Compounds chemistry
The condensation reaction of two equivalents of (1H-1,2,4-triazol-1-yl)methanol with one equivalent of 3-bromo-5-methylpyridin-2-amine, 5-bromopyridin-2-amine or 3,5-dibromopyridin-2-amine,respectively, was followed ,as reported previously in literature [44–48], to afford the new tridentate ligands L1, L2 and L3. Thus, after 4 hours of stirring, the products were isolated with good yield ranging from 70 to 76%. Furthermore, their assigned chemical structures, as depicted in Fig. 1, have been well confirmed by different spectroscopic analyses as mentioned previously.
Generally, the presence of the characteristic doublet at 7.69 and 7.70 ppm in 1H NMR spectra of L1, L2 and L3, clearly confirms the condensation between hydroxytriazole moiety and the appropriate 2-aminopyridine derivative to give suitable compounds.
Particularly, analysis of the spectral data given in experimental section of the compound L1 revealed the presence of a coupled protons system between H10 and H7 protons, which is manifested by the doublet centered at 5.70 ppm attributable to the methylene protons (N-CH2-N), and the triplet signal centered at 7.43 ppm attributable to the secondary amine proton (-NH-). It seemed that chemical reactions of primary amines with (1H-1,2,4-triazol-1-yl)methanol were leading to the formation of products resulting from a mono-condensation reaction. It has been suggested that the obtaining of the mono or bis-condensation product depends essentially on the reactivity of the amine as a nucleophilic reagent, the reaction conditions and the purification process used to isolate the products formed. Moreover, the signal of proton H3 is split into a doublet of doublets by coupling to the proton H5, and the methyl protons H8. The coupling of the three chemically non-equivalent protons H3, H5 and H8 to each other can be assimilated into an AMX system (Fig. 2).
Additionally, and in order to confirm the mono-condensation reaction and find out all the interactions between different nuclei, further analyses were performed using 2D NMR spectroscopy. COSY NMR spectrum of L1 (Fig. 3) showed a strong correlation between the methylene protons H10 and the secondary amine proton H7. The COSY correlation is also seen for three nuclei coupled to each other, indicated by the two protons H3 and H5 on the pyridine ring and methyl group H8, it seems likely that the correlation identified corresponds well to an AMX spin system. Furthermore, HSQC NMR spectrum (Fig. 3) allowed us to distinguish very clearly the heteronuclear correlations of aromatic proton-carbon coupling nuclei. Indeed, the hydrogens H13 and H15 were found to be coupled with the triazole carbons C13 and C15 respectively. Whereas, the protons H3 and H5 coupled with the pyridine carbons C3 and C5 respectively. Interestingly, H7 proton does not give a cross signal in HSQC NMR spectrum, which means the absence of correlation between proton-carbon atoms; it certainly corresponds to the nitrogen-hydrogen bond.
On the other hand, the investigation of the solvent effect on the chemical shift values of proton resonance of the compound L1, analyzing the 1H NMR spectra recorded in CDCl3 (Fig. 4-I) and DMSO (Fig. 4-II), demonstrates the presence of declination in the chemical shift values, particularly for -NH proton, which varies from over 1 ppm (from 5.98 ppm in CDCl3 to 7.43 ppm in DMSO). In fact, the electrostatic interactions resulting from the polarizability properties between the solute and the solvent molecules appear to play only a minor role with small difference on the chemical shift values obtained for both solvents. While, the hydrogen bond interactions appear to affect the resonance signal of -NH proton by significant absolute values that can probably be explained by the attraction of electrons towards oxygen atoms of the DMSO solvent [49], causing a significant decrease of the electron density around the -NH proton, thus deshielding the protons and cause it to shift furthest downfield.
Computational studies
ADME-Tox evaluation
Early predictions of ADME-Tox profiles of chemical compounds basing on their physicochemical, pharmacokinetic, metabolism and toxicity properties play a vital role in the selection and identification of promising new drug candidates with high bioactivity at minimum concentrations and low toxicity or few side effects. These estimations avoid the waste of time and resources during the drug discovery process [42, 50]. In the context, physicochemical and drug-likeliness properties such as molecular weight, lipophilicity (Log P), number of hydrogen bond donors and acceptors, number rotatable bonds, water solubility, topological polar surface area (TPSA), and percentage of absorption, in addition to the toxicity potential were determined to evaluate the in silico ADME-Tox profiles of the synthesized compounds as well as the selected drugs. The results are represented in Tables 1 and 2.
