Life-threatening diseases such as cancer and pathogenic infections impose a huge socio-economic burden on humankind. Despite significant advances in cancer treatments annual cancer-related deaths within the European Union (EU) remain extremely high. Latest statistics indicate that cancer caused over 1.25 million deaths (accounting for 26% of all deaths) within EU28 countries in 2013 [1
]. Amongst the various cancer types breast cancer caused the third most fatalities within EU28 countries (around 93,500 deaths). The economic impact of breast cancer across the EU, in terms of healthcare resources, productivity losses because of early death, lost working days, and informal care costs, amounts to around €15.0 billion [2
]. Given these harrowing statistics, continuing to conduct basic research on new breast cancer medicines and therapies that could ultimately reduce mortality rates and lessen financial burden is of significant importance. Breast cancer relapse and metastasis, the leading cause of breast cancer associated deaths, is strongly linked to the existence of breast cancer stem cells (CSCs) [3
]. Breast CSCs are a sub-set of breast cancer cells that have the intrinsic ability to differentiate, self-renew, and form secondary tumours [5
]. Breast CSCs are known to evade current breast cancer therapy (including surgery, chemotherapy, and radiation), promote re-population of primary cancer sites, and potentially lead to fatal incidences of cancer relapse and metastasis [7
]. Therefore it is vital that anti-breast chemotherapeutics possess the ability to remove breast CSCs at therapeutically significant doses.
We and others have developed several breast CSC potent and selective metal complexes over the last six to seven years [9
]. One of the most promising classes of anti-breast CSC agents reported thus far, are copper(II) complexes bearing polypyridyl and/or Schiff base ligands [10
]. It should be noted that copper(II) complexes have been widely studied as anticancer agents (with limited studies focused on their anti-CSC potential), but none have been approved for clinic use [14
]. The most advanced copper(II) complexes, called Casiopeinas, are currently in Phase I clinical trials [16
]. The very latest studies suggest that Casiopeinas may be limited by dose-dependent cardiotoxicity [20
], probably due to speciation. The breast CSC active copper(II) complexes developed by our group induce breast CSC death by generating intracellular reactive oxygen species (ROS), often activating the p38 and JNK stress pathways and caspase-dependent apoptosis [10
]. The success of these copper(II) complexes against breast CSCs is thought to arise partly from the vulnerability of breast CSCs to subtle changes in their redox state [21
]. Very recently we reported a copper(II) complex 1
, containing a O
-Schiff base ligand L1
, and 4,7-diphenyl-1,10-phenanthroline (see Supplementary Materials Figure S1
for chemical structures of L1
), capable of killing breast CSCs via cytotoxic and immunogenic mechanisms [23
]. This was the first metal complex to induce immunogenic cell death (ICD) of breast CSCs and promote their engulfment by immune cells. This was an important step in our ongoing efforts to develop clinically viable anti-breast cancer drug candidates, as the removal of CSCs by immunological activation could serve as an effective method to eliminate residual CSCs after conventional CSC inactive treatments. In this study, we explore the breast CSC activity of structurally related copper(II) complexes (2
) where the Schiff base ligand L1
has been modified. Specifically a naphthalene moiety was incorporated into the Schiff base ligand scaffold to yield (E)-1-(((2-(methylthio)ethyl)imino)methyl)naphthalen-2-ol (L2
) which was used to prepare the corresponding copper(II) complex with 1,10-phenanthroline, [Cu(L2
) (Scheme 1
). Furthermore, the methyl group on the sulphur atom in L2
was replaced with an ethyl group to yield (E)-1-(((2-(ethylthio)ethyl)imino)methyl)naphthalen-2-ol (L3
), which was subsequently used to prepare the corresponding copper(II) complex with 1,10-phenanthroline, [Cu(L3
) (Scheme 1
). These structural modifications were expected to modulate breast CSC uptake and intracellular ROS generation. Herein we report the synthesis, characterisation, and anti-breast CSC properties of the copper(II) complexes, [Cu(L2
) and [Cu(L3
3. Materials and Methods
3.1. General Procedures
All synthetic procedures were performed under normal atmospheric conditions. Fourier transform infrared (FTIR) spectra were recorded with an IRAffinity-1S Shimadzu spectrophotometer. Electron spray ionisation mass spectra were recorded on a Micromass Quattro spectrometer. UV-Vis absorption spectra were recorded on a Cary 3500 UV-Vis spectrophotometer. 1H and 13C NMR spectra were recorded on a BrukerAvance 400 MHz Ultrashield NMR spectrometer. 1H NMR spectra were referenced internally to residual solvent peaks, and chemical shifts are expressed relative to tetramethylsilane, SiMe4 (δ = 0 ppm). Elemental analysis of the compounds prepared was performed commercially by London Metropolitan University or the University of Cambridge. 2-hydroxy-1-naphthaldehyde, 2-(methylthio)ethylamine, 2-(ethylthio)ethylamine, 1,10-phenanthroline, Cu(NO3)2∙3H2O, CuCl2·2H2O, and NaPF6 were purchased from Sigma Aldrich or Alfa Aesar and used as received.
