Synthesis, Characterization and Anticancer Activities of Zn²⁺, Ni²⁺, Co²⁺, and Cu²⁺ Complexes of 4-Benzopyranone-2-carboxylic Acid

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "Synthesis, Characterization and Anticancer Activities of Zn²⁺, Ni²⁺, Co²⁺, and Cu²⁺ Complexes of 4-Benzopyranone-2-Carboxylic Acid" by authors Qian Qian Kang, Qasim Umar, Zhang Wei Jie, Meng Xiang Gao, Hao Yin, Mei Luo, and Yanmin Zhang is well written, compounds are fully characterized using adequate methods, results are correct and well presented. Only few things should be corrected:

1. If it is possible, authors should add or comment cytotoxicity results comparing to some referent compound (cisplatin, carboplatin...). In my opinion, IC50 values are relative and not such informative if they are not compared to some well-known drug as reference.
2. Some typo mistakes should be corrected: in elemental analysis data values for H should be presented with two decimal places as it was for C and N; line 338 C=O, label for O is now 0, change it to O; line 340 Fig. / number of Fig is missing.

 

Author Response

Thank you very much for your valuable time reviewing the manuscript. We appreciate your clear and detailed feedback and hope that the explanation has fully addressed all of your concerns. In this letter, we discuss each of your comments individually. In addition, in accordance with the format adjustment requirements of the journal editorial office, the positions of the “Results and Discussion” section and the “Experimental Section” in this paper have been swapped. After the adjustment, the core content of the original “Results and Discussion” section is located on pages 2–13, and the experimental details of the original “Experimental Section” are located on pages 13–16. To fully align with the journal’s guidelines, we have also made corresponding adjustments to the subsequent content of the manuscript, all of which have been highlighted in yellow for your convenience. All figures, tables, and data citations in the paper have been checked and verified to be accurate and consistent. This format adjustment has not altered the core conclusions of the study.

 

Comment 1:

If it is possible, authors should add or comment cytotoxicity results comparing to some referent compound (cisplatin, carboplatin...). In my opinion, IC50 values are relative and not such informative if they are not compared to some well-known drug as reference.

Response 1:

We sincerely appreciate the valuable comment to compare the cytotoxicity of our complexes with cisplatin/carboplatin. We fully agree that reference compounds are essential for evaluating the informativeness of cytotoxicity data. Unfortunately, objective constraints precluded a direct numerical comparison of inhibition rates or IC₅₀ values between our complexes and these reference drugs.

To align with your suggestion and improve data transparency, we have supplemented the original cytotoxicity testing data in Table 1 of the revised manuscript (page 12,lines 355–356), which we hope will facilitate a more comprehensive assessment of our complexes’ biological activity. All modified content has been highlighted in yellow in the revised manuscript to facilitate your review.

 

Comment 2:

Some typo mistakes should be corrected: in elemental analysis data values for H should be presented with two decimal places as it was for C and N; line 338 C=O, label for O is now 0, change it to O; line 340 Fig. / number of Fig is missing.

Response 2:

We were really sorry for our careless mistakes. Thank you for your reminder. And the detailed revisions are listed as follows, with the modified parts highlighted in yellow:

1) For the elemental analysis data, the values of H have been adjusted to retain two decimal places, consistent with the formatting of C and N data (page 14, line 434; page 15, lines 447–448; line 460; line 471).

2) The "0" for O in C=O was corrected to "O" (page 8, line 254).

3) The missing figure number has been supplemented (page 8, line 256).

The modified content has been highlighted in yellow in the revised manuscript for your easy reference.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Review report_inorganics-4071475

This manuscript (Manuscript ID: inorganics-4071475) entitled "Synthesis, Characterization and Anticancer Activities of Zn2+, Ni2+, Co2+, and Cu2+ Complexes of 4-Benzopyranone-2-Carboxylic Acid" reports the synthesis of four metal complexes following a direct synthesis method and characterizes them using infrared spectroscopy, UV–vis spectroscopy, electrospray ionization mass spectrometry (ESI–MS), and elemental analysis. Notably, authors have confirmed the structure of the synthesized complexes using single-crystal X-ray diffraction studies. Furthermore, the author conducted molecular docking studies and evaluated the cytotoxic activities of the complexes against human tumor cell lines. Overall, the work reported in this manuscript is of good quality and meets the standards of the Journal; however, a major revision is necessary to further enhance the overall quality of the manuscript (see critiques below). Thus, in my opinion, this paper can be accepted for publication in the Inorganics Journal after addressing the following concerns and comments.

