Computational, Molecular Docking, ADME/T And AIM Investigations Of 7-(4-Nitrophenyl) Benzo [6,7] Chromeno[3,2-E] Pyrido[1,2-A] Pyrimidin-6(7H)-One

Pyrimidines represent a versatility in N-heterocyclic compounds and displayed a verity of advantages such as low toxicity, good solubility, simple synthetic methods and low cost [1]. From past decayed pyrimidine derivatives have received considerable attention in biological and pharmacological applications, such as anticancer [2], antitumor [3], antiproliferative [4], anti-HIV [5], anti-inflammatory [6], antioxidant, anticonvulsant [7], antihypertensive [8], anti-Alzheimer's [9], antitubercular [10], anticoagulant [11], antiviral [12], antifungal [13], antimicrobial [14], and antimalarial [15]. Pyrimidines have fluorescence sensors, optical chemosensors, photosensitizers, photocatalysis as well as latent fingerprint applications because of its photophysical properties (Fig.1) [16]. In current scenario Multicomponent reactions (MCRs) have recently gained considerable economic and ecological interest as they address basic principles of synthetic efficiency and reaction design. Multi-component reactions have been endorsed to be a very exquisite and expeditious way to access complex structures in a single synthetic operation from simple building blocks, featuring high atom economy and high selectivity [17]. As a one-pot reaction, MCRs generally afford good yields, and they entirely differ from two-component reactions in several facet [18,19]. On the other hand, conceptual DFT is a branch of Density Functional Theory that has been assembled and enforced in distinct branches of chemistry [20]. Using computational approaches to explore various aspects of a molecule offers a significant time and financial advantage. Thus, atomic charges, molecular shape, dipole moment, reactive descriptors, NBO, etc are all calculated using DFT [21].  Some geometrical and structural parameters were computed and calculated for any pyridopyrimidine derivatives [22], but no work was published for the theoretical calculation about heteroannulated 2H-pyrido[1,2-a] pyrimidin-2-one. On the basis of mathematical and statistical relationships pathways are decisive for anticipating the biological effects of chemical stuffs [23]. The ongoing work is directed to utilize multi component reactions of 2H-pyrido[1,2-a] pyrimidin-2-one (1) for building a new series of 2H-pyrido[1,2-a] pyrimidin-2-one and their related compounds, in addition to evaluate their antimicrobial activity [24]. Further, DFT at the B3LYP/6-311G(d,p) level was used to correlate the theoretical data from the DFT calculations with the experimental results of synthesized 7-(4-nitrophenyl)benzo[6,7]chromeno[3,2e]pyrido[1,2-a]pyrimidin-6(7H)-one  using Gaussian 09programme. Moreover, Molecular docking, AIM and ADME, experimentally and theoretically based on DFT as well as predicting their pharmacokinetics and drug-likeness properties using efficient in silico methods [25].

Merck and Sigma Aldrich branded chemicals were purchased from the local seller for the completion of current research work. The synthesized compound was conveniently dissolved in chloroform (CHCl₃) and this solution led to initial confirmation of synthesis by thin layer chromatography. Ethyl acetate and n-hexane in wavering proportion collectively made the mobile phase while coated silica on aluminum plate incorported the stationary phase and was observed under ultraviolet light of 254 nm wavelength. The FT-IR characterization was performed by KBr pellet method in Jasco FTIR spectrometer was used for the confirmation of functional groups present in different compounds. The further characterization was performed by ¹HNMR and ¹³CNMR using Bruker spectrometers, operated at 600 and 150 MHz, respectively. The reference used was tetramethylsilane (TMS) while chloroform was used as solvent. Furthermore, the confirmation was also substantiated by EI-MS spectra using Jasco-320-A spectrophotometer.

2.1 Synthesis of 7-(4-nitrophenyl) benzo[6,7]chromeno[3,2-e]pyrido[1,2-a]pyrimidin-6(7H)-one: The synthesis of 2H-pyrido[1,2-a]pyrimidine-2,4(3H)-dione (3), m.p. 296–298 ?   by the reported method. The ascertained analytical and spectral data were found in entire obedience with the literature values. The reaction of p-nitrobenzaldehyde 5 (1.0 mmol), 2-naphthol (4) (1.0 mmol), DMSO (1-2 drops) and 2H-pyrido[1,2-a] pyrimidine-2,4(3H)-dione (3) (1.2 mmol) in refluxing toluene for about 24-27 hrs was performed as a model reaction in the presence of catalyst P₂O₅ (20 mol). The desired product resulted in good yields. To explore the generality of the reaction, we extended our study using P₂O₅ (20 mol %) as catalyst in toluene solvent [26] (Scheme 1.).

The Gaussian 09 software package was used for all DFT computations and the Gauss View 5.0.8 tool was used to frame-up the outputting. At the B3LYP (Becke’s three parameter hybrid model adopting the Lee-Yang Parr correlation functional) level of DFT with 6-311G(d,p) basis set [27], quantum chemical calculations such as optimized geometries and corresponding molecule orbitals (MOs) energies, molecular electrostatic potential (MEP), and Fukui functions were accomplished. ¹H NMR chemical shifts were performed with the Gauge-Including Atomic Orbital (GIAO) approach by using the aforesaid basis theory level and compared with the experimental ¹H NMR chemical shift values. Subtracting the calculated absolute chemical shielding of TMS, the ¹H NMR chemical shifts were transferred into the TMS scale B3LYP/6-311G(d,p) [28]. The molecular electronic dipole moment, polarizability, and the first hyper-polarizability were all described using the same level of the DFT.

2.4. ADME analysis

Pharmacokinetics and physicochemical features of formed stuffs were approximated using computational analysis. ADME is enforced for the development of drugs to be successful. This tool offers a wide range of factors, including drug-likeness rules, lipophilicity, molar refractivity, molecular weight, number of rotatable bonds, hydrogen bond acceptor, hydrogen bond donor, and medicinal chemistry access [29].

3.1 RESULTS AND DISCUSSION

3.1. Characterization of the synthesized compound (6)

Dark brown solid; m.p.: 263-265 ? ; Yield: ~67%; R_f: 0.57; [Hexane: Ethyl acetate] (9.0:1.0 v/v) as mobile phase; IR (KBr) cm^-1: 3062 (=CH stretching); 2922 (-CH stretching), 1795 (C=O stretching), 1595 (-C=C- stretching), 1514 (-NO₂ stretching); 1340 (-C-N stretching); 1107 (-C-O stretching), ¹H NMR (CDCl_3, 300 MHz) δ (ppm,): 8.20-8.17 (d, 3H), 7.91-7.89 (d, 1H), 7.75-7.73 (d, 2H), 7.60-7.43 (m, 6H), 7.39-6.32 (d, 1H), 7.16-6.50 (d, 1H), 4.13 ( s, 1H),  ¹³C NMR (CDCl_3, 75 MHz) δ (ppm): 67.00, 114.59, 116.89, 120.86, 122.70, 123.41, 126.02, 127.64, 127.80, 127.89,128.43, 129.74, 129.89, 131.27, 145.11, 147.59, 150.85, 166.64; ESI-MS: m/z 422.7 [M+1]⁺, Calculated m/z=421, molecular formula: C₂₅H₁₅N₃O₄.

3.2. Theoretical studies

3.2.1 ¹H and ¹³C NMR spectroscopy

¹H and ¹³CNMR chemical shifts were calculated with Gauge Independent Atomic Orbital (GIAO) approach using DFT method and B3LYP/6-311G(d,p) as basis sets [30]. The experimental and theoretical values of ¹H and ¹³C NMR chemical shifts of the studied compound are given in Table 1. The correlation graph between the experimental and calculated chemical shifts for ¹H and ¹³C NMR are shown in Fig. 2 and Fig. S1, S2, S3 displayed ¹H, ¹³C NMR and mass spectra respectively. The correlation graph follows the linear equation, y=0.501x+61.36  using HF for ¹H NMR and y=0.696x+2.384  for ¹³C NMR where ‘y’ is the ¹H NMR and ¹³C NMR experimental chemical shift and ‘x’ is the calculated ¹H NMR and ¹³C NMR chemical shift (in ppm). The correlation values (R² = 0.624 using B3LYP/6-311G(d,p)) for ¹H NMR and (R² = 0.727 using B3LYP/6-311G(d,p)) for ¹³C NMR shows that the correlations between experimental and the calculated chemical shifts are very good.

