Deep eutectic solvent for an expeditious sono-synthesis of novel series of bis-quinazolin-4-one derivatives as potential anti-cancer agents

To produce a new category of anti-cancer compounds, a facile and environmentally sustainable method for preparing diversified bis-quinazolinones was demonstrated using recyclable deep eutectic solvent (DES) under ultrasonic irradiation. The reactions were performed smoothly with a wide scope of substrates affording the desired derivatives in good-to-excellent yields under an atom-economical pathway. Particularly, halogen substituents that are amenable for further synthetic elaborations are well tolerated. Furthermore, the ‘greenness’ of the protocol was assessed within the scope of several green metrics and found to display an excellent score in the specified parameters. Cytotoxic activity of all novel bis-quinazolinones was investigated utilizing two cancer cell lines: breast (MCF-7) and lung (A549) cell lines and their IC50 values were determined. Most of the prepared derivatives displayed fascinating inhibitory activity with IC50 values in a low micromolar range. Remarkably, the derivative 7e [3,3'-(sulfonylbis(4,1-phenylene))bis(2-methyl-6-nitroquinazolin-4(3H)-one)] showed superior potency against MCF-7 and A549 cancer cell lines, with IC50 values of 1.26 µM and 2.75 µM, respectively. Moreover, this derivative was found to have low toxicity to the normal breast cell line (MCF-10A) and could serve as a promising lead candidate for further development.

6. The SI -The 1H NMR spectra do not have integrations. I also highly recommend including a structure of the molecule on the spectrum. This will facilitate data interpretation and confirm purity.

03-Jan-2019
Dear Professor Arafa: Title: Deep eutectic solvent for an expeditious sono-synthesis of novel series of bis-quinazolin-4one derivatives as potential anticancer agents Manuscript ID: RSOS-182046 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
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In addition to those comments, I have a couple of suggestions: (1) Table 3 does not add to the discussion as the same information can be found in Figure 3. Please move Table 3 to the supporting information.
(2) Please add structures to the 1H NMR spectra in the supporting information and add copies of the 13C NMR spectra.

RSC Subject Editor:
Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The manuscript entitled "Deep eutectic solvent for an expeditious sono-synthesis of novel series of bis-quinazolin-4-one derivatives as potential anticancer agents " by Arafa, describes a green protocol for synthesis of bis-quinazolin-4-one derivatives using deep eutectic solvent under ultrasonic irradiation. The new methodology performs for activating and deactivating starting substrates. The author describes a hypothetical mechanism presented in Scheme 5 that does not agree with me. In the first step the benzoxazine is activated by the components of DEES through the protonation of its carbonyl by tartaric acid. Hydrogen bonding is the main factor that influences the reactivity and selectivity of the process. The reversible hydrogen bonding between tartaric acid and carbonyl groups of benzoxazine make substrate-solvent complex activated. The next condensation of activated carbonyl group with the amine in the DES leads to the formation of a cationic intermediate with the following loss of a water molecule (Di Gioia et al. Molecules 2018, 23, 1891. Council to the author to completely review the hypothetical mechanism before publishing it, reporting the correct corrections with the bibliographic reference (Di Gioia et al. Molecules 2018Molecules , 23, 1891. The reference 21 is not relevant to the hypothesised reaction mechanism.
Reviewer: 2 Comments to the Author(s) See attached file Reviewer: 3 Comments to the Author(s) Arafa report the condensation of diamines with acetanthranil with the main novelty being the use of a "deep eutectic solvent." Admittedly, The key condensation reaction between diamines and acetanthranils has been known since as early as 1911, this work was not cited and should be, considering not only it's key precedent, but many of the same molecules are prepared in this work. Please note that the reference below does have the wrong structure for acetanthranils, an error likely due to the lack of spectroscopic methods from this time (early 1900's).
Journal of the American Chemical Society (1911), 33, 949-62 This age-old reaction appears to work well in DESs, and they author reports an improved scope.

RSOS-182046.R1 (Revision)
Review form: Reviewer 1 Is the manuscript scientifically sound in its present form? Yes

Comments to the Author(s) I recommend the publication of the manuscript as it is
Review form: Reviewer 2

Comments to the Author(s)
The author made all corrections/changes I suggested and this version seems to be better formatted and correct. The syntax problems also seemed to be solved. In my opinion, Figure 3 must show the values for cytotoxic activity against MCF-7 and A549 cell lines. Page 6, lines 36 and 37 "The obtained results conclude..."; please correct this sentence as "the results cannot conclude anything".

