New N-acyl Thiourea Derivatives: Synthesis, Standardized Quantification Method and In Vitro Evaluation of Potential Biological Activities

Round 1

Reviewer 1 Report

Title: “The Synthesis and in vitro Evaluation of Antimicrobial and Antioxidant Activity for a Series of New N-acyl Thiourea Derivatives” has been thoroughly reviewed.

There are many concerns/flaws and areas which should be improved before publishing it. I suggest major revision:

1.      Title: The title presented antioxidant activity as well beside antimicrobial activity of the synthesized thiourea derivatives but the author sticks to mentioning anti-biofilm activity in the abstract, in my opinion the title must be on the basis of key results.

2.      Abstract of the study is too lengthy; it should be concise according to the summarized key features of the manuscript.

3.      The abstract should be initiated with sentences that reflect the significances of thiourea or antimicrobial and antioxidants.

4.      The author had mentioned here the synthesis procedure in the manuscript which is not suit here, it should be removed.

5.      Line 33; put space before the word ”Considering”.

6.      The abstract is consisted mostly on methods section and we didn’t see any results of antioxidant or antimicrobial studies.

7.      Conclusion of abstract is missing.

8.      In my opinion the author should rewrite the abstract in a scientific way taking key features from the manuscript.

9.      Line 59; correct the spellings mistake, phisico-chemical.

10.   Introduction section is written in unscientific way and describe mostly methods of thiourea synthesis and too lengthy.

11.   The author should provide rationale for synthesizing these derivatives in the last para of introduction.

12.   The discussion section is written without mentioning any reference from previous literature study to support the current work, in my opinion discussion section should be re-written in scientific way on the basis of previous reported studies.

13.   Methods section lack references which looks someone own comments, please support methods with updated references.

14.   The central theme of the conclusion of this study is somewhat that it should be one the bases of key results and ends on a para for future perspectives.

15.   There are many typos and grammatical mistakes found that should be corrected.

16.   In my opinion, the author should revisit the whole manuscript carefully and addresses all the concerns.  

Author Response

 

REVIEWER 1

We thank the referee for the valuable comments to which we tried to respond and complete the article so that its scientific level increases.

1. Title: The title presented antioxidant activity as well beside antimicrobial activity of the synthesized thiourea derivatives but the author sticks to mentioning anti-biofilm activity in the abstract, in my opinion the title must be on the basis of key results.

The title has been reformulated to better reflect the most important features of the manuscript:

New N-acyl Thiourea Derivatives: Synthesis, Standardized Quantification Method and In vitro Evaluation of Potential Biological Activities.

 

2. Abstract of the study is too lengthy; it should be concise according to the summarized key features of the manuscript.

The abstract has been rewritten, considering the observations.

 

3. The abstract should be initiated with sentences that reflect the significances of thiourea or antimicrobial and antioxidants.

The abstract has been rewritten, considering the observations.

 

4. The author had mentioned here the synthesis procedure in the manuscript which is not suit here, it should be removed.

The abstract has been rewritten, considering the observations.

 

5. Line 33; put space before the word ”Considering”.

We thank the reviewer for pointing out the error. We have now adjusted it.

 

6. The abstract is consisted mostly on methods section and we didn’t see any results of antioxidant or antimicrobial studies.

The abstract has been rewritten, considering the observations.

 

7. Conclusion of abstract is missing.

The abstract has been rewritten, considering the observations.

 

8. In my opinion the author should rewrite the abstract in a scientific way taking key features from the manuscript.

The abstract has been rewritten, considering the observations.

 

9. Line 59; correct the spellings mistake, phisico-chemical.

The spelling mistake has been corrected.

 

10. Introduction section is written in unscientific way and describe mostly methods of thiourea synthesis and too lengthy.

The introduction has been rewritten and completed as suggested.

 

11. The author should provide rationale for synthesizing these derivatives in the last para of introduction.

To continue our efforts for the development of novel antimicrobial agents as bioactive lead candidates, we designed and synthesized thiourea derivatives carrying a heterocyclic nucleus (1a – 1g).

