Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.5b00405

The bromodomain containing proteins TRIM24 (tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis, and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of N,N-dimethylbenzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC K_d = 31 nM and ITC K_d = 14 nM, respectively). With its excellent cellular potency (EC₅₀ = 50 nM) and favorable pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo.

Full text of DOI:10.1021/acs.jmedchem.5b00405

Article
pubs.acs.org/jmc
Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual
TRIM24-BRPF1 Bromodomain Inhibitor
Wylie S. Palmer,*,† Guillaume Poncet-Montange,‡ Gang Liu,† Alessia Petrocchi,† Naphtali Reyna,†
Govindan Subramanian,† Jay Theroff,† Anne Yau,† Maria Kost-Alimova,† Jennifer P. Bardenhagen,†
Elisabetta Leo,† Hannah E. Shepard,† Trang N. Tieu,† Xi Shi,† Yanai Zhan,† Shuping Zhao,†
Michelle C. Barton,§ Giulio Draetta,† Carlo Toniatti,† Philip Jones,† Mary Geck Do,†
and Jannik N. Andersen†
‡
†Institute for Applied Cancer Science, and Core for Biomolecular Structure and Function, The University of Texas MD Anderson
Cancer Center, 1881 East Road, Unit 1956, Houston, Texas 77054, United States
§Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, 1515 Holcombe
Boulevard, Houston, Texas 77030, United States
S
* Supporting Information
ABSTRACT: The bromodomain containing proteins
TRIM24 (tripartite motif containing protein 24) and BRPF1
(bromodomain and PHD finger containing protein 1) are
involved in the epigenetic regulation of gene expression and
have been implicated in human cancer. Overexpression of
TRIM24 correlates with poor patient prognosis, and BRPF1 is
a scaffolding protein required for the assembly of histone
acetyltransferase complexes, where the gene of MOZ
(monocytic leukemia zinc finger protein) was first identified
as a recurrent fusion partner in leukemia patients (8p11
chromosomal rearrangements). Here, we present the structure
guided development of a series of N,N-dimethyl-
benzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode
enabled the design of a potent and selective inhibitor 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1
(ITC Kd = 31 nM and ITC Kd = 14 nM, respectively). With its excellent cellular potency (EC50 = 50 nM) and favorable
pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1
bromodomain function in vitro and in vivo.
probes6,14−17 are now beginning to emerge, which should
help unravel their biological role in normal development and
INTRODUCTION
■
Bromodomains are protein interaction modules found within a
diverse set of chromatin-regulator proteins and specifically
recognize, or “read”, acetylated lysine (KAc) residues on the
human disease. Equally encouraging are inhibitors that have
now been identified for more difficult to drug bromodomains,18
such as BAZ2B19,20 and ATAD2A.21−23 Unlike the BET
histone tails of chromatin as well as KAc residues on other
proteins.1,2 Inhibitors of bromodomains, and in particular the
subfamily, which contains two bromodomains that primarily
drive their binding and therefore function, a large proportion of
the other BCPs contain auxiliary domains, many with diverse
and sometimes unknown functions. These multidomain
containing BCPs can also be part of multiprotein complexes.
Accordingly, the relative contribution of the bromodomain
within these complexes has yet to be elucidated. For example,
the bromodomain-PHD finger protein (BRPF) family acts as
scaffolding proteins to assemble complexes of MYST-family
histone acetyltransferases (HATs).24 BRPF1 is a subunit of the
BET (bromodomain and extra-terminal) subfamily, have gained
much attention with the development of selective chemical
probes3−6 and the advancement of several inhibitors into the
clinic for various oncology indications.7−10 Additionally,
targeting bromodomains is being explored for the treatment
of inflammatory, neurodegenerative, and cardiovascular dis-
eases.11,12 The development of this class of inhibitors has
demonstrated the potential utility of treating diseases by the
disruption of the protein−protein interaction network of
chromatin reader modules and is enabling pharmacological
studies into how bromodomain-containing proteins (BCPs)
regulate gene transcription and cell signaling.1,13
Special Issue: Epigenetics
Received: March 11, 2015
The functions of BCPs, other than BET bromodomains, are
much less well understood; however, selective chemical
© XXXX American Chemical Society
A
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Figure 1. Chemotypes for TRIM24 identified by three different hit-finding approaches: in silico virtual screening (1), acetyllysine mimetic
(highlighted in red) library building and SAR exploration (2−5), and HTS screening (5f and 5g). TRIM24 IC50 SEM are from at least three
determinations.
Figure 2. TRIM24 (PHD-bromo) X-ray complexes of initial virtual screening hit and lead templates. (A) Compound 1 bound to TRIM24 (1.9 Å
resolution) bromodomain (gray ribbon), PHD domain (black), N980 (cyan), and zinc (magenta) is depicted (PDB code 4YAB). (B) Acetyllysine
binding site with 1, lipophilic residues L922, A923, and F924 on the ZA loop, and V986 on the BC α-helix form an “LAF/V-shelf” (yellow sticks)
(PDB code 4YAB). (C) Complex of 3b (1.7 Å resolution) shown in two poses based upon partial occupancy, second pose (purple) in ZA channel
(PDB code 4YAD). (D) Complex of 5b (2.2 Å resolution) with arylsulfonamide interacting with LAF/V-shelf (PDB code 4YAT).
monocytic leukemic zinc finger (MOZ) complex whose
translocations are associated with an aggressive form of acute
myeloid leukemia.25 The BRPF1 protein contains multiple
conserved domains involved in gene transcription, chromatin
binding, and remodeling. The BRPF1 bromodomain has been
demonstrated to bind to several histone peptides, including
H2AK5ac, H4K12ac, H4K8ac, H4K5ac, and H3K14ac; knowl-
edge of the importance of these observations and the biological
function of the BRPF1 bromodomain is largely unknown.26
The tripartite motif containing proteins (TRIMs) are a large
family of proteins whose structures are characterized by a highly
conserved sequence of domains in the N-terminal region:
RING domain, B-box zinc-fingers, and a coiled-coil region (also
collectively known as an RBCC domain).27 Many functions
have been ascribed to TRIM proteins, including their ability to
act as E3 ligases for ubiquitin and small-ubiquitin-like modifier
(SUMO) activities.28,29 A subgroup of TRIM proteins
(TRIM24, -28, and -33) also contain a dual C-terminal plant
homeodomain and bromodomain motif (PHD-bromo), which
makes these interesting targets for exploring epigenetic
regulation through bromodomain inhibition.30 TRIM24 was
originally identified as a transcriptional intermediary factor
B
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(TIF1α) which acts as a co-repressor of the retinoic acid
receptor that interacts with multiple nuclear receptors via an
LXXLL motif.31 The C-terminal PHD-bromo domain of
TRIM24 acts as a dual “reader” of unmodified H3K4 and
acetylated H3K23 marks within the same histone tail;32
additionally, TRIM24 can also bind to non-histone KAc
marks on other proteins, including p53.33 Genetic knock-
down of TRIM24 in cancer cells results in antiproliferative
phenotypes, and overexpression of TRIM24 has also been
linked to poor prognosis for several cancers, including
breast,32,34 head and neck,35 non-small-cell lung,36 hepatocel-
lular,37 and glioblastoma.38 Because of the interest in TRIM24
here at MD Anderson and the druggability of BCPs,18 we set
out to develop a potent (cellular EC50 < 100 nM) and selective
TRIM24 chemical probe in order to interrogate the role of the
bromodomain in human disease.
We explored a variety of template replacements for the
indolinone and constructed a KAc mimetic library of 5,6- and
6,6-fused heterocycles containing a sulfonamide linkage to an
aryl group. In contrast to the indolinone sulfonamide 2 which
lost potency (IC50 = 17 μM), both the tetrahydroisoquinolin-2-
one 3 and the N,N-dimethylbenzimidazol-2-one 5 scaffolds
were identified as viable chemotypes with micromolar
inhibition for TRIM24 (Figure 1). A summary of structure−
activity relationships (SARs) for the scaffolds is shown in Table
1. Both 3a and 5a have comparable potencies of 4.8 and 4.9
Table 1. Structure−Activity Relationship for Sulfonamides
3a−e and 5a−g
a
compd
R
X
TRIM-24 IC50 (μM)
3a
3b
3c
3d
3e
5a
5b
5c
5d
5e
5f
4-SMe
H
4.8 2.9 (5)
2.5 0.56 (3)
3.7 3.2 (3)
2.8 1.3 (6)
36 24 (9)
2,4-di-OMe
4-O-isobutyl
4-cyclohexyl
H
H
H
RESULTS AND DISCUSSION
■
H
Identification of Scaffolds. Since there were no known
small-molecule TRIM24 inhibitors39 at the start of our study,
we explored three different hit identification approaches for
finding novel scaffolds: virtual in silico high-throughput
screening (HTS), construction of a focused KAc mimetic
library, and a traditional small-molecule library HTS. Three
different chemotypes, as shown in Figure 1, were identified
from this diverse approach: an N-methylindolinone (1 and 2),
the N-methyltetrahydroquinolin-2-one (3 and 4), and the N,N-
dimethylbenzimidazolone (5).14
OMe
H
4-SMe
4.9 0.82 (5)
9.3 5.1 (3)
23 2.6 (3)
4.0 1.3 (5)
1.7 0.69 (5)
10 2.7 (6)
1.5 0.79 (11)
4-OMe
H
H
H
4-O-isobutyl
4-cyclohexyl
3-CN
H
H
OMe
5g
H
O(4-MeO-Ph)
a
Mean SEM (number of measurements).
The in silico virtual screening was performed using the
“Virtual Screening Workflow” implemented within Schro-
dinger’s Maestro modeling software suite and the reported
TRIM24 X-ray crystal structures (PDB codes 3O33 and 3O34,
apo and peptide substrate-bound complexes, respectively).
Commercially available compounds within the ZINC library40
that bound to the KAc binding pocket, with a constraint of
making at least one hydrogen bond (H-bond) interaction with
N980 side chain, were docked, scored, and ranked. After visual
inspection of the top ranked poses and applying appropriate
physicochemical property filters, the highest scoring hits were
evaluated for binding to the bromodomain of TRIM24 using a
biochemical bromodomain-H3K23Ac peptide displacement
assay (AlphaScreen).41 Fragment 1 (Figure 1) was identified
as a TRIM24 inhibitor with IC50 = 8.5 μM, and subsequent X-
ray cocrystal structure determination of 1 with TRIM24(PHD-
bromo) (Figure 2A) validated the initially proposed docking
hypothesis. Compound 1 was verified to be a KAc mimetic,
whereby the carbonyl of the methyloxindole moiety interacts
with the conserved N980 side chain through an H-bond
(Figure 2B). Although 1 was ligand efficient (LE = 0.41), the
linked-thiazole group is coplanar with the oxindole template
and is primarily solvent exposed, which made further
modifications unlikely to interact with the protein. We sought
to improve potency by interacting with the lipophilic sequence
of residues 922-LAF-924 on the ZA-loop and V986 on the B-
loop. This LAF/V sequence highlighted in yellow (Figure 2B)
forms a lipophilic shelf, or “LAF/V-shelf”, akin to the “WPF”
shelf of the BET-family of bromodomains (BRD2−4 and
BRDT) whereby ligand interactions within this region have
been demonstrated to be a way to improve potency for a wide
variety of inhibitors.42,43 In order to vector off orthogonally
from the template and interact with the LAF/V-shelf, we
reasoned that a sulfonamide linkage to an aromatic substituent,
as in PFI-1,5 would be a way to achieve this.
μM, respectively, and were reasonable starting points from
which to optimize. Interestingly, the regioisomeric derivative 4,
which has the same sulfonamide attachment point relative to
the KAc mimetic (red) compared to 5a, was less potent with an
IC50 = 14 μM. Both the template and the positioning of the
sulfonamide-linker appeared to be important for obtaining
potency; therefore, evaluation of the relative binding modes
between the two templates would be important for selecting a
series to progress.
