Atropisomeric 1-phenylbenzimidazoles affecting microtubule organization: influence of axial chirality.

Benzimidazoles are frequently used in medicinal chemistry. Their anticancer effect is among the most prominent biological activities exhibited by this scaffold. Although numerous benzimidazole derivatives have been synthesized, possible atropisomerism of ortho-substituted 1-phenylbenzimidazoles has been largely overlooked. The aim of this research was to synthesize a small library of novel atropisomeric benzimidazole derivatives and explore their biological activity in various cancer and normal human cell lines. The new unique structural motif provides an interesting 3D architecture with axial chirality, which further contributes to molecular complexity and specificity. Racemates and their separated atropisomers arrested the cell cycle, caused apoptosis, and affected microtubule organization in cancer cells in vitro at different intensities. Moreover, this phenomenon was also verified by the inhibition of endothelial cell migration. These results showed that (+)-atropisomers, especially 5n, exhibit a stronger effect and show promise as agents for cancer therapy.

Axially Chiral Sulfonic Acids for Brønsted Acid Catalysis: 8-Benzoimidazolylnaphthalene-1-sulfonic Acids and Their Derivatives.

A new type of axially chiral sulfonic acid was developed. The synthesis is based on cheap commercially available materials and a practical method for optical resolution via diastereomeric salt formation, which can provide both enantiomers. Eleven benzoimidazolylnaphthalenesulfonic acids were prepared and four of them were isolated as pure and stable atropisomers. Moreover, several of these sulfonic acids were transformed into triflyl imides to further expand the range of dissociation constants.

Synthesis and Structural Optimization of 2,7,9-Trisubstituted purin-8-ones as FLT3-ITD Inhibitors.

Therapy of FLT3-positive acute myeloid leukemia still remains complicated, despite the availability of newly approved kinase inhibitors. Various strategies to avoid the reduced efficacy of therapy have been explored, including the development of dual targeting compounds, which inhibit FLT3 and another kinase necessary for the survival and proliferation of AML cells. We have designed new 2,7,9-trisubstituted 8-oxopurines as FLT3 inhibitors and report here the structure-activity relationship studies. We demonstrated that substituents at positions 7 and 9 modulate activity between CDK4 and FLT3 kinase, and the isopropyl group at position 7 substantially increased the selectivity toward FLT3 kinase, which led to the discovery of compound 15a (9-cyclopentyl-7-isopropyl-2-((4-(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one). Cellular analyses in MV4-11 cells revealed inhibition of autophosphorylation of FLT3 kinase in nanomolar doses, including the suppression of downstream STAT5 and ERK1/2 phosphorylation. We also describe mechanistic studies in cell lines and activity in a mouse xenograft model in vivo.

Limitations of Trifluoromethylbenzoimidazolylbenzoic Acid as a Chiral Derivatizing Agent to Assign Absolute Configuration for ß-Chiral Aminoalcohols.

Axially chiral 2-(2-(trifluoromethyl)-1H-benzo[d]imidazole-1-yl)benzoic acid (TBBA) was used as a chiral derivatizing agent to evaluate the limits of absolute configuration assignment for ß-chiral aminoalcohols. Seven Boc-aminoalcohols and eight variously N-substituted (S)-phenylglycinols were prepared, and their TBBA esters were analyzed by NMR spectroscopy. Diverse substitution at the ß-position was employed to demonstrate the effect of structure on the general conformational model and reliability of the absolute configuration assignment. It was concluded that hydrogen bond formation and steric hindrance were the main factors affecting the correct assignment for Boc-aminoalcohols.

Novel Screen-Printed Sensor with Chemically Deposited Boron-Doped Diamond Electrode: Preparation, Characterization, and Application.

New screen-printed sensor with a boron-doped diamond working electrode (SP/BDDE) was fabricated using a large-area linear antenna microwave chemical deposition vapor system (LA-MWCVD) with a novel precursor composition. It combines the advantages of disposable printed sensors, such as tailored design, low cost, and easy mass production, with excellent electrochemical properties of BDDE, including a wide available potential window, low background currents, chemical resistance, and resistance to passivation. The newly prepared SP/BDDEs were characterized by scanning electron microscopy (SEM) and Raman spectroscopy. Their electrochemical properties were investigated by cyclic voltammetry and electrochemical impedance spectroscopy using inner sphere ([Fe(CN)6]4-/3-) and outer sphere ([Ru(NH3)6]2+/3+) redox probes. Moreover, the applicability of these new sensors was verified by analysis of the anti-inflammatory drug lornoxicam in model and pharmaceutical samples. Using optimized differential pulse voltammetry in Britton-Robinson buffer of pH 3, detection limits for lornoxicam were 9 × 10-8 mol L-1. The oxidation mechanism of lornoxicam was investigated using bulk electrolysis and online electrochemical cell with mass spectrometry; nine distinct reaction steps and corresponding products and intermediates were identified.

