Synthesis of Novel Sesamol-Based Hybrids—In Silico Analysis and In Vitro Evaluation of Radical Scavenging Activity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript under review is devoted to the synthesis and study of the antiradical properties of sesamol derivatives containing a benzothiazoline or benzimidazoline moiety. Additionally, toxicity was studied computationally using the VEGA program. This study is relevant and offers scientific novelty. The originality of the compounds was confirmed by searching in SciFinder. The structure of the synthesized compounds was reliably confirmed by H and C NMR spectroscopy. I recommend the manuscript for publication after corrections. Comments and questions that arose while reading the manuscript:

1. The article title, keywords, and text contain the term «regioselective». This term is typically used when there is an alternative reaction pathway and mixtures of regioisomers are formed with a significant predominance of one. However, in this case (as expected based on steric hindrance and literature data on the alkylation of sesamol), the formation of the other isomer is unlikely, so this term is not entirely correct. I do not recommend its use.
2. Line 95-96, «To average out the rotamers reaching adequate assignment of peaks and structure determination, the spectra were measured at 80 °C». Did you observe a doubling of the signals when recording NMR spectra at room temperature? In my opinion, the formation of rotamers is a very interesting scientific fact that I would like to discuss in more detail.
3. Line 102-103, «2,2,2-trichloroethyl chloroformate (Troc-Cl)». If an abbreviation is introduced, it should be used throughout the text of the article. I see no point in duplicating information on lines 113-114, 331.
4. Line 118-119, «The product was isolated by extraction, first with 30 mL of hydrochloric acid–water solution (1:1, v/v), followed by 50 mL of water». In this case, the term "extraction" is not entirely correct, since the target substance is already in an organic solvent and in this case is washed away from by-products, rather than extracted.
5. Line 124, «Methyl 2-(5-hydroxybenzo[d][1,3]dioxol-4-yl)benzo[d]thiazole-3(2H)-carboxylate». There's an error in the benzoxazole substituent locant. It should be «Methyl 2-(6-hydroxybenzo[d][1,3]dioxol-5-yl)benzo[d]thiazole-3(2H)-carboxylate».
6. Line 128-129, «5.81 (d, J = 0.9, 1H, -OCH₂O-), 5.86 (d, J = 1.0, 1H, -OCH₂O-)». Why do the methylene group signals split this way? Typically, in the presence of a chiral center, a diastereotopic methylene appears as two doublets with a geminal J-constant greater than 10 Hz. In this case, it's 1 Hz. Are there any published data with similar J-constants? (similar on line 147). Line 135, «129.1» - replace with «129.0» (incorrect rounding, see SI).
7. Line 142, « Ethyl-2-(5-hydroxybenzo[d][1,3]dioxol-4-yl)benzo[d]thiazole-3(2H)-carboxylate ». There's an error in the benzoxazole substituent locant. It should be « Ethyl-2-(6-hydroxybenzo[d][1,3]dioxol-5-yl)benzo[d]thiazole-3(2H)-carboxylate». Line 147, «4.15–4.29 (m, 2H, -COOCH₂CH₃)» - Typically, such methylene protons of the ethyl fragment, due to diastereotopy, manifest as two closely spaced (overlapping) quartet doublets with ²J at approximately -11 Hz and ³J at approximately 7 Hz. It may be possible to isolate these signals. Line 151-152, «62.3(*CH), 151 62.6(-COOCH₂CH₃)» - How were these closely spaced signals assigned to specific fragments (three tenths of a ppm difference)?
8. Line 164, «4.95–5.00 (m, 2H, -COOCH₂CCl₃)» - Why does this signal split? If it's due to diastereotopy, then can this multiplet be defined as two doublets of doublets with a geminal J-constant. The ¹H NMR spectrum of compound 6c clearly contains extraneous signals in the 7.1-7.3 ppm region. Is this unreacted benzothiazole?
9. Line 183, «6.37» - replace with «6.36» (incorrect rounding, see SI). Line 189, «151.2(-C-Ar), 151.8(2x-C=O)» - How were these closely spaced signals assigned to specific fragments (six tenths of a ppm difference)?