1. The introduction is informative and well-structured, but requires little more depth to succinctly convey the research focus and innovative aspects of this study rather than just stating the reported literature, necessitating meticulous revision by the author.
2. Authors should provide the NMR spectrum of the Zn-complex (i.e., complex III) and the EPR spectrum of the Cu-complex (e.g., complex IV).
3. Authors are encouraged to calculate the magnetic moment (µ_eff) of these complexes using the Evans method if possible.
4. I strongly encourage authors to provide a better-resolution picture for the crystal structure of complexes (I)-(IV) in Figure 1. Please also add an ORTEP-style plot in Figure 1. Also, change the figure 1 caption to ‘ORTEP diagram of presentation and atom numbering scheme of complexes (I)-(IV)’. Also, please mention the probability level at which the ORTEP picture in Fig. 3 was rendered
5. Authors are suggested to submit the checkcif file of the crystals reported here during revision of this manuscript, or else provide the CCDC number according to the journal guidelines.
6. X-ray Crystallography: In general, the X-ray work seems to be OK. However, this reviewer believes that some of the tables (for example, Tables 2 and 3) could be moved to the Supporting Information file.
7. On page 8, lines 219-220: Authors are suggested to compare the Ni-N(py) or, M-N/M-O bond lengths with the relevant literature and cite them wherever appropriate (Dalton Trans., 2020, 49, 6816–6831; J. Am. Chem. Soc., 2009, 131, 15582–15583; Inorg. Chem. 2019, 58, 5433–5445 etc.).
8. On page 8, lines 232: The Authors mentioned Fig. S1. However, no supporting information file was submitted with this submission.
9. I suggest that authors show the full range of mass spectra (also assign the main peak in Figure 2) and FT-IR spectra in the Supporting Information file to provide a better understanding for readers.
10. The author should explicitly mention the concentration of the complexes used for the optical absorption spectra of solutions, which is crucial for understanding the results.
11. Finally, I suggest that authors perform a thorough proofreading of the manuscript, as there are a lot of typographical errors throughout the manuscript.

Author Response

We are grateful for the time you dedicated to reviewing our manuscript. Your clear and detailed feedback has been immensely helpful in improving our work. In this letter, we discuss each of your comments individually. In addition, in accordance with the format adjustment requirements of the journal editorial office, the positions of the “Results and Discussion” section and the “Experimental Section” in this paper have been swapped. After the adjustment, the core content of the original “Results and Discussion” section is located on pages 2–13, and the experimental details of the original “Experimental Section” are located on pages 13–16. To fully align with the journal’s guidelines, we have also made corresponding adjustments to the subsequent content of the manuscript, all of which have been highlighted in yellow for your convenience. All figures, tables and data citations in the paper have been checked and verified to be accurate and consistent. This format adjustment has not altered the core conclusions of the study. Additionally, we have made corresponding adjustments to the subsequent content of the manuscript to align with the journal’s formatting guidelines.

 

Comment 1:

The introduction is informative and well-structured, but requires little more depth to succinctly convey the research focus and innovative aspects of this study rather than just stating the reported literature, necessitating meticulous revision by the author.

Response 1:

We sincerely appreciate your insightful comment on the introduction section. Following your suggestion, we have meticulously revised the introduction to enhance its depth and focus. The modified content has been highlighted in yellow in the revised manuscript for your easy reference (pages 1–2, lines 39–64).

 

Comment 2:

Authors should provide the NMR spectrum of the Zn-complex (i.e., complex III) and the EPR spectrum of the Cu-complex (e.g., complex IV).

Response 2:

We sincerely appreciate your constructive suggestion, which has significantly improved the completeness of our manuscript. As requested, we have supplemented the required spectroscopic data in the main text of the revised version, with specific arrangements as follows:

1) The NMR spectrum of Zn-complex (Complex III) and its peak analysis are added to Section 2.6 (page 10, lines314–327).

2) The EPR spectrum of Cu-complex (Complex IV) and key parameter analysis are added to Section 3.2 (pages 10–11, lines 328–338).

To ensure logical consistency and interpretability of results, corresponding introductions and discussions of these spectra have also been added to the relevant sections of the paper (page 1, lines 28–29; page 2, line 69–70; page 14, lines 395–399; page 14, line 421; page 16, line 502).

All modified content has been highlighted in yellow in the revised manuscript to facilitate your review.

 

Comment 3:

Authors are encouraged to calculate the magnetic moment (µeff) of these complexes using the Evans method if possible.

Response 3:

We sincerely appreciate your insightful suggestion on calculating the magnetic moment (µeff) of the complexes via the Evans method. Unfortunately, due to current experimental constraints, we are not able to carry out this calculation within the scope of the present work. We fully recognize that magnetic moment data would greatly enhance the comprehensiveness of the complexes’ characterization, and we will pay more attention to it in our future research. Thank you again for your constructive input.

