3,5-dimethylisoxazoles act as acetyl-lysine-mimetic bromodomain ligands

Article abstract of DOI:10.1021/jm200640v

Histone-lysine acetylation is a vital chromatin post-translational modification involved in the epigenetic regulation of gene transcription. Bromodomains bind acetylated lysines, acting as readers of the histone-acetylation code. Competitive inhibitors of this interaction have antiproliferative and anti-inflammatory properties. With 57 distinct bromodomains known, the discovery of subtype-selective inhibitors of the histone-bromodomain interaction is of great importance. We have identified the 3,5-dimethylisoxazole moiety as a novel acetyl-lysine bioisostere, which displaces acetylated histone-mimicking peptides from bromodomains. Using X-ray crystallographic analysis, we have determined the interactions responsible for the activity and selectivity of 4-substituted 3,5-dimethylisoxazoles against a selection of phylogenetically diverse bromodomains. By exploiting these interactions, we have developed compound 4d, which has IC₅₀ values of <5 μM for the bromodomain-containing proteins BRD2(1) and BRD4(1). These compounds are promising leads for the further development of selective probes for the bromodomain and extra C-terminal domain (BET) family and CREBBP bromodomains.

Full text of DOI:10.1021/jm200640v

ARTICLE
pubs.acs.org/jmc
3,5-Dimethylisoxazoles Act As Acetyl-lysine-mimetic
Bromodomain Ligands†
David S. Hewings,‡,§ Minghua Wang,‡ Martin Philpott,‡ Oleg Fedorov,‡ Sagar Uttarkar,‡
Panagis Filippakopoulos,‡ Sarah Picaud,‡ Chaitanya Vuppusetty,‡ Brian Marsden,‡ Stefan Knapp,‡
||
Stuart J. Conway,*,§ and Tom D. Heightman*,‡,
‡Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford,
Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.
§Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA, U.K.
S Supporting Information
b
ABSTRACT: Histoneꢀlysine acetylation is a vital chromatin post-
translational modification involved in the epigenetic regulation of gene
transcription. Bromodomains bind acetylated lysines, acting as readers
of the histone-acetylation code. Competitive inhibitors of this interac-
tion have antiproliferative and anti-inflammatory properties. With 57
distinct bromodomains known, the discovery of subtype-selective
inhibitors of the histoneꢀbromodomain interaction is of great im-
portance. We have identified the 3,5-dimethylisoxazole moiety as a
novel acetyl-lysine bioisostere, which displaces acetylated histone-
mimicking peptides from bromodomains. Using X-ray crystallographic analysis, we have determined the interactions responsible
for the activity and selectivity of 4-substituted 3,5-dimethylisoxazoles against a selection of phylogenetically diverse bromodomains.
By exploiting these interactions, we have developed compound 4d, which has IC50 values of <5 μM for the bromodomain-
containing proteins BRD2(1) and BRD4(1). These compounds are promising leads for the further development of selective probes
for the bromodomain and extra C-terminal domain (BET) family and CREBBP bromodomains.
’ INTRODUCTION
Although the biological roles of most bromodomains in the
human genome remain elusive, those that have been character-
ized are fundamental. For example, the BET bromodomain-
containing protein (BRD) 4 plays a key role in several cellular
processes, including mitosis.6 Expression levels of BRD4 corre-
late with breast cancer survival rates,7 and in a subset of malignant
squamous carcinomas, the N-terminal bromodomains of BRD4
are fused in frame to the NUT genegivingrise to extremely aggres-
sive tumor growth.8 Knockdown experiments have implicated
BRD4 in the transcriptional regulation of viruses such as HIV9
and EBV,10 as well as the degradation of HPV.11 BRD4 was also
shown to be required for transcriptional coactivation of NF-kB,
regulating the transcription of P-TEFb-dependent pro-inflam-
matory target genes.12 A second BET-family protein, the testis-
specific BRDT, is essential for male germ cell differentiation in
selective domain knockout mice that were viable but sterile.13
The development of small molecule inhibitors of bromodo-
main binding to histones and other acetyl-lysine-containing proteins
is in its infancy. Weakly potent small molecule ligands of the
cAMP response element-binding protein (CREB) binding pro-
tein (CREBBP) bromodomain, discovered by NMR screening of
acetyl-lysine (KAc) mimics, have been shown to modulate p53
Genetic information is encoded in DNA by the specific linear
sequence of its bases. The expression of this information is regulated
by protein scaffolds that are capable of reading the complex code of
post-translational modifications that occur on chromatin. Acetyla-
tion of histones lysine residues is one such essential component in
the epigenetic regulation of gene expression.1 Additionally, recent
proteomics studies have demonstrated that lysine acetylation occurs
in over 1750 cellular proteins involved in diverse roles such as cell
cycle, splicing, nuclear transport, and actin nucleation.2 Lysine
acetylationonhistonesandotherproteinsiseffected by the dynamic
interplay of acetyltransferase (HAT) and deacetylase (HDAC)
enzymes, which is analogous to the regulation of serine, threo-
nine, and tyrosine phosphorylation by kinases and phosphatases.
Bromodomains are a family of conserved ∼110 amino acid
modules that bind selectively to acetylated lysines present in
proteins, notably histones,3 and are thereby thought to partici-
pate in deciphering the histone code.4 Bromodomains have been
classified into several distinct subgroups according to the func-
tion of their parent protein: (i) histone acetyltransferases (HATs),
including CREBBP, GCN5, PCAF, and TAFII250; (ii) in com-
ponents of ATP-dependent chromatin-remodeling complexes
such as Swi2/Snf2; and (iii) the BET (bromodomain and extra
C-terminal domain) family, a class of transcriptional regulators
carrying tandem bromodomains and an extra terminal domain.5
Received: May 19, 2011
Published: August 18, 2011
r
2011 American Chemical Society
6761
dx.doi.org/10.1021/jm200640v J. Med. Chem. 2011, 54, 6761–6770
|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. (A) Structure of the BET probe (+)-JQ1.18 (B) Structure of the BET probe I-BET.19a (C) Structure of dihydroquinazolinone-containing 3,5-
dimethylisoxazole derivative 1. (D) The concept employed in the design of the 3,5-dimethylisoxazole-based bromodomain inhibitors.
stability and function in response to DNA damage in cells.14
More recently, a 4-hydroxyphenylazobenzenesulfonic acid deri-
vative was shown to bind to the CREBBP bromodomain, with
KD = 19 μM, and consequently blocks p53-dependent apoptosis
in cardiomyocytes under DNA-damage stress.15
We demonstrate that the 3,5-dimethylisoxazole moiety is an
effective KAc mimic and show how differential substitution of the
3,5-dimethylisoxazole core gives derivatives that bind with some
selectivity and useful affinity to either the first bromodomain of
human BRD4 [BRD4(1)] or the bromodomain of human CREBBP.
Ligands have also been developed for the BET family of bromo-
domains. A benzimidazole-derived BRD2(1) ligand with KD = 28
μM reduced the transcription of BRD2-dependent genes.16 Anti-
proliferative effects against a number of cell lines have been claimed in
a patent for a series of thienotriazolodiazepine BRD2/3/4 blockers
with nanomolar in vitro peptide displacement activity.17 The structu-
rally related (+)-JQ1 (Figure 1A), recently characterized as a
BET-family selective chemical probe, shows specific antiproli-
ferative effects in BRD4-dependent cell lines and patient-derived
xenograft models.18 In addition, a recently identified BET bromo-
domain blocker (I-BET, Figure 1B) suppressed lipopolysaccharide-
inducible genes in macrophages and showed anti-inflammatory
effects in mice.19 This work demonstrates the feasibility of inhibiting
KAc binding to bromodomains with small molecules that occupy
the well-defined KAc-binding pocket. It is possible that targeting this
pocket might prove more tractable than developing inhibitors of
catalytic activity in certain enzymes. In addition, small molecule
inhibition of bromodomainꢀprotein interactions might elicit subtly
different pharmacological responses to the inhibition of the catalytic
domain of the same protein.20
The findings outlined above demonstrate clearly the need for
systematic generation of high affinity subfamily selective bromo-
domain-binding small molecules that can be used as chemical
probes to explore further the roles of individual bromodomains.
