Chiral Analogues of PFI-1 as BET Inhibitors and Their Functional Role in Myeloid Malignancies

Article abstract of DOI:10.1021/acsmedchemlett.9b00625

Structural analogues of PFI-1 varying at the sulfur core were prepared, and their activities as BET inhibitors in myeloid cell lines and primary cells from patients with acute myeloid leukemia were studied. Docking calculations followed by molecular dynam

Full text of DOI:10.1021/acsmedchemlett.9b00625

pubs.acs.org/acsmedchemlett
Letter
Chiral Analogues of PFI‑1 as BET Inhibitors and Their Functional
Role in Myeloid Malignancies
#
#
#
̈
Bianca Altenburg, Marcus Frings, Jan-Hendrik Schobel, Jonas Goßen, Kristina Pannen,
Kim Vanderliek, Giulia Rossetti, Steffen Koschmieder, Nicolas Chatain,∇ and Carsten Bolm*,∇
Cite This: https://dx.doi.org/10.1021/acsmedchemlett.9b00625
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Structural analogues of PFI-1 varying at the sulfur
core were prepared, and their activities as BET inhibitors in
myeloid cell lines and primary cells from patients with acute
myeloid leukemia were studied. Docking calculations followed by
molecular dynamics simulations revealed the binding mode of the
newly prepared inhibitors, suggesting explanations for the observed
high enantiospecificity of the inhibitory activity.
KEYWORDS: BET inhibitors, acute myeloid leukemia (AML), sulfoximine, sulfondiimide, stereochemistry
he bromodomain and extra-terminal (BET) motif protein
TBRD4 was first linked to cancer after the discovery of the
BRD4−nuclear protein in testis (NUT) gene translocation
resulting in NUT midline carcinoma, which is a very aggressive
squamous cell carcinoma occurring primarily in adolescents.1
Importantly, clinical responses were seen in selected patients
using BET inhibitors that targeted BRD4.2,3
respective NH-sulfur derivatives (for details, see the Support-
ing Information). The latter were prepared by standard
synthetic protocols leading to N-unfunctionalized sulfoximines
617,18 and sulfondiimides 7.19 Enantiopure compounds 4 and
5a were obtained by preparative CSP-HPLC (for 4c and 7a),
resolution of diastereomeric salts (for 6a), and asymmetric
synthesis (for 6b).
Since the initial description of the BRD4−NUT fusion,
BRD4 has been examined in a variety of cancer types and
found to be a potential therapeutic target in AML and
myeloproliferative neoplasms.20,21 AML is a heterogeneous
disease characterized by a differentiation block and uncon-
trolled proliferation of hematopoietic stem and progenitor
cells.22 Approximately 97% of patients harbor clonal somatic
abnormalities, including mutations in the FMS-like tyrosine
kinase 3 (FLT3) of up to 38%.23 Two main variations of
mutations are described: the more frequent internal-tandem
duplications (ITDs) and tyrosine kinase domain (TKD)
mutations.22 Recently, the FLT3 inhibitor midostaurin has
been approved as targeted therapy in combination with
chemotherapy for the treatment of FLT3-mutant AML on
the basis of a phase 3 clinical trial.24 However, additional
BET inhibitors are structurally diverse.4−8 For example, 1
[(+)-JQ1]7 and 2 (OTX-015, MK8628, birabresib)8 are
triazolodiazepines that show potencies in the nanomolar
range with pronounced selectivities in the BET family.
Following work by Conway and co-workers9 and Prinjha and
co-workers,10 Pfizer introduced 3,4-dihydro-3-methyl-2(1H)-
quinazolinone 3 (PFI-1) as a BRD4-selective BET inhibitor.11
The latter compound caught our attention because we
hypothesized that its sulfonamidoyl group could be modified
by (formal) atom exchange reactions leading to unprecedented
sulfoximines 4 and sulfondiimides 5 (Chart 1).12−14 In other
systems, such sulfur core modifications had led to products
with improved properties, including, for example, increased
water solubility.15 Furthermore, in contrast to 3, compounds 4
and 5 are chiral, allowing studies of individual enantiomers.16
Here we report on the preparation of these compounds and
their characterization as new BET inhibitors in myeloid cell
lines and primary cells from patients with acute myeloid
leukemia (AML).
Special Issue: Medicinal Chemistry: From Targets to
Therapies
The syntheses of compounds 4 and 5 are summarized in
Scheme 1. In general, they involved metal-catalyzed Buchwald/
Hartwig- or Chan/Lam-type cross-coupling reactions of 3,4-
dihydro-3-methyl-2(1H)-quinazolinones 8a and 8b with
Received: December 19, 2019
Accepted: April 2, 2020
Published: April 2, 2020
https://dx.doi.org/10.1021/acsmedchemlett.9b00625
© XXXX American Chemical Society
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
A

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Hence, BET-BRD proteins are termed epigenetic readers.27
Hexamethylene bisacetamide-inducible protein 1 (HEXIM1)
inhibits the p-TEFb complex through binding to 7SK small
nuclear RNA. BRD4 displaces HEXIM1 from p-TEFb, which
then becomes activated and subsequently phosphorylates the
carboxy-terminal domain of RNA polymerase II to start
transcription of BRD4-dependent genes.27 Together, induction
of HEXIM1 and reduction of MYC expression proved to be a
potential biomarker in a clinical trial of BET.28
Chart 1. Known BET Inhibitors 1−3 and Newly Prepared
Compounds 4 and 5
The activity of BRD4 in regulating and accelerating cell cycle
progression through the regulation of gene expression in
myeloid malignancies makes it a promising pharmaceutical
target. Although BET inhibition is a matter of intensive
ongoing research, recent clinical trials have encountered
unexpected dose-limiting toxicity of BET inhibitors, including
vomiting, headaches, and back pain,28 as well as thrombocy-
topenia.29 Thus, novel BET inhibitors that show optimized
BET family member specificity are of interest to improve the
tolerability in order to include long-term improvements of
prognosis and quality of life in patients with AML and other
myeloid malignancies. Optimization of dosing regimens and
provision of treatment options in case of resistance emergence
are conceivable.
