New Synthetic Routes to Triazolo-benzodiazepine Analogues: Expanding the Scope of the Bump-and-Hole Approach for Selective Bromo and Extra-Terminal (BET) Bromodomain Inhibition

Article abstract of DOI:10.1021/acs.jmedchem.5b01135

We describe new synthetic routes developed toward a range of substituted analogues of bromo and extra-terminal (BET) bromodomain inhibitors I-BET762/JQ1 based on the triazolo-benzodiazepine scaffold. These new routes allow for the derivatization of the me

Full text of DOI:10.1021/acs.jmedchem.5b01135

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Article
New Synthetic Routes to Triazolo-Benzodiazepine
Analogues: Expanding the Scope of the Bump-and-
Hole Approach for Selective BET Bromodomain Inhibition
Matthias G. J. Baud, Enrique Lin Shiao, Michael Zengerle, Cynthia Tallant, and Alessio Ciulli
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01135 • Publication Date (Web): 14 Sep 2015
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New Synthetic Routes to TriazoloꢀBenzodiazepine
Analogues: Expanding the Scope of the Bumpꢀandꢀ
Hole Approach for Selective BET Bromodomain
Inhibition
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Matthias G. J. Baud,a,b,† Enrique LinꢀShiao,a,b,≠ Michael Zengerle,a Cynthia Tallantb,§ and
Alessio Ciulli*a,b
a Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of
Dundee, James Black Centre, Dow Street, Dundee, DD1 5EH, UK.
b Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW,
UK.
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ABSTRACT. We describe new synthetic routes developed towards a range of substituted
analogues of BET bromodomain inhibitors IꢀBET762/JQ1 based on the triazoloꢀbenzodiazepine
scaffold. These new routes allow for the derivatization of the methoxyphenyl and chlorophenyl
rings, in addition to the diazepine ternary centre and the side chain methylene moiety.
Substitution at the level of the side chain methylene afforded compounds targeting specifically
and potently engineered BET bromodomains designed as part of a bump and hole approach. We
further demonstrate that marked selectivity for the second over the first bromodomain can be
achieved with an indole derivative that exploits differential interaction with a Asp/His
conservative substitution on the BC loop of BET bromodomains.
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INTRODUCTION
The 1,4ꢀbenzodiazepine scaffold occupies a place of choice in the toolbox of medicinal
chemists, and is often referred to as a “privileged scaffold” in drug discovery. A larger number of
biologically active small molecules containing a 1,4ꢀbenzodiazepine scaffold have been
approved by the FDA for the treatment of various disease states, although most of them are well
known for their psychotropic effects.1 Well known examples include Diazepam, Alprazolam or
Prazepam. The therapeutic potential of 1,4ꢀbenzodiazepines has fuelled the interest of synthetic
chemists in developing new routes to a range of substituted analogues for biological evaluation.2ꢀ
4
Recently, this scaffold has attracted particular attention in the field of epigenetics, with the
discovery of a class of potent small molecules inhibitors of the interaction between Bromo and
ExtraꢀTerminal (BET) bromodomain proteins with their acetylated histone substrates. BET
proteins Brd2, Brd3, Brd4 and Brdt are key transcriptional coꢀregulators. Crucial to their activity
are paired and highly homologous bromodomains located in their aminoꢀterminal regions. The
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individual function of the first bromodomain (e.g. Brd2(1)) versus second bromodomain (e.g.
Brd2(2)) of BET proteins is however unclear. A number of BET bromodomain inhibitors are
currently in clinical trials for the treatment of cancer,5 including representative molecules Iꢀ
BET762 (1),6 JQ1 (2),7 GW841819X (3),8 OTX015 (4),9 and RVXꢀ208 (5)10 (Figure 1A). In
particular, compounds 1ꢀ4 are based on a triazoloꢀaryldiazepine scaffold (aryl = methoxyphenyl
or dimethylthiophene) and bind to the acetylꢀlysine (KAc) pocket of BET bromodomains with
high affinity (Kd of 1: 50ꢀ370 nM).11 These compounds display activity in vivo12 against a
number of disease states, including NUTꢀmidline carcinoma,13 multiple myeloma,14 mixedꢀ
lineage leukemia,15 and acute myeloid leukemia.16,17 Despite selectively targeting the BET
bromodomain family with high potency over other bromodomains, these compounds are panꢀ
BET selective thus do not significantly discriminate between individual bromodomains of the
four BET members. This lack of selectivity within the BET subfamily so far has prevented
accurate deconvolution of the biological function of individual BET proteins and of their tandem
bromodomains. To address this problem, we recently developed a chemical genetics approach to
engineer the selectivity of the BET bromodomain inhibitor IꢀBET762/JQ1 within the BET
proteins family.11 This soꢀcalled “bump and hole” approach is based on the generation of
orthogonal and highꢀaffinity protein:ligand pairs and involves introducing a single point
mutation (large to smaller amino acid i.e. the “hole”) onto the BET bromodomain of interest
together with making a synthetic modification (bulky substituent i.e. the “bump”) onto the parent
BET bromodomain binder to complement the newly created protein subpocket (Figure 1B). As a
result, the bulky ligand is expected to bind with high affinity to the mutated BET protein, while
exhibiting weak to no binding to wildꢀtype (WT) proteins due to a steric clash occuring between
the “bump” and the naturally occuring residue (Figure 1B). This approach was previously shown
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to aid selective targeting of protein kinases through engineering of the ATP binding site and ATP
cofactor as well as ATPꢀcompetitive inhibitors.18,19 In our study, we demonstrated for the first
time that the bump and hole approach can be used to selectively disrupt proteinꢀprotein
interactions within the BET family of proteins.11 Compound ET (6) (Figure 1CꢀD), a derivative
of IꢀBET762/JQ1 bearing an ethyl functional group at the level of the side chain methylene
moiety, bound to leucine/alanine mutant BET bromodomains with low nanomolar affinity and
displayed up to 540ꢀfold and no less than 40ꢀfold (160ꢀfold on average) selectivity relative to
WT BET bromodomains across the entire subfamily. This orthogonal bromodomain:ligand pair
was used within cancer cells to show that selective blockade of the first bromodomain of a given
BET protein, Brd4, is sufficient to displace it from chromatin.11 The exquisite selectivity
provided by ET for engineered bromodomains is currently exploited in our laboratory to probe
the biology of individual BET proteins through selective modulation of their interaction with
their histone substrates. Selective modulation of individual BET bromodomains is important for
accurate and reliable target validation in the different disease states that are associated with
unbalanced activity of BET proteins.
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The ET:L/A orthogonal inhibitor:protein pair was discovered and optimised within the
framework of an extended study in which we explored several mutations (“holes”) and Iꢀ
BET762 substitution patterns (“bumps”). In the current manuscript, we report the full journey
that led to that discovery. In doing so, we also describe our synthetic efforts towards 6 and other
novel analogues aimed at targeting the mutant proteins. In particular, we report new synthetic
routes that we developed towards this aim, including IꢀBET762 analogues bearing substitution
patterns at the level of the methoxyphenyl and chlorophenyl rings, in addition to the side chain
methylene. Finally, we present biophysical evaluation of the compounds within the context of
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our bumpꢀandꢀhole project, and highlight useful isoform selectivity criteria for the design of the
next generation of BET bromodomain inhibitors.
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[Insert Fig. 1 here]
Figure 1. A) Structures of BET bromodomain probes IꢀBET762 (1), JQ1 (2), GW841819X (3),
OTXꢀ015 (4) and RVXꢀ208 (5), currently in clinical trials; B) Bump and hole approach to
engineer the selectivity of BET bromodomain probes against individual BET bromodomains; C)
Structure of ET (6); D) Coꢀcrystal structure of Brd2(2)L383A (blue, surface representation) in
complex with ET (6) (stick representation, yellow carbons), PDB code 4QEW.11 The L/A
mutation is shown in red.
RESULTS AND DISCUSSION
1. Design of engineered BET bromodomain:ligand pairs and synthetic targets
Analyses of sequence alignments (Figure S1) and inhibitorꢀbound crystal structures11
guided us to focus on eleven residues that are strictly conserved within the BET subfamily and
are in close contact with the ligand. Keeping in mind that the introduced mutations should not
significantly disrupt protein stability and substrate binding, residues Y97, C136, Y139 and N140
(Brd4(1) numbering) were discarded, as they are known to be important for KAc recognition20,21
and for preserving a key network of bound water molecules.22 Buried residues P82 and F83 from
the bottom of the soꢀcalled WPF shelf were also discarded as their mutation was predicted to
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destabilize the integrity of the hydrophobic core.23 Residues L92 and M149 looked promising,
but were not pursued further due to a lack of suitable vectors arising from the inhibitor scaffold
that could be exploited to complement potential mutations. The remaining three residues i.e. the
more peripheral, hydrophobic W81 from the top of the WPF shelf, and V87 and L94 from the
ZA loop, were selected for site directed mutagenesis (Figure 2A). Mutants W/F and W/H, V/A,
L/I and L/A were constructed within Brd2(1), recombinantly expressed, purified from E. coli and
biophysically characterized in order to assess their stability and histone binding capacity (Table
S1). All mutants maintained melting temperatures (Tm) above 37 °C, and most had comparable
stabilities to the WT proteins, as assessed by differential scanning fluorimetry (DSF).
Importantly, all mutants retained competence to bind a tetraꢀacetylated H4 derived peptide24 as
assessed by ITC albeit to varying degrees (Table S1). Most mutants exhibited comparable
peptide binding affinities relative to WT, while the V/A proved the most disruptive mutation.
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With three positions identified and corresponding mutants characterized, IꢀBET762 (1)
was selected as the starting scaffold because more synthetically tractable and better suited to all
required vectors than JQ1. Molecular modeling studies suggested that i) a “bump” R1 originating
from the methoxyphenyl ring could target the hole introduced by the V87A mutation; ii) R2
functionalization at the level of the side chain methylene, in a (R) configuration, could target L94
mutations; and iii) the pꢀchlorophenyl ring could provide suitable vectors for R3 substituents to
explore W81 mutations (Figure 2BꢀC). A methyl group was elected as the bump of choice to
explore the engineered holes because it represents the smallest hydrophobic functional group that
at the same time introduces minimal alteration of the ligand scaffold in terms of electronics,
conformation and physicochemical properties. We therefore performed a “methyl scan” around
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the IꢀBET762 scaffold by synthesizing analogues functionalized with methyl groups at R1ꢀR3
(Figure 2C) to target mutations at the respective positions.
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[Insert Fig. 2 here]
Figure 2. A) Crystal structure of IꢀBET762 (1, yellow carbons) bound to Brd4(1) (PDB code
3P5O,2 surface representation, red negative, blue positive electrostatic potential). W81, V87 and
L94 are highlighted; BꢀC) IꢀBET762 chemical structure and positions selected for derivatization
to target the corresponding mutations.
2. Chemical strategies to target the V87A mutation: R1 = Me
The ester derivative of IꢀBET762 was chosen as the parent scaffold for efficient enolate
generation and substitution of the methylene side chain (R2, see later). Compound 14 was
prepared as previously described, with significant yield improvement (55% overall) compared to
those reported by Chung et al. (22% overall) (Scheme 1).8 Chlorination of protected acid 725 and
Nꢀacylation of the appropriate aminoꢀbenzophenones 8ꢀ9, followed by Fmoc deprotection and
subsequent cyclisation afforded 10ꢀ11 in excellent yields in a fourꢀstep, oneꢀpot sequence.
Further thionation afforded thioamide derivatives 12ꢀ13. Treatment of thioamides 12ꢀ13 with
hydrazine monohydrate, followed by acetylation and further cyclisation in acidic conditions
afforded triazoles 14ꢀ15 in high yield in a threeꢀstep, oneꢀpot procedure. Starting from
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benzophenone derivative 9 allowed us to ultimately introduce the R1 methyl group (Figure 2C).
While yields for the condensation and thionation reactions were excellent, the triazole formation
towards 15 proceeded in only 38% yield, much lower than in the case of 14. This reflects the
lower yield for the final cyclisation (Scheme 1, step h) which only proceeded in reflux
conditions, along with significant degradation. We attributed the latter to the steric demand
imposed by the R1 methyl group and the triazole methyl group in the cyclisation process.
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Scheme 1. Synthesis of 14 and its methylated derivative 15. Conditions: a) SOCl2, CH2Cl2,
reflux, 2.5h, b) benzophenone, CHCl3, reflux, 3h, c) Et3N, CHCl3, reflux, 16h, d) AcOH, 1,2ꢀ
DCE, 60°C, 3h, e) Lawesson's reagent, toluene, reflux, 4h, f) Hydrazine.H2O, THF, 0°C, 5h, g)
AcCl, Et3N, 0°C to rt, 16h, h) AcOH, rt, 2 days (R = H) or reflux, 3h (R = Me).
3. Chemical strategies to target the L94 mutations: R2 = Me
We envisaged that introduction of alkyl substituents on the methylene side chain would be
achievable through the generation of the enolate of 14 followed by reaction with an alkyl halide
(Scheme 2). Among the various bases explored for enolate generation, including LDA, NaH, and
KHMDS, the latter proved the most efficient and provided the cleanest reaction and best yields.
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Treatment of (+ꢀ)ꢀ14 with 1.2 equivalents of KHMDS at ꢀ78°C, followed by addition of methyl
iodide afforded a diastereomeric mixture of (+ꢀ)ꢀ(1S,2S)ꢀ16 and (+ꢀ)ꢀ(1S,2R)ꢀ16 (Scheme 2A).
The reaction provided (+ꢀ)ꢀ(1S,2S)ꢀ16 as the major alkylation product, and proved to be highly
diastereoselective e.g. up to 25:1 with MeI. Such selectivity was strongly dependent on the
temperature gradient, in certain cases, down to 3:1. The structure of the major diastereomer
resulting from the alkylation of (+ꢀ)ꢀ14 with MeI could be unambiguously assigned as (+ꢀ)ꢀ
(1S,2S)ꢀ16 on the basis of previous crystallographic studies.11 We refer to the active stereomer
(+ꢀ)ꢀ(1S,2R)ꢀ16 as ME for clarity.11 The reason for this observed diastereoselectivity is unclear.
The potassium countercation might provide conformational restriction to the (Z)ꢀenolate in a 6ꢀ
membered ring transition state, via coordination to the sp2 nitrogen of the diazepine ring
(Scheme 2C). However, in such a transition state the observed preference for the attack on the
proꢀS face is not evident simply based on sterics, suggesting other factors come into play. Further
mechanistic studies will be needed to address this point. Nevertheless, when a high
diastereomeric ratio did not allow for the isolation of reasonable amounts of the desired active
(+ꢀ)ꢀ(1S,2R)ꢀ16 diastereomer in pure form, the diastereomeric mixture could be readily
epimerised with sodium methoxide under microwave irradiation to afford a 1:1 mixture of
diastereomers, which could then be separated by flash column chromatography.
