Synthesis, molecular docking and anti-inflammatory evaluation of new trisubstituted pyrazoline derivatives bearing benzenesulfonamide moiety

Abstract A new series of trisubstituted pyrazoline bearing benzenesulfonamide moiety 6a,b–10a,b were designed, synthesised and evaluated for their anti-inflammatory in vitro. Before starting the synthesis, docking study has been used to insert compounds within the COX-2 structure active site using celecoxib drug as a reference. Final compounds 6a,b–10a,b were synthesised by condensing chalcones bearing pyridine moiety 1a,b–5a,b with 4-hydrazinyl benzenesulfonamide hydrochloride. In vitro, their anti-inflammatory activity was assessed using egg-white paw edema method, they showed moderate to strong inhibitory activity. Notably, Compounds 6a (29.78%), 7a (28.43%), 9a (27.92%) and 10a (27.92%) exhibited significant percentage inhibition at 300 min and results are comparable with percentage inhibition drug celecoxib (22.67%) and this result is highly agreement with docking scoring study. Graphical abstract


Introduction
The strong interest in the anti-inflammatory properties of small molecules, particularly for selective targeted compounds with the aid of molecular docking gains the attention of the larger research community.Non-steroidal anti-inflammatory drugs (NSAIDs) are the most often recommended class of therapeutic drugs for pain and inflammation (da Costa et al. 2017).Modulation of well-known non-selective NSAIDs is one of the primary strategies to disclose novel compounds (Takahashi et al. 2018).Most of the selective COX-2 inhibitors are linked to heterocyclic ring system containing a SO 2 NH 2 as a crucial group at the para position of an aryl ring that plays an essential role as a pharmacophore-based moiety of COX-2 selective inhibitors; as, celecoxib and rofecoxib (Pal et al. 2003;Al-Turki et al. 2017).The amino acid sequences of COX-1 and COX-2 are 63% identical and 78% similar (Kadu sevi cius 2021).1,3,5-Trisubstitutedpyrazoline are important five membered heterocyclic compounds (Chai et al. 2006).It has a very important role in the development of theory in heterocyclic chemistry and is also used as a template structure in organic synthesis (Sobhi et al. 2008).Pyrazole derivatives have been reported to possess activities like anti-inflammatory (Marella et al. 2013), antidepressant (Johnson et al. 2007), anticancer (Rostom 2006) and anti-microbial activities (Raauf et al. 2019).So, constant effort is being put in to discover newer anti-inflammatory agents with improved efficacy and safety.Therefore, in the view of the above information and based on the selective anti-inflammatory drug celecoxib, and after good results of anti-inflammation activity that obtained from in silico docking studies of synthesised compounds.We undertook the synthesis of new substituted pyrazoline derivatives bearing benzenesulfonamide and evaluation of its anti-inflammatory activity in vivo results using egg white paw edema method to induce acute inflammation.

Molecular docking
The term 'GOLD' refers to a 'genetic strategy for docking flexible ligands into protein binding sites' (Verdonk et al. 2003).The results of docking study of the celecoxib and newly designed ligands 6a,b-10a,b were represented in (Table S4) using the GOLD Suite program.Docking data revealed that all the designed ligands demonstrate good binding energies in the active pocket of the enzyme COX-2 (Figure S22), as it bind to the amino acid residue of the active side through H-bonds along with other short contacts that reinforce the binding.The Piecewise Linear Potential PLP fitness of the celecoxib (86.16) and the ligands 7a, 9a, and 10a (85.33-81.14),while, ligand 6a gave promising docking results with the COX-2 complex on average PLP fitness (87.12).The molecular binding pattern of celecoxib with COX-2 include hydrogen bonds with (PHE 504, GLN 178 and LEU 338) residues of the enzyme (Figure 1a), while the ligands 6a, 7a, 9a and 10a interact by hydrogen bond via (ARG 106, PHE 504 and TYR 371) residues as shown in (Figures 1b-e).Finally, there's compatible correlation between docking analysis and the experimental of the antiinflammation study results.

Anti-inflammatory activity
All the synthesized compounds 6a,b-10a,b were screened for anti-inflammatory activity by using egg-white paw edema method.Celecoxib has been used as a reference drug because the synthesised compounds have structural similarities to it.The anti-inflammatory activity of the final compounds was evaluated and exert significant inhibition of inflammation in rats as compared to the control and celecoxib the result shown in (Figures S23, S24 and Table S5).Compounds 6a (29.78%), 7a (28.43%), 9a (27.92%) and 10a (27.92%) exhibit strong anti-inflammatory activity to that of reference celecoxib (22.67%) at 300 min, while compound 9b (11.84%) exhibited lower anti-inflammatory effect.These results are consistent with the docking study on enzyme COX-2 that responsible for inflammation by strong binding these compounds 6a, 7a, 9a and 10a on enzyme COX-2 comparable with celecoxib.To a stirred solution of 10% potassium hydroxide (10 mL) and of the appropriate aldehydes (1.0 mmol) [4-nitrobenzaldehyde 2 or 4-chlorobenzaldehyde 4] in (5 mL) of ethanol was added drop wise over 2-3 h of 3-acetylpyridine (a) or 4-acetylpyridine (b) (1.0 mmol, 121.1 mg) at room temperature.The temperature was kept at 0 C and stirred for another 2 h.The resulting solid was filtered, washed with water, dried and recrystallized from ethanol.

