Substituted benzocoumarin derivatives: synthesis, characterization, biological activities and molecular docking with ADME studies

Herein, we report an efficient and convenient method for the synthesis of 4-(substitutedphenyl)-1,2-dihydro-2-oxo-6-(2-oxo-2H-benzo[g]chromen-3-yl)pyridine-3-carbonitrile derivatives using ammonium acetate as a catalyst. The structures of the synthesized compounds were confirmed using FT-IR, 1 H, 13 C-NMR and LC-MS spectroscopic techniques. The synthesized compounds were evaluated for antibacterial activity against bacterial strains by disc diffusion method at different concentrations. Further, all the targeted compounds were screened for anti-oxidant and anti-cancer studies by DPPH and MTT assay methods respectively at different concentrations. Compound 4b displayed good antioxidant and anticancer (against MCF-7 cell line) activity. The binding capability for the synthesized compounds ( 4a – j ) was analyzed by molecular docking studies using human peroxiredoxin 5 (PDB ID: 1HD2) and P38 MAP kinase (PDB ID: 1OUK) protein. The physicochemical properties were analyzed using absorption, distribution, metabolism and excretion (ADME) studies.


Introduction
Coumarin is a well-known naturally occurring organic compound, and it has been utilised in various fields such as pharmaceuticals. Around the world, research is being conducted on this compound due to its properties [1]. Coumarins are naturally occurring active constituents of various plants such as Dipteryxodorata, Anthoxanthumodoratum, Galiumodoratum, etc, [2]. These are found in higher plants like Rutaceaeand Umbelliferae and essential oils of cinnamon bark, cassia leaf, and lavender oil [3]. Coumarin is an aromatic compound that has a bicyclic structure with lactone carbonyl groups. The presence of an electronegative atom is effective for hydrogen bond formation and for solubility, to some extent, and the aromatic ring is responsible for having hydrophobicity. These phenomena are the cause of better interaction of the molecule with a receptor site. The substitution of coumarins activates their bioactivity. For thousands of years, natural products have been utilized in the traditional medicines, in addition to their use as a promising source of components for discovery and development of new therapies [4]. Numerous types of coumarins have been synthesized with different types of substitutions or pharmacophore in their basic nuclei, which are significant in showing effective and diverse classes of biological activity [5]. Based on the substitution pattern, coumarins show anticancer [6], anti-HIV [7], anticoagulant [8], antimicrobial [9], antioxidant [10], antihypertensive [11], antihyperglycemic [12], antituberculosis [13], and anti-inflammatory [14] activities.
Cancer is a chronic disease that is associated with multiple syndromes and, hence, its treatment requires close attention. World Health Organization (WHO) reported in 2013 that a not-contagious illness such as cancer is responsible for 60% deaths worldwide. Among them, 80% of deaths (28 million) occur in low and middle-income countries like India [15]. A number of anticancer agents are currently used in clinical practice for treatment of various kinds of cancers. Coumarin and its derivatives possess anticancer activity against different types of cancers such as prostate, renal, breast, laryngeal, lung, colon, CNS, leukemia, malignant melanoma [16][17][18]. Breast cancer is the most frequent in females, and it is one of the leading causes of cancer death for women. Breast cancer can also occur in men, but it is far less common. Yet new treatment methods provide more reason for optimism than ever before [19]. Coumarin derivatives readily interact with a variety of enzymes and receptors in breast cancer cells. Moreover, the coumarin-based irosustat drug is known to interact with sulfatase enzyme, indicating that coumarin derivatives are potential materials for anti-breast cancer agents [20]. In the last 30 years, doctors have made great strides in early diagnosis and treatment of the disease and in reducing breast cancer deaths. There are many chemotherapy agents available for the treatment of breast cancer. Still, the current therapeutic options have not fulfilled the desired outcomes for breast cancer therapy [21]; thus, there is an urgent need to develop novel synthetic coumarin derivatives with anti-breast cancer potential.

Materials and Method
The reagents, solvents and chemicals were purchased from Sigma-Aldrich and were used without further purification. Alumina TLC plates were used to check the progress of the reaction. The spots were identified in a 360 nm UV chamber. The melting points were determined by the electro-thermal apparatus using open capillary tubes and are uncorrected. FTIR spectra were recorded on a Bruker spectrophotometer using KBr pellets in the region of 400-4000 cm −1 . 1 H-NMR and 13 C-NMR (400 MHz and 100 MHz) spectra were recorded using a Jeol instruments and estimated with the Delta software in DMSO-d6 solvent system; chemical shifts (δ) were recorded in ppm relative to tetramethyl silane as a standard reference. The molecular weight of synthesized compounds was confirmed by a LC-MS 2010, SHIMADZU, mass analyzer. Elemental analysis was performed by using the unique method. The anti-bacterial activity was carried out against two Gram-negative and Gram-positive bacterial strains using the agar diffusion method and anti-oxidant by DPPH scavenging activity at different concentrations. The anti-cancer activity was tested against MCF-7 cell line by the MTT method at different concentrations and compared with a standard drug.

Pharmacological activities 2.3.1. Antibacterial activity
The four bacterial strains Bacillus subtilis, Staphylococcus aureus (gram-positive bacteria) and Escherichia coli, Pseudomonas aeruginosa (Gram-negative bacteria) were used to investigate the antibacterial activity of the synthesized compounds (4a-j) by the disk diffusion method [22]. Briefly, all the compounds were dissolved in DMSO in two different concentrations (25 and 50 mg/mL), and to this test solution, the previously cultured Mueller Hinton Agar Sabouraud's dextrose agar medium was added and autoclaved at ±37 °C for about 24 h. Streptomycin was used as a standard drug and 10% DMSO used as a negative control. The antimicrobial assay of the title compounds was measured by the formed zone of inhibition against pathogenic strains. The test was performed in triplicate and the average was taken as a final reading. The minimum inhibitory concentration (MIC) was determined by the serial broth-dilution method [23].

Antioxidant activity
The synthesized compounds (4a-j) were screened for DPPH scavenging activity [24], and DPPH methods were carried out according to the reported procedure [25,26]. The compounds at different concentrations (5 μg/mL, 10 μg/mL, 20 μg/mL, 40 μg/mL, 80 μg/mL) were used for the analysis. Ascorbic acid was choosing for comparison as a standard drug and Radical scavenging activities were calculated using the formula [27]: where Acontrol is the absorbance of the control reaction and Atest is the absorbance of the synthesized compound. IC50 value was calculated using the formula: where ƩC is the sum of synthesized compound concentrations used to test and ƩI is the sum of % of inhibition at different concentration. Each value is expressed as a mean ±SD of three replicates.

Cytotoxicity
The in vitro cytotoxicity was assessed by MTT assay by following the procedure of Kumbar et al. [28], against the MCF-7 (Breast cancer) cell line. The cells were seeded in a 96-well flat-bottom microplate and maintained at 37 °C in 95% humidity and 5% CO2 overnight. Different concentrations (200, 100, 50, 25, 12.5 and 6.25 μg/mL) of the samples were treated. The cells were incubated for another 48 h, and the wells were washed twice with PBS. 20 μL of the MTT staining solution was added to each well, and the plate was incubated at 37 °C. After 4 h, 100 μL of DMSO was added to each well to dissolve the formazan crystals, and absorbance was recorded at 570 nm using a microplate reader. The percentage of cell survival was calculated by using the following formula [29,30].
% of cell survial = Mean OD of Test compound Mean OD of Negative control · 100 (2)

In silico Molecular docking study
The molecular interactions of the synthesized compounds at the binding pocket of human peroxiredoxin 5 protein and P38 MAP kinase proteins were studied using automated docking by employing the Autodock Vina program [31]. The co-crystallized structure of human peroxiredoxin 5 protein (PDB ID: 1HD2) and P38 MAP kinase (PDB ID: 1OUK) were retrieved from the protein databank, and their substrate binding sites were identified using the pdb sum server [32]. A grid box of dimensions 20x20x20 Å with X, Y and Z coordinates at 7.654, 40.848 and 34.184 for human peroxiredoxin 5 and 20x20x20 Å with X, Y and Z coordinates at 44.438, 32.882 and 32.692 for P38 MAP kinase were created, respectively. The grid box was set around the residues forming the active pocket. The binding interactions were visualized using Biovia Discovery Studio Visualizer V.20.1.

In silico oral bioavailability assessment and ADME
Various physicochemical features and pharmacokinetic descriptors were calculated through the online web tool Swiss ADME [33]. The oral bioavailability of the synthesized compounds (4a-j) was predicted using the Lipinski rule-of-five (RO5) filter [34] to derive the candidate drug pharmacokinetic (PK). The structural properties used in the RO5 filter are derived using Osiris Data warrior V.4.4.3 software [35]. The bioavailability scores were predicted using the molinspiration server [36].

Antibacterial activity
The in vitro antibacterial activity of the synthesized compounds was screened against four pathogenic strains and the results are tabulated in Table 2. The result revealed that all the test compounds exerted a varied degree of antibacterial activity against the tested strains. Further, to quantify the lowest concentration at which the growth of the organism was prevented, we determined the minimum inhibitory concentration (MIC). The results are given in Table 3. Compounds 4a, 4c, 4h and 4i exhibited equipotent activity against E. coli, Pseudomonas aeruginosa and S. Aureus with MIC 2.1-2.3 mg/ml. From the structure activity relationship studies, it was observed that compounds 4e and 4j, having methoxy groups on the aromatic ring, showed excellent activity among the series when compared to the standard drug. Compounds 4a, 4c, 4f and 4g, having -NO2 and -Cl groups, respectively, displayed promising activity against B. subtilis. The rest of the synthesized compounds showed moderate activity against selected bacterial strains.

Antioxidant activity
The synthesized compounds (4a-j) were screened for their free radical scavenging activity by the DPPH method as shown in Figure 1. All the compounds showed varied free radial scavenging capacity in assessment with the standard Ascorbic acid. Among all the compounds, compound 4b exhibited most effective antioxidant efficacy with IC50 value 34.66±2.43 μg/mL as compared to the reference standard drug (IC50 8.88±1.19 μg/mL). Compounds 4a, 4c, 4f and 4j with IC50 in the range of 41.54-44.11 μg/mL showed promising antioxidant activity, and the rest of the compounds showed moderate scavenging activity by the DPPH method. Therefore, the synthesized compounds exhibited more effective antioxidant efficacy due to the presence of the electron donating group (OH) at para position of the phenyl ring [37]. The results are shown in Table 4.

Cytotoxicity study
The in vitro cytotoxicity of the synthesized compounds (4a-f) was evaluated against the MCF-7 (Breast cancer) cell line and shown in Figure 2. A graph detailing the concentration versus survival fraction of the compounds was plotted in Figures 3  and 4, and the results were expressed as the half-maximal inhibitory concentrations (IC50) (

In silico molecular docking studies
The study of intermolecular interactions between the synthesized compounds (4a-j) and enzymes is necessary for the development of novel therapeutic drugs [38]. Therefore, we screened the synthesized compounds for in silico molecular docking, which helps to predict the binding modes of the compounds with enzymes. From the results of the docking study, the synthesized compounds established good binding modes with docking receptors of Human peroxiredoxin 5 (Figures 5, 6) and P38 MAP kinase (Figures 7, 8), respectively. The docking of anti-oxidant and cytotoxicity investigations revealed that the synthesized compounds (4a-j) had significant docking scores in the range of -7.3 to -8.5 kcal/mol with respect to standards Ascorbic acid (-6.5 kcal/mol) and -9.8 to -11.0 kcal/mol with respect to standard Doxorubicin (-8.4 kcal/mol), respectively. The docked structures with Human peroxiredoxin 5 results suggested that compound 4h established the lowest binding energy of -8.5 kcal/mol. The remaining compounds also established good binding modes and one or more hydrogen bonds with amino acid residues THR147, ARG127, ASN76, ARG124, GLY46, CYS47, THR44 (Table S1). The docked structures with P38 MAP kinase protein results revealed that compound 4d demonstrated the lowest binding energy of -11.0 kcal/mol, forming one or more hydrogen bonds with three amino acid residues LYS53, VAL38, TYR35, VAL30, ALA40, LEU108. The remaining compounds also established encouraging binding energies and formed two or more hydrogen bonds with amino acid residues in their active pockets. The results are given in Table S2.