10. Line 199, « 4.12 (q, J = 3.4, 5.9 Hz, 4H, 2x-COOCH2CH3) » - If this is a true quartet, then there should be one vicinal J-constant of the order of 7 Hz. Line 206, «151.2(-C-Ar), 151.3(2x-C=O) » - How were these closely spaced signals assigned to specific fragments (one tenths of a ppm difference)?
11. Line 215, «4.92–4.97 (m, 4H, -COOCH2CCl3)» - Why is the signal split? There are no chiral centers in the molecule and no diastereotopy. Line 219, «75.3(2x-COOCH2CCl3), 76.3(*CH) » - How were these closely spaced signals assigned to specific fragments? Line 220, «109.9» - replace with «110.0» (incorrect rounding, see SI).
12. Line 235, «8.97» - replace with «9.03» (see SI). Line 239, «151.0(-C-Ar), 151.7(2x-C=O)» - How were these closely spaced signals assigned to specific fragments (seven tenths of a ppm difference)?
13. Line 254, « 107.0» - replace with « 106.9» (incorrect rounding, see SI). Line 255, «151.1(-C-Ar), 151.2(2x-C=O) » - How were these closely spaced signals assigned to specific fragments (one tenths of a ppm difference)?
14. Line 265, «4.91–4.96 (m, 4H, 2x-COOCH2CCl3)» - Why is the signal split? There are no chiral centers in the molecule and no diastereotopy. Line 269, «75.2(2x-COOCH2CCl3), 76.0(*CH)» - How were these closely spaced signals assigned to specific fragments?
15. P. 9, Table 1. It is incorrect to write R1 = Troc, since «Troс» denotes a 2,2,2-trichloroethoxycarbonyl group, that is, it contains CARBONYL!
16. P. 10, Scheme 2. Move the dot up in the designation of TEA hydrochloride salt.
17. Line 361-363, «…indicated substitution at the less sterically hindered “ortho” position relative to the phenolic hydroxyl group. Also, the methylenedioxy substituent likewise directed subtitution to 6th position, clearly corresponding to already published analogues [24,27]». The presence of the methylenedioxy substituent directs the attack to the less sterically congested ortho-position! These two sentences should be combined and rephrased. Line 374, «indicating substitution at the „ortho” position relative to the phenolic hydroxyl group» - In the original sesamol there are two ortho positions relative to the free hydroxyl group - position 5 (sterically busy) and position 6. In this regard, the phrase requires clarification.
18. Line 422-423, «Therefore, DPPH-examined IC₅₀ values for these compounds are not included in Table 2.». This phrase is meaningless and should be removed. Data are not included in Table 2 because no testing was conducted.
19. Line 431-434, «The use of such in silico approaches is essential for early-stage safety assessment in the development of new synthetic substances or cosmetic products and is in accordance with the 3R principles aimed at reducing animal experimentation [33–35,38,39].» and line 451-454, « The in silico analyses proposed herein enable early-stage evaluation of the biological profiles of these structures while avoiding initial animal experimentation, in accordance with the 3R principles formulated by Russell and Burch [38,39]». These sentences essentially duplicate each other. Leave one sentence.
20. Notes on the list of references. Remove spaces between author initials in references [3, 11, 13, 19, 23, 33]. Check authors in reference [3]. Place hyphens between initials for authors in references [5, 24]. Add missing initials for authors in references [7, 11]. In reference [27], replace «Suponitsky, K.Y.» with «Suponitsky, K.Yu.». In my opinion, the abbreviations of the journals should be deciphered: «JFST» (line 535), «Pharm.» (line 565), «PAC» (line 571). Reference [38] is a book review, not the book itself! Also, the year for a journal should be «1960». I believe that the authors of the manuscript should indicate their earlier article:Synthetic Communications (2020), 50(19), 3007-3015, https://doi.org/10.1080/00397911.2020.1791342

