Discovery of 1,2,4-triazole-1,3-disulfonamides as dual inhibitors of mitochondrial complex II and complex III

Article abstract of DOI:10.1039/c5nj00215j

Respiratory chain succinate-ubiquinone oxidoreductase (SQR or complex II) and ubihydroquinone-cytochrome (cyt) c oxidoreductase (cyt bc₁ or complex III) have been demonstrated as the promising targets of numerous antibiotics and fungicides. As a continuation of our research work on the development of new fungicides, a series of 1,2,4-triazole-1,3-disulfonamide derivatives with dual functions targeting both SQR and cyt bc₁ were designed and synthesized by coupling diverse diphenyl ether moieties with triazolesulfonamide units. These newly synthesized compounds were characterized by elemental analyses, ¹H NMR and ESI-MS spectrometry. The in vitro assay indicated that most of the synthesized compounds displayed good inhibition against porcine succinate-cytochrome reductase (SCR) with IC₅₀ values ranging from 3.2 to 81.8 μM, revealing much higher activity than that of the commercial control amisulbrom whose IC₅₀ value is 93.0 μM. Further evaluation against the respective SQR and cyt bc₁ indicated that most compounds exhibited SQR-inhibiting activity as well as cyt bc₁-inhibiting activity, but the inhibition potency against SQR is much higher than that against cyt bc₁, showing that the SCR inhibition might be contributed greatly by the SQR inhibition. The further antibacterial evaluation against Xanthomonas oryzae pv. oryzae revealed that four compounds showed excellent potency at the concentration of 20 μg mL^-1. In particular, compounds 6h and 6j exhibited much better antibacterial activity than the commercial control bismerthiazol in terms of their EC₅₀. Impressively, 6j has an EC₉₀ of 33.62 μg mL^-1, more than 10-fold higher than that of bismerthiazol.

Full text of DOI:10.1039/c5nj00215j

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Discovery of 1,2,4-Triazole-1,3-Disulfonamides as Dual InhibitDoOrI:s10o.10f39/C5NJ00215J
Mitochondrial Complex II and Complex III
Hua Cheng,1 Yan-Qing Shen,1 Xia-Yan Pan,2 Yi-Ping Hou,2 Qiong-You Wu, 1,*
Guang-Fu Yang1,*
1Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, College of
Chemistry Central China Normal University, Wuhan 430079, P. R. China.
2Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural
University, Nanjing 210095, P. R. China
Corresponding author:
Prof. Guang-Fu Yang
College of Chemistry, Central China Normal University
Key Laboratory of Pesticide & Chemical Biology of Ministry of Education,
Wuhan 430079, P. R. China
Tel: +86-27-67867800
Fax: +86-27-67867141
E-mail: gfyang@mail.ccnu.edu.cn

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ABSTRACT The respiratory chain succinate-ubiquinone oxidoreductase (SQR or
complex II) and ubihydroquinone-cytochrome (cyt) c oxidoreductase (cyt bc1 or
complex III) have been demonstrated as the promising targets of numerous antibiotics
and fungicides. As a continuation of our research work on the development of new
fungicides, a series of 1,2,4-triazole-1,3-disulfonamide derivatives with dual function
targeting both SQR and cyt bc1 were designed and synthesized by coupling diverse
diphenyl ether moiety with triazolesulfonamide unit. These newly synthesized
1
compounds were characterized by elemental analyses, H NMR and ESI-MS
spectrometry. The in vitro assay indicated that most of the synthesized compounds
displayed good inhibition against porcine succinate-cytochrome reductase (SCR) with
IC50 values ranging from 3.2 to 81.8 µM , revealing much higher activity than that of
the commercial control amisulbrom whose IC50 value is 93.0 µM . Further evaluation
against respective SQR and cyt bc1 indicated that most compounds exhibited
SQR-inhibiting activity as well as cyt bc1-inhibiting activity, but the inhibition
potency against SQR is much higher than that towards cyt bc1, showing the SCR
inhibition might be contributed greatly from the SQR inhibition. The further
antibacterial evaluation against Xanthomonasoryzae pv. oryzae revealed that four
compounds showed excellent potency at the concentration of 20 µg/mL. In particular,
compounds 6h and 6j exhibited much better antibacterial activity than the commercial
control bismerthiazol in terms of their EC50. Impressively, 6j has an EC90 of 33.62
µg/mL, more than 10-fold higher than that of bismerthiazol.
Key words: Cytochrome bc1, 1,2,4-Triazole-1,3-Disulfonamide, Fungicides,
Molecular docking

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1. Introduction
Oxidative phosphorylation and tricarboxylic acid (or Krebs) cycle are two
bioprocesses which coupled with electron transport and proton pump flow constituted
the highly effective mitochondrial respiration. In animals and bacteria, the oxidative
phosphorylation system comprises five multiprotein complexes (complexes I to V)
and two mobile electron carriers (ubiquinone and cytochrome c) embedded in the
lipid bilayer of the mitochondrial inner membrane. Among them, succinate-
ubiquinone oxidoreductase (SQR or complex II) and ubihydroquinone-cytochrome
(cyt) c oxidoreductase (cyt bc1 or complex III) are two essential and central
components of the cellular respiratory chain and of the photosynthetic apparatus in
photosynthetic bacteria. SQR specifically catalyzes the oxidation of succinate to
fumarate with concomitant reduction of ubiquinone to ubiquinol. This enzyme is a
four subunit membrane-bound dehydrogenase that comprises a FAD-containing
flavoprotein, a subunit containing multiple iron-sulfur clusters and two smaller
hydrophobic cytochrome b containing subunits that anchor the catalytic portion to the
mitochondrial membrane. Whereas, the function of bc1 complex is to catalyze the
electron transfer (ET) from ubiquinol (hydroxyquinones, QH2) to a water-soluble
cytochrome c (cyt c) and couples this electron transfer to the translocation of protons
across the membrane to generate a proton gradient and membrane potential for ATP
synthesis.[1–5] Though their subunit composition varied among different organisms
from three or more subunits in bacterial to ten or eleven subunits in mitochondrial
forms, the catalytic central of bc1 complexes usually contain three redox-active
subunits: a cyt. b possessing two b-type hemes, bH and bL; a cyt. c1 bearing one
c-type heme; and an ISP containing a 2Fe-2S cluster.[6] In case of the electron
transport process of complex II and/or cyt bc1 was disturbed or disrupted, the cellular
respiration will be blocked and resulted in cell death, thus complex II and III have
been identified as the promising action of target for numerous antiparastic agents,
antibiotics as well as agricultural fungicides.

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So far, approximately 30 crystal structures of SQR, including native structures and
inhibitor-bound complexes, have been reported since the first crystal structure of E.
coli SQR was solved at 2.6 resolution.[7] The small molecules involved in these
structures include ubiquinone (UQ) and some ubiquinone binding site (Q-site)
inhibitors. X-ray crystallographic studies of Q-site inhibitors bound to SQR have
indicated that the essential bonding residues were absolutely conserved and the amino
acid residues of the IP and of the transmembrane subunits constitute the putative
Q-site[8,9]. Under these circumstances, 18 specific SQR inhibitors have been
developed and commercialized as agricultural fungicides. These structurally diverse
inhibitors are categorized into carboxamide fungicides binding to Q-site.[10,11] Though
the first generation of carboxamide fungicides such as carboxin and benodanil have
manifested a narrow fungicidal spectrum, continuous efforts have led to a range of
chemical structures such as boscalid and bixafen, which exhibit broadened biological
spectrum and improved potency to match the requirement for modern agricultural
protection as well.
Unlike SQR system, in which the binding site is formed by a pocket near the
cytoplasmic interface with residues from chains B, C and D, the catalytic core of cyt
bc1 comprises of two discrete binding sites according to the Q-cycle reaction
mechanism[12], termed the quinone reduction site near the negative side of the
membrane (Qi) and the quinol oxidation site close to the positive side of the
membrane (Qo).[13,14] Consequently, the inhibitors targeted at bc1 complex are divided
into two types according to the point of action:[15,16] type I for Qo site inhibitors and
type II for the Qi site inhibitors. The strobilurin-type fungicides like azoxystrobin and
kresoxim-methyl are typical representatives of Qo site agrochemical class.[17,18] Over
15 strobilurin fungicides were commercially available since the first strobilurin
fungicide azoxystrobin was developed and launched in 1996. In comparison, only two
Qi inhibitors namely cyazofamid and amisulbrom[19–20] have been launched into the
agricultural fungicide market. Though strobilurin type fungicides have achieved great
success, the significant resistance issues were observed in a wide range of important

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plant pathogens after a short period of field applications. Thus, discovery of novel
fungicides targeting Qi site of cyt bc1 represents an attractive approach to fight against
the explosive development of resistance that the Qo site inhibitors are facing.
Antimycin A,[21] a nature product isolated from streptomyces sp. showing a
dissociation constant with bovine heart mitochondrial particles of 32 pM,[22] was
shown to bind to the Qi site of bc1 to block the mitochondrial electron transfer
between cytochrome b and c. It is a valuable starting point for the development of
agricultural fungicides with the action of Qi site of mitochondrial.[23–25] Bolgunas and
Tokutake have successfully simplified antimycin scaffold by replacing the dilactone
portion of the molecule with biphenyl and biphenyl ether group (Figure 1).[26] These
analogues possess comparable in vitro activity as potent Qi site inhibitors. On the
other hand, cyazofamid and amisulbrom, the two commercialized Qi site inhibitors are
featured with a sulfonamide azolyl pharmacophore.[27-28] Initially, we envisioned that
the integration of the biphenyl ether, which has been demonstrated their inherent
importance in agrochemistry, with the sulfonamide azolyl pharmacophore may result
in a novel class of structure as depicted in Figure 1 with efficient Qi site inhibition.
Unexpectedly, the bioassay results indicated that these inhibitors showed not only
significant bc1-inhibiting activity but also remarkable SQR-inhibiting activity. To the
best of our knowledge, this is the first observation of the inhibitors possessing dual
target inhibition toward mitochondrial complex II and complex III. These inhibitors
may serve as novel lead for further fungicide discovery in terms of the dual-targeting
inhibitors have the advantageous to improve the potency by synergistic effect and
provide a new approach to overcome the resistance issues that affect marketing
fungicides.