ADME studies
Physiochemical descriptors can be evaluated through the parameters such as molecular weight, number of heavy atoms, hydrogen bond acceptors, hydrogen bond donors, rotatable bonds, molar refractivity and topological polar surface area (TPSA). These parameters were evaluated for the synthesized compounds (4a-j), and the results are shown in Table 6.
The drug-likeness profiles were calculated based on Lipinski's (MW ≤ 500; HBA ≤ 10 and HBD ≤ 5) [39], Ghose (MW between 160 and 480; log P between -0.4 and 5.6; molar refractivity between 40 and 130, and the total number of atoms between 20 and 70), Veber (rotatable bonds ≤ 10 and TPSA ≤ 140), Egan (TPSA ≤ 131.6 Å²) and Muegge (MW between 200 and 600; log P between -2 and 5; TPSA ≤ 150; number of aromatic rings ≤ 7; number of rotatable bonds ≤ 15; HBA ≤ 10 and HBD ≤ 5) [40]. All the synthesized compounds obeyed Lipinski, Ghose, Veber, Egan and Muegge rules. The rule-based score divides the compounds into four probability score classes, i.e., 11%, 17%, 55% and 85%. The acceptable probability score is 55%, which indicates that it passed the rule of five [41,42]. The synthesized compounds (4a-e) showed a score of 55%, indicating that they all the five rules without any violations with good bioavailability. Further, synthetic accessibility of the compounds was assessed to quantify the complexity of the molecular structure. The results showed that the synthetic accessibility score was in the range of 3.44 to 3.67. It shows that no compound has a complex synthetic route (Table S3).
The predicted lipophilicity parameters were evaluated to study the solubility of the compounds either in aqueous or in non-aqueous medium, and they were calculated by considering the consensus logPo/w. According to this, if the logPo/w values are more negative, then the molecules are more soluble in nature [43]. The results showed that all the compounds have positive logPo/w values. Hence, they are less soluble in non-aqueous medium. The values of logS are related to the solubility as follows if logS are between -10 to -6 -poorly soluble, -6 to -4 -moderately soluble, -4 to -2 -soluble, -2 to 0 -very soluble and less than 0 -highly soluble. For our synthesized compounds (4a-j), the logS value is in the range of -5.21 to -6.33; this shows that the compounds are soluble in aqueous media. The results are presented in Table S4.
The pharmacokinetic parameters such as absorption, skin permeation, distribution, metabolism and excretion were predicted. The predicted absorption and distribution parameters of the compounds are in Table S5, indicating that, all the synthesized compounds (4a-j) have high gastrointestinal absorption (except for 4a and 4f) with no blood-brain barrier crossing.   Three-dimensional and two-dimensional representations of molecular interactions between human peroxiredoxin 5 protein and synthesized compounds (4g-j) and reference drug Ascorbic acid.
Hence, there was no possibility of causing harmful toxicants to appear in the brain and blood stream. If the molecules have more negative logKp value, it means that they possess lower skin permeation [44]. This is true for our synthesized compounds, which have more negative logKp values in the range of -5.41 to -6.46 cm/s (Table S5).
Metabolism plays an important role in the bioavailability of drugs as well as drug-drug interactions [45]. Metabolism parameters are important to understanding whether the compounds act as a substrate or a non-substrate of the certain proteins. Hence, all the synthesized compounds were evaluated for the metabolism parameters, and the results showed that the compounds (4a-j) are nonsubstrates of permeability glycoprotein (P-gp), CYP1A2, CYP2C19, CYP2D6 and CYP3A4 inhibitors. The P-gp is an efflux membrane transporter, which is widely distributed throughout the body and is responsible for limiting cellular uptake and the distribution of xenobiotic and toxic substances [46]. The compounds (except for 4c and 4e) were found to be non-substrates of CYP1A2 inhibitors. Compounds 4b, 4d, 4f, 4g and 4i were found to be substrates of CYP2C19 inhibitors, and the remaining compounds were non-substrates of CYP2C19 inhibitors.  All the compounds were found to be substrates of CYP2C9 inhibitors. The compounds (except for 4a, 4c, 4f, 4g, 4h) are non-substrates to CYP2D6 inhibitors. All the compounds are non-substrates of CYP3A4 inhibitors. The data are listed in Table S6.

Conclusions
In this present work, we synthesized some novel 4-(substitutedphenyl)-1,2-dihydro-2-oxo-6-(2-oxo-2Hbenzo[g]chromen-3-yl) pyridine-3-carbonitrile derivatives 4(a-j) through the one-pot reaction. The antibacterial ac-tivity results show good efficacy against four bacterial strains. The antioxidant activity results suggested that compound 4d exhibited the lowest IC50 value of 22.60±0.30. Compound 4b displayed significant cytotoxic effect with an IC50 value of 34.66±2.43 as compared to the other compounds. The in silico docking studies suggested that the synthesized compounds interacted effectively with Human peroxiredoxin 5 and P38 MAP kinase proteins with good binding energy. In that, compounds 4h and 4d show the lowest binding energy values of -8.5 kcal/mol and -11.0 kcal/mol, respectively. Doxorubicin 4j 4i 4h 4g ADME studies explained that our synthesized compounds obeyed all the five rules with good bioavailability. Pharmacokinetic parameters suggested that the compounds have GI absorption, do not cross the blood-brain barrier, possess low skin permeation, and that there is no possibility of creating harmful toxicants. Based on the obtained results, the synthesized compounds are promising materials in the pharmacological fields.

Supplementary materials
Supplementary materials are available.

Funding
None.