N-(3,5-Dichloro-4-(2,4,6-trichlorophenoxy)phenyl)benzenesulfonamide: A new dual-target inhibitor of mitochondrial complex II and complex III via structural simplification

Article abstract of DOI:10.1016/j.bmc.2019.115299

Mitochondrial complex II and complex III are two promising targets for the development of numerous pharmaceuticals and pesticides. Although tremendous inhibitors of either complex II or complex III were identified, compounds which are capable of prohibiting the activities of both complexes have been rarely reported. Since multi-target drugs can interact with several drug targets simultaneously, we were keen on discovering new and potent dual-target inhibitors of both complex II and complex III. Therefore, a new series of structurally simplified sulfonamides bearing a diaryl ether scaffold were designed and synthesized in this paper. Afterwards, the biological activities of the newly synthesized compounds were evaluated. The results implied that several compounds demonstrated outstanding potency against succinate-cytochrome c reductase (SCR, a mixture of complex II and complex III). Further studies confirmed that N-(3,5-Dichloro-4-(2,4,6-trichlorophenoxy)phenyl)benzenesulfonamide (3f), a representative compound herein, was identified as a dual-target inhibitor of both complexes. Furthermore, computational simulations were also performed to have a better understanding about binding of 3f to the enzyme complexes, which concluded that 3f should bind to complex II and the Q_o site of complex III. Consequently, we harbor the idea that this work can be beneficial for the synthesis and discovery of more dual- or multi-target inhibitors.

Full text of DOI:10.1016/j.bmc.2019.115299

Journal Pre-proofs
N-(3,5-Dichloro-4-(2,4,6-trichlorophenoxy)phenyl)benzenesulfonamide: A
new dual-target inhibitor of mitochondrial complex II and complex III via
structural simplification
Hua Cheng, Hong-Fu Liu, Lu Yang, Cheng Chen, Yuan Wu, Wen Jiang, Rui
Zhang
PII:
S0968-0896(19)31515-9
https://doi.org/10.1016/j.bmc.2019.115299
BMC 115299
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
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19 December 2019
26 December 2019
Please cite this article as: H. Cheng, H-F. Liu, L. Yang, C. Chen, Y. Wu, W. Jiang, R. Zhang, N-(3,5-Dichloro-4-
2,4,6-trichlorophenoxy)phenyl)benzenesulfonamide: A new dual-target inhibitor of mitochondrial complex II
and complex III via structural simplification, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/
0.1016/j.bmc.2019.115299
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Bioorganic & Medicinal Chemistry
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N-(3,5-Dichloro-4-(2,4,6-trichlorophenoxy)phenyl)benzenesulfonamide: A new
dual-target inhibitor of mitochondrial complex II and complex III via structural
simplification
a
a
a
b,
c
c
a,
Hua Cheng, Hong-Fu Liu, Lu Yang, Cheng Chen, Yuan Wu, Wen Jiang and Rui Zhang *
a
Department of Chemical Engineering and Food Science, Hubei University of Arts and Science, Xiangyang 441053, P. R. China;
State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan 430070, P. R. China.
Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, P. R. China.
b
c
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Mitochondrial complex II and complex III are two promising targets for the development of
numerous pharmaceuticals and pesticides. Although tremendous inhibitors of either complex II
or complex III were identified, compounds which are capable of prohibiting the activities of
both complexes have been rarely reported. Since multi-target drugs can interact with several
drug targets simultaneously, we were keen on discovering new and potent dual-target inhibitors
of both complex II and complex III. Therefore, a new series of structurally simplified
sulfonamides bearing a diaryl ether scaffold were designed and synthesized in this paper.
Afterwards, the biological activities of the newly synthesized compounds were evaluated. The
results implied that several compounds demonstrated outstanding potency against succinate-
cytochrome c reductase (SCR, a mixture of complex II and complex III). Further studies
confirmed that N-(3,5-Dichloro-4-(2,4,6-trichlorophenoxy)phenyl)benzenesulfonamide (3f), a
representative compound herein, was identified as a dual-target inhibitor of both complexes.
Furthermore, computational simulations were also performed to have a better understanding
about binding of 3f to the enzyme complexes, which concluded that 3f should bind to complex II
Available online
Keywords:
Dual-target inhibitor
Diaryl ether
Sulfonamide
Mitochondrial complex II and complex III
Structural simplification
and the Q
o
site of complex III. Consequently, we harbor the idea that this work can be beneficial
for the synthesis and discovery of more dual- or multi-target inhibitors.
2
016 Elsevier Ltd. All rights reserved.
Introduction
synthesis will be blocked, resulting in cell death. Therefore, these
two complexes have been identified as promising targets for
It is well documented that oxidative phosphorylation and the
Krebs cycle constitute the mitochondrial respiration. In general,
the oxidative phosphorylation is composed of five multiprotein
complexes (complexes I-V) and two mobile electron carriers
[
11, 12]
[13]
numerous medicines
handful of complex II
and agrochemicals. In recent years, a
[
1, 13-22]
and complex III[1, 23-46] inhibitors
were successfully discovered. Despite these reported inhibitors,
novel complex II or/and complex III inhibitors with improved
potency and new structures are still in high demand.
(
ubiquinone and cytochrome c).[1] Among these five complexes,
complex II (or succinate ubiquinone oxidoreductase) and
complex III (also known as the cytochrome bc complex) are of
The natural product Antimycin A, a highly active inhibitor of
complex III, is a valuable starting point for developing novel and
1
great significance due to their central roles in the life cycle.
Complex II is responsible for the electron transfer from succinate
to fumarate, while ubiquinone is reduced to ubiquinol
[
47]
potent inhibitors (as shown in Fig. 1a). Later, Bolgunas et al.
synthesized a series of Antimycin A analogues (Series A) by
introducing a diaryl ether scaffold to replace the dilactone portion
[
2-6]
simultaneously.
In terms of complex III, it is associated with
[
29]
of the natural product.
It was found that some of these
the electron transfer from ubiquinol to cytochrome c,
hydroquinone oxidation and cytochrome c reduction.
the activities of complex II and/or complex III are inhibited, ATP
analogues displayed comparable activities with Antimycin A,
demonstrating the attractiveness of the diaryl ether moiety in a
[
7-10]
Once
—
——

Corresponding authors. Tel.: +86-27-88016035; fax: +86-27-67867141; e-mail: chengchen@whut.edu.cn (C. Chen); rzhangccnu@163.com
R. Zhang).
(

2
Fig. 1. The design strategy of this work.
Fig. 2. The design strategy of our target molecules.
multitude of bioactive molecules (Fig. 1b).[29, 38, 48-61] In our
previous work, by retaining the diaryl ether moiety, we
successfully prepared a novel series of 1,2,4-triazole-1,3-
disulfonamides (Series B). Interestingly, some compounds were
identified as dual inhibitors targeting both complex II and
coupling of an amine and an arylsulfonyl chloride. Expectedly, a
number of sulfonamides 3 were directly prepared by reactions of
4-aryloxy anilines 1 and commercial arylsulfonyl chlorides 2 in
pyridine at ambient temperature (as depicted in the scheme of
Table 1). Afterwards, inhibitory activities of these newly
prepared compounds against succinate-cytochrome c reductase
(SCR, a mixture of complex II and complex III) were tested at a
concentration of 10 μM, and those with inhibitory percentages
higher than 50% were further chosen to measure their IC50 values
(as listed in Table 1). Moreover, Penthiopyrad (a commercialized
complex II-inhibiting fungicide) and Azoxystrobin (a commercial
fungicide of complex III) were used as the positive controls. To
facilitate the explanations of our bioassay results, the three
phenyl rings of 3 were assigned as A, B and C, respectively. The
first and foremost, an inhibition rate of 32% was observed for the
unsubstituted sulfonamide 3a at the concentration of 10 μM
(entry 1). To our delight, if a 2,4-dichlorophenyl group was
employed as ring A, the corresponding product 3b exhibited
marginally improved activity (entry 2 vs. entry 1). Meanwhile,
various substituents were introduced on both ring A and ring B
(entries 3-9). When both rings were substituted with one or
several Cl groups, most of the resulting products manifested high
potency (entries 3-6). Especially, a nanomolar inhibitor (3f), which
displayed even much higher inhibition ability than the two
commercial fungicides, was identified (entry 6 vs. entries 36-37).
Unfortunately, poor levels of inhibition were produced if a pyridine
or naphthyl-based group was utilized as ring A (entries 7-9).
Afterwards, compounds 3b, 3c, 3e, 3f, with their IC50 values smaller
than 10 μM, were selected to further investigate the substituent
[
1]
complex III (Fig. 1c). Multi-target drugs are capable of
interacting with several drug targets simultaneously, leading to
new and more effective medications for a variety of complex
[
62, 63]
diseases, even with relatively weak activities.
Therefore,
Series B may bring new chances to the discovery of highly
efficient drugs and green pesticides. However, the synthesis of
these compounds, especially the triazole-containing sulfonyl
chloride, was reported to be relatively tedious (as shown in Fig.
2a), which prompted us to conduct further structural
modifications. Since structural simplification of natural products
[
64]
has been regarded to be crucial in lead optimization, we aimed
to simply Series B by the replacement of the triazole fragment
with a substituted phenyl group (as shown in Fig. 1d). With
commercial arylsulfonyl chlorides, plenty of target molecules
could be easily obtained (Fig. 2b). In addition, the inhibitory
activities of the target compounds were evaluated against
complex II and complex III. Gratifyingly, one of these
structurally simplified compounds was also identified as a dual
inhibitor of both complexes, with even better inhibitory activities.
Furthermore, molecular docking experiments of this compound
provided more valuable information about the potential mode of
action.
2
. Results and discussion
2 2 3
effects on ring C. At the outset, 4-NO , 2,4-Cl , 2-Cl-4-CF and
Aiming at constructing a broad range of target molecules in a
facile and efficient manner, we focused on the easily-accessible
2
,4,6-Cl were incorporated into ring C, and satisfactory inhibition
3

