Design and Synthesis of EZH2-Based PROTACs to Degrade the PRC2 Complex for Targeting the Noncatalytic Activity of EZH2

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Design and Synthesis of EZH2-Based PROTACs to Degrade the PRC2

Complex for Targeting the Noncatalytic Activity of EZH2

Zhihao Liu,^∥Xi Hu,^∥Qiwei Wang, Xiuli Wu, Qiangsheng Zhang, Wei Wei, Xingping Su, Hualong He,

Shuyan Zhou, Rong Hu, Tinghong Ye, Yongxia Zhu, Ningyu Wang,* and Luoting Yu *

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ABSTRACT: EZH2 mediates both PRC2-dependent gene silencing via

catalyzing H3K27me3 and PRC2-independent transcriptional activation

in various cancers. Given its oncogenic role in cancers, EZH2 has

constituted a compelling target for anticancer therapy. However, current

EZH2 inhibitors only target its methyltransferase activity to downregulate

H3K27me3 levels and show limited eﬃcacy because of inadequate

suppression of the EZH2 oncogenic activity. Therefore, therapeutic

strategies to completely block the oncogenic activity of EZH2 are urgently

needed. Herein, we report a series of EZH2-targeted proteolysis targeting

chimeras (PROTACs) that induce proteasomal degradation of PRC2

components, including EZH2, EED, SUZ12, and RbAp48. Preliminary

assessment identiﬁed E7 as the most active PROTAC molecule, which

decreased PRC2 subunits and H3K27me2/3 levels in various cancer cells.

Furthermore, E7 strongly inhibited transcriptional silencing mediated by EZH2 dependent on PRC2 and transcriptional activation

mediated by EZH2 independent of PRC2, showing signiﬁcant antiproliferative activities against cancer cell lines dependent on the

enzymatic and nonenzymatic activities of EZH2.

function of enzyme activity, resulting in hypertrimethylated

H3K27 status and promotion of tumorigenesis in several types

of tumors. In view of the important role of dysfunctional EZH2

in tumorigenesis, the development of EZH2-speciﬁc inhibitors

has been an important research area.⁶Several S-adenosyl-L-

methionine (SAM)-competitive compounds that target EZH2,

including GSK126,²⁰EPZ6438,^21,22PF-06821497,²³and CPI-

1205,²⁴are currently in clinical trials, and these compounds

have demonstrated eﬃcacies in treating hematological

malignancies, sarcomas, and malignant rhabdoid tumors.

Recently, the U.S. FDA has granted accelerated approval of

TAZVERIK (tazemetostat, EPZ6438) for the treatment of

patients with metastatic or locally advanced epithelioid

sarcoma.²⁵

INTRODUCTION

■

Polycomb repressive complex 2 (PRC2) is a conserved

multicomponent transcriptional repressive complex with

histone methyltransferase activity that catalyzes the dimethy-

lation and trimethylation of lysine residue 27 on histone H3

(H3K27me2/3) and silences target genes that are involved in

fundamental cellular processes by facilitating chromatin

compaction.¹⁻⁴The mammalian PRC2 complex mainly

possesses four key subunits: enhancer of the zeste homolog

1/2 (EZH1/2), embryonic ectoderm development (EED),

suppressor of the zeste 12 protein homolog (SUZ12), and

retinoblastoma (Rb)-associated proteins 46/48 (RbAp46 or

RBBP7/RbAp48 or RBBP4).^1,5Overactivation of the PRC2

complex induces the occurrence of malignant tumors by

silencing tumor suppressor genes.⁶

As the core catalytic subunit of PRC2, EZH2 is frequently

overexpressed in many cancer types including breast, prostate,

lung, and ovarian cancers, and overexpression of EZH2 is

implicated in tumorigenesis and poor prognosis in several

cancers.⁷⁻¹⁴In germinal center B-cell (GCB) diﬀuse large-cell

B-cell lymphomas (DLBCLs), EZH2 point mutations at Y641

(Y641F, Y641N, Y641S, Y641C, and Y641H) occur in about

22% cases, and these Y641 mutations are also identiﬁed in 7−

12% of follicular lymphomas.¹⁵⁻¹⁷In addition, A677 and A687

mutations in EZH2 have also been found in non-Hodgkin’s

lymphomas (NHLs).^18,19All of these mutations confer gain of

However, the clinical eﬃcacy of EZH2 inhibitors remains

unsatisfactory and is limited to certain cancers.²⁶As a

multifunctional protein, EZH2 not only mediates gene

silencing via recruitment of the PRC2 complex to catalyze

H3K27me3 but also mediates transcriptional activation

Received: December 24, 2020

Published: February 19, 2021

https://dx.doi.org/10.1021/acs.jmedchem.0c02234

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Figure 1. Schematic of PROTAC inducing POI degradation (A). Proposed model for PROTAC inducing degradation of a protein complex (B).

independent of EZH2/PRC2 catalytic activity in some

cancers.²⁷⁻²⁹In estrogen receptor (ER)-negative basal-like

breast cancer cells, EZH2 interacts with RelA and RelB and

induces continuous activation of the NF-κB downstream

signaling pathway independent of its catalytic activity.³⁰In

castration-resistant prostate cancer (CRPC), the PI3K-Akt

pathway mediates the phosphorylation of EZH2 at Ser21 and

switches its function from a polycomb repressor to a PRC2-

independent transcriptional coactivator of AR.^31,32Kim et al.³³

showed that the growth and survival of SWItch/sucrose

nonfermentable (SWI/SNF) mutant tumors rely on both the

catalytic and noncatalytic activities of EZH2, and the EZH2

inhibitor GSK126 only showed limited eﬃcacy in SWI/SNF-

mutant cancer cells. Therefore, EZH2 inhibitors that target

EZH2 methyltransferase activity are only eﬃcacious in a few

cancers because of their inadequate suppression of EZH2

oncogenic activity, and therapeutic strategies to completely

block the oncogenic functions of EZH2 are urgently needed.

Regulation of EZH2 homeostasis may be an eﬀective

therapeutic approach to treat cancers dependent on and

independent of EZH2/PRC2 catalytic activity. Hydrophobic

tagging (HyT) and proteolysis targeting chimeras (PRO-

TACs) are practical and feasible approaches for regulation of

protein homeostasis via degradation of the target protein.³⁴

HyT technology uses bifunctional molecules to induce protein

degradation by appending a hydrophobic tag such as

adamantane on the target protein to mimic protein

misfolding.³⁵Recently, Ma et al.³⁶reported a ﬁrst-in-class

EZH2 selective degrader MS1943 based on HyT. MS1943

eﬀectively reduced EZH2 and SUZ12 protein levels without

aﬀecting EED protein levels in triple-negative breast cancer

(TNBC) cells and showed signiﬁcant antiproliferative activities

in multiple TNBC cells that are insensitive to EZH2 inhibitors.

PROTACs apply a linker to concatenate a ligand of E3

ubiquitin ligase and a ligand for a protein of interest (POI) and

function by recruiting the E3 ubiquitin ligase close to the POI

to promote consequent degradation of the POI (Figure

1A).³⁷⁻³⁹Almost all reported PROTACs just induce the

degradation of their direct interactive proteins, while few

studies have mentioned the possibility of PROTACs degrading

their indirect interactive proteins, for instance, the components

of a protein complex. Actually, Hsu et al.⁴⁰and Potjewyd et

al.⁴¹recently described the discovery of EED-targeted

PROTACs that indeed led to the degradation of multiple

components of the PRC2 complex such as EED, EZH2, and

SUZ12 although the in-depth mechanism was not fully

interpreted. Since the PROTAC molecule plays a binder role

in drawing the E3 ubiquitin ligase close to the POI, we

hypothesize that if the POI is capable of forming a protein

complex with other proteins, then the PROTAC molecule

could also induce the degradation of other components of the

complex by drawing the E3 ubiquitin ligase close to the

complex or downregulating the stability of these proteins due

to the inability to form a protein complex (Figure 1B).

Based on the above assumptions, we hypothesized that

EZH2-targeted PROTACs, like EED-targeted PROTACs,

could induce the depletion of the PRC2 complex as well,

leading to inhibition of both the catalytic and noncatalytic

functions of EZH2 and providing extensive anticancer activity.

In this study, we designed and synthesized the EZH2-targeted

PROTACs to investigate their ability and speciﬁc mechanism

to degrade the PRC2 complex and examined whether the

EZH2-targeted PROTACs could inhibit both the catalytic and

noncatalytic functions of EZH2 and exhibit more extensive

antitumor eﬀects.

RESULTS AND DISCUSSION

■

The degradation eﬃciency of a PROTAC molecule typically

hinges on the spatial orientation of the POI and the E3 ligase

upon PROTAC conjugation, the aﬃnity of the PROTAC with

the POI, and the ﬁtness of the connectome part.⁴²⁻⁴⁵To

identify a suitable modiﬁcation site on an EZH2 inhibitor that

can join to a linker moiety without a signiﬁcant loss in potency,

we ﬁrst analyzed the binding mode between the EZH2

inhibitor GSK126 and the PRC2 complex with a reported

cocrystal structure (PDB: 5WG6)⁴⁶and found that the

piperazinyl fragment in GSK126 extended into the solvent

region and did not touch the other subunits of the PRC2

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Figure 2. Design and proof-of-concept experiments for degrading the PRC2 complex of EZH2-targeted PROTACs. (A) Structure of GSK126

bound to PRC2 (PDB: 5WG6). (B) Spatial orientation of GSK126 toward the solvent region (PDB: 5WG6). (C) Structure of G12. (D) Levels of

PRC2 subunits and H3K27me3 after treatment with various concentrations of G12 or GSK126, 72 h.

a

Scheme 1. Syntheses of Compounds G4−G12

a

Reagents and conditions: (a) Ac₂O, 3-aminopiperidine-2,6-dione, 140 °C, 6 h, 71%; (b) DMF, dibromoalkane, N,N-diisopropylethylamine

(DIPEA), 85−100 °C, 3−6 h, 34−63%; (c) GSK126, NaHCO₃, 85−100 °C, 3−8 h, 14−41%.

complex (Figure 2A,B). We also found that the orientation of

the morpholine moiety in EPZ6438 also extended into the

solvent region after the molecular superposition of EPZ6438

and GSK126. 4-Hydroxythalidomide, a well-studied ligand of

the cereblon (CRBN) component of E3 ligase, was used as the

ligand to conjugate to the piperazinyl N-atom in GSK126

through a long alkyl linker (G12, Figure 2C). We then

investigated the potential degradation ability of G12 to PRC2

subunits including EZH2. GSK126 only inhibited H3K27

trimethylation without detectable eﬀects on the PRC2 subunit

expression (Figure 2D). By contrast, G12 dose-dependently

decreased both EZH2 and H3K27me3 levels in the DLBCL

cell line WSU-DLCL-2. Intriguingly, G12 also demonstrated

strong degradation eﬃciency against EED and SUZ12,

implying the possibility of degrading multiple subunits of a

complex by a speciﬁc subunit-targeted PROTAC.

Inspired by the preliminary proof-of-concept experiments,

we conducted an in-depth structure−activity relationship study

to optimize the PROTAC molecules. Various alkyl linkers with

diﬀerent lengths were incorporated to connect selective EZH2

inhibitors, such as EPZ6438 or GSK126, with the CRBN

ligand 4-hydroxythalidomide to investigate their degradation

eﬃciency against PRC2 subunits. The general synthetic route

of compounds G4−G12 is illustrated in Scheme 1. Racemic

hydroxythalidomide (1b) was obtained through the ammo-

nolysis reaction of 4-hydroxyisobenzofuran-1,3-dione (1a) and

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a

Scheme 2. Syntheses of Compounds E4−E12

a

Reagents and conditions: (a) NH₄Cl, iron powder, MeOH/H₂O = 3:1, 80 °C, 92%; (b) tetrahydro-4-pyrone, AcOH, sodium

triacetoxyborohydride, CHCl₃, rt, 10 h, 57.8%; (c) anhydrous acetaldehyde, 1,2-dichlorethane, AcOH, rt, 10 h, 71%; (d) 50% NaOH, MeOH,

rt, overnight, 2 M HCl, 91.2%; (e) EDCI, 1-hydroxy-7-azabenzotriazole (HOAT), dimethyl sulfoxide (DMSO), 45 °C, 20 h, 68%; (f) K₂CO₃,

Pd(dppf)Cl₂, 1,4-dioxane/H₂O = 4:1, 100 °C, 8 h; (g) triﬂuoroacetyl (TFA), dichloromethane (DCM), 25 °C, 1 h, Na₂CO₃neutralization, 74% (f,

g, two-step yield); (h) 1c−k, NaHCO₃, DMF, 85−100 °C, 3−8 h, 15−47%.

a

Table 1. EZH2 Inhibitory Potencies of G4−G12 and E4−E12

Cpd

G4

G5

G6

G7

EZH2 (IC₅₀, nM)

Cpd

E4

E5

E6

E7

EZH2 (IC₅₀, nM)

5.5

5.8

4.0

6.3

7.3

16

3.7

4.5

3.9

2.7

3.4

15

G8

G9

E8

E9

G10

G11

G12

GSK126

144

165

61

E10

E11

E12

EPZ6438

17

30

53

3.7

2.7

a

IC₅₀values reported as an average of ≥ two determinations; see the Supporting Information for further details.

3-amino-2,6-piperidinedione at 140 °C for 6 h with 71% yield.

1b was treated with the corresponding dibromoalkane and

sodium bicarbonate in N,N-dimethylformamide (DMF) at

85−100 °C for 3−6 h to provide 4-hydroxythalidomide

analogues containing an alkyl bromide group 1c−k (34−63%

yield). Then, GSK126 reacted with 1c−k by SN2 nucleophilic

substitution reactions similar to the preparation procedure for

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Figure 3. PROTACs degrade PRC2 subunits and decrease H3K27me3 levels. (A) PRC2 subunits and H3K27me3 levels in WSU-DLCL-2 cells

treated with 1 μM PROTACs, GSK126, or EPZ6438 for 48 h were analyzed. A representative western blot (WB) of three experiments is presented.

GAPDH served as the internal control. (B) Quantitative results of the EZH2, SUZ12, EED, and RbAp48 levels detected by western blot. (C)

Quantitative results of H3K27me3 levels detected by western blot. Protein levels were quantiﬁed by densitometry analysis using Image J and

normalized against GAPDH levels. Data are expressed as mean SD from three independent experiments, *p < 0.05, **p < 0.01, and ***p <

0.001, compared with the DMSO-treated group by the t-test.

1c−k to obtain PROTAC molecules G4−G12 based on

GSK126 (14−41% yield).

Compounds E4−E8 and G4−G8 with a linker length of 4−8

carbon atoms demonstrated comparable inhibitory activity

against EZH2 to that of the positive control EPZ6438 and

GSK126. When the length of linkers was further extended to

9−12 carbon atoms (E9−E12 and G9−G12), the inhibitory

activities against EZH2 were 5−80 times less potent than

Scheme 2 shows the synthetic route for compounds E4−

E12. The key intermediate of EPZ6438-based PROTACs 2g

was accomplished according to a previously published method

for the preparation of EPZ6438.²¹Brieﬂy, E4−E12 were

synthesized starting from methyl 5-bromo-2-methyl-3-nitro-

benzoate (2a) with reduction, two steps of reductive

amination, hydrolysis, and amide condensation to aﬀord the

intermediate 2f. Suzuki coupling of 2f with aryl boronate esters

using Pd(dppf)Cl₂and cesium carbonate and subsequent

deprotection aﬀorded 2g. PROTACs E4−E12 with varying

linker lengths were obtained by SN₂reactions between 1c−k

and 2g.

GSK126. In general, E7 showed the highest potency (IC₅₀

=

2.7 nM) for inhibiting the EZH2 methyltransferase activity and

exhibited >66-fold selectivity for EZH2 over its paralog EZH1

(EZH1 IC₅₀= 179.0 nM, Table S1).

We then evaluated the degradation eﬃciency of all EZH2-

targeted PROTACs against EZH2, EED, SUZ12, and RbAp48

upon treatment with title compounds in a ﬁxed concentration

and time (1 μM, 48 h) in the WSU-DLCL-2 cancer cell line

(Figures 3A,B and S1). As expected, EPZ6438 and GSK126

did not alter the levels of all PRC2 subunits. Among the G-

series PROTACs, G4−G7 with a linker length of 4−7 carbon

atoms showed poor degradation abilities against PRC2

subunits, while PROTACs with a linker length of 8−12

The title compounds were evaluated for their EZH2

inhibitory potency in an Alpha-Screen assay, and EPZ6438

and GSK126 were used as positive controls. As shown in Table

1, PROTACs G4−G12 and E4−E12 showed similar overall

inhibition properties with nanomolar IC₅₀against EZH2.

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Figure 4. Binding of EZH2 (A), SUZ12 (B), EED (C), and RbAp48 (D) to E7 in WSU-DLCL-2 cells was detected by the thermo shift assay.

Bands in immunoblotting were quantiﬁed by densitometry analysis using Image J. WSU-DLCL-2 cells were pretreated with MG-132 (5 μM for 1

h) before treatment with E7 (60 μM for 2 h). Cell lysates were examined by western blot. Data are expressed as mean

SD from three

independent experiments.

carbon atoms (G8−G12) demonstrated signiﬁcant degrada-

tion eﬃciency against all PRC2 subunits to diﬀerent extents.

The degradation of SUZ12 and EED was much more eﬃcient

than that of the primary target EZH2 across this series. Among

EZH2-targeted PROTACs based on EPZ6438, E4 with a 4-C

linker demonstrated a strong degradation eﬃciency against

EZH2, EED, SUZ12, and RbAp48. A possible explanation for

this phenomenon may be that E4 with a shorter linker could

induce an alternative ternary complex conformation between

EZH2 and CRBN, which is diﬀerent from other EZH2-

PROTACs, as the previously reported BRD4-PROTACs.⁴⁷E5

and E6 also degraded PRC2 subunits to some extent, but their

degradation abilities were signiﬁcantly weaker than that of E4.

E7−E12 signiﬁcantly degraded PRC2 subunits, but the

degradation abilities became weaker as the length of the linker

was prolonged from 7 to 12 carbon atoms.

levels was consistent with the decreasing level of PRC2

complex subunits after PROTAC treatment. PROTACs that

induced the degradation of PRC2 subunits (G8−G12, E4,

E7−E11) also signiﬁcantly decreased H3K27me3 levels in

WSU-DLCL-2 cancer cells. Among the CRBN-recruiting

PROTACs, E7 demonstrated the best degradation eﬃcacies

against all PRC2 subunits (EZH2 72%, SUZ12 81%, EED 75%,

RbAp48 74%) and a maximum decrease in H3K27me3 levels

(reduced by 83%) at 1 μM for 48 h. Hence, E7 was ﬁnally

selected for further study.

Cellular thermal shift assay is a technology for evaluating

ligand binding to target proteins in cells that uses the shift in

protein thermal stability caused by drug binding to directly

monitor target protein−drug interactions in cells.⁴⁸To

investigate which subunit of PRC2 could bind to E7, we

conducted thermal shift assays in WSU-DLCL-2 cells. E7

increased the thermal stability of EZH2, with a temperature

shift of approximately 5 °C, indicating a direct binding of E7

and the EZH2 protein (Figure 4A). However, E7 did not shift

the thermal denaturation curves of EED, SUZ12, and RbAp48,

suggesting that E7 might not directly bind to SUZ12, EED,

We also investigated the eﬀects of synthesized EZH2-

targeted PROTACs on H3K27me3 levels. We treated WSU-

DLCL-2 cells with PROTACs, GSK126, or EPZ6438 at 1 μM

for 48 h to monitor H3K27me3 levels by western blot. As

shown in Figure 3A,C, the trend of decreasing H3K27me3

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Figure 5. E7 eﬀectively degrades PRC2 subunits and speciﬁcally inhibits PRC2. (A) Decrease in PRC2 subunits and H3K27me3 levels is

dependent on E7 concentrations. WSU-DLCL-2 cells were incubated with E7 for 48 h at indicated concentrations. (B) Time course of PRC2

subunits’ degradation and H3K27me3 decrease by E7 (1 μM) in WSU-DLCL2 cells. (C) Eﬀects of E7 on histone H3 methylation in WSU-DLCL-

2 cells were detected by western blot. (D) Western blot analysis of PRC2 subunits and H3K27me3 following treatment of E7 (1 μM for 48 h) in

DLBCL (WSU-DLCL-2, Pfeiﬀer), prostate cancer (LNCaP, DU 145), and ovarian cancer (A2780, SKOV3) cell lines.

Figure 6. E7 induces the degradation of the PRC2 complex via the ubiquitin-proteasomal pathway. (A) RT-qPCR analysis of the mRNA levels of

PRC2 subunits in WSU-DLCL-2 cells following treatment with E7 (1 μM for 48 h). The mRNA levels of PRC2 components were normalized to

levels in the DMSO-treated group. Error bars represent mean

SD analyzed by the t-test. (B) WSU-DLCL-2 cells were pretreated with

lenalidomide or MLN4924/MG-132 for 4 h, followed by treatment with E7 for an additional 48 h. (C) Competitive eﬀects of EZH2 or EED

inhibitors in WSU-DLCL-2 cells on E7 were determined by western blot. Cells were treated with indicated compounds for 48 h.

and RbAp48 protein (Figure 4B−D). Overall, these data

conﬁrmed that our bivalent molecules were potent binders of

EZH2 but not EED, SUZ12, or RbAp48. The EZH2-

PROTACs might induce the degradation of EZH2 by direct

engagement and subsequently initiate the E3 ligase-mediated

proteasomal degradation pathway while inducing degradation

of other PRC2 subunits by the EZH2-mediated indirect

interaction.

To further investigate the degradation eﬃciency of E7, we

evaluated the levels of PRC2 subunits and H3K27me3 in

WSU-DLCL-2 cells exposed to graded concentrations of E7

(0, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, and 10.0 μM) for 48 h. E7

slightly downregulated PRC2 subunit levels at low concen-

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a

Scheme 3. Syntheses of Compounds E7-NC-1 and E7-NC-2

a

Reagents and conditions: (a) NaH, CH₃I, DMF, 56%; (b) TFA, DCM, 1 h; (c) Ac₂O, 4-hydroxyisobenzofuran-1,3-dione, 140 °C, 6 h, 39% (b, c,

two-step yield); (d) 1,1-dibromoheptane, DIPEA, DMF, 85 °C, 3 h, 42%; (e) NaHCO₃, 95°C, 6 h, 29%; (f) K₂CO₃, CH₃I, DMF, 86%; (g) TFA,

DCM, 25 °C, 1 h, Na₂CO₃neutralization; (h) NaHCO₃, DMF, 95 °C, 3−8 h, 35% (g, h, two-step yield).

trations, but at concentrations over 1 μM, all PRC2 subunits

and H3K27me3 levels were signiﬁcantly decreased in a dose-

dependent manner. Moreover, the PRC2 subunits and

H3K27me3 levels rebounded at 10 μM (Figure 5A), which

is consistent with a unique phenomenon of the PROTAC’s so-

called “hook” eﬀect related to a ternary-complex-mediated

mechanism.⁴⁹Then, we evaluated the levels of PRC2 subunits

and H3K27me3 levels in WSU-DLCL-2 cells upon treatment

with 1 μM E7 for various times (0, 0.25, 0.5, 1, 2, 4, 12, 24, 48,

72, and 96 h) by western blot analysis. As shown in Figure 5B,

at the early time points (0−2 h), E7 only caused a slight

downregulation of the EZH2 protein level but had no obvious

eﬀect on other PRC2 subunits. One possible explanation was

that E7 may induce rapid degradation of the free EZH2

protein at the beginning of treatment, while a longer time was

needed to form the ternary complex to induce the degradation

of the PRC2 complex by E7, and the change in H3K27me3

also indirectly conﬁrmed this speculation. All PRC2 subunits

and H3K27me3 levels were signiﬁcantly decreased after 48 h

and were completely abolished after 72 h. These results

showed that E7 had a powerful and lasting degradation

eﬃciency against PRC2 subunits and the ability to inhibit

PRC2 activity.

aﬀected across all tested concentrations, highlighting the

speciﬁcity of E7 for PRC2 inhibition (Figure 5C). To exclude

the possibility that the downregulation of PRC2 subunits by

E7 is cell-speciﬁc, we next tested the degradation abilities of E7

in other cancer cell lines. WSU-DLCL-2, SU-DLCL-6, LNCaP,

DU 145, A2780, and SKOV3 cancer cell lines were exposed to

E7 (1 μM for 48 h). As illustrated in Figure 5D, E7

signiﬁcantly decreased PRC2 subunits and H3K27me3 levels

in all of the tested cancer cell lines.

Quantitative real-time polymerase chain reaction (PCR)

showed that E7 had little eﬀect on the mRNA levels of PRC2

complex subunits including EZH2, SUZ12, EED, and RbAp48

in WSU-DLCL-2 cells (Figure 6A), indicating that E7 induced

downregulation of PRC2 subunits without aﬀecting the

transcription of PRC2 complex subunits. To further demon-

strate that EZH2-targeted PROTACs induced the degradation

of the PRC2 complex via the ubiquitin-proteasomal degrada-

tion pathway induced by CRBN E3 ligase recruitment, we

treated WSU-DLCL-2 cells with E7 in the presence of the

CRBN ligand lenalidomide that can compete with PROTAC-

mediated binding to the E3 ubiquitin ligase. Lenalidomide

treatment alone did not aﬀect the levels of PRC2 complex

subunits, but pretreatment with lenalidomide before addition

of E7 eﬀectively blocked E7-induced PRC2 subunit degrada-

tion. Moreover, proteasome inhibitor MG-132 or the Nedd8-

activating enzyme E1 (NAE) inhibitor MLN4924 alone had no

eﬀect on PRC2 subunits, while MG-132 or MLN4924

pretreatment before addition of E7 recovered the down-

regulation of PRC2 subunits (Figure 6B). Furthermore, the

To investigate the speciﬁcity of E7 for PRC2 inhibition, we

examined other histone H3 methylation modiﬁcations. E7

dose-dependently decreased H3K27me2 and H3K27me3

levels, which rebounded at 10 μM in WSU-DLCL-2 cells.

However, other histone H3 methylation modiﬁcations

including H3K27me1, H3K9me3, and H3K4me3 were not

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Figure 7. (A) EZH2 inhibitory potencies of E7, E7-NC-1, and E7-NC-2. (B) Levels of PRC2 subunits and H3K27me3 after treatment with

thalidomide (10 μM), EPZ6438 (10 μM), E7 (2.5 μM), E7-NC-1 (2.5 μM), or E7-NC-2 (2.5 μM) in WSU-DLCL-2 cells, for 48 h.

Figure 8. Ubiquitin modiﬁcation of EZH2, SUZ12, and EED was determined by immunoprecipitation assays. WSU-DLCL-2 cells were collected

and lysed after treatment with E7 (1 μM for 48 h).

Figure 9. E7 regulates the transcription of EZH2-targeted genes. (A) mRNA levels of genes silenced by the catalytic function of EZH2 in WSU-

DLCL-2, Pfeiﬀer, and A549 cells after treatment with the indicated compounds for 48 h. (B) mRNA levels of genes activated by the noncatalytic

function of EZH2 in A549, NCI-H1299, and MDA-MB-468 cells after treatment with the indicated compounds for 48 h. The mRNA levels of

EZH2-targeted genes were normalized to levels in the DMSO-treated group. Data are expressed as mean SD for three independent experiments,

*p < 0.05, **p < 0.01, compared with the DMSO-treated group by the t-test.

EZH2 inhibitor EPZ6438 or GSK126 eﬀectively recovered the

PRC2 subunit depletion by E7, whereas the EED inhibitor

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Figure 10. Proliferation curves and optical microscopy images of WSU-DLCL-2 (A), A549 (B), and NCI-H1299 (C) cells treated with E7,

EPZ6438, or GSK126 at 10 μM. Data are expressed as mean SD of three independent experiments, *p < 0.05, **p < 0.01, and ***p < 0.001,

compared with the DMSO-treated group by the t-test.

EED226 had no eﬀect (Figure 6C). These results suggest that

the PRC2 subunit depletion induced by E7 resulted from its

direct interaction with EZH2 rather than other PRC2 subunits.

We further tested the speciﬁcity of E7 by synthesizing E7-

NC-1, which resembles E7 but could not bind to CRBN due

to a methyl group at the glutarimide ring of thalidomide, and

E7-NC-2 with decreased aﬃnity for EZH2 due to an N-methyl

residue at the pyridone moieties of E7. Synthetic routes of E7-

NC-1 and E7-NC-2 are shown in Scheme 3. Brieﬂy, E7-NC-1

was synthesized from 3b, the methylation product of 3-Boc-

amino-2,6-dioxopiperidine (3a), according to a similar

synthetic route for the preparation of E7. Methylation of 3e

with iodomethane and potassium carbonate in DMF aﬀorded

3f, which underwent deprotection of the Boc group and

subsequent nucleophilic substitution reaction with 1h to

obtain E7-NC-2.

The EZH2 inhibitory potencies of E7, E7-NC-1, and E7-

NC-2 were tested by the Alpha-Screen assay. E7-NC-1

demonstrated comparable inhibitory activity against EZH2 to

that of E7 (Figure 7A, IC₅₀= 1.9 nM), but E7-NC-2 showed

about 10-fold decreased activity against EZH2 compared with

E7 (Figure 7B). As expected, the EZH2 inhibitor EPZ6438

and thalidomide did not decrease the PRC2 subunit levels, nor

did E7-NC-1 and E7-NC-2 in WSU-DLCL-2 cells (Figure

7C).

To verify the ubiquitination status of all PRC2 complex

subunits upon E7 treatment, immunoprecipitation (IP)

experiments were carried out with lysates prepared from

WSU-DLCL-2 cells treated with E7 at 1 μM for 48 h. As

shown in Figure 8, E7 increased the ubiquitination of EZH2,

SUZ12, and EED in WSU-DLCL-2 cells, suggesting that

degradation of PRC2 subunits by E7 occurs via the ubiquitin

proteasome pathway. Overall, these data indicated that the

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Figure 11. Immunoblot analysis of PRC2 subunits and H3K27me3 expression in A549 (A, B) and H1299 cells (D, E) after treatment with control

or EZH2-targeting shRNA. The proliferation curves are for A549 (C) and NCI-H1299 (F) cells after treatment with E7 or E7-NC-1 at 10 μM

compared with cells after inducing control or EZH2-targeting shRNA. Data are expressed as mean SD of three independent experiments, *p <

0.05, **p < 0.01, and ***p < 0.001, compared with the control shRNA group by the t-test.

decrease of PRC2 subunit levels induced by E7 is mediated by

the E3-ligase proteasomal degradation pathway.

DLBCL cells WSU-DLCL-2 (which harbors EZH2-Y641F)

and Pfeiﬀer (which harbors EZH2-A677G) were used to

evaluate the antiproliferative eﬀects of E7. Treatment of WSU-

DLCL-2 cells with E7 for 3, 5, and 7 days decreased the IC₅₀

value from 4.92 to 3.69 μM (Figure S2-A). Better results were

obtained for Pfeiﬀer cells; treatment with E7 for 3, 5, and 7

days decreased the IC₅₀values from 0.49 to 0.21 μM (Figure

S2-D). Then, WSU-DLCL-2 cells were chosen to evaluate

proliferation for 9 days treated with E7, GSK126, or EPZ6438

alone. EPZ6438 and GSK126 showed potent antiproliferative

eﬀects against WSU-DLCL-2 cells (Figure 10A). Remarkably,

E7 almost completely inhibited the growth of WSU-DLCL-2

cells, demonstrating the superior antiproliferative eﬀect of

EZH2-PROTAC E7 over the other EZH2 inhibitors (Figure

10A).

