New Schiff bases of 2-(quinolin-8-yloxy)acetohydrazide and their Cu(ii), and Zn(ii) metal complexes: their in vitro antimicrobial potentials and in silico physicochemical and pharmacokinetics properties

2.4.1 Synthesis of N′-[(E)-(2-hydroxyphenyl)methylidene]-2-[(quinolin-8-yl)oxy]acetohydrazide (SL₁)

For the preparation of SL₁ (Figure 2), a mixture of 2-(quinolin-8-yloxy)acetohydrazide 0.01 mol was reacted with 0.01 mol salicylaldehyde in ethanol with stirring using a hot-stage magnetic stirrer and refluxed for nearly 2 hours. The whitish-yellow solid precipitate was filtered, washed with ethanol, dried, and finally recrystallized in hot ethanol. The yield obtained was 82%, and the measured melting point was 132°C. Elemental analysis for C₁₈H₁₅N₃O₃: cal. (found), %C 67.34 (67.02), %H 4.67 (4.77), %N 13.08 (13.36), and %O 14.95 (14.63).

Figure 2

Structure of SL₁ ligand.

2.4.2 Synthesis of N′-[(E)-(2-hydroxy-3-methoxyphenyl) methylidene]-2-[(quinolin-8-yl)oxy]acetohydrazide (SL₂)

For the preparation of SL₂ (Figure 3), 0.01 mol of 2-(quinolin-8-yloxy)acetohydrazide was mixed with 0.01 mol o-vaniline (2-hydroxy-3-methoxybenzaldehyde) in 30 mL ethanol in a round-bottomed flask. The mixture was refluxed with continuous stirring using a hot-stage magnetic stirrer for nearly 2 hours. The off-white solid precipitate was filtered, washed with ethanol, dried, and finally recrystallized in hot ethanol. The yield obtained was 89%, and the measured melting point was 117°C.

Figure 3

Structure of SL₂ ligand.

Elemental analysis for C₁₉H₁₇N₃O₄: cal. (found), %C 65.01 (64.95), %H 4.84 (4.71), %N 11.97 (12.22), and %O 18.23 (17.86).

2.4.3 Synthesis of N′-[(E)-(2-hydroxynaphthalen-1-yl)methylidene]-2-[(quinolin-8-yl)oxy]acetohydrazide (SL₃)

SL₃ (Figure 4) was synthesized by condensation reaction using 0.01 mol of 2-(quinolin-8-yloxy)acetohydrazide and mixed with 0.01 mol 2-hydroxy-1-naphthaldehyde in 30 mL ethanol in a round-bottomed flask. The mixture was refluxed with continuous stirring using a hot-stage magnetic stirrer for nearly two and a half hours. The yellow solid precipitate was filtered, washed with ethanol, dried, and finally recrystallized in hot ethanol. The yield obtained was 94%, and the measured melting point was 180°C.

Figure 4

Structure of SL₃ ligand.

Elemental analysis for C₂₂H₁₇N₃O₃: cal. (found), %C 71.22 (71.63), %H 4.58 (4.54), %N 11.32 (11.59), and %O 12.94 (12.64).

2.4.4 Synthesis of N′-[(E)-(pyridin-3-yl)methylidene]-2-[(quinolin-8-yl)oxy]acetohydrazide (SL₄)

SL₄ (Figure 5) was synthesized by condensation reaction using 0.01 mol of 2-(quinolin-8-yloxy)acetohydrazide as a primary amine and mixed with 0.01 mol pyridine-3-carboxaldehyde as an aldehydic compound in 30 mL ethanol in a round-bottomed flask. The reaction mixture was refluxed with continuous stirring using a hot-stage magnetic stirrer for nearly 2 hours. The yellowish-white (beige) solid precipitate was filtered, washed with ethanol, dried, and finally recrystallized in hot ethanol. The yield obtained was 78%, and the measured melting point was 105°C.

