Vanadium(V) Complexes with Siderophore Vitamin E-Hydroxylamino-Triazine Ligands

Round 1

Reviewer 1 Report

The manuscript presents a topic of interest, obtaining compounds with antitumor activity. Regarding the experimental part and the studies performed, my observations are:

1. The attribution of the structure of complexes 3 and 4 cannot be done only through elementary analysis studies. This must be completed by spectroscopy studies, NMR, EPR, etc.

2. A major problem with compounds with antitumor potential is their solubility in liquids compatible with biological environments. So the solubility of the obtained compounds must be discussed. Also, to test the cytotoxic activity on cell lines, another solvent/mixture of compounds must be found that contains a maximum of 1% DMSO because this has cytotoxic activity and can influence the results. Solvents examples for dissolution the compounds: phosphate buffer, cacodylate buffer, glycol: propylene glycol, etc. If necessary, the compounds can be dissolved in a small volume of DMSO or DMF but their percentage in the final solution should not exceed 1%.

3. I suggest that before discussing the possible cytotoxic effect of vanadium complexes on tumor cells, their toxicity to normal cells be tested to see how their viability is affected.

Author Response

We thank the reviewer for his suggestions,

1. The attribution of the structure of complexes 3 and 4 cannot be done only through elementary analysis studies. This must be completed by spectroscopy studies, NMR, EPR, etc.

Additional characterization of 3 and 4 were added (MS, IR, UV titration)

2. A major problem with compounds with antitumor potential is their solubility in liquids compatible with biological environments. So the solubility of the obtained compounds must be discussed. Also, to test the cytotoxic activity on cell lines, another solvent/mixture of compounds must be found that contains a maximum of 1% DMSO because this has cytotoxic activity and can influence the results. Solvents examples for dissolution the compounds: phosphate buffer, cacodylate buffer, glycol: propylene glycol, etc. If necessary, the compounds can be dissolved in a small volume of DMSO or DMF but their percentage in the final solution should not exceed 1%.

The maximum quantity of DMSO does not exceed 1% of DMSO. After reviewer notice, we clarify this in the experimental.

3. I suggest that before discussing the possible cytotoxic effect of vanadium complexes on tumor cells, their toxicity to normal cells be tested to see how their viability is affected.

The studies were added

Reviewer 2 Report

This paper by Loizou et al. is one of a long series of studies from this research group on potentially bioactive vanadium complexes. It is a long and complex story describing the attempts to make and functionally characterize a series of V and Fe(III) complexes of two new ligands that combine a previously studied coordination of a triazine derivative (the main donor groups) to Vitamin E side groups. Apparently, there was an idea that some of the known benefits of Vitamin E in biology – primarily its antioxidant capability, but also significant lipophilicity – might enhance the activity of the metal complexes.

The rather complex structural assessments in this paper are dependent on complex and demonstrated fluxional NMR data (H1 and V51). The structure is presumed to be analogous to that reported from this research group in 2008 (cited as Ref. [42]) wherein an unusual dimeric oxygen-bridged crystal structure was obtained.

While this work describes some valiant efforts to wrest results from a very demanding and difficult system, I find that these complexities overwhelm the provided evidence. Furthermore, the paper as written is very confusing, and in particular the Introduction and the relationship of the mentioned species to Scheme 1 to be completely obscure. Reading later down into the paper, the actual nature of the attempted experiments becomes somewhat clearer. Nevertheless, significant questions remain:

1. Was EPR used to conclusively confirm that the complexes are V(V) and hence not showing the strong EPR signals of the vanadyl system? Or was there always a strong vanadyl EPR signal from trace unoxidized materials present?
2. Why/how can NMR titration studies be undertaken when Fe(III) salts – strongly paramagnetic ions – are added to the V complexes? Are the authors completely certain that the iron complexes simply did not precipitate and hence get removed from solution? I find it difficult to understand how else the NMR spectra could remain so unaffected.

As a bare minimum, the paper needs to be thoroughly re-written to explain all the confusing terminology. For example, the term ‘cromanol’ is mentioned in the introduction without context or explanation. The whole introduction section needs to be reconceived to make it intelligible to readers who are not ready to read through the dozens of previous papers from this author group.

Secondly, the aims and claims of the paper need to be revised downward, and it needs to be made explicitly clear what is clearly knowable from this work and what is not. I would seriously advise the removal of the whole ‘iron replacement’ story and await strong evidence.

X-ray structures to confirm more of the V and Fe complexes would be highly desirable.

Author Response

We thank the reviewer for his suggestions,

1. Was EPR used to conclusively confirm that the complexes are V(V) and hence not showing the strong EPR signals of the vanadyl system? Or was there always a strong vanadyl EPR signal from trace unoxidized materials present?

There were not any epr signals originated to vanadium(IV) either of the frozen solutions of the complexes at 110 ^oC ot at room temperature. A sentence has been added.

2. Why/how can NMR titration studies be undertaken when Fe(III) salts – strongly paramagnetic ions – are added to the V complexes? Are the authors completely certain that the iron complexes simply did not precipitate and hence get removed from solution? I find it difficult to understand how else the NMR spectra could remain so unaffected.

we remove this part

As a bare minimum, the paper needs to be thoroughly re-written to explain all the confusing terminology. For example, the term ‘cromanol’ is mentioned in the introduction without context or explanation. The whole introduction section needs to be reconceived to make it intelligible to readers who are not ready to read through the dozens of previous papers from this author group.

Chromanol is common chemical nomenclature. However, it is important in the discussion of this manuscript. We agree with the reviewers comment, therefore we show on Scheme 1 what is the chromanyl ring.

Secondly, the aims and claims of the paper need to be revised downward, and it needs to be made explicitly clear what is clearly knowable from this work and what is not. I would seriously advise the removal of the whole ‘iron replacement’ story and await strong evidence.

we remove this part

X-ray structures to confirm more of the V and Fe complexes would be highly desirable.

Additional characterization were added according to reviewers 1 and 3

Reviewer 3 Report

In this manuscript, the authors synthesized two new vitamin E compounds that bind V(V) and Fe(III) ions. They showed using UV-Vis and NMR the binding of the metal to the complex. However, the applicability of these complexes as anti-cancer drugs was found law, which limits the novelty of this work.

I think few changes need to be carried out in this manuscript before it can be considered for publication:

1. X-ray structures or mass spec of these complexes need to be reported.
2. The EPR spectra are missing, and it is not clear how Fig. 13 was derived. Which EPR intensity? what is the time of one scan? how the experiments were performed? a lot of experimental parameters are missing from the text, specifically the EPR data itself.
3. In most of the graph, the authors used very wired "ticks labels" with different spaces, and not whole numbers - this need to be changed.
4. Why the authors did not perform the cell experiments also on normal cells for control, and not just cancer cells

Author Response

We thank the reviewer for his suggestions,

1. X-ray structures or mass spec of these complexes need to be reported.

Additional characterization were added

1. The EPR spectra are missing, and it is not clear how Fig. 13 was derived. Which EPR intensity? what is the time of one scan? how the experiments were performed? a lot of experimental parameters are missing from the text, specifically the EPR data itself.

Epr were added and explained. Details of the experiment is in the experimental

2. In most of the graph, the authors used very wired "ticks labels" with different spaces, and not whole numbers - this need to be changed.

corrected

3. Why the authors did not perform the cell experiments also on normal cells for control, and not just cancer cells

added

Round 2

Reviewer 2 Report

Some of the revisions suggested have been made.

The final version has numerous spelling and wording errors, and mistakes in e.g. writing chemical and physical equations. These have been noted in Comments on the PDF file. These are uploaded here for the Authors to use in revision.

Comments for author File: Comments.pdf

Author Response

All has been corrected according to the comments of the reviewer 

Reviewer 3 Report

I have no more comments

Author Response

The reviewer has not any comments