Synthesis of 3-Trifluoromethyl-5,6-dihydro-[1,2,4]triazolo Pyrazine Derivatives and Their Anti-Cancer Studies

Round 1

Reviewer 1 Report

the authors have been reported the synthesis of a variety of 3-(trifluoromethyl 5, 6-dihydro-[1, 2, and 4] triazolo pyrazine derivatives using mild condition in excellent yields. All the synthesized compounds were characterized using 1H NMR, IR, and LC-MS analyses. All the synthesized compounds were utilized for
257 anti-cancer studies. Also, they are screened for the up-regulation of BAX and down-regulation of Bcl2 in HT 29 colon cancer cell lines. 

I recommeded  publishing this paper after minor revision

* NMR data: all the peaks should be assigned.

 

Author Response

# Reviewer 1

The authors have been reported the synthesis of a variety of 3-(trifluoromethyl 5, 6-dihydro-[1, 2, and 4] triazolo pyrazine derivatives using mild condition in excellent yields. All the synthesized compounds were characterized using 1H NMR, IR, and LC-MS analyses.

Thanks for the valuable comments. As suggested by the reviewer.

All the synthesized compounds were utilized for
257 anti-cancer studies. Also, they are screened for the up-regulation of BAX and down-regulation of Bcl2 in HT 29 colon cancer cell lines. 

Thanks for the valuable comments. As suggested by the reviewer. We have done up-regulation and down-regulation of HT29 cells for apoptotic examination.

I recommended publishing this paper after minor revision

* NMR data: all the peaks should be assigned.

Reviewer 2 Report

e manuscript presents result on synthesis and biological activity evaluation of several [1,2,4]-triazolo pyrazine derivatives. Authors tested antiproliferative activity against two colon cancer cell lines and their cytotoxicity has been tested against chang liver cell line. This work can be published but it needs some serious attention to improve its present form. The editorial part is very poor which make it difficult to read and assess the text correctly. For example the experimental part for chemistry is written often without spaces and sub- or superscript - just as a plain text. This can happen during automatic formation of pdf but it is still duty of corresponding author to check the text before submitting.

-What about purity of these compounds? There are only 1H NMR, LCMS (not high resolution) and IR without statement of purity which is important for further investigation e.g. biological screening.

-experimental for biological part is written even worse. This is crucial part to understand what really had been done. For example: authors state that “cells were developed in an ideal media” or were seeded  “in 104 cell/well” again in mtt test the “supernatant and crystals was recorded” What does it mean? What solubilistation was used in the procedure?.

RT-PCR procedure the same: authors described that the cDNA was synthesized according to manufacturer’s suggestions but what with cells? What was incubation time, concentration…?

Chang liver cell line is known to be problematic. This line is probably derived from HeLa cells or was contaminated. Did authors corrected this?  

 

Author Response

# Reviewer 2

The manuscript presents result on synthesis and biological activity evaluation of several [1, 2, 4]-triazolo pyrazine derivatives. Authors tested antiproliferative activity against two colon cancer cell lines and their cytotoxicity has been tested against chang liver cell line. This work can be published but it needs some serious attention to improve its present form. For example the experimental part for chemistry is written often without spaces and sub- or superscript - just as a plain text. This can happen during automatic formation of pdf but it is still duty of corresponding author to check the text before submitting.

Thanks for the valuable comments. As suggested by the reviewer. Spacing will be corrected during the edited part of the production team because when we convert word to file format this tiny things will happens. We have provided separate file for figures for this issues.

-What about purity of these compounds? There are only 1H NMR, LCMS (not high resolution) and IR without statement of purity which is important for further investigation e.g. biological screening.-experimental for biological part is written even worse. This is crucial part to understand what really had been done. For example: authors state that “cells were developed in an ideal media” or were seeded “in 10⁴ cell/well” again in Mtt test the “supernatant and crystals was recorded” What does it mean? What solubilisation was used in the procedure?

Thanks for the valuable comments. As suggested by the reviewer. Media used for HT29 and HCT116 was McCoy’s media and crystal was dissolved by using DMSO in our experiments. Purity of the compound was more than 90% of all the compounds.

RT-PCR procedure the same: authors described that the cDNA was synthesized according to manufacturer’s suggestions but what with cells? What was incubation time, concentration…? Chang liver cell line is known to be problematic. This line is probably derived from HeLa cells or was contaminated. Did authors correct this?  

Thanks for the valuable comments. As suggested by the reviewer. Total reaction volume to 20 μl and carry out the synthesis of cDNA for 1 h at 42°C, pursued by R.T inactivation for 5 min at 85°C. Samples of resultant products were examined in 1.5% agarose gel.

Chang liver cell lines is normal liver cell lines, entire metabolism will take place in the liver only hence we have used chang liver for cytotoxic examinations.

Round 2

Reviewer 2 Report

manuscript has been improved according to the comments. It can be accepted in present form.