Discovery of 2-(3-(3-Carbamoylpiperidin-1-yl)phenoxy)acetic Acid Derivatives as Novel Small-Molecule Inhibitors of the β-Catenin/B-Cell Lymphoma 9 Protein-Protein Interaction

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Discovery of 2‑(3-(3-Carbamoylpiperidin-1-yl)phenoxy)acetic Acid

Derivatives as Novel Small-Molecule Inhibitors of the β‑Catenin/B-

Cell Lymphoma 9 Protein−Protein Interaction

Zhen Wang,^∥Min Zhang,^∥Wen Luo, Yongqiang Zhang, and Haitao Ji*

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ABSTRACT: The β-catenin/B-cell lymphoma 9 (BCL9) protein−protein interaction (PPI) is a potential target for the suppression

of hyperactive Wnt/β-catenin signaling that is vigorously involved in cancer initiation and development. Herein, we describe the

medicinal chemistry optimization of a screening hit to yield novel small-molecule inhibitors of the β-catenin/BCL9 interaction. The

best compound 30 can disrupt the β-catenin/BCL9 interaction with a K_iof 3.6 μM in AlphaScreen competitive inhibition assays.

Cell-based experiments revealed that 30 selectively disrupted the β-catenin/BCL9 PPI, while leaving the β-catenin/E-cadherin PPI

unaﬀected, dose-dependently suppressed Wnt signaling transactivation, downregulated oncogenic Wnt target gene expression, and

on-target selectively inhibited the growth of cancer cells harboring aberrant Wnt signaling. This compound with a new chemotype

can serve as a lead compound for further optimization of inhibitors for β-catenin/BCL9 PPI.

Wnt pathway also activates the Wnt/β-catenin signal and has

been reported for various types of cancers.¹⁶⁻²²In addition,

aberrant β-catenin signaling plays a critical role in cancer

immune evasion and immunotherapy resistance.²³⁻²⁹This

collective evidence strongly supports the suppression of

hyperactive Wnt/β-catenin signaling as a new direction for

developing anticancer drugs.

The upstream eﬀector proteins of the Wnt/β-catenin cascade

have been extensively targeted by various therapeutic

agents.³⁰⁻³⁵However, these agents are expected to be ineﬀective

against cancer cells with more downstream genetic or epigenetic

mutations, such as loss-of-function mutations of APC and AXIN

or activation mutations of CTNNB1 (β-catenin target gene).

Furthermore, some of these agents might cause oﬀ-pathway

eﬀects given that their targets (GSK3β, tankyrase, and CK1α)

are upstream and involved in multiple cellular processes. The β-

catenin/BCL9 complex, located in the β-catenin-containing

transcriptional supercomplex, appears to be a promising target

to suppress hyperactive β-catenin signaling. The protein−

protein interaction (PPI) between β-catenin and BCL9 is

signiﬁcantly upregulated in tumor tissues, and murine gut

INTRODUCTION

■

The canonical Wnt pathway participates in various biological

processes including embryogenesis, tissue homeostasis, and

stem cell renewal.^1,2In unstimulated cells, the protein β-catenin,

a central hub of this pathway, undergoes constitutive

degradation guided by the destruction complex consisting of

Axin, casein kinase 1α (CK1α), adenomatous polyposis coli

(APC), protein phosphatase 2A (PP2A), and glycogen synthase

kinase 3β (GSK3β), and thus, free β-catenin is maintained at the

undetectable-to-baseline levels. Upon activation of the Wnt

signal by a Wnt ligand, the destruction complex disassembles

and β-catenin is liberated. Free β-catenin accumulates in the

cytosol in its dephosphorylated state and enters the cell nucleus,

where β-catenin interacts with the lymphoid enhancer-binding

factor (LEF)/T-cell factor (TCF) and binds with B-cell

lymphoma 9 (BCL9)/BCL9-like (BCL9L), p300/CREB-bind-

ing protein (CBP), and Pygopus (Pygo 1 or Pygo 2) and among

other co-activators to activate β-catenin target genes.

Aberrant activation of Wnt/β-catenin signaling has been

recorded in many cancers.³⁻⁶For example, APC mutations and

truncations that lead to β-catenin accumulation were detected in

∼80% of colorectal cancer.⁷AXIN with loss-of-function

mutations was observed in esophageal squamous cell carcinoma,

hepatocellular carcinoma, and colorectal cancer.⁸⁻¹⁰Oncogenic

β-catenin activation mutations were described in colorectal

cancer, hepatocellular carcinoma, prostate cancer, thyroid

tumor, and ovarian endometrioid adenocarcinoma.¹¹⁻¹⁵The

autocrine/paracrine secretion of upstream eﬀectors or the

epigenetic silencing of the genes of negative regulators of the

Received: January 11, 2021

Published: April 27, 2021

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BCL9/BCL9L elimination did not lead to overt phenotypic

consequences or aﬀect normal intestinal homeostasis, indicating

that targeting this PPI might have no or very low toxicity.^36,37

BCL9/BCL9L knockdown by siRNAs and shRNAs signiﬁcantly

suppressed the growth of cancer cells that depend upon β-

catenin signaling in vitro and in vivo.^36,38BCL9/BCL9L

conditional ablation induced diﬀerentiation and totally

abolished tumorigenicity of murine intestinal organoids, while

exhibiting no eﬀect on cell proliferation.³⁹More importantly,

BCL9/BCL9L loss blocks tumorigenesis driven by Wnt

signaling and extends disease-free survival in models that

recapitulate human cancer.^40,41These data suggest that targeting

of β-catenin/BCL9 PPI holds great promise for treatment of

cancer.⁴⁰

eﬀorts were made focusing on the amide bond, and after

synthesis, the inhibitory activity results of the derivatives are

summarized in Table 1. Compound 4 with a methylated amide

showed a slightly improved potency over 2. Increasing the size of

substituents on the amide [from methyl (4) to ethyl (5),

isopropyl (6), and cyclopropyl (7)] resulted in further

improvement on inhibitory activity, with compound 7 being

the most potent derivative. The K_ivalue for 7 is 36 μM and

twofold better than that of 2. Compound 3 with one more

methylene group between 4-isopropylbenzyl and amide was also

synthesized, and it turned out to be slightly less potent than 2.

To further improve inhibitory activity, various heterocycles

were introduced in the position of the isopropyl substituent to

explore more interactions with β-catenin. The results showed

that compounds bearing nitrogen-containing heterocycles (8, 9,

10, and 11) displayed potency loss when compared with 7

(Table 2). Compounds with the furan moiety (14 and 15) were

also less potent than 7, while compounds 12 and 13 bearing

thiophene showed increased activities (Table 2). For example,

compound 12 with 2-thiophene inhibited the β-catenin/BCL9

interaction with a K_iof 13 μM, which is threefold more potent

than 7. Notably, 12 is as potent as the hit compound 1 in the

AlphaScreen assay.

Apart from peptide-based inhibitors,⁴²⁻⁴⁵two series of small-

molecule disruptors have been reported for β-catenin/BCL9

PPI.⁴⁶⁻⁴⁹Carnosic acid (Figure 1) was reported as an inhibitor

Although compound 1 inhibited the β-catenin/BCL9

interaction with a K_iof 11 μM in AlphaScreen assays, it was

inactive in cell-based experiments.⁵⁰Careful examination on the

chemical structure of 1 suggested that the inferior cellular

activities might be caused by the carboxylic acid moiety, which is

known to negatively impact cell membrane permeability.

Consistently, the MTS assay revealed that 12 bearing the

same carboxylic acid group showed very low inhibitory activities

on viability of cancer cells (Table 5), although it displayed a K_iof

13 μM in AlphaScreen assays. Therefore, further optimization

was conducted on this carboxylic acid group to improve both

biochemical and cellular activities. Compounds 16 and 17

featured with methyl and tert-butyl esters, respectively, were

synthesized (Table 3), and both compounds showed signiﬁcant

potency loss. This result indicated that the negative-charge

character of the carboxylic acid group was important for

maintaining the inhibitory activity. Based on this observation, a

bioisosteric replacement strategy was adopted, and a series of N-

acyl sulfonamide derivatives were designed (Table 3). N-Acyl

sulfonamide is a well-known bioisostere for carboxylic acid and

often employed to obtain derivatives with improved cell

permeability, higher binding aﬃnity, and better metabolic

stability.⁵³The ﬁrst designed compound 18 with a N-acyl

methanesulfonamide group inhibited the β-catenin/BCL9

interaction with a K_iof 21 μM and was twofold less active

than 12. Encouragingly, the substitution of the methyl group in

the N-acyl methanesulfonamide moiety with larger substituents

[such as cyclopropyl (19), isopropyl (20), and phenyl (21)] led

to derivatives with improved activities. For example, compound

21 with N-acyl benzenesulfonamide displayed a K_iof 6.4 μM,

and it is twofold more potent than 12. To improve the

physicochemical properties of these inhibitors, hydrophilic

substituents including methoxy, hydroxy, and cyano moieties

were introduced to the phenyl group of N-acyl benzenesulfo-

namide. As shown in Table 3 and Figure S2, these compounds

showed increased activities when compared with 21. Speciﬁ-

cally, compounds 22 and 26 displaying K_is of 4.1 and 3.9 μM,

respectively, were the two most potent inhibitors. Finally, the R-

and S-isomers of 22 and 26 were synthesized, and their

preliminary activities were determined in AlphaScreen assays.

Figure 1. Reported small-molecule β-catenin/BCL9 disruptors.

of β-catenin/BCL9 PPI after screening two compound libraries

by Bienz and coworkers.⁴⁶Recently, our group reported 3-(4-

ﬂuorophenyl)-N-phenylbenzamide (PNPB) derivatives in

Figure 1 as β-catenin/BCL9 inhibitors by rational design and

optimization.^47,48While the existing scaﬀolds contain some

challenges for further inhibitor optimization, it is highly

desirable to discover new small-molecule inhibitors with novel

scaﬀolds to disrupt β-catenin/BCL9 PPI for chemical biology

and anti-cancer drug discovery studies. Herein, we disclose a

class of new selective small-molecule disruptors of the β-

catenin/BCL9 interaction.

RESULTS

■

Inhibitor Design and Optimization. Our medicinal

chemistry program started with a hit compound, CP-868388

(1), discovered by compound screen using the AlphaScreen

competitive inhibition assay.⁵⁰Compound 1 after resynthesis

inhibited the PPI between full-length β-catenin and BCL9 with a

K_iof 11 μM in AlphaScreen assays. The robustness of this

AlphaScreen assay using full-length β-catenin is described in

Figure S1. Given that the carbamate group in 1 is liable to

metabolic hydrolysis⁵¹and the chiral carbon is diﬃcult to

construct,⁵²the scaﬀold-hopping strategy was adopted to obtain

a new scaﬀold, as shown in compound 2 (Table 1). Inhibitor 2

displayed a K_iof 71 μM for inhibiting the β-catenin/BCL9

interaction. Although 2 is about sixfold less potent than 1 for β-

catenin/BCL9 inhibition, the amide group in 2 is more stable

and the synthetic accessibility toward 2 is much higher, and thus,

2 is a reasonable starting point for further modiﬁcation. Initial

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a

Table 1. AlphaScreen Assay Results of 1−7

a

b

The data were shown in the form of mean standard deviation (n = 3). The K_iof CP-868388 from the Sigma-Aldrich 90 LOPAC^Pfizerlibrary

was reported to be 1.2 0.21 μM using the truncated β-catenin (residue 138−686).⁵⁰

a

Table 2. AlphaScreen Assay Results of 8−15

a

The data were shown as mean standard deviation (n = 3).

The results in Table 4 show for both compounds that the S-

isomer (28 and 30) is slightly superior to the R-isomer (27 and

29).

Cell-Based Studies. The eﬀects of 12, 17, 28, and 30 on the

viability of diﬀerent cancer cells with hyperactive Wnt signaling

were evaluated in MTS assays. The cell lines used include

colorectal cancer HCT116 and SW480 cells and breast cancer

MDA-MB-468 and MDA-MB-231 cells. The normal breast

epithelial MCF10A cells were assessed in parallel for

comparison. The results are shown in Table 5. The

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a

Table 3. AlphaScreen Assay Results of 16−26

a

The data were shown as mean standard deviation (n = 3).

a

Table 4. AlphaScreen Assay Results of 27−30

a

The data were shown as mean standard deviation (n = 3).

a

Table 5. MTS Assay Results of 12, 17, 28, and 30 on Cell Viability

MTS IC₅₀SD (μM)

Wnt hyperactive

Wnt normal

compounds

SW480

HCT116

MDA-MB-231

MDA-MB-468

MCF10A

12

17

28

30

137 8.20

>200

28.2 1.08

16.3 2.04

112 9.25

>200

29.9 1.99

29.6 1.02

103 7.44

>200

19.8 1.59

14.3 0.570

95.9 8.37

>200

15.4 2.49

17.8 1.01

98.5 7.66

>200

30.4 2.47

139 3.16

a

The data were shown as mean standard deviation (n = 3).

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Figure 2. Wnt-dependent TOPFlash and FOPFlash experiment results of 28 and 30 in SW480 cells. Each experiment was performed in triplicate (n =

3).

Figure 3. qPCR experiment results of Axin2, cyclin D1, and LEF1 when treated with 30 (24-h incubation) in Wnt 3a-stimulated MDA-MB-231 (A) and

SW480 cells (B). The negative control is the house-keeper gene HPRT. Each experiment was performed in triplicate (n = 3). *P < 0.05, **P < 0.01, as

determined by Student t tests.

representative compound 30 inhibited the growth of Wnt

hyperactive cancer cells with IC₅₀s between 14 and 30 μM, while

sparing normal mammary epithelial MCF10A cells with 5- to 10-

fold selectivity. Although 28 displayed similar IC₅₀s for Wnt

hyperactive cancer cells, it was much less selective over

MCF10A cells. In addition, an inactive analogue 17 in

AlphaScreen assays (K_i> 90 μM, Table 3) did not show any

inhibitory eﬀects on all tested cells (IC₅₀> 200 μM, Table 5).

One reason why 30 is more selective for Wnt-dependent breast

cancer cells over normal mammary epithelial cells is perhaps

because the CN derivative 30 is more hydrophilic than the OMe

derivative 28, and thus, 30 may have less-nonspeciﬁc oﬀ-target

eﬀects.

The inhibitory eﬀects of 28 and 30 on transactivation of Wnt/

β-catenin signaling were examined using the TOPFlash/

FOPFlash luciferase reporter assay. For the TOPFlash assay,

the reporter construct harbors three wild-type Tcf4 binding

DNA sequences, while in the FOPFlash assay, Tcf4 binding sites

were mutated. These tandem Tcf binding sites control the

expression ratio of the ﬁreﬂy luciferase in TOPFlash/FOPFlash

assays. The Renilla luciferase reporter construct (pCMV-RL)

was used as the internal control to normalize luciferase reporter

signals and eliminate any possible systematic errors. As shown in

Figure 2, both 28 and 30 concentration-dependently suppressed

the TOPFlash activity with the IC₅₀values of 7.8 and 6.6 μM,

respectively. However, in this concentration range, compound

30 did not show any inhibition on the FOPFlash activity,

indicating that 30 is speciﬁc for Wnt/β-catenin signaling. This

selectivity trend is consistent with that from MTS cell viability

assays. Therefore, further cell-based studies were focused on

compound 30.

Quantitative real-time PCR (qPCR) experiments were

performed to evaluate the eﬀects of 30 on transcription of the

Wnt-speciﬁc target gene Axin2 and two highly important

oncogenic Wnt target genes, LEF1 and cyclin D1. The results

showed that 30 inhibited transcription of these Wnt target genes

while sparing the house-keeper gene HPRT in both Wnt 3a-

activated MDA-MB-231 and SW480 cells (Figure 3).

Western blot experiments were performed to determine the

expression levels of the proteins Axin2 and cyclin D1. As shown

in Figure 4A, treatment of 30 dose-dependently decreased the

expression of both Axin2 and cyclin D1. To evaluate the binding

of 30 to β-catenin in the cellular environment, we developed a

protein pull-down assay using the N-terminally biotin-labeled

BCL9 peptide (the homology domain 2 (HD2), residues 350−

375) and the whole cell lysates of colorectal cancer SW480 cells.

As shown in Figure 4B, β-catenin was pulled down by

biotinylated BCL9, and the unlabeled BCL9 HD2 peptide

served as a positive control to evaluate β-catenin/BCL9

disruption. Incubation of 30 resulted in the disruption of the

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Figure 4. (A) Western blot results of Axin2 and cyclin D1 when treated with 30 (24 h incubation) in SW480 cells. The internal control is β-tubulin.

The original Western blot images are provided in Figure S3. (B) Biotin−streptavidin endogenous β-catenin pulldown assay using SW480 cell lysates

and the biotinylated BCL9 HD2 peptide (residues 350−375) after treatment with 1 μM BCL9 HD2 peptide (residues 350−375) and diﬀerent

concentrations of 30. The original Western blot images are shown in Figure S4. (C) co-IP assays to examine the inhibitory eﬀects 30 on β-catenin/

BCL9 and β-catenin/E-cadherin interactions using HCT116 cells. The internal control is β-tubulin. IP, immunoprecipitation; IB, immunoblotting.

Each experiment was conducted in triplicate. The original Western blot images are provided in Figure S5.

binding of β-catenin with biotinylated BCL9 in a dose-

dependent manner, demonstrating that 30 can bind to the

HD2 domain-binding groove of the endogenous β-catenin

protein. Co-immunoprecipitation (co-IP) studies were further

performed to examine eﬀects of 30 on disruption of the full-

length β-catenin/BCL9 interaction in cells. The results

indicated that 30 concentration-dependently disrupted the β-

catenin/BCL9 interaction (Figure 4C). Additional studies

showed that 30 did not interfere with the β-catenin/E-cadherin

interaction. This is important because the surface areas of β-

catenin for β-catenin/BCL9 PPI are also used to bind E-

cadherin region V, and the β-catenin/E-cadherin complex is

critical for cell−cell adherens junctions.

To further evaluate cellular on-target eﬀects of 30, a β-catenin

rescue experiment was performed following the recommenda-

tion by Dr. Kaelin.⁵⁴Transfection with the empty pcDNA3.1

vector had no eﬀect on the growth of SW480 cells. Compound

30 exhibits similar growth inhibition activities against SW480

cells with or without the empty pcDNA3.1 vector (IC₅₀= 19 μM

vs 16 μM). As shown in Figure 5, the transfection of the

constitutively active β-catenin rescued growth inhibition of

compound 30-treated SW480 cells, and the IC₅₀values of 30

were increased by fourfold after rescuing β-catenin. This rescue

experiment, the β-catenin pull-down experiment using BCL9

HD2 peptide as the positive control (Figure 4B), the co-IP

studies to examine disruption of cellular β-catenin-containing

PPI complex structures (Figure 4C), and the TOPFlash/

FOPFlash assays (Figure 2) demonstrated the cellular on-target

eﬀects of compound 30.

Figure 5. Results of β-catenin rescue experiments. The MTS assays

were used to determine eﬀects of 30 on growth inhibition of SW480

cells after 72 h treatment. The cells were transfected with a pcDNA3.1

empty vector or the pcDNA3.1−β-catenin vector. (A) Results when

compound 30 was at the concentration of 25 μM. (B) IC₅₀s of 30 in

MTS assays to inhibit the growth of SW480 cells that were transfected

with pcDNA3.1 (empty vector) or pcDNA3.1−β-catenin. Each

experiment was performed in triplicate (n = 3). The original dose−

response curves are shown in Figure S6. **p < 0.01 calculated by

Student t tests.

prepared 32a−c or other commercially available amines went

through the amide coupling reaction with 1-Boc-piperidine-3-

carboxylic acid to produce 33. Removal of the Boc group of 33

and then Buchwald−Hartwig amination with 31 yielded

compound 34. The ﬁnal products 2−7 were obtained by

removing the tert-butyl group in 34 under acidic conditions.

The synthetic route for ﬁnal products 8−15 is shown in

Scheme 2. The intermediate 35 was synthesized through the

reductive amination reaction using 4-bromobenzaldehyde and

CHEMISTRY

■

The synthetic route for compounds 2−7 is shown in Scheme 1.

The important intermediates 31 and 32a−c were obtained

through the nucleophilic substitution reaction and the reductive

amination reaction using 4-isopropylbenzaldehyde and 3-

bromophenol as starting materials, respectively. The newly

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Scheme 1. Synthesis of Compounds 2−7

Scheme 2. Synthesis of Compounds 8−15

Scheme 3. Synthesis of Compounds 16−26

cyclopropylamine as the starting materials. Buchwald−Hartwig

amination between ethyl piperidine-3-carboxylate and 31

yielded 36. Hydrolysis of the ethyl ester in 36 and then amide

coupling with 35 produced key intermediate 37, which

underwent Suzuki coupling with diﬀerent boronic acids to

generate 38. Removal of the tert-butyl group in 38 with

triﬂuoroacetic acid (TFA)/CH₂Cl₂yielded ﬁnal products 8−15.

The synthetic route for ﬁnal products 16−26 is shown in

Scheme 3. Compounds 16 and 17 were obtained by the ester

formation reactions between 12 and methanol or between 12

and tert-butanol, respectively. The other compounds (18−26)

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Scheme 4. Synthesis of Compounds 27 and 29

Scheme 5. Synthesis of Compounds 28 and 30

were achieved by the reactions between 12 and various

sulfonamides.

this PPI might be too diﬃcult to be disrupted by small

molecules.⁵⁷β-Catenin interacts with Tcf, APC, and E-cadherin

using the same surface areas, indicating that it would also be

challenging to achieve high inhibitor selectivity.⁵⁷On the other

hand, BCL9 binds with β-catenin via a single α-helical structure

(the HD2 domain).⁵⁷β-Catenin and BCL9 have much smaller

contacting interface areas (1450 Å²), and their binding aﬃnity is

moderate (K_D= 0.47 μM). The only reported additional binding

partner at this PPI area of β-catenin is E-cadherin. Therefore, the

β-catenin/BCL9 PPI emerges as a promising alternative target

to inhibit hyperactive Wnt/β-catenin signaling. In this work,

structural optimization based on screening hit 1 (CP-868388)

successfully yielded a series of novel selective β-catenin/BCL9

inhibitors. Co-IP studies show that the representative

compound 30 not only disrupts β-catenin/BCL9 PPI in cells

but also is selective for β-catenin/BCL9 over β-catenin/E-

cadherin interactions.

This small-molecule β-catenin/BCL9 disruptor 30 we

identiﬁed is structurally diﬀerent from carnosic acid and

PNPB series of β-catenin/BCL9 inhibitors and oﬀers a new

direction for future disruptor optimization. It is worth noting

that both this series of inhibitors and carnosic acid contain an

acidic functional group. In carnosic acid, it is a carboxylic acid

group, while in 30, it is a N-acyl sulfonamide group. The natural

product scaﬀold of carnosic acid makes it diﬃcult for further

inhibitor optimization. The catechol group of carnosic acid has

been identiﬁed as the pan-assay interference compound

(PAINS) moiety. PNPB-22 contains multiple aromatic rings

and two positively charged centers (Figure 1). This lipophilic

base character makes PNPB-22 toxic to normal cells at high

concentrations.⁴⁷PNPB-29 has two positively charged centers

and one negatively charged center (Figure 1). This zwitterion

has low cellular activity.⁴⁸Compound 30 with a novel N-(2-(3-

The synthetic route for ﬁnal products 27 and 29 is shown in

Scheme 4. Ethyl (R)-piperidine-3-carboxylate underwent

Buchwald−Hartwig amination with 31 yielded 39. Hydrolysis

of the ethyl ester in 39 and then amide coupling with 35 gave

intermediate 40. The Suzuki coupling reaction between 40 and

thiophen-2-ylboronic acid produced 41. Removing the tert-butyl

group in 41 under acidic conditions and then reacting with 3-

methoxybenzenesulfonamide and 4-cyanobenzenesulfonamide

yielded ﬁnal products 27 and 29, respectively.

The synthetic route for ﬁnal products 28 and 30 is shown in

Scheme 5. Ethyl (S)-piperidine-3-carboxylate went through

Buchwald−Hartwig amination with 31 to yield 42. Hydrolysis of

the ethyl ester in 42 and then amide coupling with 35 gave

intermediate 43, which underwent the Suzuki coupling reaction

with thiophen-2-ylboronic acid to produce 44. Final products 28

and 30 were obtained by removing the tert-butyl group in 44 and

then reacting with 3-methoxybenzenesulfonamide or 4-

cyanobenzenesulfonamide, respectively.

DISCUSSION AND CONCLUSIONS

■

Hyperactive Wnt/β-catenin signaling is frequently observed in

various cancers. Targeting the downstream β-catenin-containing

transcriptional complex, rather than the upstream eﬀectors,

appears to be the most eﬀective approach for suppression of

aberrant β-catenin signaling. However, targeting β-catenin to

disrupt a β-catenin-related transcriptional complex is highly

desirable but has proven to be a formidable challenge.^1,55,56

Most β-catenin-directed programs aimed at β-catenin/Tcf PPI,

but very limited progress has been made. On one hand, the

contacting area between β-catenin and Tcf is very large (3500

Å²) and the binding constant (K_D) is 7−10 nM, suggesting that

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(3-carbamoylpiperidin-1-yl)phenoxy))acetyl sulfonamide scaf-

fold displays low micromolar activity for disrupting the β-

catenin/BCL9 interaction. This compound is PAINS-free,

exhibits on-target cellular activities, and is ready for further

optimization. This compound inhibits the viability of Wnt-

dependent cancer cells and displays selectivity over normal

breast epithelial MCF10A cells. Compared with the carboxylic

acid (e.g., the original hit CP-868388), the additional

substituent of N-acyl sulfonamide (in compound 30) allows

exploration of new binding space at the PPI interface while

maintaining the necessary negative charge, resulting in a more

potent inhibitor with desired cell-based activities. Indeed, the N-

acyl sulfonamide moiety has been used to design PPI

inhibitors.⁵³ABT-199 (Venetoclax), a N-acyl sulfonamide-

containing Bcl-2 PPI inhibitor was approved by FDA as a

second-line treatment with chronic lymphocytic leukemia

(CLL) with 17p deletion.^58,59To reason why the racemic

compound 26 displayed similar activities with its R-isomer 29

and S-isomer 30 in AlphaScreen assays, molecular modeling was

carried out for 29 and 30. The potential binding models of 29

and 30 with β-catenin are shown in Figure 6. For both 30 and 29,

compound for further optimization of small-molecule disruptors

of the β-catenin/BCL9 interaction.

EXPERIMENTAL SECTION

■

Chemical Synthesis. General Methods, Reagents, and Materials.

All chemical reagents were obtained from commercial sources (Combi-

Blocks, Inc.; Oakwood Products, Inc.; Fisher Scientiﬁc; and VWR

1

International, LLC) and used without further puriﬁcation. H NMR

and ¹³C NMR spectra were collected on Bruker AVANCEIIIHD 500

(500 MHz) spectrometers (125.7 MHz for ¹³C NMR spectra) in

CDCl₃, DMSO-d₆, and d₄-methanol. Chemical shifts were reported as

values in parts per million (ppm). The reference resonance peaks were

set at 7.26 ppm (CHCl₃), 2.50 ppm [(CD₂H)₂SO], and 3.31 ppm

(CD₂HOD) for ¹H NMR spectra and 77.23 ppm (CDCl₃), 39.52 ppm

(DMSO-d₆), and 49.00 ppm (CD₃OD) for ¹³C NMR spectra. High-

resolution mass spectra were recorded using an Agilent G6230BA TOF

LCMS mass spectrometer with a TOF mass detector. Low-resolution

mass spectra were determined on an Agilent 6120 single quadrupole

MS with 1220 inﬁnity LC system (HPLC-MS) and an ESI source.

Thin-layer chromatography was carried out on E. Merck pre-coated

silica gel 60 F254 plates with a UV−visible lamp. Column

chromatography was performed with SilicaFlash^@F60 (230−400

mesh). HPLC analyses were used to determine the purity of ﬁnal

compounds 2−30. The instrument was an Agilent 1260 Inﬁnity II

HPLC system with a quaternary pump, a DAD detector, and a vial

sampler. A Phenomenex C18 column (Luna 5 μL C18(2) 100 Å, 4.6 ×

250 mm) was used in analytical HPLC analyses. The DAD detector was

set to 254 nm. The purity of all tested compounds was determined to be

>95%.

General Procedure for the Reductive Amination Reaction. A

solution of aldehyde (1 mmol) and amine (2 mmol) in anhydrous

methanol (5 mL) was stirred under Ar at room temperature overnight.

NaBH₄(1.5 mmol) was then added portion-wise to the reaction

mixture at 0 °C. The mixture was stirred at 0 °C for 1 h. Saturated

NH₄Cl was added, followed by the addition of ethyl acetate. The

organic layer was collected and washed with brine, dried over

anhydrous Na₂SO₄, concentrated under reduced pressure, and puriﬁed

by column chromatography to yield the product.

General Procedure for the Amide Coupling Reaction. At 0 °C, to a

mixture of carboxylic acid (1 mmol), amine (1.1 mmol), and HATU

(1.5 mmol) in CH₂Cl₂(10 mL) was added dropwise N,N-

diisopropylethylamine (DIPEA) (2 mmol). The reaction mixture was

warmed to room temperature and stirred overnight. After the reaction is

completed, more CH₂Cl₂was added, and the organic phase was washed

with 1 M HCl, saturated NaHCO₃, and brine, dried over anhydrous

Na₂SO₄, and concentrated in vacuo. Column chromatography was

employed to purify the target compound.

Figure 6. Docking models of 30 (A) and 29 (B) with the β-catenin

armadillo repeat domain (PDB ID, 2GL7, residues 142−663). The

limitation of this docking study was that the N-terminally disordered

region (residues 1−141) of β-catenin was not observed in PDB ID

2GL7. If the N-terminally disordered region of β-catenin is involved in

binding with these β-catenin/BCL9 inhibitors, these docking models

might not be accurate.

General Procedure for Deprotection of Boc using 50% TFA in

CH₂Cl₂. To a solution of Boc-protected amine (1 mmol) in 3 mL of

CH₂Cl₂was added TFA (3 mL) at room temperature. The reaction was

then stirred for 3 h at room temperature. TFA was evaporated under the

reduced pressure by adding CH₂Cl₂three times to aﬀord the desired

product, which was used directly in the next step.

the thiophene moiety can occupy the hydrophobic pocket

surrounded by L159, V167, and A171 of β-catenin, while the N-

acyl sulfonamide group was predicted to form polar interactions

with K181.

General procedure for the Buchwald−Hartwig amination

Reaction. A solution of bromobenzene derivatives (1 mmol), amine

(1.1 mmol), Pd₂(dba)₃(0.1 mmol), RuPhos (0.2 mmol), and Cs₂CO₃

(2 mmol) in toluene (10 mL) was heated to 80 °C under argon and

stirred overnight. The reaction mixture was cooled to room

temperature. The solid was removed, and the ﬁltrate was concentrated

under reduced pressure and puriﬁed by column chromatography to

yield the target compound.

General Procedure for Deprotection of tert-Butyl Using 50% TFA

in CH₂Cl₂. At 0 °C, to a solution of tert-butyl ester (1 mmol) in CH₂Cl₂

(5 mL) was added TFA (5 mL) drop-wise. The reaction mixture was

stirred at 0 °C for 6 h. Upon completion, the solvent was evaporated

under the reduced pressure. TFA was removed by adding CH₂Cl₂three

times to aﬀord the desired product, which was further puriﬁed by

column chromatography.

In conclusion, the β-catenin/BCL9 interaction is an appealing

therapeutic target to block hyperactive β-catenin signaling for

cancer drug discovery. Our medicinal chemistry optimization on

a screening hit successfully yielded novel small-molecule β-

catenin/BCL9 disruptors. The representative compound 30

disrupts the β-catenin/BCL9 interaction with the K_iof 3.6 μM in

AlphaScreen assays. It is noted that although the initial hit 1

inhibits the β-catenin/BCL9 interaction with a K_iof 11 μM in

AlphaScreen assays, it does not show any cellular activity.

Encouragingly, cell-based studies revealed that new inhibitor 30

disrupted β-catenin/BCL9 PPI, while sparing the β-catenin/E-

cadherin complex, dose-dependently inhibited Wnt signaling

transactivation, decreased the Wnt target gene expression, and

selectively suppressed the growth of Wnt hyperactive cancer

cells. This inhibitor with a novel scaﬀold can serve as a lead

General Procedure for Hydrolysis of Ethyl Ester. To a solution of

ethyl ester (1 mmol) in THF (4 mL) and methanol (2 mL) was added a

5894

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J. Med. Chem. 2021, 64, 5886−5904

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Article

solution of LiOH (2 mmol) in H₂O (1 mL). The mixture was stirred at

room temperature for 3 h. After the completion of the reaction, the

solvent was removed under reduced pressure, and the residue was re-

dissolved in H₂O and was acidiﬁed using 1 M HCl. Ethyl acetate was

added, and the organic phase was washed with brine, dried over

anhydrous Na₂SO₄, and concentrated under reduced pressure to yield

the target compound, which was used directly in the next step.

General Procedure for the Suzuki Coupling Reaction. To a

solution of bromobenzene derivatives (1 mmol) in dioxane/water (12/

4 mL) were added boronic acid (1.2 mmol), Pd(PPh₃)₄(0.1 mmol),

and K₂CO₃(2 mmol). The reaction mixture was heated to 90 °C under

argon and stirred overnight. The reaction mixture was cooled to room

temperature, concentrated under reduced pressure, then re-dissolved

with ethyl acetate, washed with water and brine, and dried over Na₂SO₄.

The solution obtained was concentrated under vacuum. The residue

was puriﬁed by column chromatography to yield the target compound.

General Procedure for the Coupling Reaction between Carboxylic

Acid and Alcohol or Sulfonamide. To a solution of carboxylic acid (1

mmol) and alcohol (1 mmol) or sulfonamide (1 mmol) in CH₂Cl₂were

added EDC^.HCl (1.5 mmol) and DMAP (2 mmol). The mixture was

stirred at room temperature overnight. More CH₂Cl₂was added to the

mixture, and the organic phase was washed with 1 M HCl and

concentrated under reduced pressure. The pure compound was

obtained by column chromatography.

Synthesis of tert-Butyl 2-(3-bromophenoxy)-2-methylpropa-

noate. A solution of 3-bromophenol (1 mmol), tert-butyl 2-bromo-2-

methylpropanoate (5 mmol), K₂CO₃(4 mmol), and MgSO₄(1 mmol)

in DMF was stirred at 100 °C overnight. The DMF was removed under

reduced pressure. Water and ethyl acetate were added. The obtained

organic layer was washed with brine, dried over Na₂SO₄, and

concentrated under vacuum. The residue was puriﬁed by column

chromatography to yield the target compound.