Experimental
Method B: synthesis of compounds 1a,b, 3a,b and 5a,b To equimolar amounts of 3-acetylpyridine (a) or 4-acetylpyridine (b) (1.0 mmol, 121.1 mg) and of the appropriate benzaldehydes (1.0 mmol) [4-methoxybenzaldehyde 1,4-dimethylamino benzaldehyde 3 or benzaldehyde 5] were added to (100 mL) of water at temperatures below 5 C. The mixture was stirring thoroughly in order to obtain a finely dispersed emulsion.(10 mL) of a 10% potassium hydroxide solution was added.The mixture was stirred for 30 min and left overnight at 4 C.The resulting solid was filtered, washed with water, dried and recrystallized from ethanol.

Molecular docking
The molecular docking was performed by using the full license version of Genetic Optimization for Ligand Docking (GOLD) (v.2020.3.0)(Jones et al. 1997) include Hermes visualizer software (v.1.10.1),used for visualization of receptors, ligands, type of interaction (H-bond, short contact … etc.), active site, bond length calculation.The GOLD docking binding site was defined as all of the protein residues within the 10 Å reference ligands that accompanied the downloaded protein structure complexes.COX-2 protein was downloaded from the PDB website (PDB: 3LN1) (Wang et al. 2010).The molecular docking studies are a beneficial tool for the evolution of new compounds with prediction of ligand-receptor interaction.The protein reference ligand has been used to determine the active site radius (10 Aᵒ).Chemscore kinase was used as a configuration template.The scoring function performed using Chemplp.All parameters' values used in the GOLD docking process remained default, and all solutions are graded according to Fitness function of Piecewise Linear Potential (CHEMPLP).The docking results such as the docked pose, binding mode, and binding free energy was used to assess the ligands interaction with the protein residues of the active site of COX-2 (Figure S22).
3.4.Anti-inflammatory activity (Domiati et al. 2016) In vivo, the acute anti-inflammatory activity for the chemically synthesized derivatives 6a,b-10a,b had been screened in egg-white paw edema.The assessment of their anti-inflammatory effect was based on measuring the decreases of paw thickness.
Albino rats of male sex (170 ± 10 g) was supplied by Iraqi Center for Cancer and Medical Genetic Research and were housed in College of Pharmacy, Mustansiriyah University under standardized conditions for 10 days for acclimatization.Animals were deprived of food for 12 h prior to the experiment and only water was given.Animals were brought to the laboratory, one hour before the experiment, and were divided into twelve groups (each group consist of 6 rats) as follows: First group: control group; they were injected with the vehicle intraperitoneally (propylene glycol 50%, v/v).Second Group: reference group celecoxib in a dose of 50 mg/kg suspended in propylene glycol.Tested Groups: the tested compounds 6a,b-10a,b at dose that determined below.Propylene glycol was used as a vehicle.S6.

Experimental design
The anti-inflammatory activity of the tested compounds 6a,b-10a,b was studied using the egg-white induced edema model.The paw thickness was measured by Vernia at seven-time intervals (0, 30, 60, 120, 180, 240, and 300 min.)after drug administration.Acute inflammation was produced by a sub-cutaneous injection of (0.05 mL) of undiluted egg-white into the plantar side of the left hind paw of the rats; (30 min.)after intra-peritoneal administration of the drugs or their vehicle.

Statistical analysis
Statistical analysis of data was performed using SAS (Statistical Analysis Systemversion 9.1).One-way ANOVA and Dunnett's post-hoc test were performed to assess significant differences among means.Least significant test was used to compare the differences among inhibition percentages.P < 0.05 is considered statistically significant.
The percentage inhibition of edema is calculated using following formula.
% Inhibition ¼ TₒÀT Tₒ x 100 where T is the thickness of paw of rats given tested compounds at corresponding time and T o is the paw thickness of rats of control group at the same time (mInc.SI 1987).

Conclusion
The synthesis of the designed compounds 6a,b-10a,b has been successfully achieved them evaluation give excellent anti-inflammatory activity.Docking data revealed that all the designed ligands demonstrate good binding energies in the active pocket of the receptor and expected promising binds to the amino acids residue of the active site of COX-2 enzyme through hydrogen bonds along with other short contacts that reinforce the binding.The compounds 6a, 7a, 10a and 6b exhibited more inflammation reduction than celecoxib.The synthesised ligand 6a gave promising docking results with the COX-2 complex showed the best docking on average PLP fitness for 10 conformations of flexible docking which was (87.12).

3. 1 .
Apparatus and chemicals All reagents and anhydrous solvents were of analytical type and generally used as received from the commercial suppliers (Zhejiang Medicine Co. Ltd., Xinchang Pharmaceutical Factory (China).4-Hydrazinylbenzenesulfonamide hydrochloride from hyper-chem, Hangxing RD., Hangzhou, China.Melting points were determined by open capillary method on Stuart/Electro thermal an electric melting point apparatus (U.K.) and IR spectra were recorded on a FT-IR spectrophotometer Shimadzu-6100 as KBr disks at Mustansiriyah University/College of Pharmacy. 1 H NMR and 13 C NMR spectra were recorded using DMSO-d 6 as solvent and tetramethyl silane (TMS) as internal standard on a Varian (500 MHz) spectrometer (Chemical shifts represented in d, ppm) and a Varian (125.65 MHz)) spectrometer, respectively.Elemental analysis was carried out on by Mustansiriyah University/College of Pharmacy CHNS instrument (Vario MICRO CUBE).

Figure 1 .
Figure 1.H bond and short contact interaction profile.
: of the tested compoundsCompounds with their molecular weight and dose shown in Table