Journal of Medicinal Chemistry
Article
1-(4-Fluorobenzyl)-N-(pyrrolidin-3-yl)-1H-benzo[d]imidazole-2-
amine (32). A solution of 31 (1.44 g, 3.51 mmol) in CH2Cl2 (10 mL)
was treated with TFA (2.61 mL, 35.1 mmol). The mixture was stirred
overnight at rt. The resulting solution was diluted with water, basified
with sodium bicarbonate, and extracted with CH2Cl2 (60 mL × 3).
The organic layers were combined and washed with brine, dried over
sodium sulfate, filtered, and concentrated. The residue was purified by
flash silica chromatography (0−8% MeOH in CH2Cl2) to give 32 as a
liquid nitrogen, the crystals were immersed into a mixture consisting
of reservoir solution plus 10% glycerol (v/v).
Data Collection and Structure Determination. X-ray
diffraction data for EED were collected at 100 K at beamline 19D
of Advanced Photon Source (APS), Argonne National Laboratory,
and reduced with XDS61 and AIMLESS.62 The structure was solved
by molecular replacement with coordinates from PDB entry 3JZN.63
The model was iteratively refined with REFMAC64 and PHENIX,65
and rebuilt with COOT.66 Model geometry was evaluated with
MOLPROBITY.67
Fluorescence Polarization Competition Assay. A previously
described method was used to determine the inhibition activity of
EZH2−EED interaction inhibitors.47,51 Briefly, the compounds were
2-fold serial diluted in DMSO, and added into a mixture of 0.6 μM
EED protein and 20 nM FITC-labeled EZH2 (aa 40-63) peptide
tracer in 40 μL of FP buffer (25 mM PIPES pH 6.2, 150 mM NaCl,
0.1 mg/mL BSA, and 0.01% NP40). The final concentrations of
compounds range from 0.49 to 500 μM. After 2 h incubation at room
temperature (25 °C), the FP values were recorded on a EnVision
multimode plate reader (PerkinElmer). The IC50 values were
calculated by GraphPad Prism 6 using the mode of 4-parameter
logistic nonlinear regression.
NMR-Based Binding Assay. The procedure of ligand T1ρ and
STD NMR data acquisition was as previously described.51 Briefly, 200
μM compound in the absence or in the presence of 5 μM EED (aa
81-441) protein without or with 20, 50, or 100 μM EZH2 peptide
(residues 39-68) were prepared in phosphate buffer (20 mM NaPO4,
100 mM NaCl, pH 7.4, 10% DMSO) and measured at 25 °C on a 600
MHz Bruker Avance III NMR equipment (Bruker BioSpin,
Germany).
Surface Plasmon Resonance (SPR)-Based Binding Assay. To
determine the binding affinities of compounds to EED protein, the
surface plasmon resonance platform Biacore T200 (GE Healthcare)
was applied. Freshly purified EED (aa 81-441) proteins were
immobilized on a CM5 sensor chip with a 10,000 response unit
(RU). First, the compound stock 10 mM in DMSO was 100-fold
diluted in HBS buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.1%
Tween-20), and then 2-fold serial diluted in HBS buffer containing
1% DMSO. The final concentrations of compound range from 0.78 to
100 μM. The binding and dissociation times of compound injection
were set as 120 and 240 s, respectively. The DDKd values of the
compounds were calculated by Biacore T200 evaluation software (GE
Healthcare) using the kinetic modeling approach.
1
yellow solid (875 mg, 80.4%), mp 202 °C. H NMR (400 MHz,
DMSO-d6): δ 9.44 (s, 1H), 7.47 (s, 1H), 7.32−7.25 (m, 3H), 7.15 (d,
J = 8.9 Hz, 2H), 7.13−7.11 (m, 1H), 6.98 (td, J = 7.6, 1.2 Hz, 1H),
6.89 (td, J = 7.6, 1.2 Hz, 1H), 5.36 (s, 2H), 4.67−4.45 (m, 1H), 3.48
(dd, J = 11.9, 6.5 Hz, 1H), 3.40 (dt, J = 11.5, 7.6 Hz, 1H), 3.34−3.23
(m, 2H), 2.32−2.20 (m, 1H), 2.16−2.03 (m, 1H). ESI-MS m/z: [M +
H]+ 311.2.
4-Methoxyphenethyl 4-Methylbenzenesulfonate (33). A mixture
of phenethyl alcohol (5.00 g, 32.9 mmol), tosyl chloride (9.39 g, 49.3
mmol), and triethylamine (13.7 mL, 98.6 mmol) in CH2Cl2 (80 mL)
was stirred overnight at rt. The resulting solution was concentrated
and purified by flash silica chromatography (0−8% EtOAc in PE) to
provide 33 as a white solid (9.98 g, 99.2%), mp 58 °C. 1H NMR (300
MHz, DMSO-d6): δ 7.67 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 8.0 Hz,
2H), 7.05 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.6 Hz, 2H), 4.17 (t, J = 6.6
Hz, 2H), 3.72 (s, 3H), 2.80 (t, J = 6.5 Hz, 2H), 2.41 (s, 3H). ESI-MS
m/z: [M + Na]+ 329.1.
