Inhibition of the histone demethylase JMJD2E by 3-substituted pyridine 2,4-dicarboxylates

Article abstract of DOI:10.1039/c0ob00592d

Based on structural analysis of the human 2-oxoglutarate (2OG) dependent JMJD2 histone N^ε-methyl lysyl demethylase family, 3-substituted pyridine 2,4-dicarboxylic acids were identified as potential inhibitors with possible selectivity over other human 2OG oxygenases. Microwave-assisted palladium-catalysed cross coupling methodology was developed to install a diverse set of substituents on the sterically demanding C-3 position of a pyridine 2,4-dicarboxylate scaffold. The subsequently prepared di-acids were tested for in vitro inhibition of the histone demethylase JMJD2E and another human 2OG oxygenase, prolyl-hydroxylase domain isoform 2 (PHD2, EGLN1). A subset of substitution patterns yielded inhibitors with selectivity for JMJD2E over PHD2, demonstrating that structure-based inhibitor design can enable selective inhibition of histone demethylases over related human 2OG oxygenases.

Full text of DOI:10.1039/c0ob00592d

View Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 127
www.rsc.org/obc
PAPER
Inhibition of the histone demethylase JMJD2E by 3-substituted pyridine
2,4-dicarboxylates†‡§
Armin Thalhammer,a Jasmin Mecinovic´,b Christoph Loenarz,a Anthony Tumber,c Nathan R. Rose,a
Tom D. Heightmanc and Christopher J. Schofielda
Received 17th August 2010, Accepted 7th October 2010
DOI: 10.1039/c0ob00592d
Based on structural analysis of the human 2-oxoglutarate (2OG) dependent JMJD2 histone Ne-methyl
lysyl demethylase family, 3-substituted pyridine 2,4-dicarboxylic acids were identified as potential
inhibitors with possible selectivity over other human 2OG oxygenases. Microwave-assisted
palladium-catalysed cross coupling methodology was developed to install a diverse set of substituents
on the sterically demanding C-3 position of a pyridine 2,4-dicarboxylate scaffold. The subsequently
prepared di-acids were tested for in vitro inhibition of the histone demethylase JMJD2E and another
human 2OG oxygenase, prolyl-hydroxylase domain isoform 2 (PHD2, EGLN1). A subset of
substitution patterns yielded inhibitors with selectivity for JMJD2E over PHD2, demonstrating that
structure-based inhibitor design can enable selective inhibition of histone demethylases over related
human 2OG oxygenases.
zymes are of clinical relevance.9 The best characterised subfamily
Introduction
of the human 2OG dependent histone demethylases are the
Covalent modifications to histone proteins play important roles in
Jumonji domain containing 2 (JMJD2) enzymes, the genes for
cell differentiation, embryonic development and cancer. The activ-
four of which are expressed (JMJD2A-D), whereas JMJD2E and
JMJD2F are annotated as pseudogenes.10 The JMJD2 enzymes
appear to be selective for demethylation of histone H3 at lysine 9
(H3K9) and, in the case of JMJD2A-C, additionally for lysine 36
of histone H3 (H3K36).9 JMJD2 histone lysyl demethylases differ
in their preference for the methylation state of their substrates,
catalysing the selective demethylation of tri- and di- and, to a lesser
extent, the mono-Ne-methylated forms of their lysine substrates,
producing formaldehyde as a byproduct11 (Scheme 1A).
There is significant interest in the development of selective
histone demethylase inhibitors, both for biomedicinal studies and
as functional probes aimed at defining the roles of these enzymes
in epigenetic regulation.12 The design of inhibitor scaffolds with
selectivity for inhibition of individual histone demethylases or
subfamilies of histone demethylases is hampered by the five
2OG dependent histone demethylase subfamilies being members
of the wider 2OG oxygenase family, which has ~60–80 human
members with diverse biochemical roles in oxygen sensing, fat
mass regulation, mRNA splicing, DNA demethylation, and 5-
methylcytosine hydroxylation.13,14
JMJD2E is closely related to the catalytic domain of the
other JMJD2 enzymes (~65% sequence identity with JMJD2A
over the catalytic domain) and can be prepared in an active
form, which is amenable to kinetic analysis.15 The availability
of recombinant JMJD2E enabled the identification of several
“template” inhibitors, most of which work by Fe(II) chelation at
the active site and compete with the 2OG cosubstrate for binding.15
Recently, Hamada et al.16 reported that hydroxamate derivatives
ities of enzymes that catalyse such modifications can regulate gene
expression and have also been associated with diseases, including
cancer (for reviews, see1–3). Histone deacetylases have been success-
fully targeted using small-molecule histone deacetylase (HDAC)
inhibitors such as suberoylanilide hydroxamic acid4 (SAHA) and
the natural product Romidepsin,5 which are approved for use
in clinical cancer treatment. Similar approaches might be viable
for the modulation of histone methyl transferase and histone
demethylase activities; Indeed, some histone methylation patterns
have been observed to correlate with disease pathology (reviewed
in6–8).
In humans, there are five identified subfamilies of 2-oxoglutarate
(2OG) dependent histone demethylases and several of these en-
aDepartment of Chemistry and the Oxford Centre for Integrative Sys-
tems Biology, Chemistry Research Laboratory, University of Oxford, 12
Mansfield Road, Oxford, OX1 3TA, United Kingdom. E-mail: Christopher.
schofield@chem.ox.ac.uk; Fax: +44 1865 285002; Tel: +44 1865 275625
bDepartment of Chemistry and Chemical Biology, Harvard University, 12
Oxford St, Cambridge, MA, 02138, USA
cStructural Genomics Consortium, University of Oxford, Headington, OX3
7DQ, United Kingdom
† We dedicate this paper to Robin G. Procter in the year of his retirement
with thanks for many years of expert technical assistance in mass
spectrometry.
‡ This paper is part of an Organic & Biomolecular Chemistry web theme
issue on chemical biology.
§ Electronic supplementary information (ESI) available: Additional assay
protocols, detailed inhibition data, and synthetic procedures for all
compounds. See DOI: 10.1039/c0ob00592d
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 127–135 | 127
©

View Online
Scheme 1 A. The Ne-methyl lysine demethylation reaction catalysed by the 2OG-dependent JMJD2 histone demethylases, which preferentially act
on tri- and di-Ne-methylated lysines. Demethylation of Ne-trimethyllysyl residues (1) proceeds via hydroxylation to give an unstable hemiaminal
intermediate, which collapses spontaneously to release the demethylation product 2. The formation of the by-product formaldehyde can be measured
spectrophotometrically using formaldehyde dehydrogenase and NAD+ in a coupled enzymatic assay. B. Structures of the 2OG cosubstrate (3), its
analogues NOG (4) and 2,4-PDCA (5), and the targeted C-3 substituted 2,4-PDCA derivatives. The ligand atoms involved in chelation of the ferrous
iron are shown in red and blue.
were JMJD2A inhibitors; In cell-penetrating form, some of the
compounds were shown to inhibit cancer cell growth when used in
Results and Discussion
Synthesis of 3-bromo-2,4-PDCA dimethyl ester
combination with an inhibitor of the pyridoxal phosphate lysine-
specific class of histone demethylases. Another type of JMJD2
subfamily inhibitors works by disrupting a Zn(II) binding site
separate from the active site.17 Transition metal ions also display
differential inhibition selectivities for JMJD2A, JMJD2E and the
hypoxia-inducible prolyl hydroxylase domain 2 (PHD2, EGLN1),
a somewhat structurally different 2OG oxygenase that is involved
in the adaptation of cells to hypoxia.18 In addition to N-oxalyl
amino acid derivatives,12,15,19 a previous study identified pyridine
dicarboxylates as potential JMJD2 inhibitors;15 however, these
pyridine dicarboxylates are known to inhibit all other studied
members of the 2OG oxygenase family.15 Interestingly, potent
inhibition of JMJD2 enzymes has been observed with 2,4-pyridine
dicarboxylic acid (2,4-PDCA, Scheme 1A), but not with its other
regioisomers that were also tested, suggesting that differential
substitution of 2,4-PDCA derivatives may help to enable the devel-
opment of JMJD2-selective inhibitors.15 Crystallographic analyses
of JMJD2A in complex with 2,4-PDCA have shown that it binds
in a similar manner to the 2OG cosubstrate,15 being positioned
to bind to the active site metal ion in a bidentate manner, while
the C-4 acid is positioned for electrostatic interactions with the
sidechain of Lys-206, as also occurs with the C-5 carboxylate
of 2OG (Fig. 1C,D). One advantage of a substituted 2,4-PDCA
scaffold is that its methyl and ethyl esters have been shown to
be cell-penetrating and to inhibit histone demethylase activity in
cells,20,21 thus suggesting that in vivo applications might be possible.
Here we report the preparation of C-3 substituted 2,4-PDCA
derivatives, and their evaluation as JMJD2 inhibitors. The results
demonstrate how structure-guided substitution of a generic in-
hibitor template can be used to develop selective inhibitors of
JMJD2E over PHD2.
To obtain insights as to which inhibitor scaffold modifications are
most likely to achieve selectivity between different 2OG oxygenase
families, we compared reported crystal structures of human 2OG
oxygenases. Amongst others, co-crystal structures of complexes
with inhibitors have been reported for JMJD2A (JMJD2A·2,4-
PDCA, PDB ID 2VD715) and for PHD2 in complex with a
fragment of its hypoxia inducible factor (HIF) peptide substrate
(PHD2·NOG·HIF-1a558–574, PDB ID 3HQR22). These analyses
suggested that substitution of 2,4-PDCA at the C-3 position,
rather than the C-5 or C-6 positions, would enable functional
groups to form additional interactions with the substrate binding
pocket of the JMJD2s, and could potentially abolish binding to
the more restricted 2OG binding site of PHD2 (Fig. 1B and 1D).
We therefore targeted 2,4-PDCA derivatives substituted at C-3
with an additional phenyl ring that could bear a diverse set of
substituents (Scheme 1B).
The 3-bromopyridine 14 was considered to be a potential inter-
mediate for the preparation of several C-3 substituted 2,4-PDCA
derivatives (Scheme 2). Preparation of 14 was initially attempted
via 3-hydroxy-2,4-PDCA 9, which could also serve as a parent
compound for the preparation of O-modified derivatives itself. 3-
Hydroxypyridine (6) was O-alkylated with tetrahydropyran-2-ol
under Mitsunobu conditions23 or with methoxymethyl chloride
to furnish derivatives 7 and 8 as reported.24 However, attempted
directed metallation-carboxylation25 by treatment with n-BuLi
and quenching with CO2(s), after hydrolytic workup, only gave
4-carboxy-3-hydroxypyridine in low yields. Conditions employing
either s-BuLi or t-BuLi as a base resulted in complex product
mixtures.
128 | Org. Biomol. Chem., 2011, 9, 127–135
This journal is
The Royal Society of Chemistry 2011
©

View Online
Fig. 1 Crystallographic analyses on JMJD2A and PHD2. A. View from a crystal structure of JMJD2A in complex with NiII (substituting for FeII) and
the inhibitor 2,4-PDCA (derived from PDB ID 2VD715). B. Active site of JMJD2A showing chelation of the active site metal (green) by the conserved
iron-binding Hx[D/E] . . . H motif (His188, Glu190 and His276). Selected active site residues are shown. C. View from a crystal structure of the prolyl
hydroxylase PHD2 in complex with MnII, a fragment of the hypoxia inducible factor 1a peptide substrate (not shown), and the 2OG cosubstrate analogue
N-oxalyl glycine (NOG), a relatively non-specific 2OG oxygenase inhibitor (PDB ID 3HQR22). D. Closeup view of the PHD2 active site showing chelation
of MnII (green) by the conserved iron-binding Hx[D/E] . . . H motif (His313, Asp315 and His374).
Scheme 2 Synthesis of 3-bromopyridine 14. Reagents and conditions: (i) Br2 (0.9 eq.), 20% oleum, 165 ◦C, 24 h, 11 (12%), 12 (22%), 13 (35%); (ii)
KMnO4 (5 eq.), NaOH (0.7 eq.), H2O, 110 ◦C, 3 h; (iii) cat. H2SO4, MeOH, reflux, 24 h, 53%; (iv) n-BuLi (1.1 eq.), THF, -78 ◦C, 1 h, or i-PrMgCl
(1.1 eq.), THF, RT; then MeOH workup; (v) tetrahydropyran-2-ol (1.1 eq.), PPh3 (1.1 eq.), diisopropyl azodicarboxylate (1.1 eq.), RT, 2 h, 46%;23 (vi)
methoxymethyl chloride (1.05 eq.), KOtBu (1.1 eq.), DMF–CH3CN, RT, 1 h;26 (vii) base (1.5 eq.), then excess CO2(s); base (1.5 eq.), then excess CO2(s);
then HCl/dioxane; *Ratios as determined by 1H NMR.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 127–135 | 129
©

View Online
Scheme 3 Synthesis of pyridine dicarboxylate derivatives 34–36. Reagents and conditions: (i) Pd(OAc)2 (10 mol%), PPh3 (0.2 eq.), Cs2CO3 (1.1 eq.),
DMF, 70 ◦C, 3–5 h, 60–75%; (ii) CuI (0.1 eq.), N-(n-butyl)imidazole (0.5 eq.), Cs2CO3 (2 eq.), toluene, MW, 140 ◦C, 1 h, 44%; (iii) NaOH, MeOH, H2O,
6 h-overnight, 49–75%.
In an alternative approach, electrophilic bromination of 2,4-
dimethylpyridine 10 in 20% oleum for 24 h at 165 ◦C as described27
gave a mixture of 3,5-dibromo-2,4-dimethylpyridine 11 (12%),
5-bromo-2,4-dimethylpyridine 12 (22%), and unreacted starting
material, in addition to the desired 3-bromo-2,4-dimethylpyridine
13 (35% yield after chromatography). When a larger excess of
bromine was used (2 eq.), the reaction went to completion;
however, the yield of the dibromopyridine 11 increased at the
expense of 13. When the oleum concentration was decreased to
10% (w/v), in an attempt to enhance the selectivity of bromination,
no conversion was observed. To improve the overall yield of the
desired isomer 13, the regioselective debromination of 11 was
investigated. Treatment of the dibrominated compound 11 with
n-BuLi, or i-PrMgCl, followed by protonation of the metallated
intermediates, gave mixtures of isomeric mono- (12 and 13)
and dibromo (11) 2,4-dimethylpyridines. 1H NMR analysis of
the crude products indicated a preference for the formation of
the desired 3-bromo substituted isomer, suggesting that the C-
5 position of 2,4-dimethylpyridine is sterically more accessible
to halogen-metal exchange. By this procedure, 11 was recy-
cled to increase the overall yield of 13. Permanganate oxida-
tion and esterification then afforded the protected intermediate
scaffold 14.
Transition-metal catalysed coupling reactions
We then investigated the formation of C–C bonds at the C-3
position of 2,4-PDCA (Schemes 3 and 4). Application of cross-
coupling methodology with intermediate 14 as a substrate was
found to require optimisation, likely because of steric hindrance
at C-3 and because both 14 and the coupling products are prone
to base-catalysed hydrolysis and thermal decarboxylation.
Carbon–carbon bond formation28 with 14 was achieved in
75% yield using Suzuki–Miyaura couplings with phenyl boronic
acid/anhydride, enabling the direct incorporation of a phenyl ring
at the C-3 position of 2,4-PDCA (15) (Scheme 3). Similarly, the
styryl derivative (16) was obtained from styryl boronic acid in 60%
yield. Attempts to prepare 16 by Heck coupling of 14 with styrene
were unsuccessful, despite investigation of common catalyst and
solvent systems.
Scheme 4 Synthesis of pyridine dicarboxylate derivatives 37–50. Reagents and conditions: (i) Pd2dba3 (2 mol%), ligand L (6 mol%), base (1.4 eq.),
toluene, MW, 110–150 ◦C; L = PPh3 or BINAP or 30: <5% conversion; L = DPEPhos: 11% yield; L = XantPhos: 77% yield; (ii) NaOH, MeOH–H2O,
RT; (iii) Pd2dba3 (2 mol%), XantPhos (6 mol%), Cs2CO3 (1.4 eq.), toluene, MW, 110 ◦C, 12 h, 69%; (iv) CF3CO2H, CH2Cl2, 0 ◦C to RT, 89%; (v) NaOH,
MeOH–H2O, 2 h, RT, 67%.
130 | Org. Biomol. Chem., 2011, 9, 127–135
This journal is
The Royal Society of Chemistry 2011
©

View Online
Efforts were then directed to identify a suitable catalyst and
solvent system for the introduction of oxygen and nitrogen
nucleophiles at the C-3 position of 14, initially using phenol
and aniline as model substrates. Cu(II)-catalysed Ullmann-type
cross-coupling reactions, which are commonly employed for
diarylether formation, usually require high reaction temperatures
and long reaction times, especially on sterically demanding
scaffolds.29 As might be expected, treatment of 14 with an
excess of phenol and CuO at 200 ◦C for 24 h resulted in
decomposition of starting material. Similarly, a catalyst system
comprising Pd(OAc)2, Cs2CO3 and 4,5-bis(diphenylphosphino)-
9,9-dimethylxanthene (XantPhos) was ineffective. In contrast, the
use of N-(n-butyl)imidazole and CuI under mild phase-transfer
conditions30 with microwave heating enabled formation of the
diarylether 17 in moderate yield.
afforded the targeted amino derivative 31 in 69% yield;
subsequent deprotection with CF3CO2H in CH2Cl2 gave
ester 32.
The methyl esters thus obtained could further be employed
to obtain cell-penetrable inhibitors.20,21 For in vitro experiments,
the desired dicarboxylic acids 33–50 were prepared by alkaline
hydrolysis of the corresponding dimethyl esters and typically did
not require further purification, being >95% pure by HPLC and
NMR analyses.
Biological results
An initial series of compounds was prepared to investigate the
preferred atoms at the C-3 position of 2,4-PDCA. The JMJD2E
inhibitory activities of compounds 33–50 against JMJD2E were
measured as IC50 values in a formaldehyde dehydrogenase (FDH)
coupled, spectrophotometric turnover assay, which measures
production of formaldehyde (Scheme 1A, Table 1 and ESI§).15
Compounds were then counterscreened against FDH to con-
firm genuine JMJD2E inhibition. To investigate potential se-
lective inhibition of JMJD2s by some of the synthesised 3-
substituted 2,4-PDCA derivatives, they were tested for inhibi-
tion of PHD2. Residual activities of PHD2 were determined
at 400 mM inhibitor concentration, using a fluorescence-based
assay.35,36
Replacement of hydrogen in the 3-position of 2,4-PDCA by
bromine or phenyl moieties resulted in a significant reduction of
potency against JMJD2E (IC50 > 100 mM) and also of potency
against PHD2, possibly due to steric effects on the geometry of
the carboxylate groups, causing sub-optimal Fe(II) coordination,
and/or binding to the arginyl (PHD2) and lysyl (JMJD2A)
residues that bind the C-4 carboxylate of 2,4-PDCA (Table 1,
entries 1–3, Fig. 1B and 1D). In support of this proposal was the
observation that upon installation of an ethylene linker between
the two aromatic rings (styryl derivative 35), some recovery of
inhibitory potency was observed (Table 1, entry 4).
Interestingly, 3-amino-2,4-PDCA 50 was a highly active
JMJD2E inhibitor (IC50 ~100 nM), although it displayed little
selectivity for JMJD2E over PHD2 (Table 1, entry 19). Although
50 was fluorescent at the initial assay concentration tested, it
gave an acceptable dose-response curve. These results reveal that
the presence of a C-3 amino group promotes the binding of
the 2,4-PDCA template to JMJD2A (and PHD2). It is possible
that the additional amino group enhances potency, at least in
part by enabling pre-organisation of the two carboxylate groups
into coplanarity, thus favouring a conformation similar to that
observed in the co-crystal structure of 2,4-PDCA with JMJD2A
(Fig. 1A and 1B). Addition of a phenyl ring to the exocyclic amino
group of 3-amino-2,4-PDCA (37) ablated inhibition of PHD2
within the limits of detection (100% residual activity at 400 mM),
whilst retaining significant activity against JMDJ2E (IC50 19 mM).
In contrast, the phenoxy-derivative 36 was inactive against both
JMJD2E and PHD2 (Table 1, entries 5–6).
Couplings with aromatic amines using Buchwald–Hartwig
conditions31 were then optimised on a model system com-
prising 14, aniline, cat. Pd(II) and
a set of phosphine
ligands (Scheme 4). With XantPhos, 18 was obtained in
77% isolated yield, whereas bis(2-diphenylphosphinophenyl)ether
(DPEPhos) catalysis gave only 11% yield. No significant
product formation was observed when using either ( )-
2,2¢-bis(diphenylphosphino)-1,1¢-binaphthalene (BINAP), triph-
enylphosphine or the bicyclo[3.3.3]undecane derivative 30 as cat-
alysts. These results are consistent with the proposal that the ‘bite
angle’ (the phosphorus-metal-phosphorus angle) of the phosphine
ligand plays a role in enabling efficient catalysis.32 Amongst a
number of solvent and base combinations investigated, toluene
and Cs2CO3 proved to be a suitable combination; significant
hydrolysis of the starting material was not observed under these
conditions. Aromatic amines with a range of functional groups,
including nitro, carboxylic acid, methoxy and fluorine moieties,
were used as coupling partners under these conditions. In general,
reactions proceeded faster and more smoothly with aryl amines
containing an ortho-substituent or an electron-donating para-
substituent than those bearing an electron-withdrawing para-
substituent. In the latter case, coupling yields were considerably
lower and longer reaction times as well as elevated reaction
temperatures were required.
We then aimed to obtain the 3-amino-2,4-PDCA dimethyl ester
32 in order to evaluate the electronic effect of an amino group at the
pyridine C-3 position on inhibitor potency, independently of steric
effects introduced by more bulky substituents. Because “classical”
Buchwald–Hartwig couplings are limited to the preparation of
secondary and tertiary amines, ammonia surrogates have been de-
veloped which also allow the preparation of primary amines using
transition-metal catalysis. For example, anilines can be prepared
by palladium-catalysed amination of aryl halides with benzophe-
none imine to afford intermediate benzophenone imines, which are
readily hydrolysed to the desired anilines.33 However, attempted
coupling of 3-bromo-2,4-PDCA 14 with benzophenone imine
was inefficient, possibly due to steric limitations. Alternatively,
cross-couplings with benzyl amine followed by hydrogenolysis
have been used to install primary amino groups, for example
in the synthesis of 3-aminoestrone.34 We therefore envisaged
that the related p-methoxybenzylamine could serve as ammonia
equivalent in a Buchwald–Hartwig type cross-coupling reaction
and could be readily removed by acid-mediated cleavage. Indeed,
reaction of 3-bromo-2,4-PDCA 14 with p-methoxybenzylamine
Having established that selectivity for JMJD2E over PHD2
can be enhanced by C-3 substitution of 2,4-PDCA, efforts
were directed at increasing inhibitory potency against JMDJ2E
whilst retaining selectivity. Thus, further analogues (38–47) were
designed and prepared to evaluate the effect of substituents
on the 3-aminophenyl ring. The para-substituted compounds
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 127–135 | 131
©

View Online
Table 1 Inhibitory potency of 3-substituted 2,4-PDCA derivatives against JMJD2E, PHD2 and formaldehyde dehydrogenase
JMJD2E
JMJD2E Residual
Activity (%)a
PHD2 Residual
Activity (%)b
FDH Residual
Activity (%)c
Entry
Compound
Template
R
IC50/mMa
1
2
3
4
5
6
7
8
5
A
A
A
A
A
B
B
B
B
B
B
B
B
B
B
B
A
A
A
H
Br
Ph
0.44
n.d.
n.d.
66
n.d.
19
164
95
66
48
48
166
56
19
0%
22%
100%
100%
100%
87%
91%
83%
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50e
67%
84%
25%
48%
0%
6%
0%
13%
12%
5%
39%
6%
100%
100%
85%
(E)-CH CHPh
OPh
NHPh
88%
100%
100%
100%
0%
100%
100%
100%
100%
100%
100%
100%
100%
71%
NH-p-C6H4-OMe
NH-p-C6H4-NO2
NH-p-C6H4-CO2H
NH-o,p-C6H3-diF
NH-o-C6H4-SMe
NH-o-C6H4-CO2H
NH-o-C6H4-NO2
NH-o-C6H4-OMe
NH-o-C6H4-Me
NH-o-C6H4–F
NH-Naphthyl
NH-CH2-p-C6H4-OMe
NH2
78%
100%
100%
100%
80%
73%
59%
9
10
11
12
13
14
15
16
17
18
19
2%
76%
41
2.5
26
41
0.11
3%
79%
d
d
0%
59%
29%
100%
d
d
23%
a Determined by FDH-coupled assay; enzyme concentration 400 nM; residual activity reported at 200 mM compound concentration. b Determined by
an HTRF-based assay; enzyme concentration 20 nM; residual activity reported at 400 mM compound concentration. c Enzyme concentration 50 mM;
spectrophotometric assay; residual activity reported at 200 mM compound concentration. d Compound was fluorescent at the tested concentration. e Stock
solutions prepared at 1 mM concentration because of limited solubility.
38–41 were less potent than the unsubstituted system 37 (Table 1,
entries 6–10). In contrast, the ortho-substituted derivatives 41–47
displayed a broad range of inhibitory activities against JMJD2E
(Table 1, entries 10–16). Compounds bearing methoxy- (45) and
fluoro- (47) substituents were significantly more potent than those
with carboxylate (43), nitro (44) or methyl (46) groups. These
results suggest that a combination of steric and electronic effects
determine potency in the 3-substituted 2,4-PDCA series. One of
the ortho-substituted 3-phenyl derivatives (40) showed substantial
inhibition of PHD2 at 400 mM (Table 1, entry 9). No significant
FDH inhibition was observed for the potent compounds at
concentrations close to their IC50 value against JMJD2E (Table 1),
confirming genuine JMJD2E inhibition. Therefore, among others,
compounds 37, 45, 46, 48, and, in particular, 47 and 50, represent
selective inhibitors of the JMJD2E histone demethylase over the
related human 2OG dependent oxygenase PHD2.
family of histone demethylases. Synthetic methods employing Pd-
catalysed couplings were developed and applied to the preparation
of C-3 substituted 2,4-PDCA compounds derived from dimethyl 3-
bromopyridine-2,4-dicarboxylate 14. The results demonstrate that
compounds such as 37, 45 and 47 are potent JMJD2E inhibitors.
Importantly, these compounds do not inhibit PHD2, as predicted
on the basis of structural analyses, and may be useful in their
esterified forms as inhibitors for the dissection of the biological
roles of different 2OG oxygenase subfamilies.19 Thus, our work
demonstrates that structural analyses of the catalytic site pocket
of human 2OG oxygenases can be used both to enhance potency
to the target demethylases and to block binding to other human
2OG oxygenases.
Experimental
Representative synthetic procedures
Conclusions
Bromo-2,4-dimethylpyridines 11, 12 and 13. Preparation of
bromo-2,4-dimethylpyridines was performed according to a pub-
lished procedure with modifications.27 To 2,4-dimethylpyridine
Based on structural analyses, we identified a series of C-3
substituted 2,4-PDCA derivatives as inhibitors of the JMJD2 sub-
132 | Org. Biomol. Chem., 2011, 9, 127–135
This journal is
The Royal Society of Chemistry 2011
©

View Online
(10, 5.39 cm3, 46.7 mmol, 1 eq.) was carefully added oleum (50
cm3, containing 20% free SO3) with stirring at 0 ◦C. The reaction
flask was fitted with a high-efficiency reflux condenser and heated
to 165 ◦C in an oil bath. Bromine (4.30 cm3, 83.4 mmol, 0.9 eq.) was
added in portions of 0.5 cm3 over 5 h, and stirring was continued at
155–175 ◦C for 20 h. The solution was cooled to room temperature,
poured onto crushed ice (200 g) and stirred for 1 h. The resulting
solution was neutralised by careful addition of solid Na2CO3,
diluted with water and extracted with EtOAc (2 ¥ 250 cm3). The
combined organic layers were dried (Na2SO4) and concentrated
in vacuo. The crude residue was purified by automated flash
column chromatography (Biotage KP-Sil SNAP 340 g cartridge,
0% to 15% EtOAc in hexane) and dried in vacuo to give the bromo-
2,4-dimethylpyridines 11, 12 and 13.
(400 MHz; CDCl3) 3.98 (3 H, s, OCH3), 4.01 (3 H, s, OCH3), 7.60
(1 H, d, J 5.0 Hz, pyH) and 8.64 (1 H, d, J 5.0 Hz, pyH); dC
(100 MHz; CDCl3) 53.2, 53.3, 115.6, 125.2, 142.1, 148.0, 152.4,
164.9 and 165.4; m/z (ESI+) 295.9524 (M + Na+. C9H8BrNNaO4
requires 295.9529).
General procedure for Buchwald–Hartwig couplings of amines
with 3-bromopyridine-2,4-dicarboxylic acid dimethyl ester.
A
stirred suspension of dimethyl 3-bromopyridine-2,4-dicarboxylate
14 (1 eq.), the requisite primary aromatic or benzylic amine (1.2
eq.), anhydrous Cs2CO3 (1.4 eq.), Pd2dba3 (2 mol%) and XantPhos
(6 mol%) in anhydrous toluene (2 cm3 per 1 mmol of 14) was
heated to 150 ◦C in a Biotage Initiator microwave oven. The
progress of the reaction was monitored by TLC, and reactions
were stopped when no further consumption of starting material
could be detected. The reaction mixture was directly purified using
automated flash column chromatography (Biotage KP-SIL SNAP
cartridges, eluting with EtOAc–hexane) to afford the desired
diarylamine.
3,5-Dibromo-2,4-dimethylpyridine 11. White solid (1.48 g,
12%); mp 28–30 ◦C (lit.,27 29–30 ◦C); dH (400 MHz; CDCl3) 2.54
(3 H, s, CH3), 2.61 (3 H, s, CH3) and 8.41 (1 H, s, pyH); dC (100
MHz; CDCl3) 23.8, 25.7, 120.3, 124.4, 146.4, 148.5 and 156.5;
n
max(film)/cm-1 3045, 2998, 2956, 2921, 1558, 1433, 1376, 1349,
General procedure for hydrolysis of pyridine-2,4-dicarboxylic
acid dimethyl esters. To a stirred solution of dimethyl ester
in MeOH (10 cm3 mmol-1) was added an equal volume of
NaOH (20% w/v). The reaction mixture was stirred overnight
at room temperature and concentrated in vacuo. The residue was
dissolved in the minimum required amount of water and acidified
with conc. HCl. If a precipitate was obtained at this stage, the
solution was cooled on ice and the precipitate was isolated by
filtration and dried in vacuo to afford the desired carboxylic acid.
If no precipitation occurred at this stage, the aqueous phases
were repeatedly extracted with EtOAc, the organic layers were
combined, dried (Na2SO4) and concentrated in vacuo to afford the
desired carboxylic acid.
1232, 1049, 993, 928 and 783; m/z (ESI+) 263.9027 (M + H+.
C7H8Br2N requires 263.9018).
5-Bromo-2,4-dimethylpyridine 12. Colourless oil (1.89 g, 22%);
n
max(film)/cm-1 2985, 2957, 2924, 2854, 1592, 1461, 1377, 1353,
1289, 1177 and 1032; dH (400 MHz; CDCl3) 2.32 (3 H, s, CH3),
2.44 (3 H, s, CH3), 7.00 (1 H, s, pyH) and 8.47 (1 H, s, pyH); dC
(100 MHz; CDCl3) 22.1, 23.6, 120.4, 125.5, 146.7, 150.5 and 157.0;
m/z (ESI+) 185.9918 (M + H+. C7H9BrN requires 185.9913).
3-Bromo-2,4-dimethylpyridine 13. Colourless oil (3.04 g, 35%);
n
max(film)/cm-1 3050, 2995, 2958, 2922, 1585, 1437, 1389, 1123,
1024, 916 and 825; dH (400 MHz; CDCl3) 2.33 (3 H, s, CH3), 2.61
(3 H, s, CH3), 6.91 (1 H, d, J 5.0 Hz, pyH) and 8.18 (1 H, d,
J 5.0 Hz, pyH); dC (100 MHz; CDCl3) 23.2, 25.7, 123.4, 124.3,
146.7, 147.3 and 157.4; m/z (ESI+) 185.9917 (M + H+. C7H9BrN
requires 185.9913).
Dimethyl 3-phenylpyridine-2,4-dicarboxylate 15. A stirred sus-
pension of 3-bromopyridine 14 (300 mg, 1.1 mmol, 1 eq.), phenyl
boronic acid/anhydride (147 mg, 1.2 mmol, 1.1 eq.), Cs2CO3 (392
mg, 1.2 mmol, 1.1 eq.), Pd(OAc)2 (25 mg, 0.11 mmol, 0.1 eq.)
and PPh3 (57 mg, 0.22 mmol, 0.2 eq.) in anhydrous DMF (10
cm3) was heated to 70 ◦C for 3 h. The reaction mixture was
cooled to room temperature, diluted with water and extracted
with EtOAc. The combined organic layers were washed with
water, dried (Na2SO4) and concentrated in vacuo. The residue
was purified by automated flash column chromatography (Biotage
KP-SIL SNAP 25 g cartridge, eluting with EtOAc–hexane) to
afford 15 (223 mg, 75%) as a light yellow oil; nmax(film)/cm-1 3059,
3027, 3003, 2952, 1740, 1434, 1320, 1288 and 1199; dH (400 MHz;
CDCl3) 3.61 (3 H, s, OCH3), 3.68 (3 H, s, OCH3), 7.23 (2 H, dd,
J 6.5, 3.0 Hz, ArHmeta), 7.39 (m, 3 H, ArH), 7.73 (1 H, d, J 5.0
Hz, pyH), 8.76 (1 H, d, J 5.0 Hz, pyH); dC (100 MHz; CDCl3)
52.6, 52.6, 124.2, 128.0, 128.2, 128.4, 135.2, 135.7, 140.5, 148.6,
150.8, 166.4, 166.5; m/z (ESI+) 294.0730 (M + Na+. C15H13NNaO4
requires 294.0737).
Dimethyl 3-bromopyridine-2,4-dicarboxylate 14. 3-Bromo-
2,4-dimethylpyridine 13 (7.00 g, 38 mmol, 1 eq.) and water (100
cm3) were heated to 100 ◦C, and a solution of NaOH (1.05 g, 26
mmol, 0.7 eq.) in water (150 cm3) was added. KMnO4 (29.7 g,
188 mmol, 5 eq.) was then added in portions over 30 min under
vigorous stirring. The resulting dark suspension was stirred under
reflux until the KMnO4 colour had disappeared completely (2–3 h,
determined in 3 independent experiments). The hot solution was
filtered and solids washed with hot water (3 ¥ 50 cm3). The filtrate
was concentrated to ca. 50 cm3 in vacuo and neutralised with conc.
H2SO4. The solution was concentrated in vacuo, the residue was
coevaporated with toluene and suspended in MeOH (200 cm3).
Conc. H2SO4 (10 cm3) was then added, the resulting suspension
was refluxed overnight, and then cooled to room temperature and
concentrated in vacuo. The residue was taken up in water, the pH
was adjusted to pH 9 by careful addition of Na2CO3 and the basic
solution was extracted with EtOAc (3 ¥ 100 cm3). The combined
organic layers were dried (Na2SO4) and concentrated in vacuo.
The residue was purified by automated column chromatography
(Biotage KP-SIL SNAP 100 g cartridge) to afford 14 (4.62 g, 45%)
as a white solid (Found: C, 39.54; H, 2.95; N, 5.02. C9H8BrNO4
requires C, 39.44; H, 2.94; N, 5.11%); mp 65–68 ◦C; nmax (KBr
disk)/cm-1 3019, 2958, 1743, 1449, 1421, 1279, 1202 and 1171; dH
(E)-Dimethyl 3-styrylpyridine-2,4-dicarboxylate 16. A stirred
suspension of 3-bromopyridine 14 (500 mg, 1.82 mmol, 1 eq.),
(E)-styryl boronic acid (300 mg, 2.01 mmol, 1.1 eq.), Cs2CO3
(650 mg, 2.01 mmol, 1.1 eq.), Pd(OAc)2 (40 mg, 0.182 mmol,
0.1 eq.) and PPh3 (100 mg, 0.365 mmol, 0.2 eq.) in anhydrous
DMF (10 cm3) was heated to 70 ◦C for 5 h in an oil bath. The
reaction mixture was diluted with EtOAc (50 cm3) and washed
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 127–135 | 133
©

View Online
with water (50 cm3). The organic layer was dried (Na2SO4) and
concentrated in vacuo. The residue was purified by automated flash
column chromatography (Biotage KP-SIL SNAP 25 g cartridge,
eluting with EtOAc–hexane) to afford 16 (322 mg, 60%) as an off-
powder; mp >140 ◦C (decomp).; nmax (KBr disk)/cm-1 3424, 2920,
2852, 1729, 1612, 1379, 1259 and 1192; dH (400 MHz; DMSO-d6)
7.26–7.36 (2 H, m, ArH), 7.38–7.42 (3 H, m, ArH), 7.80 (1 H, d, J
5.0 Hz, pyH), 8.69 (1 H, d, J 5.0 Hz, pyH); dC (100 MHz, DMSO-
d6) 123.6, 128.8 and 128.9, 129.6, 132.3, 136.4, 142.8, 149.2, 153.6,
168.4, 168.5; m/z (ESI-) 242.0453 (M - H+. C13H8NO4 requires
242.0459).
◦
white solid; mp 75–76 C (decomp.); nmax(film)/cm-1 2953, 1735,
1447, 1434, 1313, 1269, 1197, 1166, 1143 and 1130; dH (400 MHz;
CDCl3) 3.86 (3 H, s, OCH3), 3.89 (3 H, s, OCH3), 6.59 (1 H, d, J
16.5 Hz, CH CH), 7.23–7.30 (1 H, t, J 8.0 Hz, ArHpara), 7.34 (2
H, t, J 8.0 Hz, ArHmeta), 7.46 (2 H, d, J 7.0 Hz, ArHortho), 7.64 (1
H, d, J 16.5 Hz, CH CH), 7.71 (1 H, d, J 5.0 Hz, pyH) and 8.65
(1 H, d, J 5.0 Hz, pyH); dC (100 MHz; CDCl3) 52.8, 52.8, 123.5,
124.6, 126.7, 128.4, 128.7, 133.0, 135.1, 136.5, 139.0, 147.9, 149.8,
166.5 and 166.7; m/z (ESI+) 320.0893 (M + Na+. C17H15NNaO4
requires 320.0893).
(E)-3-Styrylpyridine-2,4-dicarboxylic acid 35. A solution of
dimethyl ester 16 (95 mg, 0.32 mmol, 1 eq.) in MeOH (4 cm3)
was treated with a solution of NaOH (100 mg) in water (1 cm3)
and stirred overnight at room temperature. The reaction mixture
was concentrated in vacuo, the residue dissolved in the minimum
required amount of water, acidified with conc. HCl and extracted
with EtOAc (3 ¥ 5 cm3). The combined organic layers were dried
(Na2SO4) and concentrated in vacuo to afford 35 (42 mg, 49%) as
an off-white powder (Found: C, 66.84; H, 4.09; N, 5.14; C15H11NO4
requires C, 66.91; H, 4.12; N, 5.20%); mp >170 ◦C (decomp.); nmax
(KBr disk)/cm-1 3393, 3082, 3026, 3001, 1884, 1683, 1445, 1261,
1241, 1232 and 1197; dH (400 MHz; MeOD) 6.76 (1 H, d, J 16.5
Hz, CH CH), 7.30 (1 H, t, J 7.5 Hz, ArHpara), 7.37 (2 H, t, J 7.5
Hz, ArHmeta), 7.51 (2 H, d, J 7.5 Hz, ArHortho), 7.69 (1 H, d, J 16.5
Hz, CH CH), 7.85 (1 H, d, J 5.0 Hz, pyH), 8.62 (1 H, d, J 5.0
Hz, pyH); dC (100 MHz; MeOD) 123.7, 125.0, 126.8, 128.4, 128.8,
132.5, 135.2, 137.2, 141.6, 147.5, 150.5, 168.2, 168.5; m/z (ESI-)
268.0616 (M - H+. C15H10NO4 requires 268.0615).
Dimethyl 3-phenoxypyridine-2,4-dicarboxylate 17. A stirred
suspension of 3-bromopyridine 14 (50 mg, 0.182 mmol, 1.0 eq.),
phenol (21 mg, 0.219 mmol, 1.2 eq.), CuI (3 mg, 0.018 mmol,
0.1 eq.), N-(n-butyl)imidazole (12 mL, 0.091 mmol, 0.5 eq.) and
Cs2CO3 (119 mg, 1.46 mmol, 2 eq.) in anhydrous toluene (1 cm3)
was heated to 140 ◦C for 1 h in a Biotage Initiator microwave
oven. The dark suspension was directly purified by automatic flash
chromatography (Biotage KP-SIL SNAP 25 g cartridge, eluting
with EtOAc–hexane) to afford 17 (23 mg, 44%) as a colourless oil;
n
max(film)/cm-1 2954, 1741, 1591, 1492, 1435, 1407, 1322, 1283,
1244, 1203 and 1173; dH (400 MHz; CDCl3) 3.69 (3 H, s, OCH3),
3.78 (3 H, s, OCH3), 6.73–6.84 (2 H, m, ArHortho), 7.04 (1 H, dt,
J 7.5, 1.5 Hz, ArHpara), 7.18–7.34 (2 H, m, ArHmeta), 7.84 (1 H, d,
J 5.0 Hz, pyH) and 8.65 (1 H, d, J 5.0 Hz, pyH); dC (100 MHz;
CDCl3) 52.9, 52.9, 115.7, 122.8, 127.1, 129.6, 134.3, 145.6, 145.9,
148.7, 158.5, 164.0 and 164.2; m/z (ESI+) 310.0673 (M + Na+.
C15H13NNaO5 requires 310.0686).
3-Phenoxypyridine-2,4-dicarboxylic acid 36. A solution of
dimethyl ester 17 (50 mg, 0.174 mmol, 1 eq.) in MeOH (1 cm3)
was treated with a solution of NaOH (60 mg, 1.5 mmol, 9 eq.)
in water (1 cm3) and stirred overnight at room temperature. The
reaction mixture was concentrated in vacuo, the residue dissolved
in the minimum required amount of water, acidified with conc.
HCl and extracted repeatedly with EtOAc. The combined organic
layers were dried (Na2SO4) and concentrated in vacuo to afford 36
(23 mg, 51%) as a white solid (Found: C, 60.23; H, 3.27; N, 5.31.
C13H9NO5 requires C, 60.24; H, 3.50; N, 5.40%); mp >160 ◦C
(decomp.); nmax (KBr disk)/cm-1 3426, 1731, 1591, 1491, 1458,
1242, 1193; dH (500 MHz; DMSO-d6) 6.76 (2 H, d, J 8.0 Hz,
ArHortho), 7.02 (1H, t, J 7.5 Hz, ArHpara), 7.29 (2 H, dd, J 8.0, 7.5
Hz, ArHmeta), 7.87 (1 H, d, J 5.0 Hz, pyH), 8.63 (1 H, d, J 5.0
Hz, pyH) and 13.66 (1 H, br s, CO2H); dC (125 MHz; DMSO-
d6) 115.6, 122.3, 126.0, 129.6, 135.3, 145.6, 146.3, 147.4, 158.2,
164.7 and 165.6; m/z (ESI-) 258.0401 (M - H+. C13H8NO5 requires
258.0408).
Dimethyl 3-(phenylamino)pyridine-2,4-dicarboxylate 18.
A
stirred suspension of 3-bromopyridine 14 (50 mg, 0.18 mmol, 1
eq.), aniline (20 mL, 0.22 mmol, 1.2 eq.), Cs2CO3 (83 mg, 0.26
mmol, 1.4 eq.), Pd2(dba)3 (3 mg, 0.0037 mmol, 2 mol%) and
XantPhos (6 mg, 0.011 mmol, 6 mol%) in anhydrous toluene
(1.5 cm3) was heated to 110 ◦C for 4 h in a Biotage Initiator
microwave oven. The reaction mixture was directly purified using
automated flash column chromatography (Biotage KP-SIL SNAP
25 g cartridge, eluting with EtOAc–hexane) to afford 18 (40 mg,
77%) as a yellow solid; mp 93–96 ◦C; nmax(film)/cm-1 3312, 3059,
3036, 3005, 2951, 1733, 1695, 1592, 1561 and 1504; dH (400 MHz;
CDCl3) 3.49 (3 H, s, OCH3), 3.83 (3 H, s, OCH3), 6.99–7.11 (3 H,
m, ArH), 7.24–7.34 (2 H, m, ArH), 7.66 (1 H, d, J 4.5 Hz, pyH),
8.24 (1 H, d, J 4.5 Hz, pyH) and 9.60 (1 H, s, NH); dC (100 MHz;
CDCl3) 52.3, 52.8, 119.8, 123.9, 126.6, 127.7, 129.5, 135.7, 139.1,
141.4, 142.0, 166.7 and 167.2; m/z (ESI+) 309.0846 (M + Na+.
C15H14N2NaO4 requires 309.0846).
3-(Phenylamino)pyridine-2,4-dicarboxylic acid 37. A solution
of dimethyl ester 18 (150 mg, 0.524 mmol, 1 eq.) in MeOH (5 cm3)
was treated with a solution of NaOH (150 mg, 3.75 mmol, 7.2 eq.)
in water (5 cm3) and stirred overnight at room temperature. The
reaction mixture was concentrated in vacuo, the residue dissolved
in water (1 cm3) and acidified with conc. HCl. The mixture was
cooled, the crystalline precipitate collected by filtration and dried
in vacuo to afford 37 (94 mg, 70%) as orange crystals (Found: C,
60.51; H, 3.97; N, 10.75; C13H10N2O4 requires C, 60.47; H, 3.90; N,
10.85%); mp >152 ◦C (decomp.); nmax (KBr disk)/cm-1 3472, 3197,
1703, 1637, 1519, 1257, 1221; dH (500 MHz; DMSO-d6) 6.80–7.06
(3 H, m, ArH), 7.22 (2 H, t, J 8.0 Hz, ArH), 7.76 (1 H, d, J 5.0
Hz, pyH), 8.24 (1 H, d, J 5.0 Hz, pyH), 9.41 (1 H, br s, NH) and
13.46 (1 H, br s, CO2H); dC (125 MHz; DMSO-d6) 118.2, 122.4,
3-Phenylpyridine-2,4-dicarboxylic acid 34.
A solution of
dimethyl ester 15 (171 mg, 0.63 mmol, 1 eq.) in MeOH (5 cm3)
was treated with a solution of NaOH (151 mg, 3.78 mmol, 6 eq.)
in water (5 cm3) and stirred for 6 h at room temperature. The
reaction mixture was concentrated in vacuo, the residue dissolved
in the minimum required amount of water, acidified with conc.
HCl and extracted repeatedly with Et2O. The combined organic
layers were dried (Na2SO4), concentrated in vacuo and further
dried under vacuum to afford 34 (100 mg, 75%) as an off-white
134 | Org. Biomol. Chem., 2011, 9, 127–135
This journal is
The Royal Society of Chemistry 2011
©

View Online
127, 129.1, 129.1, 138.9, 139.0, 139.6, 142.8, 167.1 and 167.5; m/z
(ESI-) 257.0568 (M - H+. C13H9N2O4 requires 257.0568).
H. Nakagawa, M. Hasegawa, R. Sasaki, T. Mizukami and N. Miyata,
J. Med. Chem., 2010, 53, 5629–5638.
17 R. Sekirnik, N. R. Rose, A. Thalhammer, P. T. Seden, J. Mecinovic´ and
C. J. Schofield, Chem. Commun., 2009, 6376–6378.
18 R. Sekirnik, N. R. Rose, J. Mecinovic and C. J. Schofield, Metallomics,
2010, 2, 397–399.
Acknowledgements
19 S. Hamada, T.-D. Kim, T. Suzuki, Y. Itoh, H. Tsumoto, H. Nakagawa,
R. Janknecht and N. Miyata, Bioorg. Med. Chem. Lett., 2009, 19, 2852–
2855.
20 G. Tschank, D. G. Brocks, L. Engelbart, J. Mohr, E. Baader, V. Gunzler
and H. Hanauske-Abel, Biochem. J., 1991, 275, 469–476.
21 M. M. Mackeen, H. B. Kramer, K.-H. Chang, M. L. Coleman, R. J.
Hopkinson, C. J. Schofield and B. M. Kessler, J. Proteome Res., 2010,
9, 4082–4092.
We thank Thomas Helleday for encouragement, John M. Brown
for helpful discussions, and the Wellcome Trust, the Biotechnology
and Biological Sciences Research Council and Cancer Research
UK for financial support.
References
22 R. Chowdhury, M. A. McDonough, J. Mecinovic, C. Loenarz, E.
Flashman, K. S. Hewitson, C. Domene and C. J. Schofield, Structure,
2009, 17, 981–989.
1 Y. Shi, Nat. Rev. Genet., 2007, 8, 829–833.
2 P. A. C. Cloos, J. Christensen, K. Agger and K. Helin, Genes Dev., 2008,
22, 1115–1140.
3 B. R. Keppler and T. K. Archer, Expert Opin. Ther. Targets, 2008, 12,
1301–1312.
4 P. A. Marks, Oncogene, 2007, 26, 1351–1356.
5 J. Tan, S. Cang, Y. Ma, R. Petrillo and D. Liu, J. Hematol. Oncol., 3.
6 S. Ng, W. Yue, U. Oppermann and R. Klose, Cell. Mol. Life Sci., 2009,
66, 407–422.
7 N. Mosammaparast and Y. Shi, Annu. Rev. Biochem., 2010, 79, 155–
179.
8 R. J. Klose and Y. Zhang, Nat. Rev. Mol. Cell Biol., 2007, 8, 307–318.
9 R. J. Klose, E. M. Kallin and Y. Zhang, Nat. Rev. Genet., 2006, 7,
715–727.
10 M. Katoh and M. Katoh, Int. J. Oncol., 2004, 24, 1623–1628.
11 R. J. Hopkinson, R. B. Hamed, N. R. Rose, T. D. Claridge and C. J.
Schofield, ChemBioChem, 2010, 11, 506–510.
12 N. R. Rose, E. C. Woon, G. L. Kingham, O. N. King, J. Mecinovic, I. J.
Clifton, S. S. Ng, J. Talib-Hardy, U. Oppermann, M. A. McDonough
and C. J. Schofield, J. Med. Chem., 2010, 53, 1810–1818.
13 C. Loenarz and C. J. Schofield, Nat. Chem. Biol., 2008, 4, 152–156.
14 C. Loenarz and C. J. Schofield, Chem. Biol., 2009, 16, 580–583.
15 N. R. Rose, S. S. Ng, J. Mecinovic, B. M. Lienard, S. H. Bello, Z. Sun,
M. A. McDonough, U. Oppermann and C. J. Schofield, J. Med. Chem.,
2008, 51, 7053–7056.
23 R. Azzouz, L. Bischoff, M.-H. Fouquet and F. Marsais, Synlett, 2005,
2808–2810.
24 M. R. Winkle and R. C. Ronald, J. Org. Chem., 1982, 47, 2101–2108.
25 R. Azzouz, L. Bischoff, C. Fruit and F. Marsais, Synlett, 2006, 1908–
1912.
26 F. Le Strat, D. C. Harrowven and J. Maddaluno, J. Org. Chem., 2005,
70, 489–498.
27 A. D. Dunn and S. Guillermic, Z. Chem., 1988, 28, 59–60.
28 N. Miyaura and A. Suzuki, Chem. Rev., 1995, 95, 2457–2483.
29 I. P. Beletskaya and A. V. Cheprakov, Coord. Chem. Rev., 2004, 248,
2337–2364.
30 T. Schareina, A. Zapf, A. Cotte, N. Muller and M. Beller, Tetrahedron
Lett., 2008, 49, 1851–1855.
31 J. F. Hartwig, Angew. Chem., Int. Ed., 1998, 37, 2046–2067.
32 P. C. J. Kamer, P. W. N. M. van Leeuwen and J. N. H. Reek, Acc. Chem.
Res., 2001, 34, 895–904.
˚
33 J. P. Wolfe, J. Ahman, J. P. Sadighi, R. A. Singer and S. L. Buchwald,
Tetrahedron Lett., 1997, 38, 6367–6370.
34 U. Scho¨n, J. Messinger, M. Buchholz, U. Reinecker, H. Thole, M.
K. S. Prabhu and A. Konda, Tetrahedron Lett., 2005, 46, 7111–
7115.
35 J. H. Dao, R. J. M. Kurzeja, J. M. Morachis, H. Veith, J. Lewis, V. Yu,
C. M. Tegley and P. Tagari, Anal. Biochem., 2009, 384, 213–223.
36 C. Loenarz, J. Mecinovic´, R. Chowdhury, L. A. McNeill, E. Flashman
and C. J. Schofield, Angew. Chem., Int. Ed., 2009, 48, 1784–1787.
16 S. Hamada, T. Suzuki, K. Mino, K. Koseki, F. Oehme, I. Flamme, H.
Ozasa, Y. Itoh, D. Ogasawara, H. Komaarashi, A. Kato, H. Tsumoto,
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 127–135 | 135
©