SYNTHESIS AND EVALUATION OF INDOLEAMINES AND INDOLEAMIDES AS ANTI-CANCER AGENTS TARGETING ISOPRENYLCYSTEINE CARBOXYL METHYLTRANSFERASE (ICMT)

Ph.DDOCTOR OF PHILOSOPH

Fragment-based whole cell screen delivers hits against M. tuberculosis and non-tuberculous mycobacteria

Reactive multi-target ‘fragment drugs’ represent critical components of current tuberculosis regimens. These compounds, such as pyrazinamide, are old synthetic antimycobacterials that are activated inside Mycobacterium tuberculosis bacilli and are smaller than the usual drug-like, single-target molecules. Based on the success of small ‘dirty’ drugs in the chemotherapy of tuberculosis, we suggested previously that fragment-based whole cell screens should be introduced in our current antimycobacterial drug discovery efforts. Here, we carried out such a screen and characterized bactericidal activity, selectivity and spectrum of hits we obtained. A library of 1725 fragments was tested at a single concentration for growth inhibitory activity against M. bovis BCG as screening strain and 38 of 116 primary hits were confirmed in dose response analyses to be active against virulent M. tuberculosis. Bacterial kill experiments showed that most hits displayed bactericidal activity at their minimal inhibitory concentration. Cytotoxicity assays established that a large proportion of hits displayed a favorable selectivity index for mammalian cells. Importantly, one third of M. tuberculosis active fragments were also active against M. abscessus and M. avium, two emerging non-tuberculous mycobacterial pathogens, opening the opportunity to develop broad spectrum antimycobacterials. Activity determination against Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli, Klebsiella pneumonia, Acinetobacter baumanii, Pseudomonas aeruginosa) bacteria, as well as fungi (Candida albicans, Cryptococcus neoformans) showed only a small overlap indicating a generally narrow spectrum of these novel antimicrobial hits for mycobacteria. In conclusion, we carried out the first fragment-based whole cell screen against bacteria and identified a substantial number of hits with excellent physicochemical properties and dual activity against M. tuberculosis and non-tuberculous mycobacterial pathogens. These hits will now be evaluated in animal models of mycobacterial infection to determine whether any of them can be moved forward as a new antimycobacterial fragment drug candidate

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

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. (C) 2019 Elsevier Masson SAS. All rights reserved

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, <b>51</b>, 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[<i>d</i>][1,3,2]oxazaphospholes with Improved Antiproliferative Activity

Hydrogen sulfide (H₂S) is now recognized as a physiologically important gasotransmitter. Compounds which release H₂S <i>slowly</i> are sought after for their potential in therapy. Herein the synthesis of a series of phosphordithioates based on <b>1</b> (GYY4137) are described. Their H₂S release profiles are characterized using 2,6-dansyl azide (<b>2</b>), 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, <b>14</b>, with 10-fold greater anticancer activity than <b>1</b>, was shown to release H₂S in MCF7 cells using a cell active probe, <b>21</b>. Both permeability and intracellular pH (pHi) were found to be significantly improved for <b>14</b> compared to <b>1</b>. Furthermore, <b>14</b> 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[<i>d</i>]­[1,3,2]­oxazaphospholes, of which the simplest member, compound <b>22</b> (FW1256), was significantly more potent in cells. The improved therapeutic window of <b>22</b> in WI38 cells was compared with three other cell types. Potency of <b>22</b> was superior to <b>1</b> 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

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 (<b>1</b>), a marketed JAK1/2 inhibitor, have been merged with the HDAC inhibitor vorinostat (<b>2</b>), leading to new molecules that are bispecific targeted JAK/HDAC inhibitors. A preferred pyrazole substituted pyrrolopyrimidine, <b>24</b>, 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 <b>24</b> is supported by demonstration of JAK-STAT and HDAC pathway blockade in hematological cell lines. Methyl analogue <b>45</b> 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