1-(4-Fluorobenzyl)-N-(1-(4-methoxyphenethyl)pyrrolidin-3-yl)-
1H-benzo[d]imidazole-2-amine (21, DC-PRC2in-01). A mixture of
32 (30 mg, 96.7 μmol), 33 (44 mg, 145.0 μmol), sodium carbonate
(31 mg, 290 μmol), and potassium iodide (3 mg, 19.3 μmol) in DMF
(2 mL) was stirred at 80 °C for 1 h. The solution was diluted with
water and extracted with EtOAc (20 mL × 3). The organic layers
were combined and washed with brine, dried over sodium sulfate,
filtered, and concentrated. The residue was purified by flash silica
chromatography (0−3% MeOH in CH2Cl2) to afford a light-yellow
solid (31 mg, 72.1%), mp 124−125 °C. 1H NMR (400 MHz,
CDCl3): δ 7.44 (d, J = 7.8 Hz, 1H), 7.15−7.06 (m, 5H), 7.02 (d, J =
3.7 Hz, 2H), 6.98 (t, J = 8.6 Hz, 2H), 6.81 (d, J = 8.5 Hz, 2H), 5.09
(s, 2H), 4.75−4.53 (m, 1H), 3.76 (s, 3H), 3.18−3.05 (m, 1H), 3.01
(d, J = 10.1 Hz, 1H), 2.84−2.71 (m, 4H), 2.66 (dd, J = 10.1, 6.2 Hz,
1H), 2.46−2.37 (m, 1H), 2.35−2.29 (m, 1H), 1.85−1.63 (m, 1H).
13C NMR (151 MHz, CDCl3): δ 162.4 (d, J = 246.7 Hz), 158.2,
153.6, 142.0, 134.6, 131.5 (2C), 129.6 (2C), 128.6, 128.5, 121.7,
120.1, 116.5, 116.1, 116.0, 114.0 (2C), 107.6, 61.1, 57.5, 55.4, 52.9,
52.4, 45.1, 34.0, 32.5. HRMS (ESI/QTOF) m/z: [M + H]+ calcd for
C27H30FN4O: 445.2398; found, 445.2409. HPLC purity: 98.5%.
Protein Expression and Purification. For the crystallization
study of EED with ligands, the gene of human EED (amino acids 40−
441) was subcloned into a modified pET28GST-LIC vector, and
transformed in Escherichia coli BL21 (DE3) Codon plus RIL. The
bacteria with recombinant plasmid were cultured at 37 °C to OD600
at 1.5, and induced by 0.2 mM IPTG to protein overexpression at 16
°C. The N-terminal GST-tagged EED protein was purified by affinity
chromatography on glutathione-sepharose (GE Healthcare). After
cleavage using thrombin (Sigma-Aldrich) on the column at 25 °C
overnight, the flow-through was collected and purified further by size
exclusion chromatography (Superdex 200, GE Healthcare). The
purified EED proteins was concentrated to 10−15 mg/mL and stored
at −80 °C in a buffer containing 20 mM Tris−HCl pH 7.5, 200 mM
NaCl, and 1 mM DTT for further use in crystallization. For the FP,
NMR, and SPR binding assays, the recombinant human EED
(residues 81-441) with N-terminal 6× His-SUMO tag was overex-
pressed and purified as previously reported.47,51
Molecular Docking. The crystal structure of EED protein (aa 40-
441) complexed with astemizole (PDB ID: 7KXT) was used to
̈
construct a molecular docking model. Glide program (Schrodinger,
LLC, New York, NY, 2015) 5.568,69 was employed to perform
molecular docking simulations. After the coordinates of protein were
prepared by the Protein Preparation Wizard Workflow with default
settings, the docking grid was generated by defining residues located
within 20 Å around astemizole with the Receptor Grid Generation
panel. Finally, the prepared compound (DC-PRC2in-01) that had
̈
been prepared with LigPrep (version 2.3, Schrodinger, LLC, New
York, NY) was docked to the aforementioned docking grid with Extra
precision (XP) mode. The top 1 docking pose ranked by the XP G-
score was selected for binding mode analysis.
Cell Culture. All cell lines were maintained in a humidified
incubator at 37 °C containing 5% CO2 and 95% air. Diffused large B-
cell lymphoma (DLBCL) cell lines (Pfeiffer, SU-DHL-4, Karpsa-422,
and DB) were obtained from the American Type Culture Collection
(ATCC) and cultured in RPMI-1640 complete medium (containing
10% FBS and 1× Pen/Strep).
Crystallization and Structure Determination. Purified EED
protein (aa 40−441) was mixed with 2-fold molar excess astemizole
and crystallized by using the sitting drop vapor diffusion method at 18
°C. The complex of EED (aa 40−441) and astemizole was crystallized
in a buffer containing 3.5 M sodium formate, 0.1 M Tris−HCl, pH
8.5. After optimized by detergents, the small needle crystals grew into
diffracting crystals for structural determination. Before freezing in
Cell Viability Assay. To determine the growth inhibition IC50 of
compounds, DLBCL cell lines were treated with a 9-point 2-fold
dilution series of compounds or 0.1% DMSO. The seeding densities
of each cell line in a 96-well format were empirically adjusted to 1−2
× 104 cells per well (in duplicate). After 3 days of culture, the
CellTiter-Glo (CTG) reagents (Promega) were used to measure the
cell viability and data were processed using GraphPad Prism 6.
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J. Med. Chem. 2021, 64, 8194−8207