Synthesis, Anticancer, Antioxidant, Anti-Inflammatory, Antimicrobial Activities, Molecular Docking, and DFT Studies of Sultams Derived from Saccharin

We thank the reviewer for his comments

• It was described in methodology section.
• There was typing mistake in table numbers in methodology and corrected accordingly.
• CCl₄ provides a case of liver fibrosis in which the antioxidant activities were decreased and that allow us to test our compounds to verify their antioxidant activities and their ability to restore the antioxidant level to be close to normal values (control).

We thank the reviewer for his comment.. 

Compounds 2 and 3a-g were tested as anti-inflammatory drugs (Table 2) where the proinflammatory cytokines IL-6 and TNF-α were investigated. Therefore, we tested the ability of these drugs to inhibit modulators of the inflammation process. Among these, cyclooxygenase (COXs) enzymes are importantly responsible for conversion of arachidonic acid into the prostaglandin E2 (PGE2), which is the main moderator of the inflammation process.

Therefore,

• Upon the request of the reviewer and to compare the inhibitory effect of these drugs, we performed molecular docking analysis of acetylsalicylic acid (ASA) as standard COX-1 inhibitor against COX-1
• .As shown in Table 5, all the tested compounds (compounds 2 & 3a-g) have more negative Gibbs free, electrostatic, and total intermolecular energies than the standard inhibitor ASA.
• These results indicate that the synthesized compounds are potent anti-inflammatory drugs as compared to standard inhibitors (i.e., ASA).
• Based on that, the manuscript was updated accordingly..

We thank reviewer for their suggestion on the electron distribution of each product involve in the reaction.

Now we have generated the electron distribution of HOMO and LUMO of the compounds  all the tested compounds. These distributions are shown and added to the updated manuscript in fig 6. 

We have added the discussion in the main paper as “The spatial electron distribution of HOMO, LUMO were computed and its clearly indicate the electron donation ability.” The spatial electron distribution of each compounds (2 ,3a-g) are shown in Fig.6

in the methodology there is no description of cell cultures - it is also necessary to add full cell line name,

source/origin/reference number

-the reference substance for the MTT test is missing (see CCl4₄ for antiox/antiinfl.) – please add.

max absorbance for formazan depend on pH and cel density and has two maxima (500 and 570 nm) why you measure absorbance in 630 nm?

-what was the cell density seeding for the MTT experiment? in 4.2.2.1 you write that 50 uM (????) is a mistake - please clarify and correct

-no information on the concentrations of compounds used in individual in vitro tests

We thank the reviewer for his comment. There is no need to use standards as we compare the results with normal cells (control).

We thank the reviewer for his comment. Corrected and updated in the manuscript (in the experimental section as 570 nm ).

Corrected and updated in the manuscript

It is mentioned in table 4 (50 µmol).

 We thank the reviewer for his comment.

Because of all the tested compounds lack of any antimicrobial activities. Some images of the results can be provided upon request.

Reviewer Comments

 Response

Still some problems for Table 3. Antioxidant activity of the tested compounds 2 and 3a-g there is no percentage of activity present in this table

The percentage have been added to table 3 and updated in the manuscript

There is no normal cell lines used for cytotoxicity activity

We followed the previously reported protocol published earlier in reference (16) (I. Elghamry, M.M. Youssef, M.A. Al-Omair, H. Elsawy. (2017) Synthesis, antimicrobial, DNA cleavage and antioxidant activities of tricyclic sultams derived from saccharin. Euro J Med Chem. 139; 107-113)

Table 2: author should be moved for calculation part below the table

It is modified and rest of calculations already described in the kit protocol to reduce repetition.

Author not give any relationship between synthesized compounds and ampicillin

As we have mentioned in the first round, Ampicillin is a broad spectrum antibiotic and is usually used as standard one. Additionally, previous published literature report have used  ampicillin as standard with different organic scaffold (ex. Sak M, Al-Faiyz YS, Elsawy H, Shaaban S.” Novel Organoselenium Redox Modulators with Potential Anticancer, Antimicrobial, and Antioxidant Activities. Antioxidants (Basel). 2022 Jun 23;11(7):1231.

Reviewer comments

Response

Reviewer 1

 Response

There is no normal cell lines used for cytotoxicity activity

Thank you for your comment. We did MTT assay on normal cell lines (WI138) and the results are displayed in the following table, and updated as supplementary file (Table S1) with the manuscript.

Author not give any relationship between synthesized compounds and ampicillin

The antibacterial activity have been repeated and Sulfamethoxazole was used instead of ampicillin. This part was updated in the discussion part of the manuscript (lines201-209).

As mentioned in the introduction of the manuscript, the compounds under investigation are considered as derivatives of sulfonamide, therefore, one of the sulfa drug antibiotic, namely Sulfamethoxazole (SMZ or SMX) was used as a positive control.

“The results showed that all the compounds under investigation have no antibacterial activity.

Some pictures of these results are available in the editor covering letter