Discovery and Characterization of GSK2801, a Selective Chemical Probe for the Bromodomains BAZ2A and BAZ2B

Article abstract of DOI:10.1021/acs.jmedchem.5b00209

Bromodomains are acetyl-lysine specific protein interaction domains that have recently emerged as a new target class for the development of inhibitors that modulate gene transcription. The two closely related bromodomain containing proteins BAZ2A and BAZ2B constitute the central scaffolding protein of the nucleolar remodeling complex (NoRC) that regulates the expression of noncoding RNAs. However, BAZ2 bromodomains have low predicted druggability and so far no selective inhibitors have been published. Here we report the development of GSK2801, a potent, selective and cell active acetyl-lysine competitive inhibitor of BAZ2A and BAZ2B bromodomains as well as the inactive control compound GSK8573. GSK2801 binds to BAZ2 bromodomains with dissociation constants (K_D) of 136 and 257 nM for BAZ2B and BAZ2A, respectively. Crystal structures demonstrated a canonical acetyl-lysine competitive binding mode. Cellular activity was demonstrated using fluorescent recovery after photobleaching (FRAP) monitoring displacement of GFP-BAZ2A from acetylated chromatin. A pharmacokinetic study in mice showed that GSK2801 had reasonable in vivo exposure after oral dosing, with modest clearance and reasonable plasma stability. Thus, GSK2801 represents a versatile tool compound for cellular and in vivo studies to understand the role of BAZ2 bromodomains in chromatin biology.

Full text of DOI:10.1021/acs.jmedchem.5b00209

Article
pubs.acs.org/jmc
Discovery and Characterization of GSK2801, a Selective Chemical
Probe for the Bromodomains BAZ2A and BAZ2B
Peiling Chen,§,○ Apirat Chaikuad,†,○ Paul Bamborough,∥ Marcus Bantscheff,# Chas Bountra,‡
Chun-wa Chung,∥ Oleg Fedorov,†,‡ Paola Grandi,# David Jung,§ Robert Lesniak,§ Matthew Lindon,⊥
Susanne Muller,†,‡ Martin Philpott,†,‡ Rab Prinjha,⊥ Catherine Rogers,†,‡ Carolyn Selenski,§
̈
Cynthia Tallant,†,‡ Thilo Werner,# Timothy M. Willson,§ Stefan Knapp,*,†,‡,∇ and David H. Drewry*,§
†Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research
Building, Roosevelt Drive, Headington, Oxford OX3 7DQ, United Kingdom
‡Nuffield Department of Clinical Medicine, Target Discovery Institute, University of Oxford, NDM Research Building,
Roosevelt Drive, Headington, Oxford OX3 7FZ, United Kingdom
§Department of Chemical Biology, GlaxoSmithKline, Research Triangle Park, 5 Moore Drive, Research Triangle Park, North Carolina
27709-3398, United States
⊥
∥Computational & Structural Chemistry, Molecular Discovery Research, and Epinova, Discovery Performance Unit,
GlaxoSmithKline R&D, Stevenage, Hertfordshire SG1 2NY, United Kingdom
#Cellzome GmbH, Molecular Discovery Research, GlaxoSmithKline, Meyerhofstrasse 1, 69117 Heidelberg, Germany
∇Institute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany
S
* Supporting Information
ABSTRACT: Bromodomains are acetyl-lysine specific protein interaction
domains that have recently emerged as a new target class for the development
of inhibitors that modulate gene transcription. The two closely related
bromodomain containing proteins BAZ2A and BAZ2B constitute the central
scaffolding protein of the nucleolar remodeling complex (NoRC) that reg-
ulates the expression of noncoding RNAs. However, BAZ2 bromodomains
have low predicted druggability and so far no selective inhibitors have been
published. Here we report the development of GSK2801, a potent, selec-
tive and cell active acetyl-lysine competitive inhibitor of BAZ2A and BAZ2B
bromodomains as well as the inactive control compound GSK8573. GSK2801
binds to BAZ2 bromodomains with dissociation constants (KD) of 136 and 257 nM for BAZ2B and BAZ2A, respectively. Crystal
structures demonstrated a canonical acetyl-lysine competitive binding mode. Cellular activity was demonstrated using fluorescent
recovery after photobleaching (FRAP) monitoring displacement of GFP-BAZ2A from acetylated chromatin. A pharmacokinetic
study in mice showed that GSK2801 had reasonable in vivo exposure after oral dosing, with modest clearance and reasonable
plasma stability. Thus, GSK2801 represents a versatile tool compound for cellular and in vivo studies to understand the role of
BAZ2 bromodomains in chromatin biology.
binding activity in addition to their previously known activity. The
rapid exploration of the biology of BET bromodomains has been
facilitated by the availability of potent and selective small molecule
chemical tools that have been widely used to study chromatin
biology.13−20 With that in mind, we embarked on efforts to
identify probes for bromodomains outside of the BET subfamily
through a public private partnership coordinated by the Struc-
tural Genomics Consortium. Here we outline the collaborative
discovery of GSK2801, a chemical probe for the bromodomain
adjacent to zinc finger domain protein(BAZ) 2A/B. Analternative
INTRODUCTION
■
Bromodomain modules selectively recognize acetylated lysine
(KAc) containing sequences, a post-translational modification
that is highly abundant in histones and other nuclear proteins.1−3
Interest in these so-called epigenetic reader proteins has risen as
it has become clear that the interaction between the proteins and
the acetylated histone tails can be inhibited with small molecules.
A number of reported selective bromodomain inhibitors are
drug-like molecules and have potential as therapeutics in a range
of diseases, including cancer and inflammation.4−7 Much of the
work to date has focused on the BET family for which inhibitors
have now entered clinical testing.4,8,9 The BET subfamily of
bromodomains (BRDT and BRD2−4) has proven to be
remarkably druggable. Interestingly, some molecules in clinical
development10−12 have been reported to have BET bromodomain
Special Issue: Epigenetics
Received: February 4, 2015
© XXXX American Chemical Society
A
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Figure 1. Initial hit and SAR strategy. (a) Structure of the initial hit compound 1. (b) SAR development strategy targeting two positions (R1/R2) of the
indolizine ring.
Figure 2. Cocrystal structure of compound 1 with BRD2 and compound 2 with BAZ2B. (a) Cocrystal structure of compound 1 with BRD2 and a model
of compound 1 in BAZ2B. These illustrate that BRD2 appears to have a narrower channel that accommodates the pendant phenyl ring, suggesting this as
an area to explore in order to enhance selectivity. (b) Chemical structure of compound 2 (left) and cocrystal structure of compound 2 with BAZ2B
(right). The main interacting side chains are shown in ball and stick representation. Hydrogen bonds are shown as dotted lines. Conserved water
molecules are depicted as spheres.
BAZ2A/B chemical probe that can be used as an additional
independent validation tool has recently been published by our
group in collaboration with an academic collaborator.21
prostate cancer, and the protein may serve as a marker for
metastatic potential.27
Little is known about the BAZ2B subtype. However, single
nucleotide polymorphisms (SNPs) in the BAZ2B gene locus
have been associated with sudden cardiac death. High
expression levels of BAZ2B have been associated with poor
outcome of pediatric B cell acute lymphoblastic leukemia
(B-ALL).28 X-ray crystal structures of the BAZ2A/B bromo-
domains showed a shallow acetyl-lysine binding pocket, and
they were predicted to be among the least druggable members
of the protein family.29 We therefore felt that identification
of inhibitors for the BAZ2A/B bromodomains would be an
interesting indicator of the chemical tractability of the broader
protein family.
BAZ1A, BAZ1B, BAZ2A, and BAZ2B constitute a family of
evolutionarily conserved and ubiquitously expressed proteins
with conserved domain structure including PHD and bromodo-
main histone tail reader motifs.22 BAZ2A is a component of the
nucleolar remodeling complex (NoRC), a member of the
“imitation switch chromatin remodeling complexes” (ISWI),23
which play a role in regulating the expression of noncoding RNAs
and in the formation of repressive heterochromatin in particular
at centromeres and telomeres.24,25 Association of BAZ2A with
acetylated histone tails is mediated by its bromodomain.26
Recently, high expression levels of BAZA have been reported in
B
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Scheme 1. General Routes to Indolizines
To optimize the indolizine hit for activity on BAZ2A/BAZ2B
and selectivity over the BET bromodomains, we explored
substituents at the 7-position of the indolizine and modified the
aryl group at the 1-position. Compounds were synthesized via
two general routes. In route 1 (Scheme 1a), an appropriately para-
substituted pyridine A was N-alkylated with 1-chloropropan-2-
one. The resulting pyridinium salt B was then reacted in a dipolar
cycloaddition reaction with an aryl alkyne and potassium
carbonate in DMF at elevated temperatures in a microwave to
give the desired ring system with the acetyl lysine mimetic methyl
ketone present in the 3-position of the indolizine. In route
2 (Scheme 1b), the appropriate pyridinium salt B was reacted
with methyl acrylate in the presence of triethylamine and
manganese dioxide in toluene at 90 °C for 1 h to give an
indolizine C with a methyl ester in the 1-position, a methyl
ketone in the 3-position, and the substituent that came from the
starting pyridine in the 7-position. Saponification of the methyl
ester with aqueous base, followed by bromination gave bromo-
substituted indolizines of general structure D. In the final step, a
Suzuki reaction was utilized to install the desired aryl group, R1,
in the 1-position.
