Design and synthesis of heterocyclic azole based bioactive compounds: Molecular structures, quantum simulation, and mechanistic studies through docking as multi-target inhibitors of SARS-CoV-2 and cytotoxicity

Abstract

Two heterocyclic azole compounds, 3-(2,3-dihydrobenzo[d]thiazol-2-yl)-4H-chromen-4-one (SVS1) and 5-(1H-indol-3-yl)-4-methyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (SVS2) were obtained unexpectedly from 2-aminothiophenol and 4-oxo-4H-chromene-3-carbaldehyde (for SVS1), and (E)-2-((1H-indol-3-yl)methylene)-N-methylhydrazine-1-carbothioamide in the presence of anhydrous FeCl₃ (for SVS2), respectively. The compounds were well characterized by analytical and spectroscopic tools. The molecular structures of both the compounds were determined by single crystal X-ray diffraction (XRD) study. The results obtained from density functional theory (DFT) study revealed the molecular geometry and electron distribution of the compounds, which were correlated well with the three-dimensional structures obtained from the single crystal XRD. DMol³ was used to calculate quantum chemical parameters [chemical potential (µ), global hardness (η), global softness (σ), absolute electronegativity (χ) and electrophilicity index (ω)] of SVS1 and SVS2. Molecular docking study was performed to elucidate the binding ability of SVS1 and SVS2 with SARS-CoV-2 main protease and human angiotensin-converting enzyme-2 (ACE-2) molecular targets. Interestingly, the binding efficiency of the compounds with the molecular targets was comparable with that of remdesivir (SARS-CoV-2), chloroquine and hydroxychloroquine. SVS1 showed better docking energy than SVS2. The molecular docking study was complemented by molecular dynamics simulation study of SARS-CoV-2 main protease-SVS1 complex, which further exemplified the binding ability of SVS1 with the target. In addition, SVS1, SVS2 and cisplatin were assessed for their cytotoxicity against a panel of three human cancer cells such as HepG-2 (hepatic carcinoma), T24 (bladder) and EA.hy926 (endothelial), as well as Vero (kidney epithelial cells extracted from an African green monkey) normal cells using MTT assay. The results showed that SVS2 has significant cytotoxicity against HepG-2 and EA.hy926 cells with the IC₅₀ values of 33.8 μM (IC₅₀ = 49.9 μM-cisplatin and 8.6 μM-doxorubicin) and 29.2 (IC₅₀ = 26.6 μM-cisplatin and 3.8 μM-doxorubicin), respectively.

Graphical abstract

Two heterocyclic azole compounds were synthesized and characterized. Both the compounds act as multi-target inhibitors of SARS-CoV-2 proteins. The compounds exhibited good cytotoxicity against cancer cells.

1. Introduction

Outbreak of SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) has led to more than 100 million cases and 2 million deaths as reported by WHO (World Health Organization), and these numbers are still counting. Due to this universal life threat to humans, a global response to prepare health systems is a priority [1].

Four types of CoVs namely α, β, γ and δ were identified. Among the four, γ and δ are known to affect birds, whereas α and β affect mammals [2,3]. The novel SARS-CoV-2 belongs to β-coronavirus lineage with four main proteins, namely, spike (S) protein (binds with bronchial epithelial cells and type-II pneumocytes after entering the host cells with the help of ACE-2 and TMPRSS2 receptors through endocytosis), membrane (M) protein (acts as an organizer of CoV assembly), envelope (E) protein (interacts with membrane protein to form an envelope) and nucleocapsid (N) protein (viral RNA genome replication) [2]. Some nucleotide analogous drugs like remdesivir and galidesivir interrupt the process of viral replication by interacting with RNA-dependent RNA polymerase (RdRp), an enzyme that plays a vital role in virus replication. Some studies demonstrated that chloroquine (CQ) and hydroxychloroquine (HCQ) can reasonably inhibit SARS-CoV-2. CQ and HCQ are also suggested to block the virus entry through glycosylation of host ACE-2 receptor. Remdesivir showed inhibition activity against a variety of non-structural proteins such as RdRp [4,5].

To date, due to its high toxicity and the virus resistance to the drug, no effective dedicated treatments are approved for treating such CoVs. The development of new therapeutic anticorona agents made so far has also been limited [4,5]. Heterocyclic compounds such as triazole, thiazole, pyrrole, pyridine, quinolone, etc., and their derivatives are characterized as important classes of organic compounds that exhibit various biological and medicinal properties such as antitumor [6,7], EGFR inhibitory [8], antioxidant [9,10], antianalgesic, antiinflammatory [11], antibacterial [12], antifungal [13], antimicrobial [14], etc. The biological properties of many nitrogen based heterocyclic compounds such as triazole and its derivatives have been investigated [15]. Detailed studies have shown that they possess significant biological properties [16], [17], [18]. Demirbas et al., reported the antimicrobial activities of the triazole derivatives against several bacterial and yeast strains. The results showed that triazole derivatives are one of the active components to increase the antimicrobial activities [19]. In addition, a series of 1,2,4‐triazole compounds showed significant antitumoral activity, and acetylcholinesterase (AChE) and human carbonic anhydrase enzymes inhibition [20]. The compounds including indole and their analogs were evaluated for their anti-hepatitis B virus (HBV), anti-inflammatory, antifungal activities, and the treatment of acute lung injury [21], [22], [23], [24], etc. Chromones and their derivatives have played a vital role in the human diet. Many biologically active compounds derived from natural sources comprise of chromone moiety as their core [25], [26], [27]. These compounds have attracted the pharmaceutical industries to a large extent owing to their antioxidant, antitumor and antibacterial activities [28], [29], [30]. Besides these, several chromone derivatives have been used as antimicrobial, DNA binding, DNA cleavage, anticancer, anti-allergic, neuroprotective and pesticidal agents [31], [32], [33].

Due to the existence of chromone- and heterocyclic azole-based structures in many drugs, we were motivated to synthesize such heterocyclic compounds for biological applications. In this connection, we report the synthesis of two heterocyclic compounds (SVS1 and SVS2), and the main objectives of the study are to solve the three-dimensional structures, and to explore the various pharmaceutical potentials of the compounds through in silico and in vitro studies. The compounds were characterized by single crystal XRD, and FT-IR, UV-Visible, ¹H NMR and ¹³C NMR spectroscopy. The quantum chemical parameters of the compounds were calculated by using Materials Studio 2017. Molecular docking studies were carried out to know their interactions with the main protease of SARS-CoV-2 and human ACE-2 receptors. The molecular docking results were further supported by molecular dynamics simulation studies. The cytotoxic potential of SVS1 and SVS2 along with the standard drugs (cisplatin and doxorubicin) was assessed using MTT assay against three cancer (HepG-2, T24 and EA.hy926) and one normal (Vero) cell lines.

