Benzodiazepines and benzotriazepines as protein interaction inhibitors targeting bromodomains of the BET family

Article abstract of DOI:10.1016/j.bmc.2011.10.080

Benzodiazepines are psychoactive drugs with anxiolytic, sedative, skeletal muscle relaxant and amnestic properties. Recently triazolo-benzodiazepines have been also described as potent and highly selective protein interaction inhibitors of bromodomain and

Full text of DOI:10.1016/j.bmc.2011.10.080

Bioorganic & Medicinal Chemistry 20 (2012) 1878–1886
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Bioorganic & Medicinal Chemistry
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Benzodiazepines and benzotriazepines as protein interaction inhibitors
targeting bromodomains of the BET family
Panagis Filippakopoulos a, Sarah Picaud a, Oleg Fedorov a, Marco Keller b, Matthias Wrobel b,
a,
Olaf Morgenstern c, Franz Bracher b, , Stefan Knapp
⇑
⇑
a University of Oxford, Nuffield Department of Clinical Medicine, Structural Genomics Consortium, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7LD, UK
b Ludwig-Maximilians University, Department of Pharmacy, Center for Drug Research, Butenandtstr. 5-13, 81377 Munich, Germany
c Ernst-Moritz-Arndt University, Institute of Pharmacy, Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 4 November 2011
Benzodiazepines are psychoactive drugs with anxiolytic, sedative, skeletal muscle relaxant and amnestic
properties. Recently triazolo-benzodiazepines have been also described as potent and highly selective
protein interaction inhibitors of bromodomain and extra-terminal (BET) proteins, a family of transcrip-
tional co-regulators that play a key role in cancer cell survival and proliferation, but the requirements
for high affinity interaction of this compound class with bromodomains has not been described. Here
we provide insight into the structure–activity relationship (SAR) and selectivity of this versatile scaffold.
In addition, using high resolution crystal structures we compared the binding mode of a series of benzo-
diazepine (BzD) and related triazolo-benzotriazepines (BzT) derivatives including clinically approved
drugs such as alprazolam and midazolam. Our analysis revealed the importance of the 1-methyl triazolo
ring system for BET binding and suggests modifications for the development of further high affinity
bromodomain inhibitors.
Keywords:
BRD4
Bromodomains
Benzodiazepines
Alprazolam
Benzotriazepines
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
synthetically accessible and established a rich body of literature
on their pharmacological properties.5–7 A variety of biological
Benzodiazepines (BzDs) are drug-like small molecules that led
to the development of a large number of approved drugs that mod-
ulate the function of the GABA (gamma-aminobutyric acid) recep-
tor. BzDs have generally sedative, anxiolytic, amnesic and muscle
relaxing properties and have been approved for the treatment of
sleeping disorders, seizures, muscle spasms and anxiety.1 For in-
stance, alprazolam (8-chloro-1-methyl-6-phenyl-4H-[1,2,4]triaz-
olo[4,3-a][1,4]benzodiazepine) is a potent short acting BzD used
for the treatment of anxiety disorders2 whereas midazolam (8-
chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]ben-
zodiazepine) is prescribed for the treatment of acute seizures,
insomnia as well as a sedative drug.3 L-655,708 (ethyl (13aS)-7-
methoxy-9-oxo-11,12,13,13a-tetrahydro-9H-imidazo[1,5-a]pyr-
rolo[2,1-c][1,4]benzodiazepine-1-carboxylate) was the first sub-
type-selective inverse agonist at the BzD binding site that has
activities and synthetic routes have also been established for the
related benzotriazepines (BzT).8
Recently, we and others described BzDs as potent protein inter-
action inhibitors that selectively bind to acetyl lysine (Kac) recog-
nition modules of the BET (bromodomain and extra-terminal)
family of transcriptional co-regulators.9–11 Importantly, the discov-
ered inhibitor JQ1 has no significant GABA receptor activity most
likely due to its bulky substitution at the 2 position of the benzo-
diazepine ring system.9
Bromodomains constitute a highly diverse family of interaction
domains that comprise 61 members in humans. All bromodomains
share a conserved fold that comprises a left-handed bundle of four
alpha helices (aZ, aA, aB, aC) linked by diverse loop regions (ZA
and BC loops) that flank the substrate binding site. The helical
bromodomain bundle creates a deep central hydrophobic cavity
been discovered. This BzD binds preferentially to the
a
5 subtype
that specifically recognizes sequences that contain e-N-acetylated
of the GABAA receptor.4 The high interest in this compound class
in medicinal chemistry and medicine made many BzD analogues
lysine residues.12 Several crystal structures with peptidic
substrates revealed a common anchor point that recognizes the
acetyl moiety: In all bromodomain substrate complexes
a
conserved asparagine residue forms a hydrogen bond with the
carbonyl oxygen of the N-acetyl group of the substrate peptide
providing a starting point for the design of acetyl lysine mimetic
and competitive ligands.13 Even though it has not been
⇑
Corresponding authors. Tel.: +49 89 2180 77301; fax: +49 89 2180 77802 (F.B.);
tel.: +44 1865 617 584; fax: +44 1865 617 575 (S.K.).
E-mail addresses: franz.bracher@cup.uni-muenchen.de (F. Bracher), stefan.
knapp@sgc.ox.ac.uk (S. Knapp).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.080

P. Filippakopoulos et al. / Bioorg. Med. Chem. 20 (2012) 1878–1886
1879
demonstrated structurally, in some bromodomains the asparagine
is substituted by a threonine or tyrosine residue providing an alter-
native hydrogen donor to the acetyl lysine carbonyl.
in testis), giving rise to an extremely aggressive untreatable sub-
type of squamous carcinoma termed NUT midline carcinoma
(NMC).24–26 The recent development of the potent and selective
inhibitor JQ1 and its evaluation in mouse models of patient derived
cancer cells provided a compelling case for targeting BET bromod-
omains in NMC.9
Bromodomain-containing proteins are reader domains of epige-
netic marks that play key roles in transcription control and chro-
matin remodeling.14 In histones,
e-N-acetylation of lysine
residues has been associated with open chromatin architecture
and transcriptional activation and the recent discovery of selective
and potent BzD inhibitors suggested that these protein interaction
modules can be efficiently targeted by drug-like molecules.9–11 The
therapeutic potential of bromodomains has recently been
reviewed.15
Here we describe the structural requirements for high affinity
interactions of BzDs and BzTs with BET bromodomains and discuss
an initial SAR (structure–activity relationship) for these compound
classes. We discovered that the clinically approved BzDs alprazo-
lam and midazolam bound with low lM affinities to BET bromod-
omain and determined several high resolution co-crystal
structures to further guide structure based design efforts. In addi-
tion, the BzTs are presented as an alternative versatile scaffold that
specifically binds with nM potency to the acetyl lysine binding site
of BET bromodomains.
The BET family of bromodomain containing proteins is repre-
sented by four proteins that express several splice isoforms in
mammals (BRD2, BRD3, BRD4 and BRDT). Proteins of this family
share a common domain architecture that comprises two N-termi-
nal bromodomains and an ET (extra terminal) domain but each
family member contains diverse C-termini.16 The discovery of the
selective BET inhibitor iBET11 provided compelling evidence for
targeting BET bromodomains in inflammatory diseases and reports
have linked BET proteins to cancer. Both BRD2 and BRD3 are over-
expressed in nasopharyngeal carcinoma17 and high expression lev-
els for the testis specific isoform BRDT have been detected in lung
cancer.18 In addition, BRD4 recruits the positive transcription elon-
gation factor complex (P-TEFb) to transcriptional start sites, a key
regulatory event controlling transcriptional elongation of many
growth promoting genes.19,20
Modulation of P-TEFb activity developed into a promising strat-
egy for the treatment of chronic lymphocytic leukemia—a core
component of the P-TEFb complex is cyclin dependent kinase-9
(CDK9) which has been successfully targeted by the ATP competi-
tive inhibitor flavopiridol.21,22 Indeed a recent RNAi screen has
identified BRD4 as a key target required for the survival and main-
tenance of acute myeloid leukemia (AML) cells.23 Importantly, the
bromodomains of both BRD3 and BRD4 have been identified in
recurrent chromosomal translocations with NUT (Nuclear protein
2. Results and discussion
The recent disclosure of the triazolo-benzodiazepine iBET as a
bromodomain inhibitor11 prompted us to investigate the interac-
tion of a number of clinically approved BzDs with these protein
interaction modules (Fig. 1). The temperature shift binding assay
has emerged as a rapid screening technology for detection of pro-
tein ligands and has been shown to correlate well with binding
constants determined by direct biophysical methods such as iso-
thermal titration calorimetry and enzymatic assays.9,27 Using this
methodology we identified alprazolam and surprisingly midazo-
lam as compounds that interact with BET bromodomains
(Fig. 2A). Selectivity screening against the BET family of bromodo-
mains and five diverse bromodomains that belong to different fam-
ilies that constitute the bromodomain phylogenetic tree revealed
that both alprazolam and midazolam maintained their selectivity
for BET BRDs. The array of Tm data also suggested a slight prefer-
ence of midazolam for the second bromodomain of BET family
members (Fig. 2A and B). In contrast, the related BzDs estazolam
Figure 1. Chemical structures of studied clinical BzDs and published BET bromodomain inhibitors.

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P. Filippakopoulos et al. / Bioorg. Med. Chem. 20 (2012) 1878–1886
Figure 2. (A) Temperature shift data measured on synthesized inhibitors as well as clinical BzDs. Synthesized compounds have been initially screened at 100
concentration and interacting compounds were re-evaluated at 10 M compound concentration. Temperature shift data are color coded as indicated in the figure. (B)
Example of a raw data trace of a temperature shift experiment. Shown are data measured on BRD4(1) alone (black line) and BRD4(1) in the presence of 10 mM alprazolam
(red line). (C) Isothermal titration calorimetry data. Shown are data measured on alprazolam (red) and BzD-7 (black). The panel shows raw binding heats of 8 L injections of
BRD4(1) into a solution of each of the two inhibitors. The first injection (2 L) was not included into the data analysis. The insert shows normalized binding heats and non-
linear least squares fit to the experimental data (solid lines). The derived thermodynamic data are compiled in Table 1.
lM compound
l
l
l
and triazolam showed only very weak interaction suggesting that
the methyl group at the triazolo and imidazolo ring is a required
component for the interaction of the scaffold with BET bromodo-
mains. Comparison of alprazolam with triazolam revealed that
the chloro substitution at the 2 position of the phenyl ring is not
simplifies synthesis by abolishing the need for a stereoselective
route or separation of enantiomers.
3. Chemical synthesis
tolerated. The GABAA receptor
a
5 specific inhibitor L655708
The 6-ethyl-triazolobenzodiazepines were prepared starting
from known28 benzodiazepinone 1 (Scheme 1). Conversion of 1
to the thiolactam 2 could not be accomplished under standard con-
ditions29 (P2S5 in pyridine or other high boiling solvents), but was
successfully realized by using Lawesson’s reagent in anhydrous
THF under nitrogen atmosphere. Target compounds 4a–e were ob-
tained by condensation of thiolactam 2 with carboxylic acid hydra-
zides 3a–e. Residual hydrazides 3a–e were found to be poorly
separable from the products 4a–e by column chromatography. This
showed no significant interaction with BET bromodomains. To ob-
tain better insight into the SAR of this compound class we initiated
a synthetic effort on the benzodiazepine scaffold. For studying the
role of the 6-aryl substitutent in alprazolam, 6-ethyl analogues
were prepared, furthermore we modified the substituents in the
triazolo ring (C-1) and investigated related triazolo-benzotriaze-
pines (BzTs) in which the chiral carbon atom present in JQ1, iBET
and related compounds has been replaced by nitrogen. This greatly
Scheme 1. Synthesis outline of target compounds 4a–e. Reagents and conditions: (i) Lawesson0s reagent (1.1 equiv), THF, rt, 24 h, 53%; (ii) hydrazides 3a–e (2 equiv), n-
butanol, 130 °C, 24 h, 17–67%.

P. Filippakopoulos et al. / Bioorg. Med. Chem. 20 (2012) 1878–1886
1881
O
H
N
R
N
O
CH3
Cl
N
H
N
H
N
N
H
S
N
N
N
N
N
N
CH3
N
CH3
N CH3
N
N
CH3
N
Cl
Cl
N
Cl
BzT-2
BzT-4
BzT-7 R = CH3
BzT-8 R = NH2
BzT-9
8a
Scheme 2. Studied benzotriazepines (BzT) prepared according to Richter et al.
problem was solved by converting the hydrazides into water-solu-
ble condensation products upon stirring with glucose solution
prior to extraction with an organic solvent. The triazolo-ben-
zotriazepines were prepared according to published procedures8a
(Scheme 2).
As expected the (thio)lactams BzD-2 and BzD-3 showed no sig-
nificant interaction with bromodomains confirming the impor-
tance of the triazolo ring system. This observation has been also
confirmed with the BzT intermediates Bz-T2 and Bz-T4. The bind-
ing pocket of BET BRDs harbors a number of conserved water mol-
ecules. With the BzD series we explored the possibility of
displacing these waters by ligand atoms employing larger substit-
uents than the methyl group at the triazolo ring that would pro-
trude deeper into the acetyl lysine pocket. However, all bulkier
4. Binding mode of tested BzDs and BzTs
In order to obtain structural insight into the binding mode of
clinically approved BzDs we determined the co-crystal structures
of the first bromodomain of BRD4 with alprazolam and midazolam
and compared them to structures of related ligands as well as the
apo-structure. As expected, the co-crystal structures revealed the
canonical alpha helical fold that is typical for bromodomains
(Fig. 3A). Comparison with the apo-structure (PDB-ID: 2oss)9 re-
vealed only minor backbone re-arrangements. However, in some
cases different conformation of side chains were observed. In par-
ticular I146 assumed a different rotamer conformation compared
to the apo-structure (Fig. 3B). In apo-BRD4(1) four structural water
molecules were observed in the acetyl lysine cavity which were
coordinated either directly or indirectly (through interaction with
directly bound water molecules) by the conserved tyrosine residue
Y97. In all BzD and BzT complexes the inhibitors assumed similar
binding modes with good shape complementarity with the
BRD4(1) acetyl lysine binding site. All structures were refined to
high resolution with low R/Rfree-values (Table 2). The ligands were
well defined by electron density (Fig. 3C–E).
The water network observed in apo-BRD4(1) was slightly rear-
ranged in structures that bound the triazolo-benzodiazepine ring
system (Fig. 4). The missing second hydrogen bond to N140 in
the alprazolam complex results most likely in the lower affinity
of this ligand compared to iBET (Fig. 4B). The complex with midaz-
olam (Fig. 4C) revealed the influence of the lack of the nitrogen in
position 3 of the ring system (annulated imidazole instead of 1,2,4-
triazole). The inability of midazolam to form a hydrogen bond with
the acetyl lysine anchoring residue N140 leads to an outward shift
of this inhibitor. In addition, 2 of the 4 structural water molecules
are not present in the midazolam complex. The 2-fluorophenyl ring
system is rotated by about 30°. This is most likely a consequence of
the fluoro substitution that requires reorientation of this aromatic
ring system. Larger halogens in that position will rotate the ring
further explaining the observed inactivity of triazolam that con-
tains a 2-chlorophenyl moiety.
groups resulted in Tm shifts not larger than 2° at high (100 lM)
compound concentration indicating very weak interaction with
BET bromodomains. In support of this observation also the 6-ethyl
analogue BzD-5 of alprazolam showed strongly reduced binding
activity. Even though the methyl substituent was not extensively
tested in the BzT series it appeared to be optimal. Substitution of
the methyl moiety (BzT-7) with a primary amine (BzT-8) led to sig-
nificantly weaker interactions and a carbonyl group at this position
(BzT-9) abolished interaction with all screened bromodomains
(Fig. 2). The open-chain synthetic precursor BzT-4 of BzT-9 showed
only very poor activity.
We used isothermal titration calorimetry (ITC) to precisely
determine the binding constants of alprazolam and the best hit
of the BzT series (BzT-7) in solution. Titration of the first bromod-
omain of BRD4 (BRD4(1)) into alprazolam resulted in large exo-
thermic binding heats (
dissociation constant (KD) of 2.5
D
H: ꢀ6.96 kcal/mol). We determined a
l
M for this interaction (Fig. 2C).
Comparison with ITC data that have been collected on JQ1 indi-
cated that the loss of the tertiary butyl ester at position 2 in the
diazepam ring leads to a significant reduction of binding affinity
probably due to the loss of a second hydrogen bond to the acetyl
lysine anchoring residue N140. ITC titrations carried out on BzT-7
revealed a binding constant of 0.64 lM indicating an excellent li-
gand efficacy of this compound. The thermodynamic data are com-
piled in Table 1.
Triazolo-benzotriazepines were discovered as a new versatile
scaffold with strong BRD4 binding affinity. The binding mode of
Table 1
Thermodynamic parameters of BzD and BzT interaction with BRD4(1)
Protein
D
Tm (°C)
Kd
(l
M)
D
Hobs (kcal/mol)
N
T
D
S (kcal/mol)
DG (kcal/mol)
(+)JQ1a
Alprazolam
BzT-7
9.3
4.7
4.2
0.049 0.02
2.46 0.11
0.64 0.03
ꢀ8.42 0.019
ꢀ6.96 0.05
ꢀ6.16 0.03
1.00 0.001
1.10 0.006
0.98 0.003
1.22
0.44
2.00
ꢀ9.64
ꢀ7.40
ꢀ8.16
a
9
According to Filippakopoulos et al. Titrations were carried out in 50 mM HEPES pH 7.4 (at 25 °C), 150 mM NaCl and 15 °C while stirring at 295 rpm. In all cases the
protein was titrated into the ligand solution (reverse titration).

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P. Filippakopoulos et al. / Bioorg. Med. Chem. 20 (2012) 1878–1886
Figure 3. Structure of BzD and BzT BRD4 complexes. (A) Structural overview of the BRD4(1) structure. The main structural elements are labeled and the acetyl lysine mimetic
inhibitor alprazolam is shown in ball and stick representation. (B) Superimposition of the apo structure of BRD4(1) (green) and the alprazolam ligand complex (blue). The
figure shows main interaction residues and details of the acetyl lysine binding site as well as conserved water molecules (shown as red balls). (C–E) Detailed view of the co-
crystallized ligands. The acetyl lysine binding site is shown as a transparent white surface. Residues interacting with the inhibitors are labeled in C and the orientation has
been maintained in C–E. The conserved acetyl lysine anchoring residue N140 is labeled in blue. A 2FoFc electron density map that has been contoured at 2
lower panel for each ligand.
r is shown in the
BzT-7 was found to be reminiscent of the related BzD alprazolam
with identical conservation of the four water molecules (Fig. 4D).
strates in most bromodomains. Surprisingly, also midazolam, that
lacks the hydrogen bond forming nitrogen in the triazolo ring bound
to BRD4(1). The binding mode of this BzD is characterized by a
reorientation of midazolam in the acetyl lysine binding pocket and
in an altered network of structural water molecules. However,
FDA-approved sedatives inhibit BRD4 at high concentrations
making it unlikely that this activity will cause side effects due to
inhibition of BRD4 mediated transcription control during therapy.
This unexpected binding mode may be explored for the develop-
ment of more potent inhibitors. A small expansion of the triazolo-
bezodiazepine scaffold and comparison with estazolam underline
the importance of the 3-methyl group in the triazolo ring system.
Furthermore, we report compounds of the triazolo-benzotriazepine
5. Conclusions
Here we describe structural requirements for the development of
benzodiazepines and benzotriazepines as protein interaction inhib-
itors targeting bromodomains of the BET family. We found that the
clinical inhibitor alprazolam binds with low lM affinity to the acetyl
lysine binding site of BRD4(1). The co-crystal structure with BRD4(1)
showed that the triazolo ring acts as an acetyl lysine mimetic
scaffold forming a hydrogen bond with the conserved residue
N140 that acts as a hydrogen bond anchor point for acetylated sub-

P. Filippakopoulos et al. / Bioorg. Med. Chem. 20 (2012) 1878–1886
1883
Table 2
Data collection and refinement statistics
Data collection
PDB ID
3U5J
3U5K
3U5L
BRD4 ligand
Space group
Cell dimensions: a, b, c (Å)
Alprazolam
Midazolam
P3(1)
BzT-7
P2(1)2(1)2(1)
P2(1)2(1)2(1)
37.30, 44.21, 78.36
90.00, 90.00, 90.00
1.60 (1.69–1.60)
17,472 (2392)
98.6 (95.3)
6.3 (5.3)
0.056 (0.225)
20.7 (6.4)
89.55, 89.55, 64.41
90.00, 90.00, 120.00
1.80 (1.90–1.80)
51,230 (6551)
95.7 (83.9)
4.1 (3.4)
0.102 (0.522)
7.8 (2.1)
37.36, 43.13, 78.38
90.00, 90.00, 90.00
1.39 (1.46–1.39)
25,967 (3709)
96.9 (96.9)
6.7 (6.9)
0.063 (0.239)
18.4 (7.4)
a
, b, c (deg)
Resolutiona (Å)
Unique observationsa
Completenessa (%)
Redundancy*
Rmergea
I/r a
I
Refinement
Resolution (Å)
1.60
1.80
1.39
Rwork/Rfree (%)
14.6/17.7
1098/38/216
11.57/15.77/26.53
0.015
1.538
97.52
20.9/25.9
4055/92/231
20.34/31.67/19.97
0.016
1.640
96.71
11.1/14.2
1125/27/218
8.67/6.76/25.16
0.013
1.700
97.30
Number of atoms (p/o/w)b
B-factors (Å2) (p/o/w)b
r.m.s.d bonds (Å)
r.m.s.d angles (o)
Ramachadran Favoured (%)
Allowed (%)
2.48
3.09
2.70
Disallowed (%)
0.00
0.21
0.00
a
Values in parentheses correspond to the highest resolution shell.
(p/o/w): Protein atoms, other (ligand) atoms, water.
b
Figure 4. Details of the BzD and BzT interaction with BRD4(1). Shown is (A) iBET, (B) alprazolam, (C) midazolam and (D) BzT-7. Conserved water molecules are shown as red
spheres.
class as submicromolar inhibitors of BET bromodomains with simi-
lar binding mode as BzD and excellent ligand efficiency.
General Analysis of chemical compounds: Melting points were
determined on a Büchi Melting Point B-540 apparatus and are
uncorrected. NMR spectra were recorded on a Jeol GSX 400 (1H
NMR: 400 MHz, 13C NMR: 100 MHz) and a Jeol JNMR-GX 500 (1H
NMR: 500 MHz, 13C NMR: 125 MHz) spectrometer, respectively.
Chemical shifts are reported in d (ppm) units relative to the inter-
nal standard tetramethylsilane (TMS). Infrared spectroscopy was
6. Experimental
Alprazolam, estazolam, midazolam, L-655,708 and triazolam
were purchased from Sigma.

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P. Filippakopoulos et al. / Bioorg. Med. Chem. 20 (2012) 1878–1886
3
done on a Perkin–Elmer FT-IR Paragon 1000. Mass spectra were ob-
tained on a Hewlett Packard 5989 A Mass Spectrometer by either
chemical ionization (CI) or electron ionization (EI). High resolution
mass spectra (HRMS) were measured on a Jeol JMS GCmate II. All
chemicals and solvents used were of analytical grade and no fur-
ther purification was needed. Flash column chromatography was
1.34 (t, JHH = 7.5 Hz, 3H, 1-CH2–CH3), 2.60–2.70 (m, 1H, 6-CHH–
CH3), 2.77–2.89 (m, 2H, 1-CHH–CH3, 6-CHH–CH3), 3.00–3.11 (m,
2
1H, 1-CHH–CH3), 3.92 (d, JHH = 13.0 Hz, 1H, 4-CHH), 5.28 (d,
2JHH = 13.0 Hz, 1H, 4-CHH), 7.37 (d, JHH = 8.6 Hz, 1H, H-10), 7.59
3
4
3
4
(dd, JHH = 2.4 Hz, JHH = 8.6 Hz, 1H, H-9), 7.67 (d, JHH = 2.3 Hz,
1H, H-7); 13C NMR (125 MHz, CDCl3): d (ppm) 11.1 (6-CH2–CH3),
11.5 (1-CH2–CH3), 19.8 (1-CH2-CH3), 32.4 (6-CH2-CH3), 45.7 (C-4),
124.6 (C-10), 128.8 (C-7), 130.9 (C-10a), 131.1 (C-9), 131.8 (C-
6a), 133.8 (C-8), 154.8 (C-1), 155.1 (C-3a), 171.0 (C-6); IR [cmꢀ1]:
performed on silica gel Si 60 (40–63 lm).
6.1. 7-Chloro-5-ethyl-1H-benzo[e][1,4]diazepine-2(3H)-thione
(2)
v = 3060, 2986, 2934, 2873, 2362, 2343, 1630, 1540, 1485, 1430,
1261, 1108, 1027, 818, 799; MS (CI): m/z (%) = 275 (100) [MH+];
MS (EI): m/z (%) = 274 (40) [M+Å], 245 (40) [M+ÅAEt], 239 (100)
[M+ÅACl]; HR-MS (EI+): calcd for C14H15ClN4 [M+Å] 274.0985; found
274.0987.
7-Chloro-5-ethyl-1H-benzo[e][1,4]diazepin-2(3H)-one (1; 2.0 g,
9.1 mmol, 1.0 equiv) and Lawesson’s reagent (4.0 g, 10 mmol,
1.1 equiv) were suspended in 19 mL of anhydrous tetrahydrofuran
and stirred for 24 h at room temperature under nitrogen atmo-
sphere. The crude product was extracted three times in a separating
funnel with a dichloromethane/water mixture. The combined or-
ganic phases were washed with water, dried over sodium sulfate, fil-
tered and evaporated. The purification was done by flash column
chromatography (ethyl acetate/isohexane, 1:2), yielding thiolactam
2 (1.1 g, 4.8 mmol, 53 %) as a yellow solid. mp: 168.7 °C; 1H NMR
(400 MHz, CD2Cl2): d (ppm) 1.13 (t, 3JHH = 7.4 Hz, 3H, CH3), 2.75 (q,
3JHH = 7.3 Hz, 2H, CH2–CH3), 4.49 (s, 2H, H-3), 7.12 (d, 3JHH = 8.6 Hz,
6.2.3. 8-Chloro-6-ethyl-1-phenyl-4H-benzo[f][1,2,4]triazolo[4,3-
a][1,4]diazepine (4c)
Yield: 61%; light yellow solid; mp: 176.8 °C; 1H NMR (400 MHz,
3
CD2Cl2): d (ppm) 1.13 (t, JHH = 7.3 Hz, 3H, CH3), 2.67–2.80 (m, 1H,
2
CHH–CH3), 2.82–2.96 (m, 1H, CHH–CH3), 4.00 (d, JHH = 13.0 Hz,
2
1H, 4-CHH), 5.21 (d, JHH = 13.1 Hz, 1H, 4-CHH), 6.87 (d,
4
3
3JHH = 8.7 Hz, 1H, H-10), 7.30 (dd, JHH = 2.4 Hz, JHH = 8.7 Hz, 1H,
H-9), 7.37–7.52 (m, 5 H, H-20, H-30, H-40), 7.69 (d, 4JHH = 2.4 Hz, 1H,
H-7); 13C NMR (100 MHz, CD2Cl2): d (ppm) 11.2 (CH3), 32.8 (CH2–
CH3), 46.2 (C-4), 126.8 (C-10), 127.2 (C-10), 128.8 (C-20), 128.9
(C-7), 129.3 (C-30), 130.7 (C-40), 131.1 (C-9), 132.0 (C-10a), 132.2
(C-6a), 133.9 (C-8), 153.6 (C-1), 157.3 (C-3a), 171.9 (C-6); IR
4
3
1H, H-9), 7.45 (dd, JHH = 2.3 Hz, JHH = 8.6 Hz, 1H, H-8), 7.58 (d,
4JHH = 2.3 Hz, 1H, H-6), 10.19 (bs s, 1H, H-1); 13C NMR (100 MHz,
CD2Cl2): d (ppm) 11.4 (CH3), 31.8 (CH2–CH3), 62.9 (C-3), 122.9 (C-
9), 128.7 (C-6), 131.1 (C-7), 131.5 (C-5a), 131.6 (C-8), 136.9 (C-9a),
172.4 (C-5), 201.6 (C-2); IR [cmꢀ1]:
v
= 3113, 3070, 2973, 2899,
[cmꢀ1]:
v = 3057, 2970, 2930, 2854, 2365, 2345, 1631, 1534, 1485,
2853, 2731, 2675, 1635, 1576, 1520, 1474, 1358, 1163, 1009, 838;
1472, 1422, 1287, 1107, 977, 821, 768, 696; MS (CI): m/z (%) = 323
(100) [MH+]; MS (EI): m/z (%) = 322 (40) [M+Å], 293 (40) [M+ÅAEt],
287 (100) [M+ÅACl]; HR-MS (EI+): calcd for C18H15ClN4 [M+Å]
322.0985; found 322.0963; Elemental analysis calcd (%) for
MS (EI): m/z (%) = 238 (100) [M+Å]; HR-MS (EI+): calcd for
C
11H11ClN2S [M+Å] 238.0331; found 238.0296.
6.2. General procedure for the preparation of triazolo-
benzodiazepines 4a–e
C18H15ClN4: C 66.98, H 4.68, N 17.36; found C 65.57, H 4.78, N 17.01.
6.2.4. 8-Chloro-6-ethyl-1-(pyridin-4-yl)-4H-
Thiolactam 2 (1.0 equiv) and carboxylic acid hydrazide 3a–e
(2.0 equiv) were dissolved in n-butanol and heated to 130 °C in a
sealed vial under nitrogen atmosphere for 24 h. The reaction mix-
ture was cooled to room temperature and stirred with an aqueous
glucose solution for 2 h. The crude product was extracted several
times with dichloromethane. The combined organic layers were
washed with water, dried over sodium sulfate, filtered and evapo-
rated. Purification was done by flash column chromatography
(dichloromethane/methanol, 9:1), giving the triazolo-benzodiaze-
pines 4a–e in 17–67% yield.
benzo[f][1,2,4]triazolo[4,3-a][1,4]diazepine (4d)
Yield: 17%; yellow solid; mp: 157.3–159.9 °C; 1H NMR
3
(500 MHz, CDCl3): d (ppm) 1.16 (t, JHH = 7.4 Hz, 3H, CH3), 2.72–
2.82 (m, 1H, CHH–CH3), 2.88–2.98 (m, 1H, CHH–CH3), 4.01 (d,
2
2JHH = 13.3 Hz, 1H, 4-CHH), 5.36 (d, JHH = 13.3 Hz, 1H, 4-CHH),
3
4
6.90 (d, JHH = 8.7 Hz, 1H, H-10), 7.39 (dd, JHH = 2.3 Hz,
3
3JHH = 8.7 Hz, 1H, H-9), 7.41 (dd, 2H, JHH = 6.2 Hz, H-20), 7.72 (d,
3
4JHH = 2.3 Hz, 1H, H-7), 8.72 (d, JHH = 5.7 Hz, 2H, H-30); 13C NMR
(125 MHz, CDCl3): d (ppm) 11.3 (CH3), 32.7 (CH2–CH3), 45.9 (C-
4), 122.3 (C-20), 126.4 (C-10), 129.0 (C-7), 131.1 (C-10a), 131.3
(C-9), 131.9 (C-6a), 134.3 (C-10), 134.7 (C-8), 150.9 (C-30), 151.2
6.2.1. 8-Chloro-6-ethyl-1-methyl-4H-
(C-1), 158.0 (C-3a), 171.7 (C-6); IR [cmꢀ1]:
v = 3036, 2971, 2927,
benzo[f][1,2,4]triazolo[4,3-a][1,4]diazepine (4a)
2853, 2366, 2344, 2225, 1633, 1604, 1531, 1484, 1467, 1434,
1290, 1110, 985, 827, 728, 576; MS (CI): m/z (%) = 324 (100)
[MH+]; MS (EI): m/z (%) = 323 (30) [M+Å], 294 (50) [M+ÅAEt], 288
(100) [M+ÅACl]; HR-MS (EI+): calcd for C17H14ClN5 [M+Å]
323.0938; found 323.0934.
Yield: 40%; yellow solid; mp: 173.5–174.9 °C; 1H NMR (500 MHz,
CDCl3): d (ppm) 1.09 (t, 3JHH = 7.3 Hz, 3H, CH2–CH3), 2.60 (s, 3H, 1-
CH3), 2.56–2.65 (m, 1H, CHH–CH3), 2.80–2.89 (m, 1H, CHH–CH3),
2
2
3.93 (d, JHH = 13.0 Hz, 1H, 4-CHH), 5.28 (d, JHH = 13.0 Hz, 1H, 4-
3
4
CHH), 7.35 (d, JHH = 8.6 Hz, 1H, H-10), 7.60 (dd, JHH = 2.4 Hz,
3JHH = 8.6 Hz, 1H, H-9), 7.68 (d, JHH = 2.3 Hz, 1H, H-7); 13C NMR
6.2.5. 8-Chloro-6-ethyl-1-(3-methoxyphenyl)-4H-benzo[f][1,2,4]
triazolo[4,3-a][1,4]diazepine (4e)
4
(125 MHz, CDCl3): d (ppm) 11.0 (CH2–CH3), 12.4 (1-CH3), 32.3
(CH2–CH3), 45.7 (C-4), 124.6 (C-10), 128.9 (C-7), 130.7 (C-10a),
131.1 (C-9), 131.9 (C-6a), 133.9 (C-8), 150.2 (C-1), 154.9 (C-3a),
Yield: 67%; yellow solid; mp: 132.6–133.7 °C; 1H NMR (400 MHz,
CDCl3): d (ppm) 1.14 (t, 3JHH = 7.4 Hz, 3H, CH2–CH3), 2.70–2.82 (m,
1H, CHH–CH3), 2.83–2.96 (m, 1H, CHH–CH3), 3.81 (s, 3H, OCH3),
170.9 (C-6); IR [cmꢀ1]:
v = 3066, 2971, 2933, 2852, 2360, 1635,
2
2
1542, 1485, 1426, 1378, 1111, 831; MS (CI): m/z (%) = 261 (100)
[MH+]; MS (EI): m/z (%) = 260 (30) [M+Å], 225 (100) [M+ÅACl]; HR-
MS (EI+): calcd for C13H13ClN4 [M+Å] 260.0829; found 260.0813.
4.00 (d, JHH = 13.4 Hz, 1H, 4-CHH), 5.33 (d, JHH = 13.2 Hz, 1H, 4-
4
4
3
CHH), 6.88 (ddd, JHH = 1.0 Hz, JHH = 1.3 Hz, JHH = 7.7 Hz, 1H, H-
3
4
60), 6.91 (d, JHH = 8.7 Hz, 1H, H-10), 7.00 (ddd, JHH = 1.0 Hz,
4JHH = 2.5 Hz, JHH = 8.4 Hz, 1H, H-40), 7.14 (dd, JHH = 1.7 Hz,
3
4
3
6.2.2. 8-Chloro-1,6-diethyl-4H-benzo[f][1,2,4]triazolo[4,3-a]
[1,4]diazepine (4b)
4JHH = 2.3 Hz, 1H, H-20), 7.30 (t, JHH = 7.8 Hz, 1H, H-50), 7.32 (dd,
4
3
4
1H, JHH = 2.4 Hz, JHH = 8.7 Hz, H-9), 7.66 (d, JHH = 2.4 Hz, 1H, H-
7); 13C NMR (100 MHz, CDCl3): d (ppm) 11.2 (CH2–CH3), 32.6
(CH2–CH3), 45.9 (C-4), 55.4 (OCH3), 113.7 (C-20), 116.4 (C-40), 120.6
Yield: 45%; yellow solid; mp: 203.6–205.9 °C; 1H NMR
3
(500 MHz, CDCl3): d (ppm) 1.07 (t, JHH = 7.4 Hz, 3H, 6-CH2–CH3),

P. Filippakopoulos et al. / Bioorg. Med. Chem. 20 (2012) 1878–1886
1885
(C-60), 126.4 (C-10), 127.6 (C-10), 128.4 (C-7), 130.0 (C-50), 130.8 (C-
9), 131.4 (C-10a), 131.5 (C-6a), 133.7 (C-8), 153.2 (C-1), 156.9 (C-3a),
UK). Coarse screens were typically setup onto Greiner 3-well plates
using three different drop ratios of precipitant to protein per con-
dition (100 + 50 nL, 75 + 75 nL and 50 + 100 nL). Initial hits were
optimized further scaling up the drop sizes. All crystallizations
were carried out using the sitting drop vapor diffusion method at
4 °C. BRD4(1) crystals with alprazolam were grown by mixing
200 nL of the protein (9.5 mg/mL and 5 mM final ligand concentra-
tion) with 100 nL of reservoir solution containing 0.20 M sodium
sulfate, 0.1 M BT-Propane pH 6.5, 20% PEG3350 and 10% ethylene
glycol. BRD4(1) crystals with midazolam were grown by mixing
200 nL of protein (9.36 mg/mL and 5 mM final ligand concentra-
tion) with 100 nL of reservoir solution containing 0.1 M magne-
sium chloride, 0.1 M MES pH 6.5, 15% PEG6000 and 10% ethylene
glycol. BRD4(1) crystals with BzT-7 were grown by mixing
200 nL of protein (9 mg/mL and 5 mM final ligand concentration)
with 200 nL of reservoir solution containing 0.1 M MES pH 6.5,
10% PEG3350 and 10% ethylene glycol. In all cases diffraction qual-
ity crystals grew within a few days.
159.9 (C-30), 171.7 (C-6); IR [cmꢀ1]:
v = 3072, 2970, 2935, 1632,
1583, 1535, 1485, 1466, 1435, 1319, 1287, 1238, 1108, 1045, 994,
753; MS (CI): m/z (%) = 353 (100) [MH+]; MS (EI): m/z (%) = 352
(80) [M+Å], 323 (50) [M+ÅAEt], 317 (100) [M+ÅACl]; HR-MS (EI+): calcd
for C19H17ClN4O [M+Å] 352.1091; found 352.1098.
The NMR spectra of all synthesized compounds are shown in
the Supplementary data of this manuscript.
6.3. Protein stability shift assay
Thermal melting experiments were carried out using an
Mx3005p Real Time PCR machine (Stratagene). Proteins were buf-
fered in 10 mM HEPES pH 7.5, 500 mM NaCl and assayed in a 96-
well plate at a final concentration of 2
lM in 20
lL volume. Com-
pounds were added at a final concentration of 10
lM or 100
lM
in order to probe weaker interactions. SYPRO Orange (Molecular
Probes) was added as a fluorescence probe at a dilution of
1:1000. Excitation and emission filters for the SYPRO-Orange dye
were set to 465 nm and 590 nm, respectively. The temperature
was raised with a step of 3 °C per minute from 25 to 96 °C and fluo-
rescence readings were taken at each interval. The temperature
dependence of the fluorescence during the protein denaturation
process was approximated by the equation
6.7. Data collection and structure solution
All crystals were cryo-protected using the well solution supple-
mented with additional ethylene glycol and were flash frozen in li-
quid nitrogen. Data were collected in-house on a Rigaku FRE
rotating anode system equipped with a RAXIS-IV detector (alpraz-
olam and midazolam complexes) or at the Diamond beamline I04.1
(BzT-7 complex). Indexing and integration was carried out using
MOSFLM30 and scaling was performed with SCALA31 or XDS.32 Ini-
tial phases were calculated by molecular replacement with PHA-
SER33 using the known models of BRD4(1) (PDB ID 2OSS). Initial
models were built by ARP/wARP34 followed by manual building
in COOT.35 Refinement was carried out in REFMAC5.36 In all cases
thermal motions were analyzed using TLSMD37 and hydrogen
atoms were included in late refinement cycles. Data collection
and refinement statistics can be compiled in Table 2. The models
and structure factors have been deposited with PDB accession
codes: 3U5J (BRD4(1)/alprazolam), 3U5K (BRD4(1)/midazolam),
3U5L (BRD4(1)/BzT-7), respectively.
yU ꢀ yF
yðTÞ ¼ yF þ
ð1Þ
uGT=RT
1 þ eD
where
DuG(T) is the difference in unfolding free energy between
the folded and unfolded state, R is the gas constant and yF and yU
are the fluorescence intensity of the probe in the presence of com-
pletely folded and unfolded protein respectively. The baselines of
the denatured and native states were approximated by a linear fit.
The observed temperature shifts, D
Tombs, were recorded as the differ-
ence between the transition midpoints of sample and reference
wells containing protein without ligand in the same plate and
determined by non-linear least squares fit.
6.4. Isothermal titration calorimetry
Supplementary data
Experiments were carried out on a VP-ITC titration microcalo-
rimeter from MicroCal™, LLC (Northampton, MA). All experiments
were carried out at 15 °C while stirring at 295 rpm, in ITC buffer
(50 mM HEPES pH 7.4 at 25 °C, 150 mM NaCl). The microsyringe
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.10.080.
was loaded with a solution of the protein sample (150
BRD4(1) in ITC buffer). All titrations were conducted using an ini-
tial control injection of 2 L followed by 34 identical injections of
L with a duration of 16 s (per injection) and a spacing of 250 s
between injections. The heat of dilution was determined by inde-
pendent titrations (protein into buffer) and was subtracted from
the experimental data. The collected data were evaluated using a
single binding site model and the MicroCal™ Origin software.
lM
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