The focus of this research is on the study of a series of copper (II) benzoylpyridine thiosemicarbazone complexes. Of the six benzoylpyridine thiosemicarbazone ligands used in this study, two are reported for the first time; 2-benzoylpyridine tert-butyl thiosemicarbazone (BZP-tBTSC), and 2-benzoylpyridine benzyl thiosemicarbazone (BZP-BzTSC). Once characterized by NMR, melting point, and MS, these mono-anionic tridentate ligands were then reacted with Cu 2+ to form the new square planar metal complexes [Cu(BZP-tBTSC)Cl] and [Cu(BZP-BzTSC)Cl]. All of the copper complexes display marked inhibition of human topoisomerase IIα. The [Cu(BZP-tBTSC)Cl] complex shows marked activity against human breast cancer cell lines.
Thiosemicarbazones are an interesting class of chemical compounds that have active biological and medicinal qualities and have been used as drugs against leukemia [
A specific subset of thiosemicarbazone ligands that have been referred to as α-(N)-heterocyclic thiosemicarbazones have potent antiproliferative properties against a wide variety of microbes and cancer cells. Specifically, α-(N)-heterocyclic thiosemicarbazones have been shown to have activity against ribonucleotide reductase (RNR) [
In a fortuitous coincidence, the copper complexes of α-(N)-heterocyclic thiosemicarbazones appear to have high antineoplastic activity through an entirely different mechanism [
An important subset of the α-(N)-heterocyclic thiosemicarbazones is the benzoylpyridine thiosemicarbazones (BZP-TSC’s), based on 2-benzoylpyridine. One problem with the use of many of these α-(N)-heterocyclic thiosemicarbazones as anti-cancer agents is methemoglobin formation, but one group has found that BZP-TSC ligands limit methemoglobin formation [
All reagents and solvents used to synthesize the ligands and the copper complexes were purchased from Sigma- Aldrich or Alfa-Aesar. Recombinant human Topoisomerase IIα was overexpressed and purified from yeast Saccharomyces cerevisiae as described [
plasmid was amplified and purified following the protocol of Qiagen™ Plasmid Mega Kit.
The melting points were taken with a Stanford Research Systems Digimelt MPA160, and TLCs were taken with Whatman 250 μm layer PE SIL G/UV polyester-backed plates. All mass spectrometry data was taken with a Varian 300/310/320-MS LC/MS Quadrupole Mass Spectrometer in negative mode using APCI. The corona current was set to −5.00 μA, while the shield potential was set to −600.00 volts. The housing, drying gas, and vaporizer gas temperatures were set to 50˚C, 150˚C, and 350˚C respectively. The drying, nebulizing, and vaporizer gas pressures were each set to 12.0 psi, 55.0 psi, and 17.0 psi. For the mass spectrometry data, each sample was dissolved in minimal amounts of dimethylsulfoxide and then diluted to 10 ppm in methanol. All Nuclear Magnetic Resonance (NMR) data were collected using an Oxford/Varian 300 MHz NMR. Each 1HNMR was taken over a range of −1 to 16 ppm, with 32 - 64 total scans. The 13CNMR were taken over a range of −20 to 240 ppm and between 1000 - 2000 scans, with an acquisition time of 1.6s and a relaxation delay of 2.0 s. The original data was processed in Vnmrj software, then transferred over to Mestre Nova. UV-Vis data was gathered using a Cary Varian 3E UV-Vis Spectrophotometer. Each sample was diluted to 10 ppm and scanned over a range of 200 - 800 nm.
The ligands BZP-MTSC [
Both ligands were synthesized by the following procedure: In a 50 ml round-bottomed 14/20 necked flask with a magnetic stir bar was added 5.50 mmol of 2-benzoylpyridine and 5.50 mmol of the appropriate thiosemicarbazide (either 4-tertbutyl-3-thiosemicarbazide or 4-benzyl-3-thiosemicarbazide). The reagents were slurried in 25 mL of 2-propanol, and one drop of concentrated sulfuric acid was added to catalyze the reaction. A reflux condensor was attached to the flask, and the reaction mixture was heated to 70˚C and stirred for 24 hours. The off- white precipitate formed was filtered, washed with ethyl ether, and dried under vacuum.
