Discovery of HN37 as a Potent and Chemically Stable Antiepileptic Drug Candidate

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Discovery of HN37 as a Potent and Chemically Stable Antiepileptic

Drug Candidate

Yang-Ming Zhang,^⊥Hai-Yan Xu,^⊥Hai-Ning Hu,^⊥Fu-Yun Tian, Fei Chen, Hua-Nan Liu, Li Zhan,

Xiao-Ping Pi, Jie Liu, Zhao-Bing Gao,* and Fa-Jun Nan *

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ABSTRACT: We previously reported that P-retigabine (P-RTG),

a retigabine (RTG) analogue bearing a propargyl group at the

nitrogen atom in the linker of RTG, displayed moderate

anticonvulsant eﬃcacy. Recently, our further eﬀorts led to the

discovery of HN37 (pynegabine), which demonstrated satisfactory

chemical stability upon deleting the ortho liable −NH₂group and

installing two adjacent methyl groups to the carbamate motif.

HN37 exhibited enhanced activation potency toward neuronal

Kv7 channels and high in vivo eﬃcacy in a range of pre-clinical

seizure models, including the maximal electroshock test and a 6 Hz

model of pharmacoresistant limbic seizures. With its improved

chemical stability, strong eﬃcacy, and better safety margin, HN37

has progressed to clinical trial in China for epilepsy treatment.

neuronal KCNQs, KCNQ2 and KCNQ3 subunits can

assemble into KCNQ2/3 heterotetramers, which have been

identiﬁed as the main molecular entities of M-currents. As M-

current can function as a brake to ﬁring in neurons, activators

of neuronal KCNQ channels may have valuable applications

for developing novel AEDs.⁸⁻¹⁰This concept was validated by

the approval of retigabine (RTG) in 2011, the ﬁrst neuronal

Kv7 agonist, marketed as Potigar in USA and Trobalt in

Europe for adjunctive treatment of partial-onset seizures.⁸In a

key clinical trial, RTG was eﬃcacious in patients who suﬀered

from inadequate seizure control using other AEDs. The

average seizure frequency was reduced by 23−28, 29−40, and

35−44% at 600, 900, and 1200 mg/day, respectively,

compared with the placebo group (13−18%).^9,10This drug

was particularly eﬃcacious for a small group of children with

an aberrant KCNQ2 gene.¹¹

INTRODUCTION

■

Epilepsy is one of the most common neurological disorders

that is characterized by recurrent, unprovoked seizures and

aﬀects up to 70 million people worldwide. It usually manifests

as disturbances of perception, motor control, consciousness, or

autonomic control and frequently results in serious morbidity

and high mortality. Epilepsy is a severe threat to patients’

quality of life and well-being and exerts a heavy burden on

their families.¹

Nowadays, antiepileptic drugs (AEDs) are the mainstay

treatment for epilepsy with over 30 AEDs approved for clinical

use. Unfortunately, about 30% of patients do not respond to

these AEDs despite the practice of increasing drug dosage or

using combination therapies.^2,3While the mechanisms of

epilepsy pharmacoresistance continue to be investigated, more

eﬀective AEDs, particularly with novel mechanisms of actions

and reduced side eﬀects are still in high demand as an

additional medication option, especially for patients with

treatment-resistant epilepsy or treatment-refractory seiz-

ures.⁴⁻⁶

Most clinical adverse events of RTG include dizziness,

fatigue, confusion, vertigo, and tremor.^9,12In addition, RTG

may cause a unique adverse eﬀect, urinary retention,

presumably due to potentiating KCNQ channels expressed in

bladder tissues.¹³⁻¹⁵However, in 2013, case reports of skin

discolorations on peripheral tissues aroused much safety

Voltage-gated K⁺channels (Kv) play crucial roles in

regulating intrinsic electrical properties in excitable cells. As

the seventh member of Kv channels, the K_V7 family includes

ﬁve members (Kv7.1-5) encoded by KCNQ1−5 genes and has

gained widespread interest due to its link to diseases and

overall tractability as drug targets.⁷While KCNQ1 is conﬁned

in cardiac tissues and co-assembled with KCNE1, KCNQ2−5

are mainly expressed in central and peripheral nervous systems

and therefore often referred to as neuronal KCNQs. Among

Received: December 29, 2020

Published: April 30, 2021

https://doi.org/10.1021/acs.jmedchem.0c02252

J. Med. Chem. 2021, 64, 5816−5837

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Figure 1. Structures of retigabine, P-retigabine, and HN37 (Pynegabine).

Scheme 1. Synthesis of Deaminated Analogues of P-RTG (L1-1 to L1-8) with a Variety of N-Acyl Groups

concern with its long-term use.¹⁶⁻¹⁹Though most discolor-

ations are reversible, the potential loss of vision due to retinal

discoloration eventually led to a black box warning on RTG

medication. Eye examination every 6 months is recommended

under RTG treatment.¹⁸Although the European Medicines

Agency (EMA) and the US FDA acknowledge that the beneﬁts

of RTG therapy outweigh its potential risks, Potigar and

Trobalt have been discontinued in all markets since June 2017

on account of decreased commercial prospects.

Research on RTG-related discolorations suggests that

melanin accelerates the pigmentation process caused by

dimerization of RTG and its N-acetyl metabolite

(NAMR).^16,20−22Through its binding to both RTG and

NAMR, melanin enriches their concentrations and provides

appropriate structural conformations to initiate their dimeriza-

tion.²¹Pigmented dimers of RTG and NAMR were found in

the eye and skin of pigmented rats as well as in biopsy samples

from human subjects.²¹As the pigmentation seems unrelated

to its mechanism of action, attention has been turned toward

its polyaminobenzene structure. It is reported that RTG tends

to dimerize through forming two possible p-quinone diimines,

the potential reactive intermediates playing key roles in

forming dimers and readily reacting with a variety of substrates

in vivo.²³⁻²⁵Self-coupling of polyaminobenzenes has long been

applied in oxidative hair dye formation. Earlier, ﬂupirtine, a

non-opiate analgesic having a similar structure, was reported to

produce some dimeric metabolites.²⁶

To address these undesirable side eﬀects, novel candidates

are developed by substitution on the primary −NH₂group,

forming piperidines to increase the steric hindrance, synthesis

of sulﬁde analogues, enhancement of subtype selectivity toward

the KCNQ family, and providing conformationally restricted

RTG analogues.^{20,24,27−32}In particular, several di-ortho-

methyl-substituted RTG analogues have been reported as

promising novel Kv7 activators. For example, Yang et al.

developed a series of piperidine-based di-ortho-methyl-

substituted butanamides using a hybridization drug design

strategy.^33,34Recently, Zhang et al. reported a novel potent

neuronal Kv7 channel opener SCR2682 also containing two

ortho methyl substituents.^35,36Besides, compound XEN1101

(1OP-2198), one of a series of 4-(N-azacycloalkyl) anilides

with two ortho methyl substituents, has progressed into Phase

2 clinical trial to treat focal epilepsy (ClinicalTrials.gov

Identiﬁer: NCT03796962).³⁷⁻³⁹

Both RTG and ﬂupirtine share a motif of arene ortho-

diamines with one acylated and another as an unsubstituted

primary amine. This motif seems critical for potentiating

KCNQ channels as the deaminated version of RTG

(compound K3) shows much reduced potency.⁴⁰This SAR

(structure−activity relationship) information, on the other

hand, presents an opportunity for improving chemical stability

by deleting the adjacent liable −NH₂group, if potency is

maintained or even improved.

In our previous work, we discovered a RTG derivative, P-

retigabine (P-RTG, ethyl N-[2-amino-4-((4-ﬂuorobenzyl)

(prop-2-ynyl)amino)phenyl]carbamate, Figure 1), which in-

corporated a propargyl group at the nitrogen atom in the linker

of RTG.⁴¹P-RTG (ED₅₀= 6.5 mg/kg) displayed more potent

antiepileptic eﬃcacy in the maximal electroshock (MES)

mouse model than RTG (ED₅₀= 15.0 mg/kg). In addition, P-

RTG exhibited an inverted brain-to-blood drug distribution

probably due to increased lipophilicity. This inverted brain-to-

blood drug distribution might be beneﬁcial for CNS drugs, if it

could enhance eﬃcacy and reduce side eﬀects. However, the

concern about its chemical instability for the triaminobenzene

structure impedes its further pre-clinical development.^20,21,23,24

Enlightened by the maintained anticonvulsant potency of P-

RTG, we therefore undertook further structural explorations

and modiﬁcations on the basis of retaining the propargyl

group. The propargyl group enhanced the tolerance toward

structural modiﬁcations probably due to its potential for π−π

stacking with aromatic moieties in its binding pocket, thus

opening a new avenue for modifying RTG structures. Herein,

we describe our recent eﬀorts which led to the discovery of

methyl (4-((4-ﬂuorobenzyl) (prop-2-yn-1-yl) amino)-2, 6-

dimethylphenyl) carbamate (HN37, pynegabine, Figure 1) as

a promising antiepileptic drug candidate. It displays satisfactory

chemical stability by deleting the liable ortho −NH₂and

installing two neighboring methyl groups to the carbamate

functionality. In its pre-clinical study, HN37 exhibits enhanced

eﬃcacy and a better safety margin than RTG. As a new

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Scheme 2. Synthesis of Deaminated Analogues of P-RTG (L2-1 to L2-8) with a Variety of Substituents on the Left Benzene

Ring

generation of Kv7 activator, HN37 is an ideal drug candidate

for treating epilepsy and pursuing other indications.

scanning and modiﬁcations on the basis of retaining the

propargyl group in order to address RTG-associated issues of

discoloration through enhancing its chemical stability while

maintaining or improving anticonvulsant potency.

DESIGN AND CHEMISTRY

■

In our ﬁrst approach, the ortho liable −NH₂group

neighboring the carbamate functionality was removed to

improve chemical stability. The eﬀects of the deaminated

analogues (L1-1 to L1-8) bearing various N-acyl groups on

Kv7.2 currents were assessed by using a standard whole-cell

recording in CHO cells; the results are summarized in Table 1.

Compared with P-RTG, compound L1-2 without the ortho

−NH₂group displayed much reduced potency, with the values

of −7.26 2.50 and 1.99 0.37 for ΔV_1/2(mV) and I/I₀,

respectively. The carbamate chain replacement from ethyl to

isobutyl (L1-3), tert-butyl (L1-4), and allyl (L1-5) led to

further reduced potencies as indicated by lower ratios of I/I₀.

Compound L1-7 with a hydrophobic benzyl chain displayed

much better potency than L1-6 bearing a hydrophilic glycerol-

derived chain with a more left value of ΔV_1/2(mV). By

contrast, methyl carbamate, L1-1 (CF312) displayed the best

potency among these analogues (L1-1 to L1-8) with values of

As our ﬁrst approach to improve chemical stability, the liable

ortho −NH₂substituent in P-RTG was ﬁrst removed and a

series of analogues with a variety of N-acyl motifs were

designed and synthesized (Scheme 1). Intermediate 2, readily

obtained by the reductive amination of aniline 1 with p-

ﬂuorobenzaldehyde, was reacted with propargyl bromide to

aﬀord the corresponding compound L1-4, which was further

transformed into amine and reacted with various acylating

agents to aﬀord the deaminated analogues L1-1 to L1-3 and

L1-5 to L1-8.

Next, modiﬁcations of the left benzene ring of P-RTG were

carried out through the synthesis of compounds L2-1 to L2-8

(Scheme 2). Intermediate 4, prepared by the reaction of

methyl (4-aminophenyl) carbamate 3 with propargyl bromide,

was subsequently reacted with a variety of benzyl bromides to

aﬀord compounds L2-1 to L2-8.

Structural scanning and optimization toward the substituents

on the right benzene ring of P-RTG were achieved through the

synthesis of o-, m-, and p-methoxycarbonylaminio-substituted

analogues (L3-1 to L3-15) using various diaminobenzenes or

nitro-aminobenzenes (5a−5k) through regular functional

group protections and transformations (Schemes 3 and 4).

−24.21

2.35 and 3.27

0.10 for ΔV_1/2(mV) and I/I₀,

respectively. It is worth noting that the corresponding acetyl

amide analogue L1-8 with the absence of only one oxygen

atom displayed much reduced potency compared with CF312.

Here, the lack of potency of short-chain amides missing the

ortho −NH₂group is consistent with the report by Ostacolo et

al. that longer and ﬂexible hydrophobic chain amides more

readily interact with a possible whole, large, and hydrophobic

pocket on Kv7.2.³²We assume that methyl carbamate, instead

of ethyl carbamate existing in both RTG and P-RTG, is more

compatible with the removal of the neighboring −NH₂

substituent when incorporating a propargyl group. Therefore,

further methyl carbamate analogues were examined in our

subsequent work.

RESULTS AND DISCUSSION

■

Structure−Activity Relationship Study. The chemical

instability of RTG mainly arises from its electron-rich

polyaminobenzene structure which is further exacerbated by

an ortho −NH₂substituent. A similar motif of this ortho-

diamine arene with one acylated amine and one primary amine

is also observed in some CNS drugs, such as ﬂupirtine, a

nonopiate analgesic with decades of clinical use in Germany.

This motif seems to be a critical pharmacophore for

potentiating KCNQ channels as the deaminated version of

RTG (compound K3) displays much reduced potency

compared with RTG.⁴⁰The identiﬁcations of some pigmented

RTG-related dimers in melanin-rich tissues suggest an

association with the polyaminobenzene structure of RTG

which tends to form active intermediates and produces

pigmented dimers in an oxidative environment. Actually,

some dark-blue precipitates could be observed several hours

later when aqueous solution of RTG dihydrochloride was

stirred in an open ﬂask at ambient temperatures (unpublished

data). Enlightened by our previous work of incorporating a

propargyl group into RTG, we initiated further structure

The pharmacokinetic parameters of compound CF312 (30

mg/kg, p.o.) were then assessed in KM mice. Compared to

RTG, an inverted brain-to-blood distribution was observed,

with C_maxvalues of 2197 ng/mL and 4421 ng/g and AUC_0−t

values of 1865 ng·h/mL and 3565 ng·h/g for the blood and

brain, respectively. The brain exposure of CF312 is 1.9 times

greater than its blood exposure, and this is in accordance with

the result of P-RTG whose ratio of brain-to-blood exposure is

2.3. This inverted brain-to-blood drug distribution probably

resulted from its increased lipophilicity after introducing a

propargyl group as reported in our previous work.⁴¹

The in vivo anticonvulsant eﬀects of CF312 (30 mg/kg, p.o.)

were evaluated in the MES mouse model and subcutaneous

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Scheme 3. Synthesis of Deaminated Analogues of P-RTG (L3-1 to L3-13) with a Variety of Substituents on the Right Benzene

Ring

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Scheme 4. Synthesis of Deaminated Analogues of P-RTG (L3-14 and L3-15) with Two Methyl Substituents on the Right

Benzene Ring

a

Table 1. Electrophysiological Potencies of RTG, P-RTG, and Deaminated Analogues (L1-1 to L1-8)

a

b

c

All values are expressed as mean

SEM. The shift of half voltage of maximal activation after administration of 10 μM compound. The

amplitude change of outward current: I₀, amplitude of the outward current without the compound; I, amplitude of the outward current with the

compound; I/I₀, eﬀect of 10 μM compound on the amplitude of the outward current of KCNQ2 channels.

pentylenetetrazol (sc-PTZ, 85 mg/kg) seizure mouse model.

Given orally 1 h prior to the test, CF312 protected 50% of KM

mice from tonic-clonic seizures in the MES test, and 60% of

KM mice from clonic seizures in the sc-PTZ test, and the

protection of RTG (30 mg/kg, p.o.) against seizures in the

MES and sc-PTZ test was 60% and 50%, respectively.

Demonstrating that CF312 maintained considerable eﬃcacy

in vitro and in vivo, our data suggest that the deletion of the

ortho −NH₂group is feasible as a result of enhanced tolerance

against structural modiﬁcations upon incorporating a propargyl

group.

CF312, diﬀerent from other deaminated P-RTG analogues,

maintained considerable in vitro and in vivo potencies. We next

undertook a scanning of substituents on the left benzene ring

with the right part of the CF312 structure remaining

unchanged. We synthesized a series of CF312 analogues

bearing various substituents at diﬀerent positions on the left

benzene ring. The test results of their electrophysiological

potencies are summarized in Table 2.

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benzene ring. We synthesized and examined a series of methyl

carbamate analogues together with a variety of substituents on

the right benzene ring; the test results are summarized in Table

3. In contrast to CF312, in which the carbamate group takes a

para-position, the meta-analogue L3-2 displayed much reduced

potency and the ortho-analogue L3-1 lost potency completely.

These results indicated that a para-substituted methyl

carbamate functionality on the right benzene ring was critical

for maintaining potency. Therefore, a series of CF312

derivatives with a variety of substituents on the right benzene

ring were further examined (Table 3).

Table 2. Electrophysiological Potencies of CF312 Analogues

(L2-1 to L2-8) with a Variety of Substituents on the Left

Benzene Ring

a

The replacement of H atoms with F atoms is a common

strategy to modify the structures of many biologically active

molecules due to additional beneﬁcial eﬀects of ﬂuoro

substitution. In the case of compound L3-3, a considerable

improvement in potency was observed after introducing a

ﬂuoro substituent adjacent to the meta-carbamate functionality

in L3-2. This result prompted us to further examine the

inﬂuence of ﬂuoro substitution in CF312 with a para-

carbamate functionality. Unfortunately, L3-4 displayed no

improvement in potency; by contrast, compound L3-5 showed

considerable reduction in potency. Meanwhile, most other

CF312 derivatives, L3-6, L3-7, L3-9, and L3-10 to L3-13,

demonstrated much reduced potency, regardless of the

electronic nature or the position of these substitutes on the

right benzene ring. A surprising exception to this was

compound L3-8, which had a methyl substituent adjacent to

the para-methyl carbamate group and displayed much

enhanced potency, with values of −23.28 1.37 and 4.80

0.73 for ΔV_1/2(mV) and I/I₀, respectively. Compared to

compound L3-9 with almost loss of potency, compound L3-8

displaying notable potency enhancement is impressive.

Because the meta-methyl substitution is not beneﬁcial for

potency, it is rational to assume that the position of the methyl

substitute is responsible for the diﬀerence of the observed

contrasting potency. We speculate that the huge potency

discrepancy between compounds L3-8 and L3-9 probably arise

from their diﬀerent conformations rendered by ortho-methyl

substitution, which imposes a torsion along the C−N bond

due to the increase of steric crowding on the phenyl ring near

the carbamate motif. This twisted conformation in which the

carbonyl rotates out the plane of the phenyl ring probably

favors target binding. Constrained conformation resulting from

ortho-methyl substituents also occurs in lidocaine salts where

the amide and the phenyl rings are rotated to between 66 and

71° depending on its salt.⁴²This is also in accordance with the

conformation study on 2,6-dimethyl-acetanilide where the

amide and the phenyl ring are rotated to 62.50° based on its X-

ray crystallography.⁴³Besides, additional possible interactions

of ortho-methyl groups with surrounding residues in the large

and hydrophobic pockets on K_V7.2, to some extent, may also

contribute to the advantage of ortho-methyl substitution.

Di-ortho-methyl-substituted anilines are well-established

building blocks in Kv7 activators. Similar motifs with one or

two ortho-methyl-substituted anilines also occur in many CNS

drugs such as some anesthetics, as shown in Figure 2.⁴⁴With

this in mind, we next synthesized and examined L3-14 and L3-

15, two CF312 analogues with di-methyl groups on diﬀerent

positions. Compound L3-14 (HN37, pynegabine), with two

methyl groups neighboring the carbamate group, showed the

best potency among our compounds with values of −38.52

1.67 and 4.75 1.29 for ΔV_1/2(mV) and I/I₀, respectively. By

a

b

All values are expressed as mean

SEM. The shift of the half

voltage of maximal activation after administration of 10 μM

compound. The amplitude change of the outward current: I₀,

amplitude of the outward current without the compound; I, amplitude

of the outward current with the compound; I/I₀, eﬀect of 10 μM

compound on the amplitude of the outward current of KCNQ2

channels.

c

The results showed that electrophysiological potencies were

very susceptible to substituents on the left benzene ring for

reasons of both electronic properties and substitution position.

p-Fluoro substitution was more beneﬁcial for potency than the

respective o- and m-ﬂuoro counterparts (L2-1 and L2-2). Its

m-ﬂuoro analogue L2-2 displayed almost the same level of the

left shift value as CF312 (−21.10 1.90 vs −24.21 2.35,

ΔV_1/2, mV), yet it had a lower activation amplitude (2.33

0.17 vs 3.27 0.10, I/I₀). Further introduction of an electron-

withdrawing group adjacent to the p-ﬂuoro group led to much

reduced potency, as illustrated by compounds L2-3 and L2-4,

in which a triﬂuoromethyl group and another ﬂuoro

substituent were introduced, respectively. Compared with

compounds L2-2 and CF312, the substitution of an electron-

donating group (e.g., a methoxy group in L2-6 and a methyl

group in L2-8) was detrimental for potency. Interestingly, the

p-bromo-substituted analogue L2-7 displayed the same level of

potency as CF312 with values of −25.15 1.33 and 3.75

0.63 for ΔV_1/2(mV) and I/I₀, respectively. In our subsequent

work, we maintained p-ﬂuoro substitution on the left benzene

ring as in CF312 due to the proved in vivo eﬃcacy of CF312.

Guided by the results of structure scanning of the carbamate

functionality on the right benzene ring and substituents on the

left benzene ring, we focused further work on the positions of

methyl carbamate functionality and substituents on the right

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Table 3. Electrophysiological Potencies of CF312 Analogues (L3-1 to L3-15) with a Methyl Carbamate Functionality Taking

a

Diﬀerent Positions and Various Substituents on the Right Benzene Ring

a

b

c

All values are expressed as mean SEM. The shift of the half voltage of maximal activation after administration of 10 μM compound. The

amplitude change of the outward current: I₀, amplitude of the outward current without the compound; I, amplitude of the outward current with the

d

compound; I/I₀, eﬀect of 10 μM compound on the amplitude of the outward current of KCNQ2 channels. Left shift value was not tested due to

its very weak activating potency.

contrast, compound L3-15, with two methyl groups on meta-

positions, lost potency with an I/I₀value of 0.72 0.05.

Chemical Stability Evaluations. According to the

chemistry reviews, RTG is a nonhygroscopic, white to slightly

colored powder. Although pure RTG was reported to be a

colorless compound, its crude product was intensively colored

and changed quickly from red to dark violet. The darkening of

the active substance may be observed when RTG is exposed to

light. These indicate that some quinone diimine species could

be involved when RTG is exposed to air or light. Actually, two

dimeric impurities have been identiﬁed as RTG dimers which

were probably produced during the later work up procedures

or storage.²³Recently, pigmented dimers of both RTG and its

N-acetyl metabolite NAMR were identiﬁed by the in vivo

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Figure 2. Some anesthetics containing similar motifs with one or two methyl-substituted anilines.

Table 4. Pharmacokinetic Parameters of HN37 in KM Mice

a

b

c

d

e

f

g

dose

T_max

C_max

AUC_0−t

t_1/2

CL

V_ss

F

ratio of AUC_0−t

brain/plasma

2.23

mg/kg

5 (p.o)

sample

(h)

(ng/mL)

(ng·h/mL)

(h)

L/h/kg

L/kg

%

plasma

brain

plasma

0.25

134

263

321

716

531

1.95

0.80

24.2

2 (i.v)

3.68

2.89

a

b

c

Time of maximum concentration. Maximum observed plasma concentration. Area under the plasma concentration−time curve from time zero

d

e

f

to the last measurable concentrations calculated by the trapezoidal method. Half-life. Clearance. Steady-state distribution volume.

g

Bioavailability.

study, further suggesting the linkage between discoloration and

RTG’s chemical structure vulnerability.²¹Though RTG’s NDA

stability packaging includes long-term stability data of 18

months and accelerated data of 6 months, an expiration period

of only 15 months for all of its tablet strengths can be

supported.

Due to the removal of the liable −NH₂group, compound

HN37 exhibited satisfactory stability with or without packaging

when exposed to a high temperature (60 °C), high humidity

(25 °C, RH 92.5%), and strong light (5500 Lx). Further

stability data include accelerated data stability of 6 months (40

2 °C, RH 75 5%) and long-term stability data up to 36

months (25 2 °C, RH 60 10%). So far, an ongoing drug

product stability study can support an expiration period of 36

months for HN37 tablets with strengths of 5 and 20 mg, which

is a much longer shelf life than RTG.

Table 5. Pharmacokinetic Parameters of HN37 in SD Rats

via Oral Administration

a

b

c

dose

(mg/kg)

T_max

C_max

AUC_0−t

d

(h)

(ng/mL) (ng·h/mL)

t_1/2(h)

2

6

20

6

0.50

0.67

3.67

0.25

88.2

264

323

578

247

1130

1921

1524

1.72

2.36

brain

ratio of AUC_0−t

brain/plasma

blood

0.25

162

533

2.86

a

b

Time of maximum concentration. Maximum observed plasma

c

concentration. Area under the plasma concentration−time curve

from time zero to the last measurable concentrations calculated by the

trapezoidal method. Half-life.

d

respectively. In accordance with the results in KM mice, HN37

tended to distribute in brain with the calculated ratio of brain

to blood exposure as 2.86 in rats.

The metabolic stability of HN37 toward human, monkey,

dog, rat, and mice liver microsomes is examined, and the

results are showed in Table 6. According to these data, HN37

Pharmacokinetic Study. The pharmacokinetic properties

of HN37 were then examined in KM mice; the results are

summarized in Table 4. HN37 was quickly absorbed showing a

T_maxof 0.25 h in both the blood and brain, and C_maxvalues of

134 and 263 ng/mL, respectively. The blood and brain

distributions were observed with AUC_0−tof 321 and 716 ng·h/

mL for the plasma and brain, respectively. The calculated ratio

of the brain to blood exposure for HN37 was 2.23 times, a

sharp contrast to a ratio of 0.16 for RTG.⁴¹This inverted

brain-to-blood distribution for HN37 likely arises from its

increased lipophilicity due to the installation of one propargyl

group and two methyl substituents. Without regard to the

brain distribution, HN37 showed a moderate bioavailability

(24.2%, F) in KM mice.

Table 6. Metabolic Stability Data of HN37 toward Human,

Monkey, Dog, Rat, and Mice Liver Microsomes

liver microsomes

T_1/2(min)

CL_int(mL/min/kg)

human

monkey

dog

rat

mice

27.7

18.1

35.5

23.7

12.5

62.7

112

97.2

105

438

Further pharmacokinetic studies of HN37 are carried out in

SD rats in a dose-escalating manner (2, 6 and 20 mg/kg, p.o.),

and the results are summarized in Table 5. In rats, HN37

reached its highest concentration after 0.50, 0.67, and 3.67 h,

with C_maxvalues of 88.2, 264, and 323 ng/mL at the oral dose

of 2, 6, and 20 mg/kg, respectively. Almost in proportion to

the dosages, the corresponding AUC_0−tvalues in the blood

were 247, 1130, and 1921 ng·h/mL, respectively. In another

set of experiments, the pharmacokinetic parameters of HN37

(6 mg/kg, p.o.) in both the blood and brain were examined at

the same time. HN37 reached its highest concentration in both

the brain and blood at 0.25 h after oral administration, and

AUC_0‑tvalues were found to be 1524 and 533 ng·h/mL,

belongs to a class of high clearance with the highest clearance

rate in mice among these species. Its CL_intin mice is up to 438

mL/min/kg together with a short T_1/2of 12.5 min, which may

explain its high clearance in mice.

A major cytochrome P450s inhibition assay was carried out

using speciﬁc probe substrates in the presence of human liver

microsomes and the results are summarized in Table S1. Data

showed that HN37 at a concentration ranging from 0.10 to

50.0 μM had no signiﬁcant inhibition (<25% in all cases)

toward main human CYP isoforms, including CYP1A2,

CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and

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a

Table 7. Subtype Selectivity of HN37 and RTG

compound

HN37

RTG

b

c

b

c

KCNQ subtype

ΔV_1/2(mV)

I/I₀

ΔV_1/2(mV)

I/I₀

KCNQ1

KCNQ2

KCNQ3 (A278T)

KCNQ2/KCNQ3

KCNQ4

−5.47 2.86

−35.60 4.14

−43.11 4.42

−60.21 4.42

−56.63 9.76

−9.03 1.29

0.99 0.04

3.40 0.26

2.10 0.29

2.77 0.18

3.76 0.56

3.14 0.46

−8.03 2.59

−46.10 3.66

−46.83 5.87

−34.32 4.07

−34.09 6.90

0.09 8.61

1.01 0.05

1.79 0.11

2.46 0.20

1.97 0.06

2.85 0.35

3.04 0.32

KCNQ5

a

b

All values are expressed as mean SEM (n = 3−5). The shift of the half voltage of maximal activation after administration of 0.1 μM HN37 or

c

10 μM RTG. The amplitude change of the outward current: I₀, amplitude of the outward current without the compound; I, amplitude of the

outward current with the compound; I/I₀, eﬀect of 0.1 μM HN37 or 10 μM RTG on the amplitude of the outward current of KCNQ channels.

CYP3A4. In view of the potential metabolic liability of HN37

due to the propargyl group containing a triple bond, potentials

of time-dependent inhibition (TDI) for HN37 on cytochrome

P450s are assessed using human liver microsomes, and the

results are summarized in Figure S1; TDI test results showed

that HN37 was not a time-dependent inhibitor toward

CYP2C9, CYP2C19, CYP2D6, CYP3A4 (midazolam 1′-

hydroxylation), and CYP3A4 (testosterone 6β-hydroxylation),

+

−

with both IC_50NADPHand IC_50NADPHabove 50 μM and the

−

+

ratio of IC_50NADPH/IC_50NADPHless than 1.5; HN37 displayed

time-dependent inhibition toward CYP1A2 (IC_50NADPH

2.53, IC_50NADPH= 142 μM), CYP2B6 (IC_50NADPH= 5.76 μM,

IC_50NADPH= 141 μM), and CYP2C8 (IC_50NADPH= 35.2 μM,

+

=

−

+

−

+

Figure 3. Eﬀects of HN37 and RTG on KCNQ2 (A) and KCNQ2/3

−

IC_50NADPH= 225 μM), where the ratio of IC_50NADPH

/

(B). Data are expressed as mean

concentration).

SEM (n = 4−6 at each

+

IC_50NADPHin each case was higher than 1.5. The potential of

HN37 to cause time-dependent inhibition of CYP1A2,

CYP2B6, and CYP2C8 will be further evaluated in its clinical

study.

We next carried out a series of pre-clinical experiments, and

the results are summarized below.

nM, which is 55-fold more potent than that of RTG (2.04

0.25 μM) (Figure 3A). For KCNQ2/KCNQ3 channels, HN37

had an EC₅₀value of 33.07 4.36 nM, showing a nearly 125-

fold increased potency compared to RTG (4.15 0.54 μM)

(Figure 3B). The above results suggest that HN37 has a

greater ability to dampen neuronal excitability than RTG.

Anticonvulsant Eﬃcacy. To test and evaluate the

anticonvulsant activities of compound HN37, the eﬃcacy

was tested in a range of seizure models according to the

Epilepsy Therapy Screening Program (ETSP) sponsored by

the National Institute of Neurological Disorders and Stroke

(NINDS). ETSP is the new name for the Anticonvulsant

Screening Program (ASP), which has tested tens of thousands

of compounds from dozens of countries⁵¹and has made an

important contribution to the identiﬁcation and/or character-

ization of several FDA-approved drugs for epilepsy for over 40

years, including topiramate, lacosamide, felbamate, and

retigabine.^51,52The current work ﬂow for the ETSP focuses

on compound evaluation by using pharmacoresistant models

and begins with the assessment in two acute seizure models,

the maximal electroshock (MES) seizure test and the 6 Hz

seizure test of pharmacoresistant epilepsy. The MES test is

considered as a predictive model for generalized tonic−clonic

seizures, and it may also predict antiepileptic drugs with

eﬃcacy against partial seizures.^53,54The 6 Hz psychomotor

seizure is similar to human limbic epilepsy and shows

resistance to numerous current AEDs especially under the

high stimulus intensity (44 mA).^52,55These two seizure models

were used in this study. In addition, another classic seizure

model, the subcutaneous pentylenetetrazol (sc-PTZ) seizure

Selectivity Among KCNQ Channels. Previous studies

have shown that RTG mainly serves as a pan-agonist at Kv7.2−

Kv7.5 channels, particularly the KCNQ2/3 heterotetramer.⁴⁵

To determine the subtype selectivity and agonist potency of

HN37 among KCNQ channels, the amplitude changes of the

outward current (I/I₀) and the shift of the half voltage of

maximal activation (ΔV_1/2) were measured using patch-clamp

assays. CHO cells transiently transfected with hKCNQ1,

rKCNQ2, rKCNQ3* (an expression-optimized KCNQ3-

A278T mutant at the pore region that ensures robust currents

to ascertain the observation),⁴⁶⁻⁴⁸hKCNQ4, and hKCNQ5

were used for the recording in this study. As shown in Table 7,

after 0.1 μM HN37 application, the outward currents were

potentiated in all the tested KCNQ isoforms except KCNQ1

(cardiac isoform). The voltage-dependent activation curves

showed left shift eﬀects in neuronal isoforms, and the most

obvious eﬀect was KCNQ2/KCNQ3 (V_1/2with a left-shift of

60.21

4.42 mV). These results indicate that compound

HN37 is a potent neuronal Kv7 activator with a subtype

selectivity similar to RTG.

Given that the neuronal M-current, mainly mediated by

KCNQ2/KCNQ3 and KCNQ2 channels, plays an important

role in the regulation of neuronal excitability,^49,50we further

examined the dose-eﬀect relationship of compound HN37 and

RTG on KCNQ2/KCNQ3 and KCNQ2 channels. The

median eﬀective concentration (EC₅₀) was determined by a

dose−response curve analysis, the results are shown in Figure

3. For KCNQ2 channels, the EC₅₀of HN37 was 36.98 6.17

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Figure 4. (A,B) Eﬀects of HN37 and RTG in the MES seizure model in KM mice. % seizure protection is a percentage of animals not displaying

hindlimb tonic extension. The data for each dose are presented as N/T (N = number of animals protected, T = number of animals tested) and

stated in each bar. Fisher’s exact probability test was used for statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001 vs vehicle. (C,D)

Concentration−response curves of HN37 and RTG for seizure protection in the MES seizure model. Data are expressed as mean brain

concentration SEM. The EC₅₀values are calculated by nonlinear regression using GraphPad Prism 5.

