Molecular Docking, ADME Studies, Synthesis, and Anti-oxidant Activity of Novel Quinazoline-4(3H)-one Derivatives

Introduction

Quinazoline derivatives are classes of fused heterocyclic of extensive interest due to their varied biological activity including anti-inflammatory,^1,2 antibacterial,^3-8 antituberculosis,⁹ antimalarial,¹⁰ anti-HIV,¹¹ antiviral,¹² antiobesity,¹³ antipsychotic,¹⁴ antidiabetic,¹⁵ anticytotoxin,¹⁶ antispasmodic activities.¹⁷ The look for new molecules with antioxidant property is a popular area of research due to the fact that they could protect the human body from free radicals and retard the development of many continual diseases, including vascular diseases. Anti-oxidant activity is effective in the preventing complicated diseases like atherosclerosis, stroke, diabetes, Alzheimer’s disease and cancer. Flavonoids and phenolic compounds are broadly available in plants which have been found to exert organic effects such as antioxidant, free radical scavenging abilities, anti-inflammatory and anticarcinogenic activities. It has attracted a great deal of interest in the research of herbal antioxidants. A wide variety of synthetic compounds such as quinazolinones have been explored for antioxidants abilities. This inspired our interest to synthesize a group of compounds containing quinazolin‐4 (3H) ‐one derivatives associated with various primary hetero aromatic amines moiety and to evaluate their antioxidant potency. The antioxidant activity of synthesized compound turned into defined on the premise of general antioxidant interest with the aid of using scavenging activity of free radicals such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide radical scavenging activity.

Materials and Method

Design of compounds through in silico docking studies

R=(1a-5a) Anthranilic acid, (6b-10b) 5 Iodo Anthhranilic acid (11c-15c) 3 Iodo Anthranilic acid

Molecular docking

Before the docking analysis, ligands were prepared from the optimized compounds and saved in pdb file format using spartan, 14. The 3D compound of Dihydrofolate reductase (DHFR) tyrosine kinase was downloaded from the protein bank (with pdb ID:6DE4) (Table 1). 

The enzyme was prepared with the help of discovery studio visualizer for the docking analysis. In the course of preparation, hydrogen was added and water molecule, heteroatoms and co-ligands were eliminated from the crystal compound saved in pdb file.

The docking of the ligands to the active site of DHFR was achieved with the help of pyrex software using Autodock vina. After successful docking protocol, reformation of the complexes (ligand-receptor) for further investigation was also achieved utilizing chimera software. Discovery studio visualizer and pyMOL were used to investigate the interactions of the complexes (Figure 1 to Figure 15).

Synthesis of designed compounds and their characterization

A series of 15 compounds were designed through in silico docking and high docking score (3a, 4a, 8b, 9b, 10b) synthesis by one pot synthesis of quinazolinone derivatives from the reaction of anthranilic acid and primary heterocyclic amines with Vilsmeier reagent (DMF/POCl3) was carried out. The reaction occurred in few minutes under microwave irradiation providing excellent yields (Table 2). Various 4-(3H)- quinazolinones were synthesized by treating anthranilic acid and substituted anthranilic acid with Vilsmeier reagent (DMF/POCl3) at 0° followed by the addition of primary heterocyclic amines. The reaction-mixture was supported on anhydrous sodium sulphate and exposed to microwave irradiation for 2-4 min, resulting in the formation of 3-substituted quinazolinones. The products were characterized by infrared spectroscopy (IR)), (Nuclear magnetic resonance (NMR) and mass spectra. All the synthesized compounds were screened for their in vitro antioxidant activity. 

Characterized by IR, NMR, mass spectra

3a: Brown crys-tals, ¹H NMR (CDCl₃ , 500 MHz): δ 7.39 (d, J = 6.9 Hz, 2H), 7.46 (t, J = 7.6 Hz, 1H), 7.50 (t, J = 6.8 Hz,3H), 7.74 (t, J = 7.5 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H),8.11 (s, 1H), 8.35 (d, J = 7.6 Hz, 1H); IR (KBr): 3056,1668, 1591 cm^-1; MS: m/z 223 (M⁺ +1). Anal. Calcd. for C₁₇H₁₀BrN₃OS C,53.13, H 2.61, Br. 20.78,N 10.94 O ,4.15 S,8.34 ,

4a: Yellow crystals, ¹H NMR (CDCl₃ , 500 MHz): δ 7.43- 7.47 (m, 3H), 7.53 (t, J = 6.9 Hz, 1H), 7.59 (d, J = 6.9Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.79 (t, J = 8.4 Hz,1H), 7.94 (s, 1H), 8.35 (d, J = 7.65 Hz1H); IR (KBr): 3061, 1677, 1603, 1082 cm^-1; MS: m/z 257(M⁺ +1), 259 (M⁺ +3). Anal. Calcd. for C₁₇H₁₃N₃ O: C, 65.16; H, 4.75; N, 15.262. Found: C,65.60; H, 3.56; N, 10.89%.

8b: yellowish brown crystal, ¹H NMR (CDCl3 , 500 MHz): δ 7.28 (d, J = 9.2 Hz, 2H), 7.49 (t, J = 6.9 Hz,1H), 7.65 (d, J = 8.3 Hz, 2H), 7.74 (d, J = 8.4 Hz,1H), 7.77 (t, J = 6.9 Hz, 1H), 8.06 (s, 1H), 8.32 (d, J =8.4 Hz, 1H); IR (KBr): 3092, 1692, 1603, 1071 cm^-1;MS: m/z 301 (M⁺ +1), 302 (M⁺ +3). Anal. Calcd. for C₁₇H₉ BrIN₃OS: C, 55.84; H, 3.01; N, 9.30. Found: C,55.92; H, 2.89; N, 9.31%.

9b: Brown liquid, ¹H NMR (CDCl₃ , 500 MHz): δ 2.13 (s, 3H), 2.32 (s, 3H), 7.10 (q, J = 7.62 Hz, 2H), 7.18 (s,1H), 7.51 (t, J = 8.4 Hz, 1H), 7.75 (m, 2H), 7.97 (s,1H), 8.34 (d, J = 8.4 Hz, 1H); IR (neat): 1921, 1682,1603 cm^-1; MS: m/z 251 (M⁺ +1). Anal. Calcd. for C₁₇H₁₀IN₃O: C, 74.14; H, 5.64; N, 11.19. Found: C,76.85; H, 5.63; N, 11.21%.

10b: White crystals, ¹H NMR (CDCl₃ , 500 MHz): δ 3.83 (s, 3H), 7.01 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 9.1 Hz,2H), 7.50 (t, J = 6.9 Hz, 1H), 7.72 (d, J = 7.65 Hz,1H), 7.77 (d, J = 8.4 Hz, 1H), 8.11 (s, 1H), 8.34 (d, J= 8.4 Hz, 1H); IR (KBr): 3044, 2981, 1682, 1609, 1261, 1032 cm^-1; MS: m/z 252 (M⁺ +1). Anal. Calcd. for C₁₇H₁₀IN₃O: C, 71.42; H, 4.79; N, 11.10. Found: C, 71.35; H, 4.80; N, 11.08%

In vitro antioxidant activity

The synthesized compounds were screened for in vitro antioxidant activity.

The antioxidant capacity of quinazolinones was studied using different in vitro analytical methodologies such as DPPH scavenging, total reducing ability determination using Fe3+, Fe2+ transformation method, and hydrogen peroxide scavenging. These were used as the reference antioxidant radical scavenger compounds.

DPPH method

Preparation of control (DPPH) solution

10 mg of DPPH was dissolved in 10 mL of methanol. From this stock solution, dilutions were made to obtain concentrations of 10 µg/mL ,50 µg/mL, 100µg/mL and 250 µg/mL. The absorbance was recorded at 517 nm.

Preparation of standard solution (Ascorbic acid)

10 mg of ascorbic acid was dissolved in 10 mL of methanol. From this stock solution, dilutions were made to obtain concentrations of 10 µg/mL, 50 µg/mL, 100 µg/mL and 250 µg/mL to which 1 mL of DPPH solution was added and volume was made up to 10 mL. The absorbance was recorded at 517 nm after a duration of 30 min.

Preparation of test or sample solutions

The test solutions were prepared in a similar manner as that of standard ascorbic acid and the absorbance was recorded at 517 nm after a duration of 30 min.

% inhibition was calculated by Scavenging (%) = AS blank (DPPH) - AS (Sample) X 100/ AS control (DPPH) AS denotes absorbance (Figure 16).

Hydrogen peroxide radical scavenging method

Reaction mixture containing test samples /standard (Ascorbic acid) at different concentrations of 10 – 250 μg/mL was added to 0.6 mL of hydrogen peroxide solution in phosphate buffer (pH 7.4). After incubating for 10 minutes at 37^o C, the absorbance was measured at 230 nm. Appropriate blanks were taken. The experiment was carried out three times at 230 nm, and hydrogen peroxide absorbance in phosphate buffer was measured as control. Using below equation, the scavenging effect (%) was calculated. Hydroxyl radicals are created by hydrogen peroxide in cells. The test drug’s ability to scavenge these free radicals was utilised to determine its antioxidant activity. The decrease in absorbance at 230 nm with increasing concentration of the test medication as shown in Figure 17 indicates the reduction of these radicals.

Scavenging (%) = Absorbance - Absorbance (Sample) X 100 / Absorbance

Results and Discussion

All the synthesized compounds (3a, 4a, 8b, 9b, 10b) were evaluated for their anti-oxidant activity by DPPH, H₂O₂ assays. All of them showed significant anti-antioxidant activity, with 8b showing the maximum activity compared to others. In vitro antioxidant activities of synthesized compounds were comparable to that of the standard drug used in the study.

Absorption, Distribution, Metabolism, and Excretion (ADME) studies

ADME properties and drug-likeness prediction of some of the selected anti-microbial agents among the data set was carried out using Swiss ADME, a free web tool used in evaluating ADME properties and drug-likeness of molecules (Table 3).

Conclusion

According to data obtained from the present study, quinazolinone derivatives were found to possess anti-oxidant activity by using in vitro assay including DPPH radical and hydrogen peroxide scavenging activities. We compared standard antioxidant compounds such as ascorbic acid, butylated hydroxytoluene (BHT) respectively. Based on the discussion above, these quinazoline analogues could be considered as useful templates for further development to obtain more potent antioxidant activity. Quinazolinone derivatives could be very useful for virtual screening in the development of anticancer agents.

Conflict of interest

The authors declare that they have no conflicts of interest. 

Acknowledgement

The authors are thankful to the Management, Director, Principal, and Faculties of R. R. College of Pharmacy, Chikkabanavaram, Bangalore, for rendering the requirements in this work.