Since SWI/SNF-mutant cancers depend on the enzymatic

and nonenzymatic activities of EZH2 and displayed discrepant

responses to EZH2 enzymatic inhibitors, we selected the A549

cell line (SWI/SNF-mutant), which is sensitive to EZH2

inhibitors, and the NCI-H1299 cell line (SWI/SNF-mutant),

which is relatively resistant to EZH2 inhibitors, and conducted

proliferation assays for 9 days. EPZ6438 had little eﬀect on the

proliferation of A549 and NCI-H1299 cells. GSK126 showed a

certain degree of antiproliferative activity against A549, with

less potency against NCI-H1299 cells (Figure 10B,C), which

was consistent with previous reports.³³E7 demonstrated

signiﬁcant antiproliferative eﬀects against both A549 and NCI-

H1299 cell lines (Figures 10B,C and S2-B,C). These results

suggested that EZH2-targeted PROTAC E7 showed signiﬁcant

antiproliferative activities against cancer cells dependent on the

catalytic and noncatalytic functions of EZH2.

Several reported H3K27me3-related genes including

ADRB2, CDKN2A, TXINP, and TNFRSF21, mediated

silencing by EZH2/PRC2, were selected for quantitative

mRNA expression analysis.^22,52In lymphoma cell lines WSU-

DLCL-2 and Pfeiﬀer, the upregulation of ADRB2 and

TNFRSF21 by E7 treatment was stronger than that of

EPZ6438 and GSK126, respectively, while the upregulation

of CDKN2A and TXINP was not as good as that of EPZ6438

and GSK126. In addition, in the lung cancer cell line A549,

only CDKN2A and TXINP were signiﬁcantly upregulated after

E7 treatment (Figure 9A). With a similar extent as those

induced by EPZ6438 and GSK126, E7 strongly upregulated

the expression of these genes to various extents in diﬀerent

cancer cell lines (Figure 9A).

EZH2 also mediates gene activation including BRIC5,

ARL6IP, CEP76, CENPK, CHEK1, and TACC3 genes in

many cancers, such as lung cancer and breast cancer, which is

independent of the catalytic function of EZH2/PRC2.^6,52We

next analyzed the mRNA levels of these genes in A549, NCI-

H1299, and MDA-MB-468 cells after treatment with E7. All of

these examined genes were downregulated after treatment with

E7 at indicated concentrations (Figure 9B). However,

treatment with EPZ6438 or GSK126 did not aﬀect the

mRNA expression of these genes (Figure 9B). These data

suggested that EZH2 enzymatic inhibitors such as EPZ6438 or

GSK126 did not fully block the oncogenic activity of EZH2,

whereas EZH2-targeted PROTACs such as E7 completely

abolished the oncogenic function of EZH2 implied by the

inhibition of the EZH2 noncatalytic function.

The abovementioned results demonstrated that E7 aﬀected

both the catalytic and noncatalytic roles of EZH2. Hence, we

compared the antiproliferative eﬀects of E7 as well as EZH2

inhibitors EPZ6438 and GSK126 on various cancer cell lines.

Previous studies showed that knockdown of EED can lead to

concomitant downregulation of EZH2 and SUZ12.^50,51Thus,

EED-targeted PROTACs reported by Hsu et al.⁴⁰and

Potjewyd et al.,⁴¹which induced the downregulation of

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EZH2 and SUZ12, may be ascribed to the decreased stability

of the PRC2 complex induced by EED degradation. It has also

been shown in the literature that genetic knockdown of EZH2

caused the depletion of SUZ12 and EED besides EZH2 due to

the loss of integrity of the PRC2 complex.⁵¹To verify whether

the degradation of PRC2 subunits by E7 results from the loss

of integrity of the PRC2 complex, we knocked down EZH2 by

small hairpin RNA (shRNA) in A549 and NCI-H1299 cells.

We found that EZH2 was downregulated in all examined cell

lines, and the downregulation of SUZ12 depended on the

degree of EZH2 knockdown. However, the change in the EED

level by EZH2-shRNA was cell-line-dependent. In addition, the

eﬀect of EZH2-shRNA on RbAp48 was not as signiﬁcant as

that of EZH2, EED, and SUZ12. Similar results were also

observed in A549 and NCI-H1299 cells in which EZH2 was

knocked down by small interfering RNA (siRNA) (Figure

11A,B,D,E). Thus, the mechanism of E7-mediated down-

regulation of PRC2 subunits may be partially diﬀerent from

that for the genetic knockdown of EZH2. We speculated that

E7 primarily degrades EZH2 and EED, and the down-

regulation of EED by E7 might be partially via its recruitment

of CRBN to approach EED to initiate subsequent ubiquitin-

proteasomal degradation, while the decreased level of SUZ12

may be an indirect outcome of the instability of the PRC2

complex induced by E7.

To verify that the antiproliferative activities of E7 are caused

by the degradation of the PRC2 complex, antiproliferative

activities of E7 and E7-NC-1 and genetic knockdown of EZH2

in A549 and H1299 cell lines were evaluated. As shown in

Figure 11C,F, E7 exhibited the strongest antiproliferative

activity in A549 and NCI-H1299 cell lines, and EZH2-

shRNA#1 and EZH2-shRNA#2 also showed moderate to good

antiproliferative activity in both cell lines, while the negative

control E7-NC-1 (EZH2 IC₅₀= 1.73 nM, Figure 7B) was

almost completely inactive. These results indicate that the

proliferation inhibitory activity exhibited by PROTAC E7 is

related to the degradation of PRC2 subunits.

EXPERIMENTAL SECTION

■

Chemistry. All reactions were carried out with magnetic stirring

and in dried glassware. Unless otherwise indicated, all chemicals and

solvents were purchased from commercial sources and used without

puriﬁcation treatment. Analytical thin-layer chromatography was

carried out on 0.20 mm silica gel plates (Haiyang, Qingdao,

Shandong, CN) with the QF-254 UV indicator. Column chromatog-

raphy was conducted using Haiyang silica gel 60 (300−400 mesh).

High-resolution mass spectra (HRMS) of all target compounds were

performed by a Waters Q-TOF Premier spectrometer with

acetonitrile and water as solvents. Nuclear magnetic resonance

(NMR) spectra for proton (¹H NMR) and 101 MHz for carbon (¹³C

NMR) were acquired on Bruker AVANCE_III400 spectrometers (400

MHz). Peak multiplicity of NMR signals was as follows: s, singlet; d,

doublet; t, triplet; q, quartet; m, multiplet. Chemical shift (δ): ppm

relative to Me₄Si (internal standard). Coupling constant: J (Hz).

High-performance liquid chromatography (HPLC) analysis was

performed on the Dionex 1996−2006 Version 6.80 (Phenomenex

C18 reversed-column, 4.6 mm × 150 mm, 5 μm; gradient elution of

methanol/H₂O = 35/65; ﬂow rate, 1.0 mL/min; detection wave-

length, 254 nm; temperature, 30 °C), and all of the title compounds

were of >95% purity.

2-(2,6-Dioxopiperidin-3-yl)-4-hydroxy-2,3-dihydro-1H-isoindole-

1,3-dione (1b). A solution of 1a (3.28 g, 20 mmol, 1.0 equiv) and 3-

aminopiperidine-2,6-dione (3.1 g, 24 mmol, 1.2 equiv) in acetic

anhydride (50 mL) was stirred at 140 °C for 6 h. Then, acetic

anhydride was evaporated under reduced pressure, and the residue

was poured into water (50 mL). The resulting precipitate was

collected by ﬁltration and washed with water to yield 1b (3.84 g,

71%). HRMS m/z calculated for C₁₃H₁₀N₂O₅[M + H]⁺: 275.0662,

found: 275.0686.

General Method to Synthesize 1c−k. A solution of 1b (1 mmol,

1.0 equiv), DIPEA (3 mmol, 3.0 equiv), and linkers (1.2 mmol, 1.2

equiv) in DMF (10 mL) was stirred at 85−100 °C for 3−6 h. The

reaction mixture was poured into water and then extracted with ethyl

acetate. The organic layer was washed with a saturated solution of

NaHCO₃and brine, dried over Na₂SO₄, concentrated, and puriﬁed by

column chromatography to aﬀord the corresponding products.

4-[(4-Bromobutoxy)]-2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-1H-

isoindole-1,3-dione (1c). White solid (195.8 mg, 48% yield). ¹H

NMR (400 MHz, DMSO-d₆) δ 11.08 (s, 1H), 7.82 (m, J = 8.5, 7.2

Hz, 1H), 7.52 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 5.08 (m, J

= 12.8, 5.4 Hz, 1H), 4.26 (t, J = 6.1 Hz, 2H), 3.66 (t, J = 6.7 Hz, 2H),

2.88 (m, J = 17.1, 13.9, 5.4 Hz, 1H), 2.57 (m, J = 16.4, 12.1 Hz, 2H),

2.03 (m, J = 12.5, 8.5, 5.8 Hz, 3H), 1.90 (m, J = 8.8, 6.1 Hz, 2H).

HRMS m/z calculated for C₁₇H₁₇BrN₂O₅[M + Na]⁺: 431.0213,

found: 431.0219.

CONCLUSIONS

■

Here, we describe the design and synthesis of a potent EZH2-

targeted PROTAC molecule E7, which induced proteasomal

degradation of PRC2 subunits, including EZH2, EED, SUZ12,

and RbAp48. Given that EZH2 mediates both gene silencing

via recruitment of the PRC2 complex to catalyze H3K27me3

and PRC2-independent transcriptional activation in various

cancers, current EZH2 inhibitors cannot completely abolish

the oncogenic function of EZH2. E7 fully suppressed the

oncogenic activity of EZH2 and showed signiﬁcantly

antiproliferative activities of cancers dependent on the catalytic

and noncatalytic activities of EZH2. In addition, our data

suggest that E7 recruits the E3 ubiquitin ligase to the vicinity

of the PRC2 complex and results in the indiscriminate

ubiquitination and degradation of all PRC2 subunits. It is

worth noting that more direct evidence is needed to interpret

the mechanism underlying how E7 induces the degradation of

proteins such as SUZ12, EED, and RbAp48. Overall, our

results shed light on the fact that pharmacological degradation

of EZH2 may oﬀer a potential therapeutic strategy for fully

blocking the oncogenic activity of EZH2. In general, E7 would

be a valuable chemical probe to investigate the biological

functions of EZH2, and it could also be a promising starting

point to develop more potent anticancer drugs for targeting the

EZH2 or PRC2 complex.

4-[(5-Bromopentyl)oxy]-2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-

1H-isoindole-1,3-dione (1d). White solid (236.3 mg, 56% yield). H

1

NMR (400 MHz, DMSO-d₆) δ 11.08 (s, 1H), 7.81 (m, J = 8.5, 7.2

Hz, 1H), 7.52 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 5.08 (m, J

= 12.8, 5.4 Hz, 1H), 4.22 (t, J = 6.3 Hz, 2H), 3.56 (t, J = 6.7 Hz, 2H),

2.94−2.82 (m, 1H), 2.67−2.53 (m, 2H), 2.03 (m, J = 13.5, 6.0, 3.4,

2.9 Hz, 1H), 1.90 (m, J = 6.8 Hz, 2H), 1.80 (m, J = 6.7 Hz, 2H), 1.59

(m, J = 9.6, 6.2 Hz, 2H). HRMS m/z calculated for C₁₈H₁₉BrN₂O₅

[M + Na]⁺: 445.0370, found: 445.0367.

4-((6-Bromohexyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-

1,3-dione (1e). White solid (161.3 mg, 37% yield). H NMR (400

1

MHz, DMSO-d₆) δ 11.09 (s, 1H), 7.81 (m, J = 8.5, 7.3 Hz, 1H), 7.51

(d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 5.08 (m, J = 12.8, 5.4

Hz, 1H), 4.21 (t, J = 6.3 Hz, 2H), 3.54 (t, J = 6.7 Hz, 2H), 2.96−2.82

(m, 1H), 2.67−2.53 (m, 2H), 2.03 (m, J = 13.5, 6.5, 6.0, 3.4 Hz, 1H),

1.84 (q, J = 6.8 Hz, 2H), 1.76 (q, J = 6.7 Hz, 2H), 1.47 (m, J = 7.8,

4.5, 4.1 Hz, 4H). HRMS m/z calculated for C₁₉H₂₁BrN₂O₅[M +

Na]⁺: 459.0526, found: 459.0523.

4-[(7-Bromoheptyl)oxy]-2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-

1H-isoindole-1,3-dione (1f). White solid (284.0 mg, 63% yield). H

NMR (400 MHz, DMSO-d₆) δ 11.09 (s, 1H), 7.81 (t, J = 7.9 Hz,

1H), 7.51 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 5.08 (m, J =

12.9, 5.4 Hz, 1H), 4.21 (t, J = 6.4 Hz, 2H), 3.53 (t, J = 6.7 Hz, 2H),

1

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2.89 (m, J = 19.1, 14.4, 5.4 Hz, 1H), 2.59 (d, J = 17.3 Hz, 2H), 2.11−

1.96 (m, 1H), 1.79 (m, J = 20.7, 6.6 Hz, 4H), 1.54−1.30 (m, 6H).

HRMS m/z calculated for C₂₀H₂₃BrN₂O₅[M + H]⁺: 451.0863,

found: 451.0852.

puriﬁed by column chromatography, aﬀording the title compound

(3.3 g, 57.8%). H NMR (400 MHz, CDCl₃) δ 7.23 (d, J = 2.0 Hz,

1H), 6.84 (d, J = 2.0 Hz, 1H), 4.01 (m, J = 11.9, 3.6 Hz, 2H), 3.87 (s,

3H), 3.66 (d, J = 7.4 Hz, 1H), 3.54 (m, J = 11.6, 2.3 Hz, 2H), 3.48

(m, 1H), 2.23 (s, 3H), 2.10−1.99 (m, 2H), 1.51 (m, J = 13.3, 10.6,

4.3 Hz, 2H). HRMS m/z calculated for C₁₄H₁₈BrNO₃[M + H]⁺:

328.0542, found: 328.0547.

1

4-[(8-Bromooctyl)oxy]-2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-

1

1H-isoindole-1,3-dione (1g). White solid (232.4 mg, 50% yield). H

NMR (400 MHz, DMSO-d₆) δ 11.08 (s, 1H), 7.80 (m, J = 8.5, 7.2

Hz, 1H), 7.51 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 5.07 (m, J

= 12.9, 5.4 Hz, 1H), 4.20 (t, J = 6.4 Hz, 2H), 3.52 (t, J = 6.7 Hz, 2H),

2.94−2.81 (m, 1H), 2.69−2.52 (m, 2H), 2.03 (m, J = 14.1, 6.7, 3.3,

2.9 Hz, 1H), 1.77 (m, J = 12.4, 6.7 Hz, 4H), 1.52−1.28 (m, 8H).

HRMS m/z calculated for C₂₁H₂₅BrN₂O₅[M + H]⁺: 465.1020,

found: 465.0994;

5-Bromo-3-[ethyl(oxan-4-yl)amino]-2-methylbenzoic Acid (2d).