We first explored the structure−activity relationships (SARs)
on the pendant phenyl group in the R1-position of the indolizine.
In an AlphaScreen assay (Table 1), compounds 3−6 showed
weaker activity at both BAZ2A and BAZ2B compared to 1.
Replacing the pendant 2-pyridyl group with a meta-methoxy
phenyl (compound 3) obliterated activity at both BAZ2A and
BAZ2B. Compound 7, harboring an ortho-CH2OH, maintained
activity at BAZ2A and gave a good increase in potency at BAZ2B,
moving from 7 to 1 μM. Compound 12, which incorporates an
ortho-methyl sulfone, improved potency on BAZ2A to 720 nM,
and BAZ2B stayed in the low micromolar IC50 range.
RESULTS AND DISCUSSION
■
A few weak binding acetyl-lysine mimetic fragments have been
recently reported30 in addition to a chemical tool compound,
which showed an unusual aromatic π-stacking interaction of two
aromatic moieties present in the inhibitor that efficiently filled
out the binding pocket.21 In our hands, screening of the BAZ2A
bromodomain against a set of molecules that contained acetyl-
lysine mimetics led to identification of a low molecular weight,
chemically tractable indolizine, compound 1, which had been
previously reported as an inhibitor of BET bromodomain
subfamily proteins. Indolizine 1 had a potency of 1.5 μM in a
BAZ2A AlphaScreen assay, which was comparable to its reported
activity against the BET bromodomains (Figure 1).31,32
A crystal structure of compound 1 bound to BRD2 (PDB ID:
4A9I) revealed that the methyl ketone is serving as an acetyl
lysine mimetic (Figure 2A). Compound 1 was modeled into the
acetyl lysine binding site of BAZ2B (Figure 2A). The channel
that accommodates the indolizine R1 substituent appears
wider in BAZ2B, suggesting that modification in this region
will modulate selectivity. To facilitate the optimization of the
indolizine hit, we cocrystallized an early representative
compound (2) with BAZ2B (Figure 2B). Compound 2 has a
2-pyridyl group at R1 and a morpholine substituent in the R2
position. As is the case for compound 1 bound to BRD2,2 the
methyl ketone of compound 2 functions as an acetyl-lysine
mimetic. The carbonyl forms a hydrogen bond with the con-
served asparagine N1944 and with a network of water molecules
that are typically found at the bottom of the acetyl-lysine binding
site. The indolizine ring forms a number of hydrophobic interac-
tions, most notably to I1950 and V1893. The aromatic ring in
position R1 forms an aromatic stacking interaction with W1887.
The R2 morpholino group was oriented toward the solvent
and formed no significant interactions with BAZ2B. Details
describing protein purification, crystallization, and crystallog-
raphy as well as in vitro screening methods are available in the
Supporting Information.
Based on the improved potency of the compounds on both
BAZ2A and BAZ2B with the ortho-CH2OH compound 7 and
the ortho-methyl sulfone 12, we explored substitution on the
indolizine ring with these groups in place. In the context of the
ortho-CH2OH substituted pendant phenyl (Table 2), a methyl
C
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Table 1. Substitution on the Pendant Phenyl Ring; Shown Are
Averaged Values of Three Replicates and the Standard Error
of the Mean (SEM)
Table 3. Seven-Position Indolizine Substitution in the
Context of the ortho-SO2Me on the Pendant Phenyl Ring;
Shown Are Averaged Values of Three Replicates and the
Standard Error of the Mean (SEM)
BAZ2A IC50
BAZ2B IC50
compd
R1
(μM)
(μM)
1
2-pyridyl
1.5 0.06
>30
7.0 0.03
>30
compd
R2
BAZ2A IC50 (μM)
BAZ2B IC50 (μM)
3
m-methoxy-phenyl
2-thiophene
12
18
19
20
21
22
H
0.72 0.07
0.16 0.05
0.40 0.03
0.55 0.00
0.58 0.05
0.89 0.00
1.3 0.04
0.67 0.02
0.43 0.02
0.84 0.04
0.82 0.01
1.9 0.04
4
8.6 0.01
7.2 0.06
3.3 0.05
1.8 0.05
1.8 0.02
1.4 0.01
1.2 0.07
1.1 0.07
0.72 0.07
34.1 0.19
22.7 0.5
12.5 0.04
1.0 0.02
9.8 0.06
1.5 0.03
4.1 0.02
3.8 0.03
1.3 0.04
CH2NH-Boc
OPr
5
o-OH-phenyl
6
m-OH-phenyl
OPh
7
o-CH2OH-phenyl
m-CONH2-phenyl
o-CH2OH, p-OMe-phenyl
p-CH2OH-phenyl
p-NMe2-phenyl
OMe
8
Me
9
10
11
12
o-methyl sulfone-phenyl
indolizines 7 and 12 were quite weak on BRD4 and BRD9. The
Boc-protected aminomethyl compound 18, however, has low
micromolar BRD9 activity and thus is not suitable as a BAZ2B/A
probe. The ethers (19−21) were inactive or very weakly active
against BRD4(1) and BRD9.
Modeling suggested that the improved selectivity of the
bulkier ortho-substituted phenyls for BAZ2B over BET resulted
from reduced planarity of these compounds with respect to
ortho-H and 2-pyridyl analogues. The binding site of BRD2 is
narrower than BAZ2B and binds compound 1 in essentially a
planar conformation (Figure 2A). Docking of compounds 17
and 20 into the BRD2 site gave strained, high-energy poses con-
sistent with their lower in vitro potency. In contrast, the more
open site of BAZ2B was able to accommodate docked
o-substituted phenyl indolizines with ease.
We cocrystallized compounds 17 and 20 with BAZ2B
(Figure 3). The structure confirmed that the introduction of
the 2-hydroxyl-methyl group at the phenyl ring produced a 90°
rotation of the phenyl ring incompatible with potent BET
binding. In BAZ2B, the rotated phenyl ring makes an end-on
π-stacking interaction with W1887 and orients the 2-hydoxyl-
methyl group to produce a hydrogen-bond with the ZA-loop
backbone (N1894 amine). The methyl-sulfonyl substitution at
position-2 of the phenyl ring produces an even more dramatic
rotation to accommodate the more bulky sulfonyl group. In
addition, the sulfonyl oxygen groups also make a pair of hydrogen
bonds to the backbone NH of N1894.
Based on the potency and excellent selectivity over the BET
bromodomains, we chose to look at compound 19, GSK2801,
in more detail. We wanted to determine the potency of
the compound in orthogonal assay systems. The interaction be-
tween the small molecule GSK2801 and the bromodomain was
examined using biolayer interferometry (Octet-RED BLI)
and isothermal titration calorimetry (ITC) (Figure 4a,b). Steady
state fits of the measured dose response BLI data resulted in a
dissociation constant of 60 nM (KD). The inhibitor showed fast
on- and off-rate binding (Kon, 1.57 0.02 × 105 1/(M s); Koff,
6.95 0.058 × 10−3 1/s). ITC confirmed this value determining
a KD of 136 and 257 nM for BAZ2B and BAZ2A and a stoi-
chiometry of 1.0 0.1, respectively. The cocrystal structure with
GSK2801 showed that the observed binding mode of the probe
is conserved when compared with the related compound 20
(Figure 4c).
Table 2. Influence of the 7-Position Indolizine Substitution in
the Context of the ortho-CH2OH on the Pendant Phenyl Ring;
Shown Are Averaged Values of Three Replicates and the
Standard Error of the Mean (SEM)
compd
R2
BAZ2A IC50 (μM)
BAZ2B IC50 (μM)
7
H
1.8 0.05
1.0 0.09
1.2 0.01
1.7 0.13
4.1 0.09
4.1 0.06
1.0 0.02
0.37 0.02
0.41 0.03
0.54 0.05
1.90 0.01
0.61 0.01
13
14
15
16
17
CH3
OCH3
morpholine
CONH2
OPh
group 13 and a methoxy group 14 improved BAZ2A activity
relative to the unsubstituted parent, compound 7. Both the
primary carboxamide substituted indolizine 16 and the phenoxy
substituted compound 17 were weaker against BAZ2A. BAZ2B
activity was improved in methyl substituted indolizine 13,
methyl ether substituted indolizine 14, morpholine substituted
indolizine 15, and phenyl ether substituted indolizine 17. With
amide substituted compound 16, however, we saw a potency
decrease on BAZ2A relative to unsubstituted compound 7.
Within a series of ortho-methyl sulfone phenyl analogues
(Table 3), several indolizine substituents improved activity at
both BAZ2A and BAZ2B relative to unsubstituted compound 12.
The Boc-protected aminomethyl substituent 18 gave good
potency increases. In the ortho-hydroxymethyl series described
in Table 2, ethers off of the indolizine helped BAZ2B activity but
in general were less helpful for BAZ2A activity. In the ortho-
methylsulfone context, however, the methyl ether 21, propyl
ether 19, and phenyl ethers 20 enhanced both BAZ2A and
BAZ2B activity.
Following identification of a handful of compounds with
improved potencies, we sought to characterize their activity at
other bromodomains. Table 4 shows that the two unsubstituted
D
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Table 4. Cross Screening of Most Potent BAZ Compounds on BRD4(1) and BRD9; Shown Are Averaged Values of Three
Replicates and the Standard Error of the Mean (SEM)
compd
indolizine substituent
pendant phenyl substituent
BAZ2A IC50 (μM)
BAZ2B IC50 (μM)
BRD4 IC50 (μM)
BRD9 IC50 (μM)
7
H
o-CH2OH
o-SO2Me
o-SO2Me
o-SO2Me
o-SO2Me
o-SO2Me
1.8 0.05
0.72 0.07
0.16 0.11
0.40 0.03
0.55 0.0
0.58 0.05
1.0 0.02
1.3 0.04
0.67 0.03
0.43 0.02
0.84 0.04
0.82 0.01
38.9 13.3
>50
23.7 0.16
17.9 3.85
1.4 0.04
>50
12
18
19
20
21
H
CH2NH-Boc
OPr
>50
>50
OPh
>50
19.9 1.84
>50
OMe
>50
Figure 3. Cocrystal structure of compounds 17 and 20 withBAZ2B. (a) Cocrystal structure with compound17. (b) Cocrystal structure with compound20.
Orientation and labeling are identical to those in Figure 2. (c) Superimposition of cocrystal structures with compounds 2, 17, and 20. Compounds were
well-defined by electron density. Ligand omitted electron density maps have been included in the Supporting Information.
Figure 4. Affinity and binding mode of the BAZ2A/B chemical probe GSK2801. (a) Dose response biolayer interference (BLI) data measuring the
binding kinetics of GSK2801 to BAZ2B. Steady state fitting resulted in a Kd value of 60 nM. (b) Isothermal titration calorimetry (ITC) binding
experiments for the interaction of GSK2801 with BAZ2A (blue in top panel and circles in lower panel) and BAZ2B (black in top panel and squares in
lower panel), respectively. Raw data heats are shown in the top panel. The lower panel shows normalized binding heats and the fitted function to a single
binding site model. (c) Cocrystal structure with BAZ2B. The structure is depicted in similar orientation to that in Figure 3.
Next we assessed the wider selectivity of GSK2801 against
other bromodomains in thermal stability assays. BAZ2A/B
bromodomains are exceptionally stable interaction domains, a
property that resulted in small temperature shifts in thermal
stability assays even in the presence of tightly binding ligands.
At an inhibitor concentration of 10 μM, GSK2801 resulted in a
temperature shift of 4.1 and 2.7 °C for BAZ2A and BAZ2B,
respectively. Significant Tm shifts were also observed for the
second bromodomain in TAF1L(2) (3.4 °C) and BRD9 (2.3 °C)
(Figure 5a).
Because of the limited sensitivity of BAZ2A/B in the thermal
stability assay we evaluated the selectivity of GSK2801 using
an alternative assay format. Forty-two bromodomains modified
with a Bir A biotin ligase targeting sequence were coexpressed
in bacteria with the enzyme. This coexpression strategy resulted
in homogeneously biotin-labeled bromodomains in vivo.33
The successful labeling with biotin was verified using mass
spectrometry (data not shown). We used the biotinylated pro-
teins in BLI experiments probing the interaction of GSK2801 at
two concentrations (0.2 and 1.0 μM) (Figure 5b). In agreement
E
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Figure 5. GSK2801 selectivity against the bromodomain family. (a) Temperature shift data (ΔTm) cross screening against a panel of 46 human
bromodomains. Screened targets are shown in bold. Temperature shifts are shown as indicated in the figure. (b) Selectivity screening using biolayer
interferometry and a panel of 40 biotin-labeled proteins. Screening was carried out at two inhibitor concentrations (1.0 and 0.2 μM) as indicated in the
figure. ITC KD data for the most significant hits are also shown.
Figure 6. Structure of the inactive control compound GSK8573 and family wide screening of biotin labeled bromodomains using BLI. BLI experiments
were performed at two concentrations (1.0 and 0.2 μM). The scale of the y-axis is identical to the one used in Figure 5b.
with the ΔTm data, BRD9 and TAF1(L) were detected as the
major off-targets, while no other significant interactions were
detected within the bromodomain family.
TAF1L has been reported to be mutated and expressed in a
subset of melanoma cases.35 BRD9 is expressed in most tissues
and is an off-target of GSK2801.
We quantified the off-target interactions using ITC. These
experiments showed that GSK2801 bound TAF1L(2) with
an affinity KB of 0.31 ( 0.02) × 106 M−1 (KD: 3.2 μM) and a
binding enthalpy change ΔH of −8.6 ( 0.02) kcal/mol. ITC
experiments using the bromodomain of BRD9 resulted in the
determination of an affinity KB of 0.826 ( 0.02) × 106 M−1 (KD:
1.1 μM) and ΔH of −9.8 ( 0.01) kcal/mol. TAF1L is a retro-
transposed gene only present in the primate lineage that has a
very restricted expression to the testis compartment.34 However,
Because of the off-target activity with BRD9, we developed a
structurally highly related control compound that lacked activity
on BAZ2A/B. Based on the low activity on BAZ2A and BAZ2B
with a meta-methoxy substituted pendant phenyl (compound 3),
we designed a compound that utilized this moiety in conjunc-
tion with the propyl ether in the indolizine 7-position. Using
the established BLI cross-screening assay format, this com-
pound (compound 23, GSK8573) proved to be inactive against
BAZ2A/B and all other bromodomains except BRD9 (Figure 6).
F
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Figure 7. Chemoproteomic profiling of BAZ inhibitor selectivity to endogenous bromodomain proteins. (a) pKds of GSK2801 and GSK8573 (negative
control) for BAZ2A, BAZ2B, and 16 additional BRD proteins captured on the GSK2801 affinity matrix from HuT-78 cell lysate. (b) Dose response
curves of GSK2801 for BAZ2A (top) and BAZ2B (bottom) in the same assay.
Figure 8. BAZ2A fluorescence recovery after photobleaching assay. (a) Images of nuclei of BAZ2A-GFP transfected cells. The bleached area is indicated
by a red circle. Shown is the time dependence of the fluorescent recovery of WT BAZ2A and the bromodomain inactivating mutant N1873F in the
presence and absence of GSK2801 and the BAZ2A/BAZ2B inactive control compound GSK8573. (b) Half times of fluorescence recovery (t1/2) for
BAZ2A are shown as bars representing the mean t1/2 calculated from individual recovery curves of at least 10 cells per group, and error bars depict the
standard error of the mean (SEM). (c) Raw data fluorescent recovery curves corresponding to the fluorescence recovery shown in panel a. (d) FRAP
measured on the control compound GSK8573. (e) Corresponding fluorescent recovery curves. *p < 0.05 compared to WT treated with 2.5 μM SAHA.
Using ITC we determined an affinity for interaction of GSK8573
with BRD9 of 1.04 μM (KD) associated with a favorable binding
enthalpy change ΔH of −8.98 ( 0.152) kcal/mol. Thus,
GSK8573 shared similar affinity for the off-target BRD9 while
losing all activity against the rest of the bromodomain family.
These results suggest that GSK8573 can be used as a structurally
related negative control compound in biological experiments.
To test whether GSK2801 would also bind to endogenous
BAZ2 proteins, we devised a chemoproteomic competition
binding assay. The Boc-protecting group of compound 18
was removed to provide an amine functionalized analogue of
GSK2801 that was immobilized on sepharose beads. The
resulting affinity matrix was incubated with nuclear and
chromatin enriched HuT78 extracts containing increasing con-
centrations of GSK2801 or the structurally related GSK8573.
Bound proteins were eluted and quantified using an isobaric mass
tagging strategy and targeted mass spectrometry.36,37 Among the
18 endogenous full-length bromodomain proteins that bound to
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the matrix, only BAZ2A and BAZ2B displayed a dose-dependent
reduction binding by GSK2801 suggesting a low micromolar
potency (pKd = 5.5) (Figure 7a,b). Conversely, GSK8573 did not
show any binding to BAZ2 proteins up to a concentration of
50 μM, but limited affinity to BRD9 (pKd = 4.5) and to
SMARCA2 and 4 (pKd = 4.8), which is likely to be indirect due
to the association of SMARCA2 and 4 to BRD9 in the BAF
complex38 (Figure7a).
In order to determine if GSK2801 could displace the
BAZ2A/B from chromatin in cells we utilized FRAP experi-
ments. Treatment of U2OS cells with the HDAC inhibitor
SAHA induced hyperacetylated chromatin and a better activity
window in the FRAP assay. SAHA-treated U2OS cells were
transfected with a GFP-BAZ2A fusion construct. In parallel,
U2OS cells were transfected with a GFP fusion with a BAZ2A
mutant construct (N1873F) in which the conserved asparagine
essential for recognizing the acetylated lysine had been mutated
(Figure 8). GSK2801 accelerated FRAP half-recovery time to the
same extent as observed for the mutant construct indicating that
the compound was able to displace BAZ2A from chromatin.
Conversely, the inactive GSK-8573 did not have any effect on the
half-recovery time of GFP-BAZ2A.
selectivity over BRD4. X-ray crystallography has provided a
convincing rationalization of the steric features resulting in the
BET selectivity. The resulting molecule, GSK2801, is a potent
and selective ligand for the BAZ2A and BAZ2B bromodomains,
suitable for use as a small molecule chemical probe. GSK2801
has excellent selectivity for BAZ2A and BAZ2B, with only
low micromolar residual activity at BRD9 and TAF1L. The
compound displaces full length GFP-BAZ2A from chromatin
at concentrations below 1 μM. We have also identified a
closely related inactive control compound to help inform
cellular screening results. In order to accelerate discoveries
on the roles of BAZ2B and BAZ2A in physiology and
pathophysiology, we are sharing these compounds with the
research community.
EXPERIMENTAL SECTION
■
Chemistry. All solvents and commercial chemicals were reagent
grade and used without purification. The purity of final compounds was
determined by HPLC/MS and 1H NMR and determined to be >95%.
1H NMR spectra were recorded on Bruker BioSpin spectrometer
operating at 400 mHz in CDCl3, methanol-d4, or DMSO-d6. The
chemical shifts (δ) reported are given in parts per million (ppm), and
the coupling constants (J) are in hertz (Hz). The spin multiplicities
are reported as s = singlet, br s = broad singlet, d = doublet, t = triplet,
q = quartet, m = multiplet, and br = broad.
The LC/MS analysis was performed on a Waters SQD with a
Phenomenex Kinetex 1.7 μm XB-C18 column at 40 °C, using water +
0.2% v/v formic acid and acetonitrile + 0.15% v/v formic acid as mobile
phase, and performed on Waters Acquity BEH C18 2 × 50 mm 1.7 m
column at 50 °C, using water + 0.20% v/v formic acid, water + 0.20% v/v
formic acid, and acetonitrile + 0.15% v/v formic acid. Microwave
irradiated reactions were carried out in sealed glass vessels in a Biotage
Initiator. Flash chromatography purifications were carried out on a
Biotage SP1 instrument.
In order to determine the suitability of GSK2801 for in vivo
experiments we measured pharmacokinetic parameters after
intraperitoneal and oral dosing to male CD1 mice. This pharma-
cokinetics study showed that GSK2801 has reasonable in vivo
exposure after oral dosing, modest clearance, and reasonable
plasma stability (Figure 9). These properties should allow
GSK2801 to be used as a BAZ2A/B bromodomain inhibitor
in vivo.
CONCLUSION
■
In this article, we started from an unselective micromolar
BAZ/BET inhibitor and optimized it through iterative medicinal
chemistry to a potent inhibitor of BAZ2A/B with >50-fold
Synthesis of Compound 19 (GSK2801): 1-(1-(3-(Methylsulfonyl)-
phenyl)-7-propoxyindolizin-3-yl)ethanone. Route 1:
Synthesis of Compound 19 (GSK2801) Intermediate A. A mixture
of 4-methoxypyridine (5 g, 45.8 mmol) and 1-chloropropan-2-one
(18.43 mL, 229 mmol) in ethyl acetate (50 mL) was stirred at 80 °C for
2 h. The mixture was concentrated to dryness to give 4-methoxy-1-(2-
oxopropyl)pyridin-1-ium chloride (9 g, 44.6 mmol, 97% yield) as a
yellow solid.
Synthesis of Compound 19 (GSK2801) Intermediate B. To a sus-
pension of 4-methoxy-1-(2-oxopropyl)pyridin-1-ium chloride (5.14 g,
H
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Figure 9. Pharmacokinetic properties of GSK2801 in mice. The figure shows the mean plasma concentration of GSK2801 in mice (n = 3) after oral (PO)
(circles) and intraperitoneal (IP) (squares) dosing (30 mg/kg).
25.5 mmol) in toluene (60 mL) was added methyl acrylate (22.93 mL,
255 mmol), TEA (5.33 mL, 38.2 mmol), and then manganese dioxide
(17.73 g, 204 mmol). The mixture was stirred at 90 °C for 1 h. The solid
was filtered off over Celite and washed with acetone. The combined
filtrate was concentrated and purified by silica gel chromatography
eluting with EtOAc in hexanes (5 to 50%) to give methyl 3-acetyl-7-
methoxyindolizine-1-carboxylate (4.4 g, 17.80 mmol, 69.8% yield) as a
yellow solid.
Synthesis of Compound 19 (GSK2801) Intermediate C. To a
solution of methyl 3-acetyl-7-methoxyindolizine-1-carboxylate (4.4 g,
17.80 mmol) in methanol (60 mL) was added NaOH (44.5 mL,
178 mmol). The mixture was stirred at 80 °C for 30 min. The solution
was concentrated and acidified with 6 N HCl to pH = 1. The solid
product was filtered, washed with water, and dried to give 3-acetyl-7-
methoxyindolizine-1-carboxylic acid (2.4 g, 10.29 mmol, 57.8% yield) as
a yellow solid. The product was used in the next step without further
purification.
Synthesis of Compound 19 (GSK2801) Intermediate D. To a slurry
of 3-acetyl-7-methoxyindolizine-1-carboxylic acid (2.4 g, 10.29 mmol)
in N,N-dimethylformamide (DMF) (100 mL) was added sodium
bicarbonate (2.59 g, 30.9 mmol) and then NBS (1.923 g, 10.81 mmol)
portionwise at 0 °C. The mixture was stirred at ambient temperature for
1.5 h. The solution was treated with water and extracted with EtOAc. The
combined organics were washed with water, brine, dried over Na2SO4, and
filtered. The filtrate was concentrated to dryness to give 1-(1-bromo-7-
methoxyindolizin-3-yl)ethanone (2.5 g, 9.32 mmol, 91% yield). The prod-
uct was used in the next step without further purification.
Synthesis of Compound 19 (GSK2801) Intermediate E. Nitrogen
gas was bubbled through a mixture of 1-(1-bromo-7-methoxyindolizin-
3-yl)ethanone (515 mg, 1.921 mmol), PdCl2(dppf)−CH2Cl2 adduct
(110 mg, 0.134 mmol), K2CO3 (796 mg, 5.76 mmol), and (2-
(methylsulfonyl)phenyl)boronic acid (768 mg, 3.84 mmol) in DMF
(4 mL) and water (0.5 mL) for 10 min. Then the mixture was heated in a
sealed tube in the microwave (120 °C, 25 min). The solution was
extracted with EtOAc, and the combined organics were washed with
water, brine, dried over Na2SO4, and filtered. The filtrate was con-
centrated, then purified using silica gel chromatography, eluting
with EtOAc in hexanes (20 to 60%) to give 1-(7-methoxy-1-(2-
(methylsulfonyl)phenyl)indolizin-3-yl)ethanone (368 mg, 1.072 mmol,
55.8% yield) as a light yellow solid.