Table 1
Pharmacokinetic analysis of the studied compounds.
Entry | MW | Log P | HBD | HBA | nrotb | Violations | TPSA | %Abs | Log S | SA |
| ≤ 500 | ≤ 5 | ≤ 5 | ≤ 10 | ≤ 10 | ≤ 1 | ≤ 140 | ≥ 80 | - | - |
L1 | 268.11 | 1.61 | 1 | 3 | 3 | 0 | 55.63 | 89.80 | -2.83 | 2.32 |
L2 | 254.09 | 1.29 | 1 | 3 | 3 | 0 | 55.63 | 89.80 | -2.45 | 2.32 |
L3 | 332.98 | 1.91 | 1 | 3 | 3 | 0 | 55.63 | 89.80 | -3.17 | 2.40 |
Ribavirin | 244.20 | -2.20 | 4 | 7 | 3 | 0 | 143.73 | 59.41 | -0.65 | 3.89 |
Arbidol | 477.41 | 4.20 | 1 | 4 | 8 | 0 | 80.00 | 81.10 | -5.85 | 3.57 |
MW: Molecular weight, Log P (consensus): Calculated partition coefficient (n-octanol/water), HBD: Number of hydrogen bond donors, HBA: Number of hydrogen bond acceptors, nrotb: number of rotable bonds,, Violations: Number of violations from Lipinski’s rule of five, TPSA: Topological polar surface area (Å2), %Abs: Percentage of absorption, Log S: Water solubility (insoluble < − 10 ; poorly soluble < − 6 ; moderately soluble < −4 ; soluble < − 2 < very soluble ; 0 < highly soluble), SA: Synthetic accessibility [From 1 (very easy) to 10 (very difficult)] |
Analysis of ADME data (Table 1) clearly revealed that all of our compounds satisfying the established Lipinski's criteria. The molecular weights were found to be ranging from 254.09 to 332.98 (< 500), Log P values from 1.29 to 1.91 (< 5). For the three compounds, HBD was found to equal one (< 5), HBA equal three (< 10) and with zero violations from Lipinski’s rule of five. Hence, they should theoretically exhibit good oral absorption profiles. These parameters were also found to be in agreement with Lipinski’s rules when investigating the selected drugs Ribavirin and Arbidol. Besides, the prediction of the topological polar surface area (TPSA), which is a good factor to estimate the intestinal absorption (TPSA ≤ 140 Å2) and blood-brain barrier (BBB) permeability (TPSA ≤ 60 Å2) [51], showed that compounds L1, L2 and L3 have very good intestinal absorption and BBB penetration proprieties, with the same TPSA value of 55.63 Å2. This value was smaller than that shown by the standards. Arbidol showed a value of 80.00, while Ribavirin showed a value equal to 143.73 Å2 greater than the limit value (140 Å2). In addition, the number of rotatable bonds was found to equal three for L1, L2 and L3. This parameter describing the conformational flexibility of a molecule, and must be not more than ten to increase the probability that the functional groups in the molecule match the desired pharmacophore. The three synthesized compounds exhibit the same human intestinal absorption percentage of 89.80%, which is higher than that shown by Ribavirin (59.41%) and Arbidol (80.00%). These findings clearly confirming good permeability, absorption rate and transport via biological membranes of our compounds. Moreover, the aqueous solubility (Log S) is another important indicator to determine the bioactivity of a drug. Compounds that are highly water-soluble are more easily metabolized and eliminated from the organism, reducing the probability of adverse effects. The obtained outcomes, indicating that our compounds showed solubility values range from − 2.45 to -3.17 and were more soluble in comparison with Arbidol which showed a moderate solubility of -4.37. Synthetic accessibility of L1 and L2 was 2.32 and 2.40 for L3, suggesting that these compounds are easier to prepare than the selected standards, which is in accordance with our principal goal to produce small molecules with minimum steps and resources. The results also present a good advantage to use these molecules as precursors to develop new drugs.
On the other hand, in order to keep away from the experimental test of possibly harmful substances, the knowledge of the toxic potential of a particular compound remains one of the basic steps for the continuation of research in drug discovery and development. In this work, toxicological profiles of the studied compounds were predicted as shown in Table 2. The results illustrated that none of the synthesized ligands poses any mutagenicity, tumorgenicity, carcinogenicity, cytotoxicity, hepatotoxicity, immunotoxicity or irritant effects. The results also indicating that Ribavirin had the potential to be carcinogenic. Furthermore, from the predictions of rat acute toxicity, it was observed that all of the derivatives showed weak median lethal doses (LD50) of 1500 mg/kg for L1 and 1800 mg/kg for L2 and L3. In general, compounds with these LD50 values fall into the “class III’’ of toxicity scale (LD50 < 5000 mg/kg) and are typically thought to be druggable.
Overall, the ADME-Tox predictions suggest that the prepared triazole derivatives can be utilized as potent and promising drug candidates with optimal oral bioavailability and safety features.
Table 2
In silico toxicity risk prediction of investigated compounds.
Entry | Mutagenicity | Tumorigenicity | Irritant | Carcinogenicity | Cytotoxicity | Hepatotoxicity | Immunotoxicity | LD50 |
L1 | none | none | none | inactive | inactive | inactive | inactive | 1500 |
L2 | none | none | none | inactive | inactive | inactive | inactive | 1800 |
L3 | none | none | none | inactive | inactive | inactive | inactive | 1800 |
Ribavirin | none | none | none | active | inactive | inactive | inactive | 2700 |
Arbidol | none | none | none | inactive | inactive | inactive | inactive | 340 |
LD50: Oral rat acute toxicity in mg/kg |
Molecular docking
To get an atomistic insight of the binding mode of the synthesized derivatives to the SARS-CoV-2 spike protein, computational docking simulations using two different and separate blind docking approaches were carried out: the first was performed using only the SARS-CoV-2-RBD structure; while in the second model, the entire SARS-CoV-2-RBD- hACE2 complex had chosen. The binding affinity for each ligand to the receptor was evaluated calculating the binding energy (ΔG).
Analysis of the interface region of SARS-CoV-2-RBD (S- RBD) and hACE2 complex remain very important step to understand the interactions between these two proteins and to identify the key residues involving in this binding. Currently, several studies demonstrated that the S-protein ( via S-RBD) of the SARS-CoV-2 virus binds to the human ACE2 receptor protein through many important interface residues such as Lys417, Gly446, Tyr449, Tyr453, Leu455, Ala475, Phe486, Asn487, Tyr489, Gln493, Gly496, Gln498, Thr500, Asn501 and Gly502 [52–55]. These residues were identified to make significant interactions with the hACE2 residues Ser19, Glu24, Lys31, Asp30, Lys31, Glu35, Asp38, Tyr41, Gln42, Tyr83, Lys353, Asp355, Leu455 and Gln493, which contributing to the stability of the complex. Hence, design of molecules that can form direct interactions with S-RBD interacting surface amino acid residues (or at the interface region) seems to be an applicable strategy to block or reduce the binding of the virus to hACE2 receptor surface.
First approach: Docking against SARS-CoV-2-RBD structure (Chain B)
As mentioned previously, the interaction surface of S-RBD protein contains characteristic residues involved in hACE2 binding and facilitate the attachment of the virus to the host cell. In order to identify possible inhibitors that could interact with these residues before the attachment to the host cell receptors, we performed blind docking simulations against SARS-CoV-2-RBD structure (Chain B). The docking results as binding free energy score (ΔG), involved residues and interactions’’ type were summarized in Table 3.
Amongst the three synthesized compounds, L1 showed the highest binding energy (ΔG) of -5.5 kcal/mol compared to L2 (-5.2 kcal/mol) and L3 (-5.0 kcal/mol). Ribavirin showed a binding free energy score of -5.8 kcal/mol. Unfortunately, these two ligands (L1 and Ribavirin) were found to bind out the interaction surface pocket of S-RBD protein that interacts with hACE2 receptor (see Figure S1 in Supporting information). In contrast, L2, L3 and Arbidol (ΔG = -5.7 kcal/mol) were found to form several bonds at the S-RBD interaction surface region.
Table 3
Docking results of the studied ligands.
Compound | SARS-CoV-2-RBD |
ΔG (kcal/mol) | Involved residues | Type of interactions |
L1 | -5.5 | Ser373, Trp436, Phe342, Phe374, Leu441, Leu368, Ser371, Asn343, Gly339 | CHB, Pi-Pi stacked, Alkyl Pi-Alkyl, VDW |
L2 | -5.2 | Gly496, Asn501, Arg403, Tyr505, Tyr453, Tyr495, Glu406, Phe497, Gln498, Gly502 | HB, CHB, Pi-Pi T-shaped, Pi-Alkyl, VDW |
L3 | -5.0 | Gly496, Tyr505, Tyr495, Phe497, Gly502 | Pi-Donor HB, Pi-Pi stacked, Pi-Pi T-shaped, Pi-Alkyl, VDW |
Ribavirin | -5.8 | Arg346, Arg509, Asp442, Leu441, Asn448, Asn450, Tyr451, Thr345, Phe347 | HB, VDW |
Arbidol | -5.7 | Ser494, Tyr505, Tyr449, Tyr495, Phe497, Gly502 | HB, CHB, Pi-Pi T-shaped, VDW |
HB: Hydrogen Bond; VDW: Van der Waals forces; CHB: Carbon Hydrogen Bond. |
For L2, L3 and Arbidol that predicted to bind to the interaction surface region of S-RBD, the 2D representations of their interactions with S-RBD protein residues were illustrated in Fig. 5. Analysis of the interactions revealed that these compounds bind to S-RBD residues through numerous hydrogen, hydrophobic and electrostatic interactions. Compound L2 had the highest number bonds with side chains of residues in the S-RBD binding pocket. It forms ten interactions: two conventional hydrogen bonds with Gly496 and Asn501, one carbon hydrogen bond with Arg403 residue, one Pi-Pi T-shaped interaction with Tyr505, two Pi-Alkyl interactions with Tyr453 and Tyr495, and interacts with Glu406, Phe497, Gln498 and Gly502 via four VDW forces. Compounds L3 displayed Pi-Donor hydrogen bond with Gly496, Pi-Pi stacked, Pi-Pi T-shaped and Pi-Alkyl interactions with the amino acid Tyr505, and forms VDW interactions with Tyr495, Phe497 and Gly502 residues. While, Arbidol interacts with Ser494 through hydrogen bond and with Tyr505 through carbon hydrogen bond and Pi-Pi T-shaped interactions. The compound also forms Pi-Alkyl interaction with Tyr449 residue and three electrostatic interactions with Tyr495, Phe497 and Gly502. Analysis of these findings reveals that all these three compounds exhibited interactions with the key interacting residues involved in the binding of S-RBD with the hACE2 receptor. Chowdhury et al. [56] demonstrate by computational studies that some of these key residues have strong impacts on the binding of hACE2 ,particularly the residues Asn501, Gly502 and Tyr505. The studies indicate that the substitution of Asn501, Gly502 and Tyr505 amino acids can reduce the binding of S-RBD with the hACE2 by more than 62%, 63% and 40%, respectively. Overall, the obtained data showed that our ligands are able to involve in different kinds of interactions with many key residues of S-RBD, which can make it as promising SARS-CoV-2 inhibitors.
Second approach: Docking against the entire protein
In order to identify the capacity of the prepared derivatives to interfere and prevent the binding between the S-RBD and hACE2 proteins, we performed blind docking screening using the entire S-RBD - hACE2 complex. Table 4 resume the obtained results.
According to the in silico calculations, only L1, Ribavirin and Arbidol were found to bind to the binding interface pocket between the S-RBD and hACE2 proteins. With a ΔG score of -6.8 kcal/mol, compound L1 exhibited the best score compared to Ribavirin and Arbidol (-6.7 and − 6.1 kcal/mol, respectively), which suggest it to be better complexed to the studied receptor and more active than the two standard drugs. Surprisingly, compounds L2 and L3 did not exhibit any considerable interactions with the interface pocket residues (see Figure S2) despite their relatively good binding energies of -5.8 and − 6.2 kcal/mol, respectively.
Table 4
In silico docking outcomes against S-RBD - hACE2 complex.
Compound | ΔG (kcal/mol) | Interactions with the binding interface |
S-RBD residues (chain B) | hACE2 residues (chain A) |
L1 | -6.8 | Tyr453, Tyr495, Tyr505, Phe497, Arg403, Ser494, Gly496 | His34, Glu37, Asn33, Asp38, Lys353 |
L2 | -5.8 | No interaction | Glu375, Ala348, His378, Asp382, Glu402, His374, His401, Trp349, Asn394, Thr347 |
L3 | -6.2 | No interaction | Ala348, Arg393,, Phe40 (Pi-Alkyl), Phe390, Trp349, Thr347, Glu375, Asp382, Asn394, His401 |
Ribavirin | -6.7 | Arg403, Tyr453, Tyr495, Tyr505, Glu406 | Asn33, Glu37, His34, Gln388, Pro389, Phe390, Arg393 |
Arbidol | -6.1 | Arg403, Glu406, Gln409, Tyr505, Asp405, Arg408, Gly416, Ile418 | Glu37, Asn33, Arg393 |
HB: Hydrogen Bond; VDW: Van der Waals forces; CHB: Carbon Hydrogen Bond. |
Figure 6 depicted the binding modes of L1, Ribavirin and Arbidol into the interface pocket. Inspecting these binding modes revealed that hydrophobic and electrostatic interactions with the catalytic pocket residues mainly drive the molecular recognition of the three compounds. The most promising in silico inhibitor, compound L1, was recorded to interact through Pi-Pi T-shaped, Alkyl ,Pi-Alkyl and VDW interactions with the SARS-CoV-2-RBD Tyr453, Tyr495, Tyr505, Phe497, Arg403, Ser494, Gly496 residues and hACE2 His34, Glu37, Asn33, Asp38 Lys353 amino acids via hydrogen bonds, Pi-Pi stacked, Pi-Anion and VDW interactions. As previously reported in the literature, a single mutation of Lys353 residue is sufficient to revoke the interactions at the interface [57]. Interestingly, compound L1 was found to interact with this residue, which provides an interesting way that can destabilize the virus–receptor combination. In addition, L1 also interacts with some important S-RBD residues such as Tyr495, Phe497 and Arg403 which reported by Unni et al. [58] do not indulge in interactions with the hACE2 receptor but can present an efficient strategy to develop possible drug inhibitors. Ribavirin forms one hydrogen bond with Arg403 and four VDW interactions with the key residues Tyr453, Tyr495, Tyr505 and Glu406 of S-RBD. In the same time interacts with hACE2 Asn33 (HB and CHB), Glu37 (CHB and Pi-Anion), His34 (Pi-Pi stacked), Gln388 (VDW), Pro389 (VDW), Phe390 (VDW) and Arg393 (VDW) residues. Arbidol binds S-RBD Arg403 via a combination of HB and Pi-Cation, Glu406 and Gln409 via carbon hydrogen bonds, Tyr505 through a Pi-Sulfur interaction and Asp405, Arg408, Gly416 and Ile418 using VDW forces.
Taken together these in silico observations from both the computational approaches, it can be said that the synthesized compounds could be putative inhibitors of SARS-CoV-2 that can inhibit the S-protein function by abolishing or interfering the interactions of the key residues, which initialize the entry of the virus to its host cell in both cases of first or second approaches. However, it is worth mentioning that the theoretical calculations do not always reflect the biological response. For this, appropriate experimental assays are required to determine the real potential of these active compounds.
Catechol oxidation studies
In order to develop new model systems for mimicking catecholase enzyme activity [41, 59, 60], a series of copper(II), manganese(II) and nickel(II) complexes was prepared in situ from several metallic salts and the ligands L1, L2 and L3, and investigated toward the aerobic oxidation of catechol at room temperature.
Additionally, catechol oxidizes very slowly to its quinone form, in contact with air oxygen under standard conditions of pressure and temperature (25°C, 1 bar). Under the experimental conditions used, the spectrum obtained for catechol alone showed practically no absorbance as a function of time, the same result was observed with ligands alone and with salts of metals alone in the presence of catechol, which means the absence of o-quinone formation and any significant catalytic activity.
In some cases, it was not possible to obtain the results of the activity of complex compounds; for this reason, the catecholase activity of ligands that leads to a precipitation in the cuvette with the transition metals has not been carried out.
Oxidation of catechol in the presence of in situ complexes formed with ligands L1–L3
The catechol-quinone oxidation reaction was followed spectrophotometrically by monitoring the absorption increase of the o-quinone characteristic band in methanol over time. Blank experiments indicated that the oxidation reaction of catechol to o-quinone was quite slow in the absence of in situ complex. Special measurements of various catalysts prepared by mixing the metal salt with ligands L1, L2 or L3 at the molar ration 1:1 in methanol are represented in Fig. 7 below. Catechol oxidation rates were calculated and collected in Table 5.
The experimental results (Fig. 7) indicate the gradual increase in the catalytic activity appropriate for each particular catalyst towards catechol oxidation. The different efficiency of catecholase activity from one complex to another is certainly related to several factors required for good catalytic activity. One of the important roles is that the chelate effect of the ligands, the structural and electronic properties of the ligand type should have the ability to improve both the redox properties of the catalyst and its structural organization in order to favor the interactions between the catechol and the catalytic site. The complexing power of these tridentate ligands was attributed to the coexistence of the triazolyl - pyridyl sp2 nitrogen groups and the sp3 hybridized amine N-donor sites and the N–C–N flexibility junction of ligands. This system of N-coordinating groups could allow them an effective coordination behavior towards transition metal sites. Other factors such as metal ion, counter ion, and solvent can be determinant for the definitive structure and properties of the complex.
It is important to note that, with certain complexes, a precipitate apparently formed due to o-quinone polymerization, which prevents the absorbance measurements and hinders the comparative study, especially with those expected showing better catalytic activities.
Investigation of catecholase activities has shown oxidation rate values (V) ranging from 0.04 to 2.44 𝜇mol.l−1.min−1. While, catalytic activity concentration (b) values varying from 0.29 to 18.69 𝜇mol.l−1.min−1. In general, we notice that the rates of the complexes with metal salts CuSO4, Cu(NO3)2, CuBr2 and CuCl2, are displayed at a lower reactivity and are located in the same gap, with a slight advantage observed for the catalysts of ligand L1. However, the high level of catalytic activity was displayed with the L3 catalyst; it seems that the pyridine substituent affects the geometry and reactivity of the complex by changing the electron density distribution. In fact, the presence of the mesomeric effect as electron-donating groups (2Br) of ligand L3 could stabilize the intermediate species, and subsequently promote the oxidation reaction of catechol, compared to that of the ligand complexes showing a mesomeric effect caused by one substituent group (1Br) of L1 and L2. Then, the complexes with CH3COO− anion have shown a high efficiency for catecholase activity for most ligands, a result, which is most probably related to the weaker bond of acetate with the metal center as leaving group, and consequently can provide the vacant coordination site for the substrate during the catalytic cycle.
On the other hand, copper metal complex has shown greater activity than the analogous of manganese and nickel metal complexes respectively. The best trend in the catecholase activities observed for the copper metal catalyst may be attributed to the fact that : (i) the best electrostatic attraction between the copper ions and the catechol moiety due to the strong Lewis acid properties of the metal ions [61]; (ii) and the acceptance of the electron by the metal ion, as a redox center, after formation of substrate-complex species.
Effect of metal salts concentration on the oxidation activity
In order to study the effect of metal salts concentration on the catalytic activity, the dependence of the reaction rate on the metal salts concentration was carried out under identical conditions and illustrated in Figure S3 (see supplementary material). The corresponding data are given in Table 6. The study was conducted using two equivalents of metal salt for one equivalent of ligand.
A different increase in reaction rate was observed in response to increasing the concentrations of metal salts in the solution. These differences are thought to result from variations in the concentration of the complexes in the solution, which probably increases the formation of substrate-complex species, and improves its reactivity efficiency.
The case of Mn(CH3COO−)2 complex with the ligand having a mesomeric effect, caused by substituent groups (2Br) gives the best oxidation rate of 1.61 𝜇mol l−1 min−1, which exceeds the rate of the complexes with a ligand containing substituent groups (Br and CH3) in L1 or (Br) in L2 respectively 1.46 and 1.35 𝜇mol l−1 min−1.
Effect of ligand L3 concentration on the oxidation activity
This study consists of determining the influence of the ligand concentration on the kinetics of the oxidation reaction. In this part, we take two equivalents of ligand for one equivalent of metal salt. The absorbance graphs as well as the kinetics results are displayed in Fig. 8.
The analysis of the outcomes shows no significant increase in the rate as a function of concentration of L3 ligand in the solution. This result suggests that, in this case, the nature of the complexes formed are similar to those obtained in the equimolar case, or probably that the nature of the complexes formed was changing (mono- di- and/or poly-nuclear complexes), but that they exhibited the same activities as those obtained in the equimolar case.
In general, comparing the activity of our complexes with those reported in previous similar studies shows that the best rate is smaller than that reported for analogous complexes with pyrazole and pyridine based ligands[59], and much lower than some others reported for complexes with tripodal pyrazolyl ring [37, 39, 40]. Important to note that pyridine and 1,2,4-triazole are more basic than pyrazole, therefore the combination of both triazolyl and pyridyl ring in one tridentate ligand expected that caused an increase in affinity of ligand towards the metal ion, compared to that ligands containing pyrazolyl ring. Rigid geometries offer fewer options for complex dynamics and binding events, which negatively affects the reactivity of the catalyst complexes during the catalytic cycle.