Dynamics-Based Discovery of Novel, Potent Benzoic Acid Derivatives as Orally Bioavailable Selective Estrogen Receptor Degraders for ERα+ Breast Cancer

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Dynamics-Based Discovery of Novel, Potent Benzoic Acid

Derivatives as Orally Bioavailable Selective Estrogen Receptor

Degraders for ERα+ Breast Cancer

Xiaomeng Zhang,^§Yazhou Wang,^§Xue Li, Jie Wu, Liwen Zhao,* Wei Li,* and Jian Liu*

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ABSTRACT: The estrogen receptor α (ERα) is identiﬁed as an

eﬀective target for the treatment of ERα+ breast cancer; thus, discovery

of novel selective estrogen receptor degraders (SERDs) are developed as

an eﬀective method to overcome the resistance of breast cancer. Herein,

the hot-spot residues for protein−ligand interaction between SERDs and

ERα are analyzed by molecular dynamic simulation technology, focusing

on the hot-spot residues for four series of designed and synthesized

SERDs. SAR studies revealed that while the acrylic acid moiety of

AZD9496 is scaﬀold hopping into benzoic acid, compound D24 exhibits

potent binding aﬃnity with ERα, good degradation eﬃcacy of ERα, and

inhibitory eﬀect against the MCF-7 breast cancer cell line. Besides, D24

also displays good antitumor eﬃcacy in the MCF-7 human breast cancer

xenograft model in vivo, favorable pharmacokinetic properties, excellent

druggability, and good safety property, making D24 as a promising drug candidate of SERD for further evaluation.

androstenedione into estrogen in vivo; thus, aromatase

inhibitors (1, letrozole; 2, anastrozole; Figure 1) indicate

antitumor activity by inhibiting the synthesis of estrogen which

is responsible for the key biosynthetic step in the formation of

mitogenic molecules.¹¹SERMs can bind to the ligand binding

region (LBD) of ER competitively and form a dimer complex,

which changes the conformation of ERα and results in

blocking of the interaction between coactivator and activating

function-2 (AF2) of ERα, and ﬁnally can block the estrogen

signaling pathway and inhibit tumor formation.¹²SERMs,

which are represented by tamoxifen and its active metabolite 4-

hydroxytamoxifen, have been proved to stabilize an antagonist

conformation in breast cancer cells, leading to antiproliferative

signaling.^13,14Unfortunately, these compounds are not pure

antagonists of ERα, which often exhibit partial activation of

ERα in the uterus, resulting in serious side eﬀects after long-

term administration, such as endometrial hyperplasia and

endometrial cancer.¹⁵Moreover, although many patients

initially respond to AIs and SERMs, drug resistance often

emerges. Resistance to endocrine therapy is multifactorial,

involving mutation of ESR1 and upregulation of growth factor

INTRODUCTION

■

Cancer is characterized by uncontrolled growth and spread of

abnormal cells, and it is one of the most serious health hazards

that may occur in diﬀerent tissues.¹Breast cancer (BC) is one

of the most common malignant tumors in women, which has

the highest incidence rate.²In 2019, according to the annual

report released by China National Cancer Registration Center,

breast cancer accounted for 16.5% of all cancer cases in women

and 1.2 million women died from that. Breast cancer is

classiﬁed in a clinical setting by its molecular subtypes, which

can be divided into estrogen receptor positive (ERα+), human

epidermal growth factor receptor 2 positive (HER2+), and

triple-negative subtype.^3,4As members of the nuclear receptor

family, ERα and ERβ are transcription factors mediating the

estrogen eﬀects most known and regulating gene expression.⁵

ERα distributes in many tissues, such as breast, uterus, bone,

and aorta, etc.; approximately 74% of breast cancer patients

have high expression of ERα, while the overexpression of ERα

is the main driving factor of breast cancer cell proliferation.^6,7

Therefore, ERα has become an important target for the

treatment of breast cancer.

Nowadays, endocrine therapy is the main treatment to

estrogen receptor positive breast cancer.⁸Conceptually, on the

basis of related targets of breast cancer and estrogen signaling

pathways, the antiestrogen drugs can be mainly divided into

three types: aromatase inhibitors (AIs), selective estrogen

receptor modulators (SERMs), and selective estrogen receptor

degraders (SERDs).^9,10The aromatase can catalyze the

Received: February 13, 2021

Published: May 31, 2021

https://doi.org/10.1021/acs.jmedchem.1c00280

J. Med. Chem. 2021, 64, 7575−7595

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Figure 1. Representative selective aromatase inhibitors (AIs), selective estrogen receptor modulators (SERMs), and selective estrogen receptor

degraders (SERDs).

Figure 2. Analysis of binding “hot spots” of ERα. (a) Crystal structure of AZD9496 (blue; PDB ID 5ACC) superimposed on reference structure of

GW-5638 (white; PDB ID 5AVV) bound to ERα pocket. The red circle revealed that the acryl acid group interacted with hot spot 1: Trp383,

Leu525, and Val534. The blue rectangle revealed that the pyrido[3,4-b]-indole scaﬀold interacted with hot spot 2: Leu346, Met388, Leu391,

Phe404, Met421, and Ile424. (b) Backbone RMSD of the protein along with simulation time. The blue line represents GW-5638; the red line

represents AZD9496. (c) Hot-spot residue contribution for protein−ligand interaction. (d) Calculation results of hot-spot residue mutation to

alanine for AZD9496 (red) and GW-6838 (blue) with ERα mutants.

signaling.¹⁶⁻¹⁸Therefore, the development of novel, high-

eﬃciency and low-toxicity ERα antagonists might provide an

alternative therapeutic option for drug-resistance breast cancer

therapy.

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Fortunately, selective estrogen receptor degraders (SERDs)

are small molecules that target ERα for proteasome-dependent

degradation. Fulvestrant (5) not only antagonizes the ERα-

driven tumor cell growth but also induces the degradation of

ERα via ubiquitinoylation, which has been approved by the

FDA.¹⁹⁻²¹Furthermore, the clinical success of fulvestrant

reveals that the degradation of ER is beneﬁcial to women with

ER+ breast cancer, especially those who have progressed under

endocrine therapies such as SERMs or AIs.²²However, due to

poor physicochemical properties and low oral bioavailability,

the utilization of fulvestrant is limited in clinic, which is

administered as a monthly intramuscular implant at a dose of

up to 500 mg.²³The poor physicochemical properties of

fulvestrant are due to the steroidal structure, which led to an

eﬀort in designing a nonsteroidal scaﬀold of SERDs in order to

enhance the oral bioavailability.

Several companies are engaged in the exploration of novel

orally available SERDs, and several new compounds have

entered clinical trials recently, including GW-5638 (6), GW-

7604 (7), GDC-0810 (8), AZD9496 (9), and LSZ-102

(10).²⁴⁻²⁸When the basic side chain of tamoxifen is

substituted by acrylic acid group, GW-5638 can be obtained,

which improves the antagonistic activity and induces the

degradation of ER. GW-5638 is the ﬁrst oral SERD, which is a

prodrug and can be metabolized into GW-7604 with better

activity in vivo. The crystal structure of GW-5638 bound to

the ER LBD demonstrates that the interaction between the

acrylate side chain and Leu536 and Tyr537 at the N-terminus

of H12 was mediated by a structural water molecule; as a

result, GW-5638 shows potential inhibitory eﬀect against

tamoxifen-resistant breast cancer tumor cells.²⁹However, GW-

5638 failed in clinical phase I trial, which may be due to the

poor metabolic properties. Meanwhile, the GW-5638 analogue

GDC-0810 is also exhibiting the same problem. Furthermore,

AZD9496, a novel tricyclic scaﬀold with an acrylic acid side

chain, also possesses ER degradation mechanism, but it has

side eﬀects of uterine hyperplasia.^30,31LSZ-102, the systematic

structural optimization from Arzoxifene has some drawbacks,

such as poor metabolic properties, low degradation activity of

ERα in vivo, and so on. Hence, seeking novel scaﬀold SERDs

with potent degradation activity and excellent physicochemical

properties is crucial to the success of SERDs design.

Additionally, ERα contains two activating regions, AF1 and

AF2, which control transcription activation. AF2 is located in

the C-terminus of the LBD, and its function is dependent on

the positioning of H12.³²As a result, most SERDs in clinical

trials caused the degradation of ERα, because of the

displacement and destabilization of H12 caused by forming a

hydrogen bond between the acrylate side chain and H12

(Figure 2). The interaction between H12 and degraders will

aﬀect the position of H12, as the means to promote ERα

degradation. Herein, focusing on the hydrogen-bond and

hydrophobic interaction with key amino acids nearby H12

(called hot spots), we present the dynamics-simulation-based

discovery of novel SERDs, containing a benzoic acid side chain

instead of acrylic group. Through the systematic structure

optimization, the potent biological activity and good oral

bioavailability of compound D24 indicated it can serve as a

good candidate.

characterization of binding aﬃnity are the basis of rational drug

design. The small molecular drugs usually prefer binding to

several key amino acids (called hot spots) in protein−ligand

and protein−protein structures, rather than all of the amino

acids in the active sites of target proteins.³³⁻³⁶To quantify the

contribution of the amino acid residue to the protein−ligand

binding aﬃnity, free energy perturbation (FEP), thermody-

namic integration (TI), and the molecular mechanics−

generalized Born surface area (MM-GBSA) methods are

employed to assess the contribution of each amino acid in

the protein−ligand interaction.³⁷⁻⁴³The MM-GBSA method

is the most practical application in computing protein−ligand

binding free energy, and it has been successfully used to

predict hot-spot protein−ligand interaction in recent years.

Besides, the computational alanine scanning approach also can

be used to evaluate the speciﬁc residue that contributes to the

protein−ligand binding free energy.⁴⁴⁻⁴⁶Thus, the hot spot of

ERα can be identiﬁed by combining use of MM-GBSA and

alanine scanning approaches, and as a consequence, the

binding interaction with the active sites of the target protein

can be explored, which lead to the discovery of novel SERDs

with improved aﬃnity and unique structure.

In order to better understand the binding mode of SERDs

and ERα, we superimposed two ER crystal structures of 5acc

and 5AAV, which were complexed with AZD9496 and GW-

5638, respectively (Figure 2a). The two crystal structures

indicated that the binding modes of AZD9496 and GW-5638

with ERα were similar, and a few of the key interactions were

found from the crystal structure of AZD9496. Once the

benzenoid ring of the indole was replaced by the phenyl ring of

GW-5638, the indole NH formed a critical hydrogen bond

with the carbonyl of Leu346. Meanwhile, the ERα structures

(PDB ID 6B0F) that contain ligand LSZ-102 with a hydroxyl

moiety instead of an indole scaﬀold possessed a hydrogen-

bonding network among residues Glu353, Arg394, and a

hydroxyl group.²⁸Besides this H-bond interaction, the indole

ring and its hydrophobic side chain of AZD9496 were located

at a lipophilic pocket, which were constituted by several

hydrophobic residues, involving Leu346, Met388, Leu391,

Phe404, Met421, and Ile424, etc. Furthermore, examining the

interface between AZD9496 and H12 of ERα in the crystal

structure, it showed that the acrylic acid moiety was situated

adjacent to Trp383, Leu525, and Val534, and the pendent aryl

substituent also oriented toward the H12 region of the ERα;

thus, the formation of hydrogen-bond and hydrophobic

interaction with target were observed as the key for ERα

downregulation.

To better explore the hot-spot residues and design novel

SERDs, molecular dynamic simulations for compounds

AZD9496 and GW-5638 in complex with ERα (5ACC and

5AAV) were carried out for 100 ns, using the Desmond

module in Schrodinger Maestro 2019. The root-mean-square-

deviation (RMSD) values of protein backbone atoms relative

to the initial structure were calculated to identify the protein

stability during the simulation period. As a result, compounds

AZD9496 and GW-5638 were converged to 1.30 and 1.48 Å

(Figure 2b), respectively, which revealed that the two

compounds steadily combined with ERα. Subsequently, the

binding free energies were calculated by using MM-GBSA

method. The binding free energies were less than zero,

implying the binding interaction between ERα and SERDs

could be spontaneous. The total binding energies were −85.90

and −79.87 kcal/mol for the complexes of AZD9496−ERα

RESULTS AND DISCUSSION

Analysis of Binding “Hot Spot” of ERα. The essential

understanding of protein−ligand interaction and quantitative

■

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and GW-5638−ERα (Table 1). Besides, the contributions

with respect to the complex AZD9496−ERα, Leu346 (−2.30

kcal/mol), Trp383 (−4.68 kcal/mol), Met388 (−1.32 kcal/

mol), Leu391 (−1.86 kcal/mol), Phe404 (−1.56 kcal/mol),

Met421 (−1.20 kcal/mol), Ile424 (−1.94 kcal/mol), Leu525

(−3.56 kcal/mol), and Val534 (−2.28 kcal/mol) were

regarded as hot-spot residues (Figure 2c).

favoring ligand binding were van der Waals energy, electro-

Table 1. Total Binding Free Energy (kcal/mol) and Its

Component (kcal/mol)

contribution

AZD9496

GW-5638

In order to further evaluate the hot-spot residues, the

computational alanine mutation approaches were also utilized.

In computational alanine mutation approach to protein−ligand

binding, a speciﬁc residue was mutated to alanine and the

diﬀerences in binding free energies before and after the

mutation were calculated (Figure 2d). The results demon-

strated that once the corresponding hot-spot residues were

mutated to alanine, the binding aﬃnity of ligand−protein

became weaker. The detailed protein−ligand interactions

could be concluded as follows: the acryl acid group formed

H-bond interaction with Val534 and interacted with Trp383,

Leu525, and Val534 by lipophilic contacts (hot spot 1).

Meanwhile, the indole ring and 2-ﬂuoro-2-methylpropane

fragment of AZD9496 underwent hydrophobic interactions

with Leu346, Met388, Leu391, Phe404, Met421, and Ile424,

and the NH atoms also formed a hydrogen bond with Leu346

(hot spot 2). Therefore, all of those interactions were

beneﬁcial to stabilize the conformation and combination of

ligand lying in the binding pocket of ERα. The structural

design of novel SERDs should focus on enhancing the

interaction with hot-spot residues.

a

ΔE_VDW

ΔE_ele

ΔE_GB

ΔE_GA

−52.88 2.66

−40.58 1.92

38.84 3.08

−30.98 1.26

−85.60 4.22

−47.11 2.00

−35.64 1.72

32.66 2.66

−29.78 1.12

−79.87 3.30

b

c

d

e

ΔG_bind

a

Contribution to the free energy of binding from the van der Waals

energy. Contribution to the free energy of binding from the

electrostatic energy. Contribution to the free energy of binding from

the polar solvation energies. Contribution to the free energy of

b

c

d

e

binding from the nonpolar solvation energies. Free energy of binding.

static interaction energy, and the nonpolar solvation

interaction, whereas the polar solvation interaction was

detrimental to bind with targets. As the structures of

AZD9496 and GW-5638 mainly consist of hydrophobic

groups and acrylic acid moiety, they could form hydrophobic

and electrostatic interactions with ERα. Therefore, the

electrostatic interaction energy and van der Waals energy

were the two essential components of total binding free energy.

Meanwhile, through analyzing the MD simulation result, one

could also obtain the hydrophobic and hydrogen-bond

contributions of each residue in the binding pocket of ERα.

Then, the hydrophobic interaction between AZD9496 and

each residue was found, and the hydrophobic interaction of the

AZD9496−ERα complex could be attributed to Leu346,

Trp383, Met388, Leu391, Phe404, Met421, Ile424, Leu525,

and Val534. Meanwhile, the hydrogen bond between the

indole ring and acryl acid group of AZD9496 and Leu346,

Val534 were also dispensable for binding aﬃnity of ERα. Thus,

Dynamic Simulation-Based Structure Design. To

explore novel SERDs, four diﬀerent series of compounds

were designed and virtually screened (Figure 3). To verify the

rationality of the design strategy and estimate the binding

aﬃnity, the molecular docking and dynamic simulation study

were combined which allowed the evaluation of the binding

energies of the designed compounds (F1−F9) toward ERα

(PDB ID 5ACC). Figure 4 demonstrated the binding mode of

Figure 3. Design strategy of target compounds.

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a compound (F1−F9) to the hot spots of ERα, and Table 2

listed the docking score and binding free energies.

compound lost key interactions with hot spot 1 residue,

Trp383, Leu525, and Val534; thus, the binding conformation

changed dramatically, and the binding aﬃnity was signiﬁcantly

reduced. Besides, the 2-ﬂuoro-2-methylpropyl substituent

surrounded by Met421 and Ile424 could be replaced by

other groups, such as 3-ﬂuoro-3-methylbutyl (F4), (1-

(triﬂuoromethyl)cyclopropyl)methyl (F5), and phenyl (F6).

As a result, although the binding abilities of those three

compounds were similar to that of AZD9496, their

pharmacological activity might be decreased due to the partial

loss of binding with hot-spot residue. Finally, the three

fragments of the D series (F7−F9) were obtained by

bioisoterism and scaﬀold hopping method. When the benzene

ring was substituted by pyridine ring (F7), there was no

diﬀerence between F7 and AZD9496. The docking

orientations and interactions of F8 were ﬁtted as well as the

AZD9496. Meanwhile, F8 exhibited the favorable calculation

results and binding aﬃnity with SERDs through the strong

hydrogen-bond and hydrophobic interaction with the hot spot

1 residue at H12 of ERα. Therefore, F8 could be selected as a

candidate for further structure optimization and pharmaco-

logical evaluation.

