Discovery of Arylsulfonamides as Dual Orexin Receptor Agonists

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Dehui Zhang, David A. Perrey, Ann M. Decker, Ti ﬀ any L. Langston, Vijayakumar Mavanji,

Danni L. Harris, Catherine M. Kotz, and Yanan Zhang *

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ABSTRACT: Loss of orexin-producing neurons results in narcolepsy with cataplexy, and orexin agonists have been shown to

increase wakefulness and alleviate narcolepsy symptoms in animal models. Several OX2R agonists have been reported but with little

or no activity at OX1R. We conducted structure−activity relationship studies on the OX2R agonist YNT-185 (2) and discovered

dual agonists such as RTOXA-43 (40) with EC₅₀’s of 24 nM at both OX2R and OX1R. Computational modeling studies based on

the agonist-bound OX2R cryogenic electron microscopy structures showed that 40 bound in the same binding pocket and

interactions of the pyridylmethyl group of 40 with OX1R may have contributed to its high OX1R potency. Intraperitoneal injection

of 40 increased time awake, decreased time asleep, and increased sleep/wake consolidation in 12-month old mice. This work

provides a promising dual small molecule agonist and supports development of orexin agonists as potential treatments for orexin-

deﬁcient disorders such as narcolepsy.

enhances wakefulness and consolidates sleep/wake states.²⁵

INTRODUCTION

■

Consistent with orexin’s multifaceted role, orexin deﬁciency

Orexin A and B (also known as hypocretins 1 and 2) are two

has also been linked to abnormalities in energy homeostasis,

hypothalamic neuropeptides and the endogenous ligands for

stress-related behavior, and reward systems²⁶and is believed to

two G protein-coupled receptors (GPCRs), orexin-1 (OX1R),

be associated with pathophysiologies such as obesity and age-

and orexin-2 (OX2R).^1,2Orexin-expressing neurons are limited

related disorders.²⁷A loss of orexin neurons and/or orexin

in number and located predominantly in a small area of the

peptides has been found in Alzheimer’s and Parkinson’s

lateral hypothalamus;²⁻⁵however, the nerve ﬁbers of orexin

patients,²⁸⁻³²as well as in aged humans and mice.¹⁷⁻¹⁹

neurons project throughout the central nervous system and

Medication development targeting the orexin system has

their aﬀerents are sent to many brain regions in cortical, limbic,

largely focused on antagonists thus far as over- or abnormal

and brainstem circuits.^3,6−8The orexin system has been shown

activation of the orexin system leads to insomnia.^33,34A large

to modulate a variety of important behavioral and physiological

number of orexin receptor antagonists, dual- or subtype-

processes, including sleep/wakefulness,^9,10arousal,^11,12feed-

selective, have been developed.³⁵⁻³⁹Two dual antagonists,

ing,²energy homeostasis,²stress and anxiety,¹³⁻¹⁶and learning

suvorexant and more recently lemborexant, have been

and memory.¹⁷⁻²⁰

approved by the FDA for the treatment of insomnia. Orexin

Loss of orexin-producing neurons results in narcolepsy with

cataplexy, an incurable chronic neurological disorder charac-

terized with sleep disruptions (excessive daytime sleepiness,

Received: May 9, 2021

Published: June 8, 2021

fragmented sleep and intrusions of sleep episodes during the

active phase, etc.).²¹⁻²⁴Narcolepsy aﬀects an estimated

200,000 Americans and approximately 3 million worldwide

and severely impacts the day-to-day lives of aﬀected

individuals. Central orexin-A administration successfully

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Figure 1. Small-molecule orexin agonists reported in the literature.

antagonism has also shown early promises in the treatment of

drug addiction and anxiety disorders.^{13,14,16,36,40−42}In contrast,

activation of the orexin receptors is primarily accomplished

using orexin peptides, particularly the more stable orexin-A (33

AA with two disulﬁde bridges). Only a limited number of

small-molecule orexin agonists have been disclosed thus far.

Yan7874 (1) was ﬁrst reported as a small-molecule OX2R

agonist in a 2010 patent (Figure 1);⁴³however, it was later

were shown to be eﬃcacious in promoting sleep,⁵⁷a number of

preclinical studies have suggested that antagonizing both

orexin receptors was more eﬀective.^54,58,59Given the role of

the OX1R in many functions and the equal potency of the

endogenous peptide orexin-A, agonists that also have OX1R

activity are desired. We conducted structure−activity relation-

ship (SAR) studies on 2 with structural modiﬁcations at several

sites (Figure 2) and developed a series of new orexin agonists

conﬁrmed to be a weak agonist of both OX receptors (EC₅₀

>

3.2 μM) and also showed OX receptor independent

cytotoxicity.⁴⁴YNT-185 (2) was reported in 2015 and

displayed good OX2R potency and selectivity over OX1R

(EC₅₀= 28 nM vs 2750 nM at OX1R).⁴⁵Intracerebroven-

tricular (i.c.v., 30−300 nmol) and/or intraperitoneally (i.p., 40

mg/kg) administration of 2 (hydrochloride salt) promoted

wakefulness without aﬀecting body temperature in wild-type

mice, whereas in orexin knock-out (KO) and orexin neuron-

ablated mice, 2 suppressed cataplexy-like episodes.⁴⁶In

another study, 2 attenuated morphine-induced sedative eﬀects

in rats, as assessed by electroencephalogram (EEG) changes

and behavioral measures including locomotor activity and

startle response latency, without aﬀecting the analgesic eﬀect of

morphine.⁴⁷More recently, a series of substituted piperidines

as OX2R agonists was reported, and Tak-925 (3) is currently

in phase 1 clinical trials for the treatment of narcolepsy.⁴⁸

Another small-molecule orexin agonist by Takeda, TAK-994, is

in a phase 2 clinical trial in type 1 and 2 narcoleptic patients

(www.clinicaltrials.gov), although its structure is not disclosed.

A series of structurally similar OX2R agonists based on a

pyrrolidine core (e.g., 4) were reported in 2020 with high

potencies at OX2R, but no data were reported on OX1R

activity.⁴⁹Finally, the cryogenic electron microscopy (cryo-

EM) structure of the ﬁrst agonist-bound OX2R using agonist 5

was recently reported, providing structural insights for

agonist−receptor interactions.⁵⁰

Figure 2. SAR strategies on 2.

(6−44). One of the most potent compounds (RTOXA-43, 40)

showed good potencies at both OX1R and OX2R (EC₅₀= 24

at both OX1R and OX2R) when evaluated in calcium

mobilization assays using cells overexpressing these receptors.

Excitingly, 40 (40 mg/kg, i.p.) increased time awake, decreased

time asleep, and increased sleep/wake consolidation when

measured using continuous EEG/electromyogram (EMG)

recordings in 12-month-old mice. Hereby, we describe the

design, synthesis, and pharmacological characterization of

these orexin receptor agonists.

RESULTS AND DISCUSSION

■

Chemistry. The syntheses of all target compounds were

accomplished following procedures shown in Schemes 1−6.

Compounds 2 and 6−16 with modiﬁcations on ring-A were

synthesized following the procedures described by Nagahara

and co-workers.⁴⁵Commercially available 1-ﬂuoro-3-nitro-

benzene was reacted with excess ethylenediamine at 120 °C for

12 h to aﬀord the substituted aniline, which was immediately

reacted with Boc₂O to give 45 in 63% over two steps. The

aniline group in 45 was then protected by treatment with

benzyl bromide in the presence of potassium carbonate in

dimethylformamide (DMF) to aﬀord the key intermediate 46

The two orexin receptors (and their mRNAs) have

overlapping, but sometimes distinct, patterns of distribution

in the brain,^3,5,51,52suggesting that they may play diﬀerential

physiological roles. For instance, mice lacking OX2R display

several abnormalities similar to human narcolepsy;^18,53

however, the behavioral phenotype of OX2R KO mice appears

less severe than orexin null mice, and double-orexin-receptor

KO mice displayed sleep/wake disturbances most similar to

human narcolepsy.⁵⁴⁻⁵⁶Similarly, although OX2R antagonists

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a

Scheme 1. Syntheses of Compounds 2 and 6−16

a

Reagents and conditions: (a) ethylenediamine, 120 °C, sealed tube, overnight; (b) Boc₂O, K₂CO₃, THF/H₂O, 63% over two steps; (c) BnBr,

K₂CO₃, DMF, 60 °C, 62%; (d) Fe, NH₄Cl, EtOH, reﬂux, 98%; (e) 2-methoxy-5-bromobenzenesulfonyl chloride, pyridine, DCM, 0 °C to rt, 90%

(f) boronic acid, Pd(PPh₃)₄, K₂CO₃, 1,4-dioxane/H₂O (4:1), 90 °C; (g) 4 N HCl in 1,4-dioxane; (h) 2-dimethylamino benzoic acid, HATU,

DIPEA, DMF; (i) H₂, Pd/C, MeOH, 29−45% over four steps.

a

Scheme 2. Syntheses of Compounds 17−21

a

Reagents and conditions: (a) appropriate diamine, 120 °C, sealed tube, overnight; (b) Boc₂O, K₂CO₃, THF/H₂O, 72% for 51a, 30% for 51b over

two steps; (c) BnBr, K₂CO₃, DMF, 60 °C, 84% for, 52a, 81% for 52b; (d) Fe, NH₄Cl, EtOH, reﬂux, 92% for 53a, 92% for 53b; (e) 2-methoxy-5-

bromobenzenesulfonyl chloride, pyridine, DCM, 0 °C to rt, 85% for 54a, 83% for 54b; (f) N,N-dimethylbenzamide-3-boronic acid, Pd(PPh₃)₄,

K₂CO₃, 1,4-dioxane/H₂O (4:1), 90 °C; (g) 4 N HCl in 1,4-dioxane; (h) substituted benzoic acid, HATU, DIPEA, DMF; (i) H₂, Pd/C, MeOH,

19−32% over our steps.

in 62% yield. Reduction of the nitro group using iron gave

amine 47 almost quantitatively (98%). Reaction of 5-bromo-2-

methoxybenzenesulfonyl chloride in tetrahydrofuran (THF)

with 47 in dichloromethane (DCM) in the presence of

pyridine led to intermediate 48 in excellent yield (90%).

Suzuki coupling of 48 with diﬀerent boronic acids gave

intermediate 49, which was immediately submitted to acidic

Boc deprotection and amide coupling to provide 50. Finally,

deprotection of the benzyl group of 50 using hydrogenation

catalyzed by palladium on carbon aﬀorded target products 2

and 6−16 in 29−45% yield over four steps.

Similarly, compounds 17 to 21 that examine the importance

of the ethyl linker were synthesized following a similar

sequence, as shown in Scheme 2, using diﬀerent diamines

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a

Scheme 3. Syntheses of Compounds 22 and 23

a

(a) Piperazine, 100 °C, sealed tube,overnight; (b) Boc₂O, K₂CO₃, THF/H₂O, 49% over two steps; (c) Fe, NH₄Cl, EtOH/H₂O, reﬂux, 86%; (d)

2-methoxy-5-bromobenzenesulfonyl chloride, pyridine, DCM, 0 °C to rt, 80%; (e) N,N-dimethylbenzamide-3-boronic acid, Pd(PPh₃)₄, K₂CO₃,

1,4-dioxane/H₂O (4:1), 90 °C; (f) 4 N HCl in 1,4-dioxane; (g) substituted benzoic acid, HATU, DIPEA, DMF, 36% for 22, 41% for 23 over three

steps.

a

Scheme 4. Synthetic Route to Compounds 24−32

a

Reagents and conditions: (a) 4 N HCl in 1,4-dioxane, 78%; (b) 3-methyl benzoic acid, HATU, DIPEA, DMF; (c) Fe, NH₄Cl, EtOH, reﬂux, 62%

over three steps; (d) 2-methoxy-5-bromobenzenesulfonyl chloride, NEt₃, DMAP_(cat.), DCM/THF, 0 °C to rt, 76%; (e) B₂pin₂, PdCl₂dppf, KOAc,

1,4-dioxane, 90 °C, 87%; (f) R−Br or R−I, Pd(PPh₃)₄, K₂CO₃, 1,4-dioxane/H₂O (4:1), 63−88%.

a

Scheme 5. Synthesis of Compounds 33−42

a

Reagents and conditions: (a) aryl halide, Pd(PPh₃)₄, K₂CO₃, 1,4-dioxane, H₂O, 62−85%.

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a

Scheme 6. Synthesis of Compounds 43 and 44

a

Reagents and conditions: (a) 2-methoxy-5-bromobenzenecarboxylic chloride, NEt₃, DIPEA, DCM, 0 °C to rt, 60%; (b) B₂pin₂, PdCl₂dppf, KOAc,

1,4-dioxane, 90 °C, >99%, (c) aryl halide, Pd(PPh₃)₄, K₂CO₃, 1,4-dioxane/H₂O (4:1), 78% for 43, 72% for 44.

instead of ethylenediamine. 1-Fluoro-3-nitrobenzene under-

went ﬂuoro displacement with diamines, Boc protection, and

benzyl protection to give 52, which was then reduced to give

amine 53 in excellent yield (92%). Coupling of 53 with 5-

bromo-2-methoxybenzenesulfonyl chloride aﬀorded 54, which

was subjected to Suzuki coupling to give intermediate 55. Boc

deprotection followed by amide coupling furnished 56,

deprotection of which provided ﬁnal compounds 17−21 in

19−32% yield over four steps.

For compounds 22 and 23 with a piperidine group, since the

protection of nitrogen was not needed, a simpliﬁed synthetic

route was followed (Scheme 3). 1-Fluoro-3-nitrobenzene was

reacted with excess piperazine followed by Boc protection to

give 57 in 49% yield. Reduction of the nitro group provided

amine 58, which was then reacted with 5-bromo-2-methox-

ybenzenesulfonyl chloride to aﬀord 59 in 80% yield. Suzuki

coupling furnished 60, followed by Boc deprotection and

amide coupling to give ﬁnal compounds 22−23 in 36 and 41%

yields, respectively, over three steps.

Compounds 24−32, in which ring-A was replaced with

other aromatic or alkyl groups, were synthesized following

modiﬁed procedures as shown in Scheme 4. In order to

facilitate the examination of the SAR on the left-hand side, the

amide on the right was installed ﬁrst. In addition, to simplify

the synthetic route, we attempted to not protect the aniline

using a benzyl group as previously described but to install the

sulfonamide selectively at the more accessible primary amino

group. Thus, 45 was treated with 4 N HCl in dioxane to aﬀord

the amine 61. Amide coupling between compound 61 and 3-

methylbenzoic acid led to 62, which was reduced using iron to

give 63 in 62% over two steps. Slow addition of a diluted

solution of 5-bromo-2-methoxybenzenesulfonyl chloride in

THF into a diluted solution of 63 in DCM in the presence of

triethylamine and catalytic 4-dimethylaminopyridine (DMAP)

at 0 °C aﬀorded 64 in 76% yield, with the selective acylation of

the less hindered amino groups. This sequence avoided the

protection/deprotection steps and aﬀorded the desired

product in good yields. The subsequent Miyaura borylation

reaction gave boronic acid pinacol ester 65, which was readily

reacted with diﬀerent aromatic bromides or iodides to furnish

the ﬁnal products 24−32 in 63−88% yield.

yield. Miyaura borylation of 66 using bis(pinacolato)diboron

provided 67 quantitatively, which then underwent a Suzuki

coupling reaction with the halides to provide compounds 43

and 44 in good yield (78 and 72%, respectively). In these two

compounds, the sulfonamide was replaced with an amide

functionality.

