayed for their anti-inflammatory activity utilizing the Winter et al model that known as Carrageenan-induced paw edema model⁴⁵. Albino rats of either sex, weighing 120-150g, were undergone to the experiments by dividing them into 6 groups with 4 animals in each cage following the Research Ethical Committee of Faculty of Pharmacy, Beni-Suef University. Each group of animals was treated with a suspension of novel compounds 13a-d and celecoxib that prepared by their dissolution in 10% DMSO followed by administration at a dose of 50 mg/kg orally. Only one group whose animals were administered 10% DMSO aqueous solution (v/v) to act as a control group. After 1 h, 100µL of freshly prepared 1% carrageenan-sodium gel, which was obtained from Sigma-Aldrich (USA), has been given to the animals. Subsequently, measuring the induced edema was carried out in both hind paws of each rat after 1, 3 and 5 h of triggering of inflammation using Vernier calipers (SMIEC). The effectiveness of the compounds was expressed as the percentage of reduction in the carrageenan induced edema thickness and compared to the tested and celecoxib received groups.

Analgesic screening:

The ability of the synthesized compounds to act as analgesic was evaluated using the writhing technique in mice that was induced by acetic acid as described by Koster et al ⁴⁶. The animals were separated under 7 groups about 4 mice every. The tested compounds (equimolar to the standard drug (celecoxib)) were suspended in 10% DMSO and administered orally (10 mg/kg) 1 hour before induction of pain by intra-peritoneal injection of 0.01mL/g of 0.6%v/v acetic acid. The writhing episodes number has been counted and recorded after 5 minutes of induction of pain and for 20 min in the control group which received 10% DMSO only. On the other hand, the positive group received acetic acid, reference group received celecoxib and the tested groups were administered the newly synthesized compounds. The efficacy of the evaluated compounds was estimated by counting the number of writhing in each group.

Molecular modeling:

Molecular modeling of the synthesized compound 13a inside COX-2 active site was achieved using Molecular Operating Environment (MOE) version 2008.10. The 3D crystallized structure of COX-2 isozyme with a co-crystalized ligand, celecoxib (PDB ID: 3LN1) was downloaded from protein data bank (www.rcsb.org). All bounded water molecules were eliminated, and all hydrogen atoms were added to the protein. The tested compound 13a was inserted into the ligand-binding pocket of COX-2 with MOE-DOCK using the triangle matcher placement method and the London DG scoring function. Finally, analysis of the types of interaction of docked COX-2 pocket with the tested compound was performed.

RESULTS AND DISCUSSION:

Chemistry:

All the targeted final compounds were prepared as stated by the illustrated synthetic pathways, Schemes 1-3. The starting material 4 was synthesized by diazotization of primary amine of sulfanilamide 3 followed by reduction using SnCl₂/ HCl to furnish the corresponding hydrazine salt 4 ⁴⁷. On the other hand, β-diketone 2 was prepared through Claisen condensation with diethyl oxalate in sodium ethoxide as a base ⁴⁸. A cyclization reaction between hydrazine salt 4 and the β-diketone 2 was performed in ethanol affording the diarylpyrazol derivative 5 in a good yield ⁴⁹. Finally, treatment of the ethyl ester 5 with hydrazine hydrate provided the key intermediate 6 as reported ⁵⁰.

Pharmacology:

In vitro inhibitory COXs assay:

Compounds 13a-d were synthesized firstly and subsequently, tested for their capability to inhibit COX-2 activity using human COX-1/COX-2 enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI, USA). The efficacy of the tested compounds was expressed as IC₅₀ where the obtained results were tabulated in Table 1. Four compounds 13a-d showed moderate inhibitory COX-2 activity with IC₅₀ ranging from 2.52 to 13.72 µM when compared with celecoxib which used as a standard. The in vitro enzyme inhibitory activity submitted that 13a is more selective than other compounds towards COX-2 over COX-1 (SI = 3.94) when compared with the reference drug celecoxib (SI = 6.44). It was obvious that the introduction of 1,3,4-Oxadiazole moieties to the diaryl compounds (13a-d) showed less remarkable effect on COX-2 selectivity compared to their adamantyl analogues (13a and 13b), unlike others attached to trifluoromethyl aniline residues (13c and 13d).

Table 1: In vitro COX-1/COX-2 results of the evaluated final compounds

Compound code	COX Inhibition (IC₅₀ µM)^a		Selectivity Index^b
Compound code	COX-1 COX-2		Selectivity Index^b
13a	9.93	2.52	3.94
13b	8.98	3.67	2.44
13c	13.96	9.77	1.42
13d	14.33	13.72	1.04
celecoxib	6.12	0.95	6.44

^a Selectivity index(COX-1 IC₅₀/COX-2 IC₅₀)

^b Values are expressed as mean ± SEM (n = 3)

In vivo results:

Anti-inflammatory activity screening:

The anti-inflammatory activity of the newly synthesized compounds 13a-d were assayed using carrageenan-induced paw edema technique in rats as described by Winter et al. ⁴⁵. The change in carrageenan-induced edema thickness was reported and tabulated as shown in Table 2. The results manifested, that all the evaluated compounds performed slight anti-inflammatory activity at 3h ranging from 31.54 to 39.18 % in comparison to celecoxib, which showed 82.71 % in edema thickness after 3h from carrageenan induction. After 5 hours, the anti-inflammatory activity of all compounds showed a slight increase ranging from 39.23 to 45.35%. On comparing with celecoxib, it was noticeable that compound 13a elucidated anti-inflammatory activity approximately half that was shown under treatment with celecoxib (EI% = 45.35% of 13a; EI% = 88.3% of celecoxib). From aforementioned results, diaryl derivatives that were attached to the adamantyl moiety showed better anti-inflammatory activity than those attached to trifluoromethyl aniline moiety. As it was shown, compound 13a showed the highest reduction in edema thickness among all the evaluated compounds.