2. Experimental

2.1. Materials and methods

Chromone-3-carboxaldehyde, 2-aminothiophenol, anhydrous FeCl₃ and solvents were purchased from commercial suppliers (Sigma Aldrich or Alfa Aesar) and used as received. (E)-2-((1H-indol-3-yl)methylene)-N-methylhydrazine-1-carbothioamide was prepared according to the previously reported procedure by us [34], [35], [36]. The melting points were measured on a Lab India instrument and are uncorrected. CHNS analyses were carried out using a Vario EL III CHNS instrument. FT-IR spectra were obtained as KBr pellets using a Ncolet-iS5 spectrophotometer. UV-Visible spectra were recorded using a Shimadzu-2600 spectrophotometer. NMR spectra were recorded in DMSO‑d ₆/CDCl₃ solvent using TMS as an internal standard on a Bruker 500 MHz spectrometer.

2.2. Synthesis

2.2.1. Synthesis of 3-(2,3-dihydrobenzo[d]thiazol-2-yl)-4H-chromen-4-one (SVS1)

The compound was synthesized by mixing an equimolar amount of 4-oxo-4H-chromene-3-carbaldehyde (174 mg, 1 mmol) with 2-aminothiophenol (125 mg, 1 mmol) in 10 mL of ethanol, and the reaction mixture was refluxed for 4 h. The yellow color solid was filtered off from the reaction mixture, washed with cold ethanol and diethyl ether, and then dried at room temperature. The yellow block crystals of SVS1 suitable for single crystal XRD were obtained by slow evaporation of the compound in chloroform and dimethylformamide mixture (8:2).

Yield: 87%. M. pt.: 180–184 °C. Yellow color solid. Anal. Calc. for C₁₆H₁₁NO₂S (%): C, 68.31; H, 3.94; N, 4.98; S, 11.40. Found: C, 68.45; H, 4.12; N, 4.82; S, 11.29. UV–Vis (DMSO): λ_max, nm (ε, dm³ mol⁻¹ cm⁻¹) 259 (22,900), 341 (11,000). FT-IR (KBr, cm⁻¹): 3327 (N−H), 1608 (C=O), 840 (C−S). ¹H NMR (500 MHz, CDCl₃): δ, ppm 9.29 (s, 1H, OCH), 8.39 (d, J = 8.0 Hz, 1H, NH), 8.03 (d, J = 8.1 Hz, 1H CHNH), 7.99 (d, J = 8.0 Hz, 1H, aromatic-H), 7.75 (t, J = 6.9, Hz, 1H, aromatic-H), 7.61–7.47 (m, 4H, aromatic-H), 7.43–7.36 (m, 2H, aromatic-H). ¹³C NMR (125 MHz, CDCl₃): δ. ppm 174.8 (C=O), 158.6 (C−O), 156.5 (C−S), 155.9 (C−NH), 151.6, 136.1, 134.3, 126.3, 126.2, 126.2, 124.8, 123.8, 122.4, 121.6 (aromatic carbons).

2.2.2. Synthesis of 5-(1H-indol-3-yl)-4-methyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (SVS2)

(E)-2-((1H-indol-3-yl)methylene)-N-methylhydrazine-1-carbothioamide (232 mg, 1 mmol) was added to methanol (7 mL) and refluxed for 10 min. To this, 1 mmol of anhydrous FeCl₃ (160 mg in 5 mL methanol) was added. The resulting solution was stirred at 60–65 °C for 6 h. Upon cooling the reaction solution in deep freezer, brown needle crystals suitable for XRD were formed.

Yield: 62%. M. pt.: 257–260 °C. Brown color solid. Anal. Calc. for C₁₁H₁₀N₄S (%): C, 57.37; H, 4.38; N, 24.33; S, 13.92. Found: C, 57.22; H, 4.51; N, 24.18; S, 13.76. UV–Vis (DMSO): λ_max, nm (ε, dm³ mol⁻¹ cm⁻¹) 263 (24,300), 329 (12,050). FT-IR (KBr, cm⁻¹): 3423 (N−H), 3219 (N−H), 1540 (C=N), 1215 (C=S). ¹H NMR (500 MHz, DMSO‑d ₆): δ, ppm 13.77 (s, 1H, NH), 11.93 (s, 1H, indole NH), 8.08 (s, 1H, indole CH), 7.99 (d, J = 7.5 Hz, 1H, aromatic-H), 7.52 (d, J = 7.7 Hz, 1H, aromatic-H), 7.24 (t, J = 6.6 Hz, 1H, aromatic-H), 7.17 (t, J = 6.7 Hz, 1H, aromatic-H), 3.72 (s, 1H, CH₃). ¹³C NMR (125 MHz, DMSO‑d ₆): δ. ppm 185.4 (C=S), 166.7 (C=N), 148.5, 136.4, 127.3, 125.5, 123.1, 121.0, 112.5, 101.4 (aromatic carbons), 32.1 (methyl carbon).

2.3. X-ray crystallography

Yellow block and brown block crystals of SVS1 and SVS2 of dimensions 0.56 × 0.54 × 0.44 mm and 0.54 × 0.21 × 0.16 mm, respectively, were chosen for the three-dimensional structural studies. Single crystal X-ray diffraction (XRD) data were collected at 110 K on an APEX K_α diffractometer using graphite monochromated MoK _α (λ = 0.70173 Å) radiation. The structure was solved using XT/XS in APEX2 [37,38] and refined by full matrix least squares on F² using Olex2 [39].

2.4. Quantum calculations, and molecular docking with SARS-CoV-2 M^pro and ACE-2, and molecular dynamics simulation

Materials Studio (DMol³) software was used for optimization, and energy, HOMO (highest-occupied molecular orbital) and LUMO (lowest-unoccupied molecular orbital) calculations [40] of the compounds (SVS1 and SVS2), and remdesivir, chloroquine and hydroxychloroquine drugs (Fig. 1). AutoDock-4.2 [41] was used for molecular docking of the compounds (SVS1, SVS2, remdesivir, chloroquine and hydroxychloroquine) at the active site of the enzymes, SARS-CoV-2 main protease and human ACE-2. For the preparation of ligands, the structures of the compounds were generated, Gasteiger charges were added, and polar hydrogen bonds were merged. These coordinates were saved in pdbqt format that contains the information about partial charges, atoms type and rotatable bonds. The 3D crystal structural coordinates of main protease of SARS-CoV-2 and human ACE-2 enzymes were retrieved from RCSB protein data bank with PDB IDs 6Y2F and 6M0J, respectively, and subjected to protein preparation (heteroatoms and water molecules were removed followed by assigning of polar hydrogen atoms and Kollman charges). The coordinates were saved in pdbqt format, and grid map for the active site was generated using Autogrid program. Docked complexes were analyzed using AutoDock tools. Active site interactions were generated using Ligplot plus [42].

