α,β-Pipitzols and α,β-Isopipitzols from Natural Quinone Perezone: Quantum Chemistry, Docking, Chemoinformatic, and Pharmacological Studies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Although the study addresses an interesting topic, substantial revisions are required to improve its scientific quality:

1. Introduction section

The sentence “On the other hand, theoretical methods employing DFT calculations have consolidated their importance in chemical research, as they have been proven to achieve close similarities with experimental data, due to robustness, greater accuracy, and more efficient approximations; in addition, to the convenient acquisition of geometrical properties, electronic structures, and spectroscopic properties” would benefit from appropriate literature support. The authors are encouraged to cite representative theoretical studies in which various computational methods have been successfully applied to investigate compounds similar to those examined in the present work.

 

2. The logP values should be provided for the investigated structures, as these parameters are relevant to the discussion of physicochemical properties.
3. Introduction section

The authors should clarify the rationale for choosing the B3LYP functional. It would be helpful to indicate whether similar compounds have previously been studied using this functional and to include relevant references.

4. Figure 1: The caption “Figure 1. Perezone and isoperezone, and their derivatives 2, 3, 5, and 6, with assigned numeration” should be clarified. Please specify the purpose of the assigned numeration (e.g., for discussion of structural features, spectroscopic assignments, or computational analysis).
5. Results section title: The title “Optimization of the target molecules 2, 3, 5, and 6” raises a question regarding compounds 1 and 4. Please clarify whether these compounds were also optimized and, if so, why they are not included in the title.

6: The sentence “The energies of the optimized structures (2–3, 5–6) are summarized in Table 1, Figure 2” should be revised. The energies are presented in Table 1, whereas Figure 2 shows the geometrical representations of the optimized structures.

7. Table 1 Table 1 requires revision. Molecules 2 and 3 represent conformers of the same compound, as do molecules 5 and 6; therefore, energy comparisons should be made only within each conformational pair (2 vs. 3 and 5 vs. 6), not across different molecular systems. Since compounds 5 and 6 contain an isopropyl group, differences in their energetic values are expected. It is recommended to move total energy values to the ESI and instead report RMSD values calculated with respect to molecule 2 (for the 2/3 pair) and molecule 5 (for the 5/6 pair).
8. The reported relative energies appear to be negative. Relative energies should be positive values; according to recalculation, these values are in fact positive and should be corrected accordingly.

 

9. Geometrical parameters describing hydrogen bonds (e.g., bond lengths and angles) should be included. Furthermore, hydrogen bonds should be classified according to their strength based on these geometrical criteria.
10. In my opinion, detailed technical parameters such as bond lengths and bond angles could be moved to the ESI in tabular form, as they do not contribute significantly to the main discussion.
11. The Cartesian coordinates of all optimized compounds should be provided in the ESI. This is essential to ensure reproducibility and allow independent verification of the results.
12. Comparison of theoretical and experimental results

The are differences between theoretical and experimental bond lengths (e.g., C2–C3, C3–C4).This results from the fact that experimental structures are obtained in the solid state, whereas the calculations appear to have been performed in the gas phase, representing fundamentally different conditions. If a direct comparison is intended, the authors may consider using solid-state computational approaches (e.g., the Crystal program or the PBEsol functional) or performing calculations with several functionals to identify those that best reproduce experimental data.

13. Regarding HOMO–LUMO energies and atomic charges, comparisons between different molecules cannot be done.
14. The term “IR activity” requires clarification. Please specify whether the authors refer to IR intensities or transmittance, as “IR activity” is not commonly used in this context.
15. The comparison of calculated NMR chemical shifts with experimental data should be reconsidered. Experimental NMR measurements were probably performed in solution, whereas the calculations were carried out in the gas phase. The implications of this difference should be discussed, or solvent effects should be included in the calculations.

16.The manuscript should clearly explain how the physicochemical properties were calculated, including the theoretical methods and descriptors used.

17. The authors state in both the Abstract and Introduction that a conformational search was performed. However, the Methods section describes only geometry optimization starting from a single structure using an older semiempirical method, followed by reoptimization at the DFT level. This procedure does not constitute a conformational search, which typically involves extensive sampling of the potential energy surface using dedicated tools such as Open Babel, RDKit, or HyperChem.
18. The choice of an older semiempirical method should be justified. The authors should also specify the program used (e.g., MOPAC). More modern methods such as PM7 or PM6-D3H4, which are better suited for systems involving hydrogen bonding, are recommended.

19.The authors should further justify the use of the B3LYP functional. In addition, the absence of dispersion corrections should be addressed, as empirical dispersion schemes (e.g., D3 or D4) are commonly recommended to improve the accuracy of standard DFT methods.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this paper, the authors report a combined theoretical and experimental study on four compounds, two α,β-pipitzols and two α,β-isopipitzols. In short, the authors carried out extensive ab initio calculations, including natural atomic charges, HOMO LUMO distribution, several chemical parameters, IR and NMR spectra. For biological activity prediction, the authors use only the Lipinski rule of five prediction and PASS biological targets prediction. This resulted in two predicted targets: COX-2 and PARP-1. The following workflow was driven toward the molecular docking study of their compounds against the two targets, using the reference compounds as a standard for interpreting docking poses and energies. Finally, the authors conducted in vitro cytotoxicity experiments using five tumour cell lines: MDA-MB-231, MCF-7, U-87, A549, and U-373.

There are several significant concerns regarding this study:

1. Ab initio and electronic parameters are too extensively reported, without a reasonable need for interpretation in experiments
2. On the other hand, physicochemical parameters are too scantily described. One would expect at least the ADMET calculations. 
3. Regarding target prediction, the authors do not explicitly report the two reported targets, but it sounds as if it is their interpretation of the PASS predictions. A complete list of PASS output in the supplementary material would be appreciated. 
4. Molecular docking experiments are not a sufficient rationale for experiments, as there are significant discrepancies, suggesting a strong multitargeting problem (should be interpreted with PASS predictions).  The paper would benefit more from a simpler SAR study that maps the molecular areas of influence on activity, rather than from specific structure-based calculations.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have carried out a substantial amount of work to improve the manuscript, and in its current version it is suitable for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

The additional calculations and rationale done by the authors are satisfactory, but they still do not fully explain the experimental results. I suggest more structurally tailored calculations in future research. The manuscript is acceptable for publishing in this form.