Structure and Neuroprotector Properties of a Complex Compound of Lithium with Comenic Acid

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

In the work of a team of authors, a lithium compound with comenic acid anion was synthesized, its structure was studied using a set of methods, including X-ray diffraction for the solid phase.  Neuroprotective activity and antioxidant effect of the lithium salt were studied.  Despite the fact that only one compound is described, it has been carefully and comprehensively studied.  The presented material has interdisciplinary character (inorganic chemistry, structural studies, biological activity) and corresponds to the scientific field of the journal.

 

In my understanding, the authors are aimed at creating a drug that has a combined effect - antioxidant and neuroprotective.  For this purpose, they combine the comenic acid anion (an antioxidant) and lithium ion (compounds of which can be neuroprotective).  The summation of effects leads to an increase in the neuroprotective effect of the salt in the presence of glutamate.

 

The authors comprehensively studied lithium comenate, studying its structure and its multiple biological effects, this is the novelty of the work.  There are publications by the same group of authors on the biological activity of lithium comenate, but these works (https://pubmed.ncbi.nlm.nih.gov/24228481/) study individual aspects of bioactivity (the effect of salt on the state of oxidative processes).

 

It can be recommended to expand the number of objects being compared - expanding to other cations and organic anions for comparison under the same conditions.

 

The conclusions for the work are drawn up in accordance with the results obtained.

A sufficient number of references are provided, they are appropriate.

 

The work can be published after minor issues are corrected.

 

Abstract.  When first mentioning comenic acid, it is advisable to give its abbreviation (in line 2 - H2Com), which is mentioned below (HCom in line 9 as the monodeprotonated form).

 

Page 4, the end of section 2.1.2: it should be explained what signals 81, 82, 83, which are not observed in the mass spectrum, should correspond to.

Author Response

The authors are grateful to the reviewer for his high assessment of our work.

Below are responses to the comments:

Comment: Abstract.  When first mentioning comenic acid, it is advisable to give its abbreviation (in line 2 - H2Com), which is mentioned below (HCom in line 9 as the monodeprotonated form).

Answer: Thank you, we added “H2Com” in line 2.

 

Comment: Page 4, the end of section 2.1.2: it should be explained what signals 81, 82, 83, which are not observed in the mass spectrum, should correspond to.

 

Answer: The authors would like to note that in work [21], which we refer to in the text, it is noted that it is difficult definitely determine the decomposition products of comenic acid due to the large number of possible options for the formation of dehydration fragments having mass peaks 81, 82, 83.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Authors reported the structure, antioxidant and neuroprotective properties of lithium comenate (lithium 5-hydroxy-4-oxo-4H-pyran-2-carboxylate) in the current work. This study is well-established and well-presented. It can be accepted after minor revision.

Here are some points:

·      It was noticed that authors recently published the studies on “magnesium comenate” (Int. J. Mol. Sci. 2023, 24(9), 8046; https://doi.org/10.3390/ijms24098046). This paper is about the studies on “lithium comenate”. Examining the studies of the both papers, this study looks less comprehensive.

·      There is not a drug called “Baliz-2” in the market. This should be corrected or if it is confirmed it is a drug, then it should be indicated with ®.

·      What is the superiority of neuroprotective effects of “lithium comenate” to “magnesium comenate”? Authors should discuss bot results in “Discussion” part.

·      Introduction is too short authors should expand it with more appropriate references.

·      The word “Continuing,….” should be changed as “subsequently” or “later on”,etc. and dot should be added before it.

·      “Lithium ions, due to their similarity to magnesium ions, can probably also lead to blocking of NMDA receptor ion channels and a decrease in Ca2+ currents.” This phrase is a very strong statement to conclude.

Author Response

The authors would like to thank the reviewer for his very thoughtful consideration of our work.

 

Comment: It was noticed that authors recently published the studies on “magnesium comenate” (Int. J. Mol. Sci. 2023, 24(9), 8046; https://doi.org/10.3390/ijms24098046). This paper is about the studies on “lithium comenate”. Examining the studies of the both papers, this study looks less comprehensive.

 

Answer:  The authors are grateful for the comment, but we believe that the study of the structure of the substance was carried out using similar methods. From the data obtained, the structure of lithium comenate has been completely studied. The neuroprotective potential of the substance has also been established in biological studies.

 

Comment: There is not a drug called “Baliz-2” in the market. This should be corrected or if it is confirmed it is a drug, then it should be indicated with ®.

Answer:  The drug Baliz 2 is not currently produced and is not commercially available, but it is included in the register of medicines https://www.rlsnet.ru/drugs/baliz-2-5980.

