Novel Derivatives of Deoxycholic Acid Bearing Linear Aliphatic Diamine and Aminoalcohol Moieties and their Cyclic Analogs at the C3 Position: Synthesis and Evaluation of Their In Vitro Antitumor Potential

Round 1

Reviewer 1 Report

The authors report a very interesting article, which should be published in the journal molecules. However, minor corrections must be made, which are indicated below:

       Authors should write the steroidal structures as indicated in the figure:

2.       Letter in the atoms must be Palatino Linotype.

3.       The position of the CH₃O- group should not be represented as 25, but rather as MeO or CH₃O-, because it is not part of the original skeleton on molecule.

4.       Line 422: 61.51 (t, C-26) without bold!

5.       Line 441: 60.75 (t, C-26) without bold!

6.       Line 460: 69.52 (t, C-26) without bold!

7.       Line 608: write reference pages according to the journal format

8.     Line 685: write reference pages according to the journal format

Comments for author File: Comments.pdf

Author Response

Dear Reviewer #1,

Thank you for careful study of our manuscript and for your very useful remarks. We revised the manuscript according to your comments.

1. Authors should write the steroidal structures as indicated in the figure.

Authors: corrected. Steroidal structures were modified according to your figure.

2. Letter in the atoms must be Palatino Linotype.

Authors: corrected.

3. The position of the CH3O- group should not be represented as 25, but rather as MeO or CH3O-, because it is not part of the original skeleton on molecule.

Authors: corrected. “25” was deleted from CH₃O- group. Additionally, we deleted numbering of atoms from side moieties because it seems to give excessive information.

4. Line 422: 61.51 (t, C-26) without bold!

Authors: corrected.

5. Line 441: 60.75 (t, C-26) without bold!

Authors: corrected.

6. Line 460: 69.52 (t, C-26) without bold!

Authors: corrected.

7. Line 608: write reference pages according to the journal format

Authors: corrected.

8. Line 685: write reference pages according to the journal format

Authors: corrected.

All mentioned corrections were marked in the text by using reviewing tool in MS Word.

Reviewer 2 Report

In the manuscript “molecules-544701” the authors reported the synthesis, and the antitumor activity of new deoxycholic acid derivatives. The synthesized molecules have been the subject of preliminary studies with the aim to evaluate their cytotoxicity and to verify if the structural modification, respect to the molecules of their previous work, could improve antitumor activity. A SAR was made even if it is based only on three new compounds. Compound 9, which showed the best pharmacological profile, was selected for further studies to reveal the mechanism of action. Apparently the work seems to foresee the synthesis and the biological evaluation of eleven compound but, as referred by the same authors, seven of ten are already synthesized in a previously work (ref. 21). The cytotoxicity data are also known from ref. 21-23 and novel data are reported only for the three new derivatives. The sole exception is the inhibitory activity against NO synthesis by macrophages as new data for all the compounds. However, the manuscript provides new information about the mechanism of action of these molecules and for this reason can be interesting for those working in this field. The paper represents the scope of the journal, the synthesis look sound and the reactions are well described. For this reason it may be accepted for publication with some modification.

 Some modification:

It is no clear for me the link between the mechanism of action of BA derivatives and the molecular docking simulation reported in the manuscript. The authors stated that VDR can be  considered as a primary intracellular target for compound 9 but they not describe how this    interaction could influence the proposed mechanism.

Pag. 5 row 152 please      substitute 39.8 with 34.8

Author Response

Dear Reviewer #2,

We are sincerely thankful to you for your deep analysis of our manuscript and highly valuable remarks. We revised the manuscript and, please, let us respond to your questions.

1. It is no clear for me the link between the mechanism of action of BA derivatives and the molecular docking simulation reported in the manuscript. The authors stated that VDR can be considered as a primary intracellular target for compound 9 but they not describe how this interaction could influence the proposed mechanism.

Authors: The part of manuscript describing the links between VDR activation and triggering of apoptosis and autophagy was reinforced by additional references:

“Moreover, activation of VDR was shown to be able to induce apoptosis and autophagy that correlates well with ability of compound 9 to trigger these processes. Particularly, a range of VDR ligands displays a pro-apoptogenic activity – an active metabolite of vitamin D, 1α,25(OH)₂D₃, was found to increase Bax/Bcl-2 ratio in leukemia cells [1] and trigger cytochrome c release from mitochondria with subsequent activation of caspase cascade in prostate cancer cells [2]. Apoptosis-inducing activity of VDR ligands was also shown for Gemini vitamin D analogue of calcitriol [3] and diarylmethane skeleton-containing VDR agonist [4] in colon and breast cancer cells, respectively. Beside this, activation of VDR can stimulate autophagy – previously, it was found that 1α,25(OH)₂D₃ and calcipotriol induce autophagy in breast and colorectal cancer [5][6] and cervical carcinoma [7] cells, respectively”.

References:

1.        Kizildag, S.; Ates, H.; Kizildag, S. Treatment of K562 cells with 1,25-dihydroxyvitamin D3 induces distinct alterations in the expression of apoptosis-related genes BCL2, BAX, BCLXL, and p21. Ann. Hematol. 2010, 89, 1–7.

2.        Guzey, M.; Kitada, S.; Reed, J.C. Apoptosis induction by 1alpha,25-dihydroxyvitamin D3 in prostate cancer. Mol. Cancer Ther. 2002, 1, 667–677.

3.        Ferronato, M.J.; Alonso, E.N.; Gandini, N.A.; Fermento, M.E.; Villegas, M.E.; Quevedo, M.A.; Arévalo, J.; López Romero, A.; Rivadulla, M.L.; Gómez, G.; et al. The UVB1 Vitamin D analogue inhibits colorectal carcinoma progression. J. Steroid Biochem. Mol. Biol. 2016, 163, 193–205.

4.        Wang, C.; Wang, B.; Hou, S.; Xue, L.; Kang, Z.; Du, J.; Li, Y.; Liu, X.; Wang, Q.; Zhang, C. Discovery of novel nonsteroidal VDR agonists with novel diarylmethane skeleton for the treatment of breast cancer. Eur. J. Med. Chem. 2019, 163, 787–803.

5.        Tavera-Mendoza, L.E.; Westerling, T.; Libby, E.; Marusyk, A.; Cato, L.; Cassani, R.; Cameron, L.A.; Ficarro, S.B.; Marto, J.A.; Klawitter, J.; et al. Vitamin D receptor regulates autophagy in the normal mammary gland and in luminal breast cancer cells. Proc. Natl. Acad. Sci. 2017, 114, E2186–E2194.

6.        Abu el Maaty, M.A.; Strassburger, W.; Qaiser, T.; Dabiri, Y.; Wölfl, S. Differences in p53 status significantly influence the cellular response and cell survival to 1,25-dihydroxyvitamin D3-metformin cotreatment in colorectal cancer cells. Mol. Carcinog. 2017, 56, 2486–2498.

7.        Wang, R.C.; Levine, B. Calcipotriol Induces Autophagy in HeLa Cells and Keratinocytes. J. Invest. Dermatol. 2011, 131, 990–993.

Inserted text and novel references were marked in the manuscript by yellow color.

2. Pag. 5 row 152 please substitute 39.8 with 34.8

Authors: corrected. Corrected number was colored by yellow.