Biased multicomponent reactions to develop novel bromodomain inhibitors

Article abstract of DOI:10.1021/jm501120z

BET bromodomain inhibition has contributed new insights into gene regulation and emerged as a promising therapeutic strategy in cancer. Structural analogy of early methyl-triazolo BET inhibitors has prompted a need for structurally dissimilar ligands as probes of bromodomain function. Using fluorous-tagged multicomponent reactions, we developed a focused chemical library of bromodomain inhibitors around a 3,5-dimethylisoxazole biasing element with micromolar biochemical IC₅₀. Iterative synthesis and biochemical assessment allowed optimization of novel BET bromodomain inhibitors based on an imidazo[1,2-a]pyrazine scaffold. Lead compound 32 (UMB-32) binds BRD4 with a K_d of 550 nM and 724 nM cellular potency in BRD4-dependent lines. Additionally, compound 32 shows potency against TAF1, a bromodomain-containing transcription factor previously unapproached by discovery chemistry. Compound 32 was cocrystallized with BRD4, yielding a 1.56 ? resolution crystal structure. This research showcases new applications of fluorous and multicomponent chemical synthesis for the development of novel epigenetic inhibitors.

Full text of DOI:10.1021/jm501120z

Article
pubs.acs.org/jmc
Biased Multicomponent Reactions to Develop Novel Bromodomain
Inhibitors
Michael R McKeown,† Daniel L Shaw,† Harry Fu,‡ Shuai Liu,‡ Xiang Xu,§,∥ Jason J Marineau,†
Yibo Huang,‡ Xiaofeng Zhang,‡ Dennis L Buckley,† Asha Kadam,‡ Zijuan Zhang,‡ Stephen C Blacklow,§,∥
Jun Qi,† Wei Zhang,*,‡ and James E Bradner*,†,⊥
†Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, Massachusetts 02215, United
States
‡Department of Chemistry, University of Massachusetts Boston, 100 Morrissey Boulevard, Boston, Massachusetts 02125, United
States
§Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, Massachusetts 02215, United States
∥Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 45 Shattuck Street, Boston,
Massachusetts 02115, United States
⊥Department of Medicine, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, United States
S
* Supporting Information
ABSTRACT: BET bromodomain inhibition has contributed new insights into
gene regulation and emerged as a promising therapeutic strategy in cancer.
Structural analogy of early methyl-triazolo BET inhibitors has prompted a need for
structurally dissimilar ligands as probes of bromodomain function. Using fluorous-
tagged multicomponent reactions, we developed a focused chemical library of
bromodomain inhibitors around a 3,5-dimethylisoxazole biasing element with
micromolar biochemical IC50. Iterative synthesis and biochemical assessment
allowed optimization of novel BET bromodomain inhibitors based on an
imidazo[1,2-a]pyrazine scaffold. Lead compound 32 (UMB-32) binds BRD4
with a Kd of 550 nM and 724 nM cellular potency in BRD4-dependent lines. Additionally, compound 32 shows potency against
TAF1, a bromodomain-containing transcription factor previously unapproached by discovery chemistry. Compound 32 was
cocrystallized with BRD4, yielding a 1.56 Å resolution crystal structure. This research showcases new applications of fluorous and
multicomponent chemical synthesis for the development of novel epigenetic inhibitors.
transcriptional proteins, perhaps owing to perceptions regard-
ing the difficulty of disrupting protein−protein interactions.7
Recent work from our laboratory established the feasibility of
targeting the acetyl-lysine (Kac) recognition module, or
bromodomain.8 A bromodomain is a conserved structural
module comprised of four antiparallel α-helices which creates a
Kac-recognition pocket between two interhelical loops.9−13
Molecular recognition of Kac is often facilitated by a conserved
asparagine residue and a network of structured water molecules.
In 2010, we reported a first, methyl-triazolo bromodomain
inhibitor, compound 1 (JQ1), which selectively displaces the
BET (bromodomain and extra-terminal domain) subfamily of
human bromodomains from chromatin.8 The basic research
guiding our understanding of BET bromodomains has been
empowered by the availability of this chemical tool. We now
appreciate that the BET family (BRD2, BRD3, BRD4, and
BRDT) functions as transcriptional coactivator proteins, which
relay signals from master regulatory transcription factors, such
as MYC in cancer and NFkB in inflammation, to RNA
INTRODUCTION
■
Cellular identity is imprinted on the genome and epigenome
via covalent chemical modification. Post-translational modifi-
cation of the DNA and histone proteins around which the
genome is structured is dynamically maintained by chromatin-
modifying enzymes, which “write” or “erase” an expansive and
diverse set of marks.1−4 Some of these marks are retained
through cell division, contributing to epigenetic mitotic
memory. Others mediate open (actively transcribed) or closed
(actively repressed) conformations of chromatin, enforcing
gene expression programs that govern specialized cellular
functions. Interpretation of epigenomic marks is accomplished
by families of “reader” proteins which are recruited to sites of
context-specific modification where they nucleate higher-
ordered assemblies that influence gene expression and
chromatin condensation.2
As end-effectors of signaling pathways, regulators of
transcription, and emerging unrecognized cancer dependencies,
epigenetic reader proteins are attractive therapeutic targets in
cancer.5 However, the function of these agents via protein−
protein interaction poses a challenge to the discipline of ligand
discovery.6 Indeed, few high-quality chemical probes exist for
Received: July 23, 2014
© XXXX American Chemical Society
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Figure 1. Chemical strategies to inhibit BET bromodomain proteins. (A) Existing BET bromodomain inhibitors with biasing moiety in red. (B)
Synthetic strategy for the creation of small fragments based on dimethyl isoxazole based BET bromodomain biasing moiety (red). X signifies varied
heteroatoms and R signifies varied substituents. (C) Perfluoroalkyl synthetic strategy with reaction conditions for small fragments shown. (D) Small
fragment dimethylisoxazole inhibitors with potency values indicated. (E) Representative inhibitory curves for small fragment inhibitors. Error is
shown based on duplicate technical replicates. (F) Docking of compound 9 into BRD4 crystal structure (PDB: 3MXF). The conserved asparagine
interaction is indicated. (G) Ligand interaction diagram of compound 9.
polymerase II (RNA Pol II).14−16 BET proteins function to
promote release of RNA Pol II from promoters, leading to
effective transcriptional elongation.17−19 Beyond the central
role in chromatin-dependent transcriptional signaling, BET
bromodomains are also important mediators of cell cycle
progression13 and facilitate developmental transitions such as
spermiogenesis.20
Deregulation of BET bromodomain function is observed in
numerous malignancies, and two BET bromodomains (BRD3
and BRD4) are proto-oncogenes in aggressive epithelial
cancers.13,21 BET-rearranged lung and head and neck cancer
demonstrates in an in-frame fusion to the NUT gene, resulting
in aberrant expression of a chimeric oncoprotein that retains
the tandem N-terminal bromodomains.21 Exposure of patient-
derived cell lines from BET-rearranged lung cancers to
compound 1 results in immediate squamous differentiation
and subsequent cell death, establishing a compelling rationale
for the development of BET-targeted therapy in this disease.8
Beyond these solid tumors, compound 1 has demonstrated
potent antiproliferative efficacy in models of multiple myeloma,
acute lymphoid leukemia, and acute myeloid leukemia.8,14,22,23
Informed by this research, first-generation methyl-triazolo BET
inhibitors analogous to compound 1 have already been
translated to human clinical investigation by at least four
pharmaceutical companies.24
Beyond BETs, there are 38 additional bromodomain-
containing proteins for which high-quality small-molecule
probes are urgently needed. Transcription initiation factor
TFIID subunits 1 (TAF1) and 1L (TAF1L) are two such
proteins. As components of the STAGA complex, which
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Table 1. Exploration of Compound Scaffold Region
contains TRRAP, GCN5, TFIID, CBP/P300, mediator,25 and
Sp1,26 TAF1 is susceptible to oncogenic activation by MYC.
Moreover, TAF1 has been shown to block p53 activity,27 and
inactivation of TAF1 triggers a DNA damage response.28 In
addition, the TFIID complex, of which TAF1 is a significant
member, is vital to stem cell reprogramming.29 Inhibitors of
TAF1 may help further elucidate its biological role and
potentially function to inhibit cancer cell growth and survival.
Toward the development of a next-generation of bromodo-
main inhibitors, we have endeavored to build focused libraries
of novel small molecules possessing one of several biasing
elements with structural or electronic analogy to the methyl-
triazolo warhead of compound 1. Iterative synthesis and
biochemical testing is employed to efficiently compare chemical
cores and to explore appending groups. Complex, nonscalable,
and wasteful reactions can significantly impede iterative
screening efforts. Techniques involving the use of fluourous
reagents have shown great versatility, high-yield, rapid deploy-
ment, and are relatively eco-friendly. Complex molecules may
be synthesized in multicomponent reactions (MCRs)30 with
perfluoroalkyl “phase tags” which can be used to facilitate
purification by fluorous solid-phase extraction (F-SPE).31
Subsequent Suzuki-type reactions may replace the fluorous
tag to form a biaryl compound.32 Benefits of such reactions
include high yielding reactivity with facile purification.
Reactions have proven viable to create substituted proline
analogues,33 imidazo[1,2-a]-pyridines,34 diazepines,35 and
others.36,37 Such heterocyclic compounds form easily substitut-
able, drug-like scaffolds for the discovery of probe compounds.
Structurally dissimilar chemical tools are valuable reagents for
biological research when utilized in parallel, allowing observed
biological phenotypes to be unambiguously linked to common
target function.7 Today, coordinated efforts in ligand discovery
are working to develop additional BET bromodomain
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Scheme 1. Synthesis of Compound 32 and Close Analogues
inhibitors within our group, and beyond.38−40 Drawing upon
robust fluorous chemical synthesis and previously developed
screening capabilities, we have rapidly diversified and optimized
a set of potent and highly specific inhibitors. Building from the
dimethylisoxazole biasing element, we sought to determine
preferred regiochemistry, optimal scaffold (accessible via
MCR), and favorable appending groups. We report here a
novel class of bromodomain inhibiting compounds using the
recently identified 3,5-dimethylisoxazole biasing ele-
ment.38,39,41,42 In addition to demonstrating a new approach
to targeting BET bromodomains, these compounds and near
derivatives could create opportunities to drug other bromodo-
main-containing proteins.
functional groups around the phenyl ring. ortho- and meta-
Aldehyde substitutions were poorly tolerated, while a para-
aldehyde (compound 8) resulted in potency of 1.70 μM with a
ligand efficiency (as defined by Hopkins et al.)45 of 0.54.
Substitution with the more chemically stable para-acetyl group
(compound 9) did not affect potency. Previous study of similar
compounds had only explored variation at meta ring
positions.38
We performed computational modeling to establish hypoth-
eses regarding the mode of molecular recognition and to guide
further medicinal chemistry (Figure 1F). Using previously
published crystal structures of both compounds 1 and 3, we
were able to dock fragments from our emerging chemical
library into the Kac binding site of BRD4 domain 1
(BRD4(1)).8,43 As is the case with 3, compound 9 is thought
to bind through a conserved hydrogen bond with asparagine-
140 through the ring oxygen, while the ring nitrogen
coordinates through a structured water that interacts with the
hydroxyl group of tyrosine-97 (Figure 1G). On the basis of this
interaction model, we proceeded to build off the 4-position of
the phenyl ring to improve potency by establishing protein
surface interactions near the BC loop region.
We sought to explore a variety of chemical scaffolds while
maintaining the dimethyl isoxazole biasing moiety. A diverse set
of molecules was synthesized using reactions compatible with
fluorous-tagged multicomponent synthesis (Table 1). These
reactions create structural diversity by changing the substituent
groups on each fractional component, allowing generation of
diverse small-molecule libraries around a biasing element. The
perfluoroalkyl tags can substituted with a binding motif of
choice via Suzuki coupling (see Figure 1C).32 Synthesized
compounds included tertiary amines, pyrimidines, and fused
heterocyclic ring systems in order to explore the optimal shape
for exploiting protein−inhibitor contour interactions. In
addition to biochemical IC50, these compounds were selectively
evaluated in a BRD4-dependent cell viability study. Effects on
cell proliferation (EC50) were assessed using a surrogate
measure of cellular viability (ATP content; PerkinElmer
ATPlite) in the BET-rearranged 797 cell line and reported as
an average value of multiple experiments. The use of a fluorous-
tagged Groebke−Blackburn−Bienayme34,46 multicomponent
reaction was used to develop the para-imidazo[1,2-a]pyridine
scaffold (compound 11), which was found to be a promising
lead with biochemical and cellular inhibitory values of 11.0 and
14.1 μM, respectively. In addition, the compound 11 scaffold is
accessible at a variety of positions for diversification to drive
potency and develop a further understanding of structure−
activity relationships (SAR).
