Anticancer Study on IrIII and RhIII Half-Sandwich Complexes with the Bipyridylsulfonamide Ligand.

Organometallic half-sandwich complexes [(η5-Cp)IrCl(L)]PF6 (1) and [(η5-Cp)RhCl(L)]PF6 (2) were prepared using pentamethylcyclopentadienyl chloride dimers of iridium(III) or rhodium(III) with the 4-amino-N-(2,2'-bipyridin-5-yl)benzenesulfonamide ligand (L) and ammonium hexafluorophosphate. The crystal structures of L, 1, and 2 were analyzed in detail. The coordination reactions of the ligand with the central ions were confirmed using various spectroscopic techniques. Additionally, the interactions between sulfaligand, Ir(III), and Rh(III) complexes with carbonic anhydrase (CA), human serum albumin (HSA), and CT-DNA were investigated. The iridium(III) complex (1) did not show any antiproliferative properties against four different cancer cell lines, i.e., nonsmall cell lung cancer A549, colon cancer HCT-116, breast cancer MCF7, lymphoblastic leukemia Nalm-6, and a nonmalignant human embryonic kidney cell line HEK293, due to high binding affinity to GSH. The sulfonamide ligand (L) and rhodium(III) complex (2) were further studied. L showed competitive inhibition toward CA, while complexes 1 and 2, uncompetitive. All compounds interacted with HSA, causing a conformational change in the protein's α-helical structure, suggesting the induction of a more open conformation in HSA, reducing its biological activity. Both L and 2 were found to induce cell death through a caspase-dependent pathway. These findings position L and 2 as potential starting compounds for pharmaceutical, therapeutic, or medicinal research.

New 5-Substituted SN38 Derivatives: A Stability Study and Interaction with Model Nicked DNA by NMR and Molecular Modeling Methods.

The new 5-substituted SN-38 derivatives, 5(R)-(N-pyrrolidinyl)methyl-7-ethyl-10-hydroxycamptothecin (1) and its diastereomer 5(S) (2), were investigated using a combination of nuclear magnetic resonance (NMR) spectroscopy and molecular modeling methods. The chemical stability, configuration stability, and propensity to aggregate as a function of concentration were determined using 1H NMR. The calculated self-association constants (Ka) were found to be 6.4 mM-1 and 2.9 mM-1 for 1 and 2, respectively. The NMR experiments were performed to elucidate the interaction of each diastereomer with a nicked decamer duplex, referred to as 3. The calculated binding constants were determined to be 76 mM-1 and 150 mM-1 for the 1-3 and 2-3 complexes, respectively. NMR studies revealed that the interaction between 1 or 2 and the nicked decamer duplex occurred at the site of the DNA strand break. To complement these findings, molecular modeling methods and calculation protocols were employed to establish the interaction mode and binding constants and to generate molecular models of the DNA/ligand complexes.

Factors Affecting the Stability of Platinum(II) Complexes with 1,2,4-Triazolo[1,5-a]pyrimidine Derivatives and Tetrahydrothiophene-1-Oxide or Diphenyl Sulfoxide.

The platinum(II) complexes of general formula [PtCl2(dstp)(S-donor)] were dstp 5,7-dimethyl-1,2,4-triazolo[1,5-a]-pyrimidine (dmtp), 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp), 5-methyl-7-isobutyl-1,2,4-triazolo[1,5-a]pyrimidine (ibmtp) or 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine (dptp), whereas S-tetrahydrothio-phene-1-oxide (TMSO) or diphenyl sulfoxide (DPSO) were synthesized in a one-pot reaction. Here, we present experimental data (1H, 13C, 15N, 195Pt NMR, IR, X-ray) combined with density functional theory (DFT) computations to support and characterize structure-spectra relationships and determine the geometry of dichloride platinum(II) complexes with selected triazolopyrimidines and sulfoxides. Based on the experimental and theoretical data, factors affecting the stability of platinum(II) complexes have been determined.

Insight on the Interaction between the Camptothecin Derivative and DNA Oligomer Mimicking the Target of Topo I Inhibitors.

