Discovery of C-3 Tethered 2-oxo-benzo[1,4]oxazines as Potent Antioxidants: Bio-Inspired Based Design, Synthesis, Biological Evaluation, Cytotoxic, and in Silico Molecular Docking Studies

Abstract

The discovery of C-3 tethered 2-oxo-benzo[1,4]oxazines as potent antioxidants is disclosed. All the analogs 20a-20ab have been synthesized via "on water" ultrasound-assisted irradiation conditions in excellent yields (upto 98%). All the compounds have been evaluated for their in vitro antioxidant activities using DPPH free radical scavenging assay as well as FRAP assay. The result showed promising antioxidant activities having IC₅₀ values in the range of 4.74 ± 0.08 to 92.20 ± 1.54 μg/mL taking ascorbic acid (IC₅₀ = 4.57 μg/mL) as standard reference. In this study, compounds 20b and 20t, the most active compound of the series, showed IC₅₀ values of 6.89 ± 0.07 μg/mL and 4.74 ± 0.08 μg/mL, respectively in comparison with ascorbic acid. In addition, the detailed SAR study shows that electron-withdrawing group increases antioxidant activity and vice versa. Furthermore, in the FRAP assay, eight compounds (20c, 20j, 20m, 20n, 20r, 20u, 20z, and 20aa) were found more potent than standard reference BHT (C_0.5FRAP = 546.0 ± 13.6 μM). The preliminary cytotoxic study reveals the non-toxic nature of active compounds 20b and 20t in non-cancerous 3T₃ fibroblast cell lines in MTT assay up to 250 μg/mL concentration. The results were validated via carrying out in silico molecular docking studies of promising compounds 20a, 20b, and 20t in comparison with standard reference. To the best of our knowledge, this is the first detailed study of C-3 tethered 2-oxo-benzo[1,4]oxazines as potential antioxidant agents.

Keywords: 2-oxo-benzo[1,4]oxazines; BHT; DPPH; FRAP; antioxidant; ascorbic acid.

Figure 1

Structures of some natural as well as synthetic compounds (1-6) having antioxidant activity.

Figure 2

Design strategy for the target compound 2-oxo-benzo[1,4]oxazine 15 as an antioxidants.

Scheme 1

Synthesis of starting substrate functionalized diketo-acid (18a-h).

Scheme 2

Ultrasound-assisted green synthesis of C-3 tethered 2-oxo-benzo [1,4]oxazine analogs (20a-20ab).

Figure 3

Structures of all synthesized C-3 tethered 2-oxo-benzo [1, 4]oxazines (20a-20ab).

Figure 4

SAR analysis of synthesized 2-oxo-benzo[1,4]oxazines.

Figure 5

Percentage cell viability test.

Figure 6

Binding interactions of compound 20a, 20b, 20t and reference drug Ascorbic acid upon docking onto human antioxidant enzyme target peroxiredoxins (Prxs) (PDB ID: 3MNG). The formation of a H-bond of length 1.8Å to residue Gly-46 in the binding site was predicted in the case of 20a, 20b, and 20t along with the formation of four H-bond of length 2.2, 1.8 and 2.1 Å to residue Thr-147, Gly-46 and Thr-44 in the binding site was predicted in the case of reference drug Ascorbic acid. (A) A top docking energy (total score) of 3.8470 was predicted for 20a; (B) A top docking energy (total score) of 3.6567 was predicted for 20b. (C) A top docking energy (total score) of 4.2709 was predicted for 20t. (D) A top docking energy (total score) of 3.4829 was predicted for Ascorbic acid.