Andriy Mokhir

Study of complex formation with 2-hydroxyiminocarboxylates: specific metal binding ability of 2-(4-methylthiazol-2-yl)-2-(hydroxyimino)acetic acid

Abstract Complex formation properties of a novel water soluble thiazolyloxime 2-(4-methylthiazol-2-yl)-2-(hydroxyimino)acetic acid (H3L1) with Cu2+ and Ni2+ were investigated in solution by potentiometrical and spectral (UV–Vis, EPR, NMR) methods. All Cu2+ and most of Ni2+ complex species detected in solution were found to have square-planar MN4 core with oxime and heterocyclic nitrogen atoms which was rationalized in terms of destabilizing effect of repulsive interaction between oxygen atom of carboxylic group and nitrogen atom of thiazole ring in N,O-coordinated ligand conformation. It has been found that stability of metal complexes in a series of oxime ligands is dependent upon basicity of nitrogen atom of oxime group. The thiazolyloxime forms less stable complexes with Cu2+ but stronger ones with Ni2+ ions when compared to parent 2-(hydroxyimino)propanoic acid. The lower stability obtained for Cu2+ complexes was elucidated in terms of negative inductive effect of the thiazole and nitrile substituents as well as an effect of intramolecular attractive interaction between thiazolyl sulfur and oxime oxygen atoms in thiazolyloxime. In the case of Ni2+ the complexes formed are square-planar and it is why thiazolyl ligand is more effective in metal ion binding than simple 2-(hydroxyimino)propanoic acid forming only octahedral species. The solid state structure of the Co3+ complex K3[Co(HL1)3]·5.5H2O (1) was studied by X-ray analysis. The thiazolyloxime ligand is coordinated to Co3+ via oxime nitrogen and carboxylate oxygen atoms forming five-membered chelate rings.

Improved synthesis of N-Benzylaminoferrocene-based prodrugs and evaluation of their toxicity and antileukemic activity

We report on an improved method of synthesis of N-benzylaminoferrocene-based prodrugs and demonstrate its applicability by preparing nine new aminoferrocenes. Their effect on the viability of selected cancer cells having different p53 status was studied. The obtained data are in agreement with the hypothesis that the toxicity of aminoferrocenes is not dependent upon p53 status. Subsequently the toxicity of a selected prodrug (4) was investigated ex vivo using rat precision cut liver slices and in vivo on hybrid male mice BDF1. In both experiments no toxicity was observed: ex vivo, up to 10 μM; in vivo, up to 6 mg/kg. Finally, prodrug 4 was shown to extend the survival of BDF1 mice carrying L1210 leukemia from 13.7 ± 0.6 days to 17.5 ± 0.7 days when injected daily 6 times at a dose of 26 μg/kg starting from the second day after injection of L1210 cells.

Study of anti-fibrillogenic activity of iron(II) clathrochelates

The macrocyclic compounds mono- and bis-iron(II) clathrochelates were firstly studied as potential anti-fibrillogenic agents using fluorescent inhibitory assay, atomic force microscopy and flow cytometry. It is shown that presence of the clathrochelates leads to the change in kinetics of insulin fibrillization reaction and reduces the amount of formed fibrils (up to 70%). The nature of ribbed substituent could determine the activity of clathrochelates-the higher inhibitory effect is observed for compounds containing carboxybenzenesulfide groups, while the inhibitory properties only slightly depend on the size of complex species. The mono- and bis-clathrochelate derivatives of meta-mercaptobenzoic acid have close values of IC₅₀ namely 16 ± 2 and 24 ± 5 μM, respectively. The presence of clathrochelates decreases the fibril diameter from 5-12 nm for free insulin fibrils to 3-8 nm for these formed in the clathrochelate presence, it also prevents the lateral aggregation of mature fibrils and formation of superfibrillar clusters. However the addition of clathrochelate results in more heterogeneous (both by size and structure) insulin aggregates population as compared to the free insulin. This way, cage complexes-iron(II) clathrochelates are proposed as efficient agents able to suppress the protein aggregation processes.

Activity of aminoferrocene-based prodrugs against prostate cancer.

We tested cytotoxicity of aminoferrocene-based prodrugs towards human androgen-responsive and unresponsive prostate cancer cell lines LNCaP and DU-145 correspondingly. Two prodrugs were selected, which are both activated at elevated concentrations of ROS with generation of quinone methide (antioxidant system inhibitor) and iron-containing compounds (N-benzylaminoferrocene (prodrug 1) and Fe salts (2)). We observed that only prodrug 1 is active against the selected prostate cancer cells (IC50=11-27 μM) and its activity correlates with the high cell-membrane permeability and increased production of intracellular ROS.

Cytotoxicity of electrophilic iron(II)–clathrochelates in human promyelocytic leukemia cell line

We observed that electrophilic iron(II)-clathrochelates exhibit significant cytotoxicity in human promyelocytic leukemia cells (IC50=6.5±4.6μM), which correlates with the enhancement of intracellular oxidative stress (17-fold increase with respect to the cells treated with the solvent only). Based on in vitro studies we suggested that this effect is caused by alkylation of glutathione leading to inhibition of the cellular antioxidative system and by catalytic generation of reactive oxygen species by products of the alkylation reaction.

