Discovery and Characterization of Benzimidazole Derivative XY123 as a Potent, Selective, and Orally Available RORγ Inverse Agonist

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Discovery and Characterization of Benzimidazole Derivative XY123

as a Potent, Selective, and Orally Available RORγ Inverse Agonist

Xishan Wu,^#Hui Shen,^#Yan Zhang,^#Chao Wang, Qiu Li, Cheng Zhang, Xiaoxi Zhuang, Chenchang Li,

Yudan Shi, Yanli Xing, Qiuping Xiang, Jinxin Xu, Donghai Wu, Jinsong Liu, and Yong Xu *

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ABSTRACT: Receptor-related orphan receptor γ (RORγ) has emerged as an

attractive therapeutic target for the treatment of cancer and inﬂammatory diseases.

Herein, we report our eﬀort on the discovery, optimization, and evaluation of

benzothiazole and benzimidazole derivatives as novel inverse agonists of RORγ.

The representative compound 27h (designated as XY123) potently inhibited the

RORγ transcription activity with a half-maximal inhibitory concentration (IC₅₀)

value of 64 nM and showed excellent selectivity against other nuclear receptors.

27h also potently suppressed cell proliferation, colony formation, and the

expression of androgen receptor (AR)-regulated genes in AR-positive prostate

cancer cell lines. In addition, 27h demonstrated good metabolic stability and a

pharmacokinetic property with reasonable oral bioavailability (32.41%) and

moderate half-life (t_1/2= 4.98 h). Signiﬁcantly, oral administration of compound

27h achieved complete and long-lasting tumor regression in the 22Rv1 xenograft

tumor model in mice. Compound 27h may serve as a new valuable lead compound for further development of drugs for the

treatment of prostate cancer.

treatment of inﬂammatory diseases.¹³⁻¹⁵Consequently, RORγ

1. INTRODUCTION

inverse agonists covering a large diversity of chemical structures

Prostate cancer (PC) has long been the most common solid

cancer and remains the ﬁfth leading cause of cancer-related

have been described by many research groups (Figure 1).¹⁶⁻²²

T0901317, originally reported by Tularik as a potent liver X

death in men worldwide.¹⁻³Since Huggins and Hodges’s

receptor (LXR) agonist, was identiﬁed later as a RORγ inverse

discovery in the early 1940s that androgens promote PC growth,

agonist by Scripps Florida.^23,24Further eﬀorts led to the

androgen-deprivation therapy (ADT) is the standard treatment

discovery of selective RORγ inverse agonist SR2211 (1).²⁵

for prostate cancer.⁴However, after the initial response, the

Other potent and selective RORγ inverse agonists were later

disease eventually progresses to castration-resistant prostate

disclosed, including GSK805 (2)²⁶and BMS-986313 (3).²²

cancer (CRPC) within 2−3 years. The abnormal activation of

Among them, some compounds such as ABBV-553, ESR114,

the androgen receptor (AR) pathway is the major driver for

AZD-0284 (4), AUR-101, and JTE-451 have advanced into

progression to CRPC, including overexpression of the AR gene,

clinical studies.²⁷⁻³¹Currently, JTE-451 is the most advanced

AR mutation, AR splice variants, and others.⁵⁻⁷Clinically,

compound in clinical development, and the phase II clinical trial

abiraterone and enzalutamide, the second-line agents targeting

of JET-451 in plaque psoriasis had completed in 2020.

the AR pathway, have been widely used as novel therapies for the

Previously, we found that RORγ is key regulator of AR-, ERG-,

treatment of metastatic CRPC. Despite the success of these

and c-Myc-mediated transcription in CRPC, and targeting

second-generation AR-targeted therapies, acquired or inherent

RORγ represents an alternative strategy for the treatment of

resistance mechanisms lead to disease recurrence and ultimately

CRPC.^32,33RORγ inverse agonists XY018 and XY101 (5, Figure

death.⁸⁻¹²Clinically, there is an urgent and unmet need for a

1) could eﬀectively reduce the expression of full-length AR and

new and more eﬀective CRPC treatment strategy that can

AR splice variants, induce downregulation of AR-regulated

overcome aberrant AR signaling and improve patient prognosis.

Retinoic acid receptor-related orphan receptor γ (RORγ) is a

ligand-dependent transcriptional factor that belongs to the

nuclear receptor (NR) superfamily. RORγ has been shown to

Received: April 27, 2021

Published: June 14, 2021

regulate the diﬀerentiation of Th17 cells and the secretion of

inﬂammatory cytokines such as interleukin 17 (IL-17). Given its

crucial role in multiple inﬂammatory pathways, RORγ has

attracted signiﬁcant attention as a molecular target for the

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Figure 1. Structures of representative RORγ inverse agonists.

genes, and inhibit tumor growth in CRPC xenograft mouse

models. However, reports on the eﬃcacy in CRPC of the

pharmacologic inhibition by RORγ inverse agonists are still

limited. Thus, potent and selective RORγ inverse agonists with

diﬀerent chemotypes are still urgently needed to explore the

therapeutic space of RORγ inhibition in CRPC.

In this study, we report the structure-based virtual screening,

optimization, and biological evaluation of a new class of potent,

speciﬁc, and bioavailable small-molecular RORγ inverse

agonists, the benzimidazole derivatives, for the treatment of

prostate cancer.

2. RESULTS AND DISCUSSION

2.1. Discovery and Validation of 4-(Benzo[d]thiazol-2-

yl)aniline Scaﬀold by Virtual Screening. In the present

work, we used a common virtual screening strategy (Figure 2) to

obtain a new scaﬀold of RORγ inverse agonists.^34,35The

MayBridge database containing approximately 70 000 com-

pounds was screened by molecular docking. In the molecular

docking screening approach, compounds were docked into the

ligand-binding pocket of RORγ (PDB: 4QM0) and evaluated by

a standard precision (SP) docking score. A cutoﬀ of −9.0 kcal/

mol was used as the ﬁrst round ﬁlter. After cluster analysis and

visual inspection, 12 structurally diverse compounds were

selected and purchased for activity assessment (Figure S1 and

Table S1, Supporting Information).

A luciferase reporter assay and a thermal shift assay (TSA)

were initially used to evaluate the activity of compounds. In the

presence of compounds, the luciferase reporter assay can test the

activity of compounds on RORγ transcription, and the thermal

shift assay can determine the thermodynamic stability of the

RORγ-ligand-binding domain (LBD). Using the thermal shift

assay, 2 of the 12 compounds showed a stabilized eﬀect for the

RORγ protein with a temperature shift of more than 1.0 °C

(Table S1, Supporting Information). The luciferase assay further

demonstrated that only compound 6 (Figure 1) showed low

micromolar level activity with a half-maximal inhibitory

concentration (IC₅₀) value of 3.93 μM. To investigate the

protein−ligand interaction, we predicted the binding mode of 6

bound to RORγ-LBD (PDB code: 4QM0) by molecular

docking (Figure 3A). As shown in Figure 3A, the NH of the

sulfonamide group forms H-bonding interaction with the

backbone carbonyl of Phe377. One of the oxygen atoms of the

sulfonamide group forms indirect H-bonding interactions with

Figure 2. Schematic representation of the virtual screening strategy

used for RORγ inverse agonist discovery.

residues Gln286 and His323 via a water molecule. Moreover, the

middle phenyl ring forms face-to-edge π−π interactions with

Phe378. The p-methyl phenyl group attached to the sulfonamide

group extends toward the Arg367 pocket, which forms van der

Waals interaction with the hydrophobic residues of the central

portion of the pocket. The 6-methylbenzo[d]thiazole moiety

accessed a lipophilic pocket surrounded by residues Leu324,

Ile400, and His479. From the predicted binding mode analysis

by molecular docking, we can see that the 4-(6-methylbenzo-

[d]thiazol-2-yl)aniline moiety ﬁts snugly into the hydrophobic

LBD. Therefore, compound 6 bearing 4-(benzo[d]thiazol-2-

yl)aniline scaﬀold was chosen as the starting point for further

structural optimization.

To develop compounds with improved potency for RORγ, we

focused on three regions to perform an extensive structure−

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Figure 3. Three-dimensional (3D) presentation of predicted binding modes of ligands with RORγ-LBD (PDB code: 4QM0). (A) Predicted binding

mode of compound 6 (blue) in complex with RORγ-LBD. (B) Predicted binding mode of compound 11a (green) in complex with RORγ-LBD. Water

molecules are shown as spheres in red. H-bonding and π−π interactions are shown as yellow and green dashed lines, respectively. Figures were

prepared using PyMOL.

a

Scheme 1. Synthesis of Benzothiazole Derivatives 10a−l, 10n−q, 11a, and 11b

a

Reagents and conditions: (a) KOH, H₂O, reﬂux, overnight, 80%; (b) polyphosphoric acid, 4-aminobenzoic acid, 220 °C, 4 h, 80%; (c) R₁SO₂Cl,

pyridine, 80 °C, overnight, 20−50%; (d) R₁COOH, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexaﬂuor-

ophosphate (HATU), N,N-diisopropylethylamine (DIPEA), dichloromethane (DCM), room temperature (rt), overnight, 20−50%.

activity relationship (SAR) study to enhance the protein−ligand

interactions. The ﬁrst region is the polar region lined by Arg367

and Leu287. The second region is the lipophilic pocket

surrounded by Val376, Phe388, Ile400, and Phe401. The deep

part of this region is a little polar due to the presence of the

Ser404 side chain. The third region is the area near helices 11

and 12. The H-bond between His479 (on helix 11) and Tyr502

(on helix 12) is important for the active conformation of RORγ.

2.2. Chemistry. The derivatives of benzothiazole and

benzimidazole were designed and synthesized as shown in

Schemes 1 and 2. In Scheme 1, commercially available 2-amino-

6-methylbenzothiazole (7) was used to prepare the 2-amino-5-

methybenzenethiol (8), which was cyclized with 4-amino-

benzoic acid at 220 °C to yield the 4-(6-methylbenzo[d]thiazol-

2-yl)aniline (9). Compound 9 was treated with the appropriate

sulfonyl chlorides or carboxylic acids to generate ﬁnal

sulfonamides 10a−l, 10n−q, and amides 11a, 11b. Carboxylic

ester analogue 10l was hydrolyzed under basic conditions to

yield the corresponding carboxylic acid (10m).

benzaldehyde and yielded compounds 19a−p, 20a−h, 20j−l,

and 21a−h. Subsequently, the corresponding nitro groups were

reduced to anilines 22a−p, 23a−h, 23j−l, and 24a−h, followed

by coupling with 2-(4-(ethylsulfonyl)phenyl)acetic acid to give

ﬁnal products 25a−p, 26a−h, 26j−l, and 27a−h. Compound

26i was prepared from compound 26h by hydrolysis of

carboxylic ester.

2.3. SAR Studies of the Benzothiazole Derivatives. To

ﬁnd more potent benzothiazole derivatives, we designed various

substituents pointing to the polar region around Arg367 with a

sulfonamide linker to explore the chemical space (Table 1).

Based on the predicted binding mode of 6 with RORγ-LBD, 4-

methyl on the phenyl was close to Arg367. First, the substituents

at the 4-position of the aryl of 6 were preferentially evaluated.

Introduction of a tert-butyl moiety (10a) resulted in a signiﬁcant

loss of activity. Replacement of the methyl group (6) with

ﬂuorine (10b) and triﬂuoromethoxy (10c) led to a slight

decrease of activity. The meta and ortho positions of the aryl

group were also explored. The meta-substituent (10d) and

ortho-substituent (10e) nitro group were detrimental to RORγ

inhibitory activity. Introduction of a meta-position methyl-

sulfonyl group (10f) led to an approximately 3-fold loss in

potency compared to the initial hit. Compound 10g bearing a

In Scheme 2, commercial compounds 12a−g were reacted

with diﬀerent amines to give the amine-substituted nitrobenzene

intermediates 13a−p, 14a−h, 14j−l, and 15a−h. Reduction of

the nitro groups led to the corresponding anilines 16a−p, 17a−

h, 17j−l, and 18a−h, which was cyclized with 4-nitro-

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a

Scheme 2. Synthesis of Benzimidazole Derivatives 25a−p, 26a−h, 26j−l, and 27a−h

a

Reagents and conditions: (a) R₂NH₂, DIPEA, dimethyl sulfoxide (DMSO), 95 °C, overnight, 80−90%; or R₂NH₂, K₂CO₃, dimethylformamide

(DMF), 95 °C, 1 h, 70%; (b) Fe, AcOH, NH₄Cl, H₂O, 80 °C, 1 h, 65−95%; (c) 4-nitrobenzaldehyde, oxone, DMF, rt, 1 h, 35−90%; (d) 2-(4-

(ethylsulfonyl)phenyl)acetic acid, HATU, DIPEA, DCM, rt, overnight, 25−75%; or 2-(4-(ethylsulfonyl)phenyl)acetic acid, hydroxybenzotriazole

(HOBT), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), DIPEA, DCM, rt, overnight, 10−70%.

2,4-diﬂuorophenyl group exhibited a slight increase of activity

compared to 10f.

the carbamidine of Arg367 and backbone NH of Leu287. The

carbonyl oxygen atom of the amide interacts with residues

Gln286 and His323 through indirect H-bonds via a water

molecule. This demonstrates that formation of a H-bond with

Arg367 through ethylsulfonyl group is more critical for activity

improvement.

Further SAR investigation focused on various substituents on

the benzyl ring system to investigate the eﬀect of the position

and property of substitution through relaxing the conformation

rigidity. First, a series of compounds with diverse polar groups at

the 4-position were developed and evaluated (Table 1).

Disappointingly, introduction of the ﬂuorine (10h) and

triﬂuoromethyl (10i) groups led to a complete loss of activity

in the luciferase assay. When nitro (10j) and methylsulfonyl

(10k) groups were introduced, compounds showed weak

activity. The methyl ester analogue (10l) and its hydrolysis

product (10m) were not active. Compound 10n, bearing an

ethyl ester group, showed slightly improved potency compared

to 6. We also explored the eﬀects of substitutions at the meta or

ortho position of the benzyl group. However, these compounds

(10o−q) showed a signiﬁcant loss of inhibition. Overall, the

substituents pointing to the polar region with sulfonamide

linkers near the polar region did not improve the potency. To

obtain more potent compounds, we further explore the chemical

space with an amide linker. Signiﬁcantly, the ethylsulfonyl

derivative 11a displayed a largely increased potency with a

thermal shift of 8.6 °C and an IC₅₀value of 0.95 μM. When

ortho-substituent (11b) nitro was introduced, a sharp decrease

in the activity was found.

2.4. SAR Studies of the Benzimidazole Derivatives. To

further improve the potency of compounds, we next explored

the SAR at the lipophilic pocket lined with Val376, Phe388,

Ile400, and Phe401. From the predicted binding mode of 11a

and RORγ-LBD, we could ﬁnd that there is still some space near

the lipophilic pocket for further optimization. As shown in Table

2, we replaced the benzothiazole moiety in 11a with

benzimidazole and designed various hydrophobic and ﬂexible

substituents at the R₂position to explore the chemical space for

activity improvement. When the alkyl groups (25a−f) were

attached at the R₂position, changing the chain from propyl to

pentyl, a signiﬁcant increase in inhibition was found. Among

them, compound 25c stood out with nanomolar potency in the

luciferase assay with an IC₅₀of 4 nM, approximately 240-fold

more potent than 11a, and a thermal shift of 11.8 °C. When the

substituents were cyclobutyl or cyclohexyl groups, the resulting

compounds (25g−i) exhibited encouraging activities with IC₅₀

values ranging from 0.11 to 0.19 μM. Most of the compounds

showed a maximum inhibitory rate of approximately 90%. The

molecular docking study (Figure 4A) suggested that the n-pentyl

group of 25c occupies the lipophilic pocket and forms extensive

van der Waals interactions with the hydrophobic residues

Val376, Phe388, Ile400, and Phe401. The rest of 25c adopts a

similar binding mode to that of 11a.

Analysis of the predicted binding mode of compound 11a

(Figure 3B) in complex with RORγ-LBD suggested that the 4-

(6-methylbenzo[d]thiazol-2-yl)aniline scaﬀold in 11a adopted

the same binding mode as that of 6. As expected, the benzyl

moiety of 11a extends to the Arg367 pocket and the two oxygen

atoms of ethylsulfonyl group form H-bonding interactions with

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c

Table 1. Structure−Activity Relationship of the R₁Substitution on Benzothiazole Derivatives

a

b

c

IC₅₀values were calculated from the luciferase assay. ΔT_mvalues were calculated from the thermal shift assay. All assay results are reported as

mean standard deviation (SD) from at least two independent experiments.

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with 25k. To better understand the inﬂuence of the

conﬁguration of the methyl position on potency, we also

synthesized the enantiomers of compound 25n, including the

(R)-enantiomer 25o and (S)-enantiomer 25p. 25o and 25p

demonstrated excellent inhibitory activity with IC₅₀values of 9

and 11 nM, respectively. The TSA also demonstrated that both

25o and 25p stabilized the RORγ-LBD with temperature shifts

of 16 and 12 °C, respectively. As shown in Figure 4B, the

predicted binding mode of compound 25p was similar to that of

compound 25c. The benzyl moiety of 25p also binds to and

forms extensive hydrophobic interactions with the hydrophobic

pocket formed by residues Val376, Phe388, Ile400, and Phe401.

Overall, the results demonstrated that the modiﬁcation at the R₂

position with hydrophobic substituents could improve the

potency.

Table 2. Structure−Activity Relationship of the R₂

c

Substitution on Benzimidazole Derivatives

Since the deep part of the lipophilic pocket has a polar residue

Ser404, we next investigate the eﬀect of aromatic groups with

various polar substituents (Table 3). The introduction of a

ﬂuorine atom at the para, meta, or ortho position of the benzyl

moiety leads to 26a, 26b, and 26c. It was shown that ﬂuorine

atoms at any positions were tolerated. The corresponding

compounds exhibited similar activity as that of 25j. Compound

26d, with a ﬂexible linker of two methylene carbon, exhibited

slightly weak inhibitory activities compared to 26a. When

ﬂuorine atoms were presented as 2,6-substitutions, compound

26e also exhibited good inhibitory activity with an IC₅₀of 30

nM. Compound 26f, with a 4-triﬂuoromethylbenzyl group,

demonstrated a similar potency to 26e, whereas methylsulfonyl

analogue 26g was less active. When 4-position substitutions

were varied from methylsulfonyl (26g) to methyl ester (26h),

the compound potency was improved. However, the corre-

sponding carboxyl acid form of 26h showed a signiﬁcant loss in

potency. Next, we then turned our attention to heteroaromatic

substituents at the R₂position. Compounds 26j−l containing

pyridine groups were designed and synthesized. Compounds

26k and 26l exhibited desirable inhibitory activities with IC₅₀

values of 26 and 28 nM, respectively. The corresponding

compound 26j showed approximately 3-fold potency loss.

These ﬁndings demonstrated that some polar substituents in this

region are tolerated.

The area near helices 11 and 12 is also an active site. To ﬁnd

more potent analogues based on the predicted binding mode of

25p, we designed various substituents at the R₃and R₄positions

to explore the chemical space for activity improvement (Table

4). When the methyl group at the R₃position of compound 25o

was replaced by triﬂuoromethyl and methoxyl groups, the

corresponding compounds 27a and 27b exhibited approx-

imately 4- and 5-fold potency loss with IC₅₀values of 32 and 41

nM, respectively. Expanding the size to isopropylformamide

(27c) resulted in an approximately 9-fold decreased activity.

