INTRODUCTION:

The effective and quick preparation of biologically active compounds has encouraged researchers to identify new strategies which could be beneficial to the pharmaceutical industry. Pyrazole analogues are a class of bioactive nitrogenous heterocycles, playing an essential role in the medicinal chemistry fields¹. Incorporation of different aryl onto pyrazole nucleus have resulted in Celecoxib and Rimonabant which are anti-inflammatory drugs (Figure 1). Alegaon et al. (2014) have reported that some trisubstituted pyrazole derivatives are potent anti-inflammatory activity and COX-2 selective inhibition^2,3.

They inhibit COX enzymes because of their similarity to celecoxib (figure 1), which is a selective COX-2 inhibitor in terms of the chemical structure and also carrying a 4, 5-dihydro-1Hpyrazole ring in their structure⁴. In addition, numerous reports have appeared in the literature describing different bioactivities and good safety profiles of 1, 3, 4-trisubstituted pyrazole derivatives including anti-inflammatory^5-9, analgesic¹⁰, lipid peroxidation^11,12, antipyretic¹³, antioxidant¹⁴, antimicrobial^15,16, antiviral^17-19, anticancer^20-23, antimitotic²⁴, and immunosuppressive agents²⁵. In addition, some pyrazole compounds have gained great attention as antibacterial and fungicidal by inhibition of isoforms of human cytosolic carbonic anhydrase I or II and antitumor properties^26-29. Extension of research towards the identification of an efficient synthesis of biologically active pyrazole compounds^30-34, this study reports the synthesis of novel derivatives of 4, 5-dihydro-1H- pyrazole-1-yl acetate derivatives depending on the docking study as cyclooxygenase inhibitors.

Celecoxib

Rimonabant

Figure 1: Structure of Celecoxib and Rimonabant

MATERIALS AND METHODS:

Compounds IV_a-iderivatives structures were designed depending on previous kinds of literature and the 3D conformations structures of all derivatives were separately drawn by using ChemDraw18.0 software (ChemDraw, 2018). Next, geometry optimization calculation was applied by MM+ force field mechanics by using Hyperchem version 8.0 and saved as a separate mol file format³⁵. Then, extra geometry optimization by semi-empirical mechanics was performed by RM1 (Recife Model 1) calculation³⁶. The best lowest energy conformation of each derivative was saved in separate SDF format for the final optimization step by using Spartan 14.0 program under windows (Spartan, 2014) by including Monte Carlo mechanics method with 100 cycles of optimization and 1000 interactions panels³⁷. Molecular docking study was applied by using Glide docking tool under Schrodinger (Maestro 11.1) software (Schrodinger, 2017) for the proper preparation, energy minimization and docking calculation running on Windows 7 operating system service pack 1 on Dell Precision T-1572PC workstation (Intel(R) Core(TM) i7 CPU 868 @ 3.64GHz, 16 GB RAM, 2 TB HD). The crystal structures of the COX-2 active site (PDB code: 3LN1) were downloaded from Protein Data Bank with a crystallographic resolution of 1.6. For receptor preparation, (ProPrep) tool is used for cleaning, optimization and energy minimization of the crystal. This tool is also used to fill missing loops and crystal preparation with an ideal high-quality receptor structure. For ligand or derivative preparation, (LigPrep) tool is used before docking calculation to identify the accurate ionization level, adding missing hydrogen atom and to achieve the lowest energy conformations of each ligand using OPLS 2005 force field mechanics. For the docking study, the grid box was adjusted to 1.00 Å with an atomic charge of 0.25. Then, the essential ligand docked back in a flexible method by using Glide-extra precision (XP) simulations prosses to the active binding pocket in the receptor. During all docking calculation prosses, the receptor was kept rigid while ligands were left flexible. Finally, the best-docked orientation poses and lowest RMSD between the crystal structure of the receptor and the ligands were saved³⁸.

Instruments:

The melting point was determined by an open capillary method by using electric melting points apparatus, IR bands were recorded using FTIR Shimadzu (IR-Affinity-1) (Japan), ¹HNMR bands (solvent DMSO-d6) were documented on 500 MHZ spectrometer (Bruker, Germany) with TMS as internal standard, thin layer chromatography (TLC) to check the purity and progress of the reaction was run was run on TLC plate [silica gel (60) F₂₅₄] coated aluminium plates, (Merck, Germany), The identification of compounds was done using IR spectra were recorded on an FTIR- spectrophotometer Shimadzu by direct placing the sample on the sensitive lens of the spectrophotometer, in the range 4000 - 400 cm^-1 at BPC analysis center-Baghdad. ¹H-NMR spectra were performed on instrument Inova-Varian 500 MHz spectrometer frequency using DMSO as solvent at Tehran University. CHN analysis was performed at Tehran University examination was done via Vario macro cube-the art of elemental analysis.

Chemical Synthesis:

General Procedure for Synthesis of Chalcone Derivatives (I_a-c):

Acetophenone (1.18mL, 0.01mol) and one of the aromatic aldehyde derivatives (a-c) showed in the table (1) 0.01mol were added to absolute ethanol (22mL), and then sodium hydroxide (40%, 10mL) solution was added dropwise over 2min., The mixture was irradiated by an ultrasonic generator in a water bath at 30-35^oC for 25 min., turbidity appeared in the mixture, which was then neutralized with 2N HCl. The solid product formed was filtered, washed with cold water and recrystallized by ethanol^39-42.

Chalcone (I_a): yield = 86.43%. M.P = 56-57^◦C. FT- IR spectrum characteristics absorption bands of CH st.v neighboring to the olefinic group at 3060 cm^-1, CH st.v of aromatic at 3030 cm^-1, C=O st.v of conjugated ketone at 1662 cm^-1, C=C st.v of olefinic at 1602 cm^-1, C=C st.v of aromatic at 1575 cm^-1.