Table 8. Mean Brain or Plasma Concentration of HN37 and RTG in MES and Rotarod Tests in KM Mice

a

compound

test

brain conc. EC₅₀or TC₅₀(μM)

plasma conc. EC₅₀or TC₅₀(μM)

brain/plasma conc. ratio

brain conc. TC₅₀/EC₅₀ratio

b

HN37

RTG

HN37

RTG

MES

0.07 0.02

2.23 0.87

3.47 0.26

27.32 12.84

0.05 0.01

20.04 6.73

1.83 0.26

1.4

0.11

1.9

N/A

b

N/A

rotarod

49.6

12.3

141.33 64.48

0.19

a

b

Values are expressed as mean SEM; EC₅₀or TC₅₀values are determined by nonlinear regression using GraphPad Prism 5. Not Applicable.

test, was also used to detect the anticonvulsant eﬃcacy against

clonic seizures.⁵⁴

administration, and the signiﬁcant eﬀects were observed at

the low doses of 3 and 5 mg/kg, with seizure protections of

62.5 and 100%, respectively (Figure 4B). The ED₅₀value of

HN37 calculated by the dose−response curve in this test was

1.9 mg/kg, which was much lower than that of RTG (38.6 mg/

kg). Further PK studies showed that RTG achieved a maximal

brain concentration of 8.29 μM and a maximal plasma

concentration of 40.85 μM at the highest dose of 100 mg/

kg, and the EC_50,brainvalue of 2.23 0.87 μM was calculated by

a concentration−response curve, with a corresponding

First, the time eﬀect of compound HN37 was tested in the

MES seizure model by oral gavage administration at a dose of

2.5 mg/kg in KM mice, and the animals, whether displaying

hindlimb tonic extension, were observed at 0.25, 0.5, 1, and 2 h

after administration. Compound HN37 showed 40−80%

constant seizure protection from 0.25 to 2 h and exhibited

its peak eﬀect at 1 h (Table S2). Then, on the basis of the

above results, dose−response quantiﬁcation studies were

further carried out at 1 h to elucidate the median eﬀective

dose (ED₅₀) in the MES test. Subsequently, the median

neurotoxic dose (TD₅₀) at a peak time of 1 h was determined

using the rotarod test. After completion of both tests, animals

were euthanized immediately, and plasma and brain samples

were collected to detect the levels of compound HN37 and

RTG.

EC_50,plasmavalue of 20.04

6.73 μM (Figure 4C, Table 8).

The average brain/plasma concentration ratio (B/P) for RTG

was 0.11, which was consistent with the previous study.⁴²The

maximal brain and plasma concentration of HN37 was 0.12

and 0.11 μM at 5 mg/kg, respectively. The concentration−

response curves showed that the EC_50,brainand the correspond-

ing EC_50,plasmavalues for HN37 were 0.07 0.02 and 0.05

0.01 μM, respectively, and the average B/P was 1.4, much

higher than RTG (Figure 4D, Table 8).

Meanwhile, the neurotoxicity eﬀects of compound HN37

and RTG were observed in the rotarod test under the same

experimental conditions. After 1 h oral administration,

compound RTG produced a dose-dependent increase in the

In the MES test, compound RTG (10−100 mg/kg, p.o.)

showed a dose-dependent eﬃcacy against seizures, and the

seizure protections were 62.5 and 87.5% at the 60 and 100 mg/

kg doses, respectively, with statistically signiﬁcant diﬀerences

from the vehicle (Figure 4A). Compound HN37 displayed

improved eﬃcacy compared to RTG after 1 h oral

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Figure 5. (A,B) Eﬀects of HN37 and RTG in the rotarod test in KM mice. % motor impairment is a percentage of animals falling oﬀ the rotating

rod. The data for each dose are presented as N/T (N = number of animals exhibiting toxicity, T = number of animals tested) and stated in each bar.

Fisher’s exact probability test was used for statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001 vs vehicle. (C,D) Concentration−response

curves of HN37 and RTG for motor impairment in the rotarod test. Data are expressed as mean brain concentration SEM. The TC₅₀values are

calculated by nonlinear regression using GraphPad Prism 5.

Table 9. Anticonvulsant Eﬀects of HN37 and Three Reference Drugs (p.o.) in the KM Mouse-Induced MES and PTZ Seizure

a

Models

b

c

d

compound

time of test (h)

MES ED₅₀(mg/kg)

PTZ ED₅₀(mg/kg)

TD₅₀(mg/kg)

HN37

1.0

1.9 (1.3−2.8)

38.6 (25.1−59.3)

35.5 (23.3−54.1)

>500

9.8 (4.5−21.3)

>56

>200

82.0 (74.8−89.8)

187.7 (116.7−301.9)

379.2 (287.6−499.9)

e

RTG

e

TPM

e

LEV

>500

a

b

Values in parentheses are 95% conﬁdence intervals determined by nonlinear regression using GraphPad Prism 5. ED₅₀(MESmaximal

c

d

electroshock seizure model). ED₅₀(sc-PTZpentylenetetrazol seizure model). TD₅₀(NTacute neurological toxicity determined in the

e

rotarod test). Reference AEDs: retigabine (RTG), levetiracetam (LEV), and topiramate (TPM) tested under the same conditions.

motor impairment and had signiﬁcant eﬀects on the ability to

remain on the rotarod at the 250 and 300 mg/kg, with 62.5

and 100% motor impairment rate, respectively (Figure 5A).

Compound HN37 also showed a dose-dependent injury to the

motor behavior, and it exhibited signiﬁcant eﬀects on motor

coordination at the doses of 90, 150, and 200 mg/kg with

motor impairment rates of 62.5, 87.5, and 100%, respectively

(Figure 5B). Both HN37 and RTG did not produce obvious

eﬀects on motor coordination at 60 mg/kg, and the TD₅₀

values determined by dose−response curves were 82.0 and

187.7 mg/kg, respectively. Further PK studies showed that

RTG had a maximal brain concentration of 41.62 and 134.89

μM in the plasma at the highest dose of 300 mg/kg. The

concentration−response curves demonstrated that the

TC_50,brainand the corresponding TC_50,plasmavalues of RTG

values were 3.47

0.26 and 1.83

0.26 μM, respectively

(Figure 5D, Table 8). HN37 had a much higher average B/P

ratio (1.9) than that of RTG, this result was consistent with the

results in the MES test and PK studies in mice. To better

understand the tolerability, the safety margins for HN37 and

RTG based on the brain exposures were calculated, and the

TC_50,brain/EC_50,brainratio values of HN37 and RTG were 49.6

and 12.3, respectively (Table 8). The above results suggest that

compound HN37 has a better safety margin than RTG

between behavior eﬃcacy and neurotoxicity.

To obtain more data to assess HN37’s eﬃcacy against

seizures, the anticonvulsive eﬀects and neurotoxicity of

reference AEDs, including commonly used drug Levetiracetam

(LEV) and Topiramate (TPM), were tested in MES and

rotarod tests under the same experimental conditions, and the

ED₅₀values were also determined in the sc-PTZ seizure model

at the 1 h peak time. The data tested in KM mice are

summarized in Table 9. Although LEV was well tolerated in

the rotarod test, it did not produce any anticonvulsant eﬀects

in MES and PTZ tests; and TPM also showed limited eﬃcacy

were 27.32

12.84 and 141.33

64.48 μM, respectively

(Figure 5C, Table 8), and the average B/P was 0.19, consistent

with the results in the MES test and previous study.⁴²Both

TC_50,brainand the corresponding TC_50,plasmaof HN37 were also

calculated from the concentration−response curves, and the

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in the PTZ test but had potent anticonvulsant eﬃcacy in the

MES test with an ED₅₀value of 35.5 mg/kg. These were in line

with those reported in the literature.^56,57As mentioned earlier,

RTG was very eﬀective for tonic seizure but not for clonic

seizure even at the highest oral dose of 56 mg/kg in the PTZ

test, this might be due to the use of a higher dosage of PTZ

(100 mg/kg). Unlike the reference AEDs, compound HN37

showed a higher eﬃcacy both in MES and PTZ tests, with the

ED₅₀values of 1.9 and 9.8 mg/kg, respectively. Notably, the

TD₅₀/ED₅₀ratios of HN37 and RTG (43.2, 4.9) were actually

close to the mean TC_50,brain/EC_50,brainratios (49.6, 12.3) based

on brain exposure, so we think that HN37 has a better safety

margin (8.4-fold in the PTZ test) than that of the other three

AEDs in this study.

In addition, the quantiﬁcation study of the ED₅₀was also

examined in the rat MES test. Given orally 0.5 or 1 h prior to

testing, the ED₅₀values determined for HN37 were 2−4-fold

more beneﬁcial than that of RTG and were also better than

that for TPM (18.7-fold greater) at 1 h (Table S3). These

results indicate that compound HN37 has potent anticonvul-

sant eﬃcacy in rats.

CONCLUSIONS

■

Kv7 channels hold great promise for developing novel AEDs to

fulﬁll unmet clinical demands. Unfortunately, the supply of the

ﬁrst and only launched drug retigabine has been discontinued

for commercial reasons due to safety issues such as skin and

retina discolorations, although these are unrelated to its

mechanism of action. The incorporation of a propargyl group

at the N position of the RTG linker opened a new avenue for

its structural modiﬁcations and optimization, which eventually

led to the discovery of HN37 (compound L3−14). HN37 is a

new generation of Kv7 activator that avoids the potential of

forming pigmented dimers. Encouraged by its in vivo eﬃcacy,

PK characteristics, improved safety margin, and satisfactory

chemical stability, we have completed its IND application and

received approval for clinical study from the National Medical

Products Administration (NMPA). The potential of HN37 to

cause time-dependent inhibition of CYP1A2, CYP2B6, and

CYP2C8 will be further evaluated during the clinical study, and

its ﬁrst-in-human (FIH) results will be reported in due course.

EXPERIMENTAL SECTION

■

The 6 Hz psychomotor seizure model is an acute model for

drug-resistant epilepsy and is used for the pharmacoresistant

drug screen in the ETSP work ﬂow as with the MES seizure

model. Therefore, compound HN37 and the reference drugs

were also tested in the 6 Hz seizure test in C57BL/6 mice. As

shown in Table 10, except TPM, the other two reference drugs

Synthetic Materials and Methods. Starting materials, reagents,

and solvents were purchased from commercial suppliers and used

without further puriﬁcation. Anhydrous toluene, THF, and DCM

were obtained from a distillation over a sodium wire or CaH₂.

Reactions were carried out with magnetic stirring in round-bottomed

ﬂasks unless otherwise noted. Moisture-sensitive reactions were

conducted in oven-dried glassware using a freshly distilled solvent

under a nitrogen atmosphere with rigid exclusion of moisture. Thin-

layer chromatography (TLC) was carried out on pre-coated TLC

plates with silica gel HSGF 254. Spots were visualized under UV at

254 nm. Flash chromatography was performed on glass columns

packed with silica gel using PE/EA with varying proportions as the

Table 10. Anticonvulsant Eﬀects of HN37 and Three

Reference Drugs (p.o.) in the C57BL/6 Mouse-Induced 6

a

Hz Seizure Model

time of test

(h)

6 Hz 32 mA ED₅₀

6 Hz 44 mA ED₅₀

b

1

mobile phase. H NMR and ¹³C NMR spectra were measured on a

compound

(mg/kg)

Varian Mercury-VX 300, Varian MR 400, AVANCE III 500, or

AVANCE III 600 spectrometer using deuterated chloroform (CDCl₃)

as the solvent. Chemical shifts are expressed in δ (ppm) relative to

HN37

1.0

16.8 (14.7−19.1)

65.9 (58.6−74.0)

>500

29.9 (23.0−38.8)

102.5 (91.6−114.8)

>500

c

RTG

c

TPM

1

internal Me₄Si for H and ¹³C NMR. HR-MS were measured on a

c

LEV

41.5 (17.9−96.1)

376.4 (341.2−415.3)

Micromass Ultra Q-Tof. The purity of all ﬁnal compounds was ≥95%

conﬁrmed by analytical HPLC chromatograms using an Agilent 1200

series LC system equipped with a degasser, a quaternary pump, an

auto sampler, a column oven, and a diode array detector. Analytes

were separated on a Zorbax SB C18 column (4.6 × 150 mm, 5 μm).

Solvent A was 0.1% triﬂuoroacetic acid in H₂O and solvent B was

100% methanol. Gradient elution: 20% B for 2 min, then 20−80% B

from 2 to 20 min, and 80% B was maintained for 5 min, then 80−20%

B from 25 to 30 min. All compounds were monitored at 262 nm at

room temperature. Flow rate: 1.0 mL/min.

General Procedure A: Reductive Amination Reaction. To a

solution of commercially available anilines (1.0 equiv) in toluene was

added p-ﬂuorobenzaldehyde and catalytic amount p-TsOH (0.025

equiv). The resulting mixture was heated to reﬂux with a Dean−Stark

trap until completion. After cooling to room temperature, the mixture

was concentrated to dryness in vacuo. The obtained residue was re-

dissolved in dioxane/MeOH (4/1, v/v) or MeOH, and solid NaBH₄

was added in portions at room temperature. The mixture was allowed

to stir until completion, quenched and diluted with water, and

extracted with EA. The combined organic phase was washed with

brine, dried over anhydrous Na₂SO₄, ﬁltered, and concentrated to

dryness in vacuo. Further puriﬁcation by ﬂash chromatography gave

the desired product.

a

Values in parentheses are 95% conﬁdence intervals determined by

b

nonlinear regression using GraphPad Prism 5. ED₅₀(6 Hz

psychomotor seizure model, 32 mA or 44 mA). Reference AEDs:

retigabine (RTG), levetiracetam (LEV), and topiramate (TPM)

tested under the same conditions.

c

LEV and RTG demonstrated potent anticonvulsant eﬀects on

the 6 Hz seizure at both 32 and 44 mA stimulus intensities,

which was in conformity to the published report.^56,57

Compound HN37 was also highly eﬀective in the 6 Hz

seizure test at both 32 and 44 mA, with ED₅₀values of 16.8

and 29.9 mg/kg, respectively. As the 6 Hz stimulus current

increases from 32 to 44 mA, most AEDs lose eﬃcacy and few

are only eﬀective at the neurotoxic doses, just like the three

reference AEDs in the present study.^53,57Under the higher

stimulus intensity (44 mA), LEV was only eﬀective at very high

doses (ED₅₀= 376.4 mg/kg), and RTG produced visible

sedative eﬀects at the eﬀective doses of 80 mg/kg and higher in

this animal strain. However, HN37 showed much more

eﬃcacy and did not produce obvious motor impairment at the

eﬀective doses. Thus, based on the eﬃcacy and tolerability,

HN37 is a promising candidate for epilepsy and has potential

for eﬃcacy in clinical pharmacoresistant patients.

General Procedure B: N-Alkylation Reactions of Anilines. To

a solution of anilines in DMF was added proper bromide and DIPEA.

The mixture was stirred at 65 °C until completion. After cooling to

room temperature, the mixture was diluted with water and extracted

with EA. The combined organic phase was washed with water, brine,

dried over anhydrous Na₂SO₄, ﬁltered, and concentrated to dryness in

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vacuo. Further puriﬁcation by ﬂash chromatography gave the desired

product.

Procedure D, the deprotected intermediate was reacted with methyl

chloroformate (40 μL, 0.52 mmol) and DIPEA (146μL, 0.84 mmol)

in DCM (2 mL) at room temperature for 2 h. Puriﬁcation by ﬂash

chromatography (PE/EA, 8/1, v/v) gave the title compound L1-1 as

General Procedure C: Deprotection Reactions of Boc-

Protected Anilines. To a stirred solution of Boc-protected anilines

in DCM was added TFA dropwise with stirring at 0 °C using an ice-

water bath. The mixture was allowed to stir at room temperature until

completion. Then, the mixture was concentrated to dryness in vacuo

and was used in the next reaction without puriﬁcation.

General Procedure D: N-Acylation Reactions of Anilines.

DIPEA was added to a solution of anilines in dioxane or DCM. After

cooling to 0 °C using an ice-water bath, proper acyl chloride was

added dropwise. The reaction mixture was stirred at room

temperature until completion. Then, the mixture was quenched

with water and extracted with EtOAc. The combined organic phase

was washed with brine, dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated to dryness in vacuo. Further puriﬁcation by ﬂash

chromatography gave the desired product.

General Procedure E: Boc Protection Reactions of Anilines.

TEA and DMAP were added to a solution of anilines in DCM. After

cooling to 0 °C using an ice-water bath, Boc₂O was added. The

mixture was allowed to stir at room temperature until completion.

Then, the reaction mixture was poured into water and extracted with

EA. The combined organic phase was washed with 1 N HCl, water,

brine, dried over anhydrous Na₂SO₄, ﬁltered, and concentrated to

dryness in vacuo. Further puriﬁcation by ﬂash chromatography gave

the desired product.

General Procedure F: Nitro Reduction Reactions of Nitro

Compounds. Method A. Under a nitrogen atmosphere, a catalytic

amount of 10% Pd/C was added to a solution of the nitro compound

in EA. Then, the reaction atmosphere was changed to hydrogen using

a H₂balloon and the mixture was allowed to stir at room temperature

until completion. The mixture was ﬁltered under a nitrogen

atmosphere, and the ﬁltrate was concentrated to dryness in vacuo

and was used in the next reaction without puriﬁcation.