Fig. 1.

Structure of remdesivir, hydroxychloroquine and chloroquine.

Molecular dynamics simulation was performed for the best pose obtained from the docking of 6Y2F and SVS1 using GROMACS version 2018.3 [43]. The protein complex was solvated using simple point-charge (SPC) water model in a cubic solvation box. The physiological pH in the protein structure was balanced by adding four sodium ions to the solvent molecules, bringing the entire system to a neutral charge. By using the steepest descent algorithm, the energy minimization was carried out for a maximum of 50,000 steps. After the model structures were converged to a maximum force, a two-step equilibration via the NVT (where N is amount of substance, V is volume and T is temperature) canonical ensemble (isothermal-isochoric) equilibration was performed for 100 picoseconds (ps) at 300 K with Berendsen thermostat temperature coupling. This was followed by a constant number of particles, pressure and temperature (NPT) ensemble equilibration for another 100 ps. The LINCS (linear constraint solver) algorithm was used to constraint the covalent bonds. To treat the long-range coulombic interactions, the particle mesh Ewald (PME) method [44] was used. Production MD was carried out at a constant temperature of 300 K using the NPT ensemble for 50 ns and the MD trajectories were saved for every 10 ps. MD analysis of trajectories was done using Gromacs in-built utilities like gmx RMS, RMSF, gyrate and cluster. The conformations were visualized and images were generated using Pymol [45].

2.5. In vitro cytotoxicity

HepG-2 (hepatic carcinoma), T24 (bladder), EA.hy926 (endothelial) and Vero (kidney epithelial normal cells extracted from an African green monkey) cells were used for testing cytotoxicity of the compounds (SVS1 and SVS2) and most commonly used anticancer agents (cisplatin and doxorubicin) using MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetraazolium bromide] assay. HepG-2 and EA.hy926 cancer cells were maintained in DMEM (Dulbecco's modified Eagle's medium), T24 cancer cells in McCoy's 5a medium and Vero normal cells in RPMI-1640 (Roswell Park Memorial Institute) medium. Cells were plated in 96-well plates at a concentration of 1 × 10⁴ cells per well in suitable medium and incubated for 24 h at 37 °C. Then compounds to be tested with the known concentration (0–50 or 100 µM) were seeded into the wells after dissolving them in DMSO. At the end of the incubation (24 h) of plates, the MTT dye solution (10 µL, 5 mg/mL) was added per well in dark at 37 °C for 4 h, and 100 µL of DMSO was added, and the absorbance of compounds was measured at 570 nm. IC₅₀ values were determined as the concentration of corresponding compound that produced a 50% reduction of cell viability [46].

3. Results and discussion

3.1. Synthesis of azole-based heterocyclic compounds

As seen in Scheme 1 , 3-(2,3-dihydrobenzo[d]thiazol-2-yl)-4H-chromen-4-one (SVS1) was synthesized by reacting 2-aminothiophenol with 4-oxo-4H-chromene-3-carbaldehyde in C₂H₅OH under reflux condition for 4 h. 5-(1H-indol-3-yl)-4-methyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (SVS2) was prepared from (E)-2-((1H-indol-3-yl)methylene)-N-methylhydrazine-1-carbothioamide [[34], [35], [36],47] in methanol at 60–65 °C in the presence of anhydrous FeCl₃ at reflux condition for 6 h, as indicated in Scheme 2 . These types of cyclization reactions are rarely reported in the literature, and it might be a key reaction for developing new bioactive heterocyclic drugs in the future. Both the compounds were adequately characterized using UV-Visible, FT-IR and NMR spectroscopic techniques. The three-dimensional structures of SVS1 and SVS2 were confirmed by single crystal XRD and validated by DFT calculations. SVS1 was soluble in most of organic solvents such as CH₂Cl₂, CHCl₃, DMF, DMSO, etc., whereas SVS2 was soluble in DMF and DMSO solvents.

Scheme 1.

Synthetic route for chromone-based thiazole compound (SVS1).

Scheme 2.

Synthetic route for indole-based triazole compound (SVS2).

3.2. FT-IR and UV-Visible spectroscopy

In the FT-IR spectrum of SVS1, two bands observed at 3327 and 1608 cm⁻¹ were assigned to ʋN−H and ʋC=O, respectively (Fig. S1). In addition, a band appeared at 840 cm⁻¹ due to C−S stretching. The spectrum of SVS2 showed indole and triazole N−H stretching frequencies at 3423 and 3219 cm⁻¹, respectively. Also, two bands at 1215 and 1540 cm⁻¹ characteristic of ʋ(C=S) and ʋ(C=N) stretching frequencies, respectively, were detected [24]. The compounds showed two bands in their UV-Visible spectra corresponding to π→π* and n→π* electronic transitions in the ranges of 259–263 and 329–341 nm, respectively [22,23].

3.3. ¹H and ¹³C NMR spectroscopy

The NMR (¹H and ¹³C) spectra of SVS1 and SVS2 were recorded in CDCl₃/DMSO‑d ₆ solvent, and spectra are shown in Figs. S2 and S3. In the ¹H NMR spectrum of SVS1, the signals of OCH, CHNH and NH were observed at 9.29, 8.03 and 8.0 ppm, respectively. In addition, a doublet and triplet signals were seen in the region 7.99–7.47 ppm, corresponding to the aromatic protons. In the ¹H NMR spectrum of SVS2, the signals due to the NH protons (from triazole and indole) and indole CH proton appeared at 13.77 (triazole) / 11.93 (indole) and 8.08 ppm, respectively. The signals of phenyl ring protons of SVS2 were displayed at 7.99–7.17 ppm. A signal at 3.72 ppm is related to the protons of methyl group [48].

The ¹³C NMR spectra of SVS1 and SVS2 are shown in Figs. S4 and S5, respectively. The C=O carbon resonated at 174.8 ppm in the spectrum of SVS1. Moreover, the C−S, C−O and C−NH carbons resonated at 156.5, 158.6 and 155.9 ppm, respectively. In the ¹³C NMR spectrum of SVS2, a signal was observed at 185.4 ppm for carbon of C=S, while a peak related to carbon of C=N was observed at 166.7 ppm. In addition, signal of methyl carbon was observed at 32.1 ppm. The aromatic carbon signals of both the compounds were observed in their corresponding region.