Potency in OX1R and OX2R Calcium Mobilization

Assays. All target compounds were evaluated for their agonist

potencies in calcium mobilization assays using CHO RD-

HGA16 cells (Molecular Devices) engineered to stably express

either the human OX2R or OX1R as previously described.⁶⁰In

this assay platform, receptor activation is measured by an

increase in ﬂuorescence, which is directly proportional to an

increase in internal calcium. The EC₅₀values are listed in

Tables 1−5. YNT-185 (2) was previously reported to have

EC₅₀values of 28 nM at OX2R and 2750 nM at OX1R.⁴⁵

However, in our orexin calcium mobilization assays, 2

displayed potencies of 165 and 824 nM at OX2R and

OX1R, respectively. The potency diﬀerences may have resulted

from the diﬀerently engineered cell lines. While both groups

use calcium mobilization assays to measure receptor activation,

Nagahara and colleagues used CHO cells coexpressing the

orexin receptors and a luciferase reporter,⁴⁵and our group

used Gα₁₆-expressing CHO cells. In their assays, orexin-A had

EC₅₀’s = 1.0 and 1.5 nM at OX2R and OX1R, respectively,

whereas the EC₅₀values are 0.6 and 0.3 nM in our assays,

respectively.

In our SAR studies, we ﬁrst examined substitutions on

aromatic ring-A on the left-hand side of the structure (Table

1). The dimethylamino amide on 2 was replaced with several

diﬀerently substituted alkyl amides including diethylamino (6),

cyclic (7, 8), or secondary amides (9). However, most of the

compounds showed similar or even reduced potencies at

OX2R and/or OX1R, with 9 showing no activity at OX1R. We

then attempted to remove the carbonyl functionality, which

resulted in a decrease in potency at OX2R (10, EC₅₀= 560

nM), whereas no activity was observed at OX1R. None of the

other amino or alkyl groups (11−16) showed activity at either

receptor. Together, these results conﬁrmed the importance of

the carbonyl functionality for orexin receptor activation.

We next examined substitutions on aromatic ring-B on the

right-hand side as well as the ethyl linker (Table 2). Consistent

with results reported by Nagahara et al.,⁴⁵replacement of the

2-dimethylamino with a 3-methyl group (17) slightly improved

potencies at both OX2R and OX1R (56 vs 165 nM at OX2R;

326 vs 826 nM at OX1R). Elongation of the ethyl group to the

3-carbon propyl group (18−19) or addition of a methyl group

Compounds 33−42 with diﬀerent amide functionalities on

ring-A were prepared in good yields following a Suzuki

coupling of intermediate 63 with the appropriate aryl halides

under standard conditions in 62−85% yield.

Amide coupling between intermediate 63 and 5-bromo-2-

methoxybenzoic acid chloride aﬀorded bromide 66 in 60%

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and OX2R. We then investigated several ﬁve-membered

heteroaromatic rings (28−30), including thiophenes and a

thiazole, and they were similar or less potent than 2 at OX2R,

with little activity at OX1R. Interestingly, these ﬁve-membered

analogues (28−30) all acted as partial agonists at both OX1R

and OX2R. The ethylenyl (31) and the ethyl (32) analogues,

which were designed to provide structural ﬂexibility, were both

inactive at both receptors. These results suggest that an

aromatic group is preferred at this position.

Table 1

Given the importance of the carbonyl group of the amide

functionality on ring-A, we retained the amide and explored

further substitutions at this position (Table 4). First, we

replaced one of the methyl groups on the amide with a

dimethylaminoethyl group (33), which oﬀers a site for salt

formation to improve solubility. However, a slight reduction in

potency was observed (EC₅₀= 293 nM at OX2R and 638 nM

at OX1R). Notably, removal of the other methyl on the

nitrogen resulted in a sharp decrease of potency at OX2R (33

vs 34) and complete loss of potency at OX1R. This is

consistent with earlier observations that primary amides were

not favored (9). Introduction of a pyridyl (35) group in place

of a methyl on the dimethylamino group led to a signiﬁcant

decrease in potency at both receptors. Interestingly, while a

benzyl (36) showed lowered potency at OX2R and no activity

at OX1R, 2-pyridylmethyl group (37) led to better potencies

than 2 at both OX receptors (EC₅₀= 107 nM at OX2R and

235 nM at OX1R). This suggests that hydrogen bonding or

polar−polar interaction between the pyridyl group and the OX

receptors may be present. Similarly, removal of the other

methyl on the dimethylamino group again led to complete loss

of the OX1R potency (38). Considering the potency

enhancement by the pyridyl group, we began to ﬁnely tune

the substitution and examine diﬀerent pyridylmethyl groups.

While 3-pydidylmethyl (39) showed a slight drop in potency

(EC₅₀= 134 nM at OX2R; 460 nM at OX1R), excitingly, 40

with a 4-pyridylmethyl group showed high and equal potencies

at both OX2R and OX1R (EC₅₀= 24 nM), signiﬁcantly more

potent than 2. Compound 40 was a full agonist at both OX2R

and OX1R, with E_maxof ∼100% orexin-A (Figure 3).

Replacement of the pyridylmethyl with longer pyridylethyl

groups resulted in decrease on potency at both receptors (41

and 42).

Finally, we examined whether the sulfonamide could be

replaced with an amide (43−44, Table 5). Unfortunately, this

structural modiﬁcation resulted in signiﬁcant reduction in

potency at OX2R and total loss of activity at OX1R, similar to

a previous report.⁴⁵This clearly indicates the importance of the

sulfonamide for activity at the orexin receptors.

a

EC₅₀and E_maxvalues (% of orexin-A control) are the means SEM

of at least three independent experiments conducted in duplicate.

b

Values are from two independent experiments conducted in

c

duplicate. Concentration−response curve does not have a top

plateau.

Computational Modeling Studies. We built a full-length

OX2R model based on the cryo-EM structures of agonist 5

(PDBID: 7L1V) and orexin-B-bound OX2R (PDBID: 7L1U)

recently reported by Hong and colleagues⁵⁰and a homology

OX1R model based on the backbone and fold templates of the

human OX2R structure, which shares 64% sequence identity

with human OX1R.²The initial models were processed

through a series of reﬁnement steps employing MODELLER

(simulated annealing with topological constraints),⁶¹

AMBER18 low-mode exploration,⁶²⁻⁶⁴energy minimization,

and dynamical equilibration in a lipid/KCl/water system

employing the LIPID14 and ﬀ14SB force ﬁelds.^65,66We then

explored agonists 5, 2, and 40 with OX2R and/or OX1R

models via GLIDE-XP or SP docking,⁶⁷distilling 120

minimized docked poses to the best 5, followed by induced

(20−21) resulted in little change in potency. However, when

the ethyl group was converted to a rigid piperazine group (22−

23), all activities were lost. In general, in this series, the 3-

methyl analogues appeared to provide better potencies at

OX1R than the corresponding 2-dimethylamino analogues (2,

18, and 20 vs 17, 19, and 21), and we therefore used 3-

methylamino in the subsequent SAR studies.

Phenyl ring-A was then replaced with other aromatic rings or

a rigid alkenyl or an ethyl group (Table 3). Out of the four

pyridine groups (24−27), the 2,6-substituted pyridine (27)

aﬀorded the best potencies at both OX1R and OX2R (EC₅₀’s =

74 nM at OX2R and 226 nM at OX1R), with EC₅₀values

lower than 2, and appeared to be a full agonist at both OX1R

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Table 2

a

b

EC₅₀and E_maxvalues (% of orexin-A control) are the means SEM of at least three independent experiments conducted in duplicate. Values are

c

from two independent experiments conducted in duplicate. Concentration−response curve does not have a top plateau.

ﬁt modeling.⁶⁸Molecular dynamics (MD) simulations were

conducted with 40 in both OX2R and OX1R to identify key

receptor−ligand interactions and probe their stability.

Consistent with the cryo-EM OX2R structure, all compounds

(5, 2, and 40) formed a “Z” shape in our OX2R model, kinked

around the sulfonamide functionality with the two adjacent

aromatic rings perpendicular to each other. The right end of

these molecules inserted into the hydrophobic bottom of the

pocket with the left end extending toward extracellular space

(Figure 4A−C). This is consistent with the orexin-B bound

cryo-EM structure showing the largely hydrophobic C-

terminus residues of orexin-B, G24-I25-L26-T27-M28, bound

at the hydrophobic bottom of the binding pocket.⁵⁰

and persistent hydrogen bond interactions of the Q134^3.32

amide group with the sulfonamide, sometimes bidentate as

reported by Hong et al., were observed within 15 ns of the 180

ns MD simulation equilibration at 300 K, with additional

hydrogen bond interactions, although slightly more distant,

with the methoxy on the neighboring phenyl group (Figure

4C). These results clearly demonstrated the importance of the

sulfonamide and are consistent with the SAR results where

replacement of the sulfonamide with the corresponding amide

(43 and 44) resulted in signiﬁcant drop on potency at OX2R.

In addition, it should be noted that all the active orexin

agonists reported thus far (e.g., 2−5, Figure 1) have a

sulfonamide functionality.

Q134^3.32(Ballesteros−Weinstein numbering in superscript)

was identiﬁed as the residue essential for binding and receptor

activation in the agonist 5 and orexin-B-bound OX2R cryo-EM

structures.⁵⁰In both agonist-bound structures, the side chain

of Q134^3.32was extended and projected upward toward the

extracellular side, as opposed to pointing more downward

toward the cytoplasm in the antagonist-bound OX2R

structures.^50,69,70We observed the agonist-like orientation of

the Q134^3.32side chain in our OX2R model and such a

conformation was largely maintained during 180 ns MD

simulations of 40 in OX2R. Q134^3.32was in close proximity of

the sulfonamide group, as observed by Hong et al. in the 5-

bound OX2R cryo-EM structure⁵⁰but was also close in space

to the methoxy group on the neighboring aromatic ring in the

initial induced ﬁt docked poses in these agonists (Figure

4A,B). When MD simulations were performed on 40, intimate

In the OX1R homology model, 40 formed a similar “Z”

shape, although slightly rotated (Figure 4D). Similar to the

OX2R model, Q126^3.32of OX1R, the equivalent residue of

Q134^3.32in OX2R, formed hydrogen bonding with the

sulfonamide group and/or the neighboring methoxy group

during the 180 ns MD simulations. In addition, hydrogen

bonding between the sulfonamide group and N318^6.55was also

observed (Figure 4D). Markedly, as opposed to only transient

interactions with R339^7.28at the top of helix 7 in OX2R, the

pyridylmethyl group on the left-hand side of 40 formed

persistent hydrogen bonds or π-cation interactions with

R333^7.29in OX1R during the 180 ns MD simulations.

Although additional studies are clearly needed, these

receptor−ligand interactions, particularly via the pyridylmethyl

group, may have contributed to the high potency of 40 in

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Table 3

a

b

EC₅₀and E_maxvalues (% of orexin-A control) are the means SEM of at least three independent experiments conducted in duplicate. Values are

c

from two independent experiments conducted in duplicate. Concentration−response curve does not have a top plateau.

OX1R, in contrast to OX2R agonists 2 and 5, in which the

pyridylmethyl motif is absent.

and reduced sleep/wake fragmentation, as indicated by

reduced transitions between states (Figure 5D). In addition,

40-induced enhancement of wake time was associated with an

increase in average duration of wake episodes (Figure 5E).

Whereas the agonist reduced number of wake episodes (Figure

5F), the increased duration of wake episodes resulted in overall

enhancement of wake time following treatment. Similar to

wake episodes, 40 increased the duration of NREM sleep

episodes (Figure 5E). Reduced number of NREM sleep

episodes (Figure 5F) following agonist administration resulted

in overall reduction in time spent in NREM sleep. Surprisingly,

40 did not aﬀect the duration of REM sleep episodes (Figure

5E), but fewer episodes of REM sleep (Figure 5F) resulted in

overall reduction in REM sleep time following agonist

administration. Together, these data demonstrate that 40

enhances wake, suppresses sleep, and reduces sleep/wake

fragmentation. In particular, 40 enhances wake by increasing

episode duration and decreases NREM and REM sleep by

reducing the number, but not the duration, of episodes of

NREM and REM sleep.

Sleep Modulation Studies. It has been previously shown

that upon i.p. administration at 40 or 60 mg/kg, 2 increased

wake time, as expected with OX2R agonists and decreased

direct transitions from wakefulness to rapid eye movement

(REM) sleep, a measure that is used to assess cataplexy, for 3

h^45,46To test whether our OX agonists inﬂuence time spent

awake and the transitions between sleep/wake states, we

injected 40 into 12-month old female mice (40 mg/kg, i.p.

during the quiet, lights-on phase) and measured sleep/wake

parameters using continuous EEG/EMG recordings for 4 h

postinjection. Compound 40 signiﬁcantly increased active

wakefulness and reduced nonrapid eye movement (NREM)

and REM sleep relative to that after the vehicle injection

(Figure 4A−C). There was a main eﬀect of treatment on active

wakefulness (Figure 4A, P < 0.01), NREM sleep (Figure 4B, P

< 0.05), and REM sleep (Figure 4C, P < 0.001) during the 0−

4 h postinjection time period.

To test whether 40 inﬂuences sleep patterns and quality, the

total number of transitions between states, number, and the

mean duration of episodes of each vigilance state were

determined. Our data demonstrated that 40 stabilized behavior

The wake-promoting eﬀects observed with 40 were similar

to those of orexin-A and the OX2R agonist 2 reported earlier.

We have shown increased wakefulness and reduced NREM

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Table 4

a

b

EC₅₀and E_maxvalues (% of orexin-A control) are the means SEM of at least three independent experiments conducted in duplicate. Values are

c

from two independent experiments conducted in duplicate. Concentration−response curve does not have a top plateau.

and REM sleep following ventrolateral preoptic area injection

of orexin-A in rats, which was associated with decreased

NREM sleep and wake episodes 1 h postinjection.²⁵i.c.v.

administration of orexin-A (3 nmol) enhanced wakefulness and

suppressed both NREM and REM sleep in mice up to 3 h

following treatment.⁵³Similarly, i.c.v. administered 2 (30, 100,

and 300 nmol doses) during the light phase decreased percent

time in NREM sleep and increased time in wakefulness up to 3

h postinjection. In addition, i.p. injected 2 (40 and 60 mg/kg, 6

h into the light phase) increased percent time in wakefulness

and decreased percent time in REM sleep for up to 3 h

following injections and the 40 mg/kg (i.p.) dose also reduced

the percent time in NREM sleep in mice.⁴⁶Notably, both the

40 and 60 mg/kg doses had identical wake-promoting eﬀects,

whereas the 20 mg/kg dose (i.p.) did not have any eﬀect on

the sleep/wake parameters. The relatively high doses required

to aﬀect sleep/wake cycles suggest that while 2 and 40 are able

to reach the OX2R in the brain to modulate sleep, their brain

penetration may be limited.

At 40 mg/kg, 40 and 2 produced similar wake-promoting

eﬀects upon i.p. administration, despite the signiﬁcantly higher

OX1R potency of 40. This may reﬂect that OX2R plays a

pivotal role in the modulation of sleep/wakefulness. It has been

reported that OX2R KO mice exhibited a narcoleptic

phenotype, whereas OX1R KO mice showed only a mild

fragmentation of sleep and awake states.⁷¹In addition, while

orexin-A eﬀects on wake promotion and NREM sleep

suppression were attenuated in both OX2R and OX1R KO

mice, substantially greater reductions in OX2R KO mice

(relative to OX1R KO mice) were observed.⁵³Together, these

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Table 5

a

b

EC₅₀and E_maxvalues (% of orexin-A control) are the means SEM of at least three independent experiments conducted in duplicate. Values are

from two independent experiments conducted in duplicate.

Figure 3. Activity of 40 in the OX1R and OX2R calcium mobilization

assays. Each data point is the mean

experiments conducted in duplicate.

SEM of three independent

studies indicate that sleep appears to be primarily regulated by

OX2R and to a lesser extent by OX1R; however, the role of the

OX1R in sleep/wakefulness regulation may be best addressed

when selective OX1R agonists become available.

Figure 4. Induced ﬁt top Emodel poses for (A) 5 in OX2R, (B) 2 in

OX2R, (C) 40 in OX2R, and (D) 40 in OX1R full-length models

based on the agonist-bound cryo-EM OX2R structures (7L1U and

7L1V). Ligands are in green and residues in gray.

CONCLUSIONS

■

The orexin system is implicated in many physiological

processes, and loss or decline has been associated with

narcolepsy and other neurological diseases. Orexin agonists

suitable for systemic administration have been suggested as the

most promising strategy for the treatment of orexin deﬁciency-

associated conditions among all orexin replacement thera-

pies.^72,73Compound 2 was one of the ﬁrst small-molecule

orexin agonists reported thus far, although it mainly activates

OX2R with limited agonist activity at OX1R. Our SAR studies

at multiple sites suggested that an amide functionality is

required on ring-A at the left-hand side. Excitingly,

introduction of a pyridylmethyl group at this amide increased

potency at both OX1R and OX2R. Computational modeling

studies based on the recently reported cryo-EM structure of

OX2R bound with agonist 5 showed that 40 formed hydrogen

bonding or π-cation interactions via the pyridylmethyl group

with R333^7.29in OX1R, which may have contributed to the

observed high OX1R potency of 40. Through this eﬀort, we

have identiﬁed dual OX agonists, including 40 (RTOXA-43),

which acted as a full agonist with EC₅₀values of 24 nM at both

OX receptors. These are the ﬁrst and only small-molecule dual

orexin agonists discovered thus far. When measured using

continuous EEG/EMG recordings in 12-month-old mice, 40

(40 mg/kg, i.p.) increased time awake by increasing episode

duration, decreased NREM and REM sleep, and improved

sleep/wake consolidation by reducing the number, but not the

duration, of episodes of NREM and REM sleep. The current

results provide a promising lead for the discovery of small-

molecule agonists with OX1R activities and support develop-

ment of orexin agonists as potential treatments for orexin-

deﬁcient disorders such as narcolepsy.

EXPERIMENTAL SECTION

Chemistry. All solvents and chemicals were reagent-grade. Unless

otherwise mentioned, all reagents and solvents were purchased from

■

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tert-Butyl (2-(Benzyl(3-nitrophenyl)amino)ethyl)carbamate (46).

Compound 45 (6.28 g, 22.30 mmol) was dissolved in DMF (110

mL), followed by the addition of potassium carbonate (6.17 g, 44.65

mmol) and benzyl bromide (3.2 mL, 26.79 mmol). The reaction was

then heated at 60 °C overnight. Water (500 mL) and ethyl acetate

(200 mL) were added, and the organic layer was separated and dried.

The solvent was removed under reduced pressure, and the residue

was puriﬁed by ISCO to aﬀord the pure desired product. 5.11 g

orange syrup, yield: 62%. ¹H NMR (300 MHz, chloroform-d): δ

7.46−7.59 (m, 2H), 7.34−7.40 (m, 1H), 7.26−7.34 (m, 3H), 7.17 (d,

J = 7.16 Hz, 2H), 7.02 (d, J = 6.22 Hz, 1H), 4.58−4.76 (m, 3H),

3.57−3.72 (m, 2H), 3.37 (q, J = 6.47 Hz, 2H), 1.35−1.48 (m, 9H).

MS (ESI) m/z: 372.4 [M + H]⁺.

tert-Butyl (2-((3-Aminophenyl) (benzyl)amino)ethyl)carbamate

(47). Compound 46 (5.11 g, 13.76 mmol) was dissolved in a mixture

of ethanol and water (55/22 mL), followed by the addition of

ammonium chloride (7.36 g, 137.6 mmol) and iron powder (5.38 g,

96.3 mmol). The reaction was then heated at reﬂux for 3 h. After

cooling down, DCM (100 mL) was added, and the mixture was

ﬁltered through Celite. The organic layer was then separated and

dried. The solvent was then removed under reduced pressure and the

residue was puriﬁed by ISCO to aﬀord the desired product. 4.61 g

Figure 5. Intraperitoneal injection of 40 (40 mg/kg) during the quiet

phase (lights-on) increased time spent in wakefulness (A), reduced

time spent in NREM sleep (B), and REM sleep (C) 4 h postinjection.

Agonist 40 also reduced the number of state transitions compared to

saline injections (D), increased the duration of wake and NREM sleep

episodes, without aﬀecting the duration of REM sleep episodes (E),

and reduced the number of episodes of wake, NREM, and REM sleep

(F) 4 h postinjection. n = 5. Data represented as mean SEM. *P <

0.05, **P < 0.01 and ***P < 0.001. Note diﬀerent scaling on y-axes.

1

brown oil, yield: 98%. H NMR (300 MHz, chloroform-d): δ 7.08−

7.46 (m, 6H), 6.98 (t, J = 8.19 Hz, 1H), 6.02−6.26 (m, 2H), 4.62−

4.77 (m, 1H), 4.40−4.60 (m, 2H), 3.41−3.58 (m, 2H), 3.22−3.39

(m, 2H), 1.55−2.23 (m, 2H), 1.32−1.53 (m, 9H). MS (ESI) m/z:

342.2 [M + H]⁺.

tert-Butyl (2-(Benzyl(3-((5-bromo-2-methoxyphenyl)-

sulfonamido)phenyl)amino)ethyl)carb-amate (48). Under the

protection of nitrogen, compound 47 (3.52 g, 10.31 mmol) was

dissolved in anhydrous DCM (50 mL) at 0 °C. Pyridine (1 mL, 12.37

mmol) was added to the reaction, followed by the addition of 2-

methoxy-5-bromobenzenesulfonyl chloride (3.24 g, 11.34 mmol).

The reaction was warmed up to room temperature and stirred

overnight. The reaction was quenched by saturated NaHCO₃(30

mL), and DCM (100 mL) was added. The organic layer was

separated and dried. The solvent was removed under reduced

pressure and the residue was puriﬁed by ISCO to aﬀord the desired

product. 5.46 g oﬀ-white solid, yield: 90%. ¹H NMR (300 MHz,

chloroform-d): δ 7.87 (d, J = 2.45 Hz, 1H), 7.55 (dd, J = 2.45, 8.85

Hz, 1H), 7.18−7.35 (m, 4H), 7.10 (d, J = 6.59 Hz, 2H), 7.00 (t, J =

8.38 Hz, 1H), 6.90 (br s, 1H), 6.78 (d, J = 8.85 Hz, 1H), 6.49 (d, J =

8.48 Hz, 1H), 6.39 (d, J = 4.71 Hz, 2H), 4.59−4.73 (m, 1H), 4.48 (s,

2H), 3.84 (s, 3H), 3.41−3.53 (m, 2H), 3.19−3.34 (m, 2H), 1.36−

1.47 (m, 9H). MS (ESI) m/z: 592.2 [M + H]⁺.

commercial vendors and used as received. Flash column chromatog-

raphy was carried out on a Teledyne ISCO CombiFlash Rf system

using prepacked columns. Solvents used include hexane, ethyl acetate

(EtOAc), dichloromethane, methanol, and chloroform/methanol/

ammonium hydroxide (80:18:2) (CMA-80). Purity and character-

ization of compounds were established by a combination of high-

performance liquid chromatography (HPLC), thin-layer chromatog-

raphy (TLC), mass spectrometry, and NMR analyses. The melting

point was recorded by the Mel-Temp II instrument (Laboratory

1

Devices Inc., U.S.). H and ¹³C NMR spectra were recorded on a

Bruker AVANCE DPX-300 (300 MHz) spectrometer and were

determined in chloroform-d, DMSO-d₆, or methanol-d₄with

tetramethylsilane (0.00 ppm) or solvent peaks as the internal

reference. Chemical shifts are reported in parts per million relative

to the reference signal, and coupling constant (J) values are reported

in hertz (Hz). TLC was performed on EMD precoated silica gel 60

F254 plates, and spots were visualized with UV light or iodine

staining. Low-resolution mass spectra were obtained using a Waters

Alliance HT/Micromass ZQ system (ESI). All test compounds were

greater than 95% pure as determined by HPLC on an Agilent 1100

system using an Agilent Zorbax SB-Phenyl, 2.1 mm × 150 mm, 5 μm

column using a 15 min gradient elution of 5−95% solvent B at 1 mL/

min, followed by 10 min at 95% solvent B (solvent A, water with 0.1%

TFA; solvent B, acetonitrile with 0.1% TFA and 5% water; absorbance

monitored at 220 and 280 nm).

General Procedure for the Synthesis of Compounds 2 and

6−16. Compound 48 (1.0 equiv), boronic acid (1.2 equiv),

Pd(PPh₃)₄(0.1 equiv), and potassium carbonate (2.0 equiv) were

placed in a round-bottomed ﬂask with a condenser. The system was

ﬂushed with nitrogen, and a mixture of 1,4-dioxane/water (4/1, 0.1

M) was then added. The reaction was reﬂuxed for 2 h. After cooling

down, DCM (50 mL) was added and the organic layer was separated

and dried. The solvent was removed to give crude compound 49 that

was then dissolved in 4 N HCl in 1,4-dioxane (10 equiv). The

reaction was stirred for 2 h at room temperature, and the solvent was

then removed under reduced pressure. The residue was then dissolved

in DMF (0.1 M), followed by the addition of 2-dimethylamino

benzoic acid (1.1 equiv), HATU (1.2 equiv), and DIPEA (1.5 equiv).

The reaction was stirred overnight at room temperature and

quenched by saturated NaHCO₃. DCM (50 mL) was added, and

the organic layer was separated and dried. The solvent was removed

under reduced pressure to aﬀord the crude product (compound 50),

which was then mixed with Pd/C (0.1 equiv) in MeOH (0.1 M)

under the atmosphere of hydrogen (40 psi) for 12 h. The reaction

mixture was ﬁltered, and the solvent of the ﬁltrate was removed under

reduced pressure. The residue was puriﬁed by ISCO to aﬀord the

desired ﬁnal products (2, 6−16).

tert-Butyl (2-((3-Nitrophenyl)amino)ethyl)carbamate (45). 3-

Nitroﬂuorobenzene (5.0 mmol, 35.4 mmol) and ethylenediamine

(11.8 mL, 177.2 mmol) were mixed in a sealed tube, and the reaction

was heated at 120 °C overnight. After cooling down, the volatile was

evaporated under reduced pressure at 60 °C. The residue was then

redissolved in a mixture of THF (30 mL) and water (30 mL),

followed by the addition of potassium carbonate (14.7 g, 106.2 mmol)

and di-tert-butyl ecarbonate (19.3 g, 88.5 mmol). The reaction was

then stirred overnight and diluted by brine (150 mL). Ethyl acetate

(150 mL) was then added, and the organic layer was separated and

dried. The solvent was removed under reduced pressure, and the

residue was puriﬁed by ISCO to aﬀord the pure desired product. 6.28

1

g brown oil, yield: 63%. H NMR (300 MHz, chloroform-d): δ 7.51

(dd, J = 1.60, 8.01 Hz, 1H), 7.37 (t, J = 2.26 Hz, 1H), 7.22−7.31 (m,

1H), 6.87 (dd, J = 2.07, 8.10 Hz, 1H), 4.78−4.94 (m, 1H), 4.56−4.73

(m, 1H), 3.36−3.50 (m, 2H), 3.21−3.34 (m, 2H), 1.38−1.50 (m,

9H). MS (ESI) m/z: 282.3 [M + H]⁺; LCMS: >95% purity.

3′-(N-(3-((2-(2-(Dimethylamino)benzamido)ethyl)amino)-

phenyl)sulfamoyl)-4′-methoxy-N,N-dimethyl-[1,1′-biphenyl]-3-car-

boxamide (2). Yield: 45% over four steps. ¹H NMR (300 MHz,

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chloroform-d): δ 9.89 (br s, 1H), 8.00−8.17 (m, 2H), 7.64 (dd, J =

1.88, 8.67 Hz, 1H), 7.48−7.58 (m, 2H), 7.25−7.46 (m, 3H), 7.10−

7.23 (m, 3H), 7.01 (d, J = 8.67 Hz, 1H), 6.94 (t, J = 8.01 Hz, 1H),

6.45 (br s, 1H), 6.34 (dd, J = 8.01, 14.79 Hz, 2H), 4.33 (br s, 1H),

3.94−4.09 (m, 3H), 3.54−3.73 (m, 2H), 3.28 (br s, 2H), 2.86−3.20

(m, 6H), 2.42−2.66 (m, 6H). MS (ESI) m/z: 616.3 [M + H]⁺.

3′-(N-(3-((2-(2-(Dimethylamino)benzamido)ethyl)amino)-

phenyl)sulfamoyl)-N,N-diethyl-4′-methoxy-[1,1′-biphenyl]-3-car-

boxamide (6). Yield: 29% over four steps. ¹H NMR (300 MHz,

chloroform-d): δ 9.90 (br s, 1H), 8.07−8.16 (m, 1H), 7.97−8.06 (m,

2H), 7.65 (dd, J = 2.26, 8.67 Hz, 1H), 7.46−7.54 (m, 2H), 7.41 (t, J =

7.54 Hz, 2H), 7.30 (d, J = 7.35 Hz, 1H), 7.12−7.22 (m, 2H), 7.03 (d,

J = 8.67 Hz, 1H), 6.95 (t, J = 8.01 Hz, 1H), 6.44 (s, 1H), 6.34 (d, J =

5.09 Hz, 2H), 4.31 (t, J = 5.27 Hz, 1H), 4.07 (s, 3H), 3.65 (q, J = 5.84

Hz, 2H), 3.55 (br s, 2H), 3.17−3.36 (m, 4H), 2.75−2.99 (m, 6H),

1.26 (br s, 3H), 1.11 (br s, 3H). MS (ESI) m/z: 644.2 [M + H]⁺.

2-(Dimethylamino)-N-(2-((3-((4-methoxy-3′-(piperidine-1-car-

bonyl)-[1,1′-biphenyl])-3-sulfonamido)phenyl)amino)ethyl)-

2.87−2.99 (m, 1H), 2.54 (s, 6H), 1.27 (d, J = 6.97 Hz, 6H). MS

(ESI) m/z: 587.2 [M + H]⁺.

N-(2-((3-((3′-(Diethylamino)-4-methoxy-[1,1′-biphenyl])-3-

sulfonamido)phenyl)amino)ethyl-)-2-(dimethylamino)benzamide

(13). Yield: 32% over four steps. ¹H NMR (300 MHz, chloroform-d):

δ 9.93 (br s, 1H), 8.11 (dd, J = 1.70, 7.91 Hz, 1H), 8.04 (d, J = 2.26

Hz, 1H), 7.64 (dd, J = 2.26, 8.67 Hz, 1H), 7.36−7.46 (m, 1H), 7.07−

7.24 (m, 3H), 7.00 (d, J = 8.67 Hz, 1H), 6.87−6.96 (m, 2H), 6.69−

6.77 (m, 2H), 6.65 (d, J = 8.10 Hz, 1H), 6.45 (d, J = 2.07 Hz, 1H),

6.27−6.38 (m, 2H), 4.05 (s, 3H), 3.64 (q, J = 5.97 Hz, 2H), 3.38 (q, J

= 7.16 Hz, 3H), 3.29 (t, J = 5.75 Hz, 2H), 2.43−2.60 (m, 6H), 1.17

(t, J = 7.06 Hz, 6H). MS (ESI) m/z: 616.2 [M + H]⁺.

2-(Dimethylamino)-N-(2-((3-((3′-(dipropylamino)-4-methoxy-

[1,1′-biphenyl])-3-sulfonamido)phenyl)amino)ethyl)benzamide

(14). Yield: 33% over four steps. ¹H NMR (300 MHz, chloroform-d):

δ 9.93 (br s, 1H), 8.11 (dd, J = 1.70, 7.72 Hz, 1H), 8.03 (d, J = 2.45

Hz, 1H), 7.63 (dd, J = 2.35, 8.57 Hz, 1H), 7.40 (dt, J = 1.70, 7.72 Hz,

1H), 7.10−7.24 (m, 3H), 7.01 (d, J = 8.67 Hz, 1H), 6.93 (t, J = 8.01

Hz, 1H), 6.88 (s, 1H), 6.65−6.73 (m, 2H), 6.61 (dd, J = 2.26, 8.29

Hz, 1H), 6.44 (t, J = 2.07 Hz, 1H), 6.27−6.36 (m, 2H), 4.05 (s, 3H),

3.64 (q, J = 5.84 Hz, 1H), 3.16−3.33 (m, 4H), 2.49−2.58 (m, 6H),

1.57−1.64 (m, 4H), 0.93 (t, J = 7.44 Hz, 6H). MS (ESI) m/z: 644.2

[M + H]⁺.

N-(2-((3-((3′-(tert-Butyl)-4-methoxy-[1,1′-biphenyl])-3-

sulfonamido)phenyl)amino)ethyl)-2-(dimethylamino)benzamide

(15). Yield: 39% over four steps. ¹H NMR (300 MHz, chloroform-d):

δ 9.77−10.00 (m, 1H), 8.11 (dd, J = 1.60, 7.82 Hz, 1H), 8.04 (d, J =

2.26 Hz, 1H), 7.65 (dd, J = 2.35, 8.57 Hz, 1H), 7.47 (s, 1H), 7.39

(dd, J = 1.70, 7.54 Hz, 1H), 7.32−7.37 (m, 1H), 7.28−7.31 (m, 1H),

7.26 (s, 1H), 7.15−7.22 (m, 1H), 7.12 (d, J = 8.10 Hz, 1H), 7.03 (d, J

= 8.67 Hz, 1H), 6.88−6.99 (m, 2H), 6.43−6.49 (m, 1H), 6.27−6.38

(m, 2H), 4.06 (s, 3H), 3.57−3.70 (m, 2H), 3.29 (t, J = 5.84 Hz, 2H),

2.42−2.60 (m, 6H), 1.29−1.38 (m, 9H). MS (ESI) m/z: 601.2 [M +

H]⁺.

1

benzamide (7). Yield: 36% over four steps. H NMR (300 MHz,

chloroform-d): δ 9.67−9.90 (m, 1H), 8.03 (d, J = 2.07 Hz, 2H),

7.65−7.74 (m, 1H), 7.56−7.65 (m, 1H), 7.36−7.56 (m, 6H), 7.31 (s,

2H), 7.07 (s, 2H), 6.92 (br s, 2H), 6.79−6.87 (m, 1H), 4.04 (s, 3H),

3.79 (br s, 4H), 3.44 (br s, 4H), 3.14 (s, 6H), 1.69 (br s, 4H), 1.44−

1.59 (m, 2H). MS (ESI) m/z: 656.3 [M + H]⁺.

2-(Dimethylamino)-N-(2-((3-((4-methoxy-3′-(pyrrolidine-1-car-

bonyl)-[1,1′-biphenyl])-3-sulfonamido)phenyl)amino)ethyl)-

1

benzamide (8). Yield: 39% over four steps. H NMR (300 MHz,

chloroform-d): δ 9.90 (br s, 1H), 7.99−8.15 (m, 2H), 7.59−7.71 (m,

2H), 7.49−7.55 (m, 1H), 7.35−7.47 (m, 3H), 7.10−7.22 (m, 2H),

6.86−7.07 (m, 3H), 6.44 (s, 1H), 6.34 (dd, J = 4.71, 7.54 Hz, 2H),

4.23−4.39 (m, 1H), 4.06 (s, 3H), 3.56−3.72 (m, 8H), 3.42 (t, J =

6.50 Hz, 2H), 3.29 (t, J = 5.65 Hz, 2H), 2.82 (d, J = 8.10 Hz, 2H),

2.56 (s, 6H), 1.94−2.03 (m, 2H), 1.85−1.91 (m, 2H). MS (ESI) m/z:

642.2 [M + H]⁺.

3′-(N-(3-((2-(2-(Dimethylamino)benzamido)ethyl)amino)-

phenyl)sulfamoyl)-4′-methoxy-N-p-ropyl-[1,1′-biphenyl]-3-carbox-

amide (9). Yield: 32% over four steps. ¹H NMR (300 MHz,

chloroform-d): δ 9.87 (br s, 1H), 7.97−8.17 (m, 3H), 7.90 (s, 1H),

7.63−7.75 (m, 2H), 7.57 (d, J = 7.72 Hz, 1H), 7.33−7.49 (m, 2H),

7.10−7.23 (m, 2H), 7.02 (d, J = 4.33 Hz, 1H), 6.88−6.98 (m, 1H),

6.51 (br s, 1H), 6.44 (s, 1H), 6.32 (t, J = 6.59 Hz, 2H), 4.30 (br s,

1H), 4.05 (s, 3H), 3.60 (q, J = 5.78 Hz, 2H), 3.43 (q, J = 6.47 Hz,

2H), 3.26 (br s, 2H), 2.76−2.83 (m, 6H), 1.60−1.71 (m, 2H), 0.98

(t, J = 7.44 Hz, 3H). MS (ESI) m/z: 630.2 [M + H]⁺.

2-(Dimethylamino)-N-(2-((3-((4-methoxy-3′-(triﬂuoromethyl)-

[1,1′-biphenyl])-3-sulfonamido)phenyl)amino)ethyl)benzamide

(16). Yield: 31% over four steps. ¹H NMR (300 MHz, chloroform-d):

δ 9.91 (br s, 1H), 8.11 (dd, J = 1.70, 7.91 Hz, 1H), 8.04 (d, J = 2.45

Hz, 1H), 7.70 (s, 1H), 7.61−7.68 (m, 2H), 7.55−7.60 (m, 1H), 7.52

(d, J = 7.54 Hz, 1H), 7.42 (dt, J = 1.79, 7.68 Hz, 1H), 7.20 (d, J =

7.72 Hz, 1H), 7.14 (d, J = 8.10 Hz, 1H), 7.06 (d, J = 8.67 Hz, 1H),

6.89−6.99 (m, 2H), 6.39−6.49 (m, 1H), 6.34 (d, J = 8.10 Hz, 2H),

4.21−4.57 (m, 1H), 4.06−4.13 (m, 3H), 3.65 (q, J = 5.97 Hz, 2H),

3.29 (t, J = 5.75 Hz, 2H), 2.48−2.62 (m, 6H). MS (ESI) m/z: 613.2

[M + H]⁺.

General Procedure for the Synthesis of Compound 51a−b.

3-Nitroﬂuorobenzene (1 equiv) and the appropriate diamine (5

equiv) were mixed in a sealed tube, and the reaction was heated at

120 °C overnight. After cooling down, the volatile was evaporated

under reduced pressure at 60 °C. The residue was then redissolved in

a mixture of THF (30 mL) and water (30 mL), followed by the

addition of potassium carbonate (3.0 equiv) and di-tert-butyl

dicarbonate (2.5 equiv). The reactant was then stirred overnight

and diluted by brine (150 mL). Ethyl acetate (150 mL) was then

added, and the organic layer was separated and dried. The solvent was

removed under reduced pressure, and the residue was puriﬁed by

ISCO to aﬀord the pure desired product.

2-(Dimethylamino)-N-(2-((3-((3′-((dimethylamino)methyl)-4-

methoxy-[1,1′-biphenyl])-3-sulfonamido)phenyl)amino)ethyl)-

1

benzamide (10). Yield: 26% over four steps. H NMR (300 MHz,

chloroform-d): δ 9.89 (br s, 1H), 8.01−8.16 (m, 2H), 7.67 (dd, J =

2.35, 8.57 Hz, 1H), 7.22−7.51 (m, 6H), 7.06−7.22 (m, 3H), 6.81−

7.04 (m, 2H), 6.46 (d, J = 1.88 Hz, 1H), 6.25−6.42 (m, 2H), 4.14−

4.41 (m, 1H), 3.98−4.11 (m, 3H), 3.63 (q, J = 5.78 Hz, 2H), 3.47 (s,

2H), 3.28 (t, J = 5.65 Hz, 2H), 2.47−2.62 (m, 6H), 2.26 (s, 6H). MS

(ESI) m/z: 602.4 [M + H]⁺.

2-(Dimethylamino)-N-(2-((3-((3′-(dimethylamino)-4-methoxy-

[1,1′-biphenyl])-3-sulfonamido)phenyl)amino)ethyl)benzamide

(11). Yield: 35% over four steps. ¹H NMR (300 MHz, CDCl₃): δ 9.92

(br s, 1H), 8.11 (d, J = 7.72 Hz, 1H), 8.05 (d, J = 2.26 Hz, 1H), 7.66

(dd, J = 2.26, 8.48 Hz, 1H), 7.31−7.51 (m, 1H), 7.06−7.24 (m, 3H),

6.84−7.05 (m, 3H), 6.73−6.84 (m, 2H), 6.70 (d, J = 10.17 Hz, 1H),

6.45 (s, 1H), 6.31 (d, J = 7.91 Hz, 2H), 3.97−4.09 (m, 3H), 3.53−

3.69 (m, 2H), 3.29 (t, J = 5.75 Hz, 2H), 2.29−3.06 (m, 12H). MS

(ESI) m/z: 588.2 [M + H]⁺.

tert-Butyl (3-((3-Nitrophenyl)amino)propyl)carbamate (51a).

1

Yield: 72%. H NMR (300 MHz, chloroform-d): δ 7.50 (dd, J =

1.60, 8.01 Hz, 1H), 7.39 (t, J = 2.26 Hz, 1H), 7.25−7.30 (m, 1H),

6.88 (dd, J = 2.07, 8.10 Hz, 1H), 4.56−4.69 (m, 1H), 3.25 (dq, J =

3.58, 6.34 Hz, 4H), 2.72 (d, J = 7.16 Hz, 1H), 1.79 (t, J = 6.50 Hz,

2H), 1.45 (s, 9H). MS (ESI) m/z: 296.2 [M + H]⁺.

tert-Butyl (1-((3-Nitrophenyl)amino)propan-2-yl)carbamate

(51b). Yield: 30%. ¹H NMR (300 MHz, chloroform-d): δ 7.47−

7.52 (m, 2H), 7.35 (t, J = 2.26 Hz, 2H), 7.24−7.29 (m, 1H), 6.87

(dd, J = 1.88, 8.10 Hz, 1H), 4.78−4.87 (m, 1H), 4.46−4.59 (m, 1H),

3.92−4.03 (m, 1H), 3.21 (s, 2H), 3.06−3.15 (m, 1H), 2.74−2.92 (m,

1H), 1.40−1.47 (m, 9H), 1.24−1.28 (m, 3H). MS (ESI) m/z: 296.2

[M + H]⁺.

2-(Dimethylamino)-N-(2-((3-((3′-isopropyl-4-methoxy-[1,1′-bi-

phenyl])-3-sulfonamido)phenyl)amino)ethyl)benzamide (12).

1

Yield: 41% over four steps. H NMR (300 MHz, chloroform-d): δ

9.91 (br s, 1H), 8.11 (dd, J = 1.70, 7.91 Hz, 1H), 8.05 (d, J = 2.26 Hz,

1H), 7.65 (dd, J = 2.35, 8.57 Hz, 1H), 7.37−7.45 (m, 1H), 7.28−7.36

(m, 3H), 7.20 (d, J = 7.35 Hz, 2H), 7.08−7.16 (m, 1H), 7.02 (d, J =

8.67 Hz, 1H), 6.94 (t, J = 8.10 Hz, 1H), 6.89 (s, 1H), 6.45 (t, J = 2.07

Hz, 1H), 6.33 (td, J = 2.28, 8.05 Hz, 2H), 4.17−4.54 (m, 1H), 4.05−

4.09 (m, 3H), 3.64 (q, J = 5.97 Hz, 2H), 3.29 (t, J = 5.75 Hz, 2H),

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General Procedure for the Synthesis of Compound 52a−b.

Compound 51a or 51b (1 equiv) was dissolved in DMF (0.2 M),

followed by the addition of potassium carbonate (2 equiv) and benzyl

bromide (1.2 equiv). The reaction was then heated at 60 °C

overnight. Water and ethyl acetate were added, and the organic layer

was separated and dried. The solvent was removed under reduced

pressure, and the residue was puriﬁed by ISCO to aﬀord the desired

product 52a−b.

tert-Butyl (3-(Benzyl(3-nitrophenyl)amino)propyl)carbamate

(52a). Yield: 84%. ¹H NMR (300 MHz, chloroform-d): δ 7.46−

7.54 (m, 2H), 7.28−7.39 (m, 3H), 7.25−7.27 (m, 1H), 7.18 (d, J =

6.78 Hz, 2H), 6.86−6.96 (m, 1H), 4.60 (s, 2H), 3.45−3.58 (m, 1H),

3.21 (d, J = 6.40 Hz, 1H), 1.82−1.94 (m, 2H), 1.44 (s, 9H). MS

(ESI) m/z: 386.2 [M + H]⁺.

(m, 3H), 3.58 (br s, 1H), 3.18 (br s, 1H), 1.37 (s, 9H), 1.16 (d, J =

6.59 Hz, 2H). MS (ESI) m/z: 606.2 [M + H]⁺.

General Procedure for the Synthesis of Compounds 17−21.

Compound 48, 54a, or 54b (1.0 equiv), boronic acid (1.2 equiv),

Pd(PPh₃)₄(0.1 equiv), and potassium carbonate (2.0 equiv) were

placed in a round-bottomed ﬂask with an eﬃcient condenser. The

system was then ﬂushed with nitrogen, and a mixture of 1,4-dioxane/

water (4/1, 0.1 M) was added. The reaction was reﬂuxed for 2 h.

After cooling down, DCM (50 mL) was added and the organic layer

was separated and dried. The solvent was then removed, and the

residue (intermediate compound 53) was dissolved in 4 N HCl in

1,4-dioxane (10 equiv). The reaction was stirred for 2 h at room

temperature, and the solvent was then removed under reduced

pressure to give a brown solid that was then dissolved in DMF (0.1

M), followed by the addition of the corresponding benzoic acid (1.1

equiv), HATU (1.2 equiv), and DIPEA (1.5 equiv). The reaction was

stirred overnight at room temperature and quenched by saturated

NaHCO₃. DCM (50 mL) was added and the organic layer was

separated and dried. The solvent was removed under reduced

pressure to aﬀord the crude product (compound 54a−c), which was

then mixed with Pd/C (0.1 equiv) in MeOH (0.1 M) under the

atmosphere of hydrogen (40 psi) for 12 h. The reaction mixture was

ﬁltered, and the solvent of the ﬁltrate was removed under reduced

pressure. The residue was puriﬁed by ISCO to aﬀord the desired ﬁnal

products 17−21.

tert-Butyl (1-(Benzyl(3-nitrophenyl)amino)propan-2-yl)-

1

carbamate (52b). Yield: 81%. H NMR (300 MHz, chloroform-d):

δ 7.58 (br s, 1H), 7.48 (dd, J = 1.51, 7.91 Hz, 1H), 7.37 (d, J = 4.33

Hz, 1H), 7.25−7.32 (m, 4H), 7.17 (s, 1H), 6.97−7.12 (m, 1H),

4.59−4.77 (m, 3H), 4.27−4.50 (m, 1H), 4.09 (d, J = 6.97 Hz, 1H),

3.21−3.82 (m, 2H), 1.31−1.46 (m, 9H). MS (ESI) m/z: 386.1 [M +

H]⁺.

General Procedure for the Synthesis of Compound 53a−b.

Compound 52a or 52b (1 equiv) was dissolved in the mixture of

ethanol and water (5:2, 0.2 M), followed by the addition of

ammonium chloride (10 equiv) and iron powder (7 equiv). The

reaction was then heated at reﬂux for 3 h. After cooling down, DCM

(100 mL) was added and the mixture was ﬁltered through Celite. The

organic layer was then separated and dried. The solvent was then

removed under reduced pressure, and the residue was puriﬁed by

ISCO to aﬀord the desired product 53a−b.

4′-Methoxy-N,N-dimethyl-3′-(N-(3-((2-(3-methylbenzamido)-

ethyl)amino)phenyl)sulfamoyl-)-[1,1′-biphenyl]-3-carboxamide

1

(17). H NMR (300 MHz, chloroform-d): δ 9.89 (br s, 1H), 8.00−

8.17 (m, 2H), 7.64 (dd, J = 1.88, 8.67 Hz, 1H), 7.48−7.58 (m, 2H),

7.25−7.46 (m, 3H), 7.10−7.23 (m, 3H), 7.01 (d, J = 8.67 Hz, 1H),

6.94 (t, J = 8.01 Hz, 1H), 6.45 (br s, 1H), 6.34 (dd, J = 8.01, 14.79

Hz, 2H), 4.33 (br s, 1H), 3.94−4.09 (m, 3H), 3.54−3.73 (m, 2H),

3.28 (br s, 2H), 2.86−3.20 (m, 6H), 2.42−2.66 (m, 6H). MS (ESI)

m/z: 587.2 [M + H]⁺.

tert-Butyl N-{3-[(3-Aminophenyl)(benzyl)amino]propyl}-

1

carbamate (53a). Yield: 92%. H NMR (300 MHz, chloroform-d):

δ 7.27−7.40 (m, 2H), 7.18−7.25 (m, 3H), 6.97 (t, J = 8.01 Hz, 1H),

5.98−6.21 (m, 3H), 4.49 (m, 3H), 3.29−3.45 (m, 2H), 3.15 (d, J =

6.22 Hz, 2H), 1.74−1.88 (m, 2H), 1.43 (s, 9H). MS (ESI) m/z: 356.2

[M + H]⁺.

3′-(N-(3-((3-(2-(Dimethylamino)benzamido)propyl)amino)-

phenyl)sulfamoyl)-4′-methoxy-N,N-dimethyl-[1,1′-biphenyl]-3-car-

1

tert-Butyl N-{1-[(3-Aminophenyl)(benzyl)amino]propan-2-yl}-

boxamide (18). Yield: 29% over four steps. H NMR (300 MHz,

1

carbamate (53b). Yield: 92%. H NMR (300 MHz, chloroform-d):

chloroform-d): δ 9.71 (br s, 1H), 7.97−8.15 (m, 2H), 7.62−7.80 (m,

2H), 7.50−7.57 (m, 2H), 7.30−7.48 (m, 4H), 7.14−7.22 (m, 2H),

7.02−7.10 (m, 1H), 6.88−6.97 (m, 2H), 6.39−6.53 (m, 1H), 6.20−

6.37 (m, 2H), 4.03−4.10 (m, 3H), 3.31−3.54 (m, 2H), 2.78−3.21

(m, 12H), 2.38−2.64 (m, 2H), 1.74−1.90 (m, 2H). MS (ESI) m/z:

630.2 [M + H]⁺.

δ 7.37 (d, J = 4.33 Hz, 1H), 7.26−7.32 (m, 2H), 7.18 (t, J = 6.69 Hz,

2H), 6.96 (t, J = 8.10 Hz, 1H), 6.23 (dd, J = 2.45, 8.29 Hz, 1H),

6.12−6.19 (m, 1H), 6.06 (dd, J = 1.51, 7.72 Hz, 1H), 4.49−4.73 (m,

2H), 4.35−4.48 (m, 1H), 3.95−4.09 (m, 1H), 3.45−3.69 (m, 2H),

3.08−3.22 (m, 1H), 1.29−1.48 (m, 9H), 1.18 (d, J = 6.78 Hz, 3H).

MS (ESI) m/z: 356.2 [M + H]⁺.

General Procedure for the Synthesis of Compound 54a−b.

Under the protection of nitrogen, compound 53a or 53b (1 equiv)

was dissolved in anhydrous DCM (0.2 equiv) at 0 °C, pyridine (1.2

equiv) was added, followed by the addition of 2-methoxy-5-

bromobenzenesulfonyl chloride (1.1 equiv). The reaction was

warmed up to room temperature and stirred overnight. The reaction

was quenched with saturated NaHCO₃(10 mL), and DCM (30 mL)

was added. The organic layer was separated and dried. The solvent

was removed under reduced pressure, and the residue was puriﬁed by

ISCO to aﬀord the desired products 54a−b.

tert-Butyl (3-(Benzyl(3-((5-bromo-2-methoxyphenyl)-

sulfonamido)phenyl)amino)propyl)carbamate (54a). Yield: 85%.

¹H NMR (300 MHz, chloroform-d): δ 7.88 (d, J = 2.64 Hz, 1H), 7.55

(dd, J = 2.64, 8.85 Hz, 1H), 7.28 (d, J = 7.54 Hz, 2H), 7.23 (s, 1H),

7.11 (d, J = 6.59 Hz, 2H), 6.97 (d, J = 8.10 Hz, 1H), 6.92 (s, 1H),

6.79 (d, J = 8.85 Hz, 1H), 6.37−6.46 (m, 2H), 6.33 (d, J = 8.67 Hz,

1H), 4.54−4.64 (m, 1H), 4.44 (s, 2H), 3.85 (s, 3H), 3.30−3.42 (m,

2H), 3.15 (d, J = 6.22 Hz, 2H), 1.70−1.83 (m, 2H), 1.44 (s, 9H). MS

(ESI) m/z: 606.2 [M + H]⁺.

4′-Methoxy-N,N-dimethyl-3′-(N-(3-((3-(3-methylbenzamido)-

propyl)amino)phenyl)sulfamoyl)-[1,1′-biphenyl]-3-carboxamide

(19). Yield: 32% over four steps. ¹H NMR (300 MHz, chloroform-d):

δ 8.08 (d, J = 2.45 Hz, 1H), 7.71−8.04 (m, 1H), 7.67 (dd, J = 2.35,

8.57 Hz, 1H), 7.57 (s, 1H), 7.52 (td, J = 1.53, 3.53 Hz, 3H), 7.29−

7.45 (m, 3H), 7.04 (d, J = 8.85 Hz, 2H), 6.85−6.98 (m, 2H), 6.48 (d,

J = 1.88 Hz, 1H), 6.25−6.35 (m, 2H), 4.13 (s, 1H), 4.02−4.08 (m,

4H), 3.36 (q, J = 6.22 Hz, 2H), 2.76−3.23 (m, 14H), 2.35 (s, 3H),

1.69−1.74 (m, 3H). MS (ESI) m/z: 601.2 [M + H]⁺.

3′-(N-(3-((2-(2-(Dimethylamino)benzamido)propyl)amino)-

phenyl)sulfamoyl)-4′-methoxy-N,N-dimethyl-[1,1′-biphenyl]-3-car-

1

boxamide (20). Yield: 22% over four steps. H NMR (300 MHz,

chloroform-d): δ 9.70−9.79 (m, 1H), 8.01−8.11 (m, 2H), 7.64 (dd, J

= 2.45, 8.67 Hz, 1H), 7.49−7.56 (m, 2H), 7.38−7.47 (m, 2H), 7.30−

7.37 (m, 1H), 7.11−7.23 (m, 2H), 6.97−7.06 (m, 2H), 6.93 (t, J =

8.01 Hz, 1H), 6.42 (t, J = 1.98 Hz, 1H), 6.25−6.38 (m, 2H), 4.35−

4.46 (m, 1H), 4.06 (s, 3H), 3.13−3.20 (m, 2H), 2.85−3.06 (m, 7H),

2.80 (s, 6H), 1.28 (d, J = 6.78 Hz, 3H). MS (ESI) m/z: 630.2 [M +

H]⁺.

4′-Methoxy-N,N-dimethyl-3′-(N-(3-((2-(3-methylbenzamido)-

propyl)amino)phenyl)sulfamoyl)-[1,1′-biphenyl]-3-carboxamide

(21). Yield: 19% over four steps. ¹H NMR (300 MHz, chloroform-d):

δ 8.09 (d, J = 2.45 Hz, 1H), 8.01 (s, 1H), 7.65 (dd, J = 2.45, 8.67 Hz,

1H), 7.48−7.59 (m, 4H), 7.41 (t, J = 7.72 Hz, 1H), 7.30−7.35 (m,

1H), 7.24 (s, 1H), 7.11 (s, 1H), 7.01 (d, J = 8.67 Hz, 1H), 6.90 (t, J =

8.01 Hz, 1H), 6.72 (d, J = 8.10 Hz, 1H), 6.46 (t, J = 1.98 Hz, 1H),

6.29 (dt, J = 1.79, 7.86 Hz, 2H), 4.39−4.47 (m, 1H), 4.02 (s, 3H),

tert-Butyl (1-(Benzyl(3-((5-bromo-2-methoxyphenyl)-

sulfonamido)phenyl)amino)propan-2-yl)carbamate (54b). Yield:

1

83%. H NMR (300 MHz, chloroform-d): δ 7.86 (d, J = 2.64 Hz,

1H), 7.54 (dd, J = 2.45, 8.85 Hz, 1H), 7.25−7.30 (m, 3H), 7.07 (d, J

= 6.59 Hz, 2H), 6.99 (t, J = 8.10 Hz, 1H), 6.90 (br s, 1H), 6.70−6.83

(m, 1H), 6.52 (d, J = 8.48 Hz, 1H), 6.34−6.46 (m, 2H), 4.42−4.64

(m, 2H), 4.33 (d, J = 12.06 Hz, 1H), 3.90−4.05 (m, 1H), 3.79−3.88

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3.00 (s, 2H), 2.80 (s, 6H), 2.32 (s, 3H), 1.25 (d, J = 6.78 Hz, 3H).

MS (ESI) m/z: 601.2 [M + H]⁺.

7.42 (t, J = 7.54 Hz, 1H), 7.30−7.37 (m, 2H), 7.22 (d, J = 7.16 Hz,

1H), 7.02−7.12 (m, 2H), 6.89−6.99 (m, 3H), 6.77 (s, 1H), 6.62 (d, J

= 8.10 Hz, 1H), 6.48 (d, J = 7.54 Hz, 1H), 4.08 (s, 3H), 3.93 (br s,

1H), 3.84 (br s, 1H), 2.94−3.22 (m, 10H), 2.73−2.86 (m, 8H). MS

(ESI) m/z: 642.2 [M + H]⁺.

tert-Butyl 4-(3-Nitrophenyl)piperazine-1-carboxylate (57). 3-

Nitroﬂuorobenzene (5.0 mmol, 35.4 mmol) and piperazine (9.16 g,

106.31 mmol) were mixed in a sealed tube, and the reaction was

heated at 120 °C overnight. After cooling down, the volatile was

evaporated under reduced pressure at 60 °C. The residue was then

redissolved in the mixture of THF (30 mL) and water (30 mL),

followed by the addition of potassium carbonate (14.7 g, 106.2 mmol)

and di-tert-butyl dicarbonate (19.3 g, 88.5 mmol). The reaction was

then stirred overnight and diluted by brine (150 mL). Ethyl acetate

(150 mL) was then added, and the organic layer was separated and

dried. The solvent was removed under reduced pressure, and the

residue was puriﬁed by ISCO to aﬀord the desired product. 5.30 g as a

yellow solid, yield: 49%. ¹H NMR (300 MHz, chloroform-d): δ 7.64−

7.76 (m, 2H), 7.40 (t, J = 8.10 Hz, 1H), 7.16−7.24 (m, 1H), 3.54−

3.67 (m, 2H), 3.19−3.30 (m, 4H), 1.49 (s, 9H). MS (ESI) m/z: 308.2

[M + H]⁺.

tert-Butyl 4-(3-Aminophenyl)piperazine-1-carboxylate (58).

Compound 57 (5.35 g, 17.41 mmol) was dissolved in the mixture

of ethanol and water (70/30 mL), followed by the addition of

ammonium chloride (9.31 g, 174.1 mmol) and iron powder (6.81 g,

121.8 mmol). The reaction was then heated at reﬂux for 3 h. After

cooling down, DCM (100 mL) was added, and the mixture was

ﬁltered through Celite. The organic layer was then separated and

dried. The solvent was then removed under reduced pressure, and the

residue was puriﬁed by ISCO to aﬀord the pure desired product. 4.15

g brown oil, yield: 86%. ¹H NMR (300 MHz, chloroform-d): δ 6.98−

7.11 (m, 1H), 6.32−6.39 (m, 1H), 6.20−6.30 (m, 2H), 3.58−3.85

(m, 2H), 3.49−3.58 (m, 4H), 3.07−3.14 (m, 4H), 1.47−1.50 (m,

9H). MS (ESI) m/z: 278.2 [M + H]⁺.

tert-Butyl 4-(3-((5-Bromo-2-methoxyphenyl)sulfonamido)-

phenyl)piperazine-1-carboxylate (59). Under the protection of

nitrogen, compound 58 (3.04 g, 10.96 mmol) was dissolved in

anhydrous DCM (55 mL) at 0 °C, pyridine (1.06 mL, 13.15 mmol)

was added, followed by the addition of 2-methoxy-5-bromobenzene-

sulfonyl chloride (3.44 g, 12.06 mmol). The reaction was warmed up

to room temperature and stirred overnight. The reaction was

quenched by saturated NaHCO₃(30 mL), and DCM (100 mL)

was added. The organic layer was separated and dried. The solvent

was removed under reduced pressure, and the residue was puriﬁed by

ISCO to aﬀord the desired product 57. 4.61 g brown solid, yield: 80%.

¹H NMR (300 MHz, chloroform-d): δ 7.94 (d, J = 2.45 Hz, 1H), 7.58

(dd, J = 2.54, 8.76 Hz, 1H), 7.26 (s, 3H), 7.07 (t, J = 8.10 Hz, 1H),

6.82−6.93 (m, 2H), 6.69−6.76 (m, 1H), 6.64 (d, J = 8.29 Hz, 1H),

6.41 (d, J = 7.91 Hz, 1H), 4.01 (s, 3H), 3.48−3.62 (m, 4H), 3.00−

3.16 (m, 4H), 1.48 (s, 9H). MS (ESI) m/z: 528.2 [M + H]⁺.

General Procedure for the Synthesis of 22 and 23.

Compound 59 (1.0 equiv), boronic acid (1.2 equiv), Pd(PPh₃)₄

(0.1 equiv), and potassium carbonate (2.0 equiv) were placed in a

round-bottomed ﬂask with an eﬃcient condenser. The system was

then ﬂushed with nitrogen, and a mixture of 1,4-dioxane/water (4/1,

0.1 M) was added. The reaction was reﬂuxed for 2 h. After cooling

down, DCM (50 ml) was added and the organic layer was separated

and dried. The solvent was then removed, and the residue

(compound 58) was dissolved in 4 N HCl in 1,4-dioxane (10

equiv). The reaction was stirred for 2 h at room temperature, and the

solvent was then removed under reduced pressure. The residue was

then dissolved in DMF (0.1 M), followed by the addition of the

corresponding benzoic acid (1.1 equiv), HATU (1.2 equiv), and

DIPEA (1.5 equiv). The reaction was stirred overnight at room

temperature and quenched by saturated NaHCO₃. DCM (50 mL)

was added, and the organic layer was separated and dried. The solvent

was removed under reduced pressure to aﬀord the crude product that

was puriﬁed by ISCO to give the desired products 22−23.

4′-Methoxy-N,N-dimethyl-3′-(N-(3-(4-(3-methylbenzoyl)-

piperazin-1-yl)phenyl)sulfamoyl)-[1,1′-biphenyl]-3-carboxamide

1

(23). Yield: 41% over three steps. H NMR (300 MHz, chloroform-

d): δ 8.04 (d, J = 2.26 Hz, 1H), 7.71 (dd, J = 2.35, 8.57 Hz, 1H),

7.48−7.60 (m, 2H), 7.42 (t, J = 7.54 Hz, 1H), 7.31−7.37 (m, 1H),

7.21−7.30 (m, 4H), 7.17 (d, J = 7.16 Hz, 1H), 7.08 (d, J = 8.85 Hz,

2H), 6.97−7.04 (m, 1H), 6.78 (s, 1H), 6.62 (d, J = 8.29 Hz, 1H),

6.49 (d, J = 7.72 Hz, 1H), 4.07 (s, 3H), 3.83 (br s, 2H), 3.54 (br s,

2H), 2.90−3.25 (m, 10H), 2.38 (s, 3H). MS (ESI) m/z: 613.2 [M +

H]⁺.

N¹-(3-Nitrophenyl)ethane-1, 2-Diamine Hydrochloride (61).

Compound 45 was dissolved in minimum of ethyl acetate, and 4 N

HCl in dioxane (50 mL) was added. The mixture was then stirred for

2 h until no bubbles were released. Hexane (100 mL) was added to

precipitate any solid, and the suspension was ﬁltered. The solid

collected was rinsed with diethyl ether and dried in vacuum overnight

to give the desired product. 17 g tan solid, yield: 78%. ¹H NMR (300

MHz, DMSO-d₆): δ 8.12 (br s, 4H), 7.29−7.50 (m, 3H), 6.91−7.17

(m, 1H), 3.38 (t, J = 6.40 Hz, 2H), 2.87−3.06 (m, 2H). MS (ESI) m/

z: 182.2 [M + H]⁺.

N-(2-((3-Aminophenyl)amino)ethyl)-3-methylbenzamide (63). 3-

Methyl benzoic acid (2.73 g, 20 mmol) and 1,1′-carbonyldiimi-dazole

(3.25 g, 20 mmol) were mixed in DCM and stirred for 15 min.

Compound 59 (2.62 g, 10 mmol) was then added in one portion,

followed by the addition of DIPEA (10.5 mL, 60 mmol). The reaction

was then monitored by TLC. After completion, sat. NaHCO₃was

added to quench the reaction. The organic layer was then separated

and dried by anhydrous MgSO₄. The solvent was removed under

reduced pressure, and the residue (compound 62) was then

redissolved in the mixture of EtOH (80 mL) and water (30 mL),

followed by the addition of ammonium chloride (10.7 g, 0.2 mol) and

iron powder (7.84 g, 0.14 mol). The reaction was then brought to

reﬂux for 2 h. After cooling down to room temperature, the reaction

mixture was ﬁltered and the ﬁltrate was concentrated. Ethyl acetate

(200 mL) and brine (200 mL) were added to the residue, and the

organic layer was separated and dried. The solvent was removed

under reduced pressure, and the residue was subjected to ISCO to

give the desired product. 3.33 g, brown oil, yield: 62%. ¹H NMR (300

MHz, DMSO-d₆): δ 8.56 (m, 2H), 7.54−8.22 (m, 3H), 7.10−7.50

(m, 2H), 7.02 (br s, 2H), 3.60−3.20 (m, 4H), 2.35 (s, 3H). MS (ESI)

m/z: 270.2 [M + H]⁺.

N-(2-((3-((5-Bromo-2-methoxyphenyl)sulfonamido)phenyl)-

amino)ethyl)-3-methylbenzamide (64). Compound 63 (3.33 g, 12.4

mmol) was dissolved in DCM (100 mL) and triethylamine (3.5 mL,

24.7 mmol) was added, followed by the addition of catalytic DMAP

(302 mg, 2.47 mmol). The mixture was then cooled to 0 °C, and 5-

bromo-2-methoxy benzenesulfonyl chloride (3.86 g, 13.0 mmol) in

THF (10 mL) was added slowly over a period of 20 min. The reaction

was warmed up to room temperature and stirred overnight. The

reaction was quenched by sat. NaHCO₃(50 mL), and ethyl acetate

(100 mL) was added. The organic layer was separated and dried. The

solvent was removed under reduced pressure, and the residue was

subjected to ISCO to give the desired product. 4.84 g, light-yellow

foam, yield: 76%. ¹H NMR (300 MHz, chloroform-d): δ 7.94 (d, J =

2.45 Hz, 1H), 7.47−7.60 (m, 3H), 7.29−7.36 (m, 2H), 6.97 (t, J =

8.01 Hz, 1H), 6.80−6.92 (m, 2H), 6.45 (d, J = 2.07 Hz, 2H), 6.33−

6.40 (m, 1H), 6.29 (d, J = 9.23 Hz, 1H), 4.21 (br s, 1H), 4.00 (s, 3H),

3.66 (q, J = 5.90 Hz, 2H), 3.32 (t, J = 5.65 Hz, 2H), 2.39 (s, 3H). MS

(ESI) m/z: 519.2 [M + H]⁺.

General Procedure for the Miyaura Borylation Reaction.

Aromatic bromides or iodides (1.0 equiv) were dissolved in 1,4-

dioxane (0.1 M), and bis(pinacolato))diboron (1.5 equiv) was added,

followed by PdCl₂(dppf) (0.1 equiv) and KOAc (2 equiv). The

reaction was then heated at 90 °C overnight. After cooling to room

temperature, the reaction mixture was ﬁltered by Celite and the

3′-(N-(3-(4-(2-(Dimethylamino)benzoyl)piperazin-1-yl)phenyl)-

sulfamoyl)-4′-methoxy-N,N-dimethyl-[1,1′-biphenyl]-3-carboxa-

mide (22). Yield: 36% over three steps. ¹H NMR (300 MHz,

chloroform-d): δ 8.04 (d, J = 2.07 Hz, 1H), 8.03 (d, J₁= 9.0 Hz, J₂=

177 Hz, 1H), 7.72 (d, J = 8.67 Hz, 1H), 7.52 (d, J = 12.06 Hz, 2H),

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Article

ﬁltrate was concentrated under reduced pressure. The residue was

subjected to ISCO to aﬀord the desired product or used in the next

Suzuki reaction without further puriﬁcation.

1H), 6.79−6.83 (m, 1H), 6.58−6.66 (m, 1H), 6.46−6.50 (m, 1H),

6.31−6.37 (m, 1H), 6.24−6.30 (m, 1H), 4.21−4.34 (m, 1H), 4.05 (s,

3H), 3.25−3.33 (m, 2H), 3.10−3.24 (m, 2H), 2.38 ppm (s, 3H). MS

(ESI) m/z: 593.1 [M + H]⁺.

General Procedure for the Suzuki Coupling Reaction. The

boronic acid pinacol ester (1.0 equiv), aryl halide (1.0 equiv), and

K₂CO₃(2.0 equiv) were dissolved in a mixture of 1,4-dioxane and

water (v/v = 4:1, 0.04 M). The mixture was degassed and purged with

nitrogen three times. Pd(PPh₃)₄(0.1 equiv) was then added, and the

reaction was stirred at 90 °C for 1 h. The reaction was then cooled

down and quenched with brine. Ethyl acetate was then added, and the

organic layer was separated and dried. The solvent was removed

under reduced pressure, and the residue was subjected to ISCO to

give the pure desired product.

N-(2-((3-((2-Methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)phenyl)sulfonamido)p-henyl)amino)ethyl)-3-methylbenza-

mide (65). 65 was prepared according to the general procedure for

the Miyaura borylation reaction using compound 64 as the starting

material. Yield: 87%. ¹H NMR (300 MHz, chloroform-d): δ 8.29 (d, J

= 1.51 Hz, 1H), 8.15−8.27 (m, 1H), 7.85−7.95 (m, 1H), 7.45−7.65

(m, 2H), 7.28−7.34 (m, 2H), 6.89−7.00 (m, 3H), 6.84 (s, 1H),

6.20−6.53 (m, 3H), 4.10−4.22 (m, 1H), 4.03 (s, 3H), 3.64 (d, J =

6.03 Hz, 2H), 3.33 (br s, 2H), 2.34−2.41 (m, 3H), 1.26−1.31 (m,

12H). MS (ESI) m/z: 566.2 [M + H]⁺.

5-(4-Methoxy-3-(N-(3-((2-(3-methylbenzamido)ethyl)amino)-

phenyl)sulfamoyl)phenyl)-N,N-dimethylthiophene-3-carboxamide

1

(29). Yield: 88%. H NMR (300 MHz, chloroform-d): δ 8.08 (d, J =

2.26 Hz, 1H), 7.64 (dd, J₁= 2.35 Hz, J₂= 8.57 Hz, 1H), 7.56 (s, 1H),

7.46−7.52 (m, 1H), 7.35 (s, 1H), 7.33−7.34 (m, 1H), 6.99 (d, J =

8.67 Hz, 1H), 6.92 (d, J = 8.29 Hz, 1H), 6.83−6.88 (m, 1H), 6.81 (s,

1H), 6.48 (s, 1H), 6.30−6.37 (m, 1H), 6.21−6.27 (m, 1H), 4.34−

4.47 (m, 1H), 4.04 (s, 3H), 3.64 (d, J = 5.84 Hz, 2H), 3.26 (s, 2H),

3.11 (br s, 6H), 2.37 (s, 3H). MS (ESI) m/z: 593.2 [M + H]⁺.

4-(4-Methoxy-3-(N-(3-((2-(3-methylbenzamido)ethyl)amino)-

phenyl)sulfamoyl)phenyl)-N,N-dimethylthiazole-2-carboxamide

1

(30). Yield: 70%. H NMR (300 MHz, chloroform-d): δ 8.31 (d, J =

2.26 Hz, 1H), 7.99−8.07 (m, 1H), 7.63−7.73 (m, 1H), 7.57 (s, 1H),

7.51−7.56 (m, 1H), 7.45−7.50 (m, 2H), 7.28−7.33 (m, 2H), 7.06 (d,

J = 8.67 Hz, 1H), 6.95 (m, 1H), 6.84 (s, 1H), 6.48 (s, 1H), 6.37−6.43

(m, 1H), 4.08 (s, 3H), 3.57−3.67 (m, 5H), 3.24−3.33 (m, 2H), 3.17

(s, 3H), 2.38 (s, 3H). MS (ESI) m/z: 594.2 [M + H]⁺.

(E)-N-(2-((3-((5-(3-(Dimethylamino)-3-oxoprop-1-en-1-yl)-2-

methoxyphenyl)sulfonamido)-phenyl)amino)ethyl)-3-methylben-

zamide (31). Yield: 63%. ¹H NMR (300 MHz, chloroform-d): δ 8.03

(d, J = 2.26 Hz, 1H), 7.46−7.59 (m, 5H), 7.28−7.34 (m, 2H), 6.91−

7.01 (m, 2H), 6.73−6.85 (m, 2H), 6.50−6.58 (m, 1H), 6.44 (s, 1H),

6.24−6.38 (m, 2H), 4.13−4.32 (m, 1H), 4.04 (s, 3H), 3.65 (d, J =

5.84 Hz, 2H), 3.29 (s, 2H), 3.14 (s, 3H), 3.04 (s, 3H), 2.39 (s, 3H).

MS (ESI) m/z: 537.2 [M + H]⁺.

Compound 24−32 and 33−42 were prepared according to the

general procedure for the Suzuki coupling reaction using compound

65 and the corresponding halogenated aromatic amide as starting

materials.

2-(4-Methoxy-3-(N-(3-((2-(3-methylbenzamido)ethyl)amino)-

phenyl)sulfamoyl)phenyl)-N,N-dimethylisonicotinamide (24).

1

N-(2-((3-((5-(3-(Dimethylamino)-3-oxopropyl)-2-

Yield:78%. H NMR (300 MHz, chloroform-d): δ 8.64 (d, J = 4.90

methoxyphenyl)sulfonamido)phenyl)amino)ethyl)-3-methylbenza-

Hz, 1H), 8.43 (d, J = 2.45 Hz, 1H), 8.25 (dd, J = 2.35, 8.76 Hz, 1H),

7.67 (s, 1H), 7.55 (s, 1H), 7.47 (d, J = 6.59 Hz, 1H), 7.33−7.20 (m,

2H), 7.15 (dd, J = 1.41, 4.99 Hz, 1H), 7.08 (d, J = 8.85 Hz, 1H),

6.91−6.98 (m, 2H), 6.84−6.90 (m, 1H), 6.46−6.52 (m, 1H), 6.31 (d,

J = 8.10 Hz, 1H), 6.23 (d, J = 9.04 Hz, 1H), 4.27−4.44 (m, 1H), 4.07

(s, 3H), 3.61 (d, J = 5.46 Hz, 2H), 3.25 (t, J = 5.56 Hz, 2H), 3.15 (s,

3H), 2.98 (s, 3H), 2.35 (s, 3H). MS (ESI) m/z: 588.2 [M + H]⁺.

5-(4-Methoxy-3-(N-(3-((2-(3-methylbenzamido)ethyl)amino)-

phenyl)sulfamoyl)phenyl)-N,N-dimethylnicotinamide (25). Yield:

1

mide (32). Yield: 67%. H NMR (300 MHz, chloroform-d): δ 7.64

(d, J = 2.26 Hz, 1H), 7.58 (s, 1H), 7.52 (br s, 1H), 7.28−7.36 (m,

3H), 6.95 (t, J = 8.01 Hz, 1H), 6.89 (d, J = 8.29 Hz, 1H), 6.83 (s,

1H), 6.67−6.75 (m, 1H), 6.43 (s, 1H), 6.35 (s, 1H), 6.28 (d, J = 7.54

Hz, 1H), 3.98 (s, 3H), 3.64 (d, J = 5.65 Hz, 2H), 3.30 (t, J = 5.75 Hz,

2H), 2.81−2.92 (m, 8H), 2.49−2.57 (m, 2H), 2.39 (s, 3H). MS (ESI)

m/z: 539.2 [M + H]⁺.

N-(2-(Dimethylamino)ethyl)-4′-methoxy-N-methyl-3′-(N-(3-((2-

1

(3-methylbenzamido)ethyl)amino)phenyl)sulfamoyl)-[1,1′-biphen-

80%. H NMR (300 MHz, chloroform-d): δ 8.78 (d, J = 2.26 Hz,

1

yl]-3-carboxamide (33). Yield: 72%. H NMR (300 MHz, chloro-

1H), 8.60 (d, J = 1.88 Hz, 1H), 8.08 (d, J = 2.45 Hz, 1H), 7.89 (t, J =

2.17 Hz, 1H), 7.70 (dd, J = 2.45, 8.67 Hz, 1H), 7.57 (s, 1H), 7.50 (d,

J = 6.41 Hz, 1H), 7.09 (d, J = 8.67 Hz, 1H), 6.95−7.03 (m, 1H),

6.86−6.95 (m, 1H), 6.46−6.51 (m, 1H), 6.34 (d, J = 8.10 Hz, 1H),

6.25 (d, J = 7.72 Hz, 1H), 4.07 (s, 3H), 3.60−3.70 (m, 2H), 3.26 (t, J

= 5.75 Hz, 2H), 3.15 (s, 3H), 3.06 (s, 3H), 2.35 (s, 3H). MS (ESI)

m/z: 588.2 [M + H]⁺.

form-d): δ 8.12 (d, J = 2.26 Hz, 1H), 7.69 (dd, J₁= 2.35 Hz, J₂= 8.57

Hz, 1H), 7.58 (br s, 3H), 7.53 (s, 2H), 7.38−7.47 (m, 2H), 7.30−

7.37 (m, 2H), 7.15 (br s, 2H), 7.04 (d, J = 8.67 Hz, 1H), 6.93 (t, J =

8.01 Hz, 2H), 6.46 (s, 1H), 6.32 (d, J = 7.91 Hz, 1H), 6.23 (d, J =

9.23 Hz, 1H), 4.04 (s, 3H), 3.65−3.74 (m, 1H), 3.55−3.64 (m, 2H),

3.31−3.42 (m, 1H), 3.17−3.26 (m, 2H), 3.11 (br s, 1H), 3.04 (br s,

2H), 2.61−2.71 (m, 1H), 2.41−2.50 (m, 1H), 2.34 (s, 6H), 2.08 (br

s, 3H). MS (ESI) m/z: 644.2 [M + H]⁺.

4-(4-Methoxy-3-(N-(3-((2-(3-methylbenzamido)ethyl)amino)-

phenyl)sulfamoyl)phenyl)-N,N-dimethylpicolinamide (26). Yield:

1

N-(2-(Dimethylamino)ethyl)-4′-methoxy-3′-(N-(3-((2-(3-

83%. H NMR (300 MHz, chloroform-d): δ 8.59 (d, J = 5.27 Hz,

methylbenzamido)ethyl)amino)phe-nyl)sulfamoyl)-[1,1′-biphen-

1H), 8.19 (d, J = 2.26 Hz, 1H), 7.77 (d, J = 2.07 Hz, 1H), 7.74 (d, J =

2.45 Hz, 1H), 7.58 (s, 1H), 7.51 (d, J = 6.41 Hz, 1H), 7.47 (dd, J =

1.88, 5.27 Hz, 1H), 7.08 (d, J = 8.67 Hz, 1H), 6.92 (t, J = 8.01 Hz,

1H), 6.86 (s, 1H), 6.48 (s, 1H), 6.32 (d, J = 8.29 Hz, 1H), 6.20 (d, J =

9.23 Hz, 1H), 4.43−4.59 (m, 1H), 4.07 (s, 3H), 3.61−3.71 (m, 2H),

3.24 (t, J = 5.56 Hz, 2H), 3.15 (d, J = 3.58 Hz, 6H), 2.34 (s, 3H). MS

(ESI) m/z: 588.1 [M + H]⁺.

1

yl]-3-carboxamide (34). Yield: 75%. H NMR (300 MHz, chloro-

form-d): δ 8.14 (d, J = 2.45 Hz, 1H), 8.02 (s, 1H), 7.78−7.84 (m,

1H), 7.66−7.73 (m, 2H), 7.63 (s, 1H), 7.55 (br s, 2H), 7.49 (d, J =

3.20 Hz, 2H), 7.20 (br s, 2H), 7.04 (d, J = 8.67 Hz, 1H), 6.93 (t, J =

7.91 Hz, 2H), 6.55 (s, 1H), 6.31 (d, J = 8.10 Hz, 1H), 6.23 (d, J =

6.22 Hz, 1H), 4.03 (s, 3H), 3.65 (d, J = 5.65 Hz, 2H), 3.47−3.58 (m,

2H), 3.26 (br s, 2H), 2.57 (t, J = 5.75 Hz, 1H), 2.52 (d, J = 6.03 Hz,

1H), 2.31 (s, 6H), 2.25 (s, 3H). MS (ESI) m/z: 630.2 [M + H]⁺;

LCMS.

6-(4-Methoxy-3-(N-(3-((2-(3-methylbenzamido)ethyl)amino)-

phenyl)sulfamoyl)phenyl)-N,N-dimethylpicolinamide (27). Yield:

1

85%. H NMR (300 MHz, chloroform-d): δ 8.58 (d, J = 2.45 Hz,

4′-Methoxy-N-methyl-3′-(N-(3-((2-(3-methylbenzamido)ethyl)-

1H), 8.15 (d, J = 8.67 Hz, 1H), 7.77 (d, J = 7.72 Hz, 1H), 7.70 (s,

1H), 7.56 (s, 1H), 7.48 (d, J = 6.41 Hz, 2H), 7.06 (d, J = 8.85 Hz,

1H), 6.86−6.98 (m, 1H), 6.82 (s, 2H), 6.46 (s, 1H), 6.21−6.35 (m,

2H), 4.28−4.44 (m, 1H), 4.06 (s, 3H), 3.57 (d, J = 5.65 Hz, 2H),

3.22 (d, J = 5.46 Hz, 2H), 3.17 (s, 3H), 3.11 (s, 3H), 2.37 (s, 3H).

MS (ESI) m/z: 588.2 [M + H]⁺.

amino)phenyl)sulfamoyl)-N-(pyridin-2-yl)-[1,1′-biphenyl]-3-car-

1

boxamide (35). Yield: 78%. H NMR (300 MHz, chloroform-d): δ

8.36−8.40 (m, 1H), 8.24−8.33 (m, 2H), 8.18−8.22 (m, 1H), 7.70−

7.76 (m, 1H), 7.59−7.62 (m, 1H), 7.56−7.59 (m, 1H), 7.53−7.56

(m, 1H), 7.50−7.52 (m, 1H), 7.47−7.50 (m, 1H), 7.44−7.47 (m,

1H), 7.30−7.39 (m, 2H), 7.21 (s, 1H), 7.18 (s, 1H), 7.02−7.07 (m,

1H), 6.89−6.96 (m, 1H), 6.79−6.83 (m, 1H), 6.49−6.58 (m, 2H),

6.27−6.33 (m, 1H), 6.14−6.22 (m, 1H), 4.04 (s, 3H), 3.90 (s, 3H),

3.63−3.73 (m, 2H), 3.23−3.35 (m, 2H), 2.28 (s, 3H). MS (ESI) m/z:

650.2 [M + H]⁺.

4-(4-Methoxy-3-(N-(3-((2-(3-methylbenzamido)ethyl)amino)-

phenyl)sulfamoyl)phenyl)-N,N-dimethylthiophene-2-carboxamide

1

(28). Yield: 68%. H NMR (chloroform-d, 300 MHz): δ 8.01−8.04

(m, 1H), 7.62−7.68 (m, 1H), 7.55 (d, J = 1.5 Hz, 2H), 7.47−7.52 (m,

1H), 7.45 (s, 1H), 7.30 (s, 2H), 7.00−7.05 (m, 1H), 6.91−6.99 (m,

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N - B e n z y l - 4 ′ - m e t h o x y - N - m e t h y l - 3 ′ - ( N - ( 3 - ( ( 2 - ( 3 -

methylbenzamido)ethyl)amino)phenyl)sulfa-moyl)-[1,1′-biphen-

mL) was added slowly. The reaction was then stirred at this

temperature for 1 h and allowed to warm up to room temperature

overnight. Saturated sodium bicarbonate (50 mL) was added to

quench the reaction, and the reaction was extracted with ethyl acetate

(50 mL). The organic layer was separated and dried. The solvent was

removed under reduced pressure, and the residue was subjected to

ISCO to give the desired product 66.

1

yl]-3-carboxamide (36). Yield: 81%. H NMR (300 MHz, chloro-

form-d): δ 8.13 (br s, 1H), 7.48−7.75 (m, 5H), 7.30−7.46 (m, 6H),

6.99−7.21 (m, 3H), 6.93 (t, J = 7.91 Hz, 1H), 6.85 (s, 1H), 6.45 (s,

1H), 6.32 (d, J = 8.10 Hz, 1H), 6.23 (d, J = 7.72 Hz, 1H), 4.49−4.82

(m, 2H), 4.04 (s, 3H), 3.58 (br s, 2H), 3.21 (br s, 2H), 2.76−3.11 (m,

3H), 2.33 (br s, 3H). MS (ESI) m/z: 663.2 [M + H]⁺.

5-Bromo-2-methoxy-N-[3-({2-[(3-methylphenyl)formamido]-

1

4′-Methoxy-N-methyl-3′-(N-(3-((2-(3-methylbenzamido)ethyl)-

ethyl}amino)phenyl]benzamide (66). 579 mg, yield: 60%. H NMR

amino)phenyl)sulfamoyl)-N-(-pyridin-2-ylmethyl)-[1,1′-biphenyl]-

(300 MHz, chloroform-d): δ 9.61 (s, 1H), 8.35 (d, J = 2.64 Hz, 1H),

7.47−7.62 (m, 3H), 7.21−7.35 (m, 4H), 7.12 (t, J = 8.01 Hz, 1H),

6.90 (d, J = 8.85 Hz, 1H), 6.75 (d, J = 7.91 Hz, 1H), 6.65 (br s, 1H),

6.43 (dd, J = 1.70, 8.10 Hz, 1H), 4.15−4.27 (m, 1H), 4.02 (s, 3H),

3.69 (q, J = 5.97 Hz, 2H), 3.32−3.50 (m, 2H), 2.36 (s, 3H). MS

(ESI) m/z: 484.2 [M + H]⁺.

Compound 67 was synthesized according to the general procedure

for the Miyaura borylation reaction using 66 as the starting material.

Yield: >99%.

1

3-carboxamide (37). Yield: 84%. H NMR (300 MHz, chloroform-

d): δ 8.56−8.69 (m, 1H), 8.00−8.17 (m, 1H), 7.67−7.77 (m, 2H),

7.47−7.66 (m, 5H), 7.42 (br s, 3H), 7.01−7.13 (m, 2H), 6.93 (t, J =

8.01 Hz, 1H), 6.84 (br s, 1H), 6.46 (s, 1H), 6.32 (d, J = 8.10 Hz, 1H),

6.17−6.27 (m, 1H), 4.88 (s, 1H), 4.62 (s, 1H), 4.04 (br s, 3H), 3.59

(br s, 2H), 3.21 (br s, 2H), 3.09 (d, J = 15.82 Hz, 3H), 2.34 (br s,

3H). MS (ESI) m/z: 664.2 [M + H]⁺.

4′-Methoxy-3′-(N-(3-((2-(3-methylbenzamido)ethyl)amino)-

phenyl)sulfamoyl)-N-(pyridin-2-ylmethyl)-[1,1′-biphenyl]-3-carbox-

amide (38). Yield: 62%. ¹H NMR (300 MHz, chloroform-d): δ 8.52−

8.60 (m, 1H), 8.14 (d, J = 2.45 Hz, 1H), 8.04 (s, 1H), 7.78 (d, J =

7.72 Hz, 2H), 7.61−7.72 (m, 3H), 7.44−7.56 (m, 3H), 7.06−7.14

(m, 1H), 6.98−7.05 (m, 2H), 6.88−6.98 (m, 1H), 6.55 (s, 1H),

6.27−6.36 (m, 1H), 6.19−6.26 (m, 1H), 4.72 (d, J = 4.90 Hz, 2H),

4.25−4.48 (m, 1H), 4.00 (s, 3H), 3.65 (d, J = 5.46 Hz, 2H), 3.29 (br

s, 2H), 2.30 (s, 3H). MS (ESI) m/z: 650.2 [M + H]⁺.

4′-Methoxy-N-methyl-3′-(N-(3-((2-(3-methylbenzamido)ethyl)-

amino)phenyl)sulfamoyl)-N-(pyridin-3-ylmethyl)-[1,1′-biphenyl]-3-

carboxamide (39). Yield: 68%. ¹H NMR (300 MHz, methanol-d₄): δ

8.40−8.62 (m, 1H), 7.86−8.07 (m, 1H), 7.31 (s, 12H), 7.10−7.23

(m, 1H), 6.84−6.92 (m, 1H), 6.45−6.51 (m, 1H), 6.35−6.42 (m,

1H), 6.24−6.35 (m, 1H), 3.99 (s, 3H), 3.40−3.48 (m, 2H), 3.21 (m,

2H), 2.94−3.08 (m, 3H), 2.36 (s, 3H). MS (ESI) m/z: 664.1 [M +

H]⁺.

4′-Methoxy-N-methyl-3′-(N-(3-((2-(3-methylbenzamido)ethyl)-

amino)phenyl)sulfamoyl)-N-(pyridin-4-ylmethyl)-[1,1′-biphenyl]-3-

carboxamide (40). Yield: 80%. ¹H NMR (300 MHz, methanol-d₄): δ

8.49 (br s, 2H), 7.91−8.09 (m, 1H), 7.02−7.78 (m, 12H), 6.86 (t, J =

8.10 Hz, 1H), 6.50 (br s, 1H), 6.40 (d, J = 6.59 Hz, 1H), 6.23−6.33

(m, 1H), 4.49−4.83 (m, 2H), 3.95 (br s, 3H), 3.36−3.49 (m, 2H),

3.18 (t, J = 6.03 Hz, 2H), 2.89−3.10 (m, 3H), 2.33 (s, 3H). ¹³C NMR

(75 MHz, chloroform-d): δ 174.1 (1C, J = 45 Hz), 170.7, 157.9,

150.7, 150.5, 148.9, 141.0, 139.8, 139.5, 137.6, 135.6, 134.3, 133.3,

130.6, 130.4, 130.2, 129.5, 129.3, 128.8, 128.5, 126.9, 126.4, 126.2,

125.8, 125.4, 124.3, 123.4, 114.2, 110.5, 110.2, 105.6, 56.9, 53.3 (1 C,

J = 307.5 Hz), 44.1, 40.4, 36.4 (1C, J = 307.5 Hz), 21.4. MS (ESI) m/

z: 664.2 [M + H]⁺.

2-Methoxy-N-[3-({2-[(3-methylphenyl)formamido]ethyl}amino)-

phenyl]-5-(tetramethyl-1,3-,2-dioxaborolan-2-yl)benzamide (67).

1

Yield: > 99%. H NMR (300 MHz, chloroform-d): δ 9.57 (s, 1H),

8.70 (d, J = 1.51 Hz, 1H), 7.91 (dd, J = 1.60, 8.19 Hz, 1H), 7.51−7.66

(m, 2H), 7.42 (s, 1H), 7.22−7.35 (m, 3H), 7.12 (t, J = 8.01 Hz, 1H),

7.01 (d, J = 8.29 Hz, 1H), 6.73 (d, J = 7.91 Hz, 1H), 6.63 (br s, 1H),

6.44 (d, J = 7.91 Hz, 1H), 4.05 (s, 3H), 3.72 (q, J = 5.65 Hz, 2H),

3.37−3.52 (m, 2H), 2.37 (s, 3H), 1.33 (s, 12H). MS (ESI) m/z:

530.2 [M + H]⁺.

Compounds 43 and 44 were synthesized according to the general

procedure for the Suzuki coupling reaction using compound 67 as the

starting material.

4-Methoxy-N³′,N³′-dimethyl-N³-(3-((2-(3-methylbenzamido)-

ethyl)amino)phenyl)-[1,1′-biphenyl]-3,3′-dicarboxamide (43).

Yield: 78%. ¹H NMR (300 MHz, chloroform-d): δ 9.77 (s, 1H),

8.53 (d, J = 2.26 Hz, 1H), 7.73 (dd, J = 2.45, 8.48 Hz, 1H), 7.66 (s,

2H), 7.58 (s, 1H), 7.54 (br s, 1H), 7.47 (t, J = 7.91 Hz, 1H), 7.34−

7.42 (m, 3H), 7.29 (d, J = 4.71 Hz, 2H), 7.09−7.21 (m, 3H), 6.79 (d,

J = 8.48 Hz, 1H), 6.46 (d, J = 8.10 Hz, 2H), 4.13−4.22 (m, 1H), 4.10

(s, 3H), 3.68−3.78 (m, 2H), 3.47 (t, J = 5.75 Hz, 3H), 2.93−3.19 (m,

7H), 2.38 (s, 3H). MS (ESI) m/z: 551.2 [M + H.

4-Methoxy-N³′-methyl-N³-(3-((2-(3-methylbenzamido)ethyl)-

amino)phenyl)-N³′-(yridine-4-ylmethyl)-[1,1′-biphenyl]-3,3′-dicar-

1

boxamide (44). Yield: 72%. H NMR (300 MHz, chloroform-d): δ

9.75 (br s, 1H), 8.62 (d, J = 6.03 Hz, 2H), 8.40−8.57 (m, 1H), 7.69

(d, J = 7.72 Hz, 3H), 7.36−7.60 (m, 5H), 7.28−7.34 (m, 3H), 7.06−

7.22 (m, 3H), 6.72−6.83 (m, 1H), 6.46 (d, J = 7.91 Hz, 1H), 4.78 (br

s, 2H), 4.10 (s, 3H), 3.74 (q, J = 5.84 Hz, 2H), 3.41−3.53 (m, 2H),

2.90−3.16 (m, 3H), 2.37 (s, 3H). MS (ESI) m/z: 628.2 [M + H]⁺.

OX1R and OX2R Calcium Mobilization Assays. Activity of the

target compounds at the human OX₁and OX₂receptors was

determined utilizing CHO RD-HGA16 cells (Molecular Devices)

engineered to stably express either the human OX₁or the human OX₂

receptor. Cells were maintained in Ham’s F12 supplemented with

10% fetal bovine serum, 100 units of penicillin and streptomycin, and

100 μg/mL normocin. To conduct the assay, cells were plated at

25,000 cells/well into 96-well black-wall/clear-bottom assay plates

and incubated overnight at 37 °C, 5% CO₂. The next day prior to the

assay, Calcium 5 dye (Molecular Devices) was reconstituted

according to the manufacturer’s instructions. The reconstituted dye

was diluted 1:40 in prewarmed (37 °C) assay buﬀer (1× Hank’s

balanced salt solution, 20 mM 4-(2-hydroxyethyl)-1-piperazineetha-

nesulfonic acid, 2.5 mM probenecid, pH 7.4 at 37 °C). Growth

medium was removed and the cells were gently washed with 100 μL

of prewarmed (37 °C) assay buﬀer. The cells were incubated for 45

min at 37 °C, 5% CO₂in 200 μL of the diluted Calcium 5 dye. Serial

dilutions of test compounds and the agonist control orexin A were

prepared at 10x the desired ﬁnal concentration in 0.25% bovine serum

albumin/1% dimethyl sulfoxide (DMSO)/assay buﬀer, aliquoted into

96-well polypropylene plates and warmed to 37 °C for 15 min. After

the dye-loading incubation period, cells were pretreated with 25 μL of

9% DMSO/assay buﬀer and incubated for 15 min at 37 °C. After the

4′-Methoxy-N-methyl-3′-(N-(3-((2-(3-methylbenzamido)ethyl)-

amino)phenyl)sulfamoyl)-N-(2-(pyridin-2-yl)ethyl)-[1,1′-biphenyl]-

3-carboxamide (41). Yield: 85%. ¹H NMR (300 MHz, methanol-d₄):

δ 8.25 (dd, J₁= 159.0 Hz, J₂= 3.0 Hz, 1H), 7.95 (d, J = 33.0 Hz, 1H),

7.73−7.84 (m, 1H), 7.58 (dd, J₁= 63.0 Hz, J₂= 9.0 Hz, 1H), 7.18−

7.62 (m, 9H), 7.03 (t, J = 6.0 Hz, 1H), 6.91 (t, J = 6.0 Hz, 1H), 6.48−

6.83 (m, 2H), 6.42 (t, J = 9.0 Hz, 1H), 6.26−6.36 (m, 1H), 4.03 (d, J

= 12.0 Hz, 3H), 3.91 (t, J = 6.0 Hz, 1H), 3.71 (t, J = 6.0 Hz, 1H),

3.35−3.58 (m, 2H), 3.08−3025 (m, 7H), 2.37 (s, 3H). MS (ESI) m/

z: 678.2 [M + H]⁺.

4′-Methoxy-N-methyl-3′-(N-(3-((2-(3-methylbenzamido)ethyl)-

amino)phenyl)sulfamoyl)-N-(2-(pyridin-4-yl)ethyl)-[1,1′-biphenyl]-

1

3-carboxamide (42). Yield: 70%. H NMR (300 MHz, chloroform-

d): δ 8.55 (br s, 1H), 8.25−8.36 (m, 1H), 7.95−8.11 (m, 1H), 7.30−

7.75 (m, 6H), 6.68−7.22 (m, 8H), 6.49 (br s, 1H), 6.31 (br s, 2H),

4.04 (s, 3H), 3.83 (br s, 1H), 3.59 (br s, 4H), 3.12−3.29 (m, 3H),

3.03 (br s, 1H), 2.90 (br s, 3H), 2.34 (s, 3H). MS (ESI) m/z: 678.1

[M + H]⁺.

General Procedure for the Synthesis of Compounds 43 and

44. Intermediate 63 (539 mg, 2.0 mmol) was dissolved in DCM (20

mL), and triethylamine (0.56 mL, 4.0 mmol) was added. Then,

catalytic DMAP (25 mg, 0.4 mmol) was introduced and the reaction

mixture was cooled down to 0 °C. At this temperature, 5-bromo-2-

methoxybenzenecarboxylic chloride (499 mg, 2.0 mmol) in THF (10

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pretreatment incubation period, the plate was read with a FlexStation

II (Molecular Devices). Calcium-mediated changes in ﬂuorescence

were monitored every 1.52 s over a 60 s time period, with the

FlexStation II adding 25 μL of the test compound/control serial

dilutions at the 19 s time point (excitation at 485 nm, detection at 525

nm). Peak kinetic reduction (SoftMax, Molecular Devices) relative

ﬂuorescent units were plotted against the log of compound

concentration, and nonlinear regression analysis was used to generate

EC₅₀values (GraphPad Prism, GraphPad Software, Inc., San Diego,

CA).

to the cryo-EM structures and adequate pose reproduction. In all

remaining cases, we employed induced ﬁt and/or MD equilibration

starting with initial GLIDE-XP poses. The top Emodel docked pose

was then used as the seed structure for Schrodinger-Prime induced-ﬁt

modeling with a ﬂexibility of 5−7 Å around any atom in the ligand.

We used the 7 Å extended region, in particular, to include Arg and Lys

residues at the top of the OX2R and OX1R full-length models and

explicitly included R339^7.28(OX2R) and R333^7.29(OX1R) in order to

enhance sampling of variable conformations including those residues

in GLIDE-XP redocking into the Prime variable backbone/sidechain

conformation sampled OX2R and OX1R conﬁgurations in an

induced-ﬁt procedure.⁷⁸Top scoring induced ﬁt poses were then

analyzed for salient interactions, and these were employed in

following MD studies. All ligand models for use in Schrodinger

GLIDE SP/XP docking were prepared with the LIGPREP module.

Sleep Studies. Animals. Twelve-month-old male mice (bred at

Minneapolis VA Health Care System, n = 6) were housed individually

in solid-bottom cages with corncob bedding and a chewing substrate

(Nylabone, natural ﬂavor, BioServ, Frenchtown, NJ). Throughout the

study, a 12 h light/12 h dark cycle (lights on at 06:00) in a

temperature-controlled environment (21−22 °C) was used for

experiments. Rodent chow (Harlan Teklad 8604) and water were

allowed ad libitum. Studies were approved by the Institutional Animal

Care and Use Committee at the Minneapolis VA Health Care System.

Surgery. Mice were anesthetized with a ketamine/xylazine mixture

(15 mg/kg; 1.5 mg/kg) and implanted with a radiotelemetric

transmitter and EEG/EMG electrodes to record vigilance states

(F20-EET, Data Sciences International [DSI], St. Paul, MN).

Stereotaxic coordinates for the EE electrodes were as follows: −0.1

mm anterior, 2.0 mm lateral to bregma, and inserted to predrilled

holes to touch the dura. The EMG leads were secured in the nuchal

musculature. Animals were allowed to recover from surgery for at least

7−10 days before experimental trials began.

Injections. Mice were injected with either 40 (RTOXA-43, 40 mg/

kg i.p. in 0.3 mL saline) or vehicle (saline, n = 5) in a counter-

balanced design. Doses were chosen based on results from sleep

studies on YNT-185.⁴⁶Mice were randomly assigned to a treatment

group; each animal received each treatment once, and all treatments

were represented on each day. Injections were performed between

10:00 and 10:30 (zeitgeber time 4−4.5) with ≥48 h between

treatments. Continuous EEG/EMG recordings were obtained for 24 h

postinjection.

EEG/EMG Recording and Behavioral-State Determination. To

allow freely moving polysomnogram recordings, a receiver (PhysioTel

RPC-1, DSI, St. Paul, MN) was placed beneath the testing cage to

detect EEG/EMG signals from the implanted transmitter. Brieﬂy,

signals were digitized by a Data Exchange Matrix connected to a PC

with Dataquest A.R.T 4.1 software (DSI, St. Paul, MN). Electro-

encephalogram signals (0.3−30.0 Hz band pass) and EMG signals

(1.0−100.0 Hz bandpass) were stored on a computer, visualized with

Neuroscore software (version 2.0.1, DSI, St. Paul, MN), and sleep and

wakefulness states were scored manually in accordance with

previously described methods. In brief, consecutive 10 s epochs of

EEG and EMG signals were classiﬁed into one of the following three

behavioral states: NREM sleep, REM sleep, or wakefulness, and

percent time spent in each state was then calculated from the scored

data. The following dependent variables were quantiﬁed for each

recording session: (a) percent time spent in wakefulness, NREM

sleep, REM sleep; (b) total number of episodes for each vigilance

state; (c) mean duration of episodes for each vigilance state; and (d)

total number of state transitions.

Computational Methods. Preparation of Initial Full-Length

OX2R and OX1R Models. Initial full-length models of human OX2R

were prepared based on the reported coordinates for 5 (PDBID:

7L1V) and orexin-B-bound OX2R (PDBID: 7L1U) cryo-EM

structures,⁵⁰with restoration of missing residues in loops and reversal

of truncation of ICL3 and the N- and C-termini. In addition, the

miniGsqi/Gβ1γ2 segments that were absent in the 5 and orexin-B-

bound cryogenic structural solutions were completed using

MODELLER.⁶¹Thus, the initial conformations of the ICL3/N- and

C-termini of the OX2R were generated self-consistently with the

initial full-length G-protein. To accomplish these steps, a sequence

alignment between the sequence present in the cryogenic construct

and the human OX2R sequence was performed, employing

CLUSTALX⁷⁴and a GONNET 250 scoring matrix and a GAP

penalty of 10. This sequence and the cryogenic coordinates of OX2R

were input to MODELLER and the best zDOPE scored models

collected to select the top scored model for input into SCWRL,⁷⁵

where new (nonclashing) rotameric states of nonconserved residues

were generated for the newly modeled residues. We then employed

the Epik function embodied in the Schrodinger small-molecule suite

to make self-consistent protonation assignments along with

Schrodinger protein preparation steps. The model was then energy

minimized for 2500 conjugate gradients (PBCG) steps following

implementation of a disulﬁde constraint between cysteine127 and

cysteine210. The completed model then underwent nudged elastic

band pulls of the N-/C-termini to generate compact initial

coordinates (primarily to move away from fully extended initial N-/

C-terminal conformations) and the full model immersed in an initial

200 × 200 × 200 Å³1,2-dioleoyl-sn-glycero-3-phosphocholine

(DOPC) lipid box with 50 Å layer of water/KCl above and below

the lipid bilayer. The system was then parameterized using the

AMBER LIPD14⁶⁵and the AMBER18⁷⁶ﬀSB14 force ﬁelds including

TIP3P model parameters. The initial simulation membrane system/

GPCR complex was obtained by immersing the GPCR complex

(excluding the G-protein portion for this study) in the membrane/

water/ion system and deleting all lipids/water/ions within 3.5 Å of

any GPCR-complex atom. The system was then energy minimized

using 8000 steps of AMBER sander conjugate gradient steps, heated

with all atoms constrained for 5 ns and then equilibrated

unconstrained for ca. 30 ns. In the case of 40, we continued with

production MD out to 180 ns using AMBER (particle mesh Ewald

MD) for the purpose of examining time-dependent interactions

between the Schrodinger-induced ﬁt poses and residues in the

orthosteric binding site.

Docking, Induced Fit, and MD Studies. The initial full-length

models following equilibration and energy minimization were

employed in GLIDE SP/XP and induced-ﬁt studies. Our docking

protocol was to collect 120 post docking energy-minimized poses,

which were then distilled to the top ﬁve poses. We found simple

GLIDE SP/XP docking with a rigid receptor was often inadequate to

get good self-docking (i.e., docking the native ligand from the crystal

or cryo-EM structures) with GPCRs such as OX1R and OX2R, which

is often improved with introduction of receptor ﬂexibility. To his end,

we employed MODELLER annealing of initial cryogenic structural

solution coordinates and veriﬁed adequate docking and root-mean-

square deviations (RMSD’s) using GLIDE SP, which has a more

exhaustive conﬁguration sampling than GLIDE-XP which uses a more

conservative anchor and grow (CONFGEN⁷⁷) sampling approach. A

simple GLIDE-SP docking of 5 in the OX2R gave a RMSD of 2.5 Å

for the best Emodel scored pose, demonstrating structural similarity

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00841.

■

sı

HPLC analysis results of target compounds and

summary table of molecular formula strings with

biological data (PDF)

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REFERENCES

Molecular strings for the target compounds (CSV)

Coordinates for OX1R model (PDB)

■

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Coordinates for OX2R model (PDB)

AUTHOR INFORMATION

■

Corresponding Author

Yanan Zhang − Research Triangle Institute, Research Triangle

Park, North Carolina 27709, United States; orcid.org/

0000-0001-7153-4358; Phone: 1-919-541-1235;

Email: yzhang@rti.org; Fax: 1-919-541-6499

Authors

Dehui Zhang − Research Triangle Institute, Research Triangle

Park, North Carolina 27709, United States

(3) Peyron, C.; Tighe, D. K.; van den Pol, A. N.; de Lecea, L.;

Heller, H. C.; Sutcliffe, J. G.; Kilduff, T. S. Neurons containing

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David A. Perrey − Research Triangle Institute, Research

Triangle Park, North Carolina 27709, United States

Ann M. Decker − Research Triangle Institute, Research

Triangle Park, North Carolina 27709, United States

Tiﬀany L. Langston − Research Triangle Institute, Research

Triangle Park, North Carolina 27709, United States

Vijayakumar Mavanji − Research Service, Veterans Aﬀairs

Health Care System, Minneapolis, Minnesota 55417, United

States

Danni L. Harris − Research Triangle Institute, Research

Triangle Park, North Carolina 27709, United States

Catherine M. Kotz − Research Service, Veterans Aﬀairs

Health Care System, Minneapolis, Minnesota 55417, United

States; Department of Integrative Biology and Physiology,

University of Minnesota, Minneapolis, Minnesota 55455,

United States; Geriatric, Research, Education and Clinical

Center, Minneapolis Veterans Aﬀairs Health Care System,

Minneapolis, Minnesota 55417, United States

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M.; Goto, K. Distribution of orexin neurons in the adult rat brain.

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system: Implications for sleep and sleep disorders. Trends Neurosci.

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Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00841

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(12) de Lecea, L. Optogenetic control of hypocretin (orexin)

neurons and arousal circuits. Curr. Top. Behav. Neurosci. 2015, 25,

367−378.

Author Contributions

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript. We thank Emma Tonetti for technical

assistance.

(13) Furlong, T. M.; Vianna, D. M. L.; Liu, L.; Carrive, P.

Hypocretin/orexin contributes to the expression of some but not all

forms of stress and arousal. Eur. J. Neurosci. 2009, 30, 1603−1614.

(14) Johnson, P. L.; Truitt, W.; Fitz, S. D.; Minick, P. E.; Dietrich,

A.; Sanghani, S.; Träskman-Bendz, L.; Goddard, A. W.; Brundin, L.;

Shekhar, A. A key role for orexin in panic anxiety. Nat. Med. 2010, 16,

111−115.

(15) Johnson, P. L.; Federici, L. M.; Fitz, S. D.; Renger, J. J.;

Shireman, B.; Winrow, C. J.; Bonaventure, P.; Shekhar, A. Orexin 1

and 2 receptor involvement in co2 -induced panic-associated behavior

and autonomic responses. Depress. Anxiety 2015, 32, 671−683.

(16) Bonaventure, P.; Yun, S.; Johnson, P. L.; Shekhar, A.; Fitz, S.

D.; Shireman, B. T.; Lebold, T. P.; Nepomuceno, D.; Lord, B.;

Wennerholm, M.; Shelton, J.; Carruthers, N.; Lovenberg, T.; Dugovic,

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352, 590−601.

Funding

This work was supported by the National Institute on Drug

Abuse, the National Institutes of Health, U.S. (grants

DA040693 to Y.Z.), and the US Department of Veteran

Aﬀairs (grants 1IO1BX003004-01A2 and 1IO1BX003687-

01A1 to C.M.K.).

Notes

The authors declare no competing ﬁnancial interest.

ABBREVIATIONS

■

1,2-DCE, 1,2-dichloroethane; DCM, dichloromethane; DME,

1,2-dimethoxyethane; DOPC, 1,2-dioleoyl-sn-glycero-3-phos-

phocholine; EC₅₀, half-maximal eﬀective concentration;

FLIPR, ﬂuorometric imaging plate reader; GPCR, G protein-

coupled receptor; HPLC, high-performance liquid chromatog-

raphy; KCl, potassium chloride; MD, molecular dynamics; MS,

mass spectrometry; NMR, nuclear magnetic resonance; OX1R,

orexin-1 receptor; OX2R, orexin-2 receptor; RMSD, root-

mean-square deviation; SAR, structure−activity relationship;

TLC, thin-layer chromatography

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