The understanding of the mechanism of Topo I inhibition by organic ligands is a crucial source of information that has led to the design of more effective and safe pharmaceuticals in oncological chemotherapy. The vast number of inhibitors that have been studied in this respect over the last decades have enabled the creation of a concept of an 'interfacial inhibitor', thereby describing the machinery of Topo I inhibition. The central module of action of this machinery is the interface of a Topo I/DNA/inhibitor ternary complex. Most of the 'interfacial inhibitors' are primarily kinetic inhibitors that form molecular complexes with an "on-off" rate timing; therefore, all of the contacts between the inhibitor and both the enzyme and the DNA are essential to keep the complex stable and reduce the "off rate". To test this hypothesis, we designed the compound using a C-9-(N-(2'-hydroxyethyl)amino)methyl substituent in an SN38 core, with a view that a flexible substituent may bind inside the nick of a model of the DNA and stabilize the complex, leading to a reduction in the "off rate" of a ligand in a potential ternary complex in vivo. Using docking analysis and molecular dynamics, free energy calculations on the level of the MM-PBSA and MM-GBSA model, here we presented the in silico-calculated structure of a ternary complex involving the studied compound 1. This confirmed our suggestion that compound 1 is situated in a groove of the nicked DNA model in a few conformations. The number of hydrogen bonds between the components of a ternary complex was established, which strengthens the complex and supports our view. The docking analysis and free energy calculations for the receptor structures which were obtained in the MD simulations of the ternary complex 1/DNA/Topo I show that the binding constant is stronger than it was for similar complexes with TPT, CPT, and SN38, which are commonly considered as strong Topo I inhibitors. The binary complex structure 1/DNA was calculated and compared with the experimental results of a complex that was in a solution. The analysis of the cross-peaks in NOESY spectra allowed us to assign the dipolar interactions between the given protons in the calculated structures. A DOSY experiment in the solution confirmed the strong binding of a ligand in a binary complex, having a Ka of 746 mM-1, which was compared with a Ka of 3.78 mM-1 for TPT. The MALDI-ToF MS showed the presence of the biohybrid, thus evidencing the occurrence of DNA alkylation by compound 1. Because of it having a strong molecular complex, alkylation is the most efficient way to reduce the "on-off" timing as it acts as a tool that causes the cog to brake in a working gear, and this is this activity we want to highlight in our contribution. Finally, the Topo I inhibition test showed a lower IC50 of the studied compound than it did for CPT and SN38.

New camptothecin derivatives for generalized oncological chemotherapy: Synthesis, stereochemistry and biology.

Derivatives of SN38 were synthesized that were either monosubstituted at C-5 or C-9 or disubstituted at both C-5 and C-9. Substitution to C-5 led to the generation of pairs of diastereomers (2c-2 h) in a one-pot reaction and was readily separable by HPLC. The absolute configurations of C-5 were established by electronic circular dichroism experiments. Compounds were tested in vitro against human cancer cell lines as well as a normal cell line. The impact of compounds 2a-2j on cancer cells is significant and the IC50 values against the normal cell line are several times higher than that of SN38. Using the Mannich reaction we obtained a new innovative group of derivatives with unique biological properties that preserves the high cytotoxicity in cancer cells and eliminates the acute toxicity to non-neoplastic cells, which can be considered a breakthrough in chemotherapy with the use of topoisomerase I inhibitors from the camptothecin family.

A NMR study of binding the metabolite of SN38 derivatives to a model nicked DNA decamer mimicking target of Topo I inhibitors.

In this account we present NMR based results of the interaction of 7-ethyl-9-hydroxymethyl-10-hydroxycamptothecin (1), a derivative of SN38, with a model nicked DNA decamer mimicking the wild type DNA target of Topoisomerase I inhibitors from the camptothecin family. The title compound 1 can be considered a main metabolite of phase I in the metabolic pathway of camptothecin derivatives bearing the alkylamino substituent. Therefore, its pharmacodynamic properties are of interest. It was established by DOSY (Diffusion Ordered Spectroscopy) that compound 1 forms a fairly stable molecular complex with a model nicked DNA decamer with affinity constant Ka 3.02 mM-1. The analysis of NOESY experiments revealed intermolecular cross peaks and mutual induced shifts on both interacting components allowing the conclusion that guest molecule 1 is stacking the nitrogen bases inside the nick. MD (Molecular Dynamics) analysis of four possible inclusions of 1 inside the nick allows establishing the detailed geometry of a complex. Two conformations are suggested as the ones best representing the results of molecular modeling reconciled with experimental NOESY results. The aromatic core of both structures is stacking the nitrogen bases in a nick facing the unbroken strand with ring A. The protons in ring E interact with ribose protons of edge bases of a nick. In conclusion, it can be asserted that SN38 derivative 1 can effectively bind the molecular target of Topo I enzyme and play a role as a Topo I inhibitor.

