STRUCTURAL DOCKING STUDIES AND BIOLOGICAL EVALUATION OF SOME NEWLY SYNTHESIZED SCHIFF BASE DERIVED QUINAZOLINONE DERIVATIVES

STRUCTURAL DOCKING STUDIES AND BIOLOGICAL EVALUATION OF SOME NEWLY SYNTHESIZED SCHIFF BASE DERIVED QUINAZOLINONE DERIVATIVES

Poonam Arora ¹, Kuljit Kaur ¹, Rajanpreet Kaur ¹ and Neelima Dhingra^*2

GHG Khalsa College of Pharmacy ¹, Gurusar Sadhar, Ludhiana - 141104, Punjab, India.

University Institute of Pharmaceutical Sciences ², Panjab University, Chandigarh - 160014, Punjab, India.

ABSTRACT: Present studies describe the synthesis of a series of Schiff base derived quinazolinone derivatives (6-14) with the aim to have better anti inflammatory agents. The prepared compounds were characterized using chemical and spectral (IR, NMR and Mass) studies. Pharmacological evaluation of the newly synthesized derivatives  indicated that compounds 8, 11 and 14 exhibit significant anti-inflammatory activity in carrageenan-induced rat paw oedema assay, and their effects are found to be comparable that of standard drug Indomethacin. Molecular docking studies were further employed to investigate the binding interactions to COX-2 active site residues and to study the stability of docked conformation in detail. The experimentally determined COX-2 inhibitory activity was found to be well correlating with binding modes predicted for compounds by GLIDE dock scoring function. The 2,3-diaryl-2,3-dihydro-1H-quinazolin-4-one pharmacophore reported herein represents a new lead for further development of novel non-steroidal anti-inflammatory agents.

Quinazolinone, COX-2, Docking, Anti-inflammatory, GLIDE

INTRODUCTION: The molecular modifications of a promising lead compound are still a major line of approach for the discovery of new drugs. Quinazolin-4(3H)-one represents a versatile lead molecule for the design of potential bioactive agents ^1-7 and the pharmaceuticals containing quinazolinone nucleus show varied biological activities as different substitution patterns results in diverse biological properties ^8-10. Number of quinazoline derivatives with different substitutions at 2,3,4,6 positions have been reported and studied as antitumor ¹¹, antibacterial ¹², anticonvulsant ¹³, antimalarials ¹⁴ and antiviral agents ¹⁵ as shown in Table 1.

Further, it has been reported that substitution pattern by different moieties at 2/3 position of quinazoline nucleus markedly influences the anti-inflammatory activity ^{16, 17} (Fig. 1).

The commonly proposed mechanism of action of compounds used in inflammation is by lowering prostaglandin production through inhibition of cyclo-oxygenase (COX) enzyme ^{18, 19}. Two different forms of COX as COX-1 and COX-2 has been isolated and identified. This enzyme plays a crucial role in both pathological and physiological actions during inflammation. COX-1 is known as housekeeping enzyme, while COX-2 has been found to be expressed in pathological conditions, thus triggers inflammatory signals. Potent and selective COX-2 inhibitors by blocking the production of prostaglandin in inflammatory cells, without affecting in the homeostatic and gastro-protective actions mediated by COX-1, would be a logical approach towards treatment of inflammation ²⁰.

Thus aiming at the discovery of newer, safer potent and selective COX-2 inhibitors with anti-inflammatory activity, and to investigate the influence of structural variation, quinazolinone nucleus was used as a template. Further given the significance of different substitutions at varied position in reported compounds (1 and 2), here present study describes the synthesis and evaluation of a series of novel compounds or derivatives of 4(3H)-quinazolinone, with structure modifications involving phenyl moiety at position 2 and formation of schiff base derived from acetophenones at position 3 ^{21, 22}. The structures of the compounds synthesized were assigned on the basis of ¹H NMR, IR, and mass spectral data. Further, the selectivity of the target molecules towards COX-2 has been predicted by docking studies ^21-23. The molecular interactions and binding mode of the compounds was proposed using GLIDE program. Moreover, it was considered of interest to investigate the influence of structural variation on the anticipated biological activities, through anti-inflammatory activity and is compared with indomethacin activity.

TABLE 1: DIFFERENTLY SUBSTITUTED QUINAZOLINONE DERIVATIVES WITH VARIED ACTIVITIES

Position	Structure	Activity	Reference
2,4		Anti-tumor	11
2,3		Antibacterial	12
2,3		Anti-convulsant and CNS depressant	13
3		Antimalarial	14
2,3,6		Antiviral	15

A                                                                                              B

FIG. 1: COMPOUNDS WITH DIFFERENT SUBSTITUTIONS AT 2/3 POSITION OF QUINAZOLINONE NUCLEUS SHOWING ANTI-INFLAMMATORY ACTIVITY

MATERIALS AND METHODS:

Chemistry:

Materials and Equipment: Melting points (mp) of products were determined by open capillary method using digital melting point apparatus and were uncorrected. FTIR spectra were recorded on a Perkin Elmer FT-IR RXI spectrophotometer using potassium bromide discs. Proton Nuclear Magnetic Resonance (¹H NMR) spectra were recorded on Bruker Advance II 400MHz spectrometer in dimethyl sulphoxide (DMSO-d₆) using trimethyl-silane (TMS) as internal standard. All the reactions were monitored by thin layer chromatography (TLC) on precoated silica gel 60 F₂₅₄ (E. Merck, Mumbai) and spots were visualized under UV light (254nm).

Electron spray ionization mass spectrometry (ESI-MS) study was performed using a Q-ToF quadrupole time of flight mass spectrometer (Waters) equipped with an electronspray source. Silica gel from E. Merck, Mumbai (60-120 mesh) was used for column chromatography. Anthranilic acid, benzoyl chlorides and acetophenones were purchased from Sigma Chemical (Berlin, Germany). All chemicals used were of analytical grade and commercially available.

General Procedure for the Synthesis of 2-phenyl benzoxazin-4(3H)-one (4a-4c): Anthranilic acid (0.01mol) was dissolved in 30ml of anhydrous pyridine with stirring at room temperature. The mixture was cooled to 0 °C and a solution of different substituted benzoyl chlorides (3a-3c) (0.01mol) in anhydrous pyridine (30ml) was slowly added to this solution with constant stirring for 3 h. The pasty mass obtained was diluted with water and the resulting mixture was treated with 5% sodium bicarbonate solution to remove the unreacted acid, when the effervescence ceased. The precipitate were filtered and washed with water to remove adhered pyridine. The 2-phenyl benzoxazine (4a-4c) thus obtained was dried and recrystallized from ethanol.

2-(4-Nitrophenyl)-benzoxazin-4(3H)-one (4a): Yellow crystalline solid, Yield: 69%, M.p. 157-159 °C. IR (KBr) cm^-1: 1768, 1682, 1064, 1595, 3078, 1352, 1519. ¹H NMR (400MHz, DMSO-d₆): δ 8.68-8.64 (1H, m, quinazolinone), 8.39-8.35 (2H, m, Ar-NO₂), 8.19-8.12 (2H, m, Ar-NO₂), 7.83-7.79 (1H, m, quinazolinone), 7.54-7.50 (1H, m, quinazolinone), 7.19-7.15 (1H, m, quinazolinone). ESI-MS (m/z): 268 (C₁₄H₈N₂O₄) (100%), 146 (C₈H₄NO₂), 122 (C₆H₄NO₂).

2-(2-Chlorophenyl)-benzoxazin-4(3H)-one (4b): Yellow crystalline solid, Yield 66%, M.p. 155-157 °C. IR (KBr) cm^-1: 1768, 1620, 1090, 3075, 764. ¹H NMR (400 MHz, DMSO-d₆): δ 8.20-8.18 (1H, m, quinazolinone), 8.01-7.97 (1H, m, quinazolinone) 7.94-7.92 (1H, m, quinazolinone), 7.75-7.61 (4H, m, Ar-H), 7.57-7.54 (1H, m, quinazolinone). ESI-MS (m/z): 258 (100%) (C₁₄H₈NO₂Cl), 146 (C₈H₄NO₂).

2-(4-Methylphenyl)-benzoxazin-4(3H)-one (4c): White crystalline solid, Yield 70%, M.p. 152-153 °C. IR (KBr) cm^-1: 1757, 1607, 1009, 1569, 3031. ¹H NMR (400 MHz, DMSO-d₆): δ 8.15-8.15 (1H, d, quinazolinone), 8.10-8.08 (2H, d, Ar-H), 7.96-7.92 (1H, m, quinazolinone), 7.71-7.69 (1H, m, quinazolinone), 7.63-7.59 (1H, m, quinazolinone), 7.41-7.39 (2H, d, Ar-H), 2.41 (3H, s, CH₃). ESI-MS (m/z): 238 (100%) (C₁₅H₁₁NO₂), 146 (C₈H₄NO₂), 119 (C₇H₄O₂).

General Procedure for the Synthesis of 2-phenyl-3-amino quinazolin-4-one (5a- 5c): For the synthesis of the compounds (5a- 5c), various 2-phenyl benzoxazine derivatives (4a- 4c) (0.005mol) were treated with hydrazine hydrate (0.01mol). Anhydrous pyridine (25ml) was added drop wise, and the mixture was stirred for 30 min at room temperature, followed by refluxing for 6-9 h. The reaction mixture was allowed to cool at room temperature and poured into ice cold water containing dilute hydrochloric acid. On standing for 1 h, solidification occur, the product was filtered off, washed with water and dried.

3-Amino- 2- (4-nitrophenyl)- quinazolin- 4(3H)-one (5a): Yellow crystalline solid, Yield 62%, M.p. 157-160 °C. IR (KBr) cm^-1: 3302, 3220, 1449, 1672, 1598, 1256, 1524, 3076, 1346, 1524. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ  3.58 (2H, s, NH₂), 8.67-8.65 (1H, m, quinazolinone), 8.38-8.34 (2H, m, Ar-H), 8.20-8.13 (2H, m, quinazolinone), 7.82-7.80 (1H, m, quinazolinone), 7.55-7.51 (1H, m, quinazolinone), 7.19-7.15 (1H, m, quinazolinone). ESI-MS (m/z): 283 (C₁₄H₁₀N₄O₃) (100%), 122 (C₆H ₄NO₂), 160 (C₈H₆N₃O).

3-Amino-2- (2-chlorophenyl)- quinazolin- 4(3H)-one (5b): White crystalline solid; Yield 67%, M.p. 157-159 °C. IR (KBr) cm^-1: 1676, 1598, 1256, 1598, 2920, 764, 3484. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 3.58 (2H, s, NH₂), 8.65-8.64 (1H, m, quinazolinone), 8.36-8.32 (2H, m, Ar-H), 8.10-8.18 (2H, m, quinazolinone), 7.80-7.78 (1H, m, quinazolinone), 7.53-7.49 (1H, m, quinazoli none), 7.17-7.13 (1H, m, quinazolinone). ESI-MS (m/z): 146 (C₈H₄NO₂), 272 (100%) (C₁₄H₁₀N₃OCl), 236 (C₁₄H₁₀N₃O), 255 (C₁₄H₉ N₂O), 160 (C₈H₆N₃O), 112 (C₆H₄Cl).

3-Amino-2-(4-methylphenyl)- quinazolin- 4(3H)-one (5c): Yellow crystalline solid, Yield 65%, M.p. 155-157 °C. IR (KBr) cm^-1: 1668, 1598, 1256, 2920, 3488. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 3.55 (2H, s, NH₂), 8.15-8.15 (1H, d, quinazolinone), 8.10-8.08 (2H, d, Ar-H), 7.96-7.92 (1H, m, quinazolinone), 7.71-7.68 (1H,  m, Ar-H), 7.62-7.55 (1H, m, Ar-H), 7.40-7.38 (2H, d, quinazolinone) 2.45 (3H, s, CH₃). ESI-MS (m/z): 251 (base peak), 236 (C₁₄H₁₀N₃O), 144 (C₁₅H₁₃N₃O), 160 (C₈H₆N₃O) (fragmentation).

General Procedure for the Synthesis of Quinazolinone Fused Schiff Bases (6-14): To a solution of the appropriate acetophenones (0.01mol) dissolved in ethanol (20ml), was added different substituted 3-amino-2-phenylquinazolin-4-one derivative (5a-5c) (0.01mol) and pH of the resultant solution was adjusted to 4.0-4.5 using glacial acetic acid. The resulting mixture was refluxed for 2-3 h. The solid thus obtained was filtered and purified by column chromatographic method using n-hexane/ethyl acetate (75:25) as eluents.

3- [2- (4- Chlorophenyl)- ethylimino]- 2- (4-nitrophenyl)- quinazolin- 4(3H)-one (6): Yellow crystalline solid, Yield 65%, M.p. 162-164 °C. IR (KBr) cm^-1: 1765, 1351, 1520, 1685, 1253, 775, 2870, 1598, 3075. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 8.36-8.34 (1H,  m, quinazolinone ), 8.30-8.26 (2H, m, Ar-H NO₂ containing), 8.24-8.18 (2H, d, Ar-NO₂), 8.03-7.91 (2H, m, Ar-Cl), 7.87-7.84 (2H, m, Ar-Cl), 7.83-7.79 (1H, m, quinazolinone), 7.77-7.71 (1H, m, quinazolinone), 7.44-7.39 (1H, m, quinazolinone), 2.12 (3H, s, CH₃). ESI-MS (m/z):  283 (100%) (C₁₄H₁₀N₄O₃), 126 (C₆H₄NO₂), 237 (C₁₄H₁₀N₃O). Anal. Calculated for C₂₂H₁₅ N₄O₃Cl: N, 13.38%. Found: N, 16.53%.

3- [2- (3- Nitrophenyl)- ethylimino]- 2- (4-nitro phenyl)- quinazolin- 4(3H)-one (7): Yellow crystalline solid, Yield 62%, M.p. 158-160 °C. IR (KBr) cm^-1: 1679, 1343, 1520, 1641, 1251, 2923, 1581, 3088. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 8.68-8.69 (1H, m, quinazolinone), 8.44-8.41 (2H, m, Ar-H), 8.35-8.33 (2H, m, Ar-H), 8.30-8.28 (1H, m, Ar-H), 8.24-8.22 (1H, m, Ar-H), 8.11-8.08 (1H, m, quinazolinone), 7.83-7.76 (2H, m, Ar-H), 7.73-7.71 (1H, m, quinazolinone), 7.57-7.53 (1H, m, quinazolinone), 2.69 (3H, s, CH₃). ESI-MS (m/z): 283 (100%) (C₁₄H₁₀N₄O₃), 254 (C₁₄H₁₀N₃O₂), 237 (C₁₄H₁₁N₃O), 126 (C₆H₄NO₂). Anal. Calculated for C₂₂H₁₅N₅O₅: N, 16.31%. Found: N, 16.85%.

3- [2- (3- Hydroxyphenyl)- ethylimino]- 2-(4-nitrophenyl)-quinazolin- 4(3H)- one (8): Yellow crystalline solid, Yield 67%, M.p. 140-141 °C. IR (KBr) cm^-1: 1684, 1343, 1524, 1638, 1275, 1594, 3115, 3291. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 8.35-8.32 (1H, d, quinazolinone), 8.23-8.20 (2H, m, Ar-H), 8.07-8.04 (1H, m, Ar-H), 7.90-7.85 (2H, m, Ar-H), 7.76-7.74 (2H, m, Ar-H), 7.63-7.59 (1H, m, Ar-H), 7.58-7.55 (2H, m, quinazolinone), 7.54-7.53 (1H, m, quinazolinone), 2.42 (3H, s, CH₃). ESI-MS (m/z): 283 (100%), 254 (C₁₄H₁₀N₃O₂), 237 (C₁₄H₁₁N₃O), 126 (C₆H₄NO₂). Anal. Calculated for C₂₂H₁₆N₄O₄: N, 13.99%. Found: N, 17.57%.

3- [2- (4- Chlorophenyl)- ethylimino]- 2- (2-chlorophenyl)- quinazolin-4(3H)-one (9): Yellow compound, Yield: 70%, M.p. 198-200 °C. IR (KBr) cm^-1: 1663, 748, 1590, 1272, 3055, 1521, 3245. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 8.35-8.33 (1H, m, quinazolinone), 7.97-7.95 (1H, d, Ar-H), 7.87-7.86 (2H, m, quinazolinone), 7.76-7.74 (1H, m, quinazolinone), 7.67-7.47 (6H, m, Ar-H), 7.35-7.29 (1H, m, Ar-H), 2.36 (3H, s, CH₃). ESI-MS (m/z): 258 (C₁₄H₉ClN₂O) (100%), 146 (C₈H₅N₂O), 272 (C₁₄H₁₀ClN₃O), 236 (C₁₄H₉N₃O), 312 (C₁₆H₁₁N₄O₃). Anal. Calculated for C₂₂H₁₅N₃OCl₂: N, 10.39%. Found: N, 10.29%.

3- [2- (3- Nitrophenyl)- ethylimino]- 2- (2-chloro phenyl)- quinazolin- 4(3H)- one (10): Lemon coloured solid, Yield 67%, M.p. 178-180 °C. IR (KBr) cm^-1: 1645, 1348, 1524, 1524, 1348, 755, 2962, 3093. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 8.59-8.58 (2H, m, Ar-NO₂), 8.36-8.34 (2H, m, Ar-Cl), 8.44-8.41 (2H, m, Ar-Cl-NO₂), 8.24-8.22 (2H, m, Ar-H quinazolinone), 8.21-8.20 (2H, m, quinazolinone), 7.32-7.30 (1H, m, Ar-Cl) 7.82-7.78 (2H, m, Ar-H) 7.87-7.85 (1H, m, Ar-Cl), δ 2.44 (3H, s, CH₃). ESI-MS (m/z): 258 (C₁₄H₉ClN₂O), 272 (C₁₄H₁₀ClN₃O) (100%), 236 (C₁₄H₉N₃O), 312 (C₁₆H₁₁N₄O₃). Anal. Calculated for C₂₂H₁₅N₄O₃Cl: N, 13.38%. Found: N, 11.94%.

3- [2- (3- Hydroxyphenyl)- ethylimino]- 2-(2-chlorophenyl)- quinazolin- 4(3H)- one (11): Whitish yellow solid, Yield 67%, M.p. 218-220 °C. IR (KBr) cm^-1: 1647, 1599, 1270, 753, 2924, 3060, 3289. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 8.54-8.52 (1H, m, quinazolinone), 7.87-7.86 (1H, m, Ar-OH), 7.81-7.43 (8H, m, Ar-H and quinazolinone), 7.26-7.22 (1H, m, Ar-Cl), 6.84-6.78 (1H, m, Ar-Cl), 2.31 (3H, s, CH₃), 11.35 (1H, s, OH). ESI-MS (m/z): 258 (100%) (C₁₄H₉ClN₂O), 146 (C₈H₆N₂O), 272 (C₁₄H₁₀N₃ClO). Anal. Calculated for C₂₂H₁₅N₄O₃Cl: N, 10.78 %. Found: N, 11.33 %.

Step 1: 

Step 2: 

Step 3:   

6. R₁₌4-NO_2, R_{2 =}4-Cl, 7. R₁₌4-NO_2, R₂ =3-NO₂, 8. R₁₌4-NO_2, R₂ = 3-OH, 9. R₁₌ 2-Cl_, R_{2 =}4-Cl, 10. R₁₌ 2-Cl_, R₂ =3-NO₂,
7. R₁₌ 2-Cl_, R₂ = 3-OH, 12. R₁₌ 4-CH_3, R_{2 =}4-Cl, 13. R₁₌ 4-CH_3, R₂ =3-NO₂, 14. R₁₌ 4-CH_3, R₂ = 3-OH

SCHEME 1S: STEPS INVOLVED IN THE SYNTHESIS OF SCHIFF BASE DERIVED QUINAZOLINONE DERIVATIVE

3- [2- (4- Chlorophenyl)- ethylimino]- 2- (4-methylphenyl)-quinazolin-4(3H)-one (12): White crystalline solid, Yield 70%, M.p. 203-205 °C. IR (KBr) cm^-1: 1653, 1529, 1312, 760, 3054, 1603, 3235. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 8.60-8.58 (1H, m, quinazolinone), 7.90-7.84 (5H, m, Ar-H and quinazolinone), 7.60-7.55 (1H, m, Ar-H), 7.46-7.44 (2H, m, quinazolinone), 7.36-7.34 (2H, m, Ar-H), 7.25-7.21 (1H, m, Ar-H), 2.41-2.39 (6H, s, CH₃). ESI-MS (m/z): 238 (100%) (C₁₅H₁₂N₂O), 252 (C₁₅H₁₃N₃O), 126 (C₇H₇Cl), 292 (C₁₆H₁₁ClN₃O). Anal. Calculated for C₂₃H₁₈N₃OCl: N, 10.83 %. Found: N, 11.36 %.

3- [2- (3- Nitrophenyl)- ethylimino]- 2- (4-methylphenyl)- quinazoline- 4(3H)- one (13): Yellow crystalline solid, Yield 70%, M.p. 216-218 °C. IR (KBr) cm^-1: 1644, 1344, 1526, 1606, 1102, 2921, 3093. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 8.63-8.62 (1H, m, quinazolinone), 8.48-8.45 (1H, d, Ar-NO₂), 8.39-8.37 (1H, d, Ar-NO₂), 8.29-8.27 (1H, m, quinazolinone), 7.85-7.81 (2H, m, Ar-NO₂), 7.76-7.75 (2H, m, quinazolinone), 7.64-7.61 (2H, m, Ar-CH₃), 7.39-7.37 (2H, m, Ar-CH₃), 2.68-2.36 (6H, s, (CH₃)₂). ESI-MS (m/z): 238 (100%) (C₁₅H₁₁N₂O), 252 (C₁₅H₁₁N₃O), 337 (C₂₂H₁₅N₃O), 119 (C₈H₈N). Anal. Calculated for C₂₃H₁₈N₄O₃: N, 14.06%. Found: N, 12.67%.

3- [2- (3- Hydroxyphenyl)- ethylimino]- 2- (4-methylphenyl)- quinazolin- 4(3H)- one (14): Whitish yellow solid, Yield 68%, M.p. 208-210 °C. IR (KBr) cm^-1: 1646, 1599, 1101, 2949, 1513, 3060, 3289. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 8.58-8.56 (1H, d, quinazolinone), 7.93-7.92 (1H, d, Ar-OH), 7.86-7.80 (2H, d, Ar-OH), 7.80-7.75 (2H, d, Ar-CH₃), 7.63-7.57 (1H, m, Ar-H), 7.40-7.38 (2H, d, Ar-H), 7.27-7.22 (1H, m, Ar-H), 6.87-6.83 (2H, d, Ar-H), 2.38 (3H, s, CH₃), 2.33 (3H, s, CH₃), 11.91 (1H, s, OH). ESI-MS (m/z): 238 (100%) (C₁₅H₁₂N₂O), 119 (C₈H₈N), 276 (C₁₅H₁₂ N₂O). Anal. Calculated for C₂₂H₁₅N₄O₃Cl: N, 12.03%. Found: N, 12.03%.

Pharmacology: The pharmacological evaluation or anti-inflammatory activity of the prepared compounds was performed by carrageenan-induced rat paw edema assay ²⁴.

Animals: Wistar rats (8-10 weeks old; 200-250g) of both sexes were housed in plastic cages under standard laboratory conditions and maintained on rat chow and water. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) and conducted according to the guidelines of Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), New Delhi, India.

Anti-inflammatory Activity: Anti-inflammatory activity of the newly synthesized compounds was determined using carrageenan-induced foot paw edema assay method in rats and indomethacin was used as a standard drug ⁹. Unless otherwise stated, the following conditions were employed in all experiments. Target compounds (6-14) were suspended in aqueous solution of carboxymethyl cellulose (CMC, 0.5% w/v) and administered orally at a dose level of 10mg/kg of synthesized compounds (6-14), respectively. Control animals were similarly treated with aqueous solution of carboxymethyl cellulose (CMC, 0.5% w/v). After 30 min, 0.1ml of freshly prepared 1% carrageenan solution in normal saline was injected into the subplantar region of the right hind paw of each rat. The increase in paw volume was measured by using plethysmometer (water displacement, UGO BASILE, Italy) at 2, 3 and 4 h after carrageenan challenge. The percentage inhibition of inflammation was calculated in comparison to the control group by the given formula:

Where V_c is the increase in paw volume of control (without drug) and V_t is the increase in paw volume after administration of the test compound.

Molecular Docking Study: Docking studies were performed to find the hypothetical binding of quinazolinone containing Schiff bases with the target protein COX-2 by using the program GLIDE (Grid-based Ligand Docking with Energetic) module version Schrodinger 9.2, Schrodinger, LLC, New York, NY (Schrodinger Inc.).

Preparation of Protein: The crystal structure of COX-2 enzyme (PDB ID: 1CX2) was obtained from the RCSB Protein Data Bank, where the selective COX-2 inhibitor SC-558 houses in its active site. This PDB structures was downloaded, refined and prepared using Schrodinger protein preparation wizard tool (GLIDE), which performs the following steps: assigning of bond orders, addition of hydrogen, and optimization of hydrogen bonds by flipping amino side chains, correction of charges, and minimization of the protein complex. All the bound water molecules, ligands and cofactors were removed (pre-process) from the protein and were taken in mae format. The tool neutralized the side chains that are not close to the binding cavity and do not participate in salt bridges.

Preparation of Ligands: Structures of the ligands 2,3-disubstituted quinazolinone derivatives containing Schiff bases (6-14) were drawn by using chemdraw ultra 8.0 and the three-dimensional structures of the compounds were constructed using the Maestro interface. All 3D ligands were subjected to energy minimization and optimization using Ligprep module. LigPrep is a utility of Schrodinger software suit that combines tools for generating 3D structures from 1D (Smiles) and 2D (SDF) representation, searching for tautomers, steric isomers and perform a geometry minimization of the ligands.

Validation of the Docking Protocol in Glide: The most suitable method of evaluating the accuracy of a docking procedure is to determine how closely the lowest energy pose predicted by the scoring function resembles an experimental binding mode as determined by X-ray crystallography. In the present study, the docking of protein with its already present ligand was performed to test the reliability and reproducibility of the docking protocol for our study. The root mean square deviation (RMSD) between the predicted conformation and the observed X-ray crystallographic conformation of the ligand by Glide (3A) was analyzed. This indicates the reliability of the docking method in reproducing the experimentally observed binding mode for target proteins.

GLIDE Docking: The impref script runs a series of restrained impact energy minimizations using the Impact utility. Minimizations were run until the average root mean square deviation (rmsd) of the non-hydrogen atoms reached 0.3 Å. Glide uses two boxes that share a common centre to organize its calculations: a larger enclosing box and a smaller binding box. The grids themselves are calculated within the space defined by the enclosing box. The binding box defines the space through which the centre of the defined ligand will be allowed to move during docking calculations. It provides a measure of the effective size of the search space.

The only requirement on the enclosing box is that it should be large enough to contain all ligand atoms, even when the ligand centre is placed at an edge or vertex of the binding box. Grid files were generated using the cocrystallized ligand at the centre of the two boxes. The size of the binding box was set at 20 Å in order to explore a large region of the protein. The initial geometry of the structures was optimized using the OPLS-2005 force field performing 1000 steps of conjugate gradient minimization. The compounds were subjected to flexible docking using the pre-computed grid files. For each compound the 100 top-scored poses were saved and analyzed and only the best scoring poses were selected for the study.

Prediction of Pharmacokinetic Parameter (in silico): The in silico pharmacokinetic properties of the designed quinazoline analogues (6-14) were estimated by means of molecular weight, dipole moment, H-bond donors, H-bond acceptors, QPlogP o/w, QPlogS, QlogBB, human oral absorption, and polar surface area (PSA), molecular surface descriptors (solvent accessible surface area) and other parameters such as wiener index (WI) were also determined using QikProp (version9.2) module of Schrödinger. As well as the Lipinski' rule of five was to filter molecules with filter for drug-like properties has also been estimated.

RESULTS AND DISCUSSIONS:

Chemistry: Present work described the synthesis of nine analogous of 2,3-diaryl-3H-quinazolin-4-ones (6-14) and the synthetic strategy adopted for the synthesis of target compounds is outlined in Scheme 1. Benzoxazinones (4a-4c) were prepared from commercially available anthranilic acid and its treatment with differently substituted benzoyl chloride (3a, 3b, 3c). 3-Amino-2-phenyl-quinazolinones (5a-5c) were synthesized from various benzoxazinones by treatment with hydrazine hydrate in anhydrous pyridine. Final target compounds (6-14) were prepared from 3-amino-2-phenylquinazolinones (5a-5c), by adding different substituted acetophenones in acidic medium. Progress of the reaction was monitored by TLC using chloroform: methanol (9:1) as mobile phase. The structures of the newly synthesized compounds were confirmed by IR, NMR and mass spectral analysis followed by their physicochemical properties determination.

Anti-inflammatory Activity: The antiinflammatory activity of the newly synthesized compounds (6-14) was evaluated by applying carrageenan-induced paw edema bioassay in rats ^{1, 22} and indomethacin was used as standard drug. Inflammation was produced by injecting 0.1ml of 1% w/v of carrageenan into the subplanatar region, and reproducible and substantial inflammation was observed in all the groups. Plethysmometer was used to measure to paw volume before and after carrageenan treatment at 1 hour time interval upto 4 hours after carrageenan challenge and percentage inhibition of inflammation was calculated in comparison to the control. Rats of either sex were randomized into control, standard and test groups of five rats each. Indomethacin (10mg/kg body weight) was administered orally as suspension (0.5% w/v carboxymethyl cellulose) to standard group whereas 1% w/v carboxymethyl cellulose (vehicle alone) was given to control group. The results were expressed as mean ± S.E (n = 3). Statistically significant difference between vehicle control group and treatment group data was analyzed by one way ANOVA test followed by the least significant difference (L.S.D.). The observations for the paw volume are given in Table 2.

TABLE 2: ANTI-INFLAMMATORY DATA OF 2, 3-DIARYL-3H-QUINAZOLIN-4-ONES DERIVATIVES (6-14) ON CARRAGEENAN-INDUCED PAW EDEMA IN RATS

All values are expressed as mean ± S.E.M

Compounds were found to exhibit anti-inflammatory activity in the range of 25 to 61%, however, the significant decrease in the paw volume was observed at third hour when administered at doses of 10mg/kg body weight (Table 2). Among the newly synthesized series of 2,3- diaryl- 3H- quinazolin- 4- ones, the tested compounds 8, 11, 14 showed  more than 50% edema inhibition and the difference in paw thickness was comparable to the standard drug indomethacin (Fig. 2). The (OH) hydroxyl group at R₂ position of 2, 3-diaryl-3H-quinazolin-4-ones in compounds 8, 11 and 14 appears to have a positive effect on the anti-inflammatory potency of these compounds.

On the other hand, compounds 7 and 10 demonstrated 29% and 25% inhibition (after 3 h) and were found to be the least active towards carrageenan-induced edema in comparison to the standard drug indomethacin. Further, anti-inflammatory activity was not only found to be comparable with standard drug, but with a longer duration as activity was retained upto 24 hr.

This phenomenon may be partly be due to the low systemic bioavailability of test compounds following oral dosing, efficient first pass metabolism and some degree of intestinal metabolism ¹⁰.

FIG. 2: VOLUME OF PAW EDEMA AFTER 2, 3, 4 hr

Molecular Docking Study: The final compounds (6-14) were evaluated in silico (docking) to recognize their hypothetical binding mode using the X-ray crystal structure of COX-2 (PDB ID: 2OYE) and also to rationalize its structure activity relationship.

To investigate the ability of molecular docking to reproduce an experimentally observed ligand-binding mode, the co-crystallized ligand IM8-700 (a selective COX-2 inhibitor) has been used as reference ligand. Hundreds of generated conformations of the compounds 6-14 were docked back into its binding site of the crystal structure of COX-2 using the program GLIDE (Grid-based Ligand Docking with Energetic) module version 9.2, Schrödinger. As many as top-scored poses were saved for different conformers and analyzed, but the top docked conformations (poses) closely resembled the co-crystallized conformation of non-hydrogen atomic positions of the ligand IM8-700 was selected for docking studies to recognize their hypothetical binding mode using the X-ray crystal structure of COX-2 (PDB ID:1CX2). Table 3 summarizes the result of the docking study presented as Glide energies and Glide score.

Docking studies of the indomethacin, a nonselective COX-2 inhibitor, indicates the strong salt bridge interaction of carboxylate ion with the Arg120 in COX-2 isoenzymes (Fig. 3a) that gives a more generalized anchoring point for all the classical NSAIDs thus limiting their selectivity due to curtailed freedom of movement of the ligand ²⁵. Further, acetyl CH₂ of indomethacin formed a strong hydrogen bond with the phenolic OH group of Tyr355 at a distance of 1.5829 Å, whereas the chlorophenyl group showed significant pep stacking interaction with the phenyl ring of Phe518.

In comparison to indomethacin, all the newly synthesized compounds have been found to interacted and fitted into the known classical sites with best scores configurations at COX- 2. Further present studies revealed compounds 8, 11 and 14 posses better docking score because of  their strong direct interactions with amino acids HIS 90 of COX-2 (Fig 3b, 3c, 3d) (Table 3). 4-hydroxyl group at R₂ of quinazolinone ring of all these compounds showed strong H-bond with HIS -90 at a distance of 1.788, 1.843 and 2.000 Å (Fig. 3b, 3c and 3d) respectively. Compound 11 was found to be more active than the compound 8 and 14. The favourable binding conformation and higher interaction energy of 8, 11 and 14 (Table 3) suggest their prominent anti-inflammatory activity and are in agreement with the biological data. The Table 3 also suggests that these compounds require less energy for their interaction with protein molecule. Overall all the docking results are in agreement with the biological data. The G-score is also indicative of their higher anti-inflammatory activity in comparison to other compounds.

A                                                              B                                                                C

D                                                    E

FIG. 3: COMPOUNDS SHOWING INTERACTION WITH HIS-90; 3A- INTERACTION OF INDOMETHACIN WITH ARG-120; 3B, 3C AND 3D- COMPOUNDS 8, 11 AND 14 SHOWING INTERACTIONS WITH HIS-90 AT THE H-BOND DISTANCE OF 1.788, 1.843 AND 2.000 Å RESPECTIVELY

TABLE 3: ENERGY CONTRIBUTIONS FOR COMPOUND 6-14

Prediction of Pharmacokinetic Parameter (in silico): High oral bioavailability is an important factor for the development of bioactive molecules as therapeutic agents and the significant descriptors and pharmaceutically relevant properties which could influence the cell permeability and bioavailability for the synthesized compounds (6-14) were evaluated using QikProp (version9.2) module of Schrödinger and are given in Table 4. These parameters include molecular weight, H-bond donors, and H-bond acceptors, log P (octanol/water), log Kp (skin permeability) and humoral absorption. Some of the recognized parameters important for prediction of drug transport properties such as QPlogP o/w, QPlogS, and polar surface area (PSA) ²⁵ were also determined. Further, steric and molecular surface descriptors, e.g. solvent accessible surface area (SASA) and other parameters such as wiener index (WI) were calculated. ADME prediction methods were used to assess the bioavailability of the test compounds (6-14). Herein, we also calculated the compliance of all the compounds to the Lipinski’s ‘rule of five’²⁶ which has been widely used to determine if a chemical compound with a certain pharmacological or biological activity has properties that would make it a likely orally active drug in humans. Lipinski et al., ²⁶ used these molecular properties in formulating his ‘Rule of Five’ and as a filter for drug-like properties. The rule states that most molecules with good membrane permeability have log P ≤ 5, MW ≤ 500, the number of hydrogen bond acceptors ≤ 10, and the number of hydrogen bond donors ≤ 5. A compound that fulfils at least three out of the four criteria is said to adhere to ‘Lipinski’s Rule of Five’.

A poor permeation or absorption is more likely when there are more than five H-bond donors and ten H-bond acceptors. The series (6-14) under investigation has not only the most of the compounds possessing less number of hydrogen bond donors (<5) but also does possess considerable number of acceptors (<10). Table 4 enlists the values of these properties for the newly synthesized molecules. None of the molecules has been found to violating Lipinski rule thus not only associated with good bioavailability but also suggesting that the active compounds can be used as templates for further drug discovery effort.

TABLE 4: MOLECULAR PROPERTIES OF THE SYNTHESIZED COMPOUNDS (6-14) USING QIKPROP SOFTWARE

CONCLUSION: Molecular modelling studies confirm biological data. All our compounds 6-14 were docked into the active site of COX-2 protein and the result showed that the compounds 8, 11, 14 were more active than other compounds. Compound 8, 11 and 14 showed hydrogen bond interaction with the HIS-90 which is required to show COX-2 selectivity.

Further, the compounds comply with Lipinski’s rule of five which signifies a good absorption and hence, good bioavailability. Compound 11 was found to be most active quinazolinone derivative.

ACKNOWLEDGEMENT: We thank UGC for providing financial help through UGC networking Resource centre A1 module programme at Panjab University, Chandigarh, India.

CONFLICT OF INTEREST: The authors declared that there are no any conflicts of interest.

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    How to cite this article:

    Arora P, Kaur K, Kaur R and Dhingra N: Structural docking studies and biological evaluation of some newly synthesized schiff base derived quinazolinone derivatives. Int J Pharm Sci Res 2017; 8(12): 5088-99.doi: 10.13040/IJPSR.0975-8232.8(12).5088-99.

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