Cancer-Specific, Intracellular, Reductive Activation of Anticancer PtIV Prodrugs

Because cellular uptake of anticancer PtII and PtIV drugs occurs by different mechanisms, the latter ones can exhibit substantial activity towards cells, which have either intrinsic or acquired resistance towards PtII drugs. However, this positive effect is diminished due to reductive activation of PtIV drugs in extracellular space that can be one of the reasons why they have not yet been approved for clinical use despite over 60 clinical trials conducted worldwide. Herein, we suggest a solution to this problem by achieving highly specific intracellular versus extracellular prodrug reduction. In particular, we prepared a hybrid PtIV prodrug containing two pro-reductants. This hybrid was uptaken by cells, the pro-reductants were activated in the cancer-specific microenvironment (high H2 O2 ), and reduced PtIV by two one-electron transfers. The drug formed in this way induced cell death both in cisplatin-sensitive and resistant cell lines, but remained nontoxic to normal cells.

Drug-perturbation-based stratification of blood cancer

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non–BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.

Tuning the structure of aminoferrocene-based anticancer prodrugs to prevent their aggregation in aqueous solution

Aminoferrocene-based prodrugs are activated in cancer cells by reactive oxygen species (ROS). They were shown to exhibit high cytotoxicity towards a variety of cancer cell lines and primary cancer cells, but remain not toxic towards non-malignant cells. However, these prodrugs have rather high lipophilicity leading to relatively low water solubility. In particular, an n-octanol/water partition coefficient for the best aminoferrocene-based prodrug (2) was found to be 4.51±0.03. Though the approaches for decreasing lipophilicity are straightforward and include the addition of polar residues to the drug structure, these modifications also lead to dramatic decrease of cell permeability and, correspondingly, lower the activity of the drug. Therefore, a delicate balance of polar and unpolar groups should be found to reduce lipophilicity without compromising the useful drug properties. In this study we optimized an N-alkyl substituent, which is a key element responsible for the stabilization of the aminoferrocene drug released in cancer cells from prodrug 2. We found that an N-propargyl residue is an optimal replacement for the N-benzyl fragment. In particular, such a substitution (prodrug 7a) leads to reduction of prodrug lipophilicity down to logP=3.78±0.05, improvement of its water solubility, decrease of its propensity towards aggregation and dramatic increase of its ROS-generating properties. Finally, we demonstrated that the optimized prodrug strongly suppresses growth of Guerins carcinoma (T8) in vivo at the dose of 30mg/kg.

Oxo-Functionalized Graphene as a Cell Membrane Carrier of Nucleic Acid Probes Controlled by Aging

We applied a fluorescein-containing oligonucleotide (ON) to probe surface properties of oxidized graphene (oxo-G) and observed that graphene-like patches are formed upon aging of oxo-G, indicated by enhanced probe binding and by FTIR spectroscopic analysis. By using a recently developed fluorogenic endoperoxide (EP) probe, we confirmed that during the aging process the amount of EPs on the oxo-G surface is reduced. Furthermore, aging was found to strongly affect cell membrane carrier properties of this material. In particular, freshly prepared oxo-G does not act as a carrier, whereas oxo-G aged for 28 days at 4 °C is an excellent carrier. Based on these data we prepared an optimized oxo-G, which has a low-defect density, binds ONs, is not toxic, and acts as cell membrane carrier. We successfully applied this material to design fluorogenic probes of representative intracellular nucleic acids 28S rRNA and β-actin-mRNA. The results will help to standardize oxidized graphene derivatives for biomedical and bioanalytical applications.

Radiosynthesis of an 18F-fluoroglycosylated aminoferrocene for in-vivo imaging of reactive oxygen species activity by PET

The imaging of reactive oxygen species (ROS) at the molecular level with high sensitivity and specificity by positron emission tomography (PET) could be of enormous interest to increase our knowledge about ROS activity and signalling, especially in tumours. The aim of this research was to optimise the click chemistry-based radiosynthesis of an 18 F-labelled aminoferrocene glycoconjugate that was derived from an N-alkylaminoferrocene lead structure known to have anticancer activity in vitro. Applying the solvent system phosphate buffer/THF (12/5), Cu(OAc)2 and sodium ascorbate as reducing agent at 60°C, the alkyne 1 reacted with the 18 F-labelled glycosyl azide [18 F]2 in the presence of carrier 3 (47μM) to obtain carrier-added [18 F]4 in a radiochemical yield of 85%. Interestingly, the addition of carrier was essential for sufficient radiochemical yield, because it suppressed the oxidation of no-carrier-added (n.c.a.) [18 F]4. Future work will include the formulation of c.a. [18 F]4 for studying its biodistribution in tumour-bearing mice.