Figure 5

Structure of SL₄ ligand.

Elemental analysis for C₁₇H₁₄N₄O₂: cal. (found), %C 66.72 (66.33), %H 4.58 (4.85), %N 18.30 (18.71), and %O 10.46 (10.12).

3.1.1 Characterization of the Schiff base ligands

The isolated Schiff bases were purified by recrystallization using hot ethanol and were in good yields (78–94%). They were mostly yellow colored and were soluble in hot ethanol, N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO). The isolated Schiff base ligands were characterized, and the proposed structures were confirmed before proceeding for the preparation of the targeted Cu(ii) and Zn(ii) metal complexes step. The elemental analysis results for C, H, N, and O found were in good agreement with the theoretically calculated percentage values for the proposed molecular formulae, in favor of the expected structures of the Schiff base ligands. The obtained mass spectra (m/z) for each of the prepared ligand compounds (SL₁–SL₄) were also in agreement with the molecular formulae of the prepared Schiff bases.

The IR spectrum of the organic Schiff base compounds exhibited characteristic peaks (Table 2), which were helpful in proving the proposed structures of the ligands. The sharp bands appeared at 1,500, 1,502, 1,504 and 1,505 in the spectrum of the ligands SL₁–SL₄, respectively, designed to the characteristic azomethine group (–CH═N–), which indicates the formation of the Schiff base ligands [20]. The presence of the aromatic (–OH) group in the spectrum of the ligands exhibited peaks at 3,500, 3,513, 3,570, and 3,606 cm⁻¹, respectively [21]. The intense bands observed in the range 1,225–1,255 in the IR spectrum of the ligands (SL₁–SL₃) are due to the phenolic C–O linkage [22]. The sharp transmission bands at 1,660, 1,665, 1,685, and 1,671 cm⁻¹ in the spectra of SL₁, SL₂ SL₃, and SL₄, respectively, are due to the presence of the stretching carbonyl C═O group [9,12,23]. Weak transmission bands observed in the range 2,985–2,988 cm⁻¹ may be assigned to the –N–H amide group [9,23]. The transmission bands observed at 1,115, 1,114, 1,110, and 1,112 cm⁻¹ in the spectra of SL₁, SL₂ SL₃, and SL₄, respectively, can be assigned to the –N–N– linkage [24].

The observed UV-visible absorption spectra for the free organic Schiff base ligands (SL₁–SL₄) show two main bands. The first observed at lower wavelength bands at 295, 284, 262, and 301 nm in the spectrum of the ligands SL₁, SL₂, SL_3, and SL₄, respectively, that can be assigned to π → π^* transitions within the benzene and quinoline rings [25,26]. The second bands were observed at 324, 326, 322, 317 nm in the spectrum of the ligands SL₁, SL₂, SL₃, and SL₄, respectively, which may involve π → π* and n → π^* transitions of the (–C═N–) group [26].

In the ¹H NMR Spectra of all the Schiff base ligands (SL₁–SL₄), the imine protons were observed at 8.58–8.98 ppm as a singlet. Moreover, the peaks at 11.05–12.23 ppm, which are singlets, can be attributed to aromatic –OH protons in the ligand SL₁, SL₂, and SL₃ [27]. This shift in the position of the singlet may be due to the possibility effect of the formation of intramolecular hydrogen bonding interaction. The singlets appeared in the range of 12.18–12.52 ppm in the ¹H NMR spectrum of all Schiff base ligands are due to the –NH– linkage [28]. The peaks due to the aromatic hydrogens appeared in the range of 7.00–8.98 ppm [29].

In ¹³C NMR spectra of the ligands SL₁, SL₂, SL₃, and SL₄, the peaks appeared at 148.75, 148.48, 147.62, and 145.69 ppm, respectively, are assigned to the carbon atom of the azomethine group [27]. The singlet peaks observed in the range of 164.81–169.57 ppm is due to the carbon atom of the C═O group [30]. Moreover, the peaks observed in the region 109.00–154.49 ppm are because of the carbon atoms of the aromatic ring [9].

3.1.2 Synthesis and characterization of metal complexes

Copper(ii) and Zinc(ii) from the first transition elements series of the d-block elements were chosen for the synthesis of the designed metal complexes in this research project. These metals are familiar, important metals and form interesting coordination compounds.

The targeted coordination metal complexes were prepared successfully by reacting to the metal salts of the selected metals with the prepared Schiff base ligands (SL₁–SL₄) in (1:2) stoichiometric ratio according to the described template method. The separated metal complexes were stable solids at room temperature and nonhygroscopic. They were solids insoluble in common organic solvents but soluble in DMF and DMSO. They were purified by recrystallization from hot DMF and DMSO solvents. All prepared metal complexes had high melting points (above 300°C).

The elemental analysis measurements suggested 1:2 metal to ligand stoichiometric ratio forming tetra-coordinate monomeric Cu(ii) and Zn(ii) metal complexes. The measured percentages of the carbon, hydrogen, nitrogen, oxygen, and metal ion are in agreement with the calculated ones according to the suggested structure of the complexes as shown in Figure 6(a–d). The Schiff base quinoline acetohydrazone ligands containing NO coordination sites behave as monobasic bidentate ligands. The general formula for the prepared complexes suggested to be [M(SL_1–4)₂] for all metal complexes

Figure 6

Structure of the synthesized complexes with (a) SL₁, (b) SL₂, (c) SL₃, and (d) SL₄ where M = Cu(ii) or Ni(ii).

According to the above-proposed structures, the prepared Cu(ii) and Zn(ii) complexes are suggested to be mononuclear complexes. The ligands SL₁–SL₃ bind to metal ions through the nitrogen atom of the azomethine (–CH═N–) group and the oxygen atom of the phenolic (–OH) group forming square planar geometry. Moreover, the bidentate Schiff base ligands SL₁, SL₂, and SL₃ forms six-membered chelate rings at the Cu(ii) and Zn(ii) acceptor centers. The ligand SL₄ binds to metal ions through the N atom of –C═N– group and the O atom of the carbonyl group forming square planar geometry. The bidentate SL₄ ligand forms a five-membered ring at the metal acceptor center.

To prove the proposed structures for the prepared metal complexes, we performed different analytical techniques. Mass spectrometry analysis was carried out to help in quantifying and structure elucidation of the obtained complexes and confirm the molecular weights. The measured (m/z) peaks indicated that the observed molecular peaks for all prepared metal complexes are equivalent to the theoretically calculated formula weights according to the proposed structures. These observations support and prove the suggested structures for the prepared metal complexes shown in Figure 6(a–d).

The comparison between the IR spectral data of the free organic ligands (SL₁–SL₄) and that of the corresponding Cu(ii) and Zn(ii) metal complexes demonstrate that the ligands were binding to the metal ions. The coordination of the ligands through the nitrogen atom of the azomethine group was indicated through the shift (blue shift) of the νC═N band in the IR spectrum of the complexes when compared with the free organic ligands. This also was supported by the appearance of the new weak band in the spectrum of the metal complexes at the range of 437–488 cm⁻¹ attributed to the formation of M–N bonds [1]. The formation of M–N bond leads to the weakening of the C═N band due to the donation of electrons from the nitrogen atom to the vacant d-orbitals in the metal atoms, which causes the shift of the azomethine band in the metal complexes [16].

The bands (3,500, 3,513, 3,570, and 3,606 cm⁻¹) shown in the IR spectrum of the free Schiff bases (SL₁–SL₃) due to the phenolic (νOH) group were disappeared in the IR spectrum of the corresponding Cu(ii) and Zn(ii) complexes. This is a sign of the binding of the oxygen atom of the phenolic group to the metal atoms [22,31]. Moreover, the intense bands observed in the IR spectrum of the ligands (SL₁–SL₃) due to the phenolic C–O linkage were shifted and appeared in the range of 1,211–1,265 cm⁻¹ is another proof for the involvement of the OH group in bonding with the metal ions [22]. In addition, a weak band was observed in the IR spectrum of these complexes at the range of 542–595 cm⁻¹ which is assigned to the νM–O mode, evidence for the formation of M–O bonds [32,33].

It was also observed that the band 1,671 cm⁻¹ due to the νC═O of the carbonyl group in the IR spectrum of SL₄ ligand was shifted slightly in the spectrum of the corresponding metal complexes, which indicate the binding of the oxygen atom to the metal ions [33].

The most important bands observed in the IR spectrum of the metal complexes are shown in Table 2.

The UV-visible and magnetic moment susceptibility (μ_eff) data are helpful tools to deduce the nature of the ligand field around the metal ion and the geometry of the obtained metal complexes. The observed UV-visible transmission bands for the prepared Cu(ii) and Zn(ii) complexes are shown in Table 3. The UV-visible spectra indicate that there is a relative shift to lower wavelengths (bathochromic shift) of the transmission bands due to π–π* and n–π* transitions that observed in the spectrum of the free prepared Schiff base ligand. This observation may be because of the coordination of the ligands to the metal core, which causes a change in the distribution of electrons between the ligands and the metal ions [25,26,34,35].

The discussion for each metal complexes is discussed in the following sections.

3.1.3 Cu(ii) metal complexes

The observed UV-visible spectra of the synthesized mononuclear copper(ii) complexes showed transmission bands in the range λ_max = 648–675 nm, which may be accountable for the 2B_1g → 2A_1g transitions that are consistent with square-planar geometry around the Cu(ii) centers [36,37]. Moreover, the magnetic moment values (Table 3) were between 1.906 and 2.261 BM which lies within the permissible values reported for one unpaired electron, and it is an evident that supports the square-planar geometry of Cu(ii) complexes [37]. This geometry is achieved through the coordination of two bidentate ligands (SL₁–SL₄) to the metal center in trans-geometry with respect to each other as shown in Figure 6(a–d).

3.1.4 Zn(ii) complexes

The transmission bands observed in the range 269–277 nm in the UV-visible spectrum of Zn(ii) complexes can be assigned to the π → π^* transitions. The weak bands in the range of 305–318 nm were assigned to n → π* transitions. Moreover, the bands observed in the range 423–438 nm may be attributed to the ligand-to-metal charge transfer transitions [27,38]. The lowest energy bands at the range 610–685 nm were due to the d → d transition of Zn²⁺. These observations are consistent with square-planar geometry. The observed magnetic moment (Table 3) for all Zn(ii) complexes were diamagnetic, which is in support of the square-planar geometry [27,38].

The four-coordinated mononuclear complexes of Zn(ii) are achieved through the coordination of the bidentate (SL₁–SL₄) ligands to the metal center in trans-geometry with respect to each other as shown in Figure 6(a–d). The coordination is through two N atoms and two oxygen atoms from two same Schiff base ligands in a square-planar geometry.

The ¹H NMR analysis was further performed to achieve concrete evidence of the binding of the Cu²⁺ and Zn²⁺ to the ligands. Upon complexation, the peaks due to the proton of the imine group in the Schiff base ligands were showed field up shift slightly by Δ_δ = 0.40–0.80 ppm. The singlet peaks due to the phenolic proton in the ¹H NMR spectra of SL₁, SL₂, and SL₃ were disappeared indicating the deprotonation of this group. These observations proved that the N atom of the azomethine (>CH=N–) group and the O atom of the phenolic (–OH) group participate in coordination and formation of the metal complexes [27]. The peaks observed for the –NH– proton did not show any shift and remained at the same position in the spectra of the metal complexes, which were an indication of no-participation of this group in bonding to the metal ions.

Thermal analysis studies of some prepared metal complexes were investigated at the heating rate of 10 (°C/min) under the nitrogen atmosphere from room temperature to 800°C. These thermogravimetric studies are helpful tools and good supports in the characterization of the prepared metal complexes.

The results of representative TG/DTA plots of the analyzed metal complexes are shown in Figure 7 and 8. The results show good harmony with the proposed chemical structures suggested from the analytical data as discussed earlier. It is obvious from the decomposition pattern that the studied metal complexes were decomposed in three successive stages. The TGA curves of anhydrous complexes do not display any mass losses up to 260°C, which indicate the thermal stability of the metal complexes and confirm the absence of water molecules in the complex structures. In Figure 7, the first step for the complex [Zn(SL₁)₂] (molecular weight = 706.3) started decomposition between 256 and 331°C and weight loss observed was 28.416% which is corresponding to two molecules of the salicylaldehyde part from the ligand (i.e., C₁₄H₁₂) (calculated weight percentage 30.02%) may be due to breaking the azomethine linkage during heating. The second step of decomposition was between 421.02 and 569.41°C with an observed weight loss of 54.243%, which can correspond to the ligand and aromatic ring (C₆H₄) (calculated weight percentage 54.44%). The third step on further heating above 600°C, metal complexes decompose to stable zinc oxide. In Figure 8, the first step for the decomposition of [Zn(SL₃)₂] (molecular weight = 806.15) started decomposition between 230 and 310°C and weight loss observed was 52.37% which is corresponding to two molecules of the amine part (i.e., C₂₂H₁₈O₄N₆) from the organic ligand (calculated weight percentage 53.35%). In the second step, the observed weight loss was 44.83% which corresponds to the organic ligand (C₂₂H₁₇N₃O₃) (calculated weight percentage = 46.02%). Final step above 630°C decompose the complex to the stable zinc oxide. In the DTA curve of the metal complexes, the heat flow in the 90–600°C temperature range was shown as endothermic peaks.

Figure 7

TGA–DTA curves of [Zn(SL₁)₂].

Figure 8

TGA–DTA curves of [Zn(SL₃)₂].

3.4.1 Physicochemical properties

The pharmacological or therapeutic effect is related to the influence of various physicochemical properties of the chemical on the biomolecules that it interacts with. Different physicochemical parameters of a drug candidate play a crucial role in their pharmacokinetic behavior [43]. In view of this, their calculation and measurement help in prioritizing compounds for screening as an efficient drug candidate and enable preliminary decisions in drug discovery [44]. The prepared ligands and their metal complexes were subjected to in silico physicochemical studies such as number of rotatable bonds, hydrogen bond acceptor, hydrogen bond donor, molecular reactivity, water-solubility, lipophilicity, and topological polar surface area (TPSA), which were calculated for understanding the drug transport properties. All results obtained are presented in Table 6. The results of in silico absorption percentage calculations indicated that the percentage of absorption was in the range of 76.90–82.61% among ligands, where the ligand SL₄ was the highest with an absorption percentage of 82.61%. The in silico absorption percentage (42.84–56.23) among complexes observations showed that the complexes [Cu(SL₄)₂] and [Zn(SL₄)₂] showed the highest absorption percentage with the value of 56.23% for both complexes. On comparing the ligands and their corresponding metal complexes, it is clear that the ligands showed a much higher absorption percentage. Moreover, the obtained TPSA values for the ligands were below 140 Ų (the accepted limit of polar surface area), indicating that ligands had considerable permeability into the cellular plasma membrane [45]. While the TPSA values for the examined metal complexes were very high (>140 Ų) marking low permeability into the cellular plasma membrane. The log S (that is the coefficient of solubility determined by the estimated solubility (ESOL) method computed on Swiss ADME) for many known drugs available in the market showed values more than −4.00 [45]. In our investigation, the obtained values of log S (Table 6) for the ligands SL₁, SL₂, and SL₄ were little more than −4.00, indicating that these compounds are considered moderate soluble. While SL₂ was having a value of (−4.81) which is less and hence this compound considered insoluble. In the case of the metal complexes, the log S values are much less than (−4.00), and hence, the metal complexes are considered highly insoluble.

