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Discovery of ‘click’ 1,2,3-triazolium salts as potential anticancer drugs

Ivana Steiner
Ivana Steiner
, 
Nikolina Stojanovic
Nikolina Stojanovic
, 
Aljosa Bolje
Aljosa Bolje
, 
Anamaria Brozovic
Anamaria Brozovic
, 
Denis Polancec
Denis Polancec
, 
Andreja Ambriovic-Ristov
Andreja Ambriovic-Ristov
, 
Marijana Radic Stojkovic
Marijana Radic Stojkovic
, 
Ivo Piantanida
Ivo Piantanida
, 
Domagoj Eljuga
Domagoj Eljuga
, 
Janez Kosmrlj
Janez Kosmrlj
 e 
Maja Osmak
Maja Osmak
   | 19 lug 2016
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Article Category: Research Article
Pubblicato online: 19 lug 2016
Pagine: 280 - 288
Ricevuto: 23 nov 2015
Accettato: 17 mar 2016
DOI: https://doi.org/10.1515/raon-2016-0027
© Radiol Oncol 2016
This content is free.

Figure 1

The structures of triazoles 1 and triazolium salts 2.
The structures of triazoles 1 and triazolium salts 2.

Figure 2

Effects of 2b on the cell cycle of H460 cells. Logarithmically growing H460 cells were treated with 29.7 and 110 μM of 2b for indicated period of time. Afterwards they were harvested for cell cycle analysis measured by FACS as described in Materials and methods. Representative data of three experiments are shown.
Effects of 2b on the cell cycle of H460 cells. Logarithmically growing H460 cells were treated with 29.7 and 110 μM of 2b for indicated period of time. Afterwards they were harvested for cell cycle analysis measured by FACS as described in Materials and methods. Representative data of three experiments are shown.

Figure 3

DNA as possible target of compound 2b. Thermal denaturation curves of ct-DNA (c(ct-DNA) = 2 x 10–5 M, r[compound]/[ct-DNA] =0.3) at pH 7.0 (sodium cacodylate buffer, I = 0.05 M) upon addition of compound 2b. Error in DTm values: ±0.5°C.
DNA as possible target of compound 2b. Thermal denaturation curves of ct-DNA (c(ct-DNA) = 2 x 10–5 M, r[compound]/[ct-DNA] =0.3) at pH 7.0 (sodium cacodylate buffer, I = 0.05 M) upon addition of compound 2b. Error in DTm values: ±0.5°C.

Figure 4

Formation of ROS by 2b in H460 cells. Logarithmically growing H460 cells were stained for 1 hour with 10 mM CM-H2DCFDA and then either treated with 110 μM 2b during indicated time points (A) or treated with indicated concentrations of 2b for 180 min (3 hours) (B). Afterward ROS formation was determined by flow cytometry as described in Materials and methods section. Dose-dependent formation of ROS was additionally presented by cell count and fluorescence intensity of CMH2DCFDA. M1 line is positioned to designate MFI value of the non-treated sample (white histogram) compared to signals obtained upon cell treatment with indicated concentrations of 2b.
Formation of ROS by 2b in H460 cells. Logarithmically growing H460 cells were stained for 1 hour with 10 mM CM-H2DCFDA and then either treated with 110 μM 2b during indicated time points (A) or treated with indicated concentrations of 2b for 180 min (3 hours) (B). Afterward ROS formation was determined by flow cytometry as described in Materials and methods section. Dose-dependent formation of ROS was additionally presented by cell count and fluorescence intensity of CMH2DCFDA. M1 line is positioned to designate MFI value of the non-treated sample (white histogram) compared to signals obtained upon cell treatment with indicated concentrations of 2b.

