Synthesis, Bioevaluation, Structure-Activity Relationship and Docking Studies of Natural Product Inspired (Z)-3-benzylideneisobenzofuran-1(3H)-ones as Highly Potent antioxidants and Antiplatelet agents

For the first time, a series of highly potent natural product inspired substituted (Z)-3-benzylideneisobenzofuran-1(3H)-ones 28a-t, embraced with electron-withdrawing groups (EWG) and electron-donating groups (EDG) at site I and site II, were prepared and assessed for their in vitro antioxidant activities (DPPH free radical scavenging assay) and arachidonic acid (AA)-induced antiplatelet activities using ascorbic acid (IC50 = 4.57 µg/mL) and aspirin (IC50 = 21.34 µg/mL), as standard references, respectively. In this study, compounds 28f-g, 28k-l and 28q have shown high order of in vitro antioxidant activity. Infact, 28f and 28k were found to show 10-folds and 8-folds more antioxidant activity than ascorbic acid, respectively and was found to be the most active analogues of the series. Similarly, Compounds 28c-g, 28k-l, 28o and 28q-t were recognized as highly potent antiplatelet agents (upto 6-folds) than aspirin. Furthermore, in silico studies of the most active antioxidants 28f, 28k and 28l and very active antiplatelet molecules 28f, 28k, 28l and 28s were carrying out for the validation of the biological results. This is the first detailed study of the discovery of several (Z)-3-benzylideneisobenzofuran-1(3H)-ones as highly potent antioxidants and antiplatelet agents.

In the present study, we report the synthesis a series of functionalized (Z)-3-benzylidineisobenzofuran-1(3H)-ones 28a-t, their antioxidant and AA-induced antiplatelet activities, and structure-activity relationship (SAR) studies. Although compounds 28a-j, 28m-r and 28t have been prepared earlier by other routes; 30 including our route; 31 however, for the first time, substrate-controlled silver oxide nanoparticle (Ag 2 ONPs)-catalyzed synthesis of compound 28a-t have been prepared. Various substituted (Z)-3-benzylidineisobenzofuran-1(3H)-ones 28a-t have shown high order of antioxidant and AA-induced antiplatelet activities using ascorbic acid and aspirin taken as standard reference, respectively. We also perform the in silico studies of most active compounds 28f, 28k, 28l and 28s for the validation of biological results.
The SAR analysis revelaed that the five compounds i.e. 28f-g, 28k-l and 28q, exhibited promising antioxidant activity with the IC 50 values in the range 0.41-3.83 µg/mL. Further, no substitution at C-6 position of site I and m-OMe group at site II (i.e. compound 28f), showed best antioxidant potency than ascorbic acid. However, EDG substitution at site I (Br, CH 3 ) and H/p-OMe/m-OMe substitution on site II also accounts for promising activity (i.e. compound 28g, 28k-l and 28q). EWG group (fluoro group) at site I do not show favourable effects on the antioxidant activity [strong EWG-containing phenyl acetylenes (F, NO 2 , CN etc.) do not undergoes reaction under our optimized reaction conditions]. In addition, m-OMe group at site II augment antioxidant activity. Therefore, to examine the effect of OMe group at site I, we prepared 28t. It was found that compound 28t (IC 50 = 21.21 ± 0.12 μg/mL), having OMe group at C-6 position of site I and no substitution at site II showed diminished activity in comparison with the standard reference (Table 1, entry 20). Thus, OMe group showed advantageous effect on site II rather than site I. Finally, the structure-activity relationship studies illustrates that the two compounds, 28f and 28k, showed 10-folds and 8-folds higher antioxidant potency than commercially used antioxidant refered in the present study.
Since this scaffold have shown promising inhibition of platelet aggregation; compounds 28a-t were also tested for their AA-induced inhibition of platelet aggregation using aspirin as the standard reference (Table 1) 33,34 . As it has been observed from Table 1, compounds 28a-f were prepared having no substitutions at C-6 position of site I and H/CH 3 /Cl/Br/p-OMe/m-OMe substitutions at site II, respectively; 28a (IC 50 = 64.57 ± 0.58 μg/mL) demonstrated significant AA-induced antiplatelet activity in comparison with the standard reference aspirin www.nature.com/scientificreports www.nature.com/scientificreports/ (IC 50 = 21.34 ± 1.09 μg/mL; Table 1, entry 1). Shifting to CH 3 group at p-position of site II (28b) improves IC 50 value to 44.66 ± 0.41 μg/mL (Table 1, entry 2). However, the antiplatelet activity was improved tremendously when Cl/Br/OMe groups at site II were introduced. Compounds 28c-e showed IC 50 value of 19.57 ± 0.28 μg/ mL, 12.86 ± 0.11 μg/mL and 14.00 ± 0.17 μg/mL, respectively which were found to be more active than aspirin ( Table 1, entries 3-5). As also been noticed in the case of antioxidant activities of these compounds, 28f having m-OMe group at site II exhibited five-folds more efficacious (IC 50 = 4.20 ± 0.28 μg/mL) than the standard reference (Table 1, entry 6). Furthermore, 28g-l were analyzed having bromo substitution at C-6 position of site I along with H/CH 3 /Cl/Br/p-OMe/m-OMe groups at p-/m-position of site II (Table 1, entry 7-12). It has been interpreted that introduction of bromine group at site I improves antiplatelet activity (IC 50 = 16.28 ± 0.25 μg/ mL) upto four-folds than 28a and more active than aspirin (Table 1, entry 7). In contrast, compounds 28h-j having bromo substitution at C-6 position of site I along with CH 3 /Cl/Br groups at p-position of site II showed decreased activity i.e., IC 50 values of 29.32 ± 0.38 μg/mL, 31.34 ± 0.36 μg/mL and 79.57 ± 0.64 μg/mL, respectively, than the standard reference ( Table 1, entry 8-10). The activity has been increased effectively if OMe groups at p-/m-position of site II is introduced. Compounds 28k (IC 50 = 7.28 ± 0.48 μg/mL) and 28l (IC 50 = 4.20 ± 0.28 μg/ mL) having bromo substitution at C-6 position of site I along with p-/m-OMe substitutions on site II showed 3-5 folds greater antiplatelet potency than the standard reference, respectively (Table 1, entry [11][12]. Similarly, we analyzed the effect of EDG group at site I and prepared 28m-q having H/CH 3 /Cl/OMe groups at p-/m-position of site II (Table 1, entry 13-17). Introduction of CH 3 group at site I (28m) showed more aggregation inhibitory activity (IC 50 = 47.93 ± 0.44 μg/mL) than 28a and less activity than 28g (Table 1, entry 13). The activity has been found comparable to aspirin if CH 3 /Cl substitutions on site II were also introduced ( Table 1, entry [14][15]. While 28p (IC 50 = 24.64 ± 0.42 μg/mL) with methyl (CH 3 ) group at C-6 position of site I and p-OMe substitutions on site II exhibited comparable activity; Compound 28p (IC 50 = 24.64 ± 0.42 μg/mL) with methyl (CH 3 ) group at C-6 position of site I and m-OMe substitutions on site II showed two-folds more potency than the standard reference (Table 1, entry [16][17]. Sequentially, (EWG) group i.e., fluoro groups at C-6 position of site I were also analyzed (28r-s) and it was observed that both the compounds, 28r (IC 50 = 11.37 ± 0.67 μg/mL) and 28s (IC 50 = 3.25 ± 0.18 μg/mL), exhibited ~two-to seven-folds more potency than aspirin, respectively (Table 1, entry [18][19]. Infact, Compound 28s showed highest potency among the series. Compound 28t (IC 50 = 5.16 ± 0.32 μg/ mL), in contract to the antioxidant activity, showed four-folds more antiplatelet potency than the standard reference (Table 1, entry 20). Overall, it interprets that introduction of EWG or EDG at site I improves antiplatelet activity than aspirin. Similarly, introduction of p-/m-OMe on site II leads to either more activity or comparable to the standard reference. www.nature.com/scientificreports www.nature.com/scientificreports/ Molecular docking studies. All the most active compounds (28f, 28k and 28l) along with one inactive compound 28d were further analyzed for their in silico studies using the reported protocol where the antioxidant target (PDB ID: 3MNG) were taken to explore the orientations and binding affinities of the target compounds in order the observe the difference in the docking score of active and inactive compounds 35,36 . Wild type human antioxidant enzyme Peroxiredoxins (Prdxs) was chosen, containing essential cysteine residues as catalyst and thioredoxin as an electron donor, which help in scavenging peroxide and are involved in the metabolic cellular response to reactive oxygen species 36 . It has been confirmed that the ascorbate-mediated reduction of protein sulfenic acids represents a modification of the peroxiredoxin-thiol-specific antioxidant paradigm, which directly confirms the interlinking of peroxiredoxins with standard drug ascorbic acid (vitamin C) 36 . Therefore, the interlinking of standard reference ascorbic acid with peroxiredoxins direct us to perform molecular docking studies on this enzyme.
Similarly, to study the binding modes of the five active molecules (28k, 28s, 28f, 28l and 28j) in the cyclooxygenase-1 (COX-1) enzyme against platelet aggregation inhibitory activity, we performed molecular docking study with aspirin (reference compound) on COX-1 domain antiplatelet target (PDB ID: 2OYE) using Surflex-Dock using the reported procedure (see details in supporting information) 37 .
Antioxidant molecular docking studies. The docking outcomes for the ascorbic acid against antioxidant target reflected a high binding affinity (docking score = 3.1764) as shown in Fig. 4. The active compound 28k, 28f and 28l displayed docking results against antioxidant target (PDB ID: 3MNG) exhibited a docking score of 3.9321, 4.6899 and 3.4080, respectively, therby reflecting their high binding affinity. These values were found to be more than that of ascorbic acid. Therefore, 28k, 28f and 28l showed elevated binding affinity and hydrophobic interaction which are responsible for more stability and activity ( Fig. 5A-C). Likewise, the inactive compound 28d (Fig. 5D) was found to show less binding affinity than the ascorbic acid as indicated by its docking score of 2.9813. This showed low binding affinity and weak hydrophobic interaction which may be responsible for less stability and activity (see details in supporting information).
Antiplatelet molecular docking studies. Likewise, the antiplatelet docking outcomes for aspirin against target (PDB ID: 2OYE) revealed low docking score (total score of 4.4803) thereby reflecting its lower binding affinity (Fig. 6A). The docking outcomes for 28k, 28s 28f, and 28l against PDB ID: 2OYE reflected docking score designated by a total score of 5.1953, 5.7131, 5.1010 and 5.1184, respectively thereby indicating a high binding affinities which were found to be more than standard reference aspirin. Therefore, 28k, 28s, 28f and 28l elevated binding affinity and hydrophobic interaction which are responsible for more stability and activity (Fig. 6B-E). In contrast, the antiplatelet docking outcomes for the inactive compound 28j against target (PDB ID: 2OYE) showed lower docking score (total score = 4.1714) thereby reflecting lower binding affinity which, in turn, is found to be lesser than the standard reference. Thus, the bound compound 28j showed weak hydrophobic interaction which leads to low binding affinity thereby responsible for less stability and activity of the molecule (Fig. 6F).

conclusions
We disclose the first detailed study for the identification of (Z)-3-benzylideneisobenzofuran-1(3H)-one analogues 28a-t as highly potent antioxidant and AA-induced antiplatelet agents. Five isobenzofuran-1(3H)-one analogues 28f-g, 28k-l and 28q were found to be the active compounds of the series in DPPH assay. Infact, two compounds, 28f and 28k, showed 10-folds and 8-folds more antioxidant activity than ascorbic acid, respectively. Similarly, twelve isobenzofuran-1(3H)-one analogues 28c-g, 28k-l, 28o, and 28q-t exhibited highly potent (upto 6-folds) platelet aggregation inhibitors as compared to aspirin in AA-induced antiplatelet biological assay. Furthermore, compounds 28f, 28k, 28l and 28s were analyzed through docking studies for the justification of the obtained results. These highly active potent molecules were found commendable of additional structural optimization and advancement as potential antioxidant and/or antiplatelet representatives. capillaries on Sisco melting point apparatus and are presented uncorrected. All the AR grade chemicals were used as supplied from commercial source (Sigma Aldrich, TCI, Alpha Aesar, Spectrochem etc.) and used without further purification. Laboratory grade commercial reagents and solvents were purified by standard procedures prior to use. The silica gel (100-200 Mesh) used for column chromatography were supplied either from QualigensTM (India) or Rankem (India), unless otherwise noted. UV fluorescence and Iodine vapor served as the visualizing agent for thin layer chromatography (Merck silica gel 60 F 254 precoated plates (0.25 mm). 1 H NMR and 13 C NMR spectral data were recorded on a JEOL ECS-400 (2-channel support with an adaptable broadband RF execution) spectrometer working at 400 MHz for 1 H and 100 MHz for 13 C) utilizing CDCl 3 as a solvent. The 1 H-NMR (400 MHz) chemical shifts were measured relative to CDCl 3 as the internal reference (CDCl 3 : δ = 7.249 ppm). Tetramethylsilane (δ 0.00 ppm) served as an internal standard in 1 H NMR and CDCl 3 (δ 77.0 ppm) in 13 C NMR. Chemical shifts are reported in parts per million. Splitting patterns are described as singlet (s), doublet (d), double doublet (dd), triplet (t), multiplet (m), and broad (br). Infrared spectra were recorded on a FT-IR Spectrum 2 (Perkin-Elmer) spectrophotometer. Electron Impact Mass Spectroscopy (HR-EIMS) data were obtained from Xevo G2-S Q-Tof (Waters, USA) compatible with ACQUITY UPLC ® and nano ACQUITY UPLC ® systems. The BUCHI Rotavapor R-210 was used for drying and concentration of the solvents. All animal experiments were performed in compliance with the relevant laws and guidelines of Suresh Gyan Vihar University, and approved by the institutional animal ethical committee(s).
The detailed spectral data of compounds 28a-j, 28m-r and 28t are given in the supporting information.

Biological methods.
In vitro antioxidant DppH radical scavenging activity: 38 See Si.

Data availability
All data generated or analyzed during this study are included in this published article.