Discovery of N-Ethyl-4-[2-(4-fluoro-2,6-dimethyl-phenoxy)-5-(1-hydroxy-1-methyl-ethyl)phenyl]-6-methyl-7-oxo-1 H-pyrrolo[2,3-c]pyridine-2-carboxamide (ABBV-744), a BET Bromodomain Inhibitor with Selectivity for the Second Bromodomain

Article abstract of DOI:10.1021/acs.jmedchem.0c00628

The BET family of proteins consists of BRD2, BRD3, BRD4, and BRDt. Each protein contains two distinct bromodomains (BD1 and BD2). BET family bromodomain inhibitors under clinical development for oncology bind to each of the eight bromodomains with similar affinities. We hypothesized that it may be possible to achieve an improved therapeutic index by selectively targeting subsets of the BET bromodomains. Both BD1 and BD2 are highly conserved across family members (>70% identity), whereas BD1 and BD2 from the same protein exhibit a larger degree of divergence (a?40% identity), suggesting selectivity between BD1 and BD2 of all family members would be more straightforward to achieve. Exploiting the Asp144/His437 and Ile146/Val439 sequence differences (BRD4 BD1/BD2 numbering) allowed the identification of compound 27 demonstrating greater than 100-fold selectivity for BRD4 BD2 over BRD4 BD1. Further optimization to improve BD2 selectivity and oral bioavailability resulted in the clinical development compound 46 (ABBV-744).

Full text of DOI:10.1021/acs.jmedchem.0c00628

Subscriber access provided by BIU Pharmacie | Faculté de Pharmacie, Université Paris V
Article
Discovery of N-ethyl-4-[2-(4-fluoro-2,6-dimethyl-phenoxy)-5-(1-
hydroxy-1-methyl-ethyl)phenyl]-6-methyl-7-oxo-1H-pyrrolo[2,3-
c]pyridine-2-carboxamide (ABBV-744), a BET bromodomain
inhibitor with selectivity for the second bromodomain.
George S. Sheppard, Le Wang, Steven D. Fidanze, Lisa A. Hasvold, Dachun Liu, John K.
Pratt, Chang H. Park, Kenton L. Longenecker, Wei Qiu, Maricel Torrent, Peter Kovar, Mai
Bui, Emily J Faivre, Xiaoli Huang, Xiaoyu Lin, Denise Wilcox, Lu Zhang, Yu Shen, Daniel
H Albert, Terrance J. Magoc, Ganesh Rajaraman, Warren M Kati, and Keith F. McDaniel
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00628 • Publication Date (Web): 23 Apr 2020
Downloaded from pubs.acs.org on April 24, 2020
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Discovery of N-ethyl-4-[2-(4-fluoro-2,6-dimethyl-phenoxy)-5-(1-hydroxy-1-methyl-
ethyl)phenyl]-6-methyl-7-oxo-1H-pyrrolo[2,3-c]pyridine-2-carboxamide (ABBV-744), a BET
bromodomain inhibitor with selectivity for the second bromodomain.
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,†
,†
George S. Sheppard,* Le Wang,* Steven D. Fidanze, Lisa A. Hasvold, Dachun Liu, John K.
‡
Pratt, Chang H. Park, Kenton Longenecker, Wei Qiu, Maricel Torrent, Peter J. Kovar, Mai Bui,
‡
Emily Faivre, Xiaoli Huang, Xiaoyu Lin, Denise Wilcox, Lu Zhang, Yu Shen, Daniel H. Albert,
‡
‡
Terrance J. Magoc, Ganesh Rajaraman, Warren M. Kati and Keith F. McDaniel
Oncology Discovery, AbbVie Inc., 1 North Waukegan Road, North Chicago, IL 60064, United
States
Abstract
The BET family of proteins consists of BRD2, BRD3, BRD4, and BRDt. Each protein contains
two distinct bromodomains (BD1 and BD2). BET family bromodomain inhibitors under clinical
development for oncology bind to each of the eight bromodomains with similar affinities. We
hypothesized that it may be possible to achieve an improved therapeutic index by selectively
targeting subsets of the BET bromodomains. Both BD1 and BD2 are highly conserved across
family members (>70% identity), whereas BD1 and BD2 from the same protein exhibit a larger
degree of divergence (~40% identity), suggesting selectivity between BD1 and BD2 of all family
members would be more straightforward to achieve. Exploiting the Asp144/His437 and
Ile146/Val439 sequence differences (BRD4 BD1/BD2 numbering) allowed the identification of
compound 27 demonstrating greater than 100-fold selectivity for BRD4 BD2 over BRD4 BD1.
Further optimization to improve BD2 selectivity and oral bioavailability resulted in the clinical
development compound 46 (ABBV-744).
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Introduction
The BET (Bromodomain and ExtraTerminal) family of proteins, consisting of BRD2,
BRD3, BRD4 and BRDt, has attracted interest as an epigenetic target for drug discovery.1,2
Family members each contain two structural elements known as bromodomains which comprise
a bundle of 4 helices with connecting loops. The bromodomains bind to acetylated lysine
sidechains on chromatin and other proteins, with this recognition element playing a part in the
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assembly of multi-component protein complexes involved in gene transcription. The
involvement of BET family proteins in super enhancers is emerging as a biologically significant
role for these proteins.4,5 Many of the interacting transcription factors and their target genes are
associated with disease pathways, leading to considerable drug discovery efforts against these
6
targets and the clinical development of multiple compounds for a variety of indications. Results
reported to date provide some encouraging signals of clinical activity, but with toxicities
believed to be mechanism-based at similar doses. There is thus a need for improved agents and
or treatment protocols to provide greater benefits to patients.6
The two bromodomains of each BET protein differ in sequence, and when clustered by
sequence the set of four N-terminal or BD1 bromodomains display much higher similarity with
each other than to the set of four BD2 bromodomains. Understanding the roles and binding
partners for each bromodomain is an active area of research, limited in part by a lack of widely
available molecular probes with selectivity between the BET bromodomains. Some biologically
important interactions have been established to involve interactions of both BET bromodomains
with the same partner protein, for example the interaction of BRD4 with the acetylated Lys310
7
of RelA in recruitment of NF-B. In other contexts, the roles can be demonstrated to be
distinct. Selective inhibition of BD1 has been reported to promote differentiation of
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oligodendrocyte progenitors, while selective inhibition of BD2 had no effect, and inhibition of
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both hindered differentiation. BD1-selective inhibition is also reported to selectively block
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Th17 cell differentiation over Th1, Th2 and Treg cells. Detailed biochemical and genetic
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studies of the interaction of BRD4 with diacetylated Twist indicated that the Twist interaction
occurs through BD2, while BD1 interacts with acetylated histone H4.10 A recent study with
“
clickable” probes confirmed the differential relative affinity of BRD4 BD1 and BD2 for
chromatin.11 The greater role for BD1 in chromatin binding was also indicated in studies using a
1
2
‘
bump and hole” approach, which in addition found differing dependencies on BD2 for gene
expression among the BET-family members. Another study has reported that only BD1 of
BRD3 leads to association of BRD3 with acetylated transcription factor GATA1.13 In light of
the potential for differentiated biology and better tolerated agents, we were interested in
discovering BET-BD2 selective compounds with appropriate properties for pharmaceutical
development.
Most reported BET bromodomain inhibitors have similar affinity for all bromodomains
of the family; in contrast, they exhibit limited binding to non-BET family bromodomains. These
will be referred to as Dual-bromodomain BET inhibitors throughout the text. The first
generation of BET bromodomain inhibitors that have entered clinical development for oncology
1
4
15
indications are Dual-bromodomain BET inhibitors, including OTX-015 1, Ten-010 2, I-
1
6
17
18
19
20
21
BET762 3, CPI-610 4, ABBV-075 5, BMS-986158 6, INCB054329 7, INCB057643 8,
2
2
and PLX51107 9. (Figure 1) An exception to this trend is the clinical development compound
RVX-208 10 (Figure 2), being developed for cardiovascular indications and reported to be
selective for the BD2 bromodomains. A small number of compounds reported to display
selectivity within the BET bromodomains have been reported in the scientific literature. Among
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these are compounds reported to be selective for the bromodomains of BRD4 over other BET
family proteins,23, 24 as well as compounds reported to be selective for the first bromodomain of
each BET-family member.8, 9,25-28 Compounds reported to be selective for the second
bromodomain of each BET family member have begun to appear in the literature, (Figure 2,
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Table 1) such as 10, 11, 12, 13, 14, 15 and 16. Additional BD1-selective
compounds36-41 and BD2-selective compounds42-46 have been reported in the patent literature,
although the basis for selectivity of these compounds is yet to be described. Interestingly,
PROTAC AT-1 has been reported to achieve a degree of selectivity in degrading BRD4 in
4
7
preference to other BET proteins, potentially providing a different approach to bromodomain
selectivity.
While the BD2-selective compounds reported in the literature demonstrate that some
degree of selectivity is achievable (Table 1), we sought to identify compounds with both greater
potency and selectivity, as well as ADME properties suitable for in vivo evaluation. In this paper
we describe our successful efforts to identify a BD2-selective BET bromodomain inhibitor from
the pyrrolopyridone chemotype used in our clinical Dual-bromodomain BET inhibitor ABBV-
0
75.
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N
N
N
N
N
N
OH
N
N
N
O
O
O
S
S
N
NH2
N
H
N
N
MeO
N
H
1
1
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Cl
Cl
Cl
1 (OTX-015)
2 (TEN-010)
3 (I-BET762)
N
O
O
O
H
N
N
N
N
N
NH2
N
N
N
HO
O
F
O O
S
O
N
F
H
Cl
4
(CPI-610)
N
5 (ABBV-075, mivebresib)
6 (BMS-986158)
N
O
O
H
O
N
N
N
HO C
2
O
HN
O
N
N
O
N
N
S
N
O
O
O
7
(INCB054329)
8 (INCB057643)
9 (PLX51107)
Figure 1. Dual-bromodomain BET inhibitors of known structure in clinical development.
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N
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O
O
OH
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H CO
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O
H CO
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O
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3
N
NH
NH
H N
N
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CH O
CH O
3
3
Cl
10 (RVX-208)
11 (RVX-297)
12
N
N
N
O
O
OH
N
N
N
N
O
O
NH
N
O
NH
O
N
N
MeO
N
N
HN
O
N
H
Cl
13
14
15
16
Figure 2. BD2-selective BET bromodomain inhibitors described in the literature.
Table 1. BD2-Selective BET Bromodomain Inhibitors
Published BRD4 bromodomain affinity
Compound
Selectivity
BD1 (nM)
BD2 (nM)
(
fold)
2
5a
10
11
2.8
1,142
1,160
13
2,020
3,200
520
1.3
135
20
6
610
63
b
8.5
58
2
3.3
50
10
11
c
d
1
1
1
2
3
e
4f
g
1
5
50
7.3
h
16
80
a
b
c
d
K by TR-FRET, ref 18; K by ITC, ref 29; IC by AlphaScreen, ref 30; IC by
i
D
50
50
e
f
g
AlphaScreen, ref 31; IC by TR-FRET, ref 32; IC by TR-FRET, ref 33; K by ITC, ref 34;
5
0
50
D
h
K by FP, ref 35.
i
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Compound Evaluation
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Final compounds were evaluated for binding to the isolated first and second
bromodomains of BRD4 using a time-resolved fluorescence energy transfer (TR-FRET) assay to
measure K values as described in the Experimental Section. Results for this assay reported in
i
the tables are the geometric mean ± standard error of three independent measurements unless
otherwise noted. Cellular activity was evaluated in 5-day cell growth assays using SKM-1 and
H1299 cells. During the course of the project, the growth of AML-derived SKM-1 cells was
found to be inhibited following 5-day treatment with BD2-selective inhibitors. In contrast,
H1299 cells were observed to be minimally impacted by BD2 inhibition, and growth inhibition
was measurable only in the presence of Dual-bromodomain inhibitors or when the concentration
of the BD2-selective inhibitor reached a level wherein BD1 was also engaged.48 The activity
differential between cell lines was more pronounced at later time points, and 5 day assays were
chosen as a practical balance between greater differentiation and experimental efficiency. The
ratio of activity in these cell lines was used as a rough measure of cellular selectivity to inform
iterative compound optimization. Results from the cellular assays are reported as the average of
two independent determinations unless otherwise noted. Compounds were also evaluated for
stability in the presence of human, rat and mouse liver microsomes, and evaluated in a PAMPA
permeability assay to provide a high throughput survey of potential ADME properties and
inform selection of compounds for pharmacokinetic analysis.
Results and Discussion
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Examination of the protein sequences of the small-molecule binding site of the BET
family bromodomains (Figure 3) revealed that many of the positions have identical amino acids
across the family, with very few residues unique to a single family member. This observation
suggested that targeting a single bromodomain in the family would be challenging. There are,
however, a number of positions where the BD1 bromodomains have a common composition
while the BD2 bromodomains have a different common composition. We hoped these positions
would represent an opportunity to achieve selectivity for the set of BD2 bromodomains relative
to the BD1 bromodomains. As shown for the two bromodomains of BRD4 (Figure 3), many of
these amino acid differences are distal to the binding site occupied by compound 5 and other
BET inhibitors, or have the sidechains directed away from the binding site. Due to their
proximity, the Asp144/His 437 and Ile146/Val439 differences (BRD4 BD1/BD2 numbering)
were of particular interest.
0
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9
0
Figure 3. Left: BET bromodomain sequence comparison in the binding site region. Residues
constant for a given position are underlined, residues unique for the position among the family
are in bold italic, residues consistent among the 4 BD1 domains are in blue, residues consistent
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among the BD2 domains are shown in red. The Asp144/His 437 and Ile146/Val439 differences
(
BRD4 BD1/BD2 numbering) are boxed for emphasis Right: Location of amino acid pair
differences in the binding site region, with residues Asp144/His 437 and Ile146/Val439 again
1
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boxed for emphasis. Compound 5 bound to BRD4 BD1 (salmon, resolution 2.14 Å, PDB code
5
UVW) superimposed with the BRD4 BD2 protein (green, resolution 1.53 Å, PDB code 5UVX).
Sidechains of identical residues are omitted for clarity.
During the medicinal chemistry campaign which led to the identification of the Dual-
bromodomain BET inhibitor ABBV-075 (mivebresib, 5), a number of substitutions at the C-2
position on the pyrrolopyridone ring were examined. When reviewing the selectivity of
compounds prepared for the project, amide 17 stood out as one of the few analogs observed to
display reproducible BD2-selective binding beyond the MSR of the assay (Table 2). After
mono-alkylation of the primary amide of compound 17 with an ethyl group, the resulting
compound 18 showed slightly better BD2 selectivity. However, di-alkylation of the amide of
compound 17 to provide 19 completely changed the BD1/BD2-selectivity landscape. In fact,
compound 19 exhibited slight BD1 selectivity. Compound 20, bearing a slightly longer ethyl
sulfone, showed similar selectivity to 17 and 18. These initial amide-containing compounds
have excellent cellular activity against the SKM-1 cell line, and compounds 17 and 18
demonstrated differential activity between the sensitive and insensitive cell lines. The
compounds also exhibited suitable in vitro unbound clearance and permeability despite the
addition of additional polar functionality to the pyrrolopyridone core.
Table 2. Initial Amide SAR Around 2-Position of Pyrrolopyridone Core
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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
O
R1
N R2
H
N
N
A
D
O
O
F
B
C
R3
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
S
F
O
O
TR-FRET (BRD4)
a
Selectivity
BD1/BD2
Clint,u in liver microsomes
K
i
, (nM)
SKM-1
H1299
PAMPA
(L/(h·kg))c
Compound
R
1
R
2
R
3
IC50
(µM)
IC50
(µM)
-6
(10
b
b
BD1
BD2
(
fold)
cm/s)
Human
4.6
Mouse
<7.2
Rat
2
.4
1
1
7
8
H
H
H
-Me
8.6 ± 0.6
1.2 ± 0.1
7
0.010
0.60
<4.1
(MDCK)
-Et
-Et
-Et
-Me 21.6 ± 14
1.3 ± 0.3
40 ± 1.9
0.7 ±0.1
17
0.3
9
0.0066
0.056
0.031
0.37
0.39
0.11
3.6
3.8
3.0
<5.5
17.3
<6.2
<3.1
16.9
11.3
10.8
19
-Me -Me
-Et
13 ± 1.2
6.6 ± 1.0
16.0
3.18
2
0
H
a
TR-FRET K values are reported as the geometric mean derived from 3 independent
i
b
measurements unless otherwise indicated by the number of replicates in parentheses. IC
5
0
c
values are reported as the mean derived from two independent measurements. For reference,
average in-house positive control values for dextromorphan and verapamil, respectively, are
.
.