These molecules will provide complementary data to studies using
knockdown, peptide and antibody approaches, and will enable
prediction of the likely phenotypes of eventual small molecule
drugs. As part of our ongoing goal to generate cell-penetrant
chemical probes for bromodomains, wehave previously developed
bromodomainꢀhistone peptide binding assays to allow the iden-
tification of fragment and small molecule ligands.21 Herein, we
describe the use of these assays, together with a structure-based
design approach, to identify and develop the structureꢀactivity
relationships (SAR) of a novel series of 3,5-dimethylisoxazole-
containing bromodomainꢀhistone binding inhibitors.
’ RESULTS AND DISCUSSION
In the course of our work, we discovered that the solvent
N-methyl pyrrolidinone (NMP) interacts with the KAc-binding
pocket of a range of bromodomains, showing weak affinity but,
because of its low molecular weight, high ligand efficiency
(0.30ꢀ0.53, see Supporting Information, Table S1), with IC50
values ranging from 34 mM for the bromodomain adjacent to
zinc finger domain 2B (BAZ2B) to 2.3 mM for CREBBP.21
During subsequent studies on methyl-bearing heterocycles, we
purchased and tested the dihydroquinazolinone-containing 3,
5-dimethylisoxazole derivative 1 (Figure 1C) and observed unex-
pectedly low IC50 values of ∼3 μM against the first bromodo-
main of BRD2 [BRD2(1)] and ∼7 μM against BRD4(1). Docking
studies suggested that two binding modes were possible for this
compound, with either the dihydroquinazolinone or 3,5-dimethyl-
isoxazole moiety acting as a KAc bioisostere (data not shown).
To explore the possibility that the 3,5-dimethylisoxazole might
occupy the KAc-binding pocket, we obtained an X-ray crystal
structure of the complex between human BRD4(1) and 1.
The X-ray crystal structure of the BRD4(1) complex was
solved at 1.98 Å (Figure 2A, PDB ID: 3SVF). It demonstrated
that the 3,5-dimethylisoxazole was indeed acting as a KAc
bioisostere, accepting a hydrogen bond from the conserved N
residue within the binding pocket [N140 in BRD4(1)], which
mimics the key interaction made with the carbonyl oxygen of
KAc. Additionally, we observed additional unexplained electron
density at the 4-position of the dihydroquinazolinone that was
interpreted as a substitution of one hydrogen atom by an ethylene
glycol unit (Figure 2B). As dihydroquinazolinones are liable to
benzylic oxidation under laboratory conditions (Supporting Infor-
mation, Figure S1),22 we hypothesized that this product 2 might
arise from oxidation at this position to form the corresponding
iminium species, followed by the addition of ethylene glycol,
6762
dx.doi.org/10.1021/jm200640v |J. Med. Chem. 2011, 54, 6761–6770

Journal of Medicinal Chemistry
ARTICLE
Figure 2. (A) The X-ray crystal structure of 2 (carbon = yellow, PDB ID, 3SVF) bound to human BRD4(1). The conserved N140 residue is highlighted
in green. (B) 2Fo ꢀ Fc map at 2σ of the observed ligand 2. (C) Overlay of crystal structures of 2 (carbon = yellow) and (+)-JQ1 (carbon = orange, PDB
ID: 3MXF) bound to human BRD4(1), showing that the ethylene glycol unit binds in the same region as the chlorophenyl moiety of (+)-JQ1. The
images were generated and structure alignment conducted using PyMOL.
which is present in the crystallization buffer (see Supporting
Information, Figure S1 for mass spectrometry analysis). Inter-
estingly, only one enantiomer was observed in the X-ray crystal
structure; the ethylene glycol unit attached to the stereogenic
center occupies a hydrophobic groove formed by W81 and P82
from the ZA loop and I146 and M149 from helix C. Occupation
of this site by the 4-chlorophenyl substituent of (+)-JQ1 and
I-BET (Figure 1A,B) is believed to contribute to the selectivity of
the these compounds for the BET subfamily over other bromo-
domains since a similar groove is not available in other bromo-
domains (Figure 2C).18 In addition, we observed that a sample of
1 displayed significant cytotoxicity in the HeLa human cell line,
while (+)-JQ1 showed no cytotoxicity over the same concentra-
tion range (Supporting Information, Figure S2). Therefore, the
development of analogues that do not possess a readily oxidized
benzylic methylene unit, but that could form interactions with
this groove, was pursued. The mono- and di-meta-substituted 3,
5-dimethyl-4-phenylisoxazole scaffolds (3aꢀd and 4aꢀd) were
chosen to allow extension from the aromatic core in two directions
(Figure 1B). Initial docking studies suggested that a single meta-
substituent would occupy a position similar to that of the ethylene
glycol moiety of 2 (Supporting Information, Figure S3A). A second
meta-substituent would be directed into a groove (referred to by
Nicodeme et al.19a and Chung et al.19b as the ZA channel) defined
by W81 on one side and L92 on the other (Supporting Information,
Figure S3A). Consequently, the 3,5-dimethylisoxazole derivatives
3aꢀd and 4aꢀd were selected as target molecules that contain
a range of substituents to enable exploration of the KAc-binding
pocket of the bromodomains.
Scheme 1. Synthesis of the 30-Substituted 3,5-Dimethyl-
4-phenylisoxazole Derivatives 3aꢀca
a Conditions: (a) R = H: Na2CO3, Pd(OAc)2, RuPhos, EtOH, 85 °C, 4 h,
85%. R = Ac: Na2CO3, Pd(OAc)2, RuPhos, EtOH, 85 °C, 13 h, 86%.
R = OEt: Na2CO3, Pd(OAc)2, RuPhos, EtOH, 85 °C, 2 h, 87%.
react efficiently with both electron-rich and electron-deficient
aryl halides, with low catalyst loadings and minimal protodeboro-
nation.24 Therefore, we prepared potassium (3,5-dimethylisox-
azol-4-yl)trifluoroborate 8 from (3,5-dimethylisoxazol-4-yl)boronic
acid.24 Coupling of 8 to commercially available aryl bromides 5
and 6, and aryl bromide 7 (easily prepared from 3-bromophenol,25
Supporting Information, Scheme S2) afforded 3,5-dimethyl-
4-phenylisoxazole 3a and the 30-substituted 3,5-dimethyl-4-phe-
nylisoxazoles 3b and 3c in excellent yields (Scheme 1). Although
the coupling reactions did proceed when conducted with (3,5-
dimethylisoxazol-4-yl)boronic acid (data not shown), the yields
observed were lower than those in the route described above.
The 30,50-disubstituted 3,5-dimethyl-4-phenylisoxazoles 4aꢀc
were prepared from 3-bromo-5-hydroxybenzoic acid 9 (Scheme 2).
Alkylation of 9 afforded the ethyl ester 10, which was coupled to
8, yielding 4a. Subsequent LiOH-mediated hydrolysis furnished
the carboxylic acid 4b. Alternatively, the ethyl ester of 10 was
converted to the acetyl group via the Weinreb amide 12 in three
steps; coupling, under the same conditions employed above, yielded
the isoxazole 4c. The methyl ketones 3b and 4c were converted to
the racemic secondary alcohols 3d and 4d by reduction of the
ketone with NaBH4 (Scheme 3).
The syntheses of 3aꢀd and 4aꢀd are shown in Schemes 1ꢀ3.
Initial work focused on the direct arylation of 3,5-dimethylisox-
azole using Pd-catalyzed CꢀH bond activation.23 While thisreaction
proceeded well with bromobenzene 5, lower yields were obtained
on reaction with 6 and 10 (Supporting Information, Scheme S1).
Furthermore, the reaction has been reported to be less successful
with electron-rich aryl bromides.23 Conversely, Molander et al.
have demonstrated that potassium heteroaryltrifluoroborates
6763
dx.doi.org/10.1021/jm200640v |J. Med. Chem. 2011, 54, 6761–6770

Journal of Medicinal Chemistry
ARTICLE
Scheme 2. Synthesis of the 30,50-Substituted 3,5-Dimethyl-
Scheme 3. Synthesis of the Secondary Alcohols 3d and 4da
4-phenylisoxazole Derivatives 4aꢀca
a Conditions: (a) NaBH4, MeOH, rt, 2 h, R = H: 77%. R = OEt: 81%.
