Optimisation of a triazolopyridine based histone demethylase inhibitor yields a potent and selective KDM2A (FBXL11) inhibitor

Article abstract of DOI:10.1039/c4md00291a

A potent inhibitor of the JmjC histone lysine demethylase KDM2A (compound 35, pIC₅₀ 7.2) with excellent selectivity over representatives from other KDM subfamilies has been developed; the discovery that a triazolopyridine compound binds to the active site of JmjC KDMs was followed by optimisation of the triazole substituent for KDM2A inhibition and selectivity. This journal is

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Optimisation of a triazolopyridine based histone
demethylase inhibitor yields a potent and selective
KDM2A (FBXL11) inhibitor†
Cite this: Med. Chem. Commun., 2014,
5, 1879
Katherine S. England,‡ab Anthony Tumber,‡ac Tobias Krojer,a Giuseppe Scozzafava,ac
Stanley S. Ng,a Michelle Daniel,a Aleksandra Szykowska,a KaHing Che,a Frank von
Delft,ad Nicola A. Burgess-Brown,a Akane Kawamura,be Christopher J. Schofieldb
ac
*
and Paul E. Brennan
Received 4th July 2014
Accepted 14th September 2014
A potent inhibitor of the JmjC histone lysine demethylase KDM2A (compound 35, pIC50 7.2) with excellent
selectivity over representatives from other KDM subfamilies has been developed; the discovery that a
triazolopyridine compound binds to the active site of JmjC KDMs was followed by optimisation of the
triazole substituent for KDM2A inhibition and selectivity.
DOI: 10.1039/c4md00291a
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residues on the N-terminal tails of histone H3, H4 and to a
lesser extent H1);8 selectivity is mainly engendered through
Introduction
differences in the substrate binding pockets and by the pres-
ence of other recognition domains in addition to the JmjC
domain.
The dynamic methylation of histone lysine residues is an
important process in transcriptional regulation. The introduc-
tion of N3-methyl lysine methylation marks is catalysed by
histone methyl transferases and their removal is catalysed by
histone lysine demethylases (KDMs). Aberrant histone lysine
methylation is associated with a variety of disease states,
including cancer and ageing.1,2 Human KDMs are classied into
two families according to their mechanisms; the lysine specic
KDMs (KDM1A and B) which employ avin adenine dinucleo-
tide (FAD) as a cofactor and the 2-oxoglutarate (2OG) dependent
KDMs, the JumonjiC (JmjC) KDMs,3 which are part of the wider
2OG oxygenase superfamily and which use 2OG and molecular
oxygen as cofactors.4,5
KDM2A (FBXL11) is a member of the KDM2/7 subfamily,
human members of which include KDM2B, PHF8 and KDM7A.
KDM2A demethylates histone H3 residues, i.e. N3-mono- and di-
methyllysine-36 (H3K36me1/2),9 and has a role in cellular
differentiation,10 regulation of NF-kB11 and cell proliferation.12,13
KDM2A is overexpressed in some non-small cell lung cancers
(NSCLCs); knockdown of KDM2A with siRNA has been shown to
reduce the proliferation of KDM2A-overexpressing NSCLC cell-
lines indicating that KDM2A activity may promote proliferation
of NSCLCs.13
JmjC KDM inhibitors of varying selectivity have been
described (Fig. 1A and Table 1; compounds 2–6).14–16
Compounds with some degree of selectivity for the KDM2,
KDM3 and KDM4 subfamilies have been discovered (Fig. 1B
and Table 1); notably compound 7 which is approximately ten-
fold selective for the KDM4 subfamily,17 compound 8 (GSK-J1)
which is a KDM6A/B/C inhibitor,18,19 and the KDM2/7 inhibitor
9 (KDM2A pIC50 5.8).20 Daminozide 10, an agrochemical used to
regulate plant growth, is the most potent KDM2/7 subfamily
selective inhibitor reported in the literature to date (KDM2A
pIC50 5.8).21
The JmjC KDMs are grouped into ve subfamilies (KDM2/7,
KDM3, KDM4, KDM5 and KDM6).6 They have conserved 2OG
and Fe(II) binding sites, the precise nature of which are
subfamily specic.7 The different members of the JmjC KDM
family accept different substrates (typically methylated lysine
aStructural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt
Drive, Headington OX3 7DQ, UK. E-mail: paul.brennan@sgc.ox.ac.uk
bChemistry Research Laboratory, University of Oxford, Manseld Road, Oxford, OX1
3TA, UK
cTarget Discovery Institute, University of Oxford, NDM Research Building, Roosevelt
Most inhibitors of the JmjC KDMs described to date are 2OG
mimetics,7 as indeed for 2OG oxygenases in general.22 For
example a series of inhibitors has been developed based on the
2,20-bipyridine scaffold where one of the pyridine rings bears a
carboxylate group at the 4-position. X-ray crystallography of a
derivative of compound 4 in complex with KDM4A demon-
strates that the pyridine-N atoms bind to Fe(II) in a bidentate
Drive, Oxford, OX3 7FZ, UK
dDiamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, OX11
0QX, UK
eDivision of Cardiovascular Medicine, Radcliffe Department of Medicine, University of
Oxford, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN,
UK
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4md00291a
‡ These authors contributed equally to this work.
