Strategies toward Discovery of Potent and Orally Bioavailable Proteolysis Targeting Chimera Degraders of Androgen Receptor for the Treatment of Prostate Cancer

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Strategies toward Discovery of Potent and Orally Bioavailable

Proteolysis Targeting Chimera Degraders of Androgen Receptor for

the Treatment of Prostate Cancer

Xin Han, Lijie Zhao, Weiguo Xiang, Chong Qin, Bukeyan Miao, Donna McEachern, Yu Wang,

Hoda Metwally, Lu Wang, Aleksas Matvekas, Bo Wen, Duxin Sun, and Shaomeng Wang *

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ABSTRACT: Proteolysis targeting chimera (PROTAC) small-

molecule degraders have emerged as a promising new type of

therapeutic agents, but the design of PROTAC degraders with

excellent oral pharmacokinetics is a major challenge. In this study,

we present our strategies toward the discovery of highly potent

PROTAC degraders of androgen receptor (AR) with excellent oral

pharmacokinetics. Employing thalidomide to recruit cereblon/

cullin 4A E3 ligase and through the rigidiﬁcation of the linker, we

discovered highly potent AR degraders with good oral

pharmacokinetic properties in mice with ARD-2128 being the

best compound. ARD-2128 achieves 67% oral bioavailability in

mice, eﬀectively reduces AR protein and suppresses AR-regulated genes in tumor tissues with oral administration, leading to the

eﬀective inhibition of tumor growth in mice without signs of toxicity. This study supports the development of an orally active

PROTAC AR degrader for the treatment of prostate cancer and provides insights and guidance into the design of orally active

PROTAC degraders.

molecule degraders can achieve a more complete target

inhibition than traditional small-molecule inhibitors through

reducing target protein levels in disease tissues and are predicted

to be therapeutically more eﬃcacious.³⁷⁻⁴³To date, a number of

classes of PROTAC AR degraders have been reported (Figure

1).⁴⁴⁻⁵⁹In 2008, Crews et al. reported a class of AR degraders

designed using a ligand to recruit the MDM2 E3 ligase.⁴⁴In

2017, Naito et al. reported speciﬁc and non-genetic IAP-

dependent protein eraser (SNIPER) compounds that degrade

AR using a ligand to recruit the cellular inhibitor of apoptosis

protein 1 (cIAP1) E3 ligase.⁴⁹Salami et al. reported ARCC-4 as

a potent AR degrader that operates by recruiting the VHL-1/

cullin 2 E3 ligase complex.⁵²Our laboratory has reported the

discovery of potent AR degraders by recruiting the VHL-1/

cullin 2 E3 ligase complex.⁵⁴⁻⁵⁷Takwale et al. reported the

discovery of TD-802 as a potent AR degrader with good liver

microsomal stability and pharmacokinetic properties.⁶⁰Scien-

tists from Arvinas has disclosed a number of classes of PROTAC

INTRODUCTION

■

Prostate cancer is the second most common malignancy among

men in the US and the second most common cause of cancer-

related death worldwide. The development of eﬀective

therapeutics for the treatment of prostate cancer has been a

major focus of scientiﬁc research.¹⁻⁵The androgen receptor

(AR) and its downstream signaling play a critical role in the

development and progression of both localized and metastatic

prostate cancer.⁶⁻¹⁰A number of AR-targeted agents (Figure 1)

have been developed to treat advanced prostate cancer,

including abiraterone (1), which blocks androgen synthesis

and enzalutamide (2) and apalutamide (3, ARN-509), which

bind to AR and function as AR antagonists,¹¹⁻¹⁴but resistance

to these AR targeted agents typically develops within 18 months

of treatment. In the majority of prostate cancers resistant to

these AR targeted agents, AR signaling remains functionally

active and continues to play a key role in tumor progression. AR

gene ampliﬁcation, point mutations, and alternate splicing¹⁵⁻¹⁸

have been identiﬁed as some of the major mechanisms of

resistance to these agents targeting AR. There is an urgent need

to develop new therapeutic strategies targeting AR for the

treatment of prostate cancer, particularly metastatic castration-

resistant prostate cancer (mCRPC).^10,19,20

Received: May 16, 2021

In recent years, protein degradation based on the proteolysis

targeting chimera (PROTAC) strategy has emerged as a

promising approach to drug discovery.²¹⁻³⁶PROTAC small-

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Figure 1. Representative AR antagonists and PROTAC AR degraders.

AR degraders in patents and patent applications⁶¹⁻⁶³and has

advanced ARV-110 (9) into Phase I clinical development,^58,59

whose chemical structure was recently disclosed.⁶⁴

A PROTAC degrader molecule consists of a ligand for the

target protein tethered through a linker to a second ligand, which

binds to and recruits an E3 ligase, and consequently, it has a

much higher molecular weight than a typical oral drug. Despite

the potential promise of PROTAC degraders as a completely

new class of therapeutics, the design of PROTAC degraders to

achieve excellent oral pharmacokinetics has been a major

challenge for the entire PROTAC ﬁeld. In the present study, we

present our strategies toward discovering potent PROTAC

degraders of AR with excellent oral pharmacokinetics in mice.

Our eﬀorts yielded a number of highly potent PROTAC AR

degraders with excellent oral pharmacokinetics.

RESULTS AND DISCUSSION

■

Design and Synthesis of New AR Degraders with a

Cereblon Ligand. We previously reported a series of highly

potent AR degraders, as exempliﬁed by ARD-61, which employ

VHL E3 ligands.⁵⁴⁻⁵⁷ARD-61 has a molecular weight of 1095.8,

a large polar surface area (calculated tPSA = 189.2 Å²), and high

lipophilicity (calculated CLogP = 8.2) (Figure 2), which are

properties all detrimental to oral bioavailability. While ARD-61

potently and eﬀectively induces AR degradation in AR-positive

(AR+) prostate cancer cells in vitro and in prostate cancer

xenograft tumor tissues in vivo by intraperitoneal injection, it has

no oral bioavailability in mice (data not shown).

Figure 2. Design of new AR degraders employing a cereblon ligand.

obtain potential orally bioavailable AR degraders, we decided to

employ thalidomide, which is a cereblon ligand with a low

molecular weight (258) and excellent druglike properties.

We ﬁrst designed and synthesized compounds 10−15 (Table

1) using the AR antagonist employed in ARD-61 (shown in red

in Figure 2), tethered to thalidomide through a linear, ﬂexible

linker. The aim was to identify the linker length optimal for AR

degradation. We evaluated these compounds by Western

blotting to determine their ability to reduce the level of AR

protein in the VCaP prostate cancer cell line, which has a very

In the design of ARD-61, we employed a VHL ligand, which

has a molecular weight of nearly 500 and is a peptidomimetic. To

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Table 1. Determination of Optimal Linker Lengths Using AR

Degraders with Flexible and Semi-Rigid Linkers

Table 2. Investigation of Diﬀerent Linking Positions of the

Cereblon ligand

a

All the data are the average of three independent experiments.

degraders is critical for the potent and eﬀective reduction of AR

protein.

Compounds 12, 13, 16, and 17 potently and eﬀectively

reduce AR protein in the VCaP cell line. We next assessed the

plasma exposure for compounds 12, 13, 16, and 17 in mice with

a single oral administration at 10 mg/kg with the data

summarized in Table 3. The data show that while compounds

a

All the data were the average of three independent experiments.

high expression level of wild-type AR protein due to AR gene

ampliﬁcation. The AR protein levels were quantiﬁed by

densitometry, providing the data summarized in Table 1.

Compound 10, with a linker of three methylene groups,

eﬀectively reduces the AR protein level at 0.1 and 1 μM by 63

and 70%, respectively, but has a minimal eﬀect on the AR protein

at 1 and 10 nM. Compound 11, containing a linker with four

methylene groups, eﬀectively reduces the AR protein by 42% at

1 nM and by 76% at 10 nM. Interestingly, compound 11 at 0.1

and 1 μM reduces the AR protein level by 51 and 21%,

respectively, which are less than the reduction at 10 nM. This

reveals the classical “hook” eﬀect that is often observed with

PROTAC degraders.⁶⁰Compounds 12−15 containing a linker

with 5−8 methylene groups all eﬀectively reduce AR protein at

all the four concentrations tested (1, 10, 100, and 1000 nM).

Next, we synthesized compounds 16−19, which employ

linkers with more conformational constraints and improved

solubility. Western blotting showed that compounds 16−19 are

all very potent and eﬀective in reducing the AR protein level in

the VCaP cells, indicating that soluble and conformationally

constrained linkers can be employed for the design of potent AR

degraders.

Table 3. Assessment of Oral Exposure of Compounds 12, 13,

16, and 17 in Mice

a

plasma drug concentration (mean SD, ng/ml)

compound

1 h

3 h

6 h

12

13

16

17

111.2 41.2

16.9 6.9

1242.1 467.2

267.0 104.5

55.9 42.0

8.0 7.7

919.3 256.7

254.5 39.2

2.9 1.3

1.6 1.4

603.5 231.9

96.9 33.9

a

Each compound was administered with a single dose at 10 mg/kg via

oral gavage using 100% PEG 200 as the formulation. Plasma samples

were collected at 1, 3, and 6 h time points with three mice for each

time point and analyzed by LC-MS/MS. Plasma concentrations are

presented as mean standard deviation (SD).

12 and 13 with a very ﬂexible linker have low oral exposures,

compounds 16 and 17 with a semi-rigid linker display an

improved oral plasma exposure over that of compounds 12 and

13. In particularly, compound 16 has an excellent oral exposure

with plasma concentrations of 1242.1, 919.3, and 603.5 ng/mL

with 10 mg/kg PO dosing at 1, 3, and 6 h time points or with

plasma concentrations of 1.6, 1.2, and 0.8 μM, respectively.

Encouraged by the oral exposure data for compound 16, we

synthesized compounds 25−42 by employing even more

conformational constrained linkers with lengths similar to that

in compound 16 (Table 4). All the linkers in compounds 25−40

also retain a positively charged amine group to maintain good

physiochemical properties in the degrader molecules. These

We synthesized compounds 20−24 by changing the linking

position from the meta- to the ortho-position in the phenyl ring

of the cereblon ligand in compounds 12, 13, 16, 17, and 18

(Table 2). Compounds 20−24 are much less potent and

eﬀective than compounds 12, 13, 16, 17, and 18 in reducing AR

protein in the VCaP cell line. These data indicate that the

tethering position in the cereblon ligand for our designed AR

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Modiﬁcation of the AR Antagonist Portion in AR

Degraders. Compound 28 is a highly potent AR degrader. We

next performed modiﬁcations of the AR antagonist portion in

compound 28 and obtained compounds 43−47 (Table 5).

Table 4. Investigation of the Eﬀect of Diﬀerent Rigid Linkers

on the AR Degraders

a

Table 5. Investigation of the Eﬀect of Diﬀerent AR

a

Antagonists on the AR Degraders

a

All the data are the average of three independent experiments..

These compounds were also evaluated for AR degradation in

VCaP cells by Western blotting at four diﬀerent concentrations

(1, 10, 100, and 1000 nM) with a 24 h treatment time, delivering

the data summarized in Table 5.

Removal of dimethyl or tetramethyl groups from the

tetramethylcyclobutanyl moiety in compound 28 resulted in

compounds 43 or 44, respectively. While compound 43

eﬀectively reduces the AR protein at 100 nM and 1 μM, it is

approximately 100 times less potent than compound 28.

Compound 44 has no eﬀect on the level of AR protein at 1

nM and reduces AR protein level by 25−39% at 10−1000 nM.

We replaced 2-Cl substituent on the phenyl with 2-F or 2-CF₃in

compound 28, yielding compounds 45 or 46, respectively.

Compound 45 eﬀectively reduces AR protein at 100 nM and 1

μM, but it is ineﬀective at 1 and 10 nM. Compound 46 is less

potent than compound 45. Both compounds 45 and 46 are

therefore much less potent than compound 28 in reducing the

level of AR protein. We generated compound 47 by replacing

the substituted phenyl group in compound 46 with a substituted

pyridine, which had been used in apalutamide (3, ARN-509,

Figure 1). Compound 47 is much less potent and eﬀective than

compound 46 in reducing AR protein and is thus a weak AR

degrader. These data establish that the AR antagonist portion

plays a critical role for the highly potent and eﬀective AR protein

degradation by compound 28.

a

All the data are the average of three independent experiments.

compounds were evaluated for AR degradation in VCaP cells by

Western blotting at four diﬀerent concentrations (1, 10, 100,

and 1000 nM) with a 24 h treatment time. The data are

summarized in Table 4.

Compounds 25−33 eﬀectively reduce the AR protein level by

>50% at 1 nM and achieve a maximum AR degradation of >90%.

Compounds 34−37 are less potent than compounds 25−33 but

are still capable of reducing the AR protein level by >50% at 10

nM. Compounds 38−41 are less potent than compounds 34−

37 and reduce the AR protein level by 50% at 100 nM.

Compound 42 is the least potent degrader among this series of

compounds.

Further Assessment of the Degradation of AR Protein

in AR+ Prostate Cancer Cell Lines. Based on our initial

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Figure 3. Western blotting analysis of AR protein in the AR+ VCaP cell line.

Figure 4. Western blotting analysis of AR protein in the AR+ LNCaP cell line.

screening data in the VCaP cell line, compounds 26, 27, 28, 33,

and 34 are highly potent and eﬀective AR degraders. The

potency of these compounds in the VCaP prostate cancer cell

line was further evaluated.

Western blotting data showed that these compounds are

highly potent and eﬀective in reducing AR protein in a dose-

dependent manner in the VCaP cell line (Figure 3). Compounds

26, 27, 28, 33, and 34 achieve DC₅₀values of 0.2, 0.8, 0.3, 0.1,

and 0.4 nM, respectively, in the VCaP cell line. Compounds 26,

27, 28, 33, and 34 are capable of reducing AR protein by >90%

with DC₉₀values of 3, 2, 3, 2, and 2 nM in the VCaP cell line,

respectively. The hook eﬀect was observed at high concen-

trations for compounds 26 and 27.

We next tested these compounds for their potencies in

inducing AR degradation in the LNCaP cell line carrying an AR

T878A mutation (Figure 4). Compounds 26, 27, 28, 33, and 34

achieve DC₅₀values of 5.1, 1.4, 8.3, 1.4, and 3.4 nM, respectively,

in the LNCaP cell line. Furthermore, compounds 26, 27, 28, 33,

and 34 are capable of reducing AR protein by >90% with DC₉₀

values of 41, 42, 25, 10, and 10 nM, respectively, in the LNCaP

cell line. No hook eﬀect was observed for all these compounds at

concentrations up to 1 μM.

We evaluated their degradation kinetics in both VCaP and

LNCaP cell lines (Figure 5). Compounds 26, 27, 28, 33, and 34

eﬀectively reduce the AR protein level within 3 h and achieve

near-complete AR depletion after a 6 h treatment. The kinetic

data show that induced AR degradation by these PROTAC

degraders in both LNCaP and VCaP cells is fairly rapid.

Evaluation of Potent AR Degraders for Cell Growth

Inhibition in VCaP and LNCaP Cell Lines. We evaluated a

number of highly potent AR degraders for their cell growth

inhibition in the VCaP and LNCaP cell lines. We synthesized 64

(Scheme 3), in which a methyl group was installed on the amino

group of the piperidine-2,6-dione moiety in the cereblon ligand

portion in 28 and included 64 as a control compound in the cell

growth assay. We synthesized compound 65 (ARi-184, Scheme

3), which is the AR antagonist used for the design of these potent

AR degraders and included it as an additional control compound

in the cell growth assay. We also included enzalutamide as

another control compound in the cell growth assay. The results

are shown in Figure 6.

In the VCaP cell line, enzalutamide has an IC₅₀value of 394

nM, while 64 and 65 have IC₅₀values of 783 and 49 nM,

respectively. Compounds 26, 27, 28, 33, and 34 are all highly

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Figure 7. Mechanistic investigation of AR degradation induced by 28

(ARD-2128) in VCaP and LNCaP cells. Cells were pretreated with AR

antagonist 65 (ARi-184), thalidomide, MG132, and MLN4924

followed by 3 h treatment with ARD-2128 at 100 nM.

concentrations as low as 10 nM. In direct comparison,

compound 28 at 10 nM is more eﬀective in reducing the

mRNA levels of PSA and TMPRSS2 genes than the

corresponding AR antagonist (65) at 1 μM in both VCaP and

LNCaP cell lines.

Figure 5. Western blotting analysis of AR protein in the AR+ VCaP and

LNCaP cell lines.

Pharmacokinetic Studies of AR Degraders in Mice. We

evaluated the pharmacokinetics (PK) of ﬁve highly potent AR

degraders (compounds 26, 27, 28, 33, and 34) in mice with both

intravenous and oral administration, obtaining the data

summarized in Table 6.

potent and eﬀective in the inhibition of cell growth in the VCaP

cell line and achieve IC₅₀values of 7, 8, 4, 4, and 5 nM,

respectively.

In the LNCaP cell line, enzalutamide has an IC₅₀value of 133

nM, whereas 64 and 65 have IC₅₀values of 237 and 81 nM,

respectively. Compounds 26, 27, 28, 33, and 34 are also highly

potent and eﬀective in the inhibition of cell growth in the

LNCaP cell line and achieve IC₅₀values of 10, 11, 5, 14, and 13

nM, respectively.

We investigated the mechanism of AR degradation induced

by 28 in LNCaP and VCaP cells. Our data showed that AR

degradation induced by 28 can be eﬀectively blocked by

pretreatment with the AR antagonist (65), cereblon ligand

(thalidomide), proteasome inhibitor (MG132), or NEDD8

activating E1 enzyme inhibitor (MLN4924) in both VCaP and

LNCaP cell lines (Figure 7). These mechanistic data

demonstrate that 28 is a bona fide PROTAC AR degrader.

We next investigated the ability of 28 to suppress AR-

regulated gene expression in the VCaP and LNCaP cell lines,

with a potent AR antagonist (65) included as the control (Figure

8). Our data showed that compound 28 eﬀectively suppresses

the expression of PSA and TMPRSS2 genes in both VCaP and

LNCaP cell lines in a dose-dependent manner. It is eﬀective in

reducing the mRNA levels of PSA and TMPRSS2 genes at

The PK data show that 26, 27, 28, 33, and 34 achieve good to

excellent overall PK proﬁles. They all have low clearance (1.2−

3.2 mL/min/kg) and a moderate to high steady-state volume of

distribution (V_ss) of between 1.5 and 6.8 L/kg. Each compound

has a long T_1/2following intravenous administration, ranging

between 11.5 and 27.6 h, and oral administration, ranging

between 11.2 and 67.8 h. To compare their C_maxand AUC in oral

administration, we converted their C_maxand AUC values to per

mg/kg dosing. For each mg/kg oral administration, 26, 27, 28,

33, and 34 achieve C_maxvalues of 278, 196, 261, 69, and 45 ng/

mL, respectively, and AUC_{0−24 h}of 4120, 2918, 4472, 1042, 850

h·ng/mL, respectively. Compounds 26, 27, 28, 33, and 34

achieve overall oral bioavailabilities of 75, 37, 67, 24, and 33%,

respectively.

Pharmacodynamic Evaluation of Several AR De-

graders in VCaP Xenograft Tumors. Based on their excellent

PK proﬁles, we evaluated 26, 27, and 28 for their

pharmacodynamics (PD) eﬀect in reducing AR protein in the

VCaP Xenograft tumor tissue in mice with a single oral

administration. The PD results are shown in Figure 9.

Figure 6. Cell growth inhibition in LNCaP and VCaP cells treated with AR degraders. LNCaP and VCaP cells were treated with diﬀerent compounds

in a charcoal-stripped medium in the presence of 0.1 nM of AR agonist R1881 for 4 days. Cell viability was determined with a WST-8 assay.

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Figure 8. Suppression of AR-regulated gene expression in the VCaP and LNCaP cell lines by the AR degrader 28 (ARD-2128) and the AR antagonist

(65) (ARi-184). VCaP and LNCaP cells were treated for 24 h, and a quantitative real-time polymerase chain reaction (qRT-PCR) analysis was

performed to determine the mRNA levels for AR and AR-regulated genes.

Table 6. Summary of PK Data for Compounds 26, 27, 28, 33, and 34 in Male ICR Mice

dose

T_1/2

AUC_0−t

Cl

V_ss

dose

T_1/2

T_max

C_max

AUC_0−t

F

compound

26

27

28 (ARD-

2128)

route (mg/kg)

(h)

(h·ng/ml)

(ml/min/kg) (L/kg) route (mg/kg)

(h)

(ng/ml)

(h·ng/ml)

(%)

IV

2

17.8

11.5

27.6

11,035

15,759

13,299

1.9

1.7

1.2

2.7

1.5

2.7

PO

5

12.0

11.2

18.8

4.0

4.7

1389

980

1304

20,600

14,588

22,361

75

37

67

33

34

IV

1

21.0

25.5

4334

2565

2.2

3.2

3.8

6.8

PO

3

12.4

67.8

6.0

4.7

207

134

3127

2550

24

33

a

C

_max, maximum drug concentration; AUC_{0−24 h}, area-under-the-curve between 0 and 24 h; Cl = plasma clearance rate; V_ss= steady state volume of

distribution; T_1/2= terminal half-life, T_max= the time take to reach C_max; F = oral bioavailability; IV, intravenous administration; PO, oral

administration.

Our Western blotting analysis of the VCaP tumor tissues

showed that a single oral administration of 26 and 28 at 20 mg/

kg is eﬀective in reducing the levels of AR protein in mice after

24 h but has a modest eﬀect at the 6 h time point. However,

compound 27 reduces the level of AR protein only modestly at

both time points.

To gain further insights into the PD data, we determined the

drug concentrations in both plasma and tumor tissue for

compounds 26, 27, and 28. The data obtained are summarized

in Table 7. Our data showed that with a single dose PO

administration, all three compounds achieve good drug

exposures in both plasma and tumor tissues. However,

compounds 28 and 26 have two times higher drug

concentrations than 27 at both 6 and 24 h time points in both

plasma and tumor tissue. Furthermore, both compounds 28 and

26 have higher drug concentrations at the 24 h time point than at

Figure 9. Western blot analysis of AR protein in VCaP xenograft

the 6 h time point. These data suggest that with daily

tumors in SCID mice. SCID mice bearing VCaP tumors were treated

administration, these AR degraders could be expected to

with a single oral administration with each compound (26, 27, and

accumulate in both plasma and tumor tissues.

ARD-2128) at 20 mg/kg. Mice were euthanized at indicated time

points, and tumor tissues were collected for Western blot analysis of AR

protein. GAPDH was used as the loading control.

We next evaluated 28 (ARD-2128) for its pharmacodynamics

(PD) eﬀect in reducing AR protein in the VCaP xenograft tumor

tissue in mice with daily oral administration for 3 consecutive

days. The PD results are shown in Figure 10.

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The key intermediates 55 were synthesized by the amidation of a

series of benzoic acids (51) by compound 54. Finally, the target

compounds were obtained through the substitution reaction of

intermediate 55 with 2-(2,6-dioxopiperidin-3-yl)-5-ﬂuoroisoin-

doline-1,3-dione and compound 56 with 2-(2,6-dioxopiperidin-

3-yl)-4-ﬂuoroiso-indoline-1,3-dione.

Compounds 16−19, 22−28, and 35−40 were synthesized

according to the method shown in Scheme 2. Amidation of

compound 54 with 58 gave the key intermediate 59 after

subsequent deprotection by TFA. The intermediate 61 was

made by the reductive amination of compound 59 with diﬀerent

aldehydes and ketones (60) and subsequent deprotection by

TFA. The target compounds were obtained by the substitution

reaction of compound 61 with either 2-(2,6-dioxopiperidin-3-

yl)-5-ﬂuoroisoindoline-1,3-dione (56) or 2-(2,6-dioxopiperi-

din-3-yl)-4-ﬂuoro-isoindoline-1,3-dione (57).

The synthesis of negative control compounds (64 and 65) is

shown in Scheme 3. Compound 63 was produced by

methylation of the intermediate 56. Compound 64 was made

by the reaction of compound 61 with 63. As shown in Scheme 3,

intermediate 67 was synthesized from the methylation of

intermediate 62 and MeI with subsequent hydrolysis by NaOH.

Compound 65 was made by the amidation of compounds 67

and 54.

The synthesis of compounds 29−32 is shown in Scheme 4.

Compound 68 was produced by the amidation reaction between

54 and 66 and subsequent hydrolysis by NaOH. The key

intermediate 70 was synthesized from the amidation of

compound 68 by 69. The intermediates 71 were made by the

reductive amination of compound 70 with diﬀerent aldehydes or

ketones. Finally, the target compounds were obtained by a

substitution reaction between 71 and 2-(2,6-dioxopiperidin-3-

yl)-5-ﬂuoroisoindoline-1,3-dione (56).

Compounds 33 and 34 were synthesized according to the

method in Scheme 5. The key intermediate (72) was

synthesized with the substitution reaction of compound 56

with 69 and subsequent deprotection by TFA. Intermediate 76

was synthesized from the substitution reaction of compound 73

with the alcohol (74) and hydrolysis by NaOH. Intermediate 77

was synthesized by the amidation of compound 76 by

compound 54. The oxidation of compound 77 gave

intermediate 78, the reductive amination of which produced

the target compounds 33 and 34.

The synthesis of compounds 41 and 42 is shown in Scheme 6.

Compound 81 was produced by the substitution reaction of

compounds 73 and 79 followed by hydrolysis by NaOH.

Intermediate 82 was synthesized by the amidation of

compounds 54 with 81 followed by deprotection with TFA.

Finally, the target compounds were obtained by the substitution

reaction of 82 with 2-(2,6-dioxopiperidin-3-yl)-5-ﬂuoroisoindo-

line-1,3-dione (56).

Table 7. Analysis of Drug Concentrations in Plasma and

VCaP Tumor Tissue in SCID Mice for 26, 27, and 28 (ARD-

2128)

a

plasma concentration

(ng/ml)

tumor concentration

(ng/kg)

time point

(h)

compound

26

mean SD

6

24

6

24

6

1365.0 1102.9

1666.0 691.6

803.3 203.0

775.7 346.6

1513.3 41.6

718.3 110.6

1743.3 448.6

314.8 93.7

685.0 232.5

635.0 83.2

27

28 (ARD-

2128)

24

1659.3 846.0

1506.7 705.0

a

Each compound was administered with a single dose at 20 mg/kg

with 100% PEG200 as the formulation in mice bearing VCaP tumors

with one tumor per mouse. Plasma and tumor tissue were collected at

6 and 24 h time points for each compound with three mice for each

time point.

Western blotting analysis of the VCaP tumor tissues showed

that daily oral administration of 28 at 10 mg/kg for 3 days

eﬀectively reduces the levels of AR protein in mice at 3 and 6 h

but has only a modest eﬀect at the 24 h time point. We also

investigated the ability of 28 to suppress AR regulated gene

expression in vivo. Our data showed that 28 eﬀectively

suppresses the expression of PSA, TMPRSS2, and FKBP5

genes in vivo at 3 and 6 h and is capable of reducing the mRNA

level in all three genes by >50% at 3 h with daily oral

administration for 3 days at 10 mg/kg (Figure 10).

Antitumor Activity of ARD-2128 in the VCaP Xeno-

graft Model in Mice. Based on the PD data and also the drug

exposure data in the VCaP tumors, we evaluated 28 (ARD-

2128) for its antitumor eﬃcacy in the VCaP xenograft tumor

model in mice. We included enzalutamide, a FDA approved AR

antagonist as a control compound in this eﬃcacy experiment.

The eﬃcacy data are summarized in Figure 11.

Enzalutamide at 40 mg/kg oral administration inhibits tumor

growth by 39% over the vehicle control at the end of the 21 day

treatment (p = 0.0075). In comparison, ARD-2128 at 10, 20, and

40 mg/kg inhibits tumor growth by 46, 69, and 63%,

respectively, at the end of the 21 day treatment over the vehicle

control (p < 0.0001 for all three doses vs control). While ARD-

2128 at 10 mg/kg is slightly more eﬀective than enzalutamide at

40 mg/kg, it is signiﬁcantly more eﬀective at both 20 and 40 mg/

kg, than enzalutamide at 40 mg/kg in the inhibition of tumor

growth at the end of treatment (p < 0.05 for ARD-2128 at both

doses vs enzalutamide).

At all the three dose levels, ARD-2128 and enzalutamide are

well tolerated in mice, causing no more than 5% of maximum

weight loss during the entire experiment.

Evaluation of ARD-2128 for Its Plasma Stability and

Microsomal Stability. We evaluated ARD-2128 for its plasma

and microsomal stability in mouse, rat, dog, monkey, and

humans. The data showed that ARD-2128 has excellent plasma

and microsomal stability in all the ﬁve species (Table 8).

The synthesis of compounds 43 and 44 is shown in Scheme 7.

Compound 84 was produced by the substitution reaction of

compounds 52 and 83 with subsequent deprotection by TFA.

The key intermediate 85 was synthesized by the amidation of

compound 84 with 58 followed by deprotection by TFA.

Compound 87 was obtained through the reductive amination of

intermediates 86 and 85 and subsequent deprotection by TFA.

Finally, the target compounds were obtained by a substitution of

87 with 2-(2,6-dioxopiperidin-3-yl)-5-ﬂuoroisoindoline-1,3-

dione 56.

CHEMISTRY

■

The synthesis of compounds 10−15, 20, and 21 is shown in

Scheme 1. Compound 51 was synthesized from the reaction of

compounds 48 and 49 with subsequent hydrolysis by NaOH.

Compound 54 was produced by the substitution reaction of 52

with 53 followed by deprotection by TFA of the NHBoc group.

Compounds 45−47 were synthesized according to the

method in Scheme 8. Compound 89 was synthesized from the

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Figure 10. Pharmacodynamic analysis for 28 (ARD-2128) in VCaP xenograft tumors. SCID mice bearing VCaP tumors were treated with daily, PO

dose of ARD-2128 at 10 mg/kg for 3 consecutive days. Mice were euthanized at indicated time points after the last dose, and tumor tissues were

collected for Western blot and qRT-PCR analysis. GAPDH was used as the loading control in the Western blot analysis.

substitution reaction of compounds 88 and 53 followed by

deprotection by TFA. Amidation of compound 89 with 58 with

subsequent deprotection by TFA gave intermediate 90.

Intermediate 91 was synthesized from the reductive amination

of compound 90 with 86. Finally, the target compounds were

obtained from the substitution reaction of 91 with 2-(2,6-

dioxopiperidin-3-yl)-5-ﬂuoroisoindoline-1,3-dione (56).

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Figure 11. Antitumor activity of ARD-2128 in the VCaP xenograft tumor model in SCID mice. Enzalutamide was included as a control. Each

compound was dosed via oral gavage daily for a total of 21 days.

discovering potent and orally bioavailable AR degraders.

Following determination of the optimal linker lengths using

ﬂexible linkers, a series of AR degraders were designed and

synthesized by employing conformational constrained linkers

containing a positively charged amine. This led to identiﬁcation

of a number of highly potent AR degraders, which display

excellent pharmacokinetics and oral bioavailability with ARD-

2128 being the best compound. Mechanistic studies showed that

ARD-2128 is a bona fide PROTAC AR degrader and strongly

suppresses AR-regulated genes in a dose- and time-dependent

manner in AR+ prostate cancer cell lines. ARD-2128 also

potently inhibits cell growth in the VCaP prostate cancer cell

line with AR gene ampliﬁcation and the LNCaP cell line with an

AR mutation. As compared to our previously reported AR

Table 8. Liver Microsomal and Plasma Stability of ARD-2128

in Five Species (Human, Mouse, Rat, Dog, and Monkey)

species

liver microsomal stability (T_1/2, min) plasma stability (T_1/2, min)

mouse

rat

dog

monkey

human

>120

DISCUSSION AND CONCLUSIONS

■

In this study, we report the design, synthesis, and evaluation of a

series of PROTAC AR degraders using a potent AR antagonist

and thalidomide as the cereblon ligand with the objective of

a

Scheme 1. Synthesis of Compounds 10−15, 20, and 21

a

(a) DIPEA, DMSO, 100 °C; (b) NaOH, MeOH/H₂O, rt; (c) NaH, DMF, 0 °C to rt; TFA, DCM, rt; (d) HATU, DIPEA, DMF, rt; TFA, DCM,

rt; (e) DIPEA, DMSO, 100 °C; (f) DIPEA, DMSO, 100 °C.

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a

Scheme 2. Synthesis of Compounds 16−19, 22−28, and 35−40

a

(a) HATU, DIPEA, DMF, rt; TFA, DCM, rt; (b) NaBH(OAc)₃, AcOH, DCE, rt; TFA, DCM, rt; (c) DIPEA, DMSO, 100 °C; (d) DIPEA,

DMSO, 100 °C.

degrader ARD-61, which has a molecular weight of 1095.8, a

calculated tPSA of 189.2 Å², and a calculated CLogP of 8.2,

ARD-2128 has a much reduced molecular weight (820.4), a

reduced calculated tPSA (155.4 Å²), and a lower calculated

CLogP (6.9). ARD-2128 achieves an overall excellent

pharmacokinetic proﬁle and has excellent plasma and tissue

exposures in both native prostate tissue and xenograft tumor

tissue after a single oral administration in mice. A single oral

administration of ARD-2128 eﬀectively reduces AR protein in

the VCaP tumor tissue. Importantly, oral administration of

ARD-2128 is eﬀective in the inhibition of tumor growth in the

VCaP xenograft tumor model without any signs of toxicity. In a

direct comparison, ARD-2128 at 20 or 40 mg/kg is more

eﬃcacious than enzalutamide at 40 mg/kg. ARD-2128 is

therefore a potent and orally eﬃcacious PROTAC AR degrader.

While ARD-2128 achieves a DC₉₀of 3.5 nM in the VCaP cell

line in vitro, much higher concentrations of ARD-2128 are

needed to achieve eﬀective AR degradation in the VCaP tumor

tissue in mice. ARD-2128 was found to have a plasma protein

binding of 99.8% in mouse plasma (data not shown), suggesting

that signiﬁcant improvement in mouse plasma protein binding

for ARD-2128 may further improve its in vivo potency in AR

degradation and tumor growth inhibition.

degraders.⁶¹⁻⁶⁴In fact, the chemical structure for ARD-2128

and associated chemical data were disclosed in one of these

patents (example 316).⁶¹However, although AR degradation

activity in the VCaP cell line for ARD-2128 was reported in the

patent,⁶¹detailed biological characterization and pharmaco-

logical data were not reported.

In summary, we presented our strategies toward discovering

highly potent AR degraders with excellent oral pharmacokinetics

in mice. These general strategies employed in our study provide

insights and guidance for the design of potent and orally

bioavailable PROTAC degraders for other therapeutically

important protein targets.

EXPERIMENTAL SECTION

■

Chemistry. General Experiment and Information. Unless

otherwise noted, all purchased reagents were used as received without

further puriﬁcation. ¹H NMR and ¹³C NMR spectra were recorded on a

Bruker Advance 400 MHz spectrometer. ¹H NMR spectra are reported

in parts per million (ppm) downﬁeld from tetramethylsilane (TMS).

All ¹³C NMR spectral peaks are reported in ppm and measured with ¹H

decoupling. 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, and m =

multiplet. Mass spectrometric (MS) analysis was carried out with a

Waters UPLC mass spectrometer. The ﬁnal compounds were all

puriﬁed by a C18 reverse phase preparative HPLC column with solvent

A (0.1% TFA in H₂O) and solvent B (0.1% TFA in CH₃CN) as eluents.

It is important to note that Arvinas scientists have employed

the same class of AR antagonist and thalidomide as the cereblon

ligand for the synthesis of a large number of PROTAC AR

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a

Scheme 3. Synthesis of Compounds 64 and 65

a

(a) MeI, K₂CO₃, DMF, 60 °C; (b) DIPEA, DMSO, 100 °C; (c) MeI, K₂CO₃, DMF, 60 °C; (d) NaOH, MeOH/H₂O, rt; (e) HATU, DIPEA,

DMF, rt.

a

Scheme 4. Synthesis of Compounds 29−32

a

(a) HATU, DIPEA, DMF, rt; (b) NaOH, MeOH/H₂O, rt; (c) HATU, DIPEA, DMF, rt; TFA, DCM, rt; (d) NaBH(OAc)₃, AcOH, DCE, rt;

TFA, DCM, rt; (e) DIPEA, DMSO, 100 °C.

The purity of all the ﬁnal compounds was shown to be >95% by

UPLC−MS or UPLC.

NaH (1.2 equiv) was added to a solution of 53 (10 mmol) in dry

DMF at 0 °C. After stirring the mixture at 0 °C for 20 min, 52 (1.0

equiv) was added and the mixture was stirred at rt for 4 h. After UPLC-

MS demonstrated full conversion of the starting materials, H₂O was

added and the mixture was extracted three times with EtOAc. The

combined organic layers were washed with brine and then dried over

anhydrous Na₂SO₄. The solvent was removed on a rotary evaporator.

The Boc-protected intermediate was obtained by ﬂash column

chromatography. The desired intermediate (54) was obtained by

deprotection with TFA in DCM in 86% yield.

General Procedure for the Synthesis of Compounds 10−15, 20,

and 21. DIPEA (3 equiv) was added to a solution of compounds 48 (1

mmol) and 49 (1.1 equiv each) in DMSO. After stirring at 100 °C for 2

h, water was added and the reaction mixture was extracted with EtOAc;

it was then washed with water, and the organic phase was dried over

Na₂SO₄. Compound 50 was obtained by removing the solvent under

vacuum and puriﬁed by ﬂash column with 84% yield. Then, the desired

intermediate (51) was obtained by hydrolysis with NaOH solid (2.0

equiv) in MeOH/H₂O at r.t. in 88% yield.

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a

Scheme 5. Synthesis of Compounds 33 and 34

a

(a) DIPEA, DMSO, 100 °C; TFA, DCM, rt; (b) DIPEA, DMSO, 100 °C; (c) NaOH, MeOH/H₂O, rt; (d) HATU, DIPEA, DMF, rt; (e) DMP,

DCM, rt; (f) NaBH(OAc)₃, AcOH, DCE, rt.

a

Scheme 6. Synthesis of Compounds 41 and 42

a

(a) DIPEA, DMSO, 100 °C; (b) NaOH, MeOH/H₂O, rt; (c) HATU, DIPEA, DMF, rt; TFA, DCM, rt; (d) DIPEA, DMSO, 100 °C.

DIPEA (5 equiv) and HATU (1.2 equiv) were added to a solution of

compounds 54 (1 mmol) and 51 (1.1 equiv) in DMF (2 mL). After

stirring at rt for 1 h, water was added and the reaction mixture was

extracted by EtOAc; it was then washed by water, and the organic phase

was dried by Na₂SO₄. Compound 55 was obtained by removing the

solvent under vacuum and puriﬁed by ﬂash column with subsequent

deprotection with TFA with 80% yield.

equiv) in DMSO (2 mL). After 4 h at 100 °C, the mixture was subject

to HPLC puriﬁcation to aﬀord compounds 10−15 with 70−85% yields.

DIPEA (5 equiv) was added to a solution of the compounds 55 (0.5

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

(10) (1.1 equiv) in DMSO (2 mL). After 4 h at 100 °C, the mixture was

subject to HPLC puriﬁcation to aﬀord compounds 20−21 in yields of

75−86%.

4-((1r,3r)-3-Amino-2,2,4,4-tetramethylcyclobutoxy)-2-chloro-

benzonitrile (INT-54). ¹H NMR (400 MHz, DMSO-d₆) δ 7.87 (d, J =

8.7 Hz, 1H), 7.19 (d, J = 2.3 Hz, 1H), 6.99 (dd, J = 8.8, 2.3 Hz, 1H), 4.37

DIPEA (5 equiv) was added to a solution of 55 (0.8 mmol) and 2-

(2,6-dioxopiperidin-3-yl)-5-ﬂuoroiso-indoline-1,3-dione 56 (1.1

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a

Scheme 7. Synthesis of Compounds 43 and 44

a

(a) NaH, DMF, 0 °C to rt; TFA, DCM, rt; (b) HATU, DIPEA, DMF, rt; TFA, DCM, rt; (c) NaBH(OAc)₃, AcOH, DCE, rt; TFA, DCM, rt; (d)

DIPEA, DMSO, 100 °C.

a

Scheme 8. Synthesis of Compounds 45−47

a

(a) NaH, DMF, 0 °C to rt; TFA, DCM, rt; (b) HATU, DIPEA, DMF, rt; TFA, DCM, rt; (c) NaBH(OAc)₃, AcOH, DCE, rt; TFA, DCM, rt; (d)

DIPEA, DMSO, 100 °C.

(s, 1H), 3.76 (s, 1H), 3.11 (s, 1H), 1.29 (s, 6H), 1.08 (s, 6H). UPLC−

MS calculated for C₁₅H₂₀ClN₂O [M + H]⁺: 279.13, found: 279.07.

UPLC-retention time: 2.0 min, purity >95%.

N-((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(piperazin-1-yl)benzamide (INT-59). ¹H NMR (400

MHz, DMSO-d₆) δ 9.08 (s, 1H), 7.89 (d, J = 8.7 Hz, 1H), 7.81 (s,

1H), 7.60 (d, J = 9.2 Hz, 1H), 7.18 (d, J = 2.3 Hz, 1H), 7.05 (d, J = 8.9

Hz, 2H), 7.01−6.98 (m, 1H), 4.33 (s, 1H), 4.07 (d, J = 9.1 Hz, 1H),

3.54−3.44 (m, 4H), 3.26 (s, 4H), 1.23 (s, 6H), 1.13 (s, 6H). UPLC−

MS calculated for C₂₆H₃₂ClN₄O₂[M + H]⁺: 467.22, found: 467.24.

UPLC-retention time: 4.1 min, purity >95%.

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N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-((3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

yl)amino)propyl)amino)benzamide (10). ¹H NMR (400 MHz,

MeOH-d₄) δ 7.74 (d, J = 8.7 Hz, 1H), 7.67 (d, J = 8.8 Hz, 2H), 7.57

(d, J = 8.4 Hz, 1H), 7.14 (d, J = 2.4 Hz, 1H), 7.03−6.98 (m, 2H), 6.87

(dd, J = 8.4, 2.2 Hz, 1H), 6.68 (d, J = 8.8 Hz, 2H), 5.05 (dd, J = 12.4, 5.4

Hz, 1H), 4.30 (s, 1H), 4.14 (s, 1H), 3.37 (t, J = 6.9 Hz, 3H), 2.90−2.66

(m, 4H), 2.14−2.08 (m, 1H), 2.04−1.95 (m, 2H), 1.29 (s, 6H), 1.23 (s,

6H). UPLC−MS calculated for C₃₈H₄₀ClN₆O₆[M + H]⁺: 711.27,

found: 711.30. UPLC-retention time: 5.9 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-((4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

yl)amino)butyl)amino)benzamide (11). ¹H NMR (400 MHz,

MeOH-d₄) δ 7.73−7.67 (m, 3H), 7.54 (d, J = 8.4 Hz, 1H), 7.12 (d, J

= 2.4 Hz, 1H), 6.97 (td, J = 4.6, 2.5 Hz, 2H), 6.82 (dd, J = 8.4, 2.2 Hz,

1H), 6.79−6.74 (m, 2H), 5.03 (dd, J = 12.4, 5.5 Hz, 1H), 4.27 (s, 1H),

4.12 (s, 1H), 3.28−3.21 (m, 4H), 2.87−2.68 (m, 3H), 2.65 (s, 1H),

2.12−2.05 (m, 1H), 1.77 (d, J = 2.8 Hz, 4H), 1.27 (s, 6H), 1.21 (s, 6H).

UPLC−MS calculated for C₃₉H₄₂ClN₆O₆[M + H]⁺: 725.29, found:

725.23. UPLC-retention time: 5.9 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-((5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

yl)amino)pentyl)amino)benzamide (12). ¹H NMR (400 MHz,

MeOH-d₄) δ 7.71 (dd, J = 8.7, 4.9 Hz, 3H), 7.53 (dd, J = 8.3, 3.7

Hz, 1H), 7.12 (d, J = 2.4 Hz, 1H), 6.97 (dd, J = 9.1, 2.3 Hz, 2H), 6.81

(ddd, J = 19.2, 11.4, 6.1 Hz, 3H), 5.03 (dd, J = 12.4, 5.4 Hz, 1H), 4.28 (s,

1H), 4.13 (s, 1H), 3.25−3.17 (m, 4H), 2.88−2.65 (m, 3H), 2.09 (ddd, J

= 12.7, 6.7, 3.8 Hz, 1H), 1.69 (dt, J = 13.9, 7.0 Hz, 4H), 1.56 (dd, J =

15.0, 8.3 Hz, 2H), 1.41 (dd, J = 11.0, 4.7 Hz, 1H), 1.28 (s, 6H), 1.22 (s,

6H). UPLC−MS calculated for C₄₀H₄₄ClN₆O₆[M + H]⁺: 739.30,

found: 739.28. UPLC-retention time: 6.3 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

yl)amino)hexyl)amino)benzamide (13). ¹H NMR (400 MHz,

MeOH-d₄) δ 7.75−7.70 (m, 2H), 7.64 (d, J = 8.7 Hz, 1H), 7.47 (d, J

= 8.4 Hz, 1H), 7.05 (d, J = 2.4 Hz, 1H), 6.98−6.93 (m, 2H), 6.90 (dd, J

= 9.3, 2.2 Hz, 2H), 6.75 (dd, J = 8.4, 2.2 Hz, 1H), 5.00−4.94 (m, 1H),

4.21 (s, 1H), 4.07 (s, 1H), 3.17 (dt, J = 22.5, 7.1 Hz, 4H), 2.81−2.60

(m, 3H), 2.05−1.99 (m, 1H), 1.62 (dd, J = 11.8, 5.4 Hz, 4H), 1.43 (d, J

= 6.9 Hz, 4H), 1.21 (s, 6H), 1.15 (s, 6H). UPLC−MS calculated for

C₄₁H₄₆ClN₆O₆[M + H]⁺: 753.32, found: 753.30. UPLC-retention

time: 5.4 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-((7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

yl)amino)heptyl)amino)benzamide (14). ¹H NMR (400 MHz,

MeOH-d₄) δ 7.76−7.69 (m, 3H), 7.53 (d, J = 8.4 Hz, 1H), 7.11 (d, J

= 2.4 Hz, 1H), 7.00−6.95 (m, 2H), 6.89−6.85 (m, 2H), 6.81 (dd, J =

8.4, 2.2 Hz, 1H), 5.03 (dd, J = 12.4, 5.5 Hz, 1H), 4.27 (s, 1H), 4.13 (s,

1H), 3.20 (td, J = 7.0, 5.3 Hz, 4H), 2.77 (qdd, J = 14.4, 8.6, 6.3 Hz, 3H),

2.12−2.03 (m, 1H), 1.65 (d, J = 5.3 Hz, 4H), 1.44 (d, J = 2.2 Hz, 6H),

1.27 (s, 6H), 1.21 (s, 6H). UPLC−MS calculated for C₄₂H₄₈ClN₆O₆

[M + H]⁺: 767.33, found: 767.30. UPLC-retention time: 6.8 min, purity

>95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-((8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

yl)amino)octyl)amino)benzamide (15). ¹H NMR (400 MHz, MeOH-

d₄) δ 7.73 (dd, J = 8.7, 3.9 Hz, 3H), 7.55 (d, J = 8.4 Hz, 1H), 7.13 (d, J =

2.4 Hz, 1H), 6.98 (dd, J = 8.6, 2.3 Hz, 2H), 6.88−6.81 (m, 3H), 5.04

(dd, J = 12.4, 5.5 Hz, 1H), 4.29 (s, 1H), 4.14 (s, 1H), 3.21 (t, J = 7.2 Hz,

4H), 2.88−2.67 (m, 3H), 2.09 (ddd, J = 10.1, 5.3, 2.4 Hz, 1H), 1.66 (dd,

J = 14.1, 7.1 Hz, 4H), 1.43 (d, J = 8.5 Hz, 8H), 1.29 (s, 6H), 1.23 (s,

6H). UPLC−MS calculated for C₄₃H₅₀ClN₆O₆[M + H]⁺: 781.35,

found: 781.22. UPLC-retention time: 6.9 min, purity >95%.

UPLC−MS calculated for C₄₀H₄₄ClN₆O₆[M + H]⁺: 739.30, found:

739.31. UPLC-retention time: 6.6 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

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

yl)amino)hexyl)amino)benzamide (21). ¹H NMR (400 MHz,

MeOH-d₄) δ 7.71 (dt, J = 9.5, 2.4 Hz, 3H), 7.54 (dd, J = 8.6, 7.1 Hz,

1H), 7.13 (d, J = 2.4 Hz, 1H), 7.06−7.01 (m, 2H), 6.98 (dd, J = 8.8, 2.4

Hz, 1H), 6.84−6.80 (m, 2H), 5.05 (dd, J = 12.5, 5.5 Hz, 1H), 4.28 (s,

1H), 4.14 (s, 1H), 3.21 (t, J = 7.2 Hz, 2H), 2.77 (dddd, J = 17.6, 13.2,

4.8, 3.4 Hz, 3H), 2.14−2.07 (m, 1H), 1.75−1.65 (m, 4H), 1.50 (dd, J =

8.7, 5.3 Hz, 4H), 1.28 (s, 6H), 1.22 (s, 6H). UPLC−MS calculated for

C₄₁H₄₆ClN₆O₆[M + H]⁺: 753.32, found: 753.31. UPLC-retention

time: 6.4 min, purity >95%.

General Procedure for the Synthesis of Compounds 16−19, 22−

28, and 35−40. DIPEA (5 equiv) and HATU (1.2 equiv) were added

to a solution of compounds 54 (10 mmol) and 58 (1.1 equiv) in DMF

(2 mL). After stirring at rt for 1 h, water was added and the reaction

mixture was extracted with EtOAc; it was then washed with water, and

the organic phase was dried over Na₂SO₄. Compound 59 was obtained

by removing the solvent under vacuum and puriﬁed by ﬂash column

chromatography with subsequent deprotection by TFA with 84% yield.

AcOH (10%) in DCE was added to a solution of compounds 59 (1

mmol) and various aldehydes or ketones. After the mixture was stirred

at rt for 10 min, NaBH(OAc)₃(1.2 equiv) was added and the mixture

was stirred at rt for a further 2 h. The Boc-protected intermediates 61

were obtained by ﬂash column chromatography. The desired

intermediates (61) were obtained by deprotection with TFA in

DCM in 70−80% yields.

DIPEA (5 equiv) was added to a solution of the compounds 61 (0.5

mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-ﬂuoroisoindoline-1,3-dione

56 (1.1 equiv) or 2-(2,6-dioxopiperidin-3-yl)-5-ﬂuoroisoindoline-1,3-

dione 57 (1.1 equiv) in DMSO (2 mL). After stirring at 100 °C for 4 h,

the mixture was subject to HPLC puriﬁcation to aﬀord compounds

16−19, 22−28, and 35−40 with 70−90% yields.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

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

5-yl)amino)ethyl)piperazin-1-yl)benzamide (16). ¹H NMR (400

MHz, DMSO-d₆) δ 11.07 (s, 1H), 7.91 (d, J = 8.7 Hz, 1H), 7.82 (d,

J = 8.4 Hz, 2H), 7.64 (d, J = 8.3 Hz, 1H), 7.59 (d, J = 9.1 Hz, 1H), 7.21

(s, 1H), 7.10 (s, 2H), 7.03−6.96 (m, 2H), 5.08 (d, J = 5.2 Hz, 1H), 4.34

(s, 1H), 4.07 (d, J = 9.1 Hz, 2H), 3.67 (d, J = 17.3 Hz, 4H), 3.43−3.38

(m, 2H), 3.17 (dd, J = 28.7, 21.7 Hz, 4H), 2.88 (dd, J = 21.8, 9.5 Hz,

1H), 2.60 (d, J = 16.7 Hz, 1H), 2.51 (d, J = 1.4 Hz, 2H), 2.08−1.98 (m,

1H), 1.23 (s, 6H), 1.14 (s, 6H). UPLC−MS calculated for

C₄₁H₄₅ClN₇O₆[M + H]⁺: 766.31, found: 766.18. UPLC-retention

time: 4.6 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

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

5-yl)amino)propyl)piperazin-1-yl)benzamide (17). ¹H NMR (400

MHz, DMSO-d₆) δ 11.07 (s, 1H), 9.71 (d, J = 104.2 Hz, 2H), 7.91 (d, J

= 8.7 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 9.3 Hz, 1H), 7.21 (s,

1H), 7.07 (d, J = 8.5 Hz, 2H), 7.02 (d, J = 8.5 Hz, 1H), 4.34 (s, 1H),

4.11−3.98 (m, 3H), 3.59 (s, 4H), 3.32−3.27 (m, 3H), 3.18 (s, 3H),

3.07 (s, 1H), 2.09 (s, 4H), 1.95 (d, J = 7.9 Hz, 2H), 1.23 (s, 6H), 1.14 (s,

6H). UPLC−MS calculated for C₄₂H₄₇ClN₇O₆[M + H]⁺: 780.33,

found: 780.20. UPLC-retention time: 4.6 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

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

5-yl)amino)butyl)piperazin-1-yl)benzamide (18). ¹H NMR (400

MHz, DMSO-d₆) δ 11.06 (s, 1H), 7.90 (d, J = 8.7 Hz, 1H), 7.82 (d,

J = 8.7 Hz, 2H), 7.64−7.57 (m, 2H), 7.21 (d, J = 2.3 Hz, 1H), 7.07 (d, J

= 8.8 Hz, 2H), 7.03−6.97 (m, 2H), 5.04 (dd, J = 12.9, 5.3 Hz, 1H), 4.34

(s, 1H), 4.12−3.99 (m, 3H), 3.39 (s, 1H), 3.18 (ddd, J = 15.7, 13.7, 6.9

Hz, 8H), 2.89 (ddd, J = 17.5, 14.3, 5.2 Hz, 1H), 2.62−2.54 (m, 1H),

2.02 (dd, J = 19.0, 13.7 Hz, 1H), 1.80 (d, J = 6.8 Hz, 1H), 1.71−1.61 (m,

2H), 1.54 (dd, J = 14.5, 7.4 Hz, 1H), 1.22 (s, 6H), 1.14 (s, 6H). UPLC−

MS calculated for C₄₃H₄₉ClN₇O₆[M + H]⁺: 794.34, found: 794.18.

UPLC-retention time: 4.9 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-((5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-

yl)amino)pentyl)amino)benzamide (20). ¹H NMR (400 MHz,

MeOH-d₄) δ 7.73−7.68 (m, 3H), 7.51 (dd, J = 8.5, 7.2 Hz, 1H),

7.10 (d, J = 2.4 Hz, 1H), 7.04−6.99 (m, 2H), 6.96 (dd, J = 8.8, 2.4 Hz,

1H), 6.83 (d, J = 8.7 Hz, 2H), 5.07−5.01 (m, 1H), 4.26 (s, 1H), 4.11 (s,

1H), 3.21 (t, J = 7.1 Hz, 2H), 2.88−2.65 (m, 3H), 2.13−2.04 (m, 1H),

1.75−1.65 (m, 4H), 1.58−1.49 (m, 2H), 1.26 (s, 6H), 1.20 (s, 6H).

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(4-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-

5-yl)amino)pentyl)piperazin-1-yl)benzamide (19). ¹H NMR (400

O

https://doi.org/10.1021/acs.jmedchem.1c00882

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

MHz, DMSO-d₆) δ 11.06 (s, 1H), 7.90 (d, J = 8.7 Hz, 1H), 7.82 (d, J =

8.3 Hz, 2H), 7.59 (dd, J = 8.2, 6.3 Hz, 2H), 7.20 (d, J = 1.4 Hz, 1H), 7.07

(d, J = 8.4 Hz, 2H), 7.00 (dd, J = 14.2, 6.8 Hz, 2H), 5.04 (dd, J = 12.8,

5.2 Hz, 1H), 4.34 (s, 1H), 4.07 (d, J = 9.1 Hz, 2H), 3.99 (s, 2H), 3.91 (s,

3H), 3.60 (s, 2H), 3.17 (d, J = 12.1 Hz, 5H), 2.87 (d, J = 13.0 Hz, 1H),

2.59 (d, J = 17.0 Hz, 1H), 2.04−1.97 (m, 1H), 1.74 (d, J = 7.0 Hz, 2H),

1.63 (d, J = 7.1 Hz, 2H), 1.57−1.52 (m, 1H), 1.44 (d, J = 6.8 Hz, 1H),

1.23 (s, 6H), 1.14 (s, 6H). UPLC−MS calculated for C₄₄H₅₁ClN₇O₆

[M + H]⁺: 808.36, found: 808.19. UPLC-retention time: 4.8 min, purity

>95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

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

4-yl)amino)ethyl)piperazin-1-yl)benzamide (22). ¹H NMR (400

MHz, DMSO-d₆) δ 7.82 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.7 Hz,

1H), 7.64 (dd, J = 8.3, 7.3 Hz, 1H), 7.14 (ddd, J = 16.4, 9.7, 5.1 Hz, 5H),

7.01−6.97 (m, 1H), 5.09 (dd, J = 12.5, 5.4 Hz, 1H), 4.30 (s, 1H), 4.16

(s, 1H), 3.89 (t, J = 6.2 Hz, 2H), 3.81−3.76 (m, 1H), 3.51 (ddd, J =

25.6, 16.0, 9.9 Hz, 9H), 2.80 (tdd, J = 9.6, 8.0, 4.3 Hz, 3H), 2.17−2.10

(m, 1H), 1.30 (s, 6H), 1.24 (s, 6H). UPLC−MS calculated for

C₄₁H₄₅ClN₇O₆[M + H]⁺: 766.31, found: 766.18. UPLC-retention

time: 4.7 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

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

4-yl)amino)propyl)piperazin-1-yl)benzamide (23). ¹H NMR (400

MHz, MeOH-d₄) δ 7.81 (d, J = 2.3 Hz, 2H), 7.72 (s, 1H), 7.61 (dd, J =

8.5, 7.1 Hz, 1H), 7.12 (ddd, J = 5.8, 5.4, 2.3 Hz, 5H), 7.00 (d, J = 6.4 Hz,

1H), 5.08 (dd, J = 12.5, 5.5 Hz, 1H), 4.31 (s, 1H), 4.16 (s, 1H), 3.93

(dd, J = 31.9, 25.4 Hz, 2H), 3.69 (s, 2H), 3.52 (dt, J = 6.8, 4.2 Hz, 3H),

3.45 (d, J = 6.8 Hz, 1H), 3.29 (d, J = 2.9 Hz, 1H), 2.90−2.68 (m, 3H),

2.15 (dddd, J = 11.2, 9.6, 9.0, 4.4 Hz, 4H), 1.30 (s, 6H), 1.24 (s, 6H).

UPLC−MS calculated for C₄₂H₄₇ClN₇O₆[M + H]⁺: 780.33, found:

780.18. UPLC-retention time: 4.8 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

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

4-yl)amino)butyl)piperazin-1-yl)benzamide (24). ¹H NMR (400

MHz, MeOH-d₄) δ 7.84−7.79 (m, 2H), 7.73 (d, J = 8.7 Hz, 1H),

7.58 (dd, J = 8.6, 7.1 Hz, 1H), 7.15−7.07 (m, 5H), 6.99 (dd, J = 8.8, 2.4

Hz, 1H), 5.07 (dd, J = 12.5, 5.5 Hz, 1H), 4.30 (s, 1H), 4.16 (s, 1H), 4.06

(d, J = 34.1 Hz, 2H), 3.69 (s, 2H), 3.46 (t, J = 6.7 Hz, 2H), 3.22 (ddd, J =

59.9, 21.4, 19.4 Hz, 6H), 2.92−2.67 (m, 3H), 2.16−2.10 (m, 1H), 1.93

(ddd, J = 11.5, 10.2, 6.8 Hz, 2H), 1.83−1.74 (m, 2H). UPLC−MS

calculated for C₄₃H₄₉ClN₇O₆[M + H]⁺: 794.34, found: 794.18. UPLC-

retention time: 4.9 min, purity >95%.

yl)piperidin-4-yl)piperazin-1-yl)benzamide (27). ¹H NMR (400

MHz, MeOH-d₄) δ 10.82 (s, 1H), 7.83 (d, J = 8.9 Hz, 2H), 7.74

(dd, J = 8.6, 1.0 Hz, 2H), 7.44 (d, J = 2.2 Hz, 1H), 7.32 (dd, J = 8.6, 2.3

Hz, 1H), 7.13 (dd, J = 11.8, 5.7 Hz, 3H), 7.00 (dd, J = 8.8, 2.4 Hz, 1H),

5.10 (dd, J = 12.5, 5.5 Hz, 1H), 4.33−4.22 (m, 3H), 4.16 (s, 1H), 4.07

(d, J = 34.3 Hz, 1H), 3.63 (ddd, J = 11.9, 8.3, 3.8 Hz, 5H), 3.31−3.04

(m, 4H), 2.96−2.62 (m, 4H), 2.33 (d, J = 10.8 Hz, 2H), 2.18−2.08 (m,

1H), 1.89 (tt, J = 12.2, 6.0 Hz, 2H), 1.30 (s, 6H), 1.24 (s, 6H). ¹³C

NMR (100 MHz, MeOH-d₄) δ 173.23 (s), 170.24 (s), 169.14 (s),

167.89 (s), 167.43 (s), 163.00 (s), 154.72 (s), 152.00 (s), 137.57 (s),

135.33 (s), 134.35 (s), 128.78 (s), 125.80 (s), 124.81 (s), 119.83 (s),

118.49 (s), 116.57 (s), 115.74 (s), 114.85 (s), 114.33 (s), 108.48 (s),

104.35 (s), 84.42 (s), 63.49 (s), 58.88 (s), 49.09 (s), 48.64 (s), 46.17

(s), 45.47 (s), 40.32 (s), 30.79 (s), 25.74 (s), 23.02 (s), 22.26 (s).

UPLC−MS calculated for C₄₄H₄₈ClN₇O₆[M + H]⁺: 806.35, found:

806.32. UPLC-retention time: 4.9 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

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

5-yl)piperidin-4-yl)methyl)piperazin-1-yl)benzamide (28). ¹H NMR

(400 MHz, MeOH-d₄) δ 10.81 (s, 1H), 7.83 (d, J = 8.9 Hz, 2H), 7.72

(dd, J = 14.6, 8.6 Hz, 2H), 7.38 (d, J = 2.2 Hz, 1H), 7.26 (dd, J = 8.6, 2.3

Hz, 1H), 7.13 (dd, J = 10.5, 5.7 Hz, 3H), 7.00 (dd, J = 8.8, 2.4 Hz, 1H),

5.09 (dd, J = 12.5, 5.5 Hz, 1H), 4.31 (s, 1H), 4.17 (s, 1H), 4.09 (t, J =

20.0 Hz, 4H), 3.70 (d, J = 32.5 Hz, 3H), 3.24 (dd, J = 20.6, 12.6 Hz,

4H), 3.08 (t, J = 11.7 Hz, 2H), 2.91−2.82 (m, 1H), 2.80−2.65 (m, 2H),

2.27 (ddd, J = 11.1, 7.4, 4.0 Hz, 1H), 2.18−2.08 (m, 1H), 2.03−1.93

(m, 2H), 1.53−1.43 (m, 3H), 1.31 (s, 6H), 1.25 (s, 6H). ¹³C NMR

(100 MHz, MeOH-d₄) δ 173.24 (s), 170.29 (s), 169.15 (s), 168.08 (s),

167.55 (s), 163.00 (s), 155.39 (s), 152.05 (s), 137.57 (s), 135.34 (s),

134.33 (s), 128.79 (s), 125.72 (s), 124.77 (s), 118.92 (s), 118.05 (s),

116.58 (s), 115.74 (s), 114.84 (s), 114.33 (s), 108.10 (s), 104.35 (s),

100.00 (s), 84.42 (s), 61.64 (s), 58.88 (s), 51.98 (s), 49.05 (s), 44.99

(s), 40.33 (s), 30.84 (d, J = 8.2 Hz), 28.84 (s), 23.02 (s), 22.33 (d, J =

13.2 Hz). UPLC−MS calculated for C₄₅H₅₀ClN₇O₆[M + H]⁺: 820.36,

found: 820.24. UPLC-retention time: 4.7 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

yl)-2-azaspiro[3.3]heptan-6-yl)piperazin-1-yl)benzamide (35). ¹H

NMR (400 MHz, DMSO-d₆) δ 11.33 (s, 1H), 8.16 (d, J = 8.7 Hz,

1H), 8.08 (d, J = 8.7 Hz, 2H), 7.92 (d, J = 8.3 Hz, 1H), 7.86 (d, J = 9.2

Hz, 1H), 7.46 (d, J = 2.3 Hz, 1H), 7.34 (d, J = 8.8 Hz, 2H), 7.27 (dd, J =

8.8, 2.3 Hz, 1H), 7.06 (d, J = 1.6 Hz, 1H), 6.93 (dd, J = 8.4, 1.8 Hz, 1H),

5.32 (dd, J = 12.8, 5.3 Hz, 1H), 4.68−4.57 (m, 3H), 4.32 (s, 3H), 4.07

(dd, J = 16.1, 8.1 Hz, 3H), 3.77 (t, J = 32.8 Hz, 3H), 3.24−3.07 (m, 2H),

2.96−2.78 (m, 7H), 2.48−2.24 (m, 2H), 1.48 (s, 6H), 1.40 (s, 6H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 173.28 (s), 170.56 (s), 167.94 (s),

167.64 (s), 167.09 (s), 163.10 (s), 158.67 (s), 155.42 (s), 152.01 (s),

137.34 (s), 136.53 (s), 134.23 (s), 129.47 (s), 125.74 (s), 125.28 (s),

117.68 (s), 117.27 (s), 116.75 (s), 115.04 (d, J = 24.4 Hz), 114.74−

114.42 (m), 105.27 (s), 104.09 (s), 84.37 (s), 63.61 (s), 62.65 (s),

58.58 (s), 53.99 (s), 49.21 (s), 48.33 (s), 45.15 (s), 35.56 (s), 31.64 (s),

24.44 (s), 23.58 (s). UPLC−MS calculated for C₄₅H₄₉ClN₇O₆[M +

H]⁺: 818.34, found: 818.16. UPLC-retention time: 4.8 min, purity

>95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

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

5-yl)-2-azaspiro[3.3]heptan-6-yl)methyl)piperazin-1-yl)benzamide

(36). ¹H NMR (400 MHz, DMSO-d₆) δ 11.26 (s, 1H), 8.09 (d, J = 8.7

Hz, 1H), 8.01 (d, J = 8.8 Hz, 2H), 7.83 (s, 1H), 7.78 (d, J = 9.1 Hz, 1H),

7.39 (d, J = 2.4 Hz, 1H), 7.25 (d, J = 8.9 Hz, 2H), 7.21−7.19 (m, 1H),

6.97 (d, J = 1.9 Hz, 1H), 6.84 (dd, J = 8.4, 2.0 Hz, 1H), 5.25 (dd, J =

12.8, 5.4 Hz, 1H), 4.52 (s, 1H), 4.35−4.17 (m, 9H), 3.46 (d, J = 6.6 Hz,

3H), 3.12−3.03 (m, 1H), 2.92−2.71 (m, 5H), 2.37−2.14 (m, 4H), 1.41

(s, 6H), 1.32 (s, 6H). UPLC−MS calculated for C₄₆H₅₁ClN₇O₆[M +

H]⁺: 832.36, found: 832.19. UPLC-retention time: 4.9 min, purity

>95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

yl)azetidin-3-yl)piperazin-1-yl)benzamide (25). ¹H NMR (400 MHz,

MeOH-d₄) δ 10.74 (s, 1H), 7.77 (d, J = 8.9 Hz, 2H), 7.65 (dd, J = 8.4,

7.3 Hz, 2H), 7.06 (t, J = 5.8 Hz, 3H), 6.93 (dd, J = 8.8, 2.4 Hz, 1H), 6.88

(d, J = 2.0 Hz, 1H), 6.72 (dd, J = 8.3, 2.1 Hz, 1H), 5.07−5.01 (m, 1H),

4.40−4.30 (m, 5H), 4.24 (s, 1H), 4.11 (s, 1H), 3.55 (d, J = 57.2 Hz,

8H), 2.82−2.62 (m, 3H), 2.08 (ddd, J = 10.2, 8.9, 5.4 Hz, 1H), 1.25 (s,

6H), 1.18 (s, 6H). ¹³C NMR (100 MHz, MeOH-d₄) δ 173.24, 170.32,

169.15, 167.68, 167.65, 163.00, 154.40, 151.98, 137.56, 135.35, 134.12,

128.81, 125.78, 124.66, 119.54, 116.61, 115.83, 115.02, 114.95, 114.32,

105.05, 104.31, 84.42, 58.90, 54.84, 53.12, 49.26, 49.09, 45.18, 40.34,

30.82, 23.07, 22.37, 22.31. UPLC−MS calculated for C₄₂H₄₆ClN₇O₆

[M + H]⁺: 777.30, found: 777.25. UPLC-retention time: 4.8 min, purity

>95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

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

1

5-yl)azetidin-3-yl)methyl)piperazin-1-yl)benzamide (26). H NMR

(400 MHz, DMSO-d₆) δ 11.11 (s, 1H), 7.92 (d, J = 8.7 Hz, 1H), 7.86

(dd, J = 8.7, 3.8 Hz, 2H), 7.63 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 2.4 Hz,

1H), 7.11 (dd, J = 9.6, 4.4 Hz, 3H), 7.05−7.02 (m, 1H), 5.14−5.03 (m,

1H), 4.40−4.24 (m, 2H), 4.20−3.85 (m, 5H), 3.63 (ddd, J = 44.7, 25.1,

5.8 Hz, 5H), 3.23−3.14 (m, 2H), 2.97−2.86 (m, 1H), 2.66−2.49 (m,

5H), 2.43−2.32 (m, 1H), 2.11−2.00 (m, 1H), 1.26 (s, 6H), 1.17 (s,

6H). UPLC−MS calculated for C₄₃H₄₆ClN₇O₆[M + H]⁺: 792.33,

found: 792.23. UPLC-retention time: 4.7 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(4-(6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

yl)-6-azaspiro[3.4]octan-2-yl)piperazin-1-yl)benzamide (37). ¹H

NMR (400 MHz, DMSO-d₆) δ 11.08 (s, 1H), 7.93−7.88 (m, 1H),

7.83 (d, J = 8.6 Hz, 2H), 7.66 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 9.2 Hz,

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

P

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1H), 7.20 (s, 1H), 7.08 (d, J = 8.6 Hz, 2H), 7.01 (d, J = 8.8 Hz, 1H),

6.90 (d, J = 9.9 Hz, 1H), 6.80 (t, J = 7.7 Hz, 1H), 5.09−5.04 (m, 1H),

4.34 (s, 2H), 4.07 (d, J = 9.1 Hz, 2H), 3.95−3.89 (m, 1H), 3.55−3.40

(m, 6H), 3.08 (s, 3H), 2.90 (dd, J = 15.3, 10.5 Hz, 1H), 2.61 (d, J = 8.5

Hz, 1H), 2.51 (s, 1H), 2.40 (d, J = 7.5 Hz, 3H), 2.07 (ddd, J = 18.8, 12.9,

5.9 Hz, 3H), 1.23 (s, 6H), 1.14 (s, 6H). UPLC−MS calculated for

C₄₆H₅₁ClN₇O₆[M + H]⁺: 832.36, found: 832.21. UPLC-retention

time: 5.0 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

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

5-yl)-6-azaspiro[3.4]octan-2-yl)methyl)piperazin-1-yl)benzamide

(38). ¹H NMR (400 MHz, DMSO-d₆) δ 11.05 (s, 1H), 7.87 (d, J = 8.7

Hz, 1H), 7.79 (d, J = 8.7 Hz, 2H), 7.62 (dd, J = 8.4, 4.3 Hz, 1H), 7.57 (d,

J = 9.2 Hz, 1H), 7.17 (d, J = 2.3 Hz, 1H), 7.04 (d, J = 8.7 Hz, 2H), 6.98

(dd, J = 8.8, 2.4 Hz, 1H), 6.87 (dd, J = 12.7, 1.7 Hz, 1H), 6.81−6.72 (m,

1H), 5.03 (dd, J = 13.0, 4.9 Hz, 1H), 4.31 (s, 1H), 4.04 (d, J = 9.1 Hz,

1H), 3.97 (d, J = 8.5 Hz, 2H), 3.58−3.24 (m, 9H), 3.07 (d, J = 25.8 Hz,

4H), 2.83 (ddd, J = 22.7, 13.2, 8.3 Hz, 2H), 2.58 (d, J = 7.3 Hz, 1H),

2.48 (dd, J = 3.5, 1.7 Hz, 1H), 2.22−2.07 (m, 3H), 2.01−1.87 (m, 4H),

1.20 (s, 6H), 1.11 (s, 6H). UPLC−MS calculated for C₄₇H₅₃ClN₇O₆

[M + H]⁺: 846.37, found: 846.22. UPLC-retention time: 4.9 min, purity

>95%.

Methyl 4-(piperazin-1-yl)benzoate (62) (1 mmol) and methyl

iodide (1.1 equiv) were dissolved in DMF. K₂CO₃(1.2 equiv) was

added to the solution, and the reaction mixture was stirred at 60 °C for 2

h. Water was added; the reaction mixture was extracted by EtOAc and

washed by water; and the organic phase was dried by Na₂SO₄. The

product, methyl 4-(4-methylpiperazin-1-yl)benzoate (66) can be

obtained by removing the solvent under vacuum and puriﬁed by ﬂash

column in 83% yield.

NaOH (2 equiv) was added to a solution of methyl 4-(4-

methylpiperazin-1-yl)benzoate (66) (0.8 mmol) in MeOH/H₂O, and

the mixture was stirred at rt for 2 h. Then, the MeOH was removed

under reduced pressure; the pH was adjusted to acidity with 2 M HCl;

and the mixture was extracted with EtOAc. The solvent was removed to

aﬀord the product 4-(4-methylpiperazin-1-yl)benzoic acid (67), which

was used without further puriﬁcation.

DIPEA (5 equiv) and HATU (1.2 equiv) were added to a solution of

compounds 4-((1R,3R)-3-amino-2,2,4,4-tetramethylcyclobutoxy)-2-

chlorobenzonitrile (54) (0.5 mmol) and 4-(4-methylpiperazin-1-

yl)benzoic acid (67) (1.1 equiv) in DMF (2 mL). After stirring at rt

for 1 h, water was added and the reaction mixture was extracted by

EtOAc; it was then washed by water, and the organic phase was dried by

Na₂SO₄. The product (65) can be obtained by removing the solvent

under vacuum and puriﬁed by ﬂash column chromatography with 88%

yield.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(4-(3-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

1

yl)-3-azaspiro[5.5]undecan-9-yl)piperazin-1-yl)benzamide (39). H

5-Fluoro-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-

dione (INT-63). ¹H NMR (400 MHz, DMSO-d₆) δ 8.01 (dd, J = 8.3, 4.5

Hz, 1H), 7.84 (dd, J = 7.4, 2.2 Hz, 1H), 7.73 (ddd, J = 9.5, 8.3, 2.3 Hz,

1H), 5.24 (dd, J = 13.1, 5.4 Hz, 1H), 3.03 (s, 3H), 2.99−2.91 (m, 1H),

2.78 (ddd, J = 17.2, 4.3, 2.5 Hz, 1H), 2.62−2.53 (m, 1H), 2.10 (dtd, J =

12.9, 5.3, 2.5 Hz, 1H). UPLC−MS calculated for C₁₄H₁₁FN₂O₄[M +

H]⁺: 291.08, found: 290.94. UPLC-retention time: 3.1 min, purity

>95%.

NMR (400 MHz, DMSO-d₆) δ 11.18 (s, 1H), 8.00 (d, J = 8.7 Hz, 1H),

7.92 (d, J = 8.7 Hz, 2H), 7.77 (d, J = 8.5 Hz, 1H), 7.70 (d, J = 9.2 Hz,

1H), 7.42 (s, 1H), 7.36−7.29 (m, 2H), 7.18 (d, J = 8.9 Hz, 2H), 7.11

(dd, J = 8.8, 2.4 Hz, 1H), 5.17 (dd, J = 12.8, 5.4 Hz, 1H), 4.15 (dd, J =

17.4, 10.7 Hz, 3H), 3.75 (d, J = 10.3 Hz, 2H), 3.58 (s, 4H), 3.35 (d, J =

12.6 Hz, 3H), 3.19 (t, J = 11.8 Hz, 2H), 3.05−2.94 (m, 1H), 2.74−2.65

(m, 2H), 2.61 (dd, J = 5.5, 3.7 Hz, 2H), 2.18−1.89 (m, 6H), 1.80−1.69

(m, 4H), 1.54 (s, 2H), 1.33 (s, 6H), 1.24 (s, 6H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 173.29 (s), 170.59 (s), 168.14 (s), 167.46 (s), 167.10 (s),

163.10 (s), 159.00 (s), 158.65 (s), 155.41 (s), 152.02 (s), 137.34 (s),

136.53 (s), 134.47 (s), 129.47 (s), 125.74 (s), 125.44 (s), 117.83 (s),

117.27 (s), 116.75 (s), 115.16 (s), 114.83 (s), 104.09 (s), 84.37 (s),

58.58 (s), 49.22 (s), 48.32 (s), 45.46 (s), 40.78 (s), 33.94 (s), 30.48 (s),

24.44 (s), 23.58 (s), 21.86 (s). UPLC−MS calculated for

C₄₉H₅₇ClN₇O₆[M + H]⁺: 874.41, found: 874.25. UPLC-retention

time: 5.0 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

yl)-2-azaspiro[3.5]nonan-7-yl)piperazin-1-yl)benzamide (40). ¹H

NMR (400 MHz, MeOH-d₄) δ 10.80 (s, 1H), 7.85−7.82 (m, 2H),

7.74 (d, J = 8.7 Hz, 1H), 7.66 (d, J = 8.3 Hz, 1H), 7.15−7.10 (m, 3H),

7.01−6.98 (m, 1H), 6.84 (s, 1H), 6.68 (dd, J = 8.3, 1.7 Hz, 1H), 5.07

(dd, J = 12.4, 5.4 Hz, 1H), 4.31 (s, 1H), 4.17 (s, 1H), 4.10 (d, J = 11.4

Hz, 1H), 3.93−3.86 (m, 2H), 3.78 (s, 2H), 3.68 (d, J = 12.7 Hz, 2H),

3.38 (dd, J = 8.2, 3.2 Hz, 2H), 3.27−3.16 (m, 2H), 2.90−2.82 (m, 1H),

2.75 (dt, J = 14.2, 4.0 Hz, 2H), 2.23 (d, J = 9.4 Hz, 3H), 2.12 (ddd, J =

10.0, 5.3, 2.4 Hz, 1H), 1.85−1.61 (m, 5H), 1.31 (s, 6H), 1.25 (s, 6H).

UPLC−MS calculated for C₄₇H₅₃ClN₇O₆[M + H]⁺: 846.37, found:

846.19. UPLC-retention time: 5.0 min, purity >95%.

General Procedure for the Synthesis of Compounds 64 and 65.

Compounds 2-(2,6-dioxopiperidin-3-yl)-5-ﬂuoroisoindoline-1,3-dione

(56) (0.5 mmol) and methyl iodide (1.1 equiv) were dissolved in DMF.

K₂CO₃(1.2 equiv) was added to the solution, and the reaction mixture

was stirred at 60 °C for 2 h. Water was added; the reaction mixture was

extracted by EtOAc and washed with water; and the organic phase was

dried over Na₂SO₄. The compound Me-protected (63) (5-ﬂuoro-2-(1-

methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione) can be obtained

by removing the solvent under vacuum and puriﬁed by ﬂash column in

85% yield.

DIPEA (5 equiv) was added to a solution of the compound 61 (0.3

mmol) and 5-ﬂuoro-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-

1,3-dione 63 (1.1 equiv) in DMSO (2 mL). After 4 h at 100 °C, the

mixture was subject to HPLC puriﬁcation to aﬀord compounds 64 with

an 81% yield.

Methyl 4-(4-methylpiperazin-1-yl)benzoate (INT-66). ¹H NMR

(400 MHz, DMSO) δ 7.82−7.75 (m, 2H), 6.97 (d, J = 9.0 Hz, 2H),

3.78 (s, 3H), 2.25 (s, 3H), 1.92 (s, 1H). UPLC−MS calculated for

C₁₃H₁₄N₂O₂[M + H]⁺: 235.15, found: 235.08. UPLC-retention time:

0.6 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(4-((1-(2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoi-

soindolin-5-yl)piperidin-4-yl)methyl)piperazin-1-yl)benzamide

(64). ¹H NMR (400 MHz, MeOH-d₄) δ 7.83 (d, J = 8.9 Hz, 2H), 7.72

(dd, J = 12.7, 8.6 Hz, 2H), 7.39 (d, J = 2.2 Hz, 1H), 7.27 (dd, J = 8.6, 2.3

Hz, 1H), 7.13 (dd, J = 10.6, 5.7 Hz, 3H), 7.00 (dd, J = 8.8, 2.4 Hz, 1H),

5.12 (dd, J = 12.9, 5.4 Hz, 1H), 4.31 (s, 1H), 4.19−4.07 (m, 4H), 3.21

(d, J = 6.9 Hz, 2H), 3.16 (s, 3H), 3.09 (t, J = 11.7 Hz, 2H), 2.90 (dd, J =

7.0, 3.8 Hz, 2H), 2.77−2.70 (m, 1H), 2.68 (s, 1H), 2.32−2.23 (m, 1H),

2.14−2.08 (m, 1H), 1.98 (d, J = 12.6 Hz, 2H), 1.54−1.36 (m, 3H), 1.30

(s, 6H), 1.24 (s, 6H). UPLC−MS calculated for C₄₆H₅₃ClN₇O₆[M +

H]⁺: 834.37, found: 834.36. UPLC-retention time: 4.8 min, purity

>95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(4-methylpiperazin-1-yl)benzamide (65). ¹H NMR (400

MHz, MeOH-d₄) δ 7.75−7.69 (m, 2H), 7.61 (d, J = 8.7 Hz, 1H), 7.00

(dd, J = 9.5, 5.7 Hz, 3H), 6.88 (dd, J = 8.8, 2.4 Hz, 1H), 4.20 (s, 1H),

4.06 (s, 1H), 3.94 (d, J = 12.7 Hz, 2H), 3.57 (dd, J = 13.3, 8.8 Hz, 2H),

3.22−3.06 (m, 4H), 2.89 (s, 3H), 1.20 (s, 6H), 1.13 (s, 6H). UPLC−

MS calculated for C₂₇H₃₄ClN₇O₇[M + H]⁺: 481.24, found: 481.26.

UPLC-retention time: 3.5 min, purity >95%.

General Procedure for the Synthesis of Compounds 29−32.

DIPEA (5 equiv) and HATU (1.2 equiv) were added to a solution of

compounds 54 (2 mmol) and 66 (1.1 equiv) in DMF (2 mL). After

stirring at rt for 1 h, water was added and the reaction mixture was

extracted by EtOAc; it was then washed by water, and the organic phase

was dried by Na₂SO₄. Compound 67 was obtained by removing the

solvent under vacuum and puriﬁed by ﬂash column with 84% yield.

NaOH (2 equiv) was added to a solution of 67 (1 mmol) in MeOH/

H₂O and stirred at rt for 2 h. Then, the MeOH was removed under

reduced pressure; the pH was adjusted to <7 with 2 M HCl; and the

mixture was extracted with EtOAc. The solvent was removed to aﬀord

the product 68, which was used without further puriﬁcation.

Q

https://doi.org/10.1021/acs.jmedchem.1c00882

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Article

Compounds 68 (0.8 mmol) and 69 (1.2 equiv) were dissolved in

DMF. DIPEA (5 equiv) and HATU (1.2 equiv) was added to the

solution, and the reaction mixture was stirred at rt for 1 h. Water was

then added,; the reaction mixture was extracted by EtOAc and washed

with water; and the organic phase was dried by Na₂SO₄. The

compound, Boc-protected-70 was obtained by removing the solvent

under vacuum and puriﬁed by ﬂash column chromatography. This

desired intermediate 70 was obtained by deprotection with TFA in

DCM in 89% yield.

A solution of compounds 70 (0.5 mmol) and a series of aldehydes or

ketones was added AcOH (10%) in DCE. After the mixture was stirred

at rt for 10 min, NaBH(OAc)₃(1.2 equiv) was added and the mixture

was stirred at rt for a further 2 h. The compound Boc-protected-71 was

obtained by removing the solvent under vacuum and puriﬁed by ﬂash

column chromatography. Intermediate 71 was obtained by depro-

tection with TFA in DCM in 70−85% yields in total.

162.96 (s), 154.70 (s), 137.57 (s), 136.91 (s), 136.53 (s), 135.35 (s),

134.32 (s), 127.74 (s), 127.13 (s), 124.80 (s), 119.80 (s), 118.48 (s),

116.60 (s), 115.74 (s), 114.31 (s), 108.47 (s), 104.39 (s), 84.37 (s),

63.82 (s), 59.14 (s), 49.08 (s), 48.41 (s), 46.16 (s), 40.33 (s), 30.79 (s),

25.67 (s), 23.04 (s), 22.33 (d, J = 7.3 Hz), 7.80 (s). UPLC−MS

calculated for C₄₄H₄₇ClN₇O₇[M + H]⁺: 820.32, found: 820.19. UPLC-

retention time: 4.4 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

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

5-yl)piperidin-4-yl)methyl)piperazine-1-carbonyl)benzamide (32).

¹H NMR (400 MHz, MeOH-d₄) δ 10.80 (s, 1H), 7.96 (d, J = 8.0

Hz, 2H), 7.75 (d, J = 8.7 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.64 (d, J =

8.1 Hz, 2H), 7.36 (d, J = 1.9 Hz, 1H), 7.25 (dd, J = 8.6, 2.0 Hz, 1H), 7.15

(d, J = 2.3 Hz, 1H), 7.01 (dd, J = 8.7, 2.3 Hz, 1H), 5.09 (dd, J = 12.4, 5.4

Hz, 1H), 4.32 (s, 1H), 4.21 (s, 1H), 4.09 (d, J = 13.3 Hz, 2H), 3.86 (d, J

= 30.7 Hz, 3H), 3.42 (d, J = 26.0 Hz, 3H), 3.27−3.22 (m, 1H), 3.18 (d, J

= 6.8 Hz, 2H), 3.05 (dd, J = 14.6, 9.3 Hz, 2H), 2.92−2.83 (m, 1H),

2.80−2.68 (m, 2H), 2.29−2.20 (m, 1H), 2.18−2.10 (m, 1H), 2.01−

1.93 (m, 2H), 1.45 (dq, J = 20.7, 9.1 Hz, 3H), 1.33 (s, 6H), 1.26 (s, 6H).

¹³C NMR (100 MHz, MeOH-d₄) δ 173.26 (s), 170.21 (d, J = 19.5 Hz),

168.85 (s), 168.07 (s), 167.55 (s), 162.96 (s), 155.37 (s), 137.57 (s),

136.96 (s), 136.53 (s), 135.36 (s), 134.31 (s), 127.77 (s), 127.07 (s),

124.77 (s), 118.86 (s), 118.03 (s), 116.61 (s), 114.31 (s), 108.09 (s),

104.38 (s), 84.37 (s), 61.81 (s), 59.14 (s), 51.75 (s), 49.05 (s), 40.34

(s), 30.82 (s), 28.81 (s), 23.05 (s), 22.35 (d, J = 8.4 Hz), 7.80 (s).

UPLC−MS calculated for C₄₆H₅₁ClN₇O₇[M + H]⁺: 848.35, found:

848.19. UPLC-retention time: 4.6 min, purity >95%.

DIPEA (5 equiv) was added to a solution of compounds 71 (0.3

mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-ﬂuoroisoindoline-1,3-dione

56 (1.1 equiv) in DMSO (2 mL). After 4 h at 100 °C, the mixture was

subject to HPLC puriﬁcation to aﬀord compounds 29−32 with 70−

90% yields.

4-(((1r, 3r)-3-(3-Chloro-4-cyanophenoxy)-2, 2, 4, 4-

1

tetramethylcyclobutyl)carbamoyl)benzoic acid (INT-68). H NMR

(400 MHz, DMSO-d₆) δ 13.22 (s, 1H), 8.03 (t, J = 5.8 Hz, 3H), 7.95−

7.90 (m, 2H), 7.21 (d, J = 2.3 Hz, 1H), 7.01 (dd, J = 8.8, 2.3 Hz, 1H),

4.33 (s, 1H), 4.09 (d, J = 9.1 Hz, 1H), 1.25 (s, 6H), 1.15 (s, 6H).

UPLC−MS calculated for C₂₃H₂₄ClN₂O₄[M + H]⁺: 427.14, found:

427.18. UPLC-retention time: 5.4 min, purity >95%.

N-((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(piperazine-1-carbonyl)benzamide (INT-70). ¹H NMR

(400 MHz, DMSO-d₆) δ 9.18 (s, 1H), 7.99−7.91 (m, 3H), 7.57 (d, J =

8.3 Hz, 2H), 7.20 (d, J = 2.4 Hz, 1H), 7.01 (dd, J = 8.8, 2.4 Hz, 1H), 4.34

(s, 1H), 4.10 (d, J = 9.1 Hz, 1H), 3.69 (d, J = 106.1 Hz, 4H), 3.20 (s,

4H), 1.25 (s, 6H), 1.15 (s, 6H). UPLC−MS calculated for

C₂₇H₃₂ClN₄O₃[M + H]⁺: 495.22, found: 495.24. UPLC-retention

time: 3.8 min, purity >95%.

General Procedure for the Synthesis of Compounds 33 and 34.

DIPEA (5 equiv) was added to a solution of compounds 69 (2 mmol)

and 2-(2,6-dioxopiperidin-3-yl)-5-ﬂuoroisoindoline-1,3-dione (56)

(1.1 equiv) in DMSO. After stirring at 80 °C for 4 h, water was

added; the reaction mixture was extracted by EtOAc and washed by

water; and the organic phase was dried by Na₂SO₄. The product (72)

can be obtained by removing the solvent under vacuum and puriﬁed by

ﬂash column chromatography with following deprotection by TFA with

82% yield.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

yl)azetidin-3-yl)piperazine-1-carbonyl)-benzamide (29). ¹H NMR

(400 MHz, DMSO-d₆) δ 11.17 (s, 1H), 8.03 (dd, J = 14.4, 6.2 Hz, 3H),

7.81 (d, J = 8.3 Hz, 1H), 7.67 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 2.3 Hz,

1H), 7.10 (dd, J = 8.8, 2.4 Hz, 1H), 7.00 (d, J = 1.7 Hz, 1H), 6.85 (dd, J

= 8.4, 1.9 Hz, 1H), 5.16 (dd, J = 12.8, 5.3 Hz, 1H), 4.53−4.25 (m, 6H),

4.18 (d, J = 9.0 Hz, 1H), 4.06−3.89 (m, 1H), 3.71 (s, 2H), 3.30 (d, J =

30.5 Hz, 3H), 3.06−2.90 (m, 1H), 2.79−2.51 (m, 3H), 2.18−2.06 (m,

1H), 1.45−1.35 (m, 1H), 1.33 (s, 6H), 1.24 (s, 6H). UPLC−MS

calculated for C₄₃H₄₅ClN₇O₇[M + H]⁺: 806.31, found: 806.23. UPLC-

retention time: 5.0 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

yl)piperidin-4-yl)piperazine-1-carbonyl)enzamide (30). ¹H NMR

(400 MHz, MeOH-d₄) δ 10.79 (s, 1H), 7.96−7.92 (m, 2H), 7.72

(dd, J = 11.5, 6.1 Hz, 2H), 7.64−7.60 (m, 2H), 7.40 (d, J = 2.2 Hz, 1H),

7.28 (dd, J = 8.6, 2.3 Hz, 1H), 7.13 (d, J = 2.4 Hz, 1H), 6.99 (dd, J = 8.8,

2.4 Hz, 1H), 5.11−5.06 (m, 1H), 4.31−4.18 (m, 4H), 3.77 (dd, J =

36.2, 29.7 Hz, 2H), 3.65−3.36 (m, 5H), 3.21 (d, J = 7.3 Hz, 1H), 3.06

(t, J = 12.2 Hz, 2H), 2.93−2.82 (m, 1H), 2.79−2.67 (m, 2H), 2.27 (d, J

= 10.5 Hz, 2H), 2.17−2.06 (m, 1H), 1.85 (td, J = 12.1, 8.4 Hz, 2H),

1.32 (d, J = 7.3 Hz, 2H), 1.30 (s, 6H), 1.24 (s, 6H). UPLC−MS

calculated for C₄₅H₄₉ClN₇O₇[M + H]⁺: 834.34, found: 834.18. UPLC-

retention time: 4.3 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

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

5-yl)azetidin-3-yl)methyl)piperazine-1-carbonyl)benzamide (31).

¹H NMR (400 MHz, MeOH-d₄) δ 7.77 (d, J = 8.9 Hz, 2H), 7.65

(dd, J = 8.4, 7.3 Hz, 2H), 7.06 (t, J = 5.8 Hz, 3H), 6.93 (dd, J = 8.8, 2.4

Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.72 (dd, J = 8.3, 2.1 Hz, 1H), 5.07−

5.01 (m, 1H), 4.40−4.30 (m, 5H), 4.24 (s, 1H), 4.11 (s, 1H), 3.55 (d, J

= 57.2 Hz, 8H), 2.82−2.62 (m, 3H), 2.08 (ddd, J = 10.2, 8.9, 5.4 Hz,

1H), 1.25 (s, 6H), 1.18 (s, 6H). ¹³C NMR (100 MHz, MeOH-d₄) δ

173.23 (s), 170.25 (s), 170.02 (s), 168.85 (s), 167.89 (s), 167.45 (s),

DIPEA (5 equiv) was added to a solution of compounds 73 (1.6

mmol) and 74 (1.1 equiv) in DMSO. After stirring at 80 °C for 4 h,

water was added and the reaction mixture was extracted by EtOAc,

washed by water and the organic phase was dried by Na₂SO₄.

Compound 75 can be obtained by removing the solvent under vacuum

and puriﬁed by ﬂash column chromatography with 85−90% yields.

NaOH (2 equiv) was added to a solution of 75 (1 mmol) in MeOH/

H₂O and stirred at rt for 2 h. Then, the MeOH was removed under

reduced pressure; the pH was adjusted to <7 with 2 M HCl; and the

mixture was extracted with EtOAc. The solvent was removed to aﬀord

the product (76), which was used without further puriﬁcation.

DIPEA (5 equiv) and HATU (1.2 equiv) were added to a solution of

compounds 54 (0.8 mmol) and 76 (1.1 equiv) in DMF. After stirring at

rt for 1 h, water was added; the reaction mixture was extracted with

EtOAc and washed by water; and the organic phase was dried by

Na₂SO₄. Compound 77 can be obtained by removing the solvent under

vacuum and puriﬁed by ﬂash column with 88% yield.

DMP (2 equiv) was added to a solution of compound 77 (0.5 mmol)

in DCM. After the reaction was stirred at rt for 2 h, compound 78 was

obtained by removing the solvent under vacuum and puriﬁed by ﬂash

column with 80−90% yields.

A solution of compounds 78 (0.5 mmol) and 72 (1.2 equiv) was

added to AcOH (10%) in DCE. After the mixture was stirred at rt for 10

min, NaBH(OAc)₃(1.2 equiv) was added and the mixture was stirred at

rt for another 2 h. Then, the desired compounds 33 and 34 can be

obtained by removing the solvent under vacuum and puriﬁed by ﬂash

column chromatography with 85−90% yields.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

yl)piperazin-1-yl)piperidin-1-yl)benzamide (33). ¹H NMR (400

MHz, MeOH-d₄) δ 10.76 (s, 1H), 7.75−7.64 (m, 4H), 7.40 (d, J =

2.2 Hz, 1H), 7.28 (dd, J = 8.5, 2.3 Hz, 1H), 7.07 (d, J = 2.4 Hz, 1H), 7.01

(d, J = 9.0 Hz, 2H), 6.93 (dd, J = 8.8, 2.4 Hz, 1H), 5.05 (dd, J = 12.5, 5.5

Hz, 1H), 4.24 (s, 1H), 4.09 (d, J = 0.5 Hz, 1H), 4.06 (d, J = 13.3 Hz,

R

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3H), 3.78−3.26 (m, 8H), 2.95−2.85 (m, 2H), 2.84−2.77 (m, 1H),

2.74−2.61 (m, 2H), 2.25 (d, J = 11.5 Hz, 2H), 2.07 (ddd, J = 10.0, 5.3,

2.5 Hz, 1H), 1.84 (qd, J = 12.1, 3.7 Hz, 2H), 1.24 (s, 6H), 1.17 (s, 6H).

¹³C NMR (100 MHz, MeOH-d₄) δ 173.21 (s), 170.23 (s), 169.25 (s),

UPLC−MS calculated for C₄₅H₅₀ClN₆O₆[M + H]⁺: 805.35, found:

805.20. UPLC-retention time: 7.0 min, purity >95%.

General Procedure for the Synthesis of Compounds 43−47. NaH

(1.2 equiv) was added to a solution of 83 (10 mmol) in dry DMF at 0

°C. After stirring the mixture at 0 °C for 20 min, 52 was added and the

mixture was stirred at rt for 4 h. After UPLC-MS demonstrated the full

conversion of starting materials, H₂O was added; the mixture was

extracted three times with EtOAc; and the combined organic layers

were washed with brine and then dried over anhydrous Na₂SO₄. The

solvent was removed on a rotary evaporator. The Boc-protected

intermediate was obtained by ﬂash column chromatography. The

desired intermediate (84) was obtained by deprotection with TFA in

DCM in 85% yield.

DIPEA (5 equiv) and HATU (1.2 equiv) were added to a solution of

compounds 84 (2 mmol) and 58 (1.1 equiv) in DMF (2 mL). After

stirring at rt for 1 h, water was added; the reaction mixture was extracted

by EtOAc and washed by water; and the organic phase was dried by

Na₂SO₄. The Boc protected compound (85) can be obtained by

removing the solvent under vacuum and puriﬁed by ﬂash column

chromatography with 80% yield. Then, the desired intermediate (85)

was obtained by deprotection with TFA in DCM in 70−80% yields in

total.

AcOH (10%) was added to a solution of compounds 85 (1 mmol)

and aldehyde 86 in DCE. After the mixture was stirred at rt for 10 min,

NaBH(OAc)₃(1.2 equiv) was added and the mixture was stirred at rt

for another 2 h. The Boc-protected intermediates 87 were obtained by

ﬂash column chromatography. Then, the desired intermediate 87 were

obtained by deprotection with TFA in DCM in 73−84% yields in total.

DIPEA (5 equiv) was added to a solution of the compounds 87 (0.5

mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-ﬂuoroisoindoline-1,3-dione

(56) (1.1 equiv) in DMSO (2 mL). After stirring at 100 °C for 4 h, the

mixture was subject to HPLC puriﬁcation to aﬀord compounds 43 and

44 with 70−80% yields. Following the procedures used to prepare

compounds 43 and 44, compounds 45−47 were obtained using the

same methods.

167.59 (s), 167.30 (s), 163.01 (s), 154.32 (s), 152.50 (s), 137.56 (s),

135.33 (s), 134.14 (s), 128.76 (s), 124.74 (s), 124.28 (s), 121.55 (s),

119.15 (s), 116.59 (s), 115.79 (s), 114.56 (s), 114.32 (s), 109.08 (s),

104.32 (s), 84.43 (s), 63.76 (s), 58.83 (s), 49.16 (s), 48.33 (s), 46.72

(s), 44.93 (s), 40.33 (s), 30.79 (s), 25.80 (s), 23.03 (s), 22.30 (d, J = 6.1

Hz). UPLC−MS calculated for C₄₄H₄₉ClN₇O₆[M + H]⁺: 806.34,

found: 806.18. UPLC-retention time: 4.5 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

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

5-yl)piperazin-1-yl)methyl)piperidin-1-yl)benzamide (34). ¹H NMR

(400 MHz, MeOH-d₄) δ 10.84 (s, 1H), 7.84−7.72 (m, 4H), 7.51 (d, J =

2.2 Hz, 1H), 7.38 (dd, J = 8.5, 2.3 Hz, 1H), 7.15 (d, J = 2.4 Hz, 1H), 7.06

(d, J = 9.0 Hz, 2H), 7.01 (dd, J = 8.8, 2.4 Hz, 1H), 5.14−5.10 (m, 1H),

4.32 (s, 1H), 4.16 (s, 1H), 3.98 (d, J = 13.0 Hz, 3H), 3.87−3.35 (m,

7H), 3.22 (d, J = 7.0 Hz, 2H), 3.01−2.85 (m, 3H), 2.83−2.70 (m, 2H),

2.26−2.11 (m, 2H), 2.06−1.94 (m, 2H), 1.59−1.44 (m, 2H), 1.31 (s,

6H), 1.25 (s, 6H). UPLC−MS calculated for C₄₅H₅₁ClN₇O₆[M + H]⁺:

820.36, found: 820.22. UPLC-retention time: 4.7 min, purity >95%.

General Procedure for the Synthesis of Compounds 41 and 42.

DIPEA (5 equiv) was added to a solution of compounds 73 (2 mmol)

and 79 (1.1 equiv) in DMSO. After stirring at 80 °C for 4 h, water was

added; the reaction mixture was extracted by EtOAc and washed by

water; and the organic phase was dried by Na₂SO₄. Compound 80 was

obtained by removing the solvent under vacuum and puriﬁed by ﬂash

column chromatography with 88% yield.

NaOH (2 equiv) was added to a solution of 80 (1 mmol) in MeOH/

H₂O and stirred at rt for 2 h. Then, the MeOH was removed under

reduced pressure; the pH was adjusted to acidity with 2 M HCl; and the

mixture was extracted with EtOAc. The solvent was removed to aﬀord

product 81, which was used without further puriﬁcation.

DIPEA (5 equiv) and HATU (1.2 equiv) were added to a solution of

compounds 54 (0.8 mmol) and 81 (1.1 equiv) in DMF. After stirring at

rt for 1 h, water was added; the reaction mixture was extracted by

EtOAc and washed by water; and the organic phase was dried by

Na₂SO₄. Compound 82 can be obtained by removing the solvent under

vacuum and puriﬁed by ﬂash column with following deprotection with

80−90% yield.

DIPEA (5 equiv) was added to a solution of compounds 82 (0.5

mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-ﬂuoroisoindoline-1,3-dione

(56) (1.1 equiv) in DMSO (2 mL). After 4 h at 100 °C, the mixture was

subject to HPLC puriﬁcation to aﬀord compounds 41 and 42 with 70−

80% yields.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

yl)amino)-7-azaspiro[3.5]nonan-7-yl)benzamide (41). ¹H NMR

(400 MHz, MeOH-d₄) δ 10.80 (s, 1H), 7.83 (d, J = 8.9 Hz, 2H),

7.74 (d, J = 8.7 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.9 Hz,

2H), 8.09−6.16 (m, 14H), 7.15 (d, J = 2.4 Hz, 1H), 7.01 (dd, J = 8.8, 2.4

Hz, 1H), 6.96 (d, J = 2.0 Hz, 1H), 6.83 (dd, J = 8.4, 2.1 Hz, 1H), 5.09−

5.05 (m, 1H), 4.31 (s, 1H), 4.17 (s, 1H), 4.13−4.07 (m, 1H), 3.50−

3.45 (m, 2H), 3.40 (d, J = 5.4 Hz, 2H), 2.94−2.67 (m, 4H), 2.59−2.51

(m, 2H), 2.16−2.08 (m, 1H), 1.96 (dd, J = 6.3, 4.4 Hz, 2H), 1.89−1.82

(m, 4H), 1.31 (s, 6H), 1.25 (s, 6H). UPLC−MS calculated for

C₄₃H₄₆ClN₆O₆[M + H]⁺: 777.32, found: 777.16. UPLC-retention

time: 6.6 min, purity >95%.

N-((1S,3S)-3-(3-Chloro-4-cyanophenoxy)-2,2-dimethylcyclobu-

tyl)-4-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-

piperidin-4-yl)methyl)piperazin-1-yl)benzamide (43). ¹H NMR (400

MHz, MeOH-d₄) δ 7.77 (d, J = 8.9 Hz, 2H), 7.65 (dd, J = 8.4, 7.3 Hz,

2H), 7.06 (t, J = 5.8 Hz, 3H), 6.93 (dd, J = 8.8, 2.4 Hz, 1H), 6.88 (d, J =

2.0 Hz, 1H), 6.72 (dd, J = 8.3, 2.1 Hz, 1H), 5.07−5.01 (m, 1H), 4.40−

4.30 (m, 5H), 4.24 (s, 1H), 4.11 (s, 1H), 3.55 (d, J = 57.2 Hz, 8H),

2.82−2.62 (m, 3H), 2.08 (ddd, J = 10.2, 8.9, 5.4 Hz, 1H), 1.25 (s, 6H),

1.18 (s, 6H). UPLC−MS calculated for C₄₃H₄₇ClN₇O₆[M + H]⁺:

792.33, found: 792.22. UPLC-retention time: 4.5 min, purity >95%.

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)cyclobutyl)-4-(4-((4-(2-

(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)-

methyl)piperazin-1-yl)benzamide (44). ¹H NMR (400 MHz, DMSO-

d₆) δ 11.08 (s, 1H), 8.14 (d, J = 7.2 Hz, 1H), 7.90 (dd, J = 8.7, 4.4 Hz,

1H), 7.83 (d, J = 8.7 Hz, 2H), 7.68 (t, J = 7.0 Hz, 1H), 7.37 (s, 1H), 7.27

(dd, J = 11.0, 6.4 Hz, 2H), 7.09−7.02 (m, 3H), 4.52 (t, J = 7.2 Hz, 1H),

4.10 (d, J = 12.7 Hz, 2H), 3.95 (dd, J = 16.9, 8.0 Hz, 3H), 3.69−3.57 (m,

2H), 3.12 (d, J = 5.5 Hz, 3H), 3.02 (t, J = 11.8 Hz, 2H), 2.90 (t, J = 13.0

Hz, 1H), 2.75 (dt, J = 11.5, 7.7 Hz, 1H), 2.65−2.46 (m, 6H), 2.33−2.17

(m, 2H), 2.05−1.99 (m, 1H), 1.88 (d, J = 11.6 Hz, 2H), 1.29−1.25 (m,

2H). UPLC−MS calculated for C₄₁H₄₂ClN₇O₆[M + H]⁺: 764.30,

found: 764.24. UPLC-retention time: 3.8 min, purity >95%.

N-((1R,3R)-3-(4-Cyano-3-ﬂuorophenoxy)-2,2,4,4-tetramethylcy-

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

5-yl)piperidin-4-yl)methyl)piperazin-1-yl)benzamide (45). ¹H NMR

(400 MHz, MeOH-d₄) δ 7.77 (d, J = 8.9 Hz, 2H), 7.65 (dd, J = 8.4, 7.3

Hz, 2H), 7.06 (t, J = 5.8 Hz, 3H), 6.93 (dd, J = 8.8, 2.4 Hz, 1H), 6.88 (d,

J = 2.0 Hz, 1H), 6.72 (dd, J = 8.3, 2.1 Hz, 1H), 5.07−5.01 (m, 1H),

4.40−4.30 (m, 5H), 4.24 (s, 1H), 4.11 (s, 1H), 3.55 (d, J = 57.2 Hz,

8H), 2.82−2.62 (m, 3H), 2.08 (ddd, J = 10.2, 8.9, 5.4 Hz, 1H), 1.25 (s,

6H), 1.18 (s, 6H). UPLC−MS calculated for C₄₅H₅₁FN₇O₆[M + H]⁺:

804.39, found: 804.30. UPLC-retention time: 5.6 min, purity >95%.

N-((1R,3R)-3-(4-Cyano-3-(triﬂuoromethyl)phenoxy)-2,2,4,4-tetra-

methylcyclobutyl)-4-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoi-

soindolin-5-yl)piperidin-4-yl)methyl)piperazin-1-yl)benzamide

(46). ¹H NMR (400 MHz, MeOH-d₄) δ 7.77 (d, J = 8.9 Hz, 2H), 7.65

N-((1R,3R)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-

clobutyl)-4-(9-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-

1

yl)amino)-3-azaspiro[5.5]undecan-3-yl)benzamide (42). H NMR

(400 MHz, DMSO-d₆) δ 11.08 (s, 1H), 7.91 (dd, J = 11.9, 8.9 Hz, 1H),

7.79 (s, 1H), 7.56 (d, J = 5.5 Hz, 2H), 7.21 (d, J = 11.5 Hz, 1H), 7.11−

6.93 (m, 4H), 6.89 (d, J = 11.8 Hz, 1H), 5.04 (d, J = 5.2 Hz, 1H), 4.33

(d, J = 12.0 Hz, 2H), 4.11−4.04 (m, 1H), 3.46 (s, 1H), 3.33 (s, 3H),

3.03 (d, J = 12.1 Hz, 1H), 2.90 (d, J = 12.0 Hz, 1H), 2.66−2.49 (m, 3H),

2.02−1.91 (m, 1H), 1.75 (dd, J = 37.5, 23.0 Hz, 5H), 1.52 (s, 1H), 1.37

(dd, J = 24.9, 12.3 Hz, 3H), 1.22 (s, 6H), 1.15 (s, 6H), 1.03 (s, 1H).

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(dd, J = 8.4, 7.3 Hz, 2H), 7.06 (t, J = 5.8 Hz, 3H), 6.93 (dd, J = 8.8, 2.4

Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.72 (dd, J = 8.3, 2.1 Hz, 1H), 5.07−

5.01 (m, 1H), 4.40−4.30 (m, 5H), 4.24 (s, 1H), 4.11 (s, 1H), 3.55 (d, J

= 57.2 Hz, 8H), 2.82−2.62 (m, 3H), 2.08 (ddd, J = 10.2, 8.9, 5.4 Hz,

1H), 1.25 (s, 6H), 1.18 (s, 6H). UPLC−MS calculated for

C₄₆H₅₁F₃N₇O₆[M + H]⁺: 854.39, found: 854.28. UPLC-retention

time: 6.2 min, purity >95%.

N-((1R,3R)-3-((6-Cyano-5-(triﬂuoromethyl)pyridin-3-yl)oxy)-

2,2,4,4-tetramethyl-cyclobutyl)-4-(4-((1-(2-(2,6-dioxopiperidin-3-

yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methyl)piperazin-1-yl)-

benzamide (47). ¹H NMR (400 MHz, MeOH-d₄) δ 7.77 (d, J = 8.9 Hz,

2H), 7.65 (dd, J = 8.4, 7.3 Hz, 2H), 7.06 (t, J = 5.8 Hz, 3H), 6.93 (dd, J =

8.8, 2.4 Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.72 (dd, J = 8.3, 2.1 Hz, 1H),

5.07−5.01 (m, 1H), 4.40−4.30 (m, 5H), 4.24 (s, 1H), 4.11 (s, 1H),

3.55 (d, J = 57.2 Hz, 8H), 2.82−2.62 (m, 3H), 2.08 (ddd, J = 10.2, 8.9,

5.4 Hz, 1H), 1.25 (s, 6H), 1.18 (s, 6H). UPLC−MS calculated for

C₄₅H₅₀F₃N₈O₆[M + H]⁺: 855.38, found: 855.30. UPLC-retention

time: 5.1 min, purity >95%.

Cell Lines and Cell Culture. All the LNCaP and VCaP cells used

were purchased from American Type Culture Collection (ATCC).

LNCaP and VCaP cells were grown in RPMI 1640 (Invitrogen), and

VCaP cells were grown in DMEM with Glutamax (Invitrogen). All of

the cells were supplemented with 10% fetal bovine serum (Invitrogen)

at 37 °C in a humidiﬁed 5% CO₂incubator. Cell viability was evaluated

by a WST-8 assay (Dojindo) following the manufacturer’s instructions.

Western blot analysis was performed as previously described.^55,57

Quantitative Real-Time Polymerase Chain Reaction (qRT-

PCR). Real-time PCR was performed using a QuantStudio 7 Flex Real-

Time PCR system as described previously.^55,57RNA was puriﬁed using

the Qiagen RNase-Free DNase set, and then after quantiﬁcation, the

extracted RNA was converted to cDNA using a High Capacity RNA-to-

cDNA Kit from Applied Biosystems (Thermo Fisher Scientiﬁc). The

levels of AR, TMPRSS2, FKBP5, PSA (KLK3), and GAPDH were

quantiﬁed using a TaqMan Fast Advanced Master Mix from Applied

Biosystems. The level of gene expression was evaluated using a

comparative CT method, which compares the CT value to GAPDH

(ΔCT) and then to vehicle control (ΔΔCT).

Western Blotting. Treated cells were lysed by RIPA buﬀer

supplemented with protease and phosphatase inhibitors. The cell

lysates were separated by 4−12% SDS-PAGE gels and blotted into

PVDF membranes. Software ImageJ was used to quantify the

percentage of AR degradation. The net protein bands and loading

controls are calculated by deducting the background from the inverted

band value. The ﬁnal relative quantiﬁcation values are the ratio of net

band to net loading control.⁶⁵

PK, PK/PD, and Eﬃcacy Studies in Mice. All in vivo studies were

performed under animal protocol (PRO00009463) approved by the

Institutional Animal Care & Use Committee (IACUC) of the

University of Michigan, in accordance with the recommendations in

the Guide for the Care and Use of Laboratory Animals of the National

Institutes of Health.

To grow VCaP xenograft tumors, male CB17 SCID mice (Charles

River Laboratories) were injected subcutaneously with 5 × 10⁶VCaP

cells (ATCC) in 5 mg/mL Matrigel (Corning).

For the determination of oral exposures for AR degraders, each

compound was administered in non-tumor-bearing male mice via oral

gavage using 100% PEG200 as the dosing vehicle. Animals were

sacriﬁced at indicated time points with three mice for each time point

for each compound, and 300 μL of blood was collected from each

animal and were stored at −80 °C until analysis.

For PK/PD studies in tumor-bearing male SCID mice, each

compound was administered in animals via oral gavage using 100%

PEG200 as the dosing vehicle when the VCaP tumors reached

approximately 200 mm³. Animals were sacriﬁced at indicated time

points with three mice for each compound at each time point, and blood

(300 μL) and tumor were collected from each animal for analysis.

Isolated tumor samples were placed in a Precellys tube (CK28-R) and

immediately frozen in liquid nitrogen. All plasma and tumor samples

were stored at −80 °C until analysis. For the analysis of AR protein

levels in tumor samples, resected VCaP xenograft tumor tissues were

ground into powder in liquid nitrogen and lysed in CST lysis buﬀer with

halt proteinase inhibitors. Twenty micrograms of whole tumor clariﬁed

lysates were separated on 4−20% or 4−12% Novex gels. Western blots

were performed as detailed in the previous section.

All animal experiments in this study were approved by the University

of Michigan Committee on Use and Care of Animals and Unit for

Laboratory Animal Medicine (ULAM). The pharmacokinetics of ARD-

2128 and analogs was determined in normal male SCID mice or with

VCaP tumor following oral gavage (PO) single dosing at 10 or 20 mg/

kg. The solid compounds were dissolved in a vehicle containing 100%

PEG200. The animals (total 9 mice/compound or 6 mice/compound)

were sacriﬁced at 1, 3, and 6 h, or 6 and 24 h after the ﬁnal

administration of the chemicals and then followed by the collection of

blood samples (300 μL) and tumor samples. The blood samples were

centrifuged at 15000 rpm for 10 min, and then the supernatant plasma

was saved for analysis. Isolated tumor samples were immediately frozen

and ground with a mortar and pestle in liquid nitrogen. All plasma and

tumor samples were stored at −80 °C until analysis. To prepare tumor

samples for LCMS analysis, mixed ultrapure water and acetonitrile

solution (4:1) were added to the defrosted tumor tissue samples 5:1, v/

w, in order to facilitate homogenization with a Precellys evolution

homogenizer under 4 °C. The homogenized tissues solution was

denatured using cold acetonitrile (1:3, v/v) with vortex and centrifuged

at 13000 rpm and 4 °C for 10 min. Following protein precipitation, the

ﬁnal supernatants were collected for LC-MS analysis.

To determine drug concentrations in plasma and tumor samples, an

LC−MS/MS method was developed and validated. The LC−MS/MS

method consisted of a Shimadzu HPLC system, and chromatographic

separation of a test compound was achieved using a Waters XBridge-

C18 column (5 cm × 2.1 mm, 3.5 μm). An AB Sciex QTrap 5500 mass

spectrometer equipped with an electrospray ionization source (Applied

biosystems, Toronto, Canada) in the positive-ion multiple reaction

monitoring (MRM) mode was used for detection. For example, the

precursor/product ion transitions were monitored at m/z 820.3−542.2

and 455.2−425.2 for ARD-2128 and internal standard, respectively, in

the positive electrospray ionization mode. The mobile phases used on

HPLC were 0.1% formic acid in puriﬁed water (A) and 0.1% formic acid

in acetonitrile (B). The gradient (B) was held at 10% (0−0.3 min),

increased to 95% at 0.7 min, then stayed at isocratic 95% B for 2.3 min,

and then immediately stepped back down to 10% for 2 min re-

equilibration. The ﬂow rate was set at 0.4 mL/min. All pharmacokinetic

parameters were calculated by noncompartmental methods using

WinNonlin, version 3.2 (Pharsight Corporation, Mountain View, CA,

USA).

For the in vivo eﬃcacy experiments, when VCaP tumors reached an

average volume of 150 mm³, mice were tumor size matched and

randomly assigned to diﬀerent experimental groups with seven mice for

each group. Drugs or vehicle control were given at the dose schedule as

indicated using 100% PEG200 as the dosing vehicle. Tumor sizes and

animal weights were measured 2−3 times per week. Tumor volume

(mm³) = (length × width²)/2. Tumor growth inhibition was calculated

as TGI (%) = (Vc − Vt)/(Vc − Vo) × 100, where Vc and Vt are the

medians of the control and treated groups at the end of the treatment,

respectively, and Vo is that at the start. The tumor volumes at the end of

treatment were statistically analyzed using a two-tailed, unpaired t-test

(GraphPad Prism 8.0).

PK Studies in Mice. Pharmacokinetic (PK) studies were performed

in Shanghai Medicilon Inc. Shanghai, 201200, China. Male ICR mice,

weighing 18−20 g, were purchased from Sino-British SIPPR/BK Lab

Animal Ltd., Shanghai, China. One group of three mice was dosed

intravenously (IV) via a bolus in the tail vein with a dose level of 2 mg/

kg, and a second group of three mice was dosed orally as a single

esophageal gavage with a dose level of 5 mg/kg. The drug solution was

freshly prepared before administration. For the IV route, each

compound was formulated in 5% DMSO, 10% solutol, 85% saline as

a clear solution, a dosage volume of 5 mL/kg, and a theoretical

concentration of 0.4 mg/mL. For the oral route, each was formulated in

5% DMSO, 10% solutol, 85% saline as a clear solution, a dosage volume

of 10 mL/kg, and a theoretical concentration of 0.5 mg/mL. Blood

samples were collected at the following time points: IV, 0.083, 0.25, 0.5,

T

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1, 2, 4, 6, 8, and 24 h after dosing (n = 3 mice for ach per sampling time);

PO, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after dosing (n = 3 mice for

each per sampling time). Each mouse was sampled once and then

euthanized. Blood was kept on ice. Within 1 h after sampling, blood was

centrifuged at 3900 rpm for 15 min, and the supernatant was diluted

three times with water. A total of 5 μL of diluted supernatant was

injected into the LC/MS/MS system for quantitative analysis.

Microsomal Metabolic Stability Studies. In vitro metabolism

study of a test compound was performed in human, mouse, rat, dog, and

monkey liver microsomes to evaluate its cytochrome P450-mediated

metabolism. The metabolic stability was assessed using pooled mouse,

rat, dog, monkey, and human liver microsomes, which were purchased

from XenoTech (Lenexa, Kansas).

A total of 1 μM of the test compound was incubated with 0.5 mg/mL

of the respective liver microsome and 1.7 mM cofactor-NADPH in 0.1

M K-phosphate buﬀer (pH = 7.4) containing 5 mM MgCl₂at 37 °C,

with the acetonitrile concentration less than 0.1% in the ﬁnal incubation

solution. After 0, 5, 10, 15, 30, and 45 min of incubation, the reaction

was stopped immediately by adding 150 μL cold acetonitrile containing

IS to each 45 μL incubation solution in the wells of the corresponding

plates. The incubation without the addition of NADPH was used as the

negative control. Ketanserin was incubated similarly as the positive

control. After quenching, the plate was shaken for 10 min (600 rpm/

min) and centrifuged at 6000 rpm for 15 min. A total of 80 μL of the

supernatant was then transferred from each well into a 96-well plate

containing 140 μL of water for LC−MS/MS analysis, from which the

remaining amount of the test compound was determined. The natural

log of the remaining amount of the test compound was plotted against

time to determine the disappearance rate and the half-life of the test

compound.

Plasma Stability Studies. The in vitro stability of a test compound

was studied in human, mouse, rat, dog, and monkey plasmas in

Medicilon Inc. (Shanghai, China). Human plasma was purchased from

ZenBio (Durham, NC, USA), and other plasmas were prepared in-

house. A test compound was dissolved in DMSO to a ﬁnal

concentration of 10 mM and then diluted to 10 μM in 0.1 M K/Mg

buﬀer. A total of 90 μL of pre-warmed plasma at 37 °C was added to the

wells of a 96-well plate before spiking them with 10 μL of 10 μM test

compound to make the ﬁnal concentration of the test compound at 1

μM. The spiked plasma samples were incubated at 37 °C for 2 h.

Reactions were terminated at 0, 5, 15, 30, 60, and 120 min by adding

400 μL of acetonitrile containing IS. After quenching, the plates were

shaken for 5 min at 600 rpm and stored at −20 °C if necessary before

analysis by LC/MS. Before LC/MS analysis, the samples were thawed

at room temperature and centrifuged at 6000 rpm for 20 min. A total of

100 μL of the supernatant from each well was transferred into a 96-well

sample plate containing 100 μL of water for LC/MS analysis. Procaine

was used as the reference control compound for human, mouse, dog,

and monkey plasma stability studies, and Benﬂuorex was used as the

reference control compound for rat plasma stability studies. The in vitro

plasma half-life (t_1/2) was calculated using the expression t_1/2= 0.693/b,

where b is the slope found in the linear ﬁt of the natural logarithm of the

fraction remaining of the test compound vs incubation time.

Molecular string ﬁle for all the ﬁnal target compounds

(CSV)

AUTHOR INFORMATION

Corresponding Author

■

Shaomeng Wang − The Rogel Cancer Center,, Department of

Internal Medicine, Department of Pharmacology, and

Department of Medicinal Chemistry, University of Michigan,

Ann Arbor, Michigan 48109, United States; orcid.org/

0000-0002-8782-6950; Email: shaomeng@umich.edu

Authors

Xin Han − The Rogel Cancer Center, and Department of

Internal Medicine, University of Michigan, Ann Arbor,

Michigan 48109, United States; orcid.org/0000-0003-

0904-220X

Lijie Zhao − The Rogel Cancer Center, and Department of

Internal Medicine, University of Michigan, Ann Arbor,

Michigan 48109, United States

Weiguo Xiang − The Rogel Cancer Center, and Department of

Internal Medicine, University of Michigan, Ann Arbor,

Michigan 48109, United States

Chong Qin − The Rogel Cancer Center, and Department of

Internal Medicine, University of Michigan, Ann Arbor,

Michigan 48109, United States

Bukeyan Miao − The Rogel Cancer Center, and Department of

Internal Medicine, University of Michigan, Ann Arbor,

Michigan 48109, United States

Donna McEachern − The Rogel Cancer Center, and

Department of Internal Medicine, University of Michigan, Ann

Arbor, Michigan 48109, United States

Yu Wang − The Rogel Cancer Center, and Department of

Internal Medicine, University of Michigan, Ann Arbor,

Michigan 48109, United States

Hoda Metwally − The Rogel Cancer Center, and Department of

Internal Medicine, University of Michigan, Ann Arbor,

Michigan 48109, United States

Lu Wang − Department of Pharmaceutical Sciences, College of

Pharmacy, University of Michigan, Ann Arbor, Michigan

48109, United States

Aleksas Matvekas − Department of Pharmaceutical Sciences,

College of Pharmacy, University of Michigan, Ann Arbor,

Michigan 48109, United States

Bo Wen − Department of Pharmaceutical Sciences, College of

Pharmacy, University of Michigan, Ann Arbor, Michigan

48109, United States

Duxin Sun − Department of Pharmaceutical Sciences, College of

Pharmacy, University of Michigan, Ann Arbor, Michigan

48109, United States; orcid.org/0000-0002-6406-2126

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00882.

■

sı

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00882

Author Contributions

X.H., L.Z., and W.X. contributed equally to this work.

Western blotting of AR protein for compounds 10−47 in

VCaP cells; cell growth inhibition curves of selected AR

degraders in LNCaP and VCaP cell lines; concentrations

of compounds 12, 13, 16, and 17 in plasma in SCID mice

with a single oral administration; concentrations of

compounds 26, 27, and 28 (ARD-2128) in plasma and

tumor tissues in SCID mice; ¹H and ¹³C NMR spectra for

ARD-2128; HPLC purity spectra for representative AR

degraders; and microsomal metabolic and plasma stability

data of ARD-2128 (PDF)

Notes

The authors declare the following competing ﬁnancial

interest(s): The University of Michigan has ﬁled patent

applications on these AR degraders, which have been licensed

to Oncopia Therapeutics, Inc. X.H., L.Z., W.X., C.Q., B.M., and

B.W. are co-inventors on these patent applications and receive

royalties from the University of Michigan. S.W. was a co-founder

and served as a paid consultant to Oncopia. S.W. and the

U

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Article

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Welsbie, D.; Chen, C. D.; Higano, C. S.; Beer, T. M.; Hung, D. T.;

Scher, H. I.; Jung, M. E.; Sawyers, C. L. Development of a second

generation antiandrogen for treatment of advanced prostate cancer.

Science 2009, 324, 787−790.

University of Michigan also owned equity in Oncopia, which

was acquired by Roivant Sciences. S.W. is a paid consultant to

Roivant Sciences. The University of Michigan has received a

research contract from Oncopia and Roivant for which S.W.

serves as the principal investigator.

(9) Pal, S. K.; Patel, J.; He, M.; Foulk, B.; Kraft, K.; Smirnov, D. A.;

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abiraterone acetate or enzalutamide in patients with castration-resistant

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Chen, Y. Discovery and biological evaluation of novel androgen

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Dorow, S.; Arora, V.; Shao, G.; Qian, J.; Zhao, H.; Yang, G. B.; Cao, C.

Y.; Sensintaffar, J.; Wasielewska, T.; Herbert, M. R.; Bonnefous, C.;

Darimont, B.; Scher, H. I.; Smith-Jones, P.; Klang, M.; Smith, N. D.; De

Stanchina, E.; Wu, N.; Ouerfelli, O.; Rix, P. J.; Heyman, R. A.; Jung, M.

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ACKNOWLEDGMENTS

■

This study is supported in part by funding from Oncopia

Therapeutics, Inc. (now part of Roivant Sciences), the National

Cancer Institute, NIH (P50 CA186786), and the University of

Michigan Comprehensive Cancer Center Core Grant from the

National Cancer Institute, NIH (P30CA046592).

ABBREVIATIONS USED

■

AR, androgen receptor; mCRPC, metastatic castration-resistant

prostate cancer; PROTAC, proteolysis targeting chimera;

cIAP1, cellular inhibitor of apoptosis protein 1; V_ss, steady-

state volume of distribution; C_max, maximum drug concen-

tration; AUC_{0−24 h}, area-under-the-curve between 0 and 24 hr.;

Cl, plasma clearance rate; T_1/2, terminal half-life; F, oral

bioavailability; IV, intravenous administration; PO, oral

administration; PD, pharmacodynamics; CYP, cytochrome

P450; ATCC, American-type culture collection; qRT-PCR,

quantitative real-time polymerase chain reaction; SCID, severe

combined immunodeﬁcient

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Strategies toward Discovery of Potent and Orally Bioavailable Proteolysis Targeting Chimera Degraders of Androgen Receptor for the Treatment of Prostate Cancer

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