To a solution of 2c (3 g, 9.1 mmol, 1.0 equiv) and anhydrous

acetaldehyde (2.3 g, 27.3 mmol, 3.0 equiv) in 1,2-dichloroethane (25

mL), acetic acid (1.1 g, 18.2 mmol, 2.0 equiv) was added and reacted

at 30 °C for 1 h. Then, NaBH(OAc)₃(1.48 g, 23 mmol, 2.5 equiv)

was added and stirred overnight at room temperature. The reaction

mixture was concentrated under reduced pressure to obtain the crude

material, which was puriﬁed by column chromatography to aﬀord 2d

4-[(9-Bromononyl)oxy]-2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-

1

1H-isoindole-1,3-dione (1h). White solid (292.0 mg, 61% yield). H

1

NMR (400 MHz, DMSO-d₆) δ 11.09 (s, 1H), 7.81 (m, J = 8.5, 7.3

Hz, 1H), 7.51 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 5.08 (m, J

= 12.8, 5.4 Hz, 1H), 4.20 (t, J = 6.4 Hz, 2H), 3.52 (m, J = 6.7, 2.0 Hz,

2H), 2.89 (m, J = 16.8, 13.9, 5.3 Hz, 1H), 2.58 (m, J = 16.9, 12.4 Hz,

2H), 2.09−1.99 (m, 1H), 1.84−1.72 (m, 4H), 1.46 (t, J = 7.6 Hz,

2H), 1.37 (d, J = 7.2 Hz, 4H), 1.33−1.25 (m, 4H). HRMS m/z

calculated for C₂₂H₂₇BrN₂O₅[M + H]⁺: 479.1176, found: 479.1164.

4-[(10-Bromodecyl)oxy]-2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-

(2.3 g, 71.0%). H NMR (400 MHz, CDCl₃) δ 7.71 (d, J = 2.0 Hz,

1H), 7.37 (d, J = 2.0 Hz, 1H), 3.96 (d, J = 12.1 Hz, 2H), 3.89 (s, 3H),

3.32 (m, J = 11.3, 2.9 Hz, 2H), 3.05 (q, J = 7.1 Hz, 2H), 2.99−2.85

(m, 1H), 2.45 (s, 3H), 1.80−1.54 (m, 4H), 0.87 (t, J = 7.0 Hz, 3H).

HRMS m/z calculated for C₁₆H₂₃BrNO₃[M + H]⁺: 356.0861, found:

356.0854.

5-Bromo-3-[ethyl(oxan-4-yl)amino]-2-methylbenzoic Acid (2e).

A solution of 2d (2 g, 5.6 mmol) in 20 mL of MeOH containing 50%

aqueous NaOH solution (2 M) was stirred at room temperature

overnight and then neutralized by HCl (2 M). The resulting white

precipitate was collected by ﬁltration and washed with water to yield

2e (1.75 g, 91.2%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.15 (s, 1H),

7.62 (d, J = 2.1 Hz, 1H), 7.49 (d, J = 2.1 Hz, 1H), 3.83 (m, J = 9.5, 2.3

Hz, 2H), 3.26 (m, J = 11.6, 2.1 Hz, 2H), 3.04 (q, J = 7.1 Hz, 2H),

3.01−2.92 (m, 1H), 2.40 (s, 3H), 1.66−1.58 (m, 2H), 1.50 (m, J =

11.7, 4.3 Hz, 2H), 0.80 (t, J = 7.0 Hz, 3H). HRMS m/z calculated for

C₁₅H₂₀BrNO₃[M + H]⁺: 342.0699, found: 342.0706.

1

1H-isoindole-1,3-dione (1i). White solid (201.7 mg, 41% yield). H

NMR (400 MHz, DMSO-d₆) δ 11.09 (s, 1H), 7.84−7.75 (m, 1H),

7.51 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 5.07 (m, J = 12.8,

5.4 Hz, 1H), 4.20 (t, J = 6.4 Hz, 2H), 3.52 (m, J = 6.7, 2.2 Hz, 2H),

2.88 (m, J = 17.0, 13.9, 5.4 Hz, 1H), 2.58 (m, J = 16.3, 12.2 Hz, 2H),

2.03 (m, J = 8.0, 4.4 Hz, 1H), 1.76 (m, J = 14.1, 11.4, 6.7 Hz, 4H),

1.45 (q, J = 7.4 Hz, 2H), 1.36 (q, J = 6.3 Hz, 4H), 1.30 (d, J = 18.4

Hz, 6H). HRMS m/z calculated for C₂₃H₂₉BrN₂O₅[M + Na]⁺:

515.1152, found: 515.1147.

4-[(11-Bromoundecyl)oxy]-2-(2,6-dioxopiperidin-3-yl)-2,3-dihy-

dro-1H-isoindole-1,3-dione (1j). White solid (293.5 mg, 58% yield).

¹H NMR (400 MHz, DMSO-d₆) δ 11.08 (s, 1H), 7.80 (t, J = 7.9 Hz,

1H), 7.51 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 7.3 Hz, 1H), 5.07 (m, J =

13.0, 5.4 Hz, 1H), 4.20 (t, J = 6.4 Hz, 2H), 3.51 (t, J = 6.7 Hz, 2H),

2.88 (s, 1H), 2.61 (s, 2H), 2.03 (d, J = 13.5 Hz, 1H), 1.76 (t, J = 9.2

Hz, 4H), 1.45 (t, J = 7.8 Hz, 2H), 1.41−1.33 (m, 4H), 1.27 (s, 8H).

HRMS m/z calculated for C₂₄H₃₁BrN₂O₅[M + Na]⁺: 529.1308,

found: 529.1298.

4-[(12-Bromododecyl)oxy]-2-(2,6-dioxopiperidin-3-yl)-2,3-dihy-

dro-1H-isoindole-1,3-dione (1k). White solid (176.8 mg, 34% yield).

¹H NMR (400 MHz, DMSO-d₆) δ 11.09 (s, 1H), 7.81 (t, J = 7.8 Hz,

1H), 7.47 (m, J = 27.8, 7.8 Hz, 2H), 5.08 (m, J = 13.0, 5.5 Hz, 1H),

4.20 (t, J = 6.5 Hz, 2H), 3.51 (t, J = 6.7 Hz, 2H), 2.98−2.80 (m, 1H),

2.60 (d, J = 17.2 Hz, 2H), 2.15−1.96 (m, 1H), 1.77 (q, J = 8.6, 8.2

Hz, 4H), 1.53−1.41 (m, 2H), 1.35 (s, 4H), 1.26 (s, 10H). HRMS m/

z calculated for C₂₅H₃₃BrN₂O₅[M + Na]⁺: 543.1465, found:

543.1472.

Methyl 3-Amino-5-bromo-2-methylbenzoate (2b). Methyl 3-

bromo-5-nitrobenzoate 2a (5.5 g, 20 mmol, 1.0 equiv) and

ammonium chloride (5.6 g, 100 mmol, 5.0 equiv) were dissolved in

water/ethanol mixtures (60 mL, H₂O/methanol = 1:3). When the

mixtures were heated to 80 °C, iron powder (11.2 g, 200 mmol, 10.0

equiv) was added under stirring for 1 h. The reaction mixture was

ﬁltered through diatomite and washed with methanol. Then, the

combined ﬁltrate was evaporated to get a residue, which was dissolved

in DCM, washed with water, dried, and concentrated to give 2b

without further puriﬁcation (4.41 g, 92%). ¹H NMR (400 MHz,

CDCl₃) δ 7.31 (d, J = 2.6 Hz, 1H), 6.92 (d, J = 2.6 Hz, 1H), 3.87 (s,

3H), 3.82 (s, 2H), 2.26 (s, 3H). HRMS m/z calculated for

C₉H₁₀BrNO₂[M + H]⁺: 243.9967, found: 243.9958.

Methyl 5-Bromo-2-methyl-3-[(oxan-4-yl)amino]benzoate (2c).

To a solution of 2b (4 g, 17.4 mmol, 1.0 equiv) and tetrahydro-4-

pyrone (4.4 g, 52.2 mmol, 3.0 equiv) in CDCl₃(50 mL), acetic acid

(2.1 g, 34.8 mmol, 2.0 equiv) was added dropwise to the reaction

mixture and stirred at 30 °C for 3 h. Then, NaBH(OAc)₃(2.7 g, 43.6

mmol, 2.5 equiv) was added slowly and stirred overnight at room

temperature. The reaction solvent was evaporated and the residue was

5-Bromo-N-[(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)-

methyl]-3-[ethyl(oxan-4-yl)amino]-2-methylbenzamide (2f). The

acid 2e (1.5 g, 4.4 mmol, 1.1 equiv) and 3-(aminomethyl)-4,6-

dimethylpyridin-2(1H)-one (4 mmol, 0.61 g, 1.0 equiv), HOAT (3

mmol, 1.1 g, 0.75 equiv), and EDCI (3 mmol, 1.14 g, 0.75 equiv)

were added to DMSO (10 mL). Then, the mixture was reacted at 45

°C for 20 h and then poured into ice-cold water (100 mL). The

resulting precipitate was collected and air-dried. The precipitate was

1

puriﬁed by column chromatography to aﬀord 2f (1.42 g, 68%). H

NMR (400 MHz, DMSO-d₆) δ 11.47 (s, 1H), 8.23 (t, J = 5.0 Hz,

1H), 7.31 (d, J = 2.0 Hz, 1H), 7.09 (d, J = 2.0 Hz, 1H), 5.86 (s, 1H),

4.25 (d, J = 4.9 Hz, 2H), 3.83 (m, J = 10.8, 3.5 Hz, 2H), 3.28−3.18

(m, 2H), 3.01 (q, J = 7.0 Hz, 2H), 2.97−2.89 (m, 1H), 2.54 (s, 1H),

2.19 (s, 3H), 2.15 (s, 3H), 2.11 (s, 3H), 1.60 (d, J = 12.4 Hz, 2H),

1.49 (m, J = 11.7, 4.2 Hz, 2H). HRMS m/z calculated for

C₂₃H₃₀BrN₃O₃[M + H]⁺: 476.1543, found: 476.1552.

N-[(4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl]-5-

[ethyl(oxan-4-yl)amino]-4-methyl-4′-[(piperazin-1-yl)methyl]-[1,1′-

biphenyl]-3-carboxamide (2g). To a solution of 2f (1.2 g, 2.5 mmol,

1.0 equiv), 4-[(4-boc-1-piperazinyl)methyl]phenylboronic acid pina-

col ester (1.21 g, 3 mmol, 1.2 equiv) in 1,4-dioxane, and H₂O (4:1, 30

mL) were added K₂CO₃(3.75 mmol, 0.52 g, 1.5 equiv) and

Pd(dppf)Cl₂(0.2 mmol, 146 mg, 8% equiv). The mixture was

degassed for 5 min and successively reacted at 100 °C for 8 h. The

mixture was diluted with ethyl acetate and ﬁltered through diatomite.

Then, the ﬁltrate was evaporated to obtain the residue, which was

successively puriﬁed by chromatography eluting with DCM/CH₃OH

= 10:1−20:1 to aﬀord the product. To a mixture of coupling products

in CDCl₃(25 mL), TFA (1 mL) was added and stirred at 25 °C for 1

h. The mixture was neutralized by the saturated Na₂CO₃solution (2

M, 5 mL); then, the mixture was extracted and washed with water (25

mL) and brine, dried over Na₂SO₄, and evaporated, and the residue

was puriﬁed by column chromatography to give 2g (1.04 g, 74%

yield). ¹H NMR (400 MHz, DMSO-d₆) δ 11.47 (s, 1H), 8.63 (s, 1H),

8.20 (t, J = 5.0 Hz, 1H), 7.60 (d, J = 7.9 Hz, 2H), 7.38 (d, J = 8.0 Hz,

3H), 7.28−7.16 (m, 1H), 5.86 (s, 1H), 4.30 (d, J = 4.9 Hz, 2H),

3.89−3.77 (m, 2H), 3.57 (s, 2H), 3.25 (t, J = 11.4 Hz, 2H), 3.09 (d, J

= 6.2 Hz, 6H), 3.04−2.98 (m, 1H), 2.57 (t, J = 4.8 Hz, 4H), 2.25 (s,

2841

https://dx.doi.org/10.1021/acs.jmedchem.0c02234

J. Med. Chem. 2021, 64, 2829−2848

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

3H), 2.21 (s, 3H), 2.11 (s, 3H), 1.66 (d, J = 12.3 Hz, 2H), 1.53 (m, J

= 12.4, 12.0, 4.1 Hz, 2H), 0.84 (t, J = 6.9 Hz, 3H). HRMS m/z

calculated for C₃₄H₄₅N₅O₃[M + H]⁺: 572.3595, found: 572.3601.

General Method to Synthesize Target Compounds E4−E12 and

G4−G12. To a solution of GSK126 or 2g (0.25 mmol, 1 equiv) and

4-hydroxythalidomide analogues containing the alkyl group 1c−k (0.3

mmol, 1.2 equiv) in DMF (5 mL), NaHCO₃(0.5 mmol, 2.0 equiv)

was added and stirred at 85−100 °C for 3−8 h. When the reaction

was complete, the residue was extracted with ethyl acetate and water;

then, the organic layer was washed successively with water and brine,

dried over Na₂SO₄, and evaporated, and the crude mixture was

puriﬁed by column chromatography to aﬀord the corresponding

products.

149.8, 146.0, 143.1, 138.2, 137.5, 136.4, 133.7, 131.2, 130.2, 126.5,

124.9, 123.4, 122.2, 120.3, 116.7, 116.5, 115.6, 110.2, 108.1, 107.8,

107.4, 69.3, 58.3, 53.1, 52.1, 49.2, 45.4, 35.5, 31.4, 30.0, 28.9, 27.0,

26.7, 25.7, 22.5, 21.3, 19.4, 18.7, 12.2, 11.2. HRMS m/z calculated for

C₅₀H₅₈N₈O₇[M + H]⁺: 883.4501, found: 882.4502.

1-[(2S)-Butan-2-yl]-N-[(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-

yl)methyl]-6-{6-[4-(7-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-di-

hydro-1H-isoindol-4-yl]oxy}heptyl)piperazin-1-yl]pyridine-3-yl}-3-

methyl-1H-indole-4-carboxamide (G7). Gray solid (73.6 mg, 33%

1

yield). H NMR (400 MHz, DMSO-d₆) δ 11.45 (s, 1H), 11.09 (s,

1H), 8.49 (d, J = 2.5 Hz, 1H), 8.12 (t, J = 5.1 Hz, 1H), 7.90 (m, J =

8.8, 2.6 Hz, 1H), 7.81 (m, J = 8.5, 7.3 Hz, 1H), 7.72 (d, J = 1.5 Hz,

1H), 7.52 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.25 (s, 1H),

7.17 (d, J = 1.5 Hz, 1H), 6.89 (d, J = 8.9 Hz, 1H), 5.86 (s, 1H), 5.08

(m, J = 12.9, 5.4 Hz, 1H), 4.60 (q, J = 6.9 Hz, 1H), 4.35 (d, J = 5.1

Hz, 2H), 4.21 (t, J = 6.4 Hz, 2H), 3.50 (s, 4H), 2.88 (m, J = 17.3,

14.0, 5.4 Hz, 1H), 2.69−2.54 (m, 2H), 2.49−2.40 (m, 4H), 2.31 (d, J

= 7.6 Hz, 2H), 2.24 (s, 3H), 2.16 (s, 3H), 2.11 (s, 3H), 2.03 (m, J =

11.9, 6.1, 3.5 Hz, 1H), 1.78 (m, J = 19.7, 6.9 Hz, 4H), 1.47 (q, J = 7.6

Hz, 4H), 1.40 (d, J = 6.6 Hz, 3H), 1.34 (m, J = 9.1, 4.3 Hz, 2H), 1.24

(d, J = 4.8 Hz, 2H), 0.73 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 173.2, 170.4, 169.2, 167.3, 165.8, 163.6, 158.5, 156.5,

149.8, 146.0, 143.1, 138.2, 137.5, 136.4, 133.7, 131.2, 127.9, 126.5,

123.4, 122.2, 120.3, 116.7, 116.5, 115.6, 110.2, 108.1, 107.8, 107.4,

69.28, 58.4, 53.1, 52.1, 49.2, 45.4, 35.5, 31.4, 30.0, 29.0, 28.9, 27.4,

25.74, 22.5, 21.3, 19.4, 18.7, 12.1, 11.2. HRMS m/z calculated for

C₅₁H₆₀N₈O₇[M + H]⁺: 897.4658, found: 897.4656.

