Discovery of I-BRD9, a Selective Cell Active Chemical Probe for Bromodomain Containing Protein 9 Inhibition

Article abstract of DOI:10.1021/acs.jmedchem.5b00256

Acetylation of histone lysine residues is one of the most well-studied post-translational modifications of chromatin, selectively recognized by bromodomain "reader" modules. Inhibitors of the bromodomain and extra terminal domain (BET) family of bromodomains have shown profound anticancer and anti-inflammatory properties, generating much interest in targeting other bromodomain-containing proteins for disease treatment. Herein, we report the discovery of I-BRD9, the first selective cellular chemical probe for bromodomain-containing protein 9 (BRD9). I-BRD9 was identified through structure-based design, leading to greater than 700-fold selectivity over the BET family and 200-fold over the highly homologous bromodomain-containing protein 7 (BRD7). I-BRD9 was used to identify genes regulated by BRD9 in Kasumi-1 cells involved in oncology and immune response pathways and to the best of our knowledge, represents the first selective tool compound available to elucidate the cellular phenotype of BRD9 bromodomain inhibition.

Full text of DOI:10.1021/acs.jmedchem.5b00256

This is an open access article published under an ACS AuthorChoice License, which permits
copying and redistribution of the article or any adaptations for non-commercial purposes.
Article
pubs.acs.org/jmc
Discovery of I‑BRD9, a Selective Cell Active Chemical Probe for
Bromodomain Containing Protein 9 Inhibition
Natalie H. Theodoulou,†,‡ Paul Bamborough,§ Andrew J. Bannister,∥ Isabelle Becher,⊥ Rino A. Bit,†
Ka Hing Che,∥ Chun-wa Chung,§ Antje Dittmann,⊥ Gerard Drewes,⊥ David H. Drewry,#
Laurie Gordon,∇ Paola Grandi,⊥ Melanie Leveridge,∇ Matthew Lindon,† Anne-Marie Michon,⊥
Judit Molnar,† Samuel C. Robson,∥ Nicholas C. O. Tomkinson,‡ Tony Kouzarides,∥ Rab K. Prinjha,†
and Philip G. Humphreys*,†
†Epinova Discovery Performance Unit, GlaxoSmithKline R&D, Stevenage, Hertfordshire SG1 2NY, U.K.
‡WestCHEM, Department of Pure and Applied Chemistry, Thomas Graham Building, University of Strathclyde, 295 Cathedral
Street, Glasgow G1 1XL, U.K.
§Computational & Structural Chemistry, Molecular Discovery Research, GlaxoSmithKline R&D, Stevenage, Hertfordshire SG1 2NY,
U.K.
∥Department of Pathology, Gurdon Institute, Tennis Court Road, Cambridge CB2 1QN, U.K.
⊥Cellzome GmbH, Molecular Discovery Research, GlaxoSmithKline R&D, Meyerhofstrasse 1, 69117 Heidelberg, Germany
#Department of Chemical Biology, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, United States
∇Biological Sciences, Molecular Discovery Research, GlaxoSmithKline R&D, Stevenage, Hertfordshire SG1 2NY, U.K.
S
* Supporting Information
ABSTRACT: Acetylation of histone lysine residues is one of
the most well-studied post-translational modifications of
chromatin, selectively recognized by bromodomain “reader”
modules. Inhibitors of the bromodomain and extra terminal
domain (BET) family of bromodomains have shown profound
anticancer and anti-inflammatory properties, generating much
interest in targeting other bromodomain-containing proteins
for disease treatment. Herein, we report the discovery of I-
BRD9, the first selective cellular chemical probe for
bromodomain-containing protein 9 (BRD9). I-BRD9 was
identified through structure-based design, leading to greater
than 700-fold selectivity over the BET family and 200-fold over
the highly homologous bromodomain-containing protein 7 (BRD7). I-BRD9 was used to identify genes regulated by BRD9 in
Kasumi-1 cells involved in oncology and immune response pathways and to the best of our knowledge, represents the first
selective tool compound available to elucidate the cellular phenotype of BRD9 bromodomain inhibition.
transcription.4 Bromodomains are highly conserved protein
modules, comprising approximately 110 amino acids. The
majority are characterized by four alpha-helices, linked by two
INTRODUCTION
■
Chromatin has a highly complex structure composed of
nucleosomes, the fundamental packaging elements of genetic
flexible loop regions, which form the basis of the Kac binding
information. These structural subunits consist of DNA wrapped
pocket.5
around an octameric core of histones, which facilitate the
There are at least 56 human bromodomains in 42
arrangement of DNA in the nucleus of the cell.1 Histone tails are
bromodomain containing proteins (BCPs) identified to date,
subject to a range of post-translational modifications, aiding the
neglecting splice variants, each of which has been classified into a
regulation of chromatin accessibility.2 Acetylation of lysine
distinct subgroup based on sequence homology.6−8 The
residues is an example of one such modification and is regulated
bromodomain and extra terminal domain (BET) family of
by the dynamic action of histone acetyltransferases (HATs) and
BCPs (BRD2, BRD3, BRD4, and BRDT) contain two
histone deacetylases (HDACs), which “write” and “erase”
acetylation marks respectively.3 Bromodomain “reader” modules
recognize specific acetyl-lysine (Kac) residues located on histone
Special Issue: Epigenetics
tails and other proteins. These recognition motifs act together
with other chromatin factors to modulate regulation of gene
Received: February 13, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Chemical structures for selected bromodomain inhibitors.
bromodomains each, both capable of binding to Kac.3 The most
potent and selective bromodomain inhibitors reported to date
target all eight bromodomains of the BET family.4,9,10 Examples
include I-BET762 (1),11−13 (+)-JQ1 (2),14,15 I-BET151
(3),16−18 I-BET726 (4),19 PFI-1 (5),20 and OXF BD 02
(6)21,22 (Figure 1). There are also examples of compounds
biased toward binding at either the N-terminal bromodomain
(BD1) or C-terminal bromodomain (BD2) of the BET family
such as MS436 (7)23 and RVX-208 (8).24,25
The profound biology associated with the BET family of BCPs
suggests there may be therapeutic potential in targeting other
members of the bromodomain phylogenetic tree for the
treatment of disease. Protein knock-down studies, which block
the action of an entire complex, offer a method for probing
protein function and linking a BCP to a disease.3 However, as
BCPs are multidomain proteins and often exist as part of
multibody complexes, a chemical probe, which only inhibits the
action of the bromodomain, may show different effects.26 The
therapeutic potential of bromodomain inhibition remains less
B
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
well explored outside of the BET family,3 in part, due to the
limited availability of suitable chemical probes for each member
of the human bromodomain phylogenetic tree.27 Therefore, the
development of high quality, selective small molecule probes for
bromodomains will be a key step in facilitating validation of these
reader modules as therapeutic targets. Toward this goal, several
groups have generated high quality non-BET bromodomain
inhibitors from a diverse set of chemotypes: PFI-3 (9)28 for
SMARCA2/4 and PB1(5) domains; I-CBP112 (10),29 SGC-
CBP300 (11),30,31 and ischemin (12)32 for CREBBP and
EP300;33 GSK2801 (13)34 for BAZ2B/A, and compound 14 for
BRPF1 (Figure 1).35 NMR-based screening has been used to
identify fragment-sized small molecule inhibitors of PCAF,
BAZ2B, and ATAD2 providing further evidence for the
tractability of non-BET bromodomains.36−38 Additionally, a
fluorous-tagged multicomponent reaction biased around the 3,5-
dimethylisoxazole Kac motif was used to generate the first
compounds capable of binding to TAF1.39 There are also several
probes available from the Structural Genomics Consortium for
the following bromodomains that have not yet had their
structures disclosed: BAZ2B/A, BRPF1/2/3, and BRPF1.40
Although BRD9 has been reported as a component of the
chromatin remodeling SWI/SNF BAF complex,41−43 further
information regarding its role within disease is somewhat
limited.44 As such, a chemical probe for the BRD9 bromodomain
would be invaluable in better understanding the biological role
and therapeutic potential of this reader module. The Structural
BRD9 and BRD4 BD1 in complex with I-BET762 (1) revealed a
distinct contrast in the architecture of their ZA channels, due to
differences in their constituent amino acids. In BRD9, the
Tyr106 gate keeper residue blocks access to the lipophilic “WPF”
shelf, providing a key difference to the Ile146 gate keeper residue
present at the equivalent position of BRD4 BD1 (Figure 2, Table
Genomics Consortium have disclosed bromosporine (15),45
a
Figure 2. X-ray crystal structures of (A) BRD4 BD1 bromodomain
(orange) with I-BET762 (1) (cyan); (B) BRD9 bromodomain (blue);
(C) BRD4 BD1 surface, and (D) BRD9 surface. In each, the conserved
asparagine residue is colored green (BRD4 Asn140/BRD9 Asn100),
and the gatekeeper residue is colored blue (BRD4 Ile146/BRD9
Tyr106). BRD4 Leu94 and BRD9 Ala54 are colored red.
broad spectrum bromodomain inhibitor, which shows a thermal
shift of +3.9 °C at BRD9, suggesting bromodomain binding. In
addition, structurally similar compound 16 was reported as the
first nonselective inhibitor of BRD9.46,47 This compound shows
mixed bromodomain pharmacology, with submicromolar
activity against BRD9, BRD4, CECR2, and CREBBP. To the
best of our knowledge, there are no BRD9 selective chemical
probes described in the literature to date.48 Herein, we report the
discovery of I-BRD9, the first selective cellular chemical probe for
BRD9. The development of I-BRD9 was driven by iterative
medicinal chemistry, utilizing structure based design to deliver
nanomolar potency at BRD9, >700-fold selectivity over the BET
family and >70-fold against a panel of 34 bromodomains. I-BRD9
meets and exceeds the chemical probe criteria we defined at the
outset of this project, inspired by literature from Bunnage and co-
workers,49 who highlighted the importance of high quality
chemical probes for target validation: 100 nM or greater potency
against the bromodomain of BRD9 as determined by a
biochemical assay; 100-fold selectivity over the BET family of
bromodomains; 30-fold selectivity over other bromodomain
families; cellular activity; and broader selectivity over a range of
receptor sites, ion channels, transporters, and enzymes.
1). At this stage, it was unclear whether or not selectivity could be
gained over the highly homologous BRD7, given its proximity to
BRD9 on the phylogenetic tree and sequence similarity within
the Kac binding pocket (Figure 3, Table 1).50,51
Nonetheless, in order to identify inhibitors of the BRD9
bromodomain, a cross-screening strategy of GSK internal
compounds was conducted. Thienopyridone 17 was identified
from this effort as a potent binder to BRD9 (TR-FRET assay),
with pIC50 values of 6.7 and 4.7 against BRD9 and BRD4 BD1,
respectively (Figure 4).52
To explain the BRD9 potency of 17 and its selectivity over
BRD4, 1.3 and 1.2 Å resolution X-ray crystal structures were
solved in BRD9 and BRD4 BD1, respectively. The interactions of
the thienopyridone ring are similar in both bromodomains
(Figure 4B−D). In BRD9, the carbonyl moiety of the pyridone
ring forms a direct hydrogen-bond to the side chain NH2 group
of Asn100, the conserved asparagine residue important for Kac
recognition. The pyridone carbonyl also makes a shorter
hydrogen-bond to a conserved water molecule bound by
Tyr57. The pyridone N-methyl group sits in a small cavity
surrounded by four crystallographically conserved water
molecules, Phe45 and Val49. The N-methyl pyridone moiety
acts as a direct structural analogue of Kac (Figure 4D), whose
interactions with BRD9 and BRD4 are typical of those reported
for fragments in the corresponding site of BRD4.50,51 The
thienopyridone rings fill a tight hydrophobic groove formed by
Val49 and Ala54 of the ZA loop on one side, and Tyr106 (the
gatekeeper residue) on the other. The 7-dimethoxyphenyl
substituent extends outward through the ZA channel, in close
RESULTS AND DISCUSSION
■
Throughout the course of the investigations to identify a
chemical probe for BRD9, selectivity over the BET family of
bromodomains was considered of utmost importance. It was felt
that the profound biology associated with BET bromodomain
inhibition9−12 required at least 100-fold selectivity (as
determined using TR-FRET analysis, see Supporting Informa-
tion), to ensure that any phenotype observed through the use of a
BRD9 chemical probe was driven by BRD9 inhibition alone.
BRD4 BD1 (N-terminal, bromodomain 1) was used as a
representative member of the BET family, driving investigations
into selectivity. Analysis of the X-ray crystal structures of apo
C
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 1. Sequence Similarity between the Residues of the
Acetyl-Lysine Binding Sites of BRD4 BD1, BRD9, and BRD7
a
a
Colors represent residue properties: dark green = aromatic, light
green = hydrophobic, red = acidic, blue = basic, orange = polar, purple
= proline, white = small. INS = indel.
Figure 4. (A) pIC50 values for compound 17 in the bromodomains of
BRD9 and BRD4 BD1 as determined by TR-FRET assays.52 (B)
Differences between X-ray crystal structures of 17 in complex with the
bromodomain of BRD9 (green) and BRD4 BD1 (blue). (C) Binding
mode of 17 in BRD9. Hydrogen-bonds are shown as gray dashed lines
and the four conserved water molecules as red spheres. Unfavorable
interactions are marked as yellow dashed lines. (D) Binding mode of 17
in BRD4 BD1 (blue) with the position of acetyl-lysine (orange,
bromodomains superimposed, from protein data bank entry 2wp2).53
(E,F) Two different orientations of the complex of 17 with BRD9.
(G,H) The complex of 17 with BRD4 BD1.
At the other end of the molecule, the piperazine amide
presents a complex pattern of both desirable and undesirable
interactions (Figure 4E). The first unfavorable interaction
involves the amide carbonyl oxygen of the linker, which lies 2.7
Å from the carbonyl oxygen of Ile53, a close interaction for two
electronegative atoms. The second involves the piperidinyl 2-
methylene group, which lies 2.9 Å from the carbonyl oxygen of
the conserved asparagine (Asn100) side chain, preventing its
solvation by water. In compensation, the piperazine amide acts as
Figure 3. Bromodomain phylogenetic tree.
contact with Phe44 and Ile53 on either face. The dimethoxy
groups participate in a network of hydrogen-bonded water
molecules, the 4-methoxy in particular, in a water-mediated
interaction with the carbonyl of His42.
D
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of Amides 17, 27−29, and 32
a
Reagents and conditions: (a) n-BuLi, THF, −78 °C, then DMF, THF, −60 °C to rt, 65%; (b) NBS, THF, rt, 79%; (c) MeI, Cs2CO3, THF, rt, 71%;
(d) NaClO2, NaH2PO4, H2O2, DMSO, H2O, rt, 42−72%; (e) HATU, amine, DIPEA, DMF, rt, 13−79%; (f) (3,4-dimethoxyphenyl)boronic acid,
PdCl2(PPh3)2, K2CO3, DME, H2O, microwave, 120 °C, 24−62%.
a conformationally restricted linker, directing the terminal
methyl sulfonamide to a position where one of its oxygen
atoms accepts a hydrogen bond from the backbone NH of
Arg101.
of 17 for BRD4 compared to BRD9. The hydrogen-bonding
potential of Lys141 is satisfied not by the sulfonamide but instead
by a water molecule, which also interacts with the side chains of
Asp144 and Asp140 (Figure 4G,H).
The general binding mode of the thienopyridone to BRD4 is
the same as to BRD9, but more detailed interactions of this
selective compound differ. One difference arises from the double
substitution of BRD9 Ala54/Tyr106 for BRD4 Leu94/Ile46 on
either side of the thienopyridone ring, leading to a differently
shaped pocket illustrated in Figure 2. These result in a tilt of the
plane of the thienopyridone ring by approximately 10° away from
Leu94 in BRD4 (Figure 4B). The close proximity of Leu94 in
BRD4 also forces the thiophene 2-position carbonyl bond
torsion into an almost completely planar conformation, in
contrast to the ∼30° angle seen in BRD9. For a model
thienopyridone with a dimethyl amide substituent, the planar
BRD4-bound conformation is calculated to be slightly
unfavorable (∼0.7 kcal/mol) with respect to the energy
minimum, which corresponds to the BRD9-bound conforma-
tion, being ∼30° out-of-plane (see Supporting Information,
Figure S4). Another consequence is a knock-on effect on the
pendant piperidinyl sulfonamide, which must adopt a different
position, and as a result the sulfonamide is unable to hydrogen-
bond to the backbone of Lys141 (the equivalent of Arg101 in
BRD9). The electron density for this group is not well-defined
(Figure S2, Supporting Information), but the best placement of
the sulfonamide group is in a region predicted to be
electrostatically unfavorable, close to the side chain oxygen of
Tyr139. We believe that this is consistent with the lower potency
With compound 17 as an encouraging hit, key learnings from
the crystallography were taken forward to guide the design of
compounds with greater potency and improved selectivity over
BRD4. With the hypothesis that the selectivity over BRD4 was
driven by the amide carbonyl of compound 17, amide
modifications were explored to test our idea.
A synthetic route was developed to access the amide analogues
from known bromothienopyridone 1854 (Scheme 1). Halogen-
lithium exchange of bromide 18 with n-BuLi at −78 °C, followed
by the addition of DMF afforded aldehyde 19. Subsequent
electrophilic substitution with NBS, followed by pyridone N-
methylation provided key intermediate 21. Amides 17 and 27−
29 were synthesized by an initial Pinnick oxidation of aldehyde
21 to give acid 22, followed by amide formation. A subsequent
Suzuki−Miyaura coupling with compounds 23−26 was utilized
to install the dimethoxyphenyl motif. In contrast, amide 32 was
synthesized first by a Suzuki−Miyaura coupling of intermediate
21, followed by a Pinnick oxidation to afford acid 31. Final amide
formation using standard peptide coupling conditions furnished
compound 32. This alternative route proved more useful,
providing access to late stage intermediate 31. Functionalization
at the 2-position of 31, through a single amide coupling, allowed
for the introduction of structural diversity in the final step of the
synthesis, providing straightforward access to a range of amides.
E
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 2. SAR for BRD9 and BRD4 BD1 Activity of the Thienopyridone Amide Series
a
All results are n ≥ 3 as determined by TR-FRET analysis.
Analysis of the resulting data relative to compound 17
indicated that the methyl sulfonamide moiety was important for
BRD9 potency and, as a result, selectivity over BRD4 BD1
(Table 2). Consistent with the hydrogen-bonding interaction
made by the sulfonamide with Arg101 in BRD9 but not Lys141
in BRD4, truncating to piperidine 27 or dimethyl amide 28
reduced BRD9 potency, with little effect on BRD4 activity
(compare compounds 17 to 27 and 28). Interestingly, although
not selective over BRD4, secondary methyl amide 29 regained
the BRD9 potency observed for 17, suggesting that the amide
NH makes positive interactions in both BRD4 and BRD9.
