Review Reports - Development of Isoniazid–Pyrazole Hybrids as Potential Antitubercular Agents

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper entitled “Development of Isoniazid-Pyrazole Hybrids as Potential Antitubercular Agents” describes a molecular hybridization approach directed for connection of isoniazid and pyrazole units through a hydrazone linkage. This investigation was aimed to prepare a new series of hybrids with enhanced anti-tubercular activity. The main question addressed by the research was to synthesize complex molecules holding some pharmacophors, including isoniazid, as the possible next-generation anti-tubercular agents. I consider that the topic of the presented paper is original and relevant to the development of new antimicrobial compounds, especially against MDR strains. This paper addresses a specific gap in the field because the authors have suggested a new group of isoniazid derivatives with inhibitory activity comparable to or exceeding that of INH. The presented paper added new findings concerning the antitubercular properties of new isoniazid-based heterocycles. The conclusions are consistent with the evidence and necessary arguments are presented, also addressing the main question posed. The references are appropriate. So, the presented paper could be recommended for publication after revisions:

- Compounds 6(a-o) are presented as mixtures of E/Z-isomers – please explain why these mixtures were not separated for individual isomers. Please explain the role of each isomer in antitubercular action. Compare own results with literature data for other isoniazid-hydrazone derivatives in the synthesis and antitubercular activity parts.

- please include more information about the possible mechanism of action of synthesized compounds and the differences in the target for INH and presented compounds.

- please explain the rationale in the choosing of substituents in the phenyl ring.

- it seems that all information about NMR and other data (title, mp, IR, elemental analysis etc) of new compounds should be given in the Experimental section.

Author Response

Reviewer 1

Compounds 6(a-o) are presented as mixtures of E/Z-isomers – please explain why these mixtures were not separated for individual isomers. Please explain the role of each isomer in antitubercular action. Compare own results with literature data for other isoniazid-hydrazone derivatives in the synthesis and antitubercular activity parts.

Response: We appreciate this important observation. The synthesized hydrazone derivatives 6(a–o) indeed exist as E/Z isomeric mixtures due to the presence of the C=N double bond, which allows geometrical isomerism. Attempts to separate the individual E and Z isomers using conventional chromatographic techniques (e.g., column chromatography and preparative TLC) were not successful, as the isomers exhibited very similar physicochemical properties and interconversion in solution was observed, which is consistent with literature reports on hydrazone systems (https://doi.org/10.1002/cmdc.202400949, https://doi.org/10.1016/j.tube.2023.102363, https://doi.org/10.1002/cmdc.202500398).

Regarding their biological activity, the antitubercular assays were conducted on the equilibrated mixtures, which likely represent the biologically relevant form under physiological conditions. At present, it is difficult to assign the specific contribution of each isomer to the observed activity without successful isolation or advanced spectroscopic or computational studies. However, literature suggests that in many hydrazone-based compounds, one isomer (often the E-isomer) is thermodynamically more stable and may predominate, potentially contributing more significantly to activity (https://doi.org/10.1111/cbdd.70171, https://doi.org/10.1016/j.bmc.2009.07.068). The manuscript is revised accordingly.

please include more information about the possible mechanism of action of synthesized compounds and the differences in the target for INH and presented compounds.

Response: INH is a prodrug that requires activation by the mycobacterial catalase–peroxidase enzyme KatG. Upon activation, it forms an isonicotinoyl–NAD adduct that inhibits InhA (enoyl-acyl carrier protein reductase), a key enzyme in the fatty acid synthase II (FAS-II) pathway. This inhibition disrupts the biosynthesis of mycolic acids, essential constituents of the mycobacterial cell wall. However, the efficacy of INH may be influenced by structural modifications that affect its activation or target interaction. Resistance to INH most commonly arises from mutations in the katG gene, impairing prodrug activation, or in the inhA gene, reducing target susceptibility. Accordingly, chemical modification of INH represents a valuable strategy to enhance its activity, either by bypassing KatG-dependent activation or by directly targeting InhA.

In this context, we report a series of INH–pyrazole hybrids, a promising class of molecular hybrids that integrate the antitubercular properties of INH with the favorable bioactivity of pyrazole scaffolds, including antimicrobial potential and improved drug-like characteristics. These hybrids may exert their effects through one or multiple mechanisms, such as direct inhibition of InhA or circumvention of KatG-mediated activation, ultimately leading to disruption of mycolic acid biosynthesis.

please explain the rationale in the choosing of substituents in the phenyl ring.

Response: The choice of substituents on the phenyl ring was guided by established SAR principles for Isoniazid-based hydrazones. A range of electron-donating groups (e.g., -CH₃, -OCH₃, -NH₂) and electron-withdrawing groups (e.g., halogens, -CF₃, -NO₂) were systematically introduced at different positions (meta and para) to evaluate their electronic and steric effects on antimycobacterial activity. This design enables assessment of how substituent polarity, electronic distribution, and positional variation influence interaction with the target enzyme (e.g., InhA), as well as membrane permeability and overall biological activity.

it seems that all information about NMR and other data (title, mp, IR, elemental analysis etc) of new compounds should be given in the Experimental section.

Response: Thank you for the observation. The data of the synthesized compounds have been provided in the experimental section.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents the design, synthesis, and biological evaluation of novel hybrid compounds with potential antitubercular activity. The topic is relevant and aligns well with the scope of the journal, particularly in the context of developing new therapeutic strategies against Mycobacterium tuberculosis. The study combines synthetic chemistry with biological assays, which is a strength. However, several important issues should be addressed before the manuscript can be considered for publication.

First, the abstract requires clarification. The acronym “INH” should be explicitly defined upon first use to ensure accessibility to a broader scientific audience. While this may seem minor, clarity at this level is essential for proper comprehension and indexing.

Second, the cytotoxicity results raise important concerns regarding the therapeutic window of the proposed compounds. The authors report that the hybrid molecules are non-cytotoxic at low concentrations but exhibit cytotoxic effects at higher concentrations. However, the manuscript does not adequately relate these findings to the concentrations required for antitubercular activity. A critical discussion is needed to determine whether the effective antimycobacterial concentrations fall within a safe, non-cytotoxic range. Without this comparison, it is difficult to assess the true pharmacological potential of the compounds. Including selectivity index (SI) values would significantly strengthen the biological evaluation.

Third, the mechanistic rationale requires deeper analysis. The antitubercular activity of isoniazid (INH) is known to depend on its activation by the KatG enzyme and its subsequent inhibition of the enoyl-acyl carrier protein reductase (InhA). Resistance mechanisms frequently involve mutations in either katG or inhA. In this context, the manuscript does not sufficiently address how the synthesized hybrid compounds interact with these key enzymatic targets. It remains unclear whether these molecules retain dependence on KatG activation, directly inhibit InhA, or potentially bypass classical resistance mechanisms. The inclusion of molecular docking, enzymatic inhibition assays, or discussion supported by literature would greatly enhance the mechanistic understanding and impact of the study.

Additionally, the manuscript would benefit from improvements in the discussion section. The current version is largely descriptive and does not critically compare the obtained results with previously reported compounds. A more in-depth structure–activity relationship (SAR) analysis is recommended to highlight the advantages or limitations of the hybrid design.

From a methodological perspective, the experimental section appears generally adequate, but more detail is needed in the biological assays to ensure reproducibility. Information regarding controls, number of replicates, and statistical analysis should be clearly stated.

Finally, the English language is generally understandable but would benefit from careful editing to improve grammar, sentence structure, and overall readability, particularly in the discussion section.

Comments on the Quality of English Language

The English language is generally understandable but would benefit from careful editing to improve grammar, sentence structure, and overall readability, particularly in the discussion section.

Author Response

Reviewer 2

First, the abstract requires clarification. The acronym “INH” should be explicitly defined upon first use to ensure accessibility to a broader scientific audience. While this may seem minor, clarity at this level is essential for proper comprehension and indexing.

Response: The abstract has been revised accordingly, the acronym “INH” has now been explicitly defined as isoniazid (INH) at its first occurrence in the abstract.

Second, the cytotoxicity results raise important concerns regarding the therapeutic window of the proposed compounds. The authors report that the hybrid molecules are non-cytotoxic at low concentrations but exhibit cytotoxic effects at higher concentrations. However, the manuscript does not adequately relate these findings to the concentrations required for antitubercular activity. A critical discussion is needed to determine whether the effective antimycobacterial concentrations fall within a safe, non-cytotoxic range. Without this comparison, it is difficult to assess the true pharmacological potential of the compounds. Including selectivity index (SI) values would significantly strengthen the biological evaluation.

Response: We agree with the reviewer that our original discussion did not address whether the effective antimycobacterial concentrations fall within a safe, non-cytotoxic range. In line with the reviewer’s recommendation, we have now modified Table 2, which presents the selectivity index (SI), thus providing a comparative measure of cytotoxicity versus antimycobacterial activity.

Third, the mechanistic rationale requires deeper analysis. The antitubercular activity of isoniazid (INH) is known to depend on its activation by the KatG enzyme and its subsequent inhibition of the enoyl-acyl carrier protein reductase (InhA). Resistance mechanisms frequently involve mutations in either katG or inhA. In this context, the manuscript does not sufficiently address how the synthesized hybrid compounds interact with these key enzymatic targets. It remains unclear whether these molecules retain dependence on KatG activation, directly inhibit InhA, or potentially bypass classical resistance mechanisms. The inclusion of molecular docking, enzymatic inhibition assays, or discussion supported by literature would greatly enhance the mechanistic understanding and impact of the study.

Response: We agree with the reviewer that our original manuscript did not address the interactions between the compounds and the key enzymatic targets associated with isoniazid activity. Investigating this aspect—for example, by determining MIC values in strains overexpressing InhA, would provide valuable insight into whether these molecular hybrids operate via similar mechanisms. We feel that such studies would require substantial additional experimental work beyond the scope of the current study.

However, based on the MIC values of some of the compounds in clinical strains with mutations in KatG (Table 2), we provide evidence that the hybrids are likely dependent on KatG.

Additionally, the manuscript would benefit from improvements in the discussion section. The current version is largely descriptive and does not critically compare the obtained results with previously reported compounds. A more in-depth structure–activity relationship (SAR) analysis is recommended to highlight the advantages or limitations of the hybrid design.

Response: The discussion section has been revised to include a more in-depth SAR analysis with direct comparison to previously reported Isoniazid-based hydrazone derivatives. The updated version now critically evaluates the influence of electronic effects, substitution patterns, and the role of the INH moiety, highlighting both the advantages (enhanced potency in series 6, surpassing INH) and limitations (reduced activity in series 4 without INH) of the hybrid design.

From a methodological perspective, the experimental section appears generally adequate, but more detail is needed in the biological assays to ensure reproducibility. Information regarding controls, number of replicates, and statistical analysis should be clearly stated.

Response: As described previously, the cytotoxicity of the five most active compounds was evaluated in THP-1 cells using the resazurin assay. Untreated cells and cells exposed to the compound solvent, dimethyl sulfoxide (DMSO), served as viability controls (i.e., non-cytotoxic conditions), while 20% sodium dodecyl sulfate (SDS) was used as a cytotoxic control. Isoniazid (INH) was included as an internal reference compound, and rifabutin (RFB) was employed as an additional control in cytotoxicity assays. All experiments were performed in duplicate and independently repeated three times. Dose–response curves were generated, where applicable, and analyzed by nonlinear regression using GraphPad Prism 10.

Finally, the English language is generally understandable but would benefit from careful editing to improve grammar, sentence structure, and overall readability, particularly in the discussion section.

Response: Thank you. The entire manuscript has been revised thorougly.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The paper was seriously revised, but some points should be clarified. If compounds 6a-o are mixtures of E/Z isomers why only E-isomer is mentioned in the title of these compounds (line 544 for 6a as an example)? please indicate the signals for E and Z and their ratio in the NMR spectra.

Author Response

Reviewer 1

Response: Thank you for your valuable comment. The possible formation of the E-isomer as the major isomer is likely due to its greater thermodynamic stability relative to the corresponding Z-isomer. However, the configuration cannot be assigned with certainty based solely on the proton NMR data. Accordingly, the E/Z (or major/minor) isomer ratio for compound 6a was determined to be 93:7 based on the proton NMR spectrum. The discussion is revised accordingly in the revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed all the comments and suggestions raised by this reviewer.

Comments on the Quality of English Language

The English language is generally understandable but would benefit from careful editing to improve grammar, sentence structure, and overall readability, particularly in the discussion section.

Author Response

Reviewer 2

The authors have satisfactorily addressed all the comments and suggestions raised by this reviewer.

The English language is generally understandable but would benefit from careful editing to improve grammar, sentence structure, and overall readability, particularly in the discussion section.

Response: Thank you for the positive feedback. The relevant discussion has been revised to improve its grammatical accuracy and scientific presentation.