3.4. Description of crystal structures

Three-dimensional crystal structures of SVS1 and SVS2 were refined to good R factor of 3.6 and 2.4%, respectively. Thermal ellipsoidal plots of SVS1 and SVS2 are given in Figs. 2 and 3, respectively . All the bond lengths and angles were in the allowed ranges (Table 1, Table 2, Table 3 ). In SVS1, O2 is planar with the ring (C1−C2−C3−C4−C5−O1) as evident from the torsion angle O2−C3−C2−C1 [−179.8(1)°]. The five membered ring (C10−N1−C11−C12−S1) adopted twisted conformation with twist on S1−C10, and the puckering parameters are Q(2) = 0.208(1) Å and φ(2) = 205.0(4)°. The molecular structure was stabilized by C−H⋅⋅⋅N intramolecular interaction which generates S(5) pattern (Fig. 4 , Table 4 ). Crystal packing was stabilized by N−H1⋅⋅⋅O2 (x, -y + 3/2, z + 1/2) intermolecular hydrogen bond, generating chain C(6) (Fig. 4). As compound SVS2 crystallized in non-centrosymmetric space group P2₁2₁2₁, the absolute structure was relevant and the Flack parameter, x [0.046(16)], was determined using 929 quotients [(I+) − (I−)] / [(I+) + (I−)]. The crystal structure of SVS2 was nearly planar. Torsion angles C11−N4−C10−N3 [176.6(2)°] and S1−C10−N3−N2 [−179.1(1)°] indicated that C11 and S1 were almost in-plane with a five membered ring to which they were attached. The molecular structure of SVS2 was stabilized by C−H⋅⋅⋅S intramolecular interaction, generating S(5) pattern (Fig. 4b). The crystal packing was stabilized by N−H⋅⋅⋅S and N−H⋅⋅⋅N intermolecular hydrogen bonds. Atoms N1 and N3 in the molecule at x, y, z act as donors to atoms S1 and N2 at -x + 1, y-1/2, -z + 1/2 and x-1/2, -y + 3/2, -z + 1, generating C(8) and C(3) chains, respectively (Fig. 4b).

Fig. 2.

Molecular structure of SVS1 drawn at 30% probability level. Intra-molecular interaction is represented as dashed line.

Fig. 3.

Molecular structure of SVS2 drawn at 30% probability level. Intra-molecular interaction is represented as dashed line.

Table 1.

X-ray diffraction data collection, structure determination and refinement details for SVS1 and SVS2.

	SVS1	SVS2
CCDC number	2053105	2053106
Chemical formula	C16H11NO2S	C11H10N4S
Radiation type, Wavelength	MoKα, 0.71073
Mr	281.32	230.29
Crystal system, Space group	Monoclinic, P21/c	Orthorhombic, P212121
Temperature (K)	110
Unit cell dimensions (Å, °)	a = 7.2434(15), b = 14.400(3), c = 12.084(3), β = 92.683(3)	a = 4.9842(7), b = 12.2295(17), c = 17.006(2)
V (Å3)	1259.0(5)	1036.6(3)
Z, Dx (Mg m−3)	4, 1.484	4, 1.476
Absorption coefficient (μ) (mm−1)	0.26	0.287
F(000)	584	480
Color, Crystal size (mm)	Yellow, 0.56 × 0.54 × 0.44	Brown, 0.54 × 0.21 × 0.16
θmin, θmax (°)	2.2, 27.4	2.1, 27.4
No. of measured, independent and observed [I > 2σ(I)] reflections	22,959, 2875, 2580	22,333, 2357, 2303
Index ranges	h = −9→9	h = −6→6
	k = −18→18	k = −15→15
	l = −15→15	l = −22→21
Absorption correction	Multi-scan
Tmin, Tmax	0.649, 0.746	0.690, 0.746
Refinement method	Full matrix least-squares on F2
No. of reflections, restraints, parameters	2875, 0, 182	2357, 0, 146
Rint	0.040	0.024
sin θ/λmax (Å−1)	0.648	0.647
H-atom treatment	Mixed	Constrained
R [F2>2σ(F2), wR(F2)]	0.036, 0.093	0.024, 0.063
Final R indices [I>2sigma(I)]	R1 = 0.036, wR2 = 0.090	R1 = 0.024, wR2 = 0.063
R indices (all data)	R1 = 0.041, wR2 = 0.093	R1 = 0.025, wR2 = 0.063
Goodness of fit on F2 (S)	1.08	1.06
Absolute structure parameter	−	0.046(16)
Δρmax, Δρmin (e Å−3)	0.28, −0.34	0.23, −0.14

Table 2.

Selected geometric parameters (Å, °).

Bond length (Å)/angle (°)	SVS1	Bond length (Å)/angle (°)	SVS2
S(1)−C(10)	1.8599(15)	S(1)−C(10)	1.6903(18)
S(1)−C(12)	1.7624(15)	N(1)−C(8)	1.358(2)
O(1)−C(1)	1.3746(18)	N(2)−N(3)	1.375(2)
O(1)−C(5)	1.356(2)	N(3)-C(10)	1.332(2)
O(2)−C(3)	1.2368(18)	N(4)−C(10)	1.371(2)
N(1)−C(10)	1.4495(19)	N(4)−C(9)	1.384(2)
N(1)−C(11)	1.393(2)	N(2)−C(9)	1.319(2)
C(12)−S(1)−C(10)	90.60(7)	N(4)−C(11)	1.458(2)
C(5)−O(1)−C(1)	118.32(12)	C(8)−N(1)−C(1)	109.18(15)
O(1)−C(1)−C(2)	121.90(14)	C(9)−N(2)−N(3)	104.41(13)
O(1)−C(1)−C(6)	116.24(13)	N(3)−C(10)−N(4)	104.31(15)
N(1)−C(10)−S(1)	105.44(10)	C(10)−N(3)−N(2)	113.21(14)
N(1)−C(10)−C(4)	113.86(13)	N(4)−C(10)−S(1)	128.25(13)
C(4)−C(10)−S(1)	108.38(10)	C(9)−N(4)−C(11)	128.03(15)
N(1)−C(11)−C(12)	113.94(13)	C(10)−N(4)−C(9)	108.06(13)
C(16)−C(11)−N(1)	126.48(14)	N(1)−C(1)−C(2)	128.95(17)
C(11)−C(12)−S(1)	111.74(11)	N(1)−C(1)−C(6)	107.94(16)
C(11)−N(1)−C(10)	114.51(12)	N(1)−C(8)−C(7)	110.26(16)
C(13)−C(12)−S(1)	127.03(12)	N(2)−C(9)−N(4)	110.00(14)
		N(3)−C(10)−S(1)	127.45(13)

Table 3.

Selected torsion angles (°).

