Synthesis, Reductive Reactivity and Anticancer Activity of Cobalt(III)– and Manganese(III)–Salen Complexes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, Zhang and co-workers report the synthesis, structural characterization, reductive reactivity, and in vitro anticancer activity of two metal-salen complexes: Mn(III)-salen (Mn-1) and Co(III)-salen (Co-2). Novel insights are presented regarding their reduction behavior with KHBEt₃ and HBpin, leading to the isolation of CoK-3 (a heterobimetallic complex) and Co-4 (a reduced Co(II)-salen complex). Cytotoxicity tests on breast cancer cell lines (MCF-7 and MDA-MB-468) and a healthy breast epithelial cell line (MCF-10A) reveal promising anticancer potential for Co-2, particularly with selectivity for MCF-7 cells. In my opinion, the manuscript is well-structured and the results are particularly interesting; nonetheless, there are some minor issues that must be addressed prior to its publication in this journal.

• There are numerous typos and grammatical errors in this version (e.g., "characterzied",  "ethanole", inconsistent spacing). The manuscript would be benefited with an extensive revission in order to detect this kind of errors. 
• In the introduction, it is necessary to justify the use of Co(III) and Mn(III) as potential anticancer agents.  What are the advantages of first row transition metals as anticancer agents compared with Pt? 
  I suggest to read the following papers: https://doi.org/10.1016/j.ccr.2024.215698
  https://doi.org/10.1002/cmdc.202200367 
• The HNMR spectra of all ligands is required in order to compare the deprotonation of the hydroxil groups when coordinated to Co or Mn.  Moreover, the discussion of HNMR and 13C NMR shifts and signals is poor.
• Suplementary information is only limited with NMR and UV spectras. What happened with IR and EIS information?
• The mechanism for ligand reduction (partial imine hydrogenation) in the presence of KHBEt₃ is briefly mentioned. Expanding on plausible mechanisms or referring to similar literature.

• CoK-3 has a unique structure. Could its reduced activity be explained by lower solubility, reduced uptake, or stability differences?

• The discussion on Co-2’s selectivity is promising—further discussion on why MCF-7 is more susceptible than MDA-MB-468 would be valuable

Author Response

Comments 1: In this manuscript, Zhang and co-workers report the synthesis, structural characterization, reductive reactivity, and in vitro anticancer activity of two metal-salen complexes: Mn(III)-salen (Mn-1) and Co(III)-salen (Co-2). Novel insights are presented regarding their reduction behavior with KHBEt₃ and HBpin, leading to the isolation of CoK-3 (a heterobimetallic complex) and Co-4 (a reduced Co(II)-salen complex). Cytotoxicity tests on breast cancer cell lines (MCF-7 and MDA-MB-468) and a healthy breast epithelial cell line (MCF-10A) reveal promising anticancer potential for Co-2, particularly with selectivity for MCF-7 cells. In my opinion, the manuscript is well-structured and the results are particularly interesting; nonetheless, there are some minor issues that must be addressed prior to its publication in this journal.

 

Response 1: Thank you very much for a brief summary of this work and your kind comments!

 

Comments 2: There are numerous typos and grammatical errors in this version (e.g., "characterzied",  "ethanole", inconsistent spacing). The manuscript would be benefited with an extensive revission in order to detect this kind of errors. 

 

Response 2: Thank you for pointing out the issues. This has been fixed and we also checked the text carefully and corrected any mistakes throughout the manuscript. All the changes have been highlighted in yellow background.

 

Comments 3: In the introduction, it is necessary to justify the use of Co(III) and Mn(III) as potential anticancer agents.  What are the advantages of first row transition metals as anticancer agents compared with Pt? I suggest to read the following papers: https://doi.org/10.1016/j.ccr.2024.215698
https://doi.org/10.1002/cmdc.202200367 

 

Response 3: This is a very good point, thank you! We have added a few sentences justifying the use of Co(III) and Mn(III) as potential anticancer agents in the paragraph 3 of page 2, as highlighted in yellow background. The suggested references have been included as new refs. 24 and 25 in the revised version.

 

Comments 4: The HNMR spectra of all ligands is required in order to compare the deprotonation of the hydroxil groups when coordinated to Co or Mn.  Moreover, the discussion of HNMR and 13C NMR shifts and signals is poor.

 

Response 4: Thanks for the comments that helped us improve the manuscript! According to your suggestion, we have independently conducted the synthesis of the free salen ligand and collected its NMR spectra in DMSO-d6 (the same solvent used for the NMR spectra of complexes for comparison). The synthesis and NMR data are included to the end of section 2.1 and the copies of spectra in the Supplementary Information. A brief discussion on the signal shifts from the comparison of NMR spectra of ligand and complexes has been included in the first paragraph of section 3.1.

 

Comments 5: Supplementary information is only limited with NMR and UV spectra. What happened with IR and EIS information?

 

Response 5: According to the suggestion, all IR and ESI-MS spectra have now been included in the Supplementary Information, along with the ligand NMR spectra.

 

Comments 6: The mechanism for ligand reduction (partial imine hydrogenation) in the presence of KHBEt₃ is briefly mentioned. Expanding on plausible mechanisms or referring to similar literature.

 

Response 6: Thank you! We mentioned relevant but different reduction processes of transition metal complexes induced by KHBEt₃ in the literature to help better understand the mechanism in the end of the first paragraph of section 3.2. The relevant references Nos. 45-48 are cited.

 

Comments 7: CoK-3 has a unique structure. Could its reduced activity be explained by lower solubility, reduced uptake, or stability differences?

 

Response 7: CoK-3 displays indeed a novel structure that includes reduction in both ligand and metal center. Its isolation by crystallization could be attributed to its stability and also its lower solubility than other species.

 

Comments 8: The discussion on Co-2’s selectivity is promising—further discussion on why MCF-7 is more susceptible than MDA-MB-468 would be valuable

 

Response 8: Thanks for the helpful comments! We have included a few sentences as “This discrepancy could be due to the fact that MCF-7 cells have functional tumor suppressor p53, whereas MDA-MB-468 cells have dysfunctional p53. As many anticancer drugs target the p53 pathway, further investigation into the role of p53 in Co-2-induced cell death is necessary to fully elucidate the molecular pharmacological mechanism of this compound.” onto the Line 347 in the section 3.4.

 

Reviewer 2 Report

Comments and Suggestions for Authors

In their manuscript, Kanina et al report on two Co(III)- and Mn(III)-salen complexes (Mn-1 and Co-2) focusing on their crystal structures, and reactivities towards potassium triethyl borohydride and pinacolborane. Their in-vitro antiproliferative activities against breast cancer cells (MCF-7 and MDA-MB 468) along with a novel heterometallic complex CoK-3 have been evaluated.

The study is interesting and well conducted. However, a few concerns must be addressed before proceeding further with the evaluation of the manuscript.

Salen-based complexes of manganese and cobalt are well-known and extensively studied in the literature. In this regard, what are the advantages stemming from the ethoxy substituents in the ligand design?

Reactions of Mn-1 and Co-2 with the two reducing agents, potassium triethylborohydride (KHBEt3) and pinacolborane (HBpin) have been studied. What are the reasons about the choice of these specific reagents.

Despite being low, in the biological studies it is important to mention the percentage of DMSO in the final solutions tested in the experiments, due to the intrinsic toxicity of DMSO itself.

Still in the biological part, the tested compounds possess variable anticancer activities. This could be related to different cell-uptake abilities. ICP-MS measurements could be performed to this aim.

Biological results are presented somewhat superficially, without providing sufficient mechanistic insight or hypotheses about mechanisms of action.  On the basis of relevant literature and obtained results, which are plausible mechanisms of action explaining the different outcomes obtained?

While the introduction appropriately highlights the antitumor potential of metal complexes, it could benefit from acknowledging their more vaste application. In particular, complexes of trace biological metals such as manganese are also of interest for their antioxidant properties, owing to the redox activity of the metal centers (10.1016/j.jinorgbio.2022.112060; 10.3109/10715762.2014.979168).

It would be more appropriate moving section 2.5 on complex stability before the section discussing the biological measurements.

