Potent, Selective, and CNS-Penetrant Tetrasubstituted Cyclopropane Class IIa Histone Deacetylase (HDAC) Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.5b00302

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.

Full text of DOI:10.1021/acsmedchemlett.5b00302

Subscriber access provided by UNIV OF SOUTHERN QUEENSLAND
Letter
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
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Medicinal Chemistry Letters is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Potent, selective and CNS-penetrant tetrasubstituted cyclopropane
class IIa histone deacetylase (HDAC) inhibitors
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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; cyclopropa-
nation, Huntington ’s disease.
ABSTRACT: Potent and selective class IIa HDAC tetrasubstituted cyclopropane hydroxamic acid inhibitors were identi-
fied with high oral bioavailability that exhibited good brain and muscle exposure. Compound 14 displayed suitable prop-
erties for assessment of the impact of class IIa HDAC catalytic site inhibition in preclinical disease models.
Inhibition of class IIa HDAC enzymes has been sug-
gested as a therapeutic strategy for a number of indica-
tions, including Huntington’s disease (HD) and muscular
atrophy. Class IIa HDACs are large proteins with multiple
functions including transcription factor binding and N-
acetyl lysine recognition.[1,2] Of most interest to our labor-
atory is the role of class IIa HDAC biology in HD, in par-
ticular the beneficial effect which has been observed fol-
lowing 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 ra-
tionale for small molecule therapy. Currently there are no
marketed HDAC class IIa-selective inhibitors, whereas
four pan-HDAC inhibitors, vorinostat (SAHA), romidep-
sin, 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 Boc-
Lys(Ac)-AMC (class I/IIb-specific).[6-10] The majority of
class IIa HDAC activity in the Jurkat E6.1 cell line is de-
rived from HDAC4.[6] Biochemical HDAC isoform selec-
tivity 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)
with trisubstituted cyclopropane 13 (4CBT)[6] 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 struc-
ture-based design of trisubstituted cyclopropane class IIa-
selective 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 cyclo-
propane hydroxamic acid class IIa HDAC inhibitors, with
additional substitution at C1 (Figure 1). These com-
pounds exhibited improved pharmacokinetic profiles, and
so may provide a further means for evaluating efficacy in
preclinical in vivo HD disease models.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 8
In the trisubstituted cyclopropane series reported pre- curonidation would be their principal route of clearance,
1
2
3
4
5
6
7
8
viously, 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 cap-
ping 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 fluori-
nation 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 trisubsti-
tuted 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.
presumably via O-glucuronidation of the hydroxamic ac-
id, 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 glu-
curonidation process. Some of the previously reported
trisubstituted cyclopropanes had demonstrated high pas-
sive permeability and low P-gp efflux in MDR1-MDCK
monolayers. We anticipated that reduced clearance,
whilst maintaining high passive permeability and low P-
gp efflux, would lead to higher plasma and CNS expo-
sures. The C1-substituent on the cyclopropane ring was
selected to maintain the favorable CNS-compliant physi-
cochemical properties of the compounds.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Trisubstituted cyclopropane hydroxamic acids previ-
ously profiled in mouse hepatocytes displayed high in-
trinsic clearance values.[6] It was assumed that direct glu-
Table 1. Impact of cyclopropane substitution alpha to the hydroxamic acid upon potency and in vitro ADME
properties.
Clint
(mL/min/kg
body weight)b
Kinetic
MDCK-MDR1
Cell
solubili-
HDAC4 IC50
(µM)a
liver micro-
somes
Entry
Lys(TFA)
ty
IC50 (µM)a
(µM)
Papp
(nm/s)
R
X
mouse human
EERc
9
H
Me
Cl
F
0.18 0.09
0.05 0.01
0.11 0.03
0.51 0.16
0.73 0.13
1.15 0.28
170
150
140
160
45
>1297
281
ND
<36
ND
37
375
391
2.0
2.0
ND
2.0
1.4
10
11
ND
570
688
306
73
ND
369
294
418
129
192
380
<10d
114
12
13
14
15
16
17
18
19
0.02±0.006
0.05 0.02
0.01±0.001
0.15 0.02
0.03 0.01
0.14±0.03
0.04±0.006
0.08 0.04
0.13 0.05
0.33 0.08
0.12 0.03
0.55 0.02
0.15 0.04
0.43±0.09
0.23±0.005
0.68 0.20
H
F
42
35
<36
<36
<36
<36
<36
<36
1.4
H
F
120
100
<5
2.2
1.3
<65
<65
73
H
F
2.8
NDd
2.1
<5
H
45
>1297
20
F
0.04±0.009
0.28 0.10
55
258
56
70
2.0
21
H
F
0.03 0.01
0.23 0.06
0.12 0.03
140
65
89
<36
ND
442
487
2.4
1.3
22
0.01±0.001
<67
a Arithmetic mean and standard error of ≥3 measurements.
