Design and Synthesis of a Highly Selective and in Vivo-Capable Inhibitor of the Second Bromodomain of the Bromodomain and Extra Terminal Domain Family of Proteins

Article abstract of DOI:10.1021/acs.jmedchem.0c00605

Pan-bromodomain and extra terminal domain (BET) inhibitors interact equipotently with the eight bromodomains of the BET family of proteins and have shown profound efficacy in a number of in vitro phenotypic assays and in vivo pre-clinical models in inflam

Full text of DOI:10.1021/acs.jmedchem.0c00605

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Article
The Design and Synthesis of a Highly Selective and In Vivo
Capable Inhibitor of the Second Bromodomain (BD2) of the
Bromodomain and Extra Terminal Domain (BET) Family of Proteins
Alex Preston, Stephen J. Atkinson, Paul Bamborough, Chun-wa Chung, Peter D Craggs,
Laurie J. Gordon, Paola Grandi, James Gray, Emma J Jones, Matthew Lindon, Anne-Marie
Michon, Darren J Mitchell, Rab K. Prinjha, Francesco Rianjongdee, Inmaculada Rioja, Jon Seal,
Simon Taylor, Ian D. Wall, Robert J Watson, James M. Woolven, and Emmanuel H Demont
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00605 • Publication Date (Web): 21 Jul 2020
Downloaded from pubs.acs.org on July 21, 2020
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The Design and Synthesis of a Highly Selective
and In Vivo Capable Inhibitor of the Second
Bromodomain (BD2) of the Bromodomain and
Extra Terminal Domain (BET) Family of Proteins
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†
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Alex Preston* , Stephen Atkinson* , Paul Bamborough , Chun-wa Chung , Peter D. Craggs ,
§
‡
§
†,¥
Laurie Gordon , Paola Grandi ͋, James R. J. Gray , Emma J. Jones , Matthew Lindon ,
†
†
†
Anne-Marie Michon ͋, Darren J. Mitchell , Rab K. Prinjha , Francesco Rianjongdee ,
†
†
‡
͌
§
†
Inmaculada Rioja , Jonathan Seal , Simon Taylor , Ian Wall , Robert J. Watson , James
§
†
Woolven and Emmanuel H Demont
†
‡
Epigenetics Discovery Performance Unit; Quantitative Pharmacology,
§
Immunoinflammation Therapy Area Unit; Platform Technology and Science,
GlaxoSmithKline, Medicines Research Centre, Stevenage, Hertfordshire, SG1 2NY, U.K;
IVIVT Cellzome, Platform Technology and Science, GlaxoSmithKline, Meyerhofstr. 1,
6
9117 Heidelberg, Germany
Abstract
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Pan-BET inhibitors interact equipotently with the eight bromodomains of the BET family of
proteins and have shown profound efficacy in a number of in vitro phenotypic assays and in
vivo pre-clinical models in inflammation or oncology. A number of these inhibitors have
progressed to the clinic where pharmacology-driven adverse events have been reported. To
better understand the contribution of each domain to their efficacy and to improve from their
safety profile, selective inhibitors are required. This article discloses the profile of GSK046,
also known as iBET-BD2, a highly selective inhibitor of the second bromodomains of the BET
proteins that has undergone extensive pre-clinical in vitro and in vivo characterisation.
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Introduction
The bromodomain and extra terminal domain (BET) family of proteins consist of four
isoforms: BRD2, BRD3, BRD4 and BRDT. Each of these BET proteins contain two
bromodomains, known as bromodomain 1 (N terminus, BD1) and bromodomain 2 (C terminus,
BD2). The profound anti-proliferative and anti-inflammatory effects of pan-BET inhibitors,
acting at both BD1 and BD2 of all 4 BET proteins, has now been well documented.1-22 Indeed,
a number of pan-BET inhibitors are progressing through phase I-II clinical trials for oncology
indications.23-24 However, associated pharmacology driven toxicities with pan-BET inhibitors
have also been reported.24,25,26 Our strategy was therefore to target the rational design of
selective BET inhibitors in order to elucidate the functional contribution of each domain to the
phenotype observed with pan-BET inhibitors and with the ambition of improving on the
adverse event profile observed in human. Due to the high homology across the 4 bromodomain-
containing proteins, it was envisaged that obtaining a single isoform selective inhibitor would
be complex. However, the homology between the BD2 and the BD1 domains is lower, with
key residues close to the acetylated lysine (AcK) recognition pocket altered (see supplementary
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figure S2) which it was hypothesised would give the opportunity to deliver pan BD2
selectivity.27-32 Herein, we describe our approach to identify a tool molecule with second
bromodomain (BD2) pIC >7 at BRD2, 3, 4 and T combined with selectivity over the first
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domain (BD1) of at least 100 fold. In addition, we wanted to explore the opportunity of using
BD2-selective molecules as tools to understand the in vivo capabilities and wider
developability of our series.
At the time this work was carried out there were no highly potent BD2-selective inhibitors
known. The best-studied molecule reported to show bias in favour of BD2 was RVX-208
(Figure 1), which has Kd of 8.9 µM against BD1 and 300nM against BD2 by ITC, a selectivity
of around 30-fold.33 Independent ITC measurements showed greater potency but lower
27
selectivity (Kd of 1.1µM for BRD4 BD1 and 135nM for BRD4 BD2) . We have subsequently
reported a series of quinoxaline-based inhibitors such as GSK340 which show greater potency
and selectivity (Kd in the DiscoveRx BROMOscan assays of 1.5µM against BRD4 BD1 and
7
nM against BRD4 BD2).29 Very recently, ABBV-744 was reported as a nanomolar BD2
inhibitor and clinical candidate, with >290-fold selectivity over the BD1 domains of
BRD2/3/4.34-35
HO
OH
O
HO
NH
O
N
N
N
N
O
O
F
H
HN
O
HN
O
N
O
O
RVX-208
GSK340
ABBV-744
Figure 1 – Literature BD2- selective inhibitors RVX-208, GSK340 and ABBV-744
In order to identify hits which showed selectivity for the BD2 domain over the BD1 domain
we initiated a high-throughput screen (HTS) of over 2M compounds. The compounds were
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screened initially at a single concentration of 10µM versus the BRD4 dual domain protein
construct using a TR-FRET assay. In this construct, in order to evaluate binding at the BD2
domain only, the conserved tyrosine of the BD1 acetyl-lysine binding site was mutated to
alanine (Y97A) to inactivate the binding site leaving just the BD2 domain able to bind the
fluoro-ligand. A corresponding Y390A construct was used to assess BD1 binding. The BRD4
isoform was considered to be representative of all the BET isoforms. A thorough triage was
undertaken, including an orthogonal BD2 biochemical assay, full curve follow up against
BRD4 BD2 and BD1 and SPR biophysical characterisation to provide robust and progressible
hits. Acetamide 1 was identified as a hit compound from this screening process (Figure 2). The
difficulties in obtaining highly domain selective compounds were well known to us so we were
attracted to 1, due to the good selectivity of this hit, being over 10 fold selective for BRD4 BD2
over BD1. Notably, this translated to similar selectivity for BRD 2, 3 and T. The molecule was
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also in good physicochemical space with a clogP of 1.1, chromLogD of 2.2, and a property
forecast index (PFI) value of 5.2. The later value is a metric used originally as a prediction of
3
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solubility, with a value <6 seen as optimal. Pleasingly, 1 also had good kinetic solubility, as
3
6
measured by chemiluminescent nitrogen detection (CLND) , of 125 µg/mL. Encouragingly
for an unoptimised hit, the microsomal clearance in both rat and human was moderate, giving
us confidence that there were no major metabolic vulnerabilities with the template.
N
O
1
3
O
N
N
H
H
NH
4
O
(1)
BRD4 BD1 / BD2 pIC (n)
4.4 (2/9) / 5.5 (9) a
> 12
0.27 / 4.4
1.1 / 378
2.2 / 5.2
125
5
0
Selectivity (fold)
BD2 LE /LLE
Clogp / MW
ChromLog / PFI
7
.4
CLND solubility (µg/mL)
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AMP7.4 permeability (nm/s) 8.0
Cli (Rm / Hm)
1.6 / 1.8
a
Some results below the curve-fitting threshold of 4.3 could not be included in mean. In these cases, the numbers
included in the mean / number of times tested are both shown.
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Figure 2 – Profile of HTS hit 1
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Figure 3. A) Crystal Structure of BRD4 BD1 complex with 1 (green, PDB 6z7g) superimposed
on the BRD2 BD1-bound structure of paracetamol (magenta, PDB 4a9j). B) BRD4 BD1
complex with 1. C) BRD2 BD2 complex with 1 showing alternative conformations (PDB 6z7f).
To better understand the selectivity of this molecule we obtained crystal structures of 1 in
the BD1 domain of BRD4 and the BD2 domain of BRD2. Since the conservation within BD1
and BD2 acetyl-lysine sites is very high (Figure S2, Supporting Information), these two
structures are very likely to represent binding to all bromodomains of the family. Compound 1
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contains an acetyl-lysine mimetic core closely related to paracetamol, and its binding mode is
very similar to the published structure of paracetamol in the BRD2 BD1 bromodomain (Figure
3A).38 The N-acetyl group mimics the acetylated lysine of the histone tail, its carbonyl group
hydrogen bonding to the conserved asparagine (BRD4 BD1 Asn140) and the conserved
network of water molecules at the bottom of the binding site.
1
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Moving beyond the core of 1, the 3-position benzyloxy substituent turns sharply to position
the terminal phenyl ring on the WPF shelf of BRD4 BD1. This is a hydrophobic region created
by the sidechains of Ile146 and the Trp81-Pro82-Phe83 sequence (Figure 3B). This geometry
is encouraged by the formation of an internal hydrogen-bond between the 3-position ether
oxygen and the 4-position acetamido NH group. The 1-position amide NH is involved in a
water-bridged interaction with Asn140. The imidazole group is visible in the electron density
and forms a water-bridged hydrogen bond network with the acid sidechain of Asp96.
The binding mode of 1 in BRD2 BD2 is comparable to that in BRD4 BD1 (Figure 3C), with
similar interactions between the inhibitor and the analogous BD2 residues Asn429, Val435 and
Trp370-Pro371-Phe372. These interactions are common to many pan-BET39-41 inhibitors and
do not on their own offer an insight into the BD2 bias of 1. The main difference from the BD1-
bound complex in BD2 lies around the 4-position imidazole substituent, where the electron
density is complex, suggesting this group can adopt at least two conformations. The
neighbouring His433 sidechain also exists in two alternative rotameric states. In one trans chi1
rotamer the histidine sidechain points away from the inhibitor and allows two water molecules
to enter the interface, while in the other gauche(+) rotamer the sidechain points towards the
inhibitor and displaces these waters. This mobility of His433 contrasts with the well resolved
position of the equivalent BRD4 BD1 residue, Asp144, which is constrained by a hydrogen-
bond to the backbone NH of Lys141.
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Embarking on optimisation of this template, the crystal structures suggested that the core of
the molecule, including both amide groups, should be retained. It was hoped that further
optimisation of the shelf and amide vectors would be possible, and also that the methylene
linker of the benzyl shelf group could offer a vector from which to explore the ZA channel.
This is a narrow cleft present in both BD2 and BD1 domains, flanked by Leu381 and Trp370
in BRD2 BD2. This region has been accessed by a number of pan-BET inhibitors leading to
higher affinity.31
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Results & Discussion
Initial SAR investigations focussed on the amide-imidazole vector and attempted to improve
the artificial membrane permeability (AMP), which is correlated with oral bioavailability, as
well as ligand efficiency (LE)42-43, BD2 potency and domain selectivity (Figure 2). It was
reasoned that the basic imidazole group could be responsible for limiting the permeability,
whilst reoptimisation of this substituent could also lead to LE improvements.
