Synthesis, spectroscopic characterization, molecular modeling and eukaryotic DNA degradation of new hydrazone complexes

3.1.1 Molecular geometry of H₄L

The atomic numbering scheme and the theoretical geometric structure for H₄L are shown in Scheme 1. Reading the values of the bond lengths and angles, one can observe the formation of intramolecular hydrogen bond [(49)H–O(27)…. H(40)–N(10)] from one side with a bond length of 1.89 Ǻ; the other side has no hydrogen bonding. The molecular parameters of the ligand are: total energy (−104472), binding energy (−5309), electronic energy (−785.537), heat of formation (−55.09) kcal/mol; HOMO (−9.1591), LUMO (−0.4474) and the dipole moment (4.024 D).

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Scheme 1

Molecular modeling of H₄L using HyperChem 7.5 program optimized using molecular mechanics (MM+) at PM3 using the Polak-Ribiere algorithm.

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3.1.2 Effect of solvents and buffer on the ligand

The spectrum of H₄L shows the π → π^∗ and n → π^∗ bands at 38760, 36495, 29940 and 31445 cm⁻¹ for the C⚌O and C⚌N groups. In water, the main bands are found at 42370, 33555 and 30675 cm⁻¹. The spectrum in benzene (nonpolar molecule) has three bands at 38170, 32465 and 26455 cm⁻¹; the spectrum is changed from polar to nonpolar solvent due to the formation of hydrogen bonding with H₂O. The bands at (43105 and 33110), (41320, 38760, 36765 and 33110 cm⁻¹), and (40000, 32050, 28900 and 26665 cm⁻¹) are observed in CH₃OH, C₂H₅OH, and CH₃Cl, respectively. The spectra are similar in DMF and DMSO with strong bands at 29760 and 29240 cm⁻¹, respectively. In acetone, two weak bands are observed due to the weak solubility.

Examining the spectra at different pHs, the main band due to the nonionic form of the ligand observed at 300 nm is unaltered in the pH range of 2–7. A new band at 326 nm is observed in the pH 10–12 due to the ionic form. The spectrum at pH 7–9 has two bands at 300 and 326 nm representing the pH at which the two forms exist in equilibrium. The values [7.42 (7.33), 9.62 (10.49)] are nearly similar at the two wavelengths 326 (300 nm). The first may be due to the liberation of the amidic proton (CONH) while the second is due to the phenoxy proton (OH). The relation between the absorbance and pH at two wavelengths (Fig. 1) was used to calculate the pK’s of the ligand. The curves are characterized by two well defined s-shapes denoting the presence of two pK’s. The pK in each s-shape is calculated by the half height method (Issa et al., 1973).

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Figure 1

Absorbance-pH relation for the ligand at 300 and 326 nm.

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3.1.3 IR, ¹H NMR and MS spectra of the ligand

The IR assignments of H₄L bands are included in Table 2. The spectrum shows bands at 3290, 3090, 1644, 1626 and 1368 cm⁻¹ assigned to υ(OH), υ(NH), υ(C⚌O), υ(C⚌N) and δ(OH) vibrations, respectively. The ¹H NMR spectrum shows signals at 12.44 and 11.19 ppm for the OH protons (due to the presence of the two −OH in different planes) one in the plane while the other is out-of-plane. The signal at 10.02 ppm is due to the NH proton while the multiple signals at 7.88–6.82 ppm are due to the phenyl groups. The CH₃ and CH₂ protons appeared at 2.04 and 3.32 ppm. On deuteration, the signals of OH and NH disappeared. The mass spectrum of H₄L reveals the molecular ion peak [M−4]⁺ at m/z = 378 (Mol. Wt. = 382). The fragmentation pattern of the ligand is represented in Scheme 2.

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Scheme 2

Fragmentation pattern of the ligand.

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3.2.1 IR and ¹H NMR spectra of the complexes

Examining the IR spectra of the complexes (Table 2) one may conclude the following:

1. In all complexes, the υ(C⚌N) in the ligand is shifted to lower wavenumber with the appearance of a new band due to υ(M–N) indicating the participation of this group in bonding.

2. In most complexes, the υ(OH) appeared as medium or broad band at higher wavenumbers proving the destruction of the hydrogen bond accompanied by new bands attributed to the hydroxo bridge (OH) in [Cr₂(H₂L)(OAc)₂(OH)₂]·2H₂O and [Mn₂(H₂L)(OH)₂]·H₂O (Kumar et al., 2009) as well as the hydrate H₂O. This observation is supported by the appearance of δ(OH) more or less at the same position with another band at 1328 cm⁻¹ due to the δ(OH) of hydroxo group.

3. In [VO(H2L)]·2H2O, the υ(OH) and δ(OH) bands disappear revealing the deprotonation of the phenolic OH and its participation in bonding.

4. In most complexes, the (C⚌O) and (NH) bands disappear indicating the enolization of CONH and the CO group is participating in coordination. Evidence is the appearance of new bands at 1531–1577 and 1223–1258 cm⁻¹ due to υ(C⚌N^∗) and υ(CO) vibrations. In [VO(H₂L)]·2H₂O and [Ni(H₄L)Cl₂]·2H₂O, the C⚌O and NH bands still existing at the same position.

5. In [Cu(H₃L)₂(EtOH)₂]·2H₂O, the (C⚌O) and (NH) bands appear very weak at the same position indicating that the ligands coordinate in the keto-enol form. In [Cr₂(H₂L)(OAc)₂(OH)₂]·2H₂O, [Co₂(H₂L)(OAc)₂]·H₂O and [Cu₂(H₂L)(OAc)₂(H₂O)₆], the acetate groups act as bridged bidentate confirming by the appearance of two bands in the regions 1599–1605 and 1435–1447 cm⁻¹ with difference ≈150 cm⁻¹ (Adams et al., 2002).

6. The appearance of bands due to υ(M–O), υ(M–OH) and υ(M–OH₂) at ≈528 and ≈3347 due to the hydrated water. The bands at 3292, 930 and 600 cm⁻¹ in [Cu₂(H₂L)(OAc)₂(H₂O)₆] and [Co₂(H₂L)(H₂O)₄Cl₂]·2H₂O are attributed to υ(OH), ρ_r(H₂O) and ρ_w(H₂O) of coordinated water (El-Asmy and Al-Hazmi, 2009).

7. The ¹H NMR spectrum of [Zn(H₂L)₂] shows a signal at 13.65 ppm attributed to the OH proton with the disappearance of the NH proton. On deuteration, the OH signal disappeared.

3.2.2 Spectral and magnetic studies

The magnetic moments and the significant electronic absorption bands of the complexes, recorded in DMF or Nujol mull, are given in Table 3 (DMF has no effect on the color).

The spectrum of H₄L shows the π → π^∗ and n → π^∗ bands at 36495, 38780, 31445 and 29940 cm⁻¹ for the C⚌O and C⚌N groups. A change is observed on the spectra of its complexes. The bands at 25250–28405 and 23040–25905 cm⁻¹ in the spectra of the complexes may be due to LMCT from N and O donors.

The two bands at 23040 and 19530 cm⁻¹ in the spectrum of [VO(H₂L)]·2H₂O in DMF is assigned to the d_xz → d_xy transition (El-Metwally et al., 2005) in a square-pyramidal geometry. In Nujol, multiple bands are appeared at 17360, 19375, 20745, 21920, and 23580 cm⁻¹ with additional broad band centered at 24390 cm⁻¹ due to a charge-transfer. Its magnetic moment (0.71 BM) is anomalous for one unpaired electron which may be due to interaction with neighboring molecules.

The spectrum of [Cr₂(H₂L)(OAc)₂(OH)₂]·2H₂O, in DMSO, shows bands at 16775 and 20325 cm⁻¹ attributed to ⁴A_2g (F) → ⁴T_2g (P) (ν₁) and ⁴A_2g (F) → ⁴T_1g (F) (ν₂), in an octahedral geometry (Aranha et al., 2007) (Scheme 3). In Nujol, these bands are observed at 17855, 21095 and 22830 cm⁻¹. In addition, a band at 24035 cm⁻¹ is due to a charge-transfer. The ligand field parameters, in DMSO (Nujol) [Dq = 1676.7 (1785.8) cm⁻¹, B = 335.3 (457.9) cm⁻¹ and β = 0.365 (0.498)] further supported the proposed geometry. The lower value of β indicates more covalency. Its magnetic moment (3.92 BM) is normal for three unpaired electrons with no orbital contribution.

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Scheme 3

Structures of [Cr₂(H₂L)(OAc)₂(OH)₂]·2H₂O, [Co₂(H₂L₁)(H₂O)₄Cl₂]·2H₂O and [Ni((H₂L)]·3H₂O.

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The electronic spectrum of [Co₂(H₂L)(H₂O)₄Cl₂]·2H₂O, in Nujol, shows bands at 18725 and 20745 cm⁻¹ assigned to ⁴T_1g → ⁴A_2g (ν₂) and ⁴T_1g → ⁴T_1g (P) (ν₃) characteristic for an octahedral geometry (Chandra et al., 2006). In DMF solution, only band is observed at 20660 cm⁻¹. The band due to ⁴T_1g → ⁴T_1g (ν₁) is not observed and calculated to be 9785 cm⁻¹. The B, β and 10Dq values (Table 3) agree with those reported for octahedral Co²⁺ complexes. The value of β indicates a strong covalent bond. The μ_eff value for each metal ion is 4.53 BM is slightly lower than the values reported for complexes having the proposed geometry (Scheme 3) may be due to interaction between the two metal atoms.

The spectra of [Co₂(H₂L)(OAc)₂]·H₂O in Nujol (DMF) show bands at 22025 (22620) cm⁻¹ attributed to ⁴A₂ → ⁴T₁ (ν₃) similar to those reported for tetrahedral complexes (Efthimiadou et al., 2007]. The B, β and 10Dq values (Table 3) agree with those reported for tetrahedral Co²⁺ complexes. Reduction in the B and β values indicates a covalent character of L–M bonds. The value of β indicates a strong covalent bond. The ν₁ (⁴A₂ → ⁴T₂) is calculated to be 6889 cm⁻¹. The complex shows additional bands at 17540 and 19530 cm⁻¹ in a tetrahedral structure (El-Shazly et al., 2006) around the Co²⁺ ion.

The magnetic moment (3.21 BM) of [Ni((H₄L)Cl₂]·2H₂O is expected for octahedral structure with ³A_2g ground term (Singh and Singh, 2001). Its electronic spectrum shows broad bands in Nujol at 23255 (ν₃) and 14880 cm⁻¹ (ν₂) assigned to ³A_2g → ³T_1g (F) (ν₂) (Lal et al., 2009). The values of B (454 cm⁻¹) and 10Dq (10442 cm⁻¹) are used to calculate β and ν₁, ³A_2g → ³T_2g (F), to be 0.44 and 10442 cm⁻¹, respectively (Abd El-Wahab, 2007). The spectra in DMF and DMSO are similar showing only band at ≈21100 cm⁻¹.

The magnetic moment (3.47 BM) of [Ni((H₂L)]·3H₂O lies within the range reported for a tetrahedral structure. Its electronic spectrum in Nujol shows bands at 20325, 19155 and 17120 cm⁻¹ falling in the range reported for the proposed geometry (Scheme 3) (El-Shazly et al., 2005).

The electronic spectrum of [Cu(H₃L)₂(EtOH)₂]·2H₂O exhibits bands at 17120 19155, 22220 cm⁻¹ in Nujol assigned to ²T_2g → ²E_g and a symmetric forbidden ligand–metal charge-transfer. In DMF, no observed bands for the d–d transitions but the band appearing at 25905 cm⁻¹ is mainly due to a charge-transfer. The band positions with magnetic moment of 1.77 BM are consistent with an octahedral geometry (Manoj et al., 2009). The spectrum of [Cu₂(H₂L)(OAc)₂(H₂O)₆] in Nujol exhibits bands at 18180 and 20080 cm⁻¹ assigned to ²T_2g → ²E_g and a symmetric forbidden ligand–metal charge-transfer. In DMSO, these bands are observed at 16285 and 21735. The broadness of the first band may be due to Jahn–Teller effect which enhances the distortion in octahedral geometry (Al-Hazmi et al., 2005). The magnetic moment (2.09 BM) for each copper atom is consistent with one unpaired electron in an octahedral geometry.

The electronic spectrum of [Mn₂(H₂L)(OH)₂]·H₂O exhibits multiple weak bands at 17120, 21830 23580 and 24630 cm⁻¹ assignable to the transitions in a tetrahedral structure. The magnetic moment (6.38 BM) is corresponding to five unpaired electrons with orbital contribution in a high spin geometry (PUI, 2007).

3.2.3 Thermal studies

The thermogravimetric data considering the decomposition temperature range, the removed species and the weight losses for most complexes are presented in Table 4. Example for one mononuclear complex and binuclear complex is described here in detail.

The steady part of the thermogram of [Zn(H₂L)] (Scheme 4) till 167 °C indicates the absence of any solvent outside the sphere. The 1st decomposition step starting at 168–360 °C is due to the removal of two phenolic rings {2C₆H₅O} (% Found 41.9; Calcd. 41.7). The 2nd step (361–481 °C) indicates the decomposition of {C₆H₁₀} (% Found 18.7; Calcd. 18.4). The last step (482–555 °C) shows the decomposition of {2N₂ + CO + C} (% Found 21.8; Calcd. 21.5). ZnO is the stable residue at 800 °C (% Found 18.0; Calcd. 18.3).

In [Cr₂(H₂L)(OAc)₂(OH)₂]·2H₂O, the TG curve is characterized by steps at 36–137, 138–327, 328–431 and 432–800 °C corresponding to the removal of outside water (% Found 6.0; Calcd. 5.4), C₆H₁₀ + 2OH (% Found 17.4; Calcd. 17.3) and C₁₄H₁₀N₄O + 2OAc + OH (% Found 55.0 Calcd. 54.7) after which the residue is Cr₂O₃ (% Found 21.6; Calcd. 22.6).

3.2.4 ESR spectra

To obtain further information about the stereochemistry of VO²⁺ and Cu²⁺ complexes, ESR spectra were recorded and their spin Hamiltonian parameters were calculated.

The room temperature solid state ESR spectra of the copper complexes [Fig. 2(a and b)] exhibit an axially symmetric g-tensor parameters with g_|| > g_⊥ > 2.0023 indicating that the copper site has a $d_x^2-d_y^2$ ground state (Kasumov et al., 2004). In axial symmetry, the g-values are related by the expression, G = (g−2)/(g_⊥−2) = 4. According to Hathaway (Hathaway and Billing, 1970; Hathaway, 1984), as value of G is greater than 4, the exchange interaction between Cu²⁺ centers in the solid state is negligible, whereas when it is less than 4, a considerable exchange interaction is indicated in the solid complex. The calculated G values for the copper complexes are less than 4 suggesting copper–copper exchange interactions. The ESR spectrum of [Cu₂(H₂L)(OAc)₂(H₂O)₆] is similar to the ESR spectra of the reported binuclear Cu²⁺ complexes (Khan et al., 1989). The ESR spectrum of [Cu(H₃L)₂(EtOH)₂]·2H₂O exhibits a single line centered at g_// = 2.25 and g// = 2.07, respectively, (Fig. 1b) attributable to dipolar broadening and enhanced spin lattice relaxation. This line is probably due to insufficient spin-exchange narrowing toward the coalescence of four copper hyperfine lines to a single line. Note that, the same kind of powder ESR line shapes has been observed for many tetrahedral or square-planar binuclear Cu²⁺ complexes with a strong intranuclear spin-exchange interaction.

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Figure 2

The ESR spectra of (a) [Cu₂(H₂L)(OAc)₂(H₂O)₆]; (b) [Cu(H₄L)(H₂L)(EtOH)₂]·2H₂O and (c) [VO(H₂L)]·2H₂O.

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Molecular orbital coefficients, α² and β² were calculated (John et al., 2003):

The values of α² and β² for the complexes indicate that the in-plane σ-bonding and in-plane π-bonding are appreciably covalent, and are consistent with very strong in-plane σ-bonding in these complexes. For the Cu²⁺ complexes, the high values of β² compared to α² indicate that the in-plane π-bonding is less covalent than the in-plane σ-bonding. These data are well consistent with other reported values.

The ESR spectrum of [VO(H₂L)]·2H₂O (Fig. 1c) provides a characteristic octet ESR spectrum showing the hyperfine coupling to the ⁵¹V nuclear magnetic moment and similar to those reported for mononuclear vanadium molecule (Thaker et al., 1994). In the powdered form for the mono vanadium complex, the spectrum showed the parallel and the perpendicular features which indicate axially symmetric anisotropy with well resolved sixteen-lines hyperfine splitting characteristic for the interaction between the electron and the vanadium nuclear spin (I = 7/2) (Khasa et al., 2003). The spin Hamiltonian parameters are calculated to be g_//(1.93), g_⊥(1.96), A_//(200 × 10⁻⁴ cm⁻¹) and A_⊥(60). The calculated ESR parameters indicate that the unpaired electron (d¹) is present in the d_xy-orbital with square-pyramidal or octahedral geometry (Raman et al., 2003). The values obtained agree well with the g-tensor parameters reported for square pyramidal geometry.

The molecular orbital coefficients α² and β² for complex (2) are calculated using the reported equations (Warad et al., 2000) and found to be 0.92 and 0.83, respectively. The low value of β² than α² indicates that the in-plane σ-bonding is less covalent and consistent with other reported data (Georgieva et al., 2006).

3.2.5 Antimicrobial activity

The ligand and its metal complexes were tested against Gram-positive, Bacillus thuringiensis (BT), H₄L is more effective on BT than the complexes.

3.2.6 Eukaryotic DNA degradation test

Examining the DNA degradation assay of H₄L and its metal complexes, one can conclude variability on their immediate damage on the calf thymus (CT) DNA. The complexes have higher effect on the calf thymus DNA than the ligand. [VO(H₂L)]·2H₂O, [Cr₂(H₂L)(OAc)₂(OH)₂]·2H₂O, [Mn₂(H₂L)(OH)₂]-·H₂O, [Co₂(H₂L)(H₂O)₄Cl₂]·2H₂O, and [Co₂(H₂L)(OAc)₂]·H₂O have little effect while the [Ni(H₄L)Cl₂]·2H₂O and [Zn(H₂L)] complexes degrading the CT DNA completely (Fig. 3). [Cr₂(H₂L)(OAc)₂(OH)₂]·2H₂O and the ligand have similar effect. The results suggest that direct contact of [Ni(H₄L)Cl₂]·2H₂O and [Zn(H₂L)] is necessary to degrade the DNA of Eukaryotic subject.

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Figure 3

Biological effect of H₄L and its complexes on the Calf Thymus DNA Lanes arranged as: C-control DNA, 1-Ligand, 2-[VO(H₂L)]·2H₂O, 3-[Cr₂(H₂L)(OAc)₂(OH)₂]·2H₂O, 4-[Mn₂(H₂L)(OH)₂]·H₂O, 5-[Co₂(H₂L)(H₂O)₄Cl₂]·2H₂O, 6-[Co₂(H₂L)(OAc)₂]·H₂O, 7-[Ni(H₄L)Cl₂]·2H₂O and 8-[Zn(H₂L)].

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3.2.7 Molecular modeling of the complexes

The molecular modeling of the complexes is presented. The molecular parameters of the complexes are shown in Table 5. Inspection of the data, it is observed that:

1. All complexes have dipole moment values except [Mn₂(H₂L)(OH)₂]·H₂O and [Zn(H₂L)] which have zero moment; this may be due to the trans-form of the complexes; the highest one is [Cr₂(H₂L)(OAc)₂(OH)₂]·2H₂O which is considered as a strong polar molecule.

2. Comparing the data of the two Ni(II) complexes, one can observe that [Ni(H₂L)]·3H₂O has a lower moment than [Ni(H₄L)Cl₂]·2H₂O suggesting that the oxygen and nitrogen atoms exist in opposite side (Scheme 8).

3. The shape of [Zn(H₂L)] (Scheme 10) is found tetrahedral rather than a square-planar and this expected for the d¹⁰-system with sp³ hybridization.

4. All heats of formation are positive and arranged the complexes as: [Zn(H₂L)] > [Ni(H₂L)]·3H₂O > [Ni(H₄L)Cl₂]·2H₂O > [Mn₂(H₂L)-(OH)₂]·H₂O > [Cr₂(H₂L)(OAc)₂(OH)₂]·2H₂O > [Co₂(H₂L)(OAc)₂]·H₂O.

The bond lengths and angles of the compounds are listed in Supplementary Tables 1S–7S.