Synthesis of Lasalocid-Based Bioconjugates and Evaluation of Their Anticancer Activity

Using rationally designed bioconjugates is an attractive strategy to develop novel anticancer drugs with enhanced therapeutic potential and minimal side effects compared to the native structures. With respect to the promising activity of lasalocid (LAS) toward various cancer cells, this polyether ionophore seems to be an ideal candidate for bioconjugation. Herein, we describe the synthetic access to a cohort of nine conjugated products of LAS, in which the ionophore biomolecule was successfully combined via covalent bonds with selected anticancer therapeutics or other anticancer active components. The in vitro screening of a series of cancer cell lines allowed us to identify three products with improved anticancer activity profiles compared to those of the starting materials. The results indicate that human prostate cancer cells (PC3) and human primary colon cancer cells (SW480) were essentially more sensitive to exposure to LAS derivatives than human keratinocytes (HaCaT). Furthermore, the selected products were stronger inducers of late apoptosis and/or necrosis in PC3 and SW480 cancer cells, when compared to the metastatic variant of colon cancer cells (SW620). To establish the anticancer mechanism of LAS-based bioconjugates, the levels of interleukin 6 (IL-6) and reactive oxygen species (ROS) were measured; the tested compounds significantly reduced the release of IL-6, while the level of ROS was significantly higher in all the cell lines studied.


General procedures
All reagents and solvents were obtained from Merck or Trimen Chemicals S.A. (Poland), and were used as received without further purification. CDCl 3 , CD 2 Cl 2 and CD 3 CN spectral grade solvent was stored over 3Å molecular sieves for several days. All manipulations were carried out under nitrogen atmosphere in oven-dried glassware. Reaction mixtures were stirred using teflon-coated magnetic stir bars. Reaction mixtures were monitored by thin layer chromatography (TLC) using aluminium-backed plates (Merck 60F 254 ). TLC plates were visualized by UV-light (254 nm), after treated with phosphomolybdic acid (PMA, 5% in absolute EtOH) and gentle heating. Products of the reactions were purified using CombiFlash Rf + Lumen Flash Chromatography System (Teledyne Isco) with integrated ELS and UV detectors. All solvents used in flash chromatography were of HPLC grade (Merck), and were used as received. Solvents were removed using a rotary evaporator.
NMR spectra were recorded on a Varian 400 ( 1 H NMR at 400 MHz, 13 C NMR at 101 MHz, 19 F NMR at 282 MHz, and 31 P NMR at 162 MHz) magnetic resonance spectrometer. 1 H NMR spectra are reported in chemical shifts downfield from TMS using the respective residual solvent peak as internal standard (CDCl 3 δ 7.26 ppm, CD 2 Cl 2 δ 5.32 ppm, or CD 3 CN δ 1.94 ppm). 1 H NMR spectra are reported as follows: chemical shift (δ, ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, dt = doublet of triplets, dq = doublet of quartets, td = triplet of doublets, pd = pentet of doublets, ddd = doublet of doublet of doublets, ddt = doublet of doublet of triplets, tdd = triplet of doublet of doublets, m = multiplet), coupling constant(s) in Hz, and integration. Significant peaks are reported within the overlapping ~2.10-0.60 ppm region of the 1 H NMR spectra. 13 C NMR spectra are reported in chemical shifts downfield from TMS using the respective residual solvent peak as internal standard (CDCl 3 δ 77.16 ppm, CD 2 Cl 2 δ 53.84 ppm, or CD 3 CN δ 1.32 ppm and 118.26 ppm). 19 F NMR spectra are reported in chemical shifts upfield from TMS using CFCl 3 as internal standard. Line broadening parameters were 0.5 or 1.0 Hz, while the error of chemical shift value was 0.1 ppm.
Infrared spectra in the mid infrared region were recorded for KBr tablets on an IFS 113v FT-IR spectrophotometer (Bruker) equipped with a DTGS detector, and are reported as follows: wavenumbers (cm -1 ), description (w = weak, m = medium, s = strong, br = broad). The spectra were taken at a resolution 2 cm -1 , NSS = 64. The Happ-Genzel apodization function was used.
Electrospray ionization (ESI) mass spectra were recorded on a Waters/Micromass ZQ mass spectrometer (Waters Alliance) equipped with a Harvard syringe pump. Samples were prepared in dry acetonitrile, and were infused into the ESI source using a Harvard pump at a flow rate of 20 mL min -1 . The ESI source potentials were: capillary 3 kV, lens 0.5 kV, and extractor 4 V. Standard ESI mass spectra were recorded at the cone voltages of 10 and 30 V. The source temperature was 120 °C and the desolvation temperature was 300 °C.
Nitrogen was used as the nebulizing and desolvation gas at flow-rates of 100 dm 3 h -1 . Mass spectra were acquired in the positive ion detection mode with unit mass resolution at a step of 1 m/z unit. The mass range for ESI experiments was from m/z = 300 to m/z = 1100, or m/z = 300 to m/z = 1300. High-resolution mass spectra (HRMS) were recorded on a QTOF mass spectrometer (Impact HD, Bruker Daltonics).         -S9 - Figure S14. The 13 C NMR spectrum of bioconjugate 9 in chloroform-d. Figure S15. The 1 H NMR spectrum of bioconjugate 9 in chloroform-d. Figure S16. The 19 F NMR spectrum of bioconjugate 9 in chloroform-d.