Consequently, we decided to characterize the functional role
of the aforementioned PFI-1 analogues in myeloid cell lines
and primary cells from patients with AML. To analyze whether
4 and 5 had activity in cellular assays, MTT viability assays
with HEL, Molm-14, and K562 cells, which are erythroleuke-
mia, AML, and chronic myeloid leukemia (CML) (terminal
blast phase) cell lines that are positive for JAK2 V617F-, FLT3-
ITD-, and BCR-ABL, respectively, were performed. Initially,
only racemates of the potential inhibitors were applied. This
study showed that several compounds were active, and among
them, sulfoximine 4b and sulfondiimide 5a were most efficient
in reducing the relative metabolic activity of HEL and Molm-
14 cells at a concentration of 10 μM (Figure S1).
Sulfondiimides 5b and 5c showed lower efficacies. None of
the compounds significantly affected the growth of the BET-
inhibitor-insensitive cell line K562.
Scheme 1. Cross-Coupling Partners
With the intention to take advantage of the stereochemistry
at sulfur, the inhibitory activity of racemic 4a on HEL, Molm-
14, and K562 was compared to the effects induced by the
individual enantiomers of 4a. PFI-1 (3) was applied as a
control. As envisaged, (S)-4a and (R)-4a behaved very
differently. While treatment with 1 μM (S)-4a led to a
significant decrease in relative metabolic activity of the HEL
cells in comparison with 1 μM PFI-1 (37 6% vs 73 13%)
(Figure 1A), no effect was observed with 1 μM (R)-4a, for
which the result was comparable to the DMSO control (Figure
1A,B). Thus, the stereogenic center at sulfur significantly
affected the efficacy of 4a, with the S enantiomer being
superior to both the racemic mixture of 4a and the standard
compound PFI-1. Similar effects were observed in the Molm14
cell line (55 1% with 1 μM (S)-4a vs 80 1% with 1 μM
PFI-1) (Figure 1C). The IC50 values calculated in HEL and
Molm-14 cells showed that (S)-4a was the most efficient
compound, with concentrations of 0.5 μM (PFI-1: 2.0 μM)
and 1.0 μM (PFI-1: 2.7 μM), respectively (Figure 1B,D). In
contrast to HEL and Molm-14 cells, K562 cells remained
mostly unaffected and never reached an IC50 (Figure 1E,F).
Applying single enantiomers of 4b and 5a in MTT assays
revealed that also for 4b the S enantiomer was superior over
the R-configured compound (Figure S2). For 5a, however, the
mutations increase the risk of therapy failure in FLT3-mutant
and other subtypes of AML.
The BET motif family consists of four members in
mammals: BRD2, BRD3, BRD4, and the testis-specific
BRDT.4−6 They share a conserved structure of two
bromodomains (BRDs), an extraterminal recruitment (ET)
domain, and some other motifs, like A, B, or SEED motifs.
Furthermore, BRD4 and BRDT have a C-terminal motif
(CTM).5 While the ET domain takes part in protein−protein
interactions, the C-terminal domain interacts with the positive
transcription elongation factor b (p-TEFb).5 The bromodo-
mains contain around 110 amino acids and structurally form
two loops (ZA and BC) and four α-helices (αZ, αA, αB, and
αC).25 The four α-helices build a hydrophobic pocket that
recognizes acetylated lysines (Kac) on proteins, such as
histones.26
BRD4 and the other BET-BRDs can bind to acetylated
lysine residues of N-terminal histone tails through their
bromodomains and recruit the mediator and p-TEFb complex.
B
https://dx.doi.org/10.1021/acsmedchemlett.9b00625
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Figure 1. Marked differences in the efficacy of enantiomeric
compounds. (A) HEL, (C) Molm-14 and (E) K562 cells were
treated with PFI-1 (3), rac-4a, (S)-4a, and (R)-4a at concentrations of
10 nM, 100 nM, 1 μM, 10 μM, and 20 μM for 72 h. IC50 values
calculated from MTT assays with (B) HEL, (D) Molm-14, and (E)
K562 cells after compound treatment for 72 h. All data are normalized
to the DMSO control and shown as mean
SD. Graphs show
combinations of three independent experiments. Statistics refer to 3.
**, p < 0.01; ***, p < 0.001.
absolute configuration had only a minor effect on the
compound efficacy.
Next, we analyzed the selectivities of (S)-4a and PFI-1 for 76
bromodomain-containing proteins in a thermal shift assay
(TSA) (BromoMELT assay, Reaction Biology Corp., Malvern,
PA, USA). The TSA demonstrated a slightly higher selectivity
of (S)-4a versus PFI-1 for BRD2/4 (ΔTm of 5 vs 4 °C for
BRD4 and 3.5 vs 2.5 °C for BRD2-1) (Figure 2A,B; see the
Supporting Information for the full data sets for PFI-1 and (S)-
4a). At the same time, the ΔTm is reduced for BRDT-2 and
slightly for BRD3 [(S)-4a vs PFI-1]. Therefore, we confirmed
the binding of (S)-4a to BET proteins and demonstrated a
better selectivity profile of (S)-4a for BRD2 and BRD4 in
comparison with PFI-1.
Several studies confirmed induction of growth arrest and
apoptosis using the BET inhibitors JQ1 (1) and OTX015
(2).8,20,30 Therefore, we evaluated whether (S)-4a and further
compounds led to reduced proliferation and disturbed cell
cycle progression. Indeed, proliferation of HEL and Molm-14
cells was significantly reduced by (S)-4a compared with PFI-1
(Figures 2C and S3A−C), whereas the number of K562 cells
was only slightly affected by (S)-4a treatment, in line with data
on another BET inhibitor.31
Figure 2. Higher selectivity and potency of (S)-4a. (A) Thermal shift
assay (TSA) (BromoMELT assay) of PFI-1 (3) and (S)-4a in
duplicate at 10 μM. Shown are the values of ΔTm vs control for the
BET domain protein family. Full analyses for PFI-1 and (S)-4a are
provided in the Supporting Information. (B) TSA data for PFI-1 (3)
and (S)-4a. Temperature shifts are indicated by reddish circles with
increasing radii for higher Tm values as indicated.32 (C) HEL, Molm-
14 and K562 cells were treated with either DMSO or 5 μM of
compounds or (S)-4a. Comparison of cell proliferation after 72 h of
treatment. Three independent experiments combined. All data
displayed as mean SD. Statistics: 5 μM of PFI-1 compared to 5
μM of (S)-4a. (D) Bar graph illustrating significant viability loss by 5
μM (S)-4a treatment of HEL and Molm-14 cells after 72 h of
treatment. All data are shown as mean SD. Statistics: 3 compared to
(S)-4a. *p < 0.05, **p < 0.01, ***p < 0.001.
μM PFI-1 reduced the cell viability to 85 2% or 85 12%,
respectively, while 5 μM (S)-4a diminished it to 55 7% or 57
16%, respectively, after 72 h of treatment (Figures 2D and
S4A,B). PARP1 cleavage, as a marker of apoptosis, was
confirmed in (S)-4a-treated HEL cells and more pronounced
in Molm-14 cells (Figure S4D). In the DMSO control, PARP1
cleavage was partly detected, mostly as a result of massive
proliferation of cells without BRD4 inhibitor and therefore a
lack of nutrients (Figure S4D),33 which was also supported by
the higher amount of uncleaved PARP1 protein (upper band)
in DMSO- and (R)-4a-treated cells (Figure S4D; prominent in
Molm14 cells). As expected, no reduction of viability and
PARP1 cleavage was observed in K562 cells (Figure S4C,D).
Taken together, the results show that (S)-4a was more
efficient in reducing the viability of Molm-14 and HEL cells
Analysis of the cell cycle progression confirmed that PFI-1
and (S)-4a led to an increase in the G0/G1 phase, a decrease
in the S phase, and in some cases an increase in the sub-G1
phase (Figures S3D and S2E). Again, (S)-4a was more
effective than PFI-1 in perturbing the transition from the G1
phase to the S phase, and an increase in the sub-G1 peak was
observed.
Next, the direct effects of the novel compounds on viability
and apoptosis were determined. In HEL and Molm-14 cells, 55
C
https://dx.doi.org/10.1021/acsmedchemlett.9b00625
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
than PFI-1. The major effect of BRD4 inhibition was to block
proliferation and cell cycle progression.
PBMCs or CD34+ cells were used to analyze general
cytotoxicity.
Clonogenic growth of healthy donor cells (PBMCs and
CD34+) was reduced in both 1 μM and 5 μM compound
treatments (Figure 4A), as demonstrated already for PFI-1 and
BET protein inhibition is correlated with decreased
expression of Aurora kinase B (AURKB) and increased
expression of HEXIM1, and HEXIM1 participates in the
subsequent growth arrest of AML cell lines.34,35 Therefore, we
performed Western blot analysis and observed that HEXIM1
protein was increased in HEL and Molm-14 cells after 24 h of
treatment with 5 μM PFI-1 and even more after treatment with
(S)-4a (Figure 3A,B). In contrast, (R)-4a treatment did not
Figure 4. (S)-4a reduces clonogenic growth of primary AML patient
cells more efficiently than PFI-1. (A) CFU assays with (top) healthy-
donor peripheral blood mononuclear cells (PBMCs) or (bottom)
CD34+ cells (# indicates only two treatments at 1 μM). (B) CFU
assays with bone marrow monocuclear cells (BMMCs) of three
FLT3-ITD- or -TKD-mutated AML patient samples combined and
normalized to the DMSO control. Experiments were performed in
triplicate. All of the data are shown as mean SD. **, p < 0.01; ***,
p < 0.001.
OTX015.31,36 Importantly, (S)-4a at 1 and 5 μM reduced the
colony number of bone marrow mononuclear cells (BMMCs)
isolated from AML patients more efficiently than the standard
compound PFI-1 (Figure 4B). More HD and AML samples
need to be analyzed in the future.
