The design and synthesis of 5- and 6-isoxazolylbenzimidazoles as selective inhibitors of the BET bromodomains

Article abstract of DOI:10.1039/c2md20189e

Simple 1-substituted 5- and 6-isoxazolyl-benzimidazoles have been shown to be potent inhibitors of the BET bromodomains with selectivity over the related bromodomain of CBP. The reported inhibitors were prepared from simple starting materials in two steps

Full text of DOI:10.1039/c2md20189e

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CONCISE ARTICLE
The design and synthesis of 5- and 6-
isoxazolylbenzimidazoles as selective inhibitors of the
BET bromodomains†‡
Cite this: Med. Chem. Commun., 2013,
4, 140
Duncan Hay,a Oleg Fedorov,a Panagis Filippakopoulos,a Sarah Martin,a
Martin Philpott,a Sarah Picaud,a David S. Hewings,b Sagar Uttakar,a
Tom D. Heightman,§ Stuart J. Conway, Stefan Knapp and Paul E. Brennan
a
b
a
a
*
Received 29th May 2012
Simple 1-substituted 5- and 6-isoxazolyl-benzimidazoles have been shown to be potent inhibitors of the
BET bromodomains with selectivity over the related bromodomain of CBP. The reported inhibitors were
prepared from simple starting materials in two steps followed by separation of the regioisomers or
regioselectively in three steps.
Accepted 10th August 2012
DOI: 10.1039/c2md20189e
www.rsc.org/medchemcomm
Bromodomains are discrete protein domains that selectively
recognize acetyl lysine in proteins.1 There are 61 bromodomains
in proteins that have a variety of functions including histone
acetyl transferases such as CBP (cyclic AMP response element-
binding protein, binding protein), methyl transferases, tran-
scriptional regulators such as BRD4 (bromodomain-containing
protein 4) and chromatin remodelling complexes.2 The BET
family of bromodomain containing proteins is comprised of
BRDT, BRD2, BRD3 and BRD4 each of which has two bromo-
domains that bind to acetylated histone tails.3 Recently BET
inhibitors have been shown to have potential for use in
inammatory disease, atherosclerosis, NUT midline carcinoma,
acute leukaemia and lymphoma.4–9
Triazoloazepines such as (+)-JQ1 1, iBET762 (structure not
shown) and isoxazoles such as compound 2 have been identi-
ed as potent BET inhibitors (Fig. 1).4,9,10 Since then, a number
of other templates incorporating the privileged isoxazole moiety
such as in compounds 3 and 4 have been identied by
researchers in the EpiNova group at GlaxoSmithKline.6,11,12 As
most known BET inhibitors are complex stereogenic molecules
it would be advantageous to nd simple, rapidly accessible
inhibitors that would be selective for the BET family as
Fig. 1 BET bromodomain inhibitors.
exemplied by BRD4 over proteins containing similar bromo-
domains, such as CBP.
It was thought that fusing a 5-membered ring to the 4-aryl-
3,5-dimethylisoxazole moiety of compound 2 (ref. 10) would
give access to previously unexploited substitution patterns in
known isoxazole-containing bromodomain inhibitors. Simple
5,6-bicyclic bromides 5a–c were transformed into isoxazoles by
either direct arylation of 3,5-dimethylisoxazole or Suzuki reac-
tion of the isoxazolylboronic acid to give compounds 6–8
(Scheme 1).10,13 When tested in an AlphaScreenꢀ assay using
isolated bromodomains, compounds 6 and 7 were modest
inhibitors of the rst bromodomain of BRD4 (BRD4(1)) with no
affinity for the CBP bromodomain whereas compound 8 had
comparable affinity for both bromodomains (Table 1).14
aStructural Genomics Consortium, Nuffield Department of Clinical Medicine,
University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford
OX3 7DQ, UK. E-mail: paul.brennan@sgc.ox.ac.uk
bDepartment of Chemistry, Chemistry Research Laboratory, University of Oxford,
Manseld Road, Oxford, OX1 3TA, UK
† This article is part of a MedChemComm ‘New Talents’ issue highlighting the
work of outstanding rising scientists in medicinal chemistry research.