3
performance was obtained for these compounds (entries 10-26). It
appeared that a NO group normally triggered lower activities than
the other groups (entries 10, 14, 18, 22 vs. entries 11-13, 15-17, 19-
1, 23-26). Based on the above observations, compounds 3f, 3v-3y
which have identical ring A and ring B, were found to be more active
than the other compounds (entries 6, 22-25 vs. other entries).
Therefore, varied groups were assembled into ring C at the para
position, and it seemed that products with electron-withdrawing
substituents showed better activities than the electron-donating
counterparts (entries 29-31 vs. entries 26-28). Moreover, a
2
substituent at the para, meta or ortho position of ring C gave rise to
different activities (entries 26, 32-33). It was indicated that
compound 3f’, with 3-Me group on the phenyl ring of ring C,
exhibited higher activities than the 2-Me and 4-Me analogues (entry
32 vs. entries 26, 33). Finally, replacing Cl in compound 3f with F
or Me at positions 2,4,6 and 3’,5’ of the diphenyl ether moiety
provided target compounds 3h’ and 3i’, which demonstrated much
lower activity than 3f (entries 34-35).
2
Table 1. Inhibitory activities of the synthesized compounds against porcine SCR.
a
b
c
Entry
Compound No.
R
1
R
2
R
3
Yield (%)
I (%) or IC50 (μM)
b
c
1
2
3
4
5
6
3a
3b
3c
3d
3e
3f
H
H
H
H
H
H
H
H
91
71
70
45
69
88
>10 (32% )
b
2,4-Cl
2,4-Cl
2
2
H
9.28 ± 0.95
b
3-Cl
3-Cl
3,5-Cl
3,5-Cl
1.98 ± 0.08
b
c
2,4,6-Cl
2,4-Cl
2,4,6-Cl
3
3
>10 (47% )
b
2
2
2
1.10 ± 0.10
b
0.08 ± 0.01
b
c
7
3g
3,5-Cl
2
2
H
70
>10 (9% )
d
d
d
b
c
8
9
3h
3i
2-naphthyl
2-naphthyl
3,5-Cl
3-Cl
H
72
92
88
83
82
85
61
63
71
74
69
50
66
45
82
83
66
64
71
70
80
78
76
75
71
55
48
92
>10 (48% )
b
c
H
>10 (47% )
b
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
3j
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2
2
2
2
2
2
2
2
2
2
2
2
H
4-NO
2
4.86 ± 0.26
b
3k
3l
H
2,4-Cl
2-Cl-4-CF
2,4,6-Cl
4-NO
2,4-Cl
2-Cl-4-CF
2,4,6-Cl
4-NO
2,4-Cl
2-Cl-4-CF
2,4,6-Cl
4-NO
2,4-Cl
2-Cl-4-CF
2
1.62 ± 0.21
1.37 ± 0.04
1.49 ± 0.13
2.24 ± 0.07
1.30 ± 0.10
1.08 ± 0.05
1.33 ± 0.12
3.57 ± 0.30
1.38 ± 0.14
1.17 ± 0.07
1.12 ± 0.06
1.58 ± 0.13
1.03 ± 0.10
1.08 ± 0.05
0.22 ± 0.02
1.66 ± 0.13
1.87 ± 0.17
0.99 ± 0.01
0.63 ± 0.04
0.83 ± 0.08
0.53 ± 0.02
0.38 ± 0.04
0.80 ± 0.03
6.44 ± 0.40
b
b
b
b
H
3
3
3
3
3m
3n
3o
3p
3q
3r
H
3
3-Cl
2
3-Cl
2
b
3-Cl
b
b
3-Cl
3
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
b
3s
2
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
3t
3u
3v
3w
3x
3y
3z
3
2,4,6-Cl
2,4,6-Cl
2,4,6-Cl
2,4,6-Cl
2,4,6-Cl
2,4,6-Cl
2,4,6-Cl
2,4,6-Cl
2,4,6-Cl
2,4,6-Cl
2,4,6-Cl
2,4,6-Cl
3
3
3
3
3
3
3
3
3
3
3
3
2
2
2,4,6-Cl
4-Me
4-OMe
4-tBu
4-Cl
3
3a’
3b’
3c’
3d’
3e’
3f’
3g’
3h’
3i’
4-F
4-CF
3
3-Me
2-Me
H
2,4,6-F
3
3,5-F
2
b
c
2,4,6-Me
3
3,5-Me
2
H
>10 (3% )
1.32 ± 0.11
0.29 ± 0.01
b
b
Penthiopyrad
Azoxystrobin
b
c
d
Isolated yields. IC50 values against SCR. Inhibition rates against SCR at a concentration of 10 μM. The whole ring A was replaced with this group.

2
With the consideration of confirming whether these compounds were complex II, complex III or dual-target inhibitors, we further tested the
inhibition potency of 3f (the most active inhibitor herein) against SCR, complex II and complex III (as shown in Table 2). Expectedly,
Penthiopyrad and Azoxystrobin demonstrated outstanding activities against complex II and complex III, respectively (entries 3-4, Table 2).
In addition, a comparison between 3f and compound 4, a representative dual inhibitor in our previous work,[1] was also made (entry 2 vs.
entry 1, Table 2). Similar to 4, 3f displayed high activities for both complex II and complex III. Notably, 3f demonstrated comparable
activity with 4 against complex II, but noticeably higher potency against SCR and complex III. Therefore, 3f has been validated as a potent
and dual-target inhibitor of both complex II and complex III.
Since representative inhibitor 3f exhibited inhibitory activities against both complexes, computational simulation studies were carried out
to elucidate its possible modes of action (as shown in Fig. 3). For the binding mode of 3f towards complex II, one oxygen atom of the
sulfonamide functionality could form strong hydrogen bonds with residues B_W173 and D_Y91 (as depicted in Fig. 3A). In addition, the
phenyl group of 3f formed an ion-π interaction with C_R46 and a π-π stacking interaction with B_H126. Interestingly, the diaryl ether
moiety can bind at the hydrophobic pocket surrounded by residues B_P169, C_I30, C_W35 and C_M39, with concurrent formation of
strong π-π interactions with C_W35 and B_W173. More significantly, the Cl atoms intensified the above hydrophobic interactions.
Accordingly, 3f displayed a common binding mode, which was analogous with the classic complex II inhibitor, Penthiopyrad (as shown in
o i
and Q
sites),[65, 66] it is of great necessity to ascertain the binding site of 3f by
Fig. 3B). Since complex III has two binding sites (the Q
docking it into both sites. The docking scores in AutoDock 4.2 were used to calculate the binding free energies. When 3f was docked into
the Q site, the calculated binding free energy was -7.3 kcal/mol for the best binding conformation. Nevertheless, the binding energy was
calculated as -6.9 kcal/mol if it bound into the Q site, which suggested that 3f was probably a Q site inhibitor. Consequently, the binding
o
i
o
m
o
d
e
o
f
3
f
Table 2 The inhibition effect of the selected inhibitors against SCR, complex II and complex III
Entry
Compound No.
IC50 (µM), SCR
0.08 ± 0.01
6.44 ± 0.40
3.20 ± 0.10
1.32 ± 0.11
0.29 ± 0.01
IC50 (µM), complex II
0.51 ± 0.05
IC50 (µM), complex III
3.06 ± 0.30
1
2
3f
b
3h’
2.02±0.21
40%
a
3
4
0.50 ± 0.01
35.5 ± 1.00
b
4
5
a
Penthiopyrad
Azoxystrobin
1.19 ± 0.12
<10%
b
23%
0.53 ± 0.02
[1] b
The data was obtained from our previous work. Inhibition rates at a concentration of 10 μM.

