Synthesis and elaboration of N-methylpyrrolidone as an acetamide fragment substitute in bromodomain inhibition

Article abstract of DOI:10.1016/j.bmc.2019.115157

N-Methylpyrrolidone is a solvent molecule which has been shown to compete with acetyl-lysine-containing peptides for binding to bromodomains. From crystallographic studies, it has also been shown to closely mimic the acetamide binding motif in several bromodomains, but has not yet been directly pursued as a fragment in bromodomain inhibition. In this paper, we report the elaboration of N-methylpyrrolidone as a potential lead in fragment-based drug design. Firstly, N-methylpyrrolidone was functionalised to provide points for chemical elaboration. Then, the moiety was incorporated into analogues of the reported bromodomain inhibitor, Olinone. X-ray crystallography revealed that the modified analogues showed comparable binding affinity and structural mimicry to Olinone in the bromodomain binding site.

Full text of DOI:10.1016/j.bmc.2019.115157

Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and elaboration of N-methylpyrrolidone as an acetamide fragment
substitute in bromodomain inhibition
J.P. Hilton-Proctor , O. Ilyichova , Z. Zheng , I.G. Jennings , R.W. Johnstone , J. Shorttd,e,
a
a
a
a
b,c
a
a
a,⁎
S.J. Mountford , M.J. Scanlon , P.E. Thompson
b
c
a
Medicinal Chemistry, Monash Institute of Pharmaceutical Science, Monash University, 381 Royal Parade, Parkville, Victoria, Australia
Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, Victoria, Australia
The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia
Blood Cancer Therapeutics Laboratory, School of Clinical Sciences at Monash Health, Monash University, 246 Clayton Road, Clayton, Victoria, Australia
Monash Haematology, Monash Health, 246 Clayton Road, Clayton, Victoria, Australia
d
e
A R T I C L E I N F O
A B S T R A C T
Keywords:
N-Methylpyrrolidone is a solvent molecule which has been shown to compete with acetyl-lysine-containing
peptides for binding to bromodomains. From crystallographic studies, it has also been shown to closely mimic
the acetamide binding motif in several bromodomains, but has not yet been directly pursued as a fragment in
bromodomain inhibition. In this paper, we report the elaboration of N-methylpyrrolidone as a potential lead in
fragment-based drug design. Firstly, N-methylpyrrolidone was functionalised to provide points for chemical
elaboration. Then, the moiety was incorporated into analogues of the reported bromodomain inhibitor, Olinone.
X-ray crystallography revealed that the modified analogues showed comparable binding affinity and structural
mimicry to Olinone in the bromodomain binding site.
BRD4
Bromodomain
Acetyl-lysine
NMP
N-Methylpyrrolidone
Olinone
Fragment-based drug design
1
. Introduction
effectively targeted by various bromodomain inhibitors, with the bro-
modomain and extra-terminal (BET) family of BCPs serving as the
Bromodomains are protein domains that are recognition elements
prototype for inhibitor design. Initially, the methyltriazolodiazepines
23 11
for acetyl-lysine (K-ac) residues, and act as “readers” to regulate the
cellular functions of bromodomain-containing proteins (BCPs). In total,
(+)-JQ1 and I-BET762 were identified as K-ac mimetics among a
host of other chemotypes. Since then, it has become a fruitful subject
2
4
4
6 BCPs have been reported in humans that comprise 61 different
for fragment-based drug design (FBDD) approaches. Chung
and
2
5
bromodomains divided into 8 subfamilies. With established roles in
Bamborough both described a FBDD approach that identified an array
of low molecular weight compounds, including the 3,5-dimethylisox-
1
transcriptional activation, BCPs have been associated with a number of
2
2,13
–4
5–9
10,11
26
disease states relating to cancer,
viral diseases,
inflammation
azoles, which were also described by Hewings et al. Virtual screening
1
and cardiovascular diseases.
Bromodomain inhibitors have there-
approaches using fragment substructures have also yielded K-ac mi-
1
4–16
27
fore been identified as potential therapeutic agents
and numerous
metics. Additionally, the alkylacetamide function of K-ac has been
1
7–21
28
compounds are currently under clinical investigation.
mimicked directly in the inhibitor Olinone, which selectively targets
Bromodomains have a conserved three-dimensional structure con-
sisting of a series of four α-helices (αZ, αA, αB, αC) linked by two
the first bromodomain of BET proteins (Fig. 1).
The solvent, N-methylpyrrolidone (NMP) also interacts with the
2
2
31
interhelical loops (ZA, BC) where the space between these loops
creates the cavity where K-ac is recognised. This binding site has been
binding pocket of a range of bromodomains, and was shown to be a
mimetic of K-ac in crystallographic studies where it binds to the
Abbreviations: ATAD2, ATPase Family AAA Domain-containing Protein 2; BCP, Bromodomain-containing Protein; BET, Bromodomain and Extra-terminal; BRD#,
Bromodomain-containing Protein #; BRD4 BD1, First Bromodomain of BRD4; CBP, Cyclic AMP Response Element Binding Protein Binding Protein; FBDD, Fragment-
based Drug Design; FRET, Fluorescence Resonance Energy Transfer; GST, Glutathione S-transferase; K-ac, Acetyl-lysine; IRF-4, Interferon Regulatory Factor 4; LE,
Ligand Efficiency; LPS, Lipopolysaccharide; NHA, Non-hydrogen Atom; NMP, N-Methylpyrrolidone; NMR, Nuclear Magnetic Resonance; PB1, Polybromo-1; PHIP,
Pleckstrin Homology Domain Interacting Protein; RMS, Root Mean Square; SAR, Structure-Activity Relationship; SMARCA4, SWI/SNF Related Matrix Associated
Actin Dependent Regulator of Chromatin Subfamily A Member 4; vdW, van der Waals
⁎
Corresponding author.
E-mail address: philip.thompson@monash.edu (P.E. Thompson).
https://doi.org/10.1016/j.bmc.2019.115157
Received 14 August 2019; Received in revised form 7 October 2019; Accepted 8 October 2019
0968-0896/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: J.P. Hilton-Proctor, et al., Bioorganic & Medicinal Chemistry, https://doi.org/10.1016/j.bmc.2019.115157

J.P. Hilton-Proctor, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Figure 1. Reported bromodomain inhibitors and probes.11,23,28–31.
conserved areas of the bromodomain binding pocket in bromodomain-
containing protein 1 (BRD1, PDB: 3RCW), BRD2 (PDB: 4A9F), ATPase
family AAA domain-containing protein 2 (ATAD2, PDB: 4QSS), cyclic
AMP response element binding protein binding protein (CBP, PDB:
potential in fragment elaboration. Here, we report our expansion of the
NMP template with a range of functional groups and the subsequent
elaboration by mimicry of Olinone as an exemplar of the broader ap-
proach to bromodomain inhibitor design. X-ray crystal structures of the
NMP derivatives show the successful mimicry of the native acetamide
binding pose.
3
P1D), SWI/SNF related matrix associated actin dependent regulator of
chromatin subfamily A member 4 (SMARCA4, PDB: 3UVD), polybromo-
, (PB1, PDB: 3MB4) and pleckstrin homology domain interacting
1
protein (PHIP, PDB: 3MB3). NMP inhibits the binding of acetylated
2
. Results and discussion
3
2
histone peptides to a range of BCPs at millimolar concentrations while
its affinity, despite being modest, was comparable to that of K-ac, giving
In crystal structures of NMP bound to different bromodomains, a
3
1
it good ligand efficiency (LE) because of its low molecular weight.
conserved mode of binding is observed. The carbonyl moiety of NMP
forms a hydrogen bond with the highly conserved asparagine residue,
as well as a water-mediated hydrogen bond to a conserved tyrosine in
the K-ac binding pocket, while the N-methyl substituent sits adjacent to
the water-lined pocket. We posited that the 4-position of NMP would be
a suitable point to elaborate the NMP structure (Supplementary Video
S1).
The pleiotropic bromodomain inhibition shown by NMP may be
pharmacologically relevant and contribute to its anti-myeloma and
immunomodulatory activity, showing hallmark reduction in transcrip-
tion factors c-Myc and interferon regulatory factor 4 (IRF-4) in multiple
3
2
myeloma models. This activity has seen it advance into a phase I
3
3
clinical trial for multiple myeloma (NCT02468687). Additionally,
NMP has been suggested as an anti-bone resorptive therapy in diseases
such as osteoporosis where bromodomain inhibition mediates the pre-
3
3
4
2.1. Synthesis
vention of osteoclast differentiation,
inhibits lipopolysaccharide
5
(
LPS)-induced inflammatory mediators and reduces the effects of
36
We developed two series of NMP derivatives with functional groups
from which to elaborate using two synthetic routes. The first series was
adipocyte accumulation.
Despite the structural and functional evidence of NMP’s bromodo-
main inhibitor mimicry, its use as a fragment in FBDD has not been
reported. It was described as the inspiration behind other mimetic
derived from methyl 1-methyl-5-oxopyrrolidine-3-carboxylate
2
(
Scheme 1). Dimethyl itaconate 1 was treated with methylamine and
38,39
2
6,37
then cyclised in situ to give 2 in 91% yield as previously reported.
warheads such as the 3,5-dimethylisoxazole,
but while NMP has a
The ester 2 was then reduced to the alcohol 3 with excess sodium
high LE, additional functionalisation would be necessary to explore its
borohydride in 88% yield.38 The final step was the conversion of the
Scheme 1. Formation of 4, a mesylate NMP derivative. (a): CH
3
NH
2
, MeOH, 0 °C → rt, o/n; (b): NaBH
4
, EtOH; rt, o/n; (c): N
2
, MsCl, DIPEA, CH
2
Cl , 0 °C, 4 h.
2
2

J.P. Hilton-Proctor, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Scheme 2. Formation of 12,
a
di-
methylene NMP mesylate. (a): CH
3
NO
2
,
1
,1,3,3-tetramethylguanidine, 42 h, rt;
(
b): H
2
, Pd/C, CH
N, MeOH, o/n, reflux; (d): N
CO
d, reflux; (f):
CO acetone,
SiH, CF COOH,
, o/n, rt; (h): NaBH , EtOH; rt, o/
n; (i): N , MsCl, DIPEA, CH Cl , 0 °C,
h.
3
COOH, 5 d, rt; (c):
Et
3
2
, t-
BuOK, MeI, THF, 6 h, 0 °C; (e): K
2
3
,
MeI, CH
Paraformaldehyde,
O, 4 h, rt; (g): Et
CHCl
3
CN,
6
K
2
3
3
,
H
2
3
3
4
2
2
2
4
Scheme 3. Preparation of 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indol-1-one 16 and coupling to NMP derivatives 4 and 12. (a): EtOH, rf, o/n; (b): 70% H
c): N , NaHMDS, DMF, –78 °C → 90 °C, o/n.
2
SO , 0 °C, 4 h;
4
(
2
3

J.P. Hilton-Proctor, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
alcohol to a mesylate through the use of methanesulfonyl chloride and
Table 1
4
0,41
DIPEA under an inert atmosphere to give 4 in 59% yield.
Preliminary SAR data on NMP derivatives.
In the second series, compounds 10, 11 and 12 were prepared as
shown in Scheme 2. Dimethyl glutaconate 5 was treated with ni-
4
2
tromethane and 1,1,3,3-tetramethylguanidine to afford the nitroester
6
in 47% yield. The nitroester 6 was reduced to the amine 7 with the
use of palladium on carbon under a hydrogen atmosphere with acetic
acid as the solvent, which gave the amine 7 as an acetate salt. The
amine 7 was refluxed in a mixture of methanol and triethylamine to
neutralise the acetate salt and catalyse the cyclisation to form the
lactam 8 in 68% yield over two steps.
Compound
Number
R=
IC50
LE (kcal/mol
(µM ± SEM)a
NHA)
c
H
2660 ± 480
6880 ± 300
0.50
0.27
2
The lactam derivative 8 was N-methylated by conversion to the
hemiaminal intermediate 9 through treatment with paraformaldehyde
and potassium carbonate, which was then reduced with triethylsilane
3
4110 ± 380
2120 ± 270
0.36
0.30
4
3
and trifluoroacetic acid to give 10 in 89% yield across the two steps.