Synthesis of 4-styrylpyrazoles and Evaluation of their Inhibitory Effects on Cyclin-dependent Kinases.

BACKGROUND: Cycle-regulating and transcriptional cyclin-dependent kinases (CDKs) are attractive targets in cancer drug development. Several CDK inhibitors have already been obtained or are close to regulatory approval for clinical applications. OBJECTIVE: Phenylazopyrazole CAN508 has been described as the first selective CDK9 inhibitor with an IC50 of 350 nM. Since the azo-moiety is not a suitable functionality for drugs due to pharmacological reasons, the preparation of carbo-analogues of CAN508 with similar biological activities is desirable. The present work is focused on the synthesis of carbo-analogues similar to CAN508 and their CDK inhibition activity. METHODS: Herein, the synthesis of 21 novel carbo analogues of CAN508 and their intermediates is reported. Subsequently, target compounds 8a - 8u were evaluated for protein kinase inhibition (CDK2/cyclin E, CDK4/cyclin D, CDK9/cyclin T) and antiproliferative activities in cell lines (K562, MCF-7, MV4-11). Moreover, the binding mode of derivative 8s in the active site of CDK9 was modelled. RESULTS: Compounds 8a - 8u were obtained from key intermediate 7, which was prepared by linear synthesis involving Vilsmeier-Haack, Knoevenagel, Hunsdiecker, and Suzuki-Miyaura reactions. Styrylpyrazoles 8t and 8u were the most potent CDK9 inhibitors with IC50 values of approximately 1 µM. Molecular modelling suggested binding in the active site of CDK9. The flow cytometric analysis of MV4-11 cells treated with the most active styrylpyrazoles showed a significant G1-arrest. CONCLUSION: The prepared styrylpyrazoles showed inhibition activity towards CDKs and can provide a novel chemotype of kinase inhibitors.

The Assignment of the Absolute Configuration of ß-Chiral Primary Alcohols with Axially Chiral Trifluoromethylbenzimidazolylbenzoic Acid.

Axially chiral trifluoromethylbenzimidazolylbenzoic acid (TBBA) was used as a chiral derivatization agent for the assignment of the absolute configuration of ß-chiral primary alcohols. The structures varied from simple aliphatic alcohols to complex cyclic systems and highly substituted sugar derivatives. The NMR-based method was successfully implemented to evaluate 17 compounds and displayed ΔδPM values higher than 0.1 ppm in most cases, which makes TBBA superior to MTPA and MPA and comparable to 9-AMA.

Axially Chiral Trifluoromethylbenzimidazolylbenzoic Acid: A Chiral Derivatizing Agent for α-Chiral Primary Amines and Secondary Alcohols To Determine the Absolute Configuration.

Racemic 2-(2-trifluoromethyl)-1H-benzo[d]imidazol-1-yl)benzoic acid (TBBA) was synthesized in three steps from 1-fluoro-2-nitrobenzene. Target (P)- and (M)-TBBA atropisomers were stable with a racemization barrier above 30 kcal/mol. As a chiral derivatizing agent, TBBA showed much higher differences in chemical shifts (ΔδPM) than the conventional Mosher's acid.

Synthesis of [15 N4 ] purine labeled cytokinin glycosides derived from zeatins and topolins with 9-ß-d, 7-ß-d-glucopyranosyl, or 9-ß-d-ribofuranosyl group.

Synthesis of [15 N4 ] purine labeled cytokinine glycosides derived from zeatins and topolins containing a 9-ß-d, 7-ß-d-glucopyranosyl, or 9-ß-d-ribofuranosyl group is described. These N6 -substituted adenine derivatives are intended as internal analytic standards for phytohormone analysis. All labeled compounds were prepared from 6-chloro[15 N4 ]purine (1). The equilibrium reaction of 1 with acetobromo-α-d-glucose gave isomeric 7-ß-d (3) and 9-ß-d (4) chloro glucosyl precursors, which were treated with the corresponding amines to get desired labeled cytokinin 7-ß-d (6) and 9-ß-d (5) glucopyranosides. Cytokinins containing 9-ß-d-ribofuranosyl group (8) were obtained by direct enzymatic transglycosylation reaction of cytokinins (7) prepared from 6-chloro[15 N4 ] purine (1).

