Discovery of High-Affinity Inhibitors of the BPTF Bromodomain

Article abstract of DOI:10.1021/acs.jmedchem.1c00721

The dysfunctional bromodomain PHD finger transcription factor (BPTF) exerts a pivotal influence in the occurrence and development of many human diseases, particularly cancers. Herein, through the structural decomposition of the reported BPTF inhibitor TP-

Full text of DOI:10.1021/acs.jmedchem.1c00721

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Discovery of High-Affinity Inhibitors of the BPTF Bromodomain
Published as part of the Journal of Medicinal Chemistry special issue “Epigenetics 2022”.
Tian Lu,∇ Haibo Lu,∇ Zhe Duan,∇ Jun Wang, Jie Han, Senhao Xiao, HuanHuan Chen, Hao Jiang,
Yu Chen, Feng Yang, Qi Li, Dongying Chen, Jin Lin, Bo Li, Hualiang Jiang, Kaixian Chen, Wenchao Lu,*
Hua Lin,* and Cheng Luo*
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ABSTRACT: The dysfunctional bromodomain PHD finger
transcription factor (BPTF) exerts a pivotal influence in the
occurrence and development of many human diseases, particularly
cancers. Herein, through the structural decomposition of the
reported BPTF inhibitor TP-238, the effective structural fragments
were synthetically modified to obtain our lead compound DC-BPi-
03. DC-BPi-03 was identified as a novel BPTF-BRD inhibitor with
a moderate potency (IC50 = 698.3 21.0 nM). A structure-guided
structure−activity relationship exploration gave rise to two BPTF
inhibitors with much higher affinities, DC-BPi-07 and DC-BPi-11.
Notably, DC-BPi-07 and DC-BPi-11 show selectivities 100-fold
higher than those of other BRD targets. The cocrystal structures of
BPTF in complex with DC-BPi-07 and DC-BPi-11 demonstrate
the rationale of chemical efforts from the atomic level. Further study showed that DC-BPi-11 significantly inhibited leukemia cell
proliferation.
observations indicate that BPTF could be an important
therapeutic target in cancer treatment.
INTRODUCTION
■
Being the core subunit of the ATP-dependent nucleosome
remodeling factor (NuRF) complex,1 BPTF has 3046 amino
acid residues in total, including a bromodomain and two plant
homeodomain (PHD) finger domains commonly found in
histone and DNA-binding proteins.2 As a histone-binding
component of NuRF, BPTF functionalizes the catalysis of
ATP-dependent nucleosome sliding and the promotion of
chromatin transcription.3 BPTF is a BRD-containing protein,
which has been classified as an important research target of the
non-BET family.4 It plays an essential role in remodeling
chromatin,5 regulating transcription,6 cell differentiation,7 and
embryogenesis.8
So far, many effective and specific inhibitors of BET family
proteins have been discovered and widely used for the
treatment of various cancers.18−22 Meanwhile, more and
more attention has been paid to the discovery and develop-
ment of non-BET bromodomain inhibitors.23 Inspiringly, the
CBP/p300-BRD inhibitor CCS1477,24 which was developed
by Cell Centric, is the first non-BET small-molecular inhibitor
that has entered clinical trials, demonstrating that targeting the
non-BET family could provide more therapeutic options.
Although a variety of inhibitors of BPTF-BRD have been
reported (Figure 1), the discovery of selective BPTF-BRD
inhibitors with high affinities is conspicuously lacking. Among
them, Mishra et al. reported, in 2014 that the Plk1 inhibitor
Recently, evidence showed that the dysfunction of BPTF
may lead to the occurrence and development of malignant
tumors,9 including melanoma,10 leukemia,11 colorectal can-
cer,12 and bladder cancer.13 BPTF is overexpressed in a variety
of tumor tissues compared with its levels in normal tissue cells,
which promotes the proliferation of cancer cells, and is an
important prognostic marker in melanoma, lung cancer, and
leukemia.10 BPTF can interact with c-Myc in oncogenic
chromatin remodeling.5,14,15 Upon the silencing of BPTF, c-
BI2536 can bind to 5FW-BPTF (KD = 37 μM) through the 19
F
NMR method.9 In 2015, Andrew K. Urick et al. identified the
first BPTF-BRD small-molecule inhibitor that showed a
Received: April 21, 2021
Published: August 10, 2021
Myc transcriptional activation is completely interrupted.11,16
A
knockdown of BPTF can also enhance antitumor immunity
mediated by CD8+ T cells or NK cells.17 These previous
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Figure 1. Representative BPTF-BRD inhibitors.
Figure 2. The discovery and preliminary validation of the hit compound DC-BPi-03. (A−C) The structures and biochemical inhibitory activities of
hit compounds DC-BPi-01−DC-BPi-03. (D) The melting curves of BPTF-BRD with DC-BPi-03 at different concentrations. Fluorescence signals
from protein thermal shift assays were normalized and plotted as a function of the temperature. (E) SPR assay demonstrating the binding between
DC-BPi-03 and BPTF-BRD.
selectivity over BRD4, namely, AU1, using protein-observed
cells using a BPTF-specific reporter assay.26 In 2016, Sarah et
al. reported a broad-spectrum inhibitor, bromosporine, that
fluorine NMR.25 AU1 was further demonstrated to be active in
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Figure 3. The cocrystal structure of BPTF-BRD with DC-BPi-03. (A) Illustration of BPTF-BRD in complex with DC-BPi-03 (PDB ID 7F5D). (B)
The overlap between the acetylated H4 peptide binding pocket (PDB ID 3QZS)1 and the DC-BPi-03 binding site. (C) The 2FO − FC electron
density map for DC-BPi-03 contoured at 1.0σ. (D and E) Residues involved in BPTF and DC-BPi-03 recognition. Important residues involved in
the binding between DC-BPi-03 and BPTF are shown in stick structures. (F) Potential optimization direction for DC-BPi-03.
also inhibits BPTF (KD = 1.8 μM).27 In 2017, GSK discovered
the PCAF and GCN5 inhibitor GSK4027. The Ki values for
PCAF and GCN5 are both 1.4 nM, and it was found that
GSK4027 can also bind to BPTF with a Ki value of 130 nM.28
In 2018, the Structural Genomics Consortium (SGC) showed
on its official website the non-peer-reviewed compound TP-
238, which has a higher activity (KD = 120 nM). Novartis
collaborated with the SGC to develop NVS-BPTF-1 as a BPTF
inhibitor with IC50 = 71 nM. Previously, by using virtual
screening, our group also identified BPTF-BRD inhibitors that
were alkoxy benzamide derivatives.29 In 2020, the Pomerantz
group reported compound 8, which is similar in structure to
GSK4027 but has an equilibrium dissociation constant (KD) of
03 as the lead compound and successfully obtained a high-
resolution cocrystal structure, which provided a starting point
for guiding the next step of structural modification. Through
structural biology and subsequent rational medicinal chemistry
design, we discovered two more potent derivatives, DC-BPi-07
and DC-BPi-11. We then successfully obtained two cocrystal
structures of DC-BPi-07 and DC-BPi-11 with BPTF-BRD.
Furthermore, DC-BPi-11 effectively inhibited the proliferation
of human leukemia MV-4-11 cells through downregulating
downstream oncogene expression while exhibiting minimal
effects on normal cells, demonstrating its safety. These results
indicated that this novel BPTF-BRD inhibitor is a promising
molecule for further optimization and development as a
therapy for BPTF-related cancers.
6.0
2 μM as determined by surface plasmon resonance
technology (SPR). Additionally, the team used SPR to retest
the compounds TP-238 and GSK4027, with KD values of 200
nM and 1.7 μM, respectively.30 There is a highly urgent need
to discover novel BPTF inhibitors along with a cocrystal
structure that may facilitate further medicinal chemistry
optimization and function as chemical probes for BPTF-
BRD-related biological studies.
In the present work, based on the known BPTF inhibitor
TP-238 we fragmentized its structure and demonstrated that
its effective structural fragment is the DC-BPi-01 compound.
We further modified the benzene ring structure of its side
chain. The DC-BPi-02 and DC-BPi-03 compounds were
modified and synthesized, and the indole structure of the
DC-BPi-03 compound was more active. We selected DC-BPi-
RESULTS AND DISCUSSION
■
Discovery of DC-BPi-03 through the Screening of
Privileged Structural Fragments. Previously, our group
applied computer-aided virtual screening to find potential
BPTF inhibitors and established a stable and reproducible
detection method based on HTRF for compound character-
ization.29 Herein, we analyzed the structure of the BPTF
inhibitor TP-238, which was published on the SGC Web site,
and decomposed it into fragments to identify privileged
substructures and motifs. The results showed that DC-BPi-01
is an effective fragment that binds to BPTF (Figure 2A).
Further structural decoration led to the discovery of DC-BPi-
02 and DC-BPi-03 with improved activities, among which DC-
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a
Scheme 1. Synthesis of BPTF Compounds
a
Reaction conditions are as follows: (a) halide, NaH, DMF, 0 °C-80 °C; (b) B2Pin2, KOAc, Pd(dppf)Cl2, 1,4-dixoane, 90 °C; (c) amine, Et3N,
DMSO, 60 °C; (d) m-CPBA, DCM, 0 °C to rt; (e) Pd(PPh3)4, Na2CO3, 1,4-dixoane/H2O, 90 °C; (f) (Boc)2O, NaH, THF, 0 °C to rt; (g)
Pd(dppf)Cl2, K3PO4, 1,4-dixoane/H2O, 90 °C.
BPi-03 with an indole scaffold showed an optimal inhibitory
activity with an IC50 value of 698.3 21.0 nM (Figure 2B and
C). To further confirm the binding between BPTF-BRD and
DC-BPi-03, we next carried out protein thermal shift assays.
Compared to that of the DMSO control, the melting
temperature of BPTF-BRD evidently increased upon the
addition of DC-BPi-03, indicating that the ligand may interact
with BPTF-BRD and strengthen its thermal stability in vitro
(Figure 2D).
We also used SPR to confirm the binding between DC-BPi-
03 and BPTF-BRD. DC-BPi-03 directly bound to BPTF-BRD
with a KD value of 2.8 μM, which was consistent with in vitro
HTRF data (Figure 2E). These results suggested DC-BPi-03
indeed binds to BPTF-BRD in vitro.
Chemistry and Structure−Activity Relationship
Study. Guided by the cocrystal structure in hand, we designed
and synthesized a series of derivatives (Scheme 1).
Commercially available 5-bromo indole or 6-bromo indole 1
was alkylated by the corresponding halide in the presence of
NaH to provide N-alkyl products 2, which were coupled with
B2Pin2 to give rise to intermediate 3. Commercially available
pyrimidine 4 was reacted with the corresponding amines,
following oxidization by m-CPBA, to afford methylsulfonyl or
methylsulfinyl products 6. The coupling of 3 and 6 gave DC-
BPi-1−10 and DC-BPi-12−16 in moderate yields. Commer-
cially available 7H-pyrrolo[2,3-d]pyrimidine 7 was Boc
protected before being oxidated to its corresponding
methylsulfonyl intermediate 9. Then, the intermediate 9 was
coupled with 3 to form DC-BPi-11 and DC-BPi-17.
Cocrystal Structure Determination. To explore the
molecular basis of the inhibitory activity of DC-BPi-03 against
BPTF-BRD for further chemistry optimization, we obtained
high-resolution cocrystal structures of BPTF-BRD with DC-
BPi-03 at 1.58 Å to illustrate the structural basis for the binding
of BPTF-BRD with DC-BPi-03 (PDB ID 7F5D). The result
showed the very complete electron density of DC-BPi-03 in
the substrate pocket (Figure 3A−C). DC-BPi-03 fit well within
the histone binding pocket of BPTF-BRD and formed various
hydrogen bonds and hydrophobic interactions with neighbor-
ing residues (Figure 3D). The sulfonyl group and the N-
methylpiperidine group formed tridentate hydrogen bonds
with the primary amide group of residue N148, which was
previously found to be a crucial residue in the recognition of
acetylated lysine.20 DC-BPi-03 also formed a pair of water-
bridged hydrogen bonds with Y105, indicating the important
role of structural water within the binding pocket. In addition,
DC-BPi-03 formed a variety of hydrophobic interactions with
adjacent residues in the crystal structure, including W91, P92,
F93, V97, Y147, and F154, through the N-methylpiperidine
group and the cyclopentyl group (Figure 3E). These
interactions were the dominant driven forces for the binding
between BPTF and DC-BPi-03.
According to the cocrystal structure of DC-BPi-03 and the
BPTF protein, there was a negatively charged channel below
the indole group and an extra pocket near the pyrimidin-4-
amine group (Figure 3F). Thus, the incorporation of an
alkylamine substitution to the indole nitrogen group might
introduce new hydrogen bonds. Both N,N-dimethyl ethyl
amine and propyl amine exhibited a 20-fold increase of their
activities (DC-BPi-04 and DC-BPi-05, respectively). These
results suggested that a new hydrogen bonding interaction was
formed, which enhanced the binding affinity. Prolonging of the
alkyl substitution at the pyrimidin-4-amine group in the
bottom part might benefit hydrophobic interactions between
the alkyl group and surrounding residues. An ethyl or propyl
substituent showed more potency (DC-BPi-06 or DC-BPi-07,
respectively). However, the n-butyl substituent was too long
for the activity (DC-BPi-08). The branched alkyl group was
too bulky (DC-BPi-09), which decreased the activity. The free
NH of the pyrimidine ring was critical for the potency due to it
forming a hydrogen bond with residue N148. Thus, it is
rational to find the N,N-dimethyl group substituent diminished
the activity (DC-BPi-10). Interestingly, 7H-pyrrolo[2,3-d]-
pyrimidine was well tolerated with an IC50 value at 26.9 3.2
nM (DC-BPi-11). Mono-oxidated compounds exhibited less
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Figure 4. The cocrystal structures of DC-BPi-07 and DC-BPi-11 with BPTF-BRD. (A and B) Crystal structures of BPTF-BRD with DC-BPi-07
(PDB ID 7F5C) and DC-BPi-11 (PDB ID 7F5E). The 2FO − FC electron density maps of DC-BPi-07 and DC-BPi-11 are contoured at 1.0σ. (C)
The alignment of the binding conformation of DC-BPi-03 with DC-BPi-07. DC-BPi-03 and DC-BPi-07 are depicted as yellow and salmon sticks,
respectively. (D) The binding pocket of BPTF for DC-BPi-07 and DC-BPi-11. (E) Residues involved in BPTF inhibitor recognition. (F) The
cocrystal structure of DC-BPi-07 and BPTF-BRD (PDB ID 7F5C) shows important hydrogen bonding and hydrophobic interactions between the
ligand and the receptor.
potency in two cases (DC-BPi-12 and DC-BPi-13). A 60-fold
potency loss occurred when the pyrimidine moiety was moved
from the 6 position to the 5 position of indole ring (DC-BPi-
14). It suggested the loss of a hydrogen bond interaction due
to a mismatched conformation. Little negative effect was
observed when a different substituent was installed to the
indole ring or the 3 or 7 positions (DC-BPi-15−17,
respectively). These results indicated that the indole ring
could tolerate some substituents.
Cocrystal Structure of BPTF in Complex with
Compounds. To further clarify the molecular basis of the
improved activities of DC-BPi-07 and DC-BPi-11, we
successfully cocrystallized the BPTF with DC-BPi-07 and
DC-BPi-11 (Figure 4A and B, respectively, PDB IDs 7F5C and
7F5E, and Tables S2 and S3). DC-BPi-07 and DC-BPi-11 were
inserted deeply into the pocket and occupied the negatively
charged binding site of the acetylated H4 peptide with slight
conformation changes comparing with DC-BPi-03 (Figure 4C
and D, respectively). DC-BPi-07 formed direct and water-
bridged hydrogen bonds and hydrophobic contacts with
surrounding residues within the binding pocket of the BPTF
bromodomain, as DC-BPi-03 did (Figure 4D). Direct
hydrogen bonds were observed between DC-BPi-07 and DC-
BPi-11 and N148 and C144, while water-bridged hydrogen
bonds were formed with Y105 (Figure 4E and F). As expected,
the free NH of the pyrimidine moiety of DC-BPi-07 and DC-
BPi-11 formed an important hydrogen bond interaction with
the aspartic acid carbonyl group of N148. Importantly, another
hydrogen bond was found between the N,N-dimethyl alkyl-
amine group of DC-BPi-07 and DC-BPi-11 with D101. The
conformation of W91 was slightly shifted due to the better
occupancy of DC-BPi-07, demonstrating the rationale of
chemical efforts on the indole group (Figure 4E). In addition
to polar interactions, DC-BPi-07 and DC-BPi-11 formed
hydrophobic interactions with residues P92, V97, Y105, and
F154. When the hydrogen on the free NH of the pyrimidine
part of the compound is replaced, the activity is greatly
reduced, such as for DC-BPi-10.
