Design and nonlinear optical properties (NLO) using DFT approach of new Cr(III), VO(II), and Ni(II) chelates incorporating tri-dentate imine ligand for DNA interaction, antimicrobial, anticancer activities and molecular docking studies

2.10.1 Electronic spectra for interaction of the prepared complexes with DNA

Spectrophotometric scan test was carried out by conservation the molar concentration of the investigated metal imine chelates constant while change the (DNA) in the surrounding conditions. The absorption due to free CT-DNA was removed by accession an equimolar CT-DNA to refine buffer solution in the blank compartment and the forming spectra were considered to result from the complexes and the DNA – metal complex assemblages (Abdel-Rahman et al., 2013a,b; 2014a; 2015a,b; 2016a,b,c; 2017a,b,c,d,e,f,g). From the absorption data, the intrinsic binding invariable (K_b) was determined by plotting (DNA)/(ε_a-ε_f) vs. (DNA) according to the following equation:

2.10.2 Hydrodynamic measurements

Hydrodynamic measurements were performed utilizing an Oswald micro viscometer, keeped at constant temperature at 25 °C. The fluidity times were recorded for various concentrations of the complex (5–50 µM), maintaining the concentration of DNA constant (260 µM). Mixing of the solution was achieved by bubbling the nitrogen gas through the viscometer. The mean value of the three measures was utilized for determining the viscosity of the samples. The buffer fluidity time in seconds was recorded as t°. The relative viscosities for DNA in the presence (η) and absence (η°) of the complex were evaluated utilizing the relation η = (t − t°)/t°. Where, t is the recorded fluidity time in seconds and the amounts of the relative viscosity (η/η°) were plotted against 1/R (R = (DNA)/(Complex)) (Abdel-Rahman et al., 2013a,b; 2014a; 2015a,b; 2016a–c, 2017a–g).

2.10.3 Agarose gel electrophoresis

The DNA binding products were checked by agarose gel electrophoresis method (Abdel-Rahman et al., 2016b; 2017b,c,d,e,f,g). Agarose (600 mg) was dissolved in hot tris-acetate-EDTA (TAE) buffer (60 mL) (4.84 g Tris base, pH-8.0, 0.5 M EDTA L^_1) and heated to boil for few minutes. When the gel attains approximately 55 °C, it was then poured into the gas cassette fitted with comb. Slowly the gel was allowed to solidify by cooling to room temperature and then carefully the comb was removed. The solidified gel was placed in the electrophoresis chamber containing TAE buffer. The stock solution of complexes was prepared by solve 20 mg of the compounds in 20 ml of DMF. The sample (25 μg/ml) was added up to the separated DNA of Calf-thymus (CT-DNA) and incubated for 1 h at 37 ± 1 °C and then 30 μl of DNA sample (mixed with bromophenol blue dye at a 1:1 ratio) was charged on attention into the electrophoresis chamber wells along with a standard DNA marker in TBE buffer (50 mM Tris base, pH 7.2; 1 mM EDTA/1 L) and then loaded onto the agarose gel, then a constant electricity (60 V) was left for about 45 min. Finally, the gel was removed and spotted with 20 μg/ml of ethidium bromide for 10–20 min. The bands obtained was monitored under UV light using a transilluminator directed by photography with DMC-LZ5 Lumix Digital Camera to determine the extent of DNA binding as contrasted with standard DNA marker (Abdel-Rahman et al., 2013b; 2014a; 2015a,b; 2016a–c; 2017b–e).

