Discovery of Tetrahydroquinoxalines as Bromodomain and Extra-Terminal Domain (BET) Inhibitors with Selectivity for the Second Bromodomain

Article abstract of DOI:10.1021/acs.jmedchem.7b01666

The bromodomain and extra-terminal domain (BET) family of proteins bind acetylated lysine residues on histone proteins. The four BET bromodomains - BRD2, BRD3, BRD4, and BRDT - each contain two bromodomain modules. BET bromodomain inhibition is a potential therapy for various cancers and immunoinflammatory diseases, but few reported inhibitors show selectivity within the BET family. Inhibitors with selectivity for the first or second bromodomain are desired to aid investigation of the biological function of these domains. Focused library screening identified a series of tetrahydroquinoxalines with selectivity for the second bromodomains of the BET family (BD2). Structure-guided optimization of the template improved potency, selectivity, and physicochemical properties, culminating in potent BET inhibitors with BD2 selectivity.

Full text of DOI:10.1021/acs.jmedchem.7b01666

Subscriber access provided by University of Pennsylvania Libraries
Discovery of Tetrahydroquinoxalines as Bromodomain and Extra-Terminal
Domain (BET) Inhibitors with Selectivity for the Second Bromodomain
Robert P. Law, Stephen J. Atkinson, Paul Bamborough, Chun-wa Chung, Emmanuel H Demont,
Laurie J. Gordon, Matthew Lindon, Rab K. Prinjha, Allan J. B. Watson, and David Jonathan Hirst
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01666 • Publication Date (Web): 15 Apr 2018
Downloaded from http://pubs.acs.org on April 15, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Discovery of Tetrahydroquinoxalines as
Bromodomain and ExtraꢀTerminal Domain (BET)
Inhibitors with Selectivity for the Second
Bromodomain
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
†‡*
†
§
§
Robert P. Law , Stephen J. Atkinson , Paul Bamborough , Chun-wa Chung , Emmanuel H.
†
§
†
†
‡
Demont , Laurie J. Gordon , Matthew Lindon , Rab K. Prinjha, Allan J.B. Watson , David J.
†
Hirst .
†
§
Epigenetics Discovery Performance Unit, Platform Technology and Science,
GlaxoSmithKline, Medicines Research Centre, Stevenage, Hertfordshire, SG1 2NY, U.K.
‡
WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, Thomas
Graham Building, 295 Cathedral Street, Glasgow, G1 1 XL, U.K.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
The bromodomain and extraꢀterminal domain (BET) family of proteins bind acetylated lysine
residues on histone proteins. The four BET bromodomains, BRD2, BRD3, BRD4 and BRDT,
each contain two bromodomain modules. BET bromodomain inhibition is a potential therapy
for various cancers and immunoinflammatory diseases, but few reported inhibitors show
selectivity within the BET family. Inhibitors with selectivity for the first or second
bromodomain are desired to aid investigation of the biological function of these domains.
Focused library screening identified a series of tetrahydroquinoxalines with selectivity for the
second bromodomains of the BET family (BD2). Structureꢀguided optimization of the
template improved potency, selectivity and physicochemical properties, culminating in potent
BET inhibitors with BD2 selectivity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INTRODUCTION
Bromodomain containing proteins (BRDs) are epigenetic ‘reader’ proteins, an important
class of translation mediators which bind to acetylated lysine (KAc) residues on the Nꢀ
1
ꢀ4
terminal tails of histones and recruit transcriptional regulator complexes to chromatin. To
date, 46 human proteins have been found to contain one or more bromodomain motifs, with
5
61 distinct bromodomains identified. The bromodomain and extraꢀterminal (BET) family of
proteins consists of BRD2, BRD3, BRD4, and BRDT, each of which contain two
bromodomains (BD1 and BD2). The four BD1 domains show a high degree of structural
2
homology, as do the four BD2 domains.
BRD2, 3, and 4 are ubiquitously expressed, whereas BRDT is confined to the testes and
6
ꢀ8
ovaries and is crucial for spermatogenesis. BET BRDs recruit RNA polymerase II to
9
ꢀ10
chromatin and polymeraseꢀassociated factor complex (PAFc) complexes,
thus initiating
2
ACS Paragon Plus Environment

Page 3 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
transcription. BRD4 also binds positive transcription elongation factor (PTEFꢀb) through its
11
Cꢀterminal domain, causing transcriptional elongation.
Dysregulation of BET family members has been linked to diverse disease phenotypes.
BET proteins facilitate the transcription of tumor growthꢀpromoting and antiꢀapoptotic
1
ꢀ2, 5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
12
13
genes, including cꢀMyc, which is activated by superꢀenhancer regions that recruit BRD4
1
4
at higher than normal levels. The squamous cell epithelial cancer nuclear protein in testis
(NUT) midline carcinoma (NMC) is driven by the formation of a BRD4ꢀNUT fusion
1
5
oncogene; smallꢀmolecule BRD4 inhibition caused growth arrest and apoptosis in NMC
16
17ꢀ20
cells. Smallꢀmolecule BET inhibitors have shown efficacy against a range of cancers,
21
and several inhibitors have entered clinical trials in oncology.
BET inhibition has been shown to potently modulate inflammatory autoimmune gene
22ꢀ23
expression.
BET bromodomains control the differentiation of CD4+ T cells to
inflammatory Th17 cells through genomic localization and their effects on cytokine
24
regulation. Treatment of naïve CD4+ cells with BET bromodomain inhibitors suppressed
expression of proinflammatory cytokines whilst also upregulating antiꢀinflammatory gene
25
products, mimicking cMyc inhibition. In human monocytes, BET inhibition suppressed the
transcriptional response of the Janus kinase/signal transducers and activators of transcription
2
6
(
JAKꢀSTAT) pathway to cytokine stimulation, downregulated proꢀinflammatory cytokine
2
7
28
29
production in dendritic cells, macrophages, and rheumatoid arthritis synovial fibroblasts.
In vivo, BET inhibitors have shown therapeutic antiꢀinflammatory effects in rodent models of
30
31
32
33
34
acute sepsis, periodontitis, psoriasis, arthritis and pathologic bone loss. Furthermore,
BET inhibitors have been shown to reactivate human immunodeficiency virus (HIV) from
11, 35ꢀ37
38ꢀ39
latency,
and are in clinical investigation for the treatment of atherosclerosis.
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Selected BET inhibitors. Acetylated lysine mimetics are highlighted.
Diverse BET inhibitor scaffolds have been reported to interact with the KAc binding site
5, 21, 40ꢀ42
(Figure 1).
All feature a KAc mimic which binds to a conserved Asn residue in a
similar manner to the native Nꢀacetyl peptide. Among BET inhibitors, the triazolodiazepine
chemotype has been extensively explored, as exemplified by the clinical compound Iꢀ
3
0, 43ꢀ44
16
BET762 (1)
and widelyꢀused probe JQ1 (2). Additionally, the tetrahydroquinoline Iꢀ
4
5
BET726 (3) was discovered through phenotypic screening of Apolipoprotein A1 (ApoA1)
upregulators, and subsequently shown to function as a BET bromodomain inhibitor. 1ꢀ3 are
panꢀBET inhibitors which exhibit little or no selectivity between the 8 bromodomains of the
BET family. Whilst more selective inhibitors are less well explored, there has been recent
progress in this field (vide infra).
4
ACS Paragon Plus Environment

Page 5 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
While the sequence homology of the four dualꢀBRD BET bromodomains is high,
differences exist between the BD1 and BD2 domains which allow selectivity to be achieved
46
(
Figure 2). For BRD4, the ‘gatekeeper’ residue, which restricts access to the TrpꢀProꢀPhe ꢀ
bordered region termed the WPF shelf, switches between I146 in BD1 and V439 in BD2,
with a Q85/K378 switch at the base of the ZA channel. The greatest differences lie in the BC
loop, with D144 in BD1 exchanged for H437 in BD2 and an aspartic to glutamic acid switch
of the adjacent residue. Similar differences exist between BD1 and BD2 amongst BRD2/3/T,
as shown in the sequence alignment in Figure 2.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Top: Comparison of BRD4 BD1 (2YEL, cyan) and BD2 (2YEM, magenta) with
47
triazolodiazepane GW841819X bound. Surface over the BD1 protein (pale cyan) has been
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
shown to highlight key residue differences in the BC loop (D144/H437; D145/E438), ZA
channel (Q85/K378) and the gatekeeper residues (I146/V439) between BRD4 BD1 (blue
residue labels) and BD2 (magenta residue labels). Bottom: Sequence alignment of BET
bromodomains with the residues highlighted.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Olinone (4) utilizes a clash with the BD2ꢀspecific H437 to obtain 3.4 µM affinity for BRD4
48
BD1 with no BRD4 BD2 activity detected, while the acylpyrrole 5 showed 0.24 µM affinity
4
9
for BRD4 BD1 with 10ꢀfold selectivity over other members of the BET family. The
diazobenzene MS611 (6), in which the phenol forms the KAc mimetic, also showed BRD4
46, 48
BD1 K = 0.41 µM and 100ꢀfold selectivity over BRD4 BD2.
RVXꢀ208 (7), currently in
D
39
Phase III clinical trials for cardiovascular diseases, was elucidated to be a BET inhibitor
38, 50
with K
D
= 4 µM and ~20ꢀfold selectivity for BRD3 BD2 over BRD3 BD1;
additional
51ꢀ52
analogues of this series have also shown BD2ꢀselectivity and therapeutic effects in vivo
.
The selectivity is postulated to be due to differences in the structural dynamics of the
BD1/BD2 ZA loop regions, with 7 increasing contact between the ZA and BC loops in BD2,
5
3ꢀ54
and interaction of 7 with the BD2ꢀspecific His.
Using a ‘bumpꢀandꢀhole’ chemical
biology strategy with mutant bromodomain modules and suitably modified inhibitors, Ciulli
55ꢀ56
et al. engineered domain selective inhibition.
During this work, it was observed that the
indole 8 displayed 20ꢀfold selectivity for wildꢀtype BRD2 BD2 over BRD2 BD1, though this
was reduced to ~10ꢀfold at BRD4. The domain selectivity was attributed to improved edgeꢀ
toꢀface interactions of the indole ring with the BD2ꢀspecific His, compared to the
5
6
chlorophenyl of 1. Weak (~6ꢀfold) BRD4 BD2 bias was also observed for the
5
7
benzoisoxazoloazepine inhibitor CPIꢀ0610, currently in Phase I clinical trials. In the patent
58
literature, Abbvie have disclosed compounds with selectivity for both BRD4 BD1 and BD2,
59
with structures related to the pyrrolopyridone ABBVꢀ075/mivebresib.
6
ACS Paragon Plus Environment

Page 7 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
In an intriguing example of single BET bromodomain specificity, the xanthine derivative 9
exhibited 5 µM affinity for BRD4 BD1 with 10ꢀfold bias over other BD1 domains and no
detectable BD2 binding. Differences in the dynamic movement of the ZA loop were
60
hypothesized as the cause of selectivity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
These chemical tools have begun to provide insight into the roles of individual domains.
Using a mutated BRD4 bromodomain, the bumpꢀandꢀhole strategy demonstrated that
5
6
inhibition of BD1 alone was able to prevent chromatin binding to BRD4. Selective BD1
inhibition with 4 promoted oligodendrocyte progenitor differentiation; whilst in contrast, panꢀ
48
BET inhibition hindered it. In a HepG2 cell line 7 weakly displaced BRD3 from chromatin,
and affected only a small subset of genes compared to JQ1 (2). Furthermore, 7 was unable to
38
produce a strong transcriptional response in a subset of these genes, and had no effect on
48
oligodendrocyte progenitor differentiation.
These initial studies indicate that domain selective inhibition may exhibit differentiated
pharmacology compared to panꢀBET inhibition, yet BD2 selective inhibitors are poorly
explored. In addition, the phenotypic effect of a small molecule target should ideally be
61
confirmed with multiple chemically distinct series, but the potency and selectivity of 7ꢀ8 are
limited. To corroborate these emerging results and further investigate the biological role of
BD2, additional BD2ꢀselective BET inhibitors are desired from alternative chemotypes with
improved potency and selectivity.
RESULTS AND DISCUSSION
Discovery of the Tetrahydroquinoxaline Scaffold.
A previous fragmentꢀbased screen carried out in our laboratories identified
tetrahydroquinoline 10 (Figure 3) which bound weakly but with high ligand efficiency (LE)
6
2
to BRD2ꢀ4. Separately, optimization of the tetrahydroquinoline scaffold via phenotypic
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
assays identified appropriate vectors for potency, culminating with 3, which was
4
5
subsequently identified as a panꢀBET inhibitor (pIC50 BRD4 BD1 = 7.8 and BD2 = 8.2).
This work led to the investigation of similar chemotypes for BET bromodomain binding.
Tetrahydroquinoxalines 11ꢀ13, also upregulators of ApoAꢀ1, were screened against both
bromodomains of BRD4 in fluorescence resonance energy transfer (FRET) assays, and
showed encouraging potency, LE and BD2 selectivity. At the outset of this work, these
represented novel BET inhibitor chemotypes; the acyltetrahydroquinoxaline template
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
63
including 12 was also independently optimized by Forma Theraputics, culminating in a
6
4
series of 1,4ꢀdiacyl tetrahydroquinoxaline panꢀBET inhibitors. In addition, the structurally
distinct tetrahydroquinoline scaffold of 10 has been investigated by both ourselves and others
21, 65ꢀ66
to afford BET inhibitors with variable selectivity towards the BD2 domains.
Figure 3. Structures and potencies of initial hit compounds. pIC50 data is n=4 or greater
unless stated. Ranges shown are standard deviations. a) n=2.
We obtained an Xꢀray structure of 13 bound to BRD4 BD2 (Figure 4), which showed the
acetyl group of 13 behaving as the KAc mimetic, as seen for 3. The amide carbonyl formed
hydrogen bonds to N433 and a throughꢀwater interaction to Y390. The tolyl group is placed
between the WPF stack (W374, P375 and F375) and the BD2ꢀspecific H437 mimetic in a
similar manner to 3. Given that 3 occupies the WPF shelf yet is a panꢀBET inhibitor, the
8
ACS Paragon Plus Environment

Page 9 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
differing angle of the ring in 13 may be crucial for BD2 selectivity. The unsubstituted
tetrahydroquinoxaline ring of 13 can be seen to pucker towards a small lipophilic pocket, as
occupied by the methyl group of 3. Finally, THQ 3 derives additional potency by deploying a
6ꢀaryl group into the ZA channel. This is almost identical in BD1 and BD2 and additional
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
lipophilic binding and πꢀinteractions with the WPF tryptophan led to a similar increase in
potency at both domains.
a.
b.
Figure 4. a) Xꢀray crystal structures of 13 bound to BRD4 BD2 (cyan, PDB code:6FFD, 1.83
Å, blue residue labels), overlaid with IꢀBET726 (3) bound to BRD2 BD2 (orange, PDB code:
4UYG, 2.5 Å, orange residue labels). b) 13 bound to BRD4 BD2 (cyan, 1.83 Å, blue residue
labels), overlaid with IꢀBET726 (3) bound to BRD2 BD1 (yellow, PDB code: 4UYF, 1.6Å,
orange residue labels).
As such, there appeared three main vectors for optimization of 11ꢀ13 into a BD2 selective
tool. Firstly, the small lipophilic pocket would be explored with a screen of small alkyl
groups at the 2ꢀposition of the tetrahydroquinoxaline ring. Alternative substitution patterns of
the phenyl ring interacting with the WPF shelf would be screened, to probe their influence on
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
selectivity. Lastly, the ZA channel would be explored. It was expected that targeting this site
45
would not increase selectivity, but could enhance potency, as seen for 3.
We set out to investigate these vectors individually, before combining the optimal
substituents to give final compounds. Optimization was initially carried out on racemic
compounds, with the role of individual enantiomers investigated once potent and selective
compounds were obtained (vide infra).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthetic Chemistry
A strategy of lateꢀstage diversification was chosen to enable rapid library synthesis
1
(
Scheme 1). To access the tetrahydroquinoxaline core where R = Me, Et or cPr, a S Ar
N
reaction between fluorobenzene 14 and amino acids 15 gave the amino esters 17 after
esterification. Reduction of the nitro group and cyclisation with SnCl gave quinoxalinones
8, which could be reduced to tetrahydroquinoxalines 19 with borane. The steric bulk of the
ꢀsubstituent and electronꢀwithdrawing nature of the bromide allowed differentiation between
2
1
2
the two amines, with Bocꢀprotection affording 20 as the major product. Acetylation followed
by Boc deprotection gave 22, with synthetic handles at the 4ꢀ and 6ꢀpositions.
a
Scheme 1. Representative synthesis of building blocks 22aꢀc.
1
0
ACS Paragon Plus Environment

Page 11 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
Reagents and conditions: (i) K
two steps; (iii) SnCl , EtOH, reflux, 90ꢀ97%; (iv) BH
DMAP, Et N, DCM, rt, 53ꢀ69%; (vi) Ac O, Et N, DCM, reflux, 51ꢀ89%; (vii) HCl, DCM, ꢀ
0 ˚C, 62ꢀ90%.
2
CO
3
, DMF, 80 ˚C; (ii) SOCl
2
, MeOH, reflux, 70ꢀ82% over
2
3
•THF, THF, 60 ˚C, 90ꢀ95%; (v) Boc
2
O,
3
2
3
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To access compounds lacking a 6ꢀsubstituent with a variety of alkyl groups at the
2
ꢀposition, the 6ꢀBr moiety was removed by hydrogenation to give 23aꢀc (Scheme 2). The 2ꢀ
H analogue 23d was synthesized by acetylation of tetrahydroquinoxaline 24. Reductive
amination or S 2 displacement of a benzylic bromide gave the target compounds. For
N
examples bearing a benzoic acid, the coupling reaction was undertaken using the
corresponding ethyl esters and the product hydrolyzed to afford the final compounds 28aꢀd.
a
Scheme 2. Representative synthesis of 6ꢀH compounds.
a
Reagents and conditions: (i) Flow hydrogenation, 10% Pd/C, H , MeOH, rt, 93ꢀ97%; (ii)
2
Ac
2
O, EtOH, 0 ˚C – rt, 95%; (iii) Benzylic bromide, K
2
3
CO , DMF, 90 ꢀ 110 ˚C, 17ꢀ79%; (iv)
Aryl aldehyde, NaBH(OAc)
3
, DCM, rt, 43%; (iv) LiOH, H
2
O, MeOH, THF, rt, 40ꢀ73%.
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To explore the WPF shelf, a set of analogues bearing a 2ꢀcPr substitution were designed.
These could be accessed from 23c in a similar manner (Scheme 3). The trisubstituted
benzoic acid 31 was synthesized by Pdꢀcatalyzed carbonylationꢀesterification of the
corresponding bromide 29 with Mo(CO) , followed by saponification of the resulting ester to
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
give 31. The nitrile 36 was converted to the primary amide 37 using H O and K CO and to
2
2
2
3
the benzylamine 38 by hydrogenation over Raney Ni. Phenol 40 was alkylated with 2ꢀ
bromoethanol to afford the hydroxyethanol analogue 41.
a
Scheme 3. Synthesis of WPF shelf array.
a
Reagents and conditions: (i) Benzylic bromide, K
, DCM, rt, 21ꢀ85%; (iii) Mo(CO)
DIPEA, 1ꢀbutanol, 1,4ꢀdioxane, µW, 150 ˚C, 26%; (iv) LiOH, H O, MeOH, THF, rt, 58ꢀ
2
CO
3
, DMF, 90ꢀ110 ˚C, 42ꢀ85%; (ii)
Benzylic aldehyde, NaBH(OAc)
3
6
, Herrmann′s catalyst,
2
83%; (v) H
2
O
2
, K
2
CO
3
, DMSO, rt, 77%; (vi) Flow hydrogenation, Raney Ni, 50 bar H
2
,
EtOH, 50 ˚C, 32%; (vii) 2ꢀBromoethanol, K
2
CO , DMF, 110 ˚C, 50%.
3
6
ꢀAryl compounds were synthesized via benzylation of 2ꢀEt,6ꢀBr tetrahydroquinoxaline
2b to give 46, followed by SuzukiꢀMiyaura coupling (Scheme 4). Amidopyrimidine 57 was
synthesized by H /K CO oxidation of nitrile 56. For 5ꢀmembered heterocycles yields with
2
2
O
2
2
3
1
2
ACS Paragon Plus Environment

Page 13 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Pd(dppf)Cl
2
/Cs
2
CO
3
were low, and modified conditions were used for isoxazole 58 and
pyrazole 59. The use of the Bocꢀprotected pyrazole improved the stability of the boronic
ester, with the Bocꢀgroup being removed from the product under the reaction conditions. The
N-Boc tetrahydropyridine 61 was deprotected with TFA to give 62.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 4. Synthesis of 6ꢀaryl compounds.
a
Reagents and conditions: (i) BnBr, K CO , DMF, 90 ˚C, 56%; (ii) ArB(OH) or ArBPin,
2
3
2
Pd(dppf)Cl
0%; (iv) TFA, DCM, rt, 96%; (v) Isoxazoleꢀ4ꢀboronic acid pinacol ester, Pd(dppf)Cl
DIPEA, 1,4ꢀdioxane, H O, µW, 110 ˚C, 43%; (vi) (1ꢀ(Boc)ꢀ1 ꢀpyrazolꢀ4ꢀyl)boronic acid
pinacol ester, Pd (dba) , XPhos, K PO , 1ꢀbutanol, µW, 115 ˚C, 38%.
2 2 3 2 2 2 2 3
, Cs CO , 1,4ꢀdioxane, H O, µW, 110 ˚C, 7ꢀ96%; (iii) H O , K CO , DMSO, rt,
7
2
,
2
H
2
3
3
4
62
Only one enantiomer of fragment hit 10 was seen to crystallize in BRD4 BD1, and
separation of the two enantiomers of 44 by preparative chiral high performance liquid
chromatography (HPLC) showed only one enantiomer had significant activity (Table 2). This
was assumed to be the (
Figure 4), as such single enantiomer quinoxalinones were synthesized (Scheme 5). Cuꢀ
S)ꢀenantiomer based on the structure of 3 and crystallography of 10
(
catalyzed Ullman couplingꢀcyclisation of bromoaniline 63 and (S)ꢀcyclopropylglycine (S)ꢀ
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
7
68
15c gave the quinoxalinone core (64), which was reduced to 65. To confirm enantiopurity
at this stage, 65 was hydrogenated to 66 for comparison with racemic material, and exhibited
9
7:3 er by chiral HPLC. Protection followed by acetylation and deprotection gave 69,
followed by alkylation with benzylic bromide 70. This route necessitated the use of a 6ꢀCl
substituent to avoid regiochemical issues during the Ullmann coupling, consequently a
reassessment of the SuzukiꢀMiyaura coupling conditions was required. BrettPhos G3
palladacycle produced efficient coupling to afford pyrimidine 72 and tetrahydropyridine 73.
Removal of the TBDMS protecting group from 72 with TBAF, followed by nitrile oxidation
gave the pyrimidine amide 74, while global deprotection of 73 with HCl afforded the
tetrahydropyridine 75. Chiral HPLC of 75 (see Supporting Information) showed no
racemization had occurred during the synthesis.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 5. Synthesis of single enantiomer products.
1
4
ACS Paragon Plus Environment