1-[(2S)-Butan-2-yl]-N-[(4,6-dimethyl-2-oxo-1,2-dihydropyridine-

3-yl)methyl]-6-{6-[4-(4-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-

dihydro-1H-isoindol-4-yl]oxy}butyl)piperazin-1-yl]pyridine-3-yl}-3-

methyl-1H-indole-4-carboxamide (G4). White solid (72.1 mg, 34%

1

yield). H NMR (400 MHz, DMSO-d₆) δ 11.45 (s, 1H), 11.10 (s,

1H), 8.50 (d, J = 2.6 Hz, 1H), 8.12 (t, J = 5.2 Hz, 1H), 7.91 (m, J =

8.8, 2.7 Hz, 1H), 7.81 (t, J = 7.9 Hz, 1H), 7.72 (s, 1H), 7.53 (d, J =

8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.25 (s, 1H), 7.17 (s, 1H), 6.89

(d, J = 8.9 Hz, 1H), 5.86 (s, 1H), 5.09 (m, J = 12.9, 5.4 Hz, 1H), 4.59

(m, J = 7.0 Hz, 1H), 4.35 (d, J = 5.1 Hz, 2H), 4.26 (t, J = 6.3 Hz, 2H),

3.51 (d, J = 4.9 Hz, 4H), 2.90 (m, J = 17.3, 15.5, 5.3 Hz, 1H), 2.70−

2.53 (m, 2H), 2.47 (s, 2H), 2.41 (t, J = 6.8 Hz, 2H), 2.24 (s, 3H),

2.16 (s, 3H), 2.11 (s, 3H), 2.08−1.96 (m, 1H), 1.82 (q, J = 6.9 Hz,

4H), 1.69 (q, J = 7.4 Hz, 2H), 1.40 (d, J = 6.6 Hz, 3H), 1.23 (s, 2H),

0.73 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.2,

170.4, 169.2, 167.3, 165.8, 163.6, 160.6, 158.5, 158.5, 156.5, 149.8,

146.0, 143.1, 138.2, 137.5, 136.4, 133.7, 131.2, 130.2, 126.5, 124.9,

123.4, 122.2, 120.3, 116.7, 116.5, 115.6, 110.2, 108.2, 107.2, 107.4,

69.2, 57.8, 53.0, 52.1, 49.2, 45.4, 35.5, 31.4, 30.0, 26.9, 22.9, 22.5,

21.3, 19.4, 18.7, 12.2, 11.2. HRMS m/z calculated for C₄₈H₅₄N₈O₇

[M + H]⁺: 855.4188, found: 855.4185.

1-[(2S)-Butan-2-yl]-N-[(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-

yl)methyl]-6-{6-[4-(8-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-di-

hydro-1H-isoindol-4-yl]oxy}octyl)piperazin-1-yl]pyridine-3-yl}-3-

methyl-1H-indole-4-carboxamide (G8). White solid (41.6 mg, 18%

1

yield). H NMR (400 MHz, DMSO-d₆) δ 11.45 (s, 1H), 11.10 (s,

1H), 8.49 (d, J = 2.6 Hz, 1H), 8.13 (t, J = 5.1 Hz, 1H), 7.90 (m, J =

8.8, 2.6 Hz, 1H), 7.80 (m, J = 8.5, 7.2 Hz, 1H), 7.72 (d, J = 1.5 Hz,

1H), 7.51 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.25 (s, 1H),

7.17 (d, J = 1.4 Hz, 1H), 6.89 (d, J = 8.9 Hz, 1H), 5.86 (s, 1H), 5.08

(m, J = 12.9, 5.4 Hz, 1H), 4.60 (q, J = 6.9 Hz, 1H), 4.35 (d, J = 5.0

Hz, 2H), 4.21 (t, J = 6.3 Hz, 2H), 3.50 (t, J = 5.7 Hz, 4H), 2.88 (m, J

= 17.3, 14.1, 5.4 Hz, 1H), 2.58 (m, J = 20.8, 6.8 Hz, 2H), 2.45 (s,

4H), 2.31 (d, J = 7.4 Hz, 2H), 2.24 (s, 3H), 2.16 (s, 3H), 2.11 (s,

3H), 2.07−1.98 (m, 1H), 1.78 (m, J = 13.8, 6.7 Hz, 4H), 1.48 (d, J =

7.1 Hz, 4H), 1.40 (d, J = 6.7 Hz, 3H), 1.31 (d, J = 10.9 Hz, 4H), 1.23

(s, 2H), 0.73 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ

173.2, 170.4, 169.2, 167.3, 165.8, 163.6, 158.5, 156.5, 149.8, 146.0,

143.1, 138.2, 137.5, 136.4, 133.7, 131.2, 130.2, 126.5, 124.9, 123.4,

122.2, 120.3, 116.7, 116.5, 115.6, 108.1, 107.8, 107.4, 69.3, 58.5, 53.1,

52.1, 49.2, 45.4, 35.5, 31.4, 30.0, 29.4, 29.1, 28.9, 27.4, 26.7, 25.7,

22.5, 21.3, 19.4, 18.7, 12.1, 11.2. HRMS m/z calculated for

C₅₂H₆₂N₈O₇[M + H]⁺: 911.4814, found: 911.4819.

1-[(2S)-Butan-2-yl]-N-[(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-

yl)methyl]-6-{6-[4-(5-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-di-

hydro-1H-isoindol-4-yl]oxy}pentyl)piperazin-1-yl]pyridine-3-yl}-3-

methyl-1H-indole-4-carboxamide (G5). White solid (45.6 mg, 21%

1

yield). H NMR (400 MHz, DMSO-d₆) δ 11.45 (s, 1H), 11.10 (s,

1H), 8.50 (d, J = 2.4 Hz, 1H), 8.12 (t, J = 5.1 Hz, 1H), 7.91 (m, J =

8.8, 2.6 Hz, 1H), 7.81 (t, J = 7.9 Hz, 1H), 7.72 (s, 1H), 7.52 (d, J =

8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.25 (s, 1H), 7.18 (d, J = 1.4

Hz, 1H), 6.89 (d, J = 8.9 Hz, 1H), 5.86 (s, 1H), 5.08 (m, J = 12.9, 5.4

Hz, 1H), 4.59 (m, J = 6.9 Hz, 1H), 4.36 (d, J = 5.1 Hz, 2H), 4.23 (t, J

= 6.3 Hz, 2H), 3.51 (t, J = 4.7 Hz, 4H), 2.89 (m, J = 17.3, 14.0, 5.4

Hz, 1H), 2.66−2.53 (m, 2H), 2.48 (s, 2H), 2.34 (d, J = 7.4 Hz, 2H),

2.24 (s, 3H), 2.17 (s, 3H), 2.11 (s, 3H), 2.07−1.99 (m, 1H), 1.80 (m,

J = 8.4, 4.8 Hz, 4H), 1.53 (m, J = 19.9, 7.1 Hz, 4H), 1.41 (d, J = 6.6

Hz, 3H), 1.24 (d, J = 5.1 Hz, 2H), 0.73 (t, J = 7.3 Hz, 3H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 173.2, 170.4, 169.2, 167.3, 165.8, 163.6,

158.5, 156.5, 149.8, 146.0, 143.1, 138.2, 137.5, 136.4, 133.7, 131.2,

130.2, 126.5, 124.9, 123.4, 122.24, 120.3, 116.7, 116.5, 115.6, 110.2,

108.2, 107.8, 107.4, 69.3, 58.3, 53.0, 52.0, 49.2, 45.4, 35.5, 31.44, 30.0,

28.8, 26.4, 23.8, 22.5, 21.3, 19.4, 18.7, 12.2, 11.2. HRMS m/z

calculated for C₄₉H₅₆N₈O₇[M + H]⁺: 869.4345, found: 869.4343.

1-[(2S)-Butan-2-yl]-N-[(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-

yl)methyl]-6-{6-[4-(6-{[2-(2,6-dioxopiperidin3yl)-1,3-dioxo-2,3-dihy-

dro-1H-isoindol-4-yl]oxy}hexyl)piperazin-1-yl]pyridine-3-yl}-3-

methyl-1H-indole-4-carboxamide (G6). Light-gray solid (63.3 mg,

1-[(2S)-Butan-2-yl]-N-[(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-

yl)methyl]-6-{6-[4-(9-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-di-

hydro-1H-isoindol-4-yl]oxy}nonyl)piperazin-1-yl]pyridine-3-yl}-3-

methyl-1H-indole-4-carboxamide (G9). White solid (85.6 mg, 37%

1

yield). H NMR (400 MHz, DMSO-d₆) δ 11.45 (s, 1H), 11.11 (s,

1H), 8.49 (d, J = 2.5 Hz, 1H), 8.13 (t, J = 5.1 Hz, 1H), 7.90 (m, J =

8.8, 2.6 Hz, 1H), 7.80 (t, J = 7.9 Hz, 1H), 7.72 (s, 1H), 7.51 (d, J =

8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.25 (s, 1H), 7.22−7.13 (m,

1H), 6.89 (d, J = 8.9 Hz, 1H), 5.86 (s, 1H), 5.08 (m, J = 12.9, 5.4 Hz,

1H), 4.60 (q, J = 6.9 Hz, 1H), 4.35 (d, J = 5.1 Hz, 2H), 4.20 (t, J = 6.3

Hz, 2H), 3.50 (s, 4H), 2.88 (m, J = 17.8, 14.4, 5.3 Hz, 1H), 2.68−2.54

(m, 2H), 2.45 (s, 4H), 2.30 (s, 2H), 2.24 (s, 3H), 2.16 (s, 3H), 2.11

(s, 3H), 2.03 (m, J = 11.6, 5.8 Hz, 1H), 1.86−1.69 (m, 4H), 1.45 (d, J

= 7.2 Hz, 4H), 1.40 (d, J = 6.6 Hz, 3H), 1.38−1.27 (m, 6H), 1.23 (s,

2H), 0.73 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ

173.2, 170.4, 169.2, 167.3, 165.8, 163.6, 158.5, 156.5, 149.8, 146.0,

143.1, 138.2, 137.5, 136.4, 133.7, 131.2, 130.2, 126.5, 124.9, 123.4,

122.2, 120.3, 116.7, 116.5, 115.6, 110.2, 108.1, 107.8, 107.4, 69.3,

58.5, 56.5, 53.1, 52.1, 49.2, 45.4, 35.5, 31.4, 30.0, 29.4, 29.1, 28.9,

27.4, 26.8, 25.7, 22.5, 21.3, 19.4, 18.7, 12.2, 11.2. HRMS m/z

calculated for C₅₃H₆₄N₈O₇[M + H]⁺: 925.4971, found: 925.4975.

1

29% yield). H NMR (400 MHz, DMSO-d₆) δ 11.45 (s, 1H), 11.10

(s, 1H), 8.49 (d, J = 2.5 Hz, 1H), 8.12 (t, J = 5.1 Hz, 1H), 7.90 (m, J

= 8.8, 2.6 Hz, 1H), 7.81 (t, J = 7.9 Hz, 1H), 7.72 (d, J = 1.5 Hz, 1H),

7.52 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.25 (s, 1H), 7.17

(d, J = 1.4 Hz, 1H), 6.89 (d, J = 8.9 Hz, 1H), 5.86 (s, 1H), 5.08 (m, J

= 12.9, 5.4 Hz, 1H), 4.60 (m, J = 6.9 Hz, 1H), 4.35 (d, J = 5.1 Hz,

2H), 4.21 (t, J = 6.4 Hz, 2H), 3.50 (t, J = 4.8 Hz, 4H), 2.89 (m, J =

17.5, 14.1, 5.4 Hz, 1H), 2.66−2.54 (m, 2H), 2.46 (s, 4H), 2.32 (d, J =

7.2 Hz, 2H), 2.24 (s, 3H), 2.16 (s, 3H), 2.11 (s, 3H), 2.07−2.00 (m,

1H), 1.86−1.72 (m, 4H), 1.50 (m, J = 7.5 Hz, 4H), 1.40 (t, J = 7.3

Hz, 3H), 1.23 (s, 2H), 0.73 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 173.2, 170.4, 169.2, 167.3, 165.8, 163.6, 158.5, 156.51,

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Article

1-[(2S)-Butan-2-yl]-N-[(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-

yl)methyl]-6-{6-[4-(10-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-

dihydro-1H-isoindol-4-yl]oxy}decyl)piperazin-1-yl]pyridine-3-yl}-3-

methyl-1H-indole-4-carboxamide (G10). Light-yellow solid (54.1

Hz, 1H), 7.27−7.15 (m, 4H), 5.91 (s, 1H), 4.93 (m, J = 11.9, 5.4 Hz,

1H), 4.54 (d, J = 5.9 Hz, 2H), 4.19 (q, J = 6.2 Hz, 2H), 3.94 (m, J =

11.6, 3.3 Hz, 2H), 3.65−3.52 (m, 4H), 3.41 (s, 1H), 3.31 (m, J =

11.3, 2.9 Hz, 2H), 3.08 (q, J = 7.0 Hz, 2H), 3.04−2.95 (m, 1H),

2.88−2.68 (m, 3H), 2.58 (t, J = 5.0 Hz, 4H), 2.45 (t, J = 7.1 Hz, 2H),

2.39 (s, 3H), 2.33 (s, 3H), 2.14 (s, 3H), 2.13−2.05 (m, 1H), 1.92 (t, J

= 6.8 Hz, 2H), 1.81−1.50 (m, 9H), 0.88 (t, J = 7.0 Hz, 3H). ¹³C

NMR (101 MHz, CDCl₃) δ 171.7, 168.7, 167.1, 165.1, 158.7, 156.6,

150.7, 149.6, 145.9, 142.6, 139.3, 136.5, 135.9, 135.6, 133.9, 132.8,

125.6, 123.2, 122.1, 120.1, 118.8, 115.7, 109.9, 106.9, 69.3, 67.3, 58.4,

58.3, 52.8, 49.1, 45.3, 41.6, 36.1, 31.4, 30.5, 28.8, 26.3, 24.0, 22.7,

19.7, 18.7, 14.7, 12.8. HRMS m/z calculated for C₅₁H₆₂N₇O₈[M +

H]⁺: 900.4654, found: 900.4653.

1

mg, 23% yield). H NMR (400 MHz, DMSO-d₆) δ 11.46 (s, 1H),

11.12 (s, 1H), 8.49 (d, J = 2.5 Hz, 1H), 8.13 (t, J = 5.2 Hz, 1H), 7.90

(m, J = 8.8, 2.6 Hz, 1H), 7.84−7.76 (m, 1H), 7.72 (d, J = 1.5 Hz,

1H), 7.50 (d, J = 8.6 Hz, 1H), 7.43 (d, J = 7.2 Hz, 1H), 7.25 (s, 1H),

7.18 (d, J = 1.4 Hz, 1H), 6.89 (d, J = 8.9 Hz, 1H), 5.86 (s, 1H), 5.08

(m, J = 12.8, 5.4 Hz, 1H), 4.59 (m, J = 6.8 Hz, 1H), 4.36 (d, J = 5.1

Hz, 2H), 4.20 (t, J = 6.4 Hz, 2H), 3.50 (t, J = 4.9 Hz, 4H), 2.89 (m, J

= 17.3, 14.0, 5.3 Hz, 1H), 2.65−2.53 (m, 2H), 2.45 (t, J = 5.0 Hz,

4H), 2.29 (t, J = 7.4 Hz, 2H), 2.24 (s, 3H), 2.17 (s, 3H), 2.11 (s, 3H),

2.03 (m, J = 13.3, 6.3, 5.8, 3.2 Hz, 1H), 1.86−1.71 (m, 4H), 1.45 (d, J

= 7.7 Hz, 4H), 1.40 (d, J = 6.7 Hz, 3H), 1.29 (s, 8H), 1.23 (s, 2H),

0.73 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.3,

170.4, 169.3, 167.4, 165.8, 163.7, 158.5, 156.5, 150.1, 145.9, 143.2,

138.2, 137.5, 136.4, 133.6, 131.0, 130.2, 126.5, 124.9, 123.4, 122.1,

120.2, 116.6, 116.4, 115.6, 110.1, 108.2, 107.5, 69.3, 58.4, 53.0, 52.1,

49.2, 45.3, 35.5, 31.4, 29.9, 29.4, 29.1, 28.8, 27.4, 26.6, 25.7, 22.5,

21.3, 19.4, 18.6, 12.1, 11.2. HRMS m/z calculated for C₅₄H₆₆N₈O₇

[M + H]⁺: 939.5127, found: 939.5124.