Author Response

Response to Reviewer’s Comments

Dear Reviewer, thank you for your effort to provide timely review and scientific decision on our manuscript.

The manuscript under review is devoted to the synthesis and study of the antiradical properties of sesamol derivatives containing a benzothiazoline or benzimidazoline moiety. Additionally, toxicity was studied computationally using the VEGA program. This study is relevant and offers scientific novelty. The originality of the compounds was confirmed by searching in SciFinder. The structure of the synthesized compounds was reliably confirmed by H and C NMR spectroscopy. I recommend the manuscript for publication after corrections. Comments and questions that arose while reading the manuscript:

Author’s comment: Thank you for the positive point of view, scientific opinion and additional corrections on our work. The deep spectroscopic insights and precise recommendations are particularly impressive. The revision made significantly improved the text and scientific appearance of the manuscript.

 

 

 

Reviewer’s comments and suggestions:

1. The article title, keywords, and text contain the term «regioselective». This term is typically used when there is an alternative reaction pathway and mixtures of regioisomers are formed with a significant predominance of one. However, in this case (as expected based on steric hindrance and literature data on the alkylation of sesamol), the formation of the other isomer is unlikely, so this term is not entirely correct. I do not recommend its use.

Author’s response: Thank you for the correct remark. It is deleted in the revised version of the manuscript.

 

2. Line 95-96, «To average out the rotamers reaching adequate assignment of peaks and structure determination, the spectra were measured at 80 °C». Did you observe a doubling of the signals when recording NMR spectra at room temperature? In my opinion, the formation of rotamers is a very interesting scientific fact that I would like to discuss in more detail.

Author’s response: Thank you for the observation. The recorded NMR spectra of the amidoalkylated Sesamol derivatives at 20 ^oC or CDCl₃ as solvent, indicated slow rotameric interconversions with registration of strongly broadened or doubled signals, which made their correct interpretation impossible. This specificity was previously observed from us in the context of the synthesis of various phenolic and indole hybrids. [Y. Stremski, S. Statkova-Abeghe, P. Angelov, I. Ivanov, Synthesis of Camalexin and related analogues, Journal of Heterocyclic Chemistry, 7, 55, 2018, 1589-1595; https://doi.org/10.1002/jhet.3192]; [Y. Stremski, D. Kirkova, S. Statkova-Abeghe, P. Angelov, I. Ivanov, D. Georgiev, "Synthesis and antibacterial activity of hydroxylated 2-arylbenzothiazole derivatives", Synthetic Communications, 50, 19, 2020, 3007-3015; https://doi.org/10.1080/00397911.2020.1791342]; [D. Kirkova, Y. Stremski, M. Bachvarova, M. Todorova, B. Goranov, S. Statkova-Abeghe and M. Docheva, Molecules, 30(1), 2025, 636; https://doi.org/10.3390/molecules30030636]

 

As example:

 

Figure 1. ¹H-NMR spectrum in CDCl₃ at room temperature, 600 MHz

 

Figure 2. ¹H-NMR spectrum in DMSO-d₆ at 80 ^oC, 600 MHz

According to your recommendation, the additional text is added and specified in the revised version of the manuscript (3. Results and Discussion).

 

3. Line 102-103, «2,2,2-trichloroethyl chloroformate (Troc-Cl)». If an abbreviation is introduced, it should be used throughout the text of the article. I see no point in duplicating information on lines 113-114, 331.

Author’s response: It is clearly revised in the corrected version of the manuscript.

 

4. Line 118-119, «The product was isolated by extraction, first with 30 mL of hydrochloric acid–water solution (1:1, v/v), followed by 50 mL of water». In this case, the term "extraction" is not entirely correct, since the target substance is already in an organic solvent and in this case is washed away from by-products, rather than extracted.

Author’s response: It is corrected in the revised version of the manuscript.

 

5. Line 124, «Methyl 2-(5-hydroxybenzo[d][1,3]dioxol-4-yl)benzo[d]thiazole-3(2H)-carboxylate». There's an error in the benzoxazole substituent locant. It should be «Methyl 2-(6-hydroxybenzo[d][1,3]dioxol-5-yl)benzo[d]thiazole-3(2H)-carboxylate».

Author’s response: Thank you for the correct remark. It is corrected in the revised version of the manuscript for compounds 6a and 6b.

 

6. Line 128-129, «5.81 (d, J = 0.9, 1H, -OCH2O-), 5.86 (d, J = 1.0, 1H, -OCH2O-)». Why do the methylene group signals split this way? Typically, in the presence of a chiral center, a diastereotopic methylene appears as two doublets with a geminal J-constant greater than 10 Hz. In this case, it's 1 Hz. Are there any published data with similar J-constants? (similar on line 147). Line 135, «129.1» - replace with «129.0» (incorrect rounding, see SI).

Author’s response:

Compound 6a - ¹H-NMR spectrum - DMSO-d₆, 80 ^oC, 600 MHz, in the range of 5.79–5.88 ppm

 

The published data in this context with similar J-constants is cited as [24, Zhang, H.; Chen, M.G.; Lian, C.X.; Yuan, W.C.; Zhang, X.M. Remarkably Mild and Efficient Cetrimonium Bro-mide-Catalyzed Friedel–Crafts Amidoalkylation of Sesamols with N-Boc Imines Generated in Situ in Aqueous Medium. Synlett, 9, 2010, 1415–1417; https://doi.org/10.1055/s-0029-1219802];

Thank you for the remark. The value is rounded in the revised version of the manuscript.

 

7. Line 142, « Ethyl-2-(5-hydroxybenzo[d][1,3]dioxol-4-yl)benzo[d]thiazole-3(2H)-carboxylate ». There's an error in the benzoxazole substituent locant. It should be « Ethyl-2-(6-hydroxybenzo[d][1,3]dioxol-5-yl)benzo[d]thiazole-3(2H)-carboxylate». Line 147, «4.15–4.29 (m, 2H, -COOCH2CH3)» - Typically, such methylene protons of the ethyl fragment, due to diastereotopy, manifest as two closely spaced (overlapping) quartet doublets with 2J at approximately -11 Hz and 3J at approximately 7 Hz. It may be possible to isolate these signals. Line 151-152, «62.3(*CH), 151 62.6(-COOCH2CH3)» - How were these closely spaced signals assigned to specific fragments (three tenths of a ppm difference)?

Author’s response:

According to our previous expertise, we observed such methylene protons of the ethyl fragment as multiplet. Unfortunately, it is not possible to isolate signals.

Compound 6b - ¹H-NMR spectrum - DMSO-d₆, 80 ^oC, 600 MHz, in the range of 4.14–4.26 ppm

This observation and the additional precise determination of hydrogen from the asymmetric centre have been studied and claimed according to HSQC-NMR analysis for (benzo)thiazoline containing compounds in our previous studies [such as: Y. Stremski, S. Statkova-Abeghe, P. Angelov, I. Ivanov, Synthesis of Camalexin and related analogues, Journal of Heterocyclic Chemistry, 7, 55, 2018, 1589-1595; https://doi.org/10.1002/jhet.3192, supporting information];

 

8. Line 164, «4.95–5.00 (m, 2H, -COOCH2CCl3)» - Why does this signal split? If it's due to diastereotopy, then can this multiplet be defined as two doublets of doublets with a geminal J-constant. The 1H NMR spectrum of compound 6c clearly contains extraneous signals in the 7.1-7.3 ppm region. Is this unreacted benzothiazole?

Author’s response: Thank you for the correct comment. The extraneous signals in the 7.1-7.3 ppm region probably correspond to impurity from unreacted benzothiazole. In this regard, the NMR spectrum was repeated and the new one leads to more clean visualization.

The signals are redefined as two doublets with a geminal J-constant. The corrections are made in the revised version of the manuscript and Supporting information file.

 

9. Line 183, «6.37» - replace with «6.36» (incorrect rounding, see SI). Line 189, «151.2(-C-Ar), 151.8(2x-C=O)» - How were these closely spaced signals assigned to specific fragments (six tenths of a ppm difference)?

Author’s response: Thank you for the remark. The value is rounded in the revised version of the manuscript.

The signal at 151.8 ppm is assigned for (2x-C=O) fragments according to our previous studies regarded to N,N-dimethoxycarbonyl-benzimidazolines [Y. Stremski, M. Bachvarova, D. Kirkova, E. Milinova and S. Statkova-Abeghe, A convenient one-pot approach to the synthesis of novel benzimidazole-indole molecular hybrids as benzazacamalexin related analogues, Current Organic Synthesis, 22(5), 2025, 631-638; https://doi.org/10.2174/0115701794364219241228094932];

 

10. Line 199, « 4.12 (q, J = 3.4, 5.9 Hz, 4H, 2x-COOCH2CH3) » - If this is a true quartet, then there should be one vicinal J-constant of the order of 7 Hz. Line 206, «151.2(-C-Ar), 151.3(2x-C=O) » - How were these closely spaced signals assigned to specific fragments (one tenths of a ppm difference)?

Author’s response: Thank you for the correct observation. It is typo in the interpretation of 7b. It is revised in the corrected text.

The signal at 151.3 ppm is assigned for (2x-C=O) fragments according to our previous studies regarded to N,N-diethoxycarbonyl-benzimidazolines [Y. Stremski, M. Bachvarova, D. Kirkova, E. Milinova and S. Statkova-Abeghe, A convenient one-pot approach to the synthesis of novel benzimidazole-indole molecular hybrids as benzazacamalexin related analogues, Current Organic Synthesis, 22(5), 2025, 631-638; https://doi.org/10.2174/0115701794364219241228094932]; [Y. Stremski, M. Bachvarova, D. Kirkova and S. Statkova-Abeghe, New 2-(2,4-Dihydroxyphenyl)benzimidazolines, Molbank, 2023(1), 2023, M1602; https://doi.org/10.3390/M1602];

 

11. Line 215, «4.92–4.97 (m, 4H, -COOCH2CCl3)» - Why is the signal split? There are no chiral centers in the molecule and no diastereotopy. Line 219, «75.3(2x-COOCH2CCl3), 76.3(*CH) » - How were these closely spaced signals assigned to specific fragments? Line 220, «109.9» - replace with «110.0» (incorrect rounding, see SI).

Author’s response: Thank you for the correct observation. It is corrected in the revised version of the manuscript.

The signal at 75.3 ppm is assigned to (2x-COOCH2CCl3) according to our previous investigations regarded to N,N-di-2,2,2-trichloroethoxycarbonyl-benzimidazolines [Y. Stremski, M. Bachvarova, D. Kirkova, E. Milinova and S. Statkova-Abeghe, A convenient one-pot approach to the synthesis of novel benzimidazole-indole molecular hybrids as benzazacamalexin related analogues, Current Organic Synthesis, 22(5), 2025, 631-638; https://doi.org/10.2174/0115701794364219241228094932];

 

12. Line 235, «8.97» - replace with «9.03» (see SI). Line 239, «151.0(-C-Ar), 151.7(2x-C=O)» - How were these closely spaced signals assigned to specific fragments (seven tenths of a ppm difference)?

Author’s response: Thank you for the correct observation. It is replaced in the revised manuscript version.

The signal at 151.7 ppm is assigned for (2x-C=O) fragments according to our previous studies regarded to N,N-dimethoxycarbonyl-5,6-dimethylbenzimidazolines [Y. Stremski, M. Bachvarova, D. Kirkova, E. Milinova and S. Statkova-Abeghe, A convenient one-pot approach to the synthesis of novel benzimidazole-indole molecular hybrids as benzazacamalexin related analogues, Current Organic Synthesis, 22(5), 2025, 631-638; https://doi.org/10.2174/0115701794364219241228094932];

 

13. Line 254, « 107.0» - replace with « 106.9» (incorrect rounding, see SI). Line 255, «151.1(-C-Ar), 151.2(2x-C=O) » - How were these closely spaced signals assigned to specific fragments (one tenths of a ppm difference)?

Author’s response: Thank you for the correct observation. It is replaced in the revised version of the manuscript.

The signal at 151.2 ppm is assigned for (2x-C=O) fragments according to our previous studies regarded to N,N-diethoxycarbonyl-5,6-dimethylbenzimidazolines [Y. Stremski, M. Bachvarova, D. Kirkova, E. Milinova and S. Statkova-Abeghe, A convenient one-pot approach to the synthesis of novel benzimidazole-indole molecular hybrids as benzazacamalexin related analogues, Current Organic Synthesis, 22(5), 2025, 631-638; https://doi.org/10.2174/0115701794364219241228094932]; [Y. Stremski, M. Bachvarova, D. Kirkova and S. Statkova-Abeghe, New 2-(2,4-Dihydroxyphenyl)benzimidazolines, Molbank, 2023(1), 2023, M1602; https://doi.org/10.3390/M1602];

 

14. Line 265, «4.91–4.96 (m, 4H, 2x-COOCH2CCl3)» - Why is the signal split? There are no chiral centers in the molecule and no diastereotopy. Line 269, «75.2(2x-COOCH2CCl3), 76.0(*CH)» - How were these closely spaced signals assigned to specific fragments?

Author’s response: Thank you for the correct observation. It is corrected in the revised version of the manuscript.

The signal at 75.2 ppm is assigned to (2x-COOCH2CCl3) according to our previous investigations regarded to N,N-di-2,2,2-trichloroethoxycarbonyl-5,6-dimethylbenzimidazolines [Y. Stremski, M. Bachvarova, D. Kirkova, E. Milinova and S. Statkova-Abeghe, A convenient one-pot approach to the synthesis of novel benzimidazole-indole molecular hybrids as benzazacamalexin related analogues, Current Organic Synthesis, 22(5), 2025, 631-638; https://doi.org/10.2174/0115701794364219241228094932];

 

15. 9, Table 1. It is incorrect to write R1 = Troc, since «Troс» denotes a 2,2,2-trichloroethoxycarbonyl group, that is, it contains CARBONYL!

Author’s response: Thank you for the correct remark. The Table 1 is corrected in the revised version of the manuscript.

 

16. 10, Scheme 2. Move the dot up in the designation of TEA hydrochloride salt.

Author’s response: Thank you for the correct remark. The TEA hydrochloride salt is corrected in the revised version of Scheme 2.

 

17. Line 361-363, «…indicated substitution at the less sterically hindered “ortho” position relative to the phenolic hydroxyl group. Also, the methylenedioxy substituent likewise directed subtitution to 6th position, clearly corresponding to already published analogues [24,27]». The presence of the methylenedioxy substituent directs the attack to the less sterically congested ortho-position! These two sentences should be combined and rephrased. Line 374, «indicating substitution at the „ortho” position relative to the phenolic hydroxyl group» - In the original sesamol there are two ortho positions relative to the free hydroxyl group - position 5 (sterically busy) and position 6. In this regard, the phrase requires clarification.

Author’s response: Thank you for the exact remark. The phrase is correctly clarified and simplified in the revised version of the manuscript.

 

18. Line 422-423, «Therefore, DPPH-examined IC₅₀ values for these compounds are not included in Table 2.». This phrase is meaningless and should be removed. Data are not included in Table 2 because no testing was conducted.

Author’s response: The sentence is removed in the revised version of the manuscript.

 

19. Line 431-434, «The use of such in silico approaches is essential for early-stage safety assessment in the development of new synthetic substances or cosmetic products and is in accordance with the 3R principles aimed at reducing animal experimentation [33–35,38,39].» and line 451-454, « The in silico analyses proposed herein enable early-stage evaluation of the biological profiles of these structures while avoiding initial animal experimentation, in accordance with the 3R principles formulated by Russell and Burch [38,39]». These sentences essentially duplicate each other. Leave one sentence.

Author’s response: The one of the sentences is removed in the revised version of the manuscript and the text is simplified.

 

20. Notes on the list of references. Remove spaces between author initials in references [3, 11, 13, 19, 23, 33]. Check authors in reference [3]. Place hyphens between initials for authors in references [5, 24]. Add missing initials for authors in references [7, 11]. In reference [27], replace «Suponitsky, K.Y.» with «Suponitsky, K.Yu.». In my opinion, the abbreviations of the journals should be deciphered: «JFST» (line 535), «Pharm.» (line 565), «PAC» (line 571). Reference [38] is a book review, not the book itself! Also, the year for a journal should be «1960». I believe that the authors of the manuscript should indicate their earlier article: Synthetic Communications (2020), 50(19), 3007-3015, https://doi.org/10.1080/00397911.2020.1791342

Author’s response: Thank you for the correct recommendations. The references are correctly revised in the corrected version of the manuscript. Our previous work is indicated and cited as [28].

 

The all remarks are taken into account. In this regard, the additional corrections of manuscript are made and marked (track changes).

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Bachvarova et al. describes the synthesis of nine novel sesamol-based hybrids. Some of the prepared compounds were evaluated using DPPH and ABTS assays. The chemistry is solid, the yields are good, the compounds were thoroughly characterized, and the spectroscopic data are of high quality. The biological evaluation is the weaker aspect of the study, as the improvement in antioxidant activity is modest. Overall, the paper is suitable for publication in AppliedChem but, in my opinion, requires major revision.

Comments and Suggestions:

1) I suggest that the authors rewrite the Introduction as it is heavily cancer-focused, yet only antioxidant activity was investigated in the manuscript.

2) Schemes showing the prepared compounds in Materials and Methods section are unnecessary as these structures are already shown in Scheme 1. I suggest moving the NMR data (together with the individual structures) to Supplementary Information file.

3) Line 107: "petroleum ether/diethyl ether" instead of "petroleum/diethyl ether"; Line 98: there is a typo "... (TLC) was performed done...".

4) Line 99: please provide the particle size and manufacturer of silica gel. 

5) Sections 2.1.2 and 2.1.3: Please specify which compounds were prepared by each method (e.g., 6a-c with 2.1.2 and the rest with 2.1.3).

6) Line 118: would the term "wash" be more appropriate than "extraction" since the organic layer was washed to remove Et₃N and residual imidazole? E.g., "The organic phase was washed with 30 mL of hydrochloric acid–water solution (1:1, v/v), followed by 50 mL of water.".

7) The authors mention NMR measurement at 80 degrees to average rotamers. This is chemically very interesting, and even though it was not the focus of the paper, I would suggest, if possible, that the authors include one representative room-temperature spectra and provide a brief discussion.

8) Section 3.1.1: concentration ranges should be provided.

9) "... and it was obtained by interpolation from linear regression analysis". Isn't the DPPH curve sigmoidal?

10) Line 377: "broad" instead of "brought".

11) Lines 373-375: perhaps the authors could add a sentence that would corroborate the observed regioselectivity (since the reaction occurred only on the less hindered ortho position of sesamol).

12) Have the authors used a positive control in the assays (other than the parent compound, sesamol)? It seems that the IC₅₀ values for quercetin and rutin were taken from previous work (refs. 28, 31), but these compounds should have been tested together with the prepared hybrids. Also, why were compounds 6c–8c not evaluated in the antioxidant assays?

13) Lines 405-412: the claim that 6a and 6b show better activity than sesamol (this claim is repeated in the Conclusions) is too optimistic; the improvement is very modest (9.20 ± 1.0 and 11.0 ± 1.0 vs 11.60 ± 1.5) and might not be even statistically significant.

Author Response

Response to Reviewer’s Comments

Dear Reviewer, thank you for your effort to provide timely review and scientific decision on our manuscript.

The manuscript by Bachvarova et al. describes the synthesis of nine novel sesamol-based hybrids. Some of the prepared compounds were evaluated using DPPH and ABTS assays. The chemistry is solid, the yields are good, the compounds were thoroughly characterized, and the spectroscopic data are of high quality. The biological evaluation is the weaker aspect of the study, as the improvement in antioxidant activity is modest. Overall, the paper is suitable for publication in AppliedChem but, in my opinion, requires major revision.

Author’s comment: Thank you for the positive point of view, scientific opinion and additional corrections on our work. The revision made significantly improved the text and scientific appearance of the manuscript.

 

 

 

 

Reviewer’s comments and suggestions:

1. I suggest that the authors rewrite the Introduction as it is heavily cancer-focused, yet only antioxidant activity was investigated in the manuscript. Author’s response: Thank you for the correct remark. The Introduction is clearly revised in the corrected version of the manuscript.

 

2. Schemes showing the prepared compounds in Materials and Methods section are unnecessary as these structures are already shown in Scheme 1. I suggest moving the NMR data (together with the individual structures) to Supplementary Information file.

Author’s response: Thank you for the clear suggestion. The structures of the products are removed in the revised version of the manuscript and pointed in figures of the supplementary information file. We corrected the manuscript according to your recommendation and journal AppliedChem requirements for data conceptualization.

 

3. Line 107: "petroleum ether/diethyl ether" instead of "petroleum/diethyl ether"; Line 98: there is a typo "... (TLC) was performed done...".

Author’s response: It is clearly revised in the corrected version of the manuscript.

 

4. Line 99: please provide the particle size and manufacturer of silica gel. 

Author’s response: It is corrected in the revised version of the manuscript.

 

5. Sections 2.1.2 and 2.1.3: Please specify which compounds were prepared by each method (e.g., 6a-c with 2.1.2 and the rest with 2.1.3).

Author’s response: Thank you for the correct remark. It is revised in the corrected manuscript version.

 

6. Line 118: would the term "wash" be more appropriate than "extraction" since the organic layer was washed to remove Et₃N and residual imidazole? E.g., "The organic phase was washed with 30 mL of hydrochloric acid–water solution (1:1, v/v), followed by 50 mL of water.".

Author’s response: The sentence is revised in the corrected version of the manuscript.

 

7. The authors mention NMR measurement at 80 degrees to average rotamers. This is chemically very interesting, and even though it was not the focus of the paper, I would suggest, if possible, that the authors include one representative room-temperature spectra and provide a brief discussion.

Author’s response: Thank you for the observation. The recorded NMR spectra of the amidoalkylated Sesamol derivatives at 20 ^oC or CDCl₃ as solvent, indicated slow rotameric interconversions with registration of strongly broadened or doubled signals, which made their correct interpretation impossible. This specificity was previously observed from us in the context of the synthesis of various phenolic and indole hybrids. [Y. Stremski, S. Statkova-Abeghe, P. Angelov, I. Ivanov, Synthesis of Camalexin and related analogues, Journal of heterocyclic chemistry, 7, 55, 2018, 1589-1595; https://doi.org/10.1002/jhet.3192]; [Y. Stremski, D. Kirkova, S. Statkova-Abeghe, P. Angelov, I. Ivanov, D. Georgiev, "Synthesis and antibacterial activity of hydroxylated 2-arylbenzothiazole derivatives", Synthetic Communications, 50, 19, 2020, 3007-3015; https://doi.org/10.1080/00397911.2020.1791342]; [D. Kirkova, Y. Stremski, M. Bachvarova, M. Todorova, B. Goranov, S. Statkova-Abeghe and M. Docheva, Molecules, 30(1), 2025, 636; https://doi.org/10.3390/molecules30030636];

 

 

 

As example:

 

Figure 1. ¹H-NMR spectrum in CDCl₃ at room temperature, 600 MHz

 

Figure 2. ¹H-NMR spectrum in DMSO-d₆ at 80 ^oC, 600 MHz

According to your recommendation, the additional text is added and specified in the revised version of the manuscript (3. Results and Discussion).

 

8. Section 3.1.1: concentration ranges should be provided.

Author’s response: Thank you for the recommendation. The radical scavenging activity of the tested compounds was evaluated over a concentration range of 1–250 ppm using a series of two-fold dilutions. The linear range of each compound differed, but in all cases it fell within the tested concentration interval. The text is added in the revised version of the manuscript.

 

9. "... and it was obtained by interpolation from linear regression analysis". Isn't the DPPH curve sigmoidal?

Author’s response: It is clear observation. The method is also described in [refs. 29-32]. The text of the manuscript is revised and simplified according to the recommendation.

 

10. Line 377: "broad" instead of "brought".

Author’s response: The word is corrected in the revised version of the manuscript.

 

11. Lines 373-375: perhaps the authors could add a sentence that would corroborate the observed regioselectivity (since the reaction occurred only on the less hindered ortho position of sesamol).

Author’s response: It is done in the revised version of the manuscript.

 

12. Have the authors used a positive control in the assays (other than the parent compound, sesamol)? It seems that the IC₅₀values for quercetin and rutin were taken from previous work (refs. 28, 31), but these compounds should have been tested together with the prepared hybrids. Also, why were compounds 6c–8c not evaluated in the antioxidant assays?

Author’s response: All compounds, including quercetin and rutin, were evaluated using the same experimental methodology [refs. 29-32] as applied to the newly prepared hybrids. To avoid unnecessary repetition and to facilitate direct comparison, we have cited previously reported IC₅₀ values for these reference compounds (refs. 29, 32). We emphasize that the cited data are fully compatible with our assay conditions and provide a reliable assessing the activity of the new hybrids.

The structures of compounds 6c, 7c, and 8c differ from those of the other compounds in that they contain a 2,2,2-trichloroethoxycarbonyl (Troc) substituent. The presence of the three strongly electronegative chlorine atoms significantly decreases the electron density in the aromatic system, which destabilizes the radical and could reduce the antioxidant activity. Additionally, the bulky nature of the Troc group may introduce steric hindrance and limit the access of free radicals to the active site.

Тhe following article was referenced for information on the antioxidant activity of phenolic compounds: [Brigati, G.; Lucarini, M.; Mugnaini, V.; Pedulli, G.F. Determination of the Substituent Effect on the O–H Bond Dissociation Enthalpies of Phenolic Antioxidants by the EPR Radical Equilibration Technique. J. Org. Chem. 2002, 67, 4828–4832. https://doi.org/10.1021/jo025755y]

 

13. Lines 405-412: the claim that 6a and 6b show better activity than sesamol (this claim is repeated in the Conclusions) is too optimistic; the improvement is very modest (9.20 ± 1.0 and 11.0 ± 1.0 vs 11.60 ± 1.5) and might not be even statistically significant.

Author’s response: Thank you for the correct observation. The radical-scavenging activity examined with DPPH assay of the obtained products is comparable to Sesamol. The claim is revised in the corrected version of the text in the manuscript.

 

The all remarks are taken into account. In this regard, the additional corrections of manuscript are made and marked (track changes).

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I would like to thank the authors for addressing the points from the review.