The UV-Visible spectrum of compound has been studied by TD-DFT method using B3LYP/6-311G(d,p)  basis sets and solvent effect has been taken into consideration by implementing Integral Equation Formalism Polarisable Continuum Model (IEFPCM). The vertical excitation energies, oscillator strengths (f), percentage contribution of probable transitions and corresponding absorption wavelengths along with simulated UV data have been tabulated in Table 2 and compared with experimental results [31]. The intense electronic transitions at 334, 246 and 189 nm with oscillator strengths ƒ = 0.7965, 0.2392 and 1.1334 in CHCl₃ are anticipated, showing an agreement with the measured experimental data (λexp. = 326, 260 and 205 nm in CHCl₃) as shown in Fig. 3. Electronic transitions from HOMO to LUMO, HOMO-1 to LUMO+1, HOMO-1 to LUMO+5, HOMO-2 to LUMO+2, HOMO-2 to LUMO+3 and HOMO-2 to LUMO+4 with 34%, 48%, 40%, 36%, 28% and 42% contributions respectively. The corresponding theoretical peak in the TD-DFT UV spectrum is at 226nm. These transitions are due to n→ π* and π →π*transition. Molecular orbitals HOMO-LUMO, (HOMO-1)-LUMO, (HOMO-1)-(LUMO+1) and (HOMO-1)-(LUMO+3) of the compound are shown in Fig 4.

The vibrational analysis of molecule shows the presence of 47 atoms which belong to C1 point group possessing 138 normal modes of vibrations. Representative experimental FT-IR bands together with calculated wave numbers (scaled) and their assignments are given in Table 3. The value of correlation coefficient (R²=0.998 using DFT) showed an excellent correlation between experimental and calculated wave numbers. Fig. 5 represents the correlation graph and FT–IR spectra (experimental and calculated) are shown in Fig. 6. The hetero aromatic organic compounds and their derivatives are structurally very close to benzene and commonly exhibit multiple weak bands in the region 3100–3000 cm^-1 which is the characteristic region for the identification of C?H stretching vibrations and these vibrations are not found to be affected due to the nature and position of the substituents [32]. In the present work, the hydrogen atoms around the pyridopyrimidine ring of molecule give rise three C?H stretching mode at 3062 cm⁻¹ experimentally. The calculated frequencies for these vibrations were obtained in the range of 2994 cm⁻¹ using the B3LYP/6-311G(d,p) methods. A strong band at 1795 cm^-1 in FT-IR spectrum corresponds to -C=O stretching vibrations. The wave number calculated at 1715 cm^-1 for B3LYP/6-311G(d,p) level assigned to the -C=O stretching vibration in the molecule. The stretching mode of the -C-N group is found to be at 1340 cm^-1 which shows good agreement with the experimental value i.e. 1343 cm^-1. The -C-O stretching vibrations are generally observed at around 1150-1050 cm^-1. In the experimental spectrum, the band observed at 1107 cm^-1 has been assigned for -C-O stretching vibration and is in good agreement with the calculated value at 1062 cm^-1.

MESP provides a visual method to understand the relative polarity of a molecule and used to identify the sites for hydrogen bonding interactions [33]. MESP of the molecule are shown in Fig. 7. The range of total electron density lies from -2.28 x 10^-2 to +2.28 x 10^-2 electrons per cubic Bohr.

Fig. 7 3D plot of the molecular electrostatic potential of molecule

The Natural Bond Analysis (NBO) [34] has been performed using Gaussian09 package at the B3LYP/6-311G(d,p). It explains charge transfer or conjugative interactions in molecular systems, intra and intermolecular bonding and interaction among bonds. The corresponding results are presented in Table 4.

NBO Analysis Showed That Intramolecular Charge Transfer Is:

5. From n(C5-N10) to π*(C1-C6) antibonding orbitals with stabilization energy of 42.69 kJ/mol.
5. From π (C2-C3) to π*(C1-C6) antibonding orbitals with stabilization energy of 26.92 kJ/mol.
5. From n(C5-N10) to π*(C1-C6) antibonding orbitals with stabilization energy of 11.62 kJ/mol.
5. From n(C9-O29) to π*(C7-C8) antibonding orbitals with stabilization energy of 35.78 kJ/mol.
5. From π (C12-C15) to π*(C13-C18) antibonding orbitals with stabilization energy of 34.23 kJ/mol.
5. From π (C13-C18) to π*(C12-C15) antibonding orbitals with stabilization energy of 35.97 kJ/mol.
5. From π (C19-C20) to π*(C21-C22) antibonding orbitals with stabilization energy of 31.18 kJ/mol.
5. From π (C21-C22) to π*(C19-C20) antibonding orbitals with stabilization energy of 62.73 kJ/mol.
5. Electron donated from LP(1) of N10 to π* (C13-C18) leads to the stabilization energy of 52.92 kJ/mol.
5. Electron donated from LP(1) of N4 to π* (C21-C22) leads to the stabilization energy of 62.73 kJ/mol.

3.2.6 Nonlinear optical analysis

The first hyperpolarizability of the studied compound was computed using the B3LYP/6-311G(d,p) basis set and the total dipole moment μ, the average polarizability α_tot and the first hyperpolarizability β_tot were calculated [35].

Since the values of the polarizabilities α and the hyperpolarizability of Gaussion output are reported in atomic mass units (a.u.), the calculated values have been converted into electrostatic units (esu) (α: 1 a.u = 0.1482×10⁻²⁴ esu; β: 1 a.u. = 0.0086393×10⁻³⁰ esu). The results of electronic dipole moment μi (i = x, y, z), polarizability αij and first order hyperpolarizability βijk are presented in Table 5. The calculated dipole moment, polarizability α_tot and first hyper polarizability for the title compound are equal to 10.9928 D, 46.35×10⁻²⁴ esu and 50.7636 ×10^-30 esu respectively for DFT level.

Thermodynamic parameter of title compound, including zero-point vibrational energy, rotational temperatures, rotational constants, energies at standard temperature (298.15 K) were obtained at B3LYP/6-311G(d,p) basis set and are presented Table 6 [36]. Many useful information’s can be generated from these thermodynamic data and can be used to compute other thermodynamic energies, according to the relationships of thermodynamic functions and estimate directions of chemical reactions according to the second law of thermodynamics in thermo chemical field. All thermodynamic calculations were done in gas phase and they could not be used in solution.

3.2.8 Global Reactivity descriptors

A good approach to predict global reactivity trends is to compute reactivity descriptors such as electronegativity (χ)=–1/2(εLUMO+εHOMO), chemical potential (μ)=1/2(εLUMO+ εHOMO), global hardness (η)=1/2 (εLUMO – εHOMO), global softness (S)=1/2η,        ?Nmax =–μ/η and electrophilicity index (ω)=μ2/2η. In molecular systems, Koopman’s theorem is fundamentally used to ascertain chemical reactivity and site selectivity [37]. The above reactivity descriptors have been calculated and listed in Table 7.

AIM approach shows the molecular graph of compound at B3LYP/6-311G(d,p) level is presented in Fig 8. The strong, medium, weak H-bonds and their covalent, partially covalent and electrostatic nature can be denoted by ρ(BCP) < 0 and HBCP < 0, ρ (BCP) > 0 and HBCP < 0 and ρ(BCP) > 0 and HBCP > 0 respectively [48]. ρ(BCP) and HBCP are Laplacian of electron density and total electron density at bond critical point respectively. Parameters (geometrical and topological) for bonds of interacting atoms are given in Table 8. As all the (BCP) and HBCP parameters were greater than zero hence C24—H44···O29 is weak interactions. The ellipticity (ε) at BCP is a sensitive index to monitor the n character of bond. The lower values of ellipticity confirm that there is delocalization of electron in aromatic ring [38].

ADME is significant for inspecting the pharmacodynamics of material that could be used as target agents in drug revelation and design efforts. Numerous factors are provided by this tool, including lipophilicity, drug-likeness guidelines, and medicinal chemistry techniques [39]. Further, ADME is a balance of discrete structural and molecular properties which tested to attain efficient results to aid in drug discovery and formulation. So, it is the most novel method recommended to identify compounds that are contemplated for use in drugs that must respect certain rules that are significant. In addition, it plays a very substantial role in the very early phase of drug discovery to diminish the failing rate of drug candidates in clinical trials. By using the pharmacokinetics and physicochemical properties of compounds, we can predict pre- determine their drug-likeness and oral bioavailability, which would relate to their drug bioactivities. The synthesized molecule shows druglike properties without straying from either Lipinski’s rule of five, Ghose or Veber’s criteria, according to an analysis of these features (Table 9) [40]. Physicochemical properties are in the following: Firstly, a crucial factor in determining molecular lipophilicity is logP. In this study, logP value of the synthesized compound is 3.28, which is also within an acceptable range. Therefore, the current molecule endorsed strong permeability and absorption across the cell membrane with a lipophilicity "log P" (octanol-water partition coefficient) character 5.0 [41]. Secondly, molecule have molecular weights that are more than 160 g/mol and less than 500 g/mol, which makes it simpler for these substances to be absorbed than substances with high molecular weights. Thirdly, the current compounds having H-bond donors (HBDs) and H-bond acceptors (HBAs) less than 1 and 5, respectively. Fourth, a usually studied component identical with H- bonding (O and N atom counts) is total polar surface area (TPSA). All existing compounds have TPSA values that are lower than 140 Ų. Fifth, the bioactivity score of compounds occurs at 0.55 and, which is a critical criterion for drug-likeness property. Finally, molar refraction value of the synthesized compounds is 117.37, which is within an acceptable range (Fig. 9). Therefore, the current compound has effective drug delivery properties and may be preferred for oral administration. Since all compounds comply with the Lipinski (often known as the “rule of five”), Ghose and Veber rules, they should theoretically not pose oral bioavailability problems. According to pharmacokinetics properties from ADME results: Some of the present compounds have good responses for blood brain barrier (BBB) criteria, indicating that medications can cross the BBB. Consequently, these substances might be appropriate for the central nervous system. Additionally, the gastrointestinal absorption (GI) of all compounds was high except 8 and 9. The ability of a molecule to dissolve in both aqueous and non-aqueous media is crucial for the drug development process. Finally, skin permeability was measured by log Kp i.e. -6.09. The aforementioned data revealed that these compounds have a favorable ADME profile and drug resemblance.

In Molecular docking, structure-based drug design that simulates molecular and forecasts the binding affinities between receptors and ligands has been performed [42]. The molecule 6 interpretated with molecular docking inspection with the help of AutoDock 4.2.6 version of software. From the protein data bank, two proteins from the gene regulation-inhibitor complex (BL21(DE3)) with the PDBIDs 7DKP was invented. The binding energy of compounds 6 and the selected proteins of the 7DKP cell line was calculated using the Lamarckian Genetic Algorithm (LGA) in the AutoDock 4.2.6 programme [43]. 7DKP is an essential transcriptional and epigenetic regulator that influences the growth of microbes. According to molecular docking analyses, 6 reacted with several amino acids electrostatically, via Vander Waal interactions, and via hydrogen bonds. Two hydrogen bonds were formed with different amino acid residues at binding region of Asn33(b). The oxygen atom of pyridopyrimidine formed two hydrogen bonds with amino acid residue His8(B) and Asn33(B) at 3.32 Å and 3.37 Å respectively. The compound 6 showed highest affinity with protein of gene regulation-inhibitor complex (7DKP) (Fig. 13) with the total binding energy of -8.12 kcal/mol (Table 8). Hydrogen bonds and hydrophobic interactions presents between different amino acid residues of protein are shown in Fig. 14. Compound 6 formed ten hydrophobic interactions with the selected protein as Gln47(B), Tyr11(B), Val48(B), Cys9(B), Pro10(B), Phe105(B), Tyr6(B), Tyr121(B), Lys125(B), Lys126(B) (Table 10). The best docked conformations are dogged by scaling the RMSD (Root Mean Square Deviation) between the predicted AutoDock4 conformation and the real framework with low binding energy conformations and using the clustering histogram with various RMSD values. Therefore, as compared to the conventional medication (bevacizymab), 6 demonstrated high potential binding energy against certain proteins. The examination of particular amino acid residues and their interactions with researched compounds supports the hypothesis that 6 might be employed as a medication in the future following completion of other crucial investigations (Fig. 10, 11). This is due to the fact that while approved medications are still necessary for the more specialized proteins of the gene regulation-inhibitor complex, the binding affinities towards the chemical are comparable. This provides more proof that the investigated compounds alter the normal shape of protein and alter the active site when they bind to the suggested sites.

Fig. 10. The best docked conformation of compound with 7DKP of Escherichia coli representing different interactions.