The TRIM24 cocrystal structure complexes of 3b (Figure
2C) and 5b (Figure 2D) informed on the importance of the
relative regiochemistry of the sulfonamide-linker attachment
and the role in defining how the aryl group interacts with the
lipophilic LAF/V-shelf. Both templates interact with N980 and
bind similarly within the KAc binding site; however, the
positioning of the aryl group was remarkably different between
the two compounds. The complex with 3b also revealed the
conformational mobility of the arylsulfonamide; two poses of
the aryl group were observed within the same chain of the
complex based upon partial occupancy. The relative atomic
densities suggested an approximately 60:40 ratio between the
two conformations (Figure 2C) with the aryl group (green)
only partially interacting with V986 of the BC loop and in the
less populated conformation (purple) directing the aryl group
toward the ZA channel. In contrast, the complex of 5b showed
a single conformation of the arylsulfonamide, which makes
lipophilic contacts with both A923 and F924 of the LAF/V-
shelf (Figure 2D). Another attractive feature of scaffold 5
relative to 3 is the symmetrical nature of the two N-methyl
groups, which allows the scaffold to always place a methyl
group deep into the pocket. Scaffold 5 was also attractive
synthetically, as it allowed for a more rapid development of this
series relative to the regiochemical control necessary for the
prosecution of 3.
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Figure 3. New unexpected binding mode enabled the design of interactions with the “upper pocket”. (A) Cocrystal structure of 5g (2.3 Å resolution)
depicting a “flipped” binding mode with the aryl ether group now interacting with the LAF/V-shelf (PDB code 4YAX). (B, C) Cocrystal structure of
7b (1.5 Å resolution) depicted in two binding conformations in the asymmetric unit (PDB code 4YBM): (B) chain A, showing the benzyl group
filling the “upper pocket”; (C) chain B, with the benzyl group occupying the ZA channel. Depicted in blue are three residues of chain A TRIM24
protein with L930′ occupying the “upper pocket”. (D) Cocrystal structure of 7g (1.8 Å resolution) with the isobutyl ether group occupying the
“upper pocket” (PDB code 4YBS). (E) Cocrystal stucture of 7l (1.8 Å resolution) with the 3-tetrahydropyranyl group H-bonded to a conserved
water in the “upper pocket” and the imidazole H-bonded to a water that is π-stacked with the aryl ether group (PDB code 4YBT). (F) 2Fo − Fc map
of 7l and the π-stacked water molecule contoured at 1.0σ (PDB code 4YBT).
The positioning of the aryl group in the complex of 5b
looked ideal for targeting the lipophilic region just beyond the
LAF/V-shelf or “upper pocket” (depicted as a yellow surface in
Figure 2D). The “upper pocket” is defined by lipophilic
residues A989 and M920, which we thought we could improve
potency by exploiting this region; therefore, we evaluated a
series of para-substituted aryl derivatives for both templates
(Table 1). The SAR with substituents in the para-position
showed very little improvement going from the thiomethyl
group of 3a and 5a (IC50 of 4.8 and 4.9 μM, respectively) to the
much larger cyclohexyl group of 3d and 5e (IC50 of 2.8 and 1.7
μM, respectively). The poor ligand binding efficiency of adding
the larger cyclohexyl group (LE = 0.28 for 5e) was
discouraging. After evaluating a library of aryl sulfonamides,
there did not appear to be a likelihood of achieving the desired
nanomolar TRIM24 potency through this approach.
Concurrent with the lead optimization of this series, an HTS
screen was performed at the Texas Screening Alliance for
Cancer Therapeutics with a 61,000 compound library using the
same AlphaScreen conditions we employed for our in vitro
assay. Derivative 5f, containing a 5-methoxy subsituent, was
identified as a hit and verified as a modest TRIM24 inhibitor
(IC50 = 10 μM). This is in contrast to 3e with IC50 = 36 μM.
Knowledge of the binding mode of the unsubstituted series, as
previously discussed, made substitution at the 5-position of the
dimethylbenzimidazolone a nonobvious vector from which to
explore further changes. We subsequently performed a hit
expansion of 4-arylsulfonamide-5-substituted compounds and
identified 5g as a promising new lead with IC50 = 1.5 μM.
New Binding Mode Allowed for Significant Improve-
ments in Potency. The cocrystal structure of 5g with
TRIM24 quite surprisingly revealed a new “flipped’ binding
mode in which the aryl ether group now interacted with the
LAF/V-shelf and the arylsulfonamide group was oriented
towards the conserved asparagine (Figure 3A). The aryl ether
ring of 5g makes lipophilic contacts with A923 (3.75 Å between
the Cα and the center of the aryl ring), while the
phenylsulfonamide makes lipophilic contact with V986. The
lone pairs of the oxygen of the methoxy group directed towards
L922 would appear to be an unfavorable interaction and may
influence the positioning of this residue. The meta position
appeared to be more favorable, as it allows more room for
additional derivatizations, directing groups towards the “upper
pocket”. Although the ligand efficiency of 5g was lower (LE =
0.26), we recognized that this unexpected binding mode offered
a new opportunity to optimize interactions with the LAF/V-
shelf and target the “upper pocket” in order to improve
potencies.
D
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Table 2. Structure−Activity Relationships for Improved in Vitro Cellular Potencies and Solubility
a
ab
,
c
compd
R
X
TRIM-24 IC50 (μM)
cell EC50 (μM)
solubility (μM)
6
benzyl
benzyl
benzyl
benzyl
Et
A
A
B
2.0 0.93 (6)
>33 (4)
nt
7a
7b
7c
7d
7e
7f
0.22 0.041 (5)
0.14 0.056 (29)
0.27 0.094 (6)
0.18 0.045 (5)
0.043 0.0037 (6)
0.053 0.015 (9)
0.057 0.016 (65)
0.16 0.055 (4)
0.11 0.023 (7)
2.4 1.6 (11)
6.2 2.0 (4)
59
0.6
nt
3.9 0.87 (13)
4.9 1.3 (4)
2.3 1.1 (4)
0.83 0.17 (7)
0.95 0.31 (9)
1.3 0.27 (53)
3.0 0.81 (5)
5.0 0.53 (7)
>36 (5)
C
C
B
74
1.1
62
87
nt
n-propyl
n-propyl
C
C
C
D
H
C
A
B
7g
7h
7i
CH2CH(Me)2
n-butyl
CH2CH(Me)2
CH2CH(Me)2
(CH2)3OMe
0.78
2.6
100
77
66
7j
7k
7l
0.13 0.035 (5)
0.10 0.038 (5)
2.7 0.36 (6)
1.9 0.41 (5)
3.2 0.44 (2)
CH2(tetrahydrofuran-3-yl)
7m
(CH2)4NMe2
0.27 0.12 (4)
a
b
c
Mean SEM (number of measurements). TRIM24 AlphaLisa assay in HeLa cells. Kinetic solubility in phosphate buffer, pH = 7.0. nt = not
tested.
We evaluated the SAR of a series of aryl ether substitutions
focused on improving both the potency and the cell shift, since
this would best reflect improvements in overall physical
properties of the molecule.
to improve the potency and assess the role of the sulfonamide
functionality on enhancing the solubility of the molecule (Table
2). In order to develop a potent cellular molecular probe,
compounds were evaluated in an AlphaLisa cellular target
engagement assay, which measured the displacement of
ectopically expressed TRIM24(PHD-bromo) from endogenous
histone H3 in HeLa cells. The effect of substituents on the
kinetic solubility of the compound at pH 7.0 was also
monitored. The larger p-benzyl substituent of 6 (IC50 = 2.0
μM) did not improve upon the potency of 5g and had no
cellular activity; however, the meta-regioisomer 7a was 10-fold
In the presence of 7b, TRIM24 cocrystallized with two
molecules in the asymmetric unit. In the first TRIM24 protein
(chain A), the 1.5 Å resolution structure confirmed that the
benzyl ether targeted the “upper pocket” as we initially
envisioned (Figure 3B) and that the conformation of the
arylsulfonamide now undergoes an offset π−π interaction with
the aryl ether ring, positioning the other aryl group to interact
with the LAF/V-shelf (3.8 Å from the methoxy group of the
arylsulfonamide and the center of aryl ether ring). Conversely,
in the second TRIM24 protein (chain B), the complex revealed
a second conformation with 7b bound, such that the benzyl
ether occupies the ZA channel (Figure 3C). Interestingly,
although the ligand was not occupying the “upper pocket” in
this alternative binding conformation, residue L930′ from chain
A involved in the crystal packing does occupy the “upper
pocket”. The excellent density and high resolution of the
leucine showed an ideal and tight fit with the lipophilic residues
of the “upper pocket”. This observation suggested that alkyl
ether substituents could be more ligand efficient replacements
for the benzyl group. Therefore, a series of alkyl ether
substituents were evaluated, and we found that a three-carbon
atom length appeared to be optimal for potency, exemplified by
the n-propyl (7e,f) and isobutyl (7g) analogues, which
improved intrinsic potencies by 3- to 5-fold with respect to
7a. Improved ligand efficiency was also achieved comparing 7e
(IC50 = 0.043 μM, LE = 0.27) with 7b (IC50 = 0.14 μM, LE =
0.23).
more potent than 6 with IC50 = 0.22 μM and cellular EC50
=
6.2 μM, thereby confirming the meta position to be the best
vector for further exploration.
We found that a wide variety of aryl- and heteroaromatic
sulfonamides were tolerated and had minor effects on the
intrinsic potency of the molecules. The sulfonamide appears to
play a structural role in positioning the aryl ether group, since
few contacts with the protein are made by this functional group.
In fact, this moiety could be used to modify physical properties,
such as solubility and permeability, without adversely affecting
the binding affinity of the molecule. For example, imidazole 7a
had an improved aqueous solubility of 59 μM compared with
arylsulfonamide 7b (0.6 μM) while maintaining similar in vitro
potencies of 0.22 versus 0.14 μM, respectively. In this example,
the improved solubility was accompanied by a reduction in
apparent permeability (Papp), as determined by a Caco-2
permeability assay; compound flux from the apical to
basolateral side was determined to be 4.0 × 10−6 cm·s−1 for
7a versus 21 × 10−6 cm·s−1 for 7b. The cellular EC50 of 6.2 and
3.9 μM for 7a and 7b, respectively, resulted in identical 28-fold
cell shifts for both compounds. For further development, we
The TRIM24 cocrystal complex of 7g (Figure 3D) nicely
recapitulated the leucine interactions previously observed in
Figure 3C. Since the aryl ether ring appeared to be driving the
E
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Table 3. Structure−Activity Relationship of Disubstituted Aryl Ethers
a
ab
,
compd
Y
n
Z
TRIM-24 IC50 (μM)
cell EC50 (μM)
cell shift
8a
8b
8c
8d
8e
8f
A
A
A
B
A
A
B
B
B
6
6
6
6
5
4
4
3
4
NHBoc
NH2
OH
0.16 0.026 (6)
5.5 0.32 (5)
34
17
30
13
10
15
7
0.010 0.0025 (14)
0.060 0.017 (8)
0.013 0.0020 (15)
0.011 0.0021 (6)
0.0079 0.0029 (8)
0.0083 0.0026 (4)
0.013 0.0048 (4)
0.17 0.069 (11)
1.8 0.40 (8)
NH2
NH2
NH2
NH2
NH2
NMe2
0.17 0.059 (18)
0.11 0.023 (5)
0.12 0.0032 (8)
0.059 0.011 (4)
0.12 0.0066 (4)
0.050 0.013 (14)
8g
8h
8i
9
0.0076 0.0029 (16)
7
a
b
Mean SEM (number of measurements). TRIM24 AlphaLisa assay in HeLa cells.
Figure 4. Selectivity profile of 8i against a panel of 32 bromodomains (DiscoveRx) at 1 μM test concentration. The larger is the circle size, the
greater is the inhibition: TRIM24(PHD-bromo) in blue; BRPF1−3, BAZ2B, and TAF1(2) in red; 26 bromodomains with percent of control of >35
in green. Bromodomains in gray are not tested. See also Table S2, Supporting Information.
binding affinity and the interactions with the “upper pocket”
appeared to be optimized, we sought to minimize or completely
remove the sulfonamide group. However, replacement of the
arylsulfonamide with a smaller cyclopropyl group as in 7i (IC50
= 0.11 μM) resulted in a 3-fold loss in cellular potency (EC50 =
5.0 μM) relative to 7g and complete removal of the
sulfonamide (7j, IC50 = 2.4 μM) resulted in a 44-fold loss in
intrinsic potency and no cellular activity. Other polar functional
F
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groups on the alkyl ether chain were also well tolerated, such as
the methyl ether (7k, IC50 = 0.13 μM), tetrahydrofuran (7l,
IC50 = 0.10 μM), and dimethylamine (7m, IC50 = 0.27 μM)
providing good solubilities; however, cellular potencies were
weaker with respect to 7e.
The dimethylamine analogue 8i (IACS-9571) is one of the
most potent TRIM24 inhibitors within this series, with an IC50
= 7.6 nM in the biochemical AlphaScreen and a cellular EC50
=
50 nM in the AlphaLisa cellular target engagement assay. 8i has
excellent solubility (76 μM) and a low cell shift (7-fold).
Selectivity profiling of 8i against a panel of 32 bromodomains
(DiscoveRx) at 1 μM confirmed the TRIM24 interaction
specificity and also revealed strong interactions with family IV
bromodomains, BRPF1−3 (Figure 4 and Table S2, Supporting
Information).44 Subsequent dose−response determinations
demonstrated 8i to be a selective dual TRIM24/BRPF1
inhibitor (Kd = 1.3/2.1 nM) with 9- and 21-fold selectivity
against BRPF2 and BRPF3, respectively (Table 4). Much
Although the sulfonamide group makes minimal contact with
the protein, the SAR and structural data demonstrate that the
arylsulfonamide is an important functional group that helps fill
the much more open and wider binding pocket of TRIM24
compared to the smaller more defined pocket of the BET
family bromodomains. This is also reflected by the lower
druggability scores (Dscore) calculated for TRIM24 (0.67)
compared to those for the BET family (0.86−0.94).18
Furthermore, the cocrystal complex of 7l with TRIM24 (Figure
3E) also supports the notion that the sulfonamide plays an
important structural role in positioning the aryl ether ring in
order to direct substituents into the “upper pocket”. The basic
nitrogen of the imidazole is H-bonded to a structural water
molecule which is positioned directly above the aryl ether
group. A different orientation of 7l (Figure 3F) shows the
folded U-shape conformation of the molecule, and the ligand
density map clearly demonstrates this solvent exposed water
undergoing a π-stacking interaction with the aryl ether. The
importance of the sulfonamide stacking interaction with the
aryl ether may also explain the observed loss of potency for 7j
which has no sulfonamide group. It is interesting to note that a
similar intramolecular aromatic stacking interaction was also an
important feature in the design of a potent BAZ2A/B molecular
probe.20
Targeting the ZA-Channel for Improved Cellular
Potencies. Both 7f and 7g displayed the best balance between
potency and solubility reflected by their submicromolar cell
potencies (EC50 of 0.95 and 1.3 μM, respectively); however,
they exhibited large cell shifts of 18- and 23-fold, respectively.
We sought to further improve cell potencies by improving the
intrinsic potencies and/or lowering the cell shift. Interactions of
the n-propyl ether group with the “upper pocket” already
appeared optimal, so we sought another region of the protein
to interact with, in order to further improve potencies. The ZA
channel of TRIM24 offered a second site to pick up further
interactions. The two different bound conformations of the
benzyl group in the 7b cocrystal (Figure 3B and Figure 3C)
inspired the strategic design of a bidirectional aryl ether,
whereby one vector was used to interact with D926 in the ZA
channel, while the other vector maintained the interaction with
the “upper pocket”.
Table 4. Binding Affinity Data for 8i
a
bromodomain
Kd (nM)
TRIM24(PHD-bromo)
BRPF1
1.3
2.1
b
BRPF2
12
BRPF3
27
BAZ2B
400
1800
>10000
TAF1(domain 2)
BRD4(domains 1, 2)
a
Mean of four determinations in bromoELECT recombinant protein
binding assays performed at DiscoveRx (http://www.discoverx.com).
Also known as BRD1.
b
weaker affinities with BAZ2B (Kd = 400 nM) and the second
domain of TAF1 (Kd = 1800 nM) were also observed.
Importantly, 8i does not interact with the BET subfamily of
bromodomains, displaying greater than 7700-fold selectivity
versus BRD4(1,2) relative to TRIM24. No interactions were
detected against 26 other bromodomains (green circles) at 1
μM concentration of 8i. The dual TRIM24/BRPF1 inhibitor
profile of 8i was also confirmed by ITC resulting in Kd values of
31 and 14 nM, respectively (Figure 5). The cocrystal structure
of 8i with TRIM24(PHD-bromo) ultimately confirmed the
bidirectional design principle we envisioned, with the n-propyl
ether occupying the “upper pocket” as well as the salt-bridge
interaction of the dimethylamine group with D926 in the ZA
channel (Figure 6). The dual TRIM24/BRPF1 potency of 8i is
not surprising since potent BRPF1 dimethylbenzimidazolone
inhibitors have been described,14 and BRPF1 also contains an
acidic residue (E655) in the ZA channel which the dimethyl-
amine could potentially interact with.
The pharmacokinetics of 8i was studied in female CD1 mice,
and after iv administration of a 1 mg/kg dose, a moderate
clearance of 43 mL·min−1·kg−1 was observed, representing
approximately half the rate of hepatic blood flow. The terminal
iv half-life was 0.7 h, and after oral dosing of a 10 mg/kg dose,
the bioavailability of 8i was 29%, making the inhibitor useful for
in vivo studies.
A series of bidirectional aryl ethers were then evaluated, and
their SARs are shown in Table 3. The substantial improvement
in potency (16-fold) going from the Boc-protected amine 8a
(IC50 = 160 nM) to the basic amine analogue 8b (IC50 = 10
nM), as well as the excellent cellular potency of 8b (EC50 = 170
nM), suggested that the bidirectional design principles were
realized. The importance of the basic amine is also further
demonstrated by the alcohol analogue 8c which is 11-fold less
potent in cells compared to 8b. Shortening of the chain length
to four carbons, as in 8f, improved both the in vitro and cellular
potencies (IC50 of 7.9 and 120 nM, respectively) as well as the
observed cell shift (15-fold). We also found that with the use of
the basic amine on the bidirectional molecules, the imidazole
group was no longer needed for solubility. In fact, the
dimethoxyphenylsulfonamide 8g, with a kinetic solubility of
42 μM (at pH 7.0), displayed a lower cell shift compared to the
imidazole 8f (7-fold versus 15-fold, respectively).
CONCLUSIONS
■
Iterative use of structural biology and medicinal chemistry
optimization allowed us to develop a series of highly potent and
selective dual inhibitors of TRIM24 and BRPF1 starting from a
diverse hit-finding approach that utilized in silico virtual and
small-molecule HTS screens as well as KAc mimetic library
design. The unique binding mode observed while investigating
new substitution patterns on the benzimidazolone template
enabled the design of potent and selective dual inhibitors. Also,
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Article
Figure 5. ITC data for 8i: (A) release of energy for the titration with TRIM24(PHD-bromo); (B) release of energy for the titration with BRPF1B;
(C) integrated binding heats for (PHD-Bromo), Kd = 31 nM; (D) integrated binding heats for BRPF1B, Kd = 14 nM.
utilization of a TRIM24 cellular target engagement assay and
targeting low cell shifts allowed for the optimization of
compounds that displayed the best physicochemical properties.
The excellent cellular potency, bromodomain selectivity, and
desirable physical properties displayed by 8i provide an ideal
chemical probe to investigate the biological and pharmaco-
logical role of TRIM24 and/or BRPF1 bromodomain inhibition
in vitro and in vivo. The biological activity of 8i and related
compounds will be reported in a subsequent manuscript.
CHEMISTRY
■
The sulfonamides 5a−e were synthesized from aniline 9 using
the corresponding sulfonyl chlorides (Scheme 1). The 5-
phenylether derivatives 6 and 7a−f were synthesized from
either bromide 10a or 10b and a phenol, through either one of
two different Ullmann coupling methods45 to give intermedi-
ates 11a−d. Use of the more electrophilic trifluoroacetamide
10b and milder Ullmann coupling conditions46 with copper
Figure 6. Cocrystal structure of 8i with TRIM24(PHD-bromo) (1.8 Å
resolution) showing the n-propyl ether group occupying the “upper
iodide, N,N-dimethylglycine, and cesium carbonate in dioxane
pocket” and the dimethylamino group forming a salt bridge to D926 in
at 80 °C was the preferred method for generating intermediates
the ZA channel (PDB code 4YC9).
11a−d. An alternative and more convergent route was utilized
a
Scheme 1. Synthesis of Sulfonamides 5a−e, 6, 7a−f
a
Reagents and conditions: (a) sulfonyl chloride, pyr, DCM; (b) Br2, AcOH, CHCl3, 0 °C; (c) phenol intermediate, CuCl, quinolin-8-ol, potassium
phosphate, diglyme, 120−130 °C; (d) TFA2O, DMAP, Et3N, DCM; (e) phenol intermediate, CuI, N,N-dimethylglycine, Cs2CO3, dioxane, 80 °C;
(f) sulfonyl chloride, pyr, DCM.
H
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Article
for the synthesis of 7g−l via alkylation of the intermediate
phenol 12 (Scheme 2). The bidirectional derivatives 8a−c were
EXPERIMENTAL SECTION
■
Synthetic Methods. Column chromatography was performed on
a Biotage system using Biotage SNAP columns with Biotage KP-Sil
silica or Biotage Zip Si columns with Biotage KP-Sil silica or a
Teledyne ISCO system with RediSep Rf normal phase silica cartridges
(unless otherwise stated). All NMR spectra were recorded on Bruker
instruments operating at 300, 500, or 600 MHz. NMR spectra were
obtained as CDCl3, CD3OD, D2O, (CD3)2SO, (CD3)2CO, C6D6, or
CD3CN solutions (reported in ppm), using tetramethylsilane (0.00
ppm) or residual solvent (CDCl3, 7.26 ppm; CD3OD, 3.31 ppm; D2O,
4.79 ppm; (CD3)2SO, 2.50 ppm; (CD3)2CO, 2.05 ppm; C6D6, 7.16
ppm; CD3CN, 1.94 ppm) as the reference standard. When peak
multiplicities are reported, the following abbreviations are used: s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br-s
(broadened singlet), dd (doublet of doublets), dt (doublet of triplets).
Coupling constants, when reported, are reported in hertz (Hz). All
temperatures are reported in °C. The purity of all final compounds was
>95% and was confirmed by LC/MS analysis. Low-resolution mass
spectral data were obtained on either a Waters H class UPLC with a
Waters Acquity UPLC BEH C18 1.7 μm, 2.1 mm × 50 mm column,
UV detection between 200 and 400 nm, evaporating light scattering
detection, and a SQ detector mass spectrometer with ESI ionization or
a Water I class UPLC with a Waters Acquity UPLC CSH C18 1.7 μm,
2.1 mm × 50 mm column, UV detection at 254 and 290 nm,
evaporating light scattering detection, and a SQ detector 2 mass
spectrometer with ESI ionization. Preparative HPLC was performed
using a Waters Autopurify system with a Waters Xbridge Prep C18 5
μm OBD, 19 mm × 150 mm or 50 mm × 100 mm column and SQ
detector mass spectrometer with ESI ionization. Reagents were
purchased from commercial suppliers such as Sigma-Aldrich, Alfa
Aesar, TCI, or Acros and were used without further purification unless
otherwise indicated. Anhydrous solvents (e.g., THF, DMF, DMA,
DMSO, MeOH, DCM, toluene) were purchased from Sigma-Aldrich
and used directly. The following compounds were obtained from
commercial vendors: 1, Enamine catalog no. Z118580532; 3b, Life
Chemicals catalog no. F2278-0147; 3c, Life Chemicals catalog no.
F2278-0177; 5f, ChemDiv catalog no. G433-0348; 5g, ChemDiv
catalog no. G433-0914.
a
Scheme 2. Synthesis of Sulfonamides 7g−l
a
Reagents and conditions: (a) BBr3, DCM, −78 °C; (b) R-Br, K2CO3,
DMF; (c) sulfonyl chloride, pyr, DCM.
synthesized through iterative alkylation of phloroglucinol to
give the bifunctionalized phenols 14a and 14b, then Ullmann
coupling with 10b to give intermediates 15a and 15b, followed
by sulfonamide formation to give 8a and 8c (Scheme 3).
a
Scheme 3. Synthesis of Derivatives 8a−c
General Procedure for the Synthesis of 2, 3a, 3d, and 5a−e
(Method A: Sulfonamide Coupling). The following commercially
available intermediates were used: 5-amino-1-methyl-1,3-dihydro-2H-
indol-2-one (Combi-Blocks catalog no. ST-1422 for 2); 6-amino-1-
methyl-3,4-dihydroquinolin-2(1H)-one (Enamine catalog no. EN300-
69848 for 3a and 3d); 5-amino-1,3-dimethyl-1H-benzo[d]imidazol-
2(3H)-one (Enamine catalog no. EN300-59698 for 5a−e) (0.1 mmol)
in DCM (1 mL), treated with the sulfonyl chloride (0.1−0.20 mmol)
and pyridine (20 μL, 0.2 mmol). The reaction mixture was stirred for
2−24 h at ambient temp, then concentrated and purified by mass-
directed preparative HPLC to give sulfonamides 2, 3a, 3d, and 5a−e.
N-(1-Methyl-2-oxo-2,3-dihydro-1H-indol-5-yl)-4-
(methylsulfanyl)benzene-1-sulfonamide (2). 1H NMR (600
MHz, DMSO-d6) δ 9.95 (br-s, 1H), 7.58 (dt, J = 8.6, 2.0 Hz, 2H),
7.35 (dt, J = 8.6, 2.0 Hz, 2H), 7.01 (s, 1H), 6.93 (dd, J = 8.3, 2.1 Hz,
a
Reagents and conditions: (a) 1-bromopropane, K2CO3, DMF; (b)
Z(CH2)6Br, K2CO3, DMF; (c) 10b, CuI, N,N-dimethylglycine,
Cs2CO3, dioxane, 80 °C; (d) 1-methyl-1H-imidazole-4-sulfonyl
chloride, pyr, DCM; (e) TFA, DCM.
Alternatively, the advanced phenol intermediate 16 was
alkylated and transformed in a similar manner to give amines
8d−h (Scheme 4). Reductive alkylation of 8h using form-
aldehyde and sodium cyanoborohydride gave the dimethyl-
amine 8i.
a
Scheme 4. Synthesis of Amines 8d−i
a
Reagents and conditions: (a) Z(CH2)nBr, K2CO3, DMF; (b) sulfonyl chloride, pyr, DCM; (c) TFA, DCM; (d) hydrazine hydrate, MeOH, 80 °C;
(e) formaldehyde, AcOH, NaBH(OAc)3, Et3N, MeOH.
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Article
1H), 6.82 (d, J = 8.3 Hz, 1H), 3.48 (s, 2H), 3.03 (s, 3H), 2.99 (s, 3H).
MS (ESI) m/z 349 [M + H]+.
mmol). The mixture was stirred at rt for 48 h. The reaction mixture
was diluted with water and the resulting suspension was filtered and
collected solids were dried to give 2-(1-methyl-2-oxo-1,2,3,4-
tetrahydroquinolin-7-yl)isoindoline-1,3-dione as a white solid (120
mg, 100%). MS (ESI) m/z 307 [M + H]+.
N-(1-Methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)-4-
(methylsulfanyl)benzene-1-sulfonamide (3a). 23% yield. 1H
NMR (500 MHz, DMSO-d6) δ 10.01 (s, 1H), 7.95 (q, J = 7 Hz,
4H), 6.88 (s, 1H), 6.80 (q, J = 5 Hz, 1H), 6.67 (d, J = 6 Hz, 1H), 3.33
(s, 3H), 3.15 (s, 3H), 2.76 (t, J = 6 Hz, 2H), 2.37 (t, J = 8 Hz, 2H).
MS (ESI) m/z 363 [M + H]+.
4-Cyclohexyl-N-(1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-
6-yl)benzene-1-sulfonamide (3d). 81% yield. 1H NMR (600 MHz,
DMSO-d6) δ 10.10 (s, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.39 (d, J = 8.3
Hz, 2H), 6.99−6.91 (m, 3H), 3.15 (s, 3H), 2.75 (t, J = 6.9 Hz, 2H),
2.59−2.53 (m, 1H), 2.46 (t, J = 6.9 Hz, 2H), 1.81−1.72 (m, 4H),
1.73−1.64 (m, 1H), 1.43−1.29 (m, 4H), 1.27−1.15 (m, 1H). MS
(ESI) m/z 399 [M + H]+.
Synthesis of N-(7-Methoxy-1-methyl-2-oxo-1,2,3,4-tetrahy-
droquinolin-6-yl)benzenesulfonamide (3e). Step 1. To a solution
of 7-hydroxy-3,4-dihydroquinolin-2(1H)-one (1.64 g, 10 mmol) in 30
mL of H2SO4 was added water (7.6 mL) dropwise with stirring. The
reaction mixture was cooled to 0 °C and HNO3 (0.76 mL) added
dropwise while stirring. The reaction mixture was stirred for 15 min at
0 °C and then quenched by adding reaction mixture to a slurry of 100
mL of H2O and ice. The resulting solution was extracted with EtOAc
(3 × 10 mL) and the combined organic layers were concentrated
under reduced pressure to afford 7-hydroxy-6-nitro-3,4-dihydroquino-
lin-2(1H)-one (1.3 g, 63%) which was used without further
purification. MS (ESI) m/z 209 [M + H]+.
Step 3. To a solution of 2-(1-methyl-2-oxo-1,2,3,4-tetrahydroquino-
lin-7-yl)isoindoline-1,3-dione (120 mg, 0.39 mmol) in MeOH (5 mL)
was added hydrazine hydrate (122 mg, 1.95 mmol). The mixture was
stirred at rt for 1 h and then concentrated and purified by reverse-
phase HPLC to give 7-amino-1-methyl-3,4-dihydroquinolin-2(1H)-
one (50 mg, 72%). MS (ESI) m/z 177 [M + H]+.
1
Step 4. Synthesized 4 by method A (28 mg, 45%). H NMR (500
MHz, CDCl3) δ 7.67 (d, J = 8.5 Hz, 2H), 7.23 (d, J = 9 Hz, 2H), 6.99
(d, J = 7.5 Hz, 1H), 6.99 (br-s, 1H), 6.80 (d, J = 2.5 Hz, 1H), 6.60 (dd,
J = 7.5, 2.5 Hz, 1H), 3.26 (s, 3H), 2.80 (t, J = 7.5 Hz, 2H), 2.61 (t, J =
7.5 Hz, 2H), 2.49 (s, 3H). MS (ESI) m/z 363[M + H]+.
N-(1,3-Dimethyl-2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-
yl)-4-(methylsulfanyl) benzene-1-sulfonamide (5a). 1H NMR
(500 MHz, MeOD-d4) δ 7.56 (d, J = 8.5 Hz, 2H), 7.26 (d, J = 8.5 Hz,
2H), 6.93−6.97 (m, 2H), 6.76 (dd, J = 8.0, 1.5 Hz, 1H), 3.35 (s, 3H),
3.34 (s, 3H), 2.47 (s, 3H). MS (ESI) m/z 364 [M + H]+.
N-(1,3-Dimethyl-2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-
1
yl)-4-methoxybenzene-1-sulfonamide (5b). 39% yield; H NMR
(600 MHz, DMSO-d6) δ 9.87 (s, 1H), 7.62 (d, J = 8.9 Hz, 2H), 7.02
(d, J = 8.9 Hz, 2H), 6.96 (d, J = 8.2 Hz, 1H), 6.86 (d, J = 1.9 Hz, 1H),
6.68 (dd, J = 8.3, 1.9 Hz, 1H), 3.77 (s, 3H), 3.24 (s, 3H), 3.23 (s, 3H).
MS (ESI) m/z 348 [M + H]+.
N-(1,3-Dimethyl-2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-
yl)benzenesulfonamide (5c). 53% yield; 1H NMR (600 MHz,
DMSO-d6) δ 10.02 (s, 1H), 7.71−7.68 (m, 2H), 7.60−7.57 (m, 1H),
7.54−7.49 (m, 2H), 6.96 (d, J = 8.3 Hz, 1H), 6.86 (d, J = 1.9 Hz, 1H),
6.68 (dd, J = 8.3, 1.9 Hz, 1H), 3.24 (s, 3H), 3.23 (s, 3H). MS (ESI) m/
z 318 [M + H]+.
Step 2. To a solution of 7-hydroxy-6-nitro-3,4-dihydroquinolin-
2(1H)-one (1.1 g, 5 mmol) in 10 mL of DMF were added
iodomethane (2.5 mL, 20 mmol) and K2CO3 (5.47 g, 20 mmol).
The suspension was stirred at rt overnight. The reaction mixture was
then diluted with water (20 mL) and extracted with EtOAc (3 × 10
mL). The combined organic layers were washed with water (10 mL),
brine (10 mL) and then dried over anhydrous sodium sulfate,
concentrated under reduced pressure, then the residue was purified by
flash chromatography on silica (EtOAc/hexanes = 1:2) to afford 7-
methoxy-1-methyl-6-nitro-3,4-dihydroquinolin-2(1H)-one as a yellow
solid (950 mg, 81%). MS (ESI) m/z 237 [M + H]+.
N-(1,3-Dimethyl-2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-
yl)-4-(2-methylpropoxy)benzene-1-sulfonamide (5d). 29%
1
yield; H NMR (600 MHz, DMSO-d6) δ 9.86 (s, 1H), 7.60 (d, J =
8.9 Hz, 2H), 7.01 (d, J = 8.9 Hz, 2H), 6.96 (d, J = 8.2 Hz, 1H), 6.86
(d, J = 1.9 Hz, 1H), 6.68 (dd, J = 8.3, 1.9 Hz, 1H), 3.76 (d, J = 6.5 Hz,
2H), 3.24 (s, 3H), 3.23 (s, 3H), 2.03−1.94 (m, 1H), 0.95 (d, J = 6.7
Hz, 6H). MS (ESI) m/z 390 [M + H]+.
4-Cyclohexyl-N-(1,3-dimethyl-2-oxo-2,3-dihydro-1H-1,3-
benzodiazol-5-yl)benzene-1-sulfonamide (5e). 40% yield; 1H
NMR (600 MHz, DMSO-d6) δ 9.97 (s, 1H), 7.61 (d, J = 8.3 Hz, 2H),
7.37 (d, J = 8.3 Hz, 2H), 6.97 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 1.9 Hz,
1H), 6.71 (dd, J = 8.3, 1.9 Hz, 1H), 3.24 (s, 3H), 3.22 (s, 3H), 2.57−
2.50 (m, 1H), 1.81−1.65 (m, 5H), 1.41−1.28 (m, 4H), 1.25−1.15 (m,
1H). MS (ESI) m/z 400 [M + H]+.
5-Amino-6-bromo-1,3-dimethyl-1H-benzo[d]imidazol-
2(3H)-one (10a). To a 0 °C solution of 5-amino-1,3-dimethyl-1H-
benzo[d]imidazol-2(3H)-one (4.0 g, 23 mmol) in 25 mL of CHCl3
and 25 mL of AcOH was carefully added bromine (3.5 g, 23 mmol)
dropwise. The mixture was stirred at rt for 30 min, then concentrated
and purified by silica gel chromatography (pet ether/EtOAc 1:1) to
afford 10a as a yellow solid (3.2 g, 69%). 1H NMR (500 MHz, DMSO-
d6) δ 7.18 (s, 1H), 6.59 (s, 1H), 4.96 (s, 2H), 3.22 (s, 3H), 3.21 (s,
3H). MS (ESI) m/z 257 [M + H]+.
Step 3. To a solution of 7-methoxy-1-methyl-6-nitro-3,4-dihydro-
quinolin-2(1H)-one (900 mg, 3.8 mmol) in 30 mL of EtOH and 15
mL of H2O were added iron power (640 mg, 11.4 mmol) and
ammonium chloride (2.1 g, 38 mmol). The mixture was heated at
reflux for 2 h and then cooled to rt. The resulting suspension was
filtered through Celite, and the filtrate was diluted with 50 mL of
water. The filtrate was extracted with EtOAc (3 × 10 mL) and washed
with water (10 mL) and brine (10 mL), dried over anhydrous sodium
sulfate, then the combined organic layers were concentrated under
reduced pressure to afford a crude product. The residue was purified
by flash chromatography (hexanes/EtOAc = 1:1) on silica to afford 6-
amino-7-methoxy-1-methyl-3,4-dihydroquinolin-2(1H)-one as a white
1
solid (700 mg, 81%). H NMR (500 MHz, CDCl3) δ: 6.54 (s, 1 H),
6.48 (s, 1 H), 3.87 (s, 3 H), 3.33 (s, 3 H), 2.77−2.74 (m, 2H), 2.61−
2.58 (m, 2H). MS (ESI) m/z 207 [M + H]+.
Step 4. Sulfonamide formation with 6-amino-7-methoxy-1-methyl-
3,4-dihydroquinolin-2(1H)-one using method A afforded 3e as a white
1
solid (100 mg, 58%). H NMR (500 MHz, DMSO) δ: 9.39 (s, 1H),
N-(6-Bromo-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzo[d]-
imidazol-5-yl)-2,2,2-trifluoroacetamide (10b). To a 0 °C solution
of 10a (1.50 g, 5.9 mmol) in DCM (45 mL) were added DMAP (72
mg, 0.59 mmol), triethylamine (1.63 mL, 11.7 mmol), and
trifluoroacetic anhydride (0.91 mL, 6.4 mmol). The reaction mixture
was stirred for 2 h and warmed to ambient temperature. The reaction
mixture was then quenched with water and the organic phase was
washed with brine, dried over sodium sulfate, filtered, and evaporated
7.68 (d, J = 7.0 Hz, 2H), 7.59 (m, 1H), 7.53 (m, 2H), 6.99 (s, 1H),
6.79(s, 1H), 3.31 (s, 3H), 3.27 (s, 3H), 3.26 (s, 3H). MS (ESI) m/z
347 [M + H]+.
N-(1-Methyl-2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)-4-
(methylsulfanyl)benzene-1-sulfonamide (4). Step 1. A mixture of
7-amino-3,4-dihydroquinolin-2(1H)-one (100 mg, 0.62 mmol) and
phthalic anhydride (110 mg, 0.74 mmol) in AcOH (1 mL) was stirred
at 100 °C overnight, cooled to rt, and poured into water, and the
resulting suspension was filtered. Collected solids were dried to give 2-
(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)isoindoline-1,3-dione as a white
solid (120 mg, 67%). MS (ESI) m/z 293 [M + H]+.
1
to give 10b as a yellow solid (2.20 g, 100%). H NMR (600 MHz,
DMSO-d6) δ 11.23 (br-s, 1H), 7.57 (s, 1H), 7.31 (s, 1H), 3.34 (s, 3H),
3.32 (s, 3H). MS (ESI) m/z 353 [M + H]+.
Representative Procedure for the Synthesis of 6 and 7a−c
(Method B: Ullmann Coupling). Step 1. A mixture of 3-
(benzyloxy)phenol (156 mg, 0.78 mmol), quinolin-8-ol (30 mg, 0.21
mmol), copper(I) chloride (10 mg, 0.10 mmol), potassium phosphate
Step 2. To a mixture of 2-(2-oxo-1,2,3,4-tetrahydroquinolin-7-
yl)isoindoline-1,3-dione (120 mg, 0.41 mmol) and iodomethane (300
mg, 2.05 mmol) in DMF (2 mL) was added K2CO3 (113 mg, 0.82
J
DOI: 10.1021/acs.jmedchem.5b00405
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(200 mg, 0.94 mmol), and 10a (200 mg, 0.78 mmol) in diglyme (10
mL) was degassed under a nitrogen atmosphere. Then the reaction
mixture was heated to 130 °C for 72 h. The cooled reaction mixture
was filtered through a pad of silica gel. The collected filtrate was then
concentrated and purified by column chromatography (0−100%
EtOAc in hexanes and then 0−40% methanol in EtOAc) to give 5-
amino-6-(3-(benzyloxy)phenoxy)-1,3-dimethyl-1H-benzo[d]imidazol-
2(3H)-one (11b) as a solid (213 mg, 73%). MS (ESI) m/z 376 [M +
H]+. 1H NMR (600 MHz, CDCl3) δ 7.34 (m, 5H), 7.18 (t, J = 7.8 Hz,
1H), 6.65 (d, J = 2.8 Hz, 1H), 6.58 (s, 1H), 6.55 (m, 2H), 6.48 (s,
1H), 4.98 (s, 2H), 3.66 (br-s, 2H), 3.34 (s, 3H), 3.27 (s, 3H).
Step 2. Sulfonamide formation, using method A.
Representative Procedure for the Synthesis of 7d−f
(Method C: Ullmann Coupling).46 Step 1. A mixture of 10b (50
mg, 0.14 mmol), 2-(dimethylamino)acetic acid (15 mg, 0.14 mmol), 3-
ethoxyphenol (29 mg, 0.21 mmol), copper(I) iodide (8 mg, 0.04
mmol), and cesium carbonate (139 mg, 0.43 mmol) was charged in a
flask with dioxane (1 mL). The reaction mixture was heated to 80 °C
and stirred for 16 h. The reaction was monitored for complete
deprotection of trifluoroacetamide (addition of methanol was used to
facilitate this step). The cooled reaction mixture was diluted in
methanol, filtered and the filtrate was evaporated and purified by
column chromatography (hexanes/EtOAc 1:1) to give 5-amino-6-(3-
ethoxyphenoxy)-1,3-dimethyl-1H-benzo[d]imidazol-2(3H)-one (11c)
as an orange solid (13 mg, 29%). MS (ESI) m/z 314 [M + H]+.
Step 2. Sulfonamide formation, using method A.
N-[1,3-Dimethyl-2-oxo-6-(3-propoxyphenoxy)-2,3-dihydro-
1H-1,3-benzodiazol-5-yl]-1,2-dimethyl-1H-imidazole-4-sulfo-
1
namide (7f). 93% yield; H NMR (600 MHz, DMSO-d6) δ 9.32 (s,
1H), 7.49 (s, 1H), 7.17−7.13 (m, 2H), 6.76 (s, 1H), 6.61 (dd, J = 7.7,
2.2 Hz, 1H), 6.25 (dd, J = 8.0, 2.2 Hz, 1H), 6.23 (t, J = 2.2 Hz, 1H),
3.86 (t, J = 6.5 Hz, 2H), 3.45 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H), 2.08
(s, 3H), 1.73−1.66 (m, 2H), 0.96 (t, J = 7.5 Hz, 3H). MS (ESI) m/z
486 [M + H]+.
5-Amino-6-(3-hydroxyphenoxy)-1,3-dimethyl-1H-benzo[d]-
imidazol-2(3H)-one (12). To a −78 °C solution of 11b (400 mg,
1.07 mmol) in DCM (20 mL) was added tribromoborane (5.3 mL, 5.3
mmol). The reaction mixture was allowed to gradually warm to rt,
then quenched by the dropwise addition of methanol, concentrated,
and purified by column chromatography (20−100% EtOAc/hexanes
and then 0−40% methanol/EtOAc) to give 12 as a solid (240 mg,
1
79%). H NMR (600 MHz, DMSO-d6) 9.38 (s, 1H), 7.06 (t, J= 8.1,
1H), 6.78 (s, 1H), 6.58 (s, 1H) 6.39 (dd, J = 8.0, 2.2 Hz, 1H), 6.33
(dd, J = 8.1, 2.3, 1H), 6.23 (t, J= 2.3, 1H), 4.57 (br-s, 2H), 3.24 (s,
3H), 3.20 (s, 3H). MS (ESI) m/z 286 [M + H]+.
General Procedure for the Synthesis of Sulfonamides 7g−i,
7k, 7l. To a solution of 12 (50 mg, 0.18 mmol) in anhydrous DMF (1
mL) were added potassium carbonate (0.2−0.4 mmol) and alkyl
bromide (0.18−0.25 mmol). The reaction mixture was stirred at
ambient temperature for 1 day, and then the reaction mixture was
diluted with water and extracted with EtOAc. The separated organic
layer was dried over sodium sulfate, filtered, and concentrated to give
the intermediate 5-amino-6-(3-(alkyloxy)phenoxy)-1,3-dimethyl-1H-
benzo[d]imidazol-2(3H)-one as a crude residue which was used
without further purification in the sulfonamide formation step using
method A to give 7g−i, 7k, 7l.
N-{6-[4-(Benzyloxy)phenoxy]-1,3-dimethyl-2-oxo-2,3-dihy-
dro-1H-1,3-benzodiazol-5-yl}-1-methyl-1H-imidazole-4-sulfo-
1
namide (6). 6% yield. H NMR (600 MHz, DMSO-d6) δ 9.32 (s,
1H), 7.57 (d, J = 3.3 Hz, 2H), 7.46 (d, J = 7.5 Hz, 2H), 7.38 (t, J = 7.1
Hz, 2H), 7.30 (t, J = 6.7 Hz, 1H), 7.08 (s, 1H), 6.95 (d, J = 9.5 Hz,
2H), 6.82 (d, J = 9.5 Hz, 2H), 6.67 (s, 1H), 5.06 (s, 2H), 3.57 (s, 3H),
3.28 (s, 3H), 3.17 (s, 3H). MS (ESI) m/z 520 [M + H]+.
N-{1,3-Dimethyl-6-[3-(2-methylpropoxy)phenoxy]-2-oxo-
2,3-dihydro-1H-1,3-benzodiazol-5-yl}-1,2-dimethyl-1H-imida-
1
zole-4-sulfonamide (7g). 3% yield; H NMR (600 MHz, DMSO-
d6) δ 9.27 (s, 1H), 7.47 (s, 1H), 7.17−7.14 (m, 2H), 6.77 (s, 1H), 6.63
(d, J = 8.9 Hz, 1H), 6.27 (s, 1H), 6.24 (d, J = 8.2 Hz, 1H), 3.69 (d, J =
6.5 Hz, 1H), 3.40 (s, 1H), 3.29 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H),
2.09 (s, 3H), 2.01−1.97 (m, 1H), 0.96 (d, J = 6.7 Hz, 6H). MS (ESI)
m/z 501 [M + H]+.
N-{6-[3-(Benzyloxy)phenoxy]-1,3-dimethyl-2-oxo-2,3-dihy-
dro-1H-1,3-benzodiazol-5-yl}-1-methyl-1H-imidazole-4-sulfo-
1
namide (7a). 21% yield; H NMR (600 MHz, DMSO-d6) δ 9.35 (s,
1H), 7.56 (d, J = 3.5 Hz, 2H), 7.41 (d, J = 7.5 Hz, 2H), 7.36 (t, J = 7.1
Hz, 2H), 7.31 (t, J = 6.7 Hz, 1H), 7.17 (t, J = 8.7 Hz, 1H), 7.09 (s,
1H), 6.75 (s, 1H), 6.70 (dd, J = 2.4, 8.3 Hz, 1H), 6.29−6.31 (m, 2H),
5.05 (s, 2H), 3.55 (s, 3H), 3.29 (s, 3H), 3.19 (s, 3H). MS (ESI) m/z
520 [M + H]+.
N-[6-(3-Butoxyphenoxy)-1,3-dimethyl-2-oxo-2,3-dihydro-
1H-1,3-benzodiazol-5-yl]-1,2-dimethyl-1H-imidazole-4-sulfo-
1
namide (7h). 13% yield; H NMR (600 MHz, DMSO-d6) δ 9.33 (s,
1H), 7.50 (s, 1H), 7.16 (t, J = 7.3 Hz, 2H), 6.77 (s, 1H), 6.62 (dd, J =
8.3, 2.5 Hz, 1H), 6.25 (m, 2H), 3.91 (t, J = 6.4 Hz, 3H), 3.29 (s, 3H),
3.20 (s, 1H), 2.09 (s, 3H), 1.68−1.65 (m, 2H), 1.43−1.39 (m, 2H),
0.93 (t, J = 7.5 Hz, 3H). MS (ESI) m/z 501 [M + H]+.
N-{1,3-Dimethyl-6-[3-(2-methylpropoxy)phenoxy]-2-oxo-
2,3-dihydro-1H-1,3-benzodiazol-5-yl}cyclopropane-
sulfonamide (7i). 28% yield; 1H NMR (600 MHz, DMSO-d6) δ 9.11
(s, 1H), 7.21 (t, J = 8.2 Hz, 1H), 7.17 (s, 1H), 6.92 (s, 1H), 6.66−6.63
(m, 1H), 6.53−6.47 (m, 2H), 3.69 (d, J= 6.4 Hz, 2H), 3.33 (s, 3H),
3.25 (s, 3H), 1.97 (m, 1H), 0.95 (d, J= 6.7 Hz, 6H), 0.85−0.80 (m,
4H). MS (ESI) m/z 446 [M + H]+.
N-{6-[3-(Benzyloxy)phenoxy]-1,3-dimethyl-2-oxo-2,3-dihy-
dro-1H-1,3-benzodiazol-5-yl}-3,4-dimethoxybenzene-1-sulfo-
1
namide (7b). 13% yield; H NMR (600 MHz, CDCl3) δ 7.30−7.40
(m, 5H), 7.24 (dd, J = 8.3, 2.0 Hz, 1H), 7.06 (m, 2H), 6.82 (s, 1H),
6.70 (d, J = 8.6 Hz, 1H), 6.65 (dd, J = 8.3, 2.0 Hz, 1H), 6.42 (s, 1H),
6.17 (t, J = 2.6 Hz, 1H), 6.05 (dd, J = 8.3, 2.0 Hz, 1H), 4.97 (s, 2H),
3.79 (s, 3H), 3.60 (s, 3H), 3.45 (s, 3H), 3.25 (s, 3H). MS (ESI) m/z
576 [M + H]+.
N-{6-[3-(Benzyloxy)phenoxy]-1,3-dimethyl-2-oxo-2,3-dihy-
dro-1H-1,3-benzodiazol-5-yl}-1,2-dimethyl-1H-imidazole-4-
1
sulfonamide (7c). 47% yield; H NMR (600 MHz, DMSO-d6) δ
9.31 (s, 1H), 7.48 (s, 1H), 7.42−7.34 (m, 4H), 7.33−7.29 (m, 1H),
7.18 (t, J = 8.3 Hz, 1H), 7.15 (s, 1H), 6.73 (s, 1H), 6.70 (dd, J = 8.3,
2.2 Hz, 1H), 6.31−6.26 (m, 2H), 5.04 (s, 2H), 3.42 (s, 3H), 3.29 (s,
3H), 3.19 (s, 3H), 2.07 (s, 3H). MS (ESI) m/z 534[M + H]+.
N-[6-(3-Ethoxyphenoxy)-1,3-dimethyl-2-oxo-2,3-dihydro-
1H-1,3-benzodiazol-5-yl]-1,2-dimethyl-1H-imidazole-4-sulfo-
1,3-Dimethyl-5-[3-(2-methylpropoxy)phenoxy]-2,3-dihydro-
1H-1,3-benzodiazol-2-one (7j). Synthesized from 5-bromo-1,3-
dimethyl-1,3-dihydro-2H-benzo[d]imidazol-2-one using method C,
1
32% yield. H NMR (500 MHz, DMSO-d6) δ 7.21 (t, J = 8.5 Hz,
1H), 7.14 (d, J = 8.5 Hz, 1H), 6.98 (d, J = 2.2 Hz, 1H), 6.77 (dd, J =
8.4, 2.2 Hz, 1H), 6.65−6.60 (m, 1H), 6.48−6.44 (m, 2H), 3.69 (d, J =
6.5, 2H), 3.33 (s, 3H), 3.29 (s, 3H), 1.97 (m, 1H), 0.94 (d, J = 6.7 Hz,
6H). MS (ESI) m/z 327 [M + H]+.
1
namide (7d). 36% yield; H NMR (600 MHz, DMSO-d6) δ 9.34 (s,
1H), 7.50 (s, 1H), 7.18−7.14 (m, 2H), 6.76 (s, 1H), 6.61 (dd, J = 7.7,
2.2 Hz, 1H), 6.26 (dd, J = 8.0, 2.2 Hz, 1H), 6.19 (t, J = 2.2 Hz, 1H),
3.96 (q, J = 6.9 Hz, 2H), 3.45 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H), 2.09
(s, 3H), 1.30 (t, J = 7.0 Hz, 3H). MS (ESI) m/z 472 [M + H]+.
N-[1,3-Dimethyl-2-oxo-6-(3-propoxyphenoxy)-2,3-dihydro-
1H-1,3-benzodiazol-5-yl]-3,4-dimethoxybenzene-1-sulfona-
N-{6-[3-(3-Methoxypropoxy)phenoxy]-1,3-dimethyl-2-oxo-
2,3-dihydro-1H-1,3-benzodiazol-5-yl}-1,2-dimethyl-1H-imida-
1
zole-4-sulfonamide (7k). 13% yield; H NMR (600 MHz, DMSO-
d6) δ 9.35 (s, 1H), 7.50 (s, 1H), 7.18−7.15 (m, 2H), 6.78 (s, 1H), 6.63
(d, J = 8.3 Hz, 1H), 6.25 (dd, J = 8.4 Hz, 2H), 3.96 (t, J = 6.4 Hz, 2H),
3.46−3.43 (m, 5H), 3.29 (s, 3H), 3.23 (s, 3H), 3.20 (s, 3H), 2.09 (s,
3H), 1.92 (m, 2H). MS (ESI) m/z 516 [M + H]+.
1
mide (7e). 66% yield; H NMR (600 MHz, DMSO-d6) δ 9.51 (s,
1H), 7.19−7.14 (m, 2H), 7.11 (s, 1H), 7.08−7.02 (m, 1H), 6.89 (d, J
= 9.0 Hz, 1H), 6.74 (s, 1H), 6.55 (dd, J = 8.2, 2.2 Hz, 1H), 6.08 (dd, J
= 8.1, 2.0 Hz, 1H), 6.05−6.03 (m, 1H), 3.79 (t, J = 6.5 Hz, 2H), 3.77
(s, 3H), 3.57 (s, 3H), 3.30 (s, 3H), 3.19 (s, 3H), 1.73−1.64 (m, 2H),
0.95 (t, J = 7.4 Hz, 3H). MS (ESI) m/z 528 [M + H]+.
N-{1,3-Dimethyl-2-oxo-6-[3-(oxolan-3-ylmethoxy)phenoxy]-
2,3-dihydro-1H-1,3-benzodiazol-5-yl}-1-methyl-1H-imidazole-
1
4-sulfonamide (7l). 5% yield; H NMR (600 MHz, DMSO-d6) δ
9.32 (s, 1H), 7.56 (d, J = 3.5 Hz, 2H), 7.17 (t, J = 8.7 Hz, 1H), 7.09 (s,
K
DOI: 10.1021/acs.jmedchem.5b00405
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1H), 6.78 (s, 1H), 6.63 (dd, J = 8.3, 2.4 Hz, 1H), 6.28 (m, 2H), 3.88
(dd, J = 9.3, 6.4 Hz, 1H), 3.83 (t, J = 7.7 Hz, 1H), 3.73 (m, 1H), 3.65
(dd, J = 15.3, 7.4 Hz, 1H), 3.03 (s, 3H), 3.52 (dd, J = 8.3, 5.4 Hz, 1H),
3.27 (s, 3H), 3.20 (s, 3H), 2.63 (m, 1H), 1.99 (m, 1H), 1.64 (m, 1H).
MS (ESI) m/z 515 [M + H]+.
3.19 (s, 3H), 3.14−3.06 (m, 2H), 2.77 (d, J = 4.9 Hz, 6H), 1.79−1.64
(m, 4H). MS (ESI) m/z 585 [M + H]+.
Synthesis of tert-Butyl (6-(3-((1,3-Dimethyl-6-(1-methyl-1H-
imidazole-4-sulfonamido)-2-oxo-2,3-dihydro-1H-benzo[d]-
imidazol-5-yl)oxy)-5-propoxyphenoxy)hexyl)carbamate (8a).
Step 1. 5-Propoxybenzene-1,3-diol (13). A solution of benzene-1,3,5-
triol (1.0 g, 7.9 mmol) in DMF (10 mL) was treated with potassium
carbonate powder (1.2 g, 8.7 mmol) and 1-bromopropane (0.80 mL,
8.7 mmol), and the mixture was stirred at 50 °C for 16 h. The cooled
reaction mixture was diluted with water, carefully quenched with 1 N
HCl until acidic, then extracted with EtOAc. The organic layer was
washed with brine and dried over sodium sulfate, concentrated, and
purified by column chromatography (EtOAc/hexanes 3:7) to give 5-
Synthesis of N-(6-{3-[4-(Dimethylamino)butoxy]phenoxy}-
1,3-dimethyl-2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-yl)-3,4-
dimethoxybenzene-1-sulfonamide (7m). Step 1. To a solution of
resorcinol (585 mg, 5.32 mmol) in DMF (15 mL) were added K2CO3
(735 mg, 5.32 mmol) and 2-(4-bromobutyl)isoindoline-1,3-dione
(500 mg, 1.772 mmol). The mixture was stirred at 50 °C overnight
and then diluted with water (50 mL) and then adjusted to acidic pH
with 1 N HCl. The product was extracted with EtOAc (2 × 50 mL),
washed with brine (20 mL), dried over Na2SO4, and concentrated.
Purification was by silica gel chromatography (0% EtOAc/hexane to
100% EtOAc/hexane). Purification was by prep-HPLC using a
gradient of 40−80% ACN/water containing 0.1% TFA to afford 2-
(4-(3-hydroxyphenoxy)butyl)isoindoline-1,3-dione (263 mg, 48%
1
propoxybenzene-1,3-diol (13) as a yellow liquid (0.49 g, 39%). H
NMR (600 MHz, CDCl3) δ 6.00 (d, J = 2.1 Hz, 2H), 5.96 (t, J = 2.1
Hz, 1H), 5.48 (br-s, 2H), 3.84 (t, J = 6.5 Hz, 2H), 1.80−1.72 (m, 2H),
1.00 (t, J = 7.4 Hz, 3H). MS (ESI) m/z 169 [M + H]+.
Step 2. To a solution of 13 (344 mg, 2.05 mmol) and tert-butyl (6-
bromohexyl)carbamate (268 mg, 0.956 mmol) in 3 mL of DMF was
added potassium carbonate (326 mg, 2.36 mmol), and the resulting
mixture was stirred at 50 °C for 16 h. The cooled reaction mixture was
neutralized with 1 M HCl, diluted with EtOAc, and the separated
organic layer was washed with sat. aq NaCl solution, dried over
sodium sulfate, filtered, and concentrated under reduced pressure. The
residue was purified via silica gel chromatography (1:9 to 1:1 EtOAc/
hexanes) to give 14a (174 mg, 23%) as a light-brown viscous liquid.
1H NMR (600 MHz, CDCl3) δ 6.05 (m, 1H), 6.01 (m, 2H), 5.15 (br-
s, 1H), 4.53 (br-s, 1H), 3.90 (m, 2H), 3.86 (t, J = 6.5 Hz, 2H), 3.12
(m, 2H), 1.81−1.72 (m, 4H), 1.54−1.33 (m, 6H), 1.44 (s, 9H), 1.01
(t, J = 7.5 Hz, 3H). MS (ESI) m/z 368 [M + H]+.
1
yield) as a white solid. H NMR (600 MHz, DMSO-d6) δ 9.32 (s,
1H), 7.90−7.80 (m, 4H), 7.05−6.97 (m, 1H), 6.34−6.29 (m, 2H),
6.30−6.25 (m, 1H), 3.90 (t, J = 6.1 Hz, 2H), 3.63 (t, J = 6.6 Hz, 2H),
1.79−1.65 (m, 4H). MS (ESI) m/z 312 [M + H]+.
Step 2. 2-(4-(3-((6-Amino-1,3-dimethyl-2-methylene-2,3-dihydro-
1H-benzo[d]imidazol-5-yl)oxy)phenoxy)butyl)isoindoline-1,3-dione
was prepared from 10b using method C to give the product as as an
orange liquid (118 mg, 35% yield). 1H NMR (600 MHz, DMSO-d6) δ
7.89−7.79 (m, 4H), 7.19−7.12 (m, 1H), 6.79 (s, 1H), 6.58 (s, 1H),
6.55 (dd, J = 2.2, 8.3 Hz, 1H), 6.42−6.37 (m, 2H), 4.57 (br-s, 2H),
3.92 (t, J = 5.9 Hz, 2H), 3.61 (t, J = 6.5 Hz, 2H), 3.24 (s, 3H), 3.20 (s,
3H), 1.76−1.64 (m, 4H). MS (ESI) m/z 487 [M + H]+.
Step 3. N-(6-(3-(4-(1,3-Dioxoisoindolin-2-yl)butoxy)phenoxy)-1,3-
dimethyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-3,4-
dimethoxybenzenesulfonamide was synthesized using method A to
Step 3. A solution of 10b (100 mg, 0.284 mmol), 14a (147 mg,
0.400 mmol), and 2-(dimethylamino)acetic acid (51 mg, 0.50 mmol)
in diethylene glycol dimethyl ether (6 mL) was degassed with
nitrogen. Copper(I) iodide (34 mg, 0.18 mmol) and Cs2CO3 (410 mg,
1.26 mmol) were added, and the mixture was degassed with nitrogen
for an additional 2 min. The reaction mixture was heated to 80 °C for
1.5 days. The cooled reaction mixture was filtered through a pad of
Celite, concentrated, then purified by flash chromatography (EtOAc/
hexanes 1:9 to 1:1) to give 15a as a solid (54 mg, 35%). 1H NMR (600
MHz, CDCl3) δ: 6.63 (s, 1H), 6.47 (s, 1H), 6.15 (t, J = 2.1 Hz, 1H),
6.08 (t, J = 2.1 Hz, 1H), 6.06 (t, J = 2.1 Hz, 1H), 4.50 (br-s, 1H), 3.87
(t, J = 6.4 Hz, 2H), 3.84 (t, J = 6.6 Hz, 2H), 3.64 (br-s, 2H), 3.37 (s,
3H), 3.31 (s, 3H), 3.11 (m, 2H), 1.80−1.70 (m, 4H), 1.54−1.34 (m,
6H), 1.44 (s, 9H), 1.00 (t, J = 7.4 Hz, 3H). MS (ESI) m/z 543 [M +
H]+.
1
give the product as a light-orange liquid (160 mg, 99% yield). H
NMR (600 MHz, DMSO-d6) δ 9.53 (s, 1H), 7.90−7.80 (m, 4H),
7.18−7.13 (m, 2H), 7.12 (s, 1H), 7.06−7.00 (m, 1H), 6.87 (d, J = 9.0
Hz, 1H), 6.73 (s, 1H), 6.53 (dd, J = 8.2, 2.1 Hz, 1H), 6.06 (dd, J = 7.9,
2.0 Hz, 1H), 6.06−6.02 (m, 1H), 3.86 (t, J = 5.9 Hz, 2H), 3.75 (s,
3H), 3.63 (t, J = 6.5 Hz, 2H), 3.56 (s, 3H), 3.30 (s, 3H), 3.18 (s, 3H),
1.78−1.65 (m, 4H). MS (ESI) m/z 687 [M + H]+.
Step 4. To a solution of N-(6-(3-(4-(1,3-dioxoisoindolin-2-yl)-
butoxy)phenoxy)-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzo[d]-
imidazol-5-yl)-3,4-dimethoxybenzenesulfonamide (160 mg, 0.233
mmol) in MeOH (4 mL) was added hydrazine hydrate (29 μL, 0.47
mmol), and the reaction mixture was heated to 80 °C for 90 min. The
cooled reaction mixture was concentrated and then purified by prep-
HPLC using a gradient of 20−60% ACN/water containing 0.1% TFA
to afford the TFA salt of N-(6-(3-(4-aminobutoxy)phenoxy)-1,3-
dimethyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-3,4-
dimethoxybenzenesulfonamide (103 mg, 66% yield) as a white solid.
1H NMR (600 MHz, DMSO-d6) δ 9.52 (s, 1H), 7.68 (br-s, 3H),
7.20−7.15 (m, 2H), 7.09 (s, 1H), 7.09−7.04 (m, 1H), 6.89 (d, J = 9.0
Hz, 1H), 6.73 (s, 1H), 6.55 (dd, J = 8.2, 2.1 Hz, 1H), 6.12 (dd, J = 8.2,
2.1 Hz, 1H), 6.08−6.04 (m, 1H), 3.85 (t, J = 5.9 Hz, 2H), 3.77 (s,
3H), 3.59 (s, 3H), 3.30 (s, 3H), 3.19 (s, 3H), 2.89−2.81 (m, 2H),
1.78−1.62 (m, 4H). MS (ESI) m/z 557 [M + H]+.
1
Step 4. Method A gave 8a as an amorphous solid (68% yield). H
NMR (600 MHz, CDCl3) δ: 7.97 (br-s, 1H), 7.60 (s, 1H, overlapped
with CHCl3), 7.35 (s, 1H), 7.31 (s, 1H), 7.20 (s, 1H), 6.57 (s, 1H),
6.14 (s, 1H), 5.77 (br-s, 1H), 5.72 (br-s, 1H), 3.86 (t, J = 6.4 Hz, 2H),
3.83 (t, J = 6.5 Hz, 2H), 3.62 (s, 3H), 3.45 (s, 3H), 3.31 (s, 3H), 3.12
(br-t, J = 6.3 Hz, 2H), 1.76 (m, 4H), 1.51 (m, 2H), 1.46 (m, 2H), 1.44
(s, 9H), 1.37 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H). MS (ESI) m/z 687 [M
+ H]+.
N-(6-{3-[(6-Aminohexyl)oxy]-5-propoxyphenoxy}-1,3-di-
methyl-2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-yl)-1-methyl-
1H-imidazole-4-sulfonamide (8b). To a solution of 8a (18 mg,
0.026 mmol) in DCM (4 mL) was added TFA (500 μL, 6.49 mmol),
and the resulting mixture was stirred at 23 °C for 16 h. The reaction
mixture was concentrated to give the TFA salt of 8b as a brown-
colored viscous liquid (17 mg, 93%). 1H NMR (DMSO-d6) δ 8.52 (br-
s, 1H), 7.94 (br-s, 3H), 7.48 (br-s, 1H), 7.24 (s, 1H), 7.18 (s, 1H),
6.56 (s, 1H), 6.11 (s, 1H), 5.85 (s, 1H), 5.56 (s, 1H), 3.89 (t, J = 6.1
Hz, 2H), 3.79 (t, J = 6.6 Hz, 2H), 3.61 (s, 3H), 3.41 (s, 3H), 3.28 (s,
3H), 2.97 (br-s, 2H), 1.77−1.65 (m, 6H), 1.46 (m, 2H), 1.42 (m, 2H),
0.98 (t, J = 7.3 Hz, 3H). MS (ESI) m/z 587 [M + H]+.
Step 5. To a solution of N-(6-(3-(4-aminobutoxy)phenoxy)-1,3-
dimethyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-3,4-
dimethoxybenzenesulfonamide TFA salt (50 mg, 0.075 mmol) in
MeOH (3 mL) was added triethylamine (10 μL, 0.08 mmol), acetic
acid (9 μL, 0.15 mmol), formaldehyde (16 μL, 0.60 mmol), and
sodium triacetoxyborohydride (40 mg, 0.19 mmol). The reaction
mixture was stirred at rt for 3 h and then was quenched with a few
drops of TFA and concentrated. Purification by prep-HPLC using a
gradient of 20−60% acetonitrile/water containing 0.1% TFA afforded
1
the TFA salt of 7m (14 mg, 27% yield) as a yellow liquid. H NMR
Synthesis of N-(6-{3-[(6-Hydroxyhexyl)oxy]-5-propoxyphe-
noxy}-1,3-dimethyl-2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-
yl)-1-methyl-1H-imidazole-4-sulfonamide (8c). Step 1. Prepara-
tion was in a similar manner to 14a from 13 using 6-bromohexan-1-ol
(600 MHz, DMSO-d6) δ 9.50 (s, 1H), 9.34 (br-s, 1H), 7.21−7.15 (m,
2H), 7.12−7.05 (m, 2H), 6.89 (d, J = 9.0 Hz, 1H), 6.73 (s, 1H), 6.56
(dd, J = 8.2, 2.0 Hz, 1H), 6.14 (dd, J = 8.1, 1.9 Hz, 1H), 6.09−6.04 (m,
1H), 3.86 (t, J = 5.9 Hz, 2H), 3.77 (s, 3H), 3.59 (s, 3H), 3.29 (s, 3H),
1
to give 14b as a viscous brown liquid (39% yield). H NMR (600
L
DOI: 10.1021/acs.jmedchem.5b00405
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
MHz, CDCl3) δ 6.06 (m, 1H), 6.03−5.98 (m, 2H), 5.25 (br-s, 1H),
3.90 (t, J = 6.5 Hz, 2H), 3.86 (t, J = 6.7 Hz, 2H), 3.67 (t, J = 6.5 Hz,
2H), 1.82−1.73 (m, 4H), 1.64−1.57 (m, 2H), 1.52−1.36 (m, 5H),
1.01 (t, J = 7.4 Hz, 3H). MS (ESI) m/z 269 [M + H]+.
N-{6-[3-(4-Aminobutoxy)-5-propoxyphenoxy]-1,3-dimethyl-
2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-yl}-3,4-dimethoxy-
benzene-1-sulfonamide (8g). 8g was prepared in a similar manner
to 8d. 26% yield (3 steps). 1H NMR (600 MHz, DMSO-d6) δ 9.47 (s,
1H), 7.59 (s, 3H), 7.25 (d, J= Hz, 1H), 7.09−7.06 (m, 2H), 7.00 (s,
1H), 6.81 (s, 1H) 6.00 (s, 1H), 5.71 (s, 1H), 5.62 (s, 1H), 3.84 (s,
3H), 3.75 (t, J = 6.5 Hz, 2H), 3.70 (s, 3H), 3.31 (s, 3H), 3.25 (s, 3H),
1.69−1.65 (m, 3H), 1.38−1.35 (m, 2H), 0.95 (t, J= 7.5 Hz, 4H). MS
(ESI) m/z 615 [M + H]+.
N-{6-[3-(3-Aminopropoxy)-5-propoxyphenoxy]-1,3-dimeth-
yl-2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-yl}-3,4-dimethoxy-
benzene-1-sulfonamide (8h). 8h was prepared in a similar manner
to 8d except using a BOC-protected intermediate. 13% yield (3 steps).
1H NMR (600 MHz, DMSO-d6) δ 9.44 (s, 1H), 7.68 (br-s, 3H),
7.20−7.17 (m, 2H), 7.06 (s, 1H), 6.90 (d, J = 9.0 Hz, 1H) 6.73 (s,1H),
6.13 (t, J = 2.1 Hz, 1H), 5.72 (t, J = 2.1 Hz, 1H), 5.67 (t, J = 2.1 Hz,
1H), 3.89 (t, J = 6.1 Hz, 2H), 3.775 (t, J = 6.5 Hz, 2H), 3.770 (s, 3H),
3.61 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H), 2.95−2.89 (m, 2H), 1.98−
1.90 (m, 2H), 1.71−1.63 (m, 2H), 0.94 (t, J = 7.5 Hz, 3H). MS (ESI)
m/z 601 [M + H]+.
Steps 2 and 3. Preparation was done using method C to give 15b,
used without further purification. MS (ESI) m/z 444 [M + H]+. 15b
1
was converted to 8c using method A (38% yield). H NMR (600
MHz, DMSO-d6) δ 9.30 (s, 1H), 7.60 (m, 1H), 7.58 (m, 1H), 7.07 (s,
1H), 6.80 (s, 1H), 6.17 (s, 1H), 5.83 (m, 2H), 4.38 (t, J = 6.5 Hz, 1H),
3.86 (t, J = 6.5 Hz, 2H), 3.83 (t, J = 6.5 Hz, 2H), 3.37 (t, J = 6.5 Hz,
2H), 3.59 (s, 3H), 3.27 (s, 3H), 3.21 (s, 3H), 1.67 (m, 4H), 1.45−1.28
(m, 6H), 0.94 (t, J = 7.5 Hz, 3H). MS (ESI) m/z 588 [M + H]+.
Synthesis of N-(6-(3-((6-Aminohexyl)oxy)-5-propoxyphe-
noxy)-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-
yl)-3,4-dimethoxybenzenesulfonamide (8d). Step 1. Preparation
was from 13 using method C to give 16 as a brown solid (43% yield).
1H NMR (600 MHz, DMSO-d6) δ 9.40 (s, 1H), 6.81 (s, 1H), 6.66
(br-s, 1H), 5.99 (s, 1H), 5.93 (s, 1H), 5.85 (s, 1H), 3.80 (t, J = 6.6 Hz,
2H), 3.26 (s, 3H), 3.21 (s, 3H) 1.66 (m, 2H), 0.93 (t, J = 7.5, 3H). MS
(ESI) m/z 344 [M + H]+.
N-(6-{3-[4-(Dimethylamino)butoxy]-5-propoxyphenoxy}-
1,3-dimethyl-2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-yl)-3,4-
dimethoxybenzene-1-sulfonamide (8i). To a solution of 8h TFA
salt (180 mg, 0.25 mmol) in methanol (3 mL) were added
triethylamine (34 ul, 0.25 mmol), acetic acid (28 uL, 0.49 mmol),
formaldehyde (0.054 mL, 1.98 mmol), and sodium triacetoxyborohy-
dride (131 mg, 0.618 mmol). The reaction mixture was stirred at rt for
3 h and then concentrated under reduced pressure. The residue was
purified by prep-HPLC using a gradient of 20−60% ACN/water
containing 0.1% TFA to afford the TFA salt of 8i (106 mg, 57%) as a
white solid. 1H NMR (600 MHz, DMSO-d6) δ 9.46 (s, 1H), 9.30 (br-
s, 1H), 7.19 (m, 2H), 7.07 (s, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.75 (s,
1H), 6.13 (t, J = 2.2 Hz, 1H), 5.71 (t, J = 2.0 Hz, 1H), 5.67 (t, J = 2.0
Hz, 1H), 3.84 (t, J = 5.9 Hz, 2H), 3.77 (m, 5H), 3.62 (s, 3H), 3.29 (s,
3H), 3.20 (s, 3H), 3.12−3.05 (m, 2H), 2.78 (d, J = 4.7 Hz, 6H), 1.77−
1.63 (m, 6H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (600 MHz, DMSO-
d6) δ 160.3, 160.0, 159.3, 154.1, 152.0, 148.4, 143.9, 131.8, 128.2,
126.0, 121.9, 120.5, 110.4, 109.4, 106.4, 100.6, 95.9, 95.8, 95.2, 68.9,
66.7, 56.3, 55.6, 55.4, 42.1, 27.1, 27.0, 25.6, 21.9, 20.7, 10.4. MS (ESI)
m/z 644 [M + H]+.
Step 2. To a solution of 16 (800 mg, 2.33 mmol) in anhydrous
DMF (60 mL) were added potassium carbonate (1.29 g, 9.32 mmol)
and 2-(6-bromohexyl)isoindoline-1,3-dione (2.89 g, 9.32 mmol) in a
vial. The reaction mixture was stirred at 80 °C for 1 h. To the cooled
reaction mixture was added water (50 mL), the aqueous phase was
extracted with EtOAc (3 × 50 mL), the combined organic layers were
concentrated under reduced pressure, and the residue was purified by
silica gel chromatography (EtOAc/hexanes 1:4 to 100% EtOAc) to
give 2-(6-(3-((6-amino-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzo[d]-
imidazol-5-yl)oxy)-5-propoxyphenoxy)hexyl)isoindoline-1,3-dione
1
17a as a viscous liquid (800 mg, 60%). H NMR (600 MHz, DMSO-
d6) δ 7.87−7.82 (m, 4H), 6.83 (s, 1H), 6.69 (br-s, 1H), 6.17 (s, 1H),
6.00 (s, 2H), 3.86 (t, J = 6.2 Hz, 2H), 3.84 (t, J = 6.5 Hz, 2H), 3.56 (t,
J = 7.1 Hz, 2H), 3.26 (s, 3H), 3.21 (s, 3H), 1.70−1.56 (m, 6H), 1.39
(m, 2H), 1.31 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H). MS (ESI) m/z 573
[M + H]+.
Step 3. To a solution of 17a (150 mg, 0.262 mmol) in anhydrous
DCM (1 mL) were added pyridine (0.042 mL, 0.524 mmol) and 3,4-
dimethoxybenzene-1-sulfonyl chloride (74.4 mg, 0.314 mmol). The
mixture was stirred for 1 h before quenching with MeOH (0.5 mL).
The resulting mixture was treated with hydrazine hydrate (8.22 μL,
0.262 mmol) and then concentrated and purified by mass-triggered
preparative HPLC (mobile phase, A = 0.1% TFA/H2O, B = 0.1%
TFA/ACN; gradient, B = 20−60%, 12 min; column, C18) to give 8d
as a white solid, 30% yield (over 2 steps). 1H NMR (600 MHz,
DMSO-d6) δ 9.44 (s, 1H), 7.61 (s, 2H), 7.19−7.18 (m, 2H), 7.07 (s,
1H), 6.90 (d, J = 8.9 Hz, 1H) 6.75 (s, 1H), 6.11 (s, 1H), 5.69 (s, 1H),
5.66 (s, 1H), 3.81 (t, J = 6.4 Hz, 2H), 3.78−3.75 (m, 5H), 3.35 (s,
3H), 3.29 (s, 3H), 3.20 (s, 3H), 2.80−2.76 (m, 2H), 1.69−1.64 (m,
4H), 1.55−1.52 (m, 2H), 1.40−1.33 (m, 4H), 0.95 (t, J = 7.5 Hz, 3H).
MS (ESI) m/z 643 [M + H]+.
ASSOCIATED CONTENT
■
S
* Supporting Information
Assay conditions, materials and methods, crystallographic data
collection and refinement statistics (Table S1), bromodomain
1
profiling data for 8i (Table S2), H NMR spectral data for 8i,
and a csv file containing molecular formula strings. The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.5b00405.
Accession Codes
N-(6-{3-[(5-Aminopentyl)oxy]-5-propoxyphenoxy}-1,3-di-
methyl-2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-yl)-1-methyl-
1H-imidazole-4-sulfonamide (8e). 8e was prepared in a similar
manner to 8d except using a BOC-protected intermediate. 27% yield
The models and structure factors of TRIM24 ligand cocrystal
structures have been deposited with the PDB with the following
accession codes: 1, 4YAB; 3b, 4YAD; 5b, 4YAT; 5g, 4YAX; 7b,
4YBM; 7g, 4YBS; 7l, 4YBT; 8i, 4YC9.
1
(3 steps). H NMR (600 MHz, DMSO-d6) δ 9.28 (s, 1H), 7.60 (s,
4H), 7.63 (s, 1H), 7.05 (s, 1H), 6.80 (s, 1H) 6.17 (s, 1H), 5.87 (t, J =
2.1 Hz, 2H), 3.91 (t, J = 6.1 Hz, 2H), 3.84 (t, J = 6.5 Hz, 2H), 3.61 (s,
3H), 3.26 (s, 3H), 3.22 (s, 3H), 2.85−2.82 (m, 2H), 1.68 (m, 4H),
1.56 (m, 2H), 1.42 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H). MS (ESI) m/z
573 [M + H]+.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: wpalmer@mdanderson.org. Telephone: (001) 713-
745-3022. Fax: (001) 713-745-8865.
N-{6-[3-(4-Aminobutoxy)-5-propoxyphenoxy]-1,3-dimethyl-
2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-yl}-1-methyl-1H-imi-
dazole-4-sulfonamide (8f). 8f was prepared in a similar manner to
Author Contributions
1
8d. 50% yield (3 steps). H NMR (600 MHz, DMSO-d6) δ 9.27 (s,
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
1H), 7.64 (s, 3H), 7.61 (s, 1H), 7.07 (s, 1H), 6.80 (s, 1H) 6.18 (s,
1H), 5.89−5.87 (m, 2H), 3.91 (t, J= 6.1 Hz, 2H), 3.84 (t, J= 6.5 Hz,
2H), 3.61 (s, 3H), 3.27 (s, 3H), 3.22 (s, 3H), 2.85−2.82 (m, 2H),
1.74−1.64 (m, 7H), 0.95 (t, J = 7.3 Hz, 3H). MS (ESI) m/z 559 [M +
H]+.
Notes
The authors declare no competing financial interest.
M
DOI: 10.1021/acs.jmedchem.5b00405
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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, 750 1−7515.
ACKNOWLEDGMENTS
■
We thank Gilbert Lee IV at the CBSF (Core for Biomolecular
Structure and Function) for providing BRPF1 purified protein.
The authors also thank James Holton and George Meigs for
technical help at the Advance Light Source synchrotron facility
at the Lawrence Berkeley National Laboratory. We thank Eun
Jeong Cho and Kevin N. Dalby at the University of Texas at
Austin and the TxSACT (Texas Screening Alliance for Cancer
Therapeutics) program for performing the TRIM24 HTS
screen. The TxSACT-New Drug Discovery program is
supported by CPRIT (Cancer Prevention Research Institute
of Texas) Grant RP110532. We thank Stefan Knapp at the
Structural Genomics Consortium for providing the template for
Figure 4, and Richard Lewis for feedback and proofreading of
the manuscript.
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ABBREVIATIONS USED
■
ACN, acetonitrile; AcOH, acetic acid; Alpha, amplified
luminescence proximity homogeneous assay; BET, bromodo-
main and extra-terminal; BCP, bromodomain-containing
protein; BOC, tert-butyl carbonate; BRD4, bromodomain
containing protein 4; BRPF1, bromodomain and plant
homeodomain finger containing 1; DCM, dichloromethane;
DMF, dimethylformamide; EtOAc, ethyl acetate; H-bond,
hydrogen bond; HTS, high throughput screening; ITC,
isothermal calorimetry; KAc, acetylated lysine; PHD, plant
homeodomain; rt, room temperature; SAR, structure−activity
relationship; SEM, standard error of the mean; SUMO, small-
ubiquitin-like modifier; TBAF, tetrabutylammonium fluoride;
TFA, trifluoroacetic acid; TRIM24, tripartite motif containing
protein 24
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Journal of Medicinal Chemistry
Article
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DOI: 10.1021/acs.jmedchem.5b00405
J. Med. Chem. XXXX, XXX, XXX−XXX