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Potent, selective and CNS-penetrant tetrasubstituted cyclopropane class IIa histone deacetylase (HDAC) inhibitors Christopher Andrew Luckhurst, Perla Breccia, Andrew J Stott, Omar Aziz, Helen L Birch, Roland Burli, Samantha J Hughes, Rebecca E Jarvis, Marieke Lamers, Philip M Leonard, Kim L Matthews, George McAllister, Scott Pollack, Elizabeth Saville-Stones, Grant Wishart, Dawn Yates, and Celia Dominguez ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.5b00302 • Publication Date (Web): 10 Dec 2015 Downloaded from http://pubs.acs.org on December 17, 2015
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ACS Medicinal Chemistry Letters
Potent, selective and CNS-penetrant tetrasubstituted cyclopropane class IIa histone deacetylase (HDAC) inhibitors Christopher A. Luckhurst;† Perla Breccia;† Andrew J. Stott;† Omar Aziz;† Helen L. Birch;† Roland W. Bürli;† Samantha J. Hughes;† Rebecca E. Jarvis;† Marieke Lamers;† Philip M. Leonard;† Kim L. Matthews;† George McAllister;† Scott Pollack;† Elizabeth Saville-Stones;† Grant Wishart;† Dawn Yates;† Celia Dominguez.*,‡ †BioFocus, Charles River, Chesterford Research Park, Saffron Walden, Essex, CB10 1XL, United Kingdom ‡CHDI Management/CHDI Foundation Inc., 6080 Center Drive, Suite 100, Los Angeles, California 90045, United States KEYWORDS: Class IIa HDAC inhibitors; hydroxamic acid; CNS exposure; tetrasubstituted cyclopropane; cyclopropanation, Huntington ’s disease.
ABSTRACT: Potent and selective class IIa HDAC tetrasubstituted cyclopropane hydroxamic acid inhibitors were identified with high oral bioavailability that exhibited good brain and muscle exposure. Compound 14 displayed suitable properties for assessment of the impact of class IIa HDAC catalytic site inhibition in preclinical disease models.
Inhibition of class IIa HDAC enzymes has been suggested as a therapeutic strategy for a number of indications, including Huntington’s disease (HD) and muscular atrophy. Class IIa HDACs are large proteins with multiple functions including transcription factor binding and Nacetyl lysine recognition.[1,2] Of most interest to our laboratory is the role of class IIa HDAC biology in HD, in particular the beneficial effect which has been observed following HDAC4 genetic suppression.[3-5] Replication of these effects in preclinical models of HD via occupancy of the class IIa HDAC catalytic domain would provide a rationale for small molecule therapy. Currently there are no marketed HDAC class IIa-selective inhibitors, whereas four pan-HDAC inhibitors, vorinostat (SAHA), romidepsin, belinostat and panobinostat are on the market.
Determination of the biochemical and Jurkat E6.1 cell potencies of compounds against the HDAC isoforms has been described previously, employing artificial substrates Boc-Lys(TFA)-AMC (class IIa/HDAC8-specific) and BocLys(Ac)-AMC (class I/IIb-specific).[6-10] The majority of class IIa HDAC activity in the Jurkat E6.1 cell line is derived from HDAC4.[6] Biochemical HDAC isoform selectivity was compared to the difference in activity in the cell assays between the Lys-Ac (class I/IIb-specific) and the Lys-TFA (class IIa/HDAC8-specific) substrates. Figure 1. X-ray structure of HDAC4cd (class IIa HDAC) [6] with trisubstituted cyclopropane 13 (4CBT) showing the presence of a selectivity pocket due to a Tyr-His substitution. Residue numbering from PDB files.
Class IIa-selective HDAC inhibitors would represent important tools for elucidating the therapeutic potential of this protein family. We recently reported the structure-based design of trisubstituted cyclopropane class IIaselective HDAC inhibitors as potential therapeutics in HD.[6] This improved selectivity was driven by exploiting a selectivity pocket (Figure 1, shown with compound 13) that is not present in the class I HDAC isoforms. This pocket is formed as a consequence of a tyrosine-histidine substitution.[7] We now report the discovery of tetrasubstituted cyclopropane hydroxamic acid class IIa HDAC inhibitors, with additional substitution at C1 (Figure 1). These compounds exhibited improved pharmacokinetic profiles, and so may provide a further means for evaluating efficacy in preclinical in vivo HD disease models.
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In the trisubstituted cyclopropane series reported previously, the most potent compounds in the biochemical and cell assays, albeit with a significant drop-off in cell potency, had comprised either pyrimidine or oxazole capping groups.[6] Further improvements in biochemical potency and pharmacokinetics were targeted. The design of these molecules was inspired by the expectation that a fluorine atom alpha to the hydroxamic acids may increase their acidity. It has previously been proposed that fluorination of known HDAC inhibitors at the carbon bearing the hydroxamic acid moiety enhanced HDAC biochemical activity.[11] Analysis of the X-ray structures of the trisubstituted cyclopropanes, and docking studies for the new tetrasubstituted cores indicated that there was sufficient space for small substituents to be accommodated at the C1-cyclopropane position. Trisubstituted cyclopropane hydroxamic acids previously profiled in mouse hepatocytes displayed high intrinsic clearance values.[6] It was assumed that direct glu-
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curonidation would be their principal route of clearance, presumably via O-glucuronidation of the hydroxamic acid, and that the contribution of oxidative metabolism to the overall clearance of these molecules would be small. Compounds with very short half-lives (< 5 min) in mouse liver microsomes were not progressed. Substitution of the cyclopropane carbon atom bearing the hydroxamic acid moiety (C1), with either a methyl group or a halogen might limit, either via steric or electronic means, the glucuronidation process. Some of the previously reported trisubstituted cyclopropanes had demonstrated high passive permeability and low P-gp efflux in MDR1-MDCK monolayers. We anticipated that reduced clearance, whilst maintaining high passive permeability and low Pgp efflux, would lead to higher plasma and CNS exposures. The C1-substituent on the cyclopropane ring was selected to maintain the favorable CNS-compliant physicochemical properties of the compounds.
Table 1. Impact of cyclopropane substitution alpha to the hydroxamic acid upon potency and in vitro ADME properties.
HDAC4 IC50 a (µM)
Entry R
a
Cell Lys(TFA) a IC50 (µM)
Clint (mL/min/kg b body weight)
Kinetic solubility
liver microsomes
(µM)
X
MDCK-MDR1
mouse
human
Papp (nm/s)
EER
c
9
H
0.18±0.09
0.51±0.16
170
>1297
ND
375
2.0
10
Me
0.05±0.01
0.73±0.13
150
281
