A Unique Approach to Design Potent and Selective Cyclic Adenosine Monophosphate Response Element Binding Protein, Binding Protein (CBP) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b01372

The epigenetic regulator CBP/P300 presents a novel therapeutic target for oncology. Previously, we disclosed the development of potent and selective CBP bromodomain inhibitors by first identifying pharmacophores that bind the KAc region and then building into the LPF shelf. Herein, we report the "hybridization" of a variety of KAc-binding fragments with a tetrahydroquinoline scaffold that makes optimal interactions with the LPF shelf, imparting enhanced potency and selectivity to the hybridized ligand. To demonstrate the utility of our hybridization approach, two analogues containing unique Asn binders and the optimized tetrahydroquinoline moiety were rapidly optimized to yield single-digit nanomolar inhibitors of CBP with exquisite selectivity over BRD4(1) and the broader bromodomain family.

Full text of DOI:10.1021/acs.jmedchem.7b01372

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Article
A Unique Approach to Design Potent and Selective
Cyclic Adenosine Monophosphate Response Element
Binding Protein, Binding Protein (CBP) Inhibitors
Sarah M. Bronner, Jeremy M. Murray, F. Anthony Romero, Kwong Wah Lai, Vickie Tsui, Patrick Cyr, Maureen
H. Beresini, Gladys de Leon Boenig, Zhongguo Chen, Edna F. Choo, Kevin R. Clark, Terry D. Crawford,
Hariharan Jayaram, Susan Kaufman, Ruina Li, Yingjie Li, Jiangpeng Liao, Xiaorong Liang, Wenfeng Liu,
Justin Q. Ly, Jonathan Maher, John S. Wai, Fei Wang, Aijun Zheng, Xiaoyu Zhu, and Steven R. Magnuson
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01372 • Publication Date (Web): 20 Nov 2017
Downloaded from http://pubs.acs.org on November 20, 2017
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A Unique Approach to Design Potent and Selective Cyclic Adenosine Monophosphate
Response Element Binding Protein, Binding Protein (CBP) Inhibitors
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†
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Sarah M. Bronner, * Jeremy Murray, F. Anthony Romero, Kwong Wah Lai, Vickie Tsui,
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Patrick Cyr, Maureen H. Beresini, Gladys de leon Boenig, Zhongguo Chen, Edna F. Choo,
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Kevin R. Clark, Terry D. Crawford, Hariharan Jayaram, Susan Kaufman, Ruina Li, Yingjie
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‡
Li, Jiangpeng Liao, Xiaorong Liang, Wenfeng Liu, Justin Ly, Jonathan Maher, John Wai,
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Fei Wang, Aijun Zheng, Xiaoyu Zhu, Steven Magnuson
†
Genentech, Inc. 1 DNA Way, South San Francisco, California 94080, United States
‡
Wuxi Apptec Co., Ltd., 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131,
People’s Republic of China
§
Editas Medicine, Inc., 11 Hurley Street, Cambridge, Massachusetts 02141, United States
Abstract
The epigenetic regulator CBP/P300 presents a novel therapeutic target for oncology. Previously,
we reported the development of potent and selective CBP bromodomain inhibitors by first
identifying pharmacophores that bind the KAc region, and then building into the LPF shelf.
Herein we report the “hybridization” of a variety of KAcꢀbinding fragments with a
tetrahydroquinoline scaffold that makes optimal interactions with the LPF shelf, imparting
enhanced potency and selectivity to the hybridized ligand. To demonstrate the utility of our
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hybridization approach, two analogues containing unique Asn binders and the optimized
tetrahydroquinoline moiety were rapidly optimized to yield singleꢀdigit nanomolar inhibitors of
CBP with exquisite selectivity over BRD4(1) and the broader bromodomain family.
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Keywords: Epigenetics, CBP, CREBBP, P300, bromodomain, bromodomain inhibitor
Introduction
1
,2
Epigenetics is an exciting field of research with unharnessed therapeutic potential.
Applications to human health offered by the ability to modify gene expression include cancer,
–
3 9
aging, neuropsychiatric disease, and obesity.
While prospective therapies that result in
1
0
permanent and potentially inheritable modification of chemical marks on DNA are associated
11
with greater risk, epigenetic mechanisms that modify histones and alter rates of transcription
1
2
represent attractive therapeutic targets.
