Antimelanoma activities of chimeric thiazole–androstenone derivatives

The discovery of chimeric anti-melanoma agents is reported. These molecules are potent growth suppressors of melanoma cells in vitro with growth inhibition of 50% (GI50) values as low as 1.32 µM. Compounds were more toxic to melanoma cells in vitro than commonly used anti-melanoma agent dacarbazine as measured by TUNEL assay. They induced both caspase-independent apoptosis evident by colocalization of TUNEL with endonuclease G (EndoG) and caspase-mediated apoptosis measured by colocalization of TUNEL with caspase-activated DNase (CAD). In addition, compounds 3 and 5 strongly induced oxidative injury to melanoma cells as measured by TUNEL colocalization with heme oxygenase-1 (HO1). Dacarbazine induced only caspase-independent apoptosis, which may explain why it is less cytotoxic to melanoma cells than compounds 3, 4 and 5.


Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
The present manuscript describes biological evaluation as antimelanoma agents of a series of steroidal thiazole derivatives previously prepared in the Alam group. The manuscript presents interesting results, and provides much information regarding the investigations on the mechanism of action of the thiazole derivatives. Therefore, I would like to recommend this manuscript for publication in Royal Society Open Science, after the following comments are addressed: • The authors synthesized and submitted 50 different thiazole derivatives to the NCI, and from there, compounds 1-8 were selected for further testing. Please include what criteria was used to include compounds 1-8 and exclude the rest? Was this based on a given GI50 cutoff? • A follow-up question arises upon examination of the data in table 1, as compound 6 clearly presents a much narrower activity profile. Why was compound 6 selected for further testing in the first place? • The authors could provide a more concise medicinal chemistry analysis, for example choosing a representative melanoma cell line, they could provide information on any structureactivity trends. Do more lipophilic or more polar aromatic rings increase acitivity? If no clear relationships are identified, please state it. • Which compounds are taken to next stage looking at ADME and in-vivo antimelanoma? Why? Is compound 6 still being considered?

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? Yes

Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
This is an interesting work. This manuscript reports the GI50, TGI and LC50 of some thiazoloandrostenone derivatives on melanoma cell lines, for studying the structure-activity relationship. The authors proved that some of these compounds can induce caspase-independent apoptosis, and compounds (3 and 5) strongly induced oxidative injury to melanoma cells as measured by TUNEL colocalization with heme oxygenase-1 (HO1). The authors claim that they have found effective anti-melanoma drugs. However, there are still some concerns here.

1.
On page 4, line 31, the author says that "Overall, amino-thiazole derivatives (Table 1, 1-7)15 were more toxic to melanoma cell lines than phenyl thiazole derivatives." However, no compound has a lower IG50 than compound 8 in all melanoma cell lines. And more importantly, there is no intuitive data to explain that amino-thiazole derivatives (Table 1, 1-7)15 were more toxic to melanoma cell lines than phenyl thiazole derivatives.

2.
On page 4, line 47, the author says that" All eight compounds inhibited this cell line with GI50 values as low as 1.64 µM", however, if the author can use the highest IG50 3.22 µM, the article might be more reliable.

3.
On page 6, line 19, the author says that" Phenyl-substituted compound (2) inhibited the growth of HFF-1 cells with an IC50 value of 21 µM, which was much higher than the GI50 values against the melanoma cell lines." However, the IC50 value of compound (2) inhibiting HFF-1 cells is only significantly different from the GI50 value of melanoma cell lines LOX IMVI and MDA-MB-435.

Decision letter (RSOS-210395.R0)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Dr Alam: Title: Antimelanoma Activities of Chimeric Thiazole-Androstenone Derivatives Manuscript ID: RSOS-210395 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
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RSC Associate Editor: 2
Comments to the Author: The reviewers have raised several valid questions during this review. Addressing these questions in a revised manuscript would strengthen the paper and would be helpful to future readers.

