Anticancer Studies of Newly Synthesized Thiazole Derivatives: Synthesis, Characterization, Biological Activity, and Molecular Docking

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript, entitled “ Anticancer studies of newly synthesized thiazole derivatives: 2 Synthesis, Characterization, Biological activity, and Molecular  docking”deals with the synthesis  and anticancer, as well molecular docking studies of synthesized compounds.
The manuscript is written casually.

1.       Introduction is confusing and needs to be reorganized and enlarged.

2.       Discussion is poor.

3.       There is no structure-activity relationships

4.       Molecular docking was performed on 4 proteins but there is no conclusion what was the target for these compound.

5.       No discussion at all on molecular docking regarding binding with amino acids and comparison of activity between compounds tested.

6.       Authors in the scheme mentioned compound 6, but did not evaluate it. What for they synthesized it and why it was not tested?

7.       References should be with abbreviations.

Page 1. Line 24. Should be:  synthesized via using of thiosemicarbazones, or by using thiosemicarbazones,

Page 1. Line 30. Should be: a standard drug, with IC5₀  6.77±0.41
Page 1. Lines 33,35. Should be: compound 4c
Page 1. Line 34. Should be: phase

Page 2. Line 56. Should be: An effective

Page 2. Line 62. The following sentence needs revision . It is not completed:” Providing inhibitors for EGFR 62 signaling help in the treatment of cancer [6,7].

Page 2. Line 75. Should be: 2-substituted

Page 99. Should be: 4-hydroxy, hydrazinyl

Page 3. Line 108. Should be: [5H] and in other places of the manuscript.

Page 3. Line 109. Should be: 4-hyroxybenzaldhyde

Page 3. Line 110. Should be: thiosemicarbazone

Page 5. Line 142. Authors wrote :” while the proton of aromaticics?

Page 5. Line 145. Should be : due to acetyl group

Page 5. Line 145: Should be: -4-hyroxybenzaldehyde and across all manuscript.

Page 5. Line 155. Should be: anti-proliferative activity

Page 5. Lines 154-156. The following  sentence  needs revision . It is not clear at all.;’ Our findings revealed that all synthesized 154 thiazole derivatives (4a-c) and 5 exhibited anti-proliferation activity of MCF-7 and/or 155 HepG2 cells compared to the standard drug because of thiazole moiety in their structure 156 which contains sulfur and nitrogen mentioned in the introduction part.”

Page 5. Line 160. Should be: 4a-c) and 5 as shown in Table 1.

Page 5. Line 170. Authors wrote : toxic activity as shown in (Scheme 1 and Table 1). What is connection of scheme 1  with results on cytotoxicity?

Page 6. Line 190. Should be: of compound 4c propidium iodide

Page 8. Line 226.Should be: a molecular

Page 20. Compound 5. In NMR spectra authors gave 7 protons, while compound has 11

Page 21. Line 443. Should be: hydrazine]-thiazol- -4(5H) – ones

Page 21. Line 445. Should be:chloroacetyl

Page 21. Line 451. Should be: cell cycle

Comments on the Quality of English Language

Language should be improved.

Author Response

Reviewer 1:

Response: We are most grateful to the reviewer for the constructive evaluations rendered to our manuscript. We have addressed all your concerns and are convinced that these suggestions have helped to improve the quality of the manuscript. Our point-by-point responses are listed below. The major changes have been highlighted in the revised manuscript.

1. Introduction is confusing and needs to be reorganized and enlarged.

Response: We thank the reviewer for his valuable comment. We agree with the reviewer that the introduction is short, but in fact it covers and explains our analysis and measurements. At the beginning of the introduction, we talk about cancer as a fatal disease and about different types of proteins that help cancer cells survive and grow. We also went on to explain the biological importance of the thiazole derivative towards various diseases such as cancer. The introductory part seems short but useful and fruitful.

2. Discussion is poor.

Response: We thank the reviewer for his/her valuable comment. In fact, the discussion part covers in detail the interpretation of our results.

For example, the chemistry part of synthesized compounds covers the IR and NMR spectra analyses of synthesized compounds in detail. The evaluation of biological activity part covers the In vitro cytotoxic activity against breast cancer and liver cell lines (MCF-7 and HepG2); respectively that exhibited anti-proliferative activity of MCF-7 and/or HepG2 cells based on different substitutions and replacements on  2-(4-hydroxybenzylidene) hydrazinyl which bond with 2-position of thiazole ring  (Scheme 1). Also, Cell cycle assessment showed that Compound 4c (Most active compound)  induces cell cycle arrest at the G1/S phase by the increase of cell percentage and accumulation of MCF-7 cells at the pre-G1 peak from 2.02% to 37.36%. this in turn reflects the ability of compound 4c to delay and stop the progression and growth of cancer cells. The same findings were in detail interpreted and clarified in apoptosis, programmed cell death, and in silico, molecular docking part.

 

3. There is no structure-activity relationships.

Response: We thank the reviewer for his comment and suggestion regarding the molecular docking study. We believe that molecular docking analysis is a powerful computational analysis that could be applied to explore the binding affinity of bioactive compounds toward a targeted protein. This computational tool has succeeded in myriad studies to discover novel and potent pharmacological inhibitors and to explore the mode of action of certain bioactive agents. In the current study, we have applied molecular docking analysis to explore whether the anti-cancer activity of this class of compounds against cancer could be attributed to their ability to inhibit target proteins that have vital role in growth, proliferation and cell cycle of cancer cells, especially, breast (MCF-7) and liver cancer (HepG2). molecular docking analyses were conducted with four key proteins [Aromatase, epidermal growth factor receptor (EFGR), Cyclin-dependent kinase 2 (CDK2), and B-cell lymphoma 2 (Bcl-2)] that stimulate growth, proliferation, and development of cancer cells. aromatase protein. As previously shown, upregulation of estrogen receptors has been implicated in breast cancer and inhibition of estrogen production by aromatase inhibitors has been reported to efficiently suppress the proliferation of breast cancer. Therefore, examining the binding affinity of our compounds toward aromatase and other kinds of proteins would provide a piece of beneficial information that could be followed up in future studies.  

 

4. Molecular docking was performed on 4 proteins but there is no conclusion what was the target for these compound.

Response: We thank the reviewer for his comment and suggestion regarding the molecular docking study

We have performed a molecular docking study for the most active compounds (4a-c and 5) toward a broad range of target proteins that help cancer survive and grow, especially breast and liver cancer that included in our study, the [Aromatase, epidermal growth factor receptor (EFGR), Cyclin-dependent kinase 2 (CDK2), and B-cell lymphoma 2 (Bcl-2)]. The target for these compounds (4a-c and 5) to   exhibit good docking scores that reflect a promising and potential binding affinity toward the active site of selected docked proteins, binding site aiming at affirming the in vitro inhibitory activity of these compounds and exploring the binding mode/affinity of these compounds toward the targeted protein. The data for molecular docking study toward target proteins were summarized in tables (3-6) and figures (4-7). in tables (3-6) the higher negative value of docking score reflect the higher affinity of these compounds toward the key amino acids of active site of target proteins (i.e, CYS 20, SER 2, GLY 5 and ASN 1) and the type of binding (H-donor , H-acceptor and pi-H). Moreover, RMSD value reflect the resolution of binding. RMSD value around 1.5 and not more than 3 mean good docking of synthesized compounds toward docked proteins. Furthermore figures (4-7) are 2D and 3D shows the interaction of (4a-c and 5) with the active site of docked proteins and the type of bond with the key amino acids. All docking results were interpreted and clarified in results and discussion part.

 

5. No discussion at all on molecular docking regarding binding with amino acids and comparison of activity between compounds tested.

Response: We thank the reviewer for his comment and suggestion regarding the molecular docking study. As mentioned in response 3 and 4 about molecular docking, tables (3-6) and figures (4-6) contain all docked data compared to our previous published papers and other published articles.

Mourad, A. A., Rizzk, Y. W., Zaki, I., Mohammed, F. Z., & El Behery, M. (2021). Synthesis and cytotoxicity 693 screening of some synthesized hybrid nitrogen molecules as anticancer agents. Journal of Molecular 694 Structure, 1242, 130722. https://doi.org/10.1016/j.molstruc.2021.130722. 

 Abu Almaaty, A. H., Elgrahy, N. A., Fayad, E., Abu Ali, O. A., Mahdy, A. R., Barakat, L. A., & El Behery, M. (2021). Design, synthesis and anticancer evaluation of substituted cinnamic acid bearing 2-quinolone 697 hybrid derivatives. Molecules, 26(16), 4724. http://doi.org/10.3390/molecules26164724.

 

 

6. Authors in the scheme mentioned compound 6, but did not evaluate it. What for they synthesized it and why it was not tested?

Response: We thought that compound 6 not active based of molecular docking studies (data not shown in the manuscript) so, we did not conduct further biological measurements on compound 6

7. References should be with abbreviations.

Response: References were modified in the revised manuscript according to the MDPI guidance

We really thank the reviewer for his/her constructive evaluation of our manuscript. We really thank the reviewer for highlighting these critical corrections. We totally agree with the respected reviewer, and we apologize for these mistakes in our manuscript. We have corrected and clarified these mistakes in the revised manuscript as follow.

Responses:

Page 1. Line 24. Should be:  synthesized via using of thiosemicarbazones, or by using thiosemicarbazones, correction=  synthesized via using of thiosemicarbazones

Page 1. Line 30. Should be: a standard drug, with IC5₀  6.77±0.41 Correction =  with IC₅₀=6.77±0.41
Page 1. Lines 33,35. Should be: compound 4c   Correction =  compound 4c
Page 1. Line 34. Should be: phase  Correction =  Phase

Page 2. Line 56. Should be: An effective      correction = An effective

Page 2. Line 62. The following sentence needs revision. It is not completed:” Providing inhibitors for EGFR signaling help in the treatment of cancer [6,7].

Response: We thought the sentence Providing inhibitors for EGFR signaling help in the treatment of cancer [6,7] is correct where cancer cells produce large amount of EGFR. Cancer cells use EGFR for survival and growth. Design inhibitors for EGFR in cancer cells help in cancer treatment.

Page 2. Line 75. Should be: 2-substituted correction =  2-substituted

Page 99. Should be: 4-hydroxy, hydrazinyl    correction = 4-hydroxy, hydrazinyl   

Page 3. Line 108. Should be: [5H] and in other places of the manuscript. Correction = 5H

Page 3. Line 109. Should be: 4-hyroxybenzaldhyde  correction = 4-hyroxybenzaldhyde  

Page 3. Line 110. Should be: thiosemicarbazone  correction = thiosemicarbazone

Page 5. Line 142. Authors wrote :” while the proton of aromaticics?

Correction = proton of aromatic was observed at δ7.24 and 7.81 ppm as doublet signals

Page 5. Line 145. Should be : due to acetyl group correction = due to acetyl group

Page 5. Line 145: Should be: -4-hyroxybenzaldehyde and across all manuscript. Correction = 4-hyroxybenzaldehyde

Page 5. Line 155. Should be: anti-proliferative activity correction = anti-proliferative activity

Page 5. Lines 154-156. The following  sentence  needs revision . It is not clear at all.;’ Our findings revealed that all synthesized  thiazole derivatives (4a-c) and 5 exhibited anti-proliferation activity of MCF-7 and/or HepG2 cells compared to the standard drug because of thiazole moiety in their structure which contains sulfur and nitrogen mentioned in the introduction part.”

Response: In fact, all synthesized compounds were thiazole derivatives that contain thiazole ring with nitrogen (N) and sulfur (S) inside the ring and oxygen (O) connected to the ring with the rest of molecule structure. based on molecular docking studies as shown in tables (3-6). Most of binding and linkage between these compounds (Ligand) and docked proteins (key amino acids, receptors) were between N, S and O atoms from thiazole ring as shown in tables (3-6), (i.e, S 22  N 13 and O 18). Also, other interactions were attributed to phenyl ring and nitrogen atoms connected between rings of molecules.