O
O
HN
HN
O
O
O
R2
(2)
(3)
Com R2
BRD4 BD1 BRD4
BRD4
BRD4
CHROMLo AMP (nm/sec)
poun
d
pIC (n) BD2 pIC50 BD2 Sel BD2
gD @ pH /
cLND
5
0
(n)
(fold)
> 2
LE
7.4
4.9
2.9
3.2
3.5
2.3
(µg/mL)
2
<4.3 (3)
5.0 (6)
4.6 (3)
5.5 (6)
4.5 (4)
5.2 (3)
5.3 (3)
0.35
0.35
0.27
0.32
0.30
780 / 113
3
4
5
6
NHMe
4
220 / 135
280 / 109
320 / 143
68 / 162
NMe2
NHEt
<4.3 (4)
<4.3 (4)
> 1.5
> 8
NH(CH ) O <4.3 (3)
> 10
2
2
H
7
NH(CH ) N 4.4 (2)
5.4 (2)
10
0.31
1.4
35 / <3.0
2
2
H2
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1
NH(CH ) N 4.5 (3)
H2
5.8 (3)
5.4 (4)
5.7 (5)
20
0.32
0.30
0.29
1.6
2.4
3.3
<3 / 123
94 / 109
190 / 155
2
3
NH(CH ) O <4.3 (4)
> 13
> 25
2
3
H
H
N
0
<4.3 (5)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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4
4
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4
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4
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4
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5
5
5
5
5
5
5
5
6
0
1
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5
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7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
1
1
1
2
<4.3 (4)
<4.3 (2)
<4.3 (3)
5.6 (4)
6.1 (2)
5.8 (3)
> 20
> 63
> 31
0.27
0.30
0.28
3.5
1.7
4.0
190/ 174
170 / <3.0
104 / 200
H
N
NH
H
N
H
N
13
O
H
N
1
4
<4.3 (4)
5.9 (4)
> 40
0.29
2.7
79 / 174
OH
Table 1: Optimisation of the Amide Vector of 1
In order to understand the importance of the amide group to potency we removed this group
altogether to give the fragment 2 (Table 1) which had a poor BD2 pIC of 4.6, although
5
0
interestingly an improved LE vs 1, suggesting the core was inherently an efficient BD2 binder.
It should also be noted that deletion of the amide causes a significant and undesired increase in
lipophilicity. Truncating the amide back to the methyl amide 3 saw potency return and LE
maintained vs 2, albeit with poor selectivity, indicating that the amide was important for
potency, and that the imidazole linked moiety was offering little additional binding to BD2.
The importance of the amide NH suggested by the crystal structure of 1 (Figure 3) was
confirmed by reduced potency of the tertiary amide 4 (BRD4 BD2 pIC = 4.5). Homologating
5
0
the methyl amide 3 to ethyl amide 5 brought selectivity back, but BD2 potency and LE were
reduced. Introduction of a hydroxyl to give ethyl alcohol 6 maintained potency and selectivity,
but was detrimental to permeability. This OH was replaced with an NH group 7 with no real
2
effect on potency / selectivity observed, but a further reduced AMP (<3 nm/s) resulted, likely
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5
6
due to the highly polar nature of the protonated amine group. Further extending the OH to
propanol 9 again showed no real benefit in terms of potency. Replacing this OH with a NH2
group 8 resulted in an increase in potency (BRD4 BD2 pIC = 5.8), selectivity was also
5
0
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
increased (20 fold) but a reduced AMP was observed. In order to address the reduced
permeability seen with 6, 7 and 8, examples were made where the heteroatom was constrained
into a ring system such as a THP 10 where a boost in BD2 potency was observed. Pushing this
ring out further towards solvent (11, 13) had little impact on both potency and selectivity but
an increase in ChromLogD was observed. We also looked at replacing the THP with piperidine
1
2; here an increase in potency and selectivity was seen, but introduction of a basic centre once
again had a negative impact on permeability as measured by the AMP assay. Finally,
modification of THP 10 to trans-cyclohexanol 14 provided a compound with a good balance
of potency, selectivity, solubility and permeability, albeit the additional H-bond donor vs 10
was likely responsible for the moderate permeability measured.
In parallel to the amide exploration, the shelf region of the molecule was investigated, which
is accessed from the phenolic position of the central ring. It was decided to do this SAR
exploration with the propylamine amide group as this had been identified early in our
investigations as a group with good potency, LE and selectivity (Table 2, 8). After conducting
an array, it was determined that aromaticity was important in this region with substituents such
as THP 15 being inactive. Introduction of a heteroatom into the ring also gave reduced potency
(compounds 16-18). Electron withdrawing substituents such as chloride were tolerated in all
three positions (compounds 19-21) but offered no advantage to inhibitor 8. Replacing the para-
Cl with a para-Me 22 was equally well tolerated, expanding this group to a para-iPr 23 gave a
slight increase in potency of 0.1 log units but was accompanied with a 1.5 log unit increase in
ChromLogD. Introduction of the OMe at the para position 24 again had a similar profile to the
para-Cl 19, however more polar groups at this position such as the para-CONH 25 were not
2
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well tolerated. A key breakthrough arrived with the introduction of an alpha-methyl group at
the benzylic position to provide 26. This gave a 0.5 log increase in potency, maintained LE and
also improved BD2 selectivity to 40 fold.
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
O
HN
O
R3
O
N
H
NH2
Cmpd
No
R3
BRD4 BD1 BRD4
pIC (n) BD2
pIC (n) (fold)
BRD4
BD2 Sel BD2
BRD4 CHROMLo AMP
gD @ pH (nm/sec)
/
5
0
LE
0.32
-/-
7.4
1.6
0.4
CLND
(µg/mL)
50
8
4.5 (3)
5.8 (3)
20
-
<3 / 123
<3 / 144
15
O
<4.3 (4)
<4.3 (4)
1
1
1
1
2
2
2
6
7
8
9
0
1
2
P - <4.3 (4) 4.5 (3/4) > 2
0.26
0.26
0.28
0.30
0.30
0.30
0.30
0.1
0.2
0.3
2.2
2.1
1.9
2.0
3 / 112
N
m - <4.3 (4) 4.8 (4)
o - <4.3 (4) 5.0 (4)
> 3
> 5
13
8
<3 / 130
<3 / 80
p - 4.6 (4)
m – 4.6 (4)
o – 4.4 (5)
4.5 (5)
5.7 (4)
5.5 (4)
5.6 (5)
5.7 (5)
<3 / 108
11 / 96
Cl
16
16
15 / 124
6.5 / 114
2
3
4.7 (3)
5.9 (3)
16
0.29
3.1
7.7 / 114
O
24
4.4 (2/3)
<4.3 (3)
5.6 (3)
5.0 (3)
16
0.28
0.24
1.3
0.0
<3 / 114
<3 / 151
O
2
2
5
6
> 5
H N
2
4.7 (8)
6.3 (8)
40
0.33
1.7
<3 /159
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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
a
Some results below the curve-fitting threshold of 4.3 could not be included in mean and SD. In these cases, the
numbers included in the mean / number of times tested are both shown.
Table 2 – Optimisation of the Benzylic Group of 8.
O
O
O
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
HN
HN
HN
O
O
O
O
O
OH
O
N
O
N
H
O
N
H
H
2
7 (R)
28 (S)
29
a
BRD4 BD1 / BD2 pIC50
Selectivity (fold)
BD2 LE
<4.3 (3) / 4.9 (3) 4.5 (6/8) / 6.6 (8)
> 4
4.4 (6) / 6.8 (6)
250
125
0.24
0.32
0.32
Clogp / MW
1.4 / 382
3.8 / 5.8
159
1.4 / 382
3.7 / 5.7
215
1.6 / 396
3.1 / 5.1
170
ChromLog / PFI
7
.4
CLND solubility (µg/mL)
AMP permeability (nm/s)
300
340
93
7
.4
a
Some results below the curve-fitting threshold of 4.3 could not be included in mean and SD. In these cases, the
numbers included in the mean / number of times tested are both shown.
Figure 4 – Profile of Lead Molecules 28 and 29
To establish which enantiomer of the alpha-methyl benzyl was the more potent, both the R
and S enantiomers 27 and 28 (Figure 4) were prepared using S Ar chemistry with the
N
corresponding chiral alcohols (scheme 4). The (S) enantiomer was found to be the more potent
of the two enantiomers. This can be rationalized using the BRD2 BD2 crystal structure of 1.
Substituents with (S)-stereochemistry can grow freely towards the ZA channel of the site,
whereas even a methyl group with (R)-stereochemistry would bump with the phenyl ring of
the core as well as with the benzylic ring (Figure S6, Supporting Information). The torsional
changes needed to relax this bump would prevent the benzylic ring from occupying the WPF
shelf.
Combining our favoured cyclohexanol amide groups (cf. Table 1, 14) with the (S)-alpha-
methyl benzyl shelf group, gave 29 (Figure 4). 29 in particular showed a highly encouraging
overall profile with good levels of BD2 potency, nearing 100 nM, coupled with exquisite
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selectivity for BD2 over BD1 of 250 fold. This compared favourably with 28 which had BD2
potency of 250 nM and selectivity of 125 fold. Importantly, the good physicochemical
properties of the initial hit are maintained in both with PFI <6. The compounds are permeable
and soluble as measured by AMP and CLND respectively, whilst the LE has been improved
from 1 and is now >0.3. 29 was preferred over 28 due to its better overall profile.
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
Chemistry
Preparation of the amide variants 3-14 was achieved using the synthetic route outlined in
Scheme 1. A key late stage acid intermediate 33 was prepared by acetylation of the
commercially available methyl 4-amino-3-hydroxybenzoate 30 followed by alkylation of the
phenol with benzyl bromide to give 32; hydrolysis of the methyl amide using lithium hydroxide
gave 33 in good yield. This acid intermediate was then used to vary the amide R using standard
2
amide coupling conditions to give examples 3-14.
Similar chemistries could be utilised to access the shelf variants 15- 26 as detailed in scheme
2
. Here the strategy of synthesising a late stage intermediate which allowed variation of the
shelf vector R using standard alkylation chemistry was employed. Acetylation of 4-amino-3-
3
hydroxybenzoic acid 34 to give 35 was followed by amide coupling of a suitably protected
(boc, trifluoroacetamide) diaminopropyl group to yield 36 or 37. Alkylation of the phenol with
commercially available benzyl bromides and subsequent deprotection of the amide side chain
gave 15 – 26.
Scheme 1: Synthetic route for variance of amide group
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8
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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
O
O
O
O
NH2
HN
HN
HN
HN
HO
HO
O
i
ii
iii
O
iv, v
O
O
O
O
O
O
R1
O
OH
O
N
30
31
R2
32
33
3
-14
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
o
Reagents and conditions: i.Acetyl chloride, THF:Xylene (1:2), 70 C, 16 h (92%). ii. BnBr,
o
K CO , DMF, 70 C, 16 h (71%). iii. LiOH, THF:MeOH:H O (2:2:1), rt, 16 h (83%). iv.
2
3
2
R N(R )H, HATU or T P, DIPEA, DMF, 16 h, rt. (4 – 67%) v. HCl or TFA, 1,4-dioxane or
1
2
3
o
CH Cl , 0 C, 2 h (21-40%).
2
2
Scheme 2: Synthetic route for variance of shelf group R3
O
O
O
NH2
HN
HN
HN
HO
HO
HO
O
i
ii
iv
R3
PG
O
OH
O
OH
O
N
H
N
O
N
H
NH2
H
34
35
3
6, PG = Boc
15-26
24
iii
v
3
7, PG = Trifluoroacetamide
o
Reagents and conditions: i.Acetyl chloride, THF:Xylene (1:2), 70 C, 5 h (75%). ii.Amine,
EDC, HOBt, DMF 16 h, rt (56%).iii. EDC, HOBt, TEA, DMF, rt (44%) iv. R Br, K CO ,
3
2
3
o
o
DMF, 60-70 C, 2-3 h, then: HCl, 1,4-dioxane, rt, 3-16 h (24-69%) v. K CO , DMF, 70 C, 3
2
3
h, then K CO , MeOH, 12 h, rt (40%).
2
3
A modified synthetic strategy (Scheme 3) was used when accessing the chirally pure example
29. Again a late stage intermediate 42 was prepared which would then allow variation of the
amide as the final step. A S Ar reaction was performed with the commerically available 3-
N
fluoro-4-nitrobenzoic acid 38 and (S)-1-phenylethanol followed by addition of MeI to the
reaction gave the methyl ester 39. Reduction of the nitro group using Pt/C as catalyst followed
by acetylation of the resulting amine 40 gave 41. The ester of 41 could then be hydrolysed to
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give the acid 42. Coupling of this acid with the appropriate amine using standard amide
coupling conditions gave the desired compound 29, in acceptable yields.