Table 1. Effect of 30- and 50-Substituents on Competitive
Displacement of Histone Peptides from the BRD4(1) and
BRD2(1), and CREBBP Bromodomaina
a Conditions: (a) EtBr, K2CO3, DMF, 100 °C (microwave), 15 min,
96%; (b) 8, Na2CO3, Pd(OAc)2, RuPhos, EtOH, 100 °C, 48 h, 70%;
(c) LiOH, THF, H2O, rt, 16 h, 83%; (d) LiOH, THF, H2O, rt, 23 h,
a Protein and peptide concentration: 50 nM. Protein and peptide
concentration: 100 nM. Heat map shows relative IC50 values; red
indicates low IC50 values, and green indicates high IC50 values. Ranges
in parentheses represent 95% confidence intervals resulting from
sigmoidal curve fitting to duplicate data. n.d. = not determined.
i
95%; (e) NHMe(OMe) HCl, HBTU, Pr2NEt, DMF, rt, 14 h, 80%;
3
(f) MeMgBr, THF, rt, 15 h, 86%; (g) 8, Na2CO3, Pd(OAc)2, RuPhos,
EtOH, 85 °C, 64 h, 67%.
To enable accurate comparison with our new compounds, a
sample of 1was resynthesized (Supporting Information, Scheme S3).
Biological evaluation of 1 was conducted using freshly prepared
DMSO solutions to minimize the possibility of oxidation (see
Supporting Information for details).
the exception of R1 = CO2H in which the positioning of a
negatively charged substituent in a hydrophobic site is likely to
disfavor binding. IC50 values were generally higher against
CREBBP, except for 4b (R1 = CO2H) where the IC50 for
CREBBP was lower than that for BRD4(1) and similar to
that for BRD2(1). Gratifyingly, 4b and 4d did not show
any cytotoxicity in a human cell line (Supporting Information,
Figure S2).
To investigate the selectivity of these compounds against
other bromodomains, we employed a binding assay based on
protein stability shift (Table 2).26 Of the compounds that produced
substantial changes in melting temperature (ΔTmobs), most dis-
played selectivity for BET bromodomains. However, 4bꢀd also
produce an appreciable ΔTmobs with CREBBP, in agreement with
the IC50 values. Indeed, 4b showed selectivity, albeit modest, for
CREBBP over the BET bromodomains.
To determine whether the compounds inhibited bromodo-
mainꢀhistone binding, we utilized a peptide displacement
assay based on AlphaScreen technology.21 For the first bromo-
domain of BRD2 [BRD2(1)] and BRD4(1), a histone H4
peptide acetylated at K5, 8, 12, and 16 was employed, while
for CREBBP an H3 peptide acetylated at K56 was used. IC50
values for compounds 1, 3aꢀd, and 4aꢀd are shown in Table 1
(doseꢀresponse curves for 1, compounds 4b and 4d in BRD4(1),
BRD2(1), and CREBBP are shown in Supporting Information,
Figure S4). With the exception of 3a, all compounds inhibited
BRD4(1) and BRD2(1) with IC50 e 50 μM, the most potent
compounds showing low micromolar activity. Introduction of an
ethoxy substituent at R2 increases potency against BRD4(1) and
BRD2(1). A variety of substituents at R1 are well tolerated, with
In order to confirm that the dimethylisoxazole moiety was acting
as a KAc mimic and to elucidate structureꢀactivity profiles for
6764
dx.doi.org/10.1021/jm200640v |J. Med. Chem. 2011, 54, 6761–6770

Journal of Medicinal Chemistry
ARTICLE
Table 2. ΔTmobs against Selected Bromodomainsa
a Compound concentration, 100 μM; protein concentration, 2 μM. Heat map shows relative ΔTmobs; red indicates large ΔTmobs, and green indicates
small ΔTmobs. Experiments were carried out in duplicate; average values are reported.
Figure 3. (A) X-ray crystal structure of 4d (carbon = yellow) bound to human BRD4(1) (PDB ID: 3SVG). The conserved N140 residue is highlighted
in green. Black dashed lines represent hydrogen bonds between 4d, structured water molecules (red spheres), and interacting residues (labeled). Image
generated using PyMOL. (B) A schematic representation of the key interactions formed between 4d and human BRD4(1).
these compounds, we obtained cocrystal structures of BRD4(1)
with 4d and the CREBBP bromodomain with 4b (Figures 3
and 4).
hydrophobic pocket (pocket 2, Figure 3B) that is defined by
I146, M149, and W81, the latter forming part of the WPF shelf
(described by Nicodeme et al. as W81, P82, and F83).19a This
is the same pocket that binds the ethylene glycol unit of 2
(Supporting Information, Figure S3C) and the chlorophenyl
moieties of I-BET and (+)-JQ1 (Supporting Information, Figure
S3D). The ethyl ether binds within the ZA channel (pocket 3,
Figure 3B) that accommodates the thiophene moiety of (+)-JQ1
and the methylated phenol of I-BET. Therefore, analysis of the
X-ray crystal structure of 4d in complex with BRD4(1) shows
that this compound is forming interactions in many of the same
regions as (+)-JQ1 and I-BET, perhaps explaining how a
compound with relatively low molecular weight (261) still shows
good affinity for BRD4(1) and reasonable selectivity for the BET
subfamily of bromodomains. Indeed, 4d has ligand efficiencies of
0.39 for BRD4(1) and 0.43 for BRD2(1), which compare
favorably with the value of 0.32 for (+)-JQ1 at BRD4(1) (see
Supporting Information, Table S1 for details of ligand efficiency
calculations). These ligand efficiencies suggest that compound 4d
is a promising lead for the development of BET bromodomain
The costructure of 4d bound to BRD4(1) was obtained at 1.68 Å
resolution (PDB ID: 3SVG). Globally, the ligand-bound and apo
structures (PDB ID: 2OSS) are very similar, with a rmsd of 0.17.
This structure confirms that 4d binds with the dimethylisoxazole
oriented into the KAc-binding pocket as anticipated by our
docking studies (Supporting Information, Figure S3B). It is
predicted that the isoxazole oxygen atom forms the expected
hydrogen bond with the amine group of the conserved N140
(green, Figure 3A). The isoxazole nitrogen atom interacts with
the phenol group of Y97 via the structured water molecule A
(Figure 3B).
Although 4d was synthesized as a racemate, the electron density
for this compound implies that the (R)-enantiomer crystallizes
preferentially with BRD4(1). The secondary alcohol forms a network
of hydrogen bonds with water molecules BꢀE, which interact
with D144, D145, and the carbonyl oxygen atom of N140. The
methyl group attached to the secondary alcohol binds within the
6765
dx.doi.org/10.1021/jm200640v |J. Med. Chem. 2011, 54, 6761–6770

Journal of Medicinal Chemistry
ARTICLE
Figure 4. (A) X-ray crystal structure of 4b (carbon = yellow) bound to the bromodomain of human CREBBP (PDB ID: 3SVH). The conserved N1168
residue is highlighted in green. Black dashed lines represent hydrogen bonds among 4b, structured water molecules (red spheres), and interacting
residues (labeled). Image generated using PyMOL. (B) Schematic representation of the key interactions formed between 4b and the bromodomain of
human CREBBP.
ligands that display potencies similar to those of (+)-JQ1 and
I-BET but with lower molecular mass than those of these
compounds.
explain, at least in part, the preferential binding of this compound to
CREBBP over the BET bromodomains. It should be noted that
CREBBP crystallized as a dimer, with a second molecule of 4b
bound at the dimer interface. This phenomenon is thought to result
from the relatively high concentration of 4b used in obtaining the
structure and is unlikely to be responsible for this compound’s ability
to prevent the CREBBPꢀKAc interaction.
Identification of CREBBP bromodomain inhibitors has so far
proved challenging, with only two studies identifying small-mole-
cule ligands for this bromodomain.14,15 Therefore, given that
compound 4b shows some selectivity for CREBBP bromodo-
main over other subfamilies, the costructure of 4b bound to the
CREBBP bromodomain was obtained at 1.80 Å resolution (PDB
ID: 3SVH). Compound 4b, which possesses a carboxylic acid
group in place of the secondary alcohol of 4d, binds to the
CREBBP bromodomain in an orientation similar to that adopted
by 4d when binding to BRD4(1). The dimethylisoxazole moiety
again acts as a KAc mimic and occupies the KAc-binding pocket.