b
Intrinsic clearance (Clint) values of > 257 mL/min/kg in mouse liver microsomes (MLM) and > 52 mL/min/kg for human liver microsomes
(HLM) indicate a rapid rate of oxidative metabolism. Under the assay conditions used Clint values < 65 mL/min/kg in MLM and < 36 mL/min/kg
in HLM indicate low rate of metabolism by CYP450 enzymes.
c
Based upon previous work in our laboratory, an effective efflux ratio (EER) value of > 4 suggests that a compound is a substrate for P-gp,
whereas values < 2 suggest the compound is unlikely to be a P-gp substrate. For delivery to the CNS, a drug should ideally have an in vitro pas-
sive permeability > 150 nm/s and should not be a good P-gp substrate (B to A/A to B ratio <2.5).[12]
ACS Paragon Plus Environment

Page 3 of 8
ACS Medicinal Chemistry Letters
d Poor A to B permeability in both cell types. Low A to B mass balance.
1
2
3
4
5
6
7
8
9
The effect of C1-substitution on potency was investigat-
ed initially with compounds comprising the 4-methyl
pyrimidin-2-yl capping group (Table 1). Methyl and fluo-
ro substitution at C1 (10 and 12) conferred an improve-
ment in HDAC4 biochemical activity over trisubstituted
cyclopropane 9, whilst the potency values for the chloro
analog 11 and compound 9 were similar. In the cellular
HDAC class IIa assay, fluoro derivative 12 exhibited a sig-
nificant potency enhancement over compounds 9, 10 and
11, with no improvement in cellular activity for methyl
substituted 10 versus 9. All of the 1-fluoro derivatives (12,
14, 16, 18, 20, 22) demonstrated a consistent positive effect
on biochemical and cell activities when compared to the
trisubstituted cyclopropanes (9, 13, 15, 17, 19, 21).
formation). At steady state, the KD for compound 14 was
determined to be 0.011 µM (Table 2), with a Kon of 196800
/M.s and a Koff of 0.0044 /s, i.e. moderate on and off rates.
The corresponding des-fluoro compound 13 displayed
lower affinity, consistent with the biochemical assay, with
a KD value of 0.118 µM, driven by larger Koff of 0.0186 /s
(the Kon value was 190270 /M.s).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Comparison of SPR KD values with bio-
chemical activity values for compounds 13, 14 and 16.
Biochemical
SPR
Entry
HDAC4 IC50
(µM)a
KD (µM)a
0.11 0.01
0.02 0.01
0.08 0.06
13
14
16
0.05 0.02
0.01±0.001
0.03 0.01
The opposite enantiomer of 14, prepared via the syn-
thetic route in Scheme 1 (vide infra), was 40-fold less po-
tent in the HDAC4 biochemical and cell assays (com-
pound 26, see Supporting Information).
The tetrasubstituted cyclopropanes were assessed for in
vitro ADME properties (Table 1). As an indication of oxi-
dative metabolism, compounds 16 and 22 were more sta-
ble in mouse liver microsomes (Clint values of <65 and <67
mL/min/kg BW) than 14 (306 mL/min/kg BW). A mouse
microsomal preparation incubated with compound 14
showed the presence of a metabolite that was assigned as
the 5-hydroxypyrimidin-2-yl derivative, which may, at
least in part, account for the increased microsomal turno-
ver compared to 16 or 22. Compound 18 was moderately
stable in mouse liver microsomes but showed poor solu-
bility and poor passive permeability in MDCK monolay-
ers, whilst 20 also exhibited low passive permeability.
a Arithmetic mean and standard error of ≥3 measurements
.
An X-ray co-crystal structure of 14 with the cdHDAC4
(L728A mutant)[6] was obtained confirming the absolute
configuration and demonstrating the key interactions of
the hydroxamate molecule with the protein binding site.
The binding mode of 14 was similar to that of trisubstitut-
ed cyclopropane 13 (Figure 2B). Comparison with the
class I HDACs (Figure 2A, with SAHA) shows the tyro-
sine residue of the class I HDACs projecting into the re-
gion of the selectivity pocket in the class IIa isoforms
formed by the tyrosine-histidine switch.