RESULTS AND DISCUSSION
■
Analysis of existing small-molecule bromodomain inhibitors,
shown in Figure 1A, has defined a structure−function model in
which the competitive small molecule binds through two
chemical features: an acetyl-lysine mimetic warhead (red) and a
core region with appending surface-recognition features
(black). Nonobvious Kac mimetics in existing BET inhibitors,
such as the triazole ring in 18 and 2 (I-BET),40 the 3,5-
dimethylisoxazole ring in 3 (iBET-151),43 and the quinazoli-
none in 4 (PFI-1),44 form a highly specific hydrogen bond to
the conserved asparagine in bromodomains. The core of the
molecule establishes shape complementarity with the contour
of the binding pocket to increase binding affinity primarily
through hydrophobic interactions. Appending groups provide
an opportunity to enhance potency and selectivity via surface
and loop region interactions. The 3,5-dimethylisoxazole
headgroup of 3 was identified as a promising and chemically
accessible biasing moiety for further development as a chemical
probe. Our screening strategy created an extensive series of
diverse chemical features attached to the 4-position of the 3,5-
dimethylisoxazole (Figure 1B). As a starting point, we focused
on small, ligand efficient compounds that could serve as leads
for further derivatization.
This approach to library development was empowered by
recent advances in use of perfluoroalkyl substituents to enable
efficient Suzuki-type coupling reactions, shown in Figure 1C.
Purification of reaction intermediates is greatly facilitated by F-
SPE. In this case, aryl perfluorooctylsulfonates (ArO-
SO2(CF2)7CF3) were reacted with 3,5-dimethylisoxazole-4-
boronic acid pinacol esters to produce a low-molecular-weight
chemical series. The resulting compounds were purified using
F-SPE. Initial compounds included positional isomers of
carbonyl functionalities around the phenyl ring system (Figure
1D). Compound potency was initially assessed by relative
average IC50 determined by an AlphaScreen competition assay
(representative assay shown in Figure 1E). IC50 values were
found to be highly dependent on the steric arrangement of
Having selected a core scaffold in compound 11, we
assembled a focused library of compounds to optimize
placement of heteroatoms and appending groups. Synthesis
D
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of the eventual lead compound 32 (UMB-32) is shown in
Scheme 1 as an exemplar of the route used to assemble the
focused SAR series. Bromobenzaldehyde reagents were
substituted for fluorosulfonylbenzaldehydes in the subsequent
MCRs. The compound was synthesized in a two-step reaction.
The first step included combination of t-butylisocyanide, 2-
aminopyrazine, and 4-bromobenzaldehyde. The reaction is
catalyzed by scandium triflate and microwaved at 150 °C for 30
min in 3:1 CH2Cl2/MeOH. Flash chromatography yielded the
pure product 80%. This intermediate was then coupled to 3,5-
dimethylisoxazole-4-boronic acid pinacol ester in a Suzuki
coupling. The resulting crude mixture was purified by flash
chromatography with yield 57% and purity of >95% as
determined by liquid chromatography.
and 32 upon substitution of a 7-N. For this reason, compound
32 emerged as a lead compound.
A limited structural study around the linking phenyl ring is
shown in Table 3. Substitution of the phenyl ring for pyridine
Table 3. Variations to UMB-32 Linker
compd
R1
R2
X
BRD4 IC50 (μM) 797 EC50 (μM)
40
41
42
43
44
t-Bu
t-Bu
t-Bu
t-Bu
i-Pr
H
H
2-N
3-N
CH
CH
CH
51.9
12.9
13.0
>100
8.29
12.8
Analogues were synthesized similarly to explore the func-
tional consequence of steric and electronic modification to the
fused bicyclic scaffold, shown in Table 2. Variations of R1 from
2-F
3-OMe
3-OMe
0.562
0.474
Table 2. Elaboration of Imidazopyridine Scaffold
resulted in decreased potency (compound 40 and 41), and the
addition of electron-withdrawing fluorine (compound 42) was
detrimental as well. Electron-donating methoxy substituents
appear to be mildly beneficial in biochemical assays. Additional
work regarding this linking region of the molecule may be a
focus of ongoing medicinal chemistry.
BRD4 IC50 797 EC50
compd
R1
R2
X
(μM)
(μM)
The potency of 32 was accomplished by two key changes
from compound 11 (Figure 2A). Compound 32 has substituted
t-Bu for benzyl and inserted a 7-N. Comparison of compound
11, 19, and 32 show the critical substitutions in the
development of 32, which led to an improved biochemical
IC50 (Figure 2B, top). Biochemical potency was supported by
cellular screening of compounds again in the BET-rearranged
797 cell line (Figure 2B, bottom). The addition of 7-N and the
benzyl to t-butyl substitution clearly establish 32 as a front-
runner with dramatically improved biological activity. While
there is often a drop in potency when compounds shift from
biochemical to cellular assays, we observed similar potency for
compound 32 in both experiments. This likely reflects high
target availability of free drug, intrinsic cell permeability of this
chemical series, and high sensitivity of the genetically leveraged
BRD4-NUT cells (797). Notably, we previously observed
parity between biochemical and cellular potency during the
initial assessment of compound 1,8 which were confirmed in
the present study (IC50 of 81.5 nM and EC50 of 71.7 nM, data
not shown). Characterization of compound 32 binding kinetics
to BRD4(1) by isothermal titration calorimetry (ITC) further
supported the improved biochemical affinity (Supporting
Information, Figure S1). The resultant Kd was shown to be
550 nM, in good agreement with the biochemical measure-
ments. The binding process is shown to be exothermic and
positively entropic with a 1:1 binding ratio.
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
H
H
CH
CH
CH
7-N
CH
CH
CH
6-N
7-N
7-N
7-N
7-N
7-N
CH
6-N
8-N
7-N
CH
CH
CH
CH
CH
7-N
7-N
3.89
Bn
Bn
Bn
6-Me
>100
6-Cl
11.8
18.8
18.4
49.1
5.07
7.36
20.7
11.8
32.8
H
cyclohexyl
4-(OMe)Ph
4-(OMe)Ph
4-(OMe)Ph
4-(OMe)Ph
(S)-1-PhEt
−CH2CO2H
−CH2CO2Et
−CH2CO2tBu
t-Bu
H
0.904
2.76
31.5
H
6-Cl
7-OMe
2.89
7.86
2.86
H
H
H
>100
H
4.53
4.96
0.479
H
H
2.04
2.06
t-Bu
H
20.7
t-Bu
H
0.860
0.637
1.17
t-Bu
H
0.724
2.19
t-Bu
6-Cl
t-Bu
6-Me
8-CF3
6-CO2CH2
6-COOH
H
3.17
t-Bu
11.9
t-Bu
1.62
t-Bu
0.968
0.807
1.66
i-Pr
0.494
n-Bu
H
Lead compounds 11 (Supporting Information, Table S1)
and 32 (Figure 2C) were analyzed for bromodomain selectivity
using a commercially available, phage-based, multiplexed
bromodomain displacement assay (BromoScan; DiscovRx).
We have noted somewhat increased sensitivity of the
BromoScan assay compared to AlphaScreen. Compound 11
demonstrates broad activity against the tested bromodomains
with some selectivity for the BET family. In contrast, 32
showed markedly increased potency for the BET proteins
associated with a dramatic improvement in selectivity.
Interestingly, 32 revealed potent binding to the TAF1 (560
nM) and TAF1L (1.3 μM) bromodomains. Albeit a
the compound 11 benzyl group to bulky, nonaromatic groups
like cyclohexyl (compound 20) and t-Bu (compound 29)
improved potency. An acid at the R1 position (compound 26)
completely inactivated the ligand. Appending R2 groups at the
6-, 7-, and 8-positions of the fused ring system universally
decreased potency compared to parent compounds. Ring-
nitrogens at the 7- and 8-positions (compound 32 and 31,
respectively) had little effect on biochemical potency. A 6-
position ring-nitrogen reduced compound potency from 479
nM (compound 29) to 20 μM (compound 30). Cellular
potency, however, modestly improved between compound 29
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Figure 2. Compound 32 is a potent, selective inhibitor of BRD4. (A) Selected compounds leading to compound 32. (B) Representative biochemical
inhibitory curves (top, duplicate technical replicates) and 797 cellular viability (bottom, quadruplicate technical replicates). (C) Evaluation of
compound 32 selectivity against a panel of bromodomains. (D) Crystal structure of 32 bound to BRD4(1). Blue is oxygen and red is nitrogen (PDB:
4WIV). (E) Ligand interaction diagram of 32 from the cocrystal structure with BRD4. (F) Crystal structure of 32 overlaid on 1 (left) and rotated
90° (right).
significantly weaker interaction, this observation suggests a
starting point for development of inhibitors of the previously
undrugged TAF1 bromodomain.
To understand the mode of molecular recognition,
cocrystallization of 32 and BRD4(1) was pursued, yielding a
rather high-resolution structure of the cocomplex (PDB: 4WIV;
F
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a 100 nL 384-well pin transfer manifold on a Janus workstation. Stocks
were arrayed in 10 point quadruplicate dose response in DMSO stock
in 384-well Greiner compound plates. After addition of compound,
plates were incubated for 3 days in a 37 °C incubator. Cell viability was
read out using ATPlite from PerkinElmer. Plates were removed from
the incubator and brought to room temperature prior to use.
Lyophilized powder was resuspended in lysis buffer and diluted 1:2
with DI water. Then 25 μL of this solution was added to each well
using the Biotek liquid handler. Plates were sealed with adherent
aluminum seals prior to vortexing and spinning down at 1000g for 1
min. Plates were incubated for 15 min at room temperature before a
signal was read on an Envision plate reader. Reported EC50 values are
based on averages of multiple experiments, except where noted.
Computational Methods. All computational work was performed
in Schrodinger Suite (Schrodinger, LLC). Conformational analysis of
lead compounds was performed using Schrodinger’s Conformational
Search function. Possible poses were prepared for docking by Ligprep.
In both cases, default settings were used (OPLS2005 force field, water
solvent). Docking was conducted using Glide. The cocrystal of BRD4
and compound 1 (PDB: 3MXF) was used to define the ligand
receptor grid. Water molecules outside the binding pocket were
excluded, and hydrogen bonding interactions were optimized prior to
docking.
1.56 Å; Figure 2D,E). Compound 32 interacts with BRD4(1)
via the expected hydrogen bond to the conserved asparagine.
Hydrogen bonding to structural waters and π-stacking with
tryptophan-81 illustrate the contributions to binding of the 32
region farthest from the warhead over other similar compounds
in the SAR series. The additional heteroatom at 7-N is solvent
exposed. In addition, the t-Bu group rests in the groove
between the WPF shelf and the BC loop similar to the
chlorophenyl in compound 1 and other diazepine bromodo-
main inhibitors. An analysis the crystal structure of 32 overlaid
on 1 (PDB: 3MXF; Figure 2F) underscores this feature. The
central phenyl ring in 32 occupies the same region as the fused
thienodiazepine ring system of 1. As previously mentioned, the
t-Bu group of 32 takes the place of chlorophenyl group of 1. In
contrast, 32 has no bulky appending group analogous to the t-
Bu in 1, suggesting a means of differential binding of 32 to
TAF1.
CONCLUSION
■
Bromodomain proteins have emerged as master regulators of
development and disease. Numerous functional connections to
cancer and inflammation pathophysiology has established a
fervent interest in the development of direct-acting inhibitors.
Here we report a new functional class of bromodomain
inhibitors that potently inhibits BET bromodomains and
TAF1/TAF1L. Further optimization to develop a selective
probe to the bromodomain of TAF1 is ongoing in our
laboratories. In sum, we illustrate a facile synthetic strategy and
biochemical platform capable of efficiently optimizing selective
inhibitors from small organic biasing features. Compound 32
emerges from this research as a chemical probe available to the
scientific community for mechanistic and translational research.
General Synthetic Information. All chemicals and solvents were
purchased from commercial suppliers and used as received. All
biologically evaluated compounds were found to be >95% pure as
1
determined by NMR and LCMS. H NMR (300 or 400 MHz) and
13C NMR (75 MHz) spectra were recorded on a Varian NMR
spectrometer. CDCl3 was used as the solvent unless otherwise
specified. LC-MS were performed on an Agilent 2100 system with a
C18 (5.0 μm, 6.0 mm × 50 mm) LC column. The mobile phase is
MeOH and water both containing 0.01% trifluoroacetic acid. A linear
gradient was started from 75:25 MeOH/H2O to 100% MeOH in 5.0
min at a flow rate of 0.7 mL/min. The chromatograms were recorded
at UV 210, 254, and 365 nm and subsequently used to determine
compound purity. Low resolution mass spectra were recorded in APCI
(atmospheric pressure chemical ionization). Flash chromatography
separation was performed on YAMAZEN AI-580 system with Agela
silica gel (12 or 20 g, 230−400 μm mesh) cartridges. The microwave
reactions were performed on a Biotage Initiator 8 system.
General Procedures for the Synthesis of Compounds 11,
16−25, and 28−39. The synthesis of these compounds was
accomplished by a reported two-step synthesis shown in Scheme
1.34 The three-component reaction (Groebke−Blackburn−Bienayme
reaction) was followed by the Suzuki coupling.
Representative Procedure for the Three-Component Reaction:
Synthesis of 2-(4-Bromophenyl)-N-(t-butyl)imidazo[1,2-a]pyrazin-
3-amine (X = N, R1 = t-Bu, R2 = H).34 A sealed 10 mL microwave tube
charged with t-butylisocyanide (0.043 g, 0.52 mmol), 2-aminopyrazine
(0.05 g, 0.52 mmol), 4-bromobenzaldehyde (0.074 g, 0.40 mmol), and
Sc(OTf)3 (0.010 g, 0.02 mmol) in 2 mL of 3:1 CH2Cl2/MeOH was
heated under microwave at 150 °C for 30 min. The mixture was
filtered and washed with EtOAc (4 mL). Concentration of the organic
phase gave a crude product which was purified by flash
chromatography eluted with 2:8 EtOAc/hexane to provide product
as a yellow solid (110 mg, 80%).
EXPERIMENTAL SECTION
■
AlphaScreen BRD Binding Assay. Assays were performed with
minor modifications from the manufacturer’s protocol (PerkinElmer,
USA). All reagents were diluted in 50 mM HEPES, 150 mM NaCl,
0.1% w/v BSA, 0.01% w/v Tween20, pH 7.5, and allowed to
equilibrate to room temperature prior to addition to plates. After
addition of Alpha beads to master solutions, all subsequent steps were
performed in low light conditions. A 2× solution of components with
final concentrations of BRD (see Supporting Information) at 40 nM,
Ni-coated acceptor bead at 25 μg/mL, and 20 nM biotinylated-JQ1
(Supporting Information, Figure S2, synthesized as previously
described)47 was added in 10 μL to 384-well plates (AlphaPlate-384,
PerkinElmer, USA). After a 1 min 1000 rpm spin-down, 100 nL of
compounds in DMSO from stock plates were arrayed in duplicate dose
response by pin transfer using a Janus Workstation (PerkinElmer,
USA). The streptavidin-coated donor beads (25 μg/mL final) were
added as with previous solution in a 2×, 10 μL volume. Following this
addition, the plates were sealed with foil to block light exposure and to
prevent evaporation. The plates were spun down again at 1000 rpm for
1 min. Next, the plates were incubated in the room with the plate
reader (for temperature equilibration) for 1 h prior to reading the
assay. Signal is stable for up to 3 h after donor bead addition.
AlphaScreen measurements were performed on an Envision 2104
(PerkinElmer, USA) utilizing the manufacturer’s protocol. Reported
IC50 values are based on averages of multiple experiments, except
where noted.
