Discovery of a potent histone deacetylase (HDAC) 3/6 selective dual inhibitor.

Herein, we report the discovery of a dual histone deacetylase inhibitor displaying a unique HDAC3/6 selectivity profile. An initial strategy to merge two epigenetic pharmacophores resulted in the discovery of potent HDAC6 inhibitors with selectivity over HDAC1. Screening in an HDAC panel revealed additional low nanomolar inhibition only against HDAC3. Low micromolar antiproliferative activities against two breast cancer and four hematological cancer cell lines was supported by pharmacodynamic studies on a preferred molecule, 24c, substantiating the HDAC inhibitory profile in cells. Apoptosis was identified as one of the main cell death pathways. Modelling studies of 24c against HDAC1,2,3 and 6 further provided insights on the orientation of specific residues relevant to compound potency, explaining the observed HDAC3/6 selectivity. A subset of the compounds also exhibited good antimalarial activities, particularly against the chloroquine-resistant strain K1 of P.falciparum. In vitro studies revealed a favourable DMPK profile warranting further investigation of the therapeutic potential of these compounds.

Merging of ruxolitinib and vorinostat leads to highly potent inhibitors of JAK2 and histone deacetylase 6 (HDAC6).

Inhibition of more than one pathway in a cancer cell with a single molecule could result in better therapies with less complex dosing regimens. In this work multi-component ligands have been prepared by joining together key pharmacophores of two different enzyme inhibitors in a way which increases potency against the individual pathways. Selective JAK1/2 inhibitor, ruxolitinib (3), and pan-HDAC inhibitor vorinostat (4) were linked together by a single nitrogen atom to create a new series of compounds with very potent JAK2 and HDAC6 inhibition with selectivity against HDAC1. A preferred compound, 13b, had unprecedented sub-nanomolar JAK2 potency with an IC50 of 41 pM and a sub-nanomolar IC50 against HDAC6 of 200 pM. Binding models show a good fit into both JAK2 and HDAC6.

Design and Synthesis of Ligand Efficient Dual Inhibitors of Janus Kinase (JAK) and Histone Deacetylase (HDAC) Based on Ruxolitinib and Vorinostat.

Concomitant inhibition of multiple oncogenic pathways is a desirable goal in cancer therapy. To achieve such an outcome with a single molecule would simplify treatment regimes. Herein the core features of ruxolitinib (1), a marketed JAK1/2 inhibitor, have been merged with the HDAC inhibitor vorinostat (2), leading to new molecules that are bispecific targeted JAK/HDAC inhibitors. A preferred pyrazole substituted pyrrolopyrimidine, 24, inhibits JAK1 and HDACs 1, 2, 3, 6, and 10 with IC50 values of less than 20 nM, is <100 nM potent against JAK2 and HDAC11, and is selective for the JAK family against a panel of 97 kinases. Broad cellular antiproliferative potency of 24 is supported by demonstration of JAK-STAT and HDAC pathway blockade in hematological cell lines. Methyl analogue 45 has an even more selective profile. This study provides new leads for assessment of JAK and HDAC pathway dual inhibiton achieved with a single molecule.

Discovery of medium ring thiophosphorus based heterocycles as antiproliferative agents.

Hydrogen sulfide (H2S) has been investigated for its potential in therapy. Recently, we reported novel H2S donor molecules based on a thiophosphorus core, which slowly release H2S and have improved anti-proliferative activity in cancer cell lines compared to the most widely studied H2S donor GYY4137 (1). Herein, we have prepared new thiophosphorus organic H2S donors with different ring sizes and evaluated them in two solid tumor cell lines and one normal cell line. A seven membered ring compound, 17, was found to be the most potent with sub-micromolar IC50s in breast (0.76µM) and ovarian (0.76µM) cancer cell lines. No significant H2S release was detected in aqueous solution for this compound. However, confocal imaging showed that H2S was released from 17 inside cells at a similar level to the widely studied H2S donor GYY4137, which was shown to release 10µM H2S after 12h at a concentration of 400µM. Comparison of 17 with its non-sulfur oxygen analogue, 26, provided evidence that the sulfur atom is important for its potency. However, the significant potency observed for 26 (5.94-11.0µM) indicates that the high potency of 17 is not entirely due to release of H2S. Additional mechanism(s) appear to be responsible for the observed activity, hence more detailed studies are required to better understand the role of H2S in cancer with potent thiophosphorus agents.

Design and Synthesis of Janus Kinase 2 (JAK2) and Histone Deacetlyase (HDAC) Bispecific Inhibitors Based on Pacritinib and Evidence of Dual Pathway Inhibition in Hematological Cell Lines.

Blockage of more than one oncoprotein or pathway is now a standard approach in modern cancer therapy. Multiple inhibition is typically achieved with two or more drugs. Herein, we describe a pharmacophore merging strategy combining the JAK2/FLT3 inhibitor pacritnib with the pan-HDAC inhibitor, vorinostat, to create bispecific single molecules with both JAK and HDAC targeted inhibition. A preferred ether hydroxamate, 51, inhibits JAK2 and HDAC6 with low nanomolar potency, is <100 nM potent against HDACs 2 and 10, submicromolar potent against HDACs 1, 8, and 11, and >50-fold selective for JAK2 in a panel of 97 kinases. Broad cellular antiproliferative potency is supported by demonstration of JAK-STAT and HDAC pathway blockade in several hematological cell lines, inhibition of colony formation in HEL cells, and analysis of apoptosis. This study provides new tool compounds for further exploration of dual JAK-HDAC pathway inhibiton achieved with a single molecule.

Discovery of New H2S Releasing Phosphordithioates and 2,3-Dihydro-2-phenyl-2-sulfanylenebenzo[d][1,3,2]oxazaphospholes with Improved Antiproliferative Activity.

Hydrogen sulfide (H2S) is now recognized as a physiologically important gasotransmitter. Compounds which release H2S slowly are sought after for their potential in therapy. Herein the synthesis of a series of phosphordithioates based on 1 (GYY4137) are described. Their H2S release profiles are characterized using 2,6-dansyl azide (2), an H2S specific fluorescent probe. Most compounds have anticancer activity in several solid tumor cell lines and are less toxic in a normal human lung fibroblast, WI38. A preferred compound, 14, with 10-fold greater anticancer activity than 1, was shown to release H2S in MCF7 cells using a cell active probe, 21. Both permeability and intracellular pH (pHi) were found to be significantly improved for 14 compared to 1. Furthermore, 14 was also negative in the AMES test for genotoxicity. Cyclization of these initial structures gave a series of 2,3-dihydro-2-phenyl-2-sulfanylenebenzo[d][1,3,2]oxazaphospholes, of which the simplest member, compound 22 (FW1256), was significantly more potent in cells. The improved therapeutic window of 22 in WI38 cells was compared with three other cell types. Potency of 22 was superior to 1 in MCF7 tumor spheroids and the mechanism of cell death was shown to be via apoptosis with an increase in cleaved PARP and activated caspase-7. Evidence of H2S release in cells is also presented. This work provides a "toolbox" of slow-release H2S donors useful for studies of H2S in biology and as potential therapeutics in cancer, inflammation, and cardiovascular disease.

Thieno[3,2-d]pyrimidin-4(3H)-one derivatives as PDK1 inhibitors discovered by fragment-based screening.

Ligand efficient fragments binding to PDK1 were identified by an NMR fragment-based screening approach. Computational modeling of the fragments bound to the active site led to the design and synthesis of a series of novel 6,7-disubstituted thienopyrimidin-4-one compounds, with low micromolar inhibitory activity against PDK1 in a biochemical enzyme assay.