In mouse hepatocytes, all compounds exhibited high
intrinsic clearance values (data not shown).
The previously reported trisubstituted cyclopropanes
are selective for the class IIa versus class I and IIb HDAC
isoforms. HDAC isoform biochemical selectivity was in-
vestigated with the Boc-Lys(TFA)-AMC and Boc-Lys(Ac)-
AMC substrates (vide supra). The potency values against
each of the HDAC isoforms for the trisubstituted cyclo-
propane 13 and its 1-fluoro derivative 14 are shown in Ta-
ble 3.
C1-substitution of 9 with either fluorine, chlorine or a
methyl group retained the low P-gp efflux (measured in
MDR1-MDCK cell monolayers) characteristic of the par-
ent trisubstituted cyclopropanes, with moderate to good
passive permeability across wild type MDCK cells. The
methyl analog 10 also displayed some improvement in
mouse microsomal stability.
Compound 14 maintained a good class IIa HDAC selec-
tivity profile, demonstrating, for example, at least 100-fold
selectivity over HDACs 1, 2 and 3.
A direct measure of compound binding to the target
was determined by surface plasmon resonance (SPR)
where the HDAC4 catalytic domain was immobilized via
amine coupling to the sensor surface (see Supporting In-
Figure 2. A. X-ray structure of HDAC2 (class I HDAC) with SAHA (4LXZ).[13] B. Protein structure superposition of
HDAC4cd X-ray structures with compounds 13 and 14 (PDB codes: 4CBT, 5A2S, respectively). C. Enlarged region
of the HDAC4 active site with compound 14. Residue numbering from PDB files.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. HDAC isoform biochemical activity dataa for compounds 13 and 14.
Class IIa (cd)
Class I
Class IIb
HDAC6
1.9 0.4
En-
try
HDAC4
HDAC5
HDAC7
HDAC9
HDAC1
21c
HDAC2
ND
HDAC3b
HDAC8
13
14
0.05 0.02
0.03 0.01
0.12 0.06
0.20 0.09
9.7 2.2
1.4 0.2
0.01±0.00
4
0.01±0.001
0.03 0.01
0.06 0.03
14 4
>50
7.4 2.1
0.28 0.03
3.1 0.4d
a
Inhibition IC50 values quoted in µM. Arithmetic mean and standard error of ≥3 measurements; b HDAC3-NCoR1; c Less than three independ-
d
ent replicates; Determined from a separate assay utilising HDAC6 over-expression in a HEK cell line, due to a lack of availability of active
HDAC6 protein. Lysates from these over-expressing cells demonstrated activity that was HDAC6 expression-dependent with only a small back-
ground signal due to endogenous HDAC activity.
Compounds 14 and 16 were progressed to in vivo PK
studies on account of their high potency, MDCK data, and
kinetic solubility. At the time of writing, compound 22
had not yet been advanced to further studies.
pounds exhibited biphasic elimination profiles and high
oral bioavailability (Table 4). In addition to high volumes
of distribution, these compounds displayed high plasma
clearance, as expected from the mouse hepatocyte data.
Following oral dosing, a broad secondary peak was ob-
served for 14 and 16. This may be a result of slow dissolu-
tion of compound following gavage.
Trisubstituted cyclopropane 13, and fluoro cyclopro-
panes 14 and 16 were dosed intravenously and via oral
gavage in fed male C57Bl/6 mice. Following an oral dose,
all three compounds were rapidly absorbed (Figure 3).
Compound 14 demonstrated a linear increase in expo-
sure with dose in both plasma and brain matrices across
10, 30 and 100 mg/kg oral doses.
Compounds 14 and 16, incorporating fluoro- or difluo-
romethoxy pyrimidine capping group substituents respec-
tively, demonstrated higher distribution to brain tissue
versus the trisubstituted cyclopropane 13. All three com-
Figure 3. Mouse plasma and brain pharmacokinetic profiles for compounds 13, 14 and 16 post oral dose (10
mg/kg).
Table 4. Pharmacokinetic parameters of 13, 14 and 16 following administration to fed male C57Bl/6 mice.
