Discovery of MD-224 as a First-in-Class, Highly Potent, and Efficacious Proteolysis Targeting Chimera Murine Double Minute 2 Degrader Capable of Achieving Complete and Durable Tumor Regression

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Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

Discovery of MD-224 as a First-in-Class, Highly Potent, and

Eﬃcacious Proteolysis Targeting Chimera Murine Double Minute 2

Degrader Capable of Achieving Complete and Durable Tumor

Regression

Yangbing Li,^{†,‡,∥,⊥}Jiuling Yang,^{†,§,∥,⊥}Angelo Aguilar,^†,∥,⊥Donna McEachern,^†,∥Sally Przybranowski,^†,∥

Liu Liu,^†,∥Chao-Yie Yang,^†,∥Mi Wang,^†,∥Xin Han,^†,∥and Shaomeng Wang*^,†,‡,∥

^†The Rogel Cancer Center, ^‡Departments of Medicinal Chemistry, ^§Pharmacology, ^∥Internal Medicine, University of Michigan, Ann

Arbor, Michigan 48109, United States

S

* Supporting Information

ABSTRACT: Human murine double minute 2 (MDM2) protein is a primary

endogenous cellular inhibitor of the tumor suppressor p53 and has been

pursued as an attractive cancer therapeutic target. Several potent, nonpeptide,

small-molecule inhibitors of MDM2 are currently in clinical development. In

this paper, we report our design, synthesis, and evaluation of small-molecule

MDM2 degraders based on the proteolysis targeting chimera (PROTAC)

concept. The most promising compound (MD-224) eﬀectively induces rapid

degradation of MDM2 at concentrations <1 nM in human leukemia cells. It

achieves an IC₅₀value of 1.5 nM in inhibition of growth of RS4;11 cells and also

low nanomolar IC₅₀values in a panel of leukemia cell lines. MD-224 achieves

complete and durable tumor regression in vivo in the RS4;11 xenograft tumor

model in mice at well-tolerated dose schedules. MD-224 is thus a highly potent

and eﬃcacious MDM2 degrader and warrants extensive evaluations as a new

class of anticancer agent.

Mechanistically, MDM2 inhibitors induce accumulation and

transcriptional activation of p53 protein by blocking p53

degradation by MDM2. Because MDM2 is a p53-targeted

gene, activation of p53 results in transcription of MDM2 mRNA,

leading to robust MDM2 protein accumulation. However,

MDM2 protein accumulated in vivo can eﬃciently and rapidly

degrade p53 upon clearance of an MDM2 inhibitor because of

the pharmacokinetic eﬀect, and this is predicted to reduce the

therapeutic eﬃcacy of the MDM2 inhibitor. Indeed, our

previous study has shown that p53 protein is accumulated for

only a few hours in xenograft tumor tissues following

administration of a single dose of an MDM2 inhibitor.¹³

Furthermore, accumulation of MDM2 protein in normal tissues

might have unwanted eﬀects because MDM2 itself is oncogenic.

To overcome these potential limitations of MDM2 inhibitors,

new strategies are needed to more eﬀectively target MDM2.

The proteolysis targeting chimera (PROTAC) concept¹⁷⁻¹⁹

was formally proposed 15 years ago, with the objective of

hijacking the powerful cellular ubiquitin degradation systems to

achieve targeted protein degradation. The PROTAC strategy

uses a heterobifunctional small molecule containing a small-

molecule ligand binding to the target protein of interest and

another small-molecule ligand binding to an E3 ubiquitin ligase

INTRODUCTION

■

The tumor suppressor p53 plays a critical role in the prevention

of tumor development.¹⁻³The TP53 gene, which encodes the

p53 protein, is mutated or deleted in about 50% of human

cancers, resulting in inactivation of the tumor suppressor

function of p53.⁴In human cancers retaining wild-type p53, the

tumor suppressor function of p53 can also be suppressed by a

variety of mechanisms. One major mechanism of p53 inhibition

stems from its primary, endogenous cellular inhibitor, the

murine double minute 2 (MDM2) protein. By functioning as an

E3 ligase, MDM2 binds to and ubiquitinates p53, leading to

eﬃcient p53 degradation.^5,6The binding of MDM2 to p53 also

blocks the interaction of p53 with targeted DNA molecules and

transports p53 from the nucleus to the cytoplasm, both of which

decrease the transcriptional activity of p53.⁷Indeed, over-

expression of MDM2 protein has frequently been detected in

human cancers carrying wild-type p53.⁸

Because MDM2 eﬀectively inhibits the tumor suppressor

function of p53 through a direct protein−protein interaction,

intense eﬀorts have been made to develop small-molecule

inhibitors of the MDM2-p53 protein−protein interaction

(hereafter called MDM2 inhibitors).⁹⁻¹⁵A number of highly

potent small-molecule MDM2 inhibitors,¹⁶including two

discovered in our laboratory,^11,13have been advanced into

clinical development for the treatment of human cancers.

Received: June 7, 2018

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complex, tethered together by a chemical linker to achieve

targeted protein degradation. The PROTAC approach has

recently gained tremendous momentum because in part of the

availability of potent and druglike ligands for the cullin 2 and

cullin 4A E3 degradation systems. This strategy has successfully

been employed in the design of potent small-molecule

PROTAC degraders for a number of proteins, including

bromodomain and extra-terminal (BET) proteins,²⁰⁻²⁴the

androgen receptor,²⁵Bcr-Abl,^26,27and the estrogen-related

receptor α (ERRα),²⁸among others.

In the present study, we have employed the PROTAC

strategy to design small-molecule degraders of MDM2 based

upon our previously reported, potent MDM2 inhibitors. This

has led to the discovery of MD-224, which induces rapid

degradation of MDM2 at low nanomolar concentrations in

leukemia cells. MD-224 is a very potent inhibitor of cell growth

in a panel of leukemia cell lines carrying wild-type p53 and is

capable of achieving complete and durable tumor regression in

the RS4;11 xenograft model in mice at well-tolerated dose

schedules. It is therefore a highly potent and promising

PROTAC MDM2 degrader.

position similar to that of the solvent-exposed 4-hydroxyl group

on the cyclohexyl ring in MI-77301 (Figure 1). Accordingly, we

proposed to use the carboxylic acid group in MI-1061 as the

tethering site for the design of bifunctional PROTAC MDM2

degraders.

The design of PROTAC MDM2 degraders requires a small-

molecule ligand for an E3 ligase degradation system.

Thalidomide and lenalidomide are potent, small-molecule

ligands which bind to cereblon (CRBN),²⁹which is an adaptor

protein in the cullin 4A E3 ligase degradation system.

Thalidomide and lenalidomide have been successfully used in

the design of PROTAC degraders for BET proteins^20,21and a

number of other proteins.^30,31

Accordingly, we employed the MDM2 inhibitor MI-1061 and

thalidomide/lenalidomide for the design of two initial, putative

MDM2 degraders 1 and 2 (Table 1), using linkers similar to

those in our previously reported BET degraders ZBC260 and

ZBC246.^22,23To facilitate the synthesis of these putative MDM2

degraders, we converted the carboxylic acid group in MI-1061

into a methyl amide, obtaining MI-1242 (Figure 2). Our binding

experiments showed that MI-1242 binds to MDM2 with a high

aﬃnity (K_i= 2.7 nM), which is weaker than MI-1061 but is

consistent with the predicted strong interactions between the

negatively charged carboxylic acid in MI-1061 and the Lys94

and His73 in MDM2 protein (Figure 1). However, MI-1242

potently inhibits cell growth in the RS4;11 cell line, with an IC₅₀

value of 89 nM, comparable to the potency of MI-1061,

suggesting an enhanced cell permeability for MI-1242 over that

of MI-1061.

We examined the eﬀect of 1 and 2 on MDM2 and p53

proteins in the RS4;11 leukemia cells carrying wild-type p53,

with MI-1061 and MI-1242 used as controls. Our data show that

consistent with our previous report,²³the MDM2 inhibitor MI-

1061 is very eﬀective in inducing accumulation of both MDM2

and p53 in a dose-dependent manner (Figure 3). Similarly, MI-

1242 is equally eﬀective in inducing accumulation of both

MDM2 and p53 in a dose-dependent manner (Figure 3). In

contrast, both 1 and 2 are highly potent and eﬀective in inducing

accumulation of p53 protein in a dose-dependent manner

without increasing the MDM2 protein level (Figure 3). In fact,

both 1 and 2 eﬀectively reduce the level of MDM2 protein in the

RS4;11 cells over the control treatments, even at concentrations

as low as 3 nM. These western blotting data thus suggest that 1

and 2 do not function as regular MDM2 inhibitors but are very

potent MDM2 degraders.

RESULTS AND DISCUSSION

■

In our previous study, we reported the discovery of MI-1061 as a

potent MDM2 inhibitor (K_i= 0.16 nM).¹¹MI-1061 eﬀectively

activates wild-type p53, potently inhibits cell growth in cancer

cell lines harboring wild-type p53, and strongly suppresses

tumor growth in vivo.¹¹We have therefore employed MI-1061

as a potent MDM2 inhibitor in the design of PROTAC MDM2

degraders.

The design of MI-1061 was based upon one of our ﬁrst-

generation MDM2 inhibitors, MI-77301.¹³Although we have

not obtained a co-crystal structure for MI-1061 complexed with

MDM2, we have successfully determined the co-crystal

structure of MI-77301 in complex with MDM2¹³(Figure 1A).

Because MI-1061 and MI-77301 are structurally similar, we

predicted that in binding to the MDM2 protein, the carboxylic

acid group on the phenyl ring in MI-1061 would be located in a

Consistent with their high potency in induction of p53

accumulation, 1 and 2 achieve IC₅₀values of 10 or 7 nM,

respectively, in inhibition of cell growth in the RS4;11 acute

leukemia cell line in the WST cell growth assay (Table 1), and

they are therefore about 10 times more potent than MI-1061

and MI-1242.

Our previous study showed that the length and chemical

composition of the linker in our BET protein degraders have a

major eﬀect on their cellular potencies in both BET degradation

and cell growth inhibition.²³Accordingly, we performed further

modiﬁcations of the linker in 1 and 2. We evaluated each

synthesized compound for its potency in cell growth inhibition

in the RS4;11 cell line as an initial assessment of the cellular

eﬀect of the linker in our designed MDM2 degraders, and

obtained the data summarized in Table 1.

Figure 1. (A) Co-crystal structure of MDM2 in a complex with MI-

77301 (cyan) (PDB ID: 5TRF); (B) modeled structure of MDM2

complexed with MI-1061 (yellow); (C) chemical structures of MDM2

inhibitors MI-77301 and MI-1061.

We synthesized compound 3 (Table 1), in which the acid

group in MI-1061 is directly coupled to the amino group in

lenalidomide, to examine the importance of the linker.

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Table 1. MDM2 Degraders Designed with Various Linkers to Pomalidomide

Compound 3 has an IC₅₀value of 68.4 nM in inhibition of cell

growth in the RS4;11 cell line, similar to that for MI-1061 and

MI-1242. Our western blotting analysis showed that 3 fails to

induce MDM2 degradation in the RS4;11 cells, indicating that

this compound works as an MDM2 inhibitor, but not as an

MDM2 degrader (Figure 4). Changing the linker from the 4

methylene groups in 1 to 2, 3, 5, 6, and 7 methylene groups

resulted in 4, 5, 6, 7, and 8, respectively. Interestingly,

compounds with 2−6 methylene groups in their linker have

similar cellular potencies, all within a factor of 2, in inhibition of

cell growth, but 8 with 7 methylene groups for the linker is 10

times less potent than 1. Western blotting analysis showed that

these compounds all induce degradation of MDM2 and

accumulation of p53 in the RS4;11 cells, indicating that they

function as MDM2 degraders (Figure 4).

very similar potencies in inhibition of cell growth in the RS4;11

cell line, with IC₅₀values of 8, 5, and 7 nM, respectively. Western

blotting analysis conﬁrmed that these compounds eﬀectively

induce degradation of MDM2 and accumulation of p53 in the

RS4;11 cells, indicating that they function as MDM2 degraders

(Figure 4).

Our previous studies of BET degraders demonstrated that

modiﬁcations of the CRBN binding portion of the degrader

molecule can signiﬁcantly enhance the degradation of target

proteins, cell growth inhibition activity against cancer cells, and

in vivo antitumor eﬃcacy.²³We therefore performed

modiﬁcations of the CRBN ligand portion in 1 (Table 2).

Changing the NH group in 1 to a CH₂group yielded 11, which

has an IC₅₀value of 29 nM and is therefore 3 times less potent

than 1. Conversion of one of the carbonyl groups in 1 into a CH₂

group generated 12, which has an IC₅₀value of 5 nM and is thus

twice as potent as 1. Changing the NH linking group in 12 to a

We investigated the eﬀect of the linker length in 2 by

increasing or decreasing it by one ethylene glycol group,

producing 9 and 10, respectively. Compounds 9, 10, and 2 have

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Figure 2. Design of PROTAC MDM2 degraders using MDM2 inhibitors and CRBN ligands.

Figure 3. Western blotting analysis of MDM2 and p53 proteins in

RS4;11 cells treated with MDM2 inhibitors MI-1061 and MI-1242, and

MDM2 degraders 1 and 2. RS4;11 cells were treated for 2 h with each

compound at indicated concentrations, and proteins were probed by

speciﬁc antibodies. Glyceraldehyde 3-phosphate dehydrogenase

(GAPDH), a constitutively expressed housekeeping protein, was used

as the loading control. MDM2 and p53 levels were quantiﬁed using an

imager and were normalized over the GAPDH loading control.

CH₂group generated 13 (MD-222), which has an IC₅₀value of

2.8 nM and is therefore 4 times more potent than 1.

Compounds 12 and MD-222 have a ﬂexible linker between

Figure 4. Western blotting analysis of the eﬀect of potential MDM2

degraders with various linkers on induction of MDM2 and p53 proteins.

RS4;11 cells were treated for 2 h with each individual compound at

indicated concentrations and proteins were probed by speciﬁc

antibodies. GAPDH was used as the loading control. MDM2 and

p53 levels were quantiﬁed using an imager and were normalized over

the GAPDH loading control.

the MDM2 inhibitor and the CRBN ligand portion. We next

investigated the eﬀect of linker rigidiﬁcation in 12 and MD-222

on cell growth inhibition and MDM2 degradation (Table 2).

Conversion of two CH₂groups in MD-222 into an alkyne group

resulted in MD-224, which has an IC₅₀value of 1.5 nM in the

RS4;11 cell line and is 2 times more potent than MD-222.

Shortening the linker in MD-224 by one CH₂group yielded 15,

which has an IC₅₀value of 3.9 nM and is thus 2 times less potent

than MD-224.

The co-crystal structure of lenalidomide in a complex with

CRBN (PDB ID: 4CI2)³²suggests that in addition to the linking

position in MD-224, two other positions on the isoindolin-1-one

ring could be used for linker tethering in the design of PROTAC

degraders (Figure 5). On the basis of MD-224, we designed and

synthesized 16 and 17 (Table 3), which use two other possible

tethering positions in the phenyl ring of the CRBN ligand.

Compounds 16 and 17 both have IC₅₀values of 5 nM in

inhibition of cell growth in the RS4;11 cell line, which is 3 times

less potent than MD-224. Our western blotting data conﬁrmed

that 16 and 17 eﬀectively induce degradation of MDM2 and

accumulation of p53 in the RS4;11 cells, indicating that they

function as MDM2 degraders (Figure 6). The data therefore

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Table 2. MDM2 Degraders with Modiﬁcations of the CRBN

Ligand Portion in Compound 1

Table 3. MDM2 Degraders Linked from Diﬀerent Positions

of Lenalidomide

Figure 6. Western blotting analysis of the eﬀect of MDM2 degraders

linked from three diﬀerent tethering sites of lenalidomide on MDM2

and p53 protein levels in the RS4;11 cell line. The MDM2 inhibitor MI-

1061 and the MDM2 degrader MD-224 were included as controls.

RS4;11 cells were treated for 2 h with each individual compound at the

indicated concentrations and proteins were probed by speciﬁc

antibodies. GAPDH was used as the loading control. MDM2 and

p53 levels were quantiﬁed using an imager and were normalized over

the GAPDH loading control.

Figure 5. Three possible linker tethering positions in the CRBN ligand

(shown in red arrows) based upon a previously published co-crystal

structure of DDB1-CRBN E3 ubiquitin ligase complexed with

lenalidomide (yellow, PDB ID: 4CI2).

indicate that all three tethering positions in the isoindolin-1-one

ring in the CRBN ligand can be used successfully for the design

of potent and eﬀective MDM2 degraders.

In addition to the CRBN/cullin 4A E3 ligase complex, the von

Hippel−Lindau protein 1 (VHL-1)/cullin 2 E3 ligase system

has been employed for the design of PROTAC degraders of a

number of proteins.^25,33We investigated if the VHL-1/cullin 2

E3 ligase system can be employed for the successful design of

PROTAC MDM2 degraders. In the co-crystal structure of VHL-

1 in a complex with a potent, peptidomimetic VHL-1 ligand

(Figure 7), the terminal acetyl group is exposed to solvent,

making this site suitable for the design of potential PROTAC

MDM2 degraders. Indeed, this site has been successfully used

for the design of PROTAC BET protein degraders^24,33and

other targets.^25,26,28We designed and synthesized three putative

MDM2 degraders (18, 19, and 20) using the VHL-1 ligand and

MI-1061, with linkers of diﬀerent lengths and chemical

compositions (Table 4).

Figure 7. Proposed tethering position in the VHL-1 ligand based upon

a previously published co-crystal structure of a VHL-1 ligand in

complex with pVHL:EloB:EloC (PDB ID: 4W9H).

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Table 4. Potential MDM2 Degraders Designed Using an VHL-1 Ligand

In the cell growth assay in the RS4;11 cell line, 18, 19, and 20

display IC₅₀values of 1.2, 0.5, and 0.34 μM, respectively, and

thus all are less potent than the corresponding MDM2 inhibitor

MI-1061 (Table 4). Western blotting analysis showed that in

contrast to 1 and 2, compound 20 failed to reduce the level of

MDM2 protein in the RS4;11 cells (Figure 8). Collectively, our

data suggest that the VHL-1/cullin 2 E3 ligase system may not

be suited to the design of eﬀective PROTAC MDM2 degraders,

although more extensive investigations are needed to conﬁrm

this initial ﬁnding.

Further Evaluation of Two Potent PROTAC MDM2

Degraders, MD-222, and MD-224. We performed extensive

evaluations to further investigate the mechanism of action and

therapeutic potential of MD-224 and MD-222, two potent

PROTAC MDM2 degraders, with MI-1061 included as the

MDM2 inhibitor control.

We ﬁrst examined their potency in induction of MDM2

depletion and p53 activation in the RS4;11 and MV4;11

leukemia cell lines. Treatment of the RS4;11 cells with either

MD-222 or MD-224 for 2 h eﬀectively induces depletion of

MDM2 protein and concurrently accumulation of p53 protein in

a dose-dependent manner (Figure 9A). Consistent with their

high potency in inhibition of cell growth, both MD-222 and

MD-224 eﬀectively induce marked depletion of MDM2 protein

at concentrations as low as 1 nM, and MD-224 is more potent

than MD-222. The MDM2 inhibitor MI-1061 induces

accumulation of both MDM2 and p53 proteins, consistent

with its mechanism of action as a potent MDM2 inhibitor. Both

Figure 8. Western blotting analysis of MDM2, p53, p21, and cleaved

PAPR proteins in RS4;11 cells treated with the MDM2 inhibitor MI-

1061 and a potential MDM2 degrader 20. RS4;11 cells were treated for

12 h with each individual compound at indicated concentrations, and

each protein was probed by a speciﬁc antibody. GAPDH was used as the

loading control. MDM2, p53, p21, and cleaved PARP levels were

quantiﬁed using an imager and were normalized over the GAPDH

loading control.

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of MD-222 or MD-224 compared to MI-1061 is achieved

through the eﬃcient MDM2 degradation by MD-222 or MD-

224. In the MV4;11 AML cell line, pretreatment with

lenalidomide also eﬀectively reduces the cell growth inhibitory

activity of MD-222 or MD-224 and has no eﬀect on the activity

of MI-1061 (Figure S1). Pre-incubated with proteasome

inhibitors, MD-132 and PR-171 or a neddylation inhibitor

MLN4924 in RS4;11 cells can block MDM2 degradation

induced by MD-224 (Figure 10d). These data showed that the

MDM2 degradation was signiﬁcantly reduced by the protea-

some inhibitor and the neddylation inhibitor, indicating that the

MDM2 degradation by MD-224 is proteasome and neddylation-

dependent.

Together, these data showed that MD-224 and MD-222 are

highly eﬀective and potent in induction of MDM2 degradation

and accumulation of p53 in both RS4;11 and MV4;11 cell lines.

MD-224 is >10−50 times more potent than the MDM2

inhibitor, MI-1061, in induction of p53 activation and in

inhibition of cell growth in the RS4;11 and MV4;11 cell lines.

The induction of MDM2 degradation by MD-224 can be

eﬀectively blocked by the CRBN ligand lenalidomide,

proteasome inhibitors and a neddylation inhibitor. These data

establish that MD-224 is a highly potent, eﬀective, and bona ﬁde

PROTAC MDM2 degrader.

To further investigate the mechanism of action of MDM2

degradation induced by MD-224, we synthesized two control

compounds 21 and 22 (Figure 11A). Compound 21 is an

enantiomer of MD-224 with all the chiral centers in the MDM2

inhibitor portion inverted. Our data showed that compound 21

fails to induce any MDM2 degradation in the RS4;11 cell line

(Figure 11B), and has a minimal activity in inhibition of cell

growth in the RS4;11 cell line (Figure 11C). The amino group of

the glutarimide in lenalidomide forms a strong hydrogen bond

with CRBN as shown in the co-crystal structure^29,32and

methylation of the amino group of the glutarimide in

lenalidomide completely abrogates the binding to CRBN.^21,32

We therefore synthesized 22 by methylation of the amino group

of the glutarimide in MD-224 as an additional control

compound. Compound 22 was found to be completely inactive

in inducing MDM2 degradation (Figure 11B) and is >100 times

less potent than MD-224 in inhibition of cell growth in the

RS4;11 cells (Figure 11C). These data ﬁrmly establish that the

induced degradation of MDM2 by MD-224 requires its strong

binding to MDM2 and to CRBN, consistent with its PROTAC

design.

