Benzoisoquinolinediones as Potent and Selective Inhibitors of BRPF2 and TAF1/TAF1L Bromodomains

Article abstract of DOI:10.1021/acs.jmedchem.7b00306

Bromodomains (BD) are readers of lysine acetylation marks present in numerous proteins associated with chromatin. Here we describe a dual inhibitor of the bromodomain and PHD finger (BRPF) family member BRPF2 and the TATA box binding protein-associated factors TAF1 and TAF1L. These proteins are found in large chromatin complexes and play important roles in transcription regulation. The substituted benzoisoquinolinedione series was identified by high-throughput screening, and subsequent structure-activity relationship optimization allowed generation of low nanomolar BRPF2 BD inhibitors with strong selectivity against BRPF1 and BRPF3 BDs. In addition, a strong inhibition of TAF1/TAF1L BD2 was measured for most derivatives. The best compound of the series was BAY-299, which is a very potent, dual inhibitor with an IC₅₀ of 67 nM for BRPF2 BD, 8 nM for TAF1 BD2, and 106 nM for TAF1L BD2. Importantly, no activity was measured for BRD4 BDs. Furthermore, cellular activity was evidenced using a BRPF2- or TAF1-histone H3.3 or H4 interaction assay.

Full text of DOI:10.1021/acs.jmedchem.7b00306

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Article
Benzo-isoquinoline-diones As Potent and Selective
Inhibitors of BRPF2 and TAF1/TAF1L Bromodomains
Léa Bouché, Clara D. Christ, Stephan Siegel, Amaury E. Fernández-Montalván, Simon J. Holton,
Oleg Fedorov, Antonius ter Laak, Tatsuo Sugawara, Detlef Stöckigt, Cynthia Tallant, James M.
Bennett, Octovia P Monteiro, Laura Díaz-Sáez, paulina siejka, Julia Meier, Vera Puetter, Joerg
Weiske, Susanne Müller, Kilian Veit Marcus Huber, Ingo V. Hartung, and Bernard Haendler
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00306 • Publication Date (Web): 12 Apr 2017
Downloaded from http://pubs.acs.org on April 14, 2017
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Journal of Medicinal Chemistry
Benzo-isoquinoline-diones as Potent and Selective Inhibi-
tors of BRPF2 and TAF1/TAF1L Bromodomains
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,†
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Léa Bouché,* Clara D. Christ, Stephan Siegel, Amaury E. Fernández-Montalván, Simon J.
†
ǂ,§
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Holton, Oleg Fedorov, Antonius ter Laak, Tatsuo Sugawara, Detlef Stöckigt, Cynthia
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Tallant, James Bennett, Octovia Monteiro, Laura Díaz-Sáez, Paulina Siejka, Julia
ǂ,ǁ
†
†
ǂ,§
ǂ,§
Meier,
Vera Pütter, Jörg Weiske, Susanne Müller,
Kilian V. M. Huber,
Ingo V.
†
†
Hartung, and Bernard Haendler
†
Drug Discovery, Bayer AG, Müllerstrasse 178, 13353 Berlin, Germany
ǂ
Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Old Road
Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.
§
Target Discovery Institute, Nuffield Department of Clinical Medicine, University of Oxford, Roosevelt Drive, Ox-
ford OX3 7FZ, U.K.
ǁ
Present address: Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany
Supporting Information
ABSTRACT: Bromodomains (BD) are readers of lysine acetylation marks present in numerous proteins
associated with chromatin. Here we describe a dual inhibitor of the bromodomain and PHD finger (BRPF) family
member BRPF2 and the TATA box binding protein-associated factors TAF1 and TAF1L. These proteins are
found in large chromatin complexes and play important roles in transcription regulation. The substituted benzo-
isoquinoline-dione series was identified by high-througput screening and subsequent structure-activity
relationship optimization allowed generation of low nanomolar BRPF2 BD inhibitors with strong selectivity against
BRPF1 and BRPF3 BDs. In addition, a strong inhibition of TAF1/TAF1L BD2 was measured for most derivatives.
The best compound of the series was BAY-299 which is a very potent, dual inhibitor with an IC50 of 67 nM for
BRPF2 BD, 8 nM for TAF1 BD2 and 106 nM for TAF1L BD2. Importantly, no activity was measured for BRD4
BDs. Furthermore, cellular activity was evidenced using a BRPF2- or TAF1-histone H3.3 or H4 interaction assay.
KEYWORDS: BRPF2, TAF1, bromodomain, chemical probe
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INTRODUCTION
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Proteins involved in chromatin modulation usually contain several functional regions that serve to recruit and
tether binding partners, ultimately leading to the formation of large multiprotein complexes. A clear understanding
of the function of these complexes will only be possible through the dissection of the interactions between the
individual protein partners and the analysis of the exact role of their interactive modules. Different approaches are
currently being used to address this. One method is the selective deletion of gene exons by CRISPR-Cas9 fol-
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lowed by phenotypic analyses. Another, complementary approach is the identification of specific domain inhibi-
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tors and evaluation of their functional impact in cellular, and in some cases, animal models.
Here, great ad-
vances have recently been reported in the area of bromodomains (BDs), the best-characterized readers of acetyl-
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-4
lysine (KAc) marks which are found on histones and other proteins. BDs are potentially amenable to small mol-
ecule inhibition as impressively shown in the case of bromo and extra-terminal (BET) BD inhibitors which have
been extensively used in recent years to understand the role of this family in normal physiology and pathology,
3
-6
ultimately paving the way for their clinical evaluation in cancer indications. In the wake of this success, several
other BDs have been successfully targeted by inhibitors with very different scaffolds, further highlighting the drug-
7-14
gability of this target family.
The bromodomain and PHD finger (BRPF) family encompasses three paralogs, BRPF1, BRPF2 and BRPF3,
1
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which are found in histone acetyltransferase (HAT) complexes. BRPFs possess different functional modules
including C2H2, plant homeodomain-linked fingers, bromo and PWWP domains. Mouse studies show that loss of
either BRPF1 or BRPF2 leads to embryonic lethality at E9.5 and E15.5, respectively, due to different develop-
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mental defects.
In sharp contrast, BRPF3 is not needed for mouse development or survival, and no compen-
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satory expression change is observed for BRPF1 or BRPF2, in line with non-overlapping functions. BRPF1 is
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involved in hematopoietic stem cell development and BRPF2 in embryonic stem cell differentiation.
Little is
known about the implication of BRPFs in disease but a critical role of BRPF1 in the leukemia subgroup harboring
2
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the MOZ-TIF fusion has been reported. First BRPF BD inhibitors have been published (Figure 1). Compounds 1
21
10
OF-1) and 2 (NI-57) are pan-inhibitors recognizing all three BRPF paralogs and derived from two distinct
(
structure classes, benzimidazolones and quinolinones, which make them valuable complementary chemical
probes. Two additional inhibitors, 3 (PFI-4) and 4 (close analog of IACS-9571), as well as (R)-N-[1,3-dimethyl-6-
(
2-methylpiperazin-1-yl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl]-2-methoxybenzamide (GSK6853) and
N-
[
2,3-dihydro-1,3-dimethyl-2-oxo-6-(1-piperidinyl)-1H-benzimidazol-5-yl]-2-methoxybenzamide (GSK5959), are
22-24
derived from the 1,3-dimethyl-benzimidazolone scaffold and exhibit potent and selective binding to BRPF1.
Compound 4 shows additional strong activity on TRIM24, a nuclear receptor cofactor associated with different
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cancer types, being therefore a dual BRPF1/TRIM24 inhibitor.
Altogether, these findings demonstrate the
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high usefulness of the 1,3-dimethyl-benzimidazolone head group as a KAc mimetic binding motif and also sug-
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gest that the addition of novel decorations to this scaffold may result in a different selectivity profile.
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best of our knowledge however, no compounds showing selectivity for BRPF2 BD over its two paralogs have yet
been reported.
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Several TATA-binding protein-associated factors (TAFs) have been identified in the basal transcription factor
TFIID. TAF1 is the largest subunit of the complex and represents the essential scaffold to which the other TAFs
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and the TATA-binding protein attach to. Recent structural data indicate that TAF1 makes essential interactions
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with the core promoter DNA. It contains multiple modules including a kinase motif, a ubiquitin-conjugating activi-
ty, a HAT region and two related BDs. This modular organization is also found in the highly related paralog
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TAF1L, which functionally replaces TAF1 in male germ cells. Mutations in the TAF1 gene are associated with
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neurological pathologies in affected males.
However little is known about the implication of individual TAF1
domains in these diseases or about a role of TAF1 in cancer. The recent progress reported in the area of BD
inhibitors suggests that addressing this module may be a good starting point for functional studies. Indeed, a first
paper describing a BET inhibitor with additional submicromolar TAF1 BD2 activity, named UMB-32, has recently
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been presented. This compound is derived from an imidazo-pyrazine scaffold bearing a dimethyl-isoxazole
however its usefulness is restricted by its limited on-target activity and cross-reactivity with BET BDs, which are
2, 13, 37, 38
known to exert multiple cellular effects.
Very recently, TAF1 inhibitors derived from a methyl pyr-
39, 40
rolopyridone fragment were described.
The most selective example interacts mainly with TAF1 and TAF1L
BD1, but also with BRD9, BRD7 and BET BDs.
Here, we present the results of a screening and optimization strategy which allowed discovery of the 1,3-
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dimethylbenzimidazolone-substituted benzo-isoquinoline-dione 5 (BAY-299) which exhibits potent dual inhibito-
ry activity for both BRPF2 BD and TAF1/TAF1L BD2 in biochemical and cellular assays. This compound is the
first reported BRPF2 inhibitor with high selectivity against the BRPF1 and BRPF3 paralogs. It represents a power-
ful chemical tool suited for investigation of the role of two little-explored BD-containing proteins. Importantly, 5 is
inactive against BET BDs so that the generated cellular results will not be blurred by residual interference with
38
BRD4 or its paralogs, which are known to possess numerous biological functions.
RESULTS AND DISCUSSION
Screening and Hit Identification. Our original aim was to identify inhibitors of BRPF BDs with cellular activity
and without any activity on BET BDs. High-throughput screening (HTS) of a 3.5-million compound library was
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performed using a BRPF1 time-resolved fluorescence resonance energy transfer (TR-FRET) assay (Figure 2A).
Confirmed hits were evaluated by dose-response analysis (IC50 determination) in BRPF1, BRPF2 and BRPF3
TR-FRET (Figure 2A). BRD4 TR-FRET was used as counter screen. This exercise yielded several overlapping hit
clusters with nanomolar pan-BRPF BD activities and, surprisingly, allowed the identification of a single hit (6, Fig-
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ure 2A) containing a 1,3-dimethyl-benzimidazolone scaffold with nanomolar activity for BRPF2 while being inac-
tive up to concentrations of 20 µM for BRPF1 (Figure 2B), BRPF3 (Figure 2C) and BRD4 (Figure 2D). The bind-
ing specificity and selectivity profile of the compound were confirmed by Thermal Shift Assays (TSA), in which hit
6 stabilized BRPF2 with a ꢀTm of 0.8 K (Figure 2E), whereas only a ꢀTm of 0.2 K was measured for BRPF1 (not
shown). After confirmation of sample identity and based on its favorable binding efficiency index and lipophilic
ligand efficiency (BEI: 17.3; LLE: 3.7), hit 6 was selected as a starting point for further optimization cycles using
additional assays (Figure 2A).
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Chemistry. One structural feature of 6 is the tricyclic decoration of the benzimidazolone at position 5 (naph-
thalimide substituent). Surprisingly, the first analyses revealed that 6 had a logD of 2.1 and an excellent permea-
bility profile in Caco2 cells; furthermore, a low solubility (3.7 mg/L) was measured, which was not unexpected.
Docking studies revealed that the naphthalimide moiety was responsible for the strong BRPF2 selectivity as it
forms key interactions which are not possible with BRPF1 (see Figure 3 co-crystal structure discussion). That this
bulky substituent is indeed essential was confirmed by our early attempts to reduce its molecular size or aromatic
architecture which always led to less potent and less selective compounds (data not shown). Based on this bind-
ing mode hypothesis, we initiated an optimization program around the BRPF2-selective HTS hit 6.
Structure-Activity Relationships. We first focused on improving in vitro BRPF2 activity. Therefore, the influence
of substituents at position 6 of the 1,3-dimethyl-benzimidazolone core was studied (Table 1). A significant, four-
fold improvement of the BRPF2 activity was achieved by introducing small groups which lock the central dihedral
angle in its bioactive conformation; however, this was also accompanied by an increased interaction with BRPF1.
The best results were obtained for the methyl- and methoxy-derivatives (compounds 7 and 8) which offered the
highest selectivity for BRPF2 over BRPF1. Compounds 9 and 10 were also potent BRPF2 inhibitors but less se-
lective towards BRPF1. Addition of the larger methylpiperazine moiety (compound 11) did not lead to an activity
improvement. Importantly, all compounds demonstrated a high selectivity against BRD4 BDs. Furthermore, a
strong activity was also observed for TAF1 BD2.
42
The cellular activity of selected compounds was then determined using the NanoBRET™ assay to measure the

interaction between the BRPF BD fused to the NanoLuc luciferase and full-length histone H4 fused to the Halo-
Tag (Supporting Information Table S1). Compound 7 inhibited the interaction of BRPF2 BD and histone H4 with
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an IC50 of 470 nM, whereas no effect on the BRPF1 BD-histone H4 interaction was observed (IC50 >10000 nM).
Compound 8 was also highly selective for BRPF2 BD (IC50 = 1500 nM), compared to BRPF1 BD (IC50 >10000
nM). In addition, inhibition of TAF1 BD2-histone H4 interaction was also measured (Supporting Information Table
S1).
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The co-crystal structure of 7 with BRPF2 BD revealed the binding mode within the KAc binding pocket (Figure
3A). The expected hydrogen bond is formed between the carbonyl moiety of the benzimidazolone backbone of 7
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and residue Asn as well as the pi-stacking with the Phe . As predicted by our in silico analyses, a second
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hydrogen bond of the carbonyl moiety of the naphthalimide moiety to the backbone nitrogen of Ser is formed,
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which is key for the BRPF1 vs. BRPF2 selectivity profile. A proline residue (Pro ) at the equivalent position in
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BRPF1 is unable to form this hydrogen bond (Figure 3B). The crystal structure also revealed that the Gln side-
chain in BRPF2 unexpectedly adopted an alternative conformation in the presence of 7, forming an additional
Van-der-Waals type interaction by stacking underneath the tricyclic ring system (Figure 3A). Overlay with the
BRD4 apo-structure revealed a number of key residue differences that contribute to the strong selectivity of our
compounds (Figure 3C).
In order to enrich our structure-activity relationship (SAR) knowledge, we then focused on the naphthalimide moi-
ety and explored two different positions (5´ and 6´) (Table 2). For compounds bearing an electron-withdrawing
group at position 6´ (compounds 12 and 13), a low, two-digit nanomolar biochemical potency for BRPF2 was
reached. Aside from 15 which displayed an IC50 of 63 nM for BRPF2, analogues with a modification at position 5´
(
compounds 14, 16-18) displayed no significant improvement over 7. Except for 14, all modified compounds pos-
sessed strong additional TAF1 BD2 in vitro activity and unchanged selectivity against BRPF1. The best TAF1
BD2 IC50 values were obtained for 12 and 13 which possess an electron-withdrawing group at position 6´.
Encouraged by these data, we characterized 13 in more detail. Whilst cellular activity in the NanoBRET™ assay
was determined as IC50 675 nM for BRPF2, there was no activity for BRPF1 (Supporting Information Table S1).
Inhibition of TAF1 BD2-histone H4 interaction was furthermore observed (Supporting Information Table S1). In
addition, the selectivity of 13 was assessed using TSA with a panel of 48 BDs (Supporting Information Table S2).
The strongest stabilization was observed for BRPF2 (ꢀTm = 6.2 K) and weaker stabilization was seen for TAF1
BD2, TAF1L BD2, CECR2 BD and adenoviral E1A binding protein (EP300) BD. As the low solubility of 13 limited
further characterization, we elected to optimize this parameter by tuning the polarity via modifications at position
6
´ of the naphthalimide moiety (Table 3). A range of amines with diverse degrees of basicity were introduced by
nucleophilic aromatic substitution (SN ) or Buchwald-Hartwig coupling to provide 19-28 (Table 3). A significant
Ar
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served for 21, 22 and 26-28. One acyclic amine displaying high selectivity for BRPF2 over BRPF1 BD (118-fold)
and strong TAF1 BD2 activity could also be synthesized (compound 26). Interestingly, synthesis of its corre-
sponding C-substituted terminal alkoxy analogues (5 and 29) led to more selective compounds. We opted for
further investigation of 5 (BEI: 16.72, LLE: 4.72) since it showed the strongest selectivity and cellular activity for
BRPF2 with a reasonable solubility and a logD of 2.0. The physicochemical profile and characteristics of 5 are
summarized in the Supporting Information (Table S3). Overall, these SAR investigations revealed a high tolerabil-
ity to amines with respect to the BRPF2 BD or TAF1 BD2 in vitro activity.
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General Synthesis of 1,3,6-Trimethylbenzimidazolone-Substituted Naphthalimides. The 1,3,6-
trimethylbenzimidazolone-substituted naphthalimides were synthesized via a linear and straightforward reaction
pathway, as outlined in Scheme 1. Condensation of amine 30 with various commercially available 1,8-naphthalic
anhydrides resulted in 7, 12–14 and 16–18. Subsequent reaction of aryl bromide 12 with the appropriate amines
via nucleophilic aromatic substitution provided 19–25, 27 and 28. Compound 26 was synthesized from aryl bro-
mide 12 and propylamine via Buchwald–Hartwig amination. Sonogashira coupling of aryl bromide 12 with propar-
gyl alcohol or but-3-yn-1-ol, followed by hydrogenation of the resulting coupled alkynes with palladium on carbon,
resulted in 5 and its analogue 29.
Atropisomerism. Introduction of the methyl group at position 6 of the benzimidazolone core of the substituted
4
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naphthalimides hinders the rotation around the central torsion (axial chirality). Compound 5 is therefore a race-
mic mixture of the two atropisomers shown in Figure 4A. Several compounds containing C–N-coupled heterobiar-
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yls with related atropisomerism have already been described.
The calculated rotational energy barrier of 25
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kcal/mol for a representative fragment (Figure 4B) classifies the atropisomers of 5 as ‘class two’. For this class
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of atropisomers, interconversion might occur depending on the medium the compound is exposed to. In the
case of 5 the atropisomers 5a and 5b could be separated using chiral high-performance liquid chromatography
,
(HPLC) and individually characterized (Table 4). Compound 5 and its individual atropisomers showed similar
biochemical potency, selectivity and cellular NanoBRET™ activity for BRPF BD, TAF1 BD2 and BRD4 BDs. Fur-
thermore, the isothermal titration calorimetry (ITC) and Caco2 cell permeability profiles were comparable. Stirring
experiments with the individual atropisomers revealed slight racemization in acetonitrile and DMSO after one
week at 37 °C, and 30% racemization after two days at 80 °C (data not shown). The racemate 5 was therefore
selected as the chemical probe.
Biochemical Potency, Binding and Selectivity. TR-FRET assays showed that 5 was a potent inhibitor of
BRPF2 BD with an IC50 of 67 nM, and a selectivity of 47- and 83-fold over BRPF1 and BRPF3 BDs (Table 5). The
profile of 5 was further confirmed by AlphaScreen assay where an IC50 of 97 nM and a selectivity of 23- and 25-
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fold over BRPF1 and BRPF3 BDs were measured (Table 5). Expanded selectivity studies using TSA with a panel
of 48 BDs (Figure 5A) confirmed the selectivity of 5 within the BRPF family with a thermal shift for BRPF2 BD
more than five degrees higher than for BRPF1 BD and BRPF3 BD. Results from the BROMOscan panel further
confirmed the high selectivity with the BD family (Figure 5B). TR-FRET assays also disclosed a potent inhibition
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of TAF1 BD2 and the highly related TAF1L BD2 (Table 5). Finally ITC was performed and K values of 45 nM and
D
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
17 nM were determined for BRPF2 and TAF1 BD2, respectively (Figure 6A-B). A co-crystal structure of 5 in com-
plex with TAF1 BD2 revealed a highly conserved binding mode reminiscent of the one obtained for the precursor
7 in BRPF2 BD (Supporting Information Figure S1). In addition, interactions of 5 with the BDs of cyclic-AMP re-
sponse element binding protein (CREBBP) and the related EP300 were also detected by ITC (Figure 6A-B).
However, with the BROMOscanTM panel and in the AlphaScreen assays, only a weak activity was recorded
(30% and 41% inhibition in presence of 100 nM compound; IC50 (CREBBP) = 1710 nM) (Supporting Information
Table S4 and Table 5). ITC also revealed only modest interaction of 5 with CREBBP BD (KD = 1390 nM) (Figure
6B). Compound 5 was also tested at 10 µM in a panel containing over 300 kinases and in all cases, inhibition was
®
less than 50% (Table 5). Finally, 5 was evaluated in the LeadProfilingScreen which contains 68 potential targets
and no inhibition or stimulation exceeding 25% was noted in the presence of 10 µM compound (Table 5).
Cellular Activity. NanoBRET™ experiments (Figure 7A-C) showed that the interaction of BRPF2 BD with his-
t
o
n
e
s
H
4
a
n
d
H
3
.
3
w
a
s
b
l
o
c
k
e
d
b
y
5
w
i
t
h
I
C
v
a
l
u
e
s
o
f
5
7
5
a
n
d
8
2
5
n
M
,
r
e
s
p
e
c
t
i
v
e
l
y
.
F
o
r
T
A
F
1
B
D
2
,
t
h
e
I
C
5
0
5
0
values were 970 and 1400 nM, respectively. No inhibitory effect was observed for the interaction between BRPF1
or BRD4 and histone H4 up to 10 µM for 5.
Antiproliferative Activity. Cell viability experiments were performed with 5 to determine a potential link between
BRPF2 or TAF1 with proliferative diseases. Altogether little or no effect was observed. A weak inhibition was
seen in cell lines originating from hematological tumors (Supporting Information Table S5). Interestingly, a newly
described quinoline-2-one inhibitor targeting mainly BRPF1 and BRPF2 BDs also shows low micromolar activity
48
in leukemia cell lines.
In vitro and in vivo Pharmacokinetics. The metabolic stability of 5 was high after incubation with mouse, rat,
dog or human liver microsomes (Table 6A). In contrast, metabolic stability was low after incubation with rat or dog
hepatocytes. This difference may result from phase II metabolism and indeed corresponding metabolites have
been identified in vitro. Permeability through Caco2 cell monolayers was high, with no hint of active transport.
Plasma protein binding was low in mouse and moderate in human.