3
Fig. 3. (A) The binding mode of 3f in complex II (PDB ID:3AEB); (B) the binding mode of Penthiopyrad in complex II; (C) the binding
mode of 3f in in the Q pocket of complex III (PDB ID:1SQB); (D) the binding mode of Azoxystrobin in the Q pocket of complex III; (E)
the binding mode of 4 in complex II; (F) the binding mode of 4 in the Q pocket of complex III.
o
o
o
o
into the Q pocket of complex III was investigated (as shown in Fig. 3C). It was found that non-polar interactions played a crucial role
between this inhibitor and the target enzyme. With regard to the phenyl ring of 3f, it could enter a hydrophobic pocket surrounded by
residues P270, F274, F128 and Y131. Besides, π-π interactions were observed between the phenyl group and the corresponding residues
(
F274 and Y131). Meanwhile, the diaryl ether scaffold formed hydrophobic interactions with L294, I298, L121, I146, M124, F274 and
F128. It was worth noting that the Cl atoms were also found to enhance the hydrophobic interactions. The docking result also indicated that
the binding mode of 3f in the Q pocket was similar with that of Azoxystrobin (Fig. 3D). Based on the above results, we concluded that 3f
could bind well into complex II and the Q site of complex III. Notably, the binding mode of 3f elaborated the importance of the Cl atoms
o
o
in the synthesized molecule. In order to clearly illustrate what the structural simplification led in terms of the binding modes and types of
interactions, we also docked 4 into both complexes and compared the results with 3f (Fig. 3E and 3F). When binding to complex II, 4
showed a similar binding mode with 3f, with the triazole ring of 4 forming hydrogen bonds with B_W173 and D_Y91 (Fig. 3E). In
addition, the terminal N, N-dimethyl sulfonamide group formed an electrostatic interaction with the positive charged C_R46 and a
hydrophobic interaction with B_H126. Moreover, despite the change in the molecule size, the diaryl ether moiety of 4 could successfully
bind to the hydrophobic pocket, thus leading to similar binding capacity and comparable potency towards complex II in comparison with
3
o
f. Afterward, the binding mode of 4 toward the Q site of complex III showed significant difference with 3f (Fig. 3F). The diaryl ether
moiety of 4 demonstrated a similar function with that of 3f, but the sulfonamide group switched to the opposite side of the pocket due to the
clash with the inner hydrophobic pocket, which made 4 a weaker binder to complex III and provided a much higher IC50 value compared
with 3f for complex III.
3
. Conclusion
In summary, a new series of sulfonamides bearing a diaryl ether scaffold were prepared through structural simplification from our
previously reported 1,2,4-triazole-1,3-disulfonamides. With these newly prepared target molecules in hand, their inhibition capability
against SCR were originally examined. To our delight, the majority of compounds demonstrated good inhibition potency against SCR.
Notably, compound 3f, a nanomolar inhibitor, was also discovered. Further investigations were carried out to verify whether 3f was an
inhibitor of complex II and/or complex III. Interestingly, 3f was identified as a dual-target inhibitor of both complex II and complex III.
Furthermore, computational simulations were utilized to clarify the binding mode of 3f. It was indicated that 3f bound well to complex II

4
o
and the Q site of complex III, and it was also worth mentioning that the Cl atoms in this target molecule played a pivotal role in exhibiting
the high potency. Hopefully, this work could be valuable for the development of other dual- or multi-target inhibitors.
4
4
. Experimental
.1 General considerations
1
H NMR spectra were recorded on a Bruker Avance 500 or 400 spectrometer in CDCl
3
or DMSO-d with TMS as the internal
6
1
3
reference, C NMR spectra were recorded in CDCl on a Bruker Avance 500 (126 MHz) or 400 (101 MHz) spectrometer. The
3
following abbreviations were used to designate multiplicities: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q =
quartet, m = multiplet. HR-MS was taken on a Bruker Daltonics microTOF-QII instrument. Melting points were taken on a Buchi
B-560 melting point apparatus and were uncorrected. Ordinary reagents including the arylsulfonyl chlorides and solvents were
commercially available and directly used unless otherwise mentioned. Besides, 4-phenoxyaniline (1a) and all the arylsulfonyl
chlorides were purchased from commercial suppliers. For compounds 4-(2,4-dichlorophenoxy)aniline (1b), 3-chloro-4-(2,4-
dichlorophenoxy)aniline (1c), 3-chloro-4-(2,4,6-trichlorophenoxy)aniline (1d), 3,5-dichloro-4-(2,4-dichlorophenoxy)aniline (1e),
3,5-dichloro-4-(2,4,6-trichlorophenoxy)aniline (1f), 4-((3-chloro-5-(trifluoromethyl)pyridin-2-yl)oxy)aniline (1g), 3-chloro-4-
(
naphthalen-2-yloxy)aniline (1h), 3,5-dichloro-4-(naphthalen-2-yloxy)aniline (1i), they were synthesized using our previously
[
45, 60]
reported methods.
4.2. General procedure for the synthesis of 1h’ and 1i’
A mixture of 1,2,3-trifluoro-5-nitrobenzene (0.89 g, 5.0 mmol), 2,4,6-trifluorophenol (1.48 g, 10.0 mmol) and potassium
carbonate (1.38 g, 10.0 mmol) in DMF (5.0 mL) was stirred at 100 ºC. After the reaction was complete (as monitored by TLC),
diethyl ether (20 mL) was added and the resulting mixture was washed with 2 mol/L NaOH (3 × 20 mL), water (20 mL), brine (20
mL) and dried over Na
used directly for the next step without characterization. Afterward, 10% Pd/C (15% by weight based on 5.0 mmol) was added to a
solution of the above crude compound in CH Cl (10 mL) at room temperature, and
balloon was bubbled into the reaction mixture. When the reaction was completed (as monitored by TLC), the reaction mixture
2
SO
4
. Finally, the organic layer was concentrated under reduced pressure to give a crude mixture, which was
2
2
a
H
2
was filtered. Then the filtrate was concentrated and purified through silica gel column chromatography to give 1.07 g (3.89 mmol)
of pure 1h’ in 78% overall yield in two steps.
Compound 1i’ was prepared in a similar procedure with 1h’, affording 1.11 g (4.35 mmol) of pure 1i’ in 87% overall yield in two
steps.
4
.2. General procedure for the synthesis of target compounds 3a-3i’
To a solution of 1 (0.5 mmol) and pyridine (1.5 mL) in THF (3 mL) was added 2 (0.75 mmol) slowly at 25°C. After the complete
consumption of 1 (as monitored by TLC), the reaction was quenched by the addition of 2 N HCl (5 mL) and then extracted with
ethyl acetate (20 mL × 3). Afterwards, the combined organic layers were washed with brine (20 mL), dried over with Na
2
SO , and
4
concentrated. The crude was purified by column chromatography on silica gel to afford the desired products (3a-3i’).
4
.3. Analysis data of 1h’, 1i’ and 3a-3i’
.3.1. 3,5-Difluoro-4-(2,4,6-trifluorophenoxy)aniline (1h’). White solid, m.p. 100.8-101.2°C. Isolated yield: 78%. H NMR (500
1
4
1
3
MHz, DMSO-d
6
) δ 7.33 (t, J = 8.9 Hz, 2H), 6.32 – 6.18 (m, 2H), 5.63 (s, 2H). C NMR (126 MHz, DMSO-d
6
) δ 157.54 (dt, J =
2
1
2
44.2, 14.6 Hz), 155.25 (dd, J = 243.1, 7.0 Hz), 154.56 (ddd, J = 249.2, 15.7, 6.6 Hz), 147.42 (t, J = 13.3 Hz), 131.74 (td, J = 14.2,
+
3.7, 6.1 Hz), 123.07 (t, J = 15.2 Hz), 102.63 – 101.47 (m), 97.47 – 96.34 (m). HRMS (ESI): calc. for C12
H
7
5
F NO [M+H] :
76.04423; Found: 276.04415.
1
4
.3.2. 4-(Mesityloxy)-3,5-dimethylaniline (1i’). White solid, m.p. 73.5-74.8°C. Isolated yield: 87%. H NMR (500 MHz, CDCl
3
) δ
1
3
6
1
2
.79 (s, 2H), 6.37 (s, 2H), 3.42 (s, 2H), 2.27 (s, 3H), 2.05 (s, 6H), 2.02 (s, 6H). C NMR (126 MHz, CDCl
3
+
) δ 152.31, 147.42,
41.15, 131.14, 129.94, 129.23, 127.39, 115.82, 20.46, 17.38, 17.27. HRMS (ESI): calc. for C17
56.16950.
H
22NO [M+H] : 256.16959; Found:
1
4
.3.3. N-(4-Phenoxyphenyl)benzenesulfonamide (3a). White solid, m.p. 112.8-114.2°C. Isolated yield: 91%. H NMR (500 MHz,
CDCl
7.4 Hz, 1H), 7.02 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 8.0 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 6.64 (s, 1H). C NMR (126 MHz, CDCl
δ 156.82, 155.45, 138.90, 133.02, 131.11, 129.80, 129.01, 127.26, 124.85, 123.55, 119.41, 118.86. HRMS (ESI): calc. for
3
) δ 7.76 (d, J = 7.6 Hz, 2H), 7.55 (d, J = 7.5 Hz, 1H), 7.46 (dd, J = 7.8, 7.6 Hz, 2H), 7.32 (dd, J = 8.0, 7.8 Hz, 2H), 7.11 (d, J
1
3
=
3
)
-
C
18
H14NO
3
S [M-H] : 324.06999; Found: 324.06959.
1
4
.3.4. N-(4-(2,4-Dichlorophenoxy)phenyl)benzenesulfonamide (3b). White solid, m.p. 134.8-136.1°C. Isolated yield: 71%. H
NMR (500 MHz, CDCl ) δ 7.75 (d, J = 7.8 Hz, 2H), 7.55 (d, J = 7.4 Hz, 1H), 7.45 (dd, J = 8.9, 6.4 Hz, 3H), 7.18 (dd, J = 8.7, 2.2
3

