Development and optimization of halogenated vinyl sulfones as Nrf2 activators for the treatment of Parkinson's disease

European Journal of Medicinal Chemistry 212 (2021) 1 13103

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Development and optimization of halogenated vinyl sulfones as Nrf2

activators for the treatment of Parkinson’s disease

,

b

,

c

,

Ji Won Choi ^{a 1}, Siwon Kim ^{a 1}, Jong Seok Yoo ^{a 1}, Hyeon Jeong Kim ^{a d}, Hyeon Ji Kim ^a,

Byung Eun Kim ^{a e}, Elijah Hwejin Lee ^{a b}, Yong Sup Lee ^{c **}, Jong-Hyun Park ^a

,

e,

, ***

, b, c, *

Ki Duk Park ^a

^aConvergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology (KIST), Seoul, 02792,

Republic of Korea

^bDivision of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul, 02792, Republic of Korea

^cKHU-KIST Department of Converging Science and Technology, Kyung Hee University, Seoul, Republic of Korea

^dDepartment of Biotechnology, Yonsei University, Seoul, 03722, Republic of Korea

^eDepartment of Fundamental Pharmaceutical Sciences, Graduate School, Kyung Hee University, Seoul, 02447, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history:

The Kelch-like ECH-associated protein 1 (Keap1)-Nuclear factor erythroid 2-related factor 2 (Nrf2)

signaling pathway plays a pivotal role in the cellular defense system against oxidative stress by inducing

antioxidant and anti-inﬂammatory effects. We previously developed Nrf2 activators that potentially

protect the death of dopaminergic (DAergic) neuronal cells against oxidative stress in Parkinson’s disease

(PD). In this study, we designed and synthesized a class of halogenated vinyl sulfones by inserting

halogens and pyridine to maximize Nrf2 activation efﬁcacy. Among the synthesized compounds, (E)-3-

chloro-2-(2-((2-chlorophenyl)sulfonyl)vinyl)pyridine (9d) signiﬁcantly exhibited potent Nrf2 activating

efﬁcacy (9d: EC₅₀¼ 26 nM) at least 10-fold compared with the previous developed compounds (1 and 2).

Furthermore, treating with 9d remarkably increased Nrf2 nuclear translocation and Nrf2 protein levels in

microglial BV-2 cells. 9d was shown to induce the expression of antioxidant response genes HO-1, GCLC,

GCLM, and SOD-1 at both the mRNA and protein levels and suppress proinﬂammatory cytokines and

enzymes. Also, 9d remarkably protected DAergic neurons and restored the PD-associated motor

dysfunction in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model.

Received 7 September 2020

Received in revised form

8 December 2020

Accepted 10 December 2020

Available online 25 December 2020

Keywords:

Halogenated vinyl sulfones

Nrf2 activator

Antioxidant

Parkinson’s disease

MPTP-induced mouse model

1. Introduction

Until now, the clear cause of PD has not been widely known, but

the main pathophysiology of PD is mainly caused by the loss of

Parkinson’s disease (PD) is one of the most common neuro-

degenerative diseases characterized by rigidity, resting tremor,

bradykinesia, and postural instability in motor system [1e3].

dopaminergic (DAergic) neurons in the substantia nigra pars

compacta (SNpc) [4,5]. Currently, the mainstay strategy is by

administering drugs that mimic the action of dopamine [6,7]. A

representative therapeutic agent is dopamine precursor L-3,4-

dihydroxy phenylalanine (

mine levels. Over the years,

most effective drug for alleviating the symptoms of PD patients.

Unfortunately, long-term treatment with -DOPA induces side

effects of -DOPA-induced dyskinesia (LID) [8,9]. Another strategy

is developing therapeutic agents to prevent the death of DAergic

neurons. Oxidative stress is caused by an imbalance between the

generation of reactive oxygen species (ROS) and antioxidant

defense system, inducing damages and death of the DAergic cells

involved in dopamine production [10,1 1]. Furthermore, oxidative

L

-DOPA) that directly increases dopa-

L

-DOPA has been considered the

* Corresponding author. Convergence Research Center for Diagnosis, Treatment

and Care System of Dementia, Korea Institute of Science and Technology, 39-1

Hawolgok-dong, Seongbuk-gu, Seoul, Republic of Korea.

** Corresponding author. Department of Fundamental Pharmaceutical Sciences,

Graduate School, Kyung Hee University, Seoul, 02447, Republic of Korea.

*** Corresponding author. Convergence Research Center for Diagnosis, Treatment

and Care System of Dementia, Korea Institute of Science and Technology, 39-1

Hawolgok-dong, Seongbuk-gu, Seoul, Republic of Korea.

L

E-mail addresses: kyslee@khu.ac.kr (Y.S. Lee), jhyunprk@kist.re.kr (J.-H. Park),

kdpark@kist.re.kr (K.D. Park).

1

These authors contributed equally to this work.

https://doi.org/10.1016/j.ejmech.2020.1 13103

J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

stress is a critical factor in the pathophysiology of inﬂammation

because endothelial dysfunction of immune cells involves free

radical production. Therefore, oxidative stress and inﬂammation

are commonly known to contribute to the major signs of various

diseases, including PD, Alzheimer’s disease (AD), and other

neurodegenerative disorders [12,13].

Nrf2 translocation and upregulation of the Nrf2-dependent anti-

oxidant genes [32,33]. Bardoxolone methyl (CDDO-Me), derived

by modifying the natural product oleanolic acid, has a,b-unsatu-

rated scaffold that currently acts as an electrophilic Nrf2 activator

with the most superior Nrf2 activation efﬁcacy. CDDO-Me has

been reported to protect cells from oxidative stress by inhibiting

ROS production and diminishing proinﬂammatory signaling at

low nanomolar concentrations [34e36].

Generally, the induction of nuclear factor E2-related factor 2

(Nrf2), which resists environmental stress, seems to be a promising

strategy for maintaining cell homeostasis. Nrf2 is a member of the

cap’n’collar family of transcription factors and plays a crucial role in

antioxidant stress. Without oxidative stress, Nrf2 remains in the

cytoplasm by binding to the Kelch-like ECH-associated protein 1

(Keap1) and is directed to proteasomal degradation by ubiquiti-

nation [14,15]. Upon exposure to oxidative stress and electrophiles,

the cysteine residues of Keap1 are modiﬁed to alter the interaction

between Nrf2 and Keap1, and release Nrf2 into the nucleus. This

phenomenon causes Nrf2 to accumulate in the nucleus, allowing

Nrf2 to interact with a small Maf protein and bind to a promoter,

including an antioxidant response element (ARE) sequence [16,1 7].

Therefore, Nrf2 is known to be a major regulator of oxidative stress

that induces the expression of antioxidant enzyme genes such as

heme oxygenase-1 (HO-1), glutamate-cysteine ligase (GCL), and

superoxide dismutase 1 (SOD1) [18e21]. Furthermore, the Keap1-

Nrf2-ARE signaling pathway mainly regulates the expression of

proinﬂammatory genes such as interleukin-6 (Il-6) and tumor ne-

Structure activity analyses of different Nrf2 activators have

shown that an

a,b-unsaturated carbonyl group, a key structure

for the Michael addition, is vital for exhibiting the potent anti-

oxidant and anti-inﬂammatory effects [24e26,28]. Chalcone can

also activate Nrf2 by modifying the cysteine residues of Keap1 by

Michael addition [37e39]. In our previous study, compound 1

was developed by introducing vinyl sulfone based on the chal-

cone structure and exhibited both antioxidant and anti-

inﬂammatory effects [40,41]. Recently, compound 2 was devel-

oped with potent Nrf2 activation compared to compound 1 (1:

EC₅₀¼ 530 nM vs 2: EC₅₀¼ 326 nM) and dose-dependently

induced the expression of Nrf2-dependent antioxidant enzymes

in DAergic cells. Compound 2 also protected DAergic neuronal

cells from oxidative damages in vitro and in vivo and attenuated

PD-related movement deﬁcits in the 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine (MPTP)-induced mouse model of PD [42].

crosis factor-a (TNF-a), and then inhibits inﬂammation progression

[22,23].

It has recently been demonstrated that the introduction of

halogen atoms at a speciﬁc position on active molecules can sub-

stantially enhance its biological activity [43,44]. Furthermore, pyr-

idine ring is the second most commonly introduced aromatic

nitrogen heterocycle among all U.S. FDA-approved drugs and has

moderate activities against a number of biological targets [45e47].

In this study, we attempted to maximize Nrf2 activating effect of

vinyl sulfones by introducing halogens and nitrogen heterocycle.

The optimized vinyl sulfones were evaluated for Nrf2 activation in

cell-based assays and showed dramatically superior Nrf2 activation

than the previous compounds (1 and 2). The most potent Nrf2

activator was evaluated for its ability to induce expression of the

Nrf2-dependent antioxidant enzyme gene and suppress inﬂam-

matory responses in BV-2 microglial cells. We also examined

Accordingly, Nrf2 pharmacological activation is a promising

therapeutic approach to neurodegenerative diseases associated

with oxidative stress and inﬂammation [24,25]. Sulforaphane

(SFN), a representative well-known Nrf2 activator, is a type of

isothiocyanate derived from broccoli and other vegetables. It is

being extensively studied because it has antioxidant, anti-

inﬂammatory, and antitumor effects through various mecha-

nisms [26e29]. One of the major targets of sulforaphane is the

Nrf2 that regulates the expression of numerous cytoprotective

enzymes with antioxidant effects [30,31]. The most successful

case of Nrf2 targeting is the dimethyl fumarate (Teﬁdera™), which

is already used as an oral drug for multiple sclerosis (MS).

Dimethyl fumarate modiﬁes Keap1 to covalent bonds to induce

2

J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

Scheme 1. General procedure for the preparation of halogenated vinyl sulfone derivatives 6e21.

whether the optimized vinyl sulfone effectively protect DAergic

neurons and attenuate PD-associated behavioral deﬁcits in the

MPTP-induced mouse model.

substituted sulﬁdes were oxidized with 2.2 equivalents of m-

chloroperoxybenzoic acid (mCPBA) at room temperature to give the

desired sulfones (5). Finally, the target compounds 6e21 were

obtained from Horner-Wadsworth-Emmons oleﬁnation reaction

with commercially available desired pyridinecarboxaldehyde in the

presence of 2.0 M n-butyllithium solution (n-BuLi).

2. Results and discussion

2.1. Chemistry

2.2. Structure activity relationship (SAR) studies through cell-based

Nrf2 functional assay

Scheme 1 shows the synthetic route to access the target com-

pounds (6e21). Synthesis was conducted by coupling with

commercially available benzenethiols and (diethoxyphosphoryl)

methyl-4-methylbenzenesulfonate (3) in the presence of cesium

carbonate (Cs₂CO₃) to obtain substituted sulﬁdes (4). The

To evaluate the Nrf2 activation efﬁcacy of the synthesized

halogenated vinyl sulfones, we tested their ability to translocate

Nrf2 into the nucleus using our previously reported cell-based

3

J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

Table 1

Effects of halogenated vinyl sulfones 5e20 on Nrf2 activation.

Comp.

isomer

R¹

R²

Nrf2 EC₅₀

(m

M)^a

Comp.

isomer

R¹

R²

Nrf2 EC₅₀(m

M)^a

6a

6b

6c

6d

6e

6f

6g

7a

7b

7c

7d

8a

8b

8c

8d

8e

8f

(E)

(Z)

(E)

(Z)

(E)

(Z)

(E)

2-F

3-F

4-F

2-Cl

H

1.046 0.066

0.176 0.029

0.482 0.040

0.030 0.017

1.027 0.057

1.036 0.088

0.898 0.031

1.683 0.021

0.474 0.021

0.084 0.003

2.503 0.097

3.102 0.013

0.542 0.023

0.231 0.010

2.157 0.036

4.371 0.097

2.788 0.101

0.618 0.033

0.122 0.032

9c

9d

9e

9f

(E)

(Z)

(E)

(Z)

(E)

(Z)

(E)

(Z)

(E)

(Z)

(E)

2-Cl

3-Cl

4-Cl

3-F

0.583 0.034

0.026 0.019

1.103 0.043

2.942 0.113

0.147 0.018

1.427 0.020

0.198 0.021

0.252 0.044

0.046 0.002

0.290 0.010

1.512 0.050

1.074 0.099

1.732 0.037

0.286 0.014

0.065 0.009

1.745 0.028

3.162 0.062

1.459 0.056

3-F

3-Cl

5-Cl

6-Cl

H

3-F

3-Cl

5-Cl

H

3-F

3-Cl

5-Cl

6-Cl

H

3-Cl

5-Cl

6-Cl

H

9g

10a

10b

10c

10d

10e

10f

10g

11a

11b

11c

11d

11e

11f

3-F

3-Cl

5-Cl

6-Cl

H

3-F

3-Cl

5-Cl

6-Cl

9a

9b

3-F

Comp.

isomer

R¹

R²

Nrf2 EC₅₀

(

m

M)^a

Comp.

isomer

R¹

R²

Nrf2 EC₅₀(m

M)^a

12a

12b

12c

12d

13a

13b

13c

13d

14a

14b

15a

15b

15c

15d

16a

16b

16c

16d

17a

17b

(E)

(Z)

(E)

(Z)

(E)

(Z)

(E)

(Z)

(E)

(Z)

(E)

2-F

3-F

H

6.855 0.065

0.217 0.018

3.386 0.038

2.548 0.030

4.820 0.039

0.328 0.012

4.869 0.049

0.965 0.060

0.339 0.013

>10

2.848 0.028

0.384 0.010

0.192 0.041

1.626 0.028

3.522 0.011

0.213 0.018

2.206 0.026

3.386 0.080

0.305 0.008

>10

18

19

20

21

(E)

2-F

3-F

2-Cl

3-Cl

H

0.760 0.050

0.950 0.021

0.781 0.008

0.528 0.032

2-Cl

6-Cl

H

2-Cl

6-Cl

2-Cl

6-Cl

H

2-Cl

6-Cl

H

2-Cl

6-Cl

2-Cl

6-Cl

4-F

2-Cl

3-Cl

4-Cl

1^b

0.530 0.025

0.346 0.050

0.580 0.024

0.010 0.002

2^c

SFN^d

CDDO-Me^e

a

The activation-dependent nuclear factor (erythroid-derived 2)-like 2 (Nrf2) translocation was quantitatively detected using PathHunter U2OS Keap1-Nrf2 functional assay

(DiscoveryRx, Fremont CA) by chemiluminescence, with mean SEM of EC₅₀values.

b

Compound 1 was previously developed as an Nrf2 activator.

Compound 2 was recently developed as an Nrf2 activator.

SFN, sulforaphane: a well-known Nrf2 activator.

CDDO-Me, baldoxolone methyl: the most potent Nrf2 activator.

c

d

e

assay system [42,48,49]. The potency of Nrf2 translocation activity

is shown in Table 1 as half maximal effective concentration (EC₅₀

replaced its right benzene with pyridine. The compounds with the

Cl group surpassed the compounds with the F group in their ability

to activate Nrf2 (Cl > F). The halogenated compounds substituted at

the R¹position and with pyridine on the right ring showed mod-

)

values. Several effective structure-based strategies have been used

to optimize the previously developed compounds (1 and 2) [40,42].

Inserting electron-withdrawing group and pyridine attached to the

ring was considered a way to improve Nrf2 activating efﬁcacy by

increasing the H atom acidity of the vinyl group. We tried inserting

halogens in R¹instead of the methoxy group in compound 1, and

erate Nrf2 activation efﬁcacy (6a: EC₅₀

¼

1.046

M, 9a: EC₅₀¼ 0.618

M). Among them, the halo-

m

M, 7a:

EC₅₀¼ 1.683

mM, 8a: EC₅₀¼ 3.102

m

mM, 10a:

EC₅₀¼ 1.427

m

M, 11a: EC₅₀¼ 1.732

m

genated compounds at the ortho-position of R¹had higher Nrf2

4

J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

Fig. 1. Dose-dependent translocation of Nrf2 activated by 9d. (a) Engineered U2OS cells were treated with 9d at various concentrations for 6 h to measure Nrf2 activation efﬁcacy.

(b) Cell viability was performed at various concentrations of 9d for 6 h in BV-2 cells. (c) Nrf2 levels in whole BV-2 cells were measured by treating with 9d at various concentrations

for 6 h using Western blotting. The data are presented as mean SEM (n ¼ 3). All data were repeated in three separate experiments. **p < 0.01, ***p < 0.001, vs vehicle-treated

control. (student’s t-test).

activation efﬁcacy than those in the meta and para positions (R¹:

ortho > meta > para). Furthermore, the Nrf2 potency was compared

by changing the pyridine position on the right ring. The haloge-

nated compounds, where pyridine as heterocycle on the right ring

was substituted at ortho and para positions (6a and 18), showed

higher Nrf2 activation than the meta position (12a) (p-pyridine > o-

pyridine > m-pyridine). Unfortunately, most of the compounds

with p-pyridine showed cytotoxicity at 10 M in cell viability assay

(data not shown). So far, studies on structure activity relationships

have been shown that the above halogenated compounds slightly

reduce Nrf2 activation efﬁcacy relative to previous compounds.

Based on these ﬁndings, an electron-withdrawing group was

m

Table 2

Drug like properties of lead compound 9d.

Molecular weight (g/mol)

Actiavtion of the Nrf2

308.78

0.530 0.025

442.93

0.346 0.050

314.18

0.026 0.019

response(EC₅₀

Cell viability in 10

,

m

M)

M (%)

90.1

99.0

100.0

CYP activity (% remaining)

5.4 (2C19), 88.1 (2D6), 100.0 (2C9), 41.7 77.4 (2C19), 99.7 (2D6), 96.7 (2C9),

27.8 (2C19), 88.0 (2D6), 69.1 (2C9), 88.4

(1A2), 42.3 (3A4)

20.0

87.2 (1A2), 97.4 (3A4)

84.0

(1A2), 91.2 (3A4)

66.0

Microsomal stability (%

remaining)

Plasma stability (% remaining) 86.1

98.2

97.0

PAMPA-BBB (P_e, cm/s)

hERG toxicity (IC₅₀M)

nd

12

15.63 (CNSþ)

65.16 (CNSþ)

,

m

112

110

All tests were performed triply except for human microsomal and plasma stability tests. PAMPA-BBB, parallel artiﬁcial membrane permeability assay-blood-brain barrier; CYP,

cytochrome P; hERG, human ether-a-go-go-related gene; Nrf2, nuclear factor (erythroid-derived 2)-like 2; IC₅₀, half maximal inhibitory concentration; P_e, permeability; nd,

not determined.

5

J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

further inserted in R²on the pyridine to enhance the efﬁcacy of

Nrf2 activation. Similarly, the halogenated compounds with the

halogen group in R²on the pyridine showed potent Nrf2 activation

efﬁcacy by inserting closer to the vinyl group (S(O)₂C]C) (e.g., 6d:

bloodebrain barrier permeability using a PAMPA (parallel artiﬁcial

membrane permeability assay) kit. Consequently, the permeability

of 9d was to be found useful in the central nervous system (CNS)

drugs (P_e: 65.16 ꢀ 10^ꢁ6, Table 2). The above experiments were

performed according to the previously described methods [42].

EC₅₀¼ 0.030

m

M, 6g: EC₅₀¼ 0.898

mM, 6f: EC₅₀¼ 1.036

mM). Among

them, 9d showed extremely potent Nrf2 activation efﬁcacy at

nanomolar concentration (9d: EC₅₀¼ 0.026

mM; Fig. 1a) compared

2.4. 9d activated Nrf2 translocation into the nucleus

with the previous Nrf2 activators.

Subsequently, (Z)-isomers corresponding to some compounds

were synthesized, all of which resulted in a lower Nrf2 activation

efﬁcacy than (E)-isomers. Among them, the activity of (Z)-isomer

9e signiﬁcantly decreased 42-fold compared with that of repre-

sentative (E)-isomer 9d.

The most potent among halogenated vinyl sulfones, compound

9d was selected for further biological studies on Nrf2 efﬁcacy as a

lead compound.

To measure Nrf2 response activation of the target compounds,

we used the previously reported Keap1-Nrf2 functional cell-based

assay system [42,48,51]. This assay is a direct and rapid system

for measuring the ability of Nrf2 translocation into the nucleus after

Nrf2 is released from Keap1 by electrophile (active compounds).

The selected compound 9d showed the most potent effects on Nrf2

nuclear translocation at the nanomolar concentration levels (9d:

EC₅₀¼ 26 nM; Fig. 1a). Several studies have shown that the acti-

vated Nrf2 is not degraded when released from Keap1 by the

Keap1-mediated ubiquitin proteasome system [15e1 7]. Therefore,

Nrf2 levels were measured in whole cell lysates after 6 h of expo-

sure to various concentrations of 9d in BV-2 microglial cells.

Compound 9d considerably increased the total amount of Nrf2

2.3. In vitro pharmacological studies of 9d

The drug proﬁles of 9d were performed through the cell

viability, CYP activity, human microsomal and plasm stability,

PAMPA-BBB and hERG toxicity compared with previously devel-

oped compounds (1 and 2) [42].

protein dose-dependently (11.0-fold at 0.5 mM; 22.6-fold at 10 mM;

Fig. 1c). Collectively, compound 9d potently induced Nrf2 trans-

location into the nucleus.

The cytotoxicity of 9d revealed cell survival of 100% up to 10 mM

(100.0 0.2% at 0.1

mM, 100.0 1.6% at 0.3

mM, 98.9 1.7% at 1

mM,

92.4 3.1% at 3 M, and 100.0 3.5% at 10

m

mM; Table 2 and Fig. 1b).

2.5. 9d induced gene expression of Nrf2-dependent antioxidant

The CYP enzymes activity of 9d, expressed in percentage of

remaining CYP enzymes, was all more than 60%, except for 2C19

enzyme (2C19: 27.8%, 2D6: 88.0%, 2C9: 69.1%, 1A2: 88.4%, 3A4:

91.2%; Table 2). The microsomal and plasma stability of 9d were

measured as percentage of the amount of parent remaining after

30 min incubation in human microsome and plasma. 9d showed a

notable microsomal and plasma stability with ~66% and ~97% of the

intact remaining, respectively (Table 2). Finally, we assessed a

membrane permeability of compound 9d to predict the

enzymes

We investigated whether the Nrf2 activated by 9d induces the

expression of Nrf2-dependent antioxidant enzymes. BV-2 cells

were treated with 9d for 6 h and the expression of antioxidant

enzymes was assessed by RT-qPCR and Western blotting. RT-qPCR

analysis showed that the mRNA levels of Ho-1, Gclc, Gclm, and

Sod1 were dose-dependently increased by 9d (6.0, 2.6, 6.9, and 2.5

folds at 1

mM, respectively; Fig. 2). 9d also strikingly induced

Fig. 2. Effects of 9d on induction of antioxidant-related gene in microglial cells. BV-2 cells were exposed to various concentrations of 9d for 6 h followed by quantitative RT-qPCR

analysis. The mRNA levels of Ho1, Gclc, Gclm, and Sod1 were determined by RT-qPCR using their sequence. RT-qPCR results show the relative mRNA levels of Ho1, Gclc, Gclm, and

Sod1 at various concentrations of 9d. All values are expressed as mean SEM (n ¼ 3). *p < 0.1, **p < 0.01, ***p < 0.001, vs vehicle-treated control. (student’s t-test).

6

J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

Fig. 3. Dose-dependent increase in Nrf2-dependent antioxidant enzymes after treating with 9d. (a) Expression of antioxidant proteins were performed by Western blotting in BV-

2 cells exposed to various concentrations of 9d for 6 h. (b) The levels of HO-1, GCLC, GCLM, and SOD1 proteins were determined using -actin as a loading control. The data are

b

presented as mean SEM (n ¼ 3). Fold of control: the intensity of the band versus the control band. All data was repeated three in separate experiments. ***p < 0.001, vs vehicle-

treated control. (student’s t-test).

protein levels reaching 17.6 (HO-1), 8.5 (GCLC), 26.1 (GCLM), and

6.6 (SOD1) folds at 10 M, respectively, by Western blotting (Fig. 3).

as a NO-synthesizing enzyme was also measured by pretreating

with 9d in the LPS-stimulated BV-2 cells and nearly complete in-

m

Collectively, 9d signiﬁcantly induced the expression of Nrf2-

dependent antioxidant genes by activating Nrf2 in BV-2 micro-

glial cells. To conﬁrm the selectivity for Nrf2 activation of some of

the synthesized vinyl sulfone compounds, we compared the HO-1

expression efﬁcacies between 9d (EC₅₀¼ 26 nM) and two less

active compounds (8c: EC₅₀¼ 231 nM & 9f: EC₅₀¼ 2942 nM). We

observed that the most potent Nrf2 activating compound (9d)

showed the highest ability for induction of HO-1 enzyme genes

(Fig. S1 in the Supporting Information).

hibition at 2 mM relative to the LPS-untreated controls was

observed (Fig. 4b). Also, the effects of 9d on proinﬂammatory

cytokine production were investigated. LPS-induced elevation of

TNF-a protein was suppressed by 9d dose-dependently. IL-6 pro-

tein levels were strikingly downregulated by 9d dose-dependently

(Fig. 4c). These results indicated that treating with 9d effectively

downregulates LPS-induced inﬂammatory response by suppressing

inﬂammatory enzymes and cytokines in BV-2 microglial cells. We

performed additional experiments using small interfering RNA

(siRNA) to determine whether the anti-inﬂammatory effects of 9d

was correlatated with on Nrf2 signaling pathway. First, we

conﬁrmed that Nrf2 siRNA effectively reduced Nrf2 mRNA and

protein level (Fig. 5a and b). Next, we measured NO production

levels after transfecting Nrf2 siRNA or scrambled siRNA in BV-

2 cells, followed by co-treatment with 9d and LPS for 24 h. We

observed that NO production stimulated by LPS was not reduced by

9d in Nrf2 siRNA-transfected cells (Fig. 5c). These results indicated

that 9d attenuated the inﬂammatory response via Nrf2 activation in

LPS-induced BV-2 microglial cells.

2.6. 9d suppressed production of proinﬂammatory enzymes and

cytokines

Studies have shown that the Nrf2 signaling pathway plays a

critical role in the anti-inﬂammatory process by suppressing in-

ﬂammatory mediators, proinﬂammatory cytokines, and enzymes

[25,35]. We ﬁrst stimulated BV-2 cells with lipopolysaccharide

(LPS) to induce inﬂammatory responses and tested whether

treating with 9d could suppress inﬂammatory mediators, proin-

ﬂammatory cytokines, and enzymes. The production of NO, in-

ﬂammatory mediators, was signiﬁcantly increased by LPS

stimulation compared with LPS-untreated controls, and treating

with 9d strikingly blocked dose-dependently increased NO pro-

duction (Fig. 4a). The expression of the inﬂammatory enzyme iNOS

2.7. 9d improved abnormal movement in acute MPTP-Induced PD

mice

We investigated whether 9d effectively inhibited oxidative

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J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

Fig. 4. Dose-dependent decrease in inﬂammatory cytokines and enzyme after treating with 9d. (a) NO production was measured from culture medium of LPS-stimulated BV-2 cells

pretreated with 9d by the Griess assay. (b) iNOS protein levels from LPS-stimulated cells pretreated with 9d were measured using Western blotting. The levels of iNOS enzyme were

quantitated using b-actin as an internal control. (c) The levels of proinﬂammatory cytokines were measured from culture medium of LPS-stimulated cells pretreated with 9d by

using an ELISA analysis. The data are presented as mean SEM (n ¼ 3). All data were repeated in three separate experiments. **p < 0.01, ***p < 0.001, vs LPS-stimulated group.

(student’s t-test).

stress and mitigated ROS-induced damages in vivo. Neurotoxin 1-

methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its me-

tabolites 1-methyl-4-phenylpyridinium ion (MPP^þ) induce mito-

chondrial dysfunction and cellular oxidative stress, which are

widely used to establish a model of the PD [42,50,51]. A week after

the last MPTP administration, mice showed limb incoordination

and motor deﬁciency. We conducted a coat-hanger and vertical grid

tests to assess motor dysfunction (Fig. 6a). In the coat-hanger test

[42,51], motor coordination can be measured by assessing mouse

ability to rise above the safe area (score 5) when the mouse is

suspended on a wire bar. The MPTP-treated mice (vehicle-treated)

only showed the ability to hang at the wire end (score 1.89 0.51),

whereas the mice treated with 9d showed the ability to pull and

climb to the top of the hanger (score 3.11 0.93) (Fig. 6b). Addi-

tionally, vertical grid tests showed that the 9d-treated mice

ameliorated MPTP-induced abnormal movements. For the 9d-

treated mice placed on a vertical grid, the mouse quickly turned

and descended without slipping (Fig. 6c). We also observed that the

behavioral deﬁcit was signiﬁcantly improved in the experiment in

which 9d was administered 3 days after injection of MPTP (Fig. S2

in the Supporting Information). Collectively, these results indicated

9d effectively ameliorated motor deﬁcits in the MPTP-induced

mouse model in the pre-treatment and post-treatment regimes.

membrane potential and causes oxidative stress. Therefore, MPTP-

treatment damaged dopamine neurons in the SNpc, resulting in a

signiﬁcant tyrosine hydroxylase (TH)-positive cell loss. Meanwhile,

65% less damages of TH-positive cells were observed for mice

administered 9d for 3 consecutive days with MPTP injection (Fig. 7a).

The TH-immunoreactive ﬁber density in 9d-treated mice also

recovered compared to that of MPTP-treated mice (Fig. 7b). These

results suggested that 9d protects against neurotoxin in vivo.

3. Conclusions

Optimization of potency on Nrf2 was achieved through SAR data

collected during our hit-to-lead medicinal chemistry on the key

structure of vinyl sulfone that contributed to the efﬁcacy of the

previously developed compounds (1 and 2). These optimizations

included the following: (1) the halogen substitution in the ortho

position of the benzene ring, (2) substitution of the vinyl group in

beta-position with a pyridine ring, and (3) insertion of a halogen in

R²on pyridine ring.

As a result of SAR study, the most promising compound 9d was

identiﬁed and it showed excellent proﬁle: (1) the molecular size

was below 400 Da, (2) potent Nrf2 activation efﬁcacy at nanomolar

concentrations compared with previous developed compounds,

and (3) favorable drug-like properties. 9d signiﬁcantly increased

Nrf2 levels, upregulated antioxidant enzymes, and suppressed in-

ﬂammatory response in BV-2 microglial cells. Furthermore, 9d

attenuated the motor deﬁciency and rescued the loss of DAergic

neurons SNpc and striatum in MPTP-induced mice. Collectively, 9d

is presented as an improved candidate for therapy against PD.

2.8. 9d protected dopaminergic neurons from oxidative stress

induced by MPP^þin the striatum and SNpc

MPP^þis speciﬁcally transported to dopaminergic neurons and

then accumulates in the mitochondria, which reduces mitochondrial

8

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European Journal of Medicinal Chemistry 212 (2021) 113103

Fig. 5. Anti-inﬂammatory responses by 9d are dependent on Nrf2. (a) BV-2 cells were transfected with 83 nM scramble- or Nrf2-siRNA for 24 h, and then subjected to RTeqPCR

analysis of relative Nrf2 mRNA levels; Hprt was used as an internal control. All experiments were performed in duplicate. (b) After transfection, BV-2 cells were exposed to 100 nM

9d for 24 h, and then Nrf2 protein levels were measured by using Western blotting. Data are shown as the mean SEM and analyzed by Student’s t-test. **p < 0.01, ***p < 0.001 vs

the Scramble-siRNA transfected group. (c) BV-2 cells transfected with siRNA (83 nM) were cotreated with 100 nM 9d and 0.5 mg/ml LPS for 24 h. NO levels in the cell culture medium

were measured using the Griess assay. The data are shown as the mean SEM and analyzed by Student’s t-test. All experiments were performed in duplicate. ***p < 0.001 vs the

LPS-stimulated group with scramble-siRNA transfection; ^###p < 0.001 vs the LPS-stimulated group with Nrf2-siRNA transfection. (student’s t-test).

Fig. 6. The MPTP-induced mice restored motor deﬁcit by oral treatment of 9d. (a) Behavioral schedule and images for coat-hanger and vertical grid tests from vehicle and MPTP-

induced mice (20 mg/kg, i.p.) with or without treatment of 9d (30 mg/kg, p.o.). (b) The coat-hanger test was started with mice in the middle of the bottom wire and scored according

to their position. (c) The mice were placed from the top of the vertical grid apparatus, and their time to total, time to turn, and time to climb down were observed. The data are

shown as mean SEM (n ¼ 9e10 per group). *p < 0.1, **p < 0.01, vs MPTP-treated group. (one-way ANOVA with Tukey’s multiple comparison test).

9

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European Journal of Medicinal Chemistry 212 (2021) 113103

Fig. 7. Rescue of dopaminergic neurons in the MPTP-induced mouse model by oral treatment of 9d. The mice were treated with saline or 9d, and their midbrain slices (sacriﬁced 8

days later) were observed by immunohistochemistry with anti-TH antibody. (a) Representative immunohistochemical images in the substantia and striatal tissue sections of mice

are shown. (b) Quantitative analysis of the number of TH-positive neurons in substantia and the density of TH-positive neurons in the striatum were identiﬁed. The results are

shown as the mean SEM. **p < 0.01, ***p < 0.001, vs the MPTP-treated group. (one-way ANOVA with Tukey’s multiple comparison test).

4. Experimental section

H₂O (2 ꢀ ~200 mL). The organic layer was dried with sodium sulfate

(anhydrous Na₂SO₄) and the resulting residue was puriﬁed by

column chromatography on SiO₂.

4.1. General methods

Commercially available chemicals, solvents, and reagents were

purchased from chemical suppliers and used without further pu-

riﬁcation. The desired compounds were puriﬁed by column chro-

matography using silica gel (Merck, Cat No. 1.07734 and 1.09385).

¹H and ¹³C NMR spectra were obtained using Bruker spectrometers

(400 MHz or 300 MHz, respectively) with deuterated solvents

(DMSO‑d₆and CDCl₃). All NMR spectra were reported in ppm

downﬁeld from tetramethylsilane (TMS). Analytical HPLC for ﬁnal

compounds was performed on a Waters E2695 system. A Capcell

4.3. (E)-2-(2-((2-Fluorophenyl)sulfonyl)vinyl)pyridine (6a)

Diethyl

(((2-ﬂuorophenyl)

sulfonyl)methyl)phosphonate

(0.25 g, 0.80 mmol), 2.0M n-BuLi solution in cyclohexane (0.44 mL,

0.88 mmol) and 2-pyridinecarboxaldehyde (0.08 mL, 0.88 mmol)

gave 0.11 g (53%) of 6a as a pink powder; R_f¼ 0.32 (CH₂Cl₂/MeOH

49/1); mp: 75.0e76.0 ^ꢂC; ¹H NMR (400 MHz, DMSO‑d₆)

d 8.67 (d,

J ¼ 3.4 Hz, ArH), 7.83e8.00 (m, 5ArH), 7.80 (s, J ¼ 15.2 Hz, (E)-

isomeric H), 7.72 (s, J ¼ 15.0 Hz, (E)-isomeric H), 7.50e7.54 (m,

Pak C18 column (4.6 mm ꢀ 75 mm; 3-

mm particle size) was used

according to the following methods: ﬂow rate of 0.5 mL/min, H₂O/

3ArH); ¹³C NMR (100 MHz, CDCl₃)

d

159.9 (d, J_C-F¼ 248.8 Hz), 151.2,

150.4, 142.6, 137.0, 136.1, 130.8, 129.8, 128.3, 125.7, 125.2, 124.7, 117.2

(d, J_C-F¼ 20.8 Hz); HPLC purity: 9.2 min, 100.0%; HRMS (M þ H)^þ

(ESI^þ) 264.0486 [M þ H]^þ(calcd for C₁₃H₁₀FNO₂SH^þ264.0489).

acetonitrile,100/0 / 0/100 in 20 min, þ5 min isocratic, ¼ 254 and

l

280 nm. All ﬁnal compounds exhibited >95% purity by HPLC

analysis. The mass spectra were obtained by electron scatter ioni-

zation (ESI) using a LTQ Orbitrap (Thermo Electron Corp.) instru-

ment to perform accurate mass measurements. Melting points

were observed in open capillary tubes using OptiMelt melting point

equipment (Stanford Research Systems, Inc.).

4.4. (E)-3-Fluoro-2-(2-((2-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(6b)

Diethyl (((2-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.25 g,

0.80 mmol), 2.0M n-BuLi solution in cyclohexane (0.44 mL,

0.88 mmol) and 3-ﬂuoro-2-pyridinecarboxaldehyde (0.11 g,

0.88 mmol) gave 0.07 g (71%) of 6b as a pink solid; R_f¼ 0.33

(CH₂Cl₂/MeOH 49/1); mp: 96.8e98.2 ^ꢂC; ¹H NMR (400 MHz,

4.2. General Procedure for the Halogenated Vinyl Sulfone

Derivatives 6e21

2.0 M n-BuLi solution in cyclohexane (1.2 equiv) was added to a

cooled solution of phosphonate derivatives in anhydrous tetrahy-

drofuran solution (ꢁ78 ^ꢂC). The reaction mixture was stirred

at ꢁ78 ^ꢂC (1 h) and then the desired substituted pyridinecarbox-

aldehyde (1.2 equiv) was added at ꢁ78 ^ꢂC. The reaction mixture was

stirred at room temperature for (E)-isomer or at ꢁ78 ^ꢂC for (Z)-

isomer (1 h). The resulting mixiture was quenched with H₂O

(~50 mL) and diluted with EtOAc (~200 mL), and then washed with

DMSO‑d₆)

d

8.54 (d, J ¼ 4.1 Hz, ArH), 7.98 (t, J ¼ 7.5 Hz, ArH),

7.84e7.93 (m, 2ArH), 7.86 (d, J ¼ 15.6 Hz, (E)-isomeric H), 7.71 (d,

J ¼ 15.0 Hz, (E)-isomeric H), 7.63e7.66 (m, ArH), 7.50e7.55 (m,

ArH); ¹³C NMR (100 MHz, DMSO‑d₆)

d

159.7 (d, J_C-F¼ 255.7 Hz),

158.6 (d, J_C-F¼ 264.3 Hz), 146.0 (d, J_C-F¼ 5.0 Hz), 139.5 (d, J_C-

¼ 11.1 Hz), 136.2 (d, J_C-F¼ 8.5 Hz), 135.1, 132.2 (d, J_C-F¼ 3.3 Hz),

F

129.9, 128.2 (d, J_C-F¼ 14.0 Hz), 126.9 (d, J_C-F¼ 4.4 Hz), 124.8 (d, J_C-

¼ 3.8 Hz), 124.2 (d, J_C-F¼ 19.0 Hz), 117.2 (d, J_C-F¼ 20.7 Hz),; HPLC

F

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J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

purity: 8.9 min, 99.2%; HRMS (M þ H)^þ(ESI^þ) 282.0393 [M þ H]^þ

(m, ArH, (E)-isomeric H), 7.61e7.67 (m, ArH), 7.58 (d, J ¼ 14.8 Hz,

ArH, (E)-isomeric H), 7.39 (d, J ¼ 8.3 Hz, ArH), 7.34 (t, J ¼ 7.7 Hz, ArH)

(calcd for C₁₃H₉F₂NO₂S^þ282.0395).

7.20 (t, J ¼ 9.1 Hz, ArH); ¹³C NMR (100 MHz, CDCl₃)

d 159.7 (d, J_C-

4.5. (Z)-3-Fluoro-2-(2-((2-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(6c)

¼ 255.0 Hz), 149.3 (d, J_C-F¼ 50.0 Hz), 148.1, 141.2, 136.7, 136.2 (d, J_C-

F

¼ 9.0 Hz), 133.7, 131.3, 129.9, 128.2 (d, J_C-F¼ 14.0 Hz), 126.1, 124.8

F

(d, J_C-F¼ 4.0 Hz), 117.3 (d, J_C-F¼ 21.0 Hz); HPLC purity: 11.3 min,

97.4%; HRMS (M þ H)^þ(ESI^þ) 298.0093 [M þ H]^þ(calcd for

Diethyl (((2-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.20 g,

0.64 mmol), 2.0M n-BuLi solution in cyclohexane (0.38 mL,

0.77 mmol) and 3-ﬂuoro-2-pyridinecarboxaldehyde (0.096 g,

0.77 mmol) gave 0.023 g (13%) of 6c as a white solid; R_f¼ 0.36 (n-

hexane/EtOAc 1/1); mp: 44.6e45.8 ^ꢂC; ¹H NMR (400 MHz, DMSO)

C

₁₃H₉ClFNO₂SH^þ298.0099).

4.9. (E)-2-Chloro-6-(2-((2-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(6g)

d

8.30e8.29 (m, ArH), 8.02e7.98 (m, ArH), 7.61e7.55 (m, 2ArH),

7.41e7.26 (m, 3ArH, (Z)-isomeric H), 7.17 (t, J ¼ 9.2 Hz, ArH), 6.94

Diethyl (((2-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.3 g,

0.96 mmol), 2.0M n-BuLi solution in cyclohexane (0.58 mL,

1.16 mmol) and 6-chloro-2-pyridinecarboxaldehyde (0.16g,

1.16 mmol) gave 0.10 g (35%) of 6g as a ivory solid; R_f¼ 0.32 (n-

hexane/EtOAc 3/1); mp: 151.8e152.7 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

(dd, J ¼ 1.9, 11.7 Hz, (Z)-isomeric H); ¹³C NMR (100 MHz, CDCl₃)

d

159.4 (d, J_C-F¼ 253.8 Hz), 157.7 (d, J_C-F¼ 261.8 Hz), 144.6 (d, J_C-

¼ 5.1 Hz), 139.8 (d, J_C-F¼ 12.4 Hz), 135.5 (d, J_C-F¼ 8.5 Hz), 135.2,

F

133.3, 130.0 (d, J_C-F¼ 18.0 Hz), 125.7 (d, J_C-F¼ 4.1 Hz), 124.1 (d, J_C-

¼ 3.6 Hz), 123.1 (d, J_C-F¼ 18.8 Hz), 116.9, 116.7; HPLC purity:

d

8.01 (td, J ¼ 1.5, 7.5 Hz, ArH), 7.58e7.77 (m, 2ArH, (E)-isomeric

2H), 7.32e7.37 (m, 3ArH), 7.18e7.23 (m, ArH); ¹³C NMR (100 MHz,

CDCl₃)

F

6.4 min, 96.6%; LRMS (M þ H)^þ(ESI^þ) 282.03 [M þ H]^þ(calcd for

C

₁₃H₉F₂NO₂SH^þ282.05).

d

159.7 (d, J_C-F¼ 256.0 Hz), 152.1, 151.4, 140.7, 139.7, 136.3 (d,

J_C-F¼ 8.5 Hz), 132.2, 130.0, 128.0 (d, J_C-F¼ 14.0 Hz), 126.1, 124.8,

124.2, 117.3 (d, J_C-F¼ 20.8 Hz); HPLC purity: 10.3 min, 99.2%; LRMS

(M þ H)^þ(ESI^þ) 297.00 [M þ H]^þ(calcd for C₁₃H₉ClFNO₂SH^þ

298.00).

4.6. (E)-3-Chloro-2-(2-((2-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(6d)

Diethyl

(((2-ﬂouorophenyl)sulfonyl)methyl)phosphonate

(0.35 g, 1.13 mmol), 2.0M n-BuLi solution in cyclohexane (0.62 mL,

1.24 mmol) and 3-chloro-2-pyridinecarboxaldehyde (0.18 mL,

1.24 mmol) gave 0.30 g (90%) of 6d as a pale yellow powder;

R_f¼ 0.37 (n-hexane/EtOAc 2/1); mp: 88.0e90.7 ^ꢂC; ¹H NMR

4.10. (E)-2-(2-((3-Fluorophenyl)sulfonyl)vinyl)pyridine (7a)

Diethyl (((3-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.75 g,

2.40 mmol), 2.0M n-BuLi solution in cyclohexane (1.32 mL,

2.64 mmol) and 2-pyridinecarboxaldehyde (0.25 mL, 2.64 mmol)

gave 0.48 g (76%) of 7a as a beige solid; R_f¼ 0.41 (n-hexane/EtOAc

(400 MHz, DMSO‑d₆)

d

8.63 (d, J ¼ 4.4 Hz, ArH), 8.09 (d, J ¼ 8.2 Hz,

ArH), 8.02 (d, J ¼ 15.0 Hz, (E)-isomeric H), 7.98 (d, J ¼ 7.6 Hz, ArH),

7.84(q, J ¼ 7.2, 13.8 Hz, ArH), 7.76 (d, J ¼ 14.7 Hz, (E)-isomeric H),

2/1); mp: 86.6e87.7 ^ꢂC; ¹H NMR (400 MHz, DMSO‑d₆)

d 8.65 (d,

7.50e7.58 (m, 3ArH); ¹³C NMR (100 MHz, DMSO‑d₆)

d

159.2 (d, J_C-

J ¼ 4.3 Hz, ArH), 7.92 (t, J ¼ 7.4 Hz, ArH), 7.71e7.85 (m, 4ArH, (E)-

¼ 253.1 Hz), 149.2, 147.2, 138.9, 137.8 (d, J_C-F¼ 8.6 Hz), 137.5, 133.5,

isomeric 2H), 7.64 (t, J ¼ 7.7 Hz, ArH), 7.47 (d, J ¼ 4.9 Hz, ArH); ¹³

C

F

132.9, 130.1, 127.8, 127.6 (d, J_C-F¼ 13.8 Hz), 126.1 (d, J_C-F¼ 3.5 Hz),

118.0 (d, J_C-F¼ 20.6 Hz); HPLC purity: 8.7 min, 100.0%; HRMS

(M þ H)^þ(ESI^þ) 298.0091 [M þ H]^þ(calcd for C₁₃H₉ClFNO₂SH^þ

298.0099).

NMR (100 MHz, DMSO‑d₆)

142.3, 142.1, 137.4, 132.2, 130.8, 125.7, 125.5, 123.6, 121.1 (d, J_C-

d

162.0 (d, J_C-F¼ 247.9 Hz), 150.6, 150.1,

¼ 26.1 Hz), 114.6 (d, J_C-F¼ 32.6 Hz); HPLC purity: 9.1 min, 100.0%;

F

HRMS (M

C

þ

H)^þ(ESI^þ) 264.0486 [M

þ

H]^þ(calcd for

₁₃H₁₀FNO₂SH^þ264.0489).

4.7. (Z)-3-Chloro-2-(2-((2-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(6e)

4.11. (E)-3-Fluoro-2-(2-((3-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(7b)

Diethyl (((2-ﬂuorophenyl)sulfonyl)methyl)phosphonate (3.0 g,

9.66 mmol), 2.0M n-BuLi solution in cyclohexane (4.64 mL,

11.6 mmol) and 3-chloro-2-pyridinecarboxaldehyde (1.44 g,

11.6 mmol) gave 0.30 g (10%) of 6e as a pale yellow solid; R _f¼ 0.23

(n-hexane/EtOAc 2/1); mp: 98.1e99.7 ^ꢂC; ¹H NMR (400 MHz,

Diethyl (((3-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.74 g,

2.38 mmol), 2.0M n-BuLi solution in cyclohexane (1.31 mL,

2.62 mmol) and 3-ﬂuoro-2-pyridinecarboxaldehyde (0.28 g,

2.62 mmol) gave 0.42 g (62%) of 7b as a wihte powder; R_f¼ 0.41 (n-

hexane/EtOAc 1/1); mp: 70.3e72.1 ^ꢂC; ¹H NMR (300 MHz,

CDCl₃)

d

8.35 (dd, J ¼ 1.2, 4.6 Hz, ArH), 7.94e7.90 (m, ArH), 7.66 (dd,

J ¼ 1.3, 8.2 Hz, ArH), 7.60e7.54 (m, ArH), 7.42 (d, J ¼ 11.5 Hz, (Z)-

isomeric H), 7.31e7.27 (m, ArH), 7.26e7.15 (m, ArH, (Z)-isomeric H),

6.94 (dd, J ¼ 2.4,11.5 Hz, (Z)-isomeric H); ¹³C NMR (100 MHz, CDCl₃)

DMSO‑d₆)

d

8.52e7.54 (m, ArH), 7.60e7.93 (m, 8ArH, (E)-isomeric

161.7 (d, J_C-F¼ 273.9 Hz), 158.3

2H); ¹³C NMR (75 MHz, DMSO‑d₆)

d

(d, J_C-F¼ 288.2 Hz), 146.3 (d, J_C-F¼ 4.8 Hz), 141.7 (d, J_C-F¼ 6.7 Hz),

138.4 (d, J_C-F¼ 10.9 Hz), 133.7, 132.1, 132.1 (d, J_C-F¼ 13.5 Hz), 128.0

(d, J_C-F¼ 4.7 Hz), 124.8 (d, J_C-F¼ 18.8 Hz), 123.8 (d, J_C-F¼ 3.0 Hz),

121.2 (d, J_C-F¼ 21.1 Hz), 114.7 (d, J_C-F¼ 24.4 Hz); HPLC purity:

11.4 min, 99.6%; HRMS (M þ H)^þ(ESI^þ) 282.0395 [M þ H]^þ(calcd

for C₁₃H₉F₂NO₂SH^þ282.0395).

d

159.0 (d, J_C-F¼ 252.9 Hz), 148.7, 146.3, 136.9, 136.4, 135.1, 135.0 (d,

J_C-F¼ 7.4 Hz), 131.2, 129.8, 129.5, 124.5, 123.7, 116.5 (d, J_C-

¼ 20.9 Hz); HPLC purity: 7.5 min, 100.0%; LRMS (M þ H)^þ(ESI^þ)

F

298.00 [M þ H]^þ(calcd for C₁₃H₉ClFNO₂SH^þ298.00).

4.8. (E)-5-Chloro-2-(2-((2-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(6f)

4.12. (E)-3-Chloro-2-(2-((3-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(7c)

Diethyl (((2-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.5 g,

1.61 mmol), 2.0M n-BuLi solution in cyclohexane (0.16 mL,

0.85 mmol) and 5-chloro-2-pyridinecarboxaldehyde (0.25 g,

1.77 mmol) gave 0.10 g (21%) of 6f as a white powder; R_f¼ 0.37 (n-

hexane/EtOAc 4/1); mp: 138.3e140.1 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

Diethyl (((3-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.74 g,

2.38 mmol), 2.0M n-BuLi solution in cyclohexane (1.31 mL,

2.62 mmol) and 3-chloro-2-pyridinecarboxaldehyde (0.37 g,

2.62 mmol) gave 0.48 g (68%) of 7c as a white powder; R_f¼ 0.58 (n-

hexane/EtOAc 1/1); mp: 75.1e77.1 ^ꢂC; ¹H NMR (300 MHz,

d

8.58 (d, J ¼ 2.4 Hz, ArH), 8.02 (td, J ¼ 1.6, 7.1 Hz, ArH), 7.70e7.75

11

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European Journal of Medicinal Chemistry 212 (2021) 113103

DMSO‑d₆)

d

8.61 (dd, J ¼ 1.2, 4.5 Hz, ArH), 7.53e8.10 (m, 8ArH, (E)-

161.9 (d, J_C-

2.12 mmol) gave 0.34 g (58%) of 8c as a white solid; R_f¼ 0.36 (n-

hexane/EtOAc 2/1); mp: 92.6e94.2 ^ꢂC; ¹H NMR (300 MHz,

isomeric 2H); ¹³C NMR (75 MHz, DMSO‑d₆)

d

¼ 248.0 Hz), 148.5, 146.8,141.6 (d, J_C-F¼ 6.7 Hz),138.4,135.9, 133.3,

DMSO‑d₆)

d

8.60 (d, J ¼ 4.5 Hz, ArH), 8.06e8.11 (m, 3ArH), 7.87 (dd,

F

132.3, 132.1 (d, J_C-F¼ 7.9 Hz), 127.1, 123.8 (d, J_C-F¼ 3.1 Hz), 121.3 (d,

J_C-F¼ 21.2 Hz), 114.7 (d, J_C-F¼ 24.4 Hz); HPLC purity: 12.4 min,

99.8%; HRMS (M þ H)^þ(ESI^þ) 298.0096 [M þ H]^þ(calcd for

J ¼ 14.8 Hz, (E)-isomeric 2H), 7.49e7.55 (m, 3ArH); ¹³C NMR

(75 MHz, DMSO‑d₆)

d

165.1 (d, J_C-F¼ 251.8 Hz), 148.5, 146.9, 138.4,

135.8 (d, J_C-F¼ 2.8 Hz), 135.1, 133.9, 132.2, 131.0, 130.9, 127.0, 117.1,

116.8; HPLC purity: 11.7 min, 100.0%; HRMS (M þ H)^þ(ESI^þ)

298.0092 [M þ H]^þ(calcd for C₁₃H₉ClFNO₂SH^þ298.0099).

C

₁₃H₉ClFNO₂SH^þ298.0099).

4.13. (E)-5-Chloro-2-(2-((3-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(7d)

4.17. (Z)-3-Chloro-2-(2-((4-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(8d)

Diethyl (((3-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.23 g,

0.74 mmol), 2.0M n-BuLi solution in cyclohexane (0.46 mL,

0.91 mmol) and 5-chloro-2-pyridinecarboxaldehyde (0.13 g,

0.91 mmol) gave 0.13 g (59%) of 7d as a ivory solid; R_f¼ 0.35 (n-

hexane/EtOAc 4/1); mp: 100.7e102.7 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

Diethyl (((4-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.15 g,

0.48 mmol), 2.0M n-BuLi solution in cyclohexane (0.29 mL,

0.58 mmol) and 3-chloro-2-pyridinecarboxaldehyde (0.082 g,

0.58 mmol) gave 0.02 g (14%) of 8d as a white solid; R_f¼ 0.36 (n-

hexane/EtOAc 2/1); mp: 93.5e96.5 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

d

8.56 (d, J ¼ 2.4 Hz, ArH), 7.76 (qd, J ¼ 0.9, 8.0 Hz, ArH), 7.72 (dd,

J ¼ 2.5, 8.3 Hz, ArH), 7.61e7.67 (m, ArH, (E)-isomeric H), 7.53e7.58

(m, ArH), 7.42 (d, J ¼ 14.8 Hz, (E)-isomeric H), 7.31e7.38 (m, 2ArH);

d

8.53 (d, J ¼ 4.6 Hz, ArH), 8.07e8.03 (m, 2ArH), 7.71 (d, J ¼ 8.2 Hz,

ArH), 7.32e7.17 (m, 3ArH, (Z)-isomeric H), 6.69 (d, J ¼ 11.6 Hz, (Z)-

isomeric H); ¹³C NMR (100 MHz, CDCl₃)

167.0, 139.5, 146.9, 137.0,

¹³C NMR (100 MHz, CDCl₃)

d

162.6(d, J_C-F¼ 250.9 Hz), 149.5, 148.9,

d

142.2 (d, J_C-F¼ 6.5 Hz), 140.0, 136.7, 133.7, 131.7, 131.3 (d, J_C-

136.8, 134.8, 131.4, 131.2, 131.1, 124.8, 116.3, 116.1; HPLC purity:

¼ 7.5 Hz), 126.1, 123.7, 121.0 (d, J_C-F¼ 21.1 Hz), 115.2 (d, J_C-

6.2 min, 100.0%; LRMS (M þ H)^þ(ESI^þ) 298.00 [M þ H]^þ(calcd for

F

¼ 24.3 Hz); HPLC purity: 13.3 min, 99.8%; LRMS (M þ H)^þ(ESI^þ)

C

₁₃H₉ClFNO₂SH^þ298.00).

F

298.00 [M þ H]^þ(calcd for C₁₃H₉ClFNO₂SH^þ298.05).

4.18. (E)-5-Chloro-2-(2-((4-ﬂuorophenyl)sulfonyl)vinyl)pyridine

4.14. (E)-2-(2-((4-Fluorophenyl)sulfonyl)vinyl)pyridine (8a)

(8e)

Diethyl (((4-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.51 g,

1.65 mmol), 2.0M n-BuLi solution in cyclohexane (0.90 mL,

1.81 mmol) and 2-pyridinecarboxaldehyde (0.17 mL, 1.81 mmol)

gave 0.44 g (100%) of 8a as a brown solid; R_f¼ 0.28 (n-hexane/

Diethyl (((4-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.3 g,

0.96 mmol), 2.0M n-BuLi solution in cyclohexane (0.58 mL,

1.39 mmol) and 5-chloro-2-pyridinecarboxaldehyde (0.19g,

1.39 mmol) gave 0.15 g (56%) of 8e as a ivory solid; R_f¼ 0.37 (n-

hexane/EtOAc 4/1); mp: 107.1e108.3 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

EtOAc 1/7); mp: 71.8e72.8 ^ꢂC; ¹H NMR (400 MHz, DMSO‑d₆)

d 8.65

(d, J ¼ 4.2 Hz, ArH), 8.04e8.07 (m, 2ArH), 7.91 (td, J ¼ 1.5, 7.6 Hz,

ArH), 7.82 (d, J ¼ 7.7 Hz, ArH), 7.74 (d, J ¼ 15.1 Hz, (E)-isomeric H),

7.69 (d, J ¼ 15.1 Hz, (E)-isomeric H), 7.52 (t, J ¼ 8.8 Hz, 2ArH),

d

8.55 (d, J ¼ 2.4 Hz, ArH), 7.95e7.99(m, 2ArH), 7.71 (dd, J ¼ 2.4,

8.2 Hz, ArH), 7.60 (d, J ¼ 14.8 Hz, (E)-isomeric H), 7.35e7.43 (m, ArH,

(E)-isomeric H), 7.26e7.35 (m, 2ArH); ¹³C NMR (100 MHz, CDCl₃)

7.45e7.48 (m, ArH); ¹³C NMR (100 MHz, DMSO‑d₆)

d

161.9 (d, J_C-

d

165.8 (d, J_C-F¼ 255.0 Hz), 149.5, 149.0, 139.3, 136.7, 136.2 (d, J_C-

¼ 3.02 Hz), 133.6, 132.2, 130.8, 130.7, 126.0, 116.9, 116.6; HPLC

¼ 247.8 Hz), 150.5, 150.1, 142.2 (d, J_C-F¼ 6.6 Hz), 142.1, 137.4, 132.1

F

(d, J_C-F¼ 7.8 Hz), 130.8, 125.7, 125.5, 123.6 (d, J_C-F¼ 3.2 Hz), 121.2 (d,

J_C-F¼ 21.1 Hz), 114.5 (d, J_C-F¼ 24.3 Hz); HPLC purity: 6.4 min,

100.0%; HRMS (M þ H)^þ(ESI^þ) 264.0486 [M þ H]^þ(calcd for

purity: 11.8 min, 99.6%; LRMS (M þ H)^þ(ESI^þ) 298.00 [M þ H]^þ

(calcd for C₁₃H₉ClFNO₂SH^þ298.00).

C

₁₃H₁₀FNO₂SH^þ264.0489).

4.19. (E)-2-Chloro-6-(2-((4-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(8f)

4.15. (E)-3-Fluoro-2-(2-((4-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(8b)

Diethyl (((4-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.96 mmol), 2.0M n-BuLi solution in cyclohexane (0.58 mL,

1.16 mmol) and 6-chloro-2-pyridinecarboxaldehyde (0.16 g,

1.16 mmol) gave 0.16 g (57%) of 8f as a white solid; R_f¼ 0.27 (n-

hexane/EtOAc 3/1); mp: 129.5e130.0 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

Diethyl (((4-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.46 g,

1.47 mmol), 2.0M n-BuLi solution in cyclohexane (0.81 mL,

1.62 mmol) and 3-ﬂuoro-2-pyridinecarboxaldehyde (0.21 mL,

1.62 mmol) gave 0.34 g (74%) of 8b as a white powder; R_f¼ 0.72 (n-

hexane/EtOAc 1/3); mp: 73.0e74.0 ^ꢂC; ¹H NMR (300 MHz,

d

7.96e7.99 (m, 2ArH), 7.69e7.73 (m, ArH), 7.57 (d, J ¼ 14.8 Hz, (E)-

isomeric H), 7.47 (d, J ¼ 14.8 Hz, (E)-isomeric H), 7.34 (dd, J ¼ 1.8,

DMSO‑d₆)

d

8.51e8.53 (m, ArH), 7.59e7.92 (m, 8ArH, (E)-isomeric

161.7 (d, J_C-F¼ 273.9 Hz), 158.3

7.8 Hz, 2ArH), 7.21e7.26 (m, 2ArH); ¹³C NMR (100 MHz, CDCl₃)

2H); ¹³C NMR (75 MHz, DMSO‑d₆)

d

165.9 (d, J_C-F¼ 255.1 Hz), 152.1, 151.3, 139.7, 138.8, 136.0, 133.1,

(d, J_C-F¼ 288.1 Hz), 146.3 (d, J_C-F¼ 4.8 Hz), 141.7 (d, J_C-F¼ 6.7 Hz),

138.4 (d, J_C-F¼ 10.9 Hz), 133.7, 132.1, 132.1 (d, J_C-F¼ 13.7 Hz), 128.0

(d, J_C-F¼ 4.8 Hz), 124.8 (d, J_C-F¼ 18.9 Hz), 123.8 (d, J_C-F¼ 3.0 Hz),

121.2 (d, J_C-F¼ 21.1 Hz), 114.7 (d, J_C-F¼ 24.4 Hz); HPLC purity:

11.0 min, 99.6%; HRMS (M þ H)^þ(ESI^þ) 282.0395 [M þ H]^þ(calcd

for C₁₃H₉F₂NO₂SH^þ282.0395).

130.9, 130.8, 125.9, 124.0, 116.9, 116.6; HPLC purity: 12.4 min, 99.4%;

LRMS (M þ H)^þ(ESI^þ) 298.00 [M þ H]^þ(calcd for C₁₃H₉ClFNO₂SH^þ

298.05).

4.20. (E)-2-(2-((2-Chlorophenyl)sulfonyl)vinyl)pyridine (9a)

Diethyl (((2-chlorophenyl)sulfonyl)methyl)phosphonate (0.25 g,

0.76 mmol), 2.0M n-BuLi solution in cyclohexane (0.42 mL,

0.84 mmol) and 2-pyridinecarboxaldehyde (0.08 mL, 0.84 mmol)

gave 0.21 g (98%) of 9a as a beige powder; R_f¼ 0.34 (n-hexane/

4.16. (E)-3-Chloro-2-(2-((4-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(8c)

Diethyl (((4-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.60 g,

1.93 mmol), 2.0M n-BuLi solution in cyclohexane (1.06 mL,

2.12 mmol) and 3-chloro-2-pyridinecarboxaldehyde (0.30 g,

EtOAc 1/1); mp: 87.0e89.0 ^ꢂC; ¹H NMR (400 MHz, DMSO‑d₆)

d 8.67

(d, J ¼ 4.2 Hz, ArH), 8.16 (d, J ¼ 7.9 Hz, ArH), 7.92 (td, J ¼ 1.5, 7.9 Hz,

ArH), 7.87 (d, J ¼ 7.7 Hz, 2ArH), 7.81 (d, J ¼ 15.1 Hz, (E)-isomeric H),

12

J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

7.66e7.78 (m, 4ArH, (E)-isomeric H), 7.47e7.50 (m, ArH); ¹³C NMR

NMR (100 MHz, CDCl₃) d 148.8, 146.4, 140.4, 136.9, 136.9, 135.4,

(100 MHz, DMSO‑d₆)

132.0, 131.1, 129.8, 128.8, 126.4, 126.2; HPLC purity: 9.3 min, 99.4%;

HRMS (M

H)^þ(ESI^þ) 280.0190 [M H]^þ(calcd for

₁₃H₁₀ClNO₂SH^þ280.0194).

d

150.9, 150.7, 144.2, 138.0, 137.7, 136.2, 132.6,

133.9, 132.2, 131.7, 131.3, 130.6, 126.6, 124.9; HPLC purity: 4.1 min,

99.9%; LRMS (M þ H)^þ(ESI^þ) 313.97 [M þ H]^þ(calcd for

þ

C

₁₃H₉Cl₂NO₂SH^þ313.95).

C

4.25. (E)-5-Chloro-2-(2-((2-chlorophenyl)sulfonyl)vinyl)pyridine

(9f)

4.21. (E)-3-Fluoro-2-(2-((2-chlorophenyl)sulfonyl)vinyl)pyridine

(9b)

Diethyl (((2-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.91 mmol), 2.0M n-BuLi solution in cyclohexane (0.55 mL,

1.10 mmol) and 5-chloro-2-pyridinecarboxaldehyde (0.15 g,

1.10 mmol) gave 0.17 g (60%) of 9f as a white solid; R_f¼ 0.32 (n-

hexane/EtOAc 3/1); mp: 165.7e167.3 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

Diethyl (((2-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.92 mmol), 2.0M n-BuLi solution in cyclohexane (0.51 mL,

1.01 mmol), 3-ﬂuoro-2- pyridinecarboxaldehyde (0.13 g,

1.01 mmol) gave 0.21 g (75%) of 9b as a pale yellow solid; R_f¼ 0.34

(n-hexane/EtOAc 2/1); mp: 101.6e103.6 ^ꢂC; ¹H NMR (400 MHz,

d

8.23 (d, J ¼ 2.5 Hz, ArH), 8.21 (dd, J ¼ 1.6, 7.8 Hz, ArH), 7.78 (dd,

DMSO‑d₆)

7.62e7.93 (m, 5ArH, (E)-isomeric 2H); ¹³C NMR (100 MHz,

DMSO‑d₆)

d

8.55 (d, J ¼ 4.4 Hz, ArH), 8.18 (dd, J ¼ 1.4, 7.8 Hz, ArH),

J ¼ 2.5, 8.4 Hz, ArH), 7.74 (d, J ¼ 15.5 Hz, (E)-isomeric H), 7.48e7.61

(m, 3ArH), 7.39 (d, J ¼ 8.4 Hz, ArH), 7.14 (d, J ¼ 15.5 Hz, (E)-isomeric

d

158.7 (d, J_C-F¼ 262.0 Hz), 147.0 (d, J_C-F¼ 4.7 Hz), 138.7

H); ¹³C NMR (100 MHz, CDCl₃)

d 154.0, 150.0, 140.1, 137.5, 137.2,

(d, J_C-F¼ 10.9 Hz), 137.2, 136.4, 135.9, 132.6, 132.0, 131.3 (d, J_C-

135.0, 132.9, 132.0, 131.0, 128.2, 127.6, 127.3, 124.8; HPLC purity:

¼ 4.3 Hz), 131.2, 128.9, 128.7 (d, J_C-F¼ 4.7 Hz), 125.4 (d, J_C-

10.4 min, 98.3%; LRMS (M þ H)^þ(ESI^þ) 313.97 [M þ H]^þ(calcd for

F

¼ 18.9 Hz); HPLC purity: 9.3 min, 99.1%; HRMS (M þ H)^þ(ESI^þ)

C

₁₃H₉Cl₂NO₂SH^þ314.00).

F

298.0092 [M þ H]^þ(calcd for C₁₃H₉ClFNO₂SH^þ298.0099).

4.26. (E)-2-Chloro-6-(2-((2-chlorophenyl)sulfonyl)vinyl)pyridine

(9g)

4.22. (Z)-3-Fluoro-2-(2-((2-chlorophenyl)sulfonyl)vinyl)pyridine

(9c)

Diethyl (((2-chlorophenyl)sulfonyl)methyl)phosphonate (0.25 g,

0.76 mmol), 2.0M n-BuLi solution in cyclohexane (0.42 mL,

0.85 mmol) and 6-chloro-2-pyridinecarboxaldehyde (0.12 g,

0.85 mmol) gave 0.23 g (94%) of 9g as a white solid; R_f¼ 0.30 (n-

hexane/EtOAc 1/1); mp: 74.2e75.2 ^ꢂC; ¹H NMR (400 MHz,

Diethyl (((2-chlorophenyl)sulfonyl)methyl)phosphonate (0.3 g,

0.92 mmol), 2.0M n-BuLi solution in cyclohexane (0.55 mL,

1.10 mmol) and 3-ﬂuoro-2-pyridinecarboxaldehyde (0.137 mL,

1.10 mmol) gave 0.017 g (6%) of 9c as a yellow solid; R_f¼ 0.50 (n-

hexane/EtOAc 1/1); mp: 112.0e114.0 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

DMSO‑d₆)

d

8.39 (dd, J ¼ 1.6, 7.8 Hz, ArH), 8.18 (dd, J ¼ 1.4, 7.8 Hz,

d

8.24e8.22 (m, 2ArH), 7.54e7.46 (m, ArH, (Z)-isomeric H),

ArH), 7.93 (d, J ¼ 15.4 Hz, (E)-isomeric H), 7.79e7.83 (m, J ¼ 15.2 Hz,

ArH, (E)-isomeric H), 7.76 (td, J ¼ 1.4, 7.3 Hz, ArH), 7.69 (td, J ¼ 1.4,

7.8 Hz, ArH), 7.53e7.56 (m, ArH); ¹³C NMR (100 MHz, DMSO‑d₆)

7.42e7.25 (m, 3ArH), 7.01 (d, J ¼ 11.8 Hz, (Z)-isomeric H); ¹³C NMR

(100 MHz, CDCl₃)

d

157.7 (d, J_C-F¼ 262.6 7 Hz), 144.4 (d, J_C-

¼ 5.3 Hz), 140.2, 139.7 (d, J_C-F¼ 11.6 Hz), 135.2 (d, J_C-F¼ 1.4 Hz),

d 152.4, 150.8, 139.2, 138.7, 137.2, 136.4, 132.7, 132.0, 131.5, 131.3,

F

134.0, 132.8 (d, J_C-F¼ 1.4 Hz), 132.2, 131.3, 130.9, 126.6, 125.9 (d, J_C-

128.9, 127.4, 124.3; HPLC purity: 9.6 min, 99.4%; HRMS (M þ H)^þ

¼ 4.2 Hz), 123.2 (d, J_C-F¼ 18.7 Hz); HPLC purity: 6.0 min, 100.0%;

(ESI^þ) 313.9802 [M þ H]^þ(calcd for C₁₃H₉Cl₂NO₂SH^þ313.9804).

F

LRMS (M þ H)^þ(ESI^þ) 298.00 [M þ H]^þ(calcd for C₁₃H₉ClFNO₂SH^þ

298.00).

4.27. (E)-2-(2-((3-Chlorophenyl)sulfonyl)vinyl)pyridine (10a)

4.23. (E)-3-Chloro-2-(2-((2-chlorophenyl)sulfonyl)vinyl)pyridine

(9d)

Diethyl (((3-chlorophenyl)sulfonyl)methyl)phosphonate (0.50 g,

1.55 mmol), 2.0M n-BuLi solution in cyclohexane (0.82 mL,

1.63 mmol) and 2-pyridinecarboxaldehyde (0.18 mL, 1.71 mmol)

gave 0.37 g (82%) of 10a as a brown solid; R_f¼ 0.35 (n-hexane/EtOAc

Diethyl (((2-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.92 mmol), 2.0M n-BuLi solution in cyclohexane (0.51 mL,

1.01 mmol) and 3-chloro-2-pyridinecarboxaldehyde (0.14 g,

1.01 mmol) gave 0.20 g (70%) of 9d as a pink powder; R_f¼ 0.37 (n-

hexane/EtOAc 2/1); mp: 109.0e111.0 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

3/1); mp: 51.3e51.6 ^ꢂC; ¹H NMR (400 MHz, DMSO‑d₆)

d 8.65 (d,

J ¼ 4.0 Hz, ArH), 8.03 (t, J ¼ 1.7 Hz, ArH), 7.89e7.96 (m, 2ArH),

7.69e7.85 (m, 3ArH, (E)-isomeric 2H), 7.45e7.48 (m, ArH); ¹³C NMR

(100 MHz, CDCl₃)

132.2, 131.3, 127.5, 126.6, 126. 3, 126.1; HPLC purity: 11.5 min, 95.2%;

HRMS (M

H)^þ(ESI^þ) 280.0188 [M H]^þ(calcd for

C

₁₃H₁₀ClNO₂SH^þ280.0194).

d 151.0, 150.6, 142.7, 142.6, 138.0, 134.7, 134.3,

d

8.51 (dd, J ¼ 1.4, 4.52 Hz, ArH), 8.20e8.24 (m, ArH, (E)-isomeric H),

7.73e7.76 (m, ArH, (E)-isomeric H), 7.46e7.58 (m, 3ArH), 7.26e7.29

(m, ArH); ¹³C NMR (100 MHz, CDCl₃)

148.1, 148.0, 138.3, 138.0,

þ

d

137.6, 134.8, 133.3, 133.2, 132.4, 132.0, 131.1, 127.5, 126.0; HPLC pu-

rity: 12.8 min, 99.2%; HRMS (M þ H)^þ(ESI^þ) 313.9802 [M þ H]^þ

(calcd for C₁₃H₉Cl₂NO₂SH^þ313.9804).

4.28. (E)-3-Fluoro-2-(2-((3-chlorophenyl)sulfonyl)vinyl)pyridine

(10b)

4.24. (Z)-3-Chloro-2-(2-((2-chlorophenyl)sulfonyl)vinyl)pyridine

(9e)

Diethyl (((3-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.92 mmol), 2.0M n-BuLi solution in cyclohexane (0.51 mL,

1.01 mmol) and 3-ﬂuoro-2-pyridinecarboxaldehyde (0.13 mL,

1.01 mmol) gave 0.27 g (97%) of 10b as a white powder; R_f¼ 0.38 (n-

hexane/EtOAc 2/1); mp: 81.7e82.7 ^ꢂC; ¹H NMR (400 MHz,

Diethyl (((2-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.92 mmol), 2.0M n-BuLi solution in cyclohexane (0.55 mL,

1.10 mmol) and 3-chloro-2-pyridinecarboxaldehyde (0.55 mL,

1.10 mmol) gave 0.03 g (10%) of 9e as a white solid; R_f¼ 0.50 (n-

hexane/EtOAc 1/1); mp: 128.1e129.3 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

DMSO‑d₆)

J ¼ 7.8 Hz, ArH), 7.69e7.92 (m, 5ArH, (E)-isomeric 2H), 7.60e7.64

(m, ArH); ¹³C NMR (100 MHz, DMSO‑d₆)

d

8.52 (d, J ¼ 4.4 Hz, ArH), 8.08e8.09 (m, ArH), 7.98 (d,

d

158.1 (d, J_C-F¼ 262.1 Hz),

d

8.25 (dd, J ¼ 1.4, 4.6 Hz, ArH), 8.10 (dd, J ¼ 1.5, 7.7 Hz, ArH), 7.64

146.3 (d, J_C-F¼ 4.7 Hz), 141.6, 138.4 (d, J_C-F¼ 10.9 Hz), 134.3, 134.0,

133.8, 132.2 (d, J_C-F¼ 4.1 Hz), 131.7, 128.4 (d, J_C-F¼ 4.6 Hz), 127.3,

126.3,124.8 (d, J_C-F¼ 18.9 Hz); HPLC purity: 11.8 min,100.0%; HRMS

(dd, J ¼ 1.4, 8.2 Hz, ArH), 7.51e7.41 (m, 3ArH, (Z)-isomeric H), 7.16

(q, J ¼ 4.6, 8.16 Hz, ArH), 7.00 (d, J ¼ 11.6 Hz, (Z)-isomeric H); ¹³C

13

J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

(M þ H)^þ(ESI^þ) 298.0096 [M þ H]^þ(calcd for C₁₃H₉ClFNO₂SH^þ

C

₁₃H₉Cl₂NO₂SH^þ314.00).

298.0099).

4.33. (E)-2-Chloro-6-(2-((3-Chlorophenyl)sulfonyl)vinyl) pyridine

(10g)

4.29. (Z)-3-Fluoro-2-(2-((3-chlorophenyl)sulfonyl)vinyl)pyridine

(10c)

Diethyl (((3-chlorophenyl)sulfonyl)methyl)phosphonate (0.08 g,

0.24 mmol), 2.0M n-BuLi solution in cyclohexane (0.15 mL,

0.29 mmol) and 6-chloro-2-pyridinecarboxaldehyde (0.04 mL,

0.29 mmol) gave 0.045 g (59%) of 10g as a white solid; R_f¼ 0.32 (n-

hexane/EtOAc 3/1); mp: 116.3e117.5 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

Diethyl (((3-chlorophenyl)sulfonyl)methyl)phosphonate (0.15 g,

0.46 mmol), 2.0M n-BuLi solution in cyclohexane (0.27 mL,

0.55 mmol) and 3-ﬂuoro-2-pyridinecarboxaldehyde (0.078 g,

0.55 mmol) gave 0.020 g (15%) of 10c as a white solid; R_f¼ 0.26 (n-

hexane/EtOAc 1/1); mp: 109.2e110.3 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

d

7.94 (d, ArH), 7.85 (d, J ¼ 7.8 Hz, ArH), 7.72 (t, J ¼ 7.7 Hz, ArH),

d

8.44e8.43 (m, ArH), 8.06e8.05 (m, ArH), 7.90e7.88 (m, ArH),

7.58e7.62 (m, ArH, (E)-isomeric H), 7.46e7.53 (m, ArH, (E)-isomeric

7.57e7.55 (m, ArH), 7.48e7.40 (m, ArH, (Z)-isomeric H), 7.36e7.31

(m, ArH), 7.26e7.23 (m, ArH), 6.73 (d, J ¼ 11.7 Hz, (Z)-isomeric H);

H), 7.34 (d, J ¼ 8.0 Hz, 2ArH); ¹³C NMR (100 MHz, CDCl₃)

d 152.1,

¹³C NMR (100 MHz, CDCl₃)

d

156.4, 144.8 (d, J_C-F¼ 5.1 Hz), 142.9,

151.2, 141.7, 139.7, 139.5, 135.7, 133.9, 132.6, 130.7, 128.1, 126.1, 126.1,

124.2; HPLC purity: 13.6 min, 100%; LRMS (M þ H)^þ(ESI^þ) 313.97

[M þ H]^þ(calcd for C₁₃H₉Cl₂NO₂SH^þ313.95).

139.7, 135.0, 134.8, 133.5, 130.2, 128.5, 126.2, 125.9 (d, J_C-F¼ 4.2 Hz),

123.3, 123.1; HPLC purity: 11.7 min, 95.0%; LRMS (M þ H)^þ(ESI^þ)

298.00 [M þ H]^þ(calcd for C₁₃H₉ClFNO₂SH^þ297.95).

4.34. (E)-2-(2-((4-Chlorophenyl)sulfonyl)vinyl)pyridine (11a)

4.30. (E)-3-Chloro-2-(2-((3-chlorophenyl)sulfonyl)vinyl)pyridine

(10d)

Diethyl (((4-chlorophenyl)sulfonyl)methyl)phosphonate (0.25 g,

0.76 mmol), 2.0M n-BuLi solution in cyclohexane (0.43 mL,

0.85 mmol) and 2-pyridinecarboxaldehyde (0.08 mL, 0.85 mmol)

gave 0.15 g (70%) of 11a as a white solid; R_f¼ 0.35 (n-hexane/EtOAc

Diethyl (((3-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.92 mmol), 2.0M n-BuLi solution in cyclohexane (0.51 mL,

1.01 mmol) and 6-chloro-2-pyridinecarboxaldehyde (0.14 g,

1.01 mmol) gave 0.20 g (71%) of 10d as a white solid; R_f¼ 0.50 (n-

hexane/EtOAc 2/1); mp: 81.1e82.4 ^ꢂC; ¹H NMR (400 MHz,

2/1); mp: 87.5e89.5 ^ꢂC; ¹H NMR (400 MHz, DMSO‑d₆)

d 8.65 (d,

J ¼ 4.3 Hz, ArH), 7.99 (d, J ¼ 8.6 Hz, 2ArH), 7.91 (td, J ¼ 1.3, 7.7 Hz,

ArH), 7.82 (d, J ¼ 7.7 Hz, ArH), 7.72e7.76 (m, 4ArH), 7.44e7.47 (m,

DMSO‑d₆)

d

8.60 (dd, J ¼ 0.7, 4.0 Hz, ArH), 8.06e8.08 (m, 2ArH),

ArH); ¹³C NMR (100 MHz, DMSO‑d₆)

d 151.1, 150.6, 142.2, 139.4,

7.96e7.99 (m, ArH, (E)-isomeric H), 7.84e7.88 (m, ArH, (E)-isomeric

137.9,131.5,130.3,129.9,126.1,126.0; HPLC purity: 10.1 min,100.0%;

HRMS (M

H)^þ(ESI^þ) 280.0188 [M H]^þ(calcd for

₁₃H₁₀ClNO₂SH^þ280.0194).

H), 7.71 (t, J ¼ 7.9 Hz, ArH), 7.52e7.55 (m, ArH); ¹³C NMR (100 MHz,

þ

CDCl₃)

d 149.0, 147.4, 142.0, 138.9, 136.6, 134.8, 134.5, 133.9, 132.8,

C

132.2, 127.8, 127.7, 126.8; HPLC purity: 12.9 min, 100.0%; HRMS

(M þ H)^þ(ESI^þ) 313.9800 [M þ H]^þ(calcd for C₁₃H₉Cl₂NO₂SH^þ

313.9804).

4.35. (E)-3-Fluoro-2-(2-((4-chlorophenyl)sulfonyl)vinyl)pyridine

(11b)

4.31. (Z)-3-Chloro-2-(2-((3-chlorophenyl)sulfonyl)vinyl)pyridine

(10e)

Diethyl (((4-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.92 mmol), 2.0M n-BuLi solution in cyclohexane (0.51 mL,

1.01 mmol) and 3-ﬂuoro-2-carboxaldehydepyridine (0.13 g,

1.01 mmol) gave 0.19 g (70%) of 11b as a white powder; R_f¼ 0.32 (n-

hexane/EtOAc 3/1); mp: 91.7e93.7 ^ꢂC; ¹H NMR (400 MHz,

Diethyl (((3-chlorophenyl)sulfonyl)methyl)phosphonate (0.15 g,

0.46 mmol), 2.0M n-BuLi solution in cyclohexane (0.27 mL,

0.55 mmol) and 3-chloro-2-pyridinecarboxaldehyde (0.078 g,

0.55 mmol) gave 0.012 g (8%) of 10e as a white solid; R_f¼ 0.34 (n-

hexane/EtOAc 1/1); mp: 76.8e77.5 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

DMSO‑d₆)

d

8.52 (d, J ¼ 4.4 Hz, ArH), 8.02 (d, J ¼ 8.6 Hz, 2ArH), 7.89

(t, J ¼ 9.2 Hz, ArH), 7.60e7.76 (m, 5ArH); ¹³C NMR (100 MHz,

d

8.53 (d, J ¼ 4.6 Hz, ArH), 8.04 (s, ArH), 7.89 (d, J ¼ 7.8 Hz, ArH), 7.70

DMSO‑d₆)

d

158.1 (d, J_C-F¼ 261.9 Hz), 146.4 (d, J_C-F¼ 4.8 Hz), 139.1,

(d, J ¼ 8.2 Hz, ArH), 7.56 (d, J ¼ 8.0 Hz, ArH), 7.46 (t, J ¼ 7.9 Hz, ArH),

7.36 (d, J ¼ 11.6 Hz, (Z)-isomeric H), 7.28e7.25 (m, ArH), 6.71 (d,

138.5 (d, J_C-F¼ 3.4 Hz), 138.4, 133.3, 132.5 (d, J_C-F¼ 4.2 Hz), 129.8,

129.6, 128.0 (d, J_C-F¼ 4.7 Hz), 124.8 (d, J_C-F¼ 18.8 Hz); HPLC purity:

7.4 min, 99.7%; HRMS (M þ H)^þ(ESI^þ) 298.0091 [M þ H]^þ(calcd for

J ¼ 11.5 Hz, (Z)-isomeric H); ¹³C NMR (100 MHz, CDCl₃)

d 149.2,

₁₃H₉ClFNO₂SH^þ298.0099).

146.9, 142.8, 137.6, 136.8, 135.0, 134.6, 133.5, 131.5, 130.2, 128.5,

126.2, 125.0; HPLC purity: 11.7 min, 95.0%; LRMS (M þ H)^þ(ESI^þ)

313.97 [M þ H]^þ(calcd for C₁₃H₉Cl₂NO₂SH^þ313.95).

C

4.36. (E)-3-Chloro-2-(2-((4-chlorophenyl)sulfonyl)vinyl)pyridine

(11c)

4.32. (E) -5-Chloro -2-(2-((3-chlorophenyl)sulfonyl)vinyl)pyridine

(10f)

Diethyl (((4-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.92 mmol), 2.0M n-BuLi solution in cyclohexane (0.51 mL,

1.01 mmol) and 3-chloro-2-pyridinecarboxaldehyde (0.14 g,

1.01 mmol) gave 0.22 g (74%) of 11c as a white powder; R_f¼ 0.37 (n-

hexane/EtOAc 3/1); mp: 106.2e107.4 ^ꢂC; ¹H NMR (400 MHz,

Diethyl (((3-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.91 mmol), 2.0M n-BuLi solution in cyclohexane (0.55 mL,

1.10 mmol) and 5-chloro-2-pyridinecarboxaldehyde (0.15 mL,

1.10 mmol) gave 0.16 g (57%) of 10f as a white solid; R_f¼ 0.39 (n-

hexane/EtOAc 4/1); mp: 129.0e130.5 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

DMSO‑d₆)

d

8.61 (d, J ¼ 4.4 Hz, ArH), 8.08 (d, J ¼ 8.2 Hz, ArH), 8.03

d

8.56 (d, J ¼ 2.4 Hz, ArH), 7.94 (t, J ¼ 1.8 Hz, ArH), 7.85 (dt, J ¼ 1.3,

(d, J ¼ 7.2 Hz, 2ArH), 7.96 (d, J ¼ 14.7 Hz, (E)-isomeric H), 7.81 (d,

7.8 Hz, ArH), 7.73 (dd, J ¼ 2.4, 8.2 Hz, ArH), 7.59e7.65 (m, ArH, (E)-

isomeric H), 7.50 (t, J ¼ 7.9 Hz, ArH), 7.36e7.43 (m, ArH, (E)-isomeric

J ¼ 14.8 Hz, (E)-isomeric H), 7.76 (d, J ¼ 7.2 Hz, 2ArH), 7.53e7.56 (m,

ArH); ¹³C NMR (100 MHz, DMSO‑d₆)

d 149.1, 147.4, 139.7, 138.9,

H); ¹³C NMR (100 MHz, CDCl₃)

d

149.5, 148.8, 141.9, 140.1, 136.7,

138.8, 136.1, 134.2, 132.8, 130.4, 130.1, 127.6; HPLC purity: 8.1 min,

135.6, 133.8, 133.7, 131.7, 130.7, 128.0, 126.1, 126.0; HPLC purity:

99.7%; HRMS (M þ H)^þ(ESI^þ) 313.9802 [M þ H]^þ(calcd for

14.0 min, 99.7%; LRMS (M þ H)^þ(ESI^þ) 313.97 [M þ H]^þ(calcd for

C

₁₃H₉Cl₂NO₂SH^þ313.9804).

14

J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

4.37. (Z)-3-Chloro-2-(2-((4-chlorophenyl)sulfonyl)vinyl)pyridine

(11d)

4.41. (E)-2-Chloro-3-(2-((3-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(12b)

Diethyl (((4-chlorophenyl)sulfonyl)methyl)phosphonate (0.15 g,

0.46 mmol), 2.0M n-BuLi solution in cyclohexane (0.27 mL,

0.55 mmol) and 3-chloro-2-pyridinecarboxaldehyde (0.078 g,

0.55 mmol) gave 0.016 g (11%) of 11d as a white solid; R_f¼ 0.36 (n-

hexane/EtOAc 2/1); mp: 133.9e134.8 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

Diethyl (((2-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.3 g,

0.96 mmol), 2.0M n-BuLi solution in cyclohexane (0.62 mL,

1.25 mmol) and 2-chloro-3-pyridinecarboxaldehyde (0.18g,

1.25 mmol) gave 0.17 g (58%) of 12b as a white solid; R_f¼ 0.41 (n-

hexane/EtOAc 1/1); mp: 139.8e140.2 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

d

8.53e8.51 (m, ArH), 7.99e7.95 (m, 2ArH), 7.71 (dd, J ¼ 1.4, 8.2 Hz,

d

8.45 (dd, J ¼ 1.7, 4.7 Hz, ArH), 8.01e8.10 (m, ArH, (E)-isomeric H),

ArH), 7.52e7.49 (m, 2ArH), 7.33 (d, J ¼ 11.5 Hz, (Z)-isomeric H),

7.89 (dd, J ¼ 1.7, 7.8 Hz, ArH), 7.63e7.69 (m, ArH), 7.36 (t, J ¼ 7.61 Hz,

ArH), 7.30e7.33 (m, ArH), 7.21e7.26 (m, ArH), 7.13 (d, J ¼ 15.5 Hz,

7.28e7.25 (m, ArH), 6.70 (d, J ¼ 11.6 Hz, (Z)-isomeric H); ¹³C NMR

(100 MHz, CDCl₃)

d

149.4, 146.9, 140.1, 139.5, 137.2, 136.8, 134.7,

(E)-isomeric H); ¹³C NMR (100 MHz, CDCl₃)

d 159.6 (d, J_C-

131.4, 129.7, 129.2, 124.8; HPLC purity: 6.4 min, 98.9%; LRMS

(M þ H)^þ(ESI^þ) 313.97 [M þ H]^þ(calcd for C₁₃H₉Cl₂NO₂SH^þ

313.95).

¼ 255.2 Hz), 151.7, 151.5, 139.1, 136.9, 136.4 (d, J_C-F¼ 8.5 Hz), 131.1,

F

129.8, 128.0 (d, J_C-F¼ 13.9 Hz), 127.6, 124.9 (d, J_C-F¼ 3.7 Hz), 122.9,

117.3 (d, J_C-F¼ 20.9 Hz); HPLC purity: 12.5 min, 100%; HRMS

(M þ H)^þ(ESI^þ) 298.0095 [M þ H]^þ(calcd for C₁₄H₁₂FNO₃SH^þ

298.0099).

4.38. (E)-5-Chloro-2-(2-((4-chlorophenyl)sulfonyl)vinyl)pyridine

(11e)

4.42. (Z)-2-Chloro-3-(2-((2-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(12c)

Diethyl (((4-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.91 mmol), 2.0M n-BuLi solution in cyclohexane (0.55 mL,

1.10 mmol) and 5-chloro-2-pyridinecarboxaldehyde (0.15 g,

1.10 mmol) gave 0.16 g (57%) of 11e as a white solid; R_f¼ 0.34 (n-

hexane/EtOAc 4/1); mp: 154.0e155.4 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

Diethyl (((2-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.96 mmol), 2.0M n-BuLi solution in cyclohexane (0.58 mL,

1.16 mmol) and 2-chloro-3-pyridinecarboxaldehyde (0.16 g,

1.16 mmol) gave 0.02 g (7%) of 12c as a white solid; R_f¼ 0.46 (n-

hexane/EtOAc 2/1); mp: 109.2e110.3 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

d

8.58 (d, J ¼ 2.4 Hz, ArH), 7.90e7.93 (m, 2ArH), 7.74 (dd, J ¼ 2.5,

d

8.35e8.33 (m, ArH), 8.00 (d, J ¼ 7.6 Hz, ArH), 7.64e7.56 (m, 2ArH),

7.29e7.15 (m, 3ArH, (Z)-isomeric H), 6.89 (dd, J ¼ 1.1, 11.7 Hz, (Z)-

isomeric H); ¹³C NMR (100 MHz, CDCl₃)

8.3 Hz, ArH), 7.63 (d, J ¼ 14.8 Hz, (E)-isomeric H), 7.56 (dt, J ¼ 2.2,

8.6 Hz, 2ArH), 7.37e7.45 (m, ArH, (E)-isomeric H); ¹³C NMR

d

159.6 (d, J_C-F¼ 255.4 Hz),

(100 MHz, CDCl₃)

d 149.5, 148.9, 140.5, 139.6, 138.6, 136.7, 133.6,

150.2, 149.3, 141.0, 137.3, 136.4 (d, J_C-F¼ 8.6 Hz), 133.9, 129.6, 128.0

(d, J_C-F¼ 14.1 Hz), 127.6, 124.4 (d, J_C-F¼ 3.7 Hz), 121.7, 117.3 (d, J_C-

132.0, 129.8, 129.4, 126.0; HPLC purity: 14.0 min, 100%; LRMS

(M þ H)^þ(ESI^þ) 313.97 [M þ H]^þ(calcd for C₁₃H₉Cl₂NO₂SH^þ

314.00).

¼ 20.8 Hz); HPLC purity: 11.7 min, 95.0%; LRMS (M þ H)^þ(ESI^þ)

F

313.97 [M þ H]^þ(calcd for C₁₃H₉Cl₂NO₂SH^þ313.95).

4.43. (E)-2-Chloro-5-(2-((2-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(12d)

4.39. (E)-2-Chloro-6-(2-((4-clorophenyl)sulfonyl)vinyl)pyridine

(11f)

Diethyl (((2-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.96 mmol), 2.0M n-BuLi solution in cyclohexane (0.58 mL,

1.16 mmol) and 6-chloro-3-pyridinecarboxaldehyde (0.16 g,

1.16 mmol) gave 0.15 g (53%) of 12d as a ivory solid; R_f¼ 0.32 (n-

hexane/EtOAc 3/1); mp: 170.0e170.6 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

Diethyl (((4-clorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.96 mmol), 2.0M n-BuLi solution in cyclohexane (0.58 mL,

1.16 mmol) and 6-chloro-2-pyridinecarboxaldehyde (0.16g,

1.16 mmol) gave 0.17g (60%) of 11f as a white solid; R_f¼ 0.32 (n-

hexane/EtOAc 3/1); mp: 164.8e166.5 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

d

8.53 (d, J ¼ 2.4 Hz, ArH), 8.04 (td, J ¼ 1.5, 7.5 Hz, ArH), 7.82 (dd,

d

7.92e7.90 (m, 2ArH), 7.73 (t, J ¼ 7.8 Hz, ArH), 7.54e7.62 (m, ArH,

J ¼ 2.5, 8.4 Hz, ArH), 7.74 (d, J ¼ 15.4 Hz, (E)-isomeric H), 7.63e7.68

(m, ArH), 7.34e7.41 (m, 2ArH), 7.20e7.26 (m, ArH), 7.10 (d,

(E)-isomeric H), 7.49 (d, J ¼ 14.8 Hz, (E)-isomeric H), 7.36 (q,

J ¼ 3.7 Hz, 2ArH); ¹³C NMR (100 MHz, CDCl₃)

d 152.1, 151.3, 140.5,

J ¼ 15.5 Hz, (E)-isomeric H); ¹³C NMR (100 MHz, CDCl₃)

d 159.6 (d,

139.7, 139.1, 138.5, 132.8, 129.8, 129.5, 126.0, 124.1; HPLC purity:

J_C-F¼ 255.1 Hz), 154.0, 150.1, 139.4, 137.2, 136.3 (d, J_C-F¼ 8.6 Hz),

129.8, 129.1, 128.1 (d, J_C-F¼ 14.0 Hz), 127.2; HPLC purity: 10.3 min,

100%; LRMS (M þ H)^þ(ESI^þ) 298.00 [M þ H]^þ(calcd for

14.9 min, 100.0%; LRMS (M þ H)^þ(ESI^þ) 313.97 [M þ H]^þ(calcd for

C

₁₃H₉Cl₂NO₂SH^þ314.00).

C

₁₃H₉ClFNO₂SH^þ298.05).

4.40. (E)-3-(2-((2-Fluorophenyl)sulfonyl)vinyl)pyridine (12a)

4.44. (E)-3-(2-((3-ﬂuorophenyl)sulfonyl)vinyl)pyridine (13a)

Diethyl (((2-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.97 mmol), 2.0M n-BuLi solution in cyclohexane (0.53 mL,

1.06 mmol) and 3-pyridinecarboxaldehyde (0.10 mL, 1.06 mmol)

gave 0.22 g (84%) of 12a as a yellow powder; R_f¼ 0.36 (n-hexane/

EtOAc 2/1); mp: 107.7e109.1 ^ꢂC; ¹H NMR (400 MHz, DMSO‑d₆)

Diethyl (((3-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.50 g,

1.61 mmol), 2.0M n-BuLi solution in cyclohexane (0.97 mL,

1.93 mmol) and 3-pyridinecarboxaldehyde (0.2 mL, 1.93 mmol)

gave 0.27 g (64%) of 13a as a brown solid; R_f¼ 0.26 (n-hexane/

EtOAc 1/1); mp: 89.9e91.9 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

d 8.74 (d,

d

8.96 (s, ArH), 8.64 (d, J ¼ 4.6 Hz, ArH), 8.26 (d, J ¼ 7.8 Hz, ArH), 7.96

J ¼ 1.8 Hz, ArH), 8.64 (dd, J ¼ 1.3, 4.8 Hz, ArH), 7.64e7.81 (m, 4ArH),

(t, J ¼ 7.5 Hz, ArH), 7.46e7.88 (m, (E)-isomeric 2H, 4ArH); ¹³C NMR

7.54e7.59 (m, ArH), 7.32e7.37 (m, ArH, (E)-isomeric H), 6.95 (d,

(100 MHz, DMSO‑d₆)

d

159.2 (d, J_C-F¼ 253.4 Hz), 152.3, 151.0, 141.2,

J ¼ 15.5 Hz, (E)-isomeric H); ¹³C NMR (100 MHz, CDCl₃)

d 162.6 (d,

137.5 (d, J_C-F¼ 8.7 Hz), 136.1, 129.9, 129.6, 128.7, 128.4 (d, J_C-

J_C-F¼ 251.0 Hz), 152.1, 150.1, 142.2 (d, J_C-F¼ 6.5 Hz), 139.7, 134.9,

131.4 (d, J_C-F¼ 7.7 Hz), 128.9, 128.1, 123.9, 123.6 (d, J_C-F¼ 3.2 Hz),

121.0 (d, J_C-F¼ 21.1 Hz), 115.1 (d, J_C-F¼ 24.3 Hz); HPLC purity:

6.3 min, 100%; HRMS (M þ H)^þ(ESI^þ) 264.0488 [M þ H]^þ(calcd for

¼ 13.9 Hz), 126.0 (d, J_C-F¼ 3.6 Hz), 124.5, 118.0 (d, J_C-F¼ 20.7 Hz);

F

HPLC purity: 6.2 min,100.0%; HRMS (M þ H)^þ(ESI^þ) 264.0486 [M þ

H]^þ(calcd for C₁₃H₁₀FNO₂SH^þ264.0489).

15

J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

C₁₃H₁₀FNO₂SH^þ264.0489).

C

₁₃H₉ClFNO₂SH^þ298.05).

4.45. (E)-2-Chloro-3-(2-((3-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(13b)

4.49. (E)-2-Chloro-5-(2-((4-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(14b)

Diethyl (((3-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.96 mmol), 2.0M n-BuLi solution in cyclohexane (0.58 mL,

1.16 mmol) and 2-chloro-3-pyridinecarboxaldehyde (0.16 g,

1.16 mmol) gave 0.17 g (60%) of 13b as a white powder; R_f¼ 0.26 (n-

hexane/EtOAc 2/1); mp: 118.1e119.3 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

Diethyl (((4-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.92 mmol), 2.0M n-BuLi solution in cyclohexane (0.55 mL,

1.10 mmol) and 6-chloro-3-pyridinecarboxaldehyde (0.15g,

1.10 mmol) gave 0.17 g (63%) of 14b as a white solid; R_f¼ 0.32 (n-

hexane/EtOAc 4/1); mp: 177.3e180.1 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

d

8.44 (t, J ¼ 2.3 Hz, ArH), 8.01 (d, J ¼ 15.5 Hz, (E)-isomeric H), 7.79

d

8.51 (d, J ¼ 2.4 Hz, ArH), 7.95e8.99 (m, 2ArH), 7.75 (dd, J ¼ 2.5,

(dd, J ¼ 7.8, 16.8 Hz, 2ArH), 7.66e7.68 (m, ArH), 7.55e7.61 (m, ArH),

7.26e7.38 (m, 2ArH), 6.94 (d, J ¼ 15.5 Hz, (E)-isomeric H); ¹³C NMR

8.4 Hz, 2ArH), 7.64 (d, J ¼ 15.5 Hz, (E)-isomeric H), 7.38 (d,

J ¼ 8.4 Hz, ArH), 7.23e7.27 (m, 2ArH), 6.91 (d, J ¼ 15.5 Hz, (E)-

(100 MHz, CDCl₃)

d

162.6 (d, J_C-F¼ 251.4 Hz),151.6, 151.5, 141.9 (d, J_C-

isomeric H); ¹³C NMR (100 MHz, CDCl₃)

d

165.9 (d, J_C-F¼ 255.3 Hz),

¼ 6.5 Hz), 137.6, 136.8, 131.6, 131.5 (d, J_C-F¼ 7.7 Hz), 127.5, 123.8 (d,

F

153.8, 149.9, 137.4, 137.1, 136.0 (d, J_C-F¼ 3.2 Hz), 130.8, 130.7, 130.1,

127.1, 124.8, 117.0, 116.8; HPLC purity: 7.3 min,100%; LRMS (M þ H)^þ

(ESI^þ) 298.00 [M þ H]^þ(calcd for C₁₃H₉ClFNO₂SH^þ298.00).

J_C-F¼ 3.4 Hz), 122.9, 121.2 (d, J_C-F¼ 21.1 Hz), 115.3 (d, J_C-F¼ 24.4 Hz);

HPLC purity: 10.5 min, 98.5%; LRMS (M þ H)^þ(ESI^þ) 298.00 [M þ

H]^þ(calcd for C₁₃H₉ClFNO₂SH^þ298.00).

4.46. (E)-2-Chloro-5-(2-((3-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(13c)

4.50. (E)-3-(2-((2-chlorophenyl)sulfonyl)vinyl)pyridine (15a)

Diethyl (((2-chlorophenyl)sulfonyl)methyl)phosphonate (0.25 g,

0.76 mmol), 2.0M n-BuLi solution in cyclohexane (0.42 mL,

0.83 mmol) and 3-pyridinecarboxaldehyde (0.10 mL, 0.83 mmol)

gave 0.12 g (56%) of 15a as a white solid; R_f¼ 0.38 (n-hexane/EtOAc

Diethyl (((3-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.20 g,

0.64 mmol), 2.0M n-BuLi solution in cyclohexane (0.38 mL,

0.77 mmol) and 6-chloro-3-pyridinecarboxaldehyde (0.11 g,

0.77 mmol) gave 0.10 g (52%) of 13c as a white solid; R_f¼ 0.20 (n-

hexane/EtOAc 4/1); mp: 149.9e150.9 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

2/1); mp: 108.0e110.0 ^ꢂC; ¹H NMR (400 MHz, DMSO‑d₆)

d 8.97 (d,

J ¼ 1.3 Hz, ArH), 8.64 (d, J ¼ 3.9 Hz, ArH), 8.25 (d, J ¼ 8.0 Hz, ArH),

d

8.52 (d, J ¼ 2.5 Hz, ArH), 7.74e7.77 (m, 2ArH), 7.64e7.69 (m, ArH,

8.16e8.18 (m, ArH), 7.65e7.87 (m, (E)-isomeric 2H, 3ArH),

(E)-isomeric H), 7.55e7.60 (m, ArH), 7.33e7.40 (m, 2ArH), 6.92 (d,

7.47e7.50 (m, ArH); ¹³C NMR (100 MHz, DMSO‑d₆)

d 152.3, 151.0,

J ¼ 15.5 Hz, (E)-isomeric H); ¹³C NMR (100 MHz, CDCl₃)

d 162.6 (d,

142.2, 137.8, 136.1, 136.0, 132.6, 132.1, 131.0, 128.7, 128.5, 124.5; HPLC

purity: 7.1 min, 99.4%; HRMS (M þ H)^þ(ESI^þ) 280.0190 [M þ H]^þ

(calcd for C₁₃H₁₀ClNO₂SH^þ280.0194).

J_C-F¼ 251.3 Hz), 154.0, 150.0, 142.0 (d, J_C-F¼ 6.4 Hz), 138.1, 137.2,

131.4 (d, J_C-F¼ 7.8 Hz), 129.5, 127.1, 124.9, 121.1 (d, J_C-F¼ 3.2 Hz),

115.2 (d, J_C-F¼ 24.2 Hz); HPLC purity: 9.8 min,100%; LRMS (M þ H)^þ

(ESI^þ) 298.00 [M þ H]^þ(calcd for C₁₃H₉ClFNO₂SH^þ298.00).

4.51. (E)-2-Chloro-3-(2-((2-chlorophenyl)sulfonyl)vinyl)pyridine

(15b)

4.47. (Z)-2-Chloro-5-(2-((3-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(13d)

Diethyl (((2-chlorophenyl)sulfonyl)methyl)phosphonate (0.25 g,

0.76 mmol), 2.0M n-BuLi solution in cyclohexane (0.43 mL,

0.85 mmol) and 2-chloro-3-pyridinecarboxaldehyde (0.12 g,

0.85 mmol) gave 0.23 g (96%) of 15b as a white powder; R_f¼ 0.30

(n-hexane/EtOAc 2/1); mp: 116.9e118.4 ^ꢂC; ¹H NMR (400 MHz,

Diethyl (((3-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.2 g,

0.64 mmol), 2.0M n-BuLi solution in cyclohexane (0.38 mL,

0.77 mmol) and 6-chloro-3-pyridinecarboxaldehyde (0.11 g,

0.77 mmol) gave 0.020 g (10%) of 13d as a white solid; R_f¼ 0.28 (n-

hexane/EtOAc 2/1); mp: 108.2e108.5 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

DMSO‑d₆)

d

8.16 (dd, J ¼ 1.4, 7.9 Hz, ArH), 7.99 (t, J ¼ 7.8 Hz, ArH),

d

8.54e8.53 (m, ArH), 7.83e7.78 (m, 2ArH), 7.71 (dd, J ¼ 1.2, 8.1 Hz,

7.89 (d, J ¼ 7.4 Hz, ArH), 7.66e7.82 (m, (E)-isomeric 2H, 3ArH), 7.61

(d, J ¼ 7.8 Hz, ArH); ¹³C NMR (100 MHz, DMSO‑d₆)

d 151.6, 151.1,

ArH), 7.54e7.49 (m, ArH), 7.36 (d, J ¼ 11.6 Hz, (Z)-isomeric H),

7.33e7.26 (m, 2ArH), 6.71 (d, J ¼ 11.5 Hz, (Z)-isomeric H); ¹³C NMR

142.4, 141.6, 137.3, 136.3, 132.6, 132.1, 131.3, 131.2, 128.8, 126.8,

125.5; HPLC purity: 8.0 min, 97.6%; HRMS (M þ H)^þ(ESI^þ) 313.9802

[M þ H]^þ(calcd for C₁₃H₉C₂lNO₂SH^þ313.9804).

(100 MHz, CDCl₃)

d

162.3 (d, J_C-F¼ 250.0 Hz), 149.3, 146.9, 143.1 (d,

J_C-F¼ 6.9 Hz), 137.6, 136.8, 134.4, 131.4, 130.7 (d, J_C-F¼ 7.6 Hz), 124.9,

123.9 (d, J_C-F¼ 3.4 Hz), 120.7 (d, J_C-F¼ 21.2 Hz), 115.6 (d, J_C-

¼ 24.4 Hz); HPLC purity: 5.9 min, 95.1%; LRMS (M þ H)^þ(ESI^þ)

F

298.00 [M þ H]^þ(calcd for C₁₃H₉ClFNO₂SH^þ298.00).

4.52. (Z)-2-Chloro-3-(2-((2-chlorophenyl)sulfonyl)vinyl)pyridine

(15c)

4.48. (E)-2-Chloro-3-(2-((4-ﬂuorophenyl)sulfonyl)vinyl)pyridine

(14a)

Diethyl (((2-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.92 mmol), 2.0M n-BuLi solution in cyclohexane (0.55 mL,

1.10 mmol) and 2-chloro-3-pyridinecarboxaldehyde (0.156 g,

1.10 mmol) gave 0.02 g (7%) of 15c as a white solid; R_f¼ 0.27 (n-

hexane/EtOAc 2/1); mp: 169.0e169.5 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

Diethyl (((4-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.96 mmol), 2.0M n-BuLi solution in cyclohexane (0.58 mL,

1.39 mmol) and 2-chloro-3-pyridinecarboxaldehyde (0.19 g,

1.39 mmol) gave 0.16 g (57%) of 14a as a white solid; R_f¼ 0.26 (n-

hexane/EtOAc 2/1); mp: 153.1e155.0 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

d

8.31 (dd, J ¼ 1.5, 4.8 Hz, ArH), 8.00 (dd, J ¼ 1.3, 7.6 Hz, ArH),

7.75e7.72 (m, ArH), 7.50e7.49 (m, 2ArH), 7.28e7.24 (m, 2ArH, (Z)-

isomeric H), 6.93 (d, J ¼ 11.7 Hz, (Z)-isomeric H); ¹³C NMR

d

8.43 (dd, J ¼ 1.8, 4.7 Hz, ArH), 7.96e8.00 (m, 2ArH, (E)-isomeric H),

7.82 (dd, J ¼ 1.8, 7.7 Hz, ArH), 7.24e7.31 (m, 3ArH), 6.93 (d,

(100 MHz, CDCl₃) d 150.1, 149.3, 141.0, 137.5, 137.3, 134.9, 133.8,

J ¼ 15.5 Hz, (E)-isomeric H); ¹³C NMR (100 MHz, CDCl₃)

d

165.9 (d,

133.1, 131.9, 130.5, 127.6, 126.9, 121.7; HPLC purity: 7.5 min, 100.0%;

LRMS (M - H)^-(ESI^ꢁ) 311.97 [M - H]^-(calcd for C₁₃H₈Cl₂NO₂S^ꢁ

311.95).

J_C-F¼ 255.3 Hz), 151.5, 151.4, 137.0, 136.8, 135.9; HPLC purity:

11.5 min, 99.6%; LRMS (M þ H)^þ(ESI^þ) 298.00 [M þ H]^þ(calcd for

16

J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

4.53. (E)-2-Chloro-5-(2-((2-chlorophenyl)sulfonyl)vinyl)pyridine

(15d)

4.57. (Z)-2-Chloro-5-(2-((3-chlorophenyl)sulfonyl)vinyl)pyridine

(16d)

Diethyl (((2-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.91 mmol), 2.0M n-BuLi solution in cyclohexane (0.55 mL,

1.10 mmol) and 6-chloro-3-pyridinecarboxaldehyde (0.15g,

1.10 mmol) gave 0.19 g (67%) of 15d as a white solid; R_f¼ 0.28 (n-

hexane/EtOAc 3/1); mp: 163.3e164.7 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

Diethyl (((3-chlorophenyl)sulfonyl)methyl)phosphonate (0.15 g,

0.46 mmol), 2.0M n-BuLi solution in cyclohexane (0.27 mL,

0.55 mmol) and 6-chloro-3-pyridinecarboxaldehyde (0.078 g,

0.55 mmol) gave 0.035 g (25%) of 16d as a white solid; R_f¼ 0.26 (n-

hexane/EtOAc 4/1); mp: 167.7e168.7 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

d

8.53 (d, J ¼ 2.4 Hz, ArH), 8.25 (dd, J ¼ 1.6, 7.8 Hz, ArH), 7.72e7.80

d

8.52 (d, J ¼ 2.2 Hz, ArH), 7.93 (s, ArH, (Z)-isomeric H), 7.54e7.50

(m, ArH), 7.39 (d, J ¼ 8.36 Hz, (Z)-isomeric H), 6.92 (d, J ¼ 15.5 Hz,

ArH); ¹³C NMR (100 MHz, CDCl₃)

154.0, 150.0, 141.7, 138.2, 137.2,

(m, ArH, (E)-isomeric H), 7.48e7.61 (m, ArH), 7.39 (d, J ¼ 8.4 Hz,

ArH), 7.14 (d, J ¼ 15.5 Hz, (E)-isomeric H); ¹³C NMR (100 MHz,

d

CDCl₃)

d

153.9, 150.0, 140.1, 137.5, 137.2, 135.0, 132.9, 132.0, 131.0,

135.8, 134.0, 130.9, 129.5, 127.9, 127.1, 126.0, 124.8; HPLC purity:

128.2, 127.6, 127.3, 124.8; HPLC purity: 7.1 min, 99.5%; LRMS

(M þ H)^þ(ESI^þ) 313.97 [M þ H]^þ(calcd for C₁₃H₉Cl₂NO₂SH^þ

313.95).

7.0 min, 100.0%; LRMS (M - H)^-(ESI^ꢁ) 311.97 [M - H]^-(calcd for

C

₁₃H₈Cl₂NO₂S^ꢁ311.90).

4.58. (E)-2-Chloro-3-(2-((4-chlorophenyl)sulfonyl)vinyl)pyridine

(17a)

4.54. (E)-3-(2-((3-chlorophenyl)sulfonyl)vinyl)pyridine (16a)

Diethyl (((4-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.91 mmol), 2.0M n-BuLi solution in cyclohexane (0.55 mL,

1.10 mmol) and 2-chloro-3-pyridinecarboxaldehyde (0.15g,

1.10 mmol) gave 0.17 g (61%) of 17a as a white solid; R_f¼ 0.33 (n-

hexane/EtOAc 2/1); mp: 152.8e154.8 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

Diethyl (((3-chlorophenyl)sulfonyl)methyl)phosphonate (0.20 g,

0.61 mmol), 2.0M n-BuLi solution in cyclohexane (0.36 mL,

0.73 mmol) and 3-pyridinecarboxaldehyde (0.07 mL, 0.73 mmol)

gave 0.10 g (58%) of 16a as a ivory powder; R_f¼ 0.27 (n-hexane/

EtOAc 1/1); mp: 88.1e90.1 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

d 8.74 (s,

d

8.43 (dd, J ¼ 1.8, 4.7 Hz, ArH), 8.00 (d, J ¼ 13.5 Hz, (E)-isomeric H),

ArH), 8.65 (d, J ¼ 4.8 Hz, ArH), 7.94 (d, J ¼ 1.4 Hz, ArH), 7.83 (dd,

J ¼ 7.9, 17.9 Hz, 2ArH), 7.71 (d, J ¼ 15.5 Hz, (E)-isomeric H), 7.61 (d,

J ¼ 8.0 Hz, ArH), 7.51 (t, J ¼ 7.9 Hz, ArH), 7.34e7.37 (m, ArH), 6.94 (d,

7.91 (dt, J ¼ 2.1, 8.6 Hz 2ArH), 7.83 (dd, J ¼ 1.8, 7.8 Hz, ArH), 7.56 (dt,

J ¼ 2.2, 8.6 Hz, 2ArH), 7.30 (q, J ¼ 4.1 Hz, ArH), 6.92 (d, J ¼ 15.5 Hz,

(E)-isomeric H); ¹³C NMR (100 MHz, CDCl₃)

d 151.6, 151.5, 140.7,

J ¼ 15.5 Hz, (E)-isomeric H); ¹³C NMR (100 MHz, CDCl₃)

d 152.1,

138.3, 137.4, 136.8, 131.9, 129.9, 129.4, 127.5, 122.9; HPLC purity:

13.4 min, 99.9%; LRMS (M þ H)^þ(ESI^þ) 313.97 [M þ H]^þ(calcd for

150.1,141.9,139.8,135.7,134.9,133.8,130.8,128.9,128.1,127.8,125.9,

123.9; HPLC purity: 6.9 min, 100%; HRMS (M þ H)^þ(ESI^þ) 280.0189

[M þ H]^þ(calcd for C₁₃H₁₀ClNO₂SH^þ280.0194).

C

₁₃H₉Cl₂NO₂SH^þ313.95).

4.59. (E)-2-Chloro-5-(2-((4-chlorophenyl)sulfonyl)vinyl)pyridine

(17b)

4.55. (E)-2-Chloro-3-(2-((3-chlorophenyl)sulfonyl)vinyl)pyridine

(16b)

Diethyl (((4-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.92 mmol), 2.0M n-BuLi solution in cyclohexane (0.55 mL,

1.10 mmol) and 6-chloro-3-pyridinecarboxaldehyde (0.15 g,

1.10 mmol) gave 0.18 g (64%) of 17b as a white solid; R_f¼ 0.35 (n-

hexane/EtOAc 4/1); mp: 210.6e212.9 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

Diethyl (((3-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.91 mmol), 2.0M n-BuLi solution in cyclohexane (0.55 mL,

1.10 mmol) and 2-chloro-3-pyridinecarboxaldehyde (0.15g,

1.10 mmol) gave 0.15 g (53%) of 16b as a light yellow powder;

R_f¼ 0.32 (n-hexane/EtOAc 2/1); mp: 175.8e176.8 ^ꢂC; ¹H NMR

d

8.50 (d, J ¼ 2.4 Hz, ArH), 7.89 (dt, J ¼ 2.2, 8.7 Hz, 2ArH), 7.75 (dd,

J ¼ 2.5, 8.4 Hz, ArH), 7.65 (d, J ¼ 15.5 Hz, (E)-isomeric H), 7.55 (dt,

J ¼ 2.2, 8.6 Hz, 2ArH), 7.38 (d, J ¼ 8.4 Hz, ArH), 6.91 (d, J ¼ 15.5 Hz,

(400 MHz, CDCl₃)

d

8.43 (dd, J ¼ 1.7, 4.7 Hz, ArH), 8.02 (d,

J ¼ 15.5 Hz, (E)-isomeric H), 7.95 (d, J ¼ 1.7 Hz, ArH), 7.83e7.86 (m,

2ArH), 7.63 (d, J ¼ 8.1 Hz, ArH), 7.53 (t, J ¼ 7.9 Hz, ArH), 7.31 (q,

J ¼ 4.2 Hz, ArH), 6.93 (d, J ¼ 15.5 Hz, (E)-isomeric H); ¹³C NMR

(E)-isomeric H); ¹³C NMR (100 MHz, CDCl₃)

d 150.1, 140.7, 138.4,

137.7, 137.7, 129.9, 129.8, 129.4, 127.1, 124.8; HPLC purity: 11.7 min,

99.6%; LRMS (M þ H)^þ(ESI^þ) 313.97 [M þ H]^þ(calcd for

(100 MHz, CDCl₃)

d 149.5, 148.8, 141.9, 140.1, 136.7, 135.6, 133.8,

C

₁₃H₉Cl₂NO₂SH^þ314.10).

133.7, 131.7, 130.7, 127.6, 126.1, 126.0; HPLC purity: 13.0 min, 100%;

LRMS (M þ H)^þ(ESI^þ) 313.97 [M þ H]^þ(calcd for C₁₃H₉Cl₂NO₂SH^þ

313.95).

4.60. (E)-4-(2-((2-ﬂuorophenyl)sulfonyl)vinyl)pyridine (18)

Diethyl (((2-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.97 mmol), 2.0M n-BuLi solution in cyclohexane (0.53 mL,

1.06 mmol) and 4-pyridinecarboxaldehyde (0.10 mL, 1.06 mmol)

gave 0.22 g (87%) of 18 as a violet solid; R_f¼ 0.32 (n-hexane/EtOAc

4.56. (E)-2-Chloro-5-(2-((3-chlorophenyl)sulfonyl)vinyl)pyridine

(16c)

Diethyl (((3-chlorophenyl)sulfonyl)methyl)phosphonate (0.30 g,

0.91 mmol), 2.0M n-BuLi solution in cyclohexane (0.55 mL,

1.10 mmol) and 6-chloro-3-pyridinecarboxaldehyde (0.15 g,

1.10 mmol) gave 0.13 g (46%) of 16c as a white solid; R_f¼ 0.28 (n-

hexane/EtOAc 4/1); mp: 168.7e169.4 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

2/1); mp: 107.3e109.3 ^ꢂC; ¹H NMR (400 MHz, DMSO‑d₆)

d 8.67 (d,

J ¼ 4.9 Hz, ArH), 7.74e7.99 (m, (E)-isomeric 2H, 4ArH), 7.52 (t,

J ¼ 8.3 Hz, 2ArH); ¹³C NMR (100 MHz, DMSO‑d₆)

d 159.3 (d, J_C-

¼ 253.4 Hz), 151.0, 141.7, 139.8, 137.7 (d, J_C-F¼ 8.7 Hz), 132.4, 130.0,

F

128.0 (d, J_C-F¼ 13.8 Hz), 126.0 (d, J_C-F¼ 3.5 Hz), 123.2, 118.1 (d, J_C-

d

8.52 (d, J ¼ 2.4 Hz, ArH), 8.94 (t, J ¼ 1.8 Hz, ArH), 7.84 (d, J ¼ 7.6 Hz,

¼ 20.6 Hz); HPLC purity: 6.4 min, 100.0%; HRMS (M þ H)^þ(ESI^þ)

F

ArH), 7.76 (dd, J ¼ 2.8, 8.4 Hz, ArH), 7.62e7.69 (m, J ¼ 7.9 Hz, ArH,

(E)-isomeric H), 7.53 (t, J ¼ 7.8 Hz, ArH), 7.39 (d, J ¼ 8.4 Hz, ArH),

264.0486 [M þ H]^þ(calcd for C₁₃H₁₀FNO₂SH^þ264.0489).

4.61. (E)-4-(2-((3-ﬂuorophenyl)sulfonyl)vinyl)pyridine (19)

6.92 (d, J ¼ 15.6 Hz, ArH); ¹³C NMR (100 MHz, CDCl₃)

d 154.0, 150.0,

141.7, 138.2, 137.2, 135.8, 134.0, 130.9, 129.5, 127.9, 127.1, 126.0,

124.9; HPLC purity: 12.4 min, 100%; LRMS (M þ H)^þ(ESI^þ) 313.97

[M þ H]^þ(calcd for C₁₃H₉Cl₂NO₂SH^þ314.00).

Diethyl (((3-ﬂuorophenyl)sulfonyl)methyl)phosphonate (0.4 g,

0.28 mmol), 2.0M n-BuLi solution in cyclohexane (0.77 mL,

17

J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

1.54 mmol) and 4-pyridinecarboxaldehyde (0.14 mL, 1.54 mmol)

4.66. Cytotoxicity assay

gave 0.19 g (57%) of 19 as a dark blue solid; R_f¼ 0.33 (n-hexane/

EtOAc 2/3); mp: 99.4e100.8 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

d

8.69 (t,

The cell viability assay was performed according to previously

reported methods [42,48,49]. The BV-2 cells (1.0 ꢀ 10⁴cells/well)

were grown in 96-well plates and incubated for 24 h at 37 ^ꢂC

containing 5% CO₂. 9d with various concentrations (0.1, 0.3, 1, 3 and

J ¼ 2.02 Hz, 2ArH), 7.79 (d, J ¼ 17.8 Hz, ArH), 7.55e7.65 (m, 2ArH,

(E)-isomeric H), 7.34e7.38 (m, 2ArH, (E)-isomeric H), 7.04 (dd,

J ¼ 0.9, 15.4 ArH); ¹³C NMR (100 MHz, CDCl₃)

d 162.6 (d, J_C-

¼ 251.3 Hz), 150.8, 141.8 (d, J_C-F¼ 6.4 Hz), 140.3, 139.4, 131.5 (t, J_C-

¼ 6.2 Hz), 123.7 (d, J_C-F¼ 3.2 Hz), 122.1, 121.2 (d, J_C-F¼ 21.1 Hz),

10

reagent (10

m

M) were added for 6 h at 37 ^ꢂC in cells. After that, Ez-Cytox

F

m

L) was added and incubated for 1 h at 37 ^ꢂC. The

F

115.2 (d, J_C-F¼ 24.4 Hz); HPLC purity: 6.5 min, 100%; HRMS

(M þ H)^þ(ESI^þ) 264.0486 [M þ H]^þ(calcd for C₁₃H₁₀FNO₂SH^þ

264.0489).

signal for viability was measured by absorbance at 450 nm using a

SpectraMax®i3 microplate reader.

4.67. RT-qPCR

4.62. (E)-4-(2-((2-chlorophenyl)sulfonyl)vinyl)pyridine (20)

RT-qPCR was carried out using previously reported protocols

[40,46,47]. The antioxidant-related genes were ampliﬁed using the

following primers: Gclc (forward: 5⁰-TGTGGTATTCGTGGTACTGCTC-

3⁰, reverse: 5⁰-GGGCCACTTTCATGTTCTCG-3⁰); Gclm (forward: 5⁰-

GGAGCTTCGGGACTGTATCC-3⁰, reverse: 5⁰-TCGGGATTTATCTTC

TCCACTGC-3⁰); Ho-1 (forward: 5⁰-CAGCCACACAGCACTATG-3⁰,

reverse: 5⁰-GCAATC TTCTTCAGGACCT-3⁰); Sod1 (forward: 5⁰-

Diethyl (((2-chlorophenyl)sulfonyl)methyl)phosphonate (0.25 g,

0.76 mmol), 2.0M n-BuLi solution in cyclohexane (0.42 mL,

0.83 mmol) and 4-pyridinecarboxaldehyde (0.10 mL, 0.83 mmol)

gave 0.15 g (73%) of 20 as a brown powder; R_f¼ 0.24 (n-hexane/

EtOAc 2/1); mp: 113.0e115.0 ^ꢂC; ¹H NMR (400 MHz, DMSO‑d₆)

d

8.68 (d, J ¼ 5.9 Hz, ArH), 8.17 (dd, J ¼ 1.3, 7.8 Hz, ArH), 7.90 (d,

J ¼ 15.4 Hz, (E)-isomeric H), 7.67e7.80 (m, (E)-isomeric H, 5ArH),

GCATGGGTTCCACGTCCATC-3⁰,

reverse:

5⁰-CCAGCAGTCA-

7.52 (t, J ¼ 8.3 Hz, 2ArH); ¹³C NMR (100 MHz, DMSO‑d₆)

d 150.8,

CATTGCCCAG-3⁰); Nrf2 (forward: 5⁰-GCTCTCCATATTCCATT’C

CCTGTC-3⁰, reverse: 5⁰-CTTTTAAGTGGCCCAAGTCTTGC T-3⁰),

hypoxanthine-guanine phosphoribosyltransferase (Hprt) (forward:

5⁰-CAGGAGAGAAAGATGTGATTGA TA-3⁰, reverse: 5⁰-GCCAA-

CACTGCTGAAACA-3⁰); Hprt was used as an internal control. All RT-

qPCR reactions were performed on a Bio-Rad CFX™ Connect Real-

Time PCR Detection system. The 2 DDC_Tmethod was used to

calculate the fold changes in gene expression.

142.6, 140.1, 137.4, 136.3, 132.6, 132.1, 131.6, 131.2, 128.8, 123.2; HPLC

purity:7.0 min, 98.8%; HRMS (M þ H)^þ(ESI^þ) 280.0189 [M þ H]^þ

(calcd for C₁₃H₁₀ClNO₂SH^þ280.0194).

4.63. (E)-4-(2-((3-chlorophenyl)sulfonyl)vinyl)pyridine (21)

Diethyl (((3-chlorophenyl)sulfonyl)methyl)phosphonate (0.25 g,

0.76 mmol), 2.0M n-BuLi solution in cyclohexane (0.46 mL,

0.91 mmol) and 4-pyridinecarboxaldehyde (0.09 mL, 0.91 mmol)

gave 0.14 g (66%) of 21 as a dark green solid; R_f¼ 0.38 (n-hexane/

4.68. Western blot

EtOAc 1/2); mp: 105.2e107.2 ^ꢂC; ¹H NMR (400 MHz, CDCl₃)

d 8.69

(dd, J ¼ 1.6, 4.5 Hz, 2ArH), 7.94 (t, J ¼ 1.8 Hz, ArH), 7.85 (dt, J ¼ 1.28,

7.9 Hz, ArH), 7.62e7.67 (m, ArH, (E)-isomeric H), 7.53 (t, J ¼ 7.9 Hz,

ArH), 7.35 (dd, J ¼ 1.5, 4.5 Hz, 2ArH), 7.03 (d, J ¼ 15.4 Hz, (E)-

Western blotting was evaluated following previously reported

protocols [42,49,51]. The primary antibodies used were Nrf2

(1:500; Cell signaling Technology, Danvers, MA, USA, 1:1000;

Abcam, Cambridge, MA, USA), HO-1 (1:2000; Enzo Life Sceince,

Ann Arbor, MI, USA), GCLC (1:1000; Novus Biologicals, Littleton, CO,

USA), GCLM (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA,

isomeric H); ¹³C NMR (100 MHz, CDCl₃)

d 150.9, 141.5, 140.4, 139.3,

135.8, 134.1, 131.6, 130.9, 128.0, 126.0, 122.1; HPLC purity: 6.5 min,

100%; HRMS (M þ H)^þ(ESI^þ) 280.0194 [M þ H]^þ(calcd for

C

₁₃H₁₀ClNO₂SH^þ280.0194).

USA), SOD1 (1:1000; Abcam, Cambridge, MA, USA),

b-actin

(1:4000; Santa Cruz Biotechnology), and iNOS (1:500; Abcam,

Cambridge, MA, USA). The secondary antibodies used were either

anti-mouse or anti-rabbit horseradish peroxidase conjugated

(1:10000; GeneTex, Irvine, CA, USA). Immunoblotted proteins were

visualized with an Amersham Imager 600 (GE Healthcare, Arling-

ton Heights, IL, USA). Densitometric analyses were observed using

ImageJ software (National Institutes of Health), and the results

4.64. Cell culture

Engineered U2OS cell lines coexpressing Keap1-Nrf2 and

b-

galactosidase (93e0821C3, DiscoverX, Fremont, CA, USA) were

cultured in AssayComplete™ cell medium (92-3103G, DiscoverX) at

37 ^ꢂC under 5% CO₂. BV-2 cells were cultured in RPMI-1640 growth

media (Biowest, Nuaille, France) supplemented with 10% (v/v) fetal

bovine serum (Biowest), 100 U/mL penicillinestreptomycin (Gibco,

ꢀ

were normalized to b-actin as a control.

Thermo Fisher Scientiﬁc, Waltham, MA, USA), and 2 mM L-gluta-

mine (Gibco) at 37 ^ꢂC in an incubator containing 5% CO₂.

4.69. Griess assay

4.65. Keap1-Nrf2 functional cell-based assay

The production of nitric oxide (NO) was evaluated using the

Griess test that measured the nitrite ion (NO^ꢁ) in cell culture. The

BV-2 microglial cells were dose-dependently pretreated with 9d for

The nuclear translocation activity of Nrf2 was performed ac-

cording to previously reported procedure [42,48,51]. The ﬁnal

compounds were dissolved in DMSO and prepared in a range of

concentrations. The U2OS cells (1.3 ꢀ 10⁴cells/well) were plated

onto white 96-well plates and incubated overnight at 37 ^ꢂC in an

incubator containing 5% CO₂. The cells were then treated with

target compounds in dark conditions at room temperature for 6 h.

Subsequently, the cells were added with working solution and

incubated for 1 h in the dark conditions. Finally, a luminescence

signal was measured using a SpectraMax®i3 microplate reader

(Molecular Devices, San Jose, CA, USA).

6 h and stimulated with LPS (0.2 mg/mL) for 24 h in an incubator

containing 5% CO₂. Then the culture medium from cells were

treated with sulfanilamide solution (1% sulfanilamide in 5% phos-

phoric acid) and incubated for 5 min at room temperature in the

dark

conditions.

The

NED

solution

(0.1%

N-1-

napthylethylenediamine dihydrochloride in water) was added

and incubated for 5 min at room temperature in the dark condi-

tions. Absorbance of the reaction solution was measured at 540 nm

using a SpectraMax®i3 microplate reader and the nitrite levels

were calculated with nitrite standard.

18

J.W. Choi, S. Kim, J.S. Yoo et al.

European Journal of Medicinal Chemistry 212 (2021) 113103

4.70. Enzyme-linked immunosorbent (ELISA) assay

4.75. Statistical analysis

The BV-2 cells were seeded in 12-well plates and dose-

Data are analyzed by GraphPad Prism 7.0 software (San Diego,

CA, USA) and presented as the mean SEM. Differences between

groups were calculated using the two-tailed Student’s t-test or one-

way analysis of variance (ANOVA) analysis followed by Tukey’s

multiple comparison test. P value < 0.05 was considered statisti-

cally signiﬁcant.

dependently pretreated with 9d (0.02e2

for 6 h, the cells were stimulated with LPS (0.2

Proinﬂammatory cytokines (TNF- and IL-6) released in culture

mM). After incubation

mg/mL) for 24 h.

a

medium were measured using an ELISA kit (eBioscience, San Diego,

CA) according to the manufacturer’s instructions.

4.71. Small interfering RNA (siRNA)

Declaration of competing interest

siRNA transfection was performed as described previously [51].

Brieﬂy, BV2-cells (1 ꢀ 10⁵cells/well) were plated onto a 24-well

plates and incubated for 24 h. Cells were then transfected with

83 nM scramble or Nrf2-siRNA (Bioneer Inc., Daejeon, South Korea)

by using Viromer Blue® kit (Lipocalyx, Halle, Germany) for 24 h.

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

Acknowledgments

Cells were co-treated with 9d (100 nM) and LPS (0.5 mg/ml) for 24 h.

The culture medium was collected and the Griess assay was per-

formed to determine the NO concentration.

This study was supported by a National Research Council of

Science & Technology grant by the South Korean government

(MSIP, No. CRC-15-04-KIST), the National Research Foundation of

Korea (NRF-2018M3A9C8016849).

4.72. PD animal model and treatment

All animal experiments were performed in accordance with the

guidelines of the Institutional Animal Care and Use Committee of

the Korea Institute of Science and Technology (Seoul, South Korea).

10-week-old male C57BL/6 mice (weight, 23e26 g) were obtained

from Daehan Biolink (Eumseong, South Korea). Acute Parkinson’s

animal model by MPTP was performed similar to the paper [52],

only the drug administration method was modiﬁed. The vehicle for

oral administration was a solution containing 10% N-methyl-2-

pyrrolidone and 20% Tween-80 in distilled water. Vehicle or 9d

(30 mg/kg) was orally administered for 3 consecutive days; on the

second day, MPTP-HCl (20 mg/kg) or saline was injected intraper-

itoneally four times at 2 h intervals (n ¼ 10 per group). Six days after

MPTP injection, mice were examined for behavioral test for motor

dysfunction and sacriﬁced for further study the next day. For post-

treatment test, vehicle or 9d (30 mg/kg) was orally administered 3

days after MPTP injction (Fig. S2 in the Supporting Information).

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2020.1 13103.

Abbreviations

AD

Alzheimer’s disease

ARE

CNS

antioxidant response element

central nervous system

DAergic

EC₅₀

GCLC

GCLM

HO-1

IL-6

dopaminergic

half maximal effective concentration

glutamate-cysteine ligase catalytic subunit

glutamate-cysteine ligase modiﬁer subunit

heme oxygenase 1

interleukin-6

Iba-1

Keap1

ionized calicium binding adaptor molecule 1

Kelch-like ECH-associated protein 1

L-3,4-dihydroxyphenylalanine

lipopolysaccharide

L-DOPA-induced dyskinesia

multiple sclerosis

nuclear factor (erythroid-derived 2)-like 2

parallel artiﬁcial membrane permeability assay

Parkinson’s disease

substantia nigra pars compacta

tyrosine hydroxylase

4.73. Behavioral tests

L-DOPA

Coat-hanger test. Based on our previous paper [42,51], the

mouse was placed in the center of the coat-hanger and examined to

ascend to the safety zone for 3 min. When the mouse reaches that

location, it scores: 1, hang on and move sideways; 2, reaching the

hanger side; 3, climbing one top of the hanger; 4, moving toward

the top of the hanger; and 5, climb the hanger loop to reach the

safety zone. The mice that fell off the hanger within 20 s were not

scored. The test was repeated twice.

The vertical grid test. All mice were acclimated to the vertical grid

for 3 days before the drug administration. Mice are trained to rotate

and lower within 15 s when placed in the upper position in the

anterior direction. The test was repeated twice and video taken to

measure the time to turn, total time, and time to climb down after

the turn.

LPS

LID

MS

Nrf2

PAMPA

PD

SNpc

TH

TNF-

a

tumor necrosis factor-a.

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Development and optimization of halogenated vinyl sulfones as Nrf2 activators for the treatment of Parkinson's disease

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