3.2. Synthesis of (E)-1-(((2-(Methylthio)ethyl)imino)methyl)naphthalen-2-ol, L2
A mixture of 2-hydroxy-1-naphthaldehyde (344 mg, 2.0 mmol) and 2-(methylthio)ethylamine (191 mg, 2.1 mmol) were refluxed in ethanol (20 mL) for 16 h. The reaction mixture was then evaporated under vacuum to afford L2 as an orange solid (472 mg, 96%); 1H NMR (400 MHz, DMSO-d6): δ 13.93 (s, 1H, OH), 9.11 (d, 1H, N=CH), 8.06 (d, 1H, Ar–H), 7.73 (d, 1H, Ar–H), 7.64 (d, 1H, Ar–H), 7.43 (ddd, 1H, Ar–H), 7.19 (ddd, 1H, Ar–H), 6.71 (d, 1H, Ar–H), 3.84 (dd, 2H, CH2), 2.83 (t, 2H, CH2), 2.13 (s, 3H, CH3); 13C NMR (162 MHz, DMSO-d6): δ 177.85 (N=CH), 159.74 (Ar), 137.60 (Ar), 134.85 (Ar), 129.34 (Ar), 128.34 (Ar), 126.05 (Ar), 125.66 (Ar), 122.62 (Ar), 118.95 (Ar), 106.13 (Ar), 50.33 (CH2), 34.47 (CH2), 15.05 (CH3); IR (solid, ATR, cm−1): 3021, 2971, 2917, 1616, 1541, 1529, 1491, 1439, 1399, 1354, 1314, 1282, 1254, 1193, 1177, 1137, 1040, 996, 955, 924, 879, 855, 827, 743, 723, 686, 654, 541, 513, 497, 481, 437, 413; HR ESI-MS Calcd. for C14H16NOS [M+H]+ 246.0953 a.m.u. Found [M+H]+: 246.0954 a.m.u.; Anal. Calcd. for C14H15NOS·0.25H2O (%): C, 67.30; H, 6.25; N, 5.61. Found: C, 67.62; H, 6.06; N, 5.99.