Page 15 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
3
Reagents and conditions: (i) CuCl, DMEDA, DBU, DMSO, 110 ˚C, 73%; (ii) BH •THF,
THF, 60 ˚C, 79%; (iii) 10% Pd/C, H
5%; (v) Ac O, Et N, 2ꢀMeTHF, reflux; (vi) TFA, DCM, rt , 82% over two steps; (vii) 70,
NaH, DMF, 0 ˚C – rt, 80%; (viii) RꢀBPin, BrettPhos Pd G3, Cs CO , DME, 110 ˚C, ꢁW, 2 h,
2 2 3
, EtOH, rt, 100%; (iv) Boc O, DMAP, Et N, DCM, rt,
8
2
3
2
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
4
3ꢀ69%; (ix) TBAF, THF, rt, 88%; (x) H O , K CO , DMSO, rt, 46%; (xi) HCl, MeOH, rt,
2 2 2 3
7%.
Medicinal Chemistry
We began our optimization campaign by altering the 2ꢀsubstituent and WPFꢀshelf binding
motif. We postulated that a small alkyl substituent at the 2ꢀposition of the core could improve
potency due to the proximity of the WPF shelfꢀoccupying aryl ring to the basic histidine
(
Figure 4), and an acidic group could boost selectivity through an ionic interaction. To test
these hypotheses, 2ꢀsubstituents were screened, each with a phenyl, m-tolyl or oꢀbenzoic acid
WPF shelf group (Table 1).
In our hands, RVXꢀ208 (7) exhibited ~4ꢀfold selectivity for BRD4 BD1 over BRD4 BD2
38
(
Table 1 and Table S1), consistent with previous reports, while the tetrahydroquinoxaline
examples showed good potency and higher selectivity at BRD4 BD2. Initially, it was
pleasing to observe a 0.5 log increase in BD2 potency with the addition of a
mꢀmethyl group
1
to the shelf aryl group (13 vs 25d) in the absence of an R substituent. Furthermore, the
1
addition of an R = Me vs H, substituent also led to a similar increase in potency when the
shelf group was phenyl (12 vs 25d). Disappointingly, combining the methyl groups of 12 and
13 to give 26a did not improve potency, suggesting these two positions could not always be
optimized independently. The benzoic acid moiety (28aꢀd) generally did not produce a
significant increase in BD2 potency but did decrease BD1 potency. Furthermore, increasing
1
the size of the R substituent proved beneficial ꢀ methyl (a) and ethyl (b) groups were broadly
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
similar, with ethyl examples showing marginally higher potency but lower selectivity.
However, a cyclopropyl group (c) conferred good selectivity across all three substituents; the
2ꢀcyclopropyl acid 28c had BD1 potency below the detectable limit of the FRET assay (50
µM), giving a BD2 selectivity of >63ꢀfold. Despite the unusually high potency and selectivity
of 13 amongst the 2ꢀH examples, in general substitution at the 2ꢀposition was required for
acceptable potency. Apart from benzoic acids 28aꢀd, all compounds were highly lipophilic at
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
69
pH 7.4 as measured by chromatographic LogD (ChromLogD7.4), and we set reduction of
lipophilicity as an additional optimization goal in order to improve developability
70
parameters.
Table 1. Biochemical potency and lipophilicity of initial screen of 2ꢀsubstituents and WPF
shelf binding groups.
a
BRD4 FRET pIC50
BD2
1
2
Cmpd
R
R
Selectivity ChromLogD7.4
BD1
BD2
(fold)
(
+)JQ1 (2)
ꢀ
ꢀ
ꢀ
ꢀ
7.4 ± 0.23
7.8 ± 0.10
7.3 ± 0.19
8.2 ± 0.13
ꢀ
7.3
3.9
IꢀBET726
3)
RVXꢀ208
7)
3
4
(
ꢀ
ꢀ
5.2 ± 0.01
5.8 ± 0.06
2.9
(
1
6
ACS Paragon Plus Environment

Page 17 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
25d
H
5.0 ± 0.07
4.8 ± 0.14
5.6 ± 0.04
6.3 ± 0.54
5.3 ± 0.07
6.3 ± 0.25
6.0 ± 0.08
5.8 ± 0.05
6.2 ± 0.03
6.1 ± 0.03
6.1 ± 0.07
6.2 ± 0.05
6.1 ± 0.08
6.1 ± 0.04
4
32
>10
32
20
25
16
16
40
32
32
>63
5.6
5.9
1.2
5.9
6.5
1.5
6.5
7.1
1.9
6.7
7.2
2.2
13
H
mꢀMe
b
28d
oꢀCO
2
H
<4.3
12
H
4.8 ± 0.16
4.7 ± 0.05
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
6a
8a
Me
Et
mꢀMe
b
2
oꢀCO
2
H
4.4
25b
H
5.0 ± 0.15
4.9 ± 0.06
4.5 ± 0.17
4.7 ± 0.07
4.6 ± 0.03
2
6b
8b
mꢀMe
2
2
oꢀCO H
2
5c
6c
H
2
cPr
mꢀMe
oꢀCO
c
28c
2
H
<4.3
Ranges shown are standard deviations. a) All compounds are n = 3 or greater. b) Below
assay detection limit (50 µM). c) <4.3 on two additional test occasions.
a)
b)
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
c)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5 a) Xꢀray crystal structure of 28c (purple, PDB code:6FFE) bound to BRD2 BD2
purple), overlaid with 13 (cyan) bound to BRD4 BD2 (cyan); b) View looking down into the
(
KAc pocket with surface of BRD2 BD2 of 28c complex shown in grey; bound 28c (purple)
and 13 (cyan); c). Hydrogen bonding of acid of 28c with D434 shown as yellow dashed lines.
An Xꢀray structure of 28c bound to BRD2 BD2 was obtained and compared to that of 13
(Figure 5a). The larger 2ꢀcPr group of 28c reduces the tetrahydroquinoxaline ring pucker,
flattening it and pushing the core closer to the opposite edge of the KAc pocket, which is
smaller in BD1 due to the larger I146. Although 28c was tested as the racemate, only the (S)ꢀ
62
enantiomer was observed bound to the protein (as also seen with 10 ). The acid of 28c forms
a throughꢀwater interaction with D434, though in BRD4 BD2 this residue is a glutamic acid
and so may be unable to form this interaction. As such, the selectivity gain from this
interaction may be minimal (Figure 5c). The BD2ꢀspecific His causes the aryl to adopt a
vertical alignment on the WPF shelf, with better contact against W370 of the WPF stack.
This subtle cooperativity between the 2ꢀsubsitituent and the WPF shelfꢀbinding group
1
8
ACS Paragon Plus Environment

Page 19 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
rationalizes the nonꢀadditive elements of the initial SAR, such as the lower potency of 2ꢀ
methyl mꢀtolyl analogue 26a compared to 12 and 13.
With these encouraging results in hand, further substitution of the aryl moiety was
investigated with the 2ꢀcyclopropyl group in place (Table 2). The ethyl ester of 28c (27c)
displayed similar BD2 potency but increased BD1 potency, indicating that a polar or charged
group at C2 confers low BD1 potency. Combination of the tolyl and benzoic acid
functionalities (31) was detrimental to BD2 potency, while addition of the p-Cl functionality
seen in 1 (33) or a methylene spacer (35) had no significant effect on binding. While the
nitrile 36 showed poor selectivity and reduced potency, the corresponding amide 37 was
pleasingly potent and selective due to a significant reduction in BD1 potency, indicating that
the beneficial effect of the orthoꢀsubstituent is not ionic in nature. Indeed, the basic
compounds 38 and 39 showed high BD2 potencies and reasonable selectivity, as did the
neutral phenols 40, 42 and methoxy 43.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Moving the phenol to the mꢀposition (42) reduced BD2 potency. The benzylic alcohol 44
gave high selectivity and BD2 potency, again with potency reduced on moving substitution to
the m-position (45). Separation and testing of the two enantiomers of 44 (44a and 44b)
showed the biological activity is derived solely from one enantiomer, the Xꢀray structures of
similar compounds (not shown) and single enantiomer synthesis support this as the 2ꢀ(S)
enantiomer. Due to concerns about the metabolic stability of the benzylic position, the
ethylene glycol derivative 41 was tested, and found to maintain the high potency and
selectivity of 44. Importantly, the inclusion of hydrogen bond donor functionality
successfully reduced lipophilicity compared to the cyclopropylꢀphenyl analogue 25c, but the
lipophilicity of the highly selective examples 41 and 44 was still high, and further reductions
were desired.
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Optimization of the WPF shelf binding group.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
BRD4 FRET pIC50
BD2
Cmpd
R
ChromLogD7.4
BD1
BD2
Selectivity
27c
4.9 ± 0.03
6.0 ± 0.08
13
>63
20
7.2
2.2
2.4
2.9
2.4
6.0
3.9
2.7
7.5
5.2
b
28c
<4.3
6.1 ± 0.04
5.6 ± 0.06
5.9 ± 0.13
6.0 ± 0.08
5.6 ± 0.01
5.9 ± 0.17
6.2 ± 0.04
6.3 ± 0.03
6.3 ± 0.05
b
31
33
35
<4.3
4.4 ± 0.05
32
b
<4.3
>50
10
36
4.6 ± 0.10
b
37
38
39
<4.3
>40
32
4.7 ± 0.03
4.7 ± 0.07
4.7 ± 0.18
40
40
40
41
4.6 ± 0.07
6.5 ± 0.06
79
4.9
4
4
2
3
4.8 ± 0.12
4.8 ± 0.07
4.5 ± 0.10
6.2 ± 0.08
6.4 ± 0.08
6.3 ± 0.08
25
40
63
4.9
6.9
4.9
(±)ꢀ44
2
0
ACS Paragon Plus Environment

Page 21 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
4
4a
4.8 ± 0.06
6.7 ± 0.05
79
ꢀ
4.7
4.7
4.7
b
b
44b
<4.3
<4.3
4
5
4.6 ± 0.13
6.1 ± 0.09
32
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a) All compounds are n = 3 or greater. Ranges shown are standard deviations. b) Below
assay detection limit (50 µM).
With increased selectivity achieved, attention turned to increasing potency and
investigating the effect of substitution at the 6ꢀposition, which had been shown to increase
potency for 3. With the shelf binder and 2ꢀsubstituent set as phenyl and ethyl groups,
substitution at the 6ꢀposition was explored with the goal of protruding through the ZA
channel and establishing πꢀinteractions with the WPF tryptophan (Table 3). We focused on
aryls and partially saturated rings, in particular heteroaryls, to further reduce lipophilicity and
71ꢀ72
improve the developability profile.
Table 3. Optimization of the 6ꢀaryl substituent.
a
BRD4 FRET pIC50
BD2
Cmpd
R
ChromLogD7.4
BD1
BD2
Selectivity
25b
H
5.0 ± 0.15
5.6 ± 0.10
6.2 ± 0.03
6.5 ± 0.02
16
8
6.5
7.4
46
Br
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
47
48
Ph
5.8 ± 0.16
6.4 ± 0.18
6.6 ± 0.14
7.3 ± 0.14
6
8
7.9
4.4
49
6.3 ± 0.11
7.2 ± 0.12
8
4.7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
1
5.8 ± 0.13
6.1 ± 0.15
7.0 ± 0.17
7.1 ± 0.07
16
10
5.6
3.5
5
52
6.2 ± 0.07
5.3 ± 0.16
7.2 ± 0.08
6.3 ± 0.18
10
10
4.5
5.4
5
3
4
5
5.7 ± 0.11
5.5 ± 0.14
6.6 ± 0.25
6.9 ± 0.07
6.8 ± 0.19
7.0 ± 0.17
16
20
3
6.1
4.9
6.3
55
56
57
5.7 ± 0.05
7.0 ± 0.05
20
3.6
58
5.8 ± 0.06
6.4 ± 0.04
5.8 ± 0.07
5.5 ± 0.10
6.9 ± 0.03
7.3 ± 0.06
6.8 ± 0.13
6.8 ± 0.12
13
8
5.9
4.2
6.5
2.8
59
6
6
0
2
10
20
a) All compounds are n = 3 or greater. Ranges shown are standard deviations.
2
2
ACS Paragon Plus Environment

Page 23 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Introduction of a bromide at the 6ꢀposition (46) increased potency at both bromodomains
compared to 25b, though more so at BD1. The phenyl analogue 47 was less selective and
highly lipophilic. With the paraꢀposition believed to be solvent exposed, polar groups were
incorporated here to reduce unfavorable interactions with solvent and reduce lipophilicity.
Primary amides 48 and 49 gave a considerable improvement in potency to nanomolar levels,
with a concomitant large drop in ChromLogD. Pyridine 50 maintained high potency but
regained some BD2 selectivity compared to the phenyl 47, as such our attention turned to
heterocycles. Pyridone 51 showed a marginal drop in selectivity compared to 47, though
lipophilicity was significantly reduced. Combination of the pyridine and amide functionalities
gave 52, which maintained the BD2 potency of 48 and selectivity of 50. oꢀSubstitution (53)
was detrimental, possibly due to disruption of the water network at the base of the ZA
channel or unfavorable increase of the dihedral angle between the core and 6ꢀsubstituent. It
was thought that the lone pair of pyridines 50ꢀ52 may either interact with the water network
at the base of the ZA channel or point upwards into solvent, reducing unfavorable
interactions with aqueous solvent. With this in mind, pyrazine 54 and pyrimidine 55 were
tested and found to improve selectivity compared to 50, though with reduced BD2 potency.
The pyrimidine 55 showed better selectivity and was further examined. Introduction of a
nitrile substituent (56) boosted potency but almost completely abolished selectivity, whereas
conversion of 56 to the primary amide 57 reꢀestablished selectivity with BD2 potency
maintained. The reasons for this dramatic effect are unclear, though several residues beyond
the ZA channel differ between BD1 and BD2 and the highly directional lone pair of nitrile 56
may be making a BD1ꢀspecific interaction. 5ꢀMembered heterocycles (58 and 59) were
potent and polar but less selective compared to 6ꢀmembered variants. The partially saturated
analogues 60 and 62 broadly maintained potency compared to the aryls, with
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
tetrahydropyridine 62 also being reasonably selective and exhibiting reduced ChromLogD7.4
due to the introduction of a basic center.
With good potency, selectivity and ChromLogD7.4 profiles, the amidopyrimidine group of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
57 and the tetrahydropyridine group of 62 were combined with the previously identified (S)ꢀ
2
ꢀcyclopropyl and ortho-CH OH substituents. After synthesis as single enantiomers (Scheme
2
5), the resulting compounds 74 and 75 were profiled in biochemical, physicochemical and
cellular assays (Table 4).
a
Table 4. Extended profiles of lead compounds 74 and 75.
GSK268, 74
GSK340, 75
5.5 ± 0.13 / 7.2 ±
0.07 (x50)
5
5
.7 ± 0.11 / 7.3 ±
BRD4 BD1/BD2 FRET pIC50 (xSel)
BRD2 BD1/BD2 FRET pIC50 (xSel)
BRD3 BD1/BD2 FRET pIC (xSel)
0
.21 (x40)
.8 ± 0.09 / 7.0 ±
.10 (x16)
5.6 ± 0.20 / 6.5 ±
0.06 (x8)
0
6.1 ± 0.17 / 7.5 ±
5.9 ± 0.11 / 7.3 ±
0.10 (x25)
5
0
0.11 (x25)
2
4
ACS Paragon Plus Environment

Page 25 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
6.3 ± 0.10 / 7.0 ±
5.8 ± 0.11 / 6.7 ±
BRDT BD1/BD2 FRET pIC50 (xSel)
BRPF1 FRET pIC50
0.03 (x5)
<4
0.05 (x8)
4.7 ± 0.10
8.7
Rat Hepatocyte Cl
i
(mL/min/g liver)
22.9
1.6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Human Hepatocyte Cl (mL/min/g liver)
1.8
i
ChromLogD7.4
Artificial Membrane Permeability (nm/s)
CLND Aqueous Solubility (µM)
FaSSIF Solubility (ꢁg/mL)
3.0
2.3
135
87
110
418
18
386
hPBMC LPSꢀinduced MCPꢀ1 Release
pIC50
7.2 ± 0.23 (n=6)
7.4 ± 0.19 (n=6)
6.0 ± 0.04 (n=4)
hWB LPSꢀinduced MCPꢀ1 Release pIC50
6.3 ± 0.15 (n=4)
a) pIC50 data are n=3 or greater. Ranges shown are standard deviations. The human
biological samples were sourced ethically and their research use was in accord with the
terms of the informed consents.
Whilst both 74 and 75 showed good BRD4 BD2 potency, tetrahydropyridine 75 was
marginally more selective than amidopyrimidine 74 at BRD4. Across the BET family
selectivity was lower for both examples, with high potency and reasonable selectivity for
BRD3 BD2 but reduced affinity and selectivity for BRD2 and BRDT. This is in contrast to 7,
which exhibits significantly higher domain selectivity at BRD3 compared to BRD2, 4 and T.
The lower selectivity at BRD2 and BRD3 may be due to subtle changes in the shape of the
KAc binding site and WPF shelf, caused by minor residue differences in the ZA and BC
8
loops. BRDT is expressed only in the testes and ovaries and so is of lesser relevance to
cellular phenotype studies. Regardless, in our hands 74 and 75 exhibit improved BD2
2
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
potency and selectivity compared to 7 (Table 1), provide an alternative chemotype of BD2ꢀ
selective BET inhibitors, and offer insights into the rational design of BD2ꢀselective BET
inhibitors.
The ChromLogD7.4 and artificial membrane permeability (AMP) of both compounds were
good, though 75 was significantly more soluble in both thermodynamic and kinetic solubility
assays. 74 exhibited high and moderate clearance in rat and human hepatocytes respectively,
while for 75 in vitro clearance was improved, though still high in rat, and similar to 74 in
human. The generally high in vitro clearance likely limits the use of 74 and 75 to in vitro
experiments. Both compounds were cell permeable and active in inhibiting MCPꢀ1 release
from human peripheral blood mononuclear cells (PBMCs) and whole blood (hWB)
stimulated with lipopolysaccharide (LPS), consistent with reported effects of BET
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
2
bromodomain inhibition. The level of human whole blood inhibition is noteworthy as it
exceeds the biochemical and biophysical BD1 potencies that have been observed for both 74
and 75. The decreased potency in hWB compared to the FRET and PBMC potency can be
attributed to plasma protein binding, and is of a typical magnitude for these assays. Taking
plasma protein binding into account, these data offer good evidence to suggest that inhibition
of the BD2 domain is sufficient to drive this response. The opposite enantiomers (R)-74 and
(R)-75 showed significantly reduced binding at both bromodomains (see Supporting
Information). Binding to BRD4 BD1 and BD2 was also confirmed by surface plasmon
resonance (SPR), which showed good correlation with FRET potencies (see Supporting
Information).
Xꢀray crystal structures of 74 and 75 bound to BRD2 BD2 were obtained and showed very
similar binding (Figure 6). As expected, the 6ꢀsubstituent occupies the ZA channel and packs
against the WPF motif, making πꢀinteractions with W370, while the primary amide of 74 and
the tetrahydropyridine nitrogen of 75 interact with the water network at the exit of the ZA
2
6
ACS Paragon Plus Environment

Page 27 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
channel. The benzylic alcohol is capable of forming a hydrogen bond to the BD2 conserved
H433, in addition to solvent. The lack of this interaction in the Xꢀray structure of 74 indicates
this may be a relatively weak interaction.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a)
b)
Figure 6. Xꢀray structure of a) 74 (green, PDB code:6FFF) bound to BRD2 BD2 and b) an
overlay of the two binding modes of 75 (yellow, PDB code:6FFG) found in the crystal
structure with BRD2 BD2. Water molecules are shown as red spheres and hydrogen bonds as
yellow lines. Key residues are highlighted.
75 was screened against a panel of 35 bromodomains using the DiscoveRx BROMOscan™
73
assay platform (Figure 7, Table 5). The screen showed lowꢀnanomolar potency at each of
the BET BD2 domains and good selectivity against BD1, particularly BRD4 where over 220ꢀ
fold selectivity was recorded. Domainꢀselectivity was lesser against the other BET BRDs,
particularly BRD2 and BRD3, as seen in the FRET assays. Selectivity over nonꢀBET
bromodomains was excellent, with the only significant interaction being bromodomain and
PHD fingerꢀcontaining protein 1 (BRPF1), with a ~360ꢀfold window of selectivity compared
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
to BRD4 BD2. A pIC of 4.7 (316ꢀfold selectivity) was obtained when 75 was screened in a
50
45
BRPF1 FRET assay (Table 4). As has been reported previously for BET inhibitors, the
BromoSCAN assay appeared more sensitive than the FRET assays, differences in the
selectivity ratios may be attributed to differences in the protein constructs utilized, and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
d
inherent differences between pIC50 and K measurements.
Figure 7. DiscoveRx BROMOscan™ bromodomain crossꢀscreen of 75, showing K as
d
circles. Targets with activity <3000 nM are shown. Targets with grayed out names were not
screened.
2
8
ACS Paragon Plus Environment

Page 29 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 5. DiscoveRx BROMOscan™ pK values for 75.
D
Target
BRD4 BD1 / BD2 (xSel)
pK
D
5.82 / 8.18 (x229)
5.89 / 7.49 (x40)
6.04 / 7.72 (x48)
5.72 / 7.68 (x91)
5.62 (x363)
4.82
BRD2 BD1 / BD2 (xSel)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
BRD3 BD1 / BD2 (xSel)
BRDT BD1 / BD2 (xSel)
BRPF1 (xSel vs BRD4 BD2)
TAF1(2)
WRD9(2)
4.82
ATAD2A/B, BAZ2A/B, BRD1, BRD7, BRD8, BRD9,
BRPF3, CECR2, CREBBP, EP300, FALZ, GCN5L2,
PBRM1(2/5), PCAF, SMARCA2/4, TAF1L(2),
TRIM24(Bromo/PHD), TRIM33(Bromo/PHD),
<
4.52
To further probe the functional cellular effects of BD2ꢀselective inhibition, selected
compounds with BD2 selectivity >30ꢀfold were profiled in LPSꢀstimulated human PBMCs
(
Table 6). Compounds inhibited the release of the proinflammatory cytokine MCPꢀ1 with
pIC50 values significantly higher than the observed BRD4 BD1 potencies, and a general
correlation with BRD4 BD2 activity. Notably, 35 and 28c exhibited MCPꢀ1 inhibition with
no detectable BRD4 BD1 activity. These data are indicative of BD2ꢀdomainꢀdriven
pharmacology and show that BD2ꢀselective BET inhibitors are able to modulate aspects of an
immunoꢀinflammatory response in human cells.
Table 6. Biochemical FRET potency and cellular activity for BD2ꢀselective compounds.
a
Compound
BRD4 FRET pIC50
BD2 Selectivity (Fold)
PBMC LPS
2
9
ACS Paragon Plus Environment