Synthesis, In Silico, In Vivo, and Ex Vivo Evaluation of a Boron-Containing Quinolinate Derivative with Presumptive Action on mGluRs

Round 1

Reviewer 1 Report

In this work, Trujillo-Ferrara et al. reported the design, synthesis, and detailed biological studies of a boron-containing quinolinate derivative. Although the content is extensive, the reviewer thinks this paper is too long and the overall organization is very poor. After concise rewriting the whole manuscript, it might be publishable after addressing the concerns and comments outlined below:

1. Page 8, the authors claimed that for the first time, the BZQuin was obtained in its acid form, did they verify that the acid or salt form doesn't affect its potency?

2. For the intensity of seizures (figure 7), it seems that a higher dose of BZQuin resulted in less efficiency, can the authors explain the phenomenon?

3. For most in vitro and in vivo studies, Quin or BZQuin were combined with Rlz and Mem, how can they conclude that BZQuin exerts a positive influence on glutamatergic neurotransmission by interacting with certain mGluRs?

4. The authors should carefully read the grammatical errors and inappropriate scientific language throughout the text.

Author Response

In this work, Trujillo-Ferrara et al. reported the design, synthesis, and detailed biological studies of a boron-containing quinolinate derivative. Although the content is extensive, the reviewer thinks this paper is too long and the overall organization is very poor. After concise rewriting the whole manuscript, it might be publishable after addressing the concerns and comments outlined below:

R: Thank you very much for suggestion and comments. We have expressed some sentences in a brief form. Also, we deleted some sentences for clarity, avoiding redundance.  The entire manuscript was edited for improvement.

1. Page 8, the authors claimed that for the first time, the BZQuin was obtained in its acid form, did they verify that the acid or salt form doesn't affect its potency?

R: We have edited that section being clear regarding the features of our synthesized compound and the difference with that structurally related which was reported 10 years ago, the comparison of biological effects among these compounds is beyond the aim of this work.

2. For the intensity of seizures (figure 7), it seems that a higher dose of BZQuin resulted in less efficiency, can the authors explain the phenomenon?

R: Now it is Fig. 9.  We observe the induction of seizures. Quin induces high score seizures, while BZQuin induced less effect albeit if high concentration is used. At low doses, poor inducing data related to seizures was observed. 

3. For most in vitroand in vivo studies, Quin or BZQuin were combined with Rlz and Mem, how can they conclude that BZQuin exerts a positive influence on glutamatergic neurotransmission by interacting with certain mGluRs?

R: That is proposed due to clear effects of well-known mGluR-antagonists (Rlz or Mem) disrupting the effect of BZQuin.

4. The authors should carefully read the grammatical errors and inappropriate scientific language throughout the text.

R: We have revised and edited the entire manuscript to avoid mistakes. Maggie Brunner, M.A. has revised for using the adequate language. The title now indicates the presumption of the action in the mGluR. Additional experiments are required to confirm or discard it.

Reviewer 2 Report

The paper prepared by Cuevas-Galindo et al. is quite interesting. It describes many experimental studies (synthesis, in vivo, ex-vivo) as well as in silico calculations. The results are described in detail, presented in figures and tables although the discussion is sometimes long. Prior to publication, it requires thorough improvement. First of all, references are missing in many places when citing or providing information. This should be improved. The most important of these deficiencies is listed in detail below. The same as all remarks, comments or suggestions:

1. Abstract “In the present article, encouraged by reports of bioactive organoboron compounds, a diphenylboroxazolidone derived from quinolinate (BZQuin), a kynurenine, was evaluated.- The sentence is not clear. Remove the word “a kynurenine " or add an explanation that Quinoliate is a metabolite of kynurenine.

2.  Introduction line 64-66

“As it can be expected from this broad family of receptors, L-glutamate is not the only ligand for them, there also exist other endogenous substances with affinity for them, such as D-aspartate, an EAA, and quinolinate, a L-tryptophan metabolite.

Put “an EAA” and “L-tryptophan metabolite” in brackets not between commas

3.  Introduction line 81

Write serotonin instead of 5-hydroxytryptamine (5-HT) because in Figure 1 it is signed as serotonin

4.   Figure 1 – it should be corrected prepared in this way is not very readable. The information on the action of Quin is not necessary here, because it is listed in the text and the addition of this makes the drawing not very readable. 

- Why is serotonin with NH₃⁺? Why are 3-hydroxyanthranilate, quinolinate and BZQuin shown as anions with COO^- groups?

5.      Introduction line 90-91:

„Quinolinate at harmful excitotoxic concentrations….” - is it known which doses these are? No reference

6.      Introduction line 94-95

“In Figure 1, the quinolinate mechanisms are summarized.” – remove this sentence (see point 4)

7.      Introduction line 99-101

Redraft. Not very clear on first reading. I think it should be split into 2 independent sentences.

8.      Introduction line 112-122

These compounds (mentioned in lines 112-122) are interesting and their structures should be presented in Figure which should be placed in the main text and not in supplementary materials.

9.      Results & Discussion line 138-139

“Therefore, they do not interfere with drug metabolism in the liver.” - Cannot be written that way. Theoretical calculations are only calculations and give a suggestion but not 100% certainty that this is the case.

10.   Figure 2 – could be in Supplementry material it is not so interesting

11.   Results & Discussion line 155

Some information should be given here about these calculations. What is the relationship between delta G and binding affinity?

12.   Results & Discussion line 179-180:

On the first, the ΔG changed from -6 kcal/mol to -10.49 kcal/mol, after the structural modifications leading to BZQuin.”

It should be -6.8 kcal/mol not -6 kcal/mol or if you mean for mGluR1 it should be -6 kcal/mol to -8.66 kcal/mol (mGluR1) or -6.8 kcal/mol to -10.49 kcal/mol (mGluR2)

13.   Figure 4.

 Co-crystallized ligands interactions with amino acid residues. –write name of these ligands

14.   2.2. Spectroscopic characterization line 212-239

For a better understanding of the course of the synthesis, it would be good to draw a synthetic way of this compound. Thus, it is completely unclear why the signals from ethanolamine are missing here and where they could have come from.

15. Line 214 - 216

“To the authors’ knowledge, this is the first time that BZQuin is reported in its acid form, since it has only been previously isolated and characterized as a salt; formed by the amine group from ethanolamine as cation and the remaining carboxylate from BZQuin as anion.” – lack of reference

16.   2.3. In vivo evaluation: Acute toxicity line 260-261

….“For the acute toxicity evaluation, the lethal dose 50 (LD50) for BZQuin was estimated in male CD-1 mice with an in-house modified version of Lorke’s method”- – lack of reference

17.   Line 280-281

“Despite this, the published report from which Quin data was taken includes the experimental LD50 which is 360 mg/kg.” - lack of reference

18. Lline 295-302

Information on the doses of tested coumpounds used and the reference compounds should be given, not only in the description of the method (paragraph 3.5)

19.   I do not really understand where the dose of 593 mg/kg came from. Why is it 3.4 times higher than the toxic dose described earlier? What was the point of using such a dose? What did the authors want to test? It should be explained.

20.   Why dose of 593 mg/kg for BZQiun is as authors write is “an equimolar dose for 300 mg/kg of Quin?

21.   Table 1 – lack explanation for N/A below Table

22.   Line 541-543

These results complement with the molecular docking results, which propose the mGluR1 receptor as a possible target for BZQuin, with its binding site located at the L-glutamate site. – it should be one of possible targets

23.   Lines 638

Lack of reference (information about the website and when it was used)

24.   Line 678-708

this part should be corrected:

Synthesis of BZQiun (give full chemical name)

description of the synthesis; 2-APB (full name not abbreviation)

analytical data from lines 696-702

why is reference 33 quoted here if BZQiun was not described there?

 Synthesis of Rlz (full name not abbreviation)

description of the synthesis

analytical data from line704-708

25.   Line 697 & line 705 it should be DMSO-d₆

26.   Supplementary materials 1H NMR spectra for BZQuin and Rlz why you show only a part of this spectra. It should be shown the whole range (0-10 ppm at least for 1H NMR)

Author Response

The paper prepared by Cuevas-Galindo et al. is quite interesting. It describes many experimental studies (synthesis, in vivo, ex-vivo) as well as in silico calculations. The results are described in detail, presented in figures and tables although the discussion is sometimes long. Prior to publication, it requires thorough improvement. First of all, references are missing in many places when citing or providing information. This should be improved. The most important of these deficiencies is listed in detail below. The same as all remarks, comments or suggestions:

R: Thank you for your accurate suggestion and comments. All they were considered to improve our manuscript.

1. Abstract“In the present article, encouraged by reports of bioactive organoboron compounds, a diphenylboroxazolidone derived from quinolinate (BZQuin), a kynurenine, was evaluated.- The sentence is not clear. Remove the word “a kynurenine " or add an explanation that Quinoliate is a metabolite of kynurenine.

R: We have removed it.

2. Introduction line 64-66

“As it can be expected from this broad family of receptors, L-glutamate is not the only ligand for them, there also exist other endogenous substances with affinity for them, such as D-aspartate and EAA and quinolinate, a L-tryptophan metabolite.

Put “an EAA” and “L-tryptophan metabolite” in brackets not between commas

R: We have edited for appropriateness.

3. Introduction line 81

Write serotonin instead of 5-hydroxytryptamine (5-HT) because in Figure 1 it is signed as serotonin

R: We have added ‘(serotonin)’.

4. Figure 1 – it should be corrected prepared in this way is not very readable. The information on the action of Quin is not necessary here, because it is listed in the text and the addition of this makes the drawing not very readable. 

- Why is serotonin with NH₃⁺? Why are 3-hydroxyanthranilate, quinolinate and BZQuin shown as anions with COO^- groups?

R: The figure 1 was edited as you request. The charges were depicted as the moieties are expected at physiological pH (7.35).

5. Introduction line 90-91:

„Quinolinate at harmful excitotoxic concentrations….” - is it known which doses these are? No reference

R: The concentrations and reference were added.

6. Introduction line 94-95

“In Figure 1, the quinolinate mechanisms are summarized.” – remove this sentence (see point 4)

R: It was done as suggested.

7. Introduction line 99-101

Redraft. Not very clear on first reading. I think it should be split into 2 independent sentences.

R: It was edited for clarity.

8. Introduction line 112-122

These compounds (mentioned in lines 112-122) are interesting and their structures should be presented in Figure which should be placed in the main text and not in supplementary materials.

R: We have added them.

9. Results & Discussion line 138-139

“Therefore, they do not interfere with drug metabolism in the liver.” - Cannot be written that way. Theoretical calculations are only calculations and give a suggestion but not 100% certainty that this is the case.

R: Thank you. We are in agreement is just a suggestion from prediction. We have edited for appropriateness.

10. Figure 2 – could be in Supplementary material it is not so interesting

R: It was moved to supplementary material.

11. Results & Discussion line 155

Some information should be given here about these calculations. What is the relationship between delta G and binding affinity?

R: We have sentenced ‘more negative delta G is related to higher affinity’.

12. Results & Discussion line 179-180:

On the first, the ΔG changed from -6 kcal/mol to -10.49 kcal/mol, after the structural modifications leading to BZQuin.”

It should be -6.8 kcal/mol not -6 kcal/mol or if you mean for mGluR1 it should be -6 kcal/mol to -8.66 kcal/mol (mGluR1) or -6.8 kcal/mol to -10.49 kcal/mol (mGluR2)

R: We have edited for accuracy.

13. Figure 4.

 Co-crystallized ligands interactions with amino acid residues. –write name of these ligands

R: Name of ligands was added.

14. 2.2. Spectroscopic characterization line 212-239

For a better understanding of the course of the synthesis, it would be good to draw a synthetic way of this compound. Thus, it is completely unclear why the signals from ethanolamine are missing here and where they could have come from.

R: The figure of synthesis is figure 10 in the updated version. As you can see ethanolamine is irrelevant in this way.

15. Line 214 - 216

“To the authors’ knowledge, this is the first time that BZQuin is reported in its acid form, since it has only been previously isolated and characterized as a salt; formed by the amine group from ethanolamine as cation and the remaining carboxylate from BZQuin as anion.” – lack of reference

R: Reference [32] is reallocated to the correct site in this sentence.

16. 2.3. In vivo evaluation: Acute toxicity line 260-261

….“For the acute toxicity evaluation, the lethal dose 50 (LD50) for BZQuin was estimated in male CD-1 mice with an in-house modified version of Lorke’s method”- – lack of reference

R: We have edited, the modification is just in dose rank as is expressed in the Table 2. Reference is for the classic work of Lorke’s method.

17. Line 280-281

“Despite this, the published report from which Quin data was taken includes the experimental LD50 which is 360 mg/kg.” - lack of reference.

R: Reference [35] is here.

18. Line 295-302
    Information on the doses of tested compounds used and the reference compounds should be given, not only in the description of the method (paragraph 3.5)

R: It was done. Doses are in this 2.4 section now.

19. I do not really understand where the dose of 593 mg/kg came from. Why is it 3.4 times higher than the toxic dose described earlier? What was the point of using such a dose? What did the authors want to test? It should be explained.

R: We have declared in the methods section 593 mg/kg as equimolar to 300 mg/kg (0.001795 mol/kg).

20. Why dose of 593 mg/kg for BZQiun is as authors write is “an equimolar dose for 300 mg/kg of Quin?

R: This was clarified in methods section. The dose of Quin (300 mg/kg = 0.001795 mol/kg) …Meanwhile, for BZQuin, which is an unexplored compound, two doses were evaluated, one at a 1:1 molar ratio to that of Quin (593 mg/kg = 0.001795 mol/kg)…

21. Table 1– lack explanation for N/A below Table

R: It was added.

22. Line 541-543

These results complement with the molecular docking results, which propose the mGluR1 receptor as a possible target for BZQuin, with its binding site located at the L-glutamate site. – it should be one of possible targets

R: It was edited. These results propose the mGluR1 receptor as one of the possible targets for BZQuin, with its binding site located at the L-glutamate site.

23. Lines 638

Lack of reference (information about the website and when it was used)

R: It was added.

24. Line 678-708

this part should be corrected:

Synthesis of BZQiun (give full chemical name)

description of the synthesis; 2-APB (full name not abbreviation)

analytical data from lines 696-702

why is reference 33 quoted here if BZQiun was not described there?

 Synthesis of Rlz (full name not abbreviation)

description of the synthesis

analytical data from line704-708

R: All these was edited considering your request.

25. Line 697 & line 705it should be DMSO-d₆

R: It was done.

26. Supplementary materials 1H NMR spectra for BZQuin and Rlz why you show only a part of this spectra. It should be shown the whole range (0-10 ppm at least for 1H NMR)

R: All these are in supplementary material now.

Reviewer 3 Report

In this manuscript, the authors designed and synthesized the organoboron compound BZQuin, with the purpose of influencing neurotransmission in the CNS. BZQuin exerted a reduced ability to cause seizures, when compared against its precursor quinolinate, suggesting that it does not directly stimulates the ionotropic NMDA receptors or other ionic channels. In molecular docking experiments, BZQuin binds to the positive sites of mGluR1, mGluR2 and mGluR7 receptors, showing higher affinity for molecular pairing forces than quinolates. Meanwhile, BZQuin showed more obvious effect on the brain regions with high expression of mGluRs, and showed neuroprotective effect on them. The experiments of the in silico, in vivo, and ex vivo evaluations were carried out to prove that BZQuin exerts a positive influence on glutamatergic neurotransmission through interaction with the mGluR1, mGluR2, and mGluR7 receptors. In my opinion, this manuscript can be considered for publication in Inorganics after revisions.

1. When evaluating the toxicity of the drug in mice, some hematological test data can added to evaluate the effect of the drug on mice more comprehensively.

2. There are still small mistakes in the manuscript, please check it carefully. For example, the word "intraperitonially" in line 284 is misspelled.

3. Related studies and reviews about boron-containing organic derivatives are suggested to refer, like Journal Of Organic Chemistry, 2022, 87, 5437–5441; Symmetry, 2022, 14, 182; Tetrahedron Letters, 2022, 92, 153657 et al.

Author Response

Reviewer 3:

In this manuscript, the authors designed and synthesized the organoboron compound BZQuin, with the purpose of influencing neurotransmission in the CNS. BZQuin exerted a reduced ability to cause seizures, when compared against its precursor quinolinate, suggesting that it does not directly stimulates the ionotropic NMDA receptors or other ionic channels. In molecular docking experiments, BZQuin binds to the positive sites of mGluR1, mGluR2 and mGluR7 receptors, showing higher affinity for molecular pairing forces than quinolates. Meanwhile, BZQuin showed more obvious effect on the brain regions with high expression of mGluRs, and showed neuroprotective effect on them. The experiments of the in silico, in vivo, and ex vivo evaluations were carried out to prove that BZQuin exerts a positive influence on glutamatergic neurotransmission through interaction with the mGluR1, mGluR2, and mGluR7 receptors. In my opinion, this manuscript can be considered for publication in Inorganics after revisions.

1. When evaluating the toxicity of the drug in mice, some hematological test data can added to evaluate the effect of the drug on mice more comprehensively.

R: We have not hematological test data. However, we have declared as a limitation of this study in the end of acute toxicity results.

2. There are still small mistakes in the manuscript, please check it carefully. For example, the word "intraperitonially" in line 284 is misspelled.

R: We have revised the entire manuscript to avoid mistakes.

3. Related studies and reviews about boron-containing organic derivatives are suggested to refer, like Journal Of Organic Chemistry, 2022, 87, 5437–5441; Symmetry, 2022, 14, 182; Tetrahedron Letters, 2022, 92, 153657 et al.

R: We have added the mentioned references where they could be adequate on the manuscript.

Round 2

Reviewer 1 Report

I consider that the authors did all the corrections and that the article can be now published in Inorganics.