Clint,u (human) = 7.4 L/(h kg) (low) and Cl (human) = 30 L/(h kg) (high).
int,u
The X-ray co-crystal structures of compound 18 in the binding site of BRD4 BD1 and
BD2 were solved, providing hypotheses for the origin of the modest selectivity between the two
bromodomains (Figure 4). Many of the interactions with the bromodomain proteins are shared,
including hydrogen bonding interactions with the key Asn sidechain, positioning of the methyl
group at the 6-position of the pyrrolopyridone in an amphipathic water network, occupation of
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the WPF shelf region by the aryl ether ring and positioning of the sulfone in a cleft adjacent to
the ZA-loop where it accepts a hydrogen bond from a backbone NH. As highlighted in the space
filling views in Figure 4, bottom panel, the ethyl amide of compound 18 is making van der
Waals contact with the hydrophobic sidechains of amino acids Tyr432 and His437 in BRD4
BD2, which form a cleft. In BRD4 BD1, the position corresponding to His437 is Asp144 whose
sidechain points away from the ligand, leading to a less favorable interaction. Based on the
structure of 18 bound to BRD4 BD2, a rationalization for the reduced BD2 affinity of the tertiary
amide 19 is that while methylation of the amide would disrupt the hydrogen bond to the
asparagine amide, forcing the amide to rotate significantly to avoid a clash in either
bromodomain, this would position the alkyl groups in a more crowded region adjacent to His437
in BD2 vs a more open area adjacent to Asp144 in BD1.
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
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2
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 4. X-ray co-crystal structures of 18 with BRD4 BD1 (top, resolution 2.45 Å, PDB code
6VIW) and BRD4 BD2 (bottom, resolution 2.13 Å, PDB code 6VIX). The left panels show
hydrogen bonds predicted by Schrödinger Maestro between ligand and protein as dotted lines.
The hydrogen bonding network of the amphipathic water network is shown in the lower left
panel. The right panels show a space filling representation.
An additional observation from these X-ray co-crystal structures concerned the position
of the 2,4-difluorophenoxy portion of 18, which differs slightly in the two co-crystal structures.
In the BD1 structure, the aryl moiety is canted toward the tryptophan of the WPF shelf and away
from Ile146, whereas in the BD2 structure the aryl group is located closer to His437 and Val439.
As noted in Figure 3, the Ile/Val difference is consistent across the BET family, and this
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particular position is a part of the rigid pocket and buried deeply in the binding site. We
explored this subtle residue difference for the improvement of the BD2 selectivity by placing
appropriate substituents in the aryl ether region of the lead structure. While the size differential
is modest, a favorable precedent exists in the exploitation of the difference between valine and
isoleucine in COX-2 inhibitors related to SC-58125.50 The results of varying this portion of the
molecule are summarized in Table 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
Table 3. C-ring SAR
O
H
N
D
HN Et
O
N
A
O
Ar
B
R1
S
O
O
TR-FRET (BRD4)
a
Clint,u in liver microsomes
(L/(h·kg))
c
K
i
, (nM)
Selectivity
BD1/BD2
(fold)
SKM-1
H1299
PAMPA
-6
Compound
R
1
Ar
IC50
(nM)
IC50
(nM)
(10
b
b
BD1
BD2
cm/s)
3.68
13.8
Human
8.5
Mouse
66
Rat
2
2
1
2
-Et
-Et
8.4 ± 1.2
6.5 ± 0.4
0.8 ± 0.2
0.5 ± 0.1
11
13
3.3
3.3
95
NV
72
Cl
340
12
97
72
2
2
3
4
-Et
22 ± 0.7
2.1 ± 0.1
10
21
460
NV
86
5.6
-Et
-Et
-Et
-Et
13 ± 0.7
30 ± 0.9
45 ± 1.4
0.9 ± 0.1
0.9 ± 0.1
1.0 ± 0.1
15
35
2.5
6.2
9.8
5.2
260
670
730
450
9.0
15.3
16
NV
NV
113
42
152
127
81
15.4
2.73
10.5
7.68
F
25
Cl
Br
2
2
6
7
45
1
20 ± 48 1.1 ±0.48
112
5.4
64
(10)
(8)
2
8
-Et
400 ± 36
15 ±0.6
27
290
3,200
NV
NV
NV
NV
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4
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
29
-Et
-Et
69 ± 2.9
3.2 ± 0.1
5.8 ± 0.8
0.7 ± 0.1
21
81
37
28
730
41
49
54
92
12
15.3
15.2
NC
4
70 ± 20
2)
3
3
0
1
1,200
4.5
(
F3C
-Et
8.3 ± 0.5
12
120
67
2.6
12
20
94
32
5.5
18
96
33
97
39
3.1
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
32
-Me
140 ± 9.5 1.2 ± 0.1
38 ± 0.5 0.6 ±0.1
790
730
10.7
8.33
3
3
103
107
a
TR-FRET K values are reported as the geometric mean derived from 3 independent
i
b
measurements unless otherwise indicated by the number of replicates in parentheses. IC
5
0
c
values are reported as the mean derived from two independent measurements. For reference,
average in-house positive control values for dextromorphan and verapamil, respectively, are
.
.
Clint,u (human) = 7.4 L/(h kg) (low) and Cl (human) = 30 L/(h kg) (high).
int,u
When the 2-position of the phenyl C-ring is substituted with a small group, such as
methyl (21) or chlorine (22), compounds maintained potent activity against BRD4 BD2 and
modest BD2 selectivity. Incorporation of a larger t-butyl group (23) caused slight loss of both
BD1 and BD2 activity. It was quickly appreciated, because of unrestricted rotation of the C-ring
around the oxygen, that both the 2- and 6-positions of the phenoxyl C-ring needed to be occupied
to ensure there would be always one substituent pointing into the floor of the pocket formed by
the Ile (BD1) and Val (BD2) sidechains. Thus, several 2,6-disubstituted phenyl C-ring analogs
were synthesized and tested. When a fluorine atom was attached to the 6-postion of the C-ring
(24), no improvement for BRD4 BD2 selectivity was noted. When either a 2-chlorine (25) or 2-
bromine (26) atom was introduced paired with a methyl at the 6-position, better BD2 selectivity
was achieved. Gratifyingly, introduction of the 2,6-dimethyl phenyl C-ring (27) provided BD2
selectivity of more than 100-fold. More importantly, compound 27 maintained potent cellular
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3
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5
6
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activity against the SKM-1 cell line and adequate microsomal stability. Using a larger t-butyl
group (28) or a polar nitrile substituent (29) to replace one methyl group of 27 resulted in a
significant erosion of BD2 activity, BD2 selectivity, and cellular activity. It was clear that the
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
,6-disubstitution pattern of the substituents on the phenyl moiety is critical to achieve BD2
selectivity in this context, because when a 3,5-dimethyl phenyl C-ring was incorporated,
compound 31 lost significant BD2 selectivity. When a methyl sulfone replaced the ethyl sulfone
(32), BD2 selectivity was comparable to that of compound 27, but its cellular activity against
SKM-1 dropped slightly. However, when the slightly bigger cyclopropyl sulfone is used (33),
decreases in both BD2 selectivity and cellular activity against SKM-1 were noted.
Table 4. Examination of Linker Between B- and C-rings
O
H
N
O
N
A
D
HN
X
Ar
B
R1
TR-FRET (BRD4)
a
Clint,u in liver microsomes
K
i
, (nM)
Selectivity
BD1/ BD2
(fold)
SKM-1
H1299
PAMPA
(10
c
-6
Compound
R
1
Ar
X
IC50
IC50
(L/(h·kg))
b
b
BD1
BD2
(nM)
(nM)
cm/s)
10.7
3.59
3.81
9.0
Human Mouse
Rat
39
3
3
2
4
-SO
-SO
2
Me
Me
O
140 ± 9.5
250 ± 8.6
1.2 ± 0.1
4.3 ± 0.1
120
58
11
68
790
3,600
4,200
>10,000
2,600
3,900
5.5
7.0
320
33
33
29
2
NH
26
1,300
37
35
H
CH
2
2,100 ± 100 25 ± 1.5
83
150
1,300
9.5
2,300
NV
45
3
3
3
6
7
8
-SO
-SO
-SO
2
Et
Et
Et
-
-
-
>12,000
180 ± 8.5
170 ± 11
22 ± 0.4
1.1 ± 0.1
0.9 ± 0.1
>580
160
178
2
2
6.7
8.4
21
10.3
3.5
HN
34
31
14
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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
3
9
-SO
-SO
2
2
Et
Et
N
N
N
-
-
140 ± 3.3
380 ± 7.3
1.8 ± 0.1
1.1 ± 0.1
78
50
23
1,300
1,900
27
42
130
110
38
93
15.9
14.7
40
344
4
1
-SO
2
Et
-
9.0 ± 0.7
0.5 ± 0.1
18
4.2
340
NV
NV
NV
NV
a
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
TR-FRET K values are reported as the geometric mean derived from 3 independent
i
b
measurements unless otherwise indicated by the number of replicates in parentheses. IC
5
0
c
values are reported as the mean derived from two independent measurements. For reference,
average in-house positive control values for dextromorphan and verapamil, respectively, are
.
.
Clint,u (human) = 7.4 L/(h kg) (low) and Cl (human) = 30 L/(h kg) (high).
int,u
Having identified 2,6-dimethyl phenyl as an optimal C-ring, the linker between the B-
and C-rings was also examined, with the results listed in Table 4. When the oxygen of
compound 32 was replaced with an NH group, the resulting compound 34 showed a modest drop
in BD2 activity and selectivity. However, its cellular activity showed a 6-fold decrease. Using a
methylene group (35) to replace the oxygen did not offer any benefit, as overall activity
weakened substantially and microsomal stability was negatively impacted. Interestingly, when a
biphenyl moiety was incorporated, completely removing the ether linkage, compound 36
exhibited excellent BD2 selectivity compared to compound 27, albeit at the expense of its BD2
binding affinity and subsequent cellular activity. However, removal of one methyl group
resulted in compound 37 with restored BD2 activity and cellular anti-proliferative activity in
SKM-1. The BD2 selectivity of compound 37 was also on par with that of compound 27,
indicating that in contrast to the observations with diaryl ethers, 2,6-disubstitution was not
required for differentiation with the more rigid biaryl. Several biaryl heterocycles were also
investigated. Although compound 40 showed improved BD2 selectivity over compound 27, its
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cellular activity and microsomal stability were adversely impacted. Reducing the indole of
compound 40 to indoline rendered compound 41 with much diminished BD2 selectivity. In
general, bi-aryl compounds 37-41 also tended to demonstrate poorer solubility compared to the
bi-aryl ether counterparts.
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
Table 5. Pharmacokinetics of Compound 27 in Mouse, Rat, Dog, and Monkey
IV
PO
Cl mic, u
Species
Dose
t1/2 Vss
AUC
CLp
Dose
t1/2
AUC
F
(L/(h·kg))
(
mg/kg) (h) (L/kg) (ngh/mL) (L/(h·kg)) (mg/kg) (h) (ngh/mL) (%)
Mouse
Rat
42
64
12
32
1
1.0
1.4
3.7
1.5
0.8
3.5
1.7
2.3
1,440
384
291
89
0.69
2.71
0.34
1.2
1
1
1
760
27
53
7
1
1
Dog
0.1
0.1
0.3
0.3
0.3
644
0
74
0
Monkey
Since compound 27 stood out as a BD2-selective analog with acceptable ADME
properties, its pharmacokinetic properties were studied in multiple species (Table 5). While
compound 27 showed adequate PK profiles in mouse and dog, its PK profiles in rat and monkey
were rather disappointing. Specifically, 27 demonstrated high clearance, poor oral exposure, and
single digit bioavailability in dog. Due to the fact that compound 27 was metabolized
extensively by enzyme CYP3A4, it showed high clearance and no oral exposure in monkey.
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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 5. In vivo SKM-1 MDS model with compound 27
In spite of its pharmacokinetic shortcomings, compound 27 was utilized as a tool
compound to establish that BD2-selective BET inhibitors could maintain strong anti-tumor
activity in vivo. Compound 27 dosed orally twice a day for three weeks in an SKM-1 flank
xenograft model of AML resulted in efficacy comparable to that achieved with the Dual-
bromodomain BET inhibitor 5 given at its maximum tolerated dose (Figure 5). Both compounds
produced sustained tumor growth inhibition throughout the dosing period, thus confirming the
potential antitumor activity of BD2-selective inhibitors.
To evaluate our design hypothesis, we sought co-crystal structures of 27 with both
bromodomains. We were able to obtain structures of 27 co-crystalized with BRD4 BD1 and
BRD2 BD2 (Figure 6). Superimposing the proteins (left panel) highlights the displacement of
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the C-ring engendered by the additional methyl group of the Ile sidechain in BD1. The binding
mode of 27 bound to BRD2 BD2 was in accordance with that observed for compound 18 with
BRD4 BD2, with the ligand making an edge to face interaction with the His433 sidechain as well
as filling a cleft formed by the His433 and Pro430 with the ethyl amide, both features being
present in BD2 and not BD1.
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 6. Left panel, X-ray co-crystal structures of 27 with BRD4 BD1 (salmon protein, cyan
ligand, 2.37 Å, PDB code 6VIY) and BRD2 BD2 (green protein, orange ligand, 1.90 Å, PDB
code 6VIX). Right panel, space filling representation of 27 with BRD2 BD2.
Further improvements on compound 27 were desired, both in terms of selectivity as well
as physical/ADME properties. In some respects these needs worked against each other, as
growing the molecule to add additional differentiating interactions with the bromodomains
would add molecular weight to a compound already nearing the limits of what is typically
considered to be the preferred property space for orally active drugs (MW 507, cLogP 3.88,
eLogD 4.08, TPSA 108.57).
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0
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
One successful approach to improving PK properties identified during the discovery of
Dual-bromodomain BET inhibitor 5 was based on the observation that oxidative metabolism of
aryl ether rings corresponding to the 2,6-dimethylphenyl ether of 27 presented a significant
limitation, particularly in rats. Accordingly, we examined the addition of electron-withdrawing
substituents to the aryl ether while retaining 2,6-disubstitution to maintain selectivity (Table 6).
Introduction of a 4-F substituent was observed to improve microsomal stability vs rat liver
microsomes, while the performance of 42 in the binding and cellular assays was slightly weaker
but within assay variability. The corresponding 4-Cl analog 43 maintained selectivity, but was
less permeable and more prone to microsomal metabolism. Introduction of fluorines on the
benzylic carbons (30, Table 3, and 44) led to reduced BD2 affinity and loss of potency in the
SKM-1 assay.
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
Table 6. Additional Examination of the C-Ring and Linker
O
H
N
D
HN
O
N
A
OAr
B
S
O
O
TR-FRET (BRD4)
Clint,u in liver
a
K
i
, (nM)
Selectivity SKM-1 H1299
BD1/BD2
(fold)
PAMPA
microsomes
-6
Compound
OAr
IC50
(nM)
IC50
(nM)
(10
c
(L/(h·kg))
b
b
BD1
BD2
cm/s)
Human Mouse Rat
2
4
7
2
O
120 ± 49
79 ± 16
1.1 ± 0.5
1.3 ± 0.1
110
64
5.2
14
450
810
12
10
42
55
64
28
7.68
O
F
6.12
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1
1
2
3
4
5
6
7
8
9
4
3
3
0
O
Cl
82 ± 5
0.8 ± 0.1
5.8 ± 0.8
100
81
110
28
700
47
14
120
110
95
18
1.46
6.31
F3C
470 ± 20
1,200
O
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
F
F
44
160 ± 3
2.1 ± 0.1
74
24
880
4.5
54
12
15.2
O
a
TR-FRET K values are reported as the geometric mean derived from 3 independent
i
b
measurements unless otherwise indicated by the number of replicates in parentheses. IC
5
0
c
values are reported as the mean derived from two independent measurements. For reference,
average in-house positive control values for dextromorphan and verapamil, respectively, are
.
.
Clint,u (human) = 7.4 L/(h kg) (low) and Cl (human) = 30 L/(h kg) (high).
int,u
Another area of investigation was replacement of the sulfone substituent, in some cases in
combination with the introduction of the 4-F substituent found in 42 (Table 7). As observed for
compound 18, analysis of the crystal structures showed that the ethyl sulfone occupies a cleft
adjacent to the ZA-loop and accepts a hydrogen bond from a backbone amide. We hoped that
these features could be achieved by other substituents that would offer improvements in the
ADME properties of the molecule. Use of a sulfonamide group as a hydrogen-bond acceptor
provided compounds such as 47-48 that maintained potent BD2 binding, but were less selective
due to improved binding to BD1. The reversed sulfonamide 49 showed similar selectivity to
ethyl sulfone 42, but displayed poor solubility to such an extent that it could not be tested in the
in vitro ADME assays. Compounds 51-54 and 58 with a carbonyl hydrogen-bond acceptor such
as an acid, amide or carbamate showed similar activity profiles to 27 and 42, but offered no
advantage over the sulfone and generally displayed reduced stability toward mouse liver
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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
microsomes. In contrast, introduction of a dimethyl carbinol moiety provided compounds 45 and
6 with similar potency, a slight improvement in selectivity and in the case of 46 improved in
4
vitro ADME properties. The related primary alcohol 50 was less active in the SKM-1 cellular
assay. Cyclic tertiary alcohol analogs 55-57 were examined, but proved inferior in metabolic
stability to the gem-dimethyl. Similarly, larger alkyl groups in place of one or both methyls
provided compounds with greater BD2 selectivity, at the expense of microsomal stability (data
not shown). Of these analogs, 46 provided the best combination of activity and in vitro ADME
properties.
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
Table 7. Variations of the B-Ring Substituent
O
H
N
D
HN
O
N
A
O
B
C
R
X
TR-FRET (BRD4)
Selectivity
BD1/BD2
(fold)
SKM-1
H1299
Clint,u in liver microsomes
PAMPA
a
i
K , (nM)
c
-6
Compound
R
X
IC50
(nM)
IC50
(nM)
(L/(h·kg))
(10
b
b
BD1
BD2
cm/s)
Human Mouse
Rat
64
2
4
7
2
-SO
-SO
2
2
CH
2
2
CH
3
3
H
F
120 ± 49
79 ± 16
1.1 ± 0.5
1.3 ± 0.1
110
64
5.2
14
450
810
12
10
42
55
7.68
6.12
CH
CH
28
4
4
5
6
H
F
160 ± 9
0.6 ± 0.1
1.6 ± 0.2
280
330
11
1,200
1,600
37
170
45
65
6.3
2.0
OH
OH
520 ± 26
6.7
8.6
12
47
-NHSO
2
CH
2
2
CH
3
3
H
F
17 ± 1
49 ± 1
66 ± 3
0.8 ± 0.1
1.1 ± 0.1
1.1 ± 0.1
22
44
62
1.7
42
300
970
11
19
79
880
NV
NV
0.22
0.23
NV
4
4
8
9
-NHSO
2
CH
CH
120
NV
-SO
2
NH
2
H
7.0
3,000
NV
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1
2
3
4
5
6
7
8
9
5
5
5
0
1
2
-CH
-CONHCH
-NHC(O)OCH
2
OH
H
H
F
270 ± 51
130 ± 16
290 ± 19
76 ± 6
1.7 ± 0.1
1.8 ± 0.2
2.9 ± 0.3
0.5 ± 0.1
1.1 ± 0.1
160
71
65
19
50
3.5
14
3,000
1,500
2,400
730
25
13
33
5.0
11
140
19
NV
NV
NV
40
1.69
2.63
0.92
1.96
1.06
2
CH
3
3
100
160
82
380
120
140
53
-NHC(O)CH
3
3
H
F
5
4
-NHC(O)CH
90 ± 1
1,600
15
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
5
5
F
F
220 ± 9
1.5 ± 0.1
1.1 ± 0.1
140
150
26
24
2,200
2,700
350
21
940
32
380
16
1.56
1.66
OH
O
5
5
6
7
170 ± 13
OH
O
F
300 ± 4
1.2 ± 0.1
2.8 ± 0.2
250
110
6.7
2,100
520
NV
100
NV
49
1.47
0.74
OH
58
-CO
2
H
H
300 ± 24
3,100
>10,000
NV
a
TR-FRET K values are reported as the geometric mean derived from 3 independent
i
b
measurements unless otherwise indicated by a number of replicates in parentheses. IC values
5
0
c
are reported as the mean derived from two independent measurements. For reference, average
in-house positive control values for dextromorphan and verapamil, respectively, are Clint,u
.
.
(
human) = 7.4 L/(h kg) (low) and Cl (human) = 30 L/(h kg) (high).
int,u
Metabolite studies on compound 27 had indicated that oxidative dealkylation of the
secondary amide was a route of metabolism. Accordingly, several secondary amides were
examined (Table 8). Although several examples with large alkyl groups showed good
selectivity, the desired improvement in microsomal stability was not achieved. Compound 46
exhibited the best overall profile of potency, selectivity and in vitro ADME properties, and was
selected for more thorough characterization.
Table 8. Amide Variations
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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
O
A
H
N
HN R
O
N
D
O
B
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
X
OH
i
TR-FRET (BRD4) K ,
a
Selectivity SKM-1 H1299
BD1/BD2
(fold)
Clint,u in liver microsomes PAMPA
(
nM)
c
-6
#
R
X
IC50
(nM)
IC50
(nM)
(L/(h·kg))
(10
b
b
BD1
BD2
cm/s)
Human Mouse
Rat
65
45
-CH
-CH
2
2
CH
CH
3
3
H
F
160 ± 9
0.6 ± 0.1
1.6 ± 0.2
1.1 ± 0.1
1.3 ± 0.1
280
330
200
240
11
6.7
14
25
1,200
1,600
1,400
1,500
37
8.6
47
170
45
6.3
2.0
4
5
6
6
9
0
520 ± 26
220 ± 16
310 ± 37
12
-C(CH
-C(CH
3
3
)
3
3
H
F
260
520
180
87
1.26
NV
)
130
6
1
F
440 ± 68
1.3 ± 0.1
340
10
1,100
120
710
NV
0.97
6
6
2
3
-CH
3
F
F
F
140 ± 5
230 ± 9
1.0 ± 0.1
0.5 ± 0.1
6.9 ± 1.7
140
490
680
13
6.2
80
1,200
1,900
2,600
24
48
33
10
20
0.55
1.13
0.57
200
64 -C(CH
3
)
2
CF
3
4,700 ± 2,100
570
2,800
1,100
F
65
F
780 ± 49
2.0 ± 0.1
400
66
2,900
67
210
84
0.76
F
a
TR-FRET K values are reported as the geometric mean derived from 3 independent
i
b
measurements unless otherwise indicated by the number of replicates in parentheses. IC
5
0
c
values are reported as the mean derived from two independent measurements. For reference,
average in-house positive control values for dextromorphan and verapamil, respectively, are
.
.
Clint,u (human) = 7.4 L/(h kg) (low) and Cl (human) = 30 L/(h kg) (high).
int,u
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5
1
2
3
4
5
6
7
8
9
Compound 46 was tested for binding against the first and second bromodomains of
BRD2, BRD3 and BRDt in the TR-FRET binding assay and displayed similarly potent and
selective binding to that observed with BRD4 (Table 9). This result was unsurprising, given the
sequence similarities in the binding site discussed previously. Binding selectivity between the
two bromodomains of BRD4 was also examined in a cellular setting using a bioluminescence
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
9
resonance energy transfer-based method, which showed greater than 700-fold BD2 selectivity
(
Table 9). Selectivity was also demonstrated against a panel of non-BET family bromodomains
using the DiscoveRx BROMOSCAN assay (see Table S1 in SI for data).
Table 9. Compound 46 Selectively Inhibits BD2 in Biochemical and Cellular Assays
TR-FRET
NanoBRET
BRD2 K (nM) BRD3 K (nM) BRD4 K (nM)
BRDt K (nM)
Cellular IC (nM)
i
i
i
i
50
BD1
,162
BD2
4.6
BD1
BD2
4.9
BD1
521
BD2
1.6
BD1
917
BD2
1.0
BRD4 BD1 BRD4 BD2
1
3,140
20,700
27.5
BRD = bromodomain; BRET = bioluminescence resonance energy transfer; IC = half maximal
5
0
inhibitory concentration; K = inhibitory constant; TR-FRET = time resolved fluorescence
i
resonance energy transfer
To confirm the structural basis for bromodomain selectivity, X-ray co-crystal structures
were obtained for 46 with both the first and second bromodomains of BRD2 (Figure 7). As
observed for other compounds from the pyrrolopyridone series, the heterocyclic core exhibits
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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
key hydrogen bonding interactions with the conserved asparagine. As intended, the tertiary
alcohol of 46 was able to accept the same hydrogen bond from the amide backbone of the ZA-
loop observed with the sulfones of 18 and 27. Superposition of the BD1 and BD2 structures
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 7, lower left panel) shows the displacement of the 2,6-disubstituted aryl ether in the BD1
complex induced by the Ile162 sidechain. When 46 is positioned in the pose observed in the
BD2 complex, one of the methyl groups would lie inside of the solvent accessible surface of the
Ile in BD1. The additional buried surface area from interactions with His433 discussed earlier
rationalizes the selectivity between BRD2 BD1 and BD2. These key residues are identical
across the BET family, consistent with the observed uniformly good selectivity between the two
bromodomains of each family member.
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7
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 7. Upper left panel, co-crystal structure of 46 with BRD2 BD1(2.0 Å, PDB code 6ONY)
showing the distances observed between selected heavy atoms in Angstroms. Upper right panel,
co-crystal structure of 46 with BRD2 BD2 (2.3 Å, PDB code 6E6J) showing the distances
observed between selected heavy atoms in Angstroms. Lower left panel, superimposed aligned
X-ray co-crystal structures of 46 with BRD2 BD1 (salmon protein, cyan ligand) and BRD2 BD2
(green protein, orange ligand). The solvent accessible surface of Ile162 on BRD2 BD1 shown in
gray excludes the space occupied by a methyl in the pose when bound to BRD2 BD2. Lower
right panel, space filling representation of 46 with BRD2 BD2.
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8
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
High throughput screening assays for microsomal stability and permeability predicted
that 46 would display improved pharmacokinetic behavior over the initial lead 27. Experimental
data showed that the preclinical pharmacokinetic profiles of 46 are characterized by low
clearance in mouse and dog, with moderate clearance in rat and monkey, which correlates well
with the intrinsic metabolic clearance measured in hepatocytes (IVIVE ratio between 2-5-fold).
Moderate to high volume of distribution is observed across species, with half-life ranging from
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
.0 to 4.4 h (Table 10). Following oral administration, mono-exponential disposition profiles are
observed in all of the preclinical species tested. The oral bioavailability of 46 in preclinical
species ranges from 7% in monkey to 77% in dog, with fa*fg ranging from 19% to 87%. With
the exception of reduced AUC on oral dosing in dogs, these values were superior to those
observed for 27, and allowed examination of 46 in preclinical models to compare efficacy and
preclinical safety of a BD2-selective BET bromodomain inhibitor with Dual BET bromodomain
inhibitors.
Table 10. DMPK Data
IV Dose
PO Dose
Species Dose
t
1/2
V
ss
AUC
3.80
0.50
0.46
0.20
CL
p
Dose
1
1
0.3
0.3
t
1/2
T
max
C
max
AUC
2.35
0.12
1.05
0.014
F
fa*fg
81
52
87
19
Mouse
Rat
1
1
3.0
2.9
4.4
2.0
1.3
5.3
1.5
3.4
0.29
0.67
0.24
1.54
2.8
3.4
4.2
2.5
6.0
7.5
2.8
1.5
0.21
58
24
77
7
0.012
0.116
0.005
Dog
0.1
Monkey 0.3
Units: Dose (mg/kg); t1/2 (h); Vss (L/kg); AUC(g•h/mL); CL (L/(h·kg)); Cmax (g/mL); F (%)
po formulation (Rat, Dog, Mouse): 10:30:60 Ethanol:PEG-400: Phosal 53 MCT
po formulation (Monkey): 10:90 (v/v) DMSO:PEG-400
p
51
As has been discussed elsewhere in more detail, selective inhibition of the second
bromodomain results in a more restricted range of antiproliferative activity relative to Dual-
bromodomain BET inhibitors. Whereas potent Dual-bromodomain BET inhibitors such as 5
48
show potent anti-proliferative effects against a wide range of cancer cell lines, 46 shows
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Page 29 of 135
Journal of Medicinal Chemistry
2
9
1
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9
substantially higher relative potency against cell lines derived from prostate cancer and AML,
implying that BD1 inhibition is not required to inhibit proliferation in a subset of cancer
indications.51 The potent in vitro activity against the SKM-1 cell line and low clearance in mice
were reflected in the activity of 46 in a xenograft tumor growth inhibition model, with a 18.8
mg/kg po q.d. dose providing 83% tumor growth inhibition with minimal (2%) weight loss over
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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5
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0
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0
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0
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0
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0
2
1 days of dosing (Figure 8). In comparison, in the same study Dual-bromodomain BET
inhibitor 5 dosed at the maximally tolerated dose of 1 mg/kg provided similar tumor growth
inhibition; however, 7% weight loss was observed, and one animal had to be removed from the
study. This trend toward improved tolerability, albeit modest in magnitude, supported the
premise underlying our project that a more selective BET bromodomain inhibitor might achieve
a better therapeutic index in settings sensitive to the mechanism of action. A more systematic
evaluation supporting this hypothesis in AML models will be published separately in due
course.52
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