Evaluation of non-covalent interactions of chlorambucil (monomer and dimer) and its interaction with biological targets: Vibrational frequency shift, electron density topological and automated docking analysis

4.1 Geometry optimization

The spatial conformations around all the possible rotational bonds have been searched using MMFF94s force field and full-matrix Newton-Raphson optimization method available in CONFLEX (Goto et al., 2012; Goto and Osawa, 1989,1993). Further optimization has been done using wB97XD and B97D functional with Grimme's D2 (GD2) dispersion correction and M06-2x functional with Grimme's D3 (GD3) dispersion correction so as to consider the long-range interactions into account. The optimized structural bond lengths of monomer and dimer structures of chlorambucil are given in Table 1 and their bond angles and dihedral angles are depicted in Table S2 (Supplementary Material). The lowest computed energy obtained for the conformer A shows its dominant stability than the conformers B and C. The relative energies of the conformers with respect to the most stable conformer A at different levels of theories are depicted in Table 2. The optimized molecular structures of these three conformers and the overlapping of one over another are shown in Figs. 1 and 2, respectively. It is clear from the Fig. 2 that all the studied conformers are structurally independent. Hence, we may expect distinct physicochemical properties, intramolecular bond patterns and spectroscopic fingerprints from each conformer. When we look at the Tables 1 and S2 (Supplementary Material), we can easily observe the noticeable changes where the non-covalent interactions taking place. For example, due to the diverse orientations, the bond length of C9C16 of conformer A and B is deviated from the conformer C by 0.0120 Å. Likewise, the significant changes were also identified in conformer A with respect to other bonds C16H32 and C16H33. Furthermore, the bond angle parameters C16C9H22, C16C9H23, and C9C16C19 of each conformer are found to be deviated by an angle 2–4° from one another because of different orientations and intramolecular bonding patterns. To avoid the complexity in understanding the three-dimensional structures for the readers, the Cartesian coordinates of all the conformers are depicted in Tables S3 (Supplementary Material).

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Fig. 1

Optimized structure of chlorambucil.

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Fig. 2

Overlapping structure of one conformer over another (Conformer A, B and C are shown in Red, Yellow and Green, respectively.)

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4.1.2

4.1.2 Two dimensional NMR spectrum of chlorambucil

¹H–¹³C HSQC spectra provides correlation between the aliphatic and aromatic carbon in the chlorambucil and its attached protons. To make the precise assignments, the experimental contour plots were assigned on the basis of predicted chemical shifts of ¹³C and ¹H NMR. In the spectrum shown in Fig. 3, proton chemical shifts are recorded along x-axis and carbon chemical shifts are recorded along y-axis in order to correlate the protein with their directly attached carbon nuclei. The ¹H signals at 6.61 and 6.63 ppm correlates with ¹³C signal at 112 ppm which is ascribed to C14-H30 and C15-H31 bonds. Another two ¹H signals at 7.06 and 7.08 ppm correlates with ¹³C signals at 130 and 129 ppm, respectively are assigned to C13-H29 and C12-H28 bonds. Multiple ¹H signals near 3.7 and 3.6 ppm correlates with ¹³C signals at 54 and 41 ppm, respectively are attributed to the hydrogen atoms attached to carbon nuclei of —CH₂CH₂Cl group. The downfield ¹H signals in the spectrum coincides with the downfield ¹³C signals are found to be the signals of C—H bonds of CH₂ groups linked with —COOH group.

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Fig. 3

2D-HSQC NMR spectrum of chlorambucil.

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4.2 Intramolecular charge delocalization

In case of electronic structure calculations, the intramolecular charge delocalization plays a vital role in the chemical stability. The stabilization energies obtained in the NBO calculations clearly demonstrate the patterns of charge delocalization from various lone pair atomic orbitals (Lewis base) to antibonding orbitals (Lewis acid). Recent contributions from Saeed and his co-workers proposed the importance of “remote” donor-acceptor interactions for explaining the conformational properties of molecular systems via the resonance interactions between lone pair (LP) electrons and anti-bonding (BD^∗) orbitals (Saeed et al., 2014; Saeed et al., 2015; Saeed et al., 2016). In chlorambucil conformers, the LP → BD^∗, BD → BD^∗ transitions exists and the strength of these interactions in LP → BD^∗ is more significant than the BD → BD^∗. It is seen from the Table 3 that the charge delocalization patterns of the conformers A and C are almost equal qualitatively and conformer B has different patterns of transitions. In both the conformers A and C, the major stabilization energies of about 52.68, and 131.29 kcal/mol, respectively drawn from the transition LP(1)O3 → BD^∗(1)C18-H37. The charges transferred from O3 to the remote part (C18-H37) in this case may cause a geometrical rearrangement in the molecule. When rearrangement of molecule occurs, one can expect the shortening of X-H bond lengths (Coates, 2006). Since the C18 is attached to highly electronegative Cl2 and its natural charge is just half time that of the O3, the possibility of an improper hydrogen bond is limited. While in conformer B, the major stabilization energy of about 36.2 kcal/mol is obtained for LP(2)O3 → BD^∗(2)O4-C19. Besides, the charge delocalization from the bonding to anti-bonding orbitals (BD → BD^∗) shows a significant contribution to the stability of a molecule. The charge transfers from BD(1)O3-H38 to BD^∗(1)C18-H37 stabilizes the chlorambucil conformers A and C by 39.41 and 86.31 kcal/mol, respectively. In conformer B, the delocalization charge within the aromatic ring stabilizes the molecule by an energy 17.06 kcal/mol.

The predicted stabilization energies of interacting intermolecular species from unit 1 to unit 2 and vice versa for the chlorambucil dimer of conformer A are given in Table S4 (Supplementary Material). The charge delocalization from the lone pair oxygen (O4 and O41) and chlorine (Cl2 and Cl39) atoms of both the units to the anti-bonding species in the remote parts stabilizes the dimer to a greater extent. The stabilization energy of about 21.45 kcal/mol predicted due to an interaction between the lone pair LP(2)O42 and BD^∗(1)O3-H38 indicates the hydrogen bonding of O42⋯O3-H38 as shown in Fig. S1 (Supplementary Material).

4.3 Electron density topological and isodensity surface analysis

The theory of atoms in molecules enables us to determine the volume of electron density of atoms in a system and the changes in these volumes caused by a chemical reaction. Atoms in molecule (AIM) theory is a convenient method used to analyze the H-bonding and other interactions in a various molecular system in terms of critical points. The formation of a hydrogen bond is the result of an interaction between two closed-shell systems, and this is reflected in the properties of the charge distribution, particularly in its Laplacian, the quantity ∇²ρ(r) (Bader and Essen, 1984). The Laplacian of the electron density measures locally concentrated ∇²ρ < 0 and depleted ∇²ρ > 0 within a molecular system. QTAIM study the concept of chemical bond and bond strength in terms of topological features, electron density distribution function and energetic parameters. Geometrical as well as topological parameters are a useful tool to characterize the strength of hydrogen bond. The geometrical and topological parameters corresponding to the critical points which are connecting two non-bonded atoms in all three conformers of chlorambucil are given in Table 4. The geometrical parameters for the hydrogen bonds in all conformers are given in Table 5. On the basis of these parameters, there exist three non-conventional bonds in chlorambucil, two H H bonds which are different from dihydrogen bond (Reyes-Márquez et al., 2008; Hernandez-Trujillo and Matta, 2007) and the other O H bond such as H29 H24, H26 H28, H20 O4 in conformer A and H28 H24, H29 H26, H21 O4 in conformer B and H29 H26, H28 H24, H30 O4 in conformer C as shown in Fig. 4. The obtained results indicate that the strength of the hydrogen bonds are medium in nature as they satisfy the criteria ∇²ρ_BCP>0, HBCP < 0 proposed (Rozas et al., 2000). The relation between H-bond energy (E) and potential energy density (V_BCP) at H O/N contact: E = 1/2(V_BCP) is proposed (Espinosa et al., 1998). According to this equation, the interaction energy of H20 O4, H21 O4 and H30 O4 is calculated as −7.85, −7.98 and −5.6446 kJ/mol in conformer A, B and C which shows that hydrogen bonds (O H) are stronger in conformer A and B with comparison to conformer C. The bond ellipticity (ε) is a measure of nature and strength of the bond between the non-bonded atoms. The predicted values of ε in atomic units confirm that the strength of the O—H bonds such as H20⋯O4 (0.8691) in conformer A and H21⋯O4 (0.7389) in conformer B are stronger than the H30⋯O4 (0.0205) in conformer C. The ellipticity ε (0.7–0.8 a.u) at the (3,−1) line critical points (LCPs) of the non-bonded atoms shows the dynamical stability of a molecule (Brovarets et al., 2017). To validate the AIM topology results, the isosurface plot has been illustrated (see Fig. 5). The cube files for the functions λ₂ρ (the product of second largest eigen value of Hessian matrix and electron density) and RDG were generated and used them to plot colored isosurface which directly indicates the strength of the non-bonded interactions present in the molecule. In all the three conformers, the space between the hydrogen atoms in the alkyl chain and aromatic hydrogens are filled with green colored isosurface which illustrates the weak H—H bonding between them. The slight red shaded green isosurfaces found between the Oxygen in the carboxylic acid group and the hydrogen atom in the alkyl chain (conformer A and B) or in the aromatic ring (conformer C) indicates the C—O⋯H hydrogen bonds.

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Fig. 4

Molecular graph illustrating the non-bonded interactions of chlorambucil.

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Fig. 5

Isosurface density plot illustrating the non-bonded interactions of chlorambucil.

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4.4 Vibrational assignments

In this section, the detailed vibrational assignments of normal modes of chlorambucil conformers and the vibrational frequency shift due to the non-covalent interactions upon orientation changes have been explained. Moreover, the intermolecular bond formation enthalpy changes (also known as H-bond energy) between dimer and monomer structure of chlorambucil have been estimated using following Iogansen’s relationship (Iogansen, 1999).

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Fig. 6

Comparison of Experimental and simulated (wB97XD/6-311++G(d,p) level of theory) IR spectrum of chlorambucil.

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Fig. 7

Comparison of Experimental and simulated (wB97XD/6-311++G(d,p) level of theory) Raman spectrum of chlorambucil.

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4.5 Interactive sites of chlorambucil

The feasible interactive sites which are appropriate for the electrophilic and nucleophilic attack have been determined using Fukui function analysis. In order to find the site selectivity precisely, the values of relative electrophilicity (s_k⁺/s_k^-) and nucleophilicity (s_k⁻/s_k⁺) that derived from the hirshfeld charges corresponding to anion (N+1), cation (N−1) and neutral radical species have been computed by wB97XD and B97D methods with 6-311++G(d,p) as proposed (Roy et al., 1998). The atomic center or site having the highest s_k⁺/s_k⁻ is the most feasible site to be attacked by a nucleophile, and having the highest s_k⁻/s_k⁺ ratio is the most feasible site to be attacked by an electrophile (Karthick and Tandon, 2016). The results obtained from both the functional (wB97XD and B97D) show that the atomic centers N5 and C7 which are attached to the ring carbon atoms are the most suitable sites to be attacked by an electrophile in all the conformers (Refer the Table S10 (Supplementary Material)). In the calculations obtained for the relative electrophilicity of all the conformers using the functional wB97XD are quantitatively not comparable as we acquire the negative value of f_k⁻ in conformer A and B. Fortunately, the positive Fukui functions (f_k⁺, f_k⁻) obtained using the functional B97D show that the carbon atom C19 is the most prominent to be attacked by nucleophile as it has higher value of s_k⁺/s_k⁻ 11.7019 e, while in conformer B and C, the atoms O3 and C19 are found to be more probable sites to be attacked by nucleophile. In many chemical systems, the interactions between the electrophiles and nucleophiles play an important role in the chemical stability. In charge transfer process, the electrophiles act as a Lewis acid and the nucleophile act as a Lewis base (Cedillo and Contreras, 2012). In order to find the exact picture of charge distribution, the normalized Parr functions P⁻ and P⁺ (Domingo et al., 2013) based on respective hirshfeld atomic spin densities (ASDs) of cation ρ_s^rc (k) and anion ρ_s^ra (k) radicals also have been considered (depicted in Table S11 (Supplementary Material)). In results of both the functional, the N5 and C8 are found to be the most electrophilic center. This validates the results of relative nucleophilicity as suggested by Roy et al. (Roy et al., 1998). From the values of Parr function of cation radical (P⁻) obtained by wB97XD/6-311++G(d,p), the atoms O3, C14 and C9 are identified as the most nucleophilic centers of conformer A, B and C respectively. The results obtained in this case are in good correlation with the Fukui functions (s_k⁺/s_k⁻) of the conformers except in conformer B where the carbon atom C14 is identified as the most nucleophilic center rather than C16.

4.6 Automated docking analysis for binding sites

Since the chlorambucil is already a well-known drug, the probable active binding sites on the unexamined biological targets were found using the automated docking tool. The selection of targets has been done by cross-validation probabilities based on shaping the interaction landscape of molecules using Swiss target prediction (Gfeller et al., 2013; Gfeller et al., 2014). The chlorambucil is found to be more active against the targets in the family of Aldo-keto reductase family 1 proteins (AKR1B1, AKR1B10) and FAD-linked sulfhydryl oxidase ALR (GFER). The docking results obtained from Autodock 4.2 are depicted in Table S12 (Supplementary Material) and are summarized as follows.

4.6.1

4.6.1 Docked state parameters of chlorambucil and AKR1B1

The target AKR1B1 is found to interact with carboxylic acid derivatives which involve in oxidative stress diseases, cell signal transduction and cell proliferation process (Maccari and Ottanà, 2015). In the previous literature, β-glucogallin inhibited AKR1B1 with an IC₅₀ value of 58 ± 3 μM (Puppala et al., 2012). The estimated free binding energy of about −5.8 kcal/mol and inhibition constant of 55.72 μM in this study shows that chlorambucil is a potential inhibitor of AKRB1 (PDB ID: 5H7A). In the docked state as shown in Fig. 8a, it is found that the conventional hydrogen bond (O⋯H—N) of 2.0122 Å present between the Oxygen O4 atom in the ligand and —N—H species in the target residue LEU 618 in chain B. Also, the strong interactions are also identified between the —OH in the carboxylic acid of the ligand and oxygen donors in the target residues SER580, THR582 with bond distances of 1.9725 and 1.6225 Å, respectively.

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Fig. 8

Docked state conformers of chlorambucil with (a) 5H7A, (b) 1ZUA (c) 4LDK.

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