Synthesis of Oxidized 3ß,3'ß-Disteryl Ethers and Search after High-Temperature Treatment of Sterol-Rich Samples.

It was proven that sterols subjected to high-temperature treatment can be concatenated, which results in polymeric structures, e.g., 3ß,3'ß-disteryl ethers. However, it was also proven that due to increased temperature in oxygen-containing conditions, sterols can undergo various oxidation reactions. This study aimed to prove the existence and perform quantitative analysis of oxidized 3ß,3'ß-disteryl ethers, which could form during high-temperature treatment of sterol-rich samples. Samples were heated at 180, 200 and 220 °C for 0.5 to 4 h. Quantitative analyses of the oxidized 3ß,3'ß-disteryl ethers were performed with liquid extraction, solid-phase extraction and liquid chromatography coupled with mass spectrometry. Additionally, to perform this analysis, the appropriate standards of all oxidized 3ß,3'ß-disteryl ethers were prepared. Eighteen various oxidized 3ß,3'ß-disteryl ethers (derivatives of 3ß,3'ß-dicholesteryl ether, 3ß,3'ß-disitosteryl ether and 3ß,3'ß-distigmasteryl ether) were prepared. Additionally, the influence of metal compounds on the mechanism of ether formation at high temperatures was investigated.

Novel Nontoxic 5,9-Disubstituted SN38 Derivatives: Characterization of Their Pharmacological Properties and Interactions with DNA Oligomers.

Novel nontoxic derivatives of SN38 with favorable antineoplastic properties were characterized in water solution using NMR. The phenomena observed by NMR were linked to basic pharmacological properties, such as solubility, bioavailability, chemical and stereochemical stability, and binding to natural DNA oligomers through the terminal G-C base pair, which is commonly considered a biological target of Topo I inhibitors. Compound 1, with bulky substituents at both C5(R) and C20(S) on the same side of a camptothecin core, manifests self-association, whereas diastereomers 2, with bulky C5(S) and C20(S) substituents are mostly monomeric in solution. The stereogenic center at C5 is stable in water solution at pH 5-6. The compound with an (N-azetidinyl)methyl substituent at C9 can undergo the retro Mannich reaction after a prolonged time in water solution. Both diastereomers exhibit different abilities in terms of binding to DNA oligomers: compound 1 is strongly bound, whereas the binding of compound 2 is rather weak. Molecular modeling produced results consistent with NMR experiments. These complementary data allow linking of the observed phenomena in NMR experiments to basic preliminary information on the pharmacodynamic character of compounds and are essential for planning further development research.

The Mode of SN38 Derivatives Interacting with Nicked DNA Mimics Biological Targeting of Topo I Poisons.

The compounds 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (2) and 7-ethyl-9-(N-morpholino)methyl-10-hydroxycamptothecin (3) are potential topoisomerase I poisons. Moreover, they were shown to have favorable anti-neoplastic effects on several tumor cell lines. Due to these properties, the compounds are being considered for advancement to the preclinical development stage. To gain better insights into the molecular mechanism with the biological target, here, we conducted an investigation into their interactions with model nicked DNA (1) using different techniques. In this work, we observed the complexity of the mechanism of action of the compounds 2 and 3, in addition to their decomposition products: compound 4 and SN38. Using DOSY experiments, evidence of the formation of strongly bonded molecular complexes of SN38 derivatives with DNA duplexes was provided. The molecular modeling based on cross-peaks from the NOESY spectrum also allowed us to assign the geometry of a molecular complex of DNA with compound 2. Confirmation of the alkylation reaction of both compounds was obtained using MALDI-MS. Additionally, in the case of 3, alkylation was confirmed in the recording of cross-peaks in the 1H/13C HSQC spectrum of 13C-enriched compound 3. In this work, we showed that the studied compounds-parent compounds 2 and 3, and their potential metabolite 4 and SN38-interact inside the nick of 1, either forming the molecular complex or alkylating the DNA nitrogen bases. In order to confirm the influence of the studied compounds on the topoisomerase I relaxation activity of supercoiled DNA, the test was performed based upon the measurement of the fluorescence of DNA stain which can differentiate between supercoiled and relaxed DNA. The presented results confirmed that studied SN38 derivatives effectively block DNA relaxation mediated by Topo I, which means that they stop the machinery of Topo I activity.

Similarities and differences in d6 low-spin ruthenium, rhodium and iridium half-sandwich complexes: synthesis, structure, cytotoxicity and interaction with biological targets.

In this paper, we discussed the similarities and differences in d6 low-spin half-sandwich ruthenium, rhodium and iridium complexes containing 2,2'-biimidazole (H2biim). Three new complexes, {[RuCl(H2biim)(η6-p-cymene)]PF6}2·H2O (1), [(η5-Cp)RhCl(H2biim)]PF6 (2), and [(η5-Cp)IrCl(H2biim)]PF6 (3), were fully characterized by CHN, X-ray diffraction analysis, UV-Vis, FTIR, and 1H, 13C and 15N NMR spectroscopies. The complexes exhibit a typical pseudooctahedral piano-stool geometry, in which the aromatic arene ring (p-cymene or Cp) forms the seat, while the bidentate 2,2'-biimidazole and chloride ion form the three legs of the piano stool. Moreover, the cytotoxic activities of the compounds were examined in the LoVo, HL-60, MV-4-11, MCF-7 human cancer cell lines and BALB/3T3 normal mouse fibroblasts. Notably, the investigated complexes showed no cytotoxic effects towards the normal BALB/3T3 cell line compared to cisplatin, which has an IC50 value of 2.20 µg. Importantly, 1 displayed the highest activity against HL-60 (IC50 4.35 µg). To predict a binding mode, we explored the potential interactions of the metal complexes with CT-DNA and protein using UV absorption and circular dichroism. The obtained data suggest that the complexes could interact with CT-DNA via an outside binding mode. Moreover, binding of the complexes with the GSH via UV-Vis and ESI mass spectra was determined. Comparative studies have shown that the rhodium complex (2) is the most GSH reactive, which is probably responsible for its deactivation towards LoVo and MCF-7 tumour cells. The influence of the metal ion on the biological activity of isostructural Rh(III) and Ir(III) complexes was an important goal of the presented investigation.

Identification of Lysine Misincorporation at Asparagine Position in Recombinant Insulin Analogs Produced in E. coli.

PURPOSE: Identification of human insulin analogs' impurity with a mass shift +14 Da in comparison to a parent protein. METHODS: The protein sequence variant was detected and identified with the application of peptide mapping, liquid chromatography, tandem mass spectrometric analysis, nuclear magnetic resonance spectroscopy (NMR) and Edman sequencing. RESULTS: The misincorporated lysine (Lys) at asparagine (Asn) position A21 was detected in recombinant human insulin and its analogs. CONCLUSIONS: Although there are three asparagine residues in the insulin derivative, the misincorporation of lysine occurred only at position A21. The process involves G/U or A/U wobble base pairing.

Insight into human insulin aggregation revisited using NMR derived translational diffusion parameters.

The NMR derived translational diffusion coefficients were performed on unlabeled and uniformly labeled 13C,15N human insulin in water, both in neat, with zinc ions only, and in pharmaceutical formulation, containing only m-cresol as phenolic ligand, glycerol and zinc ions. The results show the dominant role of the pH parameter and the concentration on aggregation. The diffusion coefficient Dav was used for monitoring the overall average state of oligomeric ensemble in solution. The analysis of the experimental data of diffusion measurements, using the direct exponential curve resolution algorithm (DECRA) allows suggesting the two main components of the oligomeric ensemble. The 3D HSQC-iDOSY, (diffusion ordered HSQC) experiments performed on 13C, 15N-fully labeled insulin at the two pH values, 4 and 7.5, allow for the first time a more detailed experimental observation of individual components in the ensemble. The discussion involves earlier static and dynamic laser light scattering experiments and recent NMR derived translational diffusion results. The results bring new informations concerning the preparation of pharmaceutical formulation and in particular a role of Zn2+ ions. They also will enable better understanding and unifying the results of studies on insulin misfolding effects performed in solution by diverse physicochemical methods at different pH and concentration.

Preliminary study of mechanism of action of SN38 derivatives. Physicochemical data, evidence of interaction and alkylation of DNA octamer d(GCGATCGC)2.

The synthesis of water-soluble SN38 derivatives is presented, and their stability in solutions used during drug development studies has been investigated. A preliminary study of mechanism of action of 9-aminomethyl SN38 is presented. Using NMR techniques, the interaction of the oligomer d(GCGATCGC)2 is studied, showing that the terminal GC base pairs are the main site of interaction. Using pulsed field gradient spin echo and mass spectroscopy, evidence of a spontaneous alkylation reaction of the DNA oligomer with SN38 derivatives is presented. A proposed mechanism of reaction is suggested. Copyright © 2016 John Wiley & Sons, Ltd.

DOSY NMR and MALDI-TOF evidence of covalent binding the DNA duplex by trimethylammonium salts of topotecan upon near UV irradiation.

Using DOSY NMR and MALDI-TOF MS techniques, we present evidence that quaternary trimethylammonium salts of topotecan, [TPT-NMe3 ](+) X(-) (X = CF3SO3, HCOO), bind covalently the natural DNA oligomer upon near UV irradiation in water under physiological conditions. It is shown that formate salt is very reactive at pH 7 and requires short irradiation time. This weak irradiation at 365 nm paves the way for a new application of TPT derivatives in clinical use, which can dramatically increase the therapeutic effects of a medicine.

Insights into the tautomerism in meso-substituted corroles: a variable-temperature 1H, 13C, 15N, and 19F†NMR spectroscopy study.

Tris(pentafluorophenyl)corrole and its (15)N-enriched isotopomer were studied in [D8]toluene solution by 1D and 2D variable-temperature NMR techniques to establish the mechanisms of tautomerization of the NH protons inside the interior of the corrole macrocycle. Three such rate processes could be identified of which two modulate the spectral line shapes at temperatures above 205 K and the third is NMR-inaccessible as it is very fast. The latter involves the proton engaged in an unsymmetrical proton sponge unit formed by two pyrrole nitrogen atoms. Temperature and concentration dependences of the two remaining processes were determined. One of them is purely intramolecular and the other is intermolecular at low temperatures, with growing contribution of an intramolecular mechanism at elevated temperatures. The proposed microscopic mechanisms of all these processes are semi-quantitatively confirmed by quantum chemical calculations using density functional theory.

Synthesis, photophysical characterisation, quantum-chemical study and in vitro antiproliferative activity of cyclometalated Ir(III) complexes based on 3,5-dimethyl-1-phenyl-1H-pyrazole and N,N-donor ligands.

In this paper, we present the synthesis of four new complexes: the dimeric precursor [Ir(dmppz)2(µ-Cl)]2 (1) (Hdmppz - 3,5-dimethyl-1-phenyl-1H-pyrazole) and heteroleptic bis-cyclometalated complexes: [Ir(dmppz)2(Py2CO)]PF6·½CH2Cl2 (2), [Ir(dmppz)2(H2biim)]PF6·H2O (3), and [Ir(dmppz)2(PyBIm)]PF6 (4), with auxiliary N,N-donor ligands: 2-di(pyridyl)ketone (Py2CO), 2,2'-biimidazole (H2biim) and 2-(2'-pyridyl)benzimidazole (PyBIm). In the obtained complexes, SC-X-ray analysis revealed that Ir(III) has an octahedral coordination sphere with chromophores of the type {IrN2C2Cl2} (1) or {IrN4C2} (2-4). The complexes obtained, which have been fully characterised by physicochemical methods (CHN, TG, FTIR, UV-Vis, PL and 1H, 13C, 15N NMR), were used to continue our studies on the factors influencing the cytotoxic properties of potential chemotherapeutic agents (in vitro). To this end, the following studies are presented: (i) comparative analysis of the effects on the biological properties of N,N-donor ligands and C,N-donor ligands in the studied complexes, (ii) studies of the interactions of the compounds with the selected molecular target: DNA and BSA (UV-Vis, CD and PL methods), (iii) and the reactivity towards redox molecules: GSH, NADH (UV-Vis and/or ESI-MS methods), (iv) cytotoxic activity (IC50) of potential chemotherapeutics against MCF-7, K-562 and CCRF-CEM cell lines.

