Structural, Vibrational and Electrochemical Analysis and Antibacterial Potential of Isomeric Chalcones Derived from Natural Acetophenone

Round 1

Reviewer 1 Report

The authors have fully characterized the chalcone related molecules with intriguing spectroscopical techniques, as in this same design present in their other works reported below:

1.T.R. Garcia et al. / Journal of Molecular Structure 1216 (2020) 128358 Structural, vibrational and electrochemical analysis and antibiotic
activity study of chalcone (2E)-1-(3ʹ,-methoxy-4ʹ,-hydroxyphenyl)-3-
(3-nitrophenyl)prop-2-en-1-one

2. A.M.R. Teixeira et al. / Journal of Molecular Structure 1179 (2019) 739e748747
Structural, spectroscopic and microbiological characterization of the chalcone 2E-1-(2ʹ-hydroxy-3ʹ,4ʹ,6ʹ-trimethoxyphenyl)-3-(phenyl)-prop-2-en-1-one derived from the natural product 2-hydroxy-3,4,6-trimethoxyacetophenone

 

Although the originality of work is incomprehensibly spliced and lost on three identical papers, some points can be clarified:

1. The experimental spectrum ATR-FTIR profile of chalcone 2 in figure 7: the peaks in the region between 1200-1000 cm-1 shows a low intensity appearing different from the experimental spectra of chalcone 1. Conversely, the signal of the hydroxyl group is more strong in the experimental spectrum of chalcone 2 and slightly pronounced in the profile of chalcone 1. This phenomenon is not explained in the text or it is difficult to capture; none correspondence of signal intensity (strong, medium, low etc.) is reported in Tables 3 and 4  next to the numerical data, even if their description in the corresponding legenda.
2. The authors have done an electrochemical characterization of compounds 1 and 2 without any correlation with the other experimental part of the work. Probably they performed these experiments to prove the possible mechanism of action of this class of compounds (ref 38 e 39), but any discussion is present in the work. The authors must integrate the electrochemical data in the paragraph related to antimicrobial activities (lines 323-329) with a fruitful discussion. On the other hand, the author can eliminate the electrochemical experiments and insert them in a future work, to support more targeted and specific biological tests.
3. The molecules are structural isomers, but compound 2 seems to be more active than compound 1 associated with gentamicin. Again the authors have not added any comment of this "strange behaviour". The authors affirmed that: "On the other hand, the addition of chalcone 1 to the growth medium resulted in an antagonistic action against both multidrug-resistant strains, increasing the MIC values of the Gentamicin from 16 to 20.15 µg mL-1 308 for S. aureus SA10 and from 32 to 256 µg mL-1 309 for E. coli EC06. This antagonism could be explained by a possible chelating of Gentamicin by chalcone 1, reducing the entry of this antibiotic into the bacterial cell, where is located the Gentamicin ribosomal target. However, new studies are needed to confirm this hypothesis"; The same part of chalcone 1 able to chelate the antibiotic gentamicin (it is no specificated by the authors, but it is supposed to be the aromatic ring and /or the ketone group) is present as well as in chalcone 2. The unique structural difference between chalcone 1 and 2 is the position of nitro group. The authors should revise this affirmation or support it with target assays/ specific references. 

Author Response

1) The experimental spectrum ATR-FTIR profile of chalcone 2 in figure 7: the peaks in the region between 1200-1000 cm-1 shows a low intensity appearing different from the experimental spectra of chalcone 1. Conversely, the signal of the hydroxyl group is more strong in the experimental spectrum of chalcone 2 and slightly pronounced in the profile of chalcone 1. This phenomenon is not explained in the text or it is difficult to capture; none correspondence of signal intensity (strong, medium, low etc.) is reported in Tables 3 and 4  next to the numerical data, even if their description in the corresponding legenda.

Answer: Tables 3 and 4 were corrected and transferred to the supplementary material. We also added the Raman and infrared intensities that were observed in the vibrational spectra of the chalcones 1 and 2. New sentences were added to the manuscript to answer the queries of this item. Below are given the explanations for these queries.

It is noted that the intensities of the most infrared bands in the region between 1200 cm^-1 and 1000 cm^-1 are more intense in the chalcone 1 than those observed in the chalcone 2. The differences in the infrared intensities are expected, since this spectral region is within the fingerprint region, which is unique to each one of those chalcones. As the chalcones 1 and 2 are polycrystalline, their molecules are periodically arranged in their crystals. It is known that the intermolecular interactions in molecular crystal depending of the how the packing of molecules are arranged within of its unit cell, and how the electronegativity affects the intermolecular forces [34, 35]. Since, the chalcone 2 has higher electronegativity than chalcone 1, it has intermolecular interactions more intense than the chalcone 1 (4.30 eV for chalcone 2 against 4.09 eV for chalcone 1). The values of the electronegativity (c) were calculated by the expression c = (I+A)/2, where I is the vertical ionization energy that is equal to minus the energy of the HOMO (I = - E_HOMO), and A is vertical electron affinity that is equal to minus the energy of the LUMO (A = - E_LUMO) [28]. Variations in the intermolecular interactions produce changes in the dipole moment during the vibration, and therefore, they affect the infrared bands. In crystals, the infrared bands tend to be more intense when the fingerprinting intermolecular interactions are weak [35]. For this reason, as the chalcone 2 has stronger fingerprinting intermolecular interactions than the chalcone 1, it is expected that the infrared bands have lower intensities when compared with those of the chalcone 1.

It is noteworthy that the stretching modes of the OH hydroxyl in the infrared spectra have lower intensity in the chalcone 2 than those observed in the chalcone 1. This effect is expected, since the intensity of the OH stretching band can increase when hydroxyl group is making hydrogen bond [36].

2) The authors have done an electrochemical characterization of compounds 1 and 2 without any correlation with the other experimental part of the work. Probably they performed these experiments to prove the possible mechanism of action of this class of compounds (ref 38 e 39), but any discussion is present in the work. The authors must integrate the electrochemical data in the paragraph related to antimicrobial activities (lines 323-329) with a fruitful discussion. On the other hand, the author can eliminate the electrochemical experiments and insert them in a future work, to support more targeted and specific biological tests.

Answer: Previous studies have reported that composite nitro substances, depending on the molecular structure, are potential therapeutic agents due to their mode of action involving the biotransformation of the nitro group, releasing intermediates in the redox process and causing changes in the stability of membrane structures of several microorganisms and cells [30]. This result is directly related to the results of the electrochemical experiments. The computational chemical calculations showed that chalcone 2 has a greater electrophilic character than chalcone 1 due to the higher value of the electrophilicity index. The data obtained by the cyclic voltammetry of chalcones reveal that chalcone 2 undergoes the oxidation process before chalcone 1, which indicates a greater electrophilic character and greater chemical reactivity.  The higher reactivity and electrophilicity of the synthetic chalcone 2, can be responsible for intermediated interactions between the nitro group and the bacterial cell wall and when combined with the gentamicin drug proved to be more efficient

3) The molecules are structural isomers, but compound 2 seems to be more active than compound 1 associated with gentamicin. Again the authors have not added any comment of this "strange behaviour". The authors affirmed that: "On the other hand, the addition of chalcone 1 to the growth medium resulted in an antagonistic action against both multidrug-resistant strains, increasing the MIC values of the Gentamicin from 16 to 20.15 µg mL-1 308 for S. aureus SA10 and from 32 to 256 µg mL-1 309 for E. coli EC06. The same part of chalcone 1 able to chelate the antibiotic gentamicin (it is no specificated by the authors, but it is supposed to be the aromatic ring and /or the ketone group) is present as well as in chalcone 2. The unique structural difference between chalcone 1 and 2 is the position of nitro group. The authors should revise this affirmation or support it with target assays/ specific references. 

Answer: The sentence “This antagonism could be explained by a possible chelating of Gentamicin by chalcone 1,reducing the entry of this antibiotic into the bacterial cell, where is located the Gentamicin ribosomal target. However, new studies are needed to confirm this hypothesis”   has been removed from the manuscript.

The sentence “This antagonism could be explained by a possible chelation of the Norfloxacin by synthetic chalcones tested, as already discussed for Gentamicin”  has been removed from the manuscript.

Reviewer 2 Report

Reviewed manuscript entitled “Natural acetophenone-derived isomeric chalcones: assessment of antibacterial potential following spectroscopic analysis” prepared by Priscila Teixeira da Silva et al. describe information about synthesis of two chalcones, their identification  and antimicrobial properties.

Reviewed manuscript is very poor prepare and need major revision.

Title

The title must be change. The main subject of this manuscript is identification of synthetized chalcones.

Introduction

Authors must delete abstract from the last part of introduction (lines 88-108). The goal of research is not clear.  Authors did not determine the toxicity of synthetized chalcones (line 86-88).