Method B. The Fe powder was added to a solution of the nitro

compound in EtOH/AcOH (40/1, v/v). The mixture was heated to

reﬂux until completion. After cooling to room temperature, the

mixture was ﬁltered, and the ﬁltrate was concentrated in vacuo. The

obtained residue was re-dissolved in EA, washed with saturated

NaHCO₃aqueous solution, brine, dried over anhydrous Na₂SO₄,

ﬁltered, and concentrated to dryness in vacuo. Further puriﬁcation by

ﬂash chromatography gave the desired product.

tert-Butyl (4-((4-Fluorobenzyl)amino)phenyl)carbamate (2). Fol-

lowing general procedure A, tert-butyl (4-aminophenyl)carbamate 1

(2.57 g, 12.3 mol) was condensed with p-ﬂuorobenzaldehyde (1.69 g,

13.6 mmol) catalyzed by p-TsOH (53 mg, 0.3 mmol) in toluene (50

mL) after reﬂux for 3 h, followed by reduction by NaBH₄(0.7 g, 18.4

mmol) in dioxane/MeOH (10 mL/2.5 mL) at room temperature for

2 h. Puriﬁcation by ﬂash chromatography (PE/EA, 6/1, v/v) gave the

title compound as a pale yellow solid (3.5 g, 90% over two steps). ¹H

NMR (300 MHz, CDCl₃): δ 7.31 (t, J = 8.4 Hz, 2H), 7.14 (d, J = 8.4

Hz, 2H), 7.01 (t, J = 8.7 Hz, 2H), 6.56 (d, J = 9.0 Hz, 2H), 6.22 (s,

1H), 4.27 (s, 2H), 1.50 (s, 9H).

tert-Butyl (4-((4-Fluorobenzyl)(prop-2-yn-1-yl)amino)phenyl)-

carbamate (L1-4). Following general procedure B, compound 1

(316 mg, 1.0 mmol) was reacted with propargyl bromide (102 μL, 1.3

mmol) and DIPEA (383 μL, 2.2 mmol) in DMF (15 mL) at 65 °C for

2 h. Puriﬁcation by ﬂash chromatography (PE/EA, 8/1, v/v) gave the

title compound as a yellow solid (330 mg, 93%). ¹H NMR (300 MHz,

CDCl₃): δ 7.21−7.30 (m, 4H), 7.00 (t, J = 8.4 Hz, 2H), 6.86 (d, J =

9.0 Hz, 2H), 6.32 (s, 1H), 4.42 (s, 2H), 3.92 (d, J = 2.4 Hz, 2H), 2.20

(t, J = 2.4 Hz, 1H), 1.50 (s, 9H); ¹³C NMR (75 MHz, CDCl₃): δ

163.29, 160.86, 153.25, 145.08, 133.99, 130.23, 129.12, 120.61,

115.96, 115.49, 115.28, 80.17, 79.40, 72.47, 54.77, 40.43, 28.38;

HRMS (TOF ESI) m/z: calcd for C₂₁H₂₄FN₂O₂[M + H]⁺, 355.1822;

found, 355.1814; HPLC purity: 95.2%.

1

a pale yellow solid (100 mg, 76% over two steps). H NMR (300

MHz, CDCl₃): δ 7.23−7.28 (m, 4H), 6.99 (t, J = 8.4 Hz, 2H), 6.83

(d, J = 8.7 Hz, 2H), 4.41 (s, 2H), 3.91 (s, 2H), 3.72 (s, 3H), 2.22 (s,

1H); ¹³C NMR (75 MHz, CDCl₃): δ 163.74, 160.49, 145.36, 134.04,

129.15, 120.79, 115.74, 115.62, 115.34, 79.52, 72.68, 54.78, 52.31,

40.37; HRMS (TOF ESI) m/z: calcd for C₁₈H₁₈FN₂O₂[M + H]⁺,

313.1352; found, 313.1342; HPLC purity: 98.5%.

Ethyl (4-((4-Fluorobenzyl) (prop-2-yn-1-yl)amino)phenyl)-

carbamate (L1-2). Following procedures similar to that described

for compound L1-1 gave compound L1-2 as a yellow solid (73% over

two steps). ¹H NMR (300 MHz, CDCl₃): δ 7.23−7.29 (m, 4H), 7.00

(t, J = 8.4 Hz, 2H), 6.85 (d, J = 9.0 Hz, 2H), 6.59 (s, 1H), 4.42 (s,

2H), 4.18 (q, J = 7.2 Hz, 2H), 3.92 (d, J = 2.1 Hz, 2H), 2.21 (t, J = 2.4

Hz, 1H), 1.28 (t, J = 7.2 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃): δ

163.82, 160.57, 145.40, 134.11, 129.23, 120.87, 115.91, 115.67,

115.39, 79.56, 72.68, 61.19, 54.87, 40.48, 14.74; HRMS (TOF ESI)

m/z: calcd for C₁₉H₂₀FN₂O₂[M + H]⁺, 327.1509; found, 327.1501;

HPLC purity: 99.1%.

Isobutyl(4-((4-ﬂuorobenzyl) (prop-2-yn-1-yl)amino)phenyl)-

carbamate (L1-3). Following procedures similar to that described

for compound L1-1 gave compound L1-3 as yellow oil (88% over two

steps). ¹H NMR (300 MHz, CDCl₃): δ 7.23−7.29 (m, 4H), 6.99 (t, J

= 8.7 Hz, 2H), 6.83 (d, J = 9.3 Hz, 2H), 6.67 (s, 1H), 4.42 (s, 2H),

3.93 (s, 2H), 3.91 (d, J = 4.2 Hz, 2H), 2.21 (s, 1H), 1.92−1.97 (m,

1H), 0.94 (d, J = 6.6 Hz, 6H); ¹³C NMR (75 MHz, CDCl₃): δ

163.77, 160.53, 145.32, 134.04, 129.20, 129.09, 120.72, 115.86,

115.64, 115.36, 79.54, 72.67, 71.30, 54.82, 40.42, 28.10, 19.20; HRMS

(TOF ESI) m/z: calcd for C₂₁H₂₄FN₂O₂[M + H]⁺, 355.1822; found,

355.1817; HPLC purity: 99.2%.

Allyl(4-((4-ﬂuorobenzyl) (prop-2-yn-1-yl)amino)phenyl)-

carbamate (L1-5). Following procedures similar to that described

for compound L1-1 gave compound L1-5 as yellow oil (68% over two

steps). ¹H NMR (300 MHz, CDCl₃): δ 7.24−7.33 (m, 4H), 7.00 (t, J

= 8.4 Hz, 2H), 6.85 (d, J = 9.0 Hz, 2H), 6.65 (s, 1H), 5.88−5.99 (m,

1H), 5.33 (d, J = 17.1 Hz, 1H), 5.22 (d, J = 10.2 Hz, 1H), 4.63 (d, J =

5.7 Hz, 2H), 4.43 (s, 2H), 3.92 (s, 2H), 2.21 (s, 1H); ¹³C NMR (75

MHz, CDCl₃): δ 163.82, 160.58, 145.50, 134.08, 132.79, 129.21,

118.16, 115.85, 115.69, 115.41, 79.54, 72.69, 65.85, 54.85, 40.46;

HRMS (TOF ESI) m/z: calcd for C₂₀H₂₀FN₂O₂[M + H]⁺, 339.1509;

found, 339.1504; HPLC purity: 95.2%.

(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl(4-((4-ﬂuorobenzyl)

(prop-2-yn-1-yl)amino)phenyl)carbamate (L1-6). Following proce-

dures similar to that described for compound L1-1 gave compound

L1-6 as yellow oil (82% over two steps). ¹H NMR (300 MHz,

CDCl₃): δ 7.23−7.29 (m, 4H), 7.00 (t, J = 8.4 Hz, 2H), 6.84 (d, J =

8.4 Hz, 2H), 4.43 (s, 2H), 4.29−4.36 (m, 1H), 4.25 (dd, J = 4.2, 0.9

Hz, 1H), 4.12 (d, J = 8.1 Hz, 1H), 4.07 (d, J = 6.9 Hz, 1H), 3.93 (s,

2H), 3.75 (dd, J = 7.2, 0.9 Hz, 1H), 2.23 (s, 1H), 1.44 (s, 3H), 1.37

(s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 160.08, 156.41, 145.06,

133.58, 128.70, 120.40, 115.29, 115.22, 114.93, 109.54, 79.05, 73.58,

72.25, 65.80, 65.14, 54.36, 39.96, 26.40, 25.00; HRMS (ESI) m/z:

calcd for C₂₃H₂₆FN₂O₄[M + H]⁺, 413.1877; found, 413.1869; HPLC

purity: 95.0%.

Benzyl(4-((4-ﬂuorobenzyl) (prop-2-yn-1-yl)amino)phenyl)-

carbamate (L1-7). Following procedures similar to that described

for compound L1-1 gave compound L1-7 as yellow oil (60% over two

1

steps). H NMR (300 MHz, CDCl₃): δ 7.35−7.39 (m, 4H), 7.25−

7.30 (m, 5H), 7.03 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 5.18

(s, 2H), 4.44 (s, 2H), 3.94 (s, 2H), 2.22 (s, 1H); ¹³C NMR (75 MHz,

CDCl₃): δ 163.34, 160.91, 145.38, 136.40, 134.03, 134.00, 129.15,

129.07, 128.63, 128.30, 128.28, 120.76, 115.79, 115.58, 115.37, 79.53,

72.67, 66.88, 54.77, 40.38; HRMS (TOF ESI) m/z: calcd for

C₂₄H₂₂FN₂O₄[M + H]⁺, 389.1665; found, 389.1663; HPLC purity:

99.7%.

Methyl (4-((4-Fluorobenzyl)(prop-2-yn-1-yl)amino)phenyl)-

carbamate (L1-1). Following general procedure C, compound L1-4

(150 mg, 0.42 mmol) was deprotected using TFA (0.2 mL) in DCM

(2 mL) at room temperature for 2 h; further following General

N-(4-((4-Fluorobenzyl)(prop-2-yn-1-yl)amino)phenyl)acetamide

(L1-8). Following procedures similar to that described for compound

5828

https://doi.org/10.1021/acs.jmedchem.0c02252

J. Med. Chem. 2021, 64, 5816−5837

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1

L1-1 gave compound L1-8 as yellow oil (20% over two steps). H

NMR (300 MHz, CDCl₃): δ 7.26 (d, J = 6.9 Hz, 2H), 7.05 (d, J = 8.4

Hz, 2H), 6.99 (d, J = 9.0 Hz, 2H), 6.90 (d, J = 9.0 Hz, 2H), 4.55 (s,

2H), 4.04 (s, 2H), 2.30 (s, 3H), 2.26 (t, J = 2.1 Hz, 1H); ¹³C NMR

(75 MHz, CDCl₃): δ 163.13, 163.18, 148.65, 133.49, 133.47, 129.65,

128.61, 115.68, 115.51, 114.61, 79.07, 72.56, 54.55, 39.87, 26.94;

HRMS (TOF ESI) m/z: calcd for C₁₈H₁₈FN₂O₄[M + H]⁺, 297.1403;

found, 297.1398; HPLC purity: 99.4%.

Methyl (4-(Prop-2-yn-1-ylamino)phenyl)carbamate (4). Follow-

ing general procedure B, methyl (4-aminophenyl)carbamate (6.64 g,

40.0 mmol) was reacted with propargyl bromide (3.13 mL, 40.0

mmol) and DIPEA (10.48 mL, 60.0 mmol) in DMF (150 mL) at 65

°C for 4 h. Puriﬁcation by ﬂash chromatography (PE/EA, 6/1, 4/1, v/

v) gave the title compound as yellow oil (4.60 g, 56%). ¹H NMR (300

MHz, CDCl₃): δ 7.20 (d, J = 7.2 Hz, 2H), 6.69 (d, J = 7.2 Hz, 2H),

6.58 (br s, 1H), 3.89 (d, J = 2.1 Hz, 2H), 3.74 (s, 3H), 2.21 (t, J = 2.1

Hz, 1H); MS (ESI) m/z: 227.1 [M + Na]⁺.

m/z: calcd for C₁₈H₁₈ClN₂O₂[M + H]⁺, 329.1057; found, 329.0873;

HPLC purity: 99.4%.

Methyl (4-((3-Methoxybenzyl) (prop-2-yn-1-yl)amino)phenyl)-

carbamate (L2-6). Following procedures similar to that described

1

for compound L2-1 gave compound L2-6 as yellow oil (27%). H

NMR (300 MHz, CDCl₃): δ 7.22−7.26 (m, 4H), 6.85−6.90 (m, 4H),

6.43 (br s, 1H), 4.42 (s, 2H), 3.93 (d, J = 2.1 Hz, 2H), 3.80 (s, 3H),

3.78 (s, 3H), 2.20 (t, J = 2.4 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃):

δ 159.96, 145.51, 140.16, 129.63, 120.73, 119.61, 115.33, 112.84,

112.62, 79.61, 72.35, 55.42, 55.20, 52.22, 40.35; HRMS (TOF ESI)

m/z: calcd for C₁₉H₂₀N₂NaO₃[M + Na]⁺: 347.1372; found,

347.1373; HPLC purity: 97.5%.

Methyl (4-((4-Bromobenzyl) (prop-2-yn-1-yl)amino)phenyl)-

carbamate (L2-7). Following procedures similar to that described

1

for compound L2-1 gave compound L2-7 as yellow oil (15%). H

NMR (300 MHz, CDCl₃): δ 7.45 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 8.4

Hz, 2H), 7.19 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 6.71 (s,

1H), 4.43 (s, 2H), 3.95 (d, J = 2.1 Hz, 2H), 3.75 (s, 3H), 2.24 (t, J =

2.1 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 154.47, 137.41, 131.68,

129.11, 128.57, 121.40, 120.97, 115.64, 79.28, 72.57, 54.91, 52.25,

40.53; HRMS (TOF ESI) m/z: calcd for C₁₈H₁₈BrN₂O₂[M + H]⁺,

373.0552; found, 373.0352; HPLC purity: 98.2%.

Methyl (4-((2-Fluorobenzyl) (prop-2-yn-1-yl)amino)phenyl)-

carbamate (L2-1). Following general procedure B, compound 4

(102 mg, 0.5 mmol) was reacted with 2-ﬂuorobenzyl bromide (74 μL,

0.6 mmol) and DIPEA (0.17 mL, 1.0 mmol) in DMF (3 mL) at 65

°C for 4 h. Puriﬁcation by ﬂash chromatography (PE/EA, 15/1, 10/1,

1

Methyl (4-((4-Methylbenzyl) (prop-2-yn-1-yl)amino)phenyl)-

v/v) gave the title compound as yellow oil (150 mg, 96%). H NMR

carbamate (L2-8). Following procedures similar to that described

(300 MHz, CDCl₃): δ 7.22−7.34 (m, 4H), 7.04−7.11 (m, 2H),

6.84−6.88 (m, 2H), 6.60 (br s, 1H), 4.56 (s, 2H), 4.01 (d, J = 2.4 Hz,

2H), 3.75 (s, 3H), 2.25 (t, J = 2.4 Hz, 1H); ¹³C NMR (101 MHz,

CDCl₃): δ 161.91, 159.96, 154.48, 145.19, 129.27, 128.82, 125.27,

124.14, 120.75, 115.42, 115.25, 79.46, 72.47, 52.23, 49.42, 40.52;

HRMS (TOF ESI) m/z: calcd for C₁₈H₁₇FN₂NaO₂[M + Na]⁺:

335.1172; found, 335.1166; HPLC purity: 97.0%.

1

for compound L2-1 gave compound L2-8 as yellow oil (81%). H

NMR (300 MHz, CDCl₃): δ 7.15−7.23 (m, 4H), 6.99 (t, J = 8.7 Hz,

2H), 6.63 (d, J = 9.0 Hz, 2H), 6.53 (s, 1H), 4.44 (s, 2H), 4.18 (t, J =

2.1 Hz, 2H), 3.74 (s, 3H), 2.34 (s, 3H), 2.25 (t, J = 2.1 Hz, 1H); ¹³C

NMR (125 MHz, CDCl₃): δ 145.65, 136.82, 135.18, 129.27, 127.37,

120.72, 115.43, 79.60, 72.23, 55.07, 52.23, 40.13, 21.08; HRMS (TOF

ESI) m/z: calcd for C₁₉H₂₁N₂O₂[M + H]⁺, 309.1603; found,

309.1546; HPLC purity: 99.9%.

Methyl (4-((3-Fluorobenzyl) (prop-2-yn-1-yl)amino)phenyl)-

carbamate (L2-2). Following procedures similar to that described

1

Methyl (2-Aminophenyl)carbamate (6a). To a solution of o-

phenylenediamine 5a (1.08 g, 10.0 mmol) in DCM (50 mL) was

added DIPEA (3.49 mL, 20.0 mmol). At −20 °C, a solution of methyl

chloroformate (0.78 mL, 10.0 mmol) DCM (10 mL) was added

dropwise via a syringe. The reaction mixture was allowed to warm to

room temperature and stirred for further 2 h. After completion, the

mixture was washed with water, brine, dried over anhydrous Na₂SO₄,

and ﬁltered. The ﬁltrate was concentrated to dryness in vacuo.

Puriﬁcation by ﬂash chromatography (PE/EA, 4/1, 2/1, v/v) gave

for compound L2-1 gave compound L2-2 as yellow oil (88%). H

NMR (300 MHz, CDCl₃): δ 7.23−7.32 (m, 3H), 7.10 (d, J = 8.7 Hz,

1H), 7.04 (d, J = 9.0 Hz, 1H), 6.97 (td, J = 8.7, 1.8 Hz, 1H), 6.85 (d, J

= 9.0 Hz, 2H), 6.57 (s, 1H), 4.48 (s, 2H), 3.98 (d, J = 2.1 Hz, 2H),

3.81 (s, 3H), 2.24 (t, J = 2.4 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃):

δ 164.18, 162.22, 145.25, 141.33, 130.13, 122.81, 120.71, 115.48,

114.22, 114.05, 79.32, 72.55, 55.09, 52.24, 40.58; HRMS (TOF ESI)

m/z: calcd for C₁₈H₁₇FN₂NaO₂[M + Na] ⁺: 335.1172; found,

335.1168; HPLC purity: 97.5%.