Figure 5

The effects of ROS scavenger on survival of H460 cells treated with 2b determined by MTT assay. H460 cells were seeded and next day pretreated for 2 hours with 5 mM of NAC or 1 mM tempol. Afterwards different concentrations of 2b were added. The cell survival was determined 72 hours later by MTT assay as described in Materials and methods section. Each point represents the mean ±SD of at least three independent experiments. All data are expressed as the average percentage of survival values relative to an untreated control ± SD or samples treated with antioxidants alone. The significance in differences is indicated (*, P < 0.05; **, P < 0.01).
The effects of ROS scavenger on survival of H460 cells treated with 2b determined by MTT assay. H460 cells were seeded and next day pretreated for 2 hours with 5 mM of NAC or 1 mM tempol. Afterwards different concentrations of 2b were added. The cell survival was determined 72 hours later by MTT assay as described in Materials and methods section. Each point represents the mean ±SD of at least three independent experiments. All data are expressed as the average percentage of survival values relative to an untreated control ± SD or samples treated with antioxidants alone. The significance in differences is indicated (*, P < 0.05; **, P < 0.01).

Figure 6

The effect of ROS scavenger on survival of HEp-2 (A) and H460 (B) cells treated with 2b determined by colony-forming assay. HEp-2 and H460 cells were seeded and next day pretreated for 2 hours with 5 mM of NAC or 1 mM (HEp-2 cells) or 0.125 mM (H460 cells) tempol. Afterwards different concentrations of 2b were added. Ten days later the colonies were counted. Non-treated cells and cells treated with antioxidants alone were used as controls. Each point represents the mean ±SD of at least three independent experiments. All data are expressed as the average percentage of survival values relative to an untreated control ± SD or samples treated with antioxidants alone. The significance in differences is indicated (*, P < 0.05; **, P < 0.01).
The effect of ROS scavenger on survival of HEp-2 (A) and H460 (B) cells treated with 2b determined by colony-forming assay. HEp-2 and H460 cells were seeded and next day pretreated for 2 hours with 5 mM of NAC or 1 mM (HEp-2 cells) or 0.125 mM (H460 cells) tempol. Afterwards different concentrations of 2b were added. Ten days later the colonies were counted. Non-treated cells and cells treated with antioxidants alone were used as controls. Each point represents the mean ±SD of at least three independent experiments. All data are expressed as the average percentage of survival values relative to an untreated control ± SD or samples treated with antioxidants alone. The significance in differences is indicated (*, P < 0.05; **, P < 0.01).

IC50 values of triazolium salts and some parent triazoles against cervical carcinoma HeLa cells

CmpdIC50 (μM)

IC50 is the concentration of the triazoles and triazolium salts inducing 50% cell growth inhibition after 72 h incubation. The results are shown as mean values of at least three experiments (± SD).

1a> 100
1b> 100
1f> 100
2a91.6 ± 3.9
2b57.0 ± 12.9
2c> 100
2d> 100
2e55.4 + 9.4
2f–

Triazolium salt precipitated promptly after the addition to the growth medium and thus the cytotoxicity could not be measured accurately.

2g–

The range of concentrations 10 – 1000 μM reduced survival from about 60 to 40%, and therefore the exact IC50 was difficult to determine.

2h88.9 + 7.5
2i54.4 + 14.7
2j54.9 + 3.5
2k> 100
2l–

The range of concentrations 10 – 1000 μM reduced survival from about 60 to 40%, and therefore the exact IC50 was difficult to determine.

Effect of compound 2b on the cell cycle of H460 cells

Conc. (μM)24 h48 h72 h
G1SG2/MsubG1G1SG2/MsubG1G1SG2/MsubG1
0543115259261527313142
29.7612415261261356517186
11078913378913157951635

Cytotoxic activity of 2b against different cell lines

Cell lineIC50 (μM)

IC50 is the concentration of 2b that induces 50% cell growth inhibition after 72 h incubation.

T.I.

T.I. refers to therapeutic index, calculated from the ratio of cytotoxicity (IC50) on normal fibroblasts and cytotoxicity (IC50) on tumour cells.

HeLa57.0 ± 12.94.01
HEp-287.0 ± 28.52.63
7T111.7 ± 7.62.05
H46029.7 + 4.57.69
HCT-11651.9 ± 7.24.40
Fibroblasts228.5 ± 5.6–
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