Finally, cyclic sulfone amide 32 provided a 10-fold improvement
in BRD9 activity relative to 29, albeit with little selectivity over
BRD4. X-ray crystallography of 32 in the bromodomains of both
BRD9 and BRD4 was used to rationalize the observed data
(Figure 5).
The binding mode of the thienopyridone ring of 32
superimposes closely with that seen for 17, making identical
interactions (Figure 5A). However, the secondary amide of 32
can move closer to the side chain of Asn100 than is possible for
the tertiary amine of 17, forming a direct hydrogen-bond to the
asparagine side chain carbonyl group. This additional interaction
may explain the increased potency of 29 relative to 28 and
contributes to the greater potency of 32. The electron density for
the tetrahydro-2H-thiopyran 1,1-dioxide substituent is quite
weak, but interestingly the best fit is with the amide at the axial
rather than the equatorial position of the ring (Supporting
Information, Figure S3). Another unexpected feature is that
when complexed to 32 the ZA loop moves away from the
Figure 5. (A) X-ray crystal structures of BRD9 with 32 (orange) and 17
(green). (B) X-ray crystal structure of 32 in the bromodomains of BRD9
(orange) and BRD4 BD1 (blue). Hydrogen bonds are shown as gray
dashed lines and water molecules as red spheres.
inhibitor compared to its position in the complex with 17 (Figure
5A). This hinge-like motion, involving residues 50−56, results
from the tetrahydro-2H-thiopyran ring pushing on the loop
through its contacts with Tyr99.
Unexpectedly, the selectivity window for BRD9 over BRD4 is
much reduced for 32 than for 17. This can be rationalized by the
crystal structures of the compound bound to the two
bromodomains (Figure 5B). In contrast to the situation for 17
described above, where different thiophene-amide bond torsion
angles in each bromodomain give rise to a selectivity window, the
F
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 2. Synthesis of Amidines 38−44
a
Reagents and conditions: (a) MeI, Cs2CO3, THF, rt, quant.; (b) Zn(CN)2, Pd(PPh3)4, DMF, microwave, 115 °C, 68%; (c) NBS, THF, rt, 86%; (d)
NaOMe, MeOH, 75 °C, then amine hydrochloride salt, 87%−quant.; (e) R2B(OH)2, K2CO3, PEPPSI-iPr, IPA, H2O, microwave, 120 °C, 4−52%.
a
Table 3. SAR for BRD9 and BRD4 BD1 Activity of Thienopyridone Amidines and Their Amide Analogues
a
All results are n ≥ 3 as determined by TR-FRET.
thiophene and amide of 32 are coplanar when bound to either
bromodomain. As a result, the sulfone of 32 is able to form a
hydrogen bond in each case, to the corresponding backbone NH
atoms of Arg101 in BRD9 and Lys141 in BRD4. The similar
geometry and interactions in each bromodomain account for the
relatively small selectivity window between BRD9 and BRD4.
Despite the low selectivity of 32, the BRD9 potency
encouraged further effort to widen the selectivity window. One
approach suggested by the X-ray crystal structure of 32 in
complex with the bromodomain of BRD9 arose from the
observation that the amide carbonyl lies close (within 5 Å) to the
backbone carbonyl group of Ile53 (Figure 5A). Although the
vectors appeared challenging, it was hypothesized that the
replacement of the amide carbonyl group with a hydrogen-bond
donor moiety, such as an amidine, could increase activity, either
through a direct interaction with the carbonyl of Ile53 or via a
through-water hydrogen-bond. Furthermore, it was postulated
that selectivity could be gained through exploitation of a key
residue difference between BRD9 and BRD4 BD1. Considering
the basic nature of the amidine moiety, it was proposed that when
charged it would sit more favorably in the less hydrophobic
environment alongside Ala54 of BRD9 than beside Leu94 of
BRD4 BD1 (Figure 5B). To this end, the amidine analogues of
secondary amides 29 and 32 were designed and accessed
(Scheme 2).
N-Methylation of known thienopyridone 1854 afforded
intermediate 33. Following this, a palladium catalyzed Negishi
cyanation gave nitrile 34. Subsequent bromination at the 7-
G
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 4. SAR for BRD9 and BRD4 BD1 Activity of Thienopyridone Amidines
a
All results are n ≥ 3 as determined by TR-FRET.
position of the thienopyridone core with NBS provided 35.
Conversion of the nitrile moiety to substituted amidines 36 and
37 was achieved via a Pinner reaction, using NaOMe in MeOH,
followed by addition of the appropriate amine.55 With these key
intermediates in hand, a Suzuki−Miyaura coupling was utilized
to install the different aryl motifs, employing the appropriate
boronic acid.
Pleasingly, amidines 38 and 39 retained the BRD9 activity of
their direct amide analogues 29 and 32, respectively, with
improved levels of selectivity over BRD4 BD1 (Table 3).
Transformation of methyl amide 29 to amidine 38 produced a
substantial increase in selectivity from 2 to 16-fold. In addition,
amidine 39 was 50-fold selective over BRD4, an improvement on
the four-fold seen for its amide analogue, 32.
These exciting results provided confidence in our hypothesis
that the charged amidine was favored in the less hydrophobic
environment of BRD9. With this in mind, further work was
conducted in order to gain additional selectivity. The 7-position
of the thienopyridone core was selected as an appropriate vector
for modification, given its location in the ZA channel. Reaction of
advanced intermediate 37 with various boronic acids provided 7-
aryl substituted compounds containing both electron-donating
and electron-withdrawing substituents (Scheme 2).
Gratifyingly, all amidines (39−44) delivered ≥40-fold
selectivity over the first bromodomain of BRD4 (Table 4).
These data indicated that both the amidine and phenyl moieties
were important for selectivity, with the electronics of the phenyl
ring substituents perhaps also playing a role. Unfunctionalized
phenyl substituted compound 40 and other electron-donating
substituents such as 41 and 42 delivered no significant
improvement in selectivity over BRD4 BD1 compared to
dimethoxyphenyl derivative 39. However, greater selectivity
resulted from replacement of the electron-donating group by
electron-withdrawing substituents. Nitrile substituted com-
pound 43 delivered a 60-fold selectivity window, while the
trifluoromethyl group of compound 44 provided a 160-fold
window, satisfying the BET selectivity56 and BRD9 potency
probe criteria we defined at the outset of this project.
X-ray crystal structures of 44 in the bromodomains of BRD9
and BRD4 BD1 provided insights into the interactions
responsible for the observed selectivity and supported our
hypothesis that led to the synthesis of the amidine. Figure 6A
shows the binding mode in BRD9 of 44 compared to that of 32.
Altering the substituents of the ZA-channel phenyl ring from 3,4-
dimethoxy to 3-trifluoromethyl makes essentially no difference
to the observed binding mode of the core of the molecule. The
subtle change of the amide of 32 to the amidine of 44 maintains
its hydrogen-bond to the conserved Asn100 side chain (Figure
6A) but leads to more notable changes that suggest significant
electrostatic differences between the two molecules. The
stronger electrostatics of the amidine NH group allow it to
move closer to the backbone carbonyl of Ile53 than was possible
with the carbonyl of the amide, resulting in a direct hydrogen-
bond between these groups as we had hoped (Figure 6A). One
result of this is a ∼30° rotation of the thiophene−amidine bond
of 44. This allows the tetrahydro-2H-thiopyran 1,1-dioxide
H
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
groups larger than a methyl can be accommodated in the
bromodomain binding pocket.29,57 With this in mind, we sought
to investigate the effect of alternative Kac mimetic alkyl groups.
To this end, N-ethyl analogue 45 and N-isopropyl analogue 46
were synthesized via a similar route to that described in Scheme
2.58
Increasing the Kac mimetic alkyl chain length from N-methyl
to N-ethyl maintained ≥100-fold selectivity over BRD4 BD1
(Table 5). However, the introduction of the more sterically
Table 5. SAR for BRD9 and BRD4 BD1 Activity of 3-
Trifluoromethylphenyl Substituted Thienopyridone
a
Amidines
Cmpd
BRD9 pIC50
BRD4 BD1 pIC50
Fold Selectivity
44
45
46
7.8 0.21
7.3 0.18
5.5 0.26
5.6 0.03
5.3 0.07
<4.3
×160
×100
≥×15
a
All results are n ≥ 3 as determined by TR-FRET analysis.
demanding isopropyl substituent, as in compound 46, reduced
BRD9 and BRD4 activities, suggesting that extending beyond N-
ethyl is not well tolerated in either of these bromodomains
(compare compounds 44 and 46). X-ray crystallography of
compound 45 in complex with BRD9 was obtained in order to
confirm its binding mode. As expected, 45 adopts a similar
conformation to compound 44 (Figure 7A), making identical
interactions to Asn100, Ile53, and Arg101. The N-ethyl Kac
mimetic is comfortably accommodated in the hydrophobic
binding pocket with a slight movement of Phe45, having little
effect on the conformation of the remainder of the site.
In order to assess selectivity over non-BET bromodomains,
compounds 44 and 45 were profiled in a selection of internal TR-
FRET assays, the results of which indicated that compound 45
displayed a superior selectivity profile. Considering this result,
compound 45 was progressed for BROMOscan59 profiling
against 34 bromodomains to evaluate the effect of the N-ethyl
Kac mimetic (Figure 7B). Pleasingly, the results from this
broader bromodomain screen indicated nanomolar affinity
binding at BRD9, with pKd = 8.7 (for full data, see Supporting
Information). Compound 45 showed >700-fold selectivity over
the BET family of bromodomains and 200-fold over the highly
homologous BRD7 bromodomain (pKd = 6.4). Furthermore, 45
displayed >70-fold selectivity against every other bromodomain
tested.
The selectivity of compound 45 over BRD7 is particularly
surprising considering the 85% sequence homology between the
bromodomains of BRD9 and BRD7 (Table 1). Although an
NMR structure of BRD7 has been published,60 there is no crystal
structure reported to date. Therefore, it was challenging to
rationalize the observed selectivity over this bromodomain.
Presumably, selectivity arises from the differences in the
constituent amino acids of the WPF shelf (BRD4 nomenclature,
see Table 1) and ZA channels of these bromodomains. In BRD9,
Gly43 is replaced by Ala154 in the WPF region of BRD7, and
Ala46, by Ser157 in the ZA channel. These key changes result in a
Figure 6. X-ray structure of (A) 32 (orange) and 44 (green) in BRD9,
showing the rotation around the thiophene−amidine bond of 44. The
significant difference in conformation of the ZA loop that brings the
carbonyl group of Ile53 close to the amidine of 44 is also visible. (B)
Compounds 32 (orange) and 44 (green) in BRD4 BD1 with the 3.6 Å
distance between the amidine nitrogen and Leu94 shown as a yellow
dashed line.
amide substituent to resume its equatorial geometry while
maintaining the hydrogen bond between the cyclic sulfone
oxygen and the backbone NH of Arg101. The combined effect
allows both Tyr99 and residues 50−56 of the ZA loop to relax to
a position similar to that seen in the complex with 17, rather than
the conformation seen with 32.
In contrast, the binding mode of 44 in BRD4 BD1 differs little
from that of 32, with no movement of the ZA loop (Figure 6B). A
very slight movement of the amidine closer to Asn140 probably
reflects a tighter charged hydrogen-bonding interaction. The best
fit to the density of the tetrahydro-2H-thiopyran 1,1-dioxide
amide substituent has equatorial geometry, but this maintains the
hydrogen bond between cyclic sulfone and the backbone NH of
Lys141 (Figure S2, Supporting Information). An explanation for
the reduced BRD4 potency of 44 with respect to 32 is our
original hypothesis that a charged amidine group in close
proximity to the hydrophobic BRD4 Leu94 side chain is
unfavorable (in the X-ray structure, the distance is 3.6 Å). The
basic nature of the amidine functionality was confirmed by
measurement: the pKa of 44 was determined to be 8.4, so the
group would be protonated at neutral pH.
Given the promising activity and selectivity profile displayed
by compound 44, this molecule was taken forward for further
structure−activity relationship (SAR) exploration. It has
previously been reported that Kac mimetics containing alkyl
I
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
difference in the architecture of these binding regions, potentially
altering the conformation of other residues in the ZA channel.
The observed potency was higher in the BROMOscan format
than in house TR-FRET assays. This may be due to use of the
alternative detection system, protein construct, and sample
preparation methods. As the rank order is maintained between
formats, numerical differences in the reported affinities did not
affect decision making.
Based on the high BRD9 affinity and excellent broader
bromodomain selectivity results, compound 45 (I-BRD9), was
chosen as the chemical probe for BRD9.
As all measurements of binding affinity of I-BRD9 to date had
been carried out with truncated bromodomain proteins, we were
keen to confirm these findings were consistent with full-length
targets in their native context. Using a chemoproteomic
competition binding assay in HUT-78 cell lysate, the binding
of I-BRD9 to endogenous BRD9 displayed >625-fold selectivity
against BET family member BRD3 (Figure 8A).58 These data
confirm potency at BRD9, and selectivity over the BET family is
maintained with endogenous proteins.
Critically for a bromodomain probe, cellular target engage-
ment of BRD9 and disruption of chromatin binding was
demonstrated through a NanoBRET assay measuring displace-
ment of NanoLuc-tagged BRD9 bromodomain from Halo-
tagged histone H3.3 (Figure 8B).58
Having established endogenous protein binding, cell and
nuclear permeability, and excellent selectivity over the BET
family and other non-BET bromodomains, further profiling of I-
BRD9 was conducted. These experiments aimed to evaluate the
selectivity of the compound over a broad range of pharmaco-
Figure 7. (A) Superimposed X-ray crystal structures of the
bromodomain of BRD9 complexed with compounds 45 (magenta)
and 44 (green), showing the subtle movement of Phe45 in response to
the N1-methyl/ethyl difference between the two compounds. (B)
BROMOscan data for compound 45 (generated at DiscoveRx Corp.).
Figure 8. (A) Dose−response binding of compound 45 (I-BRD9) for endogenous BRD9 and BRD3 from HuT-78 cell lysates, measured in a
chemoproteomic competition binding assay followed by Western blot analysis. (B) BRD9 bromodomain cellular NanoBRET dose−response curve of
compound 45 (I-BRD9). (C) qPCR validation of CLEC1, DUSP6, FES, and SAMSN1 genes selectively regulated by compound 45 (I-BRD9) (10 μM),
but not by I-BET151 (1 μM), mean SD; n = 3. Genes were previously identified by full gene transcriptomics in Kasumi-1 cells.
J
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
logical targets including various receptors, transporters, ion
channels, kinases, and other enzymes. Pleasingly, I-BRD9
showed no activity at less than 5 μM against a panel of 49
targets.58 A summary of the properties of I-BRD9 is given in
Table 6.
requirements necessary for a high quality chemical probe. I-
BRD9 was used in the identification of a number of BRD9
inhibitor sensitive genes, many of which are involved in immune
function and cancer, which might indicate the biological role of
the BRD9 bromodomain, and provides insight and direction for
further investigation.
Table 6. Summary of Properties of I-BRD9
EXPERIMENTAL SECTION
■
All solvents were purchased from Sigma-Aldrich (Hy-Dry anhydrous
solvents), and commercially available reagents were used as received. All
reactions were followed by TLC analysis (TLC plates GF254, Merck) or
liquid chromatography mass spectrometry (LCMS) using a Waters ZQ
instrument. IR spectra were obtained on a PerkinElmer Spectrum 1
machine. Melting points were measured with a Stuart SMP10, 230 V
appartus and were uncorrected.
BRD9 pIC50 (TR-FRET)
7.3
NMR spectra were recorded at ambient temperature using standard
pulse methods on any of the following spectrometers and signal
frequencies: Bruker AV-400 (1H = 400 MHz, 13C = 101 MHz) and
Bruker AV-500 (1H = 500 MHz, 13C = 125 MHz). Chemical shifts are
reported in ppm and are referenced to tetramethylsilane (TMS) or the
following solvent peaks: CDCl3 (1H = 7.27 ppm, 13C = 77.00 ppm),
DMSO-d6 (1H = 2.50 ppm, 13C = 39.51 ppm), and MeOH-d4 (1H = 3.31
ppm, 13C = 49.15 ppm). Coupling constants are quoted to the nearest
0.1 Hz, and multiplicities are given by the following abbreviations and
combinations thereof: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), br (broad). Column chromatography was performed on
prepacked silica gel columns (30−90 mesh, IST) using a biotage SP4.
For detailed LCMS/MDAP/HRMS methodology, see Supplementary
Methods.
BRD4 BD1 pIC50 (TR-FRET)
BRD9 NanoBRET pIC50
5.3
6.8
BROMOscan selectivity over other bromodomains58
BETs: >×700
BRD7: ×200
all others: >×70
3.7
61
ChromLogDpH7.4
aqueous solubility (CLND)
359 μM (high)
210 nm/s (high)
artificial membrane permeability
With a high quality soluble and permeable bromodomain
probe in hand, we sought to investigate genes selectively
modulated by BRD9 bromodomain inhibition. Accordingly,
Kasumi-1 cells were treated with I-BET15116−18 and I-BRD9 for
6 h, the concentrations of which were chosen to pharmacolog-
ically match the BET activity of the two probes. RNA was
harvested, reverse transcribed, and hybridized on an Illumina
expression array. While there was an expected overlap, the
majority of genes were selectively regulated by I-BRD9.62
Analysis of four representative genes (CLEC1,63 DUSP6,64
FES,65 and SAMSN166) that were strongly down-regulated by I-
BRD9 and not by I-BET151, by qPCR further validated the
microarray data and gave insights into the effect of BRD9
inhibition (Figure 8C). These four genes are implicated in cancer
and immunology pathways, suggesting for the first time a role for
the BRD9 bromodomain in their regulation. This down
regulation also implies potential utility for synergistic disease
modulation by BRD9 inhibition: for example, DUSP6 expression
has been implicated in modulating drug sensitivity in cancers,
which could be important for chemotherapy.64
The purity of all compounds tested was determined by LCMS and 1H
NMR to be >95%.
Selected Experimental Procedures. 2-Bromo-5-ethylthieno-
[3,2-c]pyridin-4(5H)-one (47). A single portion of ethyl iodide (1.40
mL, 17.39 mmol) was added to a solution of bromothieno[3,2-
c]pyridin-4(5H)-one (18)54 (2.00 g, 8.69 mmol) and Cs2CO3 (8.50 g,
26.10 mmol) in THF (35 mL) at room temperature. The reaction
mixture was heated at 60 °C for 18 h, then allowed to cool to room
temperature and diluted with EtOAc (50 mL) and H2O (50 mL). The
separated aqueous phase was extracted with EtOAc (3 × 50 mL), and
the combined organic layers were passed through a hydrophobic frit and
concentrated in vacuo to give 47 (2.30 g, quant.) as a light brown solid.
Fifty milligrams of 47 was taken forward for purification by mass
directed autopreparation (formic acid), providing suitably pure material
(44 mg, 0.17 mmol) for full characterization; mp 124−125 °C; νmax
(solid)/cm−1 1632, 1577, 1082, 846, 761; 1H NMR (500 MHz,
(CD3)2SO) δ ppm 7.62 (s, 1H), 7.56 (d, J = 7.2, 1H), 6.89 (d, J = 7.2,
1H), 3.99 (q, J = 7.2, 2H), 1.24 (t, J = 7.2, 3H); 13C NMR (125 MHz,
(CD3)2SO) δ ppm 156.4, 148.4, 133.8, 130.2, 127.0, 111.7, 100.8, 43.3,
14.6; HRMS (M + H)+ calculated for C9H9BrNOS 257.9583; found
257.9585. LCMS (formic acid) (M + H)+ = 258.0, 260.0, Rt = 0.89 min
(98%).
CONCLUSION
■
5-Ethyl-4-oxo-4,5-dihydrothieno[3,2-c]pyridine-2-carbonitrile
(48). A mixture of 47 (1.10 g, 4.26 mmol), zinc cyanide (1.00 g, 8.52
mmol), and Pd(PPh3)4 (0.44 g, 0.3 mmol) in DMF (10 mL) was heated
to 115 °C in a microwave reactor for 3.5 h. This process was repeated to
provide a second identical batch. The two batches were allowed to cool
to room temperature, diluted with EtOAc (50 mL each), and combined.
The solution was filtered through Celite and concentrated in vacuo. The
resulting residue was purified by silica gel chromatography (0−3%
MeOH in CH2Cl2). The appropriate fractions were combined and
concentrated in vacuo to give 48 (1.70 g, 67%) as a light brown solid.
Fifty milligrams of 48 was taken forward for purification by mass
directed autopreparation (formic acid), providing suitably pure material
(33 mg, 0.16 mmol) for full characterization; mp 157−158 °C; νmax
Through several iterations of structure-based design, starting
from tertiary amide 17, we have identified I-BRD9 as the first
selective cellular chemical probe for the non-BET bromodomain
BRD9. This key contribution provides for the first time the ability
to selectively target BRD9 and further enhances the tool set
available for targeting human bromodomains. Critical to the
success of this endeavor was the selectivity enhancing amidine,
which drove unfavorable interactions between I-BRD9 and the
BET family. This optimized compound is highly potent at BRD9
and displays >700-fold selectivity over the BET family and >70-
fold against the panel of 34 bromodomains tested. It is of note
that I-BRD9 is selective for a single bromodomain, whereas the
vast majority of bromodomain probes bind to ≥2. With proof of
bromodomain cellular target engagement, binding to endoge-
nous BRD9, and broader selectivity against a variety of
pharmacological targets, we believe this molecule satisfies the
1
(solid)/cm−1 2211, 1637, 1595, 1247, 1078, 879, 780, 769; H NMR
(400 MHz, (CD3)2SO) δ ppm 8.32 (s, 1H), 7.82 (d, J = 7.2, 1H), 7.03
(d, J = 7.2, 1H), 4.00 (q, J = 7.2, 2H), 1.25 (t, J = 7.2, 3H); 13C NMR
(100 MHz, (CD3)2SO) δ ppm 157.1, 151.1, 137.1, 136.7, 128.9, 114.2,
106.4, 100.9, 43.4, 14.5; HRMS (M + H)+ calculated for C10H9N2OS
K
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
205.0430; found 205.0429. LCMS (formic acid) (M + H)+ = 205.0, Rt =
0.73 min (98%).
statistics, and computed thienopyridone amide torsional energy
profiles. The Supporting Information is available free of charge
on the ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b00256.
7-Bromo-5-ethyl-4-oxo-4,5-dihydrothieno[3,2-c]pyridine-2-car-
bonitrile (49). To a solution of 48 (1.17 g, 5.73 mmol) in THF (20 mL),
NBS (1.53 g, 8.59 mmol) was added portion-wise. The reaction mixture
was left to stir at room temperature for 48 h. The volatile components
were removed in vacuo. The resulting solid was suspended in Et2O (50
mL), filtered under reduced pressure, washed with Et2O (50 mL),
collected, and dried under vacuum at 40 °C to give 49 (1.69 g, quant.) as
an off-white solid; mp 197−198 °C; νmax (solid)/cm−1 3041, 2220, 1654,
1584, 1250, 1090, 766; 1H NMR (500 MHz, (CD3)2SO) δ ppm 8.50 (s,
1H), 8.26 (s, 1H), 4.02 (q, J = 7.2, 2H), 1.26 (t, J = 7.2, 3H); 13C NMR
(125 MHz, (CD3)2SO) δ ppm 156.1, 152.3, 138.2, 137.6, 128.3, 113.7,
107.0, 90.4, 43.9, 14.4; HRMS (M + H)+ calculated for C10H8BrN2OS
282.9535; found 282.9534. LCMS (formic acid) (M + H)+ = 283.0,
285.0, Rt = 0.93 min (100%).
7-Bromo-N-(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-5-ethyl-4-
oxo-4,5-dihydrothieno[3,2-c]pyridine-2-carboximidamide (50). To a
suspension of 49 (1.00 g, 3.53 mmol) in MeOH (30 mL) at room
temperature, sodium methoxide (25% weight in MeOH, 0.81 mL, 3.53
mmol) was added in a single portion. The reaction mixture was heated at
75 °C for 5 h. 4-Aminotetrahydro-2H-thiopyran-1,1-dioxide hydro-
chloride (1.98 g, 10.60 mmol) was added, and the solution was heated
for a further 18 h. The volatile components were removed in vacuo, and
the resulting solid was suspended in Et2O (50 mL), filtered under
reduced pressure, washed with Et2O (50 mL), collected, and dried under
vacuum at 40 °C to give 50 (1.55 g, quant.) as a white solid. Fifty
milligrams of 50 was taken forward for purification by mass directed
autopreparation (high pH), providing suitably pure material (25 mg,
0.06 mmol) for full characterization; mp 284−285 °C; νmax (solid)/
cm−1 1646, 1578, 1284, 1124, 853, 769; 1H NMR (500 MHz,
(CD3)2SO) δ ppm 8.18 (s, 1H), 8.01 (s, 1H), 6.80 (br.s, 2H), 4.01 (q, J =
7.2, 2H), 3.68−3.61 (m, 1H), 3.27−3.20 (m, 2H), 3.10−3.03 (m, 2H),
2.09−2.00 (m, 2H), 1.99−1.90 (m, 2H), 1.26 (t, J = 7.2, 3H); 13C NMR
(125 MHz, (CD3)2SO) δ ppm 156.9, 149.5, 134.6, 129.7, 123.2, 109.7,
92.0, 48.6, 43.7, 30.2, 14.8; HRMS (M + H)+ calculated for
C15H19BrN3O3S2 432.0046; found 432.0046. LCMS (formic acid) (M
+ H)+ = 432.1, 434.1, Rt = 0.53 min (100%).
N-(1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-5-ethyl-4-oxo-7-(3-
(trifluoromethyl)phenyl)-4,5-dihydrothieno[3,2-c]pyridine-2-car-
boximidamide (45, I-BRD9). A mixture of 50 (150 mg, 0.35 mmol), (3-
(trifluoromethyl)phenyl)boronic acid (79 mg, 0.42 mmol), PEPPSI-iPr
(21 mg, 0.03 mmol), and K2CO3 (115 mg, 0.83 mmol) in H2O (1 mL)
and IPA (3 mL) was heated at 120 °C in a microwave reactor for 0.5 h.
The reaction mixture was allowed to cool to room temperature, diluted
with EtOAc (40 mL), filtered through Celite, and concentrated in vacuo.
The resulting residue was purified by mass directed autopreparation
(formic acid). The appropriate fractions were combined and
concentrated in vacuo. The resulting residue was dissolved in MeOH
(5 mL) and passed through a preconditioned (MeOH, 20 mL) amino
propyl column (10 g). The appropriate fractions were combined and
concentrated in vacuo to give 45 (50 mg, 29%) as a white solid; mp 228−
229 °C; νmax (solid)/cm−1 1640, 1587, 1340, 1303, 1117; 1H NMR (500
MHz, (CD3)2SO) δ ppm 8.15 (s, 1H), 8.03−7.98 (m, 1H), 7.98−7.94
(m, 1H), 7.91 (s, 1H), 7.82−7.76 (m, 2H), 6.76 (br.s, 2H), 4.10 (q, J =
7.0, 2H), 3.64−3.57 (m, 1H), 3.21−3.12 (m, 2H), 3.10−3.00 (m, 2H),
2.06−1.96 (m, 2H), 1.96−1.85 (m, 2H), 1.32 (t, J = 7.0, 3H); 13C NMR
(125 MHz, (CD3)2SO) δ ppm 157.6, 149.7, 147.7, 142.2, 137.5, 134.1,
131.9, 130.7, 130.5, 130.3 (q, JC−F = 31.7), 125.0 (q, JC−F = 3.7), 124.60
(q, JC−F = 3.7), 124.59 (q, JC−F = 272.6), 123.1, 114.4, 49.9, 49.0, 44.0,
30.6, 15.2; 19F NMR (376 MHz, (CD3)2SO) δ ppm −61.0; HRMS (M +
H)+ calculated for C22H23F3N3O3S2 498.1128; found 498.1110. LCMS
(formic acid) (M + H)+ = 498.1, Rt = 0.81 min (100%).
Accession Codes
Atomic coordinates of BD4 BD1 bound to compounds 17
(4uiz), 32 (4uix), 44 (4uiy) and BRD9 bound to compounds 17
(4uit), 32 (4uiu), 44 (4uiv) and 45 (4uiw) have been deposited
with the Protein Data Bank.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: philip.g.humphreys@gsk.com. Phone: +44 (0)1438
764252. Fax: +44 (0)1438 764799.
Notes
The authors declare the following competing financial
interest(s): All authors except N.H.T., N.C.O.T., H.K.C., A.B.,
S.C.R. and T.K. were GlaxoSmithKline and Cellzome full-time
employees at the time when this study was performed.
ACKNOWLEDGMENTS
■
N.H.T. is grateful to GlaxoSmithKline R&D, Stevenage for Ph.D.
́
studentship funding. We thank Jacqui Mendez and Danette
Daniels of Promega Corporation for facilitating the NanoBRET
assay. We also thank Robert Watson and Darren Mitchell for the
synthesis of useful building blocks; Matthew Crane for the
synthesis of compound 32; Iain Reid for pKa determination; Sean
Lynn for assistance with NMR; Marco Smith for high resolution
mass spectrometry, and Paul White, Keir Sims, and Abigail Lucas
for crystallization support. We appreciate helpful discussions
with Emmanuel Demont during the course of this work.
ABBREVIATIONS USED
■
BAF, BRG1/brm-associated factor; BAZ2A, bromodomain
adjacent to zinc finger domain, 2A; BAZ2B, bromodomain
adjacent to zinc finger domain, 2B; BCP, bromodomain
containing protein; BD, bromodomain; BET, bromodomain
and extra terminal domain; BRD2, bromodomain containing
protein 2; BRD3, bromodomain containing protein 3; BRD4,
bromodomain containing protein 4; BRD7, bromodomain
containing protein 7; BRD9, bromodomain containing protein
9; BRDT, bromodomain containing protein, testis-specific;
CECR2, cat eye syndrome chromosome region, candidate 2;
CREBBP, cAMP response element-binding protein binding
protein; DIPEA, N,N-diisopropylethylamine; EP300, E1A bind-
ing protein p300; HAT, histone acetyltransferase; HATU, O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluor-
ophosphate; HDAC, histone deacetylase; IPA, isopropanol; Kac,
acetyl lysine; LCMS, liquid column mass spectrometry; LPS,
lipopolysaccharide; PB1(5), PolyBromo 1 (5); PCAF, P300/
CREBBP-associated factor; PEPPSI-iPr, pyridine-enhanced
precatalyst preparation stabilization and initiation; PK, pharma-
cokinetics; SMARCA2, SWI/SNF related, matrix associated,
actin dependent regulator of chromatin, subfamily A, member 2;
SMARCA4, SWI/SNF related, matrix associated, actin-depend-
ent regulator of chromatin, subfamily A, member 4; SWI/SNF,
SWItch/sucrose nonfermentable; TR-FRET, time-resolved
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional text describing all biological methods and results,
chemistry experimental procedures, full BROMOscan and
selectivity data for I-BRD9, X-ray data collection and refinement
Forster (fluorescence) resonance energy transfer
̈
L
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Discovery and characterisation of small molecule inhibitors of the BET
family bromodomains. J. Med. Chem. 2011, 54, 3827−3838.
(14) Filippalopoulos, 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.
REFERENCES
■
(1) Sanchez, R.; Zhou, M. M. The role of human bromodomains in
chromatin biology and gene transcription. Curr. Opin. Drug Discovery
Dev. 2009, 12, 659−665.
(2) Gardner, K. E.; Allis, C. D.; Strahl, B. D. Operating on chromatin, a
colourful language where context matters. J. Mol. Biol. 2011, 409, 36−46.
(3) Chung, C. W.; Tough, D. F. Bromodomains: a new target class for
small molecule drug discovery. Drug Discovery Today: Ther. Strategies
2012, 9, 111−120.
(15) Miyoshi, S.; Ooike, S.; Iwata, K.; Hikawa, H.; Sugahara, K. Int. Pat.
Appl. WO 2009/084693, 2009.
(4) Chung, C. W. Small molecule bromodomain inhibitors: extending
the druggable genome. Prog. Med. Chem. 2012, 51, 1−55.
(5) Mujtaba, S.; Zeng, L.; Zhou, M. M. Structure and acetyl-lysine
recognition of the bromodomain. Oncogene 2007, 26, 5521−5527.
(6) Filippakopoulos, P.; Knapp, S. The bromodomain interaction
module. FEBS Lett. 2012, 586, 2682−2704.
(16) Dawson, M. A.; Prinjha, R. K.; Dittmann, A.; Giotopoulos, G.;
Bantscheff, M.; Chan, W. I.; Robertson, S.; Chun, C. W.; Hopf, C.;
Savitski, M. M.; Huthmacher, C.; Gudgin, E.; Lugo, D.; Beinke, S.;
Chapman, T. D.; Roberts, E. J.; Soden, P. E.; Auger, K. R.; Mirguet, O.;
Doehner, K.; Delwel, R.; Burnett, A. K.; Jeffrey, P.; Drewes, G.; Lee, K.;
Huntly, B. J. P.; Kouzarides, T. Inhibition of BET recruitment to
chromatin as an effective treatment for MLL-fusion leukaemia. Nature
2011, 478, 529−533.
(17) Mirguet, O.; Lamotte, Y.; Donche, F.; Toum, J.; Gellibert, F.;
Bouillot, A.; Gosmini, R.; Nguyen, V. L.; Delannee, D.; Seal, J.; Blandel,
F.; Boullay, A. B.; Boursier, E.; Martin, S.; Brusq, J. M.; Krysa, G.; Riou,
A.; Tellier, R.; Costaz, A.; Huet, P.; Dudit, Y.; Trottet, L.; Kirilovsky, J.;
Nicodeme, E. From ApoA1 upregulation to BET family bromodomain
inhibition: Discovery of I-BET151. Bioorg. Med. Chem. Lett. 2012, 22,
2963−2967.
(18) Seal, J.; Lamotte, Y.; Donche, F.; Bouillot, A.; Mirguet, O.;
Gellibert, F.; Nicodeme, E.; Krysa, G.; Kirilovsky, J.; Beinke, S.;
McCleary, S.; Rioja, I.; Bamborough, P.; Chung, C. W.; Gordon, L.;
Lewis, T.; Walker, A. L.; Cutler, L.; Lugo, D.; Wilson, D. M.;
Witherington, J.; Lee, K.; Prinjha, R. K. Identification of a novel series of
BET family bromodomain inhibitors: binding mode and profile of I-
BET151 (GSK1210151A). Bioorg. Med. Chem. Lett. 2012, 22, 2968−
2972.
(19) Gosmini, R.; Nguyen, V. L.; Toum, J.; Simon, C.; Brusq, J.-M. G.;
Krysa, G.; Mirguet, O.; Riou-Eymard, A. M.; Boursier, E. V.; Trottet, L.;
Bamborough, P.; Clark, H.; Chung, C. W.; Cutler, L.; Demonet, E. H.;
Kaur, R.; Lweis, A. J.; Schilling, M. B.; Soden, P. E.; Taylor, S.; Walker, A.
L.; Walker, W. D.; Prinjha, R. K.; Nicodeme, E. The discovery of I-
BET726 (GSK1324726A), a potent tetrahydroquinoline ApoA1 up-
regulator and selective BET bromodomain inhibitor. J. Med. Chem.
2014, 57, 8111−8131.
(20) Fish, P. V.; Filippakopoulos, P.; Bish, G.; Brennan, P. E.; Bunnage,
M. E.; Cook, A. S.; Fedorov, O.; Gerstenberger, B. S.; Jones, H.; Knapp,
S.; Marsden, B.; Nocka, K.; Owen, D. R.; Philpott, M.; Picaud, S.;
Primiano, M. J.; Ralph, M. J.; Sciammetta, N.; Trzupek, J. D.
Identification of a chemical probe for bromo and extra C-terminal
bromodomain inhibition through optimization of a fragment-derived
hit. J. Med. Chem. 2012, 55, 9831−9837.
(21) Hewings, D. S.; Wang, M.; Philpott, M.; Fedorov, O.; Uttarkar, S.;
Filippakopoulos, P.; Picaud, S.; Vuppusetty, C.; Marsden, B.; Knapp, S.;
Conway, S. J.; Heightman, T. D. 3,5-dimethylisoxazoles act as acetyl-
lysine-mimetic bromodomain ligands. J. Med. Chem. 2011, 54, 6761−
6770.
(22) Hewings, D. S.; Fedorov, O.; Filippakopoulos, P.; Martin, S.;
Picaud, S.; Tumber, A.; Wells, C.; Olcina, M. M.; Freeman, K.; Gill, A.;
Ritchie, A. J.; Sheppard, D. W.; Russell, A. J.; Hammond, E. M.; Knapp,
S.; Brennan, P. E.; Conway, S. J. Optimization of 3,5-dimethylisoxazole
derivatives as potent bromodomain ligands. J. Med. Chem. 2013, 56,
3217−3227.
(23) Zhang, G.; Plotnikov, A. N.; Rusinova, E.; Shen, T.; Morohasshi,
K.; Joshua, J.; Zeng, L.; Mujtaba, S.; Ohlmeyer, M.; Zhou, M.-M.
Structure-Guided design of potent diazobenzene inhibitors for the BET
bromodomains. J. Med. Chem. 2013, 56, 9251−9264.
(7) Arrowsmith, C. H.; Bountra, C.; Fish, P. V.; Lee, K.; Schapira, M.
Epigenetic protein families: a new frontier for drug discovery. Nat. Rev.
Drug Discovery 2012, 11, 384−400.