Author Response

Comments 1: In their manuscript, Kanina et al report on two Co(III)- and Mn(III)-salen complexes (Mn-1 and Co-2) focusing on their crystal structures, and reactivities towards potassium triethyl borohydride and pinacolborane. Their in-vitro antiproliferative activities against breast cancer cells (MCF-7 and MDA-MB 468) along with a novel heterometallic complex CoK-3 have been evaluated.

The study is interesting and well conducted. However, a few concerns must be addressed before proceeding further with the evaluation of the manuscript.

 

Response 1: Many thanks for the kind comments! We have addressed your concerns as follows.

 

Comments 2: Salen-based complexes of manganese and cobalt are well-known and extensively studied in the literature. In this regard, what are the advantages stemming from the ethoxy substituents in the ligand design?

 

Response 2: The ethoxy substituents on salen could provide an extra O₄ environment potentially suitable for the incorporation of a larger metal ion (such as K) as demonstrated in the case of complex CoK-3. A sentence has been added to the end of the 2^nd paragraph of section 3.1.

 

Comments 3: Reactions of Mn-1 and Co-2 with the two reducing agents, potassium triethylborohydride (KHBEt3) and pinacolborane (HBpin) have been studied. What are the reasons about the choice of these specific reagents.

 

Response 3: Potassium triethylborohydride (KHBEt₃) has been a popular choice for the reduction of transition metal complexes in the literature as newly cited in refs. 45-48, in response to one of the comments by reviewer 1. However, pinacolborane (HBpin) has not been widely studied as reducing reagent for metal complexes. We chose it purely for a comparison purpose.

 

Comments 4: Despite being low, in the biological studies it is important to mention the percentage of DMSO in the final solutions tested in the experiments, due to the intrinsic toxicity of DMSO itself.

 

Response 4: A sentence “The highest final concentration of DMSO in the cells was 0.1%, which is considered a safe dosage for in vitro studies.” And a new ref. 30 https://pmc.ncbi.nlm.nih.gov/articles/PMC11654018/ regarding the DMSO usage in in vitro experiments was added onto Line 160 in section 2.7.

 

Comments 5: Still in the biological part, the tested compounds possess variable anticancer activities. This could be related to different cell-uptake abilities. ICP-MS measurements could be performed to this aim.

 

Biological results are presented somewhat superficially, without providing sufficient mechanistic insight or hypotheses about mechanisms of action.  On the basis of relevant literature and obtained results, which are plausible mechanisms of action explaining the different outcomes obtained?

 

Response 5: Thank you very much for the helpful comments! The mechanistic work would be indeed much valuable for the improvement of the current work. However, it would take several more months for us to complete the experiments and data analysis. Nevertheless, mechanistic hypotheses have been included as a new paragraph at the end of section 3.4 to provide some insights based on the current results and relevant literature (refs. 53-55).

 

Comments 6: While the introduction appropriately highlights the antitumor potential of metal complexes, it could benefit from acknowledging their more vaste application. In particular, complexes of trace biological metals such as manganese are also of interest for their antioxidant properties, owing to the redox activity of the metal centers (10.1016/j.jinorgbio.2022.112060; 10.3109/10715762.2014.979168).

 

Response 6: Thank you for the helpful comments! We have included a brief introduction about this in the third paragraph of page 2 in the Introduction section. New references 26-28 as you suggested are added too.

 

Comments 7: It would be more appropriate moving section 2.5 on complex stability before the section discussing the biological measurements.

 

Response 7: This has been fixed.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Synthesis, reductive reactivity and anticancer activity of cobalt(III) and manganese(III) salen complexes” is a good but rather routine study in the field of the coordination chemistry of the new complexes tested on anticancer activity and does not introduce any significant novelties in this field. The presented work could be improved by additional investigation and can be published if the following corrections/queries are addressed.

• The author claims that in the obtained Mn-1 and Co-2 complexes, the metal ions are in the +III oxidation state. However, they did not show enough evidence for that, such as magnetic measurements or what should be crucial to confirm the oxidation state, especially in the solution, EPR studies. A similar situation occurs for the CoK-3 compound. Comparison of the UV-Vis spectra for Co-2 and CoK-3 did not show any specific changes in the d-d transition region, which should be seen after reduction, in the spectrum of CoK-3 with Co(II) ions. Are cobalt ions in complex CoK-3 still in the +II oxidation state after dissolving in DMSO or DMSO/H₂O solution? In my opinion, the statement of the oxidation state of the metal ions in the solution requires EPR measurements for all the complexes.
• In the 3.1 paragraph (line 191), the Authors say about diamagnetic??? Mn(III) ion??? Is it possible for octahedral Mn(III) to be diamagnetic?
• Are Figures 5 and S6 the same? Please check it.
• There are also a few editorial corrections which were marked in the PDF file of the manuscript.

Comments for author File: Comments.pdf

Author Response

Comments 1: The manuscript entitled “Synthesis, reductive reactivity and anticancer activity of cobalt(III) and manganese(III) salen complexes” is a good but rather routine study in the field of the coordination chemistry of the new complexes tested on anticancer activity and does not introduce any significant novelties in this field. The presented work could be improved by additional investigation and can be published if the following corrections/queries are addressed.

 

Response 1: Thanks for the comments! Even though metal salen complexes have been widely explored in the literature, this work is a good contribution to the literature as agreed by the reviewers 1 & 2. This work reveals the novel reductive reactivity of metal salen complexes as demonstrated by the isolation and characterization of the unique complex CoK-3. The reduction of Co-2 to Co-4 by HBpin provides also a novel reaction pathway. In addition, we were able to compare the anticancer activities between the original and reduced metal complexes for the first time.

 

Comments 2: The author claims that in the obtained Mn-1 and Co-2 complexes, the metal ions are in the +III oxidation state. However, they did not show enough evidence for that, such as magnetic measurements or what should be crucial to confirm the oxidation state, especially in the solution, EPR studies. A similar situation occurs for the CoK-3 compound. Comparison of the UV-Vis spectra for Co-2 and CoK-3 did not show any specific changes in the d-d transition region, which should be seen after reduction, in the spectrum of CoK-3 with Co(II) ions. Are cobalt ions in complex CoK-3 still in the +II oxidation state after dissolving in DMSO or DMSO/H₂O solution? In my opinion, the statement of the oxidation state of the metal ions in the solution requires EPR measurements for all the complexes.

 

Response 2: Thank you for the comments! Actually, similar type of trivalent complexes as Mn-1 and Co-2 have been largely reported in the literature and our cases are not exceptional. The cited references from No. 34 through 43 in the revised manuscript include a large number of such complexes with slightly different substituents on the salen ligand or the axial ligands (acetate and water in our cases). In general, we used similar synthetic procedure and characterization techniques. As revealed in Figure 5, the UV-Vis spectra between Co-2 (red line) and CoK-3 (green line) do show a significant difference in the d-d transition region. The UV spectra of CoK-3 in DMSO/H₂O solution also showed relatively good stability of this complex as seen in Figure S15. In addition, the observed deprotonation of hydroxyl group in the ligand revealed by the comparison of NMR spectra between Mn-1 and the free ligand, along with the presence of an axial acetate ligand evidenced by both X-ray crystallography and NMR spectroscopy further prove the +3 oxidation state of Mn and Co complexes. We agree that EPR measurements for these complexes could be helpful for better understanding the oxidation states of metal ions, however, we are currently unable to perform those experiments due to insufficient instrumental resources.

 

Comments 3: In the 3.1 paragraph (line 191), the Authors say about diamagnetic??? Mn(III) ion??? Is it possible for octahedral Mn(III) to be diamagnetic?

 

Response 3: Thank you for pointing out this. We realized this is a mistake and the relevant statement has been adjusted now.

 

Comments 4: Are Figures 5 and S6 the same? Please check it.

 

Response 4: Yes, they were the same and it was accidentally placed as a duplicate in the SI. Now the Figure S6 is removed.

 

Comments 5: There are also a few editorial corrections which were marked in the PDF file of the manuscript.

 

Response 5: Thank you very much for the very careful corrections! We have made all the changes as you pointed out in the PDF file.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have properly addressed all the concerns raised by this reviewer. Accordingly, the Ms. is now suitable for publication.

Best regards

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is well-revised, and it can be published in the present form.