3
1
3
Hz, 1H), 7.03 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 8.7 Hz, 1H), 6.82 (d, J = 8.8 Hz, 2H), 6.58 (s, 1H). C NMR (126 MHz, CDCl
3
) δ
1
54.61, 151.03, 138.78, 133.09, 131.70, 130.56, 129.50, 129.05, 128.09, 127.24, 126.56, 124.69, 121.36, 118.63. HRMS (ESI): calc.
-
for C18
H12Cl
2
NO S [M-H] : 391.99204; Found: 391.99203.
3
4
.3.5. N-(3-Chloro-4-(2,4-dichlorophenoxy)phenyl)benzenesulfonamide (3c). White solid, m.p. 105.0-106.1°C. Isolated yield:
1
7
1
0%. H NMR (500 MHz, CDCl
H), 7.23 (s, 1H), 7.16 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.82 (s, 1H), 6.75 (d, J = 8.7 Hz, 1H), 6.71 (d, J = 8.7 Hz, 1H).
C NMR (126 MHz, CDCl
3
) δ 7.79 (d, J = 7.6 Hz, 2H), 7.59 (dd, J = 7.4, 7.2 Hz, 1H), 7.49 (dd, J = 7.6, 7.4 Hz, 2H), 7.45 (s,
1
3
3
) δ 150.91, 149.59, 138.59, 133.40, 133.12, 130.63, 129.44, 129.23, 128.01, 127.22, 125.91, 125.72,
-
1
24.65, 121.87, 120.18, 119.80. HRMS (ESI): calc. for C18
H11Cl
3
NO
3
S [M-H] : 425.95307; Found: 425.95300.
4
.3.6. N-(3-Chloro-4-(2,4,6-trichlorophenoxy)phenyl)benzenesulfonamide (3d). White solid, m.p. 205.7-206.5°C. Isolated yield:
1
4
7
1
5%. H NMR (500 MHz, DMSO-d
.4 Hz, 2H), 7.23 (d, J = 2.6 Hz, 1H), 6.92 (dd, J = 8.9, 2.6 Hz, 1H), 6.56 (d, J = 9.0 Hz, 1H). C NMR (126 MHz, DMSO-d
48.70, 145.71, 139.60, 133.97, 133.58, 131.54, 130.06, 129.81, 129.64, 127.08, 123.20, 121.81, 121.28, 115.69. HRMS (ESI): calc.
6
) δ 10.35 (s, 1H), 7.87 (s, 2H), 7.73 (d, J = 7.1 Hz, 2H), 7.68 – 7.61 (m, 1H), 7.57 (dd, J = 7.6,
1
3
6
) δ
-
for C18
H10Cl
4
NO
3
S [M-H] : 459.91410; Found: 459.91396.
4
.3.7. N-(3,5-Dichloro-4-(2,4-dichlorophenoxy)phenyl)benzenesulfonamide (3e). White solid, m.p. 140.5-141.2°C. Isolated yield:
1
6
9%. H NMR (500 MHz, CDCl
3
) δ 7.86 (d, J = 7.8 Hz, 2H), 7.64 (dd, J = 7.6, 7.4 Hz, 1H), 7.55 (dd, J = 7.8, 7.6 Hz, 2H), 7.45 (d,
1
3
J = 2.3 Hz, 1H), 7.18 (s, 2H), 7.10 (s, 1H), 7.06 (dd, J = 8.8, 2.3 Hz, 1H), 6.30 (d, J = 8.8 Hz, 1H). C NMR (126 MHz, CDCl ) δ
50.87, 144.03, 138.26, 135.01, 133.81, 130.57, 130.16, 129.48, 128.05, 127.54, 127.20, 123.49, 121.38, 114.92. HRMS (ESI): calc.
for C18
3
1
-
H10Cl
4
NO S [M-H] : 459.91410; Found: 459.91396.
3
4
.3.8. N-(3,5-Dichloro-4-(2,4,6-trichlorophenoxy)phenyl)benzenesulfonamide (3f). White solid, m.p. 201.9-203.3°C. Isolated
1
yield: 88%. H NMR (500 MHz, DMSO-d
6
) δ 10.70 (s, 1H), 7.80 – 7.72 (m, 4H), 7.66 (d, J = 7.1 Hz, 1H), 7.59 (dd, J = 7.4, 7.2 Hz,
H), 7.15 (s, 2H). C NMR (126 MHz, DMSO-d ) δ 146.53, 143.70, 138.39, 135.40, 133.43, 129.46, 129.44, 129.04, 126.68,
25.99, 125.81, 120.50. HRMS (ESI): calc. for C18
1
3
2
1
6
-
H
9
Cl
5
NO
3
S [M-H] : 493.87513; Found: 493.87487.
4
.3.9. N-(3,5-Dichloro-4-((3-chloro-5-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)benzenesulfonamide (3g). White solid, m.p.
1
2
15.8-216.7°C. Isolated yield: 70%. H NMR (500 MHz, CDCl ) δ 7.91 (d, J = 7.7 Hz, 2H), 7.87 (d, J = 2.4 Hz, 1H), 7.63 (t, J = 7.4
Hz, 1H), 7.54 (t, J = 7.7 Hz, 2H), 7.51 – 7.43 (m, 1H), 7.22 (s, 2H). C NMR (126 MHz, CDCl
3
1
3
3
) δ 157.87, 140.27, 139.04, 135.74
(
q, J = 5.5 Hz), 134.76, 133.73, 133.49, 130.19, 129.35, 128.62, 127.13, 122.21 (q, J = 270.8 Hz), 120.19, 111.21 (q, J = 36.1 Hz).
-
HRMS (ESI): calc. for C18
H
9
Cl
3
F
3
N
2
3
O S [M-H] : 494.93570; Found: 494.93546.
4
.3.10. N-(3,5-Dichloro-4-(naphthalen-2-yloxy)phenyl)benzenesulfonamide (3h). White solid, m.p. 173.0-173.3°C. Isolated yield:
1
7
1
1
1
2%. H NMR (500 MHz, CDCl
H), 7.59 – 7.51 (m, 5H), 7.24 (d, J = 9.7 Hz, 1H), 7.21 (s, 2H), 6.29 (d, J = 7.6 Hz, 1H). C NMR (126 MHz, CDCl
44.94, 138.37, 134.77, 134.43, 133.74, 130.54, 129.46, 127.58, 127.24, 126.77, 125.87, 125.26, 124.73, 122.51, 121.88, 121.63,
3
) δ 8.46 – 8.38 (m, 1H), 7.89 (d, J = 7.8 Hz, 2H), 7.87 – 7.81 (m, 1H), 7.64 (dd, J = 7.6, 7.4 Hz,
1
3
3
) δ 152.30,
-
06.58. HRMS (ESI): calc. for C22
H14Cl
2
NO
3
S [M-H] : 442.00769; Found: 442.00756.
4
.3.11. N-(3-Chloro-4-(naphthalen-2-yloxy)phenyl)benzenesulfonamide (3i). White solid, m.p. 119.7-120.6°C. Isolated yield:
1
9
2%. H NMR (500 MHz, CDCl ) δ 7.82 (dd, J = 7.2, 7.0 Hz, 4H), 7.66 (d, J = 8.1 Hz, 1H), 7.60 (dd, J = 7.6, 7.4 Hz, 1H), 7.51 (dd,
3
J = 7.8, 7.6 Hz, 2H), 7.45 (dd, J = 7.6, 7.4 Hz, 1H), 7.41 (dd, J = 7.6, 7.4 Hz, 1H), 7.20 (dd, J = 8.9, 1.9 Hz, 1H), 7.13 (s, 1H), 6.96
1
3
(
dd, J = 8.7, 2.2 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H). C NMR (126 MHz, CDCl
3
) δ 154.50, 150.40, 138.73, 134.14, 133.37, 132.71,
1
30.27, 130.11, 129.23, 127.78, 127.28, 127.10, 126.73, 126.53, 124.93, 124.80, 122.14, 121.40, 118.99, 113.00. HRMS (ESI): calc.
-
for C22
H
15ClNO S [M-H] : 408.04667; Found: 408.04651.
3
4
.3.12. N-(4-(2,4-Dichlorophenoxy)phenyl)-4-nitrobenzenesulfonamide (3j). White solid, m.p. 148.6-150.1°C. Isolated yield:
1
8
8%. H NMR (500 MHz, CDCl
3
) δ 8.30 (d, J = 7.6 Hz, 2H), 7.90 (d, J = 7.7 Hz, 2H), 7.47 (s, 1H), 7.22 (d, J = 8.7 Hz, 1H), 7.03 (d,
1
3
J = 7.8 Hz, 2H), 6.93 (d, J = 8.7 Hz, 1H), 6.84 (d, J = 8.7 Hz, 2H), 6.49 (s, 1H). C NMR (126 MHz, CDCl ) δ 155.63, 150.55,
50.33, 144.76, 130.75, 130.48, 130.14, 128.55, 128.26, 127.08, 125.42, 124.26, 122.08, 118.42. HRMS (ESI): calc. for
3
1
C
-
18
H
11Cl
2
N
2
O
5
S [M-H] : 436.97712; Found: 436.97706.
4
.3.13. 2,4-Dichloro-N-(4-(2,4-dichlorophenoxy)phenyl)benzenesulfonamide (3k). White solid, m.p. 167.9-168.1°C. Isolated
1
yield: 83%. H NMR (500 MHz, CDCl ) δ 7.90 (d, J = 8.5 Hz, 1H), 7.56 (d, J = 2.1 Hz, 1H), 7.47 (d, J = 2.5 Hz, 1H), 7.34 (dd, J =
.5, 2.1 Hz, 1H), 7.21 (dd, J = 8.7, 2.5 Hz, 1H), 7.14 – 7.07 (m, 2H), 6.91 (s, 1H), 6.88 (d, J = 8.7 Hz, 1H), 6.86 – 6.79 (m, 2H). C
3
1
3
8
NMR (126 MHz, CDCl ) δ 155.09, 150.74, 140.05, 134.67, 132.93, 132.19, 131.39, 130.60, 129.76, 128.14, 127.64, 126.78, 124.63,
3
-
121.67, 118.51. HRMS (ESI): calc. for C18
H10Cl
4
NO
3
S [M-H] ; Exact Mass: 459.91410; Found: 459.91399.
4
.3.14. 2-Chloro-N-(4-(2,4-dichlorophenoxy)phenyl)-4-(trifluoromethyl)benzenesulfonamide (3l). White solid, m.p. 149.9-
1
1
7
8
1
51.1°C. Isolated yield: 82%. H NMR (500 MHz, CDCl
3
) δ 8.12 (d, J = 8.2 Hz, 1H), 7.81 (s, 1H), 7.62 (dd, J = 8.2, 1.7 Hz, 1H),
.47 (d, J = 2.6 Hz, 1H), 7.21 (dd, J = 8.7, 2.5 Hz, 1H), 7.11 (d, J = 8.9 Hz, 2H), 7.02 (s, 1H), 6.88 (d, J = 8.7 Hz, 1H), 6.83 (d, J =
1
3
.9 Hz, 2H). C NMR (101 MHz, CDCl
3
) δ 155.27, 150.60, 139.62, 135.87 (q, J = 33.9 Hz), 132.63, 132.19, 130.62, 130.21,
29.85, 128.67 (q, J = 3.7 Hz), 128.16, 126.83, 124.75, 124.27 (q, J = 3.6 Hz), 122.29 (q, J = 272.0 Hz), 121.78, 118.47. HRMS
-
(ESI): calc. for C19
H10Cl
3
F
3
NO S [M-H] : 493.94046; Found: 493.94021.
3

6
4
.3.15. 2,4,6-Trichloro-N-(4-(2,4-dichlorophenoxy)phenyl)benzenesulfonamide (3m). White solid, m.p. 101.3-102.0°C. Isolated
1
13
yield: 85%. H NMR (500 MHz, CDCl
NMR (126 MHz, CDCl ) δ 155.11, 150.82, 138.64, 136.14, 132.88, 131.25, 130.63, 130.51, 129.77, 128.17, 126.79, 124.07,
21.67, 118.68. HRMS (ESI): Chemical Formula: C18
3
) δ 7.47 (s, 3H), 7.21 (d, J = 8.9 Hz, 1H), 7.15 (d, J = 8.5 Hz, 2H), 6.96 – 6.82 (m, 3H). C
3
-
1
H
9
Cl
5
NO
3
S [M-H] ; Exact Mass: 493.87513; Found: 493.87504.
4
.3.16. N-(3-Chloro-4-(2,4-dichlorophenoxy)phenyl)-4-nitrobenzenesulfonamide (3n). White solid, m.p. 165.5-166.3°C. Isolated
1
yield: 61%. H NMR (500 MHz, CDCl ) δ 8.33 (d, J = 8.9 Hz, 2H), 7.95 (d, J = 8.9 Hz, 2H), 7.47 (d, J = 2.5 Hz, 1H), 7.26 (d, J =
.7 Hz, 2H), 7.20 (dd, J = 8.7, 2.5 Hz, 1H), 6.92 (dd, J = 8.8, 2.6 Hz, 1H), 6.80 (d, J = 8.7 Hz, 1H), 6.73 (d, J = 8.8 Hz, 1H). C
3
1
3
1
NMR (126 MHz, CDCl ) δ 150.72, 150.44, 150.38, 144.34, 131.54, 130.78, 130.08, 128.53, 128.19, 126.23, 125.83, 125.49, 124.47,
3
-
122.48, 120.69, 119.49. HRMS (ESI): calc. for C18
H
10Cl
3
N
2
O
5
S [M-H] : 470.93815; Found: 470.93796.
4
.3.17. 2,4-Dichloro-N-(3-chloro-4-(2,4-dichlorophenoxy)phenyl)benzenesulfonamide (3o). White solid, m.p. 132.1-133.3°C.
1
Isolated yield: 63%. H NMR (400 MHz, CDCl
3
) δ 7.94 (d, J = 8.5 Hz, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.45 (d, J = 2.5 Hz, 1H), 7.36
(
(
1
4
dd, J = 8.5, 2.0 Hz, 1H), 7.28 (d, J = 2.5 Hz, 1H), 7.16 (dd, J = 8.7, 2.5 Hz, 1H), 7.11 (s, 1H), 6.97 (dd, J = 8.8, 2.6 Hz, 1H), 6.72
d, J = 8.8 Hz, 2H). 1 C NMR (101 MHz, CDCl
) δ 150.65, 150.07, 140.41, 134.40, 132.92, 132.20, 131.98, 131.56, 130.66, 129.67,
3
3
-
28.06, 127.78, 125.92, 125.86, 124.61, 121.71, 120.15, 119.90. HRMS (ESI): calc. for C18
93.87495.
H
9
Cl
5
NO S [M-H] : 493.87513; Found:
3
4
.3.18. 2-Chloro-N-(3-chloro-4-(2,4-dichlorophenoxy)phenyl)-4-(trifluoromethyl)benzenesulfonamide (3p). White solid, m.p.
1
1
7
(
32.4-133.5°C. Isolated yield: 71%. H NMR (500 MHz, CDCl
.48 (d, J = 2.5 Hz, 1H), 7.32 (d, J = 2.7 Hz, 1H), 7.19 (dd, J = 8.7, 2.5 Hz, 1H), 7.03 (s, 1H), 6.99 (dd, J = 8.9, 2.7 Hz, 1H), 6.76
d, J = 8.8 Hz, 1H), 6.74 (d, J = 8.7 Hz, 1H). C NMR (126 MHz, CDCl
3
) δ 8.16 (d, J = 8.3 Hz, 1H), 7.83 (s, 1H), 7.67 (d, J = 8.3 Hz, 1H),
1
3
3
) δ 150.51, 150.37, 139.40, 136.29 (q, J = 33.8 Hz),
1
=
32.61, 132.19, 131.54, 130.69, 129.83, 128.83 (q, J = 3.7 Hz), 128.09, 126.04, 125.86, 124.88, 124.43 (q, J = 3.6 Hz), 122.25 (q, J
-
272.5 Hz), 121.91, 120.34, 119.74. HRMS (ESI): calc. for C19
H
9
Cl
4
F
3
NO
3
S [M-H] ; Exact Mass: 527.90148; Found: 527.90108.
4
.3.19. 2,4,6-Trichloro-N-(3-chloro-4-(2,4-dichlorophenoxy)phenyl)benzenesulfonamide (3q). White solid, m.p. 175.0-176.0°C.
1
Isolated yield: 74%. H NMR (500 MHz, CDCl ) δ 7.50 (s, 2H), 7.48 (d, J = 3.0 Hz, 1H), 7.33 (d, J = 2.7 Hz, 1H), 7.20 (dd, J = 8.8,
.5 Hz, 1H), 7.05 (dd, J = 8.7, 2.7 Hz, 1H), 6.77 (dd, J = 8.9, 8.8 Hz, 2H). C NMR (126 MHz, CDCl
36.11, 132.52, 131.82, 131.39, 130.68, 129.70, 128.08, 126.00, 125.93, 124.09, 121.22, 120.16, 120.04. HRMS (ESI): calc. for
3
1
3
2
1
C
3
) δ 150.66, 150.12, 138.99,
-
18
H
8
Cl
6
NO
3
S [M-H] ; Exact Mass: 527.83615; Found: 527.83585.
4