Chemistry. The synthesis routes of the designed ﬁnal

compounds were demonstrated in Schemes 1−4. Initially,

Figure 4. Docking result of design compounds (F1−F9) toward ERα

(PDB ID 5ACC) and drug candidate AZD9496. (a) AZD9496

(blue) and F1 (green); (b) AZD9496 (blue), F2 (white), and F3

(pink); (c) AZD9496 (blue), F4 (white), F5 (yellow), F6 (pink); (d)

AZD9496 (blue), F7 (white), F8 (yellow), and F9 (pink).

a

Scheme 1. Synthesis of A Series of Compounds

With respect to A series compounds, the indole ring was

substituted by N-methylindole (F1); however the docking

energies and binding free energies decreased (Table 1), mainly

because of the lack of hydrogen-bond interaction with Leu346

(F1, Figure 4). By use of scaﬀold hopping strategy, the B series

compounds (F2, F3) were designed and obtained. Owing to

the absence of hydrogen bond with Val534 and the

hydrophobicity with Trp383 (hot spot 1), the binding free

energy of F2 was lower than that of AZD9496. Meanwhile, the

theory calculation results illustrated that once the piperidine

ring was cyclized with its linked methyl group (F3), the

a

Reagents and conditions: MeI, NaH, THF, 0 °C, 4 h, 66%.

Table 2. Predicted Docking Energies, Binding Free Energies, and Binding Sites of Compounds F1−F9

binding site

H-bond

hydrophobic

docking

score

binding free energy

(kcal/mol)

compd

F1

hot spot 1 hot spot 2

Val534

hot spot 1

hot spot 2

−9.77

Trp383, Leu525, Val534 Leu346, Met388, Leu391, Phe404, Met421,

Ile424

−68.69 3.50

F2

−10.82

Leu346

Leu525, Val534

Leu346, Met388, Leu391, Phe404, Met421,

Ile424

Leu346, Met421, Ile424

−75.22 3.16

F3

F4

F5

F6

F7

−3.80

−11.31

−11.24

−10.80

−12.50

−36.60 2.65

−80.54 1.84

−79.96 2.74

−74.72 2.40

−80.35 3.38

Val534

Trp383, Leu525, Val534 Leu346, Met388, Leu391, Phe404, Met421

Trp383, Leu525, Val534 Leu346, Met388, Leu391, Phe404, Ile424

Trp383, Leu525, Val534 Leu346, Met388, Leu391, Phe404, Met421,

Ile424

F8

−13.49

−12.66

−12.29

Val534

Leu346

Trp383, Leu525, Val534 Leu346, Met388, Leu391, Phe404, Met421,

Ile424

Trp383, Leu525, Val534 Leu346, Met388, Leu391, Phe404, Met421,

Ile424

Trp383, Leu525, Val534 Leu346, Met388, Leu391, Phe404, Met421,

Ile424

−92.35 1.98

−87.52 2.53

−85.60 4.22

F9

AZD9496

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a

Scheme 2. Synthesis of B Series of Compounds

a

Reagents and conditions: (a) TBAF, THF, 80 °C, 4 h, 73%; (b) Pd(OH)₂, H₂, EtOH, rt, 8 h, 98%; (c) MeTi(i-PrO)₃, EtMgBr, BF₃·Et₂O, THF, 2

h, 76%; (d) NaOH, H₂O, MeOH, 2 h, 80%; (e) Na₂CO₃, 2,2,2-triﬂuoroethanol, 48 h, 65%; (f) NaBH₃CN, ammonium acetate, MeOH, rt, 8 h,

54%; (g) 2-ﬂuoro-2-methylpropyl triﬂuoromethanesulfonate (11), DIPEA, dioxane, 90 °C, 2 h, 35−90%; (h) methyl (E)-3-(3,5-diﬂuoro-4-

formylphenyl)acrylate (13), HOAc, toluene, 80 °C, 8 h, 41−70%; (i) NaOH, H₂O, THF, MeOH, rt, 75−83%.

a

triﬂuoroethanol as solvent at room temperature for 48 h. By

exploring the classical reductive amination strategy, the ketone

9 was smoothly converted into amine 10 in moderate yield.

Then, owing to the excellent reaction activity of the

triﬂuoromethanesulfonate moiety, the C−N coupling reaction

was easily carried out among amines 3, 6, 10, and the other

material 2-ﬂuoro-2-methylpropyl triﬂuoromethanesulfonate

(11) to provide coupling products 12a−12c. Finally, through

the Mannich reaction of the intermediate 12a−12c with other

building block methyl (E)-3-(3,5-diﬂuoro-4-formylphenyl)-

acrylate (13) and relay ester hydrolysis under base environ-

ment, the desired carboxylic acid compounds B1−B3 could be

aﬀorded.

Scheme 3. Synthesis of C Series of Compounds

In a similar way, the C series compounds C1−C4 were

prepared (Scheme 3). The critical C−N bond formation was

performed by the free amine 14 with substituted alkyl-OTf or

PhI via CuI-catalyzed coupling reaction. Then, the ﬁnal

product C1−C4 could be obtained through consecutive

reaction involving Mannich reaction between 15a−15d and

methyl (E)-3-(3,5-diﬂuoro-4-formylphenyl)acrylate (13) and

base-mediated ester hydrolysis.

a

Reagents and conditions: (a) (i) alkyl triﬂuoromethanesulfonate,

DIPEA, dioxane, 90 °C, 8 h, 63−72% and (ii) iodobenzene, CuI,

Cs₂CO₃, DMF, microwave, 135 °C, 2 h, 34%; (b) methyl (E)-3-(3,5-

diﬂuoro-4-formylphenyl)acrylate (13), HOAc, toluene, 80 °C, 8 h,

41−72%; (c) NaOH, H₂O, THF, MeOH, rt, 72−86%.

Furthermore, the D series of compounds D1−D42 could be

synthesized by the analogous synthesis protocol (Scheme 4).

The Heck coupling reaction of 16 and methyl acrylate,

exploring Pd(OAc)₂as catalyst, Et₃N as base, and DMF as

solvent, generated the various aldehyde derivatives 17.

Meanwhile, the readily available bromine-substituted hetero-

cyclic compound 18 was smoothly converted into the

corresponding borate ester 19 by palladium(II)-catalyzed

coupling reaction; then the Suzuki coupling reactions of 19

and 4-bromo-2,6-diﬂuorobenzaldehyde generated the other

aldehyde derivatives 20. Through cascade reaction sequences

including Mannich reaction, transition-metal-catalyzed cou-

pling, and ester hydrolysis reaction, the ﬁnal products of D1−

D40 were obtained in moderate to high yield.

treatment of the N-methyl of AZD9496 in the mixture of

NaH, MeI, and THF could aﬀord the compound A1 in 60%

yield. With respect to the synthesis strategies of series B, the

primary amine intermediates of 3, 6, and 10 with various

scaﬀolds were ﬁrst synthesized (Scheme 2). Because the

commercial starting material 1 contained a good leaving group

OTs, it proceeded as a nucleophilic substitution reaction with

the ﬂuorine donor TBAF. Then, under the hydrogenation

conditions the protecting group Cbz was removed, aﬀording

the free amine intermediate 3 to be obtained in high yields.

Accordingly, to access the desired intermediate 6, the

commercial reagent 4 was cyclized with the Grignard reagent

EtMgBr, followed by the elimination of Ts from nitrogen

atoms to furnish the intermediate 6 bearing a three-member

ring in 76% yield. Meanwhile, the C−H alkylation reaction

could be achieved from readily available alkene 7 and

haloalkane 8 in the presence of Na₂CO₃as base and 2,2,2-

Initial Screen of ERα Binding, ERα Degradation, and

MCF-7 Antiproliferative Activities. With respect to the

large number of compounds synthesized, the three biological

assays including the ERα binding, ERα degradation, and MCF-

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a

Scheme 4. Synthesis of D Series of Compounds

a

Reagents and conditions: (a) methyl acrylate, Pd(OAc)₂, Et₃N, DMF, 80 °C, 8 h, 72−96%; (b) bis(pinacolato)diboron, PdCl₂(dppf), KOAc, 80

°C, 12 h, 80−88%; (c) 4-bromo-2,6-diﬂuorobenzaldehyde, PdCl₂(dppf)₂, Na₂CO₃, toluene, H₂O, 100 °C, 16 h, 75−86%; (d) 17 or 20, HOAc,

toluene, 8 h, 45−78%; (e) NaOH, H₂O, THF, MeOH, rt, 2 h, 70−92%; (f) 4-bromo-2,6-diﬂuorobenzaldehyde, HOAc, toluene, 80 °C, 8 h, 84%;

(g) NHR₁R₂, Pd₂(dba)₃, XPhos, Cs₂CO₃, toluene, 80 °C, 8 h, 60−81%;

Table 3. ER Binding, ER Downregulation Degradation, and MCF-7 Antiproliferative Activities for A, B, and C Series

Compounds

ERα binding

a

ERα degradation

MCF-7

ERα binding

a

ERα degradation

MCF-7

c

b

c

b

compd

A1

B2

C1

C3

IC₅₀(nM)

EC₅₀(nM)

IC₅₀(nM)

compd

IC₅₀(nM)

EC₅₀(nM)

IC₅₀(nM)

320.4 10.9

720.6 15.2

113.8 3.55

>1000

14.8 0.82

32.9 1.26

4.48 0.02

>1000

30.8 1.40

39.3 1.20

20.9 0.56

B1

B3

C2

C4

20.6 0.06

8.6 0.12

135.4 5.0

>1000

0.86 0.05

0.35 0.02

5.39 0.08

365.0 3.68

0.83 0.09

0.54 0.11

35.3 1.83

d

ND

AZD9496

4.60 0.04

0.23 0.05

0.76 0.06

a

Relative binding activity was determined by a Polar Screen ERα competitor Assay system from Invitrogen (no. A15883) with 150 nM full-length

b

human ERα. ERα In-Cell Western in MCF-7 breast cancer cells cultured with DMEM containing 10% hormone-free FBS; readings taken after 4 h

of incubation using an anti-ERα rabbit monoclonal antibody (n ≥ 3). MCF-7 proliferation assay in MEM medium containing 10% FBS; 5 day

incubation (n ≥ 3). ND = not determined.

c

d

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Table 4. ERα Binding, ERα Downregulation Degradation, and MCF-7 Antiproliferative Activities for D Series Compounds

ERα binding

a

ERα degradation

MCF-7

ERα binding

a

ERα degradation

MCF-7

c

b

c

b

compd

D1

D3

D5

D7

IC₅₀(nM)

EC₅₀(nM)

IC₅₀(nM)

compd

IC₅₀(nM)

EC₅₀(nM)

IC₅₀(nM)

100.8 2.80

120.7 3.06

130.8 2.45

72.9 2.28

29.5 0.94

25.2 0.56

15.6 0.58

3.2 0.08

3.23 0.09

3.48 0.06

0.48 0.02

6.85 0.05

1.47 0.03

0.98 0.10

0.22 0.01

0.10 0.01

0.55 0.06

>1000

6.27 0.13

1.56 0.08

0.49 0.06

0.26 0.02

0.70 0.04

0.25 0.02

0.66 0.06

0.58 0.03

0.43 0.02

0.34 0.01

0.23 0.05

16.0 0.19

16.3 0.62

1.23 0.06

21.0 0.28

8.63 0.16

7.48 0.52

2.03 0.12

1.54 0.07

4.34 0.21

D2

D4

D6

D8

25.4 0.19

46.4 1.06

50.8 0.98

19.8 0.45

88.9 2.14

>1000

17.1 0.52

21.8 0.44

17.6 0.06

>1000

28.8 0.96

17.1 0.52

26.2 1.03

19.6 0.65

19.8 0.70

32.9 0.85

32.2 1.26

31.2 0.32

26.6 1.04

19.8 0.40

1.00 0.06

2.51 0.11

4.67 0.25

0.53 0.03

7.80 0.52

>1000

0.23 0.02

0.61 0.04

1.51 0.12

>1000

1.28 0.10

0.30 0.01

1.08 0.15

0.50 0.02

0.54 0.01

1.92 0.08

1.70 0.06

1.61 0.10

0.85 0.05

0.61 0.04

10.2 0.12

2.07 0.08

12.8 1.40

0.89 0.07

1.03 0.05

ND

1.69 0.04

2.60 0.09

2.47 0.11

ND

0.66 0.03

0.27 0.02

1.48 0.04

1.27 0.06

1.22 0.04

10.8 0.16

5.91 0.09

5.60 0.07

2.85 0.05

1.34 0.16

D9

D10

D12

D14

D16

D18

D20

D22

D24

D26

D28

D30

D32

D34

D36

D38

D40

D11

D13

D15

D17

D19

D21

D23

D25

D27

D29

D31

D33

D35

D37

D39

AZD9496

19.8 0.42

>1000

d

ND

110.4 4.20

30.4 0.98

19.0 0.60

17.4 0.18

20.0 0.16

17.1 0.32

22.4 0.72

19.8 0.50

18.4 0.36

18.3 0.20

4.60 0.04

6.98 0.45

0.53 0.10

1.19 0.08

2.47 0.32

2.91 0.28

1.08 0.10

3.22 0.08

2.35 0.05

2.05 0.02

1.49 0.04

0.76 0.06

a

Relative binding activity was determined by a Polar Screen ERα competitor Assay system from Invitrogen (no. A15883) with 150 nM full-length

b

human ERα. ERα In-Cell Western in MCF-7 breast cancer cells cultured with DMEM containing 10% hormone-free FBS; readings taken after 4 h

of incubation using an anti-ERα rabbit monoclonal antibody (n ≥ 3). MCF-7 proliferation assay in MEM medium containing 10% FBS; 5 day

incubation (n ≥ 3). ND = not determined.

c

d

7 antiproliferative activities were conducted to evaluate the

biological properties of these four series of compounds (Tables

3 and 4). Compound AZD9496 was identiﬁed as a positive

control. As a result, once the N-methylation of the indole

scaﬀold of AZD9496 had occurred, the biological activities of

compound A1 were decreased signiﬁcantly, the reason being

that the amino group was not performed as a H-bond donor to

form H-bond interaction with Leu346.

Meanwhile, when the various fragments were incorporated

into the piperidine region of AZD9496, compounds B1−B3

exhibited potent ER binding aﬃnity, ERα degradation

eﬃciency, and MCF-9 antiproliferative activity. Because B3

contained a large volume heterocycle, it strengthened the

hydrophobic interaction with the hot-spot residues, which

revealed the best biological activities among the tested

compounds (ERα binding IC₅₀= 8.6 nM; ERα degradation

EC₅₀= 0.351 nM; MCF-7 IC₅₀= 0.544 nM). This ﬁnding was

consistent with the MM-GBSA calculation results. However,

compared to the reference compound AZD9496, the bio-

logical activity of B3 was relatively lower; thus, we did not

select it for further biological evaluation.

Besides, the 2-ﬂuoro-2-methylpropyl group was substituted

by other linkers at the same region, and the biological results

demonstrated that the linker had notable eﬀects on the ERα

binding aﬃnity. For example, compound AZD9496 with 2-

ﬂuoro-2-methylpropyl exhibited the strongest binding aﬃnity

with protein (IC₅₀= 4.6 nM), while replacing with bulkier

cyclopropyl (C1) and long butyl (C2) led to the decrease of

interaction between ligand and protein. Especially, compounds

C3 and C4 almost lost the biological activities and were not

inhibited by the growth and proliferation of MCF-7 cancer

cells.

expanded the search for alternative degradation motifs by

investigating the 2,6-diﬂuorophenyl fragment of AZD9496

replaced by various heterocyclic substituents (Table 4). Once

the 2,6-diﬂuorophenyl was substituted by a pyridine group, the

binding aﬃnity and degradation eﬃciency of compound D1

were signiﬁcantly decreased, leading to the cellular activity

being reduced (IC₅₀= 100.8 nM; EC₅₀= 3.23 nM; MCF-7

IC₅₀= 15.97 nM). The ﬂuorine atom at phenyl was beneﬁcial

for the protein−ligand interaction and protein degradation.

Moreover, the ﬂuorine also enhanced the membrane

permeability. As a result, adding the ﬂuorine into the pyridine

ring (D2) restored the biological activity (IC₅₀= 25.4 nM;

EC₅₀= 1.0 nM; MCF-7 IC₅₀= 10.23 nM). On the contrary,

while the ﬂuorine was replaced by other groups such as

chlorine or methoxy, the potency was lost slightly compared to

positive control AZD9496 (D4, D5, or D6 vs AZD9496).

Furthermore, the positions of ﬂuorine atoms were also critical

to protein−ligand interaction; the potency of compound D3

was obviously weaker than that of AZD9496 (IC₅₀= 120.7

nM; EC₅₀= 3.48 nM; MCF-7 IC₅₀= 16.25 nM). Thus, the 2,6-

diﬂuorophenyl scaﬀold was essential to the ERα binding and

degradation.

Because the acrylate side chain forms a critical hydrogen-

bond interaction with Leu536 and Tyr537 (PDB ID 5ACC),

which led to the conformational change to the AF2 domain

and promotion of ERα degradation, most SERDs in clinical

trials are acrylates. However, the potential reaction ability of

acrylate to Michael addition and the metabolism problem

stimulated us to explore novel scaﬀolds.⁴⁷First, we modiﬁed

the acrylate acid chain to a diverse library of aliphatic acids

(D7−D11); the four-membered cyclobutene moiety was

favorable for the ERα degradation and MCF-7 antiproliferation

through the investigation of ring size (D8 vs D7, D9, and

D10). Then, the diazaspiro heptane fragment occupying a

Initially, the 2,6-diﬂuorophenyl was surrounded by hydro-

phobic residues Trp383, Leu525, and Val534; thus, we further

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space similar to that of the piperazine ring was introduced on

the 2,6-diﬂuoro-phenyl, which enabled the exploration of the

novel heterocycle scaﬀold binding with ERα LBD. Compound

D11 containing diazaspiro heptane demonstrated better ERα

degradation eﬃciency and MCF-7 cellular inhibitory eﬀect

than the corresponding compound D10 (D11: IC₅₀= 25.2

nM; EC₅₀= 0.98 nM; MCF-7 IC₅₀= 7.48 nM). Subsequently,

we envisioned that the acrylate acid could be identiﬁed as a

negative center to form electrostatic interaction with H12.

However, when the carboxylic acid was replaced by another

electron-deﬁcient group (D12), the ERα degradation ability

was almost lost. Most importantly, if acrylate acid chain was

replaced by other electron-withdrawing groups D13−D15, the

ERα degradation eﬃciencies of D13, D14, and D15 were

residues (Table 2: F11). Thus, considering the excellent in

vitro ERα degradation activity and inhibitory activity of cell

viability of compound D24, we carried out further SAR studies

and structure optimization of D24.

The benzoic acid moiety of D24 was considered to extend

out of the ligand binding pocket between α-helices 3 (H3) and

11 (H11); thus the binding aﬃnity was increased.⁴⁸To enrich

the structure types of D24 and investigate the structure−

activity relationship, we supposed that other aromatic rings

should occupy similar hydrophobic interaction with target as

benzene ring on the benzoic acid fragment. Diﬀerent

heterocycles involving thiazole (D25), amino thiazole (D26),

pyrazole (D27), and pyridine (D28) were examined; as a

result, once the benzene ring was replaced by another aromatic

fragment, the antitumor activities were not improved (D25,

D26, D27, and D28 vs D24). Therefore, the benzoic acid

moiety was beneﬁcial to ERα degradation, and it could not be

replaced by other substituents.

To enrich the SAR of D24, the compound libraries of D29−

D40 were designed, synthesized, and biologically evaluated. In

general, the substitution of the aryl ring (D30−D32, D34), the

substitution of the carboxyl to the meta-position (D29), and

the carboxyl bioisostered to amide (D33) led to slight

reductions of the degradation abilities and MCF-7 antiprolifer-

ative activities. Furthermore, the biological activity of D35−

D40 also revealed that 2,6-diﬂuorophenyl was essential to

potencies of SERDs. When the acrylate side chain of

AZD9496 was replaced by the benzoic acid moiety of D24,

D24 also possessed good ERα binding aﬃnity, ERα

degradation eﬃcacy, and antiproliferative activity; thus D24

was selected for further biological evaluation.

equivalent to reference compound AZD9496 (D13, EC₅₀

=

0.22 nM; D14, EC₅₀= 0.23 nM; D15, EC₅₀= 0.10 nM), which

indicated the importance of exploring novel groups at this

region. Besides, other non-carboxylic acid substituents linked

to 2,6-diﬂuorophenyl (D16−D23) were not improving the

potencies, and some of them (D19 and D20) were inactivated

by the ERα degradation. In conclusion, the biological activity

of AZD9496 containing an acrylate side chain was more

favorable than other degraders D7−D23 without an acrylate

substituent.

On the basis of scaﬀold hopping strategy, we further

expanded the searches for alternative degradation motifs by

investigating the acrylic acid side chain of AZD9496 replaced

by various heterocyclic substituents. Gratifyingly, as the acrylic

acid was cyclized to benzoic acid, the potency of compound

D24 was signiﬁcantly enhanced. Although the interaction

between ERα and D24 was weaker than AZD9496, the

degradation ability of D24 was close to that of AZD9496; even

the MCF-7 antiproliferative activity of D24 was better than

that of AZD9496 (D24: IC₅₀= 17.1 nM; EC₅₀= 0.30 nM;

MCF-7 IC₅₀= 0.27 nM). To better understand the binding

pattern of D24 and ERα, the molecular docking analysis of

D24 in the binding pocket of ERα was performed (PDB ID

5ACC, Figure 5). As expected, the docking orientation and

interaction of D24 ﬁtted as well as the AZD9496; D24 formed

stronger binding aﬃnity with hot spot 1 and hot spot 2

Pharmacokinetics and Pharmacodynamics of D24.

Western Blot Analysis of ERα Degradation In Vitro. To

validate the possible mechanisms of ERα degradation action

mediated by compound D24, the Western blot analyses of

AZD9496, D24, and estradiol (E2) were performed (Figure

6). The results indicated that both D24 and AZD9496 were

Figure 6. Degradation of ERα by compound AZD9496, D24, and

estradiol (E2) by Western blot analysis. MCF-7 cells were incubated

with AZD9496, D24, or estradiol at the doses above. Total protein

was extracted at 48 h incubation with compound and immunoblotting

performed to detect eﬀects on ERα and PR (progesterone receptor)

levels.

identiﬁed as potent degraders of ERα in MCF-7 cells at 5 nM.

In addition, D24 possessed potent degradation activity at 0.6

nM, and the degradation mechanism of D24 was similar to that

of AZD9496, which indicated that D24 and AZD9496 could

not fully degrade ERα in MCF-7 cells even at a higher

biochemical concentration, resulting in no signiﬁcant diﬀer-

ence of degradation eﬃciency diﬀerence at concentrations of 5

and 40 nM.

In Vivo Eﬃcacy in the MCF-7 Breast Cancer Xenograft

Model. To further evaluate the antitumor activity of D24,

eﬃcacy was assessed in the MCF-7 human breast cancer

Figure 5. Docking conformation (PDB ID 5ACC) of D24 (green)

superimposed onto cocrystal structure of AZD9496 (pink).

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xenograft model in nude. Treatment of the nude with D24 at 2

mg/kg daily led to the signiﬁcant tumor growth inhibition as

compared to the AZD9496 group dose at 6 mg/kg (Figure

7a). Notably no signiﬁcant weight ﬂuctuations were observed

Table 6. In Vitro DMPK Evaluation of AZD9496 and D24

compd AZD9496

compd D24

Metabolic Stability in Liver Microsomes (T_1/2(min))

mouse

rat

human

dog

<38

<17.3

19.8

<13.8

27

27.4

20.4

38.3

25.1

monkey

31.1

Plasma Stability (T_1/2min)

mouse

rat

human

dog

>289.1

Figure 7. In vivo eﬃcacy and ERα by AZD9496 and D24 in the

MCF-7 xenograft model. (a) Tumor growth inhibition (TGI) dose

response upon oral administration of compounds AZD9496 and D24

(n = 6 per dose group, QD × 21days); (b) Nude mouse body weight

measured every 2 days.

monkey

>289.1

Binding Rate of Plasma Protein (%)

human

mouse

rat

dog

monkey

99.79

99.73

99.66

99.91

99.69

during the whole course (Figure 7b). The results indicated that

D24 could be developed as a potent SERD for the therapy

agents of breast cancer.

99.1

a

CYP Inhibition IC₅₀(μM)

1A2

2B6

2C8

2C9

2C19

2D6

3A4-M

3A4-T

>30

21.78

4.7

>30

19.2

9.22

>30

In Vivo Pharmacokinetic Study of D24. In view of the

excellent in vitro ERα degradation ability and antiproliferative

inhibitory of D24, and in vivo Eﬃcacy of D24 in the MCF-7

Breast Cancer Xenograft Model, the pharmacokinetic (PK)

parameters of D24 were evaluated in rats (Table 5) to assess

whether suﬃcient exposure for toxicological studies could be

achieved. Compound D24 was performed in rats via IV (1 mg/

kg) and PO (2 mg/kg) administration. While dosing of 2 mg/

kg solution of D24 to rats, the bioavailability of D24 was

attained to 80.5% that was twice of the positive control

AZD9496. Meanwhile, the plasma exposure (AUC) of D24

was 16073.7 h*ng/mL, and the half-life period was 12.1 h,

which indicated that D24 possessed better pharmacokinetic

properties than AZD9496. As for the intravenous admin-

istration, D24 also exhibited favorable drug exposure and low

clearance, revealing that D24 also had better pharmacokinetic

proﬁles than AZD9496. Those results revealed that D24 had

an acceptable PK proﬁle.

In Vitro DMPK Proﬁles of D24. In order to systematically

assess the druggability, the physicochemical properties of D24

was assayed (Table 6). To evaluate the metabolic stability,

compound AZD9496 and D24 were incubated with liver

microsomes of diﬀerent species (mouse, rat, dog, monkey and

human). As for human, dog and rat, D24 possessed stronger

metabolic stability than AZD9496, which proved that the

druggability of benzoic acid motifs better than acrylic acid

group. Meanwhile, the plasma stability and binding rate of

plasma protein for D24 in diﬀerent species were similar, and

the data of D24 was also approximately to AZD9496. By

screening a panel of eight cytochrome P450 enzymes, the

compound D24 revealed no inhibition activity (IC₅₀> 30 μM)

24.19

>30

b

hERG IC₅₀(μM)

>30

a

b

Inhibition of cytochrome P450 enzymes IC₅₀. Inhibition of the

hERG tail current was measured using a plate-based planar patch-

clamp system.

against six isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6,

CYP3A4-M, CYP3A4-T), however, the modest inhibition

against CYP2B6 and CYP2C8 were found in both D24 and

AZD9496.

To further understand the metabolism of D24, we employed

an in vitro model of liver microsomes to incubate D24 and

then analyzed the metabolites of D24 in diﬀerent species with

UPLC-UV/Q-TOF MS (Supporting Information). The results

of the metabolism of D24 revealed that the main metabolic

pathways of D24 were oxidation and amino dealkylation; the

proposed metabolic pathways of D24 were shown in

Supporting Information. The metabolism of D24 in human

model was appropriate to the monkey, and it was also found

that few variations among hepatic metabolic pathways of D24

in dogs, monkeys, and humans.

Toxicity Study of Compound D24. As a preclinical

evaluation of D24, the toxicity study of D24 was performed.

Because the potential cardiotoxicity was detrimental for the

Table 5. Pharmacokinetic Study of Compounds AZD9496 and D24

a

b

c

compd

dose (mg/kg)

t_1/2(h)

t_max(h)

C_max(ng/mL)

AUC_0−t(h·ng/mL) MRT_0−t(h) Cl ((L/h)/kg)

V_d(L/kg)

F (%)

AZD9496

2 p.o.

1 i.v.

2 p.o.

1 i.v.

1.6 0.1

0.1

12.1 2.7

13.8 1.5

0.6

532.8 77.2

2988.9 414.0

1277.7 91.6

2913.4 904.7

1355.7 210.8

1627.6 214.8

16073.7 835.9

9986.9 332.6

1.9

0.8

8.7

8.8

41.6 6.5

1

0.6 0.1

0.1

0.9 0.1

1.4

D24

2

80.5 4.2

a

b

c

Area under the curve. Mean retention time. Total body clearance.

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0.1%TFA:ACN; ﬂow rate, 1.2 mL/min; detection, UV 254 nm

(unless otherwise noted).

drug candidate into clinic trial, thus it was incredible to

evaluate the hERG toxicity of compound in the preclinical

phase, whereafter the patch-clamp experiment was utilized to

determine hERG inhibitory eﬀect of compounds AZD9496

and D24 with cisapride as the positive control. As shown in

Table 6, the hERG inhibitory values of AZD9496 and D24

were higher than 30 μM, which indicated that D24 had a

relatively low risk of hERG toxicity, even no cardiotoxicity

(IC₅₀> 10 μM, very weak inhibition or no inhibition).^49,50

Besides, to further assess of the safety of D24 in vivo, we

treated SD rats with D24 and carried out maximum tolerated

dose (MTD) determination. The animals were given 17.5 or

35 mg/kg D24 by gavage in a single dose for 14 days. No

animal death, obvious pathological changes, and weight loss

were observed after high-dose administration (Supporting

Information). Thus, compound D24 exhibited lower toxicity

by oral administration, and the drug safety window of D24 also

proved that D24 had relatively good druggability.

General Procedures for the Synthesis of Compound A1. (E)-

3-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3,9-dimeth-

yl-2,3,4,4a,9,9a-hexahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-

acrylic acid (A1). To a suspension of NaH (0.0394 g, 1 mmol) in

tetrahydrofuran (5 mL) was added a solution of AZD9496 (0.293 g,

0.66 mmol) in THF (5 mL) dropwise in an ice bath. The mixture was

stirred for 0.5 h followed by addition of MeI (50 μL, 0.66 mmol). The

mixture was stirred for a further 0.5 h, quenched by water, acidiﬁed

with 2 N HCl to pH ∼ 6, and extracted with ethyl acetate. The

organic layers were combined, dried with anhydrous sodium sulfate,

ﬁltered, and concentrated and puriﬁed by silica gel column

chromatography to give A1 as a yellow solid (0.200 g, 66%). ¹H

NMR (400 MHz, DMSO-d₆) δ 7.54 (d, J = 16.0 Hz, 1H), 7.50−7.48

(m, 3H), 7.35 (d, J = 8.0 Hz, 1H), 7.12−7.09 (m, 2H), 6.68 (d, J =

16.0 Hz, 1H), 5.33 (s, 1H), 3.42−3.41 (m, 1H), 3.25 (s, 3H), 2.92−

2.82 (m, 2H), 2.65−2.60 (m, 1H), 2.48−2.45 (m, 1H), 1.37 (d, J =

16.0 Hz, 3H), 1.21 (d, J = 16.0 Hz, 3H), 1.09 (d, J = 6.5 Hz, 3H).

HPLC purity: 97.8% (t_R= 9.59 min). ESI-MS m/z: [M + H]⁻= 457.2

(Calcd: 457.21).

General Procedures for the Syntheses of Compounds B1−

B3. Benzyl (S)-(1-Fluoro-3-(1H-indol-3-yl)propan-2-yl)carbamate

(2). (S)-2-(((Benzyloxy)carbonyl)amino)-3-(1H-indol-3-yl)propyl 4-

methylbenzenesulfonate (2.0 g, 4.18 mmol) was dissolved in TBAF

solution (1.0 M in THF, 12 mL) and stirred at 80 °C for 4 h. The

reaction was cooled to room temperature, quenched by water (20

mL), and extracted with ethyl acetate. The organic layers were

combined, washed with brine (50 mL × 2), dried with anhydrous

sodium sulfate, ﬁltered, and concentrated and puriﬁed by silica gel

column chromatography to give the ﬂuorinated compound 2 as a

yellow oil (1.0 g, 73%). ESI-MS m/z: [M + H]⁺= 327.1 (Calcd:

327.14).

(S)-1-Fluoro-3-(1H-indol-3-yl)propan-2-amine (3). The suspen-

sion of the intermediate 2 (1.0 g, 3.06 mmol) and Pd(OH)₂(0.05 g,

5%) in EtOH (30 mL) was stirred at hydrogen atmosphere (balloon)

overnight. The mixture was ﬁltered, and concentrated and puriﬁed by

silica gel column chromatography to give the primary amine 3 as a

yellow oil (quantitative yield). ESI-MS m/z: [M + H]⁺= 193.1

(Calcd: 193.11).

(S)-2-Fluoro-N-(1-ﬂuoro-3-(1H-indol-3-yl)propan-2-yl)-2-methyl-

propan-1-amine (12a). To a solution of the primary amine 3 (4.81 g,

25 mmol) and N,N-diisopropylethylamine (8.7 mL, 50 mmol) in

dioxane (50 mL) was added 2-ﬂuoro-2-methylpropyl triﬂuorometha-

nesulfonate (6.72 g, 30 mmol). The solution was heated to 90 °C for

2 h, cooled to room temperature, and concentrated and puriﬁed by

silica gel column chromatography to give the secondary amine 12a as

a brown oil (6.0 g, 90%). ESI-MS m/z: [M + H]⁺= 267.1 (Calcd:

267.16).

Methyl (E)-3-(3,5-Diﬂuoro-4-((1R,3S)-2-(2-ﬂuoro-2-methylprop-

yl)-3-(ﬂuoromethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-

phenyl)acrylate. To a solution of (S)-2-ﬂuoro-N-(1-ﬂuoro-3-(1H-

indol-3-yl)propan-2-yl)-2-methylpropan-1-amine (12a; 0.532 g, 2

mmol) and glacial acetic acid (1 mL) in toluene (20 mL) was

added methyl (E)-3-(3,5-diﬂuoro-4-formylphenyl)acrylate (0.543 g,

2.4 mmol) under argon. The solution was heated to 80 °C overnight,

cooled to room temperature, diluted with ethyl acetate (100 mL),

washed with saturated sodium bicarbonate (50 mL) and brine (50

mL), dried with anhydrous sodium sulfate, ﬁltered, and concentrated

and puriﬁed by silica gel column chromatography to give the ester as a

yellow solid (0.656 g, 70%). ESI-MS m/z: [M + H]⁻= 475.1 (Calcd:

475.19).

CONCLUSIONS

■

Most breast cancers are estrogen receptor α positive (ERα+);

however, resistance to current endocrine therapy has been

emerging. Although a variety of resistance mechanisms have

been elucidated, there is evidence that ERα still plays the

central role in many cases; thus the utilization of selective

estrogen receptor degraders (SERDs) to block ERα is an

eﬀective method to overcome resistance. The steroid-based

antiestrogen fulvestrant, the only approved SERD, is eﬀective

in metastatic breast cancer, but limited by poor pharmacoki-

netics, prompting the discovery of favorable pharmacokinetics

and orally bioavailable SERDs, which are still in demand.

In this study, we carry out molecular dynamic simulation

technology to analyze the hot-spot residues for protein−ligand

interaction between SERDs and ERα; subsequently focusing

on the hot-spot residues, diversity of the compound library is

designed and synthesized. Among them, while the acrylic acid

moiety of AZD9496 is modiﬁed to benzoic acid, D24 exhibits

better antitumor activity than AZD9496 in vitro (IC₅₀= 17.1

nM; EC₅₀= 0.30 nM; MCF-7 IC₅₀= 0.27 nM). Western blot

analysis proves the antitumor mechanism of D24; additionally,

D24 also displays good antitumor eﬃcacy in the MCF-7

human breast cancer xenograft model in vivo. The favorable

pharmacokinetic properties, especially good oral bioavailability

and eﬃcacious exposures, together with its excellent

druggability and good safety property, provide a favorable

proﬁle of D24. Thus, D24 can be selected as a drug candidate

of SERDs for further evaluation; the related research works will

be published in due course.

EXPERIMENTAL SECTION

■

General Procedures. Unless otherwise speciﬁed, reagents were

purchased from commercial suppliers and used without further

puriﬁcation. The reaction ﬂask is dried at 100 °C in an oven, and then

the reaction device was set up with hot and cooled under an argon

atmosphere. All reactions proceeded under the protection of an argon

stream. The reaction process was monitored by analytical thin layer

chromatography and a UV lamp (254 and 365 nm). A Bruker Biospin

(E)-3-(3,5-Diﬂuoro-4-((1R,3S)-2-(2-ﬂuoro-2-methylpropyl)-3-(ﬂu-

oromethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-

acrylic Acid (B1). To a solution of the ester (0.474 g, 1 mmol) in

MeOH (3 mL) and THF (6 mL) was added dropwise a solution of

NaOH (2 M, 2 mL) in an ice bath. The solution was stirred for 2 h,

acidiﬁed with 2 N HCl to pH ∼ 6, and extracted with ethyl acetate

(50 mL). The organic layers were combined, dried with anhydrous

sodium sulfate, ﬁltered, and concentrated and puriﬁed by silica gel

column chromatography to give B1 as a yellow solid (0.381 g, 83%).

