Experimental spectra, electronic properties (liquid and gaseous phases) and activity against SARS-CoV-2 main protease of Fluphenazine dihydrochloride: DFT and MD simulations.

The Gaussian 09 DFT tool is used to investigate the formational electronic behaviour, reactivity analysis and biological properties of fluphenazine dihydrochloride (FDD). The quantum computation is used to determine the spectroscopic and vibrational assignments of FDD. The NBO method explains charge transfer and molecular interactions. Energy gap values are determined using FMO analysis in different solvents and toluene is a better solvent due to higher value of solvation energy. The UV-visible spectra are investigated in various solvents using the TD-DFT method. Electrostatic potential, the wave function related properties such as LOL, NCI and RDG are determined in gaseous phase. Furthermore, the drug likeness is analyzed. At last, a docking study with MD simulation is used to investigate FDD's antiviral activity against SARS-CoV-2 main protease.

Introduction

Fluphenazine is a phenothiazine antipsychotic drug with pipearzine derived substituent that is used to treat schizophrenia and bipolar disorder [1]. This drug also has a number of novel and important biological properties [2]. It is worth noting that fluphenazine and melamine generate complexes that have both positive and negative pharmacological side effects [3]. Ding et al. reported a high efficiency phenothiazine for solar cell [4]. Petrus et al. reported XRD structure of fluphenzine dichloride dimethanol solvate [5]. Phenothiazine is a non planar confirmation of the butterfly or bent kind [6]. Drugs of phenothiazine family have a wide range of biological characteristics [7], [8], [9], [10], [11], [12], [13], [14]. Promazine has been determined using a variety of methods, including electrophoresis, spectro-photometry and electrochemistry [15], [16], [17], [18]. Promazine is used to treat psychiatric disorders and cancer [19], [20], [21], [22], [23], [24]. The charge transfer properties, detection by graphene sensors and molecular recognition of phenothiazine derivatives are reported [25], [26], [27], [28], [29], [30]. Trifluoperazine, an antipsychotic drug has recently been confirmed to interact with bovine serum albumin [31]. The production of a nanocomposite for promazine removal from water was reported [32]. Recently, researchers reported sulphide-metal nanocomposites synthesis for the detection of promazine and trifluoperazine [33,34]. The uses of trifluoperazine in medicine have recently been reported [35,36].

Drug research and development rely heavily on quantum chemistry theory, DFT, molecular models and vibrational spectroscopy [37]. Docking studies give the binding and interaction between ligand and receptor, as well as drug mechanism [38]. To understand the action of drugs and proteins, MD simulations can show the oscillations of receptors and ligands [39]. As a result, docking with MD simulation may be utilized to examine the structural properties and binding mechanism of ligands and receptors in a complete and systematic manner. DFT studies of FDD cover new information about their electronic, structural features and reactivity in addition to reactivity analysis and MD simulations. DFT is also used to achieve the highest precision results of FDD's properties in variety of utilizable eco-solvents [40].

Methods

FDD is obtained as a gift [5] and vibrational spectra (Figs. S1 and S2) were recorded according to literature [41]. Gaussian 09 package was used to perform all quantum chemical calculations with B3LYP/6-311++G* for FDD (Fig. 1 ) [42], [43], [44]. The FMO was used to calculate the DFT-based reactivity descriptors by obtaining ionization energy and electron affinity. NBO analysis was carried out in order to predict the important intra molecular interactions, nature and charge transfers within FDD [45]. Various solvents are being used in the current study to examine the properties of electrons in FDD [46]. HOMO-LUMO and UV analyses are done in different solvents using the IEFPCM model.

Fig. 1

Optimized molecular structure of FDD.

Molecular Docking studies are carried out for FDD with SARS-CoV-2 main protease (PDB ID: 6LU7) with highest resolution 1.25 Å [47] and subsequently cleaned for any steric clashes, intrinsic water and co-crystallized molecules. Then the protein structure was subjected to energy minimization using 1000 steps of conjugate gradient algorithm. All the hydrogen are properly added and saved in pdb format for molecular docking purpose. Docking studies were carried out in patch dock web server [48]. Grid centre was provided as 0.35 Å x 6.0 Å x 3.0 Å. Docking parameters were set for protein small ligand with clustering RMSD 0.5 Å. Ligand binding site information was uploaded in the server advanced parameter option and energy minimized protein and geometrically. The MD simulations studies were carried on the dock complex for FDD with protein (PDB ID: 6LU7) using the Desmond 2020.2 from Schrödinger, LLC as reported earlier [49], [50], [51], [52], [53], [54], [55]. The RMSD, Rg, RMSF and SASA) were calculated to monitor the stability of the MD simulations.

Results and discussion

. Spectroscopic and electronic properties

The phenyl ring modes (Table S1) are assigned at: 3075 (IR), 3082 (Raman), 3100, 3093, 3082, 3074 cm−1 (DFT) (υCH), 1587, 1448 (IR), 1594, 1590, 1469 (Raman), 1596, 1589, 1470, 1447, 1268, 1049 cm−1 (DFT) (υRA); 1041,1241 (IR), 1128, 1040 (Raman), 1244, 1150, 1126, 1039 cm−1 (DFT)(δCH) and at 959, 854, 736 (IR), 961, 922, 850, 738 cm−1 (DFT) (γCH) for 1,2-substituted ring RA; 3111 (IR), 3112, 3111, 3088 cm−1 (DFT) (υCH); 1623, 1605, 1424, (IR), 1627 (Raman), 1622, 1609, 1500, 1422, 1291, 1047 cm−1 (DFT) (υRC); 1284 (IR), 1283, 1139, 1047 cm−1 (DFT)(δCH) and at 886, 818 (IR), 888 (Raman), 886, 874, 820 (DFT) cm−1 (γCH) for 1,2,4-substituted phenyl ring RB [56], [57], [58].

The stretching modes of piperazine ring RD are at 1009, 945, 931 (IR), 1006, 947, 931 (Raman) and 1141, 1074, 1062, 1007, 943, 930 cm−1 (DFT). The associated CH2 modes are at: 2993 (IR), 2986 (Raman), 3069-2983 cm−1 (DFT) (υCH2); 1394, 1356, 1302, 1027, 969, 796, 750 (IR), 1190, 822, 782 (Raman), 1408-750 cm−1 (DFT) (δCH2). The bands at 1356 and 1027 cm−1 in IR are characteristic modes of bending of CH2. The other CH2 modes of FDD are at: 2868 (IR), 3062, 2980, 2890 (Raman), 3061-2886 cm−1 (DFT) (υCH2); 1474, 1335, 1312, 1261, 1241, 1217, 1120, 868 (IR), 1333, 1310, 1278, 1241, 1228, 865 (Raman), 1484-866 cm−1 (DFT) (δCH2) [56,59,60].

Other important functional group modes are: υNH…Cl – 2910, 2129 cm−1 (DFT), 2219 (IR), 2125 (Raman); δNH…Cl: 1508, 1495, 1490, 1472 cm−1 (DFT); υOH – 3487, 3465, 3350 cm−1 (DFT), 3433, 3274 (IR), 3404, 3345 (Raman); υCS – 728, 678 cm−1 (DFT), 727, 677 (IR), 727, 678 (Raman); υCF at 1163, 1137, 1077 cm−1 (DFT), 1164, 1137, 1078 (IR), 1077 cm−1 (Raman); δCF at 562, 513, 430, 382, 274 cm−1 (DFT); υCO – 1063, 1057, 1033 cm−1 (DFT), 1064 (IR), 1055 cm−1 (Raman) [56,61,62]. The other deformation modes are also identified.

The solvation energies, changes in enthalpy and Gibbs energy of FDD are, respectively, -5.85, -5.97, -6.76 (acetone), -1.42, -1.45, -1.68 (DMSO) and -2.94, -3.01, -3.44 (methanol) and -53.46, -59.15, -44.31 kJ/mol (toluene) and the values show that the toluene is the best solvent for FDD [63]. The quantum chemical parameters were determined though theoretical calculations using the DFT method (Table 1 ). Fig. 2 shows that the FDD's HOMO and LUMO are distributed almost throughout the rings except piperazine, indicating a high ability to donate and receive electrons [64,65]. The orbitals of the FDD differ noticeably between the gas and solvent phases. The observed higher hardness value (2.3166) in the gas phase of FDD shows the chemical stability. In the gas state of FDD, energy gap, ionization potential and electron affinity are 4.6332, 5.5140, 0.8808 eV, respectively. When comparing descriptors in solvent state, toluene has high energy gap of 4.6380 eV and a hardness value of 2.3290. The highest and lowest ionization potential values are for toluene (5.5285) and for acetone (5.5119) [66].

Table 1

Calculated chemical descriptors (eV) of FDD.

Molecular descriptors	Gas	Water	Acetone	DMSO	Methanol	Toluene
EHOMO	-5.5140	-5.5140	-5.5119	-5.5132	-5.5127	-5.5285
ELUMO	-0.8808	-0.8808	-0.8762	-0.8797	-0.8784	-0.8705
Energy gap	4.6332	4.6332	4.6357	4.6335	4.6343	4.6580
Ionization potential	5.5140	5.5140	5.5119	5.5132	5.5127	5.5285
Electron affinity	0.8808	0.8808	0.8762	0.8797	0.8784	0.8705
Chemical hardness	2.3166	2.3166	2.3179	2.3168	2.3172	2.3290
Chemical potential	-3.1974	-3.1974	-3.1941	-3.1965	-3.1956	-3.1995
Electrophilicity index	2.2065	2.2065	2.2007	2.2050	2.2034	2.1977

Fig. 2

HOMO-LUMO plots of FDD.

UV-Vis spectroscopy is used to investigate the electronic transitions [67] and the electron spectrum of FDD was computed using the IEFPC solvation model in different solvents (Fig. S3) at the B3LYP/6-311++G* level of theory. It is used to calculate FDD's band gap and higher values indicate that tightly bound valence electrons with nucleus [68]. Gas and water get the same wavelength 276.72 nm, band 4.4805 eV, energy 36137.67 cm−1 and oscillatory strength 0.065 with contribution 85% (Table 2 ). Toluene has highest absorption at 279.09 nm, band gap 4.4425 eV and a transition from HOMO to LUMO+2 (87%) giving aromaticity and biological activity of FDD. The conventional hybrid functionals, like B3LYP are known to underestimate excitation energies in organic systems according to literature [69], [70], [71]. The range-separated TDDFT methods give more accurate calculations compared to conventional hybrid DFT methods.

Table 2

Theoretical electronic transition parameters of FDD.

solvents	Wavelength (nm)	Band gap (eV)	Energy (cm−1)	Oscillator strength (f)	Symmetry	Major contributions
Gas	276.72	4.4805	36137.67	0.065	Singlet-A	HOMO→L+2 (85%)
	322.86	3.8402	30973.30	0.0116	Singlet-A	HOMO→L (92%)
Water	276.72	4.4805	36137.67	0.065	Singlet-A	HOMO→L+2 (85%)
	322.86	3.8402	30973.30	0.0116	Singlet-A	HOMO→L (92%)
DMSO	276.91	4.4774	36112.67	0.070	Singlet-A	HOMO→L+2 (86%)
	322.91	3.8396	30968.46	0.0122	Singlet-A	HOMO→L (92%)
Acetone	277.03	4.4755	36097.34	0.0672	Singlet-A	HOMO→L+2 (86%)
	322.72	3.8419	30987.01	0.0118	Singlet-A	HOMO→L (92%)
Toluene	279.09	4.4425	35831.18	0.0800	Singlet-A	HOMO→L+2 (87%)
	321.37	3.8580	31116.87	0.0133	Singlet-A	HOMO→L (91%)
Methanol	276.85	4.4784	36120.73	0.0651	Singlet-A	HOMO→L+2 (85%)
	322.77	3.8413	30982.18	0.0116	Singlet-A	HOMO→L (92%)

Due to lone pair electrons strong NBO interactions (Table S2) occurred in FDD as: N12→(C9-C11, C13-C21) with energies, 23.23 and 23.25 kcal/mol; O65→(O57-H58) with energy 23.47; Cl71→(N43-H44) with energy 29.98 and other donor (acceptor) orbials of C2-C3 (C9-C11), C5-C7 (C2-C3), C9-C11 (C5-C7), C13-C21 (C17-C19), C14-C16 (C13-C21), C17-C19 (C13-C21), C17-C19 (C14-C16) with energies, 20.85, 22.90, 20.85, 20.51, 21.20, 20.15, 22.40 kcal/mol and 100% p-character in F23, F24, F25, Cl72, O62 atoms of FDD.

Topological properties of electron density

Bader the pioneer proposed the Atoms in Molecules theory [72] in order to get abundant details about the atoms, molecules, chemical bonding and their topological properties such as electron density (ED) and Laplacian of the electron density (LED). The ED values are given in Table S3. Bond (3, -1), ring (3, +1) and cage critical points (3, +3) present in FDD (Fig. 3 ) and the range of ED and LED for C—C bonds is 1.549 - 2.024 e/Å3 and -13.877- -22.001 e/Å5. The varying charge density values suggest that nature of single and double bonds of the C atoms present in FDD and the charge environments around all the carbon atoms might be different [73].

Fig. 3

Molecular graph of the molecule showing critical points.

In FDD, among the C—O bonds, the C54-O57 bond has the slightly high value of charge density (1.631 e/Å3) when compared to the other bonds. Similarly, among C—N bonds, the C11-N12 and C26-N12 bonds have higher electron density (1.751 e/Å3, 1.774 e/Å3) than the remaining C-N bonds. All the C-H bonds have slight variation in their charge density and Laplacian values which suggests that the charge concentrations at the bond path between the atoms might be same. The two C—S bonds are having almost equal charge density value which suggests the similar accumulation of charges in between the atoms. The Cl-H atoms exhibiting closed shell interactions since their Laplacian values are positive. All the three C—F bonds are having similar ED as well as LED. The bonding regions between C and F atoms are visible through the Laplacian, ELF, electron density and LOL maps (Fig. 4 (a–d)).

Fig. 4

(a) Laplacian of the electron density (b) view of electron localization function of the plane F23-F24-F25 (c) Electron density (d) LOL of the electron density of the plane F23-F24-F25.

The cylindrical nature of the C—H and C—O bonds has been revealed through their corresponding ellipticity values whereas the π character of the C—C, C—F and C—S bonds are visible through the increased ellipticity values [74].

Atomic charges and electrostatic potential

The distributions of charges among the atoms present in FDD have been calculated through AIM analysis and listed in the Table S4. In FDD, C22 possesses the highest positive charge of 1.646e since it is attached with F23, F24 and F25 (electronegative atoms). The H44 atom possesses the highest negative charge value of -5.070e which is attached with the N43 atom. Among the Cl atoms, the Cl72 atom has the highest negative charge value (-0.830e) when compared to the Cl71 atom which has the charge value of -0.199e.

The regions of positive and negative electrostatic potentials are helpful to find out the sites of electrophilic as well as nucleophilic attack which is likely happened between the biomolecule and the target protein [75]. A large region of electronegative is seen around the O atoms and small electronegative cloud is seen around N atoms. The remaining other atoms have been surrounded by large electropositive region. The electrostatic potential map is given in Fig. 5 [76].

Fig. 5

Electrostatic potential of FDD.

Reduced density gradient

The RDG provides non-covalent and space weak interactions in the molecule [77]. The Multiwfn program [78] has been employed to sketch the RDG and scatter graph and for FDD which is given in Fig. S4. The 2-dimensional scatter plots of RDG versus (λ2) ρ(r) integrated on Fig. S4 shows fingerprints of non-covalent interactions in FDD. On left region of Fig. S4, strong attraction, H bond and halogen bond regions are portrayed, whereas the Van der Waals interactions are visualized in the central part and steric repulsion is visible at right hand side.

Pharmacology, docking and molecular dynamics simulations

FDD's drug resemblance is investigated to assess its potential for use as a functional ingredient in pharmacological products. Lipinski's rule, also known as the rule of five, has been used to investigate the relationship between the drug similarity of FDD and the oral effects. The FDD is validated according to rule 5 (Table S5). The hydrogen bond donors (5) and acceptors (6), TPSA (113.29Å2), molecular weight (576.54 g/mol), refractivity (156.09), Lipinski violation (1) and biological score (0.55) are parameters of FDD which are within the threshold values. These parameters of FDD lead to the conclusion that it has a high potential for use as a drug [79,80].

Molecular docking studies of FDD with 6LU7 are displayed in Fig. S5. Surface view of FDD with 6LU7 displayed that FDD well accommodated within in the binding pocket (Fig. S5). The major residues interacted with FDD at the binding pocket of protein Arg222 forming conventional hydrogen bond, halogen bonds with ARG217 and TRP218 and rest are in non bonded vander Waal's interaction. The dock score is calculated within 0.5 Å RMSD clusters tolerence is -8.7 kcalmol−1 within the binding pocket area, 586.40.

To determine the stability and convergence of 6LU7+FDD complex, MD simulation studies were performed for 100 ns. When the RMSD values were compared, the simulation of 100 ns revealed stable conformation. A deviation of 0.5 Å is observed for the Cα-backbone of 6LU7 bound to FDD (Fig. 6 a). The RMSD plots are in the acceptable range, indicating that 6LU7 is stable in the FDD bound state before and after simulation, and quite stable due to FDD's higher affinity. The RMSF plot gives a significant spike of fluctuation (2.0 Å) at amino acid residue 50 and 275 in 6LU7 while remaining residues show less fluctuation during 100 ns which gives a stable amino acid conformation for the entire period of simulation (Fig. 6b). It means 6LU7 structures are stable during simulation in FDD bound conformations. Radius of gyration of 6LU7 Cα-backbone is lowered from 26.45 Å to 26.40 Å in the 6LU7+FDD complex and this is an indication of the compactness of 6LU7+FDD complex (Fig. 6c). The stable Rg peak thus confirms compactness of 6LU7 in FDD bound state. According to the overall quality analysis based on RMSD and Rg, FDD bound to the 6LU7 target posthumously in the binding cavities and gives stability. The number of H-bonds showed significant numbers between 6LU7 and FDD (Fig. 6d). The complex's stability is aided by a consistent numbers of hydrogen bonds formed between protein and FDD. The accessible solvent surface area gives information about the compactness of FDD-6LU7. The lowering of SASA in case of FDD bound to 6LU7 as compared to unbound state signifies the achievement of stable converged structures due to high compactness of both the systems (Fig. 6e) [81], [82], [83], [84].

Fig. 6

(a) RMSD plot displaying the molecular vibration of Cα backbone of 6LU7+FDD (b) RMSF plots showing the fluctuations of respective amino acids throughout the simulation time 100 ns for 6LU7+FDD (C) Radius of gyration plots for the deduction of compactness of protein 6LU7+FDD (d) Number of hydrogen bonds formed between 6LU7 and FDD during 100 ns simulation time scale (e) Solvent accessible surface area (SASA) displaying the ligand bound and unbound area at the binding pocket 6LU7+FDD.

Conclusion

We used theoretical and experimental methods to investigate the spectroscopic properties of FDD in this study. The FDD drug contains reactive sites are identified on O and N atoms. The UV-Vis spectrum shows maximum absorption wavelengths with solvent toluene, which is superior to other solvents. In FDD, C54-O57 bond has the slightly high value of charge density when compared to the other bonds and among the C-N bonds, the C11-N12 and C26-N12 bonds have higher electron density. The ADMET and drug-likeness properties of FDD demonstrate its intoxicating nature.Molecular docking studies confirm the protein-FDD H bond interaction with binding energy of -8.70 kcal/mol. The lowering of SASA for FDD-6LU7 as compared to unbound state gives a stable converged structure. Hence the FDD compound can be a potential drug candidate for SARS-CoV-2 main protease.

CRediT authorship contribution statement

Jamelah S. Al-Otaibi: Conceptualization, Methodology, Data curation, Writing – original draft, Software, Validation. Y. Sheena Mary: Conceptualization, Methodology, Data curation, Writing – original draft, Software, Validation. Y. Shyma Mary: Conceptualization, Methodology, Data curation, Writing – original draft, Software, Validation. R. Niranjana Devi: Conceptualization, Methodology, Data curation, Writing – original draft, Software, Validation. Sreejit Soman: Conceptualization, Methodology, Data curation, Writing – original draft, Software, Validation.

Declaration of Competing Interest

The authors declare no conflict of interest.