Separation of menaquinone-7 geometric isomers by semipreparative high-performance liquid chromatography with silver complexation and identification by nuclear magnetic resonance.

Dietary supplements containing vitamin K2 are often used to prevent osteoporosis, vascular calcification and coronary heart disease. It has been shown that some of these products contain a mixture of menaquinone-7 geometric isomers. Since the geometric shape may influence biological activity, there was a need for a semipreparative method to isolate single compounds for further studies. Here, we present an argentation chromatographic method for the separation of menaquinone-7 isomers and an nuclear magnetic resonance (NMR) methodology for the configuration assignment of isoprenoid side chain. The DFT calculations were performed to determine more energetically favorable complexes between the cis or trans menaquinone-7 isomers and the silver cation. Seventeen components were resolved, and fractions were collected and subjected to NMR study. Structures and chemical shifts for thirteen new compounds were assigned, and the identity of three known compounds was confirmed.

Sawhorse-type ruthenium complexes with triazolopyrimidine ligands - what do they represent in terms of cytotoxic and CORM compounds?

Three sawhorse-type ruthenium(I) complexes containing purine analogs such as triazolopyrimidines of the general formula [Ru2(CO)4(µ-OOCCH3)2(L)2], where L is 1,2,4-triazolo[1,5-a]pyrimidine (tp for 1), 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp for 2) and 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine (dptp for 3), have been synthesized and characterized by elemental analysis, infrared analysis, multinuclear magnetic resonance spectroscopic techniques (1H, 13C, 15N), and single-crystal X-ray diffraction (for 1 and 2). By assay with myoglobin, the photo-activated CO-releasing molecule (PhotoCORM) character of (1-3) has been confirmed, thus indicating the possibility of use in CO-based therapies. The importance of UV-induced modification has been investigated in the context of anticancer properties. Complexes (1) and (2) have been thoroughly screened for their in vitro cytotoxicity against various cancer cell lines: MCF-7 (breast cancer), HeLa (cervical cancer) and C32 (melanoma), as well as L929 normal fibroblasts in the dark and presence of UV-A light (365 nm). The results were compared with those for cisplatin and two reference ruthenium complexes, namely NAMI-A and KP1019. The most hydrophilic [Ru2(CO)4(µ-OOCCH3)2(tp)2] (1) (log P = -1.12) was found to be more cytotoxic than (2), despite the lower cellular uptake measured by ICP-MS toward HeLa cells. Importantly, photo-induced stimulation of cells with (1) resulted in a lower decrease in the viability of L929 normal cells (IC50 = 154.7 ± 6.5 µM) in comparison with HeLa cancer cells (IC50 = 66.7 ± 3.4 µM). The photo-induced stimulation of (1) and (2) increases ROS generation, and their anticancer activity may be a partially ROS-dependent phenomenon.

A dumbbell double nicked duplex dodecamer DNA with a PEG6 tether.

A hairpin dodecamer DNA motif with a dangling end composed of four bases was studied in order to find conditions which promote a dumbbell structure as the sole form in solution. It could be used as a model of a DNA duplex with two nicks on opposite strands, mimicking a target for topo II poisons. We have established two alternative means of obtaining a dumbbell in solution as the only form present at 0 °C. The first one is to use a relatively high concentration of a hairpin motif, ca. 3.5 mM, at low ionic strength, and second is to use a moderate hairpin motif concentration of ca. 2 mM at high ionic strength, 200 mM and 15% of methanol. An NMR-derived structure in a buffered water solution is presented. A representative structure ensemble of 10 structures was obtained from MD calculations utilizing the AMBER protocol and using NOESY-derived experiment cross peak volumes transferred to experimental restraints by the MARDIGRAS algorithm.