1

AV400 (400 MHz, H NMR; 100 MHz, ¹³C NMR) instrument was

used to measure the ¹H NMR and ¹³C NMR spectra. Chemical shifts

are reported in ppm (parts per million) and are referenced to either

TMS or the solvent. The purity of all compounds for biological testing

was determined using an HPLC and was identiﬁed as >95% purity.

HPLC conditions: Xselect CSH C18 (4.6 × 150 mm², 3.5 μm); 1−2

min, 90:10 (%) 0.1%TFA:ACN, gradient, 15−18 min, 10:90 (%)

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¹H NMR (400 MHz, DMSO-d₆) δ 10.80 (s, 1H), 7.65−7.61 (m, 2H),

amine (12c) as a brown oil (1.7 g, 35%). ESI-MS m/z: [M + H]⁺

= 275.2 (Calcd: 275.18).

7.49 (s, 1H), 7.23 (d, J = 16.0 Hz, 1H), 7.15−7.13 (m, 2H), 7.08 (d, J

= 8.0 Hz, 1H), 6.45 (d, J = 16.0 Hz, 1H), 5.12 (s, 1H), 4.99−4.72 (m,

2H), 3.73 (d, J = 12.6 Hz, 1H), 3.42 (s, 1H), 2.86−2.73 (m, 2H),

2.52−2.42 (m, 1H), 1.24−1.16 (m, 6H). HPLC purity: 98.0% (t_R=

14.96 min). ESI-MS m/z: [M + H]⁺= 461.1 (Calcd: 461.18).

1-((1-Tosyl-1H-indol-3-yl)methyl)cyclopropan-1-amine (5). To a

solution of 2-(1-tosyl-1H-indol-3-yl)acetonitrile (4; 3.83 g, 12.4

mmol) in THF (100 mL) was added dropwise MeTi(i-PrO)₃(18.8

mL, 1 M in THF) under argon. After the solution was stirred for 0.5

h, ethylmagnesium bromide (18.8 mL, 1 M in THF) was added

dropwise. After stirring for a further 1 h, boron triﬂuoride etherate

(3.3 mL) was added. After additional stirring for a further 2 h, HCl

(100 mL, 1 M (aq)) and ethyl acetate (200 mL) were added

successively. NaOH (100 mL, 3 M (aq)) was added. The aqueous

phase was extracted with ethyl acetate. The organic layers were

combined, dried with anhydrous sodium sulfate, ﬁltered, and

concentrated and puriﬁed by silica gel column chromatography to

give 1-((1-tosyl-1H-indol-3-yl)methyl)cyclopropan-1-amine (5) as a

yellow oil (3.2 g, 76%). ESI-MS m/z: [M + H]⁺= 341.2 (Calcd:

341.12).

Methyl (E)-3-(3,5-Diﬂuoro-4-((5R)-4-(2-ﬂuoro-2-methylpropyl)-

1,2,3,3a,4,5,6, 10c-octahydrocyclopenta[5,6]pyrido[3,4-b]indol-5-

yl)phenyl)acrylate. To a solution of N-(2-ﬂuoro-2-methylpropyl)-2-

(1H-indol-3-yl)cyclopentan-1-amine (12c; 0.30 g, 1.1 mmol) and

glacial acetic acid (0.6 mL) in toluene (20 mL) was added methyl

(E)-3-(3,5-diﬂuoro-4-formylphenyl)acrylate (0.251 g, 1.1 mmol)

under argon. The solution was heated to 90 °C for 6 h, cooled to

room temperature, concentrated and puriﬁed by silica gel column

chromatography to give the ester as a yellow solid (0.215 g, 41%).

ESI-MS m/z: [M + H]⁺= 483.2 (Calcd: 483.21).

(E)-3-(3,5-Diﬂuoro-4-((5R)-4-(2-ﬂuoro-2-methylpropyl)-

1,2,3,3a,4,5,6,10c-octahydrocyclopenta[5,6]pyrido[3,4-b]indol-5-

yl)phenyl)acrylic Acid (B3). To a solution of the ester (0.215 g, 0.45

mmol) in MeOH (5 mL) and THF (10 mL) was added dropwise a

solution of NaOH (2 N, 0.5 mL) in an ice bath. The solution was

stirred for 2 h at room temperature, acidiﬁed with 1 N HCl to pH ∼

6, and extracted with ethyl acetate (50 mL). The organic layers were

combined, dried with anhydrous sodium sulfate, ﬁltered, and

concentrated and puriﬁed by silica gel column chromatography to

give B3 as a yellow solid (0.158 g, 75%). ¹H NMR (400 MHz,

DMSO-d₆) δ 10.76 (s, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.51−7.44 (m,

3H), 7.23 (d, J = 8.0 Hz, 1H), 7.01−6.94 (m, 2H), 6.66 (d, J = 16.0

Hz, 1H), 5.35 (s, 1H), 2.75−2.51 (m, 5H), 1.77−1.76 (m, 2H),

1.50−1.44 (m, 3H), 1.38−1.36 (m, 3H), 1.24−1.21 (m, 3H), 1.20−

1.10 (m, 2H). HPLC purity: 98.2% (t_R= 16.79 min). ESI-Ms m/z:

[M + H]⁺= 469.2 (Calcd: 469.20).

General Procedures for the Syntheses of Compounds C1−

C4. (R)-1-(1H-Indol-3-yl)-N-((1-(triﬂuoromethyl)cyclopropyl)-

methyl)propan-2-amine (15a). A solution of (1-(triﬂuoromethyl)-

cyclopropyl)methyl triﬂuoromethanesulfonate (0.296 g, 1 mmol),

(R)-1-(1H-indol-3-yl)propan-2-amine (0.270 g, 1.2 mmol), and N,N-

diisopropylethylamine (0.26 mL, 1.5 mmol) in dioxane (10 mL) was

heated to 90 °C for 8 h. The mixture was diluted with ethyl acetate

(100 mL) and washed with water (50 mL × 2) and brine. The organic

phase was dried with anhydrous sodium sulfate, ﬁltered, and

concentrated and puriﬁed by silica gel column chromatography to

give (R)-1-(1H-indol-3-yl)-N-((1-(triﬂuoromethyl)cyclopropyl)-

methyl)propan-2-amine (15a) as a yellow oil (0.212 g, 72%). ESI-

MS m/z: [M + H]⁺= 297.2 (Calcd: 297.15).

1-((1H-Indol-3-yl)methyl)cyclopropan-1-amine (6). To a solution

of 1-((1-tosyl-1H-indol-3-yl)methyl)cyclopropan-1-amine (0.50 g,

1.47 mmol) in MeOH (25 mL) was added NaOH (5.0 mL, 10%

(aq)) dropwise. The solution was heated for reﬂux for 12 h. After

cooling to room temperature, brine (30 mL) was added. The mixture

was extracted with ethyl acetate. The organic layers were combined,

dried with anhydrous sodium sulfate, ﬁltered, and concentrated to

aﬀord 1-((1H-indol-3-yl)methyl)cyclopropan-1-amine 6 as a light

yellow oil for direct use. ESI-MS m/z: [M + H]⁺= 187.2 (Calcd:

187.12).

(E)-3-(3,5-Diﬂuoro-4-(2′-(2-ﬂuoro-2-methylpropyl)-1′,2′,4′,9′-

tetrahydrospiro[cyclopropane-1,3′-pyrido[3,4-b]indol]-1′-yl)-

phenyl)acrylic Acid (B2). Prepared using the same procedure as that

1

for B1, B2 was a light yellow solid (0.026 g, 76%). H NMR (400

MHz, DMSO-d₆) δ 10.81 (s, 1H), 7.49 (d, J = 16.0 Hz, 1H), 7.39−

7.36 (m, 3H), 7.26 (d, J = 16.0 Hz, 1H), 7.04 (t, J = 7.2 Hz, 1H), 6.99

(t, J = 16.0 Hz, 1H), 6.65 (d, J = 16.0 Hz, 1H), 5.47 (d, J = 16.0 Hz,

1H), 3.31−3.27 (m, 1H), 3.00 (t, J = 12.0 Hz, 1H), 2.81−2.77 (m,

1H), 2.20 (d, J = 16.0 Hz, 1H), 1.31 (s, 6H), 0.50−0.46 (m, 2H),

0.34−0.30 (m, 2H). HPLC purity: 97.0% (t_R= 10.37 min). ESI-MS

m/z: [M + H]⁺= 455.2 (Calcd: 455.19).

2-(1H-Indol-3-yl)cyclopentan-1-one (9). To a solution of 1H-

indole (1.98 g, 16.9 mmol) and 2-chlorocyclopentan-1-one (2 g, 16.9

mmol) in 2,2,2-triﬂuoroethanol (35 mL) was added anhydrous

sodium carbonate (2.15 g, 20.28 mmol). The solution was stirred for

48 h, ﬁltered, and concentrated to aﬀord 2-(1H-indol-3-yl)-

cyclopentan-1-one (9) for direct use in the next step. ESI-MS m/z:

[M + H]⁺= 200.1 (Calcd: 200.10).

2-(1H-Indol-3-yl)cyclopentan-1-amine (10). To a solution of 2-

(1H-indol-3-yl)cyclopentan-1-one (9; 4.78 g, 24.2 mmol) in

methanol (60 mL) was added ammonium acetate (20.18 g, 262

mmol) and sodium cyanoborohydride (1.67g, 26.42 mmol). The

mixture was stirred overnight before acidiﬁcation to pH ∼ 2 by 2 N

HCl. The solution was concentrated and extracted with dichloro-

methane. The aqueous phase was basiﬁed to pH ∼ 12 by 4 M NaOH.

The solution was extracted with dichloromethane. The organic layers

were combined, washed with brine, dried with anhydrous sodium

sulfate, ﬁltered, and concentrated to aﬀord 2-(1H-indol-3-yl)-

cyclopentan-1-amine (10 ; 4.13 g, 85%). ESI-MS m/z: [M + H]⁺=

201.1 (Calcd: 201.13).

(E)-3-(3,5-Diﬂuoro-4-((1R,3R)-3-methyl-2-((1-(triﬂuoromethyl)-

cyclopropyl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-

yl)phenyl)acrylic Acid (C1). Prepared using the same procedure as

1

that for B1, C1 was a white solid (0.033 g, 79%). H NMR (400

MHz, DMSO-d₆) δ 10.55 (s, 1H), 7.59 (d, J = 16.0 Hz, 1H), 7.51−

7.49 (m, 2H), 7.42 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H),

7.04−6.83 (m, 2H), 6.58 (d, J = 16.0 Hz, 1H), 5.06 (s, 1H), 3.65−

3.63 (m, 1H), 3.00−2.96 (m, 1H), 2.80−2.71 (m, 2H), 2.65−2.63

(m, 1H), 1.42−1.36 (m, 2H), 1.43−1.29 (m, 2H), 1.10 (d, J = 7.5 Hz,

3H). HPLC purity: 96.4% (t_R= 14.96 min). ESI-MS m/z: [M + H]⁺

= 491.2 (Calcd: 491.17).

(E)-3-(4-((1R,3R)-2-Butyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido-

[3,4-b] indol-1-yl)-3,5-diﬂuorophenyl)acrylic Acid (C2). Prepared

using the same procedure as that for B1, C2 was a white solid (0.045

g, 83%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.54 (s, 1H), 7.57−7.43

(m, 3H), 7.40 (d, J = 7.5 Hz, 1H), 7.18 (d, J = 7.5 Hz, 1H), 7.02−

6.92 (m, 2H), 6.66 (d, J = 16.0 Hz, 1H), 5.11 (s, 1H), 3.47−3.41 (m,

1H), 2.71−2.54 (m, 1H), 2.43−2.25 (m, 2H), 1.38−1.35 (m, 1H),

1.33−1.30 (m, 2H),1.28−1.26 (m, 2H), 1.22 (d, J = 7.5 Hz, 3H), 0.75

(t, J = 7.5 Hz, 3H). HPLC purity: 95.9% (t_R= 10.37 min). ESI-MS

m/z: [M + H]⁺= 425.2 (Calcd: 425.19).

N-(2-Fluoro-2-methylpropyl)-2-(1H-indol-3-yl)cyclopentan-1-

amine (12c). To a solution of 2-(1H-indol-3-yl)cyclopentan-1-amine

(10; 3.5 g, 17.5 mmol) and N,N-diisopropylethylamine (4.52 g, 35

mmol) in dioxane (50 mL) was added 2-ﬂuoro-2-methylpropyl

triﬂuoromethanesulfonate (5.0 g, 22.32 mmol). The solution was

heated to 90 °C for 2 h, cooled to room temperature, and

concentrated and puriﬁed by silica gel column chromatography to

give N-(2-ﬂuoro-2-methylpropyl)-2-(1H-indol-3-yl)cyclopentan-1-

(E)-3-(3,5-Diﬂuoro-4-((1R,3R)-2-(3-ﬂuoro-3-methylbutyl)-3-meth-

yl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic

Acid (C3). Prepared using the same procedure as that for B1, C3 was a

white solid (0.090 g, 81%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.56

(s, 1H), 7.53−7.46 (m, 3H), 7.41 (d, J = 7.5 Hz, 1H), 7.19 (d, J = 7.5

Hz, 1H), 7.02−6.92 (m, 2H), 6.65 (d, J = 16.0 Hz, 1H), 5.10 (s, 1H),

3.49−3.48 (m, 1H), 2.75−2.73 (m, 1H), 2.71−2.69 (m, 1H), 2.66−

2.58 (m, 2H), 1.81−1.72 (m, 2H), 1.30−1.11 (m, 6H), 1.02 (d, J =

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7.5 Hz, 3H). HPLC purity: 95.9% (t_R= 10.30 min). ESI-MS m/z: [M

+ H]⁺= 457.3 (Calcd: 457.20).

Hz, 3H). HPLC purity: 96.6% (t_R= 9.90 min). ESI-MS m/z: [M +

H]⁺= 426.1 (Calcd: 426.19).

(E)-3-(2,6-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-

methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-

acrylic Acid (D3). Prepared using the same procedure as that for B1,

(R)-N-(1-(1H-Indol-3-yl)propan-2-yl)aniline (15d). To a solution

of (R)-1-(1H-indol-3-yl)propan-2-amine (0.500 g, 2.87 mmol) and

iodobenzene (0.702 g, 4 mmol) in DMF (5 mL) was added cesium

carbonate (1.87 g, 3.7 mmol) and copper(I) iodide (0.055 g, 0.28

mmol) under argon. The sealed vial was irradiated in the microwave

on a Biotage Smith Synthesis apparatus at 135 °C for 120 min. The

mixture was diluted with ethyl acetate (50 mL) and ﬁltered over

Celite. The ﬁltrate was washed with water (30 mL × 3) and brine,

dried with anhydrous sodium sulfate, ﬁltered, and concentrated and

puriﬁed by silica gel column chromatography to give (R)-N-(1-(1H-

indol-3-yl)propan-2-yl)aniline (15d; 0.243 g, 34%). ESI-MS m/z: [M

+ H]⁺= 251.1 (Calcd: 251.15).

Methyl (E)-3-(3,5-Diﬂuoro-4-((1R,3R)-3-methyl-2-phenyl-2,3,4,9-

tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate. To a solu-

tion of (R)-N-(1-(1H-indol-3-yl)propan-2-yl)aniline (0.20 g, 0.8

mmol) and glacial acetic acid (0.5 mL) in toluene (20 mL) was

added methyl (E)-3-(3,5-diﬂuoro-4-formylphenyl)acrylate (0.202 g,

0.89 mmol) under argon. The solution was heated to 80 °C overnight,

cooled to room temperature, and concentrated and puriﬁed by silica

gel column chromatography to give the ester as a brown oil (0.150 g,

41%). ESI-MS m/z: [M + H]⁺= 459.1 (Calcd: 459.18).

1

D3 was a light yellow solid (0.077 g, 79%). H NMR (400 MHz,

DMSO-d₆) δ 10.87 (s, 1H), 7.53 (d, J = 16.0 Hz, 1H), 7.45 (d, J = 4.0

Hz, 1H), 7.33 (d, J = 4.0 Hz, 1H), 7.08−7.06 (m, 3H), 7.01−6.98 (m,

1H), 6.55 (d, J = 16.0 Hz, 1H), 5.04 (s, 1H), 3.16 (m, 1H), 2.92−

2.83 (m, 1H), 2.69−2.66 (m, 1H), 2.54−2.53 (m, 1H), 2.53−2.51

(m, 1H), 1.29 (d, J = 16.0 Hz, 3H), 1.10 (d, J = 16.0 Hz, 3H), 1.07

(d, J = 8.0 Hz, 3H). HPLC purity: 96.7% (t_R= 12.15 min). ESI-MS

m/z: [M + H]⁺= 443.1 (Calcd: 443.19).

(E)-3-(3,5-Dichloro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-

methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-

acrylic Acid (D4). Prepared using the same procedure as that for B1,

1

D4 was a white solid (0.069 g, 73%). H NMR (400 MHz, DMSO-

d₆) δ 10.25 (s, 1H), 7.79 (s, 1H), 7.54 (s, 1H), 7.44−7.29 (m, 2H),

7.15 (d, J = 16.0 Hz, 1H), 7.04−6.83 (m, 2H), 6.58 (d, J = 16.0 Hz,

1H), 5.58 (s, 1H), 3.67 (s, 1H), 3.14−2.83 (m, 2H), 2.65−2.58 (m,

1H), 2.29−2.24 (m, 1H), 1.14−1.10 (m, 6H), 1.03−1.01 (m, 3H).

¹³C NMR (101 MHz, CDCl₃) δ 170.78, 142.53, 137.32, 136.28,

135.76, 130.57, 127.45, 121.40, 121.12, 119.31, 118.15, 110.77,

109.30, 97.13 (d, J = 166.0 Hz), 58.17 (d, J = 22.0 Hz), 55.71, 52.16

(d, J = 5.0 Hz), 27.72, 25.73 (d, J = 24.0 Hz), 24.35 (d, J = 24.0 Hz),

9.80. HPLC purity: 96.4% (t_R= 12.15 min). ESI-MS m/z: [M + H]⁺

= 475.1 (Calcd: 475.13).

(E)-3-(3,5-Diﬂuoro-4-((1R,3R)-3-methyl-2-phenyl-2,3,4,9-tetrahy-

dro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic Acid (C4). To a

solution of the ester (0.150 g, 0.33 mmol) in MeOH (2 mL) and

THF (8 mL) was added dropwise a solution of NaOH (5 N, 2 mL) in

an ice bath. The solution was stirred for 2 h at room temperature,

acidiﬁed with 2 N HCl to pH ∼ 6, and extracted with ethyl acetate

(50 mL). The organic layers were combined, dried with anhydrous

sodium sulfate, ﬁltered, and concentrated and puriﬁed by silica gel

column chromatography to give compound C4 as a light yellow solid

(E)-3-(3-Fluoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-5-methoxyphenyl)-

acrylic Acid (D5). Prepared using the same procedure as that for B1,

1

D5 was a white solid (0.045 g, 61%). H NMR (400 MHz, DMSO-

d₆) δ 10.36 (s, 1H), 7.56 (d, J = 16.0 Hz, 1H), 7.38 (d, J = 8.0 Hz,

1H), 7.28 (s, 1H), 7.15 (d, J = 4.0 Hz, 1H), 7.06 (d, J = 16.0 Hz, 1H),

6.97−6.92 (m, 2H), 6.98−6.64 (d, 1H), 5.33 (s, 1H), 3.89 (s, 3H),

3.58−3.52 (m, 1H), 2.98−2.81 (m, 2H), 2.58−2.53 (m, 1H), 2.35−

2.25 (m, 1H), 1.29 (d, J = 16.0 Hz, 3H), 1.10 (d, J = 16.0 Hz, 3H),

1.07 (d, J = 8.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 169.88,

160.69 (d, J = 180.0 Hz), 144.07, 136.04, 133.25, 126.67, 120.98,

119.93, 119.13, 118.15, 114.10, 110.73, 106.08, 97.15 (d, J = 166.0

Hz), 62.36 (d, J = 22.0 Hz), 59.26, 56.11, 54.96, 27.72, 25.91 (d, J =

14.0 Hz), 24.32 (d, J = 25.0 Hz), 19.35. HPLC purity: 96.3% (t_R=

10.74 min). ESI-MS m/z: [M + H]⁺= 455.2 (Calcd: 455.21).

(E)-3-(4-((1R,3R)-2-(2-Fluoro-2-methylpropyl)-3-methyl-2,3,4,9-

tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-dimethoxyphenyl)-

acrylic Acid (D6). Prepared using the same procedure as that for B1,

1

(0.125 g, 86%). H NMR (400 MHz, DMSO-d₆) δ 7.53−7.52 (m,

1H), 7.37 (d, J = 16.0 Hz, 1H), 7.25−7.17 (m, 2H), 7.12−7.06 (m,

5H), 6. 96−6.94 (m, 1H), 6.87 (s, 1H), 6.59 (s, 1H), 6.54 (d, J = 16.0

Hz, 1H), 5.06 (s, 1H), 3.47−3.41 (m, 1H), 3.00−2.94 (m, 1H),

1.24−1.22 (m, 1H), 1.19−1.17 (m, 3H). HPLC purity: 97.1% (t_R=

10.31 min). ESI-MS m/z: [M + H]⁺= 445.0 (Calcd: 445.16).

General Procedures for the Syntheses of Compounds D1−

D40. Methyl (E)-3-(6-Formylpyridin-3-yl)acrylate (17). To a

solution of 5-bromopicolinaldehyde (1.46 g, 7.84 mmol) and

palladium acetate (0. 239 g, 0.78 mmol) in DMF (30 mL) was

added methyl acrylate (1.01 g, 11.76 mmol) and triethylamine (1.59

g, 15.69 mmol) under argon. The solution was heated to 80 °C

overnight. After cooling to room temperature, brine (60 mL) was

added. The mixture was extracted with ethyl acetate. The organic

layers were combined, dried with anhydrous sodium sulfate, ﬁltered,

and concentrated and puriﬁed by silica gel column chromatography to

give methyl (E)-3-(6-formylpyridin-3-yl) acrylate (17) as a yellow

solid (1.45 g, 96%). ESI-MS m/z: [M + H]⁺= 192.1 (Calcd: 192.06).

(E)-3-(6-((1S,3R)-2-(2-Fluoro-2-methylpropyl)-3-methyl-2,3,4,9-

tetrahydro-1H-pyrido [3,4-b]indol-1-yl)pyridin-3-yl)acrylic Acid

(D1). Prepared using the same procedure as that for B1, D1 was a

white solid (0.077 g, 68%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.68

(s, 1H), 8.72 (s,1H), 8.11 (d, J = 16.0 Hz, 1H), 7.55 (m, 2H), 7.50

(d, J = 16.0 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.05−6.97 (m, 2H),

6.64 (d, J = 8.0 Hz, 1H), 5.10 (s, 1H), 3.19−3.12 (m, 1H), 2.93−2.86

(m, 1H), 2.52−2.43 (m, 2H), 1.23 (d, J = 20.0 Hz, 3H), 1.15 (d, J =

20.0 Hz, 3H), 1.07 (d, J = 8.0 Hz, 3H). HPLC purity: 97.8% (t_R=

9.59 min). ESI-MS m/z: [M + H]⁺= 408.3 (Calcd: 408.21).

1

D6 was a white solid (0.045 g, 61%). H NMR (400 MHz, DMSO-

d₆) δ 10.1 (s,1H), 7.52 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 16.0 Hz, 1H),

7.14 (d, J = 4.0 Hz, 1H), 6.94−6.85 (m, 4H), 6.63 (d, J = 16.0 Hz,

1H), 5.45 (s, 1H), 3.41−3.93 (m, 2H), 3.38 (s, 6H), 3.05 (m, 1H),

2.85 (d, 1H), 2.55 (m, 1H), 1.09 (d, J = 5.0 Hz, 3H), 1.04 (d, J = 5.0

Hz,3H), 1.03−1.01 (m, 3H). HPLC purity: 97.0% (t_R= 9.73 min).

ESI-MS m/z: [M + H]⁺= 467.3 (Calcd: 467.23).

(1R,3R)-1-(4-Bromo-2,6-diﬂuorophenyl)-2-(2-ﬂuoro-2-methyl-

propyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (22).

To a solution of (R)-N-(1-(1H-indol-3-yl)propan-2-yl)aniline (1.0

g, 4.03 mmol) and glacial acetic acid (0.5 mL) in toluene (10 mL)

was added 4-bromo-2,6-diﬂuorobenzaldehyde (0.934 g, 4.23 mmol)

under argon. The solution was heated to 80 °C overnight, cooled to

room temperature, quenched by saturated sodium bicarbonate, and

extracted with ethyl acetate. The organic layers were combined, dried

with anhydrous sodium sulfate, ﬁltered, and concentrated and puriﬁed

by silica gel column chromatography to give (1R,3R)-1-(4-bromo-2,6-

diﬂuorophenyl)-2-(2-ﬂuoro-2-methyl propyl)-3-methyl-2,3,4,9-tetra-

hydro-1H-pyrido[3,4-b]indole (22) as a white solid (1.50 g, 83%).

ESI-MS m/z: [M + H]⁺= 451.1 (Calcd: 450.09).

(E)-3-(5-Fluoro-6-((1S,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-yl)acrylic

Acid (D2). Prepared using the same procedure as that for B1, D2 was

a light yellow solid (0.030 g, 64%). ¹H NMR (400 MHz, DMSO-d₆) δ

10.52 (s, 1H), 8.49 (s, 1H), 8.11 (d, J = 12.0 Hz, 1H), 7.54 (d, J =

16.0 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H),

7.02−6.92 (m, 2H), 6.69 (d, J = 16.0 Hz, 1H), 5.44 (s, 1H), 3.41 (s,

1H), 2.91−2.86 (m, 2H), 2.60−2.53 (m, 1H), 2.46−2.33 (m, 1H),

1.23 (d, J = 20.0 Hz, 3H), 1.15 (d, J = 20.0 Hz, 3H), 1.07 (d, J = 8.0

Methyl 1-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-

methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-

azetidine-3-carboxylate (23). To a mixture of (1R,3R)-1-(4-bromo-

2,6-diﬂuorophenyl)-2-(2-ﬂuoro-2-methyl propyl)-3-methyl-2,3,4,9-

tetrahydro-1H-pyrido[3,4-b]indole (0.10 g, 0.22 mmol), Pd₂(dba)₃

(0.010 g, 0.011 mmol), Xphos (0.010 g, 0.022 mmol), and Cs₂CO₃

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(0.202 g, 0.62 mmol) in toluene (5 mL) was added methyl azetidine-

3-carboxylate (0.029 g, 0.25 mmol) under argon. The reaction was

heated to 110 °C for 10 h. After cooling to room temperature, the

mixture was concentrated and puriﬁed by silica gel column

chromatography to give the desired ester 23 as a yellow solid

(0.050 g, 47%). ESI-MS m/z: [M + H]⁺= 486.1 (Calcd: 486.23).

1-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)azetidine-3-

carboxylic Acid (D8). Prepared using the same procedure for

hydrolysis aforementioned, D8 was a white solid (0.035 g, 74%).

¹H NMR (400 MHz, DMSO-d₆) δ 10.45 (s, 1H), 7.38 (d, J = 8.0 Hz,

1H), 7.18 (d, J = 8.0 Hz, 1H), 7.00−6.91 (m, 2H), 6.18 (d, J = 8.0

Hz, 2H), 5.12 (s, 1H), 4.05−4.00 (m, 2H), 3.90−3.86 (m, 2H),

3.55−3.51 (m, 2H), 2.90−2.81 (m, 2H), 2.59−2.55 (m, 1H), 2.41−

2.28 (m, 1H), 1.14−1.10 (m, 6H), 1.03−1.01 (m, 3H). HPLC purity:

96.3% (t_R= 10.74 min). HPLC purity: 96.4% (t_R= 10.78 min). ESI-

MS m/z: [M + H]⁺= 472.3 (Calcd: 472.21).

1H), 2.92−2.80 (m, 2H), 2.60−2.50 (m, 1H), 2.12 (s, 6H), 1.43−

1.38 (m, 6H), 1.12−1.09 (m, 3H). HPLC purity: 96.5% (t_R= 8.23

min). ESI-MS m/z: [M + H]⁺= 471.3 (Calcd: 471.27).

(1R,3R)-1-(2,6-Diﬂuoro-4-(3-(ﬂuoromethyl)azetidin-1-yl)phenyl)-

2-(2-ﬂuoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-

pyrido[3,4-b]indole (D13). Prepared using the same procedure as that

1

for D8, D13 was a white solid (0.041 g, 65%). H NMR (500 MHz,

DMSO-d₆) δ 10.47 (s, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 8.0

Hz, 1H), 7.02−6.91 (m, 2H), 6.07 (d, J = 11.5 Hz, 2H), 5.09 (s, 1H),

4.67 (d, J = 5.0 Hz, 1H), 4.58 (d, J = 5.0 Hz, 1H), 3.95 (s, 2H), 3.66

(s, 2H), 3.32 (s, 1H), 3.13−3.04 (m, 2H), 2.93−2.86 (m, 2H), 2.43−

2.38 (m, 2H), 1.23−1.14 (m, 6H), 1.06 (d, J = 6,5 Hz, 3H). ¹³C

NMR (101 MHz, DMSO-d₆) δ 176.23, 164.23 (d, J = 8.0 Hz), 161.77

(d, J = 11.0 Hz), 152.68, 136.67, 133.68, 127.49, 120.61, 118.47,

117.82, 111.35, 106.57, 105.02, 97.56 (d, J = 165.0 Hz), 94.76 (d, J =

27.0 Hz), 84.63 (d, J = 164.0 Hz), 56.28, 53.44 (d, J = 7.0 Hz), 50.92,

40.62, 29.78 (d, J = 20.0 Hz), 27.15, 25.44 (d, J = 24.0 Hz), 24.95 (d,

J = 24.0 Hz). HPLC purity: 94.6% (t_R= 12.75 min). ESI-MS m/z: [M

+ H]⁺= 460.3 (Calcd: 460.23).

1-((3,5-Diﬂuoro-4-((3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)amino)-

cyclopropane-1-carboxylic Acid (D7). Prepared using the same

6-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-2-oxa-6-

azaspiro[3.3]heptane (D14). Prepared using the same procedure as

1

procedure as that for D8, D7 was a white solid (0.041 g, 64%). H

NMR (400 MHz, DMSO-d₆) δ 10.54 (s, 1H), 7.38 (d, J = 8.0 Hz,

1H), 7.20 (d, J = 8.0 Hz, 1H), 7.00−6.93 (m, 2H), 6.18−6.12 (m,

2H), 5.06 (s, 1H), 3.47−3.44 (m, 1H), 3.18−3.04 (m, 1H), 2.82−

2.73 (m, 2H), 2.38−2.34 (m, 1H), 1.43−1.40 (m, 4H), 1.22−1.10

(dd, J = 16.0 Hz, 12.0 Hz, 6H), 1.08 (d, J = 8.0 Hz, 3H). HPLC

purity: 99.3% (t_R= 10.13 min). ESI-MS m/z: [M + H]⁺= 472.3

(Calcd: 472.21).

1

that for D8, D14 was a white solid (0.046 g, 51%). H NMR (400

MHz, DMSO-d₆) δ 10.47 (s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.17 (d, J

= 8.0 Hz, 1H), 6.99−6.91 (m, 2H), 6.05 (d, J = 8.0 Hz, 2H), 5.06 (s,

1H), 4.70 (s, 4H), 3.98 (s, 4H), 2.84−2.75 (m, 2H), 2.43−2.38 (m,

3H), 1.22−1.10 (m, 6H), 1.05−1.02 (m, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 164.35 (d, J = 10.0 Hz), 161.87 (d, J = 10.0 Hz), 151.81 (t,

J = 13.0 Hz), 136.11, 132.88, 128.95 (d, J = 165.0 Hz), 127.86,

121.13, 119.12, 118.11, 110.60, 108.21, 105.57 (t, J = 16.0 Hz), 97.45

(d, J = 165.0 Hz), 96.14, 94.82 (d, J = 26.0 Hz), 80.95, 61.29 (d, J =

6.0 Hz), 56.94 (d, J = 21.0 Hz), 51.28 (d, J = 4.0 Hz), 50.16, 38.80,

27.22, 25.43 (d, J = 25.0 Hz), 24.44 (d, J = 25.0 Hz). HPLC purity:

98.3% (t_R= 11.30 min). ESI-MS m/z: [M + H]⁺= 470.3 (Calcd:

470.23).

1-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)pyrrolidine-3-

carboxylic Acid (D9). Prepared using the same procedure as that for

1

D8, D9 was a white solid (0.055 g, 73%). H NMR (400 MHz,

DMSO-d₆) δ 10.43 (s, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.19 (d, J = 8.0

Hz, 1H), 6.95 (m, 2H), 6.09 (d, J = 4.0 Hz, 2H), 5.05 (s, 1H), 3.50 (s,

1H), 3.40 (s, 2H), 3.39−3.33 (m, 2H), 3.27−3.20 (m, 2H), 2.81−

2.73 (m, 2H), 2.37−2.32 (m, 1H), 2.19−2.12 (m, 2H), 1.23−1.16

(dd, J = 21.5 Hz, 16.0 Hz, 6H), 1.08 (d, J = 6.5 Hz, 3H). ¹³C NMR

(101 MHz, CDCl₃) δ 178.17, 164.43, 161.92 (d, J = 11.0 Hz), 148.24,

136.11, 133.00, 127.86, 120.09 (d, J = 200.0 Hz), 118.08, 110.63,

108.03, 97.50 (d, J = 165.0 Hz), 95.14 (d, J = 28.0 Hz), 56.83 (d, J =

22.0 Hz), 51.35, 50.20, 49.85, 47.09, 42.81, 29.71, 28.44, 27.19, 25.51

(d, J = 24.0 Hz), 24.50 (d, J = 24.0 Hz). HPLC purity: 98.0% (t_R=

11.00 min). ESI-MS m/z: [M + H]⁺= 486.3 (Calcd: 486.23).

1-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)piperidine-4-

carboxylic Acid (D10). Prepared using the same procedure as that for

1-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)azetidine-3-

carboxamide (D15). Prepared using the same procedure as that for

1

D8, D15 was a white solid (0.077 g, 43%). H NMR (400 MHz,

DMSO-d₆) δ 10.47 (s, 1H), 7.47 (s, 1H), 7.37 (d, J = 8.0 Hz, 1H),

7.17 (d, J = 8.0 Hz, 1H), 7.02 (s, 1H), 6.99−6.90 (m, 2H), 6.06 (d, J

= 12.0 Hz, 2H), 5.05 (s, 1H), 3.97−3.93 (m, 2H), 3.83−3.78 (m,

2H), 3.54−3.50 (m, 1H), 3.45−3.40 (m, 1H), 2.90−2.77 (m, 2H),

2.54−2.29 (m, 2H), 1.20−1.10 (dd, J = 20.0 Hz, 16.0 Hz, 6H), 1.03

(d, J = 8.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 173.80, 164.32

(d, J = 10.0 Hz), 161.84 (d, J = 22.0 Hz), 152.21 (t, J = 13.0 Hz),

136.14, 132.94, 127.84, 121.10, 118.50 (d, J = 101.0 Hz), 110.69,

108.09, 105.67, 97.56 (d, J = 165.0 Hz), 96.14, 94.86 (d, J = 27.0 Hz),

77.35, 77.04, 76.72, 56.94 (d, J = 22.0 Hz), 54.42, 51.28 (d, J = 4.0

Hz), 50.08, 34.10, 27.27, 25.52 (d, J = 25.0 Hz), 24.37 (d, J = 25.0

Hz). HPLC purity: 96.2% (t_R= 11.26 min). ESI-MS m/z: [M + H]⁺

= 471.3 (Calcd: 471.23).