Modification of Boc-Protected CAN508 via Acylation and Suzuki-Miyaura Coupling.

The cyclin-dependent kinase inhibitor, CAN508, was protected with di-tert-butyl dicarbonate to access the amino-benzoylated pyrazoles. The bromo derivatives were further arylated by Suzuki-Miyaura coupling using the XPhos Pd G2 pre-catalyst. The coupling reaction provided generally the para-substituted benzoylpyrazoles in the higher yields than the meta-substituted ones. The Boc groups were only utilized as directing functionalities for the benzoylation step and were hydrolyzed under conditions of Suzuki-Miyaura coupling, which allowed for elimination of the additional deprotection step.

Synthesis of 4-substituted pyrazole-3,5-diamines via Suzuki-Miyaura coupling and iron-catalyzed reduction.

A general and efficient synthesis of 4-substituted-1H-pyrazole-3,5-diamines was developed to access derivatives with an aryl, heteroaryl, or styryl group, which are otherwise relatively difficult to prepare. The first step is based on the Suzuki-Miyaura cross-coupling reaction utilizing the XPhos Pd G2 precatalyst. The coupling reactions of 4-bromo-3,5-dinitro-1H-pyrazole with the electron-rich/deficient or sterically demanding boronic acids enabled the production of the corresponding dinitropyrazoles. The subsequent iron-catalyzed reduction of both nitro groups with hydrazine hydrate accomplished the synthesis. The additional demethylation of the 4-methoxystyryl derivative allowed the production of the carboanalog of CAN508 reported as a selective CDK9 inhibitor.

Ring-Opening Reactions of the N-4-Nosyl Hough-Richardson Aziridine with Nitrogen Nucleophiles.

Dinosylated α-d-glucopyranoside was directly transformed into α-d-altropyranosides via in situ formed N-4-nosyl Hough-Richardson aziridine with nitrogen nucleophiles under mild conditions in fair to excellent yields. The scope of the aziridine ring-opening reaction was substantially broadened contrary to the conventional methods introducing solely the azide anion at high temperatures. If necessary, the N-4-nosyl Hough-Richardson aziridine can be isolated by filtration in a very good yield and high purity.

The Suzuki-Miyaura Cross-Coupling Reaction of Halogenated Aminopyrazoles: Method Development, Scope, and Mechanism of Dehalogenation Side Reaction.

The efficient Suzuki-Miyaura cross-coupling reaction of halogenated aminopyrazoles and their amides or ureas with a range of aryl, heteroaryl, and styryl boronic acids or esters has been developed. The method allowed incorporation of problematic substrates: aminopyrazoles bearing protected or unprotected pyrazole NH, as well as the free amino or N-amide group. Direct comparison of the chloro, bromo, and iodopyrazoles in the Suzuki-Miyaura reaction revealed that Br and Cl derivatives were superior to iodopyrazoles, as a result of reduced propensity to dehalogenation. Moreover, the mechanism and factors affecting the undesired dehalogenation side reaction were revealed.

Novel arylazopyrazole inhibitors of cyclin-dependent kinases.

Here, we describe new 4-arylazo-3,5-diamino-1H-pyrazole derivatives developed from CAN508, one of the first inhibitors to show preference for transcriptional regulator cyclin-dependent kinase 9. By substituting nitrogen in the pyrazole ring and employing a heteroatom in the 4-aryl ring, we obtained more potent derivatives differing in their CDK-selectivity profiles. The antiproliferative and anti-CDK kinase activities of the novel arylazopyrazoles were examined. The cellular effect of compound IVc was studied on MCF-7 cells synchronized by various methods and compared with other selective CDK inhibitors. The results demonstrated that IVc shows a preference for CDK4 and CDK1. In contrast to cytostatic effects induced by IVc in MCF-7 and K562 cells, we observed apoptotic activities in the RPMI-8226 cell line, which were confirmed by detecting active caspases by different biochemical assays.

Arylazopyrazole AAP1742 inhibits CDKs and induces apoptosis in multiple myeloma cells via Mcl-1 downregulation.