Because p53 is a powerful transcription factor, we used

quantitative reverse transcription polymerase chain reaction

(qRT-PCR) analysis to examine the transcriptional activation of

p53 by MD-224, MD-222, and MI-1061 (Figure 12). A 6 h

treatment with the MDM2 inhibitor MI-1061 eﬀectively induces

marked transcriptional upregulation of p53 target genes in the

RS4;11 cell line, including MDM2, the cell cycle regulator gene

p21, and pro-apoptotic PUMA gene but not TP53, which is the

gene encoding p53 (Figure 12). MD-224 and MD-222 are >10

times more potent than MI-1061 in induction of transcriptional

upregulation of these p53 target genes but have no eﬀect on

TP53 itself in RS4;11 cells (Figure 12). Despite the robust

upregulation of MDM2 mRNA by MD-224 and MD-222, the

MDM2 protein is eﬀectively depleted by both MD-224 and

MD-222, indicating that they are highly eﬃcient degraders of the

MDM2 protein. Very similar data were obtained in the MV4;11

cell line for MD-224, MD-222, and MI-1061 (Figure S2), when

compared to the data obtained in the RS4;11 cell line.

Figure 9. Western blotting analysis for MDM2 and p53 protein levels

after treatment with the MDM2 inhibitor MI-1061 and the MDM2

degraders MD-222 and MD-224 in (A) RS4;11 and (B) MV-4-11 cell

lines. RS4;11 or MV-4-11 cells were treated with each individual

compound at the indicated concentrations for 2 h and proteins were

probed by speciﬁc antibodies. GAPDH was used as the loading control.

MDM2 and p53 protein levels were quantiﬁed using an imager and

were normalized over the GAPDH loading control.

MD-222 and MD-224 are similarly potent and eﬀective in

inducing depletion of MDM2 protein and accumulation of p53

protein in the MV4;11 cell line (Figure 9B), when compared to

the RS4;11 cell line.

As bona ﬁde PROTAC degrader molecules, depletion of

MDM2 protein by MD-224 and MD-222 should require their

binding to the CRBN/cullin 4 E3 ligase complex through their

lenalidomide segment. Accordingly, we predicted that excessive

amounts of lenalidomide should eﬀectively block the MDM2

degradation and reduce p53 activation induced by MD-224 and

MD-222 and should have no eﬀect on the upregulation of

MDM2 and p53 proteins induced by the MDM2 inhibitor MI-

1061. Western blotting analysis showed that lenalidomide

indeed eﬀectively blocks MDM2 degradation and accumulation

of p53 protein induced by MD-224 and MD-222 and has no

eﬀect on the MDM2 and p53 accumulation induced by the

MDM2 inhibitor MI-1061 (Figure 10A).

Because lenalidomide can eﬀectively block MDM2 degrada-

tion induced by MD-224 and MD-222, we predicted that

lenalidomide would be eﬀective in reducing the potent cell

growth inhibitory activity of these MDM2 degraders but would

have no eﬀect on the activity of the MDM2 inhibitor MI-1061.

Indeed, pretreatment with lenalidomide greatly reduces the cell

growth inhibitory activity of MD-222 and MD-224 to the same

level observed for the MDM2 inhibitor MI-1061 (Figure

10B,C). These data indicate that when the binding of MD-222

or MD-224 to CRBN is eﬀectively blocked by lenalidomide,

both MD-222 and MD-224 behave as an MDM2 inhibitor in

cells. As expected, pretreatment with lenalidomide has no

signiﬁcant eﬀect on the cell growth inhibitory activity of MI-

1061. These data clearly show that the superior cellular potency

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Figure 10. Investigation of activity dependence of the MDM2 degraders MD-222 and MD-224 and the MDM2 inhibitor MI-1061 on CRBN-binding,

proteasome, and neddylation. (A). Western blotting analysis of MDM2 and p53 proteins in RS4;11 cells. RS4;11 cells were treated with the MDM2

inhibitor MI-1061 and the MDM2 degrader MD-222 or MD-224 for 2 h in the presence or absence of excess lenalidomide. MDM2, p53, and GAPDH

(loading control) proteins were probed with speciﬁc antibodies. (B,C) Cell growth inhibition activity of the MDM2 inhibitor MI-1061 and the MDM2

degraders MD-222 and MD-224 in the absence or presence of lenalidomide in the RS4;11 cell line. Cells were treated for 4 days with MI-1061, MD-

222, or MD-224 alone or in combination with indicated concentrations of lenalidomide for 4 days, and cell viability was determined by a WST-8 assay.

(D). Western blotting analysis of MDM2 and p53 proteins in RS4;11 cells. RS4;11 cells were treated with DMSO, MG-132 (10 μM), PR-171 (1 μM),

or MLN4924 (3 μM) for 4 h, followed by treatment with DMSO control, MDM2 inhibitor MI-1061, or the MDM2 degrader MD-224 for additional 2

h. MDM2, p53, and GAPDH (loading control) proteins were probed with speciﬁc antibodies.

Because both the MDM2 degrader MD-224 and the MDM2

inhibitor MI-1061 eﬀectively induce upregulation of PUMA, a

potent pro-apoptotic gene, we investigated induction of

apoptosis by both compounds in the RS4;11 cells by ﬂow

cytometry (Figure 13). While both compounds are eﬀective in

inducing apoptosis in a dose-dependent manner upon a 24 h

treatment, MD-224 is >10 times more potent than MI-1061. In

fact, MD-224 induces robust apoptosis at ≤10 nM, consistent

with its high potency in induction of p53 activation and of

PUMA mRNA upregulation.

We further evaluated MD-222, MD-224, and MI-1061 in

additional human leukemia cell lines containing wild-type p53

and mutated p53 for their growth inhibitory activity and

obtained the data shown in Table 5.

Both MI-1061 and MD-224 potently inhibit cell growth in

these human leukemia cell lines with wild-type p53, but MD-224

is much more potent than MI-1061. MD-224 has IC₅₀values of

4.4−33.1 nM in these cell lines and is >10 times more potent

than MI-1061. Both MI-1061 and MD-224 are highly selective

over leukemia cell lines with mutated p53, displaying IC₅₀> 10

μM, indicating that the cell growth inhibition by both MDM2

inhibitors and degraders depends upon wild-type p53.

We next investigated the pharmacodynamic (PD) eﬀect of

MD-224 in vivo in mice bearing the RS4;11 xenograft tumor

(Figure 14). A single, intravenous dose of MD-224 eﬀectively

depletes MDM2 protein at the 3 h time point, with the eﬀect

persisting for >24 h (Figure 14). MD-224 induces strong

upregulation of p53 and p21 at the 3 h time point in the tumor

tissue with the eﬀect lasting for >24 h. PARP cleavage is evident

at the 24 h time point, indicating induction of apoptosis. The PD

data thus show that by degradation of MDM2 protein, a single

dose of the MDM2 degrader MD-224 can achieve robust and

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Figure 11. Control compounds 21 and 22 fail to induce MDM2 degradation and show much weaker activity in cell growth inhibition in the RS4;11 cell

line: (A) chemical structures of 21 and 22; (B) Western blotting analysis of p53 and MDM2 proteins in RS4;11 cells treated with MI-1061, MD-224,

21, and 22; (C) cell growth inhibition of MI-1061, MD-224, 21, and 22 in the RS4;11 cell line.

sustained MDM2 degradation and p53 upregulation for >24 h.

Our previous data showed that a single dose of our potent

MDM2 inhibitor MI-77301 can upregulate p53 protein for only

a few hours from tumor tissue in mice,¹³likely because of robust

accumulation of MDM2 protein, which can bind to and rapidly

degrade p53 once the MDM2 inhibitor is cleared from the

tumor tissue. The sustained p53 protein accumulation by MD-

224 suggests that infrequent (e.g., weekly) administration may

be suﬃcient to achieve strong antitumor activity.

On the basis of these promising PD data, we tested MD-224

for its antitumor activity in the RS4;11 xenograft model in two

experiments. In the ﬁrst experiment (Figure 15), we tested MI-

1061 at a near-maximum tolerated dose (100 mg/kg, oral, daily

dosing) and 25 mg/kg of MD-224, administered intravenously.

The treatments started on day 26 when the average tumor

volumes reached 100 mm³with 5 mice/tumors in each group.

The MDM2 inhibitor MI-1061 eﬀectively retards tumor growth

over the control, but MD-224 can achieve much stronger

antitumor activity than MI-1061 (Figure 15A). MD-224 at a 25

mg/kg weekly, intravenous dose, or at a dose of 10 mg/kg three

times per week has similar antitumor activity as MI-1061 at 100

mg/kg, 5 days a week via oral gavage. MD-224 at 25 mg/kg,

three times a week, can regress the tumors by 50%. MI-1061 and

MD-224 cause no signiﬁcant weight loss or other signs of

toxicity in this experiment.

In the second experiment, we investigated if MD-224 at

higher doses or more frequent dosing schedules can achieve

complete tumor regression in the RS4;11 model (Figure 16).

Treatments were initiated when the tumor volume reached 100

mm³at day 33. It was found that MD-224 at 25 mg/kg, daily, 5

days a week for 2 weeks, or at 50 mg/kg, every other day for 3

weeks achieves complete tumor regression. Consistent with its

strong and sustained p53 activation following a single dose in

RS4;11 tumor tissues, MD-224 with weekly dosing at 50 mg/kg

achieves complete tumor growth inhibition (Figure 16A), while

causing no weight loss or any other signs of toxicity in mice

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Figure 12. qRT-PCR analysis mRNA levels of p53 target genes and TP53 after treatment with the MDM2 inhibitor, MI-1061, and the MDM2

degraders MD-222 and MD-224 in RS4;11 cells. RS4;11 cells were treated for 6 hr and mRNA levels of MDM2, p21, TP53, and PUMA were analyzed

by qRT-PCR.

Figure 13. Flow cytometric analysis of apoptosis induction using annexin V/propidium iodide (PI) double staining after treatment with the MDM2

inhibitor, MI-1061, and the MDM2 degrader MD-224 in RS4;11 cells.

(Figure 16B). These data establish that MD-224 is highly

eﬃcacious against the RS4;11 xenograft tumors even with

weekly dosing and can achieve complete tumor regression with

either daily or every other day (three times per week) dosing

schedules.

Taken together, these two eﬃcacy experiments clearly

demonstrate that the MDM2 degrader MD-224 can achieve

complete tumor regression at well-tolerated dose schedules and

is much more eﬃcacious than the MDM2 inhibitor MI-1061.

SYNTHESIS

■

The synthesis of MDM2 degraders containing thalidomide or

lenalidomide is shown in Scheme 1. The intermediates (24a−

24i) were synthesized by a nucleophilic substitution reaction

with 2-(2,6-dioxopiperidin-3-yl)-4-ﬂuoroisoindoline-1,3-dione

(23), and the Boc-protecting groups were removed under acidic

conditions. The intermediate (24j) was synthesized by reductive

amination reaction with lenalidomide, followed by removing the

Boc-protecting group under acidic conditions. MDM2 de-

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Table 5. Cell Growth Inhibition Activity of MI-1061, MD-222, and MD-224 in a Panel of Human AML or ALL Cell Lines

IC₅₀(nM) in cell growth inhibition assay

cell line

leukemia type

p53 status

MI-1061

MD-222

MD-224

MOLM-13

MOLM-14

SIG-M5

AML

WT

MUT

76.8 40

143.3 64

105.6 73

150.7 51

374.5 143

>10 μM

11.7

22.5

29.9 16

11.5

58.2 29

>10 μM

4

8

7.3

10.5

3

19.8 11

4.4

33.1 18

>10 μM

ML-2

6

2

OCL-AML-5

mono-Mac-6

KG-1

>10 μM

leukemia cell lines and is highly eﬀective in inducing activation

of p53 in leukemia cells carrying wild-type p53. MD-224 is

highly potent in inhibition of cell growth and in induction of

apoptosis in p53 wild-type leukemia cells and is >10−100 times

more potent than the MDM2 inhibitor MI-1061. A single dose

of MD-224 eﬀectively induces MDM2 degradation and p53

activation in the RS4;11 leukemia xenograft tissue with the eﬀect

persisting for >24 h. Consequently, MD-224 is highly eﬃcacious

and is capable of achieving complete and long-lasting tumor

regression in the RS4;11 xenograft model in mice at well-

tolerated dose schedules. Collectively, our data demonstrate that

MD-224 is a highly potent, eﬃcacious, and promising MDM2

degrader which warrants further evaluation as a potential new

therapy for the treatment of human leukemia and other types of

human cancer.