Analgesic activity screening:

To evaluate the analgesic activity of the new compounds, acetic-acid induced writhing assay was used ⁴⁶. Counting and recording the change in the number of writhing was presented in Table 3. The results indicated that the evaluated compounds revealed remarkable analgesic activity in range (inhibition% = 1.49-10.44%) in comparison with celecoxib (inhibition% = 13.43). Interestingly, compounds 13a and 13b showed the best analgesic protection among the rest of compounds compared to celecoxib as a reference. It could be concluded from the aforementioned results that the analgesic protection of the adamantyl moieties paired with diaryl derivatives exceeded those of trifluoromethyl aniline moieties.

Table 3: Analgesic activity results of the final compounds (13a-d) using acetic-acid induced writhing method in mice.

Compound code	No. of Writhes in 5–15 min after treatment (Mean ± SE)^a	% Inhibition
13a	30.00±0.2	10.44
13b	30.00±0.6	10.44
13c	32.00±0.4	4.47
13d	33.00±0.3	1.49
Celecoxib	29.00±0.6	13.43
Control	33.50±0.8	--

^a Values are expressed as mean ± SE

Molecular Docking:

The binding interactions of the most active compound 13a within the active site of COX-2 was discussed through molecular modelling study using Molecular Operating Environment (MOE) version 2008.10. The docking study was performed using the 3D crystal structure of COX-2 complex (PDB code: 3LN1) that was co-crystallized with celecoxib (https://www.rcsb.org/structure/3ln1). Obviously, replacement of His513 in COX-1 with Arg513 provides additional side pocket for COX-2, allowing it to be able for accommodation with more bulky structures. It was notified that binding of celecoxib with COX-2 was carried out through forming two H-bonds between TRP 371 and SER 516 amino acids and –SO₂NH₂ group in distances 2.01 and 2.41 A^ᵒ with E score -15.47 Kcal/mol as shown in Fig.1. The tested compound 13a showed good COX-2 selectivity by applying one H-bond between SER516 amino acid and –SO₂NH₂ group in distances 3.03 A^ᵒ with E score -15.35 Kcal/mol as shown in Fig.2. Consequently, the docking results of compound 13a were consistent with that of the in vivo anti-inflammatory, analgesic and in vitro COX-2 inhibitory activities when compared to celecoxib (Edema inhibition% = 39.18, Writhing inhibition% = 10.44 and IC₅₀ = 2.52 µM for compound 13a and Edema inhibition% = 82.71, Writhing inhibition% = 13.43 and IC₅₀ = 0.95 µM for celecoxib).

Fig.3. Docking and binding pattern of Celecoxib into the active binding site of COX-2 enzyme. (A) 2D interactions diagram; (B) 3D interactions of compound 13a showing its interactions with TRP371 and SER516 amino acids of COX-2 binding site.

Fig.4. Docking and binding pattern of compound 13a into the active binding site of COX-2 enzyme. (A) 2D interactions diagram; (B) 3D interactions of compound 13a showing its interactions with SER516 amino acid of COX-2 binding site.

CONCLUSION:

Novel diarylpyrazole/triazole compounds bearing 1,3,4-oxadiazole were prepared and their anti-inflammatory and selectivity against COX-2 isozyme were evaluated. The study concluded that compound 13a, with adamantyl residue linked to 1,5- diarylpyrazole, elucidated the most elevated anti-inflammatory activity among the rest of the compounds (Edema Inhibition % (EI) = 39.18 and COX-2 IC₅₀ = 2.52 µM). On the other hand, compound 13d, with trifluoromethyl aniline residue linked to diaryl-1,2,4-triazolyl, elucidated the least in vitro /in vivo evaluation results (Edema Inhibition % (EI) = 31.52 and COX-2 IC₅₀ = 13.72 µM).

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Received on 09.03.2020 Modified on 14.04.2020

Research J. Pharm. and Tech 2020; 13(9):4255-4262.

DOI: 10.5958/0974-360X.2020.00751.9

Compound code	Change in paw volume in (ml) after drug digestion (±SEM)			Anti-inflammatory activity (% Inhibition)
Compound code	1h	3h	5h	1h	3h	5h
13a	10.60±0.45	9.95±0.30	8.94±0.18	35.15	39.18	45.35
13b	11.13±0.02	10.09±0.05	8.74±0.12	31.97	38.29	46.56
13c	11.54±0.03	10.63±0.15	9.42±0.08	29.43	34.98	42.41
13d	12.47±0.02	11.20±0.05	9.94±0.09	23.74	31.54	39.23
Celecoxib	3.08±0.15	2.94±0.08	2.28±0.14	81.17	82.71	88.3
Control	16.36±0.24	17.00±0.28	19.48±0.26	--	--	--