Design, synthesis, characterizing and DFT calculations of a binary CT complex co-crystal of bioactive moieties in different polar solvents to investigate its pharmacological activity

Abstract Imidazole (IM) and salicylic acid (SA) have a significant class among the medical compound. These are widely used as topical drugs like antifungal, antibacterial, anticancer, immunosuppressive agent, etc. These two bioactive organic moieties are combined by a weak hydrogen bond formed by hydrogen transfer. The charge transfer (CT) complex of acceptor (SA) and donor (IM), has been synthesized at room temperature in methanol and confirmed by signal-crystal XRD, conductance and UV–visible spectroscopy. The X-ray crystallography provides the original structural information of CT complex and displays the existence of N+—H––O– bond between IM and SA. The physical properties such as (ECT ), (RN ), (ID ), (f), (D) and ( G0) along with molar extinction coefficient (εCT ) and formation constant (KCT ) were estimated through UV–visible spectroscopy. Job’s method and Benesi-Hildebrand equation suggested 1:1 stoichiometry of ([IM]+[SA]−). The results indicate a complete transfer of hydrogen atom and CT complex formation with 1:1 molar ratio of IM and SA. Antimicrobial activity was veiled against different bacteria like Escherichia coli, Bacillus subtilis and Staphylococcus aureus; and different fungi as Fusarium oxysporum, Candida albicans and Aspergillus niger by disc diffusion method. CT complex was also tested for cytotoxic activity against lung cancer cell lines in comparison to breast cancer cell lines. Molecular docking provides the information of binding of [(IM)+(SA)−] with the cancer marker (1M17), which has substantial application for drug designing. The investigational studies were supplemented through time-dependent density functional theory (TD-DFT) using basis set B3LYP/6-311G**. Through DFT calculations, HOMO→LUMO electronic energy gap ( was obtained. A new organic charge transfer complex co-crystal has been prepared, characterized and explored for, Antioxident, Anticancer, antibacterial and antifungal activities. The experimental results are supported by theoretical analysis. Communicated by Ramaswamy H. Sarma


Introduction
Nitrogen having heterocyclic compounds is of unusual interest as electron donors since they can purpose as n-and p-donors, while carboxylic acid having benzene compound is of interest as electron acceptor since they can purpose as acceptor.By the interaction of this type of donor and acceptor, the charge transfer (CT) complex was formed.CT complexes are electron donor-acceptor moieties that are designed over an intermolecular shift of electronic charge.The creation of CT complex by organic species is conveyed through transfer of hydrogen among acceptor and donor moieties (Al-Ahmary et al., 2018;Adam et al., 2013;Adam, 2014;Fakhroo et al., 2010;Issa et al., 2011;Refat et al., 2017;Khan, Alam, et al., 2020;Khan, Islam, et al., 2020;Khan, Shakya, Akhtar, et al., 2020;Khan, Shakya, Islam, et al., 2020;Khan & Ahmad, 2010).CT complexation procedures weak hydrogen bonds (Mulliken, 1950;Mulliken & Pearson, 1969) among acceptor and donor.The structures of CT complexes among p-and r-acceptors with n-and p-donors were observed (Gutmann et al., 1997;Khan, Alam, et al., 2020;Khan, Islam, et al., 2020;Khan, Shakya, Akhtar, et al., 2020;Khan, Shakya, Islam, et al., 2020;Mazumder & Lahiri, 2004).The CT complexation occurs in a chemical equilibrium between free acceptor (A) and donor (D) molecules: In this case, it is categorized as a resonance among dative state Dþ … A À and non-bonded state D & A. The formation of the region state is an electronic transformation that gives rise to color-absorbing bands.CT complex has a wide range of advantages in human life due to its acquisition of various physicochemical and biological properties beyond a single molecule.CT complexes have many applications in drugs like insecticide, anti-HIV, anti-inflammatory, anticancer (Selvam et al., 2015;Tunc et al., 2015), and show potent antibacterial and antifungal properties (Khan, Alam, et al., 2020;Khan, Islam, et al., 2020;Khan, Shakya, Akhtar, et al., 2020;Khan, Shakya, Islam, et al., 2020).Moreover, CT complex has wide applications in the area of organic semiconductors (Eychm€ uller & Rogach, 2000), optical activity (Sathya et al., 2018), photo catalysts (Khan et al., 2019), chemosensors (Shakya & Khan, 2021;Shakya et al., 2020;Khan & Shakya, 2019;Zhang et al., 2017Zhang et al., , 2018) ) and micro-emulsion (Khan et al., 2017).The idea of the formation of CT complex via hydrogen bond was first proposed by Mulliken and further studies by Foster (Mulliken, 1952;Foster, 1969).
Imidazole (IM) is a significant heterocyclic molecule that is powerfully hydrogen-bonded in the solid state.It is a very exciting class of compounds because of its extensive applications in several biological areas (Narasimhan et al., 2011).Also, they are used medically as anti-pyretic, anti-rheumatoid agents, as well as herbicides and fungicides (El-Shabouri et al., 1998).Salicylic acid (SA) is a strong acceptor organic moieties that are broadly used in plant growth regulator, organic synthesis, food products preservative, anti-fungal, antiseptic agents (Suresh et al., 2014;Wen et al., 2008) and skincare products for the treatment of acne, corns, psoriasis, keratosis pilaris, calluses and warts (Karabacak et al., 2010;Karabacak & Kurt, 2009).Such a wide range of importance of IM and SA makes us keen to synthesize their CT complex.
In this study, we synthesized antimicrobial drugs in the bio-crystalline charge transfer complex between IM and SA.Many CT complexes having IM as donor were studied with 3, 5-dinitrobenzoic acid (Alam & Khan, 2018), p-acceptors (DDQ, TCNE and CHL) (Mostafa & Bazzi, 2011), TTF derivatives (Murata et al., 2010) and 2,4-dinitro-1-naphthol (Miyan et al., 2017), but no article was found with SA as an acceptor to our knowledge.Before exploring its pharmaceutical application, single crystal XRD, conductance and UV-visible spectroscopy to confirm the formation of CT complex (Figure S1, ESI).SC-XRD helps to examine the structural data and hydrogen bonding (N þ � � �H À O -) among IM and SA.The conductivity increases as the density of the donor increases, and the conductivity of the CT complex between different solvents also increases with the increase in the polarity of the solvent.UV-visible spectroscopy with Job's method and Benesi-Hildebrand equation was used to study and calculate stoichiometry and different physical parameters (molar extinction coefficient, formation constant, etc.) of CT complex.Density functional theory (DFT) calculations and studies provide theoretical information about CT complex.One of the goals of this article is to investigate the interaction of CT complex with cancer protein marker through molecular docking and calculation of its free binding energy.Antibacterial and antifungal activities were investigated and compared with standard drugs, Nystatin and Gentamicin, respectively.

Material
Donor (IM) and acceptor (SA) were obtained from Sigma-Aldrich (St. Louis, MO).These organic moieties are of high purity and are used without further purification.Solvents like methanol, dimethylformamide (DMF), ethanol, carbon tetrachloride and dimethyl sulfoxide (DMSO) were of Merck analytical grand.

Solubility and preparation of standard solution
IM is rightly soluble in DMSO, methanol, DMF, acetone, ethanol and acetonitrile.SA is also soluble in the same mentioned solvents.The 1 M standard solution of IM and 1 M standard solution of SA were obtained in 25 ml of methanol.

Synthesis and element analysis
CT complex of IM and SA was synthesized through mixing of 25 ml standard solution of IM (1.7019 g. 1 mmol) and SA (3.4530 g. 1 mmol) in methanol.The mixture of IM and SA was stirred for 45-55 min at room temperature to find good solid CT complex.The mixture was filtered using filter paper (Whatmann-41 grade) for the removal of impurities and washed with a little methanol, and dried below vacuum.

Single crystal growth
Synthesis of crystals was done by the dissolution of solid CT Complex in acetonitrile by gentle evaporation process.The solution was then filtered via filter paper (Whatmann).Filtrate was left undisturbed in a clean environment.A noble array of white glowing transparent crystals was grown in a time span of 40-45 d, shown in Figure S1 (d), ESI.

Single crystal X-Ray diffraction
A single crystal of the obtained CT complex was prepared in a glass-capillary for collecting data through graphite-monochromated Mo-Ka radiation (k ¼ 0.71073 Å) at 293 K on a Bruker SMART APEX CCD diffractometer.For integration and data reduction, the SAINT Software was used.SHELXL-97 package was used to resolve structures, which was refined to F2 by the full-matrix least-squares method (Singh et al., 2017).

Conductivity measurement
To extent the conductivity of the CT complex, 3 ml donor and 3 ml acceptor solutions were mixed.The subsequent mixture was kept overnight at room temperature to achieve a stable CT complex.In the response mixture, the concentration of the donor was kept larger than the acceptor, [Do] � [Ao] (Hasani & Rezaei, 2006;Ghosh et al., 2006).The concentration of acceptor (SA) was kept fixed at 0.01 M (Hasani & Rezaei, 2006), while the concentration of donor ranged from 0.1 M to 0.4 M. The conductance will then be measured for each set of mixture solutions (Khan & Ahmad, 2009).

Electronic spectra
The electronic absorption spectra of the donor (IM), acceptor (SA) and the resulting CT complex in ethanol and acetonitrile were recorded in the UV region (200-500 nm) using a spectrophotometer PERKINELMER UV Lambda45-visible spectrophotometer.IM and SA were scanned individually through a spectrophotometric titration (Bhattacharya et al., 2004) to their wavelength of maximum absorption at room temperature.To obtain a stable CT complex the resulting mixture of donor and acceptor left standing over night at room temperature.The wavelength of maximum absorption of the resulting solution was determined.Pure solvent was used as a reference solution.

Antioxidant
Radical Scavenging Activity of '1, 1-Diphenyl-2-picrylhydrazyl' (DPPH) -DPPH assay is a quick screening procedure for radical scavenging activity of precisecomplexes (Al-Amiery et al., 2012).The free radical scavenging effects of CT complex with DPPH radical were evaluated with different concentrations (50,100,200,300,400 and 500 lg/ml) of the CT complex was added to 1.0 ml of 0.4 mM in Me-OH solution of DPPH and were stirred strongly.After an incubation period of 45 min at room temperature, the scavenging ability determines the antiradical strength of an antioxidant by reducing the absorption of DPPH at 517 nm.Ascorbic acid (Vit-C) was used as a typical drug.The negative control contained DPPH and methanol but no CT complex.As a result of the color change, the absorption in the formation of a stable DPPH molecule is reduced by the addition of hydrogen when DPPH is bounded by the antioxidant.All test samples were achieved with triplicates to obtain mean ± SD.The presence of free radical product inhibition (I %) from DPPH was calculated by using the following equation.
Here, acontrol is the absorbance of the control, and a sample is the absorbance of the sample.

Anticancer activity
The cytotoxicity of the CT complex was evaluated against human breast carcinoma (MCF-7) and human lung carcinoma (A549) by following standard MTT assay protocols (Shi et al., 2014).With a high rate of occurrence, these cancer cell lines represent some of the most common types of cancer.
The cytotoxicity assay, in brief, A549 and MCF-7 (total cell count of 5 � 104 cells/ml) were seeded into flat bottomed 96-well plates and allowed to attach overnight.After incubating overnight, media containing 5 ml of different concentrations of the CT complex were added in triplicate well (resulting concentration 0, 25,30,40,55,70,85,100,115 and 130 mg/ml).The medium was removed after treatment, and 200 ml phenol red-free medium with MTT (1 mg/ml), was added to wells and incubated for 4 h.Further, 100 ml of DMSO was used to replace the culture medium, and the absorbance of each well was measured at 570 nm.The amount of formazan produced is directly proportional to the number of living cells in each of the wells.

Antibacterial analysis
Antibacterial activity of the CT complex of IM and SA was explored in-vitro against Escherichia coli, Bacillus subtilis and Staphylococcus aureus using agar well plate diffusion method (Cruickshank et al., 1995).Through developing a single colony in Mueller-Hinton (MH) broth at overnight bacterial inocula was obtained.100mL of bacterial test pathogen strains were blow-out on MHagarplates and various concentrations of newly synthesized CT complex of IM and SA (10, 20, 40 and 80 mg/ml) were introduced on the agar plate.These plates were lastly incubated at 35 � C for 20 h, and zone of inhibition was measured.Gentamicin on a concentration of 80 mg/ml served as a control.All the described experiments were conducted in triplicate and under sterilized conditions.

Antifungal analysis
The newly synthesized CT complex of IM and SA also verified on behalf of this one antifungal property against Candida albicans, Fusarium oxysporumin and Aspergillus niger in DMSO by typical agar disc diffusion method.Now, the CT complex was prepared at different concentrations as 10, 20, 40 and 80 mg/ml.The antifungal activity of the complex was associated with the slandered antifungal drug Nystatin(80 mg/ml).Antifungal activity was determined by evaluating the inhibition (mm) region.All the described experiments were performed in triplicate under the sterilized condition.

Molecular docking studies
Software HEX 8.0 (Ritche et al., 2010) was used to achieve docking poses of cancer protein marker with CT complex (Figure S2, ESI).The center x ¼ À 26.224, y ¼ 12.599, z ¼ 52.965 and exhaustiveness ¼ 8 was used.Protein data bank (PDB) file of the CT complex was obtained through mercury software, as shown in Figure S3 (a), ESI.The conjugate gradient optimization algorithm and MMFF94 force field were used to minimize the energy of the CT complex structure through PyRx-Python prescription 0.8 at 500 steps.The cancer protein marker having PDB ID-1M17 was downloaded from the online PDB shown in Figure S3 (b), ESI.The native ligand and other heteroatoms, including water attached to the protein were removed using Discovery Studio Visualizer version 19.1.0.18287 (developed and distributed by Dassault Systemes BIOVIA) to prepare the protein for docking.Polar hydrogen atoms were added and Kollman charges of the receptors have also been determined using Autodock Tool.Discovery studio software was used to obtain the data and visualization.Overall docking experiment was run on processor (Intel(R) Core(TM) i5-4200U CPU @ 1.60 GHz 2.10 GHz 2.30 GHz, 64 bit).

Single crystal
The obtained empirical formula for the crystal structure is 'C 10 H 10 N 2 O 3 ' which forms the N þ À H-O À bond through the weak hydrogen bonding and p-p interaction among IM and SA.It was confirmed that an IM binds to a molecule of SA that forms the donor-acceptor complex [(IM) þ (SA) À ], which displays 1:1 ratio of the CT complex.This outlines the process of proton transfer mechanism, as shown in Scheme 1.The obtained crystal with CCDC number 1,984,786 was observed to be monoclinic with P21/c space group and the crystallographic data along with bond angle and bond lengths were found to be comparable with some slight differences with the crystal obtained by Martins et al. (2019) and Zie R ba et al. ( 2019) which we have used for further significant studies.
Interaction between the reactants occurs among the COOH group of SA and the N atom of IM, which contains lone pair of electrons when the proton of OH group of SA remains isolated in the lattice, which is shown in Figure 1(a).
The hydrogen bond between IM and SA was found to be 2.562 Å.The ORTEP estimation of [(IM) þ (SA) À ] crystal is assumed in Figure 1(b) (Farrugia, 1997).The crystallographic data are provided in Table 1.The selected bond length, bond angle, hydrogen atom coordinates, isotropic displacement parameters, fractional atomic coordinates and anisotropic displacement parameters for crystal are summarized in Tables S1-S5.

Conductivity study
The measured conductivity of CT complexes in different solvents is shown in Table 2.This can be described by the possible formation of complex among the reaction follows in the solutions (Hasani & Shamsipur, 2005).It was reported that the conductivity increases as the concentration of the donor increases, and also, the conductivity of the CT complex in different solvents increases with increasing the Scheme 1. Reaction mechanism for the formation of CT complex.polarity of the solvent.This increase in conductivity may be due to fact that the derivative structure should be stable at D þ -A À in less polar solvent (Mohamed, 2008).Thus, it shows that the complex structure of low polar solvents is extra frequent.

Electronic spectra
The electronic spectra of the 1 � 10 À 4 M donor (IM), 1 � 10 À 4 M acceptor (SA), and the resulting CT complex in ethanol and acetonitrile were observed at room temperature in the ultra violet region 200-500 nm.The new absorption peaks were observed in the ultra violet region due to n !p* transition.The maximum absorbance was analyzed at 276 and 304 nm in ethanol and acetonitrile, respectively.The maximum absorbance of IM and SA in ethanol was obtained at 305 and 310 nm, respectively, and in acetonitrile it was observed at 297 and 330 nm, respectively.Change in color was observed as the IM (donor) and SA (acceptor) solutions were mixed, and new absorption band appeared in regions where neither donor (IM) nor acceptor (SA) had any absorption.This is a clear indication of the formation of CT complex of SA and IM.Furthermore, the new wave length absorption bands are imputed to the formation of IM radical anion resulting from complete charge transfer from SA to IM (Scheme 1).The transfer of lone pair of electrons from donor to acceptor is indicated by the existence of CT band.This interaction occurred through H-bonding between donor and acceptor moieties.The CT absorption bands exhibited by the spectra of the systems mentioned as above are depicted in Figure 2. When in fixed concentration of acceptor, the various concentration of donor was added the absorption intensity was changed to higher side, which is reported in Table 3.With the help of fitting to the Gaussian function, the CT absorption spectra are analyzed, where x and y indicate wavelength and absorbance, respectively.The results of the Gaussian analysis for all systems under study are shown in Table 4.The wavelength at these new absorption maximum (k CT ¼ XC) are summarized in Table 3 and the absorbance peaks in the spectra are depicted in Figure 2. According to the Benesi-Hildebrand (Hindawey et al., 1980;Issa et al., 1981) equation, the charge transfer complexes formation were measured and plotted as a function of the ratio of the concentration of the donor: acceptor ([IM]:[SA]).To calculate the formation constant (K CT ) and molar extinction coefficient (e CT ) of the charge transfer complex, the straight-line method was applied by using Benesi-Hildebrand (El-Sayed & Agrawl, 1982) equation.With the addition of the donor (IM) to a constant concentration solution of the acceptor (SA), change in absorbance takes place, which follows the Benesi-Hildebrand (El-Sayed & Agrawl, 1982) equation in the following form.
From the slope and the intercept, K CT and e CT of the complex were calculated, which are summarized in Table 3.It was observed that with the increasing dielectric constant of the solvents, the value of formation constant decreases significantly because of a strong CT-complex, the dative structure D þ -A À should be stabilized in a less polar solvent.Ionization potential (IP) (I D ), energy of interaction (E CT ), resonance energy (R N ), free energy (DG � ), oscillator strength (f) and dipole moment (l EN ) for the synthesized CT complex were also determined using following equations: e CT ¼ 1243:667 kCT nm (Briegleb & Czekalla, 1960a, 1960b), e CT ¼ ð7:7X10 À 4 Þ=½hm CT =jR N j À 3:5� (Briegleb & Czekalla, 1960a, 1960b), DG � ¼ À 2:303 RT log K CT (Martin et al., 1969), f ¼ 4:32 � 10 À 9 e CT Dv 1=2 (Lever, 1985), The calculated values are reported in Table 5.

Job's plot for determination of stoichiometry
The stoichiometric ratio of the CT complex was determined by applying Job's method in two solvents (ethanol and acetonitrile).Keeping the mole fraction of solution constant, different solutions of acceptor to donor ratio were prepared.Further, the plot of absorbance v/s mole ratio (Figure 4) gives information that stoichiometry is 1:1 (D: A) with the maximum at 0.5 mole fraction in all the cases.It was also shown from the crystallographic data that the ratio of CT complex was 1:1, i.e. one molecule of donor interacts with one molecule of acceptor by COOH groups of SA forming N þ À H-O -bond.

Antioxidant
The absorbance of stable radical DPPH� for different concentrations of newly synthesized CT complex was measured at 517 nm.Antioxidant activity results of the tested compound are shown in Table 6.Complex in low concentration showed moderate antioxidant activity, which was comparable to that of the standard ascorbic acid displayed in Figure 5(a).The synthesized complexes showed Antioxidant activity in a dose-dependent manner.
Since the antioxidant effects of IM could be mainly due both to free-radical scavenging and chelating activities, in this study, the direct superoxide anion scavenging capacity of these compounds was investigated using a method which excludes the Fenton-type reaction and xanthine/xanthine oxidase system (Russo et al., 2000).On the other side, SA protects against oxidative stress by decreasing MDA and reactive oxygen species (ROS) production and acting directly as an antioxidant to scavenge ROS and/or indirectly modulating redox balance through the activation of the antioxidant responses (Moustafa-Farag et al., 2020).Antioxidants react with free radicals (reactive oxygen species) by different mechanisms like, hydrogen atom transfer (HAT) or single electron transfer mechanism (SET); or the mixture of both HAT and SET mechanisms.The HAT reaction is a rigorous movement of a proton and an electron in a single kinetic step.In HAT mechanisms, the free radical eliminates one hydrogen atom of antioxidant, and the antioxidant itself becomes a radical.In this mechanism, the bond dissociation enthalpy (BDE) is an important parameter in assessing the Table 6.Antioxidant activity results of the tested compound.
Concentration (mg/ml) AA CT complex 50 45.4 ± 0.9 17.5 ± 0.2 100 56.9 ± 0.5 31.7 ± 0.9 200 62.4 ± 0.5 38.2 ± 0.8 300 70.1 ± 0.9 41.1 ± 0.6 400 76.2 ± 0.6 42.1 ± 0.5 500 76.1 ± 0.9 44.9 ± 0.4 antioxidant action (Mader et al., 2007).The lower the BDE of the H-donating group in the potential antioxidant, the easier it will be for the reaction of free radical inactivation.The SET reaction is copied by single-electron transfer from the nucleophile to the substrate, producing a radical intermediate, whose fate can then be involved in any number of events.In SET mechanisms, the antioxidant provides an electron to the free radical and itself then becomes a radical cation.In this mechanism, the IP of the antioxidant is the most important energetic factor in evaluating the antioxidant action.The lower the IP, the easier is the electron abstraction (Ashby, 1988).It is very difficult to distinguish between HAT and SET reactions.In most situations, these two reactions take place simultaneously, and the mechanism of the reaction is determined by the antioxidant's structure and solubility, the partition coefficient and solvent polarity.The DPPH assay is based on both electron transfer (SET) and HAT reactions.Our results revealed that the CT complex had a similar free radical scavenging activity.Phenolic compounds scavenge the DPPH radicals by their hydrogen donating ability (Benzie & Strain, 1999;Singleton & Rossi, 1965).The results obtained in this study suggest that the CT complex showed radical scavenging activity by their electron transfer or hydrogen donating ability.However, a smaller difference was observed for antioxidant potentials of CT complex when compare with IM and SA.Nevertheless, these results confirm that synthesized complex is less effective scavenging agents than the parent compounds.We can include the possibility of the deprotonation of the OH group in solvent and sequential proton loss-electron transfer (Musialik & Litwinienko, 2005), as SA have carboxyl groups being stronger acids than the phenolic hydroxyl group.Thus, any deprotonation of the OH group is shifted back.Supposedly, the observed difference in reactivity of the CT complex might result from a different strength of H-bond.At 500 lg/ml concentration CT complex produced is antioxidant activity similar to donor and acceptor (Table 7 and  Figure

Anticancer activity
In vitro cytotoxicity's of CT complex of IM and SA were experienced by MTT colorimetric analyze against MCF-7 human breast cancer cell and A549 human lung cancer cell.The control containing no complex has 100% cell viability.With an increase in the concentration of the CT complex showed a reduction of cell viability.In the 40 mg/ml concentration, the cell viability was reduced at 50% in the case of A549 cell line shown in Figure 6(a).On the other hand, the MCF-7 cell line reduced 50% in the concentration of 70 mg/ml of CT complex shown in Figure 6(b).After 100 mg/ml concentration, the decline in cell viability virtually stopped in lung cancer cells when it took 115 mg/ml of CT complex in the case of breast cancer cells.Overall, it is clear that the CT complex has good cytotoxic activity against lung cancer cell lines in comparison to breast cancer cell lines.
At 50 lg/ml concentration CT complex showed better results compared to IM in MCF7 cell line (Table 8 and Figure 7).
Table 7. Antioxidant activity results of the donor (IM), acceptor (SA) and CT complex.

Antibacterial studies
Inhibitory activity of newly designed IM and SA, CT complex was observed against E. coli, B. subtilis and S. aureus bacterial strains by dose-dependent manner.The complex at 80mg/ml (D4) concentration showed inhibition more than gentamicin (80 mg/ml) against the B. subtilis bacterial strain.The highest zone inhibition was observed in D4 (80 mg/ml) against bacterial strain of B. subtilis followed by E. coli>S.aureus, which are shown in Table 9 and observed activity against the bacteria strains growth, as shown in Figure 8(a).The antibacterial activity of the complex may be due to the disruption of cell membrane resulting in increases in ROS generation, which leads to oxidative stress (Khan, Alam, et al., 2020;Khan, Islam, et al., 2020;Khan, Shakya, Akhtar, et al., 2020;Khan, Shakya, Islam, et al., 2020).
Puupponen-Pimi€ a et al. reported that different bacterial species (Gram-positive and Gram-negative) display different sensitivities towards phenolic compound (Puupponen-Pimi€ a et al., 2001).Our newly synthesized CT complex has a single H-donating site (H-bond), which might be favorable for the passage of this molecule through the bacteria membrane.Moreover, previous studies found that IM inhibits bacterial DNA synthesis (Helmick et al., 2005) and SA would cause damage to the plasma membrane and lead to protein leakage, while SA can give rise to a change in the transmembrane pH gradient between the plasma membrane and membranes of organelles, causing cellular energy loss and leading to the death (Fang et al., 2020).A similar effect could occur in the bacterial cell membrane, affecting their energy metabolism.
In the case of B. subtilis, CT complex performed better antibacterial activity compared to donor and acceptor.Represented in Table 10 and Figure 9(a).

Antifungal studies
In the fungi case, the inhibition in growth was observed C. albicans, Fusarium oxysporum, and A. niger, when treated with different concentrations as 10, 20, 40, and 80 mg/mL of newly synthesized IM and SA complex as shown in Figure 8(b).The visible inhibition of growth was found to be at a low concentration of 10 mg/ml.The inhibition was followed in the dose-dependent manner, with the increase in the concentration of newly synthesized IM and SA concentration, the zone of inhibition was also increased.And at the higher dose, the complex significantly inhibited the fungal strain C. albicans, F. oxysporum, and A. niger compared to the control.The CT complex displayed potent antifungal effects, probably through the obliteration of membrane integrity (Khan, Alam, et al., 2020;Khan, Islam, et al., 2020;Khan, Shakya, Akhtar, et al., 2020;Khan, Shakya, Islam, et al., 2020).Obtained results showed that the CT complex has outstanding antifungal properties in comparison to that of standard drug (Table 11).
One of the anticipated mechanisms for antifungal agents is their binding to membrane ergosterol (Kobayashi et al., 1995) which leads to fungal membrane disruption and loss of intracellular content.Two mechanisms of antifungal action involving ergosterol are known, so the compounds may: (i)  bind to membrane ergosterol creating pores in this structure (Campoy & Adrio, 2017) or (ii) inhibit enzymes involved in the synthesis of ergosterol, thereby reducing the content of that macromoleculei.eergosterol (Ahmad et al., 2015).Thus, antifungal action of CT complex occurs might be these reasons.Moreover, the previous research indicated that the principal molecular target of IM is cytochrome P450-Erg11p or Cyp51p, which catalyses the oxidative removal of the 14amethyl group of lanosterol and/or eburicol in fungi by a typical P450 mono-oxygenase activity.The active site of this protein has an iron protoporphyrin moiety, and IM bind to the iron atom.Moreover, the exact conformation of the active site differs between fungal species and amongst the many mammalian P450 mono-oxygenases.The precise nature of the interaction between IM molecule and each kind of P450 therefore determines the extent of the inhibitory effect in different fungal species (Odds et al., 2003).On the other hand, SA can cause cell membrane damage.The cell membrane is essential to the cell viability of fungal strains, where damage to the lipid bilayer may result in cellular collapse, leading to the leakage of intracellular compounds (Da Rocha Neto et al., 2015).Antifungal activity was carried out between donor, acceptor and charge transfer complex.In the case of C. albicans and Fusarium oxysporum, our synthesized CT complex showed better activity (Table 12 and Figure 9(b)).

Molecular docking observations
The molecular docking study predicts the orientation of one molecule to a second when interacted together.Molecular docking is the technique that has been used for drug designing.This study has played a great role in exploring CT complex-protein interaction in rational drug design.The most acceptable docked poses of ([IM] þ [SA] À ) with cancer protein marker with PDB id 1M17 is shown in Figure 10(a).Where the CT complex is used as a ligand and1M17 is used as a receptor.Synthesized complex fits perfectly in 1M17 groove and provides free energy of binding (FEB) value to be À 103.96 kcal mol À 1 , shown in Figure S3, ESI.Higher value of binding energy shows the stronger interaction between protein and synthesized CT complex.It was well explained that a compound having lower binding energy is preferred to be a possible drug nominee (Alam et al., 2021;Khan, Islam, et al., 2021;Khan, Shakya, et al., 2021).The approximate binding distance of globulin protein and CT-complex found to be Tyr 84 ¼ 2.3 Å, Asn 82 ¼ 1.9 Å, Phe 67 ¼ 2.6 Å and Thr 60 ¼ 2.8 Å (Figure 10(b)).The possible binding site between protein and CT complex supports the observed experimental results.Figure 11 shows the hydrogen bonding surface, aromatic surface and hydrophobic surface of interacting protein and CT complex (Akram et al., 2020;Shakya et al., 2019).

DFT/TD-DFT studies
The stabilization energy obtained from MM2 calculations for IM (donor) ¼ À 225.91 a.u., SA (acceptor) ¼ À 495.72a.u and for [(IM) þ (SA) À ] is À 721.78 a.u.confirming more stability of the synthesized CT complex as compared to reactant moieties.The strength of electrostatic potentials of the reactant and synthesized moieties can be presented by molecular electrostatic potential map (MEP) is shown in Figure 12.The electronegative region is shown with red color regions while electropositive with blue regions.MEP gives evidence of the experimental interaction between IM and SA theoretically, as the electron-rich region lies at COOH of SA and electron-deficient at N of SA.The optimized structure of the synthesized [(IM) þ (SA) À ] complex showing Mulliken atom numbering is presented in Figure 13, and Mulliken charges are given in Table 13.From the obtained Mulliken charges, it was observed that the negative charge on 1 O atom considerably increased to 0.6257e (CT complex) from 0.5769e (free SA) is because of the dissociation of 1 O-17H bond in [(IM) þ (SA) À ].Additionally, the þ ve charge on 17H significantly decreased to 0.3052e from 0.3869 (free SA), which is due to proton transferred to 16 N atom whose -ve atomic charge is increased to 0.5130e form À 0.3734 of reactant SA.Huge obtainability of charge on 1 O ¼ À 0.6257e proposes the probability of N þ -H-O À interaction between the reactants, which can also be seen in the SC-XRD results.Mulliken charges of [(IM) þ (SA) À ] are in agreement with MEP surface.From DFT calculations, FTIR spectrum of the synthesized complex [(IM) þ (SA) À ] was originated to be in noble agreement with the experimental FTIR spectrum, shown in Figure S4, ESI.The nature of the electronic transitions observed has been studied by TD-DFT method in the gas phase as shown in Figure S5.Two electronic absorption bands are  obtained from TD-DFT at 287 and 466 nm.The computed UV-vis absorption bands at 287 and 466 nm are assigned to HOMO-1 !LUMO and HOMO !LUMO.Similarly, the electronic absorption band for IM and SA at 310 and 340 nm, respectively, was simulated.The obtained HOMO (À 4.9176 eV) !LUMO (À 2.2593 eV) electronic energy gap (DEÞ is 2.6583 eV and HOMO-1 (6.5793 eV)!LUMO (À 2.2593 eV), DE ¼ 4:32 eV: The pictorial presentation of frontiers molecular orbital (FMO) is given in Figure 14.The electronic band gap of the synthesized material [(IM) þ (SA) -] is observed to be reduced as compared to the material moieties.This is due to the H-bond interaction between the reactant acceptor (SA) and donor (IM) moieties (Bhattacharya, 2007;Shukla et al., 2012).

Conclusion
The synthesized CT complex interaction between IM and SA was studied in methanol as solvent.Synthesized CT Complex was confirmed through Single crystal XRD, and theoretical studies.It ensures that the CT complex is thermally more stable as compared to the IM and SA.Single crystal X-ray crystallography displayed 1:1 (IM: SA) complexation through 'N þ -H--O -' weak intermolecular hydrogen bonding among IM and SA.The nature of electronic transitions in CT complex was explained through computations, HOMO to LUMO and LUMO þ 1 to HOMO-1 energy gap were calculated to be 2.6583 and 4.9857 eV, respectively.The CT complex shows amazing pharmacology as antioxidant, anticancer, antibacterial, and antifungal activity.Molecular docking supports the interaction of CT complex and cancer protein marker (1M17), showing the hydrogen bonding and hydrogen bonding surface.The biological activities of CT complex were more than the free reactants.It can be supposed that the synthesized CT complex can be further explored and will be helpful for drug achievement approaches.

Figure 1 .
Figure 1.(a) Crystal packing of the synthesized CT complex showing hydrogen bonding and (b) ORTEP view of the CT crystal.
[D O ] and [A O ] are the initial concentration of the donor (IM) and acceptor (SA) respectively, A is the absorbance of the donor-acceptor mixture at kCT; eCT is the molar extinction coefficient, and KCT is the formation constant.The Benesi-Hildebrand equation is valid under the condition [D O ] � [A O ] (Abu-Eittah & Al-Sugeir

Figure 8 .Figure 9 .
Figure 8.(a) Graphical representation of antibacterial response in various concentrations and (b) Graphical representation of antifungal response in various concentrations.

Figure 10 .
Figure 10.(a) Docking poses of CT complex ([IM] þ [SA] À ) with cancer protein marker with PDB id 1M17 and (b) docking pose showing interactions between CT complex and protein.

Figure 11 .
Figure 11.Representation of (A) hydrophobic surface (B) interpolated charge surface (C) hydrogen bonding surface and (D) aromatic surface between CT complex and protein.

Figure 13 .
Figure 13.Optimized structure of synthesized CT complex showing Mulliken atomic numbers.

Table 1 .
Crystal data and structure refinement for charge transfer complex.

Table 2 .
Conductivity of charge transfer complex in different polar solvents at room temperature.

Table 8 .
Anticancer activity results of the donor (IM), acceptor (SA) and CT complex.

Table 9 .
Antibacterial activity of CT complex at various concentrations.

Table 11 .
Antifungal activity of CT complex at various concentrations.

Table 12 .
Antifungal activity results of the donor (IM), acceptor (SA) and CT complex.

Table 13 .
Mulliken charges of the synthesized complex.