ACS Paragon Plus Environment

Page 5 of 8
ACS Medicinal Chemistry Letters
poa
ivb
1
t
F
AUCnorm
Cmax norm
tmax
(h)
Clp
Vdss
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
½
Entry Compartment
Plasma
(%)
(nM.h.kg/mg) (nM.kg/mg)
(L/h/kg)
(L/kg)
(h)
0.3
ND
ND
2.2
ND
ND
3
~100c
ND
ND
79
110d
830
330
97
570
320
180
780
290
150
120
56
0.5
0.5
0.5
0.25
0.5
0.5
0.25
1
11
4.1
ND
ND
5.1
13
14
16
Brain
Muscle
Plasma
Brain
ND
ND
4.5
480
1900
640
660
580
300
ND
ND
~100
ND
ND
ND
ND
4.4
ND
ND
ND
ND
10
Muscle
Plasma
Brain
ND
ND
ND
ND
Muscle
0.5
a13, 14, & 16 dosed at 10 mg/kg. b13, 14, & 16 dosed at 5 mg/kg, 1 mg/kg and 2.5 mg/kg respectively. cOral bioavailability may be underestimated
for the 10 mg/kg po dose; AUCpo = AUC0-24 h whilst AUCiv was extrapolated to infinity. dCalculated using AUClast.
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
From HDAC4 SPR studies (vide supra) the dissociation
half-life for compound 14 is approximately 160 s, consid-
erably shorter than its in vivo half-life of ~ 2 h. Binding
kinetics are therefore unlikely to impact the duration of
any pharmacodynamic effect of 14.
were estimated using the CSF to brain ratio determined in
rat, assuming this remained constant across species. The-
se gave very similar estimates of concentration over the
time points (at 10 mg/kg po dose the Cmax measured in rat
CSF was at approximately the cell IC50 value).
In the absence of known intracellular concentrations of
compound in the brain, we considered that the best esti-
mate of compound available to interact with the target is
likely to be the unbound fraction of compound that may
be determined either by microdialysis, CSF exposure, or
correction of the total exposure from in vitro equilibrium
dialysis.
Compounds were synthesized according to Scheme 1.
Cyclopropanation of the methyl cinnamate derived from 1
as previously described,[6] followed by deprotonation of
the racemic intermediate 2 using LDA (1.1-3 eq) and
quenching with an appropriate electrophile (MeI, CCl4 or
NFSI)[14] afforded the tetrasubstituted cyclopropane. The
desired isomer was isolated by flash chromatography.
The heterocyclic capping groups were introduced by Su-
zuki coupling and the enantiopure hydroxamic acid
products were obtained from chiral HPLC purification.
Unbound brain concentrations of 14 were estimated by
correcting total brain concentrations for the fraction un-
bound (0.41%) determined in mouse brain homogenate
by equilibrium dialysis. CSF concentrations in mouse
Scheme 1. Synthesis of 1-substituted cyclopropanes and alternative route towards compound 14.
Reagents and conditions: (a) 12-crown-4, LiHMDS, DCM, -20 °C, 1 h; (b) LDA in THF, -78°C, 30 min then MeI or CCl4 or NFSI,
2 h; (c) (i) bis(pinacolato)diboron, KOAc, Pd(dppf)Cl2, dioxane, 90 °C, 4 h; (ii) heteroaryl halide, Pd(dppf)Cl2, CsF, dioxane, 100
°C, 17 h; (d) NH2OH 50% aq, KOH, THF:MeOH (1:1), r.t., 16 h, chiral HPLC; (e) LDA in THF, -78°C, 30 min then NFSI, 2 h, (rac)-5
separated by flash chromatography; (f) SFC chiral chromatography; (g) (i) bis(pinacolato)-diboron, KOAc, Pd(dppf)Cl2, dioxane,
90 °C, 4 h; (ii) 2-chloro-5-fluoropyrimidine, Pd(dppf)Cl2, CsF, dioxane, 100 °C, 17 h; (h) NH2OH 50% aq, KOH, THF:MeOH (1:1),
r.t., 16 h.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 8
SAHA, suberoylanilide hydroxamic acid; cd, catalytic do-
main; ND, not determined; po, per os, oral administration; iv,
intravenous route of administration; CSF, cerebrospinal fluid.
In order to access larger quantities of 1-fluoro cyclopro-
pane 14, a modification of the general route was employed
(Scheme 1, lower branch). Herein, the bromophenyl
fluoro precursor (7) was obtained as the single desired
stereoisomer. After the key electrophilic fluorination re-
action of the cyclopropane ester (rac)-2, a 1:2 diastereo-
meric ratio of epimers (rac)-5 and (rac)-6 respectively was
achieved using THF and LDA (3 eq.) at -78 °C with NFSI
(N-fluorobenzene sulfonimide), on a multi-gram scale.
This resulted in a 25% isolated yield of (rac)-5 following
separation by flash chromatography. The addition of
LiCl, previously reported to increase the yields of 1-
fluorination of cyclopropyl esters,[15] was discovered to
favor further the formation of the undesired diastereoi-
somer (rac)-6. The enantiomerically pure methyl ester 7
was isolated by chiral supercritical fluid chromatography
(SFC). Subsequently the heterocyclic capping group was
introduced by Suzuki coupling, followed by hydroxamic
acid formation to deliver compound 14.
1
2
3
4
5
6
7
8
REFERENCES
(1) Martin, M.; Kettmann, R.; Dequiedt, F. Class IIa histone
deacetylases: regulating the regulators. Oncogene 2007, 26, 5450-
5467.
(2) Bertos, N. R.; Wang, A. H.; Yang, X. J. Class II histone
deacetylases: structure, function, and regulation. Biochem. Cell.
Biol. 2001, 79, 243-252.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(3) Mielcarek, M.; Benn, C. L.; Franklin, S. A.; Smith, D. L.;
Woodman, B.; Marks, P. A.; Bates, G. P. SAHA decreases
HDAC2 and 4 levels in vivo and improves molecular phenotypes
in the R6/2 mouse model of Huntington’s disease. PLoS One
2011, 6, e27746.
(4) Mielcarek, M.; Landles, C.; Weiss, A.; Bradaia, A.;
Seredenina, T.; Inuabasi, L.; Osborne, G. F.; Wadel, K.; Touller,
C.; Butler, R.; Robertson, J.; Franklin, S. A.; Smith, D. L.; Park, L.;
Marks, P. A.; Wanker, E. E.; Olson, E. N.; Luthi-Carter, R.; van
der Putten, H.; Beaumont, V.; Bates, G. P. HDAC4 reduction: a
novel therapeutic strategy to target cytoplasmic huntingtin and
ameliorate neurodegeneration. PLoS Biol. 2013, 11, e1001717.
(5) Mielcarek, M.; Zielonka, D.; Carnemolla, A.; Marcinkowski,
J. T.; Guidez, F. HDAC4 as a potential therapeutic target in neu-
rodegenerative diseases: a summary of recent achievements.
Front. Cell. Neurosci., published online March 23, 2009: DOI:
10.3389/fncel.2015.00042.
(6) Bürli, R. W.; Luckhurst, C. A.; Aziz, O.; Matthews, K. L.;
Yates, D.; Lyons, K. A.; Beconi, M.; McAllister, G.; Breccia, P.;
Stott, A. J.; Penrose, S. D.; Wall, M.; Lamers, M.; Leonard, P.;
Muller, I.; Richardson, C. M.; Jarvis, R.; Stones, L.; Hughes, S.;
Wishart, G.; Haughan, A. F.; O’Connell, C.; Mead, T.; McNeil, H.;
Vann, J.; Mangette, J.; Maillard, M.; Beaumont, V.; Munoz-
Sanjuan, I.; Dominguez, C. Design, synthesis, and biological
evaluation of potent and selective class IIa histone deacetylase
(HDAC) inhibitors as a potential therapy for Huntington’s dis-
ease. J. Med. Chem. 2013, 56, 9934-9954.
(7) Lahm, A.; Paolini, C.; Pallaoro, M.; Nardi, M. C.; Jones, P.;
Neddermann, P.; Sambucini, S.; Bottomley, M. J.; Lo Surdo, P.;
Carfí, A.; Koch, U.; De Francesco, R.; Steinkühler, C.; Gallinari, P.
Unraveling the hidden catalytic activity of vertebrate class IIa
histone deacetylases. Proc. Natl. Acad. Sci. U.SA. 2007, 104, 17335-
17340.
In conclusion, we have discovered potent and selective
class IIa HDAC hydroxamic acid inhibitors comprising a
tetrasubstituted cyclopropane scaffold. Compounds such
as 14 displayed high oral bioavailability with good brain
and muscle exposure. Such compounds exhibit suitable
properties for assessment of the impact of class IIa HDAC
catalytic site inhibition in preclinical HD disease models.
ASSOCIATED CONTENT
Supporting Information. The Supporting Information is
available free of charge on the ACS Publications website at
DOI: XXXXXXX.