Synthesis of Compound 19 (GSK2801) Intermediate F. To a
solution of 1-(7-methoxy-1-(2-(methylsulfonyl)phenyl)indolizin-3-yl)-
ethanone (368 mg, 1.072 mmol) in DMF (5 mL) was added iodo-
cyclohexane (0.693 mL, 5.36 mmol). The mixture was stirred at 155 °C
for 12 h. The mixture was treated with sodium thiosulfate (Na2S2O3)
and extracted with EtOAc. The combined organics were washed with
water, brine, dried over Na2SO4, and filtered. The filtrate was
concentrated to dryness to give 1-(7-hydroxy-1-(2-(methylsulfonyl)-
phenyl)indolizin-3-yl)ethanone (325 mg, 0.987 mmol, 92% yield). The
crude product was used in the next step without further purification.
Compound 19 (GSK2801): 1-(1-(3-(Methylsulfonyl)phenyl)-7-
propoxyindolizin-3-yl)ethanon. A mixture of 1-(7-hydroxy-1-(3-
(methylsulfonyl)phenyl)indolizin-3-yl)ethanone (38 mg, 0.115 mmol),
K2CO3 (19.13 mg, 0.138 mmol), and 1-bromopropane (0.210 mL,
2.307 mmol) in acetone (1.5 mL) was stirred at 75 °C for 2 h. The
product was purified by reverse phase HPLC (35 to 100% acetonitrile in
water for 6 min and 100% acetonitrile for 1 min, with 0.05% TFA as
additive) to give 1-(1-(3-(methylsulfonyl)phenyl)-7-propoxyindolizin-
3-yl)ethanone (30 mg, 0.081 mmol, 70.0% yield) as a colorless solid.
1H NMR (400 MHz, chloroform-d) δ ppm 1.04 (t, J = 7.28 Hz, 3 H),
1.70−1.92 (m, 2 H), 2.57 (s, 3 H), 2.68 (s, 3 H), 3.89 (t, J = 6.27 Hz,
2 H), 6.60 (s, 1 H), 6.68 (d, J = 7.53 Hz, 1 H), 7.51 (d, J = 7.28 Hz, 1 H),
7.56−7.64 (m, 1 H), 7.67−7.74 (m, 1 H), 7.81 (s, 1 H), 8.34 (d, J = 7.78
Hz, 1 H), 9.81 (d, J = 7.53 Hz, 1 H). LCMS: Rt = 0.90 min, MS m/z =
372 (M + H)+.
Synthesis of Compound 19 (GSK2801). Route 2:
Synthesis of Compound 19 (GSK2801) Intermediate G. 4-Chloropyr-
idine hydrochloride (10 g, 66.7 mmol) in dimethyl sulfoxide (DMSO)
(10 mL) was treated with NaOH (2.93 g, 73.3 mmol). The mixture was
stirred at ambient temperature for 30 min. To this mixture was added
propan-1-ol (35.1 mL, 467 mmol) and then NaOH (2.93 g, 73.3 mmol).
The mixture was stirred at 80 °C for 3 days. The mixture was allowed to
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cool to room temperature, treated with water, and extracted with
EtOAc. The organics were washed with water and brine and then
dried over Na2SO4. The filtrate was concentrated and dried to give
4-propoxypyridine (8.3 g, 60.5 mmol, 91% yield) as a yellow oil.
Synthesis of Compound 19 (GSK2801) Intermediate H. To a
solution of 4-propoxypyridine (8.2 g, 59.8 mmol) in ethyl acetate
(20 mL) was added 1-chloropropan-2-one (14.28 mL, 179 mmol). The
mixture was stirred at ambient temperature overnight. The precipitated
solid was filtered, washed with diethyl ether, and dried to give the
indicated product quantitatively as a colorless solid.
Synthesis of Compound 19 (GSK2801) Intermediate I. To a
suspension of 1-(2-oxopropyl)-4-propoxypyridin-1-ium chloride
(6.03 g, 26.4 mmol) and manganese dioxide (9.17 g, 105 mmol) in DMF
(30 mL) was added methyl acrylate (14.23 mL, 158 mmol) and TEA
(4.41 mL, 31.6 mmol). The mixture was stirred at 90 °C for 3 h. The
mixture was allowed to cool to room temperature, filtered through
Celite, washed with EtOAc, and filtered. The combined filtrate was
washed with water and brine, dried over Na2SO4, and filtered. The
filtrate was concentrated, then purified by silica gel chromatography
(0 to 60% EtOAc in heptane) to give methyl 3-acetyl-7-propoxyindo-
lizine-1-carboxylate (5 g, 18.16 mmol, 68.9% yield) as a yellow solid.
LCMS suggested that the product was 75% pure, and it was taken to the
next step without further purification.
Synthesis of Compound 19 (GSK2801) Intermediate J. To a
solution of methyl 3-acetyl-7-propoxyindolizine-1-carboxylate (3 g,
10.90 mmol) in methanol (15 mL), tetrahydrofuran (THF) (15.00 mL),
and water (15.00 mL) was added 4 N NaOH (8.17 mL, 32.7 mmol).
The mixture was stirred at ambient temperature for 3 days. The solution
was concentrated and then acidified with 1 N HCl to pH = 1. The
precipitated solid was filtered, washed with water, and dried to give
3-acetyl-7-propoxyindolizine-1-carboxylic acid (2.35 g, 8.99 mmol, 83%
yield) as a yellow solid.
Synthesis of Compound 19 (GSK2801) Intermediate K. To a
solution of 3-acetyl-7-propoxyindolizine-1-carboxylic acid (0.95 g,
3.64 mmol) in DMF (15 mL) was added sodium bicarbonate
(0.916 g, 10.91 mmol) and then NBS (0.712 g, 4.00 mmol) portion-
wise at 0 °C. The cooling bath was removed, and the mixture was
allowed to stir at ambient temperature for 1 h. The solution was treated
with water and extracted with EtOAc. The combined organics were
washed with water, then brine, and dried over Na2SO4 and filtered. The
filtrate was concentrated to dryness to give 1-(1-bromo-7-propox-
yindolizin-3-yl)ethanone (1.05 g, 3.55 mmol, 98% yield) as an off-white
solid. The product was used in the next step without further purification.
Compound 19 (GSK2801). Nitrogen gas was bubbled through
a mixture of 1-(1-bromo-7-propoxyindolizin-3-yl)ethanone (534 mg,
1.803 mmol), (2-(methylsulfonyl)phenyl)boronic acid (721 mg, 3.61
mmol), K2CO3 (748 mg, 5.41 mmol), and PdCl2(dppf)−CH2Cl2
adduct (118 mg, 0.144 mmol) in DMF (7 mL) and water (0.5 mL)
for 10 min. Then the mixture was irradiated in a sealed tube in the
microwave (120 °C, 15 min).
The mixture was treated with EtOAc and filtered to remove solids.
The filtrate was washed with water and brine, dried over Na2SO4, and
filtered. The filtrate was concentrated, then purified with silica gel
chromatography (0 to 50% EtOAc in heptane) to give 1-(1-(2-
(methylsulfonyl)phenyl)-7-propoxyindolizin-3-yl)ethanone (535 mg,
1.442 mmol, 80% yield) as an off-white solid, which was identical to the
GSK2801 prepared by Scheme 1 (above).
Synthesis of Inactive Control Compound 23 (GSK8573): 1-(1-
(3-Methoxyphenyl)-7-propoxyindolizin-3-yl)ethanone. Com-
pound 23 (GSK8573) was synthesized from intermediate K, which
was made as described in the synthesis of probe compound 19
(GSK2801). Nitrogen gas was bubbled through a mixture of 1-(1-
bromo-7-propoxyindolizin-3-yl)ethanone (544 mg, 1.837 mmol),
(3-methoxyphenyl)boronic acid (419 mg, 2.76 mmol), K2CO3 (762 mg,
5.51 mmol), and PdCl2(dppf)−CH2Cl2 adduct (120 mg, 0.147 mmol)
in 1,4-dioxane (8 mL) and water (0.08 mL) and heated in a sealed tube
in the microwave (100 °C, 6 min). The solid was filtered off and washed
with EtOAc. The combined filtrate was concentrated and then purified
with silica gel chromatography, eluting with 0 to 35% EtOAc in heptane
to give 1-(1-(3-methoxyphenyl)-7-propoxyindolizin-3-yl)ethanone
(364 mg, 1.126 mmol, 61.3% yield) as a yellow solid.
1H NMR (400 MHz, DMSO-d6) δ ppm 1.00 (t, J = 7.40 Hz, 3 H),
1.77 (q, J = 6.69 Hz, 2 H), 2.50 (s, 3H), 3.83 (s, 3 H), 4.07 (t, J = 6.40 Hz,
2 H), 6.84 (ddd, J = 18.95, 7.78, 2.38 Hz, 2 H), 7.17 (t, J = 2.64 Hz, 2 H),
7.23 (d, J = 7.78 Hz, 1 H), 7.33−7.43 (m, 1 H), 7.94 (s, 1 H), 9.69 (d,
J = 7.78 Hz, 1 H). LCMS Rt = 0.97.