Representative Procedure for the Suzuki-Coupling Reaction:
Synthesis of N-(tert-Butyl)-2-(4-(3,5-dimethylisoxazol-4-yl)phenyl)-
imidazo[1,2-α]pyrazin-3-amine (32).34 A sealed 10 mL microwave
tube charged with 2-(4-bromophenyl)-N-(t-butyl)imidazo[1,2-a]-
pyrazin-3-amine (X = N, R1 = t-Bu, R2 = H, 0.110 g, 0.32 mmol),
3,5-dimethylisoxazole-4-boronic acid pinacol ester (0.093 g, 0.42
mmol), Pd(dppf)Cl2·CH2Cl2 (0.021 g, 0.026 mmol, 8% mol), and
K2CO3 (0.088 g, 0.64 mmol) in 2 mL of 4:4:1 acetone/toluene/H2O
was heated under microwave at 130 °C for 40 min. The mixture was
filtered and washed with EtOAc (4 mL). Concentration of the organic
phase gave a crude product which was purified by flash
chromatography eluting with 3:7 EtOAc/hexane to give 32 as
Cell Viability Assay. Assays were performed with endogenous
BRD4-NUT-expressing midline carcinoma cell lines, 797.48 Cells were
counted and adjusted to 60000 cells/mL. Using a Biotek EL406, 50 μL
of cells are media were distributed into 384-well white plates from
Thermo. Immediately after plating, compound in DMSO was
distributed to plates. For large plate sets, cells were returned to 37
°C incubator while not in use. Compounds were added to plates using
1
brownish solid (>95% purity, 66 mg, 57%). H NMR (300 MHz,
CDCl3) δ 9.01 (s, 1H), 8.16 (d, J = 4.8 Hz, 1H), 8.05 (d, J = 6.6 Hz,
G
dx.doi.org/10.1021/jm501120z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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2H), 7.88 (d, J = 4.8 Hz, 1H), 7.38 (d, J = 6.6 Hz, 2H), 3.18 (s, 1H,
NH), 2.46 (s, 3H), 2.33 (s, 3H), 1.11 (s, 9H). 13C NMR (75 MHz,
CDCl3) δ 165.0, 158.34, 143.0, 141.2, 137.1, 133.2, 129.8, 128.7,
128.6, 128.3, 124.9, 116.1, 116.1, 56.7, 30.2, 11.4, 10.6. MS (APCI) m/
z 362.2 (M+ + 1).
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Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.;
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Thangue, N.; French, C. A.; Wiest, O.; Kung, A. L.; Knapp, S.;
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ASSOCIATED CONTENT
* Supporting Information
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S
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M.; Schinzel, A. C.; McKeown, M. R.; Heffernan, T. P.; Vakoc, C. R.;
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Accession Codes
4WIV: co-crystal of compound 32 and BRD4(1).
AUTHOR INFORMATION
Corresponding Authors
*For J.E.B.: phone, 1.617.632.6629; E-mail, james_bradner@
dfci.harvard.edu.
■
*For W.Z.: phone, 1.617.287.6147; E-mail, wei2.zhang@umb.
edu.
Author Contributions
Authors M.R.M., D.L.S., H.F., and S.L. contributed equally to
this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge undergraduate students Yuan Xia and Shiva
Dastjerdi for their assistance. The work in this study was
supported by National Institutes of Health U54 grant
CA156732 (J.E.B. and W.Z.), NIH U01 grant HD076508
(J.E.B.), the Damon Runyon Cancer Research Foundation
(J.E.B.), and The William Lawrence and Blanche Hughes
Foundation (J.E.B.). D.L.B. is a Merck Fellow of the Damon
Runyon Cancer Research Foundation (DRG-2196-14).
(17) Schroder, S.; Cho, S.; Zeng, L.; Zhang, Q.; Kaehlcke, K.; Mak,
L.; Lau, J.; Bisgrove, D.; Schnolzer, M.; Verdin, E.; Zhou, M. M.; Ott,
M. Two-pronged binding with bromodomain-containing protein 4
liberates positive transcription elongation factor b from inactive
ribonucleoprotein complexes. J. Biol. Chem. 2012, 287, 1090−1099.
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Ma, L.; Agno, J. E.; Lemieux, M. E.; Picaud, S.; Yu, R. N.; Qi, J.;
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ABBREVIATIONS USED
■
Kac, acetyl lysine; BET, bromodomain and extraterminal
domain; BRD, bromodomain containing protein; BRD4(1),
first bromodomain of bromodomain containing protein 4; TAF,
transcription initiation factor TFIID subunits; F-SPE, fluorous
solid phase extraction
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