‡ Electronic supplementary information (ESI) available: Synthetic details for
selected compounds and crystallographic data for compound 15. See DOI:
10.1039/c2md20189e
Scheme 1 Synthesis of indanone, indole and benzimidazole containing inhibi-
tors. Reagents and conditions: (a) 5a, 3,5-dimethylisoxazole, PdCl2, KOAc, N,N-
dimethylacetamide, 130 ꢀC, 50%; (b) 3,5-dimethylisoxazolylboronic acid,
Pd(PPh3)4, Na2CO3, DMF, H2O, 140 ꢀC mwave, 5b (12% yield) or 5c (13% yield).
§ Current address: Astex Pharmaceuticals, 436 Cambridge Science Park,
Cambridge CB4 0QA, UK, tom.heightman@astx.com.
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Table 1 Inhibition of BRD4 and CBP bromodomains by target compounds in
AlphaScreenꢀ
a
pIC50
Cpd
BRD4(1)
CBP
6
7
8
4.2 ꢁ 0.3 (2)
4.7 ꢁ 0.2 (4)
5.2 ꢁ 0.1 (9)
<4.6 (2)
<5.0 (2)
5.4 ꢁ 0.3 (3)
a
Mean pIC50 ꢁ standard error of the mean (number of determinations).
Scheme 2 Derivitization of indanone 6. Reagents and conditions: (a) NaBH4,
EtOH, 90%; (b) ArCH2Br, NaH, 76–81% or (i) Br(CH2)3OH, pTsOH, CH2Cl2, reflux,
45%; (ii) 2ꢀ amine, TEA, THF, reflux, 22–100%; (c) Mg, PhBr, Et2O, 60%.
Table 2 BRD4(1) and CBP bromodomain affinity of indanes 9–15 in a peptide
displacement AlphaScreenꢀ assay
Fig. 2 Compound 15 bound to BRD4(1). (A) Overlay of compound 15 (yellow,
PDB ID: 4GPJ) with H4K5AcK8Ac (purple, PDB ID: 3UVW);1 (B) residues and
conserved waters in BRD4(1) binding to compound 15; (C) surface view of
BRD4(1) in the protein–ligand complex overlaid with (+)-JQ1 (orange, PDB ID:
3MXF).
a
pIC50
Cpd
R
BRD4(1)
CBP
9
Bn
4.5 ꢁ 0.1 (4)
4.7 ꢁ 0.1 (2)
<5.0 (2)
ND
10
obtained when the indanone 6 was reacted with phenyl-
magnesium bromide to give the racemic tertiary indanol 15
(Scheme 2).
11
12
13
5.2 ꢁ 0.4 (2)
5.2 ꢁ 0.5 (2)
5.2 ꢁ 0.5 (2)
ND
ND
ND
The increase in affinity of compound 15 for BRD4(1) was
explained when it was co-crystallized with this bromodomain
(Fig. 2). As expected from previous X-ray structures of BRD4(1)-
bound isoxazole ligands,6,10 the isoxazole oxygen atom formed
one hydrogen bond to the conserved asparagine (N140) and the
nitrogen atom formed a water-mediated hydrogen bond to Y97.
The aryl ring occupied the hydrophobic groove of the WPF shelf,
dened by a W81, M149 and I146. Interaction with these three
hydrophobic residues is important for the affinity of all known
BET inhibitors (Fig. 1).
14
5.4 ꢁ 0.5 (2)
5.9 ꢁ 0.1 (6)
ND
15
NA
4.7 ꢁ 0.2 (3)
a
Mean pIC50 ꢁ standard error of the mean (number of determinations).
Encouraged by the excellent potency, selectivity and simple
The indanone 6 presented an attractive intermediate for synthesis of indanol 15, further effort was made to optimize the
further derivitization (Scheme 2). Reduction to the racemic compound. A range of substituted aryl Grignard reagents were
indanol followed by alkylation with benzyl bromide or 2-bro- reacted with the ketone, but yields were generally low and the
momethyl quinolone gave compounds 9 and 10. The amines products appeared to decompose during or following isolation.
10–14 were prepared by SN1 alkylation of the indanol with 3- Re-examination of stock solutions of the rst indanol 15 by
bromo-n-propanol followed by bromide substitution. The basic LCMS showed decomposition of the parent compound and a
centres of varying pKa in compounds 10–14 were designed with mass loss of 18 amu from the molecular ion peak. It is likely that
the potential to interact with an acidic residue on the edge of the doubly benzylic tertiary alcohols dehydrate to give the
the BRD4(1) binding pocket, D145.
indenes 16 (Scheme 3).15
Addition of an O-benzyl group in compound 9 did not
In an effort to prepare compounds that were as potent as 15
increase the affinity for either BRD4(1) or CBP bromodomains but without the chemical instability problem, attention returned
compared to the indanone 6 (Table 2). In this series only the to the benzimidazole 8. To mimic the potency enhancement of 15
piperazine derived compound 14 with the piperazine group was compared to 6, an aryl group was added to N1 of the benzimid-
more potent than compound 9. A large increase in BRD4(1) azole (Scheme 4). Ullman coupling with phenyl iodide gave a
affinity, without a corresponding increase in CBP affinity, was mixture of arylated benzimidazoles 17a and 17b that could be
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Table 3 Inhibition of BRD4 and CBP bromodomains by benzimidazoles in a
peptide-displacement AlphaScreenꢀ assay
a
pIC50
Cpd
R1
R2
BRD4(1)
CBP
Routec
Scheme 3 Decomposition of aryl indanols.
19ab
19bb
20
H
H
H
3-F
3-Cl
4-Cl
4-Cl
3-Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
5.0 ꢁ 0.3 (3)
5.5 ꢁ 0.2 (2)
6.0 ꢁ 0.1 (2)
6.2 ꢁ 0.1 (2)
5.9 ꢁ 0.1 (2)
6.1 ꢁ 0.1 (9)
5.6 ꢁ 0.1 (2)
6.0 ꢁ 0.1 (3)
5.8 ꢁ 0.4 (2)
6.1 ꢁ 0.3 (2)
6.1 ꢁ 0.5 (2)
6.2 ꢁ 0.4 (2)
4.9 ꢁ 1.0 (2)
6.7 ꢁ 0.3 (4)
6.0 ꢁ 0.6 (2)
5.4 ꢁ 0.2 (2)
5.0 ꢁ 0.2 (2)
5.9 ꢁ 0.0 (2)
6.2 ꢁ 0.3 (3)
5.7 ꢁ 0.1 (2)
5.7 ꢁ 0.1 (2)
5.8 ꢁ 0.2 (2)
5.4 ꢁ 0.1 (2)
<4.6 (2)
ND
4.9 ꢁ 0.3 (2)
4.7 ꢁ 0.4 (2)
<4.7 (2)
NA
NA
NA
21a
22a
23a
23b
24a
24b
25a
26a
27a
27b
28a
29a
30a
30b
31a
32a
33a
34b
35a
sep
sep
sep/reg
sep/reg
sep
sep
sep
sep
sep
sep
sep
sep
reg
5.7 ꢁ 0.2 (2)
<5.0 (2)
3-Br
4-MeO
2-Cl
5.6 ꢁ 0.3 (3)
<5.0 (2)
<5.0 (2)
2-NO2
2-NO2
4-CN
2-CN
3,4-Cl2
3,4-Cl2
4-CO2Me
3-CON(Me)2
4-CON(Me)2
4-OH
4-MeO
<4.0 (2)
4.8 ꢁ 0.9 (3)
<4.0 (2)
5.9 ꢁ 0.5 (2)
<4.0 (2)
Scheme 4 Synthesis of substituted benzimidazoles. Reagents and conditions: (a)
PhI, KOH, TBAB, CuI, 110 ꢀC 44% 17a and 22% 17b; (b) 3,5-dimethylisox-
azolylboronic acid, Pd(dppf)Cl2, NaHCO3, DME, 120 ꢀC, 50% (19a) or 26% (19b);
(c) BnBr, K2CO3, MeCN, reflux, 52%; (d) 3,5-dimethylisoxazole, PdCl2, KOAc, N,N-
dimethyl-acetamide, 120 ꢀC, 52% (20); (e) Boc2O, TEA, CH2Cl2, 100%; (f) 3,5-
dimethylisoxazolylboronic acid, Pd(PPh3)4, Na2CO3, 1,4-dioxane, reflux, 48%; (g)
RBr, K2CO3, MeCN, reflux, 18–74%.