(8) Filippakopoulos, P.; Picaud, S.; Mangos, M.; Keates, T.; Lambert, J.
P.; Barsyte-Lovejoy, D.; Felletar, I.; Volkmer, R.; Muller, S.; Pawson, T.;
̈
Gingras, A. C.; Arrowsmith, C. H.; Knapp, S. Histone recognition and
large-scale structural analysis of the human bromodomain family. Cell
2012, 149, 214−231.
(9) For recent reviews of BET bromodomain inhibitors and their
therapeutic potential, see: (a) Hewings, D. S.; Rooney, T. P. C.;
Jennings, L. E.; Hay, D. A.; Schofield, C. J.; Brennan, P. E.; Knapp, S.;
Conway, S. J. Progress in the development and application of small
molecule inhibitors of bromodomain-acetyl-lysine interactions. J. Med.
Chem. 2012, 55, 9393−9413. (b) Furdas, S. D.; Carlino, L.; Sippl, W.;
Jung, M. Inhibition of bromodomain-mediated protein-protein
interactions as a novel therapeutic strategy. MedChemComm 2012, 3,
123−134. (c) Prinjha, R. K.; Witherington, J.; Lee, K. Place your BETs:
the therapeutic potential of bromodomains. Trends Pharmacol. Sci. 2012,
33, 146−153. (d) Knapp, S.; Weinmann, H. Small-molecule modulators
for epigenetic targets. ChemMedChem 2013, 3, 1885−1891. (e) Sweis,
R. F.; Michaelides, M. R. Recent advances in small-molecule modulation
of epigenetic targets: Discovery and development of histone
methyltransferase and bromodomain inhibitors. Annu. Rep. Med.
Chem. 2013, 48, 185−199. (f) Jennings, L. E.; Measures, A. R.;
Wilson, B. G.; Conway, S. J. Phenotypic screening and fragment-based
approaches to the discovery of small-molecule bromodomain ligands.
Future Med. Chem. 2014, 6, 179−204. (g) Garnier, J. M.; Sharp, P. P.;
Burns, C. J. BET bromodomain inhibitors: a patent review. Expert Opin.
Ther. Pat. 2014, 24, 185−199.
(10) For an elegant solution to targeting individual bromodomains of
the BET family, see Baud, M. G. J.; Lin-Shiao, E.; Cardote, T.; Tallant,
C.; Pschibul, A.; Chan, K.-H.; Zengerle, M.; Garcia, J. R.; Kwan, T. T.-L.;
Ferguson, F. M.; Ciulli, A. A bump-and-hole approach to engineer
controlled selectivity of BET bromodomain chemical probes. Science
2014, 346, 638−641.
(11) Nicodeme, E.; Jeffrey, K. L.; Schaefer, U.; Beinke, S.; Dewell, S.;
Chung, C. W.; Chandwani, R.; Marazzi, I.; Wilson, P.; Coste, H.; White,
J.; Kirilovsky, J.; Rice, C. M.; Lora, J. M.; Prinjha, R. K.; Lee, K.;
Tarakovsky, A. A. Suppression of inflammation by a synthetic histone
mimic. Nature 2010, 468, 1119−1123.
(12) Mirguet, O.; Gosmini, R.; Toum, J.; Clement, C. A.; Barnathan,
M.; Brusq, J. M.; Mordaunt, J. E.; Grimes, R. M.; Crowe, M.; Pineau, O.;
Ajakane, M.; Daugan, A.; Jeffrey, P.; Cutler, L.; Haynes, A. C.; Smithers,
N. N.; Chung, C. W.; Bamborough, P.; Uings, I. J.; Lewis, A.;
Witherington, J.; Parr, N.; Prinjha, R. K.; Nicodeme, E. Discovery of
epigenetic regulator I-BET762: lead optimization to afford a clinical
candidate inhibitor of the BET bromodomains. J. Med. Chem. 2013, 56,
7501−7515.
(13) Chung, C. W.; Coste, H.; White, J. H.; Mirguet, O.; Wilde, J.;
Gosmini, R. L.; Delves, C.; Magny, S. M.; Woodward, R.; Hughes, S. A.;
Boursier, E. V.; Flynn, H.; Bouillot, A. M.; Bamborough, P.; Brusq, J. M.
G.; Gellibert, F. J.; Jones, E. J.; Riou, A. M.; Homes, P.; Martin, S. L.;
Uings, I. J.; Toum, J.; Clement, C. A.; Boullay, A. B.; Grimley, R. L.;
Blandel, F. M.; Prinjha, R. K.; Lee, K.; Kirilovsky, J.; Nicodeme, E.
(24) McLure, K. G.; Gesner, E. M.; Tsujikawa, L.; Kharenko, O. A.;
Attwell, S.; Campeau, E.; Wasiak, S.; Stein, A.; White, A.; Fontano, E.;
Suto, R. K.; Wong, N. C.; Wagner, G. S.; Hansen, H. C.; Young, P. R.
RVX-208, an inducer of ApoA-I in humans, is a BET bromodomain
antagonist. PLoS One 2013, 8, e83190.
M
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(25) Picaud, S.; Wells, C.; Felletar, I.; Brotherton, D.; Martin, S.;
Savitsky, P.; Diez-Dacal, B.; Philpott, M.; Bountra, C.; Lingard, H.;
(42) Scotto, L.; Narayan, G.; Nandula, S. V.; Subramaniyam, S.;
Kaufmann, A. M.; Wright, J. D.; Pothuri, B.; Mansukhani, M.; Schneider,
A.; Arias-Pulido, H.; Murty, V. V. Integrative genomics analysis of
chromosome 5p gain in cervical cancer reveals target over-expressed
genes, including Drosha. Mol. Cancer 2008, 7, 58−67.
(43) Kadoch, C.; Hargreaves, D. C.; Hodges, C.; Elias, L.; Ho, L.;
Ranish, J.; Crabtree, G. R. Proteomic and bioinformatic analysis of
mammalian SWI/SNF complexes identifies extensive roles in human
malignancy. Nat. Genet. 2013, 45, 592−601.
(44) Hohmann, A. F.; Vakoc, C. R. A rationale to target the SWI/SNF
complex for cancer therapy. Trends Genet. 2014, 30, 356−363.
(45) Bromosporine. http://www.thesgc.org/chemical-probes/
bromosporine (accessed Feb 6, 2015).
(46) Fedorov, O.; Lingard, H.; Wells, C.; Monteiro, O. P.; Picaud, S.;
Keates, T.; Yapp, C.; Philpott, M.; Martin, S. J.; Felletar, M. I.; Marsden,
Fedorov, O.; Muller, S.; Brennan, P. E.; Knapp, S.; Filippakopoulos, P.
̈
RVX-208, an inhibitor for BET transcriptional regulators with selectivity
for the second bromodomain. Proc. Natl. Acad. Sci. U.S.A. 2013, 110,
19754−19759.
(26) Muller, S.; Brown, P. J. Epigenetic chemical probes. Clin.
̈
Pharmacol. Ther. 2012, 92, 689−693.
(27) Brand, M.; Measures, A. M.; Wilson, B. G.; Cortopassi, W. A.;
Alexander, R.; Hoss, M.; Hewings, D. S.; Rooney, T. P. C.; Paton, R. S.;
̈
Conway, S. J. Small molecules inhibitors of bromodomain-acetyl-lysine
interactions. ACS Chem. Biol. 2015, 10, 22−39.
(28) PFI-3: Selective chemical probe for SMARCA domains. http://
www.thesgc.org/chemical-probes/PFI-3 (accessed Feb 6, 2015).
(29) I-CBP112: A CREBBP/EP300-selective chemical probe. http://
www.thesgc.org/chemical-probes/ICBP112 (accessed Feb 6, 2015).
(30) SGC-CBP300: A CREBBP/EP300-selective chemical probe.
http://www.thesgc.org/chemical-probes/SGC-CBP30 (accessed Feb
6, 2015).
B.; Filippakopoulos, P.; Muller-Knapp, S.; Knapp, S.; Brennan, P. E.
̈
[1,2,4]Triazolo[4,3-a]phthalazines: inhibitors of diverse bromodo-
mains. J. Med. Chem. 2014, 57, 462−476.
(47) An structurally similar compound with BRD9 activity was also
identified, see: Guetzoyan, L.; Ingham, R. J.; Nikbin, N.; Rossignol, J.;
Wolling, M.; Baumert, M.; Burgess-Brown, N. A.; Strain-Damerell, C.
M.; Shrestha, L.; Brennan, P. E.; Fedorov, O.; Knapp, S.; Ley, S. V.
Machine-assisted synthesis of modulators of the histone reader BRD9
using flow methods of chemistry and frontal affinity chromatography.
MedChemComm 2014, 5, 540−546.
(31) Hay, D. A.; Fedorov, O.; Martin, S.; Singleton, D. C.; Tallant, C.;
Wells, C.; Picaud, S.; Philpott, M.; Monteiro, O. P.; Rogers, C. M.;
Conway, S. J.; Rooney, T. P. C.; Tumber, A.; Yapp, C.; Filippakopoulos,
P.; Bunnage, M. E.; Muller, S.; Knapp, S.; Schofield, C. J.; Brennan, P. E.
̈
Discovery and optimization of small-molecule ligands for the CBP/p300
bromodomains. J. Am. Chem. Soc. 2014, 136, 9308−9319.
(32) Borah, J. C.; Mujtaba, S.; Karakikes, I.; Zeng, L.; Muller, M.; Patel,
J.; Moshkina, N.; Morohashi, K.; Zhang, W.; Gerona-Navarro, G.;
Hajjar, R. J.; Zhou, M.-M. A small molecule binding to the co-activator
CREB-binding protein blocks apoptosis in cardiomyocytes. Chem. Biol.
2011, 18, 531−541.
(33) For inhibitors of CREBBP, EP300 and BRD4, see: Rooney, T. P.
C.; Filippakopoulos, P.; Fedorov, O.; Picaud, S.; Cortopassi, W. A.; Hay,
D. A.; Martin, S.; Tumber, A.; Rogers, C. M.; Philpott, M.; Wang, M.;
Thompson, A. L.; Heightman, T. D.; Pryde, D. C.; Cook, A.; Paton, R.
(48) During manuscript preparation, the Structural Genomics
Consortium disclosed the availability of LP99, a chemical probe for
BRD9 and BRD7 via their website. http://www.thesgc.org/node/9361
(accessed Feb 6, 2015).
(49) Bunnage, M. E.; Piatnitski Chekler, E. L.; Jones, L. H. Target
validation using chemical probes. Nat. Chem. Biol. 2013, 9, 195−199.
(50) Bamborough, P.; Diallo, H.; Goodacre, J. D.; Gordon, L.; Lewis,
A.; Seal, J. T.; Wilson, D. M.; Woodrow, M. D.; Chung, C. W. Fragment-
based discovery of bromodomain inhibitors part 2: optimization of
phenylisoxazole sulfonamides. J. Med. Chem. 2012, 55, 587−596.
(51) Chung, C. W.; Dean, A. W.; Woolvern, J. M.; Bamborough, P.
Fragment-based discovery of bromodomain inhibitors part 1: inhibitor
binding modes and implications for lead discovery. J. Med. Chem. 2012,
55, 576−586.
S.; Muller, S.; Knapp, S.; Brennan, P. E.; Conway, S. A series of potent
̈
CREBBP bromodomain ligands reveals an induced-fit pocket stabilized
by a cation-π interaction. Angew. Chem. Int. Ed. 2014, 53, 6126−6130.
(34) GSK2801: A selective chemical probe for BAZ2B/A bromodo-
mains. http://www.thesgc.org/chemical-probes/GSK2801 (accessed
Feb 6, 2015).
(52) On two of seven test occasions compound 17 showed pIC50 < 4.3
against BRD4 BD1. As such, the mean and standard deviation are
calculated from five of seven test occasions.
(35) Demont, E. H.; Bamborough, P.; Chung, C. W.; Craggs, P. D.;
Fallon, D.; Gordon, L. J.; Grandi, P.; Hobbs, C. I.; Hussain, J.; Jones, E.
J.; Le Gall, A.; Michon, A. M.; Mitchell, D. J.; Prinjha, R. K.; Roberts, A.
D.; Sheppard, R. J.; Watson, R. 1,3-Dimethyl benzimidazolones are
potent, selective inhibitors of the BRPF1 bromodomain. ACS Med.
Chem. Lett. 2014, 5, 1190−1195.
(36) Zeng, L.; Li, J.; Muller, M.; Yan, S.; Mujtaba, S.; Pan, C.; Wang, Z.;
Zhou, M. M. Selective small molecules blocking HIV-1 Tat and
coactivator PCAF association. J. Am. Chem. Soc. 2005, 127, 2376−2377.
(37) Ferguson, F. M.; Fedorov, O.; Chaikaud, A.; Philpott, M.; Muniz,
J. R. C.; Felletar, I.; von Delft, F.; Heightman, T.; Knapp, S.; Abell, C.;
Ciulli, A. Targeting low-drugability bromodomains: fragment based
screening and inhibitor design against the BAZ2B bromodomain. J. Med.
Chem. 2013, 56, 10183−10187.
(53) The acetyl lysine is superimposed from a BRDT BD1 structure,
see: Moriniere, J.; Rousseaux, S.; Steuerwald, U.; Soler-Lopez, M.;
̀
́
Curtet, S.; Vitte, A. L.; Govin, J.; Gaucher, J.; Sadoul, K.; Hart, D. J.;
Krijgsveld, J.; Khochbin, S.; Muller, C. W.; Petosa, C. Cooperative
̈
binding by acetylation marks on a histone tail by a single bromodomain.
Nature 2009, 461, 664−668.
(54) Gentile, G.; Bernasconi, G.; Pozzan, A.; Merlo, G.; Marzorati, P.;
Bamborough, P.; Bax, B.; Bridges, A.; Brough, C.; Carter, P.; Cutler, G.;
Neu, M.; Takada, M. Identification of 2-(4-pyridyl)thienopyridones as
GSK-3β inhibitors. Biol. Org. Med. Chem. Lett. 2011, 21, 4823−4827.
(55) Mathews, T. P.; Kennedy, A. J.; Kharel, P. C.; Kennedy, P. C.;
Nicoara, O.; Sunkara, M.; Morris, A. J.; Wamhoff, B. R.; Lynch, K. R.;
Macdonald, T. L. Discovery, biological evaluation, and structure-activity
relationship of amidine based sphingosine kinase inhibitors. J. Med.
Chem. 2010, 53, 2766−2778.
(38) Harner, M. J.; Chauder, B. A.; Phan, J.; Fesik, S. W. Fragment-
based screening of the bromodomain of ATAD2. J. Med. Chem. 2014,
57, 9687−9692.
(39) McKeown, M. R.; Shaw, D. L.; Fu, H.; Liu, S.; Xu, X.; Marineau, J.
J.; Huang, Y.; Zhang, X.; Buckley, D. L.; Kadam, A.; Zhang, Z.; Blacklow,
S. C.; Qi, J.; Zhang, W.; Bradner, J. E. Biased multicomponent reactions
to develop novel bromodomain inhibitors. J. Med. Chem. 2014, 57,
9019−9027.
(40) BAZ2-ICR: a chemical probe for BAZ2A/B bromdomains. OF-1:
A chemical probe for BRPF bromodomains. PFI-4: A chemical probe for
BRPF1B. NI-57: A chemical probe for BRPF bromodomains. http://
www.thesgc.org/chemical-probes/epigenetics (accessed Feb 6, 2015).
(41) Middeljans, E.; Wan, X.; Jansen, P. W.; Sharma, V.; Stunnenberg,
H. G.; Logie, C. SS18 Together with animal-specific factors defines
human BAF-type SWI/SNF complexes. PLoS One 2012, 7, e33834.
(56) Compound 44 showed pIC50 values of 5.1 0.03 against BRD2
BD1; 5.2 0.05 against BRD3 BD1; and 4.9 0.09 against BRDT BD1.
(57) Vollmuth, F.; Geyer, M. Interaction of propionylated and
butyrylated histone H3 lysine marks with BRD4 bromodomains. Angew.
Chem. Int. Ed. 2010, 49, 6768−6772.
(58) See Supporting Information for details.
(59) BROMOscan recombinant protein binding assays were carried
out at DiscoveRx. http://www.discoverx.com.
(60) Hongbin, S.; Jiangxin, L.; Jiahai, Z.; Weiqun, S.; Hongda, H.;
Chao, X.; Haiming, D.; Jihui, W. Biochem. Solution structure of BRD7
bromodomain and its interaction with acetylated peptides from histone
H3 and H4. Biophys. Res. Commun. 2007, 358, 435−441.
N
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(61) Young, R. J.; Green, D. V. S.; Luscombe, C. N.; Hill, A. Getting
physical in drug discovery II: The impact of chromatographic
hydrophobicity measurements and aromaticity. Drug Discovery Today
2011, 16, 822−830.
(62) See Supporting Information Figure S5 for a volcano plot
illustrating gene expression changes induced by I-BRD9 relative to a
DMSO control; Supporting Information Table S3 for all 579 genes
affected by I-BRD9 and not by I-BET151; and Supporting Information
Table S4 for all 193 genes affected by both I-BRD9 and I-BET151.
(63) C-type lectin-like receptor-1 (CLEC-1). For a role in the immune
response, see Sattler, S.; Reiche, D.; Sturtzel, C.; Karas, I.; Richter, S.;
Kalb, M. L.; Gregor, W.; Hofer, E. The human C-type lectin-like
receptor CLEC-1 is upregulated by TGF-ss and primarily localized in
the endoplasmic membrane compartment. Scand. J. Immunol. 2012, 75,
282−292.
(64) Dual specificity phosphatase 6 (DUSP6). For a role in cancer, see
Bagnyukova, T. V.; Restifo, D.; Beeharry, N.; Gabitova, L.; Li, T.;
Serebriiskii, I. G.; Golemis, E. A.; Astsaturov, I. DUSP6 regulates drug
sensitivity by modulating DNA damage response. Br. J. Cancer 2013,
109, 1063−1071.
(65) Feline sarcoma oncogene (FES); For a role in cancer, see Zhang,
S.; Chitu, V.; Stanley, E. R.; Elliott, B. E.; Greer, P. A. Fes tyrosine kinase
expression in the tumor niche correlates with enhanced tumor growth,
angiogenesis, circulating tumor cells, metastasis, and infiltrating
macrophages. Cancer Res. 2011, 71, 1465−1473.
(66) SAM domain, SH3 domain and nuclear localization signals 1
(SAMSN1); For a role in cancer, see Yan, Y.; Zhang, L.; Xu, T.; Zhou, J.;
Qin, R.; Chen, C.; Zou, Y.; Fu, D.; Hu, G.; Chen, J.; Lu, Y. SAMSN1 is
highly expressed and associated with a poor survival in glioblastoma
multiforme. PLoS One 2013, 8, e81905.
O
DOI: 10.1021/acs.jmedchem.5b00256
J. Med. Chem. XXXX, XXX, XXX−XXX