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Cl
O
N
Me
O
OHC NH OH
CN
O
H
O
O
N
N
SO2N(CH3)2
O
H
n-Hexyl
H3C
R
O
O
Me
N
S
O
Cyazofamid
Br
N
Antimycin
R
N
N
O
SO2N(CH3)2
Target Molecule
CH3
O
N
F
OH
O
S
H
N
A
A
O
N
N
N
Cl
O
SO2N(CH3)2
H
Amisulbrom
Figure 1. Antimycin, cyazofamid, amisulbrom and the designed compounds.
2. Experimental
2.1 General Techniques
All chemical reagents were commercially available and treated with standard methods
before use. Solvents were dried and redistilled before use. Silica gel column
chromatography (silica gel 200-300 mesh, Qingdao Makall Group Co., Ltd, Qingdao,
China).1H NMR spectra were recorded on a VARIAN Mercury-Plus 600 or 400
spectrometer in CDCl3 or DMSO-d6 with TMS as the internal reference, 13C NMR
spectra were recorded in CDCl3 or DMSO-d6 on a VARIAN Mercury-Plus 600 (151
MHz) or 400 (101 MHz) spectrometer, and chemical shifts (δ) are given in ppm
relative to the center line of a triplet at 77.0 ppm of CDCl3. The following
abbreviations are used to designate multiplicities: s = singlet, d = doublet, t = triplet,
m = multiplet, br = broad. Elementary analysis was taken with a Vario EL III
elementary analysis instrument. MS spectra were determined using a Trace MS 2000
organic mass spectrometry, and the signals were given in m/z. Melting points were
taken on a Buchi B-545 melting point apparatus and are uncorrected.
2.2 Preparation of the Designed Compounds
Preparation of 1,2-di(1H-1,2,4-triazol-3-yl)disulfane (1): To a mechanically stirred
slurry of 1.01 g (10 mmol) 3-mercapto-1,2,4-triazole in 5 mL dichloromethane was
added 0.79 g (10 mmol) dry pyridine. The resulting mixture was cooled in an ice bath
and 0.88 g (5 mmol) benzenesulfonyl chloride was added dropwise over a period of 1
h. The ice bath was removed and the mixture was stirred for 16 h at room temperature.
Dichloromethane was then evaporated and the resulting residue was mechanically

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stirred with a mixture of 5 mL water and 3 mL ethyl acetate for 1 h. The mixture was
filtered to isolate the resulting precipitate, which was then washed sequentially with
20 mL water and 20 mL ethyl acetate. Drying the precipitate under vacuum at
60-70 ℃ gave 0.92 g (92%) of the desired 3,3'-(dithiobis)-1,2,4-triazole as a white
solid.29
Preparation of 3,3'-disulfanediylbis(N,N-dimethyl-1H-1,2,4-triazole-1- sulfonamide)
(2): To a mixture of 0.2 g (1 mmol) of bis[1,2,4-triazole-3-yl]disulfide and 5 mL of
DMF was added 0.276 g (2 mmol) of potassium carbonate. The temperature was
raised to 30 ℃, and 0.317 g (2.2 mmol) of N, N-dimethylsulfamoyl chloride was
added dropwise at a temperature between 28 to 32 ℃ over 2 hours. After the
completion of the reaction (monitored by TLC), 30 mL of 1,2-dichloroethane was
added, and the resulting solution was added in a mixture of 10 mL of 35%
hydrochloric acid and 40 mL of water at a temperature ranging from 20 to 25 ℃. The
organic phase was collected to obtain a 1,2-dichloroethane solution containing 0.373
g bis[1-(N,N-dimethylsulfamoyl) -1,2,4-triazole -3-yl]disulfide, yield 90%.30
Preparation of 1-(N,N-dimethylsulfamoyl)-1H-1,2,4-triazole-3-sulfonyl chloride (3):
To 10 mL of a 1,2-dichloroethane solution containing 0.829 g (2 mmol) of
bis[1-(N,N-dimethyl-sulfamoyl)-1,2,4-triazole-3-yl]disulfide was added 20 mL of
water, and the mixture was cooled to 0 ℃. 10 mL of formic acid was added and then
chlorine gas was bubbled at a temperature ranging from 15 to 20 ℃ over 3 hours.
Thereafter, the resulting mixture was stirred at 15 to 20 ℃ for 0.5 hour. After the
completion of the reaction, the solution was subjected to phase seperation, washed
with water (30 mL x 3) to obtain a 1,2-dichloroethane solution containing 0.986 g of
3-chlorosulfonyl-1-(N,N-dimethyl-sulfamoyl)-1,2,4-triazole, yield 91%.30
General procedure for the preparation of compounds (5): Under N2 atmosphere, a
three-neck round bottom flask was charged with nitroarenes (1.1 mmol), phenols (1.0
mmol), and K2CO3 (1.5 mmol) in DMF (5 mL) at room temperature, the mixture was
stirred constantly at 60 ℃ (oil bath temperature) for 8 hours. After the completion of

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the reaction, as monitored by TLC, the reaction mixture was cooled to room
temperature, diluted with ethyl acetate, and filtrated. The filtrate was concentrated
under vacuum, and the resulting residue was purified by silica gel column
chromatography to afford compounds 4. Then to a solution of 4 (5 mmol ) in
dichloromethane (25 mL) was added 10% Pd-C (15% by weight based on 4) at room
temperature. When the reaction was complete (monitored by TLC), the reaction
mixture was filtered and the solvent was evaporated under reduced pressure to give
the residue, which was then purified through flash chromatography to give compound
5.
Data for 5a31: White solid (92%), mp: 239-240 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 7.45 (d, J=8.4 Hz, 2H), 6.81 (d, J=8.4 Hz, 2H), 6.77 (d, J=8.4 Hz, 2H), 6.59 (d,
J=8.4 Hz, 2H), 5.10 (s, 2H). EI-MS; m/z = 265.20 (M+).
1
Data for 5b: White solid (42%); mp: 55-56 ℃; H NMR (600 MHz, CDCl3):
δ=7.69 (s, 1H), 7.36 (d, J=9.0 Hz, 1H), 6.88 (d, J=8.4 Hz, 2H), 6.82 (d, J=9.0 Hz, 1H),
6.71 (d, J=8.4 Hz, 2H), 3.68 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 157.64,
146.45, 143.95, 127.40, 125.67, 124.87, 123.27, 122.94, 122.61, 122.43, 122.17,
121.10, 116.44, 115.07; EI-MS: m/z = 287.21 (M+).
1
Data for 5c: White solid (90%); mp: 184-185 ℃; H NMR (600 MHz, DMSO-d6):
δ 7.67 (d, J=2.3 Hz, 1H), 7.32 (dd, J=8.9, 2.5 Hz, 1H), 6.78 (m, 3H), 6.61 (d, J=8.7
Hz, 2H), 5.11 (s, 2H).; 13C NMR (100 MHz, DMSO-d6): δ 153.24, 145.46, 145.31,
129.61, 128.15, 126.19, 123.44, 120.34, 118.46, 115.43; EI-MS: m/z = 255.16(M+).
Data for 5d32: White solid (95%); mp: 88-89 ℃; 1H NMR (600 MHz, CDCl3): δ
7.09 – 6.99 (m, 1H), 6.96 (d, J=7.8 Hz, 1H), 6.88 – 6.77 (m, 4H), 6.64 (d, J=8.4 Hz,
2H), 3.87 (s, 3H), 3.47 (s, 2H). EI-MS: m/z = 215.19 (M+).
1
Data for 5e33: White solid (65%); mp: 63-64℃; H NMR (600 MHz, DMSO-d6): δ
7.36 – 7.26 (m, 1H), 7.10 (t, J=7.8 Hz, 1H), 7.06 (s, 1H), 6.88 (t, J=8.4 Hz, 1H), 6.76
(d, J=8.4 Hz, 2H), 6.59 (d, J=8.0 Hz, 2H), 5.02 (s, 2H); EI-MS: m/z = 203.19 (M+).