5.9 ꢁ 0.0 (2)
reg
sep
reg
reg
reg
<4.0 (2)
5.2 ꢁ 0.2 (2)
5.2 ꢁ 0.2 (2)
5.1 ꢁ 0.7 (2)
4.6 ꢁ 0.3 (2)
reg
a
Mean pIC50 ꢁ standard error of the mean (number of determinations).
b
The regioisomeric identity of compounds 19a and 19b could not be
c
conrmed. Synthetic route sep: separation of regioisomers as per
Scheme 4; reg: regioselective synthesis as per Scheme 5.
A regioselective route to both N-benzylbenzimidazoles was
developed in order to determine the identity of the regioisomers
and prepare additional analogues.17 Substitution of 2,4- and 2,5-
dibromonitrobenzene 36a and 36b with benzyl amines was
followed by reduction of the nitro group and cyclization to the
benzimidazoles 37a and 37b with formic acid. Coupling of the
3,5-dimethylisoxazole group completed the synthesis.
Compounds 23a, and 23b were synthesized by both non-selec-
tive and regioselective routes in order to correlate the elution
order with the identity of the regioisomers.18
Scheme 5 Regioselective synthesis of benzimidazoles. Reagents and conditions:
(a) R1PhCH2NH2, Na2CO3, EtOH, reflux 18–75%; (b) Fe, HCO2H or (MeO)3CMe,
33–76%; (c) 3,5-dimethylisoxazolylboronic acid, Pd(PPh3)4, Na2CO3, 1,4-dioxane,
reflux, 22–43%.
separated although the identity of the regioisomers could not be
conrmed. The benzyl substituted compounds 18 were prepared
in a similar manner by alkylation of N1 with benzyl bromide but
could not be separated. Isoxazole coupling furnished the rst
substituted benzimidazoles 19a,b and 20. The mixture of
regioisomers 20 was tested against both BRD4(1) and CBP and
was shown to be at least three-fold more potent than the either of
the directly arylated compounds 19a or 19b.
Table 3 summarizes the affinity data for the target compounds
against BRD4(1) and CBP. In general all compounds were more
potent against BRD4(1) than CBP. The 6-isoxazolyl-benzimid-
azoles were more potent against BRD4(1) than the 5-substituted
Analogues of 20 were prepared using the non-selective
alkylation chemistry (21a–31a, 23b–30b). By coupling the iso-
xazole group rst, diversication was possible in the last step.
The low yield of compound 8 in Scheme 1 was improved by Boc-
protection of the benzimidazole followed by Suzuki reaction
and in situ deprotection. In the case of the substituted benzyl
compounds separation was achieved by silica gel chromatog-
raphy. Comparison of the 1H-NMRs of the rst and second
eluting regioisomers showed consistent chemical shi changes
between the two isomers. This observation conrmed that if the
absolute identity of a single regioisomeric pair could be deter-
mined, the structures of the others could be correlated by
elution order from silica gel.16
Table 4 DSF thermal shift of bromodomains induced by compound 28a
Bromodomain
Tm shia ꢀC
BRD4(1)
CBP
3.2
1.1
ATAD2
BRD9
ꢂ0.1
0.1
PB1(5)
PCAF
PHIP(2)
TAF1(2)
TIF1a
ꢂ0.1
ꢂ0.2
ꢂ0.4
0.0
ꢂ0.2
a
Compound concentration 10 mM, protein concentration 2 mM.
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(see compounds 23a,b, 24a,b, 27a,b and 30a,b). This observation
is consistent with an expected binding mode where the benzyl
group lls the same pocket as the phenyl group of compound 15.
The opposite is true of CBP potency, where the 5-substitued
regioisomers were more potent. The nature of the benzyl
substituent did not have a dramatic effect on the potency with
both polar compounds (25a, 27a, 28a, 31a–33a) and non-polar
compounds (21a–24a, 26a, 30a) showing similar potency. A
methyl group was incorporated at the 2-position of the benz-
imidazole but it had little effect on either BRD4(1) or CBP potency
as shown by comparing compounds 25a and 35a.
The most potent inhibitor 28a was further screened against a
panel of diverse bromodomains using a differential scanning
uorimetry (DSF) assay (Table 4).14 Compound 28a showed no
stabilization of seven other bromodomains and only minimal
stabilization of CBP, conrming its selectivity for BRD4(1).
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Conclusions
Simple 3,5-dimethylisoxazole substituted benzimidazoles are
potent and selective inhibitors of the rst bromodomain of
BRD4 over the bromodomain of CREB-binding protein. The
most potent compound, 28a, has a BRD4(1) pIC50 of 6.7 (IC50
¼
180 nM) and is at least 100-fold selective over CBP. The addition
of the bicyclic benzimidazole ring system to the previously
reported phenylisoxazole (compound 2) has improved both
potency for BRD4(1) and selectivity over CBP without loss of
selectivity over other bromodomains. With a simple two-step
synthesis and regioisomer separation, or three-step regiose-
lective synthesis and multiple positions for modication, this is
an attractive template for bromodomain lead discovery projects.
Acknowledgements
10 D. S. Hewings, M. Wang, M. Philpott, O. Fedorov, S. Uttarkar,
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The SGC is a registered charity (number 1097737) that receives
funds from the Canadian Institutes for Health Research,
Genome Canada, GlaxoSmithKline, Lilly Canada, the Novartis
Research Foundation, Pzer, Takeda, AbbVie, the Canada
Foundation for Innovation, the Ontario Ministry of Economic
Development and Innovation, and the Wellcome Trust [092809/
Z/10/Z]. The authors would like to thank Yue Zhu at Changchun
Discovery Sciences, Ltd.
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15 Although the identity of compound 15 as the active
compound in the AlphaScreenꢀ assay conditions could
not be unequivically conrmed, the presence of the
compound in the co-crystal structure with BRD4(1) is
evidence for its role as the pharmacologically active
4 E. Nicodeme, K. L. Jeffrey, U. Schaefer, S. Beinke, S. Dewell,
C.-W. Chung, R. Chandwani, I. Marazzi, P. Wilson, 16 H-NMR resonance differences of the two isoxazole methyl
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1
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shied in all pairs of regioisomers and were used to make 18 Final compounds were puried by ash chromatography
compound assignments.
17 E. H. Sessions, M. Smolinski, B. Wang, B. Frackowiak,
S. Chowdhury, Y. Yin, Y. T. Chen, C. Ruiz, L. Lin, J. Pocas,
(25 : 1 CH2Cl2/EtOAc). Correlating the identity of
compounds 23a, 23b, 30a and 30b made via the separation
and regioselective routes showed that the 6-isoxazolyl-
substituted compounds 23a and 30a eluted before 5-
isoxazolyl-substituted compounds 23b and 30b.
¨
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