Solvent-free mechanochemical synthesis of Zn(II), Cd(II), and Cu(II) complexes with 1-(4-methoxyphenyl)-4-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)-1H-pyrrole-3-carbonitrile

2.1 Equipment

The Retsch MM 2000 swing ball-mill (Retsch, Haan, Germany) (at a frequency of 20,225 Hz) was used for the preparation of solid complexes. The Mercury 300 MHz is an nuclear magnetic resonance (NMR) spectrometer synthetic by Varian (Varian, Palo Alto, CA, USA) that was used to detect the ¹H and ¹³C NMR spectra in dimethyl sulfoxide (DMSO). Fourier transform infrared spectroscopy was done with a Mattson 5000 (Madison, WI, USA), and the spectra was determined in the 4000–400 cm⁻¹ region. A Philips X-Pert X-ray diffractometer (Philips, Eindhoven, the Netherlands) was used to detect the patterns of X-ray diffraction (XRD) for the isolated compounds. The Bruker EMX Spectrometer (Bruker, Karlsruhe, Germany) (modulation frequency=100 kHz working at 9.78 GHz in the X-band) was used to record the electron spin resonance (ESR) spectrum. A scanning electron microscope (SEM; JEOL JSM 6510lv) (JEOL, Tokyo, Japan) was used to investigate the surface structures using its high-quality stereoscopic image clarity and magnification capabilities. The mass spectra of prepared ligands was determined via “DI analysis Shimadzu QP-2010” with ion source (200°C) temperature scan speed 526, start end (m/z) (50–300), (70 eV) & ionization mode (EI). Magnetic susceptibility balance (Johnson Matthey, Wayne, PA, USA) was used to detect the magnitude of the magnetic moment. A Shimadzu UV 240 spectrophotometer (P/N 204-58000, Kyoto, Japan) was used to determine the electronic spectra in the 200–900 nm region of Cu(II) complex in DMSO as a solvent. A Perkin-Elmer 2400 Series II Analyzer (Perkin-Elmer, Waltham, MA, USA) was used to decide the percentages of carbon, hydrogen, and nitrogen. All reagents used were supplied by Sigma-Aldrich, Cairo, Egypt and software used was ChemDraw Professional 15.0, Perkin-Elmer, USA.

2.2 Synthesis of 1-(4-methoxyphenyl)-4-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)-1H-pyrrole-3-carbonitrile (HP)

HP was synthesized by refluxing the 4-hydrazinyl-1-(4-methoxy phenyl)-1H-pyrrole-3-carbonitrile (2.28 g; 10.0 mmol) with 2-acetylpyridine (1.22 g; 10.0 mmol) in 1 ml glacial acetic acid (1 m) for 3 h. The buff ppt formed was then filtered off and washed with ethyl alcohol.

2.3 Synthesis of complexes

A mix of HP (0.342 g; 1.00 mmol) and 1.00 mmol of [Zn(CH₃COO)₂]·H₂O, CdCl₂, or [CuCl₂]·2H₂O metal salts was exposed to ball milling at a speed of 40 Hz for 5–9 min. Thin-layer chromatography established the reaction progress (Figure 1).

Figure 1:

The synthesis of HP and its [Zn(HP)(OAc)₂], [Cd(HP)Cl₂], and [Cu(P)(H₂O)Cl] complexes.

2.4 Experimental data

1-(4-methoxyphenyl)-4-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)-1H-pyrrole-3-carbonitrile (HP) (1, C₁₉H₁₇N₅O) Found (buff solid): C=68.85; H=5.12; N=21.10. Calc.: C=68.87; H=5.12; N=21.13. Yield: 78%. m.p.: 208°C. ¹H NMR: d=10.46 (s, NH), 7.18–8.74 (m, 10H/aromatic protons), 2.32 (s, 3H, −N=C–CH₃/methyl group), 3.83 (s, 3H/–OCH₃). ¹³C NMR (200 MHz, DMSO-d6): d (ppm) main signals: 147 (C=N)_Azo); 156 (C=N)_py); 13 (CH₃). IR: ῡ=3250 (NH), 1652 (C=N)_azo, 802 (C=N)_py. MS: C₁₉H₁₇N₅O, Calc.: m/z=331.19; found: m/z=332.14.

[Zn(HP)(OAc)₂] (2, ZnC₂₃H₂₃N₅O₅) Found (yellowish-white solid): %C=53.64; %H=4.49; %N=21.12, %Zn=12.66 Calc.: %C=53.68; %H=4.51; %N=21.15, %Zn=12.70. Yield: 88%. m.p.: >300°C. ¹H NMR: d=10.57 (s, NH), 7.24–8.83 (m, 10H/aromatic protons), 2.30 (s, 3H, −N=C–CH₃/methyl group), 3.78 (s, 3H/–OCH₃). ¹³C NMR: d (ppm) main signals: 140 (C=N)_Azo; 149 (C=N)_py); 18 (CH₃COO); 10 (CH₃). IR: ῡ=3253 (NH), 1640 (C=N)_azo, 803 (C=N)_py, 511 (Zn-O), 464 (Zn-N). Molar conductivity: Λ_m=6 ohm⁻¹ cm² mol⁻¹ (in DMSO).

[Cd(HP)Cl₂] (3, CdC₁₉H₁₇N₅OCl₂) Found (yellowish-white solid): %C=44.40; %H=3.32; %N=13.61, %Cd=21.85; %Cl=13.80; Calc.: %C=44.42; %H=3.34; %N=13.45; %Cd=21.88; %Cl=13.82. Yield: 83%. m.p.: >300°C. IR: ῡ=3250 (NH), 1641 (C=N)_azo, 802 (C=N)_py, 509 (Cd-O), 446 (Cd-N). ¹H NMR: d=10.60 (s, NH), 7.24–8.45 (m, 10H/aromatic protons), 2.30 (s, 3H, −N=C–CH₃/methyl group), 3.75 (s, 3H/–OCH₃). ¹³C NMR: d (ppm) main signals: 139 (C=N)_Azo; 150 (C=N)_py); 10 (CH₃). Molar conductivity: Λ_m=10 ohm⁻¹ cm² mol⁻¹ (in DMSO).

[Cu(P)(H₂O)Cl] (4, CuC₁₇H₁₈N₅O₂Cl) Found (brown solid): %C=45.60; %H=4.11; %N=15.62; %Cu=14.18; %Cl=7.97, Calc.: %C=45.63; %H=4.05; %N=15.65, %Cu=14.20; %Cl=7.93. Yield: 83%, m.p.: >300°C; IR: ῡ=disappear (NH), disappear (C=N)_azo, 832 (C=N)_py, 1636 (N=N), 529 (Cu-O), 448 (Cu-N). μ_eff=2.1 B.M.; band position: 14,643 cm⁻¹. ESR: g_//=2.27, g_⊥=2.06, A_//×10⁻⁴=162 cm⁻¹, g_///A_//=140, G=4.2, α²=0.78, β²=0.84. Molar conductivity: Λ_m=9 ohm⁻¹ cm² mol⁻¹ (in DMSO).

2.5 Molecular modeling

Density function theory (DFT) was applied to predict the optimized geometry of prepared complexes by using the GAUSSIAN 09 program package [18]. The DMOL3 program in Materials Studio package [19] was used to determine the cluster calculations. PBEPBE [20] is a good exchange correlation functional that is based on generalized gradient approximation [21]. Additionally, the molecular electrostatic potential (MEP) was estimated using the B3LYP/6-31G(d) [22] level of theory.

2.6 Antibacterial activity

The disc diffusion technique [23] was used to decide the minimum inhibition concentration of examined compounds against Pseudomonas aeuroginosa and Escherichia coli as a Gram (−) bacteria and Staphylococcus aureus and Bacillus subtilis as a Gram (+) bacteria; as shown in Supplementary Figure 1S. In this method, the paper discs (Whatman filter paper, no. 42, uniform diameter 2 cm) soaked in the desired concentration of the complex solutions (2 mg/ml) were placed aseptically in petri dishes containing nutrient agar media (20 g agar+3 g beef extract+5 g peptone) seeded with different types of bacteria. The petri dishes were incubated at 37°C and the inhibition zones were recorded after 24 h. Each treatment was replicated six times. Note that the antibacterial activity of a common standard antibiotic Ampicillin was also recorded using the same procedure as above at the same concentrations and solvent. The % activity index for the investigated complexes was calculated by applying this equation:

% activity index=zone of inhibition by test compound (diameter)/(zone of inhibition by standard (diameter)×100.

2.7 Colorimetric assay for compounds that bind DNA

The colorimetric assay steps [24] for the investigated compounds are shown in Figure 2. The results improved for primary absorbance and stabilized as the percentage of the untreated standard.

Figure 2:

The steps for colorimetric assay for the investigated compounds.

3.1 Infrared, MS, ¹H, and ¹³C-NMR spectra

The significant infrared (IR) bands of the HP and its Zn(II), Cd(II), and Cu(II) complexes were chosen to identify the effect of the coordination of metal to the vibration of the ligand. The infrared spectrum of the HP revealed a medium-intensity broad band due to ν(NH) at 3250 cm⁻¹ [25]. Also, there are two bands related to ν(C=N)_azo [26] and ν(C=N)_py [27] at 1652 and 802, respectively.

The ¹H-NMR spectrum of HP in DMSO displayed peaks at 10.46, 2.32, 3.83 and 7.18–8.74 ppm movable to protons of NH, −N=C–CH₃, −OCH₃, and aromatic protons, respectively.

The MS of HP exhibited a molecular ion peak [M]˙⁺ of HP at m/z=331.14 (100.0%), that is, corresponding to its molecular weight and formula (C₁₉H₁₇N₅O).

The ¹³C-NMR spectrum of HP was detected in DMSO (Supplementary Figure 2S). The signals for the (C=N)_Azo and 156 (C=N)_py were shown at the downfield position (147 and 156, respectively).

In the IR spectra of [Zn(HP)(OAc)₂] and [Cd(HP)Cl₂] complexes, the ν(C=N)_py shifted to a higher frequency but the ν(C=N)_azo shifted to a lower frequency, representing the coordination of pyridine nitrogen and azomethine nitrogen. Therefore, HP worked as a neutral bidentate ligand. Likewise, new bands were detected at 464 (446) and 511 (509) cm⁻¹ movable to ν(M-N) and ν(M-O), correspondingly [28]. Moreover, the [Zn(HP)(OAc)₂] complex has two bands at 1345 and 1433 cm⁻¹ movable to the ν_s(O-C-O) and ν_as(O-C-O) of the acetate group with a difference (80 cm⁻¹) that points to bidentate bonding for the acetate group [29]. In ¹H-NMR spectra of [Zn(HP)(OAc)₂] and [Cd(HP)Cl₂] complexes in DMSO, the signal was ascribed to the NH proton, representing that these groups did not participate in coordination. Furthermore, the ¹³C-NMR spectra of Cd(II) and Zn(II) complexes showed signals at 140 (149) and 139 (150) ppm attributed to (C=N)_Azo and (C=N)_py, correspondingly.

However, in the case of Cu(II), the HP worked as a mononegative bidentate ligand through ν(C=N)_py and ν(−N=N–C−)_azo. This manner of coordination was proposed by (a) the shift of ν(C=N)_py to a higher frequency; (b) the fading of (C=N)_azo with the immediate presence of a new band at 1636 related to (N=N); and (c) the presence of new bands at 448 and 529 cm⁻¹ attributable to ν(Cu-N) and ν(Cu-O), respectively [28].

3.2 Magnetic moment and electronic spectra

The magnetic moment magnitude was 2.1 BM in [Cu(P)(H₂O)Cl] complex, which represented the existence of Cu(II) ion. The electronic spectrum displayed a broad band with a maximum at 14,643 cm⁻¹ that related to ²B₁⁻_g→²A₁⁻_g transitions in a square planar structure [30].

3.3 ESR studies

The ESR spectrum of the [Cu(P)(H₂O)Cl] complex at room temperature (Supplementary Figure 3S) exhibited axially symmetric g tensor parameters (g_||>g>2.0023) wherever the magnitude of g showed a square planar around the copper (II) center where the unpaired electron existed in the d_x²−_y² orbital [31]. In axial symmetry, the G magnitude (4.4), which estimates the exchange interaction between Cu(II) centers in the isolated complex, was stated by

G=(g//–2)/(g⊥–2)=4.

Therefore, according to Hathaway and Billing [32], if G>4, the exchange interaction between Cu(II) centers was excluded.

The molecular orbital coefficients (β² and α²) were estimated by applying these equations [33]:

β2=(g//−2.0023)E−8λα2

α2=(A//0.036)+(g//−2.0023)+3(g⊥−2.0023)7+0.04

where β² is a measure of the covalent in-plane π bonding; α² is a covalent in-plane σ bonding; λ=−828 cm⁻¹ for the free Cu(II) ion and E is the energy of electronic transition. The outcomes data indicated that the in-plane σ bonding and in-plane π bonding are noticeably ionic. These outcomes expected because there are suitable ligand orbitals to associate with the d_xy orbital of the Cu(II) ion. For the square planar geometry complex, the minor magnitude of β² related to α² indicated that the in-plane π bonding is more covalent than the in-plane σ bonding. This information fit reliably with other stated magnitudes [34].

3.4 SEM and energy-dispersive X-rays

The surface morphologies of [Zn(HP)(OAc)₂], [Cd(HP)Cl₂] and [Cu(P)(H₂O)Cl] complexes were estimated by using SEM, which is a potent technique for the study of surface structures. The complexes in Figure 3 show irregular broken ice rock morphologies with particle sizes of approximately a micrometer, which were randomly distributed over these ice rock–shaped structures [35].

Figure 3:

EDX and SEM views of (A) [Zn(HP)(OAc)₂], (B) [Cd(HP)Cl₂], and (C) [Cu(P)(H₂O)Cl] complexes.

Furthermore, the chemical compositions of Zn(II), Cd(II), and Cu(II) complexes are diverse according to energy-dispersive X-ray (EDX) spectrometer analysis, as displayed in Figure 3. The EDX figure represents the presence of carbon, nitrogen, oxygen, chloride atoms and zinc, cadmium, and copper ion in the complexes’ skeletons, evidencing chelation between the ligand and the metal ion.

3.5 XRD

The Zn(II), Cd(II), and Cu(II) complexes were examined by XRD at room temperature using Cu, Kα radiation. The diffraction patterns (Supplementary Figure 4S) of the examined compounds were attained in a region of (10°<2θ<80°) [36]. The sharp peaks displayed in the patterns indicate the formation of a well-defined distorted crystalline construction. The size of crystalline intended by using equation of Debye–Scherrer at full width at half maximum of the characteristic peaks:

β=0.94 λ/(S cos θ)

B=the line width at half maximum height

Cu/Kα (λ)=1.5406 Å

S=the crystallite size

θ=the diffraction angle

Furthermore, using a Bragg equation, the magnitude of the inner crystal plane d spacing was estimated as

nλ=2dsin(θ) at n=1

The magnitudes of the particle size and lattice parameters of isolated complexes are collected in Table 1. The obtained particle sizes were found in the 7.283–7.683 Å region.

3.6 DFT

The DFT was applied to optimize the geometry (Figure 4) and the quantum chemical parameters, for instance, “E_LUMO, E_HOMO, exchange-correlation, dipole moment, kinetic energy, electrostatic energy, binding energy, sum of atomic energies, spin polarization, binding energy, and total energy” (Table 2) of isolated compounds [37]. The results showed that

Figure 4:

Molecular modeling of (A) HP, (B) [Zn(HP)(OAc)₂], (C) [Cd(HP)Cl₂], and (D) [Cu(P)(H₂O)Cl].

1. The negative magnitude of E_LUMO and E_HOMO indicated the stability of isolated complexes (Supplementary Figure 5S) [38].

2. The greater magnitude of the binding energy for isolated complexes compared with the free ligand (HP) pointed to the larger stability of the complexes compared with the ligand alone [39].

3. The higher dipole moment of complexes compared with the ligand alone indicated the potent activities of the isolated Zn(II), Cd(II), and Cu(II) complexes [39]. In addition, the electric dipole moment is an assessment of the separation of electrical charges in a molecular system.

3.7 MEP of HP

The MEP is a suitable method for the determination of good sites for nucleophilic and electrophilic attack [40]. Therefore, to obtain the MEP of isolated compounds, we should optimize the geometry of the molecular system by first applying the Becke 3LYP/6-31 G(d) level of theory. The three dimensional plots of MEP were outlined for HP and its Zn(II), Cd(II), and Cu(II) complexes as shown in Figure 5. In MEP, the green area pointed to the neutral electrostatic potential, the red area indicated the more attractive potential atoms, and the blue area indicated the more repulsive potential atoms [41].

Figure 5:

Molecular electrostatic potential of (A) HP, (B) [Zn(HP)(OAc)₂], (C) [Cd(HP)Cl₂], and (D) [Cu(P)(H₂O)Cl].

3.8 Antibacterial activity

The investigated compounds, Ampicillin (standard drug) and DMSO (solvent control), were screened individually for their antibacterial activity against P. aeuroginosa and E. coli as Gram (−ve) bacteria and B. subtilis and S. aureus as Gram (+ve) bacteria [42], [43], [44]. The activity of the investigated compounds compared with the activity of Ampicillin as well as the intended percentage of activity index. The outcomes proposed that the Zn(II) and Cd(II) complexes (Table 3) had higher activity against various kinds of bacteria. We conclude that the complexes showed better antibacterial activity than the ligand (HP) alone.

3.9 Colorimetric assay for compounds that bind DNA

At pH 7, methyl green was reversibly binding to DNA and the colored complex was stable wherever methyl green was absent at this pH. However, the binding active compounds with DNA substituted the DNA from its methyl green complex. The movement was determined by spectrophotometric studies as a reduction in the absorbance at 630 nm. The results indicated that Zn(II) and Cd(II) complexes displayed the maximum affinity to DNA, which was recognized by absorption of the complex at the origin or by transferring for minor spaces and then evaluating the IC50 magnitude (Table 4), whereas HP and Cu(II) complex showed moderate activity.

3.10 Drug-likeness and molecular property prediction

The HP and its Zn(II), Cd(II), and Cu(II) complexes were examined for agreement with the rule of five (Table 5). The rule of Lipinski stated that “a molecule probable to develop as an orally active drug applicant presented no further than one violation of the following standards: hydrogen bond acceptors (HBA) <10, hydrogen bond donors (HBD) <5, an octanol-water partition coefficient <5, and molecular weight <500 Da” [45]. The MolSoft software was used to evaluate the drug-likeness model score and the molecular properties of the isolated compounds [46]. The calculated values of HBD and HBA for the studied compounds agreed with the Lipinski rule. Therefore, the prepared compounds had permeability properties through the living membrane. Furthermore, (log P) is a vital factor leading to inert membrane partitioning and stimulating permeability opposite its influence on solubility. Moreover, molecular weight has a great effect on drug action; however, the molecular weight increased outside a limit and the bulkiness of the compounds also increases. This will affect the drug receptor/DNA interactions. Additionally, the molecular polar surface area (PSA) is a sum of the surface polar atoms such as oxygen and nitrogen devoted hydrogen atoms in the investigated molecule.

3.11 Structure activity relationship

The inverse relationship between the dipole moment and the activity of the investigated compounds compared with the experienced microbe was considered a good structure activity relationship study. In fact, the dipole moment is a proper parameter for detecting the excretion speed and penetration over the cell membrane. However, the lipophilic nature of the compound increases as the dipole moment decreases and thus the polarity also decreases where the permeation is stronger over the lipid film of the microbe [47], thus abolishing them further hostilely.

The outcomes presented in Tables 2, 3 and 4 show that the Zn(II) and Cd(II) complexes have a minor magnitude of dipole moment compared with the HP and Cu(II) complex, which points to their potent activities [48]. In addition, the E_HOMO and E_LUMO orbital energies correlated to the free radical scavenging activities of the antioxidant species [49]. Hypothetically, the electrophiles and nucleophiles were closely credited with radical scavenging activities recognized under the relative energy influence of the (HOMO/LUMO) orbitals. The investigated compounds that have low ionization energy gave up electrons simply and therefore probably share chemical reactions. The investigated compound had a sizable E_HOMO and insignificant E_LUMO values and, therefore, little ΔE (energy gab) ordered as good electron-releasing species. The potent antioxidants in this research for Zn(II) and Cd(II) complexes showed the lowest ΔE (E_LUMO–E_HOMO) values [50].