With the goal to gain a more fundamental understanding of
the binding mode of the newly prepared inhibitors and to
develop a comprehension for the enantiospecific inhibitory
activity, in silico models of the structural determinants of
BRD4−ligand complexes were obtained by performing
docking calculations followed by molecular dynamics (MD)
simulations. For this purpose, the X-ray structure of the first
bromodomain of human BRD4 in complex with the inhibitor
PFI-1 (PDB ID 4E96) was used. The best-performing ligand
according to the Glide XP scoring function (see the
Supporting Information for details) is (S)-4a, while (R)-4a is
the worst (see Table S8).
In their best binding poses (see Methods in the Supporting
Information for details), the anchoring hydrogen-bonding
interactions with ASN140 and the water-mediated interaction
with TYR97 of BRD4, observed in the X-ray complex of PFI-1
(Figure 5E), are preserved for both (S)-4a (Figures 5A,B) and
(R)-4a (Figure 5C,D). However, (S)-4a orients the anchoring
3,4-dihydro-3-methyl-2(1H)-quinazolinone core toward the
BC loop, and the sulfoximidoyl group points toward the
hydrophobic patch consisting of ILE146, PRO82, and TRP81
(Figure 5A,B).37 Differently, in (R)-4a the latter substituent
points toward the opposite direction, suggesting the loss of the
hydrophobic-patch interactions and the exposure of the apolar
phenyl moiety to the solvent (Figures 5C,D). Therefore, we
expect (R)-4a to be a weaker binder with respect both (S)-4a
and PFI-1.
Figure 3. Western Blot analysis of HEXIM1 protein in compound-
treated HEL, Molm14, and K562 cells. (A) HEL, (B) Molm14, and
(C) K562 cells were incubated with DMSO or the indicated
compounds at 5 μM for 24 or 48 h. Specific antibodies detecting
HEXIM1, Aurora kinase B (AURKB), and GAPDH were used.
GAPDH served as a loading control.
increase the HEXIM1 protein level. Surprisingly, (R)-4a
reduced AURKB in Molm-14 cells and K562 cells after 48 h,
but at least the result in K562 cells was DMSO-dependent.
Nevertheless, (S)-4a most efficiently reduced AURKB protein
levels in HEL and Molm-14 cells (Figure 3).
Although HEXIM1 protein was upregulated in K562 cells
treated with PFI-1 and (S)-4a (Figure 3C), cell viability was
not reduced by BRD4 inhibition (Figures 1 and 2). Hence,
HEXIM1 alone cannot ablate CML cell line proliferation and
viability. Interestingly, HEXIM1 protein decreased already 48
h after treatment, demonstrating a relatively short half-life of
the used BRD4 inhibitors and probably explaining relatively
low induction of apoptosis. On the other hand, AURKB
protein behaved in the opposite way, most likely demonstrat-
ing the protein half-life before its proteasomal degradation.
Preincubation of PFI-1 and (S)-4a in medium and subsequent
treatment of HEL, K562, and Molm-14 cells demonstrated
compound stability and robust induction of HEXIM1,
especially by (S)-4a (Figure S5).
To support our cell line data, AML patient peripheral blood
mononuclear cells (PBMCs), carrying FLT3-ITD or tyrosine
kinase domain (FLT3-TKD) mutations were isolated and
applied into colony formation (CFU) assays. Healthy-donor
D
https://dx.doi.org/10.1021/acsmedchemlett.9b00625
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
orientation of the N−S bond in (S)-4a causes a lower loss of
entropy with respect to PFI-1 upon binding. These
observations together point to (S)-4a having a higher binding
affinity toward BRD4 compared with PFI-1.
In summary, we found a new BET inhibitor with activity in
myeloid cell lines and primary cells from patients with acute
myeloid leukemia (AML). The most effective compound, 4a,
has a stereogenic center at sulfur, and its absolute configuration
dictates its efficacy. The data show that (S)-4a is superior to
well-studied PFI-1. The R enantiomer of 4a is essentially
inactive.
Although the BET inhibitors, including PFI-1 and our newly
prepared (S)-4a, showed some degree of activity on healthy
control cells, the effects on AML cells were more pronounced.
Therapy with BET protein bromodomain antagonists is of high
interest and may be beneficial for particular patients. In
addition, combination therapy of BRD4 inhibitors with specific
tyrosine kinase inhibitors should be further evaluated. Thus,
subsequent research is needed to investigate whether (S)-4a
can be used as a single therapeutic agent or in combination
therapy for AML.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00625.
■
sı
BromoMELT assay for PFI-1 (PDF)
BromoMELT assay for (S)-4a (PDF)
Chemical syntheses, NMR spectra, HPLC traces,
biological testings, and computational studies (PDF)
Figure 5. (A−D) Best binding poses of (A) (S)-4a and (C) (R)-4a in
three dimensions and (B, D) the corresponding two-dimensional
(2D) representations of nonbonded interactions. In (A) and (C),
hydrogen bonds are shown in blue, water-mediated hydrogen bonds
are denoted as green dashed lines, and the position of water 380 is
shown as a red sphere. The phenyl moiety of (S)-4a shows a distinct
kink toward ILE146 (dihedral angle φ(CNSC) = 56.6°). The R
enantiomer does not show this kink (φ(CNSC) = 175.5°). In (B) and
(D), hydrogen bonding is denoted by the magenta arrows. The
hydrophobic surface area is shown as a green line, and the color
coding of the pocket residues is explained in the legend of (B). (E)
Direct comparison of the crystal pose orientation of PFI-1 (3) and the
best binding poses of (S)-4a and (R)-4a, with the van der Waals
surface of BRD4 shown as a gray surface. (F) 2D representation of the
binding conformation of 3 in the crystal structure (PDB ID 4E96).
The same color code as in (B) and (D) was implemented.
AUTHOR INFORMATION
Corresponding Author
■
Carsten Bolm − Institute of Organic Chemistry, RWTH Aachen
University, 52074 Aachen, Germany; orcid.org/0000-0001-
9415-9917; Email: Carsten.Bolm@oc.rwth-aachen.de
Authors
Bianca Altenburg − Department of Hematology, Oncology,
Hemostaseology, and Stem Cell Transplantation, Faculty of
Medicine, RWTH Aachen University, 52074 Aachen, Germany
Marcus Frings − Institute of Organic Chemistry, RWTH Aachen
University, 52074 Aachen, Germany; orcid.org/0000-0002-
6228-1229
̈
Interestingly, (S)-4a performed better than PFI-1 in our in
silico experiments. A significant difference between (S)-4a and
PFI-1 is the hybridization state of the nitrogen atom on passing
from sulfonamide to sulfoximine (sp3 to sp2, respectively).
Increasing the s character of the nitrogen lone pair decreases
the hydrogen-bond acceptor strength (sp3 > sp2 > sp).38 The
sp3-hybridized nitrogen in PFI-1 is therefore a stronger
hydrogen-bond acceptor than the sp2-hybridized nitrogen in
(S)-4a, suggesting slightly better solvation and higher hydro-
philicity. Therefore, the slightly more hydrophobic (S)-4a
would be better stabilized than PFI-1 in the hydrophobic
binding pocket of BRD4. Also, the sp2-hybridized nitrogen in
the sulfoximine moiety reduces the flexibility of (S)-4a, since
the free rotation around the N−S bond (dihedral angle
φ(−CNSC)) is lost. As a result, (S)-4a is restricted in its
orientation. The restriction of a small molecule’s motion on
binding to a protein usually causes a loss of configurational
entropy and thus a penalty in binding affinity.39,40 The single
Jan-Hendrik Schobel − Institute of Organic Chemistry, RWTH
Aachen University, 52074 Aachen, Germany
Jonas Goßen − Institute of Neuroscience and Medicine (INM-
9)/Institute for Advanced Simulation (IAS-5),
̈ ̈
Forschungszentrum Julich, 52425 Julich, Germany; Faculty of
Mathematics, Computer Science and Natural Sciences, RWTH
Aachen University, 52074 Aachen, Germany
Kristina Pannen − Department of Hematology, Oncology,
Hemostaseology, and Stem Cell Transplantation, Faculty of
Medicine, RWTH Aachen University, 52074 Aachen, Germany
Kim Vanderliek − Department of Hematology, Oncology,
Hemostaseology, and Stem Cell Transplantation, Faculty of
Medicine, RWTH Aachen University, 52074 Aachen, Germany
Giulia Rossetti − Department of Hematology, Oncology,
Hemostaseology, and Stem Cell Transplantation, Faculty of
Medicine, RWTH Aachen University, 52074 Aachen, Germany;
Institute of Neuroscience and Medicine (INM-9)/Institute for
Advanced Simulation (IAS-5) and Julich Supercomputing
̈
E
https://dx.doi.org/10.1021/acsmedchemlett.9b00625
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Centre (JSC), Forschungszentrum Ju
pubs.acs.org/acsmedchemlett
Letter
Li, F.; Vedadi, M.; Dilworth, J.; Lupien, M.; Brennan, P. E.;
Arrowsmith, C. H.; Muller, S.; Fedorov, O.; Filippakopoulos, P.;
̈
lich, 52425 Ju
Germany; orcid.org/0000-0002-2032-4630
̈
lich,
̈
Knapp, S. A chemical toolbox for the study of bromodomains and
Steffen Koschmieder − Department of Hematology, Oncology,
Hemostaseology, and Stem Cell Transplantation, Faculty of
Medicine, RWTH Aachen University, 52074 Aachen, Germany
Nicolas Chatain − Department of Hematology, Oncology,
Hemostaseology, and Stem Cell Transplantation, Faculty of
Medicine, RWTH Aachen University, 52074 Aachen, Germany
epigenetic signaling. Nat. Commun. 2019, 10, 1915.
́
́
(5) Jung, M.; Gelato, K. A.; Fernandez-Montalvan, A.; Siegel, S.;
Haendler, B. Targeting BET bromodomains for cancer treatment.
Epigenomics 2015, 7, 487−501.
(6) Filippakopoulos, P.; Knapp, S. Targeting bromodomains:
epigenetic readers of lysine acetylation. Nat. Rev. Drug Discovery
2014, 13, 337−356.
Complete contact information is available at:
(7) Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W. B.;
Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.;
Philpott, M.; Munro, S.; McKeown, M. R.; Wang, Y.; Christie, A. L.;
West, N.; Cameron, M. J.; Schwartz, B.; Heightman, T. D.; La
Thangue, N.; French, C. A.; Wiest, O.; Kung, A. L.; Knapp, S.;
Bradner, J. E. Selective inhibition of BET bromodomains. Nature
2010, 468, 1067−1073.
(8) Coude, M. M.; Braun, T.; Berrou, J.; Dupont, M.; Bertrand, S.;
Masse, A.; Raffoux, E.; Itzykson, R.; Delord, M.; Riveiro, M. E.;
Herait, P.; Baruchel, A.; Dombret, H.; Gardin, C. BET inhibitor
OTX015 targets BRD2 and BRD4 and decreases c-MYC in acute
leukemia cells. Oncotarget 2015, 6, 17698−17712.
(9) Hewings, D. S.; Wang, M.; Philpott, M.; Fedorov, O.; Uttarkar,
S.; Filippakopoulos, P.; Picaud, S.; Vuppusetty, C.; Marsden, B.;
Knapp, S.; Conway, S. J.; Heightman, T. D. 3,5-Dimethylisoxazoles
act as acetyl-lysine-mimetic bromodomain ligands. J. Med. Chem.
2011, 54, 6761−6770.
(10) Dawson, M. A.; Prinjha, R. K.; Dittmann, A.; Giotopoulos, G.;
Bantscheff, M.; Chan, W.-I.; Robson, S. C.; Chung, C.-w.; Hopf, C.;
Savitski, M. M.; Huthmacher, C.; Gudgin, E.; Lugo, D.; Beinke, S.;
Chapman, T. D.; Roberts, E. J.; Soden, P. E.; Auger, K. R.; Mirguet,
O.; Doehner, K.; Delwel, R.; Burnett, A. K.; Jeffrey, P.; Drewes, G.;
Lee, K.; Huntly, B. J. P.; Kouzarides, T. Inhibition of BET recruitment
to chromatin as an effective treatment for MLL-fusion leukaemia.
Nature 2011, 478, 529−533.
https://pubs.acs.org/10.1021/acsmedchemlett.9b00625
Author Contributions
#B.A., M.F., and J.-H.S. contributed equally (shared first
authorship). The manuscript was written through contribu-
tions of all authors. All of the authors approved the final
version of the manuscript.
Author Contributions
∇N.C. and C.B. have shared last authorship.
Funding
This work was supported by RWTH Aachen University
through Seed Funds Project OPSF496.
Notes
The authors declare no competing financial interest.
Part of this work was generated within the medical thesis work
of B.A., and part of this work was generated within the Master’s
thesis of K.V. A patent application was submitted.
ACKNOWLEDGMENTS
We are grateful to Cornelia Vermeeren (RWTH Aachen
University) for the preparative CSP-HPLC separation of two
■
compounds. The support of Susanne Grunebaum (RWTH
̈
Aachen University) in compound syntheses is highly
appreciated.
(11) Fish, P. V.; Filippakopoulos, P.; Bish, G.; Brennan, P. E.;
Bunnage, M. E.; Cook, A. S.; Federov, O.; Gerstenberger, B. S.; Jones,
H.; Knapp, S.; Marsden, M. J.; Nocka, K.; Owen, D. R.; Philpott, M.;
Picaud, S.; Primiano, M. J.; Ralph, M. J.; Sciammetta, N.; Trzupek, J.
D. Identification of a chemical probe for bromo and extra C-terminal
bromodomain inhibition through optimization of a fragment-derived
hit. J. Med. Chem. 2012, 55, 9831−9837.
ABBREVIATIONS
■
AML, acute myeloid leukemia; BET, bromodomain and extra-
terminal; BMMC, bone marrow mononuclear cell; CML,
chronic myeloid leukemia; CSP, chiral stationary phase; CTM,
C-terminal motif; FLT3, FMS-like tyrosine kinase 3; GAPDH,
glyceraldehyde-3-phosphatedehydrogenase; HEL, human er-
ythroleukemia cell line; HEXIM1, hexamethylene bisaceta-
mide-inducible protein 1; HPLC, high-performance liquid
chromatography; MCAP, mitotic chromosome associated
protein; NUT, nuclear protein in testis; PMF, primary
myelofibrosis; PBMC, peripheral blood mononuclear cell;
PV, polycythemeia vera; p-TEFb, positive transcription
elongation factor b; SD, standard deviation; TKD, tyrosine
kinase domain
(12) Lucking, U. Neglected sulfur(VI) pharmacophores in drug
̈
discovery: exploration of novel chemical space by the interplay of drug
design and method development. Org. Chem. Front. 2019, 6, 1319−
1324.
(13) Lucking, U. Sulfoximines: a neglected opportunity in medicinal
̈
chemistry. Angew. Chem., Int. Ed. 2013, 52, 9399−9408.
(14) Frings, M.; Bolm, C.; Blum, A.; Gnamm, A. Sulfoximines from a
medicinal chemist’s perspective: physicochemical and in vitro
parameters relevant for drug discovery. Eur. J. Med. Chem. 2017,
126, 225−245.
(15) Foote, K. M.; Nissink, J. W. M.; McGuire, T.; Turner, P.;
Guichard, S.; Yates, J. W. T.; Lau, A.; Blades, K.; Heathcote, D.;
Odedra, R.; Wilkinson, G.; Wilson, Z.; Wood, C. M.; Jewsbury, P. J.
Discovery and characterization of AZD6738, a potent inhibitor of
ataxia telangiectasia mutated and Rad3 related (ATR) kinase with
application as an anticancer agent. J. Med. Chem. 2018, 61, 9889−
9907.
(16) Park, S. J.; Baars, H.; Mersmann, S.; Buschmann, H.; Baron, J.
M.; Amann, P. M.; Czaja, K.; Hollert, H.; Bluhm, K.; Redelstein, R.;
Bolm, C. N-Cyano sulfoximines: COX inhibition, anticancer activity,
cellular toxicity, and mutagenicity. ChemMedChem 2013, 8, 217−220.
(17) Bull, J. A.; Degennaro, L.; Luisi, R. Straightforward strategies
for the preparation of NH-sulfoximines: a serendipitous story. Synlett
2017, 28, 2525−2538.
(18) Bizet, V.; Hendriks, C. M. M.; Bolm, C. Sulfur imidations:
access to sulfimides and sulfoximines. Chem. Soc. Rev. 2015, 44,
3378−3390.
REFERENCES
■
(1) French, C. A.; Miyoshi, I.; Kubonishi, I.; Grier, H. E.; Perez-
Atayde, A. R.; Fletcher, J. A. BRD4−NUT fusion oncogene: a novel
mechanism in aggressive carcinoma. Cancer Res. 2003, 63, 304−307.
(2) Stathis, A.; Zucca, E.; Bekradda, M.; Gomez-Roca, C.; Delord, J.-
P.; de La Motte Rouge, T.; Uro-Coste, E.; de Braud, F.; Pelosi, G.;
French, C. A. Clinical response of carcinomas harboring the BRD4−
NUT oncoprotein to the targeted bromodomain inhibitor OTX015/
MK-9628. Cancer Discovery 2016, 6, 492−500.
(3) Liu, Z.; Wang, P.; Chen, H.; Wold, E. A.; Tian, B.; Brasier, A. R.;
Zhou, J. Drug discovery targeting bromodomain-containing protein 4.
J. Med. Chem. 2017, 60, 4533−4558.
̈
(4) Wu, Q.; Heidenreich, D.; Zhou, S.; Ackloo, S.; Kramer, A.;
Nakka, K.; Lima-Fernandes, E.; Deblois, G.; Duan, S.; Vellanki, R. N.;
F
https://dx.doi.org/10.1021/acsmedchemlett.9b00625
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
(19) Candy, M.; Guyon, C.; Mersmann, S.; Chen, J.-R.; Bolm, C.
Synthesis of sulfondiimines by N-chlorosuccinimide-mediated oxida-
tive imination of sulfiliminium salts. Angew. Chem., Int. Ed. 2012, 51,
4440−4443.
(20) Zuber, J.; Shi, J.; Wang, E.; Rappaport, A. R.; Herrmann, H.;
Sison, E. A.; Magoon, D.; Qi, J.; Blatt, K.; Wunderlich, M.; Taylor, M.
J.; Johns, C.; Chicas, A.; Mulloy, J. C.; Kogan, S. C.; Brown, P.;
Valent, P.; Bradner, J. E.; Lowe, S. W.; Vakoc, C. R. RNAi screen
identifies Brd4 as a therapeutic target in acute myeloid leukaemia.
Nature 2011, 478, 524−528.
(21) Kleppe, M.; Koche, R.; Zou, L.; van Galen, P.; Hill, C. E.;
Dong, L.; De Groote, S.; Papalexi, E.; Hanasoge Somasundara, A. V.;
Cordner, K.; Keller, M.; Farnoud, N.; Medina, J.; McGovern, E.;
Reyes, J.; Roberts, J.; Witkin, M.; Rapaport, F.; Teruya-Feldstein, J.;
Qi, J.; Rampal, R.; Bernstein, B. E.; Bradner, J. E.; Levine, R. L. Dual
targeting of oncogenic activation and inflammatory signaling increases
therapeutic efficacy in myeloproliferative neoplasms. Cancer Cell
2018, 33, 29−43.
interaction inhibitor, targeting BET Bromodomains. Cancer Res. 2013,
73, 3336−3346.
(32) Adopted from Reaction Biology Corp.: http://www.
reactionbiology.com/webapps/site/BromoMelt.aspx.
(33) Krampe, B.; Al-Rubeai, M. Cell death in mammalian cell
culture: molecular mechanisms and cell line engineering strategies.
Cytotechnology 2010, 62, 175−188.
(34) Devaraj, S. G.; Fiskus, W.; Shah, B.; Qi, J.; Sun, B.; Iyer, S. P.;
Sharma, S.; Bradner, J. E.; Bhalla, K. N. HEXIM1 induction is
mechanistically involved in mediating anti-AML activity of BET
protein bromodomain antagonist. Leukemia 2016, 30, 504−508.
(35) Sahni, J. M.; Gayle, S. S.; Bonk, K. L. W.; Vite, L. C.; Yori, J. L.;
Webb, B.; Ramos, E. K.; Seachrist, D. D.; Landis, M. D.; Chang, J. C.;
Bradner, J. E.; Keri, R. A. Bromodomain and extraterminal protein
inhibition blocks growth of triple-negative breast cancers through the
suppression of aurora kinases. J. Biol. Chem. 2016, 291, 23756−23768.
(36) Saenz, D. T.; Fiskus, W.; Qian, Y.; Manshouri, T.; Rajapakshe,
K.; Raina, K.; Coleman, K. G.; Crew, A. P.; Shen, A.; Mill, C. P.; Sun,
B.; Qiu, P.; Kadia, T. M.; Pemmaraju, N.; DiNardo, C.; Kim, M. S.;
Nowak, A. J.; Coarfa, C.; Crews, C. M.; Verstovsek, S.; Bhalla, K. N.
Novel BET protein proteolysis-targeting chimera exerts superior lethal
activity than bromodomain inhibitor (BETi) against post-myelopro-
liferative neoplasm secondary (s) AML cells. Leukemia 2017, 31,
1951−1961.
(22) Kindler, T.; Lipka, D. B.; Fischer, T. FLT3 as a therapeutic
target in AML: still challenging after all these years. Blood 2010, 116,
5089−5102.
(23) Coombs, C. C.; Tallman, M. S.; Levine, R. L. Molecular therapy
for acute myeloid leukaemia. Nat. Rev. Clin. Oncol. 2016, 13, 305−
318.
(37) Vidler, L. R.; Brown, N.; Knapp, S.; Hoelder, S. Druggability
analysis and structural classification of bromodomain acetyl-lysine
binding sites. J. Med. Chem. 2012, 55, 7346−7359.
(24) Stone, R. M.; Mandrekar, S. J.; Sanford, B. L.; Laumann, K.;
̈
Geyer, S.; Bloomfield, C. D.; Thiede, C.; Prior, T. W.; Dohner, K.;
Marcucci, G.; Lo-Coco, F.; Klisovic, R. B.; Wei, A.; Sierra, J.; Sanz, M.
A.; Brandwein, J. M.; de Witte, T.; Niederwieser, D.; Appelbaum, F.
R.; Medeiros, B. C.; Tallman, M. S.; Krauter, J.; Schlenk, R. F.;
Ganser, A.; Serve, H.; Ehninger, G.; Amadori, S.; Larson, R. A.;
(38) Møller, K. H.; Kjaersgaard, A.; Hansen, A. S.; Du, L.;
Kjaergaard, H. G. Hybridization of nitrogen determines hydrogen-
bond acceptor strength: gas-phase comparison of redshifts and
equilibrium constants. J. Phys. Chem. A 2018, 122, 3899−3908.
̈
Dohner, H. Midostaurin plus chemotherapy for acute myeloid
̈
(39) Klebe, G.; Bohm, H. J. Energetic and entropic factors
leukemia with a FLT3 mutation. N. Engl. J. Med. 2017, 377, 454−464.
(25) Taniguchi, Y. The bromodomain and extra-terminal domain
(BET) family: functional anatomy of BET paralogous proteins. Int. J.
Mol. Sci. 2016, 17, 1849.
determining binding affinity in protein-ligand complexes. J. Recept.
Signal Transduction Res. 1997, 17, 459−473.
(40) Chang, C. A.; Chen, W.; Gilson, M. K. Ligand configurational
entropy and protein binding. Proc. Natl. Acad. Sci. U. S. A. 2007, 104,
1534−1539.
(26) Filippakopoulos, P.; Picaud, S.; Mangos, M.; Keates, T.;
̈
Lambert, J.-P.; Barsyte-Lovejoy, D.; Felletar, I.; Volkmer, R.; Muller,
S.; Pawson, T.; Gingras, A.-C.; Arrowsmith, C. H.; Knapp, S. Histone
recognition and large-scale structural analysis of the human
bromodomain family. Cell 2012, 149, 214−231.
(27) Yang, Z.; Yik, J. H.; Chen, R.; He, N.; Jang, M. K.; Ozato, K.;
Zhou, Q. Recruitment of P-TEFb for stimulation of transcriptional
elongation by the bromodomain protein Brd4. Mol. Cell 2005, 19,
535−545.
(28) Postel-Vinay, S.; Herbschleb, K.; Massard, C.; Woodcock, V.;
Soria, J.-C.; Walter, A. O.; Ewerton, F.; Poelman, M.; Benson, N.;
Ocker, M.; Wilkinson, G.; Middleton, M. First-in-human phase I
study of the bromodomain and extraterminal motif inhibitor BAY
1238097: emerging pharmacokinetic/pharmacodynamic relationship
and early termination due to unexpected toxicity. Eur. J. Cancer 2019,
109, 103−110.
(29) Amorim, S.; Stathis, A.; Gleeson, M.; Iyengar, S.; Magarotto, V.;
Leleu, X.; Morschhauser, F.; Karlin, L.; Broussais, F.; Rezai, K.;
Herait, P.; Kahatt, C.; Lokiec, F.; Salles, G.; Facon, T.; Palumbo, A.;
Cunningham, D.; Zucca, E.; Thieblemont, C. Bromodomain inhibitor
OTX015 in patients with lymphoma or multiple myeloma: a dose-
escalation, open-lable, pharmacokinetic, phase 1 study. Lancet
Haematol. 2016, 3, e196−e204.
(30) Fiskus, W.; Sharma, S.; Qi, J.; Shah, B.; Devaraj, S. G. T.;
Leveque, C.; Portier, B. P.; Iyer, S.; Bradner, J. E.; Bhalla, K. N. BET
protein antagonist JQ1 is synergistically lethal with FLT3 tyrosine
kinase inhibitor (TKI) and overcomes resistance to FLT3-TKI in
AML cells expressing FLT-ITD. Mol. Cancer Ther. 2014, 13, 2315−
2327.
(31) Picaud, S.; Da Costa, D.; Thanasopoulou, A.; Filippakopoulos,
P.; Fish, P. V.; Philpott, M.; Fedorov, O.; Brennan, P.; Bunnage, M.
E.; Owen, D. R.; Bradner, J. E.; Taniere, P.; O’Sullivan, B.; Muller, S.;
Schwaller, J.; Stankovic, T.; Knapp, S. PFI-1, a highly selective protein
G
https://dx.doi.org/10.1021/acsmedchemlett.9b00625
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX