Discovery of novel small-molecule inhibitors of BRD4 using structure-based virtual screening

Article abstract of DOI:10.1021/jm4011302

Bromodomains (BRDs) are epigenetic readers that recognize acetylated-lysine (KAc) on proteins and are implicated in a number of diseases. We describe a virtual screening approach to identify BRD inhibitors. Key elements of this approach are the extensive design and use of substructure queries to compile a set of commercially available compounds featuring novel putative KAc mimetics and docking this set for final compound selection. We describe the validation of this approach by applying it to the first BRD of BRD4. The selection and testing of 143 compounds lead to the discovery of six novel hits, including four unprecedented KAc mimetics. We solved the crystal structure of four hits, determined their binding mode, and improved their potency through synthesis and the purchase of derivatives. This work provides a validated virtual screening approach that is applicable to other BRDs and describes novel KAc mimetics that can be further explored to design more potent inhibitors.

Full text of DOI:10.1021/jm4011302

Article
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Discovery of Novel Small-Molecule Inhibitors of BRD4 Using
Structure-Based Virtual Screening
Lewis R. Vidler,† Panagis Filippakopoulos,‡,§ Oleg Fedorov,‡,∥ Sarah Picaud,‡ Sarah Martin,‡,∥
Michael Tomsett,† Hannah Woodward,† Nathan Brown,† Stefan Knapp,*,‡,∥ and Swen Hoelder*,†
†Division of Cancer Therapeutics, Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold
Road, Sutton, Surrey SM2 5NG, United Kingdom
‡Nuffield Department of Clinical Medicine, Structural Genomics Consortium, Oxford University, Old Road Campus Research
Building, Headington, Oxford OX3 7DQ, United Kingdom
§Ludwig Institute for Cancer Research, Old Road Campus Research Building, Headington, Oxford OX3 7DQ, United Kingdom
∥Nuffield Department of Clinical Medicine, Target Discovery Institute, Oxford University, NDM Research Building, Oxford OX3
7FZ, United Kingdom
S
* Supporting Information
ABSTRACT: Bromodomains (BRDs) are epigenetic readers that recognize acetylated-lysine (KAc) on proteins and are
implicated in a number of diseases. We describe a virtual screening approach to identify BRD inhibitors. Key elements of this
approach are the extensive design and use of substructure queries to compile a set of commercially available compounds featuring
novel putative KAc mimetics and docking this set for final compound selection. We describe the validation of this approach by
applying it to the first BRD of BRD4. The selection and testing of 143 compounds lead to the discovery of six novel hits,
including four unprecedented KAc mimetics. We solved the crystal structure of four hits, determined their binding mode, and
improved their potency through synthesis and the purchase of derivatives. This work provides a validated virtual screening
approach that is applicable to other BRDs and describes novel KAc mimetics that can be further explored to design more potent
inhibitors.
interactions being formed between the acetyl carbonyl of KAc
and a family-conserved asparagine residue as well as a
structurally conserved water molecule. Mimicking the inter-
action of this acetyl group has been the basis for generating
small-molecule inhibitors of the readout function of the
bromodomain proteins (Figure 1B), which was exemplified
by the discovery of (+)-JQ1 (Figure 1C). Outside of the highly
enclosed base of the pocket, inhibitors of the BET family have
shown that occupying the adjacent regions, known as the
hydrophobic shelf (occupied by phenyl in Figure 1C) and ZA
channel (occupied by thiophene in Figure 1C), leads to
INTRODUCTION
■
The bromodomain (BRD) family of proteins recognize
acetylated-lysine (KAc) in proteins and represent a set of
protein−protein interaction modules that are becoming
increasingly explored in the field of drug discovery.1 The
BET family of BRDs is a subset of this larger bromodomain
family and is made up of four members: BRD2, BRD3, BRD4,
and BRDT in humans, with each containing two BRD modules
that share high sequence similarity2 and highly similar binding
sites.3
The BET family shares the same conserved tertiary structure
of bromodomain proteins,4−6 with the KAc binding site being
formed as a central cavity by an atypical left-handed four-helix
bundle flanked by the ZA loop and the BC loop (Figure 1A).
This binding site is primarily hydrophobic, with key polar
Received: July 25, 2013
Published: October 3, 2013
© 2013 American Chemical Society
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Figure 1. (A) Structure of first bromodomain of BRD4 bound to an acetylated peptide (PDB ID: 3UVW). (B) Interaction of KAc with BRD4 (PDB
ID: 3UVW). (C) (+)-JQ1 bound to BRD4 with a chlorophenyl ring occupying the hydrophobic shelf and thiophene occupying the ZA channel
(PDB ID: 3MXF). Surface colors were generated using the pocket colors in MOE. Green represents an enclosed surface, and white, exposed.
Figure 2. Structure, activity, and LE of published BET-family inhibitors classified by KAc mimetic.
nanomolar potency and a high degree of selectivity toward
other bromodomains (Figures 1C and 2).7−12
The inhibition of the BET family of bromodomains has been
proposed as a therapeutic strategy in multiple disease areas
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including cancer, inflammation, and obesity.1,13 Here, we have
focused on BRD4, which was identified as a therapeutic target
in AML,14,15 other cancers,16−18 and inflammatory disease,12 as
a representative member of the BET subfamily. A number of
small-molecule inhibitors of BET family members have now
been published.19 A key feature of these inhibitors is a KAc
mimetic that anchors the molecule into the BRD binding site
via hydrogen bonds and hydrophobic interactions, which is
critical for potent binding. A limited number of chemotypes
that possess these features had been published when this work
started and has been increasing (Figure 2). These include
triazolodiazepine (e.g., (+)-JQ1),7,8,12,20 isoxazole (e.g.,
GSK1210151A),9,10,21−25 dihydroquinazolinone (e.g., PFI-
1),11,26,27 tetrahydroquinoline,26 benzimidazole (e.g., BIC1),28
indolizine,26 thiazolidinone,29 triazolopyridine,29 and tetrahy-
drothienopyridine29 scaffolds.
Upon starting this work, only the triazolodiazepine,
dihydroquinazolinone, and benzimidazole KAc mimetics had
been disclosed. To offer more possibilities for drug-design
efforts against BRD4 and other bromodomains, we sought to
identify novel chemotypes that can act as KAc mimetics.
Different chemotypes binding to the same protein often show
different physicochemical properties, distinct biological profiles,
and offer additional opportunities for intellectual property
generation.
Our work is relevant to scientists working on bromodomain
inhibitors because it addresses three goals: first, through the
focus on KAc mimetics, it efficiently preselects a subset of
commercially available compounds and ensures that a large
number of KAc mimetics are included in the docking step;
second, through the docking step, it meets the tight geometric
requirements of the KAc binding site; and third, this approach
is applicable to other bromodomains for which crystal
structures are available. Furthermore, we report an extensive
compilation of novel chemotypes that can act as KAc mimetics
and will thus be useful for other design approaches and scaffold
hopping, and we report novel inhibitors of BRD4 that have the
potential to be developed into significantly more potent
compounds.
METHODS
■
Virtual Screening Approach. Our approach centered on
identifying a series of chemotypes that had the potential to mimic
KAc and fit the geometric constraints of the BRD4(1) binding pocket.
These were then converted into substructure searches that were used
to mine commercially available compounds for inhibitors of BRD4
using the eMolecules database.30 We intended to generate these from
two different branches. One branch we will refer to as the “Literature
Substructures” branch, which was based on the KAc mimetic
substructures extracted from published bromodomain inhibitors.
These were also modified to generate structurally related substructures
that maintained the key pharmacophore (e.g., 1,2,4-triazole to
isoxazole). The other branch we will refer to as the “Similarity
Searching” branch, which was based on pharmacophore, shape, and 2D
fingerprint similarity searches derived from the published (+)-JQ1 and
the crystal structure of it bound to BRD4(1) (PDB ID: 3MXF). The
results of each of these branches was submitted to docking, and
through a series of filters designed to reduce docking false positives,
compounds were selected for purchase (Figure 3).
Here, we describe a virtual screening approach that focuses
on KAc mimetics and identifies novel scaffolds that fit this
profile. Furthermore, we designed the virtual screen in a fashion
largely independent of the bromodomain targeted, and we
present the validation of the approach against BRD4.
The enclosed KAc binding site of bromodomains imposes
strict geometric constraints on inhibitors, requiring excellent
shape complementarity in this part of the pocket. To meet
these constraints, we wanted to take advantage of the
availability of BRD4 and other bromodomain crystal structures
by utilizing molecular docking. However, the docking of several
million commercially available compounds and in particular the
processing of the results is still a time-consuming and labor-
intense task. Frequently, methods of higher throughput, such as
similarity- or pharmacophore-based searching, are used to select
a smaller set of compounds that is then subjected to docking.
Our approach to preselect a set of compounds for which
docking can be managed recognizes the key role of the KAc
mimetic in all known bromodomain inhibitors. We initially
selected commercially available compounds that feature an KAc
mimetic chemotype, that is, compounds that feature a moiety
that has the potential to match the hydrogen-bond pattern and
the steric constraints of the KAc binding site. Specifically, we
created an extensive set of chemotpyes by exploring published
data, intuitive design, and similarity searches. This set consisted
of known chemotypes and, critically, also many chemical
structures not yet described as bromodomain inhibitors. Next,
we converted these chemotypes into substructures searches and
retrieved commercially available compounds that feature these
KAc mimetic substructures. This set of compounds was then
subjected to docking against the first bromodomain of BRD4
(BRD4(1)). After an extensive filtering of the results and the
purchasing and testing of selected compounds, we identified six
inhibitors, including four unprecedented KAc mimetics. To
validate our results further, we generated crystal structures of
selected compounds bound to BRD4(1) and established early
SAR around the novel chemotypes through synthesis and the
purchase of derivatives.
Literature Substructures Branch. We generated a library of
bromodomain-focused substructures through the identification of the
KAc mimetic substructure from published bromodomain inhibitors.
To expand the number of substructures considered, we also included
structurally related queries that maintained the key pharmacophore.
Figure 3. Flowchart summarizing the selection of compounds taken
through to biochemical screening.
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Figure 4. Example of the generation of substructures from (+)-JQ1 for the Literature Substructures approach.
An example of this approach starting from (+)-JQ1 is highlighted in
Figure 4. The substructures carried forward to the rest of the approach
are shown in the Supporting Information, Figure S1.
Similarity Searching Branch. With this branch, we were looking
for novel KAc mimetics featuring chemotypes distinct from already
known inhibitors. To identify such chemotypes, we used similarity
searches (e.g., shape or pharmacophore) that allowed the identification
of distinct chemotypes that nevertheless share the pharmacophoric
features critical for binding of the probe compound (+)-JQ1. These
similarity searches gave us a rich set of putative KAc mimetics from 2.4
million commercially available compounds. However, we reasoned that
as a result of the tight geometric constraints of the BRD4 binding site
many of these would not be able to bind, despite a high similarity,
because even a simple change (e.g., from a methyl to an ethyl group)
may lead to clashes within the KAc pocket. To enrich for compounds
that fit the tight geometric constraint of the binding site, we thus
performed a docking step and extracted KAc mimetics with sufficient
shape complementarity. It is important to note that this docking step
serves the purpose of creating a virtual library of KAc mimetics in
conjunction with the similarity search and is distinct from the final
docking step that will be described later and served the purpose of
selecting compounds for purchase. The Similarity Searching branch is
summarized in Figure 5.
Figure 5. Flowchart summarizing the Similarity Searching branch of
the virtual screen.
Specifically, pharmacophore, shape, and 2D fingerprint searches
were performed on 2.4 million commercially available compounds for
the initial similarity search. Three pharmacophore searches were used,
probing the base of the acetyl-lysine binding site, the hydrophobic
shelf, and the ZA channel, using the structure of BRD4(1) as an
excluded volume (PDB ID: 3MXF). The first search contained all of
the features known to contribute to the potent binding of inhibitors:
two acceptors within the acetyl-binding site at the base of the pocket as
well as hydrophobic substituents in the ZA channel and the
hydrophobic shelf positions. This placed fairly tight constraints on
the molecules being screened against the probe, and as a result only a
few molecules were able to match these criteria. To allow for more
molecules to match and to focus on the KAc mimetic parts of the
molecules, these constraints were relaxed by removing the requirement
for the molecule to occupy the hydrophobic shelf. One further search
was performed that required only one of the two acceptors within the
acetyl binding site at the base of the pocket because examples of single-
acceptor-atom KAc mimetics are known (e.g., dihydroquinazolinone
derivatives). One shape-based and one 2D fingerprint similarity search
were also applied to find molecules with a similar shape and similar
functional groups to the entire (+)-JQ1 molecule. One additional
shape-based search was also applied to (+)-JQ1, with the chlorophenyl
and tertiary-butyl ester removed to focus on the KAc part of the
binding site. Each of these similarity searches was expected to identify
different compounds and therefore maximize the chances of finding
hits. Precise details of these similarity searches can be found in the
Experimental Section.
Substructure Searches. A key step of our virtual screening
approach was the use of an extensive set of substructures that we
obtained from published data, intuitive design, and similarity searches,
as already described. These substructures were consequently used to
identify all commercially available examples of each of the
substructures, employing substructure searches on the eMolecules
database.30 The results were then submitted to a second docking step
and subsequent selection for purchase, as detailed below. This docking
step was performed in the same manner as the one from the Similarity
Searching branch, but it was necessary because of the different ligands
present. It is important to note that this substructure search also
ensured that all examples of a particular substructure of interest were
identified and fed into the final docking step. This is in contrast to our
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Figure 6. Flowchart summarizing the control experiment.
Table 1. Compounds Identified as Hits against BRD4(1) from the Virtual Screen
a
b
See the Experimental Section for details. Following HPLC purification of a commercial sample.
experience with similarity searches where a number of representatives
of a particular chemotype are frequently missed, for example, because
of different tautomers, protomers, or conformers of the starting library
compounds.
Docking against BRD4(1), Reducing Docking False Positives,
and Selecting Compounds for Purchase. The results of each of
the substructure searches were subsequently docked against BRD4(1)
to predict the binding mode and to calculate a score, from which
examples of each substructure can be selected for purchase. One of the
limitations of using docking as a part of a virtual screen is that it
generates false positives, reducing the enrichment of actives.31,32
Docking comprises two key components: pose prediction and
scoring.33 Many of the false positives, however, involve the scoring
of the molecules, with the pose generation considered to be sufficiently
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accurate to be useful.32,34 A recent report by Ferreira et al.35 identified
some of the underlying causes of false positives from docking. These
include failure to penalize high-energy conformations of the molecules
as well as the presence of heteroatoms that are not engaged in
favorable interactions with the receptor.
Using this knowledge, we defined the following criteria that are
necessary to be fulfilled by a molecule and its docking pose to be
considered for purchase:
screen was performed detailing derivatives of this isoxazole
scaffold.9,10,21−25 The structures of these hits and the data
gathered for each of the compounds are summarized in Table 1.
The docking poses used to initially select these six compounds
can be found in the Supporting Information, Figure S2.
Crystallography. Following the identification of six
compounds as inhibitors of BRD4(1), crystal structures of
four of the compounds in the KAc binding site were obtained
(Figure 7). This allows the binding mode to be used to design
more potent analogues and to compare the docking poses to
(1) No obvious high-energy conformations (using the CSD as
validation) (see the Experimental Section for details).
(2) The majority of the heteroatoms form interactions with the
receptor or are satisfied by internal hydrogen bonds, and key
interactions defined by the hypothesis are formed.
(3) No clashes with the receptor or vacant sites unlikely to be filled
by water molecules or protein movement.
(4) No reactive groups or groups known to interfere in biochemical
assays.36
To implement these criteria, we manually inspected the docking
poses as a pragmatic approach. This allowed for the defined criteria to
be implemented quickly and each individual molecule to be considered
on its own merit. At least 500 of the top ranked ligands from each
docking run were inspected, and representative compounds from each
scaffold that fulfilled these criteria were selected, resulting in a set of
150 compounds.
Control Experiment. Our approach is heavily based on docking
and extensively takes advantage of human input for the final selection
of compounds. To benchmark our approach, we sought a valid control
experiment to compare our results to. Many retrospective analyses
suggest that ligand-based methods frequently outperform the
equivalent structure-based approaches when it comes to discriminating
between active and inactive compounds;37,38 therefore, we chose an
experiment based on these approaches as a control. We based our
selection on the same 2D fingerprint and shape-based similarity
searches as the Similarity Searching part of our virtual screening
approach starting from the entire (+)-JQ1 ligand and its bound
conformation to BRD4(1), but in this experiment docking and
subsequent visual inspection were not utilized.
To select compounds and to ensure that a minimum level of
diversity was achieved, the top 50 compounds from each method,
ranked by similarity to (+)-JQ1 with unique Murcko scaffolds,39 were
selected for purchase. One hundred compounds in total were selected
using this approach in a fully automated way, and there was no overlap
between the compounds selected by the control experiment and those
from the virtual screen. The control experiment is summarized in
Figure 6.
RESULTS AND DISCUSSION
■
Using our virtual screening protocol detailed above and
including the compounds selected from the control experiment,
250 compounds were purchased and prepared as DMSO stock
solutions. Ten of these compounds were insoluble in DMSO,
and as such they were not carried forward to biochemical
screening. The remaining 240 compounds were initially
screened using the AlphaScreen assay format at a single
concentration up to 250 μM,40 depending on compound
solubility. Six compounds exhibited an inhibition of BRD4(1),
but only two of these compounds yielded an IC50: 1 had an
IC50 of 4.7 μM, and 2 had an IC50 of 80.9 μM. The IC50 of the
remaining compounds was higher than the solubility and as
such could not be determined. Despite the modest activity of
some of the hits, we next sought to cocrystallize all novel KAc
mimetics that showed significant binding, and we obtained
crystal structures of four compounds bound to BRD4(1).
Finally, we investigated derivatives of five of these compounds
through purchase and synthesis, yielding compounds that
showed significantly improved activity. 2 was not followed up
further because of publications appearing after the virtual
Figure 7. Crystal-structure binding modes of four of the identified
inhibitors bound to BRD4(1). The left panel gives an overview of the
binding mode. The right panel gives a through-protein view
highlighting the binding mode of each KAc mimetic. Surface colors
were generated using the pocket colors in MOE. Green represents an
enclosed surface, and white, exposed. (A) Compound 1, (B)
compound 3, (C) compound 4, and (D) compound 5.
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the experimentally observed binding mode that the docking was
trying to predict. Extremely high accuracy was attained for the
docking of 3 and 4, for which the docking poses were within
experimental error of the crystal structure poses (rmsd ≤ 0.30
Å). In the case of these two compounds, the protein showed
very limited movement from its conformation bound to
(+)-JQ1 with an rmsd of 0.37 Å between the model used
(PDB ID: 3MXF) and each of the triazolopyrimidine bound
structures, which may have contributed to the success of the
docking experiment. In the case of 1 and 5, the docking
predicted the experimentally observed pose of the ligand (rmsd
< 2.0 Å) but with a few subtle differences. Some of these
differences were the result of water molecules bridging
interactions that the docking was unable to consider.
Comparisons between the predicted and experimentally
determined poses can be seen in Figure 8. Details of all of
the crystallographic binding modes will be discussed in the
context of each of the series below.
involved the removal of the phenyl ring, and we hypothesized
that the chloropyridone motif would be able to mimic the
remaining dihydropyrimidone substructure (Figure 9). The
carbonyl and NH donor were maintained through this
modification, with the chlorine atom occupying the same
location as the methyl group it replaced. A substructure search
performed on the eMolecules database yielded 82 commercially
available examples of this substructure. Following the docking
and selection process detailed above, 1 and 7a were selected for
purchase.
1 was the most potent of the 240 compounds initially tested,
with an IC50 of 4.7 μM and associated LE of 0.28. Structurally
related 7a, also tested in the original batch of 240 compounds,
was found to be inactive. The only other commercially available
compounds containing the chloropyridone and triazole
functionalities were 7b and 7c. These were subsequently
purchased and tested (Table 2) and found to be significantly
less active. A crystal structure of 1 bound to BRD4(1) was
obtained with the carbonyl of the pyridone occupying a similar
position to the carbonyl oxygen of the acetyl-group of KAc,
forming the same interactions with the NH donor of the
Asn140 side chain and conserved water molecule. The chlorine
atom occupies the base of the pocket that the methyl group of
KAc would usually occupy, and the NH donor of the pyridone
forms a water-mediated hydrogen bond to the carbonyl of the
conserved Asn140 side chain. This is the first example of a
chlorine substituent occupying the base of the KAc binding
pocket, which can potentially be incorporated into other
templates, replacing the methyl group commonly used at this
position. The remaining interactions of 1 involve the triazole
that forms an interaction with one of the conserved water
molecules and occupies the ZA-channel region of the pocket as
well as hydrophobic interactions of the phenyl and pyridine
rings (Figure 7A).
Triazolopyrimidines. 3 and 4 both contain the same
triazolopyrimidine scaffold and were initially selected on the
basis of the hypothesis that they mimic KAc. The observation
that 3 and 4 show significant inhibition as well as, importantly,
the cocrystal structures of these compounds with BRD4(1)
validated this hypothesis and confirmed the triazolopyrimidine
as a novel KAc mimetic. The triazolopyrimidine engages in the
same interactions in both structures as the carbonyl of the
acetyl group of KAc, albeit through two nitrogen atoms
compared to each lone pair of the carbonyl (Figure 7). This
strong complementarity is likely the reason for the accurate
prediction of the docking experiment as well as for the minimal
movement of the protein from the reference model (PDB ID:
3MXF).
Figure 8. Overlay of the observed crystallographic pose (cyan
carbons) with the docking pose in the model from PDB ID: 3MXF
(yellow carbons) following alignment of the two proteins using MOE.
(A) Compound 1, (B) compound 3, (C) compound 4, and (D)
compound 5.
Compounds for which more information has been gathered
will now be described.
Chloropyridones. The chloropyridone scaffold was
selected using the Literature Substructures branch derived
from the known dihydroquinazolinone inhibitors. The
evolution of the initial dihydroquinazolinone substructure
Figure 9. Evolution of 1 from known dihydroquinazolinone inhibitors.
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significant improvement over 3 and 4. Furthermore, the
improved activity of 8c over 4 is likely due to an interaction
of the benzylamino group of 8c with the hydrophobic shelf, as
observed from the crystal structures of 3 bound to BRD4(1).
This interaction is known to provide improvements in potency
in other series of BET-family inhibitors, including (+)-JQ1, and
is not found in the complex of 4 with BRD4(1). Compound 8d
shared comparable activity to 8c, showing that the methylation
of the benzylic amine is not required for activity. Finally,
introduction of an amino group into 8b to yield 8e maintained
the activity but in this case did not lead to an improvement in
potency. However, compounds 8b−e demonstrate that there is
scope for further improvement of the initial hits 3 and 4 and
that a reasonable LE (∼0.3) can be achieved.
Table 2. Activity of Commercially Available Derivatives of
the Chloropyridone Scaffold
Quinolines. 5 demonstrated inhibition of BRD4(1), but its
IC50 was lower than its solubility and could not be determined;
however, the crystal structure bound to BRD4(1) revealed two
interesting features. First, the acetyl group was not acting as the
KAc mimetic but formed an interaction with the backbone NH
donor of Asp88, an interaction that has not been observed in
any published bromodomain crystal structure to date (Figure
10). Although the crystallographic orientation of the molecule
was predicted by the docking pose, this particular interaction
with the backbone NH donor of Asp88 was not, suggesting
another way in which potency and potentially selectivity can be
achieved from the BRD4(1) binding site.
The second interesting feature of this binding mode is that
the quinoline nitrogen does not appear to be forming a direct
interaction with the protein. From the crystal structure,
electron density could be observed in close proximity to the
quinoline nitrogen (Figure 11), suggesting an interaction
through a bridging water molecule. Interestingly, the distance
between the oxygen of the water molecule and the ligand
appeared to be very short (2.14 Å), and we speculated that the
active ingredient of this compound was in fact the quinoline N-
oxide; however, mass spectrometry did not detect the N-oxide.
We nevertheless decided to synthesize the N-oxide 14a, and 5
was resynthesized along with it (Scheme 2), and both
compounds were tested for BRD4(1) inhibition.
The resynthesized batch of 5 demonstrated activity similar to
the original sample, however, the pure N-oxide generated a
significantly improved IC50 of 42 μM (Table 4). This is a good
example of a single-atom change in a ligand resulting in the
displacement of a single water molecule and achieving an
increase in activity.
Pyrrolopyridones and Pyrrolopyrimidones. Although
pyrrolopyridone 6 demonstrated the lowest activity of the
compounds initially identified as hits, it also contained the
fewest heavy atoms, suggesting decent ligand efficiency despite
its modest activity. In addition, it represented another example
of a novel KAc mimetic, and we sought to demonstrate that
somewhat larger derivatives show improved inhibition.
Although few pyrrolopyridones were commercially available, a
number of related pyrrolopyrimidones could be sourced.
Gratifyingly, the phenyl substituted compounds 15a and 15b
showed substantially improved activities, with an LE above 0.3
(Table 5). These compounds represent attractive starting
points for further development. This improvement in potency
is consistent with the phenyl ring occupying the ZA channel, as
would be expected from the docking pose of 6 (Supporting
Information, Figure S2).
a
See the Experimental Section for details.
A number of commercial derivatives of this scaffold were
subsequently purchased (Supporting Information, Figure S3).
Gratifyingly, 8b demonstrated a substantially improved activity
toward BRD4(1), with an IC50 of 24 μM and associated LE of
0.33, demonstrating that this class of compounds can be further
improved.
The comparison of the matched pair compounds 4 and 8a,
which only differ by the presence of an amino substitution at
the triazolopyrimidine, suggested that introduction of the
amino group into compounds 3 and 8b may lead to an
improvement of activity. The improvement in potency can be
rationalized by the observation that the amino group forms an
interaction with the conserved asparagine, as seen in the crystal
structure of 4 with BRD4(1) (Figure 7C), and completes the
acceptor−acceptor−donor motif that is observed for published
inhibitors (e.g., dihydroquinazolinones) as well as some of the
other inhibitors identified here (2, but also 1 and 6 if the
carbonyl is considered as 2 acceptors). We thus prepared
(Scheme 1) and tested compounds 8c−e. 8c showed indeed a
a b
,
Scheme 1. Synthetic Route to 8e−g
a
b
Comparison between the Branches of Our Virtual
R1 corresponds to the functional group from Table 5. Reagents and
conditions: (a) POCl3, reflux, 2 h; (b) R1 amine, EtOH, reflux, 2 h.
Screen. Within our virtual screening approach, two branches
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Table 3. SAR around the Triazolopyrimidine Scaffold
a
See the Experimental Section for details.
were used: the Literature Substructures and Similarity
Searching branches. As hypothesized, both branches success-
fully yielded inhibitors of BRD4(1). Overall, a hit rate of 2.5%
was achieved for all of the compounds tested, and a hit rate of
4.2% was achieved for the 143 compounds selected by our
virtual screening approach (Table 6). Strikingly, all of the active
compounds had been identified from our virtual screen rather
than from the control experiment, highlighting the success of
the approach. We believe that the superiority of our virtual
screening approach compared to the control branch is due to
several factors. First of all, our KAc mimetic approach reduced
the number of commercially available compounds sufficiently
to enable docking, yet it ensured that a large number of KAc
mimetics were taken forward into the docking step. Second, the
application of docking rather than just a similarity search
ensured that the purchased compound have a high likelihood of
meeting the tight geometric constraint of the KAc binding site.
Finally, our extensive manual filtering and human input after
docking removed several potential false positives.
Within the compounds selected by the virtual screen, 76
came from the Literature Substructures approach and two hits
were identified. Sixty seven compounds came from the
Similarity Searching approach, from which four of the six
actives were identified. Four KAc mimetics that were
unprecedented for bromodomain inhibition were discovered,
fulfilling the main goal of this work.
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Table 4. Activity of 5 and Its N-Oxide Derivative 14a
Figure 10. Interaction of the acetyl group of 5 with backbone donor of
Asp88 in BRD4. Surface colors were generated using the pocket colors
in MOE. Green represents an enclosed surface, and white, exposed.
Alternate views of the binding mode of 5 can be seen in Figure 7D.
a
See the Experimental Section for details.
turned out not to be the case, with a higher hit rate being
achieved by the Similarity Searching branch. A possible reason
for this observation is that the published inhibitors are
optimized for the BRD4(1) binding site and possess good
shape-complementarity as well as a KAc mimetic warhead.
Compounds selected for the Literature Substructures branch
possessed this warhead, but without optimization suboptimal
binding in the upper part of the pocket may have abrogated any
activity.
CONCLUSIONS
■
We describe a structure-based virtual screening approach to
identify inhibitors of bromdomains together with the validation
of this approach, resulting in the discovery of novel inhibitors of
BRD4(1). The key elements of this approach are the extensive
design and use of substructure queries to compile a set of
commercially available compounds featuring novel putative
KAc mimetics followed by subjection of this set to docking for
final compound selection. Using this approach, we selected and
tested 143 compounds and identified novel hits, including four
unprecedented acetyl-lysine mimetics. A control experiment
that failed to identify hits was also performed, further
Figure 11. Electron density of 5 including a water molecule in close
proximity to the quinoline nitrogen (2Fo−Fc map).
We initially expected that the Literature Substructures branch
would yield the higher hit rate because of the presence of
validated and structurally related warheads. Interestingly, this
a
Scheme 2. Synthetic Route to 5 and 14a
a
Reagents and conditions: (a) Tf2O, Et3N, CHCl3, rt, 2 h; (b) acetamidophenylboronic acid, Pd(PPh3)4, CsCO3, dioxane, 120 °C, 30 min; (c)
mCPBA, DCM, reflux, 3 h.
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ligand-bound structures were with triazolodiazepines. For this reason,
the structure used for the virtual screen was that of (+)-JQ1 bound to
the BRD4(1) (PDB ID: 3MXF), which was used in the structure-
based pharmacophore searches and the bound pose of (+)-JQ1 for
shape-based similarity searches, and it was also the structure used for
the docking experiments. Water molecules with the following UIDs
were treated as part of the pocket for docking experiments and
pharmacophore excluded volumes: 9, 12, 15, 23, 33, and 209.
Pharmacophore Searches. We performed three pharmacophore
searches on the basis of the (+)-JQ1 molecule bound to BRD4(1)
(PDB ID: 3MXF) using the receptor as an excluded volume. The first
pharmacophore was the most complex containing five points,
including two acceptors from the triazole ring of (+)-JQ1, a
hydrophobic substituent for the methyl group at the base of the
pocket, and two further hydrophobic substituents at the ZA-channel
and hydrophobic shelf positions of the binding site. This
pharmacophore characterized each feature known to significantly
contribute to the potency of (+)-JQ1 and found just 6540 matching
ligands. The next pharmacophore was a derivative of the first, relaxing
the requirement for both acceptors on the triazole ring to just one of
the two. This increased the number of matching ligands significantly to
268 474. The third pharmacophore was also a derivative of the first
one, but this time with the pharmacophoric point corresponding to the
hydrophobic shelf removed. Again this increased the number of
matching ligands but not as significantly as for the second
pharmacophore to 41 890. The pharmacophore searches employed
can be seen in Figure 12. All pharmacophore searches were performed
using the Pharmacophore module in MOE.45
Table 5. Activity of Derivatives of 6
a
See the Experimental Section for details.
Table 6. Summary of the Hit Rates for the 240 Compounds
Screened against BRD4
approach
compounds tested
hits
hit rate (%)
all compounds
virtual screen
240
143
76
6
6
2
4
0
2.5
4.2
2.6
6.0
0
literature substructures
similarity searching
control set
67
97
confirming the success of our approach. The potency of many
of the hits was initially modest. However, through use of
cocrystal structures and structure-based design, we were able to
improve the potency considerably into a range frequently found
for screening hits. This work is therefore a good example of
how novel but initially modestly potent compounds are
identified with a fast and very limited screening effort and
then quickly optimized through the use of cocrystal structures.
Because of the similarity between bromodomain binding sites
and the focus on the KAc mimetic, this approach is applicable
to the large fraction of bromodomains for which structural
information of sufficient resolution is available. Furthermore,
we believe that the novel KAc mimetics disclosed here will
serve as valuable starting points for the development of potent
inhibitors of BRD4(1) and other related bromodomains.
Figure 12. (A) Pharmacophore searches 1 and 2 (with 2 requiring
only one acceptor). (B) Pharmacophore search 3.
Shape-Based Similarity Searches. We performed two shape-based
similarity searches: one on the full (+)-JQ1 ligand in its bound
conformation from PDB ID 3MXF and one on a reduced version of
the ligand with the ester and chlorophenyl groups removed to focus on
the KAc mimetic part of the binding site (Figure 13). Both searches
were performed with two acceptor pharmacophore points from the
triazole part of the (+)-JQ1 ligand. The top 100 000 compounds
ranked by the TanimotoCombo scoring function from each search
EXPERIMENTAL SECTION
■
Computational Methods. Ligands for Similarity Searches. We
used the ICR Vendor Collection, which is a library of 2.4 million
commercially available compounds from 11 vendors for which details
have been described by Langdon et al.41
Conformer and Protonation State Generation for Pharmaco-
phore and Shape-Based Similarity Searching. Up to 100
conformations were generated using OMEGA.42,43 Single protonation
states were generated using FILTER.44
Choice of Template for Virtual Screen. At the time of performing
the virtual screen against BRD4(1), the only inhibitors published were
the triazolodiazepines, BIC1, and simple dihydroquinazolinones. A
number of published structures were available, but the only suitable
Figure 13. Shape-based similarity searches used with acceptor
pharmacophoric points included (red spheres). (A) Full (+)-JQ1
ligand. (B) Reduced (+)-JQ1 ligand.
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were taken through to subsequent docking steps. All shape-based
similarity searches were performed in vROCS.46,47
Two-Dimensional Similarity Searches. A single 2D similarity
search was performed using the ECFP4 fingerprint and the (+)-JQ1
ligand as a template in Pipeline Pilot.48,49 The top 100 000 ligands
ranked by Tanimoto similarity were taken through to subsequent
docking steps.
Protein Preparation for Docking. Protons were added to PDB
3MXF using Protonate3D in MOE.45 The structure was preprocessed
using the protein preparation wizard50 in Maestro,51 with the assign
bond orders, create disulfide bonds, and convert selenomethionines to
methionines options selected. Grids for Glide docking were then
generated using the receptor grid generation tool in Maestro using an
enclosing box of size 20 Å around the center of (+)-JQ1 and other
settings left at the default.
nL of the protein (9.0 mg/mL and 10 mM final ligand concentration)
with an equal volume of reservoir solution containing 0.20 M Na2SO4,
20.0% PEG3350 and 10.0% ethylene glycol. BRD4(1) crystals with 5
were grown by mixing 150 nL of protein (9.9 mg/mL and 5 mM final
ligand concentration) with an equal volume of reservoir solution
containing 0.2 M sodium sulfate, 0.20 M LiCl, 0.1 M Tris pH 8.0,
20.0% PEG6K, 10.0% ethylene glycol. In all cases, diffraction-quality
crystals grew within a few days.
Data Collection and Structure solution. All crystals were cryo-
protected using the well solution supplemented with additional
ethylene glycol and were flash frozen in liquid nitrogen. Data were
collected in-house on a Rigaku FRE rotating anode system equipped
with a RAXIS-IV detector at 1.52 Å. Indexing and integration was
carried out using MOSFLM,57 and scaling was performed with
SCALA.58 Initial phases were calculated by molecular replacement
with PHASER59 using the known model of BRD4(1) (PDB ID:
2OSS). Initial models were built by ARP/wARP60 followed by manual
building in COOT.61 Refinement was carried out in REFMAC5.62 In
all cases, thermal motions were analyzed using TLSMD,63 and
hydrogen atoms were included in late refinement cycles. Data
collection and refinement statistics can be found in the Supporting
Information, Table S1. The models and structure factors have been
deposited with PDB accession codes 4MEP (BRD4(1)/ compound
1), 4MEN (BRD4(1)/ compound 3), 4MEQ (BRD4(1)/ compound
4), and 4MEO (BRD4(1)/ compound 5).
Ligand Preparation for Docking. Compounds were prepared using
LigPrep52 with default settings. Otherwise, Epik53 was used for
protonation-state assignment and tautomer generation.
Docking. Docking was performed using Glide with default
settings.54,55 For more than 5000 ligands, the compounds were
initially docked using Glide in HTVS mode and the 5000 top ranked
compounds by GlideScore were docked using Glide in SP mode. For
fewer than 5000 ligands, the compounds were submitted directly to
Glide in SP mode. Water molecules included were treated as rigid,
with hydrogen positions used as defined by the Protonate3D step.
CSD Use. To analyze the observed dihedral angle of certain
substructures in the CSD,56 initial substructure searches and the
dihedral in question were defined using Conquest and the data output
and analyzed using Vista. A high-energy conformation was defined as
one that possessed a dihedral angle not observed in the CSD for
sufficiently populated substructures.
Biochemistry. AlphaScreen. Assays were performed as previously
described.40 All reagents were diluted in 50 mM HEPES, 100 mM
NaCl, 0.1% BSA, pH 7.4 supplemented with 0.05% CHAPS and
allowed to equilibrate to room temperature prior to addition to plates.
A 24-point 1:2 serial dilution of the ligands was prepared over the
range of 150−0 μM, and 4 μL was transferred to low-volume 384-well
plates (ProxiPlateTM-384 Plus, PerkinElmer) followed by 4 μL of
HIS-tagged protein (BRD4(1), 250 nM). The plates were sealed and
incubated at room temperature for 30 min before the addition of 4 μL
of biotinylated peptide at an equimolar concentration to the protein
(the peptide used for BRD4(1): H-SGRGK(Ac)GGK(Ac)GLGK-
(Ac)GGAK(Ac)RHRK(Biotin)-OH; Cambridge Research Biochem-
icals, UK). The plates were sealed and incubated for a further 30 min
before the addition of 4 μL of streptavidin-coated donor beads (25 μg/
mL) and 4 μL of nickel chelate acceptor beads (25 μg/mL) under low-
light conditions. The plates were foil sealed to protect from light,
incubated at room temperature for 60 min, and read on a PHERAstar
FS plate reader (BMG Labtech) using an AlphaScreen 680 excitation/
570 emission filter set. IC50 was calculated in Prism 5 (GraphPad
Software) after normalization against corresponding DMSO controls,
and they are given as the final concentration of compound in the 20
μL reaction volume.
Chemistry. General Experimental. Commercially available starting
materials, reagents, and dry solvents were used as supplied. Column
chromatography was performed on a Biotage SP1 purification system
using Biotage Flash silica cartridges. Reverse-phase chromatography
was performed using isolute C18 silica columns. Semiprep HPLC
purification was achieved using 1000 μL standard injections (with
needle rinse) of the sample at a 20 mg/mL concentration in DMSO
onto a Phenomenex Gemini column (10 μm, 250 × 21.2 mm, C18,
Phenomenex). Chromatographic separation at room temperature was
carried out using a Gilson GX-281 liquid handler system combined
with a Gilson 322 HPLC pump (Gilson) over a 15 min gradient
elution (Grad15 min20mlsLipo.m) from 40:60 to 100:0 methanol/
water (both modified with 0.1% formic acid) at a flow rate of 20 mL/
min. UV−vis spectra were acquired at 254 nm on a Gilson 156 UV−
vis detector (Gilson). Collection was triggered by UV signal and
collected using a Gilson GX-281 liquid handler system (Gilson). Raw
data was processed using Gilson Trilution Software. LC−MS and
HRMS analysis was performed on an Agilent 1200 series HPLC and
diode array detector coupled to a 6210 time-of-flight mass
spectrometer with dual multimode APCI/ESI source. Analytical
separation was carried out at 30 °C on a Merck Purospher STAR
column (RP-18e, 30 × 4 mm) using a flow rate of 1.5 mL/min in a 4
min gradient elution with detection at 254 nm. The mobile phase was
a mixture of methanol (solvent A) and water containing formic acid at
0.1% (solvent B). Gradient elution was as follows: 1:9 (A/B) to 9:1
(A/B) over 2.5 min, 9:1 (A/B) for 1 min, and then reversion back to
1
1:9 (A/B) over 0.3 min, and finally 1:9 (A/B) for 0.2 min. H NMR
spectra were recorded on a Bruker Avance-500. Samples were
prepared as solutions in a deuterated solvent and referenced to the
appropriate internal nondeuterated solvent peak or tetramethylsilane.
Purchased compounds were prepared from DMSO stocks with
DMSO-d6 added. Chemical shifts were recorded in ppm (δ) downfield
of tetramethylsilane.
Compound 1. 3-Chloro-5-(1-(3-methylpyridin-2-yl)-3-phenyl-1H-
1,2,4-triazol-5-yl)pyridin-2(1H)-one (1). Compound 1 was purchased
as a 5 mg solid sample from ChemBridge (supplier ID 25722975). 1H
NMR (500 MHz, DMSO) δ 2.21 (s, 3H), 7.45 (s, 1H), 7.47−7.55 (m,
4H), 7.66 (t, 1H), 7.72 (s, 1H), 8.07 (d, 1H), 8.11 (d, 2H), 8.51 (d,
1H). HRMS m/z calcd for C19H15ClN5O [M + H]+, 364.0960; found,
364.0961.
Protein Expression and Purification. Proteins were cloned,
expressed, and purified as previously described.7
Crystallization. Aliquots of the purified proteins were set up for
crystallization using a mosquito crystallization robot (TTP Labtech).
Coarse screens were typically set up onto Greiner 3-well plates using
three different drop ratios of precipitant to protein per condition (100
+ 50, 75 + 75, and 50 + 100 nL). Initial hits were optimized further by
scaling up the drop sizes. All crystallizations were carried out using the
sitting-drop vapor-diffusion method at 4 °C. BRD4(1) crystals with 1
were grown by mixing 150 nL of protein (9.9 mg/mL and 5 mM final
ligand concentration) with an equal volume of reservoir solution
containing 0.2 M sodium sulfate, 0.20 M NaBr, 0.1 M BTProp pH 8.5,
20.0% PEG6K, 10.0% ethylene glycol. BRD4(1) crystals with 3 were
grown by mixing 100 nL of protein (9.0 mg/mL and 5 mM final ligand
concentration) with 200 nL of reservoir solution containing 0.20 M
Na(malonate), 0.1 M BTProp pH 8.5, 20.0% PEG3350, 10.0%
ethylene glycol. BRD4(1) crystals with 4 were grown by mixing 150
Compound 2. 3-Methyl-4-(3-(trifluoromethyl)phenyl)isoxazol-5-
amine (2). Compound 2 was purchased as a 5 mg solid sample
from Key Organics Ltd. (supplier ID 10T-0381). 1H NMR (500 MHz,
DMSO) δ 2.13 (s, 3H), 6.93 (s, 2H), 7.54−7.65 (m, 4H). HRMS m/z
calcd for C11H10F3N2O [M + H]+, 243.0740; found, 243.0741.
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Compound 3. N,5-Dimethyl-N-(4-methylbenzyl)-[1,2,4]triazolo-
[1,5-a]pyrimidin-7-amine (3). Compound 3 was purchased as a 50
mg solid sample from Enamine Ltd. (supplier ID T6839820) and
7.57 (m, 2H), 7.66 (s, 1H). HRMS m/z calcd for C14H11ClFN4O [M
+ H]+, 305.0600; found, 305.0592.
Compound 8a. 5-Methyl-7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine
(8a). Compound 8a was purchased as a 5 mg solid sample from
Enamine Ltd. (supplier ID T0510-0868). 1H NMR (500 MHz,
DMSO) δ 7.56 (s, 1H), 7.62−7.69 (m, 3H), 8.15−8.20 (m, 2H), 8.63
(s, 1H). The methyl peak was not observed and was expected to be
below the DMSO peak. HRMS m/z calcd for C12H11N4 [M + H]+,
211.0978; found, 211.0976.
1
purified by semiprep HPLC. H NMR (500 MHz, CDCl3) δ 2.37 (s,
3H), 2.60 (s, 3H), 3.20 (s, 3H), 5.31 (s, 2H) 6.07 (s, 1H), 7.13−7.20
(m, 4H), 8.34 (s, 1H). HRMS m/z calcd for C15H18N5 [M + H]+,
268.1557; found, 268.1558.
Compound 4. 5-Methyl-7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-
2-amine (4). Compound 4 was purchased as a 5 mg solid sample from
Princeton Biomolecular Research Inc. (supplier ID OSSK_397127).
1H NMR (500 MHz, DMSO) δ 6.34 (s, 2H), 7.18 (s, 1H), 7.57−7.63
(m, 3H), 8.14 (d, 2H). The methyl peak was not observed and was
expected to be below the DMSO peak. HRMS m/z calcd for C12H12N5
[M + H]+, 226.1087; found, 226.1091.
Compound 8b. N-((5-Chlorothiophen-2-yl)methyl)-N,5-dimethyl-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (8b). Compound 8b was
purchased as a 5 mg solid sample from Enamine Ltd. (supplier ID
1
Z64611040). H NMR (500 MHz, DMSO) δ 3.13 (s, 3H), 5.39 (s,
2H), 6.44 (s, 1H), 7.00 (s, 2H), 8.49 (s, 1H). The methyl peak was not
observed and was expected to be below the DMSO peak. HRMS m/z
calcd for C12H13ClN5S [M + H]+, 294.0575; found, 294.0571.
Synthesis of Compound 8c. 7-Chloro-5-methyl-[1,2,4]triazolo[1,5-
a]pyrimidin-2-amine (10). 2-Amino-5-methyl-[1,2,4]triazolo[1,5-a]-
pyrimidin-7(3H)-one 9 (500 mg, 3.028 mmol) was added to cooled (0
°C), stirring POCl3 (3 mL). The reaction mixture was allowed to
warm to rt and was heated to reflux for 2 h. The reaction mixture was
concentrated in vacuo, and the residue was dissolved in DCM (20
mL). Water (20 mL) was added, and the resultant precipitate was
isolated by filtration. The residue was purified by triturating with DCM
and used in subsequent reactions without further purification (345 mg,
Compound 5. N-(3-(2-Methylquinolin-4-yl)phenyl)acetamide (5).
Compound 5 was initially purchased as a 5 mg solid sample from
ChemBridge (supplier ID 50866471). 1H NMR (500 MHz, DMSO) δ
2.07 (s, 3H), 7.21 (d, 1H), 7.36 (s, 1H), 7.48−7.56 (m, 2H), 7.70
(d,1H), 7.75 (t, 1H), 7.81 (s, 1H), 7.84 (d, 1H), 8.01 (d, 1H), 10.14
(s, 1H). HRMS m/z calcd for C18H16N2O [M + H]+, 277.1335; found,
277.1333. Resynthesis of Compound 5. 2-Methylquinolin-4-yl
trifluoromethanesulfonate (13). Triflic anyhydride (0.95 mL, 5.65
mmol) was added to a solution of 2-methylquinolin-4-ol 12 (750 mg,
4.71 mmol) and triethylamine (0.66 mL, 4.71 mmol) in anhydrous
DCM (5 mL) under nitrogen and stirred for 2 h. The reaction mixture
was concentrated in vacuo. The resulting purple oil was purified by
column chromatography (EtOAc to 5% MeOH in EtOAc) to afford
title compound (863 mg, 63%). LC−MS (ESI, m/z) tR 2.82 min,
1
62%). LC−MS (ESI, m/z) tR 1.15 min, did not ionize [M + H]+. H
NMR (500 MHz, TFA) δ 2.91 (s, 3H), 7.72 (s, 1H).
N-Methyl-1-(p-tolyl)methanamine. 4-Methylbenzyl chloride (0.933
mL, 7.112 mmol) was added to a solution of 40% aqueous
methylamine (10 mL, 18.031 mmol) in EtOH (10 mL) and stirred
at rt for 12 h. The reaction mixture was concentrated in vacuo, the
residue was dissolved in water (20 mL), and the pH adjusted to 12
with 2 M NaOH. The aqueous layer was extracted with EtOAc (2 ×
20 mL). The combined organic layers were dried (MgSO4) and
concentrated in vacuo. The resulting yellow oil was purified by reverse-
phase C18 flash column chromatography (water) to afford the title
compound (112 mg, 12%). LC−MS (ESI, m/z) tR 0.85 min, 136.11
[M + H]+. 1H NMR (500 MHz, CDCl3) δ 2.36 (s, 3H), 2.46 (s, 3H),
3.73 (s, 2H), 7.15 (d, 2H), 7.22 (d, 2H).
1
292.18 [M + H]+. H NMR (500 MHz, CDCl3) δ 2.87 (s, 3H), 7.35
(s, 1H), 7.68 (t, 1H), 7.86 (t, 1H), 8.05 (d, 1H), 8.17 (d, 1H).
N-(3-(2-Methylquinolin-4-yl)phenyl)acetamide (5). To a solution
of 2-methylquinolin-4-yl trifluoromethanesulfonate 13 (280 mg, 0.79
mmol) in dioxane/water (2:1, 4.5 mL) were added acetamidophe-
nylboronic acid (283 mg, 1.58 mmol), tetrakis triphenylphosphine
palladium (45.6 mg, 0.04 mmol), and cesium carbonate (386 mg,
1.185 mmol). The reaction mixture was heated at 120 °C for 30 min
under microwave conditions. The reaction mixture was diluted with
EtOAc (20 mL) and washed with water (20 mL). The aqueous layer
was re-extracted with EtOAc (20 mL), and the combined organic
layers were dried (MgSO4) and concentrated in vacuo. The residue
was purified by column chromatography (20% EtOAc in hexane to
100% EtOAc) to afford the title compound (141 mg, 65%). LC−MS
N7,5-Dimethyl-N7-(4-methylbenzyl)-[1,2,4]triazolo[1,5-a]-
pyrimidine-2,7-diamine (8c). 7-Chloro-5-methyl-[1,2,4]triazolo[1,5-
a]pyrimidin-2-amine 10 (50 mg, 0.275 mmol) was added to a stirred
solution of N-methyl-1-(p-tolyl)methanamine (74 mg, 0.55 mmol)
and K2CO3 (38 mg, 0.275 mmol) in anhydrous ethanol (0.5 mL). The
reaction mixture was heated to reflux for 2 h. The reaction mixture was
concentrated in vacuo, and the residue was dissolved in EtOAc (10
mL) and water (10 mL). The aqueous layer was re-extracted with
EtOAc (10 mL), and the combined organic layers were dried
(MgSO4) and concentrated in vacuo. The residue was purified using
an SCX-2 column (MeOH) to afford the title compound (5.63 mg,
1
(ESI, m/z) tR 1.97 min, 277.13 [M + H]+. H NMR (500 MHz,
CDCl3) δ 2.23 (s, 3H), 2.81 (s, 3H), 7.23−7.29 (m, 2H), 7.44−7.57
(m, 3H), 7.64−7.74 (m, 3H), 7.89 (d,1H), 8.15 (d, 1H). HRMS m/z
calcd for C18H17N2O [M + H]+, 277.1335; found, 277.1336.
Compound 6. 1-Methyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-one (6).
Compound 6 was purchased as a 5 mg solid sample from Enamine
1
Ltd. (supplier ID EN300−74511). H NMR (500 MHz, DMSO) δ
4.05 (s, 3H), 6.24 (d, 1H), 6.39 (d, 1H), 6.81 (t, 1H), 7.26 (d, 1H),
10.81 (s, 1H). HRMS m/z calcd for C8H9N2O [M + H]+, 149.0709;
found, 149.0709.
1
42%). LC−MS (ESI, m/z) tR 2.63 min, 283.17 [M + H]+. H NMR
(500 MHz, CDCl3) δ 2.36 (s, 3H), 2.49 (s, 3H), 3.08 (s, 3H), 4.66
(brs, 2H), 5.19 (s, 2H), 5.94 (s, 1H), 7.12 (d, 2H), 7.15 (d, 2H).
HRMS m/z calcd for C15H19N6 [M + H]+, 283.1666; found, 283.1665.
Compound 8d. 5-Methyl-N7-(4-methylbenzyl)-[1,2,4]triazolo[1,5-
a]pyrimidine-2,7-diamine (8d). 7-Chloro-5-methyl-[1,2,4]triazolo[1,5-
a]pyrimidin-2-amine 10 (50 mg, 0.275 mmol) was added to a stirred
solution of p-tolylmethanamine (1.36 mmol) and K2CO3 (38 mg,
0.275 mmol) in anhydrous ethanol (0.5 mL). The reaction mixture
was heated to reflux for 2 h, with reaction progress monitored by LC−
MS. The reaction mixture was concentrated in vacuo. The residue was
dissolved in ethyl acetate (10 mL) and water (10 mL). The aqueous
layer was re-extracted with ethyl acetate, and the combined organic
layers were dried (MgSO4) and concentrated in vacuo. The residue
was purified by column chromatography (0% MeOH in EtOAc to 20%
MeOH in EtOAc) to afford the title compound (31 mg, 42%). LC−
Compound 7a. 3-Chloro-5-(3-isopropyl-1-(3-methylpyridin-4-yl)-
1H-1,2,4-triazol-5-yl)pyridin-2(1H)-one (7a). Compound 7a was
purchased as a 5 mg solid sample from ChemBridge (supplier ID
1
90638099). H NMR (500 MHz, DMSO) δ 1.24 (m, 1H), 1.32 (d,
6H), 2.08 (s, 3H), 7.34 (s, 1H), 7.47 (d, 1H), 7.68 (s, 1H), 8.59 (d,
1H), 8.71 (s, 1H). HRMS m/z calcd for C16H17ClN5O [M + H]+,
330.1116; found, 330.1114.
Compound 7b. 3-Chloro-5-(1-(2,3-dihydrobenzo[b][1,4]dioxin-5-
yl)-3-isobutyl-1H-1,2,4-triazol-5-yl)pyridin-2(1H)-one (7b). Com-
pound 7b was purchased as a 5 mg solid sample from ChemBridge
1
(supplier ID 95921162). H NMR (500 MHz, DMSO) δ 0.93−0.99
(m, 7H), 2.07 (s, 2H), 4.29 (s, 4H), 6.90 (d, 1H), 6.97−7.03 (m, 2H),
7.41 (s, 1H), 7.69 (s, 1H). HRMS m/z calcd for C19H20ClN4O3 [M +
H]+, 387.1218; found, 387.1207.
1
Compound 7c. 3-Chloro-5-(1-(4-fluorophenyl)-3-methyl-1H-1,2,4-
triazol-5-yl)pyridin-2(1H)-one (7c). Compound 7c was purchased as a
5 mg solid sample from ChemBridge (supplier ID 26575894). 1H
NMR (500 MHz, DMSO) δ 2.34 (s, 3H), 7.36−7.42 (m, 3H), 7.51−
MS (ESI, m/z) tR 2.47 min, 269.15 [M + H]+. H NMR (500 MHz,
CDCl3) δ 2.38 (s, 3H), 2.48 (s, 3H), 4.51 (d, 2H), 4.61 (brs, 2H) 5.94
(s, 1H), 6.14 (s, 1H), 7.21 (d, 2H), 7.24 (d, 2H). HRMS m/z calcd for
C14H17N6 [M + H]+, 269.1509; found, 269.1510.
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Compound 8e. N7-((5-Chlorothiophen-2-yl)methyl)-N7,5-dimeth-
yl-[1,2,4]triazolo[1,5-a]pyrimidine-2,7-diamine (8e). 7-Chloro-5-
methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine 10 (50 mg, 0.275
mmol) was added to a stirred solution of 1-(5-chlorothiphen-2-yl)-
N-methylmethanamine (111 mg, 0.688 mmol) and K2CO3 (38 mg,
0.275 mmol) in anhydrous ethanol (0.5 mL). The reaction mixture
was heated to reflux for 2 h, with reaction progress monitored by LC−
MS. The reaction mixture was concentrated in vacuo. The residue was
dissolved in ethyl acetate (10 mL) and water (10 mL). The aqueous
layer was re-extracted with ethyl acetate, and the combined organic
layers were dried (MgSO4) and concentrated in vacuo The residue was
purified by column chromatography (0% MeOH in DCM to 10%
MeOH in DCM) to afford the title compound (4.32 mg, 42%). LC−
no. C309/A8274. S.K., S.P., S.M., and O.F. received funding
from the Structural Genomics Consortium registered charity
(no. 1097737) that receives funds from AbbVie, Boehringer
Ingelheim, the Canada Foundation for Innovation, the
Canadian Institutes for Health Research, Genome Canada,
GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research
Foundation, the Ontario Ministry of Economic Development
and Innovation, Pfizer, Takeda, and the Wellcome Trust. P.F. is
supported by a Wellcome Trust Career-Development Fellow-
ship (095751/Z/11/Z). We thank Mark Stubbs and Yvette
Newbatt for assistance with compound preparation, Gary
Nugent for dotmatics assistance, and Meirion Richards and
Maggie Liu for assistance with compound QC.
1
MS (ESI, m/z) tR 2.71 min, 309.07 [M + H]+. H NMR (500 MHz,
CDCl3) δ 2.50 (s, 3H), 3.07 (s, 3H), 4.75 (brs, 2H), 5.29 (s, 2H), 5.95
(s, 1H), 6.77 (m, 2H). HRMS m/z calcd for C12H14ClN6S [M + H]+,
309.0684; found, 309.0684.
ABBREVIATIONS USED
■
Compound 14a. 4-(3-Acetamidophenyl)-2-methylquinoline 1-
oxide (14a). mCPBA (37.5 mg, 0.317 mmol) was added slowly to a
stirring solution of N-(3-(2-methylquinolin-4-yl)phenyl)acetamide 5
(50 mg, 0.181 mmol) in anhydrous DCM (1.3 mL). The reaction
mixture was heated at 45 °C for 3 h. The reaction mixture was cooled
to room temperature and quenched with aq sat. sodium thiosulfate
solution (1.5 mL) and aq sat. sodium bicarbonate solution (4.5 mL).
The reaction mixture was extracted with DCM (2 × 10 mL), dried
(MgSO4), and concentrated in vacuo. The residue was purified by
column chromatography (0% MeOH in DCM to 10% MeOH in
DCM) to afford the title compound (7.2 mg, 14%). LC−MS (ESI, m/
z) tR 2.61 min, 293.13 [M + H]+. 1H NMR (500 MHz, CDCl3) δ 2.17
(s, 3H), 3.04 (s, 3H), 7.19 (d, 1H), 7.28 (s, 1H), 7.46 (t, 1H), 7.60−
7.66 (m, 2H), 7.69 (d, 1H), 7.86 (t, 1H), 7.97 (d, 1H), 8.10 (brs, 1H),
8.82 (d, 1H). HRMS m/z calcd for C18H17N2O2 [M + H]+, 293.1285;
found, 293.1287.
2D, two dimensional; 3D, three dimensional; ATAD, AAA
domain-containing protein; BET, bromodomain and extra
terminal; BRD, bromodomain-containing protein; brs, broad
singlet; CSD, Cambridge Structural Database; dd, doublet of
doublets; ECFP, extended connectivity fingerprint; Glide, grid-
based ligand docking with energetics; HA, heavy atom; HTVS,
high-throughput virtual screening; KAc, acetyl-lysine; LE,
ligand efficiency; MOE, molecular operating environment;
ROCS, rapid overlay of chemical structures; SP, standard
precision; VS, virtual screen
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ASSOCIATED CONTENT
* Supporting Information
■
S
Substructures used in the “Literature Substructures” approach,
docking poses used to select active compounds, triazolopyr-
imidine compounds purchased, and protein crystallography
data collection and statistics. This material is available free of
charge via the Internet at http://pubs.acs.org.
(8) Chung, C.; Coste, H.; White, J. H.; Mirguet, O.; Wilde, J.;
Gosmini, R. L.; Delves, C.; Magny, S. M.; Woodward, R.; Hughes, S.
A.; Boursier, E. V.; Flynn, H.; Bouillot, A. M.; Bamborough, P.; Brusq,
J.-M. G.; Gellibert, F. J.; Jones, E. J.; Riou, A. M.; Homes, P.; Martin, S.
AUTHOR INFORMATION
Corresponding Authors
*Phone: +44 1865617584; E-mail: stefan.knapp@sgc.ox.ac.uk
(S.K.).
*Phone: +44 2087224353; E-mail: swen.hoelder@icr.ac.uk
(S.H.).
Notes
■
́
L.; Uings, I. J.; Toum, J.; Clement, C. A.; Boullay, A.-B.; Grimley, R. L.;
Blandel, F. M.; Prinjha, R. K.; Lee, K.; Kirilovsky, J.; Nicodeme, E.
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McCleary, S.; Rioja, I.; Bamborough, P.; Chung, C.-W.; Gordon, L.;
Lewis, T.; Walker, A. L.; Cutler, L.; Lugo, D.; Wilson, D. M.;
Witherington, J.; Lee, K.; Prinjha, R. K. Identification of a novel series
of BET family bromodomain inhibitors: Binding mode and profile of I-
BET151 (GSK1210151A). Bioorg. Med. Chem. Lett. 2012, 22, 2968−
2972.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
L.V. is funded by Cancer Research UK grant no. C309/A11369.
We acknowledge NHS funding to the NIHR Biomedical
Research Centre and funding from Cancer Research UK grant
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