Structure-Based Design of Highly Selective Inhibitors of the CREB Binding Protein Bromodomain

Article abstract of DOI:10.1021/acs.jmedchem.6b01839

Chemical probes are required for preclinical target validation to interrogate novel biological targets and pathways. Selective inhibitors of the CREB binding protein (CREBBP)/EP300 bromodomains are required to facilitate the elucidation of biology associa

Full text of DOI:10.1021/acs.jmedchem.6b01839

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Article
Structure-based design of highly selective inhibitors
of the CREB binding protein bromodomain
Rajiah Aldrin Denny, Andrew C. Flick, Jotham Wadsworth Coe, Jonathan Langille, Arindrajit
Basak, Shenping Liu, Ingrid A Stock, Parag Sahasrabudhe, Paul D Bonin, Duncan Alexander
Hay, Paul E Brennan, Mathew T. Pletcher, Lyn H. Jones, and Eugene L. Piatnitski Chekler
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01839 • Publication Date (Web): 04 Apr 2017
Downloaded from http://pubs.acs.org on April 5, 2017
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Structureꢀbased design of highly selective inhibitors of the
CREB binding protein bromodomain
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a
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R. Aldrin Denny,* Andrew C. Flick, Jotham Coe, Jonathan Langille, Arindrajit Basak, Shenping
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e,f
Liu, Ingrid Stock, Parag Sahasrabudhe, Paul Bonin, Duncan A. Hay, Paul E. Brennan, Mathew
g
*a
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Pletcher, Lyn H. Jones, and Eugene L. Piatnitski Chekler*
ABSTRACT: Chemical probes are required for preclinical target validation to interrogate novel
biological targets and pathways. Selective inhibitors of the CREB Binding Protein (CREBBP)/EP300
bromodomains are required to facilitate the elucidation of biology associated with these important
epigenetic targets. Medicinal chemistry optimization, that paid particular attention to physiochemical
properties, delivered chemical probes with desirable potency, selectivity and permeability attributes.
An important feature of the optimization process was the successful application of rational structureꢀ
based drug design to address bromodomain selectivity issues (particularly against the structurally
related BRD4 protein).
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Introduction
Postꢀtranslational modifications of chromatin regulate cellular transcription. The dynamic
processes of remodeling histone tails are performed using epigenetic proteins called ‘writers’, ‘readers’
and ‘erasers’. Epigenetic writers such as histone acetyltransferases, histone methyltransferases, protein
arginine methyltransferases and kinases install one or more postꢀtranslational marks, including acetyl,
methyl and phosphate groups, on the histone tail. Histone deacetylases, lysine demethylases and
phosphatases are epigenetic erasers that remove these marks and proteins containing bromodomains,
chromodomains and Tudor domains read epigenetic marks to trigger downstream signaling.
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Epigenetic aberrations are recognized as key drivers for several human diseases and drug
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discovery efforts in this area have primarily focused on writer and eraser proteins. Recently, there has
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been an increased interest in histone reader proteins for several indications. Proteins that possess a
bromodomain are able to read the acetyl lysine marks associated with epigenetic information transfer.
Small molecule inhibitors that perturb acetyl lysine reading interactions are used to decipher their
1
associated biology. One such example that of BET bromodomain inhibitors, has also illustrated the
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ꢀ3
therapeutic potential of this class.
CREB Binding Protein (CREBBP or CBP) is a master transcriptional regulator primarily
involved in the activation of gene transcription. It is a ubiquitously expressed target that plays a key
role in transcriptional activation in human cells. CREBBP possesses many domains including a
nuclear receptor interaction domain, the CREB and MYB interaction domain, the cysteine/histidine
region, an interferon response binding domain and a single bromodomain. The bromodomain acetylꢀ
lysine reader is responsible for the recruitment of the transcription factor, CREB, to its promoter
binding sites. CREBBP is also known to interact with many proteins, including p53 to induce the
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transcription of p53 target genes. BET bromodomain biology has been significantly illuminated
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through the use of selective chemical probes. We reasoned that learnings from the medicinal
chemistry of BET bromodomains could be applied to the development of selective CREBBP
bromodomain inhibitors to further interrogate the biology and therapeutic potential of this target. We
recently reported the culmination of our efforts towards this goal that included the development and
transcriptional profiling of a selective CREBBP/EP300 inhibitor that highlighted the opportunities for
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this pharmacological approach in neurodegenerative diseases. Here we detail our broader medicinal
chemistry efforts that have successfully yielded multiple novel and selective lead series that should
considerably accelerate drug discovery in this area. A significant effort to discover CREBBP selective
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tools and to study them in various biological systems has been launched by several research groups.
The Structural Genomics Consortium (SGC) team has recently published a 40ꢀfold selective probe in
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c, 7
Figure 1 and demonstrated an interesting CREBBP/p53 associated biology.
Working concurrently
with the structural genomics consortium (SGC) team, it became our goal to obtain highly selective
molecules to help further elucidate differences in the biology of CREBBP and BET bromodomains.
Results and Discussion
Achieving selectivity across the bromodomain family is a challenging task, but a recent
7
publication by the SGC demonstrated the possibility of designing CREBBP chemical tools. The SGC
had previously disclosed the isoxazole CREBBP inhibitor 1 which was used by our group as a starting
3
c, 6c, 7ꢀ8
point to design novel CREBBP selective probe molecules (Table 1).
The project’s objectives
were to identify potent CREBBP tool compounds with 100ꢀfold selectivity over other bromodomains,
particularly BRD4. Here we report the design of selective CREBBP inhibitors that are structurally
3
c
distinct from the recently reported compounds GNEꢀ272 ((S)ꢀ1ꢀ(3ꢀ((2ꢀFluoroꢀ4ꢀ(1ꢀmethylꢀ1Hꢀ
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pyrazolꢀ4ꢀyl)phenyl)amino)ꢀ1ꢀ(tetrahydrofuranꢀ3ꢀyl)ꢀ6,7ꢀdihydroꢀ1Hꢀpyrazolo[4,3ꢀc]pyridinꢀ5(4H)ꢀ
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b
yl)ethanone) and CPIꢀ637 ((R)ꢀ4ꢀmethylꢀ6ꢀ(1ꢀmethylꢀ3ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)ꢀ1Hꢀindazolꢀ5ꢀ
yl)ꢀ1,3,4,5ꢀtetrahydroꢀ2Hbenzo[b][1,4]diazepinꢀ2ꢀone). The myriad of small moleculeꢀBRD4 crystal
structures, and our own work to generate crystal structures with CREBBP, provided confidence that
structureꢀbased design could be used to achieve the desired selectivity profile in leadꢀlike series for the
project.
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Figure 1. Chemical structure of SGC chemical probe SGCꢀCBP30.
Bromodomains are comprised of 4 helix bundles that can arrange either in a right or a left
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handed fashion. The helices are labeled as Z, A, B and C in going from the Nꢀ to Cꢀ terminus (Figure
2
A). The spatial orientation of these 4 helix bundles selectively recognizes the monoacetylated
terminal lysine in histones. The conserved Asn1168 residue in the αB helix makes a key hydrogen
bond interaction with the acetyl lysine. The loops connecting ZA and BC bundles surround the
binding site of the acetyl lysine binding pocket. A network of 5 structural water molecules provides
opportunities for hydrophilic interactions in the deep end of the binding pocket as shown in Figure 2A.
The isoxazole headpiece oxygen in 1 makes a hydrogen bonding interaction with the Asn1168 amide
moiety and the isoxazole nitrogen points towards water A which bridges with the Tyr1125 (Figure 2B).
The benzimidazole connected to the isoxazole core acts as a spacer and the phenethyl substitution
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makes a key cationꢀπ interaction with Arg1173. The morpholine substitution on the benzimidazole
acts as a solubilizing group and it points towards solvent. A comparison of the Arg1173 orientation in
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the compound 1 crystal structure and crystal structures of other ligands (PDB IDs 3SVH and 3P1C)
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identified a different orientation of this critical binding residue. When CREBBP is crystallized with
small ligands that are incapable of making cationꢀπ interactions (Figure 3A) the guanidine moiety of
Arg1173 points in the NorthꢀSouth orientation and all the carbon atoms of the Arg1173 side chain exist
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in the low energy staggered orientation. This is an expected orientation in the apoꢀprotein crystal
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structure (such as 3DWY). However, compound 1 induces a conformational change to rotate the
guanidine in Arg1173 to an EastꢀWest orientation, and also introduces a higher energy gauche
conformation along the Cγ − Cδ bond as shown in Figure 3B. An additional illustration of CREBBP
and BRD4 binding pockets is shown in the supplemental material section (Figure S1 and Table S1).
This ligandꢀinduced residue rotation enables the cationꢀπ interaction to be made. In addition, a careful
investigation of the binding orientation of the phenethyl substituent in compound 1 reveals a higher
energy gauche orientation along the ethyl bond. This observation emphasizes the importance of
capturing an energetically favorable cationꢀπ interaction in the design of new ligands that compensates
for two unfavorable gauche orientations observed in the proteinꢀligand complex. The enhancement of
this cationꢀπ interaction thus constitutes a plausible design strategy to achieve requisite potency and
selectivity in novel CREBBP bromodomain inhibitors.
A:
B:
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Figure 2. A: CREBBP protein domains with deep pocket water molecules labelled Wat A to E are
shown; B: Crystal structure of 1 with CREBBP (PDB Code 5CGP). Cationꢀpi interaction between the
Arg1173 and (4ꢀmethyoxy)phenethyl moiety is shown.
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Figure 3. Water molecules around Arg1173 as predicted by 3D RISM. A: Crystal structure with Arg
in the staggered orientation (PDB IDs 3SVH and 3P1C) with predicted bulk water molecules around
them (green sphere); B: Arg1173 is in a gauche orientation making an Argꢀphenyl interaction.
Predicted ∆G values for displacing water molecules are depicted on the green sphere.
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Electron donating groups on the phenyl ring are expected to enhance cationꢀπ interactions
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according to quantum mechanics calculations.
The unsubstituted phenyl analog 2 is 2ꢀfold less
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active than the paraꢀsubstituted compound 1 which was first disclosed by the SGC.
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corresponding metaꢀcompound 3 turned out to be even less active which is consistent with quantum
12d
mechanics calculations for cationꢀπ interactions.
However, the orthoꢀsubstituted analog 4 showed a
significant drop in potency which may indicate an effect of orthoꢀsubstitution on the orientation of the
phenethyl chain. Orthoꢀ and metaꢀ polar substitution may also lead to undesired solvation effects. Polar
substituents on the phenyl ring must interact with the water molecules that solvate the Arg1173
residue. Orthoꢀ and metaꢀmethoxy groups may hinder water molecules from solvating Arg1173, thus
creating unfavorable interactions (Figure 3). 3,4ꢀDimethoxy analog 5 is comparable to 1 in terms of
1
4
lipophilic efficiency (LipE = pIC ꢀ LogP) which is a useful parameter in drug design as it describes
50
desirable attributes related to potency and lipophilicity (high quality drug candidates possess a high
LipE). 2,4ꢀDimethoxy compound 6 demonstrated even greater selectivity but with some loss of
CREBBP potency. Another approach to enrich electron density within the aromatic system was to
install an ether linkage between the benzimidazole and benzene ring, but the corresponding derivatives
7
and 8 lost CREBBP potency either due to a suboptimal tether geometry or the abovementioned
solvation effect. The most successful approach to improve selectivity within this scaffold was to
expand on the substitution at the paraꢀposition. While the hydroxylꢀcontaining derivative 9 showed no
change in its selectivity profile, ethoxy and propoxy analogs 10 and 11 had a remarkable improvement
in selectivity (51 and 62ꢀfold respectively). These compounds were followed up in an isothermal
titration calorimetry (ITC) assay to confirm their selectivity. Propoxy analog 11 showed no thermal
signature in the BRD4 ITC assay which correlates with the weak biochemical BRD4 potency (IC 18
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ꢀ
M, Table S2 in supplemental material section). Compound 11 (that we named PFꢀCBP1) was the
most selective and potent CREBBP tool compound discovered at that time, and was used as a chemical
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probe for transcriptional profiling experiments.
Additional paraꢀposition modifications 12ꢀ20
demonstrated either a loss of CREBBP potency or selectivity. Changing the propoxy group in 11 to iꢀ
Pr substituent (12) led to a loss of selectivity, and a similar effect was observed by extending the alkyl
chain to butyl (13) or pentyl (14) groups. A remarkable loss in CREBBP potency was observed for the
electron withdrawing trifluoromethoxy analog 15, while the retention of activity for the nitrileꢀ
containing compound 16 indicates a more complex picture than a simple cationꢀπ interaction
paradigm. The loss in potency of 15 can be attributed to the torsional preference of trifluroanisole,
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where the trifluoromethoxy moiety orients 90° from the plane of the phenyl ring which creates a van
der Waals clash with the side chain residues. The influence of an electron donating group on the SAR
was further explored with paraꢀamino derivatives 17ꢀ19 but the activity and/or selectivity suffered
significantly. In the course of the SAR exploration, compound 20 was found to possess dual
CREBBP/BRD4 activity, which served as a useful chemical probe to help delineate differences in
5
inhibiting these bromodomains in our chemical biology studies. The compounds within this series
display acceptable cellular permeability, thus making them suitable for cellꢀbased studies. For
ꢀ6
example, the passive permeability rate for the chemical probes 11 and 20 are 7.9x10 cm/sec and
ꢀ6
4
.5x10 cm/sec at 2 ꢀM, respectively. However, the microsomal clearance for the majority of the
compounds from the series is relatively high limiting their applications to predominantly in vitro work.
Intrinsic clearance in human microsomal assay for compounds 11 and 20 are >300 ꢀL/min/mg and 206
ꢀL/min/mg at 1 ꢀM dose, respectively. The tool molecule 11 also demonstrated a relatively clean
polypharmacology profile determined in a broad CEREP panel where the activity was observed only
for the adrenergic A1a receptor (antagonist, IC 0.62 ꢀM, Supporting Information Table S3) and Lꢀ
5
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type calcium channel (antagonist, IC 3.0 ꢀM).
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Table 1. Potency and physicochemical data of the 4ꢀ(2ꢀ(5ꢀ(3,5ꢀdimethylisoxazolꢀ4ꢀyl)ꢀ2ꢀphenethylꢀ
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5
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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5
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a
1
Hꢀbenzo[d]imidazolꢀ1ꢀyl)ethyl)morpholine chemical class.
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CREBBP
LipE
BRD4 FP CREBBP/BRD
Compound
R
X
FRET
(CREBBP
IC (ꢀM) 4 fold selectivity
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0
a
IC (ꢀM)
FRET)
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4ꢀOMe
H
CH
CH
CH
CH
CH
CH
O
2
2
2
2
2
2
0.12
2.4
4.1
4.6
20
17
15
14
20
35
10
36
18
51
62
23
43
10
3.6
3.5
2.9
2.2
3.6
2.8
2.8
3.7
3.6
2.8
3.5
2.6
1.9
1.5
0.24
0.31
0.98
0.47
0.24
0.92
0.40
0.24
0.13
0.13
0.20
0.26
0.69
3ꢀOMe
2ꢀOMe
3,4ꢀ(OMe)
2,4ꢀ(OMe)
4ꢀOMe
H
13.9
9.4
2
2
8.3
9.2
O
14.4
4.4
OH
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
1
1
0
1
4ꢀOEt
4ꢀOPr
4ꢀOꢀiꢀPr
4ꢀOBu
4ꢀOꢀPn
6.6
18.1
4.5
12
1
3
11.2
6.9
14
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5
5
5
5
6
15
4ꢀOCF
3
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
ND
0.30
0.21
0.09
0.20
0.65
18.5
4.9
4.3
3.4
1.4
1.5
ND
16
20
38
7
0.5
3.2
3.6
3.5
1
6
4ꢀCN
17
4ꢀNMe
4ꢀNH
4ꢀNHAc
(CH NMe
2
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
1
1
2
8
9
0
2
4.2
2.6
)
3
3
2
2
a
Experimental shakeꢀflask LogD values were used.
One of our strategies to improve CREBBP potency was to establish ligand interactions such as
a shielded salt bridge or a hydrogen bond with the Arg145 residue in the CREBBP binding pocket.
This residue appeared to be accessible from the phenethyl tail present in the chemotype. Various
heterocyclic derivatives were prepared to probe hydrogen bonding interactions with the Arg145
residue. Complicated structureꢀactivity relationships in the phenethyl region of the molecule were
further demonstrated by exploring heterocyclic substituents in place of the phenyl ring (Table 2). The
2
ꢀfold loss of CREBBP activity in 3ꢀpyridyl analog 22 compared to 2ꢀpyridyl (21) and 4ꢀpyridyl (23)
derivatives is consistent with the lone pair of 22 making unfavorable interactions with the water
molecule, similar to that seen for metaꢀsubstituted derivative 3. Another BRD4/CREBBP selectivity
approach was to take advantage of the residue differences between these two bromodomain binding
sites (Table S1, supplemental material section). Trp81 in BRD4 (Figure 4A) is significantly larger
than the corresponding Leu81 in CREBBP, suggesting that adding appropriate substitution to the
ligand template in this region may establish a van der Waals clash. Indolyl compound 24 was intended
to support the hypothesis that electron rich heterocycles may improve CREBBP binding, as well as
decrease BRD4 potency by increasing steric bulk, but the secondary binding effects besides the cationꢀ
π interaction, likely played a role in decreasing CREBBP activity, and the drop in BRD4 potency was
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not significant. Quinolinyl analogs 25 and 26 represent an interesting example of the complexity of the
SAR in this region. The CREBBP potency of 25 is more than 10ꢀfold higher than the regioisomer 26
which cannot be explained by stereoelectronic considerations related to the key cationꢀπ interactions.
The BRD4 activity of 25 turned out to be similar to the parent molecule 2 thus indicating a significant
flexibility of the BRD4 binding pocket around the Trp81 residue. A substitution in this region was
further explored by preparing a library of alkyl and aryl derivatives with a direct bond to the
benzimidazole core. Compound 27 was discovered as a hit with appreciable CREBBP potency (and
impressive LipE) even though CREBBP/BRD4 selectivity did not improve over the phenethyl
chemotype. The binding mode of 27 is not able to adopt a cationꢀπ interaction similar to the phenethyl
derivatives, thus indicating a potentially novel binding mode that would need to be further elucidated
using Xꢀray crystallography.
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
Table 2. Structureꢀactivity relationships relating to modifications of the phenethyl substituent.
CREBBP
FRET
CREBBP
/BRD4 fold
selectivity
BRD4 FP
Compound
Y
LipE
IC (ꢀM)
5
0
IC (ꢀM)
5
0
2
2
2
1
2
3
CH
CH
CH
2
2
2
CH
CH
CH
2
2
2
ꢀ2ꢀpyridyl
ꢀ3ꢀpyridyl
ꢀ4ꢀpyridyl
0.33
3.2
5.8
4.1
10
7.4
10
4.7
4.3
4.6
0.78
0.40
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
24
CH
2
CH
2
ꢀ3ꢀindolyl
0.74
0.16
10.1
4.9
14
31
2.9
3.3
CH CH ꢀ2ꢀ
2
2
2
2
5
6
quinolinyl
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
CH CH ꢀ4ꢀ
2
2
2.5
16.8
1.6
7
2.7
5.3
isoquinolinyl
2
7
0.13
12
Figure 4. A: Crystal structure of 1 with BRD4 (PDB Code 5CFW). B: Compound 28;
Crystal structure of 1 determined that the morpholine group points towards the Trp81 region of
the BRD4 binding pocket (Figure 4A). Although the keto group of 28 (Figure 4B) decreased BRD4
activity, CREBBP potency generally suffered upon changes in this region. However, a single methyl
group with the right stereochemistry in the SGC probe in Figure 1 apparently provided a better
compromise with respect to both target activities. A crystal structure of BRD4 with compound 1
demonstrated that Trp81 of BRD4 is rotated around the CalphaꢀCbeta bond by about 130 degrees and also
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Cbeta is shifted by about 1Å (Figure 4A). This movement and rotation makes the pocket wider, thus
providing a hydrophobic enclosure for compounds 1, 2 and their analogs. The phenethyl moiety
occupies a small pocket in the BRD4 active site and the morpholine group points straight out towards
solvent. It is important to note that this orientation is a different binding mode compared to the
CREBBP binding pose shown in Figure 2B. This observation provides opportunities for further
selectivity enhancements through the development of constrained derivatives (Figure 5). For instance,
the selectivity was improved to 67ꢀfold for compound 29. Conformational constraints in the ethyl
tether were further explored (Figure 6). According to the dihedral computation, it was proposed that
the 5ꢀ and 6ꢀmembered ring systems are predicted to have less strain than other ring sizes, and are
closer to the crystal structure dihedral angle as shown in Figure 6. The remarkable drop in selectivity
for compound 30 supports the hypothesis that a specific orientation is crucial to retain binding potency
and selectivity. Further exploration of this strategy to provide 31 and 32 led to a loss of CREBBP
potency likely due to the change in orientation of the morpholine ring.
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
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2
3
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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
Figure 5. Constrained analogs at both morpholine and phenethyl chains (absolute stereochemistry
arbitrarily assigned).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. A: The dihedral angle of 1; B: The dihedral angle table for the lowest energy conformation
in the crystal structure of 1 and cyclization to provide the 3ꢀ to 6ꢀmembered ring systems at the
phenethyl group.
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3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 7. Modifications to the acetyl lysine mimic headpiece.
The dimethylisoxazole head group is a common acetyl lysine mimic used in various
1
3, 17
bromodomain inhibitors,
and it became our goal to replace this fragment with alternative head
pieces. Pyrazole derivative 34 demonstrated that one of the hydrogen bond acceptors of the isoxazole
ring cannot be substituted with a hydrogen bond donor to reverse an interaction with water A (Figure
7
). Compound 35 suggested that a single hydrogen bond acceptor atom is also not sufficient for potent
binding to either BRD4 or CREBBP. Nꢀethylpyridone analog 36 and tropolone derivative 37 were
prepared as part of a library with various heterocyclic moieties. These compounds prove that the
dimethylisoxazole group can be substituted with alternative head pieces though with some loss of
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
potency. We have previously described the utility of tropolone bromodomain inhibitors from this work
6
a
as photoaffinity probes of cellꢀbased target occupancy.
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
An additional structureꢀbased drug design strategy to improve selectivity was to restrict the
inhibitors into their desired conformation by forming an atropisomer. To determine whether a
compound would exist as an atropisomer we performed a dihedral scan using molecular mechanics and
quantum mechanics calculations. A systematic dihedral scan of the rotatable bond was performed in
18
MacroModel using OPLS 2005 force field and energy barriers were calculated. If the energy barrier
for the dihedrals were found to be <15 kcal/mol, the compound was classified as not being
atropisomeric and no further QM calculations were performed. If the energy barrier is calculated to be
above 15 kcal/mol, additional dihedral scans were performed using quantum mechanics using
1
9
Terachem Program from Petachem LLC.
Quantum mechanics dihedral scans were performed at the B3LYP/6ꢀ31G level of theory.
Torsional scan around the bond were performed using a rotational step of 30 degrees. After each
rotation, the resulting structure was optimized using a relaxed termination criterion with Etolerance of 5 x
ꢀ4
1
0 hartree. During optimization, coordinates of the four atoms that define the given dihedral angle
were kept fixed and all other atoms were allowed to move. On the optimized structures, single point
energies were calculated. If the barrier height based on single point energies was calculated to be > 20
kcal/mol then the compound was classified as very likely to exhibit atropisomerism. The torsional
profile scan using molecular mechanics and QM calculations showed an energy barrier of 24.2 and
2
2.8 kcal/mol respectively for the rotation around the isoxazole/benzimidazole bond by adding methyl
5
substituents to positions 4 and 6 of the benzimidazole 38 (ISOXꢀINACT, Figure 8). This atropisomer
rigidifies the benzimidazole ring at 90° from the isooxazole ring there by clashing with Trp81 in BRD4
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in both its orientations. This negative interaction with Trp81 explains the reduced potency of binding
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
to BRD4 (IC >120ꢀM) as expected. The loss of CREBBP potency was significant due to poor
5
0
conserved interactions with the Asn1168 residue and cationꢀπ interactions with the Arg1173 residue.
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
This compound in fact served as a useful negative control for this chemotype in our previously
5
described cellꢀbased experiments.
Finally, a successful selectivity approach was discovered by converting the benzimidazole core
into an indole moiety (Figure 8). The benzoimide Nꢀ3 nitrogen, as seen in the BRD4 crystal structure
acts as a hydrogen bond acceptor to Water E, thus mediating an interaction with Pro82 (Figure 4B).
Our strategy was to convert the benzimidazole into an indole group therefore preventing the hydrogen
bonding interaction. Indole analog 39 showed a significant decrease in BRD4 potency. However, the
CREBBP binding mode would cause there to be an unfavorable orientation of the indole position 3
hydrogen towards the solvent exposed region of the pocket. Therefore, a more polar substituent might
be favored in this area of the molecule and cyano group in 40 was expected to be pointing to the
solvent exposed region in CREBBP. Incorporation of a nitrile moiety to yield derivative 40 resulted in
a complete loss of BRD4 activity i.e., greater than 240ꢀfold selectivity, with CREBBP potency
remaining within several fold of compound 1 activity. To accommodate a more polar substitution in
this area of the indole chemotype, we converted indole analog 39 into Azoindole 41 (that we named
PFꢀCBP2) where the additional nitrogen atom would be pointing towards solvent. Azaindole 41
showed an improvement in CREBBP potency to a level comparable to compound 1 activity (170 nM).
BRD4 potency remained weak (18.4 uM) resulting in >100ꢀfold selectivity in the biochemical assays.
Lead molecule 41 also possessed a clean polypharmacology profile (IC > 5 ꢀM in a broad CEREP
5
0
ꢀ6
panel, Supporting Information Table S3), good cellular permeability (6.8x10 cm/sec at 2 ꢀM), and
shake flask LogD (3.29 at pH 7.4). The discovery of this very selective and potent chemotype using
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2
3
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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
structureꢀbased design provides confidence in the future medicinal chemistry development of this
series.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. Benzimidazole core modifications
The bromodomain selectivity of lead compounds was assessed using the DiscoveRx
1
5
BROMOScan platform. A screening panel of more than 30 bromodomains was utilized. The ultraꢀ
sensitive qPCR based technology demonstrated a correlation with internally generated data. Even
though the assay formats of BROMOScan and biochemical/biophysical assays may account for the
differences in potency values, general trends support the selectivity of the isoxazoleꢀbenzimidazole
compound class (Figure 9). Both compounds 11 and 41 demonstrated activity for the closely related
target EP300 that possesses significant homology to CREBBP with respect to the bromodomain
binding pocket. It is interesting to note that the bromodomain selectivity profile of compound 41
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surpasses that of compound 11, thus suggesting 41 would serve as a useful lead series for future drug
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
discovery efforts.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 9. Bromodomain selectivity profile of lead molecules 11 and 41. Green, yellow and red dots
indicate the inhibition <30%, 30ꢀ80% and >80% respectively in the DiscoveRX assay at 10µM dose.
Conclusion
In conclusion, we have developed highly selective inhibitors of the CREBBP/EP300
bromodomains using structureꢀbased drug design. These compounds will serve as useful lead series for
future drug discovery efforts that should focus on addressing other important features such as
metabolic stability. Through this work we identified chemical probes 11 and 41 that have found utility
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2
3
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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
in cell biology and transcriptional profiling experiments that furthers our understanding of CREBBP
5
bromodomain pharmacology. Novel acetyl lysine mimics were also identified that should be of broad
interest to those working in this area.
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
Experimental Section
Synthesis
Starting materials and other reagents were purchased from commercial suppliers and were used
without further purification unless otherwise indicated. All reactions were performed under a positive
pressure of nitrogen, argon, or with a drying tube, at ambient temperature (unless otherwise stated), in
anhydrous solvents, unless otherwise indicated. Analytical thinꢀlayer chromatography was performed
on glassꢀbacked Silica Gel 60_F 254 plates (Analtech (0.25 mm)) and eluted with the appropriate
solvent ratios (v/v). The reactions were assayed by highꢀperformance liquid chromatography (HPLC)
or thinꢀlayer chromatography (TLC) and terminated as judged by the consumption of starting material.
The TLC plates were visualized by UV, phosphomolybdic acid stain, or iodine stain. Microwave
assisted reactions were run in a Biotage Initiator. 1H NMR spectra were recorded on a Bruker
instrument operating at 400 MHz unless otherwise indicated. 1H NMR spectra are obtained as DMSOꢀ
d6 or CDCl3 solutions as indicated (reported in ppm), using chloroform as the reference standard (7.25
ppm) or DMSOꢀd6 (2.50 ppm). Other NMR solvents were used as needed. When peak multiplicities
are reported, the following abbreviations are used: s = singlet, d = doublet, t = triplet, m = multiplet, br
=
broadened, dd = doublet of doublets, dt = doublet of triplets. Coupling constants, when given, are
reported in Hertz. The mass spectra were obtained using liquid chromatography mass spectrometry
LCꢀMS) on an Agilent instrument using atmospheric pressure chemical ionization (APCI) or
electrospray ionization (ESI). High resolution mass measurements were carried out on an Agilent TOF
(
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200 series with ESI. All test compounds showed > 95% purity as determined by either combustion
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
analysis, highꢀperformance liquid chromatography (HPLC), liquid chromatography/mass spectrometry
1
1
analysis (LCMS), or HNMR integration. When HNMR is employed HPLC or LCMS is additional
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
used to confirm compound purity. HPLC conditions were as follows: XBridge C18 column @ 80 °C,
4
.6 mm x 150 mm, 5 ꢁm, 5%ꢀ95% MeOH/H2O buffered with 0.2% formic acid/0.4% ammonium
formate, 3 min run, flow rate 1.2 mL/min, UV detection (λ = 254, 224 nm). Combustion analyses were
performed by Atlantic Microlab, Inc. (Norcross, Georgia).
a
Scheme 1. General procedure A: benzimidazole formation.
a
Reaction conditions: (a) R NH , Et N, DMSO, 80 °C, 18 h; (b) R CHO, Na S O , 4:1 EtOH/H O, 70
2
2
3
3
2
2
4
2
°C, 18 h; (c) R B(OH) , PdCl (dppf)CH Cl , Cs CO , pꢀdiox.100 °C, 18 h;
1
2
2
2
2
2
3
5
-(3,5-dimethylisoxazol-4-yl)-2-[2-(4-methoxyphenyl)ethyl]-1-(2-morpholin-4-ylethyl)-1H-
1
benzimidazole (1): (1.3 g, 60%) as yellow liquid. H NMR (400 MHz, CDCl ) δ 2.30 (s, 3H), 2.43 (s,
3
3H), 2.44ꢀ2.47 (m, 4H), 2.58 (t, J = 6.8 Hz, 2H), 3.15ꢀ3.26 (m, 4H), 3.66ꢀ3.68 (m, 4H), 3.79 (s, 3H),
4
.11 (t, J = 6.8 Hz, 2H), 6.84 (d, J = 8.0 Hz, 2H), 7.11ꢀ7.16 (m, 3H), 7.34 (d, J = 8.0 Hz, 1H), 7.63 (d,
+
J = 0.8 Hz, 1H); LCꢀMS m/z 461 (M+1) .
4
-(2-(5-(3,5-dimethylisoxazol-4-yl)-2-phenethyl-1H-benzo[d]imidazol-1-yl)ethyl)morpholine (2).
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A mixture of 5ꢀbromoꢀ2ꢀfluoronitrobenzene (81.4 g, 0.37 mol), Nꢀ(2ꢀaminoethyl)morpholine (60.0 g,
.46 mol) and triethylamine (46.5 g, 0.46 mol) in DMSO (1.2 L) was heated to 80 °C and stirred for 2
0
hours, when TLC (petroleum ether/EtOAc = 1:1) showed the reaction was complete. To the cooled
reaction was added water (500 mL) and the mixture was extracted with ethyl acetate (500 mL × 4).
The combined organic layer was washed with water (500 mL) and brine (500 mL × 2), dried over
Na SO , filtered, and concentrated in vacuo. The residue was purified by flash chromatography (0ꢀ50%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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0
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2
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4
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8
9
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5
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8
9
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2
4
ethyl acetate in petroleum ether) to afford the titled 4ꢀBromoꢀNꢀ(2ꢀmorpholinoethyl)ꢀ2ꢀnitroaniline
1
(
145 g, 95%) as an orange solid. H NMR (400 MHz, CDCl ): δ 2.46ꢀ2.58 (m, 4H), 2.69ꢀ2.76 (m, 2H),
3
3.30ꢀ3.38 (m, 2H), 3.71ꢀ3.80 (m, 4H), 6.73 (d, J = 9.0 Hz, 1H), 7.50 (dd, J = 9.0, 1.6 Hz, 1H), 8.33 (d,
+
J = 2.4 Hz, 1H), 8.51 (br s, 1H); LCꢀMS m/z 330 (M+1) .
4
ꢀ(2ꢀ(5ꢀbromoꢀ2ꢀphenethylꢀ1Hꢀbenzo[d]imidazolꢀ1ꢀyl)ethyl)morpholine. To a mixture of 4ꢀBromoꢀNꢀ
2ꢀmorpholinoethyl)ꢀ2ꢀnitroaniline (2.01 g, 6.09 mmol) and 3ꢀphenylpropionaldehyde (2.50 g, 19.0
mmol) in 4:1 ethanol/H O (60 mL, 0.10M) was added Na S O (3.95 g, 19.0 mmol). The mixture was
(
2
2
2
4
stirred at 70 °C for 18 hours under nitrogen. The resulting mixture was diluted with saturated sodium
bicarbonate (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic layers were
washed with brine (50 mL), dried over Na SO , filtered and concentrated in vacuo. The residue was
2
4
purified by flash silica chromatography (0ꢀ100% heptane in ethyl acetate) and the major fraction
corresponding to the desired product was collected and concentrated to give the titled 4ꢀ(2ꢀ(5ꢀbromoꢀ2ꢀ
1
phenethylꢀ1Hꢀbenzo[d]imidazolꢀ1ꢀyl)ethyl)morpholine (1.41 g, 56%) as a light yellow oil: H
NMR(400 MHz, CDCl ) δ 2.40ꢀ2.46 (m, 4H), 2.51 (t, J = 7.0 Hz, 2H), 3.16ꢀ3.20 (m, 2H), 3.24ꢀ3.29
3
(m, 2H), 3.64ꢀ3.69 (m, 4H), 4.06 (t, J = 6.6 Hz, 2H), 7.17ꢀ7.24 (m, 4H), 7.26ꢀ7.30 (m, 2H), 7.35 (dd, J
=
8.6, 2.0 Hz, 1H), 7.89 (d, J= 1.6 Hz, 1H).
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To a round bottom flask charged with 4ꢀ(2ꢀ(5ꢀbromoꢀ2ꢀphenethylꢀ1Hꢀbenzo[d]imidazolꢀ1ꢀ
yl)ethyl)morpholine (506 mg, 1.22 mmol), was added sequentially (3,5ꢀdimethylisoxazolꢀ4ꢀyl)boronic
acid (260 mg, 1.28 mmol), palladium acetate (17 mg, 0.08 mmol), RuPHOS (CAS 787618ꢀ22ꢀ8; 73
mg, 0.15 mmol), sodium carbonate (262 mg, 2.47 mmol), and ethanol (6.7 mL, 0.18 M). The mixture
was sparged with argon for 5 mins, then heated to 85 °C for 1.5 hours under nitrogen. The mixture was
then cooled to rt and then filtered through a pad of celite and rinsed with ethanol. The filtrates were
then concentrated and then purified by flash silica chromatography (0ꢀ100% heptane in ethyl acetate)
6
7
8
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1
1
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2
3
4
5
6
7
8
9
0
and the major fraction corresponding to the desired product was collected and concentrated to give the
1
titled compound 2 (333 mg, 63%) as a viscous yellow oil: H NMR(400 MHz, CDCl ) δ 2.30 (s, 3H),
3
2
.43 (s, 3H), 2.45ꢀ2.61 (m, 6H), 3.24ꢀ3.35 (m, 4H), 3.63ꢀ3.81 (m, 4H), 4.07ꢀ4.25 (m, 2H), 7.17 (dd, J=
.2, 1.2 Hz, 1H), 7.22ꢀ7.26 (m, 3H), 7.29ꢀ7.33 (m, 2H), 7.43 (br s, 1H), 7.67 (s, 1H).
8
5-(3,5-Dimethylisoxazol-4-yl)-2-[2-(3-methoxyphenyl)ethyl]-1-(2-morpholin-4-ylethyl)-1H-
benzimidazole (3). Prepared according to general procedure A using 4ꢀ(3,5ꢀDimethylisoxazolꢀ4ꢀyl)ꢀNꢀ
2
0
(
2ꢀmorpholinoethyl)ꢀ2ꢀnitroaniline (100 mg, 0.29 mmol) and 3ꢀ(3ꢀmethoxyphenyl)propionaldehyde
1
giving 3 (10.6 mg, 8%) as yellow oil. H NMR (400 MHz, CDCl ): δ 2.29 (s, 3H), 2.42 (2, 3H), 2.44ꢀ
3
2
.47 (m, 4H), 2.59 (t, J = 6.8 Hz, 2H), 3.18ꢀ3.22 (m, 2H), 3.25ꢀ3.30 (m, 2H), 3.66ꢀ3.68 (m, 4H), 3.74
(s, 3H), 4.11 (t, J = 6.8 Hz, 2H), 6.77ꢀ6.79 (m, 2H), 6.84 (d, J = 7.6 Hz, 1H), 7.11 (dd, J = 8.0, 1.6 Hz,
H), 7.21ꢀ7.25 (m, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 1.2 Hz, 1H).
1
5-(3,5-Dimethylisoxazol-4-yl)-2-[2-(2-methoxyphenyl)ethyl]-1-(2-morpholin-4-ylethyl)-1H-
benzimidazole (4). Prepared according to general procedure A using 4ꢀ(3,5ꢀDimethylisoxazolꢀ4ꢀyl)ꢀNꢀ
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2
1
(
2ꢀmorpholinoethyl)ꢀ2ꢀnitroaniline (125 mg, 0.36 mmol) and 3ꢀ(2’ꢀmethoxyphenyl)propionaldehyde
1
giving 4 (5.3 mg, 4%) as a brown oil. H NMR (400 MHz, CD OD): δ 2.27 (s, 3H), 2.43 (s, 3H), 2.47
3
(
t, J = 4.8 Hz, 4H), 2.63 (d, J = 6.8 Hz, 2H), 3.17 (s, 2H), 3.24 (d, J = 8.0 Hz, 2H), 3.63 (t, J = 4.4 Hz,
0
1
2
3
4
5
6
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8
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1
2
3
4
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4H), 3.81 (s, 3H), 4.21 (d, J = 6.4 Hz, 2H), 6.84 (d, J = 0.8 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 7.08 (s,
+
1
H), 7.22 (dd, J = 4.0, 0.8 Hz, 2H), 7.54ꢀ7.58 (m, 2H); LCꢀMS m/z 461 (M+1) .
2-[2-(3,4-Dimethoxyphenyl)ethyl]-5-(3,5-dimethylisoxazol-4-yl)-1-(2-morpholin-4-ylethyl)-1H-
benzimidazole (5). Prepared according to general procedure A using 4ꢀ(3,5ꢀDimethylisoxazolꢀ4ꢀyl)ꢀNꢀ
(2ꢀmorpholinoethyl)ꢀ2ꢀnitroaniline
(52
mg,
0.15
mmol)
and
3ꢀ(3,4ꢀ
2
2
1
dimethoxyphenyl)propionaldehyde giving 5 (21.2 mg, 29%) as a brown solid. H NMR (400 MHz,
CDCl ): δ 2.30 (s, 3H), 2.43 (s, 3H), 2.45ꢀ2.47 (m, 4H), 2.57 (t, J = 6.8 Hz, 2H), 3.18ꢀ3.25 (m, 4H),
3
3.67ꢀ3.69 (m, 4H), 3.72 (s, 3H), 3.86 (s, 3H), 4.08 (t, J = 6.8 Hz, 2H), 6.67 (s, 1H), 6.77ꢀ6.82 (m, 2H),
7
.12 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.64 (s, 1H).
2
-[2-(2,4-Dimethoxyphenyl)ethyl]-5-(3,5-dimethylisoxazol-4-yl)-1-(2-morpholin-4-ylethyl)-1H-
benzimidazole (6). Prepared according to general procedure A using 4ꢀ(3,5ꢀDimethylisoxazolꢀ4ꢀyl)ꢀNꢀ
2ꢀmorpholinoethyl)ꢀ2ꢀnitroaniline (107 mg, 0.31 mmol) and 3ꢀ(2’,4’ꢀ
(
2
3
1
dimethoxyphenyl)propionaldehyde giving 6 (6.3 mg, 5%) as a brown oil. H NMR (400MHz,
CD OD): δ 2.28 (s, 3H), 2.45 (s, 3H), 3.22 (s, 4H), 3.43 (s, 6H), 3.57 (s, 4H), 3.77 (s, 4H), 4.02 (s,
3
+
4
H), 6.44ꢀ6.52 (m, 2H), 7.19 (s, 1H), 7.65ꢀ7.73 (m, 2H), 8.20 (s, 1H); LCꢀMS m/z 491 (M+1) .
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-(3,5-Dimethylisoxazol-4-yl)-2-[(4-methoxyphenoxy)methyl]-1-(2-morpholin-4-ylethyl)-1H-
3
4
5
6
7
8
9
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1
1
1
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benzimidazole (7). Prepared according to general procedure A using 4ꢀ(3,5ꢀDimethylisoxazolꢀ4ꢀyl)ꢀNꢀ
23
(
2ꢀmorpholinoethyl)ꢀ2ꢀnitroaniline (93 mg, 0.27 mmol) and (4ꢀmethoxyphenoxy)acetaldehyde giving
1
0
1
2
3
4
5
6
7
8
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0
1
2
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9
0
7 (9 mg, 7%) as a colorless solid. H NMR (400MHz, CD OD): δ 2.27 (s, 3H), 2.43 (s, 3H), 2.51 (m,
3
4
H), 2.79ꢀ2.83 (m, 2H), 3.64 (m, 4H), 3.75 (s, 3H), 4.53ꢀ4.56 (m, 2H), 5.42 (s, 2H), 6.88 (d, J = 8.8
Hz, 2H), 7.04 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.0 Hz, 1H), 7.60 (s, 1H), 7.69 (d, J = 8.4Hz, 1H); LCꢀ
+
MS m/z 463 (M+1) .
5
-(3,5-Dimethylisoxazol-4-yl)-2-phenoxymethyl-1-(2-morpholin-4-ylethyl)-1H-benzimidazole (8).
Prepared according to general procedure A using using 4ꢀ(3,5ꢀDimethylisoxazolꢀ4ꢀyl)ꢀNꢀ(2ꢀ
morpholinoethyl)ꢀ2ꢀnitroaniline (127 mg, 0.37 mmol) and phenoxyacetaldehyde giving 8 (9 mg, 6 %)
1
as a light brown solid. H NMR (400MHz, CD OD): δ 2.29 (s, 3H), 2.46 (s, 3H), 3.48 (m, 4H), 3.76ꢀ
3
3
2
.80 (m, 2H), 4.04 (m, 4H), 5.12ꢀ5.15 (m, 2H), 5.83 (s, 2H), 7.11 (t, J = 7.5 Hz, 1H), 7.28ꢀ7.30 (m,
H), 7.40ꢀ7.44 (m, 2H), 7.66 (d, J = 8.0 Hz, 1H), 7.80 (s, 1H), 8.24 (d, J = 8.4 Hz, 1H); LCꢀMS m/z
+
433 (M+1) .
4
-{2-[5-(3,5-Dimethylisoxazol-4-yl)-1-(2-morpholin-4-ylethyl)-1H-benzimidazol-2-yl]ethyl}-
phenol (9). A mixture of 4ꢀ(2ꢀ(5ꢀ(3,5ꢀDimethylisoxazolꢀ4ꢀyl)ꢀ2ꢀ(4ꢀ((tetrahydroꢀ2Hꢀpyranꢀ2ꢀ
yl)oxy)phenethyl)ꢀ1Hꢀbenzo[d]imidazolꢀ1ꢀyl)ethyl)morpholine (80 mg, 0.15 mmol) in ethanol (1.3
mL) was added concentrated HCl (0.76 mL). The mixture was stirred at ambient temperature for 4
hours. The resulting mixture was concentrated in vacuo and purified by prepꢀTLC to give 9 (55.8 mg,
1
8
3%) as a yellow solid. H NMR (400MHz, CDCl ): δ 2.27 (m, 3H), 2.40 (s, 3H), 2.49 (m, 4H), 2.64ꢀ
3
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2
.65 (m, 2H), 3.18 (m, 4H), 3.69 (m, 4H), 4.15 (m, 2H), 6.73 (d, J = 7.6 Hz, 2H), 7.01 (d, J = 8.0 Hz,
2
H), 7.12 (d, J = 7.6 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.59 (s, 1H), phenolic proton not observed;
+
LCꢀMS m/z 447 (M+1) .
0
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0
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2
3
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5-(3,5-Dimethylisoxazol-4-yl)-2-[2-(4-ethoxyphenyl)ethyl]-1-(2-morpholin-4-ylethyl)-1H-
benzimidazole (10). A mixture of 9 (45 mg, 0.10 mmol) and iodoethane (19 mg, 0.12 mmol) and
K CO (41 mg, 0.30 mmol) in acetonitrile (2 mL) was stirred at 50 °C for 5 hours. The reaction
2
3
1
mixture was filtered and purified by prepꢀHPLC to give 10 (1.6 mg, 3%) as a light yellow solid. H
NMR (400MHz, CDCl ): δ 1.42 (t, J = 7.2 Hz, 3H), 2.31 (s, 3H), 2.44 (s, 3H), 2.46ꢀ2.49 (m, 4H), 2.60
3
(t, J = 7.2 Hz, 2H), 3.18ꢀ3.24 (m, 4H), 3.67ꢀ3.69 (m, 4H), 4.02 (q, J = 7.2 Hz, 2H), 4.07ꢀ4.13 (m, 2H),
6
.84 (d, J = 8.8 Hz, 2H), 7.12ꢀ7.16 (m, 3H), 7.35 (d, J = 8.0 Hz, 1H), 7.64 (s, 1H); LCꢀMS m/z 475
+
(M+1) .
5
-(3,5-Dimethylisoxazol-4-yl)-1-(2-morpholin-4-ylethyl)-2-[2-(4-propoxyphenyl)ethyl]-1H-
benzimidazole (11). A mixture of 9 (45 mg, 0.10 mmol) and iodopropane (20 mg, 0.12 mmol) and
K CO (41 mg, 0.30 mmol) in acetonitrile (2 mL) was stirred at 50 °C overnight. The reaction mixture
2
3
1
was filtered and purified by prepꢀHPLC to give 11 (4.9 mg, 10%) as a yellow solid. H NMR
(
(
(
400MHz, CDCl ): δ 1.04 (t, J = 7.6 Hz, 3H), 1.80ꢀ1.83 (m, 2H), 2.31 (s, 3H), 2.44 (s, 3H), 2.47ꢀ2.49
3
m, 4H), 2.58ꢀ2.61 (m, 2H), 3.18ꢀ3.24 (m, 4H), 3.67ꢀ3.69 (m, 4H), 3.91 (t, J = 6.8 Hz, 2H), 4.10ꢀ4.13
t, J = 7.2 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 7.12ꢀ7.15 (m, 3H), 7.35 (d, J = 8.4 Hz, 1H), 7.64 (s, 1H);
+
LCꢀMS m/z 489 (M+1) .
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a
Scheme 2. General procedure B: Synthesis of 3-arylpropionaldehydes.
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
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3
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5
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5
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0
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2
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5
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7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
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5
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7
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2
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5
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8
9
0
a
o
Reaction conditions: (a) allyl alcohol, Pd(OAc) , nꢀBu4Cl, NaHCO , DMF, 80 C, 8 h
2
3
Allyl alcohol (2.0 eq) was added to a solution of the aryl iodide (1.0 eq), Pd(OAc) (0.25 eq), Bu NCl
2
4
(
1.33 eq) and NaHCO (3.0 eq) in DMF (0.2M). The mixture was allowed to stir at 80 °C for 8 hours.
3
The mixture was filtered and the resulting filtrate was concentrated in vacuo. The residue was purified
by flash chromatography giving the desired 3ꢀarylpropionaldhydes as described below.
5
-(3,5-Dimethylisoxazol-4-yl)-2-[2-(4-isopropoxyphenyl)ethyl]-1-(2-morpholin-4-ylethyl)-1H-
benzimidazole (12). 3ꢀ(4ꢀIsopropoxyphenyl)propionaldehyde was prepared according to general
procedure B using 4ꢀ(isopropoxy)iodobenzene (200 mg, 0.76 mmol). Purification by flash column
chromatography
(5ꢀ10%
ethyl
acetate
in
petroleum
ether)
gave
3ꢀ(4ꢀ
Isopropoxyphenyl)propionaldehyde (55 mg, 37%) as yellow liquid which exactly matched the previous
24
data provided for this compound. The material was used directly in the next step.
Compound 12 was prepared according to general procedure A using 4ꢀ(3,5ꢀDimethylisoxazolꢀ4ꢀyl)ꢀNꢀ
(2ꢀmorpholinoethyl)ꢀ2ꢀnitroaniline (468 mg, 1.35 mmol) and 3ꢀ(4ꢀIsopropoxyphenyl)propionaldehyde
1
giving 12 (5 mg, 4%) as a yellow solid. H NMR (400MHz, CDCl ): δ 1.33 (d, J = 6.0 Hz, 6H), 2.30
3
(s, 3H), 2.43 (s, 3H), 2.44ꢀ2.48 (m, 4H), 2.58ꢀ2.61 (m, 2H), 3.15ꢀ3.25 (m, 4H), 3.65ꢀ3.69 (m, 4H),
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.09ꢀ4.12 (m, 2H), 4.51 (sept. J = 6.0 Hz, 1H), 6.83 (d, J = 8.5 Hz, 2H), 7.10ꢀ7.14 (m, 3H), 7.35 (d, J
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=
8.0 Hz, 1H), 7.63 (s, 1H); LCꢀMS m/z 489 (M+1) .
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-(3,5-Dimethylisoxazol-4-yl)-1-(2-morpholin-4-ylethyl)-2-[2-(4-butyloxyphenyl)ethyl]-1H-
benzimidazole (13). To a mixture of 9 (0.075 mmol) and potassium tertꢀbutoxide (0.15 mmol) in
DMA (0.7 mL) was added iodobutane (0.11 mmol). The mixture was allowed to stir at 60 °C for 1
hour. The reaction mixture was cooled to room temperature concentrated in vacuo. The residue was
+
purified by prepꢀHPLC to give 13. LCꢀMS m/z 502 (M+1) .
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-(3,5-Dimethylisoxazol-4-yl)-1-(2-morpholin-4-ylethyl)-2-[2-(4-pentyloxyphenyl)ethyl]-1H-
benzimidazole (14). To a mixture of 9 (0.075 mmol) and potassium tertꢀbutoxide (0.15 mmol) in
DMA (0.7 mL) was added bromopentane (0.11 mmol). The mixture was allowed to stir at 60 °C for 1
hour. The reaction mixture was cooled to room temperature concentrated in vacuo. The residue was
+
purified by prepꢀHPLC to give 14. LCꢀMS m/z 517 (M+1) .
5
-(3,5-Dimethylisoxazol-4-yl)-1-(2-morpholin-4-ylethyl)-2-{2-[4-(trifluoromethoxy)phenyl]-
ethyl}-1H-benzimidazole (15). Prepared according to general procedure A using 4ꢀ(3,5ꢀ
Dimethylisoxazolꢀ4ꢀyl)ꢀNꢀ(2ꢀmorpholinoethyl)ꢀ2ꢀnitroaniline (72 mg, 0.21 mmol) and 3ꢀ(4ꢀ
25
1
trifluoromethoxyphenyl)propionaldehyde giving 15 (18.2 mg, 17%) as yellow oil. H NMR
(400MHz, CDCl ): δ 2.29 (s, 3H), 2.42 (s, 3H), 2.43ꢀ2.46 (m, 4H), 2.60 (t, J = 6.8 Hz, 2H), 3.17ꢀ3.20
3
(m, 2H), 3.29ꢀ3.33 (m, 2H), 3.64ꢀ3.66 (m, 4H), 4.12 (t, J = 6.8 Hz, 2H), 7.11ꢀ7.15 (m, 3H), 7.21 (s,
2
H), 7.34 (d, J = 8.4 Hz, 1H), 7.62 (s, 1H).
28
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Page 29 of 51
Journal of Medicinal Chemistry
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4-{2-[5-(3,5-Dimethylisoxazol-4-yl)-1-(2-morpholin-4-ylethyl)-1H-benzimidazol-2-yl]ethyl}-
benzonitrile (16). Prepared according to general procedure A using 4ꢀ(3,5ꢀDimethylisoxazolꢀ4ꢀyl)ꢀNꢀ
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(
2ꢀmorpholinoethyl)ꢀ2ꢀnitroaniline (42 mg, 0.12 mmol) and 4ꢀ(3ꢀoxopropyl)benzonitrile giving 16
1
(
20 mg, 36%) as a brown solid. H NMR (400MHz, CDCl ): δ 2.30 (s, 3H), 2.43 (s, 3H), 2.49 (s, 4H),
3
2
.66 (t, J = 6.8 Hz, 2H), 3.22ꢀ3.26 (m, 2H), 3.38ꢀ3.42 (m, 2H), 3.67ꢀ3.69 (m, 4H), 4.18 (t, J = 6.8 Hz,
2H), 7.15 (d, J = 8.4 Hz, 1H), 7.37ꢀ3.40 (m, 3H), 7.60ꢀ7.64 (m, 3H).
a
Scheme 3: General Procedure C: Synthesis of N-arylated systems.
a
o
Reaction conditions: (a) R NH Cl, NaOtBu, Pd(OAc) , RuPHOS, THF, 70 C, 12 h.
2
2
2
4
-{2-[5-(3,5-Dimethylisoxazol-4-yl)-1-(2-morpholin-4-ylethyl)-1H-benzimidazol-2-yl]ethyl}-N,N-
dimethylaniline (17). Intermediate 42 was prepared according to general procedure A using 4ꢀ(3,5ꢀ
Dimethylisoxazolꢀ4ꢀyl)ꢀNꢀ(2ꢀmorpholinoethyl)ꢀ2ꢀnitroaniline (541 mg, 1.60 mmol) and 3ꢀ(4ꢀ
2
7
bromophenyl)propionaldehyde giving 2ꢀ[2ꢀ(4ꢀBromophenyl)ethyl]ꢀ5ꢀ(3,5ꢀdimethylisoxazolꢀ4ꢀyl)ꢀ1ꢀ
1
(2ꢀmorpholinꢀ4ꢀylethyl)ꢀ1Hꢀbenzimidazole 42 (400 mg, 50%) as a light brown solid. H NMR
(
400MHz, CDCl ): δ 2.30 (s, 3H), 2.43 (s, 3H), 2.45ꢀ2.47 (m, 4H), 2.57ꢀ2.62 (m, 2H), 3.16ꢀ3.20 (m,
3
29
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