Review Reports

Round 1

Reviewer 1 Report

The manuscript "Synthesis and anticholinesterase evaluation of cassine, spec-2 taline and analogues" by Silva et al reports synthesis and biological evaluation of twelve analogues of piperidine alkaloids against acetylcholinesterase and butyrylcholinesterase inhibition.

The introduction is well written and results are well presented. The conclusion matches the results obtained from conducted research. Aprt from that, the manuscript have some minor linguistic and structural errors that need to be corrected, such as in:

line 729 &730: "This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn." must be deleted.

Line 853 "cassine (1) and spectaline (3) prepared performed better that the parent compounds in" That should be replaced with "than"

Author Response

Dear Reviewer #1,

Thank you for your evaluation and comments on the submitted manuscript which we appreciated.

We have incorporated all the corrections you pointed out:

p. 729-730: 

"This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn." must be deleted. This sentence was deleted.  

Line 853 "cassine (1) and spectaline (3) prepared performed better that the parent compounds in" That should be replaced with "than". The replacement was incorporated.      

Reviewer 2 Report

The manuscript by Silva et al. entitled Synthesis and anticholinesterase evaluation of cassine, spec-2 taline and analogues ' reports the synthesis of twelve analogues of piperidine alkaloids (-)-cassine and (-)-spectaline. The authors tested in-vitro effect of the compounds on inhibition of acetylcholinesterase and butyrylcholinesterase. Overall manuscript is well well written; clear, precise, and easy to understand. However minor comments need to be addressed before publication.

Minor Comments

The rational of current study is poorly described, it seems that the authors just select the compounds and randomly screened for anticholinesterase activity. Authors should conceive a strong rational for the current study.

Abstract section needs more details, for example mentioned IC50 values in abstract

Place the graphs obtained for determination of IC50 for most potent compound in the item results and graphs of the other compounds in supplementary data.

Is it possible to anticipate potential side effects of these drugs? What are the risks in this domain?

The evaluation of the kinetic mechanism is poorly (and trivially) described.

The presentation of the results could be improved, add graphs for kinetic mechanism study. Add compound’s chemical structure in table with IC50

Authors are suggested to perform molecular docking and dynamic simulations.

Authors are suggested to add latest citations.

Author Response

Dear Reviewer #2,

Thanks for your evaluation and comments on the submitted manuscript. We very much appreciated your contributions to improve it. Our responses can be found below:

-"The rational of current study is poorly described, it seems that the authors just select the compounds and randomly screened for anticholinesterase activity. Authors should conceive a strong rational for the current study." Authors' answer: The rational to conduct this study was based on previous biological results described in the literature regarding the effect of mixtures of (-)-cassine and (-)-spectaline, isolated from S. spectabilis, among other species. We aimed to provide an useful synthetic approach to provide pure samples of these two natural products as well analogues to support further biological studies. The docking studies reported previously in the literature (see, reference 21) suggested that the size of the side chain may play a role in the anticholinesterase activity and therefore we were interested to probe the minimum structural requirements to preserve the anti-cholinesterase activity as well the role played by the methyl group in the piperidine ring and the presence of double bonds in the ring and in the side chain. In fact, compound 10c was shown to be more potent than both natural products (1 and 3) when one considers its activity against human butyryl cholinesterase.

- "Abstract section needs more details, for example mentioned IC50 values in abstract". Authors' answer:  IC50 values were included in the Abstract section as suggested.

- "Place the graphs obtained for determination of IC50 for most potent compound in the item results and graphs of the other compounds in supplementary data." Authors' answer: Done as suggested.

-"Is it possible to anticipate potential side effects of these drugs? What are the risks in this domain?"  Authors' answer: Pharmacological studies on cassine and spectaline are at very early stages and we are not aware of any pre-clinical or clinical studies addressing potential side effects. As to the other analogues reported here, they are reported for the first time in the literature and further studies are needed to address this question.

- "The evaluation of the kinetic mechanism is poorly (and trivially) described." Authors' answer: We respectfully disagree as the data provided for the mechanistic studies speak for themselves. We have included the graphs for the most potent compounds in the text and in the Supplementary Material for the others.

- "The presentation of the results could be improved, add graphs for kinetic mechanism study. Add compound’s chemical structure in table with IC50". Authors' answer: Done as requested.

- "Authors are suggested to perform molecular docking and dynamic simulations." Authors' answer: 

We thank the referee for this suggestion. As we explained above, our research plan was based on docking studies already reported in the literature for this family of natural products and AChE. As to dynamic simulations, we do not have the expertise to do that ourselves and we will have to approach other groups to join us in these studies.  At this point, the work reported here is an experimental assessment of the structural requirements for the inhibition of cholinesterases: it clearly demonstrates that the presence of the methyl group is not  mandatory and that the presence of double bonds in the piperidine ring and in the side chain contribute to increase the inhibition of butyrylcholinesterase. These findings set the stage for further investigations which will certainly benefit from molecular dynamics simulations.   

- "Authors are suggested to add latest citations." Authors' answer:  We have included recent relevant references in the revised version of the manuscript (references 13, 14 and 41).