3-(4-(dimethylamino)phenyl)-1-phenylprop-2-en-1-one (I_b): yield = 88.24%. M.P = 111-113^◦C. FT-IR spectrum characteristics absorption bands of CH st.v neighboring to the olefinic group at 3061 cm^-1, CH st.v of aromatic at 3050 cm^-1, CH st.v of CH₃ at 2910-2808 cm^-1, C=O st.v of conjugated ketone at 1647 cm^-1, C=C st.v of olefinic at 1599 cm^-1, C=C st.v of aromatic at 1560-1531 cm^-1, N-CH₃ st.v at 1170 cm^-1.

3-(4-nitrophenyl)-1-phenylprop-2-en-1-one (I_c): yield = 92.95%. M.P=160-162^◦C. FT- IR spectrum characteristics absorption bands of CH st.v neighboring to the olefinic group at 3059 cm^-1, CH st.v of aromatic at 3048 cm^-1, C=O st.v of conjugated ketone at 1660 cm^-1, C=C st.v of aromatic overlapped with olefinic at 1593-1516 cm^-1, NO₂asymmetric st.v at 1452 cm^-1, NO₂ symmetric st.v at 1334 cm^-1.

Table 1: Aromatic Aldehyde’s Name and Products No.

No.	Aromatic Aldehyde’s Name	Products No.	R	Quantity (g)
a	Benzaldehyde	I_a	H	1.06
b	4-Dimethylamino benzaldehyde	I_b	N(CH₃)₂	1.49
c	4-nitrobenzaldehyde	I_c	NO₂	1.51

General Procedures for the Synthesis of Pyrazole Derivatives (_IIa-c):

A mixture of chalcone (0.01mol), hydrazine hydrate (0.01mol) and acetic acid (5ml) in ethanol (20ml) was refluxed for 8 hrs. The reaction mixture was cooled and poured over ice water. The solid separated was filtered, washed with water, dried and recrystallized from ethanol gave pale yellow crystals^43-46.

3, 5-diphenyl-4, 5-dihydro-1H-pyrazole (II_a): yield = 79.44%. M.P = 50-52^◦C. Anal. Calcd. for C₁₅H₁₄N₂ (222.29): C, 80.05; H6.35; N, 12.60; Found: C, 80.28; H, 6.37; N, 12.86. FT- IR spectrum characteristics absorption bands of NH st.v of pyrazole at 3250 cm^-1, CH st.v of aromatic at 3059 cm^-1, CH₂ st.v of pyrazole ring at 2974 cm^-1, CH st.v of pyrazole ring at 2924 cm^-1, C=N st.v of pyrazole ring at 1662 cm^-1, C=C st.v of aromatic at 1597 cm^-1. ¹H-NMR spectrum showed doublet for CH₂ proton of pyrazole at 3.34δ, triplet for CH proton of pyrazole at 3.50δ, singlet for NH proton at 8.73δ.

5-(4-dimethyl amino)-3-phenyl-4, 5-dihydro-1H-pyrazole (II_b): yield = 87.22%. M.P = 78-80^◦C. Anal. Calcd. for C₁₇H₁₉N₃ (265.36): C, 76.95; H, 7.22; N, 15.84; Found: C, 76.94; H, 7.19; N, 15.78. C:, H:, N: 15.78. FT- IR spectrum characteristics absorption bands of NH st.v of pyrazole at 3289 cm^-1, C-H st.v of aromatic at 3057 cm^-1, CH & CH₂ st.v of pyrazole overlap with CH asymmetric st.v of CH₃ at 2993 cm^-1, CH symmetric st.v of CH₃ at 2802 cm^-1, C=N st.v of pyrazole at 1653 cm^-1, C=C st.v of aromatic at 1608-1570 cm^-1, N-CH₃ st.v at 1172 cm^-1.¹H-NMR spectrum showed singlet for CH₃ proton of N(CH₃)₂ at 2.85δ, doublet for CH₂ proton of pyrazole at 3.37δ, multiplet for CH proton of pyrazole at 3.79δ, singlet for NH proton of pyrazole at 9δ.

5-(4-nitrophenyl)-3-phenyl-4, 5-dihydro-1H-pyrazole (II_c): yield = 79.99%. M.P = 218-220^◦C. Anal. Calcd. for C₁₅H₁₃N₃O₂ (267.29): C, 67.40; H, 4.09; N, 15. 72; Found: C, 67.81; H, 4.12N, 5.32, N: 15.06. FT- IR spectrum characteristics absorption bands of NH st.v of pyrazole at 3228 cm^-1, C-H st.v of aromatic at 3053 cm^-1, CH₂ st.v of pyrazole ring at 2926 cm^-1, CH st.v of pyrazole ring at 2848 cm^-1, C=N st.v of pyrazole at 1662 cm^-1, C=C st.v of aromatic at 1595-1518 cm^-1, NO₂ asymmetric st.v at 1448 cm^-1, NO₂ symmetric st.v at 1346 cm^-1.¹H-NMR spectrum showed doublet for CH₂ proton of pyrazole at 3.33δ, multiplet for CH proton of pyrazole at 3.57δ, singlet for NH proton of pyrazole at 8.92δ.

General Procedures for the Synthesis of antioxidant-chloroacetyl Derivatives (III_a-c):

A mixture of one appropriate antioxidant (0.01mol) and TEA (0.01mol) in dichloromethane (25ml) was cooled in an ice salt mixture to 10°C. To this reaction mixture, chloroacetylchloride (0.014mol) in chloroform (25ml) was added dropwise with constant stirring throughout 1 h, maintaining the temperature constant. The reaction mixture was stirred overnight at room temperature, washed with 5% HCl (3×50 ml), 5% NaOH (3×50ml) and finally with brine solution (2×25ml). The organic layer was dried over anhydrous sodium sulphate, filtered and the solvent was removed under reduced pressure to obtain the corresponding antioxidant-chloroacetyl derivative. This general procedure was used with different antioxidants (thymol, guaiacol and menthol) to prepare corresponding chloroacetyl derivative (III_a-c)^{47, 48}.