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Fig. 1 Structures of A. 2OG and broad spectrum JmjC KDM inhibitors (1–6) and B. Inhibitors with some selectivity for JmjC KDM subfamilies (7–10).
copper-catalysed click reactions.24,25 This would allow rapid
Table 1 Inhibitory effect of some non-selective JmjC inhibitors (2–6)
and inhibitors that are more selective for some JmjC subfamilies (7–10)
exploration of the structure activity relationship with the aim of
exploiting differences in the substrate scope of the different
JmjC KDM family members and achieving subfamily selectivity.
Methyl 2-[(trimethylsilyl)ethynyl]isonicotinate 1226 was
prepared from the corresponding bromide via Sonogashira
coupling with trimethylsilylacetylene (TMSCCH) to give a pro-
tected alkyne intermediate as a precursor for copper-catalysed
click reactions (Scheme 1).
The reaction between azidomethyl pivalate and the trime-
thylsilyl protected alkyne intermediate 12, which was depro-
tected in situ with tetrabutylammonium uoride (TBAF), gave
pivaloyloxymethyl protected triazole 13 in a method modied
from that described by Loren et al.27 Global deprotection using
sodium hydroxide gave the N–H triazole target 14a.
Compound 14a was then screened against a panel of JmjC
KDMs using AlphaScreen technology.28 The activity of
compound 14a was compared with that of the commercially
available unsubstituted bipyridine analogue 15 in the same
screening panel. Replacing the pyridine ring with the 1,2,3-tri-
azole ring resulted in a greater than ten-fold reduction in
potency for KDM3A, 4C, 4E, 5C and 6B, but a less than two-fold
reduction in potency for KDM2A and 4A (Table 2). Although
compound 14a is very small (MW 190.2) it shows a preference
for KDM2A and 4A/C. This is likely to be due to residue differ-
ences in the active site of the different JmjC KDMs.4 We there-
fore considered that the triazolopyridine scaffold may represent
a good hit for the development of a selective KDM2A inhibitor.
Although it was not possible to crystallise compound 14a
with KDM2A it was co-crystallised with KDM4A as a surrogate.
KDM2A and KDM4A have similar substrates and are both able
to demethylate H3K36me2.8 The co-crystal structure of
compound 14a conrmed that a representative of the tri-
azolopyridine series occupies the 2OG binding site in KDM4A
(Fig. 2B) and provided insights to inform the design of KDM2A
selective inhibitors.
pIC50a KDM
2A
3A
4C
4E
5A
5C
5.1
6.3
7.5
4.6
6B
6.5
4.5
5.0
7.0
6.1
4.4
7.2
214
4.3
5.4
5.3
4.8
5.7
<4.0
5.2–6.0
5.3
6.5
6.5
314
5.1
6.0
6.8
5.6
5.5
6.2
6.0
5.5
415
514
616
6.6
717
5.2
4.7
818,19
4.4
920
5.2
5.8
4.1
4.3
<4.0
1021
<4.0
<4.0
<4.0
a
Reported values determined in amplied luminescent proximity
homogeneous assay (AlphaScreen), enzyme-linked immunosorbent
assay (ELISA), dissociation-enhanced lanthanide uorescent
immunoassay (DELFIA) or matrix-assisted laser desorption/ionization
(MALDI) assay.
manner and the 4-carboxy group mimics that of the 5-carboxy
group of 2OG (Fig. 2A).23
Thus there is a need for more selective and/or potent
inhibitors of JmjC KDMs in order to elucidate their physiolog-
ical roles in healthy and diseased organisms and as starting
points for medicinal chemistry programmes. Here we report the
development of a highly selective and potent inhibitor of the
JmjC KDM KDM2A.
Results and discussion
In order to nd selective inhibitors of JmjC KDMs, a new scaf-
fold was designed based on the 2,20-bipyridine series wherein
one of the pyridine rings was replaced with a triazole ring (e.g.
compound 14a) to give a different potential iron binding motif.
It was proposed that the triazole-N atom would be able to
coordinate the catalytic iron atom and readily enable alternative
vectors for compound elaboration. We envisaged that the
planned route to the triazole series would enable synthesis of a
range of analogues through construction of the triazole ring via
The triazolopyridine scaffold itself does not extend
substantially into the substrate pocket. However we anticipated
that through the introduction of appropriate substituents on
the triazole ring at N-1 and C-5, the activity and selectivity for
KDM2A could be improved by making interactions in the
substrate pocket. A diverse range of N-substituted triazole
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Fig. 2 A. Overlay of a 2,20-bipyridine inhibitor (orange sticks, from PDB ID: 3PDQ)23 and H3K9me3 peptide (lavender ribbon and sticks, from PDB
ID: 2OQ6)32 in KDM4A (green ribbon and sticks, from PDB ID: 3PDQ). The inhibitor forms a H-bond to Y132 and two salt bridges to K206 and
D135 (red dashed lines). The bipyridine motif coordinates the nickel atom (orange dashed lines to brown sphere) which replaces the catalytic iron
in the crystal structure. The diaminoethane substituent extends into the peptide-binding pocket. B. Overlay of compound 14a (pale sticks, from
PDB ID: 4URA) and NOG, 2 (cyan sticks, from PDB ID: 2OQ6) in KDM4A (green ribbon and sticks, from PDB ID: 4URA). The catalytic iron,
substituted by nickel in the crystal structure (brown sphere) is coordinated by the side chains of E190, H188 and H276. NOG, a close analogue of
2OG, forms one H-bond and one salt bridge to Y132 and K206 respectively (dark blue dashed lines) and bidentate coordination with the metal
(light blue dashed lines). Triazole 14a forms similar interactions with the catalytic metal (orange dashed lines), K206 and Y132 (red dashed lines)
and is further stabilised by apparent aromatic stacking between the pyridine ring and F185 and van der Waals interactions with S288 (green stick
and CPK).* Only the beta strand core of the protein is shown for clarity.