Acetylation of lysine residues on histones is a reversible postꢀtranslational modification
PTM) that loosens the DNAꢀhistone interaction, thus altering the chromatin structure and
(
–
13 15
allowing for upregulation of transcription.
Proteins that recognize PTMs such as acetylated
lysine marks are collectively referred to as readers, and are involved in the recruitment of
–
16 18
additional proteins to form large transcriptional complexes.
Bromodomains (BrDs) are
arguably the most well studied class of epigenetic readers, and have recently received
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19 23
considerable attention as promising therapeutic targets.
Cyclic adenosine monophosphate
response element binding protein, binding protein (CBP, CREBBP, or CREBꢀbinding protein)
and adenoviral E1A binding protein of 300 kDa (P300 or EP300), two closely related multiꢀ
domain transcription activating proteins containing a histone acetyltransferase (HAT) as well as
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a bromodomain, have an important role in histone acetylation.
The CBP/P300 coactivator
family (hereafter together referred to as “CBP”) has been implicated in various cancers including
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leukemia and prostate cancer.
Furthermore, CBP has been found to regulate expression of
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MYC,
relevance to cancer immunotherapy,
finding suitable CBP inhibitors. Despite the therapeutic opportunities offered by CBP, few potent
a transcription factor that is upregulated in many human cancers, and also has
3
5,36
thus contributing towards a rapidly growing interest in
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and selective CBP BrD inhibitors have been reported.
Recently, Genentech (GNE) identified CBP bromodomain inhibitor GNEꢀ781 (3, Figure 1),
5
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which has unparalleled potency and selectivity over other bromodomains. Screening hit 1
provided a starting point for the development of the pyrazolopiperidine series of CBP inhibitors,
and additional potency and stability optimization led to identification of in vivo tool compound
54
GNEꢀ272 (2). Ultimately, cell potency and selectivity over the bromodomain 1 of the
bromodomainꢀcontaining
protein
4
[BRD4(1)]
were
further
improved
via
modification/rigidification of the aniline, exploration of the pyrazole substituent, and by utilizing
a methyl urea in place of the Nꢀacetyl group (2 → 3).
O
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H
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H
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GNE-272 (2)
GNE-781 (3)
CBP IC50 = 620 nM
BRD4(1) IC50 = 60,000 nM
BRD4(1) IC50 / CBP IC = 97
CBP IC50 = 20 nM
BRD4(1) IC50 = 13,000 nM
CBP IC50 = 0.94 nM
BRD4(1) IC50 = 5,100 nM
BRD4(1) IC50 / CBP IC50 = 650
BRD4(1) IC50 / CBP IC50 = 5,400
50
Figure 1. Discovery and optimization of pyrazolopiperidine CBP inhibitors.
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We previously reported a coꢀcrystal structure of CBP BrDꢀbound 3, which is illustrative
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of the interactions that govern potency and selectivity. The interaction between Asn1168 and
the urea moiety mimics the endogenous ligands, which are acetylated lysine residues. The
selectivity over other bromodomains is attributed in part to the halfꢀchair conformation of the
piperidine ring in the tetrahydroquinoline (THQ) and the THQ sharing shape complementarity
with the base of the LPF shelf (Leu1109, Pro1110, and Phe1111). In BRD4(1), the WPF shelf
contains a more sterically demanding Trp81 compared to the Leu1109 residue of CBP, and this
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difference provides an opportunity for selectivity. Additionally, a favorable interaction between
the negatively polarized fluorine atoms in the difluoromethyl and the guanidinium group of
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Arg1173 is observed. This interaction is likely to contribute to both CBP potency and improved
selectivity over BRD4 since the equivalent residue to Arg1173 is Asp145 and Glu438 in
BRD4(1) and BRD4(2), respectively.
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Figure 2. Coꢀcrystal structure of CBP BrDꢀbound 3 (PDBꢀID 5W0E).