1

D8, D10 was a white solid (0.035 g, 57%). H NMR (400 MHz,

DMSO-d₆) δ 10.34 (s, 1H), 7.93 (s,1H), 7.64 (s, 1H), 7.49 (d, J = 8.0

Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.25 (m, 1H), 7.17 (d, J = 4.0 Hz,

1H), 6.98−6.92 (m, 2H), 6.73 (d, J = 4.0 Hz, 1H), 5.62 (s, 1H),

3.79−3.74 (m, 2H), 3.68−3.65 (m, 4H), 2.98−2.91 (m, 2H), 2.67−

2.61 (m, 2H), 2.28−2.22 (m, 1H), 1.24−1.12 (dd, J = 16.0 Hz, 12.0

Hz, 6H), 1.02 (d, J = 8.0 Hz, 3H). HPLC purity: 97.0% (t_R= 11.31

min). ESI-MS m/z: [M + H]⁺= 500.3 (Calcd: 500.24).

(1R,3R)-1-(2,6-Diﬂuoro-4-(3-(2-ﬂuoroethoxy)azetidin-1-yl)-

phenyl)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-

1H-pyrido[3,4-b]indole (D16). Prepared using the same procedure as

2-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-2-azaspiro-

[3.3]heptane-6-carboxylic Acid (D11). Prepared using the same

1

that for D8, D16 as a white solid (0.056 g, 59%). H NMR (400

1

MHz, DMSO-d₆) δ 10.45 (s, 1H), 8.23 (s, 1H),7.37 (d, J = 8.0 Hz,

1H), 7.19 (d, J = 8.0 Hz, 1H), 6.98−6.95 (m, 2H), 6.05 (d, J = 4.0

Hz, 1H), 5.07 (s, 1H), 4.50 (s, 1H), 4.47 (s, 2H), 3.71 (s, 2H), 3.62

(m, 4H), 2.91−2.80 (m, 2H), 2.41−2.34 (m, 2H), 1.23−1.14 (m,

6H), 1.07 (d, J = 6.5 Hz, 3H). HPLC purity: 96.0% (t_R= 12.34 min).

ESI-MS m/z: [M + H]⁺= 490.3 (Calcd: 490.24).

1-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)azetidine-3-

carbonitrile (D17). Prepared using the same procedure as that for D8,

D17 was a white solid (0.011 g, 31%). ¹H NMR (400 MHz, DMSO-

d₆) δ 10.49 (s, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.0 Hz, 1H),

6.98−6.93 (m, 2H), 6.15 (d, J = 8.0 Hz, 2H), 5.08 (s, 1H), 4.12−4.08

(m, 2H), 4.01−3.97 (m, 2H), 3.89−3.83 (m, 1H), 2.87−2.76 (m,

3H), 2.38−2.28 (m, 2H), 1.23−1.21 (m, 3H), 1.16−1.11 (m, 3H),

procedure as that for D8, D11 was a white solid (0.054 g, 78%). H

NMR (400 MHz, DMSO-d₆) δ 10.48 (s, 1H), 7.37 (d, J = 8.0 Hz,

1H), 7.17 (d, J = 8.0 Hz, 1H), 6.98−6.90 (m, 2H), 6.01 (d, J = 12.0

Hz, 2H), 5.04 (s, 1H), 3.83 (s, 2H), 3.75 (s, 2H), 3.52−3.45 (m, 1H),

2.99−2.92 (m, 1H), 2.87−2.77 (m, 2H), 2.39 (s, 1H), 2.45−2.30 (m,

5H), 1.14−1.10 (m, 6H), 1.03−1.01 (m, 3H). HPLC purity: 96.1%

(t_R= 11.79 min). ESI-MS m/z: [M + H]⁺= 512.2 (Calcd: 512.24).

1-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-N,N-dime-

thylazetidin-3-amine (D12). Prepared using the same procedure as

1

that for D8, D12 was a light yellow solid (0.044 g, 69%). H NMR

(400 MHz, DMSO-d₆) δ 10.47 (s, 1H), 7.40 (d, J = 4.0 Hz, 1H), 7.21

(d, J = 8.0 Hz, 1H), 7.02−6.94 (m, 2H), 6.05 (d, J = 8.0 Hz, 2H),

5.09 (s, 1H), 3.93−3.90 (m, 2H), 3.62−3.54 (m, 3H), 3.21−3.18 (m,

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1.04−1.02 (m, 3H). HPLC purity: 99.2% (t_R= 12.15 min). ESI-MS

m/z: [M + H]⁺= 453.1 (Calcd: 453.22).

acrylamide (D23). Prepared using the same procedure as that for

D24, D23 was a white solid (0.056 g, 57%). H NMR (400 MHz,

1

4-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)morpholine

(D18). Prepared using the same procedure as that for D8, D18 was a

DMSO-d₆) δ 10.59 (s, 1H), 7.60−7.47 (m, 2H), 7.41 (t, J = 4.5 Hz,

2H), 7.26 (d, J = 10.2 Hz, 2H), 7.21 (d, J = 3.7 Hz, 1H), 7.01−6.94

(m, 2H), 6.68 (d, J = 15.9 Hz, 1H), 3.52 (d, J = 6.3 Hz, 1H), 2.96−

2.88 (m, 2H), 2.61−2.53 (m, 1H), 2.38−2.28 (m, 2H), 1.23−1.15

(m, 6H), 1.05 (d, J = 8.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ

166.84, 163.49, 161.03 (d, J = 8.0 Hz), 143.41 (d, J = 8.0 Hz), 139.65,

136.33, 132.89 (d, J = 13.0 Hz), 132.10, 132.98 (d, J = 5.0 Hz),

132.05 (d, J = 10.0 Hz), 131.94 (d, J = 2.0 Hz), 131.51, 127.56,

125.56 (d, J = 12.0 Hz), 121.79 (d, J = 67.0 Hz), 119.13 (d, J = 32.0

Hz), 111.06, 110.80, 108.80, 97.14 (d, J = 166.0 Hz), 96.13, 56.97,

51.27, 26.98, 25.08 (d, J = 25.0 Hz), 24.66 (d, J = 24.2 Hz), 21.98 (d,

J = 4.0 Hz). HPLC purity: 96.8% (t_R= 12.96 min). ESI-MS m/z: [M

+ H]⁺= 442.2 (Calcd: 442.20).

Methyl 3′,5′-Diﬂuoro-4′-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-

methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-[1,1′-bi-

phenyl]-4-carboxylate. To a mixture of (1R,3R)-1-(4-bromo-2,6-

diﬂuorophenyl)-2-(2-ﬂuoro-2-methyl propyl)-3-methyl-2,3,4,9-tetra-

hydro-1H-pyrido[3,4-b]indole (22; 0.12 g, 0.265 mmol), Pd(dppf)Cl₂

(0.020 g, 0.026 mmol), and Na₂CO₃(2 M, 0.5 mL) in toluene (5

mL) was added (4-(methoxycarbonyl)phenyl)boronic acid (0.072 g,

0.4 mmol) under argon. The reaction was heated to 110 °C for 3 h.

After cooling to room temperature, the mixture was concentrated and

puriﬁed by silica gel column chromatography to give the desired ester

as a white solid (0.120 g, 45%). ESI-MS m/z: [M + H]⁺= 507.2

(Calcd: 507.22).

1

light yellow solid (0.045 g, 52%). H NMR (400 MHz, DMSO-d₆) δ

10.48 (s, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H),

7.02−6.95 (m, 2H), 6.59 (d, J = 8.0 Hz, 2H), 5.12 (s, 1H), 3.76−3.71

(m, 4H), 3.59−3.54 (m, 1H), 3.19−3.13 (m, 4H), 2.90−2.81 (m,

2H), 2.59−2.51 (m, 1H), 2.43−2.40 (m, 1H), 1.20−1.10 (m, 6H),

1.03 (d, J = 8.0 Hz, 3H). HPLC purity: 95.1% (t_R= 12.40 min). ESI-

MS m/z: [M + H]⁺= 458.3 (Calcd: 458.23).

1-(4-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-

methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-

piperazin-1-yl)prop-2-en-1-one (D19). Prepared from 1-(piperazin-

1-yl)prop-2-en-1-one, using the same procedure as that for D8, D19

1

as a white solid (0.020 g, 48%). H NMR (400 MHz, DMSO-d₆) δ

10.50 (s, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H),

7.00−6.94 (m, 2H), 6.86 (dd, J = 16.0 Hz, 10.5 Hz, 1H), 6.61 (d, J =

12.0 Hz, 1H), 6.16 (dd, J = 16.0 Hz, 2.5 Hz, 1H), 5.73 (dd, J = 10.0

Hz, 2.0 Hz, 1H), 5.12 (s, 1H), 3.70−3.66 (m, 4H), 3.56−3.53 (m,

1H), 3.26 (s, 4H), 2.91−2.81 (m, 2H), 2.58−2.57 (m, 1H), 2.43−

2.35 (m, 1H), 1.24−1.20 (m, 6H), 1.06 (d, J = 8.0 Hz, 3H). HPLC

purity: 97.0% (t_R= 11.87 min). ESI-MS m/z: [M + H]⁺= 511.2

(Calcd: 511.26).

4-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-

thiomorpholine 1,1-Dioxide (D20). Prepared using the same

procedure as that for D8, D20 was a white solid (0.030 g, 35%).

¹H NMR (400 MHz, DMSO-d₆) δ 10.50 (s, 1H), 7.41 (d, J = 8.0 Hz,

1H), 7.21 (d, J = 8.0 Hz, 1H), 7.03−6.95 (m, 2H), 6.71 (d, J = 7.5

Hz, 1H), 5.14 (s, 1H), 3.85 (s, 4H), 3.54−3.52 (m, 1H), 3.11−3.08

(m, 4H), 2.91−2.81 (m, 2H), 2.59−2.53 (m, 1H), 2.44−2.36 (m,

1H), 1.25−1.16 (m, 6H), 1.08 (d, J = 8.0 Hz, 3H). HPLC purity:

95.0% (t_R= 12.01 min). ESI-MS m/z: [M + H]⁺= 506.2 (Calcd:

506.20).

(1R,3R)-1-(4-(4,4-Diﬂuoropiperidin-1-yl)-2,6-diﬂuorophenyl)-2-

(2-ﬂuoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido-

[3,4-b]indole (D21). Prepared using the same procedure as that for

D8, D21 was a light yellow solid (0.080 g, 77%). ¹H NMR (400 MHz,

DMSO-d₆) δ 10.50 (s, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 8.0

Hz, 1H), 7.03−6.95 (m, 2H), 6.67 (d, J = 8.0 Hz, 2H), 5.13 (s, 1H),

3.85 (s, 4H), 3.54−3.53 (m, 1H), 3.43−3.42 (m, 4H), 2.91−2.81 (m,

2H), 2.58−2.53 (m, 1H), 2.40−2.35 (m, 1H), 1.26 (s, 3H), 1.20 (s,

3H), 1.16 (s, 3H). HPLC purity: 97.4% (t_R= 14.70 min). ESI-MS m/

z: [M + H]⁺= 492.3 (Calcd: 492.24).

(E)-3-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-

methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-N-

hydroxyacrylamide (D22). To a solution of hydroxylamine hydro-

chloride (1.0 g, 14.4 mmol) in MeOH (5 mL) was added a solution of

KOH (1.19 g, 21.15 mmol) in MeOH (2.5 mL) at 0 °C. The solution

was stirred at 0 °C for 30 min and ﬁltered to remove the white solid.

Methyl (E)-3-(3,5-diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-

3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-

acrylate (0.2 g, 0.43 mmol) was dissolved in the hydroxylamine

solution, stirred for 30 min, neutralized with acetic acid, and

concentrated and puriﬁed by silica gel column chromatography to

give D22 as a white solid (0.140 g, 70%). ¹H NMR (400 MHz,

DMSO-d₆) δ 10.82 (s, 1H), 10.59 (s, 1H), 9.18 (s, 1H), 7.43 (s, 1H),

7.41 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 12.0 Hz, 2H), 7.18 (d, J = 8.0

Hz, 1H), 7.02−6.95 (m, 2H), 6.53−6.49 (m, 1H), 5.21 (s, 1H),

3.52−3.50 (m, 1H), 2.92−2.83 (m, 2H), 2.60−2.55 (m, 1H), 2.39−

2.30 (m, 1H), 1.23−1.10 (dd, J = 16.0 Hz, 4.0 Hz, 6H), 1.05 (d, J =

8.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 164.29, 163.36, 160.85,

147.92, 138.48, 136.42 (d, J = 21.0 Hz), 131.59, 128.34 (d, J = 1.0

Hz), 127.42, 121.33, 118.68 (d, J = 102.0 Hz), 110.92, 108.58, 97.27

(d, J = 165.0 Hz), 77.36, 77.04, 76.72, 56.89, 51.27, 29.72, 26.72,

24.92 (d, J = 14.0 Hz), 24.67 (d, J = 14.0 Hz). HPLC purity: 95.8%

(t_R= 13.50 min). ESI-MS m/z: [M + H]⁺= 458.2 (Calcd: 458.20).

(E)-3-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-

methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-

3′,5′-Diﬂuoro-4′-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-[1,1′-biphenyl]-4-

carboxylic Acid (D24). Prepared using the same procedure for

hydrolysis aforementioned, D24 was a light yellow solid (0.085 g,

1

73%). H NMR (400 MHz, CDCl₃) δ 8.24 (s, 1H), 7.99 (d, J = 8.0

Hz, 2H), 7.78 (d, J = 8.0 Hz, 2H), 7.42−7.39 (m, 3H), 7.19 (d, J =

8.0 Hz, 2H), 7.02−6.93 (m, 2H), 5.25 (s, 1H), 3.48−3.45 (m, 1H),

2.88−2.79 (m, 2H), 2.58−2.51 (m, 1H), 2.42−2.36 (m, 1H), 1.18 (d,

J = 16.0 Hz, 3H), 1.14 (d, J = 16.0 Hz, 3H), 1.02 (d, J = 8.0 Hz, 3H).

¹³C NMR (101 MHz, CDCl₃) δ 171.36, 163.75 (d, J = 8.0 Hz),

161.25 (d, J = 9.0 Hz), 143.36, 141.85 (t, J = 10.0 Hz), 136.32,

131.61, 130.95, 129.32, 127.65, 126.93, 121.53, 118.82 (d, J = 110.0

Hz), 117.22 (d, J = 15.0 Hz), 110.78, 110.55 (d, J = 21.0 Hz), 109.02,

97.21 (d, J = 166.0 Hz), 57.00 (d, J = 21.0 Hz), 51.21, 26.97, 25.15

(d, J = 25.0 Hz), 24.72 (d, J = 25.0 Hz). ¹⁹F NMR (376 MHz,

CDCl₃) δ −110.23, −139.16 (hept). ESI-MS m/z: [M − H]⁻= 491.2

(Calcd: 491.20). HPLC purity: 98.2% (t_R= 9.88 min). HRMS m/z

calcd for C₂₉H₂₇F₃N₂O₂: 491.1952 [M − H]⁻(found: 491.1956).

Ethyl 5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)thiazole-2-

carboxylate (19). A solution of ethyl 5-bromothiazole-2-carboxylate

(4.68 g, 20 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxa-

borolane) (5.59 g, 22 mmol), potassium acetate (5.88 g, 60 mmol),

and Pd(dppf)Cl₂(1.4 g, 1.8 mmol) in dioxane (100 mL) was stirred

under argon at 80 °C for 12 h. After cooling to room temperature,

brine (120 mL) was added. The mixture was extracted with ethyl

acetate. The organic layers were combined, dried with anhydrous

sodium sulfate, ﬁltered, and concentrated and puriﬁed by silica gel

column chromatography to give the desired coupling product.

Ethyl 5-(3,5-Diﬂuoro-4-formylphenyl)thiazole-2-carboxylate

(20). A solution of ethyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)thiazole-2-carboxylate (1.45 g, 5.1 mmol), 4-bromo-2,6-diﬂuor-

obenzaldehyde (0.936 g, 4.26 mmol), Pd(dppf)Cl₂(0.062 g, 0.085

mmol), and Na₂CO₃(2 M, 4.3 mL) in toluene (10 mL) was stirred at

100 °C for 16 h. After cooling to room temperature, brine (120 mL)

was added. The mixture was extracted with ethyl acetate. The organic

layers were combined, dried with anhydrous sodium sulfate, ﬁltered,

and concentrated and puriﬁed by silica gel column chromatography to

give ethyl 5-(3,5-diﬂuoro-4-formylphenyl) thiazole-2-carboxylate 20

(1.24 g, 83%).

5-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)thiazole-2-

carboxylic Acid (D25). Prepared using the same procedure as that for

1

D24, D25 was a light yellow solid (0.044 g, 54% yield). H NMR

(400 MHz, CDCl₃) δ 10.62 (s, 1H), 8.60 (s, 1H), 7.57 (d, J = 8.0 Hz,

7589

https://doi.org/10.1021/acs.jmedchem.1c00280

J. Med. Chem. 2021, 64, 7575−7595

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1H), 7.45 (s, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H),

7.02−6.94 (m, 2H), 5.25 (s, 1H), 3.36−3.33 (m, 1H), 2.94−2.84 (m,

2H), 2.61−2.58 (m, 1H), 2.48−2.45 (m, 1H), 1.23−1.20 (m, 6H),

1.05 (d, J = 8.0 Hz, 3H). HPLC purity: 99.1% (t_R= 17.09 min). ESI-

MS m/z: [M + H]⁺= 500.0 (Calcd: 500.15).

5-((3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-meth-

yl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)amino)-

thiazole-2-carboxylic Acid (D26). Prepared using the same

procedure as that for B1, D26 was a light yellow solid (0.044 g,

54% yield). ¹H NMR (500 MHz, DMSO-d₆) δ 10.59 (s, 1H), 9.14 (s,

1H), 8.12 (s, 1H), 7.69 (d, J = 12.0 Hz, 2H), 7.42 (d, J = 12.0 Hz,

1H), 7.19 (d, J = 8.0 Hz, 1H), 7.03−6.93 (m, 2H), 5.23 (s, 1H),

3.56−3.50 (m, 1H), 2.95−2.88 (m, 2H), 2.62−2.56 (m, 1H), 2.44−

2.39 (m, 1H), 1.26−1.21 (m, 6H), 1.07−1.05 (m, 3H). HPLC purity:

96.8% (t_R= 13.11 min). ESI-MS m/z: [M + H]⁺= 515.0 (Calcd:

515.17).

5.28 (s, 1H), 3.92 (s, 3H), 3.56−3.52 (m, 1H), 2.94−2.90 (m, 2H),

2.62−2.60 (m, 1H), 2.51−2.48 (m, 1H), 1.24−1.21 (m, 6H), 1.09 (d,

J = 8.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.63, 160.48,

156.17, 139.38, 136.22, 132.57, 131.82, 130.35, 127.59, 122.90,

121.39, 118.74 (d, J = 107.0 Hz), 116.44, 112.96, 112.72, 112.55,

110.75, 108.85, 97.23 (d, J = 165.0 Hz), 96.13, 56.74 (d, J = 21.0 Hz),

55.73, 51.15 (d, J = 28.0 Hz), 26.89, 25.18 (d, J = 24.0 Hz), 24.64 (d,

J = 25.0 Hz). HPLC purity: 96.6% (t_R= 15.77 min). ESI-MS m/z: [M

+ H]⁺= 523.2 (Calcd: 523.21).

3′,5′-Diﬂuoro-4′-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3-methoxy-[1,1′-bi-

phenyl]-4-carboxylic Acid (D31). Prepared using the same procedure

as that for D24, D31 was a white solid (0.120 g, 88%). ¹H NMR (400

MHz, DMSO-d₆) δ 10.48 (s, 1H), 8.20 (d, J = 8.0 Hz, 2H), 7.68 (d, J

= 8.0 Hz, 2H), 7.57−7.51 (m, 3H), 7.25−7.18 (m, 3H), 5.73 (s, 1H),

3.88−3.86 (m, 1H), 3.26−3.23 (m, 1H), 3.07−3.00 (m, 1H), 2.72−

2.67 (m, 1H), 2.34−2.23 (m, 1H), 1.24−1.20 (m, 6H), 1.09 (d, J =

8.0 Hz, 3H). HPLC purity: 96.0% (t_R= 15.77 min). ESI-MS m/z: [M

+ H]⁺= 523.2 (Calcd: 523.21).

1-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-1H-pyrazole-

4-carboxylic Acid (D27). Prepared using the same procedure as that

1

for D9, D27 was a light yellow solid (0.057 g, 65% yield). H NMR

2-Chloro-3′,5′-diﬂuoro-4′-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-

3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-6-me-

thoxy-[1,1′-biphenyl]-4-carboxylic Acid (D32). Prepared using the

same procedure as that for D24, D32 was a white solid (0.078 g,

(300 MHz, DMSO-d₆) δ 10.65 (s, 1H), 9.14 (s, 1H), 8.34 (d, J = 8.0

Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 8.0 Hz, 2H), 7.43 (d, J

= 8.0 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H), 7.01−6.96 (m, 2H), 5.28 (s,

1H), 3.55 (s, 1H), 2.91−2.86 (m, 2H), 2.60−2.53 (m, 1H), 2.46−

2.38 (m, 1H), 1.23 (d, J = 20.0 Hz, 3H), 1.15 (d, J = 20.0 Hz, 3H),

1.07 (d, J = 8.0 Hz, 3H). HPLC purity: 96.0% (t_R= 14.67 min). ESI-

MS m/z: [M − H]⁻= 481.2 (Calcd: 481.19).

Methyl 6-(3,5-Diﬂuoro-4-formylphenyl)nicotinate. A solution of

methyl 6-bromonicotinate (0.400 g, 1.389 mmol), (3,5-diﬂuoro-4-

formylphenyl)boronic acid (0.284 g, 1.528 mmol), Na₂CO₃(2 M, 2

mL), and Pd(dppf)Cl₂(0.02 g. 0.027 mmol) in toluene (15 mL) was

heated at 110 °C under argon for 8 h. After cooling to room

temperature, brine (30 mL) was added. The mixture was extracted

with ethyl acetate. The organic layers were combined, dried with

anhydrous sodium sulfate, ﬁltered, and concentrated and puriﬁed by

silica gel column chromatography to give methyl 6-(3,5-diﬂuoro-4-

formylphenyl) nicotinate as a yellow solid (0.15 g, 39%).

1

76%). H NMR (400 MHz, DMSO-d₆) δ 10.42 (s, 1H), 8.01 (d, J =

8.0 Hz, 2H), 7.89 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 1H), 7.27

(s, 1H), 7.16 (d, J = 8.0 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.98−6.91

(m, 2H), 5.37 (s, 1H), 3.95 (s, 3H), 3.59−3.56 (m, 1H), 2.95−2.87

(m, 2H), 2.59−2.56 (m, 1H), 2.46−2.34 (m,1H), 1.24−1.20 (m,

6H), 1.09 (d, J = 8.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ

170.95, 164.38, 161.88, 159.88 (d, J = 8.0 Hz), 144.62, 141.67,

141.56, 136.10, 133.18, 130.77, 129.29, 127.90, 126.95, 120.97,

118.55 (d, J = 103.0 Hz), 117.07 (d, J = 12.0 Hz), 110.58, 107.93 (d, J

= 18.0 Hz), 105.36, 97.54 (d, J = 165.0 Hz), 57.27 (d, J = 24.0 Hz),

56.21, 51.49, 50.25, 27.39, 25.48 (d, J = 24.0 Hz), 24.43 (d, J = 25.0

Hz). HPLC purity: 97.1% (t_R= 11.13 min). ESI-MS m/z: [M + H]⁺

= 557.2 (Calcd: 557.17).

3′,5′-Diﬂuoro-4′-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-[1,1′-biphenyl]-4-

carboxamide (D33). A solution of methyl 3′,5′-diﬂuoro-4′-((1R,3R)-

2-(2-ﬂuoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido-

[3,4-b]indol-1-yl)-[1,1′-biphenyl]-4-carboxylate (0.10 g, 0.2 mmol) in

ammonia (ca. 7 mol/L, 20 mL) was stirred overnight at 65 °C. After

cooling to room temperature, the mixture was concentrated and

puriﬁed by silica gel column chromatography to give D33 as a white

6-(3,5-Diﬂuoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)nicotinic Acid

(D28). Prepared using the same procedure as that for D24, D28 was a

1

light yellow solid (0.031 g, 55%). H NMR (400 MHz, DMSO-d₆) δ

10.63 (s, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.61

(d, J = 8.0 Hz, 1H), 7.46−7.41 (m, 3H), 7.20 (d, J = 8.0 Hz, 1H),

7.02−6.94 (m, 2H), 5.28 (s, 1H), 3.56−3.52 (m, 1H), 2.94−2.90 (m,

2H), 2.62−2.60 (m, 1H), 2.50−2.45 (m, 1H), 1.21 (d, J = 12.0 Hz,

3H), 1.17 (d, J = 12.0 Hz, 3H), 1.09 (d, J = 12.0 Hz, 3H). HPLC

purity: 97.0% (t_R= 15.62 min). ESI-MS m/z: [M + H]⁺= 494.2

(Calcd: 494.20).

1

solid (0.023 g, 24%). H NMR (400 MHz, DMSO-d₆) δ 10.38 (s,

1H), 8.02 (s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H),

7.42−7.39 (m 2H), 7.39 (d, J = 8.0 Hz, 1H), 7.17 (d, J = 8.0 Hz, 1H),

7.00−6.91 (m, 2H), 5.64 (s, 1H), 3.94 (s, 3H), 3.76−3.73 (m, 1H),

3.13−3.09 (m, 1H), 3.03−2.95 (m, 1H), 2.69−2.64 (m, 1H), 2.34−

2.23 (m, 1H), 1.18 (d, J = 12.0 Hz, 3H), 1.13 (d, J = 12.0 Hz, 3H),

1.08−1.06 (m, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 167.73,

163.74 (d, J = 9.0 Hz), 161.26 (d, J = 8.0 Hz), 141.63 (d, J = 10.0

Hz), 140.17, 136.75, 134.59, 132.58, 128.67, 127.35, 126.97, 120.90,

118.33 (d, J = 64.0 Hz), 117.17, 111.42, 110.48 (d, J = 24.0 Hz),

107.16, 97.40 (d, J = 166.0 Hz), 56.73, 51.65, 51.09, 26.97, 25.16 (d, J

= 24.0 Hz), 13.85. HPLC purity: 97.1% (t_R= 14.91 min). ESI-MS m/

z: [M + H]⁺= 492.1 (Calcd: 492.22).

2-Chloro-3′,5′-diﬂuoro-4′-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-

3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-[1,1′-bi-

phenyl]-4-carboxylic Acid (D34). Prepared using the same procedure

as that for D24, D34 was a white solid (0.020 g, 76%). ¹H NMR (400

MHz, DMSO-d₆) δ 10.70 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.96 (d, J

= 8.0 Hz, 1H),, 7.59 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H),

7.23−7.21 (m, 3H), 7.01−6.95 (m, 2H), 5.32 (s, 1H), 3.54−3.50 (m,

1H), 2.95−2.85 (m, 2H), 2.63−2.59 (m, 1H), 2.46−2.37 (m, 1H),

1.25 (d, J = 20.0 Hz, 3H), 1.17 (d, J = 20.0 Hz, 3H), 1.09 (d, J = 8.0

Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.34, 162.90, 160.41,

141.45, 140.14, 136.74, 133.33, 132.35, 132.22, 131.84, 130.90,

128.70, 127.31, 120.93, 118.35 (d, J = 63.0 Hz), 117.86, 113.35 (d, J =

25.0 Hz), 111.45, 107.36, 97.38 (d, J = 166.0 Hz), 56.48, 56.26, 51.95,

3′,5′-Diﬂuoro-4′-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-[1,1′-biphenyl]-3-

carboxylic Acid (D29). Prepared using the same procedure as that for

1

D24, D29 was a light yellow solid (0.031 g, 55%). H NMR (400

MHz, DMSO-d₆) δ 10.69 (s, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.50 (d, J

= 8.0 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.25−7.20 (m, 3H), 7.01−

6.96 (m, 2H), 5.30 (s, 1H), 3.86 (s, 3H), 3.51−3.49 (m, 1H), 2.89−

2.78 (m, 2H), 2.62−2.51 (m, 1H), 2.45−2.36 (m, 1H), 1.23−1.15

(m, 6H), 1.05 (d, J = 8.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ

170.88, 163.72 (d, J = 9.0 Hz), 161.17, 142.07, 138.58, 136.29, 131.67

(d, J = 15.0 Hz), 130.75, 130.09, 129.22, 128.44, 127.65, 121.43,

119.28, 118.75 (d, J = 106.0 Hz), 110.44, 110.20, 108.82, 97.25 (d, J =

166.0 Hz), 96.13, 57.02 (d, J = 22.0 Hz), 51.13 (d, J = 15.0 Hz),

27.04, 25.23 (d, J = 24.0 Hz), 24.63 (d, J = 25.0 Hz). HPLC purity:

96.2% (t_R= 15.87 min). ESI-MS m/z: [M + H]⁺= 493.2 (Calcd:

493.20).

3′,5′-Diﬂuoro-4′-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-2-methoxy-[1,1′-bi-

phenyl]-4-carboxylic Acid (D30). Prepared using the same procedure

as that for D24, D30 was a white solid (0.049 g, 58%). ¹H NMR (400

MHz, DMSO-d₆) δ 10.62 (s, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.52 (d, J

= 12.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H),

7.20 (d, J = 8.0 Hz, 1H), 7.21−7.19 (m, 1H), 7.01−6.96 (m, 2H),

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51.03, 26.80, 25.12 (d, J = 24.0 Hz). HPLC purity: 98.9% (t_R= 16.81

min). ESI-MS m/z: [M + H]⁺= 527.3 (Calcd: 527.16).

6.98 (m, 4H), 5.13 (s, 1H), 3.79 (s, 3H), 3.52−3.49 (m, 1H), 2.91−

2.85 (m, 2H), 2.62−2.58 (m, 1H), 2.51−2.49 (m, 1H), 1.23 (d, J =

20.0 Hz, 3H), 1.15 (d, J = 20.0 Hz, 3H), 1.09 (d, J = 8.0 Hz, 3H).

HPLC purity: 97.5% (t_R= 16.84 min). ESI-MS m/z: [M + H]⁺=

506.2 (Calcd: 506.21).

3′,5′-Dichloro-4′-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3-methoxy-[1,1′-bi-

phenyl]-4-carboxylic Acid (D35). Prepared using the same procedure

as that for D24, D35 was a white solid (0.047 g, 72%). ¹H NMR (400

MHz, DMSO-d₆) δ 10.62 (s, 1H), 8.06 (s, 1H), 7.97 (d, J = 8.0 Hz,

2H), 7.86 (d, J = 8.0 Hz, 2H), 7.52−7.42 (m, 4H), 7.22−7.18 (m,

1H), 7.03−6.94 (m, 2H), 5.28 (s, 1H), 3.56−3.52 (m, 1H), 2.94−

2.89 (m, 2H), 2.63−2.58 (m, 1H), 2.46−2.37 (m, 1H), 1.26−1.19

(m, 6H), 1.08 (d, J = 8.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆)

δ 167.40, 159.12, 141.69, 141.08, 138.07 (d, J = 15.0 Hz), 136.87,

135.13, 131.91, 131.77, 129.25, 127.19, 126.76, 121.59, 120.76,

118.95, 118.28 (d, J = 60.0 Hz), 111.48, 111.27, 107.36, 97.48 (d, J =

165.0 Hz), 56.52, 55.69, 52.16 (d, J = 5.0 Hz), 27.98, 25.92 (d, J =

3′-Fluoro-4′-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-5′-methoxy-[1,1′-bi-

phenyl]-4-carboxylic Acid (D40). Prepared using the same procedure

as that for D24, D40 was a white solid (0.055 g, 71%). ¹H NMR (400

MHz, DMSO-d₆) δ 10.42 (s, 1H), 8.01 (d, J = 8.3 Hz, 2H), 7.88 (d, J

= 8.4 Hz, 2H), 7.39 (d, J = 7.3 Hz, 1H), 7.27 (s, 1H), 7.16 (d, J = 7.4

Hz, 1H), 7.07 (d, J = 11.5 Hz, 1H), 7.00−6.89 (m, 2H), 5.37 (s, 1H),

3.95 (s, 3H), 3.59 (d, J = 5.7 Hz, 1H), 2.99−2.82 (m, 2H), 2.57 (dd, J

= 14.9, 3.0 Hz, 1H), 2.42−2.31 (m, 1H), 1.27−1.21 (m, 6H), 1.02 (d,

J = 8.0 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 171.28, 164.39,

161.88, 159.94, 159.85, 144.88, 141.56, 136.10, 133.06, 130.83,

128.86, 127.87, 127.03, 121.03, 119.10, 118.04, 110.60, 108.11,

107.87, 106.72 (d, J = 265.0 Hz), 97.48 (d, J = 166.0 Hz), 57.22 (d, J

= 23.0 Hz), 56.22, 51.54, 50.34, 27.35, 25.47 (d, J = 24.0 Hz), 24.49

(d, J = 25.0 Hz). HPLC purity: 97.8% (t_R= 11.60 min). ESI-MS m/z:

[M + H]⁺= 505.3 (Calcd: 505.22).

24.0 Hz), 24.64 (d, J = 25.0 Hz), 9.99. HPLC purity: 97.5% (t_R

=

17.35 min). ESI-MS m/z: [M + H]⁺= 555.1 (Calcd: 555.15).

3′,5′-Dichloro-4′-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-[1,1′-biphenyl]-4-

carboxylic Acid (D36). Prepared using the same procedure as that for

1

D24, D36 was a white solid (0.061 g, 54%). H NMR (400 MHz,

DMSO-d₆) δ 10.49 (s, 1H), 7.77 (s, 1H), 7.64 (d, J = 8.0 Hz, 1H),

7.60 (s, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.41−7.35 (m, 2H), 7.18 (d, J

= 8.0 Hz, 1H), 7.00−6.92 (m, 2H), 5.38 (s, 1H), 3.91 (s, 3H), 3.70−

3.66 (m, 1H), 3.08−3.03 (m, 2H), 2.64−2.60 (m, 1H), 2.38−2.27

(m, 1H), 1.16 (d, J = 8.0 Hz, 3H), 1.08 (d, J = 8.0 Hz, 3H), 1.06−

1.04 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 142.56, 141.27,

136.25, 135.04, 130.94, 130.90, 129.64, 127.54, 126.95, 126.51,

121.37, 118.72 (d, J = 116.0 Hz), 110.74, 109.28, 97.28 (d, J = 166.0

Hz), 96.14, 55.58, 52.00 (d, J = 5.0 Hz), 27.76, 25.92 (d, J = 25.0 Hz),

24.34 (d, J = 24.0 Hz). HPLC purity: 97.3% (t_R= 17.41 min). ESI-

MS m/z: [M + H]⁺= 525.2 (Calcd: 525.14).

3′-Chloro-5′-ﬂuoro-4′-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-

methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3-methoxy-

[1,1′-biphenyl]-4-carboxylic Acid (D37). Prepared using the same

procedure as that for D24, D37 was a white solid (0.055 g, 81%). ¹H

NMR (400 MHz, DMSO-d₆) δ 10.51 (s, 1H), 8.01 (d, J = 8.0 Hz,

2H), 7.87 (d, J = 8.0 Hz, 2H), 7.74 (s, 1H), 7.59 (d, J = 8.0 Hz, 1H),

7.41 (d, J = 8.0 Hz, 1H), 7.18−7.15 (m, 1H), 6.99−6.94 (m, 2H),

5.38 (s, 1H), 3.70−3.66 (m, 1H), 3.09−2.98 (m, 2H), 2.64−2.60 (m,

1H), 2.37−2.26 (m, 1H), 1.14 (d, J = 8.0 Hz, 3H), 1.09 (d, J = 8.0

Hz, 3H), 1.08−1.05 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ

164.69, 158.37, 151.73, 144.82, 141.19, 136.26, 134.65, 131.22,

127.66, 124.59, 121.50, 120.80, 119.38, 118.26, 117.48, 110.39 (d, J =

60.0 Hz), 108.77, 97.05 (d, J = 166.0 Hz), 58.26, 58.03, 56.96, 52.00,

27.68, 25.54 (d, J = 25.0 Hz), 24.34 (d, J = 25.0 Hz). HPLC purity:

96.4% (t_R= 9.59 min). ESI-MS m/z: [M + H]⁺= 539.1 (Calcd:

539.18).

3′-Chloro-5′-ﬂuoro-4′-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-

methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-[1,1′-bi-

phenyl]-4-carboxylic Acid (D38). Prepared using the same procedure

as that for D24, D38 was a white solid (0.047 g, 85%).¹H NMR (400

MHz, DMSO-d₆) δ 10.65 (s, 1H), 9.14 (s, 1H), 8.34 (d, J = 8.0 Hz,

1H), 8.21 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 8.0 Hz, 2H), 7.43 (d, J =

8.0 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H), 7.01−6.96 (m, 2H), 5.28 (s,

1H), 3.55 (s, 1H), 2.91−2.86 (m, 2H), 2.60−2.53 (m, 1H), 2.46−

2.43 (m, 1H), 1.23 (d, J = 20.0 Hz, 3H), 1.15 (d, J = 20.0 Hz, 3H),

1.07 (d, J = 8.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.93,

164.29, 161.75, 142.89, 141.78 (d, J = 9 Hz), 137.02, 136.26, 131.41,

130.92, 129.56, 127.68, 126.93, 126.52 (d, J = 12.0 Hz), 124.54,

121.41, 118.77 (d, J = 108.0 Hz), 113.68 (d, J = 24.0 Hz), 110.73,

108.65, 97.24 (d, J = 165.0 Hz), 57.99 (d, J = 21.0 Hz), 53.18, 51.84

(d, J = 5.0 Hz), 27.71, 25.69 (d, J = 25.0 Hz), 24.22 (d, J = 23.8 Hz).

HPLC purity: 97.4% (t_R= 16.84 min). ESI-MS m/z: [M + H]⁺=

509.1 (Calcd: 509.17).

Cell Lines and Culture Conditions. MCF-7 cell line was purchased

from ATCC (ATCC No. HTB-22) and cultured following the

instructions. Brieﬂy, MCF-7 cell line was cultured in MEM media

(Eagle’s minimum essential medium, GIBCO, No. 11095-080)

containing 10% FBS (GIBCO, No. 10091-148), 1% penicillin−

streptomycin solution (GIBCO, No. 15140-122), 1% NEAA

(GIBCO, No. 11140-050), 1 mM sodium pyruvate (GIBCO, No.

11360-070), and 0.01 mg/mL insulin (Jiangsu Wanbang Biopharma-

ceutical Co. Ltd.) in an incubator (37 °C, 5% CO₂). Cells were

detached by 0.25% trypsin-EDTA (GIBCO No. 25200-056) when

reached to 80−90% conﬂuence for further culture or assays. For

immunoﬂuorescence staining of ERα and PR level, MCF-7 cell line

was cultured according to the protocol of Pharmaron Inc. The cells

were cultured in DMEM containing 10% hormone-free FBS and 1%

penicillin−streptomycin solution. The hormone-free FBS was

produced by incubating 50 mL of FBS with 1 g of activated carbon

for 24 h in 4 °C.

Cell Proliferation Assay. MCF-7 cell line was suspended at 2 × 10⁴

cells/mL, and 100 μL/well was plated in a 96-well plate. Then the

cells were incubated for 24 h at 37 °C and 5% CO₂under humidiﬁed

conditions. A 20 mM amount of solution of each compound was

prepared with DMSO, and the solution was diluted to a series of 2×

ﬁnal concentration with complete RPMI 1640 media. Then 100 μL of

the diluted solution was added to the well. After the cells were

incubated for 5 days, the medium was discarded and Celltiter-Glo

reagent was added to the well according to the instruction of the

manufacture. Finally, the luminescence was read on a plate reader and

the inhibition rate was calculated as %Inh = 100 − [(compd signal −

min signal)/(max signal − min signal) × 100].

ERα Binding Aﬃnity. ER binding assay was performed by

Pharmaron Inc. Brieﬂy, the compound was diluted with DMSO

(ﬁnal concentration is 0.3%) and added to the assay plate by Acho

instrument. ERα and Fluormone ES2 were diluted with ES2 screening

buﬀer, and the ﬁnal concentrations were 150 and 4.5 nM, respectively.

Then 20 μL of each solution was added to the plate and mixed well.

The plate was centrifuged 1000 rpm at room temperature for 1 min

and incubated for 60 min in dark. Finally, the ﬂuorescence

polarization value (mP) was read by Envision.

ERα Degradation Activity. ERα was measured by immunoﬂuor-

escent staining, which was conducted by Pharmaron Inc. Brieﬂy,

MCF-7 cells were suspended at 1500 cells/40 and 40 μL was

inoculated in each well of a 384-well plate. After the cells were

equilibrated at room temperature for 20 min followed by incubation

for 24 h (37 °C, 5% CO₂), the compound was diluted with DMSO

(ﬁnal concentration is 0.3%) and added to the assay plate by Acho

instrument. Then the cells were washed with PBS and ﬁxed by 3.7%

paraformaldehyde after incubation for 24 h. Then the cells were

washed two times with PBS and penetrated with 0.5% Tween-20 for 1

h at room temperature, followed by being washed two times with

6-(3-Fluoro-4-((1R,3R)-2-(2-ﬂuoro-2-methylpropyl)-3-methyl-

2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-5-methoxyphenyl)-

nicotinic Acid (D39). Prepared using the same procedure as that for

1

D24, D39 was a white solid (0.033 g, 65%). H NMR (400 MHz,

DMSO-d₆) δ 10.77 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 8.0

Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.02−

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PBST. The primary antibody of anti-ERα (CST No. 8644S) was

diluted to 1:1000 and 1:250 in 1% milk in PBST, respectively. After

incubation with primary antibody for 1.5 h in room temperature, the

cells were washed two times with PBST and incubated with diluted

secondary antibody (1:1000 in PBST) for 45 min at room

temperature. Finally, the cells were washed three times with PBST

and two times with PBS and the cells were scanned with Acumen.

Western Blot Analysis. Cells were lysed in RIPA lysis buﬀer

(Beyotime) with protease inhibitors (Solarbio) and proteins run on

4−12% precast gels (GenScript). Membranes were probed overnight

with primary antibodies (anti-ERα, CST No. 8644; anti-PR, CST No.

8757; anti-GAPDH, CST No. 5174) followed by incubation with

HRP-tagged secondary antibodies (CST No. 7074) and visualized

with Chemidoc digital imager (Bio-RAD).

In Vivo Eﬃcacy in the MCF-7 Breast Cancer Xenograft Model.

Female BALB/C nude mice (6 to 8 weeks old) in this study were

purchased from Shanghai Sippe-Bk Lab Animal Co., Ltd. and were

housed under standard speciﬁc-pathogen-free (SPF) conditions. All of

the procedures and protocols were approved by The Institutional

Animal Care and Use Committee (IACUC) of Nanjing University of

Chinese Medicine and in accordance with the National Institutes of

Health Guide for the Care and Use of Laboratory Animals. A well-

established tumorigenesis assay was used to evaluate the antitumor

eﬀect of compounds AZD9496 and D24 in female BALB/C nude

mice model. A 1 × 10⁷amount of MCF-7 cells in 50% Matrigel was

subcutaneously injected in the right hind leg of the mouse. When

tumors reached ∼170 mm³, the mice were randomly divided to 6

mice per group and assigned to compound or vehicle treatment. Six

cohorts of the mice were orally treated (QD) with AZD9496 at the

doses of 0.6 and 6 mg/kg, with D24 at the doses of 0.6, 2, and 0.6

mg/kg, and with vehicle, respectively. Mice were examined thrice a

week for the development of tumors by electronic calipers, and tumor

volume was calculated as V = 0.5 × length × width².

In Vivo Pharmacokinetic Assessment in the MCF-7 Breast

Cancer Xenograft Model. This study was conducted in BALB/C

female subjects to investigate the pharmacokinetic proﬁles of test

compounds. The mice supplied by Changzhou Cavensle Laboratory

Animal Co. Ltd. were randomized and divided into two groups

consisting of 3 mice/group. The body weights were in the range from

17 to 24 g. Prepared test compounds in solutol−physiological saline

(2:98) to make 0.1 mg/mL solutions for oral gavage and for

intravenous injection. Sample collection was performed as follows:

(1) single oral administration (p.o.) group, 2 mg/kg and 0.2 mL/10g;

(2) single tail vein injection (i.v.) group, 1 mg/kg and 0.1 mL/10g.

Blood was collected from orbital venous plexus in a heparinized EP

tube at 5, 15, and 30 min, 1, 2, 6, 10, and 24 h after intravenous or

oral administration, and the contents of the blood were analyzed by

LC-MS/MS (API 4500).

Molecular Docking. The binding mode of the ligand and protein

was simulated by the Glide-Dock module of the Schrodinger Maestro

2019. The crystal structure of ERα (PDB ID 5ACC) was obtained

from the PDB; the protein was prepared by the Protein Preparation

module in Schrodinger Maestro 2019. Waters were eliminated from

the protein, and polar hydrogen was added. Meanwhile, the

protonation states of all residues had been considered to involve

His being protonated to HID, and the Asp, Arg, Glu, and Lys residues

were preprocessed to deprotonated states and so on. Receptor grids

were generated using Receptor Grid Generation; the generated

binding site was just the binding pocket of ERα, including several key

amino acid residues. The three-dimensional structure of ligand was

constructed using ChemBio 3D Ultra software; then, it was

energetically minimized by using MMFF94 with 5000 iterations and

a minimum RMS gradient of 0.05. Furthermore, the ligands were also

prepared by the Ligand Prepare module of Schrodinger Maestro 2019.

Then, the ligands were docked into a rigid protein using Glide SP; the

docking score was set to glide score, and the number of returned

poses per ligand in each docking stage was set to 10. The other

docking parameters were left at the default settings.

Maestro 2019. The protein and ligand were assigned the OPLS3 force

ﬁeld; the protonation states of charged residues were also

preprocessed: The His residues were protonated to HID, and the

Asp, Arg, Glu, and Lys residues were preprocessed to deprotonated

states. Simulation procedure and parameter settings of the MD

simulations were similar to the previous works.⁵¹The model was

solvated in a truncated octahedron box of the transferable interaction

potential (TIP3P) water molecules with a margin distance of 10 Å.

Sodium ions were added to neutralize the systems. The particle mesh

Ewald (PME) method was used for long-range electrostatic

interactions with a cutoﬀ distance of 10 Å. Next, the entire setup

was subjected to a stepwise equilibration procedure: The ﬁrst

minimization with 2500 steps of steepest descent and 2500 steps of

conjugate gradient was performed with the protein, ligand ﬁxed by

10.0 kcal/(mol Å²). Then, 5000 steps of steepest descent and 15000

steps of the conjugate gradient were employed in the whole system

with the constraints switched oﬀ. After energy minimization, the

whole system was gradually heated from 0 to 300 K for 60 ps under

NVT, followed by a 600 ps NPT simulation at 1 atm, with harmonic

restraints of 2 kcal/(mol Å²) on the complex. Subsequently, 100 ns

MD simulations were performed in NPT ensemble for each system.

The temperature and pressure were kept constant using a Langevin

thermostat and a Langevin barostat, respectively. The time step is set

at 2 fs, and the hydrogen atoms were constrained by the SHAKE

algorithm. The resulting root-mean-square derivation (RMSD), root-

mean-square ﬂuctuation (RMSF), the hydrogen bonding, and the

hydrophobic interaction were analyzed throughout the trajectory

using Simulation Interactions Diagram module in Schrodinger

Maestro 2019. The hydrogen-bond and hydrophobic contributions

of each residue also could be found; thus, the hot-spot residues

interacting with inhibitors could be identiﬁed.

Binding Free Energy Calculation. Subsequently, the binding free

energies based on the protein−ligand complex generated by the MD

procedure were calculated by using molecular mechanics−generalized

Born surface area module in Schrodinger Maestro 2019. The binding

free energy was calculated as follows: ΔG_bind= G_complex− (G_protein

_ligand) = ΔH − TΔS = ΔE_mm+ ΔE_sol− TΔS = (ΔE_VDW+ ΔE_ele

ΔE_internal) + (ΔE_GA+ ΔE_GB) − TΔS.

+

G

The ΔG_bindincludes the enthalpic contribution (ΔH) and entropic

term (−TΔS). ΔH is the sum of intermolecular energy (ΔE_mm) and

solvation free energy (ΔE_sol). ΔE_mmrepresents the complex gas-phase

interaction energy composed of the electrostatic interaction energy

(ΔE_ele), van der Waals energy (ΔE_vdw), and internal energy

(ΔE_internal). The solvation-free energy (ΔE_sol) consists of the

electrostatic solvation energy (ΔE_GB) and the nonpolar contributions

(ΔE_GA). The former was calculated by the generalized Born (GB)

continuum model, and the latter was estimated by the solvent

accessible surface area (SASA) approach. An ensemble of 1000

snapshots extracted from the last 10 ns of MD trajectory was used to

calculate the binding free energy. The entropy was determined by

normal mode calculations with the MM-GBSA module by selecting

100 of the 1000 snapshots used in the free energy calculations at

regular intervals. The convergence criterion for the energy gradient to

stop minimization was 0.5. In total, 1000 frames were used for each

MM-GBSA calculation, while 100 frames were used for each normal

mode analysis. All other parameters were deﬁned as default values.

Alanine Mutation of Hot-Spot Residue. In computational alanine

scanning approach to protein−ligand binding, one mutates a speciﬁc

residue to alanine and computes the diﬀerence in binding free

energies before and after the mutation. The hot-spot residues were

mutated to alanine by Residue Mutation module in Schrodinger

Maestro 2019; each mutated protein was saved as a separate protein

receptor and complex. The complex was prepared by the Protein

Preparation module once again, and the MD simulation and binding

free energy calculations were carried out as mentioned above. The

change in free energy between the original and mutated complexes

(ΔΔG) is calculated as ΔΔG = ΔG_mut− ΔG_wt, where ΔG_mutis the

complex with a single alanine mutation and ΔG_wtis the wild-type

complex. A negative ΔΔG suggests that the mutation stabilizes the

complex, while a positive value suggests that the mutation destabilizes

Molecular Dynamic Simulation. The molecular dynamics

simulation (100 ns) was performed using Desmond in Schrodinger

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it.⁵²A residue was considered as a hot-spot residue if ΔΔG ≥ 1.0

12; LBD, ligand binding region; i.v., intravenous adminis-

tration; MM-GBSA, molecular mechanics−generalized Born

surface area; MTD, maximum tolerated dose; p.o., oral

administration; PR, progesterone receptor; RT, room temper-

ature; SAR, structure−activity relationship; SERDs, selective

estrogen receptor degraders; SERMs, selective estrogen

receptor modulators; t_1/2, half-life; TGI, tumor growth

inhibition; V_d, volume of distribution;

kcal/mol.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00280.

■

sı

Table S1 listing the UPLC-UV/Q-TOF MS analysis

results of compound D24 metabolites, Table S2 listing

compound D24 toxicity data, Figure S1 showing D24

putative metabolic pathway in liver cells; spectra for the

target compounds (PDF)

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Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Authors

Liwen Zhao − R&D Center, Nanjing Sanhome

Pharmaceutical Company Ltd., Nanjing 211135, China;

Email: Zhaolw@sanhome.com

■

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Wei Li − School of Pharmacy, Nanjing University of Chinese

Medicine, Nanjing 210023, China; Email: liwaii@

njucm.edu.cn

Jian Liu − School of Pharmacy, Nanjing University of Chinese

Medicine, Nanjing 210023, China; orcid.org/0000-0001-

9482-9036; Email: liujian623@njucm.edu.cn

Authors

Xiaomeng Zhang − School of Pharmacy, Nanjing University

of Chinese Medicine, Nanjing 210023, China; R&D Center,

Nanjing Sanhome Pharmaceutical Company Ltd., Nanjing

211135, China

Yazhou Wang − R&D Center, Nanjing Sanhome

Pharmaceutical Company Ltd., Nanjing 211135, China

Xue Li − R&D Center, Nanjing Sanhome Pharmaceutical

Company Ltd., Nanjing 211135, China

Jie Wu − R&D Center, Nanjing Sanhome Pharmaceutical

Company Ltd., Nanjing 211135, China

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00280

Author Contributions

^§X.Z. and Y.W. contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We gratefully acknowledge the National Science Foundation of

China (Grant Nos. 81573304 and 81703342), Natural Science

Foundation of Nanjing University of Chinese Medicine (Grant

No. NZY81703342), Natural Science Foundation of Jiangsu

(Grant No. BK20161050), and Postgraduate Research &

Practice Innovation Program of Jiangsu Province (Grant No.

KYCX19_1261).

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ABBREVIATIONS USED

■

ADMET, absorption, distribution, metabolism, excretion, and

toxicity; AF, activating function; AIs, aromatase inhibitors;

AUC, area under the curve; BC, breast cancer; Cl, clearance;

C_max, maximum concentration; CYP, cytochrome P450; ER,

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Article Doi

Dynamics-Based Discovery of Novel, Potent Benzoic Acid Derivatives as Orally Bioavailable Selective Estrogen Receptor Degraders for ERα+ Breast Cancer

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