Design, Synthesis, and Evaluation of VHL-Based EZH2 Degraders to Enhance Therapeutic Activity against Lymphoma

pubs.acs.org/jmc

Article

Design, Synthesis, and Evaluation of VHL-Based EZH2 Degraders to

Enhance Therapeutic Activity against Lymphoma

Yalin Tu,^§Yameng Sun,^§Shuang Qiao,^§Yao Luo, Panpan Liu, Zhong-Xing Jiang, Yumin Hu,

Zifeng Wang, Peng Huang,* and Shijun Wen *

Cite This: J. Med. Chem. 2021, 64, 10167 −10184

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: Traditional EZH2 inhibitors are developed to

suppress the enzymatic methylation activity, and they may have

therapeutic limitations due to the nonenzymatic functions of EZH2

in cancer development. Here, we report proteolysis-target chimera

(PROTAC)-based EZH2 degraders to target the whole EZH2 in

lymphoma. Two series of EZH2 degraders were designed and

synthesized to hijack E3 ligase systems containing either von

Hippel−Lindau (VHL) or cereblon (CRBN), and some VHL-

based compounds were able to mediate EZH2 degradation. Two

best degraders, YM181 and YM281, induced robust cell viability inhibition in diﬀuse large B-cell lymphoma (DLBCL) and other

subtypes of lymphomas, outperforming a clinically used EZH2 inhibitor EPZ6438 (tazemetostat) that was only eﬀective against

DLBCL. The EZH2 degraders displayed promising antitumor activities in lymphoma xenografts and patient-derived primary

lymphoma cells. Our study demonstrates that EZH2 degraders have better therapeutic activity than EZH2 inhibitors, which may

provide a potential anticancer strategy to treat lymphoma.

locally advanced epithelioid sarcoma.¹³These inhibitors often

bind to the SET domain of EZH2 and compete with the

cofactor S-adenosylmethionine (SAM) without aﬀecting the

INTRODUCTION

■

EZH2 is the enzymatic subunit of polycomb repressive

complex 2 (PRC2) that mainly trimethylate lysine 27 of

protein stability of EZH2.^14,15However, increasing evidence

histone H3 (H3K27) to silence the gene transcription.¹⁻³

has revealed that the oncogenic function of EZH2 is not

Either overexpression or gain-of-function mutation of EZH2 is

entirely dependent on its enzymatic activity. The whole EZH2

observed in various tumors, including lymphoma, especially

protein itself is also correlated with tumor proliferation,

diﬀuse large B-cell lymphoma (DLBCL), T-cell acute

independent of its H3K27 trimethylation activity.^5,10,16,17

lymphoblastic leukemia, breast cancer, and prostate cancer.⁴⁻⁶

Meanwhile, some drawbacks of current EZH2 inhibitors are

The oncogenic roles of EZH2 are often attributed to its end-

uncovered, for example, excessive dosage, acquired resistance,

product H3K27me3-mediated epigenetic silencing of tumor

and drug insensitivity to most solid tumors.^18,19Therefore, we

suppressor genes.⁷The loss of mutation of speciﬁc subunits of

hypothesized that the therapeutic gains from abrogating the

other chromatin remodeling complexes that antagonize PRC2

whole EZH2 protein rather than simply inhibiting its

activity, like the SWI/SNF complex, is another major reason

enzymatic activity might be better in EZH2-dependent

for the activation of EZH2.⁶For example, the loss of the SWI/

cancers, and the development of EZH2-targeting degraders is

SNF subunit INI1 (encoded by SMARCB1) leads to

demanded.

constitutive EZH2 activation in nearly all epithelioid

Cells can maintain proteins in a well-preserved homeostasis

sarcomas.^8,9A broad role for EZH2 in the progression of

using the natural ubiquitin−proteasome degradation system in

cancers with mutations of the SWI/SNF subunits ARID1A,

which ubiquitination is the initial key process. When ubiquitin,

PBRM1, and SMARCA4 has also been demonstrated in both

a small protein, tags the targeted proteins that are accumulated

cell lines and in vivo models.^10,11

abnormally, the ubiquitylated proteins will be subjected to the

Many compounds that inhibit the methylation enzymatic

activity of EZH2 are developed and show promising antitumor

eﬃcacy in preclinical and clinical trials in hematologic

malignancies as well as solid tumors that exhibit strong

Received: March 13, 2021

Published: July 1, 2021

EZH2 dependencies due to synthetic lethality with the

aforementioned SWI/SNF subunit mutations.^6,12In 2020,

the most advanced EZH2 inhibitor EPZ6438 (tazemetostat)

has been approved by FDA for the treatment of metastatic and

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

10167

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 1. Strategy to develop EZH2 degraders based on PROTAC technology. (A) MTS cell viability analysis in diﬀerent lymphoma cell lines

treated with indicated concentrations of EPZ6438 for 5 days. (B) Western blot analysis of EZH2 protein levels in diﬀerent lymphoma cell lines.

Tubulin was used as a loading control. (C) Cell numbers were measured by cell count after the transfection of control shRNA or shEZH2 in SU-

DHL-2 cells for the indicated days (left). EZH2 and H3K27me3 levels were measured by western blot assay. H3 and tubulin were used as loading

controls (right). (D) Schematic representation of the EZH2 degrader design. (E) Docking conformation of EPZ6438 in the catalytic domain of

EZH2 (PDB ID: 5LS6, left) and the chemical structure of EPZ6438 (right). Note: EPZ stands for EPZ6438 in this ﬁgure and the following ﬁgures.

proteasome for degradation to remove the protein redundancy.

Proteolysis targeting chimeras (PROTACs) can achieve

selective protein degradation by artiﬁcially hijacking natural

ubiquitin−proteasome systems. PROTAC-based degraders

have two crucial warheads: one binding to a protein of interest

(POI) and the other binding to E3 ubiquitin ligase. These

special bifunctional molecules force a handshake between an

E3 ligase and the POI to artiﬁcially ubiquitylate the POI that is

subsequently subjected to the proteasome-mediated deple-

tion.²⁰The PROTAC technology has been employed to

degrade several proteins like BRD4,^21,22AR,²³and Stat3.²⁴

PROTAC-based degraders not only intervene in the enzymatic

activities that are targeted by the traditional inhibitors but also

disturb nonenzymatic functions of the interested protein, and

they may substantially improve the therapeutic eﬃcacy and

overcome the inhibitor-mediated drug resistance.^20,25,26More-

over, the potential advantages of PROTACs in the epigenetic

context have also been attracting the attention of researchers.

Compared with the parental epigenetic inhibitors, epigenetic

PROTACs endowed with higher target selectivity, increased

potency, prolonged action, reduced side-eﬀects, and risk of

resistance would oﬀer more potential therapeutic strategies for

the epigenetic POI-dependent cancers and would be very

useful as chemical tools for dissecting the biological roles of the

epigenetic POI.^27,28

viability inhibition of primary lymphoma cells from patients.

Our work provides novel EZH2 degraders to target a broad

range of lymphomas of which EZH2 inhibitors only exhibit

their limited eﬃcacy to DLBCL.

RESULTS

■

Design and Screening of EZH2 Degraders via the

PROTAC Strategy. In our previous work, we found that the

most advanced EZH2 inhibitor EPZ6438 required high doses

to reach reasonable in vitro antiproliferative activity against

acute myeloid leukemia, although the compound was reported

to suppress EZH2 activity largely at nanomolar concentration

levels.^16,29Since lymphomas are believed to be more sensitive

to EZH2 inhibitors, EPZ6438 was used to screen our

laboratory available lymphoma cell lines, including Burkitt’s

lymphoma (BL), lymphoblastic lymphoma (LBL), mantle cell

lymphoma (MCL), and DLBCL cell lines. However, most

lymphoma cell lines and even one DLBCL cell line Toledo did

not exhibit obvious responses to EZH2 inhibition, although

EZH2 protein levels are similar in all cell lines (Figure 1A,B).

Moreover, EPZ6438 could not completely suppress the cell

growth of the tested DLBCL (SU-DHL-2, SU-DHL-4, and

SU-DHL-6), which are widely reported to be sensitive to

EZH2 inhibition, with at least 25% cells remaining viable even

at a high dose (10 μM). Meanwhile, the EZH2 knockdown

almost completely inhibited the cell growth of SU-DHL-2

(Figure 1C). These observations conﬁrmed the necessity of

the development of EZH2 degraders. Using the conventional

PROTAC strategy (Figure 1D), our molecular docking model

indicated a possibility of the morpholine end to be extended to

Herein, we report the development of speciﬁc EZH2

degraders YM181 and YM281 based on PROTAC and further

elaborate their better antitumor eﬀects against not only

DLBCL but also other types of lymphomas in vitro and in

vivo compared to the parental EZH2 inhibitor EPZ6438.

Meanwhile, YM281 caused robust cell death and substantial

10168

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 2. Chemical structures and degradation eﬃcacy of the EZH2 degraders. (A) Chemical structures and linker lengths of VHL-based

PROTACs. (B) Western blot analysis of EZH2 and H3K27me3 levels in 22Rv1 cells treated with indicated concentrations of compounds or

dimethyl sulfoxide (DMSO) for 48 h. H3 and tubulin were used as loading controls. (C, D) Scatter plots showing the relative (C) H3K27me3 and

(D) EZH2 bands abundance in B. (E) Chemical structures and degradation eﬃciency of CRBN-based PROTACs. N, no signiﬁcant eﬀects on the

related target and Y, signiﬁcant eﬀects on the related target.

an E3 ligase binder to assemble EZH2 degraders (Figure

1E).³⁰

level of EZH2 at 1 μM (Figure 2B,D). Neither compound V6

nor V7 with a longer linker at either 14 or 17 atoms length had

an impact on both levels of EZH2 and H3K27me3, indicating

that the over-long linker might sacriﬁce the binding capacity to

EZH2, the cell permeability of compounds, and ternary

complex formation. Our second series of designed EZH2

degraders G1−G6 were linked to the thalidomide motif that

binds to CRBN. However, none of them showed promising

EZH2 degradation capacity, while most of them retained the

EZH2 enzymatic inhibition activities (Figures 2E and S1). It

seemed that the VHL hijacking approach is more eﬃcient to

degrade EZH2.²⁰In some cases, a more diverse arsenal of E3

ligase ligands may maximize the opportunity for comple-

mentary surfaces between the E3 ligase and POI and thus

improve the degradation eﬃciency of PROTACs.³⁴⁻³⁶

Notwithstanding, more investigation is needed to elucidate

the exact reason for the failure of G series compounds to

degrade EZH2. Based on the EZH2 degradation eﬃciency, the

We removed the morpholine motif with a direct linker

connected to EPZ6438 to minimize the overall ﬁnal molecular

size for proper cellular permeability. Then, two series of

PROTAC-based EZH2 degraders were designed and synthe-

sized to hijack two widely studied E3 ligase systems containing

either von Hippel−Lindau (VHL) or cereblon (CRBN).

Compounds V1−V7 were linked in diﬀerent lengths to a small

peptide-like fragment that can bind to VHL (Figure 2A).^31,32

Detection of the EZH2 protein level and its catalytic activity

mark H3K27me3 by western blot was performed to screen the

synthetic compounds at a 0.3−3 μM concentration range to

avoid the so-called “hook eﬀect”.³³YM281 (also named as

V2), YM181 (also named as V3), and V4 signiﬁcantly

decreased the H3K27me3 degree at 1 μM, while V1 and V5

required high doses to reach the same level (Figure 2B,C).

However, only YM181 and YM281 depleted the half protein

10169

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 3. EZH2 degraders abrogated the EZH2 protein level and the PRC2 complex through the VHL-dependent ubiquitin−proteasome system.

(A) Chemical structures of YM181 and YM281. (B) Relative protein bands abundance in 22Rv1 cells treated with indicated concentrations of

YM181 and YM281 for 24 h. (C) Western blot analysis of EZH2 and H3K27me3 levels in 22Rv1 cells treated with YM181 (2 μM) for the

indicated exposure time. H3 and tubulin were used as loading controls. (D) Western blot analysis of the indicated protein levels in 22Rv1 and SU-

DHL-2 cells treated with YM181 (2 μM) for 24 h. H3 was used as a loading control. (E) Western blot analysis of EZH2 levels in 22Rv1 cells

treated with YM181 (2 μM) for 24 h after 2 h pretreatment with DMSO, MG132 (0.5 μM), MLN-4924 (0.4 μM), EPZ6438 (2 μM), and VH032

(2 μM). Tubulin was used as a loading control. (F) Western blot analysis of EZH2 and VHL levels after the transfection of nontargeting (NC) or

VHL siRNAs for 48 h, followed by a 24 h YM181 treatment (2 μM) in 22Rv1 cells. Tubulin was used as a loading control. (G)

Immunoprecipitation-western blot analysis of ubiquitylated EZH2 levels in 22Rv1 cells treated with 2 μM YM181 and YM281 for 12 h. Tubulin

was used as a loading control.

two best compounds, YM181 and YM281, were chosen for

further investigation.

complex due to the loss of integrity.^25,37,38Alternatively, it is

also possible that EED and/or SUZ12 in proximity to EZH2

may be ubiquitinated by the EZH2-PROTAC-mediated

ternary complex formation.^37,38In the following experiments,

as shown in Figure 3E, the degradation eﬀect of YM181 could

be rescued by adding either a proteasome inhibitor MG132 or

MLN-4924, an inhibitor of the neddylation that is essential to

activate the VHL E3 ligase system.³⁹Meanwhile, the EZH2

inhibitor EPZ6438 competed with YM181 to occupy the

catalytic pocket and subsequently prevented protein degrada-

tion. Furthermore, the addition of a synthetic VHL ligand

VH032 also reversed the EZH2 degradation caused by YM181.

To further conﬁrm the requirement of VHL for YM181-

induced EZH2 degradation, VHL was knocked down by

siRNAs in 22Rv1 cells. The exposure of the VHL knockdown

cells to YM181 did not reduce the EZH2 protein level that was

largely degraded in the control cells (Figure 3F). Additionally,

Co-IP experiments clearly displayed a signiﬁcant increase of

EZH2 ubiquitination mediated by both YM181 and YM281,

while the whole protein level was decreased within 12 h

(Figure 3G). These results conﬁrmed that the degradation of

YM181 and YM281 Degraded the EZH2 Protein

through the VHL-Dependent Ubiquitin−Proteasome

System. As shown in Figure 3A, YM181 and YM281

abrogated both the EZH2 protein level and the H3K27me3

degree in a concentration-dependent manner in 24 h, and

moreover had no signiﬁcant eﬀect on the protein level of

EZH1 that is homologous to EZH2 (Figures 3B and S2). The

maximum degradation eﬃcacy reached 80% at a concentration

of 2 μM. A time-course study demonstrated that the EZH2

degradation could be detected in 2 h, and this eﬀect reached a

maximum in 4 h (Figure 3C). It is reported that the PRC2

complex collapse in the absence of any core subunit.⁶To test

the inﬂuence of EZH2 degraders on the PRC2 complex, the

protein levels of the other two subunits, EED and SUZ12, were

examined in both SU-DHL-2 and 22Rv1 cells treated with

YM181 at 2 μM (Figure 3D). Within 24 h, YM181

substantially reduced the levels of EED and SUZ12. The

results demonstrated that YM181 and YM281 rapidly deleted

the EZH2 protein and consequently destabilized the PRC2

10170

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 4. EZH2 degraders displayed stronger anticancer abilities than EPZ6438 in lymphoma cell lines. (A) MTS cell viability curves in diﬀerent

DLBCL cell lines treated with indicated compounds for 5 days. (B) Western blot analysis of EZH2 and H3K27me3 levels in SU-DHL-2 cells

treated with the indicated compounds for 24 h. H3 and tubulin were used as loading controls. (C) Cell cycle analysis in SU-DHL-6 cells treated

with DMSO or indicated compounds for 24 h. (D) Western blot analysis of PARP, caspase-3, and cleaved-caspase-3 levels in SU-DHL-6 cells

treated with indicated compounds for 48 h. Tubulin was used as a loading control. (E) Proportions of PI⁺and/or Annexin-V⁺apoptotic cells in SU-

DHL-6 cells treated with indicated compounds for 48 h as measured by ﬂow cytometry.

