Optimization of 3,5-dimethylisoxazole derivatives as potent bromodomain ligands

Article abstract of DOI:10.1021/jm301588r

The bromodomain protein module, which binds to acetylated lysine, is emerging as an important epigenetic therapeutic target. We report the structure-guided optimization of 3,5-dimethylisoxazole derivatives to develop potent inhibitors of the BET (bromodom

Full text of DOI:10.1021/jm301588r

Article
pubs.acs.org/jmc
Optimization of 3,5-Dimethylisoxazole Derivatives as Potent
Bromodomain Ligands
David S. Hewings,†,‡ Oleg Fedorov,‡ Panagis Filippakopoulos,‡ Sarah Martin,‡ Sarah Picaud,‡
Anthony Tumber,‡ Christopher Wells,‡ Monica M. Olcina,§ Katherine Freeman,† Andrew Gill,∥
Alison J. Ritchie,∥ David W. Sheppard,∥ Angela J. Russell,† Ester M. Hammond,§ Stefan Knapp,‡
Paul E. Brennan,‡ and Stuart J. Conway*,†
†Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA, U.K.
‡Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research
Building, Roosevelt Drive, Oxford, OX3 3TA, U.K.
§Department of Oncology, Cancer Research UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Old
Road Campus Research Building, Oxford, OX3 7DQ, U.K.
∥BioFocus, Chesterford Research Park, Saffron Walden, Essex, CB10 1XL, U.K.
S
* Supporting Information
ABSTRACT: The bromodomain protein module, which
binds to acetylated lysine, is emerging as an important
epigenetic therapeutic target. We report the structure-guided
optimization of 3,5-dimethylisoxazole derivatives to develop
potent inhibitors of the BET (bromodomain and extra
terminal domain) bromodomain family with good ligand
efficiency. X-ray crystal structures of the most potent
compounds reveal key interactions required for high affinity
at BRD4(1). Cellular studies demonstrate that the phenol and
acetate derivatives of the lead compounds showed strong
antiproliferative effects on MV4;11 acute myeloid leukemia
cells, as shown for other BET bromodomain inhibitors and
genetic BRD4 knockdown, whereas the reported compounds
showed no general cytotoxicity in other cancer cell lines tested.
containing proteins (BCPs) is still unknown. However, those
BCPs that have been studied in detail have been linked to
certain diseases, and this work has been extensively
reviewed.3,11−15 As bromodomains are invariably components
of large multidomain proteins, removal of the whole BCP does
not provide information on the specific function of the
bromodomain itself. Consequently, an important strategy in
the study of bromodomain function is the development of small
molecule probes that selectively prevent the interaction of a
given bromodomain with KAc, without affecting other
functions of the BCP.6
The most significant progress has been made in developing
probes for the bromodomain and extra C-terminal domain
(BET) family of bromodomains, which comprises bromodo-
main-containing proteins 2−4 (BRD2−4) and bromodomain
testis-specific protein (BRDT).16 The first probes identified
were triazolobenzodiazepine derivatives reported by Filippako-
poulos et al. (1)17 and Nicodeme et al. (2, Figure 1).18 The
compound reported by Filippakopoulos (1) was based on
INTRODUCTION
■
Lysine acetylation has long been recognized as an important
protein post-translation modification (PTM) that regulates a
diverse array of cellular functions.1,2 Acetylation of histone
lysines, in particular, has been intensely investigated because of
its key function regulating chromatin architecture and tran-
scription.3 Mounting evidence suggests that some histone
PTMs can be maintained through multiple cell cycles, giving
rise to the proposal that the specific pattern of PTMs found on
histones represents a combinatorial code,4,5 regulating gene
expression. The concept of a histone code has resulted in the
idea that specific protein classes exist to add the PTM marks
(writer), recognize the marks (readers), and remove the marks
(erasers).3,6,7 In the case of acetylated lysine (KAc) these
proteins are well characterized: histone acetyltransferases
(HATs) add the acetyl group, histone deacetylases (HDACs)
remove the acetyl group, and bromodomains bind to and
recognize KAc, acting as “readers” of lysine acetylation state.8,9
There have been 61 bromodomains identified in the human
proteome, which are found within 46 separate proteins, and
that can be phylogenetically divided into eight distinct
families.10 The precise cellular role of most bromodomain-
Received: October 28, 2012
Published: March 21, 2013
© 2013 American Chemical Society
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Figure 1. Structures of (+)-JQ1 (1),17 I-BET762 (2),18 the 3,5-dimethylisoxazoles reported by Hewings (3, 4),24 I-BET151 (5),25 the 3,5-
dimethylisoxazole (6) reported by Hay et al.,26 PFI-1 (7),27 and the optimized BET bromodomain ligands 8 and 9. IC50 values for the compounds
are shown with the method used to obtain values given in italics. Caution must be exercised when comparing between values obtained using different
methods.
Figure 2. (A) Overlaid X-ray crystal structures of compound 3 (PDB code 3SVG, carbon = yellow)24 and compound 1 (PDB code 3MXF, carbon =
orange),17 both bound to human BRD4(1). The methyl group of 3 does not occupy the WFP shelf as effectively as the chlorophenyl moiety of 1.
(B) Overlaid X-ray crystal structures of compound 3 (PDB code 3SVG, carbon = yellow)24 and compound 5 (PDB code 3ZYU, carbon = orange),25
both bound to human BRD4(1). The 3,5-dimethylisoxazole moiety of 5 resides further into the KAc-binding than that of compound 3. Structures
were aligned using the “cealign” command in PyMOL.
structures disclosed in two Mitsubishi Pharmaceuticals
patents,19,20 while the compound reported by Nicodeme et
al. (2) was identified based on a phenotypic screen monitoring
ApoA1 expression levels.21 Further work on this chemotype has
been reported by Filippakopoulos et al.22 and Zhou et al.23
We24,26 and others25,28,29 have reported the 3,5-dimethyli-
soxazole moiety as an effective KAc mimic and employed it to
develop a second, chemically distinct class of bromodomain
ligands (3−6). We showed that compound 3 (Figure 1) binds
to the BET bromodomains, having IC50 = 4.8 μM against
BRD4(1).24 Dawson et al. also employed the 3,5-dimethylisox-
azole moiety in the development of compound 5 which showed
low nanomolar potency in vitro and in cell based assays.25 Very
recently a third chemotype of BET bromodomain inhibitor has
been reported by Fish et al. based on the dihydroquinazolinone
scaffold (7).27 Herein, we detail the structure-based optimiza-
tion of our previously reported lead compound (3)24 to furnish
potent BET bromodomain inhibitors. The affinity of the
optimum compounds (8 and 9) was rationalized by X-ray
crystallography, yielding insights into the structural require-
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a
Scheme 1. Synthesis of the Diarylmethanol Derivatives 12−16
a
Conditions: (a) EtBr, K2CO3, MeOH, 120 °C (microwave), 30 min, 69%. (b) X = H: PhMgBr, THF, rt, 17 h, 80%. X = m-F: 1-bromo-3-
fluorobenzene, Mg, Et2O, reflux, 2 h and then 11, Et2O, rt, 2 h, 92%. X = p-F: 1-bromo-4-fluorobenzene, Mg, THF, reflux, 3 h and then 11, THF, rt,
61%. X = m-Cl: 1-bromo-3-chlorobenzene, Mg, THF, reflux, 1 h and then 11, THF, rt, 2 h, 90%. X = p-Cl: 1-bromo-4-chlorobenzene, Mg, THF,
reflux, 2.5 h and then 11, THF, rt, 3 h, 67%.
Table 1. IC50 Values of Compounds 3 and 12−16 against BRD4(1) and CREBBP
a
b
Protein and peptide concentration: 50 nM. Protein and peptide concentration: 200 nM. Heat map shows relative IC50 values obtained in an
ALPHA assay.30 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. Values taken from ref 24.
c
ments for binding to BET bromodomains and the observed
SAR. In addition, compounds 8 and 9 were shown to have
cellular activity consistent with a BET bromodomain inhibitor
in an acute myeloid leukemia (AML) cell line.
including a simple phenyl derivative (12) and both meta- and
para-substituted fluoro (13 and 14) and chloro analogues (15
and 16). The aldehyde 10 was a common precursor for all
compounds reported here, and synthesis details are provided in
the Supporting Information.
Utilizing a peptide displacement-based amplified luminescent
proximity homogeneous assay (ALPHA),24,30 we evaluated the
compounds for their ability to bind the bromodomains of
BRD4(1) and cAMP response element binding protein
(CREB) binding protein (CREBBP) (Table 1). All compounds
showed improved BRD4(1) affinity compared to the lead 3. A
clear SAR trend is evident for BRD4(1) affinity, and the simple
phenyl derivative 12 is the most potent compound with an IC50
of 640 nM. The fluorophenyl substituents (13 and 14) are
better tolerated than the chlorophenyl substituents (15 and
16), with para-substitution preferred to meta-substitution.
These data suggest that increased affinity does result from a
hydrophobic interaction with the WPF shelf, as has been
observed with other BET bromodomain inhibitors.16−18,29,27 It
initially appears surprising that the optimum substituent in the
above series of compounds is the unsubstituted phenyl ring,
given that both compounds 1 and 2 possess a chlorophenyl
substituent that binds in the WPF shelf region. However, it is
possible that the ethoxy group that resides in the ZA channel is
not optimal, pushing the aryl ring further into the WPF shelf
than is the case with 1 or 2, meaning that there is less room
available for the aryl substituent to bind (Figure 2A). Our
RESULTS AND DISCUSSION
■
The X-ray crystal structure of our lead compound (3) bound to
the first bromodomain of BRD4(1) showed that the methyl
group bound in a shallow hydrophobic groove frequently
referred as the WPF shelf (Figure 2A).24 The ethoxy
substituent was directed toward a channel formed by residues
in the loop region between the Z and A helices, termed the ZA
channel. In order to develop a compound with improved
affinity for BRD4(1), we have investigated enhancing the
interactions of the compound in these two key regions. We first
chose to optimize the substituents bound in the WPF shelf. It
was hypothesized that a substituent larger than the methyl
group, such as those aromatic rings present in 1, 2, and 5,
would give an increase in BRD4(1) affinity by occupying the
WPF shelf more effectively (Figure 2A). In addition, it was
noted that the 3,5-dimethylisoxazole moiety of 3 binds further
out of the KAc-binding pocket compared to, for example, 5
(Figure 2B). It seemed likely that the addition of a larger WPF
shelf-binding substituent would push the 3,5-dimethylisoxazole
further into the KAc-binding pocket, increasing interactions in
this part of the protein as well. Therefore, a series of
diarylmethanol derivatives (12−16, Scheme 1) was designed
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Figure 3. (A) X-ray crystal structure of compound 3 (PDB code 3SVG, carbon = yellow)24 bound to human BRD4(1). The oxygen atom of the
ethoxy group does not hydrogen bond with the ZA channel water molecules. (B) X-ray crystal structure of compound 4 (PDB code 3SVH, carbon =
yellow)24 bound to human BRD4(1). The oxygen atom of the ethoxy group forms a hydrogen bond with one of the ZA channel water molecules.
observations are consistent with the data of Bamborough et al.
who synthesized sulfonamide derivatives with a range of
substituents occupying the WPF shelf.29 They noted that a
lipophilic substituent of three to five heavy atoms was optimal
for occupying the WPF shelf in their series of compounds.
Larger substituents were tolerated but not optimal. Their series
included a range of phenylsulfonamide derivatives, which would
be expected to bind to the WPF shelf in a similar manner
compared to compounds 12−16; however, structural data are
not available for these compounds. In their case, the o-chloro
substituent was preferred over the m-chloro substituent, which
was preferred over the p-chloro substituent. This disparity with
our work might result from the phenyl rings in each series
having different orientations in relation to the WPF shelf.
However, the unsubstituted phenyl ring was optimal in both
cases. An analogous series of methoxyphenyl-substituted
sulfonamides compounds showed similar potency and trends
compared to the chloro-substituted series, indicating that the
electronic properties of the substituents did not affect the
potency of the compound. In our case, we cannot rule out the
possibility that the electron-withdrawing nature of the halide
substituents is affecting the affinity of the compounds for
BRD4(1).
bound to BRD4(1), the oxygen atom of the ethoxy group was
not hydrogen bonding to this water molecule (Figure 3A).
Conversely, an X-ray structure of a similar 3,5-dimethylisox-
azole derivative (4) bound to the bromodomain of CREBBP
was forming a hydrogen bond with the corresponding ZA
channel water molecule (Figure 3B).24 The loop region that
binds the two ZA channel water molecules, which comprises
P82, Q85, P86, V87, and D88 in BRD4(1), is conserved in
CREBBP (P1110, Q1113, P1114, V1115, and D1116) (see
Supporting Information Figure S5). It should be noted that
there are some differences between the BRD4(1) and CREBBP
bromodomains. Three key residues that differ are W81, K91
and D145 in BRD4(1), which correspond to L1109, L1119 and
R1173 in CREBBP, respectively (see Supporting Figure S4).
However, the ZA channel water molecules are bound in a very
similar manner by both bromodomains, and therefore,
comparison between them is valid. It seemed possible that an
increase in affinity would be achieved if the ligand was able to
displace, or hydrogen-bond to, one of the ZA channel water
molecules.31−34
To test this hypothesis, compounds were designed to probe
the interaction with the ZA channel water molecules. The
smaller phenol (8) and acetate (9) derivatives (Scheme 2) were
The affinity of all compounds for the CREBBP bromodo-
main was also increased with compound 12 again the most
potent. However, the selectivity for BRD4(1) over CREBBP
was maintained, with compounds 3 and 12 both displaying
approximately 6-fold selectivity for the bromodomain of
BRD4(1). These data indicate that the phenyl group of 12
binds effectively to the WPF shelf region of BRD4(1), whereas
interaction of this moiety with CREEBP, which does not
possess this structural feature, is less favorable (see Supporting
Information Figure S4). One advantage of compound 3 was its
leadlike ligand efficiency of 0.39 for BRD4(1), and despite an
increase in molecular weight, compound 12 retains a
respectable ligand efficiency of 0.36 for BRD4(1).
Scheme 2. Synthesis of the Phenol 8, the Acetate 9, and the
Ketone 17
a
The next area of SAR we wished to explore was the
substituent directed toward the ZA channel. As the number of
ligand-bound structures of BET bromodomains solved has
increased, it has become evident that there is always one, and
usually two, water molecule present in the ZA channel.16 It was
observed that in the X-ray crystal structure of compound 3
a
Conditions: (a) PhMgBr, THF, rt, 1 h, 91%; (b) Ac2O, NaOH, H2O,
iPrOH, rt, 1 h, 90%; (c) MnO2, 1,4-dioxane, 80 °C, 53 h, 84%.
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intended to form hydrogen bonds with these water molecules
and hence would be expected to be the most potent if the water
molecule remained in place. Larger ethylene glycol and
methoxy derivatives (21−23, Scheme 3) were expected to be
and evaluated individually. Unexpectedly, both enantiomers
displayed similar affinity for BRD4(1). This observation was
confirmed by surface plasmon resonance using immobilized
BRD4(1). KD values of 0.36 (0.30−0.41) μM for the (R)-
enantiomer and of 0.39 (0.37−0.40) μM for the (S)-
enantiomer were in good agreement with those determined
in the ALPHA assay and demonstrated 1:1 binding
stoichiometry with rapid on and off rates (Supporting
Information Table S1 and Figure S3). Compared to the lead
compound (3), 8 maintains the ligand efficiency and improves
the lipophilic ligand efficiency (Table 3). Having confirmed
Scheme 3. Synthesis of the Methoxy and Ethylene Glycol
a
Derivatives (21−23)
Table 3. pIC50 Values, Ligand Efficiencies, clogP Values, and
Lipophilic Ligand Efficiencies for Compounds 3, (+)-8, and
9
a
Conditions. R = Me: (a) MeI, Cs2CO3, DMF, rt, 2.5 h, 92%; (b)
PhMgBr, THF, 0 °C, 1 h, 76%. R = CH2CH2OMe: (a) 1-bromo-2-
methoxyethane, K2CO3, MeOH, 110 °C (microwave), 30 min, 70%;
(b) PhMgBr, THF, rt, 1.5 h, 83%. R = CH2CH2OH: (a) 2-bromoethyl
acetate, Cs2CO3, DMF, 80 °C, 16 h and then MeOH, rt, 1.5 h, 41%;
(b) PhMgBr, THF, rt, 3 h, 62%.
a
BRD4(1)
ligand
lipophilic ligand
b
compd
IC50 (μM)
pIC50 efficiency clogP
efficiency
3
4.80
5.32
6.42
6.43
0.39
0.41
0.36
2.2
2.5
2.6
3.12
3.92
3.83
(S)-(+)-8
0.382
0.371
9
a
b
Protein and peptide concentration: 50 nM. clogP was calculated
more potent if the water molecules were displaced. We also
synthesized the ketone 17 to investigate the effect of an sp2-
hybridized carbon atom linking the two aryl rings (Scheme 2).
Compounds 8, 9, 17, 21−23 were evaluated for BRD4(1)
affinity in an ALPHA assay (Table 2). The phenol (8) and
acetate (9) showed IC50 values of 370−390 nM against
BRD4(1), which is a significant enhancement in BRD4(1)
affinity compared to the ethyl ether 12. The ketone 17 had a
slightly higher IC50 of 544 nM. The ethylene glycol and
methoxy derivatives (21−23) had affinities greater than 1 μM
for BRD4(1). As only the (R)-enantiomer of the lead
compound 3 was observed in its X-ray crystal structure in
complex with BRD4(1), the (R)- and (S)-enantiomers of
phenol 8 were separated (Supporting Information Figure S1)
using ACD/Labs (algorithm version 5.0.0.184).
that the enantiomers were stable in buffer and did not racemize
(Supporting Information Figure S2), we obtained X-ray crystal
structures of both (R)-(−) and (S)-(+)-8 in complex with
BRD4(1) (Figure 4) in order to rationalize the observed
affinities and to determine whether our SAR predictions were
correct. The absolute configurations were assigned after
obtaining X-ray crystal structures of the (−)- and (+)-enan-
tiomers of 8 in complex with BRD4(1).
The X-ray crystal structures reveal that the (R)- and (S)-
enantiomers have almost identical modes of binding to
BRD4(1). Overlaying the X-ray crystal structures of 8 [(S)-8
Table 2. IC50 Values of Compounds 8, 9, 17, 21−23 against BRD4(1)
a
b
Protein and peptide concentration: 50 nM. Protein and peptide concentration: 50 nM. Heat map shows relative IC50 values obtained in an
ALPHA assay.30 Red indicates low IC50 values, and green/yellow indicates high IC50 values. Ranges in parentheses represent 95% confidence
intervals resulting from sigmoidal curve fitting to duplicate data.
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Figure 4. (A) X-ray crystal structures of compound 3 (PDB code 3SVG, carbon = orange) and (S)-8 (PDB code 4J0S, carbon = yellow) both bound
to human BRD4(1). The 3,5-dimethylisoxazole moiety of (S)-8 resides deeper in the KAc-binding pocket than that of 3. (B) Overlaying the X-ray
crystal structure of (S)-8 (PDB code 4J0S, carbon = yellow) and 1 (PDB code 3MXF, carbon = orange) demonstrates that the phenyl group of (S)-8
binds to the WPF shelf in a similar position as the chlorophenyl group of 1. (C) Overlaying the X-ray crystal structures of (R)-8 (PDB code 4J0R,
carbon = orange) and (S)-8 (PDB code 4J0S, carbon = yellow) demonstrates that the hydroxyl group attached to the stereogenic center is solvent
exposed. (D) The phenol hydroxyl group of 8 (PDB code 4J0S, carbon = yellow) forms a hydrogen bond with one of the conserved ZA channel
water molecules. (E) Docking studies indicate that a feasible binding mode for 9 (docked, carbon = orange) is in a similar orientation as (S)-8 (PDB
code 4J0S, carbon = yellow), with the 3,5-dimethylisoxazole occupying the KAc-binding pocket and the phenyl ring residing on the WPF shelf. (F)
The phenolic oxygen atom of 9 is predicted to form a hydrogen bond with one of the conserved ZA channel water molecules. The acetate carbonyl
group is predicted to form a hydrogen bond with a side chain of Q85. The methyl of the acetate group is predicted to be located in a hydrophobic
region close to W81.
shown] with that of 3 bound to BRD4(1) shows that (S)-8
resides deeper in the KAc-binding pocket than 3 (Figure 4A).
The phenyl ring of (S)-8 occupies the WPF shelf and binds in a
similar region as the chlorophenyl moiety of 1 (Figure 4B). It is
possible that substituents on the phenyl ring will be better
tolerated in a phenol-derived series than in the ethoxy series
described above. We have not investigated this point, but this
strategy might lead to compounds with further enhanced
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affinity for BRD4(1). Overlaying the X-ray crystal structures of
(R)- and (S)-8 explains the similar affinity of these two
compounds for BRD4(1). As the phenyl group binds in the
WPF shelf and the 3,5-dimethylisoxazole occupies the KAc-
binding pocket, the secondary hydroxyl group is solvent
exposed (Figure 4C). Consequently, the configuration at the
stereogenic center does not impact the affinity of the
compounds for BRD4(1). However, given the reduction in
affinity of 17, compared to 8, it seems that a tetrahedral atom
linking the two aryl rings is favored for BRD4(1) binding. Both
enantiomers of compound 8 are observed to form a hydrogen
bond between the phenol hydroxyl group and one of the
conserved ZA-channel water molecules (Figure 4D); a similar
interaction is formed by the quinoline nitrogen atom of
compound 5.25 As we have discussed previously,16 it seems that
this water molecule is tightly bound to BRD4(1) and hence
cannot be displaced easily. It does seem, however, that forming
a hydrogen bond with this water molecule might enhance the
affinity of 8 for BRD4(1). A combination of this hydrogen
bond and binding of the phenyl group in the WPF shelf likely
pushes the 3,5-dimethylisoxazole group further into the KAc-
binding pocket.
By use of the X-ray crystal structure of (S)-8 bound to
BRD4(1), docking studies (AutoDock Vina) were performed to
rationalize the high affinity of 9 for BRD4(1). These studies
indicate that it is feasible for 9 to bind to BRD4(1) in an
orientation similar to that adopted by 8 (Figure 4E). The 3,5-
dimethylisoxazole can occupy the KAc-binding pocket, and the
phenyl ring can reside on the WPF shelf. The phenolic oxygen
atom of 9 is predicted to form a hydrogen bond with one of the
conserved ZA channel water molecules (Figure 4F). The
acetate carbonyl group is predicted to form a hydrogen bond
with side chain of Q85 and might also interact with the lower
ZA channel water molecule. The methyl of the acetate group is
predicted to be located in a hydrophobic region close to W81,
explaining how the extra steric bulk associated with the acetate
moiety might be accommodated. Consequently, the docking
studies provide a possible model for the binding of 9 to
BRD4(1). It is noted that compound 9 is both an active
BRD4(1) ligand and a possible precursor to compound 8 in a
cellular setting.
Figure 5. Selectivity of 8 displayed on a phylogenetic tree of
bromodomains. Compound 8 showed ∼100% inhibition of the
BRD4(1) and CREBBP bromodomains at 25 μM. Comparison of IC50
values from the ALPHA assays indicates that (S)-8 is ∼3-fold selective
for the bromodomain of BRD4(1) over the bromodomain of
CREBBP. Investigation of percentage inhibition of the bromodomains
from ATAD2, BAZ1A, BAZ2A, BAZ2B, BRD9, BRPF3, CECR2,
GCN5L2, PHIP(1), PB1(1), and TIF1α at 25 μM indicated that
compound 8 shows little affinity for these bromodomains. In addition,
thermal shift analysis of 8 against the bromodomains from PCAF and
TAF(1) showed a ΔTm of less than 0.5 °C, which corresponds to very
low affinity for these proteins.
a
Table 4. Effect of 8, 9, and 15 in Cancer Cell Lines
IC50 (μM)
cell line
MV4;11 (AML)
8
9
15
(+)-JQ1 (1)
0.794
>10
0.616
>10
2.9
0.242
A549 (lung adenocarcinoma)
H1975 (lung adenocarcinoma)
U2OS (osteosarcoma)
>10
>10
>100
>100
>100
>100
>100
>100
HeLa (cervical adenocarcinoma)
a
For MV4;11, A549, and H1975 cells, viability was assessed after a 72
h incubation using MTS. For U2OS and HeLa cells, viability was
assessed after a 24 h incubation using WST-1. Further details are
provided in the Supporting Information.
The ALPHA assay was also used to determine the selectivity
of 8, 9, and 17, 21−23 for BRD4(1) over the bromodomain of
CREBBP (Table 2). Comparison of IC50 values indicates that
(S)-8 is 2- to 3-fold selective while compound 9 shows ∼7-fold
selectivity. The selectivity of 8 was further evaluated across a
phylogenetically diverse range of bromodomains (Figure 5).
The ALPHA assay indicated that compound 8 displayed less
than 25% inhibition of bromodomains contained in this panel
at 25 μM (Figure 5), with the exception of BRD4(1) and
CREBBP.
BET bromodomain inhibitors have previously shown
antiproliterative effects in a variety of hematopoietic malig-
nancies, including AML25,35,36 and multiple myeloma.36,37
Consequently, we investigated the effects of compounds 8, 9,
and 15 in the AML cell line MV4;11, which harbors an MLL−
AF4 gene fusion (Table 4).25 Compounds 8 and 9 had IC50
values of 794 and 616 nM, respectively, in an MTS cytotoxicity
assay (Supporting Information Figure S4). The weaker BRD4
inhibitor 15, which has an IC50 of approximately 7 times that of
8 and 9 in the BRD4(1) ALPHA assay, was 5-fold less active
than 9 in this cytotoxicity assay. Gratifyingly, 8 and 9 showed
no appreciable cytotoxicity (>100 μM) in HeLa or U2OS cells
over a period of 24 h suggesting that the effects seen in the
MV4;11 cells result predominantly from inhibition of the BET
BCPs. Over a period of 72 h, compounds 8 and 9 showed less
toxicity than (+)-JQ1 in the HeLa and U2OS cells (see
Supporting Information Table S2).
We also investigated the effects of 8 and 9 in two lung
adenocarcinoma cell lines, A549 and H1975. Compounds 8
and 9 markedly reduced the viability of both cell lines at 100
μM, as determined by an MTS cytotoxicity assay (Supporting
Information Figure S4). H1975 appeared to be somewhat more
sensitive to these compounds, a finding confirmed by a
clonogenic survival assay (Supporting Information Figure S5).
The modest effect of 8 and 9 in these cell lines is consistent
with the findings of Mertz et al., who observed only weak
growth inhibition by BET inhibitor I-BET151 in several solid
tumor lines.36
Overall, these results suggest that compounds 8 and 9 are
cell-permeable compounds without appreciable nonspecific
cytotoxicity. These compounds show antiproliterative effects
in a leukemic cell line with known sensitivity to BET
inhibitors.25,35,36
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added either neat if a liquid or as a solution in THF (269 mM, 10 mL).
The mixture was heated gently to initiate Grignard reagent formation,
then heated under reflux until all the Mg had reacted. The mixture was
then cooled to 0 °C and added slowly to a solution of 11 (200 mg, 815
μmol, 1.0 equiv) in Et2O or THF (81.5 mM, 10 mL, as stated) at 0 °C
under a nitrogen atmosphere. The solution was warmed to room
temperature. On consumption of aldehyde (as indicated by TLC), the
reaction was quenched with H2O and HCl (1 M aqueous). The phases
were separated, and the aqueous layer was extracted with Et2O. The
combined organic layers were washed with H2O and brine, dried
(MgSO4), filtered, and concentrated in vacuo. The crude residues were
purified by silica gel chromatography (gradient elution, Et2O/
petroleum ether).
Synthetic Procedure and Characterization of Compounds 8,
9, 12−16, 21−23. (3-(3,5-Dimethylisoxazol-4-yl)-5-ethoxyphenyl)-
(phenyl)methanol 12. To a solution of 11 (79 mg, 322 μmol, 1.0
equiv) in dry THF (5 mL) under a nitrogen atmosphere was added
PhMgBr (0.9 M in THF, 716 μL, 644 μmol, 2.0 equiv) dropwise at 0
°C. The solution was warmed to room temperature over 17 h, then
quenched with HCl (1 M aqueous, 10 mL) and extracted with Et2O (3
× 10 mL). The combined organic layers were washed with H2O (2 ×
30 mL) and brine (30 mL), dried (MgSO4), filtered, and concentrated
in vacuo. Purification by silica gel column chromatography (gradient
elution, 10% → 60% Et2O/petroleum ether) gave 12 as a colorless oil
(83 mg, 80%). 1H NMR (500 MHz, CDCl3) 1.42 (3H, t, J = 7.0 Hz),
2.23 (3H, s), 2.29 (1H, d, J = 3.3 Hz), 2.37 (3H, s), 4.04 (2H, q, J =
7.0 Hz), 5.85 (1H, d, J = 3.3 Hz), 6.67 (1H, dd, J = 2.2, 1.6 Hz), 6.82
(1H, s), 6.96 (1H, dd, J = 2.2, 1.1 Hz), 7.29 (1H, tt, J = 7.2, 1.5 Hz),
7.34−7.43 (4H, m); HRMS m/z (ES+) found [M + Na]+ 346.1406;
C20H21NNaO3 requires M+ 346.1414; m/z (ES+) 324 ([M + H]+, 24),
346 ([M + Na]+, 33), 669 ([2M + Na]+, 100).
(3-(3,5-Dimethylisoxazol-4-yl)-5-ethoxyphenyl)(4-fluorophenyl)-
methanol 13. Following the general procedure, 1-bromo-4-
fluorobenzene (471 mg, 296 μL) and Mg turnings in THF were
heated under reflux for 3 h. Following addition of the resultant cloudy
yellow suspension to 11 in THF, the mixture was stirred for 4.5 h.
Then the reaction was quenched with H2O (15 mL) and HCl (1 M
aqueous, 15 mL). The phases were separated, and the aqueous layer
was extracted with Et2O (3 × 30 mL). The combined organic layers
were washed with H2O (90 mL) and brine (90 mL), dried (MgSO4),
filtered, and concentrated in vacuo. Purification by silica gel column
chromatography (gradient elution, 20% → 80% Et2O/petroleum
ether) gave 13 as a pale yellow viscous gum (170 mg, 61%). 1H NMR
(500 MHz, CDCl3) 1.41 (3H, t, J = 7.0 Hz), 2.19 (3H, s), 2.35 (3H,
s), 2.98 (1H, br s), 4.03 (2H, q, J = 7.0 Hz), 5.80 (1H, s), 6.66 (1H,
dd, J = 2.3, 1.5 Hz), 6.79 (1H, s), 6.92 (1H, s), 6.98−7.05 (2H, m),
7.32−7.38 (2H, m); HRMS m/z (ES+) found 364.1318;
C20H20FNNaO3 requires M+ 364.1319; m/z (ES+) 342 ([M + H]+,
12), 364 ([M + Na]+, 19), 396 ([M + Na + MeOH]+, 8), 705 ([2M +
Na]+, 100).
(3-(3,5-Dimethylisoxazol-4-yl)-5-ethoxyphenyl)(3-fluorophenyl)-
methanol 14. Following the general procedure, 1-bromo-3-
fluorobenzene (471 mg, 300 μL) and Mg turnings in Et2O were
heated under reflux for 2 h. Following addition of the resultant cloudy
yellow suspension to 11 in Et2O, the mixture was stirred for 2 h. Then
the reaction was quenched with H2O (10 mL) and HCl (1 M aqueous,
10 mL). The phases were separated, and the aqueous layer was
extracted with Et2O (2 × 20 mL). The combined organic layers were
washed with H2O (60 mL) and brine (60 mL), dried (MgSO4),
filtered, and concentrated in vacuo. Purification by silica gel column
chromatography (gradient elution, 20% → 80% Et2O/petroleum
ether) gave 14 as a pale yellow viscous gum (255 mg, 92%). 1H NMR
(500 MHz, CDCl3) 1.42 (3H, t, J = 7.0 Hz), 2.21 (3H, s), 2.36 (3H,
s), 2.87 (1H, d, J = 3.4 Hz), 4.03 (2H, q, J = 7.0 Hz), 5.81 (1H, d, J =
3.4 Hz), 6.68 (1H, dd, J = 2.1, 1.3 Hz), 6.80 (1H, dd, J = 1.4, 1.3 Hz),
6.92 (1H, dd, J = 2.1, 1.4 Hz), 6.93−6.99 (1H, m), 7.10−7.14 (1H,
m), 7.14−7.19 (1H, m), 7.27−7.33 (1H, m); HRMS m/z (ES+) found
364.1312; C20H20FNNaO3 requires M+ 364.1319; m/z (ES+) 342 ([M
+ H]+, 4), 364 ([M + Na]+, 14), 396 ([M + Na + MeOH]+, 10), 705
([2M + Na]+, 100).
CONCLUSION
■
We have described the structure-guided optimization of
compound 3 to give potent inhibitors of the BET
bromodomains (8 and 9). The X-ray crystal structures obtained
in this work help to demonstrate many of the structural
characteristics required for a compound to show high affinity
for the BET bromodomains. These compounds show some
selectivity over the CREBBP bromodomain and excellent
selectivity over other phylogenetically diverse bromodomain
classes. On the basis of our structural and docking studies, it
seems that constraining the acetate substituent of compound 9
into a five- or six-membered ring might lead to a stronger
hydrogen bond with Q85 and provide entropic gains in binding
affinity. A re-evaluation of the WPF-shelf-binding ring
substituents might also lead to enhanced BRD4(1) affinity
and potentially improved selectivity for the BET bromodo-
mains over the CREBBP bromodomain. Assessment of
compounds 8 and 9 in a range of cells lines reveals that the
compounds have submicromolar IC50 values in MV4;11 cells
and that the effects are predominantly due to inhibition of the
BET BCPs. These compounds will likely prove to be useful
tools in the study of the BET bromodomains and are highly
ligand-efficient lead compounds for further development.
EXPERIMENTAL SECTION
■
General Experimental. 1H NMR spectra were recorded on
Bruker DPX400 (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 (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.
13C NMR spectra were recorded on a Bruker AVII 500 (126 MHz)
spectrometer with broadband proton decoupling and 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.
19F NMR spectra were recorded on a 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).
Mass spectra were acquired on either a Micromass LCT Premier
spectrometer (low resolution) or a Bruker MicroTOF spectrometer
(high resolution), 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 Leica Galen III 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%.
General Procedure for Grignard Reaction of Aryl Bromide
and 11. To a dry two-necked flask, equipped with a condenser,
containing Mg turnings (66 mg, 2.45 mmol, 3.0 equiv) and a crystal of
iodine was added dry Et2O or dry THF (20 mL, as stated) under a
nitrogen atmosphere. Aryl bromide (2.69 mmol, 3.3 equiv) was then
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(4-Chlorophenyl)(3-(3,5-dimethylisoxazol-4-yl)-5-ethoxyphenyl)-
methanol 15. Following the general procedure, 1-bromo-4-
chlorobenzene (515 mg) and Mg turnings in THF were heated
under reflux for 2.5 h. Following addition of the resultant cloudy
yellow suspension to 11 in THF, the mixture was stirred for 3 h. Then
the reaction was quenched with H2O (10 mL) and HCl (1 M aqueous,
20 mL). The phases were separated, and the aqueous layer was
extracted with Et2O (3 × 40 mL). The combined organic layers were
washed with H2O (120 mL) and brine (120 mL), dried (MgSO4),
filtered, and concentrated in vacuo. Purification by silica gel column
chromatography (gradient elution, 20% → 80% Et2O/petroleum
ether) gave 15 as a colorless viscous gum that slowly crystallized to a
dd, J = 1.7, 1.5 Hz), 7.28−7.32 (1H, m), 7.33−7.42 (4H, m); HRMS
m/z (ES+) found 360.1206; C20H19NNaO4 requires M+ 360.1206; m/
z (ES+) 338 ([M + H]+, 35), 360 ([M + Na]+, 100), 697 ([2M + Na]+,
95).
(3-(3,5-Dimethylisoxazol-4-yl)-5-hydroxyphenyl)(phenyl)-
methanone 17. To a solution of alcohol 8 (207 mg, 701 μmol) in
anhydrous 1,4-dioxane (10 mL) was added activated MnO2 (305 mg,
3.50 mmol, 5 equiv). The mixture was stirred under nitrogen at 80 °C
for 15 h, after which time further MnO2 (152 mg, 1.75 mmol, 2.5
equiv) was added, and stirring was continued at 80 °C for 9 h. Further
MnO2 (152 mg, 1.75 mmol, 2.5 equiv) was then added, and stirring
was continued at 80 °C for 29 h. The mixture was cooled to room
temperature and filtered through Celite (eluent CH2Cl2), then
concentrated in vacuo. Purification by silica gel column chromatog-
raphy (gradient elution, gradient 20% → 50% EtOAc/40−60 °C
petroleum ether, then further purification of mixed fractions, 20% →
35% EtOAc/40−60 °C petroleum ether) gave 17 as a cream solid
(172 mg, 84%). Rf = 0.43 (50% EtOAc/40−60 °C petroleum ether);
1
colorless solid under vacuum (195 mg, 67%). Mp 103−104 °C; H
NMR (500 MHz, CDCl3) 1.42 (3H, t, J = 7.0 Hz), 2.22 (3H, s), 2.37
(3H, s), 2.60 (1H, br s), 4.03 (2H, q, J = 7.0 Hz), 5.81 (1H, s), 6.68
(1H, s), 6.80 (1H, s), 6.91 (1H, s), 7.29−7.37 (4H, m); HRMS m/z
(ES+) found 380.1018, C20H20ClNNaO3 requires M+ 380.1024; m/z
(ES+) 358 ([M + H]+, 7), 380 ([M + Na]+, 7), 412 ([M + Na +
MeOH]+, 9), 737 ([2M + Na]+, 100).
1
mp 188−190 °C (acetone); H NMR (500 MHz, acetone-d6) 2.27
(3-Chlorophenyl)(3-(3,5-dimethylisoxazol-4-yl)-5-ethoxyphenyl)-
methanol 16. Following the general procedure, 1-bromo-3-
chlorobenzene (515 mg, 316 μL) and Mg turnings in THF were
heated under reflux for 1 h. Following addition of the resultant yellow
solution to 11 in THF, the mixture was stirred for 2 h. Then the
reaction was quenched with H2O (10 mL) and HCl (1 M aqueous, 10
mL). The phases were separated, and the aqueous layer was extracted
with Et2O (3 × 40 mL). The combined organic layers were washed
with H2O (120 mL) and brine (120 mL), dried (MgSO4), filtered, and
concentrated in vacuo. Purification by silica gel column chromatog-
raphy (gradient elution, 20% → 80% Et2O/petroleum ether) gave 16
as a colorless viscous gum (262 mg, 90%). 1H (500 MHz, CDCl3) 1.42
(3H, t, J = 7.0 Hz), 2.20 (3H, s), 2.36 (3H, s), 2.88 (1H, br s), 4.04
(2H, q, J = 7.0 Hz), 5.78 (1H, s), 6.67 (1H, s), 6.79 (1H, s), 6.92 (1H,
s), 7.21−7.29 (3H, m), 7.40 (1H, s); HRMS m/z (ES+) found
380.1018, C20H20ClNNaO3 requires M+ 380.1024; m/z (ES+) 380
([M + Na]+, 4), 737 ([2M + Na]+, 100).
(3H, s), 2.45 (3H, s), 7.13 (1H, dd, J = 2.4, 1.5 Hz), 7.20 (1H, dd, J =
1.5, 1.5 Hz), 7.27 (1H, dd, J = 2.4, 1.5 Hz), 7.55−7.62 (2H, m), 7.66−
7.71 (1H, m), 7.82−7.88 (2H, m); HRMS m/z (ES+) found 316.0938;
C18H15NNaO3 requires M+ 316.0944; m/z (ES−) 292 ([M − H]−,
100), 328 ([M + Cl]−, 13).
(3-(3,5-Dimethylisoxazol-4-yl)-5-methoxyphenyl)(phenyl)-
methanol 21. To a solution of 18 (80 mg, 346 μmol, 1.0 equiv) in dry
THF (5 mL) under a nitrogen atmosphere was added PhMgBr (0.9 M
in THF, 769 μL, 692 μmol, 2.0 equiv) dropwise at 0 °C. The solution
was stirred for 1 h, then quenched with HCl (1 M aqueous, 5 mL) and
concentrated in vacuo to remove THF. The residues were extracted
with EtOAc (3 × 5 mL). The combined organic layers were washed
with H2O (20 mL) and brine (20 mL), dried (MgSO4), filtered, and
concentrated in vacuo. Purification by silica gel column chromatog-
raphy (gradient elution, 30% → 80% Et2O petroleum ether) gave 21
1
as a colorless oil (66 mg, 76%). H NMR (500 MHz, CDCl3) 2.21
(3H, s), 2.36 (3H, s), 2.68 (1H, s), 3.82 (3H, s), 5.84 (1H, s), 6.68
(1H, dd, J = 2.3, 1.3 Hz), 6.83 (1H, dd, J = 1.3, 1.3 Hz), 6.98 (1H, dd,
J = 2.3, 1.3 Hz), 7.26−7.31 (1H, m), 7.33−7.38 (2H, m), 7.38−7.42
(2H, m); HRMS m/z (ES+) found [M + Na]+ 332.1257;
C19H19NNaO3 requires M+ 332.1257; m/z (ES+) 332 ([M + Na]+,
12), 364 ([M + Na + MeOH]+, 6), 641 ([2M + Na]+, 100).
3-(3,5-Dimethylisoxazol-4-yl)-5-(hydroxy(phenyl)methyl)phenol
8. To a solution of 10 (250 mg, 1.02 mmol, 1.0 equiv) in dry THF (20
mL) under a nitrogen atmosphere was added PhMgBr (0.9 M in THF,
3.4 mL, 3.06 mmol, 3.0 equiv) dropwise at 0 °C. The solution was
warmed to room temperature over 1 h, then quenched with HCl (1 M
aqueous, 10 mL) and concentrated in vacuo to remove THF. The
residues were extracted with EtOAc (3 × 20 mL). The combined
organic layers were washed with H2O (2 × 40 mL) and brine (40 mL),
dried (MgSO4), filtered, and concentrated in vacuo. Purification by
silica gel column chromatography (gradient elution, 10% → 50%
EtOAc/petroleum ether) gave 8 as a pale yellow solid (273 mg, 91%).
(3-(3,5-Dimethylisoxazol-4-yl)-5-(2-methoxyethoxy)phenyl)-
(phenyl)methanol 22. To a solution of 19 (150 mg, 545 μmol, 1.0
equiv) in anhydrous THF (10 mL) under a nitrogen atmosphere was
added PhMgBr (0.9 M in THF, 1.2 mL, 1.08 mmol, 2.0 equiv)
dropwise at 0 °C. The solution was warmed to room temperature and
stirred for 90 min, then quenched with HCl (1 M aqueous, 5 mL) and
concentrated in vacuo to remove THF. The residues were extracted
with EtOAc (3 × 5 mL). The combined organic layers were washed
with H2O (15 mL) and brine (15 mL), dried (MgSO4), filtered, and
concentrated in vacuo. Purification by silica gel column chromatog-
raphy (gradient elution, 6% → 60% EtOAc/petroleum ether) gave 22
as a colorless oil that crystallized slowly under vacuum to give a
1
Mp 187−188 °C (CHCl3); H NMR (500 MHz, acetone-d6) 2.21
(3H, s), 2.38 (3H, s), 4.95 (1H, d, J = 3.1 Hz), 5.84 (1H, d, J = 3.1
Hz), 6.71 (1H, dd, J = 2.3, 1.6 Hz), 6.91−6.94 (1H, m), 6.96 (1H, dd,
J = 2.3, 1.4 Hz), 7.24 (1H, tt, J = 7.3, 1.3 Hz), 7.31−7.36 (2H, m),
7.46−7.50 (2H, m), 8.50 (1H, s); HRMS m/z (ES+) found 318.1102,
C18H17NNaO3 requires M+ 318.1101; m/z (ES−) 294 ([M − H]−,
48), 330 [M + Cl]−, 14), 408 ([M + TFA − H]−, 9), 589 ([2M −
H]−, 100), 884 ([3M − H]−, 18).
1
colorless solid (159 mg, 83%). Mp 70−73 °C; H NMR (500 MHz,
CDCl3) 2.20 (3H, s), 2.35 (3H, s), 2.74 (1H, d, J = 3.2 Hz), 3.44 (3H,
s), 3.74 (2H, t, J = 4.6 Hz), 4.11 (2H, t, J = 4.6 Hz), 5.82 (1H, d, J =
3.2 Hz), 6.71 (1H, dd, J = 1.9, 1.5 Hz), 6.83 (1H, dd, J = 1.6, 1.5 Hz),
6.98 (1H, dd, J = 1.9, 1.6 Hz), 7.25−7.30 (1H, m), 7.31−7.40 (4H,
m); HRMS m/z (ES+) found [M + Na]+ 376.1515; C21H23NNaO4
requires M+ 376.1519; m/z (ES+) 376 ([M + Na]+, 23), 729 ([2M +
Na]+, 100).
3-(3,5-Dimethylisoxazol-4-yl)-5-(hydroxy(phenyl)methyl)phenyl
Acetate 9. Following the procedure of Srivastava et al.,38 solid NaOH
dissolved in a minimum amount of H2O (37 mg, 915 μmol, 2.7 equiv)
was added to a stirred suspension of 8 (100 mg, 339 μmol, 1.0 equiv)
i
in PrOH (5 mL). Ac2O (93 mg, 86 μL, 915 μmol, 2.7 equiv) was
added dropwise, and the solution was stirred at room temperature for
i
1 h. The solution was then concentrated in vacuo to remove PrOH
2-(3-(3,5-Dimethylisoxazol-4-yl)-5-(hydroxy(phenyl)methyl)-
phenoxy)ethanol 23. To a solution of 20 (125 mg, 478 μmol, 1.0
equiv) in anhydrous THF (8 mL) under a nitrogen atmosphere was
added PhMgBr (0.9 M in THF, 1.6 mL, 1.44 mmol, 3.0 equiv)
dropwise at 0 °C. The solution was warmed to room temperature and
stirred for 3 h, then quenched with aqueous HCl (1 M, 10 mL) and
concentrated in vacuo to remove THF. The residues were extracted
with EtOAc (3 × 10 mL). The combined organic layers were washed
with H2O (20 mL) and brine (20 mL), dried (MgSO4), filtered, and
and diluted with 10 mL of H2O, then extracted with EtOAc (3 × 10
mL). The combined organic layers were washed with H2O (30 mL)
and brine (30 mL), dried (MgSO4), filtered, and concentrated in
vacuo. Purification by silica gel column chromatography (gradient
elution, 10% → 50% EtOAc/petroleum ether) gave 9 as a colorless oil
(104 mg, 90%). 1H NMR (500 MHz, CDCl3) 2.22 (3H, s), 2.30 (3H,
s), 2.37 (3H, s), 2.63 (1H, d, J = 2.9 Hz), 5.86 (1H, d, J = 2.9 Hz,),
6.90 (1H, dd, J = 1.7, 1.5 Hz), 7.11 (1H, dd, J = 1.7, 1.5 Hz), 7.15 (1H,
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Article
concentrated in vacuo. The residues were purified by silica gel column
chromatography (gradient elution, 40% → 80% EtOAc/petroleum
ether) to give a colorless gum that crystallized slowly under vacuum to
give a colorless solid. Repeated recrystallization from boiling CHCl3/
cyclohexane (1:1) gave 23 as a colorless solid (100 mg, 62%). Mp
133−134 °C (1:1 CHCl3/cyclohexane); 1H NMR (500 MHz, DMSO-
d6) 2.20 (3H, s), 2.38 (3H, s), 3.71 (2H, dt, J = 5.5, 5.0 Hz), 4.00 (2H,
t, J = 5.0 Hz), 4.86 (1H, t, J = 5.5 Hz), 5.73 (1H, d, J = 3.9 Hz), 5.96
(1H, d, J = 3.9 Hz), 6.76 (1H, dd, J = 2.2, 1.4 Hz), 6.93−6.96 (1H, m),
6.96−6.98 (1H, m), 7.21 (1H, t, J = 7.5 Hz), 7.31 (2H, dd, J = 7.5, 7.5
Hz), 7.43 (2H, d, J = 7.5, Hz); HRMS m/z (ES+) found [M + Na]+
362.1353; C20H21NNaO4 requires M+ 362.1363; m/z (ES+) 362 ([M
+ Na]+, 7), 701 ([2M + Na]+, 100).
Docking Studies. Preparation of the Receptor. The receptor was
initially prepared by adding polar hydrogen atoms at pH 7.4 using
Protonate3D in MOE. Gasteiger charges were then assigned using
AutoDock’s graphical user interface AutoDockTools (ADT, version
1.5.4). Following the addition of Gasteiger charges, ADT was
implemented to build a “united atom model” of the receptor by
merging nonpolar hydrogen atoms and adding their partial charges to
their parent carbon atoms.
Preparation of the Ligands. The ligands were prepared by adding
polar hydrogens atoms at pH 7.4 using Protonate3D in Molecular
Operating Environment (MOE), version 2011.10. MOE then assigned
Gasteiger charges before performing an energy minimization using an
MMFF94x force field with a 0.05 gradient. AutoDock’s graphical user
interface AutoDockTools (ADT, version 1.5.4) was subsequently used
to build a “united atom model” of the ligands by merging nonpolar
hydrogens and adding their partial charges to their parent carbon
atoms.
Individual Docking with AutoDock Vina (Version 1.1.2). The search
space was defined as a cubic box with dimensions 18 Å × 18 Å × 18 Å
and center x = 30.048, y = 16.281, z = −1.723. The exhaustiveness and
number of modes were both set to 20, and all other parameters were
kept at their default values.
Accession Codes
Atomic coordinates and structure factors for the reported
crystal structures have been deposited with the Protein Data
Bank under accession codes 4J0R [(R)-8] and 4J0S [(S)-8].
AUTHOR INFORMATION
Corresponding Author
■
*Telephone: +44 (0)1865 285 109. Fax: +44 (0)1865 285 002.
E-mail: stuart.conway@chem.ox.ac.uk.
Author Contributions
The manuscript was written with contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Cancer Research UK for a studentship
(D.S.H.). S.J.C. thanks St. Hugh’s College, Oxford, U.K., for
research support. The authors receive funding from the SGC, a
registered charity (No. 1097737) that receives funds from the
Canadian Institutes for Health Research, the Canada
Foundation for Innovation, Genome Canada, GlaxoSmithKline,
Pfizer, Eli Lilly, Takeda, AbbVie, the Novartis Research
Foundation, the Ontario Ministry of Research and Innovation,
and the Wellcome Trust. P.F. is supported by a Wellcome
Trust Career Development Fellowship (Grant 095751/Z/11/
Z). We are grateful to Dr. Pavol Jakubec for carrying out
preparative and analytical chiral HPLC and Sam Grayer for
performing the docking studies on compound 9.
Biological Evaluation. Protein Expression and Purification.
Proteins were cloned, expressed, and purified as previously
described.17
ABBREVIATIONS USED
■
ALPHA, amplified luminescent proximity homogeneous assay;
AML, acute myeloid leukemia; BET, bromodomain and extra
terminal domain; BCP, bromodomain-containing protein;
BRD2(1)/(2), first/second bromodomain of BRD2; BRD4-
(1)/(2), first/second bromodomain of BRD4; BRDT,
bromodomain, testis-specific; CREB, cAMP response ele-
ment-binding protein; CREBBP, CREB binding protein;
HAT, histone acetyltransferase; HDAC, histone deacetylase;
KAc, acetyllysine; MLL, mixed lineage leukemia; MTS, (3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium); PTM, post-translational modifica-
tion; WST-1, water-soluble tetrazolium 1
Peptides. H4Ac4 peptide (BRD4 assay, H2N-SGRGK(Ac)GGK-
(Ac)GLGK(Ac)GGAK(Ac)RHRK(biotin)-COOH) and H3K56Ac
peptide (CREBBP assay, H2N-ALREIRRYQK(Ac)STELLIRKLK-
(biotin)-COOH) were synthesized by Tufts University Core Facility.
AlphaScreen Peptide Displacement Assay (ALPHA Assay).
Bromodomain AlphaScreen assays were carried out as previously
described.24,30 All experiments were carried out in duplicate on the
same plate.
Cytotoxicity Assay. Viability of MV4;11, H549, and H1975 cells
was monitored by MTS-based CellTiter 96 AQueous nonradioactive
cell proliferation assay (Promega, Madison, WI). Viability of U2OS
and HeLa cells was monitored by WST-based cell proliferation kit
(Roche, Mannheim, Germany). Further details are provided in
Supporting Information
REFERENCES
■
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ASSOCIATED CONTENT
■
S
* Supporting Information
Enantiomeric purity determination of 8 by chiral HPLC,
stability of (+)-8 in aqueous buffer, SPR steady state affinity
analysis of (R)- and (S)-8 binding to BRD4(1), comparison of
the BRD4(1) and CREBBP bromodomains, BRD4(1) and
CREBBP bromodomain sequence alignment, MTS viability
assays for MV4;11, A549, and H1975 cells, steady state analysis
parameters for (R)- and (S)-8 binding to BRD4(1), crystallo-
graphic information, further general experimental details,
synthesis and characterization of compounds 10, 11, 18−20,
and further characterization (13C and 19F NMR, elemental
analysis) for compounds 8, 9, 12−17, 21−23. This material is
available free of charge via the Internet at http://pubs.acs.org.
3226
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