Results

Results are presented on 245 lines of manuscript (from line 110 to line 355). The antibacterial potential consist only of 65 lines. On 180 lines of paper, 4 tables and 7 figures Authors showed information about identification of synthetized chalcones. But in the title we read only about “antibacterial potential”. Authors ought to change the title of manuscript or correct the results. Additionally, synthetized chalcones are not natural but they were obtained by chemical synthesis.

Tables 3 and 4 ought to be transferred to Supplementary data.

The lack of discussion in all manuscript, especially in microbial part is confusing.

Material and methods

Lines 376-390 – What does show presented data? Are these results obtained in Authors experiment or from literature? Why is it presented in material chapter?

Chapters 3.2-3.6 – Add information what was analyzed or determined by presented methods.

Authors ought to add information how many replicates of synthesis and microbial analysis were performed.

Author Response

1) The title must be change. The main subject of this manuscript is identification of synthetized chalcones.

Answer: The title of the manuscript was modified to:

Structural, vibrational and electrochemical analysis and antibacterial potential of isomeric chalcones derived from natural acetophenone.

2) Introduction

2.1) Authors must delete abstract from the last part of introduction (lines 88-108). The goal of research is not clear.  

Answer: The last part of introduction (lines 88-108) has been removed and the goal of research was modified to make it clearer.

2.2.) Authors did not determine the toxicity of synthetized chalcones (line 86-88).

Answer: The text referring to toxicity (lines 86-88) has been removed

3) Results

3.1) Results are presented on 245 lines of manuscript (from line 110 to line 355). The antibacterial potential consist only of 65 lines. On 180 lines of paper, 4 tables and 7 figures Authors showed information about identification of synthetized chalcones. But in the title we read only about “antibacterial potential”. Authors ought to change the title of manuscript or correct the results. Additionally, synthetized chalcones are not natural but they were obtained by chemical synthesis.

Answer: The title of the manuscript was modified to Structural, vibrational and electrochemical analysis and antibacterial potential of isomeric chalcones derived from natural acetophenone.

3.2) Tables 3 and 4 ought to be transferred to Supplementary data.

Answer: Tables 3 and 4 were transferred to the supplementary material

3.3.) The lack of discussion in all manuscript, especially in microbial part is confusing.

Answer: The discussion of the results of the chalcones' antimicrobial activity it was modified and highlighted in yellow in the text.

4) Material and methods

4.1) Lines 376-390 – What does show presented data? Are these results obtained in Authors experiment or from literature? Why is it presented in material chapter?

Answer: The data presented are of structural characterization of the synthesized chalcones has been removed from the manuscript.

4.2) Chapters 3.2-3.6 – Add information what was analyzed or determined by presented methods.

Answer: The Information about what was analyzed by presented methods is present in the discussion of the results

4.3) Authors ought to add information how many replicates of synthesis and microbial analysis were performed.

Answer: The antibacterial assays were performed in triplicates, and results were expressed as an average of replicates. (The information about how many replicates of microbial analysis has been added in the manuscript).

The synthesis of chalcones was performed according to scheme 1.

Reviewer 3 Report

Research topic is important and has potential interesting results.

The antimicrobial testing section may be the most relevant in terms of the the applied part of the study-

It would important to describe the assay methods in details instead of referencing other studies 

May need in the the abstract to highlight whether the strains indicated are standard or drug resistant strains 

There is no discussion on the potential reasons  why the chalcones did not show any potentiating effect with the Ciprofloxacin and Cephalexin. What do the authors think about these results and what what are the possible mechanisms to describe the findings?Line 301-302

Results on the gentamicin MIC reduction for chalcone 2-include the values in the results example" reduced the MIC from 32ug/ml to 15ug/ul for both strains etc Line 302-304.This is mentioned in line 308 for increase in MIC for chalcone 1 

Line 313 figure B : there is a * on chalcone 1-what is the meaning?

There are strains of bacteria that were mentioned that are not described anywhere in the assay methods and results (ATCC E.coli 25922 and ATCC S.aureus 25923. Line 452-457

Some compounds and assays are also included in the results (EtBr, CPZ,CCCP) but are not mentioned anywhere in the methods or why the results are presented Line 351-354

Fig 10 A line 350: +CPZ-non signficant and the other CCCP -significant?

Antibiotic (Norfloxacin) results are discussed but not mentioned in abstract or introduction 

The conclusion mentions potential use of chalcones for prevention of infectious diseases(Line 50)-there is no basis for this potential use

Author Response

Research topic is important and has potential interesting results.

The antimicrobial testing section may be the most relevant in terms of the the applied part of the study-

It would important to describe the assay methods in details instead of referencing other studies 

Response: The assay methods were described in detail and referenced 

May need in the the abstract to highlight whether the strains indicated are standard or drug resistant strains 

Response:  It was highlighted in the abstracts that the strains used were drug resistant

There is no discussion on the potential reasons  why the chalcones did not show any potentiating effect with the Ciprofloxacin and Cephalexin. What do the authors think about these results and what what are the possible mechanisms to describe the findings?Line 301-302

Response: Dear reviewer, there are not a lonely reason for this, neither an mechanistical answer totally accepted for this. Two theories are the most interesting: the first one is based in the chelating possibility of the chalcones against the antibiotics, due the high number of hydroxil groups in these compounds; the other is possibility of the chalcones reduce the prooxidative effect of the antibiotics and doing this, reduce the effectivity of these drugs. Both putative mechanisms need more assays to identify what is occurring in the reaction, but due these assay be nos vaidated yet, we did not discussed in the text. I hope your comprehension.

Results on the gentamicin MIC reduction for chalcone 2-include the values in the results example" reduced the MIC from 32ug/ml to 15ug/ul for both strains etc Line 302-304.This is mentioned in line 308 for increase in MIC for chalcone 1 

Response: The sentence was corrected.

Line 313 figure B : there is a * on chalcone 1-what is the meaning?

Response: The * on chalcone 1 has been deleted, we are sorry for the mistake

There are strains of bacteria that were mentioned that are not described anywhere in the assay methods and results (ATCC E.coli 25922 and ATCC S.aureus 25923. Line 452-457

Response: The strains of bacteria that were not described anywhere have been deleted from methods and results, we are sorry for the mistake.

Some compounds and assays were also included in the results (EtBr, CPZ,CCCP) but were not mentioned anywhere in the methods or why the results are presented Line 351-354

Response: The compounds and assays about the results (EtBr, CPZ,CCCP) have been corrected in the methods.

Fig 10 A line 350: +CPZ-non signficant and the other CCCP -significant?

Response: The sentence was correct and referred to the different compounds Chlorpromazine (CPZ) and Carbonylcyanide m-chlorophenyl hydrazone (CCCP)

Antibiotic (Norfloxacin) results are discussed but not mentioned in abstract or introduction 

Response: The results with Antibiotic (Norfloxacin) have been added in abstract

The conclusion mentions potential use of chalcones for prevention of infectious diseases(Line 50)-there is no basis for this potential use

Response: The sentence has been deleted

Round 2

Reviewer 1 Report

The authors have proofed the manuscript following the comments and the highlighted suggestions. 

For the future works, I personally recommended the authors:

-to pay more attention to Structure-activity relationship-biological assays data discussion, and to avail of docking studies/medicinal chemist support.

-to not spread the experimental data in three different works published in the same years and related to the same topic. This "salami slicing" action belittles the work and it is self-defeating for your careers and scientific community.

Author Response

The authors have proofed the manuscript following the comments and the highlighted suggestions. 

For the future works, I personally recommended the authors:

-to pay more attention to Structure-activity relationship-biological assays data discussion, and to avail of docking studies/medicinal chemist support.

-to not spread the experimental data in three different works published in the same years and related to the same topic. This "salami slicing" action belittles the work and it is self-defeating for your careers and scientific community.

DEAR REVIEWER, THANKS FOR YOUR WORDS. HOWEVER, WE WOULD LIKE TO INFORM THAT THIS IS NOT A CASE OF “SALAMI SLICING”. OUR CHOICE TO SEPARATE THE RESULTS IS A MANNER TO MAKE EASIER THE DISCUSSION. WHEN WE WORK WITH DIFFERENT SYNTHETIC COMPOUNDS WITH NONE SIMILARITY BETWEEN THEIR SYNTHESIS PROTOCOLS AND THE STRUCTURE, THE DISCUSSION MUST BE MUCH MORE EXTENSE, BEING MUCH MORE CONFUSE TO COMPARE THE RESULTS. THIS WAS THE REASON FOR THIS SEPARATION, NOT TO MULTIPLICATE THE NUMBER OF PUBLISHED PAPERS. BUT WE ACKNOWLEDGE YOUR GRATEFUL IMPUT, THAT INDICATES YOUR PREOCUPATION WITH THE SCIENTIFIC ETHICS. THANKS AGAIN.

Reviewer 2 Report

All my suggestions were have neen corrected. I accept all answers.

Author Response

All my suggestions were have neen corrected. I accept all answers.

DEAR REVIEWER, THANKS FOR YOUR WORDS.

Reviewer 3 Report

It would have been better to reference in the revised document the Line numbers where the specific corrections were made. 

The edited part of the abstract is cut and paste from the results section. It is added to the conclusion section while it should( i think) be in the results section of the abstract. The abstract still has reference to chalcone "prevention"-Line 49-50

 

Figure 8 B still not corrected.There is still a * on the figure with no explanation.

The added methodology section is difficult to follow. Needs major English editing. Also the concentration of bacteria used is not indicated. Line 432-469

Author Response

The edited part of the abstract is cut and paste from the results section. It is added to the conclusion section while it should ( i think) be in the results section of the abstract. The abstract still has reference to chalcone "prevention"-Line 49-50

Response: The edited part was placed in the results section of the abstract (Line 47-52). The word "prevention" has been removed from the abstract (Line 52-54)

Figure 8 B still not corrected.There is still a * on the figure with no explanation.

 Response: (*) Statistically significant values (p < 0.05). The explanation was placed at the end of the legend in figure 8 (Line 302).

The added methodology section is difficult to follow. Needs major English editing. Also the concentration of bacteria used is not indicated. Line 432-469

Response: The English language of the added methodology section has been carefully checked and corrected (line 432-466). The concentration of bacteria used was added (Line 432-433)

Round 3

Reviewer 3 Report

Line 438 is incomplete

Line 462-463  you mean 96 well plate?

 

Author Response

Line 438 is incomplete 

Response:  The correction was made Line (438)

 

Line 462-463  you mean 96 well plate?

Response: The correction was made Line (461-462)

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The search of inhibitors of efflux pumps is a field of interest and the article might help in caracrherizing a new inhibitor. However, the article lacks clarity, both in the way it is written and in the information provided, and there are some doubts in the methodology. 

Specifica aspects.

• The article will benefits from an in depth editing because sometimes the messageis difficult to follow.
• The strains must be described in more detail. Maybe one of them overexpress MepA and the other one NorA, but without detailed information, the reasons why the authors state that this compound inhibits MepA remain obscure. To determine the specificity of efflux pump inhibitors their effect on mutants, either lacking either overexpressing this efflux pump must be compared with the effect on the susceptibility to antibiotics of the wild-type strain.
• If MICs have been determined by double dilutions, they cannot differ in less than twice: this is the error of the method. Hence difference in MICs of 40 to 30 (figure 5) or 200 to 175 (Figure 6) are misleading. If the authors want to analyze small differences, they can make the assay using small variations in concentrations or determine MICs by Etest.
• To determine that a given compound inhibits an efflux pump, an experiment showing the intracellular accumulation of a substrate of this efflux pump in  presence and in absence of the inhibitor an using strains that express or lack the efflux pump must be included. Otherwise, the authors only could claim that the compounds improves the activity of the antibiotic, but without knowing the mechanism involved.

 

Reviewer 2 Report

The topic being researched is of high interest in global fight against  antimicrobial resistance

This manuscript however has deficiencies that make it difficult to evaluate the reported findings and the significance in the current format.

The introduction section is very shallow and does not review much on the drugs, antibiotics and the strains used in the study. The materials and the methods are scantly written with no details on how the experiments were designed or carried or out and therefore how the results were derived. There is no explanation on why the specific drugs, strains of Staphylococcus aureus and the antibiotics were used, details on how they were used. The chalcone being evaluated is not mentioned anywhere on how it was used in the antibacterial, EPIs, MIC assays alongside the other drugs and antibiotics.

There are several terms/abbrev that are not explained (for example Mep A)

There is no meaningful discussion of the study findings although in absence of a clear introduction, methodology, results and introduction, probably it wouldn't be possible. 

Many statements in the results and discussion do not  seem to inform or have relevance in the study findings or are not fully explained. There is barely any discussion section.

These are just a few examples of statements not fully explained or are not clear what is being discussed:

line 263-267,  N-methylation and N-acetylation of what?

What is the meaning of statement in line 275

Line 283 NorA (Norfloxaxin?) or is it SA1199-B(NorA)?

In the abstract it is mentions standard and MDR S.aureus (which are the standard and which are MDR in the study)

Line 55-what is selectivity?

Line 140-why use MIC/8, is this a standard protocol(ref)?

Line 132-what is Epperndorf?

The manuscript needs rewriting in all the sections to make it clear.