These results demonstrated that the methyl group substituent at

the R₃position was optimal for RORγ transcriptional activity.

We further investigated the contribution of the R₄group to

RORγ inhibition using 25o as the template molecule. When the

methyl or methoxyl group at the R₃position was merged to the

R₄position, the corresponding compounds 27d and 27e

displayed 73- and 3.7-fold potency losses, respectively.

However, when the isopropylformamide moiety at the R₃

position was merged to the R₄position, the resulting compound

27f showed a slightly improved potency. The further

investigation suggested that when the p-methyl in compound

27a was replaced with ﬂuorine (27g), the resulting 27g

displayed a little improvement in potency with an IC₅₀value

a

b

IC₅₀values were calculated from the luciferase assay. ΔT_mvalues

c

were calculated from the thermal shift assay. All assay results are

reported as mean SD from at least two independent experiments.

To conﬁrm the importance of hydrophobic interactions, a

phenyl group with one to two methylenes was introduced at the

R₂position (Table 2). When the substituent was a benzyl group

(25j) or a 4-methylbenzyl group (25k), the compounds showed

a slight potency decrease (IC₅₀= 13 and 22 nM, respectively)

compared to 25c. Flexible linkers with two methylenes (25l and

25m) were also used to attach substituents for improved activity.

Compound 25l showed similar potency to 25j, whereas 25m

exhibited a little decrease in potency relative to 25k. From

attempts to increase activity by introducing methyl group onto

the 4-methylbenzyl moiety of 25k. Compound 25n indeed

exhibited a slightly increased potency (IC₅₀= 13 nM) compared

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Figure 4. Three-dimensional presentation of binding modes of ligands with RORγ-LBD (PDB code: 6J1L). (A) Predicted binding mode of compound

25c (pink) in complex with RORγ-LBD. (B) Predicted binding mode of compound 25p (yellow) in complex with RORγ-LBD. Water molecules are

shown as spheres in red. H-bonding and π−π stacking interactions are shown as yellow and green dashed lines, respectively. Figures were prepared

using PyMOL.

of 23 nM. The corresponding (S)-enantiomer compound 27h

was also investigated. Compound 27h displayed an IC₅₀value of

64 nM and a thermal shift of 10.5 °C, which is comparable to

(R)-enantiomer 27g. To further understand the potency of 27h

at the molecular level, we also conducted a molecular docking

study of 27h with RORγ-LBD and superimposed it with XY101

(PDB code: 6J1L, Figure S2A, Supporting information). The

docking results indicated the core phenyl group and ethyl-

sulfonyl benzyl group of 27h overlap well with that of XY101 and

make similar interactions with surrounding residues. The

triﬂuoromethyl group of 27h occupies the area near His479.

The 4-ﬂuorobenzyl group of 27h forms hydrophobic

interactions with residues Val376, Phe388, Ile400, and

Phe401. Next, the binding mode of 27g was also predicted by

molecular docking. From the overlaid binding models of 27g

and 27h, we can see that both enantiomers shared similar

binding modes (Figure S2B, Supporting information). The 4-

ﬂuorobenzyl groups of both 27g and 27h point to the same

hydrophobic pocket and contribute to the high potency.

2.5. Evaluation of Nuclear Receptor Selectivity. To

determine the selectivity against other ROR isoforms,

compounds 25c, 25o, 25p, 27g, and 27h were assessed in a

series of Gal4-ROR-LBD construct transcriptional reporter

assays in 293T cells. In addition, we also investigated the

selectivity proﬁles for these compounds against some nuclear

receptors by the same cell assay system, including human

farnesoid X receptor (FXR) and liver X receptor-α (LXR-α). As

shown in Table 5, the tested compounds did not show

signiﬁcant activity against RORα, RORβ, FXR, and LXRα

(IC₅₀> 20 μM, data not shown), demonstrating that all of the

compounds displayed excellent selectivity for RORγ versus the

other nuclear receptors in our selectivity panel.

compounds showed strong cell growth inhibitory activity against

AR-positive prostate cancer cell lines than the second-

generation antiandrogen enzalutamide. These ﬁndings impli-

cated that our compounds demonstrated reasonable antiproli-

ferative activity in the AR-positive prostate cancer cell lines.

Consistent with the inhibition of cellular antiproliferation

activity, treatment of C4-2B and 22Rv1 cells with 27h reduces

colony formation in a dose-dependent manner as compared to

treatment with vehicle (Figure 5A). 27h inhibited colony

formation signiﬁcantly in C4-2B and 22Rv1 cells, and almost no

colony was observed in both cell lines upon treatment with 8 μM

27h, consistent with its potency in cell viability assays.

We next performed quantitative reverse transcription

polymerase chain reaction (qRT-PCR) analysis to evaluate the

ability of compounds 25p, 27g, and 27h to suppress AR-

regulated genes and other oncogenes in LNCaP cells (Figure

5B,C). Our data showed that all of the compounds eﬀectively

suppressed the expression of the AR-regulated genes, prostatic-

speciﬁc antigen (PSA) (also known as KLK3), KLK2, and

TMPRSS2, in LNCaP cells. In addition, compound 25p was also

very eﬀective in suppressing the c-Myc gene, a known oncogenic

driver in prostate cancer, in LNCaP cells. Next, western blot

analysis showed that compound 27h eﬀectively induces AR-FL

degradation in LNCaP, C4-2B, and 22Rv1 cells and AR variant

degradation in 22Rv1 cells (Figures 5D and S3). These

mechanistic data clearly demonstrated that the RORγ inverse

agonists 25p, 27g, and 27h could eﬀectively suppress cell growth

and related gene expression in prostate cancer cells, which

further supports the promising therapeutic potential of RORγ

inhibition in prostate cancer.

2.7. Evaluation of Metabolic Stability. Compounds 25c,

25p, 27g, and 27h were further assessed for their metabolic

stability in human liver microsomes (HLMs) and rat liver

microsomes (RLMs), with a long-acting drug ketanserin as a

reference (Tables 7 and S4). The results showed that 25c and

25p were rapidly metabolized in the human liver microsome, as

shown by t_1/2values of 3.26 and 3.03 min at 1 μM, respectively.

Moreover, the metabolic stability of compound 25p in RLM

(t_1/2= 22.41 min, Cl_int= 110.84 mL/(min·kg)) was better than

that in HLM (t_1/2= 3.03 min, Cl_int= 573.69 mL/(min·kg)). To

our delight, compounds 27g and 27h were particularly stable in

HLM and RLM. Both compounds showed higher t_1/2(>120

min) and lower intrinsic microsome clearance (C_lint, 6.82 and

2.6. Evaluation of the Inhibitory Eﬀects on Cell Growth

and Gene and Protein Expression in Prostate Cancer

Cells. Considering their potent RORγ inhibitory activities and

encouraging selectivities, representative compounds were

evaluated for their antiproliferative activities against AR-positive

prostate cancer cell lines such as LNCaP, 22Rv1, and C4-2B

(Table 6). The RORγ inverse agonist 1 was included as the

control. The results indicated that all of the tested compounds

showed low micromolar range activities in AR-positive prostate

cancer cell lines. In AR-positive 22Rv1 cells, 27g and 27h

showed IC₅₀values of 4.62 and 8.27 μM, respectively, which are

more potent than other compounds. Note that all of the tested

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c

Table 3. Structure−Activity Relationship of the R₂Substitution on Benzimidazole Derivatives

a

b

c

IC₅₀values were calculated from the luciferase assay. ΔT_mvalues were calculated from the thermal shift assay. All assay results are reported as

mean SD from at least two independent experiments.

10.05 mL/(min·kg)) than the reference control ketanserin in

HLM and RLM.

reasonable pharmacokinetic properties in SD rats, with high

plasma exposure AUC_(0−∞)values of 16 778.29 and 41 917.70

μg/L·h. These two compounds exhibited good oral half-life

(3.65 and 4.98 h) and oral bioavailability (11.88 and 32.41%) at

an oral dose of 25 mg/kg. In summary, the potent and selective

compound 27h possessed the most favorable PK properties and

good metabolic stability, deserving further evaluation of its oral

antitumor activity (Figure 6).

2.9. Compound 27h Inhibits Prostate Cancer Tumor

Growth. We next evaluated the in vivo eﬀect of 27h on prostate

cancer tumor growth using a 22Rv1 prostate cancer cell

xenograft model (Figure 7). After the solid tumors were

established and reached approximately 100 mm³, the mice were

randomized divided into three groups (six or seven mice in each

group) and orally administrated with 27h (10 or 40 mg/kg) or

vehicle ﬁve times per week for 3 weeks. As shown in Figure 7,

treatment with 10 mg/kg 27h signiﬁcantly inhibited the growth

2.8. Pharmacokinetic (PK) Studies. To check the plasma

exposure of the compounds in vivo, the pharmacokinetic (PK)

proﬁles of compounds 25c, 25p, 27g, and 27h were investigated

in Sprague−Dawley (SD) rats, following a single oral dose (po)

of 25 mg/kg or an intravenous (iv) dose of 5 mg/kg. The

relevant PK parameters are summarized in Table 8. When

intravenously administrated at 5 mg/kg, compounds 25c and

25p displayed acceptable PK proﬁles with area under the curve

(AUC) values of 3484.88 and 6427.37 μg/L·h and maximum

plasma concentration (C_max) of 4973.96 and 11 852.17 μg/L,

respectively. Oral administration (po) of 25 mg/kg 25c and 25p

to SD rats resulted in low oral bioavailability. Encouragingly,

further PK investigation revealed that compounds 27g and 27h

exhibited improved PK parameters in SD rats compared to

compounds 25c and 25p. Both of the compounds demonstrated

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c

Table 4. Structure−Activity Relationship of the R₂, R₃, and R₄-Group Substitutions on Benzimidazole Derivatives

a

b

c

IC₅₀values were calculated from the luciferase assay. ΔT_mvalues were calculated from the thermal shift assay. All assay results are reported as

mean SD from at least two independent experiments.

a

of tumor compared to vehicle-treated mice, with a tumor growth

inhibition (TGI) value of 83%. Compound 27h treatment at a

dose of 40 mg/kg eﬀectively achieved complete and durable

tumor regression. Moreover, 27h was also well tolerated at all

doses with minimal impact on body weight during the treatment,

indicating that compound treatment caused no major toxicity to

the mice. Taken together, these results demonstrated that 27h

exhibited high eﬃcacy against prostate cancer without any

obvious toxicity.

Table 5. Selectivity Proﬁles

IC₅₀(μM)

cmpd

RORγ

RORα

RORβ

LXRα

FXR

1

0.240 0.090

0.004 0.0003

0.009 0.004

0.011 0.002

0.024 0.003

0.064 0.014

>20

25c

25o

25p

27g

27h

a

3. CONCLUSIONS

Transcriptional activities were measured using reporter gene assays

in 293T cells with Gal4-RORγ LBD, Gal4-RORα-LBD, Gal4-RORβ-

LBD, Gal4-LXRα-LBD, and Gal4-FXR-LBD expression vectors. IC₅₀

values are reported as means SD.

In the present study, we described the identiﬁcation,

optimization, and biological evaluation of novel benzothiazole

and benzimidazole derivatives as inverse agonists of RORγ

starting from the structure-based virtual screening approach for

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Table 6. Antiproliferation Eﬀects of Inverse Agonists against

the AR-Positive Prostate Cancer Cell Lines LNCaP, 22Rv1,

and C4-2B

4. EXPERIMENTAL SECTION

4.1. Virtual Screening. The MayBridge database containing

70 000 compounds was selected and downloaded from the ZINC

website (http://zinc.docking.org) for virtual screening. In this study,

structure-based virtual screening was performed with the Glide

a

cell viability IC₅₀(μM)

cmpd

LNCaP

C4-2B

22Rv1

̈

molecular docking program (version 9.4, Schrodinger, LLC, New

b

York, NY, 2013) using the standard precision (SP) score mode. For the

molecular docking, the crystal structure of RORγ (PDB code: 4QM0)

was used as the reference structure. A total of 4609 structures were

selected based on their binding mode and docking scores (lower than

−9.0 kcal/mol). These structures were further assessed with cluster

analysis and visual inspection. Finally, 12 compounds were selected and

purchased from Specs Ltd. for subsequent biological evaluation (Figure

S1 and Table S1, Supporting Information).

4.2. Molecular Docking Studies. The crystal structures of RORγ

in complex with the inverse agonists (PDB codes: 4QM0 and 6J1L)

were used for the molecular docking study. All of the ligand and protein

preparations were performed in Maestro (version 9.4, Schrodinger,

LLC, New York, NY, 2013) implemented in the Schrodinger program.

enzalutamide

1

25c

25p

27g

27h

42.37 2.37

5.38 0.73

9.22 0.02

4.82 1.08

8.07 0.66

6.33 0.47

23.56 0.61

5.38 0.27

5.76 0.05

4.40 0.18

6.20 0.60

6.82 0.72

36.66 4.21

10.13 1.99

14.36 0.46

11.10 0.72

4.62 0.91

8.27 0.75

a

The IC₅₀was calculated from the cell viability assay by Cell-Titer

b

GLO (Promega). The data were taken from ref 32. The data were

expressed as the mean SD, representing at least two independent

experiments.

̈

The proteins were prepared using the Protein Preparation Wizard

treatment against prostate cancer. Our eﬀorts have resulted in

the discovery of a number of potent and highly eﬃcacious RORγ

inverse agonists. In particular, compound 27h potently inhibited

the RORγ transcription activity with an IC₅₀value of 64 nM.

Compound 27h demonstrated an outstanding selectivity for

RORγ versus the other NRs in our selectivity panel. Compound

27h also displayed potent antiproliferative activity and sup-

pressed the colony formation and expression of AR-regulated

genes in AR-positive prostate cancer cell lines. Moreover,

Compound 27h exhibited good pharmacokinetic properties and

showed strong antitumor activity by oral administration in the

22Rv1 tumor xenograft model. Our ﬁndings demonstrated that

compound 27h (XY123) is a potent, selective, and orally

available RORγ inverse agonist and a promising candidate for

the treatment of prostate cancer.

̈

within Maestro 9.4 (Schrodinger, LLC). Hydrogens were added, bond

orders were assigned, and missing side chains for some residues were

added using Prime. The added hydrogens were subjected to energy

minimization until the root-mean-square deviation (RMSD) relative to

the starting geometry reached 0.3 Å. The Glide docking program in

Maestro 9.4 was used for docking studies. For Glide docking, the grid

was deﬁned using a 20 Å box centered on the ligand, and the important

water molecules around the ligand were kept. All parameters were kept

as default. The designed molecules were docked using the Glide SP

mode, and the predicted binding modes of all of the compounds were

ranked according to their glidescores.

4.3. General Chemistry. All commercial reagents were used

without further puriﬁcation unless otherwise speciﬁed. Enzalutamide

was purchased from Selleck. Final compounds were puriﬁed either by

silica gel chromatography (300−400 mesh) or by recrystallization. ¹H

NMR and ¹³C NMR spectra were recorded on a Bruker AV-400 or AV-

Figure 5. (A) Compound 27h inhibited the colony formations of C4-2B and 22Rv1 prostate cancer cells. Cells were cultured and treated with vehicle

(DMSO), 2, 4, or 8 μM 27h for 14 days followed by staining. (B, C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis

for mRNA expression LNCaP cells treated with vehicle (DMSO), 10 μM 25p (B), or 5 μM 27g and 27h (C) for 48 h. Data are expressed as the mean

standard error of the mean (SEM) (n = 3): **p ≤ 0.01, ***p ≤ 0.0001 by two-tailed Student’s t-test. (D) Western blot analysis of AR (AR-FL) in

LNCaP and C4-2B cells after 48 h treatment with compound 27h at indicated concentrations. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

was used as the loading control.

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Table 7. Metabolic Stability of Compounds in Human and Rat Liver Microsomes

microsomal stability in HLM

microsomal stability in RLM

a

b

a

b

cmpd

t_1/2(min)

Cl_int(mL/(min·kg))

t_1/2(min)

Cl_int(mL/(min·kg))

cLog P

ketanserin

25c

25p

27g

27h

27.11

3.26

3.03

>120

64.12

532.52

573.69

3.87

15.61

159.10

5.91

6.12

6.21

22.41

>120

110.84

6.82

10.05

5.36

a

b

Half-lives (t_1/2) in human and rat liver microsomes. Human liver microsome (HLM)- and rat liver microsome (RLM)-predicted hepatic

clearance.

1

500 spectrometer. Coupling constants (J) were expressed in hertz (Hz).

NMR chemical shifts (δ) were reported in parts per million (ppm) units

relative to the internal control (tetramethylsilane (TMS)). Low- or

high-resolution electrospray ionization mass spectrometry (ESI-MS)

were recorded on an Agilent 1200 HPLC-MSD mass spectrometer or

an Applied Biosystems Q-STAR Elite ESI-LC−MS/MS mass

spectrometer, respectively. Compound purities were determined by

reverse-phase high-performance liquid chromatography (HPLC) with

20% solvent A (H₂O) and 80% solvent B (MeOH, 0.5‰ triﬂuoroacetic

acid (TFA) in MeOH or 0.5‰ NH₃in MeOH) as eluents. HPLC

analysis uses a Dionex Summit HPLC column (Inertsil ODS-SP, 5.0

μm, 4.6 mm × 250 mm (GL Sciences Inc.)) with a UVD170U detector,

a manual injector, and a P680 pump with a detection wavelength of 254

nm and a ﬂow rate of 1.0 mL/min. The purity of all of the ﬁnal

compounds was determined by HPLC to be >95%. Optical rotations

were measured on a WZZ-2S Automatic polarimeter.

4.3.1. 4-Methyl-N-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-

benzenesulfonamide (6). 4.3.1.1. Step 1. 2-Amino-6-methylbenzo-

thiazole (7, 5 g, 30.5 mmol) was suspended in a solution of KOH (25 g,

44.6 mmol) in water (50 mL) and heated to reﬂux overnight. After

cooling to ambient temperature, the solution was adjusted to pH 6 with

acetic acid. The thick precipitate was collected by ﬁltration and rinsed

with water. The residue was dissolved in CH₂Cl₂, dried over Na₂SO₄,

and concentrated to provide the desired 2-amino-5-methybenzenethiol

(8) as a yellow solid (3.4 g, 80% yield). ¹H NMR (400 MHz, DMSO-

d₆) δ 6.91 (dd, J = 8.2, 1.7 Hz, 1H), 6.81 (d, J = 1.4 Hz, 1H), 6.64 (d, J =

8.2 Hz, 1H), 5.21 (s, 2H), 2.05 (s, 3H).

4.3.1.2. Step 2. A mixture of compound 8 (2.03 g, 14.58 mmol) and

4-aminobenzoic acid (1.99 g, 14.51 mmol) in polyphosphoric acid (20

g) was heated at 220 °C for 4 h. After cooling, the reaction mixture was

poured into ice-cold aqueous sodium carbonate (10% w/v) and stirred

until gas evolution ceased. The precipitated product was collected by

ﬁltration and washed with water. The residue was dissolved in EtOAc,

dried over Na₂SO₄, and evaporated. The residue was puriﬁed by silica

gel chromatography with petroleum ether/EtOAc (4/1, v/v) to aﬀord

4-(6-methylbenzo[d]thiazol-2-yl)aniline (9) as a brown solid (3.14 g,

90% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.80 (s, 1H), 7.77 (d, J =

8.3 Hz, 1H), 7.72 (d, J = 8.6 Hz, 2H), 7.26 (dd, J = 8.3, 1.1 Hz, 1H), 6.65

(d, J = 8.6 Hz, 2H), 5.85 (s, 2H), 2.42 (s, 3H).

4.3.1.3. Step 3. To a solution of compound 9 (73 mg, 0.3 mmol) in

pyridine (10 mL) was added 4-methylbenzenesulfonyl chloride (85.8

mg, 0.45 mmol). The mixture was stirred at 80 °C for overnight. Dilute

HCl was added; the aqueous layer was extracted with EtOAc (50 mL ×

3); and the organic layer was washed with brine, dried with Na₂SO₄, and

evaporated. The residue was puriﬁed by silica gel chromatography with

petroleum ether/EtOAc (4/1, v/v) to aﬀord 6 as a white solid (37 mg,

31% yield). ¹H NMR (500 MHz, DMSO-d₆) δ 10.71 (s, 1H), 7.93 (d, J

= 8.7 Hz, 2H), 7.90−7.85 (m, 2H), 7.72 (d, J = 8.3 Hz, 2H), 7.37 (d, J =

8.2 Hz, 2H), 7.32 (dd, J = 8.4, 1.1 Hz, 1H), 7.27 (d, J = 8.7 Hz, 2H), 2.43

(s, 3H), 2.32 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.48,

156.03, 151.70, 143.59, 140.55, 136.49, 135.09, 134.45, 129.82, 128.19,

128.02, 126.72, 122.17, 121.75, 119.32, 21.01, 20.93. MS (ESI), m/z for

C₂₁H₁₈N₂O₂S₂([M + H]⁺): calcd 394.51, found 395.0. HPLC analysis:

MeOH (1‰ TFA)−H₂O (85:15), t_R= 11.33 min, 95.31% purity.

4.3.2. 4-(tert-Butyl)-N-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-

benzenesulfonamide (10a). Compound 10a was prepared from 9 and

4-(tert-butyl)benzenesulfonyl chloride according to the general

procedure of step 3 as described for 6. White solid, 79% yield. H

NMR (400 MHz, DMSO-d₆) δ 10.75 (s, 1H), 7.94 (d, J = 8.7 Hz, 2H),

7.90−7.84 (m, 2H), 7.78 (d, J = 8.5 Hz, 2H), 7.60 (d, J = 8.6 Hz, 2H),

7.35−7.26 (m, 3H), 2.44 (s, 3H), 1.25 (s, 9H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 165.49, 156.19, 151.70, 140.58, 136.70, 135.09, 134.44,

128.26, 128.09, 128.03, 126.56, 126.29, 122.17, 121.77, 119.02, 34.88,

30.68, 21.02. MS (ESI), m/z for C₂₄H₂₄N₂O₂S₂([M + H]⁺): calcd

436.59, found 437.2. HPLC analysis: MeOH (1‰ TFA)−H₂O

(80:20), t_R= 26.74 min, 98.65% purity.

4.3.3. 4-Fluoro-N-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-

benzenesulfonamide (10b). Compound 10b was prepared from 9

and 4-ﬂuorobenzenesulfonyl chloride according to the general

1

procedure of step 3 as described for 6. White solid, 61% yield. H

NMR (400 MHz, DMSO-d₆) δ 10.76 (s, 1H), 7.95 (d, J = 8.7 Hz, 2H),

7.92−7.86 (m, 4H), 7.42 (t, J = 8.8 Hz, 2H), 7.33 (dd, J = 8.4, 0.8 Hz,

1H), 7.28 (d, J = 8.7 Hz, 2H), 2.44 (s, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 165.42, 165.38, 163.42, 151.68, 140.17, 135.67, 135.65,

135.11, 134.45, 129.78, 129.71, 128.51, 128.23, 128.02, 122.19, 121.75,

119.68, 116.71, 116.53, 20.99. MS (ESI), m/z for C₂₀H₁₅FN₂O₂S₂([M

+ H]⁺): calcd 398.47, found 398.9. HPLC analysis: MeOH (1‰

TFA)−H₂O (80:20), t_R= 15.70 min, 98.33% purity.

4.3.4. N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-4-

(triﬂuoromethoxy)benzenesulfonamide (10c). Compound 10c was

prepared from 9 and 4-(triﬂuoromethoxy)benzenesulfonyl chloride

according to the general procedure of step 3 as described for 6. White

1

solid, 74% yield. H NMR (400 MHz, DMSO-d₆) δ 10.87 (s, 1H),

8.00−7.92 (m, 4H), 7.91−7.85 (m, 2H), 7.58 (d, J = 8.3 Hz, 2H), 7.33

(d, J = 8.5 Hz, 1H), 7.29 (d, J = 8.7 Hz, 2H), 2.44 (s, 3H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 165.37, 151.68, 151.22, 140.01, 138.21,

135.16, 134.48, 129.33, 128.64, 128.30, 128.05, 122.22, 121.78, 121.54,

120.79, 119.75, 118.74, 21.02. MS (ESI), m/z for C₂₁H₁₅F₃N₂O₃S₂([M

+ H]⁺): calcd 464.48, found 465.0. HPLC analysis: MeOH (1‰

TFA)−H₂O (85:15), t_R= 13.62 min, 99.09% purity.

4.3.5. N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-3-nitrobenze-

nesulfonamide (10d). Compound 10d was prepared from 9 and 3-

nitrobenzenesulfonyl chloride according to the general procedure of

step 3 as described for 6. White solid, 38% yield. ¹H NMR (400 MHz,

DMSO-d₆) δ 11.01 (s, 1H), 8.58−8.54 (m, 1H), 8.46 (dd, J = 8.2, 1.6

Hz, 1H), 8.21 (d, J = 7.9 Hz, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.91−7.85

(m, 3H), 7.36−7.27 (m, 3H), 2.44 (s, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 165.27, 151.66, 147.97, 140.80, 139.55, 135.19, 134.49,

132.55, 131.53, 129.01, 128.34, 128.05, 127.82, 122.24, 121.77, 121.41,

120.25, 21.00. MS (ESI), m/z for C₂₀H₁₅N₃O₄S₂([M + H]⁺): calcd

425.48, found 426.0. HPLC analysis: MeOH (1‰ TFA)−H₂O

(80:20), t_R= 17.30 min, 97.51% purity.

4.3.6. N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-2-nitrobenze-

nesulfonamide (10e). Compound 10e was prepared from 9 and 2-

nitrobenzenesulfonyl chloride according to the general procedure of

step 3 as described for 6. ¹H NMR (400 MHz, DMSO-d₆) δ 11.16 (s,

1H), 8.07−8.03 (m, 1H), 8.03−7.95 (m, 3H), 7.91−7.81 (m, 4H),

7.36−7.32 (m, 1H), 7.30 (d, J = 8.7 Hz, 2H), 2.44 (s, 3H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 165.31, 151.68, 147.86, 139.36, 135.18,

134.91, 134.51, 132.73, 131.19, 129.94, 128.90, 128.28, 128.05, 124.81,

122.24, 121.77, 119.95, 21.01. MS (ESI), m/z for C₂₀H₁₅N₃O₄S₂([M +

H]⁺): calcd 425.48, found 426.0. HPLC analysis: MeOH (1‰ TFA)−

H₂O (75:25), t_R= 19.28 min, 95.43% purity.

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Figure 6. Summary of potency, selectivity, stability, and PK properties

b

of 27h. (^aIC₅₀values were calculated from the luciferase assay. ΔT_m

c

values were calculated from the thermal shift assay. The IC₅₀was

calculated from the cell viability assay by Cell-Titer GLO (Promega).

^dLiver microsome-predicted hepatic clearance (mL/(min·kg)) and

half-life(min).)

4.3.7. N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-3-

(methylsulfonyl)benzenesulfonamide (10f). Compound 10f was

prepared from 9 and 3-(methylsulfonyl)benzenesulfonyl chloride

according to the general procedure of step 3 as described for 6.

1

White solid, 34% yield. H NMR (400 MHz, DMSO-d₆) δ 10.93 (s,

1H), 8.32 (s, 1H), 8.20 (d, J = 7.7 Hz, 1H), 8.12 (d, J = 8.0 Hz, 1H),

7.96 (d, J = 8.6 Hz, 2H), 7.92−7.84 (m, 3H), 7.33 (d, J = 8.8 Hz, 1H),

7.29 (d, J = 8.6 Hz, 2H), 3.28 (s, 3H), 2.44 (s, 3H). ¹³C NMR (126

MHz, DMSO-d₆) δ 165.33, 151.68, 141.99, 140.40, 139.74, 135.20,

134.50, 131.68, 131.37, 131.21, 128.92, 128.33, 128.07, 124.99, 122.25,

121.79, 120.16, 43.22, 21.03. MS (ESI), m/z for C₂₁H₁₈N₂O₄S₃([M +

H]⁺): calcd 458.57, found 459.2. HPLC analysis: MeOH (1‰ TFA)−

H₂O (80:20), t_R= 11.31 min, 97.08% purity.

4.3.8. 2,4-Diﬂuoro-N-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-

benzenesulfonamide (10g). Compound 10g was prepared from 9

and 2,4-diﬂuorobenzenesulfonyl chloride according to the general

procedure of step 3 as described for 6. White solid, 73% yield. ¹H NMR

(400 MHz, DMSO-d₆) δ 11.13 (s, 1H), 8.03−7.92 (m, 3H), 7.90−7.85

(m, 2H), 7.58−7.50 (m, 1H), 7.36−7.24 (m, 4H), 2.44 (s, 3H). ¹³C

NMR (126 MHz, DMSO-d₆) δ 166.41, 166.32, 165.38, 164.38, 164.29,

160.04, 159.93, 157.99, 157.88, 151.69, 139.67, 135.16, 134.49, 132.63,

132.55, 128.57, 128.27, 128.06, 123.65, 123.57, 122.23, 121.78, 119.32,

112.61, 112.44, 106.45, 106.24, 106.03, 21.02. MS (ESI), m/z for

C₂₀H₁₄F₂N₂O₂S₂([M + H]⁺): calcd 416.46, found 417.1. HPLC

analysis: MeOH (1‰ TFA)−H₂O (85:15), t_R= 12.34 min, 96.04%

purity.

4.3.9. 1-(4-Fluorophenyl)-N-(4-(6-methylbenzo[d]thiazol-2-yl)-

phenyl)methanesulfonamide (10h). Compound 10h was prepared

from 9 and (4-ﬂuorophenyl)methanesulfonyl chloride according to the

general procedure of step 3 as described for 6. White solid, 26% yield.

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Figure 7. In vivo antitumor eﬃcacy of compound 27h. (A) Antitumor eﬃcacy of compound 27h against a 22Rv1 prostate cancer xenograft mouse

model and (B) body weight of mice during treatment with 27h. The mice were randomized and orally treated with 27h (10 or 40 mg/kg) or vehicle ﬁve

times per week for 3 weeks. Data are expressed as the mean tumor volume SEM (n = 6−7) of the animals in each treatment group.

¹H NMR (400 MHz, DMSO-d₆) δ 10.26 (s, 1H), 8.01 (d, J = 8.7 Hz,

57.00, 52.17, 21.02. MS (ESI), m/z for C₂₃H₂₀N₂O₄S₂([M + H]⁺):

calcd 452.54, found 453.0. HPLC analysis: MeOH (1‰ TFA)−H₂O

(85:15), t_R= 12.06 min, 98.89% purity.

2H), 7.94−7.87 (m, 2H), 7.38−7.27 (m, 5H), 7.19 (t, J = 8.9 Hz, 2H),

4.60 (s, 2H), 2.46 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.66,

163.16, 161.21, 151.76, 141.19, 135.04, 134.44, 133.04, 132.98, 128.27,

128.01, 127.65, 125.71, 125.68, 122.17, 121.76, 118.46, 115.38, 115.21,

56.32, 21.01. MS (ESI), m/z for C₂₁H₁₇FN₂O₂S₂([M − 1]⁻): calcd

412.50, found 411.1. HPLC analysis: MeOH−H₂O (75:25), t_R= 19.68

min, 96.87% purity.

4.3.14. 4-((N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-

sulfamoyl)methyl)benzoic Acid (10m). 10l (40 mg, 0.09 mmol) was

dissolved in methanol (5 mL) and 2 mol/L NaOH aqueous solution

(10 mL). The mixture was stirred at room temperature for 2 h. The

solvent was removed; then, diluted hydrochloric acid was added

dropwise, and a white precipitate was formed. The precipitate was

collected by ﬁltration and washed with water (10 mL × 2). The

resulting crude product was puriﬁed by recrystallization with petroleum

ether/EtOAc to aﬀord 10m (28.8 mg, 73%) as a white solid. ¹H NMR

(500 MHz, DMSO-d₆) δ 10.38 (s, 1H), 8.23 (d, J = 8.7 Hz, 2H), 8.02

(d, J = 8.7 Hz, 2H), 7.94−7.88 (m, 2H), 7.57 (d, J = 8.6 Hz, 2H), 7.38−

7.31 (m, 3H), 4.82 (s, 2H), 2.46 (s, 3H). ¹³C NMR (126 MHz, DMSO-

d₆) δ 165.62, 165.40, 151.77, 147.44, 140.88, 137.02, 135.09, 134.47,

132.37, 128.32, 128.04, 127.93, 123.42, 122.21, 121.79, 118.76, 56.66,

21.03. MS (ESI), m/z for C₂₂H₁₈N₂O₄S₂([M + H]⁺): calcd 438.52,

found 440.0. HPLC analysis: MeOH (1‰ TFA)−H₂O (85:15), t_R=

10.11 min, 97.83% purity.

4.3.15. 4-((N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-

sulfamoyl)methyl)phenyl Propionate (10n). Compound 10n was

prepared from 9 and 4-((chlorosulfonyl)methyl)phenyl propionate

according to the general procedure of step 3 as described for 6. White

solid, 55% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.30 (s, 1H), 8.01

(d, J = 8.7 Hz, 2H), 7.95−7.87 (m, 4H), 7.42 (d, J = 8.2 Hz, 2H), 7.38−

7.29 (m, 3H), 4.70 (s, 2H), 4.29 (q, J = 7.1 Hz, 2H), 2.46 (s, 3H), 1.28

(t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.64, 165.37,

151.77, 141.07, 135.04, 134.55, 134.45, 131.31, 129.76, 129.08, 128.28,

128.01, 127.77, 122.17, 121.75, 118.58, 60.77, 57.02, 21.02, 14.07. MS

(ESI), m/z for C₂₄H₂₂N₂O₄S₂([M + H]⁺): calcd 466.57, found 467.1.

HPLC analysis: MeOH (1‰ TFA)−H₂O (85:15), t_R= 11.60 min,

96.35% purity.

4.3.10. N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-1-(4-

(triﬂuoromethyl)phenyl)methanesulfonamide (10i). Compound

10i was prepared from 9 and (4-(triﬂuoromethyl)phenyl)-

methanesulfonyl chloride according to the general procedure of step

1

3 as described for 6. White solid, 47% yield. H NMR (500 MHz,

DMSO-d₆) δ 10.34 (s, 1H), 8.01 (d, J = 8.7 Hz, 2H), 7.94−7.88 (m,

2H), 7.74 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 7.38−7.30 (m,

3H), 4.75 (s, 2H), 2.46 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ

165.63, 151.77, 141.04, 135.05, 134.44, 134.16, 131.83, 128.96, 128.70,

128.29, 128.01, 127.79, 125.26, 125.23, 125.20, 123.03, 122.18, 121.77,

118.57, 56.72, 21.02. MS (ESI), m/z for C₂₂H₁₇F₃N₂O₂S₂([M + H]⁺):

calcd 462.51, found 463.0. HPLC analysis: MeOH (1‰ TFA)−H₂O

(85:15), t_R= 12.76 min, 99.51% purity.

4.3.11. N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-1-(4-

nitrophenyl)methanesulfonamide (10j). Compound 10j was pre-

pared from 9 and (4-nitrophenyl)methanesulfonyl chloride according

to the general procedure of step 3 as described for 6. White solid, 40%

yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.36 (s, 1H), 8.22 (d, J = 8.7

Hz, 2H), 8.02 (d, J = 8.6 Hz, 2H), 7.94−7.88 (m, 2H), 7.57 (d, J = 8.7

Hz, 2H), 7.38−7.31 (m, 3H), 4.81 (s, 2H), 2.46 (s, 3H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 165.62, 151.77, 147.45, 140.87, 136.99,

135.10, 134.47, 132.36, 128.31, 128.04, 127.95, 123.41, 122.20, 121.77,

118.79, 56.71, 21.02. MS (ESI), m/z for C₂₁H₁₇N₃O₄S₂([M + H]⁺):

calcd 439.50, found 440.0. HPLC analysis: MeOH−H₂O (75:25), t_R=

19.21 min, 95.55% purity.

4.3.16. N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-1-(3-

nitrophenyl)methanesulfonamide (10o). Compound 10o was

prepared from 9 and (3-nitrophenyl)methanesulfonyl chloride

according to the general procedure of step 3 as described for 6.

4.3.12. N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-1-(4-

(methylsulfonyl)phenyl)methansulfonamide (10k). Compound 10k

was prepared from 9 and (4-(methylsulfonyl)phenyl)methanesulfonyl

chloride according to the general procedure of step 3 as described for 6.

1

White solid, 55% yield. H NMR (400 MHz, DMSO-d₆) δ 10.33 (s,

White solid, 60% yield. H NMR (400 MHz, DMSO-d₆) δ 10.37 (s,

1H), 8.21 (d, J = 8.2 Hz, 1H), 8.16 (s, 1H), 8.00 (d, J = 8.7 Hz, 2H),

7.94−7.88 (m, 2H), 7.74 (d, J = 7.7 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H),

7.38−7.29 (m, 3H), 4.83 (s, 2H), 2.46 (s, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 165.60, 151.76, 147.55, 140.99, 137.69, 135.06, 134.46,

131.68, 129.95, 128.25, 128.01, 127.87, 125.65, 123.18, 122.19, 121.76,

118.67, 56.34, 21.01. MS (ESI), m/z for C₂₁H₁₇N₃O₄S₂([M − H]⁻):

calcd 439.50, found 438.0. HPLC analysis: MeOH (1‰ TFA)−H₂O

(80:20), t_R= 14.37 min, 96.05% purity.

1H), 8.02 (d, J = 8.7 Hz, 2H), 7.94−7.88 (m, 4H), 7.56 (d, J = 8.3 Hz,

2H), 7.39−7.30 (m, 3H), 4.76 (s, 2H), 3.20 (s, 3H), 2.46 (s, 3H). ¹³C

NMR (126 MHz, DMSO-d₆) δ 165.63, 151.76, 141.00, 140.68, 135.17,

135.07, 134.45, 131.93, 128.31, 128.02, 127.84, 126.97, 122.19, 121.77,

118.66, 56.79, 43.38, 21.02. MS (ESI), m/z for C₂₂H₂₀N₂O₄S₃([M +

H]⁺): calcd 472.59, found 472.9. HPLC analysis: MeOH (1‰ TFA)−

H₂O (85:15), t_R= 9.29 min, 95.38% purity.

4.3.13. Methyl 4-((N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-

sulfamoyl)methyl)benzoate (10l). Compound 10l was prepared

from 9 and methyl 4-((chlorosulfonyl)methyl)benzoate according to

the general procedure of step 3 as described for 6. White solid, 30%

yield. ¹H NMR (500 MHz, DMSO-d₆) δ 10.33 (s, 1H), 8.01 (d, J = 8.6

Hz, 2H), 7.95−7.89 (m, 4H), 7.43 (d, J = 8.2 Hz, 2H), 7.37−7.30 (m,

3H), 4.71 (s, 2H), 3.83 (s, 3H), 2.46 (s, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 165.89, 165.65, 151.77, 141.07, 135.05, 134.64, 134.45,

131.35, 129.47, 129.14, 128.29, 128.02, 127.77, 122.17, 121.76, 118.59,

4.3.17. N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-1-(2-

nitrophenyl)methanesulfonamide (10p). Compound 10p was

prepared from 9 and (3-nitrophenyl)methanesulfonyl chloride

according to the general procedure of step 3 as described for 6.

1

White solid, 20% yield. H NMR (400 MHz, DMSO-d₆) δ 10.47 (s,

1H), 8.04 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 8.6 Hz, 2H), 7.94−7.89 (m,

2H), 7.73 (t, J = 7.6 Hz, 1H), 7.64 (t, J = 7.5, Hz, 1H), 7.50 (d, J = 7.0

Hz, 1H), 7.35 (dd, J = 8.5, 0.8 Hz, 1H), 7.28 (d, J = 8.7 Hz, 2H), 5.05 (s,

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2H), 2.46 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.59, 151.75,

149.40, 140.69, 135.08, 134.56, 134.47, 133.53, 132.01, 130.21, 128.21,

128.02, 125.29, 123.25, 122.20, 121.77, 118.66, 53.54, 21.02. MS (ESI),

m/z for C₂₁H₁₇N₃O₄S₂([M + H]⁺): calcd 439.50, found 440.0. HPLC

analysis: MeOH (1‰ TFA)−H₂O (80:20), t_R= 13.64 min, 98.62%

purity.

4.3.18. Methyl 3-((N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-

sulfamoyl)methyl)benzoate (10q). Compound 10q was prepared

from 9 and (3-nitrophenyl)methanesulfonyl chloride according to the

general procedure of step 3 as described for 6. White solid, 25% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 10.27 (s, 1H), 8.00 (d, J = 8.6 Hz,

added, and the mixture was ﬁltered. The ﬁltrate was adjusted to pH 8−9

with saturated Na₂CO₃solution and extracted with ethyl acetate (50

mL × 3). The organic layer was washed with brine, dried over Na₂SO₄,

and concentrated under reduced pressure. The resulting crude product

was puriﬁed by silica gel chromatography with petroleum ether/EtOAc

(10/1, v/v) to obtain 5-methyl-N¹-(4-methylphenethyl)benzene-1,2-

1

diamine (16m) as a yellow liquid (2.1 g, 81% yield). H NMR (400

MHz, DMSO-d₆) δ 7.17 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 7.9 Hz, 2H),

6.43 (d, J = 7.6 Hz, 1H), 6.28 (s, 1H), 6.22 (d, J = 7.6 Hz, 1H), 4.36 (t, J

= 5.5 Hz, 1H), 4.21 (s, 2H), 3.23−3.15 (m, 2H), 2.83 (t, J = 7.4 Hz,

2H), 2.27 (s, 3H), 2.12 (s, 3H).

2H), 7.95−7.86 (m, 4H), 7.58−7.48 (m, 2H), 7.35 (d, J = 8.8 Hz, 1H),

7.32 (d, J = 8.6 Hz, 2H), 4.71 (s, 2H), 3.83 (s, 3H), 2.46 (s, 3H). ¹³C

NMR (126 MHz, DMSO-d₆) δ 165.78, 165.66, 151.77, 141.16, 135.76,

135.04, 134.45, 131.73, 130.17, 129.76, 128.98, 128.89, 128.23, 128.01,

127.73, 122.17, 121.76, 118.59, 56.80, 52.15, 21.02. MS (ESI), m/z for

C₂₃H₂₀N₂O₄S₂([M + H]⁺): calcd 452.54, found 453.0. HPLC analysis:

MeOH (1‰ TFA)−H₂O (80:20), t_R= 15.15 min, 95.41% purity.

4.3.19. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methylbenzo[d]-

thiazol-2-yl)phenyl)acetamide (11a). To a solution of compound 9

(80 mg, 0.33 mmol) in DCM (10 mL) were added HATU (188.1 mg,

0.495 mmol) and DIPEA (127.7 mg, 0.99 mmol). The mixture was

stirred at room temperature for 15 min. 2-(4-(Ethylsulfonyl)phenyl)-

acetic acid (112.86 mg, 0.495 mmol) was then added to the mixture.

The reaction mixture was stirred at room temperature for overnight.

Water was added, and the mixture was extracted with EtOAc (50 mL ×

3). The organic layer was washed with brine, dried over Na₂SO₄, and

evaporated. The resulting crude product was puriﬁed by silica gel

chromatography with petroleum ether/EtOAc (3/1, v/v) to obtain 11a

as a white solid (70 mg, 47% yield). ¹H NMR (400 MHz, DMSO-d₆) δ

10.56 (s, 1H), 8.02 (d, J = 8.7 Hz, 2H), 7.92−7.83 (m, 4H), 7.79 (d, J =

8.7 Hz, 2H), 7.63 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.4 Hz, 1H), 3.86 (s,

2H), 3.32−3.23 (m, 2H), 2.45 (s, 3H), 1.10 (t, J = 7.3 Hz, 3H). ¹³C

NMR (126 MHz, DMSO-d₆) δ 168.64, 165.77, 151.77, 141.85, 141.64,

136.88, 135.04, 134.43, 130.28, 128.65, 128.02, 127.88, 127.85, 122.16,

121.78, 119.38, 49.21, 42.95, 21.04, 7.14. MS (ESI), m/z for

C₂₄H₂₂N₂O₃S₂([M + H]⁺): calcd 450.57, found 451.1. HPLC analysis:

MeOH (1‰ TFA)−H₂O (85:15), t_R= 11.07 min, 95.31% purity.

4.3.20. N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-2-(2-

nitrophenyl)acetamide (11b). Compound 11b was prepared from 9

and 2-(2-nitrophenyl)acetic acid according to the general procedure as

described for 11a. White solid, 35% yield. ¹H NMR (400 MHz, DMSO-

d₆) δ 10.55 (s, 1H), 8.08 (d, J = 7.9 Hz, 1H), 8.02 (d, J = 8.7 Hz, 2H),

7.93−7.86 (m, 2H), 7.78−7.70 (m, 3H), 7.62−7.55 (m, 2H), 7.34 (d, J

= 8.8 Hz, 1H), 4.19 (s, 2H), 2.45 (s, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 168.06, 165.80, 151.78, 148.97, 141.70, 135.50, 134.42,

133.80, 133.61, 130.38, 128.51, 128.00, 127.89, 127.70, 124.61, 122.14,

121.76, 119.22, 40.83, 21.03. MS (ESI), m/z for C₂₂H₁₇N₃O₃S ([M +

H]⁺): calcd 403.46, found 404.3. HPLC analysis: MeOH (1‰ TFA)−

H₂O (85:15), t_R= 11.37 min, 96.66% purity.

4.3.21.3. Step 3. To a solution of 16m (1.3 g, 5.4 mmol) in DMF (20

mL) were added 4-nitrobenzaldehyde (816.0 mg, 5.4 mmol) and

OXONE (1.84 g, 3.0 mmol). The mixture was stirred at room

temperature for 1 h. Water was added, and the mixture was extracted

with EtOAc (50 mL × 3). The organic layer was washed with brine,

dried over Na₂SO₄, and evaporated. The resulting crude product was

puriﬁed by silica gel chromatography with petroleum ether/EtOAc (2/

1, v/v) to obtain 6-methyl-1-(4-methylphenethyl)-2-(4-nitrophenyl)-

1H-benzo[d]imidazole (19m) as a yellow solid (1.78 g, 86% yield). ¹H

NMR (400 MHz, CDCl₃) δ 8.21 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.2 Hz,

1H), 7.48 (d, J = 8.8 Hz, 2H), 7.27 (s, 1H), 7.19 (d, J = 8.3 Hz, 1H),

6.94 (d, J = 7.8 Hz, 2H), 6.64 (d, J = 7.9 Hz, 2H), 4.45 (t, J = 6.8 Hz,

2H), 3.03 (t, J = 6.8 Hz, 2H), 2.57 (s, 3H), 2.29 (s, 3H).

4.3.21.4. Step 4. 5-Methyl-N¹-(4-methylphenethyl)benzene-1,2-

diamine (22m) was prepared from 19m according to the general

procedure of step 2 as described for 25m. Yellow soild, 85% yield. ¹H

NMR (400 MHz, DMSO) δ 7.46 (d, J = 8.1 Hz, 1H), 7.36 (s, 1H), 7.28

(d, J = 8.5 Hz, 2H), 7.04 (d, J = 7.8 Hz, 2H), 7.00 (dd, J = 8.3, 1.0 Hz,

1H), 6.97 (d, J = 8.0 Hz, 2H), 6.65 (d, J = 8.5 Hz, 2H), 5.51 (s, 2H),

4.34 (t, J = 8.0 Hz, 2H), 2.97 (t, J = 7.6 Hz, 2H), 2.45 (s, 3H), 2.25 (s,

3H).

4.3.21.5. Step 5. To a solution of 22m (150 mg, 0.44 mmol) in DCM

(10 mL) were added HATU (250.8 mg, 0.66 mmol) and DIPEA (170.3

mg, 1.32 mmol). The mixture was stirred at room temperature for 15

min. 2-(4-(Ethylsulfonyl)phenyl)acetic acid (121.0 mg, 0.53 mmol)

was then added to the mixture. The reaction mixture was stirred at room

temperature for overnight. Water was added, and the mixture was

extracted with EtOAc (50 mL × 3). The organic layer was washed with

saturated NaHCO₃solution and brine, dried over Na₂SO₄, and

evaporated. The resulting crude product was puriﬁed by silica gel

chromatography with dichloromethane/CH₃OH (70/1, v/v) to obtain

25m as a white solid (70 mg, 29% yield). ¹H NMR (400 MHz, DMSO-

d₆) δ 10.47 (s, 1H), 7.87 (d, J = 8.3 Hz, 2H), 7.71 (d, J = 8.6 Hz, 2H),

7.64 (d, J = 8.3 Hz, 2H), 7.51 (d, J = 8.2 Hz, 1H), 7.48 (d, J = 8.7 Hz,

2H), 7.43 (s, 1H), 7.05 (dd, J = 8.2, 0.8 Hz, 1H), 6.98 (d, J = 7.7 Hz,

2H), 6.86 (d, J = 7.9 Hz, 2H), 4.39 (t, J = 7.3 Hz, 2H), 3.86 (s, 2H), 3.28

(q, J = 7.5 Hz, 2H), 2.95 (t, J = 7.3 Hz, 2H), 2.47 (s, 3H), 2.23 (s, 3H),

1.11 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.50,

152.44, 141.96, 140.72, 139.96, 136.86, 135.64, 135.53, 134.73, 131.61,

130.26, 129.61, 128.88, 128.49, 127.84, 125.27, 123.38, 118.70, 118.54,

110.63, 49.22, 45.70, 42.96, 34.55, 21.44, 20.58, 7.14. MS (ESI), m/z

for C₃₃H₃₃N₃O₃S ([M − H]⁻): calcd 551.71, found 550.4. HPLC

analysis: MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R= 18.94 min,

98.25% purity.

4.3.22. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methyl-1-propyl-1H-

benzo[d]imidazol-2-yl)phenyl)acetamide (25a). Compound 25a

was prepared according to the general procedure as described for

25m. White solid, 45% yield. ¹H NMR (500 MHz, DMSO-d₆) δ 10.51

(s, 1H), 7.86 (d, J = 8.2 Hz, 2H), 7.79 (d, J = 8.6 Hz, 2H), 7.70 (d, J =

8.8 Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 8.2 Hz, 1H), 7.42 (s,

1H), 7.05 (d, J = 8.1 Hz, 1H), 4.21 (t, J = 7.4 Hz, 2H), 3.86 (s, 2H), 3.28

(q, J = 7.5 Hz, 2H), 2.46 (s, 3H), 1.71−1.64 (m, 2H), 1.10 (t, J = 7.4 Hz,

3H), 0.73 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ

168.59, 151.94, 141.92, 140.33, 139.43, 136.87, 135.57, 132.06, 130.23,

129.77, 127.85, 124.61, 123.81, 119.01, 118.04, 110.74, 49.21, 45.61,

42.97, 22.44, 21.42, 10.86, 7.13. MS (ESI), m/z for C₂₇H₂₉N₃O₃S ([M

+ H]⁺): calcd 475.61, found 476.3. HPLC analysis: MeOH (1‰ NH₃·

H₂O)−H₂O (75:25), t_R= 14.56 min, 96.42% purity.

4.3.21. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methyl-1-(4-methyl-

phenethyl)-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (25m).

4.3.21.1. Step 1. To a solution of 3-ﬂuoro-4-nitrotoluene (12a, 2 g,

12.9 mmol) in DMSO (5 mL) were added 4-methylphenethylamine

(5.23 g, 38.7 mmol) and DIPEA (2.51 g, 19.4 mmol). The reaction

mixture was stirred overnight at 95 °C. Water was added; the reaction

mixture was extracted with ethyl acetate (50 mL × 3); and the organic

layer was washed with brine, dried with Na₂SO₄, and evaporated. The

resulting crude product was puriﬁed by silica gel chromatography with

petroleum ether/EtOAc (50/1, v/v) to aﬀord 5-methyl-N-(4-

methylphenethyl)-2-nitroaniline (13m) as a yellow solid (2.8 g, 80%

yield). ¹H NMR (400 MHz, DMSO-d₆) δ 8.09 (s, 1H), 7.94 (d, J = 8.4

Hz, 1H), 7.27−7.05 (m, 4H), 6.94−6.82 (m, 1H), 6.51 (d, J = 8.0 Hz,

1H), 3.62−3.50 (m, 2H), 2.90 (t, J = 6.8 Hz, 2H), 2.31 (s, 3H), 2.27 (s,

3H).

4.3.21.2. Step 2. A reaction mixture of Fe powder (3.46 g, 61.8

mmol) and NH₄Cl (551.1 mg, 10.3 mmol) in AcOH (1.24 g, 20.6

mmol) and water (20 mL) was heated at 50 °C for 10 min. 13m (2.8

g,10.3 mmol) was dissolved in DMF (15 mL) and added to the reaction

mixture. The reaction mixture was stirred at 50 °C for 1 h. Water was

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4.3.23. N-(4-(1-Butyl-6-methyl-1H-benzo[d]imidazol-2-yl)-

phenyl)-2-(4-(ethylsulfonyl)phenyl)acetamide (25b). Compound

25b was prepared according to the general procedure as described

C₂₉H₃₃N₃O₃S ([M + H]⁺): calcd 503.66, found 504.7. HPLC analysis:

MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R= 21.25 min, 97.24% purity.

4.3.28. N-(4-(1-Cyclobutyl-6-methyl-1H-benzo[d]imidazol-2-yl)-

phenyl)-2-(4-(ethylsulfonyl)phenyl)acetamide (25g). Compound

25g was prepared according to the general procedure as described for

25m. White solid, 30% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.52

(s, 1H), 7.87 (d, J = 8.2 Hz, 2H), 7.79 (d, J = 8.5 Hz, 2H), 7.67−7.58

(m, 5H), 7.54 (d, J = 8.2 Hz, 1H), 7.09 (d, J = 8.2 Hz, 1H), 5.13−5.01

(m, 1H), 3.87 (s, 2H), 3.28 (q, J = 7.2 Hz, 2H), 2.76−2.61 (m, 2H),

2.49 (s, 3H), 2.43−2.32 (m, 2H), 1.95−1.70 (m, 2H), 1.11 (t, J = 7.3

Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.58, 152.15, 141.95,

141.71, 140.30, 136.87, 134.55, 131.74, 130.22, 130.05, 127.85, 125.06,

123.58, 118.82, 118.59, 112.20, 50.41, 49.21, 42.98, 28.91, 21.52, 14.30,

7.14. MS (ESI), m/z for C₂₈H₂₉N₃O₃S ([M + H]⁺): calcd 487.62, found

488.5. HPLC analysis: MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R=

17.27 min, 97.43% purity.

4.3.29. N-(4-(1-(Cyclobutylmethyl)-6-methyl-1H-benzo[d]-

imidazol-2-yl)phenyl)-2-(4-(ethylsulfonyl)phenyl)acetamide (25h).

Compound 25h was prepared according to the general procedure as

described for 25m. White solid, 22% yield. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.50 (s, 1H), 7.87 (d, J = 8.1 Hz, 2H), 7.79 (d, J = 8.5 Hz,

2H), 7.70 (d, J = 8.5 Hz, 2H), 7.64 (d, J = 8.1 Hz, 2H), 7.50 (d, J = 8.2

Hz, 1H), 7.44 (s, 1H), 7.04 (d, J = 8.2 Hz, 1H), 4.35 (d, J = 7.2 Hz, 2H),

3.86 (s, 2H), 3.32−3.24 (m, 2H), 2.60−2.51 (m, 1H), 2.46 (s, 3H),

1.78− 1.57 (m, 4H), 1.54−1.44 (m, 2H), 1.11 (t, J = 7.3 Hz, 3H). ¹³C

NMR (126 MHz, DMSO-d₆) δ 168.53, 152.25, 141.94, 140.50, 140.00,

136.86, 136.04, 131.56, 130.24, 129.78, 127.84, 125.66, 123.32, 118.93,

118.45, 110.63, 49.21, 48.66, 42.97, 35.12, 25.55, 21.46, 17.57, 7.13. MS

(ESI), m/z for C₂₉H₃₁N₃O₃S ([M + H]⁺): calcd 501.65, found 502.5.

HPLC analysis: MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R= 16.17

min, 95.77% purity.

4.3.30. N-(4-(1-(Cyclohexylmethyl)-6-methyl-1H-benzo[d]-

imidazol-2-yl)phenyl)-2-(4-(ethylsulfonyl)phenyl)acetamide (25i).

Compound 25i was prepared according to the general procedure as

described for 25m. White solid, 40% yield. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.50 (s, 1H), 7.87 (d, J = 8.2 Hz, 2H), 7.78 (d, J = 8.6 Hz,

2H), 7.70 (d, J = 8.5 Hz, 2H), 7.64 (d, J = 8.1 Hz, 2H), 7.51 (d, J = 8.1

Hz, 1H), 7.42 (s, 1H), 7.04 (d, J = 8.2 Hz, 1H), 4.17 (d, J = 7.2 Hz, 2H),

3.86 (s, 2H), 3.28 (q, J = 7.2 Hz, 2H), 2.46 (s, 3H), 1.69−1.56 (m, 1H),

1.53−1.42 (m, 4H), 1.34−1.19 (m, 6H), 1.11 (t, J = 7.6 Hz, 3H). ¹³C

NMR (126 MHz, DMSO-d₆) δ 168.52, 152.60, 141.93, 140.45, 139.93,

136.87, 136.15, 131.56, 130.25, 129.76, 127.84, 125.75, 123.34, 118.92,

118.46, 110.84, 49.75, 49.21, 42.97, 37.35, 29.87, 25.59, 24.94, 21.48,

7.13. MS (ESI), m/z for C₃₁H₃₅N₃O₃S ([M + H]⁺): calcd 529.70, found

530.7. HPLC analysis: MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R=

26.25 min, 96.87% purity.

1

for 25m. White solid, 26% yield. H NMR (400 MHz, DMSO-d₆) δ

10.53 (s, 1H), 7.87 (d, J = 8.2 Hz, 2H), 7.80 (d, J = 8.6 Hz, 2H), 7.71 (d,

J = 8.6 Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H), 7.53 (d, J = 8.2 Hz, 1H), 7.45

(s, 1H), 7.08 (d, J = 8.1 Hz, 1H), 4.26 (t, J = 7.3 Hz, 2H), 3.86 (s, 2H),

3.33−3.23 (m, 2H), 2.47 (s, 3H), 1.71−1.59 (m, 2H), 1.19−1.07 (m,

5H), 0.76 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ

168.57, 152.01, 141.92, 140.24, 139.85, 136.87, 135.62, 131.90, 130.24,

129.73, 127.84, 124.86, 123.66, 118.98, 118.21, 110.61, 49.21, 43.76,

42.96, 31.08, 21.44, 19.18, 13.28, 7.13. MS (ESI), m/z for

C₂₈H₃₁N₃O₃S ([M + H]⁺): calcd 489.63, found 490.5. HPLC analysis:

MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R= 15.27 min, 96.30% purity.

4.3.24. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methyl-1-pentyl-1H-

benzo[d]imidazol-2-yl)phenyl)acetamide (25c). Compound 25c

was prepared according to the general procedure as described for

25m. White solid, 31% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.50

(s, 1H), 7.86 (d, J = 8.2 Hz, 2H), 7.79 (d, J = 8.6 Hz, 2H), 7.70 (d, J =

8.6 Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 8.2 Hz, 1H), 7.41 (s,

1H), 7.06 (d, J = 8.1 Hz, 1H), 4.24 (t, J = 7.3 Hz, 2H), 3.86 (s, 2H), 3.28

(q, J = 7.2 Hz, 2H), 2.46 (s, 3H), 1.70−1.61 (m, 2H), 1.20−1.06 (m,

7H), 0.75 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ

168.54, 152.13, 141.93, 140.36, 140.13, 136.86, 135.76, 131.71, 130.24,

129.65, 127.84, 125.23, 123.47, 118.95, 118.41, 110.49, 49.21, 43.89,

42.97, 28.68, 28.05, 21.45, 21.43, 13.70, 7.13. High-resolution mass

spectrometry (HRMS) (ESI) for C₂₉H₃₃N₃O₃S ([M + 1]⁺), calcd

503.2243, found 504.2285. HPLC analysis: MeOH (1‰ NH₃·H₂O)−

H₂O (75:25), t_R= 22.24 min, 98.40% purity.

4.3.25. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(1-isopropyl-6-methyl-

1H-benzo[d]imidazol-2-yl)phenyl)acetamide (25d). Compound

25d was prepared according to the general procedure as described

for 25m. White solid, 28% yield. ¹H NMR (400 MHz, DMSO) δ 10.52

(s, 1H), 7.86 (d, J = 8.2 Hz, 2H), 7.78 (d, J = 8.5 Hz, 2H), 7.64 (d, J =

8.2 Hz, 2H), 7.61−7.56 (m, 3H), 7.51 (d, J = 8.2 Hz, 1H), 7.03 (d, J =

8.1 Hz, 1H), 4.75−4.63 (m, 1H), 3.86 (s, 2H), 3.28 (q, J = 7.3 Hz, 2H),

2.46 (s, 3H), 1.57 (d, J = 6.9 Hz, 6H), 1.11 (t, J = 7.3 Hz, 3H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 168.53, 152.28, 141.98, 141.49, 140.06,

136.85, 133.55, 131.19, 130.20, 129.95, 127.84, 125.68, 123.04, 118.94,

118.71, 112.36, 49.20, 48.32, 42.96, 21.47, 20.87, 7.13. MS (ESI), m/z

for C₂₇H₂₉N₃O₃S ([M + H]⁺): calcd 475.61, found 476.3. HPLC

analysis: MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R= 13.39 min,

98.90% purity.

4.3.26. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(1-isobutyl-6-methyl-1H-

benzo[d]imidazol-2-yl)phenyl)acetamide (25e). Compound 25e was

prepared according to the general procedure as described for 25m.

4.3.31. N-(4-(1-Benzyl-6-methyl-1H-benzo[d]imidazol-2-yl)-

phenyl)-2-(4-(ethylsulfonyl)phenyl)acetamide (25j). Compound 25j

was prepared according to the general procedure as described for 25m.

1

White solid, 27% yield. H NMR (400 MHz, DMSO-d₆) δ 10.54 (s,

1H), 7.87 (d, J = 8.2 Hz, 2H), 7.80 (d, J = 8.6 Hz, 2H), 7.73 (d, J = 8.6

Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.2 Hz, 1H), 7.50 (s, 1H),

7.10 (d, J = 8.2 Hz, 1H), 4.16 (d, J = 7.5 Hz, 2H), 3.87 (s, 2H), 3.28 (q, J

= 7.2 Hz, 2H), 2.47 (s, 3H), 2.01−1.87 (m, 1H), 1.11 (t, J = 7.3 Hz,

3H), 0.64 (d, J = 6.6 Hz, 6H). ¹³C NMR (126 MHz, DMSO-d₆) δ

168.61, 152.11, 141.91, 140.64, 140.35, 136.88, 135.68, 132.20, 130.25,

129.91, 127.84, 124.62, 123.96, 118.97, 117.85, 111.17, 51.04, 49.21,

42.97, 26.32, 21.43, 19.51, 7.13. MS (ESI), m/z for C₂₈H₃₁N₃O₃S ([M

+ H]⁺): calcd 489.63, found 490.5. HPLC analysis: MeOH (1‰ NH₃·

H₂O)−H₂O (75:25), t_R= 15.56 min, 98.45% purity.

1

White solid, 20% yield. H NMR (400 MHz, DMSO-d₆) δ 10.48 (s,

1H), 7.85 (d, J = 8.2 Hz, 2H), 7.72 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 8.7

Hz, 2H), 7.61 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 8.2 Hz, 1H), 7.32− 7.20

(m, 4H), 7.08 (d, J = 8.1 Hz, 1H), 6.99 (d, J = 7.2 Hz, 2H), 5.55 (s, 2H),

3.84 (s, 2H), 3.32−3.22 (m, 2H), 2.39 (s, 3H), 1.10 (t, J = 7.3 Hz, 3H).

¹³C NMR (126 MHz, DMSO-d₆) δ 168.54, 152.39, 141.89, 140.65,

140.31, 136.97, 136.86, 136.09, 132.09, 130.21, 129.59, 128.77, 127.82,

127.42, 125.99, 124.67, 123.85, 118.95, 118.51, 110.66, 49.21, 47.33,

42.93, 21.41, 7.12. MS (ESI), m/z for C₃₁H₂₉N₃O₃S ([M + H]⁺): calcd

523.65, found 524.3. HPLC analysis: MeOH (1‰ NH₃·H₂O)−H₂O

(75:25), t_R= 15.73 min, 95.30% purity.

4.3.32. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methyl-1-(4-methyl-

benzyl)-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (25k). Com-

pound 25k was prepared according to the general procedure as

described for 25m. White solid, 25% yield. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.49 (s, 1H), 7.85 (d, J = 8.2 Hz, 2H), 7.73 (d, J = 8.7 Hz,

2H), 7.66 (d, J = 8.7 Hz, 2H), 7.62 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 8.2

Hz, 1H), 7.25 (s, 1H), 7.12−7.03 (m, 3H), 6.88 (d, J = 7.9 Hz, 2H),

5.49 (s, 2H), 3.84 (s, 2H), 3.27 (q, J = 7.2 Hz, 2H), 2.39 (s, 3H), 2.22 (s,

3H), 1.10 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ

168.38, 152.20, 141.74, 141.33, 140.14, 136.68, 136.43, 135.91, 133.76,

4.3.27. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(1-isopentyl-6-methyl-

1H-benzo[d]imidazol-2-yl)phenyl)acetamide (25f). Compound 25f

was prepared according to the general procedure as described for 25m.

1

White solid, 41% yield. H NMR (400 MHz, DMSO-d₆) δ 10.50 (s,

1H), 7.87 (d, J = 6.9 Hz, 2H), 7.80 (d, J = 7.4 Hz, 2H), 7.70 (d, J = 7.3

Hz, 2H), 7.64 (d, J = 7.0 Hz, 2H), 7.52 (d, J = 7.4 Hz, 1H), 7.39 (s, 1H),

7.05 (d, J = 7.2 Hz, 1H), 4.33−4.16 (m, 2H), 3.87 (s, 2H), 3.32−3.22

(m, 2H), 2.47 (s, 3H), 1.63−1.52 (m, 2H), 1.51−1.41 (m, 1H), 1.11 (t,

J = 7.4 Hz, 3H), 0.79 (d, J = 5.0 Hz, 6H). ¹³C NMR (126 MHz, DMSO-

d₆) δ 168.53, 152.16, 141.92, 140.70, 140.09, 136.86, 135.74, 131.62,

130.25, 129.65, 127.83, 125.35, 123.37, 118.91, 118.56, 110.35, 49.20,

42.95, 42.45, 37.83, 25.24, 22.04, 21.46, 7.13. MS (ESI), m/z for

8789

https://doi.org/10.1021/acs.jmedchem.1c00763

J. Med. Chem. 2021, 64, 8775−8797

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

131.86, 130.05, 129.40, 129.16, 127.66, 125.77, 124.55, 123.64, 118.79,

118.34, 110.50, 49.04, 46.95, 42.77, 21.25, 20.40, 6.96. MS (ESI), m/z

for C₃₂H₃₁N₃O₃S ([M + H]⁺): calcd 537.68, found 538.6. HPLC

analysis: MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R= 18.15 min,

95.68% purity.

found 552.2279. HPLC analysis: MeOH (1‰ NH₃·H₂O)−H₂O

(75:25), t_R= 19.75 min, 97.52% purity.

4.3.35. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methyl-1-(pyridin-4-

ylmethyl)-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (26j).

4.3.35.1. Step 1. To a solution of 12a (2 g, 12.9 mmol) in DMF (20

mL) was added 4-pyridinemethaneamine (1.68 g, 15.5 mmol) and

K₂CO₃(2.68 g, 19.4 mmol). The mixture was stirred at 80 °C for 1 h.

Water was added, and the mixture was extracted with EtOAc (50 mL ×

3). The organic layer was washed with brine, dried over Na₂SO₄, and

evaporated. The resulting crude product was puriﬁed by silica gel

chromatography with petroleum ether/EtOAc (4/1, v/v) to obtain 5-

methyl-2-nitro-N-(pyridin-4-ylmethyl)aniline (14j) as a yellow solid

(2.2 g, 70% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 8.72 (t, J = 6.2

Hz, 1H), 8.51 (d, J = 5.9 Hz, 2H), 7.99 (d, J = 8.7 Hz, 1H), 7.34 (d, J =

5.5 Hz, 2H), 6.65 (s, 1H), 6.52 (dd, J = 8.8, 0.8 Hz, 1H), 4.68 (d, J = 6.4

Hz, 2H), 2.19 (s, 3H).

4.3.33. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methyl-1-phenethyl-

1H-benzo[d]imidazol-2-yl)phenyl)acetamide (25l). Compound 25l

was prepared according to the general procedure as described for 25m.

1

White solid, 45% yield. H NMR (400 MHz, DMSO-d₆) δ 10.48 (s,

1H), 7.87 (d, J = 8.2 Hz, 2H), 7.72 (d, J = 8.5 Hz, 2H), 7.64 (d, J = 8.2

Hz, 2H), 7.55−7.47 (m, 3H), 7.45 (s, 1H), 7.22−7.13 (m, 3H), 7.06 (d,

J = 8.2 Hz, 1H), 7.02−6.95 (m, 2H), 4.43 (t, J = 7.3 Hz, 2H), 3.86 (s,

2H), 3.28 (q, J = 7.6 Hz, 2H), 3.01 (t, J = 7.3 Hz, 2H), 2.47 (s, 3H), 1.11

(t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.50, 152.40,

141.93, 140.61, 139.99, 137.83, 136.86, 135.61, 131.66, 130.26, 129.62,

128.63, 128.32, 127.83, 126.49, 125.19, 123.43, 118.76, 118.51, 110.66,

49.21, 45.57, 42.95, 34.92, 21.44, 7.13. MS (ESI), m/z for

C₃₂H₃₁N₃O₃S ([M + H]⁺): calcd 537.68, found 538.6. HPLC analysis:

MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R= 16.55 min, 98.25% purity.

4.3.34. (S)-2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methyl-1-(1-(p-

tolyl)ethyl)-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (25p).

4.3.34.1. Step 1. (S)-5-Methyl-2-nitro-N-(1-(p-tolyl)ethyl)aniline

(13p) was prepared from 12a and (S)-1-(p-tolyl)ethan-1-amine

according to the general procedure of step 1 as described for 25m.

Yellow oil, 85% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 8.33 (d, J = 6.6

Hz, 1H), 7.96 (d, J = 8.7 Hz, 1H), 7.30 (d, J = 7.8 Hz, 2H), 7.15 (d, J =

7.7 Hz, 2H), 6.69 (s, 1H), 6.50 (d, J = 8.4 Hz, 1H), 4.92−4.81 (m, 1H),

2.26 (s, 3H), 2.18 (s, 3H), 1.53 (d, J = 6.8 Hz, 3H).

4.3.35.2. Step 2. 5-Methyl-N¹-(pyridin-4-ylmethyl)benzene-1,2-

diamine (17j) was prepared from 14j according to the general

procedure of step 2 as described for 25m. Yellow liquid, 90% yield. ¹H

NMR (400 MHz, DMSO-d₆) δ 8.48 (d, J = 4.8 Hz, 2H), 7.35 (d, J = 4.8

Hz, 2H), 6.46 (d, J = 7.6 Hz, 1H), 6.21 (d, J = 7.5 Hz, 1H), 6.08 (s, 1H),

5.18 (t, J = 6.0 Hz, 1H), 4.40 (s, 2H), 4.32 (d, J = 5.0 Hz, 2H), 2.01 (s,

3H).

4.3.35.3. Step 3. 6-Methyl-2-(4-nitrophenyl)-1-(pyridin-4-ylmeth-

yl)-1H-benzo[d]imidazole (20j) was prepared from 17j according to

the general procedure of step 3 as described for 25m. Yellow solid, 35%

yield. ¹H NMR (400 MHz, DMSO-d₆) δ 8.47 (d, J = 5.9 Hz, 2H), 8.33

(d, J = 8.8 Hz, 2H), 7.97 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 8.2 Hz, 1H),

7.33 (s, 1H), 7.15 (d, J = 8.3 Hz, 1H), 6.97 (d, J = 5.7 Hz, 2H), 5.68 (s,

2H), 2.41 (s, 3H).

4.3.34.2. Step 2. (S)-5-Methyl-N¹-(1-(p-tolyl)ethyl)benzene-1,2-

diamine (16p) was prepared from 13p according to the general

procedure of step 2 as described for 25m. Yellow-purple oil, 90% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 7.24 (d, J = 7.5 Hz, 2H), 7.08 (d, J =

7.4 Hz, 2H), 6.40 (d, J = 7.6 Hz, 1H), 6.13 (d, J = 7.4 Hz, 1H), 6.02 (s,

1H), 4.71 (d, J = 6.2 Hz, 1H), 4.52−4.29 (m, 3H), 2.24 (s, 3H), 1.94 (s,

3H), 1.41 (d, J = 6.5 Hz, 3H).

4.3.34.3. Step 3. (S)-6-Methyl-2-(4-nitrophenyl)-1-(1-(p-tolyl)-

ethyl)-1H-benzo[d]imidazole (19p) was prepared from 16p according

to the general procedure of step 3 as described for 25m. Yellow solid,

79% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 8.38 (d, J = 8.8 Hz, 2H),

7.95 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.2 Hz, 1H), 7.15 (d, J = 8.1 Hz,

2H), 7.10−7.00 (m, 4H), 5.80 (q, J = 6.9 Hz, 1H), 2.32 (s, 3H), 2.27 (s,

3H), 1.96 (d, J = 7.1 Hz, 3H).

4.3.34.4. Step 4. (S)-4-(6-Methyl-1-(1-(p-tolyl)ethyl)-1H-benzo-

[d]imidazol-2-yl)aniline (22p) was prepared from 19p according to the

general procedure of step 2 as described for 25m. Yellow solid, 93%

yield. ¹H NMR (400 MHz, DMSO-d₆) δ 7.47 (d, J = 8.1 Hz, 1H), 7.31

(d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 7.05 (d, J = 8.0 Hz, 2H),

6.94 (d, J = 8.1 Hz, 1H), 6.88 (s, 1H), 6.66 (d, J = 8.4 Hz, 2H), 5.80 (q, J

= 6.8 Hz, 1H), 5.54 (s, 2H), 2.27 (s, 3H), 2.26 (s, 3H), 1.90 (d, J = 7.1

Hz, 3H).

4.3.35.4. Step 4. 4-(6-Methyl-1-(pyridin-4-ylmethyl)-1H-benzo[d]-

imidazol-2-yl)aniline (23j) was prepared from 20j according to the

general procedure of step 2 as described for 25m. Yellow solid, 69%

yield. ¹H NMR (400 MHz, DMSO-d₆) δ 8.49 (d, J = 6.0 Hz, 2H), 7.53

(d, J = 8.2 Hz, 1H), 7.32 (d, J = 8.6 Hz, 2H), 7.15 (s, 1H), 7.04 (dd, J =

8.2, 0.8 Hz, 1H), 6.98 (d, J = 5.9 Hz, 2H), 6.60 (d, J = 8.6 Hz, 2H),

5.60−5.47 (m, 4H), 2.37 (s, 3H).

4.3.35.5. Step 5. Compound 26j was prepared from 23j and 2-(4-

(ethylsulfonyl)phenyl)acetic acid according to the general procedure of

1

step 5 as described for 25p. White solid, 26% yield. H NMR (400

MHz, DMSO-d₆) δ 10.55 (s, 1H), 8.50−8.43 (m, 2H), 7.84 (d, J = 8.3

Hz, 2H), 7.72 (d, J = 8.7 Hz, 2H), 7.66−7.57 (m, 5H), 7.24 (s, 1H),

7.09 (dd, J = 8.2, 0.8 Hz, 1H), 6.97−6.93 (m, 2H), 5.58 (s, 2H), 3.84 (s,

2H), 3.26 (q, J = 7.6 Hz, 2H), 2.39 (s, 3H), 1.10 (t, J = 7.3 Hz, 3H). ¹³C

NMR (126 MHz, DMSO-d₆) δ 168.59, 152.46, 149.90, 146.18, 141.96,

140.43, 140.02, 136.83, 136.06, 132.29, 130.21, 129.50, 127.81, 124.41,

124.00, 121.13, 118.97, 118.70, 110.44, 49.20, 46.47, 42.91, 21.39, 7.12.

MS (ESI), m/z for C₃₀H₂₈N₄O₃S ([M + H]⁺): calcd 524.64, found

525.4. HPLC analysis: MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R=

10.47 min, 98.16% purity.

4.3.34.5. Step 5. To a solution of 2-(4-(ethylsulfonyl)phenyl)acetic

acid (121.0 mg, 0.53 mmol) in DCM (10 mL) were added EDCI (126.5

mg, 0.66 mol), HOBT (89.2 mg, 0.66 mmol), and DIPEA (170.6 mg,

1.32 mmol). The mixture was stirred at room temperature for 15 min.

22p (150.2 mg, 0.44 mmol) was then added to the mixture. The

reaction mixture was stirred at room temperature for overnight. Water

was added, and the mixture was extracted with EtOAc (50 mL × 3).

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

evaporated. The resulting crude product was puriﬁed by silica gel

chromatography with petroleum ether/EtOAc (1/1, v/v) to obtain 25p

as a white solid (53.4 mg, 22% yield). ¹H NMR (400 MHz, DMSO-d₆)

δ 10.51 (s, 1H), 7.85 (d, J = 8.1 Hz, 2H), 7.77 (d, J = 8.5 Hz, 2H), 7.66−

7.58 (m, 4H), 7.53 (d, J = 8.1 Hz, 1H), 7.14 (d, J = 7.8 Hz, 2H), 7.05 (d,

J = 7.9 Hz, 2H), 6.99 (d, J = 8.3 Hz, 1H), 6.95 (s, 1H), 5.82−5.72 (m,

1H), 3.85 (s, 2H), 3.31−3.22 (m, 2H), 2.29 (s, 3H), 2.26 (s, 3H), 1.92

(d, J = 7.0 Hz, 3H), 1.10 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 168.55, 152.96, 141.94, 141.44, 140.25, 137.05, 136.85,

136.53, 133.75, 131.24, 130.21, 129.91, 129.20, 127.84, 125.90, 125.37,

123.27, 119.01, 118.95, 112.42, 53.70, 49.20, 42.96, 21.46, 20.54, 18.32,

7.13. HRMS (ESI), m/z for C₃₃H₃₃N₃O₃S ([M + H]⁺): calcd 551.2243,

4.3.36. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methyl-1-(1-(p-tolyl)-

ethyl)-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (25n). Com-

pound 25n was prepared according to the general procedure as

described for 25p. White solid, 25% yield. ¹H NMR (400 MHz, DMSO-

d₆) δ 10.52 (s, 1H), 7.85 (d, J = 8.2 Hz, 2H), 7.77 (d, J = 8.6 Hz, 2H),

7.66−7.58 (m, 4H), 7.53 (d, J = 8.2 Hz, 1H), 7.14 (d, J = 8.0 Hz, 2H),

7.05 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 8.4 Hz, 1H), 6.95 (s, 1H), 5.76 (q, J

= 6.8 Hz, 1H), 3.85 (s, 2H), 3.27 (q, J = 7.2 Hz, 2H), 2.29 (s, 3H), 2.26

(s, 3H), 1.92 (d, J = 7.1 Hz, 3H), 1.10 (t, J = 7.3 Hz, 3H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 168.55, 152.96, 141.94, 141.46, 140.24,

137.05, 136.85, 136.52, 133.76, 131.22, 130.20, 129.90, 129.19, 127.84,

125.90, 125.38, 123.26, 119.01, 118.96, 112.42, 53.69, 49.20, 42.95,

21.45, 20.53, 18.32, 7.13. MS (ESI), m/z for C₃₃H₃₃N₃O₃S ([M + H]⁺):

calcd 551.71, found 552.6. HPLC analysis: MeOH (1‰ NH₃·H₂O)−

H₂O (75:25), t_R= 21.45 min, 98.20% purity.

4.3.37. (R)-2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methyl-1-(1-(p-

tolyl)ethyl)-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (25o).

Compound 25o was prepared according to the general procedure as

described for 25p. White solid, 18% yield. ¹H NMR (400 MHz, DMSO-

d₆) δ 10.52 (s, 1H), 7.85 (d, J = 8.2 Hz, 2H), 7.77 (d, J = 8.5 Hz, 2H),

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7.66−7.58 (m, 4H), 7.53 (d, J = 8.2 Hz, 1H), 7.14 (d, J = 8.0 Hz, 2H),

7.05 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 8.2 Hz, 1H), 6.95 (s, 1H), 5.76 (q, J

= 6.8 Hz, 1H), 3.85 (s, 2H), 3.27 (q, J = 7.2 Hz, 2H), 2.29 (s, 3H), 2.26

(s, 3H), 1.92 (d, J = 7.1 Hz, 3H), 1.10 (t, J = 7.4 Hz, 3H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 168.53, 152.95, 141.92, 141.45, 140.22,

137.04, 136.85, 136.51, 133.75, 131.21, 130.18, 129.88, 129.17, 127.82,

125.88, 125.38, 123.24, 119.00, 118.94, 112.40, 53.68, 49.20, 42.94,

21.44, 20.52, 18.31, 7.11. HRMS (ESI), m/z for C₃₃H₃₃N₃O₃S ([M +

H]⁺): calcd 551.2243, found 552.2293. HPLC analysis: MeOH (1‰

NH₃·H₂O)−H₂O (75:25), t_R= 21.05 min, 98.17% purity. [α]_D²⁵= + 81

(c 0.1, MeOH).

4.3.38. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(1-(4-ﬂuorobenzyl)-6-

methyl-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (26a). Com-

pound 26a was prepared according to the general procedure as

described for 25m. White solid, 26% yield. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.48 (s, 1H), 7.85 (d, J = 8.3 Hz, 2H), 7.73 (d, J = 8.7 Hz,

2H), 7.65 (d, J = 8.7 Hz, 2H), 7.62 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 8.2

Hz, 1H), 7.28 (s, 1H), 7.11 (t, J = 8.8 Hz, 2H), 7.07 (d, J = 8.4 Hz, 1H),

7.04−6.98 (m, 2H), 5.53 (s, 2H), 3.84 (s, 2H), 3.27 (q, J = 7.2 Hz, 2H),

2.40 (s, 3H), 1.10 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆)

δ 168.53, 162.29, 160.35, 152.43, 141.91, 140.81, 140.22, 136.85,

136.10, 133.25, 133.23, 131.99, 130.22, 129.55, 128.12, 128.06, 127.83,

124.93, 123.72, 118.95, 118.73, 115.67, 115.50, 110.55, 49.20, 46.61,

42.94, 21.42, 7.13. MS (ESI), m/z for C₃₁H₂₈FN₃O₃S ([M + H]⁺):

calcd 541.64, found 542.3. HPLC analysis: MeOH (1‰ NH₃·H₂O)−

H₂O (75:25), t_R= 13.69 min, 98.87% purity.

4.3.39. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(1-(3-ﬂuorobenzyl)-6-

methyl-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (26b). Com-

pound 26b was prepared according to the general procedure as

described for 25m. White solid, 29% yield. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.48 (s, 1H), 7.85 (d, J = 8.3 Hz, 2H), 7.73 (d, J = 8.7 Hz,

2H), 7.65 (d, J = 8.8 Hz, 2H), 7.62 (d, J = 8.3 Hz, 2H), 7.58 (d, J = 8.2

Hz, 1H), 7.36−7.29 (m, 1H), 7.28 (s, 1H), 7.12−7.03 (m, 2H), 6.82 (d,

J = 9.9 Hz, 1H), 6.76 (d, J = 7.8 Hz, 1H), 5.56 (s, 2H), 3.84 (s, 2H),

3.31−3.23 (m, 2H), 2.40 (s, 3H), 1.10 (t, J = 7.4 Hz, 3H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 168.53, 163.21, 161.27, 152.45, 141.90,

140.71, 140.27, 140.05, 139.99, 136.85, 136.12, 132.10, 130.91, 130.84,

130.21, 129.54, 127.82, 124.78, 123.82, 121.93, 118.97, 118.73, 114.37,

114.21, 113.08, 112.90, 110.49, 49.20, 46.81, 42.93, 21.41, 7.12. MS

(ESI), m/z for C₃₁H₂₈FN₃O₃S ([M + H]⁺): calcd 541.64, found 542.3.

HPLC analysis: MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R= 16.75

min, 99.04% purity.

4.3.40. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(1-(2-ﬂuorobenzyl)-6-

methyl-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (26c). Com-

pound 26c was prepared according to the general procedure as

described for 25m. White solid, 25% yield. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.48 (s, 1H), 7.85 (d, J = 8.2 Hz, 2H), 7.72 (d, J = 8.6 Hz,

2H), 7.67−7.55 (m, 5H), 7.34−7.25 (m, 2H), 7.20 (t, J = 8.7 Hz, 1H),

7.11−7.02 (m, 2H), 6.69 (t, J = 7.6 Hz, 1H), 5.57 (s, 2H), 3.84 (s, 2H),

3.27 (q, J = 7.2 Hz, 2H), 2.39 (s, 3H), 1.10 (t, J = 7.3 Hz, 3H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 168.53, 160.40, 158.45, 152.59, 141.90,

140.58, 140.25, 136.85, 136.07, 132.08, 130.22, 129.67, 129.61, 129.51,

127.82, 127.76, 124.78, 123.85, 123.81, 123.73, 118.93, 118.69, 115.59,

115.42, 110.47, 49.20, 42.93, 41.90, 21.41, 7.12. MS (ESI), m/z for

C₃₁H₂₈FN₃O₃S ([M + H]⁺): calcd 541.64, found 542.3. HPLC analysis:

MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R= 15.12 min, 95.06% purity.

4.3.41. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(1-(4-ﬂuorophenethyl)-6-

methyl-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (26d). Com-

pound 26d was prepared according to the general procedure as

described for 25p. White solid, 29% yield. ¹H NMR (400 MHz, DMSO-

d₆) δ 10.47 (s, 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 8.7 Hz, 2H),

7.64 (d, J = 8.4 Hz, 2H), 7.53−7.47 (m, 3H), 7.43 (s, 1H), 7.05 (dd, J =

8.2, 0.8 Hz, 1H), 6.99−6.94 (m, 4H), 4.44 (t, J = 7.3 Hz, 2H), 3.86 (s,

2H), 3.31−3.23 (m, 2H), 2.98 (t, J = 7.4 Hz, 2H), 2.46 (s, 3H), 1.11 (t, J

= 7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.51, 162.03,

160.10, 152.40, 141.94, 140.58, 139.99, 136.86, 135.59, 133.99, 133.97,

131.67, 130.49, 130.42, 130.26, 129.57, 127.84, 125.17, 123.45, 118.75,

118.52, 115.06, 114.89, 110.68, 49.22, 45.54, 42.96, 34.01, 21.44, 7.14.

MS (ESI), m/z for C₃₂H₃₀FN₃O₃S ([M − H]⁻): calcd 555.67, found

554.7. HPLC analysis: MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R=

14.47 min, 98.59% purity.

4.3.42. N-(4-(1-(2,6-Diﬂuorobenzyl)-6-methyl-1H-benzo[d]-

imidazol-2-yl)phenyl)-2-(4-(ethylsulfonyl)phenyl)acetamide (26e).

Compound 26e was prepared according to the general procedure as

described for 25m. White solid, 35% yield. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.49 (s, 1H), 7.86 (d, J = 8.2 Hz, 2H), 7.76 (d, J = 8.6 Hz,

2H), 7.69 (d, J = 8.6 Hz, 2H), 7.64 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 8.1

Hz, 1H), 7.39−7.28 (m, 1H), 7.18 (s, 1H), 7.07−6.94 (m, 3H), 5.60 (s,

2H), 3.86 (s, 2H), 3.28 (q, J = 7.6 Hz, 2H), 2.38 (s, 3H), 1.10 (t, J = 7.3

Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.50, 161.59, 161.53,

159.62, 159.55, 153.01, 141.95, 140.65, 140.08, 136.86, 135.57, 131.65,

130.85, 130.76, 130.68, 130.24, 129.82, 127.84, 125.32, 123.47, 118.76,

118.72, 112.07, 111.93, 111.82, 111.78, 111.62, 109.98, 49.21, 42.96,

36.98, 21.52, 7.13. MS (ESI), m/z for C₃₁H₂₇F₂N₃O₃S ([M + H]⁺):

calcd 559.63, found 560.3. HPLC analysis: MeOH (1‰ NH₃·H₂O)−

H₂O (75:25), t_R= 13.55 min, 95.88% purity.

4.3.43. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methyl-1-(4-

(triﬂuoromethyl)benzyl)-1H-benzo[d]imidazol-2-yl)phenyl)-

acetamide (26f). Compound 26f was prepared according to the

1

general procedure as described for 25p. White solid, 23% yield. H

NMR (400 MHz, DMSO-d₆) δ 10.48 (s, 1H), 7.85 (d, J = 8.1 Hz, 2H),

7.75−7.54 (m, 9H), 7.26 (s, 1H), 7.18 (d, J = 8.1 Hz, 2H), 7.08 (d, J =

8.1 Hz, 1H), 5.65 (s, 2H), 3.83 (s, 2H), 3.32−3.22 (m, 2H), 2.39 (s,

3H), 1.09 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ

168.54, 152.49, 141.91, 140.84, 140.28, 136.85, 136.13, 134.05, 132.15,

130.21, 129.52, 128.64, 128.38, 128.13, 127.83, 126.75, 125.74, 125.71,

125.18, 124.76, 123.86, 123.02, 122.70, 118.99, 118.82, 110.45, 49.20,

46.95, 42.94, 21.41, 7.13. MS (ESI), m/z for C₃₂H₂₈F₃N₃O₃S ([M −

H]⁻): calcd 591.65, found 590.3. HPLC analysis: MeOH (1‰ NH₃·

H₂O)−H₂O (70:30), t_R= 29.32 min, 98.06% purity.

4.3.44. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methyl-1-(4-

(methylsulfonyl)benzyl)-1H-benzo[d]imidazol-2-yl)phenyl)-

acetamide (26g). Compound 26g was prepared according to the

1

general procedure as described for 25m. White solid, 49% yield. H

NMR (400 MHz, DMSO-d₆) δ 10.49 (s, 1H), 7.90−7.81 (m, 4H), 7.72

(d, J = 8.6 Hz, 2H), 7.68−7.56 (m, 5H), 7.29−7.18 (m, 3H), 7.08 (d, J

= 8.1 Hz, 1H), 5.67 (s, 2H), 3.83 (s, 2H), 3.33−3.23 (m, 2H), 3.17 (s,

3H), 2.39 (s, 3H), 1.09 (t, J = 7.2 Hz, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 168.56, 152.49, 143.01, 141.91, 140.85, 140.29, 139.89,

136.85, 136.09, 132.18, 130.21, 129.54, 127.84, 127.54, 126.81, 124.74,

123.90, 119.02, 118.84, 110.45, 49.21, 46.98, 43.40, 42.94, 21.42, 7.14.

MS (ESI), m/z for C₃₂H₃₁N₃O₅S₂([M − H]⁻): calcd 601.74, found

600.8. HPLC analysis: MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R=

7.52 min, 95.25% purity.

4.3.45. Methyl 4-((2-(4-(2-(4-(Ethylsulfonyl)phenyl)acetamido)-

phenyl)-6-methyl-1H-benzo[d]imidazol-1-yl)methyl)benzoate

(26h). Compound 26h was prepared according to the general

1

procedure as described for 25p. White solid, 29% yield. H NMR

(400 MHz, DMSO-d₆) δ 10.48 (s, 1H), 7.88 (d, J = 8.2 Hz, 2H), 7.84

(d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.7 Hz, 2H), 7.66−7.56 (m, 5H), 7.24

(s, 1H), 7.12 (d, J = 8.2 Hz, 2H), 7.08 (dd, J = 8.2, 0.4 Hz, 1H), 5.63 (s,

2H), 3.83 (s, 2H), 3.81 (s, 3H), 3.27 (q, J = 7.2 Hz, 2H), 2.38 (s, 3H),

1.09 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.53,

165.84, 152.50, 142.57, 141.90, 140.79, 140.26, 136.85, 136.19, 132.10,

130.22, 129.68, 129.51, 128.76, 127.83, 126.29, 124.78, 123.83, 118.95,

118.78, 110.46, 52.10, 49.21, 47.18, 42.94, 21.41, 7.13. MS (ESI), m/z

for C₃₃H₃₁N₃O₅S ([M + H]⁺): calcd 581.69, found 582.4. HPLC

analysis: MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R= 37.07 min,

95.98% purity.

4.3.46. 4-((2-(4-(2-(4-(Ethylsulfonyl)phenyl)acetamido)phenyl)-6-

methyl-1H-benzo[d]imidazol-1-yl)methyl)benzoic Acid (26i). Com-

pound 26i was prepared according to the general procedure as

described for 10m. White solid, 69% yield. ¹H NMR (500 MHz,

DMSO) δ 10.78 (s, 1H), 7.87 (d, J = 8.2 Hz, 2H), 7.84 (d, J = 8.2 Hz,

2H), 7.81 (d, J = 8.3 Hz, 2H), 7.74−7.66 (m, 3H), 7.62 (d, J = 7.9 Hz,

2H), 7.43 (s, 1H), 7.26 (d, J = 7.9 Hz, 1H), 7.18 (d, J = 8.0 Hz, 2H),

5.71 (s, 2H), 3.87 (s, 2H), 3.27 (q, J = 7.5 Hz, 2H), 2.42 (s, 3H), 1.09 (t,

J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.81, 168.82,

151.46, 142.36, 141.90, 141.48, 140.96, 136.85, 136.16, 134.82, 130.23,

130.17, 130.00, 129.82, 127.79, 127.68, 126.31, 125.52, 123.38, 119.05,

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118.86, 111.36, 49.21, 47.60, 42.88, 21.34, 7.11. MS (ESI), m/z for

C₃₂H₂₉N₃O₅S ([M + H]⁺): calcd 567.66, found 568.7. HPLC analysis:

MeOH−H₂O (65:35), t_R= 3.68 min, 96.75% purity.

amine according to the general procedure of step 5 as described for

25m. Yellow solid, 95% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 8.59

(d, J = 7.2 Hz, 1H), 8.25 (t, J = 8.1 Hz, 1H), 7.97 (dd, J = 12.1, 1.5 Hz,

1H), 7.87 (d, J = 8.5 Hz, 1H), 4.14−4.02 (m, 1H), 1.18 (d, J = 6.6 Hz,

6H).

4.3.51.2. Step 2. (R)-N-Isopropyl-4-nitro-3-((1-(p-tolyl)ethyl)-

amino)benzamide (15c) was prepared from 12d according to the

general procedure of step 1 as described for 25m. Orange oil, 87% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.31 (d, J = 5.5 Hz, 1H), 8.25 (d, J =

6.0 Hz, 1H), 8.11 (d, J = 8.3 Hz, 1H), 7.31 (d, J = 6.8 Hz, 2H), 7.26 (s,

1H), 7.14 (d, J = 6.8 Hz, 2H), 7.04 (d, J = 8.1 Hz, 1H), 5.02−4.85 (m,

1H), 4.10−3.92 (m, 1H), 2.26 (s, 3H), 1.57 (d, J = 5.4 Hz, 3H), 1.13 (d,

J = 5.6 Hz, 6H).

4.3.51.3. Step 3. (R)-4-Amino-N-isopropyl-3-((1-(p-tolyl)ethyl)-

amino)benzamide (18c) was prepared from 15c according to the

general procedure of step 2 as described for 25m. Colorless oil, 91%

yield. ¹H NMR (400 MHz, DMSO-d₆) δ 7.47 (d, J = 7.8 Hz, 1H), 7.24

(d, J = 7.9 Hz, 2H), 7.07 (d, J = 7.8 Hz, 2H), 6.93 (dd, J = 8.0, 1.4 Hz,

1H), 6.77 (s, 1H), 6.46 (d, J = 8.0 Hz, 1H), 5.14 (s, 2H), 4.90 (d, J = 6.8

Hz, 1H), 4.61−4.47 (m, 1H), 4.00−3.90 (m, 1H), 2.23 (s, 3H), 1.44 (d,

J = 6.7 Hz, 3H), 1.07 (dd, J = 6.5, 2.5 Hz, 6H).

4.3.51.4. Step 4. (R)-N-Isopropyl-2-(4-nitrophenyl)-1-(1-(p-tolyl)-

ethyl)-1H-benzo[d]imidazole-6-carboxamide (21c) was prepared from

18c according to the general procedure of step 3 as described for 25m.

Yellow oil, 77% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (d, J = 8.7

Hz, 2H), 8.20 (d, J = 7.7 Hz, 1H), 7.98 (d, J = 8.7 Hz, 2H), 7.82−7.74

(m, 2H), 7.71 (s, 1H), 7.15 (d, J = 8.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H),

5.85 (q, J = 6.7 Hz, 1H), 4.07−3.98 (m, 1H), 2.26 (s, 3H), 2.00 (d, J =

7.2 Hz, 3H), 1.15 (d, J = 6.5 Hz, 6H).

4.3.47. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methyl-1-(pyridin-3-

ylmethyl)-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (26k). Com-

pound 26k was prepared according to the general procedure as

described for 25p. White solid, 44% yield. ¹H NMR (400 MHz, DMSO-

d₆) δ 10.51 (s, 1H), 8.47−8.39 (m, 1H), 8.30−8.23 (m, 1H), 7.85 (d, J

= 8.1 Hz, 2H), 7.74 (d, J = 8.5 Hz, 2H), 7.67 (d, J = 8.6 Hz, 2H), 7.62 (d,

J = 8.1 Hz, 2H), 7.58 (d, J = 8.3 Hz, 1H), 7.35 (s, 1H), 7.31−7.25 (m,

2H), 7.08 (d, J = 8.1 Hz, 1H), 5.61 (s, 2H), 3.85 (s, 2H), 3.28 (q, J = 7.6

Hz, 2H), 2.40 (s, 3H), 1.10 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 168.56, 152.42, 148.70, 147.71, 141.90, 140.64, 140.32,

136.85, 135.96, 133.94, 132.69, 132.17, 130.23, 129.63, 127.83, 124.73,

123.87, 121.13, 118.98, 118.71, 110.56, 49.20, 45.07, 42.94, 21.41, 7.13.

MS (ESI), m/z for C₃₀H₂₈N₄O₃S ([M + H]⁺): calcd 524.64, found

525.4. HPLC analysis: MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R=

8.88 min, 98.34% purity.

4.3.48. 2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methyl-1-(pyridin-2-

ylmethyl)-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (26l). Com-

pound 26l was prepared according to the general procedure as

described for 25m. White solid, 41% yield. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.46 (s, 1H), 8.49 (d, J = 4.4 Hz, 1H), 7.85 (d, J = 8.1 Hz,

2H), 7.79−7.66 (m, 5H), 7.61 (d, J = 8.1 Hz, 2H), 7.56 (d, J = 8.2 Hz,

1H), 7.31−7.24 (m, 1H), 7.20 (s, 1H), 7.09 (d, J = 7.8 Hz, 1H), 7.05 (d,

J = 8.2 Hz, 1H), 5.57 (s, 2H), 3.83 (s, 2H), 3.27 (q, J = 7.2 Hz, 2H), 2.37

(s, 3H), 1.10 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ

168.52, 156.05, 152.69, 149.52, 141.93, 140.74, 140.18, 137.23, 136.85,

136.33, 131.82, 130.22, 129.66, 127.84, 125.03, 123.61, 122.80, 121.13,

118.88, 118.61, 110.48, 49.22, 49.12, 42.94, 21.42, 7.13. MS (ESI), m/z

for C₃₀H₂₈N₄O₃S ([M + H]⁺): calcd 524.64, found 525.4. HPLC

analysis: MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R= 10.66 min,

97.71% purity.

4.3.51.5. Step 5. (R)-2-(4-Aminophenyl)-N-isopropyl-1-(1-(p-

tolyl)ethyl)-1H-benzo[d]imidazole-6-carboxamide (24c) was pre-

pared from 21c according to the general procedure of step 2 as

1

described for 25m. Yellow solid, 88% yield. H NMR (400 MHz,

4.3.49. (R)-2-(4-(Ethylsulfonyl)phenyl)-N-(4-(1-(1-(p-tolyl)ethyl)-

6-(triﬂuoromethyl)-1H-benzo[d]imidazol-2-yl)phenyl)acetamide

(27a). Compound 27a was prepared from 3-ﬂuoro-4-nitrobenzotri-

ﬂuoride (12b) and (R)-1-(p-tolyl)ethan-1-amine according to the

DMSO-d₆) δ 8.11 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.64−

7.58 (m, 2H), 7.36 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 8.1 Hz, 2H), 7.05 (d,

J = 8.0 Hz, 2H), 6.67 (d, J = 8.4 Hz, 2H), 5.88 (q, J = 6.9 Hz, 1H), 5.65

(s, 2H), 4.10−3.99 (m, 1H), 2.26 (s, 3H), 1.96 (d, J = 7.1 Hz, 3H), 1.13

(d, J = 6.4 Hz, 6H).

1

general procedure as described for 25p. White solid, 44% yield. H

NMR (400 MHz, DMSO-d₆) δ 10.57 (s, 1H), 7.89−7.84 (m, 3H), 7.82

(d, J = 8.5 Hz, 2H), 7.69 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 7.9 Hz, 2H),

7.50 (d, J = 8.5 Hz, 1H), 7.37 (s, 1H), 7.16 (d, J = 7.8 Hz, 2H), 7.10 (d, J

= 7.8 Hz, 2H), 5.95−5.83 (m, 1H), 3.86 (s, 2H), 3.31−3.22 (m, 2H),

2.26 (s, 3H), 1.94 (d, J = 6.9 Hz, 3H), 1.10 (t, J = 7.3 Hz, 3H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 168.66, 156.34, 145.69, 141.88, 140.83,

136.98, 136.87, 136.23, 132.99, 130.21, 130.13, 129.29, 127.83, 126.04,

125.77, 124.39, 123.60, 122.50, 122.25, 122.01, 120.22, 119.06, 118.60,

109.71, 54.17, 49.19, 42.95, 20.52, 18.24, 7.12. MS (ESI), m/z for

C₃₃H₃₀F₃N₃O₃S ([M − H]⁻): calcd 605.68, found 604.4. HPLC

analysis: MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R= 18.14 min,

97.44% purity.

4.3.50. (R)-2-(4-(Ethylsulfonyl)phenyl)-N-(4-(6-methoxy-1-(1-(p-

tolyl)ethyl)-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (27b).

Compound 27b was prepared from 3-ﬂuoro-4-nitroanisole (12c) and

(R)-1-(p-tolyl)ethan-1-amine according to the general procedure as

described for 25p. White solid, 27% yield. ¹H NMR (400 MHz, DMSO-

d₆) δ 10.53 (s, 1H), 7.86 (d, J = 8.2 Hz, 2H), 7.78 (d, J = 8.6 Hz, 2H),

7.66−7.59 (m, 4H), 7.54 (d, J = 8.8 Hz, 1H), 7.15 (d, J = 8.0 Hz, 2H),

7.09 (d, J = 8.0 Hz, 2H), 6.81 (dd, J = 8.8, 2.2 Hz, 1H), 6.53 (d, J = 1.9

Hz, 1H), 5.76 (q, J = 6.8 Hz, 1H), 3.85 (s, 2H), 3.62 (s, 3H), 3.28 (q, J =

7.4 Hz, 2H), 2.26 (s, 3H), 1.91 (d, J = 7.1 Hz, 3H), 1.10 (t, J = 7.3 Hz,

3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.51, 155.15, 152.69,

141.92, 140.14, 137.79, 136.85, 136.70, 136.62, 134.13, 130.17, 129.86,

129.13, 127.80, 126.10, 125.40, 119.70, 119.01, 110.34, 96.50, 55.38,

53.58, 49.20, 42.94, 20.51, 17.97, 7.10. MS (ESI), m/z for

C₃₃H₃₃N₃O₄S ([M − H]⁻): calcd 567.70, found 566.4. HPLC analysis:

MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R= 18.00 min, 96.29% purity.

4.3.51. (R)-2-(4-(2-(4-(Ethylsulfonyl)phenyl)acetamido)phenyl)-

N-isopropyl-1-(1-(p-tolyl)ethyl)-1H-benzo[d]imidazole-6-carboxa-

mide (27c). 4.3.51.1. Step 1. 3-Fluoro-N-isopropyl-4-nitrobenzamide

(12d) was prepared from 3-ﬂuoro-4-nitrobenzoic acid and isopropyl-

4.3.51.6. Step 6. 27c was prepared from 24c and 2-(4-

(ethylsulfonyl)phenyl)acetic acid according to the general procedure

of step 5 as described for 25p. White solid, 26% yield. ¹H NMR (400

MHz, DMSO-d₆) δ 10.57 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.85 (d, J =

8.3 Hz, 2H), 7.79 (d, J = 8.7 Hz, 2H), 7.76−7.60 (m, 7H), 7.14 (d, J =

8.1 Hz, 2H), 7.05 (d, J = 8.0 Hz, 2H), 5.88−5.80 (m, 1H), 4.10−4.01

(m, 1H), 3.86 (s, 2H), 3.32−3.22 (m, 2H), 2.25 (s, 3H), 1.97 (d, J = 7.1

Hz, 3H), 1.14 (d, J = 6.5 Hz, 6H), 1.10 (t, J = 7.4 Hz, 3H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 168.64, 165.29, 155.37, 145.22, 141.94,

140.63, 136.86, 136.72, 136.54, 133.30, 130.22, 130.00, 129.30, 128.73,

127.84, 125.85, 124.83, 121.03, 119.03, 118.52, 112.42, 54.05, 49.21,

42.96, 41.01, 22.38, 22.35, 20.54, 18.58, 7.14. MS (ESI), m/z for

C₃₆H₃₈N₄O₄S ([M − H]⁻): calcd 622.78, found 621.0. HPLC analysis:

MeOH (1‰ NH₃·H₂O)−H₂O (75:25), t_R= 12.93 min, 98.37% purity.

4.3.52. (R)-2-(4-(Ethylsulfonyl)phenyl)-N-(4-(5-methyl-1-(1-(p-

tolyl)ethyl)-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (27d).

Compound 27d was prepared from 4-ﬂuoro-3-nitrotoluene (12e)

and (R)-1-(p-tolyl)ethan-1-amine according to the general procedure

1

as described for 25p. White solid, 31% yield. H NMR (400 MHz,

DMSO-d₆) δ 10.54 (s, 1H), 7.85 (d, J = 8.3 Hz, 2H), 7.78 (d, J = 8.6 Hz,

2H), 7.67−7.60 (m, 4H), 7.44 (s, 1H), 7.12 (d, J = 8.0 Hz, 2H), 7.04 (d,

J = 8.0 Hz, 2H), 6.98 (d, J = 8.3 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 5.76

(q, J = 7.1 Hz, 1H), 3.85 (s, 2H), 3.28 (q, J = 7.4 Hz, 2H), 2.35 (s, 3H),

2.24 (s, 3H), 1.92 (d, J = 7.0 Hz, 3H), 1.09 (t, J = 7.3 Hz, 3H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 168.52, 153.31, 143.65, 141.90, 140.26,

136.97, 136.85, 136.53, 131.52, 130.69, 130.17, 129.90, 129.12, 127.80,

125.93, 125.32, 123.41, 119.02, 118.96, 112.22, 53.68, 49.19, 42.93,

20.98, 20.48, 18.32, 7.09. MS (ESI), m/z for C₃₃H₃₃N₃O₃S ([M −

H]⁻): calcd 551.71, found 550.4. HPLC analysis: MeOH (1‰ NH₃·

H₂O)−H₂O (75:25), t_R= 24.55 min, 95.22% purity.

4.3.53. (R)-2-(4-(Ethylsulfonyl)phenyl)-N-(4-(5-methoxy-1-(1-(p-

tolyl)ethyl)-1H-benzo[d]imidazol-2-yl)phenyl)acetamide (27e).

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Compound 27e was prepared from 4-ﬂuoro-3-nitroanisole (12f) and

(R)-1-(p-tolyl)ethan-1-amine according to the general procedure as

described for 25p. White solid, 25% yield. ¹H NMR (400 MHz, DMSO-

d₆) δ 10.53 (s, 1H), 7.85 (d, J = 7.8 Hz, 2H), 7.78 (d, J = 8.2 Hz, 2H),

7.69−7.58 (m, 4H), 7.17 (s, 1H), 7.12 (d, J = 7.5 Hz, 2H), 7.05 (d, J =

7.5 Hz, 2H), 6.98 (d, J = 8.9 Hz, 1H), 6.70 (d, J = 8.8 Hz, 1H), 5.82−

5.69 (m, 1H), 3.85 (s, 2H), 3.75 (s, 3H), 3.33−3.22 (m, 2H), 2.25 (s,

3H), 1.91 (d, J = 6.7 Hz, 3H), 1.10 (t, J = 7.2 Hz, 3H). ¹³C NMR (126

MHz, DMSO-d₆) δ 168.54, 155.35, 153.61, 144.14, 141.92, 140.27,

136.95, 136.85, 136.58, 134.18, 130.18, 129.88, 129.15, 127.82, 125.95,

125.26, 119.04, 112.95, 111.63, 101.86, 55.39, 53.74, 49.19, 42.94,

20.50, 18.39, 7.10. MS (ESI), m/z for C₃₃H₃₃N₃O₄S ([M + H]⁺): calcd

567.70, found 568.8. HPLC analysis: MeOH (1‰ NH₃·H₂O)−H₂O

(75:25), t_R= 16.04 min, 95.43% purity.

4.3.54. (R)-2-(4-(2-(4-(Ethylsulfonyl)phenyl)acetamido)phenyl)-

N-isopropyl-1-(1-(p-tolyl)ethyl)-1H-benzo[d]imidazole-5-carboxa-

mide (27f). Compound 27f was prepared according to the general

procedure as described for 27c. White solid, 29% yield. ¹H NMR (400

MHz, DMSO-d₆) δ 10.57 (s, 1H), 8.18 (s, 1H), 8.14 (d, J = 6.7 Hz, 1H),

7.86 (d, J = 7.6 Hz, 2H), 7.81 (d, J = 7.7 Hz, 2H), 7.68 (d, J = 7.3 Hz,

2H), 7.63 (d, J = 7.5 Hz, 2H), 7.57 (d, J = 7.9 Hz, 1H), 7.17−7.09 (m,

3H), 7.08−7.00 (m, 2H), 5.88−5.74 (m, 1H), 4.16−4.02 (m, 1H), 3.86

(s, 2H), 3.31−3.22 (d, J = 7.1 Hz, 2H), 2.24 (s, 3H), 1.96 (d, J = 4.8 Hz,

3H), 1.16 (d, J = 5.5 Hz, 6H), 1.13−1.07 (m, 3H). ¹³C NMR (126

MHz, DMSO-d₆) δ 168.62, 165.64, 154.82, 142.84, 141.92, 140.57,

136.86, 136.72, 136.69, 135.31, 130.22, 130.07, 129.20, 128.90, 127.85,

125.98, 124.83, 121.65, 119.07, 118.51, 112.06, 53.99, 49.20, 42.96,

40.93, 22.38, 20.51, 18.29, 7.13. MS (ESI), m/z for C₃₆H₃₈N₄O₄S ([M

+ H]⁺): calcd 622.78, found 623.3. HPLC analysis: MeOH (1‰ NH₃·

H₂O)−H₂O (75:25), t_R= 13.80 min, 99.18% purity.

4.3.55. (R)-2-(4-(Ethylsulfonyl)phenyl)-N-(4-(1-(1-(4-

ﬂuorophenyl)ethyl)-6-(triﬂuoromethyl)-1H-benzo[d]imidazol-2-yl)-

phenyl)acetamide (27g). Compound 27g was prepared from 3-

chloro-4-nitrobenzotriﬂuoride and (R)-1-(4-ﬂuorophenyl)ethylamine

according to the general procedure as described for 25p. White solid,

39% yield. ¹H NMR (500 MHz, DMSO-d₆) δ 10.58 (s, 1H), 7.91−7.84

(m, 3H), 7.82 (d, J = 7.9 Hz, 2H), 7.69 (d, J = 8.1 Hz, 2H), 7.63 (d, J =

7.5 Hz, 2H), 7.51 (d, J = 8.1 Hz, 1H), 7.35 (s, 1H), 7.30−7.23 (m, 2H),

7.22−7.15 (m, 2H), 5.96−5.86 (m, 1H), 3.86 (s, 2H), 3.32−3.23 (m,

2H), 1.96 (d, J = 6.5 Hz, 3H), 1.10 (t, J = 7.1 Hz, 3H). ¹³C NMR (126

MHz, DMSO-d₆) δ 168.66, 162.42, 160.47, 156.34, 145.69, 141.88,

140.85, 136.88, 135.47, 135.45, 132.92, 130.21, 130.17, 128.45, 128.38,

127.84, 125.75, 124.32, 123.59, 122.91, 122.66, 122.41, 122.16, 120.28,

119.06, 118.67, 115.61, 115.44, 109.57, 109.54, 53.86, 49.21, 42.96,

18.39, 7.12. HRMS (ESI), m/z for C₃₂H₂₇F₄N₃O₃S ([M + H]⁺): calcd

609.1709, found 610.1774. HPLC analysis: MeOH−H₂O (85:15), t_R=

13.02 min, 98.08% purity. [α]_D²⁵= + 84 (c 0.1, MeOH).

4.3.56. (S)-2-(4-(Ethylsulfonyl)phenyl)-N-(4-(1-(1-(4-

ﬂuorophenyl)ethyl)-6-(triﬂuoromethyl)-1H-benzo[d]imidazol-2-yl)-

phenyl)acetamide (27h). Compound 27h was prepared from 3-

chloro-4-nitrobenzotriﬂuoride and (S)-1-(4-ﬂuorophenyl)ethylamine

according to the general procedure as described for 25p. White solid,

28% yield. ¹H NMR (500 MHz, DMSO-d₆) δ 10.58 (s, 1H), 7.90−7.83

(m, 3H), 7.82 (d, J = 8.6 Hz, 2H), 7.69 (d, J = 8.6 Hz, 2H), 7.63 (d, J =

8.2 Hz, 2H), 7.51 (d, J = 8.1 Hz, 1H), 7.35 (s, 1H), 7.29−7.24 (m, 2H),

7.18 (t, J = 8.8 Hz, 2H), 5.92 (q, J = 6.8 Hz, 1H), 3.86 (s, 2H), 3.28 (q, J

= 7.4 Hz, 2H), 1.96 (d, J = 7.1 Hz, 3H), 1.10 (t, J = 7.3 Hz, 3H).¹³C

NMR (126 MHz, DMSO-d₆) δ 168.66, 162.42, 160.47, 156.34, 145.69,

141.88, 140.85, 136.88, 135.47, 135.45, 132.92, 130.21, 130.17, 128.45,

128.38, 127.84, 125.75, 124.32, 123.59, 122.91, 122.66, 122.41, 122.16,

120.28, 119.06, 118.67, 115.61, 115.44, 109.57, 109.54, 53.86, 49.21,

42.96, 18.39, 7.12. HRMS (ESI), m/z for C₃₂H₂₇F₄N₃O₃S ([M + H]⁺):

calcd 609.1709, found 610.1749. HPLC analysis: MeOH−H₂O

(85:15), t_R= 12.59 min, 98.81% purity. [α]_D²⁵= − 60 (c 0.1, MeOH).

4.4. Biological Assays. 4.4.1. Expression and Puriﬁcation of the

RORγ LBD. A pET24a (Novagen, Madiso, WI) expression vector

encoding the human RORγ LBD (residues 262−507, wild type or

C455E mutant) with a N-terminal His6-tag was transformed into

Escherichia coli BL21 (DE3) cells by heat shock. Cells were grown in

Luria−Bertani (LB) broth at 25 °C until the OD₆₀₀of the culture

reached approximately 1.0, and then, the cells were induced with 0.1

mM isopropyl-β-^D-thiogalactopyranoside (IPTG) at 16 °C overnight.

The cells were harvested by centrifugation (5000 rpm, 4 °C, 15 min)

and suspended in extract buﬀer (20 mM Tris−HCl (pH 8.0), 500 mM

NaCl, 20 mM imidazole, 10% glycerol). After lysis using a homogenizer,

the cell lysate was centrifuged at 12 000 rpm for 40 min. The

supernatant was loaded onto a 20 mL NiSO₄-loaded His Trap HP

column (GE Healthcare, Piscataway, NJ). The column was washed with

extract buﬀer (20 mM Tris−HCl (pH 8.0), 500 mM NaCl, 20 mM

imidazole, 10% glycerol), and the protein was eluted with a 50−500

mM imidazole gradient. Elution fractions containing the protein of

interest were concentrated and further puriﬁed by a gel ﬁltration

column (Hiload 16/60 Superdex 75 column GE Healthcare)

equilibrated in 10 mM N-(2-hydroxyethyl)piperazine-N′-ethanesul-

fonic acid (HEPES) (pH 7.5), 150 mM NaCl, 2 mM tris(2-

carboxyethyl)phosphine (TCEP), 5% glycerol. The puriﬁed protein

was stored at −80 °C.

4.4.2. Thermal Stability Shift Assay (TSA). Thermal stability shift

assay was carried out using the Bio-Rad CFX96 real-time PCR system.

All reactions were buﬀered in 10 mM HEPES, 150 mM NaCl, 5%

glycerol, and pH 7.5. Protein and compounds were diluted using

reaction buﬀer to obtain ﬁnal concentrations of 10 μM protein and 200

μM compounds. Reactions were performed in a 10 μL ﬁnal volume in

96-well PCR plates. SYPRO Orange (Sigma) was added as a

ﬂuorescence probe at a dilution of 1:1000 and incubated with the

compounds on ice for 30 min. The total DMSO concentration was less

than 2%. The temperature gradient was performed in steps of 0.5 °C in

the range 30−80 °C. The ﬂuorescence readings were recorded at a 0.5

°C interval. All experiments were performed in duplicate. The data were

ﬁtted to a Boltzmann sigmoid curve function, and melting temperatures

(T_m) were calculated using GraphPad Prism. ΔT_mis the diﬀerence in

T_mvalues calculated for reactions with and without compounds.

4.4.3. Cell Transient Transfection Assays. 293T cells were plated

into 96-well plates at 1.5 × 10⁴per well (100 μL/well) and grown at 37

°C under a humidiﬁed 5% CO₂atmosphere and maintained in

Dulbecco’s modiﬁed Eagle’s medium (DMEM) containing 10% fetal

bovine serum (FBS) for 24 h. For Gal4-RORγ-driven reporter assays,

293T cells were transfected with 25 ng of Gal4-RORγ-LBD plasmid, 25

ng of pG5-luc reporter plasmid, and 5 ng of Renilla luciferase expression

plasmid per well. For Gal4-RORα-driven reporter assays, 293T cells

were transfected with 25 ng of Gal4-RORα-LBD plasmid, 25 ng of pG5-

luc reporter plasmid, and 5 ng of Renilla luciferase expression plasmid

per well. For Gal4-RORβ-driven reporter assays, 293T cells were

transfected with 25 ng of Gal4-RORβ-LBD plasmid, 25 ng of pG5-luc

reporter plasmid, and 5 ng of Renilla luciferase expression plasmid per

well. For Gal4-LXRα-driven reporter assays, 293T cells were trans-

fected with 25 ng of Gal4-LXRα-LBD plasmid, 25 ng of pG5-luc

reporter plasmid, and 5 ng of Renilla luciferase expression plasmid per

well. For Gal4-FXR-driven reporter assays, 293T cells were transfected

with 25 ng of Gal4-FXR-LBD plasmid, 25 ng of pG5-luc reporter

plasmid, and 5 ng of Renilla luciferase expression plasmid per well. The

cells were transiently transfected in Opti-MEM medium using a DNA

(μg) to Lipofectamine 2000 (Invitrogen) transfection reagent (μL)

ratio of 1:3. Five hours after transfection, tested compounds were

added. Cells were harvested after another 24 h for the luciferase assay

using a dual-luciferase reporter assay system (Promega). Luciferase

activities were normalized to Renilla activity, which was cotransfected

as an internal control. All assays were performed in triplicate, and

standard deviations were calculated accordingly.

4.4.4. Prostate Cancer Cell Culture, Cell Viability, and Cell Colony

Formation Assays. Prostate cancer LNCaP, C4-2B, and 22Rv1 cells

were cultured in Roswell Park Memorial Institute (RPMI) 1640

medium supplemented with 10% fetal bovine serum (FBS), penicillin

(100 U/mL), and streptomycin (100 μg/mL) at 37 °C in a humidiﬁed

incubator containing 5% CO₂. For cell viability, cells were seeded in 384

opaque-walled plates with clear bottoms at 500−1000 cells per well

(optimum density for growth) in 10 μL of culture medium. After 12 h,

10 diﬀerent concentrations ranging from 5 nM to 100 μM for the

prepared compounds were added into the 384 opaque-walled plates.

After incubation for 96 h, 25 μL of Cell-Titer GLO reagents (Promega)

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was added into each well, and plates were shaken for 10 min.

Luminescence was measured on an EnSpire Multimode Plate Reader

(PerkinElmer), according to the manufacturer’s instructions. The

estimated in vitro half-maximal inhibitory concentration (IC₅₀) values

were calculated using GraphPad Prism 7 software.

For colony formation, C4-2B and 22Rv1 cells were seeded in six-well

plates with each well containing 1500 cells in 2 mL of medium. Then,

cells were treated with vehicle or diﬀerent 27h concentrations and

incubated for additional 14 days at 37 °C. The medium was removed,

and the plates were washed with 2 mL of phosphate-buﬀered saline

(PBS) for one time. The cell colonies were stained with 2.5% crystal

violet in MeOH for 2 h at room temperature. Cells were photographed

with an HP scanner.

The rats were placed in the SPF animal feeding room and had free

access to food and water. The testing compounds were dissolved with

DMA/Solutol/saline (5:10:85, v/v/v) as the stock solution (1 mg/mL,

iv; 2.5 mg/mL, po). The stock solution was orally administrated (po) to

three SD rats at a dose of 25 mg/kg and intravenously administrated

(iv) to three SD rats at a single dose of 5 mg/kg. The blood samples

were collected via jugular vein after oral administration at 0.25, 0.5, 1, 2,

4, 6, 8, 10, and 24 h and after intravenous administration at 0.083, 0.25,

0.5, 1, 2, 4, 6, 8, and 24 h. Each blood sample (0.2 mL) was placed in

tubes containing dipotassium ethylenediaminetetraacetic acid (K2-

EDTA) and stored on ice until centrifuged. Blood samples were

centrifuged at 6800g for 6 min at 2−8 °C to obtain plasma and stored

frozen at approximately −80 °C until LC−MS/MS analysis.

4.4.9. In Vivo Eﬃcacy Studies in the 22Rv1 Xenograft Model in

Mice. Four-week-old male NOD-SCID mice were purchased from Vital

River Laboratory Animal Technology Co., Ltd., Beijing, China. All

experiments were approved and carried out according to the guidelines

of the Care and Use of Laboratory Animals of Guangzhou Institutes of

Biomedicine and Health, Chinese Academy of Sciences. For establish-

ing tumors, 3 × 10⁶cells were suspended in 100 μL of PBS and Matrigel

(1:1) and implanted subcutaneously into the right ﬂanks of male

nonobese diabetic/severe combined immunodeﬁciency (NOD/SCID)

mice. When the tumor volume reached approximately 100 mm³, mice

were randomized and treated with 27h (40 mg/kg, n = 7; 10 mg/kg, n =

6) or vehicle (n = 6) via oral gavage (po) ﬁve times a week for 3 weeks.

Compound 27h was dissolved in 15% Cremophor EL (Calbiochem),

82.5% PBS, and 2.5% DMSO. Tumor volumes were determined

according to formula π/6 (L × l²), by measuring the tumor length (L)

and width (l) with a caliper every day. Tumor growth inhibition (TGI)

was calculated as 100 − 100 × ((T − T₀)/(C − C₀)). T and T₀are the

mean tumor volumes for the teat groups on the last day of treatment

and on the ﬁrst day of treatment, respectively. C and C₀are the mean

tumor volumes for the vehicle control group on the last day of treatment

and on the ﬁrst day of treatment, respectively. Animal activities and

body weights were also monitored during the duration of the study to

assess potential acute toxicity.

4.4.5. Analysis of mRNA Expression in Cells. LNCaP cells were

seeded into 12-well plates at 1.5 × 10⁵cells per well. After 24 h,

compounds were added at the indicated doses and treated for 48 h.

Then, total RNA was isolated with TRIzol reagent. The obtained total

RNA was further subjected to reverse transcription to obtain cDNA

with Hifair II 1st Strand cDNA Synthesis SuperMix for qPCR. The

quantitative PCR analyses were performed in triplicate using standard

SYBR green reagents (Hieﬀ qPCR SYBR Green Master Mix). Full-

length AR, PSA (KLK3), KLK2, TMPRSS2, and C-MYC gene

expression levels were assessed by real-time PCR, normalizing to β-

actin. The primers used are as follows: AR-FL_fwd, ACA TCA AGG

AAC TCG ATC GTA TCA TTG C; AR-FL_rev, TTG GGC ACT

TGC ACA GAG AT; PSA_fwd, CAC AGG CCA GGT ATT TCA

GGT; PSA_rev, GAG GCT CAT ATC GTA GAG CGG; KLK2_fwd,

CAA CAT CTG GAG GGG AAA GGG; KLK2_rev, AGG CCA AGT

GAT GCC AGA AC; TMPRSS2_fwd, CAA GTG CTC CAA CTC

TGG GAT; TMPRSS2_rev, AAC ACA CCG ATT CTC GTC CTC;

C-MYC_fwd, GGC TCC TGG CAA AAG GTC A; C-MYC_rev, CTG

CGT AGT TGT GCT GAT GT; β-actin_fwd, GAG AAA ATC TGG

CAC CAC ACC; β-actin_rev, ATA CCC CTC GTA GAT GGG CAC.

4.4.6. Western Blotting. LNCaP, C4-2B, and 22Rv1 cells were

treated with vehicle or indicated concentrations of tested compounds

for 48 h. Subsequently, cells were harvested and lysed in buﬀer

containing 50 mM Tris−HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-

40, 0.25% sodium deoxycholate, and 0.1% sodium dodecyl sulfate

(SDS). Equal amounts of extracted total proteins were separated by

SDS−polyacrylamide gel electrophoresis (PAGE) and transferred onto

poly(vinylidene diﬂuoride) (PVDF) membranes. The membranes were

incubated for 1 h in blocking buﬀer (Tris-buﬀered saline, 0.1% Tween

(TBST), 5% nonfat dry milk) and then reacted with primary antibodies

(AR, Abclonal; A2053; GAPDH, Abclonal, AC001) overnight at 4 °C.

Membranes were washed with TBST and incubated with horseradish

peroxidase (HRP)-conjugated secondary antibody (Beyotime, A0208).

Finally, the signal was detected using a Minichemi imager.

4.4.7. Liver Microsome Stability Assays. All liver microsome assays

were performed by Medicilon Corporation, Shanghai, China.

Metabolic stability of testing compounds was evaluated using human

or rat liver microsomes to predict the half-life (t_1/2) and intrinsic

clearance. Ketanserin was used as the positive control to verify assay

performance. Brieﬂy, 1 μM compounds were incubated with human

liver microsomes or rat liver microsomes (ﬁnal concentration 0.75 mg/

mL) in phosphate buﬀer 0.1 M (pH 7.4) in a 37 °C water bath and the

reaction was initiated by the addition of a reduced nicotinamide

adenine dinucleotide phosphate (NADPH) cofactor (6 mM). The

reaction was then evaluated at 0, 5, 15, 30, and 45 min and quenched by

the addition of acetonitrile. Quenched samples were centrifuged for 15

min at 6000 rpm, and the supernatant was analyzed using HPLC−

tandem mass spectrometry (MS/MS). Percentage parent remaining

was calculated considering percentage parent area at 0 min as 100%. t_1/2

values and intrinsic clearances were determined according to the

metabolism plot.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00763.

■

sı

Virtual screening results from the molecular docking;

compounds identiﬁed as hits against RORγ from virtual

screening; overlaid complex structures of compound

XY101 and compound 27h; overlaid structures of

compounds 27g and 27h; western blot analysis of the

AR and AR variants levels in 22Rv1 cells treated with

compound 27h; metabolic stability of 25c, 25p, 27g, and

27h in human and rat liver microsomes; ¹H and ¹³C NMR

spectra of the synthesized compounds; and HPLC traces

of representative compounds (PDF)

Molecular formula strings for compounds with associated

biological data (CSV)

Database ﬁles (ZIP)

AUTHOR INFORMATION

Corresponding Author

■

Yong Xu − Guangdong Provincial Key Laboratory of

Biocomputing, Joint School of Life Sciences, Guangzhou

Institutes of Biomedicine and Health, Chinese Academy of

Sciences, Guangzhou Medical University, Guangzhou 510530,

China; Bioland Laboratory (Guangzhou Regenerative

Medicine and Health Guangdong Laboratory), Guangzhou

510005, China; State Key Laboratory of Respiratory Disease,

Guangzhou Institutes of Biomedicine and Health, Chinese

Academy of Sciences, Guangzhou 510530, China;

4.4.8. Pharmacokinetic Studies. Sprague−Dawley rats were

purchased from Shanghai Sino-British SIPPR/BK Lab Animal Ltd.

and used for the pharmacokinetic studies of testing compounds. All

animal procedures described in this study were performed in

accordance with the Regulations of Experiment Animal Administration

issued by the State Committee of Science and Technology of China.

8794

https://doi.org/10.1021/acs.jmedchem.1c00763

J. Med. Chem. 2021, 64, 8775−8797

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

orcid.org/0000-0003-3601-0246; Phone: +86-20-

32093612; Email: xu_yong@gibh.ac.cn

Institutes of Biomedicine and Health, Chinese Academy of

Sciences, Guangzhou Medical University, Guangzhou 510530,

China

Authors

Jinxin Xu − Guangzhou Institutes of Biomedicine and Health,

Chinese Academy of Sciences, Guangzhou 510530, China

Donghai Wu − Guangzhou Institutes of Biomedicine and

Health, Chinese Academy of Sciences, Guangzhou 510530,

China

Jinsong Liu − State Key Laboratory of Respiratory Disease,

Guangzhou Institutes of Biomedicine and Health, Chinese

Academy of Sciences, Guangzhou 510530, China;

orcid.org/0000-0003-2317-0558

Xishan Wu − Guangdong Provincial Key Laboratory of

Biocomputing, Joint School of Life Sciences, Guangzhou

Institutes of Biomedicine and Health, Chinese Academy of

Sciences, Guangzhou Medical University, Guangzhou 510530,

China; Bioland Laboratory (Guangzhou Regenerative

Medicine and Health Guangdong Laboratory), Guangzhou

510005, China

Hui Shen − Guangdong Provincial Key Laboratory of

Biocomputing, Joint School of Life Sciences, Guangzhou

Institutes of Biomedicine and Health, Chinese Academy of

Sciences, Guangzhou Medical University, Guangzhou 510530,

China; University of Chinese Academy of Sciences, Beijing

100049, China

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00763

Author Contributions

^#X.W., H.S., and Y.Z. contributed equally to this work.

Yan Zhang − Guangdong Provincial Key Laboratory of

Biocomputing, Joint School of Life Sciences, Guangzhou

Institutes of Biomedicine and Health, Chinese Academy of

Sciences, Guangzhou Medical University, Guangzhou 510530,

China; Bioland Laboratory (Guangzhou Regenerative

Medicine and Health Guangdong Laboratory), Guangzhou

510005, China

Chao Wang − Guangdong Provincial Key Laboratory of

Biocomputing, Joint School of Life Sciences, Guangzhou

Institutes of Biomedicine and Health, Chinese Academy of

Sciences, Guangzhou Medical University, Guangzhou 510530,

China; University of Chinese Academy of Sciences, Beijing

100049, China

Qiu Li − Guangdong Provincial Key Laboratory of

Biocomputing, Joint School of Life Sciences, Guangzhou

Institutes of Biomedicine and Health, Chinese Academy of

Sciences, Guangzhou Medical University, Guangzhou 510530,

China; University of Chinese Academy of Sciences, Beijing

100049, China

Cheng Zhang − Guangdong Provincial Key Laboratory of

Biocomputing, Joint School of Life Sciences, Guangzhou

Institutes of Biomedicine and Health, Chinese Academy of

Sciences, Guangzhou Medical University, Guangzhou 510530,

China

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported in part by grants from the National Key

R&D Program of China (grants 2019YFE0123700 and

2017YFA0504104), the Key International Cooperation Projects

of the Chinese Academy of Sciences (grant

154144KYSB20180044 and 154144KYSB20180063), the Chi-

nese Academy of Sciences STS Program (grant KFJ-STS-

QYZX-090), the National Science & Technology Major Project

“Key New Drug Creation and Manufacturing Program”, China

(grant 2018ZX09711002), the State Key Laboratory of

Respiratory Disease (grant SKLRD-Z-202018), the Guangdong

Basic and Applied Basic Research Foundation (grants

2020A1515010250 and 2020A1515110793), the Science and

Technology Program of Guangzhou, China (grant

202102080215), the Guangdong Provincial Key Laboratory of

Biocomputing (grant 2016B030301007), Frontier Research

Program of Bioland Laboratory (Guangzhou Regenerative

Medicine and Health Guangdong Laboratory) (grant

2018GZR110105016), the China Postdoctoral Science Foun-

dation, the Guangdong Provincial Postdoctoral Special Funding,

and the Special Research Assistant Project of the Chinese

Academy of Sciences. The authors also gratefully acknowledge

support from the Guangzhou Branch of the Supercomputing

Center of the Chinese Academy of Sciences.

Xiaoxi Zhuang − Guangdong Provincial Key Laboratory of

Biocomputing, Joint School of Life Sciences, Guangzhou

Institutes of Biomedicine and Health, Chinese Academy of

Sciences, Guangzhou Medical University, Guangzhou 510530,

China

Chenchang Li − Guangdong Provincial Key Laboratory of

Biocomputing, Joint School of Life Sciences, Guangzhou

Institutes of Biomedicine and Health, Chinese Academy of

Sciences, Guangzhou Medical University, Guangzhou 510530,

China

Yudan Shi − Guangdong Provincial Key Laboratory of

Biocomputing, Joint School of Life Sciences, Guangzhou

Institutes of Biomedicine and Health, Chinese Academy of

Sciences, Guangzhou Medical University, Guangzhou 510530,

China

Yanli Xing − Guangdong Provincial Key Laboratory of

Biocomputing, Joint School of Life Sciences, Guangzhou

Institutes of Biomedicine and Health, Chinese Academy of

Sciences, Guangzhou Medical University, Guangzhou 510530,

China

ABBREVIATIONS

■

CRPC, castration-resistant prostate cancer; LBD, ligand-bind-

ing domain; PC, prostate cancer; PSA, prostatic-speciﬁc antigen;

RORγ, retinoic acid receptor-related orphan receptor γ; SAR,

structure−activity relationship; TSA, thermal shift assay

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Discovery and Characterization of Benzimidazole Derivative XY123 as a Potent, Selective, and Orally Available RORγ Inverse Agonist

DOI: 10.1021/acs.jmedchem.1c00763

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Full text of DOI:10.1021/acs.jmedchem.1c00763

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