.3.20. N-(3,5-Dichloro-4-(2,4-dichlorophenoxy)phenyl)-4-nitrobenzenesulfonamide (3r). White solid, m.p. 228.1-229.5°C.
1
Isolated yield: 69%. H NMR (500 MHz, DMSO-d ) δ 11.21 (s, 1H), 8.43 (d, J = 8.9 Hz, 2H), 8.08 (d, J = 8.9 Hz, 2H), 7.75 (d, J =
.5 Hz, 1H), 7.31 (s, 2H), 7.28 (dd, J = 8.9, 2.5 Hz, 1H), 6.57 (d, J = 8.9 Hz, 1H). C NMR (126 MHz, DMSO-d
44.12, 142.08, 136.38, 130.11, 128.76, 128.42, 128.28, 127.15, 124.94, 122.17, 120.55, 115.60. HRMS (ESI): calc. for
6
1
3
2
1
C
6
) δ 150.55, 150.14,
-
18
H
9
Cl
4
N
2
O
5
S [M-H] ; Exact Mass: 504.89918; Found: 504.89876.
4
.3.21. 2,4-Dichloro-N-(3,5-dichloro-4-(2,4-dichlorophenoxy)phenyl)benzenesulfonamide (3s). White solid, m.p. 155.0-156.3°C.
1
Isolated yield: 50%. H NMR (500 MHz, CDCl ) δ 8.01 (d, J = 8.5 Hz, 1H), 7.57 (d, J = 1.9 Hz, 1H), 7.45 (d, J = 2.5 Hz, 1H), 7.41
dd, J = 8.5, 2.0 Hz, 1H), 7.19 (s, 2H), 7.05 (dd, J = 8.8, 2.5 Hz, 1H), 6.29 (d, J = 8.8 Hz, 1H). C NMR (126 MHz, CDCl
3
1
3
(
3
) δ
1
50.82, 144.33, 140.78, 134.43, 134.30, 132.85, 132.24, 131.78, 130.59, 130.30, 128.11, 127.95, 127.54, 123.55, 121.45, 114.95.
-
HRMS (ESI): calc. for C18
H
8
Cl
6
NO
3
S [M-H] ; Exact Mass: 527.83615; Found: 527.83587.
4
.3.22. 2-Chloro-N-(3,5-dichloro-4-(2,4-dichlorophenoxy)phenyl)-4-(trifluoromethyl)benzenesulfonamide (3t). White solid, m.p.
1
1
7
1
3
68.3-169.7°C. Isolated yield: 66%. H NMR (500 MHz, CDCl
.45 (d, J = 2.5 Hz, 1H), 7.22 (s, 2H), 7.04 (dd, J = 8.8, 2.5 Hz, 1H), 6.28 (d, J = 8.8 Hz, 1H). C NMR (126 MHz, CDCl
44.77, 139.12, 136.53 (q, J = 31.3 Hz), 133.68, 132.60, 132.28, 130.63, 130.46, 129.06 (q, J = 3.8 Hz), 128.21, 127.53, 124.63 (J =
3
) δ 8.22 (d, J = 8.2 Hz, 1H), 7.84 (s, 1H), 7.70 (d, J = 8.3 Hz, 1H),
1
3
3
) δ 150.76,
-
.8 Hz), 123.60, 122.19 (q, J = 272.5 Hz), 121.62, 114.94. HRMS (ESI): calc. for C19
H
8
Cl
5
F
3
NO S [M-H] ; Exact Mass: 561.86251;
3
Found: 561.86204.
4
.3.23. 2,4,6-Trichloro-N-(3,5-dichloro-4-(2,4-dichlorophenoxy)phenyl)benzenesulfonamide (3u). White solid, m.p. 143.4-
1
1
6
1
5
44.0°C. Isolated yield: 45%. H NMR (500 MHz, CDCl
.34 (dd, J = 9.0, 4.5 Hz, 1H). C NMR (126 MHz, CDCl ) δ 150.81, 144.56, 139.48, 136.17, 133.89, 132.22, 131.58, 130.62,
30.50, 128.18, 127.56, 123.58, 120.96, 114.97. HRMS (ESI): calc. for C18
3
) δ 7.61 – 7.43 (m, 3H), 7.28 (d, J = 4.8 Hz, 2H), 7.09 (d, J = 7.9 Hz, 1H),
1
3
3
-
H
7
Cl
7
NO S [M-H] ; Exact Mass: 561.79718; Found:
3
61.79687.
4
.3.24. N-(3,5-Dichloro-4-(2,4,6-trichlorophenoxy)phenyl)-4-nitrobenzenesulfonamide (3v). White solid, m.p. 225.1-226.5°C.
1
Isolated yield: 82%. H NMR (500 MHz, DMSO-d
.20 (s, 2H). C NMR (126 MHz, DMSO-d
25.36, 121.46. HRMS (ESI): calc. for C18
6
) δ 11.05 (s, 1H), 8.41 (d, J = 8.9 Hz, 2H), 8.02 (d, J = 8.9 Hz, 2H), 7.76 (s, 2H),
1
3
7
1
6
) δ 150.61, 147.01, 144.65, 144.32, 135.11, 129.94, 129.65, 128.83, 126.57, 126.45,
-
H
8
Cl
5
N
2
O
5
S [M-H] : 538.86020; Found: 538.85986.
4
.3.25 2,4-Dichloro-N-(3,5-dichloro-4-(2,4,6-trichlorophenoxy)phenyl)benzenesulfonamide (3w). White solid, m.p. 145.6-
1
1
2
46.9°C. Isolated yield: 83%. H NMR (500 MHz, CDCl
H), 7.10 (s, 2H), 6.93 (s, 1H). C NMR (101 MHz, CDCl ) δ 147.12, 146.29, 140.70, 133.94, 133.01, 132.20, 132.16, 131.64,
3
) δ 7.94 (d, J = 8.2 Hz, 1H), 7.56 (s, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.29 (s,
1
3
3

3
-
1
5
29.54, 129.09, 127.82, 127.34, 126.97, 122.13. HRMS (ESI): Chemical Formula: calc. for C18
61.79718; Found: 561.79686.
H
7
Cl
7
NO S [M-H] ; Exact Mass:
3
4
.3.26. 2-Chloro-N-(3,5-dichloro-4-(2,4,6-trichlorophenoxy)phenyl)-4-(trifluoromethyl)benzenesulfonamide (3x). White solid,
1
m.p. 149.7-151.1°C. Isolated yield: 66%. H NMR (500 MHz, CDCl
3
) δ 8.19 (d, J = 8.2 Hz, 1H), 7.90 – 7.81 (m, 1H), 7.69 (dd, J =
.3, 1.7 Hz, 1H), 7.30 (s, 2H), 7.19 (s, 1H), 7.14 (s, 2H). C NMR (126 MHz, CDCl ) δ 147.11, 146.52, 138.94, 136.43 (q, J = 34.1
Hz), 132.75, 132.24, 131.87, 129.63, 129.11, 128.94 (q, J = 3.7 Hz), 127.46, 126.99, 124.48 (q, J = 3.5 Hz), 122.21 (q, J = 272.5
1
3
8
3
-
Hz), 122.29. HRMS (ESI): calc. for C19
H
7
Cl
6
F
3
NO
3
S [M-H] ; Exact Mass: 595.82354; Found: 595.82318.
4
.3.27. 2,4,6-Trichloro-N-(3,5-dichloro-4(2,4,6-trichlorophenoxy)phenyl)benzenesulfonamide (3y). White solid, m.p. 192.4-
1
13
1
1
C
93.6°C. Isolated yield: 64%. H NMR (500 MHz, CDCl
3
) δ 7.48 (s, 2H), 7.29 (s, 2H), 7.15 (s, 3H). C NMR (101 MHz, CDCl
3
) δ
47.12, 146.33, 139.28, 136.19, 132.05, 132.03, 131.43, 129.54, 129.10, 127.49, 126.97, 121.62. HRMS (ESI): calc. for
-
18
H
6
Cl
8
NO
3
S [M-H] : 595.75821; Found: 595.75801.
4
.3.28. N-(3,5-Dichloro-4-(2,4,6-trichlorophenoxy)phenyl)-4-methylbenzenesulfonamide (3z). White solid, m.p. 189.7-191.2°C.
1
Isolated yield: 71%. H NMR (500 MHz, CDCl
C NMR (126 MHz, CDCl
1.60. HRMS (ESI): calc. for C19
3
) δ 7.67 (d, J = 8.2 Hz, 2H), 7.29 (s, 3H), 7.27 (s, 1H), 7.07 (s, 2H), 2.42 (s, 3H).
1
3
3
) δ 147.27, 145.81, 144.69, 135.15, 133.46, 129.93, 129.40, 129.10, 127.28, 127.21, 126.98, 122.01,
-
2
H11Cl
5
NO
3
S [M-H] 507.89078; Found: 507.89072.
4
.3.29. N-(3,5-Dichloro-4-(2,4,6-trichlorophenoxy)phenyl)-4-methoxybenzenesulfonamide (3a’). White solid, m.p. 181.0-181.9°C.
1
Isolated yield: 70%. H NMR (500 MHz, CDCl
3
) δ 7.70 (d, J = 7.8 Hz, 2H), 7.29 (s, 2H), 7.05 (s, 2H), 6.94 (d, J = 8.8 Hz, 2H),
.51 (s, 1H), 3.86 (s, 3H). C NMR (126 MHz, CDCl ) δ 163.76, 163.59, 147.28, 145.86, 133.52, 129.70, 129.48, 129.12, 127.22,
27.00, 122.18, 114.45, 55.68. HRMS (ESI): calc. for C19
1
3
6
1
3
-
H11Cl
5
NO
4
S [M-H] 523.88569; Found: 523.88547.
4
.3.30. 4-(Tert-butyl)-N-(3,5-dichloro-4-(2,4,6-trichlorophenoxy)phenyl)benzenesulfonamide (3b’). White solid, m.p. 199.4-
1
2
2
1
01.1°C. Isolated yield: 80%. H NMR (500 MHz, CDCl
3
) δ 7.70 (d, J = 8.5 Hz, 2H), 7.49 (d, J = 8.5 Hz, 2H), 7.29 (s, 2H), 7.07 (s,
H), 6.87 (s, 1H), 1.33 (s, 9H). C NMR (126 MHz, CDCl ) δ 157.63, 147.27, 145.82, 135.04, 133.48, 129.41, 129.10, 127.22,
27.16, 126.96, 126.31, 122.08, 35.26, 30.99. HRMS (ESI): calc. for C22
1
3
3
-
H17Cl
5
NO
3
S [M-H] 549.93773; Found: 549.93751.
4
.3.31. 4-Chloro-N-(3,5-dichloro-4-(2,4,6-trichlorophenoxy)phenyl)benzenesulfonamide (3c’). White solid, m.p. 188.7-189.4°C.
1
Isolated yield: 78%. H NMR (500 MHz, CDCl
.92 (s, 1H). C NMR (126 MHz, CDCl
27.02, 122.43. HRMS (ESI): calc. for C18
3
) δ 7.72 (d, J = 8.5 Hz, 2H), 7.47 (d, J = 8.5 Hz, 2H), 7.30 (s, 2H), 7.08 (s, 2H),
1
3
6
1
3
) δ 147.21, 146.26, 140.37, 136.66, 132.80, 129.68, 129.56, 129.13, 128.64, 127.39,
-
H
8
Cl
6
NO
3
S [M-H] 527.83615; Found: 527.83593.
4
.3.32. N-(3,5-Dichloro-4-(2,4,6-trichlorophenoxy)phenyl)-4-fluorobenzenesulfonamide (3d’). White solid, m.p. 163.5-165.3°C.
1
Isolated yield: 76%. H NMR (500 MHz, CDCl
3
) δ 7.80 (dd, J = 8.8, 4.9 Hz, 2H), 7.30 (s, 2H), 7.18 (dd, J = 8.6, 8.4 Hz, 2H), 7.07
s, 2H), 6.88 (s, 1H). C NMR (126 MHz, CDCl ) δ 165.59 (d, J = 256.9 Hz), 147.21, 146.21, 134.18 (d, J = 3.2 Hz), 132.92,
30.06 (d, J = 9.5 Hz), 129.55, 129.13, 127.36, 127.00, 122.44, 116.69 (d, J = 22.7 Hz). HRMS (ESI): calc. for C18 Cl FNO S [M-
1
3
(
1
3
H
8
5
3
-
H] 511.86570; Found: 511.86546.
4
.3.33. N-(3,5-Dichloro-4-(2,4,6-trichlorophenoxy)phenyl)-4-(trifluoromethyl)benzenesulfonamide (3e’). White solid, m.p. 165.7-
1
1
2
1
5
67.5°C. Isolated yield: 75%. H NMR (500 MHz, CDCl
H), 6.84 (s, 1H). C NMR (126 MHz, CDCl
27.48, 127.01, 126.55 (q, J = 3.5 Hz), 122.96 (d, J = 273.2 Hz), 122.47. HRMS (ESI): calc. for C19
3
) δ 7.91 (d, J = 8.2 Hz, 2H), 7.78 (d, J = 8.3 Hz, 2H), 7.30 (s, 2H), 7.09 (s,
1
3
3
) δ 147.16, 146.43, 141.72, 135.34 (q, J = 33.3 Hz), 132.46, 129.63, 129.13, 127.78,
-
H
8
Cl
5
F
3
NO S [M-H]
3
61.86251; Found: 561.86230.
4
.3.34. N-(3,5-Dichloro-4-(2,4,6-trichlorophenoxy)phenyl)-3-methylbenzenesulfonamide (3f’). White solid, m.p. 170.1-171.4°C.
1
Isolated yield: 71%. H NMR (500 MHz, CDCl
H), 6.88 (s, 1H), 2.40 (s, 3H). C NMR (126 MHz, CDCl ) δ 147.26, 145.95, 139.67, 138.04, 134.45, 133.30, 129.45, 129.14,
29.10, 127.54, 127.20, 127.01, 124.37, 122.33, 21.35. HRMS (ESI): calc. for C19
07.89075.
3
) δ 7.61 (s, 1H), 7.57 (d, J = 7.5 Hz, 1H), 7.43 – 7.34 (m, 2H), 7.29 (s, 2H), 7.07 (s,
1
3
2
1
5
3
-
H11Cl
5
NO S [M-H] 507.89078; Found:
3
4
.3.35. N-(3,5-Dichloro-4-(2,4,6-trichlorophenoxy)phenyl)-2-methylbenzenesulfonamide (3g’). White solid, m.p. 172.2-172.6°C.
1
Isolated yield: 55%. H NMR (500 MHz, CDCl ) δ 7.96 (d, J = 7.8 Hz, 1H), 7.51 (dd, J = 7.6, 7.4 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H),
.31 (s, 2H), 7.03 (s, 2H), 6.87 (s, 1H), 2.69 (s, 3H). C NMR (126 MHz, CDCl
32.89, 130.09, 129.43, 129.08, 127.23, 127.03, 126.53, 121.46, 20.53. HRMS (ESI): calc. for C19
3
1
3
7
1
3
) δ 147.24, 145.67, 137.11, 136.46, 133.71, 133.21,
-
H11Cl
5
NO S [M-H] 507.89078;
3
Found: 507.89060.
4
.3.36. N-(3,5-Difluoro-4-(2,4,6-trifluorophenoxy)phenyl) benzenesulfonamide (3h’). White solid, m.p. 126.5-127.5°C. Isolated
1
yield: 48%. H NMR (500 MHz, CDCl ) δ 7.85 (d, J = 7.7 Hz, 2H), 7.63 (t, J = 7.5 Hz, 1H), 7.52 (t, J = 7.7 Hz, 3H), 6.78 (d, J =
.0 Hz, 2H), 6.72 (t, J = 8.4 Hz, 2H). C NMR (126 MHz, CDCl
54.71 (dd, J = 251.2, 5.7 Hz), 138.18, 133.70, 133.08 (t, J = 11.7 Hz), 131.32 (t, J = 14.3 Hz), 130.88 (td, J = 12.8, 3.2 Hz),
29.40, 127.18, 105.37 (dd, J = 18.9, 6.3 Hz), 101.62 – 99.95 (m). HRMS (ESI): calc. for C19
3
1
3
9
1
1
4
3
) δ 157.95 (dt, J = 247.6, 13.9 Hz), 154.83 (dd, J = 251.7, 6.3 Hz),
-
H
11
F
5
NO S [M-H] 414.02288; Found:
3
14.02281.