BZP-tBTSC
Yield: 1.53 g (89.8%) MP: 141.5˚C
1H NMR (300 MHz, DMSO-d6, δ): 12.94 (s, 3H, hydrazinic N-H), 8.87 (ddd, J = 4.9, 0.8, 0.9 Hz, 3H, Ar-H), 8.48 (ddd J = 4.8, 1.8, 0.9 Hz, 1H, Ar-H), 8.18 (dt, J = 8.1, 1.1 Hz, 1H, Ar-H), 8.06 - 7.98 (m, 5H, Ar-H), 7.91 (ddd, J = 8.1, 7.5, 1.8 Hz, 1H, Ar-H), 7.84 (s, 3H, thioamide N-H), 7.68 - 7.39 (m, 21H, Ar-H), 7.46 - 7.27 (m, 5H, Ar-H), 1.53 (s, 26H, tert-butyl C-H). 13C NMR (75 MHz, DMSO-d6, δ): 175.99 (N-C-N), 151.92 (Ar), 149.29 (Ar), 142.62 (Ar), 138.73 (tert butyl C-C-H), 137.52 (Ar), 129.76 (Ar), 129.25 (Ar), 129.07 (Ar), 126.69, 125.42 (Ar), 53.44 (tert-butyl C-H), 28.78. Anal. Found: C, 65.13; H, 6.56. Calcd. for C17H20N4S: C, 65.35; H, 6.45. Theoretical MS m/z (relative intensity): 311.14 (100%). Actual MS m/z (relative intensity): 310.9 (100%).
BZP-BzTSC
Yield: 1.57 g (82.4%) MP: 137.2˚C
1H NMR (300 MHz, DMSO-d6, δ): 12.92 (d, J = 2.2 Hz, 1H, hydrazinic N-H), 9.57 (t, J = 6.3 Hz, 0H, Thioamide N-H), 9.30 (t, J = 6.3 Hz, 2H, thioamide N-H), 8.89 - 8.81 (m, 2H, Ar-H), 8.78 (s, 0H, Ar-H), 8.52 (dtd, J = 8.1, 1.1 Hz, 1H, Ar-H), 8.48 - 8.39 (m, 0H, Ar-H), 7.69 - 7.52 (m, 6H, Ar-H), 7.58 - 7.36 (m, 6H, Ar-H), 7.39 - 7.15 (m, 13H, Ar-H), 4.85 (dd, J = 6.2, 2.4 Hz, 4H, benzyl N-C-H), 3.42 (d, J = 2.5 Hz, 15H, benzyl N-C-H). 13C NMR (75 MHz, DMSO-d6, δ): 178.36 (N-C-N), 151.85 (Ar-C), 149.61 (Ar-C), 149.30 (Ar-C), 149.09 (Ar-C), 143.79 (Ar-C), 139.46 (Ar-C), 139.24 (Ar-C), 138.65 (Ar-C), 137.28 (Ar-C), 136.92 (Ar-C), 129.69 (Ar-C), 129.42 (thioamide N-C-Ar), 129.10 (Ar-C), 128.82 (Ar-C), 128.62 (Ar-C), 127.73 (Ar-C), 127.66 (Ar-C), 127.33 (Ar-C), 127.23(Ar-C), 126.57 (Ar-C), 125.38 (Ar-C), 124.47 (Ar-C), 122.17 (Ar-C), 47.73 (Ar-C). Anal. Found: C, 69.13; H, 5.56. Calcd. for C20H18N4S:C, 69.34; H, 5.24. Theoretical MS m/z (relative intensity): 345.1 (100%). Actual MS m/z (relative intensity): 344.9 (100%).
Both Cu(II) complexes were synthesized by the following procedure: In a 50 ml round-bottomed 14/20 necked flask with a magnetic stir bar was added 2.50 mmol of cupric chloride dihydrate which was dissolved in 7.5 mL of methanol. Next a solution of 2.50 mmol of the appropriate thiosemicarbazone ligand (either the BZP-tBTSC or the BZP-BzTSC) in 20 mL of methanol was added. Reaction to form the green product was observed to occur immediately. A reflux condenser was attached to the flask, and the reaction mixture was heated to 75˚C and stirred for 4 hours. The green microcrystalline precipitate formed was filtered, washed with ethyl ether, and dried under vacuum.
[Cu(BZP-tBTSC)Cl]
Yield: 0.579 g (88.5%), TLC Silica/ethyl acetate (Rf): 0.91
Theoretical MS m/z (relative intensity): 413.02 (3.6%), 412.02 (18.1%), 411.02 (8.5%), 410.02 (81.3%), 409.03 (18.6%), 408.2 (100.0%). Actual MS m/z (relative intensity): 413.0 (19.4%), 412.0 (17.6%), 411.0 (82.3%), 410.0 (30.9%), 408.9 (100%), 408.1 (6.9%). Anal. Found: C, 49.55; H, 4.79. Calcd. for C17H19ClCuN4 S:C, 49.75; H, 4.67. UV (thiosemicarbazone complexes) λmax, nm (ε): 421 (30.85), 683 (0.6115).
