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  • Silvana Alfei 1,*,
  • Maria Grazia Signorello 1,2 and
  • Cinzia Domenicotti 3,6
  • et al.

Reviewer 1: Anonymous Reviewer 2: Anonymous

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper about the synthesis, characterization and biological of phosphonium salts contains a lot of work, and displays original and interesting results. However, it suffers from numerous important drawbacks.

  • This paper is very long (58 pages) and looks more like a chapter of a PhD thesis than a paper in a journal.
  • In the abstract, line 45, what is the meaning of “2.5forl”?
  • The discussion about NMR data is by far too long. Only the salient points should be discussed, in particular 31P NMR (chemical shift for PPH3 in the conditions used should be given) for all compounds, and the CH=CH2 linkage of compound 4 in 1H and 13C NMR. Besides, the attribution of the signals to the protons and carbons, using the numbering shown in Figures 2, 3, 4, and 5, should be given in the Materials and Methods section.
  • Looking to blue part of Scheme 3, the yield should not exceed 50%, but it is 81% after workup. It means that a part of the mechanism is missing. What happens to the compound R-O-Ph? I suppose that it can also react with NaH? This Scheme should be modified.
  • I also found the ATR-FTIR part too long.
  • The part about Principal component analysis is difficult to understand (not the principle, but the explanations, and the pact that PC1 and PC2 have not the same meaning depending on the part of this section it is discussed. And thus, the captions to Figures 6 and 7 are not informative of what they really represent.
  • In the part about optical microscopy analyses, I have been unable to find the concentration used to obtain the vesicles. It is important to know if this association occurs in the concentration range that has been tested in the biological experiments. Was it individual compounds or associated compounds that have been tested? And I not sure that all these photos are necessary in the manuscript, most of them could be added to the Supporting Information
  • In part B-b of Figure 10, for compound 3 after 72 h against the MeTRAV PLX-R cancer cells, there is a very large increase of the number of cancer cells at concentrations from 1 to 25 microMol. This is very important adverse effect, but I seen no comment about this point. This should be added. It is also observed with compound 4, but with a lower intensity.
  • All the biological data are exploited several times in this paper, presented as bars, as curves, and again as bars gathering all the data. I think that here also it is possible to shorten.
  • In Figure 18, the y scale should be limited to 10. As it is, at the first glance, it seems that less than 50 % of the cells survived, which is not the case.
  • In Figure 20, the very large difference in viability observed between the HaCAT cells and the 3T3 cells might give an information about the mechanism of action. I have seen nothing in this paper.
  • In the Materials and Methods section, in addition to the attribution of the signals to specific hydrogen and carbon atoms which should be added, the solvent used for all 31P NMR experiments is missing.

In conclusion, the work displayed in this paper is interesting, but the presentation should be substantially improved, shortened and discussed.

Author Response

The paper about the synthesis, characterization and biological of phosphonium salts contains a lot of work, and displays original and interesting results. However, it suffers from numerous important drawbacks.

We thank a lot the Reviewer for appreciating our work and for her/his suggestions. We therefore have satisfied requests or responded to her/his comments, also adding further explanations in the main text, reaching a supplemental improvement of quality and scientific rigor of this study.

 

  • This paper is very long (58 pages) and looks more like a chapter of a PhD thesis than a paper in a journal.

The work has been substantially revised according to the Reviewer suggestions and therefore shortened in several parts. Cuttings to the original text were mainly made in lines 240-271, 322-361, 392-439, 444-477, 536-548. Additionally, Figure 8 (original version) has been removed and inserted as Figure S5.1. in Supplementary Materials (SM) file

  • In the abstract, line 45, what is the meaning of “2.5forl”?

We thank the Reviewer for having signalled this typo, which was corrected in 2.5-fold (line 45).

 

  • The discussion about NMR data is by far too long. Only the salient points should be discussed, in particular 31P NMR (chemical shift for PPH3 in the conditions used should be given) for all compounds, and the CH=CH2 linkage of compound 4 in 1H and 13C NMR. Besides, the attribution of the signals to the protons and carbons, using the numbering shown in Figures 2, 3, 4, and 5, should be given in the Materials and Methods section.

We thank the Reviewer for suggestions. The detailed discussion and peak attribution of NMR spectra have been removed from the main text and has been included in the Supplementary materials (SM) under the images of related spectrum, containing the numbered structures, useful for attributions (deleted lines 240-271, 322-361, 392-439, 444-477). In the main text, only the peak lists of all NMR spectra in the Material and Methods Section have been maintained, where the numbered structures of compounds 1, 2, 3 and 4, previously present in the Discussion Section, have been moved. Chemical shift for PPh3 in the conditions used to acquire 31P NMR spectra have been given (lines 1279-1280, 1321-1322, 1359, 1397 and 1423), as well as more details have been included in both main text (lines 1409, 1413, 1415 and 1419) and SM, for the signals of CH=CH2 double bond .

  • Looking to blue part of Scheme 3, the yield should not exceed 50%, but it is 81% after workup. It means that a part of the mechanism is missing. What happens to the compound R-O-Ph? I suppose that it can also react with NaH? This Scheme should be modified.

Why did the Reviewer argue that yield should not exceed 50% for compound 2, when we have not inserted any yield in Scheme 3? Scheme 3, as explained in its caption and in the text, does not show any experimental detail, but it only shows the assumed mechanism by which compound 2 can form, as suggested in literature by Hands et al (cited in the paper). Experimental details for such reaction are in fact reported in Scheme 2. In a reaction mechanism scheme, the yields of the various steps should not be included. Schemes concerning mechanisms must show only electronic movements (curved arrows), intermediates, transition states (if necessary), reactants and products of each step, as reported in Scheme 3. They must not include individual step yields, overall yields, reaction times, which we have correctly reported in Scheme 2. A mechanism does not represent the experimental procedure, it describes how the transformation occurs, not how much product is obtained. Why did the Reviewer suppose that also just formed R-O-Ph should react with NaH, when it is an ether, whose reactivity is extremely limited, and ethers react only in acidic conditions, as organic synthesis teach us??? On these considerations, we are confident that Scheme 3 is correct and does not need modifications.

I also found the ATR-FTIR part too long.

The comment of the Reviewer about the length of this section find explanation in the fact that an unexpected double band was observed for the aliphatic C-H stretching band of benzyl methylene of compound 1. In fact, while such band is typically found at 2968 cm-1 in form of single band, it was found as a double band, being the first band at 2859 cm-1 and the second one at lower values of wavenumber (2773 cm-1). In this regard, we fought mandatory provide and explanation for this phenomenon with the related literature reference (part maintained in lines 496-536). Anyway, this part was substantially modified (lines 482-496) and shortened (deleted lines 536-548), by moving the ATR-FTIR discussion for compounds 2, 3 and 4 in the SM.

  • The part about Principal component analysis is difficult to understand (not the principle, but the explanations, and the pact that PC1 and PC2 have not the same meaning depending on the part of this section it is discussed. And thus, the captions to Figures 6 and 7 are not informative of what they really represent.

If the Reviewer is so kind to allow us to give her/him an explanation of how PCA works, the question will be easily clarified. In PCA, principal components (PC1, PC2, PC3 etc.) do not have a fixed meaning valid for all data sets. PCs take on different meanings depending on the data type and structure (FTIR, NMR or UV-Vis data), variables, and correlations. They are directions of maximum variance (explained variance as showed in the related Figures), constructed specifically for that dataset. As an example, PC1 of dataset A does not have the same meaning as PC1 of dataset B. PCA works by finding new coordinates so that, PC1 = direction along which the data variance is maximum and PC2 = direction with maximum variance orthogonal to PC1, and so on. This means that each PC is a linear combination of the original variables specific to that dataset. Since each dataset has different correlations, different scales, different variances and different structures its PCs will be different and will assume a different meaning, and therefore PCs will not represent the same "phenomenon" in different datasets. Anyway, additional explanations for the meaning of PCs have been included in the revised version of our paper (lines 579-590). Figure captions of Figure 6, 7 and 23 (now Figure 2 and 3 and 18 in revised version) have been modified adding more useful details for readers’ comprehension of their contents (lines 628-633, 648-660 and 1245-1248).

  • In the part about optical microscopy analyses, I have been unable to find the concentration used to obtain the vesicles. It is important to know if this association occurs in the concentration range that has been tested in the biological experiments. Was it individual compounds or associated compounds that have been tested?

We make kindly note to the Reviewer, that concentrations used for optical microscopy in MeOH were already present in the unrevised version of the paper expressed as mg/mL, and those in water could have been calculated from already provided numbers. Anyway, this part was completed adding concentrations for both experiments also in mM concentrations (lines 1492, and 1495-1496). On the other hand, we thank a lot the Reviewer for having raised this important question, regarding which form of compounds 1, 3 and 4 (aggregated or not), having shown to have tendency to aggregate in optical images, have been provided to cells in biological experiments. Since from new calculations, it was found that in optical experiments mM concentrations were used, while in biological experiments µM ones (three order of magnitude lower), we can assert that 1, 3 and 4 were administered to cells in not aggregated forms. Specifications on this question were added in the main text in lines 1514-1516, 1546-1548 and 1574-1576.

And I not sure that all these photos are necessary in the manuscript, most of them could be added to the Supporting Information

As suggested optical images have been moved in Supplementary Materials thus further shortening the manuscript length, new Section S5.

  • In part B-b of Figure 10, for compound 3 after 72 h against the MeTRAV PLX-R cancer cells, there is a very large increase of the number of cancer cells at concentrations from 1 to 25 microMol. This is very important adverse effect, but I seen no comment about this point. This should be added. It is also observed with compound 4, but with a lower intensity.

We thank a lot the Reviewer for having asked a more detailed discussion on this experimental observation, which was only partially already present in the unrevised version of the paper, and we thought that all this part of discussion needed improvements. We thank the Reviewer for having highlighted this important point and agreed that the marked increase in PLX-R MeTRAV cell number observed at 72 h with compound 3, and to a lesser extent with compound 4, but also observable with 1, deserved specific discussion. We have now substantially modified all this part of discussion on MCM cells responses and have explicitly described the biphasic effect observed and added a corresponding discussion (lines 786-808 inside all modified discussion in lines 744-831). In the revised manuscript, we have discussed the possibility that this response may reflect a concentration-dependent hormetic-like effect, potentially related to compensatory survival signalling in this resistant cell line. We have also clarified that this phenomenon appears to be cell line–specific and warrants further mechanistic investigation.

  • All the biological data are exploited several times in this paper, presented as bars, as curves, and again as bars gathering all the data. I think that here also it is possible to shorten.

We make kindly note to the Reviewer, that the several bars and curves graphs reported in this paper do not regard different graphical representation of same results, but regard the presentation of different results from different experiments and/or calculations, or results from comparison studies. On this consideration, we kindly ask the Reviewer to not force use to move them in SM.  

  • In Figure 18, the y scale should be limited to 10. As it is, at the first glance, it seems that less than 50 % of the cells survived, which is not the case.

We make kindly ask the Reviewer, why should we limit y scale to 10, when in several cases, available cells (%) reported on y axes were up to > 90%. Has perhaps the Reviewer made confusion between x and y scales? If so, and if Reviewer would kindly listen to our explanation, we note that the scope of the graph is making a comparison between cells viability of cancer cells and RBCs under treatment with 1, 3 and 4 at three different increasing 10, 25 and 50 µM concentrations. Reporting only the 10 µM one would loose of significance to the discussion, also because viability of RBCs was higher than that of cancer cells up to 25 µM concentrations in almost all cases and up to 50 µM in more sporadic cases, which is an overall merit of this study, worthy of being evidenced. Anyway, y scale was shorted to 120%, being this percentage more proper to our results.

  • In Figure 20, the very large difference in viability observed between the HaCAT cells and the 3T3 cells might give an information about the mechanism of action. I have seen nothing in this paper.

The mechanism by which QPSs as 1, 3 and 4 could exert their cytotoxicity against bacterial and cancer cells is reported in Section 1.1. of the not revised and revised version of the paper. The same mechanism is assumed for normal cells, towards which cytotoxic effects are usually lower than those observed towards tumoral cells, due to the lower negativity of their membrane, which improve selectivity of QPSs for malignant cells, as we have experimentally demonstrated. Anyway, the reason for which HaCaT cells could be more sensitive to QPSs than 3T3 cells was not debated in the original manuscript. Now, thanks to the relevant Reviewer suggestion, such question has been extensively discussed, thus improving the quality of our paper (lines1130-1162).

  • In the Materials and Methods section, in addition to the attribution of the signals to specific hydrogen and carbon atoms which should be added, the solvent used for all 31P NMR experiments is missing.

We make kindly note to the Reviewer that, since a detailed attribution of all signals to specific hydrogen, carbon and phosphorous atoms, for all NMR spectra acquired, was reported in the Results and Discussion Section (which it was now moved in SM on request of this Reviewer), it appear us redundant repeating it in Materials and Methods section, where a mere peaks list with numerosity of atoms giving each signal for all compounds could be sufficient. On the other hand, the solvent used for all 31P NMR experiments was added in the revised versions of main text (lines 1321, 1359, 137 and 1423).

In conclusion, the work displayed in this paper is interesting, but the presentation should be substantially improved, shortened and discussed.

Thanks to the Reviewers’ suggestions and comments, the presentation of the original manuscript has been substantially improved, shortened and better discussed. Additionally, conclusions were substantially changed and improved and SM modified (1604-1661 and 1883-1691).

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript is an extensive study devoted to the synthesis and biological evaluation of novel quaternary phosphonium salts as potential anticancer agents for the treatment of metastatic melanoma and neuroblastoma, including drug-resistant forms. The absolute advantage of the work is the use of relevant and clinically significant models of resistance (PLX-resistant melanoma and MDR neuroblastoma), as well as a comprehensive assessment of toxicity on several normal cell lines. The authors conducted a huge amount of experimental work, synthesized new compounds and characterized them in detail.

The manuscript is well structured and well written.

The methods are described in detail. The results are well illustrated and sufficiently discussed. The conclusions are supported by the results.

I just have a small comment. The authors should label the x-axis in Figures 12 and 15.

Author Response

This manuscript is an extensive study devoted to the synthesis and biological evaluation of novel quaternary phosphonium salts as potential anticancer agents for the treatment of metastatic melanoma and neuroblastoma, including drug-resistant forms. The absolute advantage of the work is the use of relevant and clinically significant models of resistance (PLX-resistant melanoma and MDR neuroblastoma), as well as a comprehensive assessment of toxicity on several normal cell lines. The authors conducted a huge amount of experimental work, synthesized new compounds and characterized them in detail.

The manuscript is well structured and well written.

The methods are described in detail. The results are well illustrated and sufficiently discussed. The conclusions are supported by the results.

I just have a small comment. The authors should label the x-axis in Figures 12 and 15.

The Reviewer has been very kind in judging our work and we trust that her/his professional opinion could be shared also by other Reviewer and journal staff, already after this first round revision. X axis in old Figure 12 and 15, now Figure 6 and 9 (revised version) have been labelled.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have satisfied almost all my queries. Only one problem is still pending:

  • Looking to blue part of Scheme 3, the yield should not exceed 50%, but it is 81% after workup. It means that a part of the mechanism is missing. What happens to the compound R-O-Ph? I suppose that it can also react with NaH? This Scheme should be modified.

Why did the Reviewer argue that yield should not exceed 50% for compound 2, when we have not inserted any yield in Scheme 3? Scheme 3, as explained in its caption and in the text, does not show any experimental detail, but it only shows the assumed mechanism by which compound 2 can form, as suggested in literature by Hands et al (cited in the paper). Experimental details for such reaction are in fact reported in Scheme 2. In a reaction mechanism scheme, the yields of the various steps should not be included. Schemes concerning mechanisms must show only electronic movements (curved arrows), intermediates, transition states (if necessary), reactants and products of each step, as reported in Scheme 3. They must not include individual step yields, overall yields, reaction times, which we have correctly reported in Scheme 2. A mechanism does not represent the experimental procedure, it describes how the transformation occurs, not how much product is obtained. Why did the Reviewer suppose that also just formed R-O-Ph should react with NaH, when it is an ether, whose reactivity is extremely limited, and ethers react only in acidic conditions, as organic synthesis teach us??? On these considerations, we are confident that Scheme 3 is correct and does not need modifications.

I persist to say that the mechanism shown in Scheme 3 is not compatible with a yield of 81% indicated in the experimental section for compound 2. In Scheme 3, you need two phosphonium alcohol sodium salts to get one phosphine alcohol. Thus undoubtedly, the yield in compound 2 cannot exceed 50%, because half of the phosphonium is waste in the form of phosphonium phenol ether. If the yield is really 81% (and I suppose that it is indeed 81%), thus the mechanism is false. 

My answer to a minor point:

  • In Figure 18, the y scale should be limited to 10. As it is, at the first glance, it seems that less than 50 % of the cells survived, which is not the case.

We make kindly ask the Reviewer, why should we limit y scale to 10, when in several cases, available cells (%) reported on y axes were up to > 90%. Has perhaps the Reviewer made confusion between x and y scales? If so, and if Reviewer would kindly listen to our explanation, we note that the scope of the graph is making a comparison between cells viability of cancer cells and RBCs under treatment with 1, 3 and 4 at three different increasing 10, 25 and 50 µM concentrations. Reporting only the 10 µM one would loose of significance to the discussion, also because viability of RBCs was higher than that of cancer cells up to 25 µM concentrations in almost all cases and up to 50 µM in more sporadic cases, which is an overall merit of this study, worthy of being evidenced. Anyway, y scale was shorted to 120%, being this percentage more proper to our results.

It is my mistake, I wanted to indicate 100, not 10. It was for the y axis (cell viability) as I indicated, and not for the x axis for which the authors answered. Anyway, the y axis is now limited to 120 (instead of 200 previously), thus it is fine.

Author Response

 

The authors have satisfied almost all my queries. Only one problem is still pending:

  • Looking to blue part of Scheme 3, the yield should not exceed 50%, but it is 81% after workup. It means that a part of the mechanism is missing. What happens to the compound R-O-Ph? I suppose that it can also react with NaH? This Scheme should be modified.

Why did the Reviewer argue that yield should not exceed 50% for compound 2, when we have not inserted any yield in Scheme 3? Scheme 3, as explained in its caption and in the text, does not show any experimental detail, but it only shows the assumed mechanism by which compound 2 can form, as suggested in literature by Hands et al (cited in the paper). Experimental details for such reaction are in fact reported in Scheme 2. In a reaction mechanism scheme, the yields of the various steps should not be included. Schemes concerning mechanisms must show only electronic movements (curved arrows), intermediates, transition states (if necessary), reactants and products of each step, as reported in Scheme 3. They must not include individual step yields, overall yields, reaction times, which we have correctly reported in Scheme 2. A mechanism does not represent the experimental procedure, it describes how the transformation occurs, not how much product is obtained. Why did the Reviewer suppose that also just formed R-O-Ph should react with NaH, when it is an ether, whose reactivity is extremely limited, and ethers react only in acidic conditions, as organic synthesis teach us??? On these considerations, we are confident that Scheme 3 is correct and does not need modifications.

I persist to say that the mechanism shown in Scheme 3 is not compatible with a yield of 81% indicated in the experimental section for compound 2. In Scheme 3, you need two phosphonium alcohol sodium salts to get one phosphine alcohol. Thus undoubtedly, the yield in compound 2 cannot exceed 50%, because half of the phosphonium is waste in the form of phosphonium phenol ether. If the yield is really 81% (and I suppose that it is indeed 81%), thus the mechanism is false. 

The Reviewer is right in wanting to persist in her/his comment. We greatly thank her/him for having persisted, giving us a second opportunity to correct our enormous error. In this regard, we firstly apologise a lot to the Reviewer for our previous unprofessional and even irrespective comment given to Reviewer about ethers reactivity, which did not consider that ether forming with 2 is an aromatic ether susceptible of nucleophilic aromatic substitution, as the same Reviewer had suggested without awakening our limited wit. Scheme 3 has been completed and related text modified. Please, see lines 274-294.

My answer to a minor point:

  • In Figure 18, the y scale should be limited to 10. As it is, at the first glance, it seems that less than 50 % of the cells survived, which is not the case.

We make kindly ask the Reviewer, why should we limit y scale to 10, when in several cases, available cells (%) reported on y axes were up to > 90%. Has perhaps the Reviewer made confusion between x and y scales? If so, and if Reviewer would kindly listen to our explanation, we note that the scope of the graph is making a comparison between cells viability of cancer cells and RBCs under treatment with 1, 3 and 4 at three different increasing 10, 25 and 50 µM concentrations. Reporting only the 10 µM one would loose of significance to the discussion, also because viability of RBCs was higher than that of cancer cells up to 25 µM concentrations in almost all cases and up to 50 µM in more sporadic cases, which is an overall merit of this study, worthy of being evidenced. Anyway, y scale was shorted to 120%, being this percentage more proper to our results.

It is my mistake, I wanted to indicate 100, not 10. It was for the y axis (cell viability) as I indicated, and not for the x axis for which the authors answered. Anyway, the y axis is now limited to 120 (instead of 200 previously), thus it is fine.

We thank a lot the Reviewer for her/his high professionality, outperforming our, and for appreciating/approving our revision.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Everything is fine now.