2-isopropyl-5-methylphenyl 2-chloroacetate (III_a): yield = 83.70%. FT- IR spectrum characteristics absorption bands of C-H st.v of aromatic at 3028cm^-1, CH st.v of CH₃ at 2962-2970 cm^-1, C=O st.v of ester at 1739 cm^-1, C=C st.v of aromatic at 1573-1504 cm^-1, C-Cl st.v overlapping with CH bending out of plain at 813 cm^-1.

2-isopropyl-5-methylcyclohexyl 2-chloroacetate (III_b): yield = 87.55^%. M.P = 60-62^◦C. FT- IR spectrum characteristics absorption bands of C-H st.v of aromatic at 2993cm^-1, CH st.v of CH₃ at 2947-2843cm^-1, C=O st.v of ester at 1762 cm^-1, C=C st.v of aromatic at 1600-1496 cm^-1, C-Cl st.v overlapping with CH bending out of plain at 755cm^-1

2-methoxyphenyl 2-chloroacetate (III_c): yield = 85.34%. M.P=30-32^◦C. FT- IR spectrum characteristics absorption bands of CH st.v of CH₃ at 2947-2862 cm^-1, C=O st.v of ester at 1738 cm^-1, C-Cl st.v at 790 cm^-1.

General Procedures for the Synthesis of Designed Derivatives (IV_a-i):

A mixture of one derivative of compound II_a-c(0.01mol) and one derivative of compound (III_a-c)(0.014mol) was mixed in 15ml of 1, 4-dioxane. To this mixture, 0.01 mol of triethylamine (TEA) solution was added and the reaction mixture was refluxed for 2-3h followed up by TLC. It was then cooled and poured into crushed ice. The solid filtered was washed with 10% K₂CO₃ and then with distilled water⁴⁹.

2-isopropyl-5-methylphenyl-2-(5-(4-(dimethylamino)phenyl)-3-phenyl 4, 5 -dihydro-1H-pyrazol-1-yl)acetate (IV_a): yield = 77.70%. M.P = 201-203^◦C. CHN Anal. Calcd. for C₂₉H₃₃N₃O₂ (455.59): C, 76.45; H, 7.30; N, 9.22; Found: C, 76.44; H, 7.32; N, 9.20. FT- IR spectrum characteristics absorption bands of CH st.v of aromatic at 3059 cm^-1, CH, CH₂ & CH₃ st.v at 2954-2850 cm^-1, C=O st.v of ester at 1732 cm^-1, C=N st.v of pyrazole at 1681 cm^-1, C=C st.v of aromatic at 1570 cm^-1, C-N st.v of pyrazole at 1361 cm^-1.¹H-NMR spectrum showed doublet for CH₃ proton of isopropyl at 1.19δ, multiplet for CH₃ protons of aromatic ring overlap with proton of CH of isopropyl at 2.84 δ, singlet for CH₃ proton of N(CH₃)₂ at 3.05 δ, doublet for CH₂ proton of pyrazole at 3.35δ, singlet for CH₂ proton adjacent to C=O group and N of pyrazole at 3.60δ, triplet for CH proton of pyrazole at 3.71δ, multiplet for CH proton of aromatic rings at 6.67-7.07δ.

2-isopropyl-5-methylcyclohexyl-2-(5-(4-(dimethylamino)phenyl)-3-phenyl-4, 5-dihydro-1H-pyrazol-1-yl)acetate (IV_b): yield = 80.60%. M.P = 110-112^◦C. CHN Anal. Calcd. for C₂₉H₃₉N₃O₂ (461.64): C, 75.45; H, 8.52; N, 9.10; Found: C, 75.44; H, 8.50; N, 9.15. FT- IR spectrum characteristics absorption bands of CH st.v of aromatic at 3055 cm^-1, CH, CH₂ & CH₃ st.v at 2954-2800 cm^-1, C=O st.v of ester at 1774 cm^-1, C=N st.v of pyrazole at 1681 cm^-1, C=C st.v of aromatic at 1566 cm^-1, C-N st.v of pyrazole at 1346 cm^-1.¹H-NMR spectrum showed doublet for CH₃ proton of cyclohexane overlap with protons of CH₃ of isopropyl at 0.85δ, multiplet for CH, CH₂proton of cyclohexane overlap with the proton of CH of isopropyl at 0.98-1.19δ, singlet for CH₃ proton of N(CH₃)₂ at 2.87δ, doublet for CH₂ proton of pyrazole at 2.99δ, singlet for CH₂ proton adjacent to C=O group and N of pyrazole at 3.55δ, multiplet for CH proton of cyclohexane adjacent to O atom at 3.77δ, multiplet for CH proton of aromatic rings at 6.54-7.19δ

2-methoxyphenyl-2-(5-(4-(dimethylamino)phenyl)-3-phenyl-4, 5-dihydro-1H-pyrazol-1-yl)acetate(IV_c): yield = 86.92%. M.P = 101-103^◦C. CHN Anal. Calcd. for C₂₆H₂₇N₃O₃ (429.51): C, 72.71; H, 6.34; N, 9.78; Found: C, 72.90; H, 6.44; N, 9.78. FT- IR spectrum characteristics absorption bands of CH st.v of aromatic at 3055 cm^-1, CH, CH₂& CH₃ st.v at 2997-2800 cm^-1, C=O st.v of ester at 1766 cm^-1, C=N st.v of pyrazole at 1651 cm^-1, C=C st.v of aromatic at 1604-1516 cm^-1, C-N st.v of pyrazole at 1350 cm^-1.¹H-NMR spectrum showed singlet for CH₃ proton of N(CH₃)₂ at 2.87δ, doublet for CH₂ proton of pyrazole at 3.36δ, singlet for CH₂ proton adjacent to C=O group and N of pyrazole at 3.58δ, Triplet for CH proton of pyrazole at 3.76δ, singlet for CH₃ proton of methoxy group at 3.79δ, multiplet for CH proton of aromatic rings at 6.71-6.99δ