In addition to preparing triazole derivatives with simple alkyl
substituents (Me, 14b and Et, 14c), derivatives bearing more
complex substituents were also selected for synthesis. Substit-
uents were selected so that the targets would have lead-like
Scheme 1 Synthesis of compound 14a. Reagents and conditions: (a)
TMSCCH, CuI, Pd(PPh3)4, Et3N, THF, 85%; (b) azidomethyl pivalate,
TBAF, CuSO4$5H2O, (+)-sodium L-ascorbate, DMF, H2O, 65 ꢀC, 53%;
(c) NaOH, H2O, MeOH, 61%.
derivatives were synthesised from the trimethylsilyl protected
alkyne intermediate 12 in a click triazole forming reaction
either directly with functionalised azides or by generating
substituted azides in situ in a one pot reaction between sodium
azide and the corresponding alkyl or aryl iodide (Scheme 2).29
Scheme 2 Synthesis of N-substituted triazoles. Reagents and condi-
tions: (a) TBAF, BnN3, DIPEA, CuI, MeOH, 69%; (b) TBAF, RI, NaN3,
CuSO4$5H2O, (+)-sodium L-ascorbate, DMF, H2O, 65 ꢀC, 20–77%; (c)
(i) KOTMS, MeCN, 81%; (ii) MeI, NaN3, CuSO4$5H2O, (+)-sodium
L-ascorbate, DMF, H2O, 65 ꢀC, 24%; (d) KOTMS, MeCN, 59–100%; (e) (i)
TBAF, RN3, DIPEA, CuI, MeOH; (ii) KOTMS, MeCN, 14–50%.
Table 2 Inhibitory effect of compounds 14a and 15 against seven JmjC KDMs
pIC50a KDM
Structure
2A
3A
4A
4C
4E
5C
6B
5.3 ꢁ 0.20 (8)
4.7 ꢁ 0.15 (2)
5.5 ꢁ 0.33 (2)
5.2 ꢁ 0.15 (4)
4.8 ꢁ 0.15 (4)
4.9 ꢁ 0.22 (4)
<4.0 (6)
5.4 ꢁ 0.17 (2)
5.9 ꢁ 0.13 (4)
5.9 ꢁ 0.07 (4)
6.7 ꢁ 0.14 (2)
6.4 ꢁ 0.19 (2)
6.2 ꢁ 0.07 (2)
5.0 ꢁ 0.13 (6)
a
Mean pIC50 ꢁ standard error of the mean (number of determinations) as determined by AlphaScreen.
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Table 3 Inhibitory effect of N-substituted 1,2,3-triazole isonicotinic acids in seven JmjC KDMs
Compound
pIC50a KDM
2A
14
R
3A
4A
4C
4E
5C
6B
14a
14b
14c
14d
H
Me
Et
5.3 ꢁ 0.20 (8)
4.9 ꢁ 0.18 (8)
4.5 ꢁ 0.13 (8)
5.2 ꢁ 0.16 (8)
4.7 ꢁ 0.15 (2)
4.6 ꢁ 0.24 (2)
5.1 ꢁ 0.21 (2)
5.1 ꢁ 0.28 (2)
5.5 ꢁ 0.33 (2)
4.7 ꢁ 0.19 (4)
ND
5.2 ꢁ 0.14 (4)
5.0 ꢁ 0.23 (4)
5.2 ꢁ 0.19 (4)
5.3 ꢁ 0.20 (4)
4.8 ꢁ 0.15 (4)
4.1 ꢁ 0.10 (4)
4.1 ꢁ 0.10 (2)
4.3 ꢁ 0.21 (2)
4.9 ꢁ 0.22 (4)
5.2 ꢁ 0.13 (4)
5.0 ꢁ 0.10 (4)
5.3 ꢁ 0.09 (4)
<4.0 (6)
<4.0 (6)
<4.0 (4)
4.3 ꢁ 0.17 (6)
ND
14e
14f
Bn
5.0 ꢁ 0.14 (2)
5.1 ꢁ 0.12 (2)
4.9 ꢁ 0.25 (2)
5.5 ꢁ 0.13 (2)
4.1 ꢁ 0.11 (4)
4.2 ꢁ 0.12 (4)
4.4 ꢁ 0.12 (2)
4.4 ꢁ 0.08 (2)
<4.0 (4)
4.0 ꢁ 0.20 (2)
5.1 ꢁ 0.13 (2)
5.1 ꢁ 0.16 (2)
<4.0 (4)
4.1 ꢁ 0.17 (4)
14g
14h
5.2 ꢁ 0.10 (2)
5.1 ꢁ 0.17 (2)
5.0 ꢁ 0.19 (2)
4.7 ꢁ 0.28 (2)
<4.0 (2)
<4.0 (2)
<4.0 (2)
5.0 ꢁ 0.09 (2)
5.4 ꢁ 0.09 (2)
4.1 ꢁ 0.12 (4)
<4.0 (4)
<4.0 (2)
<4.0 (2)
<4.0 (2)
a
Mean pIC50 ꢁ standard error of the mean (number of determinations) as determined by AlphaScreen.
KDM2A over KDM4A/C and KDM5C (Table 3). For example the
methyl derivative 14b is ca. three-fold more potent against
KDM5C than KDM2A and the ethyl derivative 14c is ca. ve-fold
more potent against KDM4C and 5C than KDM2A. The ethyl
carbamate 14h was selected as an attractive lead for substitu-
tion on the piperidine-N atom with the aim of improving
selectivity through exploiting differences between the substrate
binding pockets of KDM2A and KDM4A/C and 5C. tert-Buty-
loxycarbonyl (Boc)-protected 4-hydroxypiperidine was trans-
formed to the 4-azido derivative via the methanesulfonate, then
taken into the click reaction with the protected alkyne (Scheme
3). The Boc group was removed with CF3CO2H (TFA) to give a
late-stage intermediate for functionalization with acid chlorides
followed by pyridine ester deprotection.
Scheme
3 Synthesis of 4-piperidine derivatives. Reagents and
conditions: (a) MsCl, Et3N, DCM, 59%; (b) NaN3, DMF, 60 ꢀC, 65%; (c)
12, TBAF, DIPEA, CuI, MeOH, 56%; (d) TFA, DCM, 73%; (e) RCOCl, Et3N,
DCM; (f) LiOH (aq), MeOH.
properties (clogP < 3, MW 200–350 g molꢂ1 30 and be amenable
)
to rapid follow-up from suitable late-stage intermediates. For
example benzyl and phenethyl substituents were selected with
the view of synthesising substituted aryl systems from aryl
halides. The piperidinyl derivative was selected to enable
synthesis of substituted piperidines from the corresponding
NH piperidine. Potential targets were docked into a KDM2A
structure (PDB ID: 2YU1)31 based on the binding pose of
compound 14a in KDM4A. Compounds were selected for
synthesis that could be accommodated in the enzyme pocket.
When tested in the JmjC assay panel, these substituted tri-
azoles maintained good selectivity for KDM2A over KDM4E and
KDM6B; however, some of the compounds were not selective for
Scheme 4 Synthesis of azetidine, pyrrolidine and 3-piperidine deriv-
atives. Reagents and conditions: (a) MsCl, Et3N, DCM; (b) NaN3, DMF,
60 ꢀC; (c) 12, TBAF, DIPEA, CuI, MeOH; (d) TFA, DCM; (e) RCOCl, Et3N,
DCM; (f) LiOH (aq), MeOH.
Table 4 Inhibitory effect of the substituted 4-piperidine series in seven JmjC KDMs
a
Compound
pIC50 KDM
21
R
2A
3A
4A
4C
4E
5C
6B
21a
21b
21c
21d
21e
Et
5.2 ꢁ 0.14 (2)
5.1 ꢁ 0.14 (2)
5.1 ꢁ 0.13 (2)
5.4 ꢁ 0.11 (2)
5.6 ꢁ 0.09 (4)
4.3 ꢁ 0.13 (2)
<4.0 (2)
<4.0 (2)
ND
ND
4.8 ꢁ 0.42 (2)
4.8 ꢁ 0.19 (2)
5.0 ꢁ 0.18 (4)
4.5 ꢁ 0.19 (2)
4.1 ꢁ 0.15 (2)
<4.0 (2)
<4.0 (2)
<4.0 (2)
<4.0 (2)
<4.0 (2)
5.3 ꢁ 0.11 (2)
5.2 ꢁ 0.07 (2)
5.4 ꢁ 0.02 (2)
5.4 ꢁ 0.06 (2)
5.5 ꢁ 0.06 (2)
4.2 ꢁ 0.11 (2)
<4.0 (2)
4.4 ꢁ 0.26 (2)
4.8 ꢁ 0.09 (2)
<4.0 (2)
nPr
Ph
Bn
4.3 ꢁ 0.29 (4)
4.4 ꢁ 0.30 (2)
4.1 ꢁ 0.18 (2)
4.5 ꢁ 0.19 (2)
<4.0 (2)
a
Mean pIC50 ꢁ standard error of the mean (number of determinations) as determined by AlphaScreen.
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Table 5 Inhibitory effect of azetidine, pyrrolidine and 3-piperidine derivatives in seven JmjC KDMs
Compound
pIC50a KDM
2A
28
n
R
3A
4A
4C
4E
5C
6B
28a
28b
28c
28d
28e
28f
0
Me
Et
<4.0 (2)
4.3 ꢁ 0.17 (2)
<4.0 (2)
<4.0 (2)
4.5 ꢁ 0.07 (2)
4.0 ꢁ 0.07 (2)
<4.0 (2)
5.6 ꢁ 0.29 (2)
5.3 ꢁ 0.22 (2)
5.3 ꢁ 0.26 (2)
<4.0 (4)
5.8 ꢁ 0.19 (2)
4.9 ꢁ 0.31 (2)
5.1 ꢁ 0.19 (2)
4.7 ꢁ 0.14 (2)
4.4 ꢁ 0.13 (4)
5.1 ꢁ 0.12 (4)
5.3 ꢁ 0.07 (2)
<4.0 (2)
4.1 ꢁ 0.24 (2)
4.3 ꢁ 0.07 (2)
<4.0 (2)
4.6 ꢁ 0.11 (2)
<4.0 (4)
ND
6.2 ꢁ 0.07 (2)
5.6 ꢁ 0.04 (2)
5.2 ꢁ 0.06 (2)
5.4 ꢁ 0.03 (2)
5.2 ꢁ 0.10 (2)
5.9 ꢁ 0.05 (2)
<4.0 (2)
4.1 ꢁ 0.15 (2)
4.3 ꢁ 0.13 (2)
5.3 ꢁ 0.21 (2)
5.2 ꢁ 0.20 (2)
5.0 ꢁ 0.20 (2)
4.9 ꢁ 0.10 (2)
6.4 ꢁ 0.10 (2)
6.0 ꢁ 0.09 (2)
ND
4.3 ꢁ 0.87 (2)
4.2 ꢁ 0.83 (2)
4.0 ꢁ 0.03 (2)
<4.0 (2)
nPr
Ph
Bn
ND
5.2 ꢁ 0.25 (2)
<4.0 (2)
28g
28h
28i
28j
1
2
nPr
Ph
Bn
<4.0 (4)
ND
ND
ND
ND
ND
4.7 ꢁ 0.15 (6)
5.5 ꢁ 0.13 (4)
<4.0 (4)
ND
ND
ND
4.2 ꢁ 0.19 (2)
4.5 ꢁ 0.18 (2)
4.4 ꢁ 0.16 (2)
<4.0 (2)
<4.0 (2)
5.2 ꢁ 0.06 (2)
5.7 ꢁ 0.07 (2)
4.3 ꢁ 0.63 (2)
4.0 ꢁ 0.10 (2)
4.1 ꢁ 0.03 (2)
28k
28l
28m
28n
28o
28p
Me
Et
4.9 ꢁ 0.15 (2)
5.1 ꢁ 0.17 (2)
5.3 ꢁ 0.09 (2)
6.9 ꢁ 0.13 (6)
5.7 ꢁ 0.08 (2)
6.2 ꢁ 0.08 (2)
4.4 ꢁ 0.27 (2)
4.2 ꢁ 0.17 (2)
4.5 ꢁ 0.22 (2)
4.3 ꢁ 0.10 (2)
4.6 ꢁ 0.14 (2)
4.3 ꢁ 0.17 (2)
4.4 ꢁ 1.65 (2)
4.8 ꢁ 0.21 (2)
4.8 ꢁ 0.38 (2)
4.7 ꢁ 0.29 (2)
4.7 ꢁ 0.14 (14)
4.7 ꢁ 0.42 (2)
4.6 ꢁ 0.78 (4)
<4.0 (2)
5.6 ꢁ 0.06 (2)
5.4 ꢁ 0.05 (2)
5.3 ꢁ 0.06 (2)
5.0 ꢁ 0.11 (2)
5.1 ꢁ 0.27 (2)
5.3 ꢁ 0.07 (2)
4.0 ꢁ 0.17 (2)
4.3 ꢁ 0.16 (2)
4.1 ꢁ 0.10 (2)
4.7 ꢁ 0.13 (8)
4.9 ꢁ 0.19 (2)
4.9 ꢁ 0.13 (2)
ND
4.0 ꢁ 0.13 (2)
4.0 ꢁ 0.15 (2)
<4.0 (10)
<4.0 (2)
<4.0 (2)
nPr
Ph
Bn
ND
4.2 ꢁ 1.13 (2)
ND
ND
a
Mean pIC50 ꢁ standard error of the mean (number of determinations) as determined by AlphaScreen.
The ethyl, n-propyl and phenyl substituted amides (21a–c)
In order to investigate if the activity of compound 28n is due
retained moderate activity against KDM2A (Table 4). Gratify- to a single stereoisomer, the two enantiomers were synthesised
ingly, the benzyl (21d) and phenylethyl (21e) substituents from stereoisomerically pure Boc-protected alcohols which were
resulted in improved activity for KDM2A (pIC50 5.4 and 5.6 converted to the corresponding azides. Click triazole formation,
respectively) and reduced activity for KDM3A (pIC50 4.5 and Boc deprotection, amide formation and ester hydrolysis gave
<4.0) and KDM4A/C (pIC50 4.1–4.5), however the 4-piperidine enantiomers 35 and 36. In order to verify the stereochemical
derivatives (21a–e) all inhibit KDM5C in the same range as integrity of compounds 35 and 36, they were reconverted to
KDM2A.
the methyl ester derivatives 37 and 38 with MeI and NaHCO3
Given the differences in substrate selectivity of the KDM5 to enable analysis by chiral HPLC. The enantiomeric excesses
subfamily (H3K4me1/2/3) and the KDM2 subfamily were determined as 98%, conrming that racemisation had
(H3K36me1/2),8 we reasoned that selectivity for KDM2A over
KDM5C could be achieved through further exploration of the
histone substrate pocket. Other saturated heterocyclic ring
systems were thus synthesised as alternatives to the 4-piperi-
dine ring to explore different vectors within the pocket
(Scheme 4). The chiral 3-piperidine and 3-pyrrolidine deriva-
tives were prepared as racemates for initial screening.
The acetyl substituted azetidine 28a manifested sub-micro-
molar KDM5C activity with greater than 85-fold selectivity over
KDM2A, 3A, and 6B and three to seven-fold selectivity over the
KDM4 subfamily. Potency against KDM5C was observed to
decrease with increasing size of amide substituent on the amide
(Table 5; compounds 28b–e). KDM2A activity increased for the
phenyl, benzyl and phenethyl-amides 28d–f, but these still
suffered from a lack of selectivity over KDM5C. The 3-
substituted pyrrolidine compounds 28g–j generally showed
increased potency for KDM2A and improved selectivity over the
other KDM representatives. The 3-piperidine series resulted in a
dramatic increase in KDM2A inhibition with concurrent
reduction in KDM5C activity. Excitingly, the most potent
KMD2A inhibitor, the benzoyl 3-piperidine derivative 28n (pIC50
6.9) demonstrated greater than 50-fold selectivity for KDM2A
over all six other JmjC KDMs in our panel.
Scheme 5 Synthesis of the (R)- and (S)-enantiomers of compound
28n and conversion to the methyl ester derivatives. Reagents and
conditions: for the (R)-enantiomer: (a) tert-butyl (3S)-3-hydrox-
ypiperidine-1-carboxylate, MsCl, Et3N EtOAc, 96%; (b) (i) NaN3, DMF,
75 ꢀC; (ii) 12, TBAF, DIPEA, CuI, DMF, 48%; (c) TFA, DCM, 97%; (d)
PhCOCl, Et3N, MeCN, quant.; (e) LiOH (aq), MeCN, 85%; (f) MeI,
NaHCO3, DMF quant. For the (S)-enantiomer: (a) tert-butyl (3R)-3-
hydroxypiperidine-1-carboxylate, MsCl, Et3N EtOAc, 98%; (b) (i) NaN3,
DMF, 75 ꢀC; (ii) 12, TBAF, DIPEA, CuI, DMF, 34%; (c) TFA, DCM; (d)
PhCOCl, Et3N, MeCN; (e) LiOH (aq), MeCN, 81% over 3 steps; (f) MeI,
NaHCO3, DMF, 65%.
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Table 6 Inhibitory effect of the two enantiomers of 28n in seven JmjC KDMs
pIC50a KDM
Compound
2A
3A
4A
4C
4E
5C
6B
35
36
7.2 ꢁ 0.16 (4)
5.5 ꢁ 0.09 (2)
<4.0 (4)
ND
4.8 ꢁ 0.28 (2)
4.8 ꢁ 0.10 (4)
<4.0 (2)
<4.0 (6)
5.7 ꢁ 0.07 (2)
<4.0 (4)
4.5 ꢁ 0.24 (2)
ND
<4.0 (6)
ND
a
Mean pIC50 ꢁ standard error of the mean (number of determinations) as determined by AlphaScreen.
Table 7 Selected properties of compounds 8, 35 and 37
Physicochemical properties
PAMPA Pe (10ꢂ6 cm sꢂ1
)
a
Compound MW
H bond donors H bond acceptors c log Pa c log D at pH 7.4a Carboxylic acid c-pKa
pH 7.4 pH 6.2 pH 5.0
8
35
37
389.5
377.4
391.4
2
1
0
7
8
8
3.8
2.5
2.6
1.2
ꢂ1.3
2.6
4.2
3.0
N/A
ND
0.69
52
ND
0.41
45
ND
1.9
56
a
Properties calculated using ACD labs prediction model.33,34
not occurred to any signicant degree during the synthesis poor (0.41–1.9 ꢃ 10ꢂ6 cm sꢂ1) however the methyl ester 37 is
(Scheme 5).§
predicted to have good permeability (45–56 ꢃ 10ꢂ6 cm sꢂ1).
Gratifyingly, the (R)-enantiomer (35) is approximately 50-fold Biological investigations into the effects of inhibition of the
more potent than the (S)-enantiomer (36) indicating that the KDM2/7 subfamily JmjC KDMs by both compound 35 and its
stereochemistry of the substituted piperidine plays an impor- methyl ester pro-drug 37 are currently underway.
tant role in facilitating binding to KDM2A and that the (R)-
enantiomer positions the amide substituent in a favoured
position for binding to KDM2A (Table 6). KDM4A/C and 5C
Conclusions
activity also increased for the (R)-enantiomer relative to the
racemic mixture, but inhibitor 35 is still greater than 100-fold
selective for KDM2A over KDM4A/C/E, 30-fold selective over
KDM5C and exhibits negligible activity on KDM3A and 6B at
100 mM.
A new KDM inhibitor scaffold has been discovered through the
incorporation of an alternative triazole metal binding motif to
the known 2,20-bipyridine-4-carboxylate scaffold. A co-crystal
structure of the simplest example 14a with KDM4A demon-
strated that it binds to the JmjC KDMs via active site metal
chelation. A number of analogues were synthesised leading to
selective KDM inhibitors; the azetidine and piperidine
substituted triazoles showed promise as selective KDM5C
(e.g. compound 28b) and KDM2A (e.g. compound 28n) inhibi-
tors respectively. When prepared as a single enantiomer,
compound 35 is a potent and selective inhibitor of KDM2A. Due
to the similarity in the catalytic domain of the KDM2 and KDM7
subfamilies, it is expected that compound 35 will be a potent
inhibitor of all members of these subfamilies. Compound 35 is
signicantly more potent than other KDM2/7 subfamily
selective inhibitors reported in the literature to date,
hydroxamate 9 and daminozide 10.
Its potency and selectivity means that inhibitor 35 will be
of interest as a tool molecule for the study of KDM2A in bio-
logical systems. The physico-chemical properties of
compound 35 fall within the range predicted to give oral
bioavailability by the Lipinski Rule of Five (MW < 500,
log P < 5, H bond donors <5, H bond acceptors <10, Table 7).35
The KDM6A/B/C inhibitor 8 also complies with the Rule of
Five but cellular activity has only been observed when it is
dosed as the ethyl ester pro-drug,18 presumably due to poor
membrane permeability as a result of deprotonation of the
carboxylic acid at physiological pH (calculated pKa 4.5).
Compound 35 would also be expected to have poor membrane
permeability as it is predicted to be more acidic than
compound 8. The cellular permeability of compound 35 and
its methyl ester 37 was assessed using a parallel articial
membrane permeation assay (PAMPA, Table 7).36 The
membrane permeability of compound 35 is predicted to be
Acknowledgements
We thank Cunyu Zhang and David Drewry (GlaxoSmithKline)
and Shanghai Chempartner Co. for the synthesis of
compounds 21a–e and 28a–p; Diamond Light Source for
beamtime (proposal mx8421), and the staff of beamlines I04-1
and I03 for assistance with crystal testing and data collection,
Prof. Darren Dixon and Alistair Farley for chiral HPLC anal-
ysis and Brian Linehan (Boehringer Ingelheim) for PAMPA
determination. 1-Phenethyl azide, 2-phenoxyethyl azide and
§ 35 and 36 were synthesised from stereoisomerically pure Boc- protected alcohols
which were purchased from Sigma Aldrich Ltd. The alcohols were converted to the
methanesulfonates and then reacted with sodium azide. In assigning the
stereochemistry of 35 and 36 we have assumed that displacement with sodium
azide occurred with inversion of conguration.
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ethyl 4-azidopiperidine-1-carboxylate were gis from Pzer.
C. J. Schoeld and A. Kawamura, Chem. Sci., 2013, 4, 3110–
The SGC is a registered charity (number 1097737) that
3117.
receives funds from AbbVie, Bayer, Boehringer Ingelheim, the 15 D. Rotili, S. Tomassi, M. Conte, R. Benedetti, M. Tortorici,
Canada Foundation for Innovation, the Canadian Institutes
for Health Research, Genome Canada, GlaxoSmithKline,
Janssen, Lilly Canada, the Novartis Research Foundation, the
Ontario Ministry of Economic Development and Innovation,
G. Ciossani, S. Valente, B. Marrocco, D. Labella,
E. Novellino, A. Mattevi, L. Altucci, A. Tumber, C. Yapp,
O. N. F. King, R. J. Hopkinson, A. Kawamura,
C. J. Schoeld and A. Mai, J. Med. Chem., 2013, 57, 42–55.
Pzer, Takeda, and the Wellcome Trust [092809/Z/10/Z]. 16 L. Wang, J. Chang, D. Varghese, M. Dellinger, S. Kumar,
˜
This research was also supported in part by the Biotechnology
and Biological Sciences Research Council, the Medical
Research Council and a Royal Society Dorothy Hodgkin
fellowship (AK).
A. M. Best, J. Ruiz, R. Bruick, S. Pena-Llopis, J. Xu,
D. J. Babinski, D. E. Frantz, R. A. Brekken, A. M. Quinn,
A. Simeonov, J. Easmon and E. D. Martinez, Nat. Commun.,
2013, 4, 2035.
17 X. Luo, Y. Liu, S. Kubicek, J. Myllyharju, A. Tumber, S. Ng,
K. H. Che, J. Podoll, T. D. Heightman, U. Oppermann,
S. L. Schreiber and X. Wang, J. Am. Chem. Soc., 2011, 133,
9451–9456.
Notes and references
1 C. H. Arrowsmith, C. Bountra, P. V. Fish, K. Lee and 18 L. Kruidenier, C.-w. Chung, Z. Cheng, J. Liddle, K. Che,
M. Schapira, Nat. Rev. Drug Discovery, 2012, 11, 384–400.
2 E. L. Greer and Y. Shi, Nat. Rev. Genet., 2012, 13, 343–357.
3 S. M. Kooistra and K. Helin, Nat. Rev. Mol. Cell Biol., 2012, 13,
297–311.
4 M. A. McDonough, C. Loenarz, R. Chowdhury, I. J. Clion
and C. J. Schoeld, Curr. Opin. Struct. Biol., 2010, 20, 659–
672.
G. Joberty, M. Bantscheff, C. Bountra, A. Bridges, H. Diallo,
D. Eberhard, S. Hutchinson, E. Jones, R. Katso,
M. Leveridge, P. K. Mander, J. Mosley, C. Ramirez-Molina,
P. Rowland, C. J. Schoeld, R. J. Sheppard, J. E. Smith,
C. Swales, R. Tanner, P. Thomas, A. Tumber, G. Drewes,
U. Oppermann, D. J. Patel, K. Lee and D. M. Wilson,
Nature, 2012, 488, 404–408.
5 W. Aik, M. A. McDonough, A. Thalhammer, R. Chowdhury 19 L. J. Walport, R. J. Hopkinson, M. Vollmar, S. K. Madden,
and C. J. Schoeld, Curr. Opin. Struct. Biol., 2012, 22, 691–
700.
C. Gileadi, U. Oppermann, C. J. Schoeld and
C. Johansson, J. Biol. Chem., 2014, 26, 18302–18313.
6 R. J. Klose, E. M. Kallin and Y. Zhang, Nat. Rev. Genet., 2006, 20 T. Suzuki, H. Ozasa, Y. Itoh, P. Zhan, H. Sawada, K. Mino,
7, 715–727.
L. Walport, R. Ohkubo, A. Kawamura, M. Yonezawa,
Y. Tsukada, A. Tumber, H. Nakagawa, M. Hasegawa,
R. Sasaki, T. Mizukami, C. J. Schoeld and N. Miyata, J.
Med. Chem., 2013, 56, 7222–7231.
7 C. C. Thinnes, K. S. England, A. Kawamura, R. Chowdhury,
C. J. Schoeld and R. J. Hopkinson, Biochim. Biophys. Acta,
Gene
Regul.
Mech.,
2014,
DOI:
10.1016/
j.bbagrm.2014.1005.1009.
8 L. J. Walport, R. J. Hopkinson and C. J. Schoeld, Curr. Opin.
Chem. Biol., 2012, 16, 525–534.
9 Y. Tsukada, J. Fang, H. Erdjument-Bromage, M. E. Warren,
C. H. Borchers, P. Tempst and Y. Zhang, Nature, 2006, 439,
811–816.
10 J. Du, Y. Ma, P. Ma, S. Wang and Z. Fan, Stem Cells, 2013, 31,
126–136.
21 N. R. Rose, E. C. Y. Woon, A. Tumber, L. J. Walport,
R. Chowdhury, X. S. Li, O. N. F. King, C. Lejeune, S. S. Ng,
T. Krojer, M. C. Chan, A. M. Rydzik, R. J. Hopkinson,
K. H. Che, M. Daniel, C. Strain-Damerell, C. Gileadi,
G. Kochan, I. K. H. Leung, J. Dunford, K. K. Yeoh,
P. J. Ratcliffe, N. Burgess-Brown, D. F. von, S. Muller,
B. Marsden, P. E. Brennan, M. A. McDonough,
U. Oppermann, R. J. Klose, C. J. Schoeld and
A. Kawamura, J. Med. Chem., 2012, 55, 6639–6643.
11 T. Lu, M. W. Jackson, B. Wang, M. Yang, M. R. Chance,
M. Miyagi, A. V. Gudkov and G. R. Stark, Proc. Natl. Acad. 22 N. R. Rose, M. A. McDonough, O. N. F. King, A. Kawamura
Sci. U. S. A., 2010, 107, 46–51.
and C. J. Schoeld, Chem. Soc. Rev., 2011, 40, 4364–4397.
12 R. Gao, R. Dong, J. Du, P. Ma, S. Wang and Z. Fan, Mol. Cell. 23 K.-H. Chang, O. N. F. King, A. Tumber, E. C. Y. Woon,
Biochem., 2013, 379, 115–122.
13 K. W. Wagner, H. Alam, S. S. Dhar, U. Giri, N. Li, Y. Wei,
T. D. Heightman, M. A. McDonough, C. J. Schoeld and
N. R. Rose, ChemMedChem, 2011, 6, 759–764.
D. Giri, T. Cascone, J.-H. Kim, Y. Ye, A. S. Multani, 24 V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless,
C.-H. Chan, B. Erez, B. Saigal, J. Chung, H.-K. Lin, X. Wu, Angew. Chem., Int. Ed., 2002, 41, 2596–2599.
M.-C. Hung, J. V. Heymach and M. G. Lee, J. Clin. Invest., 25 C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem.,
2013, 123, 5231–5246.
2002, 67, 3057–3064.
14 R. J. Hopkinson, A. Tumber, C. Yapp, R. Chowdhury, W. Aik, 26 S. W. Bagley, D. A. Griffith and D. W.-S. Kung, WO Pat., 2011/
K. H. Che, X. S. Li, J. B. L. Kristensen, O. N. F. King,
058473 A1, 2011.
M. C. Chan, K. K. Yeoh, H. Choi, L. J. Walport, 27 J. C. Loren, A. Krasinski, V. V. Fokin and K. B. Sharpless,
C. C. Thinnes, J. T. Bush, C. Lejeune, A. M. Rydzik, Synlett, 2005, 2847–2850.
N. R. Rose, E. A. Bagg, M. A. McDonough, T. J. Krojer, 28 A. Kawamura, A. Tumber, N. R. Rose, O. N. F. King,
´
W. W. Yue, S. S. Ng, L. Olsen, P. E. Brennan,
M. Daniel, U. Oppermann, T. D. Heightman and
U. Oppermann, S. Muller, R. J. Klose, P. J. Ratcliffe,
C. Schoeld, Anal. Biochem., 2010, 404, 86–93.
This journal is © The Royal Society of Chemistry 2014
Med. Chem. Commun., 2014, 5, 1879–1886 | 1885

View Article Online
MedChemComm
Concise Article
29 R. J. Thibault, K. Takizawa, P. Lowenheilm, B. Helms,
T. Borowski, M. Sundstrom, C. J. Schoeld and
U. Oppermann, Nature, 2007, 448, 87–91.
´
J. L. Mynar, J. M. J. Frechet and C. J. Hawker, J. Am. Chem.
Soc., 2006, 128, 12084–12085.
30 A. Nadin, C. Hattotuwagama and I. Churcher, Angew. Chem.,
Int. Ed., 2012, 51, 1114–1122.
33 A. Petrauskas and E. Kolovanov, Perspect. Drug Discovery Des.,
2000, 19, 99–116.
´
34 M. Meloun and S. Bordovska, Anal. Bioanal. Chem., 2007,
31 Z. Han, P. Liu, L. Gu, Y. Zhang, H. Li, S. Chen and J. Chai,
Frontier Science, 2007, 52–67.
389, 1267–1281.
35 C. A. Lipinski, Drug Discovery Today: Technol., 2004, 1, 337–
32 S. S. Ng, K. L. Kavanagh, M. A. McDonough, D. Butler,
341.
E. S. Pilka, B. M. R. Lienard, J. E. Bray, P. Savitsky, 36 A. Avdeef and O. Tsinman, Eur. J. Pharm. Sci., 2006, 28, 43–
O. Gileadi, F. von Del, N. R. Rose, J. Offer, J. C. Scheinost, 50.
1886 | Med. Chem. Commun., 2014, 5, 1879–1886
This journal is © The Royal Society of Chemistry 2014