Torsion angle (°)	SVS1	Torsion angle (°)	SVS2
S(1)−C(12)−C(13)−C(14)	176.01(12)	N(1)−C(1)−C(2)−C(3)	179.81(18)
O(1)−C(1)−C(2)−C(3)	0.2(2)	N(1) −C(1)−C(6)−C(5)	178.60(16)
O(1)−C(1)−C(2)−C(9)	−179.70(12)	N(1)−C(1)−C(6)−C(7)	−1.04(19)
O(1)−C(1)−C(6)−C(7)	179.97(13)	N(2)−N(3)−C(10)−S(1)	−179.07(12)
O(2)−C(3)−C(4)−C(5)	179.21(13)	N(2)−N(3)−C(10)−N(4)	1.16(19)
O(2)−C(3)−C(4)−C(10)	−1.3(2)	N(3)−N(2)−C(9)−N(4)	0.04(18)
N(1)−C(11)−C(12)−S(1)	3.35(17)	N(3)−N(2)−C(9)−C(7)	179.88(14)
N(1)−C(11)−C(12)−C(13)	−179.73(14)	C(1)−N(1)−C(8)−C(7)	−0.3(2)
N(1)−C(11)−C(16)−C(15)	−179.76(15)	C(6)−C(7)−C(8)−N(1)	−0.39(19)
C(1)−C(2)−C(3)−O(2)	−179.75(13)	C(6)−C(7)−C(9)−N(2)	5.2(3)
C(3)−C(4)−C(5)−O(1)	0.9(2)	C(6)−C(7)−C(9)−N(4)	−174.97(16)
C(3)−C(4)−C(10)−S(1)	−64.88(14)	C(8)−N(1)−C(1)−C(2)	−179.26(18)
C(3)-C(4)-C(10)-N(1)	178.14(12)	C(8)−N(1)−C(1)−C(6)	0.8(2)
C(5)−C(4)−C(10)−S(1)	114.55(14)	C(8)−C(7)−C(9)−N(2)	−171.56(17)
C(5)−C(4)−C(10)−N(1)	−2.4(2)	C(8)−C(7)−C(9)−N(4)	8.3(3)
C(9)−C(2)−C(3)−O(2)	0.2(2)	C(9)−N(2)−N(3)−C(10)	−0.77(19)
C(9)−C(2)−C(3)−C(4)	−179.43(13)	C(9)−N(4)−C(10)−S(1)	179.16(13)
C(10)−S(1)−C(12)−C(11)	−12.31(12)	C(9)−N(4)−C(10)−N(3)	−1.07(18)
C(10)−S(1)−C(12)−C(13)	170.99(14)	C(9)−C(7)−C(8)−N(1)	176.88(16)
C(10)−N(1)−C(11)−C(12)	11.4(2)	C(10)−N(4)−C(9)−N(2)	0.66(19)
C(10)−N(1)−C(11)−C(16)	−168.96(15)	C(10)−N(4)−C(9)−C(7)	−179.17(15)
C(10)−C(4)−C(5)−O(1)	−178.50(13)	C(11)−N(4)−C(9)−N(2)	175.91(15)
C(11)−N(1)−C(10)−S(1)	−19.35(16)	C(11)−N(4)−C(9)−C(7)	−3.9(3)
C(11)−N(1)−C(10)−C(4)	99.32(16)	C(11)−N(4)−C(10)−S(1)	3.7(2)
C(12)−S(1)−C(10)−N(1)	17.36(11)	C(11)−N(4)−C(10)−N(3)	−176.58(15)
C(12)−S(1)−C(10)−C(4)	−104.91(11)	C(1)−C(2)−C(3)−C(4)	1.5(3)
C(16)−C(11)−C(12)−S(1)	−176.36(11)	C(1)−C(6)−C(7)−C(8)	0.88(18)

Fig. 4.

Packing diagram showing inter-molecular hydrogen bonding interactions in (a) SVS1 and (b) SVS2.

Table 4.

Intra- and inter-molecular hydrogen bonding interactions.

Donor−Hydrogen⋅⋅⋅Acceptor	D−H (Å)	H⋅⋅⋅A (Å)	D⋅⋅⋅A (Å)	D−H⋅⋅⋅A (°)
SVS1
C5−H5⋅⋅⋅N1	0.95	2.42	2.818(2)	105
N1−H1⋅⋅⋅O2 (x, -y+3/2, z+1/2)	0.87	2.16	2.980(2)	156
SVS2
C11−H11B⋅⋅⋅S1	0.98	2.74	3.230(2)	111
N1−H1⋅⋅⋅S1 (-x+1, y-1/2, -z+1/2)	0.88	2.44	3.309(2)	169
N3−H3⋅⋅⋅N2 (x-1/2, -y+3/2, -z+1)	0.88	2.03	2.893(2)	167

3.5. Quantum chemical calculations

We optimized the compounds to obtain energies, HOMO and LUMO in Materials Studio software. SVS1, SVS2 and the drugs (chloroquine, hydroxychloroquine and remdesivir) were optimized and are shown in Fig. 5 . DMol³ module, based on the first principles of electronic structure calculations, in the Materials Studio was used to calculate the electronic properties; basis set properties, core treatment, effective core potential and molecular orbitals – HOMO and LUMO. The electron charge distributions of the HOMO and LUMO are shown in Fig. 6 . The HOMO and LUMO energies of SVS1 were found to be −0.2112 and −0.1080 eV, respectively. The band gap energy (ΔE = E_LUMO – E_HOMO) of SVS1 was calculated to be 0.1032 eV. The HOMOs were principally localized on N of thiazole and benzene ring linked to thiazole ring as well as oxygen atoms of chromone. The LUMOs were largely localized on O, C and H atoms of benzopyran-4-one and benzene linked to benzopyran-4-one as well as, S and N of thiazole ring and carbons of benzene linked to thiazole. The total energy, kinetic energy, electrostatic energy and binding energy for SVS1 were estimated to be –1211.6113, −12.9679, 2.5631, and −6.4527 kcal/mol, respectively. Meanwhile, the HOMO and LUMO energies of SVS2 were found to be –0.1916 and –0.0735 eV, respectively. The calculated energy gap between HOMO and LUMO of SVS2 was found to be 0.1181 eV (Table 5 ). The HOMOs were largely localized on the N and S atoms of triazole as well as N and ethylene (C=C) of pyrrole, while the LUMOs were localized on the N atoms of triazole and benzene ring, as well as N and C–C of pyrrole. The total energy, kinetic energy, electrostatic energy and binding energy of SVS2 were evaluated to be –1036.7616, −10.7901, 1.9682, and −5.2075 kcal/mol, respectively. The HOMO energies of remdesivir, hydroxychloroquine and chloroquine were estimated to be −0.1865, −0.1973 and −0.2047 eV, respectively. Meanwhile, the LUMO energies of remdesivir, hydroxychloroquine and chloroquine were found to be −0.1055, −0.0798 and −0.0884 eV, respectively. The band gap energies of remdesivir, hydroxychloroquine and chloroquine were seen as 0.0810, 0.1175 and 0.1163 eV, respectively. The HOMO of remdesivir was largely localized on the cyanide, N and C atoms of pyrrole, while the LUMOs were localized on cyanide, N and C atoms of pyrrole and triazine as well as primary amine linked to triazine. The total energy, kinetic energy, electrostatic energy and binding energy of remdesivir were found to be −2321.2748, −31.2326, 6.7183, and −14.5585 kcal/mol, respectively. HOMO of hydroxychloroquine can be related to the aromatic rings and secondary amine while the LUMO can be assigned to the N linked to 7-chloroquinoline and N of quinoline. The total energy, kinetic energy, electrostatic energy and binding energy of chloroquine were found to be −1316.7732, −16.4052, 1.9865, and −8.6195 kcal/mol, respectively. HOMOs and LUMOs of chloroquine were localized on 7-chloroquinoline moiety and N linked to 7-chloroquinoline.

Fig. 5.

Optimized structures of SVS1, SVS2, remdesivir, hydroxychloroquine and chloroquine (DMol³ module in Materials Studio 2017 was used).

Fig. 6.

Perspective views of the obtained HOMO and LUMO of SVS1, SVS2, remdesivir, hydroxychloroquine and chloroquine (DMol³ module in Materials Studio 2017 was used).

Table 5.

Important thermodynamic parameters obtained by Materials Studio simulation.

	SVS1	SVS2
Global and chemical reactivity descriptor	(eV)	(eV)
Energy of HOMO	–0.211	–0.1916
Energy of LUMO	–0.108	–0.0735
Energy gap [ΔE = LUMO – HOMO]	0.103	0.1181
Ionization energy [I = –EHOMO]	0.211	0.1916
Electron affinity [A = –ELUMO]	0.108	0.0735
Chemical hardness (η) [(I–A)/2]	0.051	0.0590
Chemical potential (µ) [1/2 (EHOMO+ ELUMO)]	–0.159	–0.1325
Softness (σ) [1/η]	19.379	16.9491
Electrophilicity index (ω) [μ2/2η]	5.172	5.0102

3.6. Quantum chemical parameters

The module DMol³ in Materials Studio 2017 was used to calculate quantum chemical parameters such as chemical potential (µ), global hardness (η), global softness (σ), absolute electronegativity (χ) and electrophilicity index (ω) for the studied compounds [49].

3.6.1. Global hardness (η) and electronegativity (χ)

To describe the chemical and biological reactivity ranking of SVS1 and SVS2, various electronic parameters such as ionization potential (I), electron affinity (A), absolute electronegativity (χ) and chemical hardness (η) were calculated. I and A were calculated as the negative of energy values of HOMO and LUMO, respectively (Table 5).

The chemical reactivity of molecules increased with the increasing of I. Systems having high E_HOMO such as DNA are good electron donors, and drugs or synthesized compounds having low E_LUMO are good electron acceptors [50]. So, with more negative E_LUMO, chemical reactivity of molecules increased as electron acceptor. The electron affinity value (A) of SVS2 (0.0735 eV) was lower than that of SVS1 (0.1080 eV). So, SVS2 may be able to interact with the targeted biomolecules (DNA or protein) better than SVS1 due to more negative E_LUMO [6]. The band gap is an important parameter to determine some of the molecular properties such as global hardness (η), electronegativity (χ), chemical potential (µ) and global electrophilicity index (ω) (Table 4). If energy gap is decreased, it leads to less stability, high kinetic energy, high chemical reactivity and more polarization [51]. The hardness (η) of the compounds was calculated using Eq. (1) and values are given in Table 5 [40].

$$\eta =\left(\frac{I-A}{2}\right)$$ (1)

I and A are the ionization potential and electron affinity of the molecules, respectively. The calculated energy gap values of SVS1 and SVS2 were 0.1032 and 0.1181 eV, respectively. The energy gap of SVS2 was more than that of SVS1, indicating that the chemical reactivity of SVS2 was less compared to SVS1. To explain the chemical reactivity, the hardness and softness of compounds are important parameters. With the increasing of softness, chemical reactivity is increased. Meanwhile, increase of hardness leads to decrease of chemical reactivity. The hardness of SVS1 (0.0516 eV) was lower than that of SVS2 (0.0590 eV). As a result, the chemical reactivity of SVS1 was more. In addition, SVS2 had high stability and polarization [52].

3.6.2. Chemical potential (µ) and electrophilicity power (ω)

Chemical potential (µ) can be associated with the electron withdrawing capability [51]. The values of chemical potential (µ) of SVS1 and SVS2 were −1.5664 and −1.8860 eV, respectively. As a result, SVS2 had more chemical potential (µ) and electronegativity (χ) than SVS1 [3,51]. In addition, chemical potential (µ) is associated to the adsorptive capacity. An elevated value of chemical potential showed enhanced adsorption between molecules and biological systems as well as the adsorbent surfaces. Therefore, the high value of chemical potential can cause electron transfer from a molecule to biological systems. As a result, electrons of SVS2 can be transferred easily to the biological systems.

The electrophilicity index (ω) can be calculated using the chemical potential and chemical hardness (Eq. (2)) [52,53].

$$\omega =\frac{{\left(\eta \right)}^2}{2\eta }$$ (2)

The electron accepting ability of a molecule from the surrounding without changing its stability is referred to electrophilicity index (ω) [51]. In addition, the toxicity of compounds such as antimicrobial, anticancer, etc., can be estimated through electrophilicity index (ω) [54]. A compound with a low electrophilicity index is anticipated to have less toxicity [55]. The electrophilicity index (ω) values for SVS1 and SVS2 were 23.7752 and 30.1440 eV, respectively (Table 5). The higher value of electrophilicity index (ω) for SVS2 can be correlated with its less toxicity. So, low concentration of SVS2 is enough to kill cancer cells [29,30].

3.7. Molecular docking with SARS-CoV-2 M^pro and ACE-2, and molecular dynamics simulation

Coronaviruses have enveloped structures, their RNA is positive, and have very long single stranded RNA. SARS-CoV-2 has structural and non-structural proteins. One of the most important structural proteins is spike glycoprotein (S). For the entry of SARS-CoV into the host cell, spike glycoprotein (S) binds with the host receptor and angiotensin-converting enzyme-2 (ACE-2) [31,32]. In addition, papain-like protease (PL^pro) and 3-chymotrypsin-like protease (3CL^pro or M^pro) are non-structural proteins that are essential for the replication of the virus. SARS-CoV-2 main protease is made up of three domains. The active site of protein lies at the interface of domain I and domain II and contains a characteristic Cys-His dyad. A linker joins domain II to domain III, which is critical for the dimerization of protein.

It must be emphasized that the action of a viral spike protein is one of the most important pathophysiological mechanisms that facilitates the virus to enter into the host with the aid of ACE-2 receptor. Hepacidin, regulator of iron metabolism, interacts with ferroportin to facilitate the entry of iron into the cells. In SARS-CoV-2 infection, iron metabolism dysfunction occurs in extreme cases leading to ferroptosis. Iron metabolism dysfunction plays a major role in multi-organ failure [56]. As a result, anemic hypoxia creates lung disease. The above mentioned facts have shown ways to create effective protocols for the treatment of COVID-19 disease including (a) a drug that obstructs the life cycle of the virus by blocking ACE-2 receptors or inhibiting SARS-CoV-2 main protease (M^pro) [57], (b) an iron chelating agent to conjugate with the excess iron resulting from its dysmetabolism, and (c) anti-inflammatory drugs as phosphodiesterase-4 inhibitors for the treatment of lung inflammation and necrosis [32,34].

In our study, the synthesized heterocyclic compounds (SVS1 and SVS2) were screened against the SARS-CoV-2 main protease (M^pro) enzyme and human ACE-2 receptor through molecular docking. The structures of our compounds are similar to some commercially available drugs containing thiazole, 1,3,4-oxadiazole and heterocyclic moieties (Fig. 7 ). Amongst the coronavirus targets, the main protease (M^pro) has received a major attention [58,59], and ACE-2 plays a critical role for SARS-CoV to enter into target cells. So, ACE-2 is also one of the key targets for therapeutic development [36,37] of antiviral drugs against SARS-CoV-2. The crystal structure of SARS-CoV-2 spike receptor-binding domain bound with ACE-2 (PDB ID: 6M0J) contains two chains, where chain A represents the angiotensin-converting enzyme-2 and chain E is the spike receptor binding domain. Only chain A of 6M0J (ACE-2) was used for the present docking study. The docking results showed that both the compounds can effectively bind with the molecular targets SARS-CoV-2 main protease and human ACE-2. The docking energies of SVS1, SVS2, remdesivir, chloroquine and hydroxychloroquine with SARS-CoV-2 M^pro and human ACE-2 are shown in Table 6 . Fig. 8, Fig. 9 show the interactions of the compounds at the active site of the enzymes, SARS-CoV-2 main protease and human ACE-2, respectively. The docking results showed that both SVS1 and SVS2 had maintained comparable interactions at the active site of the enzymes when compared to remdesivir (SARS-CoV-2), chloroquine and hydroxychloroquine.

Fig. 7.

Commercially available drugs containing thiazole-based heterocyclic moiety and structures of heterocyclic compounds (SVS1 and SVS2) used in this work.

Table 6.

Docking energies (kcal/mol) of the compounds bound at the active site of the enzymes SARS-CoV-2 main protease and human ACE-2 receptor.

Compound	Docking energy (kcal/mol)
	SARS-CoV-2 main protease	Human ACE-2 receptor
SVS1	−7.79	−8.48
SVS2	−7.38	−7.28
Remdesivir	−7.16	−7.26
Chloroquine	−6.47	−8.23
Hydroxychloroquine	−6.68	−9.07

Fig. 8.

Ligplot image showing the hydrogen bonds (green dashed lines) and hydrophobic interactions (red arcs) of the compounds (SVS1, SVS2, remdesivir, hydroxychloroquine and chloroquine) at the active site of SARS-CoV-2 main protease enzyme (6Y2F).

Fig. 9.

Ligplot image showing the hydrogen bonds (green dashed lines) and hydrophobic interactions (red arcs) of the compounds (SVS1, SVS2, remdesivir, hydroxychloroquine and chloroquine) at the active site of human ACE-2 enzyme (6M0J).

50 ns molecular dynamics simulation (MDS) of the SARS-CoV-2 main protease and SVS1 complex was performed to study the stability and conformational changes. The stability of the complex was initially assessed by plotting the root mean square deviation (RMSD). From the RMSD graph (Fig. 10 ), it was observed that the RMSD values were approximately between 0.25 and 0.37 nm. The deviation in the RMSD was calculated to be ∼0.12 nm. Less RMSD deviation confers that SVS1 remained within the active site pocket of the enzyme throughout the simulation but maintained the dynamic nature within the active site. RMSF (root-mean-square fluctuation) analysis was done to study the average per-residue fluctuations during MDS. From the RMSF graph (Fig. 10), it was observed that the RMSF varied between 0.1 and 0.35 nm with the highest fluctuation of 0.5 nm for the N-terminal atoms. High fluctuation in RMSF is usually observed for the terminal atoms. The radius of gyration was plotted based on the Cα atoms of the protein, it was noted that the values ranged between 2.15 and 2.25 nm.

Fig. 10.

a) RMSD, b) RMSF and c) Radius of gyration graphs of the SARS-CoV-2 main protease (6Y2F) with SVS1 obtained from 50 ns molecular dynamics simulation.

The gmx cluster command was used to form clusters based on the RMSD over the entire 50 ns time frame, and a representative of the largest cluster was taken as the average complex structure. It was observed the SVS1 is found to interact in the active site pocket of the enzyme both in the initial pose and also in the average complex structure. This indicates that SVS1 has not deviated much from the active site pocket and has been quite stable in the active site throughout the 50 ns MDS. Fig. 11 (a) shows the cavity of binding site with SVS1 from the initial pose in red and from the average representative structure of the MD run in blue. On superimposing the initial and average representative structures, an RMSD of 1.8 Å was calculated and the image can be seen in Fig. 11(b). These results highlight the stability of SVS1 at the active site of the enzyme throughout the simulation, and it maintained the dynamic interactions at the active site of the SARS-CoV-2 main protease.

Fig. 11.

a) Binding pocket showing SVS1 in red from the initial pose and blue from the average representative structure from MDS and b) Superimposed image of the initial (red) and average representative (blue) structures of 6Y2F-SVS1 complex (RMSD = 1.8 Å).

3.8. Cytotoxic ability of the compounds

Indole derivatives induce apoptosis, and inhibit proliferation, expansion and invasion of human cancer cells via various targets such as histone deacetylases (HDACs), sirtuins, PIM kinases, DNA topoisomerases and σ receptors [[59], [60], [61]]. Chromone compounds such as PD98059 and myricetin act as highly specific MEK1/2 inhibitors in hepatocarcinoma (HCC), prostate carcinoma, non-small cell lung cancer (NSCLC), leukemia and melanoma [62]. Various small molecules of triazoles can specifically arrest tumor cells [16,63]. The cell viability (%) obtained after 24 h for the present compounds against HepG-2, T24 and EA.hy926 cancer cells showed growth-inhibition in a dose-dependent manner (Fig. 12 a and 12b). The cell viability decreased with the increasing concentrations of SVS1, SVS2, cisplatin and doxorubicin. The IC₅₀ values of compounds are listed in Table 7 . The IC₅₀ values of SVS1, SVS2, cisplatin and doxorubicin were >50, 33.8, 49.9 and 8.6 µM against HepG-2 cell line, >50, 29.2, 26.6 and 3.8 µM against EA.hy926 cell line, and 44.8 (SVS1), 46.7 (SVS2) and >50 µM (cisplatin) against T24 cell line, respectively. Cell proliferation assay results showed that SVS2 possessed good cytotoxicity against HepG-2 cancer cells. The ability of SVS1, SVS2, cisplatin and doxorubicin to kill the HepG-2 cells vary as doxorubicin > SVS2 > cisplatin > SVS1 at 24 h, and interestingly, SVS2 was significantly more cytotoxic (ANOVA, p < 0.05) against HepG-2 cells than cisplatin and showed less activity than doxorubicin. On comparing the IC₅₀ values of SVS1, SVS2 and cisplatin (Table 7), it was found that SVS1 and SVS2 were more active against T24 cells than cisplatin. Further, the cytotoxicity of SVS1 was more than that of SVS2 against T24 cells. In addition, both SVS2 and cisplatin were highly toxic against EA.hy926 cancer cells. To explore the selectivity, the cytotoxicity of SVS1, SVS2 and cisplatin was investigated in Vero normal cells. The IC₅₀ values of SVS1, SVS2, cisplatin and doxorubicin against Vero normal cells were >100, >100, 29.5 and 8.9 µM, respectively (Table 7). It appears as SVS1 and SVS2 were selective to cancer cell lines, and hence these can be tested as chemotherapeutic drugs.

Fig. 12.

a) Cytotoxicity of SVS1, SVS2 and cisplatin against HepG-2, T24 and EA.hy926 cancer cells after 24 h incubation time. Each value represents the mean ± SD for n = 3. p < 0.05. b) Cytotoxicity of SVS1, SVS2 and cisplatin against Vero normal cells after 24 h incubation time. Each value represents the mean ± SD for n = 3. p < 0.05.

Table 7.

Half maximal inhibitory concentration (IC₅₀) values of the compounds in HepG-2-hepatic carcinoma, T24-bladder, EA.hy926-endothelial cancer and Vero-kidney epithelial normal cells extracted from an African green monkey cell lines as calculated by MTT assay after 24 h incubation at 37 °C, 5% CO₂.

Compound	IC50 (µM)
	HepG-2	T24	EA.hy926	Vero
SVS1	>50	44.8	>50	>100
SVS2	33.8	46.7	29.2	>100
Cisplatin	49.9	>50	26.6	29.5
Doxorubicin	8.6	NT	3.8	8.7

NT = Not tested.

4. Conclusions

SVS1 was prepared from 2-aminothiophenol and 4-oxo-4H-chromene-3-carbaldehyde. SVS2 was synthesized from (E)−2-((1H-indol-3-yl)methylene)-N-methylhydrazine-1-carbothioamide in the presence of anhydrous FeCl₃. SVS1 and SVS2 were thioazole (with chromone) and triazole (with indole) heterocyclic compounds, respectively. They were characterized by analytical and spectroscopic methods. The solid-state structures were confirmed by single crystal X-ray diffraction technique. Quantum chemical parameters such as electronic chemical potential (µ), global hardness (η), global softness (σ), absolute electronegativity (χ) and electrophilicity index (ω) were assessed using module DMol³ in Materials Studio 2017. The calculated energy gap of SVS1 and SVS2 showed that the chemical reactivity of SVS1 was more than that of SVS2. As a result, the hardness of SVS1 was lower than that of SVS2. In addition, SVS2 had high stability and polarization. The electrons of SVS2 might be able to be transferred to the biological systems rather than those of SVS1 due to the high chemical potential of SVS2. SVS2 possessed a high value of electrophilicity index (ω) compared to SVS1, which can be correlated with its high toxicity. Hence, SVS2 can act as a better cytotoxic compound even at low concentrations. Based on quantum calculations, SVS2 as electron acceptor, was more reactive when compared with SVS1 due to its more negative E_LUMO. Binding energies of the compounds with the target enzymes based on the molecular docking results clearly showed that SVS1 had more binding potential than SVS2. The active site interactions of SVS1 and SVS2 were comparable with remdesivir, chloroquine and hydroxychloroquine. Molecular dynamics simulation of M^pro–SVS1 complex clearly showed the stability of SVS1 to interact only at the active site of the enzyme. At last, the study was complemented by the cytotoxic activity of compounds against three human cancer (HepG-2, T24 and EA.hy926) and one normal (Vero) cells. SVS2 was more active than the other compounds against liver (HepG-2) cancer cells. In addition, SVS1 and SVS2 were more active against T24-bladder cancer cells than cisplatin. Furthermore, it was found that SVS2 and cisplatin had a higher cytotoxic activity than SVS1 against EAhy926 cancer cells. The IC₅₀ values of SVS1, SVS2 and cisplatin against Vero normal cells showed that SVS1 and SVS2 were selectively toxic against the cancer cells. The mode of cell death and in vitro cytotoxicity will be analyzed to further establish its therapeutic potential as anticancer/antiviral agent.

Appendix A. Supplementary data

All the spectra and tables are provided. Crystallographic data for the structures reported in this paper have been deposited with the Cambridge Crystallographic Data Center (CCDC) as supplementary publication numbers (CCDC 2053105 and 2053106 for SVS1 and SVS2, respectively). Copies of the data can be obtained free of charge from the CCDC (12 Union Road, Cambridge CB2 1EZ, UK; Tel.: + 44–1223–336408; Fax: + 44–1223–336003; e-mail: deposit@ccdc.cam.ac.uk; web site: http://www.ccdc.cam.ac.uk).

CRediT authorship contribution statement

Jebiti Haribabu: Writing – original draft, Investigation, Methodology. Vasavi Garisetti: Software, Formal analysis, Writing – review & editing. Rahime Eshaghi Malekshah: Software, Formal analysis, Writing – review & editing. Swaminathan Srividya: Writing – original draft, Investigation, Methodology. Dasararaju Gayathri: Software, Formal analysis, Writing – review & editing. Nattamai Bhuvanesh: Software, Formal analysis, Writing – review & editing. Ramalinga Viswanathan Mangalaraja: Software, Formal analysis, Writing – review & editing. Cesar Echeverria: Writing – original draft, Investigation, Methodology, Supervision. Ramasamy Karvembu: Writing – original draft, Investigation, Methodology, Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.