3.3. Synthesis of (E)-1-(((2-(Ethylthio)ethyl)imino)methyl)naphthalen-2-ol, L3
A mixture of 2-hydroxy-1-naphthaldehyde (172 mg, 1.0 mmol) and 2-(ethylthio)ethylamine (116 mg, 1.1 mmol) were refluxed in ethanol (20 mL) for 16 h. The reaction mixture was evaporated under vacuum to afford L3 as an orange solid (259 mg, 100%); 1H NMR (400 MHz, DMSO-d6): δ 13.94 (s, 1H, OH), 9.10 (d, 1H, N=CH), 8.06 (d, 1H, Ar–H), 7.73 (d, 1H, Ar–H), 7.63 (dd, 1H, Ar–H), 7.43 (ddd, 1H, Ar–H), 7.19 (ddd, 1H, Ar–H), 6.72 (d, 1H, Ar–H), 3.82 (dd, 2H, CH2), 2.86 (t, 2H, CH2), 2.60 (q, 2H, CH2), 1.20 (t, 3H, CH3); 13C NMR (162 MHz, DMSO-d6): δ 177.84 (N=CH), 159.70 (Ar), 137.60 (Ar), 134.85 (Ar), 129.34 (Ar), 128.34 (Ar), 126.04 (Ar), 125.66 (Ar), 122.61 (Ar), 118.95 (Ar), 106.12 (Ar), 50.97 (CH2), 31.96 (CH2), 25.33 (CH2), 15.28 (CH3); IR (solid, ATR, cm−1): 3049, 3025, 2969, 2920, 1610, 1540, 1493, 1440, 1402, 1344, 1256, 1206, 1183, 1137, 1070, 1034, 995, 961, 867, 832, 740, 638, 542, 515, 504, 480, 434, 414; HR ESI-MS Calcd. for C15H18NOS [M+H]+ 260.1109 a.m.u. Found [M+H]+: 260.1112 a.m.u.; Anal. Calcd. for C15H17NOS·0.2H2O (%): C, 68.51; H, 6.67; N, 5.33. Found: C, 68.62; H, 6.38; N, 5.55.
3.4. Synthesis of [Cu(L2)1,10-Phenanthroline][PF6], 2
1,10-phenanthroline (74 mg, 0.41 mmol) and Cu(NO3)2·3H2O (99 mg, 0.41 mmol) dissolved in methanol (10 mL) were stirred at room temperature for 0.5 h. The colour of the solution changed from blue to light green. L2 (100 mg, 0.41 mmol) in methanol (10 mL) was added dropwise. The dark green mixture was stirred at room temperature for 72 h. The mixture was then filtered to remove the precipitate. The filtrate was reduced to ~10 mL. An excess of NaPF6 (250 mg, 1.5 mmol) in water (50 mL) was added and the mixture stirred for 0.5 h. The resultant precipitate was collected and washed thoroughly with water and diethyl ether to give 2 as a dark green solid (116 mg, 45%); IR (solid, ATR, cm−1): 1618, 1602, 1585, 1540, 1518, 1430, 1413, 1392, 1366, 1342, 1254, 1223, 1194, 1144, 1106, 1029, 979, 828, 752, 722, 647, 557, 523, 476, 416; HR ESI-MS Calcd. for C26H22CuN3OS [M-PF6]+ 487.0780 a.m.u. Found [M-PF6]+ 487.0775 a.m.u.; Anal. Calcd. for C26H22CuN3OSPF6 (%): C, 49.33; H, 3.50; N, 6.64. Found: C, 49.14; H, 3.34; N, 6.46.
3.5. Synthesis of [Cu(L3)1,10-Phenanthroline][PF6], 3
1,10-phenanthroline (110 mg, 0.61 mmol) and Cu(NO3)2·3H2O (147 mg, 0.61 mmol) dissolved in methanol (10 mL) were stirred at room temperature for 0.5 h. The colour of the solution changed from blue to light green. L3 (160 mg, 0.62 mmol) in methanol (10 mL) was added dropwise. The dark green mixture was stirred at room temperature for 72 h. The mixture was then filtered to remove the precipitate. The filtrate was reduced to ~10 mL. An excess of NaPF6 (400 mg, 2.4 mmol) in water (~30 mL) was added and the mixture stirred for 0.5 h. The resultant precipitate was collected and washed thoroughly with water and diethyl ether to give 3 as a green solid (272 mg, 69%); IR (solid, ATR, cm−1): 1616, 1604, 1583, 1539, 1509, 1460, 1437, 1428, 1416, 1393, 1363, 1345, 1254, 1224, 1187, 1145, 1104, 1094, 1036, 1006, 979, 828, 751, 725, 645, 555, 521, 490, 476, 450, 421, 387; HR ESI-MS Calcd. for C27H24CuN3OS [M-PF6]+ 501.0936 a.m.u. Found [M-PF6]+ 501.0936 a.m.u.; Anal. Calcd. for C27H24CuN3OSPF6 (%): C, 50.12; H, 3.74; N, 6.49. Found: C, 49.79; H, 3.45; N, 6.18.
3.6. Synthesis of [Cu(L2)Cl], 4
L2 (96.4 mg, 0.39 mmol) in methanol (20 mL) was added to CuCl2·2H2O (66.5 mg, 0.39 mmol) in methanol (5 mL). The dark green mixture was stirred overnight. The resultant precipitate was collected and washed with cold methanol and diethyl ether to yield 4 as a dark green solid (20 mg). The filtrate was reduced and left overnight in the freezer (−20 °C) to yield a second crop of 4 as a dark green solid (16 mg) (total 36 mg, 27%); IR (solid, ATR, cm−1): 1615, 1604, 1589, 1537, 1503, 1452, 1431, 1409, 1392, 1359, 1339, 1306, 1250, 1210, 1183, 1164, 1140, 1089, 1029, 976, 957, 940, 859, 825, 778, 765, 746, 646, 583, 553, 516, 474, 448, 417, 387; HR ESI-MS Calcd. for C14H14CuNOS [M-Cl]+ 307.0092 a.m.u. Found [M-Cl]+ 307.0096 a.m.u.; Anal. Calcd. for C14H14CuNOSCl (%): C, 48.98; H, 4.11; N, 4.08. Found: C, 48.77; H, 3.98; N, 4.02.
3.7. Synthesis of [Cu(L3)Cl], 5
L3 (119 mg, 0.46 mmol) in methanol (5 mL) was added to CuCl2·2H2O (78 mg, 0.46 mmol) in methanol (5 mL). The dark green mixture was refluxed for 2 h. The mixture was then put in the freezer (−20 °C) overnight. The resultant precipitate was collected and washed with cold methanol and diethyl ether to yield 5 as a dark green solid (52 mg, 32%); IR (solid, ATR, cm−1): 1614, 1604, 1537, 1507, 1454, 1433, 1412, 1391, 1361, 1340, 1308, 1253, 1206, 1183, 1172, 1162, 1141, 1093, 1077, 1033, 1000, 977, 949, 869, 830, 752, 644, 584, 558, 549, 513, 473, 449, 421, 384; HR ESI-MS Calcd. for C15H16CuNOS [M-Cl]+ 321.0249 a.m.u. Found [M-Cl]+ 321.0257 a.m.u.; Anal. Calcd. for C15H16CuNOSCl (%): C, 50.42; H, 4.51; N, 3.92. Found: C, 50.75; H, 4.25; N, 3.83.
3.8. X-ray Single Crystal Diffraction Analysis
Single crystals of complexes 2
were obtained by slow evaporation of an acetone solution of 2
and by vapour diffusion of diethyl ether into an acetonitrile solution of 3
. Crystals suitable for X-ray diffraction analysis were selected and mounted on a Bruker Apex 2000 CCD area detector diffractometer using standard procedures. Data was collected using graphite-monochromated Mo-Kα radiation (λ = 0.71073) at 150(2) K. Absorption corrections were applied using a multiscan method (SADABS) [29
]. The structures were solved using SHELXS [30
]; the datasets were refined by full-matrix least-squares on all unique F2
values, with anisotropic displacement parameters for all non-hydrogen atoms, and with constrained riding hydrogen geometries [31
(H) was set at 1.2 (1.5 for methyl groups) times Ueq
of the parent atom. The largest features in final difference syntheses were close to heavy atoms and were of no chemical significance. SHELX was employed through OLEX2 for structure solution and refinement [29
]. ORTEP-3 and POV-Ray were employed for molecular graphics [33
]. The structures have been deposited with the Cambridge Crystallographic Data Centre (CCDC 2046679 and 2046680). This information can be obtained free of charge from www.ccdc.cam.ac.uk/data_request/cif
3.9. Measurement of Water-Octanol Partition Coefficient (LogP)
The LogP values for 2–5 were determined using the shake-flask method and UV-Vis spectroscopy. The 1-octanol used in this experiment was pre-saturated with water. An aqueous solution of 2–5 (500 μL, 100 μM) was incubated with 1-octanol (500 μL) in a 1.5 mL tube. The tube was shaken at room temperature for 24 h. The two phases were separated by centrifugation and the 2–5 content in each phase was determined by UV-Vis spectroscopy.
3.10. Cell Lines and Cell Culture Conditions
R. A. Weinberg (Whitehead Institute, MIT) (Cambridge, USA) generously gave the human mammary epithelial cell lines, HMLER and HMLER-shEcad, used in this study. The cells were grown in Mammary Epithelial Cell Growth Medium (MEGM) with supplements and growth factors (BPE, hydrocortisone, hEGF, insulin, and gentamicin/amphotericin-B). Standard cell culture conditions were used to grow the cells (310 K, 5% CO2).
3.11. Cytotoxicity MTT Assay
The cytotoxicity of 2–5, L2, and L3 was determined using the colorimetric MTT assay. Five thousand HMLER and HMLER-shEcad cells were added to each well of a 96-well plate. Upon allowing the cells to attach to the bottom of each well of a 96-well plate overnight, different concentrations of the test compounds (0.2–100 µM) were added, and incubated for 72 h (total volume 200 µL). 10 mM stock solutions of the compounds in DMSO were prepared and appropriately diluted using Mammary Epithelial Cell Growth Medium (MEGM). The untreated control wells contained 0.5% DMSO, which was the final concentration of DMSO in each treated well. Upon 72 h incubation, a 4 mg mL−1 solution of MTT dissolved in PBS was added to each well (20 µL). The 96-well plate was then incubated for an additional 4 h. After aspiration of the MEGM/MTT solution in each well, 200 μL of DMSO was added to each well to dissolve any purple formazan crystals. The absorbance of the resultant solutions in each well was read at 550 nm. The absorbance values corresponding to each well were normalised to DMSO-containing control wells and plotted as concentration of test compound versus % cell viability. IC50 values were calculated from the resulting dose dependent curves. The reported IC50 values are the average of three independent experiments (n = 18).
3.12. Tumorsphere Formation and Viability Assay
Five thousand HMLER-shEcad cells were added to each well of an ultralow-attachment 96-well plate (Corning) in MEGM containing B27 (Invitrogen), 20 ng/mL EGF, and 4 μg/mL heparin (Sigma), and incubated for 120 h. The HMLER-shEcad cells were also treated with 2–5, L2, L3, and salinomycin (0–133 μM). Wells where HMLER-shEcad mammospheres were incubated with 2–5, L2, L3, and salinomycin (at 0.5 μM and their respective IC20 values for 5 days) were manually counted and imaged using an inverted microscope. The resazurin-based dye, TOX8 (Sigma) was used to determine mammosphere viability. Upon incubation for 16 h, the fluorescence of the solutions in each well was read at 590 nm (λex = 560 nm). The fluorescence values corresponding to each well were normalised to DMSO-containing controls and plotted as concentration of test compound versus % mammospheres viability. IC50 values were calculated from the resulting dose dependent curves. The reported IC50 values are the average of two independent experiments, each consisting of two replicates per concentration level.
3.13. Cellular Uptake
To determine the internalisation of 2–5 by HMLER-shEcad cells, about one million HMLER-shEcad cells were seeded in separate 60 mm Petri dishes overnight. The HMLER-shEcad cells were then dosed with 2–5 (at 5 μM) and incubated for 24 h. After the incubation period, the cells were harvested using standard procedures and the number of cells was counted using a haemocytometer. The resultant cellular pellets were dissolved in 65% HNO3 (250 μL) overnight. The solutions were then appropriately diluted using UltraPure water and analysed using inductively coupled plasma mass spectrometry (ICP-MS, ThermoScientific ICAP-Qc quadrupole ICP mass spectrometer). The copper levels found in the cellular pellets are given as Cu (ng) per million cells. The data presented corresponds to the mean of four determinations for each data point.
3.14. Intracellular ROS Assay
HMLER-shEcad cells (5 × 103) were seeded in each well of a 96-well plate. After incubating the cells overnight, they were treated with 2 and 3 (2 × IC50 value for 0.5–24 h) and incubated with 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (20 μM) for 90 min. The intracellular ROS level was determined by measuring the fluorescence of the solutions in each well at 529 nm (λex = 504 nm).