Scheme 3: Synthetic route to 29
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
O
NO2
NH2
HN
NO2
F
O
O
O
i
ii
iii
O
OH
O
O
O
O
O
O
38
39
40
41
iv
O
O
HN
HN
O
v
O
R
O
N
H
O
OH
42
29
o
Reagents and conditions: i.NaH, MeI, (S)-1-phenylethanol, DMF:THF (2:1), 0-25 C, 22 h
(
(
(
74%). ii. Pt/C, H , EtOH, rt, 1 atm (97%). iii. Ac O, H O, rt, 2 h, (81%). iv.LiOH, THF:H O
2 2 2 2
o
1:1), 50 C 3 h – rt 16 h (81%) v. Trans-4-aminocyclohexanol, HATU, DIPEA, DMF, rt, 18 h
55%).
The synthetic steps in this sequence were reordered in order to access 27 and 28 (Scheme 4).
Here the first step was an amide coupling between the acid 38 and tetrahydropyran amine using
standard amide coupling conditions to give 43. A S Ar reaction was then performed with the
N
commercially available (S) chiral alcohol to give 44 this was then followed by reduction of the
nitro 45 and subsequent acetylation as seen in the previous scheme to give 28. The enantiomer
27 was accessed using similar chemistry but with the (R) chiral alcohol in step ii.
Scheme 4: synthetic route to 27, 28
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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
NO2
NH2
NO2
NO2
F
F
O
O
R
R
i
ii
iii
O
O
O
O
OH
O
N
O
N
H
O
N
H
H
38
4
5
43
44
iv
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
O
HN
O
R
O
O
N
H
27, 28
Reagents and conditions: i. CDI, DMF, tetrahydro-2H-pyran-4-amine HCl, rt, 18 h (92%) ii.
NaH, DMF, (S)-1-phenylethanol, rt, 2 h, (72%) for 44b or 1M LiHMDS in THF, THF, (R)-1-
o
o
phenylethanol, 100 C, 30 min (81%) for 44 iii. Sn(II)Cl, EtOH, 60 C, 16 h (37% for 45b and
o
3
9% for 45) iv. Ac O, H O, 50 C, 16 h, (51% for 28 and 76% for 27).
2
2
AMES liability
Despite the promising profile of 29, this molecule contains an embedded aniline, which
brings with it the risk of a potential genotoxic liability, by virtue of the possibility of forming
4
4
a mutagenic nitrenium ion in vivo following deacetylation. Therefore, we wanted to gain a
better understanding of this risk before further progressing the molecule. An in vitro metabolic
identification study on a closely related analogue 28 in human and rat liver S9 fraction showed
that deacetylation to the aniline was the main metabolite (Figure S4, Supporting Information).
Assuming the metabolic process would be similar across the series aniline 46 would be the
main metabolite of compound 28 and 29. In-silico analysis of 28 using meteor (Figure S3,
Supporting Information) suggested that both amides would be metabolically vulnerable.
Debenzylation was also predicted. A rudimentary stability study of 29 was performed by
treatment with a 2M HCl aqueous solution for 24 h. Aniline 46 was identified as a potential
degradant. Removal of the benzyl group to give the phenol was also observed. Certain anilines
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are known to have mutagenic properties, as they are oxidised by the cytochrome P450 enzymes
in vivo to form a hydroxylamine, which can then break down to a potentially mutagenic
nitrenium species which reacts with DNA. The gold standard test for mutagenicity is the Ames
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
45
test. However, in order to predict the mutagenic liability of 46, together with another potential
degradant/predicted metabolite, phenol 47, an in silico semi-empirical calculation, the eHOMO
4
6
(
electronic Highest Occupied Molecular Orbital) model was used (Figure 5). A HOMO
energy of -0.320 atomic units (a.u.) or higher represents an elevated risk of Ames positive test
results. eHOMO results for both 46 and 47 were similar and borderline, with 46 considered as
having a reduced risk whilst aniline 47 considered as having a high risk of generating an Ames
positive result. Interestingly acetamide 29 was predicted to be negative. Given the eHOMO
findings and a finite likelihood of generating 46 and 47 in vivo, we decided to progress them
into the Ames assay to confirm these findings. Aniline 46 was found to be negative in the Ames
assay against strains TA1535, TA1537, TA98, TA100 and E.Coli WP2uvrA +/-S9.
Unfortunately, the potential degradant/predicted metabolite 47 was indeed positive in the Ames
assay against strain TA98 in the presence of S9, in agreement with the eHOMO prediction.
Therefore the potential for 29 to degrade / metabolise to 47 was considered a significant risk if
we were to consider developing this series towards a clinical candidate.
O
NH2
NH2
HN
metabolism /
degradation
metabolism / HO
degradation
O
O
OH
OH
O
OH
O
N
O
N
H
H
29
46
47
2
9
46
47
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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
HOMO
eHOMO
HOMO
energy (a.u.)
-0.3399
-0.3204
Low
-0.3198
High
Risk
Low
-
Strains (-S9)
TA1535, TA1537, TA1535,
TA98, TA100 and TA1537, TA98,
E. coli WP2uvrA
TA100 and E.
coli WP2uvrA
Ames
Strains (+S9) -
TA1535, TA1537, TA1535,
TA98, TA100 and TA1537, TA98,
E. coli WP2uvrA
TA100 and E.
coli WP2uvrA
Result
-
Negative
Positive
Figure 5: Investigating and Mitigating the Mutagenic Liabilities of Potential Degradants of
29
Strategies were investigated to mitigate the potential mutagenicity risk inherent in 29. One
option was to reduce the electron density of the aniline through the introduction of electron
withdrawing groups to the aromatic core. This was investigated by substituting with Fluorine
at the 2, 5 or 6 positions to provide 49, 51 and 53 respectively. These potential degradants /
metabolites all had reduced HOMO energies and were therefore predicted to be negative in the
AMES test (Figure 6). As a result, examples of these F-substituted compounds were prepared
(compounds 48, 50, 52). For synthetic ease, the optimal cyclohexanol amide was replaced with
an ethyl group, whilst the alpha-methyl group was not introduced on these examples initially.
