Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript by Jiang et al. describes the synthesis and the evaluation of 42 derivatives as tubulin polymerization inhibitors. The results of this study are quite interesting, since some compounds proved to possess in vitro antiproliferative activity and one of them, compound B6, in vivo antitumor efficacy, as well. However, I believe there are aspects that need to be addressed before it can be published. Therefore, I recommend its publication only after major revisions.

Major issues to be addressed are the following:

- Novelty issues. Upon research of literature, one can easily find that very similar analogues to the series B of compounds (benzimidazole class, possessing a trimethoxy phenyl ring and an indole system, as well) have been already reported as tubulin inhibitors with antiproliferative activity (Lu et al., J. Med. Chem. 2014, 57, 7355−7366). Nevertheless, the authors do not report this previous publication. Under this scope, the part of the design of the new compounds of this manuscript, where the benzimidazole class derives from bioisosteric replacement of the quinazoline core of class A compounds, should be reconsidered and the corresponding part of the design of the new compounds should be reorganized.

 - Design of compounds: the design strategy of the novel derivatives is not quite clear. Since the molecular hybridization approach includes combination of compound 32 with the trimethoxygroup of colchicine, the first analogue of the A series should be denoted as A1 and not A17. Then, the rest of these compounds should be numbered accordingly. Among the A series of compounds, only A17 possesses an indole ring, similarly to compound 32, while the rest of them are aryl/heteroaryl/cycloalkyl substituted analogues, of different size and substitution patterns. Thus, a more detailed explanation for the transition from the structure of A17 to the rest of compounds of class A should be provided. In addition, the presence of the acetamido linker group between the aryl ring III and the thienopyridine core for the third series of compounds has not been justified during the design of this class of compounds. It is quite necessary, since both groups A and B possess an aryl group (III) directly attached to the central core (part II).

 - page 3, figure 2: the structure of A17 includes an unsubstituted on the nitrogen indole ring. Nevertheless, in scheme 1 and at the experimental section, one can see that there is a methyl group present on the indole nitrogen of compound A17. Thus, which is the actual structure of derivative A17? If it is the methylated analogue, why the unsubstituted analogue was not synthesized as well, given the fact that the quinazolinone lead derivative 32 also included the N-unsubstituted indole fragment?   

- A SAR discussion part is completely missing from this manuscript. Although the authors have synthesized 42 derivatives, that belong to 3 different classes, there is not such a discussion about the effect of the substitution pattern on the observed activity (either binding affinity to tubulin, or antiproliferative activity), or the effect of the replacement of core II when comparing similarly substituted on the part III ring derivatives, for example compounds B6 with A11, C3 and C10.

- The rationale behind the selection of the substituents at core III of the new compounds is not quite clear, especially during the transition from class A compounds to classes B and C analogues. Some particular substituents, for example the o-trifluoromethyl group present on the derivative A5 was not used within B and C series, although compound A5 proved to be one of the best binders to tubulin assay.

 

 

Additional issues to be addressed / corrections:

- page 1, lines 17-18: “42 novel tubulin polymerization inhibitors via molecular hybridization techniques,…”. Part of the sentence is missing. Did the authors mean: “42 novel tubulin polymerization inhibitors were designed via molecular hybridization techniques,….”?

- page 3, lines 79-81: The authors should provide a reference for compound 32. Also, it is not quite clear why they have numbered this compound as “32”. Since arabic numerals have been used later during the description of the synthetic pathways, it is rather confusing to refer to this compound with such a number herein.

- page 3, lines 92-93: “we developed two novel derivative classes: B (thienopyridine core) and C (benzimidazole core)”. According to the figure below, this should be vice versa, class B with the benzimidazole core and class C with the thienopyridine one.

- page 3, figure 2: the upper-right arrow should denote “replacement of ring III” instead of “replacement of ring I”.

- page 4, lines 106-107: “2-Amino-3-bromobenzamide reacted with different substituted aromatic aldehydes…” should be corrected to “3-Bromo-1,2-benzenediamine reacted with different substituted aromatic aldehydes..”.

- page 4, scheme 2: according to the comment above, the diamine should be now numbered with 3, and the resulting benzimidazole intermediates as 4-1 to 4-10, both in scheme 2 and the corresponding text above (lines 108-110).

- page 4, lines 108-109: The authors say: “The reaction of intermediate 3 with ditert-butyl dicarbonate resulted in product intermediate 3”. What is the meaning of this sentence?

- page 5, section 2.2.1.: the authors should provide the relative references for the bio-layer interferometry method used for the binding affinity determination.

- page 6, table 1: the authors should explain the difference between N.D and N.B.

- page 7, figure 3: the authors should provide the concentration of colchicine used for the in vitro antiproliferative assay.

- page 7, line 172: “high antiproliferative activity against five tumor cell lines” should be corrected to “high antiproliferative activity against the four tumor cell lines”.

- page 8, lines 183-185: the statement “introducing alkyl or methoxy groups on the phenyl ring of fragment III can significantly enhance the antitumor activity of the compounds” does not sound reasonable, since the unsubstituted phenyl derivative was not synthesized and evaluated. The authors should rephrase this part.

- page 8, lines 195-197: the phrase “we further evaluated the inhibitory effects of compound B6 on tubulin polymerization with colchicine and paclitaxel as positive control” is not correct. Paclitaxel served as a negative control to this experiment, since it is not a tubulin polymerization inhibitor.

- page 8, figure 4: the authors should include the concentrations of paclitaxel and colchicine used in this figure.

- page 28, references section: references 26 and 30 are identical.

- experimental section, paragraphs 3.2.3 and 3.2.5: A more formal language should be used for the description of these experimental parts.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The paper describes the synthesis, characterization and tubulin binding activity of substituted trimethoxyquinazolines, -phenylbenzimidazoles and -thienopyridine. The study overall produced new derivatives narrowing down to one antiproliferative compound, benzimidazole B6, with moderately strong tubulin binding activity. This was complemented by molecular docking and other in vitro assays such as cell migration, apoptosis and cell cycle assays to assess the preliminary putative mechanism. In vivo assay also indicate promise for treating skin cancer. The paper is generally interesting scientifically especially in the field of medicinal chemistry. However, the authors must address the following comments.

1. The authors must refrain from using novel since the core scaffolds are no novel skeletons.
2. While I appreciate the robust number of derivatives synthesized based on three heterocyclic cores, the rationale is confusing and ambiguous. The authors can do better by inserting more profound justifications in the text for choosing such cores, substituents, etc
3. It seems that the choice for substituents/pendant structures is very random. In series A, the substituents are aliphatic and aromatic while in B and C are mostly aromatic. WHy is this?
4. In series A, only cycloaliphatics were considered. How about the unbranched/branched straight aliphatics. It would be interesting to check on polyethers. Pls justify.
5. The aromatic substituents considered for B and C series are not the same, why is this?
6. The molecular docking study must be extended to all (just put data in the SI) for comparison of experimental and in silico data. 
7. Where is the molecular dynamics simulation data? Please add (up to 100 ns please). Please put docking and MDS after binding in the results and discussion text.
8. Prior to performing the mice melanoma experiment, was LD50 determination done to determine baseline toxicity and as reference for the dosages?
9. How about selectivity tests for B6.
10. Why no further examination of the effect of alkyl group in the 4-methoxyphenyl moiety? It would have been better to check on ethoxy, propoxy, butoxy, isopropoxy, isobutoxy, etc...or perhaps a polyether!
11. In the SI, the NMR spectra are too small. Please maximize the page. One cannot clearly see the peaks/signals. 
12. What is the purity of the compounds? Any LC trace to show? 

Comments on the Quality of English Language

Please check several text. What do the authors mean by substituted aromatic piperazines? It is ambiguos. Please correct!

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

It is not clear from Scheme 2., page 4., where the structures of substituents R1 are presented.

It is also unclear from Scheme 3. page 5., how the substituents R3 in compounds C1 and C2 differ in their structures. Similarly, Scheme 3 does not clearly show how the substituents R4 in compounds C8 and C9 differ in their structures.

On page 7, in the caption of Figure 3, it is unclear what the graph represents. It is incorrect to state that it shows “the in vitro inhibition rate… on MCF-7 cells,” as the graph presents the results of a CCK-8 assay, which measures cell viability and proliferation in cell culture. Thus, the correct description should refer to inhibition of cell viability and/or proliferation in MCF-7 cells following treatment with the tested compounds. in the Figure 3 caption, should be specified whether the tested compounds were applied at an equimolar concentration of 50 µM, and the treatment time should be indicated as well.

On page 23, line 807, In the Materials and Methods section: after “CCK-8 method,” the manufacturer should be indicated if it is not mentioned elsewhere.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed most of the issues raised during the revision of the manuscript. Nevertheless, there are a few more issues to be addressed before the final acceptance. Thus, I recommend a minor revision of the manuscript.

More specifically,

• The title of the manuscript should be corrected to: “Discovery of New Quinazolinone and Benzimidazole Analogues….”.
• Page 3, line 91: The phrase “the heterocyclic core (fragment II), and the substituted aryl ring (fragment III)” should be corrected to “the heterocyclic core (fragment II), and the indole ring (fragment III)”.
• Page 3, lines 103-104: The phrase “compound B0 featuring benzimidazole cores was designed” should be corrected to “compound B0 featuring benzimidazole core was designed”.
• Page 4, lines 122-123: The phrase “3-Bromo-1,2-bromobenzamide reacted with different substituted aromatic aldehydes” should be corrected to “3-Bromo-1,2-benzenediamine reacted with different substituted aromatic aldehydes”.
• Page 6, lines 144-145: The authors say “with compounds A5, A6, A15, B3, B4, B5, and B6 demonstrating superior binding potency to tubulin compared to colchicine.” According to Table 1, compound A6 does not possess superior binding potency compared to colchicine, thus it should be removed from this sentence.
• Pages 6 and 7: There is an inconsistency between the results presented for colchicine in Figure 3 and Table 2. The IC₅₀ of colchicine against the MCF-7 cell line is in the low nM area (Table 2), thus if a concentration of 50 μM was used during the initial screening (as denoted at page 6, line 148), then the cell viability of the MCF-7 cells cannot be approximately 50%, as presented in Figure 3. This issue must be clarified by the authors. Did the authors use 50 nM concentration instead of 50 μM during the initial screening?

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The authors addressed the concerns raised in the original submission. The paper is now acceptable for publication in its present form.

Author Response

The reviewer has no comments.