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32
1
Data for 5f : White solid (93%), mp: 77-78 ℃; H NMR (600 MHz, CDCl3) δ
=7.17 (t, J=7.8 Hz, 1H), 6.88 (d, J=8.4 Hz, 2H), 6.85 (d, J=7.8 Hz, 1H), 6.83 – 6.71
(m, 4H), 4.38 (s, 2H), 2.31 (s, 3H); EI-MS: m/z = 199.25 (M+).
1
Data for 5g34: White solid (98%); Mp: 116-117℃; H NMR (600 MHz, CDCl3): δ
7.62 (d, J=7.2 Hz, 1H), 7.41 (t, J=7.6 Hz, 1H), 7.05 (t, J=7.2 Hz, 1H), 6.91 (d, J=7.2
Hz, 2H), 6.78 (d, J=8.4 Hz, 1H), 6.72 (d, J=7.2 Hz, 2H), 3.78 (s, 2H); EI-MS: m/z =
210.20 (M+).
1
Data for 5h35: White solid (94%); Mp: 111-112℃; H NMR (400 MHz, CDCl3): δ
7.56 (d, J=8.4 Hz, 2H), 6.94 (d, J=8.4 Hz, 2H), 6.88 (d, J=8.4 Hz, 2H), 6.72 (d, J=8.4
Hz, 2H), 3.80 (s, 2H); EI-MS: m/z = 210.24(M+).
1
Data for 5i36: White solid (77%); Mp: 130-131℃; H NMR (400 MHz, CDCl3): δ
7.51 – 7.35 (m, 4H), 7.33 (d, J=7.2 Hz, 1H), 6.89 (s, 4H), 6.82 (d, J=8.8 Hz, 2H), 6.65
(d, J=8.4 Hz, 2H), 5.02 (s, 2H), 3.44 (s, 2H); EI-MS: m/z = 291.32(M+).
1
Data for 5j37: Yield: 70%; Mp: 138-139℃; H NMR (600 MHz, DMSO-d6): δ
7.51-7.28 (m, 5H), 7.19 (d, J=8.2 Hz, 1H), 6.75 (d, J=5.8 Hz, 2H), .6.65 (d, J=6.7 Hz,
1H), 6.58 (d, J=5.6 Hz, 2H), 6.48 (s, 1H), 6.40 (d, J=5.6 Hz, 1H), 5.04 (s, 2H), 5.01 (s,
2H); EI-MS: m/z = 291.28(M+).
1
Data for 5k38: White solid (90%); Mp: 66 - 68 ℃; H NMR (600 MHz,
DMSO-d6): δ=8.01 (d, J=8.4 Hz, 1H), 7.54 (d, J=7.8 Hz, 2H), 7.15 (d, J=8.4 Hz, 2H),
6.54 (d, J=7.8 Hz,1H), 6.48 (d, J=7.2 Hz, 2H), 5.57 ppm (s, 2H); EI-MS: m/z =
186.08 (M+).
1
Data for 5l: White solid (73%); Mp: 127-129 ℃; H NMR (600 MHz, DMSO-d6):
δ 8.08 (s, 1H), 7.94 (s, 1H), 7.15 (d, =8.4 Hz, 1H), 6.74 (s, 1H), 6.58 (d, J=7.2 Hz,
1H), 5.77 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 156.66, 150.91, 139.21, 136.98,
130.39, 129.46, 125.54, 124.95, 113.22, 112.63, 109.44; EI-MS: m/z = 288.15 (M+).

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1
Data for 5m: White solid (68%); Mp: 131-132 ℃; H NMR (600 MHz, DMSO-d6):
δ 8.09-7.97 (m, 2H), 7.12-7.09 (m, 1H), .6.46-6.42 (m, 2H), 5.78 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) δ 158.35, 156.54, 155.89, 151.61, 139.12, 137.06, 128.99,
125.30, 114.96, 109.54, 99.97, 99.75; EI-MS: m/z = 271.11 (M+).
Data for 5n: White solid (57%); Mp: 208-210 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 8.13 (d, J=2.4 Hz, 1H), 8.04 (d, J=2.4 Hz, 1H), 6.73 (s, 2H), 6.08 (s, 2H); 13C NMR
(100 MHz, DMSO-d6): δ 156.19, 151.22, 139.81, 136.75, 132.34, 125.79, 121.81,
112.39, 110.20; EI-MS: m/z = 324.08 (M+).
1
Data for 5o: White solid (73%); Mp: 95-96 ℃; H NMR (600 MHz, DMSO-d6):
δ 7.69 (d, J=2.5 Hz, 1H), 7.31 (dd, J=9.0, 2.4 Hz, 1H), 6.95 (d, J=8.4 Hz, 1H), 6.74 (d,
J=2.4 Hz, 1H), 6.61 (d, J=8.9 Hz, 1H), 6.57 (d, J=8.4 Hz, 1H), 5.44 (s, 2H); 13C NMR
(100 MHz, DMSO-d6): δ 152.76, 147.71, 139.19, 129.68, 128.13, 126.14, 125.50,
123.22, 122.51, 116.72, 114.54, 113.74; EI-MS: m/z = 288.15(M+).
Data for 5p: White solid (59%); Mp: 99-100 ℃; 1H NMR (600 MHz, DMSO-d6): δ
8.06 (s, 1H), 7.72 (d, J=7.8 Hz, 1H), 7.24 (s, 2H), 7.17 (d, J=8.4 Hz, 1H), 5.34 (s, 2H);
13C NMR (100 MHz, DMSO-d6): δ 155.76, 151.54, 127.79, 125.92, 125.05, 124.69,
124.10, 121.98, 121.18, 119.01; EI-MS: m/z = 324.13(M+).
1
Data for 5q: White solid (86%); Mp: 96-97 ℃; H NMR (600 MHz, DMSO-d6):
δ 7.80 (s, 1H), 7.35 (s, 1H), 6.95 (s, 1H), 6.73 (s, 1H), 6.57 (s, 2H), 5.44 (s, 2H); 13C
NMR (100 MHz, DMSO-d6): δ 153.77, 147.61, 139.38, 132.38, 128.62, 126.42,
125.53, 123.20, 116.46, 114.59, 113.73, 111.59; EI-MS: m/z = 333.11(M+).
1
Data for 5r: White solid (75%); Mp: 58-60 ℃; H NMR (600 MHz, DMSO-d6): δ
7.97 (s, 1H), 7.63 (d, J=8.4 Hz, 1H), 7.05 (d, J=8.4 Hz, 1H), 6.84 – 6.69 (m, 2H), 6.61
(d, J=8.4 Hz, 1H), 5.52 (s, 2H); 13C NMR (100 MHz, DMSO-d6): δ 156.73, 148.23,
138.45, 127.51, 125.70, 124.81, 124.01, 123.66, 123.34, 122.11, 122.01, 115.41,
114.52, 113.84; EI-MS: m/z = 319.18 (M+).

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1
Data for 5s: White solid (55%); Mp: 90-92 ℃; H NMR (600 MHz, DMSO-d6): δ
8.01 (s, 1H), 7.63 (d, J=7.8 Hz, 1H), 6.76-6.70 (m, 3H), 5.81 (s, 2H); 13C NMR (100
MHz, DMSO-d6): δ 155.42, 148.57, 133.92, 127.91, 127.74, 125.89, 124.74, 124.14,
123.82, 122.04, 121.77, 114.61, 113.35; EI-MS: m/z = 355.37 (M+).
1
Data for 5t: White solid (82%); Mp: 46-47 ℃; H NMR (600 MHz, DMSO-d6): δ
7.70 (s, 1H), 7.33 (d, J=9.0 Hz, 1H), 6.96 (t, J=9.0 Hz, 1H), 6.73 (d, J=9.0 Hz, 1H),
13
6.51 (d, J=12.0 Hz, 1H), 6.41 (d, J=9.0 Hz, 1H), 5.46 (s, 2H); C NMR (100 MHz,
DMSO-d6): δ 155.33, 153.09, 152.91, 148.21, 148.11, 130.33, 130.19, 129.68, 128.26,
126.18, 123.40, 122.34, 116.58, 110.05, 101.68, 101.47; EI-MS: m/z = 271.16 (M+).
Data for 5u: White solid (55%); Mp: 102-104 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 7.74 (d, J=2.4 Hz, 1H), 7.35 (d, J=9.0 Hz, 1H), 6.81 (d, J=9.0 Hz, 1H), 6.39 (d,
J=10.8 Hz, 2H), 5.84 (s, 2H); 13C NMR (150 MHz, DMSO-d6): δ 156.40, 154.77,
152.65, 148.18, 148.08, 129.84, 128.38, 126.68, 121.97, 118.36, 118.25. 118.14,
115.47, 97.18, 97.03; EI-MS: m/z = 289.16 (M+).
1
Data for 5v39: White solid (46%); Mp: 168-169 ℃; H NMR (600 MHz,
DMSO-d6): δ 7.72 (s, 2H), 6.62 (s, 2H), 5.59 (s, 2H); EI-MS: m/z = 357.08 (M+).
Data for 5w: White solid (37%); Mp: 104-106 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 7.76 (s, 2H), 6.26 (d, J=11.4 Hz, 2H), 5.60 (s, 2H); 13C NMR (100 MHz,
DMSO-d6): δ 155.28, 155.21, 152.87, 152.80, 148.19, 146.26, 146.13, 129.21, 129.08,
127.48, 122.02, 97.01, 96.77; EI-MS: m/z = 323.35 (M+).
1
Data for 5x39: White solid (54%); Mp: 142-143 ℃; H NMR (600 MHz,
DMSO-d6): δ 7.85 (s, 2H), 6.71 (d, J=2.4 Hz, 1H), 6.38 (dd, J=8.4, 2.4 Hz, 1H), 6.29
(d, J=8.4 Hz, 1H), 5.14 (s, 2H); EI-MS: m/z = 323.08 (M+).
Data for 5y: White solid (83%); Mp: 114-115 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 8.29 (s, 1H), 7.94 (d, J=9.0 Hz, 1H), 7.58 (m, 3H), 7.35 (s, 1H), 6.99 (d, J=8.4 Hz,
1H), 6.77 (d, J=2.4 Hz, 1H), 6.63 – 6.58 (m, 1H), 6.50 (d, J=7.8 Hz, 1H), 5.39 (s, 2H);

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13
C NMR (100 MHz, DMSO-d6): δ 157.01, 156.84, 154.13, 151.47, 139.70, 139.41,
134.75, 132.52, 132.38, 126.21, 124.42, 124.18, 121.58, 112.42, 111.19, 108.50;
EI-MS: m/z = 269.57(M+).
Data for 5z: White solid (64%); Mp: 105-106 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 8.35 (m, 1H), 7.95 (t, J=8.4 Hz, 1H), 7.60 (m ,3H), 7.34 (t, J=7.8 Hz, 1H), 6.79 (s,
2H), 6.43 (d, J=7.8 Hz, 1H), 5.73 (s, 2H); 13C NMR (100 MHz, DMSO-d6): δ=152.76,
147.89, 135.19, 134.31, 128.46, 127.56, 126.73, 125.84, 124.06, 121.66, 121.37,
113.46, 106.35; EI-MS: m/z = 303.15 (M+).
Data for 5I: White solid (56%); Mp: 90-92 ℃; 1H NMR (600 MHz, DMSO-d6): δ 8.38
– 8.28 (m, 1H), 8.02 – 7.90 (m, 1H), 7.67 – 7.54 (m, 3H), 7.37 (t, J=8.4 Hz, 1H), 6.64
13
(d, J=7.8 Hz, 1H), 6.41 (d, J=10.8 Hz, 2H), 5.77 (s, 2H); C NMR (100 MHz,
DMSO-d6): δ=157.24, 154.80, 153.86, 147.90, 147.75, 134.25, 127.62, 126.87,
125.97, 125.89, 124.06, 121.92, 121.26, 106.41, 97.28, 97.07; EI-MS: m/z = 271.20
(M+).
General procedure for the preparation of compound (6):A solution of
triazole-3-sulfonyl chloride 3 (1.1 mmol) in dry THF (5 mL) was added dropwise to a
solution of aniline (1.0 mmol) in dry THF (5 mL) and NEt3 (2 mmol) at room
temperature under N2 atmosphere. After stirring overnight, the reaction mixture was
poured into water, and extracted with CH2Cl2 (30 mL). The organic layer was washed
with 2 M HCl (10 mL × 2), brine (10 mL × 2), saturated aqueous NaHCO3 (10 mL),
brine (10 mL × 2), and dried over Na2SO4. The solvent was evaporated and the crude
product was purified through flash chromatography to give the pure product 6.40
Data for 6a: White solid (86%); Mp: 209-210 ℃; 1H NMR (400 MHz, DMSO-d6):
δ=11.03 (s, 1H), 9.40 (s, 1H), 7.57 – 7.50 (m, 2H), 7.20 (d, J=8.8 Hz, 2H), 7.01 (d,
J=8.8 Hz, 2H), 6.90 (d, J=8.8 Hz, 2H), 2.84 (s, 6H); 13C NMR (150 MHz, DMSO-d6):
δ=162.62, 156.69, 153.65, 148.78, 133.19, 132.80, 123.97, 120.66, 122.21, 115.45,

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+
38.68; EI-MS: m/z = 503.14 (M ). Anal. Calcd for C16H16BrN5O5S2 (502.98): C,
38.25; H, 3.21; N, 13.94; S, 12.77; Found: C, 38.43; H, 3.337; N, 14.01; S, 12.93.
Data for 6b: White solid (63%); Mp: 160-161 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 11.14 (s, 1H), 9.42 (s, 1H), 8.03 (s, 1H). 7.86 (d, J=10.8 Hz, 1H), 7.49 (d, J=8.4 Hz,
2H), 7.10(d, J=8.4 Hz, 2H), 7.03 (d, J=8.4 Hz, 1H), 2.86 (s, 6H); 13C NMR (150 MHz,
DMSO-d6): δ 162.61, 156.03, 152.37, 148.78, 133.75, 128.25, 126.35, 125.41, 125.20,
124.58, 123.76, 122.85, 120.38, 119.45, 38.65; EI-MS: m/z = 525.27 (M+). Anal.
Calcd for C17H15ClF3N5O5S2 (525.02): C, 38.82; H, 2.87; N, 13.32; S, 12.19; Found:
C, 38.99; H, 2.755; N, 13.59; S, 12.17.
1
Data for 6c: White solid (83%); Mp: 140-142 ℃; H NMR (600 MHz, DMSO-d6):
δ 11.02 (s, 1H), 9.40 (s, 1H), 7.76 (s, 1H), 7.42 (d, J=8.7 Hz, 1H), 7.19 (d, J=8.4 Hz,
2H), 7.03 (d, J=8.4 Hz, 1H), 6.96 (d, J=8.4 Hz, 2H), 2.84 (s, 6H); 13C NMR (150
MHz, DMSO-d6): δ 162.58, 153.76, 151.23, 148.81, 132.66, 130.58, 129.26, 128.92,
125.92, 124.05, 122.24, 118.90, 38.68; EI-MS: m/z = 491.22 (M+). Anal. Calcd for
C16H15Cl2N5O5S2 (490.99): C, 39.03; H, 3.07; Cl, 14.22; S, 13.03; Found: C, 39.19; H,
3.255; N, 14.49; S, 12.93.
Data for 6d: White solid (88%); Mp: 132-134 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 10.85 (s, 1H), 9.39 (s, 1H), 7.19 (d, J=7.8 Hz, 1H), 7.16 (d, J=7.8Hz, 1H), 7.09 (d,
J=8.4 Hz, 2H), 7.00 – 6.93 (m, 2H), 6.77 (d, J=8.4 Hz, 2H), 3.72 (s, 3H), 2.82 (s, 6H);
13C NMR (150 MHz, DMSO-d6): δ 162.61, 155.94, 151.74, 148.79, 143.81, 130.79,
126.16, 124.51, 122.01, 121.56, 117.01, 113.84, 56.02, 38.63; EI-MS: m/z =
453.27(M+). Anal. Calcd for C17H19N5O6S2 (453.08): C, 45.02; H, 4.22; N, 15.44; S,
14.14; Found: C, 45.25; H, 4.326; N, 15.53; S, 13.99.
Data for 6e: White solid (72%); Mp: 168-169 ℃; 1H NMR (400 MHz, DMSO-d6):
δ 11.32 (s, 1H), 9.40 (s, 1H), 7.35 (t, J=7.6 Hz, 2H), 7.24 – 7.13 (m, 2H), 7.11 (d,
J=7.6 Hz, 1H), 7.04 (s, 1H), 6.90 (d, J=8.4 Hz, 2H), 2.86 (s, 6H); 13C NMR (150
MHz, DMSO-d6): δ 162.18, 154.34, 142.81, 142.73, 131.66, 125.68, 125.55, 123.81,

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+
122.00, 117.60, 117.31, 117.21, 38.24; EI-MS: m/z = 441.29 (M ). Anal. Calcd for
C16H16FN5O5S2 (441.06): C, 43.53; H, 3.65; F, 4.30; N, 15.86; S, 14.53; Found: C,
43.79; H, 3.605; N, 16.07; S, 14.68.
Data for 6f: White solid (83%); Mp: 117-118 ℃; 1H NMR (400 MHz, DMSO-d6):
δ 10.97 (s, 1H), 9.39 (s, 1H), 7.23 (d, J=7.6 Hz, 1H), 7.17 (d, J=8.8 Hz, 2H), 6.95 (m,
13
3H), 6.77 (s, 1H), 6.73 (d, J=7.6 Hz, 1H), 2.84 (s, 6H), 2.27 (s, 3H); C NMR (150
MHz, DMSO-d6): δ 162.62, 157.10, 154.42, 148.81, 140.21, 132.16, 130.17, 124.61,
124.09, 119.82, 119.31, 115.81, 38.66, 21.34; EI-MS: m/z = 437.31(M+). Anal. Calcd
for C17H19N5O5S2 (437.08): C, 46.67; H, 4.38; N, 16.01; S, 14.66; Found: C, 46.56; H,
4.410; N, 16.18; S, 14.41.
Data for 6g: White solid (86%); Mp: 128-130 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 11.15 (s, 1H), 9.39 (s, 1H), 7.89 (d, J=7.6 Hz, 1H), 7.65 (t, J=7.6 Hz, 1H), 7.27 (dd,
J=18.4, 8.2 Hz, 3H), 7.13 (d, J=8.4 Hz, 2H), 6.88 (d, J=8.4 Hz, 1H), 2.85 (s, 6H); 13C
NMR (150 MHz, DMSO-d6): δ 162.16, 158.80, 151.93, 148.45, 135.32, 134.19,
133.45, 123.79, 123.31, 120.43, 117.29, 115.93, 102.74, 38.31; EI-MS: m/z =
448.22(M+). Anal. Calcd for C17H16N6O5S2 (448.06): C, 45.53; H, 3.60; N, 18.74; S,
14.30; Found: C, 45.59; H, 3.468; N,18.69; S, 14.37.
Data for 6h: White solid (81%); Mp: 170-172 ℃; 1H NMR (400 MHz, DMSO-d6):
δ 11.16 (s, 1H), 9.42 (s, 1H), 7.83 (d, J=8.4 Hz, 2H), 7.26 (d, J=8.4 Hz, 2H), 7.13 (d,
J=8.4 Hz, 2H), 7.03 (d, J=8.4 Hz, 2H), 2.85 (s, 6H); 13C NMR (150 MHz, DMSO-d6):
δ 162.19, 161.18, 151.35, 148.48, 134.65, 133.60, 123.25, 121.22, 118.74, 117.76,
105.12, 38.29; EI-MS: m/z = 448.32(M+). Anal. Calcd for C17H16N6O5S2 (448.06): C,
45.53; H, 3.60; N, 18.74; S, 14.30; Found: C, 45.67; H, 3.736; N, 18.50; S, 14.49.
1
Data for 6i: White solid (78%); Mp: 151-153 ℃; H NMR (400 MHz, DMSO-d6):
δ 10.90 (s, 1H), 9.39 (s, 1H), 7.45 (d, J=7.2 Hz, 2H), 7.40 (t, J=7.2 Hz, 2H), 7.34 (d,
J=6.8 Hz, 1H), 7.17 – 7.10 (m, 2H), 7.03 (d, J=9.2 Hz, 2H), 6.97 – 6.91 (m, 2H), 6.87
(d, J=8.8Hz, 2H), 5.08 (s, 2H), 2.83 (s, 6H); 13C NMR (150 MHz, DMSO-d6): δ

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162.25, 155.41, 154.75, 149.66, 148.39, 137.04, 131.06, 128.45, 127.87, 127.73,
123.94, 120.47, 118.11, 116.03, 69.64, 38.24; EI-MS: m/z = 529.27(M+). Anal. Calcd
for C23H23N5O6S2 (529.11): C, 52.16; H, 4.38; N, 13.22; S, 12.11; Found: C, 52.51; H,
4.178; N, 13.46; S, 12.26.
Data for 6j: White solid (60%); Mp: 97-98 ℃; 1H NMR (400 MHz, DMSO-d6): δ
10.99 (s, 1H), 9.38 (s, 1H), 7.36 (m, 6H), 7.25 (t, J=8.4 Hz, 1H), 7.17 (d, J=8.8 Hz,
2H), 6.96 (d, J=8.9 Hz, 2H), 6.77 (d, J=8.0 Hz, 1H), 6.57 (s, 1H), 6.47 (d, J=8.0 Hz,
13
1H), 5.06 (s, 2H), 2.81 (s, 6H); C NMR (150 MHz, DMSO-d6): δ 162.65, 160.16,
158.39, 153.90, 148.81, 137.22, 132.51, 130.96, 128.86, 128.20, 123.94, 120.15,
110.80, 110.21, 105.57, 69.82, 38.65; EI-MS: m/z = 529.38(M+). Anal. Calcd for
C23H23N5O6S2 (529.11): C, 52.16; H, 4.38; N, 13.22; S, 12.11; Found: C, 52.18; H,
4.431; N, 13.40; S, 11.98
Data for 6k: White solid (41%); Mp: 183-185 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 11.08 (s, 1H), 9.43 (s, 1H), 8.12 (s, 1H), 7.84 (t, J=7.6 Hz, 1H), 7.20 (d, J=8.8 Hz,
13
2H), 7.12 (s, 1H), 7.08 (d, J=8.8 Hz, 2H), 7.01 (d, J=8.4 Hz, 1H), 2.83 (s, 6H); C
NMR (150 MHz, DMSO-d6): δ 163.34, 162.61, 151.35, 148.93, 147.75, 140.62,
133.25, 123.25, 122.50, 119.48, 111.87, 38.67; EI-MS: m/z = 424.85 (M+). Anal.
Calcd for C15H16N6O5S2 (424.06): C, 42.45; H, 3.80; N, 19.80; S, 15.11; Found: C,
42.36; H, 3.919; N, 19.66; S, 15.23.
Data for 6l: White solid (56%); Mp: 169-170 ℃; 1H NMR (600 MHz, DMSO-d6) δ
11.75 (s, 1H), 9.45 (s, 1H), 8.13 (s, 1H), 8.02 (s, 1H), 7.58 (d, J=8.0 Hz, 1H), 7.45 (s,
13
1H), 7.29 (s, 1H), 2.88 (s, 6H); C NMR (150 MHz, DMSO-d6): δ 162.33, 156.60,
149.16, 140.07, 139.43, 136.41, 133.58, 131.53, 130.87, 126.06, 120.38, 119.43,
110.54, 38.77; EI-MS: m/z = 528.01 (M+). Anal. Calcd for C15H13Cl3N6O5S2 (527.94):
C, 34.13; H, 2.48; N, 15.92; S, 12.15; Found: C, 34.16; H, 2.254; N, 15.97; S, 11.02.
Data for 6m: White solid (47%); Mp: 214-216 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 11.75 (s, 1H), 9.45 (s, 1H), 8.11 (s, 1H), 8.05 (s, 1H), 7.54 (d, J=8.4 Hz, 1H), 7.21

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13
(d, J=11.4 Hz, 1H), 7.14 (d, J=8.4 Hz, 1H), 2.88 (s, 6H); C NMR (150 MHz,
DMSO-d6): δ 162.30, 156.54, 156.02, 140.04, 139.87, 139.81, 136.56, 130.44, 125.84,
123.54, 123.45, 116.03, 110.65, 38.75; EI-MS: m/z = 510.02 (M+). Anal. Calcd for
C15H13Cl2FN6O5S2 (509.97): C, 35.23; H, 2.56; N, 16.44; S, 12.54; Found: C, 35.49;
H, 2.659; N, 16.71; S, 12.38.
Data for 6n: White solid (38%); Mp: 126-128 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 12.05 (s, 1H), 9.48 (s, 1H), 8.21 (s, 1H), 8.13 (s, 1H), 7.46 (s, 2H), 2.92 (s, 6H); 13C
NMR (150 MHz, DMSO-d6): δ 162.10, 156.00, 140.78, 140.05, 133.66, 133.52,
130.81, 126.30, 119.17, 118.33, 111.43, 38.78; EI-MS: m/z = 562.09 (M+). Anal.
Calcd for C15H12Cl4N6O5S2 (561.90): C, 32.04; H, 2.15; N, 14.95; S, 11.41; Found: C,
32.28; H, 2.316; N, 14.83; S, 11.44.
Data for 6o: White solid (72%); Mp: 145-146 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 11.38 (s, 1H), 9.43 (s, 1H), 7.79 (s, 1H), 7.40 (s, 2H), 7.16 (s, 1H), 7.07(d, J=7.8 Hz,
1H), 6.88 (d, J=7.2 Hz, 1H), 2.98 (s, 6H); 13C NMR (150 MHz, DMSO-d6): δ 162.38,
151.09, 148.24, 134.44, 130.64, 129.18, 128.77, 124.81, 123.14, 121.85, 121.33,
120.40,107.41, 38.68; EI-MS: m/z = 527.26 (M+). Anal. Calcd for C16H14Cl3N5O5S2
(526.95): C, 36.48; H, 2.68; N, 13.29; S, 12.17; Found: C, 37.19; H, 2.256; N, 14.43;
S, 12.93.
Data for 6p: White solid (63%); Mp: 136-137 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 11.79 (s, 1H), 9.48 (s, 1H), 7.79 (s, 1H), 7.44 (s, 2H), 7.32 – 7.28 (m, 1H), 6.58 (d,
J=9.0Hz, 1H), 2.93 (s, 6H); 13C NMR (150 MHz, DMSO-d6): δ 162.10, 151.08,
150.23, 142.42, 136.62, 130.61, 129.03, 128.82, 127.64, 122.65, 120.87, 115.92,
38.69; EI-MS: m/z = 561.08 (M+). Anal. Calcd for C16H13Cl4N5O5S2 (560.91): C,
34.24; H, 2.33; N, 12.48; S, 11.43; Found: C, 34.70; H, 2.256; N, 12.23; S, 11.93.
Data for 6q: White solid (66%); Mp: 140-141 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 11.35 (s, 1H), 9.43 (s, 1H), 7.89 (s, 1H), 7.41 (m, 2H), 7.16 (s, 1H), 7.05 (d, J=8.4
Hz, 1H), 6.85 (d, J=9.0 Hz, 1H), 2.87 (s, 6H); 13C NMR (150 MHz, DMSO-d6): δ

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162.39, 152.24, 148.95, 148.35, 134.43, 133.40, 129.72, 129.00, 124.68, 123.20,
121.87, 121.35, 120.20, 114.08 38.70; EI-MS: m/z = 571.00 (M+). Anal. Calcd for
C16H14BrCl2N5O5S2 (570.90): C, 33.64; H, 2.47; N, 12.26; S, 11.23; Found: C, 33.62;
H, 2.454; N, 12.41; S, 11.50.
Data for 6r: White solid (57%); Mp: 136-137 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 11.47 (s, 1H), 9.44 (s, 1H), 8.05 (s, 1H), 7.66 (d, J=8.4 Hz, 1H), 7.44 (s, 1H), 7.30
13
(d, J=8.4 Hz, 1H), 7.24 (s, 1H), 6.88 (d, J=8.4 Hz, 1H), 2.89 (s, 6H); C NMR (150
MHz, DMSO-d6): δ 162.39, 155.57, 148.96, 146.92, 135.59, 128.40, 126.44, 125.66,
125.40, 125.17, 124.65, 123.62, 123.23, 122.81, 121.64, 117.76, 38.68; EI-MS: m/z =
558.96 (M+). Anal. Calcd for C17H14Cl2F3N5O5S2 (558.98): C, 36.44; H, 2.52; N,
12.50; S, 11.44; Found: C, 36.35; H, 2.712; N, 12.75; S, 11.30.
Data for 6s: White solid (52%); Mp: 141-142 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 11.48 (s, 1H), 9.47 (s, 1H), 8.07 (s, 1H), 7.66 (d, J=8.4 Hz, 1H), 7.45 (s, 1H), 6.76
13
(d, J=9.0 Hz, 2H), 2.89 (s, 6H); C NMR (150 MHz, DMSO-d6): δ 162.13, 154.73,
146.56, 142.00, 141.42, 136.90, 133.84, 128.92, 128.82, 126.35, 125.14, 125.00,
124.54, 122.76, 122.44, 120.90, 120.06, 115.23, 38.68; EI-MS: m/z = 595.21 (M+).
Anal. Calcd for C17H13Cl3F3N5O5S2 (594.93): C, 34.33; H, 2.20; N, 11.77; S, 10.78;
Found: C, 35.09; H, 2.275; N, 11.49; S, 10.93.
Data for 6t: White solid (55%); Mp: 144-146 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 11.39 (s, 1H), 9.43 (s, 1H), 7.78 (s, 1H), 7.39 (d, J=7.8 Hz, 1H), 7.22 (d, J=12.0 Hz,
1H), 7.14 (d, J=9.0 Hz, 1H), 7.02 (d, J=8.4 Hz, 1H), 6.93 (d, J=8.4Hz, 1H), 2.89 (s,
6H); 13C NMR (150 MHz, DMSO-d6): δ 162.41, 153.84, 152.18, 151.49, 148.78,
139.52, 134.64, 130.46, 128.97, 128.57, 124.49, 122.07, 119.66, 117.98, 110.34,
110.20, 38.35; EI-MS: m/z = 509.09 (M+). Anal. Calcd for C16H14Cl2FN5O5S2
(508.98): C, 37.65; H, 2.77; N, 13.72; S, 12.57; Found: C, 37.73; H, 2.387; N, 13.99;
S, 12.31.

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1
Data for 6u: White solid (57%); Mp: 161-163 ℃; H NMR (600 MHz, DMSO-d6):
δ 11.80 (s, 1H), 9.46 (s, 1H), 7.78 (s, 1H), 7.33 (d, J=9.0 Hz, 1H), 7.10 (t, J=4.8 Hz,
2H), 6.86 (d, J=8.0 Hz, 1H), 2.91 (s, 6H); 13C NMR (150 MHz, DMSO-d6): δ 162.12,
156.05, 154.39, 152.00, 148.95, 135.96, 130.41, 128.80, 128.02, 126.53, 122.79,
116.53, 104.91, 104.76, 38.62; EI-MS: m/z = 527.27 (M+). Anal. Calcd for
C16H13Cl2F2N5O5S2 (526.97): C, 36.37; H, 2.48; N, 13.26; S, 12.14; Found: C, 36.36;
H, 2.549; N, 13.37; S, 12.32.
Data for 6v: White solid (36%); Mp: 193-195 ℃; 1H NMR (600 MHz, DMSO-d6):
13
δ 11.62 (s, 1H), 9.42 (s, 1H), 7.79 (s, 2H), 7.30 (s, 2H), 2.90 ppm (s, 6H); C NMR
(150 MHz, DMSO-d6): δ 162.13, 148.98, 147.05, 144.49, 134.78, 129.71, 129.61,
126.72, 126.16, 121.26, 38.63 ppm; EI-MS: m/z = 595.05 (M+). Anal. Calcd for
C16H12Cl5N5O5S2 (594.87): C, 32.26; H, 2.03; N, 11.76; S, 10.77; Found: C, 32.50; H,
2.160; N, 11.56; S, 10.94.
Data for 6w: White solid (35%); Mp: 191-193 ℃; 1H NMR (600 MHz, DMSO-d6):
13
δ 11.61 (s, 1H), 9.43 (s, 1H), 7.84 (s, 2H), 7.01 (d, J=10.1 Hz, 2H), 2.88 (s, 6H); C
NMR (150 MHz, DMSO-d6): δ 162.10, 154.07, 152.44, 149.01, 147.64, 133.72,
130.70, 129.64, 129.38, 127.91, 105.33, 107.17, 38.62; EI-MS: m/z = 563.13 (M+).
Anal. Calcd for C16H12Cl5F2N5O5S2 (562.93): C, 34.15; H, 2.15; N, 12.44; S, 11.40;
Found: C, 34.02; H, 2.326; N, 12.71; S, 11.43.
Data for 6x: White solid (43%); Mp: 212-213 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 11.21 (s, 1H), 9.42 (s, 1H), 7.92 (s, 2H), 7.37 (s, 1H), 7.04 (d, J=9.0 Hz, 1H), 6.64
13
(d, J=8.4 Hz, 1H), 2.85 (s, 6H); C NMR (150 MHz, DMSO-d6): δ 162.44, 149.19,
148.89, 145.50, 132.76, 131.67, 130.00, 129.67, 123.94, 122.18, 121.73, 115.43.
38.66; EI-MS: m/z = 561.14 (M+). Anal. Calcd for C16H13Cl3N5O5S2 (560.91): C,
34.24; H, 2.33; N, 12.48; S, 11.43; Found: C, 34.07; H, 2.348; N, 12.58; S, 11.71.
Data for 6y: White solid (75%); Mp: 141-143 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 11.35 (s, 1H), 9.44 (s, 1H), 8.11 (d, J=7.8 Hz, 1H), 7.99 (d, J=7.8 Hz, 1H), 7.72 (d,

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J=8.0 Hz, 1H), 7.60 (d, J=7.8 Hz, 2H), 7.43-7.40 (m, 2H), 7.17 (s, 1H), 7.10 (s, 1H),
6.72 (d, J=7.8 Hz, 1H), 2.88 (s, 6H); 13C NMR (150 MHz, DMSO-d6): δ 162.49,
152.52, 149.32, 148.98, 134.95, 134.12, 128.29, 127.36, 126.81, 126.38, 125.50,
125.18, 123.79, 123.37, 122.12, 121.56, 111.69, 38.68; EI-MS: m/z = 507.26.(M+).
Anal. Calcd for C20H18ClN5O5S2 (507.04): C, 47.29; H, 3.57; N, 13.79; S, 12.62;
Found: C, 47.23; H, 3.769; N, 14.04; S, 12.89.
Data for 6z: White solid (75%); Mp: 172-175 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 11.82 (s, 1H), 9.47 (s, 1H), 8.34-8.32 (m, 1H), 7.97(t, J=8.0 Hz, 1H), 7.65-7.62 (m,
3H), 7.62 (d, J=9.6 Hz, 2H), 7.32 (d, J=6.6 Hz, 1H), 6.37 (d, J=7.8 Hz, 1H), 2.86 (s,
6H); 13C NMR (150 MHz, DMSO-d6): δ 162.19, 152.23, 149.13, 143.47, 136.11,
134.83, 129.51, 128.11, 127.42, 126.62, 126.18, 124.35,122.92, 121.70, 121.16,
107.03, 38.63; EI-MS: m/z = 542.70.(M+). Anal. Calcd for C20H17Cl2N5O5S2 (541.00):
C, 44.29; H, 3.16; N, 12.91; S, 11.82; Found: C, 44.22; H, 3.010; N, 12.97; S, 12.05.
1
Data for 6I: White solid (78%); Mp: 158-160 ℃, H NMR (600 MHz, DMSO-d6): δ
11.76 (s, 1H), 9.47 (s, 1H), 8.29 (s, 1H), 7.99 (s, 1H), 7.67 (d, J=7.8 Hz, 1H), 7.62 (s,
13
2H), 7.36 (s, 1H), 7.14 (d, J=9.6 Hz, 2H), 6.63 (d, J=7.8 Hz, 1H), 2.92 (s, 6H); C
NMR (150 MHz, DMSO-d6): δ 162.19, 156.61, 154.97, 153.38, 149.10, 135.55,
134.70, 128.11, 127.47, 126.73, 126.16, 124.35, 123.19, 121.50, 107.40, 105.19,
38.65; EI-MS: m/z = 509.23.(M+). Anal. Calcd for C20H17F2N5O5S2 (509.06): C, 47.15;
H, 3.36; N, 13.75; S, 12.59; Found: C, 47.16; H, 3.592; N, 13.76; S, 12.60.
2.3 Enzyme assay
The preparation of succinate-cytochrome c reductase (SCR, mixture of respiratory
complex II and bc1 complex) from porcine heart was essentially as reported.41 The
activity of SCR was measured by monitoring the increase of cytochrome c at 550 nm,
by using the extinction coefficient of 18.5 mM-1 cm-1. The succinate–ubiquinone
reductase (complex II) activity was measured by monitoring the decrease of
2,6-dichlorophenolindophenol (DCIP) at 600 nm, by using the extinction coefficient
of 21 mM-1 cm-1. The reaction mixture may be scaled down to 1.8 mL with final

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concentrations of PBS (pH 7.4), 100 mM; EDTA, 0.3 mM; succinate, 20 mM;
oxidized cytochrome c, 60µM (or DCIP, 53 µM ); and appropriate amounts of enzyme
to start the reaction.42
2.4 Determination of median effective concentration (EC50)
EC50 was determined by bacterial growth inhibition according to literature method
with slight modification.43 The bacteria were grown in nutrient broth (NB) at 28 ℃ to
late logarithmic growth phase, and the suspension was diluted to a density of
approximately 107 CFU ⁄ mL. Aliquots (100 µL ) of the suspension were added to 25
mL of NB culture in 50-ml Erlenmeyer flasks containing various concentrations of the
test compounds, and flasks were placed on an orbital shaker (28 ℃, 170 rpm). When
the concentration of bacterial suspension in the control flask increased to
approximately 108 CFU ⁄ mL, the values of optical density of bacterial suspension in
all flasks were measured with a nephelometer (WCY-WOG; Baoli, Beijing, China).
The toxicity regression equation was deduced with the values of optical density, and
the EC50 was determined.
3. Results and Discussion
3.1 Chemistry
The designed compounds 1,2,4-triazole-1,3-disulfonamide derivatives were
synthesized as shown in Scheme 1. The target molecules consist of two parts: the
triazole-disulfonamides unit and the diphenyl ether moiety. Firstly, triazolesulfonyl
chloride was prepared in three steps via 3-mercapto-1,2,4-triazole as the starting
material. The reaction of 3-mercapto-1,2,4-triazole with benzenesulfonylchloride
affords the intermediate triazolesulfonothioate, which react with another molecular
thiol to generate the symmetric disulfide ether in the presence of the pyridine as a
base. The disulfide ether was then treated with N, N-dimethylsulfamoyl chloride in
the presence of potassium carbonate to give bis[1-(N,N-dimethylsulfamoyl)-
1,2,4-triazole-3-yl]disulfide. Thereafter, the oxidation of the disulfide was performed
by bubbling chlorine gas in the acetic acid solvent. It conveniently gives access to the
triazolesulfochloride 3.

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The diphenyl ether moiety was prepared by nucleophilic reaction of appropriate
substituted phenol with para-nitro fluorobenzene to generate the intermediate 4,
which was reduced with palladium catalytic hydrogenation to produce the
corresponding aromatic amine 5. The triazolesulfochloride and varied diphenyl ether
was assembled to furnish the designed product 6 in good to excellent yield.
Scheme 1. Synthetic route of the designed compounds.
3.2 Inhibition activities of compounds against porcine succinate-cytochrome c
reductase
(SCR),
succinate-ubiquinone
oxidoreductase
(SQR)
and
ubihydroquinone-cytochrome (cyt) c oxidoreductase (cyt bc1)
The in vitro activities of the prepared compounds were assayed against porcine
succinate-cyctochrome reductase (SCR), which compose of respiratory complex II
（SQR） and complex III (bc1 complex), and they also deemed to form complex
II-complex III supercomplexes. Complex II (SQR) firstly passes electrons from
succinate to ubiquinone, and then the cytochrome bc1 complex passes electrons from
reduced ubiquinone to cytochrome c. The activity of complex II in SCR was
selectively determined using succinate and dichlorophenolindophenol (DCIP) as
substrates, and the activity of only the cytochrome bc1 complex in SCR was
determined using decylubiquinol (DBH2) and cytochrome c as substrates, whereas the
overall activity of SCR (both complex II and bc1 complex) was determined using
succinate and cytochrome c as substrates.

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The inhibition results against SCR derived from porcine heart mitochondria were
listed in Table 1. For clarity, the two benzene rings of the biphenyl ether component
were marked as A and B, respectively. The activities of these prepared compounds
were varied significantly depending on the substituted pattern of the biphenyl ether
moiety. Although some analogues were inactive, most of them showed good to
excellent inhibitory capability toward bc1 complex compared to the commercial
control amisulbrom, which exhibited an IC50 value of 93.0 µM . Some conclusion
about the structure activity relationship can be drawn based on the biological results.
It was clear that the feature of phenyl ring B affect the inhibition activity dramatically.
When there is no further substituent on phenyl ring B other than phenoxyl and
triazolesulfonamidyl, the introduction of electron-withdrawing substituent such as
halogen (6a and 6c) or trifluromethyl (6b) but not cyano group (6h) on the para
position of the phenoxyl ring seems favorable to maintain bc1 complex inhibition
since compounds with substitution on other position (ortho or meta) all showed much
less activity regardless of electron-donating or electron-withdrawing groups were
introduced. Interestingly, substituting a halogen atom to the ortho position of
phenoxyl group on phenyl ring B leads to considerable improvement in the bc1
inhibition. For example, the activity of compound 6o (IC50 = 9.6 µM ) with chlorine
tied at the ortho position of phenoxyl group displayed 3-fold enhancement over that
of the unsubstituted compound 6c (IC50 = 28.8 µM ). Similarly, compound 6r also
showed a little higher inhibition activity (IC50 = 15.5 µM ) as compared to compound
6b (IC50 = 27.4 µM ) without chlorine substitution on the ortho position of phenyl ring
B. It was worthy to note that the activity was further improved by introducing another
halogen substituent on ring B. For instance, the activity of compound 6p and 6s
bearing dichloro-substituent of phenyl ring B increased a further 2-fold and 3-fold
over compound 6o and 6r with monochloro-substitution, respectively. When the
substituted pattern of ring B was fixed with two chlorine substituents, 2,4,6-trichloro
substituted phenoxyl was selected as ring A, the most potent compound 6v with IC50
value of 3.2 μM was discovered. In comparison, chlorine substitution is superior to
fluorine substitution on ring B. when the substituent was changed from chlorine (6o)

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to fluorine (6t), the bc1-inhibiting activity was reduced sharply with IC50 value
decreased from 9.6 μM to 37.6 μM, nearly 4-fold reduction. Similarly, difluoro
substituted compound (6u) also displayed higher activity than monofluorine
substituted counterpart (6t). These observations demonstrated that the substituted
pattern of phenyl ring B played a crucial role for their interaction with the target
enzyme. Further investigation indicated that phenyl ring A can be replaced with more
bulky group such as naphthyl without affecting their activity when compared the
activity of compounds 6z and 6I with that of compounds 6p and 6u. However, when
heterocycle such as substituted pyridyl was selected instead of phenyl ring A, the bc1
inhibition activity was decreased significantly and some of them even lost activity.
Because SCR is composed of respiratory complex II (SQR) and complex III (bc1
complex), it is very interesting to further determine which one, SQR or bc1, is
responsible for the inhibition activity against SCR system. Then, those inhibitors with
IC50 values less than 100 µM were assayed against SQR and bc1 alone. Interestingly,
apart from several compounds such as 6a, 6b, 6c, 6n and 6w, which did not show
significant inhibition effect toward bc1, most compounds showed characteristics of
dual inhibitors of SQR and bc1. Additionally, the inhibition potency against SQR is
usually over one order of magnitude higher than that to complex bc1. Their IC50
values against porcine SQR ranged from 0.2 to 43.1 µM , whereas the IC50 values
against complex bc1 varied from 35.5 to 105.0 µM . These results might indicate that
the inhibition against succinate-cyctochrome reductase (SCR) is mainly attributed to
the interruption of the electrons transfer from succinate to ubiquinone in SQR.
Considering the inhibition activity against SQR system, it can be concluded that the
IC50 values are generally smaller than those against SCR system. Nevertheless, the
interaction regularity between the inhibitors and SQR and the tendency of their
inhibitory potency are consistent with that of SCR system. For instance, introduction
of halogen atom such as chlorine or fluorine to the ortho position of phenoxyl group
on phenyl ring B is favorable to the activity against SQR. This is evidenced by the
chlorinated inhibitor 6o (IC50 = 4.3 µM ) and 6r (IC50 = 2.7 µM ) whose IC50 are 5-fold

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and 4-fold higher as compared to inhibitor 6c (IC50 = 23.0 µM ) and 6b (IC50 = 9.9
µM ). Similarly, the activity of dihalogenated inhibitor such as 6p, 6s and 6u are
superior to the counterpart monohalogenated inhibitors 6o, 6r and 6t.
Table 1 Inhibitory activity of the synthesized compounds against porcine SCR, SQR
a
and cyt bc1
O
R1
H
O
R2
O
N S
R4
N
A
B
N
N
SO2N(CH3)2
R3
6
IC50 (µM)
Entry
R1
R2 R3
SCR
SQR
cyt bc1
6a
6b
6c
6d
6e
6f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
F
81.8±2.1
27.4±1.0
28.8±1.1
>100
43.1±2.1
1%a
9.9 ± 1.1
23%a
3%a
23.0± 1.3
-
-
>100
-
-
>100
-
-
6g
6h
6i
>100
-
-
>100
-
-
>100
-
-
-
6j
>100
-
6k
6l
>100
-
-
>100
-
-
6m
6n
6o
6p
6q
6r
>100
-
-
Cl Cl
Cl
Cl Cl
37.8±3.8
9.6±1.2
5.0±0.8
17.6±2.6
15.5±2.0
5.3±1.1
37.6±1.8
12.5±1.2
3.2±0.1
15.8±1.2
14.7±1.1
16.6±1.1
10.7± 1.1
4.3 ± 1.2
0.2 ± 0.1
3.1 ± 1.5
2.7 ± 1.9
0.3 ± 0.1
9.5 ± 1.1
2.5 ± 1.2
0.5± 0.0
0.9 ± 0.1
3.8 ± 1.2
3.3 ± 1.1
5%a
40%a
H
52.6±1.5
105.0± 1.0
39%a
H
H
Cl
Cl
6s
6t
Cl Cl
51.8 ± 1.0
37%a
H
F
F
F
6u
6v
6w
6x
6y
64.9 ± 1.2
35.5± 1.0
0
Cl Cl
F
H
H
F
Cl
Cl
55.1 ± 1.1
45.7 ± 1.1

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6z
6I
Cl Cl
5.9±1.2
9.5±1.1
0.7± 0.1
49.2 ±1.1
58.2± 1.1
F
F
1.8± 1.1
93.0±1.3
0.033×10-3±0.00027
a the inhibition ratio was determined at the concentration of 100 µM .
0
-
29%a
0.26×10-3±0.046
amisulbrm
Antimycin
3.3 Inhibition activities of selected compounds against plant-pathogenic
bacterium Xanthomonasoryzae pv. oryzae
Bacterial blight of rice, caused by Xanthomonasoryzae pv. oryzae is one of the
bacterial diseases of rice in many rice-growing regions of the world including
southern China.[44] The inhibitory potency of some selected compounds against
Xanthomonasoryzae pv. oryzae were evaluated at a concentration of 20 µg/mL and,
the results listed in Table 2 indicated that these compounds displayed varied
antibacterial activity with the inhibition ratios ranging from 1.5% to 95.6% depending
on the substitution pattern of the diphenyl ether component of the inhibitors. It is
difficult to draw some reliable conclusions about the structure-activity relationship
according to these results. Evidently, substituting a cyano group on aromatic ring A is
unambiguously facilitate the antibacterial activity since compound 6g with a
ortho-cyano group and compound 6h with a para-cyano substituent are showed
94.7% and 94.3% inhibition potency, respectively. Furthermore, benzyloxyl
substituent also demonstrated its importance for conserve inhibition activity against
Xanthomonasoryzae pv. oryzae because compound 6j with benzyloxyl group on the
meta-position of phenyl-ring A exhibited 95.6% inhibition ratio. However, when the
benzyloxyl substituent was moved from the meta-position to para-position in
phenyl-ring A, the resulting compound 6i produced much lower activity. The analog
6u bearing a 2,4-dichlorinated phenyl-ring A is also favorable to antibacterial activity.
Other halogenated samples such as 6v, 6s and 6e showed moderate inhibition rate,
namely 73.0%, 58.5% and 56.5% respectively.
The preliminary antibacterial assay against Xanthomonasoryzae pv. oryzae at the
concentration of 20µg/mL revealed that compounds 6g, 6h, 6j and 6u showed
excellent potency. Therefore, these four compounds were selected for further

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evaluation. Bismerthiazol, the most frequently used effective bactericide which has
both protective and curative activity against Xanthomonasoryzae pv. oryzae, was
selected as positive control. The EC50 values were determined based on in vitro
inhibition of bacterial growth according to a previous study.[44]
As listed in Table 2, compound 6h (EC50 = 6.62 µg/mL) and 6j (EC50 = 6.76 µg/mL)
exhibited better antibacterial activity against Xanthomonasoryzae pv. oryzae than that
of bismerthiazol (EC50 = 12.46 µg/mL). Whereas compounds 6g and 6u are much less
active, their EC50 values are 45.07 µg/mL and 78.72 µg/mL, respectively.
Impressively, compound 6j showed more than 10-fold higher antibacterial activity
than bismerthiazol when compared their EC90 values. However, compound 6h, which
has the highest EC50 value, displayed more than 4-fold lower EC90 values than the
positive control bismerthiazol. The other two compounds, 6g and 6u showed much
less active with regard to the EC90 values. It is worthy to note that three of the four
compounds namely 6g, 6h and 6j, which showed good antibacterial activity against
Xanthomonasoryzae pv. oryzae, exhibited much lower inhibition potency toward SCR
in terms of their respective IC50. At the same time, some compounds such as 6p, 6s
and 6v, displaying excellent SCR inhibitory activity, are inactive against
Xanthomonasoryzae pv. oryzae. One possible reason may due to the inhibitors must
suffer from complicated biological process such as absorption, distribution,
metabolism and excretion property, resulting some inhibitors with high enzyme
inhibition inactive during in vivo evaluation, alternatively, enzymes/protein other than
SCR that interact with the inhibitors may account for the antibacterial activity.
Table 2 Inhibition effect of selected compounds against x. oryzae pv. oryzae
Compd.
Inhibition ratio
toxic regression equation EC50 (µg/mL) EC90 (µg/mL)
（%, 20 µg/mL）
6a
6b
6e
6f
11.2
1.5
56.5
74.5
94.7
94.3
95.6
6g
6h
6j
y = 0.4401x + 4.6404
y = 0.5543x + 4.5449
y = 1.8401x + 3.4724
45.07
6.62
6.76
36382.71
1357.89
33.63

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6n
8.7
6r
34.3
58.5
93.1
73.0
6s
6u
6v
y = 0.6081x + 4.2194
y = 0.8855x + 4.0297
78.72
12.46
14274.24
349.169
Bismerthiazol
4. Conclusion
In conclusion, a new series of 1,2,4-triazole-1,3-disulfonamide derivatives were
designed and synthesized by coupling diverse diphenyl ether moiety with
triazolesulfonamide unit. The in vitro bioassay results indicated that these newly
prepared compounds exhibited varied inhibition toward porcine succinate-
cyctochrome reductase (SCR) dependent on the substituted pattern of the diphenyl
ether moiety. Further evaluation against respective SQR and cyt bc1 indicated that
most of the title compounds are dual inhibitors of SQR and bc1 complex activity. In
general, the potency of these dual inhibitors against SQR is much higher than that of
cyt bc1, showing the SCR inhibition might be contributed greatly from the SQR
inhibition. Notablely, placing the halogen substituent adjacent to the phenoxyl group
on the phenyl ring B is crucial in determining the inhibitory activity. Compounds 6p,
6s, 6v and 6z, in which dichloro-substitutent was introduced in the middle phenyl ring
B displayed good succinate-cyctochrome reductase inhibition with the IC50 values of
5.0, 5.3, 3.2 and 5.9 µM, respectively. In comparison, the commercial control showed
much lower inhibition activity with the IC50 value of 93.0 µM. Further antibacterial
assay against Xanthomonasoryzae pv. oryzae indicated that four compounds 6g, 6h,
6j and 6u showed excellent potency at the concentration of 20 µg/mL. In particular,
6h and 6j exhibited much better antibacterial activity than the commercial control
bismerthiazol in terms of their EC50. Impressively, 6j has an EC90 of 33.62 µg/mL,
more than 10-fold higher than that of bismerthiazol, may recognize as one potential
fungicide for combat bacterial blight of rice, the bacterial diseases of rice caused by
Xanthomonasoryzae pv. oryzae.

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ACKNOWLEDGMENT
The research was supported by the National Key Technologies R&D Program
(2011BAE06B05) and the National Natural Science Foundation of China (No.
21332004 and 21372094).
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