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Introduction of fluorine at the 2-position (48, Table 3) gave a reduction in potency at BD2
when compared with the unsubstituted parent 5. However the 5-substituted fluoro example 50
showed an increase in potency, but this was accompanied with an unacceptable increase in
chromLogD. The 6-F substituted compound 52 was preferable, demonstrating an increase in
BD2 potency of 5 fold and also a slight decrease in ChromLogD.
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
O
O
O
HN
HN
HN
O
F
O
O
F
F
O
N
H
O
N
H
O
N
H
48
50
52
metabolism/
degradation
metabolism/
degradation
metabolism/
degradation
NH2
NH2
NH2
HO
HO
F
HO
F
F
O
OH
O
OH
O
OH
49
51
53
4
9
51
53
HOMO
eHOMO
HOMO
energy (a.u.)
-0.3280
-0.3250
-0.3353
Low
Risk
Low
-
Low
-
Strains (-S9)
TA1535,
TA1537, TA98,
TA100 and E.
coli WP2uvrA
Ames
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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
Strains (+S9)
Result
-
-
-
-
TA1535,
TA1537, TA98,
TA100 and E.
coli WP2uvrA
Positive
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: Mitigating the Genotoxic Risk of Aniline 47
Cmp BRD4 BD1 BRD4 BD2 BRD4
BRD4
CHROMLo AMP (nm/sec)
d No pIC (n)
pIC (n)
BD2 Sel BD2 LE gD
(fold)
/
cLND
5
0
50
(µg/mL)
5
<4.3 (4)
<4.3 (2)
<4.3 (2)
<4.3 (2)
5.3 (3)
4.7 (2)
5.8 (2)
6.0 (2)
10
2.5
32
50
0.31
0.27
0.33
0.34
3.5
3.9
4.4
3.1
320 / 143
460 / 119
670 / 159
110 / 18
48
50
52
Table 3 – Potency and Selectivity of Fluorinated Core Derivatives of 5.
Given these exciting data, it was decided to combine the 6-F substituent with the favoured
shelf and amide groups of 29. This gave acetamide 59 which can be prepared using the
synthetic route defined in scheme 5. A S Ar reaction was performed on 5-bromo-1,3-difluoro-
N
2
-nitrobenzene 54 with (S)-phenylethanol to give 55, reduction of the nitro and subsequent
acetylation of the resulting amine 56 gave 57 in good overall yields. A carbonylation reaction
with 57 using Xantphos and Pd(OAc) under a CO atmosphere could be used to access the
2
methyl ester 58. Hydrolysis of 58 followed by amide coupling under standard conditions was
used to give 59. Interestingly the late stage intermediate 57 (where R=Bn) could be used to
synthesise 52 directly using a amidocarbonylation reaction with Xantphos, Pd(OAc)2,
ethylamine, Co CO and microwave irradiation.
2
8
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5
9 was tested in the Brd4 assay and pleasingly resulted in a 1000 fold BD2 selective inhibitor
(
Table 4). Regretably, whilst 59 itself was Ames negative, when we tested potential metabolite
/ degradant 53, it tested Ames positive against strain WP2 uvrA in the presence and absence of
S9. Therefore whilst the eHOMO model is in general a very useful in silico predictor, on this
occasion it predicted the mutagenicity risk of 53 incorrectly. Despite the positive Ames result
with the potential metabolite, 59 had the potential to be an excellent tool molecule and so was
fully profiled for its potency, selectivity and pharmacokinetic attributes. Indeed, the high level
of potency and selectivity observed in the BRD4 FRET assay was recapitulated against the
other BET bromodomains (BRD2, 3 and T), whilst 59 was also sent to DiscoverX and profiled
in their Bromoscan platform (Figure 7, Table S2 Supporting Information). 59 shows an
excellent level of selectivity for the BET family with no measurable activity for non-BET
bromodomains
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
Scheme 5: Synthesis of 5-F substituted analogues including 59
O
O
NO2
Br
NO2
Br
NH2
Br
HN
HN
F
F
O
F
O
F
O
F
R
R
O
F
R
i
ii
iii
iv, v
R
Br
O
X
54
55
56
52, X = NHEt, R = Bn
57
58, X= OMe, R = (S)-1-
Phenylethanol
vi, vii
OH
59, X =
H2N
Reagents and conditions:i, ROH, LiHMDS, THF, rt, 30 min (99%), ii. Fe(0), NH Cl, EtOH,
4
o
8
0 C, 1 h (82%). iii.Ac O, rt, 20 h (81%) iv. Xantphos, Pd(OAc) , DMAP, Co CO , 1,4-
2
2
2
8
o
dioxane, ethylamine, 90 C, 20 min (66%). v. Xantphos, Pd(OAc) , Et N, CO, MeOH, DMF,
2
3
o
o
0 C, 1 h (73%). vi. LiOH, THF:H O 1:1, 50 C, 2 h (93%). vii Trans-4-aminocyclohexanol,
2
HATU, DIPEA, DCM, rt, 1 h (89%).
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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
Given the profound efficacy associated with pan-BET inhibitors in models of inflammation
and oncology, having such high selectivity makes delineating the respective roles of the BD2
domain of proteins BRD2, 3, 4 and T more facile. 59 was therefore tested in an LPS stimulated,
peripheral blood mononuclear cell (PBMC) cellular assay. After stimulation, these immune
cells release a range of cytokines and chemokines, including monocyte chemoattractant protein
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
7
1
(MCP-1 / CCL2). MCP-1 is a key chemokine that regulates migration and infiltration of
macrophages and as such is involved in a range of immuno-inflammatory diseases. It has been
shown previously that pan-BET and BD2-biased inhibitors are capable of inhibiting an MCP-
3
1
1
response. The BD2-selective inhibitor 59 also inhibits MCP-1 production (pIC = 7.5),
50
with minimal drop-off from the biochemical BRD4 BD2 potency observed. Not only does this
confirm cellular penetration of 59, but also strongly suggests that pan-BD2 inhibitors are
capable of maintaining an anti-inflammatory phenotype with minimal BD1 engagement.
Pleasingly, this inhibition translated into sub-micromolar inhibition in human whole blood. A
greater drop off would be expected in this assay, as a proportion of the inhibitor will be bound
to blood proteins (human blood F = 32%). 59 also has an encouraging developability profile,
ub
with a chromLogD of 2.8 and a PFI in the desired range of 4.8. This translates into a good
kinetic solubility (CLND) and perhaps more importantly, an excellent thermodynamic
solubility measured in fasted-state simulated intestinal fluid (FaSSIF) of 996 µg/mL. The
predicted permeability using the AMP assay was low, however we had already shown that this
assay was not predictive of cellular penetration using the PBMC assay so this result was not
overtly concerning. In search of an in vivo capable probe, the pharmacokinetics of 59 were
evaluated (Figure 8). Utilising an external panel of isolated rat, dog and human hepatocytes,
we were able to show that 59 was not metabolised at the level of detection possible in these in
vitro systems, which therefore was predictive of a low/moderate clearance in vivo. To explore
this prediction, 59 was characterised in vivo in the rat and dog by both intravenous and oral
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routes of administration. Pleasingly, 59 showed moderate clearance in rat and dog of 46% and
3
6% liver blood flow (LBF) respectively when dosed i.v. After oral administration, 59 had a
bioavailability in rat of 35% and dog of 66%. These data show that 59 is a suitable probe for
use in in vivo animal models. Indeed we have recently described further studies with 59 (also
known as iBET-BD2) which show that selective BD2 inhibition is efficacious in a T-cell
dependent immunisation model.48
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
O
HN
O
F
OH
O
N
H
5
9
BRD4 BD1 / BD2 pIC50
Selectivity (fold)
BD2 LE
4.2 +/- 0.44 (3/13) / 7.3 +/- 0.13 (14) a
> 1000
0.33
Clogp / MW
ChromLogD / PFI
1.3 / 414
2.8 / 4.8
91 / 996
7
.4
CLND solubility (µg/mL) / FaSSIF
µg/mL)
AMP7.4 permeability (nm/s)
(
16
hERG pIC / HSA binding (%)
CYP3A4 (MDI)
< 4.3 / 47
<4.4
50
5
9
BD1
BD2
BRD4
BRD2
BRD3
BRDT
4.2 +/- 0.44 (3/13) a 7.3 +/- 0.13 (12)
5.0 (1/11) a
4.4 +/- 0.01 (2/8) a
6.6 +/- 0.2 (9)
7.0 +/- 0.08 (8)
6.7 +/- 0.25 (8)
< 4.3 (8)
a
Some results below the curve-fitting threshold of 4.3 could not be included in mean and SD. In these cases, the
numbers included in the mean / number of times tested are both shown.
Table 4: Profile of pan-BD2 selective probe 59
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1
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2
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2
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5
5
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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: Bromoscan Profile of 59.
Hepatocyte Cli
mL/min/g
Vss
T½
(h)
Fpo
CLb
mL/min/kg)
Species
Rat
Fub
(
(
(%)
tissue)
(L/kg)
0.91
<0.80
36
20
0.5
0.7
31
66
0.44
0.32
0.33
Human <0.45
Dog <1.26
0.91
Figure 8: in vitro and in vivo pharmacokinetic profile of 59
To understand the origins of the high levels of BD2 selectivity seen with 59, crystal structures
were obtained in BRD2 BD2 and BRD4 BD1. The binding mode of the molecule to both
bromodomains is similar to that of 1, with the 4-acetamido group occupying the acetyl-lysine
pocket and hydrogen-bonding to the conserved W1 water. The 3-benzyloxy group still occupies
the WPF shelf region of the site, as shown for BRD2 BD2 in Figure 9a, including Val435 and
Trp370. A comparison to the BRD2 bound complex of 1 reveals that 59 binds with a rotation
in the site relative to 1 (Figure 9b), with a less planar conformation of the phenyl-4-acetamido
torsion (about 50° in the case of 59 versus 14° for 1). This is probably due to electrostatic
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repulsion between the 5-fluoro substituent and the 5-acetamide carbonyl, but may be the steric
constraint exerted upon the 3-position benzylic methyl group of 59 by the narrow ZA channel
region between Trp370 and Leu381 could also contribute. One consequence of the movement
is to bring the 3-position benzyl group closer to the sidechain of His433, which unambiguously
lies in a single gauche(+) conformation, the two rings making an edge-to-face contact. Another
is to bring the 1-position amide NH significantly closer to the Asn429 carbonyl oxygen than in
the complex with 1, into direct hydrogen-bonding distance. The cyclohexanol 4-position group
is clearly visible and makes predominantly hydrophobic contacts with Pro430 and His433.
The complex of 59 with BRD4 BD1 is similar to the BRD2 BD2 structure (Figure 9c). The
extra potency and selectivity for BD2 over BD1 most likely comes from the significantly
greater interactions with His433 in BD2, which include the edge-face aryl-aryl contact with the
shelf benzyl substituent as well as the cyclohexanol ring, as well as with Pro430 (Figure 9c).
These stabilise the His433 gauche(+) conformation resulting in excellent shape
complementarity. In BD1, the His433 equivalent residue Asp144 points away from the
inhibitor and makes essentially no direct interaction, resulting in a much smaller contact surface
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 9d) and explaining the lower potency. A comparison of the BRD2 BD2 bound structure
of 59 with the BRD4 BD2 bound complex with ABBV-744 shows similar positioning of the
amide substituents in each, reinforcing the importance of this group to the BD2 selectivity of
both inhibitors (Figure 9e).
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1
1
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2
2
2
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2
2
3
3
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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 9 – X-ray structures of: (A) 59 (cyan, PDB 6swp) and 1 (green, PDB 6z7f, mobile
imidazole not shown for clarity) in BRD2 BD2. (B) 59 (orange, PDB 6swq) in BRD4 BD1.
(C) 59 (cyan) with BRD2 BD2 surface shown in blue. The surface of His433 is shown in yellow
and its contacts with 59 are shown as red dashed lines. (D) 59 (orange) with the Asp144 surface
shown in yellow. (E) Superimposed crystal structures of BRD2 BD2 bound to 59 (cyan, PDB
3
4
6
swp) and ABBV-744 (magenta, PDB 6e6j, ).