1
0
In contrast, yields for direct methylation with methyl iodide in the
4
4–46
presence of a base were modest at best.
Finally, the reduction of
the ester 10 to the alcohol 11 and subsequent conversion to the me-
sylate 12 were carried out using the same methods described for 3 and
11
4810 ± 60
24 ± 2b
0.32
0.29
1
7
8
4
in similar yields. Collectively, we have built a set of six analogues of
NMP substituted at the 4-position. Each of these compounds, 2–4 and
0–12, is amenable to further elaboration.
Olinone was originally reported by Gacias et al. as a molecule that
1
2
8
showed selectivity for the first bromodomain of BET proteins with a K
d
79 ± 3b
1
0.24
of 3.4 µM. It possesses an alkylacetamide chain similar to K-ac, linked to
a 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indol-1-one scaffold which inter-
acts with residues that are unique to the first bromodomain of BRD4
(
BRD4 BD1). Olinone was a suitable starting point for trying to re-
capitulate the acetamide binding motif in an NMP-containing analogue
and we considered that either 4 or its homologue 12 would be suitable
in creating this mimetic.
a
b
c
Unless stated otherwise, all results of means are n ≥ 2.
SD reported.
2
8
Following the reported procedure (Scheme 3),
2,4-piper-
Ligand efficiency = 1.37(−log(IC50)) / NHA. Control compound used: I-
idinedione 13 was treated with phenylhydrazine 14 in ethanol to give
the hydrazone intermediate 15, which was then subjected to Fischer
indole synthesis conditions, using 70% sulfuric acid to give the desired
tricyclic scaffold 16, albeit in modest yield. Alkylation of 16 with me-
sylates 4 and 12 using sodium bis(trimethylsilyl)amide as the base was
low yielding, but both compounds 17 and 18 were successfully isolated.
The difference in yields points to some hindrance due to the proximity
BET726 (IC50: 0.01 μM). NHA, non-hydrogen atom.
mimicry of K-ac in these compounds. As shown in Fig. 2, the NMP
segment of 10 retains its mimicry of K-ac, interacting with Asn140 and
Tyr97, while the ester interacts with the WPF shelf of BRD4 BD1. The
WPF shelf is often targeted by selective inhibitors given its lack of
4
7
conservation between bromodomains. Here, the ester moiety inter-
acts with residues Trp81 and Pro82 through weak vdW interactions.
The X-ray crystal structures of 17 and 18 show that, in both cases,
the tricyclic scaffolds align with that of Olinone with a root mean
square (RMS) for the heavy atoms of 0.56 Å and 0.24 Å, respectively. As
anticipated, the NMP moiety of 17 and 18 mimics the acetamide group
of Olinone and closely overlays with the reported structures of NMP
(electron density maps can be found in Supplementary Fig. S2). While
the pharmacophore elements of 17 and 18 are identical, the linkers
differ in the conformation and stereochemical configuration at the NMP
stereocentre. Despite each NMP derivative being submitted as a racemic
mixture, density maps point to single enantiomers observed in the
crystal form. Compound 17 is found in the same relative configuration
as compound 10 and their backbones overlay closely (RMS = 0.36 Å).
In contrast, compound 18 shows the opposite relative stereochemistry
while accommodating an extended chain conformation (Fig. 3,
Supplementary Video S2 and S3). Under the Cahn-Ingold-Prelog no-
menclature, the absolute configuration is R for each of 10, 17 and 18.
1
of the NMP moiety in 4 towards 16. H NMR confirmed the selective
alkylation at the indole nitrogen of the tricycle and not the lactam, with
the disappearance of the indole NH proton resonance.
2.2. BRD4 BD1 FRET assay results
With this series of compounds in hand, the esters 2 and 10, alcohols
3
and 11, and Olinone analogues 17 and 18 were all evaluated as in-
hibitors of BRD4 BD1 binding to a tetra-acetylated histone peptide
using a fluorescence resonance energy transfer (FRET)-based assay. The
results of the assays are shown in Table 1.
The simple NMP analogues 2, 3, 10 and 11 showed comparable
binding to NMP itself showing that 4-substitution was tolerated in the
BRD4 BD1 binding site. The Olinone analogues 17 and 18 showed
much improved affinity relative to NMP, consistent with the anticipated
mimicry of the K-ac motif by the NMP functional group. Compound 17
was the higher affinity ligand with an IC50 of 24 µM, while compound
1
8 had an IC50 of 79 µM, marking an improvement of NMP’s activity by
1
10-fold and 33-fold, respectively (dose response curves of 17 and 18
are available in Supplementary Fig. S1). Olinone is reported to have a
3. Conclusion
K of 3.4 µM as measured by ITC, which we considered comparable to
28
d
1
7 given the differences in the assay formats.
.3. X-ray crystallography
We successfully obtained crystal structures of compounds 10, 17
In summary, we have demonstrated the use of NMP derivatives as
fragments that can be used in the design of bromodomain ligands. We
have successfully developed two synthetic schemes for six functiona-
lised derivatives of NMP which can be produced on multigram scales.
An X-ray crystal structure of one of these derivatives (10) in complex
with BRD4 BD1 shows that it retains a similar mode of binding to that
2
and 18 in complex with BRD4 BD1, which allowed us to assess the
4

J.P. Hilton-Proctor, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Figure 2. Comparison of binding substrates
in the bromodomain binding site of BRD4
BD1. A. K-ac (purple blue, PDB: 3UVW). B.
A
B
Tyr97
Tyr97
(
R)-10 (yellow, PDB: 6PRT, resolution: 1.3
Asn140
Å). Water molecules shown as red spheres.
Pro86, Val87 and Asp88 were hidden for
better visualisation. Distances in Å, high-
lighted by red dashes. (For interpretation of
the references to colour in this figure le-
gend, the reader is referred to the web ver-
sion of this article.)
Asn140
Pro82
Pro82
Trp81
Trp81
previously observed for NMP.
on the Total Ion Chromatogram (TIC) is an average of 13,700 tran-
sients, producing a spectrum every second. Mass spectra were created
by averaging the scans across each peak and background subtracted
against the first 10 s of the TIC. Acquisition was performed using the
Agilent Mass Hunter Data Acquisition software version B.05.00 Build
5.0.5042.2 and analysis was performed using Mass Hunter Qualitative
Analysis version B.05.00 Build 5.0.519.13.
The precursors 4 and 12 have been employed in the synthesis of two
compounds, 17 and 18, based on the inhibitor Olinone. The inhibition
of bromodomain binding to an acetylated histone peptide by 17 and 18
is comparable to the parent compound and both compounds precisely
mimic the parent compound in the BRD4 BD1 binding site.
These results should encourage the further exploration of NMP as a
fragment in bromodomain inhibition for three reasons. Firstly, the pre-
existing literature suggests that NMP is a general mimic of the acet-
amide function of K-ac. This suggests that the strategy applied here for
BRD4 BD1 could be applied equally against one or more of the other 60
bromodomains in the epigenome. Secondly, the structures of 17 and 18
show that either of the stereochemical configurations of the NMP de-
rivatives can be applied with success. Therefore, these are actually pairs
of fragments which may be expected to have their own selectivity
profiles. Further work to resolve or synthesise the individual stereo-
isomers is on-going. Finally, the variety of functionalities in the six
fragments described here opens the door for alternate reactions to make
products such as amines, ethers, amides and esters. Taken together,
these results have further proved the usefulness of NMP as a mimetic
fragment and the potential of its incorporation into other known in-
hibitors.
All LCMS analyses were carried out on an Agilent 6100 Series Single
Quad LC/MS coupled with an Agilent 1200 Series HPLC, 1260 Infinity
G1312B Binary pump, 1260 Infinity G1367E 1260 HiP ALS autosampler
and 1290 Infinity G4212A 1290 DAD detector. The liquid chromato-
graphy conditions were: reverse phase HPLC analysis fitted with a Luna
C8(2) 5 μL 50 × 4.6 mm 100 Å at a temperature of 30 °C. The sample
injection volume was 5 μL, which was run in 0.1% formic acid in
acetonitrile at a gradient of 5–100% over 10 min. Detection methods
were either 254 nm or 214 nm. The mass spectrum conditions were:
Quadrupole ion source with Multimode-ES. The drying gas temperature
was 300 °C and the vaporizer temperature was 200 °C. The capillary
voltage in positive mode was 2000 V, while in negative mode, the ca-
pillary voltage was 4000 V. The scan range was 100–1000 m/z with a
step size of 0.1 s over 10 min.
TLCs were carried out on Merck TLC Silica gel 60 F254 plates using
the appropriate mobile phase. Purification by column chromatography
was conducted with Davisil Chromatographic Silica LC60A (40–63 µm)
using the specified mobile phases.
4
. Experimental
Compound purity was determined using an Agilent 1260 Infinity
Analytical HPLC (1260 Infinity G1322A Degasser, 1260 Infinity
G1312B Binary pump, G1367E HiP ALS autosampler, 1260 Infinity
G1316A Thermostatted Column Compartment, and 1260 Infinity
G4212B DAD detector. The liquid chromatography conditions were:
reverse phase HPLC analysis fitted with a Zorbax Eclipse Plus C18 Rapid
Resolution 4.6 × 100 mm 3.5‐Micron. The sample injection volume was
4.1. Chemistry
1
13
H and C Nuclear Magnetic Resonance spectra were conducted on
a Bruker Advance III Nanobay 400 MHz spectrometer coupled to the
BACS 60 automatic sample changer and obtained at 400.1 MHz and
1
6
00.6 MHz, respectively. All spectra were processed using MestReNova
1
.0 software. The chemical shifts of all H were measured relative to the
1
μL, which was run in Solvent A (0.1% TFA in H O) and Solvent B
2
expected solvent peaks of the respective NMR solvents; CDCl
3
, 7.26;
(
0.1% TFA in CH CN), with a gradient of 5–100% Solvent B over a 10-
3
1
3
MeOD, 3.31. The chemical shifts of all C were measured relative to
minute period. All compounds submitted for assays and X-ray crystal-
lography studies were assessed for purity of 95% or greater on 214 nm
and 254 nm.
the expected solvent peaks of the respective NMR solvents; CDCl , 77.2;
3
MeOD, 49.0. The data for all spectra are reported in the following
format: chemical shift (integration, multiplicity, coupling constant,
assignment). Multiplicity is defined as; s = singlet, d = doublet,
t = triplet, q = quartet, quint. = quintet, dd = doublet of doublets,
td = triplet of doublets, qd = quartet of doublets, ddd = doublet of
doublet of doublets, m = multiplet. Coupling constants are applied as J
4.1.1. Methyl 1-methyl-5-oxo-3-pyrrolidine carboxylate (2)
40% Methylamine solution in H O (10 mL) was dissolved in me-
2
thanol (125 mL) in an ice-bath and stirred. Dimethyl itaconate 1
(15.1 g, 94.8 mmol) dissolved in methanol (275 mL) was added drop-
wise to the stirring mixture. After an hour, the ice-bath was removed
and the reaction continued to stir for a further 24 h. The mixture was
concentrated under reduced pressure and the residue purified by va-
1
13
in Hertz (Hz). For H and C spectra, refer to Supplementary Fig. S3.
All HRMS analyses were obtained using an Agilent 6224 TOF LC/MS
Mass Spectrometer coupled to an Agilent 1290 Infinity (Agilent, Palo
Alto, CA). All data were acquired and reference mass corrected via a
dual‐spray electrospray ionisation (ESI) source. Each scan or data point
1
cuum distillation to afford 2 as an oil (13.7 g, 91%). H NMR (400 MHz,
5

J.P. Hilton-Proctor, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Figure 3. Comparison of Olinone and its
Trp81
Trp81
respective analogues in BRD4 BD1. [A–C]
A
B
C
Trp81
2
1
,3,4,5-Tetrahydro-1H-pyrido[4,3-b]indol-
-one scaffold. [D–F] K-ac binding site.
Water molecules shown as red spheres.
Pro86, Val87 and Asp88 were hidden for
better visualisation. Distances in Å, high-
lighted by red dashes. [G–I] Linker group
visualisation. [A, D, G] Olinone (light blue,
PDB: 4QB3). [B, E, H] (R)-17 (warm pink,
PDB: 6PS9, resolution: 1.59 Å). [C, F, I] (R)-
Pro82
Pro82
Asp145
Ile146
Asp145
Asp145
Ile146
Ile146
1
8 (pale yellow, PDB: 6PSB, resolution: 1.21
Å). (For interpretation of the references to
colour in this figure legend, the reader is
referred to the web version of this article.)
Asp144
Asp144
Asp144
D
F
E
Tyr97
Tyr97
Tyr97
Asn140
Asn140
Asn140
G
H
I
CDCl
3
) δ 3.71 (s, 3H), 3.58 (ddd, J = 22.8, 14.4, 8.2 Hz, 2H), 3.27–3.16
C
6
H
10NO
2
[M+H]+: 130.0863; found 130.0863; HPLC (PP gradient,
1
3
(
m, 1H), 2.82 (s, 3H), 2.73–2.57 (m, 2H) ppm. C NMR (101 MHz,
MeOH): 1.75 min.
CDCl
3
) δ 173.4, 172.5, 52.6, 51.2, 35.9, 34.0, 29.6 ppm. ESI-MS, m/z
+
+
1
58.2 [M+H] ; HR-MS: m/z calcd. for C
7
H11NO
3
[M+H] : 158.0812;
4.1.3. (1-Methyl-5-oxopyrrolidin-3-yl)methyl methanesulfonate (4)
In a 50 mL round-bottom flask, 4-(hydroxymethyl)-1-methylpyrro-
lidin-2-one 3 (209 mg, 1.62 mmol) was weighed out, then dissolved in
found 158.0813; HPLC (PP gradient, MeOH): 3.27 min.
4
.1.2. 4-(Hydroxymethyl)-1-methylpyrrolidin-2-one (3)
CH
2
Cl (5 mL). The flask was placed in an ice bath, sealed with a rubber
2
Methyl 1-methyl-5-oxo-3-pyrrolidine carboxylate
2
(10.5 g,
septum and purged with N . DIPEA (565 μL, 3.24 mmol) and MsCl
2
6
7.1 mmol) was dissolved in ethanol (400 mL) and stirred at room
(150 μL, 1.95 mmol) were added via syringe and the mixture was left to
temperature. Sodium borohydride (25.3 g, 0.671 mol) was added
slowly in small portions over a 7h period. After this period, water
stir for 4 h. After this time, the mixture was quenched with water
(20 mL) and the CH
with additional CH
bined, dried with MgSO
2
2
Cl
2
layer separated. The aqueous layer was washed
(
20 mL) was added to form a cloudy mixture. The quenched mixture
Cl
2
(3 × 20 mL). The organic layers were com-
, filtered and concentrated via rotary eva-
was then filtered through Celite 545 and carefully concentrated to a
white solid by rotary evaporation. The solid was taken up in CHCl
4
3
poration to give the crude mesylate as a yellow oil. This oil was then
(
500 mL) and left to stir overnight. The mixture was dried with Na
2
SO
4
,
purified using a silica column (Mobile phase: 2% MeOH in CH
2
Cl
2
) to
1
filtered through filter paper and concentrated under reduced pressure
give 4 as a clear oil (190 mg, 59%). H NMR (400 MHz, CDCl
3
) δ 4.16
to afford a crude oil. Purification by column chromatography (Mobile
(qd, J = 10.0, 6.8 Hz, 2H), 3.51 (dd, J = 10.2, 8.2 Hz, 1H), 3.21 (dd,
J = 10.2, 5.2 Hz, 1H), 3.01 (s, 3H), 2.85–2.73 (m, 4H), 2.53 (dd,
1
phase: 5% MeOH in CHCl
3
) afforded 3 as a clear oil (6.82 g, 79%).
H
1
3
NMR (400 MHz, MeOD) δ 3.58–3.47 (m, 3H), 3.26 (dd, J = 10.1,
J = 17.1, 9.4 Hz, 1H), 2.16 (dd, J = 17.1, 6.2 Hz, 1H) ppm. C NMR
5
.2 Hz, 1H), 2.61–2.44 (m, 2H), 2.19 (dd, J = 16.5, 5.4 Hz, 1H) ppm.
C NMR (101 MHz, MeOD) δ 176.7, 64.9, 53.4, 34.77, 34.37,
9.8 ppm. ESI-MS, m/z 130.1 [M+H]+. HR-MS: m/z calcd. for
(101 MHz, CDCl
3
) δ 172.8, 70.4, 51.4, 37.5, 33.3, 30.7, 29.6 ppm. ESI-
+ +
1
3
MS, m/z 208.0 [M+H] ; HR-MS: m/z calcd. for C
7
H
13NO S [M+H] :
4
2
207.0565; found 207.0567; HPLC (PP gradient, MeOH): 2.36 min.
6

J.P. Hilton-Proctor, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
4
.1.4. Dimethyl 3-(nitromethyl)glutarate (6)
mixture was quenched with water and the solvent was evaporated off
In a 250 mL round-bottom flask, nitromethane (14.9 mL, 0.352 mol)
under reduced pressure to give a white solid. The solid was then pur-
was added to dimethyl glutaconate 5 (5.56 g, 35.2 mmol). 1,1,3,3-
Tetramethylguanidine (882 μL, 7.03 mmol) was added dropwise to the
mixture. After complete addition, the mixture was left to stir overnight.
The mixture was quenched with 5% HCl (75 mL), then washed with
diethyl ether (3 × 75 mL). The organic layers were collected, combined
ified by column chromatography (Mobile phase: 5% MeOH in CH
2
Cl
2
) δ
)
1
to obtain 11 as a clear oil (313 mg, 84%). H NMR (400 MHz, CDCl
3
3.72–3.58 (m, 2H), 3.49 (dd, J = 9.7, 8.1 Hz, 1H), 3.07 (dd, J = 9.7,
6.7 Hz, 1H), 2.80 (s, 3H), 2.58–2.25 (m, 3H), 2.13–2.01 (m, 1H),
1
3
1.76–1.61 (m, 2H) ppm. C NMR (101 MHz, CDCl
3
) δ 174.7, 60.7,
+
and dried with MgSO
4
. The solvent was filtered and evaporated under
55.5, 37.51, 37.49, 29.68, 28.79 ppm. ESI-MS: m/z 222.0 [M+H]
.
+
reduced pressure to give a brown-coloured oil. The resin was purified
using silica gel chromatography (Mobile phase: 10% EtOAc in petro-
HR-MS: m/z calc. for C
7
H
13NO
2
[M+H] : 143.0946; found 143.0951.
HPLC (PP gradient, MeOH): 2.09 min.
leum benzine, TLCs checked at 25% EtOAc in petroleum benzine) to
1
afford 7 as a clear oil (3.32 g, 47%). H NMR (400 MHz, CDCl
3
) δ 4.62
4.1.8. 2-(1-Methyl-5-oxopyrrolidin-3-yl)ethyl methanesulfonate (12)
(
d, J = 6.1 Hz, 2H), 3.71 (s, 6H), 3.13–3.00 (m, 1H), 2.56 (dd, J = 6.6,
In a 50 mL round-bottom flask, 4-(2-hydroxyethyl)-1-methylpyrro-
1
3
1
.1 Hz, 4H) ppm. C NMR (101 MHz, CDCl
3
) δ 171.6, 77.5, 52.1, 35.1,
lidin-2-one 11 (128 mg, 0.893 mmol) was weighed out, then dissolved
3
0.7 ppm.
in CH
2
Cl (10 mL). The flask was placed in an ice bath, sealed with a
2
rubber septum and purged with N . DIPEA (233 μL, 1.34 mmol) and
2
4
.1.5. Methyl 2-(5-oxopyrrolidin-3-yl)acetate (8)
MsCl (103 μL, 1.34 mmol) were added via syringe and the mixture was
left to stir for 4 h. After this time, the mixture was quenched with water
In a 250 mL three-neck round-bottom flask purged with N , 10%
2
Pd/C (332 mg) was added and submerged in CH
2
Cl
2
. Dimethyl 3-(ni-
(20 mL) and the CH
with additional CH
2
2
Cl
Cl
2
2
layer separated. The aqueous layer was washed
tromethyl)glutarate 6 (3.32 g, 15.2 mmol) was dissolved in acetic acid
(3 × 20 mL). The organic layers were com-
(
150 mL) and added to the flask, then stirring began. The flask was then
bined, dried with MgSO
4
, filtered and concentrated via rotary eva-
flushed with N
2
three times, followed by H
2
three times. Progress was
poration to give the crude mesylate 12 as a yellow oil. This oil was then
monitored using NMR and LCMS. Once the starting material and hy-
droxylamine intermediate were consumed, the flask was purged with
purified using silica gel chromatography (Mobile phase: 2% MeOH in
1
CH
CDCl
J = 9.7, 6.6 Hz, 1H), 3.01 (s, 3H), 2.82 (s, 3H), 2.61–2.47 (m, 2H),
2
Cl
2
) to give 12 as a clear oil (122 mg, 62%). H NMR (400 MHz,
N
2
and the Pd/C was filtered through glass microfiber filter paper. The
3
) δ 4.31–4.18 (m, 2H), 3.53 (dd, J = 9.7, 8.0 Hz, 1H), 3.07 (dd,
solvent was then evaporated off to give the amine 7 as a caramel-co-
1
3
loured oil. The amine was then dissolved in MeOH (75 mL), basified
2.14–2.03 (m, 1H), 1.94–1.86 (m, 2H) ppm. C NMR (101 MHz,
with Et
3
N (1 mL) and heated under reflux. The solvent and base were
CDCl ) δ 173.7, 67.7, 54.8, 37.60, 37.13, 34.1, 29.67, 28.60 ppm. ESI-
+ +
3
evaporated off under reduced pressure to give the crude lactam 8 as a
MS: m/z 222.0 [M+H] . HR-MS: m/z calc. for C
8
H
15NO S [M+H] :
4
yellow resin. The resin was purified by column chromatography
221.0726; found 221.0726. HPLC (PP gradient, MeOH): 3.28 min.
(
Mobile phase: 2% MeOH in CH
2
Cl ) to afford 8 as a white solid (1.61 g,
2
1
6
8%). H NMR (400 MHz, CDCl
3
) δ 7.07 (br s, 1H), 3.62 (s, 3H), 3.54
4.1.9. 2,3,4,5-Tetrahydro-1H-pyrido[4,3-b]indol-1-one (16)
(
dd, J = 9.9, 7.8 Hz, 1H), 3.02 (dd, J = 9.9, 6.2 Hz, 1H), 2.86–2.75 (m,
2,4-Piperidinedione 13 (500 mg, 4.42 mmol) was weighed into a
100 mL round-bottom flask, and phenylhydrazine 14 (434 μL,
4.42 mmol) was added. The mixture was dissolved in EtOH (50 mL) and
stirred at reflux overnight. The solvent was evaporated off to give the
hydrazone 15 as a crimson oil. This was cooled to 0 °C, dissolved in 70%
sulfuric acid (20 mL) and stirred for 4 h. The acid was neutralised with
2 M NaOH and the aqueous layer was washed with EtOAc (3 × 50 mL).
1
3
1
H), 2.52–2.40 (m, 3H), 1.99 (dd, J = 16.9, 7.3 Hz, 1H) ppm. C NMR
(
101 MHz, CDCl
3
) δ 177.7, 171.8, 51.3, 47.4, 38.0, 36.2, 30.6 ppm. ESI-
+
MS: m/z 158.1 [M+H]
.
4
.1.6. Methyl 2-(1-methyl-5-oxopyrrolidin-3-yl)acetate (10)
To a solution of lactam 8 (1.12 g, 7.09 mmol) in acetone (120 mL)
was added paraformaldehyde (1.07 g, 35.5 mmol) and K
2
CO
3
(132 mg),
The organic layers were combined, dried with MgSO , filtered and
4
followed by the addition of water (13 mL). The mixture was then so-
nicated for 4 h at 20-minute intervals with 10-minute rests. The mixture
was then filtered and the filtrate was concentrated under reduced
pressure to give the crude hemiaminal 9 as an opaque oil. The oil was
purified by silica column chromatography (Mobile phase: 5% MeOH in
concentrated under reduced pressure to afford a yellow/brown resin.
This was purified with silica gel chromatography (Mobile phase:
Gradient of 3–5% MeOH in CH
2
Cl ) to acquire 16 as a yellow resin
2
1
(137 mg, 17%). H NMR (400 MHz, CDCl
3
) δ 8.91 (br s, 1H), 8.17–8.09
(m, 1H), 7.38–7.31 (m, 1H), 7.25–7.17 (m, 2H), 5.53 (br s, 1H), 3.63 (t,
CH
2
Cl
2
) to give 9 as a clear oil. The hemiaminal 9 was then dissolved in
J = 6.9 Hz, 2H), 3.02 (t, J = 6.9 Hz, 2H) ppm. ESI-MS: m/z 187.1 [M
+
CHCl
3
(125 mL), to which CF
3
COOH (25 mL) and Et
3
SiH (25 mL) were
+H] . HPLC (PP gradient, MeOH): 4.36 min.
added, and the mixture was stirred at room temperature overnight. The
mixture was then quenched with water (75 mL) and the organic layer
4.1.10. 5-((1-Methyl-5-oxopyrrolidin-3-yl)methyl)-2,3,4,5-tetrahydro-1H-
pyrido[4,3-b]indol-1-one (17)
was separated. The aqueous layer was washed with additional CHCl
3
(
2 × 75 mL). The organic layers were combined, dried with MgSO
4
,
In a 25 mL round-bottom flask, 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]
indol-1-one 16 (36.5 mg, 0.196 mmol) was dissolved in DMF (3 mL).
filtered and concentrated under reduced pressure to afford a biphasic
oil. This was purified by column chromatography (neat EtOAc), giving
The flask was sealed, purged with N and then chilled to −78 °C.
2
1
1
3
2
2
1
0 as a clear oil (1.08 g, 89%). H NMR (400 MHz, CDCl
3
) δ 3.67 (s,
NaHMDS (94 μL, 0.294 mmol, 1 M in THF) was added via syringe and
the mixture was stirred for 30 min. After this time, 2-(1-methyl-5-ox-
opyrrolidin-3-yl)ethyl methanesulfonate 12 (60.9 mg, 0.294 mmol) was
weighed, dissolved in DMF (1 mL) and added via syringe. The mixture
was warmed to room temperature, then heated to 90 °C and left stirring
overnight. After this time, the mixture was quenched with a saturated
H), 3.57 (dd, J = 10.0, 7.9 Hz, 1H), 3.06 (dd, J = 10.0, 6.0 Hz, 1H),
.85–2.68 (m, 4H), 2.58 (dd, J = 16.8, 9.0 Hz, 1H), 2.53–2.38 (m, 2H),
1
3
.08 (dd, J = 16.8, 7.0 Hz, 1H) ppm. C NMR (101 MHz, CDCl ) δ
3
73.7, 172.2, 54.8, 51.9, 38.8, 37.1, 29.6, 27.9 ppm. ESI-MS: m/z 172.1
+
+
[
M+H] HR-MS: m/z calc. for C
8
H
13NO
3
[M+H] : 171.0895; found
1
71.0899. HPLC (PP gradient, MeOH): 3.31 min.
aqueous solution of NaHCO
3
(40 mL) and washed with EtOAc
(
3 × 40 mL). The combined organic layer were dried with MgSO , fil-
4
4
.1.7. 4-(2-Hydroxyethyl)-1-methylpyrrolidin-2-one (11)
tered and concentrated under reduced pressure to obtain a yellow oil.
In 100 mL round-bottom flask, methyl 2-(1-methyl-5-ox-
a
The oil was purified using a silica column (Mobile phase: 5% MeOH in
1
opyrrolidin-3-yl)acetate 10 (446 mg, 2.61 mmol) was dissolved in EtOH
25 mL). NaBH (0.99 g, 26.1 mmol) was then added over a period of
h, and the mixture was left to stir overnight. After completion, the
CH
CDCl
(d, J = 8.0 Hz, 2H), 3.70 (t, J = 6.9 Hz, 2H), 3.38 (dd, J = 10.2, 7.2 Hz,
2
Cl
2
) to obtain 17 as a yellow oil (3.80 mg, 6%). H NMR (400 MHz,
(
4
3
) δ 8.23–8.18 (m, 1H), 7.33–7.26 (m, 3H), 5.53 (br s, 1H), 4.14
4
7

J.P. Hilton-Proctor, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
1
2
1
2
H), 3.12–2.92 (m, 4H), 2.85 (s, 3H), 2.57 (dd, J = 17.1, 8.3 Hz, 1H),
sonication. The His-tagged BRD4 BD1 was purified using Ni-agarose
chromatography with elution in 50 mM Hepes pH 7.5, 300 mM NaCl,
500 mM imidazole, 5% (v/v) glycerol. The 6-His tag was then removed
by TEV protease digestion. The bromodomain was further purified
using gel filtration chromatography using a Superdex-75, 16/60 column
(GE Healthcare) in a buffer containing 50 mM Hepes pH 7.5, 0.3 M NaCl
and 5% glycerol.
1
3
.18 (dd, J = 16.9, 4.8 Hz, 1H) ppm. C NMR (101 MHz, CDCl ) δ
3
23.13, 122.58, 121.62, 109.4, 52.2, 46.7, 40.8, 35.2, 32.2, 29.9,
+
2.5 ppm. ESI-MS: m/z 297.9 [M+H]
. HR-MS: m/z calc. for
+
C
17
H
19
N
3
O
2
[M+H] : 297.1477; found 297.1475. HPLC (PP gradient,
MeOH): 4.32 min.
4
.1.11. 5-(2-(1-Methyl-5-oxopyrrolidin-3-yl)ethyl)-2,3,4,5-tetrahydro-
H-pyrido[4,3-b]indol-1-one (18)
1
4.4. Crystallisation and data collection
In a 50 mL round-bottom flask, 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]
indol-1-one 16 (45 mg, 0.241 mmol) was dissolved in DMF (3 mL). The
X-ray crystal structures were obtained using a 6-His tagged bro-
modomain. The concentrated BRD4 BD1 protein (17 mg/mL, stored at
−80 °C) was then incubated with 5 mM ligand at 4 °C for 16 h. Crystals
were obtained using the hanging drop method in 24-well plates using
1 µL drops of protein and the reservoir solution containing 0.2 M
flask was sealed, purged with N and then chilled to –78 °C. NaHMDS
2
(
362 μL, 0.362 mmol, 1 M in THF) was added via syringe and the
mixture was stirred for 30 min. After this time, 2-(1-methyl-5-ox-
opyrrolidin-3-yl)ethyl methanesulfonate 12 (80 mg, 0.362 mmol) dis-
solved in DMF (1 mL) was added via syringe. The mixture was warmed
to room temperature, then heated to 90 °C and left stirring overnight.
After this time, the mixture was quenched with a saturated aqueous
NaNO , PEG-3350 (35%) and ethylene glycol (6% v/v) concentrations.
3
The crystals were cryoprotected using mother liquor supplemented
with 5 mM ligand and then were flash frozen in liquid nitrogen. All
datasets were collected at the Australian Synchrotron, part of ANSTO,
solution of NaHCO (40 mL) and washed with EtOAc (3 × 40 mL). The
3
4
8–50
combined organic layers were dried with MgSO
4
, filtered and con-
on the MX1 and MX2 beamlines.
The datasets were processed with
centrated under reduced pressure to obtain a yellow oil. The oil was
either automated data processing pipeline implemented at the beam-
51 52 53
purified with a silica column (Mobile phase: Gradient of 2–7% MeOH in
lines, using xdsme and AIMLESS, or manually using iMOSFLM and
52 54
1
CH
2
Cl
2
) to obtain 18 as a yellow oil (24.7 mg, 33%). H NMR
AIMLESS from the CCP4 suite. 5% of reflections in each dataset
were flagged for calculation of Rfree. A summary of statistics is provided
in Supplementary Table S1. Molecular replacement was performed with
(
400 MHz, CDCl ) δ 8.24–8.18 (m, 1H), 7.33–7.24 (m, 3H), 5.68 (br s,
3
1
H), 4.21–4.05 (m, 2H), 3.69 (td, J = 6.8, 1.9 Hz, 2H), 3.46 (dd,
5
5
J = 9.7, 8.1 Hz, 1H), 3.05–2.97 (m, 3H), 2.82 (s, 3H), 2.59 (dd,
J = 16.5, 8.9 Hz, 1H), 2.42–2.28 (m, 1H), 2.14 (dd, J = 16.5, 7.2 Hz,
Phaser using a previously solved structure of BRD4 as a search model
(PDB: 5DW2). The final structures were obtained after several rounds of
1
3
56
57
1
H), 2.07–1.86 (m, 2H) ppm. C NMR (101 MHz, CDCl
3
) δ 173.5,
manual refinement using Coot and refinement with phenix.refine.
1
66.9, 143.8, 136.5, 125.7, 122.72, 122.17, 121.44, 109.2, 106.2, 55.1,
4
2.0, 40.7, 37.2, 35.1, 29.7, 29.2, 22.2 ppm. ESI-MS: m/z 312.0 [M
Declaration of Competing Interest
The authors declare no conflicts of interest.
Acknowledgements
+
+
+
H] . HR-MS: m/z calc. for C18
H
21
3
N O
2
[M+H] : 311.1634; found
3
11.1641. HPLC (PP gradient, MeOH): 4.644 min.
4.2. Fluorescence resonance energy transfer assay
The IC50s were measured using a Fluorescence Resonance Energy
This work was supported by a Research Training Program scholar-
ship from the Monash Institute of Pharmaceutical Sciences, NHMRC
project, program and fellowship grants, and a Cancer Council Victoria
Venture Grant. This research was undertaken in part using the MX2
beamline at the Australian Synchrotron, part of ANSTO, and the
Australian Cancer Research Foundation (ACRF) detector. Initial crys-
tallisation screens were carried out at the CSIRO Collaborative
Crystallisation Centre (www.csiro.au/C3) in Melbourne, Australia.
3
+
Transfer (FRET) assay. The assay is dependent on a europium (Eu
)
cryptate-conjugated antibody/glutathione S-transferase (GST) fused to
BRD4 BD1 (49–170) complex binding to a Streptavidin-D2 biotinylated,
tetra-acetylated histone H4 peptide, SGRG-K(Ac)-GG-K(Ac)-GLG-K(Ac)-
GGAK(Ac)-RHRKVGG-K-(Biotin) complex. Both Streptavidin-D2 and
3
+
the Eu
cryptate-conjugated antibody were purchased from CisBio
Assays. When both are in close proximity, a 337 nm laser light activates
3
+
the Eu donor which emits at 620 nm, resulting in D2 fluorescence at
65 nm. The assays were performed in 384-well volume microtiter
plates. The serially diluted small molecule inhibitors were diluted in
0% (v/v) DMSO resulting in a final concentration of 1% DMSO in each
well. The final reagent concentrations in each well were 10 nM of GST-
6
Appendix A. Supplementary material
1
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2019.115157.
3
+
BRD4 BD1 protein, 40 nM of histone H4 peptide, 5 nM of Eu
cryp-
tate-conjugated GST-antibody, 6.25 nM Streptavidin-D2 in 50 mM
Hepes, 50 mM NaCl, 0.5 mM CHAPS, 400 mM KF, 0.01% (w/v) BSA, pH
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