N-[(4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl]-4′-{[4-

(5-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-

4-yl]oxy}pentyl)piperazin-1-yl]methyl}-5-[ethyl(oxan-4-yl)amino]-

4-methyl-[1,1′-biphenyl]-3-carboxamide (E5). Yellow solid (61.7

1

mg, 27% yield). H NMR (400 MHz, DMSO-d₆) δ 11.45 (s, 1H),

11.08 (s, 1H), 8.17 (t, J = 5.0 Hz, 1H), 7.80 (t, J = 7.9 Hz, 1H), 7.57

(d, J = 7.9 Hz, 2H), 7.51 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 7.2 Hz, 1H),

7.42−7.38 (m, 1H), 7.35 (d, J = 7.8 Hz, 2H), 7.22 (d, J = 1.7 Hz,

1H), 5.86 (s, 1H), 5.07 (m, J = 12.9, 5.4 Hz, 1H), 4.30 (d, J = 5.0 Hz,

2H), 4.23 (t, J = 6.3 Hz, 2H), 3.88−3.78 (m, 2H), 3.46 (s, 2H), 3.25

(t, J = 11.4 Hz, 2H), 3.09 (q, J = 7.2 Hz, 2H), 3.01 (d, J = 10.6 Hz,

1H), 2.94−2.82 (m, 1H), 2.57 (m, J = 15.3, 11.4 Hz, 2H), 2.45−2.29

(m, 8H), 2.25 (s, 3H), 2.21 (s, 3H), 2.11 (s, 3H), 2.07−1.97 (m, 1H),

1.83−1.71 (m, 2H), 1.71−1.59 (m, 4H), 1.59−1.48 (m, 2H), 1.25 (d,

J = 13.0 Hz, 4H), 0.84 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 173.2, 170.4, 169.5, 167.3, 165.8, 163.5, 156.5, 149.9,

149.3, 143.2, 140.1, 138.9, 137.9, 137.5, 137.5, 133.7, 133.1, 129.9,

126.8, 123.4, 122.1, 121.3, 120.3, 116.7, 115.6, 107.8, 69.2, 66.8, 62.2,

58.3, 57.7, 53.2, 49.2, 41.7, 35.4, 31.4, 30.8, 29.5, 26.8, 22.9, 22.5,

19.4, 18.7, 15.0, 13.2. HRMS m/z calculated for C₅₂H₆₄N₇O₈[M +

H]⁺: 914.4811, found: 914.4812.

1-[(2S)-Butan-2-yl]-N-[(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-

yl)methyl]-6-{6-[4-(11-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-

dihydro-1H-isoindol4yl]oxy}undecyl)piperazin-1-yl]pyridine-3-yl}-

3-methyl-1H-indole-4-carboxamide (G11). Light-yellow solid (97.6

1

mg, 41% yield). H NMR (400 MHz, DMSO-d₆) δ 11.45 (s, 1H),

11.10 (s, 1H), 8.49 (d, J = 2.5 Hz, 1H), 8.12 (t, J = 5.1 Hz, 1H), 7.90

(m, J = 8.8, 2.6 Hz, 1H), 7.80 (t, J = 7.9 Hz, 1H), 7.72 (s, 1H), 7.50

(d, J = 8.5 Hz, 1H), 7.43 (d, J = 7.2 Hz, 1H), 7.25 (s, 1H), 7.20−7.11

(m, 1H), 6.89 (d, J = 8.9 Hz, 1H), 5.86 (s, 1H), 5.08 (m, J = 12.9, 5.4

Hz, 1H), 4.60 (m, J = 6.7 Hz, 1H), 4.35 (d, J = 5.1 Hz, 2H), 4.20 (t, J

= 6.4 Hz, 2H), 3.51 (s, 4H), 2.96−2.83 (m, 1H), 2.58 (m, J = 15.9,

11.8 Hz, 2H), 2.47 (s, 4H), 2.31 (d, J = 7.8 Hz, 2H), 2.24 (s, 3H),

2.16 (s, 3H), 2.11 (s, 3H), 2.03 (m, J = 9.3, 4.6 Hz, 1H), 1.86−1.70

(m, 4H), 1.46 (t, J = 7.5 Hz, 4H), 1.40 (d, J = 6.7 Hz, 3H), 1.28 (s,

12H), 0.73 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ

173.2, 170.4, 169.2, 167.3, 165.8, 163.6, 158.5, 156.5, 149.8, 146.0,

143.1, 138.2, 137.5, 136.4, 133.7, 131.2, 130.2, 126.5, 124.9, 123.4,

122.2, 120.3, 116.7, 116.5, 115.6, 110.2, 108.1, 107.8, 107.4, 69.3,

58.4, 53.0, 52.1, 49.2, 45.3, 35.5, 31.4, 30.0, 29.4, 29.1, 28.9, 27.4,

26.7, 25.7, 22.5, 21.3, 19.4, 18.7, 12.1, 11.2. HRMS m/z calculated for

C₅₅H₆₈N₈O₇[M + H]⁺: 953.5283, found: 953.5284.

N-[(4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl]-4′-{[4-

(6-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-

4-yl]oxy}hexyl)piperazin-1-yl]methyl}-5-[ethyl(oxan-4-yl)amino]-4-

methyl-[1,1′-biphenyl]-3-carboxamide (E6). Yellow solid (46.4 mg,

1

20% yield). H NMR (400 MHz, DMSO-d₆) δ 11.45 (s, 1H), 11.09

(s, 1H), 8.17 (t, J = 5.0 Hz, 1H), 7.80 (t, J = 7.9 Hz, 1H), 7.57 (d, J =

7.8 Hz, 2H), 7.51 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.40

(s, 1H), 7.35 (d, J = 7.9 Hz, 2H), 7.22 (s, 1H), 5.86 (s, 1H), 5.07 (m,

J = 12.9, 5.4 Hz, 1H), 4.29 (d, J = 5.0 Hz, 2H), 4.20 (t, J = 6.4 Hz,

2H), 3.83 (d, J = 11.2 Hz, 2H), 3.47 (s, 2H), 3.24 (d, J = 11.3 Hz,

2H), 3.06 (m, J = 18.9, 11.1 Hz, 3H), 2.88 (q, J = 12.5 Hz, 1H), 2.58

(d, J = 17.5 Hz, 2H), 2.38 (s, 6H), 2.25 (s, 3H), 2.21 (s, 3H), 2.11 (s,

3H), 2.03−1.99 (m, 1H), 1.75 (m, J = 7.0 Hz, 2H), 1.67 (d, J = 12.0

Hz, 2H), 1.49 (m, J = 24.8, 6.0 Hz, 4H), 1.38−1.31 (m, 2H), 1.24 (d,

J = 6.6 Hz, 6H), 0.84 (q, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 173.3, 170.4, 169.7, 167.3, 165.8, 163.5, 156.5, 150.3, 149.4,

143.3, 140.0, 137.6, 133.7, 133.1, 130.0, 126.8, 123.4, 122.0, 121.2,

121.0, 120.3, 116.6, 115.6, 108.1, 69.2, 66.8, 58.3, 52.9, 49.2, 41.7,

35.4, 31.4, 30.8, 30.7, 29.4, 29.1, 28.7, 25.6, 22.5, 19.4, 18.6, 15.0,

13.1. HRMS m/z calculated for C₅₃H₆₆N₇O₈[M + H]⁺: 928.4967,

found: 928.4982.

1-[(2S)-Butan-2-yl]-N-[(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-

yl)methyl]-6-{6-[4-(12-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-

dihydro-1H-isoindol-4-yl]oxy}dodecyl)piperazin-1-yl]pyridine-3-yl}-

3-methyl-1H-indole-4-carboxamide (G12). Yellow solid (33.8 mg,

1

14% yield). H NMR (400 MHz, DMSO-d₆) δ 11.45 (s, 1H), 11.10

(s, 1H), 8.49 (d, J = 2.5 Hz, 1H), 8.12 (t, J = 5.1 Hz, 1H), 7.90 (m, J

= 8.8, 2.6 Hz, 1H), 7.80 (m, J = 8.5, 7.3 Hz, 1H), 7.72 (d, J = 1.5 Hz,

1H), 7.50 (d, J = 8.6 Hz, 1H), 7.43 (d, J = 7.3 Hz, 1H), 7.25 (s, 1H),

7.17 (d, J = 1.4 Hz, 1H), 6.89 (d, J = 8.9 Hz, 1H), 5.86 (s, 1H), 5.08

(m, J = 12.9, 5.4 Hz, 1H), 4.59 (q, J = 6.9 Hz, 1H), 4.34 (t, J = 5.7 Hz,

2H), 4.19 (t, J = 6.4 Hz, 2H), 3.50 (t, J = 5.0 Hz, 4H), 2.88 (m, J =

17.4, 14.1, 5.4 Hz, 1H), 2.69−2.52 (m, 2H), 2.46 (d, J = 4.8 Hz, 4H),

2.30 (t, J = 7.5 Hz, 2H), 2.24 (s, 3H), 2.16 (s, 3H), 2.11 (s, 3H), 2.03

(m, J = 15.1, 7.9, 4.2 Hz, 1H), 1.85−1.70 (m, 4H), 1.51−1.43 (m,

4H), 1.40 (d, J = 6.7 Hz, 3H), 1.27 (s, 14H), 0.73 (t, J = 7.3 Hz, 3H).

¹³C NMR (101 MHz, DMSO-d₆) δ 173.2, 170.4, 169.2, 167.3, 165.8,

163.6, 158.5, 156.5, 149.8, 146.0, 138.2, 137.5, 136.4, 133.7, 131.2,

130.2, 126.5, 123.4, 122.2, 120.3, 116.7, 116.5, 115.6, 110.2, 108.1,

107.8, 107.4, 69.3, 58.6, 53.1, 52.1, 49.2, 45.4, 35.5, 31.4, 30.0, 29.5,

29.5, 29.1, 28.9, 27.4, 26.8, 25.7, 22.5, 21.3, 19.4, 18.7, 12.1, 11.2.

HRMS m/z calculated for C₅₆H₇₀N₈O₇[M + H]⁺: 967.5440, found:

967.5438.

N-[(4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl]-4′-{[4-

(7-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-

4-yl]oxy}heptyl)piperazin-1-yl]methyl}-5-[ethyl(oxan-4-yl)amino]-

4-methyl-[1,1′-biphenyl]-3-carboxamide (E7). Yellow solid (101.3

1

mg, 43% yield). H NMR (400 MHz, DMSO-d₆) δ 11.45 (s, 1H),

11.10 (s, 1H), 8.17 (t, J = 5.0 Hz, 1H), 7.80 (t, J = 7.9 Hz, 1H), 7.56

(d, J = 7.8 Hz, 2H), 7.50 (d, J = 8.6 Hz, 1H), 7.43 (d, J = 7.3 Hz, 1H),

7.40 (s, 1H), 7.35 (d, J = 7.8 Hz, 2H), 7.22 (s, 1H), 5.86 (s, 1H), 5.08

(m, J = 12.9, 5.4 Hz, 1H), 4.30 (d, J = 4.9 Hz, 2H), 4.20 (t, J = 6.4 Hz,

2H), 3.83 (d, J = 11.1 Hz, 2H), 3.47 (s, 2H), 3.28−3.20 (m, 2H),

3.09 (q, J = 7.6, 7.1 Hz, 2H), 3.01 (d, J = 10.7 Hz, 1H), 2.89 (m, J =

13.6, 12.5, 6.9 Hz, 1H), 2.59 (d, J = 17.0 Hz, 2H), 2.37 (s, 6H), 2.25

(s, 3H), 2.21 (s, 3H), 2.11 (s, 3H), 2.03 (d, J = 12.5 Hz, 1H), 1.75 (t,

J = 7.2 Hz, 2H), 1.67 (d, J = 12.0 Hz, 2H), 1.53 (m, J = 12.5, 7.6 Hz,

2H), 1.43 (m, J = 7.4 Hz, 4H), 1.33 (d, J = 6.3 Hz, 2H), 1.28 (d, J =

7.8 Hz, 2H), 1.23 (s, 4H), 0.83 (t, J = 7.0 Hz, 3H). ¹³C NMR (101

N-[(4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl]-4′-{[4-

(4-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-

4-yl]oxy}butyl)piperazin-1-yl]methyl}-5-[ethyl(oxan-4-yl)amino]-4-

methyl-[1,1′-biphenyl]-3-carboxamide (E4). Light-yellow solid (36.0

1

mg, 15% yield). H NMR (400 MHz, CDCl₃) δ 12.00 (s, 1H), 9.66

(s, 1H), 8.35 (d, J = 2.5 Hz, 1H), 7.71−7.57 (m, 2H), 7.44 (d, J = 7.2

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Article

MHz, DMSO-d₆) δ 173.3, 170.4, 169.7, 167.3, 165.8, 163.6, 156.5,

150.3, 149.4, 143.3, 140.0, 139.0, 137.6, 137.6, 137.5, 133.7, 133.1,

130.0, 126.8, 123.4, 122.0, 121.2, 120.2, 116.6, 115.6, 108.1, 69.3,

66.8, 62.0, 58.3, 58.1, 53.0, 52.6, 49.2, 41.7, 35.4, 31.4, 30.8, 29.4,

28.9, 28.8, 27.2, 26.2, 25.7, 22.5, 19.4, 18.6, 15.0, 13.1. HRMS m/z

calculated for C₅₄H₆₈N₇O₈[M + H]⁺: 942.5124, found: 942.5135.

N-[(4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl]-4′-{[4-

(8-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-

4-yl]oxy}octyl)piperazin-1-yl]methyl}-5-[ethyl(oxan-4-yl)amino]-4-

methyl-[1,1′-biphenyl]-3-carboxamide (E8). Yellow solid (69.3 mg,

18.4, 18.0, 16.8, 15.0, 14.6, 11.4. HRMS m/z calculated for

C₅₇H₇₄N₇O₈[M + H]⁺: 984.5593, found: 984.5593.

N-[(4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl]-4′-{[4-

(11-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoin-

dol-4-yl]oxy}undecyl)piperazin-1-yl]methyl}-5-[ethyl(oxan-4-yl)-

amino]-4-methyl-[1,1′-biphenyl]-3-carboxamide (E11). Yellow

solid (119.8 mg, 47% yield). ¹H NMR (400 MHz, DMSO-d₆) δ

11.44 (s, 1H), 11.09 (s, 1H), 8.17 (t, J = 5.0 Hz, 1H), 7.80 (t, J = 7.9

Hz, 1H), 7.56 (d, J = 7.8 Hz, 2H), 7.51 (d, J = 8.6 Hz, 1H), 7.43 (d, J

= 7.3 Hz, 1H), 7.39 (d, J = 1.8 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.22

(d, J = 1.8 Hz, 1H), 5.86 (s, 1H), 5.07 (m, J = 12.8, 5.4 Hz, 1H), 4.29

(d, J = 4.9 Hz, 2H), 4.19 (t, J = 6.4 Hz, 2H), 3.83 (d, J = 11.2 Hz,

2H), 3.47 (s, 2H), 3.24 (d, J = 11.3 Hz, 2H), 3.09 (q, J = 7.1 Hz, 2H),

3.01 (d, J = 10.7 Hz, 1H), 2.88 (m, J = 18.6, 14.5, 5.3 Hz, 1H), 2.58

(d, J = 17.4 Hz, 2H), 2.37 (s, 6H), 2.24 (s, 4H), 2.21 (s, 3H), 2.10 (s,

3H), 2.06−1.96 (m, 2H), 1.75 (t, J = 7.3 Hz, 2H), 1.66 (d, J = 12.4

Hz, 2H), 1.52 (m, J = 12.1, 3.9 Hz, 2H), 1.45 (s, 2H), 1.34 (s, 4H),

1.24 (d, J = 4.5 Hz, 12H), 0.83 (t, J = 7.5 Hz, 3H). ¹³C NMR (101

MHz, CDCl₃) δ 171.6, 170.1, 168.7, 167.1, 165.7, 165.2, 158.7, 156.7,

150.7, 149.6, 146.0, 142.6, 139.4, 136.4, 136.1, 135.8, 135.6, 133.9,

132.8, 125.5, 123.2, 122.1, 120.1, 118.9, 117.2, 115.6, 109.9, 106.8,

77.3, 69.5, 67.3, 58.7, 58.4, 52.9, 49.1, 45.2, 41.6, 36.2, 31.5, 30.5,

29.5, 29.4, 29.3, 29.2, 28.9, 27.5, 26.7, 25.7, 22.7, 19.7, 18.7, 14.7,

12.8. HRMS m/z calculated for C₅₈H₇₆N₇O₈[M + H]⁺: 998.5750,

found: 998.5775.

1

29% yield). H NMR (400 MHz, DMSO-d₆) δ 11.45 (s, 1H), 11.09

(s, 1H), 8.17 (t, J = 5.0 Hz, 1H), 7.80 (t, J = 7.9 Hz, 1H), 7.56 (d, J =

7.8 Hz, 2H), 7.50 (d, J = 8.6 Hz, 1H), 7.43 (d, J = 7.2 Hz, 1H), 7.40

(d, J = 2.1 Hz, 1H), 7.35 (d, J = 7.9 Hz, 2H), 7.22 (d, J = 1.9 Hz, 1H),

5.86 (s, 1H), 5.07 (m, J = 12.9, 5.4 Hz, 1H), 4.30 (d, J = 5.0 Hz, 2H),

4.19 (t, J = 6.4 Hz, 2H), 3.88−3.77 (m, 2H), 3.47 (s, 2H), 3.25 (t, J =

11.8 Hz, 2H), 3.09 (q, J = 7.1 Hz, 2H), 3.02 (q, J = 6.9, 5.4 Hz, 1H),

2.88 (m, J = 17.5, 14.2, 5.3 Hz, 1H), 2.63−2.54 (m, 2H), 2.46−2.30

(m, 6H), 2.25 (s, 5H), 2.21 (s, 3H), 2.11 (s, 3H), 2.06−2.00 (m, 1H),

1.75 (t, J = 7.3 Hz, 2H), 1.66 (d, J = 12.0 Hz, 2H), 1.54 (m, J = 11.8,

4.0 Hz, 2H), 1.43 (m, J = 11.6, 6.5 Hz, 4H), 1.36−1.20 (m, 8H), 0.83

(t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.2, 170.4,

169.5, 167.3, 165.8, 163.5, 156.5, 150.0, 149.3, 143.2, 140.1, 139.0,

137.5, 133.7, 133.1, 129.9, 126.8, 123.4, 122.1, 121.3, 120.3, 116.7,

115.6, 107.8, 69.3, 66.8, 62.2, 58.3, 53.2, 49.2, 41.7, 35.4, 31.4, 30.8,

29.3, 29.1, 28.9, 27.3, 26.6, 25.7, 22.5, 19.4, 18.7, 15.0, 13.2. HRMS

m/z calculated for C₅₅H₇₀N₇O₈[M + H]⁺: 956.5280, found:

956.5281.

N-[(4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl]-4′-{[4-

(12-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoin-

dol-4-yl]oxy}dodecyl)piperazin-1-yl]methyl}-5-[ethyl(oxan4yl)-

amino]-4-methyl-[1,1′-biphenyl]-3-carboxamide (E12). Yellow

solid (48.1 mg, 19% yield). ¹H NMR (400 MHz, DMSO-d₆) δ

11.44 (s, 1H), 11.09 (s, 1H), 8.17 (t, J = 4.9 Hz, 1H), 7.82−7.75 (m,

1H), 7.60−7.53 (m, 2H), 7.50 (m, J = 8.6, 1.9 Hz, 1H), 7.43 (m, J =

7.3, 1.6 Hz, 1H), 7.39 (d, J = 2.0 Hz, 1H), 7.38−7.31 (m, 2H), 7.22

(d, J = 1.8 Hz, 1H), 5.85 (s, 1H), 5.07 (m, J = 12.9, 5.3 Hz, 1H), 4.29

(d, J = 4.9 Hz, 2H), 4.22−4.14 (m, 2H), 3.83 (d, J = 11.1 Hz, 2H),

3.46 (s, 2H), 3.23 (d, J = 11.3 Hz, 2H), 3.08 (q, J = 7.1 Hz, 2H), 3.00

(d, J = 11.1 Hz, 1H), 2.88 (m, J = 18.9, 14.2, 5.4 Hz, 1H), 2.57 (m, J

= 15.5, 11.9 Hz, 2H), 2.37 (s, 6H), 2.24 (s, 5H), 2.21 (s, 3H), 2.10 (s,

3H), 2.06−1.97 (m, 1H), 1.74 (t, J = 7.2 Hz, 2H), 1.66 (d, J = 12.3

Hz, 2H), 1.53 (m, J = 11.9, 4.0 Hz, 2H), 1.49−1.41 (m, 2H), 1.35 (m,

J = 16.7, 5.5 Hz, 4H), 1.25 (d, J = 7.7 Hz, 14H), 0.83 (t, J = 6.9 Hz,

3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.2, 170.4, 169.5, 167.3,

165.8, 163.5, 156.5, 149.9, 149.3, 143.2, 140.1, 139.0, 137.5, 133.7,

129.9, 126.8, 122.1, 121.3, 120.3, 116.7, 115.6, 107.8, 69.3, 66.8, 62.2,

58.3, 53.3, 53.1, 49.2, 41.7, 35.4, 31.4, 30.8, 29.5, 29.4, 29.4, 29.1,

28.9, 27.4, 26.7, 25.7, 22.5, 19.4, 18.7, 15.0, 13.2. HRMS m/z

calculated for C₅₉H₇₈N₇O₈[M + H]⁺: 1012.5906, found: 1012.5910.

tert-Butyl (1-Methyl-2,6-dioxopiperidin-3-yl)carbamate

(3b). To a solution of 3a (456 mg, 2 mmol) in DMF (10 mL),

NaH (60%, 87.8 mg) was slowly added and stirred for 10 min at 0 °C.

CH₃I (0.19 mL, 3 mmol) was added dropwise by a syringe and

reacted for 2 h at 25 °C. The reaction residue was extracted with ethyl

acetate and aqueous HCl (1 N, 20 mL). The organic layer was

washed with water, dried over Na₂SO₄, and evaporated under a

vacuum. The crude product was puriﬁed by column chromatography

N-[(4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl]-4′-{[4-

(9-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-

4-yl]oxy}nonyl)piperazin-1-yl]methyl}-5-[ethyl(oxan-4-yl)amino]-4-

methyl-[1,1′-biphenyl]-3-carboxamide (E9). Yellow solid (97.1 mg,

1

40% yield). H NMR (400 MHz, DMSO-d₆) δ 11.45 (s, 1H), 11.10

(s, 1H), 8.18 (q, J = 5.6, 5.0 Hz, 1H), 7.80 (t, J = 7.9 Hz, 1H), 7.56

(d, J = 7.8 Hz, 2H), 7.51 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 7.3 Hz, 1H),

7.40 (d, J = 1.9 Hz, 1H), 7.35 (d, J = 7.9 Hz, 2H), 7.22 (d, J = 1.8 Hz,

1H), 5.86 (s, 1H), 5.07 (m, J = 12.9, 5.4 Hz, 1H), 4.29 (d, J = 4.9 Hz,

2H), 4.19 (t, J = 6.4 Hz, 2H), 3.83 (d, J = 11.1 Hz, 2H), 3.47 (s, 2H),

3.24 (d, J = 11.3 Hz, 2H), 3.09 (q, J = 7.1 Hz, 2H), 3.01 (q, J = 7.2,

5.6 Hz, 1H), 2.89 (m, J = 13.4, 12.1, 6.9 Hz, 1H), 2.57 (m, J = 15.9,

12.0 Hz, 2H), 2.37 (s, 6H), 2.25 (s, 5H), 2.21 (s, 3H), 2.11 (s, 3H),

2.05−1.99 (m, 1H), 1.75 (m, J = 6.6 Hz, 2H), 1.66 (d, J = 12.1 Hz,

2H), 1.54 (m, J = 11.9, 4.1 Hz, 2H), 1.45 (t, J = 7.7 Hz, 2H), 1.40 (d,

J = 8.9 Hz, 2H), 1.33 (d, J = 5.8 Hz, 2H), 1.25 (d, J = 11.4 Hz, 8H),

0.84 (q, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.3,

170.4, 169.6, 167.3, 165.8, 163.5, 156.5, 150.2, 149.4, 143.3, 140.0,

139.0, 137.5, 133.7, 133.1, 130.0, 126.8, 123.4, 122.0, 121.2, 120.3,

116.6, 115.6, 108.0, 69.3, 66.8, 62.1, 58.3, 53.0, 52.6, 49.2, 41.7, 35.4,

31.4, 30.8, 29.3, 29.2, 29.0, 28.8, 27.3, 25.7, 22.5, 19.4, 18.6, 15.0,

13.2. HRMS m/z calculated for C₅₆H₇₂N₇O₈[M + H]⁺: 970.5437,

found: 970.5474.

N-[(4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl]-4′-{[4-

(10-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoin-

dol-4-yl]oxy}decyl)piperazin-1-yl]methyl}-5-[ethyl(oxan-4-yl)-

amino]-4-methyl-[1,1′-biphenyl]-3-carboxamide (E10). Light-yel-

1

low solid (81.2 mg, 33% yield). H NMR (400 MHz, DMSO-d₆) δ

1

to aﬀord 3b as a white solid (272 mg, 56%). H NMR (400 MHz,

11.44 (s, 1H), 11.10 (s, 1H), 8.17 (t, J = 5.0 Hz, 1H), 7.80 (t, J = 7.9

Hz, 1H), 7.56 (d, J = 7.9 Hz, 2H), 7.50 (d, J = 8.5 Hz, 1H), 7.43 (d, J

= 7.3 Hz, 1H), 7.39 (d, J = 1.8 Hz, 1H), 7.35 (d, J = 7.9 Hz, 2H), 7.22

(d, J = 1.8 Hz, 1H), 5.85 (s, 1H), 5.07 (m, J = 12.9, 5.3 Hz, 1H), 4.29

(d, J = 5.0 Hz, 2H), 4.19 (d, J = 6.5 Hz, 2H), 3.86−3.78 (m, 2H),

3.47 (s, 2H), 3.24 (d, J = 11.3 Hz, 2H), 3.09 (q, J = 7.2 Hz, 2H), 3.01

(d, J = 11.0 Hz, 1H), 2.88 (m, J = 18.0, 14.1, 5.3 Hz, 1H), 2.68−2.54

(m, 2H), 2.35 (d, J = 16.6 Hz, 6H), 2.25 (s, 5H), 2.21 (s, 3H), 2.10

(s, 3H), 2.06−1.98 (m, 1H), 1.75 (m, J = 6.6 Hz, 2H), 1.66 (d, J =

11.4 Hz, 2H), 1.53 (m, J = 12.2, 5.9 Hz, 2H), 1.45 (t, J = 7.8 Hz, 2H),

1.42−1.31 (m, 4H), 1.25 (d, J = 8.1 Hz, 10H), 0.83 (t, J = 6.9 Hz,

3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.2, 170.4, 167.3, 166.1,

165.6, 164.0, 161.2, 156.5, 149.9, 149.3, 137.5, 133.1, 129.9, 126.8,

121.3, 120.3, 116.7, 115.6, 106.4, 76.6, 69.3, 67.7, 64.1, 58.4, 53.1,

49.2, 35.4, 34.7, 31.4, 29.4, 29.1, 28.9, 26.3, 25.5, 22.5, 22.2, 19.0,

DMSO-d₆) δ 7.19 (d, J = 8.8 Hz, 1H), 4.30 (m, 1H), 2.80 (m, Hz,

1H), 2.64 (m, 1H), 2.02−1.85 (m, 2H), 1.40 (s, 9H). HRMS m/z

calculated for C₁₁H₁₉N₂O₄[M + H]⁺: 243.1339, found: 243.1341.

4-Hydroxy-2-(1-methyl-2,6-dioxopiperidin-3-yl)-2,3-dihy-

dro-1H-isoindole-1,3-dione (3c). To a solution of 3b (242 mg, 1

mmol) in DCM (10 mL), TFA (1 mL) was added and stirred at 25

°C for 1 h. When the reaction was complete, the crude mixture was

evaporated under reduced pressure. Then, residues were used in the

next step without puriﬁcation. Then, compound 3c was prepared

according to the synthesis method of 1b on a 1 mmol scale.

Puriﬁcation by column chromatography aﬀorded the title compound

1

(112.1 mg, 39%). H NMR (400 MHz, DMSO-d₆) δ 11.18 (s, 1H),

7.66 (t, J = 7.8 Hz, 1H), 7.32 (d, J = 7.1 Hz, 1H), 7.26 (d, J = 8.4 Hz,

1H), 5.14 (dd, J = 13.0, 5.4 Hz, 1H), 2.99−2.87 (m, 1H), 2.76 (m,

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1H), 2.57 (dd, J = 13.0, 4.5 Hz, 1H), 2.04 (m, 1H). HRMS m/z

calculated for C₁₄H₁₃N₂O₅[M + H]⁺: 289.0819, found: 289.0821.

4-[(7-Bromoheptyl)oxy]-2-(1-methyl-2,6-dioxopiperidin-3-

yl)-2,3-dihydro-1H-isoindole-1,3-dione (3d). Compound 3d was

prepared according to the general method to synthesize 1c−k on a

0.35 mmol scale of 3c (69.2 mg, 42%). ¹H NMR (400 MHz,

chloroform-d) δ 7.59 (t, J = 7.9 Hz, 1H), 7.36 (d, J = 7.3 Hz, 1H),

7.14 (d, J = 8.5 Hz, 1H), 4.99−4.80 (m, 1H), 4.10 (t, J = 6.5 Hz, 2H),

3.34 (t, J = 6.8 Hz, 2H), 3.12 (s, 3H), 2.96−2.81 (m, 1H), 2.81−2.62

(m, 2H), 2.09−1.98 (m, 1H), 1.80 (m, J = 6.5 Hz, 4H), 1.39 (m, J =

36.6, 15.1, 7.9 Hz, 6H). HRMS m/z calculated for C₂₁H₂₆BrN₂O₅[M

+ H]⁺: 465.1020, found: 465.1022.

14.6, 13.2. HRMS m/z calculated for C₅₅H₇₀N₇O₈[M + H]⁺:

956.5280, found: 956.5282.

Cell Lines and Culture. All of the cells used in this study were

purchased from the American Type Culture Collection (ATCC,

Manassas, VA) or the Cell Bank of the Chinese Academy of Science

(Shanghai, China). Cells were cultured in Dulbecco’s modiﬁed Eagle’s

medium (DMEM) or Rosewell Park Memorial Institute (RPMI)

1640 media containing 1% penicillin-streptomycin and 10% fetal

bovine serum under humidiﬁed conditions with 5% CO₂at 37 °C.

Biochemical Assay. The EZH1 and EZH2 inhibition assays were

performed by the AlphaLISA immunodetection assay provided by

Shanghai ChemPartner Limited (Shanghai, China). Values were

determined using an AlphaLISA methyltransferase assay kit

(PerkinElmer, MA) according to the manufacturer’s protocol. The

compounds’ screening protocol is shown in the Supporting

Information.

Thermal Shift Assay. WSU-DLCL-2 cells were suspended at 1.5

× 10⁶cells/mL and pretreated with MG-132 for 1 h. E7 (60 μM) was

added, and cells were cultured for 2 h; an equal volume of DMSO

served as the negative control. The cells were collected, and RIPA

containing cocktail was added. After 1 h, the supernatant was

collected by centrifugation and divided into six parts. The solutions

were heated for 6 min at diﬀerent temperatures (45, 48, 51, 54, 57,

and 60 °C) and centrifuged at 13 000 rpm for 15 min. The

supernatant solutions were used for immunoblotting analysis.

Immunoblotting Analysis. Protein samples were separated by

sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-

PAGE) and transferred to a nitrocellulose (NC) membrane. Then,

the NC membrane was blocked with 5% skim milk for 2 h at room

temperature, followed by incubation with the appropriate primary

antibody at 4 °C overnight. The NC membrane was then incubated

with the corresponding secondary antibody. Speciﬁc protein bands

were obtained by chemiluminescence detection. The antibodies used

for immunoblotting are listed in Table S1.

N-[(4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl]-5-

[ethyl(oxan-4-yl)amino]-4-methyl-4′-{[4-(7-{[2-(1-methyl-2,6-

dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]-

oxy}heptyl)piperazin-1-yl]methyl}-[1,1′-biphenyl]-3-carboxa-

mide (E7-NC-1). E7-NC-1 was prepared according to the synthesis

1

method of E7 on a 0.1 mmol scale of 3d (27.8 mg, 29%). H NMR

(400 MHz, CDCl₃) δ 11.68 (s, 1H), 7.66 (t, J = 7.9 Hz, 1H), 7.44 (d,

J = 7.6 Hz, 3H), 7.34 (s, 1H), 7.32 (s, 2H), 7.28 (d, J = 1.7 Hz, 1H),

7.20 (d, J = 8.4 Hz, 1H), 7.15 (t, J = 5.9 Hz, 1H), 5.90 (s, 1H), 4.95

(dd, J = 12.2, 5.4 Hz, 1H), 4.55 (d, J = 5.8 Hz, 2H), 4.16 (t, J = 6.6

Hz, 2H), 3.94 (d, J = 11.4 Hz, 2H), 3.53 (s, 2H), 3.48 (s, 4H), 3.32

(m, J = 11.2, 3.1 Hz, 2H), 3.20 (s, 3H), 3.09 (q, J = 7.0 Hz, 2H),

2.99−2.94 (m, 1H), 2.83−2.70 (m, 1H), 2.51 (s, 6H), 2.40 (s, 3H),

2.34 (s, 4H), 2.13 (s, 3H), 1.87 (m, J = 6.7 Hz, 4H), 1.69 (d, J = 10.5

Hz, 4H), 1.50 (q, J = 7.5 Hz, 4H), 1.42−1.31 (m, 4H), 0.89 (t, J = 6.9

Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 166.5, 165.4, 164.0, 162.5,

161.1, 160.3, 151.9, 145.9, 144.7, 137.7, 134.6, 134.5, 133.6, 132.5,

131.7, 129.1, 128.5, 124.9, 122.0, 119.3, 117.4, 116.1, 114.1, 112.4,

110.9, 105.1, 64.7, 62.6, 57.9, 54.0, 53.7, 48.4, 48.2, 46.1, 45.1, 36.9,

31.4, 27.2, 25.8, 25.0, 24.5, 24.1, 22.7, 22.5, 21.9, 21.0, 17.2, 15.0,

14.0, 10.0, 8.1. HRMS m/z calculated for C₅₅H₇₀N₇O₈[M + H]⁺:

956.5280, found: 956.5283.

tert-Butyl-4-({3′-[ethyl(oxan-4-yl)amino]-4′-methyl-5′-

{[(1,4,6-trimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl]-

carbamoyl}-[1,1′-biphenyl]-4-yl}methyl)piperazine-1-carboxy-

late (3f). To a solution of 3e (120 mg, 0.25 mmol, 1 equiv), K₂CO₃

(69 mg, 0.5 mmol, 1 equiv) in DMF (2.5 mL) was added and stirred

at 25 °C for 5 min before adding iodomethane (30 μL). The mixture

was stirred at 25 °C for 24 h and puriﬁed by column chromatography

eluting to yield 3f (105.7 mg, 86%). ¹H NMR (400 MHz, DMSO-d₆)

δ 8.17 (t, J = 5.0 Hz, 1H), 7.58 (d, J = 7.9 Hz, 2H), 7.43−7.32 (m,

3H), 7.21 (d, J = 1.8 Hz, 1H), 6.02 (s, 1H), 4.33 (d, J = 5.0 Hz, 2H),

3.83 (d, J = 11.4 Hz, 2H), 3.50 (s, 2H), 3.41 (s, 3H), 3.30 (s, 4H),

3.25 (t, J = 11.5 Hz, 2H), 3.09 (q, J = 7.2 Hz, 2H), 3.01 (d, J = 11.1

Hz, 1H), 2.33 (d, J = 5.1 Hz, 4H), 2.30 (s, 3H), 2.24 (s, 3H), 2.22 (s,

3H), 1.66 (d, J = 12.5 Hz, 2H), 1.52 (m, J = 11.7, 9.9, 5.5 Hz, 2H),

1.39 (s, 9H), 0.83 (t, J = 6.9 Hz, 3H). HRMS m/z calculated for

C₄₀H₅₆N₅O₅[M + H]⁺: 686.4276, found: 686.4279.

4′-{[4-(7-{[2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihy-

dro-1H-isoindol-4-yl]oxy}heptyl)piperazin-1-yl]methyl}-5-

[ethyl(oxan-4-yl)amino]-4-methyl-N-[(1,4,6-trimethyl-2-oxo-

1,2-dihydropyridin-3-yl)methyl]-[1,1′-biphenyl]-3-carboxa-

mide (E7-NC-2). Compound E7-NC-2 was prepared according to

the synthesis method of E7 on a 0.1 mmol scale of 3f (30.6 mg, 35%).

¹H NMR (400 MHz, chloroform-d) δ 7.66 (t, J = 7.9 Hz, 1H), 7.45

(dd, J = 7.6, 5.0 Hz, 3H), 7.38−7.29 (m, 3H), 7.26 (s, 1H), 7.20 (d, J

= 8.4 Hz, 1H), 7.12 (t, J = 6.1 Hz, 1H), 5.99 (s, 1H), 4.92 (dd, J =

11.9, 5.5 Hz, 1H), 4.56 (d, J = 6.1 Hz, 2H), 4.18 (m, J = 22.3, 14.4,

7.0 Hz, 2H), 3.95 (d, 2H), 3.50 (s, 3H), 3.47 (s, 3H), 3.33 (m, J =

11.6, 6.1 Hz, 2H), 3.09 (q, J = 6.9 Hz, 2H), 3.01 (dd, J = 10.6, 5.1 Hz,

1H), 2.88−2.67 (m, 4H), 2.55 (s, 6H), 2.40 (s, 3H), 2.32 (s, 5H),

2.12 (m, 2H), 1.93−1.78 (m, 4H), 1.71 (s, 4H), 1.56−1.46 (m, 4H),

1.42 (d, 2H), 1.33 (d, J = 4.8 Hz, 2H), 0.90 (t, J = 6.9 Hz, 3H). ¹³C

NMR (101 MHz, DMSO-d₆) δ 173.2, 170.8; 170.4, 169.5, 167.3,

165.8, 162.9, 156.5, 149.3, 147.8, 145.2, 140.0, 138.9, 137.9, 137.5,

133.7, 133.1, 129.9, 126.8, 123.3, 121.5, 121.3, 120.3, 116.7, 115.6,

109.0, 69.2, 66.8, 60.2, 58.3, 53.3, 53.1; 49.2, 41.6, 36.3, 31.4, 31.2,

30.8, 29.5, 29.5, 29.0, 28.8, 27.3, 25.7, 22.5, 21.2, 20.5, 19.2, 15.0,

Immunoprecipitation. Lysates were prepared from WSU-DLCL-

2 cells treated with E7 for 48 h using the RIPA lysis buﬀer. The cell

lysates were incubated with the indicated antibodies overnight at 4

°C, and then, Protein G agarose beads (Roche) were added. After 3 h

incubation at 4 °C, the proteins were eluted from Protein G agarose

beads with IP buﬀer, and ubiquitination modiﬁcation of the target

protein was examined by immunoblotting analysis.

Real-Time qPCR Assay. Cells were treated with diﬀerent drugs

for 48 h, and then, the total RNA was extracted with Trizol, according

to the manufacturer’s instruction. RNA was reverse-transcribed by

Hiscript III RT SuperMix. RT-qPCR was carried out using the

ChamQ Universal SYBR qPCR Master Mix on the CFX96 RT-qPCR

system in accordance with the manufacturer’s instruction. The

reaction procedure was as follows: 95 °C for 30 s followed by 40

cycles of ampliﬁcation for 6 s at 95 °C, 20 s at 60 °C. The primer

sequences used for RT-qPCR are listed in Table S2.

Cell Viability Assay. Cells were seeded (1.5−8 × 10³cells per

well in 100 μL of medium) in 96-well plates for 24 h. Then, 100 μL of

medium containing various concentrations of E7 was added to each

well. After the indicated incubation time, 20 μL of a 5 mg/mL 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) sol-

ution was added to each well and cells were incubated for an

additional 2.5 h at 37 °C. For A549 and NCI-H1299 cells, the

medium was removed; 150 μL of DMSO was added to each well, and

then, the absorbance of each well was measured at 570 nm. For WSU-

DLCL-2 and Pfeiﬀer cells, 50 μL of 20% (w/v) SDS was directly

added to each well and cells were incubated at 37 °C overnight; the

absorbance of each well was measured at 570 nm wavelength.

Cell Proliferation Analysis. Cells were seeded in 12-well plates

(5 × 10³cells per well), incubated for 24 h, and treated with 10 μM

E7, EPZ6438, or GSK126 for 9 days. The cells were photographed

and counted on days 1, 3, 5, 7, and 9, and the medium was refreshed.

Statistical Analysis. All quantitative results were expressed as

mean values SD. Statistically signiﬁcant diﬀerences were obtained

via Student’s t-test. P < 0.05 can be considered statistically signiﬁcant.

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Innovation Center of Biotherapy, Sichuan University,

Chengdu 610041, China

Shuyan Zhou − State Key Laboratory of Biotherapy and

Cancer Center, West China Hospital, and Collaborative

Innovation Center of Biotherapy, Sichuan University,

Chengdu 610041, China

Rong Hu − School of Life Science and Engineering, Southwest

Jiaotong University, Chengdu 610031, China

Tinghong Ye − State Key Laboratory of Biotherapy and

Cancer Center, West China Hospital, and Collaborative

Innovation Center of Biotherapy, Sichuan University,

Chengdu 610041, China

Yongxia Zhu − Department of Pharmacy, Sichuan Cancer

Hospital & Institute, Sichuan Cancer Center, School of

Medicine, University of Electronic Science and Technology of

China, Chengdu 610041, China

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02234.

■

sı

Protein levels of PRC2 subunits and H3K27me3 in

WSU-DLCL-2 cells treated with indicated compounds

for 48 h analyzed by western blot; potencies of E7

against WSU-DLCL-2, A549, NCI-H1299, and Pfeiﬀer

cancer cell lines; compounds’ screening methods against

EZH1 and EZH2; EZH1 inhibitory activities of the

synthesized compounds; transfection of EZH2-shRNA

and siRNA; antibodies used in immunoblotting analysis;

primer sequences used in RT-qPCR; H NMR, ¹³H

1

NMR, and HRMS spectrogram data of all target

compounds including G4−G12, E4−E12, E7-NC-1,

and E7-NC-2; and HPLC for purity determination of E7

(PDF)

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c02234

Molecular formula strings (CSV)

Author Contributions

^∥Z.L. and X.H. contributed equally to this work.

AUTHOR INFORMATION

■

Corresponding Authors

Notes

Ningyu Wang − School of Life Science and Engineering,

Southwest Jiaotong University, Chengdu 610031, China;

orcid.org/0000-0003-3571-4903; Email: wangny-

swjtu@swjtu.edu.cn

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We are grateful to Guobo Li of West China School of

Pharmacy (Sichuan University) for advice on the PROTAC

molecule design, Shuhui Xu of State Key Laboratory of

Biotherapy (Sichuan University) for NMR measurements, and

Professor Lijuan Chen’s team of State Key Laboratory of

Biotherapy (Sichuan University) for HRMS measurements.

This work was supported by grants from the Sichuan

Provincial Science and Technology Program for Key Research

and Development, China (No. 2018SZ0007), the Postdoctoral

Science Foundation of Sichuan University (No.

2020SCU12020), the National Natural Science Foundation

of China (No. 81702946), and the National S&T Major

Special Project on Major New Drug Innovations (No.

2018ZX09201018).

Luoting Yu − State Key Laboratory of Biotherapy and Cancer

Center, West China Hospital, and Collaborative Innovation

Center of Biotherapy, Sichuan University, Chengdu 610041,

China; orcid.org/0000-0001-9297-6665;

Email: yuluot@scu.edu.cn

Authors

Zhihao Liu − State Key Laboratory of Biotherapy and Cancer

Center, West China Hospital, and Collaborative Innovation

Center of Biotherapy, Sichuan University, Chengdu 610041,

China; orcid.org/0000-0002-0246-8761

Xi Hu − State Key Laboratory of Biotherapy and Cancer

Center, West China Hospital, and Collaborative Innovation

Center of Biotherapy, Sichuan University, Chengdu 610041,

China

Qiwei Wang − State Key Laboratory of Biotherapy and

Cancer Center, West China Hospital, and Collaborative

Innovation Center of Biotherapy, Sichuan University,

Chengdu 610041, China

Xiuli Wu − State Key Laboratory of Biotherapy and Cancer

Center, West China Hospital, and Collaborative Innovation

Center of Biotherapy, Sichuan University, Chengdu 610041,

China

Qiangsheng Zhang − State Key Laboratory of Biotherapy and

Cancer Center, West China Hospital, and Collaborative

Innovation Center of Biotherapy, Sichuan University,

Chengdu 610041, China

Wei Wei − State Key Laboratory of Biotherapy and Cancer

Center, West China Hospital, and Collaborative Innovation

Center of Biotherapy, Sichuan University, Chengdu 610041,

China

Xingping Su − State Key Laboratory of Biotherapy and

Cancer Center, West China Hospital, and Collaborative

Innovation Center of Biotherapy, Sichuan University,

Chengdu 610041, China

ABBREVIATIONS

■

PRC2, polycomb repressive complex 2; H3K27me3, histone 3

lysine 27 trimethyl; EZH2, enhancer of zeste homolog 2; EED,

embryonic ectoderm development; SUZ12, suppressor of zeste

12 protein homolog; RbAp46/48, retinoblastoma (Rb)-

associated proteins 46/48; SAM, S-adenosyl-^L-methionine;

GCB, germinal center B-cell; DLBCL, diﬀuse large-cell B-cell

lymphomas; NHL, non-Hodgkin’s lymphomas; SWI/SNF,

switch/sucrose nonfermentable; A, alanine; Y, tyrosine; F,

phenylalanine; N, asparagine; S, serine; C, cystine; H,

histidine; AR, androgen receptor; SWI/SNF, switch/sucrose

nonfermentable; HyT, hydrophobic tagging; PROTACs,

proteolysis targeting chimeras; POI, protein of interest;

CRBN, cereblon; DMSO, dimethyl sulfoxide; Boc, tert-

butyloxycarboryl; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-di-

phenyltetrazolium bromide; RT-qPCR, quantitative real-time

polymerase chain reaction; WB, western blot; IP, immunopre-

cipitation

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NOTE ADDED AFTER ASAP PUBLICATION

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Design and Synthesis of EZH2-Based PROTACs to Degrade the PRC2 Complex for Targeting the Noncatalytic Activity of EZH2

DOI: 10.1021/acs.jmedchem.0c02234

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