Synthesis and pharmacological characterization of glucopyranosyl-conjugated benzyl derivatives as novel selective cytotoxic agents against colon cancer

Glucopyranosyl-conjugated benzyl derivatives containing a [1,2,3]-triazole linker were synthesized. Benzyl served as an important pharmacophore in anti-cancer compounds. Compound 8d inhibited the proliferation of colorectal cancer cells with the potency comparable to 5-fluorouracil (5-FU) with improved selectivity towards cancer cells. The antiproliferative activity of 8d is achieved through triggering apoptotic cell death.

[1]" 5) Introduction, page 1: You write that "Benzyl and its derivatives are important pharmacophore in many anticancer compounds". So I suggest you add a Figure showing the structures of some of these compounds. 6) Introduction, page 2: Change "by the "click chemistry"" to "by "click chemistry" (2 times) 7) Introduction, page 2: Change "compling" to "coupling" 8) Introduction, page 2: Change "conjuagate" to "conjugate"; "conjuating" to "conjugating"; "conjuate" to "conjugate" 9) Introduction, page 2: At the end of the introduction section, I would be clearer saying that your target compounds were the one with modification at C2. 10) Results and discussion, page 2: "8a-i" must be bolded; "4" must be bolded 11) Results and discussion, page 2: Change "itermediate" to "intermediate"; "avaliable" to "available"; "chromatagraphy" to "chromatography" 12) Results and discussion, page 2: Change "by the deacetylation" to "by deacetylation" 13) Results and discussion, page 2: You write that "All the products were purified by silica gel chromatography". I would add by which methods the compounds were characterized. 14) Scheme 1, page 3: For compounds 3 and 4, what was the anomeric ratio? Why was only compound 9d deprotected? 15) Scheme 1 caption: Change "Conditions of reactions" to "reaction conditions"; "4" must be bolded. Add a "space" between "4" and "h". Delete the "space" between "N," and "N" 16) Title of section 3.2: I would rather write "The cytotoxicity of the synthesized compounds against…" 17) Section 3.2, page 3: Change "coloreactal" to "colorectal" 18) Section 3.2, page 3: Please define "MTS"; Change "via MTS" to "via a MTS". Deleted "the" before "cancer cells"; Change "to HCT-116" to "toward HCT-116"; Change "antiproliferation activity is fairly" to "antiproliferative activity was fairly". Delete "," after "8b-8i"; Change "showed 23 fold" to "showed a 23-fold" 19) Table 1: It would have been interesting to test peracetylated glucose as a control. The standard deviations ±SD must be written with only one significative figures and the values of the IC50 must be rounded up accordingly. For instance, "4.23 ± 1.98" should be written as "4 ± 2" and so on for the other values. 20) Page 4: Sections 3.3 and 3.4 are identical!!! Please delete one section and changer section numbering accordingly.

Decision letter (RSOS-201642.R0)
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. ********************************************** RSC Associate Editor: Comments to the Author: The authors have expressed enthusiasm for the work as a whole, but believe the manuscript could be improved by further editing. In addition, Reviewer 2 has requested that all compounds be tested again as single anomers, rather than the anomeric mixtures that were already tested. it is not clear to me how feasible this will be, or how improved the results would be by doing this.
Rather than attempting what would likely be a tedious separation for every compound, I suggest performing this purification/reanalysis of compound 8d (much like the authors already did by testing 9d). I also agree with Reviewer 2 that judging purities by TLC is not sufficient. At the very least, HPLC purities should be reported (I do note that the provided NMR spectra do show the compounds are of relatively high purity).
In addition to the extensive grammatical and typographical corrections suggested by Reviewer 2, I suggest changing the anomer ratios provided in the experimental section so that they all have the same format, e.g. 2.7:1 instead of 100:37. This will make it easier for readers to compare the different ratios.

RSC Subject Editor:
Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The manuscript RSOS-201642 is devoted to the synthesis of novel Glucopyranosyl conjugated benzyl derivatives as potential anticancer agents and can be interested to the specialists working in this field. The reviewed article is interesting and theme of the article meets the scope of the journal. Work is performed at sufficient scientific level and has good quality; the results of investigation are professionally interpreted. However, it needs major revision before publication.
To improve the quality and perception of the manuscript I would suggest paying attention to following comments: 1) All 13C NMR should be to 1 dp, but not to 2 dp (experimental part). 2) Moderate English changes required. There are grammar and orthographical errors in the manuscript, which should be corrected.
My decision is minor revision.

Reviewer: 2
Comments to the Author(s) The paper of Fu and co-workers deals with the synthesis and cytotoxic activity of glucopyranosyl bearing benzyl derivatives. The main issue with this paper is that the synthesized compounds were tested as mixtures of anomers (in different α,β ratios) and not as pure compounds. Because of this, it is almost impossible to compare the cytotoxic activities of the different derivatives and to conclude about possible structure-activity relationships. I suggest that the authors isolate both the anomers and test them independently. Furthermore, I have found a lot of typographical errors and the manuscript bears the stamp of a non-native English speaker. I suggest the authors ask a Native-English speaker to carefully check their manuscript. I have also other corrections that are written below: 1) Title: Change "for" to "against" 2) Introduction, page 1: Change "5-fluorouracil" to "5-Fluorouracil" 3) Introduction, page 1: Change "to limit" for "limiting" 4) Introduction, page 1: Change ".1" to ".[1]" 5) Introduction, page 1: You write that "Benzyl and its derivatives are important pharmacophore in many anticancer compounds". So I suggest you add a Figure showing the structures of some of these compounds. 6) Introduction, page 2: Change "by the "click chemistry"" to "by "click chemistry" (2 times) 7) Introduction, page 2: Change "compling" to "coupling" 8) Introduction, page 2: Change "conjuagate" to "conjugate"; "conjuating" to "conjugating"; "conjuate" to "conjugate" 9) Introduction, page 2: At the end of the introduction section, I would be clearer saying that your target compounds were the one with modification at C2. 10) Results and discussion, page 2: "8a-i" must be bolded; "4" must be bolded 11) Results and discussion, page 2: Change "itermediate" to "intermediate"; "avaliable" to "available"; "chromatagraphy" to "chromatography" 12) Results and discussion, page 2: Change "by the deacetylation" to "by deacetylation" 13) Results and discussion, page 2: You write that "All the products were purified by silica gel chromatography". I would add by which methods the compounds were characterized. 14) Scheme 1, page 3: For compounds 3 and 4, what was the anomeric ratio? Why was only compound 9d deprotected? 15) Scheme 1 caption: Change "Conditions of reactions" to "reaction conditions"; "4" must be bolded. Add a "space" between "4" and "h". Delete the "space" between "N," and "N" 16) Title of section 3.2: I would rather write "The cytotoxicity of the synthesized compounds against…" 17) Section 3.2, page 3: Change "coloreactal" to "colorectal" 18) Section 3.2, page 3: Please define "MTS"; Change "via MTS" to "via a MTS". Deleted "the" before "cancer cells"; Change "to HCT-116" to "toward HCT-116"; Change "antiproliferation activity is fairly" to "antiproliferative activity was fairly". Delete "," after "8b-8i"; Change "showed 23 fold" to "showed a 23-fold" 19) Table 1: It would have been interesting to test peracetylated glucose as a control. The standard deviations ±SD must be written with only one significative figures and the values of the IC50 must be rounded up accordingly. For instance, "4.23 ± 1.98" should be written as "4 ± 2" and so on for the other values. 20) Page 4: Sections 3.3 and 3.4 are identical!!! Please delete one section and changer section numbering accordingly. 21) Page 4, section 3.3: Change "aganist" to "against"; "As showed" to "As shown"; " Figure 1A, 1C and 1D" to "Figures 1A, 1C, and 1D"; "8d" must be bolded. Change " Figure 1C &1D" to " Figures 1C and 1D" 22) Conclusion: Change "aganist" to "against" (2 times); "selectiity" to "selectivity"; "nomal" to "normal"; "subsitution" to "substitution"; "para-position" to "para position"; "influences" to "influenced"; "theapetuic" to "therapeutic"; "is from inducing" to "was shown to induce" 23) Section 5.1: Change "propargyl" to "Propargyl"; Change "and removed" by "and solvents were removed". 24) Section 5.1: You write that "purities of the intermediates were established by TLC"!!! It is only possible to assess qualitatively the purity by TLC. The proper way to do it is by HPLC and/or elemental analyses. 25) Section 5.1: Change "Thin Layer" to "Thin layer"; "water used was distilled" to "water was distilled" 26) Section 5.1: Add a "space" between "400" and "MHz" and between "100" and "MHz" (please correct this anywhere it appears in the manuscript" 27) Experimental: Please add the aspect of all compounds. The letter "D" in sugars must be written in small capital letters. The "O" must be italicized in compound names. Delete the additional "space" between "of" and "compound"; add an "s" to "compound 7b-7i" 28) Experimental: Change "3-bromopropyne" to "3-Bromopropyne"; "completeion" to "completion"; "silca gel" to "silica gel" -8i"; add a "space" between "1" and "mL"; delete the "space" between "N," and "N-dimethylformamide"; change "The organic layers were dried" by "The organic layer was dried"; change "sodium chloride" to "saturated aqueous sodium chloride solution" 30) Experimental: Delete the unneeded "spaces" in compound names. 31) Section 5.6: Change "the incubated compound 8d" to "then compound 8d was incubated"; change "after fixed" to "after being fixed" 32) Section 5.7: Change "following treated" to "following treatment" 33) Section 5.9: Add a "space" between "5.9" and "Annexin" 34) Supporting information: Change "HR-MS" to HRMS"; α,β anomeric ratios must be written for each compound such as for 9b. For 9b, the α,β anomeric ratio is written but this is an equilibrium. Was the NMR spectrum taken at equilibrium?

Decision letter (RSOS-201642.R1)
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With our best for a peaceful festive period and New Year, and we look forward to working with you in 2021. We are submitting the revised manuscript (RSOS-201642) entitled " Synthesis and Pharmacological Characterization of Glucopyranosyl-conjugated Benzyl Derivatives as Novel Selective Cytotoxic Agents for Colon Cancer ". We thank both reviewers and editors for their constructive comments and suggestions based on which the manuscript has been revised. As suggested by reviewers, we performed some new experiments on the effect of compounds 4 and 2-amino-1,3,4,6-O-acetyl-D-glucose(AOAG) as peracetylated glucose derivatives. The results showed that 4 and AOAG had almost no cytotoxicty toward HCT-116 and 293T.

Dear
We separated all the compounds by preparation thin layers. Two different anomers were isolated. When two anomers were tested by 1 H NMR, respectivly, we found that they were still the mixture of α, β anomers again in every anomer sample. The ratio of α/β anomers were fixed. There was an dynamic equilibrium among anomers. The HPLC purities of partial compounds compounds 8a-8i were attached in supporting information.
We have changed the anomer ratios according to the suggestions of Reviewer 2 provided in the experimental section. Some minor points suggested by reviewers has been revised. All changes are marked in blue. We also responded point-by-point to all questions raised by reviewers. We hope the revised version will meet the requirement for publication in Royal Society Open Science.
We will enclose our responses with the resubmission. This resubmission has been approved by all authors. We declare that this manuscript has not been published or being published previously in whole or in part elsewhere.
Thank you very much for allowing us to revise the manuscript. requested that all compounds be tested again as single anomers, rather than the anomeric mixtures that were already tested. it is not clear to me how feasible this will be, or how improved the results would be by doing this. Rather than attempting what would likely be a tedious separation for every compound, I suggest performing this purification/reanalysis of compound 8d (much like the authors already did by testing 9d). I also agree with Reviewer 2 that judging purities by TLC is not sufficient. At the very least, HPLC purities should be reported (I do note that the provided NMR spectra do show the compounds are of relatively high purity).
In addition to the extensive grammatical and typographical corrections suggested by Reviewer 2, I suggest changing the anomer ratios provided in the experimental section so that they all have the same format, e.g. 2.7:1 instead of 100:37. This will make it easier for readers to compare the different ratios.
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The manuscript RSOS-201642 is devoted to the synthesis of novel Glucopyranosyl conjugated benzyl derivatives as potential anticancer agents and can be interested to the specialists working in this field. The reviewed article is interesting and theme of the article meets the scope of the journal. Work is performed at sufficient scientific level and has good quality; the results of investigation are professionally interpreted. However, it needs major revision before publication.
To improve the quality and perception of the manuscript I would suggest paying attention to following comments: My decision is minor revision. 1) All 13 C NMR should be to 1 dp, but not to 2 dp (experimental part).
Answer: Thank you for good suggestions. The results of all the 13 C NMR have been changed into 1dp in experimental part.
2) Moderate English changes required. There are grammar and orthographical errors in the manuscript, which should be corrected.
Answer: Thank you for good suggestions. English in the manuscript has been revised. Grammar and orthographical errors in the manuscript have been corrected.
Reviewer: 2 Comments to the Author(s) The paper of Fu and co-workers deals with the synthesis and cytotoxic activity of glucopyranosyl bearing benzyl derivatives. The main issue with this paper is that the synthesized compounds were tested as mixtures of anomers (in different α,β ratios) and not as pure compounds. Because of this, it is almost impossible to compare the cytotoxic activities of the different derivatives and to conclude about possible structure-activity relationships. I suggest that the authors isolate both the anomers and test them independently. Furthermore, I have found a lot of typographical errors and the manuscript bears the stamp of a non-native English speaker. I suggest the authors ask a Native-English speaker to carefully check their manuscript. I have also other corrections that are written below: 1) Title: Change "for" to "against" Answer: Thanks the reviewer for the good suggestions, we have changed "for" to "against" in title.
3) Introduction, page 1: Change "to limit" for "limiting" Answer: Thanks the reviewer for the good suggestions, we have changed "to limit" for "limiting" in introduction.
[1]" in introduction, page 1. 5) Introduction, page 1: You write that "Benzyl and its derivatives are important pharmacophore in many anticancer compounds". So I suggest you add a Figure  showing the structures of some of these compounds.
Answer: Thanks the reviewer for the good suggestions. Figure 1 showing the structures of some anticancer compounds with benzyl as pharmacophore (page 1: ) has been inserted into in Introduction .