2-(3-(3-((4-Isopropylbenzyl)carbamoyl)piperidin-1-yl)-

phenoxy)-2-methylpropanoic Acid (2). ¹H NMR (500 MHz,

Chloroform-d): δ 7.30 (t, J = 5.9 Hz, 1H), 7.19 (t, J = 8.2 Hz, 1H), 7.14

(s, 4H), 7.08−6.96 (m, 2H), 6.84 (dd, J = 8.3, 2.1 Hz, 1H), 4.39 (dd, J =

14.7, 6.1 Hz, 1H), 4.24 (dd, J = 14.7, 5.4 Hz, 1H), 3.56−3.35 (m, 3H),

3.18 (dtd, J = 31.6, 12.0, 11.5, 3.5 Hz, 2H), 2.86 (hept, J = 6.9 Hz, 1H),

2.17−2.02 (m, 1H), 2.02−1.86 (m, 2H), 1.86−1.74 (m, 1H), 1.57 (s,

6H), 1.20 (d, J = 7.0 Hz, 6H). ¹³C NMR (126 MHz, Chloroform-d): δ

175.9, 171.8, 156.8, 148.2, 144.3, 135.1, 130.7, 127.7, 126.7, 119.0,

113.4, 111.3, 79.8, 56.8, 55.4, 43.2, 41.0, 33.8, 25.4, 25.2, 25.1, 24.0,

22.5. HRMS (ESI): calcd for C₂₆H₃₄N₂O₄(M − H)⁻, 437.2446; found,

437.2443. HPLC purity 99.7%, t_R= 12.69 min.

2-(3-(3-(Ethyl(4-isopropylbenzyl)carbamoyl)piperidin-1-yl)-

phenoxy)-2-methylpropanoic Acid (5). ¹H NMR (500 MHz,

Chloroform-d): δ 7.30−7.18 (m, 3H), 7.16 (d, J = 8.1 Hz, 1H), 7.13−

7.01 (m, 3H), 6.87 (ddd, J = 23.2, 8.2, 2.2 Hz, 1H), 4.66 (dd, J = 40.0,

15.8 Hz, 1H), 4.45 (t, J = 15.0 Hz, 1H), 3.78−3.21 (m, 6H), 3.20−2.96

(m, 1H), 2.94−2.81 (m, 1H), 2.45−1.64 (m, 4H), 1.60 (t, J = 2.7 Hz,

6H), 1.22 (dd, J = 6.9, 3.3 Hz, 6H), 1.17 (t, J = 7.1 Hz, 2H), 1.10 (t, J =

7.1 Hz, 1H). ¹³C NMR (126 MHz, Chloroform-d): δ 175.3, 172.2,

171.8, 162.3, 162.0, 156.8, 156.6, 148.6, 148.2, 144.1, 134.3, 133.9,

130.8, 130.6, 127.8, 127.0, 126.7, 126.5, 119.9, 119.2, 113.7, 113.5,

112.1, 111.8, 80.1, 80.0, 56.6, 56.4, 55.9, 55.3, 50.6, 47.9, 41.94, 41.87,

37.4, 36.9, 33.78, 33.76, 26.1, 25.8, 25.3, 24.9, 24.0, 23.9, 22.73, 22.68,

14.1, 12.6. HRMS (ESI): calcd for C₂₈H₃₈N₂O₄(M − H)⁻, 465.2759;

found, 465.2758. HPLC purity 98.9%, t_R= 13.27 min.

2-(3-(3-(Isopropyl(4-isopropylbenzyl)carbamoyl)piperidin-

1-yl)phenoxy)-2-methylpropanoic Acid (6). ¹H NMR (500 MHz,

Chloroform-d): δ 7.28−7.08 (m, 5H), 7.06 (d, J = 8.2 Hz, 1H), 7.02−

6.91 (m, 1H), 6.91−6.75 (m, 1H), 4.80 (p, J = 6.8 Hz, 0.5H), 4.58 (t, J =

16.0 Hz, 1H), 4.40 (t, J = 16.8 Hz, 1H), 4.24 (p, J = 6.6 Hz, 0.5H), 3.62

(d, J = 29.3 Hz, 2H), 3.50 (t, J = 14.6 Hz, 1H), 3.38 (t, J = 11.5 Hz,

0.5H), 3.14 (dd, J = 13.2, 10.1 Hz, 1H), 2.96 (td, J = 11.6, 4.4 Hz, 0.5H),

2.87 (dp, J = 13.9, 6.9 Hz, 1H), 2.25 (t, J = 13.6 Hz, 0.5H), 2.10−1.90

(m, 1H), 1.85−1.38 (m, 8.5H), 1.31−0.82 (m, 12H). ¹³C NMR (126

MHz, Chloroform-d): δ 175.2, 173.2, 156.7, 156.5, 148.2, 147.3, 136.0,

135.3, 130.7, 130.5, 126.8, 126.49, 126.47, 126.2, 113.7, 113.4, 112.1,

111.6, 80.2, 80.0, 56.5, 56.1, 55.8, 54.8, 49.2, 46.4, 46.1, 43.9, 38.7, 37.3,

33.70, 33.68, 26.3, 25.8, 25.24, 25.18, 25.0, 24.9, 24.0, 23.9, 22.9, 22.7,

21.8, 21.7, 20.5, 20.2. HRMS (ESI): calcd for C₂₉H₄₀N₂O₄(M − H)⁻,

479.2915; found, 479.2914. HPLC purity 100%, t_R= 13.70 min.

(R)-2-(3-(3-(Cyclopropyl(4-isopropylbenzyl)carbamoyl)-

piperidin-1-yl)phenoxy)-2-methylpropanoic Acid (7). ¹H NMR

(500 MHz, Chloroform-d): δ 7.34−7.22 (m, 1H), 7.22−7.02 (m, 6H),

6.90 (dd, J = 8.3, 2.2 Hz, 1H), 4.69 (d, J = 14.6 Hz, 1H), 4.44 (d, J = 14.4

Hz, 1H), 4.06 (dd, J = 13.3, 9.6 Hz, 1H), 3.80−3.46 (m, 3H), 3.14 (td, J

= 12.5, 3.1 Hz, 1H), 2.89 (p, J = 6.9 Hz, 1H), 2.66 (tt, J = 6.8, 4.1 Hz,

1H), 2.24 (q, J = 13.6 Hz, 1H), 2.13−1.93 (m, 2H), 1.77 (qd, J = 13.1,

3.8 Hz, 1H), 1.62 (s, 6H), 1.25 (d, J = 6.9 Hz, 6H), 1.13−0.36 (m, 4H).

¹³C NMR (126 MHz, Chloroform-d): δ 175.4, 174.7, 156.7, 147.9,

144.5, 134.9, 130.7, 127.5, 126.7, 119.6, 113.5, 111.8, 80.0, 56.1, 55.8,

49.9, 37.9, 33.8, 30.0, 25.6, 25.3, 24.9, 24.0, 22.9, 9.5, 8.7. HRMS (ESI):

calcd for C₂₉H₃₈N₂O₄(M − H)⁻, 477.2759; found, 477.2753. HPLC

purity 97.9%, t_R= 13.45 min.

2-(3-(3-((4-(1H-Pyrazol-4-yl)benzyl)(cyclopropyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoic Acid

(8). ¹H NMR (500 MHz, DMSO-d₆): δ 8.02 (s, 2H), 7.55 (d, J = 8.1

Hz, 2H), 7.16 (dd, J = 7.9, 5.9 Hz, 3H), 6.73 (s, 1H), 6.56 (s, 1H), 6.34

(s, 1H), 4.63 (d, J = 15.0 Hz, 1H), 4.40 (d, J = 15.0 Hz, 1H), 3.76−3.67

(m, 1H), 3.65 (d, J = 12.4 Hz, 1H), 3.48 (s, 1H), 2.92 (d, J = 52.1 Hz,

2H), 2.70 (tt, J = 6.8, 4.1 Hz, 1H), 1.94 (d, J = 11.3 Hz, 1H), 1.79 (d, J =

13.0 Hz, 1H), 1.70−1.57 (m, 2H), 1.49 (d, J = 3.1 Hz, 6H), 0.90−0.75

(m, 4H). ¹³C NMR (126 MHz, DMSO-d₆): δ 175.6, 156.7, 136.5,

132.0, 131.2, 130.1, 128.0, 125.7, 121.4, 78.8, 53.0, 49.0, 38.9, 29.9,

27.4, 25.6, 25.5, 23.9, 9.5, 8.7. HRMS (ESI): calcd for C₂₉H₃₄N₄O₄(M

− H)⁻, 501.2507; found, 501.2512. HPLC purity 99.1%, t_R= 10.83 min.

2-(3-(3-(Cyclopropyl(4-(1-methyl-1H-pyrazol-4-yl)benzyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoic Acid

(9). ¹H NMR (500 MHz, Chloroform-d): δ 7.52 (d, J = 2.0 Hz, 1H),

7.39−7.32 (m, 2H), 7.29 (d, J = 8.1 Hz, 2H), 7.09 (t, J = 8.2 Hz, 1H),

6.63 (dd, J = 8.2, 2.2 Hz, 1H), 6.60 (s, 1H), 6.41 (dd, J = 8.1, 2.2 Hz,

1H), 6.29 (d, J = 1.9 Hz, 1H), 4.70 (d, J = 14.9 Hz, 1H), 4.59 (d, J = 14.9

Hz, 1H), 3.87 (s, 3H), 3.65 (dd, J = 12.7, 3.5 Hz, 2H), 3.49 (s, 1H), 3.00

(t, J = 11.6 Hz, 1H), 2.81−2.70 (m, 1H), 2.66 (tt, J = 7.0, 4.0 Hz, 1H),

1.96 (d, J = 8.0 Hz, 1H), 1.87−1.67 (m, 3H), 1.59 (s, 6H), 0.96−0.78

(m, 4H). ¹³C NMR (126 MHz, Chloroform-d): δ 177.1, 176.6, 155.8,

152.3, 143.4, 138.8, 138.4, 129.5, 129.3, 128.9, 127.8, 111.4, 111.0,

108.9, 106.0, 79.5, 52.3, 50.4, 49.8, 39.8, 37.4, 30.1, 27.7, 25.3, 25.1,

24.4, 9.5, 9.1. HRMS (ESI): calcd for C₃₀H₃₆N₄O₄(M − H)⁻,

515.2664; found, 515.2673. HPLC purity 99.5%, t_R= 11.58 min.

2-(3-(3-((4-(1H-Pyrazol-5-yl)benzyl)(cyclopropyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoic Acid

2-(3-(3-((4-Isopropylphenethyl)carbamoyl)piperidin-1-yl)-

phenoxy)-2-methylpropanoic Acid (3). ¹H NMR (500 MHz,

Methanol-d₄): δ 7.35 (t, J = 8.2 Hz, 1H), 7.18−7.09 (m, 4H), 7.04 (dd, J

= 8.2, 2.2 Hz, 1H), 6.99 (t, J = 2.3 Hz, 1H), 6.82 (dd, J = 8.3, 2.2 Hz,

1H), 3.56 (ddd, J = 15.9, 11.1, 4.3 Hz, 2H), 3.49 (dt, J = 14.1, 7.2 Hz,

1H), 3.45−3.33 (m, 2H), 3.31−3.20 (m, 1H), 2.81 (dt, J = 29.7, 6.9 Hz,

4H), 1.99−1.80 (m, 3H), 1.61 (s, 6H), 1.20 (dd, J = 6.9, 1.1 Hz, 6H).

¹³C NMR (126 MHz, Methanol-d₄): δ 175.7, 173.7, 156.9, 146.8, 136.2,

130.2, 128.5, 126.0, 116.6, 112.8, 111.0, 79.3, 56.1, 53.7, 40.4, 34.6,

33.6, 25.7, 24.4, 24.3, 23.10, 23.12, 22.2. HRMS (ESI): calcd for

C₂₇H₃₆N₂O₄(M − H)⁻, 451.2602; found, 451.2602. HPLC purity

98.0%, t_R= 12.76 min.

2-(3-(3-((4-Isopropylbenzyl)(methyl)carbamoyl)piperidin-1-

1

yl)phenoxy)-2-methylpropanoic Acid (4). H NMR (500 MHz,

Chloroform-d): δ 9.01 (s, 2H), 7.26−7.19 (m, 2H), 7.19−7.12 (m,

2H), 7.11−7.08 (m, 2H), 7.08−6.98 (m, 1H), 6.86 (ddd, J = 26.7, 8.6,

1.9 Hz, 1H), 4.79−4.57 (m, 1H), 4.43 (dd, J = 20.4, 15.5 Hz, 1H),

3.70−3.44 (m, 4H), 3.20−2.95 (m, 4H), 2.88 (hept, J = 6.9 Hz, 1H),

2.28−1.67 (m, 4H), 1.65−1.55 (m, 6H), 1.23 (dd, J = 6.9, 2.0 Hz, 6H).

¹³C NMR (126 MHz, Chloroform-d): δ 175.5, 172.7, 171.9, 156.8,

156.7, 148.6, 148.3, 144.8, 144.2, 133.7, 133.5, 130.8, 130.6, 127.9,

127.0, 126.8, 126.5, 119.6, 118.8, 113.6, 113.4, 111.8, 111.5, 80.0, 56.5,

56.2, 55.8, 55.2, 53.3, 50.9, 37.2, 37.0, 34.8, 33.79, 33.76, 25.9, 25.35,

25.30, 25.26, 25.01, 24.97, 24.0, 23.9, 22.7. HRMS (ESI): calcd for

C₂₇H₃₆N₂O₄(M − H)⁻, 451.2602; found, 451.2599. HPLC purity

99.1%, t_R= 12.99 min.

1

(10). H NMR (500 MHz, DMSO-d₆): δ 7.74 (d, J = 8.1 Hz, 2H),

5895

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7.70 (d, J = 2.2 Hz, 1H), 7.22 (d, J = 8.0 Hz, 2H), 7.14 (t, J = 8.2 Hz,

1H), 6.78−6.63 (m, 2H), 6.55 (s, 1H), 6.33 (d, J = 8.1 Hz, 1H), 4.65 (d,

J = 15.2 Hz, 1H), 4.44 (d, J = 15.1 Hz, 1H), 3.78−3.69 (m, 1H), 3.65 (d,

J = 11.9 Hz, 1H), 3.47 (s, 1H), 2.91 (d, J = 53.2 Hz, 2H), 2.73 (tt, J =

6.9, 4.1 Hz, 1H), 1.95 (d, J = 11.1 Hz, 1H), 1.84−1.56 (m, 3H), 1.49 (d,

J = 3.1 Hz, 6H), 0.92−0.70 (m, 4H). ¹³C NMR (126 MHz, DMSO-d₆):

δ 176.0, 175.6, 156.7, 138.2, 133.2, 131.6, 130.1, 127.9, 125.7, 110.7,

107.8, 102.3, 78.7, 52.8, 50.4, 49.1, 38.9, 30.0, 27.4, 25.6, 25.5, 24.0, 9.5,

8.7. HRMS (ESI): calcd for C₂₉H₃₄N₄O₄(M − H)⁻, 501.2507; found,

501.2511. HPLC purity 99.5%, t_R= 10.91 min.

6.62 (m, 2H), 6.58 (t, J = 2.4 Hz, 1H), 6.41 (dd, J = 8.0, 2.2 Hz, 1H),

4.71 (d, J = 14.7 Hz, 1H), 4.50 (d, J = 14.6 Hz, 1H), 3.70−3.57 (m, 2H),

3.48 (d, J = 11.0 Hz, 1H), 3.01 (t, J = 11.7 Hz, 1H), 2.74 (td, J = 12.1,

11.6, 3.4 Hz, 1H), 2.58 (ddt, J = 9.6, 6.8, 4.0 Hz, 1H), 1.94 (d, J = 8.6

Hz, 1H), 1.84−1.70 (m, 3H), 1.58 (s, 6H), 0.90−0.77 (m, 4H). ¹³C

NMR (126 MHz, Chloroform-d): δ 177.0, 176.8, 155.6, 152.1, 143.7,

138.4, 137.0, 131.3, 129.5, 128.2, 126.1, 126.0, 111.7, 111.4, 109.2,

108.8, 79.6, 52.3, 50.5, 49.6, 39.7, 29.8, 27.6, 25.2, 25.1, 24.4, 9.5, 9.0.

HRMS (ESI): calcd for C₃₀H₃₄N₂O₅(M − H)⁻, 501.2395; found,

501.2404. HPLC purity 98.8%, t_R= 13.06 min.

2-(3-(3-(Cyclopropyl(4-(1-methyl-1H-pyrazol-5-yl)benzyl)-

Methyl 2-(3-(3-(cyclopropyl(4-(thiophen-2-yl)benzyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate (16).

¹H NMR (500 MHz, Chloroform-d): δ 7.58−7.53 (m, 2H), 7.29−7.24

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoic Acid

1

(11). H NMR (500 MHz, Chloroform-d): δ 7.79−7.72 (m, 1H),

7.58 (s, 1H), 7.46−7.36 (m, 2H), 7.20 (d, J = 7.9 Hz, 2H), 7.09 (t, J =

8.2 Hz, 1H), 6.63 (dd, J = 8.2, 2.2 Hz, 1H), 6.56 (t, J = 2.3 Hz, 1H),

6.45−6.35 (m, 1H), 4.67 (d, J = 14.6 Hz, 1H), 4.51 (d, J = 14.6 Hz, 1H),

3.93 (s, 3H), 3.64 (t, J = 8.4 Hz, 2H), 3.46 (s, 1H), 2.99 (t, J = 11.6 Hz,

1H), 2.79−2.69 (m, 1H), 2.58 (tt, J = 6.8, 4.0 Hz, 1H), 1.93 (d, J = 7.9

Hz, 1H), 1.86−1.68 (m, 3H), 1.58 (s, 6H), 0.91−0.76 (m, 4H). ¹³C

NMR (126 MHz, Chloroform-d): δ 176.9, 176.6, 155.6, 152.3, 136.6,

136.4, 131.4, 129.5, 128.3, 127.0, 125.7, 122.9, 111.5, 111.1, 109.1, 79.6,

52.3, 50.4, 49.6, 39.8, 39.0, 29.8, 27.6, 25.2, 25.1, 24.4, 9.5, 9.0. HRMS

(ESI): calcd for C₃₀H₃₆N₄O₄(M − H)⁻, 515.2664; found, 515.2674.

HPLC purity 99.8%, t_R= 11.57 min.

(m, 4H), 7.09−7.04 (m, 2H), 6.64−6.54 (m, 1H), 6.48 (s, 1H), 6.25

(dd, J = 8.2, 2.2 Hz, 1H), 4.67 (d, J = 14.7 Hz, 1H), 4.57 (d, J = 14.8 Hz,

1H), 3.74 (s, 3H), 3.71−3.66 (m, 2H), 3.45 (s, 1H), 3.01 (t, J = 11.7

Hz, 1H), 2.82−2.74 (m, 1H), 2.61 (ddt, J = 10.8, 6.9, 4.0 Hz, 1H),

2.01−1.93 (m, 1H), 1.77 (ddd, J = 20.6, 17.2, 8.3 Hz, 3H), 1.58 (s, 6H),

0.91−0.82 (m, 4H). ¹³C NMR (126 MHz, Chloroform-d): δ 176.8,

175.0, 156.4, 144.1, 137.7, 133.3, 129.4, 128.3, 128.0, 126.1, 124.7,

123.0, 110.7, 109.3, 107.9, 78.9, 52.5, 52.3, 50.1, 49.5, 39.8, 29.8, 27.8,

25.5, 25.4, 24.4, 9.5, 9.1. HRMS (ESI): calcd for C₃₁H₃₆N₂O₄S (M +

H)⁺533.2469; found, 533.2469. HPLC purity 99.0%, t_R= 14.44 min.

tert-Butyl 2-(3-(3-(cyclopropyl(4-(thiophen-2-yl)benzyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate (17).

