Synthesis and biological evaluation of salophen nickel(II) and cobalt(III) complexes as potential anticancer compounds.

Recent in vitro investigations of N,N'-bis(salicylidene)-1,2-phenylenediamine (SAP) iron(III) complexes substituted with alkyl (ethyl, propyl, butyl) carboxylates at position 4 in tumor and leukemia cells revealed strong cytotoxic activity. In continuation of this study, analogous nickel(II) and cobalt(III) complexes were synthesized and tested in HL-60 leukemia, and cisplatin-sensitive and -resistant A2780 ovarian cancer cell lines. The biological activity depended on the extent of cellular uptake and the formation of reactive oxygen species (ROS). Inactive [(Ni(II)SAP] complexes (1-3) only marginally accumulated in tumor cells and did not induce ROS. The cellular uptake of [Co(III)SAP]Cl complexes (4-6) into the cells depended on the length of the ester alkyl chain (ethyl, 4 < propyl, 5 < butyl, 6). The cytotoxicity correlated with the presence of ROS. The low cytotoxic complex 4 induced only few ROS, while 5 and 6 caused a good to outstanding antiproliferative activity, exerted high ROS generation, and induced cell death after 48 h. Necrostatin-1 prevented the biological effects, proving necroptosis as part of the mode of action. Interestingly, the effects of 5 and 6 were not reversed by Ferrostatin-1, but even enhanced upon simultaneous application to the tumor cells.

Development and validation of a LC method for the separation and determination of the anticancer-active Fe(III) (4-methoxy-salophene) using the new second-generation monolith.

LC method with the newly introduced second-generation monolithic silica RP-18e column has been developed for the separation of Fe(III) (salophene) and four methoxy-substituted Fe(III) (salophene) complexes. The method has been validated for the quantitation of Fe(III) (4-OMe-salophene), a highly active anticancer substance in vitro, bound to serum albumin. Our routinely used high-resolution continuum-source atomic absorption spectroscopy method based on the determination of the central iron atom was unsuitable in this case because serum originally contains significant amounts of iron as revealed by a blank sample of serum albumin. The developed LC method depends on detecting the whole complex rather than the bound iron. Two morphologically different first- and second-generation HPLC monolithic columns have been compared for this purpose. The newly introduced second-generation monolithic silica column Chromolith(®) HighResolution RP-18e column (100 × 4.6 mm, Merck) separated the mixture successful within 13 min. A mobile phase consisting of 25 mM phosphate buffer pH 3/methanol (60:40, v/v) was used at a flow rate of 1 mL/min. The dynamic linear working range of the calibration curve for Fe(III) (4-OMe-salophene) was found to be between 1 and 200 µg/mL. Detection and quantitation limits were 0.3 and 1 µg/mL, respectively.

Amide and ester derivatives of chlorido[4-carboxy-1,2-disalicylideneaminobenzene]iron(iii) as necroptosis and ferroptosis inducers.

In continuation of the structure-activity study about 4-substituted chlorido[N,N'-disalicylidene-1,2-phenylenediamine]iron(iii) complexes as necroptosis and ferroptosis inducers, we introduced a 4-COOH group at the 1,2-phenylenediamine moiety of the lead ([Fe(iii)salopheneCl]) and derived the resulting complex 15 to the respective ethyl, propyl, or butyl amides (16-18) and esters (19-21). The compounds 16-21 exerted concentration-dependent antiproliferative and antimetabolic effects against HL-60 cells. The esters were more active than the analogous amides. Elongation of the alkyl chain enhanced the activity of the amides, while that of the esters decreased. The complexes 16-21 induced necroptosis and/or ferroptosis but not apoptosis. Studies on protein binding and uptake into HL-60 cells indicated that the complexes mainly accumulated by passive transport. The high binding tendency of all complexes to apo-Transferrin, however, points to participation of a carrier-mediated transport into the cells, too.

A New Approach in Cancer Treatment: Discovery of Chlorido[N,N'-disalicylidene-1,2-phenylenediamine]iron(III) Complexes as Ferroptosis Inducers.

Chlorido[N,N'-disalicylidene-1,2-phenylenediamine]iron(III) complexes generate lipid-based ROS and induce ferroptosis in leukemia and neuroblastoma cell lines. The extent of ferroptosis on the mode of action is regulated by simple modifications of the substituents at the 1,2-phenylenediamine moiety. In HL-60 cells, the unsubstituted lead exclusively caused ferroptosis. For instance, a 4-F substituent shifted the mode of action toward both ferroptosis and necroptosis, while the analogously chlorinated derivative exerted only necroptosis. Remarkably, cell-death in NB1 neuroblastoma cells was solely induced by ferroptosis, independent of the used substituents. The effects were higher than that of the therapeutically applied drug cisplatin. These data clearly demonstrate for the first time that not only iron ions but also iron salophene complexes are potent ferroptosis inducers, which can be optimized as antitumor agents.

Synthesis and In Vitro Evaluation of Thiolated Carrageenan.

The aim of this study was to generate and characterize a thiolated carrageenan. Thiolated carrageenan (carrageenan-SH) was synthesized from kappa (κ)- and iota (ι)-carrageenan by bromine replacement of the hydroxyl moieties followed by substitution to thiol groups using thiourea. Thiolated κ- and ι-carrageenan exhibited 176.57 ± 20.11 and 109.51 ± 18.26 µmol thiol groups per gram polymer, respectively. The resazurin test in Caco-2 cells revealed no toxic effect of both thiolated carrageenans at a concentration below 0.1% (w/v). Regarding efflux pump inhibitory effect, cellular accumulation of multidrug-resistance protein 2 substrate, sulforhodamine 101, was 1.38- and 1.35-fold increased in cells treated with thiolated κ- and ι-carrageenan, respectively. Modification of κ- and ι-carrageenan led to 3.9- and 2.0-fold increase in dynamic viscosity of mucus-thiolated carrageenan mixture within 4 h. Furthermore, residence time of κ- and ι-carrageenan-SH on porcine intestinal mucosa was 6.4- and 1.8-fold prolonged, respectively, as demonstrated by rotating cylinder method, indicating improved mucoadhesive properties. Hence, thiolation of carrageenans led to novel pharmaceutical excipients for various applications.

Thiopyrazole preactivated chitosan: combining mucoadhesion and drug delivery.

The objective of this study was to develop a preactivated chitosan derivative by the introduction of thioglycolic acid followed by 3-methyl-1-phenylpyrazole-5-thiol (MPPT) coupling via disulfide bond formation. The newly synthesized conjugate was characterized in terms of water-absorbing capacity, cohesive properties, mucoadhesion and drug release kinetics. Further in vitro characterization was conducted regarding permeation enhancement of the model compound fluorescein isothiocyanate dextran (FD4) and cytotoxic effects on Caco-2 cells. Based on the attachment of the hydrophobic residue, chitosan-S-S-MPPT test discs showed increased stability of the polymer matrix as well as improved water uptake and liberation of fluorescein isothiocyanate dextran (FD4) compared to chitosan only. The mucoadhesive qualities on porcine intestinal mucosa could be improved 38-fold based on the enhanced bonding between chitosan-S-S-MPPT and mucus through the thiol/disulfide exchange reaction of polymer and mucosal cysteine-rich domains supported by MPPT as the leaving group. This novel biomaterial presents a disulfide conjugation-based delivery system that releases the antibacterial thiopyrazole when the polymer comes into contact with the intestinal mucosa. These properties, together with the safe toxicological profile, make chitosan-S-S-MPPT a valuable carrier for mucoadhesive drug delivery systems and a promising matrix for the development of antimicrobial excipients.