Likewise, the isoxazole oxygen atom forms a hydrogen bond with
the amine group of N1168 (equivalent to N140 in BRD4(1),
green, Figure 4A). The isoxazole nitrogen atom also interacts
with the phenol group of Y1125 [equivalent to Y97 in BRD4(1)]
via the structured water molecule A. The dihedral angle between
the 3,5-dimethylisoxazole and the phenyl ring of compounds 2,
4b, and 4d differs in the crystal structures obtained. However,
this angle is observed as 50° in the case of 2 [BRD4(1)], 45° in
the case of 4d [BRD4(1)] and 39° in the case of 4b (CREBBP),
which are all similar to the angle of 40° in the predicted lowest
energy conformation (Supporting Information, Figure S5), suggesting
that these compounds can bind in the KAc-binding pocket
without the introduction of significant strain. The oxygen atom
of the ethoxy group in 4b forms a hydrogen bond with structured
water G, which interacts with the turn-forming residues
1110ꢀ1113. Interestingly, this structured water molecule is also
present in BRD4(1), but the orientation of the ethoxy group in
compound 4d is such that it does not form a similar hydrogen
bond, at least within crystals. Residue W81, which plays a key
role in defining the binding site in BRD4(1), is not present in
CREBBP. The equivalent amino acid is L1109, which although
also hydrophobic, is oriented away from the binding site,
providing more space in this region. R1173 provides a basic
rim to the binding pocket, which is not present in BRD4.
Although these residues are not in direct contact with the carboxylic
acid of 4b, it is possible that a weak electrostatic interaction might
In summary, we have discovered an entirely new chemical class
of bromodomain ligands comprising low-molecular weight
4-phenyl-3,5-dimethylisoxazole derivatives. Using structure-based
approaches, we have demonstrated that the unstable dihydroqui-
nazolinone moiety of lead 1 can be replaced with a single aromatic
ring without significant loss of affinity. Most of the resultant
4-phenyl-3,5-dimethylisoxazoles are selective ligands for the BET
family of bromodomains. However, compounds4bꢀd produced a
marked increase in Tmobs for CREBBP, suggesting that 3,5-dimethyl-
isoxazoles are also useful leads for selective inhibitors of CREBBP-
KAc interactions, which have proved elusive to date. The costructures
that we have obtained demonstrate that the 3,5-dimethylisox-
azole moiety acts as a KAc bioisostere in two classes of bromo-
domains, corroborating the binding data and IC50 values pre-
sented. As few chemical classes of small-molecule inhibitors
of bromodomainꢀhistone interactions have been described pre-
viously,15ꢀ20 this work represents a significant advance in the
SAR of these important proteins.
In conclusion, we have exploited the serendipitous discovery
of a KAc mimic to develop a novel class of compounds that inhibit
bromodomainꢀhistone interactions. Crystallographic analysis and
rational design have provided insight into the structural require-
ments for potent, selective BRD4(1) and CREBBP bromodo-
main ligands. These compounds are invaluable leads in the
continuing search for chemical probes of epigenetic targets.
’ EXPERIMENTAL SECTION
General Experimental. 1H NMR spectra were recorded on Bruker
DPX400 or DQX400 (400 MHz) or Bruker AVII 500 (500 MHz) using
deuterochloroform (unless indicated otherwise) as a reference for the
internal deuterium lock. The chemical shift data for each signal are given
as δH in units of parts per million (ppm) relative to tetramethylsilane
6766
dx.doi.org/10.1021/jm200640v |J. Med. Chem. 2011, 54, 6761–6770

Journal of Medicinal Chemistry
ARTICLE
(TMS) where δH (TMS) = 0.00 ppm. The multiplicity of each signal is
indicated by s (singlet); br s (broad singlet); d (doublet); t (triplet);
q (quartet); dd (doublet of doublets); ddd (doublet of doublet of doublets);
or m (multiplet). The number of protons (n) for a given resonance signal is
indicated by nH. Coupling constants (J) are quoted in Hz and are recorded to
the nearest 0.1 Hz. Identical proton coupling constants (J) are averaged in
each spectrum and reported to the nearest 0.1 Hz. The coupling constants are
determined by analysis using Bruker TopSpin software.
500 μmol) and degassed EtOH (3.8 mL) were added by syringe, and
the mixture was heated at 85 °C for 13 h. The mixture was cooled to rt and
filtered through a thin pad of silica (eluent 25% MeOH/EtOAc) and
concentrated in vacuo. Purification by silica gel column chromatography
(gradient elution, gradient 6f 50% EtOAc/petroleum ether) gave3basa
pale yellow solid (93 mg, 86%); mp 107ꢀ110 °C (50% Et2O/40ꢀ60 °C
petroleum ether); 1H NMR (400 MHz, CDCl3) 2.29 (s, 3H), 2.43 (s,
3H), 2.65 (s, 3H), 7.44ꢀ7.49 (m, 1H), 7.53ꢀ7.60 (m, 1H), 7.85ꢀ7.89
(m, 1H), 7.93ꢀ7.98 (m, 1H); m/z (ES+) 216 ([M + H]+, 54), 238 ([M +
Na]+, 100), 453 ([2M + Na]+, 65). These dataare in good agreement with
the literature values.23
13C NMR spectra were recorded on Bruker DQX400 (100 MHz) or
Bruker AVII 500 (125 MHz) spectrometers with broadband proton
decoupling and a internal deuterium lock. The chemical shift data for
each signal are given as δC in units of parts per million (ppm) relative to
tetramethylsilane (TMS) where δC (TMS) = 0.00 ppm.
Method B. To a dry 2ꢀ5 mLmicrowavevialwereaddedPdCl2 (0.5 mg,
3 μmol) and KOAc (98 mg, 1.0 mmol). The vial was sealed and purged
with argon (3ꢁ evacuate/fill). Degassed DMAc (2.5 mL), 1-(3-bromo-
phenyl)ethanone 6 (66 μL, 100 mg, 502 μmol), and 3,5-dimethylisox-
azole (74 μL, 73 mg, 752 μmol) were added, and the reaction was heated
at 130 °C for 20 h. The mixture was filtered through Celite (eluent
EtOAc), and the filtrate was washed with water (2 ꢁ 25 mL) and brine
(25 mL), dried (MgSO4), filtered and concentrated in vacuo. Purification
by flash column chromatography (gradient elution, gradient 6 f 50%
EtOAc/petroleum ether) gave 3b as a pale yellow solid (55 mg, 51%).
4-(3-Ethoxyphenyl)-3,5-dimethylisoxazole 3c. To a dry 2ꢀ5 mL
microwave vial were added 8 (108 mg, 532 μmol), Pd(OAc)2 (2 mg,
8 μmol), RuPhos (7 mg, 15 μmol), and anhydrous Na2CO3 (106 mg,
1.00 mmol). The vial was sealed and purged with argon (3ꢁ evacuate/
fill). Compound 7 in degassed EtOH (178 μM, 2.8 mL, 499 μmol) was
added by syringe, and the mixture was heated at 85 °C for 2 h. The
mixture was cooled to rt and filtered through a thin pad of silica (eluent
CH2Cl2) and concentrated in vacuo. Purification by silica gel column
chromatography (gradient elution, gradient 5 f 40% Et2O/petroleum
ether) gave 3c as a pale yellow solid (94 mg, 87%); mp 51ꢀ52 °C (15%
19F NMR spectra were recorded on Bruker AVII 500 (470 MHz)
using a broadband proton decoupling pulse sequence and deuterium
internal lock. The chemical shift data for each signal are given as δF in
units of parts per million (ppm).
11B NMR spectra were recorded on a Bruker DRX500 (160 MHz).
The chemical shift data for each signal are given as δB in units of parts per
million (ppm).
Mass spectra were acquired on a VG platform spectrometer. Electro-
spray ionization spectra were obtained on Micromass LCT Premier and
Bruker MicroTOF spectrometers, operating in positive or negative
mode, from solutions of MeOH. m/z values are reported in Daltons
and followed by their percentage abundance in parentheses.