EZH2 induced by the two compounds was indeed associated

with the VHL-dependent ubiquitin−proteasome system.

EZH2 Degraders Displayed Stronger Anticancer

Eﬀects than EPZ6438 in Lymphoma Cell Lines. Given

that EZH2 inhibitors are reportedly eﬀective in the

intervention of DLBCL, anticancer eﬀects of EZH2 degraders

were ﬁrst tested in three DLBCL cell lines, SU-DHL-2, SU-

DHL-4, and SU-DHL-6. The MTS assays conﬁrmed that both

YM181 and YM281 caused cell viability to decrease stronger

than EPZ6438 in all three cell lines (Figure 4A). YM181 and

YM281 could induce nearly complete cell viability inhibition

compared to EPZ6438, although their initial eﬀective

concentrations were slightly higher. Meanwhile, EPZ6438

only achieved half inhibition in SU-DHL-2 and SU-DHL-4

cells and 70% inhibition in SU-DHL-6. Moreover, all of the

other lymphoma cell lines that were resistant to the EZH2

inhibitor also showed a complete response to the EZH2

degraders (Figure S3A). Western blot analysis further

conﬁrmed that YM181 induced substantial EZH2 degradation

(Figures 4B and S3B,C). EPZ6438 robustly reduced the

H3K27me3 levels at low nanomolar concentrations, while

YM181 required high doses. The installment of both the linker

and the VHL ligand motif might cause an EZH2 inhibitory

activity loss, explaining the reason why EZH2 degraders

needed a higher eﬀect-starting concentration. To study

whether the linker and the VHL ligand warhead would bring

oﬀ-target eﬀects, we also evaluated the cytostatic eﬀects of V6

and V7 that are structurally similar but do not induce EZH2

degradation. Neither V6 nor V7 induced signiﬁcant cell

viability inhibition (Figure S3D,E). Meanwhile, we synthesized

YM620, an isomer of YM281, with alterations of two

stereocenters in hydroxyproline, which diminish the binding

aﬃnity to VHL (Figure S3F). Indeed, YM620 did not have an

apparent eﬀect on the EZH2 protein level, while it substantially

inhibited EZH2 enzymatic activity (Figure S3G). Compared to

YM281, YM620 had a weaker antiproliferative capacity in two

tested cancer cell lines (Figure S3H). All of these data

excluded the oﬀ-target eﬀects of our EZH2 degraders.

To investigate how the EZH2 degraders intervene in cell

growth of lymphoma cells, both the cell cycle and apoptosis

analyses were conducted. After a 24 h treatment, EZP6438 at

the tested doses (1, 3, 5 μM) slightly caused a G0/G1 phase

arrest of SU-DHL-6 cells without signiﬁcant observation of

sub-G1 increase (Figures 4C and S4A). However, YM181 and

YM281 at the same doses led to a concentration-dependent

cell cycle arrest and a profound sub-G1 population increase.

The caspase-3/7 assay demonstrated that EZH2 degraders

increased the activity of caspase-3/7, and western blot also

showed that the cleaved caspase-3 and PARP were signiﬁcantly

increased, indicating the apoptotic event (Figures 4D and

S4B,C). The Annexin-V/PI assay further veriﬁed that

apoptosis was clearly induced by both EZH2 degraders in a

dose-dependent manner, while EZP6438 did not (Figures 4E

and S4D). These results demonstrated that EZH2 degraders

induced obvious cell cycle arrest and apoptosis, which might be

the reasons why YM181 and YM281 could achieve a complete

cell viability inhibition, whereas EPZ6438 could not if only

inhibiting the methylation function of EZH2.

EZH2 Degraders Reduced Tumor Growth In Vivo and

Decreased Cell Viability in Primary Lymphoma Patient

Cells. To investigate the antitumor activity of EZH2 degraders

in vivo, a xenograft mouse model of DLBCL cell line SU-DHL-

6 was ﬁrst conducted. Consistent with in vitro results, YM281

(80 mg/kg) administered by intraperitoneal injection 6 times

10171

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 5. EZH2 degraders reduced tumor growth in vivo and decreased cell viability in primary lymphoma patient cells. (A) Tumor volume of

Balb/c nude mice bearing SU-DHL-6 xenograft administrated intraperitoneally with a vehicle, YM281 (80 mg/kg) or EPZ (42.5 mg/kg), for 3

weeks. (B) Western blot analysis of EZH2 and H3K27me3 levels in the representative SU-DHL-6 model excised tumors. GAPDH was used as a

loading control. Representative tumor images were presented. (C) Immunohistochemistry analysis of EZH2, H3K27me3, and Ki67 levels in the

representative excised tumors from A. (D) Tumor volume of Balb/c nude mice bearing the Jeko-1 xenograft administrated intraperitoneally with a

vehicle, YM281 (100 mg/kg) or EPZ (50 mg/kg), for 30 days. (E) Western blot analysis of EZH2 and H3K27me3 in the representative Jeko-1

model excised tumors. Tubulin was used as a loading control. Representative tumor images were presented. (F) Immunohistochemistry analysis of

EZH2 and Ki67 levels in the representative Jeko-1 model excised tumors. (G) Western blot analysis of EZH2 and H3K27me3 levels in a DLBCL

patient sample’s cells treated with YM281 at indicated concentrations for 24 h. H3 and tubulin were used as loading controls. (H) Caspase-3/7

activity of patient-derived primary lymphoma cells treated with each compound at indicated concentrations for 48 h in 11 lymphoma patient cases.

(I) Quantitation of the present ATP level in patient-derived cells with each compound at indicated concentrations for 48 h in 11 lymphoma patient

cases.

weekly for 3 weeks remarkably suppressed the tumor volume

(Figure 5A). However, EPZ6438 at the equal molar dose to

YM281 (42.5 mg/kg) failed. Neither EPZ6438 nor YM281

caused signiﬁcant weight loss in mice (Figure S5A). Western

blot analysis of tumor tissue showed that YM281 signiﬁcantly

reduced the EZH2 protein and H3K27me3 levels (Figure 5B).

EPZ36438 did not prevent the tumor growth, although it

substantially decreased the H3K27me3 level, indicating that

the enzymatic inhibition of EZH2 might not be enough to

attenuate lymphoma cells in vivo. The signiﬁcant destabiliza-

tion of EZH2 and inhibition of tumor proliferation (indicated

by Ki67 staining) induced by YM281 was further conﬁrmed by

immunohistochemistry in the tumor slices (Figure 5C). The in

vivo anticancer eﬃcacy in DLBCL was validated in the mice

xenograft model of Jeko-1, a mantle cell lymphoma cell line

(Figures 5D−F and S5B). More importantly, the tumor weight

was clearly associated with the EZH2 protein level in YM281

treated mice (Figure S5C,D). Finally, to evaluate the potential

clinical implication of EZH2 degraders that showed promising

eﬃcacy in all diﬀerent lymphoma cell lines, as shown in Figure

S3A, primary lymphoma cells extracted from various

lymphoma patient samples were used for further tests. First,

EZH2 degrader YM281 induced dose-dependent EZH2

degradation in primary cells from one DLBCL patient (Figure

10172

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 1. Synthesis of Intermediate Compounds 6 and 7

a

Reagents and conditions: (a) Fe, NH₄Cl, MeOH, 90 °C; (b) tetrahydro-4H-pyran-4-one, AcOH, Na(AcO)₃BH, 1,2-dichloroethane; (c)

acetaldehyde, AcOH, Na(AcO)₃BH, 1,2-dichloroethane; (d) Boc₂O, H₂, Raney-Ni, MeOH; (e) HCl/MeOH; (f) NaOH, EtOH, 60 °C; (g) HOBt,

EDCI, NMM, DMSO; (h) 4-methoxycarbonylphenylboronic acid, K₂CO₃, Pd(PPh₃)₄, N,N-dimethylformamide (DMF), 90 °C; and (i) NaOH,

EtOH, 60 °C. Note: unless stated, reactions underwent at room temperature in all synthetic schemes.

a

Scheme 2. Synthesis of VHL Ligands 15, 16, and VH032

a

Reagents and conditions: (a) NaOH, Boc₂O, MeOH; (b) Pd(OAc)₂, KOAc, N,N-dimethylacetamide (DMA), 150 °C; (c) HCl/MeOH; (d) 12,

HATU, N,N-diisopropylethylamine (DIPEA), tetrahydrofuran (THF); (e) HCl/MeOH; (f) 14, HATU, DIPEA, DMF; (g) HCl/MeOH; and (h)

DIPEA, acetic anhydride, CH₂Cl₂.

5G and Table S1). Furthermore, lymphoma primary cells from

11 cases of lymphoma patients, including two cases of BLBCL,

were tested for the eﬃcacy of YM281. Compared to EPZ6438,

YM281 increased the activity of caspase-3 and -7 and

meanwhile reduced the cell viability observed in adenosine

triphosphate (ATP) assays (Figure 5H,I).

previously described (Schemes 1, 2, and 5).^31,40−43As shown

in Scheme 3, compound V2 (YM281) was used as an example

for the synthesis of series V compounds. The commercially

available propane-1,3-diol (17b) was protected by the benzyl

group on one side hydroxyl and then reacted with t-butyl

bromoacetate. The removal of the benzyl group under catalytic

hydrogenation conditions followed by a substitution reaction

of hydroxyl with tosyl chloride gave compound 19b. Alkylation

of 4-hydroxyphenylboronic acid pinacol ester with 19b

aﬀorded 20b, and consequential Suzuki coupling with

CHEMISTRY

■

The synthesis of the key intermediates 6, 7, VHL ligands 16

and VH032, and CRBN ligands 32 and 33 have been

10173

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 3. Synthesis of Series V Compounds

a

Reagents and conditions: (a) BnBr, NaH, THF; (b) tert-butyl bromoacetate, NaOH, TBACl, CH₂Cl₂; (c) Pd/C, H₂, EtOH; (d) TsCl, DMAP,

TEA, CH₂Cl₂; (e) 4-hydroxyphenylboronic acid pinacol ester, K₂CO_3,DMF, 70 °C; (f) 6, K₃PO₄, Pd(PPh)_4,DMF, 90 °C; (g) 20% TFA, CH₂Cl₂;

(h) 16, HATU, DIPEA, DMF; (i) NaH, tert-butyl bromoacetate, DMF; (j) TsCl, DMAP, TEA, CH₂Cl₂; (k) 4-hydroxyphenylboronic acid pinacol

ester, K₂CO₃, DMF, 70 °C; (l) 6, K₃PO₄, Pd(PPh)₄, DMF, 90 °C; (m) 20% TFA, CH₂Cl₂; (n) 16, HATU, DIPEA, DMF; (o) diethylamine,

CH₂Cl₂; (p) Boc₂O, NaOH, CH₂Cl₂; (q) 16, HATU, DIPEA, DMF; (r) HCl/MeOH; and (s) 7, HATU, DIPEA, DMF.

10174

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 4. Synthesis of YM620

a

Reagents and conditions: (a) 11, HATU, DIPEA, THF; (b) HCl/MeOH; (c) 14, HATU, DIPEA; (d) HCl/MeOH; and (e) 21b, HATU,

DIPEA, DMF.

compound 6 and the removal of the Boc group under acidic

conditions gave the acid 21b. The ﬁnal compound V2

(YM281) was obtained via standard amide coupling between

acid 21b and the amine 16. In a similar manner, other V series

compounds V1, V3 (YM181), V4-V7, and YM620 modiﬁed

with the varying linker were prepared, as shown in Schemes 3

and 4.

Compound G5 was used as an example for the synthesis of

series G compounds (Scheme 5). The commercially available

octane-1,8-diamine was protected by Boc on one side amine to

give 37b. Amide coupling with 36⁴¹and subsequent removal of

the Boc group under acidic conditions aﬀorded amine 38b.

The ﬁnal compound G5 was obtained via standard amide

coupling between acid 7 and amine 38b. In a similar manner,

other series G compound G1−G4 and G6 modiﬁed with the

varying linker were prepared.

that reduce the EED protein level as well.⁴⁶The concomitant

degradation of EED induced by YM281 and YM181 could

either result from collateral ubiquitination of EED by the VHL

E3 ligase due to its proximity to EZH2 or from the reduced

stability of the PRC2 complex to eject the EED subunit after

the ﬁrst EZH2 degradation.^25,39,47The exact mechanism

remains to be elucidated in the future. More recently, some

CRBN-based PROTACs were reported while our revised

manuscript was under review.⁴⁸However, their compounds

remained to be evaluated in the animal study. Meanwhile,

PROTAC-based degradation of EED, another subunit of the

PRC2 complex, was also reported recently.^37,38However, the

reported EED degraders did not show superior in vitro

anticancer activities compared with their parental EED

inhibitors, whereas more in vivo experiments are also needed

to validate their eﬃcacy.

In our observation, the therapeutic eﬃcacy of EZH2

inhibitors was limited to DLBCL cell lines among the tested

lymphoma cell lines (Figure 1A). We wonder whether the

direct EZH2 degradation via PROTAC technology could be

developed to improve their targeting capacity to the other

types of lymphoma cells. In our current study, we developed

PROTAC-based EZH2 degraders and investigated their

eﬃciency of EZH2 degradation and therapeutic eﬃcacy in

various types of lymphoma in vitro and in vivo. Our study

revealed that only VHL-targeting compounds enabled the

EZH2 degradation with an appropriate linker at 7 or 9 atoms

length. Compared to the parental EZH2 inhibitor EPZ6438,

our two best EZH2 degraders YM181 and YM281 selectively

degraded EZH2 over EZH1, and they exhibited eﬀective

antiproliferative activity both in DLBCL and other types of

lymphoma cell lines. Furthermore, the EZH2 degrader showed

an apparent advantage to prevent in vivo tumor growth in

lymphoma xenografts without obvious toxicity at the eﬃca-

cious doses.

DISCUSSION AND CONCLUSION

■

As widely conceptualized, EZH2 inhibitors that simply

suppress EZH2 enzymatic activity exhibit antitumor eﬀects

by inhibiting trimethylation of H3K27 and thereby activating

tumor suppressor genes.^6,7This class of EZH2 inhibitors

reduces H3K27 methylation levels at very low concentrations.

However, their therapeutic eﬃcacy varies largely in diﬀerent

types of tumors; for example, DLBCL is the major subtype of

lymphomas that are most sensitive to EZH2 inhibitors.

Previous research studies indicated that the whole EZH2

protein itself is involved in tumor proliferation.^5,10,16,17

Therefore, abrogating the whole EZH2 protein may have a

signiﬁcant therapeutic advantage over simply inhibiting its

enzymatic activity. Chen and co-workers have reported that

gambogenic acid derivatives could covalently bind to Cys668

within the EZH2-SET domain, triggering EZH2 degradation

to inhibit head and neck cancer cells.⁴⁴But the selectivity of

gambogenic acid derivatives for EZH2 is questionable.⁴⁵Very

recently, Jin’s group reported a hydrophobic tagging-based

EZH2 degrader (MS1943) that showed better anticancer

capacity in triple-negative breast cancer than the parental

EZH2 inhibitor.⁴⁶Surprisingly, MS1943 was reported to

reduce the protein level of SUZ12 without aﬀecting the EED

protein level, which is diﬀerent from our EZH2 PROTACs

However, the incomplete EZH2 degradation and the modest

cellular potencies for YM281 and YM181 in the inhibition of

cell viability at low concentrations suggest that there is still

room for further optimization. In the future, a structure and

activity relationship study on diﬀerent linker scaﬀoldings with

the same linking length as YM181 and YM281 may be

10175

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 5. Synthesis of Series G Compounds

a

Reagents and conditions: (a) 29, NaOAc, AcOH, 120 °C reﬂux, or 30, pyridine, 110 °C; (b) 34a−b, DIPEA, DMF 90 °C or 34c−d, Na₂CO₃,

DMF, 80 °C; (c) 20% TFA, CH₂Cl₂; (d) 7, HATU, DIPEA, DMF; (e) tert-butyl bromoacetate, K₂CO₃, DMF; (f) 20% TFA, CH₂Cl₂; (g) HATU,

DIPEA, DMF; (h) 20% TFA, CH₂Cl₂, 50 °C; and (i) 7, HATU, DIPEA, DMF.

puriﬁcation unless stated. The chemicals used were either purchased

from commercial sources or prepared according to literature

procedures. The ¹H and ¹³C nuclear magnetic resonance (NMR)

spectra were recorded on a Bruker Avance spectrometer 400 at 400

MHz and 100 MHz or a Bruker Avance spectrometer 500 at 500

MHz and 125 MHz respectively. Chemical shifts are given in ppm (δ)

warranted to obtain more potent EZH2 degraders. Meanwhile,

cancer cells that may not depend on EZH2 for their

tumorigenesis, for example, pancreatic cancer cell AsPC1 and

lung cancer cell NCI-H460, are not sensitive to YM181 and

YM181, although the compounds were able to decrease their

EZH2 levels (Figure S6). By measuring cell permeability with

Caco-2 cells, both YM181 and YM281 showed their apparent

permeability ability largely compromised compared to the

parental EHZ2 inhibitor EPZ6438 (Table S2), indicating the

importance to improve their oral bioavailability through further

structural optimization. Overall, our results demonstrate that

EZH2 degraders may have better therapeutic potential than

EZH2 inhibitors against lymphomas. The exact mechanism of

action and the application of our EZH2 degraders in other

cancers are under investigation.

1

referenced to CDCl₃with 7.26 for H and 77.10 for ¹³C, and to d₆-

DMSO with 2.50 for ¹H and 39.5 for ¹³C. In the case of multiplet, the

signals are reported as intervals. Signals are abbreviated as follows: s,

singlet; d, doublet; t, triplet; q, quartet; and m, multiplet. Coupling

constants are expressed in hertz. High-resolution mass spectra

(HRMS) were recorded on a BRUKER VPEXII spectrometer (the

ESI mode). The progress of the reactions was monitored by thin-layer

chromatography on a glass plate coated with silica gel with a

ﬂuorescent indicator (GF254). Flash column chromatography was

performed on silica gel (200−300 mesh). An Agilent 1100 HPLC

system equipped with an Eclipse XDB-C18 column was used to

determine the purity of all of the ﬁnal key products. Gradient elution:

10−90% MeOH against H₂O with 0.1% TFA over 15 min (the ratio

of MeOH/H₂O from 10 to 90% in 8 min, and ﬁnally 90% for 7 min)

at a ﬂow rate of 1.0 mL/min; detection wavelength: 254 nm. All

biologically tested compounds had a purity of more than 95%.

EXPERIMENTAL SECTION

■

Chemistry: General Experiment and Information. Synthesis

details for the key ﬁnal compounds are described here. All solvents

were commercially available and were used without further

10176

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

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

methyl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenza-

mide (6).⁴⁰To a solution of methyl 5-bromo-2-methyl-3-nitro-

benzoate (5 g, 18.24 mmol) in MeOH (50 mL), Fe (5.09 g, 91.22

mmol) and NH₄Cl (1.95 g, 36.49 mmol) were added sequentially.

The mixture was stirred at 90 °C overnight. In the end, the solid

precipitated was ﬁltered oﬀ and washed with MeOH (3 × 80 mL).

The combined ﬁltrate was dried and concentrated under reduced

pressure to give the desired crude 2 as a yellow liquid (4.34 g, 99.6%

yield). To a solution of 2 (4.34 g, 17.78 mmol) in 1,2-dichloroethane

(50 mL) were added tetrahydro-4H-pyran-4-one (2.46 mL, 26.67

mmol) and AcOH (6.10 mL, 106.68 mmol). The mixture was stirred

at r.t. for 0.5 h before Na(AcO)₃BH (11.31 g, 53.34 mmol) was added

at 0 °C. The mixture was warmed to r.t. and stirred for 24 h. EtOAc

and water were added, and the aqueous phase was extracted with

EtOAc twice. The combined organic phases were dried over Na₂SO₄,

ﬁltered, and concentrated under reduced pressure. The residue was

puriﬁed by ﬂash column chromatography with EtOAc/PE (10−35%)

to aﬀord the product as a yellow solid (5.34 g, 91.5% yield). To a

solution of the product (5.34 g, 16.27 mmol) in 1,2-dichloroethane

(50 mL) were added acetaldehyde (2.76 mL, 48.81 mmol) and AcOH

(5.58 mL, 97.62 mmol). The mixture was stirred at r.t. for 0.5 h

before Na(AcO)₃BH (10.35 g, 48.81 mmol) was added at 0 °C. The

mixture was warmed to r.t. and stirred for 24 h before EtOAc (200

mL) and water (100 mL) were added. The aqueous layer was

extracted with EtOAc (2 × 150 mL). The combined organic phases

were dried over Na₂SO₄, ﬁltered, and concentrated under reduced

pressure. The residue was puriﬁed by ﬂash column chromatography

with EtOAc/PE (10−35%) to aﬀord 3 as a yellow solid (5.18 g, 89.4%

yield).

onic acid (453 mg, 2.52 mmol), K₂CO₃(1.16 g, 8.40 mmol), and

Pd(PPh₃)₄(121 mg, 104.95 μmol). The solution was purged and

reﬁlled with argon three times. Then, the solution was stirred at 90 °C

for 9 h before EtOAc (70 mL) and water (30 mL) were added at r.t.

The aqueous phase was extracted with EtOAc (2 × 70 mL). The

combined organic phases were dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The residue was puriﬁed by

ﬂash column chromatography with MeOH/CH₂Cl₂(0−10%) to

aﬀord the coupled product as a yellow solid (0.82 g, 73.5% yield). The

obtained product was hydrolyzed under NaOH in EtOH at 60 °C to

1

give the ﬁnal acid 7. H NMR (500 MHz, CDCl₃) δ 12.96 (s, 1H),

8.17 (d, J = 8.2 Hz, 2H), 7.69 (m, 3H), 7.51 (s, 1H), 7.41 (s, 1H),

6.12 (s, 1H), 4.51 (d, J = 5.8 Hz, 2H), 3.96 (d, J = 11.3 Hz, 2H), 3.33

(t, J = 11.0 Hz, 2H), 3.15 (s, 2H), 3.06 (s, 1H), 2.50 (d, J = 15.6 Hz,

6H), 2.37 (s, 3H), 1.72 (m, 4H), 0.92 (t, J = 6.4 Hz, 3H).

tert-Butyl((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)-

benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-

carbamate (15).^31,43To a solution of (4-bromophenyl)methanamine

8 (6.79 mL, 53.75 mmol) in MeOH (80 mL) were added Boc₂O

(18.52 mL, 80.62 mmol) and NaOH (107.5 mmol, 1 M) at 0 °C. The

reaction mixture was warmed to r.t. and stirred for 5 h. MeOH was

removed under reduced pressure, and the residue was acidiﬁed by

adding aq. HCl until pH = 5. The aqueous solution was extracted with

CH₂Cl₂(3 × 150 mL). The combined organic phases were washed

with brine, dried over anhydrous Na₂SO₄, and ﬁltered. The

concentration under reduced pressure gave a colorless liquid (14.4

g, 93.6% yield), which was used in the next step without further

puriﬁcation.

To a solution of the above-obtained liquid (14.4 g, 50.42 mmol) in

DMA (50 mL) under argon were added Pd(AcO)₂(226.45 mg, 1.01

mmol), potassium acetate (9.9 g, 100.86 mmol), and 4-methylthiazole

9 (4.59 mL, 50.43 mmol). The reaction mixture was stirred at 150 °C

for 10 h. Then, CH₂Cl₂(150 mL) and water (100 mL) were added.

The aqueous phase was extracted with CH₂Cl₂(2 × 150 mL). The

combined organic phases were dried over anhydrous Na₂SO₄, ﬁltered,

concentrated under reduced pressure to give 10 as a yellow liquid

(14.35 g, 93.7% yield), which was used in the next step without

further puriﬁcation. After the removal of the Boc group in 10 (6.5 g.

21.35 mmol) under acidic conditions, a free amine residue (5.14 g,

20.77 mmol) was obtained. To a stirred solution of 12 (5.92 g, 25.62

mmol) and DIPEA (10.59 mL, 64.05 mmol) in anhydrous THF (75

mL) at 0 °C were added the above-obtained amine (5.14 g, 20.77

mmol) and HATU (9.74, 25.62 mmol). The resulting mixture was

warmed to r.t. and stirred for 2 h. After THF was removed under

reduced pressure, CH₂Cl₂(120 mL) and water (80 mL) were added.

The aqueous phase was extracted with CH₂Cl₂(2 × 100 mL). The

combined organic phases were dried over anhydrous Na₂SO₄, ﬁltered,

and concentrated under reduced pressure to give 13 as a yellow liquid

(8.67 g, 82.5% yield), which was used in the next step without further

puriﬁcation. After the removal of the Boc group in 13 (5 g, 11.98

mmol) under acidic conditions, the obtained free amine residue was

added to the stirred solution of N-Boc-^L-tert-Leucine 14 (2.76 g, 11.93

mmol), DIPEA (5.91 mL, 35.78 mmol), and HATU (6.8 g, 17.89

mmol) in DMF (45 mL). The reaction mixture was stirred at r.t. for 5

h. CH₂Cl₂(120 mL) and water (60 mL) were added, and the aqueous

phase was extracted with CH₂Cl₂(2 × 100 mL). The combined

organic phases were dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The residue was puriﬁed by

column chromatography on silica gel with MeOH/CH₂Cl₂(0−10%)

to give 15 as a yellow solid (4.5 g, 70.8% yield). ¹H NMR (400 MHz,

CDCl₃) δ 8.68 (s, 1H), 7.47 (m, 1H), 7.33 (m, 4H), 5.20 (d, J = 9.0

Hz, 1H), 4.74 (m, 1H), 4.53(m, 2H), 4.30 (dd, J = 15.0, 5.1 Hz, 1H),

4.17 (d, J = 9.1 Hz, 1H), 4.02 (d, J = 11.3 Hz, 1H), 3.60 (dd, J = 11.3,

3.4 Hz, 1H), 2.50 (s, 3H), 2.50 (m, 1H), 2.10 (m, 1H), 1.40 (s, 9H),

0.91 (s, 9H).

To a solution of 4,6-dimethyl-2-oxo-1,2-dihydro-3-pyridinecarboni-

trile 4 (3.0 g, 20.25 mmol) in MeOH (120 mL) was added a catalytic

amount of Raney nickel and Boc₂O (5.3 g, 24.30 mmol). The mixture

was purged and reﬁlled with H₂three times and stirred at r.t. for 12 h

under H₂. After ﬁltration and concentration, the residue was puriﬁed

by ﬂash column chromatography with MeOH/CH₂Cl₂(2−5%) to

aﬀord a white solid (5.1 g, 99.6% yield). Acetyl chloride (5.74 mL,

80.68 mmol) was added to MeOH (28 mL) at 0 °C for 1 h to prepare

a fresh HCl solution in methanol, and then the above-obtained

compound (5.09 g, 20.17 mmol) was added. The solution was

warmed to r.t., stirred for 3 h, and ﬁnally concentrated under reduced

pressure to give 5 (4.1 g, 100% yield) without further puriﬁcation.

To a solution of 3 (5.18 g, 14.54 mmol) in ethanol (50 mL) was

added aqueous NaOH solution. The reaction mixture was stirred at

60 °C for 3 h before it was concentrated under reduced pressure.

Then, CH₂Cl₂(100 mL) and water (150 mL) were added, and the

mixture was washed with CH₂Cl₂(2 × 100 mL). The aqueous phase

was acidiﬁed by adding aq. HCl (1 M) until pH = 3−4. Then, the

aqueous solution was extracted with CH₂Cl₂(3 × 200 mL). The

combined organic phases were concentrated under reduced pressure

to give the intermediate acid as a white solid (4.55 g, 91.4% yield).

To a solution of 5 (3.03 g, 19.93 mmol) and the above-obtained

acid (4.55 g, 13.29 mmol) in DMSO (50 mL), were added HOBt

(2.15 g, 15.94 mmol), EDCI (3.06 g, 15.94 mmol), and NMM (8.76

mL, 79.72 mmol). The mixture was stirred at r.t. overnight before

EtOAc (150 mL) and water (100 mL) were added. The aqueous

phase was extracted with EtOAc (2 × 100 mL). The combined

organic phases were dried over anhydrous Na₂SO₄and concentrated

under reduced pressure. The residue was puriﬁed by silica gel ﬂash

column chromatography with EtOAc/PE (10−35%) to aﬀord 6 as a

1

yellow solid (3.7 g, 58.3% yield). H NMR (400 MHz, CDCl₃) δ

11.12 (s, 1H), 7.22 (d, J = 1.8 Hz, 1H), 7.18 (d, J = 1.8 Hz, 1H), 7.12

(t, J = 5.6 Hz, 1H), 5.95 (s, 1H), 4.52 (d, J = 5.9 Hz, 2H), 3.95 (d, J =

11.4 Hz, 2H), 3.35−3.27 (m, 2H), 3.02 (q, J = 6.9 Hz, 2H), 2.93 (m,

1H), 2.39 (s, 3H), 2.24 (d, J = 2.3 Hz, 6H), 1.66 (m, 4H), 0.85 (t, J =

7.0 Hz, 3H).

3′-(((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-

carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-

[1,1′-biphenyl]-4-carboxylic Acid (7).⁴⁰To a solution of 6 (1 g, 2.10

mmol) in DMF (20 mL) were added 4-methoxycarbonylphenylbor-

(2S,4R)-1-((S)-2-Acetamido-3,3-dimethylbutanoyl)-4-hydroxy-N-

(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

(VH032).^31,43Removal of the Boc group in 15 (50 mg, 94.22 mmol)

under acidic conditions aﬀorded amine compound 16. To a solution

of 16 (40 mg, 94.22 μmol) in CH₂Cl₂was added DIPEA (46.06 μL,

10177

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

278.70 μmol) at 0 °C. After the mixture was stirred for 0.5 h at r.t.,

acetic anhydride (13.17 μL, 139.35 μmol) was added. The mixture

was continued to stir at r.t. for 2 h. CH₂Cl₂(10 mL) and water (5

mL) were added, and the mixture was extracted with CH₂Cl₂(2 × 10

mL). The combined organic phases were dried over anhydrous

Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

residue was puriﬁed by column chromatography on silica gel with

MeOH/CH₂Cl₂(0−10%) to give VH032 as a yellow solid (32 mg,

67.7% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.68 (s, 1H), 7.36 (q, J

= 8.3 Hz, 4H), 6.32 (d, J = 8.6 Hz, 1H), 4.70 (t, J = 8.0 Hz, 1H),

4.65−4.47 (m, 3H), 4.33 (dd, J = 15.0, 5.1 Hz, 1H), 4.10 (d, J = 11.5

Hz, 1H), 3.61 (dd, J = 11.4, 3.3 Hz, 1H), 2.51 (s, 3H), 2.51 (m, 1H),

2.14 (dd, J = 13.6, 8.4 Hz, 1H), 1.98 (s, 3H), 0.93 (s, 9H).

The reaction mixture was stirred at r.t. for 3 h before it was diluted

with CH₂Cl₂(100 mL). The organic phase was washed with water (3

× 10 mL), dried over Na₂SO₄, and concentrated under reduced

pressure. The residue was puriﬁed by ﬂash column chromatography

with EtOAc/PE (20−50%) to aﬀord 19b as a colorless oil (450 mg,

54.8% yield).

To a solution of 19b (150 mg, 435 μmol) in DMF (2 mL) were

added 4-hydroxyphenylboronic acid pinacol ester (144 mg, 653

μmol) and K₂CO₃(120 mg, 871 μmol). The reaction mixture was

stirred at 70 °C for 5 h and then cooled to r.t. before EtOAc (30 mL)

and water (10 mL) were added. The mixture was extracted with

EtOAc (2 × 30 mL). The combined organic phases were dried over

Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

residue was puriﬁed by ﬂash column chromatography with EtOAc/PE

(20−50%) to aﬀord 20b as a colorless solid (145 mg, 84.9% yield).

To a solution of 6 (121.4 mg, 254.9 μmol) in DMF (2 mL) were

added 20b (100 mg, 254.91 μmol), K₂CO₃(108.2 mg, 509 μmol),

and Pd(PPh₃)₄(14.7 mg, 12.75 μmol). The mixture was purged and

reﬁlled with argon three times. The mixture was stirred at 90 °C for 9

h before EtOAc (30 mL) and water (10 mL) were added at r.t. The

aqueous phase was extracted with EtOAc (2 × 30 mL). The

combined organic phases were dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The residue was puriﬁed by

ﬂash column chromatography with MeOH/CH₂Cl₂(0−10%) to

aﬀord the coupled product as a yellow solid (120 mg, 71.1% yield).

Then, the obtained product (120 mg, 181.32 μmol) was dissolved

in CH₂Cl₂(1 mL), and TFA (135 μL, 1.81 mmol) was added before

the mixture was stirred at r.t. for 3 h. The concentration of the solvent

provided acid 21b, which was used for the following steps without

further puriﬁcation. To a stirred solution of 21b in DMF (2 mL),

amine 16 (92.98 mg, 215.94 mmol), DIPEA (178.45 μL, 1.08 mmol),

and HATU (68.42 mg 179.95 μmol) were added sequentially. The

resulting mixture was stirred at r.t. for 10 h, and then brine (5 mL)

was added and extracted with EtOAc (3 × 15 mL). The combined

organic phases were washed with brine, dried over anhydrous

Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

residue was puriﬁed by ﬂash column chromatography with MeOH/

CH₂Cl₂(0−10%) to give V2 (YM281) as a white powder (118 mg,

2-(2,6-Dioxopiperidin-3-yl)-4-ﬂuoroisoindoline-1,3-dione (32).⁴¹

To a suspension of 4-ﬂuoroisobenzofuran-1,3-dione 29 (1 g, 6.02

mmol) and 3-aminopiperidine-2,6-dione hydrochloride 31 (0.99 g,

6.02 mmol) in AcOH (20 mL) was added sodium acetate (0.59 g,

7.22 mmol). The reaction mixture was heated to 120 °C for 12 h.

After cooling to r.t., AcOH was removed under reduced pressure. The

residue was puriﬁed by ﬂash column chromatography with MeOH/

CH₂Cl₂(0−10%) to give 32 as a white solid (1.33 g, 79.7% yield). ¹H

NMR (400 MHz, d₆-DMSO) δ 11.14 (s, 1H), 7.98−7.91 (m, 1H),

7.79 (d, J = 7.3 Hz, 1H), 7.74 (t, J = 8.9 Hz, 1H), 5.16 (dd, J = 12.9,

5.4 Hz, 1H), 2.89 (ddd, J = 17.2, 14.0, 5.5 Hz, 1H), 2.68−2.52 (m,

2H), 2.06 (ddd, J = 10.7, 5.5, 3.1 Hz, 1H).

2-(2,6-Dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione

(33).⁴²3-Hydroxyphthalic anhydride 30 (3 g, 18.28 mmol) and 3-

aminopiperidine-2,6-dione hydrochloride 31 (2.99 g, 18.28 mmol)

were dissolved in pyridine (80 mL) and heated to 110 °C. After 14 h,

the mixture was cooled to r.t. and concentrated under reduced

pressure. The residue was puriﬁed by ﬂash column chromatography

with MeOH/CH₂Cl₂(0−10%) to give 33 as a tan solid (4.02 g,

80.2% yield). ¹H NMR (500 MHz, d₆-DMSO) δ 11.18 (s, 1H), 11.09

(s, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.32 (d, J = 7.0 Hz, 1H), 7.25 (d, J =

8.4 Hz, 1H), 5.07 (dd, J = 12.8, 5.2 Hz, 1H), 2.93−2.83 (m, 1H), 2.55

(dd, J = 27.8, 11.8 Hz, 2H), 2.01 (dd, J = 13.6, 7.8 Hz, 1H).

(2S,4R)-1-((S)-2-(2-(3-((3′-(((4,6-Dimethyl-2-oxo-1,2-dihydropyri-

din-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)-

amino)-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)propoxy)acetamido)-

3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)-

benzyl)pyrrolidine-2-carboxamide (V2, YM281). To a solution of

1,3-propanediol 17b (2 g, 26.28 mmol) in THF (10 mL) was added

NaH (60%, 525.61 mg, 131.14 mmol) at 0 °C. The reaction mixture

was stirred for 1 h before BnBr (1.04 mL, 8.76 mmol) was added at 0

°C. The reaction mixture was gradually warmed to r.t., stirred

overnight, and quenched with saturated aq. NH₄Cl (30 mL). The

mixture was extracted with EtOAc (3 × 80 mL). The combined

organic phases were dried over Na₂SO₄, ﬁltered, and concentrated

under reduced pressure. The residue was puriﬁed by ﬂash column

chromatography with EtOAc/PE (10−50%) to aﬀord 3-(benzyloxy)-

propan-1-ol as a colorless oil (0.82 g, 56.3% yield).

1

64.4% yield). H NMR (500 MHz, d₆-DMSO) δ 11.47 (s, 1H), 8.95

(s, 1H), 8.60 (s, 1H), 8.18 (s, 1H), 7.51 (d, J = 7.8 Hz, 2H), 7.44 (d, J

= 8.8 Hz, 1H), 7.38 (m, 4H), 7.32 (s, 1H), 7.16 (s, 1H), 7.01 (d, J =

7.4 Hz, 2H), 5.85 (s, 1H), 5.17 (s, 1H), 4.57 (d, J = 9.3 Hz, 1H), 4.46

(t, J = 8.1 Hz, 1H), 4.38 (d, J = 15.9 Hz, 2H), 4.33−4.21 (m, 3H),

4.10 (s, 2H), 3.97 (s, 2H), 3.81 (d, J = 9.9 Hz, 2H), 3.71−3.58 (m,

4H), 3.23 (t, J = 11.2 Hz, 2H), 3.06 (d, J = 6.3 Hz, 2H), 2.99 (s, 1H),

2.41 (s, 3H), 2.23 (s, 3H), 2.20 (s, 3H), 2.10 (s, 3H), 2.07−1.98 (m,

3H), 1.91 (s, 1H), 1.64 (d, J = 11.2 Hz, 2H), 1.50 (d, J = 10.6 Hz,

2H), 0.94 (s, 9H), 0.81 (t, J = 5.9 Hz, 3H). ¹³C NMR (125 MHz, d₆-

DMSO) δ 171.7, 169.2, 169.1, 168.4, 163.0, 158.1, 151.4, 149.5,

148.8, 147.7, 142.7, 139.6, 139.4, 136.9, 132.3, 131.9, 131.1, 129.7,

128.9, 128.7, 128.2, 127.7, 127.5, 122.5, 121.6, 120.5, 114.9, 107.4,

69.5, 68.9, 67.7, 66.3, 64.6, 58.8, 57.9, 56.6, 55.7, 41.7, 41.2, 37.9,

35.8, 34.9, 30.3, 29.0, 26.2, 19.0, 18.2, 15.9, 14.5, 12.7. HRMS (ESI,

m/z) calcd for C₅₆H₇₂N₇O₉S [M + H]⁺: 1018.5107, found:

1018.5121. Purity: 98.1%.

A mixture of 3-(benzyloxy)propan-1-ol (0.81 g, 4.87 mmol), tert-

butyl bromoacetate (2.84 mL, 19.5 mmol), aqueous sodium

hydroxide (35%, 20 mL), and TBACl (1.38 mL, 4.87 mmol) in

CH₂Cl₂(20 mL) was stirred vigorously at r.t. overnight. The reaction

mixture was poured into water (15 mL) and then was extracted with

CH₂Cl₂(3 × 20 mL). The combined organic phases were washed

with water (15 mL × 2) and brine (2 × 15 mL), dried over anhydrous

Na₂SO₄, and concentrated under reduced pressure. The residue was

puriﬁed by ﬂash column chromatography with EtOAc/PE (0−20%)

to aﬀord 18b as a colorless oil (1.2 g, 87.8% yield).

(2S,4R)-1-((S)-2-(2-(2-((3′-(((4,6-Dimethyl-2-oxo-1,2-dihydropyri-

din-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)-

amino)-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)ethoxy)acetamido)-3,3-

dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-

pyrrolidine-2-carboxamide (V1). V1 was prepared following the

1

procedure for the synthesis of compound V2 (YM281). H NMR

A mixture of 18b (670 mg, 2.39 mmol) and palladium on carbon

(10%, 70 mg) in ethanol (8 mL) was stirred at r.t. overnight under a

hydrogen atmosphere. The mixture was ﬁltered and washed with

EtOAc (3 × 30 mL). The combined ﬁltrate was concentrated under

reduced pressure to aﬀord a hydroxyl product as a colorless oil, which

was used in the next step without further puriﬁcation. The hydroxyl

product (454 mg, 2.39 mmol) was dissolved in CH₂Cl₂(5 mL),

followed by the addition of 4-toluenesulfonyl chloride (901 mg, 4.73

mmol), TEA (658 μL, 4.73 mmol), and DMAP (145 mg, 1.18 mmol).

(400 MHz, d₆-DMSO) δ 11.47 (s, 1H), 8.92 (s, 1H), 8.63 (t, J = 5.9

Hz, 1H), 8.17 (t, J = 4.8 Hz, 1H), 7.53−7.46 (m, 3H), 7.42 (d, J = 8.3

Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 7.29 (s, 1H), 7.15 (s, 1H), 7.09 (d,

J = 8.7 Hz, 2H), 5.85 (s, 1H), 5.18 (d, J = 3.4 Hz, 1H), 4.61 (d, J =

9.6 Hz, 1H), 4.51−4.34 (m, 3H), 4.30 (d, J = 4.8 Hz, 2H), 4.26 (d, J

= 5.5 Hz, 1H), 4.24−4.12 (m, 2H), 4.06 (s, 2H), 3.86 (t, J = 4.3 Hz,

2H), 3.80 (d, J = 11.7 Hz, 2H), 3.69 (dd, J = 10.5, 3.7 Hz, 1H), 3.62

(d, J = 10.8 Hz, 1H), 3.22 (t, J = 11.0 Hz, 2H), 3.08−3.01 (m, 2H),

3.01−2.92 (m, 1H), 2.38 (s, 3H), 2.22 (s, 3H), 2.21 (s, 3H), 2.10 (s,

10178

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

3H), 2.09−2.02 (m, 1H), 1.96−1.88 (m, 1H), 1.63 (d, J = 11.4 Hz,

2H), 1.55−1.45 (m, 2H), 0.94 (s, 9H), 0.79 (t, J = 7.0 Hz, 3H). ¹³C

NMR (101 MHz, d₆-DMSO) δ 171.8, 169.1, 168.4, 163.0, 158.0,

151.3, 149.5, 148.8, 147.6, 142.7, 139.5, 139.4, 136.8, 132.4, 131.9,

131.1, 129.6, 128.8, 128.6, 128.1, 127.6, 127.4, 122.5, 121.6, 120.5,

115.0, 107.4, 69.6, 69.5, 68.9, 70.0, 66.3, 58.8, 57.8, 56.6, 55.7, 41.7,

41.2, 37.9, 35.8, 34.9, 30.3, 26.2, 18.9, 18.9, 18.2, 15.9, 14.5, 12.7.

HRMS (ESI, m/z) calcd for C₅₅H₇₀N₇O₉S [M + H]⁺: 1004.4805,

found: 1004.4805. Purity: 98.1%.

1H), 2.43 (s, 3H), 2.23 (s, 3H), 2.21 (s, 3H), 2.10 (s, 3H), 2.08−2.03

(m, 1H), 1.95−1.86 (m, 1H), 1.65 (d, J = 10.9 Hz, 2H), 1.57−1.45

(m, 2H), 0.94 (s, 9H), 0.82 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz,

d₆-DMSO) δ 171.8, 169.1, 168.6, 163.0, 158.0, 151.4, 149.5, 148.8,

147.7, 14.7, 139.5, 139.4, 136.9, 132.3, 132.0, 131.1, 129.7, 128.7,

127.6, 127.5, 122.5, 121.6, 120.5, 114.9, 107.4, 70.5, 69.9, 69.9, 69.6,

68.9, 68.9, 67.2, 66.3, 58.8, 57.9, 56.6, 55.7, 48.6, 41.7, 41.2, 37.9,

35.7, 34.9, 30.3, 26.2, 18.9, 18.2, 15.9, 14.5, 12.7 HRMS (ESI, m/z)

calcd for C₅₉H₇₆N₇O₁₁S [M − H]⁻: 1090.5329, found: 1090.5319.

Purity: 98.4%.

(2S,4R)-1-((S)-2-(2-(2-(2-((3′-(((4,6-Dimethyl-2-oxo-1,2-dihydro-

pyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-

yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)ethoxy)ethoxy)-

acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthia-

zol-5-yl)benzyl)pyrrolidine-2-carboxamide (V3, YM181). V3 was

prepared following the procedure for the synthesis of compound V2

(YM281). ¹H NMR (500 MHz, d₆-DMSO) δ 11.46 (s, 1H), 8.95 (s,

1H), 8.59 (t, J = 5.6 Hz, 1H), 8.17 (s, 1H), 7.51 (d, J = 8.1 Hz, 2H),

7.45 (d, J = 9.5 Hz, 1H), 7.42−7.36 (m, 4H), 7.33 (s, 1H), 7.17 (s,

1H), 6.98 (d, J = 8.0 Hz, 2H), 5.85 (s, 1H), 5.16 (s, 1H), 4.57 (d, J =

9.5 Hz, 1H), 4.45 (t, J = 8.0 Hz, 1H), 4.38 (dd, J = 16.4, 6.1 Hz, 2H),

4.29 (d, J = 4.2 Hz, 2H), 4.25 (dd, J = 16.0, 5.3 Hz, 1H), 4.13 (s, 2H),

3.97 (d, J = 16.9 Hz, 2H), 3.80 (t, J = 14.1 Hz, 4H), 3.70−3.59 (m,

4H), 3.23 (t, J = 11.4 Hz, 2H), 3.06 (d, J = 6.6 Hz, 2H), 3.01 (d, J =

10.2 Hz, 1H), 2.42 (s, 3H), 2.23 (s, 3H), 2.20 (s, 3H), 2.10 (s, 3H),

2.05 (d, J = 7.3 Hz, 1H), 1.98 (dd, J = 17.1, 10.8 Hz, 1H), 1.91 (dd, J

= 14.8, 6.5 Hz, 1H), 1.65 (d, J = 11.4 Hz, 2H), 1.57−1.44 (m, 2H),

0.94 (s, 9H), 0.82 (t, J = 6.7 Hz, 3H). ¹³C NMR (101 MHz, d₆-

DMSO) δ 171.8, 169.2, 168.7, 163.1, 158.1, 151.4, 149.6, 147.8,

142.8, 139.5, 139.4, 136.9, 132.3, 132.0, 131.2, 129.7, 128.7, 127.7,

127.5, 122.6, 121.6, 120.5, 114.9, 107.5, 70.5, 69.8, 69.7, 69.1, 68.9,

67.2, 66.4, 58.8, 57.9, 56.6, 55.8, 41.7, 37.9, 35.8, 34.9, 31.3, 30.4,

29.0, 26.2, 25.2, 22.1, 21.4, 19.0, 18.2, 15.9, 14.5, 14.0, 12.7. HRMS

(ESI, m/z) calcd for C₅₇H₇₄N₇O₁₀S [M + H]⁺: 1048.5212, found:

1048.5216. Purity: 96.3%.

N³-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-

(ethyl(tetrahydro-2H-pyran-4-yl)amino)-N⁴′-((S)-14-((2S,4R)-4-hy-

droxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-

carbonyl)-15,15-dimethyl-12-oxo-3,6,9-trioxa-13-azahexadecyl)-4-

methyl-[1,1′-biphenyl]-3,4′-dicarboxamide (V6). V6 was prepared

following the procedure for the synthesis of compound V2 (YM281).

¹H NMR (400 MHz, d₆-DMSO) δ 11.45 (s, 1H), 8.97 (s, 1H), 8.55

(t, J = 5.7 Hz, 2H), 8.21 (t, J = 4.9 Hz, 2H), 7.91 (d, J = 8.4 Hz, 2H),

7.89 (d, J = 9.5 Hz, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.46 (s, 1H), 7.40

(q, J = 8.3 Hz, 4H), 7.30 (s, 1H), 5.87 (s, 1H), 5.14 (d, J = 3.5 Hz,

1H), 4.55 (d, J = 9.4 Hz, 1H), 4.42 (m, 2H), 4.35 (s, 1H), 4.30 (d, J =

4.9 Hz, 2H), 4.22 (dd, J = 15.8, 5.4 Hz, 2H), 4.01−3.95 (m, 1H), 3.83

(d, J = 10.8 Hz, 2H), 3.70−3.41 (m, 15H), 3.29−3.20 (m, 2H), 3.15−

2.99 (m, 3H), 2.44 (s, 3H), 2.26 (s, 3H), 2.22 (s, 3H), 2.11 (s, 3H),

2.03 (m, 1H), 1.97−1.86 (m, 1H), 1.67 (d, J = 11.3 Hz, 2H), 1.54−

1.48 (m, 2H), 0.93 (s, 9H), 0.83 (t, J = 7.0 Hz, 3H). ¹³C NMR (101

MHz, d₆-DMSO) δ 171.9, 170.0, 169.6, 169.0, 166.0, 163.0, 151.4,

149.6, 149.0, 147.7, 142.8, 142.8, 142.3, 139.7, 139.5, 136.2, 133.5,

133.1, 131.2, 129.7, 128.6, 127.8, 127.4, 126.4, 124.0, 123.1, 121.6,

121.0, 107.4, 69.7, 69.6, 69.5, 68.9, 66.9, 66.3, 58.7, 57.9, 56.3, 41.7,

41.3, 41.2, 37.9, 35.7, 35.3, 34.9, 30.3, 26.3, 19.0, 18.6, 18.2, 15.9,

14.6, 12.8. HRMS (ESI, m/z) calcd for C₆₁H₇₉N₈O₁₁S [M − H]⁻:

1131.5594, found: 1131.5584. Purity: 99.1%.

N³-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-

(ethyl(tetrahydro-2H-pyran-4-yl)amino)-N⁴′-((S)-17-((2S,4R)-4-hy-

droxy-2-((4-(4-methylthiazol-5-yl) benzyl) carbamoyl) pyrrolidine-

1-carbonyl)-18,18-dimethyl-15-oxo-3,6,9,12-tetraoxa-16-azanona-

decyl)-4-methyl-[1,1′-biphenyl]-3,4′-dicarboxamide (V7). V7 was

prepared following the procedure for the synthesis of compound V2

(YM281). ¹H NMR (400 MHz, d₆-DMSO) δ 11.47 (s, 1H), 8.98 (s,

1H), 8.58 (s, 2H), 8.23 (s, 1H), 7.93 (d, J = 8.2 Hz, 3H), 7.72 (d, J =

8.0 Hz, 2H), 7.46 (s, 1H), 7.39 (dd, J = 16.2, 7.9 Hz, 4H), 7.29 (s,

1H), 5.86 (s, 1H), 5.15 (s, 1H), 4.55 (d, J = 9.3 Hz, 1H), 4.42 (d, J =

4.8 Hz, 2H), 4.35 (s, 1H), 4.30 (d, J = 4.0 Hz, 2H), 4.26−4.16 (m,

1H), 4.08−4.01 (m, 1H), 3.98 (t, J = 6.5 Hz, 1H), 3.83 (d, J = 10.2

Hz, 2H), 3.70−3.59 (m, 4H), 3.55-3.48 (m, 8H), 3.45 (d, J = 4.9 Hz,

2H), 3.25 (t, J = 11.2 Hz, 3H), 3.17−2.96 (m, 3H), 2.44 (s, 3H), 2.25

(s, 3H), 2.21 (s, 3H), 2.10 (s, 3H), 2.07−2.00 (m, 1H), 1.99 (s, 1H),

1.90 (s, 1H), 1.66 (d, J = 10.9 Hz, 2H), 1.52 (d, J = 7.4 Hz, 4H), 1.31

(d, J = 6.9 Hz, 2H), 0.93 (s, 9H), 0.83 (t, J = 7.0 Hz, 3H). ¹³C NMR

(101 MHz, d₆-DMSO) δ 172.0, 170.1, 169.6, 169.3, 169.0, 166.0,

163.1, 151.5, 149.7, 149.1, 147.7, 142.8, 142.4, 139.8, 139.5, 136.3,

133.5, 133.1, 131.2, 129.7, 128.7, 127.9, 127.5, 126.4, 123.1, 121.6,

121.1, 107.5, 72.9, 69.8, 69.8, 69.7, 69.7, 69.5, 68.9, 68.9, 66.9, 66.4,

63.8, 58.8, 57.9, 56.4, 48.6, 43.1, 41.7, 41.3, 38.0, 35.7, 35.4, 35.0,

30.4, 30.1, 30.0, 26.4, 19.0, 18.6, 18.2, 15.9, 14.7, 13.6, 12.8. HRMS

(ESI, m/z) calcd for C₆₃H₈₃N₈O₁₂S [M − H]⁻: 1175.5857, found:

1175.5874. Purity: 97.7%.

(2R,4S)-1-((S)-2-(2-(3-((3′-(((4,6-Dimethyl-2-oxo-1,2-dihydropyri-

din-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)-

amino)-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)propoxy)acetamido)-

3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)-

benzyl)pyrrolidine-2-carboxamide (YM620). YM620 was prepared

from 21b and 41 following the procedure for the synthesis of

compound V2 (YM281). ¹H NMR (400 MHz, d₆-DMSO) δ 11.46 (s,

1H), 8.96 (s, 1H), 8.35 (t, J = 5.9 Hz, 1H), 8.17 (t, J = 4.9 Hz, 1H),

7.52 (d, J = 8.6 Hz, 2H), 7.45−7.37 (m, 4H), 7.32 (t, J = 6.3 Hz, 3H),

7.18 (s, 1H), 6.97 (d, J = 8.7 Hz, 2H), 5.86 (s, 1H), 5.14 (d, J = 3.5

Hz, 1H), 4.54 (d, J = 9.0 Hz, 1H), 4.44−4.33 (m, 3H), 4.30 (d, J =

4.5 Hz, 3H), 4.25 (d, J = 5.8 Hz, 1H), 4.04 (t, J = 6.3 Hz, 2H), 3.82

(d, J = 10.5 Hz, 2H), 3.75 (dd, J = 10.3, 4.7 Hz, 1H), 3.59 (d, J = 6.5

N³-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-

(ethyl(tetrahydro-2H-pyran-4-yl)amino)-N⁴′-(2-(2-(2-(((S)-1-

((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)-

pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-

oxoethoxy)ethoxy)ethyl)-4-methyl-[1,1′-biphenyl]-3,4′-dicarboxa-

mide (V4). V4 was prepared following the procedure for the synthesis

of compound V2 (YM281). ¹H NMR (400 MHz, d₆-DMSO) δ 11.44

(s, 1H), 8.95 (s, 1H), 8.60−8.50 (m, 2H), 8.20 (t, J = 5.0 Hz, 1H),

7.91 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 9.9 Hz,

2H), 7.38 (brs, 4H), 7.29 (s, 1H), 5.86 (s, 1H), 5.16 (d, J = 3.5 Hz,

1H), 4.57 (d, J = 9.6 Hz, 1H), 4.47 (d, J = 8.1 Hz, 1H), 4.35 (d, J =

6.0 Hz, 2H), 4.30 (d, J = 4.8 Hz, 2H), 4.25 (dd, J = 15.8, 5.7 Hz, 1H),

3.97 (s, 2H), 3.82 (d, J = 10.3 Hz, 2H), 3.70−3.55 (m, 8H), 3.46 (d, J

= 5.8 Hz, 2H), 3.25 (t, J = 11.2 Hz, 2H), 3.15−2.97 (m, 3H), 2.43 (s,

3H), 2.25 (s, 3H), 2.21 (s, 3H), 2.10 (s, 3H), 2.06 (s, 1H), 1.90 (s,

1H), 1.66 (d, J = 11.7 Hz, 2H), 1.52 (d, J = 8.8 Hz, 2H), 0.93 (s, 9H),

0.83 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, d₆-DMSO) δ 171.7,

169.2, 168.9, 168.6, 165.9, 163.0, 151.4, 149.5, 149.0, 147.7, 142.3,

139.7, 139.4, 136.2, 133.1, 131.1, 129.7, 128.7, 127.8, 127.4, 126.4,

123.0, 121.0, 99.5, 70.4, 69.6, 69.4, 69.0, 68.8, 66.3, 58.7, 57.9, 56.6,

55.7, 41.7, 41.2, 37.9, 35.7, 34.9, 30.3, 26.2, 18.9, 18.2, 15.9, 14.6,

12.8. HRMS (ESI, m/z) calcd for C₅₈H₇₃N₈O₁₀S [M − H]⁻:

1073.5176, found: 1073.5195. Purity: 95.8%.

(2S,4R)-1-((S)-2-(tert-Butyl)-14-((3′-(((4,6-dimethyl-2-oxo-1,2-di-

hydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-

pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)-4-oxo-

6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-methylthia-

zol-5-yl)benzyl)pyrrolidine-2-carboxamide (V5). V5 was prepared

following the procedure for the synthesis of compound V2 (YM281).

¹H NMR (400 MHz, d₆-DMSO) δ 11.47 (s, 1H), 8.97 (d, J = 3.9 Hz,

1H), 8.60 (t, J = 5.9 Hz, 1H), 8.17 (t, J = 4.8 Hz, 1H), 7.53 (d, J = 8.6

Hz, 2H), 7.43 (d, J = 4.6 Hz, 1H), 7.41 (s, 1H), 7.38 (d, J = 9.1 Hz,

4H), 7.35 (s, 1H), 7.18 (s, 1H), 6.99 (d, J = 8.7 Hz, 2H), 5.85 (s,

1H), 5.17 (s, 1H), 4.57 (d, J = 9.6 Hz, 1H), 4.50−4.39 (m, 1H), 4.37

(d, J = 6.1 Hz, 2H), 4.33−4.20 (m, 3H), 4.13−4.06 (m, 2H), 3.97 (s,

2H), 3.82 (d, J = 10.2 Hz, 3H), 3.76−3.71 (m, 2H), 3.60 (m, 6H),

3.24 (t, J = 11.2 Hz, 2H), 3.17 (s, 2H), 3.11-3.03 (m, 2H), 3.01 (s,

10179

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Hz, 3H), 3.24 (t, J = 11.1 Hz, 3H), 3.12−3.04 (m, 2H), 3.01 (s, 1H),

2.43 (s, 3H), 2.24 (s, 3H), 2.21 (s, 3H), 2.11 (s, 3H), 2.06 (dd, J =

8.2, 4.2 Hz, 1H), 1.99−1.91 (m, 3H), 1.66 (d, J = 10.7 Hz, 2H), 1.52

(d, J = 8.6 Hz, 2H), 0.97 (s, 9H), 0.84−0.80 (m, 3H).¹³C NMR (101

MHz, d₆-DMSO) δ 171.5, 169.1, 168.9, 163.0, 158.0, 151.4, 149.4,

148.8, 147.7, 142.7, 139.5, 139.3, 136.8, 132.2, 131.9, 131.1, 129.7,

128.7, 127.6, 127.4, 122.5, 121.6, 120.4, 114.8, 107.3, 69.4, 68.5, 67.6,

66.3, 64.5, 58.8, 57.8, 55.9, 55.4, 41.5, 41.2, 37.9, 35.0, 34.9, 30.3,

29.0, 26.4, 18.9, 18.2, 15.9, 14.5, 12.7. HRMS (ESI, m/z) calcd for

C₅₆H₇₀N₇O₉S [M − H]⁻: 1016.4961, found: 1016.4929. Purity:

99.4%.

(s, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.60−7.53

(m, 1H), 7.46 (s, 1H), 7.29 (s, 1H), 7.12 (d, J = 8.6 Hz, 1H), 7.01 (d,

J = 7.0 Hz, 1H), 6.57 (s, 1H), 5.86 (s, 1H), 5.04 (dd, J = 12.8, 5.4 Hz,

1H), 4.30 (d, J = 4.9 Hz, 2H), 3.83 (d, J = 10.4 Hz, 3H), 3.14−3.03

(m, 4H), 2.89 (dd, J = 22.5, 8.5 Hz, 2H), 2.58 (d, J = 16.9 Hz, 2H),

2.25 (s, 3H), 2.21 (d, J = 5.4 Hz, 3H), 2.11 (s, 3H), 2.08−1.99 (m,

2H), 1.65 (d, J = 20.5 Hz, 6H), 1.53 (d, J = 8.2 Hz, 2H), 0.84 (t, J =

6.9 Hz, 3H). ¹³C NMR (101 MHz, d₆-DMSO) δ 172.8, 170.1, 169.0,

167.3, 165.8, 163.0, 149.6, 149.1, 146.4, 142.3, 139.8, 136.3, 133.4,

133.3, 132.2, 127.8, 126.4, 123.1, 121.6, 121.0, 117.3, 110.4, 109.1,

107.4, 83.9, 66.3, 57.9, 48.6, 41.6, 41.2, 34.9, 31.0, 30.3, 26.6, 26.3,

22.2, 19.0, 18.2, 14.6, 12.8. HRMS (ESI, m/z) calcd for C₄₇H₅₁N₆O₉

[M − H]⁻: 843.3723, found: 843.3684. Purity: 95.2%.

N³-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-N⁴′-(8-

(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-

acetamido)octyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-

methyl-[1,1′-biphenyl]-3,4′-dicarboxamide (G5). To a solution of

Boc₂O (1 g, 4.58 mmol) in CH₂Cl₂(10 mL) was added a solution of

octane-1,8-diamine (2.64 g, 18.33 mmol) in CH₂Cl₂(10 mL)

dropwise at 0 °C. Then, the reaction mixture was warmed to r.t. and

stirred overnight. Finally, CH₂Cl₂(50 mL) and water (20 mL) were

added. The mixture was separated, and the organic phase was washed

with brine (3 × 10 mL). The organic phase was dried over Na₂SO₄

and concentrated under reduced pressure. The crude residue was

puriﬁed by ﬂash column chromatography MeOH/CH₂Cl₂(0−10%)

to give compound 37b as a white solid (0.93 g, 63.5% yield).

To a solution of 36 (100 mg, 301 μmol) in DMF (3 mL) were

added a solution of 37b (110.3 mg, 451 μmol) in DMF (4.5 mL),

DIPEA (116.69 mg, 149.22 μmol), and HATU (114.44 mg, 300.96

μmol). The reaction mixture was stirred for 19 h at r.t. and was then

diluted with EtOAc (30 mL). The organic phase was washed

sequentially with 10% aq. citric acid (10 mL), saturated aq. sodium

bicarbonate (10 mL), water (10 mL), and brine (10 mL × 2). The

organic phase was dried over Na₂SO₄, ﬁltered, and concentrated

under reduced pressure. Puriﬁcation by column chromatography with

MeOH/CH₂Cl₂(0−5%) gave the desired coupling product as a

yellow solid (95 mg, 56.5% yield). Then, the obtained compound was

dissolved in TFA (1.5 mL, 0.1 M) and heated to 50 °C. After 1 h, the

mixture was cooled to r.t., diluted with MeOH, and concentrated

under reduced pressure. The crude residue was precipitated with

diethyl ether and dried under vacuum to give 38b as a yellow solid

(95 mg, 100%).

N³-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-N⁴′-(2-

(2-((2-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,3-dioxoisoindolin-4-

yl)amino)ethoxy)ethyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-

4-methyl-[1,1′-biphenyl]-3,4′-dicarboxamide (G2). G2 was pre-

pared using the similar procedure for the synthesis of compound

G5. ¹H NMR (400 MHz, d₆-DMSO) δ 11.47 (s, 1H), 11.07 (s, 1H),

8.53 (t, J = 5.5 Hz, 1H), 8.21 (t, J = 4.7 Hz, 1H), 7.89 (d, J = 8.3 Hz,

2H), 7.69 (d, J = 8.4 Hz, 2H), 7.58−7.52 (m, 1H), 7.46 (s, 1H), 7.29

(s, 1H), 7.14 (d, J = 8.6 Hz, 1H), 7.01 (d, J = 7.0 Hz, 1H), 6.62 (t, J =

5.6 Hz, 1H), 5.86 (s, 1H), 5.03 (dd, J = 12.9, 5.3 Hz, 1H), 4.30 (d, J =

4.9 Hz, 2H), 3.83 (d, J = 9.6 Hz, 2H), 3.66 (t, J = 5.3 Hz, 2H), 3.61

(d, J = 6.0 Hz, 2H), 3.47 (dd, J = 10.3, 5.3 Hz, 6H), 3.10 (dd, J =

13.9, 6.8 Hz, 2H), 3.03 (s, 1H), 2.92−2.80 (m, 1H), 2.56 (d, J = 16.1

Hz, 2H), 2.25 (s, 3H), 2.21 (s, 3H), 2.11 (s, 3H), 2.04−1.94 (m, 1H),

1.67 (d, J = 10.2 Hz, 2H), 1.60−1.46 (m, 2H), 0.83 (t, J = 6.9 Hz,

3H). ¹³C NMR (101 MHz, d₆-DMSO) δ 172.8, 170.1, 169.0, 167.3,

166.0, 163.0, 158.1, 157.8, 149.6, 149.1, 146.5, 142.8, 142.3, 139.7,

136.3, 133.5, 133.1, 132.1, 127.8, 126.4, 123.1, 121.6, 121.1, 117.5,

110.7, 109.3, 107.5, 68.8, 66.4, 57.9, 48.6, 41.7, 41.3, 34.9, 31.0, 30.4,

22.2, 19.0, 18.2, 14.7, 12.8. HRMS (ESI, m/z) calcd for C₄₇H₅₁N₆O₁₀

[M − H]⁻: 859.3672, found: 859.3682. Purity: 96.7%.

N³-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-N⁴′-(2-

(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-

ethoxy)ethoxy)ethyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-

methyl-[1,1′-biphenyl]-3,4′-dicarboxamide (G3). G3 was prepared

following the similar procedure for the synthesis of compound G5. ¹H

NMR (400 MHz, d₆-DMSO) δ 11.46 (s, 1H), 11.09 (s, 1H), 8.52 (d,

J = 5.7 Hz, 1H), 8.21 (s, 1H), 7.91 (d, J = 8.3 Hz, 2H), 7.82−7.75 (m,

1H), 7.71 (d, J = 8.2 Hz, 2H), 7.52−7.40 (m, 3H), 7.29 (s, 1H), 5.86

(s, 1H), 5.08 (dd, J = 12.8, 5.3 Hz, 1H), 4.31 (s, 4H), 3.84-3.82 (m,

3H), 3.70−3.63 (m, 2H), 3.57 (dd, J = 11.1, 5.5 Hz, 5H), 3.43 (d, J =

5.4 Hz, 2H), 3.09 (d, J = 6.8 Hz, 2H), 3.01 (d, J = 11.0 Hz, 1H),

2.94−2.79 (m, 2H), 2.63−2.53 (m, 2H), 2.26 (s, 2H), 2.21 (d, J = 5.2

Hz, 3H), 2.11 (s, 3H), 2.07 (d, J = 3.3 Hz, 1H), 2.06−1.98 (m, 2H),

1.66 (d, J = 10.2 Hz, 2H), 1.52 (d, J = 8.2 Hz, 2H), 0.83 (t, J = 6.8 Hz,

3H). ¹³C NMR (101 MHz, d₆-DMSO) δ 172.8, 170.0, 169.0, 166.8,

165.9, 165.3, 163.0, 155.8, 149.5, 149.1, 142.8, 142.3, 139.7, 136.9,

136.2, 133.5, 133.2, 133.1, 127.8, 126.4, 123.1, 121.6, 121.0, 120.0,

116.3, 115.4, 107.4, 70.1, 69.7, 68.9, 68.9, 68.7, 66.3, 57.9, 48.8, 41.2,

34.9, 30.9, 30.3, 22.0, 18.9, 18.2, 14.6, 12.8. HRMS (ESI, m/z) calcd

for C₄₉H₅₇N₆O₁₁[M + H]⁺905.4080, found: 905.4096. Purity:

95.0%.

To the solution of 7 (50 mg, 99.29 μmol) in DMF (2 mL) were

added 38b (71.6 mg, 119 μmol), DIPEA (73.84 μL, 446.79 μmol),

and HATU (36.73 mg, 99.29 μmol). The mixture was stirred at r.t. for

22 h before EtOAc and water were added. The mixture was extracted

with EtOAc twice. The combined organic phases were dried over

Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

residue was puriﬁed by ﬂash column chromatography with MeOH/

CH₂Cl₂(0−10%) to aﬀord G5 as a yellow solid (32 mg, 34.6% yield).

¹H NMR (400 MHz, d₆-DMSO) δ 11.45 (s, 1H), 11.10 (s, 1H), 8.45

(t, J = 5.5 Hz, 1H), 8.21 (t, J = 4.7 Hz, 1H), 7.91 (d, J = 8.5 Hz, 3H),

7.84−7.78 (m, 1H), 7.71 (d, J = 8.3 Hz, 2H), 7.49 (d, J = 7.2 Hz,

1H), 7.46 (s, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.29 (s, 1H), 5.86 (s,

1H), 5.12 (dd, J = 12.8, 5.4 Hz, 1H), 4.76 (s, 2H), 4.30 (d, J = 4.7 Hz,

2H), 3.83 (d, J = 10.8 Hz, 2H), 3.29−3.20 (m, 4H), 3.18−3.06 (m,

4H), 2.96−2.84 (m, 3H), 2.60−2.53 (m, 2H), 2.26 (s, 3H), 2.21 (s,

3H), 2.11 (s, 3H), 2.05 (dd, J = 12.5, 4.2 Hz, 2H), 1.67 (d, J = 11.1

Hz, 2H), 1.52 (s, 3H), 1.44 (s, 2H), 1.28 (s, 8H), 0.83 (t, J = 6.8 Hz,

3H). ¹³C NMR (101 MHz, d₆-DMSO) δ 172.7, 169.8, 169.0, 166.7,

166.6, 165.7, 165.5, 163.0, 155.0, 149.6, 149.1, 142.8, 142.2, 139.7,

136.9, 136.3, 133.4, 133.0, 127.8, 126.3, 123.1, 121.6, 121.0, 120.4,

116.9, 116.1, 107.4, 67.7, 66.3, 57.9, 48.8, 41.2, 38.3, 34.9, 31.0, 30.3,

29.1, 29.0, 28.7, 28.7, 26.5, 26.3, 22.0, 18.9, 18.2, 14.6, 12.8. HRMS

(ESI, m/z) calcd for C₅₃H₆₄N₇O₁₀[M + H]⁺: 958.4709, found:

958.4677. Purity: 96.0%.

N³-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-N⁴′-(6-

(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-

acetamido)hexyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-

methyl-[1,1′-biphenyl]-3,4′-dicarboxamide (G4). G4 was prepared

following the similar procedure for the synthesis of compound G5. ¹H

NMR (500 MHz, d₆-DMSO) δ 11.47 (s, 1H), 11.12 (s, 1H), 8.47 (t, J

= 5.3 Hz, 1H), 8.22 (t, J = 4.7 Hz, 1H), 7.95 (t, J = 5.4 Hz, 1H), 7.90

(d, J = 8.2 Hz, 2H), 7.81 (t, J = 7.9 Hz, 1H), 7.71 (d, J = 8.2 Hz, 2H),

7.51−7.43 (m, 2H), 7.39 (d, J = 8.5 Hz, 1H), 7.29 (s, 1H), 5.86 (s,

1H), 5.12 (dd, J = 12.7, 5.4 Hz, 1H), 4.77 (s, 2H), 4.30 (d, J = 4.7 Hz,

2H), 3.83 (d, J = 11.6 Hz, 2H), 3.25 (t, J = 9.3 Hz, 4H), 3.15 (dd, J =

12.5, 6.2 Hz, 2H), 3.10 (d, J = 6.9 Hz, 2H), 3.02 (t, J = 10.8 Hz, 1H),

2.90 (dd, J = 23.2, 7.3 Hz, 3H), 2.56 (dd, J = 24.7, 11.7 Hz, 2H), 2.25

(s, 3H), 2.21 (s, 3H), 2.10 (s, 3H), 2.08−2.00 (m, 1H), 1.66 (d, J =

11.1 Hz, 2H), 1.53 (d, J = 7.4 Hz, 2H), 1.45 (m, 2H), 1.31 (m, 4H),

0.83 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, d₆-DMSO) δ 172.8,

N³-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-N⁴′-(4-

((2-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,3-dioxoisoindolin-4-yl)-

amino)butyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-

[1,1′-biphenyl]-3,4′-dicarboxamide (G1). G1 was prepared using the

1

similar procedure for the synthesis of compound G5. H NMR (400

MHz, d₆-DMSO) δ 11.44 (s, 1H), 11.07 (s, 1H), 8.52 (s, 1H), 8.20

10180

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

169.9, 169.0, 166.8, 166.7, 165.8, 165.6, 163.1, 155.1, 149.6, 149.1,

142.8, 142.2, 139.8, 137.0, 136.3, 133.4, 133.1, 127.8, 126.4, 123.1,

121.6, 121.0, 120.4, 116.9, 116.1, 107.4, 67.7, 66.4, 57.9, 48.8, 41.2,

38.3, 34.9, 31.0, 30.3, 29.2, 29.0, 26.2, 26.1, 22.0, 19.0, 18.2, 14.7,

12.8. HRMS (ESI, m/z) calcd for C₅₁H₆₀N₇O₁₀[M + H]⁺: 930.4396,

found: 930.4413. Purity: 95.9%.

citrate buﬀer for 4−6 min for antigen retrieval, peroxide blocking was

performed with 3% H₂O₂at r.t. for 20 min. The sections were

incubated with EZH2, H3K27me3 (Cell Signaling Technology), and

Ki67 (Abcam), following the suggested concentration at 37 °C

overnight. Then, the samples were probed with a secondary antibody

at 37 °C for 1 h. All slides were stained with 3,3′-diaminobenzidine

and counterstained with hematoxylin.

Immunoprecipitation. The cells were harvested using NP40

(Beyotime Biotechnology, Shanghai, China) containing a protease

inhibitor cocktail. After 30 min, cell lysates were centrifuged at 4000g

for 15 min at 4 °C, and the supernatants were incubated with EZH2

antibody or IgG (Cell Signaling Technology) overnight at 4 °C.

Subsequent incubation with protein A/G-coated agarose beads

(Merck, Germany) continued for an additional 3 h at 4 °C. After

the samples were washed six times with ice-cold NP40, the

supernatants were removed by centrifugation at 800g for 2 min.

The proteins were then separated from the beads using immunoblot-

ting loading buﬀer for 5 min at 95 °C. The supernatants were

collected for subsequent immunoblotting analysis after SDS gel

separation.

Lentiviral Constructs and siRNAs. The shEZH2 lentiviral

constructs were purchased from GeneCopoeia (GeneCopoeia, Inc,

Rockville, Maryland) and transduced into SU-DHL-2 cells according

to the literature.¹⁵VHL siRNA (target sequence: GCTCTACGAA-

GATCTGGAA) was from RIBOBIO (Guangzhou, China). 22Rv1

cells were transfected with siRNA using Lipofectamine RNAiMAX

(ThermoFisher, Rockford, IL), following the manufacturer’s in-

structions.

N³-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-N⁴′-(2-

(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-

ethoxy)ethoxy)ethoxy)ethyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)-

amino)-4-methyl-[1,1′-biphenyl]-3,4′-dicarboxamide (G6). G6 was

prepared following the similar procedure for the synthesis of

1

compound G5. H NMR (400 MHz, d₆-DMSO) δ 11.45 (s, 1H),

11.09 (s, 1H), 8.52 (dd, J = 12.1, 6.3 Hz, 1H), 8.20 (t, J = 4.9 Hz,

1H), 7.90 (t, J = 9.3 Hz, 2H), 7.81−7.76 (m, 1H), 7.74−7.67 (m,

2H), 7.50 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.3 Hz, 2H), 7.30 (s, 1H),

5.86 (s, 1H), 5.08 (dd, J = 12.8, 5.4 Hz, 1H), 4.30 (dd, J = 11.9, 6.1

Hz, 4H), 3.85 (d, J = 5.9 Hz, 1H), 3.82−3.75 (m, 2H), 3.66−3.60 (m,

2H), 3.54 (m, 8H), 3.46−3.39 (m, 2H), 3.09 (d, J = 7.0 Hz, 2H), 3.03

(s, 1H), 2.94−2.80 (m, 2H), 2.66−2.53 (m, 2H), 2.25 (s, 2H), 2.21

(d, J = 5.4 Hz, 4H), 2.10 (s, 3H), 2.09−1.97 (m, 3H), 1.90 (d, J =

11.7 Hz, 1H), 1.66 (d, J = 10.6 Hz, 1H), 1.59−1.47 (m, 2H), 0.83 (t,

J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, d₆-DMSO) δ 172.7, 169.9,

166.8, 165.9, 165.2, 163.0, 155.8, 137.0, 133.2, 127.8, 126.4, 123.4,

121.5, 121.0, 120.0, 116.3, 115.4, 107.4, 99.4, 70.2, 69.8, 69.7, 69.6,

69.2, 68.9, 68.9, 68.7, 66.3, 66.1, 48.8, 34.9, 32.7, 30.9, 30.3, 22.0,

18.9, 18.9, 18.2, 14.6, 12.8. HRMS (ESI, m/z) calcd for C₅₁H₆₁N₆O₁₂

[M + H]⁺: 949.4342, found 949.4323. Purity: 95.0%.

Cell Lines. The human prostate cancer cell lines 22rv1; DLBCL

cell lines SU-DHL-2, SU-DHL-4, and SU-DHL-6; BL cell lines NCI-

BL209, Daudi, Raji, and Namalwa; LBL cell lines JVM-2; and MCL

cell lines MINO and Jeko-1 were obtained from ATCC. All cell lines

were cultured in RPMI-1640, supplemented with 10% FBS at 37 °C

and 5% CO₂. All of the cell lines used in the study had been tested for

mycoplasma contamination every three weeks.

Caco-2 Cell Permeability Assay. The Caco-2 cells were seeded

onto polycarbonate 12-well Transwell ﬁlters at a density of 2 × 10⁵

cells/well. The conﬂuent monolayers obtained at 21 days were

utilized to assess the in vitro permeability. Culture media in the apical

and basolateral compartments were replaced every 2 days, and the

integrity of the monolayer was detected by ﬂuorescein. Before the

experiments, the culture medium in both chambers was replaced with

prewarmed Hank’s balanced salt solution (HBSS). The cultures were

then stabilized at 37 °C for 30 min. For the permeation studies, 0.2

mL of drug formulation diluted with HBSS was added to the apical

side, and the basolateral side was replaced with 1 mL fresh HBSS. The

treated cells were incubated at 37 0.5 °C. The amount of permeated

drug was determined by collecting 50 μL of samples from the

basolateral compartment, followed by replacement with 50 μL of fresh

HBSS at 1, 2, and 3 h. The collected samples were evaporated and

reconstituted with methanol, then the concentrations of drugs in the

samples were determined by liquid chromatography−mass spectrom-

etry (LC-MS). The apparent permeability (P_app) of ETP in various

Cell Number Count. Cell number was counted by Cellometer

Auto T4 cell counter (Nexcelom Bioscience, Lawrence, Massachu-

setts).

Cell Viability Assay. Cell viability was determined by the MTS

assay, as we reported previously.⁴⁹

Cell Cycle and Apoptosis. The cell cycle was measured by ﬂow

cytometry according to the commercial cell cycle analysis kit

(KeyGEN BioTECH, Jiang Su, China). Annexin-V and propidium

iodide-based apoptosis analyses were measured by ﬂow cytometry

according to the commercial cell apoptosis analysis kit (BD, Franklin

Lakes, NJ). The activity of Caspase-3/7 was evaluated by the Caspase-

Glo 3/7 Assay (Promega, Madison, WI) through the manufacturer’s

instructions.

Measurement of Cellular ATP. The cellular ATP concentration

was detected using an ATP-based CellTiter-Glo Luminescent Cell

Viability Kit (Promega, Madison, WI) according to the manufac-

turer’s instructions.

Western Blot. The cells were washed with phosphate-buﬀered

saline (PBS) and then lysed in lysis buﬀer with a protease inhibitor

cocktail (Selleck). After the protein concentration normalization using

a bicinchoninic acid (BCA) protein assay (ThermoFisher, Rockford,

IL), the samples were separated by standard sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to

poly(vinylidene ﬂuoride) (PVDF) membranes (Millipore, Billerica,

MA). Subsequently, the membranes were blocked with 5% milk for 1

h at r.t. before blotting with the indicated ﬁrst antibodies overnight at

4 °C. After being washed with TBST (Tris-buﬀered saline containing

0.1% Tween-20), the membranes were probed with the horseradish

peroxidase-conjugated secondary antibodies for 1 h at r.t. Enhanced

chemiluminescence was used for signal detection. The intensities of

bands were performed using Image Lab (Bio-Rad, Hercules, CA). All

the antibodies used for western blot are from Cell Signaling

Technology.

dQ

dt

V

formulations was calculated as follows: P

=

×

where dQ/

app

A × C₀

dt is the slope of the cumulative drug permeated versus time (μg/s), A

is the surface area of the monolayer (1.12 cm²), V is the volume of

basolateral side HBSS (1 mL), and C₀is the initial concentration of

compounds on the apical side (μg/mL).

Animal Experiments. The animal experiment was conducted in

compliance with a protocol approved by the Institutional Animals

Care and Use Committee of Sun Yat-sen University Cancer Center

and was carried out in the Center of Experiment Animal of Sun Yat-

sen University (North Campus, approval no.: L102012019050K).

Balb/c nude mice (female) were bought from Beijing Vital River

Laboratory Animal Technology Co., Ltd. For SU-DHL-6 xenografts,

and ﬁve million cells were injected subcutaneously. After 2 weeks,

when tumors reached 100−200 mm³, mice were randomly divided

into three groups (6 mice per group) and administrated with a vehicle

control (80% PBS, 10% castor oil, and 10% DMSO) or indicated

doses of compounds (YM281: 80 mg/kg; EPZ6438: 42.5 mg/kg)

through intraperitoneal injection 6 times weekly. Tumor sizes and

animal weights were measured 2−3 times per week. The mice were

sacriﬁced after 3 weeks’ drug administration, and the tumor tissue was

harvested for analyses. For Jeko-1 xenografts, two million cells were

injected subcutaneously. Ten days later, most of the tumors grow

about 100 mm³, mice were then randomly divided into three groups

Immunohistochemistry. Tumors were harvested in formalin and

dehydrated and embedded in paraﬃn. Tissue slides were then

deparaﬃnized, hydrated, and rinsed in PBS. After being boiled in

10181

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(5 mice per group), and intraperitoneally administrated daily with the

control (70% PBS, 20% castor oil, and 10% DMSO) or indicated

doses of compounds (YM281: 100 mg/kg; EPZ6438: 50 mg/kg).

Tumor sizes and animal weights were measured every 3 days. The

mice were sacriﬁced after 30 days’ drug treatment, and then the tumor

tissue was harvested for analyses.

Isolation and Culture of Clinically Derived Lymphoma

Samples. Tumor tissues from patients with lymphoma at the Sun

Yat-sen University cancer center after proper informed consent were

collected for cellular Caspase-3/7 activity, ATP content detection, and

Western blot analyses (see Table S2 for the details of patient

information). The tumor tissues were immediately minced with ﬁne

scissors, and single cells were isolated through a 70 μm strainer (BD

Falcon) and cultured in RPMI-1640, supplemented with 10% FBS, 50

μM β-mercaptoethanol, and penicillin/streptomycin solution at 37 °C

and 5% CO₂. Studies using human specimen were approved (No.

GZR2018-089) by the Institutional Ethical Committee of Sun Yat-sen

University Cancer Center.

Molecular Docking. Molecular Operating Environment (MOE

2014) (Chemical Computing Group Inc, Montreal, Quebec, Canada)

was used for molecular docking. EPZ6438 was constructed using the

builder module, and energy was minimized using Force Field

MMFF94x and saved as the MDB ﬁle. The crystal structure of

EZH2 (PDB ID: 5LS6) was downloaded from the protein data bank,

and water molecules were removed. The residual crystal structure was

prepared using ligX with the default parameters of MOE [gradient:

0.1, Force Field: MMFF94X]. A 5Å radius area around the bounded

inhibitor in the crystal structure was deﬁned as the active site. The

prepared ligand structure was ﬂexibly docked into the EZH2 binding

site, with a triangle matcher as the placement methodology, London

ΔG or ASE as a scoring methodology, force ﬁeld reﬁnement was

selected, and dock calculations were run automatically. The obtained

30 conformations were generated and stored in a database, and the

best conformations were analyzed for the binding interaction analysis.

Statistical Analysis. Statistical analysis was performed by

GraphPad Prism 8 software (GraphPad Software, Inc, San Diego,

CA). All data are presented as mean standard error of the mean

(SEM). The statistical signiﬁcance of diﬀerences was determined

using Student’s t-test. Diﬀerences are considered statistically

signiﬁcant when the p values are less than 0.05.

AUTHOR INFORMATION

■

Corresponding Authors

Peng Huang − State Key Laboratory of Oncology in South

China, Collaborative Innovation Center for Cancer Medicine,

Sun Yat-sen University Cancer Center, Guangzhou 510060,

China; Phone: +86 20 87343511; Email: huangpeng@

sysucc.org.cn

Shijun Wen − State Key Laboratory of Oncology in South

China, Collaborative Innovation Center for Cancer Medicine,

Sun Yat-sen University Cancer Center, Guangzhou 510060,

China; orcid.org/0000-0002-9347-8243; Phone: +86

20 87342283; Email: wenshj@sysucc.org.cn

Authors

Yalin Tu − State Key Laboratory of Oncology in South China,

Collaborative Innovation Center for Cancer Medicine, Sun

Yat-sen University Cancer Center, Guangzhou 510060,

China

Yameng Sun − State Key Laboratory of Oncology in South

China, Collaborative Innovation Center for Cancer Medicine,

Sun Yat-sen University Cancer Center, Guangzhou 510060,

China; The School of Pharmaceutical Sciences, Wuhan

University, Wuhan 430071, China

Shuang Qiao − State Key Laboratory of Oncology in South

China, Collaborative Innovation Center for Cancer Medicine,

Sun Yat-sen University Cancer Center, Guangzhou 510060,

China

Yao Luo − State Key Laboratory of Oncology in South China,

Collaborative Innovation Center for Cancer Medicine, Sun

Yat-sen University Cancer Center, Guangzhou 510060,

China

Panpan Liu − State Key Laboratory of Oncology in South

China, Collaborative Innovation Center for Cancer Medicine,

Sun Yat-sen University Cancer Center, Guangzhou 510060,

China

Zhong-Xing Jiang − The School of Pharmaceutical Sciences,

Wuhan University, Wuhan 430071, China; orcid.org/

0000-0003-2601-4366

Yumin Hu − State Key Laboratory of Oncology in South

China, Collaborative Innovation Center for Cancer Medicine,

Sun Yat-sen University Cancer Center, Guangzhou 510060,

China

Zifeng Wang − State Key Laboratory of Oncology in South

China, Collaborative Innovation Center for Cancer Medicine,

Sun Yat-sen University Cancer Center, Guangzhou 510060,

China

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00460.

Western blot analysis of EZH2, EZH1, and H3K27me3

levels aﬀected by tested compounds; MTS cell viability

analysis in diﬀerent lymphoma cell lines treated with

YM281; the structure of YM620 and its eﬀects on EZH2

and cell proliferation; ﬂow cytometry analysis of the cell

cycle and cell apoptosis; Western blot analysis of PARP,

Casp-3, and C-casp-3 levels; the body weights of the

tested compound treated mice; nonsensitive cancer cell

lines NIC-H460 and AsPC1 treated by the tested

compounds (Figures S1−S6); patient sample informa-

tion and corresponding analytical methods; apparent

permeability of EPZ6438, YM181, and YM281 (Tables

S1−S2); NMR spectra of V1−V7, YM620, G1−G6, and

key intermediates; HRMS spectra and HPLC results of

YM181 and YM281 (PDF)

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00460

Author Contributions

^§Y.T., Y.S., and S.Q. contributed equally to this work.

Author Contributions

Conceptualization, S.W. and Y.T.; funding acquisition, S.W.

and P.H.; methodology, Y.T., Y.S., Y.H., Z.W., and S.Q.;

project administration, Y.T., Y.S., and S.Q.; resources, S.W.;

Supervision, S.W. and P.H.; validation, Y.T., Y.S., and S.Q.;

visualization, Y.T., Y.S., and S.Q.; writingoriginal draft, Y.T.

and Y.S.; writingreview & editing, Y.T., S.W., P.H., and S.Q.

The docking session for EPZ6438 in EZH2 crystal 5LS6

(PDB)

Notes

Molecular formula strings (CSV)

The authors declare no competing ﬁnancial interest.

10182

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(15) McCabe, M. T.; Ott, H. M.; Ganji, G.; Korenchuk, S.;

Thompson, C.; Van Aller, G. S.; Liu, Y.; Graves, A. P.; Della Pietra, A.,

3rd; Diaz, E.; LaFrance, L. V.; Mellinger, M.; Duquenne, C.; Tian, X.;

Kruger, R. G.; McHugh, C. F.; Brandt, M.; Miller, W. H.; Dhanak, D.;

Verma, S. K.; Tummino, P. J.; Creasy, C. L. EZH2 inhibition as a

therapeutic strategy for lymphoma with EZH2-activating mutations.

Nature 2012, 492, 108−112.

ACKNOWLEDGMENTS

■

We are grateful for the grant support from the National

Natural Science Foundation of China (81672952, 81872440),

the Guangdong Science and Technology Program

(2017A020215198), and the Guangzhou Science and Tech-

nology Program (201807010041).

(16) Bate-Eya, L. T.; Gierman, H. J.; Ebus, M. E.; Koster, J.; Caron,

H. N.; Versteeg, R.; Dolman, M. E. M.; Molenaar, J. J. Enhancer of

zeste homologue 2 plays an important role in neuroblastoma cell

survival independent of its histone methyltransferase activity. Eur. J.

Cancer 2017, 75, 63−72.

(17) Kim, J.; Lee, Y.; Lu, X.; Song, B.; Fong, K. W.; Cao, Q.; Licht, J.

D.; Zhao, J. C.; Yu, J. Polycomb- and Methylation-Independent Roles

of EZH2 as a Transcription Activator. Cell Rep. 2018, 25, 2808−

2820e4.

(18) Huang, X.; Yan, J.; Zhang, M.; Wang, Y.; Chen, Y.; Fu, X.; Wei,

R.; Zheng, X. L.; Liu, Z.; Zhang, X.; Yang, H.; Hao, B.; Shen, Y. Y.; Su,

Y.; Cong, X.; Huang, M.; Tan, M.; Ding, J.; Geng, M. Targeting

Epigenetic Crosstalk as a Therapeutic Strategy for EZH2-Aberrant

Solid Tumors. Cell 2018, 175, 186−199.e19.

(19) Gibaja, V.; Shen, F.; Harari, J.; Korn, J.; Ruddy, D.; Saenz-Vash,

V.; Zhai, H.; Rejtar, T.; Paris, C. G.; Yu, Z.; Lira, M.; King, D.; Qi, W.;

Keen, N.; Hassan, A. Q.; Chan, H. M. Development of secondary

mutations in wild-type and mutant EZH2 alleles cooperates to confer

resistance to EZH2 inhibitors. Oncogene 2016, 35, 558−566.

(20) Burslem, G. M.; Crews, C. M. Proteolysis-Targeting Chimeras

as Therapeutics and Tools for Biological Discovery. Cell 2020, 181,

102−114.

(21) Lu, J.; Qian, Y.; Altieri, M.; Dong, H.; Wang, J.; Raina, K.;

Hines, J.; Winkler, J. D.; Crew, A. P.; Coleman, K.; Crews, C. M.

Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target

BRD4. Chem. Biol. 2015, 22, 755−763.

(22) Winter, G. E.; Buckley, D. L.; Paulk, J.; Roberts, J. M.; Souza,

A.; Dhe-Paganon, S.; Bradner, J. E. DRUG DEVELOPMENT.

Phthalimide conjugation as a strategy for in vivo target protein

degradation. Science 2015, 348, 1376−1381.

(23) Han, X.; Wang, C.; Qin, C.; Xiang, W.; Fernandez-Salas, E.;

Yang, C. Y.; Wang, M.; Zhao, L.; Xu, T.; Chinnaswamy, K.;

Delproposto, J.; Stuckey, J.; Wang, S. Discovery of ARD-69 as a

Highly Potent Proteolysis Targeting Chimera (PROTAC) Degrader

of Androgen Receptor (AR) for the Treatment of Prostate Cancer. J.

Med. Chem. 2019, 62, 941−964.

(24) Bai, L.; Zhou, H.; Xu, R.; Zhao, Y.; Chinnaswamy, K.;

McEachern, D.; Chen, J.; Yang, C. Y.; Liu, Z.; Wang, M.; Liu, L.;

Jiang, H.; Wen, B.; Kumar, P.; Meagher, J. L.; Sun, D.; Stuckey, J. A.;

Wang, S. A Potent and Selective Small-Molecule Degrader of STAT3

Achieves Complete Tumor Regression In Vivo. Cancer Cell 2019, 36,

498−511e17.

(25) Farnaby, W.; Koegl, M.; Roy, M. J.; Whitworth, C.; Diers, E.;

Trainor, N.; Zollman, D.; Steurer, S.; Karolyi-Oezguer, J.;

Riedmueller, C.; Gmaschitz, T.; Wachter, J.; Dank, C.; Galant, M.;

Sharps, B.; Rumpel, K.; Traxler, E.; Gerstberger, T.; Schnitzer, R.;

Petermann, O.; Greb, P.; Weinstabl, H.; Bader, G.; Zoephel, A.;

Weiss-Puxbaum, A.; Ehrenhofer-Wolfer, K.; Wohrle, S.; Boehmelt, G.;

Rinnenthal, J.; Arnhof, H.; Wiechens, N.; Wu, M. Y.; Owen-Hughes,

T.; Ettmayer, P.; Pearson, M.; McConnell, D. B.; Ciulli, A. BAF

complex vulnerabilities in cancer demonstrated via structure-based

PROTAC design. Nat. Chem. Biol. 2019, 15, 672−680.

(26) Sun, Y.; Zhao, X.; Ding, N.; Gao, H.; Wu, Y.; Yang, Y.; Zhao,

M.; Hwang, J.; Song, Y.; Liu, W.; Rao, Y. PROTAC-induced BTK

degradation as a novel therapy for mutated BTK C481S induced

ibrutinib-resistant B-cell malignancies. Cell Res. 2018, 28, 779−781.

(27) Tomaselli, D.; Mautone, N.; Mai, A.; Rotili, D. Recent advances

in epigenetic proteolysis targeting chimeras (Epi-PROTACs). Eur. J.

Med. Chem. 2020, 207, No. 112750.

ABBREVIATIONS

■

EZH2, enhancer of zeste homologue 2; PROTAC, proteolysis-

target chimera; VHL, von Hippel−Lindau; CRBN, cereblon;

DLBCL, diﬀuse large B-cell lymphoma; PRC2, polycomb

repressive complex 2; SAM, S-adenosylmethionine

REFERENCES

■

(1) Margueron, R.; Reinberg, D. The Polycomb complex PRC2 and

its mark in life. Nature 2011, 469, 343−349.

(2) Di Croce, L.; Helin, K. Transcriptional regulation by Polycomb

group proteins. Nat. Struct. Mol. Biol. 2013, 20, 1147−1155.

(3) Velichutina, I.; Shaknovich, R.; Geng, H.; Johnson, N. A.;

Gascoyne, R. D.; Melnick, A. M.; Elemento, O. EZH2-mediated

epigenetic silencing in germinal center B cells contributes to

proliferation and lymphomagenesis. Blood 2010, 116, 5247−5255.

(4) Ntziachristos, P.; Tsirigos, A.; Van Vlierberghe, P.; Nedjic, J.;

Trimarchi, T.; Flaherty, M. S.; Ferres-Marco, D.; da Ros, V.; Tang, Z.;

Siegle, J.; Asp, P.; Hadler, M.; Rigo, I.; De Keersmaecker, K.; Patel, J.;

Huynh, T.; Utro, F.; Poglio, S.; Samon, J. B.; Paietta, E.; Racevskis, J.;

Rowe, J. M.; Rabadan, R.; Levine, R. L.; Brown, S.; Pflumio, F.;

Dominguez, M.; Ferrando, A.; Aifantis, I. Genetic inactivation of the

polycomb repressive complex 2 in T cell acute lymphoblastic

leukemia. Nat. Med. 2012, 18, 298−301.

(5) Xu, K.; Wu, Z. J.; Groner, A. C.; He, H. H.; Cai, C.; Lis, R. T.;

Wu, X.; Stack, E. C.; Loda, M.; Liu, T.; Xu, H.; Cato, L.; Thornton, J.

E.; Gregory, R. I.; Morrissey, C.; Vessella, R. L.; Montironi, R.; Magi-

Galluzzi, C.; Kantoff, P. W.; Balk, S. P.; Liu, X. S.; Brown, M. EZH2

oncogenic activity in castration-resistant prostate cancer cells is

Polycomb-independent. Science 2012, 338, 1465−1469.

(6) Kim, K. H.; Roberts, C. W. Targeting EZH2 in cancer. Nat. Med.

2016, 22, 128−134.

(7) Bonasio, R.; Tu, S.; Reinberg, D. Molecular signals of epigenetic

states. Science 2010, 330, 612−616.

(8) Bates, S. E. Epigenetic Therapies for Cancer. N. Engl. J. Med.

2020, 383, 650−663.

(9) Kohashi, K.; Oda, Y. Oncogenic roles of SMARCB1/INI1 and

its deficient tumors. Cancer Sci. 2017, 108, 547−552.

(10) Kim, K. H.; Kim, W.; Howard, T. P.; Vazquez, F.; Tsherniak,

A.; Wu, J. N.; Wang, W.; Haswell, J. R.; Walensky, L. D.; Hahn, W. C.;

Orkin, S. H.; Roberts, C. W. SWI/SNF-mutant cancers depend on

catalytic and non-catalytic activity of EZH2. Nat. Med. 2015, 21,

1491−1496.

(11) Bitler, B. G.; Aird, K. M.; Garipov, A.; Li, H.; Amatangelo, M.;

Kossenkov, A. V.; Schultz, D. C.; Liu, Q.; Shih Ie, M.; Conejo-Garcia,

J. R.; Speicher, D. W.; Zhang, R. Synthetic lethality by targeting EZH2

methyltransferase activity in ARID1A-mutated cancers. Nat. Med.

2015, 21, 231−238.

(12) Yamagishi, M.; Uchimaru, K. Targeting EZH2 in cancer

therapy. Curr. Opin. Oncol. 2017, 29, 375−381.

(13) First EZH2 Inhibitor Approved-for Rare Sarcoma. Cancer

Discovery 2020, 10, 333 334. DOI: 10.1158/2159-8290.CD-NB2020-

006.

(14) Knutson, S. K.; Wigle, T. J.; Warholic, N. M.; Sneeringer, C. J.;

Allain, C. J.; Klaus, C. R.; Sacks, J. D.; Raimondi, A.; Majer, C. R.;

Song, J.; Scott, M. P.; Jin, L.; Smith, J. J.; Olhava, E. J.; Chesworth, R.;

Moyer, M. P.; Richon, V. M.; Copeland, R. A.; Keilhack, H.; Pollock,

R. M.; Kuntz, K. W. A selective inhibitor of EZH2 blocks H3K27

methylation and kills mutant lymphoma cells. Nat. Chem. Biol. 2012,

8, 890−896.

(28) Vogelmann, A.; Robaa, D.; Sippl, W.; Jung, M. Proteolysis

targeting chimeras (PROTACs) for epigenetics research. Curr. Opin.

Chem. Biol. 2020, 57, 8−16.

10183

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(29) Wen, S.; Wang, J.; Liu, P.; Li, Y.; Lu, W.; Hu, Y.; Liu, J.; He, Z.;

Huang, P. Novel combination of histone methylation modulators with

therapeutic synergy against acute myeloid leukemia in vitro and in

vivo. Cancer Lett. 2018, 413, 35−45.

(30) Kuntz, K. W.; Campbell, J. E.; Keilhack, H.; Pollock, R. M.;

Knutson, S. K.; Porter-Scott, M.; Richon, V. M.; Sneeringer, C. J.;

Wigle, T. J.; Allain, C. J.; Majer, C. R.; Moyer, M. P.; Copeland, R. A.;

Chesworth, R. The Importance of Being Me: Magic Methyls,

Methyltransferase Inhibitors, and the Discovery of Tazemetostat. J.

Med. Chem. 2016, 59, 1556−1564.

(31) Galdeano, C.; Gadd, M. S.; Soares, P.; Scaffidi, S.; Van Molle,

I.; Birced, I.; Hewitt, S.; Dias, D. M.; Ciulli, A. Structure-guided

design and optimization of small molecules targeting the protein-

protein interaction between the von Hippel-Lindau (VHL) E3

ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit

with in vitro nanomolar affinities. J. Med. Chem. 2014, 57, 8657−

8663.

(32) Frost, J.; Galdeano, C.; Soares, P.; Gadd, M. S.; Grzes, K. M.;

Ellis, L.; Epemolu, O.; Shimamura, S.; Bantscheff, M.; Grandi, P.;

Read, K. D.; Cantrell, D. A.; Rocha, S.; Ciulli, A. Potent and selective

chemical probe of hypoxic signalling downstream of HIF-alpha

hydroxylation via VHL inhibition. Nat. Commun. 2016, 7, No. 13312.

(33) Zorba, A.; Nguyen, C.; Xu, Y.; Starr, J.; Borzilleri, K.; Smith, J.;

Zhu, H.; Farley, K. A.; Ding, W.; Schiemer, J.; Feng, X.; Chang, J. S.;

Uccello, D. P.; Young, J. A.; Garcia-Irrizary, C. N.; Czabaniuk, L.;

Schuff, B.; Oliver, R.; Montgomery, J.; Hayward, M. M.; Coe, J.;

Chen, J.; Niosi, M.; Luthra, S.; Shah, J. C.; El-Kattan, A.; Qiu, X.;

West, G. M.; Noe, M. C.; Shanmugasundaram, V.; Gilbert, A. M.;

Brown, M. F.; Calabrese, M. F. Delineating the role of cooperativity in

the design of potent PROTACs for BTK. Proc. Natl. Acad. Sci. U.S.A.

2018, 115, E7285−E7292.

(41) Remillard, D.; Buckley, D. L.; Paulk, J.; Brien, G. L.; Sonnett,

M.; Seo, H. S.; Dastjerdi, S.; Wuhr, M.; Dhe-Paganon, S.; Armstrong,

S. A.; Bradner, J. E. Degradation of the BAF Complex Factor BRD9

by Heterobifunctional Ligands. Angew. Chem., Int. Ed. 2017, 56,

5738−5743.

(42) Zhou, B.; Hu, J.; Xu, F.; Chen, Z.; Bai, L.; Fernandez-Salas, E.;

Lin, M.; Liu, L.; Yang, C. Y.; Zhao, Y.; McEachern, D.; Przybranowski,

S.; Wen, B.; Sun, D.; Wang, S. Discovery of a Small-Molecule

Degrader of Bromodomain and Extra-Terminal (BET) Proteins with

Picomolar Cellular Potencies and Capable of Achieving Tumor

Regression. J. Med. Chem. 2018, 61, 462−481.

(43) Zengerle, M.; Chan, K. H.; Ciulli, A. Selective Small Molecule

Induced Degradation of the BET Bromodomain Protein BRD4. ACS

Chem. Biol. 2015, 10, 1770−1777.

(44) Wang, X.; Cao, W.; Zhang, J.; Yan, M.; Xu, Q.; Wu, X.; Wan,

L.; Zhang, Z.; Zhang, C.; Qin, X.; Xiao, M.; Ye, D.; Liu, Y.; Han, Z.;

Wang, S.; Mao, L.; Wei, W.; Chen, W. A covalently bound inhibitor

triggers EZH2 degradation through CHIP-mediated ubiquitination.

EMBO J. 2017, 36, 1243−1260.

(45) Kasibhatla, S.; Jessen, K. A.; Maliartchouk, S.; Wang, J. Y.;

English, N. M.; Drewe, J.; Qiu, L.; Archer, S. P.; Ponce, A. E.;

Sirisoma, N.; Jiang, S.; Zhang, H. Z.; Gehlsen, K. R.; Cai, S. X.; Green,

D. R.; Tseng, B. A role for transferrin receptor in triggering apoptosis

when targeted with gambogic acid. Proc. Natl. Acad. Sci. U.S.A. 2005,

102, 12095−12100.

(46) Ma, A.; Stratikopoulos, E.; Park, K. S.; Wei, J.; Martin, T. C.;

Yang, X.; Schwarz, M.; Leshchenko, V.; Rialdi, A.; Dale, B.; Lagana,

A.; Guccione, E.; Parekh, S.; Parsons, R.; Jin, J. Discovery of a first-in-

class EZH2 selective degrader. Nat. Chem. Biol. 2020, 16, 214−222.

(47) Huang, H. T.; Dobrovolsky, D.; Paulk, J.; Yang, G.; Weisberg,

E. L.; Doctor, Z. M.; Buckley, D. L.; Cho, J. H.; Ko, E.; Jang, J.; Shi,

K.; Choi, H. G.; Griffin, J. D.; Li, Y.; Treon, S. P.; Fischer, E. S.;

Bradner, J. E.; Tan, L.; Gray, N. S. A Chemoproteomic Approach to

Query the Degradable Kinome Using a Multi-kinase Degrader. Cell

Chem. Biol. 2018, 25, 88−99.e6.

(48) Liu, Z.; Hu, X.; Wang, Q.; Wu, X.; Zhang, Q.; Wei, W.; Su, X.;

He, H.; Zhou, S.; Hu, R.; Ye, T.; Zhu, Y.; Wang, N.; Yu, L. Design and

Synthesis of EZH2-Based PROTACs to Degrade the PRC2 Complex

for Targeting the Noncatalytic Activity of EZH2. J. Med. Chem. 2021,

64, 2829−2848.

(34) Bondeson, D. P.; Smith, B. E.; Burslem, G. M.; Buhimschi, A.

D.; Hines, J.; Jaime-Figueroa, S.; Wang, J.; Hamman, B. D.;

Ishchenko, A.; Crews, C. M. Lessons in PROTAC Design from

Selective Degradation with a Promiscuous Warhead. Cell Chem. Biol.

2018, 25, 78−87.e5.

(35) Schapira, M.; Calabrese, M. F.; Bullock, A. N.; Crews, C. M.

Targeted protein degradation: expanding the toolbox. Nat. Rev. Drug

Discovery 2019, 18, 949−963.

(36) Zoppi, V.; Hughes, S. J.; Maniaci, C.; Testa, A.; Gmaschitz, T.;

Wieshofer, C.; Koegl, M.; Riching, K. M.; Daniels, D. L.; Spallarossa,

A.; Ciulli, A. Iterative Design and Optimization of Initially Inactive

Proteolysis Targeting Chimeras (PROTACs) Identify VZ185 as a

Potent, Fast, and Selective von Hippel-Lindau (VHL) Based Dual

Degrader Probe of BRD9 and BRD7. J. Med. Chem. 2019, 62, 699−

726.

(49) Yang, M.; Liu, P.; Wang, K.; Glorieux, C.; Hu, Y.; Wen, S.;

Jiang, W.; Huang, P. Chemotherapy induces tumor immune evasion

by upregulation of programmed cell death ligand 1 expression in bone

marrow stromal cells. Mol. Oncol. 2017, 11, 358−372.

(37) Potjewyd, F.; Turner, A. W.; Beri, J.; Rectenwald, J. M.; Norris-

Drouin, J. L.; Cholensky, S. H.; Margolis, D. M.; Pearce, K. H.;

Herring, L. E.; James, L. I. Degradation of Polycomb Repressive

Complex 2 with an EED-Targeted Bivalent Chemical Degrader. Cell

Chem. Biol. 2020, 27, 47−56.e15.

(38) Hsu, J. H.; Rasmusson, T.; Robinson, J.; Pachl, F.; Read, J.;

Kawatkar, S.; DH, O. D.; Bagal, S.; Code, E.; Rawlins, P.; Argyrou, A.;

Tomlinson, R.; Gao, N.; Zhu, X.; Chiarparin, E.; Jacques, K.; Shen,

M.; Woods, H.; Bednarski, E.; Wilson, D. M.; Drew, L.; Castaldi, M.

P.; Fawell, S.; Bloecher, A. EED-Targeted PROTACs Degrade EED,

EZH2, and SUZ12 in the PRC2 Complex. Cell Chem. Biol. 2020, 27,

41−46.e17.

(39) Maniaci, C.; Hughes, S. J.; Testa, A.; Chen, W.; Lamont, D. J.;

Rocha, S.; Alessi, D. R.; Romeo, R.; Ciulli, A. Homo-PROTACs:

bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to

induce self-degradation. Nat. Commun. 2017, 8, No. 830.

(40) Knutson, S. K.; Warholic, N. M.; Wigle, T. J.; Klaus, C. R.;

Allain, C. J.; Raimondi, A.; Porter Scott, M.; Chesworth, R.; Moyer,

M. P.; Copeland, R. A.; Richon, V. M.; Pollock, R. M.; Kuntz, K. W.;

Keilhack, H. Durable tumor regression in genetically altered

malignant rhabdoid tumors by inhibition of methyltransferase

EZH2. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 7922−7927.

10184

https://doi.org/10.1021/acs.jmedchem.1c00460

J. Med. Chem. 2021, 64, 10167−10184

Article Doi

Design, Synthesis, and Evaluation of VHL-Based EZH2 Degraders to Enhance Therapeutic Activity against Lymphoma

DOI: 10.1021/acs.jmedchem.1c00460

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.1c00460

Full text of DOI:10.1021/acs.jmedchem.1c00460

Products guided by the article

R&D Labs maybe for 930294-37-4

Relevant to this article

Hot Product