¹H NMR (500 MHz, Chloroform-d): δ 7.53−7.42 (m, 2H), 7.24−7.12

(m, 4H), 7.04−6.93 (m, 2H), 6.48 (dd, J = 8.2, 2.3 Hz, 1H), 6.41 (t, J =

2.3 Hz, 1H), 6.22 (dd, J = 8.1, 2.3 Hz, 1H), 4.59 (d, J = 14.7 Hz, 1H),

4.48 (d, J = 14.7 Hz, 1H), 3.69−3.51 (m, 2H), 3.37 (tt, J = 10.9, 3.0 Hz,

1H), 2.99−2.82 (m, 1H), 2.70 (td, J = 12.2, 2.8 Hz, 1H), 2.59−2.44 (m,

1H), 1.86 (d, J = 11.3 Hz, 1H), 1.75−1.63 (m, 3H), 1.47 (s, 6H), 1.35

(s, 9H), 0.85−0.66 (m, 4H). ¹³C NMR (126 MHz, Chloroform-d): δ

176.9, 173.5, 156.7, 152.4, 144.1, 137.7, 133.3, 129.2, 128.3, 128.0,

126.1, 124.7, 123.0, 110.2, 109.1, 107.6, 81.5, 79.2, 52.2, 50.0, 49.5,

39.8, 29.8, 27.9, 27.8, 25.53, 25.47, 24.5, 9.5, 9.1. HRMS (ESI): calcd

for C₃₄H₄₂N₂O₄S (M + H)⁺575.2938; found, 575.2925. HPLC purity

100%, t_R= 15.31 min.

N-Cyclopropyl-1-(3-((2-methyl-1-(methylsulfonamido)-1-

oxopropan-2-yl)oxy)phenyl)-N-(4-(thiophen-2-yl)benzyl)-

piperidine-3-carboxamide (18). ¹H NMR (500 MHz, Chloroform-

d): δ 8.93 (s, 1H), 7.52−7.45 (m, 2H), 7.22 (dd, J = 3.6, 1.2 Hz, 1H),

7.20−7.16 (m, 3H), 7.08 (t, J = 8.2 Hz, 1H), 7.00 (dd, J = 5.1, 3.5 Hz,

1H), 6.70−6.56 (m, 1H), 6.41 (t, J = 2.4 Hz, 1H), 6.28 (dd, J = 8.2, 2.2

Hz, 1H), 4.67 (d, J = 14.7 Hz, 1H), 4.43 (d, J = 14.7 Hz, 1H), 3.68−3.55

(m, 2H), 3.43−3.30 (m, 1H), 3.25 (s, 3H), 2.97 (t, J = 11.6 Hz, 1H),

2.73 (t, J = 10.6 Hz, 1H), 2.55 (tt, J = 7.0, 4.1 Hz, 1H), 1.91 (d, J = 9.8

Hz, 1H), 1.81−1.75 (m, 1H), 1.72−1.63 (m, 2H), 1.47 (s, 6H), 0.86−

0.74 (m, 4H). ¹³C NMR (126 MHz, Chloroform-d): δ 176.8, 174.2,

154.1, 152.7, 144.1, 137.6, 133.3, 129.7, 128.3, 128.0, 126.1, 124.7,

123.0, 112.3, 111.5, 109.8, 81.5, 51.9, 49.9, 49.5, 41.3, 39.8, 29.8, 27.8,

24.6, 24.5, 9.6, 9.0. HRMS (ESI): calcd for C₃₁H₃₇N₃O₅S₂(M − H)⁻,

594.2102; found, 594.2107. HPLC purity 97.3%, t_R= 13.86 min.

1-(3-((1-(Cyclopropanesulfonamido)-2-methyl-1-oxopro-

pan-2-yl)oxy)phenyl)-N-cyclopropyl-N-(4-(thiophen-2-yl)-

benzyl)piperidine-3-carboxamide (19). ¹H NMR (500 MHz,

Chloroform-d): δ 8.87 (s, 1H), 7.52−7.46 (m, 2H), 7.23−7.16 (m,

4H), 7.07 (t, J = 8.2 Hz, 1H), 7.00 (dd, J = 5.1, 3.5 Hz, 1H), 6.67−6.56

(m, 1H), 6.42 (s, 1H), 6.33−6.24 (m, 1H), 4.67 (d, J = 14.6 Hz, 1H),

4.42 (d, J = 14.7 Hz, 1H), 3.67−3.54 (m, 2H), 3.38 (d, J = 11.7 Hz, 1H),

3.01−2.88 (m, 2H), 2.72 (t, J = 11.0 Hz, 1H), 2.54 (td, J = 6.6, 5.9, 3.4

Hz, 1H), 1.90 (s, 1H), 1.81−1.75 (m, 1H), 1.67 (td, J = 11.6, 5.7 Hz,

2H), 1.47 (s, 6H), 1.35−1.32 (m, 2H), 1.08−1.02 (m, 2H), 0.85−0.73

(m, 4H). ¹³C NMR (126 MHz, Chloroform-d): δ 176.8, 173.8, 154.3,

152.7, 144.1, 137.6, 133.3, 129.7, 128.3, 128.0, 126.1, 124.7, 123.0,

112.2, 111.3, 109.6, 81.4, 51.9, 50.0, 49.5, 39.8, 31.1, 29.8, 27.8, 24.6,

24.5, 9.6, 9.0, 6.2. HRMS (ESI): calcd for C₃₃H₃₉N₃O₅S₂(M − H)⁻,

620.2258; found, 620.2262. HPLC purity 96.9%, t_R= 14.26 min.

N-Cyclopropyl-1-(3-((2-methyl-1-((1-methylethyl)-

sulfonamido)-1-oxopropan-2-yl)oxy)phenyl)-N-(4-(thiophen-

2-yl)benzyl)piperidine-3-carboxamide (20). ¹H NMR (500 MHz,

2-(3-(3-(Cyclopropyl(4-(thiophen-2-yl)benzyl)carbamoyl)-

piperidin-1-yl)phenoxy)-2-methylpropanoic Acid (12). ¹H

NMR (500 MHz, Chloroform-d): δ 7.50−7.45 (m, 2H), 7.21 (dd, J

= 3.6, 1.2 Hz, 1H), 7.19−7.17 (m, 1H), 7.14 (d, J = 8.0 Hz, 2H), 7.02−

6.97 (m, 2H), 6.56 (dd, J = 8.2, 2.3 Hz, 1H), 6.50 (d, J = 2.4 Hz, 1H),

6.33 (dd, J = 8.1, 2.3 Hz, 1H), 4.64 (d, J = 14.7 Hz, 1H), 4.42 (d, J = 14.7

Hz, 1H), 3.56 (d, J = 11.6 Hz, 2H), 3.41 (tt, J = 11.4, 3.6 Hz, 1H), 2.93

(t, J = 11.6 Hz, 1H), 2.65 (td, J = 11.9, 3.3 Hz, 1H), 2.51 (tt, J = 6.9, 4.0

Hz, 1H), 1.85 (d, J = 7.8 Hz, 1H), 1.74−1.62 (m, 3H), 1.50 (s, 6H),

0.83−0.71 (m, 4H). ¹³C NMR (126 MHz, Chloroform-d): δ 177.2,

177.1, 155.8, 152.1, 144.1, 137.5, 133.4, 129.5, 128.3, 128.0, 126.1,

124.7, 123.0, 111.5, 111.2, 109.1, 79.4, 52.4 50.6, 49.6, 39.7, 29.9, 27.6,

25.3, 25.2, 24.3, 9.6, 9.0. HRMS (ESI): calcd for C₃₀H₃₄N₂O₄S (M −

H)⁻, 517.2167; found, 517.2176. HPLC purity 99.2%, t_R= 13.56 min.

2-(3-(3-(Cyclopropyl(4-(thiophen-3-yl)benzyl)carbamoyl)-

piperidin-1-yl)phenoxy)-2-methylpropanoic Acid (13). ¹H

NMR (500 MHz, Chloroform-d): δ 7.48−7.44 (m, 2H), 7.35 (t, J =

2.2 Hz, 1H), 7.29 (d, J = 2.1 Hz, 2H), 7.18−7.15 (m, 2H), 7.00 (t, J =

8.2 Hz, 1H), 6.56 (dd, J = 8.2, 2.3 Hz, 1H), 6.51 (t, J = 2.4 Hz, 1H), 6.33

(dd, J = 8.1, 2.2 Hz, 1H), 4.64 (d, J = 14.7 Hz, 1H), 4.44 (d, J = 14.6 Hz,

1H), 3.61−3.51 (m, 2H), 3.42 (ddd, J = 11.2, 7.4, 3.5 Hz, 1H), 2.98−

2.89 (m, 1H), 2.65 (td, J = 11.8, 3.4 Hz, 1H), 2.51 (tt, J = 6.9, 4.1 Hz,

1H), 1.86 (d, J = 7.8 Hz, 1H), 1.74−1.62 (m, 3H), 1.50 (s, 6H), 0.82−

0.70 (m, 4H). ¹³C NMR (126 MHz, Chloroform-d): δ 177.1, 177.0,

155.8, 152.1, 141.9, 137.1, 134.8, 129.5, 128.2, 126.6, 126.2, 120.2,

111.5, 111.2, 109.1, 79.4, 52.4, 50.6, 49.6, 39.7, 29.9, 27.5, 25.3, 25.2,

24.3, 9.5, 9.0. HRMS (ESI): calcd for C₃₀H₃₄N₂O₄S (M − H)⁻,

517.2167; found, 517.2170. HPLC purity 99.9%, t_R= 13.43 min.

2-(3-(3-(Cyclopropyl(4-(furan-2-yl)benzyl)carbamoyl)-

piperidin-1-yl)phenoxy)-2-methylpropanoic Acid (14). ¹H

NMR (500 MHz, Chloroform-d): δ 7.64−7.58 (m, 2H), 7.45 (d, J =

1.7 Hz, 1H), 7.24 (d, J = 8.1 Hz, 2H), 7.12−7.07 (m, 1H), 6.64 (dd, J =

12.7, 5.9 Hz, 2H), 6.57 (t, J = 2.4 Hz, 1H), 6.46 (dd, J = 3.3, 1.8 Hz, 1H),

6.41 (dd, J = 8.1, 2.2 Hz, 1H), 4.72 (d, J = 14.6 Hz, 1H), 4.49 (d, J = 14.7

Hz, 1H), 3.70−3.60 (m, 2H), 3.48 (s, 1H), 3.01 (t, J = 11.6 Hz, 1H),

2.73 (dt, J = 11.9, 6.3 Hz, 1H), 2.58 (tt, J = 6.6, 4.0 Hz, 1H), 1.94 (d, J =

8.5 Hz, 1H), 1.82−1.70 (m, 3H), 1.58 (s, 6H), 0.89−0.78 (m, 4H). ¹³C

NMR (126 MHz, Chloroform-d): δ 177.0, 155.6, 153.7, 152.2, 142.0,

137.3, 129.9, 129.5, 128.1, 124.0, 113.9, 111.4, 109.2, 104.9, 79.6, 52.3,

50.5, 49.6, 39.7, 29.8, 27.6, 25.2, 25.1, 24.4, 9.6, 9.0. HRMS (ESI): calcd

for C₃₀H₃₄N₂O₅(M − H)⁻, 501.2395; found, 501.2405. HPLC purity

96.0%, t_R= 13.22 min.

2-(3-(3-(Cyclopropyl(4-(furan-3-yl)benzyl)carbamoyl)-

piperidin-1-yl)phenoxy)-2-methylpropanoic Acid (15). ¹H

NMR (500 MHz, Chloroform-d): δ 7.71 (t, J = 1.2 Hz, 1H), 7.46 (t,

J = 1.7 Hz, 3H), 7.22 (d, J = 8.0 Hz, 2H), 7.09 (t, J = 8.2 Hz, 1H), 6.72−

5896

https://doi.org/10.1021/acs.jmedchem.1c00046

J. Med. Chem. 2021, 64, 5886−5904

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Chloroform-d): δ 8.72 (s, 1H), 7.54−7.45 (m, 2H), 7.23−7.16 (m,

4H), 7.07 (t, J = 8.2 Hz, 1H), 7.00 (dd, J = 5.1, 3.6 Hz, 1H), 6.67−6.56

(m, 1H), 6.42 (s, 1H), 6.29 (dd, J = 8.2, 2.2 Hz, 1H), 4.67 (d, J = 14.7

Hz, 1H), 4.43 (d, J = 14.7 Hz, 1H), 3.77 (hept, J = 6.9 Hz, 1H), 3.66−

3.56 (m, 2H), 3.37 (t, J = 10.7 Hz, 1H), 2.97 (t, J = 11.7 Hz, 1H), 2.76−

2.68 (m, 1H), 2.59−2.51 (m, 1H), 1.91 (d, J = 10.0 Hz, 1H), 1.82−1.74

(m, 1H), 1.68 (td, J = 10.2, 8.8, 3.1 Hz, 2H), 1.47 (s, 6H), 1.35 (dd, J =

6.9, 1.4 Hz, 6H), 0.86−0.73 (m, 4H). ¹³C NMR (126 MHz,

Chloroform-d): δ 176.8, 174.1, 154.2, 152.7, 144.1, 137.6, 133.3,

129.7, 128.3, 128.0, 126.1, 124.7, 123.0, 112.3, 111.6, 109.8, 81.6, 54.1,

51.9, 49.9, 49.5, 39.8, 29.8, 27.8, 24.6, 24.5, 16.0, 15.9, 9.6, 9.0. HRMS

(ESI): calcd for C₃₃H₄₁N₃O₅S₂(M − H)⁻, 622.2415; found, 622.2419.

HPLC purity 96.9%, t_R= 14.33 min.

N - C y c l o p r o p y l - 1 - ( 3 - ( ( 2 - m e t h y l - 1 - o x o - 1 -

(phenylsulfonamido)propan-2-yl)oxy)phenyl)-N-(4-(thiophen-

2-yl)benzyl)piperidine-3-carboxamide (21). ¹H NMR (500 MHz,

Chloroform-d): δ 9.04 (s, 1H), 8.02−7.94 (m, 2H), 7.64−7.57 (m,

1H), 7.48 (dt, J = 7.8, 3.8 Hz, 4H), 7.22−7.16 (m, 4H), 7.03−6.99 (m,

1H), 6.91 (t, J = 8.2 Hz, 1H), 6.62−6.51 (m, 1H), 6.35 (t, J = 2.5 Hz,

1H), 5.99 (dd, J = 8.0, 2.3 Hz, 1H), 4.67 (d, J = 14.6 Hz, 1H), 4.43 (d, J

= 14.7 Hz, 1H), 3.64−3.52 (m, 2H), 3.36 (t, J = 10.9 Hz, 1H), 2.94 (t, J

= 11.7 Hz, 1H), 2.67 (t, J = 11.1 Hz, 1H), 2.55 (tt, J = 7.2, 4.1 Hz, 1H),

1.90 (d, J = 10.8 Hz, 1H), 1.77−1.63 (m, 3H), 1.36 (s, 6H), 0.84−0.74

(m, 4H). ¹³C NMR (126 MHz, Chloroform-d): δ 176.8, 172.9, 154.3,

152.7, 144.1, 138.2, 137.6, 134.1, 133.3, 129.6, 129.0, 128.5, 128.3,

128.0, 126.1, 124.7, 123.0, 112.0, 110.6, 109.3, 81.2, 51.9, 49.8, 49.6,

39.8, 29.8, 27.8, 24.5, 24.4, 9.6, 9.0. HRMS (ESI): calcd for

C₃₆H₃₉N₃O₅S₂(M − H)⁻, 656.2258; found, 656.2252. HPLC purity

96.2%, t_R= 14.70 min.

N-Cyclopropyl-1-(3-((1-((3-methoxyphenyl)sulfonamido)-2-

methyl-1-oxopropan-2-yl)oxy)phenyl)-N-(4-(thiophen-2-yl)-

benzyl)piperidine-3-carboxamide (22). ¹H NMR (500 MHz,

Chloroform-d): δ 9.05 (s, 1H), 7.55 (dt, J = 7.8, 1.2 Hz, 1H), 7.50−7.46

(m, 3H), 7.39−7.35 (m, 1H), 7.22−7.15 (m, 4H), 7.11 (ddd, J = 8.3,

2.7, 0.9 Hz, 1H), 6.99 (dd, J = 5.1, 3.6 Hz, 1H), 6.91 (t, J = 8.2 Hz, 1H),

6.56 (dd, J = 8.3, 2.3 Hz, 1H), 6.39−6.28 (m, 1H), 5.99 (dd, J = 8.1, 2.2

Hz, 1H), 4.66 (d, J = 14.7 Hz, 1H), 4.43 (d, J = 14.6 Hz, 1H), 3.77 (s,

3H), 3.62−3.52 (m, 2H), 3.35 (dq, J = 11.4, 5.8, 4.3 Hz, 1H), 2.94 (t, J =

11.6 Hz, 1H), 2.70−2.63 (m, 1H), 2.54 (tt, J = 6.9, 4.1 Hz, 1H), 1.89 (d,

J = 10.9 Hz, 1H), 1.76−1.63 (m, 3H), 1.36 (s, 6H), 0.85−0.73 (m, 4H).

¹³C NMR (126 MHz, Chloroform-d): δ 176.8, 172.9, 159.7, 154.3,

152.7, 144.1, 139.3, 137.6, 133.3, 130.0, 129.6, 128.3, 128.0, 126.1,

124.7, 123.0, 120.9, 120.5, 112.6, 111.9, 110.6, 109.2, 81.2, 55.7, 51.9,

49.8, 49.6, 39.8, 29.8, 27.8, 24.5, 24.4, 9.6, 9.0. HRMS (ESI): calcd for

C₃₇H₄₁N₃O₆S₂(M − H)⁻, 686.2364; found, 686.2360. HPLC purity

99.6%, t_R= 14.75 min.

N-Cyclopropyl-1-(3-((1-((4-methoxyphenyl)sulfonamido)-2-

methyl-1-oxopropan-2-yl)oxy)phenyl)-N-(4-(thiophen-2-yl)-

benzyl)piperidine-3-carboxamide (23). ¹H NMR (500 MHz,

Chloroform-d): δ 9.07 (s, 1H), 8.02−7.95 (m, 2H), 7.56 (d, J = 8.0 Hz,

2H), 7.31−7.23 (m, 4H), 7.07 (dd, J = 5.1, 3.6 Hz, 1H), 7.02−6.97 (m,

3H), 6.64 (dd, J = 8.6, 2.2 Hz, 1H), 6.41 (t, J = 2.4 Hz, 1H), 6.08 (dd, J =

8.1, 2.2 Hz, 1H), 4.74 (d, J = 14.7 Hz, 1H), 4.51 (d, J = 14.7 Hz, 1H),

3.89 (s, 3H), 3.65 (t, J = 12.7 Hz, 2H), 3.42 (d, J = 10.8 Hz, 1H), 3.01 (t,

J = 11.7 Hz, 1H), 2.75 (t, J = 11.1 Hz, 1H), 2.62 (tt, J = 7.1, 4.0 Hz, 1H),

1.97 (d, J = 10.8 Hz, 1H), 1.83 (d, J = 11.1 Hz, 1H), 1.68 (d, J = 35.2 Hz,

2H), 1.43 (s, 6H), 0.91−0.82 (m, 4H). ¹³C NMR (126 MHz,

Chloroform-d): δ 176.8, 172.9, 164.0, 154.4, 152.7, 144.1, 137.7, 133.3,

130.9, 129.6, 129.5, 128.6, 128.3, 128.0, 126.1, 124.7, 123.0, 114.1,

111.9, 110.7, 109.2, 81.2, 55.7, 51.9, 49.8, 39.8, 29.8, 27.8, 24.5, 24.4,

9.6, 9.0. HRMS (ESI): calcd for C₃₇H₄₁N₃O₆S₂(M − H)⁻, 686.2364;

found, 686.2367. HPLC purity 96.5%, t_R= 14.69 min.

N-Cyclopropyl-1-(3-((1-((3-hydroxyphenyl)sulfonamido)-2-

methyl-1-oxopropan-2-yl)oxy)phenyl)-N-(4-(thiophen-2-yl)-

benzyl)piperidine-3-carboxamide (24). ¹H NMR (500 MHz,

Chloroform-d): δ 7.53−7.48 (m, 3H), 7.37 (t, J = 2.1 Hz, 1H), 7.29 (t, J

= 8.0 Hz, 1H), 7.22−7.15 (m, 4H), 7.07−7.04 (m, 1H), 7.00−6.95 (m,

2H), 6.64−6.53 (m, 1H), 6.23 (s, 1H), 6.14 (dd, J = 8.0, 2.2 Hz, 1H),

4.72 (d, J = 14.7 Hz, 1H), 4.42 (d, J = 14.7 Hz, 1H), 3.53−3.38 (m, 3H),

2.86 (t, J = 11.5 Hz, 1H), 2.59−2.50 (m, 2H), 1.90 (d, J = 8.1 Hz, 1H),

1.75 (dt, J = 9.2, 2.9 Hz, 1H), 1.67−1.60 (m, 2H), 1.41 (s, 3H), 1.32 (s,

3H), 0.87−0.77 (m, 4H). ¹³C NMR (126 MHz, Chloroform-d): δ

177.6, 173.0, 157.1, 154.6, 152.7, 144.0, 139.0, 137.1, 133.5, 130.1,

129.8, 128.2, 128.0, 126.2, 124.8, 123.1, 122.2, 120.3, 114.6, 113.1,

112.0, 108.5, 81.3, 52.0, 50.3, 39.9, 30.1, 27.6, 25.4, 24.5, 23.9, 9.7, 8.9.

HRMS (ESI): calcd for C₃₆H₃₉N₃O₆S₂(M − H)⁻, 672.2208; found,

672.2201. HPLC purity 99.4%, t_R= 14.07 min.

1-(3-((1-((3-Cyanophenyl)sulfonamido)-2-methyl-1-oxopro-

pan-2-yl)oxy)phenyl)-N-cyclopropyl-N-(4-(thiophen-2-yl)-

benzyl)piperidine-3-carboxamide (25). ¹H NMR (500 MHz,

Chloroform-d): δ 8.30 (dq, J = 4.6, 1.4 Hz, 2H), 7.93 (dt, J = 7.8, 1.4 Hz,

1H), 7.70 (t, J = 8.2 Hz, 1H), 7.58−7.52 (m, 2H), 7.29 (dd, J = 3.6, 1.2

Hz, 1H), 7.27−7.23 (m, 3H), 7.07 (dd, J = 5.1, 3.6 Hz, 1H), 7.01 (t, J =

8.2 Hz, 1H), 6.74−6.62 (m, 1H), 6.42 (s, 1H), 6.12−5.97 (m, 1H), 4.74

(d, J = 14.6 Hz, 1H), 4.51 (d, J = 14.7 Hz, 1H), 3.70−3.62 (m, 2H), 3.43

(s, 1H), 3.02 (t, J = 11.7 Hz, 1H), 2.80−2.71 (m, 1H), 2.62 (tt, J = 6.9,

4.1 Hz, 1H), 2.01−1.95 (m, 1H), 1.86−1.81 (m, 1H), 1.74 (td, J = 10.7,

9.1, 3.2 Hz, 2H), 1.45 (s, 6H), 0.93−0.82 (m, 4H). ¹³C NMR (126

MHz, Chloroform-d): δ 176.7, 173.1, 154.1, 152.8, 144.0, 139.8, 137.6,

137.1, 133.3, 132.7, 132.1, 130.0, 129.7, 128.3, 128.0, 126.1, 124.7,

123.0, 116.9, 113.7, 112.2, 110.3, 109.1, 81.2, 51.8, 49.8, 49.6, 39.8,

29.8, 27.8, 24.5, 24.43, 24.38, 9.6, 9.0. HRMS (ESI): calcd for

C₃₇H₃₈N₄O₅S₂(M − H)⁻, 681.2211; found, 681.2219. HPLC purity

98.2%, t_R= 14.54 min.

1-(3-((1-((4-Cyanophenyl)sulfonamido)-2-methyl-1-oxopro-

pan-2-yl)oxy)phenyl)-N-cyclopropyl-N-(4-(thiophen-2-yl)-

benzyl)piperidine-3-carboxamide (26). ¹H NMR (500 MHz,

Chloroform-d): δ 8.18−8.14 (m, 2H), 7.85−7.82 (m, 2H), 7.58−7.53

(m, 2H), 7.30−7.23 (m, 4H), 7.07 (dd, J = 5.1, 3.5 Hz, 1H), 7.01 (t, J =

8.2 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 6.42 (s, 1H), 6.08 (d, J = 8.0 Hz,

1H), 4.74 (d, J = 14.7 Hz, 1H), 4.51 (d, J = 14.7 Hz, 1H), 3.65 (d, J =

11.7 Hz, 2H), 3.40 (d, J = 34.3 Hz, 1H), 3.02 (t, J = 11.7 Hz, 1H), 2.75

(t, J = 11.5 Hz, 1H), 2.62 (tt, J = 6.9, 4.1 Hz, 1H), 1.98 (d, J = 9.5 Hz,

1H), 1.88−1.83 (m, 1H), 1.74 (dd, J = 14.5, 5.8 Hz, 2H), 1.44 (s, 6H),

0.94−0.82 (m, 4H). ¹³C NMR (126 MHz, Chloroform-d): δ 176.7,

173.1, 154.1, 152.8, 144.0, 142.2, 137.6, 133.3, 132.7, 129.7, 129.2,

128.3, 128.0, 126.1, 124.8, 123.0, 117.7, 117.1, 112.3, 110.7, 109.3, 81.3,

51.9, 49.9, 49.6, 39.8, 29.8, 27.7, 24.5, 24.4, 24.3, 9.7, 8.9. HRMS (ESI):

calcd for C₃₇H₃₈N₄O₅S₂(M − H)⁻, 681.2211; found, 681.2212. HPLC

purity 98.7%, t_R= 14.62 min.

(R)-N-Cyclopropyl-1-(3-((1-((3-methoxyphenyl)-

sulfonamido)-2-methyl-1-oxopropan-2-yl)oxy)phenyl)-N-(4-

(thiophen-2-yl)benzyl)piperidine-3-carboxamide (27). [α]_D²⁵

1

−16.10° (c 0.955, MeOH). H NMR (500 MHz, Chloroform-d): δ

9.08 (s, 1H), 7.63 (dt, J = 7.8, 1.2 Hz, 1H), 7.58−7.53 (m, 3H), 7.45 (t, J

= 8.0 Hz, 1H), 7.29−7.23 (m, 4H), 7.19 (ddd, J = 8.3, 2.6, 0.9 Hz, 1H),

7.07 (dd, J = 5.1, 3.6 Hz, 1H), 6.99 (t, J = 8.2 Hz, 1H), 6.64 (d, J = 8.3

Hz, 1H), 6.42 (s, 1H), 6.12−6.01 (m, 1H), 4.74 (d, J = 14.7 Hz, 1H),

4.50 (d, J = 14.7 Hz, 1H), 3.85 (s, 3H), 3.69−3.60 (m, 2H), 3.43 (s,

1H), 3.02 (t, J = 11.6 Hz, 1H), 2.75 (t, J = 11.7 Hz, 1H), 2.62 (tt, J = 6.8,

4.0 Hz, 1H), 1.97 (d, J = 10.8 Hz, 1H), 1.83 (d, J = 11.0 Hz, 1H), 1.75−

1.72 (m, 1H), 1.64 (s, 1H), 1.44 (s, 6H), 0.93−0.81 (m, 4H). ¹³C NMR

(126 MHz, Chloroform-d): δ 176.8, 172.8, 159.7, 154.3, 152.7, 144.1,

139.2, 137.6, 133.3, 130.2, 130.0, 129.6, 128.3, 128.0, 126.1, 124.7,

123.0, 120.9, 120.6, 119.2, 118.5, 112.6, 111.9, 111.1, 110.6, 109.2, 81.2,

55.73, 55.68, 51.9, 49.8, 49.6, 39.8, 29.8, 27.8, 24.4, 24.4, 9.6, 9.0.

HRMS (ESI): calcd for C₃₇H₄₁N₃O₆S₂(M − H)⁻, 686.2364; found,

686.2348. HPLC purity 96.3%, t_R= 14.70 min.

(S)-N-Cyclopropyl-1-(3-((1-((3-methoxyphenyl)-

sulfonamido)-2-methyl-1-oxopropan-2-yl)oxy)phenyl)-N-(4-

(thiophen-2-yl)benzyl)piperidine-3-carboxamide (28). [α]_D²⁵

1

+14.38° (c 0.955, MeOH). H NMR (500 MHz, Chloroform-d): δ

9.06 (s, 1H), 7.63 (ddd, J = 7.8, 1.8, 1.0 Hz, 1H), 7.59−7.49 (m, 3H),

7.47−7.43 (m, 1H), 7.29 (dd, J = 3.6, 1.2 Hz, 1H), 7.27 (d, J = 6.1 Hz,

1H), 7.24 (d, J = 8.0 Hz, 2H), 7.19 (ddd, J = 8.3, 2.6, 0.9 Hz, 1H), 7.07

(dd, J = 5.1, 3.6 Hz, 1H), 6.99 (t, J = 8.2 Hz, 1H), 6.64 (dd, J = 8.1, 2.4

Hz, 1H), 6.42 (t, J = 2.4 Hz, 1H), 6.05 (dd, J = 7.8, 2.3 Hz, 1H), 4.74 (d,

J = 14.7 Hz, 1H), 4.50 (d, J = 14.6 Hz, 1H), 3.85 (d, J = 1.4 Hz, 3H),

3.69−3.60 (m, 2H), 3.43 (tt, J = 11.0, 3.7 Hz, 1H), 3.01 (dd, J = 12.4,

11.0 Hz, 1H), 2.75 (td, J = 12.2, 2.9 Hz, 1H), 2.66−2.58 (m, 1H), 1.97

(d, J = 11.0 Hz, 1H), 1.83 (dq, J = 11.8, 3.2, 2.7 Hz, 1H), 1.73 (d, J = 9.2

Hz, 1H), 1.62 (d, J = 6.9 Hz, 1H), 1.44 (s, 6H), 0.93−0.81 (m, 4H). ¹³C

5897

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J. Med. Chem. 2021, 64, 5886−5904

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Article

NMR (126 MHz, CDCl₃): δ 176.8, 172.8, 159.7, 154.3, 152.7, 144.1,

139.2, 137.6, 133.3, 130.0, 129.6, 128.3, 128.1, 126.1, 124.8, 123.0,

121.0, 120.6, 112.6, 111.9, 110.6, 109.2, 81.2, 55.7, 51.9, 49.8, 49.5,

39.8, 29.8, 27.8, 24.5, 24.4, 9.6, 9.0. HRMS (ESI): calcd for

C₃₇H₄₁N₃O₆S₂(M − H)⁻, 686.2364; found, 686.2345. HPLC purity

97.3%, t_R= 14.69 min.

148.1, 135.6, 127.8, 126.7, 79.8, 45.9, 44.6, 43.2, 43.0, 33.8, 28.4, 27.8,

24.2, 24.0. MS (ESI) m/z: 361.3 [M + H]⁺, 383.3 [M + Na]⁺.

tert-Butyl 3-((4-isopropylphenethyl)carbamoyl)piperidine-

1

1-carboxylate (33b). H NMR (500 MHz, Chloroform-d): δ 7.12

(dd, J = 8.2, 1.8 Hz, 2H), 7.03 (dd, J = 8.0, 1.6 Hz, 2H), 6.59−5.89 (m,

1H), 4.09−3.50 (m, 2H), 3.42 (qt, J = 13.3, 7.0 Hz, 2H), 3.04 (s, 1H),

2.87−2.82 (m, 1H), 2.74−2.71 (m, 4H), 2.16 (dt, J = 12.5, 6.2 Hz, 1H),

1.78 (d, J = 8.5 Hz, 1H), 1.56 (t, J = 12.7 Hz, 1H), 1.39 (d, J = 1.6 Hz,

10H), 1.20 (dd, J = 7.0, 1.5 Hz, 6H). MS (ESI) m/z: 375.3 [M + H]⁺,

397.3 [M + Na]⁺.

tert-Butyl 3-((4-isopropylbenzyl)(methyl)carbamoyl)-

piperidine-1-carboxylate (33c). ¹H NMR (500 MHz, Chloro-

form-d): δ 7.24−7.12 (m, 2H), 7.11−6.89 (m, 2H), 4.51 (s, 2H), 4.16

(s, 1H), 4.06 (s, 1H), 2.95 (s, 2H), 2.90−2.79 (m, 3H), 2.70−2.45 (m,

2H), 2.02−1.54 (m, 3H), 1.53−1.30 (m, 10H), 1.21 (t, J = 6.9 Hz, 6H).

¹³C NMR (126 MHz, Chloroform-d): δ 173.8, 173.1, 154.7, 154.5,

(R)-1-(3-((1-((4-Cyanophenyl)sulfonamido)-2-methyl-1-oxo-

propan-2-yl)oxy)phenyl)-N-cyclopropyl-N-(4-(thiophen-2-yl)-

benzyl)piperidine-3-carboxamide (29). [α]²_D⁵−13.90° (c 1.104,

MeOH). ¹H NMR (500 MHz, Chloroform-d): δ 8.20−8.12 (m, 2H),

7.88−7.81 (m, 2H), 7.62−7.52 (m, 2H), 7.29 (dd, J = 3.6, 1.2 Hz, 1H),

7.28−7.26 (m, 1H), 7.24 (d, J = 8.1 Hz, 2H), 7.11−6.95 (m, 2H), 6.67

(d, J = 8.3 Hz, 1H), 6.42 (s, 1H), 6.08 (d, J = 8.0 Hz, 1H), 4.74 (d, J =

14.7 Hz, 1H), 4.51 (d, J = 14.7 Hz, 1H), 3.65 (d, J = 12.1 Hz, 2H), 3.43

(s, 1H), 3.02 (t, J = 11.6 Hz, 1H), 2.76 (t, J = 11.9 Hz, 1H), 2.62 (tt, J =

6.7, 4.1 Hz, 1H), 1.98 (d, J = 9.7 Hz, 1H), 1.85 (d, J = 9.7 Hz, 1H), 1.73

(d, J = 8.7 Hz, 1H), 1.57 (d, J = 7.9 Hz, 1H), 1.44 (s, 6H), 0.95−0.82

(m, 4H). ¹³C NMR (126 MHz, Chloroform-d): δ 176.7, 173.0, 154.1,

144.0, 142.1, 137.6, 133.4, 132.7, 129.7, 129.2, 128.3, 128.0, 126.1,

124.8, 123.0, 117.7, 117.1, 112.3, 110.7, 109.3, 81.3, 51.9, 49.9, 49.6,

39.8, 29.8, 27.7, 24.5, 24.4, 24.3, 9.7, 8.9. HRMS (ESI): calcd for

C₃₇H₃₈N₄O₅S₂(M − H)⁻, 681.2211; found, 681.2217. HPLC purity

96.0%, t_R= 14.62 min.

(S)-((4-Cyanophenyl)sulfonyl)(2-(3-(3-(cyclopropyl(4-(thio-

phen-2-yl)benzyl)carbamoyl)piperidin-1-yl)phenoxy)-2-

methylpropanoyl)amide (30). [α]²_D⁵+16.92° (c 0.881, MeOH). ¹H

NMR (500 MHz, Chloroform-d): δ 8.20−8.12 (m, 2H), 7.88−7.80 (m,

2H), 7.60−7.50 (m, 2H), 7.29 (dd, J = 3.5, 1.2 Hz, 1H), 7.28−7.26 (m,

1H), 7.24 (d, J = 7.9 Hz, 2H), 7.12−6.97 (m, 2H), 6.67 (d, J = 8.3 Hz,

1H), 6.42 (s, 1H), 6.08 (d, J = 8.0 Hz, 1H), 4.74 (d, J = 14.7 Hz, 1H),

4.51 (d, J = 14.7 Hz, 1H), 3.65 (dd, J = 11.8, 3.6 Hz, 2H), 3.45 (d, J =

12.3 Hz, 1H), 3.02 (t, J = 11.6 Hz, 1H), 2.76 (t, J = 11.6 Hz, 1H), 2.62

(tt, J = 6.9, 4.1 Hz, 1H), 1.98 (d, J = 9.9 Hz, 1H), 1.89−1.81 (m, 1H),

1.75 (d, J = 8.9 Hz, 1H), 1.62 (s, 1H), 1.44 (s, 6H), 0.93−0.81 (m, 4H).

¹³C NMR (126 MHz, Chloroform-d): δ 176.7, 173.0, 154.1, 152.8,

148.4, 148.0, 134.5, 133.9, 127.9, 127.0, 126.6, 126.2, 79.6, 79.5, 52.8,

50.4, 46.5, 44.3, 39.5, 34.6, 33.83, 33.76, 28.44, 28.39, 28.1, 27.6, 24.7,

24.6, 23.97, 23.95. MS (ESI) m/z: 375.3 [M + H]⁺.

tert-Butyl 3-(ethyl(4-isopropylbenzyl)carbamoyl)piperidine-

1-carboxylate (33d). ¹H NMR (500 MHz, Chloroform-d): δ 7.20−

7.14 (m, 1H), 7.14−6.90 (m, 3H), 4.85−3.96 (m, 4H), 3.26 (s, 2H),

2.84 (dp, J = 13.7, 6.9 Hz, 2H), 2.60 (tt, J = 31.9, 17.1 Hz, 2H), 1.97−

1.55 (m, 3H), 1.38 (d, J = 30.3 Hz, 10H), 1.19 (dd, J = 8.0, 6.9 Hz, 6H),

1.08 (dt, J = 48.7, 7.1 Hz, 3H). ¹³C NMR (126 MHz, Chloroform-d): δ

173.3, 173.1, 154.6, 154.5, 148.2, 147.8, 135.0, 134.4, 127.8, 126.9,

126.56, 126.2, 79.5, 79.4, 53.4, 50.0, 47.3, 44.6, 43.9, 41.3, 40.6, 39.5,

33.7 (d, J = 2.8 Hz), 28.43, 28.37, 28.1, 24.6, 24.0, 23.9, 14.3, 12.6. MS

(ESI) m/z: 389.3 [M + H]⁺.

tert-Butyl 3-(isopropyl(4-isopropylbenzyl)carbamoyl)-

piperidine-1-carboxylate (33e). ¹H NMR (500 MHz, Chloro-

form-d): δ 7.19−7.12 (m, 1H), 7.12−6.97 (m, 3H), 4.94−3.85 (m,

5H), 3.19−2.20 (m, 4H), 2.04−1.62 (m, 2H), 1.64−1.52 (m, 1H), 1.44

(s, 4H), 1.36 (s, 6H), 1.29−1.10 (m, 9H), 1.04 (d, J = 6.8 Hz, 3H). ¹³C

NMR (126 MHz, Chloroform-d): δ 174.0, 173.3, 154.6, 154.5, 147.8,

147.0, 136.7, 136.0, 126.7, 126.6, 126.3, 125.6, 79.5, 79.4, 53.4, 48.5,

47.1, 46.5, 45.5, 44.6, 43.6, 40.7, 39.7, 33.7, 28.5, 28.4, 28.2, 24.8, 24.4,

24.0, 23.9, 22.0, 21.9, 20.2. MS (ESI) m/z: 403.3 [M + H]⁺, 425.3 [M +

Na]⁺.

tert-Butyl 3-(cyclopropyl(4-isopropylbenzyl)carbamoyl)-

piperidine-1-carboxylate (33f). ¹H NMR (500 MHz, Chloro-

form-d): δ 7.12 (t, J = 6.7 Hz, 4H), 4.91−3.94 (m, 4H), 3.19 (tt, J =

11.5, 3.8 Hz, 1H), 2.87 (dq, J = 13.8, 6.8 Hz, 2H), 2.65 (d, J = 69.8 Hz,

2H), 1.90 (d, J = 13.3 Hz, 1H), 1.85−1.63 (m, 2H), 1.45 (s, 10H), 1.22

(d, J = 6.9 Hz, 6H), 0.97−0.70 (m, 4H). ¹³C NMR (126 MHz,

Chloroform-d): δ 176.2, 154.7, 147.7, 135.5, 127.6, 126.5, 79.5, 49.4,

47.3, 44.8, 40.3, 33.7, 29.6, 28.5, 27.8, 24.7, 24.0, 9.3. MS (ESI) m/z:

401.4 [M + H]⁺, 423.3 [M + Na]⁺.

144.0, 142.1, 137.6, 133.4, 132.7, 129.7, 129.2, 128.3, 128.0, 126.1,

124.8, 123.0, 117.7, 117.1, 112.3, 110.7, 109.3, 81.3, 51.9, 49.9, 39.8,

29.8, 27.7, 24.5, 24.4, 24.3, 9.7, 8.9. HRMS (ESI): calcd for

C₃₇H₃₈N₄O₅S₂(M − H)⁻, 681.2211; found, 681.2199. HPLC purity

97.0%, t_R= 14.61 min.

tert-Butyl 2-(3-bromophenoxy)-2-methylpropanoate (31).

¹H NMR (500 MHz, Chloroform-d): δ 7.16−7.04 (m, 2H), 7.01

(dd, J = 2.7, 1.3 Hz, 1H), 6.82−6.71 (m, 1H), 1.56 (s, 6H), 1.44 (s, 9H).

¹³C NMR (126 MHz, Chloroform-d): δ 172.8, 156.5, 130.1, 124.6,

122.2, 121.8, 117.3, 82.0, 79.8, 27.8, 25.3. MS (ESI) m/z: 315.1 [M +

H]⁺.

N-(4-Isopropylbenzyl)ethanamine (32a). ¹H NMR (500 MHz,

Chloroform-d): δ 7.24 (d, J = 8.2 Hz, 2H), 7.21−7.16 (m, 2H), 3.76 (s,

2H), 2.89 (hept, J = 6.9 Hz, 1H), 2.69 (q, J = 7.1 Hz, 2H), 1.25 (d, J =

6.9 Hz, 6H), 1.13 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz,

Chloroform-d): δ 147.5, 137.9, 128.1, 126.4, 53.8, 43.7, 33.8, 24.1, 15.3.

MS (ESI) m/z: 178.2 [M + H]⁺

tert-Butyl 2-(3-(3-((4-isopropylbenzyl)carbamoyl)piperidin-

1

1-yl)phenoxy)-2-methylpropanoate (34a). H NMR (500 MHz,

Chloroform-d): δ 7.18 (d, J = 1.6 Hz, 4H), 7.06 (t, J = 8.2 Hz, 1H), 6.90

(t, J = 5.7 Hz, 1H), 6.54 (dd, J = 8.3, 2.3 Hz, 1H), 6.49 (t, J = 2.3 Hz,

1H), 6.34 (dd, J = 8.2, 2.3 Hz, 1H), 4.42 (d, J = 5.6 Hz, 2H), 3.39 (dd, J

= 12.5, 3.6 Hz, 1H), 3.31−3.15 (m, 2H), 3.02 (ddd, J = 11.8, 8.4, 3.1 Hz,

1H), 2.88 (p, J = 6.9 Hz, 1H), 2.55 (tt, J = 8.0, 4.1 Hz, 1H), 2.02−1.72

(m, 3H), 1.66 (tq, J = 8.7, 4.4 Hz, 1H), 1.55 (s, 6H), 1.42 (d, J = 0.9 Hz,

9H), 1.23 (d, J = 6.9 Hz, 6H). ¹³C NMR (126 MHz, Chloroform-d): δ

174.1, 173.5, 156.7, 152.5, 148.0, 135.8, 129.2, 127.7, 126.7, 111.1,

110.1, 108.2, 81.6, 79.3, 52.7, 50.6, 43.1, 42.4, 33.8, 27.8, 27.4, 25.5,

25.4, 24.0, 23.5. MS (ESI) m/z: 495.4 [M + H]⁺.

1

N-(4-Isopropylbenzyl)propan-2-amine (32b). H NMR (500

MHz, Chloroform-d): δ 7.26−7.21 (m, 2H), 7.20−7.16 (m, 2H), 3.75

(s, 2H), 3.00−2.65 (m, 2H), 1.24 (d, J = 6.9 Hz, 6H), 1.10 (d, J = 6.2

Hz, 6H). ¹³C NMR (126 MHz, Chloroform-d): δ 147.4, 138.2, 128.1,

126.4, 51.4, 48.1, 33.8, 24.0, 23.0. MS (ESI) m/z: 192.3 [M + H]⁺.

N-(4-Isopropylbenzyl)cyclopropanamine (32c). ¹H NMR (500

MHz, Chloroform-d): δ 7.25−7.22 (m, 2H), 7.20−7.17 (m, 2H), 3.81

(s, 2H), 2.90 (hept, J = 6.9 Hz, 1H), 2.22−2.12 (m, 1H), 1.25 (d, J = 6.9

Hz, 6H), 0.48−0.35 (m, 4H). ¹³C NMR (126 MHz, Chloroform-d): δ

147.5, 138.0, 128.2, 126.4, 53.5, 33.8, 30.1, 24.1, 6.5. MS (ESI) m/z:

190.2 [M + H]⁺.

tert-Butyl 2-(3-(3-((4-isopropylphenethyl)carbamoyl)-

1

piperidin-1-yl)phenoxy)-2-methylpropanoate (34b). H NMR

(500 MHz, Chloroform-d): δ 7.18−7.01 (m, 5H), 6.71−6.60 (m, 1H),

6.54−6.45 (m, 2H), 6.36 (dd, J = 8.1, 2.2 Hz, 1H), 3.63−3.42 (m, 2H),

3.28−3.13 (m, 2H), 3.10 (ddd, J = 11.4, 6.7, 4.2 Hz, 1H), 3.00 (ddd, J =

11.8, 7.2, 4.1 Hz, 1H), 2.91−2.81 (m, J = 6.7 Hz, 1H), 2.79 (td, J = 6.9,

5.1 Hz, 2H), 2.47 (td, J = 6.6, 3.3 Hz, 1H), 1.84 (dtd, J = 12.1, 7.5, 4.4

Hz, 1H), 1.78−1.67 (m, 1H), 1.65−1.51 (m, 8H), 1.44 (s, 9H), 1.22 (d,

J = 7.0 Hz, 6H). MS (ESI) m/z: 509.4 [M + H]⁺.

tert-Butyl 3-((4-isopropylbenzyl)carbamoyl)piperidine-1-

1

carboxylate (33a). H NMR (500 MHz, Chloroform-d): δ 7.15 (s,

4H), 4.37 (d, J = 19.2 Hz, 2H), 4.03−3.60 (m, 2H), 3.14 (dd, J = 13.5,

9.3 Hz, 1H), 2.86 (hept, J = 6.8 Hz, 2H), 2.28 (dq, J = 9.8, 5.3, 4.9 Hz,

1H), 1.91−1.77 (m, 2H), 1.69−1.54 (m, 1H), 1.38 (s, 10H), 1.21 (d, J

= 7.0 Hz, 6H). ¹³C NMR (126 MHz, Chloroform-d): δ 172.9, 154.8,

tert-Butyl 2-(3-(3-((4-isopropylbenzyl)(methyl)carbamoyl)-

piperidin-1-yl)phenoxy)-2-methylpropanoate (34c). ¹H NMR

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(500 MHz, Chloroform-d): δ 7.17−7.06 (m, 3H), 7.04−6.83 (m, 2H),

6.57−6.30 (m, 2H), 6.21 (ddd, J = 16.6, 8.1, 2.2 Hz, 1H), 4.62−4.28

(m, 2H), 3.87−3.51 (m, 2H), 3.00−2.76 (m, 6H), 2.67 (dtd, J = 18.5,

12.0, 2.7 Hz, 1H), 2.05−1.61 (m, 4H), 1.54−1.43 (m, 6H), 1.36 (d, J =

3.4 Hz, 9H), 1.17 (dd, J = 6.9, 3.2 Hz, 6H). ¹³C NMR (126 MHz,

Chloroform-d): δ 174.4, 173.8, 173.5, 156.75, 156.73, 152.3, 152.2,

148.4, 148.0, 134.7, 134.0, 129.2, 129.1, 128.0, 127.0, 126.7, 126.2,

110.2, 110.1, 109.3, 109.1, 107.6, 107.5, 81.51, 81.49, 79.3, 79.2, 53.0,

52.5, 52.2, 50.5, 50.0, 49.8, 39.1, 39.0, 34.7, 34.0, 33.79, 33.78, 28.1,

27.8, 27.7, 25.54, 25.46, 25.44, 24.4, 24.3, 24.0. MS (ESI) m/z: 509.4

[M + H]⁺.

2.0 Hz, 9H), 0.91−0.74 (m, 4H). MS (ESI) m/z: 571.2, 573.2 [M +

H]⁺.

tert-Butyl 2-(3-(3-((4-(1H-pyrazol-4-yl)benzyl)(cyclopropyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate

(38a). ¹H NMR (500 MHz, Chloroform-d): δ 7.78 (s, 2H), 7.42 (d, J =

8.1 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 7.07 (t, J = 8.2 Hz, 1H), 6.56 (d, J

= 8.2 Hz, 1H), 6.49 (t, J = 2.2 Hz, 1H), 6.30 (d, J = 8.1 Hz, 1H), 4.67 (d,

J = 14.7 Hz, 1H), 4.55 (d, J = 14.8 Hz, 1H), 3.69 (t, J = 11.3 Hz, 2H),

3.46 (t, J = 10.9 Hz, 1H), 3.04−2.97 (m, 1H), 2.77 (t, J = 11.0 Hz, 1H),

2.62 (dt, J = 6.6, 2.8 Hz, 1H), 1.94 (d, J = 10.6 Hz, 1H), 1.83−1.67 (m,

3H), 1.55 (s, 6H), 1.43 (s, 9H), 0.89−0.81 (m, 4H). MS (ESI) m/z:

559.4 [M + H]⁺.

tert-Butyl 2-(3-(3-(cyclopropyl(4-(1-methyl-1H-pyrazol-4-

yl)benzyl)carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropa-

noate (38b). ¹H NMR (500 MHz, Chloroform-d): δ 7.50 (s, 1H), 7.36

(d, J = 8.0 Hz, 2H), 7.29 (d, J = 7.8 Hz, 2H), 7.07 (t, J = 8.2 Hz, 1H),

6.55 (d, J = 8.0 Hz, 1H), 6.49 (s, 1H), 6.29 (d, J = 8.0 Hz, 2H), 4.65 (q, J

= 15.0 Hz, 2H), 3.88 (s, 3H), 3.76−3.64 (m, 2H), 3.46 (t, J = 10.5 Hz,

1H), 2.99 (t, J = 11.5 Hz, 1H), 2.77 (t, J = 11.7 Hz, 1H), 2.69−2.65 (m,

1H), 1.95 (d, J = 11.0 Hz, 1H), 1.82−1.70 (m, 3H), 1.54 (s, 6H), 1.43

(s, 9H), 0.94−0.79 (m, 4H). MS (ESI) m/z: 573.4 [M + H]⁺, 595.4 [M

+ Na]⁺.

tert-Butyl 2-(3-(3-((4-(1H-pyrazol-5-yl)benzyl)(cyclopropyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate

(38c). ¹H NMR (500 MHz, Chloroform-d): δ 7.71 (d, J = 8.0 Hz, 2H),

7.65 (d, J = 1.4 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 7.06 (t, J = 8.2 Hz,

1H), 6.59−6.51 (m, 2H), 6.48 (t, J = 2.2 Hz, 1H), 6.29 (d, J = 8.1 Hz,

1H), 4.69 (d, J = 14.7 Hz, 1H), 4.54 (d, J = 14.7 Hz, 1H), 3.68 (t, J = 9.5

Hz, 2H), 3.44 (t, J = 10.9 Hz, 1H), 3.05−2.96 (m, 1H), 2.77 (t, J = 11.9

Hz, 1H), 2.63−2.54 (m, 1H), 1.93 (d, J = 10.7 Hz, 1H), 1.75 (ddd, J =

29.2, 17.1, 8.6 Hz, 3H), 1.54 (s, 6H), 1.42 (s, 9H), 0.88−0.78 (m, 4H).

MS (ESI) m/z: 559.4 [M + H]⁺.

tert-Butyl 2-(3-(3-(cyclopropyl(4-(1-methyl-1H-pyrazol-5-

yl)benzyl)carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropa-

noate (38d). ¹H NMR (500 MHz, Chloroform-d): δ 7.72 (s, 1H), 7.57

(s, 1H), 7.39 (d, J = 7.9 Hz, 2H), 7.20 (d, J = 7.8 Hz, 2H), 7.05 (t, J = 8.2

Hz, 1H), 6.54 (d, J = 8.2 Hz, 1H), 6.47 (s, 1H), 6.28 (d, J = 8.0 Hz, 1H),

4.63 (d, J = 14.6 Hz, 1H), 4.56−4.51 (m, 1H), 3.90−3.88 (m, 3H),

3.71−3.63 (m, 2H), 3.41 (d, J = 10.7 Hz, 1H), 2.98 (t, J = 11.7 Hz, 1H),

2.75 (t, J = 11.4 Hz, 1H), 2.61−2.55 (m, 1H), 1.92 (d, J = 10.8 Hz, 1H),

1.74 (dq, J = 26.8, 15.7, 11.2 Hz, 3H), 1.53 (s, 6H), 1.41 (d, J = 1.2 Hz,

9H), 0.83 (ddd, J = 25.3, 13.1, 8.0 Hz, 4H). MS (ESI) m/z: 573.4 [M +

H]⁺.

tert-Butyl 2-(3-(3-(ethyl(4-isopropylbenzyl)carbamoyl)-

1

piperidin-1-yl)phenoxy)-2-methylpropanoate (34d). H NMR

(500 MHz, Chloroform-d): δ 7.16−7.04 (m, 3H), 7.04−6.87 (m, 2H),

6.54−6.27 (m, 2H), 6.21 (ddd, J = 19.7, 7.9, 2.2 Hz, 1H), 4.62−4.31

(m, 2H), 3.75−3.49 (m, 2H), 3.42−3.16 (m, 2H), 3.03−2.52 (m, 4H),

1.96−1.57 (m, 4H), 1.50−1.41 (m, 6H), 1.35 (d, J = 3.3 Hz, 9H), 1.16

(dd, J = 6.9, 3.5 Hz, 6H), 1.05 (dt, J = 25.1, 7.1 Hz, 3H). ¹³C NMR (126

MHz, Chloroform-d): δ 174.0, 173.8, 173.51, 173.48, 156.8, 156.7,

152.4, 152.3, 148.3, 147.9, 135.2, 134.4, 129.2, 129.1, 127.9, 126.9,

126.6, 126.2, 110.2, 110.1, 109.2, 109.0, 107.6, 107.4, 81.48, 81.47,

79.24, 79.21, 52.6, 52.5, 50.1, 50.0, 49.8, 47.3, 41.4, 40.8, 39.2, 39.1,

33.78, 33.76, 28.3, 28.1, 27.8, 25.55, 25.53, 25.48, 25.44, 24.5, 24.3,

24.0, 14.4, 12.7. MS (ESI) m/z: 523.4 [M + H]⁺.

tert-Butyl (R)-2-(3-(3-(isopropyl(4-isopropylbenzyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate

(34e). ¹H NMR (500 MHz, Chloroform-d): δ 7.19 (d, J = 8.2 Hz, 1H),

7.15−7.04 (m, 3H), 6.98 (t, J = 8.2 Hz, 1H), 6.63−6.21 (m, 3H), 5.07−

4.21 (m, 3H), 3.71 (ddt, J = 16.2, 12.0, 2.0 Hz, 1H), 3.57 (td, J = 9.8, 7.6,

3.0 Hz, 1H), 3.18−2.56 (m, 4H), 2.05−1.63 (m, 4H), 1.59−1.52 (m,

6H), 1.43 (d, J = 7.8 Hz, 9H), 1.33−1.00 (m, 12H). ¹³C NMR (126

MHz, Chloroform-d): δ 175.2, 173.2, 172.1, 156.7, 156.5, 148.2, 147.3,

136.0, 135.3, 130.7, 130.5, 126.8, 126.49, 126.47, 126.2, 113.7, 113.4,

112.1, 111.6, 80.2, 80.0, 56.5, 56.1, 55.8, 54.9, 49.2, 46.4, 46.1, 43.9,

38.7, 37.3, 33.70, 33.68, 26.3, 25.8, 25.24, 25.18, 25.0, 24.9, 24.0, 23.9,

22.9, 22.7, 21.8, 21.7, 20.5, 20.2. MS (ESI) m/z: 537.4 [M + H]⁺.

tert-Butyl (R)-2-(3-(3-(cyclopropyl(4-isopropylbenzyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate

(34f). ¹H NMR (500 MHz, Chloroform-d): δ 7.20−7.12 (m, 4H), 7.07

(t, J = 8.2 Hz, 1H), 6.55 (dd, J = 8.2, 2.3 Hz, 1H), 6.49 (t, J = 2.4 Hz,

1H), 6.30 (dd, J = 8.1, 2.3 Hz, 1H), 4.71−4.37 (m, 2H), 3.85−3.61 (m,

2H), 3.43 (ddt, J = 11.0, 7.1, 3.4 Hz, 1H), 2.99 (dd, J = 12.4, 10.9 Hz,

1H), 2.88 (p, J = 6.9 Hz, 1H), 2.77 (td, J = 12.1, 2.6 Hz, 1H), 2.59 (tt, J =

6.9, 4.1 Hz, 1H), 2.00−1.89 (m, 1H), 1.88−1.65 (m, 3H), 1.55 (s, 6H),

1.44 (s, 9H), 1.24 (d, J = 6.9 Hz, 6H), 0.97−0.75 (m, 4H). ¹³C NMR

(126 MHz, Chloroform-d): δ 176.8, 173.5, 156.7, 152.4, 147.6, 135.7,

129.1, 127.6, 126.6, 110.2, 109.1, 107.6, 81.5, 79.2, 52.2, 50.0, 49.5,

39.9, 33.8, 29.7, 27.8, 25.53, 25.48, 24.5, 24.0, 9.4, 9.1. MS (ESI) m/z:

535.4 [M + H]⁺.

tert-Butyl 2-(3-(3-(cyclopropyl(4-(thiophen-2-yl)benzyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate

(38e). ¹H NMR (500 MHz, Chloroform-d): δ 7.50−7.43 (m, 2H), 7.19

(dd, J = 3.6, 1.2 Hz, 1H), 7.18 (s, 3H), 7.02−6.92 (m, 2H), 6.47 (dd, J =

8.3, 2.3 Hz, 1H), 6.41 (t, J = 2.4 Hz, 1H), 6.22 (dd, J = 8.1, 2.2 Hz, 1H),

4.58 (d, J = 14.7 Hz, 1H), 4.47 (d, J = 14.7 Hz, 1H), 3.66−3.55 (m, 2H),

3.36 (tt, J = 10.9, 3.4 Hz, 1H), 2.92 (dd, J = 12.5, 10.9 Hz, 1H), 2.68 (td,

J = 12.1, 2.7 Hz, 1H), 2.52 (tt, J = 6.9, 4.1 Hz, 1H), 1.89−1.83 (m, 1H),

1.74−1.59 (m, 3H), 1.46 (s, 6H), 1.34 (s, 9H), 0.83−0.65 (m, 4H). MS

(ESI) m/z: 575.4 [M + H]⁺.

N-(4-Bromobenzyl)cyclopropanamine (35). ¹H NMR (500

MHz, Chloroform-d): δ 7.46−7.37 (m, 2H), 7.22−7.08 (m, 2H), 3.79

(s, 2H), 2.12 (tt, J = 6.5, 3.6 Hz, 1H), 0.49−0.39 (m, 2H), 0.39−0.25

(m, 2H). MS (ESI) m/z: 226.0, 228.0 [M + H]⁺.

tert-Butyl 2-(3-(3-(cyclopropyl(4-(thiophen-3-yl)benzyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate

(38f). ¹H NMR (500 MHz, Chloroform-d): δ 7.47−7.40 (m, 2H), 7.32

(dd, J = 2.6, 1.7 Hz, 1H), 7.29−7.22 (m, 2H), 7.19−7.10 (m, 2H), 6.97

(t, J = 8.2 Hz, 1H), 6.45 (dd, J = 8.2, 2.3 Hz, 1H), 6.39 (t, J = 2.4 Hz,

1H), 6.20 (dd, J = 8.0, 2.2 Hz, 1H), 4.57 (d, J = 14.6 Hz, 1H), 4.47 (d, J

= 14.8 Hz, 1H), 3.64−3.53 (m, 2H), 3.34 (tt, J = 11.2, 3.4 Hz, 1H), 2.90

(dd, J = 12.5, 10.9 Hz, 1H), 2.67 (td, J = 12.1, 2.7 Hz, 1H), 2.51 (tt, J =

6.9, 4.1 Hz, 1H), 1.88−1.80 (m, 1H), 1.73−1.57 (m, 3H), 1.44 (s, 6H),

1.33 (s, 9H), 0.81−0.66 (m, 4H). MS (ESI) m/z: 575.4 [M + H]⁺.

tert-Butyl 2-(3-(3-(cyclopropyl(4-(furan-2-yl)benzyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate

Ethyl 1-(3-((1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl)-

1

oxy)phenyl)piperidine-3-carboxylate (36). H NMR (500 MHz,

Chloroform-d): δ 7.04 (t, J = 8.2 Hz, 1H), 6.54 (dd, J = 8.1, 2.3 Hz, 1H),

6.45 (t, J = 2.4 Hz, 1H), 6.31−6.21 (m, 1H), 4.12 (q, J = 7.1 Hz, 2H),

3.67 (ddt, J = 12.4, 3.5, 1.5 Hz, 1H), 3.42 (ddd, J = 12.3, 4.9, 3.1 Hz,

1H), 2.95 (dd, J = 12.4, 9.9 Hz, 1H), 2.80−2.68 (m, 1H), 2.60 (tt, J =

10.0, 3.9 Hz, 1H), 2.05−1.93 (m, 1H), 1.74 (th, J = 9.2, 3.1 Hz, 1H),

1.69−1.59 (m, 2H), 1.53 (s, 6H), 1.41 (s, 9H), 1.24 (t, J = 7.2 Hz, 3H).

MS (ESI) m/z: 392.3 [M + H]⁺, 414.3 [M + Na]⁺.

tert-Butyl 2-(3-(3-((4-bromobenzyl)(cyclopropyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate (37).

¹H NMR (500 MHz, Chloroform-d): δ 7.42 (d, J = 8.3 Hz, 2H), 7.11

1

(38g). H NMR (500 MHz, Chloroform-d): δ 7.58−7.50 (m, 2H),

(d, J = 8.3 Hz, 2H), 7.07 (t, J = 8.2 Hz, 1H), 6.54 (d, J = 8.2 Hz, 1H),

6.48 (s, 1H), 6.30 (d, J = 8.1 Hz, 1H), 4.59 (d, J = 14.7 Hz, 1H), 4.49 (d,

J = 14.7 Hz, 1H), 3.67 (d, J = 12.1 Hz, 2H), 3.42 (s, 1H), 2.97 (t, J = 11.6

Hz, 1H), 2.76 (t, J = 11.0 Hz, 1H), 2.58 (s, 1H), 1.91 (d, J = 8.5 Hz,

1H), 1.76 (dt, J = 32.5, 8.6 Hz, 3H), 1.55 (d, J = 1.9 Hz, 6H), 1.43 (d, J =

7.37 (d, J = 1.7 Hz, 1H), 7.21−7.15 (m, 2H), 6.99 (t, J = 8.2 Hz, 1H),

6.57−6.51 (m, 1H), 6.48 (dd, J = 8.2, 2.3 Hz, 1H), 6.41 (t, J = 2.3 Hz,

1H), 6.37 (dd, J = 3.4, 1.8 Hz, 1H), 6.22 (dd, J = 8.1, 2.2 Hz, 1H), 4.59

(d, J = 14.7 Hz, 1H), 4.47 (d, J = 14.7 Hz, 1H), 3.65−3.56 (m, 2H), 3.36

(tt, J = 11.1, 3.5 Hz, 1H), 2.92 (dd, J = 12.5, 10.9 Hz, 1H), 2.69 (td, J =

5899

https://doi.org/10.1021/acs.jmedchem.1c00046

J. Med. Chem. 2021, 64, 5886−5904

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

12.1, 2.7 Hz, 1H), 2.51 (tt, J = 6.9, 4.1 Hz, 1H), 1.90−1.81 (m, 1H),

1.76−1.61 (m, 3H), 1.47 (s, 6H), 1.35 (s, 9H), 0.82−0.69 (m, 4H). MS

(ESI) m/z: 559.4 [M + H]⁺.

= 12.4, 11.0 Hz, 1H), 2.77 (td, J = 12.2, 2.8 Hz, 1H), 2.70−2.45 (m,

1H), 2.01−1.87 (m, 1H), 1.83−1.74 (m, 2H), 1.55 (s, 6H), 1.43 (s,

9H), 0.97−0.73 (m, 4H). MS (ESI) m/z: 575.3 [M + H]⁺.

Protein Expression and Puriﬁcation. Full-length β-catenin

(residues 1−781) was cloned into a pET-28b vector carrying a C-

terminal 6× histidine (Novagen) and transformed into Escherichia coli

BL21 DE3 (Novagen). Cells were cultured in LB medium with 50 μg/

mL kanamycin until the OD₆₀₀was approximately 0.8, and then, protein

expression was induced with 400 μM IPTG at 20 °C overnight. Cells

were lysed by sonication. The proteins were puriﬁed by three steps of

chromatography, including Ni-NTA aﬃnity chromatography (30210,

Qiagen), HiTrap Q HP anion exchange chromatography (17-1154-01,

GE Healthcare Life Science), and size-exclusion chromatography with a

HiLoad 26/600 Superdex 200 pg column (28-9893-36, GE Healthcare

Life Science) using an AKTA Pure FPLC (GE Healthcare Life Science)

system. Protein was eluted in a buﬀer containing 20 mM Tris (pH 8.5),

100 mM NaCl, and 2 mM DTT. The purity of β-catenin was greater

than 95% as determined by SDS-PAGE gel analyses. Thermal shift

assays were performed on a CFX96 real-time system (Bio-Rad) to

monitor protein stability and detect protein aggregation. Protein

unfolding was evaluated through measurement of the ﬂuorescence

changes of ﬂuorescent dye Sypro Orange when interacting with β-

catenin proteins. A temperature increment of 1°/min was applied. All

proteins were stable, and no aggregation was observed under storage or

assay conditions. Proteins were aliquoted and stored at −80 °C.

BCL9 Peptide Synthesis and Puriﬁcation. Human BCL9

(residues 350−375) and N-terminally biotinylated human BCL9

(residues 350−375) were synthesized by InnoPep Inc. (San Diego, CA,

www.innopep.com). The synthesized peptides were puriﬁed by HPLC

with purity >95%. The structures were validated by LC/MS. The

sequences of the peptides are provided in Table 6.

tert-Butyl 2-(3-(3-(cyclopropyl(4-(furan-3-yl)benzyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate

(38h). ¹H NMR (500 MHz, Chloroform-d): δ 7.74 (t, J = 1.2 Hz, 1H),

7.52−7.42 (m, 3H), 7.27 (dd, J = 8.9, 7.0 Hz, 2H), 7.09 (t, J = 8.2 Hz,

1H), 6.71 (dd, J = 1.9, 0.9 Hz, 1H), 6.58 (dd, J = 8.2, 2.3 Hz, 1H), 6.52

(t, J = 2.3 Hz, 1H), 6.33 (dd, J = 8.1, 2.3 Hz, 1H), 4.68 (d, J = 14.7 Hz,

1H), 4.59 (d, J = 14.7 Hz, 1H), 3.78−3.67 (m, 2H), 3.47 (tt, J = 11.2,

3.6 Hz, 1H), 3.03 (dd, J = 12.5, 10.9 Hz, 1H), 2.80 (td, J = 12.1, 2.8 Hz,

1H), 2.63 (tt, J = 6.9, 4.1 Hz, 1H), 2.00−1.90 (m, 1H), 1.86−1.72 (m,

3H), 1.57 (s, 6H), 1.46 (s, 9H), 0.92−0.80 (m, 4H). MS (ESI) m/z:

559.4 [M + H]⁺.

Ethyl (R)-1-(3-((1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl)-

1

oxy)phenyl)piperidine-3-carboxylate (39). H NMR (500 MHz,

Chloroform-d): δ 7.04 (t, J = 8.2 Hz, 1H), 6.54 (dd, J = 8.1, 2.3 Hz, 1H),

6.45 (t, J = 2.4 Hz, 1H), 6.31−6.21 (m, 1H), 4.12 (q, J = 7.1 Hz, 2H),

3.67 (ddt, J = 12.4, 3.5, 1.5 Hz, 1H), 3.42 (ddd, J = 12.3, 4.9, 3.1 Hz,

1H), 2.95 (dd, J = 12.4, 9.9 Hz, 1H), 2.80−2.68 (m, 1H), 2.60 (tt, J =

10.0, 3.9 Hz, 1H), 2.05−1.93 (m, 1H), 1.74 (th, J = 9.2, 3.1 Hz, 1H),

1.69−1.59 (m, 2H), 1.53 (s, 6H), 1.41 (s, 9H), 1.24 (t, J = 7.2 Hz, 3H).

¹³C NMR (126 MHz, Chloroform-d): δ 173.7, 173.4, 156.6, 152.5,

129.1, 110.4, 109.3, 107.6, 81.4, 79.1, 60.4, 52.0, 49.7, 41.3, 27.8, 27.0,

25.5, 25.4, 24.1, 14.2. MS (ESI) m/z: 392.3 [M + H]⁺, 414.3 [M + Na]⁺.

tert-Butyl (R)-2-(3-(3-((4-bromobenzyl)(cyclopropyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate (40).

¹H NMR (500 MHz, Chloroform-d): δ 7.46−7.39 (m, 2H), 7.13−7.03

(m, 3H), 6.54 (dd, J = 8.3, 2.3 Hz, 1H), 6.47 (t, J = 2.3 Hz, 1H), 6.29

(dd, J = 8.0, 2.2 Hz, 1H), 4.59 (d, J = 14.7 Hz, 1H), 4.49 (d, J = 14.7 Hz,

1H), 3.72−3.60 (m, 2H), 3.41 (tt, J = 11.0, 3.5 Hz, 1H), 2.96 (dd, J =

12.5, 10.9 Hz, 1H), 2.76 (td, J = 12.0, 2.9 Hz, 1H), 2.58 (tt, J = 7.0, 4.0

Hz, 1H), 1.94−1.89 (m, 1H), 1.79 (ddd, J = 12.4, 6.7, 3.4 Hz, 1H),

1.77−1.67 (m, 2H), 1.55 (s, 6H), 1.43 (s, 9H), 0.91−0.75 (m, 4H). MS

(ESI) m/z: 571.2, 573.2 [M + H]⁺.

Table 6. Sequences of Peptides

a

peptide

sequence

tert-Butyl (R)-2-(3-(3-(cyclopropyl(4-(thiophen-2-yl)benzyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate (41).

¹H NMR (500 MHz, Chloroform-d): δ 7.53−7.42 (m, 2H), 7.24−7.12

BCL9 26-mer

H-³⁵⁰GLSQEQLEHRERSLQTLRDIQRMLFP³⁷⁵

NH₂

-

biotinylated BCL9

26-mer

biotin-

Ahx-³⁵⁰GLSQEQLEHRERSLQTLRDIQRMLFP³⁷⁵

NH₂

-

(m, 4H), 7.04−6.93 (m, 2H), 6.48 (dd, J = 8.2, 2.3 Hz, 1H), 6.41 (t, J =

2.3 Hz, 1H), 6.22 (dd, J = 8.1, 2.3 Hz, 1H), 4.59 (d, J = 14.7 Hz, 1H),

4.48 (d, J = 14.7 Hz, 1H), 3.69−3.51 (m, 2H), 3.37 (tt, J = 10.9, 3.0 Hz,

1H), 2.99−2.82 (m, 1H), 2.70 (td, J = 12.2, 2.8 Hz, 1H), 2.59−2.44 (m,

1H), 1.86 (d, J = 11.3 Hz, 1H), 1.75−1.63 (m, 3H), 1.47 (s, 6H), 1.35

(s, 9H), 0.85−0.66 (m, 4H). MS (ESI) m/z: 575.4 [M + H]⁺.

Ethyl (S)-1-(3-((1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl)-

a

Ahx, 6-aminohexanoic acid.

AlphaScreen Assays of β-Catenin and BCL9 Interaction.

Experiments were performed in white opaque 384-well plates from

PerkinElmer (Waltham, MA), and the samples were read on a Biotek

Synergy 2 plate reader (Winooski, VT) with excitation at 680 nm and

emission at 570 nm. The standard AlphaScreen protocol was used with

a sensitivity setting of 200. All dilutions were made in 1× assay buﬀer

containing 25 mM Hepes (pH 7.4), 100 mM NaCl, 0.01% Triton X-

100, and 0.1% BSA to minimize nonspeciﬁc interactions. For the

competitive inhibition assays of the β-catenin/BCL9 PPI, the negative

control (equivalent to 0% inhibition) refers to 5.0 nM biotinylated

BCL9, 50 nM His₆-tagged β-catenin, and 10 μg/mL donor and acceptor

beads in a ﬁnal volume of 25 μL assay buﬀer, with no tested inhibitor

present. The positive control (equivalent to 100% inhibition) refers to

5.0 nM biotinylated BCL9 and 10 μg/mL donor and acceptor beads in a

ﬁnal volume of 25 μL assay buﬀer.

1

oxy)phenyl)piperidine-3-carboxylate (42). H NMR (500 MHz,

Chloroform-d): δ 7.07 (t, J = 8.2 Hz, 1H), 6.57 (ddd, J = 8.3, 2.4, 0.8 Hz,

1H), 6.48 (t, J = 2.4 Hz, 1H), 6.30 (ddd, J = 8.1, 2.3, 0.8 Hz, 1H), 4.20−

4.11 (m, 2H), 3.70 (ddt, J = 12.3, 3.5, 1.5 Hz, 1H), 3.51−3.42 (m, 1H),

2.97 (dd, J = 12.4, 10.0 Hz, 1H), 2.77 (tdd, J = 10.2, 4.5, 3.1 Hz, 1H),

2.66−2.56 (m, 1H), 2.06−1.95 (m, 1H), 1.82−1.72 (m, 1H), 1.72−

1.62 (m, 2H), 1.55 (s, 6H), 1.44 (s, 9H), 1.27 (t, J = 7.1 Hz, 3H). ¹³C

NMR (126 MHz, Chloroform-d): δ 173.8, 173.5, 156.6, 152.5, 129.1,

110.4, 109.3, 107.6, 81.5, 79.2, 60.5, 52.1, 49.7, 41.4, 27.8, 27.0, 25.5,

25.4, 24.1, 14.3. MS (ESI) m/z: 392.2 [M + H]⁺.

tert-Butyl (S)-2-(3-(3-((4-bromobenzyl)(cyclopropyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate (43).

¹H NMR (500 MHz, CDCl₃): δ 7.37−7.33 (m, 2H), 7.08−6.91 (m,

3H), 6.47 (dd, J = 8.1, 2.4 Hz, 1H), 6.40 (t, J = 2.3 Hz, 1H), 6.27−6.03

(m, 1H), 4.52 (d, J = 14.7 Hz, 1H), 4.47 (s, 1H), 3.63−3.51 (m, 2H),

3.34 (tt, J = 10.8, 3.5 Hz, 1H), 2.90 (dd, J = 12.5, 10.9 Hz, 1H), 2.69 (td,

J = 12.1, 2.8 Hz, 1H), 2.51 (tt, J = 6.9, 4.1 Hz, 1H), 1.84−1.62 (m, 4H),

1.48 (s, 6H), 1.36 (s, 9H), 0.85−0.65 (m, 4H). MS (ESI) m/z: 571.2,

573.2 [M + H]⁺.

tert-Butyl (S)-2-(3-(3-(cyclopropyl(4-(thiophen-2-yl)benzyl)-

carbamoyl)piperidin-1-yl)phenoxy)-2-methylpropanoate (44).

¹H NMR (500 MHz, CDCl₃): δ 7.61−7.43 (m, 2H), 7.31−7.19 (m,

For the β-catenin/BCL9 assay, 5 nM biotinylated BCL9 and 50 nM

His₆-tagged β-catenin were incubated in assay buﬀer for 30 min.

Diﬀerent concentrations of the tested inhibitor were added and

incubated in 20 μL assay buﬀer for another 1 h. All of the assay plates

were covered and gently mixed on an orbital shaker. Donor and

acceptor beads were then added to the plates to a ﬁnal concentration of

10 μg/mL in 25 μL of assay buﬀer. The mixture was incubated for 1 h

before detection. The IC₅₀value was determined by nonlinear least-

square analysis of GraphPad Prism 8.0. The K_ivalues were derived from

the IC₅₀values using a method reported by Wang and co-workers.⁶⁰

The equation used is K_i= [I]₅₀/([L]₅₀/K_d+ [P]₀/K_d+ 1) (where [I]₅₀

denotes the concentration of the free inhibitor at 50% inhibition, [L]₅₀

4H), 7.10−7.00 (m, 2H), 6.56 (dd, J = 7.9, 2.3 Hz, 1H), 6.49 (t, J = 2.3

Hz, 1H), 6.33−6.15 (m, 1H), 4.67 (d, J = 14.7 Hz, 1H), 4.56 (d, J = 14.7

Hz, 1H), 3.77−3.60 (m, 2H), 3.45 (tt, J = 11.0, 3.5 Hz, 1H), 3.00 (dd, J

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is the concentration of the free labeled ligand at 50% inhibition, [P]₀is

the concentration of the free protein at 0% inhibition, and K_dis the

dissociation constant of the protein−ligand complex). All of the

experiments were performed in triplicate and carried out in the

presence of 1% DMSO for small-molecule inhibitors. Each compound

was assayed at least by three independent experiments. The results were

expressed as mean standard deviation.

Western Blotting of Wnt Target Genes. SW480 cells at 1 × 10⁶

cells/mL were treated with diﬀerent concentrations of inhibitors for 24

h. Cells were lysed in buﬀer containing 50 mM Tris, pH 7.4, 150 mM

NaCl, 1% Nonidet P-40, 2 mM EDTA, and protease inhibitors. After

centrifugation at 12,000 rpm for 20 min at 4 °C, the supernatant was

loaded onto an 8% SDS polyacrylamide gel for electrophoretic analysis.

Separated proteins were transferred onto nitrocellulose membranes for

immunoblot analysis. The antibodies against Axin2 (MA5-15015,

Thermo Fisher), cyclin D1 (sc-853, Santa Cruz Biotechnology), and β-

tubulin (sc-55529, Santa Cruz Biotechnology) were incubated with the

membranes overnight at 4 °C. IRDye 680LT goat anti-mouse IgG

(827-11080, LiCOR) or IRDye 800CW goat anti-rabbit IgG (827-

08365, LiCOR) was used as the secondary antibody. The images were

detected using the Odyssey infrared imaging system (Odyssey Fc,

LiCOR). Experiments were performed in triplicate.

MTS Cell Growth Inhibition Experiments. Colorectal cancer

cells (SW480 and HCT116) and triple negative breast cancer cells

(MDA-MB-231 and MDA-MB-468) were seeded in 96-well plates at 5

× 10³cells/well in DMEM with 10% FBS, maintained overnight at 37

°C, and then incubated with the tested compounds at various

concentrations in DMEM with 5% FBS. Human mammary epithelial

MCF10A cells were seeded in 96-well plates at 1 × 10⁴cells/well in

mammary epithelial cell growth medium (MEGM) (CC-3150, Lonza)

with 100 ng/mL cholera toxin, maintained overnight at 37 °C, and

incubated with the tested compounds at various concentrations. Cell

viability was monitored after 72 h using a freshly prepared mixture of

one part phenazine methosulfate (PMS, Sigma) solution (0.92 mg/

mL) and 19 parts 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethox-

yphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, Promega) solution

(2 mg/mL). Cells were incubated in 10 μL of this solution at 37 °C for 3

h, and absorptions at 490 nm (A490) were measured. The eﬀect of each

compound is expressed as the concentration required to reduce A490

by 50% (IC₅₀) relative to DMSO-treated cells. Experiments were

performed in triplicate.

Biotin Streptavidin Pull-down Assay. SW480 colon cancer cells

were lysed in NP-40 buﬀer (50 mM Tris pH 7.4, 150 mM NaCl, 1%

NP-40, and 2 mM EDTA) with protease inhibitors. Cell lysate (2 mg/

mL) was precleared with streptavidin-agarose beads (S-1638, Sigma)

and incubated with the indicated concentrations of the compound or

BCL9 HD2 peptide (residues 350−375) for 1 h at 4 °C. Then, the cell

lysate was incubated with N-terminally biotinylated BCL9 HD2 peptide

(residues 350−375) (0.2 μM) for 2 h at 4 °C. Protein−peptide

complexes were pulled down with streptavidin-agarose beads for 2 h at

4 °C. Beads were washed four times with NP-40 buﬀer, and protein was

eluted by boiling in 4× SDS loading buﬀer. The samples were loaded

onto 8% SDS polyacrylamide gel for electrophoretic analysis. Separated

proteins were transferred onto nitrocellulose membranes for immuno-

blot analysis. The antibody against β-catenin (610153, BD Biosciences)

was incubated with membranes. IRDye 800CW goat anti-mouse IgG

(926-32210, LiCOR) was used as the secondary antibody. The images

were detected using the Odyssey infrared imaging system (LiCOR).

Experiments were performed in triplicate.

β-Catenin Rescue Experiments. SW480 cells were seeded in 96-

well plates at 1 × 10⁴cells/well in DMEM with 10% FBS and

maintained overnight at 37 °C. Then, 200 ng of pcDNA3.1−β-catenin

or 200 ng of empty vector pcDNA3.1 per well was transfected into the

cells, and 12 h later, the tested compound at various concentrations was

added into the cells in DMEM with 5% FBS. Cell viability was

monitored after 72 h using a freshly prepared mixture of one part

phenazine methosulfate (PMS, Sigma) solution (0.92 mg/mL) and 19

parts MTS (Promega) solution (2 mg/mL). Cells were incubated in 10

μL of this solution at 37 °C for 3 h, and A490 was measured. The eﬀect

of each compound is expressed as the concentration required to reduce

A490 by 50% (IC₅₀) relative to DMSO-treated cells. Experiments were

performed in triplicate.

co-IP Experiments. Colorectal cancer HCT116 cells with

hyperactive β-catenin signaling at 1× 10⁶cells/mL were treated with

diﬀerent concentrations of the inhibitor for 24 h. Cells were then lysed

in buﬀer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-

40, 2 mM EDTA, and protease inhibitors. The cell lysates were pre-

adsorbed to A/G plus agarose (sc-2003, Santa Cruz Biotechnology) at 4

°C for 1 h. Pre-adsorbed lysates were incubated with a speciﬁc primary

antibody against β-catenin (610153, BD Biosciences) overnight at 4 °C.

A/G plus agarose was then added to the lysate mixture and incubated

for 3 h. The beads were washed four times with the lysis buﬀer at 4 °C.

The bound protein was eluted by boiling in the SDS sample buﬀer and

loaded onto 8% SDS polyacrylamide gel for electrophoretic analysis.

Separated proteins were transferred onto nitrocellulose membranes for

immunoblot analysis. The antibodies against BCL9 (ab37305, Abcam)

and E-cadherin (610404, BD Biosciences) were incubated with the

membranes. IRDye 680LT goat anti-mouse IgG (827-11080, LiCOR)

and IRDye 800CW goat anti-rabbit IgG (926-32211, LiCOR) were

used as the secondary antibodies. The images were detected using the

Odyssey infrared imaging system (LiCOR). Experiments were

performed in triplicate.

Cell Transfection and Luciferase Assay. The FuGENE6

(E269A, Promega) 96-well plate format was used for the transfection

of colorectal cancer SW480 cells according to the manufacturer’s

instruction. SW480 cells were co-transfected with 60 ng of TOPFlash or

FOPFlash ﬁreﬂy luciferase reporter gene and 40 ng of renilla luciferase

pCMV-RL normalization reporter gene. Cells were cultured in

Dulbecco’s modiﬁed Eagle medium (DMEM) and 10% fetal bovine

serum (FBS) at 37 °C for 24 h. Diﬀerent concentrations of inhibitors or

DMSO were added and incubated in DMEM with 5% FBS. After 24 h,

the luciferase reporter activity was measured using the Dual-Glo system

(E2940, Promega). Normalized luciferase activity in response to the

treatment with inhibitors was compared with that obtained from the

cells treated with DMSO. Experiments were performed in triplicate.

Quantitative Real-Time PCR Analysis. SW480 cells at 1× 10⁶

cells/mL were treated with inhibitors at diﬀerent concentrations for 24

h. MDA-MB-231 cells at 1 × 10⁶cells/mL were treated with 100 ng/mL

recombinant Wnt 3a (rmW3aL-010, Time BioScience) for 30 min;

then, the inhibitors at diﬀerent concentrations were added and

incubated for 24 h. Total RNAs were extracted with TRIzol

(15596026, Life Technologies), and the cDNA was synthesized with

the superscript III ﬁrst-strand kit (18080-051, Invitrogen). qPCR

experiments were performed using the iQTM SYBR green supermix kit

(170-8880, BIO-RAD) on an CFX96 real-time system (BIO-RAD).

The threshold cycle (CT) values were normalized to those of internal

reference GAPDH. Experiments were performed in triplicate. The

primer pairs are shown in Table 7.

Table 7. Primer Pairs

human GAPDH forward 5′-GAAGGTGAAGGTCGGAGTC-3′

reverse

5′-GAAGATGGTGATGGGATTTC-3′

human HPRT

forward 5′-GCTATAAATTCTTTGCTGACCTGC

TG-3′

reverse

5′-AATTACTTTTATGTCCCCTGTTGA

CTGG-3′

human AXIN2

human LEF1

forward 5′-AGTGTGAGGTCCACGGAAAC-3′

reverse 5′-CTTCACACTGCGATGCATTT-3′

forward 5′-GACGAGATGATCCCCTTCAA-3′

reverse 5′-AGGGCTCCTGAGAGGTTTGT-3′

forward 5′-ACAAACAGATCATCCGCAAACAC-3′

Docking Studies. Crystallographic coordinates for the human β-

catenin armadillo repeat domain (PDB id 2GL7, 2.60 Å resolution,

Rcryst = 0.223, residues 142−663) were obtained from the Research

Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank

(PDB). Preparation of the crystal structure and molecular modeling

human cyclin

D1

reverse

5′-TGTTGGGGCTCCTCAGGTTC-3′

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were achieved with the commercially available Schrodinger (http://

www.schrodinger.com/) software packages. The protein was processed

using the “Protein Preparation Wizard” workﬂow in Maestro 11.4

(Schrodinger LLC) to add bond orders and hydrogens. All heteroatom

(het) residues and crystal water molecules beyond 5 Å from the het

group were removed. Small molecules were built in the LigPrep module

using the OPLS3 force ﬁeld. The Glide module was used as the docking

program. The standard precision (SP) approach of Glide was adopted

to dock small molecules into β-catenin with the default parameters.

Residues in the BCL9 L366/I369/L373 binding site of β-catenin

include D144−A146, L148, A149, A152, I153, E155−L160, D162−

A171, M174, V175, Q177, L178, K180, K181, A183, S184, A187, I188,

M194, and I198.

ACKNOWLEDGMENTS

■

This work was supported by the Department of Defense

CDMRP BCRP breakthrough award W81XWH-14-1-0083, the

Susan G. Komen Career Catalyst Research Grant

CCR16380693, and Floridian Breast Cancer Foundation

Scientiﬁc Grant (19012901). The H. Lee Moﬃtt Cancer Center

and Research Institute is an NCI-designated Comprehensive

Cancer Center, supported under NIH grant P30-CA76292. We

thank John A. Wisniewski for the initial synthesis of some

compounds.

ABBREVIATIONS

■

APC, adenomatous polyposis coli; BCL9, B-cell lymphoma 9;

BCL9L, BCL9-like; CBP, CREB-binding protein; CK1α, casein

kinase 1α; co-IP, co-immunoprecipitation; DMEM, Dulbecco’s

modiﬁed Eagle medium; Dvl, Disheveled; FBS, fetal bovine

serum; Fzd, Frizzled; GSK3β, glycogen synthase kinase 3β;

HD2, homology domain 2; K_i, inhibition constant; LEF,

lymphoid enhancer-binding factor; MEGM, mammary epithe-

lial cell growth medium; PAINS, pan-assay interference

compounds; PP2A, protein phosphatase 2A; PPI, protein−

protein interaction; Pygo, Pygopus; PNPB, 3-(4-ﬂuorophenyl)-

N-phenylbenzamide; qPCR, quantitative real-time PCR; TCF,

T-cell factor

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00046.

K_Dof full-length β-catenin/BCL9 PPI; AlphaScreen assay

curves of β-catenin/BCL9 inhibitors; full images of

Western blot, β-catenin pull-down, and co-IP experi-

ments; β-catenin rescue assay results of 30; HPLC

conditions and chromatograms; and NMR spectra (PDF)

Glide docking models of 29 and 30 with β-catenin (PDB)

Crystallographic coordinates (PDB)

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AUTHOR INFORMATION

■

Corresponding Author

Haitao Ji − Drug Discovery Department, H. Lee Moﬃtt Cancer

Center and Research Institute, Tampa, Florida 33612-9497,

United States; Departments of Oncologic Sciences and

Chemistry, University of South Florida, Tampa, Florida

33620-9497, United States; orcid.org/0000-0001-5526-

4503; Phone: (813) 745-8070; Email: Haitao.Ji@

mo ﬃ tt.org; Fax: (813) 745-4506

Authors

Zhen Wang − Drug Discovery Department, H. Lee Moﬃtt

Cancer Center and Research Institute, Tampa, Florida 33612-

9497, United States

Min Zhang − Drug Discovery Department, H. Lee Moﬃtt

Cancer Center and Research Institute, Tampa, Florida 33612-

9497, United States

Wen Luo − Drug Discovery Department, H. Lee Moﬃtt Cancer

Center and Research Institute, Tampa, Florida 33612-9497,

United States

Yongqiang Zhang − Department of Chemistry, Center for Cell

and Genome Science, University of Utah, Salt Lake City, Utah

84112-0850, United States; orcid.org/0000-0002-7210-

4787

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00046

Author Contributions

^∥Z.W. and M.Z. contributed equally to this work.

Notes

The authors declare the following competing ﬁnancial

interest(s): a provisional patent application has been ﬁled

based on these results.

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Article Doi

Discovery of 2-(3-(3-Carbamoylpiperidin-1-yl)phenoxy)acetic Acid Derivatives as Novel Small-Molecule Inhibitors of the β-Catenin/B-Cell Lymphoma 9 Protein-Protein Interaction

DOI: 10.1021/acs.jmedchem.1c00046

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