 

Comment 4:

I strongly encourage authors to provide a better-resolution picture for the crystal structure of complexes (I)-(IV) in Figure 1. Please also add an ORTEP-style plot in Figure 1. Also, change the figure 1 caption to ‘ORTEP diagram of presentation and atom numbering scheme of complexes (I)-(IV)’. Also, please mention the probability level at which the ORTEP picture in Fig. 3 was rendered.

Response 4:

We sincerely appreciate your constructive suggestion to improve the presentation of crystal structure data. As recommended, high-resolution ORTEP-style plots with complete atom numbering schemes have been incorporated into Figure 1 (page 4), and the figure caption has been revised exactly as suggested: “ORTEP diagram of presentation and atom numbering scheme of complexes (I)-(IV)” (page 4, line 121). We presume that the reference to “Figure 3” in the comment may be a minor oversight, and the probability level (30%) used for rendering the ORTEP plot in Figure 1 has been explicitly stated in the revised manuscript (page 4, line 122).

The modified content has been highlighted in yellow in the revised manuscript for your easy reference.

 

Comment 5:

Authors are suggested to submit the checkcif file of the crystals reported here during revision of this manuscript, or else provide the CCDC number according to the journal guidelines.

Response 5:

We sincerely appreciate your valuable suggestion. The CCDC deposition numbers for the crystal structures of complexes (I)–(IV) have been added in the revised manuscript (page 16, lines 532–533) and are highlighted in yellow.

 

Comment 6:

This reviewer believes that some of the tables (for example, Tables 2 and 3) could be moved to the Supporting Information file.

Response 6:

Thanks for your suggestion. As recommended, Tables 2 and 3 have been moved to the Supporting Information file (labeled as Tables S2 and S3).

 

 

Comment 7:

On page 8, lines 219-220: Authors are suggested to compare the Ni-N(py) or, M-N/M-O bond lengths with the relevant literature and cite them wherever appropriate (Dalton Trans., 2020, 49, 6816–6831; J. Am. Chem. Soc., 2009, 131, 15582–15583; Inorg. Chem. 2019, 58, 5433–5445 etc.).

Response 7:

We sincerely appreciate your valuable suggestions. In response to your comments, we have supplemented a comparison of the target bond lengths with the values reported in the recommended references and other related studies on transition metal complexes (page 4, lines 132–136). The full bibliographic information of these references has been properly cited at the corresponding positions.

The revised content has been highlighted for your convenience. We would like to express our sincere gratitude again for your rigorous and insightful guidance.

 

Comment 8:

On page 8, lines 232: The Authors mentioned Fig. S1. However, no supporting information file was submitted with this submission.

Response 8:

Thanks for your careful checks. We are sorry for our carelessness. Figure S1 shows the formation of the three-dimensional network by hydrogen bonding interactions. As requested, we have compiled and incorporated it into the supporting information file, with the relevant content highlighted in yellow.

 

Comment 9:

I suggest that authors show the full range of mass spectra (also assign the main peak in Figure 2) and FT-IR spectra in the Supporting Information file to provide a better understanding for readers.

Response 9:

We sincerely appreciate your insightful suggestion, which is crucial for improving the completeness and readability of our manuscript.​

As you recommended, we have shown the full range of mass spectra and complete FT-IR spectra in the revised Supporting Information (labeled as Figures S2 and S3), and we have also labeled the main characteristic peaks in Figure 2 of the manuscript. (pages 6–7, lines 234–239).

We have highlighted the updated parts of the Supporting Information and manuscript in yellow for your easy checking.

 

Comment 10:

The author should explicitly mention the concentration of the complexes used for the optical absorption spectra of solutions, which is crucial for understanding the results.

Response 10:

We sincerely thank you for pointing out this critical oversight in our manuscript and apologize for any confusion this may have caused. The optical absorption spectra reported herein were actually measured for solid-state samples instead of solutions, and the relevant term has been corrected to “UV-vis diffuse reflectance spectra (UV-vis DRS)” throughout the revised manuscript (page 1, line 27; page 2, line 68; page 9, lines292–293; page 10, line 313; page 14, line 421; page 16, line 502). Additionally, we have supplemented detailed experimental parameters for the solid-state measurements in the Materials and Methods section (page 14, lines 388–390). We thank you again for helping us improve the accuracy of this manuscript.

 

Comment 11:

Finally, I suggest that authors perform a thorough proofreading of the manuscript, as there are a lot of typographical errors throughout the manuscript.