[Cu(BZP-BzTSC)Cl]
Yield: 0.320 g (82.5%). TLC Silica/ethyl acetate (Rf): 0.90
Theoretical MS m/z (relative intensity): 447.01 (3.6%), 446.00 (17.8%), 445.01 (18.1%), 444.01 (79.3%), 443.01 (24.1%), 442.01 (100.0%). Actual MS m/z (relative intensity): 447.0 (19.7%), 446.0 (22.2%), 445.0 (77.3%), 444.0 (41.6%), 443.0 (100%), 441.9 (12.6%). Anal. Found: C, 53.53; H, 3.79. Calcd. for C20H17 ClCuN4S: C, 54.05; H, 3.86. UV (thiosemicarbazone complexes) λmax, nm (ε): 423 (35.11), 684 (0.6340).
Plasmid DNA relaxation assays were performed as previously described [
Cell lines used were MDA-MB-231 (cancerous, epithelial-type human breast adenocarcinoma cells), and MCF7 (cancerous, epithelial-type human breast adenocarcinoma cells). Adherent cells were cultured in 25 cm2 or 75 cm2 tissue culture flasks within a humidified incubator at 37˚C and 5% CO2. DMEM containing L-glutamine (Lonza) supplemented with penicillin/streptomycin and 10% FBS was used as the complete culture medium. Confluent cells were trypsinized, cell clumps dissociated by gentle pipetting, and split using a 1:10 ratio into fresh flasks containing pre-warmed complete media. Cell counts were performed by staining 100 µL of cells with 100 µL of trypan blue (Hyclone) and then counted on a hemocytometer. With this number determined, cells were diluted in complete media to a concentration 25,000 cells/mL, and 200 µL/well of a cell solutions was added to a 96-well plate using a multichannel pipette. Plated cells were placed in the humidified incubator at 37°C and 5% CO2 to incubate for 24 hr prior to drug addiction.
After cells were cultured for 24 hr, after which the existing culture media was replaced with complete media containing dilutions of compounds [Cu(APY-ETSC)Cl] and [Cu(BZP-tBTSC)Cl] ranging from 100 µM and 0.001 µM. The dilutions were generated using serial dilutions of prepared DMSO stock solutions of 10 mM [Cu(APY-ETSC)Cl] and 10 mM [Cu(BZP-tBTSC)Cl] in ACS-grade DMSO (Fischer) which were stored at −20°C until use. Wells were also prepared with 200 µL of complete media only or 200 µL of complete media with 1:100 dilution of DMSO as positive controls. Both positive controls were found to be statistically equivalent showing no added toxicity of DMSO at the highest applied concentration of DMSO. The 96-well plate was then incubated (humidified, 37°C, 5% CO2) for 24 hr after application of the compounds. Alamar Blue (Thermo Scientific) was then added to each well using complete media as a carrier to attain 8% alamar blue per well and applied using a multichannel pipette. The plate was then placed back into the incubator and allowed to incubate for approximately 5 hr, after which fluorescence measurements were made at 560 nm excitation and 590 nm emission using the Tecan infinite M200 Pro reader.
Initial analysis of fluorescence data were performed with Microsoft Excel 2013 normalized by the fluorescence signal from positive controls. Statistical analysis, data fitting, and plot generation was performed with GraphPad Prism 6; normalized data were fit using log (inhibitor) vs. response with variable slope (four parameter) model. This model calculated EC50 and Hill slope values for each curve.
The synthesis of the BZP-TSC ligands has been documented for over two decades, and the ligands act primarily as monoanionic tridentate chelating ligands with a variety of metal ions. Copper(II) complexes of the BZP-TSC ligands synthesized from copper (II) chloride are paramagnetic square-planar complexes in solution as has been determined previously, and we find evidence that the [Cu(BZP-tBTSC)Cl] and the [Cu(BZP-BzTSC)Cl] have the same solution structure. Because of the history of biological properties of the copper complexes, we decided to investigate the possibility that they are involved in inhibition of Topoisomerase IIα, just as the structurally similar acetylpyridine complexes of formula [Cu(APY-TSC)Cl] have recently been discovered to be inhibitors of that important enzyme.