Biophysical Binding Assays. Table 1 and Figure S1
summarized the structures and their inhibitory activities on
BPTF-H4. Among them, the derivatives DC-BPi-07 and DC-
BPi-11 had the best inhibitory activities, with IC50 values of
16.0 5.0 and 26.9 3.2 nM, respectively (Figure 5A and D,
respectively). To investigate interactions between DC-BPi-07,
DC-BPi-11, and the BPTF bromodomain, a protein thermal
shift assay was similarly carried out. As the concentrations of
DC-BPi-07 and DC-BPi-11 increased, the thermostability of
BPTF-BRD also increased accordingly (Figure 5B and E,
respectively). Besides, in SPR assays DC-BPi-07 and DC-BPi-
11 showed equilibrium dissociation constant (KD) values of
49.6 and 20.7 nM, respectively. The values were consistent
with their inhibitory activities in the HTRF assays (Figure 5C
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Table 1. Chemical Modifications on DC-BPi-03
and F). To confirm the binding between DC-BPi-11 and
BPTF, both Carr−Purcell−Meiboom−Gill (CPMG) and
saturation transfer difference (STD) NMR experiments were
performed. As shown in Figure S2, a strong signal in the STD
NMR experiment and a significantly decreased signal in the
CPMG spectrum were detected under the conditions of 5 μM
BPTF and 200 μM DC-BPi-11, indicating the mutual binding
between BPTF-BRD and the compound. Collectively, these
biophysical evidence prove that DC-BPi-11 bound to the
BPTF bromodomain in vitro.
BRD at a concentration of 1 μM. DC-BPi-11 exhibited
complete inhibition against BPTF-BRD, while it displayed
minimal activity against the other bromodomains (Figure 5G).
The selectivity was also validated using protein thermal shift
assays. The shift of the Tm value of BPTF-BRD with the DC-
BPi-11 treatment was 8.0 °C, while the Tm shift was relatively
much smaller for other bromodomain targets (Figure 5H). In
summary, DC-BPi-11 is the effective BPTF inhibitor, which is
a promising starting point for structural optimization in the
future.
Selectivity Profiling. The selectivity of our compounds
over the BRDs family in vitro was further profiled, considering
that the structures of bromodomains in BRD families are
highly conserved.29 We tested the inhibitory activities of DC-
BPi-11 using AlphaScreen against BRD4(1), BRD4(2),
BRD2(1), BRD2(2), CBP-BRD, P300-BRD and SMARCA2-
We then aligned the available crystal structures of different
BRDs to BPTF-DC-BPi-11 to investigate the molecular basis
of the selectivity (Figure S3). We found that the aspartic acid
residue D101 in BPTF that formed the hydrogen bond with
the positively charged N,N-dimethyl alkylamine group of DC-
BPi-11 was replaced by the conservative leucine in all other
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Figure 5. Biochemical and biophysical methods demonstrated the direct binding between DC-BPi-07 and DC-BPi-11 and BPTF-BRD. (A)
Inhibitory activity of DC-BPi-07 against BPTF-BRD. (B) Changes in the thermodynamic stability of BPTF-BRD upon binding of DC-BPi-07. (C)
SPR assay of DC-BPi-07. (D) Inhibitory activity of DC-BPi-11 against BPTF-BRD. (E) Changes in the thermodynamic stability of BPTF-BRD
upon binding of DC-BPi-11. (F) SPR assay of DC-BPi-11. (G) In vitro inhibitory activity of compound DC-BPi-11 against BRD proteins. (H) The
thermal stability effect of DC-BPi-11 on BRD proteins. The concentration of proteins was 5 μM, and the concentration of the compound was 50
μM.
bromodomains. Additionally, the hydrophobic residue F154 of
BPTF-BRD was replaced with the valine residue in other
BRDs, demonstrating the importance of the π−π stacking
effect in specific recognition of DC-BPi-11.
derivatives. Considering that BPTF is a key cofactor for c-Myc
transcriptional activity, the well-characterized undruggable
oncogene is essential for the progression of a variety of
malignant diseases.14 As shown in Figure 6B, DC-BPi-11
demonstrated a potent antiproliferative effect in MV-4-11
leukemia cells, with a concentration-dependent inhibitory
activity of 0.89 μM. Meanwhile, two normal human endothelial
cell lines HUV-EC-C and MRC-5 were also tested. After three
days of treatment, the proliferation rate was measured using
the CellTiter-Glo assay. Notably, the results showed that DC-
BPi-11 had almost no inhibitory effect on the growth of normal
cells even at 100 μM (Figure S4). The downregulation of c-
Myc was confirmed by Western blotting (Figure 6C). These
results indicated that the antiproliferative effect of our
compounds was, at least in part, due to the inhibition of
BPTF in leukemia cell lines. Based on these data, DC-BPi-11
Cell-Based Activity. To demonstrate that the identified
BPTF inhibitor could directly engage the target in cells, we
developed a NanoBRET assay to quantify the binding of the
histone H4-HaloTag to the NanoLuc-tagged BPTF-BRD. The
EC50 value of DC-BPi-11 is 120 nM, which also demonstrates
its good target engagement and cell permeability (Figure 6A).
Previous evidence demonstrated that the knockdown of
BPTF leads to impaired activation of the c-Myc signal in human
foreskin fibroblasts.14 Numerous clinical sample analysis also
indicated the higher expression level and mutation rate in
melanoma and leukemia cell lines.10,31,32 Thus, we further
explored the therapeutic potential of DC-BPi-03 and its
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Figure 6. Cellular effect of DC-BPi-11 in MV-4-11 leukemia cells. (A) The intracellular NanoBRET experiment verifies the inhibitory effect of DC-
BPi-11 on BPTF. (B) Compound effect on the proliferation of MV-4−11 cancer cells. (C) Depletion of c-Myc in a dose-dependent manner after
compound treatment for 24 h. (D) Inhibition of the transcription of BPTF downstream genes after DC-BPi-11 treatment for 24 h at the different
concentrations. (E and F) Cell cycle arrest and apoptosis were detected by flow cytometry after treatment with DC-BPi-11 for 24 h in MV-4-11
cells.
and its derivatives could be applied as promising therapeutic
small molecules for the treatment of leukemia via targeting the
BPTF-Myc axis.
mechanism of oncogenes such as c-Myc has been investigated
thoroughly, it has long been considered untargetable.
Bromodomain inhibitors now make it possible to target c-
Myc indirectly to treat tumors. Many BET inhibitors have
shown an impressive efficacy in preclinical and clinical studies.
However, an exploration of novel small-molecule inhibitors
targeting BPTF-Myc axis is particularly lacking.
Downregulation of the Expression of BPTF Target
Genes. BPTF plays a crucial role in the transcriptional
regulation of several oncogenes, including c-Myc,14 Bcl-2, and
CDK6.10 Thus, RT-PCR experiments were performed to
evaluate the effect of DC-BPi-11 on target gene expression at
the transcriptional level. Remarkable dose-dependent decreases
in the expression of target genes were observed at the mRNA
level, including that of c-Myc, Bcl2, and CDK6 (Figure 6D).
Cell Cycle and Apoptosis. Emerging evidence demon-
strated that the silencing of BPTF will effectively arrest the cell
cycle at the G1 phase and shorten the S phase in melanoma
and mouse embryonic fibroblasts (MEFs).2,14 Thus, we tried
to further explore if DC-BPi-11 could affect the cell cycle in
MV-4-11 cells.33 When treated with DC-BPi-11 for 24 h, a
fraction of MV-4-11 cells were arrested in the G1 or S phase in
a dose-dependent manner, which is consistent with genetic
studies (Figure 6E).14 To determine the cell-killing effect of
DC-BPi-11, we further investigated the effect of DC-BPi-11 on
apoptosis. After treatment with DC-BPi-11, cells were stained
with Annexin V FITC and PI, and apoptotic levels were
measured using flow cytometry. Treatment with DC-BPi-11
for 24 h could significantly induce apoptosis in MV-4-11
leukemia cells (Figure 6F).
Herein, the discovery and optimization of high-affinity and
selective BPTF-BRD inhibitors have been described. Through
structural decomposition, we identified the privileged fragment
DC-BPi-03 that could bind to BPTF. A variety of biochemical
and crystallographic evidence demonstrated the binding
between BPTF-BRD and DC-BPi-03. Through a combination
of crystal structure analysis and rational chemical optimization,
we increased the potency of BPTF-BRD inhibitors by more
than 40−26-fold, resulting in the highly potent compounds
DC-BPi-07 and DC-BPi-11. Based on the high-resolution
crystal structure, DC-BPi-07 and DC-BPi-11 occupy the
hydrophobic groove in the substrate pocket. The optimal
compound in this study, DC-BPi-11, is a potent inhibitor of
BPTF-BRD (KD = 20.7 nM) and is selective over the other
BRD family members screened. In the cell model of MV-4-11
leukemia cells, DC-BPi-11 reduced proliferation, blocked the
cell cycle, and induced apoptosis, which is consistent with
previous observations in BPTF genetic studies.34 DC-BPi-11 is
expected to enable the further deeper investigation of the role
of BPTF-BRD in transcriptional regulation through chemical-
genetic dissection. DC-BPi-11 could also serve to validate the
potential of BPTF-BRD inhibitors in the treatment of cancers
related to c-Myc transcription and could serve as a starting
point for developing more bioavailable clinical candidates.
CONCLUSION
■
The close correlation between BPTF dysfunction and proto-
oncogene signaling activation in human tumorigenesis suggests
that a multitude of tumors may be affected by the therapeutic
disruption of the BPTF-Myc axis. Although the pathogenesis
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(3 equiv) at 0 °C. The mixture was stirred at rt for 3 h under a N2
atmosphere. The reaction solution was poured into ice water. The
mixture was extracted with EtOAc, washed with brine, and dried over
anhydrous Na2SO4. The organic phase was filtered, concentrated, and
purified by column chromatography to give compound 8 as the
desired product.
General Synthesis Procedure for 9. To a solution of 8 (1 equiv) in
DCM (0.1 mM) was added m-CPBA (3 equiv) at 0 °C. The mixture
was stirred at rt for 2 h under a N2 atmosphere before being quenched
with DCM. The mixture was washed with sodium sulfate (sat. aq.), a
NaOH solution (1 M), and brine and then dried over anhydrous
Na2SO4. The organic phase was filtered, concentrated, and purified by
column chromatography to give compound 9 as the desired product.
General Synthesis Procedure for DC-BPi-12 and DC-BPi-16. To
a solution of 9 (1 equiv) and 3 (0.9 equiv) in 1,4-dioxane/H2O (10/
1, 0.1 mM) were added K3PO4 (3 equiv) and Pd(dppf)Cl2 (0.1
equiv). The mixture was purged with N2 and stirred at 90 °C for 12 h.
The mixture was filtered and purified by column chromatography or
preparative TLC to give the desired product.
EXPERIMENTAL PROCEDURES
■
Chemistry. All solvents and chemicals were of reagent grade.
Unless otherwise mentioned, all reagents and solvents were purchased
from commercial vendors and used as received. Flash column
chromatography was carried out on a Teledyne ISCO CombiFlash Rf
system using prepacked columns. Solvents used include petroleum
ether (PE), ethyl acetate (EtOAc; EA), dichloromethane (DCM), and
methanol. The purity and characterization of compounds were
established by a combination of HPLC, thin-layer chromatography
(TLC), mass spectrometry, and NMR analyses. H and 13C NMR
1
spectra were recorded on a Bruker AVANCE DPX-400 (400 MHz)
spectrometer, a Bruker UltraShield 500 Plus spectrometer, and an
AVANCE III 600 (600 MHz) spectrometer and were determined in
chloroform-d or methanol-d4 with solvent peaks as the internal
reference. Chemical shifts are reported in parts per million (ppm)
relative to the reference signal, and coupling constant (J) values are
reported in hertz (Hz). TLC was performed on EMD precoated silica
gel 60 F254 plates, and spots were visualized with UV light or iodine
staining. Low-resolution mass spectra were recorded using a Thermo
Scientific Ultimate 3000/LCQ Fleet system (ESI). High-resolution
mass spectra were recorded using a Thermo Scientific EXACTIVE
system (ESI). All test compounds were greater than 95% pure as
determined using a Shimadzu HPLC apparatus equipped with a
photodiode array detector (Kyoto, Japan). Chromatographic
separation was carried out using a YMC-Triart C18 column (150
mm × 4.6 mm, 3 μm) at 35 °C. The mobile phase consisted of a 5
mM ammonium carbonate aqueous solution (eluent A) and
acetonitrile (eluent B) with a gradient elution of 0−5 min 30% B,
5−15 min 30−90% B, 15−25 min 90% B, 25−27 min 90−30% B, and
27−35 min 30% B. The flow rate was 1.0 mL·min −1, the injection
volume was 10 μL, and the detection wavelength was set at 254 nm.
Data were acquired and processed using a LabSolutions software (ver.
5.51).
General Synthesis Procedure for 2. To a solution of 1 (1 equiv)
and halide (0.9 equiv) in anhydrous DMF (0.1 mM) was added NaH
(2 equiv) at 0 °C. Then, the mixture was stirred at 80 °C for 3 h
under a N2 atmosphere. The reaction solution was poured into ice
water. The mixture was extracted with EtOAc, washed with brine, and
dried over anhydrous Na2SO4. The organic phase was filtered,
concentrated, and purified by column chromatography to give
compound 2 as the desired product.
General Synthesis Procedure for 3. To a solution of 2 (1 equiv) in
anhydrous 1,4-dioxane (0.1 mM) were added B2Pin2 (1.2 equiv),
KOAc (2 equiv), and Pd(dppf)Cl2 (0.1 equiv). The mixture was
purged with N2 for 5 min and then stirred at 90 °C for 8 h. The
reaction solution was filtered, concentrated, and purified by column
chromatography to give compound 3 as the desired product.
General Synthesis Procedure for 5. To a solution of 4 (1 equiv)
and amine (2 equiv) in DMSO (0.2 mM) was added Et3N (3 equiv).
The mixture was stirred at 60 °C for 6 h under a N2 atmosphere
before being diluted with EtOAc. The mixture was washed with water
and brine and then dried over anhydrous Na2SO4. The organic phase
was filtered and concentrated to use for the next step.
N-Methyl-2-(methylsulfonyl)-6-phenylpyrimidin-4-amine (DC-
BPi-01). Pale white solid (56 mg, 68.5% yield). 1H NMR (400
MHz, CDCl3, methanol-d4) δ 7.93 (brs, 2H), 7.41 (brs, 3H), 6.76 (s,
1H), 6.52 (brs, 1H), 3.29 (s, 3H), 2.97 (d, J = 4.5 Hz, 3H). 13C NMR
(151 MHz, CDCl3, methanol-d4) δ 164.5, 135.8, 130.8, 128.7, 126.9,
38.7. HRMS (ESI) m/e calc’d for C12H14N3O2S (M + H)+ 264.0807,
found 264.0799.
N-Methyl-2-(methylsulfonyl)-6-(quinolin-5-yl)pyrimidin-4-amine
1
(DC-BPi-02). Pale white solid (45 mg, 48.5% yield). H NMR (600
MHz, methanol-d4) δ 8.88 (d, J = 3.1 Hz, 2H), 8.71 (d, J = 8.5 Hz,
1H), 8.13 (d, J = 8.3 Hz, 1H), 7.89−7.74 (m, 2H), 7.56 (dd, J = 8.6,
4.2 Hz, 1H), 6.86 (d, J = 21.0 Hz, 1H), 3.34 (s, 2H), 3.06 (s, 3H). 13C
NMR (126 MHz, methanol-d4) δ 165.8, 164.6, 162.6, 150.4, 147.8,
136.2, 135.1, 130.1, 129.3, 128.2, 126.3, 121.9, 108.3, 38.1, 26.8.
HRMS (ESI) m/e calc’d for C15H15N4O2S (M + H)+ 315.0916, found
315.0902.
6-(1H-Indol-5-yl)-N-methyl-2-(methylsulfonyl)pyrimidin-4-
amine (DC-BPi-03). Pale white solid (83 mg, 28.3% yield). 1H NMR
(600 MHz, CDCl3, MeOD) δ 8.35 (s, 1H), 7.84 (s, 1H), 7.46 (d, J =
12.2 Hz, 1H), 7.29 (d, J = 4.8 Hz, 1H), 7.00 (s, 1H), 6.56 (d, J = 4.8
Hz, 1H), 3.36 (s, 3H), 3.02 (s, 3H). 13C NMR (126 MHz, CDCl3,
MeOD) δ 164.6, 137.9, 128.2, 127.1, 125.8, 120.2, 119.8, 111.3,
102.4, 38.4. HRMS (ESI) m/e calc’d for C14H14N4O2S (M + H)+
303.0916, found 303.0912.
6-(1-(3-(Dimethylamino)propyl)-1H-indol-5-yl)-N-methyl-2-
(methylsulfonyl)pyrimidin-4-amine (DC-BPi-04). Pale yellow solid
(65 mg, 54.1% yield). 1H NMR (600 MHz, methanol-d4) δ 8.29 (brs,
1H), 7.82 (brs, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 3.6 Hz,
1H), 6.94 (s, 1H), 6.53 (d, J = 2.4 Hz, 1H), 4.20 (t, J = 7.2 Hz, 2H),
3.34 (s, 3H), 2.97 (s, 3H), 2.38−2.36 (m, 2H), 2.27 (s, 6H), 2.04−
1.99 (m, 2H). 13C NMR (126 MHz, methanol-d4) δ 165.5, 164.6,
162.8, 137.6, 129.1, 128.9, 127.1, 120.1, 109.3, 102.1, 101.3, 56.0,
43.8, 43.6, 37.9, 27.3, 26.6. HRMS (ESI) m/e calc’d for C19H26N5O2S
(M + H)+ 388.1807, found 388.1807.
6-(1-(2-(Dimethylamino)ethyl)-1H-indol-5-yl)-N-methyl-2-
(methylsulfonyl)pyrimidin-4-amine (DC-BPi-05). Pale yellow solid
(41 mg, 26.7% yield). 1H NMR (600 MHz, Chloroform-d) δ 8.32 (s,
1H), 7.86 (brs, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 2.4 Hz,
1H), 6.79 (s, 1H), 6.57 (brs, 1H), 5.94−5.67 (m, 1H), 4.23 (t, J = 6.6
Hz, 2H), 3.38 (s, 3H), 3.01 (d, J = 5.4 Hz, 3H), 2.71 (t, J = 7.1 Hz,
2H), 2.29 (s, 6H), 2.11 (brs, 2H). 13C NMR (126 MHz, chloroform-
d) δ 165.4, 164.5, 137.7, 129.4, 1288, 127.3, 120.7, 109.5, 102.7, 58.9,
45.7, 44.8, 38.9, 28.5. HRMS (ESI) m/e calc’d for C18H24N5O2S (M +
H)+ 374.1651, found 374.1647.
General Synthesis Procedure for 6. To a solution of 5 (1 equiv) in
DCM (0.1 mM) was added m-CPBA (n = 1, 1 equiv; n = 2, 3 equiv)
at 0 °C. The mixture was stirred at rt for 2 h under a N2 atmosphere
before being diluted with DCM. The mixture was washed with
sodium sulfate (sat. aq.), a NaOH solution (1 M), and brine and then
dried over anhydrous Na2SO4. The organic phase was filtered,
concentrated, and purified by column chromatography to give
compound 6 as the desired product.
General Synthesis Procedure for DC-BPi-01−10 and DC-BPi-
12−16. To a solution of 3 (1 equiv) in 1,4-dioxane/H2O (10:1) (0.1
mM) were added 6 (0.9 equiv), Na2CO3 (3 equiv), and Pd (PPh3)4
(0.1 equiv). The mixture was purged with N2 and stirred at 90 °C for
12 h. The mixture was filtered and purified by column
chromatography or a silica gel plate to give the desired product.
General Synthesis Procedure for 8. To a solution of 7 (1 equiv)
and Boc2O (2 equiv) in anhydrous THF (0.1 mM) was added NaH
6-(1-(3-(Dimethylamino)propyl)-1H-indol-5-yl)-N-ethyl-2-
(methylsulfonyl)pyrimidin-4-amine (DC-BPi-06). Pale yellow solid
(60 mg, 48.2% yield). 1H NMR (600 MHz, methanol-d4) δ 8.30 (brs,
1H), 7.84 (brs, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 3.0 Hz,
1H), 6.95 (s, 1H), 6.53 (d, J = 2.4 Hz, 1H), 4.20 (t, J = 6.6 Hz, 1H),
3.49−3.43 (m, 2H), 3.34 (s, 2H), 2.35 (t, J = 7.2 Hz, 1H), 2.25 (s,
3H), 2.03−1.98 (m, 2H), 1.24 (t, J = 7.2 Hz, 1H). 13C NMR (126
12083
https://doi.org/10.1021/acs.jmedchem.1c00721
J. Med. Chem. 2021, 64, 12075−12088

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1
MHz, methanol-d4) δ 165.5, 163.8, 162.9, 137.6, 129.1, 128.9, 127.2,
120.1, 120.0, 109.3, 102.0, 101.3, 56.1, 43.8, 43.6, 37.9, 35.5, 27.4,
13.3. HRMS (ESI) m/e calc’d for C20H28N5O2S (M + H)+ 402.1964,
found 402.1955.
mg, 51.1% yield). H NMR (600 MHz, methanol-d4) δ 8.36 (s, 1H),
7.91 (d, J = 12.6 Hz, 1H), 7.51 (d, J = 12.6 Hz, 1H), 7.29 (d, J = 4.8
Hz, 1H), 6.91 (s, 1H), 6.57 (d, J = 4.8 Hz, 1H), 4.27 (t, J = 10.2 Hz,
2H), 3.45 (brs, 2H), 2.96 (s, 3H), 2.50−2.46 (m, 2H), 2.35 (s, 6H),
2.12−2.04 (m, 2H), 1.91 (s, 1H), 1.66−1.68 (m, 2H), 1.01 (t, J =
10.8 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 172.6, 164.0, 137.6,
129.2, 128.8, 127.9, 120.7, 120.6, 109.6, 102.5, 56.1, 45.0, 44.1, 43.7,
40.1, 27.8, 22.5, 11.5. HRMS (ESI) m/e calc’d for C21H29N5OS (M +
H)+ 400.2171, found 400.2162.
6-(1-(3-(Dimethylamino)propyl)-1H-indol-5-yl)-2-(methylsulfon-
yl)-N-propylpyrimidin-4-amine (DC-BPi-07). Pale yellow solid (58
1
mg, 44.9% yield). H NMR (600 MHz, CDCl3) δ 8.32 (s, 1H), 7.83
(d, J = 5.4 Hz, 1H), 7.38 (d, J = 13.2 Hz, 1H), 7.15 (d, J = 4.2 Hz,
1H), 6.82 (s, 1H), 6.54 (d, J = 4.2 Hz, 1H), 5.73 (s, 1H), 4.20 (t, J =
9.6 Hz, 2H), 3.37 (s, 3H), 3.37 (brs, 1H), 2.29 (t, J = 10.2 Hz, 2H),
2.26 (s, 6H), 1.96−2.04 (m, 2H), 1.61−1.69 (m, 2H), 0.98 (t, J =
11.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 165.6, 163.9, 137.8,
129.4, 128.8, 127.4, 120.7, 109.7, 102.6, 56.1, 45.1, 44.1, 39.0, 27.9,
22.5, 11.5. HRMS (ESI) m/e calc’d for C21H30N5O2S (M + H)+
416.2115, found 416.2111.
6-(1-(3-(Dimethylamino)propyl)-1H-indol-6-yl)-N-methyl-2-
(methylsulfonyl)pyrimidin-4-amine (DC-BPi-14). Pale yellow solid
(25 mg, 20.7% yield). 1H NMR (400 MHz, methanol-d4) δ 8.34 (brs,
1H), 7.65−7.63 (m, 2H), 7.40 (d, J = 2.8 Hz, 1H), 7.07 (s, 1H), 6.54
(d, J = 2.8 Hz, 1H), 4.41 (t, J = 6.8 Hz, 2H), 3.37 (s, 3H), 3.10 (t, J =
8.0 Hz, 2H), 2.10 (s, 3H), 2.81 (s, 6H), 2.34−2.26 (m, 2H). 13C
NMR (151 MHz, methanol-d4) δ 165.6, 164.5, 162.6, 136.1, 131.0,
130.1, 129.3, 120.7, 117.5, 108.5, 101.9, 101.6, 55.2, 48.4, 42.7, 42.3,
37.8, 25.6. HRMS (ESI) m/e calc’d for C19H25N5O2S (M + H)+
388.1807, found 388.1808.
6-(1-(3-(Dimethylamino)propyl)-7-methoxy-1H-indol-5-yl)-2-
(methylsulfonyl)-N-propylpyrimidin-4-amine (DC-BPi-15). Pale yel-
low solid (80 mg, 42.7% yield). 1H NMR (400 MHz, methanol-d4) δ
7.88 (brs, 1H), 7.36 (brs, 1H), 7.15 (d, J = 3.2 Hz, 1H), 6.98 (s, 1H),
6.48 (d, J = 2.8 Hz, 1H), 4.45 (t, J = 6.8 Hz, 2H), 4.01 (s, 3H), 3.41
(brs, 2H), 3.34 (s, 3H), 2.728 (d, J = 7.6 Hz, 1H), 2.54 (s, 6H), 2.15−
2.08 (m, 2H), 1.69−1.61 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H). 13C NMR
(126 MHz, methanol-d4) δ 165.5, 164.0, 162.9, 147.3, 131.1, 130.0,
128.3, 127.0, 113.2, 102.5, 101.5, 100.8, 55.6, 54.7, 46.1, 43.0, 42.3,
37.8, 28.2, 22.1, 10.4. HRMS (ESI) m/e calc’d for C22H32N5O3S (M +
H)+ 446.2226, found 446.2211.
6-(1-(3-(Dimethylamino)propyl)-3-methyl-1H-indol-5-yl)-2-
(methylsulfonyl)-N-propylpyrimidin-4-amine (DC-BPi-16). Pale yel-
low solid (87 mg, 45.9% yield). 1H NMR (600 MHz, methanol-d4) δ
8.21 (brs, 1H), 7.79 (brs, 1H), 7.36 (d, J = 9.0 Hz, 1H), 7.01 (s, 1H),
6.97 (brs, 1H), 4.15 (t, J = 6.6 Hz, 2H), 3.38 (brs, 2H), 3.32 (s, 3H),
3.30−3.29 (m, 1H), 2.83 (t, J = 7.8 Hz, 2H), 2.62 (s, 6H), 2.28 (s,
3H), 2.14−2.09 (m, 2H), 1.64−1.61 (m, 2H), 0.96 (t, J = 7.2 Hz,
2H). 13C NMR (151 MHz, methanol-d4) δ 165.5, 163.9, 163.1, 137.9,
129.0, 126.7, 126.6, 120.2, 118.0, 111.7, 109.1, 101.3, 55.4, 42.6, 42.3,
37.9, 29.4, 25.9, 22.1, 10.5, 8.3. HRMS (ESI) m/e calc’d for
C22H32N5O2S (M + H)+ 430.2277, found 430.2272.
N-Butyl-6-(1-(3-(dimethylamino)propyl)-1H-indol-5-yl)-2-
(methylsulfonyl)pyrimidin-4-amine (DC-BPi-08). Pale yellow solid
1
(72 mg, 37.2% yield). H NMR (600 MHz, methanol-d4) δ 8.34 (s,
1H), 7.91 (s, 1H), 7.53 (d, J = 8.7 Hz, 1H), 7.32 (d, J = 3.1 Hz, 1H),
6.60 (d, J = 3.1 Hz, 1H), 4.32 (t, J = 7.2 Hz, 2H), 3.45 (brs, 3H), 3.35
(s, 3H), 2.79 (t, J = 7.28 Hz, 2H), 2.59 (s, 5H), 2.21−2.16 (m, 2H),
1.65 (brs, 2H), 1.46 (brs, 2H), 0.99 (t, J = 7.2 Hz, 3H). 13C NMR
(126 MHz, CDCl3) δ 165.5, 163.7, 137.5, 129.1, 128.8, 127.6, 120.8,
120.7, 109.6, 103.0, 55.6, 44.0, 43.6, 38.9, 31.2, 29.7, 26.5, 20.1, 13.8.
HRMS (ESI) m/e calc’d for C22H32N5O2S (M + H)+ 430.2277, found
430.2281.
N-(sec-Butyl)-6-(1-(3-(dimethylamino)propyl)-1H-indol-5-yl)-2-
(methylsulfonyl)pyrimidin-4-amine (DC-BPi-09). Pale yellow solid
1
(72 mg, 37.2% yield). H NMR (600 MHz, chloroform-d) δ 8.33
(brs, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 6.0 Hz, 1H), 7.17
(brs, 1H), 6.83 (s, 1H), 6.57 (brs, 1H), 5.52 (brs, 1H), 4.22 (t, J = 7.2
Hz, 2H), 3.38 (s, 3H), 2.36 (t, J = 7.2 Hz, 2H), 2.29 (s, 6H), 2.06−
2.03 (m, 2H), 1.62−1.59 (m, 2H), 1.25 (d, J = 6.6 Hz, 3H), 0.97 (t, J
= 7.8 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 165.6, 163.2, 137.7,
129.3, 128.8, 127.4, 120.6, 109.7, 102.6, 55.9, 48.9, 44.7, 44.1, 38.9,
29.5, 27.53, 20.0, 10.3. HRMS (ESI) m/e calc’d for C22H32N5O2S (M
+ H)+ 430.2277, found 430.2269.
6-(1-(3-(Dimethylamino)propyl)-1H-indol-5-yl)-N,N-dimethyl-2-
(methylsulfonyl)pyrimidin-4-amine (DC-BPi-10). Pale yellow solid
(68 mg, 54.7% yield). 1H NMR (600 MHz, CDCl3) δ 8.36 (d, J = 1.8
Hz, 1H), 7.90 (dd, J = 9.0, 1.8 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.17
(d, J = 3.0 Hz, 1H), 6.90 (s, 1H), 6.58 (d, J = 3.0 Hz, 1H), 4.23 (t, J =
7.2 Hz, 2H), 3.39 (s, 3H), 3.24 (brs, 6H), 2.20−2.25 (m, 2H), 2.22
(s, 6H), 1.97−2.02 (m, 2H); 13C NMR (126 MHz, CDCl3) δ 165.4,
164.8, 163.4, 137.8, 129.4, 128.9, 127.9, 120.8, 120.7, 109.8, 102.5,
98.6, 56.3, 45.5, 44.3, 39.0, 37.7, 28.3; HRMS (ESI) m/e calc’d for
C20H28N5O2S (M + H)+ 402.1958, found 402.1949.
N,N-Dimethyl-3-(5-(2-(methylsulfonyl)-7H-pyrrolo[2,3-d]-
pyrimidin-4-yl)-1H-indol-1-yl)propan-1-amine (DC-BPi-11). Pale
yellow solid (70 mg, 58.7% yield). 1H NMR (400 MHz, CDCl3,
MeOD) δ 8.42 (brs, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 3.6
Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.12 (d, J = 3.2 Hz, 1H), 6.96 (d, J
= 3.6 Hz, 1H), 6.54 (d, J = 3.2 Hz, 1H), 4.15 (t, J = 6.4 Hz, 2H), 3.37
(s, 3H), 2.21 (t, J = 6.8 Hz, 2H), 2.13 (s, 6H), 1.99−1.91 (m, 2H).
13C NMR (126 MHz, CDCl3, MeOD) δ 159.4, 157.5, 151.6, 137.6,
N,N-Dimethyl-3-(3-methyl-5-(2-(methylsulfonyl)-7H-pyrrolo[2,3-
d]pyrimidin-4-yl)-1H-indol-1-yl)propan-1-amine (DC-BPi-17). Pale
yellow solid (50 mg, 40.4% yield). H NMR (400 MHz, chloroform-
1
d) δ 8.30 (d, J = 2.0 Hz, 1H), 7.96 (dd, J = 8.8, 2.0 Hz, 1H), 7.56 (d, J
= 3.6 Hz, 1H), 7.35 (d, J = 8.8 Hz, 1H), 6.94 (d, J = 3.2 Hz, 1H), 6.90
(brs, 1H), 4.12 (t, J = 6.4 Hz, 2H), 3.36 (s, 3H), 3.27 (s, 1H), 2.50 (t,
J = 8.0 Hz, 2H), 2.35 (s, 6H), 2.28 (s, 3H), 2.09−2.02 (m, 2H). 13C
NMR (151 MHz, chloroform-d) δ 159.4, 157.2, 151.4, 137.8, 130.5,
129.0, 127.4, 126.7, 122.7, 121.0, 116.8, 112.0, 109.5, 102.0, 56.3,
44.7, 43.8, 40.2, 27.6, 9.5. HRMS (ESI) m/e calc’d for C21H26N5O2S
(M + H)+ 412.1807, found 412.1798.
Protein Expression and Purification. The human BPTF
bromodomain (residues 2914−3037) was expressed and purified as
previously described.29 Briefly, a BPTF2914−3037 construct containing a
PreScission protease-cleavable N-terminal GST-tag was expressed in
Escherichia coli BL21-CodonPlus (DE3) cells. For the HTRF assay,
GST-BPTF2914−3037 fusion protein was purified in a final buffer
containing 20 mM HEPES pH 8.5, 100 mM NaCl, 5% glycerol, and
1 mM DTT. The GST tag was removed by PreScission protease for
further experiments, except for the HTRF assay.
HTRF Assay. The HTRF assay was developed as previously
described with a minor optimization where the fluorescence was
measured using an Envision plate reader (excitation of 340 nm,
emission of 665 and 620 nm, and lag time of 60 μs) according to the
assay manufacturer’s recommendations.29
Protein Thermal Shift Assay. The protein thermal shift assay
was performed in a QuantStudio 6 Flex Real-Time PCR system.
Before the thermal cycle (ramped from 25 to 95 °C), 5 μM protein
was mixed with 5× SYPRO Orange (Sigma-Aldrich, catalog no.
129.8, 129.3, 128.8, 128.2, 122.9, 122.7, 116.8, 109.8, 102.7, 102.1,
56.5, 45.0, 44.3, 40.0, 27.9. HRMS (ESI) m/e calc’d for C19H26N5OS
(M + H)+ 372.1651, found 372.1646.
6-(1-(3-(Dimethylamino)propyl)-1H-indol-5-yl)-N-methyl-2-
(methylsulfinyl)pyrimidin-4-amine (DC-BPi-12). Pale yellow solid
(44 mg, 30.4% yield). 1H NMR (600 MHz, methanol-d4) δ 8.36 (brs,
1H), 7.89 (brs, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 3.0 Hz,
1H), 6.89 (s, 1H), 6.56 (d, J = 3.0 Hz, 1H), 4.24 (t, J = 7.2 Hz, 2H),
3.00 (s, 3H), 2.96 (s, 3H), 2.40 (t, J = 7.2 Hz, 2H), 2.30 (s, 6H),
2.08−1.99 (m, 2H). 13C NMR (151 MHz, methanol-d4) δ 159.4,
157.5, 151.6, 137.6, 129.8, 129.3, 128.8, 128.2, 122.9, 122.7, 116.8,
109.8, 102.7, 102.1, 56.5, 45.0, 44.3, 40.0, 27.9. HRMS (ESI) m/e
calc’d for C19H26N5OS (M + H)+ 372.1858, found 372.1849.
6-(1-(3-(Dimethylamino)propyl)-1H-indol-5-yl)-2-(methylsulfin-
yl)-N-propylpyrimidin-4-amine (DC-BPi-13). Pale yellow solid (100
12084
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Article
S5692). All compounds were tested in the assay buffer containing 20
mM HEPES pH 8.5, 100 mM NaCl, and 2.5% DMSO. The filter was
set at ROX with no quencher and a passive filter. The protein melting
temperature (Tm) was calculated with fluorescence raw data by the
Protein Thermal Shift software ver. 1.3 (Life Technologies). Data
were plotted using GraphPad Prism 7.
was reverse transcribed to cDNA with HiScript II Q Select RT
SuperMix (Vazyme, R232−01). Real-time PCR analysis was
performed in the QuantStudio 6 Flex Real-Time PCR system using
the ChamQ SYBR qPCR master mix (Vazyme, Q331−02, low ROX
premixed). The GAPDH gene was used as a normalization control for
the gene expression levels. Primer sequences are listed in Table S1.
Cell Cycle and Apoptosis Analysis. MV-4−11 cells were treated
with the indicated compounds or a DMSO control for 24 h before
being harvested by centrifugation and washed twice with ice-cold
PBS. For cell cycle detection, cells were fixed with ice-cold 70%
ethanol overnight, resuspended with a cell cycle staining solution and
RNase A, and diluted in PBS (Yeasen, no. 40301ES50). For the
apoptosis analysis, cells were resuspended in 400 μL of the binding
buffer and incubated with 5 μL of FITC Annexin V and propidium
iodide for 30 min at 37 °C in the dark. Cell cycle and apoptosis data
were acquired by flow cytometry on BD FACSAria.
Western Blotting. Cell lysate samples were heated in a 95 °C
heat block for 20 min and separated by 12% acrylamide SDS-PAGE in
a Tris-glycine buffer. The proteins were transferred to nitrocellulose
membranes (GE Healthcare) and blocked for 1 h at room
temperature in 5% nonfat milk (diluted in TBST). Blots were
incubated with shaking at 4 °C overnight with the following primary
antibodies: c-Myc (Cell Signaling Technology, catalog no. 18583)
and GAPDH (Cell Signaling Technology, no. 5174). After the
incubation of primary antibodies, blots were incubated with a
1:10 000 dilution of the secondary antibody (BBI life, catalog no.
D110058) for 1 h at room temperature. Images were detected by a
GE ImageQuant LAS 4000 system with SuperSignal WestDura
(Thermo Scientific, no. 34076) as the HRP substrate.
Protein Crystallography and Data Collection and Process-
ing. Purified BPTF2914−3037 was concentrated to 5 mg mL−1 and
crystallized at 16 °C using the sitting-drop vapor-diffusion method
with a reservoir solution containing 200 mM lithium sulfate
monohydrate, 100 mM Tris pH 8.5, and 25% w/v polyethylene
glycol 3350. The crystals were soaked with 2 mM solutions of the
indicated compounds (in a reservoir solution with 1% DMSO) for five
days and were subsequently flash-cooled in liquid nitrogen for data
collection with 15% glycerol (v/v). X-ray diffraction data were
collected at beamlines BL17U1 and BL19U1 in the Shanghai
Synchrotron Radiation Facility (SSRF). All data sets, which were
processed with HKL-3000, are summarized in Table S2. Raw data was
further processed using the HKL program suite, and the initial models
were solved by molecular replacement with the PDB entry 3QTZ as
the search template. Then, the initial structure was rebuilt using the
AutoBuild35 module of Phenix. Ligands were fit to the electron
density map with the LigandFit36 module of Phenix and Coot.37
Then, complex structures were refined for several rounds using
Phenix. Structure validation was performed using MolProbity.38
Surface Plasma Resonance (SPR). A Biacore T200 instrument
(GE Healthcare) was used to analyze the binding interactions with a
direct binding assay format. The purified protein was immobilized on
a CM5 sensor chip at the concentration of 50 μg mL−1. The system
was equilibrated by HBS buffer (25 mM HEPES pH 8.0, 150 mM
NaCl, and 0.05% (v/v) P20 surfactant) containing 0.2% (v/v)
DMSO. Compounds were diluted in the HBS buffer at the indicated
concentration. Samples were injected over the protein and reference
surfaces for 30 s and included a 120 s dissociation time. Data analysis
was conducted via the state and binding model of the T200 evaluation
software (GE Healthcare). Data were fitted globally using Biacore
T200 Evaluation software to determine the binding kinetic rate
constants, ka (M−1 s−1) and kd (s−1), and the equilibrium dissociation
constant KD (M).
NanoBRET. Halo-tagged Histone H4 and NanoLuc-tagged BPTF-
BRD plasmids were constructed, and NanoBRET assays were
performed according to the manufacturer instructions (Promega).
To elevate the assay window, 2.5 μM SAHA was added to the
mixture.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00721.
■
sı
AlphaScreen. The selectivity against the BRD-family proteins,
including BRD2(1), BRD2(2), BRD4(1), BRD4(2), p300-BRD,
CBP-BRD, and SMARCA2-BRD, was determined by an Alpha
Screen assay. In a 384-well plate, 12.5 nM BRD2(1) and 25 nM
acetylated H4, 100 nM BRD2(2) and 75 nM acetylated H4, 12.5 nM
BRD4(1) and 25 nM acetylated H4, 25 nM BRD4(2) and 50 nM
acetylated H4, 40 nM p300-BRD and 100 nM acetylated H4, 20 nM
CBP-BRD and 200 nM acetylated H4, and 100 nM SMARCA2-BRD
and 100 nM acetylated H4 were mixed and incubated for 1 h. Then,
10 μL beads were added, and the plate was incubated for another 1 h
before signal collection.
Dose dependent inhibitory activities of DC-BPi-04−17,
NMR spectra of BPTF-BRD binding with DC-BPi-11,
structural comparison of other bromodomain family
members with BPTF-BRD, compound effect on the
proliferation of normal human endothelial cells, primer
sequences, X-ray crystallography data collection and
refinement statistics, NMR data, and purity data (PDF)
Compound molecular formula strings (CSV)
BPTF-BRD in complex with DC-BPi-03 (CIF)
Cell Culture and Cell Viability Assay. The MLL-rearranged
leukemia cell line of MV-4-11 and human normal cell lines (HUV-
EC-C and MRC-5) were purchased from American Type Culture
Collection (ATCC) and were cultured in either RPMI-1640 or
DMEM medium (Gibco) supplemented with 10% FBS and 1%
penicillin/streptomycin, maintained in a humidified incubator (37 °C,
5% CO2), and checked regularly for mycoplasma contamination. Cells
(5000−10 000 cells, 100 μL per well) were dispensed into 96-well
microplates (Corning, no. 3599) in triplicate and incubated for 10 h
in a humidified incubator. Subsequently, cells were treated with the
indicated serial dilutions of the compounds or DMSO for five days.
Cell viability was assessed using a CellTiter-Glo Kit (10 μL per well,
Promega). Luminescence curves, which were recorded by an Envision
Reader, were fit using the nonlinear fit module (four parameters, log
dose versus response) to determine IC50 values in GraphPad Prism 7.
Quantitative Real-Time PCR. MV-4-11 cells were seeded in 6-
well plates (8 × 105 cells/well) in the RPMI-1640 medium overnight
before drug treatment. Cells were treated with either compounds or
DMSO for 12 h. Total RNA was extracted using a TRIzol reagent
(Vazyme, R401−01) following the manufacturer’s instructions and
BPTF-BRD in complex with DC-BPi-07 (CIF)
BPTF-BRD in complex with DC-BPi-11 (CIF)
Accession Codes
Atomic coordinates for the X-ray structures of BPTF-BRD in
complex with DC-BPi-03, DC-BPi-07, and DC-BPi-11 have
been deposited in the Protein Data Bank (PDB IDs 7F5D,
7F5C, and 7F5E, respectively) and will be released upon
publication.
AUTHOR INFORMATION
Corresponding Authors
■
Wenchao Lu − Stanford Cancer Institute, Stanford School of
Medicine, Stanford University, Stanford, California 94305,
United States; orcid.org/0000-0003-1175-365X;
Email: wclu@stanford.edu
Hua Lin − The Chemical Biology Center, Drug Design and
Discovery Center, State Key Laboratory of Drug Research,
12085
https://doi.org/10.1021/acs.jmedchem.1c00721
J. Med. Chem. 2021, 64, 12075−12088

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China; Biomedical Research
Center of South China, College of Life Sciences, Fujian
Normal University, Fuzhou 350117, China; orcid.org/
0000-0002-0840-6553; Email: hlin@fjnu.edu.cn
Cheng Luo − School of Pharmacy, Fujian Medical University,
Fuzhou 350122, China; School of Chinese Materia Medica,
Nanjing University of Chinese Medicine, Nanjing 210023,
China; School of Pharmacy, Fudan University, Shanghai
201203, China; The Chemical Biology Center, Drug Design
and Discovery Center, State Key Laboratory of Drug
Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, China; orcid.org/
0000-0003-3864-8382; Email: cluo@simm.ac.cn
Dongying Chen − Laboratory of Pharmaceutical Analysis,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China
Jin Lin − School of Pharmacy, Fujian Medical University,
Fuzhou 350122, China
Bo Li − The Chemical Biology Center, Drug Design and
Discovery Center, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China; orcid.org/0000-0002-
4279-4596
Hualiang Jiang − The Chemical Biology Center, Drug Design
and Discovery Center, State Key Laboratory of Drug
Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, China
Kaixian Chen − School of Chinese Materia Medica, Nanjing
University of Chinese Medicine, Nanjing 210023, China; The
Chemical Biology Center, Drug Design and Discovery Center,
State Key Laboratory of Drug Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China
Authors
Tian Lu − School of Pharmacy, Fujian Medical University,
Fuzhou 350122, China; Guizhou University of Traditional
Chinese Medicine, Guiyang, Guizhou 550025, China; School
of Chinese Materia Medica, Nanjing University of Chinese
Medicine, Nanjing 210023, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00721
Haibo Lu − School of Pharmacy, Fudan University, Shanghai
201203, China; The Chemical Biology Center, Drug Design
and Discovery Center, State Key Laboratory of Drug
Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, China
Zhe Duan − The Chemical Biology Center, Drug Design and
Discovery Center, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China; School of Pharmacy,
Nanchang University, Nanchang 330006, China
Jun Wang − The Chemical Biology Center, Drug Design and
Discovery Center, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China
Jie Han − The Chemical Biology Center, Drug Design and
Discovery Center, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China
Author Contributions
∇T.L., H.L., and Z.D. are co-first authors and contributed
equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the staff of the BL17UB and BL19U1 beamlines at
the National Protein Science Facility (NFPS) in the Shanghai
Synchrotron Radiation Facility for their assistance during the
data collection process. We are very grateful for the
instrumental support and technical support of the Shanghai
National Protein Science Center (Shanghai Science Research
Center Protein Expression and Purification System). We
gratefully acknowledge financial support from the National
Natural Science Foundation of China (81625022, 91853205,
and 81821005 to C.L. and 21820102008 to H.J.); the Science
and Technology Commission of Shanghai Municipality
(18431907100 to H.J., Y811298033 to Q.L., and
19XD1404700 to C.L.); and the scientific research innovation
program “Xiyuanjiang River Scholarship” of the College of Life
Sciences, Fujian Normal University, the State Key Laboratory
of Drug Research (SIMM2105KF-07 to H.L.).
Senhao Xiao − The Chemical Biology Center, Drug Design
and Discovery Center, State Key Laboratory of Drug
Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, China
HuanHuan Chen − Laboratory of Pharmaceutical Analysis,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China
Hao Jiang − The Chemical Biology Center, Drug Design and
Discovery Center, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China
Yu Chen − The Chemical Biology Center, Drug Design and
Discovery Center, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China
Feng Yang − The Chemical Biology Center, Drug Design and
Discovery Center, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China
Qi Li − The Chemical Biology Center, Drug Design and
Discovery Center, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China
ABBREVIATIONS USED
■
BET, bromodomain and extra C-terminal domain; BPTF,
bromodomain PHD finger transcription factor; BRD,
bromodomain; BRD2, bromodomain-containing protein 2;
BRD4, bromodomain-containing protein 4; CBP, cyclic
adenosine monophosphate response element binding protein
binding protein; DCM, dichloromethane; DMF, dimethylfor-
mamide; DMSO, dimethyl sulfoxide; Et3N, trimethylamine;
EtOAc, ethyl acetate; HTRF, homogeneous time-resolved
fluorescence; NMR, nuclear magnetic resonance; RT-PCR,
reverse transcription-polymerase chain reaction; SAR, struc-
ture−activity relationship; SGC, structural genomics consor-
tium; SMARCA2, SWI/SNF-related matrix-associated actin-
dependent regulator of chromatin, subfamily A, member 2;
SPR, surface plamon resonace; THF, tetrahydrofuran
12086
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J. Med. Chem. 2021, 64, 12075−12088

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
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