Response 11:

We sincerely appreciate your insightful suggestions on regarding the proofreading of our manuscript. the typographical errors, spelling mistakes, and formatting inconsistencies throughout the text have been corrected, seen in the attached file.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The article "Synthesis, Characterization and Anticancer Activities of Zn2+, 2 Ni2+, Co2+, and Cu2+ Complexes of 4-Benzopyranone-2-Carboxylic Acid" is a well-structured, methodically competent and relevant study in the field of bioorganic chemistry of metal complexes. The work meets the standards of scientific publication in a peer-reviewed journal. The study has scientific novelty and practical significance, contributing to the search for new inexpensive and selective anticancer agents based on essential metals. The authors conducted an exhaustive set of physico-chemical methods to confirm the structure and purity of the synthesized compounds: infrared spectroscopy, UV–24 Vis spectroscopy, electrospray ionization mass spectrometry (ESI–MS), elemental analysis-25 sis, and single-crystal X-ray diffraction. Cytotoxicity was tested on a panel of five cancer cell lines. A standard MTS test was used, the data is presented with an indication of the standard deviation.: Carrying out molecular docking with DNA in the form of a G-quadruplex adds depth to the work and suggests a possible mechanism of biological activity at the molecular level. Strengths of the article: Comments and disadvantages 1) It is not recommended to use gross formulas. It is better to use together on C₃₀H₂H₂N₂O₁₂ (I), C₃₀H₂₄CoN₂O₁₀ (II), 19 C₂₀H₂O₁₃Zn (III), and C₃₀H₂₂CuN₂O₉ formula [Ni(bzpyr)2(py)2(H2O)2] (I) [Co(bzpyr)2(py)2(H2O)2] (II), [Zn(bzpyr)2(H2O)3] (III), [Cu(bzpyr)2(py)2(H2O)] (IV). In addition, there are typos in formulas I and 3. 2) The synthesis scheme should be moved to the Results and Discussions section. And describe a little bit the chosen reaction scheme. 3) Tables 1-3 should be moved to Supplementary, and Table 4 should be moved to Section 3.5. Units of measurement should be added to Tables 4 and 5. 4) It is not written in which solvent and in what concentration the UV spectra were made. 5) When discussing X-ray, you need to add a comparison of the complexes with each other and explain what the differences are. Why is there no pyridine in compound 3? There is also an actual error that the complexes contain 2 benzopyranone-carboxylic acids, it is correct to write that the complexes contain benzopyranone-carboxylic acid anions. It is also necessary to add drawings reflecting the hydrogen bonds in the complexes. 6) When discussing cytotoxicity, it is advisable to compare it with toxicity to healthy cells, since the main indicator of anti-cancer compounds is the selectivity of action. 7) In addition, the authors believe that the activity of the 3-hour complex in comparison with the others is related to the role of the metal, but take into account that this complex differs from the others in the absence of pyridine. The article can be accepted after Major revision.

Author Response

Thank you very much for your valuable time involved in reviewing the manuscript. We appreciate your clear and detailed feedback and hope that the explanation has fully addressed all of your concerns. In this letter, we discuss each of your comments individually. In addition, in accordance with the format adjustment requirements of the journal editorial office, the positions of the “Results and Discussion” section and the “Experimental Section” section in this paper have been swapped. After the adjustment, the core content of the original “Results and Discussion” section is located on pages 2–13, and the experimental details of the original “Experimental Section” section are located on pages 13–16. To fully align with the journal’s guidelines, we have also made corresponding adjustments to the subsequent content of the manuscript, all of which have been highlighted in yellow for your convenience. All figures, tables and data citations in the paper have been checked and verified to be accurate and consistent. This format adjustment has not altered the core conclusions of the study.

Comment 1:

It is not recommended to use gross formulas. It is better to use together on C₃₀H₂H₂N₂O₁₂ (I), C₃₀H₂₄CoN₂O₁₀ (II), 19 C₂₀H₂O₁₃Zn (III), and C₃₀H₂₂CuN₂O₉ formula [Ni(bzpyr)₂(py)₂(H₂O)₂] (I) [Co(bzpyr)₂(py)₂(H₂O)₂] (II), [Zn(bzpyr)₂(H₂O)₃] (III), [Cu(bzpyr)₂(py)₂(H₂O)] (IV). In addition, there are typos in formulas I and 3.

Response 1:

Thank you very much for your valuable comments. We have carefully revised the manuscript according to your suggestions, and the detailed responses are as follows:

1) As recommended, the molecular formulas and corresponding structural formulas of the complexes are presented in pairs for their first introduction, as follows: C₃₀H₂₈N₂NiO₁₂ [Ni(bzpyr)₂(py)₂(H₂O)₂] (I), C₃₀H₂₄CoN₂O₁₀ [Co(bzpyr)₂(py)₂(H₂O)₂] (II), C₂₀H₂₀O₁₃Zn [Zn(bzpyr)₂(H₂O)₃] (III), and C₃₀H₂₂CuN₂O₉ [Cu(bzpyr)₂(py)₂(H₂O)] (IV) (page 1, lines 20-22).

2) We apologize for the typos in Formulas (I) and (III) in the original manuscript. These errors have been fully corrected based on the exact elemental analysis data of the synthesized complexes. The revised molecular formulas are as follows: C₃₀H₂₈N₂NiO₁₂ (I), C₂₀H₂₀O₁₃Zn (III) (page 1, lines 20-21).

All related revisions are highlighted in yellow in the revised manuscript for your convenience.

 

Comment 2:

The synthesis scheme should be moved to the Results and Discussions section. And describe a little bit the chosen reaction scheme.

Response 2:

We sincerely thank you for this constructive suggestion. As advised, we have moved the synthesis scheme to the Results and Discussion section (page 3, lines 91–92). Additionally, we have supplemented a concise description of the chosen reaction scheme in the sub-section [2.1. Synthetic Analysis of Complexes I–IV] (page 2, lines 78–90). The revised content has been highlighted in yellow in the manuscript for easy identification.

 

Comment 3:

Tables 1-3 should be moved to Supplementary, and Table 4 should be moved to Section 3.5. Units of measurement should be added to Tables 4 and 5.

Response 3:

We sincerely thank you for this meticulous and helpful suggestion. Following your instruction, we have moved Tables 1–3 to the Supporting Information, where they are relabeled as Tables S1, S2, and S3, respectively. We have also relocated the original Table 4 to Section 2.8 (formerly Section 3.5 in the initial submission) of the revised manuscript, and it has been renumbered as Table 1 (page 11, lines 354–355). Regarding the missing units of measurement, we have supplemented complete units for all data entries in the original Tables 4 and 5. Notably, these two tables have been renumbered as Table 1 and Table 2 in the revised manuscript, respectively. For the revised Table 1, we have clearly labeled the complexation concentration unit (100 μM) in the column header (page 11, line 354). For the revised Table 2, we have added the unit kcal/mol to all Docking Score entries (page 12, line 365). All the above revisions have been highlighted in yellow for your convenient check.

 

Comment 4:

It is not written in which solvent and in what concentration the UV spectra were made.

Response 4:

We sincerely thank you for pointing out this critical oversight in our manuscript and apologize for any confusion this may have caused. The optical absorption spectra reported herein were actually measured for solid-state samples instead of solutions, and the relevant term has been corrected to “UV-vis diffuse reflectance spectra (UV-vis DRS)” throughout the revised manuscript (page 1, line 27; page 2, line 68; page 9, lines292–293; page 10, line 313; page 14, line 421; page 16, line 502). Additionally, we have supplemented detailed experimental parameters for the solid-state measurements in the Materials and Methods section (page 14, lines 388–390). We thank you again for helping us improve the accuracy of this manuscript.

Comment 5:

When discussing X-ray, you need to add a comparison of the complexes with each other and explain what the differences are. Why is there no pyridine in compound 3? There is also an actual error that the complexes contain 2 benzopyranone-carboxylic acids, it is correct to write that the complexes contain benzopyranone-carboxylic acid anions. It is also necessary to add drawings reflecting the hydrogen bonds in the complexes.

Response 5:

We sincerely thank you for these insightful and constructive comments, which have significantly improved the quality of our manuscript. We have addressed all your points as follows:

1) As you suggested, we have supplemented a detailed comparison of the crystal structures of complexes 1–4 in the X-ray diffraction section (page 3, lines 96–97, lines 108–120);

2) We sincerely appreciate this insightful comment. A corresponding explanatory elaboration has been added for the absence of pyridine in complex III in the revised manuscript (Page 5, Lines 174–179).

3) We apologize for the inaccurate description of the ligand species. The relevant term has been corrected to " 4-benzopyranone-2-carboxylic acid anions " throughout the revised manuscript (page 3, lines100–103).

4) In accordance with your suggestion, we have added a figure depicting the intramolecular and intermolecular hydrogen bond interactions in the complexes to the Supporting Information (Figure S1).

All the above revisions have been highlighted in yellow for your convenient check.

 

Comment 6:

When discussing cytotoxicity, it is advisable to compare it with toxicity to healthy cells, since the main indicator of anticancer compounds is the selectivity of action.

Response 6:

We sincerely appreciate this valuable suggestion. Unfortunately, due to current experimental constraints, we are unable to supplement the comparison of cytotoxicity against healthy cells in this revision. We fully agree that selective action is a key indicator for anticancer compounds, and we will pay more attention to it in our future research. Thank you again for your constructive input.

 

Comment 7:

In addition, the authors believe that the activity of the 3-hour complex in comparison with the others is related to the role of the metal, but take into account that this complex differs from the others in the absence of pyridine.

Response 7:

We sincerely appreciate your insightful comment. We agree that the distinct activity of the 3-hour complex cannot be solely attributed to the metal center, as this complex also differs from others in the absence of the pyridine ligand. The revised content has been highlighted in yellow in the manuscript for easy identification (page 11, lines 348–351).

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Review report_inorganics-4071475-v2

In this revised version, the authors have made most of the recommended changes, resulting in substantially improved quality and readability of the manuscript. I do appreciate the changes made by the authors. But a few minor corrections, as outlined below, are required.

1. Page 4, Figure 1: It seems the previous version of Figure 1 was better than this revised Figure 1. Authors are encouraged to provide a better-resolution picture for the crystal structure of complexes (I)-(IV) in Figure 1. The selected H-atoms in the entire molecule may be omitted for a clearer representation, and this should be mentioned in the figure caption.
2. I thank the authors for providing the NMR spectrum of the Zn-complex (i.e., complex III). However, the resolution of the spectrum is very poor in Figure 5 (it is not visible even after zooming in). It is suggested to provide a clear zoomed-in spectrum in Figure 5; otherwise, consider moving Figure 5 to the supporting information file.
3. Page-11, lines 331-332: ‘………and supports a square-pyramidal coordination geometry for the Cu(II) center.’ A reference should be cited against this statement.
4. The concentration of the complexes used for the UV-vis diffuse reflectance spectra (UV-vis DRS) measurement is still missing. I suggest that the authors include this information in the relevant text or the Figure 4 caption.

Therefore, I would like to recommend its acceptance for publication in the Inorganics Journal after considering the above-mentioned corrections/modifications and without further revision from this reviewer.

Author Response

Thank you very much for your meticulous review and positive feedback on our revised manuscript. We greatly appreciate your recognition of the improvements we have made to the quality and readability of the paper, as this has been a great encouragement to us. Each of the minor corrections have been carefully addressed by your outlined, and the corresponding revisions are detailed point-by-point below. All changes have been marked in yellow in the revised manuscript for your convenience.

Once again, we would like to express our sincere gratitude for your time, effort, and constructive suggestions, which have helped us further polish this work.

 

Comment 1:

Page 4, Figure 1: It seems the previous version of Figure 1 was better than this revised Figure 1. Authors are encouraged to provide a better-resolution picture for the crystal structure of complexes (I)-(IV) in Figure 1. The selected H-atoms in the entire molecule may be omitted for a clearer representation, and this should be mentioned in the figure caption.

Response 1:

We sincerely appreciate your valuable suggestion on Figure 1. As recommended, much  higher-resolution images for the crystal structures of complexes (I)–(IV) on page 4 Meanwhile, The selected H-atoms in the molecular structures to enhance clarity, with a corresponding clarification have been added in the figure caption on page 4, lines 122–123. All of which have been marked in yellow in the revised manuscript for your convenience. We hope the updated Figure 1 meets your requirements.

 

Comment 2:

I thank the authors for providing the NMR spectrum of the Zn-complex (i.e., complex III). However, the resolution of the spectrum is very poor in Figure 5 (it is not visible even after zooming in). It is suggested to provide a clear zoomed-in spectrum in Figure 5; otherwise, consider moving Figure 5 to the supporting information file.

Response 2:

We sincerely thank you for taking the time to re-evaluate our manuscript and for your careful attention to the NMR spectrum of complex III.We fully recognize your concern regarding the resolution of the original NMR spectrum in Figure 5, and regret to note that the spectral quality cannot be further improved due to unavoidable experimental limitations encountered during the measurement process.​

To fully address your comment and further improve the quality of our manuscript, we have followed your kind suggestion and moved Figure 5 which from the NMR spectrum of complex III to the Supporting Information of the revised manuscript on page 12, labeled as Figure S5. Specifically, the original label of Figure 5 in the main text has been revised to Figure S5 (on page 10, line 320), and the corresponding descriptive entry for Figure S5 has been added in the Supplementary Materials section of the revised manuscript on page 16, line 524. All the revisions mentioned above have been highlighted in yellow in the revised manuscript.

We deeply apologize for any inconvenience that the less-than-ideal initial spectral quality may have caused during your review process. We greatly appreciate your valuable guidance, which has been instrumental in helping us optimize the data presentation of the manuscript.​

 

Comment 3:

Page-11, lines 331-332: ‘………and supports a square-pyramidal coordination geometry for the Cu(II) center.’ A reference should be cited against this statement.

Response 3:

We sincerely appreciate your insightful comment regarding the assignment. Following your suggestion, an authoritative reference [86] have been supplemented to substantiate this statement in the revised manuscript on page 11, line 334; page 21, lines 776–778). All modified content has been highlighted in yellow in the revised manuscript for your convenience. We apologize for the oversight in the initial submission and thank you again for your valuable guidance, which has significantly improved the scientific rigor of our manuscript.

 

Comment 4: 

The concentration of the complexes used for the UV-vis diffuse reflectance spectra (UV-vis DRS) measurement is still missing. I suggest that the authors include this information in the relevant text or the Figure 4 caption.

Response 4:

We would like to extend our sincere gratitude again for your meticulous attention to the experimental details of UV-vis DRS measurements, which is crucial to improving the rigor of our manuscript.

We note that this concern was initially addressed in our response to the first-round revision, and we sincerely apologize if our previous explanation failed to be sufficiently clear or comprehensive to you. For the characterization of our metal complexes, we opted for solid-state UV-vis DRS measurements using an integrating sphere accessory. We regret any confusion caused by the fact that, in contrast to liquid-phase UV-vis tests where concentration is a core parameter, this solid-based approach renders the reporting of sample concentration inapplicable.

To fully resolve this concern, the complete experimental specifications for UV-vis DRS measurements in the revised manuscript on page 13, lines 389–391 have been further refined and emphasized. Meanwhile, supplemented specific clarifying notes in the caption of Figure have been supplemented to specify the solid-state testing mode and the inapplicability of the concentration parameter on page 10, lines 314–317. And the specific clarifying have also been supplemented in the caption of Figure 4. 

We sincerely appreciate your patience and rigorous review, which have significantly enhanced the clarity and completeness of our manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

 The manuscript can be accepted 

Author Response

Thank you very much for your valuable time involved in reviewing the manuscript. We appreciate your clear and detailed feedback and hope that the explanation has fully addressed all of your concerns. In this letter, we discuss each of your comments individually. In addition, in accordance with the format adjustment requirements of the journal editorial office, the positions of the “Results and Discussion” section and the “Experimental Section” section in this paper have been swapped. After the adjustment, the core content of the original “Results and Discussion” section is located on pages 2–13, and the experimental details of the original “Experimental Section” section are located on pages 13–16. To fully align with the journal’s guidelines, we have also made corresponding adjustments to the subsequent content of the manuscript, all of which have been highlighted in yellow for your convenience. All figures, tables and data citations in the paper have been checked and verified to be accurate and consistent. This format adjustment has not altered the core conclusions of the study. 

Comment 1:

It is not recommended to use gross formulas. It is better to use together on C₃₀H₂H₂N₂O₁₂ (I), C₃₀H₂₄CoN₂O₁₀ (II), 19 C₂₀H₂O₁₃Zn (III), and C₃₀H₂₂CuN₂O₉ formula [Ni(bzpyr)₂(py)₂(H₂O)₂] (I) [Co(bzpyr)₂(py)₂(H₂O)₂] (II), [Zn(bzpyr)₂(H₂O)₃] (III), [Cu(bzpyr)₂(py)₂(H₂O)] (IV). In addition, there are typos in formulas I and 3.

Response 1:

Thank you very much for your valuable comments. We have carefully revised the manuscript according to your suggestions, and the detailed responses are as follows:

1) As recommended, the molecular formulas and corresponding structural formulas of the complexes are presented in pairs for their first introduction, as follows: C₃₀H₂₈N₂NiO₁₂ [Ni(bzpyr)₂(py)₂(H₂O)₂] (I), C₃₀H₂₄CoN₂O₁₀ [Co(bzpyr)₂(py)₂(H₂O)₂] (II), C₂₀H₂₀O₁₃Zn [Zn(bzpyr)₂(H₂O)₃] (III), and C₃₀H₂₂CuN₂O₉ [Cu(bzpyr)₂(py)₂(H₂O)] (IV) (page 1, lines 20-22).

2) We apologize for the typos in Formulas (I) and (III) in the original manuscript. These errors have been fully corrected based on the exact elemental analysis data of the synthesized complexes. The revised molecular formulas are as follows: C₃₀H₂₈N₂NiO₁₂ (I), C₂₀H₂₀O₁₃Zn (III) (page 1, lines 20-21).

All related revisions are highlighted in yellow in the revised manuscript for your convenience.

 

Comment 2:

The synthesis scheme should be moved to the Results and Discussions section. And describe a little bit the chosen reaction scheme.

Response 2:

We sincerely thank you for this constructive suggestion. As advised, we have moved the synthesis scheme to the Results and Discussion section (page 3, lines 91–92). Additionally, we have supplemented a concise description of the chosen reaction scheme in the sub-section [2.1. Synthetic Analysis of Complexes I–IV] (page 2, lines 78–90). The revised content has been highlighted in yellow in the manuscript for easy identification.

 

Comment 3:

Tables 1-3 should be moved to Supplementary, and Table 4 should be moved to Section 3.5. Units of measurement should be added to Tables 4 and 5.

Response 3:

We sincerely thank you for this meticulous and helpful suggestion. Following your instruction, we have moved Tables 1–3 to the Supporting Information, where they are relabeled as Tables S1, S2, and S3, respectively. We have also relocated the original Table 4 to Section 2.8 (formerly Section 3.5 in the initial submission) of the revised manuscript, and it has been renumbered as Table 1 (page 11, lines 354–355). Regarding the missing units of measurement, we have supplemented complete units for all data entries in the original Tables 4 and 5. Notably, these two tables have been renumbered as Table 1 and Table 2 in the revised manuscript, respectively. For the revised Table 1, we have clearly labeled the complexation concentration unit (100 μM) in the column header (page 11, line 354). For the revised Table 2, we have added the unit kcal/mol to all Docking Score entries (page 12, line 365). All the above revisions have been highlighted in yellow for your convenient check.

 

Comment 4: 

It is not written in which solvent and in what concentration the UV spectra were made.

Response 4:

We sincerely thank you for pointing out this critical oversight in our manuscript and apologize for any confusion this may have caused. The optical absorption spectra reported herein were actually measured for solid-state samples instead of solutions, and the relevant term has been corrected to “UV-vis diffuse reflectance spectra (UV-vis DRS)” throughout the revised manuscript (page 1, line 27; page 2, line 68; page 9, lines292–293; page 10, line 313; page 14, line 421; page 16, line 502). Additionally, we have supplemented detailed experimental parameters for the solid-state measurements in the Materials and Methods section (page 14, lines 388–390). We thank you again for helping us improve the accuracy of this manuscript. 

Comment 5: 

When discussing X-ray, you need to add a comparison of the complexes with each other and explain what the differences are. Why is there no pyridine in compound 3? There is also an actual error that the complexes contain 2 benzopyranone-carboxylic acids, it is correct to write that the complexes contain benzopyranone-carboxylic acid anions. It is also necessary to add drawings reflecting the hydrogen bonds in the complexes.

Response 5:

We sincerely thank you for these insightful and constructive comments, which have significantly improved the quality of our manuscript. We have addressed all your points as follows:

1) As you suggested, we have supplemented a detailed comparison of the crystal structures of complexes 1–4 in the X-ray diffraction section (page 3, lines 96–97, lines 108–120);

2) We sincerely appreciate this insightful comment. A corresponding explanatory elaboration has been added for the absence of pyridine in complex III in the revised manuscript (Page 5, Lines 174–179).

3) We apologize for the inaccurate description of the ligand species. The relevant term has been corrected to " 4-benzopyranone-2-carboxylic acid anions " throughout the revised manuscript (page 3, lines100–103).

4) In accordance with your suggestion, we have added a figure depicting the intramolecular and intermolecular hydrogen bond interactions in the complexes to the Supporting Information (Figure S1).

All the above revisions have been highlighted in yellow for your convenient check.

 

Comment 6:

When discussing cytotoxicity, it is advisable to compare it with toxicity to healthy cells, since the main indicator of anticancer compounds is the selectivity of action.

Response 6: 

We sincerely appreciate this valuable suggestion. Unfortunately, due to current experimental constraints, we are unable to supplement the comparison of cytotoxicity against healthy cells in this revision. We fully agree that selective action is a key indicator for anticancer compounds, and we will pay more attention to it in our future research. Thank you again for your constructive input.

 

Comment 7:

In addition, the authors believe that the activity of the 3-hour complex in comparison with the others is related to the role of the metal, but take into account that this complex differs from the others in the absence of pyridine.

Response 7:

We sincerely appreciate your insightful comment. We agree that the distinct activity of the 3-hour complex cannot be solely attributed to the metal center, as this complex also differs from others in the absence of the pyridine ligand. The revised content has been highlighted in yellow in the manuscript for easy identification (page 11, lines 348–351).