1H NMR (400 MHz, methanol-d4), δ ppm 1.00−1.17 (m, 3 H),
1.78−1.94 (m, 2 H), 2.56 (s, 3 H), 3.88 (s, 3 H), 4.05 (s, 2 H), 6.68−6.80
(m, 1 H), 6.85−6.95 (m, 1 H), 7.08−7.23 (m, 3 H), 7.33−7.46 (m, 1 H),
7.77 (s, 1 H), 9.68−9.81 (m, 1 H); LCMS: Rt = 1.06 min, MS m/z = 324
(M + H)+.
Example synthesis using alkyne route (Scheme 1a in main text).
Compound 15: 1-(1-(2-(hydroxymethyl)phenyl)-7-morpholinoindoli-
zin-3-yl)ethanone.
4-Morpholino-1-(2-oxopropyl)pyridin-1-ium chloride: A mixture of
4-(pyridin-4-yl)morpholine (2.037 mL, 12.18 mmol) and 1-chloropro-
pan-2-one (4.90 mL, 60.9 mmol) was heated at 85 °C for 2 h. The
precipitated solid was filtered and washed with ethyl acetate and then
diethyl ether to give 4-morpholino-1-(2-oxopropyl)pyridin-1-ium
(s, 3 H), 3.74 (dd, J = 14.49, 4.50 Hz, 8 H), 5.35 (s, 2 H), 7.32 (d, J = 7.44
Hz, 2 H), 8.17 (d, J = 7.44 Hz, 2 H).
Compound 15: 1-(1-(2-(Hydroxymethyl)phenyl)-7-morpholinoin-
dolizin-3-yl)ethanoneA slurry of 4-morpholino-1-(2-oxopropyl)-
pyridin-1-ium chloride (291 mg, 1.135 mmol), (2-ethynylphenyl)-
methanol (50 mg, 0.378 mmol), and K2CO3 (261 mg, 1.892 mmol) in
1
chloride (3.1 g, 99%). H NMR (400 MHz, DMSO-d6) δ ppm 2.26
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DMF was irradiated in microwave at 120 °C for 30 min. The product
was purified with reverse phase HPLC (10−100% acetonitrile in water
for 6 min and 100% acetonitrile for 1 min, with 0.05% TFA as additive)
to give 1-(1-(2-(hydroxymethyl)phenyl)-7-morpholinoindolizin-3-yl)-
ethanone (18 mg, 13.56%). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.43
(s, 3 H), 3.20 (br. s., 4 H), 3.72 (br. s., 4 H), 4.44 (s, 2 H), 6.55 (s, 1 H),
7.04 (s, 1 H), 7.28−7.43 (m, 3 H), 7.61 (br. s., 1 H), 7.69 (s, 1 H),
9.63 (d, J = 7.78 Hz, 1 H). LCMS: Rt = 0.71 min, MS m/z = 351
(M + H)+.
Compound 2 was prepared in a manner similar to that described for
compound 15. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.50 (s, 3 H),
3.39 (t, J = 4.50 Hz, 4 H), 3.82 (t, J = 4.60 Hz, 4 H), 7.05−7.18 (m, 1 H),
7.23−7.34 (m, 1 H), 8.00 (d, J = 15.27 Hz, 3 H), 8.32 (s, 1 H), 8.64 (d,
J = 4.50 Hz, 1 H), 9.66 (d, J = 7.83 Hz, 1 H). LCMS: Rt = 0.62 min, MS
m/z = 322 (M + H)+.
Example synthesis of indolizine compound 3: 1-(1-(3-methoxyphenyl)-
indolizin-3-yl)ethanone.
Intermediate L: (1-(2-Oxopropyl)pyridin-1-ium chloride). A
mixture of pyridine (10 mL, 124 mmol) and 1-chloropropan-2-one
(4.3 mL, 53.4 mmol) was heated at 85 °C for 1 h. The solid was
filtered and washed with diethyl ether to give 1-(2-oxopropyl)-
pyridin-1-ium chloride (9 g, 52.4 mmol, 98% yield). The product
NBS (1.839 g, 10.33 mmol) portion-wise. The mixture was stirred
at rt for 20 min. The mixture was distributed between water and
EtOAc, and the aqueous layer further extracted with EtOAc. The
combined organics were washed with water and brine, dried over
Na2SO4, and filtered. The filtrate was concentrated. The product
was purified on the Biotage eluting with EtOAc in hexanes (0−30%)
to give 1-(1-bromoindolizin-3-yl)ethanone (2.1 g, 8.82 mmol,
90% yield) as a green solid. 1H NMR (400 MHz, DMSO-d6) δ ppm
2.47−2.54 (m, 3 H), 7.12 (td, J = 6.90, 1.25 Hz, 1 H), 7.39 (ddd,
J = 8.78, 6.78, 1.00 Hz, 1 H), 7.55−7.70 (m, 1 H), 7.97 (s, 1 H),
9.74 (d, J = 7.03 Hz, 1 H). LCMS: Rt = 0.83 min, MS m/z = 238
(M + H)+.
Compound 3: (1-(1-(3-Methoxyphenyl)indolizin-3-yl)ethanone). A
mixture of 1-(1-bromoindolizin-3-yl)ethanone (65 mg, 0.273 mmol),
(3-methoxyphenyl)boronic acid (49.8 mg, 0.328 mmol), K2CO3
(113 mg, 0.819 mmol), and PdCl2(dppf)−CH2Cl2 adduct (15.61 mg,
0.019 mmol) in DMF (3 mL) and 4 drops of water were irradiated in the
microwave at 120 °C for 10 min. The product was purified with reverse
phase HPLC (50−100% acetonitrile in water for 6 min and 100%
acetonitrile for 1 min, with 0.05% TFA as additive) to give 1-(1-(3-
methoxyphenyl)indolizin-3-yl)ethanone (32 mg, 0.121 mmol, 44%
yield). 1H NMR (400 MHz, methanol-d4) δ ppm 2.61 (s, 3 H), 3.88 (s,
3 H), 6.91 (dd, J = 7.91, 2.13 Hz, 1 H), 7.02 (td, J = 6.90, 1.00 Hz, 1 H),
7.13−7.17 (m, 1 H), 7.20 (d, J = 7.78 Hz, 1 H), 7.31 (ddd, J = 8.91, 6.78,
0.88 Hz, 1 H), 7.35−7.43 (m, 1 H), 7.83 (s, 1 H), 7.91 (d, J = 8.78 Hz,
1 H), 9.86 (d, J = 7.28 Hz, 1 H). LCMS: Rt = 1.18 min, MS m/z =
266 (M + H)+.
1
was used in the next step without further purification. H NMR
(400 MHz, DMSO-d6) δ ppm 2.35 (s, 3 H), 5.93 (s, 2 H), 8.25 (t,
J = 6.95 Hz, 2 H), 8.71 (t, J = 7.73 Hz, 1 H), 8.99 (d, J = 5.87 Hz,
2 H).
Intermediate M: (Methyl 3-acetylindolizine-1-carboxylate). A
suspension of 1-(2-oxopropyl)pyridin-1-ium chloride (5 g, 29.1
mmol), methyl acrylate (21.81 mL, 291 mmol), manganese dioxide
(20.26 g, 233 mmol), and TEA (6.09 mL, 43.7 mmol) in toluene
(50 mL) was refluxed for 2 h. The suspension was filtered over
Celite and washed with acetone, and the filtrate was concentrated
to dryness to give methyl 3-acetylindolizine-1-carboxylate (5.6 g,
25.8 mmol, 88% yield). The product was used in the next step
without further purification. LCMS: Rt = 0.71 min, MS m/z = 218
(M + H)+.
Intermediate N: (3-Acetylindolizine-1-carboxylic acid). A solution
of methyl 3-acetylindolizine-1-carboxylate (7 g, 32.2 mmol) and NaOH
(4M, 81 mL, 322 mmol) in methanol (100 mL) was stirred at 90 °C for
3 h and then at rt overnight. The solution was concentrated and
distributed between DCM and water. The aqueous phase was acidified
with 6 N HCl, and the precipitate was filtered, washed with water, and
dried to give 3-acetylindolizine-1-carboxylic acid (4.3 g, 21.16 mmol,
65.7% yield). The product was used in the next step without further
purification. 1H NMR (400 MHz, Chloroform-d) δ ppm 2.59 (s, 3 H),
6.97 (t, J = 6.85 Hz, 1 H), 7.22−7.31 (m, 2 H), 7.50−7.70 (m, 2 H),
9.90 (d, J = 7.24 Hz, 1 H). LCMS: Rt = 0.55 min, MS m/z = 204
(M + H)+.