Melting points were determined using a Kofler hot stage microscope
and are uncorrected.
Compound purity for all tested compounds was determined by elemental
analysis, obtained at the Elemental Analysis Service, London Metropolitan
University, London. Elemental analysis was carried out in duplicate; average
values are reported in Supporting Information. For all tested compounds,
experimentally determined hydrogen, carbon, and nitrogen composition was
within (0.4% of the expected value, implying a purity of >95%.
1
Et2O/petroleum ether); H NMR (400 MHz, CDCl3) 1.44 (t, J =
7.0 Hz, 3H), 2.27 (s, 3H), 2.41 (s, 3H), 4.06 (q, J = 7.0, 2H), 6.78 (dd, J =
2.4, 1.6 Hz, 1H), 6.82 (ddd, J = 7.7, 1.6, 0.9 Hz, 1H), 6.89 (ddd, J = 8.2,
2.4, 0.9 Hz, 1H), 7.33 (dd, J = 8.2, 7.7 Hz, 1H); HRMS m/z (ES+) found
[M + Na]+ 240.0994, C13H15NNaO2 requires M+ 240.0995; m/z (ES+)
218 ([M + H]+, 47), 240 ([M + Na]+, 100), 457 ([2M + Na]+, 99).
(RS)-1-(3-(3,5-Dimethylisoxazol-4-yl)phenyl)ethanol 3d. To a solu-
tion of 3b (100 mg, 465 μmol) in anhydrous MeOH (2 mL) at 0 °C was
added NaBH4 (18 mg, 476 μmol). The mixture was warmed to rt and
stirred for 2 h, then quenched with saturated aqueous NH4Cl (10 mL).
The solution was extracted with CH2Cl2 (3 ꢁ 10 mL), and the combined
organic layers were washed with water (30 mL) and brine (30 mL), then
dried (MgSO4), filtered, and concentrated in vacuo. Purification by silica
gel column chromatography (gradient elution, gradient 8 f 70% EtOAc/-
40ꢀ60 °C petroleum ether) gave 3d as a colorless solid (78 mg, 77%);
Synthetic Procedures and Characterization for Compounds
3aꢀd, 4aꢀd. 3,5-Dimethyl-4-phenylisoxazole 3a27. Method A. To a
dry 2ꢀ5 mL microwave vial were added 8 (108 mg, 532 μmol), Pd(OAc)2
(2 mg, 8 μmol), RuPhos (7 mg, 15 μmol), and anhydrous Na2CO3
(106 mg, 1.00 mmol). The vial was sealed and purged with argon (3 ꢁ
evacuate/fill). Bromobenzene 5 (79 mg, 53 μL, 496 μmol) and degassed
EtOH (2.8 mL) were added by syringe, and the mixture was heated at 85 °C
for 4 h. The mixture was cooled to rt and filtered through a thin pad of silica
(eluent CH2Cl2) and concentrated in vacuo. Purification by silica gel column
chromatography (gradient elution, gradient 6 f30% Et2O/petroleum ether)
gave 3a as a colorless oil which crystallized slowly on standing (73 mg,
85%); mp 37ꢀ38 °C (lit. 42ꢀ43 °C);27 1H NMR (400 MHz, CDCl3) 2.29
(s, 3H), 2.42 (s, 3H), 7.24ꢀ7.29 (m, 2H), 7.34ꢀ7.40 (m, 1H), 7.42ꢀ7.48
(m, 2H); m/z (ES+) 174 ([M + H]+, 73), 196 ([M + Na]+, 100), 369
([2M + Na]+, 19). These data are in good agreement with the literature
values.27
Method B. To a dry 2ꢀ5 mL microwave vial were added PdCl2
(0.5 mg, 3 μmol) and KOAc (98 mg, 1.0 mmol). The vial was sealed and
purged with argon (3ꢁ evacuate/fill). Degassed DMAc (2.5 mL),
bromobenzene 5 (53 μL, 79 mg, 503 μmol), and 3,5-dimethylisoxazole
(74 μL, 73 mg, 752 μmol) were added, and the reaction was heated at
130 °C for 27 h. The mixture was filtered through Celite (eluent Et2O),
and the filtrate was washed with water (40 mL) and brine (40 mL), dried
(MgSO4), filtered and concentrated in vacuo. Purification by flash
column chromatography (gradient elution, gradient 3 f 30% Et2O/
petroleum ether) gave 3a as a pale yellow solid (60 mg, 69%).
1ꢀ3-(3,5-Dimethylisoxazol-4-yl)phenyl)ethanone 3b23. Method A.
To a dry 2ꢀ5 mL microwave vial were added 8 (108 mg, 532 μmol),
Pd(OAc)2 (2 mg, 8 μmol), RuPhos (7 mg, 15 μmol), and anhydrous
Na2CO3 (106 mg, 1.00 mmol). The vial was sealed and purged with argon
(3ꢁ evacuate/fill). 1-(3-Bromophenyl)ethanone 6 (100 mg, 66 μL,
1
mp 72ꢀ73 °C (40% EtOAc/40ꢀ60 °C petroleum ether); H NMR
(400 MHz, CDCl3) 1.53 (d, J = 6.4 Hz, 3H), 2.26 (s, 3H), 2.30 (br s,
1H), 2.40 (s, 3H), 4.95 (q, J = 6.4 Hz, 1H), 7.13ꢀ7.17 (m, 1H),
7.26ꢀ7.29 (m, 1H), 7.34ꢀ7.44 (m, 2H); HRMS m/z (ES+) found [M +
Na]+ 240.1002, C13H15NNaO3 requires M+ 240.0995; m/z (ES+) 218
([M + H]+, 26), 240 ([M + Na]+, 81), 457 ([2M + Na]+, 100).
Ethyl 3-(3,5-dimethylisoxazol-4-yl)-5-ethoxybenzoate 4a. Method
A. To a dry 10ꢀ20 mL microwave vial were added 8 (408 mg, 2.01
mmol), 10 (500 mg, 1.83 mmol), Pd(OAc)2 (4 mg, 16 μmol), RuPhos
(26mg, 56μmol), andanhydrousNa2CO3 (388 mg, 3.66mmol). The vial
was sealed and purged with argon (3ꢁ evacuate/fill). Degassed EtOH
(10 mL) was added by syringe, and the mixture was heated at 100 °C for
48 h. The mixture was cooled to rt and filtered through a thin
pad of silica (eluent CH2Cl2) and concentrated in vacuo. Purification
by silica gel column chromatography (gradient elution, gradient 6 f 50%
Et2O/petroleum ether) gave 4a as a colorless solid (370 mg, 70%); mp
102ꢀ105 °C (CH2Cl2); 1H NMR (400 MHz, CDCl3) 1.40 (t, J = 7.1 Hz,
3H), 1.46 (t, J = 7.0 Hz, 3H), 2.28 (s, 3H), 2.42 (s, 3H), 4.11 (q,
6767
dx.doi.org/10.1021/jm200640v |J. Med. Chem. 2011, 54, 6761–6770

Journal of Medicinal Chemistry
ARTICLE
J = 7.0 Hz, 2H), 4.40 (q, J = 7.1 Hz, 2H), 6.96 (dd, J = 2.3, 1.7 Hz, 1H),
7.52 (dd, J = 1.7, 1.4 Hz, 1H), 7.55 (dd, J = 2.3, 1.4 Hz, 1H); HRMS m/z
(ES+) found [M + Na]+ 312.1204, C16H19NNaO4 requires M+ 312.1206;
m/z (ES+) 290 ([M + H]+, 29), 312 ([M + Na]+, 50), 602 ([2M + Na]+,
100).
Method B. To a dry 0.5ꢀ2 mL microwave vial were added PdCl2
(0.5 mg, 3 μmol), KOAc (36 mg, 367 μmol), and 10 (28 mg, 103 μmol).
The vial was sealed and purged with argon (3ꢁ evacuate/fill). Degassed
DMAc (1 mL) and 3,5-dimethylisoxazole (15 μL, 15 mg, 154 μmol)
were added, and the reaction was heated at 130 °C for 44 h. The mixture
was filtered through Celite (eluent EtOAc), and the filtrate was washed
with water (2 ꢁ 30 mL) and brine (30 mL), dried (MgSO4), and con-
centrated in vacuo. Purification by flash column chromatography
(gradient elution, gradient 5 f 60% EtOAc/petroleum ether) gave 4a
as a colorless solid (25 mg, 44%).
3-(3,5-Dimethylisoxazol-4-yl)-5-ethoxybenzoic acid 4b. To a solu-
tion of 4a (300 mg, 1.04 mmol) in THF (5 mL) were added water
(2.5 mL) and LiOH (37 mg, 1.56 mmol), and the mixture was stirred for
16 h at rt. NaOH (1 M, 10 mL) was then added, and the mixture was
washed with Et2O (3 ꢁ 10 mL). The solution was acidified to pH 2 with
HCl (1 M) and extracted with CH2Cl2 (3 ꢁ 40 mL). The combined
organic layers were washed with brine (100 mL), dried (MgSO4),
filtered, and concentrated in vacuo. Repeated recrystallization from
cyclohexane/CHCl3 (2:1) gave 4b as a colorless crystalline solid (225 mg,
83%); mp 191ꢀ193 °C (cyclohexane/CHCl3, 2:1); 1H NMR (500 MHz,
CDCl3) 1.48 (t, J = 7.0 Hz, 3H), 2.31 (s, 3H), 2.45 (s, 3H), 4.14 (q, J =
7.0 Hz, 2H), 7.03ꢀ7.05 (m, 1H), 7.60ꢀ7.63 (m, 2H); HRMS m/z
(ES+) found [M + Na]+ 284.0886, C14H15NNaO4 requires M+ 284.0893;
m/z (ES+) 262 ([M + H]+, 32), 284 ([M + Na]+, 100), 545 ([2M +
Na]+, 75).
1-(3-(3,5-Dimethylisoxazol-4-yl)-5-ethoxyphenyl)ethanone 4c. To
a dry 2ꢀ5 mL microwave vial were added 8 (88 mg, 433 μmol), 13
(100 mg, 411 μmol), Pd(OAc)2 (1 mg, 4 μmol), RuPhos (6 mg,
13 μmol), and anhydrous Na2CO3 (87 mg, 821 μmol). The vial was
sealed and purged with argon (3ꢁ evacuate/fill). Degassed EtOH
(2.3 mL) was added by syringe, and the mixture was heated at 85 °C
for 64 h. The mixture was cooled to rt and filtered through a thin pad of
silica (eluent CH2Cl2) and concentrated in vacuo. Purification by silica
gel column chromatography (gradient elution, gradient 6 f 50%
EtOAc/petroleum ether) gave 4c as a pale yellow solid (71 mg, 67%);
mp 104ꢀ106 °C (CH2Cl2); 1H NMR (500 MHz, CDCl3) 1.47 (t, J =
7.0 Hz, 3H), 2.29 (s, 3H), 2.43 (s, 3H), 2.62 (s, 3H), 4.13 (q, J = 7.0 Hz,
2H), 6.98 (dd, J = 2.2, 1.5 Hz, 1H), 7.41 (dd, J = 1.5, 1.5 Hz, 1H), 7.46
(dd, J = 2.2, 1.5 Hz, 1H); HRMS m/z (ES+) found [M + Na]+ 282.1098,
C15H17NNaO3 requires M+ 282.1101; m/z (ES+) 260 ([M + H]+, 35),
282 ([M + Na]+, 62), 541 ([2M + Na]+, 100).
standard protocol28 implemented in ICM-Pro 3.6-1 (Molsoft LLC).
The costructure of 2 bound to BRD4(1) (PDB ID: 3SVF) was used to
define the receptor; only water molecules interacting with 2 were
included.
Conformational Analysis of Lead Compounds. Conformational
analysis was performed for the two cocrystallized compounds (4b and
4d) using MacroModel (version 9.8) in Schrodinger Suite 2010 from
Schrodinger LLC. The dihedral angle between the two ring systems was
subjected to coordination scan with 1 degree of increment. The chemical
structures from Ligprep were used as the starting structure. The default
option was used, including a force field of OPLS2005, a solvent of water,
and a PRCG minimization protocol with a convergence gradient of 0.05.
The relative energy in kcal/mol of each minimized structure was then
plotted versus the dihedral angle.
Biological Evaluation of Compounds. Protein Expression and
Purification. Proteins were cloned, expressed, and purified as previously
described.18
Peptides. H4Ac4 peptide (BRD2 and BRD4 assays, H2N-SGRGK-
(Ac)GGK(Ac)GLGK(Ac)-GGAK(Ac)RHRK(Biotin)-COOH) and
H3K56Ac peptide (CREBBP assay, H2N-ALREIRRYQK(Ac)STELLI-
RKLK(Biotin)-COOH) were synthesized by Tufts University Core
Facility.
Thermal Stability Shift Assay. Thermal melting experiments were
carried out using the Mx3005p real-time PCR machine (Agilent) and
employing a protein concentration of 2 μM and an inhibitor concentra-
tion of 100 mM. Buffer conditions were 10 mM HEPES, pH 7.5,
500 mM NaCl and 1:1000 dilution of SyproOrange (Invitrogen, CA).
The assay and data evaluation were carried out as described previously.29
Experiments were performed in duplicate; average values are reported.
AlphaScreen Peptide Displacement Assay. Bromodomain AlphaSc-
reen assays were carried out as previously described.18,21 All experiments
were carried out in duplicate on the same plate. Interexperimental IC50
values generated for individual compounds typically varied over an ∼3-
fold range. However, rank order remained highly consistent between
repeat experiments, with Spearman rank order correlations for 3aꢀd
and 4aꢀd >0.90 for repeats of each assay. Thus, for the purposes of SAR,
data in Table 1 is given from a single assay, with intraexperimental
variability expressed as 95% confidence intervals resulting from sigmoi-
dal curve fitting to duplicate data.
Cytotoxicity Assay. HeLa cells were subcultured in DMEM supple-
mented with L-glutamine and 10% fetal calf serum. 20000 cells per well
were seeded into a 96 well plate. After 24 h of incubation at 37 °C, the
medium was changed, and compounds were applied at final concentra-
tions of 10, 1, 0.1, and 0.01 μM. The cells were incubated for 24 or 48 h,
after which time the medium was changed, and MTT solution (5 mg/
mL) was added to the plate. After 2 h of incubation at 37 °C, the medium
was replaced with 200 μL of DMSO to solubilize the formazan crystals.
After a further 15 min, absorbance was measured at 550 nm using a
spectrophotometer. Experiments were performed in triplicate on the
same plate.
(RS)-1-(3-(3,5-Dimethylisoxazol-4-yl)-5-ethoxyphenyl)ethanol 4d.
To a solution of 4c (30 mg, 123 μmol) in anhydrous MeOH (1 mL)
at 0 °C was added NaBH4 (6 mg, 159 μmol). The mixture was warmed
to rt and stirred for 2 h, then quenched with saturated aqueous NH4Cl
(10 mL). The solution was extracted with CH2Cl2 (3 ꢁ 10 mL), and the
combined organic layers were washed with water (30 mL) and brine
(30 mL), then dried (MgSO4), filtered, and concentrated in vacuo.
Purification by silica gel column chromatography (gradient elution,
gradient 10 f 100% EtOAc/40ꢀ60 °C petroleum ether) gave 4d
as a pale yellow viscous oil (26 mg, 81%); 1H NMR (400 MHz, CDCl3)
1.44 (t, J = 7.0 Hz, 3H), 1.52 (d, J = 6.4 Hz, 3H), 2.14 (br s, 1H), 2.27 (s,
3H), 2.41 (s, 3H), 4.07 (q, J = 7.0 Hz, 2H), 4.91 (q, J = 6.4 Hz, 1H),
6.66ꢀ6.69 (m, 1H), 6.81ꢀ6.84 (m, 1H), 6.92ꢀ6.95 (m, 1H); HRMS
m/z (ES+) found [M + Na]+ 284.1246, C15H15NNaO3 requires
M+ 284.1257; m/z (ES+) 262 ([M + H]+, 21), 284 ([M + Na]+, 47),
545 ([2M + Na]+, 100).