Results and Discussion
During the course of development of 3, we sought to develop a structurally unique
second chemical series with comparable potency and selectivity to that of our lead in order to
minimize risk of bringing only one series forward. Intrigued with the possibility of introducing
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diversity in the Asnꢀbinding pharmacophore, we decided to reexamine the benzodiazepinone
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core of CBP inhibitor 4, which was previously developed during our program (Figure 3). The
benzodiazepinone scaffold had a clear vector for the THQꢀCF Hꢀpyrazole, and exchanging the
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indazole in 4 with the optimized LPF binder led to “THQ hybrid” 5. This modification resulted
in a ~9ꢀfold increase in CBP biochemical potency as measured by the timeꢀresolved fluorescence
energy transfer (TRꢀFRET) assay, and an increase in selectivity over all other bromodomains
assayed including bromodomainꢀcontaining protein 9 (BRD9) and cat eye syndrome critical
protein 2 (CECR2). Furthermore, this result demonstrated that attaching the THQ moiety onto an
Asnꢀbinding pharmacophore, in this case the benzodiazepinone, is an effective strategy to
improve selectivity and potency.
Figure 3. Effect of hybridization of benzodiazepinone core of 4 with THQꢀCF Hꢀpyrazole on
2
CBP potency and selectivity.
As part of our screening efforts we executed a high throughput screen (HTS) targeting
alternative Asnꢀbinding cores. Cores with high ligand efficiency (LE) were selected as possible
analogues for hybridization. In each case, the coꢀcrystal structure of the CBPꢀbound ligand was
overlaid with the coꢀcrystal structure of 3 to evaluate whether the core had an appropriate vector
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for the THQ functionality. When coꢀcrystal structures were not available, computational docking
was utilized to approximate the binding orientation of the HTS hit within the CBP
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6
bromodomain. Targets were selected based on synthetic ease and whether the THQ fragment
was predicted to have a similar spatial orientation in the receptor to that of lead molecule 3. For
example, a crystal structure of ligand efficient HTS hit 6 when compared to lead compound 3
indicated that appendage of the THQ onto C5 of the Asnꢀbinding indole core might lead to a
hybrid with the desired geometry (Figure 4a). Thiazolone 7 was identified as a hit in the primary
HTS (Figure 4b). Although this fragment had a CBP IC >100 µM in the reconfirmation screen,
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was nonetheless a core of interest because it was a frequently appearing motif in the HTS and
also because similar scaffolds are known inhibitors of the bromodomains of tripartite motifꢀ
containing 24 protein (TRIM24) and bromodomain and PHD fingerꢀcontaining protein
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(
BRPF). Docking of thiazolone 7 suggested two possible binding orientations and two potential
vectors for the THQ moiety. To our delight, THQ hybrids 16 and 17 led to increases in potency
from 99,000 nM to 9.5 nM in the case of the indole, and >100,000 nM to 69 nM with the
thiazolone (Figure 5) relative to the parent HTS hit.
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Figure 4a. Overlay of coꢀcrystal structures of CBP BrDꢀbound HTS hit 6 (purple; PDBꢀID
6AXQ) and 3 (cyan; PDBꢀID 5W0E).
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NH2
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CBP IC50 = >100,000 nM
BRD4(1) IC = >100,000 nM
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Figure 4b. Docked structures of HTS hit 7 (orange and pink) overlaid with coꢀcrystal structure
of CBP BrDꢀbound 3 (cyan; PDBꢀID 5W0E).
In a similar fashion, HTS hits 8–13 were combined with the THQ moiety to create
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hybrids containing their original Asnꢀbinging cores (Figure 5). In some cases, the Asnꢀbinding
moiety present in the core was slightly modified to provide improved potency utilizing our prior
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structure activity relationship (SAR) learnings. The resulting THQ hybrids 18
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25 demonstrated
varying magnitudes of potency and selectivity increases, but nonetheless led to improved CBP
inhibitors. Hybridization even rescued scaffolds that originally displayed selectivity for
BRD4(1); hit 10, which showed a 4.4ꢀfold preference for BRD4(1)ꢀinhibition, was transformed
into a 530ꢀfold selective CBP inhibitor via hybridization. Interestingly, the pharmacophore of 23,
although derived from the HTS hit 13, also closely resembles that of early weak CBP inhibitor
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MS2126, reported by Zhou. The same design exercise was performed with published CBP
4
3
inhibitors with an equally fruitful outcome; hybridization of 14 increased the CBP potency by
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.5ꢀfold, while modification of 15 resulted in a 34ꢀfold improvement.
Xꢀray coꢀcrystal structures of CBPꢀbound hybrids 16 and 17 were obtained, and are
depicted in Figure 6 overlaid with lead 3. The comparison of the crystal structures with different
Asnꢀbinding motifs indicates that the binding pocket of the CBP bromodomain can
accommodate some flexibility in the location of the Asnꢀbinding core, but the THQꢀCF Hꢀ
2
pyrazole substituents have very similar orientations (Figure 6). This suggests that
complementarity between THQ and the subꢀpocket on CBP enhances both activity and
selectivity in most cases. This structural comparison illustrates the crucial effect of the LPF shelf
on selectivity and potency of bromodomain inhibitors and exemplifies the reduced importance of
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the KAcꢀbinding region. While the latter is necessary and is the anchor of the inhibitor, this
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binding region only procures modest selectivity and potency.
Asn Binder
THQ Hybrid
Asn Binder
THQ Hybrid
H
O
O
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NH N
N
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N
HN
O
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16
CBP IC50 = 9.5 nM
BRD4(1) IC50 = 12,000 nM
Fold Selectivity = 1,300
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21
CBP IC50 = 99,000 nM
BRD4(1) IC50 = >100,000 nM
Fold Selectivity = >1
LE = 0.43
CBP IC50 = 270 nM
CBP IC50 = 120 nM
BRD4(1) IC50 = >20,000 nM
Fold Selectivity = >170
BRD4(1) IC50 = >20,000 nM
Fold Selectivity = >74
LE = 0.31
O
S
N
F
N
N
F
S
N
NH2
N
F
NH
N
H
F
O
N
N
N
N
N
N
H
N
N
N
O
N
O
7
17
CBP IC50 = 69 nM
BRD4(1) IC50 = 18,000 nM
Fold Selectivity = 260
12
22
CBP IC50 = 1,400 nM
BRD4(1) IC50 = >20,000 nM
Fold Selectivity = >14
CBP IC50 = >100,000 nM
BRD4(1) IC50 = >100,000 nM
Fold Selectivity = n/a
LE = n/a
CBP IC50 = 32,000 nM
BRD4(1) IC50 = >84,000 nM
Fold Selectivity = >2.6
LE = 0.35
N
O
N
F
O
N
O
O
F
N
F
O
NH
O
N
N
F
O
O
N
S
N
N
HN
H
N
N
O
F
HO
O
8
18
CBP IC50 = 34 nM
BRD4(1) IC50 = >20,000 nM
Fold Selectivity = >590
13
23
CBP IC50 = 230 nM
CBP IC50 = 3,000 nM
CBP IC50 = 450 nM
BRD4(1) IC50 = 6,900 nM
Fold Selectivity = >15
BRD4(1) IC50 = >20,000 nM
Fold Selectivity = >87
LE = 0.32
BRD4(1) IC50 = 74,000 nM
Fold Selectivity = 25
LE = 0.30
H
O
N
N
F
O
N
F
O
F
N
O
O
F
N
H
HN
N
N
N
O
N
N
Cl
O
N
N
NH
N
N
H
O
9
19
CBP IC50 = 47 nM
14
CBP IC50 = 580 nM
24
CBP IC50 = 61 nM
CBP IC50 = 540 nM
BRD4(1) IC50 = 52,000 nM
Fold Selectivity = 96
LE = 0.35
BRD4(1) IC50 = >20,000 nM
Fold Selectivity = >430
BRD4(1) IC50 = 13,000 nM
Fold Selectivity = 22
LE = 0.29
BRD4(1) IC50 = >20,000 nM
Fold Selectivity = 330
O
O
O
O
F
O
N
N
F
H
O
S
O
N
N
F
O
N
F
S
N
N
N
N
NH
O
N
N
N
O
O
1
0
20
CBP IC50 = 38 nM
15
CBP IC50 = 440 nM
25
CBP IC50 = 23,000 nM
CBP IC50 = 13 nM
BRD4(1) IC50 = 5,200 nM
Fold Selectivity = 0.23
LE = 0.30
BRD4(1) IC50 = >20,000 nM
Fold Selectivity = >530
BRD4(1) IC50 = 15,000 nM
Fold Selectivity = 34
LE = 0.26
BRD4(1) IC50 = 7,400 nM
Fold Selectivity = 570
Figure 5. Hybridization of HTS hits and known CBP inhibitors with THQ LPF binder.
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6
7
8
9
0
Figure 6. Overlay of coꢀcrystal structures of CBP BrDꢀbound hybrids 16 (magenta; PDBꢀID
6AY3), 17 (green; PDBꢀID 6AY5) and 3 (cyan; PDBꢀID 5W0E).
The hybridization studies described above provided a good starting point for rapid
development of structurally unique and potent CBP inhibitors. Hybridization of the indole and
thiazolone cores (6 → 16 and 7 → 17) resulted in two of the most dramatic increases in CBP
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biochemical potency (10,400ꢀfold and >1,450ꢀfold, respectively), while also imparting exquisite
selectivity over BRD4(1) (1,300ꢀfold and 260ꢀfold, respectively). It was anticipated that
additional SAR could easily render inhibitors with similar potency to that of our lead molecule 3.
Xꢀray coꢀcrystal structures also suggested that both hybrids had available vectors to grow into
the loop connecting helices B and C (BC loop), which previously had been found to be a
10
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17
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5
3
productive technique to improve potency (Figure 7a/b). This further supported the decision to
pursue the indole and thiazolone series.
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O
S
F
N
F
N
N
N
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5
6
7
8
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0
1
7
Figure 7a. Overlay of coꢀcrystal structures of CBP BrDꢀbound thiazolone hybrid 17
(
pink; PDBꢀID 6AY5) and 3 (cyan; PDBꢀID 5W0E).
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H
N
F
H
F
N
N
N
O
N
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17
18
19
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1
6
Figure 7b. Overlay of coꢀcrystal structures of CBP BrDꢀbound indole hybrid 16
(
magenta; PDBꢀID 6AY3) and 3 (cyan; PDBꢀID 5W0E).
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6
Improvement of potency and metabolic stability were the primary goals of optimization
of the thiazolone series; clearance of 17 as measured by mouse, rat, and human liver microsomeꢀ
predicted hepatic clearance assays (LM Clhep M / R / H) was undesirably high at 71, 39, and 17
mL/min/kg, respectively (Figure 8). Initially, SAR of the Asn binder was examined using de
novo design and ideas inspired by frequently appearing scaffolds in the HTS that had previously
been overlooked because of lower LE. While other pharmacophores did not yield enhanced
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affinity, introduction of the benzimidazolone Asn binder resulted in a CBP IC of 20 nM (26,
50
Figure 8). Expansion into the BC loop with morpholine 27 provided a good balance of
biochemical potency and selectivity over BRD4(1), while imparting modest in vitro stability
48,60
gains. Furthermore, 27 had excellent potency in a cellular assay of MYC expresssion
with an
EC of 12 nM.
50
O
N
N
O
S
O
N
O
N
F
F
F
N
F
N
F
N
F
N
N
N
N
N
N
N
N
1
7
26
CBP IC50 = 20 nM
27
CBP IC50 = 69 nM
CBP IC50 = 3.6 nM
BRD4(1) IC50 = 18,000 nM
Fold Selectivity = 260
BRD4(1) IC50 = 4,100 nM
Fold Selectivity = 210
BRD4(1) IC50 = 1,100 nM
Fold Selectivity = 310
LM Clhep M / R / H = 71 / 39 / 17 LM Clhep M / R / H = 61 / 38 / 16 LM Clhep M / R / H = 48 / 23 / 15
MYC EC50 = 1,000 nM
MYC EC50 = 12 nM
Figure 8. Optimization of the thiazoloneꢀTHQ hybrid.
Efforts to optimize the indole series also centered on improvement of potency and
metabolic stability. While indole hybrid 16 had moderate in vitro clearance (LM Clhep M / R / H
=
47 / 20 / 13 mL/min/kg), efforts to improve potency by growing into the BC loop by
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substitution at C7 of the indole resulted in decreased metabolic stability. To address these
limitations, a nitrogen walk around the indole skeleton was performed leading to the
identification of azaindole 28 that demonstrated improved CBP potency and metabolic stability
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11
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15
16
17
18
19
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53
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60
(
Figure 9). Azaindole 28 proved to be a suitable starting point for expansion into the critical BC
loop, and isopropyl substitution at C7 provided a good balance of biochemical potency (CBP
IC50 = 2.2 nM), and in vitro clearance (LM Clhep M / R / H = 50 / 25 / 13 mL/min/kg).
Furthermore, 29 displayed an impressive improvement in potency in the MYC assay relative to
hybrids 16 and 28 (EC = 53, 360, and 2,200 nM, respectively). Compound 29 was tested in an
5
0
in vivo pharmacokinetics (PK) experiment, exhibiting low clearance (18.2 mL/min/kg) and
moderate bioavailability (52%) when dosed to mice.
Figure 9. Optimization of the indoleꢀTHQ hybrid.
To probe the broader bromodomain selectivity of optimized THQ hybrids, 27 and 29
®
were submitted to the DiscoveRx BROMOscan selectivity panel, which assesses binding in a
competitive fashion by quantitating the reduction in capture of DNAꢀtagged bromodomain on
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5
5
6
61
ligandꢀcoated beads in the presence of compound. Benzimidazolone 27 proved to have
exquisite selectivity over other bromodomains. Out of 39 bromodomainꢀcontaining constructs
surveyed, only 4 had any activity other than CBP and P300, with BRD9 being the most potent
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0
off target (K = 3,600 nM). Other off target hits were BRD7, TRIM24(BrD), and BRD3(2), with
d
potencies of K = 5,100, 7,600, and 8,700 nM, respectively. Azaindole 29 also displayed
d
exceptional selectivity in the BROMOscan selectivity panel, and no activity was detected in any
potential bromodomain offꢀtargets (K > 10,000 nM).
d
Chemistry
The target compounds were synthesized as depicted in Schemes 1–15. In all cases, aryl
bromide coupling partners were obtained from either commercial sources or accessed through
diverse synthetic sequences of varying complexity. Buchwald coupling of intermediate 31 was
used to install the requisite tetrahydroquinoline, and conditions utilizing PdꢀPEPPSIꢀIPent
catalyst were found to be suitable for accessing most Buchwald adducts.
Scheme 1. Preparation of Compound 5.0
Scheme 2. Preparation of Compound 16.
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Scheme 3. Preparation of Compound 17.
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5
5
6
Scheme 4. Preparation of Compound 18.
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9
0
Scheme 5. Preparation of Compound 19.
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H
N
1. Br , AcOH
H
N
2
MeI
O
O
0
°C
rt, 3 h
LiHMDS
O
N
O
N
2
. Boc O, K CO
3
Br
DMF, 0 °C
2
2
CHCl , H O, rt
O
3
2
O
(
68% yield)
4
1
42
10
11
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16
17
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27
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48
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57
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59
60
(63% yield, 2 steps)
1. TFA
DCM, rt
N
O
N
O
O
N
N
2. Ac O, TEA
Br
2
Br
DCM, 0 °C
rt
O
O
4
3
44
(
80% yield, 2 steps)
31
N
F
Pd-PEPPSI-IPent
N
O
O
F
t-BuONa
N
N
dioxane, 120 °C
N
(
18% yield)
1
9
Scheme 6. Preparation of Compound 20.
Scheme 7. Preparation of Compound 21.
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1
2
3
4
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9
0
1
2
3
4
5
6
7
8
9
0
Scheme 8. Preparation of Compound 22.
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Scheme 9. Preparation of Compound 23.
Scheme 10. Preparation of Compound 24.
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9
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0
1
2
3
4
5
6
7
8
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0
1
2
3
4
5
6
7
8
9
0
Scheme 11. Preparation of Compound 25.
Scheme 12. Preparation of Compound 26.
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Scheme 13. Preparation of Compound 27.
Scheme 14. Preparation of Compound 28.
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9
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1
1
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1
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2
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5
5
5
5
5
6
O
H
H
N
H
N
Cl
CCl3
N
AlCl3
KOH
N
N
N
DCM, 48 °C
(81% yield)
Cl3C
MeOH, rt
(84% yield)
O
Br
O
Br
O
Br
66
67
68
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
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9
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1
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9
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
1
SEM
N
SEM
Pd-PEPPSI-IPent
F
N
Cs CO3
SEMCl, NaH
2
F
N
N
O
O
N
N
N
DMF, 0 °C
62% yield)
rt
O
O
dioxane, 110 °C
Br
(
(
42% yield)
69
70
SEM
N
F
MeNH •HCl
2
LiOH•H O
F
HATU, TEA
2
N
N
HO
N
N
MeOH, H O, rt
DMF, rt
2
O
(77% yield)
(98% yield)
71
SEM
N
H
N
F
F
H
F
TBAF
THF, 50 °C
20% yield)
H
N
F
N
N
N
N
N
N
N
O
O
N
N
(
7
2
28
Scheme 15. Preparation of Compound 29.
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6
Conclusion
This work demonstrates that a variety of unique Asn binders can be hybridized with the
0
1
2
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4
5
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8
9
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1
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9
0
THQꢀCF Hꢀpyrazole LPF fragment to create potent, selective, and structurally diverse CBP
2
probes. Structureꢀbased drug design (SBDD) provided a robust foundation for rapid optimization
of novel cores in the hybridization process, as evidenced by the development of the THQꢀ
benzimidazolone and THQꢀindole series; 43 hybrids were synthesized in the case of the
thiazolone/imidazolone series, while optimization of the indole series required the synthesis of
29 analogues. Generally in SBDD, larger structural modifications are associated with greater risk
of potency loss. The novelty in this work is that half of the inhibitor was replaced with a
completely new scaffold while potency was maintained; furthermore, the fragment necessary for
ligand recognition, the Asn binder, underwent significant alteration. While this was a highly
structurally enabled project, in many cases, crystal structures were not necessary and hybrids
could be effectively designed simply with computationally docked structures. Rarely is SBDD
executed in such a predictable manner.
Whereas previously disclosed bromodomain inhibitors were developed by first
identifying an Asnꢀbinding starting point and then building into the LPF shelf, our THQꢀ
hybridization strategy provides a complimentary and unique strategy to efficiently perform hitꢀ
toꢀlead optimization. While an Asnꢀbinding element was still necessary to maintain activity,
these findings illustrate that both CBP bromodomain potency and selectivity over other
bromodomains can be controlled via favorable interactions with the LPF shelf and adjacent
residues. Optimization of the LPF shelf/ligand interaction provides a robust potency and
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Page 29 of 84
Journal of Medicinal Chemistry
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selectivity handle for the development of CBP inhibitors that can be used to probe the biology of
this epigenetic target. Furthermore, we anticipate that this novel hybridization strategy will have
a broader application for the discovery of inhibitors of the medicinallyꢀrelevant bromodomain
family beyond CBP and P300.
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Experimental Procedures
General Methods. All solvents and reagents were used as obtained. NMR analysis performed in
a deuterated solvent with a Varian Avance 300ꢀMHz or Bruker Avance 400ꢀ or 500ꢀMHz NMR
spectrometers, referenced to trimethylsilane (TMS). Chemical shifts are expressed as δ units
using TMS as the external standard (in NMR description, s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, and br = broad peak). All coupling constants (J) are reported in Hertz.
Mass spectra were measured with a Finnigan SSQ710C spectrometer using an ESI source
coupled to a Waters 600MS high performance liquid chromatography (HPLC) system operating
in reverseꢀphase mode with an Xꢀbridge Phenyl column of dimensions 150 mm by 2.6 mm, with
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ꢀm sized particles. Preparatoryꢀscale silica gel chromatography was performed using mediumꢀ
pressure liquid chromatography (MPLC) on a CombiFlash Companion (Teledyne ISCO) with
RediSep normal phase silica gel (35−60 µm) columns and UV detection at 254 nm. Reverseꢀ
phase high performance liquid chromatography (HPLC) was used to purify compounds as
needed by elution from a Phenomenex GeminiꢀNX C18 column (30 × 50 mm, 5 micron) as
stationary phase using mobile phase indicated, and operating at a 60 mL/min flow rate on a
Interchim PuriFlash SFC UVꢀdirected prep instrument. Super critical fluid (SFC)
chromatography was used to purify compounds as needed by elution from a Phenomenex
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