Methyl (4-((4-Fluoro-3-(triﬂuoromethyl)benzyl) (prop-2-yn-1-yl)-

amino)phenyl)carbamate (L2-3). Following procedures similar to

that described for compound L2-1 gave compound L2-3 as yellow oil

(87%). ¹H NMR (300 MHz, CDCl₃): δ 7.50−7.59 (m, 2H), 7.25 (d,

J = 8.7 Hz, 2H), 7.15 (t, J = 9.0 Hz, 1H), 6.86 (d, J = 8.7 Hz, 2H),

6.49 (br s, 1H), 4.48 (s, 2H), 3.96 (d, J = 2.4 Hz, 2H), 3.69 (s, 3H),

2.27 (t, J = 2.4 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 159.95,

157.92, 145.00, 134.72, 132.71, 126.01, 123.66, 121.49, 120.67,

117.14, 1159.9, 78.99, 72.91, 54.63, 52.26, 40.81; HRMS (TOF ESI)

m/z: calcd for C₁₉H₁₆F₄N₂NaO₂[M + Na]⁺: 403.1046; found,

403.1042; HPLC purity: 97.4%.

Methyl (4-((3,4-Diﬂuorobenzyl) (prop-2-yn-1-yl)amino)phenyl)-

carbamate (L2-4). Following procedures similar to that described for

compound L2-1 gave compound L2-4 as yellow oil (15%). ¹H NMR

(300 MHz, CDCl₃): δ 7.26 (d, J = 8.7 Hz, 2H), 7.05−7.18 (m, 3H),

6.85 (d, J = 8.7 Hz, 2H), 6.47 (br s, 1H), 4.42 (s, 2H), 3.96 (d, J = 2.1

Hz, 2H), 3.75 (s, 3H), 2.23 (t, J = 2.4 Hz, 1H); ¹³C NMR (125 MHz,

CDCl₃): δ 151.59, 149.51, 148.6, 145.10, 135.57, 123.17, 120.68,

117.32, 116.30, 115.80, 79.17, 72.74, 54.66, 52.27, 40.69; HRMS

(TOF ESI) m/z: calcd for C₁₈H₁₆F₂N₂NaO₂[M + Na]⁺: 353.1073;

found, 353.1073; HPLC purity: 97.0%.

1

title compound 6a as a yellow solid (1.14 g, 69%). H NMR (300

MHz, CDCl₃): δ 7.24−7.27 (m, 1H), 7.03 (t, J = 7.2 Hz, 1H), 6.76−

6.81 (m, 2H), 6.41 (br s, 1H), 3.77 (s, 3H); MS (ESI) m/z: 189.1 [M

+ Na]⁺.

Methyl (2-(Prop-2-yn-1-ylamino)phenyl)carbamate (7a). Follow-

ing general procedure B, compound 6a (0.67 g, 4.0 mmol) was

reacted with propargyl bromide (313 μL, 4.0 mmol) and DIPEA

(1.05 mL, 6.0 mmol) in DMF (20 mL) at 65 °C for 4 h. After

completion, puriﬁcation by ﬂash chromatography (PE/EA, 4/1, 2/1,

v/v) gave the title compound 7a as yellow oil (0.74 g, 90%). ¹H NMR

(300 MHz, CDCl₃): δ 7.34 (d, J = 8.1 Hz, 1H), 7.16 (td, J = 8.4, 1.5

Hz, 1H), 6.83−6.88 (m, 2H), 6.32 (br s, 1H), 3.92 (d, J = 2.1 Hz,

2H), 3.77 (s, 2H), 2.24 (t, J = 2.1 Hz, 1H); MS (ESI) m/z: 227.0 [M

+ Na]⁺.

Methyl (2-((4-Fluorobenzyl) (prop-2-yn-1-yl)amino)phenyl)-

carbamate (L3-1). Following general procedure B, compound 7a

(0.102 g, 0.5 mmol) was reacted with p-ﬂuorobenzyl bromide (74 μL,

0.55 mmol) and DIPEA (174 μL, 1.0 mmol) in DMF (3 mL) at 65

°C for 4 h. After completion, puriﬁcation by ﬂash chromatography

(PE/EA, 15/1, 10/1, v/v) gave title compound L3-1 as yellow oil

(150 mg, 96%). ¹H NMR (300 MHz, CDCl₃): δ 8.10 (d, J = 8.4 Hz,

1H), 7.86 (s, 1H), 7.35 (dd, J = 8.4, 1.2 Hz, 1H), 7.28 (ddd, J = 8.1,

4.8, 3.0 Hz, 2H), 7.18 (dd, J = 7.8, 1.2 Hz, 1H), 7.00 (ddd, J = 9.0,

4.8, 2.7 Hz, 2H), 4.11 (s, 2H), 3.79 (s, 3H), 3.56 (d, J = 2.4 Hz, 2H),

2.27 (t, J = 2.4 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 164.14,

160.89, 156.30, 154.15, 138.32, 134.48, 133.03, 131.03, 126.69,

123.79, 122.72, 118.11, 115.52, 78.89, 74.19, 56.40, 52.48, 42.69;

HRMS (TOF ESI) m/z: calcd for C₁₈H₁₈FN₂O₂[M + H]⁺, 313.1352;

found, 313.1354; HPLC purity: 95.3%.

Methyl (4-((2-Chlorobenzyl) (prop-2-yn-1-yl)amino)phenyl)-

carbamate (L2-5). Following procedures similar to that described

1

for compound L2-1 gave compound L2-5 as yellow oil (81%). H

NMR (300 MHz, CDCl₃): δ 7.30−7.41 (m, 2H), 7.19−7.25 (m, 4H),

6.85 (d, J = 8.7 Hz, 2H), 6.46 (br s, 1H), 4.59 (s, 2H), 4.06 (d, J = 2.4

Hz, 2H), 3.75 (s, 3H), 2.25 (t, J = 2.4 Hz, 1H); ¹³C NMR (125 MHz,

CDCl₃): δ 154.52, 144.94, 135.52, 133.06, 129.58, 128.44, 126.92,

120.89, 114.54, 79.54, 72.45, 53.63, 52.23, 40.73; HRMS (TOF ESI)

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Methyl (3-Aminophenyl)carbamate (6b). Following procedures

similar to that described for 6a, using benzene-1,3-diamine 5b (1.08 g,

10.0 mmol), gave the title compound 6b as a yellow solid (1.0 g,

60%). ¹H NMR (300 MHz, CDCl₃): δ 7.05 (t, J = 8.4 Hz, 1H), 6.94

(s, 1H), 6.59 (d, J = 8.4 Hz, 1H), 6.53 (s, 1H), 6.38 (d, J = 8.4 Hz,

1H), 3.76 (s, 3H); MS (ESI) m/z: 167.1 [M + H]⁺.

described for compound 6c, using compound 8 (512 mg, 2.0 mmol),

gave intermediate 9 after ﬂash chromatography (PE/EA, 10/1, v/v) as

yellow oil (362 mg, 58%); the above-obtained oil (362 mg, 1.15

mmol) was reduced following method A of general procedure F to

give the respective amine quantitatively; following general procedure

B, the obtained amine was reacted with propargyl bromide (90 μL,

1.15 mmol) and DIPEA (0.38 mL, 2.2 mmol) in DMF (10 mL) at 65

°C for 4 h. Puriﬁcation by ﬂash chromatography (PE/EA, 5/1, 4/1, v/

v) gave yellow oil (162 mg, 44%); further following general procedure

B, the above-obtained oil (120 mg, 0.37 mmol) was reacted with p-

ﬂuorobenzyl bromide (50 μL, 0.37 mmol) and DIPEA (64 μL, 0.37

mmol) in DMF (5 mL) at 65 °C for 4 h. Puriﬁcation by ﬂash

chromatography (PE/EA, 15/1, 10/1, v/v) gave yellow oil (132 mg,

83%); following general procedure C, the above-obtained oil (120

mg, 0.28 mmol) was deprotected using TFA (0.2 mL) in DCM (2

mL) and gave compound L3-4 as yellow oil after puriﬁcation by ﬂash

Methyl (3-(Prop-2-yn-1-ylamino)phenyl)carbamate (7b). Follow-

ing procedures similar to that described for 7a, using compound 6b

(0.67 g, 4.0 mmol), gave the title compound 7b as yellow oil (205 mg,

25%). ¹H NMR (300 MHz, CDCl₃): δ 7.11 (t, J = 8.1 Hz, 1H), 6.89

(s, 1H), 6.67 (d, J = 8.1 Hz, 1H), 6.40 (d, J = 8.1 Hz, 1H), 3.99 (s,

2H), 3.75 (s, 3H), 2.21 (s, 1H); MS (ESI) m/z: 227.0 (M + Na⁺).

Methyl (3-((4-Fluorobenzyl) (prop-2-yn-1-yl)amino)phenyl)-

carbamate (L3-2). Following procedures similar to that described

for compound L3-1, using compound 7b (0.102 g, 0.5 mmol), gave

the title compound L3-2 as yellow oil (128 mg, 82%). ¹H NMR (300

MHz, CDCl₃): δ 7.28 (t, J = 8.1 Hz, 2H), 7.19 (t, J = 8.7 Hz, 2H),

6.95−7.07 (m, 3H), 6.77 (d, J = 8.7 Hz, 1H), 6.56−6.62 (m, 2H),

4.51 (s, 2H), 3.99 (d, J = 2.4 Hz, 2H), 3.75 (s, 3H), 2.23 (t, J = 2.4

Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 163.97, 160.72, 147.94,

146.00, 144.82, 134.00, 129.49, 115.00, 110.08, 109.99, 79.38, 72.85,

54.97, 51.71, 40.80; HRMS (TOF ESI) m/z: calcd for C₁₈H₁₈FN₂O₂

[M + H]⁺, 313.1352; found, 313.1344; HPLC purity: 98.0%.

Methyl (2-Fluoro-5-nitrophenyl)carbamate (6c). Under a nitro-

gen atmosphere, sodium hydride (60% dispersion in mineral oil, 200

mg, 5.0 mmol) was added in portions to a solution of 2-ﬂuoro-5-

nitroaniline 5c (785 mg, 5.0 mmol) in dry THF (25 mL) at 0 °C.

After the reaction mixture was stirred for 30 min, it was allowed to

warm to room temperature and stirred for further 1 h. A solution of

methyl chloroformate (0.39 mL, 5.0 mmol) in dry THF (25 mL) was

added dropwise via a syringe, and the resulting mixture was stirred for

further 30 min. After completion, the reaction was quenched with

water and extracted with EA. The combined organic phase was

washed with brine, dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated to dryness in vacuo. Puriﬁcation by ﬂash chromatog-

raphy (PE/EA, 4/1, v/v) gave title compound 6c as a yellow solid

(428 mg, 40%). ¹H NMR (300 MHz, CDCl₃): δ 9.08 (d, J = 6.9 Hz,

1H), 7.94 (t, J = 9.0 Hz, 1H), 7.21 (d, J = 9.0 Hz, 1H), 7.01 (s, 1H),

3.87 (s, 3H); MS (ESI) m/z: 237.0 [M + Na]⁺.

1

chromatography (PE/EA, 8/1, 6/1, v/v) (76 mg, 83%). H NMR

(300 MHz, CDCl₃): δ 7.78 (br s, 1H), 7.24−7.29 (m, 2H), 7.02 (t, J

= 8.7 Hz, 2H), 6.60−6.66 (m, 2H), 6.51 (br s, 1H), 4.45 (s, 2H), 3.96

(d, J = 2.1 Hz, 2H), 3.77 (s, 3H), 2.24 (t, J = 2.1 Hz, 1H); ¹³C NMR

(125 MHz, CDCl₃): δ 163.36, 160.93, 133.46, 128.89, 115.65, 115.44,

110.45, 102.08, 101.85, 78.99, 72.76, 54.61, 52.45, 40.27; HRMS

(TOF ESI) m/z: calcd for C₁₈H₁₆F₂N₂NaO₂[M + Na]⁺: 353.1078;

found, 353.1068; HPLC purity: 97.1%.

2-Fluoro-N-(4-ﬂuorobenzyl)-4-nitroaniline (10). Following gen-

eral procedure A, 2-ﬂuoro-4-nitroaniline 5d (624 mg, 4.0 mmol) was

condensed with p-ﬂuorobenzaldehyde (0.43 mL, 4.0 mmol) catalyzed

by p-TsOH (17 mg, 0.1 mmol) in toluene (20 mL) after reﬂux for 3

h, followed by reduction by NaBH₄(380 mg, 10.0 mmol) in MeOH

(20 mL) at room temperature for 2 h. Puriﬁcation by ﬂash

chromatography (PE/EA, 8/1, 6/1, v/v) gave the title compound

1

10 as a light yellow solid (982 mg, 93% over two steps). H NMR

(300 MHz, CDCl₃): δ 7.98−7.90 (m, 3H), 7.33−7.31 (m, 2H),

7.08−7.06 (m, 2H), 6.61 (br s, 1H), 5.03 (s, 1H), 4.45 (s, 2H); MS

(ESI) m/z: 287.0 [M + Na]⁺.

Methyl (3-Fluoro-4-((4-ﬂuorobenzyl) (prop-2-yn-1-yl)amino)-

phenyl)carbamate (L3-5). To a solution of compound 10 (264

mg, 1.0 mmol) in dry DMF (5 mL) was added sodium hydride (60%

dispersed in mineral oil, 44 mg, 1.1 mmol) in portions at 0 °C under a

nitrogen atmosphere. After the mixture was stirred for 30 min,

propargyl bromide (94 μL, 1.2 mmol) was added and the reaction

mixture was stirred at 65 °C for further 4 h. After completion, the

reaction was quenched with water and extracted with EA. The

combined organic phase was washed with brine, dried over anhydrous

Na₂SO₄, ﬁltered, and concentrated to dryness in vacuo. The

puriﬁcation of the residue by ﬂash chromatography (PE/EA, 10/1,

v/v) gave a yellow solid (260 mg, 86%).

The above-obtained yellow solid (260 mg, 0.86 mmol) was

reduced to the respective amine following method B of general

procedure F, using the Fe powder (200 mg) in EtOH/AcOH (20

mL/0.5 mL) after reﬂux for 3 h. Puriﬁcation by ﬂash chromatography

(PE/EA, 6/1, v/v) aﬀorded the desired product as a yellow solid (166

mg, 71%). ¹H NMR (300 MHz, CDCl₃): δ 7.35−7.37 (m, 2H),

6.98−7.00 (m, 3H), 6.35−6.44 (m, 2H), 4.18 (s, 2H), 3.68 (br s,

2H), 2.25 (s, 1H); MS (ESI) m/z: 273.1 [M + H]⁺.

Methyl (5-Amino-2-ﬂuorophenyl)carbamate (6d). Following

method A of general procedure F, catalytic hydrogenation of

compound 6c (428 mg, 2.0 mmol) gave compound 6d quantitatively

1

and was used without puriﬁcation. H NMR (300 MHz, CDCl₃): δ

7.49 (s, 1H), 6.81−-6.86 (m, 2H), 6.26−-6.28 (d, J = 6Hz, 1H), 3.87

(s, 3H), 3.59 (br s, 2H); MS (ESI) m/z: 207.0 [M + Na]⁺.

Methyl (2-Fluoro-5-((4-ﬂuorobenzyl) (prop-2-yn-1-yl)amino)-

phenyl)carbamate (L3-3). Following procedures similar to that

described for 7a, using compound 6d (368 mg, 2 mmol), gave

intermediate 7c as yellow oil (187 mg, 42%). MS (ESI) m/z: 245.0

[M + Na]⁺; further following a procedure similar to that described for

compound L3-1, using compound 7c (90 mg, 0.41 mmol), gave ﬁnal

1

compound L3-3 as yellow oil (112 mg, 83%). H NMR (300 MHz,

CDCl₃): δ 7.79 (br s, 1H), 7.29 (ddd, J = 9.0, 5.4, 2.4 Hz, 2H), 7.02

(ddd, J = 8.7, 5.1, 2.7 Hz, 2H), 6.93 (dd, J = 10.2, 9.0 Hz, 1H), 6.83

(br s, 1H), 6.50 (ddd, J = 9.0, 5.4, 3.3 Hz, 1H), 4.44 (s, 2H), 3.95 (d,

J = 2.1 Hz, 2H), 3.79 (s, 3H), 2.41 (t, J = 2.1 Hz, 1H); ¹³C NMR

(125 MHz, CDCl₃): δ 163.08, 161.13, 153.65, 145.75, 133.72, 129.22,

115.48, 114.96, 109.83, 107.31, 79.12, 72.59, 54.74, 52.47, 40.57;

HRMS (TOF ESI) m/z: calcd for C₁₈H₁₇F₂N₂O₂[M + H]⁺,

331.1258; found, 331.1251; HPLC purity: 96.0%.

Following general procedure D, the above-obtained amine

intermediate (100 mg, 0.37 mmol) was reacted with methyl

chloroformate (34 μL, 0.44 mmol) and DIPEA (96 μL, 0.56

mmol) in DCM (5 mL) at room temperature for 1 h. After

completion, the crude product was puriﬁed by ﬂash chromatography

(PE/EA, 10/1, v/v) to give the ﬁnal compound L3-5 as yellow oil (98

tert-Butyl (2-Fluoro-4-nitrophenyl)carbamate (8). Following

general procedure E, 2-ﬂuoro-4-nitroaniline 5d (624 mg, 4.0 mmol)

was reacted with Boc₂O (963 mg, 4.4 mmol), TEA (837 μL, 4.8

mmol), and DMAP (49 mg, 0.4 mmol) in DCM (20 mL) at room

temperature overnight. Puriﬁcation by ﬂash chromatography (PE/EA,

1

mg, 81%). H NMR (300 MHz, CDCl₃): δ 7.78 (br s, 1H), 7.24−

7.29 (m, 2H), 7.02 (t, J = 8.7 Hz, 2H), 6.60−6.66 (m, 2H), 6.51 (br s,

1H), 4.45 (s, 2H), 3.96 (d, J = 2.1 Hz, 2H), 3.77 (s, 3H), 2.24 (t, J =

2.1 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 162.17, 161.22, 157.09,

155.13, 153.95, 133.31, 130.34, 122.11, 115.31, 114.19, 78.94, 73.49,

54.71, 52.42, 40.80; HRMS (TOF ESI) m/z: calcd for C₁₈H₁₇F₂N₂O₂

[M + H]⁺, 331.1258; found, 331.1251; HPLC purity: 97.6%.

1

25/1, v/v) gave compound 8 as a yellow solid (747 mg, 73%). H

NMR (300 MHz, CDCl₃): δ 7.99−8.07 (m, 2H), 7.38 (t, J = 7.2 Hz,

1H), 1.43 (s, 9H); MS (ESI) m/z: 257.1 [M + H]⁺.

tert-Butyl (4-Amino-2-chlorophenyl)carbamate (11). Following

general procedure E, 2-chloro-4-nitroaniline 5e (692 mg, 4.0 mmol)

Methyl (2-Fluoro-4-((4-ﬂuorobenzyl) (prop-2-yn-1-yl)amino)-

phenyl)carbamate (L3-4). Following the procedure similar to that

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was reacted with Boc₂O (963 mg, 4.4 mmol) in the presence of TEA

(0.84 mL, 6.0 mmol) and DMAP (49 mg, 0.4 mmol) in DCM (20

mL), gave its N-Boc-protected product as a yellow solid after

puriﬁcation by ﬂash chromatography (PE/EA, 25/1, v/v) (400 mg,

37%).

(m, 2H), 6.52 (s, 1H), 4.23 (s, 2H), 3.77 (s, 3H), 3.73 (d, J = 2.1 Hz,

2H), 2.26 (t, J = 2.1 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ

163.35, 160.92, 153.90, 142.70, 133.38, 130.55, 130.28, 124.24,

115.30, 78.79, 73.61, 54.89, 52.49, 41.41; HRMS (TOF ESI) m/z:

calcd for C₁₈H₁₇ClFN₂O₂[M + H]⁺, 347.0963; found, 347.0981;

HPLC purity: 95.7%.

Following method A of general procedure F, catalytic hydro-

genation of the above-obtained solid (400 mg, 1.47 mmol) gave

N¹-(4-Fluorobenzyl)-3-methylbenzene-1,4-diamine (14). Follow-

ing general procedure B, 3-methyl-4-nitroaniline 5f (1.52 g, 10.0

mmol) was reacted with p-ﬂuorobenzyl bromide (1.35 mL, 10.0

mmol) and DIPEA (3.5 mL, 20.0 mmol) in DMF (25 mL) at 65 °C

overnight. Crystallization of the crude product in EA/PE (15 mL/45

mL) gave a yellow solid (1.25 g, 48%); following method A of general

procedure F, this solid was further hydrogenated to aﬀord compound

1

amine 11 quantitatively and was used without puriﬁcation. H NMR

(300 MHz, CDCl₃): δ 7.24−7.28 (s, 1H), 7.07−7.10 (d, J = 9.0 Hz,

1H), 6.80−6.83 (d, J = 9.0 Hz 1H), 4.12 (br s, 2H), 1.43 (s, 9H); MS

(ESI) m/z: 243.0 [M + H]⁺.

tert-Butyl (2-Chloro-4-((4-ﬂuorobenzyl)amino)phenyl)-

carbamate (12). Following general procedure B, compound 11

(360 mg, 1.48 mmol) was reacted with p-ﬂuorobenzyl bromide (0.2

mL, 1.48 mmol) in the presence of DIPEA (0.52 mL, 2.96 mmol) in

DMF (5 mL) at 65 °C for 4 h. Puriﬁcation by ﬂash chromatography

(PE/EA, 10/1, 6/1, v/v) gave intermediate 12 as yellow oil (430 mg,

1

14 quantitatively. H NMR (300 MHz, CDCl₃): δ 7.30−7.32 (m,

2H), 7.03−7.05 (m, 3H), 6.77 (s, 1H), 6.74−6.75 (m, 2H), 4.47 (s,

2H), 2.24 (s, 3H); MS (ESI) m/z: 231.1 [M + H]⁺.

Methyl (4-((4-Fluorobenzyl) (prop-2-yn-1-yl)amino)-2-

methylphenyl)carbamate (L3-8). To an ice-cooled solution of the

above-obtained compound 14 (1.10 g, 0.48 mmol) in THF/H₂0 (40

mL/20 mL) was added NaHCO₃(604 mg, 7.2 mmol) and Boc₂O

(1.05 g, 4.8 mmol). 30 min later, the mixture was allowed to warm to

room temperature and stirred overnight. After completion, the

mixture was poured into water (50 mL) and extracted with EA. The

combined organic phase was washed with brine, dried over anhydrous

Na₂SO₄, ﬁltered, and concentrated in vacuo. The residue was further

puriﬁed by ﬂash chromatography (PE/EA, 8/1, 6/1, v/v) to give an

oﬀ-white solid (1.42 g, 90%).

1

83%). H NMR (300 MHz, CDCl₃): δ 7.24−7.28 (m, 2H), 6.98−

7.06 (m, 3H), 6.55−6.62 (m, 2H), 4.53 (s, 2H), 1.43 (s, 9H); MS

(ESI) m/z: 351.1 [M + H]⁺.

Methyl (2-Chloro-4-((4-ﬂuorobenzyl) (prop-2-yn-1-yl)amino)-

phenyl)carbamate (L3-6). Following general procedure B, inter-

mediate 12 (35 mg, 0.1 mmol) was reacted with propargyl bromide

(11.9 μL, 0.15 mmol) in the presence of DIPEA (35.0 μL, 0.2 mmol)

in DMF (2 mL) at 65 °C for 4 h. Puriﬁcation by ﬂash

chromatography (PE/EA, 10/1, v/v) aﬀorded yellow oil (36.9 mg,

95%).

The above-obtained oil (36.9 mg, 0.095 mmol) was then

deprotected following general procedure C in DCM (1 mL) using

TFA (0.1 mL). Following general procedure C, the deprotected

product was then reacted with methyl chloroformate (12 μL, 0.15

mmol) in presence of DIPEA (35 μL, 0.2 mmol) in DCM (1 mL) at

room temperature for 1 h. Further puriﬁcation by ﬂash chromatog-

raphy (PE/EA, 8/1, v/v) gave ﬁnal compound L3-6 as yellow oil (28

mg, 85% over two steps). ¹H NMR (300 MHz, CDCl₃): δ 7.88 (d, J =

8.7 Hz, 1H), 7.24−7.29 (m, 2H), 6.98−7.06 (m, 2H), 6.89 (d, J = 2.7

Hz, 1H), 6.81 (dd, J = 9.0, 3.0 Hz, 2H), 4.44 (s, 2H), 3.94 (d, J = 2.1

Hz, 2H), 3.78 (s, 3H), 2.24 (t, J = 2.1 Hz, 1H); ¹³C NMR (125 MHz,

CDCl₃): δ 163.13, 161.18, 154.11, 145.55, 133.36, 128.95, 126.30,

115.62, 114.39, 78.85, 72.80, 54.55, 52.44, 40.21; HRMS (TOF ESI)

m/z: calcd for C₁₈H₁₇ClFN₂O₂[M + H]⁺, 347.0963; found,

347.0779; HPLC purity: 99.3%.

tert-Butyl Methyl (2-Chloro-1,4-phenylene)dicarbamate (13).

Following general procedure C, compound 11 (242 mg, 1.0 mmol)

was reacted with methyl chloroformate (117 μL, 1.5 mmol) in the

presence of DIPEA (350 μL, 2.0 mmol) in DCM (10 mL).

Puriﬁcation by ﬂash chromatography (PE/EA, 8/1) gave compound

13 as yellow oil (285 mg, 95%). ¹H NMR (300 MHz, CDCl₃): δ 7.59

(s, 1H), 7.23−7.26 (d, J = 9.0 Hz, 1H), 7.09−7.12 (d, J = 9.0 Hz 1H),

6.92 (br s, 1H), 3.78 (s, 3H), 1.43 (s, 9H); MS (ESI) m/z: 301.1 [M

+ H]⁺.

Methyl (3-Chloro-4-((4-ﬂuorobenzyl) (prop-2-yn-1-yl)amino)-

phenyl)carbamate (L3-7). The above-obtained compound 13 (285

mg, 0.95 mmol) was deprotected following general procedure C in

DCM (3 mL) and TFA (0.3 mL) at room temperature for 2 h.

Puriﬁcation by ﬂash chromatography (PE/EA, 4/1) gave the

respective amine intermediate as a pale yellow solid (148 mg, 78%).

Following general procedure B, the above-obtained amine

intermediate (148 mg, 0.74 mmol) was then reacted with p-

ﬂuorobenzyl bromide (100 μL, 0.74 mmol) in the presence of

DIPEA (0.26 mL, 1.48 mmol) in DMF (5 mL) at 65 °C for 4 h.

Puriﬁcation by ﬂash chromatography (PE/EA, 8/1, 6/1, v/v) gave a

yellow solid (143 mg, 63%).

Following general procedure B, the above-obtained solid (62 mg,

0.2 mmol) was further reacted with propargyl bromide (24 μL, 0.3

mmol) in the presence of DIPEA (70 μL, 0.4 mmol) in DMF (2 mL)

at 65 °C for 4 h. Puriﬁcation by ﬂash chromatography (PE/EA, 8/1,

v/v) gave ﬁnal compound L3-7 as yellow oil (66 mg, 95%). ¹H NMR

(300 MHz, CDCl₃): δ 7.51 (d, J = 2.4 Hz, 1H), 7.39−7.43 (m, 2H),

7.24 (d, J = 8.7 Hz, 1H), 7.18 (dd, J = 8.7, 2.4 Hz, 1H), 6.97−7.04

Following general procedure B, the above-obtained solid (660 mg,

2.0 mmol) was reacted with propargyl bromide (0.24 mL, 3.0 mmol)

and DIPEA (0.7 mL, 4.0 mmol) in DMF (2 mL) at 65 °C for 4 h.

Flash chromatography (PE/EA, 10/1, v/v) gave a white solid (662

mg, 90%). ¹H NMR (300 MHz, CDCl₃): δ 7.30−7.32 (m, 2H),

7.03−7.05 (m, 2H), 6.77 (s, 1H), 6.74−6.75 (m, 2H), 4.47 (s, 2H),

3.96 (s, 2H), 2.24 (s, 3H), 2.23 (s, 1H) 1.53 (s, 9H); MS (ESI) m/z:

369.2 [M + H]⁺.

Following general procedure C, the above-obtained solid (180 mg,

0.49 mmol) was deprotected using TFA (0.4 mL) in DCM (4 mL) at

room temperature for 1 h; further following general procedure D, the

deprotected product was further reacted with methyl chloroformate

(117 μL, 1.5 mmol) in the presence of DIPEA (174 μL, 1.0 mmol) in

DCM (1 mL) at room temperature for 1 h. Flash chromatography

(PE/EA, 6/1, v/v) gave ﬁnal compound L3-8 as pale yellow oil (135

1

mg, 85%). H NMR (300 MHz, CDCl₃): δ 7.41 (br s, 1H), 7.26−

7.31 (m, 2H), 6.99−7.05 (m, 2H), 6.73−6.76 (m, 2H), 6.23 (s, 1H),

4.46 (s, 2H), 3.96 (d, J = 2.1 Hz, 2H), 3.76 (s, 3H), 2.22−2.24 (m,

4H); ¹³C NMR (75 MHz, CDCl₃): δ 163.05, 161.10, 146.3, 133.96,

128.93, 127.14, 124.32, 116.66, 115.50, 113.18, 79.40, 72.38, 54.52,

52.35, 40.08, 18.23; HRMS (TOF ESI) m/z: calcd for C₁₉H₂₀FN₂O₂

[M + H]⁺, 327.1509; found, 327.1503; HPLC purity: 99.0%.

tert-Butyl (4-((4-Fluorobenzyl)amino)-3-methylphenyl)-

carbamate (15). Following general procedure E, 3-methyl-4-nitro-

aniline 5f (1.52 g, 10.0 mmol) was reacted in DCM (40 mL) with

Boc₂O (2.18 g, 10.0 mmol) in the presence of TEA (1.67 mL, 12.0

mmol) and DMAP (0.122 g, 1.0 mmol) to give the N-Boc-protected

intermediate as a pale yellow solid after ﬂash chromatography (PE/

EA, 10/1, v/v) (1.9 g, 75%).

Following method A of general procedure F, the above-obtained

solid was hydrogenated in EA (40 mL) using 10% Pd−C (75 mg) to

give the respective amine in a quantitative yield.

Following general procedure A, the above-obtained amine (1.62 g,

7.3 mmol) was condensed in toluene with p-ﬂuorobenzaldehyde (0.81

mL, 7.5 mmol) catalyzed by p-TsOH (32 mg, 0.19 mmol), followed

by reduction by NaBH₄(570 mg, 15.0 mmol) in MeOH (20 mL) at

room temperature for 2 h, giving compound 15 as a yellow solid after

1

ﬂash chromatography (PE/EA, 10/1, v/v) (1.78 g, 74%). H NMR

(300 MHz, CDCl₃): δ 7.31−7.35 (m, 2H), 7.15 (s, 1H), 6.96−7.05

(m, 3H), 6.49 (d, J = 8.1 Hz, 1H), 6.29 (s, 1H), 4.33 (s, 2H), 2.15 (s,

3H), 1.46 (s, 9H).

Methyl (4-((4-Fluorobenzyl) (prop-2-yn-1-yl)amino)-3-

methylphenyl)carbamate (L3-9). Following general procedure B,

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compound 15 (165 mg, 0.5 mmol) was reacted in DMF (2 mL) with

propargyl bromide (59 μL, 0.75 mmol) in the presence of DIPEA

(175 μL, 1.0 mmol) at 65 °C for 4 h, giving pale yellow oil after

puriﬁcation by ﬂash chromatography (PE/EA, 8/1, v/v) (165 mg,

90%).

(1 mL) at room temperature for 2 h. Following general procedure B,

the deprotected product was further reacted with p-ﬂuorobenzyl

bromide (134 μL, 1.0 mmol) and DIPEA (256 μL, 1.46 mmol) in

DMF (2 mL) at 65 °C overnight. Puriﬁcation by ﬂash

chromatography (PE/EA, 8/1, v/v) gave compound 18 as a pale

1

The above-obtained product (165 mg, 0.45 mmol) was

deprotected following general procedure C, using TFA (0.2 mL) in

DCM (2 mL) at room temperature for 1 h. Following general

procedure D, the deprotected amine was then reacted in DCM (2

mL) with methyl chloroformate (53 μL, 0.68 mmol) and DIPEA (157

μL, 0.9 mmol) at room temperature for 1 h. Further puriﬁcation by

ﬂash chromatography (PE/EA, 6/1, v/v) gave title compound L3-9 as

yellow solid (120 mg, 56% over two steps). H NMR (300 MHz,

CDCl₃): δ 7.70 (d, J = 8.7 Hz, 1H), 7.26−7.32 (m, 2H), 7.04 (t, J =

8.7 Hz, 2H), 6.82 (dd, J = 8.4, 2.7 Hz, 2H), 6.70 (d, J = 2.7 Hz, 1H),

4.27 (s, 2H), 4.21 (s, 1H), 3.78 (s, 3H).

Methyl (2-Cyano-4-((4-ﬂuorobenzyl) (prop-2-yn-1-yl)amino)-

phenyl)carbamate (L3-11). Following general procedure B, com-

pound 18 (120 mg, 0.40 mmol) was reacted with propargyl bromide

(48 μL, 0.62 mmmol) and DIPEA (141 μL, 0.81 mmol) in DMF (2

mL) at 65 °C for 3 h. Puriﬁcation by ﬂash chromatography (PE/EA,

10/1, v/v) gave title compound L3-11 as pale yellow oil (85 mg,

1

a pale yellow solid (122 mg, 83%). H NMR (300 MHz, CDCl₃): δ

7.35 (dd, J = 8.4, 5.7 Hz, 2H), 7.17−7.26 (m, 3H), 6.97−7.03 (m,

2H), 6.75 (s, 1H), 4.15 (s, 2H), 3.76 (s, 3H), 3.56 (d, J = 2.1 Hz,

2H), 2.33 (s, 3H), 2.25 (t, J = 2.1 Hz, 1H); ¹³C NMR (125 MHz,

CDCl₃): δ 163.04, 161.10, 154.15, 145.08, 134.80, 133.98, 130.38,

123.34, 115.22, 79.14, 73.27, 55.53, 52.29, 42.09, 18.08; HRMS (TOF

ESI) m/z: calcd for C₁₉H₂₀FN₂O₂[M + H]⁺, 327.1509; found,

327.1502; HPLC purity: 95.2%.

Methyl (4-(Prop-2-yn-1-ylamino)-2-(triﬂuoromethyl)phenyl)-

carbamate (17). Following method A of general procedure F, 4-

nitro-2-(triﬂuoromethyl)aniline 5g (1.03 g, 5.0 mmol) was hydro-

genated in EtOAc (30 mL) using 10% Pd/C (38 mg) as the catalyst

to give amine product 2-(triﬂuoromethyl)benzene-1,4-diamine in a

quantitative yield.

Following general procedure E, above-obtained 2-

(triﬂuoromethyl)benzene-1,4-diamine was reacted in DCM (40

mL) with Boc₂O (1.10 g, 5.0 mmol) in presence of TEA (0.84 mL,

6.0 mmol), and DMAP (61 mg, 0.5 mmol) at room temperature

overnight. Puriﬁcation by ﬂash chromatography (PE/EA, 10/1, v/v)

gave the product as a yellow solid (480 mg, 35%).

Following general procedure D, the above-obtained product (100

mg, 0.36 mmol) was reacted with methyl chloroformate (37 μL, 0.48

mmol) and DIPEA (128 μL, 0.72 mmol) in DCM (2 mL) at room

temperature for 1 h, giving compound 16a as a white solid after

puriﬁcation by ﬂash chromatography (PE/EA, 6/1, v/v) (88 mg,

73%).

1

63%). H NMR (300 MHz, CDCl₃): δ 7.70 (d, J = 2.1 Hz, 1H),

7.42−7.52 (m, 3H), 7.31 (d, J = 9.0 Hz, 1H), 7.02 (t, J = 8.7 Hz, 2H),

6.68 (s, 1H), 4.34 (s, 2H), 3.83 (d, J = 2.1 Hz, 2H), 3.78 (s, 3H), 2.31

(t, J = 2.1 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 162.69, 161.06,

153.37, 148.94, 132.02, 129.99, 122.52, 117.09, 115.04, 108.22, 77.58,

73.93, 55.16, 52.22, 41.64; HRMS (TOF ERI) m/z: calcd for

C₁₉H₁₆FN₃NaO₂[M + Na]⁺: 360.1124; found, 360.1114; HPLC

purity: 98.7%.

tert-Butyl (3-Cyano-4-((4-ﬂuorobenzyl) (prop-2-yn-1-yl)amino)-

phenyl)carbamate (20). Following method A of general procedure F

and general procedure E, respectively, starting material 2-amino-5-

nitrobenzonitrile 5i (820 mg, 5.0 mmol) was hydrogenated in EA (30

mL) using 10% Pd/C (38 mg) as a catalyst to give 2,5-

diaminobenzonitrile in a quantitative yield, which was further reacted

in DCM (40 mL) with Boc₂O (1.10 g, 5.0 mmol), TEA (0.84 mL, 6.0

mmol), and DMAP (61 mg, 0.5 mmol) at room temperature

overnight. Puriﬁcation by ﬂash chromatography (PE/EA, 10/1, v/v)

gave compound 19 as a pale yellow solid (0.87 g, 75% over two

steps).

Following general procedure A, the above-obtained compound 19

(200 mg, 0.86 mmol) was condensed with p-ﬂuorobenzaldehyde

(0.18 mL, 1.68 mmol) in toluene (10 mL) catalyzed by p-TsOH (3.7

mg, 0.02 mmol), followed by reduction by NaBH₄(98 mg, 2.58

mmol) in MeOH (10 mL). Puriﬁcation by ﬂash chromatography

(PE/EA, 8/1, 6/1, v/v) gave a yellow solid (243 mg, 83%).

Following general procedure B, the above-obtained solid (243 mg,

0.71 mmol) was reacted with propargyl bromide (85 μL, 1.08 mmol)

and DIPEA (253 μL, 1.45 mmol) in DMF (2 mL) at 65 °C for 3 h.

Flash chromatography (PE/EA, 10/1, v/v) gave title compound 20 as

Following general procedure C, the above-obtained compound 16a

(88 mg, 0.26 mmol) was deprotected with TFA (0.1 mL) in DCM (1

mL) at room temperature for 2 h. Further following general

procedure B, the deprotected product was then reacted with propargyl

bromide (32 μL, 0.40 mmol) and DIPEA (94.0 μL, 0.53 mmol) in

DMF (2 mL) at 65 °C for 3 h. Puriﬁcation by ﬂash chromatography

(PE/EA, 10/1, v/v) gave compound 17 as pale yellow oil (45 mg,

1

pale yellow oil (195 mg, 73%). H NMR (300 MHz, CDCl₃): δ 7.71

1

63% over two steps). H NMR (300 MHz, CDCl₃): δ 7.70 (s, 1H),

(d, J = 2.7 Hz, 1H), 7.42−7.47 (m, 2H), 7.26−7.33 (m, 2H), 7.04 (t,

J = 8.4 Hz, 2H), 6.54 (s, 1H), 4.32 (s, 2H), 3.81 (d, J = 2.1 Hz, 2H),

3.77 (s, 3H), 2.27 (t, J = 2.1 Hz, 1H), 1.51 (s, 9H).

6.82−6.88 (m, 2H), 6.55 (s, 1H), 4.03 (s, 2H), 3.95 (d, J = 2.1 Hz,

2H), 3.77 (s, 3H), 2.23 (t, J = 2.1 Hz, 1H).

Methyl (4-((4-Fluorobenzyl) (prop-2-yn-1-yl)amino)-2-

(triﬂuoromethyl)phenyl)carbamate (L3-10). Following general

procedure B, compound 17 (45 mg, 0.17 mmol) was reacted with

p-ﬂuorobenzyl bromide (36 μL, 0.27 mmmol) and DIPEA (57μL,

0.33 mmol) in DMF (2 mL) at 65 °C for 3 h. Puriﬁcation by ﬂash

chromatography (PE/EA, 10/1, v/v) gave title compound L3-10 as

Methyl (3-Cyano-4-((4-ﬂuorobenzyl) (prop-2-yn-1-yl)amino)-

phenyl)carbamate (L3-12). Following general procedure C, com-

pound 20 (195 mg, 0.51 mmol) was deprotected using TFA (0.1 mL)

in DCM (1 mL) at room temperature for 2 h. Following general

procedure D, the deprotected amine was then reacted in DCM (2

mL) with methyl chloroformate (60 μL, 0.78 mmol) and DIPEA (185

μL, 1.06 mmol) at room temperature for 1 h. Further puriﬁcation by

ﬂash chromatography (PE/EA, 6/1, v/v) gave title compound L3-12

1

pale yellow oil (45 mg, 71%). H NMR (300 MHz, CDCl₃): δ 7.73

(d, J = 7.5 Hz, 1H), 7.25−7.29 (m, 2H), 6.99−7.08 (m, 4H), 6.58 (s,

1H), 4.50 (s, 2H), 4.00 (d, J = 2.1 Hz, 2H), 3.68 (s, 3H), 2.54 (t, J =

2.1 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 163.43, 160.99, 154.69,

145.45, 133.16, 128.88, 126.11, 125.28, 122.56, 118.48, 115.75,

111.65, 78.62, 72.97, 54.61, 52.59, 40.23; HRMS (TOF ESI) m/z:

calcd for C₁₉H₁₇F₄N₂O₂[M + H]⁺, 381.1226; found, 381.1221;

HPLC purity: 99.5%.

Methyl (2-Cyano-4-((4-ﬂuorobenzyl)amino)phenyl)carbamate

(18). Following procedures similar to that described to prepare

compound 16a, starting from 2-amino-5-nitrobenzonitrile 5h (820

mg, 5.0 mmol), compound 16b was prepared as a yellow solid after

puriﬁcation by ﬂash chromatography (PE/EA, 6/1, v/v) (210 mg,

63% over three steps).

1

as a white solid (163 mg, 94% over two steps). H NMR (300 MHz,

CDCl₃): δ 7.92 (d, J = 8.7 Hz, 1H), 7.23−7.28 (m, 2H), 7.10 (dd, J =

9.3, 3.0 Hz, 1H), 6.99−7.06 (m, 3H), 6.86 (s, 1H), 4.47 (s, 2H), 3.98

(d, J = 2.1 Hz, 2H), 3.79 (s, 3H), 2.27 (t, J = 2.1 Hz, 1H); ¹³C NMR

(125 MHz, CDCl₃): δ 162.61, 160.98, 153.43, 144.31, 132.34, 128.33,

120.29, 116.47, 115.34, 77.91, 72.71, 54.20, 52.29, 39.88; HRMS

(TOF ESI) m/z: calcd for C₁₉H₁₆FN₃NaO₂[M + Na]⁺: 360.1124;

found, 360.1116; HPLC purity: 98.5%.

Methyl (4-((4-Fluorobenzyl) (prop-2-yn-1-yl)amino)-2-

(triﬂuoromethoxy)phenyl)carbamate (L3-13). Following general

procedure E, 4-bromo-2-(triﬂuoromethoxy)aniline 5j (1.28 g, 5.0

mmol) was reacted DCM (40 mL) with Boc₂O (2.4 g, 11.0 mmol),

TEA (0.84 mL, 6.0 mmol) and DMAP (0.73 g, 6.0 mmol) at room

Following general procedure C, the above-obtained compound 16b

(210 mg, 0.72 mmol) was deprotected with TFA (0.1 mL) in DCM

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temperature overnight. Puriﬁcation by ﬂash chromatography (PE/EA,

15/1, v/v) gave compound 21 as a white solid (1.48 g, 65%).

Under a nitrogen atmosphere, to a mixture of compound 21 (0.91

g, 2.0 mmol), p-ﬂuorobenzyl amine (344 μL, 3.0 mmol), Cs₂CO₃(1.3

g, 4.0 mmol), and BINAP (62 mg, 0.1 mmol) in dry toluene (20 mL)

was added Pd (dba)₂(92 mg, 10.1 mmol) quickly. The resulting

mixture was heated to reﬂux for 6 h, cooled to room temperature, and

added EA (30 mL). Then, the mixture was washed with water, brine,

dried over anhydrous Na₂SO₄, ﬁltered, and concentrated in vacuo.

The obtained residue was puriﬁed by ﬂash chromatography (PE/EA,

10/1, v/v) to give compound 22 as a yellow solid (730 mg, 73%). ¹H

NMR (300 MHz, CDCl₃): δ 7.32 (d, J = 8.7 Hz, 2H), 7.03 (t, J = 8.7

Hz, 2H), 6.96 (d, J = 8.7 Hz, 1H), 6.45−6.49 (m, 3H), 4.28 (d, J =

5.4 Hz, 2H), 4.22 (t, J = 5.4 Hz, 1H), 1.4 (s, 18H); MS (ESI) m/z:

500.2 [M + H]⁺.

160.60, 155.67, 147.75, 137.12, 134.15, 128.99, 125.13, 115.74,

114.11, 798.66, 72.49, 54.43, 52.54, 39.90, 18.94; HRMS (TOF ESI)

m/z: calcd for C₂₀H₂₂FN₂O₂[M + H]⁺, 341.1665; found, 341.1656;

HPLC purity: 99.5%.

Methyl (4-((4-Fluorobenzyl) (prop-2-yn-1-yl)amino)-3,5-

dimethylphenyl)carbamate (L3-15). Following general procedure

D, compound 23 (336 mg, 1.0 mmol) was reacted with methyl

chloroformate (117 μL, 1.5 mmol) and DIPEA (350 μL, 2.0 mmol) in

DCM (1 mL) at room temperature for 1 h, giving intermediate 25 as

pale yellow oil after puriﬁcation by ﬂash chromatography (PE/EA, 8/

1, v/v) (374 mg, 95%).

Following general procedure C, intermediate 25 (374 mg, 0.95

mmol) was deprotected using TFA (0.2 mL) in DCM (2 mL) at

room temperature for 2 h. The deprotected product was then reacted

with p-ﬂuorobenzyl bromide (128 μL, 0.95 mmol) and DIPEA (0.34

mL, 2.0 mmol) in DMF (3 mL) at 65 °C for 4 h. Puriﬁcation by ﬂash

chromatography (PE/EA, 10/1, v/v) gave compound 26 as pale

yellow oil (192 mg, 67%). ¹H NMR (300 MHz, CDCl₃): δ 7.78 (br s,

1H), 7.26 (m, 1H), 7.02 (t, J = 8.4 Hz, 2H), 6.64 (m, 2H), 6.51 (br s,

1H), 4.45 (s, 2H), 3.95 (d, J = 2.1 Hz, 2H), 3.77 (s, 3H), 2.24 (t, J =

2.1 Hz, 1H).

Following general procedure B, compound 22 (250 mg, 0.5 mmol)

was reacted with propargyl bromide (58 μL, 0.75 mmol) and DIPEA

(177 μL, 1.0 mmol) in DMF (2 mL) at 65 °C for 3 h, giving a white

solid after puriﬁcation by ﬂash chromatography (PE/EA, 10/1, v/v)

(223 mg, 83%).

Following general procedure C, the above-obtained solid (223 mg,

0.41 mmol) was deprotected using TFA (0.2 mL) in DCM (2 mL) at

room temperature for 2 h. The deprotected amine was then reacted in

DCM (2 mL) using methyl chloroformate (48 μL, 0.62 mmol) and

DIPEA (150 μL, 0.86 mmol) at room temperature for 1 h, following

general procedure D. Further puriﬁcation by ﬂash chromatography

(PE/EA, 8/1, v/v) gave ﬁnal compound L3-13 as a white solid (150

mg, 92% over two steps). ¹H NMR (300 MHz, CDCl₃): δ 7.92 (br s,

1H), 7.25−7.30 (m, 2H), 7.03 (d, J = 8.7 Hz, 2H), 6.81 (dd, J = 8.7,

2.7 Hz, 1H), 6.77 (s, 1H), 6.63 (s, 1H), 4.46 (s, 2H), 3.96 (d, J = 2.1

Hz, 2H), 3.78 (s, 3H), 2.26 (t, J = 2.1 Hz, 1H); ¹³C NMR (125 MHz,

CDCl₃): δ 163.15, 161.20, 154.07, 133.25, 128.89, 121.93, 119.52,

115.65, 113.87, 107.66, 78.66, 72.85, 54.78, 52.49, 40.38; HRMS

(TOF ESI) m/z: calcd for C₁₉H₁₇F₄N₂O₃[M + H]⁺, 397.1175, found,

397.1169; HPLC purity: 99.7%.

Methyl (4-((4-Fluorobenzyl) (prop-2-yn-1-yl)amino)-2,6-

dimethylphenyl)carbamate (L3-14). Following general procedure

E, 2,6-dimethyl-4-nitroaniline 5k (0.98 g, 5.9 mmol) was reacted with

Boc₂O (2.58 g, 11.8 mmol), TEA (1.77 mL, 12.7 mmol), and DMAP

(722 mg, 5.9 mmol) in DCM (40 mL) at room temperature

overnight. Puriﬁcation by ﬂash chromatography (PE/EA, 6/1, v/v)

gave the protected product as a pale yellow solid (1.84 g, 85%).

Following method a of general procedure F, the above-obtained

solid (1.84 g, 5.0 mmol) was hydrogenated in EA (20 mL) using 10%

Pd/C (55 mg) as a catalyst, giving compound 23 in a quantitative

yield. ¹H NMR (300 MHz, CDCl₃): δ 6.38 (s, 1H), 3.52 (s, 2H), 2.05

(s, 6H), 1.39 (s, 18H).

Following general procedure A, the above-obtained compound 23

(336 mg, 1.0 mmol) was condensed with p-ﬂuorobenzaldehyde (108

μL, 1.0 mmol) in toluene (10 mL) catalyzed by p-TsOH (4.3 mg,

0.025 mmol) after reﬂux for 3 h, followed by reduction by NaBH₄(76

mg, 2.0 mmol) in MeOH (20 mL) at room temperature for 2 h,

giving the product as a yellow solid after ﬂash chromatography (PE/

EA, 10/1, v/v) (320 mg, 72%).

Following general procedure B, the above-obtained compound

(320 mg, 0.72 mmol) was reacted with propargyl bromide (84 μL,

1.08 mmol) and DIPEA (257 μL, 1.47 mmol) in DMF (5 mL) at 65

°C for 4 h. Puriﬁcation by ﬂash chromatography (PE/EA, 15/1, v/v)

gave intermediate 24 (312 mg, 90%).

Following general procedure B, compound 26 (192 mg, 0.64

mmol) was further reacted with propargyl bromide (50 μL, 0.64

mmol) and DIPEA (226 μL, 1.3 mmol) in DMF (3 mL) at 65 °C for

4 h. Puriﬁcation by ﬂash chromatography (PE/EA, 15/1, v/v) gave

1

compound L3-15 as a white solid (165 mg, 76%). H NMR (300

MHz, CDCl₃): δ 7.36 (d, J = 8.7 Hz, 2H), 7.04 (s, 2H), 6.91 (t, J =

9.0 Hz, 3H), 4.48 (s, 2H), 4.20 (s, 2H), 3.75 (s, 3H), 2.23−2.38 (m,

7H); ¹³C NMR (125 MHz, CDCl₃): δ 163.27, 160.83, 154.16,

143.39, 138.10, 135.05, 130.47, 119.19, 115.18, 114.97, 81.52, 72.02,

56.06, 52.29, 41.21, 19.92; HRMS (TOF ESI) m/z: calcd for

C₂₀H₂₂FN₂O₂[M + H]⁺, 341.1665; found, 341.1659; HPLC purity:

95.1%.

Plasmid Construction. The plasmids encoding human KCNQ1,

rat KCNQ2, rat KCNQ3, human KCNQ4, and human KCNQ5 were

gifts from Drs. T. Jentsch (Zentrum fur Molekulare Neurobiologie,

̈

Hamburg, Germany), D. MacKinnon (State University of New York,

Stony Brook, NY), M. Sanguinetti (University of Utah, Salt Lake City,

UT), M. Shapiro (University of Texas Health Science Center, San

Antonio, TX), and Kenneth L. Byron (Loyola University, Chicago,

IL), respectively. In addition, the EGFP were oﬀered by Haijun Chen

(State University of New York, Stony Brook, NY). A278T mutation

of rKCNQ3 (AF091247) were introduced using the QuikChange II

site-directed mutagenesis kit (Stratagene).

Cell Culture and Transient Transfection. Chinese hamster

ovary (CHO) cells were cultured in DMEM/F12 (Gibco, Life

Technologies, Carlsbad, CA, USA) with 10% FBS. 24 h prior to

transfection, cells were split into 6-well dishes. Plasmids of EGFP and

KCNQ channels were cotransfected with Lipofectamine 3000 reagent

(Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the

manufacturer’s instructions. Moreover, to express KCNQ2/KCNQ3

channels, the cDNA ratio transfected is 1:1.

Electrophysiology. A standard whole-cell recording was

performed at room temperature in CHO cells with an Axopathca-

200B ampliﬁer (Molecular Devices, Sunnyvale, CA). The pipettes

were pulled from borosilicate glass capillaries (World Precision

Instruments, Sarasota, FL) and had resistances of 3−5 MΩ when

ﬁlled with the intracellular solution. The holding potential is −80 mV,

followed by a series of depolarization steps from −90 to +60 mV with

a 10 mV increment, the currents were measured at the end of

depolarization to −10 mV to determine I/I₀, and the tail currents

elicited at −120 mV were measured to obtain ΔV_1/2. The intracellular

solution contained 145 mM KCl, 1 mM MgCl₂, 5 mM EGTA, 10 mM

HEPES and 5mM MgATP (pH = 7.3), and the extracellular solution

contained 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1.5 mM MgCl₂,

10 mM Glucose (pH = 7.4), and 10 mM HEPES. During the

recording, constant perfusion of the extracellular solution was

maintained using a bath perfusion system (ALA Scientiﬁc Instru-

ments, NY). The electrical signals were ﬁltered at 1 kHz and digitized

using a DigiData 1322A with pClamp 9.2 software (Molecular

Following general procedure C, intermediate 24 (48 mg, 0.1

mmol) was deprotected using TFA (0.1 mL) in DCM (1 mL) at

room temperature for 2 h. The deprotected product was then reacted

in DCM (1 mL) with methyl chloroformate (11.7 μL, 0.15 mmol)

and DIPEA (35 μL, 0.20 mmol) at room temperature for 1 h,

following general procedure D. Further puriﬁcation by ﬂash

chromatography (PE/EA, 8/1, v/v) gave ﬁnal compound L3-14 as

a white solid (31 mg, 91%).¹H NMR (300 MHz, CDCl₃): δ 7.27 (dd,

J = 8.4, 5.4 Hz, 2H), 7.02 (t, J = 9.0 Hz, 2H), 6.63 (s, 2H), 5.95 (br s,

1H), 4.49 (s, 2H), 3.98 (d, J = 2.1 Hz, 2H), 3.74 (br s, 3H), 2.25 (t, J

= 2.1 Hz, 1H), 2.21 (s, 6H); ¹³C NMR (125 MHz, CDCl₃): δ 163.85,

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Devices, Sunnyvale, CA). Series resistance was compensated by 60−

80%.

the time-dependent inhibition potential of HN37 on cytochrome

P450s. Human liver microsomes was pre-incubated with HN37 at

various concentrations (0.10−50 μM) for 0.5 h with the presence or

absence of NADPH, followed by the addition of speciﬁc CYP450

probe substrates and further incubation. An LC−MS/MS method was

used to determine the concentration of each speciﬁc metabolite and

the value of IC₅₀was calculated to indicate the potential of TDI of

HN37 toward major CYP450. The percent of enzyme activity

remaining after incubation with HN37 is calculated by the following

equations

Patch Clamp Data Analysis. Patch clamp data were processed

using Clampﬁt 10.2 (Molecular Devices, Sunnyvale, CA, USA) and

then analyzed in GraphPad Prism 8 (GraphPad Software, San Diego,

CA, USA). Voltage-dependent activation curves were ﬁtted using the

Boltzmann equation, G = G _min+ (G_max− G_min)/(1 + exp (V−V_1/2)/

S), where G_maxis the maximum conductance, G_minis the minimum

conductance, V_1/2is the voltage for reaching 50% of maximum

conductance, and S is the slope factor. Dose−response curves were

ﬁtted with the Hill equation, E = E_max/(1 − (EC₅₀/C) P), where EC₅₀

is the drug concentration producing half of the maximum response

and P is the Hill coeﬃcient. The data are presented as the mean

SEM.

Use of Animals. In the present study, healthy male Chinese Kun

Ming (KM) mice (18−24 g) from Beijing HFK Bioscience (Beijing,

China), male C57BL/6 mice (20−25 g) obtained from Shanghai Slac

Laboratory Animal (Shanghai, China), and healthy male Sprague

Dawley (SD) rats (180−220 g) provided by Shanghai Sippr-Bk

Laboratory Animal (Shanghai, China) were used. Animals were group

housed in a temperature (22−24 °C) and relative humidity (50−

70%) controlled facility and maintained on a constant 12 h light/dark

cycle with free access to food and water. This study was approved by

the Animal Care and Use Committee of Shanghai Institute of Materia

Medica, Chinese Academy of Sciences (IACUC Numbers: 2016-04-

JHL-13 and 2020-02-GZB-05) and performed in compliance with the

National Institutes of Health Guide for the Care and Use of

Laboratory Animals.

% activity remaining (at appointed conc. )

metabolite conc. at appointed conc. of test compound

=

metabolite conc. without test compound

× 100%

The concentration of HN37 inhibited the metabolism of the

respective P450 probe substrates by 50% was determined as

−

+

IC_50NADPH/IC_50NADPHcalculated using the Probit model by Graph-

Pad Prism 8.0.1 software.

Anticonvulsant Eﬃcacy. The anticonvulsant eﬃcacy of com-

pound HN37 was evaluated in MES, sc-PTZ, and 6 Hz tests. All test

compounds including retigabine, topiramate, and levetiracetam as

references, were freshly suspended in 0.5% CMC-Na and adminis-

tered intragastrically to mice in a volume of 20 mL/kg body weight

and in a volume of 10 mL/kg body weight orally to rats 0.5 or 1 h

before the seizure tests. The pentylenetetrazol solution was prepared

freshly and dissolved in normal saline.

Pharmacokinetic Studies. PK studies were performed in KM

mice. The mice were fasted for 12 h before and 2 h after drug

administration. The test compound HN37 was prepared for an oral

formulation using 5% DMSO/5% Tween 80/90% normal saline, and

the oral formulation was diluted with normal saline containing 1%

Tween 80 to obtain the intravenous formulation. Mice (n = 3 per time

point) were given HN37 orally at 5 mg/kg or intravenously at 2 mg/

kg. Blood samples (0.5 mL) were collected at 0.25, 0.5, 1, 2, 4, 6, 8,

and 24 h from the abdominal aorta into the heparinized tubes after

oral dosing, or blood samples (0.2 mL) were collected from the

posterior venous plexus of the eyeball at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8,

and 24 h after iv injection. The plasma was separated by

centrifugation (11,000 rpm for 5 min) and then stored at −80 °C

until analyzed. Mice treated orally were sacriﬁced to obtain the whole

brain tissue which was also stored at −80 °C. Plasma samples (20 μL)

were treated with the addition of the internal standard solution (20

μL) and acetonitrile (300 μL), then vortexed for 15 min (1000 rpm,

RT), and centrifuged of 15 min (3700 rpm, 4 °C). The supernatants

were collected for analysis. The whole brain samples were

homogenized in acetonitrile with a 10-fold volume to tissue weight

(8000 rpm, 3 × 30 s), then was sonicated for 5 min. The obtained

homogenate was then treated as described for the plasma samples.

The plasma and brain levels of compound HN37 were analyzed using

LC−MS/MS. The data were processed using Analyst software version

1.6.3 (Sciex, Ontario, Canada). PK studies of HN37 were also carried

out in SD rats at the oral doses of 2, 6, and 20 mg/kg, and the

procedure was performed similarly as in the mice.

To examine the plasma and brain exposures after the oral

administration of compound HN37 and RTG, the separate PK

studies were performed in the MES test and rotarod test in KM mice.

Upon the completion of the MES test or rotarod test, mice treated

with HN37 and RTG were sacriﬁced to collect brain and plasma

samples, and the brain and plasma concentrations of compound

HN37 were measured as described above. For compound RTG, the

brain samples were processed and analyzed the same as HN37, and

the plasma samples were slightly diﬀerent. After centrifugation with

the internal standard solution and acetonitrile for plasma samples, the

supernatants (20 μL) were added into 1:1 acetonitrile/water (v/v) for

15 min of vertexing to obtain a mixture for analysis by LC−MS/MS.

Time-Dependent Inhibition Potential Assay. To determine

whether HN37 was a time-dependent inhibitor of major human

hepatic CYP enzymes, human liver microsomes were used to evaluate

Maximal Electroshock Seizure Test in mice and Rats. In this

anticonvulsant screening test, the mouse test procedure was described

in our previous papers,^41,58and the rat test was performed partly

according to the method introduced by Swinyard.⁵⁹In the mouse test,

a stimulus intensity of 160 V was applied through ear clip electrodes

for 5.4 s using an electronic stimulator (YLS-9A, Bio-will, Shanghai,

China). In the rat test, the seizures were induced by an electronic

stimulator (EC-02, Orchid Scientiﬁcs, Maharashtra, India) by the

delivery of a 200 mA current to the cornea for 0.2 s. Both

electroshock applications caused immediate hindlimb tonic extension.

The test compound which showed a complete suppression of the

hindlimb tonic seizure was thought to have a protective eﬀect in the

MES test. Compound HN37 and the reference drugs were

determined by the anticonvulsant quantiﬁcation study. Eight to ten

KM mice or SD rats were used per group per dose, and 5−6 doses

were used to establish the median eﬀective dose (ED₅₀), which were

calculated by nonlinear regression using Graph Pad Prism 5. After

completion of the test, all mice that were treated with HN37 and

RTG were sacriﬁced immediately, and the brain and plasma sample

collection, processing, and analysis, were performed according to the

above methods in the PK studies in mice.

Subcutaneous Pentylenetetrazol Test in Mice. PTZ was injected

subcutaneously to KM mice at a dose of 100 mg/kg in a volume of 10

mL/kg body weight, and this dose induced clonic and tonic seizures

ﬁrst, and then led to death in mice in our previous study.^56,60The

number and the latency of clonic seizures, tonic seizures, and death

were recorded within 1 h after PTZ injection, respectively. The mice

not displaying both clonic and tonic seizures were considered to be

protected. Ten mice were used per group per dose. Compound HN37

and reference drugs were evaluated in this test and the ED₅₀

calculation was the same with the MES test.

6 Hz Psychomotor Seizure Test in Mice. 6 Hz psychomotor

seizure was considered as the only acute model of drug-resistant

epilepsy,⁶¹and this test was performed as previously described.^56,60

The C57BL/6 mice were chosen and were stimulated through corneal

electrodes using a Grass stimulator (S48, Grass technologies, USA)

connected to a constant current unit (CCU1, Grass technologies,

USA). Focal seizures were induced by delivering a stimulus of 32 or

44 mA current (6 Hz, 0.2 ms rectangular pulse width) for 3 s

duration. The mice exhibited immobility associated with rearing,

automatisms, forelimb clonus, twitching of the vibrissae, and Straub

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Article

tail for 10−50 s duration following the electrical stimulation. The

mice resuming normal behavior within 7 s from the stimulation were

considered protected. There were ten mice per group per dose. The

determination of ED₅₀values for HN37 and reference drugs was

performed as above.

Laboratory of Drug Research, Shanghai Institute of Materia

Medica, Chinese Academy of Sciences, Shanghai 201203,

China

Hai-Ning Hu − Chinese National Center for Drug Screening,

CAS Key Laboratory of Receptor Research, State Key

Laboratory of Drug Research, Shanghai Institute of Materia

Medica, Chinese Academy of Sciences, Shanghai 201203,

China

Fu-Yun Tian − Chinese National Center for Drug Screening,

CAS Key Laboratory of Receptor Research, State Key

Laboratory of Drug Research, Shanghai Institute of Materia

Medica, Chinese Academy of Sciences, Shanghai 201203,

China

Fei Chen − Chinese National Center for Drug Screening, CAS

Key Laboratory of Receptor Research, State Key Laboratory

of Drug Research, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, Shanghai 201203, China

Hua-Nan Liu − Chinese National Center for Drug Screening,

CAS Key Laboratory of Receptor Research, State Key

Laboratory of Drug Research, Shanghai Institute of Materia

Medica, Chinese Academy of Sciences, Shanghai 201203,

China

Li Zhan − Chinese National Center for Drug Screening, CAS

Key Laboratory of Receptor Research, State Key Laboratory

of Drug Research, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, Shanghai 201203, China

Xiao-Ping Pi − Chinese National Center for Drug Screening,

CAS Key Laboratory of Receptor Research, State Key

Laboratory of Drug Research, Shanghai Institute of Materia

Medica, Chinese Academy of Sciences, Shanghai 201203,

China

Neurotoxicity in Mice. To assess the neurotoxicity eﬀects for

compound HN37 and reference drugs, the standardized rotarod test

was performed in KM or C57BL/6 mice. The mice were placed on a

rotarod appliance (YLS-4C, Bio-will, Shanghai, China) with a rod of 3

cm diameter, rotating at a constant speed of 6 rpm. The day before

the compound test, all the mice were pre-trained and only the animals

able to remain on the rod for at least 1 min every time in three

consecutive trials (3 min) were retained. On the test day, the mice

were measured in the rotarod test at 1 h after oral gavage

administration of HN37 and reference drugs, respectively. The

animal unable to remain on the rod for 3 consecutive periods was

considered motor coordination impaired. Six to ten mice were used

per group per dose, and 4−6 doses were used to establish the median

neurotoxic dose (TD₅₀) determined by nonlinear regression using

Graph Pad Prism 5. To determine the safety margin for HN37, the

plasma and brain exposures of HN37 and RTG were also detected as

described in PK studies following the ﬁnishing of the test.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02252.

■

sı

Additional information for the CYP inhibition study and

TDI assessment, time eﬀect of HN37 in the mouse MES

test, quantitative anticonvulsant eﬀects of HN37 and

RTG in rat MES test, and NMR and HPLC spectra for

ﬁnal compounds (PDF)

Molecular formula strings for all the ﬁnal compounds

(CSV).

Jie Liu − Hainan Haiyao Company Ltd., Haikou City, Hainan

570311, China

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c02252

AUTHOR INFORMATION

Corresponding Authors

■

Zhao-Bing Gao − Chinese National Center for Drug

Screening, CAS Key Laboratory of Receptor Research, State

Key Laboratory of Drug Research, Shanghai Institute of

Materia Medica, Chinese Academy of Sciences, Shanghai

201203, China; School of Chinese Materia Medica, Nanjing

University of Chinese Medicine, Nanjing City, Jiangsu

210023, China; Email: zbgao@simm.ac.cn

Fa-Jun Nan − Chinese National Center for Drug Screening,

CAS Key Laboratory of Receptor Research, State Key

Laboratory of Drug Research, Shanghai Institute of Materia

Medica, Chinese Academy of Sciences, Shanghai 201203,

China; Yantai Key Laboratory of Nanomedicine & Advanced

Preparations, Yantai Institute of Materia Medica, Yantai

City, Shandong 264000, China; orcid.org/0000-0002-

2284-3637; Email: fjnan@simm.ac.cn

Author Contributions

^⊥Y.-M.Z., H.-Y.X., and H.-N.H contributed equally.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by grants from The National Natural

Science Foundation of China (81825021), the National

Science and Technology Major Project (2013ZX09103-001-

016 and 2018ZX09711002-013-002), Chinese Academy of

Sciences (XDA12040211 and CASIMM0120151001), Minis-

try of Science and Technology of China (2013CB910604), and

Shanghai Science and Technology Committee (16431901700

and 19431906000). The authors are grateful to J-L Zhou for

TDI assessment and discussion. The authors thank Y-F Zhang

and Z-W Gao for their suggestions and discussions about the

PK study.

Authors

Yang-Ming Zhang − Chinese National Center for Drug

Screening, CAS Key Laboratory of Receptor Research, State

Key Laboratory of Drug Research, Shanghai Institute of

Materia Medica, Chinese Academy of Sciences, Shanghai

201203, China; Yantai Key Laboratory of Nanomedicine &

Advanced Preparations, Yantai Institute of Materia Medica,

Yantai City, Shandong 264000, China

Hai-Yan Xu − School of Chinese Materia Medica, Nanjing

University of Chinese Medicine, Nanjing City, Jiangsu

210023, China; Chinese National Center for Drug Screening,

CAS Key Laboratory of Receptor Research, State Key

ABBREVIATIONS

■

AED, antiepileptic drug; P-RTG, ethyl N-[2-amino-4-((4-

ﬂuorobenzyl) (prop-2-ynyl)amino)phenyl] carbamate; CNS,

central nervous system; Kv, voltage-gated potassium channel;

EC₅₀, median eﬀective concentration; IC₅₀, half-maximal

inhibitory concentration; ED₅₀, median eﬀective dose; TD₅₀,

median neurotoxic dose; DCM, dichloromethane; DIPEA,

N,N-diisopropylethylamine; DMF, N,N-dimethylformamide;

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THF, tetrahydrofuran; TFA, triﬂuoroacetic acid; TEA, triethyl

amine; DMAP, 4-dimethylaminopyridine; MES, maximal

electroshock; scPTZ, subcutaneous pentylenetetrazol; EMA,

European Medicines Agency; FDA, Food and Drug Admin-

istration; NMPA, National Medical Products Administration

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nonclinical aspects. Epilepsy Curr. 2015, 15, 336−337. (AES 2014

Annual Meeting Online Abstract Supplement)

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https://doi.org/10.1021/acs.jmedchem.0c02252

J. Med. Chem. 2021, 64, 5816−5837

Article Doi

Discovery of HN37 as a Potent and Chemically Stable Antiepileptic Drug Candidate

DOI: 10.1021/acs.jmedchem.0c02252

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Article abstract of DOI:10.1021/acs.jmedchem.0c02252

Full text of DOI:10.1021/acs.jmedchem.0c02252

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