3.1. Hirshfeld Surface Analysis
The conventional mapping of dnorm and 2D fingerprint plots of newly synthesized three different heterocyclic compounds drawn by Crystal Explorer 17.5 software with the aid of Crystallographic Information File (CIF). And, cluster of molecules within 3.8 Å radius were selected around a reference molecule to calculate pair-wise interaction energies in a crystal and energy frameworks were also carried out using CE-B3LYP/6-31G(d,p) method.
In the dnorm surface, the strong red surface observed on nitrile and hydroxyl groups which confirms O‒H‧‧‧N type hydrogen bonding interactions in the crystal phase. The small red spots are also detected around the molecules, it indicates the C‒H···N type of weak hydrogen bonding interactions. The strong white and blue surfaces distinguish sum of Van der Waals radii and short contact distances on the 3D dnorm based-Hirshfeld surface maps. The shaped index shows the blue and red colour which reflects the cycle stacking (C−H∙∙∙π and π∙∙∙π interactions). The two-dimensional fingerprint plots were created for each surface point and the data obtained from discrete intervals of di and de. In which, the different type of intermolecular interactions of the molecule in the crystal can be characterized by the shapes of fingerprint plots, the fingerprint plots of the titled compounds are shown in Fig.2. The two large spikes are noticed both the left and right sides, validates the N−H interactions and small spikes are from O−H interactions. The wing-shaped and lobes are attributed to the C‒H interactions and H−H interactions. The contribution of the total intermolecular interactions is significantly divided into the N−H, O−H C−H and H−H interactions, the values 31.2, 6.0, 12.7 and 42.4% respectively. A visualization of their energy frameworks were performed for titled compounds (Fig.2), the pair-wise interaction energies were also estimated between molecules within a standard cluster of a radius of 3.8Å at the B3LYP/6-31G(d,p) level of theory (CE-B3LYP model). Here, the total interaction energy between any nearest-neighbour molecular pairs is given in terms of four components: electrostatic, polarization, dispersion, and exchange–repulsion which gives an insight into the underlying interaction energy and leads to the organization of crystal packing into supramolecular architectures in crystalline materials, were summarized in the Table2. The representative energy frameworks diagram of Pair-wise interaction energies is shown as cylinders between molecular centroids, the thickness of each cylinder is proportional to the relative strength of interaction energies. The total energies of all interacting molecules with respect to corresponding reference molecule along with the symmetry (x, y, z: -39.2; -x, -y, -z: -30.0 and -x, y+1/2, -z+1/2: -41.8 kcal/mol) carries highest energy than the other molecules. and centroid-centroid distance. The electrostatic, polarization energies are lesser than the dispersion and exchange–repulsion energies, which reveals the topology of the molecules and it concludes that these interacting energies are playing major role in the assembly of the molecules in the crystal phase of molecules.
3.2. Analysis of quantum chemical calculation
3.2.1 Optimized Structure
The atomic charges, HOMO, LUMO and electrostatic potential were also analyzed to understand the insight beauty of the molecule. And, the natural bonding orbitals (NBO) were also carried out to reveal the interactions that take a place between the filled and vacant orbitals. The graphical interpretation of all these studies were made by Gauss view and 3plot program. The comparison of optimized geometry of titled molecule reveals the similarity with crystal structure, shown in overlaid structure, Fig. 4.
3.2.2 Global reactivity descriptors
To understand the relationship between structure, stability and global chemical reactivity, the reactive descriptors of the titled compound can be derived from the conceptual density functional theory. The ionization potential and electron affinity are related to the energy of highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO). These orbital energies are highly helpful in computational way using Koopman’s theorem for closed shell molecules to reproduce the reactivity of the molecule. The calculated value of ionization potential, electron affinity and electronegativity of the titled compound are 7.39, 1.51 and
4.45eV respectively. And, the calculated electrophilicity index (w) is 3.37 eV. The HOMO and LUMO are main part in the characterization of molecules in chemical reactions. The HOMO considers the outermost orbital filled by electrons, it acts as donor and LUMO indicates the first innermost orbital unfilled by electrons, it acts as acceptor. The 3D maps of HOMO-LUMO are shown in Fig. 5; in which, the HOMO is localized only around the phenyl rings whereas the LUMO localized on the whole molecule except methyl groups in the molecule.
Table 3
Calculated global reactivity properties of the molecule
Global Reactivity
Descriptors
|
DFT
Energy (eV)
|
Compound 1
|
Band Gap
|
5.88
|
HOMO Energy
|
-7.39
|
LUMO Energy
|
-1.51
|
Ionization Potential
I= -EHOMO
|
7.39
|
Electron Affinity
A= -ELUMO
|
1.51
|
Global Hardness
η= (I-A)/2
|
2.94
|
Electronegativity
χ= (I + A)/2
|
4.45
|
Electrophilicity
ω = µ2/2η, µ= -χ
|
3.37
|
3.2.3 Atomic charges and Electrostatic potential
The knowledge of charge distribution of the molecule is very much essential in the field of quantum chemistry to molecular system due the effect of atomic charges, dipole moment, polarizability and etc. The creation of acceptor and donor pairs placed by the distribution of atomic charges over the molecule which is also connecting the charge transfer in and between the molecules. This charge transfer helps us to comprehend the chemical reactivity, molecular electrostatic potential and the electrostatic interactions. Atomic charges of the molecule are calculated to explore the electrostatic properties of the molecule which is calculated from the spherical charge approximation. The atomic charges of carbon and nitrogen atoms in the C ≡ N bond are approximately 0.307e and − 0.269e respectively. Among all the carbon atoms, methyl group carbon atoms carried high negative charge (~ -0.607e) and oxygen bonded keto carbon (C = O) carries high positive charges (0.843e). The more positive charges of carbon atom confirmed due to the substitution of negative charge of the oxygen and nitrogen atoms in the molecule. Further, the hydroxyl group (O‒H) oxygen atom possesses high negative charge (-0.761e) than other two oxygen atoms in the molecule and hydroxyl group hydrogen atom exhibits higher charge than other hydrogen atoms in the molecule. In this molecule, hydroxyl group plays major role in the inter and intra-molecular interactions.
Molecular electrostatic potential is an important property to understand the molecular interactions, their charge distribution. Interestingly, it’s very helpful to interpret and also predict the sites of electrophilic and nucleophilic attack. Therefore, molecular electrostatic potential map plays a vital role investigate the molecular structure along with physiochemical property relationship. Figure 6 displays the electrostatic potential (ESP) map of the titled molecule; it also provides information about the role in drug-receptor interactions and the alignment of molecule in the receptor active site. A large negative ESP (red surface) corresponds to the attraction of proton by the concentrated electron density in the molecule, is found around C ≡ N bond region and C = O bond region in the molecule and it acts as a nucleophilic attack. Whereas, positive ESP (blue surface) corresponds to repulsion of the proton by the atomic nuclei and low electron density observed in these regions. Therefore, the molecular electrostatic potential map of the titled molecule confirms that the negative surface around nitrogen and oxygen atoms while positive surface around all carbon and hydrogen atoms. This may be used to predict the alignment of the molecule in the receptor active site.
3.2.4 Nonlinear optical properties (NLO)
The organic molecules with second-order nonlinear optical (NLO) properties exhibits a keen interest due to significant role in the multidiscipline research like medicine, molecular switches, telecommunication, data storage and etc. There are many reports confirmed certain electronic properties to have the desired nonlinear optical properties. In the computational way, the gaussian software gives different components of 3×3×3 matrix from dipole moment (µ) of the molecule. Here, polarizability and hyperpolarizability of the titled molecule were calculated from the DFT/B3LYP 6-311G** method (Table 4). The dipole moment, polarizability, small HOMO_LUMO energy gap and planarity of the molecule are responsible for the non-linear optical properties. The dipole moment (µ), the polarizability (αtotal) and first-order hyperpolarizability (βtotal) of the molecule are 6.287 Debye, -28.54 ×10− 24 esu and 2.07×10− 30 esu respectively. Generally, the computed NLO properties of the organic materials are compared with Urea which is also calculated at same level of basis set, the value is 0.37 ×10− 30. The hyperpolarizability (βtotal) of the titled compound is much higher than the urea which clearly confirms the title molecule is good material for the NLO applications.
Table 4
Dipole moment (µ) in Debye (D), polarizability (α) and hyperpolarizability (β) of the titled compound using base level of DFT/B3LYP 6-311G** method
|
esu (×10− 24)
|
|
|
esu (×10− 33)
|
αXX
|
-25.42
|
|
βXXX
|
591.056
|
αXY
|
-1.669
|
|
βXXY
|
674.217
|
αYY
|
-29.84
|
|
βXYY
|
900.283
|
αXZ
|
-0.509
|
|
βYYY
|
-478.387
|
αZZ
|
-30.37
|
|
βXXZ
|
-454.158
|
αYZ
|
0.706
|
|
βXYZ
|
127.729
|
αTotal
|
-28.54
|
|
βYYZ
|
118.384
|
∆α
|
4.708
|
|
βXZZ
|
348.02
|
µx
|
4.925 D
|
|
βYZZ
|
-65.64
|
µy
|
-1.078 D
|
|
βZZZ
|
604.8
|
µz
|
-3.757 D
|
|
βtotal
|
2070.00
|
µtotal
|
6.287 D
|
|
|
|
3.2.5 Natural bond orbital analysis (NBO)
The NBO analysis gives the information about the inter- and intra-molecular bonding interactions to investigate hyperconjugation or delocalization interactions or charge transfers in molecular system (Table 5). And, it also gives the details of the interactions between lone-pair filled orbitals (bonding) and Rydgberg empty orbitals (anti-bonding). The large E(2) value confirms the interaction between electrons donor and electron acceptors and greater the extent of the conjugation of the whole system. The stabilizing donor-acceptor interactions are depending on the delocalization of electron density between occupied Lewis type and unoccupied non-Lewis type orbitals. The hyperconjugative interactions are the most responsible for the stability of the molecular system. The stabilization energies associated with electron delocalization between donor and acceptor are used to understand the inter- and intra-molecular interactions in the desired molecular system. From the NBO analysis, the titled compound forms three hydrogen bonds between the nonbonding orbital of the hydrogen-bonded acceptor (nA) and the antibonding orbital of the H-Donor bond ( σH−D∗). The stabilization energy of all three hydrogen bonds is greater than 0.5 kcal/mol.
Table 5
NBO analysis of quinoxaline derivative
The type of nA
|
The electron configuration of nA
|
The type of orbital interaction
|
E(2) (in kcal/mol)
|
The occupancy of σH−D*
|
The bond order of σH−Db
|
LP2 of O3
|
s(38.01%)p(61.97%)d(0.02%)
|
LP(O2)-σ*(O4-H5)
|
2.20
|
0.01653
|
0.3941
|
LP2 of O4
|
s(0.01%)p(99.95%)d(0.04%)
|
LP(O4)-σ*(C52-H53)
|
0.89
|
0.00640
|
0.3859
|
LP2 of O3
|
s(0.05%)p(99.91%)d(0.04%)
|
LP(O3)-σ*(C20-H21)
|
1.81
|
0.01868
|
0.3699
|
3.3 Molecular docking
Molecular docking technique is highly useful in predicting intermolecular binding conformation between molecules. The docking score of the compound is -12.14 kcal/mol and binds to the minor groove of the DNA. The binding mode of the compound is as such that it positioned itself at the center of the minor groove; the ethyl formate moiety is more towards the leader while the central 2-hydroxy-2-methylcyclohexane-1,1-dicarbonitrile ring is placed straight over the nitrogen bases. The interactions of the compounds is dominated by van der Waals. The docked binding mode of the compound with DNA and its interactions are presented in Fig. 7.
3.4 Molecular Dynamics Simulations and Binding free Energies
Molecular dynamics simulation of long 100 ns run was conducted to determine biding interactions and binding mode stability of the receptor DNA molecule in the presence of the drug. It has been unveiled that both DNA and the compound attained considerable stability along the simulation time as can be seen in the Fig. 8. Conformation stability of the DNA and the compounds was evaluated via root mean square deviations (RMSD) analysis. The RMSD for the DNA and the compound fluctuates around 1 Å, which is an indication of highly equilibrium system. The RMSD of the compound near 50 ns experienced a small deviation which corresponds to a bit conformational change in order to get more stability. Afterward, the RMSD plot for the compound was seen uniform and the binding conformation rigid. The DNA molecule in the presence of the compound enjoyed enhanced level of stability. This data reflects on the high inter-molecular strength and formation of strong and stable complex. Additionally, to validate the predictions made by docking and simulation studies, MMGBSA binding free energies were estimated for the complex. As witnessed in the docking studies that the van der Waals energy contribute to bulk of the interactions, same was revealed in MMGBSA binding free energy analysis. The net binding van der Waals binding free energy of the complex is -31.54 kcal/mol while electrostatic energy is -10.84 kcal/mol. The presence of electronegative atoms in the compound structure also allow the compound to produce electrostatic energy. Both the van der Waals and electrostatic energy is contributed high to the net binding energy. The net solvation energy of the system is 15.56 kcal/mol with favorable energy from non-polar energy (-9.48 kcal/mol) and non-favorable contribution from polar solvation energy (25.04 kcal/mol). The total binding energy of the system estimated is -26.82 kcal/mol. The different binding free energy estimated for the complex is tabulated in Table 6.
Table 6
MMGBSA binding free energy estimation for DNA-compound complex.
Energy component
|
Average
|
Van Der Waals
|
-31.54
|
Electrostatic
|
-10.84
|
Polar
|
25.04
|
Non-polar
|
-9.48
|
DELTA G gas
|
-42.38
|
DELTA G solv
|
15.56
|
Delta total
|
-26.82
|
3.5 Prediction of Drug likeness
For good drug like molecule, it is necessary to be absorbed well in the host body in a given time. This supports good distribution of the compound and reaching to the target site in maximum concentration for effective action [21]. Also, the drug needs to be nontoxic as failure of a drug molecule in clinical trials is highly costly. In silico prediction of such properties allow selection of suitable drug molecule which not accelerate drug discovery process [22]. The compound was unveiled to fulfill all parameters of Lipinski rule of five, Ghose rule and Muegge rule. However, it fails by violating TPSA value in both Veber and Egan rule. The molecular weight of the compound is 438.48 g/mol. This weight affirm that the compound can get enhance absorption rate [23]. The number of rotatable bonds, hydrogen bond acceptors, hydrogen bond donors, molar refractivity and topological polar surface area (TPSA) is 5, 7, 1,117.23 and 141.69 Ų, respectively. The LogP value of the compound is 2.83, which indicates good absorption and permeation of the compound. The compound has TPSA value within the limits and would be not a substrate for p-glycoprotein for efflux from the cell [24].
3.6 ADMET properties analysis
The compound was unveiled to have better intestinal absorption with probability score of 0.99. This demonstrate good intestinal absorption of the compound after oral administration. Similarly, the compound predicted to have good blood-brain barrier (BBB) penetration. The compound is non-inhibitor of P-glycoprotein (P-gp) and acted as substrate of P-gp. From toxicity, perspective the compound is mutagenic, and hepatotoxic. Also, the compound showed oral toxicity.