Our strategy to design and synthesize some novel prototypes has been based on bringing together two pharmacophores, i.e., thiourea and a heterocyclic ring (thiazole, benzothiazole, pyridine and pyrimidine) in a single molecular backbone. 

 The benzo[d]thiazole- based N-acyl thiourea derivatives (1a – 1g) were designed and synthesized according to a previously published method [Limban, C.; Missir, A.V.; Chiriţă, I.C.; Bădiceanu, C.D.; Drăghici, C.; Balotescu, M.C.; Stamatoiu, O. New thioureides of 2-(4-methyl-phenoxymethyl)-benzoic and 2-(4-methoxy-phenoxymethyl)-benzoic acids with biological activity. Rev Roum Chim 2008, 53(8), 595 – 602], with punctual alterations, characterized by in silico and experimental approaches and evaluated for their biological properties. Furthermore, a routine quality control method for quantitative evaluation of the most promising compound (1d) has been developed and validated by reversed-phase high-performance liquid chromatography (RP- HPLC).

 

The rationale is inserted in the last paragraph of the introduction.

 

12. The discussion section is written without mentioning any reference from previous literature study to support the current work, in my opinion discussion section should be re-written in scientific way on the basis of previous reported studies.

In the present research paper, we continue the computational studies performed in our previous publication [Roman, R.; Pintilie, L.; Nuță, D.; Limban, C. A QSAR Study on Thiourea Derivatives – New Approaches in Drug Development, Farmacia, 2022, 70, 2, 228 – 240. https://doi.org/10.31925/farmacia.2022.2.7]. A series of molecular descriptors was generated based on the designed molecules, followed by the molecular docking studies, where the ligands (chemicals 1a – 1g, referred in the mentioned source as A.01 – A.07) established hydrogen bonds with the amino acids moieties of Staphylococcus aureus DNA gyrase, and Escherichia coli DNA gyrase. Similarities between evaluated ligands and implied co-crystallized were also identified.

Overall, the molecular properties linked with potential in vivo influence (determined by using Spartan 14 software), namely the predictions of substances permeability and toxicity, and detection of binding strength obtained with the aid of CLC Drug Discovery Workbench, provided means to explore the affinity of the designed molecules to the selected proteins.

 

We agree and have rewritten and expanded this section to clarify our statements based on previous studies.

 

13. Methods section lack references which looks someone own comments, please support methods with updated references.

The methods section has been completed with references that support the analyses.

 

14. The central theme of the conclusion of this study is somewhat that it should be one the bases of key results and ends on a para for future perspectives.

The conclusion paragraph has been reformulated.

 

15. There are many typos and grammatical mistakes found that should be corrected.

In response to the reviewer's comments, we have had this article edited by experienced scholarly writers.

 

16. In my opinion, the author should revisit the whole manuscript carefully and addresses all the concerns. 

We appreciate the constructive review. We hope that we have addressed all the concerns in the revised section.

Reviewer 2 Report

The presented publication concerns the synthesis, antimicrobial and antioxidant activities for a series of new N-acyl thiourea derivatives.

The publication is interesting, it is written completely correctly.

However, the biological (microbiological) activity of the final compounds are weak. Noteworthy is the fact that the compound 1d in the study showed antioxidant activity (43%).

In the experimental part (spectra data) please bold the numbers of the compounds.

I think that the Authors should synthesize a few more new products that could show better microbiological activity.

Author Response

REVIEWER 2

We would like to thank the referee for carefully reading our manuscript and for giving such constructive comments.

The presented publication concerns the synthesis, antimicrobial and antioxidant activities for a series of new N-acyl thiourea derivatives.

The publication is interesting, it is written completely correctly.

However, the biological (microbiological) activity of the final compounds are weak. Noteworthy is the fact that the compound 1d in the study showed antioxidant activity (43%).

1. In the experimental part (spectra data) please bold the numbers of the compounds.

We adjusted the text as suggested.

2. I think that the Authors should synthesize a few more new products that could show better microbiological activity.

We thank Reviewer 2 for this suggestion. We do sincerely appreciate your guidance. The present research is part of a PhD thesis in progress. We developed another synthetic strategy involving two scaffolds, namely thiourea and pyridine moieties, structures chemically derived from 2-((4-ethylphenoxy) methyl)- N-(heteroarylcarbamothioyl)benzamides. The prediction of the drug-like, absorption, distribution, metabolism, and elimination (ADME) profiles of the studied compounds was discussed in a recently published study [Roman, R.; Pintilie, L.; Nuță, D.; Avram, S.; Buiu, C.; Sogor, C.; Limban, C. In Silico Prediction, Characterization and Molecular Docking Studies on New Benzamide Derivatives. Processes 2023, 11, 479. https://doi.org/10.3390/pr11020479].

The work covers a library of 15 compounds evaluated by the determination of a series of molecular descriptors, interconnected to elucidate and predict their behavior within the human body based on the chemical structure of the scaffold. As the docking scores have suggested a potential activity towards investigated microbial strains (S. aureus and E. coli), the synthesis and evaluation of their antimicrobial properties are underway.

Reviewer 3 Report

Roman et. al., have synthesized few compounds and shown their antimicrobial and antibiofilm

effects. I have few suggestions to strengthen the work.

 

1.     I would suggest showing percentage biofilm inhibition (bar diagram) following compound 1b & 1d treatment. 

2.     Please include the image of biofilm inhibition with or without treatment and quantify the total protein concentration.

3.     It would be good if you can show the zone of inhibition with or without treatment (compound 1b & 1d).

4.     It is very important to measure the cytotoxicity of those compounds against mammalian cells as they have very high MIC concentrations.

Author Response

REVIEWER 3

We thank Reviewer 3 for carefully reading our manuscript and for giving such constructive comments. We have addressed each of them as follows.

1. I would suggest showing percentage biofilm inhibition (bar diagram) following compound 1b & 1d treatment. 

Our microtiter assay allowed us to establish the minimum biofilm inhibitory concentration, based on the value of absorbance measured at 490 nm.

2. Please include the image of biofilm inhibition with or without treatment and quantify the total protein concentration.

We thank the referee for this suggestion. The present research is part of a PhD thesis in progress. Unfortunately, we did not take pictures under a microscope of the 96 microplate wells used for performing the MBIC assays, as it was not a similar requirement in our previous tests. Also, we do not have the possibility to measure the protein content.

We understand the rationale behind including images, and we will consider this requirement in our subsequent studies. Unfortunately, we cannot afford to retake the tests at the moment, as the procedures’ costs are pretty high for our budget.

3. It would be good if you can show the zone of inhibition with or without treatment (compound 1b & 1d).

We have chosen to use the more standardized MIC assay instead of the agar diffusion assay, which is only qualitative and non-standardized for poorly water-soluble compounds.

4. It is very important to measure the cytotoxicity of those compounds against mammalian cells as they have very high MIC concentrations.

We thank Reviewer 3 for this valuable suggestion. We acknowledge the significance of the measurement. This limitation has been addressed in the perspectives section.

Again, we do sincerely appreciate your guidance.

Round 2

Reviewer 1 Report

The manuscript is improved. However needs final check for grammar and spelling mistakes.

Author Response

Thank you for your comments! The manuscript has been rewritten!

Reviewer 2 Report

The publication presented for review after the corrections and additions explains the reason for the planned syntheses of the final compounds undertaken by the Authors. Molecular docking assumed the occurrence of microbial activity of compounds. In practice, however, they are weak.

Understanding the cause of poor activity of the compounds, despite a positive explanation of the molecular docking of these compounds, will be very important in planning the synthesis of further structures with a similar structure.

Author Response

Thank you for your comments and suggestions!

 

Reviewer 3 Report

Authors did not address any of my comments. Authors have limitations to address those questions I raised. But to support the antimicrobial statement and strengthen the biological part I expect minimum experiments to be done. Even authors were unable to address the cytoxicty issue which was my major concern.

Therefore, I do not recommend the work for publication.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf