Indolyl-Derived 4H-Imidazoles: PASE Synthesis, Molecular Docking and In Vitro Cytotoxicity Assay

Round 1

Reviewer 1 Report

The manuscript describes the synthesis of novel aromatic heterocycles that can be used as inhibitors of enzymes thought to be implication in neurodegenerative diseases, their in-silico study and toxicity study on HEK-293 cell lines.

The compounds and their method of synthesis are of interest and the authors show that further studies of their ability to prevent neurodegenerative diseases is warranted.
There are a few issues that need addressing:

1- The authors should replace all comas into dots for the decimal symbol, e.g. -13,57 must become -13.57 in the intro. The issues is found in Figure 3, Table 2 for example.

2- The model of the IR instrument is not included. It should be added.

3- Scheme 2: Replace "X = H, F, Br" by "X = H (a), Br (b), F (c)" to indicate the numbering.

4- Scheme 3: Indicate the structures of R1 and R2 chosen. It's only in Figure 2 that it becomes apparent.

5- Line 378: "optimal" suggest the best reaction that can be achieved but we can never know really if a reaction is optimal. It may be better to say "With the best identified conditions in hand..."

6- There does not seem to be any consistency in labelling in Figure 2. Why not use 6a, 6b and start at 6e? The letter does not seem to match the indole ring structure as 5b and 6h have the same.

7- Scheme 5:

    Structure 3.2 should be better written as its resonance form where the C+-N is replaced by C=N+.

8- The authors report that the reaction of 3a and 4a forms a C-C on C3 of the indole. It's known that in some cases the indole's C2 carbon can react as the nucleophile. The authors should include one sentence with a reference showing that it's indeed the correct regioisomer formed.

9- Figure 3: include the scheme for ChemBL95.

10- Figure 5c seem to show a carbon atom with 5 bonds (C=O). It may be the third methyl group that is drawn over the C=O. Correct the drawing to make it look nicer.

11- Alongside the SwissADME BBB permeability estimation, the authors may want to also include the statistical BBB Score (J. Med. Chem. 2019, 62, 21, 9824).

11- Figure 6a and Table 3 say the same thing. Remove one.

Author Response

The manuscript describes the synthesis of novel aromatic heterocycles that can be used as inhibitors of enzymes thought to be implication in neurodegenerative diseases, their in-silico study and toxicity study on HEK-293 cell lines.

The compounds and their method of synthesis are of interest and the authors show that further studies of their ability to prevent neurodegenerative diseases is warranted.
There are a few issues that need addressing:

We kindly thank the reviewer for the positive feedback on our manuscript. We thoroughly revised the manuscript and corrected mistakes and inaccuracies.                

1. The authors should replace all comas into dots for the decimal symbol, e.g. -13,57 must become -13.57 in the intro. The issues is found in Figure 3, Table 2 for example.

All comas were completely replaced with dots for decimal symbols.

2. The model of the IR instrument is not included. It should be added.

The model of the IR instrument was added in the beginning of the section “Materials and methods”.

3. Scheme 2: Replace "X = H, F, Br" by "X = H (a), Br (b), F (c)" to indicate the numbering

Scheme 2 was revised and the indication was added.

4. Scheme 3: Indicate the structures of R¹ and R² chosen. It's only in Figure 2 that it becomes apparent.

The corresponding indications were added to the Scheme 3.

5. Line 378: "optimal" suggest the best reaction that can be achieved but we can never know really if a reaction is optimal. It may be better to say "With the best identified conditions in hand..."

The sentence was corrected.

6. There does not seem to be any consistency in labelling in Figure 2. Why not use 6a, 6b and start at 6e? The letter does not seem to match the indole ring structure as 5b and 6h have the same.

At first, the synthesis of compounds 5a-d and 6e-h has a small difference including the additional step for the preparation of 6e-h. It is the reason that why we did not name them from 6a to 6e. At second, if we had the similar compounds in both hydrochloric and free forms, we would name them with the same letter and different numbers. There have been no any similar structures of hydrochloric and free forms of compounds in our manuscript. This is the reason that why we chose that way of naming.

7. Scheme 5: Structure 3.2 should be better written as its resonance form where the C⁺-N is replaced by C=N⁺.

Structure 3.2 was corrected.

8. The authors report that the reaction of 3a and 4a forms a C-C on C3 of the indole. It's known that in some cases the indole's C2 carbon can react as the nucleophile. The authors should include one sentence with a reference showing that it's indeed the correct regioisomer formed.

The C(3)-carbon atom is known to be a nucleophilic center of indole. However, the C(2) nucleophilic functionalization of indole ring is also possible, but the special conditions are required in this case [Chem. - A Eur. J. 2017, 23, 10925–10930, doi:10.1002/chem.201702338.]. According to our previous work [J. Org. Chem. 2012, 77, 9087–9093, doi:10.1021/jo301618b.], which describes the similar chemical synthesis proceeded via the same Addition-Elimination (AE) S_N^H mechanism using relevant indole substrates as nucleophilic agents. In this regard, there is no doubt that C(3) atom of indole is a nucleophilic center in the reactions with 4H-imidazole-3-oxides reported in this manuscript. The relevant discussion supported with the corresponding reference was added to the revised version of the manuscript.

9. Figure 3: include the scheme for ChemBL95.

ChEMBL95 is the identifier for Tacrine. Figure 3 was revised.

10. Figure 5c seem to show a carbon atom with 5 bonds (C=O). It may be the third methyl group that is drawn over the C=O. Correct the drawing to make it look nicer.

Figure 5c was revised.

11. Alongside the SwissADME BBB permeability estimation, the authors may want to also include the statistical BBB Score (J. Med. Chem.2019, 62, 21, 9824).

The reference was added.

12. Figure 6a and Table 3 say the same thing. Remove one.

Figure 6a was removed.

Reviewer 2 Report

The author has done a good job in the synthesis and research of these compounds. Although the number of compounds is a little small, this work can provide a good idea for the future design of small molecular candidates for the treatment of neurodegenerative disorders. I think it can be published after small revisions.

1.       The compounds 5a-5d are hydrochlorides, but I found the 1H NMR data did not include these hydrogens from “hydrochloride”, and they were not marked on the 1H NMR spectra. For example, I think the hydrogen peak of 5a is mixed in the peaks around 7.5ppm, ie, 7.53 – 7.50 (m, 3H). Please correct the 5a-5d 13C NMR data and spectrum.

2.       It is possible to have fewer peaks in the carbon spectrum, but not more. Please check the 13C NMR data and spectrum of 6e. It should be wrong.

3.       5a-5d are hydrochlorides, but 6e-6h are free form. There is no problem to do docking using free form. But no compound can get both hydrochloride and free form. Without comparison, we do not know whether the two forms of the same compound have an impact on cell activity.

Author Response

The author has done a good job in the synthesis and research of these compounds. Although the number of compounds is a little small, this work can provide a good idea for the future design of small molecular candidates for the treatment of neurodegenerative disorders. I think it can be published after small revisions.

We kindly thank the reviewer for the positive feedback on our manuscript. We thoroughly revised the manuscript and corrected mistakes and inaccuracies.

1. The compounds 5a-5d are hydrochlorides, but I found the ¹H NMR data did not include these hydrogens from “hydrochloride”, and they were not marked on the ¹H NMR spectra. For example, I think the hydrogen peak of 5a is mixed in the peaks around 7.5ppm, ie, 7.53 – 7.50 (m, 3H). Please correct the 5a-5d ¹³C NMR data and spectrum.

Hydrogen from “hydrochloride” usually is not revealed by NMR spectra. To identify whether it is a hydrochloride or a free form of a substance we always look for the hydrogen peaks of water. If it is a salt form of the compound, there are no signals of hydrogens of water in a spectrum around 3.33 ppm in DMSO. We have followed the same rule in our previous publications [J. Org. Chem. 2021, 86, 19, 13702–13710 https://doi.org/10.1021/acs.joc.1c01796]. The 5a-5d ¹³C NMR have been recorded again and all the revealed signals are found to be matched with the desired compounds structures.

2. It is possible to have fewer peaks in the carbon spectrum, but not more. Please check the ¹³C NMR data and spectrum of 6e. It should be wrong.

The ¹³C NMR data and spectrum of 6e were checked and corrected. There are 17 peaks of non-equivalent ¹³C.

3. 5a-5d are hydrochlorides, but 6e-6h are free form. There is no problem to do docking using free form. But no compound can get both hydrochloride and free form. Without comparison, we do not know whether the two forms of the same compound have an impact on cell activity.

We agreed that comparison of two forms would be a better way to completely estimate an impact on cell activity. Unfortunately, at this step we are not able to obtain both hydrochloride and free form of the same substance. Anyway, both in vitro and in silico assays were performed to assess the opportunities for further possible applications of the described structures in the design of candidates in neuroprotective agents.

Reviewer 3 Report

Oleg N. Chupakhin  et al., reporting a manuscript entitled 'ndolyl-derived 4H-Imidazoles: PASE Synthesis, Molecular Docking and in vitro Cytotoxicity Assay'. In this manuscript authors disclosing the new strategy of nucleophilic substitution of hydrogen (SN H) was first applied for the metal-free C-H/C-H coupling reactions of 4H-imidazole 3-oxides with indoles. As a result, a series of novelbifunctional azaheterocyclic derivatives were obtained in yields up to 95%. In silico experiments on the molecular docking were performed to evaluate the binding possibility of the synthesized small azaheterocyclic molecules to the selected biotargets (BACE1, BChE, CK1δ, AChE) associated with the pathogenesis of neurodegenerative diseases.

I believe this manuscript is well written and the results and experimental section agreed to be accepted for publication.

Author Response

Oleg N. Chupakhin  et al., reporting a manuscript entitled 'Indolyl-derived 4H-Imidazoles: PASE Synthesis, Molecular Docking and in vitro Cytotoxicity Assay'. In this manuscript authors disclosing the new strategy of nucleophilic substitution of hydrogen (SN H) was first applied for the metal-free C-H/C-H coupling reactions of 4H-imidazole 3-oxides with indoles. As a result, a series of novel bifunctional azaheterocyclic derivatives were obtained in yields up to 95%. In silico experiments on the molecular docking were performed to evaluate the binding possibility of the synthesized small azaheterocyclic molecules to the selected biotargets (BACE1, BChE, CK1δ, AChE) associated with the pathogenesis of neurodegenerative diseases.

I believe this manuscript is well written and the results and experimental section agreed to be accepted for publication.

We are very pleased to receive these positive comments on our manuscript. We thank the referee for the kind assessment of our work.