06-Feb-2019
Dear Professor Arafa: Title: Deep eutectic solvent for an expeditious sono-synthesis of novel series of bis-quinazolin-4one derivatives as potential anticancer agents Manuscript ID: RSOS-182046.R1 Thank you for submitting the above manuscript to Royal Society Open Science. On behalf of the Editors and the Royal Society of Chemistry, I am pleased to inform you that your manuscript will be accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the reviewers' comments at the end of this email.
The reviewers and handling editors have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your manuscript.
Because the schedule for publication is very tight, it is a condition of publication that you submit the revised version of your manuscript before 15-Feb-2019. Please note that the revision deadline will expire at 00.00am on this date. If you do not think you will be able to meet this date please let me know immediately.
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When uploading your revised files please make sure that you have: 1) A text file of the manuscript (tex, txt, rtf, docx or doc), references, tables (including captions) and figure captions. Do not upload a PDF as your "Main Document".
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Supplementary files will be published alongside the paper on the journal website and posted on the online figshare repository (https://figshare.com). The heading and legend provided for each supplementary file during the submission process will be used to create the figshare page, so please ensure these are accurate and informative so that your files can be found in searches. Files on figshare will be made available approximately one week before the accompanying article so that the supplementary material can be attributed a unique DOI. ************************************* RSC Associate Editor: Comments to the Author: The reviewers are generally pleased with the changes made to the previous manuscript. One reviewer has asked that one sentence be modified. I agree with this suggestion, as the results cannot conclude anything. The authors, however, are able to reach a conclusion. The same reviewer also appears to be asking for actual IC50 values to be added to Figure 3. I don't think this is necessary as the current version conveys the comparative data quite nicely and the actual values are available in the supporting information. There is even a note in the main text directing the reader to the SI for this information.
I will gladly accept a revised manuscript if the authors simply modify the offending sentence.

RSC Subject Editor:
Comments to the Author: (There are no comments.) ************************************** Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) I recommend the publication of the manuscript as it is Reviewer: 2 Comments to the Author(s) The author made all corrections/changes I suggested and this version seems to be better formatted and correct. The syntax problems also seemed to be solved. In my opinion, Figure

Deep eutectic solvent for an expeditious sono-synthesis of novel series of bis-quinazolin-4-one derivatives as potential anticancer agents
Arafa, W.

RSOS-182046
The article describes a study on the synthesis of bis-quinazolin-4-ones using a sonochemical approach in deep eutectic mixtures as solvent/catalyst. Although the reported results can be considered as a good contribution to the area, the author failed to furnish enough and accurate evidence to support his contribution. In addition, I recommend a careful English review of the text and I suggest the author to look into consideration the following main points.
1. Page 1, lines 44 and 59 (and others); "utilize" cannot be used as a noun and it must be changed by "the use" or "the utilization".  Table 2 are isolated or conversion.

Please indicate if yields shown in
6. Page 4, lines 30,31: not all reactions were quantitative as some described yields are not >99%. In addition purification by chromatography was not necessary but the products were purified by recrystallization. The efficiency of the method is over estimated in that sentence. 7. Page 3, lines 51-53: "Owing to the fact that heterocycles bearing halogens are significant building blocks in the assembly of plentiful of pharmaceuticals and natural products…" this sentence must be reviewed because its meaning is quite broad.
8. Page 4, lines 37-39: "Finally, the required product was formed through dehydration. From the proposed mechanism, it obvious concluded that, L-(+)-tartaric acid choline chloride performs a dual function; solvent and catalyst." I do not believe this conclusion is obvious. The author has not provided any mechanistic evidence (despite of any logical suggestions) for the study of the mechanism of such transformation. 10. Concerning the MTT test, it is important to mention that this test reveals cytotoxicity profiles, The "anticancer activity" term used throughout the text must be corrected.

Appendix A
11. The cytotoxicity activity was evaluated in normal cells? It is important to check the selectivity of the title compounds, using normal cell lines such as HUVEC (endothelial cells), human mammary epithelial cell (MRF10a), among many others. In this scenario, I was intrigued by the fact that the most active compound is a nitro-derivative (7e). Once biological active nitro-compounds are recognized as highly toxic, I speculate if 7e would also exhibit high cytotoxic activity against normal cell lines. I strongly recommend the author to run in vitro cytotoxicity evaluation against normal cell lines for the most active compounds.
12. Page 8, lines 52-54: halogen atoms and NO 2 group are not "electron deficient groups". They are electron withdrawing groups, modifying the electron density of the aromatic ring. Similarly, the methyl group is not "electron rich"; in fact the electron donating ability of alkyl groups is somewhat limited. I suggest this argumentation should be entirely reviewed.
13. Finally, in the Conclusion section, the author overestimated the "potential anticancer activity" of the studied compounds in the article. Once again, the in vitro evaluation of cytotoxic activity is only the first step in the development of an anticancer drug. I think it is not appropriate to use "anticancer activity" (in vivo) as synonym for "cytotoxic activity" (in vitro).

Entry
Reviewer Comments Responses 1. RSC Associate Editor Table 3 does not add to the discussion as the same information can be found in Figure 3. Please move Table 3 to the supporting information.

2.
Please add structures to the 1 H NMR spectra in the supporting information and add copies of the 13 C NMR spectra.

14.
Concerning the MTT test, it is important to mention that this test reveals cytotoxicity profiles, The "anticancer activity" term used throughout the text must be corrected. Done.

15.
The cytotoxicity activity was evaluated in normal cells? It is important to check the selectivity of the title compounds, using normal cell lines such as HUVEC (endothelial cells), human mammary epithelial cell (MRF10a), among many others. In this scenario, I was intrigued by the fact that the most active compound is a nitro-derivative (7e). Once biological active nitro-compounds are recognized as highly toxic, I speculate if 7e would also exhibit high cytotoxic activity against normal cell lines. I strongly recommend the author to run in vitro cytotoxicity evaluation against normal cell lines for the most active compounds.
The cytotoxicity activity, for the most active products, was evaluated in normal breast cell line (MCF-10A) and the results were mentioned in the discussion part (highlighted in yellow color).

16.
Page 8, lines 52-54: halogen atoms and NO 2 group are not "electron deficient groups". They are electron withdrawing groups, modifying the electron density of the aromatic ring. Similarly, the methyl group is not "electron rich"; in fact the electron donating ability of alkyl groups is somewhat limited. I suggest this argumentation should be entirely reviewed.

Done.
Both "electron deficient" and "electron rich" have been replaced by "electron withdrawing" and "electron donating", respectively.

17.
Finally, in the Conclusion section, the author overestimated the "potential anticancer activity" of the studied compounds in the article. Once again, the in vitro evaluation of cytotoxic activity is only the first step in the development of an anticancer drug. I think it is not appropriate to use "anticancer activity" (in vivo) as synonym for "cytotoxic activity" (in vitro). Done.

Reviewer: 3
The citation referenced above Done. "within reference No. 4" (highlighted in yellow color).

19.
Syntax. This is a big problem throughout the manuscript. Done.

20.
The mechanism -(a) the DES structure seems suspect: the chloride is binding to the carboxylates only on the tartaric acid. Is this likely the case? Shown is a 9-membered chelate. I would suspect that tartrate chelates to form smaller rings? Is there evidence to back this structure up? Is this just a simplification of the DES? (b) an imine with only one long pair is shown having H-bonds to two protons. This is impossible... (c) L-(+)-Tartaric acid's stereochemistry is omitted.
a) The chloride is now binding to both the carboxylate on the tartaric acid and choline moiety. b) The proposed mechanism has been changed. c) L-(+)-Tartaric acid isomer was used in the preparation of DES while, other stereoisomers did not. For simplification, L-(+)-Tartaric acid was drawn in Chart 5 without regarding to its stereochemistry.

21.
This reviewer does not understand the temperature column in Table 1. (i.e. 50/US) I think it might be an abbreviation for Ultrasound. Why is this in the termperature column if this is the case? It is confusing.
• An abbreviation for Ultrasound has been mentioned along with Table 1. (highlighted in yellow color). • To the column of temperature, word "Method" has been added.

22.
The SI -There are only 1 H NMRs shown? If this is to the journals standard then OK. Include 13 C spectra if journal requires this.
Done, copies of 13 C NMR spectra have been attached with SI file.

23.
The SI -The 1 H NMR spectra do not have integrations. I also highly recommend including a structure of the molecule on the spectrum. This will facilitate data • All details about integrations were mentioned in details in experimental part.
• All chemical structures have interpretation and confirm purity.
been attached within the spectra.