Studies of the in vivo pharmacokinetic properties of 5 in rat revealed that blood clearance was low (ca. 17% of
hepatic blood flow), volume of distribution in steady-state high, terminal half-life long to very long, and bioavaila-
7
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bility high (F = 73%) (Table 6B). In vivo blood clearance was as anticipated based on rat liver microsome values
but lower than expected based on hepatocyte data. Bioavailability matched blood clearance.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Negative Control. In order to deliver a valuable tool, synthesis of an inactive, structurally close analogue of a
chemical probe, as a direct comparator from the same chemotype in biochemical and cellular assays is recom-
1
3
mended. For this purpose, the key potency drivers of candidate 5 were disrupted. As a first strategy, we opted
for inducing steric hindrance at the 1,3-dimethyl-part of the benzimidazolone moiety. The corresponding diverse
set of compounds had, however, remaining BRPF2 or TAF1 activity. Then a different strategy aiming at reducing
the size of the molecule was followed. Previous results suggested that a significant loss of potency could be
achieved upon removing of the methyl group at position 6 of the benzimidazolone. Additional removal of one or
both methyl groups from the 1,3-dimethylimidazolone led to a complete loss of activity on BRPF2 BD, low residu-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
1
al activity on TAF1 BD2 and no activity on TAF1L BD2. 31 (BAY-364) was selected among several candidates
due to its favorable permeability profile (Figure 8). Compound 31 showed no activity for BRPF3 or BRD4 BDs in
the AlphaScreen assay. Also, no cellular effect was seen in the NanoBRET™ assay for BRPF2 BD or TAF1 BD2,
or for BRPF1 or BRD4 BDs (Figure 7A-C). Furthermore, 31 did not show any activity up to 10 µM in a panel of 22
kinases (data not shown). Thus, 31 fulfills the requirements of an inactive, structurally close analogue of probe 5.
CONCLUSION
An HTS approach followed by medicinal chemistry optimization led to the identification of 5, a highly potent and
first selective dual inhibitor of BRPF2 BD and TAF1/TAF1L BD2, which differs from previously reported BRPF
inhibitors. Our straightforward three-step synthesis ensures 5 is an easily accessible tool. A co-crystal structure of
the close congener 7 in complex with BRPF2 BD revealed four key interactions, including a hydrogen bond be-
592
tween a carbonyl group and Ser which was essential for selectivity over BRPF1 BD. Importantly 5 was demon-
strated to engage BRPF2 and TAF1 in cells at submicromolar concentrations. Antiproliferative activity was gener-
ally low and mainly observed in leukemia cell lines. The strong biochemical and cellular activities of 5 suggest it to
be a high-quality chemical probe for the in-depth analysis of the physiological function and pathological relevance
of BRPF2 and TAF1/TAF1L, which has not been possible to date. This novel tool enlarges the repertoire of avail-
able chemical probes for epigenetic players such that new knowledge about an implication of these two BD-
49-53
containing proteins in diverse pathologies can be expected forthwith.
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
EXPERIMENTAL PROCEDURES
Chemistry. General Comments. Chemical names and numbers of the compounds are given in italics and in
bold, respectively. Chemical names were generated using ACD/Name Batch or Autonom 2000, following IUPAC
nomenclature.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Stereochemistry. In certain cases (e.g. where the substituent at position 6 is not a hydrogen atom) atropisomer-
ism could be induced, and the compounds are assumed to be obtained as racemic mixtures of atropisomers. In
the cases where the atropisomeric ratio was investigated, the ratio and the retention time of the corresponding
atropisomers are given in the analytical data part of the corresponding racemates. In some cases, the atropiso-
mers were separated using chiral HPLC. The HPLC methods used for separation are described in the individual
cases, along with the corresponding analytical data. The atropisomers with the shorter retention time (LCMS)
were defined as atropisomer 1 (atrop1) and those with the longer retention time as atropisomer 2 (atrop2). The
exact configuration of the obtained atropisomers was not determined.
1
Analytical Methods. All NMR spectroscopy results were recorded on Bruker Avance III HD spectrometers. H
spectra were obtained at 400 MHz and referenced to residual solvent signals (2.50 ppm for [D] DMSO). All spec-
6
tra were obtained at ambient temperature (22 +/– 1 °C). Peak forms and multiplicities are specified as apparent in
the spectra; potential higher-order effects have not been considered. Chemical shifts (δ) are reported in parts per
million (ppm), and coupling constants (J) in hertz (Hz). Spin multiplicities are reported as s = singlet, d = doublet, t
=
triplet, q = quartet, m = multiplet, bs = broad singlet and m = centered multiplet. Optical rotations were meas-
c
ured on a P2000 JASCO polarimeter using a 1 mL microcell (10 cm thickness, 3 mm diameter) and the alpha-D-
line of sodium at 20 °C.
Chromatographic Methods. Analytical liquid chromatography–mass spectrometry (LC-MS) methods were per-
formed using UPLCMS on a Waters Acquity UPLCMS SingleQuad instrument (with an Acquity UPLC BEH C18
1
.7 µm, 50 x 2.1 mm column or a Kinetex 2.6 µm 50 x 2.1 mm column) or on an Agilent 1290 UPLCMS 6230
time-of-flight instrument with a BEH C18 1.7 µm, 50 x 2.1 mm column. The different methods are described below.
Analytical methods with the Waters instruments:
-
Method I: column: Acquity UPLC BEH C18 1.7 µm, 50 x 2.1 mm; eluent A: water + 0.1 vol% formic acid
99%), eluent B: acetonitrile (ACN); gradient: 0–1.6 min 1–99% B, 1.6–2.0 min 99% B; flow: 0.8 mL/min;
(
temperature: 60 °C; diode array detector (DAD) scan: 210–400 nm.
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-
-
-
Method II: column: Acquity UPLC BEH C18 1.7 µm, 50 x 2.1 mm; eluent A: water + 0.2 vol% aqueous
ammonia (32%), eluent B: ACN; gradient: 0–1.6 min 1–99% B, 1.6–2.0 min 99% B; flow: 0.8 mL/min;
temperature: 60 °C; DAD scan: 210–400 nm.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Method III: column: BEH C18 1.7 µm 50 x 2.1 mm; eluent A: water + 0.05% formic acid (99%), eluent B:
ACN + 0.05% formic acid (99%); gradient: 0–0.2 min 2% B, 0.2–1.7 min 2–90% B, 1.7–1.9 min 90% B;
1.9–2.0 min 90–2% B, 2.0–2.5 min 2% B, flow: 1.3 mL/min; temperature: 60 °C; DAD scan: 200–400 nm.
Method IV: column: Kinetex 2.6 µm, 50 x 2.1 mm; eluent A: water + 0.05% formic acid (99%), eluent B:
ACN + 0.05% formic acid (99%); gradient: 0–1.9 min 1–99% B, 1.9–2.1 min 99% B; flow: 1.3 mL/min;
temperature: 60 °C; DAD scan: 200–400 nm.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Analytical methods with the Agilent instruments:
-
Method V: eluent A: water + 0.05% formic acid (99%), eluent B: ACN + 0.05% formic acid (99%); gradi-
ent: 0–1.7 min 2–90% B, 1.7–2.0 min 90% B; flow: 1.2 mL/min; temperature: 60 °C; DAD scan: 190–400
nm.
Reagents. All reagents for which the synthesis is not described below are either commercially available or were
synthesized according to literature procedures. All final products were at least 95% pure, as determined by ana-
lytical HPLC, apart from the two cases otherwise indicated.
General Procedure for the Condensation of Naphthalic Anhydrides under Acidic Conditions (GP1).
In a pressure vessel, or a round-bottom flask, the aniline (1.0 equiv) was suspended in acetic acid (3.8 mL/mmol)
under argon atmosphere. Then, the commercially available anhydride partner (0.9–1.5 equiv) was added. The
reaction mixture was heated to reflux for 3–16 h. After cooling, the mixture was concentrated under reduced
pressure. The residue was suspended in methanol (10 mL/mmol) and 4 N sodium hydroxide solution (177
µL/mmol) was added. After stirring at room temperature (RT) for 1 h, water (74 mL/mmol) was added and the
mixture was further stirred for 10 min. The precipitate was separated by filtration, washed three times with water
and methanol, and dried overnight in a vacuum oven at 40 °C. If necessary, the precipitate was purified by HPLC
or flash column chromatography to give the desired product.
General Procedure for the Nucleophilic Substitution of Amines (GP2). In a pressure vessel, the aryl bromide (1.0
equiv) was dissolved in DMSO (7.5 mL/mmol) under argon atmosphere. The commercially available amine part-
ner (1.1 equiv) and potassium carbonate (2.5 equiv) were added and the mixture was heated to 110 °C overnight.
After cooling, the precipitate was separated by filtration and purified by HPLC.
General Procedure for the SONOGASHIRA Coupling (GP3). In a pressure vessel, the aryl bromide (1.0 equiv) was
dissolved
in
tetrahydrofuran
(0.4
mL/mmol).
The
commercially
available
catalyst
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Journal of Medicinal Chemistry
bis(triphenylphosphine)palladium(II) dichloride (0.02 equiv), copper(I) iodide (0.02 equiv), triethylamine (7.4 equiv)
and the coupling partner (2.0 equiv) were added to the solution and the mixture was degassed with argon for 5
min. The mixture was heated to 65 °C for 5–16 h. After cooling, the mixture was diluted with aqueous ammonia (6
mL/mmol) and was stirred again for 5 min. Dichloromethane (60 mL/mmol) and water (15 mL/mmol) were added
and the layers were separated. The aqueous layer was extracted with dichloromethane (2 x 40 mL/mmol). The
combined organic layers were washed with brine (60 mL/mmol) and dried over sodium sulfate. After filtration, the
solvent was removed under reduced pressure. The residue was purified by HPLC to give the desired product. In
some cases, the crude product was directly used in the next step without any further purification.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General Procedure for Hydrogenation with Palladium on Carbon (GP4). In a hydrogenation flask, the alkyne (1.0
equiv) was dissolved in ethyl acetate (450–1000 mL/mmol) and Pd/C (0.02 equiv) was added. The suspension
was flushed with argon and vigorously shaken under a 1 bar hydrogen atmosphere for 2–4 h. After reaction com-
pletion, the suspension was flushed with argon again, the catalyst was removed by filtration through a pad of
Celite and the solvent was removed under reduced pressure. The residue was purified by HPLC or flash column
chromatography to give the desired product.
6
1
-(3-Hydroxypropyl)-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-
,3(2H)-dione (5). According to GP3, aryl bromide 12 (125 mg, 0.278 mmol), propargyl alcohol (32 µL, 0.555
mmol), triethylamine (288 µL, 2.07 mmol), CuI (1.1 mg, 0.006 mmol) and bis(triphenylphosphine)palladium(II
)
dichloride (3.9 mg, 0.006 mmol) in tetrahydrofuran (1.2 mL) gave the corresponding alkyne which, after purifica-
tion, was obtained as a yellow solid (83 mg, 67%). According to GP4, the alkyne (72 mg, 0.169 mmol) and Pd/C
(
36 mg, 0.034 mmol) in EtOAc (56 mL) gave 5 as a colourless solid (49 mg, 64%). 1H NMR (400 MHz,
[
D]6DMSO): δ = 1.88 (m , 2 H), 2.08, 3.29 (2 s, 3 H each), 3.30 (m , 2 H), 3.38 (s, 3 H), 3.54 (m , 2 H), 4.67 (t, J =
c
c
c
5
8
.2 Hz, 1 H), 7.17 (m , 2 H), 7.78 (d, J = 7.6 Hz, 1 H), 7.94 (dd, J = 7.4, 8.6 Hz, 1 H), 8.46 (d, J = 7.6 Hz, 1 H),
c
t
.55 (dd, J = 1.0, 7.4 Hz, 1 H), 8.69 (dd, J = 1.0, 8.4 Hz, 1 H) ppm; LCMS (method V): R = 0.87 min; m/z:
t
t
[
M + H]+ = 430; atropisomeric ratio: atrop 1/atrop 2 = 1:1; R (atrop1) = 2.64 min; R (atrop2) = 3.44 min. The at-
ropisomeric ratio was determined using the following chiral HPLC method: instrument: Agilent HPLC 1260; col-
umn: Chiralpak IA 3 µm 100 x 4.6 mm; eluent A: 2-methoxy-2-methylpropane + 0.1 vol% diethylamine (99%);
eluent B: ACN; isocratic: 50% A + 50% B; flow: 1.4 mL/min; temperature: 25 °C; DAD: 254 nm.
The atropisomers were separated using the following chiral HPLC method: instrument: Labomatic HD5000,
Labocord-5000; Gilson GX-241, Labcol Vario 4000, column: Chiralpak IA 5 µm 250 x 30 mm; eluent A: methanol
+
0.1 vol% diethylamine (99%); eluent B: 2-methoxy-2-methylpropane + 0.1% diethylamine (99%); isocratic: 50%
A + 50% B; flow: 50.0 mL/min; UV 254 nm.
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Journal of Medicinal Chemistry
Page 12 of 51
(
–)-6-(3-Hydroxypropyl)-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
20
1
1
,3(2H)-dione (5a). [α] = –1.8 (c = 1.00, DMSO); H NMR (400 MHz, [D] DMSO): δ = 1.88 (m , 2 H), 2.08, 3.29
D
6
c
(
2 s, 3 H each), 3.30 (m , 2 H), 3.38 (s, 3 H), 3.54 (m , 2 H), 4.67 (t, J = 5.2 Hz, 1 H), 7.17 (m , 2 H), 7.78 (d, J =
c c c
7
1
.6 Hz, 1 H), 7.94 (dd, J = 7.4, 8.6 Hz, 1 H), 8.46 (d, J = 7.6 Hz, 1 H), 8.55 (dd, J = 1.0, 7.4 Hz, 1 H), 8.69 (dd, J =
t
+
.0, 8.4 Hz 1 H) ppm; LCMS (method V): R = 0.87 min; m/z: [M + H] = 430.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
+)-6-(3-Hydroxypropyl)-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-
20
1
1
,3(2H)-dione (5b). [α]
= +4.8 (c = 1.00, DMSO); H NMR (400 MHz, [D] DMSO): δ = 1.88 (m , 2 H), 2.08, 3.29
D
6
c
(
2 s, 3 H each), 3.30 (m , 2 H), 3.38 (s, 3 H), 3.54 (m , 2 H), 4.67 (t, J = 5.2 Hz, 1 H), 7.17 (m , 2 H), 7.78 (d, J =
c c c
7
1
2
.6 Hz, 1 H), 7.94 (dd, J = 7.4, 8.6 Hz, 1 H), 8.46 (d, J = 7.6 Hz, 1 H), 8.55 (dd, J = 1.0, 7.4 Hz, 1 H), 8.69 (dd, J =
t
+
.0, 8.4 Hz 1 H) ppm; LCMS (method V): R = 0.87 min; m/z: [M + H] = 430.
-(1,3,6-Trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (7). Accord-
ing to GP1, 30 (150 mg, 0.784 mmol) and 1,8-naphthalic anhydride (233 mg, 1.18 mmol) in acetic acid (3 mL)
1
gave 7 as a colorless solid (40 mg, 13%). H NMR (400 MHz, [D] DMSO): δ = 2.09, 3.29, 3.38 (3 s, 3 H each),
6
t
7
.18 (m , 2 H), 7.93 (dd, J = 7.8 Hz, 2 H), 8.53–8.56 (m, 4 H) ppm; LCMS (method III): R = 1.04 min; m/z:
c
+
[
M + H] = 372. Compound 7 was assumed to be obtained as a racemic mixture of atropisomers. The atropiso-
meric ratio was not investigated at this stage.
-(6-Methoxy-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (8).
2
According to GP1, 5-amino-6-methoxy-1,3-dimethyl-1,3-dihydro-2H-benzimidazol-2-one (159 mg, 0.769 mmol)
and 1,8-naphthalic anhydride (229 mg, 1.15 mmol) in acetic acid (3 mL) gave 8 as a colorless solid (4.2 mg, 1%).
1
H NMR (400 MHz, [D] DMSO): δ = 3.29, 3.41, 3.72 (3 s, 3 H each), 7.13, 7.21 (2 m , 1 H each), 7.92 (t, J ≈ 7.8
6
c
t
+
Hz, 2 H), 8.53 (t, J ≈ 8.4 Hz, 4 H) ppm; LCMS (method III): R = 0.98 min; m/z: [M + H] = 388. Compound 8 was
assumed to be obtained as a racemic mixture of atropisomers. The atropisomeric ratio was not investigated at
this stage.
2-[6-(Dimethylamino)-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl]-1H-benzo[de]isoquinoline-1,3(2H)-
dione (9). According to GP1, 5-amino-6-(dimethylamino)-1,3-dimethyl-1,3-dihydro-2H-benzimidazol-2-one dihy-
drochloride (244 mg, 0.833 mmol) and 1,8-naphthalic anhydride (248 mg, 1.15 mmol) in acetic acid (3 mL) gave 9
1
as a colorless solid (20 mg, 6%). H NMR (400 MHz, [D] DMSO): δ = 2.47, 3.29, 3.40 (3 s, 6 H, 3 H, 3 H), 7.18,
6
t
7.26 (2 m , 1 H each), 7.92 (t, J ≈ 7.7 Hz, 2 H), 8.53, 8.55 (2 m , 2 H each) ppm; LCMS (method V): R = 0.78
c
c
+
min; m/z: [M + H] = 401. Compound 9 was assumed to be obtained as a racemic mixture of atropisomers. The
atropisomeric ratio was not investigated at this stage.
12
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Page 13 of 51
Journal of Medicinal Chemistry
2
-(6-Bromo-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (10).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
According to GP1, 5-amino-6-bromo-1,3-dimethyl-1,3-dihydro-2H-benzimidazol-2-one (150 mg, 0.586 mmol) and
1
1
,8-naphthalic anhydride (174 mg, 0.879 mmol) in acetic acid (3 mL) gave 10 as a colorless solid (9 mg, 3%). H
NMR (400 MHz, [D] DMSO): δ = 3.32, 3.40 (2 s, 3 H each), 7.46, 7.66 (2 m , 1 H each), 7.96 (t, J ≈ 7.8 Hz, 2 H),
6
c
t
+
+
8
.55–8.58 (m, 4 H) ppm; LCMS (method V): R = 1.03 min; m/z: [M + H] = 436, [(M + 2) + H] = 438 (Br isotopic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
pattern). Compound 10 was assumed to be obtained as a racemic mixture of atropisomers. The atropisomeric
ratio was not investigated at this stage.
2
-[1,3-Dimethyl-6-(4-methylpiperazin-1-yl)-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl]-1H-benzo[de]isoquinoline-
1,3(2H)-dione (11). Commercially available 5-chloro-1,3-dimethyl-6-nitro-1,3-dihydro-2H-benzimidazol-2-one (250
mg, 1.03 mmol) was suspended under an atmosphere of argon in N,N-dimethylformamide (10 mL), and sodium
carbonate (286 mg, 2.07 mmol) and 1-methylpiperazine (170 µL, 1.6 mmol) were added. The reaction mixture
was heated to 120 °C overnight. After cooling, the reaction mixture was filtered and the solvent was removed
under reduced pressure. The obtained residue was then suspended in ethanol (19.2 mL) and Pd/C (57.9 mg,
0.054 mmol) was added under argon. The suspension was flushed with argon and vigorously shaken under an
atmosphere of hydrogen (1 bar) for 4 h. After reaction completion, the suspension was flushed with argon again,
the catalyst was removed by filtration through a pad of Celite and the solvent was removed under reduced pres-
t
sure. The crude product was directly used in the next step without any further purification. LCMS (method I): R =
+
0
.48 min; m/z: [M + H] = 275. According to GP1, crude product (200 mg, 0.726 mmol) and 1,8-naphthalic anhy-
1
dride (144 mg, 0.269 mmol) in acetic acid (2.6 mL) gave 11 was obtained as a colorless solid (20 mg, 6%). H
NMR (400 MHz, [D] DMSO): δ = 1.87 (s, 3 H), 2.72 (m , 4 H), 3.30, 3.39 (2 s, 3 H each), 7.22, 7.27 (2 m , 1 H
6
c
c
each), 7.93 (t, J ≈ 7.7 Hz, 2 H), 8.53, 8.55 (2 m , 2 H each) ppm; four proton signals could not be detected; LCMS
c
t
+
(method V): R = 0.61 min; m/z: [M + H] = 456. Compound 11 was assumed to be a racemic mixture of atro-
pisomers. The atropisomeric ratio was not investigated at this stage.
6
-Bromo-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(
12). According to GP1, 30 (60 mg, 0.314 mmol) and 4-bromo-1,8-naphthalic anhydride (96 mg, 0.345 mmol) in
1
acetic acid (1.2 mL) gave 12 as a yellow solid (90 mg, 61%). H NMR (400 MHz, [D] DMSO): δ = 2.08, 3.28, 3.37
6
(
(
3 s, 3 H each), 7.17* (m , 2 H), 8.06 (dd, J = 7.4, 8.1 Hz, 1 H), 8.29, 8.37 (2 d, J = 7.9 Hz, 1 H each), 8.63–8.67
c
t
+
m, 2 H) ppm; *overlapping signals; LCMS (method V): R = 1.12 min; m/z: [M + H] = 450, [(M+2)+H]+ = 452 (Br
isotopic pattern). Compound 12 was assumed to be obtained as a racemic mixture of atropisomers. The atro-
pisomeric ratio was not investigated at this stage.
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 51
6
-Chloro-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
13). According to GP1, 30 (60 mg, 0.314 mmol) and 4-chloro-1,8-naphthalic anhydride (80 mg, 0.345 mmol) in
1
acetic acid (1.2 mL) gave 13 as a yellow solid (57 mg, 43%). H NMR (400 MHz, [D] DMSO): δ = 2.09, 3.28, 3.38
6
(
3 s, 3 H), 7.18* (m , 2 H), 8.07 (dd, J = 7.4, 8.4 Hz, 1 H), 8.11, 8.48 (2 d, J = 8.0 Hz, 1 H each), 8.63 (dd, J = 7.0
c
t
+
Hz, 1 H), 8.71 (dd, J = 8.6 Hz, 1 H) ppm; *overlapping signals; LCMS (method V): R = 1.16 min; m/z: [M + H] =
06. Compound 13 was assumed to be obtained as a racemic mixture of atropisomers. The atropisomeric ratio
was not investigated at this stage.
-Nitro-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (14).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5
According to GP1, 30 (60 mg, 0.314 mmol) and 3-nitro-1,8-naphthalic anhydride (84 mg, 0.345 mmol) in acetic
1
acid (1.2 mL) gave 14 as a yellow solid (113 mg, 78%). H NMR (400 MHz, [D] DMSO): δ = 2.11, 3.29, 3.38 (3 s,
6
3
=
4
H each), 7.19* (m , 2 H), 8.12 (t, J ≈ 7.7 Hz, 1 H), 8.72 (dd, J = 7.4 Hz, 1 H), 8.87 (dd, J = 8.4 H, 1 H), 9.00 (d, J
c
t
+
2.3 Hz), 9.57 (d, J = 2.0 Hz, 1 H) ppm; *overlapping signals; LCMS (method V): R = 1.02 min; m/z: [M + H] =
17. Compound 14 was assumed to be obtained as a racemic mixture of atropisomers. The atropisomeric ratio
was not investigated at this stage.
5-Amino-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinolone-1,3(2H)-dione
(15). Compound 14 (78 mg, 0.187 mmol) was suspended in methanol (110 mL) and Pd/C (40 mg, 0.037 mmol)
was added. The suspension was flushed with argon and vigorously shaken under an atmosphere of hydrogen (1
bar) for 3 h. After reaction completion, the suspension was flushed with argon again, the catalyst was removed by
filtration through a pad of Celite and the solvent was removed carefully under reduced pressure. The residue (23
mg) was purified by HPLC (basic) and lyophilized to give the desired aniline 15 as a yellow solid (10 mg, 12%)
1
which was stored under argon in the fridge. H NMR (400 MHz, [D] DMSO): δ = 2.06, 3.29, 3.37 (3 s, 3 H each),
6
6.05 (bs, 2 H), 7.16* (m , 2 H), 7.35 (d, J = 2.3 Hz, 1 H), 7.66 (dd, J = 7.4, 8.1 Hz, 1 H), 8.00 (d, J = 2.3 Hz, 1 H),
c
t
+
8.09 (m , 2 H) ppm; *overlapping signals; LCMS (method V): R = 0.86 min; m/z: [M + H] = 387. Compound 15
c
was assumed to be obtained as a racemic mixture of atropisomers. The atropisomeric ratio was not investigated
at this stage.
5-Hydroxy-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(
16). According to GP1, 30 (60 mg, 0.314 mmol) and 3-hydroxy-1,8-naphthalic anhydride (74 mg, 0.345 mmol) in
1
acetic acid (1.2 mL) gave 16 as a bright yellow solid (80 mg, 60%). H NMR (400 MHz, [D] DMSO): δ = 2.07,
6
3.29, 3.27 (3 s, 3 H each), 7.16*, 7.17* (2 m , 1 H each), 7.72 (d, J = 2.5 Hz, 1 H), 7.79 (dd, J = 8.1, 8.4 Hz, 1 H),
c
t
8.06 (d, J = 2.5 Hz, 1 H), 8.27–8.32 (m, 2 H), 10.63 (bs, 1 H) ppm; *overlapping signals; LCMS (method IV): R =
14
ACS Paragon Plus Environment

Page 15 of 51
.85 min; m/z: [M + H] = 388. Compound 16 was assumed to be obtained as a racemic mixture of atropisomers.
The atropisomeric ratio was not investigated at this stage.
-Bromo-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
Journal of Medicinal Chemistry
+
0
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
(
17). According to GP1, 30 (100 mg, 0.522 mmol) and 3-bromo-1,8-naphthalic anhydride (159 mg, 0.575 mmol) in
1
acetic acid (2 mL) gave 16 as a bright yellow solid (110 mg, 44%). H NMR (400 MHz, [D] DMSO): δ = 2.08, 3.27,
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
.37 (3 s, 3 H each), 7.16, 7.17 (2 m , 1 H each), 7.95 (t, J ≈ 7.6 Hz, 1 H), 8.47–8.54 (m, 3 H), 8.84 (m , 1 H)
c c
t
+
+
ppm; LCMS (method V): R = 1.17 min; m/z: [M + H] = 450, [(M+2)+H] = 452 (Br isotopic pattern). Compound 17
was assumed to be obtained as a racemic mixture of atropisomers. The atropisomeric ratio was not investigated
at this stage.
1,3-Dioxo-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2,3-dihydro-1H-benzo[de]isoquinoline-5-
carbonitrile (18). According to GP1, 30 (58 mg, 0.305 mmol) and 3-cyano-1,8-naphthalic anhydride (75 mg, 0.336
1
mmol) in acetic acid (1.2 mL) gave 18 as a yellow solid (30 mg, 22%). H NMR (400 MHz, [D] DMSO): δ = 2.09,
6
3
.28, 3.38 (3 s, 3 H each), 7.18* (m , 2 H), 8.07 (t, J ≈ 7.7 Hz, 1 H), 8.62 (d, J ≈ 8.4 Hz, 1 H), 8.68* (d, J ≈ 7.4 Hz,
c
t
+
1 H), 8.70*, 9.18 (2 m , 1 H each) ppm; *overlapping signals; LCMS (method V): R = 0.95 min; m/z: [M + H] =
c
397. Compound 18 was assumed to be obtained as a racemic mixture of atropisomers. The atropisomeric ratio
was not investigated at this stage.
6-(Morpholin-4-yl)-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione (19). According to GP2, aryl bromide 12 (150 mg, 0.333 mmol), morpholine (32 µL, 0.366 mmol) and po-
1
tassium carbonate (115 mg, 0.833 mmol) in DMSO (2 mL) gave 19 as a yellow solid (58 mg, 34%). H NMR (400
MHz, [D] DMSO): δ = 2.06 (s, 3 H), 3.26 (m , 4 H), 3.29, 3.37 (2 s, 3 H each), 3.94 (t, J ≈ 5.0 Hz, 4 H), 7.14, 7.17
6
c
(2 s, 1 H each), 7.41 (d, J = 8.1 Hz, 1 H), 7.87 (dd, J = 7.4, 8.3 Hz, 1 H), 8.45 (d, J ≈ 8.5 Hz, 1 H), 8.52 (dd, J =
t
+
1.1, 7.2 Hz, 1 H), 8.58 (dd, J = 1.1, 8.4 Hz, 1 H) ppm; LCMS (method III): R = 1.12 min; m/z: [M + H] = 457; at-
t
t
ropisomeric ratio: atrop 1/atrop 2 = 50:50; R (atrop1) = 2.74 min; R (atrop2) = 3.28 min. The atropisomeric ratio
was determined using the following chiral HPLC method: instrument: Agilent HPLC 1260; column: Chiralpak IB 3
µm 100 x 4.6 mm; eluent A: 2-methoxy-2-methylpropane + 0.1 vol% diethylamine (99%); Eluent B: methanol;
isocratic: 50% A + 50% B; flow: 1.4 mL/min; temperature: 25 °C; DAD: 254 nm.
6
1
-(4-Hydroxypiperidin-1-yl)-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-
,3(2H)-dione (20). According to GP2, aryl bromide 12 (150 mg, 0.333 mmol), 4-hydroxypiperidine (37 mg, 0.366
mmol) and potassium carbonate (115 mg, 0.833 mmol) in DMSO (2 mL) gave 20 as a yellow solid (48 mg, 28%).
1
H NMR (400 MHz, [D] DMSO): δ = 1.77, 2.02 (2 m , 2 H each), 2.06 (s, 3 H), 3.07 (m , 2 H), 3.29, 3.37 (2 s, 3 H
6
c
c
each), 3.49, 3.79 (2 m , 2 H, 1 H), 4.94 (bs, 1 H), 7.13, 7.17 (2 m , 1 H each), 7.37 (d, J = 8.3 Hz, 1 H), 7.86 (dd, J
c
c
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 51
t
+
=
4
7.5, 8.2 Hz, 1 H), 8.4 (d, J = 8.0 Hz, 1 H), 8.49 (m , 2 H) ppm; LCMS (method V): R = 0.90 min; m/z: [M + H] =
c
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
t
t
71; atropisomeric ratio: atrop 1/atrop 2 = 47:53; R (atrop1) = 4.86 min; R (atrop2) = 7.23 min. The atropisomeric
ratio was determined using the following chiral HPLC method: instrument: Agilent HPLC 1260; column: Chiralpak
IA 3 µm 100 x 4.6 mm; eluent A: methanol + 0.1 vol% diethylamine (99%); eluent B: ethanol; isocratic: 50% A +
50% B; flow: 1.4 mL/min; temperature: 25 °C; DAD: 254 nm.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6-(4-Methylpiperazin-1-yl)-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-
1,3(2H)-dione (21). According to GP2, aryl bromide 12 (150 mg, 0.333 mmol), 1-methylpiperazine (41 µL, 0.366
mmol) and potassium carbonate (115 mg, 0.833 mmol) in DMSO (2 mL) gave 21 as a yellow solid (39 mg, 22%).
1
H NMR (400 MHz, [D] DMSO): δ = 2.06, 2.32 (2 s, 3 H each), 2.67, 3.29 (2 m , 4 H, 7 H), 3.37 (s, 3 H), 7.14,
6
c
7.17 (2 m , 1 H each), 7.39 (d, J = 8.4 Hz, 1 H), 7.86 (dd, J = 7.5, 8. 3 Hz, 1 H), 8.43 (d, J = 8.1 Hz, 1 H), 8.50,
c
t
+
8.52 (2 m , 1 H each) ppm; LCMS (method V): R = 0.62 min; m/z: [M + H] = 470; atropisomeric ratio:
c
t
t
atrop1/atrop2 = 41:42 (17% impurities); R (atrop1) = 2.72 min; R (atrop2) = 4.11 min. The atropisomeric ratio
was determined using the following chiral HPLC method: instrument: Agilent HPLC 1260; column: Chiralpak IB 3
µm 100 x 4.6 mm; eluent A: 2-methoxy-2-methylpropane + 0.1 vol% diethylamine (99%); eluent B: methanol;
isocratic: 50% A + 50% B; flow: 1.4 mL/min; temperature: 25 °C; DAD: 254 nm.
6-[4-(Cyclopropylmethyl)piperazin-1-yl]-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-
benzo[de]isoquinoline-1,3(2H)-dione (22). According to GP2, aryl bromide 12 (100 mg, 0.222 mmol), 1-
(cyclopropylmethyl)piperazine (36 µL, 0.244 mmol) and potassium carbonate (77 mg, 0.555 mmol) in DMSO (2
1
mL) gave 22 as a yellow solid (16 mg, 14%). H NMR (400 MHz, [D] DMSO): δ = 0.15, 0.52, 0.91 (3 m , 2 H, 2 H,
6
c
1 H), 2.06 (s, 3 H), 2.33 (m , 2 H), 2.80 (m , 4 H), 3.29 (s, 3 H), 3.30 (m , 4 H), 3.37 (s, 3 H), 7.14, 7.17 (2 m , 1 H
c
c
c
c
each), 7.39 (d, J = 8.1 Hz, 1 H), 7.86 (dd, J = 7.4, 8.4 Hz, 1 H), 8.43 (d, J = 8.2 Hz, 1 H), 8.50–8.53 (m, 2 H) ppm;
t
+
t
LCMS (method V): R = 0.70 min; m/z: [M + H] = 510; atropisomeric ratio: atrop1/atrop2 = 50:50; R (atrop1) =
t
2
.59 min; R (atrop2) = 3.83 min. The atropisomeric ratio was determined using the following chiral HPLC method:
instrument: Agilent HPLC 1260; column: Chiralpak IB 3 µm 100 x 4.6 mm; eluent A: 2-methoxy-2-methylpropane
0.1 vol% diethylamine (99%); eluent B: Methanol; isocratic: 50% A + 50% B; flow: 1.4 mL/min; temperature: 25
C; DAD: 254 nm.
+
°
6
-(4-Methoxypiperidin-1-yl)-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-
1
,3(2H)-dione (23). According to GP2, aryl bromide 12 (100 mg, 0.222 mmol), 4-methoxypiperidine (29 µL, 0.244
mmol) and potassium carbonate (77 mg, 0.555 mmol) in DMSO (4 mL) gave 23 as a yellow solid (30 mg, 26%).
1
H NMR (400 MHz, [D] DMSO): δ = 1.82 (m , 2 H), 2.06 (s, 3 H), 2.13, 3.09 (2 m , 2 H each), 3.28, 3.37 (2 s, 3 H
6
c
c
each), 3.48 (m , 3 H), 7.13, 7.16 (2 m , 1 H each), 7.38 (d, J = 8.1 Hz, 1 H), 7.85 (t, J = 7.7 Hz, 1 H), 8.41 (d, J =
c
c
16
ACS Paragon Plus Environment

Page 17 of 51
Journal of Medicinal Chemistry
t
+
8
.4 Hz, 1 H), 8.50 (m , 2 H) ppm; LCMS (method III): R = 1.25 min; m/z: [M + H] = 485; atropisomeric ratio:
c
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
t
t
atrop1/atrop2 = 47:47 (6% impurities); R (atrop1) = 2.70 min; R (atrop2) = 3.17 min. The atropisomeric ratio was
determined using the following chiral HPLC method: instrument: Agilent HPLC 1260; column: Chiralpak IB 3 µm
1
00 x 4.6 mm; eluent A: 2-methoxy-2-methylpropane + 0.1 vol% diethylamine (99%); eluent B: methanol; isocrat-
ic: 50% A + 50% B; flow: 1.4 mL/min; temperature: 25 °C; DAD: 254 nm.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6-(Piperidin-1-yl)-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione (24). aryl bromide 12 (100 mg, 0.222 mmol), piperidine (29 µL, 0.244 mmol) and potassium carbonate (77
1
mg, 0.555 mmol) in DMSO (4 mL) gave 24 as a yellow solid (25 mg, 24%). H NMR (400 MHz, [D] DMSO): δ =
6
1
.68, 1.85 (2 m , 2 H, 4 H), 2.06 (s, 3 H), 3.24 (m , 4 H), 3.28, 3.37 (2 s, 3 H), 7.13, 7.16 (2 m , 1 H each), 7.36 (2
c c c
s, 1 H each), 7.85 (dd, J = 7.4, 8.4 Hz, 1 H), 8.41 (d, J = 8.1 Hz, 1 H), 8.49 (m , 2 H each) ppm; LCMS (method
c
t
+
t
t
III): R = 1.42 min; m/z: [M + H] = 455; atropisomeric ratio: atrop1/atrop2 = 50:50; R (atrop1) = 3.60 min; R
(atrop2) = 4.59 min. The atropisomeric ratio was determined using the following chiral HPLC method: instrument:
Agilent HPLC 1260; column: Chiralpak ID 3 µm 100 x 4.6 mm; eluent A: 2-methoxy-2-methylpropane + 0.1 vol%
diethylamine (99%); eluent B: ACN; isocratic: 50% A + 50% B; flow: 1.4 mL/min; temperature: 25 °C; DAD: 254
nm.
6-(Pyrrolidin-1-yl)-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione (25). According to GP2, aryl bromide 12 (50 mg, 0.111 mmol), pyrrolidine (10 µL, 0.122 mmol) and potassi-
1
um carbonate (38 mg, 0.278 mmol) in DMSO (1 mL) gave 25 as a yellow solid (15 mg, 29%). H NMR (400 MHz,
[D] DMSO): δ = 2.04 (m , 7 H), 3.29, 3.37 (2 s, 3 H each), 3.81 (m , 4 H), 6.94 (d, J = 8.9 Hz, 1 H), 7.09, 7.15 (2
6
c
c
m , 1 H each), 7.66 (dd, J = 7.4, 8.6 Hz, 1 H), 8.27 (d, J = 8.6 Hz, 1 H), 8.46 (dd, J = 0.9, 7.2 Hz, 1 H), 8.81 (dd, J
c
t
+
= 1.0, 8.6 Hz, 1 H) ppm; LCMS (method V): R = 1.11 min; m/z: [M + H] = 441; atropisomeric ratio: atrop 1/atrop
t
t
2 = 50:50; R (atrop1) = 2.01 min; R (atrop2) = 2.25 min.
6
-(Propylamino)-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione (26). In a pressure vessel, aryl bromide 12 (100 mg, 0.222 mmol, 1.0 eq) was suspended in toluene (2 mL)
under argon atmosphere. The commercially available catalyst tris(dibenzylideneacetone)dipalladium (8.1 mg,
0
0
.009 mmol, 0.040 equiv), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (Xantphos, 5.1 mg, 0.009 mmol,
.040 equiv), cesium carbonate (218 mg, 0.671 mmol, 3.0 equiv) and propylamine (56 µL, 0.671 mmol, 2.1 equiv)
were added and the mixture was degassed with argon for 5 min. The reaction mixture was heated to 90 °C for 18
h. After cooling, the solvent was removed under reduced pressure. The residue was purified by HPLC giving
1
product 26 as a green solid (10 mg, 10%). H NMR (400 MHz, [D] DMSO): δ = 1.00 (t, J = 7.4 Hz, 3 H), 1.74 (m ,
6
c
2
H), 2.04, 3.29, 3.37* (3 s, 3 H each), 3.14–3.37* (m, 2 H), 6.83 (d, J = 8.9 Hz, 1 H), 7.09, 7.15 (2 m , 1 H each),
c
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7
.72 (t, J = 7.9 Hz, 1 H), 7.89 (m , 1 H), 8.28 (d, J = 8.6 Hz, 1 H), 8.45 (d, J = 6.8 Hz, 1 H), 8.79 (d, J = 8.1 Hz, 1
c
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
t
+
H) ppm; *overlapping signals; LCMS (method III): R = 1.18 min; m/z: [M + H] = 429. Compound 26 was as-
sumed to be obtained as a racemic mixture of atropisomers. The atropisomeric ratio was not investigated at this
stage.
6
-{4-
[
2-(Dimethylamino)ethyl]piperazin-1-yl}-2-(1,3,6,-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
benzo
[
de isoquinoline-1,3(2H)-dione (27). According to GP2, aryl bromide 12 (300 mg, 0.666 mmol), 1-(2-
]
dimethylaminoethyl)piperazine (129 mL, 0.733 mmol) and potassium carbonate (230 mg, 1.66 mmol) in DMSO (5
1
mL) gave 27 as a yellow solid (25 mg, 7%). H NMR (400 MHz, [D] DMSO): δ = 2.06, 2.18 (2 s, 3 H, 6 H), 2.43,
6
2
.52, 2.76, 3.28 (4 m , 2 H, 2 H, 4 H, 7 H), 3.37 (s, 3 H), 7.13, 7.17 (2 s, 1 H each), 7.38 (d, J = 8.4 Hz, 1 H), 7.85
c
t
(
dd, J = 7.4, 8.2 Hz, 1 H), 8.42 (d, J = 8.1 Hz, 1 H), 8.50, 8.52 (2 m , 1 H each) ppm; LCMS (method III): R = 0.76
c
+
t
t
min; m/z: [M + H] = 527; atropisomeric ratio: atrop1/atrop2 = 50:50; R (atrop1) = 2.69 min; R (atrop2) = 4.13
min. The atropisomeric ratio was determined using the following chiral HPLC method: instrument: Agilent HPLC
1
260; column: Chiralpak IB 3 µm 100 x 4.6 mm; eluent A: 2-methoxy-2-methylpropane + 0.1 vol% diethylamine
99%); eluent B: methanol; isocratic: 50% A + 50% B; flow: 1.4 mL/min; temperature: 25 °C; DAD: 254 nm.
-{Methyl[2-(pyrrolidin-1-yl)ethyl]amino}-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-
benzo[de]isoquinoline-1,3(2H)-dione (28). According to GP2, aryl bromide 12 (50 mg, 0.111 mmol), methyl[2-
(
6
(
pyrrolidin-1-yl)ethyl]amine (17 µL, 0.122 mmol) and potassium carbonate (39 mg, 2.278 mmol) in DMSO (1 mL)
1
gave 27 as a yellow solid (18 mg, 29%). H NMR (400 MHz, [D] DMSO): δ = 1.63 (m , 4 H), 2.06 (s, 3 H), 2.44,
6
c
2
.82 (2 m , 4 H, 2 H), 3.10, 3.29, 3.37 (3 s, 3 H each), 3.51 (m , 2 H), 7.13, 7.16 (2 m , 1 H each), 7.34 (d, J = 8.4
c c c
Hz, 1 H), 7.80 (dd, J = 7.4, 8.6 Hz, 1 H), 8.38 (d, J = 8.4 Hz, 1 H), 8.49 (dd, J = 1.0, 7.4 Hz, 1 H), 8.69 (dd, J =
t
+
1
.0, 8.6 Hz, 1 H) ppm; LCMS (method III): R = 0.78 min; m/z: [M + H] = 498; atropisomeric ratio: atrop1/atrop2 =
t
t
5
0:50; R (atrop1) = 2.30 min; R (atrop2) = 3.14 min. The atropisomeric ratio was determined using the following
chiral HPLC method: instrument: Waters Acquity UPLCMS SingleQuad; column: Acquity UPLC BEH C18 1.7 µm,
50 x 2.1 mm; eluent A: water + 0.1 vol% formic acid (99%), eluent B: ACN; gradient: 0–1.6 min 1–99% B, 1.6–2.0
min 99% B; flow: 0.8 mL/min; temperature: 60 °C; DAD: 210–400 nm.
6-(4-Hydroxybutyl)-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione (29). According to GP3, aryl bromide 12 (150 mg, 0.333 mmol), but-3-yn-1-ol (50 µL, 0.666 mmol), triethyl-
amine (345 µL, 2.48 mmol), CuI (1.3 mg, 0.007 mmol) and bis(triphenylphosphine)palladium(II) dichloride (4.7
mg, 0.007 mmol) in tetrahydrofuran (1.5 mL) gave after workup, the violet residue (150 mg, 0.341 mmol) which
was directly used in the next step according to GP4 with Pd/C (145 mg, 0.034 mmol) in EtOAc (112 mL). 29 was
1
obtained as a yellow solid (48 mg, 30%). H NMR (400 MHz, [D] DMSO): δ = 1.57, 1.76 (2 m , 2 H each), 2.08 (s,
6
c
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Journal of Medicinal Chemistry
3
=
=
2
H), 3.26 (m , 2 H), 3.29, 3.38 (2 s, 3 H each), 3.46 (m , 2 H), 4.44 (t, J = 5.2 Hz, 1 H), 7.17 (m , 2 H), 7.78 (d, J
c
c
c
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7.6 Hz, 1 H), 7.94 (dd, J = 7.2, 8.5 Hz, 1 H), 8.46 (d, J = 7.4 Hz, 1 H), 8.54 (dd, J = 1.0, 7.4 Hz, 1 H), 8.69 (dd, J
t
+
0.9, 8.5 Hz, 1 H) ppm; LCMS (method V): R = 0.93 min; m/z: [M + H] = 444; atropisomeric ratio: atrop 1/atrop
t
t
= 45:47 (8% impurities); R (atrop1) = 2.90 min; R (atrop2) = 3.33 min.
The atropisomeric ratio was determined using the following chiral HPLC method: instrument: Agilent HPLC 1260;
column: Chiralpak IE 3 µm 100 x 4.6 mm; eluent A: ACN + 0.1 vol% diethylamine (99%); eluent B: methanol;
isocratic: 50% A+50% B; flow: 1.4 mL/min; temperature: 25 °C; DAD 325 nm.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6-(3-Hydroxypropyl)-2-(1-methyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione
(31).
According
to
GP1,
6-bromo-2-(1-methyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-
benzo[de]isoquinoline-1,3(2H)-dione was prepared from 5-amino-1-methyl-1,3-dihydro-2H-benzimidazol-2-one
(3.34 g, 20.5 mmol) and 4-bromo-1,8-naphthalic anhydride (5.96 g, 21.5 mmol) in acetic acid (82 mL). The corre-
sponding aryl bromide was obtained as a yellow solid (6.22 g), part of which (500 mg, 1.18 mmol) was used in the
next step, according to GP3, with propargyl alcohol (138 µL, 2.39 mmol), triethylamine (1.3 mL, 8.81 mmol), CuI
(
(
4.5 mg, 0.024 mmol) and bis(triphenylphosphine)palladium(II) dichloride (17 mg, 2.37 mmol) in tetrahydrofuran
t
+
40 mL). After workup, the residue [200 mg, LCMS (method II): R = 0.87 min; m/z: [M + H] = 397] was used di-
rectly in the next step. According to GP4, intermediate alkyne and Pd/C (214 mg, 0.101 mmol) in EtOAc (167 mL)
1
gave 31 as a yellow solid (7 mg, 3%). H NMR (400 MHz, [D] DMSO): δ = 1.87, 3.28 (2 m , 2 H each), 3.34* (s, 3
6
c
H), 3.52, 4.66 (2 m , 2 H, 1 H), 6.98–7.03 (m, 2 H), 7.18 (d, J = 8.1 Hz, 1 H), 7.76 (d, J = 7.6 Hz, 1 H), 7.92 (dd, J
c
=
7.2, 8.5 Hz, 1 H), 8.42 (d, J = 7.4 Hz, 1 H), 8.51 (dd, J = 1.0, 7.1 Hz, 1 H), 8.65 (dd, J = 1.0, 8.5 Hz, 1 H), 11.00
t
(
bs, 1 H) ppm; *overlapping with water solvent peak of [D] DMSO; LCMS (method IV): R = 0.75 min; m/z:
6
+
[
M + H] = 402.
Protein Expression and Purification. For screening and biophysical testing cDNAs encoding human BRPF2 BD
residues 563–687, UniProt O95696), human TAF1 BD2 (residues 1501-1635, UniProt P21675), ), TAF1L (resi-
(
dues 1523-1654, UniProt Q8IZX4) and human CREBBP BD (residues 1081-1197, UniProt Q92793) were cloned
into a modified pET28-derived expression vector (pNIC28-Bsa4) using ligation-independent cloning. Expression
constructs encoding the respective target proteins were then transformed into competent E. coli BL21 (DE3)-R3-
pRARE2 bacteria (phage-resistant derivative with a pRARE plasmid encoding rare codon tRNAs). Bacteria were
then grown at 37 °C in Terrific Broth from overnight cultures until the OD600 reached between 0.6-0.8, followed by
cooling and addition of 0.2 mM isopropyl-β-
for 16 h. Cells were lysed in loading buffer (20 mM Hepes pH 7.5, 500 mM NaCl, 20 mM imidazole, 1 mM TCEP,
% glycerol) in the presence of Protease Inhibitor Cocktail EDTA-free using an EmulsiFlex-C5 high-pressure
D-1-thiogalactopyranoside (IPTG) to allow protein expression at 18 °C
5
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homogenizer. Lysates were cleared by centrifugation at 14000 × g for 45 min at 4 °C after which the supernatant
was loaded onto a nickel affinity column (HisTrap Chelating FF 5 mL), followed by washing and elution with a
linear imidazole gradient. The eluted proteins were collected and treated overnight with Tobacco Etch Virus pro-
tease at 4 °C, followed by a second affinity purification step. Proteins were further purified by size exclusion
chromatography (HiLoad 16/600 Superdex 75 GE Healthcare Life Sciences). Protein identity was confirmed by
intact mass analysis.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The construct for co-crystallization of human BRPF2 encoded the residues 556-688 with two mutations P566E
and V569R integrated in the pNIC28-Bsa4 vector (Addgene, Teddington, U.K.). The expression construct was
introduced into competent E. coli BL21 (DE3) bacteria which were then grown in buffered LB medium until the
OD550 reached 1.0. This was followed by cooling and addition of 0.1 mM IPTG to allow protein expression at 17
°C for 24 h. Cells were resuspended in base buffer (10 mM Hepes pH 7.5, 500 mM NaCl, 10 mM imidazole, 0.5
mM TCEP, 5% glycerol) supplemented with 0.1% Triton X100, 0.025% NP40 in the presence of Protease Inhibi-
tor Cocktail EDTA-free and passed through a Microfluidizer (3 x 900 bar) for lysis. Lysates were cleared by cen-
trifugation at 30000 × g at 4 °C for 60 min after which the supernatant was loaded onto a nickel affinity column
(
HisTrap Chelating HF 5x5 mL), followed by a washing step with 25 mM and subsequent elution step with 250
mM imidazole in base buffer. The eluted proteins were collected, dialyzed against base buffer and treated with
Tobacco Etch Virus protease at 4 °C overnight, followed by a second affinity purification step. Proteins were fur-
ther purified by size exclusion chromatography using base buffer without imidazole (HiLoad 35/600 Superdex 75
GE Healthcare Life Sciences). Monomeric peak fractions were pooled and concentrated to 13 mg/mL. These
samples were directly used for crystallization trials. Protein identity was confirmed by intact mass analysis. For
co-crystallization of human TAF1 BD2, the same plasmid construct, expression and purification conditions as
above were used.
Computational Methods. Coordinate Scans. Optimization was performed at the B3LYP/6-31G* level followed by
54
single-point LMP2/cc-pVTZ(-f) calculations.
Predictions and Calculation Method. BEI and LLE were determined by a proprietary software tool using the fol-
lowing equations at physiological pH: LLE = -log(IC ) - clogD and BEI = [-log(IC ) / MWcorr]*1000. clogD (pH
5
0
50
55-57
7
.5) as estimated from HPLC retention times was calculated by using a highly predictive method.
Crystallography. BRPF2. Apo BRPF2 crystals were grown at 22 °C using the sitting-drop method by mixing 200
nL of protein (13 mg/mL/0.825 mM) with 100 nL of well solution (100 mM bis-tris pH 6.5, 30% (w/v) PEG 3350).
For complex formation, a single crystal was transferred into a soaked drop containing mother liquor supplement-
ed with 10 mM compound (prepared as a 100 mM stock solution in 100% ethylene glycol). After soaking for 48 h,
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Journal of Medicinal Chemistry
the crystal was briefly immersed in cryoprotection solution consisting of mother liquor supplemented with 10 mM
compound and 30% ethylene glycol and then flash-frozen in liquid nitrogen. X-ray data were collected on the
Helmholtz-Zentrum Berlin beamline 14-1 at a wavelength of 0.91814 Å and using a PILATUS detector. Data were
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
8, 59
integrated, scaled and merged using the programs XDS and SCALA.
The structure was solved by molecular
60
replacement using the program Phaser. Initial electron density maps clearly indicated binding of the compound.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The model was refined through iterative manual and maximum-likelihood refined using the programs COOT and
5
8, 61
REFMAC5.
Statistics for the final model are given in the Supporting Information Table S6. Coordinates and
structure factors have been submitted to the PDB database and are accessible with the code 5N49.
TAF1. Aliquots of purified TAF1 BD2 were set-up for crystallization using a mosquito crystallization robot (TTP
Labtech, Royston U.K.). Coarse screens were typically set up in Greiner 3-well plates using three different drop
ratios of precipitant to protein per condition (200 + 100 nL, 150 + 150 nL and 100 + 200 nL). All crystallizations
were carried out using the sitting-drop vapor diffusion method at 4 °C. TAF1 crystals with 5 were grown by mixing
100 nL of protein (11.3 mg/mL mixed with 1:3 molar ratio of ligand) with 200 nL of reservoir solution containing
2
0% PEG 10K, 0.20 M NaCl and 0.1 M citrate/phosphate pH 4.2. TAF1 complex crystals were cryoprotected
using well solution supplemented with additional 20% ethylene glycol for flash-freezing in liquid nitrogen. Data
were collected at Diamond beamline I02 at a wavelength of 0.9794 Å. Indexing, integration and scaling were car-
62
59
63
ried out using autoPROC (Global Phasing Limited - GPhL); this package uses XDS , POINTLESS ,
6
4
65
60
AIMLESS and CCP4 . Initial phases were calculated by molecular replacement with PHASER using the apo
6
6
67
TAF1 structure (PDB ID: 3UV4). Initial models were built by ARP/wARP followed by manual building in COOT .
68
69
Refinement was carried out in REFMAC5 . GRADE (GPhL) was used to generate compound coordinates and
7
0
cif files. All model validations were carried out using MolProbity . Data collection and refinement statistics can be
found in Supporting Information Table S6. The model and structure factors have been deposited with PDB acces-
sion code: 5MG2.
Time-Resolved Fluorescence Resonance Energy Transfer Assays. TR-FRET binding competition assays
were performed at RT in 1536- or 384-well black microliter plates in a final volume of 5 µL. Compounds were
diluted in DMSO at a single concentration (10 µM for primary screening, 5 and 25 µM for confirmation tests) or in
a titration series (3.5-fold, 11-points, maximal test concentration: 20 µM) and dispensed 100x concentrated onto
the plates. Then, 25 nM His-tagged BRPF1 (a.a. 626-740), BRPF2 (a.a. 563-688); BRPF3 (a.a. 592-711) or 10
nM BRD4 BD1 (a.a. 67-152) in assay buffer [50 mM Hepes pH 7.5, 50 mM NaCl, 50 mM KF, 0.01% NP40, 0.05%
bovine serum albumin (BSA) and 1 mM dithiothreitol (DTT)] were added, and the plates were incubated for 15
min. Finally, 50 nM of C-terminal biotinylated synthetic acetylated peptides derived from histone H4 a.a.1-20
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(
K5,12 di-acetylated for BRPF1, 2, 3 or K5,8,12,16 tetra-acetylated for BRD4) and detection reagents (10 nM
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
anti-6His-XL665 (Cisbio), 2.5 nM streptavidin Eu (Perkin Elmer), both in assay buffer) were dispensed and further
incubated for at least 1 h. TR-FRET signals corresponding to the number of protein-peptide complexes in equilib-
rium were acquired either with Viewlux (Perkin Elmer) or Pherastar (BMG Lab Technologies) microtiter plate
readers. The normalized ratios of the fluorescence emission at 665 nm and at 620-622 nm after excitation at 330-
350 nm, at increasing compound concentrations were used to calculate IC50 values by regression analysis based
on a four-parameter equation [minimum, maximum, IC , Hill; Y = Max + (Min - Max) / (1 + (X/IC )^Hill].
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
AlphaScreen®. Assay conditions: BRD4 BD1, BRPF1B, BRPF2, BRPF3, CECR2, CREBBP and FALZ/BPTF
7
1
assays were performed as described previously, with minor modifications of the manufacturer's protocol (Perki-
nElmer) using the same acetylated H4 peptide. All reagents were diluted in 25 mM Hepes, 100 mM NaCl, 0.1%
BSA, pH 7.4 supplemented with 0.05% CHAPS. Plates filled with 5 µL of the assay buffer followed by 7 µL of
biotinylated peptide and His-tagged protein to achieve final assay concentrations of typically 50 nM. Plates were
sealed and incubated for a further 30 min, before the addition of 8 ꢀL of the mixture of streptavidin-coated donor
beads (12.5 ꢀg/mL) and nickel chelate acceptor beads (12.5 ꢀg/mL) under low light conditions. Plates were foil-
sealed for protection from light, and incubated at RT for 60 min before reading.
Isothermal Titration Calorimetry. For BRPF2 and TAF1, experiments were performed in buffer (20 mM Hepes
pH 7.5, 100 mM NaCl) on a VP-ITC microcalorimeter (MicroCal) at 20 °C. For CREBBP and TAF1L, experiments
were done at 15 °C using a buffer containing 20 mM Hepes pH 7.5, 150 mM NaCl and 0.5 mM TCEP. Protein
concentrations were between 200-240 µM and the compound concentration was set between 18 and 25 µM. The
titrations were conducted using an initial injection of 2 µL followed by identical injections of 8 µL (between 24 and
3
5 injections). The dilution heats were determined in separate experiments and subtracted from the titration data.
Thermodynamic parameters were calculated using ∆G = ∆H - T∆S = -RTlnKB, where ∆G, ∆H and ∆S are the
changes in free energy, enthalpy and entropy of binding, respectively. In all cases a single binding site model was
employed.
Thermal Shift Assay. Thermal melting experiments were carried out using an Mx3005p Real-Time PCR ma-
72
chine (Stratagene), as described. Proteins were prepared in 10 mM Hepes pH 7.5, 500 mM NaCl, and assayed
in a 96-well plate at a final concentration of 2 ꢀM in 20 ꢀL volume. Compounds were added at a final concentra-
tion of 10 ꢀM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1:1000.
The temperature gradient was 3 °C per min. The melting temperature was calculated by fitting the Boltzmann
TM
equation using Prism 5. Thermal shift assays for hit validation were performed with a ViiA Real-Time PCR ma-
chine (Thermo Fisher Scientific) in a 384-well plate format. The thermal ramping was 4 °C per minute from 25 °C
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to 80 °C. BRPF1 protein was diluted to 2.5 ꢀM in 10 mM HEPES pH 7.5, 150 mM NaCl and BRPF2 with a protein
concentration of 2.7 µM in HEPES pH 7.5 and 300 mM NaCl. Both proteins were measured in the presence or
absence of 100 µM of ligand (1% final DMSO concentration) and a 6-fold SYPRO-Orange dye concentration.
TSA data were analyzed using Genedata Assay Analyzer.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
2
NanoBRET™ Assay. The assay principle has been described. Expression vectors were purchased from

0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Promega (Madison, Wisconsin, U.S.A). The NanoLuc luciferase was C-terminally fused to BRPF2 BD (a.a. 560-

6
66), BRPF1 BD (a.a. 626-732) or TAF1 BD2 (a.a. 1528-1640). A BRD4-NanoLuc fusion was used as control.
The HaloTag was C-terminally fused to histone H3.3 or H4. The resulting plasmids were transfected into HCT116
cells using standard procedures. Following addition of the HaloTag ligand, the cells were treated with compound
(
10 nM-10 µM) for 4 or 24 h, and the IC50 values were calculated.
®
LeadProfilingScreen and Kinase Panel. The LeadProfilingScreen containing 68 different targets and the ki-
nase panel experiment (300 kinases) were performed by Eurofins, Cerep Panlabs (Taipei, Taiwan).
Cell Proliferation Assay. Cell lines were obtained from ATCC (Manassas, Virginia, USA) or the DSMZ (Braun-
schweig, Germany), authenticated and grown following standard procedures. The cells were treated with com-
pound while in the logarithmic growth phase and their viability was determined by AlamarBlue staining (Ther-
moFisher, Waltham, Massachusetts, U.S.A.).
4
Permeability Assay. Caco2 cells purchased from the DSMZ were seeded at a density of 4.5 x 10 cells per well
and grown for 15 d in DMEM with typical supplements. Cells were kept at 37 °C in a humidified 5% CO atmos-
2
phere. Before the permeation assay was run, the culture medium was replaced by a FCS-free Hepes carbonate
transport buffer (pH 7.2). For assessment of monolayer integrity the transepithelial electrical resistance was
measured. Test compounds were predissolved in DMSO and added either to the apical or basolateral compart-
ment at a final concentration of 2 µM. Before and after 2 h incubation at 37 °C, samples were taken from both
compartments. Analysis of compound content was conducted after precipitation with methanol by LC/MS/MS
analysis. Permeability (Papp) was calculated in the apical to basolateral (A → B) and basolateral to apical (B →
A) directions. The efflux ratio basolateral (B) to apical (A) was calculated by dividing Papp B-A by Papp A-B. Ref-
erence compounds were analyzed in parallel as assay control.
Pharmacokinetics. Metabolic Stability in Hepatocytes. A hepatocyte cell suspension of the respective species
was filtered through sterile gauze in 50 mL Falcon tubes and centrifuged at 50×g for 3 min at RT. The cell pellet
®
was resuspended in 30 mL WME and centrifuged through a Percoll gradient twice at 100×g. The hepatocytes
were washed again with WME and resuspended in medium containing 5% FCS. Liver cells were distributed in
6
WME containing 5% FCS to glass vials at a density of 1.0 × 10 vital cells/mL. Compound 5 was added at a final
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Page 24 of 51
concentration of 1 µM. During incubation, the hepatocyte suspensions were continuously shaken at 580 rpm and
aliquots were taken at 2, 8, 16, 30, 45 and 90 min, to which equal volumes of cold methanol were immediately
added. Samples were frozen at -20 °C overnight, then centrifuged for 15 min at 3000 rpm and the supernatant
was analyzed by LC/MS/MS. The half-life of 5 was determined from the concentration-time plot. The intrinsic
clearances were calculated from the half-life.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Metabolic Stability in Liver Microsomes. The in vitro metabolic stability was determined by incubating compounds
at 1 µM concentration in a suspension of liver microsomes in 100 mM phosphate buffer, pH 7.4 (NaH PO x H O
2
4
2
+ Na HPO x 2H O) and at a protein concentration of 0.5 mg/mL at 37 °C. The microsomes were activated by
2
4
2
adding a co-factor mix containing 8 mM glucose-6-phosphate, 4 mM MgCl ; 0.5 mM NADP and 1 IU/mL G-6-P-
2
dehydrogenase in phosphate buffer, pH 7.4. The metabolic assay was started shortly afterwards by adding 5 to
the incubation in a final volume of 1 mL. During incubation, the microsomal suspensions were continuously shak-
en at 580 rpm and aliquots were taken at 2, 8, 16, 30, 45 and 60 min, to which equal volumes of cold methanol
were immediately added. Samples were frozen at -20 °C overnight, then centrifuged for 15 min at 3000 rpm and
the supernatant was analyzed by LC/MS/MS. The half-life of 5 was determined from the concentration-time plot.
The intrinsic clearances were calculated from the half-life.
In vivo Rat Pharmacokinetics. Compound 5 was administered to three male Wistar rats per arm, either intrave-
nously or intragastrally formulated as solutions. 5 was given as i.v. bolus and blood samples were taken at 2 min,
8
min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h after dosing. For pharmacokinetics after intragastral
administration, 5 was given intragastrally to fasted rats and blood samples were taken at 5 min, 15 min, 30 min,
®
4
5 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h after dosing. Blood was collected into lithium-heparin tubes (Monovettes ,
Sarstedt) and centrifuged for 15 min at 3000 rpm. An aliquot of 100 µL from the supernatant (plasma) was taken
and precipitated by addition of 400 µL cold ACN and frozen at -20 °C overnight. Samples were thawed and centri-
fuged at 3000 rpm, 4 °C for 20 min. Aliquots of the supernatants were submitted to LC/MS/MS analysis employ-
ing a validated method on an Agilent 1200 HPLC coupled to a Sciex API 5500 instrument. Standard pharmacoki-
netic parameters were calculated by non-compartmental analysis using a dedicated software.
Physicochemistry. Reversed-Phase HPLC Method for Lipophilicity (logD). logD (pH 7.5) was determined by
73
reversed-phase HPLC at physiological pH, as described. A homologous series of n-alkan-2-ones (C -C , 0.02
3
16
mol in ACN) was used for calibration. Test compounds were applied as 0.67 mM DMSO stock solutions in
ACN/H O 1:1. The lipophilicity of compounds was then assessed by comparison to the calibration curve.
2
High-Throughput Determination of Aqueous Drug Solubility (100 mM in DMSO). The high-throughput screening
74
method to determine aqueous drug solubility was performed as described previously. Test compounds were
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Journal of Medicinal Chemistry
applied as 1 mM solutions in DMSO. After the addition of a pH 6.5 buffer, the solution was shaken for 24 h at RT.
Undissolved material was separated by filtration. The compound dissolved in the supernatant was diluted accord-
ingly and quantified by HPLC-MS/MS.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ASSOCIATED CONTENT
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
Supporting Information
The Supporting Information is avaialble free of charge on the ACS Publication website at DOI: 10.1021/acs.jmed-
chem.6bxxxxx.
Binding mode of 5 in TAF1 BD2 (Figure S1), cellular activity of 7, 8 and 13 in NanoBRET™ assay (Table
S1), TSA results for 13 in a panel of 48 BDs (Table S2), molecular properties and physicochemical profile
of 5 (Table S3), BD selectivity profile of 5 (Table S4), anti-proliferative activity of 5 (Table S5), crystallo-
graphic data for 7 in BRPF2 BD and 5 in TAF1 BD2 (Table S6)
Molecular formula strings (CSV)
Accession Codes
PDB code for the structure of the BRPF2 BD in complex with 7 is 5N49. PDB code for the structure of the TAF1
BD2 with 5 is 5MG2. Authors will release the atomic coordinates and experimental data upon article publication.
AUTHOR INFORMATION
Corresponding Author
*Léa Bouché: Phone: +49 30 468 194438; E-mail: lea.bouche@bayer.com.
Notes
Competing financial interests: L. B., C.D. C., S.S., A.E. F.-M., S.J. H., A. t. L., T. S., D. S., J. M., V. P., J. W., I.V.
H. and B. H. are or have been employees and/or are stockholders of Bayer AG.
ACKNOWLEDGMENTS
We are indebted to M. Hitchcock, C. Scholten, U. Egner, S. Gründemann, U. Ganzer, G. Depke (Bayer AG) and
P. J. Brown, J. Porter (SGC Oxford) for their support throughout the project. We thank J. Méndez and D. Daniels
(
Promega Corporation) for excellent advice with the NanoBRET™ assays, and DiscoverX Corp. for performing
the BROMOscan screening. The expert technical assistance of J. Blaese, N. Dittmar, R. Karmauss, A. Mattstedt,
S. Wolf, L. Schaelicke, V. Trepte, C. Bölitz, F. Knoth, H. Muckwar, I. Herms and M. Wolfrum is gratefully
acknowledged. The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Phar-
ma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome
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Page 26 of 51
Canada, Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck & Co., No-
vartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foun-
dation-FAPESP, Takeda and Wellcome Trust [106169/ZZ14/Z].
1
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9
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5
5
6
ABBREVIATIONS
0
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0
ACN, acetonitrile; BD, bromodomain; BEI, binding efficiency index; BET, bromodomain and extra-terminal; BRPF,
bromodomain and PHD finger; CREBBP, cyclic-AMP response element binding protein; DAD, diode array detec-
tor; DMSO, dimethyl sulfoxide; EP300, adenoviral E1A binding protein; eq., equivalent, stoichiometric equiva-
lents; GP, general procedure; HAT, histone acetyltransferase; HPLC, high-performance liquid chromatography;
HTS, high-throughput screening; ITC, isothermal titration calorimetry; KAc, acetyl-lysine; LCMS, liquid chromatog-
raphy-mass spectrometry; LLE, lipophilic ligand efficiency; NMR, nuclear magnetic resonance spectroscopy; Rt,
retention time; RT, room temperature; SAR, structure-activity relationship; TAF1, TATA box binding protein-
associated factor; TR-FRET, time-resolved fluorescence energy transfer; TSA, thermal shift assay; UPLCMS,
ultra performance liquid chromatography-mass spectrometry; vol, volume.
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