Complexes of Zinc(II) Chloride with N-Vinyl-, N-Allyl- and N-Propargylimidazoles: Structural, Theoretical and Biological Studies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I have carefully re-evaluated the manuscript entitled “Complexes of Zinc(II) Chloride with N-Vinyl-, N-Allyl- and N-Propargylimidazoles: Structural, Theoretical and Biological Studies” submitted to Pharmaceuticals. The study represents a substantial body of work combining synthetic coordination chemistry, crystallographic and spectroscopic characterization, density functional theory calculations, and a diverse set of biological assays. The manuscript is technically sound and methodologically comprehensive, and the authors have clearly invested significant effort in addressing multiple aspects of structure, stability, and biological response.
At the same time, several important issues, primarily related to interpretation, statistical rigor, and mechanistic depth,should be explicitly acknowledged. For clarity and completeness, I summarize all major and minor points below.

Com. 1.: The interpretation of antihypoxic activity remains insufficiently supported by statistical rigor. Reported effects are largely marginal or statistically unclear (values close to control such as 99±5%), while the claim of “limited anti-hypoxic activity” for ALL-2-Cl based on a single dose showing 132% lifespan extension requires stronger validation. The manuscript should clearly specify the number of biological replicates per dose group, whether multiple-comparison corrections were applied, and whether any power analysis justifies the chosen sample size (n=10).

Com. 2.: There is still a noticeable disconnect between computational results and experimental biological outcomes. Although DFT-derived thermochemical parameters and frontier orbital analyses are competently presented, the manuscript does not provide a sufficiently developed mechanistic explanation linking these descriptors to observed biological activity. For example, the noted correlation between chemical potential and antifungal activity lacks a plausible biochemical or molecular rationale.

Com. 3.: The observed anion-dependent differences in biological activity between zinc chloride and previously reported zinc acetate complexes represent a potentially important finding but remain underexplored. While the authors acknowledge the discrepancy, the discussion does not go beyond a descriptive level. Consideration of speciation under physiological conditions, potential differences in cellular uptake, or subcellular localization is lacking and should at least be discussed as a direction for future work.

Com. 4.: The interpretation of antitumor activity overstates the relevance of the data. The cytotoxicity results (Table 6) indicate limited selectivity toward cancer cell lines compared to non-tumor controls (Vero cells), with CC50 values suggesting a narrow therapeutic window. The manuscript should adopt a more cautious tone and avoid presenting these compounds as promising antitumor agents without clear selectivity.

Com. 5.: The discussion of QSAR predictions (PASS analysis) would benefit from a clearer acknowledgment of methodological limitations. In particular, the applicability of models trained predominantly on organic compounds to metal-based systems is uncertain, and the anion-independent modeling approach may overlook important coordination effects.

Com. 6.: Nomenclature throughout the manuscript is not fully consistent. Variations such as “N-allylimidazole” versus “N-allyl imidazole” should be standardized, and formatting of complex identifiers should be unified.

Com. 7.: The presentation of spectroscopic data in the Supplementary Information requires improvement. NMR data would be clearer if chemical shifts were tabulated systematically with explicit indication of solvents for each dataset, as some current tables lack this information.

Com. 8.: Crystallographic reporting would benefit from a brief clarification that ORTEP diagrams were generated with displacement ellipsoids at 50% probability and that hydrogen atoms are shown in the conventional manner. While standard, this should be explicitly stated for reproducibility.

Com. 9.: The choice of the MN15 functional for DFT calculations is not sufficiently justified. A short rationale explaining its suitability for Zn(II) coordination systems, in comparison with more commonly used functionals such as B3LYP or PBE0, would strengthen the methodological section.

Com. 10.: Biological methodology requires additional detail. In particular, for the wound-healing assay, the manuscript should clarify whether measurements were performed in a blinded manner, define the criteria for “complete epithelialization,” and indicate whether inter-observer variability was assessed.

Com. 11.: All references should be aligned with the journal’s formatting requirements.

Com. 12.: Figure quality could be improved, particularly for Figure 4 (MEP surfaces), where a consistent color scale across all complexes would facilitate direct comparison of electronic distributions.

Com. 13.: The manuscript would benefit from stronger and more explicit mechanistic hypotheses connecting electronic descriptors to specific biological interactions, even if presented as testable assumptions rather than definitive conclusions.

Com. 14.: A more quantitative structure–activity relationship analysis would enhance the scientific rigor. Even a preliminary correlation (e.g., regression or rank-based comparison) between computed parameters and biological endpoints would strengthen the discussion.

Com. 15.: Pharmacokinetic considerations are not addressed. Given the observed anion-dependent differences, the manuscript should include at least a brief discussion of how chloride versus acetate coordination might influence stability in biological media, transport, or cellular uptake.

Com. 16.: Statistical reporting across all biological assays should be more transparent and standardized, including clear definitions of n (biological vs. technical replicates), explicit reporting of statistical tests, and, where possible, exact p-values instead of symbolic indicators.

Com. 17.: The conclusions should be framed more conservatively, emphasizing that the reported complexes represent preliminary candidates or starting points for further optimization rather than fully validated therapeutic agents.

Com. 18.: The organization of the Supplementary Information could be improved by including a summary table that consolidates key experimental and computational parameters (yields, stability constants, orbital energies, IC50/CC50 values) to facilitate comparison among complexes.

Com. 19.: The Introduction would benefit significantly from a more comprehensive integration of recent literature in the field of Zn(II) coordination compounds with imidazole-based ligands. In its current form, the background relies predominantly on older references and does not fully reflect the rapid development of this research area. Very recent studies have demonstrated the continued relevance of Zn(II)–imidazole systems in combined structural, computational, and biological investigations, including works published in Journal of Molecular Structure (2024, DOI: 10.1016/j.molstruc.2024.141120), Polyhedron (2025, DOI: 10.1016/j.poly.2025.117813), and RSC Advances (DOI: 10.1039/D5RA03626G). These contributions highlight current trends such as the integration of DFT analysis with biological evaluation, the role of ligand substitution in modulating coordination geometry and activity, and the importance of structure–property relationships in Zn(II) complexes. For instance, recent studies show that Zn(II) complexes with imidazole or benzimidazole ligands are routinely investigated using combined spectroscopic, crystallographic, and theoretical approaches, often revealing subtle correlations between coordination environment and biological behavior . Incorporating and critically discussing these up-to-date references would substantially strengthen the scientific context of the manuscript, better justify the choice of ligand systems, and clearly position the present work within the current state-of-the-art.

In summary, the manuscript presents a valuable and methodologically solid contribution, but several aspects, particularly related to interpretation and data presentation require careful attention. Addressing these points would significantly strengthen the scientific impact and clarity of the work.

Based on the overall quality of the study and the potential relevance to the field, I consider that the manuscript can be suitable for publication after appropriate revision.

Author Response

Com. 1.: The interpretation of antihypoxic activity remains insufficiently supported by statistical rigor. Reported effects are largely marginal or statistically unclear (values close to control such as 99±5%), while the claim of “limited anti-hypoxic activity” for ALL-2-Cl based on a single dose showing 132% lifespan extension requires stronger validation. The manuscript should clearly specify the number of biological replicates per dose group, whether multiple-comparison corrections were applied, and whether any power analysis justifies the chosen sample size (n=10).

Response. The absence of an appreciable antihypoxic effect for most of the compounds was stated in the manuscript. The only exception where the effect was clear is ALL-2-Cl (complex 2), showing 132% lifespan extension. The effect exceeds standard deviation about 6 times, thus the large Cohen effect size allows to use even 3 replicates for the confirmation of the effect for the compound 2. Each dose was studied in 10 biological replicates. The information about replicates was in footnotes under the table 5. We added this information to the main text for clarity. Multiple comparisons were used with the Bonferroni correction. The information about power analysis and multiple-comparison corrections has been added to the section 4.6.5. Statistical data processing.

Com. 2.: There is still a noticeable disconnect between computational results and experimental biological outcomes. Although DFT-derived thermochemical parameters and frontier orbital analyses are competently presented, the manuscript does not provide a sufficiently developed mechanistic explanation linking these descriptors to observed biological activity. For example, the noted correlation between chemical potential and antifungal activity lacks a plausible biochemical or molecular rationale.

Response. The discussion of correlation between computational and experimental biological results has been added to the paper.

Com. 3.: The observed anion-dependent differences in biological activity between zinc chloride and previously reported zinc acetate complexes represent a potentially important finding but remain underexplored. While the authors acknowledge the discrepancy, the discussion does not go beyond a descriptive level. Consideration of speciation under physiological conditions, potential differences in cellular uptake, or subcellular localization is lacking and should at least be discussed as a direction for future work.

Response. The discussion of anion-dependent differences in biological activity has been added to the paper.

 

Com. 4.: The interpretation of antitumor activity overstates the relevance of the data. The cytotoxicity results (Table 6) indicate limited selectivity toward cancer cell lines compared to non-tumor controls (Vero cells), with CC50 values suggesting a narrow therapeutic window. The manuscript should adopt a more cautious tone and avoid presenting these compounds as promising antitumor agents without clear selectivity.

Response. The cytotoxicity study could not establish CC50 values for the complexes for the Vero cells, as none of the studied compounds exhibited a toxic effect on the Vero cell line in the used concentration range, it was indicated as  > 200 M, and the selectivity might be very high. Regarding the overstatement of anti-tumor activity, we did not emphasize anti-tumor properties, but rather discussed them in a gentle and suggestive manner. We have also changed the description of anti-tumor properties in conclusions to a more conservative style.

Com. 5.: The discussion of QSAR predictions (PASS analysis) would benefit from a clearer acknowledgment of methodological limitations. In particular, the applicability of models trained predominantly on organic compounds to metal-based systems is uncertain, and the anion-independent modeling approach may overlook important coordination effects.

Response. The acknowledgment of the methodological limitations is included in the text. Although anion-independent modeling approaches may overlook important coordination effects, the lability of anions in zinc complexes can lead to their dissociation and fast exchange.

Com. 6.: Nomenclature throughout the manuscript is not fully consistent. Variations such as “N-allylimidazole” versus “N-allyl imidazole” should be standardized, and formatting of complex identifiers should be unified.

Response. Nomenclature and complex identifiers have been standardized.

Com. 7.: The presentation of spectroscopic data in the Supplementary Information requires improvement. NMR data would be clearer if chemical shifts were tabulated systematically with explicit indication of solvents for each dataset, as some current tables lack this information.

Response. NMR data in different solvents have been tabulated in the Supplementary Information.

Com. 8.: Crystallographic reporting would benefit from a brief clarification that ORTEP diagrams were generated with displacement ellipsoids at 50% probability and that hydrogen atoms are shown in the conventional manner. While standard, this should be explicitly stated for reproducibility.

Response. Corrected.

Com. 9.: The choice of the MN15 functional for DFT calculations is not sufficiently justified. A short rationale explaining its suitability for Zn(II) coordination systems, in comparison with more commonly used functionals such as B3LYP or PBE0, would strengthen the methodological section.

Response. The justification for using the MN15 functional has been included in the revised paper. According to benchmarking reports, commonly used B3LYP often performs worse than Minnesota functionals. We have also performed some benchmarking of methods, including B3LYP and other functionals, and various Minnesota functionals. Based on the comparison of calculated parameters with X-ray geometry, we found the MN15 to be the most accurate.

Com. 10.: Biological methodology requires additional detail. In particular, for the wound-healing assay, the manuscript should clarify whether measurements were performed in a blinded manner, define the criteria for “complete epithelialization,” and indicate whether inter-observer variability was assessed.

Response. Additional details of biological methodology have been added to the corresponding experimental section.

Com. 11.: All references should be aligned with the journal’s formatting requirements.

Response. Referencing has been done using the EndNote software with MDPI formatting. Some errors have been corrected.

Com. 12.: Figure quality could be improved, particularly for Figure 4 (MEP surfaces), where a consistent color scale across all complexes would facilitate direct comparison of electronic distributions.

Response. The Figure quality has been improved.

Com. 13.: The manuscript would benefit from stronger and more explicit mechanistic hypotheses connecting electronic descriptors to specific biological interactions, even if presented as testable assumptions rather than definitive conclusions.

Response. Although there is not enough data to suggest specific mechanisms, some discussion of mechanistic considerations has been included in the paper.

Com. 14.: A more quantitative structure–activity relationship analysis would enhance the scientific rigor. Even a preliminary correlation (e.g., regression or rank-based comparison) between computed parameters and biological endpoints would strengthen the discussion.

Response. The qualitative discussion of the correlation has been expanded in the paper. However, the quantitative correlation parameters are uncertain and unreliable due to the large dispersion and the small number of variables involved. Nevertheless, calculated coefficients between the toxicity of the complexes and their electronic parameters have been included in the Supplementary Information (Table S20).

Com. 15.: Pharmacokinetic considerations are not addressed. Given the observed anion-dependent differences, the manuscript should include at least a brief discussion of how chloride versus acetate coordination might influence stability in biological media, transport, or cellular uptake.

Response. The discussion about the difference between chloride and acetate complexes has been expanded.

Com. 16.: Statistical reporting across all biological assays should be more transparent and standardized, including clear definitions of n (biological vs. technical replicates), explicit reporting of statistical tests, and, where possible, exact p-values instead of symbolic indicators.

Response. The statistical reporting has been improved and provided with additional details.

Com. 17.: The conclusions should be framed more conservatively, emphasizing that the reported complexes represent preliminary candidates or starting points for further optimization rather than fully validated therapeutic agents.

Response. The conclusions have been revised to a more conservative style.

Com. 18.: The organization of the Supplementary Information could be improved by including a summary table that consolidates key experimental and computational parameters (yields, stability constants, orbital energies, IC50/CC50 values) to facilitate comparison among complexes.

Response. The tables, which consolidate key experimental and computational parameters, have been added to the Supplementary Information.

Com. 19.: The Introduction would benefit significantly from a more comprehensive integration of recent literature in the field of Zn(II) coordination compounds with imidazole-based ligands. In its current form, the background relies predominantly on older references and does not fully reflect the rapid development of this research area. Very recent studies have demonstrated the continued relevance of Zn(II)–imidazole systems in combined structural, computational, and biological investigations, including works published in Journal of Molecular Structure (2024, DOI: 10.1016/j.molstruc.2024.141120), Polyhedron (2025, DOI: 10.1016/j.poly.2025.117813), and RSC Advances (DOI: 10.1039/D5RA03626G). These contributions highlight current trends such as the integration of DFT analysis with biological evaluation, the role of ligand substitution in modulating coordination geometry and activity, and the importance of structure–property relationships in Zn(II) complexes. For instance, recent studies show that Zn(II) complexes with imidazole or benzimidazole ligands are routinely investigated using combined spectroscopic, crystallographic, and theoretical approaches, often revealing subtle correlations between coordination environment and biological behavior.  Incorporating and critically discussing these up-to-date references would substantially strengthen the scientific context of the manuscript, better justify the choice of ligand systems, and clearly position the present work within the current state-of-the-art.

Response. We have added a discussion of recent papers in the Introduction to place the present work in the context of the current state of the art.

Reviewer 2 Report

Comments and Suggestions for Authors

Please see the attachment.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English can be improved.

Author Response

Major aspects that must be addressed:

1. The manuscript does not provide any experimental data on the stability of the complexes under physiologically relevant conditions. It is unclear from the DFT data whether the complexes retain their structural integrity in solution or undergo ligand exchange prior to exhibiting biological activity. Consequently, 1H NMR studies over time in D2O are necessary. The spectra registered in different deuterated solvents do not provide stability data as the authors

Response. The NMR studies of the solutions of the compounds in D₂O showed that the spectra remain the same over time and the complexes retain their integrity. The corresponding text has been added to the paper.

2. Biological studies must be conducted in comparison with those of the free ligands and ZnCl₂ respective  These  must  also  be  compared  with  both  antimicrobial  and antitumour reference drugs. Without this information, the clinical relevance of the reported MIC or CC50 values cannot be assessed.

Response. We have presented the first results of our evaluation of the antimicrobial and antitumor activities of the studied complexes. In the future, we plan to conduct a clinical evaluation of these results and compare the minimum inhibitory concentration (MIC) and cytotoxic concentration (CC50) values of the complexes to those of free ligands, zinc chloride, and other known reference drugs.

3. The cell death mechanism for the most active compounds must be assessed using either LDH release or flow cytometry (Annexin V/PI).

Response. Thank you for the suggestion regarding the method of studying the cell death mechanism. We will utilize this approach in our future work to further explore the most active compounds.

Response. We would like to thank the reviewer for their constructive suggestion. We will address the detailed characterization of the cell biological effects and cellular toxicity of the compounds in our future study. For the present study, we are primarily focused on the structural characterization of these molecules and assessing their biological activity in order to test them as potential lead structures for further optimization and in-depth investigation.

4. Powder X-ray data must be provided and compared with data simulated from single crystal X-rays in order to prove that the bulk species has the same structure as the single crystal, and that the two compounds have the same structure where single crystals were

Response. The experimental and simulated powder X-ray diagrams have been added to the Supplementary Information.

5. The purpose of this study is not clearly explained in the

Response. The text explaining the purpose of the study has been added to the introduction.

6. The thermodynamic parameters were calculated considering both the dissociation reactions:

ZnCl2L2 + 7H2O → [ZnClL2×H2O]^- + Cl^-×6H2O (2) ZnCl2L2 + H2O → ZnCl2L×H2O + L (3)

however, there is no experimental data in the paper accounting for one of these processes. Moreover, based on the NMR data, the authors conclude that all species are stable in solution.

Response. The calculated dissociation constants are sufficiently low and the corresponding concentrations of the dissociated species are too small to be detected in the NMR spectra. Thus, the complexes are thermodynamically stable in solution at the concentration used in NMR (10 g/L). However, according to the equilibrium equation, upon a dilution the degree of dissociation increases. This factor needs to be taken into account in pharmacokinetics.

7. Why were 'anion-independent structures' used in the QSAR study analysis? What are these structures?

Response.  Zinc complexes are easily subjected to the anion exchange during transport in the body, so the key components of these complexes are the ligand and zinc. Therefore, we used more general, anion-independent structures in the analysis. These include imidazole complexes with zinc, without any anions.

Minor aspects that must be addressed:

• The complexes are not of zinc chloride, but of Zn(II). This must therefore be changed throughout the paper. The title must also be changed to mention zinc(II) complexes and imidazole derivatives, since 'N-propargylimidazoles' is not correct as it is a single

Response. The notation has been revised according to the nomenclature. Concerning the title, multiple 'imidazoles' correspond not only to ‘N-propargyl”, but also to ‘N-Vinyl-‘ and ‘N-Allyl-‘. This nomenclature practice is standard in chemistry, and this kind of pluralization occurs in published titles.

• The formula of the complexes with the corresponding notations and ligands enumeration must be provided in the abstract. The complexes must be presented as [ZnCl2L2] instead of ZnCl2L2.

Response. The notation has been revised according to the nomenclature.

• The Expression „Staphylococcus Aureus, Escherichia Coli, and the yeast-like fungus Candida Albicans” must be corrected as „Staphylococcus aureus, Escherichia coli, and the yeast-like fungus Candida albicans”.

Response. The text has been corrected accordingly.

• The oxidation state of zinc must be specified throughout the paper and it can be written as Zn(II).

Response. The text has been corrected accordingly.

• The expression 'vital metals' must be changed to 'essential metals'.

Response. The text has been corrected accordingly.

• I disagree that zinc should be prioritised for drug development; in my opinion, copper should be prioritised due to its redox properties.

Response. The text in the introduction has been corrected, and both copper and zinc have been noted as priority metals for drug development.

• The sentence “Zinc redox inertness and lack of ligand field stabilisation energy provide important advantages for coordination, allowing it to maintain diverse coordination geometries and complex stability” is incorrect, as Zn(II) complexes are highly reactive precisely as a result of its zero-field stabilisation energy.

Response. The corresponding text in the introduction has been corrected.

• The notation used for the complexes in this work can sometimes be confusing for the For example, the first compound is presented as (1), 1, (ALL-Cl) (1) and (ALL-Cl). Please use the same notation throughout the paper.

Response. The notation has been changed to numerical only.

• The arrows in Scheme 1 should be replaced with lines, as in modern coordination chemistry bonds are not considered localised.

Response. Corrected.

• The expression 'Chemical shifts of the protons of imidazole cycle were significantly low-field shifted' must be

Response. The expression has been modified.

• The expression 'short-wavelength region' must be replaced by 'higher wavelength' and angstroms by Å.

Response. Corrected.

• In an enumeration, the unit of measurement must only be presented once at the end (i.e. 80° and 133° instead of 80° and 133°).

Response. Corrected.

• The terms 'antiproliferative' and 'anti-tumour' mean the same thing, so only 'anti- tumour' activities must be

Response. Corrected.

• In vitro, the name of the microorganisms and N- must be in italics throughout the

Response. Corrected.

• The number of repeated biological experiments must be presented in the experimental section, since the standard deviation is presented in Tables 5 and

Response. The number of repeated biological experiments has been provided in the experimental section.

• The ml must be replaced by mL throughout the

Response. Corrected.

• The '50% toxic concentration' must be replaced by 'medium toxic concentration'.

Response. Corrected.

• The sentence at lines 392–394 must be modified, as 'metal complexes' appears

Response. Corrected.

• The sentence 'As shown in Table 7, A had the highest predicted probabilities of being anti-atherosclerotic (Pa = 426, 88) and anti-eczematic (Pa = 0.84)' is unintelligible. What compound is A?

Response. Corrected.

• The m.p. must be eliminated from the experimental part, since the complexes decompose at that temperature and do not melt.

Response. The complexes melt without decomposing.

• The medium used for compound solubilisation in anti-hypoxic, wound-healing and antitumour experiments must be

Response. The medium has been specified in the experimental part.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have now carefully examined the revised version of the manuscript entitled ''Complexes of Zinc(II) Chloride with N‑Vinyl‑, N‑Allyl‑ and N‑2‑Propargylimidazoles: Structural, Theoretical and Biological Studies'', and the authors’ point‑by‑point response to my earlier comments. I am pleased to report that I am substantially satisfied with the revision.
The authors have addressed all of my previous comments with commendable thoroughness, scientific care, and professionalism. The statistical methodology has been rendered fully transparent. In short, the revision reflects a high scholarly standard and genuine respect for the peer‑review process.
I recommend that the manuscript be accepted for publication subject to two minor technical corrections that require no additional experimental or computational work:
Exact p‑values in biological assays. Although the statistical methods are now fully described, Table 5 and related biological results still rely on symbolic significance markers (e.g., asterisks for p < 0.05). To achieve complete statistical transparency, the authors should replace these symbols with the exact p‑values for each comparison, either within the tables themselves or in an accompanying supplementary table.
Explicit definition of “complete epithelialization.” In 4.6.2 the authors state that measurements were performed in a blinded manner and that inter‑observer variability was assessed, which is welcome. However, the histological and morphological criteria used to judge complete epithelialization (for example, continuity of the epidermal layer, maturity of granulation tissue, regeneration of skin appendages) remain implicit. I ask that the authors insert a precise, pre‑defined description of this endpoint into the Methods section.
Once these two minor points are addressed, the manuscript will, in my view, be fully ready for publication.

Author Response

1. Exact p‑values in biological assays. Although the statistical methods are now fully described, Table 5 and related biological results still rely on symbolic significance markers (e.g., asterisks for p < 0.05). To achieve complete statistical transparency, the authors should replace these symbols with the exact p‑values for each comparison, either within the tables themselves or in an accompanying supplementary table.

 Response. The exact p‑values have been provided in the footnotes to Table 5.

2. Explicit definition of “complete epithelialization.” In 4.6.2 the authors state that measurements were performed in a blinded manner and that inter‑observer variability was assessed, which is welcome. However, the histological and morphological criteria used to judge complete epithelialization (for example, continuity of the epidermal layer, maturity of granulation tissue, regeneration of skin appendages) remain implicit. I ask that the authors insert a precise, pre‑defined description of this endpoint into the Methods section.

Response. In section 4.6.2, we have added a more detailed definition of complete epithelialization.

Reviewer 2 Report

Comments and Suggestions for Authors

The raised questions have been addressed in the revised manuscript. I recommend acceptance for publication in its current form.

Author Response

Thank you for your valuable feedback.