Inhibitors of cyclin-dependent kinases 9 have been developed as potential anticancer drugs for the treatment of multiple myeloma. We have previously prepared a library of arylazo-3,5-diaminopyrazole inhibitors of CDKs. Here, we describe a novel member, AAP1742 (CDK9 inhibition with IC(50) = 0.28 µm), that reduces the viability of multiple myeloma cell lines in low micromolar concentrations. Consistent with inhibition of CDK9, AAP1742 decreases the phosphorylation of RNA polymerase II and inhibits mRNA synthesis of anti-apoptotic proteins Mcl-1, Bcl-2, and XIAP, followed by apoptosis in the RPMI-8226 cell line in a dose- and a time-dependent manner. These results are consistent with the biochemical profile of AAP1742 and further suggest cellular inhibition of CDK9 as a possible target for anticancer drugs.

Fluorescence properties of selected benzo[c]phenanthridines.

The fluorescence properties of selected benzo[c]phenanthridines (BPs) were examined. The effect of structure, pH and solvent on the fluorescence properties has been investigated. It was found out that the presence of charged iminium nitrogen significantly decreased the fluorescence of the compounds. The fluorescence (intensity as well as emission spectra shape) of the investigated compounds was significantly dependent on pH as well as used solvent. The utilization in epigenetic modification mechanisms studies as demethylase probe and as possible pH indicator was suggested.

The selective P-TEFb inhibitor CAN508 targets angiogenesis.

Small molecule inhibitors of cyclin-dependent kinases (CDK) have been developed as anticancer drugs with cytostatic and cytotoxic properties, but some of them have also been shown to limit angiogenesis. Here, we report that the 3,5-diaminopyrazole CAN508 inhibits endothelial cell migration and tube formation. In addition, it reduces phosphorylation of the C-terminus of RNA polymerase II and inhibits mRNA synthesis in endothelial cells, in accordance with previous observations that it has high selectivity towards the positive transcriptional regulator P-TEFb. Moreover, CAN508 reduces expression of vascular endothelial growth factor by several human cancer cell lines. The findings suggest that P-TEFb may be an attractive target for anti-angiogenic therapy.

Identification of benzo[c]phenanthridine metabolites in human hepatocytes by liquid chromatography with electrospray ion-trap and quadrupole time-of-flight mass spectrometry.

The metabolism of the benzo[c]phenanthridine alkaloids was studied using human hepatocytes which are an excellent model system for biotransformation studies. For analysis of the alkaloids and their metabolites, an electrospray quadrupole ion-trap mass spectrometry (ESI ion-trap MS) connected to a reversed phase chromatographic system based on cyanopropyl modified silica was used. The optimized experimental protocol allowed simultaneous analysis of the alkaloids and their metabolites and enabled study of their uptake into and interconversion in human hepatocytes. The results show that formation of the dihydro metabolite which may be followed by specific O-demethylenation/O-demethylation processes, is probably the main route of biotransformation (detoxification) of the benzo[c]phenanthridines in human hepatocytes. The structure of the main O-demethyl metabolite (2-methoxy-12-methyl-12,13-dihydro-[1,3]dioxolo[4',5':4,5]benzo[1,2-c]phenanthridin-1-ol; 336.1 m/z,) was proposed by the multi-stage MS and quadrupole time-of-flight MS methods using chemically synthesized standard.

4-arylazo-3,5-diamino-1H-pyrazole CDK inhibitors: SAR study, crystal structure in complex with CDK2, selectivity, and cellular effects.

In a routine screening of our small-molecule compound collection we recently identified 4-arylazo-3,5-diamino-1H-pyrazoles as a novel group of ATP antagonists with moderate potency against CDK2-cyclin E. A preliminary SAR study based on 35 analogues suggests ways in which the pharmacophore could be further optimized, for example, via substitutions in the 4-aryl ring. Enzyme kinetics studies with the lead compound and X-ray crystallography of an inhibitor-CDK2 complex demonstrated that its mode of inhibition is competitive. Functional kinase assays confirmed the selectivity toward CDKs, with a preference for CDK9-cyclin T1. The most potent inhibitor, 4-[(3,5-diamino-1H-pyrazol-4-yl)diazenyl]phenol 31b (CAN508), reduced the frequency of S-phase cells of the cancer cell line HT-29 in antiproliferation assays. Further observed cellular effects included decreased phosphorylation of the retinoblastoma protein and the C-terminal domain of RNA polymerase II, inhibition of mRNA synthesis, and induction of the tumor suppressor protein p53, all of which are consistent with inhibition of CDK9.