The ligands and Cu(II) complexes of BZP-TSCs with different substituents were examined in a topoIIα-med- iated plasmid relaxation assay. Without the presence of any compounds (ND), topoIIα relaxed the supercoiled (SC) plasmid pBR322 to the relaxed (R) form as shown in lane 2 of
We also examined the dose-dependence of [Cu(BZP-tBTSC)Cl] inhibition on topoIIα as shown in
4 show inhibition of the topoIIα enzyme in a dose dependent manner from 10 μM solution of [Cu(BZP-tBTSC) Cl] up to a 50 μM solution. Our experiment shows that as little as 10 μM of [Cu(BZP-tBTSC)Cl] can partially inhibit topoIIα activity and that even at this concentration it exhibits more inhibition than does etoposide at a 50 μM solution concentration as seen in Lane 5. It should be noted that etoposide is a widely-used anticancer agent.
The in vitro viability assay of compounds [Cu(APY-ETSC)Cl)] and [Cu(BZP-tBTSC)Cl] was determined using two cancerous, epithelial-type human breast adenocarcinoma cell lines, MDA-MB-231 and MCF7. Compound concentrations ranging from 0.001 - 100 µM were used and cell viability were assayed after 24 hours of incubation with the compounds in complete DMEM media. The fluorometric, metabolic compound Alamar Blue was used to determine cellular viability. As shown in
In all cases, the viability of the cells decreased with increasing concentration of the compounds. Compound [Cu(APY-ETSC)Cl] generally had a lower effective concentration compared to [Cu(BZP-tBTSC)Cl], but the differences were not determined to be statistically significant in these assays. The MDA-MB-231 cells had an abrupt change in viability after a certain threshold concentration of applied compounds, perhaps due to the physiology of these cells. GraphPad Prism was used to calculate the (EC50) for the various cell types.
As shown in
Thiosemicarbazones that are of the a-(N)-heterocyclic thiosemicarbazone class, such as the BZP-TSC’s in this study, have proven to be a special class of ligands with many biological properties. The ligands themselves are often potent ribonucleotide reductase inhibitors (such as Triapine), but have very little inhibitory effect against topoIIa. However, we show in this study that the copper(II) complexes of those ligands with the formula [Cu(BZP-TSC)Cl] are potent topoisomerase IIa inhibitors, just as has been shown to be the case for the analogous copper(II) complexes of the formylpyridine thiosemicarbazones, [Cu(FP-TSC)Cl], acetylpyridine thiosemicarbazones, [Cu(APY-TSC)Cl], and acetylpyrazine thiosemicarbazones, [Cu(APZ-TSC)Cl]. The alkyl and aryl
substituents on the end of the thiosemicarbazone ligand, such as methyl, ethyl, tert-butyl, phenyl, etc., seem to have no real effect on the efficacy of the copper(II) complexes to inhibit the action of human topoIIα as we have determined in this study.
If the substituents on the thiosemicarbazone seem to have no real effect, and the imine portion of the copper complex can be either a formyl, acetyl, or even a benzoyl group, as we have shown here, then the question of the mechanism of action of the copper complexes to inhibit topoIIα now seems to focus on the square-planar geometry of these complexes, which is rare for copper (II), and the nature of the Cu-Cl bond. Studies in the future may address the question of whether or not the [Cu(BZP-TSC)Cl] complexes react with the topoIIα enzyme to become five or six coordinate, and whether or not the Cu-Cl bond is involved in this process.
In summary, our results demonstrate that Cu(II) BZP-TSC complexes are better topoIIα inhibitors compared with the BZP-TSC ligands and that the [Cu(BZP-tBTSC)Cl] complex inhibits topoIIα in a dose-dependent way. Additionally, the [Cu(BZP-tBTSC)Cl] complex also appears to be more effective at killing MDA-MB-231 and MCF7 breast cancer cell lines than does [Cu(APY-ETSC)Cl].
J.D.C. thanks the TTU Chemistry Department for a Student Research Grant, and M.T.S. thanks the TTU URECA! Program for a research mini-grant.
Jennifer D. Conner,Wathsala Medawala,Madison T. Stephens,William H. Morris,Joseph E. Deweese,Patrick L. Kent,Jeffery J. Rice,Xiaohua Jiang,Edward C. Lisic,1 1, (2016) Cu(II) Benzoylpyridine Thiosemicarbazone Complexes: Inhibition of Human Topoisomerase IIα and Activity against Breast Cancer Cells. Open Journal of Inorganic Chemistry,06,146-154. doi: 10.4236/ojic.2016.62010