Buravchenko et al describe the design and synthesis of a series of sulfonamide derived quinoxaline 1,4-dioxides as potential carbonic anhydrase (CA) inhibitors with an outlook on further developing their quinoxaline 1,4-dioxides as a solid tumour chemotherapy. The authors describe one aspect in the present difficulty in treating solid tumours is the occurrence of hypoxic conditions in and around the tumour cells. Carbonic anhydrase IX is implicated in assisting cancer cell survival under hypoxic conditions, with recent studies indicating that the inhibition of CA IX reduces the survival of cancer cells under hypoxic conditions. There are already CA inhibitors used clinically, and others undergoing clinical trials now.

In this manuscript the authors begin by attempting to dock sulfonamidequinoxaline 1,4-dioxides into CA IX. The authors then synthesise a small library of sulfonamide derived quinoxaline 1,4-dioxides. The antiproliferative activity of some of these analogues was then tested against a breast cancer cell line under normoxic and hypoxic conditions. A different selection of the same compounds is then tested against a broader panel of cancer cell lines under presumably hypoxic conditions? Next, the activity of the synthesised inhibitors was measured against a small panel of cytosolic and membrane-bound carbonic anhydrase isoforms revealing 7g to be the most potent inhibitor of the desired CA IX target. The authors then dock 7g into a crystal structure of CA IX in order to seek a mechanistic explanation for the observed biochemical activity. Finally, in another cell assay, the authors demonstrate that 7g’s impact on the viability of skin cancer cells is higher in hypoxic conditions than in normoxic conditions and provide evidence that this may be due to increased apoptosis of cancer cells.

I found the explanation and supporting data provided for the synthetic chemistry section of the manuscript clear and sufficient to support the author’s conclusions. However, the sections “Biology”, “antiproliferative data” and “Carbonic anhydrase inhibition assay”, relating to the discussion and analysis of the biological data was difficult to follow and I am not convinced that the stated conclusions can be fully justified by the data provided. In this section it was frequently not clear which part of the data the authors were referring to. More reference to the specific table being discussed is sorely needed. Not all the compounds prepared (listed in Table 2) were tested in all the biological assays. It was not clear why this was not done. Furthermore, it appears at multiple instances that the authors are drawing trends between inhibitors without there being data in tables for those inhibitors. For example, correlating the enzyme inhibition results with the antiproflerative data of compounds 7a, 7b, 8a, and 8b. But the authors do not provide any antiproflerative data for 8b at all, and only provide it for 8a against one cell type. In general, the quality of the figures is also not of publication quality. The figures showing the docking (Fig. 3 and 4) are borderline, and figure 5 is blurry and looks amateurish.

Despite this, the authors do present novel work, with well-designed chemistry generating novel CA inhibitors from the quinoxaline 1,4-dioxide scaffold. Several of synthesised compounds showed nanomolar activity against CA IX with many of the compounds possessing antiproliferative activity against cancer cell lines in the low micromolar range. The results showing that 7g selectively reduces cancer cell viability under hypoxic conditions is notable, and supports the authors hypothesis that inhibition of CA IX may promote cancer cell apoptosis under hypoxic conditions. Furthermore, the authors reveal structural insights into which groups are were tolerated in the 2 and 3 positions of the scaffold, useful information for others in the field to be aware of.

Corrections:
1) On Page 8: Therefore, we adapted the early described procedure for the preparation of the sulfamide analog 12 (Scheme 1).
Should read: Therefore, we adapted the previously described procedure for the preparation of the sulfamide analog 12 (Scheme 1).
2) Page 9: Treating of acetanilide 14 with dimethylacetal of dimethylformamide (DMADMF) in dimethylformamide (DMF) at room temperature yielded the key sulfamidine 15 in high yield.
Should read: Treatment of acetanilide 14 with N,N-dimethylformamide dimethyl acetal (DMADMF) in dimethylformamide (DMF) at room temperature yielded the key sulfamidine 15 in high yield.
3) Additional comment: I’m also not sure that DMADMF is the common abbreviation for this compound. It was not immediately clear to me what this was. I think the more common abbreviation is DMFDMA or DMF-DMA.
4) Page 10: did not proceed under procedures the previously described for the Beirut reaction.
Should read: did not proceed under previously described procedures for the Beirut reaction.
5) General comment: throughout the text hyphens (-) are used for ranges of numbers, this should be changed to en-dashes (–) for all ranges (i.e. 10–20, or compound 7a–h).
6) Why is the Hypoxia value for 8g reported as <3.1? Other compounds in the table report lower values.
7) The sections under the headings of “biology and antiproliferative activity” are difficult to follow. Please ensure that you make clear in the text which table you are referring to. For example the sentence: “The spectrum of antiproliferative properties of new quinoxaline 1,4-dioxide derivatives 7a-h, 8a, and 18 was studied, comparing them to docetaxel and etoposide, against an expanded panel of tumor cells, including eight lines of different histogenesis.” Please make clear in the text that you are now referring to table 4. Furthermore, you say that the properties of 8a were studied, then why is there no data for compound 8a in table 4? Why weren’t 8b, 8c and 8g also screened for activity?
8) Is the data provided in table 4 under normoxic or hypoxic conditions? This is not clear from the text nor the experimental procedure.
9) On page 12 the authors state: “Furthermore, the cytotoxicity of most synthesized derivatives increased by 1.5-7 times under hypoxic conditions.”
-I assume the authors are referring to their HCR values from table 3 here. But these range from 1.1–4.7? Could the authors either correct their statement or explain what the reviewer is overlooking?
10) On page 16 the authors state: “It was observed that the presence of a halogen atom in the phenyl ring at position 3 of quinoxaline 1,4-dioxides 7b and 8b generally enhances the activity of these derivatives.”
This sentence cannot be supported by the data provided. I see no antiproliferation data for compound 8b.
11) Page 16: “Interestingly, introducing of a sulfonamide group into the phenyl ring at the C3 carbon atom of the heterocycle (derivative 18)…”
Should read: “Interestingly, the introduction of a sulfonamide group into the phenyl ring at the C3 carbon atom of the heterocycle (derivative 18)…”
12) The data in Table 5 are all missing their margin of error. I don’t find the statement in the footnote acceptable (errors were ± 5–10% of the presented values).
13) On page 18: “It is noteworthy that the position of the sulfonamide group plays a significant role in the ability of quinoxaline 1,4-dioxides to inhibit CAIX. The introduction of a sulfonamide fragment into position 7 of quinoxaline 2-carbonitrile 1,4-dioxide results in a partial or complete loss of the ability of derivatives 8a, 8b to block CA IX activity when compared to their 6-analogues (compounds 7а, 7b). This observation correlates with the antiproliferative activity of these compounds (Table 3, 4).”
In this paragraph the authors correlate enzyme inhibition results with the antiproflerative data of compounds 7a, 7b, 8a, and 8b. But the authors do not provide any antiproflerative data for 8b at all, and only provide it for 8a against one cell type. These conclusions cannot be supported based on the data provided.
14) Section title “Mechanism of tumor cells death”
Should be: “Mechanism of tumor cell death”
15) Figure 5 is not a publication quality figure and would need to be remade.