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Conclusion
In summary, we have detailed the optimisation of a relatively weak BD2-selective HTS hit
into 59, a potent and highly selective pan-BD2 inhibitor. A screening hit with weak BD2 bias
containing a simple AcK mimetic and WPF shelf group was extended to exploit a key residue
difference between BD1 and BD2, to generate highly potent and domain-selective BD2
inhibitors. 59 has suitable physicochemical and pharmacokinetic properties to be utilised in
vivo and it has also been demonstrated that a highly selective BD2 inhibitor retains the ability
to potently inhibit MCP-1 cytokine release in a cellular and whole blood context. 59 proved
useful for further biological profiling. Indeed, we have recently published a comparison
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
49
48
between the in vitro and in vivo phenotypes of selective pan-BD1 and pan-BD2 inhibitors.
These data show that 59 is efficacious in a broad range of inflammatory pathologies and that
selective BD2 inhibition may be a useful new therapeutic strategy for immunoinflammatory
diseases.
Experimental Section
General Experimental
Unless otherwise stated, all reactions were carried out under an atmosphere of nitrogen in heat
or oven dried glassware and anhydrous solvent. Solvents and reagents were purchased from
commercial suppliers and used as received. Reactions were monitored by thin layer
chromatography (TLC) or liquid chromatography-mass spectrometry (LC-MS). TLC was
carried out on glass or aluminium-backed 60 silica plates coated with UV254 fluorescent
indicator. Spots were visualized using UV light (254 or 365 nm) or alkaline KMnO solution,
4
followed by gentle heating. LCMS analysis was carried out on a Waters Acquity UPLC
instrument equipped with a CSH C18 column (50 mm x 2.1 mm, 1.7 µm packing diameter)
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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
and Waters micromass ZQ MS using alternate-scan positive and negative electrospray.
Analytes were detected as a summed UV wavelength of 210 – 350 nm. Two liquid phase
methods were used: Formic – 40 °C, 1 mL/min flow rate. Gradient elution with the mobile
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
phases as (A) H O containing 0.1% volume/volume (v/v) formic acid and (B) acetonitrile
2
containing 0.1% (v/v) formic acid. High pH – 40 °C, 1 mL/min flow rate. Gradient elution with
the mobile phases as (A) 10 mM aqueous ammonium bicarbonate solution, adjusted to pH 10
with 0.88 M aqueous ammonia and (B) acetonitrile. Flash column chromatography was carried
out using Biotage SP4 or Isolera One apparatus with SNAP silica cartridges. Mass directed
automatic purification (MDAP) was carried out using a Waters ZQ MS using alternate-scan
positive and negative electrospray and a summed UV wavelength of 210 – 350 nm. Two liquid
phase methods were used: Formic – Sunfire C18 column (100 mm x 19 mm, 5 µm packing
diameter, 20 mL/min flow rate) or Sunfire C18 column (150 mm x 30 mm, 5 µm packing
diameter, 40 mL/min flow rate). Gradient elution at ambient temperature with the mobile
phases as (A) H O containing 0.1% volume/volume (v/v) formic acid and (B) acetonitrile
2
containing 0.1% (v/v) formic acid. High pH – Xbridge C18 column (100 mm x 19 mm, 5 µm
packing diameter, 20 mL/min flow rate) or Xbridge C18 column (150 mm x 30 mm, 5 µm
packing diameter, 40 mL/min flow rate). Gradient elution at ambient temperature with the
mobile phases as (A) 10 mM aqueous ammonium bicarbonate solution, adjusted to pH 10 with
0
.88 M aqueous ammonia and (B) acetonitrile. NMR spectra were recorded at ambient
temperature (unless otherwise stated) using standard pulse methods on any of the following
1
13
spectrometers and signal frequencies: Bruker AV-400 ( H = 400 MHz, C = 101 MHz,),
1
13
1
Bruker AV-600 ( H = 600 MHz, C = 150 MHz,) or Bruker AV4 700 MHz spectrometer ( H
1
3
= 700 MHz, C = 176 MHz). Chemical shifts are referenced to trimethylsilane (TMS) or the
residual solvent peak, and are reported in ppm. Coupling constants are quoted to the nearest
0
.1 Hz and multiplicities are given by the following abbreviations and combinations thereof: s
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(
singlet), δ (doublet), t (triplet), q (quartet), quin (quintet), sxt (sextet), m (multiplet), br.
(broad). Purity of synthesized compounds was determined by LCMS analysis. All compounds
for biological testing were >95% pure.
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Synthetic Procedures
Methyl 4-acetamido-3-hydroxybenzoate (31): To a solution of methyl 4-amino-3-
hydroxybenzoate (22 g, 132 mmol, commercially available from supplier such as Fluorochem)
in p-Xylene (440 mL) and THF (220 mL) was added acetyl chloride (9.39 mL, 132 mmol) at
rt, the reaction was stirred at 70 °C for 16 h. The reaction was cooled to rt and was partitioned
between H O and EtOAc, the organics were dried over Na SO and concentrated in vacuo, to
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2
4
afford crude methyl 4-acetamido-3-hydroxybenzoate (24 g, 106 mmol, 92 % yield) as a brown
+
1
solid. LCMS (Formic modifier) retention time 3.46 min, [M + H] = 209.9. H NMR (400 MHz,
DMSO-d ) δ ppm: 10.10-10.50 (m, 1H), 9.34 (s, 1H), 8.09 (br d, 1H, J = 8.6 Hz), 7.30-7.50 (m, 2H),
6
3
.80 (s, 3H), 2.14 (s, 3H).
Methyl 4-acetamido-3-(benzyloxy)benzoate (32): To a solution of methyl 4-acetamido-3-
hydroxybenzoate (24 g, 115 mmol) in DMF (240 mL) was added K CO (23.78 g, 172 mmol)
2
3
and stirred at rt for 20 min, to this benzyl bromide (17.74 mL, 149 mmol) in DMF (20 mL) was
added and the reacton was stirred at 70 °C for 16 h. The reaction mass cooled to rt and was
poured on crushed ice and was extracted with EtOAc. The organic phase was washed with cold
H O dried over Na SO and concentrated in vacuo to afford brown sticky solid. This solid was
2
2
4
triturated with DCM:Pentane to afford methyl 4-acetamido-3-(benzyloxy)benzoate (25 g, 82
mmol, 71 %) as a brown solid.
+
LCMS (Formic modifier) retention time 2.26 min, [M + H] = 300.0
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