Page 5. Line 160. Should be: 4a-c) and 5 as shown in Table 1. Correction (4a-c) and 5 as shown in Table 1

Page 5. Line 170. Authors wrote : toxic activity as shown in (Scheme 1 and Table 1). What is connection of scheme 1  with results on cytotoxicity?

Response: Correction has been made as follow, compounds 4b with bromide substitution and compound 5 with replacement of hydroxyl on 2-(4-hydroxybenzylidene) hydrazinyl (Scheme 1)demonstrated a moderated cytotoxic activity as shown in (Table 1).

Scheme 1 show the structure of compound 4b and 5

Compound 4b

 

 

 

 

 

 

 

 

 

Compounds 4b with bromide substitution and compound 5 with replacement of hydroxyl on 2-(4-hydroxybenzylidene) hydrazinyl,  as shown in scheme1 demonstrated a  moderated cytotoxic activity compared to other derivatives (4a and 4c) as shown in (Table 1).

Page 6. Line 190. Should be: of compound 4c propidium iodide correction = of compound 4c. Propidium iodide

Page 8. Line 226.Should be: a molecular correction = a molecular

Page 20. Compound 5. In NMR spectra authors gave 7 protons, while compound has 11

Response: we apologize for this mistake in our manuscript. We have corrected and clarified this mistake and added the missing data in the revised manuscript as follows

 

¹H-NMR (DMSO-d6): δ 2.30 (S, 3H, COCH3), 3.91 (S , 2H, CH₂ of thiazole ring), 414 7.236 - 7.256(d,2H, J= 8.00 HZ , Ar-H) , 7.802 - 7.821 (d,2H, J= 8.00HZ, Ar-H), 415 8.428 (S,1H,CH=N),11.99 (br-S, 1H, NH) ppm.

Page 21. Line 443. Should be: hydrazine]-thiazol- -4(5H) – ones

Correction has been made

Page 21. Line 445. Should be:chloroacetyl Correction has been made

Page 21. Line 451. Should be: cell cycle Correction has been made

 

We really thank the reviewer for highlighting these critical points, We have corrected and clarified these mistakes in the revised manuscript (highlighting)

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors the manuscript quoted crystals-2623279 and entitled "Anticancer studies of newly synthesized thiazole derivatives: Synthesis, Characterization, Biological activity, and Molecular docking" describes the synthesis of thiazole derivatives as promising anticancer agents. The paper is interesting but some points must be addressed before publication: -the quality of structures in figure 1 is not good for publication. please redraw figures. -the molecular target must be experimentally validated. -the non toxic profile in normal cells must be ascertained. 

Author Response

Reviewer 2:

the manuscript quoted crystals-2623279 and entitled "Anticancer studies of newly synthesized thiazole derivatives: Synthesis, Characterization, Biological activity, and Molecular docking" describes the synthesis of thiazole derivatives as promising anticancer agents. The paper is interesting, but some points must be addressed before publication: -the quality of structures in figure 1 is not good for publication. please redraw figures. -the molecular target must be experimentally validated. -the non toxic profile in normal cells must be ascertained. 

Response: We are most grateful to the reviewer for the constructive evaluations rendered to our manuscript. We have addressed all your concerns and are convinced that these suggestions have helped to improve the quality of the manuscript.

Scheme 1 was redrawn again with high quality. Moreover, the molecular target of synthesized compounds (4a-c) and 5 were interpreted in detail in the discussion part and molecular docking studies. Unfortunately, the present study was done using in vitro cancer cell lines similar to our previous publications and other publications.

1. Al-Warhi, T.; Abu Ali, O.A.; Alqahtani, L.S.; Abo-Elabass, E.; El Behery, M.; El-Baky, A.E.A.; Samir A. Zaki, M.; Fayad, E.; Radwan, E.M. Novel 3-Substituted 8-Methoxycoumarin Derivatives as Anti-Breast Cancer Drugs. Crystals 2023, 13, 1037.
2. A.H. Abu Almaaty, N.A. Elgrahy, E. Fayad, O.A. Abu Ali, A.R.E. Mahdy, L.A.A. Barakat and M. El Behery “Design, Synthesis and Anticancer Evaluation of Substituted Cinnamic Acid Bearing 2-Quinolone Hybrid Derivatives,” molecules, vol. 26, no. 16, pp. 1–14, 2021, doi: 10.3390/molecules26164724.

In fact, in the present study we did not conduct experiments using in vitro normal cell lines (not available now) however, in our previous publications we tested the synthesized compounds toward cancer cell and normal cell lines.

1.Salem, Manar G., Dina M. Abu El-Maaty, Yassmina I. Mohey El-Deen, Basem H. Elesawy, Ahmad El Askary, Asmaa Saleh, Essa M. Saied, and M. El Behery. 2022. "Novel 1,3-Thiazole Analogues with Potent Activity against Breast Cancer: A Design, Synthesis, In Vitro, and In Silico Study" Molecules 27, no. 15: 4898.

2. A. E. Mourad, Y. W. Rizzk, I. Zaki, F. Z. Mohammed, and M. El Behery, “Synthesis and cytotoxicity screening of some synthesized hybrid nitrogen molecules as anticancer agents,” J. Mol. Struct., vol. 1242, p. 130722, 2021, doi: 10.1016/j.molstruc.2021.130722.
3. F. Z. Mohammed, W. Rizzk, M. El, A. A. E. Mourad, and M. El Behery, “Design, Synthesis, Cytotoxic Screening and Molecular Docking Studies of Novel Hybrid Thiosemicarbazone Derivatives as Anticancer Agents,” Chem. Biodivers., vol. 18, no.12, pp. 1–18, 2021, doi: 10.1002/cbdv.202100580.

Moreover, we continue to perform in vivo studies (experimental animals) using the most active compound 4c to test its ability to decrease the volume and count of tumor-bearing mice. Also in vivo studies using compound 4c were done (data unpublished). In in vivo studies the tested compound injected to a group of mice (normal group does not contain tumor cells) to determine the side effect of target compounds. Furthermore other biochemical and histological analyses were conducted

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

There is many design, drafting and techno-editing mistakes. I am not familiar with the English language, but I realized that the English used is not appropriate.

Compound codes must be in bold.

Chemical names start with a capital letter.

The acetyl rest is CH₃CO. The OCOCH₃ rest is acetate or acetyloxy.

There are many other chemistry mistakes, mistakes that I highlighted in the manuscript.

Compound 5 in scheme 1 is wrong. The conditions for obtaining compound 6 are missing from the legend.

How do the authors argue the decision to synthesize compound 6 and why was it not subjected to biological studies?

In the spectral characterization of the compounds, the authors mention two isomers. Which isomers are the authors referring to?

IR, NMR and MS spectral data must be provided for all compounds.

Acronyms must be explained in the text the first time they appear.

The RMSD and Ligand lines in the tables must be explained.

 

The presentation of the chemical synthesis part is inadequate, in an unacceptable form.

Comments for author File: Comments.pdf

Author Response

 

Reviewer 3

 

There is many design, drafting and techno-editing mistakes. I am not familiar with the English language, but I realized that the English used is not appropriate.

Compound codes must be in bold.

Chemical names start with a capital letter.

The acetyl rest is CH₃CO. The OCOCH₃ rest is acetate or acetyloxy.

There are many other chemistry mistakes, mistakes that I highlighted in the manuscript.

Compound 5 in scheme 1 is wrong. The conditions for obtaining compound 6 are missing from the legend.

How do the authors argue the decision to synthesize compound 6 and why was it not subjected to biological studies?

In the spectral characterization of the compounds, the authors mention two isomers. Which isomers are the authors referring to?

IR, NMR and MS spectral data must be provided for all compounds.

Acronyms must be explained in the text the first time they appear.

The RMSD and Ligand lines in the tables must be explained.

 

Response: We are most grateful to the reviewer for the constructive evaluations rendered to our manuscript. We have addressed all your concerns and are convinced that these suggestions have helped to improve the quality of the manuscript.

We really thank the reviewer for highlighting these critical points, we have corrected and clarified these mistakes in the revised manuscript (highlighting). Our point-by-point responses are listed below.

 

1. There is many design, drafting and techno-editing mistakes. I am not familiar with the English language, but I realized that the English used is not appropriate.

the English language was revised

 

1. Compound codes must be in bold. (the correction has been made)

• Chemical names start with a capital letter. (the correction has been made)

The acetyl rest is CH₃CO. The OCOCH₃ rest is acetate or acetyloxy. (the correction has been made)

 

1. There are many other chemistry mistakes, mistakes that I highlighted in the manuscript. (the correction has been made)

 

1. Compound 5in scheme 1 is wrong. The conditions for obtaining compound 6 are missing from the legend.

Response: scheme is correct the redraw has been made

1. How do the authors argue the decision to synthesize compound 6and why was it not subjected to biological studies?

Response: Based on molecular docking studies, compound 6 was inactive (data not shown)

• In the spectral characterization of the compounds, the authors mention two isomers. Which isomers are the authors referring to?

Response: some compounds when dissolved in DMSO lead to the formation of Tautomers and stereoisomers. These isomers cannot be isolated as published before in our previous papers.

 

 Salem, Manar G., Dina M. Abu El-Maaty, Yassmina I. Mohey El-Deen, Basem H. Elesawy, Ahmad El Askary, Asmaa Saleh, Essa M. Saied, and M. El Behery. 2022. "Novel 1,3-Thiazole Analogues with Potent Activity against Breast Cancer: A Design, Synthesis, In Vitro, and In Silico Study" Molecules 27, no. 15: 4898.

 

1. El Behery, Ibrahim M. El-Deen, Manar A. El-Zend, Lamiaa A.A. Barakat,

Design, synthesis and evaluation the bioactivities of novel 8‑methoxy-1-azacoumarin-3-carboxamide derivatives as anti-diabetic agents, Journal of Molecular Structure, Volume 1294, Part 2, 2023, 136486. doi.org/10.1016/j.molstruc.2023.136486.

 

• IR, NMR and MS spectral data must be provided for all compounds.

Response: IR, NMR for all compounds were measured and included in the revised version except IR analyses of compounds 4b and 4c data were lost after measurement (technical issue). Author depends on IR and NMR spectra to confirm and interpret the structure of synthesized compounds.

. Also, mass spectrum has not been measured in the present study at the present time due to difficulty in doing so, and there is no problem in publishing using IR and NMR spectra, as happened in previous publications by us and others.

 

Previous publication with Mass spectra when available to do.

 Salem, Manar G., Dina M. Abu El-Maaty, Yassmina I. Mohey El-Deen, Basem H. Elesawy, Ahmad El Askary, Asmaa Saleh, Essa M. Saied, and M. El Behery. 2022. "Novel 1,3-Thiazole Analogues with Potent Activity against Breast Cancer: A Design, Synthesis, In Vitro, and In Silico Study" Molecules 27, no. 15: 4898.

 

Previous publication without Mass spectra when not available to do.

 

 A.H. Abu Almaaty, N.A. Elgrahy, E. Fayad, O.A. Abu Ali, A.R.E. Mahdy, L.A.A. Barakat and M. El Behery “Design, Synthesis and Anticancer Evaluation of Substituted Cinnamic Acid Bearing 2-Quinolone Hybrid Derivatives,” molecules, vol. 26, no. 16, pp. 1–14, 2021, doi: 10.3390/molecules26164724.

 

1. El Behery, Ibrahim M. El-Deen, Manar A. El-Zend, Lamiaa A.A. Barakat,

Design, synthesis and evaluation the bioactivities of novel 8‑methoxy-1-azacoumarin-3-carboxamide derivatives as anti-diabetic agents, Journal of Molecular Structure, Volume 1294, Part 2, 2023, 136486. doi.org/10.1016/j.molstruc.2023.136486.

 

1. Acronyms must be explained in the text the first time they appear.

(the correction has been made)

 

1. The RMSD and Ligand lines in the tables must be explained.

(the correction has been made)

This is the atom number in the compound (4a-c) and 5. The moe program uses a numbering system to identify which  atom in the compound forms bond with the docked protein

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript, entitled “ Anticancer studies of newly synthesized thiazole derivatives: 2 Synthesis, Characterization, Biological activity, and Molecular  docking”deals with the synthesis  and anticancer, as well molecular docking studies of synthesized compounds after revision almost did not improved..
1.Introduction is still needs to be reorganized and enlarged.

2.Discussion is poor.

3.There is no structure-activity relationships

4.Molecular docking was performed on 4 proteins but there is no conclusion what was the target for these compound.

5.No discussion at all on molecular docking regarding binding with amino acids and comparison of activity between compounds tested.

6.Authors in the scheme mentioned compound 6, but did not evaluate it. What for they synthesized it and why it was not tested?

7.Authors should take care and correct NMR spectra. For example 4b has 8 protons authors mentioned 6.

7.References should be with abbreviations. Authors did nothing , ignored almost all comments and corrected some mistakes, but did another .

All these points were mentioned in my previous review.

According to the answers it seems that authors did  not understand what I am asking about and answer what is convenient for them.

Page 2. Line 81.Should be: thiazole-4[5H]

Page 3. Line 108. Should be: [5H] and in other places of the manuscript.

Page 3,4. Line 115,142.Should be: 2-[2-[4-hydoroxy -

Page 5. Line 175. Should be: Consequently, compound

Page 5. Line 186. The following sentence needs revision: As a result, to affirm the anticancer activity, and was selected for further 187 analyses including cell cycle arrest, the vascular endothelial growth factor receptor-2 (VEGFR-2) kinase assay and apoptosis assay

Page 6. Line  205. Should be: of compound 4c propidium iodide

Page 19.Line 333. Should be: 7.01-7.89 (m ,

Page 21.Line 388. Authors wrote: 3.94 (S, CH2 of Thiazole), 3. 91, 3.94 (S, CH2 of Thiazole ring of two isomer).The number of protons should be mentioned.

 

Comments on the Quality of English Language

.

 

Author Response

Reviewer 1:# Round 2

We really thank the reviewer for highlighting these critical points, we have corrected and clarified these mistakes in the revised manuscript (highlighting). Our point-by-point responses are listed below.

1. The introduction still needs to be reorganized and enlarged.

Response: We thank the reviewer for his/her valuable comment. The introduction was enlarged and organized.

2. 2.       Discussion is poor.

Response: We thank the reviewer for his/her valuable comment. The discussion part was modified in detail in the revised manuscript.

3. There is no structure-activity relationship.

Response: We thank the reviewer for his/her comment. In fact, molecular docking study. was applied to explore the binding affinity of compounds (4a-c) and 5 toward a targeted protein. In the current study, we have applied molecular docking analyses to explore whether the anti-cancer activity of this class of compounds against cancer could be attributed to their ability to inhibit target proteins that have vital role in growth, proliferation and cell cycle of cancer cells, especially, breast (MCF-7) and liver cancer (HepG2). molecular docking analyses were conducted with four key proteins [Aromatase, epidermal growth factor receptor (EFGR), Cyclin-dependent kinase 2 (CDK2), and B-cell lymphoma 2 (Bcl-2)] that stimulate growth, proliferation, and development of cancer cells. aromatase protein. As previously shown, upregulation of estrogen receptors has been implicated in breast cancer and inhibition of estrogen production by aromatase inhibitors has been reported to efficiently suppress the proliferation of breast cancer. Therefore, examining the binding affinity of our compounds toward aromatase and other kinds of proteins would provide a piece of beneficial information that could be followed up in future studies.  

 

4. Molecular docking was performed on 4 proteins but there is no conclusion what was the target for these compound.

Response: We thank the reviewer for his/her comment and suggestion regarding the molecular docking study. The purpose of molecular docking on 4 proteins was mentioned in the revised version.

It was found that these classes of proteins [Aromatase, epidermal growth factor receptor (EFGR), Cyclin-dependent kinase 2 (CDK2), and B-cell lymphoma 2 (Bcl-2)] help cancer cell (MCF-7) to survive, grow and proliferate.

1. Breast cancer cells use Aromatase enzyme to produce estrogen as a fuel for survival and progression, consequently discovering aromatase inhibitors help in breast cancer treatment.
2. CDK2 has a role in the cell cycle of cancer cells, inhibition of CDK2 will stop the division of cancer cells.

• Bcl-2 antiapoptotic protein prevents the cancer cell from undergoing apoptosis mechanism, targeting and inhibition of Bcl-2 aid in cancer treatment.

1. EFGR has a role in the growth of cancer cells, inhibition of EFGR restricts the progression and proliferation of cancer cells.

 

5. No discussion at all on molecular docking regarding binding with amino acids and comparison of activity between compounds tested.

Response: We thank the reviewer for his/her comment and suggestion regarding the molecular docking study. discussion on molecular docking was modified in detail in the revised manuscript.

4. Authors in the scheme mentioned compound 6, but did not evaluate it. What for they synthesized it and why it was not tested?

Response: We thank the reviewer for his/her comment. Actually, we did not examine all synthesized compounds in scheme 1

We thought that compound 6 not active based of molecular docking studies (data not shown in the manuscript) so, we did not conduct further biological measurements on compound 6

7.Authors should take care and correct NMR spectra. For example 4b has 8 protons authors mentioned 6.

Response: We thank the reviewer for his/her comment.

The ¹H-NMR of compound 4b showed only 6 protons with 6 peaks between 3.91-8.46, and the two protons OH and NH were more shielded due to the presence of a withdrawing group in the compound structure like Br, hence, they did not appear on the NMR pattern. Moreover, the peaks that appeared at 2.5 and 3.5 for H₂O and DMSO, respectively.

(C=N), 1605, 1586 (C=C), 1081, 1061 (C-O) cm-1. 1H-NMR (DMSO-d6) δ: 3.91, 3.94 (s, CH2 of Thiazole ring), 7.93 (s, 1H, Ar.H), 8.13-8.21 (m, 2H, Ar-H). 8.30, 8.46 (s, 1H, CH=N of two isomer) ppm. ¹³CNMR (DMSO-d6). Anal. Calcd for C 10H8BrN 3O 2 S (M.wt=313): C, 38.34; H, 2-SS; N,13.42. Found: C, 38.11; H, 2.35; N, 13.22.

8. References should be with abbreviations.

Response: References were modified in the revised manuscript according to the Crystal journal guidance

We really thank the reviewer for his/her constructive evaluation of our manuscript. We really thank the reviewer for highlighting these critical corrections. We totally agree with the respected reviewer, and we apologize for these mistakes in our manuscript. We have corrected and clarified these mistakes in the revised manuscript as follow.

Responses: correction has been made

Page 2. Line 81.Should be: thiazole-4[5H]

Page 3. Line 108. Should be: [5H] and in other places of the manuscript.

Page 3,4. Line 115,142.Should be: 2-[2-[4-hydoroxy -

Page 5. Line 175. Should be: Consequently, compound

Page 5. Line 186. The following sentence needs revision: As a result, to affirm the anticancer activity, and was selected for further 187 analyses including cell cycle arrest, the vascular endothelial growth factor receptor-2 (VEGFR-2) kinase assay and apoptosis assay.

Page 6. Line  205. Should be: of compound 4c propidium iodide

Page 19.Line 333. Should be: 7.01-7.89 (m ,

Page 21.Line 388. Authors wrote: 3.94 (S, CH2 of Thiazole), 3. 91, 3.94 (S, CH2 of Thiazole ring of two isomer). The number of protons should be mentioned

Response: Correction has been made.

As yellow crystals, yield 71%, m.p. 258° C. IR(KBr) υmax: 3463 (br-OH), 3271

(NH), 1641 (C=N), 1605, 1586 (C=C), 1081, 1061 (C-O) cm^-1. 1H-NMR (DMSO-d₆) δ: 3.91, 3.94 (s, 2H, CH2 of Thiazole ring), 7.93 (s, 1H, Ar.H), 8.13-8.21 (m, 2H, Ar-H). 8.30, 8.46 (s, 1H, CH=N of two isomers) ppm. 13C-NMR (DMSO-d6). Anal. Calcd for C10H8BrN3O2S (M.wt. = 313): C, 38.34; H, 2-SS; N,13.42. Found: C, 38.11; H, 2.35; N, 13.22.

Reviewer 2 Report

Comments and Suggestions for Authors

ms revised accordingly.

Author Response

Reviewer 2

Response: We are most grateful to the reviewer for the constructive evaluations rendered to our manuscript. We have addressed all your concerns and are convinced that these suggestions have helped to improve the quality of the manuscript.

We really thank the reviewer for highlighting these critical points, we have corrected and clarified these mistakes in the revised manuscript (highlighting).

Reviewer 3 Report

Comments and Suggestions for Authors the observations are on the manuscript (attached file)

Comments for author File: Comments.pdf

Author Response

Reviewer 3# Round 2

Response: We are most grateful to the reviewer for the constructive evaluations rendered to our manuscript. We have addressed all your concerns and are convinced that these suggestions have helped to improve the quality of the manuscript.

We really thank the reviewer for highlighting these critical points, we have corrected and clarified these mistakes in the revised manuscript (highlighting). Our point-by-point responses are listed below.

Responses:

1. spectroscopic techniques, please mention them.

 

spectroscopic techniques (IR and NMR)  

2. 2. 2-[4-Hydroxy-3, capital letter (correction has been made)
3. CH=CH are vinylic protons

By this, we mean the protons of the aromatic ring (phenyl ring) containing (CH=CH). For this reason (CH=CH) was removed from the modified version to avoid interference.

4. the legend is missing e): reactiv and conditions

These are the reagent and reaction conditions.

5. The numbered atoms in the table must be identified in the structural formulas. In what way?

Response:

Numbered atoms of the compounds(4a-c) and 5 and types of bonds in (Tables 3-6) have been identified in the two-dimensional structural 2-D (Figures 4-7). Correction has been made in (Tables 3-6) and 2-D (Figures 4-7).

 

6. N cannot be H-donor; need NH. Likewise, the H-donor can be OH, not oxygen itself

Response:

Correction has been made in (Tables 3-6) (NH, N, OH, O, S)

 

7. in these reaction conditions, I think that NH is also acylated, the nitrogen atom being more nucleophilic than oxygen.

response

Response: reaction conditions were found correct after revision.

8. by biological testing of compound 6, it could be checked if the result of the docking study is correct.

Response: In fact, we did not conduct any biological experiment to examine compound 6, Moreover in the future we will prepare enough amount of compound 6 to examine its biological activity

 

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

 

The manuscript, entitled “ Anticancer studies of newly synthesized thiazole derivatives: 2 Synthesis, Characterization, Biological activity, and Molecular  docking”after revision improved a little . Authors addressed some points but I still don’t satisfy with the answers to some points.

Thus

The answer to point ; there is no structure -activity relationship discussion is completely wrong.

When I spoke about structure-activity relationship I did not speak about docking but influence of substituent to anticancer activity.

Regarding point 4 Molecular docking was performed on 4 proteins, again the answer is not acceptable. Authors referred to the tables 3-5. They are right, but the conclusion is wrong. According to the results presented in these tables compounds target aromatase and followed CDK2 cyclin dependent kinase-2 .

References should be with abbreviations.

 

Page 4. Line 118.Should be : 3-substituted 4-hydroxybenzaldehyde

Page 5. Scheme 1.Line 142. Should be: 2-[2-[4-hydroxy -3-subtituted

Page 6.Line 159. Should be: 3-methoxy -4-hydroxybenzaldehyde

Page 6.Line 161. Should be: 5-(4-hydroxy-3-methoxybenzylidene)-2-(2-4- hydroxybenzylidene)

Page 6.Line 4 after line 171. Should be: Consequently, compound 4c

Page 6. Line 5 after line 171. Should be: 2-(4-hydroxybenzylidene)

Page 8. Line 193.Should be: To confirm the efficacy of compound 4c as an anticancer agent,further analyses using…

Page 9.Line 143. Should be: activity, the VEGFR-2

Page 10.Line 156. Authors wrote:” For further clarification of the biological activity of synthesized thiazole derivatives used in this study, molecular docking analyses were conducted” Molecular docking don’t clarify the biological activity , it has nothing to do with this. It just predicts the binding mode to the target.

Page 10. 2.3. Line 5. The following sentence needs revision” Further, the prediction of the mechanism of action of certain biologically active compounds.

Page 11. Line 157. The subtitle 2.3.1. needs improvement Actually there is no need for subtitle. All subtitles regarding docking are useless.

Page 25. Line 404. Should be: Synthesis of 4-((2-(4-oxo-4,5-dihydrothiazo

Page 25. Line 422. Should be: Synthesis of 5-(4-hydroxy-3-methoxybenzylidene

In docking should be reference drug.

In most of references the name of journal did not mentioned with abbreviation name. For example, ref.32, 34. Should be. Eur. J.Med.Chem.

Ref.33 Should be: Bioorg Med Chem Lett and so on.

Comments on the Quality of English Language

 

Author Response

Reviewer 1:# Round 3

We really thank the reviewer for highlighting these critical points, we have corrected and clarified these mistakes in the revised manuscript (highlighting). Our point-by-point responses are listed below.

1-The answer to point ; there is no structure -activity relationship discussion is completely wrong.

When I spoke about structure-activity relationship I did not speak about docking but influence of substituent to anticancer activity.

Response:

We have interpreted the results of the compounds’ activity and the relationship of the composition to the anti-tumor activity based on IC₅₀ values that we obtained Table 1. We found a relationship between the substituents that were introduced to the compounds and these values (line 171-line 176). Further, by conducting theoretical studies (molecular docking), it was confirmed that there is a relationship between the composition of the compounds or the substitutions that were introduced and its activity and the explanation for this actually exists in the modified versions (highlighting), and this explanation is acceptable compared to our previously published research.

Salem, Manar G., Dina M. Abu El-Maaty, Yassmina I. Mohey El-Deen, Basem H. Elesawy, Ahmad El Askary, Asmaa Saleh, Essa M. Saied, and M. El Behery. 2022. "Novel 1,3-Thiazole Analogues with Potent Activity against Breast Cancer: A Design, Synthesis, In Vitro, and In Silico Study" Molecules 27, no. 15: 4898

2-Regarding point 4 Molecular docking was performed on 4 proteins, again the answer is not acceptable. Authors referred to the tables 3-5. They are right, but the conclusion is wrong. According to the results presented in these tables compounds target aromatase and followed CDK2 cyclin dependent kinase-2.

Response: Correction has been made in the revised version (highlighting)

3. References should be with abbreviations.

Response: Correction has been made

 

Page 4. Line 118.Should be : 3-substituted 4-hydroxybenzaldehyde   correction has been made

Page 5. Scheme 1.Line 142. Should be: 2-[2-[4-hydroxy -3-subtituted correction has been made

Page 6.Line 159. Should be: 3-methoxy -4-hydroxybenzaldehyde         correction has been made

Page 6.Line 161. Should be: 5-(4-hydroxy-3-methoxybenzylidene)-2-(2-4- hydroxybenzylidene) correction has been made

Page 6.Line 4 after line 171. Should be: Consequently, compound 4c correction has been made

Page 6. Line 5 after line 171. Should be: 2-(4-hydroxybenzylidene)

Response: thank you so much for this comment

 correction has been made.

Page 8. Line 193.Should be: To confirm the efficacy of compound 4c as an anticancer agent, further analyses using…  correction has been made.

Page 9.Line 143. Should be: activity, the VEGFR-2  correction has been made.

Page 10.Line 156. Authors wrote:” For further clarification of the biological activity of synthesized thiazole derivatives used in this study, molecular docking analyses were conducted” Molecular docking don’t clarify the biological activity, it has nothing to do with this. It just predicts the binding mode to the target.

Response: we apologize for this mistake; correction has been made.

Page 10. 2.3. Line 5. The following sentence needs revision” Further, the prediction of the mechanism of action of certain biologically active compounds. correction has been made. (The sentence was removed)

Page 11. Line 157. The subtitle 2.3.1. needs improvement Actually there is no need for subtitle. All subtitles regarding docking are useless. correction has been made. (Subtitles were removed and reorganized in the revised version)

Page 25. Line 404. Should be: Synthesis of 4-((2-(4-oxo-4,5-dihydrothiazo correction has been made

Page 25. Line 422. Should be: Synthesis of 5-(4-hydroxy-3-methoxybenzylidene correction has been made

In docking should be reference drug.

Response: Reference drug was added [52-56]

 

[52] Suvannang, N.; Nantasenamat, C.; Isarankura-Na-Ayudhya, C.; Prachayasittikul, V. Molecular Docking of Aromatase Inhibitors. Molecules. 2011, 16, 3597–3617.

 

[53] Mansourian, M.; Hassanzadeh, F.; Shahlaei, M. Exploring the interaction between epidermal growth factor receptor tyrosine kinase and some of the synthesized inhibitors using combination of in-silico and in-vitro cytotoxicity methods. Res Pharm Sci. 2018, 13, 509-522

 

[54] kwu, F. A.; Isyaku, Y.; Obadawo, B. S.; Lawal, H. A.; Ajibowu, S. A. In silico design and molecular docking study of CDK2 inhibitors with potent cytotoxic activity against HCT116 colorectal cancer cell line. J Genet Eng Biotechnol. 2020 15, 18, 51.

 

[55] Zheng, L.; Yang, Y.; Bao, J.; He, L.; Qi, Y.; Zhang, J. Z. H. Discovery of novel inhibitors of CDK2 using docking and physics-based binding free energy calculation. Chem Biol Drug Des. 2022, 99, 662-673.

 

[56] Swati Krishna, S.; Birendra Kumar, T.P.; Krishna, M.; Manikanta, M. Structure-based design approach of potential BCL-2 inhibitors for cancer chemotherapy. Computers in Biology and Medicine.2021, 134,104455.

In most of references the name of journal did not mentioned with abbreviation name. For example, ref.32, 34. Should be. Eur. J.Med.Chem.

Ref.33 Should be: Bioorg Med Chem Lett and so on.

 Response: Correction has been made

Reviewer 3 Report

Comments and Suggestions for Authors

Tables 3 and 4 : S22 : H-acceptor ; S23: H-acceptor;

At Scheme1, point e) must be added: i. dimethylformamide, sodium acetate, ii. HCl.

Also, the numbering of the compounds in the scheme is not complete.

To the previous observation "in these reaction conditions, I think that NH is also acylated, the nitrogen atom being more nucleophilic than oxygen." your response is that reaction conditions were found correct after revision. I do not dispute the correctness of the reagent conditions, but say that under these conditions NH is also acylated, which is more nucleophilic than OH. When interpreting the NMR spectra, you must demonstrate that you have succeeded in a selective acylation. On the other hand, acylation of phenolic hydroxyl requires acid or base catalyst.

Tables 3 and 4: S22: H-acceptor; S23: H-acceptor!

Line 371: data “0.04 (br.s, 1H, OH)” is incorect.

The expressions two isomers do not seem suitable to me for the following reasons: there are many categories of isomers and the possibility of their existence is not sufficient. Their existence must be demonstrated.

Author Response

 

Reviewer 3# Round 3

Response: We are most grateful to the reviewer for the constructive evaluations rendered to our manuscript. We have addressed all your concerns and are convinced that these suggestions have helped to improve the quality of the manuscript.

We really thank the reviewer for highlighting these critical points, we have corrected and clarified these mistakes in the revised manuscript (highlighting). Our point-by-point responses are listed below.

• Tables 3 and 4 : S22 : H-acceptor ; S23: H-acceptor;

Responses: correction has been made in tables and figures

 

• At Scheme1, point e) must be added: dimethylformamide, sodium acetate, ii. HCl.

Responses: Correction has been made

 

• Also, the numbering of the compounds in the scheme is not complete.

Responses: Correction has been made

 

• To the previous observation "in these reaction conditions, I think that NH is also acylated, the nitrogen atom being more nucleophilic than oxygen." your response is that reaction conditions were found correct after revision. I do not dispute the correctness of the reagent conditions but say that under these conditions NH is also acylated, which is more nucleophilic than OH. When interpreting the NMR spectra, you must demonstrate that you have succeeded in a selective acylation. On the other hand, acylation of phenolic hydroxyl requires acid or base catalyst.

 

Response:

The NH group is more difficult with acetylation reaction, these confirmed by the following: -

• The ¹H-NMR data of compound 4a showed the OH signal at 10.30, while NH signal at δ 11.71. The ¹H-NMR of compound 5 confirmed the acetylation reaction on the hydroxy group only, where the signal of OH of compound 4
• The NH group is difficult with acetylation because the lone pair of nitrogen group formed resonance (tautomer) with the nitrogen atom in the thiazole ring as shown.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Acylation conducted under reflux and did not require acid or base catalyst.

 

• Line 371: data “0.04 (br.s, 1H, OH)” is incorect.

Response: Correction has been made    10.04 (br.s, 1H, OH)

 

• The expressions two isomers do not seem suitable to me for the following reasons: there are many categories of isomers, and the possibility of their existence is not sufficient. Their existence must be demonstrated.

 

Response: Bromine has two isotopes (Br (79), and Br (81) have relative abundances of 50.686 % and 49.314, respectively, so compounds that contain Br in their structure display 2 isomers such as compound 4b. moreover, other compounds such as compounds 4c, 5, and 6, display Cis, and trans isomers exist in tautomerism (E and Z), they are integrated and cannot be separated. The NMR (¹H and ¹³ C) spectrum of these compounds supported the formation of two isomers (as two E and Z stereoisomers)

Round 4

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript, entitled “ Anticancer studies of newly synthesized thiazole derivatives: 2 Synthesis, Characterization, Biological activity, and Molecular  docking”after revision improved a little . Authors addressed almost all  points, but I still not regarding the reference drug and discussion for docking. Authors presented 4 papers but I need the energy of reference drug and comparison with obtained results.

Comments on the Quality of English Language

English of the manuscript more or less is acceptable.

Author Response

We really appreciate the reviewer's insightful suggestions, and we have made the necessary corrections and clarifications in the updated article. The binding energy (docking scores) of the reference drug was included, and results were compared (highlighting). Also, in the updated article, the molecular docking section's discussion was improved (highlighting).

 

This citation was added.

[55] Ramos, J.; Muthukumaran, J.; Freire, F.; Paquete-Ferreira, J.; Otrelo-Cardoso, A. R.; Svergun, D.; Panjkovich, A.; Santos-Silva, T. Shedding Light on the Interaction of Human Anti-Apoptotic Bcl-2 Protein with Ligands through Biophysical and in Silico Studies. Int J Mol Sci. 2019, 20, 860.

Author Response File: Author Response.pdf

Round 5

Reviewer 1 Report

Comments and Suggestions for Authors

Authors improved the manuscript, but nevertheless there are still some mistakes.

Page 3. Line after 104. Should be: capacity to kill

Page 4 .Line 107 : Should be:2-[2-[4-hydoroxy

Comments on the Quality of English Language

English is more or less now acceptable. I am not an expert.