EXPERIMENTAL SECTION

■

General. Unless otherwise noted, all purchased reagents were used

as received without further puriﬁcation. ¹H NMR and ¹³C NMR spectra

1

were recorded on a Bruker ADVANCE 400 MHz spectrometer. H

NMR spectra are reported in parts per million (ppm) downﬁeld from

tetramethylsilane (TMS). In reported spectral data, the format (δ)

chemical shift (multiplicity, J values in Hz, integration) was used with

the following abbreviations: s = singlet, d = doublet, t = triplet, q =

quartet, m = multiplet. MS analyses were carried out with a Waters

ultra-performance liquid chromatography (UPLC)−mass spectrom-

eter. The ﬁnal compounds were all puriﬁed by C18 reverse-phase

preparative high-performance liquid chromatography (HPLC) column

with solvent A [0.1% triﬂuoroacetic acid (TFA) in water] and solvent B

(0.1% TFA in MeCN) as eluents. The purity of all the ﬁnal compounds

was conﬁrmed to be >95% by UPLC−MS or UPLC.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((4-((2-

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

carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (1). Reaction 1. 23 (200 mg, 0.73

mmol) was added to a solution of tert-butyl-(4-aminobutyl)carbamate

(207 mg, 1.1 mmol) and DIEA (0.6 mL, 4 mmol) in dimethylforma-

mide (DMF, 2 mL). The reaction was heated to 90 °C for 12 h, and

then the mixture was cooled to room temperature and loaded onto

Celite. The crude residue was puriﬁed by reverse-phase C18 column to

aﬀord tert-butyl-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-

4-yl)amino)butyl)-carbamate without further puriﬁcation. The product

was then dissolved in 5 mL DCM and 1 mL TFA was added to the

reaction. The reaction was stirred for 2 h at room temperature until no

starting material could be detected by HPLC. The solvent was removed

to yield 24a (107 mg, yield: 43%) as a yellow solid.

Figure 14. PD analysis of MDM2, p53, p21, and PARP cleavage protein

levels by western blotting after a single dose of MD-224 administered in

the RS4;11 xenograft mouse model. A single dose of MD-224 (25 mg/

kg, IV) eﬀectively induces MDM2 degradation, p53 activation, and

PAPR cleavage in the xenograft RS4;11 tumors.

graders 1−10 and 12 were obtained by an amide condensation

reaction with MI-1061.

The synthesis of MDM2 degraders 11, 13−17, 21, and 22 is

shown in Scheme 2. Intermediates 26a−26f were synthesized by

Sonogashira coupling reactions followed by removing Boc-

protecting groups under acidic conditions from 25a−25e. The

triple bond in 26b was then reduced by H₂with Pd/C

(palladium on carbon) to form the intermediate 27. MDM2

degraders 11, 13−17, 21, and 22 were then obtained by an

amide condensation reaction with MI-1061 and its enantiomer.

The synthesis of MDM2 degraders containing a VHL ligand is

shown in Scheme 3. The VHL ligands with diﬀerent linkers

(28a−28c) were synthesized as described previously.³³

Compounds 18, 19, and 20 were then obtained by an amide

condensation reaction with MI-1061.

CONCLUSIONS

■

Reaction 2. HATU (13.3 mg, 1.2 equiv) and N,N-diisopropylethyl-

amine (0.026 mL, 0.15 mmol) were stirred with a solution of MI-1061

(20 mg, 0.029 mmol) in 0.5 mL DMF. After 10 min, 24a [0.35 mL, 0.1

M in dimethyl sulfoxide (DMSO)] was added to the reaction. After 30

min, the solvent was removed and the crude was dissolved in 3:1

MeOH/water, acidiﬁed with TFA, and puriﬁed by reverse-phase

preparative HPLC. The puriﬁed fractions were combined, concentrated

in vacuo, re-dissolved in water, frozen, and lyophilized to give 1 (18.5

mg, yield: 71%) as a yellow powder.

In this study, we report our design, synthesis, and evaluation of

the ﬁrst-in-class PROTAC small-molecule MDM2 degraders.

Our study shows that by employing our previous reported

potent MDM2 inhibitor MI-1061 and CRBN ligands

thalidomide and lenalidomide, we have successfully obtained

highly potent and eﬃcacious MDM2 degraders, as exempliﬁed

by MD-224. MD-224 achieves rapid MDM2 protein degrada-

tion even at concentrations below 1 nM with a 2 h treatment in

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Figure 15. In vivo antitumor activity of MI-1061 and MD-224 in the RS4;11 xenograft model in mice: (A) tumor growth. (B) Average tumor volume at

the end of administration.

LC−MS(ESI) m/z (M + H)⁺: 908.32; calcd for C₄₇H₄₅Cl₂FN₇O₇

(M + H)⁺: 908.27; >98% purity.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((2-

(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamoyl)phenyl)-

2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-car-

boxamide (3). 2 (3.5 mg, yield: 17% from MI-1061) was obtained as

white powder using the same condensation method for 1 with

lenalidomide.

¹H NMR (400 MHz, MeOD): δ 7.79−7.68 (m, 3H), 7.63−7.58 (m,

2H), 7.53 (dd, J = 8.3, 2.4 Hz, 1H), 7.49 (ddd, J = 8.5, 7.1, 1.4 Hz, 1H),

7.39−7.32 (m, 1H), 7.17 (t, J = 8.1 Hz, 1H), 7.11 (dd, J = 8.2, 2.0 Hz,

1H), 7.02 (d, J = 8.6 Hz, 1H), 6.98 (dd, J = 6.8, 2.8 Hz, 1H), 6.79 (d, J =

1.9 Hz, 1H), 5.30 (d, J = 10.7 Hz, 1H), 5.03 (dd, J = 12.6, 5.5 Hz, 1H),

4.96 (d, J = 10.8 Hz, 1H), 3.44−3.34 (m, 4H), 2.92−2.79 (m, 2H),

2.78−2.65 (m, 2H), 2.17 (d, J = 14.1 Hz, 1H), 2.13−2.05 (m, 1H),

2.02−1.87 (m, 3H), 1.81−1.69 (m, 6H), 1.62−1.48 (m, 1H), 1.27−

1.16 (m, 2H).

LC−MS(ESI) m/z (M + H)⁺: 823.45; calcd for C₄₃H₃₈Cl₂FN₆O₆

(M + H)⁺: 823.22; >98% purity.

¹H NMR (400 MHz, MeOH-d₄): δ 8.05−7.91 (m, 2H), 7.84−7.63

(m, 5H), 7.60 (t, J = 7.7 Hz, 1H), 7.54 (dd, J = 8.3, 2.5 Hz, 1H), 7.37 (t,

J = 7.5 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.12 (dd, J = 8.3, 2.0 Hz, 1H),

6.80 (d, J = 2.0 Hz, 1H), 5.18 (dd, J = 13.3, 5.1 Hz, 2H), 4.98−4.93 (m,

1H), 4.55 (d, J = 2.0 Hz, 2H), 3.14−3.02 (m, 1H), 2.97−2.87 (m, 1H),

2.83−2.75 (m, 1H), 2.57−2.43 (m, 1H), 2.24−2.16 (m, 1H), 2.15−

2.06 (m, 1H), 2.01−1.85 (m, 3H), 1.78 (t, J = 15.1 Hz, 2H), 1.62 (d, J =

13.2 Hz, 1H), 1.44−1.30 (m, 2H).

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((2-(2-

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

ethoxy)ethoxy)-ethyl)carbamoyl)phenyl)-2″-oxodispiro-

[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-carboxamide (2). 2

(20.8 mg, yield: 75% from MI-1061) was obtained as yellow powder

using the same synthetic strategy described for 1 with 24b.

LC−MS(ESI) m/z (M + H)⁺: 968.35; calcd for C₄₉H₄₉Cl₂FN₇O₉

(M + H)⁺: 968.30; >98% purity.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((2-((2-

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

carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (4). 4 (10.9 mg, yield: 43% from MI-

1061) was obtained as a yellow powder using the synthetic strategy

described for 1 with 24c.

¹H NMR (400 MHz, MeOD): δ 7.79−7.66 (m, 3H), 7.59−7.47 (m,

3H), 7.40−7.27 (m, 2H), 7.19 (t, J = 8.1 Hz, 1H), 7.10 (dd, J = 8.2, 1.8

Hz, 1H), 6.95−6.83 (m, 2H), 6.79 (d, J = 1.9 Hz, 1H), 5.40 (d, J = 10.9

Hz, 1H), 5.03−4.96 (m, 3H), 3.73−3.62 (m, 8H), 3.57−3.50 (m, 2H),

3.42−3.35 (m, 2H), 2.94 (d, J = 8.0 Hz, 1H), 2.85−2.57 (m, 3H), 2.21

(d, J = 13.6 Hz, 1H), 2.07−1.87 (m, 4H), 1.76 (d, J = 11.6 Hz, 2H), 1.53

(dd, J = 26.9, 13.3 Hz, 1H), 1.31−1.10 (m, 2H).

LC−MS(ESI) m/z (M + H)⁺: 880.21; calcd for C₄₅H₄₁Cl₂FN₇O₇

(M + H)⁺: 880.24; >98% purity.

¹H NMR (400 MHz, MeOH-d₄): δ 7.85−7.75 (m, 2H), 7.74−7.61

(m, 3H), 7.53 (dd, J = 8.6, 7.1 Hz, 1H), 7.45 (dd, J = 8.2, 2.4 Hz, 1H),

7.25 (t, J = 7.4 Hz, 1H), 7.19 (d, J = 8.5 Hz, 1H), 7.13−6.98 (m, 3H),

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Figure 16. In vivo antitumor activity of MD-224 in the RS4;11 xenograft model: (A) tumor volume during the administration; (B) monitoring animal

weight as an indication of tolerability. MD-224 eﬀectively induced complete tumor regression or strong tumor growth inhibition in RS4;11 xenograft

models at well tolerated dose schedules.

6.74 (d, J = 2.0 Hz, 1H), 5.05 (dd, J = 12.4, 5.5 Hz, 1H), 4.83−4.63 (m,

2H), 3.69−3.52 (m, 4H), 2.93−2.59 (m, 4H), 2.25−1.90 (m, 5H),

1.79−1.71 (m, 2H), 1.62−1.59 (m, 1H), 1.15−0.99 (m, 2H).

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((3-((2-

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

carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (5). 5 (10.9 mg, yield: 69% from MI-

1061) was obtained as yellow powder using the same synthetic strategy

described for 1 with 24d.

7.10 (dd, J = 8.2, 1.9 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H), 6.98 (dd, J = 6.7,

4.1 Hz, 1H), 6.78 (d, J = 1.9 Hz, 1H), 5.22 (d, J = 9.8 Hz, 1H), 5.04−

4.98 (m, 1H), 4.95 (dd, J = 10.6, 2.9 Hz, 1H), 3.41−3.33 (m, 4H),

2.90−2.61 (m, 4H), 2.17−2.04 (m, 2H), 1.97−1.82 (m, 3H), 1.78−

1.49 (m, 9H), 1.24−1.13 (m, 2H).

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((6-((2-

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

carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (7). 7 (14.3 mg, yield: 53% from MI-

1061) was obtained as a yellow powder using the same synthetic

strategy described for 1 with 24f.

LC−MS(ESI) m/z (M + H)⁺: 894.24; calcd for C₄₆H₄₃Cl₂FN₇O₇

(M + H)⁺: 894.26; >98% purity.

¹H NMR (400 MHz, MeOH-d₄): δ 7.87−7.74 (m, 2H), 7.72 (t, J =

7.4 Hz, 1H), 7.67−7.57 (m, 2H), 7.58−7.44 (m, 2H), 7.37 (t, J = 7.6

Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.12 (dd, J = 8.2, 2.0 Hz, 1H), 7.08−

6.94 (m, 2H), 6.79 (d, J = 2.0 Hz, 1H), 5.31 (d, J = 10.8 Hz, 2H), 5.07−

4.93 (m, 2H), 3.55−3.37 (m, 5H), 2.93−2.77 (m, 3H), 2.78−2.60 (m,

1H), 2.24−2.13 (m, 1H), 2.12−2.01 (m, 1H), 2.03−1.90 (m, 5H),

1.84−1.71 (m, 3H), 1.54 (d, J = 14.0 Hz, 1H), 1.26 (d, J = 21.4 Hz, 2H).

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((5-((2-

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

carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (6). 6 (8.6 mg, yield: 33% from MI-

1061) was obtained as a yellow powder using the same synthetic

strategy described for 1 with 24e.

LC−MS(ESI) m/z (M + H)⁺: 936.27; calcd for C₄₉H₄₉Cl₂FN₇O₇

(M + H)⁺: 936.31; >98% purity.

¹H NMR (400 MHz, MeOD): δ 7.76 (d, J = 8.7 Hz, 2H), 7.71 (t, J =

6.7 Hz, 1H), 7.60 (d, J = 8.7 Hz, 2H), 7.56−7.47 (m, 2H), 7.36 (t, J =

7.0 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.11 (dd, J = 8.2, 1.9 Hz, 1H),

7.02−6.97 (m, 2H), 6.79 (d, J = 1.9 Hz, 1H), 5.33 (d, J = 10.9 Hz, 1H),

5.03 (dd, J = 12.4, 5.3 Hz, 1H), 4.97 (d, J = 10.9 Hz, 1H), 3.40−3.27 (m,

4H), 2.93−2.65 (m, 4H), 2.19 (d, J = 11.6 Hz, 1H), 2.15−2.06 (m,

1H), 2.04−1.89 (m, 3H), 1.78 (d, J = 11.8 Hz, 2H), 1.71−1.59 (m,

4H), 1.55−1.38 (m, 5H), 1.27−1.18 (m, 2H).

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((7-((2-

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

carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (8). 8 (16.3 mg, yield: 60% from MI-

1061) was obtained as yellow powder using the same synthetic strategy

described for 1 with 24g.

LC−MS(ESI) m/z (M + H)⁺: 922.30; calcd for C₄₈H₄₇Cl₂FN₇O₇

(M + H)⁺: 922.29; >98% purity.

¹H NMR (400 MHz, MeOD): δ 7.79−7.67 (m, 3H), 7.63−7.57 (m,

2H), 7.57−7.46 (m, 2H), 7.37−7.30 (m, 1H), 7.16 (t, J = 7.9 Hz, 1H),

M

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Scheme 1. Reaction Conditions: (a) DIPEA, DMF, 80 °C; (b) NaBH(OAc)₃, HOAc, DCE; (c) TFA, DCM, rt; and (d) HATU,

DIPEA, DMF, rt

Scheme 2. Reaction Conditions: (a) Pd(PPh₃)₂Cl₂, CuI, Et₃N, DMF, 80 °C; (b) TFA, DCM; rt; (c) H₂(1 atm), Pd/C, EtOH, rt;

and (d) HATU, DIPEA, DMF, rt

¹H NMR (400 MHz, MeOD): δ 7.80−7.74 (m, 2H), 7.74−7.68 (m,

1H), 7.64−7.59 (m, 2H), 7.55−7.47 (m, 2H), 7.37−7.30 (m, 1H), 7.16

(t, J = 8.1 Hz, 1H), 7.10 (dd, J = 8.2, 1.9 Hz, 1H), 7.01 (s, 1H), 6.99 (d, J

LC−MS(ESI) m/z (M + H)⁺: 950.29; calcd for C₅₀H₅₁Cl₂FN₇O₇

(M + H)⁺: 950.32; >98% purity.

N

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Scheme 3. Reaction Conditions: (a) HATU, DIPEA, DMF, rt; (b) TFA, DCM, rt; and (c) HATU, DIPEA, DMF, rt

= 2.2 Hz, 1H), 6.79 (d, J = 1.9 Hz, 1H), 5.27 (d, J = 10.6 Hz, 1H), 5.03

(dd, J = 12.6, 5.4 Hz, 1H), 4.95 (d, J = 10.7 Hz, 1H), 3.38−3.27 (m,

4H), 2.90−2.64 (m, 4H), 2.20−2.05 (m, 2H), 2.00−1.84 (m, 3H),

1.83−1.71 (m, 2H), 1.67−1.53 (m, 5H), 1.46−1.35 (m, 6H), 1.25−

1.13 (m, 2H).

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((4-((2-

(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)-

carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (12). Reaction 1. Lenalidomide (73

mg, 0.28 mmol) was added to a solution of tert-butyl-(4-hydroxybutyl)-

carbamate (65 mg, 0.34 mmol) in 3 mL 1,2-dichloroethane (DCE) and

stirred for 30 min. Then, sodium triacetoxyborohydride (120 mg, 0.56

mmol) was added to the mixture and reaction was stirred at room

temperature overnight. The reaction was quenched by adding saturated

sodium bicarbonate solution (30 mL), and the product was extracted

with DCM (3 × 30 mL) and the combined organic solution was washed

by brine. The crude product was obtained by removing the solvent in

vacuum and was then dissolved in 10 mL DCM and 2 mL TFA. The

reaction was stirred for 30 min, and the solvent was removed in vacuo.

The residue was puriﬁed by reverse-phase chromatography over a C18

column to yield 24j as a colorless oil.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((2-(2-

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

ethoxy)ethyl)carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-

pyrrolidine-3′,3″-indoline]-5′-carboxamide (9). 9 (15.9 mg, yield:

60% from MI-1061) was obtained as a yellow powder using the same

synthetic strategy described for 1 with 24h.

LC−MS(ESI) m/z (M + H)⁺: 924.20; calcd for C₄₇H₄₅Cl₂FN₇O₈

(M + H)⁺: 924.27; >98% purity.

¹H NMR (400 MHz, MeOD): δ 7.76−7.68 (m, 3H), 7.61−7.51 (m,

3H), 7.47−7.39 (m, 1H), 7.35 (t, J = 7.6 Hz, 1H), 7.19 (t, J = 8.1 Hz,

1H), 7.11 (dd, J = 8.2, 0.9 Hz, 1H), 7.01 (d, J = 8.6 Hz, 1H), 6.91 (t, J =

7.2 Hz, 1H), 6.79 (d, J = 1.9 Hz, 1H), 5.39 (d, J = 10.9 Hz, 1H), 5.03−

4.92 (m, 3H), 3.69 (dt, J = 10.5, 5.0 Hz, 4H), 3.58−3.50 (m, 2H), 3.45

(t, J = 4.9 Hz, 2H), 3.03−2.53 (m, 4H), 2.22 (d, J = 13.4 Hz, 1H), 2.09−

1.86 (m, 4H), 1.78 (d, J = 12.1 Hz, 2H), 1.54 (dd, J = 27.2, 13.5 Hz,

1H), 1.29−1.12 (m, 2H).

Reaction 2. HATU (13.3 mg, 1.2 equiv) and N,N-diisopropylethyl-

amine (0.026 mL, 0.15 mmol) were added to a solution of MI-1061 (20

mg, 0.029 mmol) in 0.5 mL DMF and stirred. After 10 min, 24j (0.35

mL, 0.1 M in DMSO) was added to the reaction. After 30 min, the

solvent was removed and the crude was dissolved in 3:1 MeOH/water,

acidiﬁed with TFA, and puriﬁed by reverse-phase preparative HPLC.

The puriﬁed fractions were combined, concentrated in vacuo,

redissolved in water, frozen, and lyophilized to yield 12 (15.1 mg,

yield: 58%) as a white powder.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((2-(2-

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

ethoxy)ethoxy)ethoxy)-ethyl)carbamoyl)phenyl)-2″-oxodispiro-

[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-carboxamide (10).

10 (11.8 mg, yield: 41% from MI-1061) was obtained as a yellow

powder using the same synthetic strategy described for 1 with 24i.

LC−MS(ESI) m/z (M + H)⁺: 1012.30; calcd for C₅₁H₅₃Cl₂FN₇O₁₀

(M + H)⁺: 1012.32; >98% purity.

LC−MS(ESI) m/z (M + H)⁺: 894.30; calcd for C₄₇H₄₇Cl₂FN₇O₆

(M + H)⁺: 894.29; >98% purity.

¹H NMR (400 MHz, MeOH-d₄): δ 7.80−7.66 (m, 3H), 7.66−7.49

(m, 3H), 7.42−7.34 (m, 1H), 7.32−7.24 (m, 1H), 7.19 (t, J = 8.1 Hz,

1H), 7.12 (dd, J = 8.2, 2.0 Hz, 1H), 7.04 (d, J = 7.5 Hz, 1H), 6.94−6.67

(m, 2H), 5.34 (d, J = 10.9 Hz, 1H), 5.12 (dd, J = 13.3, 5.1 Hz, 1H), 4.98

(d, J = 11.0 Hz, 1H), 4.41−4.11 (m, 2H), 3.47−3.35 (m, 2H), 3.29−

3.19 (m, 2H), 2.90 (ddd, J = 18.4, 13.3, 5.6 Hz, 2H), 2.78 (ddd, J = 17.6,

4.9, 2.6 Hz, 1H), 2.43 (dddd, J = 17.1, 14.0, 10.3, 3.6 Hz, 1H), 2.28−

2.10 (m, 2H), 2.08−1.89 (m, 3H), 1.89−1.61 (m, 6H), 1.52 (t, J = 13.7

Hz, 1H), 1.30−1.19 (m, 2H).

¹H NMR (400 MHz, MeOD): δ 7.76 (d, J = 8.7 Hz, 2H), 7.72 (t, J =

6.6 Hz, 1H), 7.58 (d, J = 8.7 Hz, 2H), 7.53 (dd, J = 8.2, 2.3 Hz, 1H),

7.49−7.43 (m, 1H), 7.36 (t, J = 7.0 Hz, 1H), 7.19 (t, J = 8.1 Hz, 1H),

7.11 (dd, J = 8.2, 1.8 Hz, 1H), 6.99 (d, J = 8.0 Hz, 2H), 6.79 (d, J = 1.9

Hz, 1H), 5.37 (d, J = 10.9 Hz, 1H), 5.02 (ddd, J = 12.3, 5.4, 1.3 Hz, 1H),

4.97 (d, J = 11.0 Hz, 1H), 3.68−3.60 (m, 12H), 3.52 (t, J = 5.3 Hz, 2H),

3.42 (t, J = 5.2 Hz, 2H), 2.95−2.87 (m, 1H), 2.86−2.79 (m, 1H), 2.76−

2.61 (m, 2H), 2.21 (d, J = 13.4 Hz, 1H), 2.13−2.05 (m, 1H), 2.00−1.88

(m, 3H), 1.77 (d, J = 11.7 Hz, 2H), 1.53 (dd, J = 27.1, 13.2 Hz, 1H),

1.29−1.19 (m, 2H).

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((5-(2-

(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)pentyl)carbamoyl)-

phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indo-

line]-5′-carboxamide (13). Reaction 1. CuI (50 mg, 0.25 mmol) and

Pd(Ph₃P)₂Cl₂(90 mg, 0.13 mmol) were added to a solution of tert-

butyl pent-4-yn-1-ylcarbamate (236 mg, 1.29 mmol) and 25b (400 mg,

1.29 mmol) in Et₃N (3 mL) and DMF (3 mL). The mixture was stirred

at 80 °C under an N₂atmosphere overnight. The reaction mixture was

poured into a saturated aqueous solution of NH₄Cl and after separation

of the organic layer the aqueous layer was extracted with EtOAc. The

combined organic layers were washed with brine, dried over Na₂SO₄,

and concentrated in vacuo. The crude product was puriﬁed by ﬂash

chromatography to aﬀord 26b as a white solid (350 mg, yield: 64%).

Reaction 2. Pd/C (20 mg) was added to a solution of 26b (210 mg,

0.5 mmol) in EtOH (5 mL). The reaction was stirred under hydrogen

atmosphere for 2 h. Then, the mixture was ﬁltered through celite and

the solvent was removed in vacuo. The residue was dissolved in 10 mL

DCM and 2 mL TFA. The reaction was stirred for 30 min and then the

solvent was removed in vacuo. The residue was puriﬁed by reverse-

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((5-(2-

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

carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (11). 11 (18.2 mg, yield: 58% from

MI-1061) was obtained as a white powder using the same synthetic

strategy described for MD-222 with 26a.

LC−MS(ESI) m/z (M + H)⁺: 907.27; calcd for C₄₈H₄₆Cl₂FN₆O₇

(M + H)⁺: 907.28; >98% purity.

¹H NMR (400 MHz, MeOD): δ 7.75−7.68 (m, 3H), 7.66−7.63 (m,

2H), 7.63−7.57 (m, 3H), 7.54 (dd, J = 8.2, 2.2 Hz, 1H), 7.37 (t, J = 7.4

Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 7.11 (dd, J = 8.2, 1.4 Hz, 1H), 6.80 (d,

J = 1.5 Hz, 1H), 5.38 (d, J = 10.9 Hz, 1H), 5.09 (dd, J = 12.6, 5.4 Hz,

1H), 4.99 (d, J = 11.0 Hz, 1H), 3.34 (t, J = 7.0 Hz, 2H), 3.11 (dd, J =

17.7, 10.1 Hz, 2H), 2.98−2.79 (m, 2H), 2.79−2.62 (m, 2H), 2.22 (d, J

= 13.9 Hz, 1H), 2.10 (dd, J = 8.6, 3.5 Hz, 1H), 2.04−1.91 (m, 3H),

1.80−1.51 (m, 7H), 1.44 (dd, J = 15.0, 8.0 Hz, 2H), 1.31−1.19 (m,

2H).

O

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phase chromatography over the C18 column to give 27 as colorless oil

(180 mg, yield: 85%)

5.08 (m, 1H), 4.96 (dd, J = 11.0, 3.8 Hz, 1H), 4.55−4.28 (m, 2H),

3.66−3.44 (m, 2H), 2.97−2.84 (m, 2H), 2.84−2.72 (m, 1H), 2.55 (t, J

= 6.4 Hz, 2H), 2.53−2.40 (m, 1H), 2.27−2.10 (m, 2H), 2.04−1.87 (m,

5H), 1.78 (d, J = 12.3 Hz, 2H), 1.62−1.43 (m, 1H), 1.30−1.15 (m,

2H).

Reaction 3. HATU (13.3 mg, 1.2 equiv) and N,N-diisopropylethyl-

amine (0.026 mL, 0.15 mmol) were added to a solution of MI-1061 (20

mg, 0.029 mmol) in 0.5 mL DMF and stirred. After 10 min, 27 (0.35

mL, 0.1 M in DMSO) was added to the reaction. After 30 min, the

solvent was removed and the crude was dissolved in 3:1 MeOH/water,

acidiﬁed with TFA, and puriﬁed by reverse-phase preparative HPLC.

The puriﬁed fractions were combined, concentrated in vacuo, re-

dissolved in water, frozen, and lyophilized to give 13 (17.5 mg, yield:

68%) as a white powder.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((5-(2-

(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)pent-4-yn-1-yl)-

carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (17). 17 (15.4 mg, yield: 60% from

MI-1061) was obtained using the same synthetic strategy described for

MD-224 with 26e.

LC−MS(ESI) m/z (M + H)⁺:889.32; calcd for C₄₈H₄₄Cl₂FN₆O₆(M

+ H)⁺889.27; >98% purity.

LC−MS(ESI) m/z (M + H)⁺: 893.19; calcd for C₄₈H₄₈Cl₂FN₆O₆

(M + H)⁺: 893.30; >98% purity.

¹H NMR (400 MHz, MeOH-d₄): δ 7.83−7.76 (m, 2H), 7.76−7.68

(m, 2H), 7.63−7.57 (m, 3H), 7.54 (dd, J = 8.3, 2.5 Hz, 1H), 7.49 (dd, J

= 7.9, 1.0 Hz, 1H), 7.41−7.34 (m, 1H), 7.19 (t, J = 8.0 Hz, 1H), 7.12

(dd, J = 8.2, 1.9 Hz, 1H), 6.80 (d, J = 1.9 Hz, 1H), 5.36 (d, J = 10.9 Hz,

1H), 5.15 (ddd, J = 13.3, 5.1, 1.5 Hz, 1H), 4.97 (d, J = 11.0 Hz, 1H),

4.57−4.34 (m, 2H), 3.53 (t, J = 6.9 Hz, 2H), 2.90 (ddd, J = 15.3, 10.3,

3.6 Hz, 2H), 2.78 (ddd, J = 17.6, 4.5, 2.3 Hz, 1H), 2.59−2.43 (m, 3H),

2.26−2.12 (m, 2H), 2.04−1.86 (m, 5H), 1.78 (d, J = 11.9 Hz, 2H), 1.53

(dd, J = 27.2, 13.4 Hz, 1H), 1.30−1.18 (m, 2H).

¹H NMR (400 MHz, MeOD): δ 7.78−7.66 (m, 3H), 7.66−7.56 (m,

3H), 7.53 (dd, J = 8.2, 2.5 Hz, 1H), 7.47−7.38 (m, 2H), 7.38−7.32 (m,

1H), 7.17 (t, J = 8.1 Hz, 1H), 7.11 (dd, J = 8.2, 2.0 Hz, 1H), 6.79 (d, J =

1.9 Hz, 1H), 5.29 (d, J = 10.7 Hz, 1H), 5.14 (dd, J = 13.3, 5.2 Hz, 1H),

4.97 (d, J = 10.8 Hz, 1H), 4.46 (dd, J = 5.7, 2.5 Hz, 2H), 3.41−3.33 (m,

2H), 2.96−2.64 (m, 5H), 2.50 (qdd, J = 13.3, 4.6, 2.5 Hz, 1H), 2.22−

2.09 (m, 2H), 2.02−1.84 (m, 3H), 1.79−1.48 (m, 7H), 1.48−1.35 (m,

2H), 1.22 (td, J = 13.7, 4.0 Hz, 2H).

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((5-(2-

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

carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (14). HATU (13.3 mg, 1.2 equiv) and

N,N-diisopropylethylamine (0.026 mL, 0.15 mmol) were added to a

solution of MI-1061 (20 mg, 0.029 mmol) in 0.5 mL DMF and stirred.

After 10 min, 26b (0.35 mL, 0.1 M in DMSO) was added to the

reaction. After 30 min, the solvent was removed and the crude was

dissolved in 3:1 MeOH/water, acidiﬁed with TFA, and puriﬁed by

reverse-phase preparative HPLC. The puriﬁed fractions were

combined, concentrated in vacuo, re-dissolved in water, frozen, and

lyophilized to give MD-224 (8.7 mg, yield: 34%) as a white powder.

LC−MS(ESI) m/z (M + 2H)²⁺: 445.30; calcd for C₄₈H₄₅Cl₂FN₆O₆

(M + 2H)²⁺: 445.14; >98% purity.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((5-

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

phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-

2-yl)amino)-5-oxopentyl)carbamoyl)phenyl)-2″-oxodispiro-

[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-carboxamide (18).

28a−28c were synthesized as previously reported.²⁰Then, 18 (21.3

mg, yield: 67% from MI-1061) was synthesized with the same synthetic

strategy described for 13 with 28a.

LC−MS(ESI) m/z (M + H)⁺:1107.41, 5.765 min; calcd for

C₅₈H₆₆Cl₂FN₈O₇S (M + H)⁺: 1107.41; >95% purity.

¹H NMR (400 MHz, MeOH-d₄) δ 9.01 (s, 1H), 7.88−7.74 (m, 2H),

7.72 (t, J = 6.5 Hz, 1H), 7.69−7.60 (m, 2H), 7.53 (dd, J = 8.4, 2.6 Hz,

1H), 7.47−7.30 (m, 5H), 7.19 (t, J = 8.1 Hz, 1H), 7.14−7.05 (m, 1H),

6.80 (d, J = 1.9 Hz, 1H), 5.38 (d, J = 11.0 Hz, 1H), 5.02−4.96 (m, 2H),

4.65−4.51 (m, 2H), 4.43 (s, 1H), 3.88 (d, J = 11.2 Hz, 1H), 3.74 (dd, J

= 11.0, 3.9 Hz, 1H), 3.37 (t, J = 6.5 Hz, 2H), 2.95 (d, J = 8.1 Hz, 1H),

2.49 (s, 3H), 2.37−2.12 (m, 4H), 2.06−1.84 (m, 4H), 1.78 (d, J = 11.8

Hz, 2H), 1.72−1.59 (m, 4H), 1.55−1.42 (m, 4H), 1.36−1.14 (m, 2H),

1.03 (s, 9H).

¹H NMR (400 MHz, MeOH-d₄): δ 7.83−7.62 (m, 4H), 7.56 (dd, J =

8.3, 2.5 Hz, 1H), 7.54−7.47 (m, 2H), 7.47−7.32 (m, 3H), 7.19 (ddd, J

= 8.9, 7.6, 3.3 Hz, 1H), 7.12 (dd, J = 8.2, 2.0 Hz, 1H), 6.80 (d, J = 1.9 Hz,

1H), 5.30 (d, J = 10.8 Hz, 1H), 5.13 (ddd, J = 13.0, 7.4, 5.2 Hz, 1H),

4.98 (d, J = 10.9 Hz, 1H), 4.49−4.25 (m, 2H), 3.57 (t, J = 6.6 Hz, 2H),

2.97−2.83 (m, 2H), 2.83−2.73 (m, 1H), 2.68−2.42 (m, 3H), 2.28−

2.13 (m, 2H), 2.13−1.84 (m, 5H), 1.84−1.68 (m, 2H), 1.57 (ddd, J =

16.3, 6.2, 3.0 Hz, 1H), 1.34−1.19 (m, 2H).

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((2-(2-

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

phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-

2-yl)amino)-3-oxopropoxy)ethoxy)ethyl)carbamoyl)phenyl)-2″-

oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-carbox-

amide (19). 19 (25.0 mg, yield: 75% from MI-1061) was obtained

using the same synthetic strategy described for 18 with 28b.

LC−MS(ESI) m/z (M + H)⁺:1167.64, 5.656 min; calcd for

C₆₀H₇₀Cl₂FN₈O₉S (M + H)⁺: 1167.44; >95% purity.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((4-(2-

(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)but-3-yn-1-yl)-

carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (15). 15 (16.9 mg, yield: 67% from

MI-1061) was obtained as a white powder using the same synthetic

strategy described for MD-224 with 26c.

LC−MS(ESI) m/z (M + H)⁺: 875.36; calcd for C₄₇H₄₂Cl₂FN₆O₆

(M + H)⁺: 875.25; >98% purity.

¹H NMR (400 MHz, MeOH-d₄): δ 8.97 (s, 1H), 7.86−7.76 (m, 2H),

7.71 (dd, J = 10.4, 3.9 Hz, 1H), 7.66−7.58 (m, 2H), 7.53 (dd, J = 8.2,

2.5 Hz, 1H), 7.47−7.30 (m, 5H), 7.19 (t, J = 8.1 Hz, 1H), 7.10 (dd, J =

8.2, 1.9 Hz, 1H), 6.79 (d, J = 1.9 Hz, 1H), 5.38 (d, J = 10.9 Hz, 1H),

5.00−4.95 (m, 2H), 4.64 (s, 1H), 4.57 (dd, J = 11.9, 4.6 Hz, 1H), 4.42

(s, 1H), 3.86 (d, J = 11.2 Hz, 1H), 3.76−3.53 (m, 11H), 2.94 (d, J = 8.9

Hz, 1H), 2.62−2.40 (m, 5H), 2.26−2.09 (m, 2H), 2.04−1.86 (m, 4H),

1.78 (d, J = 11.9 Hz, 2H), 1.63−1.38 (m, 4H), 1.32−1.17 (m, 2H), 1.01

(s, 9H).

¹H NMR (400 MHz, MeOH-d₄): δ 7.89−7.80 (m, 2H), 7.76−7.69

(m, 2H), 7.69−7.61 (m, 2H), 7.59 (dt, J = 7.7, 0.8 Hz, 1H), 7.56 (dt, J =

8.3, 2.6 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.42−7.30 (m, 1H), 7.18 (td, J

= 8.1, 1.1 Hz, 1H), 7.11 (dt, J = 8.2, 1.7 Hz, 1H), 6.80 (d, J = 1.9 Hz,

1H), 5.43−5.27 (m, 1H), 5.12−4.97 (m, 2H), 4.32 (d, J = 7.8 Hz, 2H),

3.73−3.56 (m, J = 6.4 Hz, 2H), 3.02−2.76 (m, 4H), 2.67 (ddt, J = 21.0,

17.7, 3.4 Hz, 1H), 2.20 (d, J = 13.9 Hz, 1H), 2.12−1.85 (m, 5H), 1.78

(d, J = 12.9 Hz, 2H), 1.63−1.49 (m, 1H), 1.31−1.16 (m, 2H).

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((5-(2-

(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)pent-4-yn-1-yl)-

carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (16). 16 (18.1 mg, yield: 71% from

MI-1061) was obtained using the same synthetic strategy described for

MD-224 with 26d.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-(((S)-

14-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)-

ethyl)carbamoyl)pyrrolidine-1-carbonyl)-15,15-dimethyl-12-oxo-

3,6,9-trioxa-13-azahexadecyl)carbamoyl)phenyl)-2″-oxodispiro-

[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-carboxamide (20).

20 (23.9 mg, yield: 69% from MI-1061) was obtained using the same

synthetic strategy described for 18 with 28c.

LC−MS(ESI) m/z (M + H)⁺:889.38; calcd for C₄₈H₄₄Cl₂FN₆O₆(M

+ H)⁺889.27; >98% purity.

LC−MS(ESI) m/z (M

+ : 606.31; calcd for

2H)²⁺

C₆₂H₇₅Cl₂FN₈O₁₀S (M + 2H)²⁺: 606.23; >95% purity.

¹H NMR (400 MHz, MeOH-d₄): δ 7.82−7.74 (m, 2H), 7.71 (ddd, J

= 6.4, 4.0, 1.5 Hz, 1H), 7.66 (dd, J = 7.9, 4.2 Hz, 1H), 7.61−7.46 (m,

4H), 7.38 (dt, J = 9.2, 8.4 Hz, 2H), 7.19 (t, J = 8.0 Hz, 1H), 7.15−7.06

(m, 1H), 6.80 (d, J = 1.9 Hz, 1H), 5.35 (dd, J = 10.8, 8.6 Hz, 1H), 5.24−

¹H NMR (400 MHz, MeOH-d₄): δ 8.94 (s, 1H), 7.87−7.76 (m, 2H),

7.76−7.66 (m, 1H), 7.66−7.57 (m, 2H), 7.54 (dd, J = 8.2, 2.5 Hz, 1H),

7.46−7.32 (m, 5H), 7.18 (t, J = 8.1 Hz, 1H), 7.11 (dd, J = 8.2, 2.0 Hz,

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1H), 6.79 (d, J = 1.9 Hz, 1H), 5.36 (d, J = 10.9 Hz, 1H), 5.04−4.95 (m,

2H), 4.64 (s, 1H), 4.57 (dd, J = 9.9, 6.7 Hz, 1H), 4.43 (d, J = 1.8 Hz,

1H), 3.86 (d, J = 11.0 Hz, 1H), 3.76−3.53 (m, 15H), 2.94 (d, J = 8.7 Hz,

1H), 2.62−2.36 (m, 5H), 2.28−2.07 (m, 2H), 2.03−1.83 (m, 4H), 1.78

(d, J = 12.1 Hz, 2H), 1.61−1.44 (m, 4H), 1.29−1.18 (m, 2H), 1.02 (s,

9H).

blotted into polyvinylidene ﬂuoride (PVDF) membranes. Antibodies

for immunoblotting were MDM2 (SMP14) and p53 (DO-1)

purchased from Santa Cruz.

For apoptosis assays, apoptosis in RS4;11 cells was analyzed by ﬂow

cytometry with PI/annexin V-FLUOS double staining (Alexa Fluor 488

Annexin V/Dead Cell Apoptosis Kit, Invitrogen) following manufac-

turer’s protocols after treatments for 24 h as indicated.

(3′S,4′R,5′S)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((5-(2-

(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)pent-4-yn-1-yl)-

carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (21). 21 (27.9 mg, yield: 87%) was

obtained as white powder using the same synthetic strategy described

for MD-224 from the enantiomer of MI-1061,¹¹with 26b.

Eﬃcacy and PDs Studies in the RS4;11 Xenograft Model in

Mice. To develop xenograft tumors, 5 × 10⁶RS4;11 cells with 50%

Matrigel were injected subcutaneously on the dorsal side of severe

combined immunodeﬁcient mice, obtained from Charles River Co.,

one tumor per mouse. When tumors reached ∼100 mm³, mice were

randomly assigned to treatment and vehicle control groups. Animals

were monitored daily for any signs of toxicity, weighed 2−3 times per

week during the treatment, and weighed at least weekly after the

treatment ended. Tumor size was measured 2−3 times per week by

electronic calipers during the treatment period and at least weekly after

the treatment was ended. Tumor volume was calculated as V = L × W²/

2, where L is the length and W is the width of the tumor.

For PD analysis, resected RS4;11 xenograft tumor tissues were

ground into powder in liquid nitrogen and lysed in lysis buﬀer [1%

CHAPS, 150 mM NaCl, 20 mM Tris-HCl, 1 mM. ethylenediaminete-

traacetic acid, 1 mM EGTA, and COMPLETE proteinase inhibitor

(Roche)]. Whole tumor lysates were separated on 4−20% Novex gels.

The separated proteins were transferred to a PVDF membrane for

immunoblotting. The following antibodies were used: antibodies for

MDM2 (SMP14), p53 (DO-1), and GAPDH (FL-335) from Santa

Cruz Biotechnology; p21 (12D1), PARP (46D11), and caspase-3

(8G10) from Cell Signaling Technology. The secondary antibody used

was horseradish peroxidase-conjugated goat anti-rabbit (Thermo

Scientiﬁc). The Bio-Rad Clarity Western Enhanced Chemilumines-

cence Substrates and HyBlot Chemiluminescence ﬁlm were used for

signal development and detection using a SRX-101A tabletop processor

(Konica Minolta).

LC−MS(ESI) m/z (M + H)⁺: 903.35; calcd for C₄₉H₄₆Cl₂FN₆O₆

(M + H)⁺: 903.28; >98% purity.

¹H NMR (400 MHz, MeOH-d₄): δ 7.75−7.68 (m, 3H), 7.64 (ddd, J

= 12.7, 7.6, 1.1 Hz, 1H), 7.56 (dd, J = 8.2, 2.6 Hz, 1H), 7.52−7.46 (m,

2H), 7.46−7.42 (m, 1H), 7.41−7.31 (m, 2H), 7.19 (tdd, J = 8.2, 3.5, 1.1

Hz, 1H), 7.12 (dd, J = 8.2, 2.0 Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 5.36

(dd, J = 10.8, 1.4 Hz, 1H), 5.13 (ddd, J = 13.1, 7.8, 5.1 Hz, 1H), 4.99

(dd, J = 10.9, 1.7 Hz, 1H), 4.41−4.31 (m, 2H), 3.57 (t, J = 6.6 Hz, 2H),

2.98−2.84 (m, 2H), 2.84−2.70 (m, 1H), 2.62−2.43 (m, 3H), 2.29−

2.11 (m, 2H), 2.05−1.87 (m, 5H), 1.79 (d, J = 13.2 Hz, 2H), 1.62−1.48

(m, 1H), 1.25 (t, J = 13.5 Hz, 2H).

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-((5-(2-

(1-methyl-2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)pent-4-yn-

1-yl)carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrroli-

dine-3′,3″-indoline]-5′-carboxamide (22). 22 (26 mg, yield: 98%

from MI-1061) was obtained as a white powder using the same

synthetic strategy described for MD-224 with 26f.

LC−MS(ESI) m/z (M + H)⁺: 903.35; calcd for C₄₉H₄₆Cl₂FN₆O₆

(M + H)⁺: 903.28; >98% purity.

¹H NMR (400 MHz, MeOH-d₄): δ 7.76−7.68 (m, 3H), 7.68−7.62

(m, 1H), 7.56 (ddd, J = 8.3, 2.7, 1.3 Hz, 1H), 7.53−7.44 (m, 3H), 7.44−

7.33 (m, 2H), 7.23−7.15 (m, 1H), 7.11 (dd, J = 8.2, 2.0 Hz, 1H), 6.79

(d, J = 1.9 Hz, 1H), 5.32 (d, J = 10.8 Hz, 1H), 5.15 (dt, J = 13.5, 4.7 Hz,

1H), 4.98 (d, J = 10.8 Hz, 1H), 4.41−4.27 (m, 2H), 3.57 (t, J = 6.7 Hz,

2H), 3.09 (d, J = 5.9 Hz, 3H), 2.96−2.89 (m, 2H), 2.58 (t, J = 6.5 Hz,

2H), 2.55−2.38 (m, 1H), 2.29−2.08 (m, 2H), 2.04−1.87 (m, 5H), 1.78

(d, J = 13.2 Hz, 2H), 1.62−1.49 (m, 1H), 1.33−1.14 (m, 3H).

Determination of Binding Aﬃnities to MDM2 Proteins. The

binding aﬃnity of MDM2 inhibitors or degraders to MDM2 protein

was determined by an optimized, sensitive, and quantitative

ﬂuorescence polarization-based (FP-based) binding assay, as described

previously. A recombinant human MDM2 protein (residues 1−118)

and a FAM-tagged p53-based peptide as the ﬂuorescent probe were

used in the binding assays.¹³

All the animal experiments were performed under an approved

animal protocol (Protocol ID: PRO00007499, PI, Shaomeng Wang) by

the Institutional Animal Care & Use Committee of the University of

Michigan.

Molecular Modeling. Co-crystal structures of MDM2/MI-77301

(PDB entry: 5TRF) were used to model the binding poses of MI-1061

with MDM2. We extracted chain A of MDM2 from the crystal

structures and used the “protonate 3D” module in MOE³⁴to protonate

MDM2 at the physiological pH = 7.0 condition. The structure of MI-

1061 was drawn and optimized using the MOE program. The

GOLD^35,36program (version 5.2) was used in the docking simulation.

The binding site was centered at Ile99 in MDM2 with a radius of 13 Å to

cover the entire binding pocket of MDM2 structures. The default

parameters for the genetic algorithm run and the GoldScore were used

to generate and evaluate the docked poses. After analyzing the 20

binding poses of MI-1061 to MDM2, we selected the highest ranked

pose for the MDM2 structure as the binding model of MI-1061. Figures

were prepared using the PyMOL program (www.pymol.org).

Cell Growth Inhibition, Apoptosis Analysis, and Western

Blotting Assays. The human leukemia RS4;11 cell line (CRL-1873)

was purchased from the American Type Culture Collection (ATCC).

In all experiments, RS4;11 human leukemia cells were used within three

months of thawing fresh vials. RS4;11 cells were cultured in RPMI 1640

media supplemented with 10% fetal bovine serum and 1% penicillin−

streptomycin at 37 °C in a humidiﬁed atmosphere containing 5% CO₂.

In cell growth experiments, cells were seeded in 96-well cell culture

plates at a density of 10 000−20 000 cells/well in 100 μL of culture

medium. Each compound tested was serially diluted in the appropriate

medium, and 100 μL of the diluted solution containing the tested

compound was added to the appropriate wells of the cell plate. After

addition of the tested compound, the cells were incubated for 4 days at

37 °C in an atmosphere of 5% CO₂. Cell growth was evaluated by a

lactate dehydrogenase-based WST-8 assay (Dojindo Molecular

Technologies) using a Tecan Inﬁnite M1000 multimode microplate

reader (Tecan, Morrisville, NC). The WST-8 reagent was added to the

plate, incubated for at least 1 h, and read at 450 nm. The readings were

normalized to the DMSO-treated cells, and the IC₅₀was calculated by

nonlinear regression analysis using GraphPad Prism 6 software.

For Western blot analysis, 2 × 10⁶cells/well were treated with

compounds at the indicated concentrations for various times. Cells

were collected and lysed in RIPA buﬀer containing protease inhibitors.

An amount of 20 μg of lysate was run in each lane of a PAGE-SDS and

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.8b00909.

■

S

Evaluation of the eﬀect of cereblon ligand lenalidomide

on the activity of the MDM2 inhibitor and degraders in

MV4;11 cells; qRT-PCR analysis mRNA levels of p53

target genes and TP53 after treatment with the MDM2

inhibitor, MI-1061, and the MDM2 degraders MD-222

1

and MD-224 in MV4;11 cells; H NMR spectrum for

MD-224; and UPLC−MS results for MD-224 (PDF)

PDB coordinates of a computational models of MI-1224

in a complex with MDM2, and molecular string ﬁle

(PDB)

Q

DOI: 10.1021/acs.jmedchem.8b00909

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(6) Wu, X.; Bayle, J. H.; Olson, D.; Levine, A. J. The p53-mdm-2

autoregulatory feedback loop. Genes Dev. 1993, 7, 1126−1132.

(7) Juven-Gershon, T.; Oren, M. Mdm2: the ups and downs. Mol.

Med. 1999, 5, 71−83.

Molecular formula string ﬁle (CSV) and PDB coordinates

of the binding model of MI-1061 in complex with MDM2

protein (PDB)

(8) Momand, J.; Zambetti, G. P.; Olson, D. C.; George, D.; Levine, A.

J. The mdm-2 oncogene product forms a complex with the p53 protein

and inhibits p53-mediated transactivation. Cell 1992, 69, 1237−1245.

(9) Ding, Q.; Zhang, Z.; Liu, J.-J.; Jiang, N.; Zhang, J.; Ross, T. M.;

Chu, X.-J.; Bartkovitz, D.; Podlaski, F.; Janson, C.; Tovar, C.; Filipovic,

Z. M.; Higgins, B.; Glenn, K.; Packman, K.; Vassilev, L. T.; Graves, B.

Discovery of RG7388, a potent and selective p53-MDM2 inhibitor in

clinical development. J. Med. Chem. 2013, 56, 5979−5983.

AUTHOR INFORMATION

Corresponding Author

*E-mail: shaomeng@umich.edu.

ORCID

■

Shaomeng Wang: 0000-0002-8782-6950

Author Contributions

(10) Holzer, P.; Masuya, K.; Furet, P.; Kallen, J.; Valat-Stachyra, T.;

Ferretti, S.; Berghausen, J.; Bouisset-Leonard, M.; Buschmann, N.;

^⊥Y.L., J.Y., and A.A. contributed equally.

̀

Pissot-Soldermann, C.; Rynn, C.; Ruetz, S.; Stutz, S.; Chene, P.; Jeay, S.;

Funding

Gessier, F. Discovery of a dihydroisoquinolinone derivative (NVP-

CGM097): A highly potent and selective MDM2 inhibitor undergoing

Phase 1 clinical trials in p53wt tumors. J. Med. Chem. 2015, 58, 6348−

6358.

This study was supported in part by the National Cancer

Institute, NIH (R01CA219345 to S.W.), and the P30 Cancer

Center Core grant (CA046592) from the National Cancer

Institute, NIH and Oncopia Therapeutics LLC (to S.W.).

(11) Aguilar, A.; Lu, J.; Liu, L.; Du, D.; Bernard, D.; McEachern, D.;

Przybranowski, S.; Li, X.; Luo, R.; Wen, B.; Sun, D.; Wang, H.; Wen, J.;

Wang, G.; Zhai, Y.; Guo, M.; Yang, D.; Wang, S. Discovery of 4-

((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-fluorophenyl)-1′-ethyl-2″-

oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamido)bicyclo[2.2.2]octane-1-carboxylic Acid (AA-115/APG-

115): A Potent and Orally Active Murine Double Minute 2 (MDM2)

Inhibitor in Clinical Development. J. Med. Chem. 2017, 60, 2819−2839.

(12) Vu, B.; Wovkulich, P.; Pizzolato, G.; Lovey, A.; Ding, Q.; Jiang,

N.; Liu, J.-J.; Zhao, C.; Glenn, K.; Wen, Y.; Tovar, C.; Packman, K.;

Vassilev, L.; Graves, B. Discovery of RG7112: A small-molecule MDM2

inhibitor in clinical development. ACS Med. Chem. Lett. 2013, 4, 466−

469.

(13) Wang, S.; Sun, W.; Zhao, Y.; McEachern, D.; Meaux, I.; Barriere,

C.; Stuckey, J. A.; Meagher, J. L.; Bai, L.; Liu, L.; Hoffman-Luca, C. G.;

Lu, J.; Shangary, S.; Yu, S.; Bernard, D.; Aguilar, A.; Dos-Santos, O.;

Besret, L.; Guerif, S.; Pannier, P.; Gorge-Bernat, D.; Debussche, L.

SAR405838: An optimized inhibitor of MDM2-p53 interaction that

induces complete and durable tumor regression. Cancer Res. 2014, 74,

5855−5865.

(14) Sun, D.; Li, Z.; Rew, Y.; Gribble, M.; Bartberger, M. D.; Beck, H.

P.; Canon, J.; Chen, A.; Chen, X.; Chow, D.; Deignan, J.; Duquette, J.;

Eksterowicz, J.; Fisher, B.; Fox, B. M.; Fu, J.; Gonzalez, A. Z.; Gonzalez-

Lopez De Turiso, F.; Houze, J. B.; Huang, X.; Jiang, M.; Jin, L.; Kayser,

F.; Liu, J. J.; Lo, M.-C.; Long, A. M.; Lucas, B.; McGee, L. R.; McIntosh,

J.; Mihalic, J.; Oliner, J. D.; Osgood, T.; Peterson, M. L.; Roveto, P.;

Saiki, A. Y.; Shaffer, P.; Toteva, M.; Wang, Y.; Wang, Y. C.; Wortman,

S.; Yakowec, P.; Yan, X.; Ye, Q.; Yu, D.; Yu, M.; Zhao, X.; Zhou, J.; Zhu,

J.; Olson, S. H.; Medina, J. C. Discovery of AMG 232, a potent,

selective, and orally bioavailable MDM2-p53 inhibitor in clinical

development. J. Med. Chem. 2014, 57, 1454−1472.

Notes

The authors declare the following competing ﬁnancial

interest(s): The University of Michigan has ﬁled a number of

patent applications on these MDM2 degraders reported in this

study, which have been licensed to Oncopia Therapeutics, LLC.

S. Wang, Y. Li, J. Yang, A. Aguilar, D. McEachern are co-

inventors on one or more of these patents, and receive royalties

on these patents from the University of Michigan. S. Wang is a

co-founder and a paid consultant of Oncopia Therapeutics,

LLC. A. Aguilar is a paid consultant of Oncopia Therapeutics,

LLC. The University of Michigan and S. Wang own stock in

Oncopia.

ACKNOWLEDGMENTS

■

We are grateful for the ﬁnancial support from the National

Cancer Institute, NIH (1R01CA208267 to S.W.), Oncopia

Therapeutics LLC (to S.W.) and the University of Michigan

Rogel Cancer Center support grant from the National Cancer

Institute, NIH (P30 CA046592).

ABBREVIATIONS

■

MDM2, human murine double minute 2; PROTAC, proteolysis

targeting chimera; BET, bromodomain and extra-terminal;

ERRα, estrogen-related receptor α; VHL-1, von Hippel−Lindau

protein 1; CRBN, cereblon; PUMA, p53 upregulated modulator

of apoptosis; PD, pharmacodynamic; DIPEA, N,N-diisopropy-

lethylamine; DMF, Dimethylformamide; HOAc, acetic acid;

DCE, 1,2-dichloroethane; TFA, triﬂuoroacetic acid; HATU, 1-

[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-

pyridinium 3-oxid hexaﬂuorophosphate; DCM, dichlorome-

thane; TMS, tetramethylsilane; Pd/C, palladium on carbon

(15) Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.;

Filipovic, Z.; Kong, N.; Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi,

N.; Liu, E. A. In vivo activation of the p53 pathway by small-molecule

antagonists of MDM2. Science 2004, 303, 844−848.

(16) Zhao, Y.; Aguilar, A.; Bernard, D.; Wang, S. Small-molecule

inhibitors of the MDM2-p53 protein-protein interaction (MDM2

Inhibitors) in clinical trials for cancer treatment. J. Med. Chem. 2015, 58,

1038−1052.

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DOI: 10.1021/acs.jmedchem.8b00909

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery of MD-224 as a First-in-Class, Highly Potent, and Efficacious Proteolysis Targeting Chimera Murine Double Minute 2 Degrader Capable of Achieving Complete and Durable Tumor Regression

DOI: 10.1021/acs.jmedchem.8b00909

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Full text of DOI:10.1021/acs.jmedchem.8b00909

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