Additional compound data; synthesis procedures and charac-
terization of compounds, quantification of compound
binding to HDAC4cd using SPR; X-ray crystallography data
collection and refinement statistics; rat pharmacokinetics.
AUTHOR INFORMATION
Corresponding Author
*E-mail: Celia.Dominguez@CHDIFoundation.org. Phone:
+1 310-342-5503.
(8) Bottomley, M J ; Lo Surdo, P.; Di Giovine, P.; Cirillo, A.;
Scarpelli, R.; Ferrigno, F.; Jones, P.; Neddermann, P.; De Frances-
co, R.; Steinkühler, C.; Gallinari, P.; Carfí, A. Structural and
functional analysis of the human HDAC4 catalytic domain re-
veals a regulatory structural zinc-binding domain. J. Biol. Chem.
2008, 283, 26694-26704.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
(9) Beconi, M.; Aziz, O.; Matthews, K.; Moumne, L.; O'Con-
nell, C.; Yates, D.; Clifton, S.; Pett, H.; Vann, J.; Crowley, L.;
Haughan, A. F.; Smith, D. L.; Woodman, B.; Bates, G. P.;
Brookfield, F.; Bürli, R. W.; McAllister, G.; Dominguez, C.;
Munoz-Sanjuan, I.; Beaumont, V. Oral administration of the
pimelic diphenylamide HDAC inhibitor HDACi 4b is unsuitable
for chronic inhibition of HDAC activity in the CNS in vivo. PLoS
One 2012, 7, e44498.
(10) Bradner, J. E.; West, N.; Grachan, M. L.; Greenberg, E. F.;
Haggarty, S. J.; Warnow, T.; Mazitschek, R. Chemical phyloge-
netics of histone deacetylases. Nat. Chem. Biol. 2010, 6, 238-243.
(11) Bradner, J. E. Fluorinated HDAC inhibitors and uses
thereof, WO2011084991.
Notes
The authors declare no conflicts of interest.
ACKNOWLEDGMENTS
The authors thank Vahri Beaumont, Ignacio Munoz-Sanjuan,
and Michel Maillard (CHDI) for helpful discussions. The
authors also express their gratitude to the analytical chemis-
try and purification team (BioFocus), including Mark Sandle,
Nicholas Richards and Paul Bond, and also Tania Mead and
Catherine O’Connell for ADME assays.
ABBREVIATIONS
(12) Mahar Doan, K. M.; Humphreys, J. E.; Webster, L. O.;
Wring, S. A.; Shampine, L. J.; Serabjit-Singh, C. J.; Adkinson, K.
K.; Polli, J. W. Passive permeability and P-glycoprotein-
ACS Paragon Plus Environment

Page 7 of 8
ACS Medicinal Chemistry Letters
mediated efflux differentiate central nervous system (CNS) and
phenylcyclopropanecarboxylic acids: configurational stability
1
2
3
4
5
6
7
8
non-CNS marketed drugs. J. Pharm. Exp. Ther. 2002, 303, 1029-
1037.
and reaction with electrophiles. Tetrahedron 1984, 40, 5177-5180.
(15) Zhou, S.; Kern, E. R.; Gullen, E.; Cheng, Y-C.; Drach, J. C.;
Matsumi, S.; Mitsuya, H.; Zemlicka, J. (Z)- and (E)-[2-Fluoro-2-
(hydroxymethyl)cyclopropylidene]methylpurines and pyrim-
idines, a new class of methylenecyclopropane analogs of nucleo-
sides: synthesis and antiviral activity. J. Med. Chem. 2004, 47,
6964-6972.
(13) Lauffer, B. E.; Mintzer, R.; Fong, R.; Mukund, S.; Tam, C.;
Zilberleyb, I.; Flicke, B.; Ritscher, A.; Fedorowicz, G.; Vallero, R.;
Ortwine, D. F.; Gunzner, J.; Modrusan, Z.; Neumann, L.; Koth, C.
M.; Lupardus, P. J.; Kaminker, J. S.; Heise, C. E.; Steiner, P. His-
tone deacetylase (HDAC) inhibitor kinetic rate constants corre-
late with cellular histone acetylation but not transcription and
cell viability. J. Biol. Chem. 2013, 288, 26926-26943.
9
(14) Feit, B. A.; Elser, R.; Melamed, U.; Goldberg, I. Cyclopro-
pyl
carbanions
derived
from
esters
of
2-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 8 of 8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
157x79mm (150 x 150 DPI)
ACS Paragon Plus Environment