Compound 4, 1-(1-(Thiophen-2-yl)indolizin-3-yl)ethanone, was
prepared in a manner similar to that described for compound 3, using
intermediate O. The Suzuki reaction was performed using thiophen-2-
yl-boronic acid, PdCl2(dppf)−CH2Cl2 adduct as catalyst and heating in
the microwave at 120 °C for 10 min (yield = 34%). 1H NMR (400 MHz,
methanol-d4) δ ppm 2.58 (s, 3 H), 7.01 (t, J = 6.85 Hz, 1 H), 7.13
(dd, J = 4.89, 3.72 Hz, 1 H), 7.26−7.41 (m, 3 H), 7.83 (s, 1 H), 7.99
Intermediate O: (1-(1-Bromoindolizin-3-yl)ethanone). To a sol-
ution of 3-acetylindolizine-1-carboxylic acid (2 g, 9.84 mmol) in DMF
(15 mL) was added sodium bicarbonate (2.481 g, 29.5 mmol) and then
K
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(d, J = 8.81 Hz, 1 H), 9.82 (d, J = 7.05 Hz, 1 H). LCMS: Rt = 0.87 min,
MS m/z = 242 (M + H)+.
(m, 1 H), 7.49 (br. s., 1 H), 7.56−7.63 (m, 1 H), 7.85 (t, J = 8.81 Hz, 2
H), 8.05 (d, J = 9.00 Hz, 1 H), 8.13 (br. s, 2 H), 8.17 (s, 1 H), 9.85 (d, J =
7.24 Hz, 1 H). LCMS: Rt = 0.68 min, MS m/z = 279 (M + H)+.
Compound 5, 1-(1-(2-hydroxyphenyl)indolizin-3-yl)ethanone, was
prepared in a manner similar to that described for compound 3 using
intermediate O. The Suzuki reaction was performed using (2-
hydroxyphenyl)boronic acid, PdCl2(dppf)−CH2Cl2 adduct as catalyst,
and heating in the microwave at 120 °C for 10 min (yield = 43%). 1H
NMR (400 MHz, DMSO-d6) δ ppm 2.56 (s, 3 H), 6.93 (t, J = 7.34 Hz,
1 H), 7.01 (d, J = 7.83 Hz, 1 H), 7.08 (t, J = 6.56 Hz, 1 H), 7.18−7.24 (m,
1 H), 7.25−7.32 (m, 1 H), 7.38 (d, J = 6.46 Hz, 1 H), 7.64 (d, J = 8.81
Hz, 1 H), 7.85 (s, 1 H), 9.54 (s, 1 H), 9.81 (d, J = 7.05 Hz, 1 H). LCMS:
Rt = 0.85 min, MS m/z = 252 (M + H)+.
Compound 9, 1-(1-(2-(hydroxymethyl)-4-methoxyphenyl)-
indolizin-3-yl)ethanone, was prepared in a manner similar to that de-
scribed for compound 3 using intermediate O. The Suzuki reaction was
performed using (2-(hydroxymethyl)-4-methoxyphenyl)boronic acid,
PdCl2(dppf)−CH2Cl2 adduct as catalyst, and heating in the microwave
at 120 °C for 10 min (yield = 11%). 1H NMR (400 MHz, chloroform-d)
δ ppm 2.56−2.67 (m, 3 H), 3.94 (s, 3 H), 4.65 (s, 2 H), 6.92−7.01 (m,
2 H), 7.14−7.21 (m, 1 H), 7.23 (d, J = 2.15 Hz, 1 H), 7.34 (d, J = 8.42
Hz, 1 H), 7.44 (d, J = 9.00 Hz, 1 H), 7.59 (s, 1 H), 9.95 (d, J = 7.05 Hz, 1
H). LCMS: Rt = 0.79 min, MS m/z = 296 (M + H)+.
Compound 6, 1-(1-(3-hydroxyphenyl)indolizin-3-yl)ethanone, was
prepared in a manner similar to that described for compound 3 using
intermediate O. The Suzuki reaction was performed using (3-
hydroxyphenyl)boronic acid, PdCl2(dppf)−CH2Cl2 adduct as catalyst,
and heating in the microwave at 120 °C for 20 min (yield = 37%). 1H
NMR (400 MHz, CDCl3) δ 9.96 (d, 1H, J = 6.7 Hz), 7.91 (d, 1H, J = 8.6
Hz), 7.66 (s, 1H), 7.38−7.13 (m, 3H), 6.98−6.85 (m, 2H), 5.25 (s, 1H),
2.66 (s, 3H). LRMS (ESI-TOF) [M + H]+ m/z calcd for C16H13NO2H+,
252.1; found, 252.1.
Compound 10, 1-(1-(4-(hydroxymethyl)phenyl)indolizin-3-yl)-
ethanone, was prepared in a manner similar to that described for
compound 3 using intermediate O. The Suzuki reaction was performed
using (4-(hydroxymethyl)phenyl)boronic acid, PdCl2(dppf)−CH2Cl2
adduct as catalyst, and heating in the microwave at 120 °C for 10 min
(yield = 11%). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.60 (s, 3 H),
4.58 (d, J = 4.11 Hz, 2 H), 5.24 (br. s., 1 H), 7.12 (t, J = 6.66 Hz, 1 H),
7.32−7.41 (m, 1 H), 7.46 (d, J = 7.83 Hz, 2 H), 7.67 (d, J = 8.03 Hz, 2
H), 7.99 (d, J = 9.00 Hz, 1 H), 8.03 (s, 1 H), 9.84 (d, J = 7.05 Hz, 1 H).
LCMS: Rt = 0.78 min, MS m/z = 266 (M + H)+.
Compound 7, 1-(1-(2-(hydroxymethyl)phenyl)indolizin-3-yl)-
ethanone, was prepared using the alkyne route in a manner similar to
that described for compound 15. 1H NMR (500 MHz, CDCl3) δ 9.96
(d,1H, J = 7 Hz), 7.68−7.66 (m, 2H), 7.50−7.45 (m, 4H), 7.31 (s, 1H),
7.19 (t, 1H, J = 7.6 Hz), 6.97 (t, 1H, J = 7.6 Hz), 4.69 (s, 2H), 2.64 (s,
3H). LRMS (ESI-TOF) [M + H]+ m/z calcd for C17H15NO2H+, 266.1;
found, 266.1.
Compound 11, 1-(1-(4-(dimethylamino)phenyl)indolizin-3-yl)-
ethanone, was prepared in a manner similar to that described for
compound 3 using intermediate O. 1H NMR (400 MHz, CDCl3) δ 9.92
(d, 1H, J = 7 Hz), 7.83 (d, 1H, J = 9 Hz), 7.58 (s, 1H), 7.48 (d, 2H, J = 7.5
Hz), 7.17−7.16 (m, 1H), 6.90−6.86 (m, 3H), 3.03 (s, 6H), 2.62 (s, 3H).
LRMS (ESI-TOF) [M + H]+ m/z calcd for C18H18N2OH+, 279.2;
found, 279.3.
Compound 8, 3-(3-acetylindolizin-1-yl)benzamide, was prepared in
a manner similar to that described for compound 3 using intermediate O.
The Suzuki reaction was performed using (3-carbamoylphenyl)-
boronic acid, PdCl2(dppf)−CH2Cl2 adduct as catalyst, and heating in
the microwave at 120 °C for 10 min (yield = 56%). 1H NMR (400 MHz,
DMSO-d6) δ ppm 2.62 (s, 3 H), 7.15 (t, J = 6.75 Hz, 1 H), 7.36−7.44
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Compound 12, 1-(1-(2-(methylsulfonyl)phenyl)indolizin-3-yl)-
ethanone, was prepared in a manner similar to that described for
compound 3 using intermediate O. 1H NMR (400 MHz, DMSO-d6) δ
ppm 2.55 (s, 3 H), 2.85 (s, 3H), 7.14 (t, J = 6.56 Hz, 1 H), 7.27−7.35 (m,
1 H), 7.37−7.43 (m, 1 H), 7.58 (d, J = 7.05 Hz, 1 H), 7.71−7.78 (m, 1
H), 7.80−7.87 (m, 1 H), 7.96 (s, 1 H), 8.20 (d, J = 7.83 Hz, 1 H), 9.81 (d,
J = 7.05 Hz, 1 H). LCMS: Rt = 0.79 min, MS m/z = 314 (M + H)+.
Compound 18, tert-butyl ((3-acetyl-1-(2-(methylsulfonyl)phenyl)-
indolizin-7-yl)methyl)carbamate, was prepared in a manner similar to
that described for compound 3. 1H NMR (400 MHz, methanol-d4) δ
ppm 1.40 (s, 9 H), 2.58 (s, 3 H), 2.70 (s, 3 H), 4.25 (s, 2 H), 6.99 (d,
J = 7.28 Hz, 1 H), 7.24 (s, 1 H), 7.57 (d, J = 7.28 Hz, 1 H), 7.66−7.73
(m, 1 H), 7.75−7.83 (m, 1 H), 7.92 (s, 1 H), 8.27 (d, J = 8.03 Hz, 1 H),
9.80 (d, J = 7.28 Hz, 1 H). LCMS: Rt = 0.82 min, MS m/z = 443
(M + H)+.
Compound 13, 1-(1-(2-(hydroxymethyl)phenyl)-7-methylindolizin-
3-yl)ethanone, was prepared in a manner similar to that described for
compound 3. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.34 (s, 3 H), 2.50
(s, 3 H), 4.44 (s, 2 H), 5.10 (br. s., 1 H), 6.94 (dd, J = 7.15, 1.13 Hz, 1 H),
7.26 (s, 1 H), 7.31−7.44 (m, 3 H), 7.63 (d, J = 7.53 Hz, 1 H), 7.80 (s,
1 H), 9.69 (d, J = 7.28 Hz, 1 H). LCMS: Rt = 0.79 min, MS m/z = 280
(M + H)+.
Compound 20, 1-(1-(2-(methylsulfonyl)phenyl)-7-phenoxyindoli-
zin-3-yl)ethanone, was prepared in a manner similar to that described
1
for compound 3. H NMR (400 MHz, chloroform-d) δ ppm 2.62 (s,
3 H), 2.70 (s, 3 H), 6.78 (dd, J = 7.73, 2.45 Hz, 1 H), 6.85 (d, J = 2.35 Hz,
1 H), 7.11 (d, J = 7.83 Hz, 2 H), 7.20−7.27 (m, 1 H), 7.39−7.51 (m,
3 H), 7.55−7.62 (m, 1 H), 7.64−7.72 (m, 1 H), 7.92 (s, 1 H), 8.32 (d,
J = 7.63 Hz, 1 H), 9.95 (d, J = 7.63 Hz, 1 H). LCMS: Rt = 0.92 min, MS
m/z = 406 (M + H)+.
Compound 14, 1-(1-(2-(hydroxymethyl)phenyl)-7-methoxyindoli-
zin-3-yl)ethanone, was prepared in a manner similar to that described
for compound 3. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.47 (s, 3 H),
3.79 (s, 3 H), 4.45 (s, 2 H), 6.76 (s, 1 H), 6.80 (d, J = 7.53 Hz, 1 H),
7.30−7.44 (m, 3 H), 7.63 (d, J = 7.28 Hz, 1 H), 7.78 (s, 1 H), 9.68 (d,
J = 7.53 Hz, 1 H). LCMS: Rt = 0.80 min, MS m/z = 296 (M + H)+.
Compound 21, 1-(7-methoxy-1-(2-(methylsulfonyl)phenyl)-
indolizin-3-yl)ethanone, was prepared in a manner similar to that
described for compound 3. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.46
(s, 3 H), 2.82 (s, 3 H), 3.76 (s, 3 H), 6.65 (d, J = 2.51 Hz, 1 H), 6.83 (dd,
J = 7.65, 2.38 Hz, 1 H), 7.57 (d, J = 7.53 Hz, 1 H), 7.66−7.74 (m, 1 H),
7.76−7.85 (m, 2 H), 8.17 (d, J = 7.78 Hz, 1 H), 9.67 (d, J = 7.78 Hz,
1 H). LCMS: Rt = 0.80 min, MS m/z = 344 (M + H)+.
Compound 16, 3-acetyl-1-(2-(hydroxymethyl)phenyl)indolizine-7-
carboxamide, was prepared in a manner similar to that described for
compound 3. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.60 (s, 3 H), 4.48
(s, 2 H), 7.38−7.59 (m, 5 H), 7.69 (d, J = 7.44 Hz, 1 H), 7.97 (s, 1 H),
8.03 (s, 1 H), 8.23 (br. s., 1 H), 9.77 (d, J = 7.24 Hz, 1 H). LCMS: Rt =
0.64 min, MS m/z = 309 (M + H)+.
Compound 22, 1-(7-methyl-1-(2-(methylsulfonyl)phenyl)indolizin-
3-yl)ethanone, was prepared in a manner similar to that described for
compound 3. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.32 (s, 3 H), 2.49
(s, 3 H), 2.81 (s, 3 H), 6.97 (dd, J = 7.40, 1.38 Hz, 1 H), 7.16 (s, 1 H),
7.54 (d, J = 7.53 Hz, 1 H), 7.67−7.74 (m, 1 H), 7.77−7.83 (m, 1 H), 7.87
(s, 1 H), 8.16 (d, J = 8.03 Hz, 1 H), 9.69 (d, J = 7.28 Hz, 1 H). LCMS:
Rt = 0.82 min, MS m/z =328 (M + H)+.
Compound 17, 1-(1-(2-(hydroxymethyl)phenyl)-7-phenoxyindoli-
zin-3-yl)ethanone, was prepared in a manner similar to that described
for compound 3. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.50 (s, 3 H),
4.41 (d, J = 5.27 Hz, 2 H), 5.11 (s, 1 H), 6.70 (s, 1 H), 6.95 (d, J = 7.78
Hz, 1 H), 7.13−7.37 (m, 6 H), 7.44 (t, J = 7.78 Hz, 2 H), 7.57 (d, J = 7.53
Hz, 1 H), 7.85 (s, 1 H), 9.80 (d, J = 7.53 Hz, 1 H). LCMS: Rt = 0.92 min,
MS m/z = 358 (M + H)+.
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(5) Muller, S.; Filippakopoulos, P.; Knapp, S. Bromodomains as
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The cocrystal structures reported in this study have been deposited to
the protein data bank under accession codes 4IR3, 4IR4, 4IR5, 4IR6 and
4RVR.
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional method descriptions, a crystallographic data
collection and refinement table, and supplemental figures
showing the electron density around the cocrystallized ligands.
This material is available free of charge via the Internet at http://
pubs.acs.org.
(12) Ciceri, P.; Muller, S.; O’Mahony, A.; Fedorov, O.;
Filippakopoulos, P.; Hunt, J. P.; Lasater, E. A.; Pallares, G.; Picaud, S.;
Wells, C.; Martin, S.; Wodicka, L. M.; Shah, N. P.; Treiber, D. K.; Knapp,
S. Dual kinase-bromodomain inhibitors for rationally designed
polypharmacology. Nat. Chem. Biol. 2014, 10, 305−312.
(13) Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W. B.;
Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.;
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.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: Knapp@pharmchem.uni-frankfurt.de. Phone: +49 69
79 82 98 71.
*E-mail: davidhdrewry@gmail.com. Phone: +1 919 602 1327.
Author Contributions
○These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
(14) Muller, S.; Knapp, S. Discovery of BET bromodomain inhibitors
and their role in target validation. MedChemComm 2014, 5, 288−296.
(15) Picaud, S.; Da Costa, D.; Thanasopoulou, A.; Filippakopoulos, P.;
Fish, P. V.; Philpott, M.; Fedorov, O.; Brennan, P.; Bunnage, M. E.;
Owen, D. R.; Bradner, J. E.; Taniere, P.; O’Sullivan, B.; Muller, S.;
Schwaller, J.; Stankovic, T.; Knapp, S. PFI-1, a highly selective protein
interaction inhibitor, targeting BET bromodomains. Cancer Res. 2013,
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(16) Mirguet, O.; Gosmini, R.; Toum, J.; Clement, C. A.; Barnathan,
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ACKNOWLEDGMENTS
■
The SGC is a registered charity (number 1097737) that receives
funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim,
the Canada Foundation for Innovation, Genome Canada,
GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research
Foundation, the Ontario Ministry of Economic Development
and Innovation, Pfizer, Takeda, and the Wellcome Trust
[092809/Z/10/Z].
ABBREVIATIONS USED
■
BAZ2A, bromodomain adjacent to zinc finger domain protein
2A; BAZ2B, bromodomain adjacent to zinc finger domain
protein 2B; B-ALL, B cell acute lymphoblastic leukemia; DMF,
dimethylformamide; PHD, plant homology domain; BET,
bromodomain and extraterminal; ISWI, imitation switch
chromatin remodeling complexes; NoRC, nucleolar remodeling
complex; FRAP, fluorescent recovery after photobleaching;
SEM, standard error of the mean
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