Design, synthesis and antitrypanosomal activities of 2,6-disubstituted-4,5,7-Trifluorobenzothiophenes

Current treatments for Human African Trypanosomiasis (HAT) are limited in their application, have undesirable dosing regimens and unsatisfactory toxicities highlighting the need for the development of a safer drug pipeline. Our medicinal chemistry programme in developing rapidly accessible and modifiable heterocyclic scaffolds led to the design and synthesis of novel substituted benzothiophenes, with 6-benzimidazol-1-ylbenzothiophene derivatives demonstrating significant antitrypanosomal activities (IC50 <1 μM) against Trypanosoma brucei rhodesiense and no toxicity towards mammalian cells

Novel fluorinated benzimidazole-based scaffolds and their anticancer activity in vitro

A small library of twelve, structurally diverse, fluoroaryl benzimidazoles was prepared using a simple synthetic strategy employing SNAr reactions. This allowed rapid assembly of heterocyclic structures containing linked and tethered fluoroaryl benzimidazoles. X-ray crystal structures of seven compounds were obtained including those of two macrocyclic compounds containing 21- and 24-membered rings. Three tethered fluoroaryl benzimidazole derivatives demonstrated micromolar inhibition against K-562 and MCF-7 cell lines. These compounds, in addition to 1-tetrafluoropyrid-4-yl-2-tetrafluoropyrid-4-ylsulfanyl-1H-benzimidazole, also demonstrated micromolar inhibition against G361 and HOS cell lines. Two of the compounds were found to activate caspases leading to apoptosis

Inhibition of CDK9 but not cell cycle dependent CDKs increases neutrophil apoptosis.

<p>(a) Isolated human neutrophils were incubated with NU6102 (filled triangle), R-roscovitine (filled square), or LGR1406 (filled diamond) at the concentrations shown. Apoptosis was determined after 15h by assessment of DiOC₆ uptake and flow cytometry, with DiOC₆ low cells taken as apoptotic. Data are mean ± s.d. for four separate experiments. * indicates p<0.05 for treatment versus control. (b) Neutrophils were incubated with flavopiridol for 15 h at the concentrations shown and apoptosis determined by assessment of DiOC₆ uptake. Data are mean ± s.d. for three separate experiments. * indicates p<0.05 for control versus flavopiridol treated cells. (c) Images of Giemsa stained neutrophils cultured in the absence (control) or presence of 100 nM flavopiridol for 15 h.</p

Human neutrophils express only cell cycle independent CDKs.

<p>Isolated human neutrophils (N) and promyelocytic HL60 cells (H) were assessed for expression of CDK proteins by western blotting (upper panel). Blots were scanned to determine the CDK:β-actin ratio by densitometry (lower panel; black bars  =  HL60, grey bars  =  neutrophils). Data are mean ± s.d. for three separate cell extracts.</p

Flavopiridol inhibits upregulation of Mcl-1 by GM-CSF.

<p>(a). Neutrophils were incubated with medium alone, or medium containing 100 nM flavopiridol in the absence (open bars) or presence of (filled bars) of 50 ng/ml GM-CSF. Live cells were detected by their ability to retain DiOC₆ and data are mean ± s.d. of 3 separate experiments. * indicates p<0.05 for control versus flavopiridol treated cells. (b). The same cells were extracted and measured for Mcl-1 content by western blotting. Blots were probed for β-actin to confirm equal loading.</p

CDK9 activity and cyclin T1 expression decrease as neutrophils age in culture.

<p>Isolated human neutrophils were cultured for 9h and (a) CDK9 protein expression and (b) enzymatic activity were determined and compared with freshly isolated cells (0h). For CDK9 activity protein was immunoprecipitated from neutrophils with a monoclonal anti-CDK9 antibody and activity determined by incorporation of [γ ³²P]-ATP into the CTD of RNA polymerase II. The phosphorylated peptide was isolated on an SDS-PAGE gel and excised for scintillation counting. Data are expressed as % of the value for control freshly isolated cells and are mean ± s.d. for three separate experiments. * indicates p<0.05. (c). Isolated neutrophils were cultured for 0 h or 9 h and expression of cyclin T1 determined by western blotting. Blots were probed with an antibody to β-actin to confirm equal protein loading. The blot shown is representative of 4 separate experiments performed. (d) Western blots of CDK9 expression relative to β-actin were quantitated using densitometry.</p

Cdk5 is expressed in HUVECs and HMEC-1 cells, and the compounds are not cytotoxic to endothelial cells at concentrations used in functional assays.

<p>A: Cdk5 protein expression in endothelial cells in comparison to human cortex. Cdk5 protein amount was analyzed by Western blot in samples of human cortex (HC), confluent HUVECs (HU) and HMEC-1 (HM). β-actin served as a loading control and for normalization of protein amount. Relative quantification (left panel) and one representative image (right panel) of three individual blots are shown. Note the much lower protein loading in the HC sample in the right panel. (n = 3, mean ± SEM, p>0.05, One Way ANOVA, Dunnett). B: Confluent HUVECs were treated for 16 h with 10 or 30 µM of the indicated compounds or left untreated as control. After addition of CellTiter-BlueTM Reagent, cells were incubated for 4 h and fluorescence was measured at 560 nm (n = 3, mean ± SEM, * p<0.05, One Way ANOVA, Dunnett).</p

The experimental setup:

<p>1. Total RNA was extracted. 2. Reverse transcription was performed in two different setups i) using random hexamers to collect all possible types of RNA transcripts and ii) using oligo dT primers to gain polyadenylated types of RNA transcripts. 3) Real-time PCR with primers designed to specifically recognize N- and C-terminus of β-galactosidase cDNA was used to amplify sequences at both 5′-end (PCR-1) and 3′-end (PCR-2) of β-galactosidase RNA transcripts.</p

Cdk inhibition profile of LGR 1406 and 1407.

<p>The numbers given are IC₅₀ values (molar). Both compounds show an increased selectivity for Cdk2 and Cdk5.</p

LGR 1404, 1406 and 1407 decrease chemotaxis of endothelial cells.

<p>Chemotaxis of HUVECs in the presence or absence of 10 µM of the indicated compounds was determined in µ-slides Chemotaxis. A: Quantitative evaluation of accumulated and euclidean distance, velocity and y-forward migration index (n = 3, mean ± SEM, * p<0.05, One Way ANOVA, Dunnett). B: Representative cell tracking plots of untreated and treated cells.</p