2.12.1 Preparation of ligands and target protein-tyrosine kinase

The compounds in this research as ligands were studied for their binding ability into protein-tyrosin kinase (TRK). The target compounds were constructed into a 3D model using the builder interface of the MOE program after checking their structures and the formal changes on atoms by 2D depiction, the following steps were carried out: The target compounds were subjected to a conformational search. Conformers were subjected to energy minimization using MOE module until a RMSD gradient of 0.01 and RMS distance of 0.1 Å kcal mol⁻¹ Å⁻¹ with MMFF94X force-field and the partial charges were automatically evaluated. The obtained database was then saved as MDB file to be used in the docking calculations. The X-ray crystallographic structure of c-kit receptor protein-tyrosine kinase in complex with STI-571 (Imatinib or Gleevec) were uploaded from the Protein Date Bank (PDB) (http://www.rcsb.org/pdb/explore/explore.do?structureId = 1T46) (PDB code: 1t46) was obtained from Protein data bank (Mol et al., 2004). Partial charges and hydrogen atom were set on to the protein with the protonation 3D application in MOE. This implementation is carried out to appropriate position hydrogen atom in the macromolecule structures and ionization states. Then four steps were made for both active sites: checking the atoms connection and type, adding hydrogen atoms to the system, selection of the receptor an fix it’s atoms potential, MOE Alpha Site Finder was utilized for the active site search in the enzyme structure utilizing all default items. The active site was chosen to contain the residues that were bound to receptor. Dummy atoms were created from the obtained alpha Spheres.

2.12.2 Molecular modeling and analysis of the docked results

The binding affinity of the synthesized compounds to TRK protein by formation of the hydrogen bonds between the ligand and amino acid in TRK were carried out, which measured the hydrogen bond length, which does not surpass 3.7 Å. In addition, RMSD of the co-crystal ligand position compared to the docking pose was utilized in ranking. Both RMSD as well as the mode of interaction of the regional ligand within the crystal structure of c-kite tyrosin kinase receptor were carried out as the standard model.

3.1.1 ¹H NMR and ¹³C NMR spectra of the prepared ESAP imine ligand

The ¹H and ¹³C NMR spectral data of the prepared ligand are recorded in the experimental section (cf Figs. S1 and S2). The ¹H NMR spectrum of ESAP imine ligand showed two singlet signals at δ = 13.90 and 9.61 ppm, that are assigned to the two phenolic —OH. The higher value of δ for the —OH group (13.90 ppm (s, 1H, 2′-OH) can be assigned to the presence of intermolecular hydrogen bonding (Abdel-Rahman et al., 2016c). Also, it shows singlet signal at δ = 8.94 which is characteristic for azomethine (CH⚌N) proton of the ligand (Abdel-Rahman et al., 2017c,d,e). Moreover, it shows multiplet signals at 7.18–6.99 ppm for aromatic of 9-CH protons. Furthermore, it shows one proton doublet at position 10. ¹³C NMR of ESAP imine ligand exhibited signal at 162.15 ppm may be assigned to azomethine carbon. The signals observed in the region 152.70–15.28 ppm were assigned to phenyl carbons. The ¹H NMR spectra of the complexes cannot be obtained due to interference of their paramagnetic properties.

3.1.2 Elemental analysis and electrical conductivity measurements

All the prepared complexes are tinted, solid, steady at room temperature and non-hygroscopic in nature and possess high decomposition points (>300 °C).

The elemental analysis results of the prepared imine ligand and its complexes are recorded in Table 1 and suggested that ESAP imine ligand act as tridentate and form complexes with VO(II), Cr(III) and Ni(II) in 1:1 ratio metal to ligand (cf. Scheme 1). The molar conductance of all the prepared complexes was observed at room temperature in DMF as a solvent and their results in (Ω⁻¹ cm² mol⁻¹) are recorded in Table 1. The molar conductance values of ESAPNi and ESAPV complexes at room temperature are 2.55 and 10.35 Ω⁻¹ cm² mol⁻¹, respectively assigned to their non-electrolytic nature whereas ESAP with 68 Ω⁻¹ cm² mol⁻¹ is electrolyte species (Abdel-Rahman et al., 2013a,b; 2014a;2015a,b; 2016a,b,c; 2017b,c,d,e).

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Scheme 1

Synthetic strategy for the preparation of the investigated complexes.

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3.1.3 Infrared spectra

In order to study the bonding mode of ligand to metal in the complexes, IR spectrum of the free ligand was compared with the spectra of metal complexes. The fundamental infrared spectral frequencies of the ESAP ligand its complexes along with their assignments are shown in (Table 2). Bands due to —OH and —CH⚌N are distinguishable and offer proof regarding the structure of the ligand and its bonding with metal (cf. Figs. S3 and S4). A band at 1627 cm⁻¹ in the ESAP ligand is due to —C⚌N stretching vibration. On complexation, this band is dislodged to a lower frequency (1602–1620 cm⁻¹). The negative shift of this band is an obvious indication of the participation of the azomethine nitrogen atoms in complex formation (Abdel-Rahman et al., 2013a,b; 2014a; 2015a,b; 2017b,c,d,e). This is supported by the appearance of band at 430–499 cm⁻¹ corresponding to the stretching vibration of M—N bond. Bands at 537–597 cm⁻¹ correspond to M—O stretching vibrations. Band at 3424 cm⁻¹ observed in the ligand spectrum is due to stretching vibrations of free —OH. In the complexes, the recorded IR spectra of all the prepared imine complexes show broad band at 3380–3418 cm⁻¹ which have been assigned to υ(OH) stretching vibration of hydrated water molecules, in correspondence with the findings of the elemental analysis listed in (Table 2). A band at 968–976 cm^–1 (OH rocking) suggests the presence of coordinated water in all three complexes. In the low frequency zone, the band observed for ESAP imine ligand showed an absorption band at 1227 cm⁻¹ which can result from the stretching vibration of the phenolic (C—O) group. The displace of that band to lower wave number values upon complexation indicates that the oxygen atoms of the phenolic groups are coordinated to the metal ion. The characteristic frequencies of the coordinating nitrate group in ESAPCr complex possess three non-degenerated modes at 1470 cm⁻¹ ʋ(NO₂)asy, 1356 cm⁻¹ ʋ(NO₂)sy and 858 cm⁻¹ ʋ(NO) (Abdel-Rahman et al., 2017d)

3.1.4 Electronic spectra

The molecular electronic absorption spectra are often very important in the evaluation of results furnished by other methods of check. The numerical values of the maximum absorption wavelength (ε_max) and the molar absorptivity (λ_max) were listed in TableS1 and the spectra were presented in Fig. 1. The electronic spectral measurements were utilized for assigning the stereo chemistries of metal ions in the complexes relying on the sites and number of d–d transition peaks (Chen et al., 2009). The electronic absorption spectra of ligands and their complexes were registered at the wavelength range 800–200 nm and 298 K. The ligand exhibits absorption bands in UV–Vis region around 400 nm which is assigned to n → π^∗ transition originating from the imine function of the imine ligand (Silverstein and Webster., 1997). The absorption bands of complexes at λ_max = 306–471 nm is assigned to charge transfer with in ESAP imine ligand to the metal ion. These charge-transfer transitions probably occur form the p-orbitals of the Schiff bases to the d-orbitals of metal ion. Another difference observed in the electronic spectra of the prepared complexes, compared to the spectra of the corresponding free ligand, is associated with the appearance of a broad low intensity band. This a long and a broad band lying in the region 504–524 nm (ɛ_max = 230–570 mol⁻¹ cm²). This band could be mainly attributed to the d → d transition in the structure of the prepared complexes (cf. Table S1) (Dyer, 1965). These bands are of low molar absorptivity, Ɛ, being Laporte ‘forbidden’ transitions.

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Fig. 1

Molecular electronic spectra of 10⁻³ M of ESAPV complex and its components in DMF at 298 K.

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3.1.5 Magnetic susceptibility measurements

The magnetic susceptibility measurements give information considering the geometric structure of the compounds. The observed magnetic moment for complexes are generally diagnostic of the coordination geometry about the metal ion (cf. Table 1). ESAPCr complexes show magnetic moments corresponding to three unpaired electrons, i.e. (3.76 B.M.), as expected for octahedral Cr(III) complexes (Alaghaz et al., 2015). The magnetic moment values of Ni(II) complexes is 2.62 BM, indicating the presence of two unpaired electrons per Ni(II) ion and suggesting these complexes to have tetrahedral geometry (Ismail et al., 2012). The magnetic susceptibility of ESAPV complex has a magnetic moment value of 1.71 B.M., which is close to the spin-only value for d¹ and in agreement with the reported values for distorted octahedral complexes of VO(II) (Sarkar et al., 2007).

3.1.6 Thermal analysis

Thermal analysis (TGA) of the metal chelates is an important tool for (i) get information about the thermal stability of these complexes, (ii) decide whether the water molecules (if present) are inside or outside the inner coordination sphere of the central metal ion.