2-isopropyl-5-methylphenyl-2-(5-(4-nitrophenyl)-3-phenyl-4, 5-dihydro-1H-pyrazol-1-yl)acetate(IV_d): yield = 88.21%. M.P = 212-214^◦C. CHN Anal. Calcd. for C₂₇H₂₇N₃O₄ (457.52): C, 70.88; H, 5.95; N, 9.18; Found: C, 70.84; H, 5.94; N, 9.22. FT- IR spectrum characteristics absorption bands of CH st.v of aromatic at 3055 cm^-1, CH₂ st.v of pyrazole at 2958 cm^-1, CH st.v of pyrazole at 2870 cm^-1, C=O st.v of ester at 1759 cm^-1, C=N st.v of pyrazole ring at 1697 cm^-1, C=C st.v of aromatic at 1597-1516 cm^-1, NO₂ asymmetric stretching at 1454 cm^-1, NO₂ symmetric stretching at 1342 cm^-1. ¹H-NMR spectrum showed doublet for CH₃ proton of isopropyl at 1.10δ, multiplet for CH proton of isopropyl at 2.15δ, singlet for CH₃ proton of aromatic ring at 2.27δ, doublet for CH₂ proton of pyrazole at 3.12δ, singlet for CH₂ proton adjacent to C=O group and N of pyrazole at 3.32δ, triplet for CH proton of pyrazole at 3.93δ, multiplet for CH proton of aromatic rings at 7.25-7.82δ

2-isopropyl-5-methylcyclohexyl-2-(5-(4-nitrophenyl)-3-phenyl-4, 5-dihydro -1H-pyrazol-1-yl)acetate(IV_e): yield = 84.65%. M.P = 220^◦C. CHN Anal. Calcd. for C₂₇H₃₃N₃O₄ (463.57): C, 69.95; H, 7.18; N, 9.06; Found: C, 69.90; H, 7.20; N, 9.06. FT- IR spectrum characteristics absorption bands of CH st.v of aromatic at 3059 cm^-1, CH & CH₂ st.v of pyrazole at 2947-2870 cm^-1, C=O st.v of ester at 1739 cm^-1, C=N st.v of pyrazole at 1658 cm^-1,C=C st.v of aromatic at 1512 cm^-1, NO₂ asymmetric stretching at 1454 cm^-1, NO₂ symmetric stretching at1307 cm^-1.¹H-NMR spectrum showed doublet for CH₃ proton of cyclohexane overlap with protons of CH₃ of isopropyl at 0.93δ, multiplet for CH, CH₂proton of cyclohexane overlap with the proton of CH of isopropyl at 0.95-1.32δ, doublet for CH₂ proton of pyrazole at 2.94δ, singlet for CH₂ proton adjacent to C=O group and N of pyrazole at 3.15δ, triplet for CH proton of pyrazole at 3.24, multiplet for CH proton of cyclohexane adjacent to O atom at 3.50δ, multiplet for CH proton of aromatic rings at 7.27-8.31δ

2-methoxyphenyl-2-(5-(4-nitrophenyl)-3-phenyl-4, 5-dihydro-1H-pyrazol-1-yl)acetate (IV_f): yield = 95.13%. M.P = 51-52^◦C. CHN Anal. Calcd. for C₂₄H₂₁N₃O₅ (431.44): C, 66.81; H, 4.91; N, 9.74; Found: C, 66.20; H, 4.92; N, 9.87. FT- IR spectrum characteristics absorption bands of CH st.v of aromatic at 3055 cm^-1, CH₂ st.v of pyrazole at 2947 cm^-1, CH st.v of pyrazole ring at 2843 cm^-1, C=O st.v of ester at 1766 cm^-1, C=N st.v of pyrazole ring at 1681 cm^-1, C=C st.v of aromatic at 1519-1500 cm^-1, NO₂ asymmetric stretching at 1408cm^-1, NO₂ symmetric stretching at 1315 cm^-1.¹H-NMR spectrum showed doublet for CH₂ proton of pyrazole at 3.33δ, singlet for CH₂ proton adjacent to C=O group and N of pyrazole at 3.58δ, Triplet for CH proton of pyrazole at 3.76δ, singlet for CH₃ proton of methoxy group at 3.79δ, multiplet for CH proton of aromatic rings at 7.14-7.18δ

2-isopropyl-5-methylphenyl-2-(3, 5-diphenyl-4, 5-dihydro-1H-pyrazol-1-yl)acetate (IV_g): yield = 93.96%. M.P = 66-68^◦C. CHN Anal. Calcd. for C₂₇H₂₈N₂O₂ (412.52): C, 78.61; H, 6.84; N, 6.69; Found: C, 78.64; H, 6.81; N, 6.82. FT- IR spectrum characteristics absorption bands of CH st.v of aromatic at 3024 cm^-1, CH & CH₂ st.v of pyrazole at 2958-2850 cm^-1, C=O st.v of ester at 1766 cm^-1, C=N st.v of pyrazole at 1670 cm^-1, C=C st.v of aromatic at 1608 cm^-1.¹H-NMR spectrum showed doublet for CH₃ proton of isopropyl at 1.11δ, singlet for CH₃ proton of aromatic ring at 2.16δ, multiplet for CH proton of isopropyl at 2.29δ, doublet for CH₂ proton of pyrazole at 3.32δ, singlet for CH₂ proton adjacent to C=O group and N of pyrazole at 3.59δ, triplet for CH proton of pyrazole at 3.69δ, multiplet for CH proton of aromatic rings at 7.04-8.17δ

2-isopropyl-5-methylcyclohexyl-2-(3, 5-diphenyl-4, 5-dihydro-1H-pyrazol-1-yl)acetate (IV_h): yield = 81.73%. M.P = 130-132^◦C. CHN Anal. Calcd. for C₂₇H₃₄N₂O₂ (418.57): C, 77.48; H, 8.19; N, 6.69; Found: C, 77.44; H, 8.20; N, 6.72. FT- IR spectrum characteristics absorption bands of CH st.v of aromatic at 3059 cm^-1, CH & CH₂ st.v of pyrazole at 2947-2858 cm^-1, C=O st.v of ester at 1728 cm^-1, C=N st.v of pyrazole at 1662 cm^-1, C=C st.v of aromatic at 1600 cm^-1, NO₂asymmetric stretching at 1450 cm^-1, NO₂asymmetric stretching at 1365 cm^-1.¹H-NMR spectrum showed doublet for CH₃ proton of cyclohexane overlap with protons of CH₃ of isopropyl at 0.73δ, multiplet for CH₂proton of cyclohexane at 0.89δ, multiplet CHproton of isopropyl at 0.97δ, multiplet CH proton of cyclohexane at 1.31δ, doublet for CH₂ proton of pyrazole at 3.16δ, singlet for CH₂ proton adjacent to C=O group and N of pyrazole at 3.33δ, triplet for CH proton of pyrazole at 3.47 δ, multiplet for CH proton of cyclohexane adjacent to O atom at 3.57δ, multiplet for CH proton of aromatic rings at 7.19-8.19δ

2-methoxyphenyl-2-(3, 5-diphenyl-4, 5-dihydro-1H-pyrazol-1-yl) acetate (IV_i): yield = 86.22%. M.P = 52-53^◦C. CHN Anal. Calcd. for C₂₄H₂₂N₂O₃ (386.44): C, 74.59; H, 5.74; N, 7.25; Found: C, 74.61; H, 5.78; N, 7.20. FT- IR spectrum characteristics absorption bands of CH st.v of aromatic at 3059 cm^-1, CH & CH₂ st.v of pyrazole at 2951-2843 cm^-1, C=O st.v of ester at 1766 cm^-1, C=N st.v of pyrazole at 1662 cm^-1, C=C st.v of aromatic at 1597 cm^-1, C-N st.v of pyrazole at 1361 cm^-1.¹H-NMR spectrum showed doublet for CH₂ proton of pyrazole at 3.33δ, singlet for CH₂ proton adjacent to C=O group and N of pyrazole at 3.37δ, Triplet for CH proton of pyrazole at 3.75δ, singlet for CH₃ proton of methoxy group at 3.79δ, multiplet for CH proton of aromatic rings at 7.14-7.30δ

RESULTS AND DISCUSSION:

Chemistry:

The synthesis of the designed compounds (IV_a-i) through their derivatives was achieved by three steps demonstrated in the scheme (1), the first step gave 4, 5-dihydro-1H-pyrazole (II_a-c) derivatives upon reflexing chalcones (I_a-c) and hydrazine hydrate for 8 hr. while step two gave antioxidant-chloroacetyl derivatives (III_a-c) when reaction takes place between the appropriate antioxidant and the chloroacetyl chloride at -10^oC in the presence of TEA. Step three gave the final compounds 4, 5 -dihydro-1H-pyrazol-1-yl acetate (IV_a-i) by reflexing one of the compounds (II_a-c) with one of compound (III_a-c) in the presence of dioxane and TEA.

Computational study:

In the present work, the focus was on the discovery of novel molecules with high binding affinity to the active target, COX-2 active site receptor, using molecular modelling and theoretical binding affinity studies. A total of 18 molecular structures of designed derivatives were docked inside the active site of COX-2 to evaluate the binding affinity.

The calculated docking score with the molecular structure of all derivates with the active site of COX-2 was listed in the table (2). It showed that the highest docking affinity score was -9.434 kcal/mol for compound IV_iwhich give good affinity for binding inside the COX-2 receptor due to the presence of three aromatic rings and electron with drawing methoxy group in unique position on one of the aromatic rings, the lowest docking affinity score was at -6.736 kcal/mol for compound IV_e which have less affinity for binding inside the COX-2 receptor due to the changesof one aromatic rings that hold the methoxy group to isopropyl methyl cyclohexyl and the presence of electron with drawing nitro group on another rings of designed derivatives while standard drug Celecoxib was -12.049 kcal/mol. Docking score refers to the high affinity of the compound to bind the active site pocket inside COX-2 surrounded by important amino acids as shown in figure (2). Inside the active site of COX-2, compound IV_e binds by hydrophobic interaction reign with multi-interactions: TRP373 and TYR371 bind by Pi-Pi stacking interaction bond with aromatic ring and forming 1 H-bonds between GLN178 and hydroxy group. In addition, it binds with suitable surrounding amino acids, such binding interactions with surrounding amino acids giving the best fit inside the receptor pocket. While compound IV_fbinds by hydrophobic interaction reign with multi-interactions: TRP373 and TYR371 bind by Pi-Pi stacking interaction bond with aromatic ring and forming 3 H-bonds between ARG106-TYR 341 with methoxy group and ARG499 with Nitro group. In addition, it binds with suitable surrounding amino acids, such binding interactions with surrounding amino acids giving the best fit inside the receptor pocket.

Scheme (1): synthetic steps of the designed compounds

Note: Most appropriate derivatives were synthesized depending on the docking study.

Table (2): COX-2 docking affinity score of designed derivatives and standard drug Celecoxib.

Compound

Docking score

Structure

IV_a

N/A

IV_b

N/A

IV_c

-9.187

IV_d

N/A

IV_e

-6.736

IV_f

-8.977

IV_g

-9.103

IV_h

-7.345

IV_i

-9.434

Celecoxib

-12.049

CONCLUSION:

The new series of 4, 5-dihydro-1H- pyrazole-1-yl acetate derivatives were synthesized through different chemical reactions and validated utilizing physical, spectral and elemental data. The phenyl ring in the 3rd position of the 4, 5-dihydro-1H-pyrazole is essential in interaction with COX-2 enzymes, and this phenyl interacted with amino acids in all compounds. The introduction of the bulky group into the structure did not abolish the interaction and 4, 5-dihydro-1H-pyrazole groups made hydrophobic bonds with the COX-2 enzyme and are essential in interactions with the COX-2 enzyme, this binding mode of the compounds inside the COX-2 active site was predicted using a docking technique. A total of 18 molecular structures of designed derivatives were docked inside the active site of COX-2 to evaluate the binding affinity. The highest docking affinity score was -9.434 kcal/mol, the lowest docking affinity score was at -6.736kcal/mol of designed derivatives while the standard drug Celecoxib was -12.049 kcal/mol. In vivo and in vitro COX inhibition tests of these substances are required to obtain more detailed information about their inhibition properties.

LIST OF SYMBOLS AND ABBREVIATIONS:

Cyclooxygenase-2: COX-2, triethylamine: TEA, dimethyl sulfoxide: DMSO, protein data bank: PDB, thin layer chromatography: TLC, tetramethylsilane: TMS.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENT:

The authors would like to thank doctor Basma M. Abd Razik for her kind help during the docking study.

REFERENCES:

1. Godase AS, Pimpodkar NV, Indalkar YR. An Overview on A Pyrazole: Promising Moiety. Asian Journal of Pharmacy and Technology. 2015; 5(4):201-13. doi.org/10.5958/2231-5713.2015.00030.6

2. Alegaon SG, Alagawadi KR, Garg M, Dushyant K, Vinod D. 1, 3, 4-Trisubstituted pyrazole analogues as promising anti-inflammatory agents. Bioorganic Chemistry. 2014; 54:51-9. doi.org/10.1016/j.bioorg.2014.04.001

3. Alegaon S, Hirpara M, Alagawadi K, Hullatti K, Kashniyal K. Synthesis of novel pyrazole–thiadiazole hybrid as potential potent and selective cyclooxygenase-2 (COX-2) inhibitors. Bioorganic & Medicinal Chemistry Letters. 2014; 24(22):5324-9. doi.org/10.1016/j.bmcl.2014.08.062

4. Costa RF, Turones LC, Cavalcante KVN, Rosa Júnior IA, Xavier CH, Rosseto LP, et al. Heterocyclic Compounds: Pharmacology of Pyrazole Analogs From Rational Structural Considerations. Frontiers in Pharmacology. 2021; 12:1091. doi.org/10.3389/fphar.2021.666725

5. Chougala BM, Samundeeswari S, Holiyachi M, Shastri LA, Dodamani S, Jalalpure S, et al. Synthesis, characterization and molecular docking studies of substituted 4-coumarinylpyrano [2, 3-c] pyrazole derivatives as potent antibacterial and anti-inflammatory agents. European Journal of Medicinal Chemistry. 2017; 125:101-16. doi.org/10.1016/j.ejmech.2016.09.021

6. Abdel-Sayed MA, Bayomi SM, El-Sherbeny MA, Abdel-Aziz NI, ElTahir KEH, Shehatou GS, et al. Synthesis, anti-inflammatory, analgesic, COX-1/2 inhibition activities and molecular docking study of pyrazoline derivatives. Bioorganic and Medicinal Chemistry. 2016; 24(9):2032-42.doi.org/10.1016/j.bmc.2016.03.032

7. Abdelall EK, Lamie PF, Ali WA. Cyclooxygenase-2 and 15-lipoxygenase inhibition, synthesis, anti-inflammatory activity and ulcer liability of new celecoxib analogues: determination of region-specific pyrazole ring formation by noesy. Bioorganic and Medicinal Chemistry Letters. 2016; 26(12):2893-9. doi.org/10.1016/j.bmcl.2016.04.046Get

8. Abdellatif KR, Abdelall EK, Fadaly WA, Kamel GM. Synthesis, cyclooxygenase inhibition, anti-inflammatory evaluation and ulcerogenic liability of new 1, 3, 5-triarylpyrazoline and 1, 5-diarylpyrazole derivatives as selective COX-2 inhibitors. Bioorganic and Medicinal Chemistry Letters. 2016; 26(2):406-12. doi: 10.1016/j.bmcl.2015.11.105.

9. El-Feky SA, Abd El-Samii ZK, Osman NA, Lashine J, Kamel MA, Thabet HK. Synthesis, molecular docking and anti-inflammatory screening of novel quinoline incorporated pyrazole derivatives using the Pfitzinger reaction II. Bioorganic Chemistry. 2015; 58:104-16. doi.org/10.1016/j.bioorg.2014.12.003

10. Koduru BS, Shinde AR, Preeti PJ, Kumar KP, Rajavel R, Sivakumar T. Synthesis, characterization, anti-tubercular, analgesic and anti-inflammatory activities of new 2-pyrazoline derivatives. Asian Journal of Pharmacy and Technology. 2012; 2(2):47-50.

11. Hussain S, Kaushik D. Noval 1-substituted-3, 5-dimethyl-4-[(substituted phenyl) diazenyl] pyrazole derivatives: Synthesis and pharmacological activity. Journal of Saudi Chemical Society. 2015; 19(3):274-81. doi.org/10.1016/j.jscs.2012.04.002

12. Thore S, Gupta SV, Baheti KG. Novel ethyl-5-amino-3-methylthio-1H-pyrazole-4-carboxylates: Synthesis and pharmacological activity. Journal of Saudi Chemical Society. 2016; 20(3):259-64. doi.org/10.1016/j.jscs.2012.06.011

13. do Carmo Malvar D, Ferreira RT, de Castro RA, de Castro LL, Freitas ACC, Costa EA, et al. Antinociceptive, anti-inflammatory and antipyretic effects of 1.5-diphenyl-1H-Pyrazole-3-carbohydrazide, a new heterocyclic pyrazole derivative. Life sciences. 2014; 95(2):81-8. doi.org/10.1016/j.lfs.2013.12.005

14. Sharath V, Kumar HV, Naik N. Synthesis of novel indole based scaffolds holding pyrazole ring as anti-inflammatory and antioxidant agents. Journal of Pharmacy Research. 2013; 6(7):785-90. doi.org/10.1016/j.jopr.2013.07.002

15. Dawane BS, Shaikh BM, Khandare NT, Mandawad GG, Chobe SS, Konda SG. Synthesis of Some Novel Substituted Pyrazole Based Chalcones and Their In-Vitro Antimicrobial Activity. Asian Journal of Research in Chemistry. 2010; 3(1):90-3.

16. Balaji P, Prathusha K, Chandu T, Sreevani MS, Rani PJ, Harini P. Synthesis, Characterization and Antimicrobial Activity of Some Synthesised Isoxazole and Pyrazole Derivatives. Asian Journal of Research in Chemistry. 2011; 4(2):301-3.

17. Mady MF, Saleh TS, El-Kateb AA, Abd El-Rahman NM, Abd El-Moez SI. Microwave-assisted synthesis of novel pyrazole and pyrazolo [3, 4-d] pyridazine derivatives incorporating diaryl sulfone moiety as potential antimicrobial agents. Research on Chemical Intermediates. 2016; 42(2):753-69. doi.org/10.1007/s11164-015-2054-x

18. Yu L-G, Ni T-F, Gao W, He Y, Wang Y-Y, Cui H-W, et al. The synthesis and antibacterial activity of pyrazole-fused tricyclic diterpene derivatives. European Journal of Medicinal Chemistry. 2015; 90:10-20. doi.org/10.1016/j.ejmech.2014.11.015

19. Mert S, Kasımoğulları R, Iça T, Çolak F, Altun A, Ok S. Synthesis, structure–activity relationships, and in vitro antibacterial and antifungal activity evaluations of novel pyrazole carboxylic and dicarboxylic acid derivatives. European Journal of Medicinal Chemistry. 2014; 78:86-96. doi.org/10.1016/j.ejmech.2014.03.033

20. Wang F-Q, Yang H, He B, Jia Y-K, Meng S-Y, Zhang C, et al. A novel domino approach for synthesis of indolyl tetrahydropyrano [4, 3-c] pyrazole derivatives as anticancer agents. Tetrahedron. 2016; 72(38):5769-75. doi.org/10.1016/j.tet.2016.07.078

21. Reddy VG, Reddy TS, Nayak VL, Prasad B, Reddy AP, Ravikumar A, et al. Design, synthesis and biological evaluation of N-((1-benzyl-1H-1, 2, 3-triazol-4-yl) methyl)-1, 3-diphenyl-1H-pyrazole-4-carboxamides as CDK1/Cdc2 inhibitors. European Journal of Medicinal Chemistry. 2016; 122:164-77. doi.org/10.1016/j.ejmech.2016.06.011

22. Metwally NH, Abdelrazek FM, Eldaly SM. Synthesis and anticancer activity of some new heterocyclic compounds based on 1-cyanoacetyl-3, 5-dimethylpyrazole. Research on Chemical Intermediates. 2016; 42(2):1071-89. doi.org/10.1007/s11164-015-2074-6

23. Reddy TS, Kulhari H, Reddy VG, Bansal V, Kamal A, Shukla R. Design, synthesis and biological evaluation of 1, 3-diphenyl-1H-pyrazole derivatives containing benzimidazole skeleton as potential anticancer and apoptosis inducing agents. European Journal of Medicinal Chemistry. 2015; 101:790-805. doi.org/10.1016/j.ejmech.2015.07.031

24. Minu M, Singh D, Mahaddalkar T, Lopus M, Winter P, Ayoub AT, et al. Chemical synthesis, pharmacological evaluation and in silico analysis of new 2, 3, 3a, 4, 5, 6-hexahydrocyclopenta [c] pyrazole derivatives as potential anti-mitotic agents. Bioorganic & Medicinal Chemistry Letters. 2016; 26(16):3855-61. doi.org/10.1016/j.bmcl.2016.07.025

25. Lv X-H, Li Q-S, Ren Z-L, Chu M-J, Sun J, Zhang X, et al. (E)-1, 3-diphenyl-1H-pyrazole derivatives containing O-benzyl oxime moiety as potential immunosuppressive agents: Design, synthesis, molecular docking and biological evaluation. European Journal of Medicinal Chemistry. 2016; 108:586-93. doi.org/10.1016/j.ejmech.2015.12.020

26. Mert S, Alım Z, İşgör MM, Beydemir Ş, Kasımoğulları R. The synthesis of novel pyrazole-3, 4-dicarboxamides bearing 5-amino-1, 3, 4-thiadiazole-2-sulfonamide moiety with effective inhibitory activity against the isoforms of human cytosolic carbonic anhydrase I and II. Bioorganic Chemistry. 2016; 68:64-71. doi.org/10.1016/j.bioorg.2016.07.006

27. Guillén E, González A, Basu PK, Ghosh A, Font-Bardia M, Calvet T, et al. The influence of ancillary ligands on the antitumoral activity of new cyclometallated Pt (II) complexes derived from an ferrocene-pyrazole hybrid. Journal of Organometallic Chemistry. 2017; 828:122-32. doi.org/10.1016/j.jorganchem.2016.11.031

28. Zhang H, Zhu P, Liu J, Lin Y, Yao H, Jiang J, et al. Synthesis, in vitro and in vivo antitumor activity of pyrazole-fused 23-hydroxybetulinic acid derivatives. Bioorganic and Medicinal Chemistry Letters. 2015; 25(3):728-32. doi.org/10.1016/j.bmcl.2014.11.058

29. PN B, Prathusha K, Chandu T, Sreevani MS, Rani PJ, Harini P. Synthesis, Characterization and Antimicrobial Activity of Some Synthesized Isoxazole and Pyrazole Derivatives.

30. Fahmy HH, Khalifa NM, Ismail MM, El-Sahrawy HM, Nossier ES. Biological validation of novel polysubstituted pyrazole candidates with in vitro anticancer activities. Molecules. 2016; 21(3):271. doi.org/10.3390/molecules21030271

31. Ismail MM, Khalifa NM, Fahmy HH, EL-Sahrawy HM, Nossier ES. Anticancer evaluation of novel 1, 3, 4-trisubstituted pyrazole candidates bearing different nitrogenous heterocyclic Moieties. Biomedical Research (0970-938X). 2016; 27(4).

32. Fahmy HH, Khalifa NM, Ismail M, El-Sahrawy HM, Nossier ES, Ali M. Biological Evaluation of Novel 1, 3-Diaryl-1H-Pyrazoles Incorporating Different Heterocyclic Ring Systems as Potent Cytotoxic Agents. Lat Am J Pharm. 2016; 35(6):1340-7.

33. Ismail MM, Khalifa NM, Fahmy HH, Nossier ES, Abdulla MM. Design, Docking, and Synthesis of Some New Pyrazoline and Pyranopyrazole Derivatives as Anti‐inflammatory Agents. Journal of Heterocyclic Chemistry. 2014; 51(2):450-8. doi.org/10.1002/jhet.1757

34. Fahmy HH, Khalifa NM, Nossier ES, Abdalla MM, Ismai M. Synthesis and anti-inflammatory evaluation of new substituted 1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazole derivatives. Acta Pol Pharm. 2012; 69:411-21.

35. Abd Razik B, Ezzat MO, Al-Shohani A. Molecular docking and design study for anticancer activity of flavonoid derivatives against breast cancer. Indian Drugs. 2020; 57:7-14. doi.org/10.53879/id.57.04.12202

36. Rocha GB, Freire RO, Simas AM, Stewart JJ. Rm1: A reparameterization of am1 for H, C, N, O, P, S, F, Cl, Br, and I. Journal of Computational Chemistry. 2006; 27(10):1101-11. doi.org/10.1002/jcc.20425

37. Cohen NC. Guidebook on molecular modeling in drug design: Gulf Professional Publishing; 1996.

38. Otuokere I, Amaku F, Alisa C. In silico geometry optimization, excited-state properties of (2E)-N-Hydroxy-3-[3-(Phenylsulfamoyl) Phenyl] prop-2-enamide (Belinostat) and its molecular docking studies with Ebola Virus glycoprotein. Asian Journal of Pharmaceutical Research. 2015; 5(3):131-7. doi.org/10.5958/2231-5691.2015.00020.9

39. Mahdi MF, Raauf AMR, Mohammed NM. Synthesis, characterization and preliminary pharmacological evaluation of new non-steroidal anti-inflammatory pyrazoline derivatives. European Journal of Chemistry. 2015; 6(4):461-7. doi.org/10.5155/eurjchem.6.4.461-467.1321

40. Ismaeel SS, Mahdi MF, Abd Razik BM. Molecular Drug Design, Synthesis and Antibacterial study of Novel 4-Oxothiazolidin-3-yl Derivatives. Al-Mustansiriyah Journal of Pharmaceutical Sciences (AJPS). 2020; 20(2):1-10.

41. Kumar K, Singh B. Synthesis, characterization and anti-microbial activity of some 4-thiazolidinone conjugatives. Asian Journal of Pharmaceutical Analysis. 2020; 10(4).

42. Saravanan G, Alagarsamy V, Prakash C, Kumar PD, Selvam TP. Synthesis of novel thiazole derivatives as analgesic agents. Asian Journal of Pharmaceutical Research. 2011; 1(4):134-8.

43. Alam F, Dey BK, Korim R, Chakrabarty A. Synthesis, characterization, and biological evaluation of pyrazole derivatives. Journal of Global Trends in Pharmaceutical Sciences. 2015; 6(1):2411-6.

44. Muhsin YF, Alwan SM, Khan AK. Design, Molecular Docking, Synthesis of Aromatic Amino Acids Linked to Cephalexin. AJPS. 2021:32.

45. Selvam TP, Saravanan G, Prakash C, Kumar PD. Microwave-Assisted synthesis, characterization and biological activity of novel pyrazole derivatives. Asian Journal of Pharmaceutical Research. 2011; 1(4):126-9.

46. Tirlapur VK, Noubade K. Synthesis, Characterization and Biological Activities of New Pyrimidines and Pyrazoles Derivative. Asian Journal of Research in Chemistry. 2010; 3(3):659-62.

47. Ali B, Monther F, Mohammed H. Design, synthesis, and hydrolysis study of mutual prodrugs of NSAIDS with different antioxidants via glycolic acid spacer‖. International Journal of Comprehensive Pharmacy. 2012; 1(3):3.

48. Jwaid MM, Ali KF, Abd-alwahab MH. Synthesis, Antibacterial study and ADME Evaluation of Novel Isonicotinoyl Hydrazide Derivative Containing 1,3,4-Oxadiazole Moiety. AJPS. 2020:113.

49. Rudyakova E, Savosik V, Papernaya L, Albanov A, Evstaf’eva I, Levkovskaya G. Synthesis and Reactions of Pyrazole-4-carbaldehydes. Russian Journal of Organic Chemistry. 2009; 45(7):1040-4. doi.org/10.1134/S1070428009070100

Received on 15.11.2021 Modified on 23.01.2022

Research J. Pharm. and Tech. 2022; 15(8):3382-3390.

DOI: 10.52711/0974-360X.2022.00566

Design, Molecular Docking Studies, Synthesis and Characterization of some New 4,5-dihydro-1H- Pyrazole-1-yl acetate Derivatives as Cyclooxygenase Inhibitors