Discovery, design, and optimization of isoxazole azepine BET inhibitors

Article abstract of DOI:10.1021/ml4001485

The identification of a novel series of small molecule BET inhibitors is described. Using crystallographic binding modes of an amino-isoxazole fragment and known BET inhibitors, a structure-based drug design effort lead to a novel isoxazole azepine scaffold. This scaffold showed good potency in biochemical and cellular assays and oral activity in an in vivo model of BET inhibition.

Full text of DOI:10.1021/ml4001485

Letter
pubs.acs.org/acsmedchemlett
Discovery, Design, and Optimization of Isoxazole Azepine BET
Inhibitors
Victor S. Gehling,*,† Michael C. Hewitt,*,† Rishi G. Vaswani,† Yves Leblanc,† Alexandre Cote,†
̂
́
Christopher G. Nasveschuk,† Alexander M. Taylor,† Jean-Christophe Harmange,† James E. Audia,†
Eneida Pardo,† Shivangi Joshi,† Peter Sandy,† Jennifer A. Mertz,† Robert J. Sims, III,† Louise Bergeron,†
Barbara M. Bryant,† Steve Bellon,† Florence Poy,† Hariharan Jayaram,† Ravichandran Sankaranarayanan,‡
Sreegouri Yellapantula,‡ Nandana Bangalore Srinivasamurthy,‡ Swarnakumari Birudukota,‡
and Brian K. Albrecht†
†Constellation Pharmaceuticals, 215 First Street, Suite 200, Cambridge, Massachusetts 02142, United States
‡Jubilant Biosys Limited, #96, Industrial Suburb, Second Stage, Yeshwantpur, Bangalore 560 022, India
S
* Supporting Information
ABSTRACT: The identification of a novel series of small
molecule BET inhibitors is described. Using crystallographic
binding modes of an amino-isoxazole fragment and known
BET inhibitors, a structure-based drug design effort lead to a
novel isoxazole azepine scaffold. This scaffold showed good
potency in biochemical and cellular assays and oral activity in
an in vivo model of BET inhibition.
KEYWORDS: BET inhibitors, bromodomain, isoxazoles, fragments, MYC
he bromodomain and extra-C terminal domain (BET)
models, combined with their ability to modulate a compelling
Tfamily of proteins is made up of chromatin adaptors that subset of genes prompted us to initiate a program to discover
small molecule inhibitors of BET.8−20
contain tandem bromodomains that recognize specific
Our drug discovery efforts began with a fragment screen that
uncovered several compounds with potencies in the micro-
molar (μM) range. Several of these fragments were cocrystal-
lized with BRD4 BD1 to give high-resolution structures. Of the
cocrystal structures obtained, the amino-isoxazole 1/BRD4-
BD1 complex was of particular interest. It was observed that the
amino-isoxazole 1 mimicked the key interactions found
between BRD4-BD1 and the endogenous acetylated lysine
binding partner (Figure 1).
Comparison of the binding modes of 1 and the reported
diazepine inhibitor JQ13 (2) showed that both 1 and 2 made
similar hydrogen bonding contacts with the asparagine residue
critical for recognition of the acetyl lysine substrate and
suggested that an isoxazole could serve as a replacement for the
triazole of 2. Subsequent to our initial fragment work with
amino isoxazole 1, the 3,5-dimethyl isoxazole motif was
disclosed as a preferred binding motif for bromodomains (see
refs 9−11 and 16). Compounds 1 and 2 also filled a part of the
acetylated lysine residues in the N-terminal tails of histones.
Members of the BET family, including BRD2, BRD3, BRD4,
and BRDT, regulate gene expression by recruiting transcrip-
tional regulators to specific genomic locations.1 The proteins in
the BET family are known to control expression of genes
critical for proper cell growth, and reduction of their levels or
inhibition of their activity has profound effects on cell
proliferation and viability. Small molecule BET inhibitors
initially appeared in the patent literature2 and were followed by
publications detailing activity in models of cancer and
inflammation.3,4 The initial reports indicated that BET
inhibition led to antiproliferative effects in a BRD4-NUT
mediated midline carcinoma.3 In addition, it has been reported
that BET inhibition suppresses a subset of NF-κB dependent
inflammatory genes following LPS stimulation in macrophages,
with concomitant efficacy in a mouse model of endotoxic
shock.4 The mechanistic link between BET activity and cancer
was further developed when several groups showed that small
molecule inhibition of BET reduced transcription of the MYC
oncogene,5 resulting in cell cycle arrest and activity in vivo in
models of leukemia and lymphoma.6,7 The demonstrated
efficacy of BET inhibitors in cancer xenograft and immunology
Received: April 18, 2013
Accepted: July 16, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ml4001485 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Figure 1. Crystal structures of acetyl-lysine,21 amino isoxazole 1,22 JQ1 (2),3,23 and isoxazole azepine 324 in BRD4 BD1.
a
Scheme 1. Synthesis of Isoxazole Azepine 3
a
Reagents: (a) (i) Pd2(dba)3, SPhos, K3PO4, n-BuOH, 100 °C; (ii) MeONa (cat.), MeOH (88% over 2 steps); (b) (i) Swern oxidation, (ii)
Ti(OEt)4, (S)-t-butyl sulfinylamide (91% over 2 steps); (c) 2-(tert-butoxy)-2-oxoethyl)zinc(II) chloride, NMP, −10 °C (89%, d.r. 7:1 to 5:1); (d)
(i) HCl, MeOH, (ii) i-PrMgBr, THF (80% over 2 steps); (e) PCl5, CH2Cl2 (65%); (f) Pd(PPh3)4, K2CO3, 4-chlorophenylboronic acid, toluene/
water (10:1), 95 °C (82%); (g) (i) TFA (50 equiv), CHCl3, 36 °C, (ii) HATU, NH4Cl, Et3N, CH2Cl2 (80% over 2 steps).
B
dx.doi.org/10.1021/ml4001485 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Table 1. Isoxazole Azepine Side-Chain SAR31,32
acetyl binding pocket with a methyl group from either the
isoxazole or triazole moieties and acted as acetyl lysine mimics.
In addition, the LEAN of 1 was calculated to be 0.34 and
binding efficiency 25.7, which made the fragment an excellent
starting point for our drug discovery efforts.25−28 We
rationalized that incorporating the isoxazole motif into an
azepine scaffold would provide a concave shape similar to 2 and
afford compounds with increased potency against BRD4 as
compared to isoxazole 1. Herein, we report the genesis,
synthesis, structure−activity relationships (SARs), biochemical
potency, cellular potency, and in vivo activity of a series of
isoxazole azepine BET inhibitors.
Synthesis of compound 3 (Scheme 1) began with bromo-
thiophene 4 and isoxazole boronic ester 5, both readily
synthesized in three steps from commercially available starting
materials (see Supporting Information for experimental de-
tails). Union of 4 and 5 via Suzuki reaction and subsequent
acetate deprotection afforded primary alcohol 6. Oxidation and
imine formation with (S)-tert-butyl sulfinamide29 formed
sulfinyl imine 7. The addition of the Reformatsky enolate of
tert-butyl acetate to imine 7 afforded sulfinamine 8 and
established the requisite stereocenter, albeit with modest
diastereoselectivity (d.r. 7:1 to 5:1). The sulfinyl amine was
then deprotected under acidic conditions, and subsequent
treatment with i-PrMgBr provided lactam 9. Chlorination of 9
with PCl5 afforded imidoyl chloride 10, which was converted to
azepine 11 under standard Suzuki reaction conditions.
Deprotection of the tert-butyl ester and amide bond formation
provided desired compound 3.
The first synthetic entry in our SAR table was the direct
isoxazole analogue of JQ1 (2), tert-butyl ester intermediate 11,
which showed modest potency in our biochemical assay and
poor translation into cells (Table 1). The unexpected loss of
potency of 11 prompted us to focus on replacing the
suboptimal tert-butyl protecting group with a variety of amides
and amide isosteres. The first amide analogue we made was the
simple primary carboxamide 3, which showed excellent
biochemical and cellular potency.
a
An average of ≥3 determinations, with standard deviation (SD)
b
reported. An average of ≥2 determinations, with SD reported. Our
MOA cell assay used suppression of MYC expression in Raji cells; see
Supporting Information for details. Raji cells were chosen because they
are a MYC-dependent cancer cell line, showing potent growth
inhibition and MYC downregulation upon BET inhibitor treatment.
The next entries in Table 1 were ethyl amide 12, which
showed good cellular and biochemical potency, and enantiomer
13. Compound 13 was over 50-fold less potent than 12,
showing that activity was strongly dependent on the stereo-
chemistry of the side chain attached to the azepine core.30
Further exploration of the side-chain substituents was
performed to determine whether additional potency could be
gained in this area. Our attempts to change the nature or
location of the hydrogen bond donor were successful
biochemically, as the carbamate (14) and reversed amide
(15) retained biochemical potency. Other modifications of the
amide, including replacing the amide with a heterocycle (16
and 17) or incorporation of an α-methyl group (18) led to less
cellular potency, although the changes were tolerated biochemi-
cally. With the preliminary SAR pointing to the superiority of
the carboxamide side-chain, we obtained a crystal structure of
compound 3 (Figure 1). Like the previously reported dimethyl-
isoxazoles (PDB ID’s 4GPJ,9 3SVF,10 and 3SVG11), compound
3 also formed a hydrogen bond with Asn140 and a water
mediated hydrogen bond with Tyr 97. At 3.2 Å, the H-bonding
distance between the isoxazole and Asn140 was similar to that
observed in the literature structures (range 2.9−3.2 Å). The
ring system below the isoxazole in compound 3 was within van
der Waal distance of the Ile146 residue and wrapped around
c
d
Single measurement, no SD reported. Compound 18 had 4-
cyanophenyl instead of 4-Cl-phenyl substitution. n.t. = not tested.
this residue, which is also a common feature of the reported
bromodomain inhibitors.
Modifications to the 4-chlorophenyl ring was the next SAR
vector we examined, with the goal of modulating the
biophysical and 3-dimensional properties of our analogues,
while maintaining the potency we had achieved in analogues
such as 3 (Table 2). We began with a chlorine scan around the
aromatic ring. As might be predicted from the crystal structure,
para- or meta-chlorine substitution (3 and 19) afforded
similarly potent compounds in both biochemical and cellular
assays. ortho-Chloro substitution (20) led to a 10-fold drop in
biochemical potency, perhaps because this substitution induced
an unfavorable twist between the two unsaturated systems.
However, the ortho-methyl and para-chloro substitutions (23)
were tolerated, leading to a compound with potency similar to
that of the parent, suggesting that the ortho-chloro substituent
was not tolerated due to an unfavorable electrostatic
interaction, rather than a steric one. Other aromatic systems
were tolerated (21 and 22) but did not appear to offer any clear
C
dx.doi.org/10.1021/ml4001485 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Table 2. Aromatic SAR
Table 3. ADME and PK for Selected Compounds
compd
3
22
21
25
3
3
a
mCLint
140
27
b
c
rCLint
8.9
160
2.4
18
dCLint
hCLint
11
d
5.5
4.6
5.4
PPB (%)
mouse
98.1
99.2
99.4
20
e
rat
n.a.
95.9
0
n.a.
98.2
1
n.a.
99.4
20
human
f
CYP 3A4 (%)
PK species
rat
dog
0.32
1.27
3
mouse
723
g
CLint (L/h/kg)
2.77
2.45
1.39
605
31
4.35
2.22
0.393
504
44
g
Vss (L/kg)
g
t1/2 (h)
h
AUC (ng h/mL)
h
F (%)
a
b
Mouse liver microsomes clearance (μL/min/mg). Rat liver
c
microsomes clearance (μL/min/mg). Dog liver microsomes clearance
d
(μL/min/mg). Human liver microsomes clearance (μL/min/mg).
e
f
n.a. = not applicable, compounds not stable in rat plasma. In vitro
inhibition of cytochrome P450 3A4 isoforms, % inhibition at 10 μM.
IV 1 mg/kg. PO 5 mg/kg.
g
h
microsomal clearance (compared to 3). Compound 3 was also
profiled in vivo in mouse, as our PD assay was to be run in
mouse and dog. We saw adequate exposure in mouse, which
gave us confidence we could use our PD model to test target
engagement. As we were also interested in translation of this
chemotype into higher species, we were pleased that compound
3 showed excellent dog PK, with low clearance (0.32 L/h/kg),
moderate volume (1.3 L/kg), and a half-life of 3 h.
Our target engagement assay was based on the documented
binding of BRD4 at the MYC locus and known downregulation
of MYC transcription following small molecule BET inhib-
ition.6,7 On the basis of the PK of 3 in mouse and its high
clearance in MLM, doses of 10, 30, and 100 mg/kg PO were
chosen for the MYC PK-PD experiments. As shown in Figure
2a, a dose-dependent decrease in MYC expression was
observed, with highest suppression at 100 mg/kg. Additionally,
MYC suppression correlated with the amount of compound in
the plasma and tumor, with a ∼75% reduction in MYC levels
with nearly 5 μM compound in the tumor at 4 h after a 100
mg/kg dose. To better understand the dynamics of MYC
expression upon BET inhibition, a time course experiment was
run, which showed that dosing with 3 at 100 mg/kg led to
inhibition of MYC expression out to 8 h (Figure 2b). After 8 h,
MYC levels rebounded to vehicle levels. This was consistent
with elimination of 3 from both the tumor and plasma and
suggested that MYC expression could recover rapidly upon
compound elimination.
a
b
An average of ≥3 determinations, with SD reported. An average of
≥2 determinations, with SD reported. Our MOA cell assay used
suppression of MYC expression in Raji cells; see Supporting
Information for details. Raji cells were chosen because they are a
MYC-dependent cancer cell line, showing potent growth inhibition
and MYC downregulation upon BET inhibitor treatment. n.t. = not
tested.
advantages. Replacing the aromatic ring with 2-pyridine (24) or
amino-pyridine (25) was tolerated, but substitution with a
pyrimidine (26) was not. Replacement of the aromatic ring
with a saturated system and polar saturated system, as in 28, 29,
or 30, was also not tolerated in the highly lipophilic WPF shelf
region of the binding site.
We next profiled a selection of our isoxazole azepines in
several in vitro ADME screening assays (Table 3). Universally
the compounds showed good stability in human liver
microsomes (HLM) and were generally stable in rat liver
microsomes (RLM). The only exception was compound 21,
which showed very high clearance in RLM, likely due to the
completely exposed phenyl ring. In addition, these compounds
displayed high plasma protein binding in human plasma. The
single-point CYP inhibition also showed negligible inhibition
across the series. Overall, the profiles of compounds 3 and 22
supported further profiling in rat PK experiments.
In conclusion, we have described the identification of
fragment 1 as a micromolar inhibitor of the BET family of
proteins and the elaboration of 1 into novel isoxazole azepine 3.
Compound 3 is a potent and selective33 BET inhibitor in
biochemical and cellular assays, has a rat and dog
pharmacokinetic profile suitable for in vivo experiments, and
inhibits MYC mRNA expression in vivo after PO dosing in a
dose-dependent manner. Further studies detailing the opti-
mization and development of 3 and related compounds will be
reported in due course.
Of the two compounds profiled in vivo in rats, compound 3
showed the best profile: a t1/2 of 1.4 h, Vss of 2.45 L/kg, and a
bioavailability of 31% (Table 3). For compound 22, we had
seen instability in rat plasma in our PPB assay, which probably
contributed to the higher in vivo clearance for this compound
and might explain the discontinuity with the lower in vitro
D
dx.doi.org/10.1021/ml4001485 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Figure 2. MYC mRNA expression in Raji tumor in mice with compound 3 dosed 10 to 100 mpk PO (a) and a time course with compound 3 at 100
mpk PO (b).
dicyclohexylphosphino-2′,6′-dimethoxybiphenyl; TFA, tri-
fluoroacetic acid
ASSOCIATED CONTENT
* Supporting Information
Synthetic procedures, assay details, PK−PD assay description,
and DSF selectivity profile of compound 3. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
REFERENCES
■
(1) Wu, S. Y.; Chiang, C. M. The Double Bromodomain-Containing
Chromatin Adaptor Brd4 and Transcriptional Regulation. J. Biol.
Chem. 2007, 282, 13141−13145.
AUTHOR INFORMATION
Corresponding Author
*(V.S.G.) Tel: 617-714-0540. E-mail: victor.gehling@
constellationpharma.com. (M.C.H.) Tel: 617-714-0569. E-
mail: mike.hewitt@constellationpharma.com.
■
(2) Miyoshi, M.; Ooike, S.; Iwata, K.; Hikawa, H.; Sugahara, K.
Antitumor Compounds, WO 2009/084693A1, 9 Sept 2009.
(3) Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W. B.;
Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.;
Philpott, M.; Munro, S.; McKeown, M. R.; Wang, Y.; Christie, A. L.;
West, N.; Cameron, M. J.; Schwartz, B.; Heightman, T. D.; La
Thangue, N.; French, C. A.; Wiest, O.; Kung, A. L.; Knapp, S.;
Bradner, J. E. Selective Inhibition of BET Bromodomains. Nature
2010, 468, 1067−1073.
(4) 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.;
Tarakhovsky, A. Suppression of Inflammation by a Synthetic Histone
Mimic. Nature 2010, 468, 1119−1123.
(5) Vita, M.; Henricksson, M.; Myc, M. M. Oncoprotein As a
Therapeutic Target for Human Cancer. Semin. Cancer Biol. 2006, 16,
318−330.
(6) Delmore, J. E.; Issa, G. C.; Lemieux, M. E.; Rahl, P. B.; Shi, J.;
Jacobs, H. M.; Kastritis, E.; Gilpatrick, T.; Paranal, R. M.; Qi, J.; Chesi,
M.; Schinzel, A. C.; McKeown, M. R.; Heffernan, T. P.; Vakoc, C. R.;
Bergsagel, P. L.; Ghobrial, I. M.; Richardson, P. G.; Young, R. A.;
Hahn, W. C.; Anderson, K. C.; Kung, A. L.; Bradner, J. E.; Mitsiades,
C. S. BET Bromodomain Inhibition As a Therapeutic Strategy to
Target c-Myc. Cell 2011, 146, 904−917.
(7) Mertz, J. A.; Conery, A. R.; Bryant, B. M.; Sandy, P.;
Balasubramanian, S.; Mele, D. A.; Bergeron, L.; Sims, R. J.;
Targeting, M. Y. C. Dependence in Cancer by Inhibiting BET
Bromodomains. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 16669−16674.
(8) 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.
(9) Hay, D.; Fedorov, O.; Filippakopoulos, P.; Martin, S.; Philpott,
M.; Picaud, S.; Hewings, D. S.; Uttakar, S.; Heightman, T. D.; Conway,
S. J.; Knapp, S.; Brennan, P. E. The Design and Synthesis of 5- and 6-
Isoxazoylbenzimidazoles As Selective Inhibitors of the BET
Bromodomains. Med. Chem. Commun. 2013, 4, 140−144.
(10) Hewings, D. S.; Wang, M.; Philpott, M.; Federov, O.; Uttarkar,
S.; Filippakopoulos, P.; Picaud, S.; Vuppusetty, C.; Marsden, B.;
Knapp, S.; Conway, S. J.; Heightman, T. 3,5-Dimethylisoxazoles Act
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Ted Peters, Christina Lee, and the Lead Discovery
group at Constellation for compound management and plating
of test compounds, and Kerry Spillane for running the DSF
experiments to determine the selectivity profile of compound 3.
We also thank Richard Walter and Gina Ranieri at Shamrock
Structures LLC, 1440 Davey Road, Woodridge, IL 60517, for
data collection and processing. Data for compound 1 was
collected at Argonne National Laboratory Advanced Photon
Source, Life Science Collaborative Access Team (APS-LSCAT)
Beamline 21-ID-G.
DEDICATION
■
Dedicated to Professor Robert M. Williams on the occasion of
his 60th birthday.
ABBREVIATIONS
■
ADME, absorption, distribution, metabolism, excretion;
COMU, (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)-
dimethylamino-morpholino-carbenium hexafluorophosphate;
CYP, cytochrome P450; dba, 1,5-diphenyl-1,4-pentadien-3-
one; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethy-
luronium hexafluorophosphate; HLM, human liver micro-
somes; LPS, lipopolysaccharide; mg/kg, milligram per kilo-
gram; MLM, rat liver microsomes; NMP, 1-methyl-2-
pyrrolidinone; NF-κB, nuclear factor kappa-light-chain-en-
hancer of activated B cells; Pin, 2,3-dimethylbutane-2,3-diol;
PK−PD, pharmacokinetic−pharmacodynamic; RLM, rat liver
microsomes; SAR, structure−activity relationship; SPhos, 2-
E
dx.doi.org/10.1021/ml4001485 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
As Acetyl−Lysine−Mimetic Bromodomain Ligands. J. Med. Chem.
2011, 54, 6761−6770.
(25) Perola, E. An Analysis of the Binding Efficiencies of Drugs and
Their Leads in Successful Drug Discovery Programs. J. Med. Chem.
2010, 53, 2986−2997.
(26) Abad-Zapatero, C. Ligand Efficiency Indices for Effective Drug
Discovery. Expert Opin. Drug Discovery 2007, 2, 469−488.
(27) Bembenek, S. D.; Tounge, B. A.; Reynolds, C. H. Ligand
Efficiency and Fragment-Based Drug Discovery. Drug Discovery Today
2009, 14, 278−283.
(28) May, P. C.; Dean, R. A.; Lowe, S. L.; Martenyl, F.; Sheehan, S.
M.; Boggs, L. N.; Monk, S. A.; Mathes, B. M.; Mergott, D. J.; Watson,
B. M.; Stout, S. L.; Timm, D. E.; LaBell, E. S.; Gonzales, C. R.;
Nakano, M.; Jhee, S. S.; Yen, M.; Ereshefsky, L.; Lindstrom, T. D.;
Calligaro, D. O.; Cocke, P. J.; Hall, D. G.; Friedrich, S.; Citron, M.;
Audia, J. E. Robust Central Reduction of Amyloid-β in Humans with
an Orally Available, Non-Peptidic β-Secretase Inhibitor. J. Neurosci.
2011, 31, 16507−16516.
(29) Ellman, J. A.; Owens, T. D.; Tang, T. P. N-tert-Butanesulfinyl
Imines: Versatile Intermediates for the Asymmetric Synthesis of
Amines. Acc. Chem. Res. 2002, 35, 984−995.
(30) The stereochemical binding preference was also seen in the
thienodiazepines such as JQ1; see ref 3.
(31) In the isoxazole azepine scaffold, we saw pan inhibition across
the BET family (BRD2, 3, 4, and either bromodomain 1 or 2), so
screening against BRD4 BD1 was representative of BET inhibitory
activity.
(32) The in vitro potency data for the three literature reference
compounds (JQ1, I-BET151, and I-BET762) was generated in our
biochemical and cellular assays, under identical conditions to the test
compounds.
(11) Hewings, D. S.; Federov, O.; Filippakopoulos, P.; Martin, S.;
Picaud, S.; Tumber, A.; Wells, C.; Olcina, M. M.; Freeman, C.; 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 BET Bromodomain Ligands.
J. Med. Chem. 2013, 56, 3217−3227.
(12) Dawson, M. A.; Prinjha, R. K.; Dittman, A.; Giotopoulos, G.;
Bantscheff, M.; Chan, W. I.; Robson, S. C.; Chung, 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, P.;
Lee, K.; Huntly, B. J. P.; Kouzarides, T. Inhibition of BET Recruitment
to Chromatin As an Effective Treatment for MLL-Fusion Leukemia.
Nature 2011, 478, 529−533.
(13) 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.; Seal, J. 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.
(14) Mirquet, 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.
(33) See Supporting Information for selectivity data of compound 3.
(15) Chung, C. W.; Dean, A. W.; Woolven, 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.
(16) 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.
(17) Chung, C. W.; Herve, C.; White, J. H.; Olivier, M.; 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.
Discovery and Characterization of Small Molecule Inhibitors of the
BET Family Bromodomains. J. Med. Chem. 2011, 54, 3827−3838.
(18) Filippakopoulos, P.; Picaud, S.; Fedorov, O.; Keller, M.; Wrobel,
M.; Morgenstern, O.; Bracher, F.; Knapp, S. Benzodiazepines and
Benzotriazepines As Protein Interaction Inhibitors Targeting Bromo-
domains of the BET Family. Bioorg. Med. Chem. 2012, 20, 1878−1886.
(19) Fish, P. V.; Filippakopoulos, P.; Bish, G.; Brennan, P. E.;
Bunnage, M. E.; Cook, A. S.; Federov, O.; Gerstenberger, B. S.; Jones,
H.; Knapp, S.; Marsden, B. B.; Nocka, K. K.; Owen, D. R.; Philpott, M.
M.; Picaud, S. S.; Primiano, M. J.; Ralph, M. J.; Sciammetta, N. 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.
(20) Zhao, L.; Cao, D.; Chen, T.; Wang, Y.; Miao, Z.; Xu, Y.; Chen,
W.; Wang, X.; Li, Y.; Du, Z.; Xiong, B.; Li, J.; Xu, C.; Zhang, N.; He, J.;
Shen, J. Fragment-Based Drug Discovery of 2-Thiazolidinones As
Inhibitors of the Histone Reader BRD4 Bromodomain. J. Med. Chem.
2013, 56, 3833−3851.
(21) PDB ID 3uvw.
(22) PDB ID 4lr6.
(23) PDB ID 3mxf.
(24) PDB ID 4lrg.
F
dx.doi.org/10.1021/ml4001485 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX