Design, Synthesis, and Biological Activity of 1,2,3-Triazolobenzodiazepine BET Bromodomain Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.7b00389

A number of diazepines are known to inhibit bromo- and extra-terminal domain (BET) proteins. Their BET inhibitory activity derives from the fusion of an acetyl-lysine mimetic heterocycle onto the diazepine framework. Herein we describe a straightforward, modular synthesis of novel 1,2,3-triazolobenzodiazepines and show that the 1,2,3-triazole acts as an effective acetyl-lysine mimetic heterocycle. Structure-based optimization of this series of compounds led to the development of potent BET bromodomain inhibitors with excellent activity against leukemic cells, concomitant with a reduction in c-MYC expression. These novel benzodiazepines therefore represent a promising class of therapeutic BET inhibitors.

Full text of DOI:10.1021/acsmedchemlett.7b00389

Subscriber access provided by READING UNIV
Letter
Design, Synthesis and Biological Activity of 1,2,3-
Triazolobenzodiazepine BET Bromodomain Inhibitors
Phillip P. Sharp, Jean-Marc Garnier, Tamas Hatfaludi, Zhen Xu, David Segal, Kate
E. Jarman, Helene Jousset Sabroux, Alexandre Garnham, John T. Feutrill, Anthony
Cuzzupe, Peter D. Hall, Scott Taylor, Carl Walkley, Dean Tyler, Mark Dawson, Peter E
Czabotar, Andrew F. Wilks, Stefan Glaser, David C. S. Huang, and Christopher J. Burns
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00389 • Publication Date (Web): 14 Nov 2017
Downloaded from http://pubs.acs.org on November 15, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Medicinal Chemistry Letters is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 7
ACS Medicinal Chemistry Letters
1
2
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
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 7
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Design, Synthesis and Biological Activity of 1,2,3-
Triazolobenzodiazepine BET Bromodomain Inhibitors
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
a,b,†,‡
a,b,‡
a,b,‡
a,b
a,b
Phillip P. Sharp,
JeanꢀMarc Garnier,
Tamas Hatfaludi,
Zhen Xu, David Segal, Kate E.
a,b
a,b
a,b
c
c
a,b
Jarman, Hélène Jousset, Alexandra Garnham, John T. Feutrill, Anthony Cuzzupe, Peter Hall,
d
d
e
e,f,g
a,b
c
Scott Taylor, Carl R. Walkley, Dean Tyler, Mark A. Dawson, Peter Czabotar, Andrew F. Wilks,
a,b
a,b
*,a,b,h
Stefan Glaser, David C. S. Huang, Christopher J. Burns
a) Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia
b) Department of Medical Biology, The University of Melbourne, VIC 3010, Australia
c) Synthesis MedChem, 399 Royal Parade, Parkville, VIC 3052, Australia
d) St. Vincent’s Institute of Medical Research and Department of Medicine, St Vincent’s Hospital, University of Melbourne,
Fitzroy, VIC 3052, Australia
e) Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, VIC 3002, Australia
f) Department of Haematology, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia
g) Sir Peter MacCallum Department of Oncology, The University of Melbourne, East Melbourne, Victoria 3002, Australia
h) School of Chemistry, The Bio21 Institute, The University of Melbourne, VIC 3010, Australia
KEYWORDS. bromodomain, JQ1, triazole, leukemia, benzodiazepine
ABSTRACT: A number of diazepines are known to inhibit bromoꢀ and extraꢀterminal domain (BET) proteins. Their BET inhibitoꢀ
ry activity derives from the fusion of an acetylꢀlysine mimetic heterocycle onto the diazepine framework. Herein we describe a
straightforward, modular synthesis of novel 1,2,3ꢀtriazolobenzodiazepines and show that the 1,2,3ꢀtriazole acts as an effective aceꢀ
tylꢀlysine mimetic heterocycle. Structureꢀbased optimization of this series of compounds led to the development of potent BET
bromodomain inhibitors with excellent activity against AF9ꢀMLLꢀdriven leukemic cells, concomitant with a reduction in cꢀMYC
expression. These novel benzodiazepines therefore represent a promising class of therapeutic BET inhibitors
.
The bromoꢀ and extraꢀterminal domain (BET) family of
proteins are epigenetic reader proteins involved in
cells dependent on the oncogene cꢀMYC with BET inhibitors,
either in vitro or in vivo, can result in significant antiꢀ
proliferative and cytotoxic effects. As a consequence of these
and other data, at least ten BET inhibitors have now entered
clinical trials for the treatment of a range of hematological
cancers (incl. leukemia, lymphoma and myeloma), certain
solid tumors (including glioblastoma multiforme and NUTꢀ
1,2
transcription regulation and chromatin remodelling. The
family consists of BRD2, BRD3 and BRD4, which are
ubiquitously expressed, and BRDT, which is expressed only in
the testes. Each BET protein contains two bromodomain
structural motifs, here designated as D1 and D2, which have
been shown to bind acetylated lysines on histones H3 and H4,
and existing data indicates that the biology of BET proteins is
2
,11
midline carcinoma) and atherosclerosis.
3,4
The first potent BET inhibitors described in the literature were
the benzodiazepine IBETꢀ762 (1)
largely regulated through the first bromodomain.
The
12,13
and the related
acetylated histone lysine residues bind into a hydrophobic
pocket of the BET proteins making specific hydrogen bonding
interactions with a conserved asparagine and tyrosine residue,
1
4
thienodiazepine (S)ꢀJQꢀ1 (2) (Chart 1). Coꢀcrystal structures
of these compounds with BRD4(D1) revealed that the 1,2,4ꢀ
triazole within these inhibitors acts as an acetylꢀlysine mimic
by interacting with Asn140 and a conserved water molecule in
the KAc recognition pocket. Constellation Pharmaceuticals
have reported isoxazole azepines (e.g. 3)
benzotriazolo[4,3ꢀd][1,4]diazepine 4 that also inhibit BET
bromodomains, while Knapp and coworkers reported that
5
the latter through a water molecule. In the last six years a
number of research teams have identified high affinity, small
molecule ligands for this hydrophobic pocket that block
1
5
6
–10
and
binding to the cognate acetylated histones.
Such small
1
6
molecule BET inhibitors, along with genetic deletion studies,
have demonstrated that inhibition of the BET – histone
interaction can result in profound disruption of transcriptional
programs resulting in anticancer and antiꢀinflammatory
activity, amongst others. For example, treatment of cancer
17
benzotriazepine 5 binds to BRD4. Interestingly, based on
structural similarities to IBETꢀ762, a range of benzodiazepine
drugs were retrospectively tested for inhibition of BRD4(D1).
ACS Paragon Plus Environment

Page 3 of 7
ACS Medicinal Chemistry Letters
Of these, alprazolam (6) was found to be a weak inhibitor (Kd
2.5 ꢁM) and Xꢀray crystallography confirmed the 1,2,4ꢀ
functional group interconversions, we synthesized a range of
analogs shown in Table (see electronic supporting
information for full synthetic details).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
=
1
triazole acts as an acetyl lysine mimetic. Other closely related
analogues of alprazolam that were devoid of the 1,2,4ꢀtriazole
17
motif were found to be inactive, underscoring the importance
NH
2
of the acetyl lysine mimicking heterocycle for BET activity.
TMS
TBAF, CuI
Pd(PPh ) , THF
Pd(dba)
2
, xantphos
I
I
I
2
Cs CO
3
, toluene
3 4
We have been interested in understanding the impact of the
NH
NH
(84%)
(
27%)
KAc mimicking pharmacophore on bromodomain inhibition.
18
Based on previous studies
compounds based on
we designed and tested
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
8
9
10
a
5Hꢀbenzo[f][1,2,3]triazolo[1,5ꢀ
O
Cl
Cl
d][1,4]diazepine nucleus. The 1,2,3ꢀtriazole motif was
predicted to have appropriate hydrogenꢀbond accepting vector
at N2 to interact with the conserved asparagine [Asn140 in
BRD4(D1)] and molecular modeling indicated that a phenyl
substituent at N7 of the diazepine ring would project into the
WPF shelf, closely mapping over chlorophenyl (C6) of 3H-
benzoꢀ1,4ꢀdiazepines JQꢀ1 and IBETꢀ762. We found that this
is an excellent BET bromodomainꢀbinding framework and,
through subsequent structureꢀbased optimization, developed a
series of 1,2,3ꢀtriazolobenzodiazepines that display potent
BET bromodomain inhibition and excellent cellular activity.
(64%)
2 3
K CO ,
DCM
N
N
N
N2
N
O
NaN , DMF
3
N
BH
3
, THF
150 o
N
C
O
Cl
NN7
(88%)
(13%)
N
N
7
12
11
Scheme 1. Synthesis of 1,2,3ꢀtriazolobenzodiazepines:
Synthesis of 7.
Initially we assessed the BET inhibitory activity of 7 using
TM
Chart 1. Example azepine BET inhibitors
AlphaScreen , a wellꢀvalidated competition binding assay.
Gratifyingly, 7 showed good activity in this assay against all
bromodomains tested with the strongest activity against
BRD4(D2) (IC50 = 22 nM), but significantly weaker activity
against BRD2(D1) (IC50 = 264 nM) (Figure 1 and Table 1).
Control compound, JQꢀ1, gave BRD4(D2) IC50 = 12 nM and
BRD2(D1) IC50 = 26 nM (Figure 1). We also tested the
diazepinone (12) but this was essentially inactive (IC50 greater
than 10 ꢁM, data not shown).
N
N
N
N
O
N
O
O
O
N
N
NH2
NH
S
O
MeO C8
N
N
N
Cl
Cl
Cl
1
2
3
IBET-762 ref. 13
JQ-1 ref. 14
CPI-0610 ref. 15
BRD4 FP IC50 = 631 nM
BRD4(D1) AS IC50 = 77 nM BRD4(D1) TR FRET IC50 = 39 nM
N
N
N
N
N
N
N
N
ALPHAscreen dose response
IC50 (nM) ± SD
N
N
N
BRD2(D1) 263.5 ± 0.5
BRD2(D2)
Cl
N
Cl
N
1
00
50
0
3
9.0 ± 4.2
H
2
N
N
BRD3(D1)
BRD4(D1)
BRD4(D2)
29.7 ± 5.5
39.4 ± 6.9
21.5 ± 1.8
Cl
4
5
6
Compound 3 ref. 16
BRD4(D1) AS IC50 = 15 nM
TzD 7 ref. 17
BRD4(D1) K = 640 nM
Alprazolam ref. 17
BRD4(D1) K = 2.5 µM
d
d
0.0001
0.01
compound 7] (ꢁM)
1
100
Initially, we prepared 1,2,3ꢀtriazoleꢀcontaining benzodiazepine
via the route shown in Scheme 1. Starting from 1,2ꢀ
[
7
diiodobenzene (8), Buchwald coupling with aniline afforded
diarylaniline 9. The propyne was introduced at the remaining
iodinated position using Sonogashira crossꢀcoupling and the
product (10) was acetylated with chloroacetyl chloride
providing chloroacetamide 11. Based on a report by Majumdar
IC50 (nM) ± SD
BRD2(D1) 26.1 ± 3.4
BRD2(D2)
100
50
0
1
6.0 ± 6.4
BRD3(D1) 19.1 ± 1.3
BRD4(D1) 28.4 ± 8.1
BRD4(D2) 12.1 ± 5.7
1
9ꢀ21
et. al. and others,
,3ꢀdipolar cycloaddition reaction cascade to directly prepare
the 1,2,3ꢀtriazolobenzodiazepine framework by heating 11 to
we performed a oneꢀpot S 2/Hüigsen
N
1
o
0.0001
0.01
JQꢀ1] (ꢁM)
1
100
1
50 C in the presence of sodium azide. While this strategy
[
provided triazole 12 as expected, the unꢀoptimized yield in this
case was low (13%). Nevertheless, with the diazepinone in
hand, selective deletion of the carbonyl oxygen with borane
provided the desired 1,2,3ꢀtriazolobenzodiazepine 7. Using an
optimized version of this general strategy, followed by
additional lateꢀstage crossꢀcoupling reactions and other
TM
Figure 1. Dose response plots for AlphaScreen assays.
Results of at least 2 independent experiments reported as
mean ± SD.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 7
ꢀ
1
2
The good ligand efficiency [0.51 kcal.mol per nonꢀhydrogen
atom for BRD4(D2)] of 7 prompted us to expand this series
and explore structureꢀactivity relationships with the view to
improve the overall BET activity. First we briefly examined
modifications to the Dꢀring. Benzyl substitution at N7 (13)
led to a reduction in activity by greater than 50ꢀfold against all
bromodomains tested, while addition of either meta or para
substituents on the Dꢀring were tolerated in terms of overall
activity with the 4ꢀchlorophenylꢀsubstituted analog 15
provided a modest improvement over the activity of parent
compound 7 [about 2ꢀfold improvement against BRD2(D1)],
consistent with previous studies on BET inhibitors, and we
therefore retained this modification in subsequent compounds.
Next we investigated modifications to the Aꢀring. Crystal
structures of both IBETꢀ762 (pdb code 2YEK) or alprazolam
bulk, further attesting to the accomodation of sp ꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
modifications at C9. Lastly, we investigated substitutions to
the benzodiazepine ring. Methyl substitution at C5
(Compounds 36, 37) improved activity against BRD4(D1) and
the Rꢀstereogenic configuration was preferred consistent with
similar modification of previously reported benzodiazepine
16
BET inhibitors.
From this chemical series we selected compounds 18 and 20
for further study because they displayed potent activity and
were predicted to have good cell permeability (for example the
tetrazole 22, while potent, is likely an efflux transporter
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
22
substrate). First, we assessed the bromodomain selectivity of
2
0 across a panel of 32 recombinant human bromodomains in
TM
a bromoMAX assay (see electronic supporting information
for full data). In these assays, 20 showed excellent selectivity
for the BET family bromodomains. We also assessed the
binding kinetics of 20 against individual bromodomains using
surface plasmon resonance (SPR). The dissociationꢀrate
measured for compound 20 against BRD2 was approximately
(pdb code 3U5J) bound to BET bromodomains indicated that
substitution at C9 (C8 in IBETꢀ762 numbering) projected into
the ZA channel and we extrapolated that our series of
benzodiazepines would also accommodate functionalization at
this position. A set of compounds was therefore designed to
incorporate a substituent from the C9 vector. From the activity
ꢀ
1
3
=
(
ꢀfold slower for domain 2 than domain 1 (k = 0.039 s vs k
d d
ꢀ
1
3
0.13 s , respectively), whereas association rates were similar
k = 2.90 x10 M s vs k = 2.62 x10 M s , respectively)
a a
of the set of C9 analogs it was clear that sp substitution
5
ꢀ1 ꢀ1
5
ꢀ1 ꢀ1
provided no improvement in activity (compounds 16, 17 and
27), whereas heteroaryl substituents significantly improved
activity, with pyrazole 18 and aminopyridine 20 giving IC50
values as low as 7 and 6 nM respectively against BRD4(D2).
Interestingly, tetrazole 22 showed the greatest activity across
each bromodomain with IC50 values ranging from 7 nM
against BRD2(D2) to 2 nM against BRD4(D2). At this point
we established the binding mode of the aminopyridylꢀ
substituted compound (20) in BRD2(D2) by single crystal Xꢀ
ray analysis (Figure 2). The structure confirmed that the 1,2,3ꢀ
triazole motif acts as an acetylꢀlysine mimetic, accepting a
hydrogen bond from both Asn429 and the conserved water
molecule coordinated to Tyr386, as expected. Additionally,
the chlorophenyl ring lies within the WPF shelf and the
aminopyridine ring extends through the ZA channel and
towards solvent exposed space. The positioning of the
aminopyridine towards solvent indicated that modifications to
this position should be well tolerated. Based on these
(see electronic supporting information for full data).
Asn429
ZA channel
WPF shelf
Figure 2. A Xꢀray crystal structure of 20 (magenta) in
complex with BRD2(D2) PDB code = 5U6V overlayed
with coꢀcrystal structure of JQꢀ1 (grey) bound to
BRD2(D2) PDB = 3ONI.
2
structural insights we reasoned that other sp ꢀhybridized
moities, such as carbonyls, might also improve potency and
allow for greater diversity of functionalization. Initially we
compared carboxylic acids, esters and simple amides. In
general, these derivatives showed good activity. Of the initial
carbonyl series, primary amide 25 was significantly more
potent (~3ꢀfold) against all BET bromodomains tested
compared with ester 23 and prompted us to explore the amide
derivatives further. The benzyl amide series (compounds 29 to
BET inhibition has been shown to have a remarkable effect on
certain primary cancer cells and cell lines, which is thought to
be a consequence of downregulation of oncogenes cꢀMYC,
BCL-2 and others by displacement of BRD4 from
hyperacetylated histone tails near their respective promoters
23,24
and enhancers.
To test the antiꢀleukemic effect of this
3
3) were also exceptionally potent, particularly chiral benzyl
chemical series we profiled 18 and 20 against a cancer cell
panel (Table 2). Compound 20 potently inhibited proliferation
of known BRD4ꢀdependant leukemic line MLLꢀAF9, and
showed significant inhibition of leukemic macrophage cell
line MV4ꢀ11, (EC50 = 140 nM), whereas B cell lymphoma
lines DOHH2, SUꢀDHLꢀ4 and Raji, and myeloma cell line
RPMIꢀ8226 were less sensitive (EC50 > 500 nM). Consistent
with BETꢀtargeted antiꢀproliferative effects, BCRꢀAblꢀdriven
cell line K562 was completely resistant to 20 (>1000 nM).
amides 29, 30 and 31. Further, in the amide series the Sꢀ
configured amides 29 and 30 displayed significant domain
selectivity [BRD2(D2) vs BRD2(D1)] realtive to the Rꢀ
configured analog 31. In light of the potent activity of the
tetrazole, 22, we explored bioisosteric acylsulfonamides at the
C9 position. These compounds, although somewhat less active
than the tetrazole, also displayed low nanomolar activity
against BRD4(D2) despite the significantly increased steric
ACS Paragon Plus Environment

Page 5 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
TM
Table 1. Inhibition of BET bromodomains as determined by AlphaScreen assays. Numbers represent mean IC50 in nanomolar
(nM) of at least two independent experiments. A. Dꢀring modifications. B C9 sp3 functionalized derivatives. C C9 sp2ꢀ
functionalized derivatives. D C9 carbonyl derivatives. E miscellaneous C9 derivatives. F C9 benzylamide derivatives. G
Acylsulphonamides. H C5 Alkyl derivatives.
N
C
N
N
N
N
N
N
A
B
N
N
N
N
N
N
N
N
N
N
N
A
B
N
HO
N
N
N
N
N
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
N
N
N7
7
D
13
14
15
16
17
IC50
nM)
Br
Cl
Cl
Cl
(
BRD2 BRD3 BRD4
domain 1 264 19 28
>999
>999
>999
>999
129 21 29
44
129 25 35
273 52 45
147
388 36 79
40
domain 2
38
30
3
9
12
17
43
32
N
N
N
N
N
C
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
HN
HN
H
2
N
H
2
N
N
N
18
19
20
21
53 10 16
22
Cl
Cl
Cl
Cl
Cl
3
2
5
5
10 14
7
64 14 23
33
21
9
13
6
6
7
3
2
38
12
28
7
D
E
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H
N
O
O
HO
H
2
N
HO
N
N
N
Ph
N
N
N
N
O
O
O
CF
3
2
3
Cl
24
25
26
27
28
Cl
Cl
Cl
Cl
N
Cl
1
16 18 29
2 18
185 16 28
50
34
17
6
2
79 14 21
18
204
370
43
23
56
24
11
13
5
17
13
F
N
N
N
N
N
N
N
N
N
F
N
N
N
N
N
H
N
H
H
H
H
Ph
N
Ph
N
Ph
N
Ph
N
N
N
N
N
N
O
O
O
O
Ph
O
29
30
31 Cl
68 13 14
32
33
151 39 44
Cl
Cl
Cl
Cl
95
23 34
17
51 12 10
136 21 35
4
3
6
20
13
37
15
44
14
G
N
N
H
N
N
N
N
N
N
N
N
N
N
N
N
N
OH
H
H
N
N
S
N
S
N
N
N
N
O
O
O
O
O
O
36
34
35
37
38
Cl
Cl
Cl
Cl
Cl
54
21 21
10
44
25
13
7
53 13 15
34 10
38 11 14
25
340 44 52
45 36
25
7
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 7
Osteosarcoma (OS) cells are also known to be sensitive to
BET inhibition, but through MYCꢀindependent
mechanism. We were therefore interested to characterize the
optimization led to the development of analogs with singleꢀ
digit nanomolar activity. Compound 20, showed good BET
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
25
bromodomain selectivity and potent activity against a number
of BRD4ꢀdependant leukaemia cell lines and caused downꢀ
regulation of oncogene MYC. Taken together these data show
that the benzodiazepine scaffold is an useful framework on
which to base BET bromodomain inhibtors.
effect of 18 and 20 on primary mouse OS cells (fibroblastic
4
94H and osteoblastic 148I). Both compounds 18 and 20
inhibited proliferation of both primary OS cell types, serving
to highlight the broad utility of 1,2,3ꢀtriazolobenzodiazepines
derivatives in cancer studies. The cellular potency profile of
1
8 and 20 also closely correlated with positive control JQꢀ1.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
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. Treatment of various cancer cell lines with 18, 20
TM
or JQꢀ1 for 72 h. Cell viability measure by CellꢀTiterGlo
EC50 (nM)
Experimental methods, synthesis, and characterization (PDF)
Cell line
20
18
JQꢀ1
MLLꢀAF9
HLꢀ60
77
152
111
139
64
51
AUTHOR INFORMATION
MV4ꢀ11
DOHH2
RPMIꢀ8226
SUꢀDHLꢀ4
CCRFꢀCEM
Raji
140
200
39
Corresponding Author
* (C.B.) email: burns@wehi.edu.au
561
285
279
631
495
258
977
570
660
Present Addresses
1365
560
2221
780
2037
330
†
. Department of Cellular and Molecular Pharmacology, Univerꢀ
sity of California, San Francisco, United States.
HEL92.1.7
K562
400
720
180
>10000
166
>10000
194
>10000
122
Author Contributions
‡These authors contributed equally.
4
1
94H
48I
1285
973
284
Funding Sources
Our work is supported by the NHMRC (fellowship to PEC) and
the Victorian State Government Operational Infrastructure Supꢀ
port, and the Australian Government NHMRC IRIISS.
We used RNAꢀseq to compare the effect of 20 and JQꢀ1 on
global gene expression in THPꢀ1 leukemia cells. These
experiments revealed that the effect on gene expression
between these two compounds is highly correlated for both up
and down regulated genes (Figure 3A). In particular, BRD4ꢀ
Notes
The authors declare no competing financial interests.
dependent
oncogene
cꢀMYC
demonstrated
robust
ACKNOWLEDGMENT
We thank beamline staff at the Australian Synchrotron, where
diffraction data were collected. Crystallization experiments were
performed at the C3 Collaborative Crystallization Centre.
downregulation on treatment with 20 (Figure 3B) whilst
2
6
HEXIM1 levels were upregulated. Similar effects on cꢀMYC
and BCL-2 downregulation were observed for JQꢀ1, 18, 20
and nonꢀdiazepine BET inhibitor IBETꢀ151 in MV4ꢀ11 cells
(data not shown, see electronic supporting information for full
ABBREVIATIONS
BET, bromoꢀ and extra Cꢀterminal domain containing protein.
data).
B
Compare logFC for compound 20 and JQ-1
top 10 DE genes
A
REFERENCES
2
0
JQ-1
(1)
Belkina, A. C.; Denis, G. V. BET Domain Coꢀ
Regulators in Obesity, Inflammation and Cancer. Nat. Rev. Canꢀ
cer 2012, 12 (7), 465–477.
(2)
Filippakopoulos, P.; Knapp, S. Targeting Bromoꢀ
domains: Epigenetic Readers of Lysine Acetylation. Nat. Rev.
Drug Discov. 2014, 13 (5), 337–356.
Compound 20 vs control
(3)
Picaud, S.; Wells, C.; Felletar, I.; Brotherton, D.; Marꢀ
tin, S.; Savitsky, P.; DiezꢀDacal, B.; Philpott, M.; Bountra, C.;
Lingard, H.; Fedorov, O.; Müller, S.; Brennan, P. E.; Knapp, S.;
Filippakopoulos, P. RVXꢀ208, an Inhibitor of BET Transcriptionꢀ
al Regulators with Selectivity for the Second Bromodomain. Proc.
Natl. Acad. Sci. U. S. A. 2013, 110 (49), 19754–19759.
In summary, we developed
a
novel series of 5H-
benzo[f][1,2,3]triazolo[1,5ꢀd][1,4]diazepines.
Structureꢀ
Figure 3. A. GENAS plot comparing effects of 20 and JQꢀ1
on THPꢀ1 cells. B. Identity of 10 highest up and down
regulated genes by 20 and JQ-1.
(4)
Baud, M. G. J.; LinꢀShiao, E.; Cardote, T.; Tallant, C.;
Pschibul, A.; Chan, K.ꢀH.; Zengerle, M.; Garcia, J. R.; Kwan, T.
T.ꢀL.; Ferguson, F. M.; Ciulli, A. A BumpꢀandꢀHole Approach to
activity relationship studies and crystal structureꢀguided
ACS Paragon Plus Environment

Page 7 of 7
ACS Medicinal Chemistry Letters
Engineer Controlled Selectivity of BET Bromodomain Chemical
Probes. Science 2014, 12 (7), 465–477.
Discovery of Benzotriazolo[4,3ꢀd][1,4]diazepines as Orally Acꢀ
tive Inhibitors of BET Bromodomains. ACS Med. Chem. Lett.
2016, 7 (2), 145–150.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(5)
Filippakopoulos, P.; Picaud, S.; Mangos, M.; Keates, T.;
Lambert, J. P.; BarsyteꢀLovejoy, D.; Felletar, I.; Volkmer, R.;
Müller, S.; Pawson, T.; Gingras, A. C.; Arrowsmith, C. H.;
Knapp, S. Histone Recognition and LargeꢀScale Structural Analyꢀ
sis of the Human Bromodomain Family. Cell 2012, 149 (1), 214–
(17)
Filippakopoulos, P.; Picaud, S.; Fedorov, O.; Keller, M.;
Wrobel, M.; Morgenstern, O.; Bracher, F.; Knapp, S. Benzodiazꢀ
epines and Benzotriazepines as Protein Interaction Inhibitors Tarꢀ
geting Bromodomains of the BET Family. Bioorganic Med.
Chem. 2012, 20 (6), 1878–1886.
2
31.
6)
domain Inhibitors: A Patent Review. Expert Opin. Ther. Pat.
013, 24 (2), 1–15.
7) Müller, S.; Knapp, S. Discovery of BET Bromodomain
(
Garnier, J.ꢀM.; Sharp, P. P.; Burns, C. J. BET Bromoꢀ
(18)
Sharp, P. P.; Garnier, J.ꢀM.; Huang, D. C. S.; Burns, C.
J. Evaluation of Functional Groups as AcetylꢀLysine Mimetics for
BET Bromodomain Inhibition. Medchemcomm 2014, 5, 1834–
1842.
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
Inhibitors and Their Role in Target Validation. Medchemcomm
2014, 5 (3), 288.
(19)
Majumdar, K. C.; Ray, K.; Ganai, S.; Ghosh, T. Cataꢀ
lystꢀFree 1,3ꢀDipolar Cycloaddition: An Efficient Route for the
Formation of the 1,2,3ꢀTriazoleꢀFused Diazepinone Framework.
Synthesis (Stuttg). 2010, No. 5, 858–862.
(8)
Brand, M.; Measures, A. M.; Wilson, B. G.; Cortopassi,
W. A.; Alexander, R.; Höss, M.; Hewings, D. S.; Rooney, T. P.
C.; Paton, R. S.; Conway, S. J. Small Molecule Inhibitors of Broꢀ
modomain–AcetylꢀLysine Interactions. ACS Chem. Biol. 2015,
(20)
Hooyberghs, G.; De Coster, H.; Vachhani, D. D.; Ermoꢀ
lat’Ev, D. S.; Van Der Eycken, E. V. Synthesis of [1,2,3]ꢀ
triazolo[1,5ꢀa][1,4]benzodiazepines via an Unprecedented Oneꢀ
Pot CuꢀCatalyzed AzidationꢀCyclization Reaction. Tetrahedron
2013, 69 (21), 4331–4337.
1
(
0 (1), 22–39.
9) Zhang, G.; Smith, S. G.; Zhou, M. M. Discovery of
Chemical Inhibitors of Human Bromodomains. Chem. Rev. 2015,
15 (21), 11625–11668.
10) Liu, Z.; Wang, P.; Chen, H.; Wold, E. A.; Tian, B.;
1
(
(21)
Sudhapriya, N.; Nandakumar, A.; Arun, Y.; Perumal, P.
T.; Balachandran, C.; Emi, N. An Expedient Route to Highly
Diversified [1,2,3]triazolo[1,5ꢀa][1,4]benzodiazepines and Their
Evaluation for Antimicrobial, Antiproliferative and in Silico Studꢀ
ies. RSC Adv. 2015, 5 (81), 66260–66270.
Brasier, A. R.; Zhou, J. Drug Discovery Targeting Bromodomainꢀ
Containing Protein 4. J. Med. Chem. 2017, 60 (11), 4533–4558.
(11)
Gelato, K. A.; Shaikhibrahim, Z.; Ocker, M.; Haendler,
B. Targeting Epigenetic Regulators for Cancer Therapy: Modulaꢀ
tion of Bromodomain Proteins, Methyltransferases, Demethylasꢀ
es, and microRNAs. Expert Opin. Ther. Targets 2016, 20 (7),
(22)
Young, A. M.; Audus, K. L.; Proudfoot, J.; Yazdanian,
M. Tetrazole Compounds: The Effect of Structure and pH on
Cacoꢀ2 Cell Permeability. J. Pharm. Sci. 2006, 95 (4), 717–725.
7
83–799.
(23)
Chapuy, B.; McKeown, M. R.; Lin, C. Y.; Monti, S.;
(12)
Nicodeme, E.; Jeffrey, K. L.; Schaefer, U.; Beinke, S.;
Roemer, M. G.; Qi, J.; Rahl, P. B.; Sun, H. H.; Yeda, K. T.; Doꢀ
ench, J. G.; Reichert, E.; Kung, a L.; Rodig, S. J.; Young, R. a;
Shipp, M. a; Bradner, J. E. Discovery and Characterization of
SuperꢀEnhancerꢀAssociated Dependencies in Diffuse Large B
Cell Lymphoma. Cancer Cell 2013, 24 (6), 777–790.
Dewell, S.; Chung, C.; Chandwani, R.; Marazzi, I.; Wilson, P.;
Coste, H.; White, J.; Kirilovsky, J.; Rice, C. M.; Lora, J. M.;
Prinjha, R. K.; Lee, K.; Tarakhovsky, A. Suppression of Inflamꢀ
mation by a Synthetic Histone Mimic. Nature 2010, 468 (7327),
1119–1123.
(24)
Lovén, J.; Hoke, H. A.; Lin, C. Y.; Lau, A.; Orlando, D.
(13)
Mirguet, O.; Gosmini, R.; Toum, J.; Clément, C. A.;
A.; Vakoc, C. R.; Bradner, J. E.; Lee, T. I.; Young, R. A. Selecꢀ
tive Inhibition of Tumor Oncogenes by Disruption of Superꢀ
Enhancers. Cell 2013, 153 (2), 320–334.
Barnathan, M.; Brusq, J. M.; Mordaunt, J. E.; Grimes, R. M.;
Crowe, M.; Pineau, O.; Ajakane, M.; Daugan, A.; Jeffrey, P.;
Cutler, L.; Haynes, A. C.; Smithers, N. N.; Chung, C. W.; Bamꢀ
borough, P.; Uings, I. J.; Lewis, A.; Witherington, J.; Parr, N.;
Prinjha, R. K.; Nicodème, E. Discovery of Epigenetic Regulator Iꢀ
bet762: Lead Optimization to Afford a Clinical Candidate Inhibiꢀ
tor of the BET Bromodomains. J. Med. Chem. 2013, 56 (19),
7501–7515.
(25)
Baker, E. K.; Taylor, S.; Gupte, A.; Sharp, P. P.; Walia,
M.; Walsh, N. C.; Zannettino, A. C.; Chalk, A. M.; Burns, C. J.;
Walkley, C. R. BET Inhibitors Induce Apoptosis through a MYC
Independent Mechanism and Synergise with CDK Inhibitors to
Kill Osteosarcoma Cells. Sci. Rep. 2015, 5 (May), 10120.
(26)
Devaraj, S. G. T.; Fiskus, W.; Shah, B.; Qi, J.; Sun, B.;
(14)
Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith,
Iyer, S. P.; Sharma, S.; Bradner, J. E.; Bhalla, K. N. HEXIM1
Induction Is Mechanistically Involved in Mediating AntiꢀAML
Activity of BET Protein Bromodomain Antagonist. Leukemia
2016, 30 (2), 504–508.
W. B.; Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.;
Felletar, I.; Philpott, M.; Munro, S.; McKeown, M. R.; Wang, Y.;
Christie, A. L.; West, N.; Cameron, M. J.; Schwartz, B.; Heightꢀ
man, T. D.; La Thangue, N.; French, C. A.; Wiest, O.; Kung, A.
L.; Knapp, S.; Bradner, J. E. Selective Inhibition of BET Bromoꢀ
domains. Nature 2010, 468 (7327), 1067–1073.
(15)
Gehling, V. S.; Hewit, M. C.; Vaswani, G. R.; et al.;
Hewitt, M. C.; Vaswani, R. G.; Leblanc, Y.; Coîté, A.; Nasvesꢀ
chuk, C. G.; Taylor, A. M.; Harmange, J. C.; Audia, J. E.; Pardo,
E.; Joshi, S.; Sandy, P.; Mertz, J. A.; Sims, R. J.; Bergeron, L.;
Bryant, B. M.; Bellon, S.; Poy, F.; Jayaram, H.; Sankaranaꢀ
rayanan, R.; Yellapantula, S.; Bangalore Srinivasamurthy, N.;
Birudukota, S.; Albrecht, B. K. Discovery, Design, and Optimizaꢀ
tion of Isoxazole Azepine BET Inhibitors. ACS Med. Chem. Lett.
2
013, 4 (9), 835−840.
(16) Taylor, A. M.; Vaswani, R. G.; Gehling, V. S.; Hewitt,
M. C.; Leblanc, Y.; Audia, J. E.; Bellon, S.; Cummings, R. T.;
Coîté, A.; Harmange, J. C.; Jayaram, H.; Joshi, S.; Lora, J. M.;
Mertz, J. A.; Neiss, A.; Pardo, E.; Nasveschuk, C. G.; Poy, F.;
Sandy, P.; Setser, J. W.; Sims, R. J.; Tang, Y.; Albrecht, B. K.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 8 of 7
Insert Table of Contents artwork here
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
• X-ray structure
• Binding kinetics
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
Novel Kac
mimetic
R3
N
N
NaN
3
O
JQ1
IBET151
916
100
I
Cl
N
IC50 JQ1
=
=
=
32.3nM
42.6nM
32.7nM
Cl
*
R
IC50 IBET151
IC50 916
•
Leukemia cell
killing
R1
4
50
NH
2
B(OH)2
N
H
2
N
N
0
-
10
-8
-6
-4
log[drug],M
R2
Cl
IC50 (nM)
Readily available
building blocks
• Gene expression
profiling
BRD2(D1) = 33
BRD2(D2) = 21
BRD3(D1) =
BRD4(D1) = 13
BRD4(D2) =
9
6
7
ACS Paragon Plus Environment