 

Comment: What is the superiority of neuroprotective effects of “lithium comenate” to “magnesium comenate”? Authors should discuss bot results in “Discussion” part.

 Answer: The authors are grateful for your comments. We believe that lithium and magnesium cations have different therapeutic effects and can be used in different areas of neuroprotection. Therefore, comparing the effectiveness of these substances was not one of the objectives of the study.

 

Comment: Introduction is too short authors should expand it with more appropriate references.

Answer: The authors tried to write the introduction section in lapidary style so as not to distract the reader from the main idea of the article. However, the introduction section contains 21 references to literary sources and it reflects all the main motivations for performing this work.

 

 

Comment: The word “Continuing,….” should be changed as “subsequently” or “later on”,etc. and dot should be added before it.

Answer: Thanks for the comment, we put a dot and replaced the word with “subsequently”.

 

Comment: “Lithium ions, due to their similarity to magnesium ions, can probably also lead to blocking of NMDA receptor ion channels and a decrease in Ca2+ currents.” This phrase is a very strong statement to conclude.

Answer: The authors agree with the reviewer's comment. This phrase has been removed from the text.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Kozin et al. report on a lithium complex of comenic acid (5-ydroxy-4-oxo-4H-pyran-2-carboxylic acid), which was structurally characterized and whose neuroprotective activity was investigated. In my opinion, the significance of the work does not justify publication in IJMS. I suggest publication in MPDI Chemistry or so after revision.

Some routine characterization data such as IR spectra should be placed in the supplementary materials.

It struck me that assignments for 13C NMR signals have been made. This should usually only be done on the basis of 2D NMR spectra. It is not necessary to elaborate on minor differences in the NMR shifts for free comenic acid and the lithium salt (section 2.1.4). It can be expected that the complex is mostly dissociated in a D2O solution.

It is stated several times throughout that the compound crystallizes in the "chiral (non-centrosymmetric) space group Pna2₁", which is not correct. Although the space group Pna2₁ is non-centrosymmetric, it is certainly not a "chiral" (or Sohncke space group) but a polar space group. Hence, there are certainly no "independent molecules of different chirality, that is, a mixture of enantiomers" in the crystal structure, as stated in section 2.1.7. Please revise.

In Figure 5, it looks as if there is a dotted line between H atoms, which makes no sense to me.

There are some issues with the crystal structure refinement, which should be fixed ahead of publication:

- The appropriate TEMP instruction in SHELXL should be used in the final refinement (see checkCIF level B alert).

- With regard to the checkCIF level C alert concerning the poor data/parameter ratio, it is inappropriate to refine H atoms freely. Those bonded to carbon should be refined using the appropriate riding model (i.e. AFIX 43 in this case). The H atoms bonded to oxygen should be refined with the O-H distance restrained to, say, 0.84(2) A by using DFIX instructions and with U_iso(H) = 1.2 U_eq(O). This should also resolve the level C alert regarding the U_iso(max) / U_iso(min) range.

- Minor point: the atom list should be sorted before the final CIF file is written.

Author Response

Reviewer #1:

Comments and Suggestions for Authors

Kozin et al. report on a lithium complex of comenic acid (5-ydroxy-4-oxo-4H-pyran-2-carboxylic acid), which was structurally characterized and whose neuroprotective activity was investigated. In my opinion, the significance of the work does not justify publication in IJMS. I suggest publication in MPDI Chemistry or so after revision.

Some routine characterization data such as IR spectra should be placed in the supplementary materials.

Thanks for the comment! As suggested, FTIR spectra were placed in the supplementary materials file.                                                          

It struck me that assignments for 13C NMR signals have been made. This should usually only be done on the basis of 2D NMR spectra. It is not necessary to elaborate on minor differences in the NMR shifts for free comenic acid and the lithium salt (section 2.1.4). It can be expected that the complex is mostly dissociated in a D2O solution.

Thanks for the comment! Now it given as follows:

NMR spectrum ¹³С [D₂O, 298 K], (δ, ppm): 177.0, 165.1, 158.2, 146.0, 141.8, 114.3.

Also, section 2.1.4 NMR spectroscopy is given as follows:

NMR ¹Н spectra of lithium comenate 6 and the initial comenic acid 2 are almost identical, the chemical shifts of proton signals differ by no more than δ 0.05 ppm (Figure S2 in the supplementary file). In the ¹³C NMR spectra, the difference is not exceeding 1.7 ppm (Figure S3 in the supplementary file).

 

It is stated several times throughout that the compound crystallizes in the "chiral (non-centrosymmetric) space group Pna2₁", which is not correct. Although the space group Pna2₁ is non-centrosymmetric, it is certainly not a "chiral" (or Sohncke space group) but a polar space group. Hence, there are certainly no "independent molecules of different chirality, that is, a mixture of enantiomers" in the crystal structure, as stated in section 2.1.7. Please revise.

Thank you a lot for such a valuable comment! Indeed, as Pna2₁ is non-Sohncke space group, so the use of the word “chirality” is unnecessary. In addition, as indicated in the original text of the manuscript, both enantiomers are present in the cell. Therefore, as suggested we excluded the use of chirality term in the text.

In Figure 5, it looks as if there is a dotted line between H atoms, which makes no sense to me.

Thanks for the comment! Indeed, this is a structure representation error. In fact, there are O-H hydrogen bonds. Figure 5 was replaced.

There are some issues with the crystal structure refinement, which should be fixed ahead of publication:

- The appropriate TEMP instruction in SHELXL should be used in the final refinement (see checkCIF level B alert).

Thanks for the comment! TEMP instruction was included in CIF

- With regard to the checkCIF level C alert concerning the poor data/parameter ratio, it is inappropriate to refine H atoms freely. Those bonded to carbon should be refined using the appropriate riding model (i.e. AFIX 43 in this case). The H atoms bonded to oxygen should be refined with the O-H distance restrained to, say, 0.84(2) A by using DFIX instructions and with U_iso(H) = 1.2 U_eq(O). This should also resolve the level C alert regarding the U_iso(max) / U_iso(min) range.

Thanks for the comment! AFIX and DFIX instructions were included and U were fixed as 1.2x which resolved all the level C

- Minor point: the atom list should be sorted before the final CIF file is written.

Thanks for the comment! Sorting of atoms was performed. Cif was updated in CCDC.

Thanks for the comments!

All the corrections marked green.

We appreciate the efforts of the editor and referees in reviewing our manuscript.

 We believe that the revised paper can meet the journal publication requirements.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Kozin et al. reports the synthesis, structure, and the biological evaluation of a Lithium-comenic acid. The topic of the paper is of interest to the reader of the journal.  

The research is well-structured, the text is well organized, and the findings are clearly presented. The results provided seems to be of interest, and generally call no objections.

The paper needs minor revisions before finally accepting the paper for publication, considering the following:

-    1) In Figure 1, the structure of compounds 1, 3-5 are unnecessary;

- 2) In paragraph 2.1.2 “Simultaneous …” the structure of the unstable fragment should be avoided in the text.

Comments on the Quality of English Language

Only minor editing of English language is required.

Author Response

Reviewer #2:

 

The manuscript by Kozin et al. reports the synthesis, structure, and the biological evaluation of a Lithium-comenic acid. The topic of the paper is of interest to the reader of the journal.  The research is well-structured, the text is well organized, and the findings are clearly presented. The results provided seems to be of interest, and generally call no objections.The paper needs minor revisions before finally accepting the paper for publication, considering the following:

-    1) In Figure 1, the structure of compounds 1, 3-5 are unnecessary;

Thanks for the comment! Yes, we don't work any further with the above compounds in the paper. However, here we would like to show the genetic relationship between comenic acid and other γ-pyrone derivatives. Many of these compounds are of considerable interest due to their detectable biological activity.

- 2) In paragraph 2.1.2 “Simultaneous …” the structure of the unstable fragment should be avoided in the text.

Thanks for the comments! Corrected as suggested. All the corrections are marked green.We appreciate the efforts of the editor and referees in reviewing our manuscript. We believe that the revised paper can meet the journal publication requirements.

Reviewer 3 Report

Comments and Suggestions for Authors

Neuroprotective agents like Lithium comenate presented in this manuscript are the compounds that support neuron health and function and aim to prevent or slow down the progression of neurodegenerative diseases. In this manuscript, the authors came up with a novel complex compound Lithium comenate synthesized from Lithium and comenic acid and explored its neuroprotective and antioxidant properties. This is quite an interesting piece of work and worth studying.

The authors well characterized this complex compound and explored its biological role as well.  All the characteristic data and biological data provided is very reliable and clearly explained. Overall, the work is commendable and can be extended to further in-depth studies to make Lithium comenate or its derivatives to identify as a lead molecule. Hence, this work is publishable in IJMS provided after addressing or highlighting the following points in their manuscript.
  1. Firstly, the term 'excitoxic' should be replaced by 'excitotoxic' in the abstract and make sure in all over the manuscript. 2. The authors should explain briefly about the glutamate. Glutamate is the neurotransmitter which creates an excitotoxic effect through excessive stimulation on nerve cells. The authors are trying to address this question as the main objective and never highlighted anywhere in the text although provided the solution with the help of their novel complex compound Lithium comenate. 3. What are NMDA and AMPA? Need a brief explanation as a good start. Abbreviate both. May include something like the following.

