Design of a biased potent small molecule inhibitor of the bromodomain and PHD finger-containing (brpf) proteins suitable for cellular and in vivo studies

Article abstract of DOI:10.1021/acs.jmedchem.6b01583

The BRPF (bromodomain and PHD finger-containing) family are scaffolding proteins important for the recruitment of histone acetyltransferases of the MYST family to chromatin. Evaluation of the BRPF family as a potential drug target is at an early stage although there is an emerging understanding of a role in acute myeloid leukemia (AML). We report the optimization of fragment hit 5b to 13-d as a biased, potent inhibitor of the BRD of the BRPFs with excellent selectivity over nonclass IV BRD proteins. Evaluation of 13-d in a panel of cancer cell lines showed a selective inhibition of proliferation of a subset of AML lines. Pharmacokinetic studies established that 13-d had properties compatible with oral dosing in mouse models of disease (F_po 49%). We propose that NI-42 (13-d) is a new chemical probe for the BRPFs suitable for cellular and in vivo studies to explore the fundamental biology of these proteins.

Full text of DOI:10.1021/acs.jmedchem.6b01583

Article
pubs.acs.org/jmc
Design of a Biased Potent Small Molecule Inhibitor of the
Bromodomain and PHD Finger-Containing (BRPF) Proteins Suitable
for Cellular and in Vivo Studies
Niall Igoe,† Elliott D. Bayle,† Oleg Fedorov,‡ Cynthia Tallant,‡ Pavel Savitsky,‡ Catherine Rogers,‡
Dafydd R. Owen,§ Gauri Deb,∥ Tim C. P. Somervaille,∥ David M. Andrews,⊥ Neil Jones,# Anne Cheasty,#
Hamish Ryder,# Paul E. Brennan,‡ Susanne Muller,‡,∇ Stefan Knapp,‡,∇,○ and Paul V. Fish*,†,◊
̈
†UCL School of Pharmacy, University College London, 29/39 Brunswick Square, London WC1N 1AX, U.K.
‡Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research
Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.
§Pfizer Worldwide Medicinal Chemistry, 610 Main Street, Cambridge, Massachusetts 02139, United States
∥Leukemia Biology Laboratory, Cancer Research UK Manchester Institute, Manchester M20 4BX, U.K.
⊥AstraZeneca Discovery Sciences, Darwin Building, Cambridge Science Park, Cambridge CB4 0FZ, U.K.
#CRT Discovery Laboratories, Jonas Webb Building, Babraham Research Campus, Cambridge CB22 3AT, U.K.
∇Buchmann Institute for Molecular Life Sciences, Max-von-Laue-Strasse 15, D-60438 Frankfurt am Main, Germany
○Institute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main,
Germany
S
* Supporting Information
ABSTRACT: The BRPF (bromodomain and PHD finger-containing) family are scaffolding proteins important for the
recruitment of histone acetyltransferases of the MYST family to chromatin. Evaluation of the BRPF family as a potential drug
target is at an early stage although there is an emerging understanding of a role in acute myeloid leukemia (AML). We report the
optimization of fragment hit 5b to 13-d as a biased, potent inhibitor of the BRD of the BRPFs with excellent selectivity over
nonclass IV BRD proteins. Evaluation of 13-d in a panel of cancer cell lines showed a selective inhibition of proliferation of a
subset of AML lines. Pharmacokinetic studies established that 13-d had properties compatible with oral dosing in mouse models
of disease (Fpo 49%). We propose that NI-42 (13-d) is a new chemical probe for the BRPFs suitable for cellular and in vivo
studies to explore the fundamental biology of these proteins.
family in 2010,3,4 inhibitors for the major branches of the
bromodomain family tree have been generated.5 Importantly,
INTRODUCTION
■
Bromodomains (BRDs) are protein interaction modules that
bind to acetylated lysine containing sequence motifs on
chromatin and nonchromatin proteins and act as epigenetic
reader domains.1 Despite large sequence variations, bromodo-
mains share a conserved fold with a central deep, largely
hydrophobic acetyl-lysine (Ac-Lys) binding pocket, which for
many family members is considered highly druggable.2
Accordingly, since the publication of the first bromodomain
inhibitors of the bromodomain and extra-terminal (BET)
for many of the bromodomains more than one inhibitor from
alternative scaffolds are available, providing confidence in the
observed biological results.6,7 In addition, structurally related
compounds that do not bind to the respective bromodomain
Received: October 25, 2016
© XXXX American Chemical Society
A
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often complement the inhibitor package, lending credibility to
the observed findings.3,8,9
bromodomain does not prevent the acetylation of histone H3
by the HBO1/BRPF1 complex.14
Contemporaneous studies identified the 1,3-dimethyl
benzimidazolone template as binding to the BRD of
BRPF1,6,7 and this core has been independently optimized to
give three chemical probes as either the pan-BRPF BRD
inhibitor OF-1 (1)23 or as the selective BRPF1 BRD inhibitors
PFI-4 (2)24 and GSK5959 (3),25 which share a very similar
chemical structure (Figure 1). Piperidine 3 has been further
In human, 61 bromodomains are present in 46 proteins and
some exist as alternatively spliced forms.10 The bromodomain
and PHD finger-containing (BRPF) protein family of
bromodomains contains four members. BRPF1A and
BRPF1B are generated by alternative splicing, resulting in a
longer BRPF1A isoform, which has a six-residue insert in the
BRD, and the shorter BRPF1B isoform.10 The related BRPF2
(also termed BRD1) and BRPF3 proteins share the general
domain structure with BRPF1A and 1B comprising a double
PHD and zinc finger arrangement, a bromodomain, and a C-
terminal chromo/Tudor-related PWWP domain. The PWWP
domain of BRPF1 binds to trimethylated Lys36 in histone H3
(H3K36me3) and is important for targeting BRPF1 to
chromatin,11 while the BRD of BRPF1 recognizes multiple
Ac-Lys modifications on the N-terminal histone tail, in
particular H2AK5ac, H4K12ac, and H3K14ac.12 This modular
arrangement points to a role of the BRPF family members as
scaffolding proteins. Indeed, BRPFs are found in a tetrameric
complex with ING5 and Eaf6/EPC-related scaffold subunit, as
well as one of the MYST family members of the H3K9 and
H3K14 histone acetyl transferases (HATs): MOZ or MORF,
or alternatively, HBO1 in the case of BRPF2/3.13 Interestingly,
BRPF1 can also associate with HBO1, resulting in a switch
from the commonly preferred substrate H4 to H3.14
Chromosomal translocations that involve the MOZ gene are
associated with acute myeloid leukemia (AML) and over-
expression of HOXA9 and HOXA10 was observed in AML
patients with MOZ fusions.15 BRPF1 is a core component of
MOZ/MORF HAT complexes with a key role in their assembly
and activation16 and facilitates interaction with chromatin.
BRPF1 is essential for the proper formation of fetal
hematopoietic stem cells as well as expression of stem cell
genes such as Hoxa9.17 In AML, MOZ is recurrently targeted
by chromosomal translocations and the resulting fusion
oncoproteins (e.g., MOZ-TIF2) form a stable complex with
BRPF1 which interacts with, and deregulates, HOX gene loci.18
Conversely, depletion of BRPF1 decreased the MOZ local-
ization on HOX genes abrogating transformation and mutant
MOZ-TIF2 engineered to lack HAT activity was incapable of
deregulation of HOX genes or initiating leukemia.18 These data
indicate that the MOZ-TIF2/BRPF1 complex upregulates
HOX gene expression possibly mediated by MOZ-dependent
histone acetylation, leading to development of leukemia.18
Further evidence supporting BRPF1 as a credible target for
AML includes: (1) its expression is highest in acute leukemia
cell lines versus all other cell line types;19 (2) the normal role of
BRPF1 is to sustain HOX gene expression in development18
and misregulated HOX gene expression is critical to the
pathogenesis of at least 70% of AML, in particular, the MLL
mutated subtypes and AMLs with mutations in NPM1;20 (3)
BRPF1 has significant homology with AF10 and AF17, two
recurrent fusion partners of MLL in human AML;21 and (4)
the complex formed by BRPF1, ING5, and MOZ binds to, and
acetylates, histone H3 to act as a coactivator of RUNX1, which
is another critical transcriptional regulator in AML.22
Figure 1. 1,3-Dimethyl benzimidazolones as inhibitors of the
bromodomain of the BRPFs.
optimized to the α-methyl piperazine analogue GSK6853 (4),
which has significantly improved solubility, and properties
suitable for cellular and in vivo dosing, albeit by ip
administration.26 The 1,3-dimethyl benzimidazolone template
has also been used to identify selective TRIM24-BRPF1B dual
inhibitors.27
The use of selective small molecule inhibitors as chemical
probes to investigate the relationship between the target and
disease is significantly enhanced when two structurally
orthogonal chemical probes are employed. Two different
chemotypes which share the same primary pharmacology are
likely to offer the advantage of a different secondary
pharmacology fingerprint profile and so help identify any off-
target effects in cellular assays.
Our aims were to discover new small molecule inhibitors of
the BRD of the BRPFs to explore the fundamental biology of
these proteins and then build target validation to underpin new
drug discovery programs. Ideally these small molecule
inhibitors would be suitable for oral dosing in in vivo models
of disease and have drug-like properties. Here we report 13-d as
a new, structurally orthogonal chemical probe for the BRPFs
suitable for cellular and in vivo studies.
RESULTS AND DISCUSSION
■
Origin of Quinolin-2-one Hit Series. The starting point
was fragment hit N-methylquinolin-2(1H)-one (5b) which was
originally identified as binding to PCAF (Figure 2).28 In fact,
quinolin-2-ones 5a and 5b have proven to be efficient and
versatile fragment hits for a number of BRDs and further
optimization has identified potent inhibitors and chemical
probes for the BETs,29 BRD7/9,30 and ATAD2.31 However,
our analysis of quinolin-2-one 5b showed it to be a more potent
inhibitor of BRPF1 (IC50 4800 nM) when compared to other
BRDs which have been investigated (Table 1).
Hence, this emerging body of evidence supports the potential
role of BRPF1 in leukemia and prompted us to explore AML as
a pathfinder cancer disease. Targeting the MYST complex by
inhibition of BRPF1B provides a potential therapeutic
opportunity in AML, although deletion of the BRPF1
Hence 5b was selected as our starting point for several
reasons: (1) excellent “hit-like” properties (mw 159; cLogP 1.3;
LE = 0.62; LLE = 4.0) with no obvious reactive groups; (2)
evidence that selectivity between BRDs could be achieved and,
in particular, over BET family member BRD4; (3) chemically
B
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explore and establish SAR to guide optimization; and (4) a
cocrystal structure of BRPF1B was solved to support
compound design.
We determined the X-ray cocrystal structure of fragment 5b
bound to BRPF1B. As predicted, the quinolin-2-one scaffold
serves as the acetyl-lysine mimetic with the carbonyl forming a
hydrogen bond to the conserved Asn708 at 2.9 Å and forming a
water mediated hydrogen bond with Tyr665 (Figure 3). The
aromatic core is stabilized with the hydrophobic residues at the
ZA loop (Val657 and P658) and two residues at the lipophilic
shelf adjacent to the ZA loop (Ile652 and Phe653). The
quinolin-2-one fragment was not large enough to induce
conformational changes to the binding site and to the Phe714
gatekeeper residue when compared with the ligand-free
structure of BRPF1B.
On the basis of the affinity values (IC50) obtained for 5b
among these bromodomains (Table 1), we superposed
structures and aligned sequences (Supporting Information,
Figure S4) and observed that the binding specificity of 5b is
very sensitive to the nature of the gatekeeper residue, in which a
rather bulky and hydrophobic residue is favorable. Also, a
lipophilic residue at the ZA-loop (Pro658 for BRPF1B) is
preferred to stabilize and sandwich the fragment at the binding
site. The proline side chain is at 3.4 Å distance to the aromatic
core of the quinolin-2-one, whereas other side chain residues in
this position are pointing away to the binding site (Glu1014 for
ATAD2; Thr50 for BRD9) or disordered side chains (Lys753
for PCAF).
Figure 2. Evolution of N-methylquinolin-2(1H)-ones as BRPF
inhibitors.
a
Table 1. Inhibition of BRD Activity by Fragment Hit 5b
Introduction of a 6-NH2 into the quinolin-2-one template
gave 6, which retained affinity while providing a functional
group suitable for further elaboration to explore SAR. This 6-
NH2 group is positioned adjacent to the “WPF shelf” region of
the protein, which is important for establishing selectivity
between BRDs; the corresponding sequence in BRPF1 is Asn-
Ile-Phe with Phe714 as the “gate-keeper” residue. One of the
most significant modifications was the introduction of a 3-Me
group to give 7 as a potent fragment-like lead (BRPF1 IC50 190
nM; mw 188; cLogP 0.95; LE = 0.67; LLE = 5.8). This methyl
group enhanced affinity by 10-fold, which is near to the
maximum feasible from a buried Me group in an hydrophobic
environment as assessed by binding energy calculations.32
BRD
BRPF1
IC50 (nM)
4800
14000
BRD9
PCAF
70000
BRD4(BD1)
ATAD2
73000
>100000
a
BROMOscan assay. BRD IC50 values are geometric means of two
experiments. Differences of <2-fold should not be considered
significant.
enabled with sufficient synthetic chemistry precedent to
selectively functionalize each position of the template to
Figure 3. N-Methylquinolin-2(1H)-one (5b in yellow) cocrystallized with BRPF1B (gray) and solved at 1.5 Å (pdb 5T4U). (a) 2Fo − Fc electron
density map contoured at 1.2σ showing the 5b inhibitor bound to BRPF1B. The figure shows the map for the fully refined structure. (b) Binding site
details of the 5b ligand interaction.
C
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Using our cocrystal structure of BRPF1B with 5b as a model,
the 3-Me of 7 was rationalized as projecting toward Y707 with
the N1-Me fitting into the Me pocket as employed by the Me
groups of the Ac-Lys residues of the histone substrates
(Supporting Information, Figure S5).33 Alternatively, the
binding of 7 could also be accommodated by flipping the
template about the C(2)O axis and swapping the binding
orientation of the 3-Me and N1-Me groups; both orientations
would likely stabilize binding for 7 and improve affinity for
BRPF1B when compared to 5b.
Scheme 1. General Synthesis of Sulfonamides 12−16
Capping the 6-NH2 group of 6 as a phenyl sulfonamide gave
12-a. This modification introduced a bend into the molecule
which gave the sulfonamide group the opportunity to turn out
of the Ac-Lys binding pocket and wrap around the “gate-
keeper” Phe714. This binding mode would be quite different
from the linear 5-carboxamides as used in 2−4, which project
out past Phe714 toward the side chain of Glu661. Preliminary
results with the phenyl sulfonamide 12-a were encouraging
showing reasonable binding affinity for all three BRPFs with a
preferred affinity for BRPF1 (IC50 65 nM) while retaining good
physicochemical properties (mw 314; cLogP 2.0; LE 0.46; LLE
5.2). Hence, we selected 12-a as the lead for our SAR studies
with the aim of improving binding affinity for the BRPFs while
reducing affinity for other BRDs, especially BRD4.
chromatography-free, and operationally simple synthesis of 7.
3-Methylquinoline (17) was converted to the corresponding 3-
methylquinolin-2(1H)-one (18) by the two-step process of
oxidation with mCPBA to the intermediate N-oxide followed by
rearrangement and hydrolysis of the activated benzoyl ester
with aq NaOH. Selective N-methylation of 18 was achieved
with NaH and MeI in DMF to give 19, and nitration with
KNO3 in conc H2SO4 gave 20 in high yield. Finally, reduction
of the 6-NO2 group was most conveniently achieved by
catalytic hydrogenation to give amine 7.
The general method for the small-scale synthesis of
sulfonamide test compounds 12−16 (typically 20−100 mg)
was to react the amine with an excess of sulfonyl chloride (1.5
equiv) with pyridine (2.0 equiv) in CH2Cl2 or DMF at room
temperature overnight, followed by evaporation of solvents and
direct purification of the crude reaction mixture by chromatog-
raphy. For larger amounts of material, the amine was coupled
with the sulfonyl chloride (1.0 equiv) with DABCO (2.0 equiv)
as the base in DMF at room temperature for 6 h. The reaction
mixture was poured into ice-cold dilute aqueous HCl; the
sulfonamide usually precipitated from solution and was
collected by filtration.
Screening Strategy. Compounds were tested for BRPFs
BRD binding (ΔTm) with a differential scanning fluorimetry
(DSF) assay (Table 2). Selected compounds were then
screened for inhibition (IC50) of BRPF BRD activity as
measured by the BROMOscan biochemical assay (Table 3).
BRD9 and BRD4(BD1) were routinely used as BRD counter
screens to establish evidence of selectivity in both the DSF and
BROMOscan assays. Preferred compounds were then evaluated
for selectivity against a panel of 48 BRDs by DSF, and
inhibition of BRPF BRD activity was confirmed by independent
evaluation by an AlphaScreen biochemical assay (IC50). Finally,
equilibrium dissociation constants (KD) were determined by
isothermal titration calorimetry (ITC).
Structure−Activity Relationships. The SARs were
initially focused on exploring substituents on the phenyl ring
of 12 and then modification of the groups at the N1 and C3
positions of the quinolin-2-one core 13−15 (Tables 2 and 3).
Furthermore, target compounds were designed to have
molecular and physicochemical properties consistent with
drug-like space. Modulation of the lipophilicity of target
compounds was achieved by the application of the lighter
halogens (F, Cl), small alkyls (Me, Et), alkoxy (OMe), and
nitriles (CN).
All non-BET BRD inhibitors need to demonstrate excellent
selectivity over BRD4 because of the dominant phenotype of
this protein in cellular models of disease due to the central role
played in many biological pathways. The selection of quinolin-
2-one sulfonamide 12-a as our lead meant it was necessary for
us to monitor selectivity over BRD4 as 12-a shares some
structural similarity with PFI-1 (23), a chemical probe for the
BETs34 which is a potent inhibitor of BRD4 (BD1 and BD2)
(IC50 44 nM) and yet has weak BRPF1 affinity (IC50 1800
nM).29,35 We aimed to identify a prototype compound with a
1000-fold selectivity window for BRPF1 over BRD4 and a
BRD4 affinity of IC50 > 5 μM to be used in cellular
phenotypical assays at reasonable concentrations and then
adjust selectivity if required. These criteria are far more
stringent than the usual minimum selectivity requirements for
chemical probes (30-fold)8,36 but necessary to deliver a
compound that would be “fit-for-purpose” without an under-
current of BRD4 activity to potentially confound biological
results.
Chemistry. The general method for the synthesis of the
quinolin-2(1H)-one sulfonamides 12−16 is outlined in Scheme
1. The 6-amino quinolin-2(1H)-ones 6−10 were reacted with
the appropriate sulfonyl chlorides 11 in the presence of base to
give the corresponding sulfonamides 12−16. The amines 6−10
were prepared by published methods or the application of
known procedures. The sulfonyl chlorides 11 were purchased
from commercial sources or prepared by standard methods.
As the project developed, the need for larger amounts of
material to support SAR studies prompted the development of
an improved synthesis of 6-amino-1,3-dimethylquinolin-2(1H)-
one (7)37,38 (Scheme 2). This five-step sequence was
performed on a multigram scale and represents a high-yielding,
In general, there was a good correlation between the DSF
and BROMOscan data for these compounds with very similar
trends in ranked order of affinities. However, as has been noted
by others, the BROMOscan data routinely showed higher
affinities when compared to other quantified end points such as
the AlphaScreen or ITC measurements.
D
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a
Scheme 2. Synthesis of 6-Amino-1,3-dimethylquinolin-2(1H)-one (7) and 13-d
a
Reagents and conditions: (a) mCPBA (1.1 equiv), CH2Cl2, 0 °C → rt, o/n; (b) C6H5COCl (1.2 equiv), aq NaOH (1.0 M), CH2Cl2, 0 °C → rt, o/
n, 59% over two steps; (c) NaH (1.1 equiv), MeI (1.3 equiv), DMF, 0 °C → rt, 5 h, 95%; (d) conc H2SO4, KNO3 (1.05 equiv), 0−5 °C → rt, 4 h,
87%; (e) H2 (1 atmos), Pd/C (5 wt %, 0.05 equiv), EtOH, rt, 7 h, 95%; (f) 4-cyanobenzenesulfonyl chloride (1.0 equiv), pyridine (2.0 equiv), DMF,
rt, o/n, 37−61%.
Table 2. Evaluation of N-Methylquinolin-2(1H)-ones 12−15 in DSF ΔTm Shift Assay
ΔTm shift at 10 μM (°C)
compd
R1
R3
X
BRPF1
BRPF2 (BRD1)
BRPF3
1.5
BRD9
BRD4 (BD1)
12-a
12-b
12-c
12-d
12-e
12-f
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
H
H
5.3
2.4
4.4
5.2
3.6
6.5
4.3
4.9
9.7
5.6
5.2
1.9
0.9
2.1
2.4
1.3
3.4
3.5
2.9
2.5
2.3
1.1
1.3
1.1
1.4
H
2-CN
H
3-CN
H
4-CN
H
2-F; 4-CN
2-Me; 4-CN
3-Cl; 4-CN
3-Cl; 4-Cl
4-CN
1.2
2.4
H
2.2
3.5
0.4
12-g
12-h
13-d
14-d
15-d
H
H
2.5
5.1
3.3
1.5
Me
Et
Me
4.5
1.2
0.95
4-CN
4-CN
A more complete analysis of phenyl derivative 12-a
established some modest selectivity over both BRD9 and
BRD4 (BD1). Introduction of a nitrile at each of the positions
of the phenyl ring showed a preference for the 3- and 4-
positions, 12-c and 12-d, respectively, with the 2-CN 12-b
being poorly tolerated. Combining a 4-CN group with a second
substituent at either the 2- or 3-positions on the phenyl ring
(12-e−12-h) offered no significant advantage except for the 2-
Me,4-CN analogue 12-f. The 3-Cl,4-Cl compound 12-h had
similar potency to 12-d but at the expense of increased
lipophilicity (ΔcLogP + 1.5) and was not pursued.
As had been seen previously with 6 and 7, once again, a
significant improvement in BRPF1 affinity was achieved by the
introduction of a 3-Me group on the quinolin-2-one of 12-d to
give 13-d as the most potent compound so far (BRPF1 IC50 7.9
nM; mw 353; cLogP 2.5; LE = 0.45; LLE = 5.6). Additional
screening of 13-d showed reasonable binding affinity for all
three BRPFs with good selectivity over BRD9 (39-fold) and
BRD4 (BD1) (>500-fold) (Supporting Information, Figure
S2). The introduction of a 2-MeO group 13-i also gave a
potent inhibitor of BRPF1 activity but, not surprisingly,
reintroduced some unwanted BRD4 activity.29 Compound
13-i represents our closest structural analogue to 23 and yet 13-
i still shows reasonable selectivity over BRD4 (BD1) (60-fold).
This selectivity can be attributed to a combination of the
intrinsic preference of the quinolin-2-one core 5b for BRPF1
(Table 1) and the accommodation of the substituent at N1.
Limited SARs would suggest that BRD4 prefers a N1-H,
whereas BRPF1 prefers a N1-Me; this single point change in
chemical structure may represent a simple way to switch
between BRPF1 and BRD4 activity.
Increasing the size of the 3-alkyl substituent from 3-Me to 3-
Et (14-d) was poorly tolerated, whereas exchanging the N1-Me
for an N1-Et (15-d) showed similar affinity. Although these
simple modifications at C3 and N1 of the quinolin-2-one failed
to improve the BRPF1 affinity (cf. 13-d vs 14-d vs 15-d), they
did provide some evidence of how the affinity could be reduced
to identify an inactive close analogue of 13-d (vide infra).
Preliminary attempts to design a BRPF1 selective inhibitor in
the quinolin-2-one template were based upon hybrid structures
of 13-d with 2. The incorporation of a 6-benzamide and 7-
pyrrolidine gave 21 (Table 3), which proved to be a modest
inhibitor of BRPF1 (IC50 220 nM; mw 391; cLogP 3.0; LE =
0.32; LLE = 3.7) with only an incremental improvement of
selectivity over BRD1 when compared to 13-d (11- vs 6-fold,
respectively). However, amide 21 had similar potency for
BRD9 (IC50 420 nM) and may be considered as a modest
BRPF1−BRD9 dual inhibitor for further optimization.
E
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a
Table 3. Evaluation of N-Methylquinolin-2(1H)-ones 12−15, 21, and 22 in the BROMOscan Assay
IC50 (nM)
compd
R1
R3
X
BRPF1
BRPF2 (BRD1)
BRPF3
7600
BRD9
BRD4 (BD1)
1700
12-a
12-b
12-c
12-d
12-g
12-h
13-d
13-i
14-d
15-d
21
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
H
H
65
430
56
1400
1700
270
200
450
230
48
2200
H
2-CN
H
3-CN
H
4-CN
43
1000
2300
910
2800
7200
H
3-Cl; 4-CN
3-Cl; 4-Cl
4-CN
62
2500
H
44
b
Me
Me
Et
Me
7.9
260
310
4500
600
2-OMe
4-CN
9
180
15
130
4-CN
220
2600
1800
12000
9300
420
890
4600
b
22
60
17000
a
b
BRD IC50 values are geometric means of two experiments (n = 2). Differences of <2-fold should not be considered significant. n = 4.
Figure 4. Co-crystal structure of 22 (NI-48; ref 38) with BRPF1B bromodomain solved at 1.65 Å (pdb 5T4V). (a) Surface representation, (b)
cartoon representation of the binding site details of 22 (yellow) bound to BRPF1B (gray) and overlaid with ligand-free BRPF1B structure (blue).
Around this time, our initial efforts to determine the binding
mode of a representative quinolin-2-one sulfonamide to
BRPF1B was achieved by the determination of a cocrystal X-
structure of BRPF1B with 1,4-dimethylquinolin-2-one 22
(BRPF1 IC50 60 nM; Table 3, Figure 4, and Supporting
Information, Figure S6).
Compound 22 occupies part of the substrate acetylated
peptide-binding groove of the BRD in good shape
complementarity (Figure 4a). The quinolin-2-one scaffold
maintains the same conformation as shown for the complex
structure with fragment 5b. However, new features of the
binding mode arose with this novel structure. Interestingly,
upon binding, the ligand induces a conformational twist of the
Phe714 gatekeeper residue. The phenyl side chain forms a
parallel, face-centered π−π stacking interaction with the
quinolinone core ring at approximately 3.6 Å. Moreover, the
new rotamer is pointing toward the benzonitrile ring, forming a
perpendicular T-shaped C−H−π interaction. This is shown
with the structural superposition of the free-ligand BRPF1B
structure (Figure 4b). We also observed the nitrile group
accommodated to a preformed hydrophobic cleft with residue
Ile713 at the top of the αC helix. Additional points of interest
are that the network of five conserved water molecules in the
binding pocket is not altered and the NH group from the
sulfonamide is stabilized through an intrahydrogen bond to the
7-OMe group on the quinolin-2-one (Supporting Information,
Figure S6).
Selectivity of 13-d. Completing the evaluation of lead
compound 13-d against all members of the class IV family of
BRDs in the BROMOscan assay identified affinity for BRD7 but
was completely selective over the ATAD2s (Table 4). 13-d was
confirmed as a potent inhibitor of BRPF1 by independent
evaluation in an AlphaScreen biochemical assay and with a very
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photobleaching (FRAP) assay39 using a fusion protein of green
fluorescent protein with a triplicated BRD of BRPF1B linked to
a nuclear localization signal ectopically expressed in the human
osteosarcoma cell line U2OS. The histone deacetylase
(HDAC) inhibitor SAHA was used to increase overall levels
of histone acetylation, resulting in a sufficient window
measuring differences in recovery time and demonstrating the
acetylation dependence of the FRAP experiments. Compound
13-d displaced BRPF1B-BRDx3 from chromatin at 1 μM as
shown by the significantly reduced recovery time of the
BRPF1B-GFP fusion protein (Supporting Information, Figure
S7). Hence, these FRAP experiments confirm 13-d inhibits
BRPF1B BRDs in the nucleus.
Table 4. Summary of Physicochemical Properties, in Vitro
Bromodomain Inhibition, ITC Screening, and Cell Transit
Permeability of 13-d
Physicochemical Properties:
mw
353
2.5
90
clogP
tPSA (Å2)
AML Cell Based Activity. Compound 13-d has been
evaluated in a number of cellular models in order to explore a
credible link between the BRPF proteins and disease. These
cellular models include osteoclastogenesis, inflammation, and
fibrosis; results from these studies will be reported separately.
Our own interest was to explore the potential role of BRPF1 in
acute myeloid leukemia (AML) and other cancers.
Bromodomain Inhibition:
BRPF1/2/3
BRD7/9
BROMOscan
IC50 (nM)
IC50 (nM)
IC50 (nM)
IC50 (nM)
7.9; 48; 260
82; 310
BROMOscan
BROMOscan
BROMOscan
ATAD2A/2B
BRD4(1)
>100000; >100000
4500
BRPF1/2/3
BRD9
AlphaScreen
AlphaScreen
AlphaScreen
IC50 (nM)
IC50 (nM)
IC50 (nM)
140; 760; 2000
1100
We treated an indexed cell panel of 211 cancer cell lines
(CLIMB panel)40 with 13-d for 72 h and observed modest and
selective inhibition of proliferation of certain AML cell lines
(Table 5). Three of these lines (NOMO-1, THP-1. and MV-4-
11) exhibit translocations targeting MLL. All five lines either
originate from patients who presented with AML with
significant monocytic lineage differentiation at diagnosis
(OCI-AML2, NOMO-1, THP-1, and MV-4−11) or have
strong latent monocyte/macrophage lineage differentiation
potential (KG-1), in response, for example, to phorbol
ester.41 Given the original observation of BPRF1-associated
MOZ fusions in myelomonocytic leukemia,42 therapeutic
targeting of BRPF1 may, in particular, be valuable in the
myelomonocytic morphologic subtype of human AML.
Taken together, these results provide clear evidence that 13-
d is active in cellular assays although additional studies will be
required to more fully establish the role of 13-d and the BRPFs
in AML.
Pharmacokinetics. Pharmacokinetic data for 13-d was
generated in vivo in mouse (Table 6 and Supporting
Information, Table S3A). Following single intravenous
administration of 13-d, clearance was low relative to liver
blood flow and volume of distribution was moderate, resulting
in an elimination half-life of 2.0 h. Single oral administration of
13-d as a suspension showed good oral bioavailability of 49%
even with incomplete absorption. These results established that
13-d had pharmacokinetic properties compatible with oral
dosing in mouse PK−PD models of disease with a 3 mg/kg po
dose, achieving free drug levels above the BRPF1 KD (ITC) for
4−5 h (Supporting Information, Table S3B).
BRD4(1)
>20000
Bromodomain ITC Screening:
BRPF1/2/3
BRD9
ITC
ITC
KD (nM)
KD (nM)
40; 210; 940
1130
Solubility and Cell Permeability:
a
aqueous solubility
Caco2 transit AB/BA, Papp (× 10−6 cms−1
moderate (100 μM min)
)
10/50
a
Maximum concentration used in screening assays created by serial
dilution of a 100 mM stock solution of 13-d in DMSO.
similar relative rank order of activities to other screening
formats (Table 4).
Compound 13-d was then evaluated in a number of assays to
establish the broader pharmacological selectivity. 13-d was
screened for BRD binding by DSF against a panel of 48 BRDs
and showed excellent selectivity. All activity was confined to the
class IV family of BRDs with ΔTm < 1 °C stabilization for all
nonclass IV BRDs (Figure 5; Supporting Information, Table
S1). ITC data showed that 13-d bound the BRD of BRPF1
with an equilibrium dissociation constant, KD, of 40 nM and an
enthalpy change, ΔH, of −11.7 kcal mol−1 (Table 4, Figure 6).
Additional ITC studies showed 13-d bound the BRD of BRD1
with a KD of 210 nM, whereas binding to BRPF3 and BRD9
was weaker (Table 4; Supporting Information, Figure S3).
Compound 13-d has reasonable aqueous solubility and good
cell permeability as measured by transit performance in the
Caco2 cell line but with some evidence of efflux (Table 4). Any
cardiovascular risk through direct interaction with cardiac ion
channels was low, as 13-d showed minimal activity against a
panel of eight cardiac ion channels in the Eurofins
CardiacProfile with only 45.6% normalized inhibition of the
hERG current amplitudes at 30 μM (Supporting Information,
Table S2).
Inactive Control. Chemical probes are most valuable when
they are partnered with an inactive control to assist with the
interpretation of phenotypic assays. Ideally, the inactive control
will share a similar chemical structure and physicochemical
properties as close as possible to the active compound. Hence
we designed a series of close analogues of 13-d where the key
recognition interactions between the quinolone C2-carbonyl
with Asn708 and the Tyr665 water network could be disrupted
by increasing the size of the alkyl groups flanking the carbonyl
and so prevent binding. Incorporation of ethyl groups at both
N1 and C3 gave 16-d (cf 14-d and 15-d), which removed
activity as measured against the BRPFs and a representative set
Hence, 13-d is a biased, potent inhibitor of the BRD of the
BRPFs with excellent selectivity over nonclass IV BRD
proteins.
BRPF1B Target Engagement. Target engagement in
living cells was demonstrated in a fluorescence recovery after
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Figure 5. Selectivity profile of 13-d assessed against 48 BRDs (bold type) by thermal shift, ΔTm (°C), at 10 μM as measured by DSF.
of bromodomains in the BROMOscan (>100 μM) and
AlphaScreen assays (>20 μM) (Table 7).
BRPF1B-BRDx3 from chromatin at 1 μM as shown by the
significantly reduced recovery time of the BRPF1B-GFP fusion
protein.
Evaluation of 13-d in a panel of cancer cell lines, including
AML lines, showed a modest (GI50 1−10 μM) and selective
inhibition of proliferation of certain lines, particularly those
lines exhibiting monocytic lineage differentiation. These results
provide clear evidence that 13-d is active in cellular assays.
However, additional studies will be required to fully establish
the role of 13-d and the BRPFs in AML.
Pharmacokinetic data for 13-d was generated in vivo in
mouse with single oral administration of 13-d as a suspension
showing good oral bioavailability. These results established that
13-d had pharmacokinetic properties compatible with oral
dosing in mouse PK−PD models of disease with a 3 mg/kg po
dose, achieving free drug levels above the BRPF1 KD for 4−5 h.
In conclusion, we propose that 13-d is a new, structurally
orthogonal chemical probe for the BRPFs suitable for cellular
and in vivo studies to explore the fundamental biology of these
proteins. Chemical probe 13-d (NI-42)38 will be of most value
in the interpretation of phenotypic assays when used in
conjunction with the inactive control 16-d and with other
available chemical probes of the class IV BRDs.
CONCLUSIONS
■
We have designed and evaluated a number of new quinolin-2-
one derivatives as inhibitors of BRPF BRD activity. Starting
with the fragment hit N-methylquinolin-2-one (5b), which had
modest affinity for PCAF, we demonstrated that 5b had much
higher affinity for BRPF1. The preferred binding of 5b to
BRPF1 was rationalized through an X-ray cocrystal structure
determination which underpinned future inhibitor design.
Optimization of 5b by incorporation of a 6-phenyl sulfonamide
gave 12-a and preliminary results were encouraging, showing
reasonable binding affinity for all three BRPFs. SARs
established that potent BRPF1 inhibition could be retained
through incorporation of various groups on the phenyl ring of
the sulfonamide with a 4-CN group preferred (12-d). A
significant improvement in affinity was achieved by the
introduction of a 3-Me group onto the quinolin-2-one of 12-
d to give 13-d as the most potent compound prepared while
retaining good physicochemical properties.
Compound 13-d was then evaluated in a number of assays to
establish the broader pharmacological selectivity. 13-d was
screened by DSF against a panel of 48 BRDs and showed
excellent selectivity with all activity confined to the class IV
family of BRDs. ITC data showed that 13-d preferentially
bound the BRD of BRPF1 and BRD1, whereas binding to
BRPF3 and BRD9 was weaker. On the basis of these results,
13-d was identified as a biased, potent inhibitor of the BRD of
the BRPFs with excellent selectivity over nonclass IV BRD
proteins. BRPF1 target engagement in living cells was
demonstrated in a FRAP assay. Compound 13-d displaced
EXPERIMENTAL SECTION
■
Chemistry Methods and Compound Characterization Data
for 13-d and 16-d: General Methods. All anhydrous solvents and
reagents were obtained from commercial suppliers and used without
further purification. Flash chromatography refers to medium pressure
silica gel (C60 (40−60 μm)) column chromatography, unless
otherwise stated. The progress of reactions was monitored by thin
layer chromatography (TLC) performed on Keiselgel 60 F254 (Merck)
silica plates and visualized by exposure to UV light at 254 nm.
H
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Journal of Medicinal Chemistry
Article
a
Table 6. Mouse Pharmacokinetic Data for 13-d
liver microsomes, Clint (μL/min/mg)
plasma protein binding, fu
14
0.03
intravenous dose (mg/kg)
1.0
2.0
4
elimination half-life, T1/2 (h)
blood clearance, Cl (mL/min/kg)
volume of distribution, Vd (L/kg)
0.7
oral dose (mg/kg)
Cmax (μM)
3.0
4.0
1.0
Tmax (h)
AUC0−∞ (ng.h/mL)
bioavailability, Fpo (%)
fraction absorbed (%)
5700
49
51
a
Female CD-1 mouse (n = 3).
Table 7. Inhibition (IC50) of BRD Activity for Inactive
Control 16-d
IC50 (nM)
BRDs
Figure 6. ITC data for 13-d with BRPF1. The figure shows raw
binding heats for each injection (upper panel) as well as normalized
binding enthalpies and a nonlinear least-squares fit to a single binding
site model. The fit revealed KD = 40 nM, ΔH = −11.7 kcal mol−1, and
TΔS = −1.87 kcal mol−1.
a
BROMOscan
>100000 BRPF1, BRD1, BRPF3, BRD9, BRD7,
ATAD2A, ATAD2B, BRD4(BD1),
CREBBP, PCAF, BAZ2A, TRIM33, CECR2
77000 SMARCA2A
AlphaScreen
>20000 BRPF1, BRD9, BRD4(BD1), CECR2, FALZ,
TAF1A
a
Table 5. Examples of Inhibition of Proliferation of AML and
Other Cancer Cell Lines (GI50) by 13-d
BRD IC50 values are geometric means of two experiments (n = 2).
Multiplicities in 1H NMR spectra are quoted as s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, dd = double doublet,
ddd = double double doublet. 13C nuclear magnetic resonance (13C
NMR) spectra were recorded on a Bruker Advance 500 spectropho-
tometer at 125 MHz. Chemical shifts were measured in parts per
million (ppm) relative to tetramethylsilane (δ = 0) using the following
internal references: CDCl3 (δ 77.16), CD3OD (δ 49.00), and DMSO-
d6 (δ 39.52). 2D NMR techniques HSQC, HMQC, and HMBC were
a
cancer cell line
GI50 (μM)
AML Cell Lines:
OCI-AML2
Nomo-1
1.3
4.6
5.7
7.0
9.9
THP-1
KG-1
MV-4-11
1
also utilized for the assignment of H and 13C NMR signals. High-
resolution mass spectra (HRMS) were recorded on a Thermo
Navigator mass spectrometer coupled to an HPLC instrument using
electrospray ionization (ESI) and time-of-flight (TOF) mass
spectrometry. Alternatively, HRMS were recorded at the EPSRC UK
National Mass Spectrometry Facility (NMSF) at Swansea University.
Analytical reverse-phase high-performance liquid chromatography
(HPLC) was carried out on a XSELECT CSH C-18 column (2.5
μm; 6 mm × 50 mm). HPLC experiments (system A) were performed
with gradient conditions: initial fixed composition 5% B to 50% B over
20 min, then increased to 95% B over 2 min, held for 2 min at 95% B,
then returned to 5% B in 1 min. Total duration of gradient run was 25
min. Eluents used were solvent A (H2O with 0.02% TFA) and solvent
B (MeCN with 0.02% TFA). Flow rate: 1.00 mL/min.
Non-AML Cell Lines:
SW48, NUGC-4, HepG2, COLO 205, OCUM-1, HUP-T4,
LOVO, C-99, 22Rv1, SK-CO-1, MOLP-8, OGP-1, AGS, Hs
766t, SNU-5, SW948, RKO, OCI-LY-19, KG-1, Panc02.03,
SW403, HCC1187, NCI-H1437, IM-9, AsPC-1, SW620, LS180
1−10
∼100 cell lines
>30
a
AstaZeneca CLIMB panel includes 211 cancer cell lines. 72 h
proliferation assay; 9 point dose−response curve with a highest
concentration of 10 or 30 μM (n = 2). For an example of the use of
oncology cell panels in mechanistic and pathways analysis, see ref 40.
Melting points (mp) were determined in open capillary tubes on a
Stuart SMP10 apparatus and are uncorrected. Infrared (IR) analysis
was performed on a PerkinElmer Spectrum 1000 FT-IR in the 4000−
Purity of screening compounds 12−15, 21, and 22 was evaluated by
NMR spectroscopy and HPLC analysis. All compounds had purity
≥95% except 21, which had a purity of 91% by HPLC.
1
400 cm−1 range. H nuclear magnetic resonance (1H NMR) spectra
were recorded on a Bruker Advance 400 spectrophotometer at 400
MHz or Bruker Advance 500 spectrophotometer at 500 MHz.
Chemical shifts were measured in parts per million (ppm) relative to
tetramethylsilane (δ = 0) using the following internal references:
CDCl3 (δ 7.26), CD3OD (δ 3.32), and DMSO-d6 (δ 2.50).
6-Amino-1,3-dimethyl-6-quinolin-2-(1H)-one (7). A 500 mL
round-bottom flask was charged with 1,3-dimethyl-6-nitroquinolin-2-
(1H)-one (20) (5.00 g, 22.9 mmol, 1.0 equiv) and palladium on
carbon (5% wt, 2.44 g, 1.45 mmol, 0.05 equiv). The flask was sealed
then evacuated and backfilled with nitrogen three times before adding
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ethanol (300 mL). The stirred slurry was then re-evacuated until the
solvent began to bubble and backfilled with nitrogen once more. A
balloon of hydrogen was attached to the flask via a syringe fitted with a
stopcock. The flask was evacuated until the solvent began to boil and
then backfilled with hydrogen gas five times. The reaction was then
stirred for 7 h, after which point the complete consumption of starting
material was confirmed by TLC. At the end of the reaction, the
reaction flask was evacuated and backfilled with nitrogen three times
and filtered through a pad of Celite onto a glass-sintered funnel. The
Celite pad was further washed with ethanol (300 mL) and then the
filtrate concentrated under reduced pressure to give 6-amino-1,3-
dimethyl-6-quinolin-2-(1H)-one (7) (4.10 g, 95%) as a pale-yellow
solid that was not purified further. 1H NMR (500 MHz, DMSO-d6): δ
7.55 (s, 1H), 7.21 (d, J = 8.8 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 6.71
(s, 1H), 5.04 (br s, 2H), 3.56 (s, 3H) 2.08 (s, 3H). 13C NMR (125
MHz, DMSO-d6): δ 160.9, 143.6, 135.0, 130.5, 128.7, 120.9, 117.7,
115.0, 110.0, 29.2, 17.5.
4-Cyano-N-(1,3-dimethyl-2-oxo-1,2-dihydroquinolin-6-yl)-
benzenesulfonamide (13-d). To a solution of 6-amino-1,3-dimethyl-
quinolin-2(1H)-one (7) (188 mg, 1.00 mmol) and pyridine (158 mg,
2.00 mmol, 2.0 equiv) in DMF (5 mL) was added 4-
cyanobenzenesulfonyl chloride (201 mg, 1.00 mmol, 1.0 equiv) at
room temperature and the reaction mixture stirred overnight. The
resulting suspension was homogenized by addition of DMF (3 mL),
concentrated onto Celite and purified by column chromatography
(acetone:hexane, gradient elution 30:70 to 60:40) to give 13-d (129
mg, 0.37 mmol, 37%) as a white solid; melting point 279−282 °C
(acetone−hexane). 1H NMR (400 MHz, DMSO-d6): δ 10.52 (1H, s),
8.06−8.00 (2H, m), 7.90−7.84 (2H, m), 7.73 (1H, s), 7.41 (1H, d, J =
9.1 Hz), 7.33 (1H, d, J = 2.5 Hz), 7.23 (1H, dd, J = 9.1, 2.5 Hz), 3.57
(3H, s), 2.09 (3H, d, J = 1.0 Hz). 13C NMR (126 MHz, DMSO-d6): δ
161.4, 143.3, 136.3, 135.0, 133.5, 130.8, 130.0, 127.4, 123.5, 120.3,
119.9, 117.5, 115.5, 115.3, 29.4, 17.3. HRMS m/z (ESI+) found (M +
H)+ 354.0908, C18H15N3O3S requires (M + H)+ 354.0907. HPLC:
retention time (system A) tR = 8.97 min. Purity > 95%.
4-Cyano-N-(1,3-diethyl-2-oxo-1,2-dihydroquinolin-6-yl)-
benzenesulfonamide (16-d). 16-d was prepared according to general
procedure as described for 13-d from 6-amino-1,3-diethylquinolin-
2(1H)-one (40 mg, 0.18 mmol) with 4-cyanobenzenesulfonyl chloride
to give 16-d (37 mg, 0.09 mmol) as a pale-yellow solid. 1H NMR (500
MHz, DMSO-d6): δ 10.56 (1H, s), 8.04 (2H, d, J = 8.5 Hz), 7.89 (2H,
d, J = 8.5 Hz), 7.68 (1H, s), 7.46 (1H, d, J = 8.8 Hz), 7.41 (1H, d, J =
2.5 Hz), 7.23 (1H, dd, J = 9.1, 2.5 Hz), 4.22 (2H, q, J = 6.9 Hz), 1.21−
1.10 (6H, m), [1 × CH2 overlaps with DMSO]. 13C NMR (126 MHz,
DMSO-d6): δ 160.5, 143.4, 135.3, 134.9, 133.6, 133.5, 130.7, 127.4,
123.6, 120.7, 120.3, 117.5, 115.3, 115.2, 36.9, 23.5, 12.7, 12.5. HRMS
m/z (ESI−) found (M − H)− 380.1057, C20H19N3O3S requires (M −
H)− 380.1069. HPLC: retention time (system A) tR = 10.36 min.
Purity > 95%.
3-Methylquinolin-2-(1H)-one (18). To a stirred solution of 3-
methylquinoline (17) (25.0 g, 175 mmol, 1.0 equiv) in dichloro-
methane (875 mL) in a 2 L round-bottom flask at 0 °C was added 3-
chloroperbenzoic acid (70%, 47.3 g, 192 mmol, 1.1 equiv) portionwise
over 10 min. The resulting solution was allowed to warm to room
temperature and then stirred overnight. After completion of the
reaction, the solution was washed with sodium hydroxide (1.0 M, 300
mL) and the aqueous phase extracted with dichloromethane (2 × 300
mL). The organic layers were combined, dried over anhydrous MgSO4,
filtered, and the solvent removed under reduced pressure to yield
crude 3-methylquinolone-N-oxide as a white solid, which was used
without further purification.
was collected by filtration, washed with diethyl ether (200 mL) and
then water (200 mL), and dried under vacuum to give 3-
methylquinolin-2(1H)-one (18) (16.3 g 59% over 2 steps). 1H
NMR (400 MHz, DMSO-d6): δ 11.72 (br s, 1H), 7.75 (s, 1H), 7.56
(d, J = 7.8 Hz, 1H), 7.41 (t, J = 8.3 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H),
7.14 (t, J = 7.5 Hz, 1H), 2.09 (s, 3H). 13C NMR (125 MHz, DMSO-
d6): δ 162.4, 137.9, 136.3, 129.8, 129.0, 126.9, 121.6, 119.4, 114.7,
16.5.
1,3-Dimethylquinolin-2-(1H)-one (19). To a solution of 3-
methylquinolin-2(1H)-one (18) (16.3 g, 103 mmol, 1.0 equiv) in
anhydrous DMF (500 mL) in a 1 L round-bottom flask cooled to 0 °C
and under an argon atmosphere was added NaH (60% wt., 4.50 g, 113
mmol, 1.1 equiv) and the reaction mixture stirred for 0.5 h.
Iodomethane (8.3 mL, 133 mmol, 1.3 equiv) was added dropwise
via syringe and the reaction mixture stirred for 5 h. Excess sodium
hydride was quenched by the addition of water (30 mL) and the
solvents removed under reduced pressure. The resultant slurry was
taken up in the minimum volume of ethyl acetate and poured onto
crushed ice (approximately 200 g). The resultant heterogeneous
mixture was stirred rapidly for 15 min and then the precipitate
collected by filtration, washed with cold diethyl ether (100 mL), and
dried under vacuum to give 1,3-dimethylquinolin-2-(1H)-one (19)
1
(16.9 g, 95%) as a white powder. H NMR (400 MHz, DMSO-d6): δ
7.78 (s, 1H), 7.63 (dd, J = 7.8, 1.3 Hz, 1H), 7.58−7.53 (m, 1H), 7.50−
7.48 (m, 1H), 7.26−7.22 (m, 1H), 3.64 (s, 3H) 2.13 (s, 3H). 13C
NMR (125 MHz, DMSO-d6): δ 161.7, 138.7, 135.5, 129.5, 128.9,
127.7, 121.8, 120.0, 114.3, 29.3, 16.5.
1,3-Dimethyl-6-nitroquinolin-2-(1H)-one (20). A 500 mL round-
bottom flask was charged with 1,3-dimethylquinolin-2-(1H)-one (19)
(16.9 g, 97.5 mmol, 1.0 equiv) and cooled to 0 °C. Concentrated
sulfuric acid (120 mL) was added and the reaction mixture stirred for
0.5 h, during which time most of the solid dissolved. To the resultant
pale-red solution, potassium nitrate (10.4 g, 102.4 mmol, 1.05 equiv)
was added in two portions. The reaction mixture rapidly turned yellow
and was stirred for a further 4 h, after which it was poured onto
crushed ice (approximately 400 g) and stirred rapidly for 0.5 h. The
yellow precipitate was collected by filtration onto a sintered-glass
funnel and washed with water (2 × 250 mL). Still on the sintered disk,
the resultant filter cake was slurried with hot ethanol (200 mL) using a
glass rod and allowed to stand for 10 min. The solid was collected by
filtration to give 1,3-dimethyl-6-nitroquinolin-2-(1H)-one (20) (18.5
g, 87%) as a pale-yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 8.63
(s, 1H), 8.35 (d, J = 8.8 Hz, 1H), 8.03 (s, 1H), 7.7 (d, J = 8.8 Hz, 1H),
3.71 (s, 3H) 2.16 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 161.8,
142.9, 141.4, 135.3, 131.2, 123.9, 123.5, 119.7, 115.7, 30.1, 17.4.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b01583.
Materials and methods: synthetic procedures and
characterization data for compounds 6−22; DSF,
BROMOscan, AlphaScreen, and ITC determinations;
protein crystallization, data collection, and structure
solution; FRAP assay. Figures: spectroscopic and
analytical data for 13-d and 16-d; dose−response curves
for 13-d in BROMOscan assay; ITC analysis; BRD
protein structures superposition with 5b and sequence
alignment; superposition of BRPF1B histone complex
structures and costructure of BRPF1B with 5b;
experimental electron density map contoured around
22 and ligand interactions map between 22 and
BRPF1B; FRAP assay data. Tables: DSF thermal shift
selectivity values for 13-d; IonChannelProfiler data;
mouse PK determinations; crystal structure data
collection and refinement statistics (PDF)
To a 2 L three-necked round-bottom flask fitted with an overhead
mechanical stirrer was charged crude 3-methylquinolone-N-oxide
followed by dichloromethane (430 mL) and aqueous sodium
hydroxide solution (1.0 M, 400 mL). The resulting biphasic mixture
was cooled to 0 °C, and to this was added, under rapid agitation,
benzoyl chloride (24.4 mL, 210 mmol, 1.2 equiv) dropwise via a side
arm pressure equalized dropping funnel. The suspension was stirred
overnight, and the resulting heterogeneous solution was diluted with
diethyl ether and stirred rapidly for 15 min. The resultant precipitate
J
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Accession Codes
structure−activity relationship; TIF2, transcriptional media-
tors/intermediary factor 2; TRIM24, tripartite motif containing
24; WPF, Trp-Pro-Phe
Coordinates for the X-ray structures of the bromodomain of
BRPF1B cocrystallized with fragment N-methylquinoline-
2(1H)-one (5b) (5T4U) and NI-48 (22) (5T4V) have been
deposited in the Protein Data Bank.
REFERENCES
■
(1) Brown, P. J.; Muller, S. Open access chemical probes for
̈
AUTHOR INFORMATION
Corresponding Author
*Phone: +44 (0)20 7679 6971. E-mail: p.fish@ucl.ac.uk.
■
epigenetic targets. Future Med. Chem. 2015, 7, 1901−1917.
(2) Vidler, L. R.; Filippakopoulos, P.; Fedorov, O.; Picaud, S.; Martin,
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ORCID
Paul V. Fish: 0000-0002-2117-2173
Present Address
◊For P.V.F.: Alzheimer’s Research UK UCL Drug Discovery
Institute, The Cruciform Building, University College London,
Gower Street, London WC1E 6BT, UK.
Notes
The authors declare no competing financial interest.
NI-42 (13-d) is available from Tocris (catalogue no. 5786).
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Chung, C. W.; Chandwani, R.; Marazzi, I.; Wilson, P.; Coste, H.;
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ACKNOWLEDGMENTS
■
N.I. is supported by a UCL School of Pharmacy Ph.D.
studentship. P.V.F. thanks the UCL School of Pharmacy, UCL
Faculty of Life Sciences, and UCL Business for financial
support. We thank Jeremy Hunt, Aimee Allen, Dan Treiber,
and the screening team at DiscoverX Corp. for measuring
inhibitor IC50 values in the BROMOscan assay, and the
AstraZeneca Oncology screening group for evaluation of NI-42
in a panel of cancer cell lines (CLIMB panel). HRMS were
recorded at the EPSRC UK National Mass Spectrometry
Facility (NMSF) at Swansea University. The SGC is a
registered charity (no. 1097737) that receives funds from
AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada
Foundation for Innovation, Eshelman Institute for Innovation,
Genome Canada through Ontario Genomics Institute,
Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD
grant no. 115766], Janssen, Merck & Co., Novartis Pharma
AG, Ontario Ministry of Economic Development and
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ABBREVIATIONS USED
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■
AML, acute myeloid leukemia; ATAD2, ATPase family, AAA
domain containing; BRD4(BD1), bromodomain containing
protein 4, first bromodomain; BET, bromodomain and extra
terminal domain; BRD, bromodomain containing protein;
BRPF, bromodomain and PHD finger-containing; CLIMB, cell
lines indexed for molecular biology; DABCO, 1,4-diazabicyclo-
[2.2.2]octane; DSF, differential scanning fluorimetry; DMF,
N,N-dimethylformamide; EAF6, EAS1-associated factor 6;
EPC, enhancer of polycomb; FRAP, fluorescence recovery
after photobleaching; HAT, histone acyl transferase; HBO1,
histone acetyltransferase binding to ORC 1 (KAT7); HOX,
homeotic; ING5, inhibitor of growth family, member 5; ITC,
isothermal titration calorimetry; LE, ligand efficiency =
1.4(−log IC50)/number of heavy atoms; LLE, lipophilic ligand
efficiency = (−log IC50) − clogP; mCPBA, 3-chloroperbenzoic
acid; MLL, mixed-lineage leukemia; MORF, monocytic
leukemia zinc finger protein-related factor (KAT6B); MOZ,
monocytic leukemia zinc finger (KAT6A); NPM1, nucleo-
phosmin 1; PCAF, P300/CBP-associated factor; PK−PD,
pharmacokinetic−pharmacodynamic; PWWP, Pro-Trp-Trp-
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