INTRODUCTION

Coumarin is a naturally fragrant and bioactive molecule with a wide range of biological significance, including antibacterial, antifungal, antioxidant, anti-inflammatory, analgesic, herbicidal, and anticancer activity [1–9]. Due to the diversity of bioactivities, coumarin is considered an important scaffold in medicinal and agricultural chemistry [10, 11]. The coumarin derivatives integrating with pyrazole, pyridine, azetidine, and oxazole rings were also discovered to have intriguing antibacterial and antifungal properties [12]. A number of novel coumarin derivatives in combination with triazole pursue effective anticancer activities. It has been discovered that coumarin compounds are useful for photochemotherapy, anticancer, anti-HIV therapy, and inhibitor action. Additionally, coumarin derivatives are finding new uses in material science as laser dyes, nonlinear optical materials, and organic light-emitting diodes [13, 14].

Another important multifaceted nucleus is benzimidazole in the field of medicinal chemistry due to its various pharmaceutical properties. Benzimidazole template is part of many important natural products, such as histidine, purine and vitamin B1. coumarin is an important pharmacophore with high therapeutic potential as an anticancer, anti-inflammatory, antihypertensive, antiprotozoal, anthelmintic, antiviral and antitubercular medication [15].

The presence of one or more pharmacophores with diverse biological activities such as coumarin and benzimidazole improves efficacy and reduces the toxicity of the synthesized hybrid molecules [16, 17]. Owing to the compelling biological significance of coumarin derivatives in pharmaceutical chemistry and drug discovery leads us to synthesize a novel series of coumarin derivatives.

One pot synthesis is a fast solution for the formation of pharmaceutically important products with high atom economy. Therefore, in a quest for a novel agent, we design a one-pot synthesis of coumarin-benzimidazole derivatives while taking into account the structure-activity relationship at the active side [18, 19], which we design, a multi-component reaction for the formation of coumarin derivatives and further confirmation were carried out by IR, NMR, LC-MS, and elemental analysis. As computational analysis is of current interest in the field of synthetic organic chemistry, it encouraged us to perform DFT studies to analyse the structural properties, global parameters, Non-linear optical (NLO) properties and thermodynamic properties of the resulting derivatives. In addition, in silico molecular docking is performed to investigate receptor docking interactions on target molecules [20–22].

RESULTS AND DISCUSSION

Chemistry

The present study includes the synthesis of 3-{[(1-Hbenzimidazole-2-yl)sulfanyl](heterocarbaldehyde)methyl}-4-hydroxy-2-H-benzopyran-2-onederivatives (IVa–IVf) by the one-pot multicomponent reaction of 4-Hydroxycoumarin (I), heterocyclic aldehyde (IIa–IIf), and 2-mercaptobenzimidazole (III) using L-proline as a catalyst. The synthetic route is shown in Scheme 1 and further structures were characterized by various analytical techniques.

Scheme 1.
scheme 1

Synthesis of thioether-linked coumarin-benzimidazole derivatives (IVa–IVf).

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Computational Study

DFT calculations at B3YLP/6-311+G(d,p) were used to perform geometry optimization on the coumarin derivatives (IVa–IVf), and the resulting structures are shown in the supplementary information (Fig. S1, see Supplementary Information). The optimized structures’ stability (minimal energy) is confirmed by the wave numbers analysis at the same basis set level, and all the compounds displayed C1 symmetry. In comparative studies, theoretical IR and NMR data are compared with experimental data and both are in close agreement.

IR Spectral Analysis

The characteristic stretching and bending vibrations of the target compounds (IVa–IVf) between 4000 and 500 cm–1 have been studied using FT-IR spectroscopy. The theoretical frequencies and intensities of the derivatives are calculated using the vibrational analysis, which is carried out by DFT at the B3YLP/6-311+G(d,p) basis set in the gas phase. The number of atoms in the synthesized compounds (IVa–IVf) is 49, 44, 44, 45, 42, and 42, and their respective non-linear (3N-6) fundamental modes of vibration are 141, 126, 120, and 120. To reduce the overestimation of vibrational modes caused by the neglect of anharmonicity, the vibrational frequencies were scaled using the scaling factor 0.967 of the corresponding basis set, and the molecules (IVa), (IVb), (IVd), (IVe), and (IVf) of the IR spectra are displayed in Figs. S2–S6 of the supplementary information.

The experimental values were compared with the theoretical scaled frequencies(DFT) for the synthesized compounds (IVa–IVf) which are shown in Tables S1–S6 of the supplementary information. In the FT-IR spectrum, Slight deviations might be observed as the computational vibrational analysis (DFT) was conducted in the gas phase whereas FT-IR analysis was in the solid phase [23].

O–H Vibrations

The experimentally determined O–H stretching frequencies for the coumarin derivatives (IVa–IVf) are observed at 3426, 3428, 3430, 3413, 3428, and 3441cm–1, respectively, whereas DFT values were shown at 3653, 3800, 3652, 3652, 3652, and 3633cm–1.

N–H Vibrations

For all synthesized derivatives (IVa–IVf), the N–H stretching bands appeared at 3166, 3080, 3089, 3081, 3077, and 3094 cm–1 in FT-IR and the theoretically predicted peaks were found at 3200, 3233, 3208, 3236, 3237, and 3280 cm–1.

C=O Vibrations

The characteristic carbonyl stretching frequency of all derivatives (IVa–IVf) were observed at 1679, 1657, 1659, 1668, 1672, and 1676 cm–1 respectively and theoretically calculated peaks occurred at 1760, 1665, 1668, 1760, 1760, and 1680 cm–1 respectively.

C=N Vibrations

The experimental obtained C=N stretching bands for coumarin derivatives (IVa–IVf) appeared at 1570, 1563, 1573, 1563, 1577, and 1576 cm–1 and DFT analysis exhibits peaks at 1590, 1601, 1598, 1592, 1592, and 1605 cm–1.

1H NMR and 13C NMR Spectral Data

NMR spectroscopy is a key tool to confirm the structure of organic compounds. In DMSO-d6, the 1H and 13C NMR spectra of synthesized compounds (IVa), (IVb), (IVd), (IVe), and (IVf) were recorded and given in Figs. S7–S11 (supplementary information).

The CH (aliphatic) junction proton reverberated as a singlet at 5.86–6.44 ppm in the 1H NMR spectra of compounds (IVa–IVf), while the aromatic protons produced as a multiplet in the range 6.35–8.08 at 9.18–12.58 ppm, the imidazole ring NH proton was visible as a singlet.

In these derivatives’ 1H NMR spectra, there are no OH signals from protons linked to coumarin, which may be explained by interactions with the solvent DMSO-d6 [24–26] (Table 1).

Table 1. Experimental theoretical 1H NMR values of compound (IVa)
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In 13C NMR spectra, aromatic carbons resonated between 115 and 164 ppm, junction C–S group resonated between 35.75 and 36.38 ppm, and carbonyl (C=O) carbon resonated between 165.46 and 167.22 ppm, as seen in Figs. S12– S14. But woefully, the 13C NMR of compounds (IVc) and (IVd) were not recorded due to their low solubility in DMSO. By using the GaugeIndependent Atomic Orbital (GIAO) approach at B3LYP/6-311+G(d,p), the theoretical 1H NMR spectra were calculated. The experimental chemical shift values, the theoretically estimated NMR values, and the suggested structures agreed quite closely. Experimental and theoretical 1H NMR values of compound (IVa) are provided in Table S1 and for other derivatives (IVb–IVf) given supplementary information.

Mass Spectra

The synthesized compound’s mass spectra were recorded, and the molecular ion peaks of synthesized molecules observed were found at m/z 439.19, 401.17, 401.30, 420.70, 407.10, and 390.48, respectively, correlating to their molecular masses. Mass spectra of (IVa), (IVb), (IVd) and (IVf) are given in supplementary information in Figs. S15–S18.

DFT Studies

The main aim of computational studies includes the geometry optimization, calculation of global parameters, MEP and nonlinear optical properties to study the reactivity of the synthesized compounds. Analysis of thermodynamic properties and correlation graphs predicted to explain, the temperature depending nature of synthesized compounds.

HOMO-LUMO Analysis

The energies of the Frontier orbitals, HOMO and LUMO, and their band gap, were calculated using the DFT method using the B3LYP/6-311+G(d,p) basis set. In addition, global parameters including electronegativity, electron affinity, ionization energy, electrophilicity index, hardness, softness, and chemical potential were determined and summarized in Table 2. We explore the chemical reactivity and stability of molecules as a result of the energy difference between HOMO and LUMO [27, 28]. When the energy gap is small and the molecule is seen to be soft, a molecule is said to be more polar and reactive, and when the gap is high, the molecule is thought to be hard and stable [29].

$${\rm{Electronegativity}}{\rm{,}}\;\chi = {{I + A} \over 2},$$
$${\rm{Global}}\;{\rm{hardness}}{\rm{,}}\;\eta = {{I - A} \over 2},$$
$${\rm{Chemical}}\;{\rm{potential}}{\rm{,}}\;\mu = - {{I + A} \over 2},$$
$${\rm{Global}}\;{\rm{softness}}{\rm{,}}\;S = {1 \over 2}\eta ,$$
$${\rm{Electrophilicity}}\;{\rm{index}}{\rm{,}}\;\omega = {{{\mu ^2}} \over {2\eta }}.$$
Table 2. The global parameters and energies HOMO and LUMO of the synthesized compounds (IVa–IVf)
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According to the HOMO-LUMO diagram in Fig. 1, the electron density in HOMO orbitals is primarily dispersed in the benzimidazole and indole rings, whereas in LUMO, the electron density is primarily distributed in the coumarin ring. Among all compounds, compound (IVe) has the largest energy gap (4.29 eV), whereas compound (IVb) has the smallest energy gap (3.23 eV). The intramolecular electron transfer from the benzimidazole ring (HOMO) to the coumarin ring (LUMO) is accomplished by all compounds (IVa–IVf) exhibiting a smaller band gap in the range 3.23–4.34eV.

Fig. 1.
figure 1

3D representation of FMO orbitals and HOMO-LUMO energy gap of the synthesized compounds (IVa–IVf).

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Molecular Electrostatic Potential Map (MEP)

The charge distribution of a molecule’s electrophilic and nucleophilic sites is represented in three dimensions by electrostatic potential surfaces [30]. MEP provides a visual method for understanding a molecule’s reactivity. The negative ESP is due to nucleophilic sites (electron rich centres) appeared in red shades and the positive ESP is due to the electrophilic sites (electron deficient centres) appear in blue shades. The neutral sites are illustrated by green shades. The potential increases in the order of colors as red<orange<yellow<green<blue. All of the target molecule’s electrostatic potential energy maps were computed by B3YLP/6-311+G(d,p), and the MEP diagram of these compounds is shown in Fig. 2. From the MEP diagram, it can be seen that the NH group of the imidazole ring and the OH group connected to coumarin have a larger positive electrostatic potential, which is indicated by the blue color. While the remainder of the molecular structures has zero electrostatic potential, the higher negative electrostatic potential is seen in red over the carbonyl group in coumarin and green over the heterocyclic ring linked to the hydrogen atoms of all the compounds.

Fig. 2.
figure 2

The molecular electrostatic potential surface of synthesized molecules (IVa–IVf).

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Non-Linear Optical Properties

Due to its numerous applications such as frequency shifting, and optical memory in the fields of telecommunications, signal processing, and optical interconnection technologies, non-linear optical property is significant in current research. NLO study includes the interaction of intense electromagnetic fields with various media to produce different fields with changes in amplitude, phase, and frequency. The synthetic organic compounds with π conjugation were studied for their non-linear properties [31]. The non-linear parameters µα, α and β0 are the components of dipole moment, polarizability, and the first hyperpolarizabilities, respectively calculated in the DFT method at B3YLP/6-311+G(d,p) basis set. These values µ, α, ∆α, and β0 using the x, y, and z components are defined as follows:

$$\mu = {\left( {\mu _{\rm{x}}^2 + \mu _{\rm{y}}^2 + \mu _{\rm{z}}^2} \right)^{1/2}},$$
((1))
$$\alpha = {{{\alpha _{{\rm{xx}}}} + {\alpha _{{\rm{yy}}}} + {\alpha _{{\rm{zz}}}}} \over 3},$$
((2))
$$\Delta \alpha = {2^{ - 1/2}}{\left[ {{{\left( {{\alpha _{{\rm{xx}}}} - {\alpha _{{\rm{yy}}}}} \right)}^2} + {{\left( {{\alpha _{{\rm{yy}}}} - {\alpha _{{\rm{zz}}}}} \right)}^2} + {{\left( {{\alpha _{{\rm{xx}}}} - {\alpha _{{\rm{zz}}}}} \right)}^2} + 6\alpha _{{\rm{xx}}}^2} \right]^{1/2}},$$
((3))
$${\beta _{\rm{0}}} = {\left( {\beta _{\rm{x}}^2 + \beta _{\rm{y}}^2 + \beta _{\rm{z}}^2} \right)^{1/2}},$$
((4))

here,

$${\beta _{\rm{x}}} = {\beta _{{\rm{xxx}}}} + {\beta _{{\rm{xyy}}}} + {\beta _{{\rm{xzz}}}},$$
$${\beta _{\rm{y}}} = {\beta _{{\rm{yyy}}}} + {\beta _{{\rm{xxy}}}} + {\beta _{{\rm{yzz}}}},$$
$${\beta _{\rm{z}}} = {\beta _{{\rm{zzz}}}} + {\beta _{{\rm{xxz}}}} + {\beta _{{\rm{yyz}}}}.$$

In Table 3, the estimated parameters are listed. Urea is considered a prototype molecule (β0 = 0.372 × 10–30) in NLO properties of molecular systems for comparative investigations. The synthetic molecules (IVa), (IVc), and (IVf) have hyperpolarizability (β0) values that are seven times higher than those of urea, indicating a stronger NLO characteristic. Due to their high polarizability, these molecules will therefore be thought of as NLO in the future. The dipole moment is an important parameter to determine molecular stability in the polar environment [32, 33]. Among the synthesized compounds (IVb) has a high dipole moment (9.4681 Debye) due to higher dipole interaction.

Table 3. Dipole moment µ (Debye), polarizability α (esu), mean polarizability ∆α (esu), β components and first-order hyperpolarizability β0 (esu) value of title molecule calculated at B3LYP/6-311G+(d,p)
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Thermodynamic Properties

Thermodynamic characteristics were computed at various temperatures to investigate the temperature dependence of the target compounds. With the aid of MOLTRAN software, standard thermodynamic quantities such as specific heat capacity (CP and CV), internal energy, enthalpy, and entropy were calculated in the temperature range (100–1000 K), and a correlation graph was plotted for compound (IVa) in Fig. 3. The correlation graph of (IVb–IVf) compounds is provided in the supplementary information (Fig. S18). Due to an increase in molecular vibrations, as temperature rises, it has been observed that the values of CP, CV, U, H, and S increase as temperature increases from 100 to 1000 K. Based on the second rule of thermodynamics and the thermochemical field, this thermodynamic data can be used to calculate other thermodynamic energies and identify chemical reaction directions [34, 35].

Fig. 3.
figure 3

Correlation graphs of thermodynamic properties at different temperatures for the synthesized compound (IVa).

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Biological Studies Acetylcholinesterase Inhibitory Assay

In Acetylcholinesterase inhibitory assay, the HeLa cell line is treated with synthesized compounds (IVa–IVf) related to their Acetyl-CoA inhibitory activity at varying concentrations (10 to 50 µg/mL). Table 4 presents the findings with the percentage of (IVa–IVf) compounds that inhibit Acetyl-CoA. Due to the presence of indole moiety, ligand (IVa) demonstrates significant activity with 19.09 µg/mL, ligands, (IVb) and (IVc) have good activity due to the presence of the pyridine moiety, while ligands (IVe) and (IVf) have a moderate degree of activity, while ligand (IVd) molecule has a lower activity of 40.4 µg/mL. Figure 4 depicts the Acetyl-CoA standard graph and activity images of the synthetic compounds.

Table 4. Acetylcholinesterase inhibitory assay of the synthesized compounds (IVa–IVf)
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Fig. 4.
figure 4

The standard graph of acetyl CoA synthesized compounds (IVa–IVf).

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DPPH Free Radical Scavenging Assay

The discovery of natural or synthetic antioxidants is important to prevent the organism from being damaged by free radicals [36]. The DPPH radical scavenging assay was used to test the antioxidant activity of the recently synthesized compounds (IVa–IVf). In Table 5, the findings of an analysis of the stable 2,2-diphenyl1-pecrylhydrazyl (DPPH) radical scavenging activity of target compounds at different concentrations and the standard butylated hydroxytoluene (BHT) are presented. Compound (IVb) has the strongest inhibitions compared to the reference (BHT), with an IC50 value of 20.46 mg/mL. while, compounds (IVc), (IVe), and (IVf) offered better inhibition with IC50 –36.34, 35.84, 37.99, and 39.27 mg/mL respectively and the remaining compounds exhibited lesser inhibition. Figure 5 provides a standard graph for the DPPH radical assay of (IVa), (IVb) chemicals and the reference medication BHT. The scavenging radical inhibition also rises with the sample concentration, which lowers the IC50 value. This suggests that the (IVb) material has a better capacity for scavenging DPPH, which also suggests a higher level of antioxidant activity [37, 38].

Table 5. DPPH radical scavenging assay of the synthesized compounds (IVa–IVf)
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Fig. 5.
figure 5

Standard graph DPPH radical assay of (IVa), (IVb) compounds and standard drug BHT.

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Drug-Likeness Properties

The Molinspiration web tool is used to virtually screen molecules with the highest likelihood in order to explore the pharmacokinetics and drug-like features of the target compounds. Table 6 lists the values for molecular characteristics, including the partition coefficient (logP), molecular weight (MW), number of rotatable bonds, number of hydrogen bond acceptors, and number of hydrogen bond donors. It was noted that the synthetic compounds (IVa), (IVc), and (IVf) adhere to Lipinski’s rule of five with no violations, while the remaining compounds (IVa), (IVd), and (IVe) with one violation [39]. A very good descriptor for understanding drug absorption, including intestinal absorption, bioavailability, and blood-brain barrier penetration, is the topological polar surface area (TPSA) parameter, which is evaluated to analyse drug transport properties of molecules and is found in the range of 79.12–94.12 (140) [40, 41].

Table 6. Drug-likeness of synthesized compounds (IVa–IVf)
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The bioactive characteristics used to assess a molecule’s potential for drug development, such as GPCR, ion channel modulator, kinase inhibitor, nuclear receptor ligands, protease inhibitor, and enzyme inhibitor, were compiled in Table 7. A chemical is thought to be more bioactive if its bioactive score is greater than 0.00, moderately active if it is between –0.50 and 0.00, and inert if it is below –0.50. Table 7 of the results suggests that all target compounds have a moderate level of bioactivity [42].

Table 7. Bioactive score of synthesized compounds (IVa–IVf)
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Molecular Docking Studies

Molecular docking research is critical in the fields of computer-aided drug design and pharmacogenomics. It is a computer-based program that studies the interactions of ligands (synthesized compounds) with specific receptors [43, 44]. All of the synthesized compounds were docked using the acetylcholinesterase enzyme receptor 1W6R, and the docking results were given in Table 8. The table provides information about the number of hydrogen bonds, interacting residues, hydrophobic interactions, and binding affinities. According to the findings, the receptor (1W6R) and the ligand molecules (IVa–IVf) demonstrate a significant number of hydrogen bonds and a greater number of hydrophobic contacts. Molecules (IVa) and (IVc) have a greater binding affinity of –10.1 kcal/mol compared to the other synthesized compounds. The presence of an indole ring in compound (IVa) is a response for enhanced activity and best binding affinity which is supported by the experimental acetyl-CoA inhibition studies in which (IVa) demonstrates significant activity. Compound (IVb) has a moderate binding affinity of –8.3 kcal/mol, and (IVf) has a lower binding affinity of –7.8 kcal/mol. As a result, all of the recently synthesized compounds demonstrate good binding affinity in the range of –7.8 to –10.1 kcal/mol when compared to the standard drug Docetaxel, whose binding affinity is –6.03 kcal/mol. Figures 6 and 7 show the 3D and 2D diagrams of ligand molecules’ interactions with receptor residues respectively. Additionally, the 3D representation of all derivatives’ interactions with proteins and ligands as well as the active site of ligands on receptor surfaces are given in supplementary information (Figs. S19, S20).

Table 8. Findings of docking studies of the designed molecules against 1W6R
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Fig. 6.
figure 6

2D diagrams of molecular interaction (IVa–IVf) with residues of receptors and standard drug Docetaxel.

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Fig. 7.
figure 7

3D diagrams of molecular interaction (IVa–IVf) with residues of protein and standard drug Docetaxel.

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EXPERIMENTAL

Materials and equipment. All the chemicals and solvents are purchased from Sigma Aldrich, Indian Mart and used further without purification. Thin-layer chromatography (TLC) is used for confirmation of reaction completion and melting points were measured in an electrothermal apparatus. IR spectra were recorded on a Jasco 6300 FT-IR spectrometer. 1H and 13C NMR recorded in DMSO-d6 with vnmrs 400 instrument.

Computational methodology. All computational calculations.All Density functional theory (DFT)-based computer calculations were done on Gaussian 09W software. The theoretical properties of the synthesized compounds were studied through optimization using the hybrid Becke 3 Lee-Yang-Parr correlation functional with the basis set B3LYP/6-311+G(d,p). At the same gas phase basis set level, the normal mode frequency analysis was performed. The Gauge-Independent Atomic Orbital (GIAO) approach was chosen to predict NMR spectra at the B3LYP/6-311+G(d,p) basis set in the solvent phase (DMSO) [45–47]. The thermodynamic parameters, global parameters, and nonlinear optical properties were estimated at the same theoretical level.

General procedure for the synthesis of 3-(((benzo[d]imidazole-2yl)thio)(heterocarbaldehyde)methyl)-4hydroxynaphthalen-2(1H)-one derivatives (IVa–IVf). A mixture of substitute heterocyclic aldehydes, 4-hydroxy coumarin, and 2-mercapto benzimidazole was added to a 50 mL round bottom flask along with 20 mL of ethanol to make an equimolar (1 mmol) solution. L-proline (10 mol %) was added to the mixture and refluxed for roughly 8 h at 70°C. TLC plates were used to track the reaction’s progress moving forward. A solid product was precipitated and dried with absolute ethanol and the final product was obtained by recrystallizing a solid product with hot methanol.

3-(((Benzo[d]imidazole-2yl)thio)(1H-indol-3-yl)methyl)-4-hydroxynaphthalen-2(1H)-one (IVa). Brown solid; yield: 78%; mp 138–140°C; FT-IR (KBr) νmax, cm−1: 3426.49 (–OH), 3166.29 (N–H), 2840.48 (Ar–C–H), 1679.57 (C=O), 1570.84 (C=N), 1461.46 (C=C), 760.13 (C–S), 1H NMR (400 MHz, DMSO-d6), ppm: 12.6 (s, 2H, NH), 8.08–8.06 (d, 1H, J = 7.6 Hz, Ar–H), 7.98–7.92 (t, 3H, J = 8 Hz, Ar–H), 7.70–7.68 (d, 1H, J = 7.6 Hz, Ar–H), 7.647.55 (m, 3H, Ar–H), 7.40–7.32 (m, 4H, Ar–H), 6.44 (s, 1H, CH); 13C NMR (100 MHz, DMSO-d6), ppm: 164.44 (C=O), 152.44, 147.89, 143.84, 134.06, 131.94, 129.58, 124.08, 123.66, 121.33, 120.80, 118.41, 116.0, 103.36 (CH), 36.38 (C-S Junction); LC-MS: m/z 439.19 [M+]; C25H17N3O3S; Calculated, %: C, 68.32; H, 3.90; N, 9.56; O, 10.92; S, 7.30; Found, %: C, 68.35; H, 3.8; N, 9.60; O, 10.86; S, 7.32.

3-(((Benzo[d]imidazole-2yl)thio)(pyridine-2-yl)methyl)-4-hydroxynaphthalen-2(1H)-one (IVb). White solid; yield: 76%, mp 122–124°C; FT-IR (KBr) νmax, cm−1: 3428.58 (–OH), 3080.42 (N–H), 2924.95 (Ar–H), 1657.90 (C=O), 1563.20 (C=N), 1445.55 (C=C), 763.19 (C–S); 1H NMR (400 MHz, DMSO-d6), ppm: 9.18 (s, 1H, NH), 7.95–7.93 (t, 2H, J = 6.8 Hz, Ar–H), 7.62–7.60 (d, 2H, J = 8.4 Hz, Ar–H), 7.20–7.40 (m, 7H, Ar–H), 6.37 (s, 1H, CH), 13C NMR (100 MHz, DMSO-d6), ppm: 165.46 (C=O), 164.71, 152.27, 139.29, 131.97, 130.14, 128.74, 127.96, 123.95, 123.77, 117.95, 115.99, 103.92 (CH), 35.71 (C-S Junction); LC-MS: m/z 401.17 [M+]; C22H15N3O3S; Calculated, %: C, 68.32; H, 3.90; N, 9.56; O, 10.92; S, 7.30; Found, %: C, 68.26; H, 3.94; N, 9.55; O, 10.97; S, 7.28.

3-(((Benzo[d]imidazole-2yl)thio)(pyridine-4-yl)methyl)-4-hydroxynaphthalen-2(1H)-one (IVc). White solid; yield: 80%, mp 118–120°C; FT-IR (KBr) νmax, cm−1: 3430.48 (–OH) 3089.27 (N–H), 2934.22 (Ar–H), 1659.49 (C=O), 1573.50 (C=N), 1448.64 (C=C), 768.15 (C–S); 1H NMR (400 MHz, DMSO-d6), ppm: 9.24 (s,1H, NH), 7.94–7.92 (m, 3H, Ar–H), 7.61–7.59 (d, 2H, J = 8 Hz, Ar–H), 7.52–7.25 (m, 7H, Ar–H), 6.41 (s, 1H, CH); 13C NMR (100 MHz), ppm: 165.46 (C=O), 164.71, 152.27, 139.29, 131.97, 130.14, 128.74, 127.96, 123.95, 123.77, 117.95, 115.99, 103.92 (CH), 35.71 (C-S Junction); LC-MS: m/z 401.17 [M+]; C22H15N3O3S; Calculated, %: C, 68.32; H, 3.90; N, 9.56; O, 10.92; S, 7.30; Found, %: C, 68.41; H, 3.85; N, 9.52; O, 10.91; S, 7.31.

3-(((Benzo[d]imidazole-2yl)thio)(3-methylthiophen2-yl)methyl)-4-hydroxynaphthalen-2(1H)-one (IVd). Green solid, yield: 92%, mp 28–130°C; FT-IR (KBr) νmax, cm−1: 3413.31 (–OH), 3081.10 (N–H), 2925.20 (Ar–H), 1668.18 (C=O), 1563.97 (C=N), 1447.63 (C=C), 760.21 (C–S), 1H NMR (400 MHz, DMSO-d6), ppm: 9.86 (s, 1H, NH, 7.90–7.26 (m, 8H, Ar–H), 6.99–6.35 (d, 3H, J = 5.6Hz, Ar–H), 5.60 (s, 1H, CH), 2.11 (m, 3H, CH3); LC-MS: m/z 420.70 [M+]; C22H16N2O3S2; Calculated, %: C, 68.32; H, 3.90; N, 9.56; O, 10.92; S, 7.30; Found, % C, 68.36; H, 3.94; N, 9.50; O, 10.88; S, 7.32.

3-(((Benzo[d]imidazole-2yl)thio)(thiophen-2-yl)methyl)-4-hydroxynaphthalen-2(1H)-one (IVe). Green solid, yield: 86%, mp 132–134°C; FT-IR (KBr) νmax, cm−1: 3428.86 (–OH), 3077.82 (N–H), 2982.22 (Ar–H), 1672.52 (C=O), 1577.31 (C=N), 1475.59 (C=C), 770.80 (C–S); 1H NMR (400 MHz, DMSO-d6), ppm; 10.14 (s, 1H, NH), 7.90–7.32 (m, 7H, Ar–H), 7.29–6.89 (m, 4H, Ar–H), 5.56 (s, 1H, CH); 13C NMR (100 MHz, DMSO-d6), ppm; 166.74 (C=O), 164.82, 152.82, 146.60, 132.30, 126.89, 124.24, 119.01, 116.36, 104.72, 33.30 (C–S Junction); LC-MS: m/z 406.47 [M+]; Calculated, %: C, 68.32; H, 3.90; N, 9.56; O, 10.92; S, 7.30; Found, %: C, 68.38; H, 3.87; N, 9.60; O, 10.88; S, 7.27.

3-(((Benzo[d]imidazole-2yl)thio)(furon-2-yl)methyl)-4-hydroxynaphthalen-2(1H)-one (IVf). Black solid; yield: 89%, mp 118–120°C; FT-IR (KBr) νmax, cm−1: 3441.12 (–OH), 3094.48 (NH), 2980.49 (Ar–CH), 1676.28 (C=O), 1557.56 (C=N), 1471.78 (C=C), 779.88 (C–S); 1H NMR (400 MHz, DMSO-d6), ppm: 12.43 (s, 1H, NH), 7.82–7.22 (m, 7H, Ar–CH), 7.18–6.96 (m, 4H, Ar–CH), 5.56 (s, 1H, CH); 13C NMR (100 MHz, DMSO-d6), ppm: 161.27 (C=O), 131.29, 124.16, 123.70, 123.14, 123.08, 115.65, 115.55, 115.42, 103.72, 34.85 (C–S Junction); LC-MS: m/z 389.20 [M+]; C21H14N2O4S; Calculated, %: C, 68.32; H, 3.90; N, 9.56; O, 10.92; S, 7.30; Found, %: C, 68.29; H, 3.93; N, 9.53; O, 10.92; S, 7.33.

Biological activity. Sample preparation. The synthesized compounds (IVa–IVf) were tested cytotoxicity against HeLa cells associated with their Acetyl CoA inhibitory activity in 10 to 50 µg/mL concentrations. HeLa cells were grown for 24 h at OD 1.2 and treated with the above listed compound further cells were centrifuged and washed with PBS buffer and cells were sonicated and further centrifuged for 10.000 rpm. The supernatant was used for the enzyme assay.

Acetylcholinesterase enzyme assay. The enzyme assay was carried out for ACAS reaction, the following reagents were combined in a 1 mL reaction volume: 50 mM acetyl-coA assay buffer, and acetyl-CoA synthetase along with the containing sample. The control reaction contained all of the components of the reaction, except acetyl-CoA synthetase. Following incubation at 37°C for 20 min, the reaction was terminated and 380 μL of supernatant was transferred to a test tube 50 μL of 2.5% molybdate reagent, 50 μL of 0.5 M 2-mercaptoethanol and 20 μL of Eikonogen was added for a total volume of 0.5 mL. At equilibrating for 10 min, the absorbance at 580 nm was determined using a plate reader (multiplate reader). The blank was prepared by similar treatment of the samples except that the samples were denatured before being added. The amount of PPi attributed to the action of acetyl-CoA synthetase was determined by subtracting the background absorbance at 580 nm measured in the control reaction with the absence of enzyme [48]. The formula used to determine the concentration is given below:

$${\rm{Inhibitory}}\;{\rm{activity}} = {{{A_{\rm{s}}}} \over {{A_{\rm{c}}}}} \times 100,$$

where As = Absorbance sample, Ac = absorbance control.

Antioxidant studies. The antioxidant activity of synthesized compounds was assessed using the modified DPPH 2,2-diphenyl-1-picryl-hydrazyl method. A sample was prepared 25 µL makeup to 600 µL with methanol and 200µL of 0.004% DPPH solution (4 mg DPPH in 100 mL MeOH) was added, further, it was incubated for 30 min in the dark medium. UV-absorbance was recorded at 517 nm in a UV-Vis spectrophotometer, and methanol was used as a blank solution. The experiment was conducted under dark conditions due to DPPH being extremely sensitive. The percentage of antioxidants was calculated by using the formula:

$${\rm{RSA}} = {{{A_{\rm{c}}} - {A_{\rm{s}}}} \over {{A_{\rm{c}}}}} \times 100.$$

Molecular docking studies. In order to understand how to target ligands and interact with appropriate receptors, the pharmacological activity of the generated compounds (IVa–IVf) was examined in silico molecular docking experiments. By using the method in Gaussian 09W software’s energy minimization (optimizations), the stable structure of the produced molecules (ligands) was found, and the structures were then transferred into PDB format. The receptor for acetylcholinesterase, 1W6R, was selected and obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB). The protein was prepared for docking experiments using the Pymol visualization program by eliminating water molecules, organic residues, and chains. Ligand (PDBQT), receptor (PDBQT), and grid box dimensions are created using the AutoDock tool. These pdbqt files were used for docking analysis in the AutoDock Vina program [49, 50] and for visualizing the results in the Discovery studio program and Chimera software [51–53]. The ligands’ binding affinity and hydrophobic interactions with the binding site and protein surface were investigated.

CONCLUSIONS

In conclusion through one pot synthesis, we successfully designed and synthesized a novel series of coumarin-benzimidazole derivatives in the presence of an L-proline catalyst. FT-IR, 1H NMR and 13C NMR, LC-MS, and spectroscopic techniques confirm the structures of synthesized compounds. Computational investigations are performed to obtain optimized structures of synthesized molecules, global parameters and to calculate theoretical IR and NMR for comparative studies with the experimental data. In MEP analysis positive electrostatic potential was observed around the NH-group of the imidazole ring and negative electrostatic potential was observed around the carbonyl group of the coumarin ring. The smaller energy gap ranged from 3.23 to 4.23 eV, supported intermolecular electron transfer in molecules and their electrophilicity index was found in the range of 3.65 to 5.12 eV. The molecule’s NLO powerful nature is demonstrated by the nonlinear optical studies, and hyperpolarizability (β0) values, which are seven times higher than those of urea. Furthermore, to investigate thermodynamic properties such as the compound’s heat capacity, entropy and enthalpy to study the temperature depending nature of substances from correlation graphs. The drug likeness characteristics support the notion the target molecules are important from a pharmacological perspective. The synthesized compounds show promising bioactivity for the acetylcholine esterase enzyme. Among all, compound (IVa) exhibits good inhibition against Hela cancer cell lines which is also supported by the in silico docking studies in which (IVa) compound gives a good binding affinity of –10.1kcl/mol which indicates the best binding interactions compared to the target molecules with the receptor.