RSC Subject Editor:
Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The present manuscript describes biological evaluation as antimelanoma agents of a series of steroidal thiazole derivatives previously prepared in the Alam group. The manuscript presents interesting results, and provides much information regarding the investigations on the mechanism of action of the thiazole derivatives. Therefore, I would like to recommend this manuscript for publication in Royal Society Open Science, after the following comments are addressed: • The authors synthesized and submitted 50 different thiazole derivatives to the NCI, and from there, compounds 1-8 were selected for further testing. Please include what criteria was used to include compounds 1-8 and exclude the rest? Was this based on a given GI50 cutoff?
• A follow-up question arises upon examination of the data in table 1, as compound 6 clearly presents a much narrower activity profile. Why was compound 6 selected for further testing in the first place? • The authors could provide a more concise medicinal chemistry analysis, for example choosing a representative melanoma cell line, they could provide information on any structure-activity trends. Do more lipophilic or more polar aromatic rings increase acitivity? If no clear relationships are identified, please state it. • Which compounds are taken to next stage looking at ADME and in-vivo antimelanoma? Why? Is compound 6 still being considered?
Reviewer: 2 Comments to the Author(s) This is an interesting work. This manuscript reports the GI50, TGI and LC50 of some thiazoloandrostenone derivatives on melanoma cell lines, for studying the structure-activity relationship. The authors proved that some of these compounds can induce caspase-independent apoptosis, and compounds (3 and 5) strongly induced oxidative injury to melanoma cells as measured by TUNEL colocalization with heme oxygenase-1 (HO1). The authors claim that they have found effective anti-melanoma drugs. However, there are still some concerns here. 1. On page 4, line 31, the author says that "Overall, amino-thiazole derivatives (Table 1, 1-7)15 were more toxic to melanoma cell lines than phenyl thiazole derivatives." However, no compound has a lower IG50 than compound 8 in all melanoma cell lines. And more importantly, there is no intuitive data to explain that amino-thiazole derivatives (Table 1, 1-7)15 were more toxic to melanoma cell lines than phenyl thiazole derivatives. 2. On page 4, line 47, the author says that" All eight compounds inhibited this cell line with GI50 values as low as 1.64 µM", however, if the author can use the highest IG50 3.22 µM, the article might be more reliable. 3. On page 6, line 19, the author says that" Phenyl-substituted compound (2) inhibited the growth of HFF-1 cells with an IC50 value of 21 µM, which was much higher than the GI50 values against the melanoma cell lines." However, the IC50 value of compound (2) inhibiting HFF-1 cells is only significantly different from the GI50 value of melanoma cell lines LOX IMVI and MDA-MB-435.

Decision letter (RSOS-210395.R1)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Dr Alam:
Title: Antimelanoma Activities of Chimeric Thiazole-Androstenone Derivatives Manuscript ID: RSOS-210395.R1 It is a pleasure to accept your manuscript in its current form for publication in Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science and the Royal Society of Chemistry, I look forward to your continued contributions to the Journal. Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The present manuscript describes biological evaluation as antimelanoma agents of a series of steroidal thiazole derivatives previously prepared in the Alam group. The manuscript presents interesting results, and provides much information regarding the investigations on the mechanism of action of the thiazole derivatives. Therefore, I would like to recommend this manuscript for publication in Royal Society Open Science, after the following comments are addressed: • The authors synthesized and submitted 50 different thiazole derivatives to the NCI, and from there, compounds 1-8 were selected for further testing. Please include what criteria was used to include compounds 1-8 and exclude the rest? Was this based on a given GI50 cutoff?
 We appreciate the reviewer for this insightful comment. The cutoff is based on the activity of the compounds against NCI-60 cancer cell lines at 10 µM.
Compounds showing significant activity were tested at lower concentration to determine the IC50 values and these 8 compounds are in the cutoff list.
• A follow-up question arises upon examination of the data in table 1, as compound 6 clearly presents a much narrower activity profile. Why was compound 6 selected for further testing in the first place?  We want to emphasize that we focused on compounds 3, 5, and 7 in the manuscript.
• The authors could provide a more concise medicinal chemistry analysis, for example choosing a representative melanoma cell line, they could provide information on any structure-activity trends. Do more lipophilic or more polar aromatic rings increase acitivity? If no clear relationships are identified, please state it.  We thank the reviewer for this comment. We added the test for more structure-activity trends.
• Which compounds are taken to next stage looking at ADME and in-vivo antimelanoma? Why? Is compound 6 still being considered?  For the future studies, we are making more derivatives to find better compounds. Due to high selectivity, we are focusing on compounds 3 and 7 for ADMET and in vivo studies.

Reviewer: 2
Comments to the Author(s) This is an interesting work. This manuscript reports the GI50, TGI and LC50 of some thiazolo-androstenone derivatives on melanoma cell lines, for studying the structure-activity relationship. The authors proved that some of these compounds can induce caspase-independent apoptosis, and compounds (3 and 5) strongly induced oxidative injury to melanoma cells as measured by TUNEL colocalization with heme oxygenase-1 (HO1). The authors claim that they have found effective anti-melanoma drugs. However, there are still some concerns here. 1. On page 4, line 31, the author says that "Overall, amino-thiazole derivatives (Table 1, 1-7)15 were more toxic to melanoma cell lines than phenyl thiazole derivatives." However, no compound has a lower IG50 than compound 8 in all melanoma cell lines. And more importantly, there is no intuitive data to explain that aminothiazole derivatives (Table 1, 1-7)15 were more toxic to melanoma cell lines than phenyl thiazole derivatives.


We appreciate the reviewer for this insightful comments. This is a general statement for the series of 50 compounds. Only one of the ~20 phenyl derivative showed potent activity. In the amino series, we found 7 compounds in a series of ~30 compounds as potent antimelanoma agents.