Synthesis, characterization and identification of inhibitory activity on the main protease of COVID-19 by molecular docking strategy of (4-oxo-piperidinium ethylene acetal) trioxonitrate

2.1 Synthesis of (4-OPEAN) and its crystallization

The title chemical is created by adding drops gradually. an aqueous solution of HNO₃ acid (1 mmol) to an alcoholic solution (ethanol as solvent) containing 1 mmol of 1,4-dioxa-8-azaspiro [4. 5] decane under magnetic agitation. At room temperature, a gradual evaporation process is applied to the resulting solution. 4 days later forming clear, stable monocrystals of an appropriate size for structural analysis. The reaction can be represented schematically as follows:

2.2 Materials and physical data

The infrared (IR) spectrum in the 4000-400 cm⁻¹ region was recorded at room temperature using a Nicolet IR 200 FTIR spectrophotometer. 150 K was used as the temperature for the X-ray tests. By means of a diffractometer Bruker-AXS APEXII operating with a wavelength of 0.71107 Å for molibdene, collections intensities were measured on solitary crystals. The SADABS software was used to perform absorption corrections using the multi-scan method (Bruker, 2006). Using the SHELXT program (Sheldrick, 2015), the dual-space technique was used to solve the crystal structure, and then improved using F²-based full-matrix least-squares techniques (SHELXL software) (Sheldrick, 2015a)). Anisotropic atomic displacement parameters were used to refine all non-hydrogen atoms. Except Hydrogen atoms linked to Nitrogen atom that were introduced in the structural model through Fourier difference maps analysis, H atoms were finally included in their calculated positions and treated as riding on their parent atom with constrained thermal parameters. A final refinement on F² with 1159 unique intensities and 84 parameters converged at wR(F²) = 0.1762 (R_F = 0.0725) for 1113 observed reflections with I > 2σ. The X-ray data parameters, the method for determining the crystal structure, and the outcomes are all listed in Table 1. ORTEP (Farrugia, 2012) and the Diamond program (Brandenburg, 1998) are used to create the structure graphics.

2.3 Theoretical research

The theoretical computations were performed using the hybrid B3LYP/6-311++G (d,p) approach built within the Gaussian 09 program (Frisch, 2009). A 2D graph that summarizes the intricate information contained in a structure and a 3D graph that shows the area of space where the molecules are in touch form the basis of a Hirshfeld surface analysis. This allows for the identification of each type of interaction. The Crystal Explorer program was used to conduct this study (Wolff et al., 2013). The topological properties in this research were computed via means of the Multiwfn software (Lu and Chen, 2012) in accordance with Bader's theory (Kumar et al., 2016). A multipurpose wave function analyzer called Multiwfn was used to perform the RDG-NCI analysis, and the outcomes were visualized using the VMD molecular visualization tool. To investigate the molecular docking computation, iGEMDOCK software was employed (Yang and Chen, 2004), this application calculates the total energy of the basic ligand–protein interactions. Protein Data Bank (https://www.rcsb.org/pdb/) is where the enzyme blueprints can be found. The visual depictions of the docked ligands are created using molecular operating environment (Molecular, 2015).

3.1 Structure evaluation and geometry optimization

An anionic entity, NO₃⁻ and a monoprotonated cationic entity, 4-oxo-piperidinium ethylene acetal, make up the investigated structure's asymmetric unit (Fig. 1a). Medium and weak hydrogen bonds between these entities ensure their connection. The crystal structure of (4-OPEAN) is projected sequentially in the (ac) and (ab) planes in Fig. 2. In contrast to the organic groups, which are organized into dimers along the [0 0 1] direction, the NO₃⁻, nitrate groups form rows that develop in a position equal to 0 and 0.5 along the direction of the $\overrightarrow{c}$ axis. A three-dimensional of a series of hydrogen bandings, highlighting the O…H—N and O…H—C kinds of linkages, which ensures the cohesion and stability of our structure, provides the sequence for the two cationic and anionic halves. Analyzing the nitrate anion (Table S1) reveals that the nitrogen atom (N11) occupying general positions and forming a somewhat planar ionic structure with the oxygen atoms (O12, O3 and O14). In fact, the interatomic N—O bond lengths and O—N—O angle ranges are 1.231(4) – 1.254(4) Å and 119.4(3) – 121.1(3)°, respectively. The anions' geometric shape is similar to that which has been documented in the literature for homologous compounds (Gatfaoui et al., 2014a, b; Jmai et al., 2023). About the 4-oxo-piperidinium ethylene acetal's geometric properties, it is noted that this cation is made up of the combination of the two groups «1,3-dioxolane» and «piperidinium» intermingled at the carbon atom. With a mean plane deviation of zero, the first ring's planar portion contains two oxygen atoms, whereas the second ring displays a chair conformation. The organic cation then exhibits a predictable spatial arrangement, with the normal distances C—C, C—N, C—O, and angles C—C—C, C—C—N, C—C—O, O—C—O, C—O—C changing respectively on the scales [1.374(5)–1.523(4) Å] and [106.1(3)–111.8(3)°]. These distances and angles are equivalent to those in similar constructions such as Tetrakis(4-oxo-piperidinium ethylene acetal) bis sulfate (Marouani et al., 2011) and (4-oxo-piperidinium ethylene acetal) dihydrogen monophosphate monohydrate (Dhaouadi et al., 2010). The crystalline structure of the compound (4-oxo-piperidinium ethylene acetal) trioxonitrate is mostly built on the basis of interconnected system of moderately strong, average and weak hydrogen bonds (Table 2) including two to three centers of the O…H—N design involving the H1A and H1B hydrogen atoms, one with three centers of C—H…O type (C6—H6…O12, C6—H6…O13) and one with two centers involving the H3B atom (d_C3…O14 = 3.536(3) Å). Indeed the N1—H1A…O13 hydrogen bond is characterized as being moderately strong with a N1…O13 distance of 2.818(4) and a D—H…A angle that is extremely close to 180 degrees, around 177 (5)°. The crystalline edifices of (4-OPEAN) material are composed of mixed layers that are created by successive R₁²(4) and R₄²(12) cycles that are brought about by the interaction of hydrogen bonds between the organic cations and the nitrate anions.

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Fig. 1

Drawing of the 4-OPEAN using the atom-labeling method in ORTEP. At the 30% probability threshold, displacement ellipsoids are drawn. H atoms are shown as tiny spheres with variable radii (a) and the idealized molecular structure (b).

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Fig. 2

Projection of the atomic arrangement along the $\overrightarrow{b}$ and $\overrightarrow{c}$ axes and hydrogen bonding motifs of 4-OPEAN.

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Focusing now on the theoretical part, table S1 lists the geometric parameters of the 4-OPEAN that were investigated theoretically by DFT (B3LYP/6-311 ++ G(d, p)) and experimentally by X-ray diffraction, such as the bond lengths and angles. Thus Fig. 1b displays the geometrical optimization. The results show that these parameters nearly match the values obtained through experimentation. The C—C, C—O and C—N bonds in the moiety of the organic cation were calculated to be between 1.3267 and 1.5347 Å, and the C—C—N, C—N—C, O—C—O, C—C—O and C—C—C bond angles were determined to range between 105.7174 and 112.8089 degrees. With regards to the nitrate anion the N1—O24 and N1—O26 calculating distances nearly identical to those found by X-ray diffraction, as long as N1—O25 is elongated by 0.11 relative to the value in experimentation. This is comprehensible by the oxygen atom O25 being a part of the O25…H2—N1 hydrogen molecule link, which is fairly strong. Recapitulating, it can be seen that some theoretical errors exist because calculations are made in the gaseous state as opposed to experimental design is observed during the crystalline stage.

3.2 3D map projections and graphs of 2D fingerprints

Statistical evaluation and visualization of the supramolecular interactions in the title structure were done using Crystal Explorer. Fig. 3 shows the Hirshfeld surface projected over d_norm (Fig. 3a) with values ranging from −0.531 to 1.174 a.u and d_e (Fig. 3b) from 0.786 to 2.289 a.u. The relative positioning of the nearby atoms in the nitrate and 4-oxo-piperidinium ethylene acetal molecules that interact with one another can be graphically represented using the normalized contact distance. Using a color-coded surface (red, blue, white), this form of study shows the shortest intermolecular interactions detected by the red spots, which are linked to the N—H…O and C—H… O hydrogen bonds. Orange areas on the d_e map indicate this hydrogen bonding. In relation to the blue hue shown on the two graphs, she relates to the furthest intermolecular contacts in the structure in this area due to the too big of a distance between the nearby atoms and the existence of far-off connections as H… H, C… H, N… H, C… O and O… O (Gatfaoui et al., 2019, 2021; Kurbanova et al., 2023; Kansız et al., 2021 and 2021b). Fig. S1 shows the numerical results of the HS evaluation and includes the percentages of contributions from various types of connections and atoms in the crystal structure. We note that the H…O/O…H intermolecular interactions take up a significant portion of the Hirshfeld surface (55.8%) and appear in 2D fingerprint plots (Fig. 3c) shaped like wings with two equal sides with a significant long tip that is sum (d_e + d_i ̴ 1.9 Å) lower than the total of the van der Waals radii of the hydrogen (1.09 Å) and oxygen (1.52 Å) atoms. Such connections are responsible for the generation of N—H…O and C—H…O hydrogen ties. The two-dimensional fingerprint maps for H…H contacts show them as scattered points in the center, making up 35.8% of Hirshfeld's surface. A prolonged interatomic H…H contact is what causes the single round peak at d_e + d_i = 2.4 Å in Fig. 3d. By contrasting between the total van der Waals radii of the participating atoms and the sum of the components of the pairs of various contacts (d_i, d_e) existing in the structure just H…O/O…H considered to be close and involved in the production of hydrogen bonds which ensure the cohesion and maintenance of the crystalline structure. The enrichment ratio (ER), a novel term to describe a pair of chemical substances that is described as being the ratio between the ratio of actual crystal connections and the assumed ratio of random contacts, has been used to evaluate the various interactions present in our crystal. These enrichment ratios (ER) are computed and provided in Table S2. An inventory of enrichment ratios draws attention to the H…O/O…H contacts (ER_OH = 1.44), which appear to be preferred in the packing of crystal with the creation of the N—H…O and C—H…O hydrogen links. The enrichment ratio for the H…H interactions is about unity (0.84), which is in good agreement with Jlech's prediction (Jelsch et al., 2014). The ER_OO ratio is very low, close to zero, this is explained by the O … O contacts which are generally depleted, and thus the contact surface is poor in oxygen (S_O < 20%). Although the additional contributions to the Hirshfeld surface (C…O/O…C, N…C/C…N, C…H/H…C and H…N/N…H) are very modest, the ER values are not especially useful for some interactions.

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Fig. 3

d_norm (a), and d_e (b) cartography of 4-OPEAN Compound. Fingerprint plot of O…H/H…O (c) and H…H (d) connections.

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3.3 Quantum methods of topological analysis

3.3.2

3.3.2 Gradient of reduced density examination

A unique non-covalent interaction (NCI) descriptor based on a reduced density gradient (RDG) was constructed by Johnson et al (Johnson et al., 2010). It has been proven that this method can tell tiny molecules, molecular complexes, and solids apart when it comes to hydrogen bonds, Van der Waals interactions, and steric repulsion. The following expression is used to compute the RDG quantity: $$RDG\left(r\right)=\frac{1}{2{{(3\pi }^2)}^{1/3}}\frac{\left|\mathrm{\nabla }\rho (r)\right|}{{\rho (r)}^{4/3}}$$

As a function of the electron density multiplied by the sign of the second eigenvalue of the Hessian matrix, the reduced density gradient of (4-OPEAN) compound is depicted in Fig. 4a. It is easy to distinguish between the three zones thanks to the evolution of the RDG (a.u) as a function of sign λ₂*ρ according to a clear color scheme. While the areas that match to a hydrogen bond (ρ > 0, λ₂ < 0) or a steric effect (ρ > 0, λ₂ > 0) are related to high density, the van der Waals interactions usually display very modest values of the electron density (ρ ≈ 0, λ₂ ≈ 0). A closer look at Fig. 4b reveals the existence of light blue dots that indicate strong N—H…O attractive interactions between the atoms of hydrogen and oxygen. The round red spots situated in the center of the two piperidinium and 1,3-dioxolane rings are attributed to describe the destabilizing steric interaction. While the van der Waals bonds are visible as green specks that are situated between the hydrogen atoms.

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Fig. 4

Scale bar with colored data delineating the interaction boundaries for the RSDPN compound is plotted along with the decreased density gradient (a) and the density of an isosurface (b).

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3.4 Molecular electrostatic potential

Based on electronic density and the locations of a molecule's chemical reactivity, molecular electrostatic potential (MEP) provides details about the total charge distribution of the molecule. This instrument enables the identification of electrophilic (regions of positive electrostatic potential) and nucleophilic (regions of negative electrostatic potential) sites, and as a result, the determination of the likelihood that intra or intermolecular hydrogen bonds will form. Fig. 5a shows the molecular electrostatic potential (MEP) maps of the (4-OPEAN) molecule using the following color-code range: −6.100 10⁻² (red denotes the strongest repulsion) to 6.100 10⁻² (blue indicates strongest attraction), the green color indicates a neutral zone (zero potential). According to the MEP study, the oxygen atoms in the nitrate unit are where the most negative potential zone is concentrated (red and yellow hue). Contrarily, the most positive sites (in blue) are concentrated mostly around the ammonium NH₂⁺ grouping of the cationic portion and point to the sites that are electron-deficient and the most vulnerable to nucleophilic attack. Then again,the development of hydrogen bonds between the nitrate groups and the 4-oxo-piperidinium ethylene acetal cation and their significance in the stability of (4-OPEAN) are also explained by the electrophilic and nucleophilic sites.

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Fig. 5

Molecular electrostatic potential (MEP) (a) and Frontier molecular orbital (b) of 4-OPEAN crystal.

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3.5 HOMO–LUMO energy

Frontier molecular orbital theory (FMO theory) is a result of Kenichi Fukui's (Fukui, 1975) study of frontier orbitals, namely the impact of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) on reaction processes. These orbitals contribute significantly to the chemical stability of the molecule and are crucial for the electrical characteristics and UV–visible spectra The HOMO-LUMO gap, which measures the energy difference between HOMO and LUMO, is used to forecast the strength and stability of materials, therefore a large molecule's stability is indicated by a high gap, contrariwise small gap is a high-reactivity criterion. Fig. 5b depicts the (4-OPEAN) material's border orbitals (HOMO, LUMO), as well as the gap energy determined in the gas phase. The MO plot's positive and negative phases are portrayed by the colors red and green, respectively. The predicted energies of HOMO and LUMO are approximately −5.47 eV and −1.36 eV respectively, favoring a Gap in the vicinity of 4.11 eV. This high number suggests, in some ways, a low chemical reactivity toward chemical reactions and a high kinetic stability. Chemical hardness and softness are reliable predictors of a molecule's chemical stability in addition to the energy gap. Another crucial element of a material electrical structure is its dipole moment. The results of the calculations are then displayed in Table S4 along with additional quantum characteristics (electronegativity and electrophilic index). Indeed, I = −E_HOMO and A = −E_LUMO are the relationships that link the energies of the frontier orbitals HOMO and LUMO to their respective ionization potentials I and electronic affinities A. Mulliken (Mulliken, 1934) defines the electronegativity χ as follows: $\mathrm{\chi }=\frac{A+I}{2}$

The correlations shown below (Pearson, 1997) determine chemical hardness and softness: $$\mathrm{\eta }=\frac{I-A}{2};\mathrm{s}=\frac{1}{\mathrm{\eta }}$$

Parr et al. (Parr et al., 1999) defined the electrophile index ψ as: $\mathrm{\psi }=\frac{{\mu }^2}{2\mathrm{\eta }}$

The chemical potential represented by in the final statement is given by: $\mathrm{\mu }=-\frac{I+A}{2}$

The composite object of our investigation has a chemical softness of 0.49 eV and a hardness of 2.05 eV in the gaseous phase. Low is a sign of a good nucleophile, and vice versa, our chemical can be regarded as an excellent electrophile because the electrophilic index in our case is high at 2.84 eV. Furthermore, the chemical potential is negative (-3.41 eV), which indicates that our crystal is stable and does not spontaneously break down into its constituent parts, a well-known characteristic of biologically active compounds. It's important to note that these outcomes mirror those obtained with other organic nitrates (Gatfaoui et al., 2020, 2017a, b, 2020).

3.6 Infrared spectrum

The investigation of produced nitrate using IR absorption spectroscopy enabled us to identify the vibrational properties of the atomic groups in the (4-OPEAN) molecule. The experimental IR spectra and their theoretical counterpart are shown in Fig. 6. Table S5 provides a list of the estimated and experimental vibrational frequencies as well as descriptions of normal modes. The assignment efforts are based on prior findings in the literature as well as the hypotheses generated via group theoretical research (Marouani et al., 2011; Dhaouadi et al., 2010; Gatfaoui et al, 2017a and 2017b).

• o

  Vibrational modes of the 4-oxo-piperidinium ethylene acetal cation

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Fig. 6

FT-IR spectra of the 4-OPEAN compound, both theoretical and practical.

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In its protonated state, the organic group exhibits valency and bending vibrations between 3400 and 2500 and 1650 and 1300 cm⁻¹, respectively. The theoretical values that correspond to these vibration range between 3100 and 2200 and 1750 and 1300 cm⁻¹. The interaction of the intermolecular hydrogen bond N—H…O, whose mode is calculated to be approximately 3050 cm⁻¹, explains the broad band's mode of stretching, which was experimentally detected at 2842 cm⁻¹. The —CH₂ groups' stretching modes can be found in the spectral region [3100–3000 cm⁻¹]. The symmetric stretching of the —CH₂ group is responsible for the band at 3077 cm⁻¹; the matching experimental mode can be seen at 2842 cm⁻¹. At 2975 cm⁻¹, the —CH₂ asymmetric stretching peaks are visible. Calculations for these modes are done at 3100 cm⁻¹. (—CH₂) bending vibrations are responsible for frequencies in the 1700–1300 range. Theoretically, they are worth 1532 and 1309 cm⁻¹. Regarding the C—C, C—N, and C—O groupings that make up the piperidinium and dioxolane nuclei, vibration modes have been experimentally identified in a frequency range between 1200 and 1000 cm⁻¹. These modes are anticipated to range between 1200 and 1080 cm⁻¹ in theoretical terms. δ(CC), δ(CN), and δ(CO) were ultimately demonstrated by experimental peaks at the [915—500 cm⁻¹] domain. Theoretically, these are offered in the spectral space from 915 to 720 cm⁻¹.

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  Vibrational modes of the NO₃⁻ anion

The nitrate group has D3h symmetry in the free state and has the following normal vibrational modes: A_1′(ν₁), A_2′’(ν₂), and E’(ν₃ and ν₄); ν₁ is Raman active, ν₂ is IR active, ν₃ and ν₄ are both IR and Raman active. All of these vibrations are predicted to occur at respective frequencies of 1049, 830, 1355, and 690 cm⁻¹. In the IR spectrum experiment, the strong peak for the NO₃⁻ asymmetric vibration that stretches is located at 1338 cm⁻¹. At 1532 cm⁻¹, the DFT technique predicts the same stretching vibration. Experimentally and theoretically, extra stretching vibrations v_s(NO₃⁻) were observed at 1181 and 1302 cm⁻¹, respectively. The experimental band that was measured at 765 cm⁻¹ and computed at 825 cm⁻¹ is a result of the plane's distortion. Regarding the out of plane bending vibrations of NO₃⁻, the experimental peaks are located at 832 cm⁻¹ (calculated at 916 cm⁻¹).

3.7 Chemical docking research

The discipline of molecular modeling, and more specifically molecular docking, often known as rapidly brought into the field of biological study with the advent of electronic devices during the previous twenty years. This method is employed to investigate the interactions between two molecules, often a protein and a ligand. These interactions are crucial to the organization of biological systems because they permit the control of some biological processes, the signaling of events, or the catalysis of various biochemical reactions. In our study, a molecular docking analysis of the ligand (4-oxo-piperidinium ethylene acetal) trioxonitrate (4-OPEAN) with COVID-19/7EJY and COVID-19/6Y84 receptors was performed. These proteins are intriguing targets for the development of novel drugs against disease, according to recent research. The outcomes of the calculations allow for the possibility of ten ligand positions within each protein's active site, with the optimal position having the lowest energy and being associated with the most stable complex. These ideal locations are shown in Fig. 7 and Fig. S4. Table 3 compiles information about the computation of conformational interaction energies, including total energy scores, hydrogen bond energies, and VDW interactions. The following findings are obtained from a tabular examination: The overall energy scores for the inhibitor (4-OPEAN) interacting with the 6Y84 and 7EJY proteins, respectively, is −80.0643 and −73.1571 kcal/mol. Their VDW interactions are about −51.133; −54.1381 kcal/mol. High hydrogen bond energy of −20.0932 kcal/mol for (4-OPEAN/7EJY) and −29.6289 kcal/mol for (4-OPEAN/6Y84) is also produced by the molecular interaction of the ligand in the active sites of the two enzymes. Furthermore, Table S6 analysis shows that the root mean square deviation (RMSD) of this compound in each system in the order of 1.0299 (6Y84) and 1. 7153 (7EJY), so since its values are less than three, they help to stabilize the system and support the validity of our docking results. 6Y84 and 7EJY have corresponding binding score values of −3.1968 and −3.0197 kcal/mol. Visual examination of the 3D and 2D representations of the 4-OPEAN ligand in the active sites of 6Y84 and 7EJY (Fig. 7, Fig. S4 and Table S7) demonstrates that the atoms C2, O13 and O5 are connected to the binding residues A: CYS 145, A: ASN 142, TYR 140, LYS 125 and A: SER 126 via conventional hydrogen bonding interactions, with distance ranges varying between 2.74 and 3.81 Å and energy values ranging from −0.7 to −3.5 kcal/mol for the two complexes. As a result, considering the energy scores, all the findings mentioned above, and previous research, we can consider our ligand to be a potential inhibitor in the treatment of COVID-19 diseases because it penetrates well into the active regions of the receptors.

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Fig. 7

3D best docked poses and 2D interactions of 4-OPEAN compound with the 6Y84 protein.

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