"The excitotoxic effect is largely mediated through glutamate receptors known as NMDA (N-methyl-D-aspartate) receptors, as well as through AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors. Upon binding to these receptors, there's an influx of calcium (Ca^2+) into the neuron."

4. On the other hand, the authors also studied the antioxidant effect focusing on the brain tissues and the results obtained are commendable. 5. The unstable intermediate should be assagnes some number or name instead including the structure. The structure can be shown at the place the related data is presented along with the assagned number or name.  

 

Author Response

Reviewer #3:

Comments and Suggestions for Authors

Neuroprotective agents like Lithium comenate presented in this manuscript are the compounds that support neuron health and function and aim to prevent or slow down the progression of neurodegenerative diseases. In this manuscript, the authors came up with a novel complex compound Lithium comenate synthesized from Lithium and comenic acid and explored its neuroprotective and antioxidant properties. This is quite an interesting piece of work and worth studying.

The authors well characterized this complex compound and explored its biological role as well.  All the characteristic data and biological data provided is very reliable and clearly explained. Overall, the work is commendable and can be extended to further in-depth studies to make Lithium comenate or its derivatives to identify as a lead molecule. Hence, this work is publishable in IJMS provided after addressing or highlighting the following points in their manuscript.
  1. Firstly, the term 'excitoxic' should be replaced by 'excitotoxic' in the abstract and make sure in all over the manuscript. 

Thanks for the comments! Corrected as suggested

2. The authors should explain briefly about the glutamate. Glutamate is the neurotransmitter which creates an excitotoxic effect through excessive stimulation on nerve cells. The authors are trying to address this question as the main objective and never highlighted anywhere in the text although provided the solution with the help of their novel complex compound Lithium comenate. 

The authors are grateful to the reviewer for his comments. However, in the Discussion section, glutamate is discussed in sufficient detail as applied to the purpose of the work.

3. What are NMDA and AMPA? Need a brief explanation as a good start. Abbreviate both. May include something like the following.

"The excitotoxic effect is largely mediated through glutamate receptors known as NMDA (N-methyl-D-aspartate) receptors, as well as through AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors. Upon binding to these receptors, there's an influx of calcium (Ca^2+) into the neuron."

Thanks for the comments! Corrected as suggested, highlighted in green.

 

4. On the other hand, the authors also studied the antioxidant effect focusing on the brain tissues and the results obtained are commendable. 5. The unstable intermediate should be assagnes some number or name instead including the structure. The structure can be shown at the place the related data is presented along with the assagned number or name.  

 Thanks for the comments! Corrected as suggested.

We appreciate the efforts of the editor and referees in reviewing our manuscript. We believe that the revised paper can meet the journal publication requirements.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have revised their manuscript taking some of the reviewers' comments into account.

It puzzles me that the authors replied that they have updated the CIF file deposited with CCDC, but when I downloaded the CIF using CCDC 2288394, the file appears to be unchanged. According to the CCDC, it was deposited on 13/08/2023. I would also expect that some refinement parameters listed in the manuscript would have changed, which is obviously not the case.

Still the appropriate TEMP instruction must be used in the final SHELXL job, and with regard to data/parameter ratio, still the carbon-bound H atoms should be refined using a riding model and with Uiso(H)=1.2Ueq(C). Oxygen-bound H atoms should be refined using appropriate distance restraints on the O-H bond lengths and with Uiso(H)=1.2Ueq(O). And still the atom list should be sorted.

As long as these issues have not been fixed, I cannot recommend publication.

Take care that all numbers in the manuscript are updated accordingly. If the refinement has been modified, it is also necessary to replot the structure pictures.

It looks as if Figure 3 was taken from checkCIF via copy and paste. A picture of higher quality could be drawn. In light of Figures 3 and 5, Figure 4 appears to be redundant.

It is, by the way, not necessary to mention the computer programs used for structure solution and refinement in the Results section. This should belong just to the experimental part.

It is usually not necessary either to report the CCDC deposistion number in the abstract.

In Figure 2, I would omit the dots representing the lone pairs of water oxygen, because then strictly, the dots should also be on the comenate oxygen atoms.

The description of the metal coordination could be improved. The coordination sphere of Li+ appears to be best described as square pyramidal, which could be proven by calculating the geometry index: https://en.wikipedia.org/wiki/Geometry_index

Author Response

Comments and Suggestions for Authors

The authors have revised their manuscript taking some of the reviewers' comments into account. In the manuscript changes marked with turquoise.

 

It puzzles me that the authors replied that they have updated the CIF file deposited with CCDC, but when I downloaded the CIF using CCDC 2288394, the file appears to be unchanged. According to the CCDC, it was deposited on 13/08/2023. I would also expect that some refinement parameters listed in the manuscript would have changed, which is obviously not the case.

Thanks for the comment! We apologize for the error. The author who did the X-ray diffraction studies and the author who deposited the .cif file are different people. There was an unfortunate discrepancy. The updated .cif file has now been re-uploaded from the ССDC database under the same number 2288394.

 

Still the appropriate TEMP instruction must be used in the final SHELXL job, and with regard to data/parameter ratio, still the carbon-bound H atoms should be refined using a riding model and with Uiso(H)=1.2Ueq(C). Oxygen-bound H atoms should be refined using appropriate distance restraints on the O-H bond lengths and with Uiso(H)=1.2Ueq(O). And still the atom list should be sorted.

As long as these issues have not been fixed, I cannot recommend publication.

Thanks for the comment! In the new version of .cif, all the necessary changes have been made.

Take care that all numbers in the manuscript are updated accordingly. If the refinement has been modified, it is also necessary to replot the structure pictures.

Thanks for the comment! New pictures were pasted..

It looks as if Figure 3 was taken from checkCIF via copy and paste. A picture of higher quality could be drawn. In light of Figures 3 and 5, Figure 4 appears to be redundant.

Thanks for the comment! New pictures were pasted. Fig.4 was deleted.

It is, by the way, not necessary to mention the computer programs used for structure solution and refinement in the Results section. This should belong just to the experimental part.

Thanks for the comment! Done.

It is usually not necessary either to report the CCDC deposistion number in the abstract.

Thanks for the comment! CCDC deposistion number was removed from the abstract.

In Figure 2, I would omit the dots representing the lone pairs of water oxygen, because then strictly, the dots should also be on the comenate oxygen atoms.

Thanks for the comment! Corrected as suggested.

The description of the metal coordination could be improved. The coordination sphere of Li+ appears to be best described as square pyramidal, which could be proven by calculating the geometry index: https://en.wikipedia.org/wiki/Geometry_index

Thanks for the comment! Corrected as follows:

The coordination sphere of Li+ was found to be close to square pyramidal with geometry index tau = 0.16

We appreciate the efforts of the editor and referees in reviewing our manuscript.

 We believe that the revised paper can meet the journal publication requirements.

 

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have tried to revise their submission but have messed it up. The CIF deposited with the CCDC has been updated, but nonsensical distance restraints have been introduced in the re-refinement of the structure:

DFIX 0.84 O9 C6
DFIX 0.84 H6 H8b
DFIX 0.84 O8 O6
DFIX 0.84 C6 H9a
DFIX 0.84 H8a O2
DFIX 0.84 O5 O4
DFIX 0.84 C1 O8
DFIX 0.84 O1 H8a
DFIX 0.84 H7b O7
DFIX 0.84 O7 H7a

That resulted in: _refine_ls_restrained_S_all      17.182

I went ahead and corrected the DFIX instruction:

DFIX 0.84 0.02 O4 H4
DFIX 0.84 0.02 O6 H6A O6 H6B
DFIX 0.84 0.02 O7 H7A O7 H7B
DFIX 0.84 0.02 O8 H8A O8 H8B

Then I obtained:

_refine_ls_restrained_S_all       1.009

Please seek expert advice or read the SHELXL manual if you are unsure how to use DFIX instructions. Restraints do more ham than good if not used appropriately.

The TEMP instruction in SHELXL requires the temperature in degree Celsius, not Kelvin (i.e. TEMP -173 in your case, I reckon). This is quite important, because the C-H distances set by the AFIX instructions depend on that parameter.

Please also write the hydrogen bond geometry parameters into the CIF. This is achieved by using the appropriate HTAB instructions.

Aside from the fact that you need to deposit a revised CIF with the CCDC again, it struck me that the refinement data in the CIF are not consistent with those in the manuscript. For example:

_refine_ls_R_factor_gt           0.0245

and R1 = 0.0203 in the manuscript.

This was just one example. Please be more careful when you revise the manuscript again.

It also struck me the that the displacement ellipsoid plot in Figure 3 obviously does not correspond to the asymmetric unit in the CIF but to its mirror image. Please make sure that all structure pictures are drawn form the final CIF.

Figure 4 should be redrawn with atom labels and symmetry codes for the atoms involved in hydrogen bonding. O-H...O hydrogen bond parameters should be listed in a table or in the figure caption.