3.4.2 In silico pharmacokinetic/ADME and drug-likeness properties

Pharmacokinetics is used to investigate the time course of chemical absorption, distribution, metabolism, and excretion (ADME) and determines the fate of the chemical administered to the living organisms. In this paper, we have performed in silico pharmacokinetic predictions for the prepared four Schiff base ligands (SL₁–SL₄) and the prepared corresponding Cu(ii) and Zn(ii) metal complexes and the obtained results are presented in Table 7. The gastrointestinal (GI) absorption score measures the extent of absorption of a chemical from the intestine, the results pointed out that all the ligands have high GI absorption and hence had excellent absorption possibility from the intestine after oral administration, while the complexes showed low GI absorption and hence had low absorption possibility from the intestine. It was very interesting that all the ligands and complexes were unable to penetrate through the blood–brain barrier (BBB) except the ligand SL₄. Besides this drug-likeness is the other parameter, which gives the probability of the molecules for the drug candidate. In the light of the drug-likeness definition, the chemical has a logarithm of the partition (log P) (i.e., octanol/water partition coefficient used to predict the solubility of a probable oral drug) between −0.4 and 5.6 values [46]. The skin permeability coefficient (K_p) is a criterion that considers the octanol/water partition coefficient to be a direct effect on the transdermal movement of chemical molecules and used to quantitatively describe the potential of dermal absorption for the synthesized compounds. In our study, log K_p values for all examined ligands and complexes were in the range of −7.50 to −5.04, which are below −0.4 indicating low lipophilicity and cannot penetrate through the lipid bilayers of the cells and suggesting that the synthesized compounds have bad drug-likeness. In addition, to discriminate between the drug-like and non-drug-like chemical substances, the Lipinski (rule-of-five) that describes the relationship between pharmacokinetic and physicochemical parameters, Ghose, Veber, Egan, and Muegge rules were applied to predict whether the target compounds are likely to be bioactive and assess qualitatively the chance of these compounds to become oral drug candidates [19]. The number of violations to these rules is listed in Table 8. All the ligands fulfilled Lipinski rule and other rules and showed zero violation against all the rules, while all the complexes showed two violations against the Lipinski rule and different violations against the other rules. All the ligands in the screening processes showed good bioavailability with a score of 0.55, which is an indication that all organic ligands reach the circulation system, whereas the complexes showed moderate to poor bioavailability with scores in the range 0.11–0.17 and hence cannot reach the circulation system easily.

The prediction of the interaction of the tested compounds with cytochromes CYP450 major isoforms (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4) was performed in order to check which compound will cause significant pharmacokinetic interactions that lead to toxic effect through inhibition of these isoenzymes. The CYP450 inhibition prediction results (Table 7) indicated that all synthesized ligands have probable inhibition to the CYP1A2, CYP2C9 and CYP2D6 isoforms. The ligand SL₃ showed inhibition possibility for all CYP450 isoforms. The majority of metal complexes showed inhibition possibility to the CYP3A4 isoform and no inhibition to the other CYP450 isoforms. The complexes with SL₄ ligand [Cu(SL₄)₂] and [Zn(SL₄)₂] showed no inhibition to all isoforms. Moreover, the probability of the compounds to be the substrate of P-glycoprotein (P-gp) was suggesting that ligands and the majority of complexes have a probability to be nonsubstrate of P-gp. The complexes obtained from SL₃ and SL₄ showed a probability to be the substrate of P-gp. These results suggesting the more favor of non-drug-likeness of the tested compounds.