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Along with alkylation of the side chain, we observed minor albeit observable alkylation
of the ternary C1 carbon of the diazepine ring, affording derivative (+ꢀ)ꢀ17. This suggests that
the C1 position of (+ꢀ)ꢀ15 is deprotonated, at least partially, during addition of KHMDS at ꢀ
78°C. This is consistent with the intense dark colour observed following addition of KHMDS,
potentially reflecting the generation of a highly delocalised anionic species. During their study of
the memory of chirality in related 1,4ꢀbenzodiazepinꢀ2ꢀone systems, the Carlier group
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documented on the instalment of quaternary centres at C1 using enolate alkylation chemistry.28ꢀ30
Notably, they also observed superior results when using KHMDS as a base for enolate
generation. Due to the “privileged” status attributed to the 1,4ꢀbenzodiazepinꢀ2ꢀone scaffold in
drug discovery, this represented an important finding as it offered the first short and robust route
towards novel, conformationally restricted 1,4ꢀbenzodiazepinꢀ2ꢀone analogues.28ꢀ30 We here
show that instalment of a quaternary centre at C1 on a triazolo benzodiazepine scaffold can
indeed be achieved, although in low yield. This can be particularly attractive if one wants to
develop structureꢀactivity relationships of BET bromodomain binders (e.g. 1ꢀ4) through double
functionalization at C1. In particular, this would represent a real advantage to the use of
quaternary amino acid precursors, which are sterically hindered and likely to reduce the overall
yield, in addition to being expensive and providing a narrow scope for substitution. Moreover,
this would offer a late state divergent synthetic strategy towards library analogues. However, the
usefulness and general applicability of such a strategy will be contingent on more robust reaction
conditions and improved yields. Further optimization studies are ongoing in our laboratories in
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order to tune the regioselectivity, reaction times, and yields of this key reaction. Of note, the H
NMR spectrum of (+ꢀ)ꢀ17 showed two species in a ca. 2:1 ratio. This is reflective of the slow
conformational equilibrium imposed by the steric demand at C1, and in line with Carlier’s
results.28
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Scheme 2 A) Alkylation of (+ꢀ)ꢀ14 provides a mixture of diastereomers and alkylation at C1; B)
Proposed overall mechanism for the observed results; C) Proposed transition state for the enolate
alkylation.
4. Chemical strategies to target the W81 mutations: Derivatization of the chlorophenyl
ring
We envisaged that developing new synthetic routes towards the IꢀBET scaffold would be of
particular interest to gain rapid access to libraries of analogues to address other mutational
positions in the binding pocket. Specifically, we next explored the possibility to access Iꢀ
BET762 analogues with diverse substitution patterns at the level of the chlorophenyl ring (Figure
2C). While this should be potentially achievable through previously reported routes (Scheme 1),
the early stage introduction of the chlorophenyl ring makes this linear sequence very impractical
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for analogue generation. We therefore considered that a new route allowing the late stage
introduction of the substituted phenyl moiety would be valuable. We hypothesised that such
analogues would be obtained by SuzukiꢀMiyaura cross coupling of an imidoyl chloride with the
appropriate phenylboronic acid derivative31 (Scheme 3). A wide variety of phenylboronic acid
derivatives are commercially available, readily accessible and affordable. The imidoyl chloride
would be obtained by chlorination of the corresponding amide. The triazole moiety would in turn
be introduced from the corresponding amide, through a thionation/condensation/cyclisation
sequence. The diamide would be obtained through condensation of inexpensive 5ꢀ
methoxyisatoic anhydride32 and aspartic acid dimethyl ester.
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Scheme 3. Proposed retrosynthetic analysis for the functionalization of the chlorophenyl ring.
The synthesis of our library of analogues is shown in Scheme 4. 2ꢀaminoꢀ5ꢀ
methoxybenzoic acid 18 was converted to 5ꢀmethoxyisatoic anhydride 19 in quantitative yield.32
Condensation of 19 with aspartic acid dimethylester afforded the bicyclic precursor 20 in 42%
yield. Selective thionation could be achieved in modest yield by treatment with Lawesson’s
reagent in refluxing pyridine, affording thioamide 21 in 48% yield. We envisaged that a oneꢀstep
procedure for the instalment of the triazole would be particularly convenient compared to the
threeꢀsteps procedure employed previously (Scheme 1). A representative set of conditions used
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for the instalment of the triazole moiety is shown in Table 1. Reaction outcome was assessed by
NMR of crude mixtures. Thioamide 21 was poorly soluble in a variety of solvents but was
soluble in refluxing pyridine. Treatment of 21 with 2.5 eq. acethydrazide for 1 day at reflux led
to the formation of product 22, along with remaining unreacted 21 and significant formation of
the exocyclised product arising from condensation of the intermediate acylhydrazone with the
side chain ester (Table 1, entry 1). Increasing the number of equivalents of acethydrazide
resulted in low formation of 22, and afforded the exocyclised byꢀproduct as the major component
of the reaction (Table 1, entry 2). We envisaged that exocyclisation could be prevented by
lowering the reaction temperature. However, the reaction did not proceed due to the poor
solubility of 21 in pyridine at rt (Table 1, entry 3). Despite its poor solubility, activation of the
thioamide with thiophilic mercury diacetate33 allowed for rapid and quantitative consumption of
21 at rt, and afforded the intermediate acylhydrazone almost quantitatively along with trace
amount of product 22 (Table 1, entry 4). Switching the solvent to acetonitrile led to similar
results, even after reaction times up to six days (Table 1, entry 5). Pleasingly, changing the
solvent to THF/AcOH afforded the desired product 22 in 91% yield after 24h reaction. Other
methods involving chlorophosphate reagents have been previously reported for the instalment of
the triazole unit of JQ1, although in those cases the amide chlorination step required cooling
to ꢀ78°C and the subsequent cyclisation step required heating up to 90°C.7,34 Despite the toxicity
of the mercury reagent, our oneꢀstep procedure is particularly convenient because it is milder,
lowers the reaction time by ca. 3 fold compared to previously reported routes, does not require
intermediate workꢀup, proceeds smoothly at rt (and even at 0°C), while displaying similar yields.
Of note, this procedure could also be applied for the conversion of 12 to 14 (Scheme 1) with an
identical 91% yield. The amide of 22 was subsequently converted to the corresponding imidoyl
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22
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chloride 23 in 39% yield. In particular 23 proved to be moisture and nucleophile (e.g. MeOH)
sensitive, which translates into the only moderate yield obtained for its formation. Finally,
SuzukiꢀMiyaura crossꢀcoupling of imidoyl chloride 23 with a set of representative phenylboronic
acids31 afforded the final, substituted IꢀBET analogues 24ꢀ28. As a control, coupling of 23 with
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1
4ꢀchlorophenylboronic acid afforded the parent molecule 12, as compared by H NMR with a
reference sample. This synthetic route proved to be robust and reasonably scalable, allowing
preparation of imidoyl chloride precursor 23 on a 2 mmol scale after five steps. Of interest, no
chromatographic step was required prior to obtaining triazole 22. The poor solubility of 20 and
21 in a variety of solvents allowed them to be isolated in pure form by simple trituration and
filtration. Of note, the synthetic routes described here are purposely racemic in order to provide
maximum stereochemical diversity. However, non racemising conditions should in theory be
applicable to this route. In particular, the use of chlorophosphate reagents instead of Lawesson’s
reagent or P2S5 for triazole formation has been previously shown to significantly reduce
epimerization at the level of the ternary centre (final d.r. 9:1).7,34
O
O
H
H
O
S
H
N
N
N
NH2
OH
O
O
O
a
b
c
O
NH
NH
O
O
O
O
O
18
19
20
21
O
O
e
O
O
N
N
N
N
N
N
O
O
N
N
N
d
f
O
O
O
NH
N
N
O
O
O
O
Cl
Ar
22
23
Cl
Ar =
Cl
Cl
Cl
24
25
26
27
28
NH
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Scheme 4. Racemic synthesis of analogues 24ꢀ28. Conditions: a) Triphosgene, THF, rt, quant. b)
Aspꢀ(OMe)2, pyridine, reflux, 24h, 42%, c) Lawesson's reagent, pyridine, reflux, 1.25h, 48%, d)
AcNHNH2, Hg(OAc)2, THF/AcOH, rt, 24h, 91%, e) P(O)Cl3, N,Nꢀdimethylaniline, 125°C, 1h,
39%, f) ArB(OH)2, Et3N, Pd(PPh3)4, DMF, 100°C, 27ꢀ31%.
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Entry
Conditions
Outcome
1
2
3
4
5
6
2.5 equiv. AcNHNH2 , pyridine, reflux ~1d
6 equiv. AcNHNH2, pyridine, reflux ~1d
mix. 21 (32%) + 22 (43%) + exocyclisation (25%)
mix. 21 (22%) + 22 (49%) + exocyclisation (29%)
21 insoluble
3 equiv. AcNHNH2, pyridine, rt, 1d
3 equiv. AcNHNH2, 1.5eq. Hg(OAc)2, pyridine, rt, 1d
3 equiv. AcNHNH2, 1.5eq. Hg(OAc)2, MeCN, rt, 6d
3 equiv. AcNHNH2, 1.5eq. Hg(OAc)2, THF/AcOH, rt, 24h
Acylhydrazone (> 95%) + 22 (< 5%)
Acylhydrazone (87%) + 22 (13%)
91% 22
Table 1. Representative conditions for triazole formation (conversion of 21 to 22). Yields for
entries 1ꢀ5 were determined based on NMR spectra of crude samples. The yield for entry 6 is for
the isolated, purified material.
5. Biophysical evaluation
In order to determine which position/substitution combination would provide the best
selectivity profile, binding of IꢀBET762 and analogues 15, (+ꢀ)ꢀ(1S,2S)-16, ME, 24ꢀ28 against
WT and mutant Brd2(1) and Brd2(2) proteins was assessed initially by differential scanning
fluorimetry (Table 2). Brd2(1) and Brd2(2) were chosen as representatives of the first and second
bromodomain of BET proteins, respectively. Introduction of methyl “bumps” at R1 and R3
(methoxyphenyl and chlorophenyl rings respectively, cpds 15, 24ꢀ28) did not provide noticeable
thermal stabilization of the targeted Brd2 mutants compared to WT. In contrast, the methyl bump
at R2 in a 2ꢀR configuration (ME) provided the first evidence of selective stabilization in our
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engineered system.11 Compound ME induced ꢁTm of 5.7 and 9.6 °C on Brd2(1)L110I and
Brd2(2)L383I, respectively, while stabilizing the WT proteins by only 3.2 and 5.6 °C. This
selective thermal stabilization was even more pronounced in the case of the L/A mutations, with
ꢁTm of 7.9 and 9.3 °C against Brd2(1)L110A and Brd2(2)L383A, respectively. This selectivity
profile was validated by measuring dissociation constants (Kd) using ITC (Table 3). ME was
highly potent against both Leu mutants, displaying Kds of 17 and 22 nM against brd2(1)L110A and
brd2(2)L383A and Kds of 260 and 27 nM against brd2(1)L110I and brd2(2)L383I, respectively.
Crucially, ME showed between 11ꢀfold and 86ꢀfold weaker affinities to WT compared to the
Leu mutant proteins. As we anticipated, the diastereoisomer (+ꢀ)ꢀ(1S,2S)ꢀ16 did not induce a
significant stabilization of mutant or WT proteins (Table 2).
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Synthetic optimization of ME led to the highly potent and L/Aꢀmutant selective
compound ET (6) (Figure 1C), data that we have reported elsewhere.11 A complete binding
selectivity profiling by DSF and ITC against all eight WT BET bromodomains and their L/A
mutant counterparts showed that ET binds up to 540 fold stronger and not less than 30ꢀfold
(average 160ꢀfold) to L/A mutants compared to WT proteins, therefore validating our design
strategy.11 Importantly, selective targeting of engineered L/A mutants could be achieved in a
cellular context, as demonstrated using a fluorescence recovery after photoꢀbleaching assay.11
Bromodomain
IꢀBET762 (1)
15
16a
MEb
24
25
26
27
28
protein
Brd2(1)
5.4 ± 0.5
0.1 ± 0.6
6.7 ± 0.4
3.1 ± 0.4
0.4 ± 0.2
0.7 ± 0.2
0.7 ± 0.2 ꢀ0.3 ± 0.2
3.2 ± 0.2 6.3 ± 0.1 1.5 ± 0.2
1.8 ± 0.2
1.2 ± 0.2
6.8 ± 0.6
Brd2(1)V103A
Brd2(1)L110I
Brd2(1)L110A
Brd2(1)W097F
Brd2(1)W097H
0.5 ± 0.3
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
0.0 ± 0.5
5.7 ± 0.7
/
/
/
1.6 ± 0.2
7.9 ± 0.2
/
/
/
/
/
/
/
1.4 ± 0.2 ꢀ0.1 ± 0.2
0.9 ± 0.3 0.2 ± 0.2
0.1 ± 0.2
0.0 ± 0.2
1.9 ± 0.5
0.6 ± 0.3
ꢀ0.4 ± 0.3 ꢀ0.4 ± 0.2
Brd2(2)
8.3 ± 0.3
4.0 ± 0.1
0.2 ± 0.2
5.6 ± 0.1 6.6 ± 0.2 3.2 ± 0.1
3.5 ± 0.1
2.5 ± 0.2
7.7 ± 0.2
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Brd2(2)V376A
Brd2(2)L383I
Brd2(2)L383A
Brd2(2)W370F
Brd2(2)W370H
1.1 ± 0.0
9.3 ± 0.3
6.4 ± 0.2
2.1 ± 0.0
1.7 ± 0.2
1.2 ± 0.1
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
0.3 ± 0.2
9.6 ± 0.1
/
/
/
0.8 ± 0.6
9.3 ± 0.2
/
/
/
/
/
/
/
2.8 ± 0.1 1.5 ± 0.0
1.1 ± 0.2 1.0 ± 0.3
0.6 ± 0.1
0.3 ± 0.0
5.2 ± 0.1
2.7 ± 0.4
ꢀ0.1 ± 0.1 ꢀ0.4 ± 0.2
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Table 2. “Methyl scan”. Thermal stabilization (°C) of wildꢀtype and mutant Brd2 bromodomains
by IꢀBET derivatives 15, 16, 24ꢀ28, as assessed by DSF. a(+ꢀ)ꢀ(1S,2S)ꢀ16. b(+ꢀ)ꢀ(1S,2R)ꢀ16
(ME).
Bromodomain
Kd (nM)
ꢀH (kcal/mol)
protein
Brd2(1)
1470 ± 180
260 ± 40
17 ± 4
ꢀ8.6 ± 0.2
ꢀ8.5 ± 0.1
ꢀ16.8 ± 0.2
Brd2(1)L110I
Brd2(1)L110A
Brd2(2)
300 ± 80
27 ± 12
22 ± 4
ꢀ5.4 ± 0.1
ꢀ9.8 ± 0.1
ꢀ12.6 ± 0.1
Brd2(2)L383I
Brd2(2)L383A
Table 3. Affinities (Kds) and binding enthalpies (ꢀH) obtained by ITC for ME against wildꢀtype
and L/A and L/I mutant Brd2 bromodomains at 25 oC.
While indole derivative 28 could only induce moderate stabilization of W/F and W/H
mutants, we noted that 28 greatly stabilized WT Brd2(1) and Brd2(2) (Table 2). We therefore
decided to further characterize 28 and determined its binding affinity to Brd2(1) and Brd2(2) by
ITC (Table 4). 28 exhibited Kds of 800 and 40 nM against Brd2(1) and Brd2(2), respectively,
corresponding to ca. 20 fold selectivity for the second over the first bromodomain. The same
trend in selectivity was observed with the two bromodomains of Brd4 (Table 4). This selectivity
of 28 for the second BET bromodomain could result at least in part from amino acid changes in
the BC loop flanking the inhibitor binding site. In particular, an Asp residue in the BC loop
(Asp160 in Brd2(1)) is conserved amongst all first BET bromodomains and conservatively
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replaced by a His residue in the second BET bromodomains (His433 in Brd2(2), highlighted in
Figure S1). To test this hypothesis, we solved the Xꢀray crystal structures of Brd2(2)W370F in its
apo form and with both 28 and the parent IꢀBET762 (1) bound (Figure 3, see Table S1 for Xꢀray
crystallographic data collection and refinement statistics and Figure S2 for electron density map
around the bound ligands). The binding modes of 28 and 1 to Brd2(2)W370F were found to be
identical, with all atoms of the triazoloꢀbenzodiazepine scaffold superposing very well, and the
aromatic indole ring of 28 being almost coꢀplanar with the paraꢀchloroꢀphenyl ring of 1 (Figure
3A). The observed binding mode recapitulates that of IꢀBET762/JQ1 bound to WT BET
bromodomains, suggesting that it is not altered by the W/F mutation (Figure 3B). Importantly,
the side chain of His433 switches from an “open” conformation observed in the crystal structure
of Brd2(2)W370F with 1 bound, pointing away from the ligand, to a “closed” conformation when
28 is bound to form an edgeꢀtoꢀface π stacking with the indole ring of 28 (Figure 3A). Both these
“open” and “closed” His side chain conformers are observed in other crystal structures of wildꢀ
type Cꢀterminal BET bromodomain, as in Brd2 (PDB codes 2E3K and 5BT5) and Brd3 (His395,
PDB codes 2OO1 and 3S92). His433 in Brd2(2) is substituted by Asp160 in Brd2(1) (Figure
3B), which cannot engage in such interaction in a closed conformation, potentially explaining the
decreased potency of 28 against Brd2(1). Discrimination between first and second
bromodomains of BET protein has been observed to varying degrees with small molecules
RVXꢀ208 (5),10 MS436,35 and Olinone36 (Table 4), none of which are based on the triazoloꢀ
benzodiazepine scaffold. For example, RVXꢀ208 (5) displayed up to 23 fold selectivity for
Brd2(2) (Kd ca. 250 nM) compared to Brd2(1) (Kd ca 5800 nM), which could also be explained
by the flexibility of His433.10 Our data highlight that such isoform selectivity can be achieved
with the IꢀBET762/JQ1 scaffold via careful substitution of the parent chlorophenyl ring. This
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adds a useful isoform selectivity criterion that can be exploited for the design of next generation
triazoloꢀbenzodiazepine probes targeting BET proteins.
7
8
9
IꢀBET762
(1)11
Bromodomain
28
RVXꢀ208 (5)10
MS43635
Olinone36
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protein
Brd2(1)
Brd2(2)
Brd4(1)
Brd4(2)
230
100
95
780
45
5800
250
8600
> 300 000
3400
520
50
1100
140
85
65
340
> 300 000
Table 4. Binding affinities (Kds, nM) of 28 measured by ITC against wildꢀtype first and second
bromodomains of Brd2 and Brd4, and compared to the Kds reported for IꢀBET762 (1),11 RVXꢀ
208 (5),10 MS436,35 and Olinone.36
[Insert Fig. 3 here]
Figure 3. A) Coꢀcrystal structure of Brd2(2)W370F (transparent surface representation) in complex
with 28 (PDB code 5DFD, stick representation, orange carbons) superimposed with the coꢀ
crystal structure of Brd2(2)W370F in complex with IꢀBET762 (1) (PDB code 5DFC, stick
representation, yellow carbons). The side chain of His433 switches from an “open” conformation
when bound to 1 to a “closed” conformation when bound to 28, engaging in an edgeꢀtoꢀface
interaction with 28; B) Coꢀcrystal structure of Brd2(2)W370F in complex with (28) (PDB code
5DFC, stick representation, orange carbons) superimposed with the coꢀcrystal structure of
Brd2(1) (transparent surface representation) in complex with IꢀBET762 (1) (PDB code 2YEK,8
stick representation, pink carbons). All structures show a conserved scaffold binding mode in the
K(Ac) pocket.
CONCLUSIONS
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Here, we have described novel synthetic analogues of the triazoloꢀaryldiazepineꢀbased
bromodomain inhibitor IꢀBET762. We were able to introduce substitutions at the level of the
methoxyphenyl ring, the ternary carbon centre, the side chain methylene, and the chlorophenyl
moiety. The design and development of the analogue series was aimed at targeting a number of
specific BET bromodomains mutants with high selectivity compared to wildꢀtype via a bumpꢀ
andꢀhole approach. Amongst the “bumped” compounds reported, ME and ET achieved the
highest selectivity levels targeting mutations at the Leu94 position.11 Several interesting
chemistry were developed in the process that will potentially see useful applications. For
example, we showed that alkylation at the ternary centre and the side chain methylene could be
achieved, and that a high level of stereocontrol could be achieved during enolate alkylation. We
also developed a new route allowing late stage diversity introduction at the level of the
chlorophenyl ring. An indole analogue (28) was highly potent and displayed a marked BD2
selectivity profile by exploiting the Asp/His substitution in the bromodomain BC loop. Taken
together, we anticipate that our findings should be of broad interest, not only to other researchers
working in the field of epigenetics and bromodomain inhibition, but also to medicinal chemists
focusing on related benzodiazepine systems.
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ASSOCIATED CONTENT
Supporting Information.
Material and methods, supplementary figures and tables, synthetic procedures and
characterization. This material is available free of charge via the Internet at http://pubs.acs.org.
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Purity of compounds (as measured by peak area ratio) was >95%, as determined by HPLCꢀMS
(ESI).
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Accession Codes
Coordinates for the Xꢀray structures of Brd2(2)W370F apo and in complex with compounds 1 and
28 have been deposited in the Protein Data Bank (PDB) under accession codes 5DFB, 5DFC and
5DFD, respectively.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: a.ciulli@dundee.ac.uk
Present Addresses
† Present address: Medical Research Council, Laboratory of Molecular Biology, Francis Crick
Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK.
≠ Present address: Biochemistry and Molecular Biophysics Graduate Group, Perelman School of
Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
§ Present address: Nuffield Department of Clinical Medicine, Structural Genomics Consortium,
University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.
ACKNOWLEDGMENT
This work was supported by awards to A.C. from the UK Biotechnology and Biological Sciences
Research Council (BBSRC, grants BB/J001201/1 and BB/J001201/2 and David Phillips
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Fellowship BB/G023123/1 and BB/G023123/2). E.L.S. was supported by a European
Commission Erasmus work placement grant. Biophysics and drug discovery activities at Dundee
are supported by a Wellcome Trust strategic award 100476/Z/12/Z to the Division of Biological
Chemistry and Drug Discovery. We thank Prof. Stefan Knapp and his team at the Oxford
Structural Genomics Consortium for constructs and discussions.
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ABBREVIATIONS
ꢁTm, thermal shift; AcOH, acetic acid; ATP, adenosine triphosphate; BET, bromodomain and
extraꢀterminal; Brd, bromodomain; DSF, differential scanning fluorimetry; FDA, Food and Drug
Administration; ITC, isothermal titration calorimetry; K(Ac), acetylꢀlysine; Kd, dissociation
constant; KHMDS, potassium hexamethyldisilazane;MeOH, methanol; NMR, nuclear magnetic
resonance; NUT, nuclear protein in testis; THF, tetrahydrofuran; Tm, melting temperature; WT,
wildꢀtype.
REFERENCES
(1) Sternbach, L. H., The benzodiazepine story. J. Med. Chem. 1979, 22, 1ꢀ7.
(2) Sternbach, L. H.; Fryer, R. I.; Metlesics, W.; Reeder, E.; Sach, G.; Saucy, G.; Stempel, A.,
Quinazolines and 1,4ꢀBenzodiazepines. VI.1a Haloꢀ, Methylꢀ, and Methoxyꢀsubstituted 1,3ꢀ
Dihydroꢀ5ꢀphenylꢀ2Hꢀ1,4ꢀbenzodiazepinꢀ2ꢀones1b,c. J. Org. Chem. 1962, 27, 3788ꢀ3796.
(3) Sternbach, L. H., The Benzodiazepines. Raven Press: New York: 1973.
(4) Ellman, J. A., Design, Synthesis, and Evaluation of SmallꢀMolecule Libraries. Acc. Chem.
Res. 1996, 29, 132ꢀ143.
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(5) Filippakopoulos, P.; Knapp, S., Targeting bromodomains: epigenetic readers of lysine
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Insert Table of Contents Graphic and Synopsis Here
New synthetic routes towards analogues of the BET bromodomain inhibitor IꢀBET762 were
developed, allowing substitution at the level of the methoxyphenyl, chlorophenyl and methylene
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side chain moieties. A new route based on a key palladium crossꢀcoupling allows for late stage
diversity introduction at R4. Small alkyl substituents at R3 provide selectivity for engineered L/A
BET bromodomain mutants designed for a “bump and hole” study, and an indole derivative at R4
displays up to 20 fold selectivity for the second bromodomains of Brd2 and Brd4 over their first
domains, therefore providing a new isoform selectivity criteria for the design of future
generations of triazoloꢀbenzodiazepine bromodomain inhibitors.
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