Spectroscopic exploration in aggregation with in silico molecular modeling and in vitro assay on antioxidant 7-chloro-2-4-fluorophenyl-3-hydroxychromen-4-one

4.1 Recrystallization process

7-Chloro-2-(4-fluorophenyl)-3-hydroxychromen-4-one was purchased (powder) with a stated purity of greater than 94% from OTAVA Chemicals, Canada. Methanol was used as solvent for recrystallization process in order to reproduce crystal sample. Solution saturates over a period of 26 days and small needle shape grown crystals with crystal size of 0.25 × 0.25 × 0.20 mm were used for further studies.

4.2 X-ray crystallography analysis

X-ray crystal data of the compound was collected at 293 K and intensity data collected shows h from −9 to 9, for k from −10 to 10 and for l from −14 to 14 in the range of 2.7–29.2° totaling 2639 unique reflections. After so many cycles of refinement, R factor dropped down to 0.059. Table S1 (Supplementary information) illustrates the complete crystallographic data, data collection and structural refinement details of the grown crystal CFPHC that exists in triclinic system with P $\overline{1}$ space group, exhibiting lattice and unit cell parameters α = 101.87(1)°, β = 103.14 (1)°, γ = 95.09° and a = 7.19 (1) Å, b = 7.86(2) Å, c = 11.36 (2) Å, respectively. Number of molecules per unit cell (Z) and the volume (V) are 2 and 606.23(2) ų, respectively. Final anisotropic full-matrix least-squares refinement on F² with 182 variables and values of largest peak in final difference electron density synthesis are 0.29 e/Å⁻³, largest hole was −0.36 e/Å⁻³ and calculated density was 1.592 mg/m³. Crystallographic information file of CFPHC with reference number CCDC 1530535 were deposited in Cambridge crystallographic data centre.

4.3 Optimized structural geometry analysis

Bond length of C2–O11 is 1.23 Å, which has fine agreement with reported C–O bond length (1.2 Å) of related compounds with small variations (Robinet et al., 2001). Owing to hyperconjugation, the bond length of hydroxyl group in pyran ring reduces also by negative inductive effect, delocalization of lone pairs happens by releasing electrons, reduced the corresponding bond angle C3–O12–H13 = 104°. Bond lengths associated with electron donating oxygen atom (C–O) reduces from the expected value 1.43 Å (Sawant and Nirwan, 2012) due to electron delocalized ring system. C–H bond parameters vary due to the electron cloud surrounding each atom repelling each other leading to destabilization along with steric hindrance. Bond angles connecting C8 alters due to the negative inductive effect of chlorine atom. Hydrogen and fluorine forms a polar covalent bond by sharing electrons thereby electrons being attracted to one atom more strongly than other, which results in partial positive and negative charges. Separation of partial charges creates a dipole and electrons are more pulled towards fluorine forming C20–H25⋯F26 and C22–H27⋯F26 halogen bonding interactions. Simulated geometric parameters show good correlations with the crystallographic bond parameters while slight deviations arise because theoretical calculations was done for single isolated stable molecule in gas phase and experimental values predicted from crystalline solid phase and are summarized, given in Table S3. Fig. 1(a) shows ORTEP diagram while (b) represents optimized molecular structure of CFPHC and (c) crystal pack diagram of CFPHC.

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Fig. 1

(a) ORTEP diagram (b) Crystallographic view (c) Optimized geometrical structure of CFPHC.

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4.4 Spectral assignments via experimental and computational view

CFPHC has 28 atoms and according to group theory analysis, 78 normal modes of vibrations are distributed as: Γ_3N-6 = 53 À (in-plane) + 25 Ȁ (out-of-plane); harmonic wavenumbers are adjusted by the scaling factor and detailed vibrational assignments of CFPHC carried out with the aid of normal co-ordinate analysis illustrates in Table 1. Theoretical IR and Raman spectra of CFPHC are shown in Fig. 2(a,b). Slight differences concerning experimental and calculated vibrational modes observed perhaps due to hydrogen bonding leading to strong perturbation of wavenumbers and intensities of various PED modes.

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Fig. 2

(a) IR Spectrum (b) Raman Spectrum of CFPHC.

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4.5 UV–vis spectral analysis

UV–vis spectroscopy studies the changes in electronic energy levels within the molecule arising due to transfer of electrons from π- or non-bonding orbitals and provides the knowledge about π-electron systems and conjugated non-bonding electron systems. Ultraviolet spectrum of 4-chromene showed absorption peaks around 260 nm and 330 nm when measured in ethanol (Ellis, 2009). C⚌C bond prevents conjugation in γ-pyrone carbonyl group thereby reducing involvement of flurophenyl ring to UV–vis spectrum. Electron-donating groups such as –CH₃, –Cl, –Br, –OH, –OCH₃, –NH₂ etc. increases secondary peak absorption. The presence of conjugate double bond and functional group –OH also influence the conjugated systems, decreases the energy difference between HOMO and LUMO and the radiations of longer wavelength are absorbed. Consequently a peak of very low intensity in 300–330 nm range (peak I) have been assigned in chromenes. Increased electron drift from electron-donating hydroxyl group and chlorine atom in γ-pyrone ring through π-bond of benzene ring shows shoulder peak at 309 nm and bathochromic (red) shift to 347 nm occurs due to the solvent stabilization of excited or ground electronic states showing π→π^∗ transition with 56% minor contribution from HOMO-2 → LUMO excitation. UV–vis spectrum of CFPHC shows n→π^∗ transition around carbonyl chromophore undergoing hypsochromic (blue) shift showing sharp intensity peak at 253 nm in γ-pyrone moiety for ethanol solvent stabilized by hydrogen bonding having polar excited state. Table S4 shows UV absorption corresponding to experimental peaks (Fig. 3) along with calculated excitation energies; absorbance and oscillator strength (f) for title molecule for ethanol solvent. Maximum absorption wavelengths obtained, viz. 350, 301, and 265 nm, are in good agreement with experimental values of 347, 309, and 253 nm, attributed to the excitation with oscillator strengths 0.53, 0.17, 0.12, respectively.

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Fig. 3

UV–vis spectrum of CFPHC.

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4.5.1

4.5.1 HOMO and LUMO analysis

Compounds having high E_HOMO are good electron donors while those having low E_LUMO are good electron acceptors therefore a significant degree of intramolecular charge transfer (ICT) from electron donor groups to electron acceptor groups transpire through a π-conjugated pathway (Xavier et al., 2011). Frontier orbital analysis of CFPHC shows that E_HOMO value is calculated as −6.43 eV, displaying more electron accepting capability while E_LUMO value is −2.63 eV. HOMO-LUMO orbital energy gap can provide useful information about the charge transfer interaction, chemical reactivity and biological activities such as antibacterial, antioxidant as well as DNA binding (Fleming, 1976; Mu et al., 2015; Rahmounia et al., 2014). By comparing the DFT computations of fluorine and chlorine substituted compounds (Nilesh and Anil, 2017), molecular orbital energy gap of CFPHC is found to be 3.80 eV and the corresponding wavelength fall in the UV region. Frontier orbital energy values such as HOMO − 1 = −7.36 eV; LUMO + 1 = −1.47 eV; HOMO − 2 = −7.69 eV; LUMO + 2 = −1.18 eV are also calculated. In CFPHC, HOMO and HOMO-1 are delocalized and predominantly located on hydroxyl groups, chlorophenyl ring and carbonyl group showing major contribution of 93% HOMO → LUMO transition, which entails an electron density transfer from pyran ring through oxygen atoms to chlorophenyl ring, while HOMO − 2 are confined with 22% major influence on chlorophenyl ring. As the number of conjugated double bonds is increased, the gap between highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) is progressively lowered. LUMO populates on all atoms except hydrogen atoms while LUMO + 1 mainly populates on hydroxyl group oxygen atom and carbon atoms and LUMO + 2 on in both chloro- and fluro-phenyl ring carbon atoms. Molecular orbital plots for ground state of CFPHC molecule in gas phase including the HOMO, LUMO, LUMO + 1, LUMO + 2 are shown in Fig. 4.

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Fig. 4

HOMO-LUMO plot of CFPHC.

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4.6 Natural population analysis

Population analysis assign the atomic population for each atom in a molecule (Reed et al., 1985) and the summary of natural population charges from NBO output is listed in Table S5. Natural population distribution chart of CFPHC is as shown in Fig. 5. Charge distribution of optimized monomer CFPHC produces a total dipole moment of 1.61 Debye and points out that all hydrogen atoms possess a positive charge and among them H13 of hydroxyl group possess highest positive charge due to intramolecular bonding interaction sites. Oxygen draws the electrons in the bonds and shares with the hydrogen atoms towards it while effect of electronegative halogen atom in the π system is weak, because the lone pair of electrons are so tightly held, and they are strong σ acceptors (Fleming, 2010). All oxygen atoms acquire negative charge while hydroxyl oxygen atom (O12) having highest negative charge and oxygen atom of carbonyl group also possess high value of negative charge due to O–H⋯O intramolecular hydrogen bonding. Since oxygen and chlorine atoms are highly electronegative, the effective charges on oxygen and hydrogen caused by the chlorine drawing electrons from the carbon are high, thereby reducing the charge in chlorine atom Cl15.

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Fig. 5

Natural Population Charges distribution chart of CFPHC.

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4.7 Molecular electrostatic potential surface analysis

Molecular electrostatic potential surface (MESP) analysis serves to explain hydrogen bonding, reactivity and structure-activity relationship of CFPHC especially its bioactivity. Electrostatic potential at different points on the electron density surface is shown in Fig. S1 and the reported values are in Table S5. In CFPHC, γ-pyrone ring carbon atoms are blue in color characterized by a relative absence of electrons. Negative (red) potential (−22.37a.u) mostly projects above the carbonyl oxygen atom and hydroxyl group contributing hydrogen bonding interactions resulting in concentrated electron density across it. F26 with electric potential (−26.57a.u) shows red color exposing its greater electronegativity difference between atoms in the bond. Cl15 has a highest electric potential (−64.37a.u) showing slight yellow color and due to the greater electronegativity difference between atoms in a bond, makes it the more polar bond.

4.8 Natural bond orbital analysis

NBO analysis summarizes hybridization of atomic lone pairs and of the atoms involved in bond orbitals. Mixing of the donor and acceptor orbitals treated with second-order perturbation theory expounds the intramolecular interactions, rehybridization and delocalization of electron density within the molecule and second order perturbation analysis of Fock matrix using NBO basis of CFPHC is as shown in Table S6. Interaction energies from electron donating groups lone pair LP(O) to the anti-bonding acceptor σ^∗(C–C) of phenyl rings showing high stabilization energies due to resonance inside the ring as well as hyperconjugation between the electron donating groups and phenyl ring. Lone pair interactions such as LP2O11→σ^∗(C2–C3) is 20.83 kcal/mol; LP2O12→σ^∗(C2–C3) is 16.45 kcal/mol; LP2O11→σ^∗(C1–C2) is 18.40 kcal/mol; LP2O12→σ^∗(C3–C4) is 23.95 kcal/mol increases electron density (ED) in C–C antibonding orbital thereby weakening the respective bonds.

Corresponding stabilization energies associated with dihedral angles of γ-pyrone ring and phenyl ring show high values as 991.69 kcal/mol and 55.55 kcal/mol resulting charge transfer from γ-pyrone ring to flurophenyl ring. Red shift of O–H stretching wavenumber confirms O–H⋯O hydrogen bonding with stabilization energies 0.59 and 4.59 kcal/mol due to the lone pair (LP) interactions, LP₁(O11)/LP₂(O11)→σ^∗O12–H13. Intramolecular charge transfer from σC4–O5→σ^∗(C23–H28) with stabilization energy 16.71 kcal/mol exposes C–H⋯O hydrogen bonding with increase in electron density (ED) in C–H antibonding orbital weakening the respective bonds and the corresponding interactions are LP₁(O5)→σ^∗(C23–H28) is 43.06 kcal/mol and LP₁(O12)→σ^∗(C19–H24) is 0.82 kcal/mol for CFPHC. Interaction involving carbon-fluorine bond C–H⋯F having highest stabilization energies are obtained by overlapping orbital atoms LP₁(F26)→σ^∗(C22–H27), LP₁(F26)→σ^∗(C20–H25) having very high stabilization energies 7828.52 kcal/mol and 926.52 kcal/mol, correspondingly. Hydroxyl group and carbonyl group increases the number of double bonds in chromene moiety due to the presence of highly electronegative oxygen atom, stabilizing molecular π-system and distressing π-electronic system at junction σC4–C18→σ^∗C18–C19 of neighboring flurophenyl ring partly destroying its aromaticity showing less stabilization energy 1.94 kcal/mol.

4.9 NMR analysis

Nuclear magnetic resonance spectroscopy is a powerful analytical technique used to characterize organic molecules by identifying carbon-hydrogen frameworks within the molecules using ¹H, ¹³C and ¹⁹F NMR analyses. NMR solvent deuterated dimethyl sulfoxide (DMSO)-d₆ is liable to dissolve compounds of reasonably varying polarity.

4.10 Thermo gravimetric and differential thermal analysis

When sample CFPHC was heated beyond 340 °C, single stage decomposition curve shows two stages of decomposition. First weight loss occurs in the temperature range 98 °C amounting to 2.40% and the second one between 98.70 and 317.50 °C which amounts to the rest of the sample 91%. Presence of multiple weight loss steps indicates the presence of chromophore present in the sample and endothermic dip observed at 98.7 °C is due to their decomposition. DTA curve shows an endothermic dip at 196.20 °C which is due to the melting point of the compound. Complete decomposition at temperature 317.50 °C reflects in DTA thermograms. TG and DTA thermograms of CFPHC are shown in Fig. S3.

Thermodynamic functions such as heat capacity ( $C_m^0$ ), entropy ( $S_m^0$ ) and enthalpy (Δ $H_m^0$ ) changes for CFPHC molecule were computed from theoretical harmonic wavenumbers in gas phase and tabulated in the Table 3. Thermodynamic parameters calculated shows rise in temperature ranging from 100 to 1000 K due to the fact that, the molecular vibrational intensity increases with increase in temperature (Bevan and Boerio-goates, 2000). Correlation equations connecting heat capacity, entropy, enthalpy changes and temperatures are been fitted by quadratic formulas and the corresponding polynomial curve fitting factors (R²) for these thermodynamic properties are 0.99, 0.99 and 0.99, respectively. Thermochemical field can use these parameters to compute the other thermodynamic energies and estimate directions of chemical reactions according to relationships of thermodynamic functions and using second law of thermodynamics. The corresponding fitting equations ((1)–(3)) are as follows and the correlation graphics are shown in Fig. S4.

4.11 In silico study

Molecular docking, the In silico analysis is a computational structure-based design method for assessing ligand molecule to select those that are likely to bind a protein with highest affinity (Shimada, 2006) estimating the bioactivity of the ligand. Hydrogen bonding and hydrophobic interactions play an important role in stabilizing a ligand energetically at the interface of a protein structure. Ligand binding with receptor protein involves the breakage of hydrogen bonds with the release of water molecules and the formation of new hydrogen bonds between the protein and ligand. Ligand CFPHC created from the optimized ground state structure (gas phase) was successively docked with seven reported protein receptors, having PDB id: 2GG5 (Evdokimov et al., 2007), PDB id: 2KCE (Rutenber and Stroud, 1996) as Escherichia coli PDB proteins along with PDB id: 1GAL (Hecht et al., 1993), [organism: Aspergillus niger], PDB id: 4L2J (Moreno et al., 2014), PDB id: IJZQ (Nakama et al., 2001), PDB id: 2MHV (Fizil et al., 2015), (antifungal proteins) and PDB id: 2K35 (Jung et al., 2008) (antimicrobial protein), respectively. Fig. 6 shows ligand-targeted complexes with hydrogen bonding interactions and binding pockets of protein-ligand interactions was visualized using PyMOL1.2r1. Yellow colored dotted lines shows hydrogen bonding of protein-ligand interactions while green and blue color indicates the hydrophobic interactions. Binding affinity of a ligand to its target protein represented in Table 4.

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Fig. 6

Ligand CFPHC interactions with target proteins.

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Residue Asp 97 of 2GG5 attached to carbonyl group at 3.3 Å and hydroxyl group at 2.1 Å with average inhibition constant (K_i) = 23.36 µM while Gln 233 interact with ligand at 3.1 and 2.1 Å with oxygen atom of carbonyl group in chromene moiety having binding energy −6.99 kcal/mol. Interactions regarding the carbonyl and hydroxyl group of ligand CFPHC with the residue Ile 79 of 2KCE, respectively at 2.0 Å and 3.5 Å possess binding energy (ΔG) = −6.45 kcal/mol with very low inhibition constant of 18.79 µM. Hydrogen atom of hydroxyl group possess binding energy of −8.27 kcal/mol and Root-mean-square-deviation (RMSD) 78.94 Å that holds Val 250 residue of 1GAL at 1.9 Å while carbonyl oxygen interact with this residue forming two hydrogen bonds with high inhibition constant 868.8 µM at 3.4 Å and 1.7 Å, respectively. Oxygen atoms in both carbonyl and hydroxyl chromophore show the hydrogen bonding interaction with residue Gln-89 having ΔG = −5.66 kcal/mol and K_i = 70.61 µM] for 4L2J protein at bond distances of 2.0 Å and 2.3 Å. In addition, Trp 32 attached with carbonyl group at 1.9 Å with RMSD of 20.24 Å. Lys 482 residue of antifungal protein 1JZQ forms a hydrogen bond with hydrogen atom of hydroxyl group at 2.5 Å and carbonyl group at 2.4 Å having K_i = 52.18 µM and ΔG = −5.84 kcal/mol. Likewise carbonyl group forms two hydrogen bonds [2.1 and 3.2 Å] with residue Glu 499 having an inhibition constant of 52.18 µM at RMSD 67.83 Å. Amino acid protein Glu13 of antifungal protein 2MHV interacts with hydroxyl hydrogen atom at 1.8 Å with binding energy −5.49 kcal/mol also at 2.7 Å and 2.8 Å with carbonyl oxygen atom with RMSD of 20.15 Å with very high inhibition constant of 94.58 µM. Arg 37 of 2K35 antimicrobial protein interacts with both carbonyl group and hydroxyl group forming three hydrogen bond at 2.1 Å, 2.3 Å and 2.2 Å, respectively with an inhibition constant of 34.52 µM having binding energy −6.09 kcal/mol.

4.12 In vitro assays