The thermal stabilities were investigated for imine complexes have been studied as a function of temperature. The proposed stepwise thermal degradation of the complexes with respect to temperature and the formation of respective metal are depicted in Table 3. The thermograms of the prepared imine complexes confirm the presence one molecule of coordinated water in case of ESAPV complex and three coordinated water molecules in case ESAPCr complex (cf. Scheme 1). The thermal behavior of the metal complexes showed that the hydrated complexes lose water molecules of hydration in the first step; then lose coordinated water molecules with decomposition of the ligand molecules in the subsequent steps as shown in (Table 3).

The thermogram of ESAPV complex showed three stages of decomposition up to 604 °C. It dehydrates one water molecule in the first step up to 122 °C. During furthers heating, the ESAPV complex complete decomposition in two steps and shows mass losses of about 35.10% (Calc. 35.00%), 39. 92% (Calc. 40.01%) in the temperature ranges of 124–430 °C, 432–604 °C, respectively leaving VO(II) as a residue.

The TGA curve of ESAPCr complex show loss in weight within the temperature range 35–301 °C, which is due to removal of the lattice nitrate and three coordinated water molecules (Found 27.48%, Calc. 27.43%). Also, the ESAPCr complex shows mass loss within the temperature range 303–486 °C, which is due to removal of the part of ligand (C₈H₈O₂) with mass losses (Found 32.18%, Calc. 32.15%). Finally the complexes undergo decomposition of the organic ligand within the temperature range up to 635 °C with mass losses (Found 28.09%, Calc. 28.13%), leading to the formation of Cr as final product (Shakir et al., 2016; Abdel-Rahman et al., 2017b).

The thermogram of the ESAPNi complex showed three decomposition steps within temperature range 33–486 °C. The first step of decomposition within the temperature range from 33 to 149 °C correspond to the loss of coordinated ethanol molecule, with a mass loss of 12.69% (Calc. 12.79%). The second step of decomposition within the temperature range 151–301 °C with mass loss of 37.83% (Calc. 37.80%) indicated that removal of part of imine ligand (C₈H₈O₂). The third stage in the temperature range 303–486 °C, an estimated mass loss of 33.12% (calc. 33.08%). This mass correspond to loss of the remaining part of ligand (C₇H₅NO) leaving Ni as a residue (Shakir et al., 2016; Abdel-Rahman et al., 2016c; 2017b). The horizontal lines in the TGA curves beyond 650 °C have been observed in all the metal complexes indicated no further weight loss, implying that a metal residue may be the final product. The thermal analysis data agree well with the proposed stoichiometry derived from the results of elemental analysis findings.

3.1.7 Spectrophotometric determination of the stoichiometry of the prepared complexes

Stoichiometry of the prepared complexes is defined utilizing the two methods involving the use of spectrophotometry, namely, continuous-variations method and mole-ratio method. Based on the methods which were utilized and the experimental results, the stoichiometry of the prepared complexes is 1:1. The curves of the continuous variation method (cf. Fig. 2) displayed maximum absorbance at mole fraction X_ligand = 0.5–0.6 showing the complexation of metal ions to ligand in molar ratio 1:1. Moreover, the data resulted from utilizing the molar ratio method support the same metal ion to ligand ratio of the prepared complexes (cf. Fig. S5) (Abdel-Rahman et al., 2013a,b; 2014a; 2015a,b; 2016a,b,c,d; 2017a,b,d,e).

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Fig. 2

Continuous variation plots for the prepared complexes in aqueous – ethanolic medium at (ESAPV) = (ESAPCr) = (ESAPNi) = 10⁻³ M and 298 K.

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3.1.7.1

3.1.7.1 Determination of the apparent formation constants of the synthesized complexes

The formation constants (K_f) of the studied imine complexes formed in solution were obtained from the spectrophotometric measures by utilizing the continuous variation method (cf. Table 4). The obtained K_f values show the high stability of the tested complexes. The values of K_f for the checked complexes increase in the following order: ESAPNi > ESAPCr > ESAPV complex. Moreover, the values of the stability constant (pK) and Gibbs free energy (ΔG^≠) of the presented complexes are calculated. The negative values of Gibbs free energy mean that the reaction is spontaneous and preferable. The pH-profile (absorbance vs. pH) explained in (cf. Fig. 3) showed typical dissociation curves and a high stability pH extent (4−9) of the checked complexes. This indicates that the formation of the complex greatly stabilizes imine ligands. Consequently, the suitable pH range for the different applications of the tested complexes is from pH = 4 to pH = 9 rely on the finding of elemental analyses, molar conductance, magnetic measures, infrared and electronic spectra, the suggested composition of the complexes was identified.

Table 4 The formation constant (K_f), stability constant (pK) and Gibbs free energy Δ(G*) values of the synthesized complexes at 298 K.

Complex	Type of complex	Kf	Log Kf	ΔG* (kJ mol−1)
ESAPNi	1:1	4.09 × 104	4.61	−26.31
ESAPCr	1:1	2.56 × 104	4.41	−25.15
ESAPV	1:1	1.26 × 104	4.10	−23.39

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Fig. 3

Dissociation curve of the prepared imine complexes in DMF.

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3.1.8 Molecular orbital calculations

The optimized geometrical parameters (bond lengths, bond angles and dihedral angles), natural charges (valence, core, Rydberg and total) on active centers, natural configuration of the metal ions, natural population and energetic of the ground state for the studied complexes are calculated and discussed using B3LYP/GEN and B3LYP/LANL2DZ. From the elemental analysis and spectroscopic data, metal ions coordinated to the ligand via N8, O15 and O16 atoms, two water molecules and OH⁻ ion in case of Cr forming octahedral complex . In case of ESAPNi complex, the metal ion coordinated to the ligand via N8, O16 and O15-atoms and ethyl alcohol molecule forming tetrahedral complex. In ESAPV complex, the central metal ion coordinated to N8, O15 and O16-atoms and one water forming square pyramidal structure.

3.1.8.1

3.1.8.1 Structure of the prepared complexes

The optimized geometry, numbering system, vector of the dipole moment, bond lengths, bond angles and dihedral angles of all metal complexes studied in this work are presented in Table 5 and Fig. 4. In ESAPCr complex, the metal ion coordinates with O15, N8 and O16 atoms forming five membered rings namely, MO15C14C9N8, six membered rings, namely, MO16C5C6C7N8, two water molecules and OH⁻ ion to form octahedral structure. In case of ESAPNi complex, the metal ion coordinates with O15, N8 and O16 to form five membered rings namely, MO15C14C9N8, six membered rings, namely, MO16C5C6C7N8 and with one ethyl alcohol molecule to form tetrahedral structure. In case of ESAPV complex, the metal ion coordinates with O15, N8 and O16 to form five membered ring namely, MO15C14C9N8, six membered ring, namely, MO16C5C6C7N8 and water molecule to form square pyramidal structure. Therefore, distortions from regular octahedral, tetrahedral and square pyramidal geometry are expected for all the studied complexes. In the studied complexes, most of M—N and M—O bonds show elongation upon complexation. The elongation of the M—N bond length is greater than M—O bonds in the studied complexes. The length of the coordinate covalent bonds between metal and ligand site, i.e. M—N, and M—O, are too long compared to the typical MX bond lengths (Abdel-Rahman et al., 2016d). The too long M—O and M—N bonds in the complexes mean that the ionic character of these bonds is small. The bond angles between metal ion and binding sites in the coordination sphere (c.f. Tables 5) in the ESAPCr complex vary between 49° and 86° which compare nicely with the experimental data as obtained from X-ray analysis for O_h complexes (Armelao et al., 2010) which indicate a distorted octahedral geometry. Also a distortion from tetrahedral of ESAPNi complex and square pyramidal of ESAPV complex is observed from bond angles of the coordination spheres (c.f. Table 5). The values of the dihedral angles around metal ion in the coordination sphere in the studied complexes (c.f. Tables 5) are far from 0° or 180° which indicate that the metal ion is not in the same plane as the donating sites.

Table 5 Optimized bond lengths, bond angles and dihedral angles of the studied complexes using B3LYP/GEN.

Parameter	ESAPV	ESAPCr	ESAPNi
M—O16, Ǻ	1.671	1.746	1.625
M—N8	1.984	1.644	1.741
M—O15	1.912	1.659	1.688
M—O21	2.037	1.972	1.779
M—O22		2.145
M—O23		2.338
<O16MO23		87.6
<O16MN8	87.8	86.2	93.1
<N8MO15	43.4	51.3	44.4
<O15MO22		57.6
<O22MO21		49.7
<O15MO21	90.6
<O21MO16	86.3
<N8MO15	83.7
<N8MO16	87.8
<O21MO23		57.3
<O23MN8C9		−74.7
<O22MO15C14		110.2
<O21MO15C14	−170.6	26.9	−154.7
<O21MO16C5	138.7	−92.4	111.8

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Fig. 4

The optimized geometry, the vector of the dipole moment and numbering system for the studied complexes using B3LYP/GEN.

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3.1.8.3

3.1.8.3 Global reactivity descriptors

They include HOMO, LUMO, energy gap (E_g), chemical hardness (η), electronegativity (X), chemical potential (V), electron affinity (A), ionization potential (I) and global softness (S). The frontier molecular orbital (FMO) energies of the studied complexes were calculated using B3LYP/LANL2DZ and presented in Table 6 and Fig. 5. Energy gap (E_g) between HOMO and LUMO characterizes the molecular chemical stability (reactivity). The results in Fig. 5 and Table 6 indicate that the smaller the energy gap the easier the charge transfer and the polarization occurs within the molecule. The order of increasing reactivity in the studied complexes is: ESAPCr > ESAPNi > ESAPV. Using HOMO and LUMO energies, ionization potential and electron affinity can be expressed as I ∼ −E_HOMO, A ∼ −E_LUMO, the chemical hardness (η) = (I − A)/2, electronegativity (X) = (I + A)/2, chemical potential (V) = −(I + A)/2 and chemical global softness (S) = 1/2η values are calculated and presented in Table 6. The variation of electronegativity (X) values is supported by electrostatic potential, the results in Table 6 shows that the orders of decreasing X (increasing CT within the complexes) are: ESAPCr > ESAPV > ESAPNi. The small η values for the studied complexes reflect the ability of charge transfer inside the complexes. Therefore, the order of increasing the charge transfer within the studied complexes is: ESAPCr > ESAPNi > ESAPV which is the same order of reactivity.

Table 6 Total energy, E_T, energy of HOMO and LUMO, energy gap, Eg, ionization energy, I, electron affinity, A, electronegativity, X, Hardness, η, softness, s and chemical potential, v, of the studied complexes using B3LYP/LANL2DZ.

Parameter	ESAPV	ESAPCr	ESAPNi
EHOMO, a.u.	−0.2119	−0.3165	−0.1923
ELUMO, a.u.	−0.0950	−0.2110	−0.0766
Eg, eV	3.179	2.869	3.147
I, eV	5.763	8.608	5.231
A, eV	2.584	5.739	2.084
X, eV	4.174	7.174	3.657
η, eV	1.589	1.435	1.574
S, eV−1	0.315	0.348	0.318
v, eV	−4.174	−7.174	−3.65

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Fig. 5

HOMO-LUMO charge density maps and energy gap of the studied complexes using B3LYP/LANL2DZ.

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3.3.1 Electronic spectra of interaction with DNA

Titration with electronic absorption spectroscopy is an active route to check the binding mode of DNA with metal complexes. The spectra were recorded as a function of the addition of the buffer solutions of pretreated CT-DNA to the buffer solutions of the tested complexes. If the interaction mode is intercalation, the orbital of the intercalated ligand can join with the orbital of the base pairs, lowering the π–π^∗ transition energy and lead to bathochromism. If the conjunction orbital is partially filled by electrons, it gives rise to reduce the transition probabilities and lead to hypochromism (Abdel-Rahman et al., 2013a,b; 2015a,b; 2016a,b,c; 2017a,b,c,d,e). The electronic absorption spectra of ESNAPV complex in the absence and presence of various concentrations of buffered CT-DNA are given in Fig. 8. Addition of increasing amounts of CT-DNA resulted in a reduction of absorbance for a complex. The spectral parameters and binding constants (K_b) for the DNA interaction with the checked complexes which are calculated using Eq. (12) (cf. Fig. S6), are shown in Table 10. The investigated complexes could bind to DNA mainly via an intercalative and replacement modes with the sequence: ESAPCr > ESAPV > ESAPNi complex.

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Fig. 8

DNA binding results of the prepared imine complexes based on gel electrophoresis. Lane 1: DNA Ladder, lane 2: ESAPNi + DNA, lane 3: ESAPCr + DNA, lane 4: ESAPV + DNA, lane5: ESAPV complex.

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3.3.2 Viscosity measurements

For explaining the interaction nature between the checked complexes and DNA, viscosity measures were executed. Hydrodynamic methods such as viscosity measures which are sensitive to length increment or minimize of DNA are regarded as the most effective methods of studying the binding mode of compounds to DNA in the absence of crystallographic structural data and NMR. For further explaining of the binding mode, viscosity measurements were done. Under suitable conditions, a traditional intercalative mode such as intercalation of drugs like ethidium bromide leads to a obvious increment in the viscosity of DNA solution due to an increment in the segregation of base pairs at the intercalation site and hence an increment in the overall DNA length. On other hand, drug molecules attachment exclusively to the DNA grooves result in less pronounced in DNA solution viscosity (Raja et al., 2012) a partial intercalation of compound may bend the DNA helix, resulting in the reduction of its effective length and, concomitantly, its viscosity (Abdel-Rahman et al., 2016a,b,c; Satyanarayana et al., 1993, Liu et al., 2008). The relative viscosity of DNA solution enhances significantly as the amount of the compound raises, as shown in Fig. 9. This may be due to the admission of the aromatic ring in imine ligand into the DNA base pairs resulting in a crook in the DNA helix, hence, increase in the separation of the base pairs at the intercalation place and increment in DNA molecular length. Moreover, the sequence of the observed increment in the values of viscosity was renovated with the binding affinity to DNA i.e. ESAPCr complex shows the highest binding affinity to DNA and the highest viscosity. Moreover, ESAPCr complex could be bind to DNA via electrostatic binding.

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Fig. 9

Histogram showing the comparative antibacterial activities of the prepared compounds (ESAP, ESAP Cr, ESAPNi and ESAPV) against Bacillus subtilis bacteria.

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3.3.3 Gel electrophoresis

Agarose gel electrophoresis is used for the DNA binding studies. The prepared imine chelates were studied for their DNA binding activity by agarose gel electrophoresis method (cf. Fig. 10). The gel after electrophoresis clearly showed that the intensity of all the treated DNA samples has partially detracted, possibly because of the interaction with DNA. The partial binding of DNA was observed in the prepared imine chelates . The difference was clarified in the bands of the complexes compared to that of the control DNA. This indicates that the control DNA alone does not show any visible cleavage whereas the complexes show cleavage (Mishra and jain, 2012). However, the nature of reactive intermediates involved in the DNA binding by the complexes is not obvious (Yang et al., 1997). These results show that the metal ions play an important role in the interaction with isolated DNA. As the compound was observed to bind with DNA, it can be complemented that the compound minimizes the growth of the pathogenic organism by interaction with genome. The studies reveal that partial binding of DNA was observed by VO(II), Cr(III) and Ni(II) imine complexes.

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Fig. 10

Histogram showing the comparative antifungal activities of the prepared compounds (ESAP, ESAPCr, ESAPNi and ESAPV) against C. albicans fungi.

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3.3.4 The proposed mechanism for interaction of the investigated imine complexes with DNA

Correlation between spectral characteristics and hydrodynamic measurements between the investigated complexes and DNA, there are different binding modes can be suggested in our investigation. Complex interacts with DNA most likely through a mode that involves electrostatic or hydrophobic interaction. As stated before, ESAPCr, complex has a replaceable nitrate ligand in solution, which may be replaced with H₂O molecules in the solution (Abdel Rahman et al., 2017c, d). So the complex contains a positive charge in the middle of the Chromium or Mn atoms. This positive charge on the complex ion could easily interaction with negatively charged backbone in phosphate group at the periphery of the double helix CT-DNA via electrostatic interaction . Also due to the removal of nitrate ligand from the complex in solution, the investigated complexes will have a flat part in the middle. Therefore, possible interaction of the ESAPCr, complex with DNA could be as follow (cf. Schemes 2a, 2b):

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Scheme 2a

Suggested mechanism for interaction of ESAPCr with DNA via (A) intercalation binding and (B) replacement.

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Scheme 2b

Suggested mechanism for interaction of ESAPCr with DNA via groove binding (electrostatic and hydrogen bond).

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First, interaction of Cr(III) complex with base backbone of DNA or electrostatic interaction of coordination sphere with base pairs of DNA.

Second, insertion of the flat part of the complex between the base pairs and consequently coordination of Cr³⁺ with base pairs of DNA.

3.5.1 Molecular modeling: Docking study

The prepared compounds were analyzed for the binding affinity of tyrosine kinases receptor (PDB 1t46) for the purpose of both investigate the interaction between studied compounds and c-kit receptor and for lead optimization. Molecular modeling calculation was carried out to investigate the binding free energies of these inhibitor inside the target c-kit kinase receptor. Scheme 3 shows representative keys for the type of interactions between the investigated compounds and THR 670

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Scheme 3

Representative keys for the type of interactions between the substrate and THR 670.

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3.5.2 Validation of the docking performance and accuracy

Docking of the native co-crystallized STI-571 ligand (Imatinib or Gleevec) was used to establish the docking accuracy of the program. The docked ESAP imine ligand was exactly superimposed on the native co-crystallized one with RMSD being 0.40 Å and binding free energies of −20.04 kcal mol⁻¹ (cf. Fig. 12). The hydrogen bonds between the docked ESAP imine ligand and the amino acids were the same as those between the amino acid and the native ligand.

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Fig. 12

The binding mode of the native ligand STI with C-kit exhibited one H-bond donor with THR 670 at distance 2.08 and one H-bond donor with ILE 789 at distance 2.19 and one H-bond donor with CYS 673 at distance 2.85 and one H-bond acceptor with HOH 1105 at distance 2.82 its score was − 20.04 kcal mol⁻¹ (2D and 3D ligand-receptor interactions).

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3.5.3 The binding affinities of the investigated compounds into c-kit kinase receptor

To compare affinity and to investigate the interaction between the investigated ESAP imine ligand and receptor, molecular docking study was done (cf. Fig. 13). For the docking calculation, firstly, the protein structure (PDB code: 1t46) was separated from the inhibitor and hydrogen atoms were added. The binding free energy, hydrogen bond and RMSD were used to evaluate the binding affinity. All the investigated compounds were docked into the same binding site of the native co-crystallized ligand (cf. Figs. 14–16). The investigated complexes show docking score following sequence ESAPV > ESAPCr > ESAPNi complex (cf. Figs. 14–16).

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Fig. 13

The proposed binding mode of ESAP ligand docked in the active site of TRK protein; (2D and 3D ligand-receptor interactions). The binding mode of the ligand with C-kit receptor exhibited one H-bond acceptor with Ile789 1105 at distance 2.99; and one hydrophobic interaction with Arg 791 at distance 3.13.

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Fig. 14

The proposed binding mode of ESAPV complex docked in the active site of TRK protein; (3D ligand-receptor interactions). The binding mode of ESAPV complex with C-kit receptor exhibited one H-bond acceptor with HOH 1105 at distance 2.14; and one H-bond acceptor with HOH 1106 at distance 3.13 and one H-bond acceptor with HOH 1104 at distance 2.82 and the binding energy is −18.11 kcal/mol.

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Fig. 15

The proposed binding mode of ESAPCr complex docked in the active site of TRK protein; (3D ligand-receptor interactions). The binding mode of ESAPCr complex with C-kit exhibited one H-bond donor with THR 670 at distance 3.08 and one H-bond donor with ILE 789 at distance 1.99 and one H-bond acceptor with HOH 1105 at distance 2.88 and the binding energy is −17.38 kcal/mol.

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Fig. 16

The proposed binding mode of ESAPNi complex docked in the active site of TRK protein; (3D ligand-receptor interactions). The binding mode of ESAPNi complex with C-kit receptor exhibited one H-bond acceptor with Ile789 1106 at distance 1.29; and one hydrophobic interaction with Arg 791 at distance 3.23. H-bond acceptor with HOH 1105 and the binding energy is −11.95 kcal/mol.

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