’ ASSOCIATED CONTENT
S
Supporting Information. Supporting figures, schemes, and
b
tables, crystallographic information, further general experimental
details, further characterization for compounds 3aꢀd, 4aꢀd, and
synthesis and characterization of compounds 1, 7, 8, and 10ꢀ13.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Accession Codes
†Atomic coordinates and structure factors for the reported crystal
structures have been deposited with the Protein Data Bank under
accession codes 3SVF (2), 3SVG (4d), and 3SVH (4b).
Computational Procedures. Docking Procedures for Structure-
Aided Design. Docking of compounds was carried out using the
6768
dx.doi.org/10.1021/jm200640v |J. Med. Chem. 2011, 54, 6761–6770

Journal of Medicinal Chemistry
ARTICLE
’ AUTHOR INFORMATION
function of a human TAFII250 double bromodomain module. Science
2000, 288, 1422–1425. (e) Owen, D. J.; Ornaghi, P.; Yang, J. C.; Lowe,
N.; Evans, P. R.; Ballario, P.; Neuhaus, D.; Filetici, P.; Travers, A. A. The
structural basis for the recognition of acetylated histone H4 by the
bromodomain of histone acetyltransferase gcn5p. EMBO J. 2000, 19,
6141–6149.
Corresponding Author
*(S.J.C.) Tel: +44 (0)1865 285 109. Fax: +44 (0)1865 285 002.
E-mail: stuart.conway@chem.ox.ac.uk. (T.D.H.) Tel: +44 (0)1223
226270. E-mail: t.heightman@astex-therapeutics.com.
(4) (a) Strahl, B. D.; Allis, C. D. The language of covalent histone
modifications. Nature 2000, 403, 41–45. (b) Winston, F.; Allis, C. D.
The bromodomain: a chromatin-targeting module?. Nat. Struct. Biol.
1999, 6, 601–604. (c) Jenuwein, T.; Allis, C. D. Translating the histone
code. Science 2001, 293, 1074–1080. (d) Agalioti, T.; Chen, G.; Thanos,
D. Deciphering the transcriptional histone acetylation code for a human
gene. Cell 2002, 111, 381–392. (e) Loyola, A.; Almouzni, G. Bromodo-
mains in living cells participate in deciphering the histone code. Trends
Cell Biol. 2004, 14, 279–281.
(5) Florence, B.; Faller, D. V. You bet-cha: a novel family of transcrip-
tional regulators. Front. Biosci. 2001, 6, D1008–1018.
(6) Yang, Z.; He, N.; Zhou, Q. Brd4 recruits P-TEFb to chromo-
somes at late mitosis to promote G1 gene expression and cell cycle
progression. Mol. Cell. Biol. 2008, 28, 967–976.
(7) Crawford, N. P.; Alsarraj, J.; Lukes, L.; Walker, R. C.; Officewala,
J. S.; Yang, H. H.; Lee, M. P.; Ozato, K.; Hunter, K. W. Bromodomain 4
activation predicts breast cancer survival. Proc. Natl. Acad. Sci. U.S.A.
2008, 105, 6380–6385.
(8) (a) French, C. A.; Kutok, J. L.; Faquin, W. C.; Toretsky, J. A.;
Antonescu, C. R.; Griffin, C. A.; Nose, V.; Vargas, S. O.; Moschovi, M.;
Tzortzatou-Stathopoulou, F.; Miyoshi, I.; Perez-Atayde, A. R.; Aster,
J. C.; Fletcher, J. A. Midline carcinoma of children and young adults with
NUT rearrangement. J. Clin. Oncol. 2004, 22, 4135–4139. (b) Haruki,
N.; Kawaguchi, K. S.; Eichenberger, S.; Massion, P. P.; Gonzalez, A.;
Gazdar, A. F.; Minna, J. D.; Carbone, D. P.; Dang, T. P. Cloned fusion
product from a rare t(15;19)(q13.2;p13.1) inhibit S phase in vitro.
J. Med. Genet. 2005, 42, 558–564.
(9) Zhou, M.; Huang, K.; Jung, K. J.; Cho, W. K.; Klase, Z.; Kashanchi,
F.; Pise-Masison, C. A.; Brady, J. N. Bromodomain protein Brd4 regulates
human immunodeficiency virus transcription through phosphorylation of
CDK9 at threonine 29. J. Virol. 2009, 83, 1036–1044.
(10) Lin, A.; Wang, S.; Nguyen, T.; Shire, K.; Frappier, L. The EBNA1
protein of Epstein-Barr virus functionally interacts with Brd4. J. Virol.
2008, 82, 12009–12019.
(11) Gagnon, D.; Joubert, S.; Senechal, H.; Fradet-Turcotte, A.; Torre,
S.; Archambault, J. Proteasomal degradation of the papillomavirus E2
protein is inhibited by overexpression of bromodomain-containing protein
4. J. Virol. 2009, 83, 4127–4139.
(12) Huang, B.; Yang, X. D.; Zhou, M. M.; Ozato, K.; Chen, L. F. Brd4
coactivates transcriptional activation of NF-kappaB via specific binding to
acetylated RelA. Mol. Cell. Biol. 2009, 29, 1375–1387.
(13) Shang, E.; Nickerson, H. D.; Wen, D.; Wang, X.; Wolgemuth,
D. J. The first bromodomain of Brdt, a testis-specific member of the BET
sub-family of double-bromodomain-containing proteins, is essential for
male germ cell differentiation. Development 2007, 134, 3507–3515.
(14) Sachchidanand; Resnick-Silverman, L.; Yan, S.; Mutjaba, S.;
Liu, W. J.; Zeng, L.; Manfredi, J. J.; Zhou, M. M. Target structure-based
discovery of small molecules that block human p53 and CREB binding
protein association. Chem. Biol. 2006, 13, 81–90.
Present Addresses
Astex Therapeutics, 436 Cambridge Science Park, Cambridge,
CB4 0QA, U.K.
’ ACKNOWLEDGMENT
We thank Cancer Research U.K. for a studentship (to D.S.H.). S.J.
C. thanks St. Hugh’s College, Oxford, for research support. The
Structural Genomics Consortium is a registered charity (number
1097737) that receives funds from the Canadian Institutes for
Health Research, the Canadian Foundation for Innovation, Genome
Canada through the Ontario Genomics Institute, GlaxoSmithKline,
Karolinska Institutet, the Knut and Alice Wallenberg Foundation,
the Ontario Innovation Trust, the Ontario Ministry for Research and
Innovation, Merck & Co., Inc., the Novartis Research Foundation,
the Swedish Foundation for Strategic Research, and the Wellcome
Trust. We are grateful to Timothy Rooney for the preparation of
6-bromo-3-methyl-3,4-dihydroquinazolin-2(1H)-one and to Dr.
Rod Chalk and Dr. James Wickens for MS studies. We thank Dr.
Paul Brennan for useful discussions.
’ ABBREVIATION USED
BET, bromodomain and extra terminal domain; BRD, bromo-
domain-containing protein; BRD2(1)/(2), first/second bromo-
domain of BRD2; BRD4(1)/(2), first/second bromodomain
of BRD4; BRDT, bromodomain, testis-specific; CREB, cAMP
response element-binding protein; CREBBP, CREB binding
protein; DMAc, N,N-dimethylacetamide; EBV, EpsteinꢀBarr virus;
GCN5, general control of amino acid synthesis, yeast, homo-
logue-like 2; HAT, histone acetyltransferase; HBTU, O-(benzo-
triazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate;
HDAC, histone deacetylase; KAc, acetyl-lysine; MTT, 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenylltetrazolium bromide; NUT, nuclear
protein in testis; P-TEFb, positive transcription elongation
factor b; PB1(5), fifth bromodomain of PB1 (BAF180), a unique
subunit of the polybromo (PBAF) complex; PCAF, P300/
CREBBP-associated factor; RuPhos, 2-dicyclohexylphosphino-
20,60-diisopropoxybiphenyl; TAFII250, TATA box binding pro-
tein-associated factor, 250 kDa
’ REFERENCES
(1) Turner, B. M. Histone acetylation and an epigenetic code. BioEssays
2000, 22, 836–845.
(15) Borah, J. C.; Mujtaba, S.; Karakikes, I.; Zeng, L.; Muller, M.; Patel, J.;
Moshkina, N.; Morohashi, K.; Zhang, W.; Gerona-Navarro, G.; Hajjar, R. J.;
Zhou, M. M. A small molecule binding to the coactivator CREB-binding
protein blocks apoptosis in cardiomyocytes. Chem. Biol. 2011, 4, 531–541.
(16) Ito, T.; Umehara, T.; Sasaki, K.; Nakamura, Y.; Nishino, N.;
Terada, T.; Shirouzu, M.; Padmanabhan, B.; Yokoyama, S.; Ito, A.;
Yoshida, M. Real-time imaging of histone H4K12-specific acetylation
determines the modes of action of histone deacetylase and bromodo-
main inhibitors. Chem. Biol. 2011, 18, 495–507.
(17) Miyoshi, S.; Ooike, S.; Iwata, K.; Hikawa, H.; Sugahara, K.
Antitumor Agent. Int. Pat. Appl. WO 2009/084693, 2009.
(18) Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W. B.;
Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.;
(2) Choudhary, C.; Kumar, C.; Gnad, F.; Nielsen, M. L.; Rehman, M.;
Walther, T. C.; Olsen, J. V.; Mann, M. Lysine acetylation targets protein
complexes and co-regulates major cellular functions. Science 2009, 325,
834–840.
(3) (a) Dhalluin, C.; Carlson, J. E.; Zeng, L.; He, C.; Aggarwal, A. K.;
Zhou, M. M. Structure and ligand of a histone acetyltransferase
bromodomain. Nature 1999, 399, 491–496. (b) Ornaghi, P.; Ballario,
P.; Lena, A. M.; Gonzalez, A.; Filetici, P. The bromodomain of Gcn5p
interacts in vitro with specific residues in the N terminus of histone H4.
J. Mol. Biol. 1999, 287, 1–7. (c) Hudson, B. P.; Martinez-Yamout, M. A.;
Dyson, H. J.; Wright, P. E. Solution structure and acetyl-lysine binding
activity of the GCN5 bromodomain. J. Mol. Biol. 2000, 304, 355–370.
(d) Jacobson, R. H.; Ladurner, A. G.; King, D. S.; Tjian, R. Structure and
6769
dx.doi.org/10.1021/jm200640v |J. Med. Chem. 2011, 54, 6761–6770

Journal of Medicinal Chemistry
ARTICLE
Philpott, M.; Munro, S.; McKeown, M. R.; Wang, Y.; Christie, A. L.; West,
N.; Cameron, M. J.; Schwartz, B.; Heightman, T. D.; La Thangue, N.;
French, C. A.; Wiest, O.; Kung, A. L.; Knapp, S.; Bradner, J. E. Selective
inhibition of BET bromodomains. Nature 2010, 468, 1067–1073.
(19) (a) Nicodeme, E.; Jeffrey, K. L.; Schaefer, U.; Beinke, S.;
Dewell, S.; Chung, C.-w.; Chandwani, R.; Marazzi, I.; Wilson, P.; Coste,
H.; White, J.; Kirilovsky, J.; Rice, C. M.; Lora, J. M.; Prinjha, R. K.; Lee,
K.; Tarakhovsky, A. Suppression of inflammation by a synthetic histone
mimic. Nature 2010, 468, 1119–1123. (b) Chung, C.-w.; Coste, H.; White,
J. H.; Mirguet, O.; Wilde, J.; Gosmini, R. L.; Delves, C.; Magny, S. M.;
Woodward, R.; Hughes, S. A.; Boursier, E. V.; Flynn, H.; Bouillot, A. M.;
Bamborough, P.; Brusq, J.-M. G.; Gellibert, F. J.; Jones, E. J.; Riou, A. M.;
Homes, P.; Martin, S. L.; Uings, I. J.; Toum, J.; Clꢀement, C. A.; Boullay, A.-
B.; Grimley, R. L.; Blandel, F. M.; Prinjha, R. K.; Lee, K.; Kirilovsky, J.;
Nicodeme, E. Discovery and characterization of small molecule inhibitors of
the BET family bromodomains. J. Med. Chem. 2011, 54, 3827–3838.
(20) Bowers, E. M.; Yan, G.; Mukherjee, C.; Orry, A.; Wang, L.;
Holbert, M. A.; Crump, N. T.; Hazzalin, C. A.; Liszczak, G.; Yuan, H.;
Larocca, C.; Saldanha, S. A.; Abagyan, R.; Sun, Y.; Meyers, D. J.; Marmorstein,
R.; Mahadevan, L. C.; Alani, R. M.; Cole, P. A. Virtual ligand screening of
the p300/CBP histone acetyltransferase: identification of a selective
small molecule inhibitor. Chem. Biol. 2010, 17, 471–482.
(21) Philpott, M.; Yang, J.; Tumber, T.; Fedorov, O.; Uttarkar, S.;
Filippakopoulos, P.; Picaud, S.; Keates, T.; Felletar, I.; Ciulli, A.;
Knapp, S.; Heightman, T. D. Bromodomain-peptide displacement assays
for interactome mapping and inhibitor discovery. Mol. Biosyst. 2011, DOI:
10.1039/C1MB05099K.
(22) (a) Burgey, C. S.; Stump, C. A.; Nguyen, D. N.; Deng, J. Z.;
Quigley, A. G.; Norton, B. R.; Bell, I. M.; Mosser, S. D.; Salvatore, C. A.;
Rutledge, R. Z.; Kane, S. A.; Koblan, K. S.; Vacca, J. P.; Graham, S. L.;
Williams, T. M. Benzodiazepine calcitonin gene-related peptide
(CGRP) receptor antagonists: optimization of the 4-substituted piper-
idine. Bioorg. Med. Chem. Lett. 2006, 16, 5052–5056. (b) Shaw, A. W.;
Paone, D. V.; Nguyen, D. N.; Stump, C. A.; Burgey, C. S.; Mosser, S. D.;
Salvatore, C. A.; Rutledge, R. Z.; Kane, S. A.; Koblan, K. S.; Graham,
S. L.; Vacca, J. P.; Williams, T. M. Caprolactams as potent CGRP
receptor antagonists for the treatment of migraine. Bioorg. Med. Chem.
Lett. 2007, 17, 4795–4798.
(23) Fall, Y.; Reynaud, C.; Doucet, H.; Santelli, M. Ligand-free-
palladium-catalyzed direct 4-arylation of isoxazoles using aryl bromides.
Eur. J. Org. Chem. 2009, 2009, 4041–4050.
(24) Molander, G. A.; Canturk, B.; Kennedy, L. E. Scope of the
Suzuki-Miyaura cross-coupling reactions of potassium heteroaryltri-
fluoroborates. J. Org. Chem. 2008, 74, 973–980.
(25) Sarju, J.; Danks, T. N.; Wagner, G. Rapid microwave-assisted
synthesis of phenyl ethers under mildly basic and nonaqueous condi-
tions. Tetrahedron Lett. 2004, 45, 7675–7677.
(26) Niesen, F. H.; Berglund, H.; Vedadi, M. The use of differential
scanning fluorimetry to detect ligand interactions that promote protein
stability. Nat. Protocol. 2007, 2, 2212–2221.
(27) Li, X.; Du, Y.; Liang, Z.; Li, X.; Pan, Y.; Zhao, K. Simple
conversion of enamines to 2H-azirines and their rearrangements under
thermal conditions. Org. Lett. 2009, 11, 2643–2646.
(28) Abagyan, R.; Totrov, M.; Kuznetsov, D. ICM: A new method
for protein modeling and design: Applications to docking and structure
prediction from the distorted native conformation. J. Comput. Chem.
1994, 15, 488–506.
ꢀ
(29) Bullock, A. N.; Debreczeni, J. E.; Fedorov, O. Y.; Nelson, A.;
Marsden, B. D.; Knapp, S. Structural basis of inhibitor specificity of the
human protooncogene proviral insertion site in Moloney murine
leukemia virus (PIM-1) kinase. J. Med. Chem. 2005, 48, 7604–7614.
6770
dx.doi.org/10.1021/jm200640v |J. Med. Chem. 2011, 54, 6761–6770