Members of a novel class of anticancer compounds, exhibiting high antitumor activity, i.e. the unsymmetrical bisacridines (UAs), consist of two heteroaromatic ring systems. One of the ring systems is an imidazoacridinone moiety, with the skeleton identical to the structural base of Symadex. The second one is a 1-nitroacridine moiety, hence it may be regarded as Nitracrine’s structural basis. These monoacridine units are connected by an aminoalkyl linker, which vary in structure. In theory, these unsymmetrical dimers should act as double-stranded DNA (dsDNA) bis-intercalators, since the monomeric units constituting the UAs were previously reported to exhibit an intercalating mode of binding into dsDNA. On the contrary, our earlier, preliminary studies have suggested that specific and/or structurally well-defined binding of UAs into DNA duplexes might not be the case. In this contribution, we have revisited and carefully examined the dsDNA-binding properties of monoacridines C-1305, C-1311 (Symadex), C-283 (Ledakrin/Nitracrine) and C-1748, as well as bisacridines C-2028, C-2041, C-2045 and C-2053 using advanced NMR techniques, aided by molecular modelling calculations and the analysis of UV–VIS spectra, decomposed by chemometric techniques. These studies allowed us to explain, why the properties of UAs are not a simple sum of the features exhibited by the acridine monomers.

New unsymmetrical bisacridines (UAs) demonstrated high activity not only against a set of tumor cell lines but also against human tumor xenografts in nude mice. Representative UA compounds, named C-2028, C-2045 and C-2053, were characterized in respect to their physicochemical properties and the following studies aimed to elucidate the role of metabolic transformations in UAs action. We demonstrated with phase I and phase II enzymes in vitro and in tumors cells that: (i) metabolic products generated by cytochrome P450 (P450), flavin monooxygenase (FMO) and UDP-glucuronosyltransferase (UGT) isoenzymes in noncellular systems retained the compound’s dimeric structures, (ii) the main transformation pathway is the nitro group reduction with P450 isoenzymes and the metabolism to N-oxide derivative with FMO1, (iii), the selected UGT1 isoenzymes participated in the glucuronidation of one compound, C-2045, the hydroxy derivative. Metabolism in tumor cells, HCT-116 and HT-29, of normal and higher UGT1A10 expression, respectively, also resulted in the glucuronidation of only C-2045 and the specific distribution of all compounds between the cell medium and cell extract was demonstrated. Moreover, P4503A4 activity was inhibited by C-2045 and C-2053, whereas C-2028 affected UGT1A and UGT2B action. The above conclusions indicate the optimal strategy for the balance among antitumor therapeutic efficacy and drug resistance in the future antitumor therapy.

Unsymmetrical bisacridines (UAs) represent a novel class of anticancer agents previously synthesized by our group. Our recent studies have demonstrated their high antitumor potential against multiple cancer cell lines and human tumor xenografts in nude mice. At the cellular level, these compounds affected 3D cancer spheroid growth and their cellular uptake was selectively modulated by quantum dots. UAs were shown to undergo metabolic transformations in vitro and in tumor cells. However, the physicochemical properties of UAs, which could possibly affect their interactions with molecular targets, remain unknown. Therefore, we selected four highly active UAs for the assessment of physicochemical parameters under various pH conditions. We determined the compounds’ pK_a dissociation constants as well as their potential to self-associate. Both parameters were determined by detailed and complex chemometric analysis of UV-Vis spectra supported by nuclear magnetic resonance (NMR) spectroscopy. The obtained results indicate that general molecular properties of UAs in aqueous media, including their protonation state, self-association ratio, and solubility, are strongly pH-dependent, particularly in the physiological pH range of 6 to 8. In conclusion, we describe the detailed physicochemical characteristics of UAs, which might contribute to their selectivity towards tumour cells as opposed to their effect on normal cells.

In the search for new selective inhibitors of human carbonic anhydrase (hCA), particularly the cancer-associated isoforms hCA IX and hCA XII, a series of 4-substituted pyridine-3-sulfonamides was synthesized using the “click” CuAAC reaction, proven by X-ray crystallography, and evaluated for their inhibitory activity against hCA I, hCA II, hCA IX, and hCA XII. Additional molecular docking studies and cytostatic activity assays on three cancer cell lines were conducted. The compounds exhibited a broad range of inhibitory activity, with K_I reaching 271 nM for hCA II, 137 nM for hCA IX, and 91 nM for hCA XII. Notably, compound 4 demonstrated up to 5.9-fold selectivity toward the cancer-associated hCA IX over the ubiquitous hCA II, while compound 6 exhibited a remarkable 23.3-fold selectivity between transmembrane isoforms hCA IX and hCA XII. Molecular docking studies have shown the possibility of selective interaction with the hydrophilic or lipophilic half of the active site, what results from the adjacent (3,4) position of the “tail” in relation to the sulfonamide group.

This work is the next step in studying the interplay between C-2028 (anticancer-active unsymmetrical bisacridine developed in our group) and the glutathione S-transferase/glutathione (GST/GSH) system. Here, we analyzed the concentration- and pH-dependent GSH conjugation of C-2028 in rat liver microsomes and cytosol. We also applied three recombinant human GST isoenzymes, which altered expression was found in various tumors. The formation of GSH S-conjugate of C-2028 in liver subfractions followed Michaelis-Menten kinetics. We found that C-2028 was conjugated with GSH preferentially by GSTM1-1, revealing a sigmoidal kinetic model. Using a colorimetric assay (MTT test), we initially assessed the cellular GST/GSH-dependent biotransformation of C-2028 in relation to cytotoxicity against Du-145 human prostate cancer cells in the presence or absence of the modulator of GSH biosynthesis. Pretreatment of cells with buthionine sulfoximine resulted in a cytotoxicity decrease, suggesting a possible GSH-mediated bioactivation process. Altogether, our results confirmed the importance of GSH conjugation in C-2028 metabolism, which humans must consider when planning a treatment strategy. Finally, nuclear magnetic resonance spectroscopy elucidated the structure of the GSH-derived product of C-2028. Hence, synthesizing the compound standard necessary for further advanced biological and bioanalytical investigations will be achievable.

Telomerase is an interesting biological target for cancer treatment. Despite recent advances in telomerase research and the rapid development of new methodologies, significant challenges remain in identifying effective telomerase inhibitors with anticancer activity. In this manuscript, we provide computational studies to analyse how structural modification of BIBR1532, a selective and non-competitive telomerase inhibitor, may affect binding by a newly reported human telomerase model. BIBR1532 has been extensively studied in various cancer types, but analyses have shown its low anticancer activity. Further modification of BIBR1532 yielded compound BIBR1591 through a lipophilic morpholine ring extension of the benzoic acid. Interestingly, BIBR1591 showed approximately five-fold weaker inhibitory activity toward human telomerase relative to BIBR1532, but its in vivo preclinical studies have demonstrated significant anti-tumour and pro-apoptotic efficacy. Despite promising preclinical activities, neither compound has advanced to clinical trials, reportedly due to challenges with its pharmacokinetic properties. BIBR1532 suffers from poor solubility and low cellular uptake. This manuscript aims to provide an updated overview of human telomerase inhibition by BIBR1532 analogues and emphasise the importance of understanding its molecular mode of action based on the most updated literature. Using an in silico approach, we analysed how the structural modification of the BIBR scaffold influences the binding mode at the BIBR binding site recently discovered in the structure of human telomerase. We have also assessed whether reported analogues offer a measurable advantage over the parent compound.