8
4
.3.37. N-(3,5-Dimethyl-4-(2,4,6-trimethyl)phenyl) benzenesulfonamide (3i’). White solid, m.p. 224.5-225.3°C. Isolated yield:
1
9
2
1
2%. H NMR (500 MHz, CDCl
H), 6.29 (s, 1H), 2.26 (s, 3H), 1.96 (s, 6H), 1.96 (s, 6H). C NMR (126 MHz, CDCl
51.72, 138.84, 132.86, 132.03, 130.19, 129.92, 129.25, 128.81, 127.50, 127.33, 124.03, 20.47, 17.27, 17.18. HRMS (ESI): calc. for
3
) δ 7.74 (d, J = 7.4 Hz, 2H), 7.56 (t, J = 7.5 Hz, 1H), 7.45 (t, J = 7.8 Hz, 2H), 6.78 (s, 2H), 6.68 (s,
1
3
13
3
) δ C NMR (126 MHz, CDCl ) δ 152.68,
3
-
C
23
H
24NO
3
S [M-H] 394.14824; Found: 394.14819.
4
.4. Enzyme assays
SCR was isolated according to the method reported in the literature.[32] The activity of SCR, which catalyzes the oxidation of
succinate by cytochrome c, was measured by monitoring the enhancement of cytochrome c at 550 nm with an extinction coefficient
-1
-1
of 18.5 mM cm . With regard to the activity of complex II, it was measured by monitoring the decrease of 2,6-
-
1
-1
dichlorophenolindophenol (DCIP) at 600 nm, by using an extinction coefficient of 21 mM cm . DBH and cytochrome c were used
2
[
67]
to test the activity of complex III, and DBH
2
was prepared from DB using the method reported before. The IC50 (concentration at
5
0% inhibition) values with SCR, complex II and complex III were obtained from a nonlinear regression of the activity data
[
32]
according to a four parameter logistic model. The detailed procedure for the enzyme assay was described in a previous report.
4
.5. Computational investigations
The 3D structure of porcine heart complex II (PDB ID: 3AEB) was downloaded from the PDB databank as a receptor to predict the
binding mode of 3f and the reference compound (Penthiopyrad). The ligand molecule in 3AEB was extracted from the complex and
AutoDock 4.2 was utilized to dock the synthesized inhibitor 3f into the Q-site. The extracted ligand was used to define the center of
gridbox, Gasteiger charge was used for these inhibitors. The Lamarkian genetic algorithm (LGA)[68] was applied for the
conformational search. Among a series of docking parameters, the grid size and space were set at 30 × 30 × 30 Å and the default
value of 0.375 Å, respectively. The interaction energy, which was resulted from probing porcine complex II with our molecules,
was assessed with the standard Auto-Dock scoring function. Among a set of 100 candidates of the docked complex structures, the
best one was selected based on the interaction energy. In order to determine the binding modes of our compounds against complex
III, 3f, together with Azoxystrobin, were docked into both the Q
Azoxystrobin (PDB ID: 1SQB) was downloaded from the PDB databank as a receptor to discuss the binding modes of 3f and
Azoxystrobin. Azoxystrobin, which binds at the Q site of 1SQB, was extracted from the complex. Besides, AutoDock 4.2 was used
to dock these inhibitors into the Q and Q sites, and a similar docking method with complex II was applied for the docking of 3f and
sites.
o
and Q sites. The crystal structure of complex III from Bovine with
i
o
o
i
Azoxystrobin against Q
o
and Q
i
Acknowledgements
This research was supported by the National Natural Science Foundation of China (No. 21502062), Scientific Research Program
Guiding Project of Hubei Provincial Department of Education (B2019135), the Teachers' Scientific Research Ability Cultivation
Fund, Hubei University of Arts and Science (Natural Science, Grant No. 2018kypy001) and the Open Foundation of Discipline of
Hubei University of Arts and Science (Grant Nos. XK2019038, XK2019039).
Appendix A. Supplementary data
Supplementary data associated with this article (including the characterization data and spectra of all target compounds) can be
found, in the online version, at
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3
Highlights
1
2
. A new series of sulfonamides bearing a diaryl ether scaffold were designed and prepared via
structural simplification.
. N-(3,5-Dichloro-4-(2,4,6-trichlorophenoxy)phenyl)benzenesulfonamide (3f), a representative target
compound, demonstrated potent inhibitory activities against both mitochondrial complex II and
complex III.
3
. Computational simulations of 3f in the present study were carried out to clarify its possible binding
mode with mitochondrial complex II and complex III.

1
2
Graphical Abstract
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N-(3,5-Dichloro-4-(2,4,6-
Leave this area blank for abstract info.
trichlorophenoxy)phenyl)benzenesulfonamide
:
A new dual-target inhibitor of mitochondrial
complex II and complex III via structural
simplification
a
a
a
b, *
c
c
a, *
Hua Cheng, Hong-Fu Liu, Lu Yang, Cheng Chen, Yuan Wu, Wen Jiang and Rui Zhang
a
Department of Chemical Engineering and Food Science, Hubei University of Arts and Science, Xiangyang
41053, P. R. China;
State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of
4
b
Technology, Wuhan 430070, P. R. China;
c
Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, College of Chemistry, Central China
Normal University, Wuhan 430079, P. R. China.

7
Declaration of interests
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.
☐
The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests: